key: cord- -xw nz g authors: koskiniemi, marjaleena; rautonen, jukka; lehtokoski‐lehtiniemi, eeva; vaheri, antti title: epidemiology of encephalitis in children: a ‐year survey date: - - journal: ann neurol doi: . /ana. sha: doc_id: cord_uid: xw nz g four hundred five children from the helsinki area who were month to years old were treated for acute encephalitis at the children's hospital, university of helsinki, from january through december . encephalitis occurred most commonly in children to . years of age, among whom the incidence was . per , child‐years. the incidence remained quite high until the age of years, and then gradually declined to . per , child‐years at the age of years. since , when mumps, measles, and rubella vaccination eradicated the encephalitides associated with these microbes, the major associated agents have been varicella‐zoster, mycoplasma pneumoniae, and respiratory and enteroviruses. in infants younger than year of age, the major agents were enteroviruses, herpes simplex virus, and the group of “others,” whereas in older children, respiratory viruses and mycoplasma pneumoniae as well as varicella‐zoster virus, dominated. in children aged to months, the causal agent could not be identified in one‐half of all cases, whereas in children who were at least years old, the etiology remained unknown in only one‐fourth of cases. male dominance was most evident in the ‐ to ‐year age group. the difference in etiology between males and females was significant (p = . ); mumps and varicella were more common in boys, and adenovirus and mycoplasma pneumoniae were more common in girls. the overall male‐to‐female ratio was . : . characteristic seasonal variation occurred in encephalitides associated with mumps, measles, and entero‐ and respiratory viruses. in the whole series, some accumulation appeared in february and march. less than one‐half of this number appeared in july and august. for certain severe types of encephalitides, specific therapy is available, but early institution is essential for a favorable outcome. in central nervous system (cns) infections, there appears to be age-dependent and seasonal variation { , ] . knowing this variability may help us to detect the patients with encephalitis and confirm the etiological diagnosis early enough for therapy to be effective. therefore, we have analyzed the incidence and epidemiology of encephalitis in children, in the course of years. our series included children with encephalitis treated at the children's hospital, university of helsinki, during a -year period, january through december , initiated in a prospective manner by our colleagues and continued by us in . fifty-seven patients with encephalitis, transferred from other parts of finland to the children's hospital in helsinki, were excluded from all calculations. thus, only the patients originating from the helsinki area and covered by the university central hospital were included. patients with mild encephalitis may be treated at other governmental hospitals as well, but children with severe disease are referred to the children's hospital from all parts of southern finland, including from a new hospital built in the area in . thus, the incidence figures are underestimates rather than overestimates. acute encephalitis was defined by neurological symptoms lasting for not more than month before admission and not due to purulent, systemic, neoplastic, or other diseases. the most characteristic neurological symptoms were lowered consciousness (disorientation, confusion, somnolence, coma), focal or generalized seizures or both, opisthotonos, pareses, tremors, ataxia, hypotonia, mental changes (aggressiveness, staring, apathy), dizziness, impaired speech, and diplopia. meningeal symptoms and signs or cerebrospinal fluid (csf) alterations were not a prerequisite for diagnosis. neonatal infections (at - weeks) were excluded because they often are generalized and are seldom restricted to the cns. the microbiological diagnosis of encephalitis was regarded as confirmed if the virus or its antigen was detected from brain tissue or csf, or the serumicsf antibody ratio was (normal, - ) ( patients, %). the diagnosis was suggested if in the course of clinical encephalitis there appeared at least a fourfold rise in antibody titer in paired serum specimens ( patients, %) or a high titer ( , occurring in % of all specimens) in one serum specimen ( patients, %), or a positive virus culture or antigen finding in some extracranial source ( patients, %). antigens of herpes simplex virus (hsvj, varicella-zoster virus (vzv), influenza a and b, parainfluenza and , respiratory syncytial virus (rsv), and adenoviruses were detected in a biopsy or throat specimen by the immunofluorescence (if) technique. antibody titers were measured by the complement fixation (cf) technique to the following antigens: adenovirus, coronavirus, coxsackie virus , cytomegalovirus (cmv), epstein-barr virus (ebv), hepatitis b, hsv, influenza a and b, polio, and reoviruses, as well as rsv, vzv, chlamydiagroup antigen, mycoplasma pneumoniae, and toxoplasma gondii antigens. if the patient had a well-defined clinical disease (e.g., mumps, measles, varicella, or rubella) within weeks before o r week after the onset of encephalitis, the clinical disease was considered the cause ( patients, %). in some of these patients, a microbiological test was done. there were patients, males ( %) and females ( ). their ages ranged from month to years, with a mean of . k . years. the patients were divided into the following four groups, according to age: to months ( patients), to years ( patients), to years ( patients), and to years ( patients). the etiology was confirmed or suggested in patients ( %j and remained unknown in patients ( %). the following variables were analyzed: age and sex, microbiological diagnosis, month of onset of the disease, and year of the disease. different age groups and microbiological entities were analyzed both separately and together. the x test was used to assess the significance of the difference in the etiology of encephalitis between males and females. the occurrence of encephalitis was highest in the age groups of children younger than years ( patients, . % of all), whereas the number was very low in the oldest children, to years of age ( patients, . % of all). the incidence was . per , child-years at the age of . to . years, then declined, but was still quite high to the age of and even years. at the age of years, the incidence was . per , child-years. the proportion of children with encephalitis of unknown etiology was consistently at the highest level in the youngest children, among whom one-half of all causes remained unknown, whereas in children at least years of age, the proportion was only one-fourth (figs and ; table) . the known or suggested etiology varied considerably with age. in the to -month age group, the major role was played by enteroviruses and hsv, followed by the group of "others" (this group includes cmv, ebv, rotavirus and reovirus, multiple etiologies, and bacterial and vaccination-associated encephalitides) (see fig ) . the agents of the measlesparotitis-rubella (mpr) group did not occur in children younger than year of age. mumps and measles viruses were previously the major associated agents in preschool and school-aged children, but since their eradication by mpr vaccination, respiratory viruses have dominated in the age group of to years, and vzv in the age group of to years. additionally, adenovirus and m. pneumoniae appeared important agents at these ages. m . pneumoniue was quite evenly distributed over the age scale beginning at year of age (see fig l) , and years of age, but from the age of years on, it was a dominating agent besides vzv. males outnumbered females in encephalitides associated with mumps ( . :l) and vzv ( . : ), and females more frequently had encephalitides associated with adenovirus ( . : ) and m. pneumoniae ( . : ) (see fig ) . the difference was statistically significant ( p = . ). in the series as a whole, males outnumbered females ( . : ). encephalitis occurred in children throughout the year, although an increase was seen in winter and early spring. the incidence was . per , child-years in february (fig ) . the lowest numbers appeared in july and august, . and . per , child-years, respectively. mumps and measles, characteristically, were the most frequent agents in the winter and spring months. respiratory viruses were seen most frequently in february, whereas enteroviruses occurred chieffy during the summer and autumn, especially in august. the occurrence of hsv, vzv, m. pnezlmoniue, and the group of others was quite even throughout the year. vzv remained at the level of about per , child-years. february, september, and december were the primary months for the occurrence of encephalitis with unknown etiologies (see fig ) . the high level of unknown causes in the youngest children aged to months (see table) may reflect underdiagnosis of respiratory virus infections (see fig ) . in children aged to years and to years, the proportion of unknown causes is at its highest in autumn, suggescing that enteroviruses and m. pneumoniue may escape diagnosis. encephalitides are diseases of young children and young adults [i, . the multiplicity of causative and associated agents is much greater in children than in adults, and the agents are more heterogeneous - . in children, hsv is merely one of a wide array of agents, whereas in adults it is the leading cause . the incidence of childhood encephahtis in the present series reached the proportions reported by wang and bortolussi [l] . the excess of encephalitis among the youngest children is more evident in our series than in most others, but similar to that of wang and bortolussi . in our series, male dominance was seen most clearly in mumps and vzv, whereas female dominance is seen in adenovirus and m. pnetlrnoniaeassociated encephalitides. many agents can be eradicated by vaccination. in the early s, polio vanished; and in the early s, it appears that mpr vaccination has eliminated the encephalitides associated with these agents [ , , . thus, the spectrum of cns infections evidently is a changing one [ ], and the total number of encephalitides may have declined. in children younger than year old, however, the number of encephalitides may even be increasing , lo}. furthermore, encephalitis does the most damage in infants [ , . thus, encephalitis in young children needs more attention. the leading causes among childhood encephalitides are now vzv and m. pne.wnoniae. against both of them specific therapy is available, and it is especially important in m. pneurnaniae infections because they are severe [ ) . in m. pneumonzue-associated cns infections, however, we cannot be sure of the most effective therapy. vzv causes a severe disease in children younger than year old f ; our series included only one such patient, and the child made a good recovery. hsv-associated encephalitis, fortunately, is relatively rare in childhood except in infants younger than year old, and the drug of choice is currently acycloguanosine , . besides hsv, enteroviruses play a major role in this age group. for these viruses, no specific therapy is available as yet. diagnosis is important, however, because enteroviruses are common in young infants [ , . these diseases may be caused by viruses and so, basically, be encephalitides. the number of unknown causes in the winter months suggests failure to identify respiratory viruses and adenovirus, and in autumn, underdiagnosis of enteroviruses. despite this, our series reached one of the highest levels in microbiological identification. we admit that the association is not always firm ) and is sometimes coincidental rather than causative, but in cns diseases, direct evidence for causality is difficult to obtam. we need a keener interest in childhood acute encephalopathies, in general. by knowing the age, sex, and season, we may anticipate the exact diagnosis and confirm it by rapid methods. presently, we have antigen tests for respiratory viruses (influenza a and b, rsv, and parainfluenza , , and ), adenoviruses, and herpes viruses (hsv, vzv, and cmv) as well as for ~ . m. pneamoniae. by these tests, the diagnosis is obtained within day, sometimes within hours. in addition to these if techniques, tests based on dna techniques are forthcoming { ] and may be helpful, especially in csf diagnostics. acute viral infection of the central nervous system in children: an %year review acute viral encephalitis in children acute encephalitis. a survey of epidemiological, clinical and microbiological features covering a twelve-year period central nervous system syndromes of "viral" etiology. a study of cases a four-year survey studies on viral encephalitis acute encephalitis of viral origin viral disease of the central nervous system. influence of poliomyelitis vaccination on etiololy effect of measles, mumps vaccination on pattern of encephalitis in children encephalitis and aseptic meningitis clinical predictors of outcome in encephalitis virus meningitis and encephalitis varicella-zoster virus infections vidarabine versus acyclovir therapy in herpes simplex encephalitis herpes simplex encephalitis. vidarabine therapy and diagnostic problems enteroviral disease in the united states - sequelae of central-nervoussystem enteroviral infection etiology and outcome in children with acute nonbacterial meningoenceph-atis social insurance institution of finland. staristics over diseases from the period - smoldering encephalitis in children mycoplama pneumoniue encephalitis: a severe entity in children noninvasive diagnosis of herpes simplex encephalitis we thank prof mind dunner, md, and prof ossi pettay, md, for diagnosing and treating patients with encephalitis at the children's hospital in the s and s, until the years and , respectively. key: cord- -l lkj authors: brouard, j.; vabret, a.; bach, n.; toutain, f.; duhamel, j. f.; freymuth, f. title: prise en charge des pathologies respiratoires à adénovirus chez l’enfant immunocompétent À propos d’une étude rétrospective de enfants hospitalisés date: - - journal: antibiotiques doi: . /s - ( ) - sha: doc_id: cord_uid: l lkj résumé les adénovirus sont une cause commune d’atteinte respiratoire ; bien que dépendant du sérotype, ils peuvent également être la cause d’atteintes extrarespiratoires. le diagnostic positif peut en être difficile. les résultats cliniques chez enfants hospitalisés en raison d’une infection adénovirale ont été repris rétrospectivement. chez enfants, le diagnostic virologique a été obtenu par immunofluorescence directe sur les aspirations nasales, par culture virale de ces mêmes prélèvements. le tableau clinique de l’infection adénovirale est caractérisé par une fièvre élevée (moyenne ° c) et prolongée (durée moyenne , jours). l’atteinte des voies aériennes supérieures (rhinopharyngite, angine, otite) et des voies aériennes inférieures (bronchite, bronchiolite, pneumopathie) sont les plus fréquentes. douze enfants ont présenté des convulsions hyperpyrétiques, avaient une méningite lymphocytaire. les examens complémentaires ont objectivé des valeurs allant de la normalité à celles évocatrices d’infection bactérienne. cinquante-neuf enfants furent adressés pour fièvre résistante à une antibiothérapie. les symptômes de l’atteinte respiratoire dues aux infections adénovirales s’étendent de la rhinite à la pneumopathie et la bronchiolite. les adénovirus peuvent entraîner des séquelles graves même chez l’enfant sain. les recherches sur les mécanismes moléculaires de l’infection virale sur les voies aériennes amèneront d’importantes voies de réflexion sur la nature des processus inflammatoires participant à l’asthme et à la bronchite chronique obstructive. la plupart des infections sont modérées et ne nécessitent qu’un traitement symptomatique. il n’existe pas actuellement de traitement antiviral efficace pour les infections adénovirales graves. le diagnostic virologique rapide par l’étude des sécrétions nasopharyngées est d’une grande utilité clinique. abstract adenoviruses most commonly cause respiratory illness; however, depending on the infecting serotype, they may also cause various other diseases. diagnosis may be difficult to achieve. the clinical findings for children hospitalised with adenoviral infection were studied retrospectively. in children, the diagnosis was based on detection of adenovirus antigen in the nasopharyngeal specimens and in children on viral culture. the clinical picture of adenoviral infection was characterised by high-grade (mean ° c) and prolonged fever (mean duration , days). upper respiratory and lower respiratory symptoms were the most common infections. twelve had been admitted to the hospital due to febrile convulsions, had meningitis. laboratory findings varied from normal values to values seen in bacterial infections. thus it was difficult to distinguish adenoviral disease from a bacterial disease. fifty-nine children were referred to the hospital due to infection unresponsive to antimicrobial therapy. symptoms of respiratory infection caused by adenovirus may range from the common cold syndrome to pneumonia, croup and bronchiolitis. adenoviruses can be responsible for severe consequences, even in previously healthy children. studies of the molecular mechanisms of viral infections of the airways could provide important insights into the nature of the inflammatory process involved in asthma and chronic obstructive pulmonary disease. most infections are mild and require no therapy or only symptomatic treatment. there are at present time no recognised antiviral agents that are effective in treating serious adenovirus disease. the rapid detection of adenovirus antigen in nasopharygeal specimens proved to have a great clinical value in the diagnosis. prise en charge des pathologies respiratoires à adénovirus chez l'enfant immunocompétent j. brouard, a. vabret, n. bach, f. toutain, j.f. duhamel, f. freymuth les adénovirus sont une cause commune d'atteinte respiratoire ; bien que dépendant du sérotype, ils peuvent également être la cause d'atteintes extrarespiratoires. le diagnostic positif peut en être difficile. les résultats cliniques chez enfants hospitalisés en raison d'une infection adénovirale ont été repris rétrospectivement. chez enfants, le diagnostic virologique a été obtenu par immunofluorescence directe sur les aspirations nasales, par culture virale de ces mêmes prélèvements. le tableau clinique de l'infection adénovirale est caractérisé par une fièvre élevée (moyenne ° c) et prolongée (durée moyenne , jours). l'atteinte des voies aériennes supérieures (rhinopharyngite, angine, otite) et des voies aériennes inférieures (bronchite, bronchiolite, pneumopathie) sont les plus fréquentes. douze enfants ont présenté des convulsions hyperpyrétiques, avaient une méningite lymphocytaire. les examens complémentaires ont objectivé des valeurs allant de la normalité à celles évocatrices d'infection bactérienne. cinquante-neuf enfants furent adressés pour fièvre résistante à une antibiothérapie. les symptômes de l'atteinte respiratoire dues aux infections adénovirales s'étendent de la rhinite à la pneumopathie et la bronchiolite. les adénovirus peuvent entraîner des séquelles graves même chez l'enfant sain. les recherches sur les mécanismes moléculaires de l'infection virale sur les voies aériennes amèneront d'importantes voies de réflexion sur la nature des processus inflammatoires participant à l'asthme et à la bronchite chronique obstructive. la plupart des infections sont modérées et ne nécessitent qu'un traitement symptomatique. il n'existe pas actuellement de traitement antiviral efficace pour les infections adénovirales graves. le diagnostic virologique rapide par l'étude des sécrétions nasopharyngées est d'une grande utilité clinique. adenoviruses most commonly cause respiratory illness; however, depending on the infecting serotype, they may also cause various other diseases. diagnosis may be difficult to achieve. the clinical findings for children hospitalised with adenoviral infection were studied retrospectively. in children, the diagnosis was based on detection of adenovirus antigen in the nasopharyngeal specimens and in children on viral culture. the clinical picture of adenoviral infection was characterised by high-grade (mean ° c) and prolonged fever (mean duration , days). upper respiratory and lower respiratory symptoms were the most common infections. twelve had been admitted to the hospital due to febrile convulsions, had meningitis. laboratory findings varied from normal values to values seen in bacterial infections. thus it was difficult to distinguish adenoviral disease from a bacterial disease. fifty-nine children were referred to the hospital due to infection unresponsive to antimicrobial therapy. plus de virus antigéniquement distincts, appartenant à genres différents, atteignent en général tout l'arbre respiratoire. leur diagnostic est souvent retardé par l'aspect peu spécifique de leur expression. les adénovirus (adv) humains comprennent sérotypes, ils sont ubiquitaires et responsables d'un large éventail de syndrômes cliniques. quelques sérotypes sont particulièrement en cause dans la gravité de la maladie en phase aiguë même chez l'enfant immunocompétent. la littérature est relativement importante sur les atteintes adénovirales du sujet fragilisé, immunodéprimé ou porteur d'une cardiopathie. elle est par contre plus restreinte pour les enfants a priori sains : or chez eux des altérations bronchiolaires et bronchiques peuvent être à l'origine de lésions définitives parfois d'expression retardée. À partir d'une étude rétrospective personnelle cet article se focalise sur la pathologie respiratoire à adv chez l'enfant immunocompétent. les pneumopathies virales se définissent par l'existence d'une atteinte parenchymateuse : elles ne représentent qu'une faible part des infections respiratoires basses, environ %. l'épidémiologie de quelques repères épidémiologiques syncytial (vrs), à % par les adénovirus (adv), à % par les virus parainfluenza (vpi), % par les virus influenza [ ] . la biologie moléculaire amène des modifications de l'épidémiologie descriptive en raison de sa plus grande sensibilité [ ] . ces techniques permettent également la mise en évidence de co-infection virale [ ] . les atteintes respiratoires à adv les plus fréquentes sont essentiellement endémiques, observées régulièrement d'octobre à mai et liées aux sérotypes , , , et . il s'agit de rhinopharyngites, de conjonctivites, de syndrômes apc (adénopharyngo-conjonctivites), de bronchites et de pneumonies, rarement de détresse respiratoire d'évolution sévère. cependant d'autres sérotypes évoluent seulement sous forme d'épidémies, plutôt hivernales, irrégulières dans le temps, au sein de collectivités de nourrissons, parfois d'enfants plus agés : ils sont parfois marqués par l'apparition de formes graves pour certains sérotypes ( , , , ) . un aspect particulier est représenté par les épidémies d'atteintes respiratoires aiguës chez les recrues militaires, bien décrites aux usa, dont les principaux sérotypes sont le et le [ ] . la prédominance de quelques sérotypes a été soulignée dans certaines zones géographiques [ , ] . la circulation du sousgroupe b avec le génotype h a été documentée lors de bronchiolites et de pneumopathies sévères en argentine [ ] . ce même type a provoqué de redoutables infections nosocomiales avec une létalité approchant % et la présence de séquelles chroniques chez % des survivants [ , ] . récemment ce sous-type a émergé au japon [ ] . il est peut-être important d'être vigilant sur cette épidémiologie car cela pourrait annoncer un glissement du génotype circulant et les adv comptent pour environ % de tous les adv rapportés par l'oms [ ] . caractères des adv : les adv sont des virus à capside nue très résistants dans le milieu extérieur ; il sont présents dans les voies aériennes et dans les selles des sujets infectés. les modes de contamination sont donc soit directs, se faisant par les sécrétions respiratoires, soit indirects par la contamination d'objets ou d'aliments responsables de transmissions nosocomiales. le portage d'adv latents est possible, la contamination peut s'effectuer par des sujets asymptomatiques. une étude a été récemment menée par notre équipe [ ] . cent seize enfants manifestations respiratoires aiguës ches issues de la culture ( / ) a révélé que sérotypes représentent % des isolements : ce sont les sérotypes ( %), ( %), ( %), ( %), ( %), ( %). donc dans cette cohorte près d'un enfant sur cinq présente un sérotype potentiellent grave. une coinfection virale respiratoire est présente fois ( vrs, vpi, rhinovirus, coronavirus), digestive fois ( rotavirus, entérovirus). le motif principal ou le motif associé pour l'admission hospitalière a été le suivant : % fièvre réfractaire à une antibiothérapie ambulatoire (n = ), % toux persistante de plus de trois jours ( ), % malaise ou convulsion ( ), % troubles digestifs ( ), % otite moyenne aiguë ( ) et une cystite hémorragique. lorsqu'elle était présente, la fièvre était élevée (médiane ° ) et prolongée (moyenne , jours). la durée de l'hospitalisation a été en moyenne de , jours (+/- , j). au cours de l'hospitalisation les diagnostics cliniques isolés ou associés ont été : atteinte des voies aériennes supérieures chez % des enfants, atteinte respiratoire basse chez %, gastro-entérites aiguës chez %. douze enfants ont présenté des convulsions, parmi eux avaient une méningite lymphocytaire mais l'identification virale dans le lcr est restée négative. le plus souvent les examens paracliniques peuvent orienter faussement vers une infection bactérienne, par l'élévation modérée des polynucléaires neutrophiles (moyenne = /mm ) et de la c-reactive protein (moyenne = , mg/l), par la mise en évidence d'une bactérie potentiellement pathogène dans des examens de crachats effectués : l'orientation erronée a aussi été liée à la présence d'anomalies radiologiques au sein de % des clichés thoraciques pratiqués. aucune différence significative n'a été mise en évidence entre sérotypes et signes cliniques, gravité (oxygénodépendance), durée de l'hospitalisation. douze enfants ont été réhospitalisés pour atteinte respiratoire, mais les enquêtes virologiques sont restées vaines, aucun de ceux-ci n'avaient initialement un sérotype à risque. les affections respiratoires aiguës furent à l'origine de la découverte des adv et restent la manifestation la plus connue symptoms of respiratory infection caused by adenovirus may range from the common cold syndrome to pneumonia, croup and bronchiolitis. adenoviruses can be responsible for severe consequences, even in previously healthy children. studies of the molecular mechanisms of viral infections of the airways could provide important insights into the nature of the inflammatory process involved in asthma and chronic obstructive pulmonary disease. most infections are mild and require no therapy or only symptomatic treatment. there are at present time no recognised antiviral agents that are effective in treating serious adenovirus disease. the rapid detection of adenovirus antigen in nasopharygeal specimens proved to have a great clinical value in the diagnosis. de l'infection chez l'enfant. la plupart d'entre elles restent modérées et focales, elles ne peuvent être cliniquement distinguées des infections dues à d'autres virus [ ] . le spectre clinique peut être : une pharyngite, une angine, une otite moyenne aiguë, une laryngite, une bronchiolite, une pneumopathie. chez les sujets hospitalisés la fièvre est habituelle ( %), prolongée, en moyenne : , jours (extrêmes de à jours) [ ] . en l'absence d'enquête étiologique spécifique, il est impossible cliniquement ou biologiquement de distinguer une atteinte respiratoire secondaire à un adv d'une autre cause infectieuse [ , ] . les pharyngites et les angines constituent un syndrome clinique fréquent, et les adv peuvent être identifiés chez à % des enfants porteurs d'une pharyngite isolée. elle peut être exsudative et souvent fébrile. la plupart des cas sont dus aux types , , et [ ] . il est difficile de distinguer une amygdalite bactérienne d'une amygdalite virale. l'infection à adv reste une cause fréquente d'angines ou d'otites moyennes aiguës résistantes à l'antibiothérapie, fréquemment adressées alors aux admissions hospitalières [ , ] . les fièvres pharyngo-conjonctivales sont parmi les manifestations cliniques les plus typiques et fréquentes de l'infection à adv [ , ] particulièrement avec les sous-types et . elles associent des signes non spécifique : un syndrome pseudogrippal (fièvre, malaise, myalgies), une toux, une congestion nasale, une angine érythémateuse, des adénopathies cervicales, à une conjonctivite uni ou bilatérale. les adv du sous-groupe c (adv , , , ) rendent compte d'environ à % des infections respiratoires de l'enfant de moins de ans. l'adv est une cause peu fréquente de laryngite. les atteintes par les adv peuvent parfois s'exprimer par une bronchiolite mais également par une maladie plus sévère. formes graves d'infections à adv : les pneumopathies à adv intéressent principalement le nourrisson, l'enfant en bas-âge, le sujet immunodéprimé, le transplanté rénal, médullaire ou pulmonaire et le patient porteur d'une cardiopathie ou d'une atteinte respiratoire chronique [ ] [ ] [ ] . en dehors de ce contexte ces infections respiratoires sont le plus souvent bénignes, mais par-fois cette symptomatologie peut être marquée même chez l'enfant sain [ ] . les sérotypes , et en particulier peuvent être responsables des atteintes respiratoires les plus graves [ , ] . le début est alors brutal avec fièvre élevée, la symptomatologie pulmonaire comporte une toux rebelle, une dyspnée avec tachypnée et tirage, des râles bronchiques et parfois alvéolaires. les signes radiologiques sont souvent évocateurs, associant des infiltrats, une atteinte interstitielle et des adénopathies hilaires ; les épanchements pleuraux ont été décrits dans à % des cas [ , ] . de telles atteintes ont une mortalité d'environ %. après une régression initiale elles font place à une forme plus chronique de bronchiolite définissant la bronchiolite oblitérante. c'est un syndrome relativement rare caractérisé cliniquement par une pneumonie ou une bronchiolite sévère, avec une évolution chron i q u e c o n d u i s a n t s o u v e n t à d e s séquelles permanentes [ , ] . l'histologie du tissu pulmonaire révèle alors des lésions nécrotiques des bronches et de l'épithélium bronchiolaire avec atteinte des glandes bronchiques. une atteinte pulmonaire persistante après une pneumopathie adénovirale à adv est rapportée chez un tiers à deux tiers des sujets [ ] . les lésions les plus graves sont représentées par : les dilatations des bronches, la bronchiolite oblitérante, le poumon clair unilatéral (syndrome de mcleod) ou, rarement, la fibrose pulmonaire [ ] . infections disséminées : les pneumonies adénovirales s'accompagnent parfois d'une infection disséminée redoutable avec atteintes cardiaques (myocardite aiguë, péricardite aiguë), hépatiques, pancréatiques, rénales (glomérulonéphrite aiguë), et quelques signes encéphalitiques s'y associent souvent. la létalité est alors élevée, de l'ordre de % [ ] . absorbé à la surface des cellules de l'épithélium respiratoire, l'adv pénètre dans les cellules et s'y réplique. l'atteinte de l'ensemble de l'arbre respiratoire se réalise surtout de proche en proche, la plupart des infections restent locales dans les voies respiratoires supérieures voire asymptomatiques. plus rarement l'atteinte tissulaire se fait par voie lymphatique et sanguine. les lésions tissulaires résultent soit directement de l'infection par cytotoxicité, soit indirectement par la réponse inflammatoire et immunitaire. cette dernière est essentielle. les séquelles pulmonaires graves surviennent avec une particulière fréquence dans certaines populations homogènes, chez qui un facteur génétique pourrait intervenir mais le sous-type adénoviral garde un rôle essentiel [ , ] . le site préférentiel de réplication des adv est l'épithélium respiratoire, la réplication semblant limitée au tissu lymphoïde, qui représente par contre un lieu essentiel de persistance virale [ ] . les mécanismes pathogéniques de l'infection à adv sont peu connus. l'épithélium respiratoire est modérément atteint (vacuolisation, atteinte de l'épithélium cilié), sans réelle cytolyse mais avec des infiltrats péri-bronchiolaires et péri-vasculaires à prédominance lymphocytaire (t cytotoxique) ainsi que péri-alvéolaires (lymphocytes, macrophages, polynucléaires neutrophiles). il a été démontré que le facteur alpha de nécrose tumorale (tnf-α), l'interleukine (il)- et l'il- sont élaborés durant les deux à trois premiers jours de l'infection mais seul le tnf-α a un rôle majeur dans la phase précoce de la pathogénie. chez l'enfant une élévation des igg contenant des complexes immuns circulants spécifiques ou des valeurs sériques élevées d'il- , d'il- et de tnf-α sont associées significativement aux adénoviroses graves [ ] . À long terme l'infection à vrs est plutôt corrélée avec l'apparition d'un asthme [ ] alors que l'atteinte adénovirale, surtout pour le h, est corrélée avec des séquelles pulmonaires lourdes, dilatations des bronches et poumon hyperclair unilatéral. une équipe a étudié les différences de réponses immunitaires entre ces deux virus chez l'enfant. les cellules mononuclées périphériques (pbmc) produisent davantage d'il- lorsqu'elles ont été infectées par le vrs versus l'adv, alors qu'elles sécrètent plus d'interféron (ifn)-γ lorsqu'elles ont été infectées par l'adv versus le vrs. le ratio il- /ifn-γ est significativement plus bas lors des infections par l'adv, ce qui suggère que l'adv induit une réponse de type th [ ] . l'étude des mécanismes moléculaires par les mutants thermosensibles d'adv , par mutation des gènes des adn-binding protein ou de l'adn polymérase, révèle que la synthèse des protéines structurales et des virions ne sont pas nécessaires au développement des lésions pulmonaires. les pneumopathies à adv sont certainement plus liées à l'expression de certains gènes de ce virus qu'à la réplication virale [ , ] . la réplication de l'adv se déroule en deux étapes successives : phase précoce survenant avant la réplication de l'adn génomique et phase tardive débutant en même temps que cette dernière. les gènes précoces sont lus dès le début de l'infection (e a, e b, e a, e b, e , e ). le gène e a est le premier transcrit et les protéines correspondantes contrôlent la transcription des autres gènes précoces. les protéines des gènes e et e jouent un rôle important dans l'établissement de l'infection lymphocytaire latente à adv et l'échappement des adv aux réponses inflammatoires et immunitaires de l'hôte. ces protéines agissent en inhibant les deux actions antivirales du tnf-α, sur le blocage de la réplication virale et la mort des cellules infectées. l'adn adénoviral peut persister plusieurs années dans le poumon après l'infection aiguë. la protéine adénovirale e a (early region a) est exprimée dans le poumon et est capable d'assurer une dysrégulation majeure de l'expression des cytokines, avec une augmentation de l'expression de l'arn messager de la molécule d'adhésion icam- , de la cytokine inflammatoire il- et ceci implique le facteur de transcription nf-κb [ ] . les infections subaiguës des voies respiratoires par les adv pourraient être impliquées dans la genèse de certaines formes de bronchopathies chroniques obstructives chez l'enfant et chez l'adulte. macek et al. ont rapporté une étude chez des enfants ayant développé un asthme corticorésistant après une bronchiolite [ ] . dans des biopsies bronchiques par bronchoscopie, il a été révélé dans la majorité de ces cas la présence d'une protéine adénovirale de la capside, et l'isolement d'un adv sur culture de cellules a été positive dans certains de ces échantillons. hegele et al. trouvent de plus grandes quantités d'adn adénoviral chez les patients souffrant de bronchopathie chronique obstructive, suggérant le rôle possible de l'adv dans cette maladie [ ] . les observations in vivo et in vitro qui démontrent que l'infection adénovirale latente puisse être associée avec la persistance d'une inflammation à bas bruit dans le parenchyme pulmonaire périphérique, suggèrent un lien causal dans l'installation de la bronchite chronique obstructive [ ] . parallèlement l'inflammation induite par la fumée de tabac est amplifiée chez les sujets emphysèmateux par l'expression latente de la protéine adénovirale e a exprimée par les cellules épithéliales alvéolaires [ ] . récemment, une étude rapporte l'implication possible de l'adv dans le développement de la dysplasie bronchopulmonaire du prématuré [ ] . les aléas du diagnostic étiologique d'une pneumonie chez le jeune enfant conduit quasi systématiquement à l'indication de l'antibiothérapie en raison des risques potentiels de l'abstention thérapeutique [ ] : celle-ci pourra être interrompue en fonction des résultats des examens complémentaires. la disponibilité des outils de diagnostic virologique de lecture rapide, fiables et réalisables au lit du malade, actuellement possible pour le vrs et la grippe, pourrait dans l'avenir modifier cette attitude [ ] . l'abstention d'une antibiothérapie pourrait en effet être justifiée comptetenu des enquêtes épidémiologiques, mais à condition que soit assuré un suivi clinique rapproché et rigoureux. la bronchite aiguë n'associe par définition aucune atteinte du parenchyme pulmonaire. plus encore que les pneumopathies, l'origine virale prédomine, et l'abstention de toute prescription d'antibiotiques est justifiée en l'absence de risque vital de laisser une bactérie se développer. les critères d'hospitalisation lors d'une pneumopathie supposée virale sont communs avec ceux des infections bactériennes. en l'absence de score validé en pédiatrie, ils se fondent sur l'âge (inférieur à mois), l'aspect général (syn-drome toxi-infectieux), une mauvaise tolérance respiratoire (tachypnée, signes de lutte respiratoire), l'existence d'une hypoxie estimée par la détection de la saturation en oxygène par l'oxymétrie de pouls. d'autres indicateurs sont à retenir : les difficultés à s'alimenter (dyspnée lors de la prise des biberons chez le nourrisson), une aggravation rapide de la maladie, les risques liés au terrain (cardiopathie, bronchopathie chronique, déficits immunitaires…), des conditions socio-économiques précaires ou les difficultés d'accès aux soins. en raison de leur persistance durant une à plusieurs semaines dans les sécrétions nasopharyngées des sujets infectés et de leur capacité de survie de plusieurs heures sur des surfaces inertes, les virus à tropisme respiratoire peuvent se transmettre plus particulièrement aux nourrissons immunologiquement naïfs [ ] . il est essentiel de limiter ce risque au cours des hospitalisations, notamment dans les services d'immunodéprimés. la meilleure mesure de prévention reste le lavage des mains. il est recommandé le lavage régulier des surfaces susceptibles d'être contaminées (jouets, table à langer, outre le stéthoscope, otoscope etc) [ ] . l'hydratation, le contrôle thermique, l'oxygénothérapie, la kinésithérapie respiratoire restent la base de la prise en charge des infections bronchopulmonaires virales [ ] . le retentissement sur l'alimentation est directement fonction de l'intensité de la détresse respiratoire (dyspnée, essoufflement) ou secondaire aux vomissements provoqués par la toux. les risques de déshydratation sont présents chez le nourrisson et une perfusion initiale semble préférable à la mise en place d'une nutrition entérale à débit continu lors de la phase aiguë [ ] . l'oxygénothérapie sera initiée lors d'une hypoxémie ; l'oxygène doit être humidifié et administré par lunettes nasales ; son débit sera adapté afin d'obtenir une saturation pulsée en oxygène transcutanée proche de %. c'est une réelle prescription médicale dont les modalités doivent être clairement définies [ ] . l'intensité de la détresse respiratoire, l'hypercapnie, conduit parfois à la réalisation d'une intubation endotrachéale avant mise sous ventilation mécanique. la kinésithérapie respiratoire nous semble justifiée lors de la phase sécrétante, sa technique exige une compétence obtenue par une grande pratique pédiatrique. l'accélération du flux expiratoire avec expectoration provoquée est la technique la plus utilisée. peu de références étayent l'efficacité de cet acte en dehors de la mucoviscidose [ ] . malgré une prescription très large tant ambulatoire qu'hospitalière, il faut bien reconnaître la faiblesse des preuves cliniques concernant les mucomodificateurs [ ] . la toux est un phénomène réflexe protégeant les voies respiratoires basses dont le respect doit être la première attitude. comme pour les mucomodificateurs, peu de preuves existent quant à l'efficacité des antitussifs [ ] . en cas de persistance de la toux, le diagnostic d'infection bronchopulmonaire virale devra être révisé. l'inflammation bronchopulmonaire est particulièrement intense lors d'une infection virale, et elle a un double visage : d'une part, il s'agit d'une réaction normale de l'hôte contre l'infection, d'autre part l'amplification de cette réaction peut conduire à la constitution de lésions cicatricielles. peu d'études pédiatriques randomisées sont disponibles, à l'exception de quelques références concernant la corticothérapie inhalée lors des sifflements associés aux infections virales [ ] mais dont on connaît les relations étroites avec l'asthme [ ] . des vaccins inactivés et vivants expérimentaux ont été développés aux usa pour prévenir les épidémies de pneumonie par les sérotypes et chez les recrues militaires mais n'ont pas été utilisés chez l'enfant, et certaines interrogations sur un éventuel potentiel oncogène exigent de futures recherches [ ] . ces vaccins ne sont pas disponibles en france. on possède peu d'informations sur l'éventuelle efficacité de l'utilisation préventive d'immunoglobulines intraveineuses. elle semble être une aide comme traitement adjuvant lors des infections disséminées du nouveau-né ou de l'immunodéprimé. chez le sujet immunodéprimé les infections adénovirales résultent de la réactivation d'infections latentes du tissu lymphoïde (adv , , ) ou du rein ( adv , , ) . la ribavirine semble avoir une efficacité en aérosol et surtout par voie intraveineuse. les protocoles thérapeutiques ne sont pas fixés [ , ] . quelques essais d'utilisation réussis ont porté sur le cidofovir (hpmc ou cdv) parentéral [ ] . les données chez l'enfant sont encore très fragmentaires. aucune thérapeutique n'a fait ses preuves après le diagnostic de bronchiolite constrictive. les seuls arguments expérimentaux concernent la corticothérapie si celle-ci pouvait être prescrite la veille de l'agression virale [ ] . l'absence d'effet clinique des corticoïdes par voie systémique ou inhalée pourrait être due à une prescription trop tardive, les anomalies anatomiques étant fixées. les bronchodilatateurs inhalés sont également le plus souvent inefficaces [ ] . on ne peut que limiter les rechutes infectieuses, qui aggravent la situation respiratoire, grâce à des mesures de prévention. le diagnostic d'infection respiratoire à adv repose sur la mise en évidence directe du virus ou de ses constituants dans les voies aériennes. la détection de cellules spécifiquement infectées par immunofluorescence signe l'atteinte virale de l'épithélium. mais cette méthode manque de sensibilité. les méthodes d'amplification moléculaire permettent une amélioration de la détection virale mais la détection de séquences d'adv ne traduit pas toujours une infection virale active, sachant cependant, que celle-ci n'est pas indispensable pour initier et entretenir une inflammation persistante des voies respiratoires inférieures. bilan de aspirations nasales réalisées chez l'enfant en une période de six ans viral etiology in acute lower respiratory infections in children from a closed community diagnostic moléculaire des infections virales respiratoires communautaires les co-infections virales lors des bronchiolites du nourrisson immunocompétent strain variation in adenovirus serotypes and a causing acute respiratory disease analysis of different genome types of adenovirus type isolated on five continents adenovirus type associated with severe and fatal acute lower respiratory infections in argentina children adenovirus type h respiratory infections : a report of cases of acute lower respiratory disease molecular and serological characterization of adenovirus genome type h isolated in japan worldwide epidemiology of human adenovirus infections pathologie respiratoire de l'enfant immunocompétent à adénovirus : étude de sujets hospitalisés adenoviral diseases in children : a study of hospital cases comparison of clinical characteristics of adenovirus and non-adenovirus pneumonia in children adenovirus infections in young children adenovirus infection in the immunocompromised patient viral pneumonia adenovirus pneumonia in lung transplant recepients severe adenovirus bronchiolitis in children adenovirus a: a community-acquired outbreak in a children's hospital adenovirus pneumonia adenovirus type b in children's hospital lung function in infants with chronic pulmonary disease after severe adenoviral illness chronic lung damage caused by adenovirus type : a ten-year follow-up study factors predisposing to abnormal pulmonary function after adenovirus pneumonia bronchiolitis obliterans, bronchiectasis, and other sequelae of adenovirus type infection in young children pneumopathie sévère à adénovirus type chez un adulte immunocompétent group c adenovirus dna sequences in human lymphoid cells cytokines in adenoviral diseases in children: association of interleukine- , interleukine- and tumor necrosis factor alpha levels with clinical outcome respiratory syncytial virus in early life and risk of wheeze and allergy by age years differential effects of respiratory syncytial virus and adenovirus on mononuclear cell cytokine responses the molecular basis of adenovirus pathogenesis les pneumonies à adénovirus identification of glucocorticoid-and adenovirus e a-regulated genes in lung epithelial cells by differential display persistent adenoviral infection and chronic airway obstruction in children mechanisms of airway narrowing and hyperresponsiveness in viral respiratory tract infections role of latent viral infections in chronic obstructive pulmonary disease and asthma amplification of inflammation in emphysema and its association with latent adenoviral infection detection of microorganisms in the tracheal aspirates of preterm infants by polymerase chain reaction: association of adenovirus infection with bronchopulmonary dysplasia current management of community-acquired pneumonia in children: an algorithmic guideline recommendation rationalised prescribing for community acquired pneumonia: a closed loop audit nosocomial adenovirus infection in a paediatric respiratory unit l'isolement en pédiatrie les traitements non antibiotiques des pneumopathies communautaires de l'enfant why block a small hole abc of oxygen. acute oxygen therapy does chest physical therapies work? mucomodificateurs et anti-tussifs systematic review of randomised controlled trials of over the counter cough medecines for acute cough in adults effect of inhaled corticosteroids on episodes of wheezing associated with viral infection in school age children: randomised double blind placebo controlled trial rôle des infections virales et des infections à chlamydia pneumoniae et à mycoplasma pneumoniae au cours de l'asthme du nourrisson et du jeune enfant. À propos d'une étude épidémiologique chez enfants vaccines for control of respiratory disease caused by adenoviruses intravenous ribavirin therapy in a neonate with disseminated adenovirus infection undergoing extracorporeal membrane oxygenation: pharmacokinetics and clearance by hemofiltration intravenous ribavirin treatment for severe adenovirus disease in immunocompromised children successful treatment of adenovirus disease with intravenous cidofovir in an unrelated stemcell transplant recipient obliterative bronchiolitis in children key: cord- -rthnj wd authors: cheng, v. c. c.; hung, i. f. n.; tang, b. s. f.; chu, c. m.; wong, m. m. l.; chan, k. h.; wu, a. k. l.; tse, d. m. w.; chan, k. s.; zheng, b. j.; peiris, j. s. m.; sung, j. j. y.; yuen, k. y. title: viral replication in the nasopharynx is associated with diarrhea in patients with severe acute respiratory syndrome date: - - journal: clin infect dis doi: . / sha: doc_id: cord_uid: rthnj wd the role of severe acute respiratory syndrome (sars) coronavirus as an enteric pathogen was investigated in a cohort of patients with sars who were treated with a standard treatment protocol. data from daily hematological, biochemical, radiological, and microbiological investigations were prospectively collected, and the correlation of these findings with diarrhea was retrospectively analyzed. sixty-nine patients ( . %) developed diarrhea at a mean (± standard deviation [sd]) of . ± . days after the onset of symptoms. the diarrhea was most severe at a mean (±sd) of . ± . days after onset, with a maximum frequency of episodes per day (median, episodes; range, – episodes). a higher mean virus load in nasopharyngeal specimens obtained on day after the onset of symptoms was significantly associated with the occurrence of diarrhea ( . log( ) vs. . log( ) copies/ml; p = . ) and mortality ( . vs. . log( ) copies/ml; p < . ). however, diarrhea was not associated with mortality. the lung and the gastrointestinal tract may react differently to sars coronavirus infection. additional investigation of the role of sars coronavirus in the pathogenesis of diarrhea in patients with sars should be conducted. the role of severe acute respiratory syndrome (sars) coronavirus as an enteric pathogen was investigated in a cohort of patients with sars who were treated with a standard treatment protocol. data from daily hematological, biochemical, radiological, and microbiological investigations were prospectively collected, and the correlation of these findings with diarrhea was retrospectively analyzed. sixty-nine patients ( . %) developed diarrhea at a mean ‫ע(‬ standard deviation [sd]) of days after the onset of symptoms. the . ‫ע‬ . diarrhea was most severe at a mean (‫ע‬sd) of days after onset, with a maximum frequency of . ‫ע‬ . episodes per day (median, episodes; range, - episodes). a higher mean virus load in nasopharyngeal specimens obtained on day after the onset of symptoms was significantly associated with the occurrence of diarrhea ( . log vs. . log copies/ml; ) and mortality ( . vs. . log copies/ml; ). p p . p ! . however, diarrhea was not associated with mortality. the lung and the gastrointestinal tract may react differently to sars coronavirus infection. additional investigation of the role of sars coronavirus in the pathogenesis of diarrhea in patients with sars should be conducted. the severe acute respiratory syndrome (sars) pandemic has affected patients, with fatalities [ ] . sars is caused by a novel coronavirus, which was consistently isolated from patients with sars who had subsequent seroconversion [ ] [ ] [ ] . similar histopathological findings of sars and seroconversion have been reproduced in cynomolgus macaques (macaca fascicularis) that were artificially inoculated with the same sars coronavirus [ ] . subsequent detailed histopathological study of these infected macaques confirmed that the inflammatory changes were confined to the lungs [ ] . thus, sars is largely regarded as a novel viral pneumonia. however, in a prospective clinical study of a cohort of patients with sars, diarrhea was noted in % of patients at hospital admission and in % during hospitalization [ ] . viral genomes or the virus could be detected by rt-pcr or cell culture of stool samples obtained from these patients [ ] . as illustrated by the example of enterovirus infection, shedding of virus in stool does not necessarily imply the presence of disease in the intestinal tract. nevertheless, presence of numerous coronavirus particles was demonstrated in the terminal ileum and colon in a patient with sars who had diarrhea [ ] . it would be interesting to know whether the sars coronavirus indeed causes diarrhea, because histopathological examination of intestinal biopsy specimens does not reveal any inflammatory or cytolytic damages. we have previously shown that specimens obtained from the nasopharynx are useful for the diagnosis of rotavirus infection in children [ ] . this is not unexpected, because an earlier study also demonstrated the presence of rotavirus in nasopharyngeal secretions from children with upper respiratory tract infection [ ] . in this retrospective study, we attempt to correlate the virus load of sars coronavirus shedding from the nasopharynx, the upper end of the aerodigestive tract, with the presence of diarrhea in a cohort of patients with sars. demographic characteristics, clinical features, and hematological and radiological findings of patients with sars who had or did not have diarrhea were collected and analyzed. the clinical records for all patients whose cases fulfilled the modified world health organization definition of sars [ , ] and who were treated at the united christian hospital and caritas medical centre (hong kong) were analyzed. the daily clinical findings from history and physical examinations and hematological, biochemical, radiological, and microbiological investigations were prospectively collected and analyzed. in brief, the case definition includes fever (temperature, у Њc), cough or shortness of breath, and new pulmonary infiltrates noted on chest radiographs or high-resolution ct, in the absence of an alternative diagnosis to explain the clinical presentation. some of the clinical and virological results for the first patients were previously reported [ ] . all patients were treated with amoxicillin-clavulanate ( . g q h iv) and azithromycin ( mg q.d. po). in patients with a known allergy to penicillin, we administered mg of oral levofloxacin every h. as soon as the diagnosis of sars was established, ribavirin (a -g oral loading dose, followed by . g q h or mg/kg q h iv if the patient could not tolerate oral treatment) was given for days, in addition to a tailing regimen of hydrocortisone (starting dose, mg q h iv) for days, followed by oral prednisolone ( mg/kg for days, . mg/kg for days, and . mg/kg for days) for days. pulses of methylprednisolone ( - mg q.d. iv for up to g) were administered to patients with clinical deterioration. all hepatitis b surface antigen-positive patients were given prophylactic lamivudine ( mg q.d. po) while taking corticosteroids. patients were prospectively monitored for development of diarrhea during hospitalization. diarrhea was defined as у bowel movements per day for у consecutive days. the occurrence of diarrhea in relation to the onset of symptoms of sars, the frequency of bowel movements, and the duration of diarrhea were recorded. investigation for other causes of diarrhea was performed, including culture for clostridium difficile, cell culture assay for detection c. difficile cytotoxin, and elisa (ideia rotavirus; dako) for detection of rotavirus. for the diagnosis of coronavirus infection, nasopharyngeal specimens and serum samples were obtained at hospital admission. the convalescent-phase serum sample was taken - days after the onset of symptoms. for all patients, qualitative and quantitative rt-pcrs for sars coronavirus were retrospectively performed using the nasopharyngeal specimens obtained at admission and days after the onset of symptoms. stool and urine specimens were obtained for rt-pcr during the hospital stay. all virological diagnostic protocols, including performance of qualitative and quantitative rt-pcrs, rapid viral antigen detection tests, viral cultures, immunofluorescent antibody tests for detection for igg seroconversion against sars-associated coronavirus, and other microbiological diagnostic evaluations, were done in the manner described in our previous publications [ , ] . statistical analysis. all data were calculated from the day of onset of clinical symptoms. we compared the demographic characteristics and laboratory values for patients with and without diarrhea by means of fisher's exact test for categorical variables and student's t test or the mann-whitney u test for continuous variables. a -tailed p value of !. was considered to be statistically significant. we used spss software, version . (spss), for all analyses. of the patients recruited in this study, ( . %) were ethnic chinese, and the remaining patients were filipinos. the patients who presented with single-zone lesions, the right lower and left lower zones were more commonly involved than were the other zones. thoracic ct was performed for patients with initial apparently normal chest radiograph findings. the lesions were mostly confined to the retrocardiac region in the left lower lobes. with regard to the microbiological evaluation for sars coronavirus, patients ( . %) had either a -fold increase in antibody to sars coronavirus or positive results of rt-pcr of nasopharyngeal, stool, or urine specimens (table ) . fourfold increases in antibody titers to sars coronavirus were found in of patients who did not have any history of epidemiological exposure, and rt-pcr results were positive for the remaining patients. during the course of infection, diarrhea occurred in patients ( . %). the distribution of episodes of diarrhea during the first weeks of hospitalization is shown in figure . diarrhea occurred days after the onset of symptoms and be- . ‫ע‬ . came most severe on day , with a maximum frequency . ‫ע‬ . of episodes per day (median, episodes per day; range, - episodes per day). the diarrhea lasted for a duration of days in this cohort, resulting in prerenal azotemia in . ‫ע‬ . patients and electrolyte disturbances in patients. the diarrhea was rather painless, with no blood or mucus present in the stool for any patients. none of the patients had positive results of culture for c. difficile or cell culture assay for c. difficile cytotoxin. all stool samples were negative for rotavirus on elisa tests. the clinical characteristics of patients with and those without diarrhea are summarized in table . there were no differences between the groups with regard to age, sex, comorbidities, and chronic hepatitis b status. the manifestations on chest radiographs at hospital admission were also similar. there were no statistical differences in hematological and biochemical parameters between the groups at presentation and on day after the onset of symptoms. patients with diarrhea had lower potassium levels ( mmol/l) and higher mean sodium . ‫ע‬ . levels ( mmol/l). there was no difference in the mean ‫ע‬ virus loads in nasopharyngeal specimens (as measured by quantitative rt-pcr) at baseline between the groups with and without diarrhea. a higher mean virus load in nasopharyngeal specimens obtained on day after the onset of symptoms was significantly associated with the occurrence of diarrhea ( . vs. . log copies/ml; ) and mortality ( . vs. . log p p . copies/ml; ). however, there were no differences be-p ! . tween patients with and those without diarrhea with regard to the incidence of intensive care admission, receipt of mechanical ventilatory support, length of hospital stay, and overall mortality. furthermore, the severity of diarrhea had no correlation with the virus load in nasopharyngeal specimens and overall mortality (figure ). sars is predominantly a viral pneumonia with a rapid tempo of deterioration. however, extrapulmonary manifestations are not uncommon. skin rash [ ] , petechial cutaneous bleeding with prolonged activated partial thromboplastin time and increased d-dimer [ ] , impaired liver function, subclinical left ventricular diastolic dysfunction [ ] , and rhabdomyolysis [ ] were occasionally reported. one of the more common presentations other than respiratory symptoms is diarrhea [ , , , [ ] [ ] [ ] [ ] . our previous report that % of patients in the amoy garden cohort developed diarrhea showed that fecal excretion may be an important mode of shedding and transmission [ ] . subsequent epidemiological investigation confirmed the im- portance of faulty sewage systems in propagating this massive outbreak of disease. retrospective virological examination of stool samples by rt-pcr confirmed the presence of the virus in % of the samples obtained between day and after onset of symptoms, which is almost twice the positivity rate of the corresponding nasopharyngeal samples [ ] . it is therefore interesting to examine the role of sars coronavirus in the pathogenesis of diarrhea in patients with sars. diarrhea is not an uncommon symptom during hospitalization for patients with infectious diseases other than acute gastroenteritis. in fact, diarrhea has been well reported in pneumonic illnesses other than sars, such as legionnaires disease [ ] , pneumocystis carinii pneumonia [ ] , and influenza (especially in children) [ ] . moreover, diarrhea is not uncommon in association with many systemic infections. for example, the rate of diarrhea was reported to be . % among children with primary dengue fever [ ] . the exact mechanisms leading to diarrhea in these systemic or pulmonary infections were not known, but they could be related to the cytokine profiles or metabolic changes associated with sepsis. in addition to primary viral illness, other potential causes of diarrhea include the use of drugs during hospitalization and c. difficile superinfection, because most patients with sars have been empirically treated with antibiotics that cover typical and atypical pneumonia. finally, diarrhea can be the direct result of viral cytolysis, because the virus can grow and produce a cytopathic effect in a large intestinal cell line, caco- [ ] . a study of the correlation between the clinical parameters, microbiological findings, and outcomes for patients with sars with and without diarrhea may differentiate the relative importance of the suggested causative factors. in this cohort of patients with sars, only . % of the patients had diarrhea at hospital admission. during hospitalization, ( . %) of the patients had developed diarrhea at a mean time of days after the onset of symptoms. . ‫ע‬ . the most severe diarrhea occurred - days after the onset of diarrhea, with a maximum frequency of episodes per day. in patients, diarrhea was so severe that intravenous fluid hydration was warranted. the diarrhea was relatively painless and watery, as we have reported previously [ ] . no correlation was found between diarrhea and hematological changes, radiological changes, use of antibacterials, or isolation of c. difficile and rotavirus from stool specimens. the only significant correlation was that there was a higher virus load in the nasopharyngeal specimens taken from patients with diarrhea on day after the onset of symptoms. the finding that a higher virus load-but not the presence of diarrhea-predicted mortality in this group of patients is not unexpected. in previous studies of animal models of sars [ , ] , histological changes of acute respiratory distress syndrome (ards) and inflammation-characterized by diffuse alveolar damage (disruption of alveolar walls and infiltration with neutrophils and macrophages), alveolar hyaline membrane formation, as well as multinucleated syncytial cells-were found in a macaque monkey model artificially inoculated with sars coronavirus [ ] . none of the monkeys developed diarrhea over a period of days, despite the fact that rt-pcr results were positive for sars coronavirus in of the stool samples. similarly, no histopathological changes could be detected in the intestines of the monkeys, despite there being concomitant florid pathological changes in the lungs [ ] . these findings suggest that viral rep-lication triggers a different response and thus different functional impairment in the lungs and the gastrointestinal tract. a high virus load in the nasopharynx specimens either may predict severe pneumonia, ards, and mortality in some patients, or it may predict just diarrhea in other patients. this postulation is in keeping with the lack of significant difference in hematological markers of inflammation or increased cell turnover (lactate dehydrogenase level) between the groups with or without diarrhea. although suppression of inflammatory changes in the intestine resulting from steroid therapy is possible, the routine use of steroids and ribavirin as part of the treatment protocol did not allow us to control the effect of use of steroids or antivirals on diarrhea. moreover, the use of steroids has not affected the severe inflammatory reactions in the lungs of persons who have died of sars [ ] . in our previous study, we showed that rotavirus can be detected by viral isolation in cell culture and by indirect immunofluorescent antigen detection [ ] . this is not unexpected, because many viruses that affect the gastrointestinal tract also affect the respiratory tract, and vice versa. for instance, in several studies, rotavirus has been well reported to be associated with bronchiolitis and fatal pneumonitis in immunocompromised as well as immunocompetent hosts [ , , ] . others reported that respiratory symptoms were found in % of patients with gastroenteritis caused by enteric adenovirus [ ] . this virus has been found in samples from the upper respiratory tract and in stool samples obtained from these patients [ ] . similar to rotavirus and adenovirus, the sars coronavirus can multiply and shed in the mucosa of the upper aerodigestive tract. in this regard, determination of the virus load in specimens from the nasopharynx may be useful for predicting the severity of illness in both the respiratory and the gastrointestinal tracts. from the laboratory perspective, rt-pcr is more likely to be affected by the abundant inhibitors present in stool than by nasopharyngeal secretions. moreover, the volume of diarrhea varies markedly from patient to patient. this may also affect the interpretation of data on the virus load in stool. therefore, we attempted to use nasopharyngeal specimens instead of stool specimens in this study. coronaviruses can cause respiratory or diarrheal diseases in both human and animals [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . human enteric coronavirus has been seen in diarrheal stool samples, but culture methods and characterization are still problematic [ , ] . human coronavirus oc and e are known to cause one-third of cases of the common cold [ , ] . in the case of porcine coronavirus, a collection of transmissible gastroenteritis coronavirus (tgev) recombinants were generated to study the molecular basis of tgev tropism [ ] . recombinants of group had enteric and respiratory tropism, whereas group recombinants infected the respiratory but not the enteric tract. a substitution in amino acid of the s protein in group recombinant was responsible for the loss of enteric tropism [ ] . on the other hand, targeted recombination of the s gene of tgev resulted in a change from respiratory to enteric tropism and enhanced virulence [ ] . however, no major deletion or mutation of functional significance had been identified by genomic sequencing of sars coronavirus isolated from the diarrheal patients [ ] . we have clearly demonstrated the presence of a numerous virus in tissue biopsy specimens from the terminal ileum and colon of a sars patient with diarrhea [ ] . the virus is capable of active replication inside the endoplasmic reticulum and adheres to the surface of enterocytes in both small and large intestines (as shown by electron microscopy), without causing significant villous atrophy or inflammatory cell infiltration in the mucosal or submucosal regions [ ] . the absence of inflammation, cell necrosis, or microvilli atrophy does not exclude sars coronavirus as the cause of diarrhea in these patients. for astrovirus-associated diarrhea in turkeys, the animal model has demonstrated mild histological changes, with a surprising lack of inflammation due to increased activation of the potent immunosuppressive cytokine transforming growth factor b at the peak of diarrhea during astrovirus infection [ ] . additional studies should be performed to ascertain the mechanism used by sars coronavirus in the pathogenesis of diarrhea in 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n-terminus of transmissible gastroenteritis coronavirus spike protein result in the loss of enteric tropism targeted recombination demonstrates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence molecular epidemiology of the novel coronavirus that causes severe acute respiratory syndrome astrovirus induces diarrhea in the absence of inflammation and cell death key: cord- -jf dnllj authors: tang, catherine so-kum; wong, chi-yan title: factors influencing the wearing of facemasks to prevent the severe acute respiratory syndrome among adult chinese in hong kong date: - - journal: prev med doi: . /j.ypmed. . . sha: doc_id: cord_uid: jf dnllj background. the global outbreak of the severe acute respiratory syndrome (sars) in has been an international public health threat. quick diagnostic tests and specific treatments for sars are not yet available; thus, prevention is of paramount importance to contain its global spread. this study aimed to determine factors associating with individuals' practice of the target sars preventive behavior (facemask wearing). methods. a total of adult chinese residing in hong kong were surveyed. the survey instrument included demographic data, measures on the five components of the health belief model, and the practice of the target sars preventive behavior. logistic regression analyses were conducted to determine rates and predictors of facemask wearing. results. overall, . % of the respondents reported consistent use of facemasks to prevent sars. women, the – age group, and married respondents were more likely to wear facemasks. three of the five components of the health belief model, namely, perceived susceptibility, cues to action, and perceived benefits, were significant predictors of facemask-wearing even after considering effects of demographic characteristics. conclusions. the health belief model is useful in identifying determinants of facemask wearing. findings have significant implications in enhancing the effectiveness of sars prevention programs. a new and highly infectious disease in humans, the severe acute respiratory disease syndrome (sars), has created a major public health threat in many countries. within months since its first appearance in asia in mid-february of , the world health organization (who) has already received reports of the outbreak of sars in countries on all five continents [ ] . the clinical symptoms of sars are nonspecific, including high fever, dry cough, breathing difficulties, muscle pain, and generalized weakness. the incubation period can last from to days, thus enables symptomless individuals to transmit the disease through either close person-to-person contact or travel from one city to another city in the world. the mortality rate of sars is about - %. only recently has the causative agent of this disease been found. the latest multicountry laboratory findings have confirmed that a new pathogen, a member of the coronavirus family never been seen in humans, is the cause of sars [ ] . however, the exact transmission route of the disease is still unknown, and quick diagnostic tests as well as specific treatments are also not yet available. under these unknown circumstances, prevention is of particular importance in containing the global spread of this new infectious disease. this study aimed to examine factors affecting hong kong people's practice of preventive behaviors against sars. findings from this study would provide pertinent information in designing and implementing sars prevention programs not only for hong kong, but for other countries as well. hong kong was one of the hardest hit area during the global outbreak of sars in and has accounted for almost % of the probable cases and deaths of sars. at the beginning of the local outbreak of this disease in early march of , mainly health care workers who treated the index patients were infected with the disease [ ] . very soon, new cases were reported among close contacts of known patients, and the disease then quickly spread to the community. local health authorities have since stepped up various prevention and intervention activities against further spread of the disease [ ] . at the community level, health authorities have launched large-scale public health education programs about the disease, issued preventive health guidelines to health care workers and the general public, suspension of classes for schools and universities, prompt isolation of infected individuals, and ordering of probable infected individuals to quarantine themselves at home for days. at the individual level, health advice is given on ways to prevent contracting and spreading of sars. the suggested sars preventive behaviors include ( ) maintaining good personal hygiene (covering nose and mouth with a tissue when sneezing or coughing and washing hands immediately afterward with liquid soap), ( ) developing a healthy lifestyle with proper diet, regular exercises, adequate rest, and no smoking, ( ) ensuring good ventilation at home and in the office, and ( ) wearing facemasks, especially for those with respiratory tract infections or those caring for them. despite all these efforts, an average of - new infected cases and about five deaths of sars were reported daily. the disease continued to affect both health care workers, as well as individuals from the community until june . researchers have argued that the practice of preventive behaviors by individuals is one of the most effective ways in disease prevention and health promotion [ ] [ ] [ ] . with environmental and policies support, these individual preventive behaviors can pass on to effective population-level prevention efforts [ , ] . for example, public education and media campaigns that disseminate health messages and information, environmental manipulation that provide necessary facilities, and national policies that make available economic incentive or reimbursement can motivate many individuals in the community to practice the desired health behaviors. thus, various psychosocial approaches, such as the health belief model [ ] , the theory of reasoned action [ ] , the social cognitive model [ ] , the protection motivation theory [ ] , and the stages of change model [ ] , have been put forward to predict the practice of preventive behaviors at the individual level. among various psychosocial approaches, the health belief model is one of the most widely used and provides the necessary conceptual framework for this study. this model postulates that the practice of preventive behaviors is a function of the degree to which individuals perceive a personal health threat and the perception that particular preventive behaviors will be effective in reducing the threat [ ] . in applying this model to understand the practice of sars preventive behaviors, perceived health threat refers to individuals' perception of their vulnerability to contracting sars (perceived susceptibility) and that this disease has serious consequences (perceived severity). individuals' be-lief that the practice of the suggested sars preventive behaviors will prevent sars depends on ( ) whether they think these preventive behaviors will be effective (perceived benefits), ( ) whether the cost of undertaking these behaviors (perceived barriers) exceeds the benefits, and ( ) whether there are any cues (cues to action) to trigger these behaviors. cues to action can be internal, such as the perception of a body state, or external, such as the influence of mass media and social pressure. there is ample support for the health belief model in explaining individual practice of preventive behaviors. this model helps to predict the practice of preventive dental care [ ] , dieting for obesity [ ] , aids risk-reduction behaviors [ ] , breast self-examination [ - ] , sunscreen use [ ] , and participation in a broad array of health screening programs such as obtaining a mammogram to screen for breast cancer [ - ] and undergoing genetic testing for cancer susceptibility [ , ] . prevention programs that draw on this model to effect behavioral change have also yielded positive results in increasing various health behaviors to prevent dental problems [ ] , osteoporosis [ ] , and diabetes [ ] . overall, perceived benefits, perceived barriers, and perceived susceptibility are the three most powerful components of the health belief model in influencing whether individuals practice different preventive behaviors [ , , ] . it is also found that the actual risk of developing a disease is a much less important predictor of individual preventive behaviors than is perceived susceptibility [ ] . other than psychosocial predictors, there is also an accumulation of literature documenting the importance of associations between individuals' demographic characteristics and their practice of preventive behaviors [ , - ] . in general, women and more affluent and better educated individuals are more likely to practice the suggested preventive behaviors. an inverted curvilinear relationship is found between age and practice of preventive behaviors. typically, young children are often compliant in adopting various preventive behaviors, which tend to decline in adolescence and adulthood but improve again among older people. findings in relation to marital status and ethnicity are inconclusive. the purposes of this study were twofold. the first objective was to determine the rates of the target sars preventive behavior in adult chinese with different demographic background. among various sars preventive behaviors suggested by local and international health authorities, this study focused on the wearing of facemasks. this target preventive behavior was chosen for this study because it was specific to this disease and involved deliberate effort of individuals. based on past related literature, it was expected that men, the younger age group, and individuals with low educational attainment would be less likely to wear facemasks. the second objective of this study was to test the efficacy of the health belief model in predicting the practice of the target preventive behavior. based on past related literature, it was expected that perceived susceptibility, perceived severity, perceived benefits, perceived barriers, and cues to action were significant predictors of facemask wearing. this study was conducted between march and april , , in hong kong, when there was clear evidence that sars had started to spread from health care workers in hospitals to the community. at the time of the study, the exact causative agent and route of transmission of the disease were still not yet fully known [ ] . local health authorities had since implemented enhanced infection control procedures in all hospitals and cohorting of sars patients [ ] . they had also stepped up communitywide sars prevention and intervention activities. data for this study were obtained using a community telephone survey of adult chinese (aged and above) residing in hong kong during the specified period. random-digit dialing of the local residential telephone directory for was used to select respondents. this directory covered all listed telephone numbers in all regions of hong kong, where over % of the households owned at least one or more telephone lines. telephone surveys were conducted by trained telephone interviewers and took about min. when telephones were busy or there was no answer, three follow-up calls on different time or dates were attempted before substituting a new telephone number. the response rate was %, and the sampling error was . percentage points. a total of adult chinese were surveyed, and their demographic information are summarized in table . compared to the hong kong population census data [ ] , the present sample included more women as well as individuals with university education and higher monthly personal income. similar differences in demographic characteristics between telephone surveys and census data were also noted in previous local telephone surveys [ ] . the present sample comprised . % men and . % women. about half of them aged between and years, . % between and years, . % between and years, and . % were older than years. among them, % were single, % were currently married, and the remaining were either separated, divorced, or widowed. slightly more than half of the respondents completed high school education and worked either full time or part time. another . % of the respondents were homemakers, . % were students, . % were retirees, and . % were unemployed. respondents were asked to indicate how often in the past week they wore facemasks to prevent contracting and spreading sars. they responded with either ''never,'' ''occasionally,'' or ''most of the time.'' the first two responses were coded as '' '' and the last response was coded as '' '' for subsequent statistical analyses. this was assessed by three items: ( ) whether respondents felt vulnerable to contracting sars, ( ) whether they knew or had close contact with any individuals infected with sars, and ( ) whether they had respiratory infection syndromes such as sore throat, dry cough, fever, muscle ache, and shortness of breath. respondents answered with ''yes'' or ''no'' responses, and affirmative responses were then summed to form a total score. high total scores represent respondents perceiving themselves as being highly susceptible to contracting sars. respondents indicated on two -point scales the degree to which they were fearful of sars ( as ''not at all fearful'' to as ''very fearful'') and worried that hong kong would become a quarantine city because of the widespread of sars to the community ( as ''not at all worried'' to as ''very worried''). high mean scores of these two scales represent respondents perceiving sars as having very severe adverse consequences. respondents were asked to indicate on a -point scale the degree to which they agreed wearing facemasks could prevent contracting and spreading sars ( as ''strongly disagree'' to as ''strongly agree''). high scores indicate respondents perceiving great benefits in wearing facemasks. respondents were asked to rate on two -point scales the degree to which they had difficulty in obtaining facemasks ( as ''not at all difficult'' to as ''very difficult'') and the level of discomfort when wearing them ( as ''not at all uncomfortable'' to as ''very comfortable''). high mean scores of these two items indicate respondents perceiving great barriers in wearing facemasks. this was assessed by asking respondents to indicate on two -point scales the degree to which the local government and their family members encouraged them to wear facemasks ( as ''strongly disagree'' to as ''strongly agree''). high mean scores of these two scales represent respondents having great awareness of environmental cues to wear facemasks. respondents were also asked about their sex, age, educational attainment, marital status, and personal monthly income. statistical analyses in this study were conducted using spss . software. descriptive statistics for demographic characteristics of respondents were generated and compared with the hong kong population census data ( table ). the rates of wearing facemasks were determined for individuals with various demographic characteristics. bivariate logistic regression analyses were then conducted to determine whether the practice of facemask wearing differed within each demographic characteristic (table ) . a multivariate logistic regression analysis was also performed to test the health belief model and to identify significant predictors of the target preventive behavior. odds ratios (ors) for each predictor were estimated from the logistic regression (table ) . demographic variables of the respondents were entered in the logistic regression first to control for their effects before testing the health belief model. then, predictor variables were entered simultaneously in the next block of the regression. the predictor variables consisted the five components of the health belief model: perceived sus-ceptibility, perceived severity, perceived benefits, perceived barriers, and cues to action. overall, . % of the respondents reported consistent wearing of facemasks to prevent contracting and spreading sars. table . ) were more likely to wear facemasks to prevent sars within their own demographic groups. results also showed that sex (v = . , p < . ), age (v = . , p < . ), and marital status (v = . , p < . ) had significant subgroup differences in the target preventive behavior. a logistic regression with odds ratios was conducted to test the efficacy of the health belief model in predicting the wearing of facemasks to prevent sars. in block i, demographic factors of sex, age, and marital status were entered first to control for their effects. results showed that this block was significant (v = . , p < . ). the five components of the health belief model were entered in the next block, and they were significant in predicting facemask wearing, even after considering effects of demographic factors (v = . , p < . ). the final model of the logistic regression analysis is presented in table . with the exception of perceived barriers, all estimated coefficients were in the expected direction. all odds ratios were above . . in summary, respondents who were women (or = . ; ci = . , . ), who belonged to the older age group (or = . ; ci = . , . ), who were married (or = . ; ci = . , . ), who felt more susceptible to contracting sars (or = . ; ci = . , . ), who perceived sars as having more serious consequences (or = . ; ci = . , . ), who believed greater benefits in wearing facemasks (or = . ; ci = . , . ), who encountered greater barriers in wearing facemasks (or = . ; ci = . , . ), and who were more aware of environmental cues (or = . ; ci = . , . ) were more likely to wear facemasks. results showed that three of the five components of the health belief model, namely, perceived susceptibility, cues to action, and perceived benefits, were significant predictors. perceived severity and perceived barriers were not significant predictors of facemask wearing when other factors were also considered. this study examined how various psychosocial factors are associated with the practice of the target sars preventive behavior among adult chinese in hong kong. similar to previous research [ - ] , this study found the health belief model useful in identifying major determinants of the wearing of facemasks to prevent contracting and spreading sars. in particular, findings showed that three of the five components of the model, namely, perceived susceptibility, cues to action, and perceived benefits, were significant predictors. review studies of the health belief model have also found that among the five components, perceived susceptibility and perceived benefits are the more powerful components in predicting preventive behaviors [ , ] . the present results showed that compared to those with a low level of perceived susceptibility, individuals feeling personally very vulnerable to contracting sars were . times more likely to wear facemasks. it was also found that individuals who had strong beliefs in the effectiveness of wearing facemasks to prevent sars were . times more likely to wear facemasks than those who did not have these beliefs. furthermore, this study showed that cues to action were as an important predictor as perceived susceptibility. those who were more aware of environmental cues were . times more likely to wear facemasks than those who perceived few cues to action. previous studies have also indicated that cues to action in the form of advice from family members and health care professionals are also very important factors in increasing various preventive behaviors [ ] . the remaining two components of the health belief model, perceived severity and perceived barriers, were found to be nonsignificant determinants of the target sars preventive behavior in this study. in spite of previous literature indicating the powerfulness of perceived barriers in influencing the practice of preventive behaviors [ , , , ] , this component did not significantly predict the wearing of facemasks in the present sample of adult chinese. it might be that this target preventive behavior is relatively easy to perform, despite some discomfort and inconvenience. individuals can have complete control of the behavior and can take off the facemasks anytime they want or feel uncomfortable. facemasks are cheap and easy to obtain, except during the early outbreak of sars. thus, there are relatively less perceived barriers in wearing facemasks as compared to participating in screening or immunization programs. this study also found that perceived severity of sars did not significantly predict facemask wearing. earlier studies on cancer noted that perceived severity was not related to preventive behaviors, as cancer is uniformly thought of as a serious disease [ ] . on the other hand, it would also be argued that the failure of perceived severity to predict the target sars preventive behavior might be related to individuals' underestimation of the potential of this disease to become a global epidemic. during the early stage of the local outbreak, this disease affected mainly health care workers, a few index patients, and their close contacts [ ] . the serious consequences of this disease with increasing prevalence and mortality rates might have been overlooked. thus, the public was not motivated or did not anticipate the need to practice the suggested sars preventive behavior. similar to existing literature on preventive behaviors [ , - ] , this study also found that individuals' demographic characteristics were significant predictors of facemask wearing. results showed that men, single people, and the - age group were significantly less likely to wear facemasks to prevent sars than women, married people, and individuals aged between and years. although statistically nonsignificant, odds ratios also showed that individuals with less than university education and very low personal income were also less likely to wear facemasks. researchers have argued that the low rates of preventive behaviors among young, single, and low education men may be related to their inadequate knowledge about the disease, peer influences, risk-taking tendency, and perceived immortality and immunity to various disease [ , ] . this study found that about . % of the respondents consistently wore facemasks to prevent sars. in other words, despite the rapid spread, increasing death toll, and vigorous preventive activities, close to % of the respondents did not practice the target sars preventive behavior. for some subgroups, the noncompliance rates were even higher. as there are evidences that sars may emerge again both locally and across countries [ ] , there is a need for hong kong, as well as other countries, to further enhance the effectiveness of related prevention and intervention measures to contain the disease. results of this study had significant implications for sars prevention programs. it is suggested that individuals' perception of their own vulnerability to the disease should be emphasized by highlighting the global outbreak and debilitating health consequences of sars, which not only affect health care workers, older people, those with chronic illness, or individuals of a particular ethnicity. public education and media campaigns should be conducted regularly in schools, workplaces, and community settings to disseminate the latest findings of sars. the public should be made aware that there is no specific treatment and vaccination for this disease; therefore, individuals must take own precautions to prevent contracting the disease. it should also be emphasized that at the individual level, facemask wearing remains one of the most effective ways to prevent contracting and spreading sars [ ] . indeed, studies have shown that communications that highlight perceived susceptibility and simultaneously increase the perception that a particular health behavior will reduce the health threat are successful in promoting various preventive behaviors [ , , ] . the present study also showed that environmental cues to wear facemasks are very important. other researchers have argued that environmental manipulation and policies support are crucial to translate individual to collective preventive efforts [ , ] . environmental cues can be in the form of signs and posters to remind people to wear facemasks and should be placed in prominent areas in schools, at worksites, and in public places. advice, recommendation, or encouragement from health care professionals are also useful to increase individuals' likelihood to wear facemasks. policies that require the wearing of facemasks in certain locations (e.g., inside hospitals) or for those who have respiratory symptoms will also ensure individuals to practice the desired preventive behaviors. finally, it is also particularly important to target sars prevention activities at those who are less likely to practice these behaviors, namely, men, single individuals, and the younger age group. this study had several limitations. first, there were concerns about the representative of the sample. this study used telephone surveys to collect data and failed to include individuals who were without home telephone lines or not at home during the surveyed period. comparison with the census data showed that the present sample comprised more women as well as more affluent and better educated individuals, who were also more likely to practice the suggested sars behaviors. in view of this, the present sample might have higher rates of facemask wearing than the general population. second, only retrospective self-reports 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onset on young women's intentions to prevent osteoporosis preventive care in the context of men's health a meta-analysis of studies of the health belief model with adults the health belief model: a decade later decision-making about genetic testing among women at familial risk for breast cancer associations between exercise and health behaviors in a community sample of working adults a population-based study of age and gender differences in patterns of healthrelated behaviors elucidating the relationships between race, socioeconomic status, and health socio-demographic characteristics and individual health behaviors main tables of the population census the rate of physical child abuse in chinese families: a community survey in hong kong individual psychology of risktaking behaviors in non-adherence effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars) incomplete knowledge and attitude-behavior inconsistency key: cord- -eft vwgb authors: xepapadaki, paraskevi; psarras, stelios; bossios, apostolos; tsolia, maria; gourgiotis, dimitrios; liapi-adamidou, georgia; constantopoulos, andreas g; kafetzis, dimitrios; papadopoulos, nikolaos g title: human metapneumovirus as a causative agent of acute bronchiolitis in infants date: - - journal: j clin virol doi: . /j.jcv. . . sha: doc_id: cord_uid: eft vwgb background: human metapneumovirus (hmpv), has been recently isolated from children with acute respiratory tract infections (rtis), including bronchiolitis, and classified in the pneumovirinae subfamily within the paramyxoviridae family. objectives: since most bronchiolitis studies fail to detect any viral pathogen in part of the samples, we sought for the presence of hmpv in a well characterized bronchiolitis cohort. study design: nasal washes were obtained from children admitted to the hospital for acute bronchiolitis. rna extraction and subsequent rt-pcr were used to detect hmpv, and correlated the presence of the virus with clinical characteristics of the disease. results and conclusions: pcr revealed the presence of hmpv in % of bronchiolitis cases, whereas respiratory syncytial virus (rsv; . %) was the most frequently encountered viral pathogen. hmpv was identified either as a unique viral pathogen or co-existed with rsv, with whom they shared a similar seasonal distribution. there were no differences in disease characteristics, either clinical or laboratory, between bronchiolitis cases where hmpv was present and those caused by rsv or other viral pathogens. these findings suggest that hmpv is a common and important causative agent in infants with bronchiolitis, with clinical characteristics similar to that of rsv. a new respiratory virus, human metapneumovirus (hmpv), has been recently isolated from nasopharyngeal aspirates of young children in the netherlands (van den hoogen et al., ) . hmpv is the first human respiratory pathogen classified as a member of the metapneumovirus genus, which together with pneumoviruses constitute the pneumovirinae subfamily within the paramyxoviridae family (van den hoogen et al., ; boivin et al., ; viazov et al., ) . the clinical symptoms of the children from which the virus was isolated ranged from upper respiratory airway disease to severe bronchiolitis and pneumonia (van den hoogen et al., ) . subsequent studies established the association of this virus with acute rtis in all age groups (maggi et al., ; osterhaus and fouchier, ; boivin et al., ; vicente et al., ; stockton et al., ; boivin et al., ; peiris et al., ; freymouth et al., ) . however, several epidemiological as well as clinical aspects of hmpv infection are still to be firmly established and in this context the analysis of well defined respiratory disease cohorts is of particular importance (jartti et al., ; rawlinson et al., ) . we hypothesized that hmpv may be an important pathogen in the context of acute bronchiolitis of infancy. in addition to bronchiolitis cases being reported within the patients of published hmpv studies, this is further suggested by the observation that most bronchiolitis studies fail to identify a pathogen in a considerable proportion of cases. we have recently reported the results of virological evaluation of a well-characterized cohort of infants admitted to hospital with acute bronchiolitis, using reverse transcription polymerase chain reaction (pcr) for respiratory pathogens (papadopoulos et al., ) . as expected, rsv was the predominant pathogen, while human rhinovirus (rv) was implicated in almost one-third of cases. adenoviruses, influenza viruses, parainfluenza viruses and corona viruses, were involved to a smaller extent. nevertheless, no pathogen was identified in almost one quarter of the samples (papadopoulos et al., ) . therefore, the aim of the present study was to assess the presence of hmpv in stored nasal secretions of this cohort and evaluate the relationship between the presence of the virus and the clinical and epidemiological characteristics of the disease. the study was carried out in the second pediatric clinic of the university of athens, greece, at the p&a kyriakou children's hospital, and was approved by the hospital's ethics committee. children were admitted in the hospital with the diagnosis of bronchiolitis, which was defined as an acute infection of the lower airway, characterized by increased respiratory effort (tachypnea of > respirations/min and/or use of accessory respiratory muscles), and expiratory wheezing and/or crackles. a detailed description of the cohort has been reported (papadopoulos et al., ) . nasopharyngeal washes (npw) have been obtained at admission, aliquoted and kept at − • c. a new aliquot was thawed and used for hmpv detection. sufficient material for analysis was available in cases. viral rna was extracted using a phenol-guanidine isothiocyanate method as previously described (papadopoulos et al., ) . reverse transcription was performed at • c in a mm tris-hcl, mm kcl, mm mgcl buffer containing mm dtt, . mm dntps, . g random hexamer primers and units of reverse transcriptase (superscript tm , invitrogen). cdna was stored at − • c until used. primers (upper mpvnf: aggccctcagcaccagaca ; lower mpvnr: ttgaccggccccataagc ) were designed against the n (nucleocapsid) region of the - isolate of hmpv (genbank accession nr. af . ) to amplify a bp fragment corresponding to nucleotides - of the published hmpv genome (van den hoogen et al., ) . pcr reaction was conducted in the presence of . mm mgcl , m dntps, . m of each mpv primer and . units taq polymerase (invitrogen) for cycles of • c for s, • c for s and • c for min. subsequently, % of the pcr reaction volume was run in a . % agarose gel and, when present, bp amplicons were excised and further purified with the concert rapid gel extraction system kit (gibcobrl), according to the manufacturers' instructions. selected samples were vacuum dried and sequenced using the mwg ag biotech (ebersberg, germany) sequencing facilities. comparison of the sequenced fragments to the hmpv genome was done by submission to the genbank using the standard nucleotide-nucleotide blast (blastn), whereas comparison between individual amplicons was conducted using blast . npws obtained from healthy children, without symptoms or signs of upper respiratory infection were also analyzed as negative controls. one-way analysis of variance was used for the comparison of continuous variables among subjects with hmpv, without hmpv and with rsv. chi-square was performed for the analysis of ordinal or categorical data. results of continuous data are expressed as mean ± standard error of mean. probability values of < . were considered significant. hmpv was present in / samples ( . %). sequence analysis of positive pcr products and comparison with the published sequences (van den peret et al., ) , confirmed the identity of the amplicons. rsv was the predominant pathogen in this cohort, found in / samples ( . %), followed by human rhinovirus (eight cases: . %), adenoviruses (four cases), coronavirus (three cases) and parainfluenza virus (one case). in patients, two viruses were simultaneously present, while no pathogen was identified in nine cases. pcrs performed in npws of healthy control children obtained during the same period, were all negative for hmpv. among the hmpv positive samples, the virus was a unique pathogen in five cases ( . %), while in four it was present together with rsv. no double positive cases of hmpv with other respiratory viruses were observed. most hmpv cases appeared in february and persisted, though gradually diminishing, until april (fig. ) . the rsv season on that year was between december and march. there were no differences in disease characteristics between bronchiolitis cases were hmpv was identified and those caused by rsv or other pathogens (table ) . there was also no difference in disease severity between double positive rsv-hmpv cases and single positive hmpv (severity index: . ± . versus . ± . , respectively). similarly, no differences were found when components of the severity index (heart rate, respiratory rate, degree of wheezing, skin colour and oxygen saturation) were analyzed individually, or in laboratory findings (data not shown). unless otherwise indicated data are presented as number of positive/number of reported (%). a severity score was calculated by adding individual scores ( - ) for cardiac rate (< , - , > ), respiratory rate (< , - , > ), wheezing (expiratory, expiratory + inspiratory, audible without auscultation), skin color/feeding (normal, mild cyanosis/difficult feeding, cyanosis/feeding not possible) and sao (> %, - %, < %). the severity score ranges from - . although rsv is indisputably the most common cause of acute bronchiolitis, virological analyses have failed to reveal a pathogen in a considerable fraction of cases in most studies. in this context, the recently isolated hmpv (van den hoogen et al., ) , could partially account for previously undiagnosed cases. the present study provides evidence of frequent involvement of hmpv in acute bronchiolitis in infants, using pcr-based detection. hmpv was present in % of bronchiolitis cases, a percentage only second to rsv, comparable to that of human rhinovirus (papadopoulos et al., ) , and clearly higher than that of the other respiratory viruses tested. similarly, viazov et al. ( ) detected hmpv in . % (and rsv in . %) of young children with respiratory tract illness, with most the cases diagnosed as bronchiolitis (viazov et al., ) . in another study, the percentage of hmpv within the bronchiolitis subgroup was % (maggi et al., ) . hmpv was either a unique pathogen, or co-existed with rsv. greensill et al. ( ) , analyzed bronchoalveolar lavage fluids collected from infants with rsv bronchiolitis and found that % of them were co-infected with hmpv (greensill et al., ) . in contrast, vicente et al. ( ) found no hmpv co-infections with any other virus in a pediatric population with acute rti (vicente et al., ) . the simultaneous presence of hmpv with rsv may reflect the considerably overlapping seasonal distribution of the two viruses. hmpv seasonal distribution observed in this study resembles those recently reported in finland and canada (jartti et al., ; boivin et al., ) . finally, it should be noted that this is the first report of hmpv infection in greece and the east mediterranean area, further confirming the worldwide distribution of this virus (van den hoogen et al., ; stockton et al., ; peret et al., ; howe, ; jartti et al., ; freymouth et al., ; viazov et al., ; peiris et al., ; vicente et al., ; maggi et al., ) . no differences were found between cases where hmpv was identified and those caused by rsv or other viral pathogens. we also found no difference in disease severity, or laboratory tests, between double positive rsv-hmpv cases and single positive hmpv cases. all the above support the notion that hmpv is able to induce bronchiolitis which is clinically identical to that caused by rsv. current evidence suggests that hmpv may account for a fraction of respiratory infection cases whose causative agent remained unidentified in previous cohorts (osterhaus and fouchier, ) . additional studies should be undertaken in order to characterize the role of hmpv in different human respiratory tract diseases and provide important information for future development of specific antiviral therapies and vaccines. virological features and clinical manifestations associated with human metapneumovirus: a new paramyxovirus responsible for acute respiratory-tract infections in all age groups human metapneumovirus infections in hospitalized children presence of the new human metapneumovirus in french children with bronchiolitis human metapneumovirus in severe respiratory syncytial virus bronchiolitis australian find suggests worldwide reach for metapneumovirus osterhaus ad, ruuskanen o. metapneumovirus and acute wheezing in children human metapneumovirus associated with respiratory tract infections in a -year study of nasal swabs from infants in italy human metapneumovirus in the community association of rhinovirus infection with increased disease severity in acute bronchiolitis children with respiratory disease associated with metapneumovirus in hong kong characterization of human metapneumoviruses isolated from patients in north america asthma exacerbations in children associated with rhinovirus but not human metapneumovirus infection a newly discovered human pneumovirus isolated from young children with respiratory tract disease analysis of the genomic sequence of a human metapneumovirus high prevalence of human metapneumovirus infection in young children and genetic heterogeneity of the viral isolates human metapneumovirus and community-acquired respiratory illness in children key: cord- - u o authors: tan, kian teo; greaves, malcolm w. title: n acne date: - - journal: int j dermatol doi: . /j. - . . .x sha: doc_id: cord_uid: u o two women, aged and years, presented to the dermatology outpatient clinic with acne vulgaris. both had nodular acne in a similar distribution over the cheeks, chin, and perioral areas (fig. ). each had a history of acne vulgaris as a teenager. both were healthcare assistants working in the singapore general hospital throughout the severe acute respiratory syndrome (sars) crisis, had worn n masks continuously for about months whilst on the wards, and had suffered an outbreak of acne of the skin occluded by the mask. they were treated with topical retinoid and systemic antimicrobials, and both responded well. the giant porokeratosis lesion on the left hand was totally excised and grafted. fifteen of the pm lesions were treated by cryotherapy. lesions healed with macular atrophy (fig. ) . porokeratosis is a disorder of keratinization. clinical forms include porokeratosis of mibelli (pm), giant porokeratosis, linear porokeratosis, disseminated superficial actinic porokeratosis (dsap), palmoplantar porokeratosis and punctate porokeratosis. the lesions of classic porokeratosis were described by mibelli in as one or more localized, chronically progressive, hyperkeratotic, irregular plaques with central atrophy and prominent peripheral keratotic ridge. a more superficial, disseminated form was described at approximately the same time by resphigi. in disseminated superficial actinic porokeratosis was described by chernosky, and in guss et al . added disseminated porokeratosis with palmar and plantar involvement (pppd) to the spectrum. giant porokeratosis is a very rare form of porokeratosis. several reports suggest it as a morphological variant of porokeratosis of mibelli, , , but others consider it as a different clinical form. , the lesion may be - cm in diameter and the surrounding wall raised up to cm. , rarely, variants of porokeratosis may coexist in one patient. , our patient has typical mibelli lesions, - cm in size, with central atrophy surrounded by keratotic walls on his limbs, trunk, hands and feet, widely disseminated flat and small annular dsap lesions on his face and neck as well as the giant porokeratotic lesion on his left hand. he was immunocompetent and had no other triggering factors. the histopathologic examination of the biopsy samples from the three different morphological lesions confirmed the diagnosis of porokeratosis. giant and linear porokeratosis lesions may be seen with underlying bony anomaly and mutilation, , but none was present in our case. the etiology of porokeratosis is unknown. an autosomal dominant mode of inheritance has been established for pm, pppd, disseminated superficial porokeratosis (dsp) and dsap. the similarities of clinical appearance and histopathology as well as the coexistence of different variants of porokeratosis in one patient or in several members of an affected family make us consider them as different phenotypic expressions of a common genetic aberration. reed and leone have proposed a mutant clone of epidermal cells expands peripherally, leading to formation of a cornoid lamella at the boundary between the clonal population and normal keratinocytes in porokeratosis. the tendency for abnormal clones to develop is probably inherited, and additional triggering factors such as ultraviolet light, photochemotherapy and loss of immunocompetence (hiv infection, organ transplantation) lead to clinical manifestation. , an alternative hypothesis is that an inflammatory mononuclear infiltrate composed of helper t cells, suppressor t cells and langerhans cells beneath the cornoid lamella may provide a mitotic stimulus for overlying keratinocytes. the optimal treatment procedure must be selected depending on lesion size and localization, and functional and aesthetic requirements. excision and grafting, cryotherapy, electrodesiccation, dermabrasion and co laser are all options. topical -fluorouracil is effective in pm, linear porokeratosis and in dsp and dsap. oral retinoids have conflicting results. excellent results were obtained with oral retinoids in some patients with dsap, widespread pm, pppd, and linear porokeratosis. with retinoid therapy cytologic atypia has disappeared. retinoids may have an inhibitory effect on cutaneous carcinogenesis in porokeratotic lesions. , one must be aware that relapses usually follow several weeks or months after discontinuation of retinoid therapy. the giant porokeratosis lesion on the left hand of our patient was totally excised and grafted. fifteen of the porokeratosis mibelli lesions were treated by cryotherapy, and these lesions healed with macular atrophy. malignancy was not detected by histopathological examination in the excised giant porokeratosis lesion. a -year-old african-american woman was referred to the department of dermatology, mount sinal hospital, new york for evaluation of a skin lesion on her scalp which had been present for years and had recently changed in appearance. on examination, she was found to have erythematous plaques with hair loss extending from the frontal hairline on the right side in a "c-shaped" distribution, terminating behind the left ear. two years later the lesion had rapidly extended to the vertex of the scalp and left temporal area (fig. ). there was a cm × cm area of alopecia at the vertex with slight erythema, but there was no evidence of scarring, inflammation or nodularity of the scalp (fig. ) . a skin biopsy taken from the forehead revealed granulomatous dermatitis showing noncaseating granuloma with negative acid fast bacilli (afbc) and ammonical-silverstain for fungus (gms) (fig. ) . she complained of chronic nasal congestion for months. nasal cavity examination and laryngeal endoscopy showed multiple nodules on the nasal septum and vocal cord. at that time, a presumptive diagnosis of sarcoidal nodule of the nasal septum and vocal cord was made and corticosteroids were administered orally. a chest x-ray demonstrated prominence of the right peritracheal and perihilar regions consistent with sarcoid. a gallium scan disclosed increased uptake in the lung and perihilar lymph nodes. a skull x-ray showed soft tissue density of the skull, probably indicating a subcutaneous sarcoidal nodule. computed tomography (ct) scanning of her brain and skull base showed a soft tissue mass along the outer table of the calvarium within the frontal region. six years ago, she experienced visual changes: blurred vision, tearing, floaters on both eyes and a cm × cm subcutaneous, painful, firm mass on the lateral side of her left upper eyelid. a skin biopsy was performed in another hospital and revealed noncaseating granuloma. a diagnosis of sarcoidosis involving the central nervous system, lacrimal gland, nasal septum, vocal cord, lung and scalp was made, and the patient was treated with mg of methylprednisone on alternate days with intralesional triamcinolone injection for skin lesions. during the follow-up period, nasal, laryngeal, pulmonary, ocular and cutaneous lesions were slightly improved and magnetic resonance imaging (mri) scanning will be carried out for further evaluation of the brain lesion. sarcoidosis is a systemic disease with unknown etiology that is characterized by the accumulation of mononuclear phagocytes with the formation of noncaseating granulomas. multiorgan involvement, including the skin, lungs, lymphatic system, liver, spleen, eyes, parotid glands, central nervous system, bones, and joints, is possible. , sarcoidosis occurs worldwide, affecting persons of all races, both sexes, and all ages. african-american women between the ages of and years were found to have the highest annual incidence. sarcoidosis affects african-americans more acutely with more extensive cutaneous involvement. the lesions are morphologically extremely variable, are frequently atypical, and often demonstrate fibrinoid necrosis on histology. a diagnosis of sarcoidosis is generally made on the basis of a biopsy finding of noncaseating granulomas without other explanation. on average, % of sarcoidosis cases have cutaneous involvement that can occur at any stage; however, most often cutaneous involvement occurs at the onset of the disease. , scalp lesions, eyelid papules, andichthyotic lesions are also associated with severe systemic disease. although cutaneous involvement in sarcoidosis is relatively common, sarcoidosis of the scalp is rare. there are few reported cases of cicatricial alopecia due to sarcoidosis, which predominantly affects black women with evidence of intrathoracic sarcoidosis and cutaneous involvement at other sites. [ ] [ ] [ ] [ ] cases of nonscarring alopecia have also been reported, either with localized involvement or with total alopecia. the high incidence of systemic involvement in reported cases indicates that a systemic work-up is needed when scalp sarcoidosis is diagnosed. this entity is usually resistant to treatment. there are no definitive diagnostic tests. a biopsy (skin, preitracheal nodes, or salivary glands) should be performed to obtain histologic confirmation of noncaseating granulomas in addition to pulmonary function tests, electrocardiography, slit-lamp eye examination, and tuberculin /anergy testing. , because sarcoidosis of the scalp may exhibit markedly variable morphologies, we emphasize that sarcoidosis should be considered in the differential diagnosis of plaques or nodules of the scalp as well as both cicatricial and noncicatricial alopecia. sars-associated coronavirus. apart from sars infection, healthcare workers were also affected in other ways. skin disorders were seen as a consequence of the various measures instituted to curb the transmission of sars. the two patients with acne vulgaris reported here are representative of several seen during and after the sars period. acne vulgaris is a self-limiting, multifactorial disorder affecting the sebaceous glands and pilosebaceous follicles. it is a common problem in singapore. there are several clinical subgroups of acne, including tropical acne. this is a wellknown entity which occurs in hot and humid climates. it is particularly common in soldiers and affects mainly the trunk and buttocks. in the two patients reported here, there was localized exacerbation of acne on the part of the face covered by the n mask. during the sars period, n masks were recommended by the cdc and world health organization (who) for use when healthcare workers came into contact with confirmed or suspected sars patients. they provided at least % filtration against oil-free particles and needed to be worn tightly against the face to be effective. it was not surprising to see acne occurring in the regions of the face covered by the masks. donning of these masks over prolonged periods of time creates a humid "tropical" skin microclimate conducive to a flare-up of acne. alternatively, the flare-up could have been a consequence of simple pilosebaceous duct occlusion due to local pressure on the skin from the close-fitting masks. apart from the risk of contracting sars, the disease also affected healthcare workers in other physical and psychologic ways. acne vulgaris resulting from the wearing of tightfitting masks over prolonged periods of time was one of the physical hazards. we will no doubt continue to see this condition as long as we need to take precautions to prevent another sars epidemic. disorders of keratinization dermatology in general medicine porokeratosis -a mutant clonal keratosis of the epidermis porokeratosis in immunosuppressed and nonimmunosuppressed patients mibelli's porokeratosis gigantea porokeratosis mibelli gigantea: case report and literature review the coexistence of linear and giant porokeratosis associated with bowen's disease co-existence of variants of porokeratosis: a case report and a review of the literature linear porokeratosis with underlying bony abnormalities generalized linear porokeratosis treated with etretinate imaging in ophthalmology cutaneous sarcoidosis cutaneous involvement in sarcoidosis cutaneous sarcoidosis in black south africans cutaneous sarcoidal granulomas and the development of systemic sarcoidosis sarcoidosis of the scalp: a case series and review of the literature sarcoidosis: a cause of steroid-responsive total alopecia cicatricial alopecia of sarcoidosis sarcoidosis presenting as a scarring alopecia singapore government sars website diseases of the sebaceous glands. in: fitzpatrick's dermatology in general medicine tropical acne -one hundred cases fighting the sars epidemic in taiwan: a nursing perspective figure twenty-seven-year-old woman with acne papules and nodules on the face over the area occluded by the n mask two women, aged and years, presented to the dermatology outpatient clinic with acne vulgaris. both had nodular acne in a similar distribution over the cheeks, chin, and perioral areas ( fig. ). each had a history of acne vulgaris as a teenager. both were healthcare assistants working in the singapore general hospital throughout the severe acute respiratory syndrome (sars) crisis, had worn n masks continuously for about months whilst on the wards, and had suffered an outbreak of acne of the skin occluded by the mask. they were treated with topical retinoid and systemic antimicrobials, and both responded well. singapore was one of several countries affected by severe acute respiratory syndrome (sars key: cord- -vruq r z authors: guihot, amélie; bricaire, françois; li, taisheng; bossi, philippe title: syndrome respiratoire aigu sévère : une épidémie singulière de pneumonie virale date: - - journal: la presse médicale doi: . /s - ( ) - sha: doc_id: cord_uid: vruq r z résumé agent infectieux le syndrome respiratoire aigu sévère (sras) est une pneumopathie fébrile initialement observée en chine à la fin de l’année . l’agent infectieux étiologique a rapidement été identifié comme étant un nouveau coronavirus, baptisé coronavirus associé au sras (sras-cov). la transmission du virus est interhumaine, par les particules respiratoires principalement. clinique et traitement la gravité clinique est variable, allant de la simple fièvre au syndrome de détresse respiratoire aigu. il n’existe pas de traitement spécifique. cependant, la ribavirine combinée à des corticoïdes a été utilisée avec succès dans un certain nombre de cas. Épidémiologie au cours du premier semestre de l’année , la dissémination du virus a été extrêmement rapide, évoluant sur un mode pandémique, contaminant plus de patients, dont en sont décédés. le réservoir viral, probablement d’origine animale, reste inconnu à ce jour. l’épidémie semble être actuellement jugulée, mais des ré-émergences sporadiques ou épidémiques sont possibles et ont été décrites en chine dans la province de guangdong début janvier . summary infectious agent the severe acute respiratory syndrome (sars) is a febrile pneumonia initially observed in china at the end of . the infectious agent has rapidly been identified as a new coronavirus, baptised sars-associated coronavirus (cov-sars). transmission is inter-human, via respiratory particles mainly. clinical presentation and treatment the clinical presentation is highly variable, from a mild fever to an acute respiratory distress syndrome. there is no specific treatment. ribavirin associated with steroids have been used with success in numerous cases. epidemiology during the first half of , the spreading of the virus has been very fast, with a pandemic mode of evolution. more than people were infected and died. the reservoir of the virus, which may be animal, is still unknown. the epidemic seems to be controlled, but sporadic or epidemic re-emergences may occur and have been observed in china duting january . agent infectieux le syndrome respiratoire aigu sévère (sras) est une pneumopathie fébrile initialement observée en chine à la fin de l'année . l'agent infectieux étiologique a rapidement été identifié comme étant un nouveau coronavirus, baptisé coronavirus associé au sras (sras-cov).la transmission du virus est interhumaine, par les particules respiratoires principalement. clinique et traitement la gravité clinique est variable, allant de la simple fièvre au syndrome de détresse respiratoire aigu. il n'existe pas de traitement spécifique. cependant, la ribavirine combinée à des corticoïdes a été utilisée avec succès dans un certain nombre de cas. Épidémiologie au cours du premier semestre de l'année , la dissémination du virus a été extrêmement rapide, évoluant sur un mode pandémique, contaminant plus de patients, dont en sont décédés. le réservoir viral, probablement d'origine animale, reste inconnu à ce jour. l'épidémie semble être actuellement jugulée, mais des ré-émergences sporadiques ou épidémiques sont possibles et ont été décrites en chine dans la province de guangdong début janvier . n chine du sud-est, au début de l'année , une épidémie de pneumopathie hautement contagieuse et potentiellement mortelle a été signalée par les autorités sanitaires chinoises.cette entité clinique d'étiologie inconnue a été baptisée syndrome respiratoire aigu sévère (sras) par l'organisation mondiale de la santé (oms). grâce au travail de chercheurs du monde entier, l'agent infectieux responsable du sras a rapidement été identifié comme étant un nouveau coronavirus :le coronavirus associé au sras (sras-cov). le caractère a priori particulièrement contagieux de ce micro-organisme a contraint l'oms et les autorités de nombreux pays à prendre des mesures exceptionnelles de santé publique au cours du premier semestre de l'année . le premier cas de sras a été officiellement observé le novembre dans la ville de fushan, province de guangdong, dans le sud-est de la chine. plusieurs cas de sras ont été recensés au cours de cette période dans les villes adjacentes. c'est dans la ville de zhongshan qu'a été déclaré le probable cas index -un homme de ans -à l'origine d'une épidémie plus importante signalée à guangzhou (canton), capitale de la province de guangdong.ce marchand de poisson aurait transmis l'infection à plus de personnels soignants et plus de . ces résultats suggèrent que le sras-cov aurait été transmis à l'homme par passage de barrière inter-espèce. le très probable réservoir animal du virus pourrait résider au sein de plusieurs familles d'animaux domestiques ou sauvages,en particulier des mammifères (civette, chat, rat, pangolin), mais ne demeure pas totalement connu à ce jour . la transmission du sras-cov est interhumaine, par l'intermédiaire de gouttelettes de salive, et peut-être par contamination des surfaces ou de l'air ambiant par des sécrétions respiratoires ou digestives , . la contagiosité est maximale au cours de la deuxième semaine d'évolution de la maladie. ces données épidémiologiques coïncident avec les données virologiques puisque l'excrétion virale respiratoire est maximale au e jour d'évolution clinique [ ] [ ] [ ] [ ] [ ] [ ] . elle a été de , % à travers le monde. l'âge est le facteur aggravant majeur : la mortalité des patients de plus de ans est trois fois supérieure à celle des malades plus jeunes . dans une étude réalisée sur cas chez des enfants,aucun décès n'a été constaté, et seulement deux ont nécessité une ventilation au masque . sur la numération formule sanguine, une lymphopénie est l'anomalie la plus fréquente, présente dans à % des cas , .cette lymphopénie porte à la fois sur les lymphocytes t cd et cd , les lymphocytes b et nk (natural killer), et suggère une altération des défenses immunitaires des malades au cours du sras. il existe parfois une thrombopénie ( %), une leucopénie globale ( %), une anémie ( %) . parmi les anomalies biochimiques, l'élévation des lactico-déshydrogé-nases (ldh) est la plus fréquente, présente dans à % des cas , . d'autres anomalies biologiques ont également été observées : élévation des transaminases (asat/alat) ( %),des créatine phosphokinases (cpk) ( %), hyponatrémie ( %), hypokaliémie ( %), hypocalcémie, hypophosphorémie.les gaz du sang sont perturbés selon la sévérité de l'atteinte du parenchyme pulmonaire. selon la définition des cas probables de sras élaborée par l'oms, tous les patients ont des anomalies radiologiques pulmonaires (encadré ). la radiographie du thorax peut être normale au stade précoce de l'infection mais, en quelques jours, des opacités pulmonaires apparaissent. celles-ci sont constituées de condensations alvéolaires focales ou disséminées, ou d'aspects moins francs en verre dépoli. les opacités interstitielles ne sont pas habituelles. les anomalies radiologiques prédominent dans les lobes inférieurs ( %) et dans le poumon droit ( %).elles sont unilatérales dans % des cas (figure ). le scanner thoracique révèle souvent des consolidations alvéolaires sous pleurales, avec bronchogramme aérien et/ou aspect en verre dépoli . certains auteurs com-parent ces anomalies avec celles observées au cours des bronchiolites oblitérantes avec pneumonie organisée (boop). les lésions semblent prédominer dans les segments apicaux des lobes inférieurs (figure ).par ailleurs, des abcès pulmonaires, des adénopathies médiastinales ou des épanchements pleuraux n'ont pas été décrits. l'évolution radiologique se fait le plus souvent vers une progression rapide des lésions, pouvant mener à un aspect de sdra avec poumons blancs . une sérologie du sras-cov est disponible, mais ne permet qu'un diagnostic rétrospectif. en effet, la séroconversion, définie par une multiplication par au moins du taux d'anticorps igg spécifiques anti-sras-cov, survient entre le e et le e jour d'évolution clinique, de façon concomitante à la diminution de la charge virale dans les sécrétions respiratoires .la sérologie est l'outil de référence pour le diagnostic de sras. son intérêt réside essentiellement dans les enquêtes épidémiologiques. les mesures symptomatiques à prendre au cours du sras ne diffèrent pas de celles appliquées au cours des autres pneumopathies tout cas possible ayant des signes de pneumopathie à la radiographie ou au scanner pulmonaire tout cas possible pour lesquels les critères suivants sont remplis : bon état clinique, absence d'atteinte à la radiographie ou au scanner pulmonaire lors du suivi, absence de lymphopénie, absence de contact avec un cas probable. infectieuses aiguës : repos, hydratation, oxygénation. plusieurs auteurs recommandent un recours précoce à la ventilation non-invasive, afin d'éviter un collapsus alvéolaire menant au sdra. une ventilation au masque par pression positive (cpap ou bi-pap) est recommandée en cas de saturation artérielle capillaire en oxygène inférieure à %, de polypnée supérieure à /minute, ou d'aggravation rapide des lésions radiologiques pulmonaires. l'intubation est indiquée en cas d'aggravation clinique ou radiologique malgré la ventilation non-invasive,ou d'intolérance de celle-ci .l'utilisation précoce de la ventilation non-invasive semble améliorer le pronostic vital . en l'absence de certitude diagnostique, fréquente lors de la phase initiale de la maladie, certains auteurs recommandent la prescription d'une antibiothérapie empirique dirigée contre les bactéries communautaires responsables de pneumopathie (germes typiques et atypiques) . la ribavirine est un analogue nucléosidique utilisé comme anti-viral dans le traitement de l'hépatite c chronique (rébétol ® ), en association avec l'interféron α .la ribavirine possédant une activité in vitro contre plusieurs virus respiratoires (virus syncytial respiratoire, virus de la grippe), elle a été utilisée empiriquement par plusieurs équipes chez des patients atteints de sras. cependant, son utilisation dans le traitement curatif du sras n'a pas été validée par des essais randomisés contrôlés. plusieurs équipes ont noté une diminution de la mortalité des patients après introduction précoce de cet antiviral, d'autres n'ont pas observé d'amélioration de la survie , . la dose moyenne utilisée était de mg/kg/ heures en intraveineux, avec parfois une dose de charge de g/ h pendant jours.la durée du traitement variait de à jours , . des auteurs ont recommandé l'utilisation de la ribavirine en cas de non-réponse à une antibiothérapie empirique en heures, ou en première intention s'il existait une forte suspicion clinique (présence d'un cas contact) . des traitements curatifs par immunoglobulines intra-veineuses, sérothérapie ou interféron α n'ont pas prouvé leur efficacité . l'hypothèse de la participation d'une dérégulation cytokinique dans la pathogenèse des lésions pulmonaires du sras,la similarité des lésions radiologiques avec une boop, l'expérience clinique d'une équipe de hong kong ont conduit de nombreuses équipes à utiliser une corticothérapie par voie systémique . son utilisation précoce à haute dose semble être recommandée par la majorité des auteurs . les doses proposées vont de à mg/jour à le tri et le conditionnement des déchets s'effectuent dans la chambre. tous les déchets doivent être éliminés par la filière des dasri (déchets d'activité de soins à risque infectieux) en vue d'une incinération. les urines et selles doivent être évacuées dans les toilettes de la chambre. les soins de conservation (thanatopraxie) ne devraient pas être réalisés pour des malades décédés du sras. en l'état actuel des connaissances, il est légitime de recommander la crémation du corps. cependant, le libre choix de la famille doit être respecté après information. de nombreuses études ont tenté de mettre en évidence des facteurs pronostiques péjoratifs précoces afin de guider le traitement médical. en effet, des patients atteints d'une authentique infection à sras-cov sont restés pauci-symptomatiques, ou ont eu une évolution favorable spontanément en l'absence de traitement spécifique • les anomalies biologiques telles que polynucléose neutrophile, élévation des cpk, de l'urée sanguine, élévation franche des ldh, hyponatrémie , , . une équipe a pu établir un lien quantitatif entre la rt-pcr naso-pharyngée et le taux de mortalité . les effets secondaires de la ribavirine, en particulier l'anémie hémolytique, fréquente, et plusieurs désordres électrolytiques (hypokaliémie, hypomagnésémie) ont conduit certains auteurs à ne pas recommander de prophylaxie postexposition au sras-cov par cet antiviral , . la question de la résurgence d'une épidémie de sras reste entière, étant donnée l'ignorance donc le noncontrôle du probable réservoir animal du virus. quelques cas sporadiques ont été décrits à la fin de l'année : plusieurs cas de contamination en laboratoire, un autre en chine fin décembre (province de guangdong) dont le mode de contamination reste inconnu, et cas début janvier dans la province de guandong. les informations les plus récentes sur les données épidémiologiques du sras sont disponibles sur le site internet de l'oms . le sras est une pneumopathie alvéolaire aiguë virale liée au sras-cov,particulière par sa forte contagiosité. cette caractéristique a permis l'expansion d'une épidémie mondiale remarquable par plusieurs aspects. d'abord sur le plan médical, l'identification d'un nouveau virus pathogène pour l'homme et la description d'une nouvelle entité clinique ont été particulières par sa rapidité d'évolution et sa gravité, causant la mort de jeunes personnes. une collaboration internationale de chercheurs a permis l'identification du virus du sras en quelques semaines et le séquençage de son génome en une semaine. ensuite sur le plan épidémiologique, il y a eu une mobilisation sans précédent des systèmes de surveillance sanitaire internationaux et nationaux. ces structures ont permis l'observation d'une augmentation rapide du nombre de cas à travers le monde, facilitée par l'intensification des échanges aériens.l'oms a publié régulièrement des informations médicales et épidémiologiques. les mesures strictes de prévention de la transmission du virus appliquées dans les pays les plus touchés, et/ou la diminution du réservoir viral et l'évolution naturelle de l'épidémie ont permis de contrôler le nombre de nouveaux cas. sur le plan économique, durant le pic de l'épidémie, une véritable paralysie de l'activité a été observée dans les régions atteintes. les hôpitaux et les systèmes de soins, au centre de cette épidémie, ont été touchés de plein fouet par le sras.cette épidémie souligne encore une fois que les maladies infectieuses,loin d'être des maladies du passé, sont en perpétuelle adaptation à l'environnement animal et humain, et resteront toujours d'actualité. i description and clinical treatment of an early outbreak of severe acute respiratory syndrome (sars) in guangzhou, pr china sars: epidemiology, clinical presentation, management, and infection control measures introduction of sars in france identification of a novel coronavirus in patients with severe acute respiratory syndrome koch's postulates fulfilled for sars virus comparative full-length genome sequence analysis of sars coronavirus isolates and common mutations associated with putative origins of infection enteric involvement of severe acute respiratory syndrome-associated coronavirus infection lung pathology of fatal severe acute respiratory syndrome sars-coronavirus replicates in mononuclear cells of peripheral blood (pbmcs) from sars patients possible role of an animal vector in the sars outbreak at amoy gardens virology: sars virus infection of cats and ferrets clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong clinical features and short-term outcomes of patients with sars in the greater toronto area outcomes and prognostic factors in patients with severe acute respiratory syndrome in hong kong clinical presentations and outcome of severe acute respiratory syndrome in children expression of lymphocytes and lymphocyte subsets in patients with severe acute respiratory syndrome a cluster of cases of severe acute respiratory syndrome in hong kong severe acute respiratory syndrome: radiographic appearances and pattern of progression in patients a major outbreak of severe acute respiratory syndrome in hong kong evaluation of reverse transcription-pcr assays for rapid diagnosis of severe acute respiratory syndrome associated with a novel coronavirus lung pathology of severe acute respiratory syndrome (sars): a study of autopsy cases from singapore conduite à tenir pour la prise en charge des personnes présentant un syndrome ou une suspicion de syndrome respiratoire aigu sévère (sras) et des personnes contacts our strategies for fighting severe acute respiratory syndrome (sars) biochemistry and clinical applications of ribavirin severe acute respiratory syndrome: clinical outcome and prognostic correlates clinical features and short-term outcomes of patients with sars in the greater toronto area coronaviruspositive nasopharyngeal aspirate as predictor for severe acute respiratory syndrome mortality les coxibs sont-ils véritablement moins pourvoyeurs d'effets secondaires digestifs que les anti-inflammatoires non stéroïdiens classiques? entre l'éditorial remettant en cause l'étude class post-amm de l'évaluation de la tolérance digestive du célécoxib ( key: cord- -c c rfzi authors: gordon, sharon m.; jackson, james c.; ely, e. wesley; burger, candace; hopkins, ramona o. title: clinical identification of cognitive impairment in icu survivors: insights for intensivists date: - - journal: intensive care med doi: . /s - - -y sha: doc_id: cord_uid: c c rfzi background: a growing body of research has demonstrated the presence of ongoing cognitive impairment in large numbers of icu survivors. objective: this review offers a practical framework for practicing intensivists and those following patients after their icu stay for the identification of cognitive impairment in icu survivors. conclusions: early detection of cognitive impairment in critically ill patients is an important and achievable goal, but overt cognitive impairment remains unrecognized in most cases. however, it can be identified by objective (test scores) or subjective evidence (clinical judgment, patient observation, family interaction). approximately , patients are hospitalized in intensive care units (icus) each day in the united states [ ] . while research is limited regarding cognitive outcomes in patients who survive critical illness, these patients are at risk for physical, emotional, and neurocognitive morbidity [ , , , , ] . although additional research is necessary to address crucial questions regarding cognitive impairment in icu survivors, early reports are worrying, and in some respects parallel early reports of cognitive impairment following coronary artery bypass grafting (cabg). two decades ago studies on cabg and cognitive outcome were in their infancy and received relatively little attention [ ] . since that time over investigations [ ] have studied the effects of cabg on cognition and have documented the existence of pervasive and frequently severe cognitive deficits in - % of patients following surgery [ ] . the cognitive impairment reported in icu survivors is similar to that observed following elective cabg surgery [ ] and following carbon monoxide poisoning [ ] . current data suggest that approximately one-third or more of icu survivors develop ongoing and persistent cognitive impairment [ ] . among specific populations of icu survivors such as patients with acute respiratory distress syndrome (ards) the prevalence of persistent cognitive impairment is even greater and may be as high as % at year [ ] and % at years [ ] (table ) . while cross-study comparisons are difficult due to differences in study design (e.g., prospective vs. retrospective), definition of sequelae, neurocognitive tests administered, time to follow-up, patient population, and disease severity, the potential ramifications of these findings are significant, particularly if cognitive impairment goes unidentified. the purpose of this report is to highlight the problem of cognitive impairment following icu survival, to assist clinicians in identifying probable cognitive impairment in icu patients through objective as well as clinically oriented strategies, and to provide guidelines for referral of cognitively impaired patients to specialists in cognitive evaluation and rehabilitation. for a discussion of research issues with icu survivors, please refer to our companion article in this issue. defining cognitive impairment this review uses key terms that are widely understood in psychiatric, neurology, and neuropsychological settings but may be less familiar to intensivists. the term cognitive impairment, as defined here, refers to clinically significant abnormalities in one or more brain functions including memory, attention, mental processing speed, executive function, visual spatial abilities, and intellectual function. cognitive impairment can be mild, moderate, or severe and can limit an individual's ability to think, reason, and/or perform everyday tasks. the term cognitive decline refers to deterioration in cognitive abilities from baseline and is not necessarily synonymous with cognitive impairment as it does not imply an absolute level of functioning. for example, a person with an intelligence quotient in the superior range might experience significant cognitive decline and still function within the normal range, therefore not being characterized as cognitively impaired. however, this type of decline can cause significant disruption in the everyday life of a person who is used to performing at high levels in occupational and vocational areas. such was the case of the person quoted in the opening paragraph. alternatively, slight decline in a person with below average intelligence could result in the diagnosis of cognitive impairment but have a minor impact on everyday function. the impairment experienced by patients following icu hospitalization should not be equated with common dementias, such as alzheimer's disease and vascular dementia, which are typically age related, largely irreversible, progressive in nature, and characterized by significant impairments in memory and at least one other sphere of mental activity [ , ] . in contrast to common dementias, there is only limited information regarding the clinical course of icu-related cognitive impairment. for example, cognitive functioning appears to improve in many icu survivors from hospital discharge to year; however, significant numbers ( %) of icu survivors remain impaired at year [ ], with little improvement during the nd [ ] . the cognitive impairment experienced by many icu survivors varies widely with regard to severity and should be thought of as acquired disease or an exacerbation of a preexisting disease (depending upon the individual patient's situation). acquired cognitive impairment can range from mild to severe. for example, jackson et al. [ ] reported that after excluding those with detectable pre-icu baseline cognitive impairment % of patients suffered from persistent cognitive impairment of a severity similar to the cognitive impairments observed in mild to moderate dementia. although the nature of deficits differs across studies, it appears that impairment is particularly pervasive in areas of memory, visuoconstruction, processing speed, and executive functioning (fig. ) . the cause and risk factors for the development of cognitive impairment following icu hospitalization are largely unknown, although the risk factors for cognitive impairment following cardiac surgery are well documented and include advanced age, lower premorbid intelligence, cerebrovascular and peripheral-vascular disease, and hypoxia [ , ] . researchers have hypothesized that the presence of certain factors such as sepsis and ards and its associated hypoxemia [ ] , the development of delirium [ ] , and the use of sedative and narcotic medications are associated with the development of cognitive impairment after critical illness, although such mechanisms are in need of further exploration. although between one and three of every four patients experience cognitive impairment following icu treatment [ , ], little is known regarding the functional and financial impact of such impairment in these patients. cognitive impairment is generally associated with inability to return to work, decreased quality of life and independence, and generalized functional decline; an important caveat to this observation, however, is that many investigations on the consequences of cognitive impairment have been carried out in populations with alzheimer's disease, and may not be directly applicable to icu survivors [ , , , ] . cognitive impairment resulting from a host of illnesses and medical syndromes including human immunodeficiency virus, ards, trau-matic brain injury, and bacterial meningitis are associated with decreased quality of life [ , , , ] . even mild forms of cognitive impairment can be extremely problematic and may lead to significant difficulties in activities of daily living such as impaired driving, money management, and performance of basic household functions (e.g., cleaning, cooking, organizing) [ , , ] . the specific economic consequences of cognitive impairment following a stay in the icu are not yet known. however, in the general population the economic consequences of cognitive impairment are substantial and depend on factors such as the severity and nature of impairment, rate of decline, and the setting in which care is provided (e.g., nursing home vs. private residence) [ ] . for example, a -point decrease on the mini mental state examination (mmse) is associated with a $ , per year increase in overall healthcare expenditures [ ] . the "per-patient societal cost burden" of even mild forms of cognitive impairment is estimated to be over $ , per year [ ] . the costs associated with traumatic brain injury are less well known, but it appears that the wages of individuals returning to work after a brain injury decline by approximately % per year [ ] . should the icu team strive towards early identification of cognitive impairment? a consensus is emerging among neurologists, psychiatrists, and other specialists regarding the importance of early identification of cognitive impairment [ ] . the failure to identify cognitive impairment can have serious implications for patients in a variety of functional domains. for example, a person may return to work based on the erroneous assumption that he or she is "perfectly fine," only for the patient to encounter difficulties performing at the previous level due to problems with memory and disorganization. these difficulties may be wrongly attributed to "laziness" or lack of motivation and may result in the termination of employment. situations such as this are not inevitable and can often be avoided if a patient's cognitive impairment is identified as such. the identification of cognitive impairment is valuable not only to patients but also to their families and caregivers as it enables them to mobilize necessary resources before the onset of a crisis such as inability to care for self or children and to function independently. the lack of early identification of cognitive impairment delays referral for cognitive rehabilitation, which has been shown to improve cognitive function [ ] . cognitive rehabilitation may be appropriate for individuals with cognitive impairment due to a wide variety of causes (e.g., traumatic brain injury, cerebrovascular accident, hypoxia) and is considered to be effective in improving neuropsychological abilities such as attention/ concentration, memory, and executive function [ ] . despite the importance of early identification of cognitive impairment, studies consistently demonstrate that physicians fail to recognize (or assess) cognitive impairment in - % of patients in non-icu clinical practice settings [ , ] . recent data suggest that cognitive impairment is rarely evaluated in icu patients [ ] and may be overlooked in one of every two cases [ ] . reasons for limited recognition of cognitive impairment include time constraints, perception of limited treatment options, and limited knowledge regarding how to perform cognitive screening [ ] . intensivists and those caring for patients after the icu stay should be aware that there are excellent brief screening tools that can be readily used in the midst of a busy day by themselves or other members of the icu team ( table ). these measures are simple to use and do not require specialized training to administer. while the early identification of cognitive impairment is very important, the approaches to identification vary widely depending on the setting. assessing patients in various hospital and outpatient settings presents various challenges and may require the use of different tools. patients can be assessed at various stages of their illness as they move from the icu to acute care and then to the outpatient setting. cognitive impairment in these different settings can be identified in a variety of ways and can be based on objective data (e.g., test scores) or more subjective evidence (e.g., clinical judgment, patient observation, family interaction). the following section suggests a logical approach at each stage and consider advantages and limitations of tools that can be used in each setting. how do you identify cognitive impairment in critically ill icu patients? in many instances intensivists are the providers best positioned to identify possible acute cognitive impairment in critically ill patients. although it is unlikely that they have the time to assess these patients individually, evaluations can be performed by nurses and other allied healthcare professionals such as psychologists, social workers, and speech therapists [ , ] . however, due to multiple factors in icu settings such as mechanical ventilation, related communication difficulties, the high prevalence of delirium, and patient fatigue, formal in-depth assessment of critically ill patients is often not possible. sometimes the only assessment possible in such populations is related to detection of delirium, which can be rapidly and reliably assessed with the intensive care delirium screening checklist [ , ] or the confusion assessment method for the intensive care unit (cam-icu) [ , ] . for free downloads of material used to monitor delirium in the icu (including translations into multiple languages) the reader is referred to the educational website: http://www.icudelirium.org. if patients are not delirious, their cognitive function can be quickly evaluated using the mmse or another brief cognitive screening tool. the detection of delirium may be important in light of evidence suggesting an association between delirium and an increased risk of cognitive impairment and other adverse outcomes (although much remains to be discovered about this association) [ ] . how do you identify cognitive impairment following icu stay in hospitalized patients? when patients are discharged from the icu to rehabilitation or general hospital units, their cognitive status may improve, and they may be more able to interact with an evaluator or clinician. at this point neuropsychological assessment may be appropriate and the completion of such testing more realistic. in cases where cognitive screening is possible numerous suitable instruments are available [ ] . the mmse is widely considered the "gold standard" among screening tools and consists of items ( possible points) that assess a range of global abilities including orientation, memory, and attention [ ] . a score of or below on the mmse indicates the presence of moderate to severe cognitive impairment, but it should be noted that the test is susceptible to the effects of age and education and can be more reliably scored using age and education adjusted norms [ ] . other screening tools that are equally "user friendly" and, in some cases require even less time to administer are available ( table ). in general, cognitive screening instruments require little if any specialized training to administer and score, and depending on the instrument the administration time varies from to min. while the sensitivity and specificity of these instruments vary, they generally have acceptable reliability and validity and are effective at identifying moderate to severe cognitive impairment. they are less sensitive in the detection of mild forms of cognitive impairment [ ] . while more comprehensive and sophisticated instruments exist, using them with hospitalized patients may be impractical as they can be quite lengthy and may require specialized training to administer. moderate or severe forms of cognitive impairment can frequently be identified without the use of psychometric instruments or questionnaires and through reliance on more subjective methods [ , , ] . these methods include the use of clinical judgment, the direct observation of patients, and interaction with families. the perceptions of family members can be very helpful as parents, spouses, or children are often aware of even minor changes in a patient's functional abilities or personality. the following is a list of warning signals, or "red flags," that can suggest possible cognitive impairment in hospitalized or icu patients: -personality changes -increased apathy -loss of social inhibitions, display of socially inappropriate behavior with staff -increased irritability or suspiciousness toward family, visitors, or medical team -outbursts of inappropriate or unprovoked anger -memory complaints -difficulty learning new facts and information about one's medical condition -persistent word finding problems -inability to recall conversations with medical staff and recent events in the hospital such as visits by staff, family, or friends -inability to remember having eaten or what was eaten at meal time -executive dysfunction -difficulty following nurses', physicians', or therapists' directions -problems with planning and decision making related to such things as discharge planning -confusion when trying to perform multiple tasks -functional deficits -difficulty looking up telephone numbers or using the telephone or other equipment such as the television and hospital bed -decline in self-care not attributable to physical problems or limitations -inability to find one's room -inability to follow a conversation -difficulty following through with tasks caution should be exercised when drawing conclusions about cognitive functioning based on in-hospital assessments as performance may be adversely affected by factors such as fatigue and residual effects of sedative and narcotic medications. how do you identify cognitive impairment following icu stay in the outpatient clinic? patients typically return to outpatient clinics approx. - months after hospital discharge for routine follow-up. by then patients have recovered from any transient cognitive dysfunction (e.g., delirium, effects of medications) and may be functioning at levels that reflect their new baseline. generally, individuals have begun to resume their normal activities and may experience previously nonexistent functional limitations due to acquired cognitive impairment. it may be beneficial to repeat the mmse and compare the current score with those obtained during the patient's hospitalization. improvement in cognitive function is expected and a decline of more than points (or a score below the standard cutoff of ), as well as the presence of persistently abnormal scores, suggests the need for further evaluation, as the mmse is a relatively stable, reliable measure and resistant to large fluctuations in scoring in the absence of actual neuropsychological change [ ] . it is also appropriate to assess activities of daily living such as bathing and dressing or, more importantly, instrumental activities of daily living (iadls) such as cooking, following a recipe, and balancing a checkbook (which can be significantly affected by even minor neuropsychological changes) [ , ] . formal assessments of functional abilities can be carried out with instruments such as the pfeffer et al. [ ] functional activities questionnaire (faq) and the lawton and brody [ ] instrumental activities of daily living (table ) or by asking simple, targeted questions. for example, clinicians can inquire about a patient's ability to perform complex tasks such as using a telephone or a remote control, following a complex recipe, making a grocery list, or managing money or medications [ ] . an important factor to evaluate is the presence of change and the degree to which the current level of function is different from prehospital levels. poor performance on measures of functional ability are not proof of cognitive impairment but can assist a practitioner in determining whether a patient should be referred for a more comprehensive neuropsychological assessment [ ] . many icu survivors experience significant affective symptoms such as depression and anxiety [ ] . the prevalence and severity of affective disorders including symptoms of depression and anxiety in icu survivors range from less than % to % [ , , , , ] . depression has been reported to occur in up to % of icu survivors [ ], and it is estimated that % have clinically significant anxiety [ ] . indeed, it may be that the high rates of depression among icu survivors are related to the cognitive impairment they experience, although this has not been evaluated in icu cohorts. affective disorders such as depression as well as posttraumatic stress disorder and anxiety may adversely affect test performance, especially if severe [ , ] . moderate to severe depression may result in decreased effort and low motivation that may decrease neuropsychological test scores in cognitive domains such as psychomotor speed or attention [ , ] , whereas moderate to severe anxiety may result in increased distractibility and blocked thoughts or words [ , ] . in some cases severe depression may mimic symptoms of cognitive impairment, although important differences exist between these conditions. in general, individuals with depression retain the ability to learn and do not forget as rapidly, do not display significant decrements in language, are inconsistent with regard to orientation to time and date and are typically more self-aware than their cognitively impaired counterparts [ , , ] . a variety of instruments are available for use in the assessment of affective function (table ). those for assessing depression include the geriatric depression scale-short form (gds-sf) [ ] , the beck depression inventory (bdi) [ ] , the center for epidemiologic studies depression scale (ces-d) [ ] , and the hospital anxiety-depression scale (hads) [ ] . anxiety can be assessed using the hads [ ] or the beck anxiety inventory (bai) [ ] . recent guidelines ( ) for dementia screening developed by the united states preventative services task force recommend that clinicians assess cognitive function whenever cognitive impairment or deterioration is suspected [ ] . in keeping with this recommendation (given the high rates of cognitive impairment in icu survivors), it would be ideal yet impractical to screen all icu survivors at hospital discharge and subsequent follow-up visits. therefore this is not recommended. an alternative approach is to screen only those individuals with an increased likelihood of developing cognitive impairment, although, as discussed above, only limited research has assessed causal mechanisms and risk factors of cognitive impairment following critical illness. more general evi- , , , , , , , ] . cognitive screening using a tool such as the mmse or mini-cog should be performed on any individuals who answer affirmatively to questions about memory difficulties, display impaired iadls, or have signs of cognitive impairment including ongoing delirium or memory/ orientation problems (e.g., confusion, repeating the same question, losing things such as glasses, forgetting familiar names, getting lost), social problems (e.g., neglect of appearance, nutrition, hygiene, loss of interest in hobbies, social withdrawal), and/or behavioral problems (e.g., wandering, irritability, agitation, apathy) should undergo cognitive screening using a tool such as the mmse. although screening at hospital discharge may result in a high false-positive rate because of the transient effects of medication and acute illness, it is important to track the patient's cognitive status during the weeks to months following icu and hospital discharge. when patients are thought to have cognitive impairment, they should be referred to a clinical neuropsychologist for consultation and further neuropsychological evaluation. although few neuropsychologists are actively involved in the assess-ment and management of survivors of critical illness at the present time, they are the appropriate professionals to assess cognitive function in these patients. it should be noted that neuropsychologists might be unavailable in small hospitals or rural areas. neuropsychologists are typically employed in neurology, rehabilitation medicine, or psychiatry departments in most moderately sized or large medical centers. in cases where neuropsychologists are unavailable, it is appropriate to refer patients to a clinical psychologist, as they are trained in performing basic cognitive evaluations. the adverse effects of critical illness on cognitive functioning are being increasingly studied and recognized by both clinicians and investigators. although much remains unknown, it appears that a significant percentage of critically ill patients and survivors experience cognitive impairment affecting quality of life and overall daily functioning. intensivists, particularly those that follow patients after icu discharge, are uniquely positioned to initiate cognitive screening and subsequent referral of critically ill patients and survivors. cognitive screening is simple, quick, and of great potential benefit, particularly in the early detection of cognitive impairment and should be widely incorporated in 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change test screening for dementia with the memory impairment screen the gpcog: a new screening test for dementia designed for general practice tics telephone for cognitive status, professional manual. psychological assessment resources key: cord- -pmzdovna authors: pennington, hugh title: politics, media and microbiologists date: journal: nat rev microbiol doi: . /nrmicro sha: doc_id: cord_uid: pmzdovna severe acute respiratory syndrome (sars) took everybody by surprise. its emergence was one of the most significant microbiological events of . it challenged microbiologists to identify the aetiological agent and satisfy koch's postulates — in so far as they ever can be met for a virus — in real time. not only were the patients' respiratory secretions and the agents grown in cultured cells put under the microscope, but so were the actions of politicians. what lessons can we learn from sars? acquired , and of the , sars infections in hong kong, ( %) were in healthcare workers - of whom were hospital staff who were infected while on duty . a low degree of transmissibility relative to other viruses is not the only property of the sars virus that was an important factor in the success of the response to the outbreak. the ease of propagation and identification of sars by well-established, standard virological methods were also of significance. the causative virus was grown without particular difficulty or problems with detection in readily available standard cell-culture systems. within a month of the identification of sars as a new clinical entity of uncertain aetiology -in late february in hanoi, vietnam -the virus had been grown in several laboratories. researchers at the university of hong kong cultured the virus using foetal rhesus monkey kidney cells challenged with material from an open lung biopsy from a -year-old male hong kong resident, and a nasopharyngeal aspirate from a -year-old woman. researchers at the bernhard nocht institue for tropical medicine, hamburg used vero cells inoculated with sputum from a -year-old male physician who had fallen ill in new york, after travelling from singapore where he had treated a patient. finally, at the centers for disease control and prevention (cdc), atlanta , researchers cultured the virus in vero cells inoculated with oropharyngeal samples from a -year-old male physician working in vietnam, sputum from a -year-old male, a kidney sample from a -year-old male and oropharyngeal washings from an adult vietnamese patient. in hong kong and atlanta the results of thinsection and negative-staining electron microscopy showed unequivocally that the cytopathic effect was being caused by a coronavirus. in all three laboratories, reverse transcription pcr and comparison with known coronavirus sequences revealed the same sequence identity; this work also showed that the sars virus was new, because its sequences were only distantly related to those from the wide range of previously sequenced coronaviruses that infect humans, dogs, cats, pigs, bovines, rats, mice, turkeys and chickens. louis pasteur said that "chance favours the prepared mind". the relevance of this explanation of scientific success to the sars virus, and in particular to the rapid initiation of collaborative work to understand and control it, is very clear. influenza had prepared the way. ever since the - influenza pandemic -which killed many millions, and was the low r value for sars indicates that controlling the spread of infection should be easier to achieve than for many other respiratory viruses. the only other virus spread by the respiratory route with an r value approaching that of sars is smallpox. for isolated pre-twentieth century populations with negligible immunity, gani and leach estimated the r value for smallpox to be . - . and, for importations into europe between and , to be about . for community-acquired disease. although an r value of this magnitude means that without control measures an outbreak would grow exponentially, in the case of smallpox outbreaks it was always possible to rapidly reduce the value of r during the progress of an outbreak. maintaining the r value below prevented the development of secondary cases; it was achieved by locating and isolating cases, and creating a ring of immune individuals around the outbreak by vaccination. outbreaks lasted only for weeks, with case numbers rarely exceeding double figures; more than three-quarters of outbreaks ended with the generation that was infected immediately after the detection of infection. r is a function of k,b and d ; where k is the number of contacts each infectious individual has per unit time; b is the probability of transmission per contact between an infectious case and a susceptible person; and d is the mean duration of infectiousness . in the case of infectious patients in hospital, their physical proximity to other patients and regular -often close and prolonged -contact with medical staff and nurses means that k and b will be greater than in the community or in domestic settings, particularly if transmission is only effective over short distances -centimetres rather than metres. this was the case for both sars and smallpox, and nosocomial transmission was indeed a characteristic feature. in outbreaks of variola major in europe between - , reviewed by mack , transmissions in hospital settings far outnumber those in any other category. of the cases, ( . %) contracted the disease in hospital, and cases ( . %) were in staff. in singapore, ( %) sars cases were nosocomially severe acute respiratory syndrome (sars) took everybody by surprise. its emergence was one of the most significant microbiological events of . it challenged microbiologists to identify the aetiological agent and satisfy koch's postulates -in so far as they ever can be met for a virus -in real time. not only were the patients' respiratory secretions and the agents grown in cultured cells put under the microscope, but so were the actions of politicians. what lessons can we learn from sars? the common cry uttered after crises is that 'lessons must be learned' . what lessons does sars teach? the crisis is over, at least for the time being, but how certain can we be that the german philosopher hegel was wrong when he said "what experience and history teach is this -that people and governments have never learned anything from history, or acted upon principles deduced from it"? before attempting to answer these questions using sars as an appropriate example, it is necessary to consider the sars virus itself and ask another question. how severe a test did sars pose? were we lucky? the answer is 'yes, we were' . this is qualified by the number of deaths that it caused; however, it is due, at least in part, to the properties of the virus. most important is the low r value -the basic reproductive number -for the sars virus, which represents the number of secondary infectious cases that are generated by an average infectious case in a susceptible population. the r value predicts the likelihood that an infectious agent will start an outbreak, the amount of transmission that can be expected in the absence of control measures and the ability of these control measures to reduce spread. studies on the sars outbreak in hong kong -after the exclusion of two 'superspread' events where special circumstances allowed index cases to infect many individuals (at the prince of wales hospital and at the amoy gardens estate) -gave an estimated r value of . . this is much lower than for any other virus that is spread by the respiratory route; r values for such infections in england and wales have been estimated to be - for measles, - for chickenpox, - for measles and - for rubella . national influenza centres in countries also had an extremely important role . reports in early february from guandong province, china, of cases and five deaths owing to an atypical pneumonia of unknown aetiology, coupled with the isolation of an avian influenza virus a subtype h n from two members of a family that had visited fujian province, china, in january, caused the activation of the pandemic plan. from analyses of samples taken from vietnam, singapore and hong kong, laboratories in the network ruled out the possibility of infection by any of the known influenza virus strains or other established causes of pneumonia, and concluded that sars was new. on march , the who issued emergency travel advice and, using the influenza network as a model, set up a network of scientists from laboratories around the world to identify the causative agent and develop diagnostic tests. laboratories were chosen owing to their experience in detecting a wide range of microorganisms, a history of collaboration in international investigations coordinated by the who, their technical capacity to fulfil koch's postulates and their access to sars samples. rules governing the confidentiality of data were set -shared data and information would only be used to advance the project in a collaborative way, data would only be shared outside the network with the approval of the originating laboratory, and caused by a virus of subtype h n -the possibility of the emergence of a virus strain capable of causing a similar event has, of necessity, been contemplated. the world health organization (who) initiated its international cooperation coordination programme in . pandemics with significantly lower mortalities occurred in (asian flu, subtype h n ), (hong kong flu, subtype h n ) and (russian flu, subtype h n ) (table ). although none have occurred since, the influenza outbreak that occurred in hong kong in , although local and small in case numbers, had such a high mortality rate in confirmed cases that it stimulated a review of pandemic response policies both in hong kong and internationally. it meant that the hong kong department of health, hospital authority and laboratory surveillance facility , and the who, were particularly well prepared to respond to the sars outbreak. in march , an outbreak of avian influenza caused by the a virus subtype h n killed several thousand chickens in three rural hong kong chicken farms. in may , a -year-old boy in hong kong contracted an influenza-like illness and died days later from reyes' syndrome -a paediatric complication that is associated with salicylate medication, which he had received. the virus strain resisted characterization with the available reagents; by august, detailed study in the netherlands and the united states had revealed that the virus was closely related to the avian strains that were prevalent in march. in november, human cases caused by this virus began to occur; by late december, there had been cases, of which five were fatal. contact with chickens had occurred in all confirmed cases. on december , the slaughter of all chickens in hong kong (a total of . million) began, their import was stopped and the outbreak ceased. in , a group of influenza specialists reviewed the responses to the outbreak and drew several lessons from them . some were influenza-specific, but others had wider relevance. regarding the actions that were taken in response to the outbreak, they reported that, at the time the outbreak started, the who was developing formal guidelines for addressing pandemic situations. these guidelines were revised after the hong kong influenza epidemic to include two strategic steps that were important in the outbreakrisk assessment (data collection and data evaluation) and risk management (continuously considering and reviewing the stages of a response, defining the risks and benefits, and making recommendations for the next steps to be followed). these principles underpinned the who response to sars. the who global influenza surveillance network of four collaborating centres (in atlanta, london, melbourne and tokyo) and committee was charged with the identification of lessons to be learned and with making recommendations for future epidemics. they focused on seven principles: a strengthened epidemiology capacity, systems for early detection and reporting, contingency planning, clear command and control structures, integrated responses, a surge capacity and transparency and effective communication. although it is impossible to disagree with these principles and their importance in outbreak control, a striking feature of the page report is that, with the exception of a box describing the isolation of the virus in hong kong entitled 'ground-breaking discovery' early in the report, there is little comment, favourable or unfavourable, about virology, virology laboratories or virologists. it could be said that this might be a reflection of the backgrounds and interests of the committee that generated the report -the two co-chairs (both from the united kingdom) were experts in hospital management and administration and in public health, respectively. but it is very probable that it reflects another problem -the lack of regard that is paid to the importance of microbiology in the response to microbiological threats to public health. reforming and improving healthservice administrative structures and information systems, and planning for unexpected rises in the number of contagious patients are all necessary, but by themselves they are insufficient. the flaw in hegel's aphorism about lessons from history is that microorganisms evolve in real time; learning lessons from past failures (and successes) will be insufficient because evolution throws up new challenges. in their review , maclehose, mckee and weinberg state that "one of the greatest challenges facing surveillance systems is awareness of the unexpected, recognizing when things seem not quite right. nipah virus was thought to be japanese b encephalitis, west nile virus in new york was thought to be st louis encephalitis, and prions were thought not to cross species barriers. focusing surveillance systems on the diseases of today fails to address the challenges of an uncertain future". we were lucky with sars due to the existence of a network of laboratories that were looking for new influenza viruses and which were linked by the mature surveillance network run by the who. other microbiological networks also exist. for food-borne pathogens there are, for example, pulse net in the united states (which uses standardized pulsed-field gel electrophoresis (pfge) protocols to generate and compare profiles of escherichia coli o , salmonella spp. and listeria monocytogenes), and enternet more than £ billion. the conclusions of the 'lessons to be learned inquiry' regarding the role of british virologists before and during the outbreak speak for themselves. the report states that, although the pirbright laboratory is the world reference laboratory and holds data on reported outbreaks throughout the world, "there is no coherent assessment of the full range of work undertaken by pirbright in relation to the national surveillance and control strategies" and "the service arrangements in place in covered the processing of samples a year…but contained no procedure for increasing the level of provision in an emergency". in addition, "the laboratory was not consulted when the fmd contingency plans were drawn up". the same failure to consult and use scientific expertise was evident in the handling of bovine spongiform encephalopathy (bse) -politics came before science. not long after the first occurrence of bse, an attempt to describe a case in the scientific literature was firmly quashed by the head of the veterinary investigation service of england and wales owing to "possible effects on exports and the political implications" . the phillips inquiry into bse and variant creutzfeldt-jacob disease (cjd) reported the lessons that had been learned; the largest number of which addressed one topic -the improved use of scientific advisory committees. their failure to use the full range of expertise available -even within the united kingdom -contrasts sharply with the inclusive approach adopted by the who in the case of the sars epidemic. the united kingdom was lucky with sars as there were no reported outbreaks. however, the situation was very different in hong kong, which had , cases and deaths. an expert review committee appointed by the government of the hong kong special administrative region reported its findings in october (ref. ). among other things, the samples would be regularly exchanged. there were daily teleconferences and a secure who website. on april , the participating laboratories collectively announced the conclusive identification of a new coronavirus as the causative agent. the who had performed well . owing to its low r value and ease of cultivation, the challenge that sars posed to the who was less difficult than it might have been. however, not everything was straightforward; for laboratories, epidemiologists and international organizations to react they must be told that there is a problem. the reaction to sars was delayed because initially it was unclear that there was a problem. the first cases of sars probably occurred in guangdong province, china, in november . on january , the health authority in guangdong province produced an expert report on the outbreak. many of the conclusions that the health authority reached would have been of significant use to policy makers elsewhere, but circulation of this expert report was limited. neither the who nor the hong kong authorities received a copy. whether an early reversal of the denial of sars by the chinese authorities during the early part of the epidemic would have had a significant effect on later events can only be a matter for speculation. however, the who was not able to investigate the situation in china until april , by which time sars had spread worldwide. a key factor in this spread had been the overnight stay in room of the metropole hotel, hong kong, of a professor from guangzhou in guangdong, which triggered outbreaks in hong kong, singapore, canada and vietnam. the lesson for microbiologists is a stark one -they operate in a political environment, and it can be unhelpful. other examples of the poor handling of infections by political systems are, unfortunately, not hard to find. there is a tradition in the united kingdom of responding to disasters by holding rigorous inquiries afterwards, which are expected to leave the public feeling "confident that a searching investigation has been held, that nothing has been swept under the carpet and that no punches have been pulled" . although this is good, the pity is that the need for such inquiries seems to recur far too often. in , the united kingdom suffered a serious outbreak of foot and mouth disease (fmd). millions of animals were slaughtered, and the overall costs totalled "the lesson for microbiologists is a stark one -they operate in a political environment, and it can be unhelpful." (which uses phenotypic data for typing), salmgene and pulsenet europe (both of which use pfge) in europe. the european commission has proposed the creation of a european centre for disease control and prevention to standardize surveillance methods, ensure data compatability, provide scientific assessments, technical support, information to officials and to the public, and to assume responsibility for existing networks and early warning and response systems. however, it is proposed that it will only have a staff of . the european health commissioner david byrne explained that "it will be a hub, an intelligence centre. the core is already in place". in this way it will be utterly reliant on national laboratories, national microbiologists and national infrastructures. is this 'core' up to the job? consider the united kingdom. its core is at least as effective as that of any other large european country. in universities, for example, it has many microbiologists of international rank . however, recent inquiries have identified serious problems. the recent report of the house of lords select committee on science and technology, 'fighting infection' , concluded that the infectious disease services in england that are "expected to protect the population from both common and more unusual infections are under-resourced and over-stretched…there is not enough surge capacity". the report went on to predict that "without improvements we fear that this country will suffer from major epidemics". the academy of medical sciences inquiry into 'academic medical bacteriology in the twenty-first century' focused on research. the inquiry concluded that although bacteriology has recently undergone a transformation with the introduction of several new techniques, "medical microbiology departments in the united kingdom, with a few exceptions, are in a state of torpor…". so microbiologists need to get their act together, and it will not do to rely on the bursts of funding and political approval that result from the panic engendered by disease outbreaks. hegel was right; history tells us that this kind of support is short-lived. sars proves the point. in july , the who declared the world to be free of sars, and in august , klaus stöhr, its sars research coordinator, stepped down and returned to influenza work owing to a lack of money . the ability to respond rapidly to new pathogens is not cheap. the next pathogen might have a high r value and be as elusive as a prion. the past tells us to be prepared. european budgets pale into insignificance compared with the $ . billion of the us cdc. obviously, european microbiologists have lessons to learn about getting political support! but funding is not the whole answer. the united kingdom, for example, has plenty of microbiological talent. recent experiences with bse and fmd indicate the need for big improvements in how policy makers get scientific advice. this problem persists. doctors, veterinarians, civil servants, politicians and scientists must come out of their boxes, learn to speak their different languages and communicate more effectively. if they fail, sooner or later there will be a heavy price to pay. evolution says so. coronavirus as a possible cause of severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome up close and personal with sars the next influenza pandemic: lessons from hong kong world health organization multicentre collaborative network for severe acute respiratory syndrome diagnosis. a multicentre collaboration to investigate the cause of severe acute respiratory syndrome report of the uk academy of medical sciences working group on sars the merchant shipping act formal investigations . (the stationery office report of the 'lessons to be learned' inquiry report when food kills responding to the challenge of communicable disease in europe the national molecular subtyping network for foodborne disease surveillance european equivalent of us centers for disease control proposed research assessment exercise: the outcome house of lords select committee on science and technology session - fourth report. fighting infection academic medical bacteriology in the twenty-first century search for sars origins stalls transmission dynamics of the etiological agent of sars in hong kong: impact of public health interventions infectious diseases of humans transmission potential of smallpox in contemporary populations transmission dynamics and control of severe acute respiratory syndrome smallpox in europe, - severe acute respiratory syndrome: lessons from singapore sars in hong kong: from experience to action the author declares that he has no competing financial interests. http://www.bt.cdc.gov centers for disease control and prevention: http://www.cdc.gov/ pulse net: http://www.cdc.gov/pulsenet/ society for general microbiology: http://www.sgm.ac.uk access to this interactive links box is free online. key: cord- -j jpqd k authors: o'brien, stephen j. title: cats date: - - journal: curr biol doi: . /j.cub. . . sha: doc_id: cord_uid: j jpqd k nan ecological niches on every continent except australia and the poles. wild cats dominate their habitat but require vast expanses to survive, which explains the tragic depredation such that every species of felidae, except the domestic cat, is considered either endangered or threatened in the wild today by cites, iucn red book and other monitors of the world's most endangered species. there are lots of them, small and easy to keep in the laboratory. whether we love cats or hate them, biology has learned much from studies of their comparative anatomy, comparative physiology (notably neurology) and behavior. for many biological subdisciplines, the cat species are considered as three groups: large, medium and small, a testament to their amazing similarity. the exception is reproduction, where each species has evolved exquisite co-adapted strategies for ovulation, hormone level regulation, sperm production, estrous incidence, mating preference and social organization. scrutiny by behavioral ecologists has provided a rich literature of distinctive reproductive parameters for several cat species, facilitating advances in assisted reproduction such as artificial insemination, cryopreservation, embryo transfer, in vitro fertilization and the first cloning of a domestic cat in . the advanced stage of reproductive assessment will one day soon lead to feline embryonic stem cells, transgenic and gene knockout cats, and protocols for stem cell and gene therapy trials. one of the most powerful biomedical models for cats involves the interaction of deadly infectious agents and the cat host's genome. domestic cats first gave us feline leukemia virus, which allowed the discovery of scores of 'oncogenes' in the s and s. when homologous human oncogenes were rapidly discovered thereafter, their misfiring in signal transduction pinpointed the molecular basis for many aggressive cancers. more recently, feline immunodeficiency virus (fiv), a first cousin of hiv, was discovered in house cats as the cause of a depletion of the cd t-cell subset that is a prelude to immune system collapse and pathology, the only naturally occurring model of aids. interestingly, over eight free-ranging wild species of felidae are infected with their own species-specific fiv strain (based on fiv gene sequence monophyly) that in most cases seems to be attenuated by historic selection of genetically resistant survivors in today's wild places. the devastating sars human coronavirus has a feline counterpart that causes a deadly feline infectious peritonitis (fip) syndrome in domestic cats. an outbreak of fip in a genetically uniform african cheetah colony led to % morbidity and % mortality, emphasizing the sensitivity of genetically inbred hosts to viral outbreaks. cats and their wild relative have given some sobering lessons about emerging virus outbreaks. in the mid s, a feline pan-leucopenia virus cultivated in a cat vaccine factory abruptly jumped from cats to dogs, producing a hyper-virulent a persian breed domestic cat. (photo courtesy of m. calingo). strain in puppies that within a few months caused widespread puppy mortality across the world. payback from the dogs came when a strain of canine distemper, endemic in the pet dogs of masai tribesman in tanzania, jumped to hyenas and then to african lions, killing a third of the huge lion population of serengeti national park in a six month interval in . add to the list of verified catspecific agents: alpha-herpesvirus, toxoplasmosis, cryptococcus, plague, q-fever, chlamydiosis and rotavirus infections, ehrlichiosis, calicivirus infection, poxvirus infection and mycobacteriosis. cats are also highly resistant to anthrax, with obvious implications. all these infections, and more, could prove valuable to biomedical research, providing we have a better working knowledge of the innate and adaptive immune system of cats. domestic cats and dogs enjoy more medical scrutiny than any species except humans. the world's veterinary schools produce thousands of practitioners each year, providing extensive documentation of genetic and chronic diseases with relevance to human maladies. the result is a comprehensive veterinary literature, which has described some feline genetic diseases (http://www.angis.org.au/databa ses/birx/omia/). these disease models offer, not only insight into disease development, but opportunities for better diagnostics and treatment experimentations. institute (nhgri) announced its decision to support sequencing of the domestic cat genome, along with those of seven other species: elephant, armadillo, tenrec, common shrew, guinea pig, hedgehog and rabbit. these species were chosen to complement those already selected for whole genome sequencing (human, mouse, rat, cow, chimpanzee, macaque, opossum and platypus) and to reflect the diversity among the living species of mammals as a first step in annotating the human genome's largely uninterpreted coding, regulatory and evolutionary conserved sequences. the cat offers the promise of a second carnivore species (in addition to the dog, which shares a common ancestor with cats dating back to approximately million years ago) to improve human genome annotation, as well as to complement the biomedical and genomic discoveries that make the feline genome attractive. genome evolution in mammals appears to proceed at two very different rates. the common default rate of chromosomal exchange is very slow and deliberate, so that the genome organization can be inferred for the common ancestor of all primates, carnivores and placental mammals. but a more rapid mode of genome rearrangement is seen in some lineages, such as gibbons, owl monkeys, dogs, bears and murid rodents, the genomes of which appear to have been re-shuffled several times relative to the ancestral form. cats and humans both have genomes in the primitive, un-rearranged form, so the cat provides a good opportunity to study the constraints on genome organization that have characterized the million year old mammalian radiations. the conserved genome of the cat is retained in the other felidae species, as well as most of the species of the carnivora order, the only reshuffled exceptions occuring in the dog and bear families. what can we expect after the cat genome sequence becomes available? many areas of biological and medical research will benefit from the projected cat genome sequence. human genome interpretation and annotation will be augmented by, on average, a single variant for each of its three billion base pairs. cats will enjoy genomic tools for inspection of feline hereditary disease, as well as the discovery of candidate gene variants that may explain evolved genomic defenses of infectious disease that threaten cats and man. evolutionary biologists will identify specific genes contributing to survival and species formation as unabridged gene/sequence maps narrow the search for adaptations. the observed constraints on genome reorganization will become a challenge for inferring the footprints of evolutionary steps that led to the modern cat species. an increased and informed database of mechanistic developmental specializations will add yet another reason to conserve the surviving cat species that are the keystone of species and habitat conservation projects, from cheetahs in namibia, to tigers in india and the russian far east, to lions in east africa and jaguars in the amazon. the natural history of the wild cats the laboratory cat wild cats status survey and conservation action plan iucn gland switzerland tears of the cheetah and other tales from the genetic frontier the promise of comparative genomics in mammals the feline genome project wild cats of the world key: cord- - d k authors: schlesinger, sondra title: alphaviruses — vectors for the expression of heterologous genes date: - - journal: trends biotechnol doi: . / - ( ) -p sha: doc_id: cord_uid: d k dna viruses and retroviruses are well established as vectors for the expression of heterologous genes, but there is increasing interest in the possibilities of using rna viruses, which do not replicate through a dna intermediate, for this purpose. this article summarizes some of the general properties of rna viruses and concentrates on one class of rna viruses — the alphaviruses — and their potential as expression vectors. virology is celebrating its centennial in . the existence of a filtrable infectious agent was first recognized years ago, when studies in russia and the netherlands on the infectious agent that caused the mosaic disease of tobacco plants led to the concept of infectious agents that were smaller than bacteria . at the beginning of the th century, the first human virus was discovered yellow fever virus -and for much of the intervening time the study of viruses has been involved primarily in the identification and characterization of viral agents that cause disease. however, viruses are now beginning to be associated not only with the cause of disease, but also with its cure. viruses are being used as vectors for the introduction of heterologous genes into cells, which means that they can be used for the delivery of therapeutic agents into cells, as well as for gene therapy. kna viruses infect a wide range of organisms, both prokaryotic and eukaryotic. an important feature of these viruses is that many of them produce high s. schlesinger levels of specific viral proteins, which makes them good candidates as vectors for heterologous gene expression , . to use the rna viral genome as a vector necessitates first converting the rna genome into a genomic complementary dna (edna), which is then placed downstream from a promoter for a bacterial or bacteriophage dna-dependent rna polymerase. inserts ofheterologous dna that encode the proteins of interest may then be introduced into the edna downstream from a strong viral promoter by standard restriction-endonuclease digestion and ligation techniques. the whole edna is then transcribed in vitro, using the rna polymerase promoter, into the genomic viral rna, which, in many cases, can be directly transfected into cultured cells, or into plants or animals. many rna viruses possess relatively small genomes; cloning of the recombinant edna can therefore be accomplished in only a few steps. many of the rna viruses that infcct higher eukaryotic organisms replicate exclusively in the cytoplasm and hence expression of their genes will be independent of host nuclear functions and will not involve any splicing. not all rna viruses, however, have the same mode of replication and this affects the strategy that must reviews be used in engineering the viral genome to obtain expression of the heterologous genes. a key feature which divides rna viruses into two categories is the 'polarity' of the rna genome . rna genomes with positive polarity are those in which the genomic rna functions as a mrna; the naked rna genome added to cells is infectious (i.e. it is able to initiate the steps leading to the production of new virus progeny). rna gcnomes in which the sequence is complementary to the mrna (i.e. negative polarity) must be transcribed into mrna before translation to yield protein can occur. negative-strand rna viruses contain the enzymes required for transcription o f their rna within the virus particle. although this makes the engineering of such genomes more complex, the feasibility of using these genomes to express heterologous genes has been established , . two other characteristics which vary among the different rna-virus families and are relevant to their potential use as expression vectors are: ( ) whether the genome exists as a single molecule of rna or as several molecules of different structure (i.e. a 'segmented' genome); and ( ) how the genome is translated. many rna viruses have segmented genomes: examples include both the negative-strand rna myxoviruses, most notably influenza virus; and the positive-strand rna plant viruses such as the bromovimses and cucumoviruses. the genome of viruses such as togaviruses, picornaviruses and flaviviruses is a single strand of rna of positive polari w. the latter two virus families have rna genomes with a single open reading frame (orf), from which the individual viral proteins are generated by co-and posttranslational proteolytic cleavages. insertions into these genomes must be in frame, and must permit the correct proteolytic processing. efforts to insert small heterologous epitopes into the picornavirus genome have met with success . togaviruses, coronaviruses and the plant viruses, bromoviruses and cucumoviruses, are examples of virus families in which translation of the nonstructural proteins involved in replication, and the translation of the structural proteins essential for packaging of the genome can be independent. during the course of replication, the infected cell produces both genomicand subgenomic-sized mrnas. the genomic rna is translated to produce some of the viral-specific proteins, but the other viral proteins are translated from subgenomic rnas. alphaviruses are the major genus of the togavirus family . they include sindbis virus, semliki forest virus and the human pathogens eastern and western equine encephalitis viruses. sindbis virus and semliki forest vires are best known as valuable models for molecular and cell biology, and it is these two viruses that are presently being developed as vectors for the expression of heterologous genes. the alphavirus genome is a single-strand rna of positive polarity of ~ . x nucleotides, that is capped at the ' terminus and polyadenylated at the ' terminus , . the genomic rna is referred to as s rna (on the basis of its sedimentation coefficient), and is encapsidated in an icosahedral protein shell which is composed of a singleprotein subunit. this shell is surrounded by a lipid bilayer that contains two transmembranal glycoproteins, organized as trimers of heterodimeric subunits. as many of the steps in vims replication and maturation utilize the host cellular machinery, the virus proteins provide markers for analysing the synthesis and modification of membrane glycoproteins, and the localization of proteins to the plasma membrane. the ' portion (approximately two-thirds) of the alphavirus genome contains the genetic information encoding the nonstructural viral proteins that are required for transcription and replication of the viral rna , . the ' portion of the gene (approximately one-third) contains the genes encoding the viral structural proteins -the capsid protein, a hydrophobic kda protein and the two viral envelope glycoproteins. the replication cycle of the alphaviruses is shown schematically in fig. . following attachment of the virus to the cell surface, entry is effected by receptormediated endocytosis. fusion of the virus membrane with the endosomal membrane releases the viral nucleocapsid into the cytoplasm, where translation of the genomic rna occurs. only the nonstructural viral proteins are translated from the genomic rna. there is a single orf and the resulting polyprotein is co-and post-translationally cleaved to form four polypeptides. these function together in a replication complex which is required for the synthesis of the complementary, negative rna strand. the newly synthesized negative rna strand serves as a template for the synthesis of more virion rna, and also for the transcription ofa subgenomic ( s) rna which may then be translated to yield the structural proteins. the nucleotides spanning the junction between the genes for the nonstructural protein and those for the structural proteins on the negative rna strand serve as the promoter for transcription of the subgenomic rna °, . the minimal sequence required to initiate transcription ( nucleotides upstream and five nucleotides downstream from the start of the subgenomic rna) is located within a region that is highly conserved among alphaviruses . if regions flanking the minimal promoter are also present, the level of transcription of the subgenomic rna is increased. the viral structural proteins translated from s rna are also synthesized as a polyprotein, with the n-terminal capsid protein functioning as an autoprotease to cleave itself from the nascent polypeptide. the capsid protein can then interact with the genomic rna to form the nucleocapsid. there is a specific recognition signal located near the ' terminus of the rna that serves as the nucleation site for encapsidation and thus ensures that the genomic, but not the subgenomic rna, is packaged. the viral glycoproteins, synthesized on membrane-bound ribosomes, are transported through the host-cell golgi network to the plasma membrane of the infected cell. finally, interaction of the nucleocapsid with the cytoplasmic tail of a viral glycoprotein leads to budding of the virus particle and release of infectious virus from the host-cell membrane. the basic strategy for using alphaviruses as vectors for the expression of heterologous genes has been to construct cdnas of the alphavirus genome, in which the heterologous gene is placed downstream from the promoter used to transcribe a subgenomic rna (fig. a) . the cdna is transcribed in vitro using the sp i)na-dependent rna polymerase and the rna transcript is transfected into cells either by lipofection ~ or electroporation is -methods that enhance the uptake of nucleic acids into cells. the genomic rna contains the genes for the nonstructural proteins and the cis-actiug sequences important in the recognition of the rna for replication and encapsidation , . the subgenomic rna synthesized in the transfected cells is translated into the heterologous protein. the first alphavirus vector designed using this strategy replaced the viral structural-protein gencs with the gene for the bacterial enzyme chloramphenicol acetyltransferase (cat) . about cat polypeptides were produced per transfected cell, corresponding to % of the total cell protein. more recently, a similar vector was designed using the cdna of semliki forest virus -~. infection by a virus is a much more efficient means for introducing its genome into the cell than transfection, and several different strategies have been designed to permit packaging of the rna vector. hahn et al. engineered the sindbis virus cdna so that it contained two promoters for subgenomic rna synthesis; one upstream of the sindbis virus genes, the other upstream of the heterologous gene (fig. b) . cells transfected with the recombinant, in vitro transcribed rna produced vires with yields ranging from to plaque forming units (pfu) per ml. the vector studied in the greatest detail contained the heterologous gene inserted downstream from the structural genes. infection of cells with the double subgenomic rna sindbis virus vector resulted in the production of >t cat molecules seven hours after transfection. the size of the heterologous insert can affect the stability of the recombinant rna. large inserts (i.e. > kb) are much less stable than smaller ones (i.e. < kb). the level of expression of the heterologous gene is higher when it is located downstream from the structural genes, although the recombinant rna appears to be more stable when placed upstream of the structural gene. the alphavirus vector containing a single subgenomic rna promoter can be packaged by complementation with a defective helper-virus rna (fig. ) . two types of packaging helper rnas have been used: • one, first described by geigenmtiller-gnirke et al. , contains the structural genes downstream from the promoter for the subgenomic rna and also contains the cis-acting sequences required for replication and encapsidation (fig. ) . most of the released particles contain both the helper and vector rnas in a single particle; such particles are able to form plaques and appear to be stable over several passages (i.e. repeated subculture). this type of packaged vector should prove to be of value in animal experiments in which both production of the heterologous gene and spread of the virus is desired. revieues • the second type of defective helper-virus rna lacks the region required for encapsidation of the rna. in studies of the packaging of the semliki forest virus vector, liljestr m and garoff ls found that cotransfection of the latter type of helper rna with vector rna led to efficient packaging of the vector rna, but the helper rna was not packaged. electroporation of both helper and vector rnas produced yields of up to particles from x cells (as observed by immunofluorescence analysis). packaging of the sindbis virus vector using sindbis defective helper rnas has proved to be more complex (p. bredenbeck, c. rice, i. frolov and s. schlcsinger, unpublished). deletion of the packaging signal leads to a considerable reduction in the amount of helper rna in the progeny; however, its presence can still be detected by analysis of the rna synthesized in vector-infected cells, and by a low level of pfu in the media, presumably due to copackaging of the rnas. this may be due to the ability of the encapsidation process to include other rnas in the capsid in conjunction with the viral rna encoding the specific packaging signal. a second potential problem is recombination between the packaging helper virus rna and vector rnas. the two sindbis rnas can undergo recombination to produce a single molecule of rna containing the genes that encode both the nonstructural and structural proteins m. although the growth and replication of such recombinants are suppressed under conditions in which most of the cells have been transfected with both helper and vector rnas, they can be detected after passaging of the infectious particles. a completely different approach to using alphaviruses as vectors has been to insert heterologous sequences into the viral structural proteins in such a way that the virus is still viable, but the heterologous epitope is expressed and can function as an immunogen ~ . this concept was originally tested with picornaviruses, where the site of insertion was chosen on the basis of the crystallographic structure of the virus . however, the three-dimensional structures of the sindbis virus glycoproteins are not known, and therefore it has not been possible to predict the regions of the glycoprotein which form surface loops and into which insertion of additional sequence would not adversely affect glycoprotein folding. initial studies, using an epitope from rift valley fever virus, involved making random insertions into the structural genes, followed by selection of viable viruses containing the insert. the chimeric sindbis virus that could tolerate insertions and that displayed immunogenic rift valley fever virus epitopes was used as a vaccine to make mice resistant to infection by rift valley fever virus. there are now several reports in the literature in which the sindbis virus vectors have provided an the double subgenomic rna sindbis viruses have been used in two studies. hahn et al. inserted either a truncated form of the influenza hemagglutinin (ha) protein, or minigenes encoding immunodominant cytotoxic t-cell epitopes into the sindbis virus genome. these vectors were used to sensitize target cells for lysis by appropriate major histocompatibility complex-restricted, ha-specific, cytotoxic t cells. they were also used to infect mice to prime a specific t-cell response. in addition, the double subgenorrfic kna vector was used to express the insulin-regulated glucose-transporter protein in cultured cells l this protein, when expressed by the sindbis vector, was found in adipocytes and muscle cells -its normal intracellular location. the semliki forest virus vector has been used to express a variety of proteins, including dihydrofolate reductase (dhfr), the transferrin receptor, and lysozyme, proving that the vector can be used to express cytoplasmic, membrane and secreted proteins, respectively is. studies on the expression of ~ -galactosidase indicated that about txg of the heterologous protein could be produced from x cells, corresponding to approximately % of total cellular protein is. the initial studies with sindbis and semliki forest virus suggest that both viruses are promising as vectors for heterologous gene expression. the potential of using alphaviruses to stimulate production of specific subsets of t cells by infection of animals is only just beginning to be explored. the use of alphaviruses to express proteins for production is ready to be exploited. one particular aspect of using alphaviruses as vectors needs further investigation. infection by these viruses is cytopathic for most vertebrate cells that are grown in culture. there are some variants that seem to be much less cytopathic and, for most of the sindbis virus variants, infected mosquito cells in culture do not show cytopathic effects . the mechanisms of cell killing are not known. infection of cultured cells with the semliki forest virus vectors shuts offthe synthesis of host-cell proteins, demonstrating that the viral structural proteins are not required for this effect . the inhibition of host-cell protein synthesis may be due to the expression of a particular viral gene product, or it could be a consequence of the very high level of expression of the heterologous gene. it is possible to modulate the expression of the subgenomic rna by the use of temperature-sensitive mutants or by the down-regulation of the subgenomic rna promoter ~ . a second issue of importance is the possible biohazard of working with alphaviruses. the vectors derived from sernliki forest virus that are unable to provide any viral structural proteins (termed 'suicide' vectors) should be safe for laboratory procedures, although the possibility of recombination and the production of segmented genome particles has not been fully resolved. there is one report of a fatal infection by semliki forest virus , so caution in using this virus must be maintained. most strains of sindbis virus are not pathogenic and so they may be preferred for many purposes, particularly for those investigators who are less familiar with working with infectious agents. there are many ways that these viruses can be attenuated and made safe for routine work, so that in the future their use should become essentially risk-free. the initial studies on the development of alphaviruses as vectors uncovered the phenomenon of recombination. it also led to some important discoveries about these viruses (such as the identification of the packaging signal and a definition of the subgenomic rna promoter). in the future, continued work on these vectors should not only provide us with improved expression systems, but also reveal other interesting facets of the biology of these viruses. the bgaviridae and flaviviridae proc. natl acad. &i. usa proc. natl acad. &i. usa proc. natl acad. &i. usa the "lb aviridae and blavivmdae key: cord- - p g p authors: peiris, j s m; guan, y; yuen, k y title: severe acute respiratory syndrome date: - - journal: nat med doi: . /nm sha: doc_id: cord_uid: p g p severe acute respiratory syndrome (sars) was caused by a previously unrecognized animal coronavirus that exploited opportunities provided by 'wet markets' in southern china to adapt to become a virus readily transmissible between humans. hospitals and international travel proved to be 'amplifiers' that permitted a local outbreak to achieve global dimensions. in this review we will discuss the substantial scientific progress that has been made towards understanding the virus—sars coronavirus (sars-cov)—and the disease. we will also highlight the progress that has been made towards developing vaccines and therapies the concerted and coordinated response that contained sars is a triumph for global public health and provides a new paradigm for the detection and control of future emerging infectious disease threats. of these deletions, however, is not clear. similarly, sars-cov in individuals before february was genetically more diverse than the later isolates , , . the spike protein (the viral surface glycoprotein which mediates viral attachment and entry into the cell; fig. ) of early isolates contained higher rates of nonsynonymous mutations, probably reflecting the ongoing adaptation to the new host. the relative genetic homogeneity of sars-cov isolates from later in the outbreak - may reflect a virus better adapted to the new host. the fact that much of the global spread arose from one index case in hotel m in hong kong , may also contribute to this genetic homogeneity. a ban on the sale of wildlife in wet markets in guangdong imposed during the later period of the sars outbreak was lifted in september . between december and january , there were four new cases of sars, the first nonlaboratory-associated cases diagnosed in humans since the end of the sars outbreak in july . epidemiological linkage and phylogenetic data suggest that the associated viruses were new introductions from animals (y. guan, unpublished observations) , , . these human cases were relatively mild and did not lead to secondary transmission, reflecting that the animal precursor virus is probably not well adapted to efficient human-tohuman transmission. this is probably a recapitulation of events in late in the run-up to the sars outbreak in . this time, the findings led to the reintroduction of the ban on wild-game animal markets and there have been no further naturally acquired human cases since. it is likely that the precursor of sars-cov has repeatedly crossed the species barrier but only occasionally has it succeeded in adapting to human-human transmission. this adaptation clearly occurred in late and it may happen again in the future. but given the present understanding and awareness about sars, we expect that such reemergence is unlikely to lead to a global outbreak on the scale of . the major routes of transmission of sars are droplet infection, aerosolization and fomites (refs. , and world health organization, http://www.who.int/csr/sars/en/whoconsensus.pdf ) . deposition of infected droplets or aerosols on the respiratory mucosal epithelium probably initiates viral infection. whether infection can occur through the oral or conjunctival routes is unknown, but sars-cov has been detected in tears . although exposure to the animal precursor of figure the global spread of sars. the number of probable cases of sars and the date of onset of the first case in each country (or group of countries) is denoted. the countries denoted in red are those where substantial local transmission occurred. the data are based on world health organization, http://www.who.int/csr/sars/country/table _ _ /en_ /en/print.html and the figure is adapted from ref. . wet markets in guangdong: 'wet markets' selling live poultry, fish, reptiles and other mammals are commonplace across southeast asia and southern china to service the cultural demand for freshly killed meat and fish produce. in some regions (e.g., guangdong province, china), increasing affluence has led to the proliferation of markets housing a range of live 'wild' animal species, such as civet cats, pictured, linked to the restaurant trade servicing the demand for these exotic foods. , , once the virus had adapted to human-to-human transmission in the later part of the outbreak, asymptomatic infection seemed to be rare . other peculiarities about sars-cov transmission were also evident. transmission was infrequent during the first five days of illness and, unlike transmission of influenza, was relatively inefficient in the household setting . despite sars's fearsome reputation and global spread, the average number of secondary infectious cases generated by one case (r ) was low ( . - . ); in contrast, the r of influenza ranges from to (ref. ) . although not unique to sars, 'superspreading events' (in which a few affected individuals disproportionately contribute to transmission) were characteristic of the outbreak , . the factors underlying the superspreading phenomenon of sars are poorly understood but may include coinfection with other viruses and host factors, as well as behavioral and environmental factors. the clinical symptoms of sars-cov infection are those of lower respiratory tract disease [ ] [ ] [ ] [ ] . besides fever, malaise and lymphopenia, affected individuals have slightly decreased platelet counts, prolonged coagulation profiles and mildly elevated serum hepatic enzymes. chest radiography reveals infiltrates with subpleural consolidation or 'ground glass' changes compatible with viral pneumonitis. but although the main clinical symptoms are those of severe respiratory illness, sars-cov actually causes infection of other organs: some affected individuals have watery diarrhea, and virus can be cultured from the feces and urine, as well as the respiratory tract [ ] [ ] [ ] . in addition, rt-pcr has identified the virus in the serum, plasma and peripheral blood leucocytes , . individuals with sars also have a pronounced peripheral t-cell lymphocytopenia: numbers of cd + and cd + cells are both reduced, and more than one-third of individuals have a cd + t-cell count of less than cells/mm (refs. , ) , suggesting increased susceptibility to secondary infections. the mechanisms underlying the t-cell lymphopenia remain to be elucidated. around - % of individuals with sars require management in intensive care units and the overall fatality rate is ∼ % (world health organization, http://www.who.int/csr/sars/en/whoconsensus.pdf). the age dependence of disease severity and mortality is notable; during the outbreak, mortality rates of affected individuals in hong kong who were - , - , - and > -year old were %, %, % and %, respectively (world health organization, http://www.who.int/ csr/sars/en/whoconsensus.pdf). none of the - -year-olds infected with sars-cov in hong kong had disease severe enough to require intensive care or mechanical ventilation , . this progressive age dependence in mortality is not totally explained by comorbid factors and the underlying biological basis remains unclear. quantitative studies of viral load have provided insights into the pathogenesis of sars. viral load is higher in the lower respiratory tract than in the upper airways , . viral load in the upper respiratory tract and feces is low during the first days and peaks at around day of illness. in marked contrast, viral load in influenza peaks soon after onset of clinical symptoms . this unusual feature of sars-cov infection explains its low transmissibility early in the illness. it also explains the poor diagnostic sensitivity of the first-generation rt-pcr diagnostic tests on upper respiratory tract and fecal specimens collected early in the illness (reviewed in ref. ) . affected individuals with high serum viral loads have a poor prognosis . between days - of illness, high viral load in nasopharyngeal aspirates, feces and serum, as well as detection of virus in multiple anatomic sites, are independently predictive of adverse clinical outcome . serial studies of viral load throughout illness also reflect clinical outcome . taken together, these findings suggest that poor clinical outcome is associated with continued uncontrolled viral replication. sars-cov rna can be invariably detected in the lungs of individuals dying of sars, but viral load is higher in those dying earlier in the course of the illness (< days) . the respiratory tracts of affected individuals who die during the first ten days of illness show diffuse alveolar damage with a mixed alveolar infiltrate, lung edema and hyaline membrane formation. macrophages are a prominent component of the cellular exudates in the alveoli and lung interstitium , . multinucleate syncytia of macrophage or epithelial cell origin are sometimes seen later in the disease. immunohistochemistry, in situ hybridization and electron microscopy on autopsy or tissue biopsy have unequivocally demonstrated sars-cov replication in pneumocytes in the lung and enterocytes in the intestine [ ] [ ] [ ] [ ] . individual reports of virus detection by in situ hybridization or immunohistochemistry in other tissues await confirmation by electron microscopy . in the large and small intestines, the virus replicates in enterocytes . viral particles primarily are seen on the apical surface of enterocytes and rarely in the glandular epithelial cells. but there is no villous atrophy or cellular infiltrate in the intestinal epithelium and the pathogenic mechanisms responsible for watery diarrhea in individuals with sars is unclear. some human intestinal epithelial cell lines support productive replication of sars-cov and gene expression arrays have shown that virus replication is associated with the expression of an antiapoptotic host cellular response, perhaps explaining the lack of enterocyte destruction in vivo . studies using pseudotyped lentiviruses, carrying the spike, membrane and envelope surface glycoproteins of sars-cov (fig. ) separately and in combination demonstrated that the spike protein is both neces- sary and sufficient for virus attachment on susceptible cells [ ] [ ] [ ] [ ] . the sars-cov spike protein uses a mechanism similar to that of class fusion proteins in mediating membrane fusion , . there is no consensus as to whether the virus entry occurs through a ph-dependent receptor-mediated endocytosis or through direct membrane fusion at the cell surface , , . the receptor for sars-cov was identified as the metallopeptidase ace- (refs. , ) . the soluble ace- ectodomain blocks sars-cov infection , and amino acids - of the spike protein are required for interaction with ace- (ref. ) . other coronaviruses use different cell receptors and enter cells either by means of fusion at the plasma membrane or through receptor-mediated endocytosis . immunostaining techniques have identified ace- on the surface of type and pneumocytes, the enterocytes of all parts of the small intestine and the proximal tubular cells of the kidney. this localization explains the documented tissue tropism of sars-cov for the lung and gastrointestinal tract and its isolation from the urine. but it is notable that colonic enterocytes lack ace- protein expression although sars-cov replication does occur in colonic epithelium , . in contrast, whereas ace- is strongly expressed on the endothelial cells of small and large arteries and veins of all tissues studied and the smooth muscle cells of the intestinal tract, there is no evidence of virus infection at any of these sites. this lack of virus infection in tissues that express the putative receptor prompts the question of whether a coreceptor is required for successful virus infection . vasculitis is known to occur in individuals with sars but its relation to infection of endothelial cells is unknown. because only the basal layer of the nonkeratinized squamous epithelium of the upper respiratory tract expresses ace- (ref. ) , undamaged epithelium of the nasopharynx is unlikely to support sars-cov replication. other receptors for virus entry that are independent of ace- expression may exist. pseudotyped virus containing the spike protein has also been shown to bind to dendritic cell-specific intercellular adhesion molecule grabbing nonintegrin (dc-sign) . dc-sign is a type-ii transmembrane adhesion molecule found on dendritic cells consisting of a c-type lectin domain that recognizes carbohydrate residues on a variety of pathogens. unlike the ace- receptor on pneumocytes and enterocytes, dc-sign does not permit sars-cov infection of the dendritic cells. instead, binding of sars-cov to dc-sign allows dendritic cells to transfer infectious sars-cov to susceptible target cells . a similar mechanism has been described for dengue virus, human immune deficiency virus (hiv) and cytomegalovirus, and may be relevant in sars pathogenesis. many details of sars-cov pathogenesis remain to be elucidated, but the development of a full-length infectious cdna clone of sars-cov should permit precise manipulation of the virus genome and will help our understanding of the viral determinants of pathogenesis . several inflammatory cytokines (il- β, il- and il- ) and chemokines chemotactic for monocytes (mcp- ) and neutrophils (ip- ) are elevated in adults and children with sars [ ] [ ] [ ] [ ] . the increased levels of monocyte-tropic chemokines may contribute to the prominently monocytic macrophagic infiltrate observed in the lung . but increases of these same chemokines occur in other viral diseases (e.g., influenza) and are not a unique feature of sars. in addition, elispot assays of peripheral blood leukocytes have revealed prolonged immunological dysregulation in individuals with sars . it is difficult to evaluate the overall pathogenic significance of these findings because immunological markers in the peripheral blood do not always reflect the local microenvironment of the lung . genetic factors associated with susceptibility to, or severity of, sars are under investigation. hla-b* has been associated with severe sars disease in taiwan but not hong kong . hla-b* has also been associated with disease susceptibility and hla-drb * with resistance to sars. the coinheritance of b* and b was significantly higher in individuals with sars than in the general population . the mechanisms underlying these disease associations remain to be elucidated. key to the development of effective antiviral drugs and vaccines against sars-cov was the development of animal models of sars ( table ) . sars-cov seems to cause infection in cynomolgous macaques following intratracheal inoculation [ ] [ ] [ ] . but whereas some researchers find evidence of disease pathology reminiscent of that seen in individuals dying of sars and can show sars-cov antigen and viral particles in the pneumocytes of infected macaques , , others only find evidence of a mild upper-airway disease and low levels of virus by rt-pcr . these differences in outcome may reflect differences in the viral strain, pre-exposure history and age of the animals, route of inoculation, stage of infection at which necropsy was performed or other factors. other animal models include ferrets, cats, golden syrian hamsters, mice and african green monkeys ( table ) [ ] [ ] [ ] [ ] [ ] . these animal models support viral replication in the upper and lower respiratory tracts [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . ferrets and hamsters also develop notable lung pathology. infected cats and ferrets transmit sars-cov to noninfected animals held in the same cage . natural asymptomatic infection in cats was documented during the community outbreak at amoy gardens, hong kong (world health organization, http://www.who.int/csr/sars/en/ whoconsensus.pdf). these animal models of sars differ from natural human disease in that the period between infection and peak disease pathology or peak viral load is shorter than is found in human disease and because the disease pathology, when present, is self-limited and rarely progresses to a fatal outcome as occurs with sars. they also do not accurately reproduce the intestinal component of the human disease. but these models provide the only options presently available for addressing questions relevant to therapeutics and vaccine development. they can provide useful information providing their limitations are recognized. several potential antiviral agents have been evaluated in vitro, and a few have been tested in animal models. screening of currently available antiviral drugs and chemical libraries reveals that interferons, glycyrrhizin, baicalin, reserpine, niclosamide, luteolin, tetra-o-galloylβ-d-glucose and the protease inhibitors have in vitro activity against sars-cov - . differences in in vitro susceptibility of sars-cov to interferon (ifn)-β b, ifn-α and ribavirin , - probably relate to differences in the testing methods used. overall, ifn-αn /n , leukocytic ifn-α, ifn-β and the hiv protease inhibitors (especially nelfinavir) are consistently active in vitro and should be considered for animal studies and randomized placebocontrolled clinical trials. type interferons render uninfected cells refractory to sars-cov replication through a mxa-independent mechanism , whereas the hiv protease inhibitors may block the activity of the main sars-cov proteinase . so far, only interferons have been tested in animal models: in cynomolgous macaques, pegylated ifn-αn provided prophylaxis but was only marginally effective for early treatment . no randomized placebo-controlled trials have been performed for any of these antiviral drugs, although treatment studies using historical controls have suggested clinical benefit from ifn-α (infacon- ) and the combination of a protease inhibitor with ribavirin . the rapidity with which the sars-cov genome was sequenced, the determination of the structure of potential drug targets and the prediction of functional properties of sars-cov proteins based on prior knowledge of homologs from other coronaviruses have allowed identification of potential new drug targets. peptides derived from the heptad-repeat- region of the spike protein have been shown to block virus infection, albeit at much higher molar concentrations than similar inhibitors needed to prevent hiv entry , . short interfering rnas also seems to be effective in decreasing viral replication in cell lines [ ] [ ] [ ] , but this remains an experimental strategy rather than one immediately amenable to clinical application. screening of combinatorial chemical libraries has identified inhibitors of sars protease, helicase and spike-protein-mediated cell entry . for successful treatment of influenza, antiviral drugs must be administered within hours of disease onset to obtain substantial clinical effect. but because the sars-cov load increases until day of illness , and in light of the correlation of high viral load in the second week of illness with adverse outcome , the window of opportunity for antiviral therapy may be wider. much scientific effort has been focused on developing a vaccine to protect against future outbreaks of sars-cov. the commercial viability of developing a vaccine for sars-cov will ultimately depend on whether the virus re-emerges in the near future. as discussed above, it is unlikely that future outbreaks will reach global proportions, but nevertheless, vaccines or passive immunization would be relevant in the context of protecting high-risk individuals such as laboratory and health-care workers. a vaccine could also be considered in the setting of the farmed-game-animal trade, if farming of civets for human consumption continues. in the short time since the virus was identified, substantial progress has been made toward developing a vaccine. immunodominant b-and t-cell epitopes of sars-cov are being defined [ ] [ ] [ ] . natural human infection with sars-cov leads to a long-lived neutralizing antibody response and immune sera crossneutralize diverse human sars-cov , suggesting that active immu- (in the press) ( ) nization against sars may be a feasible proposition. but so far there has been no known instance of human re-exposure to sars-cov to confirm that the naturally acquired immune response confers protection from reinfection. when sars-cov spike, envelope, membrane and nucleocapsid proteins were individually expressed in an attenuated parainfluenza type vector, only the recombinants expressing the spike protein induced neutralizing antibody and protected from challenge in hamsters ( table ) . mucosal immunization of african green monkeys with this parainfluenza-spike protein chimeric virus led to neutralizing antibody and protection from viral replication in the upper and lower respiratory tracts after challenge with live sars-cov , and spike protein-encoding dna vaccines stimulated neutralizing antibody production and protection from live-virus challenge in mice . these studies confirm the assumption that the spike protein is the dominant protective antigen for sars. experiments using adoptive transfer and t-cell depletion showed that humoral immunity alone can confer protection . other vaccine strategies have included the use of naked dna - , adenoviral vectors or modified vaccinia (ankara) and inactivated whole virus , . many investigators have optimized the codon usage of the gene target to improve expression. in summary, all vaccines based on the spike protein seem to induce neutralizing antibody responses, and those carrying nucleoprotein can induce nucleoprotein-specific cellmediated immunity. but thus far only four studies have used live sars-cov to challenge immunized animals ( table ). an inactivated vaccine with alum adjuvant, which induces neutralizing antibody in mice, is entering phase human clinical trials in china . experience with coronavirus vaccines for animals is relevant for sars vaccine development . one problem facing animal coronavirus vaccines has been strain variation among field isolates, leading to variable vaccine efficacy. a further concern is the experience with feline infectious peritonitis coronavirus, in which prior immunization led to enhanced disease rather than protection . in the case of sars-cov, neither vaccination nor passive transfer of antibody has yet been reported to lead to disease enhancement, but challenge with live sars-cov has occurred soon after immunization. whether waning immunity or low titers of antibody lead to sars disease enhancement remains unclear; the recent suggestion that immunized ferrets became more ill after challenge clearly needs to be confirmed or refuted . passive transfer of immune serum protects naive mice from sars-cov infection , and hyperimmune globulin with sufficient neutralizing activity for use in humans could be prepared from pooled convalescent human plasma or from horses immunized with inactivated vaccine. alternatively, monoclonal antibodies with sufficient neutralizing antibody activity have been developed by screening phage-display antibody libraries and by immortalizing b-cell repertoires of convalescent sars individuals with epstein-barr virus ( table ) [ ] [ ] [ ] . one of these ( r) blocks the virus-ace- receptor interaction through binding to the spike protein s domain . passive immunization of ferrets and mice was effective in suppressing viral replication in lungs, but less so in the nasopharynx , . no randomized placebo control trial evaluated antibody therapy for pre-or post-exposure prophylaxis in at-risk groups during the sars outbreak. retrospective analysis of outcome in a limited human study using human sars convalescent plasma suggested that passive immunization had no obvious adverse effects . the antigenic diversity of sars-cov-like precursor viruses in the wild-animal reservoir is undefined. in the event of a new interspecies transmission event prompting another sars outbreak, the crossprotection afforded by current vaccine constructs based on the human sars-cov of is unknown and is likely to influence the efficacy of both passive and active immunization strategies. sars provided a painful reminder of the global impact of emerging infectious diseases. it illustrated how microbes, with their evolutionary drive to preserve and propagate their genes, exploit new opportunities and niches created by modern society . interspecies transmission of viruses to humans clearly has occurred throughout human history. but recent developments allowed sars-cov increased opportunity to adapt to human-to-human transmission and, subsequently, to spread globally. in particular, large centralized wet markets and hospitals proved to be venues for amplification of transmission to humans, and the burgeoning increase of international travel (currently ∼ million travelers annually) exploded the local outbreak of an emerging infection into a potential pandemic. because most recent emerging infectious disease threats have a zoonotic origin, we need to better understand the microbial ecology of livestock and wildlife. in the context of increased attention and research funding directed at preparedness to combat bioterrorism threats, it is relevant to note that nature remains the greatest 'bioterrorist.' although microbes that cause commercially important diseases in livestock are well studied, organisms that pose threats to human health are not necessarily ones known to cause disease in livestock, or for that matter, in wildlife. nipah virus, hendra virus and sars-cov all have a wildlife reservoir. furthermore, at present there is concern over the possible role played by wild birds and ducks in the maintenance and spread of avian influenza a (h n ) in asia . greater understanding of the viral ecology of apparently healthy domestic animals and wildlife is therefore important. for example, the attention on ecological studies arising from the nipah virus and sars outbreaks have already led to the identification of a number of new viruses, including tioman, menangle, australian bat lyssavirus and a novel group coronavirus , . some of these are now known to be associated with human or livestock disease. but prioritizing such research efforts and assessing the public health relevance-if any-of such findings, poses challenges. three incidents of laboratory-acquired sars have arisen from biohazard level and laboratories, with community transmission arising from one (world health organization, http://www.wpro.who.int/ sars/docs/update/update_ .asp). these incidents were associated with lapses in biohazard level and practices. sars-cov can be safely handled in biohazard level laboratories provided that biohazard level practices are rigorously complied with. but as hospital health-care workers learned to their cost, sars-cov is an unforgiving virus; one lapse may be one too many, and it is irrelevant whether the lapse occurs in a biohazard level or laboratory. despite the impressive speed of scientific understanding of the disease, the global success in containing sars owed much to traditional public health methods of clinical case identification, contact investigation, infection control at healthcare facilities, patient isolation and community containment (that is, quarantine) . but the application of such measures in modern society during the control of sars highlighted several ethical and medical dilemmas, many of which arose from the need to balance individual freedoms against the common good , . sars signaled a paradigm shift in international public health. it highlighted the need for rapid information exchange regarding unusual infectious disease outbreaks and the possibility of , and the need for , a coordinated global response to emerging infectious disease threats. during the early stages of the outbreak, the who acted independently, issuing travel alerts 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investigate the cause of severe acute respiratory syndrome germs, governance, and global public health in the wake of sars the authors declare that they have no competing financial interests. key: cord- -lvw h w authors: atzema, clare; poirier, vincent title: career options in aerospace and aviation medicine() date: - - journal: ann emerg med doi: . /j.annemergmed. . . sha: doc_id: cord_uid: lvw h w nan underlying all these issues is a thorough understanding of human physiology, basic physics, toxicology, infectious disease, and engineering principles. , in general, physicians trained in aerospace medicine practice health care in populations exposed to flight and space, consult on the physical and engineering aspects of the flight environment, and manage public safety issues at a variety of regulatory agencies. they may choose to specialize in civil aviation, military aviation, or space flight. they might work for the national aeronautics and space administration, the federal aviation administration, a commercial or corporate airline, the department of transportation, an aerospace manufacturer, the occupational safety and health administration, the centers for disease control and prevention, or in private practice. practitioners may choose to focus on medical research, they may teach, or they may consult on a variety of preventive, workplace safety, and environmental programs , or on aerospace health care system development. aerospace medicine and emergency medicine are complementary specialties, for several reasons. each requires the evaluation and stabilization of patients in a less than perfectly controlled environment. training for mass casualty incidents serves as preparation for airline crashes, and disaster medicine training, along with out-of-hospital care and toxicology, contributes to the preparation for manned space flight. the variety of clinical problems encountered in aerospace medicine overlaps with emergency medicine, ranging from treatment of multiple trauma patients after an airline accident to running a medical clinic for aircrew, which may mirror some of what is seen in the minor area of an emergency department. in addition, the problem-solving and teamwork skills that are inherent to emergency medicine are also basic tenets of astronautics. the daily practice of a physician in aerospace medicine depends on his or her area of expertise. a medical consultant for a major airline, for example, will oversee the health of the pilots, flight attendants, and ground staff. he or she is likely to run daily clinics to manage the medical problems of the airline crew, such as barotrauma, occupational injuries (back pain is a common complaint), and return to work assignment. the consultant is usually a trained aviation medical examiner (ame) and, as such, gives out the pilot medical certificates from the federal aviation administration, the canadian civil aviation, or the united kingdom civil perhaps the plane will be unable to land because of weather conditions. there is little routine, and by flying full-time, there is a risk of becoming physically exhausted. , in addition, teamwork is ubiquitous in aerospace medicine: one is likely to take part in several committees, and if one is a flight physician, one works in a confined environment with a medical team (ie, a respiratory therapist, a nurse, paramedics), often for many hours at a time. if you like to work alone, this may be a drawback. there are many topical issues in aerospace medicine, which you are likely to find yourself debating if you choose this career. traveler's thrombosis is being addressed by the wright project (world health organization research into global hazards of travel), which is a collaboration of an international research project and the world health organization. severe acute respiratory syndrome and aeromedical transport will likely remain a serious issue for anyone in this field, and the effects of cosmic radiation are a continuing concern. other issues include the use of selective serotonin reuptake inhibitors by pilots, refractive eye surgery in pilots, long-haul flight and aircrew fatigue, and long stay in the international space station. if you have a predisposition toward research, there are a plethora of opportunities in this field. the routes to becoming a specialist in aerospace medicine are through either civilian training (table ) or the military (table ) . after graduation as a medical doctor at an accredited medical school, one may: ( ) enter an internship of choice, and, after completion of the internship, enter a residency in aerospace medicine lasting an additional years; or ( ) enter the united states military to complete a military residency for to years. however, there are many other options available (tables and ) . the final pertinent issue to consider is that military programs require their trainees to fly, and in civilian programs, although it is not a requirement, flight training is encouraged through several affiliated flight schools. thus, the strong desire to fly is an important prerequisite. aviation authority. in addition, he or she will ensure that protocols are followed, including medical clearance of passengers, medical kit onboard the aircrafts, emergency procedures, and air quality control, to name a few. another example is that of a flight physician, who usually works for an aeromedical transport company. this individual is in charge of flight planning, in conjunction with the medical team. in particular, the flight physician will determine whether the patient is fit to fly, assess the patient's oxygen requirements and the aircraft's cabin pressure, and determine if any special equipment is required. the physician escorts patients onboard different aircraft types (ie, small fixed wing, commercial, helicopter) and will work to minimize the risk of barotrauma and problems resulting from the hypoxic inflight environment. often, this physician is attached to a university center and will be in charge of medical education for residents and medical staff. a third example is that of a director for a specialized aviation authority, such as the civil aerospace medical institute or the international civil aviation organization. the director will lead a professional, technical, and clerical team that is engaged in policy development, evaluation, and administration. this individual will develop aeromedical education programs for flight crews and civil aviation pilots. he or she will supervise the creation of the didactic materials used in these programs, as well as general aeromedical publications such as aviation safety brochures and research technical reports. these jobs have some distinct advantages. if one works for an airline or an aviation authority as a medical consultant, a typical workday extends from am until pm. despite additional on-call duties, one is likely to be called in only a few times a year (for emergencies such as crash investigations, emergency landings, etc). a flight physician who retrieves injured or ill passengers is afforded the opportunity to discover the world, particularly because some travelers have the desire to visit some fairly unusual places. some aerospace medicine physicians will maintain a part-time position in an emergency department or another ambulatory setting, providing both variety and the opportunity to maintain clinical skills. a career in aerospace medicine has disadvantages as well. the daily medical practice of an airline consultant is usually limited to the health care issues of a healthy population. flight physicians are on call many times a month and may be required to leave within to minutes of a telephone call. although these physicians often know when they are going to leave, they may not know when they will return: the patient may be more unstable than predicted (potentially requiring an emergency landing) or table . fundamentals of aerospace medicine aerospace medicine residency at university of texas medical branch-galveston and nasa/jsc emergency medical preparedness and response to a singapore airliner crash space shuttle operations at the nasa kennedy space center: the role of emergency medicine aviation medicine space doctor similarities of medicine and astronautics the preventative medicine physician: a national study a review of human physiological and performance changes associated with desynchronosis of biological rhythms fatigue in operational settings: examples from the aviation environment key: cord- -elzkwyz authors: day, brennan; burnice mckay, ruth; ishman, michael; chung, ed title: the new normal: lessons learned from sars for corporations operating in emerging markets date: - - journal: nan doi: . / sha: doc_id: cord_uid: elzkwyz the modern industrialized world was completely caught off guard by the recent sars outbreak. fortunately, for most organizations, the impact has been short lived, but management has been provided with a reminder of the impact of the external environment in a world of ever increasing globalization. as seen with the sars outbreak, a lack of preparedness can have devastating effects on business and warrant inclusion in a business definition of a crisis. this paper uses the recent sars epidemic as a background to highlight the importance of crisis planning, particularly in emerging economies, and suggests how organizations can address these concerns. the implementation of environmental scanning in strategic planning is critical to the survival of an organization. that businesses operate in the context of some larger system, not a vacuum, is today part of the accepted business perspective. however, we ought to recognize that scanning and the related processes of contingency planning presume our ability to discern future patterns. many well-known business mistakes, such as the big three's disastrous handling of the energy crisis in the s, can be attributed as much to inadequate managerial skills as ignorance about environmental trends. after all, it was not any existing predators that eradicated the dinosaurs, but instead it was something never before seen and hence could not have been anticipated. the fact that the meteor was not in the dinosaur's vernacular did not prevent a meteor from changing the course of earth's history. the scope of our environmental scanning activities expands as our knowledge increases. such scanning is informed by new developments that have often been prospering just below the corporate radar screen. the recent (and arguably not over) sever acute respiratory syndrome (sars) outbreak is a case in point. even though contagious diseases are well-known, and their fatal impact acknowledged, e.g. pneumonic plague, spanish-flu and bubonic plague, the world was caught completely off-guard by the sars outbreak. despite superhuman efforts by the many health-care workers around the world, it was months (and many deaths) before the world health organization pronounced the epidemic temporarily halted. the health-care establishment could not have predicted the outbreak. as devastating as it was for many businesses, strategic planners would have to be psychic to have anticipated the disease and to incorporate it in the planning process. however, it would be useful to delineate lessons that can be learned from the recent sars (and other) outbreaks, and discuss how strategic management may benefit from an appreciation of these lessons. in preparation for unforeseen external events organizations should have a plan in place to reduce the influence on the organization and provide an operation manual in the event of a crisis. however, many fortune , firms do not have a crisis-management plan at all (hickman and crandall, ) . as seen with the sars outbreak this lack of preparedness can have devastating effects on business and warrant inclusion in our business definition of a crisis. a crisis is defined as a low probability/high consequence event, caused by human actions, that threatens the most fundamental goals of an organization (weick, ) . mitroff and pearson ( ) categorized crises according to two dimensions: normal to sever and technical/economic to human/social. however, their crisis analysis included only industrial health problems, not epidemics. there is a need to broaden the scope of crisis to include epidemics. our paper aims to provide this addition. this paper will start by presenting background information on the sars epidemic and the impact on organizations, especially those operating in emerging markets. next, characteristics of countries termed emerging markets will be identified and the inherent risks and challenges organizations face operating in these markets will be discussed. third, we offer recommendations for minimizing the impact of operating in an emerging market where contagious diseases are a reality. this represents an important first step towards developing (and implementing) a robust contingency plan for purposes of business continuity. on the basis of purchasing power parity, emerging and developing economies account for percent of the world's economy (gingrich, ) . conservative predictions conclude that by the year the developing world will account for over percent of the total world output (belous et al., ) . given the substantial growth in these economies, it is not surprising that the us department of commerce's international trade administration recently predicted that the greatest commercial opportunities will be found in the big emerging markets such as the people's republic of china (china), india, singapore, mexico, vietnam, or south africa (qureshi, ) . china provides perhaps the clearest illustration. for example, in china received $ billion in direct foreign investment, overtaking the united states to make it the world's biggest recipient of direct foreign investment (buckman, ) . according to the us bureau of economic analysis in , china and hong kong accounted for nearly a quarter of the income of us overseas subsidiaries (karmin, ) . however, the recent sars outbreak has shown how quickly commerce can be impacted in the global market. according to richard miller, an economist and vice president of the world travel & tourism council: "never before has there been a massive impact of this nature other than a war shutting down an economy totally" (prystay, ) . as of june , , outbreaks of sars have occurred in countries, resulting in , individuals being infected and subsequent deaths (world health organization, ) . economists are predicting that china, as a result of sars may see gdp growth drop below percent to its lowest level in nearly a decade (leggett, ) . some areas have been hit more harshly than others. in the chinese province of guangdong, the place where sars is believed to have started, the country's largest export-trade fair saw orders drop in april nearly percent (leggett, ) . in beijing, draconian measures were imposed to try to stem the spread of sars. public schools and universities were closed, migrant workers were told to stay in the capital and make-shift roadblocks were set up in villages to prevent city dwellers from entering (kaufman and chen, ) . in may, hong kong retail sales were down to percent, restaurants and their suppliers were going broke and hotel occupancy rates were in the single digits. airlines were also severely impacted. cathay pacific airway during this time dealt with an percent decrease in air traffic from a year earlier (meredith, ) . such a decrease in travel is not surprising given the overall drop in business air travel globally. in a survey conducted by the society for human resource management in the usa, based on human resource professionals, percent reported cuts in employee travel as a result of sars (usa today, ) . kingsway international holdings limited, a hong kong investment holding company that engages in securities brokerage, corporate finance advisory services, fund and asset management, securities investments, and strategic investment in technology-related businesses has seen earning per share drop from $ . in june to $ . in june . the company attributes the results to the difficult operating conditions including the influence of sars (infomart, ) . march sales of desktop computers in hong kong plunged percent, laptop sales dropped percent and mobile-phone sales fell percent (leggett, ) . cell phone sales dropped percent in guangzhou, the shanghai auto show only drew one quarter of its expected visitors and motorola shut its -story asia pacific headquarters building in beijing (engardio et al., ) . businesses located in developed nations have also been impacted. in australia, some small-to-medium sized businesses could face losses of up to $ million due to sars. westpac bank has predicted that sars could reduce australia's economic growth by . percent (chong, ) . multinational corporations are also being impacted. for example m, the makers of the n face mask, one of the few face masks that are percent effective at filtering out airborne diseases like sars, does percent of its $ billion in annual sales in the asia pacific region. j.p. morgan lists m as well as intel and united technologies as american companies that could be hurt by sars (freedman, ) . other companies suggesting revenues will be down as a result of sars include american international group inc., yum brand inc., goodrich corp. (karmin, ) and cara operations ltd. (flavelle, ) . another industry feeling the impact of sars is the phone makers. motorola, a company holding percent of the chinese mobile phone market, has warned that it may fall short of sales and profit forecasts for the second quarter and year end citing weakening demand in asia because of sars (timmons, ) . four season's hotel inc. has a net loss in the first quarter of cents per share compared to net income of cents per share a year earlier (infomart, ) . sars is seen as a key contributor to this loss. the city of toronto also provides an indication of the economic impact of sars claiming the cost to the government for health care and income compensation for those quarantined for the first six months of totaled us$ million (chowdhury, ) . while the above figures are indicators of the effect of sars, the true impact may never be known. it is believed that china, in order to maintain a semblance of stability and economic prosperity at the time of changing government leaders, covered up the md , truth about the extent of sars. it went so far as to remove sars patients from hospitals to deceive inspectors from the world health organization (jue, ) . in april, jiang yanyong blew the whistle on chinese authorities' deceit regarding the truth about sars in beijing. in a letter to journalists, when beijing was saying that only people in the city were infected with sars, dr jiang disclosed that the true number of sars patients was at least , including ten who had died. in response to dr jiang's revelation the health minister and beijing mayor were fired. for a regime with a long history of deception and denial, this event has been viewed as a revolution in official openness (york, ) . in a step to reinforce openness the chinese government has threatened to execute anyone who knowingly spreads the sars virus (dean, ) . however, this may have the reverse impact of making people hesitant to admit they have sars for fear of punishment. by lying about the true number of sars patients, beijing damaged what little faith international economist has in its numbers. for example, repeated yearly claims of percent annual gdp may be questionable. for the new chinese leadership the incentive to cheat is even greater than usual. strong gdp growth has been the key to the communist party's hold on power. the new government would not want to be associated with instant economic failure (august, ) . in addition to the sars outbreak there is news that another deadly virus has emerged in southern china through the winter. the associated press ( ) reported an outbreak of encephalitis in southern china that has (at time of writing) affected several hundred people and resulted in more than a dozen deaths. at the same time as this new disease is spreading in china and hong kong, the government in hong kong is pushing ahead with tough new security laws which journalists fear could inhibit future reporting of crisis. under the new legislation reporters could well be jailed for publishing details of state secrets and confidential communication between hong kong and beijing. thomas crampton, president of the foreign correspondents club of hong kong stated that in a country where health statistics are a state secret the legislation "puts a pall of uncertainty over reporting in hong kong. it encourages self-censorship" (korporaal, ) . for businesses operating in this and related regions, another outbreak and the possibility of another chinese cover up could not be good news. while it is hard to beat the wage rates of to cents an hour in china, it is a constant corporate challenge to operate in an environment where propaganda and obfuscation are used as government marketing tools. given the rapid onset of sars, the presence of other contagious diseases in the world and the government and infrastructure challenges for corporations operating in emerging markets, it is essential that corporations are prepared for future contagious disease outbreaks. it is not clear if sars can be eradicated. the disease could gain a toehold in the southern hemisphere, or it could lie low until cold weather returns (regalado, ) . while sars is taking the center stage, areas in europe are dealing with another disturbing virus that is spreading among chickens and in some cases humans. the avian flu has been found in the netherlands, belgium and germany. the virus, which rarely causes serious illness in humans, infected people who came in contact with infected birds (fuhrmans, ) . it is also conceivable that corporations may be operating in regions of the world where they will face bio-terrorism. as an indication of the possibility of such sabotage the us department of justice and the federal emergency management agency staged a mock terrorist attack on chicago airports in may . this is the second such exercise, the first being staged at a denver concert hall in may . in this year's exercise, terrorists sprayed unseen germs over chicago airports, infecting thousands with a lethal pneumonia. the exercise was undertaken to determined level of preparedness in the event of bio-terrorism (chase, ) . according to the australian state chamber of commerce chief executive officer, margy osmond, "(t)he lack of preparation by many businesses in terms of handling workplace issues and risks arising from the (sars) virus is . . . a concern". she also notes that percent of corporate respondents had a risk management plan to deal with potential disruptions created by sars but only percent had made this plan known to their staff. benefits of a fast response can be seen in the fast reaction of the canadian health authorities dealing with sars preventing the outbreak from reaching unmanageable numbers. according to castillo-chavez, a mathematical epidemiologist at cornell university, if the canadian outbreak had been left to run unchecked it could easily have infected as many as , in the toronto area (brown, ) . the toronto sars experience is a warning that even a giant thriving city can be laid low by a nasty and highly contagious disease (vogel, ) . since emerging markets are increasingly important to the world economy and are at the same time susceptible to outbreaks of infectious diseases, we need to understand how we are linked together on an interdependent global level. that interdependency is highlighted in the following section. the days of national economies existing as relatively self-contained entities are rapidly disappearing. individual economies are no longer isolated from each other by barriers to cross-border trade and investment -by distance, time zones, and language -and by national differences in government regulation, culture, and business systems. national economies are merging into an interdependent global economic system and, consequently, the rich industrial countries are dominating the world economy relatively less than they used to. at the beginning of its fiscal year (ending on june ), countries had been identified by the world bank as developing economies (international bank for reconstruction and development /the world bank, , p. ). the developed world, however, currently represents more than percent of total world output, while the developing nations represent less than percent. this proportion is poised to change in the near future though, with conservative projections indicating that by the situation could be reversed, with the developing world representing well over percent of total world output (belous et al., , p. ). the developing countries that have been the frontrunners in economic advancement have been designated as emerging economies by the economist. this classification is based on both the gross domestic product of each nation and the capitalization of their stock markets. these countries have historically played an unparalleled role in the global economy -a role that is well positioned to steadily expand in international importance. the emerging economies are home to approximately percent of the world's population and the population growth rates of these economies are the highest of all countries. the five largest developing nations, home to . billion people, have a combined population that is four times that of the g- countries. the populations of china and india alone, . billion and more than billion respectively, easily outnumber those of many developed countries combined (cavusgil et al., , pp. , ) . many of the world's developed countries now have birth rates below those that are required to maintain their present population levels. furthermore, individuals in the developed world are living progressively longer and leaving the work force earlier. with fewer workers generating output, and with more of the population dependent on those who do, income growth, savings levels, and economic growth will all be lower. in contrast, the populations of emerging countries are comparatively young. the working-age population in these markets -which will be the portion of the population driving economic and consumption growth -will grow at rates three to four times that of the developed world. the emerging markets also offer a large pool of low-cost, unskilled labor and it has become increasingly commonplace for large, multinational corporations to transfer their assembly and production plants from developed locations to these areas (gingrich, ) . the size of the consumer markets in the emerging countries now rivals those of the developed markets in many cases. for instance, the combined gross domestic product of the ten largest emerging markets is now nearly two-thirds the size of that of the g- counties in purchasing power parity terms. five emerging economies -china, india, brazil, mexico, and indonesia -are among the largest economies in the world and have a combined purchasing power already half of that of the g- . according to the international monetary fund (imf), developing countries will achieve a growth rate of more than percent per year over the next two decades, whereas industrialized countries are likely to average . percent (cavusgil et al., , p. ) . if just three of the asian emerging economies -china, india, and indonesia -are able to maintain this growth rate of percent per year, the organisation for economic co-operation and development (oecd) has estimated that by approximately million people in those countries will have an average income equivalent to that of spain today. as a group, they are roughly equivalent in population to the usa, japan, and europe combined (us department of commerce, , p. ). given these trends, multinational corporations (mncs) face profound changes in the economic landscape. over the next to years, most of the total world growth in consumption of consumer goods will likely be concentrated within the emerging economies. the emerging markets have also become major suppliers of many of the natural resources that the industrialized world relies on. for example, mexico, venezuela, and indonesia, in addition to several middle eastern countries, are major sources of oilstill the most vital energy source for developed countries. similarly, brazil, russia, and china, along with developing countries papua new guinea and jamaica, supply half of the world's bauxite (aluminum ore) (day, , p. ) . looking to the future, the supplier role of the emerging countries will expand since the exploitation of natural resources in many of the wealthy countries has reached its limit. either the oil fields, mines, or forests have been tapped out, or environmental regulations reduce new exploration and development. industrialized nations are therefore becoming increasingly dependent on emerging markets to ensure that sufficient levels of natural resources are available. overall, development in the emerging markets has been steady, but none of these countries have been fortunate enough to avoid certain growing pains along the way. one historical trend of many emerging markets is the inability of their currencies to hold value. rather than attack the principal causes of currency depreciation, many governments opt instead for a short term, cosmetic solution. after several years of declining currency value, they might print a new currency that "lops off two zeros" from the denomination of the older currency (hooke, , p. ) . in , for example, mexico initiated the new peso that was worth , old pesos. these superficial actions diminish investor confidence. additionally, widespread corruption exists in virtually all emerging markets, with hong kong and singapore being the most notable exceptions. many politicians view a government career as similar to a private sector job, with one primary goal in mind: making money. with many top officials setting a poor example, low-level bureaucrats are active in demanding payments for business licenses, permits, and concessions. since public sector jobs, as compared to a private sector position, have low salaries and no stock options, the difference is often made up through bribes and insider deals. public employees and elected officials accept these prerequisites in exchange for government contracts, licenses, and privileges (hooke, , p. ) . environmental issues in the emerging world often run unchecked. due to their size, their growing industrialization, and the urgency they attach to business expansion and job creation, many emerging markets have barely begun to put in place effective environmental programs. they often lack, or claim to lack, the financing capability to address the environment at this stage of their development, especially in light of competing priorities such as infrastructure. despite these ever-present risks and challenges that exist in the emerging world, the staggering economic impact of many of these countries coupled with their substantial population growth has created a growth potential too significant to be ignored by the developed world. accordingly, many private sector and government analysts continue to point to emerging markets as a potential major source of western economic growth and profit in the coming years (day, , p. ) . over the past three decades emerging markets have consistently shown growth rates well above those of more mature economies and this trend is expected to continue. the economies that make up the emerging world will almost undoubtedly have an even greater impact on the overall global economy throughout the foreseeable future. emerging markets can present tremendous opportunities as well as unforeseen challenges for global organizations. the recent sars epidemic highlights how a disease centered in countries identified as emerging can create formidable obstacles for managers. in order to examine the potential long-term effect of sars on emerging economies, an examination of infectious disease is required. the next section provides a brief historical look at the impact of epidemics on communities and organizations. despite the advents of modern medical science, infectious diseases such as malaria continue to wreak havoc. "if seven boeing s, filled mostly with children, were crashing into mount kilimanjaro each day, something might be done about it. that, in the memorable phrase of wen kilama, a tanzanian researcher, is roughly the scale of the carnage wrought by one disease, malaria, mainly in africa" (the economist, ) . more recently, the spread of sars captured center stage, due as much to the disease's proclivity as to media's desire for the next major headline. augmented or not, that sars has been (and may continues to be) destructive is unequivocal. indeed, because md , most of the inflicted jurisdictions are important developing economies, sars poses serious implications for businesses around the world. as surowiecki ( ) noted in the new yorker magazine, "before sars, china was going to be the factory floor to the world. now it has to worry about becoming the sick ward of the east". it is important to note, however, that menacing as sars is, it is but one epidemic in a long series of infectious outbreaks that have plagued human history. and in spite of its highly vilified image, sars has caused relatively minor damage compared to past epidemics. as one commentator puts it, "sars has not yet reached the scope or virulence to be described as a pancosmic. so far it is a fleabite compared to previous plagues. you are more likely to be struck by lightning than sars" (howard, ) . a history of plagues is beyond the scope of this paper. however, it is important to offer some background on infectious diseases and their impact on humanity over the centuries. we also wish to point out that, quite aside from sars, infectious diseases (some with much more deadly and rapid effects) continue to threaten the world. while most have heard of the black death and yellow fever, perhaps the most famous plague of all happened centuries ago in athens ( - bc). though it is impossible to know with certitude what the disease actually was, it was of such devastation that even vultures refused to touch the corpses of the dead (howard, ) . fast forward to the s, black death swept across europe. as "recently" as the seventeenth century, the great plague of london killed , people, followed by the plague that killed up to , people in china and hong kong. indeed, within the next years, plagues had killed more than million people worldwide. lest we thought the plague is a thing of the past, outbreaks of bubonic and pneumonic plagues in killed more than in india (king, ) . recent reports by the washington post suggest that rotaviral infections, a virus that causes acute gastroenteritis, are fast becoming an emerging problem in the former soviet union. north america has never been exempt from the devastations of infectious diseases. the advance of european cultures over first people in north america, such as the mohawks and algonquin nations, was due to the importing of diseases such as tuberculosis (tb) that devastated the established cultures killing millions of people. yellow fever ravaged philadelphia in , killing thousands in the then capital of the usa (dalrymple, ) . in and , spanish influenza killed more than half a million people in the usa alone (king, ) , and many insurance companies saw their profits wiped out and had to reduce dividend payments because of the claims made from the illness (bell, ) . today, tb is perhaps one of the most lethal infectious diseases. even though was the tenth consecutive year of declining tb cases in the usa, each year some two million people die from tb and an estimated one-third of the world's population is infected with the bacteria (center for disease control, ) . other diseases such as malaria and ebola continue to claim the lives of thousands, and since they occur predominantly in under-developed economies with poor infrastructure and health services, their effects are staggering. a disease like ebola, for example, can have mortality rates as high as percent (washington post, ) . it is informative to note the difference between epidemics and endemic diseases, though with the ease of modern travel and human contacts, the lines are somewhat blurred. endemic diseases are those that are localized in certain regions. unlike epidemics which really have no "home base," so to speak, endemic diseases are associated with particular geographic locations (surowiecki, ) . usually because of inadequate infrastructure, these jurisdictions are ill-equipped to stem the disease. the disease not only negates current economic growth but also acts as a barrier to future economic growth since investment flows tend to avoid these regions where companies remain concerned about the health of their employees. as business becomes increasingly globalized, companies large and small will encounter infectious diseases of both the epidemic and endemic varieties. in the latter case, it may well be in business's interest to work with local and global health officials to improve local infrastructures, such as hospitals and health services, not only as a means to generate goodwill and promote good corporate citizenship, but also as a way to help create a more hospitable operational environment. for example, the state-run china national offshore oil corporation donated million renminbi (just over us$ million) to aid research on sars. one of the most widespread and publicized epidemics today is hiv/aids. while the disease may be well publicized, its connection with the business community is not often elaborated upon. in fact, otherwise innocuous actions on the part of businesses have a hand in spreading the disease. for instance, truckers working for long-distance transportation companies have been identified as a major transmitter of the disease throughout africa and asia. hiv/aids also has devastating effects on businesses themselves. barclay's bank of zambia, for example, reported losing more than a quarter of its senior managers to the disease. a mattress company in zimbabwe hires and trains three people for every position due to attrition by the disease (forstater et al., ) . aids has become south africa's biggest single killer. in a south africa's medical research council calculated that percent of the deaths of those aged between and in the previous year were due to the aids pandemic. in recognition of the impact hiv/aids is having on its work force, daimlerchrysler south africa has, since , provided funding for standardized treatment for hiv-related diseases and anti-retroviral medication for its employees (daimlerchrysler, ) . according to the african vice president of the world bank, callisto madavo, hiv is the single greatest threat to future economic development in africa. of equal concern though is the growth rate of aids in asia. aids is growing fastest in asia leading many health experts to believe that it may overtake africa in the number of people infected with hiv. this will create a vexing problem for investors in europe and north america as their economies peak and they increasingly turn to the third world for profits. inexpensive labor is appealing but what if you cannot find employees to complete an order (jeter, ) ? the above discussion seeks not to provide a chronological listing of infectious diseases, but to highlight the dangers the world continues to face. businesses, indeed the world community, cannot afford to be complacent in the efficacy of modern medicine and assume that sars is an anomaly. infectious diseases are a fact of life and will continue to impact human society in the future. it is imperative that businesses develop coherent strategies to deal with such diseases when necessary. there should be no excuse for being "caught off-guard". the following section will look at specific impacts of the sars epidemic on commerce and present suggestions for organizations to protect themselves against similar future spread epidemics. in this section a distinction is made between the impact on large and small organizations. suggestions for contingency planning in face of epidemics so far we have attempted to provide a comprehensive discussion of how infectious diseases can gravely impact a firm's ability to function smoothly, unless adequate contingency planning is incorporated into its strategic management function and crisis management plan. in this section, we attempt to provide suggestions to minimize the impact of operating in an emerging market where contagious diseases are a reality. there are several areas that a contingency plan should address, in order to deal with the kind of epidemics that we have recently witnessed. further work will be needed to more fully develop the precise parameters of these dimensions, but for our purposes, we offer below some of the key areas that we feel a contingency plan should seek to address. businesses will need to re-examine the level of inventory, so as to provide some level of buffer in case of emergency. certainly we recognize that this adds to the cost of doing business, but we suggest that operationally this merely represents an additional variable in modeling the optimal level of inventory necessary. furthermore, businesses will need to assess whether it is practicable to secure their supplies from more than one source. diversification, as we know, spreads the risk. as much as possible, we need to incorporate technology into our operations, and re-evaluate location attractiveness with an eye towards the ease of technology replication as well as emergency backups. when opportunities arise move employees around global locations. these moves can be for short stints. the objective is to share understanding of competitive advantage. this may help upper management in reestablishing their competitive advantage in an alternative location. it is also important that values central to the business be ingrained in the workers to the extent possible. in this instance, continual efforts of re-education may be a useful step to take. we posit that companies would be concerned about how its workers are affected by the epidemic in terms of health, safety, and even emotion. as major epidemics such as sars could not be foreseen, it is imperative that managers take an emergent approach to plowing through the problem. companies in the many emerging markets where government regulations and transparency are lacking may particularly need to do more than simply adopt the official government line regarding the effects of the epidemic. to the extent possible, businesses should consider hiring local management talents as well as rotating key personnel to multiple locations of operations so as to build a solid base within the company from which local knowledge and expertise can be tapped. it is critical also that this knowledge is not only the exclusive domain of senior management, but that open communication be instituted throughout the company to enable workers at all level to know what is going on and what to expect. one of the unfortunate results of secrecy is that it often leads to unfounded rumors that only serve to aggravate a dangerous situation. corporations need to revisit their health plan to consider the need to establish health policies for suppliers. the "we didn't know" defense does not work too well any more in today's world of instant communication. businesses need to not only have in place their own health policies related to epidemics, but should determine whether their suppliers have adopted effective health policies to deal with such crises. regardless of all that we know, we acknowledge the impossibility of predicting future events. consequently, companies should not waste time and resources attempting to plan for patterns that are simply unpredictable. rather, it is critical that companies pay appropriate attention to contingency planning (such as crisis management plans); the basis of which are already well understood. what we have done is to tease out the important lessons from the sars epidemic. we have looked at its impact on organizations operating in emerging markets and drawn suggestions from this event. crisis management plans must be robust enough to handle all forms of the unexpected. as events arise that give us insight into the unforeseen, it is essential that organizations reexamine their crisis management plans to see if they were designed effectively enough to handle the unique features of our evolving environment. for those organizations that have operated without a crisis management plan, sars is a reminder of the importance of designing such a plan. the sars epidemic also shows how a crisis management plan must be integrally connected with an organization's strategic planning, thus being emergent rather than static in structure. it would be informative to take stock of what has transpired since the outbreak of the sars epidemic. a year after the disease first broke, it would be of intellectual (and practical) interest to assess what, if anything, the business community has put in place to combat such medical challenges. furthermore, inasmuch as we have offered some useful suggestions in this paper to deal with similar crises, we believe the exercise would be well served by couching our recommendations in a framework based in theory. such a future study should reveal whether the business community has adopted any important lessons that might be learned from the sars outbreak, and render our proposed contingency planning recommendations more practical in light of actual corporate actions. encephalitis outbreak in mainland china statistics are first casualty flu pandemic hit insurers hard emerging markets and international development: options for us foreign policy china isn't the only alluring partner for investors: despite worries, southeast asia still draws firms doing business in emerging markets: entry and negotiation strategies exercise in terror municipal affairs minister david young says he hopes ottawa will 'rethink' its compensation offer for the province's sars outbreak how congress coped in the age of yellow fever emerging markets of the global marketplace the sars outbreak: cases flare in taiwan; china gets draconian sales take a holiday airline woes: sars take toll on cara masked peril europe struggles to eradicate avian flu outbreak five rules for winning emerging market consumers before disaster hits: a multifaceted approach to crisis management emerging markets: a practical guide for corporations, lenders, and investors infomart ( ), available at: www.infomart.ca/doss/doss_db_display.php?key ¼ corp/cfps international bank for reconstruction and development/the world bank aids plants crop of death in africa many us firms fear sars will hurt profits this quarter beijing imposes new sars curbs: city's theaters and cafés are shut down as china lists another cases epidemic disease since the black death hong kong worries about bill to curb sars reporting china's growth may fall below percent: sars causes drop in sales of electronic products, closing of stock exchanges crisis management: a diagnostic guide for improving your organization's crisis preparedness sars squeezes asia's travel sector: industry study says virus may cost economies billions of dollars, millions of jobs emerging markets and international development: options for us foreign policy infection rates might drop as spring temperatures rise the high cost of illness nokia warns of lower sales, blaming economy and sars the big emerging markets: outlook and sourcebook local health official continue to plan for crisis cumulative number of reported probable cases of sars china cool toward md who told sars truth: whistleblower treated both as a hero and political threat, writes geoffrey york in beijing key: cord- -rh e n w authors: lippens, ronnie title: viral contagion and anti-terrorism: notes on medical emergency, legality and diplomacy date: journal: nan doi: . /b:sela. . . sha: doc_id: cord_uid: rh e n w the dominant imagery in current international relations seems to betray the emergence of an imperialist imaginary that differs markedly from an earlier one. this paper traces the main outlines of this emerging imaginary that has left notions of empire as spheres of integrative production firmly behind, and is now geared towards imagining empire as a complete, organic body of free-but-organic-and-therefore-orderly flows that however needs to be kept intact by means of epidemiological interventions aimed at excluding or neutralizing viral entities. dealing with terrorism, or invading states that allegedly breed them, in this imaginary, is first and foremost a matter of medical necessity and urgency. the legal and diplomatic 'logic' of un resolutions (resolution for example), in this imaginary space, can only be imagined as being of secondary importance. cooperation and `cosmopolitan' negotiation, as alternatives, disappear in this imaginary that projects an imperialist globalism of epidemiological purity. shortly after the end of the war in (or on) iraq, the british prime minister was invited to address the us congress, at which occasion he would also be given the congressional gold medal. a few seconds into his address, delivered on july , , blair stresses how, in the past five decades or so, nations have grown ever closer because of 'technology, communication, trade and travel'. he continues with a few lines that are worth quoting here in full: we are bound together as never before. this coming together provides us with unprecedented opportunity but also makes us uniquely vulnerable. the threat comes, because, in another part of the globe, there is shadow and darkness where not all the world is free, where many millions suffer under brutal dictatorship; where a third of our planet lives in poverty beyond anything even the poorest in our societies can imagine; and where a fanatical strain of religious extremism has arisen, that is a mutation of the true and peaceful faith of islam and because in the combination of these afflictions, a new and deadly virus has emerged. the virus is terrorism, whose intent to inflict destruction is unconstrained by human feeling (. . .) . in this essay we hope to be able to demonstrate how the imagery in this quotation is indicative of a newly emerging imaginary of empire. empire here appears as the body of freedom-loving nations 'bound together', whose health, always 'vulnerable', is threatened by 'afflictions' and 'strains' of 'viruses' that are 'unconstrained', and, because of their being devoid of any 'human feeling', and because of their status as 'unconstrained' 'viruses', firmly belong outside this benign empire, i.e., outside the body of real humanity that, in recent years, had been growing together. it is worthwhile to note here that this healthy body of empire is one that combines both the freedom of flows and the order of an assemblage, 'bound together'. this empire is basically one of assembled technologies -''technology, communication, trade and travel'', says blair -where freedom flows orderly. the gaps in-between these assembled technologies is where disorderly, 'viral' 'fanaticism' can and will slip within. we will get back to the prime minister's speech later. it may suffice for our purposes here to underline the importance of this newly emerging imaginary. its particular medical imagery seems to betray a new vision of empire, i.e. one that, on the one hand, is moving beyond notions of empire as productive achievement, and one that, on the other hand, is gradually abandoning both legality and diplomacy as the main tools of empire-building. although this particular medical imaginary, in our view, already began to crystallise during the early s, i.e., shortly after the fall of the berlin wall, and, with it, the cold war, it could be argued that, in the aftermath of september , and in the months prior to the latest iraq war, events catalysed this medical imaginary and refined it. by the time of blair's congressional address, it emerged fully fledged. we will attempt to reconstruct this process of catalysis and refinement. but before we do that, we need to explore some imaginaries of empire, old and new. empire used to be considered as productive process. empire was something to be achieved, to be accomplished. it was a goal, and, as such, it was strived for. empire was not just there, it was not given. it had to be made, it had to be produced, and it had in turn to be made productive. forces had to be marshalled, populations had to be subdued and mobilised, crops had to be cultivated, peoples had to be disciplined and 'civilised', landscapes had to be changed, goods had to be transported, networks and dependencies had to be established, and ultimately, something like imperial integration or better, inclusion, had to be accomplished: 'they' had to be part of 'us'. and sometimes this accomplishment of empire seemed to have implied that 'they' must be 'like us', or 'with us', or 'under us'. referring to victorian writers such as ruskin and lord macaulay, this is how ian baucom recently read the british empire: 'a factory for the production of english identity'. let us be clear and not overstate our case here, and let us at least qualify this claim. empire, it should be stressed, was simultaneously and inevitably also exclusive; it aimed, at best, at disciplinary normalization of subdued populations (that sometimes went under the heading of 'civilization'), and, at worst, at sheer repression, even genocide. if 'they' would not be 'part of us', if 'they' would refuse to 'be like us', or be disciplined to be 'with us' or 'under us', then the options of repression and destruction were opened up. but this is not the issue of this paper. the issue here is to realise how empire used to be imagined and practiced, first and foremost, as something that had to be made, that had to be produced, as something that had to be assembled. out of fragments, incoherence, and disunity, empire had to be produced. so unnatural an enterprise, this empire, in the words of rudyard kipling, the colonialist par excellence, was literally the white man's burden. and all this required tremendous productive effort as well as discipline. one could hope that, at the end of this productive effort, empire would emerge. if ever it did emerge. the production of empire did seem to be a never-ending process of construction, mobilisation, inclusion, discipline, and integration (however repressive it may at times have been). empire never seemed to be fully accomplished, it required constant and unrelenting productive attention. in this empire, there's no room for complacency. as a 'factory', empire seemed to be an ongoing productive process. its decline threatened every minute of the day from within as well as from without (outside was the location of other empires). this th century notion of empire as an unrelenting process of productive inclusion or integration of some sort coincided with the consolidation of european nation-states and the emergence of nationalism. nation-states too were not given. they too had to be built, they had to be produced, they had to be fashioned out of fragments, incoherence, and disunity. they too were the result of productive inclusion and discipline. they too were the result of tremendous effort, of particular arrangements that kept populations and landscapes together. they too were the result of factories and of labour. they too had to be made productive in turn. however, in both cases, i.e. the production of nation-states on the one hand, and the production of empire on the other, legality and diplomacy were crucial. law and diplomacy were important technologies (however repressive at times) by which nation-states as well as empires were held together, or indeed, by which they were produced or maintained, and by which they were made to be productive. this imaginary -i.e. empire is the result of a productive process of inclusion -has, since about the fall of the berlin wall, been replaced by another. this new imaginary, we hope to be able to demonstrate below, unfolded gradually throughout the s. at first notions such as 'production', 'productivity', and 'inclusion' gradually disappeared from the imaginary of empire. later, after the events of september and the latest iraq war, a newly emerged imaginary of empire, i.e. empire as an 'exclusive' 'given', was medicalised. let us reconstruct this first phase here. the second phase, i.e. the phase of medicalisation, will be dealt with in the next section. empire does not have to be produced or maintained anymore. it is accomplished. it's just there. today, empire is a given. nothing needs to be included anymore. empire is global, universal, complete, and is, as such, 'immutable'. it may, as hardt and negri recently argued, be a 'multicentric' empire, driven and fuelled by what they call the 'multitude' of desires and hopes. it may not have a clear and obvious centre anymore. but it is, hardt and negri argue, global and complete, and it seems to have reached a point of no return. indeed, prior to september , it looked as if the global and globalizing 'multitude' and their 'multicentric' controls, i.e. empire, in hardt's and negri's view at least, were there, or here, to stay. such an empire does not need to be produced or maintained. it produces and maintains itself. it doesn't have to be made productive. its productivity is determined by this multitude of desires and controls. there is no imperial centre that arranges for particular inclusions or that works towards integration. there is a multitude of desires and controls that interact with each other or resist each other. this empire is complete. now this is how two radicals have imagined empire in the s. one does not have to agree with them of course. according to peter fitzpatrick, for example, although this 'new imperialism' assumed something like a 'complete givenness', something like the 'naturalism' or 'the implanted truth and the inevitability' of 'the neo-liberal order', while 'it assures itself that it has encompassed its own limit, achieved completeness, and marked the end of history', it also retained and continues to retain severely disciplinary features, such as 'structural adjustment plans' and other 'contractual' devices whereby states are forced to collaborate towards their own disciplined subordination to 'global capital'. fitzpatrick also amply demonstrates how the 'new imperialism' of the s tended to be imagined as that particularly western but highly pretentious universalism that hides under words like 'human rights'. and this entailed massive exclusion, as, of course, 'those who do not behave in accordance with or aspire to human rights, must not be human'. one might however agree with the claim that the imaginary of empire, after the fall of the berlin wall, say roughly from president bush sr.'s new world order onwards, has arguably undergone marked changes. empire seems to have lost much of its 'productive' and 'inclusive' connotations (however repressive at times such production and inclusion may have been). if anything, today's imagined empire seems to be in need of exclusions. in the imaginary of bush sr.'s new world order, for example, there was no room for 'rogue states'. those should be kept at bay, or neutralised, or embargoed, or just contained. this imaginary empire, i.e. an almost natural given of free-flowing forces and desires (freeflowing but orderly, new-world-orderly, that is), only needs to reproduce itself by excluding 'rogue states' (rogue-but-unfree, that is), or at least by keeping them in check. this is its imaginary legality, this is what its imaginary diplomacy is about: the exclusion, or at least the policing (with the us as the 'world's policeman') of 'rogue' elements from a given state -a given empire -of natural freedom and natural order. this shift in the imaginary of empire again coincided with a similar shift that could be detected in the imaginary of the nationstate. in an age when, as habermas once noted, life and governance are marked by an 'exhaustion of utopian energies', the project of the nation-state, or any project for that matter (habermas himself wrote about the welfare state in particular), tends to be imagined and practiced much more as a negative complex of exclusions rather than a focused, positive, indeed utopian project of inclusion. or, in a somewhat different vein, and borrowing from deleuze, discipline gradually made way for control. during the past few decades, the legality of national projects often expressed itself in images such as 'fight' and 'enemy', or 'stranger' and 'risk'. the fight against crime, for example, or the stranger at the border, or the prevention and containment of risk, all did begin to weigh heavily upon the imaginary of nation. however, in both cases, i.e. empire as well as the nation-state, legality and diplomacy remained fully part of their respective imaginary space. policing rogue states, fighting crime and criminals, and keeping strangers out did remain a matter of legality see note , at - . this emerging imaginary of empire (i.e. empire as a natural, selfsufficient, complete given of orderly freedom and exclusion of rogue elements), we will argue, got medicalised, and this medicalization also catalysed the imaginary of empire towards a higher level of coherence. now this medicalization of the imaginary of empire, one could argue, was already on its way before september . indeed, the first gulf war ( ) ( ) , the first test of bush sr.'s new world order if you wish, already saw imagery like 'surgical strikes' surge to the surface. the then us government administration may have regarded themselves as the world's policeman, operating within the bounds of legality and diplomacy (however repressive at times), they nevertheless saw no problem in using the scalpel when cutting out 'rogue' elements with surgical precision. we shall demonstrate how this embryonic medicalization further evolved, passing an oncological stage, and ending in a final, epidemiological one. again, this trend of medicalisation could also be seen on an intra-national level, where, as nikolas rose has shown, crime control, in today's 'biological culture', seems to adopt genetically informed risk prevention strategies 'that aim to identify, treat and control individuals predisposed to impulsive or aggressive conduct', and that should be implemented against (and this is important) 'those whose conduct seems to show wanton disregard for the moral constraints on the conduct of free individuals in a liberal society'. before we explore the medicalised imaginary of empire in more detail below, let us just emphasise here how, in this imaginary, particularly in the oncological and epidemiological stages (see below), empire appears as a full, self-sufficient given, as a naturally given body, as a naturally ordered body of freedom-cum-constraint, whose undoubted health should be maintained in and through the exclusion of anything -indeed: anything inhuman -that does not belong there. at this point, preoccupations with legality and diplomacy give way to a different logic of empire, to one could add here that medical imagery as such, e.g. the use of words such as ''health'' to describe the state of the nation, has th century origins. as many criminologists know, louis pasteur's work in bacteriology made sure that late th century criminologies did tend to define crime in terms of bacterial ''infection''. rose, n., ''the biology of culpability: pathological identity and crime control in a biological culture'', theoretical criminology ( ), - , at (italics r.l.). viral contagion and anti-terrorism a different logic of imperial intervention, if you wish. this is the point where imperial intervention comes to be imagined as an instance of medical urgency. in a medicalised imaginary of empire, it's the logic of medical urgency and medical necessity that is likely to start determining and structuring imperial interventions such as 'pharmacotic' wars on terrorism. . the image of terrorism as a cancer that needs to be fought (gill himself, somewhat ironically, understands this 'fighting' to consist of 'longterm' 'understanding') would prove to be rather resilient. president bush's national security adviser, condoleeza rice, e.g. about a month after the september attacks, and days into operation enduring freedom in afghanistan, admits to a cnn journalist that 'what we want to do is to work with every government in which there is a substantial al-qaeda presence to figure out a strategy for rooting it out (...) because it's like cutting out a cancer (...)'. although it moves towards further medicalization -oncologization, to be precise -rice's imaginary remains a somewhat uneasy hybrid. 'cutting out cancers' does not sit nicely and squarely within an apparently tenaciously persisting legal and diplomatic framework which implies 'working together' with 'governments' to 'figure out strategies', particularly at a time when a severe military campaign is going on. the image of 'terrorism as cancer' as such was unable to capture both the conditions and the particulars of what happened on september . cancer tends to grow inside bodies. yes, like 'evil', it 'knows no boundaries' (president bush during his trip to china), and it often grows and spreads through metastases, but it is localised, and, moreover, it appears to come from within, and as such, has something human about it -it's just human cells running wild, it's humanity running wild. but the image did persist, and surfaced on a few occasions, one as late as march (during a press conference on the eve of the invasion of iraq) when president bush, referring to 'regime change', talked about 'replacing this cancer inside of iraq'. 'viral' imagery, however, would prove to be more appropriate to capture the events of september and the conditions that led to it. viruses -at least some viruses -tend to be both outside and within. they come form 'outside' the body, but somehow, and unpredictably so, like 'evil', manage to penetrate boundaries, slipping 'within'. viruses also need 'cultures' where they are able to grow and multiply incessantly. this notion of 'cultures' is worth dwelling on for a moment. fitzpatrick, for example, critically examines the use made by proponents of 'human rights' of phrases such as 'cultural absolutism' to denominate 'culture-bound, closed, exclusionary, traditional, authoritarian, status-ridden, static, 'pure and inviolable community'', i.e. those rigid and automaton-like elements devoid of the capacity for moral, and therefore, human deliberations. in a strange and ironic twist these 'cultures' of 'absolutism' prove to be highly flexible and mobile on a global scale, and possibly more so than the allegedly culturally universal particulars that go under the name 'human rights'. indeed, and most importantly, viruses -at least some of them -tend to be airborne, just like the images of the twin towers that circulated through cnn's ether, spreading fear and terror, contagiously. as the head of a fascist terrorist conspiracy tells us (in the hollywood production the sum of all fears, which was released in ), whilst enjoying the apparent inability of both us and russian protagonists to move beyond cold war thinking, and whilst planning a nuclear attack on baltimore, 'our virus is airborne'. a french judge, in a televised programme, likened al-qaeda to the aids virus : 'this virus is totally mutant'. viruses are not human. a virus is a 'fremdkorper', an alien, inhuman entity that does not belong within (and that has no place without either). viruses ought to be tracked down in their cultures and destroyed without further ado. this is a matter of medical, or more precisely, of epidemiological necessity and urgency. the image of 'terrorism as viral contagion' surfaced fairly shortly after the september attacks, particularly in academic milieux. a few days after the attacks, arthur and marilouise kroker, the canadian editors of the pomo-zine par excellence, ctheory, published an essay under the heading 'terrorism of viral power'. the attacks on see note , at . ''the third world war: al-qaeda'', bbc two (television), tuesday th february , p.m. the virus metaphor of course had been used before. in his statement before the house of un-american activities committee, j. edgar hoover, fbi director, spoke of the ''virus of communism''. epidemiological destruction of internal communism, however, still had a seriously legal tinge about it in hoover's statement. ''i do favor unrelenting prosecution wherever they are found to be violating our country's laws. as americans, our most effective defense is a workable democracy that guarantees and preserves our cherished freedoms'' (taken from http://edition.cnn.com/specials/cold.war/episodes/ /documents/hoover). the twin towers, the krokers tell us, have taught us that, when confronted with the 'contagious logic of viral power', particularly with a suicidal viral logic, 'dissuasion is inoperative'. diplomacy and legality, in other words, are inoperative. when dealing with sickness and contagion (and how else to define suicidal mania that spreads like wildfire in fundamentalist cultures and in rigid communities of machine-like automatons?) epidemiological 'logic' has to take over. 'viral power adopts the strategy of the attacking parasite', the krokers continue, 'invading the body of the host (the american homeland), bleeding its tactical intelligence (those flight schools in florida), circulating in its commercial bloodstream (american airlines), and imploding in a violent fatal metastasis that has as its aim the infiltration of the mediascape through its apocalyptic effects'. it should come as no surprise that such imagery emerged very quickly in postmodern academia. baudrillard of course had been preparing for this for years. 'hatred, a viral passion', claimed baudrillard, in , 'is also a vital passion. against the perfection of the system, hatred is a last vital reaction'. against the pretended self-sufficiency of a naturally given, perfect, human and healthy empire, against this new world order, hatred is a viral reaction. 'terrorism as viral contagion' gradually replaced oncological imagery in official statements and documents. the when it became already clear that a number of nato member states were likely to formally object to any invasion of iraq before the complete exhaustion of legal and diplomatic possibilities (a temporary compromise would be found in and offered by un resolution a few weeks later, in november ). the image of terrorism as viral contagion appeared throughout in a host of publications and addresses, such as bruce hoffman's rand paper on al-qaeda, or terrorism expert magnus ranstorp's statement to the us national commission on terrorist attacks upon the united states, which conjures up an image of al-qaeda as an 'incontrollable virus', or a 'disease' that aims to strike 'at the heart of the west's strength -the interconnectivity of their economies'. in his statement, ranstorp seems to combine both the krokers' and blair's imaginaries when he argues for 'making suicide-bombings a crime against humanity', i.e. a crime committed by elements with a 'suicidal mindset becoming airborne missiles' (or machine-like fanaticism) against this healthy body of global interconnectivity (or orderly freedom). here is a passage taken from the president's state of the union, delivered in january , at a time when preparations for the large scale epidemiological expedition into iraq's viral culture were in full swing: Ôbefore september the th, many in the world believed that saddam hussein could be contained. but chemical agents, lethal viruses and shadowy terrorist networks are not easily contained'. it could be noted here how, in this quotation, terrorists are placed on the same line as their weapons. indeed, terrorists, in particular the alien, inhuman, suicidal, machine-like types that have placed themselves beyond legality and diplomacy -'dissuasion', however repressive, 'is inoperative'are (like) their weapons; they are (like) noxious chemicals or contagious viruses. as viruses, they don't need moral reform or social rehabilitation. they don't need punishment. they don't need mere containment. they need to be destroyed. that is ultimately a matter of medical necessity that needs to be dealt with urgently and swiftly by medical experts, and it's the latter, i.e. the experts, that will decide on this urgency and necessity. in a curious way, bush's state of the union is replete with medical images and imagery, virological and epidemiological ones in particular. the president for example announces a massive budget increase aimed at 'strengthening medicare' (the nation's healthy body), asks for a strategic budget 'to quickly make available effective vaccines and treatments against agents like anthrax, botulinum toxin, ebola, and plague', and promises developing countries substantial help in their struggle against the hiv/aids virus. as suggested above, one could argue that this epidemiological imaginary helped refine a space where medical necessity and medical urgency provided the natural 'logic' for imperial intervention, thereby gradually replacing the unnatural 'logic' of legality and diplomacy. the 'authoritarianism' that is crystallizing in today's healthy body of empire (global 'post-fordism', claims steinmetz) seems to be of a medical nature. empire's permanent 'state of emergency' feels like an epidemiological one. one could wonder why this imaginary has formed as and when it did, and whether there was a clear and conscious strategy behind it, but that is an issue we cannot hope to resolve here. what we can say here is that 'terrorism as viral contagion' seems to have facilitated a number of imperial interventions. or, to weaken down this claim a little, it seems to have coincided with a number of issues and subsequent interventions which it may have provided a facilitating imaginary space for. one of these issues and interventions came with the capture of al-qaeda and taliban fighters, the first of whom were imprisoned in guantanamo bay, in january , on a site that was aptly named 'camp x-ray'. naming those fighters 'criminals' would have triggered a whole body of international human rights charters and domestic due process provisions. naming those fighters and alleged terrorists 'soldiers' or 'warriors' would have turned them into prisoners of war, and that would at least have made the geneva convention applicable. a medical or viral problem, a clear and present problem of inhumanity, on the other hand, can and should be dealt with 'outside', i.e. 'outside' america's healthy body (or the world's, for that matter), and 'outside' the bounds of humane -or simply: human -legality and diplomacy. the natural body of empire is in no need for unnatural legality or diplomacy; precisely because it is a given, natural body, it needs medical attention and intervention instead. there is no need to build or rebuild the body of empire: once freed from the virus, it will emerge healthy, naturally. there is no need for the productive negotiations of a 'cosmopolitan globalism' either (to use mikkel rasmussen's words ), nor for reconciliatory efforts (one does not reconcile with viruses): the sanitary exclusion of viral contagion will suffice to keep the body of today's imperial new world order healthy. another issue (and subsequent intervention) that, at least to some extent, however minor, seems to have been facilitated by this epidemiological imaginary, was the invasion of iraq. invading an alleged or potential culture of viral, and therefore contagious terrorism is first and foremost a matter of medical necessity and urgency. the legal and diplomatic 'logic' of un resolutions (resolution in particular), in this imaginary, can only be of secondary importance. furthermore, epidemiological interventions allow empire to deal with contagious terrorists and their cultures (the 'rogue states' of yore) simultaneously, in one cleansing, sterilising sweep. it allows for a move beyond both the unnatural legal strictures of criminal procedure and the unnatural diplomatic constraints of international law. empire, after the end of the cold war, is imagined ever more as the medical practice of exclusion rather than a legal and diplomatic (however repressive at times) project of inclusion. against the given, naturally ordered, healthy body of empire the image of the inhuman, machinic, disorderly, virally chaotic ''fremdkorper'' looms darkly. in 'shadow and darkness', said blair in his july congressional address, somewhere 'outside' the healthy body of humanity. the events of september and their aftermath, in our view, have catalysed this shift in the imaginary of empire. however, it remains to be seen whether empire is now on a course that will take it beyond any regard for legality and diplomacy. in his address, tony blair is unclear about it. 'freedom', he says in his address, 'democracy', and 'the rule of law' are 'not western values', but 'the universal values of the human spirit and anywhere, and anytime, ordinary people are given the chance to choose'. if left on its own, the healthy body of empire (of freedom-cum-rule of law) will emerge naturally and orderly, indeed, like a body. what therefore needs to be done is to make sure it is left on its own. what is needed, in michael ignatieff's words, is an 'empire lite'. and once the virus removed, there will be no real need to start reconstructing the rule of law, or indeed, there will be no need to start reconstructing anything at all: the healthy body of empire (i.e. freedom-cum-order) will emerge or re-emerge naturally and organically. blair continues, 'our new world rests on order', the 'danger is disorder and in today's world it now spreads like contagion' through fundamentalist terrorism, whose 'weapon is chaos'. this contagion needs to be dealt with. the natural rule of law must be protected swiftly. if necessary -and it is necessary, says george w. bush, as 'there are no rules' -this must be done beyond an unnatural logic of legality and diplomacy: let us say one thing. if we are wrong, we will have destroyed a threat that, at its least is responsible for inhuman carnage and suffering. that is something i am confident history will forgive. but if our critics are wrong, if we are right as i believe with every fibre of instinct and conviction i have that we are, and we do not act, then we will have hesitated in face of this menace, when we should have given leadership. that is something history will not forgive. this is the new logic of empire. resolution was adopted at security council meeting trends in terrorism and future potentialities: an assessment statement to the us national commission on terrorist attacks upon the united states president delivers 'state of the union key: cord- - zzj ts authors: baker, robert; shelton, wayne; strosberg, martin title: ethics and epidemics date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: zzj ts nan how to promote transnational collaboration in implementing quarantine was the topic of a january [ ] [ ] [ ] [ ] [ ] , conference sponsored by the defense threat reduction agency. fifty invited participants discussed the status of quarantine planning in countries (the americas, israel, and several members of the european union [eu] nations). held in the wilton park conference centre, sussex, united kingdom, the conference, "quarantine following an international biological weapons attack: building cooperation, achieving consistency," also addressed quarantine in response to emerging infectious diseases. participants first examined the legal foundation for quarantine in their countries. federal canadian quarantine law applies only to national ports of entry or exit; provincial laws govern quarantine in the provinces. the u.s. centers for disease control and prevention has quarantine responsibilities at national ports of entry or departure; this agency may also become involved when a disease is spreading across state borders or even within a state (when invited by the governor of the state or ordered by the u.s. secretary of health and human services or the u.s. president). in general, however, quarantine in the united states is a local or state government issue. quarantine laws in these jurisdictions vary, and some public health authorities expressed reluctance to address their shortcomings through legislation for fear that skeptics of quarantine would further weaken the laws. other nations would turn to the world health organization and its international health regulations of (ihr) for guidance. a revised ihr should be available by , but currently it lists only three diseasesplague, cholera, and yellow fever-as subject to quarantine and offers scant help in planning quarantine. thus, the legal framework for quarantine varies and contributes little to the construction of a consistent approach to quarantine among nations. european public health officials have forged some bilateral cooperative agreements and are discussing establishing a regional disease control center for eu nations. they are not, however, developing and testing national or transnational plans for possible large-scale quarantine. some participants thought that consistency in developing and implementing quarantine measures was not necessarily desirable, given that each nation must deal with threats in accordance with its own culture, laws, and traditions. others thought that inconsistencies in response to the same disease threat might encourage persons to question the need for quarantine measures and choose not to comply. the united states also has not developed comprehensive quarantine plans, trained staff, or conducted quarantine exercises in local communities, despite recently issued federal quarantine guidelines. especially lacking are processes and procedures to clarify decision-making and coordination in communities with multiple jurisdictions. the heightened concern of the united states about bioterrorism was not shared by others at the conference, although all agreed that persons would likely demand a federal response to a health crisis caused by terrorists, including any required quarantine. other issues discussed included assurances of compensation for income lost while in quarantine (strongly recommended as a component of any quarantine plan) and psychosocial support to reduce the sense of isolation experienced by many persons while in quarantine. officials with information management experience during health-and nonhealthrelated crises commented on the need for caution in making public statements when faced with a new and evolving threat. the conference permitted participants to establish working relationships with one another, but it also highlighted gaps in comprehensive transnational quarantine planning. presentations ranged from case studies to health policy debates. many reviewed the history of epidemics, emphasizing their global nature and the imperative of global strategies for epidemic control. several papers examined recent epidemics and explored new strategies for dealing with epidemic control while respecting human rights. the consensus was that the old policeman model of public health needs updating. discussion focused on how best to balance public safety, professional responsibility, personal liberty, and human rights, while effectively containing epidemics. emanuel and wynia reaffirmed the responsibility of physicians and first responders to put their health and lives at risk in combating epidemics. yet, noting the vulnerability of first responders (in the toronto severe acute respiratory syndrome [sars] outbreak and elsewhere), they distinguished between bravery and foolhardiness, arguing that just as professionals have a responsibility to protect the public from disease, the public, in turn, has a responsibility to provide the training, equipment, and resources to minimize the need to take risks. virtually all conferees observed that the public health infrastructure needs substantial rebuilding to cope effectively with epidemics. annas, however, noted that in bioterrorist assaults, the control of biologic agents is a public health problem to be dealt with by public health officials, not by the u.s. department of defense or the u.s. department of homeland security. he further stated that policies on epidemic control that involve consistent, open, and truthful communication with the public-like those used in new york and toronto during the recent sars outbreak-create cooperative environments that minimize conflicts between freedom and safety and limit the effects of isolation and quarantine. however, emanuel et al. asserted that the traditional enforcement authority of public health law was essential and needed as a fallback. the result of the debate was that st century methods need to be developed to control infectious disease epidemics that reconcile the need to protect public health and respect human rights. the conference program is available on http://www.bioethics.union.edu under "news." for further information contact bioethics@union.edu or - - . email: bioethics@union.edu emerging infectious diseases • www.cdc.gov/eid • murine typhus with renal involvement in canary islands, spain" by michele hernandez-cabrera et al. was inaccurate protective behavior survey, west nile virus, british columbia beginning in january , summaries of emerging infectious disease conferences will be published online only.summaries submitted for online publication may include illustrations and relevant links. for more information on conference summary requirements, please refer to author guidelines at http://www.cdc.gov/ ncidod/eid/ instruct.htm.submit conference summaries at http:// www.eid.manuscriptcentral.com key: cord- -ghjseynl authors: arbely, eyal; khattari, ziad; brotons, guillaume; akkawi, mutaz; salditt, tim; arkin, isaiah t. title: a highly unusual palindromic transmembrane helical hairpin formed by sars coronavirus e protein date: - - journal: journal of molecular biology doi: . /j.jmb. . . sha: doc_id: cord_uid: ghjseynl abstract the agent responsible for the recent severe acute respiratory syndrome (sars) outbreak is a previously unidentified coronavirus. while there is a wealth of epidemiological studies, little if any molecular characterization of sars coronavirus (scov) proteins has been carried out. here we describe the molecular characterization of scov e protein, a critical component of the virus responsible for virion envelope morphogenesis. we conclusively show that scov e protein contains an unusually short, palindromic transmembrane helical hairpin around a previously unidentified pseudo-center of symmetry, a structural feature which seems to be unique to scov. the hairpin deforms lipid bilayers by way of increasing their curvature, providing for the first time a molecular explanation of e protein's pivotal role in viral budding. the molecular understanding of this critical component of scov may represent the beginning of a concerted effort aimed at inhibiting its function, and consequently, viral infectivity. a previously unidentified member of the coronaviridae is the etiologic agent responsible for the recent severe acute respiratory syndrome (sars) outbreak. viral genome sequencing, combined with protein phylogenetic analyses, have shown that sars coronavirus (scov) belongs to a new subfamily within the coronaviridae. , in an effort to better understand the components contributing to the pathogenic mechanism of the virus, we have structurally analyzed the transmembrane domain of scov e protein using fourier transform infrared (ftir) spectroscopy, x-ray scattering, electron microscopy and global searching molecular dynamics simulations. functionally, coronavirus e protein's pivotal role in viral morphogenesis has been studied extensively. co-expression of mouse hepatitis virus (mhv) e and m proteins on their own has been shown to result in the production of virus-like particles. , this fundamental observation proved that neither the nucleocapsid nor the viral spike are needed for viral budding. work on other viruses, such as transmissible gastroenteritis virus (tgev), bovine coronavirus (bcov) and infectious bronchitis virus (ibv) has shown this to be a general phenomena of the coronaviridea. the interaction between the cytoplasmic domains of the two proteins is thought to take place in pre-golgi compartments. the importance of the role played by e protein in virus budding and morphogenesis is further strengthened by the observation that in a number of coronaviruses, expression of m protein on its own does not produce virus-like particles. - , in contrast, expression of ibv or mhv e protein on its own causes the release of vesicles containing e protein, thereby pointing to the importance of coronavirus e protein in the budding process. , interestingly, cells infected with mhv release vesicles containing e protein as well. finally, mutations in mhv e protein cause marked morphological changes in the resulting viruses. in the cell, evidence has been accumulating suggesting that ibv e protein localizes to the golgi apparatus, the site from which coronaviruses are known to invaginate into mature virions. during the expression of e protein the golgi apparatus changes its morphology dramatically, which could explain in part e protein's ability to induce apoptosis. , structurally, e proteins are conserved within the different coronaviridea groups, yet exhibit little sequence similarity between the groups (figure ). in line with its belonging to a new group within the coronaviridea, scov e protein differs substantially from other coronavirus e proteins. whether this has any bearing upon the marked difference in pathogenicity of scov versus other coronaviruses remains to be seen. finally, scov e protein shares no significant sequence similarity with any other known protein. it is possible, however, to make the following generalizations regarding the sequence of all coronavirus e proteins: they are small proteins (, residues), with an unusually long hydrophobic stretch ( - residues) located between hydrophilic n and c termini (, and , residues, respectively). scov e protein shares the same overall characteristics, although only % identical with other coronaviridea e proteins: residues in length with a stretch of hydrophobic amino acid residues (figure ). the length of the hydrophobic segment of scov e protein has posed a problem with respect to assigning a topology to the protein (figure (a) ). the average length of a transmembrane a-helix is using a window size of and a hydrophobicity cutoff of dg water!oil ¼ kcal/mol. the region of the synthesized peptide is boxed, while gray shading indicates the extent of the hydrophobic stretch of the protein. the location of the iodinated phe residue is indicated by dark shading. (b) inversion diagram of the scov e protein hydrophobic region outlining identity and similarity between the two inverted sequence elements, in continuous and broken lines, respectively. hydrophobic amino acid residues according to the ges scale are indicated by gray shading. residues, far shorter than the hydrophobic stretch of e proteins. in contrast, if a hairpin were to be formed by the long hydrophobic stretch, the resulting helices would be much shorter than the average. protease digestion of mhv does not affect e protein's molecular mass, suggesting that no part of the protein protrudes beyond the viral membrane. antibodies raised against the n terminus of the protein could detect the protein only after treatment of the cells with triton x- , but not with digitonin. both of the above studies concluded that the protein traverses the lipid bilayer once, with its c terminus located in the viral interior. , more recent results with an epitope tagged mhv e protein are indicative of the protein traversing the lipid bilayer twice, whereby both termini of the protein reside in the virus lumen. in the current study we present the results of a detailed structural examination of scov e protein, in which we determine the topology of the protein and the effects upon the lipid bilayer thereof. the results conclusively show that scov e protein has a highly unusual topology, consisting of a very short transmembrane helical hairpin. the hairpin forms an inversion about a previously unidentified pseudo-center of symmetry which seems to be unique to scov. electron microscopy studies indicate that the protein dramatically distorts lipid bilayers, causing tubulation. taken together, the structure of the protein facilitates a molecular explanation of the effects upon the bilayer curvature underpinning the function of the protein in vivo and may represent the beginning of a concerted effort aimed at inhibiting its function and consequently viral infectivity. in order to examine the secondary structure of the transmembrane domain (tmd) of scov e protein, peptides were made which encompassed the entire hydrophobic region of the protein (glu -arg ), as depicted in figure . the peptide was purified and reconstituted into dimyristoylphosphocholine (dmpc) lipid vesicles, to be used for ftir spectroscopy. the transmission ftir spectrum of the resulting proteo-liposomes focusing on the amide i vibrational mode ( - cm ) is presented in figure (a). since the amide i vibrational mode arises mostly from the peptidic c ¼ o stretch, its frequency is well correlated with the secondary structure of the protein. peaks resonating at cm , cm and cm , correspond to a-helical, b-strand and random coil peptide segments, respectively. the amide i mode of scov e protein tmd is centered at cm with a peak-width at half height of cm , both indicative of the very high helical content of the protein, and the absence of any other secondary structure component. e protein contains residues embedded in the lipid bilayer ftir spectroscopy can be used to delineate the extent of membrane incorporation of transmembrane proteins. this is achieved by observing the reduction in any vibrational mode containing significant contributions from the amide proton (e.g ii mode : peptidic n -h deformation) upon exchanging the solvent from h o to d o. the reason is that the lipid bilayer protects the peptide from exchange, whereas amide groups exposed to the solvent undergo h þ /d þ exchange. any vibrational mode containing the "new" n -d group will resonate elsewhere and h þ /d þ exchange can be quantitated by measuring the reduction in the amide ii mode directly. as shown in figure (b), upon flushing the membrane with air saturated with d o for several hours, the reduction in the amide ii peak, centered at cm , is minimal. since the amide i peak at cm is not expected to undergo any intensity changes upon exchange from h o to d o, it is possible to calculate the extent of exchange by normalizing both spectra (in h o and d o) on the amide i peak. the resulting exchange rate of ^ % is indicative of only six out of residues in the peptide undergoing h þ /d þ exchange. in other words, residues of scov e protein are protected from exchange as a result of their being embedded in the lipid bilayer. note that this value corresponds exactly to the length of the hydrophobic stretch of the protein. after establishing the helicity of the peptide and determining that most of it is embedded in the lipid bilayer, experiments were undertaken to determine its orientation using polarized ftir spectroscopy of oriented membranes. attenuated total internal reflection (atr-ftir) spectra obtained using both parallel and perpendicular polarized light are depicted in figure (c). the dichroism (ratio of absorption of parallel and perpendicular polarized light) for the amide i peak (centered at cm ) is . ^ . . from this dichroism it is possible to calculate an exceptionally high order parameter for the peptide of s ¼ : ^ : , a value nearing the theoretical limit. when taking into account sample disorder, it is possible to convert the maximal value of the measured order parameter to a maximal tilt angle. thus, the tilt angle of scov e protein helical elements from the membrane normal must be less than or equal to . . so far three lines of evidence were obtained characterizing the transmembrane domain of scov e protein: (i) it is highly helical, (ii) it is composed of amino acid residues and (iii) the helical elements are oriented normal to the membrane plane. thus, there are two ways in which to describe the structure of the protein: either as a single long transmembrane helix, or as two short helices forming a transmembrane helical hairpin. both models however, incur a hydrophobicity mismatch between the protein and the bilayer. in the case of a single helix, the hydrophobic stretch is too long relative to the bilayer thickness. † on the other hand, the helical hairpin would have to be comprised of two very short helices in order to enable the residues to traverse the membrane twice. since the smallest possible loop is of three residues, each helix cannot contain more than - hydrophobic residues. this value is much smaller than the average of residues per transmembrane helix. in order to conclusively determine the topology of scov e protein we iodinated phe , which is located at the center of the hydrophobic stretch of the protein (see figure ) . ftir spectroscopy has shown that the iodinated and unlabeled peptides are indistinguishable in any of the experiments described above (data not shown). electron density profiles obtained using x-ray scattering should be able to pinpoint the position of the labeled electron-dense iodine. as shown in figure , the electron density of lipid vesicles containing iodinated and unlabeled scov e protein transmembrane domain exhibit normal density profiles. however, upon subtracting the densities form one another, clear density resulting from the iodine label is identified, located . Å from the bilayer core, close to the lipid head-group region. thus, one can clearly state that the labeled phe is located adjacent to the head-group region, despite the fact that it is in the middle of the hydrophobic segment of the protein. taken together, our data prove conclusively that the transmembrane domain of scov e protein forms a hairpin with unusually short hydrophobic helices. after establishing that scov e protein forms a helical hairpin, close inspection of its sequence reveals an unusual feature. as shown in figure (b), inverting the protein about itself at phe indicates that the protein is virtually symmetrical with respect to the inversion point, thereby forming a palindrome. the only deviation from palindromic symmetry is located at asn , the significance of which is discussed below. in order to estimate the statistical significance of forming a palindrome within the helical hairpin framework, a monte -carlo simulation was performed. the sequence of each helix forming the hairpin was randomized, followed by assessing the extent of palindromic symmetry. the results indicate that the degree of inverted identity between the two helices of scov e protein (thereby forming a palindrome) is a rare event, observed at a frequency of ca . this would explain why to our knowledge palindromic symmetry in helical hairpins has not been observed before. the scov e protein contains the shortest transmembrane hydrophobic helical hairpin known to date. its hydrophobic amino acid residues not only traverse the lipid bilayer as two helices, but must form a connecting turn between the helices. an immediate question then arises: what affect does the unusual structure of scov e protein transmembrane domain have upon the lipid bilayer? in order to answer the above question we undertook two lines of experiments, aimed at (i) examining the molecular structure of the lipid in detail, alongside (ii) establishing the effects upon the membrane morphology as a whole. the order of the lipid bilayer acyl chains was examined using polarized ftir spectroscopy of oriented multilayered dmpc vesicles containing scov e protein. figure (d) presents the atr-ftir spectrum of the lipid ch and ch stretching modes of the aforementioned vesicles. the measured dichroism for the lipid ch asymmetric stretching mode (centered at cm ) is . ^ . , yielding an order parameter of s ¼ : ^ : . interestingly, the order parameter of the protein ðs ¼ : ^ : Þ is much higher than that of its ordering environment, the lipid bilayer. the reduced order of the lipid acyl chains is an inherent property of the system caused by scov e protein and cannot be attributed to incomplete sample deposition. the reason is that the exceptionally high order parameter obtained for the protein would not be possible under such circumstances. moreover, the mosaicity of the membranes was measured to be below . , as determined by x-ray reflectivity rocking scans (not shown). thus, incorporation of scov e protein transmembrane domain in the lipid vesicle deforms the acyl chains of the lipid bilayer. the global change in the structure and morphology of the lipid bilayer upon incorporating scov e protein transmembrane domain was examined using negative staining electron microscopy. while vesicles without protein ( figure (a)) exhibited normal globular structure, scov e protein-containing vesicles were markedly different ( figure (c) ), exhibiting extensive tubulation and deformation. vesicles containing a control transmembrane protein, the mhc class ii-associated invariant chain transmembrane domain, were similar to vesicles without protein, and did not exhibit any tubulation ( figure (b) ). in summery, ftir spectroscopy has shown that the incorporation of scov e protein in the lipid bilayer results in a molecular disordering of the lipid acyl chains. this leads to the tubulation of the vesicles as we have shown using electron microscopy. thus, the molecular defects in the lipid molecule are translated into a distortion of the entire bilayer structure. the evidence obtained so far points conclusively to the fact that the scov e protein transmembrane domain forms an unusually short transmembrane helical hairpin: (i) the secondary structure of the transmembrane domain is highly helical. (ii) more than % of the protein is embedded in the lipid bilayer. (iii) the helical element of the protein is tilted from the membrane normal by no more than . . (iv) phe residing in the middle of the hydrophobic stretch of the protein is located in the lipid head-group region. (v) the protein contains a center of symmetry upon which it can be inverted. what might the hairpin structure of scov e protein look like? the only deviation from hairpin symmetry is at asn (figure (b) ). the groups of engelman & degrado have recently shown that asparagine residues in transmembrane helices promote strong homo-dimerization due to h-bonding. , in this instance there is only a single asparagine in the two helices and as such the hydrogen bonds that it might form are to a backbone amide group, thereby driving the two helices to close apposition. alternatively, an asparagine residue from a different protein might promote dimerization, forming a pseudo fourhelix bundle. another interaction that might take place between the helices is through a salt bridge between glu and arg . the presence of opposing charged resides at both sites is highly conserved in the coronaviridea (figure ) , thereby substantiating the possibility of an interaction between them. in order to derive a model for the protein based on its deduced topology and helix tilt angles, global searching molecular dynamics simulations were employed. multiple starting structures were generated in which both helices of scov e protein (glu -leu and val -arg ) formed a hetero-dimer. the sizes of the helices reflected the high helical content of the protein. each structure was distinguished by the rotational angle of the two helices with respect to one another, in increments of , leading to a total of £ ¼ structures. the tilt angle between the helices was set to , according to the high degree of orientation found experimentally. the stability of each structure was then determined by a molecular dynamics and energy minimization trajectory. the resulting structures were then compared in order to find regions of the configuration space to which structures from different starting configurations converged. the results of the global searching molecular dynamics simulations are shown in figure , depicting two clusters to which structures have converged. the energy of the structures and the two clusters show a clear preference to lower rotational angles of the two helices (most likely due to the formation of a salt bridge, as discussed below). thus the cluster found at rotational angles of and (helix i and ii, respectively) was taken to represent a model for the interactions between the two helices (see figure ). after the packing between the two helices forming the hairpin was obtained, the loop connecting the helices formed by amino acid residues ala -val was constructed. the entire hairpin structure containing the loop was subsequently subjected to figure ( ) , and exhibits three prominent features: (i) a salt bridge between glu and arg , (ii) an inter-helical h-bond between the amide side-chain of asn and leu peptide carbonyl and (iii) three phenylalanine residues located at the tip of the hairpin pointing outwards forming an aromatic belt. finally, based on the evidence that ibv e protein is palmitoylated, it is possible to speculate that scov e protein might be as well. in contrast, experiments attempting to label tgev e protein with palmitic acid have failed. if present in scov e protein, palmitoylation will take place on one or more of the three juxtamembranous cysteine residues. it is difficult to estimate the effects, if any of palmitoylation on the scov e protein. one might speculate that the intercalation of the palmitic acid in the cytoplasmic membrane leaflet might further affect the local membrane structure. in any in vitro study the relevance of the results to those found under native conditions is obviously a concern. however, the following facts negate any suspicions of artifactuality: comparison to the full length protein. the protein under study encompasses all the hydrophobic domain of the scov e protein. the only thing known about the endogenous protein is that it traverses the lipid bilayer twice. that is exactly what was found herein. thus it is highly unlikely that the full length protein and its membrane domain both traverse the lipid bilayer twice, but do so in different ways. structural similarity to that found in native conditions. the endogenous function of scov e protein is in viral budding by way of causing membrane tubulation. - , moreover, it is the only protein in the virus that is required for this process. , thus, the finding that the membranous segment of scov e protein (analyzed in vitro) causes membrane tubulation in an indistinguishable manner to the wild-type protein, in vivo, is proof that the membranous segment is necessary and sufficient for scov e protein role in membrane tubulation. finally, the membranous segment of scov e protein was not studied in detergent micelles (as is customary), but rather in hydrated lipid bilayers, a system that is closest to the native conditions as possible. it was shown that the protein has a defined helical secondary structure and that it is highly embedded in the lipid bilayer. it is difficult to think of an example in which a small membrane protein was reconstituted in lipid vesicles, shown that it was helical as well as highly embedded in the lipid bilayer but is nevertheless in a non-native conformation. with a structural model of scov e protein at hand, is it possible to rationalize the effects of the protein upon the lipid bilayer? the hydrophobic stretch of both hairpin helices is only residues long, which is much shorter than the average of residues. at the apex of the hairpin three amino acid residues have their amide carbonyl and amine groups unpaired, surrounded by an aromatic belt. at the other end of the hairpin a cluster of charged residues is located. the distance between these two polar elements is much shorter than the hydrophobic thickness of the lipid bilayer. the polarity of both elements is not equal however, whereby the charged residues at the opening of the hairpin predominate. thus, one can speculate that the hairpin opening is situated at the lipid headgroup region of one leaflet. therefore, the hairpin apex is situated in the hydrophobic region of the bilayer, with its aromatic belt (known for its affinity to the polar head group) and unpaired amide groups poised to attract the lipid head-group region. it is this attraction that exerts a deformation force on the bilayer, causing an increase in curvature. in comparison to other coronaviridea e proteins, only the length of the hydrophobic segment lined by opposing charged residues at the either end, is conserved ( figure ) . thus, e proteins from different coronavirus subfamilies do not exhibit any sequence similarity to those from other subfamilies. it would seem therefore, that members of the coronaviridea have "found" at least four independent sequence motifs to form a functional e protein. the effects of the unique features of scov e protein-the aromatic belt, the inter-helical h-bond of asn and the inversion symmetry on the overall virus life cycle and resulting pathogenicity, remain to be seen. viral strains to ) were synthesized by standard solid-phase n-( fluorenyl) methoxycarbonyl chemistry. the peptides were cleaved from the resin with trifluoroacetic acid (tfa, aldrich) and lyophilized. two different synthetic peptides were made: an unlabeled peptide and one containing a para-iodo phenylalanine at position of the sequence, as shown in figure . peptide purity was confirmed by mass spectrometry. the lyophilized peptide was dissolved in tfa (final concentration , mg/ml), and immediately injected on a jupiter m c Å column (phenomenex, chesire, uk), equilibrated with % data were recorded on a nicolet magna- infrared spectrometer (nicolet instrument corporation, usa) purged with dry air and equipped with an mcta detector, cooled with liquid nitrogen. a total of interferograms were collected at a resolution of cm . attenuated total reflection (atr) spectra were measured with a reflections atr accessory from grasbey specac (kent, uk) and a wire grid polarizer ( . mm, graseby specac). three hundred microlitres of sample (ca mg/ml of peptide and mg/ml of lipid) were deposited onto a ge trapezoidal internal reflection element ( £ £ mm) flowed by removal of bulk solvent. for the purpose of solvent exchange, air was followed over the sample that was bubbled through h o or h -o. transmission ftir spectra were collected by depositing ml of sample (ca mg/ml of peptide and mg/ ml of lipid) on a caf window. the dichroic ratios of the amide i band were calculated by integrating between cm and cm . all integrations were performed by using a straight baseline that contains points immediately before and after the band. reported standard deviations values represent a minimum of three data sets measured. order parameters for the transmembrane helices were calculated as described. in brief, the order parameter s is defined as follows: whereby u represents the angle between the helix director and the z axis which is coincident with the membrane normal. the order parameter can be determined experimentally from the atr dichroic ratio r atr by the following equation: whereby the electric field components of the evanescent wave for a ge internal reflection element, e x ¼ : , e y ¼ : and e z ¼ : are given by harrick, and a is the angle between the helix director and the transition dipole moment of the vibrational mode: for the amide i mode and for the amide a mode. lipid order parameters are obtained from the lipid methylene symmetric ( cm ) and asymmetric ( cm ) stretching modes using the same equation by setting a ¼ . these equations are based on the reasonable assumption that the thickness of the deposited film (. mm) is much larger than the penetration depth (ca mm) of the evanescent wave. for x-ray reflectivity measurements, samples of varied molar peptide-to lipid ratio ðp=l ¼ ; : ; : ; : ; : ; : Þ were deposited on silicon surfaces by spreading from hfip/chloroform solvent ( . ml of mg/ml solution on wafers cut to £ mm ). the orientational distribution (mosaicity) of the highly oriented membranes was measured to be below . , as determined by a rocking scan. high resolution reflectivity scans up to vertical momentum transfer q z . a were taken at the d bending magnet beamline of the doris storage ring at hasylab/desy, at a photon energy of kev, set by a si( ) monochromator. the curves were measured with a fast scintillation counter (cyberstar, oxford instruments), using motorized collimating slits on the incident and exit arms. the curves were corrected for ring current, sample illumination, and diffuse background (offset-scan). general aspects of reflectivity experiment and analysis are discussed in ref. the samples were kept in an environmental chamber at controlled relative humidity of % and a temperature of t ¼ c to assure that the bilayers were in the fluid l a state. the electron density profile was obtained by the fourier synthesis method from the integrated peak intensities, using a lorentz correction factor =q z , and phases (þ, , , , ). the integrated intensity of the first bragg peak was normalized to for the iodinated scov e protein and for the influenza a m curve, while the non-iodinated scov e protein curve was properly scaled to the iodinated curve corresponding to the respective scattering signals. full q z -range fits to determine the density profiles on an absolute scale and analysis of all samples at different r.h.% is in progress (z.k. et al., to be published). liposomes containing the transmembrane domain of the scov e protein (residues - ) and the mhc class ii-associated invariant chain (residues - ) where prepared as described. liposomes composed of pure dmpc were prepared in an identical manner, with no addition of peptide. unilamellar vesicles where obtained by two cycles of freeze-thaw sonication ( seconds) using an inverted probe sonicator (vibra cell model vc , sonics and materials inc., ct). the liposomes where passed twice through nm polycarbonate filter using an avanti mini-extruder (avanti polar lipids inc., al). negative staining for transmission electron microscopy was done by mixing the liposome suspension at : ratio with % (v/v) phosphotungstic acid (electron microscopy sciences, hatfield, pa) and incubating for one minute at room temperature. the mixed sample was absorbed onto carbon-coated grids that had been rendered hydrophilic by glow discharge in air. excess solution was removed by filter paper absorbance. after drying, the samples were imaged with a tecnai electron microscope (philips) operating at kv. molecular modeling of the transmembrane domain of scov e protein was undertaken using the chi suite of macros for cns . . briefly, bundles of helices were generated in which the angle of the helices about their director was rotated independently in increments of . the crossing angle of the helices was set close to that found experimentally: . each structure was subjected to the following simulated annealing and energy minimization protocol as follows: steps of powell energy minimization followed by torsion angle molecular dynamics at k for ps and at k for ps. finally, an additional steps of powell energy minimization were undertaken. throughout the simulations, harmonic restraints were employed to maintain helical geometry and distance restraints were employed to maintain the helix centers within . Å one another. the resulting structures were compared, whereby clusters were defined as those that contained at least seven structures and a c a rmsd , . Å . the clusters were than averaged and subjected to the same molecular dynamics/energy minimization protocol as above. are as follows, with the accession numbers in parentheses: aibv-avian infectious bronchitis virus beaudette us (cac ) cec-canine enteric coronavirus strain insavc- (p ) prc-porcine respiratory coronavirus strain rm (p ) hcv -human coronavirus strain e (p ) bcv -bovine coronavirus strain f (p ) hcv -human coronavirus strain oc (aar ) mhv -murine hepatitis virus strain a (np_ ) rsdcv-rat sialodacryoadenitis coronavirus strain sdav- (aaf ) mhv -murine hepatitis virus strain s (p ) protein synthesis, purification and reconstitution peptides encompassing the predicted transmembrane domain of sars coronavirus e protein (figure , residues references the genome sequence of the sars-associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome the production of recombinant infectious di-particles of a murine coronavirus in the absence of helper virus nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes coronavirus pseudoparticles formed with recombinant m and e proteins induce alpha interferon synthesis by leukocytes infectious bronchitis virus e protein is targeted to the golgi complex and directs release of virus-like particles the missing link in coronavirus assembly. retention of the avian coronavirus infectious bronchitis virus envelope protein in the pre-golgi compartments and physical interaction between the envelope and membrane proteins release of coronavirus e protein in membrane vesicles from virus-infected cells and e protein-expressing cells analysis of constructed e gene mutants of mouse hepatitis virus confirms a pivotal role for e protein in coronavirus assembly characterization of the coronavirus mouse hepatitis virus strain a small membrane protein e induction of apoptosis in murine coronavirus-infected cultured cells and demonstration of e protein as an apoptosis inducer induction of apoptosis in murine coronavirus-infected cl- cells statistical analysis of predicted transmembrane alpha-helices membrane topology of coronavirus e protein fourier transform infrared techniques for probing membrane protein structure site-directed dichroism as a method for obtaining rotational and orientational constraints for oriented polymers x-ray reflectivity of solid supported, multilamellar membranes a structure for the trimeric mhc class ii-associated invariant chain transmembrane domain interhelical hydrogen bonding drives strong interactions in membrane proteins asparagine-mediated self-association of a model transmembrane helix computational searching and mutagenesis suggest a structure for the pentameric transmembrane domain of phospholamban the cytoplasmic tail of infectious bronchitis virus e protein directs golgi targeting tgev coronavirus orf encodes a membrane protein that is incorporated into virions internal reflection spectroscopy infrared dichroism and molecular conformation of a-form poly-g-benzyl-l-glutamate crystallography and nmr system: a new software suite for macromolecular structure determination identifying nonpolar transbilayer helices in amino acid sequences of membrane proteins do more complex organisms have a greater proportion of membrane proteins in their genomes? transmembrane four-helix bundle of influenza a m protein channel: structural implications from helix tilt and orientation algorithm for ribbon models of proteins this research was supported in part by a grant from the israel science foundation ( / ) to ita, a grant from the deutsche forschungsgemeinschaft grant to ita, ma and ts and by a grant from niedersachsen to ita. ita wishes to thank professors a. panet, i. ohad and dr m. kosloff for helpful discussions. key: cord- - ih c v authors: guo, jian-ping; petric, martin; campbell, william; mcgeer, patrick l title: sars corona virus peptides recognized by antibodies in the sera of convalescent cases date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: ih c v we synthesized on cellulose membranes ten-amino-acid peptides which included all of the sequences predicted for the severe acute respiratory syndrome (sars) corona virus. we probed these membranes with four pairs of acute and convalescent sera from recovered sars cases. we correlated positively reacting peptides with the in vitro sars-cov neutralizing activity of the samples. we found that convalescent sera with high neutralizing activity recognized exclusively only a limited number of peptides on the membranes. this suggests that antibodies against the epitopes represented by these peptides could be responsible for much of the sars-cov neutralizing activity. the findings have implications for monitoring humoral responses to sars-cov as well as for developing a successful sars vaccine. a newly identified virus named sars-cov has been established as the etiological agent of severe acute respiratory syndrome (sars). a total of probable cases of this highly infectious disease had been reported to who by august including deaths (http:// www.who.int/csr/sars/en/). the viral genome has been sequenced (marra et al., ; rota et al., ) . it consists of nucleotides which contain identifiable open reading frames (ay ). peptides incorporating all of the sequences predicted in the open reading frames of the sars-cov genome were prepared on derivatized cellulose membranes using a robotic peptide synthesizer (autospot asp , intavis bioanalytical instruments, lagenfeld, germany). the peptides were amino acids long and overlapped by eight residues. each peptide on a membrane was therefore shifted from the one previous by two amino acids towards the c-terminal end. the arrangement of the peptides on sets of four membranes covering the open reading frames (orfs) is illustrated in fig. . membranes were probed with pairs of acute and convalescent sera from four cases who recovered from a sars infection. as controls, serum from one case that failed to survive, and one from a healthy, nonexposed volunteer was utilized. samples were diluted -fold in buffer and then applied to a membrane for h at jc. the membranes were developed with horseradish peroxidase-conjugated goat antibodies against human igg, igm, or iga. positive spots were then identified following treatment with ecl chemiluminescence reagents. sera were tested for the presence of antibodies against sars-cov in a standard virus neutralizing test. serial fold dilutions of each serum from / to / were incubated with plaque-forming units of sars-cov (tor- isolate, marra et al., ) for h and then added to monolayers of vero e cells. the cultures were examined after h for the presence of characteristic cytopathic effects. the dilution before the one at which cytopathic effects were first noted was recorded as the antivirus titer. all sera, which showed evidence of antivirus activity, were retested following heat treatment at jc for min. this process confirmed the presence of neutralizing antibodies and eliminated the possibility of nonspecific, heat-sensitive factors contributing to the neutralizing activity. the results of neutralization assays for the various sera are shown in table , along with the age and sex of the respective patients. acute sera were collected on presentation, and convalescent sera collected month later in case , and at least weeks later in cases , , and . the acute serum of case had a neutralizing antibody titer of / , which indicates the beginning of an immune response. other acute sera as well as the acute serum of the deceased case taken days after illness onset, and the healthy control, had no detectable neutralizing activity. these data indicate that the four recovered cases developed antibodies with viral neutralizing potency between the time of acute and convalescent serum sampling. therefore, those peptides strongly recognized on membranes probed with convalescent sera, but not with acute or control sera, should be the most immunodominant and may include sars-cov epitopes that are vulnerable to neutralization by antibody. this analysis is limited to linear epitopes since peptides on the membranes do not replicate conformational epitopes or those that develop from posttranslational modifications such as glycosylation. three examples of membranes reacted with acute and convalescent sera and developed with anti-iga antibodies are shown in fig. . fig. b compares the second panel of membrane probed with serum from the unexposed control, the deceased case, convalescent serum from case , and acute and convalescent serum from case . the panel has a total of peptides encoded from the orf a open reading frame (see fig. a ). no spots were strongly immunostained except for a single triad in the two convalescent sera. the peptides in the triad have the following sequences: sddyiklngp, dyiklngplt, and iklngpvg. the sequence iklngp is common to all three. fig. c compares the third panel of membrane probed with the acute and convalescent serum of case , and the deceased case. the panel has peptides encoded from the s-protein open reading frame. one triad of spots was immunopositive, in the convalescent, but not the acute serum. the triad has the following sequences: fqpfqqfgrd, pfqqfgrdvs, and qqfgrdvsdf. the common sequence is qqfgrd. fig. d compares the fourth panel of membrane probed with acute and convalescent serum of case , and the convalescent serum of case . the bottom half of the membrane has peptides encoded for the complete nucleocapsid (n) protein open reading frame. one triad was immunopositive in the two convalescent sera but was not positive in the corresponding acute serum of case . the triad has the following sequences: qlpqgttlpk, pqgttlpkgf, and gttlpkgfya with the common sequence being gttlpk. shown in table are the overlapping membrane peptides that were recognized exclusively, or much more strongly, in multiple pairs of convalescent compared with the respective acute sera. none of these peptides was recognized by the serum from the deceased case or the healthy control. there were two overlapping sequences from orf a, one from orf b, six from n-protein, eleven from s-protein, two from m-protein, one from e protein and one from orf . the remaining open reading frames were not reactive in more than one serum sample. in the table, those peptides listed with a sequence of amino acids were identified as a combination of three adjacent spots on the membranes, whereas those listed with were identified as a combination of two adjacent spots. common sequences therefore include residues - of the -amino-acid peptides and - of the -aminoacid peptides. the note to the right of m p shows the key to the orfs with peptide totals in brackets. this membrane was spotted with dye instead of amino acids. in actual membranes, each spot is a -amino-acid peptide with adjacent spots being shifted by two amino acids. characterization of the immune response against these single case epitopes promises to provide important insights into their role in the resolution of infection. however, epitopes recognized by multiple convalescent sera may be the most important targets of neutralizing antibodies. (b -d) examples of membrane panels probed with various serum samples and developed with peroxidase-labeled goat antihuman iga; (b) m p probed with control serum, acute as well as convalescent serum from case , convalescent serum from case , and serum from the deceased case. notice the triad of spots recognized only in the serum of the two convalescent cases. the peptide sequences from orf a are sddyiklngp, dyiklngplt, and iklngpltvg. (c) p m probed with acute and chronic serum from case and serum from the deceased case. panel has peptides from s-protein. notice the triad of spots recognized only by the convalescent serum. the peptide sequences from s-protein are fqpfqqfgrd, pfqqfgrdvs, and qqfgrdvsdf. (d) p m probed with acute and convalescent serum from case and convalescent serum from case . notice the triad of spots recognized in the two convalescent sera. the peptide sequences from n-protein are qlpqgttlpk, pqgttlpkgf, and gttlpkgfya. additional peptides to those shown in table were recognized only by convalescent serum from one of the four cases. among these single case examples were peptides from orf , orf , orf , orf , and orf , indicating that the virus expresses these proteins and that antibodies against certain of their epitopes are being produced. characterization of the immune response against these single case epitopes promises to provide important insights into their role in the resolution of infection. however, epitopes recognized by multiple convalescent sera may be the most important for developing antibodies that will protect against the virus. our knowledge of the functions of the sars-cov proteins is mostly derived from analogies with those of other well-characterized corona viruses. over kb of the total genome is taken up by orf a and orf b, which are presumed to encode for proteins such as proteases and polymerases that are associated with virus replication (marra et al., ; ruan et al., ) . the genome is sufficiently unique to be proposed as belonging to a separate group designated as group iv (marra et al., ; ruan et al., ) , but this view is not consensual and it has been noted that sars-cov is most closely related to group coronaviruses (snijder et al., ) . nevertheless, the structural proteins s, m, n, and e have been clearly identified in virus grown in infected cells (marra et al., ; ruan et al., ) . the spike, or s-protein, protrudes from the outer surface and is a strong candidate for facilitating viral entry into host cells. li et al. ( ) have reported that the sars-cov sprotein efficiently binds to angiotensin converting enzyme of sars-cov permissive vero e cells, suggesting that this binding is a key step in sars-cov infectivity. interaction between the s-protein of a corona virus and specific mammalian receptors can determine species sensitivity. for example, when the s-protein ectodomain of mouse corona hepatitis virus was replaced with that of the feline infectious peritonitis virus, the viral particles were no longer infective for mouse cells, but became infective for feline cells (kuo et al., ) . sars-cov has been reported to infect domestic cats and ferrets (martina et al., ) so these relationships may have relevance to this broader species susceptibility. a further example of interaction between the s-protein and mammalian receptors involves cd or aminopeptidase n. it is the main mammalian receptor for the s-protein of many coronaviruses. substitution on human cells of the human cd n-glycosylation site with the porcine n-glycosylation site resulted in a failure to bind human corona virus- e (wentworth and holmes, ) . antibodies directed against the epitopes of s-protein identified in table are therefore potential candidates for neutralizing activity. antibodies against m-protein and eprotein epitopes could also be important although they are smaller and are believed to be less exposed. the protein encoded by orf may be displayed on the viral surface and an immune response to it may contribute to the resolution of disease. antibodies against orf a, orf b, and n-protein probably are unlikely to have contributed to the in vitro neutralization results. n-protein is internal and is probably inaccessible in the brief exposure time of the neutralization test, and the products of orf a and orf b are not believed to be structural proteins. nevertheless, they may be very important to in vivo protection. given the complexity of antibodies produced in each convalescent serum, it would be difficult to predict what the relative contribution of each to the overall neutralizing activity might be, particularly when antibodies against conformational or carbohydrate-dependent epitopes might be present. nevertheless, high titers of those consistently represented in the various convalescent sera might be sufficient to confer immunity. numerous strategies have been suggested for producing a sars vaccine (degroot, ) . one possibility is to use a combination of peptides, such as those listed in table , as a sequences with amino acids represent three adjacent peptides with a common sequence from residues to . sequences with amino acids represent two adjacent peptides with a common sequence from residues to . the amino acid positions (aa) in sars-cov open reading frames are taken from blast at ncbi: http://www.ncbi.nlm.nih.gov/blast/blast.cgi. polyvalent antigen. peptide-based vaccines have heretofore had only limited success but this can be attributed to a lack of knowledge as to which peptides to use. such uncertainty is reduced by analyzing the antibody pattern in sera from cases that have successfully resolved the infection. peptides making up the antigen can be screened in advance for homologies to mammalian proteins, reducing the possibility of self-attack. they can also be easily modified to cope with viral mutations that could escape traditional vaccines. significant mutations have already been identified in sars-cov (ruan et al., ; martina et al., ) , indicating the necessity of developing adaptable vaccines. polypeptide vaccines should be safe, highly stable, and easy to administer. antibodies cloned against particularly sensitive peptides could be used as therapeutic agents. the type of analysis described here may have general utility in designing peptide vaccines and therapeutic antibodies against other infectious agents such as hiv. cellulose-bound overlapping peptides ( mers) derived from all proteins encoded by the orfs of the sars genome were synthesized using an autospot robot provided by intavis bioanalytical instruments. the software used for designing the arrays was from digen, jerini biotools gmbh, berlin, germany. membranes derivatized with a polyethylene (peg) linker and a free amino terminal, as well as -fluorenylmethyloxycarbonyl (fmoc) amino acids were obtained from intavis bioanalytical instruments. the fmoc amino acids ( . m) were dissolved in dimethylformamide (dmf, sigma, oakville, ont. canada) and then activated with -hydroxybenzotriazole (hobt, sigma) and n, n v -disopropylcarbodiimide (dipc, sigma) for at least min. they were then delivered to the membrane in -nl aliquots per spot by the robotic synthesizer. fifteen minutes after completion of each cycle, the membranes were removed from the apparatus and treated with % acetic anhydride in dmf to acetylate any free remaining amino groups. they were then washed and further treated with % piperidine in dmf to remove the fmoc protecting group. this was followed by washing with dmf and methanol, and finally by air drying. the membrane was then precisely repositioned on the robotic apparatus to initiate the next coupling cycle. after the final cycle, the side chain protecting groups were removed by treatment with a solution of the following composition: ml of % trifluoracetic acid; ml dichloromethane (dcm); al triisopropyl silane (sigma); and al water. membranes were then washed twice with dcm, twice with dmf, and twice with methanol. after air drying, membranes were stored in a sealed bag at jc until used. membranes were rehydrated by treating first with methanol, then % methanol followed by washing three times in mm tris-buffered saline/ . % tween (tbs-t). they were then blocked with % skim milk in tbs-t overnight at jc. after washing three times with tbs-t, the membranes were incubated at jc for h with / dilutions of serum samples of patients and controls. after washing, membranes were treated with / dilutions ( . ag/ml) of goat antihuman iga, igg and igm antibodies conjugated to horseradish peroxidase (sigma). after washing in tbs-t, membranes were developed with an ecl chemiluminescence kit provided by amersham pharmacia biotech (buckinghamshire, england). four milliliters of the two ecl western blotting detection reagents were mixed before being applied on the membranes. the membranes were scanned using a bio-rad fluorescent imager (hercules, ca) and saved for later analysis. the membranes were regenerated by incubation in m urea/ % sds overnight followed by min in % ethanol/ % acetic acid. after this step, membranes were washed three times in methanol, and after air drying stored at jc in a sealed bag until reuse. in initial development of the sars-cov serum neutralization test, comparisons were made between the microneutralization test and a % plaque reduction assay. testing of several sera consistently produced assay results where the % plaque reduction titer was -fold greater than in the microneutralization test. following this validation, the microneutralization test system was adopted for greater convenience despite the -fold greater sensitivity of the plaque reduction assay. typical results of this assay are illustrated in fig. . how the sars vaccine effort can learn from hivspeeding towards the future, learning from the past retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier angiotensin-converting enzyme is a functional receptor for the sars coronavirus sars virus infection of cats and ferrets characterization of a novel coronavirus associated with severe acute respiratory syndrome comparative full-length genome sequence analysis of sars coronavirus isolates and common mutations associated with putative origins of infection unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group lineage molecular determinants of species specificity in the coronavirus receptor aminopeptidase n (cd ): influence of n-linked glycosylation the authors are grateful to drs. a mcgeer and d. skowronski for help in obtaining specimens and to dr. k karunakarum for initial assistance in processing the membranes. we are also grateful to dr. mitoshi kunimatsu of nagoya city university for help in programming the robotic synthesizer. this research was supported by a cihr grant on which m.p. is a co-investigator, a grant to p.l.m. from the province of british columbia via the sars accelerated vaccine initiative (savi), and support from individual british columbians to the kinsmen laboratory of neurological research. key: cord- - bik authors: hillisch, alexander; pineda, luis felipe; hilgenfeld, rolf title: utility of homology models in the drug discovery process date: - - journal: drug discovery today doi: . /s - ( ) - sha: doc_id: cord_uid: bik abstract advances in bioinformatics and protein modeling algorithms, in addition to the enormous increase in experimental protein structure information, have aided in the generation of databases that comprise homology models of a significant portion of known genomic protein sequences. currently, d structure information can be generated for up to % of all known proteins. however, there is considerable controversy concerning the real value of homology models for drug design. this review provides an overview of the latest developments in this area and includes selected examples of successful applications of the homology modeling technique to pharmaceutically relevant questions. in addition, the strengths and limitations of the application of homology models during all phases of the drug discovery process are discussed. - / /$ -see front matter © elsevier ltd. all rights reserved. pii: s - ( ) - the majority of drugs available today were discovered either from chance observations or from the screening of synthetic or natural product libraries. the chemical modification of lead compounds, on a trial-and-error basis, typically led to compounds with improved potency, selectivity and bioavailability and reduced toxicity. however, this approach is labor-and time-intensive and researchers in the pharmaceutical industry are constantly developing methods with a view to increasing the efficiency of the drug discovery process [ ] . two directions have evolved from these efforts. the 'random' approach involves the development of hts assays and the testing of a large number of compounds. combinatorial chemistry is used to satisfy the need for extensive compound libraries. the 'rational', protein structure-based approach relies on an iterative procedure of the initial determination of the structure of the target protein, followed by the prediction of hypothetical ligands for the target protein from molecular modeling and the subsequent chemical synthesis and biological testing of specific compounds (the structure-based drug design cycle). the rational approach is severely limited to target proteins that are amenable to structure determination. although the protein data bank (pdb; http://www.rcsb.org/pdb) is growing rapidly (~ new entries daily), the d structure of only - % of all known proteins has as yet been experimentally characterized. however, advances in sequence comparison, fold recognition and protein-modeling algorithms have enabled the partial closure of the so-called 'sequence-structure gap' and the extension of experimental protein structure information to homologous proteins. the quality of these homology models, and thus their applicability to, for example, drug discovery, predominantly depends on the sequence similarity between the protein of known structure (template) and the protein to be modeled (target). despite the numerous uncertainties that are associated with homology modeling, recent research has shown that this approach can be used to significant advantage in the identification and validation of drug targets, as well as for the identification and optimization of lead compounds. in this review, we will focus on the application of homology models to the drug discovery process. homology, or comparative, modeling uses experimentally determined protein structures to predict the conformation of another protein that has a similar amino acid sequence. the method relies on the observation that in nature the structural conformation of a protein is more highly conserved than its amino acid sequence and that small or medium changes in sequence typically result in only small changes in the d structure [ ] . generally, the process of homology modeling involves four steps -fold assignment, sequence alignment, model building and model refinement ( figure ). the fold assignment process identifies proteins of known d structure (template structures) that are related to the polypeptide sequence of unknown structure (the target sequence; this is not to be mistaken with drug target). next, a sequence database of proteins with known structures (e.g. the pdb-sequence database) is searched with the target sequence using sequence similarity search algorithms or threading techniques [ ] . following identification of a distinct correlation between the target protein and a protein of known d structure, the two protein sequences are aligned to identify the optimum correlation between the residues in the template and target sequences. the next stage in the homology modeling process is the model-building phase. here, a model of the target protein is constructed from the substitution of amino acids in the d structure of the template protein and the insertion and/or deletion of amino acids according to the sequence alignment. finally, the constructed model is checked with regard to conformational aspects and is corrected or energy minimized using force-field approaches. several improvements and modifications of this general homology modeling strategy have been developed and applied to the prediction of protein structures. to subject the available structure prediction methods to a blind test, community-wide experiments on the critical assessment of techniques for protein structure prediction (casp - ) have been performed and their results presented and published. as a result, the current state-of-the-art in protein structure prediction has been established, the progress made has been documented and the areas where future efforts might be most productively concentrated have been highlighted [ , ] . homology modeling techniques are dependent on the availability of high-resolution experimental protein structure data. the development of effective protein expression systems and major technological advances in the instrumentation used for structure determination (x-ray crystallography and nmr spectroscopy) has contributed to an exponential growth in the number of experimental protein d structures. by may , the pdb contained ~ , experimental protein structures for ~ different proteins (proteins with less than % sequence identity). a recent analysis of all protein chains in the pdb shows that these proteins can be grouped into protein families ddt vol. , no. august reviews research focus www.drugdiscoverytoday.com figure . the steps involved in the prediction of protein structure by homology modeling. structure modeling of the bacterial transcriptional repressor copr is shown [ ] . although the model is based on a low-sequence identity of only . % between copr and the p c repressor, several experimental methods support this homology model. reproduced, with permission, from ref. [ ] . abbreviation: copr, plasmid copy control protein. target sequence: homolog , no d structure: homolog , no d structure: template , d structure: template , d structure: comprising unique protein folds [ ] (updates can be found at http://scop.mrc-lmb.cam.ac.uk). the majority of the structures in the pdb ( %) were determined by x-ray crystallography, with % of the structures being characterized by nmr spectroscopy. the pdb database encompasses experimental information on an extensive array of ligands (small organic molecules and ions) bound to more than , different binding sites that can be analyzed using programs including relibase (http://relibase.ebi.ac.uk) [ ] , ligbase (http://alto.compbio.ucsf.edu/ligbase) [ ] and pdbsum (http://www.biochem.ucl.ac.uk/bsm/pdbsum) [ ] . although the experimental structure database is growing rapidly, there is still a substantial gap between the number of known annotated sequences [ , , unique sequences in swiss-prot-trembl (http://www.expasy.org/ sprot) as of august ] and known protein d structures ( , ). if only significantly different proteins are considered (~ ), which omits muteins, artificial proteins and multiple structure determinations of the same proteins (e.g. hiv-protease and carbonic anhydrase ii), then less than % of the d structures of known protein sequences have been elucidated. this sequence-structure gap can partly be filled with homology models. for example, the queryable database modbase (http://alto.compbio. ucsf.edu/modbase-cgi/index.cgi) provides access to an enormous number of annotated comparative protein structure models [ ] . the program psi-blast was used to assign protein folds to all , , unique sequence entries in swiss-prot-trembl. for % of these sequences, comparative models with an average model size of amino acids could be built using the program modeller [ ] . thus, by august , , d structure models of proteins were accessible via the internet. the models are predicted to have at least % of their c α atoms superimposed within . Å of their correct positions. information on binding sites and ligands can be retrieved from this database using ligbase [ ] . however, the majority of the models are built on a low sequence identity and it should be realized that this level of accuracy is, in most cases, not sufficient for a detailed structure-based ligand design. the swiss-model repository (http://swissmodel.expasy. org/repository) [ ] is also a database of annotated comparative protein d structure models, which have been generated using the fully automated homology-modeling pipeline swiss-model. as of august , this database contained models for , different protein sequence entries ( %) from the swiss-prot-trembl databases ( , , sequences), with an average model size of ~ amino acids. researchers from eidogen (http://www.eidogen.com) have created a database system called target informatics platform™ [ ] that currently includes homology models for , proteins. homology modeling of , human protein sequences resulted in the construction of , models for , different sequences ( %). thus, putative and known ligand binding pockets can be detected, analyzed and compared and the resulting data used to support target prioritization and lead discovery and/or optimization procedures. accelrys (http://www.accelrys.com) produces discovery studio (ds) atlasstore™ as a complete oracle ® -based protein and ligand structural data management solution. currently, ds atlasstore™ contains , , homology models that have been automatically generated from the sequences of , proteins from different genomes. in conjunction with homology models, cengent therapeutics (http://www.cengent.com) offers dynamic structural information generated from molecular dynamics simulations for human drug target proteins. this structural information can be used for target prioritization and virtual screening. the quality of the homology models is dependent on the level of sequence identity between the protein of known structure and the protein to be modeled [ ] . for a sequence identity that is greater than %, homology can be assumed; the two proteins probably have a common ancestor and are, therefore, evolutionarily related and are likely to share a common d structure. in this case, pairwise and multiple sequence alignment algorithms are reliable and can be used for the generation of homology models ( figure ). if the sequence identity is below %, structure modeling becomes speculative, which could lead to misleading conclusions. when the sequence identity is between % and %, conventional alignment methods are not sufficiently reliable and only sophisticated, profile-based methods are capable of recognizing homology and predicting fold. for regions of low sequence identity, threading methods [ ] are often applied. protein models that are built on such low sequence identities can be used for the assignment of protein function and for the direction of mutagenesis experiments ( figure ). models that have a sequence identity between ~ % and % could facilitate the structure-based prediction of target drugability, the design of mutagenesis experiments and the design of in vitro test assays ( figure ). if sequence identity is greater than ~ %, the resulting models are frequently of sufficient quality to be used in the prediction of detailed protein-ligand interactions, such as structure-based drug design and prediction of the preferred sites of metabolism of small molecules ( figure ). there are numerous applications for protein structure information and, hence, homology models at various stages of the drug discovery process [ ] . the most spectacular successes are clearly those where protein structural information has helped to identify or to optimize compounds that were subsequently progressed to clinical trials or to the drug market [ ] . the applications of homology models that had an impact on target identification and/or validation, lead identification and lead optimization are reviewed here ( figure ). it is clear that only a minute fraction of the entire proteome can be affected by drug-like (preferentially orally bioavailable) small molecules. based on the total numbers of known genes, disease-modifying genes and drugable proteins, the number of drug target proteins, for humans, has been estimated at - [ ] . for small molecules, sets of properties have been established that differentiate drugs from other compounds [ , ] ; these properties can be used to identify compounds with, for example, poor oral absorption properties [ ] . drug molecules and their corresponding target proteins are highly complementary, which suggests that some rules that distinguish good target proteins from others should be deducible [ ] . deep lipophilic pockets that comprise distinct polar interaction sites are clearly superior to shallow highly charged protein surface regions. the inhibition of protein-protein interfaces as a valuable therapeutic principle has recently been shown with inhibitors of the p -murine double minute clone (mdm ) interaction [ , ] . the binding site for these inhibitors is a distinct lipophilic pocket that normally interacts with the α-helical surface patches of the p tumor suppressor transactivation domain. advances in the rapid detection, description and analysis of ligand-binding pockets [ ] [ ] [ ] , together with the availability of more than . million homology models, will open new possibilities for the prioritization of proteins with regards to drugability. in the pharmaceutical industry, structural aspects are being increasingly implemented as additional decision criteria on the drugability of potential drug targets. companies such as inpharmatica (http:// www.inpharmatica.com) have developed an integrated suite of informatics-based discovery technologies that contain software tools for the structure-based assessment of target drugability. the design of site-directed mutant proteins is one further important option for the application of homology models to target validation. introducing point mutations and subsequently studying the effects in vitro or in vivo is a common approach in molecular biology. this strategy enables the identification of amino acids that are functionally or relationship between target and template sequence identity and the information content of resulting homology models. arrows indicate the methods that can be used to detect sequence similarity between target and template sequences. applications of the homology models in drug discovery are listed to the right. the higher the sequence identity, the more accurate the resulting structure information. homology models that are built on sequence identities above ~ % can frequently be used for drug design purposes. superimpositions of x-ray crystal structures of the ligand-binding domains of members of the nuclear receptor family are shown to the left. these x-ray structures illustrate the increase in structure deviation with a decreased sequence identity. the pr is red, the gr is green, the erα is blue and the trβ is cyan. sequence identities: pr:gr, %; pr:erα, %; and pr:trβ, %. abbreviations: erα, estrogen receptor α; gr, glucocorticoid receptor; pr, progesterone receptor; trβ, thyroid receptor β. structurally important in the protein under investigation, which ultimately contributes to biological knowledge on, for example, potential target proteins. typically, the amino acids that are to be modified in these studies are selected on the basis of sequence alignments by focusing on conserved residues. however, if at least some structure information is available, the selection of the amino acids that are to be mutated can be much more precise and successful [ ] . this approach is even more powerful when applied in conjunction with pharmacologically active compounds. site-directed mutants of the target protein can be made to render that target sensitive to an existing pharmacological agent. based on homology models, some members of the mitogen-activated protein (map) kinase family were mutated to make them sensitive to a kinase inhibitor from the pyridinyl imidazole class [ ] . this enabled the use of the compound for broader target validation studies. one of the most attractive ways to validate a target protein is to administer a pharmacologically active compound that selectively acts on that protein and to study the effects in a relevant animal model. similar strategies have been described under the term 'chemogenomics' [ ] . it has recently been shown that it is possible to design small molecules based on homology models and then to use these compounds as tools to study the physiological role of the respective target protein of that particular drug [ ] . eight years after the discovery of estrogen receptor β (erβ), the distinct roles of the two er isotypes, erα and erβ, in mediating the physiological responses to estrogens are not completely understood. although knockout animal experiments have provided an insight into estrogen signaling, additional information on the function of erα and erβ was imparted by the application of isotype selective er agonists. based on the crystal structure of the erαligand-binding domain (lbd) and a homology model of the erβ-lbd ( % sequence identity to erα), hillisch et al. [ ] designed steroidal ligands that exploit the differences in size and flexibility of the two ligand-binding cavities ( figure ). compounds that were predicted to bind preferentially to either erα or erβ were synthesized and tested in vitro. this approach led directly to highly er isotype-selective ( - -fold) ligands that were also highly potent. to unravel the physiological roles of each of the two receptors, in vivo experiments with rats were conducted using the erα-and erβ-selective agonists in parallel with the natural ligand of er, β-estradiol. the erα agonist was shown to be responsible for most of the known estrogenic effects (e.g. induction of uterine growth and bone-protection), in addition to pituitary (e.g. reduction of luteinizing hormone plasma levels) and liver (e.g. increase in angiotensin i plasma levels) effects [ ] . however, the erβ agonist had distinct effects on the ovary, for example, the stimulation of early folliculogenesis [ ] , which possibly presents clinicians with a new option for tailoring classical ovarian stimulation protocols. a comparison of the homology model with the x-ray crystal structure of the erβ-lbd complexed with genistein [ ] revealed that the homology model had a root-mean-square deviation (rmsd) of the backbone atoms (not considering helix ) of . Å. the x-ray crystal structure confirmed the presence of essential interactions between the ligand and the erβ and did not reveal, at least in this case, any new aspects for the design of erβ agonists that were not covered by the homology model. these studies show that it is possible to design highly selective compounds, if structure information on all of the relevant homologs of the target is available, and ddt vol. , no. august reviews research focus www.drugdiscoverytoday.com figure . applications of homology models in the drug discovery process. the enormous amount of protein structure information currently available could not only support lead compound identification and optimization, but could also contribute to target identification and validation. reproduced, with permission, from [ ] . there are numerous examples where protein homology models have supported the discovery and the optimization of lead compounds with respect to potency and selectivity. currently, the structures of of the known different human protein kinases have been characterized by x-ray crystallography [ ] . homology model-based drug design has been applied to epidermal growth factor-receptor tyrosine kinase protein [ , ] , bruton's tyrosine kinase [ ] , janus kinase [ ] and human aurora and kinases [ ] . using the x-ray crystal structure of cyclin-dependent kinase (cdk ), honma et al. [ ] generated a homology model of cdk . this model guided the design of highly potent and selective cdk inhibitors that were targeted towards the atp binding pocket. the diarylurea class of compounds were subsequently synthesized and tested. in an in vitro inhibition assay, the most potent compound had an ic of nm. the predicted binding mode of the lead compound was verified by co-crystallization with cdk [ ] . vangrevelinghe et al. [ ] identified a cdk inhibitor using a homology model of the protein and highthroughput docking. siedlecki et al. [ ] have demonstrated the utility of homology modeling in the prediction of pharmacologically active compounds. alterations in dna methylation patterns play an important role in tumorigenesis; therefore, inhibitors of dna methyltransferase (dnmt ), which is the protein that represents the major dna methyltransferase activity in human cells, are desired. known inhibitors from the -azacytidine class were docked into the active site of a dnmt homology model, which led to the design of n -fluoroacetyl- -azacytidine derivatives that acted as highly potent inhibitors of dna methylation in vitro. thrombin-activatable fibrinolysis inhibitor (tafi) is an important regulator of fibrinolysis, and inhibitors of this enzyme have potential use in antithrombotic and thrombolytic therapy. based on a homology model of tafi (~ % sequence identity to carboxypeptidases a and b), appropriately substituted imidazole acetic acids were designed and were subsequently found to be potent and selective inhibitors of activated tafi [ ] . homology models of the voltage-gated k + -channel k v . and the ca + -activated channel ik ca were used to design selective ik ca inhibitors that were based on the polypeptide toxin charybdotoxin. comparison of the two models revealed a unique cluster of negatively charged residues in the turret of k v . that were not present in ik ca . to exploit this difference, the homology model was used to design novel analogs, which were then synthesized and tested. research demonstrated that the novel compounds blocked ik ca activity with ~ -fold higher affinity than k v . [ ] . . (a,b) comparison of the two isotypes of the estrogen receptor. in the homology model, erα (blue) and erβ (green) ligand-binding pockets are shown in complex with the natural ligand of the er, β-estradiol. the binding of β-ve , a highly potent and selective erβ agonist, modeled into the erβ ligand-binding niche is depicted to the right. reproduced, with permission, from ref. [ ] . (c,d) a model of the antiprogestin ru (mifepristone) bound to hpr. a single amino acid mutation renders this compound inactive at the cpr and hamster pr. steric clashes between ru and cpr are shown on the right side. abbreviations: er, estrogen receptor; her, human estrogen receptor; cpr, chicken progesterone receptor; hpr, human progesterone receptor; pr, progesterone receptor; rba, relative binding affinity; β-ve , β-vinylestra- , , ( )-triene- , β-diol. the key proteinase (m pro , or cl pro ) of the new coronavirus (cov) that caused the severe acute respiratory syndrome (sars) outbreak of (sars-cov) is another example of successful homology model building; in this case, success is defined as the ability to use the model to propose an inhibitor that has significant affinity for the target enzyme. x-ray crystal structures for the m pro s of transmissible gastroenteritis virus (tgev, a porcine coronavirus) and of human coronavirus e [ , ] have been characterized. these proteinases have and % sequence identity, respectively, with the key proteinase of sars-cov. following publication of the genome sequence of the new virus, first on the internet and a few weeks later in print [ , ] , the level of sequence identity between the proteinases enabled anand et al. [ ] to construct a d homology model for the m pro of human cov. however, the d homology model generated was insufficient for the design of inhibitors with reasonable confidence. to establish the structural basis of the interaction with the polypeptide substrate of the m pro , anand and co-workers [ ] synthesized a substrate-analogous hexapeptidyl chloromethylketone inhibitor that was complexed with tgev m pro . the x-ray crystal structure of the complex was then determined, which revealed that, as expected, the chloromethylketone moiety had covalently reacted with the active-site cysteine residue of the proteinase. the p , p , and p side chains of the inhibitor had bound to, and thereby defined, the specificity binding sites of the target enzyme. the experimentally determined structure of the inhibitor-tgev m pro complex was then compared with all inhibitor complexes of cysteine proteinases in the pdb, which revealed a surprisingly similar inhibitor binding mode in the complex of human rhinovirus type (hrv ) c proteinase with ag ( figure ) [ ] . at that time, ag was in late phase ii clinical trials as a drug for the treatment of the strain of the common cold that is caused by human rhinovirus. the comparison of the crystal structures of hrv in complex with ag and tgev m pro in complex with the hexapeptidyl chloromethylketone inhibitor revealed little similarity between the two target enzymes, except in the immediate neighborhood of the catalytic cysteine residue, but an almost perfect match of the inhibitors. to investigate these findings further, ag was docked into the substrate-binding site of the sars-cov m pro model without much difficulty, although it was noted that there could potentially be steric problems with the p-fluorobenzyl group in the s pocket, and also with the ethylester moiety in s ′. therefore, it was proposed that, although ag was not an ideal inhibitor, this compound should be a good starting point for the design of anti-sars drugs. indeed, only a few days after the on-line publication of these results in sciencexpress [ ] , it was confirmed that ag had anti-sars activity in vitro. derivatives of ag with modified p residues have since been shown to have k i values in the lower µmolar range (rao et al., pers. commun.) . the crystal structure of the authentic sars-cov key proteinase was determined a few months later [ ] . although the dimeric structure showed the expected similarity to the homologous enzymes of tgev and human cov e, there were interesting differences in detail. in particular, one of the monomers in the dimer was observed to be in an inactive conformation, which was thought to be the result of the low ph of crystallization. the overall rmsd for the entire dimer from the homology model of anand et al. [ ] was > . Å (i.e. no residues excluded from the comparison), which dropped to . Å when a few outliers at the carboxy terminus were excluded from the comparison, and to < . Å for each of the three individual domains of the enzyme. other homology models were generated (d. debe, unpublished and [ ] ) and virtual screening has been performed using a sars-cov m pro model [ ] . taken together, these findings confirm that homology modeling is often inadequate for the prediction of the mutual orientation of domains in multidomain proteins. however, the homology model generated by anand et al. [ ] also shows that a reasonable model of a substrate-binding site can serve to develop useful ideas for inhibitor design that can inspire medicinal chemists to start a synthesis program long before the d structure of the target enzyme is experimentally determined. in the case of g-protein-coupled receptors (gpcr), homology-modeling approaches are limited by the lack of experimentally determined structures and the low sequence similarity of those structures that have been characterized with respect to pharmacologically important target proteins. the x-ray crystal structure of only one gpcr, bovine rhodopsin, has been determined [ ] . this structure is complemented by bacteriorhodopsin, which is a transmembrane protein that comprises seven helices and is also of relevance for modeling approaches, even though this protein is not a gpcr. some examples of homology models for gpcrs and their utility have recently been reviewed [ ] . high-throughput docking has been applied to verify the ability of homology models to identify agonists (glucocorticoid receptor agonists) [ ] , antagonists of retinoic acid receptor α [ ] , d -dopamine-, m -muscarinic acetylcholine-and v a -vasopressin-receptors [ ] and inhibitors of thrombin [ ] . in the identification of thrombin inhibitors, homology models of thrombin were built retrospectively and were based on homologous serine proteases ( %- % sequence identity); the best docking solutions were yielded with those models that were derived from proteins of higher sequence identity. recently, the performance of docking studies into protein active sites that had been constructed from homology models was assessed using experimental screening datasets of cdk and factor viia [ ] . when the sequence identity between the model and the template near the binding site was greater than ~ %, there was an approximate fivefold increase in the number of active compounds identified than would have been be detected randomly. this performance is comparable to docking to crystal structures. a further application of homology models is the design of test assays for the in vitro pharmacological characterization of compounds or hts. based on the structure of the coiledcoil domain of c-jun, models for α-helical proteins were designed such that they can be used as affinity-tagged proteins that incorporate protease cleavage sites [ ] . the resulting . kda recombinant proteins were synthesized and used as molecularly defined and uniform substrates for in vitro detection of hiv- and iga endoprotease activity, which enabled the surface plasmon resonance-based screening of inhibitors. the enormous volume of structure information on entire target protein families that is available might also have an impact on screening cascades. many drug discovery projects endeavor to identify ligands that are highly selective for particular drug targets. selective compounds are supposed to be superior because such compounds typically lead to fewer adverse side effects (e.g. cox- inhibitors). however, the most important homologs that should not be targeted by the desired drug, with respect to the actual target, are not always clear, particularly within the larger target protein families. the sequence similarity of the full-length proteins or entire domains might not always be representative of the target protein when considering the conservation of the ligand-binding pockets. comparison of the shape and features of the binding pockets within a protein family could indicate which homologs should be included in the screening cascade for so-called 'counter screening'. the structure information that is currently available on entire protein families (e.g. proteases, kinases and nuclear receptors) could contribute to the design of selective compounds or better screening cascades, both of which could potentially advance the design of drugs that have fewer side effects. a detailed structural knowledge of the ligand-binding sites of target proteins was also shown to facilitate the selection of animal models for ex vivo or in vivo experiments. the proposal is that animals having target proteins with significantly different binding sites compared with human orthologs should be excluded as pharmacological models. many promising compounds showing high-potency in human in vitro assays have not reached clinical trials because efficacy could not be demonstrated in animal models. single amino acid differences between humans and animals might, in some cases, be sufficient to cause such effects. the er selectivity of ligands described by hillisch et al. [ ] was shown by homology models and in vitro assays to be crucially dependent on the interaction of ligand substituents with one particular amino acid that differs between erα and erβ (figure a ) [ ] . to ensure that this important interaction is present in estrogen receptors of all animal models that are used to characterize compounds [ ] , homology models of murine, rat and bovine erβ were built and compared with the binding pocket of human erβ (herβ). a complete conservation of amino acids within the binding pockets of human, murine and rat erβ was observed. however, bovine erβ showed one amino acid difference at the exact position that was determined to be crucial for erβ ligand selectivity. the prediction that the herβ selective compounds should not bind to bovine erβ was later verified using transactivation experiments (unpublished results). thus, the implementation of uninterpretable experiments could be avoided at an early stage and the otherwise attractive bovine tissues (later available in larger amounts) could be excluded from ex vivo investigations. similarly, information on the structure of progesterone receptors (pr) can be used to explain the abolished binding of the progesterone antagonist mifepristone (ru ) to chicken pr and hamster pr [ ] . a single point mutation (human pr gly to chicken pr cys ) prevents antiprogestins containing β-aryl substituents (e.g. ru ) from binding to chicken (and hamster) pr (figure c) , which therefore excludes hamsters, for example, from pharmacological studies with antiprogestins [ ] . in the future, such effects could be predicted and particular species could then be excluded from pharmacological studies at an early stage, which would ultimately reduce attrition rates in the drug discovery process. one of the challenges in lead optimization is to identify compounds that not only show a high potency at the desired target protein but also have adequate physical properties to reach systemic circulation, to resist metabolic inactivation for a specific time period and to avoid undesired pharmacological effects. knowledge of the structure of the proteins that are involved in these processes, such as drugmetabolizing enzymes, transcription factors or transporters, could help to design molecules that do not interact with these 'non-target' proteins. the cytochrome p s (cyp) are an extremely important class of enzymes that are involved in phase i oxidative metabolism of structurally diverse chemicals. only ~ hepatic cyps are responsible for the metabolism of % of known drugs. recently, the x-ray crystal structures of three mammalian cyps, cyp c [ ] , cyp c [ ] and cyp c [ ] , have been solved and represent a solid basis for the homology modeling of this entire superfamily. models of cyp a , cyp a , cyp b , cyp c , cyp c , cyp c , cyp d , cyp e , cyp a and cyp a have been generated using different structure templates. these models have been used to explain and to predict the probable sites of metabolic attack in a variety of cyp substrates [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, the large lipophilic and highly flexible character of some cyp binding cavities renders pure in silico approaches towards the prediction of the occurrence and site of small molecule metabolism extremely difficult. if protein structure information is combined with pharmacophoric patterns and quantum mechanical calculations, some predictions concerning the preferred sites of metabolism within small molecules are possible [ ] . regarding this aspect of homology modeling, cyp d is a particularly interesting cyp because - % of the caucasian population does not produce this polymorphic member of the cyp superfamily. the resulting deficiencies in drug oxidation can lead to severe side effects in these individuals. predictions on whether or not a lead compound could act as a cyp d substrate could help to identify problematic cases early in drug discovery. combined homology modeling and quantitative sar approaches are able to predict such cyp inhibitors [ ] . thus, in the future, protein structure information in conjunction with high-throughput docking and pharmacophore-based methods could be used to decide which compounds have the potential to inhibit particular cyps. this approach could facilitate the detection of potential drug-drug interactions early in the drug discovery process and measures could then be taken to avoid such interactions [ ] . cyp substrates and inhibitors are not the only compounds to have been studied using homology models. these approaches have been used recently to investigate cyp inducers. the induction of cyps is primarily mediated via the activation of ligand-dependent transcription factors, such as the aryl hydrocarbon receptor (ahr) for the cyp a family, the constitutive androstane receptor (car) for the cyp d family and the pregnane x receptor (pxr), glucocorticoid receptor (gr) and vitamin d receptor (vdr) for the cyp a family [ ] . in principle, the in silico prediction of drug-metabolizing enzyme induction could be reduced to predicting the binding and activation of transcription factors (e.g. ahr and car). however, recent xray structure analyses of pxr have shown that the lbd of this nuclear receptor contains a large lipophilic and flexible binding pocket [ ] . this renders pure in silico structure-based predictions concerning whether or not a small molecule will activate pxr difficult. the homology modeling of car [ , ] and other members of the nuclear receptor family involved in cyp induction [ ] have recently been described. these models predict reasonably shaped potential ligand binding pockets. however, further results on the utility of these models are needed. with respect to the structure-based prediction of adverse health effects, progress has been described with the human ether-a-go-go-related gene (herg). this tetrameric potassium channel contributes to phase three repolarization of heart muscle cells by opposing the depolarizing ca + influx during the plateau phase. inhibition of this protein results in cardiovascular toxicity (qt-prolongation) and has caused several drugs to be withdrawn from the market. therefore, in silico predictions on the probability of the formation of an interaction between a drug and herg have gained enormous attention and have recently been reviewed [ ] . homology models of herg, which are based on the x-ray crystal structures of the bacterial kcsa [ ] and mthk channels [ ] , have already shed light on some details of the molecular interactions that initiate herg inhibition. however, the complexity of this potassium channel signifies that detailed x-ray structure analyses of the protein in the open-and closed-state are required before these molecular interactions can be fully understood and predicted, which has implications for the prediction of cardiotoxicity. numerous examples for the successful application of homology modeling in drug discovery are described here. in the absence of experimental structures of drug target proteins, homology models have supported the design of several potent pharmacological agents. one of the advantages of homology models is that these models can be generated ddt vol. , no. august reviews research focus relatively easily and quickly. furthermore, such models could support the hypotheses of medicinal chemists on how to generate biologically active compounds in the important early conceptual phase of a drug discovery project. the design of compounds that are selectively directed at particular drug target proteins is one of the strengths of this technique. such selective compounds can even be applied to gain insights into the physiological role of novel drug targets. the in silico protein structure-based prediction of metabolism and toxicity of small molecules, particularly cyp inhibition and induction and herg inhibition, is currently in its infancy and predictive capabilities could be limited to classification only. however, while complete experimental structures of pharmacologically important proteins are missing, the homology modeling technique provides one approach to bridge the gap until this information becomes available. modern methods of drug discovery: an introduction the response of protein structures to amino-acid sequence changes fold recognition methods progress in protein structure prediction assessment of homology-based predictions in casp scop: a structural classification of proteins database for the investigation of sequences and structures databases for protein-ligand complexes ligbase: a database of families of aligned ligand binding sites in known protein sequences and structures pdbsum: summaries and analyses of pdb structures modbase, a database of annotated comparative protein structure models, and associated resources comparative protein modelling by satisfaction of spatial restraints the swiss-model repository of annotated threedimensional protein structure homology models supporting your pipeline with structural knowledge the relation between the divergence of sequence and structure in proteins casp assessment of fold recognition target predictions the role of protein d structures in the drug discovery process the impact of structure-guided drug design on clinical agents the druggable genome prediction of 'drug-likeness' a scoring scheme for discriminating between drugs and nondrugs drug-like properties and the causes of poor solubility and poor permeability structural bioinformatics in drug discovery inhibition of the p -hdm interaction with low molecular weight compounds inhibition of the p -mdm interaction: targeting a protein−protein interface a new method to detect related function among proteins independent of sequence and fold homology inferring functional relationships of proteins from local sequence and spatial surface patterns structural classification of protein kinases using d molecular interaction field analysis of their ligand binding sites: target family landscapes transcriptional repressor copr: structure model-based localization of the deoxyribonucleic acid binding motif use of a drug-resistant mutant of stress-activated protein kinase a/p to validate the in vivo specificity of sb chemogenomics: an emerging strategy for rapid target and drug discovery dissecting physiological roles of estrogen receptor alpha and beta with potent selective ligands from structurebased design impact of isotype-selective estrogen receptor agonists on ovarian function structure of the ligand-binding domain of oestrogen receptor beta in the presence of a partial agonist and a full antagonist the protein kinase complement of the human genome design and synthesis of novel tyrosine kinase inhibitors using a pharmacophore model of the atp-binding site of the egf-r rational design of potent and selective egfr tyrosine kinase inhibitors as anticancer agents rational design and synthesis of a novel antileukemic agent targeting bruton's tyrosine kinase (btk), lfm-a structure-based design of specific inhibitors of janus kinase as apoptosis-inducing antileukemic agents targeting aurora kinase in oncogenesis: a structural bioinformatics approach to target validation and rational drug design structure-based generation of a new class of potent cdk inhibitors: new de novo design strategy and library design discovery of a potent and selective protein kinase ck inhibitor by high-throughput docking establishment and functional validation of a structural homology model for human dna methyltransferase synthesis and evaluation of imidazole acetic acid inhibitors of activated thrombin-activatable fibrinolysis inhibitor as novel antithrombotics structure-guided transformation of charybdotoxin yields an analog that selectively targets ca + -activated over voltage-gated k + channels structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical domain coronavirus main proteinase ( clpro) structure: basis for design of anti-sars drugs characterization of a novel coronavirus associated with severe acute respiratory syndrome structure-assisted design of mechanismbased irreversible inhibitors of human rhinovirus c protease with potent antiviral activity against multiple rhinovirus serotypes the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor evaluation of homology modeling of the severe acute respiratory syndrome (sars) coronavirus main protease for structure-based drug design a d model of sars cov cl proteinase and its inhibitors design by virtual screening crystal structure of rhodopsin: a g-proteincoupled receptor modeling the d structure of gpcrs: advances and application to drug discovery nuclear hormone receptor targeted virtual screening rational discovery of novel nuclear hormone receptor antagonists protein-based virtual screening of chemical databases. ii. are homology models of g-protein-coupled receptors suitable targets? docking ligands onto binding site representations derived from proteins built by homology modelling performance of d database molecular docking studies into homology models design of helical proteins for real-time endoprotease assays a single amino acid that determines the sensitivity of progesterone receptors to ru ru is not an antiprogestin in the hamster mammalian microsomal cytochrome p monooxygenase: structural adaptations for membrane binding and functional diversity structure of human microsomal cytochrome p c . evidence for a peripheral fatty acid binding site crystal structure of human cytochrome p c with bound warfarin molecular modeling of human cytochrome p -substrate interactions modelling human cytochromes p involved in drug metabolism from the cyp c crystallographic template homology modelling of human cyp a based on the cyp c crystallographic template structure homology modelling of cyp a based on the cyp c crystallographic template: enzyme-substrate interactions and qsars for binding affinity and inhibition molecular modelling of cyp b based on homology with the cyp c crystal structure: analysis of enzyme-substrate interactions a molecular model of cyp d constructed by homology with the cyp c crystallographic template: investigation of enzyme-substrate interactions investigation of enzyme selectivity in the human cyp c subfamily: homology modelling of cyp c , cyp c and cyp c from the cyp c crystallographic template prediction of drug metabolism: the case of cytochrome p d a novel approach to predicting p mediated drug metabolism. cyp d catalyzed n-dealkylation reactions and qualitative metabolite predictions using a combined protein and pharmacophore model for cyp d competitive cyp c inhibitors: enzyme inhibition studies, protein homology modeling, and three-dimensional quantitative structure-activity relationship analysis molecular basis of p inhibition and activation: implications for drug development and drug therapy prediction of human drug metabolizing enzyme induction coactivator binding promotes the specific interaction between ligand and the pregnane x receptor a structural model of the constitutive androstane receptor defines novel interactions that mediate ligandindependent activity insights from a three-dimensional model into ligand binding to constitutive active receptor molecular modelling of the human glucocorticoid receptor (hgr) ligand-binding domain (lbd) by homology with the human estrogen receptor alpha (heralpha) lbd: quantitative structure-activity relationships within a series of cyp a inducers where induction is mediated via hgr involvement predicting undesirable drug interactions with promiscuous proteins in silico a structural basis for drug-induced long qt syndrome characterization of herg potassium channel inhibition using comsia d qsar and homology modeling approaches modern methods of drug discovery we gratefully acknowledge fruitful discussions with mario lobell (bayer healthcare; http://www.bayerhealthcare.com), derek debe, sean mullen (eidogen) and sunil patel (accelrys). key: cord- -v fzp n authors: coyle, peter v; ong, grace m; o'neill, hugh j; mccaughey, conall; de ornellas, dennis; mitchell, frederick; mitchell, suzanne j; feeney, susan a; wyatt, dorothy e; forde, marian; stockton, joanne title: a touchdown nucleic acid amplification protocol as an alternative to culture backup for immunofluorescence in the routine diagnosis of acute viral respiratory tract infections date: - - journal: bmc microbiol doi: . / - - - sha: doc_id: cord_uid: v fzp n background: immunofluorescence and virus culture are the main methods used to diagnose acute respiratory virus infections. diagnosing these infections using nucleic acid amplification presents technical challenges, one of which is facilitating the different optimal annealing temperatures needed for each virus. to overcome this problem we developed a diagnostic molecular strip which combined a generic nested touchdown protocol with in-house primer master-mixes that could recognise common respiratory viruses. results: over an month period a total of specimens were tested by both immunofluorescence and the molecular strip. the specimens came from males (median age . y), females (median age y) and quality assurance scheme specimens. viruses were recovered from a number of specimen types including broncho-alveolar lavage, nasopharyngeal secretions, sputa, post-mortem lung tissue and combined throat and nasal swabs. viral detection by if was poor in sputa and respiratory swabs. a total of viruses were detected in the study from patients and quality control specimens: by immunofluorescence and using the molecular strip. the strip consistently out-performed immunofluorescence with no loss of diagnostic specificity. conclusions: the touchdown protocol with pre-dispensed primer master-mixes was suitable for replacing virus culture for the diagnosis of respiratory viruses which were negative by immunofluorescence. results by immunofluorescence were available after an average of – hours while molecular strip results were available within hours, considerably faster than viral culture. the combined strip and touchdown protocol proved to be a convenient and reliable method of testing for multiple viruses in a routine setting. acute respiratory tract infections are major causes of morbidity and mortality. in , lower respiratory tract infections were globally the number one infectious cause of disability adjusted life-years [ ] . the commonest respiratory viruses that cause acute upper and lower respiratory tract infections and which are routinely tested for in most virus diagnostic laboratories are: influenza a virus (fla); influenza b virus (flb); respiratory syncytial virus (rsv); parainfluenza virus type (pf ); parainfluenza virus type (pf ); parainfluenza virus type (pf ) and adenovirus (adv). additionally, human rhinoviruses (hrv) and coronavirus e (cov- e) are also linked to acute respiratory infection but less commonly included in laboratory reports; human metapneumovirus (hmpv) is not yet part of most united kingdom virus laboratory test repertoires (personal feed-back from the united kingdom clinical virology network). as part of service development it was necessary to provide an alternative to virus culture for testing immunofluorescence negative respiratory specimens. historically and indeed currently immunofluorescence [ ] and virus culture [ , ] are the main methods used to diagnose acute respiratory virus infections. culture is accepted as more sensitive than immunofluorescence but slower and therefore less useful for direct patient management decisions. using a standard culture technique [ ] for the culture of respiratory viruses our median reporting times for culture positive and culture negative specimens were days (based on specimens) and days (based on specimens) respectively; virus identification by this technique required the use of monoclonal antibody staining of the cell monolayer in addition to observation for viral cytopathic effect. we therefore wished to develop a test capable of reporting on immunofluorescence negative specimens within a hour period. increasingly however, the sensitivity of nucleic acid amplification techniques for diagnosis has become recognised [ ] [ ] [ ] [ ] [ ] [ ] . however widespread concerns about contamination issues [ , ] and perceived cost [ ] have slowed their widespread adoption. an added problem for acute respiratory tract infections is the relatively large number of viruses that need to be accounted for, a problem which presents specific technical challenges. one such challenge is the different optimal annealing temperatures of the primer sets for each prospective virus target. the abi prism real-time facility from applied biosystems addresses this by using bundled software to design primer/probe combinations that use a common amplification protocol. however this approach is compromised by the inability of software to allow for target heterogeneity. in addition it does not allow users to adopt clinically validated primer sets from the literature. to address these problems we adopted an alternative approach through the development of a generic touchdown amplification protocol. touchdown protocols involve a pre-designed stepped reduction in the annealing temperature used for primer-to-template binding, which introduces a competitive advantage for specific base-pair priming over non-specific priming [ ] . a detailed knowledge of the optimum annealing temperature is therefore not required. the study protocol was empirically constructed and proved robust when applied to a large range of respiratory viral and bacterial targets, without compromising individual test sensitivity. it was designed for use with in-house primer master-mixes that recognise common respiratory viruses. before deciding on the layout of the molecular strip, as described in the methods, we undertook a wide range of preliminary validation steps for each primer set. the complexity of the strip makes it impossible to fully evaluate using the classical approach of applying an individual gold standard to each virus type. classically this approach works well where a single target is under investigation [ ] . however although the strip is putatively designed to identify viruses, the actual number of individual types targeted is over one hundred and sixty because of the inclusion of generic primer sets for hrv [ ] and adv [ ] respectively. the classical approach is further compounded for viruses (a) that cannot be grown or grown easily; (b) for which commercial if sera are not available; (c) for which specimen panels are not available. we therefore adopted a phased validation, culminating in the present study. sensitivity was ascribed by undertaking copy number determination on cloned targets and these ranged form × copies per ml for human rhinovirus type b to . × copies/ml for rsv-a. specificity was ascribed through reproducibility, i.e. specimens which were repeatedly positive, following our standard clinical reporting algorithm [ ] , were regarded as true positives; a similar approach was recently described for hmpv [ ] . in addition amplicon sequencing was used as an initial specificity check. the primers sets were tested on clinical respiratory specimens arising from a number of ethically approved studies. these included respiratory specimens from patients: (a) with chronic obstructive pulmonary disease; (b) with acute asthma; (c) on assisted ventilation in intensive care. they were also tested on respiratory specimens collected as part of an influenza spotter program as well as on laboratory specimens of known virus reactivity. to test the feasibility of its routine use we needed to clinically validate its performance in a routine setting on specimens tested in parallel with our standard immunofluorescence protocol for the diagnosis of acute virus respiratory infections. although the routine immunofluoresence panel lacked capacity for the detection of rhinoviruses, human metapneumovirus and cov- e, these were included on the strip for clinical reasons during the period of the study. these findings and their implications are reported. a total of viruses were detected in / specimens from a total of / patients and / national external quality assurance scheme (neqas) controls; immunofluorescence did not detect the parainfluenza virus type virus in one of the neqas specimens. viruses were detected in all of the specimen types processed. the molecular strip detected virus in: / ( . %) bronchoalveolar lavages, / ( . %) nasopharyngeal secretions, / ( . %) sputa and / ( . %) combined throat and nasal swabs. immunofluorescence detected virus in: / ( . %) broncho-alveolar lavages, / ( . %) nasopharyngeal secretions, / ( . %) sputa and / ( . %) combined throat and nasal swabs. the median age of male and female patients where virus was detected was y (range weeks - years) and y ( weeks - years) respectively. sixteen viruses were detected in / ( . %) specimens, confirming a respiratory virus in out of ( %) patients investigated in general practice. seventy-nine viruses were detected in / ( . %) specimens, confirming a respiratory virus in out of ( . %) patients investigated in hospital. of the viruses detected in specimens from the community, pcr detected all in contrast to a single identification, influenza a (h ), by immunofluorescence. pcr identified one or more viruses in specimens from of the patients and the neqas positive specimens, detecting a total of viruses as shown in table . the viruses detected were: influenza a (h ) virus ( ); influenza a (h ) virus ( ); influenza b virus ( ); human rhinovirus ( ); adenovirus ( ) ; parainfluenza virus type ( ); parainfluenza virus type ( ); respiratory syncytial virus type a ( ); respiratory syncytial virus type b ( );. no parainfluenza virus type , coronavirus e or human metapneumovirus were detected. dual infections were detected in / ( . %) patients. the dual infections were: influenza a (h ) and adenovirus ( ); influenza a (h ) virus and rhinovirus ( ); influenza a (h ) and adenovirus ( ); adenovirus and rhinovirus ( ); respiratory syncytial virus type b and rhinovirus ( ). nine patients had more than one specimen taken on the same day in which a virus was detected in at least one specimen by pcr. for of the patients the same virus was detected in each of the specimens. the viruses identified were rhinovirus ( ), adenovirus ( ) and parainfluenza type ( ); the latter was also immunofluorescence positive. in cases a rhinovirus was detected in only one of the specimens. as part of a separate rhinovirus validation protocol one of these specimens was subjected to retesting coupled with limited sequencing of the ' non-coding region amplicon which confirmed the presence of a rhinovirus sequence. additionally, premature twin boys admitted to intensive care on the same day with severe bronchiolitis, both had evidence of acute rhinovirus infection by pcr. limited sequencing of the ' non-coding region of these viruses as part of the rhinovirus validation protocol confirmed the presence of an identical sequence of rhinovirus in both specimens. although touchdown pcr has been used successfully to help overcome some of the uncertainties associated with the thermal amplification of microbial nucleic acid targets [ ] [ ] [ ] [ ] , its use in this study has extended its role further and in so doing brought closer the goal of undertaking molecular diagnostics in a routine setting. previously its main impact has been seen where multiplexing [ , ] or degenerate primers have been needed [ ] [ ] [ ] and where the problems of choosing correct annealing temperatures are at their most difficult. in this study the large number of targets is the main problem encountered. using an empirical approach a series of amplification steps linked to a stepped reduction in annealing temperature from °c to °c was constructed. this proved extremely resilient when used with a wide range of primer sets and included the apparent anomaly of putting adenovirus through an initial reverse transcription step to stream line all of the targets on to a single strip; we have previously reported this approach for testing group f adenovirus alongside norovirus, astrovirus and rotavirus [ ] . the touchdown surprisingly out-performed individual amplification protocols optimised for annealing temperature and thus proved suitable for use on the diverse range of respiratory viruses addressed in the study. where multiple viral targets are sought in clinical practice, we believe that it is only feasible to correlate the performance of the new assay in a head-to-head comparison with that already in routine use. unfortunately for many clinical laboratories there is an elusion of testing for a wider range of viruses than is the case, by the inoculation of cell lines with a theoretical ability to grow the respective viruses. the annual reports of most clinical laboratories of one of the commonest human respiratory viruses, human rhinovirus, is an example of this; using the touchdown protocol we now report approximately hrv infections per annum. the under reporting of adenovirus by standard methods [ ] and the paucity of hmpv reporting, further underlines this assertion. the ability to simultaneously validate the performance of multiple molecular primer sets in a routine clinical setting is a major accomplishment of the current methodological approach. the results demonstrated that a range of primers from both the medical literature and from in-house development could be amplified with a single generic touchdown protocol. it therefore confirmed the feasibility of directly incorporating primer sets into a standard operating procedure without the necessity for the individual optimisation of cycling parameters. as such the study results should facilitate primer selection and formal critical evaluation as here described. as an example of this enhanced flexibility we have recently replaced the primer sets for influenza a h and h (with respective copy number sensitivities of × and × copies per ml) with a generic matrix set (copy number sensitivity of × copies per ml). the use of strips containing pre-dispensed mastermixes facilitates their use in a routine setting where laboratory personnel have only to thaw the strip and add the specimen extract. we make and aliquot for routine use a large range of multi-reaction mastermixes which are repeatedly subjected to freeze-thaw cycles as required on a daily basis. provided the mixes are handled on ice, they remain extremely stable, over many months if so required. however the strip is designed for a single use only and thus only goes through a single freeze-thaw cycle. mix stability is not a problem and the single positive control is used only to confirm that the touchdown amplification cycle has run successfully. because the technique of using nested amplification followed by running agarose gel electrophoresis is relatively cumbersome, it was important to evaluate how the complete protocol, inclusive of report generation, would perform when introduced into a routine line-managed diagnostic setting. over the months of the study the technique fitted in well to the demands of routine service. central to this was the use of pre-dispensed and quality checked primer master-mixes which allowed the molecular strip to be adapted for use in a routine laboratory. the study confirmed that a broad based molecular approach was feasible as an alternative to virus culture to support immunofluorescence in the diagnosis of respiratory viruses. the overall superior performance of the strip and the missed neqas specimen by immunofluorescence underlines the need for a more sensitive back-up for negative specimens. while nested protocols must be regarded as a pragmatic, interim solution until perfected single round systems are available, the format of the strip reduces the concern most attached to nested formats, i.e. false positive results. in our experience there is little evidence to support contamination arising from environmental sources and that the two major points of contamination in a nested system are (a) cross-contamination during manual extraction and (b) contamination of second round adjacent wells with product from a first round positive amplification. the use of the qiagen biorobot for the extraction of all specimens reduced the former while the nature of the strip prevents the latter, since all the wells have separate mixes ( table ). with both nested and non-nested assays the most critical requirements for reliable results are the use of well trained, appropriately skilled and knowledgeable staff, operating in a managed environment. as with any service, test performance must stand up to both external and internal quality assurance and in this regard we welcome the new respiratory quality control panel soon to be made available from quality control for molecular diagnostics (qcmd), glasgow. the results obtained were very encouraging. although the strip was constructed to detect a wider range of viruses than immunofluorescence, over the period of validation it almost doubled ( versus ) the number of viruses that could have been detected by immunofluorescence, including a positive neqas specimen which was missed by immunofluorescence. of this group of viruses the detection of adenovirus showed the most dramatic increase, an observation we have also previously made in a separate study [ ] and which we continue to see both in routine respiratory specimens and in a number of respiratory studies. similar to hrv viruses we believe these common infections are underdiagnosed by the standard techniques of immunofluorescence and culture. they are the second commonest virus, after hrv, that we observe in mixed infections and it is self-evident that these additional infections are at a level below the detection thresholds of standard methods. their clinical significance when detected at these lower copy numbers remains to be determined. as mentioned in the introduction a factor which often impacts negatively on a laboratory's decision to use molecular diagnostics is one of cost. it is worth considering that no matter which assay is chosen for use, it will attract the same overheads needed to provide the infrastructure of a laboratory set-up i.e. building, utilities, staff and equipment. in this regard there are no cheap tests and to use reagent costs as the sole factor in determining which assay to use is somewhat perverse. while the reagent costs of the strip are higher that commercial immunofluorescence reagents by a factor of , including extraction, this would undoubtedly narrow if immunofluorescence were capable of closing the pathogen gaps that currently exist e.g. hrv, hmpv. currently using this approach, we have been able to replace both immunofluorescence and viral culture and this ability makes molecular diagnostics a more cost effective method for diagnosing viral infections. taking into account the superior range, sensitivity, ability to quantify and speed of molecular techniques it is incredible how little they are used in routine laboratories. with the advent of sars and the threat of avian influenza, this deficit is now beginning to disturb health care planners at the highest level. because specimen sampling was not contiguous seasonal peaks were not detected, accounting for the small numbers of respiratory syncytial virus detected and the lack of detection of human metapneumoviruses, parainfluenza virus type and coronavirus e; subsequent (unpublished) data from the routine use of the molecular strip support an important role for human metapneumovirus in acute respiratory infections and the sporadic nature of infections caused by parainfluenza type and coronavirus e. several interesting observations need highlighting. first, for immunofluorescence to perform reliably it was essential that a good nasopharyngeal specimen was available. the use of throat and/or nasal swabs with immunofluorescence alone is inappropriate. second, immunofluorescence was very poor at detecting viruses from patients in the community, again almost certainly because of the universal use of swabs in that setting. third, the rapid results of immunofluorescence were complemented by the touchdown protocol which can report definitive results within hours, considerably faster than culture. fourth, the molecular strip was better at detecting multiple infections. even allowing for the inability of immunofluorescence to detect rhinoviruses, it should have detected the mixed adenovirus and influenza virus infections. although immunofluorescence is capable of diagnosing dual infections, its routine use along with culture probably grossly underestimates their prevalence. the most plausible explanation is that the molecular technique detects infections where one of the viruses is below the positive control detection threshold of immunofluorescence. these low level viruses are either just starting or more likely reaching the end of an infectious episode (latency is less likely) and this raises the previously unaddressed question of their role in viral respiratory pathogenesis. fifth, the extent of rhinovirus infections was very significant. their clinical significance ranged from acting as a definitive respiratory pathogen to a less certain role when acting as the most frequently detected co-pathogen in mixed infections. in conclusion the use of the touchdown protocol with pre-dispensed and quality checked primer master-mixes was suitable for replacing virus culture for the diagnosis of respiratory viruses for immunofluorescence negative specimens. immunofluorescence results were available after an average of - hours while molecular strip results were available within hours, considerably faster than viral culture. the combined strip and touchdown protocol is a convenient and reliable method of testing for multiple viruses in a routine setting. its generic nature makes it especially useful for introducing test repertoire modifications e.g. incorporating primers for the newly identified coronaviruses sars-cov and hcov-nl . a total of specimens were included in the validation between january and june , including from an influenza surveillance scheme. the specimens were collected from patients including: male, median age . y ( m - y); female patients, median age y ( m - y); both male and female ages were skewed towards the lower age ranges, and national external quality assurance scheme (neqas) specimens ( positive, negative). one hundred and fifty-nine patients were in hospital and were in the community at the time of sampling. specimens tested consisted of a wide range of specimens including: broncho-alveolar lavage ( ), nasopharyngeal secretions ( ), sputum ( ) and combined throat and nasal swabs ( ) . nasopharyngeal secretions, broncho-alveolar lavage and sputum specimens were received in dry sterile containers at ambient temperature. upon receipt they were re-suspended in ml of virus transport medium (vtm) consisting of phosphate buffered saline ph . , bovine serum albumin . µg/ml, penicillin g sodium units/ml, streptomycin sulphate µg/ml and amphotericin b . µg/ml. throat and nasal swabs were received in ml of vtm and vortexed on arrival to release cells attached to the fibres of the swab. an aliquot of µl was taken off for extraction after which the specimens were centrifuged at g for min and the resulting cell deposits air-dried on glass multi-well slides and fixed in acetone prior to testing. immunofluorescence was set up on the respiratory specimens using commercial reagents according to the manufacturer's instructions, and was able to detect: influenza a, influenza b, respiratory syncytial virus, adenovirus, parainfluenza type , parainfluenza type and parainfluenza type (dako diagnostics, ely, uk). this protocol allows the co-extraction of both rna and dna simultaneously. simultaneous amplification of all targets was facilitated by using a standard well multi-well pcr strip to which all mixes were pre-dispensed and stored frozen; this format is referred to in the paper as the "respiratory strip" because of the respiratory nature of the targets. the respiratory strip targeted the following common respiratory viruses: influenza a (h ), influenza a (h ), influenza b, respiratory syncytial virus type a, respiratory syncytial virus type b, adenovirus, coronavirus e, parainfluenza virus type , parainfluenza virus type , parainfluenza virus type , human rhinovirus and human metapneumovirus. the final configuration of the single and multiplex primer mixes in the well strip are shown in table . the primer sets used were taken mainly from published studies [ , [ ] [ ] [ ] but also included primer sets validated inhouse after modification or de-novo design, including those for influenza a (h ), influenza a (h ) and the generic adenovirus primers [ ] . the primers, gene targets and expected product sizes following amplification are shown in table . each primer master-mix was made-up and titrated against a known positive control before being aliquoted and dispensed into its respective well of the -well microtube strip. the strips were stored frozen at - °c until used. a positive control was also aliquoted and stored separately at - °c until used. for the duration of the study the positive control was the cloned target of parainfluenza virus type ; a negative control was not deemed necessary. first round volumes were made-up in access rt-pcr buffer (promega, southampton, england, u.k) and in the final µl volume contained the following reagent amounts: agc aaa gct ttc agc aac tg haemagglutinin fla d (h ) gct tcc att tgg agt gat gc- fla e (h ) agt gct gaa cgt gac tat gc fla f (h ) ttt gct ggc ttc tct tgg t rsv a gtc tta cag ccg tga tta gg nucleoprotein rsv b ggg ctt tct ttg gtt act tc rsva c gat gtt acg gtg ggg agt ct rsva d gta cac tgt agt taa tca ca rsvb c aat gct aag atg ggg agttc rsvb d gaa att gag tta atg aca gc adv a gccgcagtggtcttacatgcacatc hexon adv b cagcacgccgcggatgtcaaagt adv c gacgcctcggagtacctswsycc adv dd tacgagtacgtggtgtcctckcgrtc the first round reaction added to . µl of second round primer master-mix per well; a multi-channel pipette facilitated the transfer of the volumes in one step. the positive control was run on the eighth well of each strip. the second round products were run on ethidium bromide stained % agarose gels and photographed. specimens were reported positive when respectively the correct size bands and the positive control bands were present. vaccine preventable disease comparison of conventional viral cultures with direct fluorescent antibody stains for diagnosis of community-acquired respiratory virus infections in hospitalized children sensitivity of respiratory virus culture when screening with r-mix fresh cells isolation of viruses from clinical specimens in microtitre plates with cells inoculated in suspension a comparison of nested polymerase chain reaction and immunofluorescence for the diagnosis of respiratory infections in children with bronchiolitis, and the implications for a cohorting strategy a comparison of virus isolation, indirect immunofluorescence and nested multiplex polymerase chain reaction for the diagnosis of primary and recurrent herpes simplex type and type infections nested multiplex polymerase chain reaction for the diagnosis of cutaneous herpes simplex and herpes zoster infections and a comparison with electronmicroscopy differential detection of rhinoviruses and enteroviruses rna sequences associated with classical immunofluorescence assay detection of respiratory virus antigens in nasopharyngeal swabs from infants with bronchiolitis molecular diagnosis of influenza diagnosis of acute respiratory tract infections: serology and new methods van helden pd: laboratory experience and guidelines for avoiding false positive polymerase chain reaction results evaluation of the prodesse hexaplex multiplex pcr assay for direct detection of seven respiratory viruses in clinical specimens single-step pcr optimization using touchdown and stepdown pcr programming assessment of cmv load in solid organ transplant recipients by pp antigenemia and real-time quantitative dna pcr assay: correlation with pp rna detection comparison of pcr primer pairs in the detection of human rhinoviruses in nasopharyngeal aspirates clinical assessment of a generic dna amplification assay for the identification of respiratory adenovirus infections human metapneumovirus infection among children hospitalized with acute respiratory illness a new dig-pcr-eia method for the detection of chlamydia pneumoniae dna in clinical samples diagnostic use of pcr for detection of pneumocystis carinii in oral wash samples a touchdown pcr for the differentiation of equine herpesvirus type (ehv- ) field strains from the modified live vaccine strain rach improved detection of rhinoviruses in nasal and throat swabs by seminested rt-pcr combining multiplex and touchdown pcr for microsatellite analysis combining multiplex and touchdown pcr to screen murine microsatellite polymorphisms use of degenerate primers and touchdown pcr to amplify a halogenase gene fragment from streptomyces venezuelae isp use of degenerate primers and touchdown pcr for construction of cdna libraries cloning and molecular characterization of cntef which encodes translation elongation factor alpha in cryptococcus neoformans clinical utility of nested multiplex rt-pcr for group f adenovirus, rotavirus and norwalk-like viruses in acute viral gastroenteritis in children and adults multiplex pcr for typing and subtyping influenza and respiratory syncytial viruses simultaneous detection and identification of human parainfluenza viruses , , and from clinical samples by multiplex pcr evaluation of nested polymerase chain methods for the detection of human coronaviruses e and oc we wish to thank the northern ireland chest, heart and stroke foundation for funding that contributed to the development of the molecular strip. pvc: touchdown and molecular strip design and manuscript preparation.gmo: early application of touchdown cycling to respiratory samples. key: cord- - h tg hg authors: ho, tin-yun; wu, shih-lu; cheng, shin-ei; wei, yen-chiao; huang, shan-ping; hsiang, chien-yun title: antigenicity and receptor-binding ability of recombinant sars coronavirus spike protein date: - - journal: biochem biophys res commun doi: . /j.bbrc. . . sha: doc_id: cord_uid: h tg hg severe acute respiratory syndrome (sars) is an emerging infectious disease associated with a novel coronavirus and causing worldwide outbreaks. sars coronavirus (sars-cov) is an enveloped rna virus, which contains several structural proteins. among these proteins, spike (s) protein is responsible for binding to specific cellular receptors and is a major antigenic determinant, which induces neutralizing antibody. in order to analyze the antigenicity and receptor-binding ability of sars-cov s protein, we expressed the s protein in escherichia coli using a pet expression vector. after the isopropyl-β-d-thiogalactoside induction, s protein was expressed in the soluble form and purified by nickel-affinity chromatography to homogeneity. the amount of s protein recovered was . – . mg/ ml bacterial culture. the s protein was recognized by sera from sars patients by elisa and western blot, which indicated that recombinant s protein retained its antigenicity. by biotinylated elisa and western blot using biotin-labeled s protein as the probe, we identified -kda and -kda proteins in vero cells that might be the cellular receptors responsible for sars-cov infection. taken together, these results suggested that recombinant s protein exhibited the antigenicity and receptor-binding ability, and it could be a good candidate for further developing sars vaccine and anti-sars therapy. severe acute respiratory syndrome (sars) is a newly emerging human disease, resulting globally in deaths from probable cases [ ] . a novel coronavirus has been identified as the etiological agent of sars and designated as sars coronavirus (sars-cov) after tests of causation according to koch's postulates, including monkey inoculation [ ] [ ] [ ] . sars-cov can infect african green monkey kidney (vero e ) cells and cause a similar disease in cynomolgus macaques (macaca fascicularis) [ , ] . the full-length genome sequence of sars-cov has been elucidated within weeks after the identification of this novel pathogen [ , ] . sars-cov contains a single-stranded plus-sense rna genome about kb in length that has a -cap structure and a -polyadenylation tract. the genomic organization is typical of coronaviruses, having five major open reading frames (orfs) that encode the replicase polyproteins; the spike (s), envelope (e), and membrane (m) glycoproteins; and the nucleocapsid protein (n) in the same order as those of other coronaviruses [ ] [ ] [ ] . when coronaviruses enter cells, the -region of viral genome is translated into a large polypeptide that is cleaved by viral-encoded proteases to release rna-dependent rna polymerase and adenosine triphosphatase/helicase. these proteins, in turn, are responsible for replicating the viral genome as well as generating nested transcripts that are used in the synthesis of viral proteins. viral membrane proteins, including s and m are inserted into the endoplasmic reticulum (er), while rna genome assembles with the n protein. this rnaprotein complex then associates with m proteins and buds into the lumen of the er. the virus particles then migrate through the golgi complex and exist in the cells by exocytosis [ ] . the first step in viral infection is the binding of viral proteins to certain cellular receptors. so far, the s protein of coronavirus is considered as the site of viral attachment to the host cells [ , ] . the s proteins of coronaviruses are large type i membrane glycoprotein projections from viral envelope [ ] . s proteins are responsible for both binding to receptors on host cells and for membrane fusion [ , ] . s proteins also contain important virus-neutralizing epitopes that elicit neutralizing antibody in the host species [ , ] . furthermore, mutations in this gene dramatically affect the virulence, pathogenesis, and host cell tropism [ ] [ ] [ ] . these results suggested that s protein is a good candidate for vaccine because neutralizing antibodies are directed against s. moreover, s protein is also a good target for antiviral therapies because blockade of binding of s protein to cellular receptor can prevent virus entry. therefore, in this study, we expressed the sars-cov s protein in escherichia coli (e. coli). the antigenicity and receptor-binding ability of recombinant s protein were further analyzed. construction of recombinant plasmids. sars-cov rna was provided by department of medical technology, china medical university hospital. sars-cov s gene was kindly provided by dr. p.j. chen. briefly, the rna was reverse transcribed into cdna using sars-m primer ( -ggaattcgccaaacataccaaggcc- ) and super-script iii (invitrogen) according to the manufacturer's protocol. the -bp dna fragment was then amplified from the cdna template by polymerase chain reaction (pcr) using pfuulter dna polymerase (stratagene) with sars-p primer ( -cgggatcctagtggtagt gaccttgacc- ) and sars-m primer. the pcr profile was as follows: one cycle at °c for min; cycles at °c for s, °c for s, and °c for min. the amplified s cdna fragments were cleaved by ecori and bamhi, and the -bp fragments were then inserted into pet- b(+) (novagen) to create the pet-spike expression plasmid. dna sequencing. dna sequencing was performed on doublestranded plasmids by dideoxy chain termination with the bigdye terminator cycle sequencing ready reaction kit (applied biosystems) and analyzed by abp prism (applied biosystems). dimethyl sulfoxide was introduced to the reaction to a final concentration of % to diminish the secondary structure. the primer used in the sequencing reaction was t , sars-p ( -cgggatcctaattacacctggaacaaatgc- ), t terminator, sars-m , sars-m ( -ttgatatagaacagc aacttcagatgaagc- ), or sars-m ( -ggaattccactta cacccccaaaagagc- ). dna was sequenced on both strands of at least two repeats of cloned dna fragments. expression and purification of recombinant sars-cov s protein. recombinant protein was expressed in e. coli bl (de )plyss strain by transforming the pet-spike to produce an n-terminal fusion with six histidine residues. the expression and purification of recombinant s protein were performed as described previously with modification [ ] . briefly, cells were grown in ml luria-bertani broth agitated at °c until od reached . . isopropyl-b-d d -thiogalactoside (iptg) was then added to a final concentration of . mm and the cells were pelleted h after induction. the cell pellet was washed twice with icecold binding buffer ( mm tris-hcl, ph . , mm nacl, and mm imidazole) and resuspended in ml ice-cold binding buffer containing m urea. the resuspended pellet was incubated at °c with shaking for h. after sonication, the suspension was centrifuged at , g for min at °c. the resulting supernatant was applied on nickel-affinity chromatography with m urea present throughout the procedure. the protein in the final column eluate was dialyzed overnight against phosphate buffered saline (pbs) ( mm nacl, . mm kcl, . mm na hpo , and . mm kh po ) and stored at ) °c until further analysis. protein was analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page) and was quantified with a bradford assay (bio-rad). cells and cell extracts. human lung carcinoma cell (a- ), human liver cell (chang liver), african green monkey kidney cells (vero e , vero), and human monocyte (u- ) were purchased from bioresources collection and research center (hsinchu, taiwan). cells were maintained in dulbecco's modified eagle's medium supplemented with % fetal bovine serum at °c and % co in air. the cell extracts were prepared by washing the cells once with ice-cold pbs and lysing the cells by lysis buffer ( mm nacl, % np- , mm tris-hcl, ph . , and lm phenylmethylsulfonyl fluoride). after a -min-incubation on ice, the cell lysate was collected by centrifugation. protein was quantified with a bradford assay and stored at ) °c until further analysis. biotinylation of recombinant s protein. recombinant s protein was mixed with sulfo-nhs-biotin (pierce) in a ratio of ten to one. after a -h-incubation on ice, the unincorporated biotin was removed by centricon- (amicon) and the biotinylated s protein was stored at °c until further analysis. sulfo-nhs-biotin should be prepared freshly by dissolving in ddw. western blot analysis. proteins ( lg) were separated by % sds-page and the protein bands were then transferred electrophoretically to nitrocellulose membrane (amersham pharmacia biotech). membrane was blocked in blocking buffer ( mm tris-hcl, ph . , mm nacl, . % tween , and % skim milk) and probed with rabbit anti-spike (residue - ) polyclonal antibody or human anti-sars sera at room temperature for h. the bound antibody was detected with horseradish peroxidase-conjugated secondary antibody (sigma) followed by chemiluminescence (ecl system, amersham) and exposed by x-ray films. rabbit anti-spike (residue - ) polyclonal antibody was prepared by immunizing rabbit with truncated spike and was provided kindly by dr. p.j. chen. human anti-sars sera were collected from sars patients and provided by dr. l.k. chen. for biotinylated western blot, cell extracts were separated by % sds-page and the protein bands were then transferred to membrane. after blocking, the membrane was probed with . lg/ml biotinylated s protein at °c for h. the bound protein was detected with peroxidase-conjugated avidin (sigma) followed by chemiluminescence and exposed by x-ray films. enzyme-linked immunosorbent assay (elisa). microtiter plates (nunc) were coated at °c overnight with ll of ng/ll proteins, which was diluted in . m carbonate buffer (ph . ). the wells were rinsed with ll washing buffer ( . % tween in pbs) and blocked with ll blocking buffer ( % bovine serum albumin (bsa) in washing buffer) by incubating at °c for min. the absorbed protein in each well was challenged with ll diluted human anti-sars sera and incubated at °c for h. after three washes with washing buffer, ll diluted peroxidase-conjugated secondary antibody was added to each well and incubated at °c for h. following three washes, ll chromogenic substrate, , -azinobis( -ethylbenzthiazo-line-sulfonic acid), was added to each well and incubated at °c for min. the absorbance was read at nm in an elisa plate reader. for biotinylated elisa, the procedure was performed as described previously with modification. briefly, microtiter plates were coated with lg/well cell extract which was diluted in . m carbonate buffer, blocked with blocking buffer, and challenged with . lg/well biotinylated s protein. after a -h incubation at °c, diluted peroxidase-conjugated avidin was added and incubated at °c for h. following three washes, chromogenic substrate was added to each well and incubated at °c for min. the absorbance was read at nm in an elisa plate reader. the sars-cov genome is approximately . kb long and contains five major orfs flanked by and untranslated regions of and nucleotides, respectively (fig. a) [ , ] . the predicted s gene is located from nucleotide , to , in the sars-cov genome. in order to clone s gene, the -bp dna fragment containing whole s gene was amplified by reverse transcription-pcr and inserted into prokaryotic vector pet- (+). the deduced amino acid sequence of s protein is shown in fig. . s gene had an orf of nucleotides, capable of coding for a -amino-acid polypeptide of about kda. the amino terminus of sars-cov s protein contained a short type i signal peptide (residue - ) composed of hydrophobic amino acids that are presumably removed during cotranslational transport through er. the carboxyl terminus (residue - ) consisted of a transmembrane domain and a cytoplasmic tail rich in cysteine residues. twenty-three potential n-linked glycosylation sites were predicted among sars-cov s protein. together these data predicted that sars-cov s protein is a type i membrane glycoprotein with the n-terminus and the majority of the protein (residue - ) on the outside of virus particle, in agreement with other coronavirus s protein data (fig. b) . multiple alignment and phylogenetic analysis among s proteins of mammalian coronaviruses, which cause respiratory infection, were further performed by pileup program of genetics computer group (gcg) (figs. and ). alignment of these sequences produced a low level of similarity ( - % pairwise amino acid identity) between the predicted amino acid sequence of sars-cov s protein and other coronavirus s proteins. phylogenetic analysis showed that the species formed monophyletic clusters consistent with established taxonomic groups. however, sars-cov s protein sequences segregated into a well-resolved branch, indicating that sars-cov s protein is not closely related to any of the previously characterized coronavirus s proteins. although overall sequence conservation was low, the c-terminus, consisting of a transmembrane domain and a cytoplasmic tail, was highly conserved. putative cellular receptor-binding sites (residue - ) of sars-cov s protein [ ] were not conserved compared with other coronaviruses. the antigenic sites of porcine transmissible gastroenteritis virus s protein [ , ] were also varied among these coronaviruses. these data suggested that comparison of primary amino acid sequences does not provide insight into the receptor-binding specificity or antigenic properties of sars-cov s protein. by analyzing the primary structure of sars-cov s protein, we tried to predict the putative antigenic regions of s protein. the hydrophilicity, surface probability, and chain flexibility of sars-cov s protein were calculated by peptidestructure program of gcg according to kyte and doolittle plots, emini prediction, and karplus and schulz prediction, respectively (fig. a ) [ ] [ ] [ ] . the antigenicity of sars-cov s protein was further analyzed based on three aforementioned criteria, and the regions in which antigenic index exceeds . are shown in fig. b . most of the putative antigenic sites were located at outer membrane regions, in which residue - and - exhibited the highly antigenic potential. because most t cell and b cell epitopes contain a sequence of - -amino acids [ , ] , we further analyzed the antigenicity using a window of residues. residue - , - , - , - , and - of sars-cov s protein displayed the highly antigenic potential. these data suggested that these regions might be good candidates for developing sars peptide vaccine. in order to study the antigenicity and receptorbinding ability of sars-cov s protein, we expressed and purified the full-length recombinant sars-cov s protein from e. coli. we expressed the s protein from e. coli bl (de )plyss strain transformed with a pet plasmid carrying s gene. after induction with iptg, a product with -kda was observed by sds-page fig. . phylogenetic analysis of coronavirus s proteins. amino acid sequences of sars-cov s proteins were compared with those of hcov- e, tgev, fipv, hcov-oc , mhv, hev, bcov, avian infectious bronchitis virus (ibv) (genbank accession no. p ), and turkey coronavirus (tcov) (genbank accession no. aaq ). sequence alignment and neighbor-joining tree were generated by growtree program of gcg using the blosum comparison matrix. coronavirus groups i, ii, and iii are indicated. branch lengths are proportionate to amino acid differences. analysis (fig. ) . the amount of induced s protein was consistent when the bacteria were refreshed to . - . od and the recombinant s protein was expressed in the soluble form in bacterial cells. the soluble s protein was further purified by affinity chromatography using his-bond resin. no detectable s protein was purified, suggesting that the histidine tag is folded into the interior of s protein (data not shown). we therefore dena-tured the s protein by urea, purified by nickel-affinity chromatography, and renatured the protein by dialysis. the amount of recombinant s protein recovered was approximately . - . mg/ ml of bacterial culture (fig. ) . to analyze the antigenicity of recombinant s protein, we performed western blot and elisa using sera from sars patients or from spike-immunized rabbits. fig. shows that recombinant s was detectable by both sera in both assays. additionally, truncated s protein (residue - ) exhibited the similar antigenicity compared with full-length spike (residue - ). these results indicated that recombinant s protein remains its antigenicity that could be recognized by sera from sars patients. the receptor-binding ability of recombinant s protein was analyzed by biotinylated elisa and western blot. several reports indicated that sars-cov could have succeeded in growing progeny virus in vero cells [ , , ] . the bsa and vero cell extracts were therefore coated on elisa plates and challenged with biotinlabeled s protein. the binding ability of s protein to cell extract was evaluated by od value in elisa. fig. a shows that the binding ability of s protein to bsa was very low. however, s protein significantly bound vero cell extracts with the od exceeding . . these results proved the specificity of biotinylated elisa in analyzing the receptor-binding ability of s protein. it also suggested that recombinant s protein could be served as a probe to analyze the cellular receptors involved in virus attachment. to further identify the potential cellular receptors for sars-cov attachment, we performed biotinylated western blot. the cell extracts were separated by sds-page, transferred to membrane, and detected by biotin-labeled s protein. fig. b shows that recombinant s protein interacted with several cellular proteins in different cell types. by comparison of protein patterns of different cell types, we found that two proteins with molecular masses of and kda were detectable in vero cells instead of other cell types. it is now known that vero cell is the only cell line that could be infected by sars-cov [ ] . these results suggested that -kda and -kda polypeptides in vero cells might be the cellular receptors responsible for sars-cov s protein binding. the elucidation of amino acid sequences of these proteins is now proceeded. in this study, we cloned, expressed, and purified the sars-cov s protein from e. coli. the recombinant s protein was expressed in soluble form in bacterial cells, and the amount of protein recovered was . - . mg/ ml bacterial culture. the s protein was recognized by sera from sars patients, indicating that recombinant s protein retained its antigenicity. by biotinylated elisa and western blot using recombinant s protein as the probe, we identified that -kda and -kda proteins might be the cellular receptors responsible for sars-cov infection. these results suggested that s protein could be a good candidate for further developing sars vaccine and anti-sars therapy. cumulative number of reported probable cases of severe acute respiratory syndrome (sars) koch's postulates fulfilled for sars virus the sars working group, a novel coronavirus associated with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome coronaviridae: the viruses and their replication mhv s peplomer protein expressed by a recombinant vaccinia virus vector exhibits igg fc-receptor activity further characterization of aminopeptidase-n as a receptor for coronaviruses nucleotide sequence of the gene encoding the surface projection glycoprotein of coronavirus mhv-jhm stably expressed fipv peplomer protein induces cell fusion and elicits neutralizing antibodies in mice major receptorbinding and neutralization determinants are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein monoclonal antibodies to murine hepatitis virus- (strain jhm) define the viral glycoprotein responsible for attachment and cell-cell fusion antigenic relationship of murine coronaviruses: analysis using monoclonal antibodies to jhm (mhv- ) virus the v a . envelope glycoprotein deletion mutant of mouse hepatitis virus type- is neuroattenuated by its reduced rate of spread in the central nervous system targeted recombination within the spike gene of murine coronavirus mouse hepatitis virus-a : q is a determinant of hepatotropism targeted recombination demonstrates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence recombinant pseudorabies virus dnase exhibits a recbcd-like catalytic function putative hapn receptor binding sites in sars-cov spike protein the major subunit c pg of escherichia coli cs a fibrillae as an expression vector for different combinations of two tgev coronavirus epitopes a continuous epitope from transmissible gastroenteritis virus s protein fused to e. coli heat-labile toxin b subunit expressed by attenuated salmonella induces serum and secretory immunity a simple method for displaying the hydropathic character of a protein induction of hepatitis a virus-neutralizing antibody by a virus-specific synthetic peptide prediction of chain flexibility in proteins. a tool for the selection of peptide antigens the epitopes of influenza nucleoprotein recognized by cytotoxic t lymphocytes can be defined with short synthetic peptides strategies for epitope analysis using peptide synthesis we thank dr. p.j. chen for providing the sars-cov s gene and rabbit anti-spike antibody. we also thank dr. l.k. chen for providing the sera from sars patients. this work is supported by grants from national science council (nsc - -b- - -y and nsc - -b- - -y), taiwan, roc. key: cord- -wl jk authors: totoiu, minodora o.; nistor, gabriel i.; lane, thomas e.; keirstead, hans s. title: remyelination, axonal sparing, and locomotor recovery following transplantation of glial-committed progenitor cells into the mhv model of multiple sclerosis date: - - journal: exp neurol doi: . /j.expneurol. . . sha: doc_id: cord_uid: wl jk the behavior and myelinogenic properties of glial cells have been well documented following transplantation into regions of focal experimental demyelination in animal models. however, the ability of glial cell preparations to remyelinate in such models does not necessarily indicate that their transplantation into demyelinated lesions in clinical disease will be successful. one of the precluding factors in this regard is a greater understanding of the environmental conditions that will support transplant-mediated remyelination. in this study, we determined whether the complex and reactive cns environment of the mouse hepatitis virus (mhv) model of multiple sclerosis (ms) could support transplant-mediated remyelination. striatal neural precursors derived from postnatal day mice were committed to a glial cell lineage and labeled. immunohistochemical staining indicated that this population generated > % glial cells following differentiation in vitro. transplantation of glial-committed progenitor cells into the t spinal cord of mhv-infected mice demonstrating complete hindlimb paralysis resulted in migration of cells up to mm from the implantation site and remyelination of up to % of axons. transplanted-remyelinated animals contained approximately × the number of axons within sampled regions of the ventral and lateral columns as compared to non-transplanted animals, suggesting that remyelination is associated with axonal sparing. furthermore, transplantation resulted in behavioral improvement. this study demonstrates for the first time that transplant-mediated remyelination is possible in the pathogenic environment of the mhv demyelination model and that it is associated with locomotor improvement. remyelination is a successful regenerative event within the adult central nervous system (cns), as it can restore saltatory conduction (smith et al., ) and facilitate functional recovery (jeffery and blakemore, ) . remyelination has been demonstrated in a variety of experimental demyelination/mutant models (blakemore, (blakemore, , (blakemore, , (blakemore, , duncan et al., ; gumpel et al., ; herndon et al., ; blakemore, , ; jeffery et al., ; moore et al., ; rodriguez et al., ; sasaki and ide, ; yajima and suzuki, ) and occurs spontaneously in naturally occurring demyelinating diseases such as ms (prineas et al., a) . however, remyelination failure is more prevalent than its success when considering the spectrum of pathologies that affect the cns. although remyelination is usually successful in most animal models, it is often incomplete in the theiler's model of demyelination (murray et al., ) . furthermore, remyelination is less efficient in old animals than in young animals (shields et al., ) . the failure of remyelination is illustrated within foci of chronic demyelination within the later stages of ms (prineas et al., b) . chronic ms lesions are characterized by oligodendrocyte loss (ozawa et al., ) , with remyelination limited to the borders of inactive plaques (suzuki et al., ) . repairing persistent demyelination may ameliorate clinical deterioration. in addition to restoring normal impulse conduction (utzschneider et al., ) , remyelination may decrease axonal degeneration/transection. this is suggested by the demonstration that chronically demyelinated axons are vulnerable to degeneration/transection and that axonal loss in the later stages ms contributes to clinical deterioration (bjartmar et al., ; de stefano et al., ; . these findings underscore the importance of developing therapeutic strategies to enhance remyelination. one approach is to activate or enhance the response of endogenous mechanisms for repair, as has been demonstrated following growth factor administration in the experimental autoimmune encephalomyelitis (eae) demyelination model (yao et al., ) , or passive transfer of antiserum (rodriguez et al., ) or purified immunoglobulin (rodriguez and lennon, ) from mice immunized with spinal cord homogenate into the theiler's demyelination model (miller and rodriguez, ) . an alternative approach to remyelinating areas of demyelination is the transplantation of remyelination-competent cells. stem cells and neural precursors represent attractive sources for the generation of remyelination-competent cells, as they can be readily amplified and differentiated to the oligodendrocyte lineage (ben-hur et al., ; brustle et al., ) . stem cell-derived glial precursors have been shown to myelinate following transplantation into the myelindeficient rat (brustle et al., ) , and neural precursorderived glial-committed progenitors (ben-hur et al., ; have been shown to myelinate following transplantation into regions of acute experimental demyelination . more recently, intracerebroventricular or intrathecal implantation of neural precursors into the eae demyelination model resulted in migration of transplanted cells into white matter and their differentiation to astrocytes and oligodendrocytes, although no assessment of their ability to myelinate was performed (ben-hur et al., ) . to determine whether transplantation represents a viable strategy for treating demyelination, it is necessary to better understand the range of environmental conditions that support transplantation-mediated remyelination. in this study, we investigated the ability of the complex and reactive disease state of the chronic demyelinating mhv model of ms to support transplant-mediated remyelination. intracerebral injection of the j . v- strain of mhv results in acute encephalomyelitis with demyelination, followed - days later by an immunemediated demyelinating encephalomyelitis with hindlimb paralysis and progressive cns destruction, including the initiation of new demyelination foci probably for the life of the mouse (fleming et al., ; haring and perlman, ; lane et al., ; stohlman and hinton, ; stohlman et al., ) . this model provides an environment of ongoing demyelinating pathogenesis, and is thus distinct from gliotoxin lesions. intraspinal transplantation of glial-committed progenitors into the mhv model of ms resulted in extensive migration of transplanted cells, robust remyelination, axonal sparing, and behavioral improvement. these results show that transplant-mediated remyelination is possible following intraspinal transplantation into an environment of ongoing pathogenesis resembling ms. striata from postnatal day c bl/ mice were dissected, triturated, and maintained as previously described (ben-hur et al., ) . trypan blue was used to determine cell viability. cells were grown for days as floating clusters in -well low-adherent plastic dishes (corning life sciences acton, ma) at an initial density of  cells/ ml of dmem:f , b supplement (gibco-invitrogen, carlsbad, ca) with insulin, sodium selenite, transferrin, putrescin, progesterone, t , detailed in ben-hur et al. ( ) , and . ag/ml epidermal growth factor (egf; sigma-aldrich, st. louis, mo). on day , culture supernatant containing the floating clusters was removed and centrifuged at  g for min. clusters were then resuspended in fresh media and added to new culture dishes. on day , . ag/ml egf was added to the culture dishes. on day , clusters were washed, resuspended in fresh media, and added to new culture dishes. on day , clusters were incubated with mm brdu (sigma-aldrich) in the culture medium overnight. one culture dish was not labeled with brdu to compare viability and differentiation in the presence and absence of brdu. on day , cultures were prepared for transplantation. clusters were dissociated with . % trypsin-edta (invitrogen canada inc., burlington, on) for min, triturated, centrifuged for min at  g, resuspended three times in calcium-and magnesium-free dmem (gibco-invitrogen), and concentrated to a final density of  cells/ al in the same media. the cell preparation was kept on ice for a maximum of h before transplantation. on the day of transplantation, some cells were prepared for immunocytochemistry. clusters were centrifuged for min at  g and resuspended three times in fresh media without egf, and were plated on mg/ml poly-l-lysine (sigma-aldrich) and ag/ml laminin (sigma-aldrich) coated four chamber, imaging slides (nalgene-nunc international, rochester, ny). to assess differentiation potential, cells were grown on imaging slides on adherent substrate for days, then fixed in % paraformaldehyde (fisher scientific, pittsburgh, pa) in pbs for min and immunocytochemical staining was performed using standard protocols. imaging chambers were blocked with % normal goat serum (ngs) (chemicon, temecula, ca) for min at room temperature. primary antibodies (polyclonal rabbit anti-galc, chemicon, : dilution in % ngs; monoclonal mouse anti-neun, chemicon, : dilution in % ngs; polyclonal rabbit anti-gfap, dako, denmark, : dilution in % ngs; monoclonal mouse anti-cd b, serotec, uk, : dilution in % ngs; polyclonal rat anti-brdu, accurate chemical and scientific corporation, westbury, ny, : dilution in % ngs) were applied to imaging chambers overnight at jc. imaging chambers were rinsed three times with pbs, incubated for min in % ngs, and fluorescent-conjugated secondary antibodies (alexa or , goat anti-rabbit, goat anti-rat, or goat antimouse igg h+l, : dilution in % ngs; vector laboratories, burlingame, ca) was applied and incubated for h at room temperature. chambers were rinsed three times in pbs, and nuclear staining was conducted by exposing cultures to bis-benzimide (hoechst , molecular probes, eugene, or) for min. cell quantification was conducted using an olympus ax- light microscope with a  objective. the percentage of immunopositive cells was determined by dividing the total number of immunopositive cells by the total number of hoechst-positive cells in each imaging chamber, and averaging the results from three different imaging chambers per marker. a total of hoechst-positive cells were counted for these analyses. each -chamber imaging slide had one no-primary control chamber and three stained chambers for each of the markers mentioned above. only immunopositive cells with clearly hoechstpositive nucleus were counted. age-matched, weight-matched ( - g) male c bl/ mice (h- b background; national cancer institute, bethesda, md; n = ) were anesthetized by methoxyflurane inhalation (pitman-moore inc., washington crossing, nj). mice received intracerebral injections of plaque forming units of the neurotropic corona virus mhv strain j . v- (kindly provided by j. fleming, university of wisconsin, madison, wi), suspended in al of sterile saline (lane et al., ) . intracerebral injection of mhv results in a biphasic disease: acute encephalomyelitis with myelin loss, followed - days later by an immunemediated demyelinating encephalomyelitis with hindlimb paralysis and progressive destruction of the cns (fleming et al., ; haring and perlman, ; lane et al., ; stohlman et al., ) . two of the mhv injected animals died during the first week post injection; there is an - % survival rate of animals injected with this viral strain, and the animals usually die during the first days of acute infection. if the animals survive the acute stage of disease there is > % chance of survival. control (sham) animals (n = ) were injected with al of sterile saline alone and did not develop any behavioral or histological deficits. twelve days after mhv intracerebral injection, each animal (n = ) received a single injection of , cells in al of calcium-and magnesium-free dmem (gibco-invitrogen) at t of the spinal cord, using the protocol described in blakemore and crang ( ) . briefly, animals were anesthetized with avertin and received a laminectomy at t . the spinal process cranial to the laminectomy was immobilized using a micromanipulator and the transplant needle, a -al hamilton syringe (hamilton company, reno, nv) with a silicon-coated pulled glass tip, was lowered into the spinal cord using a stereotactic manipulator arm. cell suspensions were injected into one site at the midline of the spinal cord. the needle was removed from the cord min after injection of the cells was complete. in addition, six control animals that received an mhv intracerebral injection received no transplant. one animal died during transplantation/anesthesia. animals were randomly selected for inclusion in either group and all received an incision to the skin overlying t to allow blinded analyses for the duration of the experiment. behavioral testing of all animals was conducted by a blinded observer, every other day using the -point clinical scoring scale (houtman and fleming, ) where = normal, = limp tail, = waddling gait and partial hindlimb weakness, = complete hindlimb paralysis, = death animal. this -point scale was supplemented with a single increment between each point, such that . = limp tail and partial waddling gait and no hindlimb weakness, . = hindlimb weakness and partial hindlimb paralysis, . = complete hindlimb paralysis and moribund disposition. animals were acclimated and behaviorally tested day before mhv injection. after mhv injection, animals were tested every day. after transplantation, animals were tested every other day for weeks and then every rd day until the end of the experiment. statistical significance was determined using the mann -whitney u test. animals were euthanized under chloral hydrate anesthesia (fisher scientific) days following transplantation or days after mhv infection and fixed by cardiac perfusion with % paraformaldehyde (fisher scientific) in . m pbs, ph . . the length of spinal cord extending mm cranial and mm caudal to the site of implantation was cut into -mm transverse blocks and processed so as to preserve the craniocaudal sequence and orientation. alternating tissue blocks were processed for resin and cryostat sectioning and analyzed in a blinded fashion. resin sections were used to determine the area of the ventral and lateral columns, and the number of demyelinated, remyelinated, and normally myelinated axons. cryostat sections were used to determine spread of brdu-prelabeled transplanted cells and their differentiation profile. for resin sectioning, odd numbered blocks were post fixed in % glutaraldehyde (fisher scientific), then exposed to % oso (electron microscopy sciences, fort washington, pa), dehydrated in ascending alcohols, and embedded in spurr resin (electron microscopy sciences) according to standard protocols. transverse semi-thin ( am) sections were cut from the cranial face of each block, stained with alkaline toluidine blue, cover slipped, and examined by light microscopy on an olympus ax- microscope using olympus microsuite b sv software. total areas of the ventral and lateral columns were measured with the  objective and averaged. a t test was performed to compare the areas of the ventral and lateral columns in transplanted and non-transplanted animals. the state of myelination was determined by assessing the thickness of the myelin sheath in relation to the axon diameter (guy et al., ; hildebrand and hahn, ) . demyelinated, remyelinated, and normally myelinated axons were counted within  am areas, totaling am , on each tissue section using the  objective with  optical zoom. am represents approximately % of the total area of remyelination within transplanted animals, which was determined by measuring the total area of remyelination in tissue sections from each block in each animal using the  objective, and averaging areas from all animals in each group. these quantitative assessments were conducted throughout the region extending mm cranial and mm caudal to the implantation site. the number of demyelinated axons, remyelinated axons, the total number of axons (normally myelinated plus demyelinated plus remyelinated axons), and the percentage of remyelinated axons (number remyelinated/number total axons) were determined for each of the four regions on each tissue block, averaged, then averaged across animals within each group for each tissue block. a t test was performed to compare these values for transplanted and non-transplanted groups. to determine whether the number of remyelinated axons in each animal correlated with the total number of axons in each counting area, remyelinated and total axons counts were each averaged among all four counting areas for all blocks for each animal, and the correlation coefficient between these two values was determined for both transplanted and non-transplanted groups. statistical analyses were conducted using spss software. for cryostat sectioning, even numbered blocks were cryoprotected in % sucrose solution in pbs, embedded in oct (fisher scientific) and frozen sectioned in the transverse plane at am on a jung cm leica cryostat for anti-brdu staining. for brdu staining, sections were washed in pbs, exposed to % formamide (sigma-aldrich) and n hydrochloric acid (fisher scientific) for min at jc for dna denaturation, followed by a -min exposure to . % h o in pbs, then incubated overnight at room temperature with rat anti-brdu polyclonal antibody (accurate chemical and scientific corporation) at a : dilution in % ngs. secondary antibody was biotinylated goat anti-rat heavy and light chain igg (sigma-aldrich). vectastain abc (vector laboratories) and , v-diaminobenzidine (vector laboratories) were used for signal visualization. the number of brdu-positive cells was counted on three sections -am apart from each tissue block for each animal using olympus microsuite b sv software, and averaged. the numbers of brdu-positive cells within corresponding blocks from animals within a group were then averaged. for brdu, apc-cc double staining, sections were washed in pbs, exposed to n hydrochloric acid (fisher scientific) for min at jc for dna denaturation, and primary antibodies (rat anti-brdu, : , accurate chemical and scientific corporation; mouse anti-apc-cc , : , oncogene research products, san diego, ca) were diluted in % bovine serum albumin and applied to slides overnight at room temperature. slides were rinsed three times with pbs, incubated for min in % bovine serum albumin, and secondary antibodies (alexa or , goat anti-rat or goat anti-mouse igg h+l, : dilution in % bovine serum albumin; vector laboratories) were applied and incubated for h at room temperature. trypan blue analysis indicated that approximately . - .  viable cells were obtained from each postnatal day mouse. floating cell clusters reached approximately am in diameter after days of culture (fig. a) . on day , clusters were either ( ) dissociated and transplanted into animals or ( ) transferred to an adherent substrate in the absence of growth factors, grown for an additional days and assayed for differentiation potential. as early as h after transfer of cell clusters to an adherent substrate and concomitant growth factor withdrawal, cells started to spread out from the adherent clusters and by day, displayed complex cellular morphologies (fig. b) . after days of growth on adherent substrate, oligodendrocytes and astrocytes could be identified by their morphology and immunolabeling. oligodendrocytes displayed a multipolar morphology with membranous extensions and galc immunoreactivity (fig. c) , whereas astrocytes displayed a flat or stellate morphology and gfap immunoreactivity (fig. d) . few neun immunoreactive neurons (fig. e ) and cd b immunoreactive microglia (results not shown) were detected. no-primary antibody control staining chambers had no positive staining. different cell types often occupied discrete regions of the culture dishes. a total of hoechst-positive cells were counted for the following analyses. quantification of immunolabeled cells indicated that f . % (range = - , median = ) of the total cell population was brdu immunoreactive following an overnight pulse (fig. f) , and that the differentiation protocol yielded . f . % oligodendrocytes (range = - , median = ), f . % astrocytes (range = - , median = ) and . f . % other cell types (range = - , median = ), which included neun + neurons, cd b + microglia, and other hoechst-positive cells not identified by the immunostains tested (fig. g) . no difference was observed in the viability or differentiation of brdu-labeled or -unlabeled cells. transplanted glial-committed progenitors, labeled with brdu before implantation, survived and colonized long lengths of the spinal cord during the -day survival period (fig. ) , and differentiated into mature oligodendrocytes (fig. ) . brdu-labeled cells were detected mm cranial and mm caudal to the site of implantation (the extent of tissue examined), and extended throughout the transverse plane of the spinal cord, primarily within white matter tracts. the number of brdu-labeled cells was greatest around the site of implantation, and fell off sharply - mm either side of the site of implantation. brdu-labeled cells were absent in non-transplanted animals (fig. c) and in no-primary antibody control stains of transplanted animals. non-transplanted animals infected with mhv develop a demyelinating disease accompanied by mononuclear cell infiltration, widespread myelin destruction, and progressive degeneration of the cns in the survivors (fleming et al., ; haring and perlman, ; liu et al., ; stohlman et al., ) . systematic random analyses (fig. a) indicated that numerous demyelinated axons were present (figs. b, e, g) among vacuoles, myelin debris, activated macrophages, lymphocytes, and necrotic cells throughout the region of spinal cord examined, indicative of ongoing pathogenesis. the number of demyelinated axons was not significantly different ( p > . ) in transplanted and non-transplanted animals (fig. a) , likely because mhv is an ongoing demyelinating disease generating a similar number of newly demyelinated axons in both non-transplanted and transplanted animals. the number (fig. b) and percentage (g) quantification of cell types after days of growth on adherent substrate in the absence of growth factors indicated that the differentiation protocol yielded . f . % oligodendrocytes, f . % astrocytes, and . f . % other cell types, which included neun+ neurons, cd b + microglia, and other hoechst-positive cells not identified by the immunostains tested. error bars represent standard deviation.  magnification for a and b,  for magnification for c and d,  magnification for e,  magnification for f. (fig. c ) of remyelinated axons in non-transplanted animals was significantly lower ( p < . ) than in transplanted animals, and composed less than % of the total number of axons counted (fig. c ). normally myelinated axons were exceedingly rare at this time point. control animals injected with saline only did not develop clinical or histopathological deficit. transplantation of glial-committed progenitors resulted in widespread remyelination (figs. and ). remyelinated axons were identified by their characteristically thin my- elin sheaths (figs. c, f, h) and appeared in aggregates distributed throughout the dorsal, ventral, and lateral columns among demyelinated axons. remyelination extended mm cranial and mm caudal to the implantation site in six of seven transplanted animals. counts of remyelinated axons throughout the region extending mm cranial and mm caudal to the implantation site (the extent of our quantification; fig. b ) indicated that regions of remyelination contained % to % remyelinated axons (fig. c) . normally myelinated axons were exceedingly rare at this time point; note that for each tissue block, the number of remyelinated axons (fig. b) plus the number of demyelinated axons (fig. a) approximates the total number of axons (fig. d) . one transplanted animal contained few remyelinated axons, similar to non-transplanted animals ( p > . ); immunohistological analysis of this animal revealed that it contained no brdu-positive cells. we did not observe tumor, teratoma, or non-neuronal tissue formation in transplant recipients, nor did we observe a qualitative difference in schwann cell presence or remyelination between transplanted and non-transplanted animals. counts of the total number of axons (normally myelinated, demyelinated, and remyelinated) within the region extending mm cranial and mm caudal to the transplantation site (the extent of spinal cord examined) suggest that transplanted animals had significantly more axons ( p < . ) within the ventral and lateral columns as compared to non-transplanted animals (figs. g, h and d). area measurements of the ventral and lateral columns revealed no significant difference between animals within and between the transplanted and non-transplanted groups ( p > . , t test), however, a two-sample t test power analysis indicated that an experimental n of per group was insufficient to conclude statistical significance of the differences between the groups. nonetheless, these data suggest that the higher number of total axons within transplanted animals was not a result of tissue shrinkage in the transplanted animals. comparison of the total number of axons with the number of remyelinated axons for each animal within the transplanted group indicated a statistically significant correlation between these two values (r = . ; p < . ), indicating that a greater degree of axonal sparing correlated with a greater degree of remyelination. all transplanted animals survived for the duration of the experiment. non-transplanted animals developed clinical disease typically characterized by a waddling gait and partial hindlimb weakness by - days post-infection, which progressed to complete hindlimb paralysis by days post-infection, persisting for the duration of the experiment (fig. ) . control saline-injected animals displayed normal locomotor behavior throughout the duration of the experiment (all animals scored ). transplantation of glial-committed progenitors resulted in a significant improvement in locomotor abilities (fig. ) . remyelination extended mm cranial and mm caudal to the implantation site (arrow) in transplanted animals (the extent of tissue examined) and was significantly greater than the degree of remyelination in non-transplanted animals at every point examined ( p < . ). (c) throughout this region, % to % of the total number of axons in the ventral and lateral columns of transplanted animals were remyelinated. remyelination in non-transplanted animals was significantly less at every point examined ( p < . ). (d) the total number of axons within am of white matter in transplanted animals was approximately  the total number of axons within similar regions in non-transplanted animals ( p < . for all points), suggesting that remyelination is associated with axonal sparing. error bars represent standard deviation. fig. . transplantation of glial-committed progenitor cells resulted in an improvement in locomotor abilities. transplanted animals demonstrated a significant improvement ( p < . ) in locomotor abilities beginning days after transplantation (arrow). from days after induction of disease, transplanted animals walked with a waddling gait and partial hindlimb weakness, whereas non-transplanted animals demonstrated complete hindlimb paralysis. error bars represent standard deviation. all transplanted animals had complete hindlimb paralysis at the time of transplantation. six of seven transplanted animals demonstrated a significant ( p < . ) improvement in locomotor abilities beginning days after transplantation, which progressed to weight-supported waddling locomotion with partial hindlimb weakness. one transplanted animal demonstrated no improvement in locomotor abilities; postmortem analysis of this animal revealed that it contained no brdu-positive transplanted cells and a degree of remyelination that was not significantly different ( p > . ) from non-transplanted animals. cell replacement strategies must contend with the fact that the differentiation of transplanted cells is strongly influenced by the environmental signals and cellular deficiencies operating at the site of implantation. for example, stem cells isolated from the adult hippocampus generate new hippocampal neurons and glia following transplantation into the hippocampi of other animals, while these same cells generate olfactory bulb neurons expressing appropriate neurotransmitter phenotypes following transplantation into the rostral migratory stream, or glial cells following transplantation into regions of the cns which do not normally generate neurons in the adult (gage et al., ; suhonen et al., ) . these influences present formidable challenges to cell replacement strategies aimed at promoting repair of neurodegenerative disease or injury due to the complex and reactive environment of such lesions, and point towards lineage commitment of multipotent cells before transplantation (keirstead, ) . our differentiation protocol resulted in the generation of oligodendrocytes and astrocytes in high yield ( . % and %, respectively) from postnatal day derived striatal neural precursors (fig. ) , supporting previous studies using a similar differentiation protocol (ben-hur et al., ) . cellular migration in vitro was evident as early as h, and cellular differentiation in vitro was evident as early as day, after transfer of cell clusters to an adherent substrate and concomitant growth factor withdrawal. to assess migration and differentiation in vivo, undifferentiated prelabeled glial-committed progenitors were transplanted into regions of demyelination in the mhv model of ms. intracerebral injection of the j . v- strain of mhv results in initial viral replication in the ependymal cells lining the ventricles, which subsequently extends to the central canal, gray and white matter of the spinal cord. infectious virus is usually eliminated by days - post infection, but is not completely cleared. acute encephalomyelitis with myelin loss is present by day and extends rapidly in the anterior funiculi. an immune-mediated demyelinating encephalomyelitis with hindlimb paralysis and progressive destruction of the cns then follows; although some remyelination takes place, the initiation of new foci of demyelination likely continues for the entire life of the mouse (fleming et al., ; haring and perlman, ; lane et al., ; liu et al., ; stohlman and hinton, ; stohlman et al., ) . in the current study, undifferentiated glial-committed progenitors were transplanted into the thoracic spinal cords of mice with fulminate demyelinating pathology and complete hindlimb paralysis as a result of mhv infection. transplanted cells survived for the duration of the -day post-transplantation period and spread throughout the mediolateral extent of the spinal cord as well as mm cranial and mm caudal to the site of implantation, the extent of the tissue being examined (fig. ) . these findings demonstrate that transplanted progenitor cells are capable of extensive migration throughout regions of ongoing pathogenesis. survival and extensive migration of transplanted gliogenic cell populations has been demonstrated in the developing normal cns (jacque et al., ) , myelin-deficient mutant cns (brustle et al., ; gansmuller et al., ; liu et al., ; tontsch et al., ) , chemically demyelinated cns (groves et al., ; , and in inflamed white matter tracts in the eae model of ms (ben-hur et al., ; pluchino et al., ) . however, the ability of gliogenic cell populations to survive and migrate in these environments does not indicate that they will do so in the intact normal adult cns. when glial progenitors (franklin et al., ; o'leary and blakemore, ) are transplanted into the intact normal adult cns, they fail to survive or migrate; only when the cells are placed in the immediate vicinity of active regions of demyelination, where survival factors are elevated (hinks and franklin, ) , do they enter the lesion. these findings suggest that the survival and extensive migration of cells in our study was due in part to the continuity of pathology throughout the region of migration. transplantation of glial-committed progenitors resulted in extensive remyelination, ranging from % to % of the axons present within randomly selected regions of the spinal cord (figs. and ) . the in vitro (fig. ) and in vivo (fig. ) differentiation profile of these cells, the presence of extensive remyelination only in transplanted animals (figs. b, c) , and the presence of remyelination throughout the region over which transplanted cells migrated (figs. d and b) suggest that remyelination was conducted by the transplanted cell population. nonetheless, we cannot rule out the possibility that remyelination in transplanted animals was carried out by endogenous cells that may have been activated as a result of trophic support by the transplanted population. cumulative axonal loss has recently been demonstrated in several human (bitsch et al., ; ferguson et al., ; trapp et al., ) and rodent (bjartmar et al., ; griffiths et al., ; yin et al., ) demyelinating pathologies, using in vivo magnetic resonance imaging (stevenson and miller, ; van waesberghe et al., ) , in vivo magnetic resonance spectroscopy (arnold et al., ; matthews et al., ) , morphological analysis of brain sections (ferguson et al., ; trapp et al., ) , and biochemical methods (bjartmar et al., ) . chronic demyelination is a probable cause of such axonal loss (bjartmar and trapp, ) . to determine whether remyelination was associated with axonal sparing in the current study, total axon numbers were determined within the ventral and lateral columns of transplanted and non-transplanted animal groups days after induction of disease. although the area of the ventral and lateral columns were not significantly different between the transplanted and nontransplanted groups ( p > . , t test), the total number of axons within the ventral and lateral columns of transplanted remyelinated animals was approximately  the number of axons within the ventral and lateral columns of non-transplanted animals (fig. d) . furthermore, comparison of the total number of axons with the number of remyelinated axons for each animal within the transplanted group indicated a statistically significant correlation between these two values (r = . ). these findings suggest that a greater degree of axonal sparing correlated with a greater degree of remyelination. reduced axonal loss has also been shown after intravenous or intrathecal neurosphere transplantation in the eae mouse model of ms (pluchino et al., ) . these data support the view that chronic demyelination is a cause of axonal loss, and suggest that transplant-mediated remyelination protects axons from degeneration/transection. further studies are required to determine the molecular mechanisms underlying demyelination-associated axonal degeneration/transection. transplantation of glial-committed progenitors resulted in a significant improvement in locomotor abilities beginning days after transplantation, which progressed to weightsupported waddling locomotion with partial hindlimb weakness (fig. ) . non-transplanted animals remained completely paralyzed throughout this time period (fig. ). in regions of experimental demyelination induced by lysolecithin (smith et al., (smith et al., , yezierski et al., ) or ethidium bromide (felts and smith, ) , return of secure axonal conduction occurs at the same time as spontaneous remyelination and transplanted glial cells have been shown to improve conduction velocity to near-normal values in myelin-deficient rats (utzschneider et al., ) and ethidium bromide lesions (honmou et al., ) . transplantation of clonal neural cells into the intracerebroventricular system of the shiverer mice at birth resulted in myelination of up to % of host neuronal processes and reduced the symptomatic tremor in some animals (yandava et al., ) . thus, it is intriguing that the time course of behavioral improvement following transplantation in the current study approximates the time required for remyelination (pender et al., ; smith et al., ) . our findings demonstrate that transplant-mediated remyelination is possible in the complex and reactive environment of the mhv model of multiple sclerosis. transplant-mediated remyelination has been demonstrated in regions of the cns subjected to experimental demyelination or in myelin mutants, environments that are characterized by a clear cellular deficit, a largely non-reactive cns environment, and a lack of inflammation. thus, transplantation of embryonic stem cell-derived precursors (brustle et al., ) or oligodendrocyte progenitors (zhang et al., ) into postnatal myelin-deficient rats resulted in differentiation of transplanted cells into myelinating oligodendrocytes and astrocytes. multipotential psa-ncam + neural precursors isolated from the postnatal rat brain have been shown to differentiate into oligodendrocytes, schwann cells, and astrocytes following transplantation, to completely remyelinate regions of acute demyelination in the adult rat induced by ethidium bromide injection into x-irradiated dorsal column white matter . similarly, transplantation of embryonic stem cells into regions of ethidium bromide induced demyelination in the adult rat spinal cord or into myelin-deficient shiverer mutant mice, resulted in the generation of stem cell-derived oligodendrocytes that were capable of myelinating axons (liu et al., ) . more recently, transplant-mediated remyelination has been illustrated in the eae model of ms, following intravenous or intrathecal injection of adult neural precursor cells (pluchino et al., ) . our findings confirm and extend those outlined above, in demonstrating that intraspinal transplantmediated remyelination can occur in the fulminate neurodegenerative environment of the mhv model of ms and is associated with locomotor improvement. the ability of glial cell preparations to remyelinate in such models does not necessarily indicate that their transplantation into demyelinated lesions in clinical disease will be successful (stangel, ) . firstly, transplanted cells may be subject to the same demyelinating process that led to the development of the initial pathology. notably, the viral 'trigger' of pathology in the mhv model of ms is largely cleared by days post-infection (lane et al., ; stohlman and hinton, ) , before the time of implantation in the current study. secondly, the inability of glial progenitors to migrate through normal tissue (o'leary and renders them less than ideal for remyelinating disparate foci of demyelination as seen in clinical disease (prineas et al., a) . thirdly, human transplantation necessitates a human progenitor or stem cell population that is capable of amplification and is myelinogenic. although robust amplification of human embryonic stem cells has been achieved (carpenter et al., ; reubinoff et al., ) , the conditions needed to differentiate them into a remyelinationcompetent transplant population have not yet been defined. and fourthly, the gliotic environment of clinical lesions, which may contribute to the failure of endogenous remyelination, may prohibit the transplant population from effecting repair. clearly, several key issues must be addressed before a cellular replacement strategy can be considered for the treatment of human demyelinating pathologies such as ms. use of proton magnetic resonance spectroscopy for monitoring disease progression in multiple sclerosis growth and fate of psa-ncam+ precursors of the postnatal brain transplanted multipotential neural precursor cells migrate into the inflamed white matter in response to experimental autoimmune encephalomyelitis acute axonal injury in multiple sclerosis. correlation with demyelination and inflammation axonal and neuronal degeneration in multiple sclerosis: mechanisms and functional consequences axonal pathology in myelin disorders neurological disability correlates with spinal cord axonal loss and reduced nacetyl aspartate in chronic multiple sclerosis patients remyelination of the superior cerebellar peduncle in the mouse following demyelination induced by feeding cuprizone remyelination of the superior cerebellar peduncle in old mice following demyelination induced by cuprizone remyelination by schwann cells of axons demyelinated by intraspinal injection of -aminonicotinamide in the rat ethidium bromide induced demyelination in the spinal cord of the cat transplantation of glial cells into areas of demyelination in the adult rat spinal cord embryonic stem cellderived glial precursors: a source of myelinating transplants enrichment of neurons and neural precursors from human embryonic stem cells axonal damage correlates with disability in patients with relapsing -remitting multiple sclerosis. results of a longitudinal magnetic resonance spectroscopy study transplantation of oligodendrocytes and schwann cells into the spinal cord of the myelin-deficient rat conduction properties of central nerve fibers remyelinated by schwann cells axonal damage in acute multiple sclerosis lesions experimental demyelination induced by coronavirus jhm (mhv- ): molecular identification of a viral determinant of paralytic disease transplanted cg cells (an oligodendrocyte progenitor cell line) survive, migrate, and contribute to repair of areas of demyelination in x-irradiated and damaged spinal cord but not in normal spinal cord survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain tracing transplanted oligodendrocytes during migration and maturation in the shiverer mouse brain axonal swellings and degeneration in mice lacking the major proteolipid of myelin repair of demyelinated lesions by transplantation of purified o- a progenitor cells myelination and remyelination in the central nervous system by transplanted oligodendrocytes using the shiverer model. discussion on the remyelinating cell population in adult mammals spectra of g ratio, myelin sheath thickness, and axon and fiber diameter in the guinea pig optic nerve mouse hepatitis virus regeneration of oligodendroglia during recovery from demyelinating disease relation between myelin sheath thickness and axon size in spinal cord white matter of some vertebrate species distinctive patterns of pdgf-a, fgf- , igf-i, and tgf-beta gene expression during remyelination of experimentally-induced spinal cord demyelination restoration 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alpha-and beta-chemokine expression in the central nervous system during mouse hepatitis virusinduced demyelinating disease embryonic stem cells differentiate into oligodendrocytes and myelinate in culture and after spinal cord transplantation neutralization of the chemokine cxcl reduces inflammatory cell invasion and demyelination and improves neurological function in a viral model of multiple sclerosis putting magnetic resonance spectroscopy studies in context: axonal damage and disability in multiple sclerosis spontaneous and induced remyelination in multiple sclerosis and the theiler's virus model of central nervous system demyelination dose-dependency of mbp-induced demyelination in the guinea pig spontaneous remyelination following extensive demyelination is associated with improved neurological function in a viral model of multiple sclerosis oligodendrocyte precursors survive poorly and do not migrate following transplantation into the normal adult central nervous system patterns of oligodendroglia pathology in multiple sclerosis demyelination and early remyelination in experimental allergic encephalomyelitis passively transferred with myelin basic protein-sensitized lymphocytes in the lewis rat injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis multiple sclerosis: remyelination of nascent lesions multiple sclerosis. pathology of recurrent lesions effective cryopreservation of human embryonic stem cells by the open pulled straw vitrification method immunoglobulins promote remyelination in the central nervous system remyelination by oligodendrocytes stimulated by antiserum to spinal cord demyelination and remyelination in the dorsal funiculus of the rat spinal cord after heat injury remyelination occurs as extensively but more slowly in old rats compared to young rats following gliotoxin-induced cns demyelination central remyelination restores secure conduction the restoration of conduction by central remyelination transplantation of myelinating cells as regenerative therapy for multiple sclerosis-experimental basis and present state of clinical studies magnetic resonance imaging in the monitoring of disease progression in multiple sclerosis viral induced demyelination the art of survival during viral persistence differentiation of adult hippocampus-derived progenitors into olfactory neurons in vivo ultrastructural studies of multiple sclerosis transplantation of an oligodendrocyte cell line leading to extensive myelination axonal transection in the lesions of multiple sclerosis transplantation of glial cells enhances action potential conduction of amyelinated spinal cord axons in the myelin-deficient rat axonal loss in multiple sclerosis lesions: magnetic resonance imaging insights into substrates of disability demyelination and remyelination in the rat central nervous system following ethidium bromide injection. lab global'' cell replacement is feasible via neural stem cell transplantation: evidence from the dysmyelinated shiverer mouse brain insulin-like growth factor i treatment reduces demyelination and up-regulates gene expression of myelin-related proteins in experimental autoimmune encephalomyelitis effects of dorsal column demyelination on evoked potentials in nucleus gracilis myelin-associated glycoprotein is a myelin signal that modulates the caliber of myelinated axons adult brain retains the potential to generate oligodendroglial progenitors with extensive myelination capacity we thank oswald steward for discussion and advice. we thank michael liu for assistance with viral injections, rafael gonzalez and giovanna bernal for assistance with animal surgeries, and josh kunellis for assistance with tissue processing. this project was supported by the national multiple sclerosis society, and individual donations to the reeve-irvine research center. key: cord- -dkp fwe authors: mazzulli, tony; farcas, gabriella a.; poutanen, susan m.; willey, barbara m.; low, donald e.; butany, jagdish; asa, sylvia l.; kain, kevin c. title: severe acute respiratory syndrome–associated coronavirus in lung tissue date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: dkp fwe efforts to contain severe acute respiratory syndrome (sars) have been limited by the lack of a standardized, sensitive, and specific test for sars-associated coronavirus (cov). we used a standardized reverse transcription-polymerase chain reaction assay to detect sars-cov in lung samples obtained from well-characterized patients who died of sars and from those who died of other reasons. sars-cov was detected in all postmortem lung tissues (to ( ) viral copies/g) from patients with probable sars but was not detected in any of the lung control samples (sample analysis was blinded). the sensitivity and specificity ( % confidence interval) were % ( . % to %) and % ( . % to %), respectively. viral loads were significantly associated with a shorter course of illness but not with the use of ribavirin or steroids. cov was consistently identified in the lungs of all patients who died of sars but not in control patients, supporting a primary role for cov in deaths. efforts to contain severe acute respiratory syndrome (sars) have been limited by the lack of a standardized, sensitive, and specific test for sars-associated coronavirus (cov). we used a standardized reverse transcription-polymerase chain reaction assay to detect sars-cov in lung samples obtained from well-characterized patients who died of sars and from those who died of other reasons. sars-cov was detected in all postmortem lung tissues (to viral copies/g) from patients with probable sars but was not detected in any of the lung control samples (sample analysis was blinded). the sensitivity and specificity ( % confidence interval) were % ( . % to %) and % ( . % to %), respectively. viral loads were significantly associated with a shorter course of illness but not with the use of ribavirin or steroids. cov was consistently identified in the lungs of all patients who died of sars but not in control patients, supporting a primary role for cov in deaths. province, china, severe acute respiratory syndrome (sars) has become an emerging infectious disease that has spread to areas throughout the world, including hong kong, vietnam, singapore, taiwan, and canada ( ). although controversy remains over the etiology of sars, the world health organization has declared a newly described virus known as the sars-associated coronavirus (sars-cov) as the cause ( ). this announcement has led to a rapid proliferation of different in-house laboratory tests aimed at detecting either sars-cov-specific antibodies or sars-cov nucleic acid in clinical specimens. the centers for disease control and prevention definition for a confirmed case of sars includes the results of these laboratory tests ( ) . however, because different assays are being used, comparing results from different centers has been difficult. in addition, the inability of these nonstandardized tests to detect sars-cov in all cases has led to speculation that other agents may be associated with sars. some researchers have suggested that illnesses that progress to respiratory failure and death may not be caused by uncontrolled viral replication but rather are the result of an immunopathologic process ( ). in a recent report of six fatal cases of sars, sars-cov was detected by reverse transcriptase-polymerase chain reaction (rt-pcr) in postmortem lung tissue in only four patients ( ) . the purpose of this study was to use a standardized, commercially available, rt-pcr assay to test for the presence of sars-cov rna. lung tissue obtained at autopsy from well-characterized patients with sars who died during the outbreak in canada were compared to lung samples obtained at autopsy from patients without sars who died during the outbreak and lung samples from patients who died before the outbreak. all patients who met the current world health organization case definition of probable sars and who underwent a postmortem examination in canada during the march-april outbreak were included in this study. clinical details were extracted retrospectively from hospital records. clinical descriptions of some of these cases have been published separately ( , ) . as of may , , a total of patients died of sars in canada; all died in toronto. of the patients, autopsies were performed on patients. results of ante-and postmortem examination for routine bacterial and viral respiratory pathogens from these patients, as described elsewhere, were negative ( ). a total of discrete postmortem lung samples collected from these patients were included in this analysis. an additional postmortem lung samples from patients who died during the sars outbreak but whose deaths were attributed to other causes were also included. the attributed cause of death in these patients was as follows: a -yearold woman died of invasive group a streptococcal infection; a -year-old man died of congestive heart failure; a -year-old man died of sudden death cardiovascular disease; a -year-old man died of amiodarone pulmonary toxicity; a -year-old woman died of dementia and aspiration pneumonia; a -year-old woman died of diabetes and congestive heart failure; and an -year-old man died of bladder cancer and aspiration pneumonia. in addition, lung samples collected in from patients ( women and men; age range - years) with lung cancer were also included as negative controls. all samples collected at the time of autopsy were snap frozen in a mixture of absolute ethanol and dry ice and subsequently stored at - °c until tested. the samples were coded and then processed, subjected to rt-pcr analysis, and interpreted before the identity of the samples was divulged. this study was approved by the research ethics boards at mount sinai hospital and the university health network. lung tissue samples were thawed and immediately homogenized in lysis buffer (qiagen, mississauga, canada) with disposable tissue grinders (kendall precision, mansfield, ma). the homogenate was passed through qiashredder columns (qiagen) before rna isolation by using the rneasy mini kit (qiagen). the sample was eluted in µl of rnase free water. the rt-pcr was carried out by using the realart hpa-coronavirus lightcycler rt reagents assay (artus gmbh, hamburg, germany) with a lightcycler real-time platform (roche diagnostics, laval, canada). the hpa-coronavirus master mix contains reagents and enzymes for the specific amplification of an -bp region of the sars-cov polymerase gene from µl of rna with the primer pairs published by the bernhard-nocht institute (hamburg, germany) as posted on the world health organization web site (available from: url: http://www.who.int/csr/sars/primers/en/). viral load was calculated from a standard curve based on four external positive controls (quantification standards) included in the realart hpa-coronavirus lightcycler rt reagents assay kit ( figure a and b) . the standards were treated as previously purified samples, and the same -ml volume was added per capillary. a standard preparation of sars-cov isolated from cell culture supernatants of veroe cells was used as a calibrator in each run. in addition, the kit contains a second heterologous amplification system (i.e., an internal control) to identify either pcr inhibition exclusively, if added to the extracted rna, or rna isolation quality as well as pcr inhibition, if added during the rna isolation procedure ( figure c) . although the assay insert states that the primers and probes used in the assay were checked for possible similarity to other pathogens by means of sequence comparison, randomly chosen amplicons from our sample pool were independently sequenced to confirm sars-cov-specific amplification and detection. univariate analysis comparing potential predictors of viral load (e.g., duration of illness, the use of ribavirin, the use of steroids) was completed by using fisher exact test. two-sided p values < . were considered significant. the clinical description and rt-pcr results for the patients with probable sars from whom postmortem lung tissue samples were examined are summarized in table . the mean age of the patients was years (range - ). six of the patients were men. all but of the patients had underlying coexisting conditions, the most common of which was diabetes mellitus in patients. the mean duration of illness was days (range - ). seven patients had been intubated and mechanically ventilated before death. three patients had requested not to be intubated (information on ventilation was not available for one patient). ten of the patients were treated with ribavirin; of the patients were treated with steroids. sars-cov was detected in all postmortem lung tissue samples collected from all patients who died with a diagnosis of probable sars. all postmortem lung samples from the seven non-sars fatalities that occurred during the sars outbreak were negative for sars-cov, as were all lung-tissue samples collected from patients with lung cancer years before the outbreak ( table ). the corresponding sensitivity and specificity of the realart hpa-coronavirus lightcycler rt reagent assay are both % ( % confidence interval [ci] for sensitivity . % to %; % ci for specificity . % to %) for the detection of sars-cov. the sars-cov viral load in postmortem lung tissue ranged from . x copies/g tissue to . x copies/g tissue. higher viral loads (> copies/g tissue) were associated with patients who had a shorter duration of illness (< days) (p= . , fisher exact test). the use of ribavirin or steroids was not significantly associated with viral load levels ( table ) . twenty-five randomly selected amplicons from the sample pool were sequenced to assess specificity and possible cross-reactivity to other pathogens. a blast (available from: url: http://www.ncbi.nlm.nih.gov/blast/) search performed against the sars-cov genomes in genbank, european molecular biology laboratory, dna data bank of japan, and protein data bank on the national center for biotechnology information web site (available from: url: http://www.ncbi.nlm.nih.gov/), indicated that all amplicon samples contained sars-cov polymerase gene sequence. by using a standardized rt-pcr assay, sars-cov has been unequivocally identified in the lung tissue of all patients who died with probable sars but not in any of the controls. these observations support a primary role for this virus in patients with sars who have fatal outcomes and provide additional, strong evidence to fulfill koch's postulates regarding sars-cov as the cause of sars ( ) . sars-cov was found in different lung samples from the same patient, suggesting that the virus is widely disseminated throughout the lung at the time of death. previous studies suggested that progression of disease to respiratory failure may be primarily mediated by host immune response rather than viral replication ( ) . although viral rna in lung tissue does not necessarily indicate replicating virus, virus in multiple lung lobes, often in high copy number, at the time of death suggests that sars-cov may also be contributing to disease progression. the fact that higher viral loads were significantly associated with patients with a shorter duration from onset of illness to death supports the role of viral replication as a contributor to death. ten of the patients had received therapy with ribavirin, and patients were treated with steroids. the failure to eradicate sars-cov despite ribavirin therapy and the lack of association between the use of ribavirin and sars-cov viral load are consistent with in vitro data showing that ribavirin has no activity against this agent ( ) . global efforts to contain sars have been severely impeded by the lack of a standardized, sensitive, and specific diagnostic test for sars-cov. different diagnostic strategies, including culture, serologic assays, and molecular detection methods, have been described, but each of these tests has limitations. in-house rt-pcr assays have been associated with sensitivities as low as % in patients with sars ( ) , which raises uncertainty about the role of cov versus co-pathogens in mediating severe or fatal sars. by contrast, the sensitivity and specificity of the realart hpa-coronavirus rt-pcr assay for detecting cov in lung tissue samples appear to be excellent. in addition, with the real-time lightcycler system, the assay generates quantitative results within hour, which is much shorter than traditional pcr reactions. the type of specimen tested, the timing of sample collection, (i.e., acute versus convalescent phase) the method of specimen collection, as well as the method of sample preservation may have substantial impact on the results obtained from a diagnostic test. the lower sensitivity of sars-cov detection reported by peiris et al. ( ) may be a consequence of these confounding factors. our study design of examining lung biopsies from clearly defined patient populations overcame confounding issues, such as sampling technique, nonspecific case definitions, and possible undocumented exposure to sars. given the predominance of respiratory symptoms in patients with sars, lung samples have perhaps the highest viral titers of all specimen types; yet in nonfatal cases, obtaining routine lung biopsies is not practical. other respiratory tract specimens may be satisfactory substitutes for biopsies, but further studies examining the prevalence of sars-cov in these other specimen types and in a larger population are needed. with the use of standardized commercially available assays, comparison of results from different centers may be facilitated. update: outbreak of severe acute respiratory syndrome-worldwide world health organization. coronavirus never before seen in humans is the cause of sars-update . geneva: the organization updated interim surveillance case definition for severe acute respiratory syndrome (sars)-united states clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study lung pathology of fatal severe acute respiratory syndrome identification of severe acute respiratory syndrome in canada clinical features and short-term outcomes of patients with sars in the greater toronto area koch's postulates fulfilled for sars virus severe acute respiratory syndrome (sars) and coronavirus testing-united states coronavirus as a possible cause of severe acute respiratory syndrome address for correspondence: kevin kain, tropical disease unit, eaton south - , toronto general hospital we thank allison mcgeer, karen green, poolak akhavan, sylvia pong-porter, peter faure, wayne gold, the ontario coroner's office; brendan mullen for generously providing control lung samples; h.w. doerr and m. niedrig for generously providing supernatants of veroe cells; and all of the clinicians who were involved in courageously caring for the patients described in this paper who lost their lives to severe acute respiratory syndrome.this study is supported by a grant from the canadian institute of health research (mt- to k.c.k). k.c.k. is supported by a career scientist award from the ontario ministry of health and a canada research chair. artus gmbh provided assays for this study, but neither they nor the sponsors of the study had any role in the study design, data collection, data analysis, data interpretation, or writing of the report. none of the authors has a conflict of interest in relationship to this study.dr. mazzulli is deputy chief microbiologist at toronto medical labaratories/mount sinai hospital department of microbiology, toronto canada. he is also associate professor in the department of pathology and laboratory medicine, university of toronto. key: cord- - t wpiqy authors: webby, rj; perez, dr; coleman, js; guan, y; knight, jh; govorkova, ea; mcclain-moss, lr; peiris, js; rehg, je; tuomanen, ei; webster, rg title: responsiveness to a pandemic alert: use of reverse genetics for rapid development of influenza vaccines date: - - journal: lancet doi: . /s - ( ) - sha: doc_id: cord_uid: t wpiqy background: in response to the emergence of severe infection capable of rapid global spread, who will issue a pandemic alert. such alerts are rare; however, on feb , , a pandemic alert was issued in response to human infections caused by an avian h n influenza virus, a/hong kong/ / . h n had been noted once before in human beings in and killed a third ( / ) of infected people. , the variant seemed to have been transmitted directly from birds to human beings and caused fatal pneumonia in one of two infected individuals. candidate vaccines were sought, but no avirulent viruses antigenically similar to the pathogen were available, and the isolate killed embryonated chicken eggs. since traditional strategies of vaccine production were not viable, we sought to produce a candidate reference virus using reverse genetics. methods: we removed the polybasic aminoacids that are associated with high virulence from the haemagglutinin cleavage site of a/hong kong/ / using influenza reverse genetics techniques. a reference vaccine virus was then produced on an a/puerto rico/ / (pr ) backbone on who-approved vero cells. we assessed this reference virus for pathogenicity in in-vivo and in-vitro assays. findings: a reference vaccine virus was produced in good manufacturing practice (gmp)-grade facilities in less than weeks from the time of virus isolation. this virus proved to be non-pathogenic in chickens and ferrets and was shown to be stable after multiple passages in embryonated chicken eggs. interpretation: the ability to produce a candidate reference virus in such a short period of time sets a new standard for rapid response to emerging infectious disease threats and clearly shows the usefulness of reverse genetics for influenza vaccine development. the same technologies and procedures are currently being used to create reference vaccine viruses against the h n viruses circulating in asia. in february, , two family members were admitted to intensive care wards in hong kong special administrative region with influenza-like respiratory illness. avian-like h n influenza viruses were isolated from both patients, one of whom succumbed to infection. this was the first time since that h n viruses had been identified in human beings, and who responded by issuing a pandemic alert. candidate vaccines were immediately sought. the recent outbreak of severe acute respiratory syndrome (sars) had been a striking example of the rapid and global spread of an emerging infectious disease. however, even the effects of sars could be dwarfed by those that could arise with the emergence of an influenza pandemic. infection caused by the influenza a virus is a zoonosis, and the animal reservoir of this virus is the aquatic bird populations of the world. the compelling epidemiological link between the presence of the virus in poultry in live-bird markets and the appearance of h n in human beings in suggested that influenza a viruses can be transmitted directly from avian species to man and can cause severe respiratory disease. [ ] [ ] [ ] although control of the outbreak was achieved by culling millions of birds in the hong kong markets, this episode demonstrated that the capability for an effective global response to emerging influenza threats was poor because of technical, legislative, and infrastructural limitations. a disturbing finding that emerged from this event was that the scientific community was unable to produce an effective vaccine even after several years. the inactivated human influenza vaccines in use today are derived from essentially modified viruses. by exploiting the segmented nature of the influenza a genome, vaccine manufacturers and the laboratories of the who influenza network have produced a reassortant virus carrying the circulating virus's gene segments that encode haemagglutinin and neuraminidase, the major targets of neutralising antibodies. the remaining six-gene segments are supplied from pr , a laboratoryadapted avirulent h n strain. the resulting reassortant virus has the antigenic properties of the circulating strain and the safety and high-yield properties of pr . the first batch of inactivated material against the h n virus was not ready for clinical trial until months after the second case of human infection arose, and even today the effectiveness of vaccine against this virus has not been proven. a key reason for this delay in the production of an h n -specific vaccine was the nature of the virus itself. the h n virus is highly pathogenic in human beings and poultry. the agent must be handled only under conditions of at least biosafety level (bsl ), and it can kill fertilised chicken eggs, the standard medium for the reassortment and responsiveness to a pandemic alert: use of reverse genetics for rapid development of influenza vaccines propagation of influenza virus before its inactivation and formulation for use in vaccines. these same traits are present in the h n virus. the pathogenic nature of these h n viruses is linked to the presence of additional basic residues in haemagglutinin at the site of cleavage, a step required for haemagglutinin activation and, thus, for virus entry into cells. [ ] [ ] [ ] to overcome the high pathogenicity of the virus, polybasic aminoacids have to be eliminated. a rapid, reproducible system to achieve these modifications-ie, plasmid-based reverse genetics-has been developed only in the past - years [ ] [ ] [ ] the potential benefits of reverse genetics for the generation and attenuation of vaccine candidates against highly pathogenic and low pathogenic influenza viruses are enormous. [ ] [ ] [ ] however, the host specificity of the rna polymerase i promoter used in the influenza reverse-genetics systems and the required use of an approved cell line limits the practical options for the system's use in the manufacture of human vaccines. the vaccine-candidate reference virus stock described in this report has been produced entirely on a cell substrate licensed for the manufacture of human vaccine, and as such, is-to our knowledge-the first reverse genetically derived influenza vaccine suitable for testing in clinical trials. we describe the construction of a vaccine reference virus in good manufacturing practice (gmp)-grade facilities in less than weeks from the time of virus isolation. our findings highlight the speed with which new technologies can be implemented in response to influenza pandemic alerts. we obtained who-approved vero cells (who-vero, x , p ) from the american type culture collection (manassas, virginia, usa). passage- cells (five passages since their removal from a working cell bank) were used for the rescue of the vaccine-candidate virus. the plasmids containing the genes from pr have been described elsewhere. virus propagation, rna extraction, pcr amplification, and haemagglutinin and neuraminidase gene cloning we obtained a/hong kong/ / (h n ) that had been passaged in eggs from the who influenza network. the virus was isolated and propagated in -day-old embryonated chicken eggs. total rna was extracted from infected allantoic fluid with use of the rneasy kit (qiagen, valencia, ca, usa) in accordance with manufacturer's instructions. reverse transcription was carried out with the uni primer ( Ј-agca aaagcagg- Ј) and amv reverse transcriptase (roche, indiana biochemicals indianapolis, usa). the removal of the connecting peptide of the haemagglutinin was done with use of pcr with the following primer sets: ( ) bm-ha- ( Ј-tattcgtctcagggagcaa aagcagggg- Ј) and ⌬r ( Ј-taatcgtc tcgtttcaatttgagggctatttctgagcc- Ј); and ( ) ⌬f ( Ј-taatcgtctctgaaa ctagaggattatttggagctatagc- Ј) and bm-ns- r ( Ј-atatcgtctcgtattagtag aaacaagggtgtttt- Ј). we amplified the neuraminidase gene of a/hong kong/ / using the primer pair ba-na- ( Ј-tattggtctc agggagcaaaagcaggagt- Ј) and ba-na- r ( Ј-atatggtctcgtattagtagaaacaag gagtttttt- Ј). pcr products were purified and cloned into the vector phw as described previously. the rescue of infectious virus from cloned cdna was done under gmp conditions. vero cells were grown to % confluency in a cm flask, trypsinised (with trypsin-versene), and resuspended in ml of opti-mem i (invitrogen, carlsbad ca, usa). to ml of cell suspension we added ml of fresh opti-mem i; then, we added ml of this diluted suspension to each well of a six-well tissue culture plate (about ϫ cells per well). the plates were incubated at °c overnight. the next day, g of each plasmid and l of transit lt- transfection reagent (panvera, madison, wi, usa) were added to opti-mem i to a final volume of l and the mixture incubated at room temperature for min. after incubation, the medium was removed from one well of the six-well plate, l of opti-mem i added to the transfection mix, and this mixture added dropwise to the cells. h later, the dna-transfection mixture was replaced by opti-mem i. h after transfection, ml of opti-mem i that contained g/ml l-(tosylamido- phenyl) ethyl chloromethyl ketone (tpck)-treated trypsin (worthington biochemicals, lakewood, nj, usa) was added to the cells. about h after the addition of tpck-trypsin, the culture supernatants were harvested and clarified by low-speed centrifugation; we then injected l of the clarified supernatant into the allantoic cavity of individual -day-old pathogen-free embryonated research grade eggs (charles river spafas, north franklin, ct, usa). ten -week-old chickens received intravenous injections of · ml diluted virus (dilution ratio, / ). we monitored chickens for signs of disease for days using the intravenous pathogenicity index, approved by the office of international epizooites (oie). additionally, we took tracheal and cloacal swabs (in ml of media) days and days after infection, and we did assays for the presence of virus by injection of · ml into all of three -day-old embryonated chicken eggs. haemagglutination activity in the allantoic fluid of these eggs was assessed after incubation at °c for days. pathogenicity testing in ferrets we tested pathogenicity of the vaccine in five young adult male ferrets (marshall's farms, north rose, ny, usa) aged - months (weight about · kg) that were shown by haemagglutination inhibition assays to be seronegative for currently circulating human influenza a viruses (h n , h n ) and h n viruses. we anaesthetised the ferrets with inhaled isoflurane, and they were then infected intranasally with % egg infectious dose (eid )/ml of vaccine reassortant virus or wildtype virus. we monitored the ferrets once per day for signs of sneezing, inappetence, and inactivity, and we recorded rectal temperatures and bodyweights. , , and days after infection, the ferrets were anaesthetised with ketamine ( mg/kg), and we collected nasal washes using ml of sterile phosphatebuffered saline (pbs) containing antibiotics. we measured titres of virus in these washes with eid assays. to further assess the pathogenicity of the viruses, we collected tissue samples from lungs, brain, olfactory bulb, spleen, and intestine for virus isolation and histopathological analysis at the time of death or in the case of three ferrets, after euthanasia at day after infection. the tissues were fixed in % neutral buffer formalin, processed and embedded in paraffin, sectioned at g, stained with haematoxylin and eosin and examined by light microscopy in a blinded fashion. to test the stability of the vaccine virus on propagation, we made consecutive passages of the virus in embryonated chicken eggs. a - dilution of the virus was made in pbs, and · ml of the solution was injected into the allantoic cavities of all of four -day-old embryonated chicken eggs. eggs were incubated at ºc for · - days. after incubation, each egg was candled to determine embryo viability before chilling at ºc. we harvested ml of allantoic fluid from each egg harvested, and samples were pooled together, tested for haemagglutination activity, and then reinjected into another four eggs. the sponsor had no role in study design, in the collection, analysis, and interpretation of data, in the writing of the report or decision to submit this manuscript for publication. the first challenge we faced in producing a vaccine against a/hong kong/ / (h n ) was to attenuate the virus in preparation for mass production. previous experiences have shown that removal of the basic aminoacids at the haemagglutinin cleavage site substantially attenuates pathogenic influenza viruses. [ ] [ ] [ ] using a pcr-based mutagenesis approach, we replaced the cleavage site encoded by the haemagglutinin gene of a/hong kong/ / (h n ) with that of the avirulent a/teal/hong kong/w / (h n ) (figure ); this modified haemagglutinin gene and the neuraminidase gene of a/hong kong/ / (h n ) were cloned individually into the vector phw . the two resulting plasmids and the six plasmids encoding the remaining proteins of pr were transfected into whoapproved vero cells under gmp conditions to rescue the vaccine seed virus, ⌬ /pr . - h after transfection, isolated areas of cytopathic effect could be seen on the vero monolayers. although addition of further g aliquots of tpck-treated trypsin every h led to a proportional increase in the cytopathic effect, it was not required for successful virus rescue. the candidate vaccine strain grew to high titres on subsequent amplification in eggs (haemagglutination titres of - ) and did not cause embryo death. the vaccine seed virus was unable to form plaques on madin-darby canine kidney (mdck) cells in the absence of trypsin, a trait consistent with that of influenza viruses that lack the polybasic cleavage site, and was antigenically indistinguishable from the parental h n virus in haemagglutination inhibition assays. the rescued virus was fully sequenced and was identical to the plasmids used in its creation. to assess the pathogenicity of the h n vaccine seed virus, we compared the properties of this virus with those of the wildtype a/hong kong/ / (h n ) in ferrets and in chickens. by stark contrast with the wildtype virus, which was lethal to all chickens within h of infection, intravenous administration of a / dilution of ⌬ /pr did not result in any signs of infection in chickens, and we were unable to detect any virus in swabs of cloacae or tracheae from inoculated birds. compared with a/hong kong/ / (h n ), ⌬ /pr was attenuated in ferrets that had been inoculated intranasally with eid of virus. ferrets infected with a/hong kong/ / had inappetence and weight loss (figure ), with one infected animal dying days after infection and a second killed days after infection because of hind-limb paralysis. infection in these animals was characterised by viral shedding until days after infection and replication of virus in the lower respiratory tract and olfactory bulb (as determined by virus isolation). in the a/hong kong/ / infected animals, there was a mild mononuclear cell infiltrate in the meninges and tracheal submucosal mucous glands and an extensive bronchopneumonia. the pneumatic infiltrate progressed in severity from the bronchi to the pleura. the bronchi and bronchioles contained sloughed necrotic epithelial cells, numerous mononuclear cells, and a few neutrophils. the alveoli were consolidated with inflammatory cells and fibrin (figure ). by contrast, those ferrets infected with ⌬ /pr did not lose weight (figure ) and seemed to remain healthy during the study ( days) ( figure ) . virus was detected in the nasal washes of these animals at days but not days after infection, and virus was recovered from the upper respiratory tract only. by light microscopy, the meninges and trachea of the ⌬ /pr infected ferrets did not have an inflammatory infiltrate and only a few neutrophils were noted occasionally in pulmonary bronchi. our results clearly show that ⌬ /pr was attenuated. in view of our findings, this virus can be safely handled with standard precautions in bsl containment facilities. because the mechanisms and requirements for the accumulation of basic aminoacids at the haemagglutinin cleavage site are not entirely understood, we wanted to confirm that the altered cleavage site remained stable on multiple passages in embryonated chicken eggs. such passaging in eggs would occur in transition and amplification of the reference virus to vaccine stock. the rescued virus was stable on continued serial passage in embryonated eggs, and we did not detect any change in nucleotide sequence of the haemagglutinin cleavage site after passages. there was no evidence of changing pathogenicity of the virus and we noted only one dead embryo at passage . no haemagglutination activity was evident in this egg and no embryo death was seen in passage , which strongly suggests that the death was not related to virus replication. haemagglutination titres at each passage ranged from to with no apparent trend of increasing or decreasing titres in subsequent passages. the rapid response in terms of potential vaccine reference virus production to the h n outbreak differs strikingly from the response to the episode. this difference is attributable to the new scientific technology available in and, just as importantly, to the infrastructure for virus surveillance in hong kong developed since . the first case of h n influenza in hong kong was in may, ; yet several months elapsed before this virus was finally characterised as an h n virus. in , the causative agent was identified only hours after admission of the patients to the hospital. the increased awareness, surveillance, and availability of reagents to identify influenza viruses of all subtypes bode well for the rapid identification of viruses that arise from future interspecies transfer events and for the coordination of international vaccine development by who. the timely distribution of candidate viruses is a very important step in the development of vaccines for pandemic emergencies. despite the heightened security and documentation requirements for shipping and receiving potential bioterrorism agents, the h n and sars outbreaks have shown that in true emergencies, global distribution is feasible. although it is pertinent to prepare for future pandemics by stockpiling potential vaccine strains, the h n situation in -and the ongoing h n outbreaks throughout asia in (http://www.who.int)-have highlighted the fact that some of the focus of pandemic planning must go into the implementation of technology to rapidly produce vaccines from field isolates. although viruses similar to a/hong kong/ / (h n ) had been circulating in bird populations, these viruses were antigenically distinct, despite high genetic similarities (guan y and peiris js, unpublished data). that the aminoacid differences are on the globular head of haemagglutinin and seem to be responsible for much of the antigenic difference means that even a vaccine previously prepared from genetically similar precursor viruses might not provide adequate protection. we may well be faced with potential pandemic situations in the future and the rapid production of a matched vaccine will be needed-a point again highlighted by h n outbreaks in . although the reference virus described in this report was prepared from a virus isolated in a similar geographic region and only a year earlier, it shares only limited antigenic cross-reactivity to the h n viruses. hyperimmune sheep serum samples produced against the purified haemagglutinin of ⌬ /pr has at least a six-fold reduced haemagglutination inhibitory activity against a/vietnam/ / as compared with a/hong kong/ / . as our findings show, we have the technical capabilities to respond rapidly to outbreaks with a safe and stable reference virus, but there is still much to be accomplished before such viruses can be fully used in pandemic and interpandemic influenza vaccine production. the use of reverse genetics introduces a number of new processes into influenza vaccine manufacture that are not encountered with standard reassortment methods. one of the most obvious is the need for cultured cells. although both vero and mdck , cells are in development as substrates for the growth of influenza vaccine, there are additional requirements for the use of cells in reverse genetics. unfortunately, the number of suitable cell lines is very small. in addition to the regulatory requirements, the choice of cell is also limited by the technology. the plasmid based reverse-genetics systems necessitates the use of cells from primate origin. the vero cell line is probably the only option currently able to meet both regulatory and technical demands. we have shown that vero cells can be used to successfully rescue h n , h n , h n , and h n viruses on the pr backbone using the -plasmid system. others have demonstrated the suitability of vero cells for alternative influenza virus reverse-genetics systems. although cultures of vero cells are easily obtained, only cells from fully tested and licensed cell banks are likely to be acceptable for vaccine manufacture. this issue must be acknowledged and access to such cells must be incorporated as part of future pandemic plans. that future threats of influenza pandemics will be addressed by the use of the technology described in this report seems inevitable. despite the presence of low pathogenic surrogate strains, the recent human death from influenza-like illness caused by highly pathogenic h n virus in the netherlands reinforces the fact that future outbreaks will probably occur in which this reversegenetics technology provides the logical-and, possibly, the only-way to respond rapidly and effectively. although our response to the outbreak of h n influenza in has shown that current scientific capabilities are sufficient to respond to the threat, there are still legal and infrastructural barriers to be overcome. these barriers include licensing and intellectual property issues surrounding what is, essentially, a genetically modified organism. yet, these difficulties are not insurmountable and pandemic scares such as the and ongoing h n outbreaks are forcing commercial and regulatory parties to address these issues with some urgency. with the development of the h n vaccine reference virus, and ongoing attempts to create the same for the virus, the challenge in responding to a threat of an influenza pandemic must now be supported by the largescale manufacture of the vaccine and by clinical trials of a new vaccine manipulated by reverse genetics. r j webby, d r perez, j s coleman, j h knight, e i tuomanen, r g webster designed the study; r j webby did much of the construction of the vaccine seed virus; d r perez developed and constructed plasmid templates; y guan and j s peiris characterised and isolated the initial h n virus; j e rehg participated in the design and analysis of animal safety testing of the candidate h n vaccine seed virus; e a govorkova participated in the safety testing of the candidate h n vaccine seed virus; l r mcclain-moss participated in the preparation of gmp documentation of the process and was involved in the reconstitution of the vaccine seed virus. a pandemic warning? characterization of an avian influenza a (h n ) virus isolated from a child with a fatal respiratory illness characterization of avian h n influenza viruses from poultry in hong kong interspecies transmission of influenza viruses: h n virus and a hong kong sar perspective future influenza vaccines and the use of genetic recombinants developing vaccines against pandemic influenza the structure of the hemagglutinin, a determinant for the pathogenicity of influenza viruses proteolytic cleavage of influenza virus hemagglutinins: primary structure of the connecting peptide between ha and ha determines proteolytic cleavability and pathogenicity of avian influenza viruses molecular analyses of the hemagglutinin genes of h influenza viruses: origin of a virulent turkey strain rescue of influenza a virus from recombinant dna a dna transfection system for generation of influenza a virus from eight plasmids generation of influenza a viruses entirely from cloned cdnas eight-plasmid system for rapid generation of influenza virus vaccines plasmid-only rescue of influenza a virus vaccine candidates evaluation of a genetically modified reassortant h n influenza a virus vaccine candidate generated by plasmid-based reverse genetics recombinant influenza a virus vaccines for the pathogenic human a/hong kong/ (h n ) viruses preparation of a standardized, efficacious agricultural h n vaccine by reverse genetics development of a vero cellderived influenza whole virus vaccine influvac: a safe madin darby canine kidney (mdck) cell culturebased influenza vaccine safety and immunogenicity of a trivalent, inactivated, mammalian cell culture-derived influenza vaccine in healthy adults, seniors, and children generation of high-yielding influenza a viruses in african green monkey kidney (vero) cells by reverse genetics avian influenza a virus (h n ) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome pandemic influenza and the global vaccine supply we thank todd hatchette, katherine sturm-ramirez, and scott krauss for expert advice; ashley baker, christie johnson, yolanda sims, patrick seiler, jennifer humberd, and kelly jones for excellent technical assistance; julia hurwitz for access to the vero-cell banks. editorial assistance was provided by julia cay jones. these studies were supported by grant ai from the national institute of allergy and infectious disease, by cancer center support (core) grant ca from the national institutes of health, and by the american lebanese syrian associated charities (alsac). none declared. the corresponding author has had full access to all the data in the study and has had the final responsibility for the decision to submit this manuscript for publication. key: cord- -p twx a authors: lau, arthur chun-wing; yam, loretta yin-chun; so, loletta kit-ying title: management of critically ill patients with severe acute respiratory syndrome (sars) date: - - journal: int j med sci doi: nan sha: doc_id: cord_uid: p twx a severe acute respiratory syndrome (sars) is frequently complicated with acute respiratory failure. in this article, we aim to focus on the management of the subgroup of sars patients who are critically ill. most sars patients would require high flow oxygen supplementation, – % required intensive care unit (icu) or high dependency care, and – % developed acute respiratory distress syndrome (ards). in some of these patients, the clinical course can progress relentlessly to septic shock and/or multiple organ dysfunction syndrome (mods). the management of critically ill sars patients requires timely institution of pharmacotherapy where applicable and supportive treatment (oxygen therapy, noninvasive and invasive ventilation). superimposed bacterial and other opportunistic infections are common, especially in those treated with mechanical ventilation. subcutaneous emphysema, pneumothoraces and pneumomediastinum may arise spontaneously or as a result of positive ventilatory assistance. older age is a consistently a poor prognostic factor. appropriate use of personal protection equipment and adherence to infection control measures is mandatory for effective infection control. much of the knowledge about the clinical aspects of sars is based on retrospective observational data and randomized-controlled trials are required for confirmation. physicians and scientists all over the world should collaborate to study this condition which may potentially threaten human existence. in , an outbreak of severe acute respiratory syndrome (sars) caused by the sars-associated coronavirus involved countries and patients, resulted in deaths [ ] . thereafter, sars has re-emerged sporadically in both laboratory and community settings. its clinical spectrum varies from minimal respiratory symptoms to severe respiratory failure. we have previously contributed to an overview on the contemporary treatment of sars [ ] , and the whole topic has also been reviewed elsewhere [ ] . in this article, we aim to focus on the management of a subgroup of critically ill sars patients with more significant respiratory failure. critically ill sars patients frequently demonstrate the following clinical features: persistent pyrexia (occasionally from admission but often recurring after an initial period of defervescence), tachycardia (infrequently bradycardia), tachypnoea and significant oxygen desaturation. more than onethird of all the sars patients required high flow oxygen therapy [ ] , - % required intensive care unit (icu) admission or high dependency care, and - % developed acute respiratory distress syndrome (ards) [ , ] . the clinical course of some of these patients can progress relentlessly irrespective of all attempts at pharmacological treatment, eventually resulting in septic shock and/or multiple organ dysfunction syndrome (mods). lymphocytopaenia, neutrophilia and thrombocytopenia are frequently seen in critically ill sars patients. neutrophilia could be due to sars per se, to superimposed infection or related to corticosteroid administration. pancytopaenia, if present, could be due to haemophagocytosis syndrome [ ] or reactivation of latent human parvovirus (unpublished data). prolonged activated partial thromboplastin time and picture of disseminated intravascular coagulation has been reported [ ] . coinfections with other agents including chlamydia-like agents [ ] , metapneumovirus [ ] or influenza virus (unpublished data) have been reported. persistent and increasing elevations of creatine kinase, lactate dehydrogenase, and transaminases levels are common [ , , ] . associated lung damage is believed to be the result of a virally-triggered inflammatory reaction mediated by a host of cytokines [ , ] . in sicker patients, levels of pro-inflammatory cytokines (il- beta, il- , il- , il- , tnf-α) and tgf-β were higher, with slower decline on clinical recovery [ ] . radiographic abnormalities in the chest usually progress upwards from initial unilateral or bilateral lower-to mid-zone peripheral ground-glass shadows, to focal, multifocal or diffuse consolidation. peak radiographic changes occurred at . days after fever onset, with . % showing two peaks at . and . days, and % showing relentless progression [ ] . cavitation is rare but may be associated with superimposed infection in patients with a prolonged illness course and who are mechanically ventilated [ ] . high-resolution computer tomography (hrct) of the thorax showed focal ground-glass and scattered "crazy paving" patterns at presentation, followed by development of interstitial thickening, consolidation, pleural reaction, and scarring and fibrosis in later stages [ , ] . small (< cm) pulmonary cysts may be detected even if the patient is not receiving ventilatory assistance [ ] . subcutaneous emphysema, pneumothoraces or pneumomediastinum are distinct complications of severe sars [ ] . hrct features of late-stage ards caused by sars are similar to those arising from other causes [ ] . lung biopsy and postmortem studies [ , ] showed acute-phase diffuse alveolar damage (dad), airspace edema, bronchiolar fibrin, increased numbers of interstitial macrophages (with focal haemophagocytosis) and alveolar macrophages in patients with shorter duration (< days) of illness. on the other hand, histology after > days of illness showed organizing-phase dad with increased fibrosis, hyperplasia of type ii pneumocyte, squamous metaplasia, multinucleated giant cells, and acute bronchopneumonia [ ] . in patients who died late in the course of this disease, high loads of viral rna were detectable by reverse transcriptase polymerase chain reaction (rt-pcr) in the lungs, bowel, lymph nodes, spleen, liver, and kidneys [ ] . general principles anti-bacterial therapy for community-acquired pneumonia in accordance with standard guidelines [ ] should always be administered before laboratory confirmation of sars-cov infection. where effective anti-viral therapy is available, it should be started as early as possible after diagnosis, and even empirically if suspicious clinical features and especially epidemiological links are present. since critically ill patients are deemed to have already progressed from the viral replicative phase to the immunopathological phase [ ] , concomitant institution of an immunomodulatory therapy should also be considered [ ] . since there are no consensus regarding the most optimal treatment regimen in these respects, we will thus review the more commonly used agents and discuss their relative merits based on published reports. when respiratory failure eventually sets in, oxygen supplementation, assisted ventilation and intensive supportive treatments will be required. ribavirin was the most commonly used empirical antiviral agent for sars. it is a broad-spectrum purine nucleoside analogue which inhibits both rna and dna viruses by interfering with nucleic acid synthesis. there is experimental evidence to show that it has immunomodulatory effects in the treatment of mouse coronavirus hepatitis [ ] . subsequently, it was found that ribavirin has no direct in vitro activity against sars-cov [ ] . higher doses given intravenously resulted in more frequent and severe adverse effects including haemolytic anaemia, elevated transaminase levels and bradycardia [ ] . lopinavir-ritonavir co-formulation (kaletra ® , abbott laboratories, usa) is a protease inhibitor for the treatment of human immunodeficiency virus (hiv) infection. it can inhibit the coronaviral proteases, thus blocking the processing of viral replicase polyprotein and preventing the replication of viral rna. ritonavir inhibits lopinavir metabolism thus increasing its serum concentration, but it has no activity against sars-cov. in a retrospective analysis in hong kong [ ] , patients who had received kaletra as rescue therapy together with high dose corticosteroids had no difference in rates of oxygen desaturation, intubation and mortality compared with a matched cohort. however, when given as initial treatment in combination with ribavirin in another subgroup of patients, there were significant reductions in the need for rescue pulsed corticosteroid therapy, intubation rate and overall mortality. in addition to the prevalence of diarrhoea among these patients which may render oral drugs more appropriate and useful, synergism between kaletra and ribavirin might have contributed to the benefits since either drug alone has only weak anti-viral activities. another hong kong study of sars patients treated with a combination of lopinavir/ritonavir and ribavirin compared with patients (historical controls) treated with ribavirin only showed that adverse clinical outcomes (ards or death) were significantly lower in the treatment group than in the historical controls at day after symptom onset. further randomised placebo controlled trials are required [ ] . interferons are a family of cytokines with important roles in the cellular immune response. interferon α has been used for sars treatment in china and canada [ , , ] . in an open-label uncontrolled study [ ] , nine patients treated with corticosteroids plus interferon alfacon- (infergen ® , intermune inc., usa) showed better oxygen saturation, faster radiographic resolution and lesser need for supplemental oxygen compared to given corticosteroids alone. in vitro testing showed that interferon β was more potent than interferon α or γ, being effective even when administered after sars-cov infection in cell culture [ ] . traditional chinese herbal medicine has been used concomitantly with other drugs to treat sars in mainland china with good results reported [ ] . however, its value in critically ill patients has not been reported. glycyrrhizin, an active component derived from liquorice roots, is effective against sars-cov in vitro [ ] . its clinical utility remains uncertain. another herbal compound, baicalin, also demonstrates anti-sars-cov activity in vitro (unpublished data). in the absence of an effective antiviral agent in the outbreak, most physicians had opted to use immunomodulatory agents, most commonly corticosteroids, in the treatment of sars [ , , , ] it is generally agreed that corticosteroids should not be used during the early viral replicative phase, and that its administration should best coincide with the onset of the immunopathological phase [ ] . clinicoradiological surrogate criteria have been used to indicate the onset of this immune hyperactive phase, thus providing a practical guide to the timing of starting corticosteroids [ ] . corticosteroid dosages should be high enough, especially in the severe cases, to abort the cytokine storm, and maintained for long enough to prevent the rebound phenomenon [ , , ] . this may be achieved by using a weightadjusted [ ] and radiographic extent-modified dosages [ ] for a period of - weeks. in one-third to half of sars patients, fever may recur while on immunomodulatory treatment due to superimposed infections, too rapid tailing of corticosteroids or persistently severe and uninhibited cytokine storm. empirical anti-pseudomonal antibiotics should then be given first. if there is no apparent clinical response, opportunistic infections like fungal infection should be excluded. if fever is accompanied by obvious respiratory deterioration in the absence of superimposed pulmonary or systemic infection, most patients can be presumed to be suffering from a severe recrudescence of the sars illness. in such critically ill sars patients, further escalation of immunomodulation is warranted. such deterioration could sometimes occur very rapidly; immediate administration of pulsed methylprednisolone therapy at - mg per day intravenously for days, followed by tapering doses in the subsequent weeks, has been associated with improved outcome [ , ] . up to one-third to one-half of critically ill sars patients may benefit from this strategy [ , , ] . because radiographic abnormalities may lag behind clinical improvement, persistent radiographic shadows per se, when accompanied by clinical improvement, do not warrant additional corticosteroids [ ] . human gamma immunoglobulins have been used in selected sars patients who continued to deteriorate despite treatment [ , ] . an igm-enriched immunoglobulin product (pentaglobin ® , biotest pharma gmbh, germany) has been used in hong kong and mainland china [ , , ] . pentaglobin at mg/kg/day for three days given to patients who deteriorated despite repeated rescue methylprednisolone and ribavirin therapy had shown some improvement in radiographic scores and oxygen requirement [ ] . it has been reported that the use of combined methylprednisolone and highdose intravenous immunoglobulin ( . g/kg) daily for three consecutive days in probable sars patients with acute lung injury (ali) or ards had resulted in lower mortality and a trend towards earlier recovery [ ] . randomized controlled trials in larger numbers of patients are required to confirm its efficacy. based on the assumption that the neutralizing immunoglobulins in convalescent plasma can curb increases in viral load, convalescent plasma collected from recovered sars patients has been used in hong kong to treat severely ill patients not responding to corticosteroids. some clinical benefits were reportedly observed in a small number of patients [ ] . despite all efforts, at least % of sars patients would still develop acute hypoxemic respiratory failure, with up to % requiring supplemental oxygen [ ] overall, - % of patients had been admitted into icu, and - % eventually required intubation and mechanical ventilation [ ] . both non-invasive and invasive ventilatory support has been applied to critically ill sars patients. niv delivers continuous positive airway pressure (cpap) or bi-level pressure support through a tight-fitting facial or nasal mask. it was commonly employed in many chinese hospitals [ , , , , ] and our own centre in hong kong [ , , ] . early application may be beneficial because it could rapidly improve vital signs, oxygenation and tachypnoea [ , ] , and may reduce the need for increasing dosages of corticosteroids for progressive respiratory failure. it could avoid intubation and invasive ventilation in up to two-thirds of critically ill sars patients [ , , ] . use of niv in immunocompromised subjects of other diseases has reported similarly reduced rates of endotracheal intubation and serious complications [ ] . niv in sars may be of particular benefit, since high dose corticosteroids per se would already predispose to ventilator-associated pneumonia, and risks to healthcare workers (hcw) could also be markedly reduced through obviating the need for intubation, a potentially highly infectious procedure. patients who respond to niv will usually do so within hours, non-responders who will eventually need endotracheal intubation can thus be identified early [ ] . niv is indicated in the presence of ali and early ards when oxygen saturation (spo ) could not improve to more than % despite > litres per minute of oxygen; persistent tachypnoea of at least breaths per minute; and progressive radiographic deterioration in the lungs [ ] . the usual contraindications to niv apply, including impaired consciousness, uncooperative patient, high aspiration risk, and haemodynamic instability [ ] . sars-related respiratory failure responds readily to niv given at low pressures. cpap of - cm h o, or bi-level pressure support with inspiratory positive airway pressure (ipap) of < cm h o and expiratory positive airway pressure (epap) of - cm h o are reasonable starting pressures [ ] . higher pressures should be avoided whenever possible, because it may increase the risk of pneumothorax and pneumomediastinum, which are frequently spontaneous complications of sars even without assisted positive pressure ventilation [ ] . when patients do not improve within one to two days of niv or continue to deteriorate, or if niv is contraindicated, endotracheal intubation and mechanical ventilation should be considered. most centres [ ] adopted a ventilatory strategy similar to that recommended for ards from other causes [ ] . both pressure and volume control ventilation may be employed [ ] . the tidal volume should be kept low (e.g. - ml/kg predicted body weight), and plateau pressures maintained below cm h o. because of a higher risk of barotraumas in sars, the lowest positive end-expiratory pressure (peep) which could achieve satisfactory alveolar recruitment and oxygenation, usually - cm water, should be employed. other adjunctive measures employed in the usual ards cases had been tried in sars, including: prone positioning [ , ] , high frequency oscillatory ventilation [ , ] , nitric oxide [ ] , high peep and regular lung recruitment [ ] , but their efficacy is uncertain. tracheostomy is required in patients requiring prolonged mechanical ventilation and icu stay. strict adherence to infection control guidelines is mandatory in performing tracheostomy in the icu or operating room, as well as during subsequent changes of the tracheostomy tube [ , ] . critically ill sars patients on high dose corticosteroids and mechanical ventilation are particularly susceptible to superimposed bacterial and opportunistic infections. their peripheral blood cd +, cd + and cd + were also lower than normal [ , ] . ventilator-associated infection with organisms like pseudomonas aeruginosa, methicillin-resistant staphylococcus aureus, acinetobacter baumanii, as well as invasive mucor sp [ ] and aspergillosis [ , ] have been reported. strict control of hyperglycaemia during corticosteroid administration is essential to reduce the chance of septic complications [ ] . spontaneous subcutaneous emphysema, pneumothoraces and pneumomediastinum are common complications that are potentially aggravated by noninvasive or invasive ventilation [ ] . while chest drain insertion is useful to relieve pneumothoraces, prolonged air leak may sometimes occur. by itself, sars predominantly results in single organ failure of the lungs. other complications reported are more likely the result of sepsis and its attending problems, including acute renal failure ( %), acute liver failure ( %), rhadomyolysis, cardiovascular dysfunction, or of prolonged immobilization and underlying co-morbidities, including deep vein thrombosis, pulmonary embolism, ischaemic strokes, etc [ ] . the case-fatality ratio (cfr) of sars has been estimated to range from % to > % depending on the age group affected. the overall cfr is approximately % [ ] . variability may be due to different host and viral factors as well as treatment strategies. cfr may also be significantly affected by the duration of follow-up and inclusion of different mixes of suspected, probable and laboratory confirmed cases in different series [ ] . based on the treatment principles presented above, we have developed a standard treatment protocol early on in the outbreak, comprising initially high (but not pulsed) dose methylprednisolone with tapering over three weeks [ ] . this protocol was eventually applied to consecutively admitted sars patients [ ] . their mean age was years, with % having laboratory-confirmed sars. a low overall mortality of . % ( / ) was obtained, with all three deaths occurring in patients over the age of years. twenty four percent required icu admission: % received niv (bi-level pressure support) alone and % had both niv and invasive mechanical ventilation. hrct thorax in all survivors taken days after commencement of treatment showed most did not have clinically significant lung scarring. another multi-centered study comparing four treatment regimens in guangzhou, china, also found that a regimen of high dose corticosteroids adjusted according to clinical and radiological severity, coupled with nasal cpap ventilation, produced the best result: zero mortality in all clinically-defined sars patients, mean age . years. with % treated with cpap and none requiring mechanical ventilation. subsequently, very low mortality was again recorded among a further patients treated with the same regimen [ ] . many prognostic factors have been reported to independently predict adverse outcome in sars. they include advanced age [ , , , ] , diabetes [ , , ] , heart disease [ , ] , other significant coexisting conditions [ , , ] , shortness of breath on admission [ ] , degree of hypoxaemia [ ] , high total leukocyte count on admission [ , , ] , high initial lactate dehydrogenase [ , , ] , low platelet counts [ ] , and use of pulsed doses of corticosteroid [ , ] . compared to patients with nasopharyngeal aspirates negative for sars-cov by rt-pcr, pcr-positive ones are more likely to require icu care and mechanical ventilation, develop acute renal failure and die [ ] . in particular, mortality was high among icu patients: -day icu mortality was variously reported to be - % [ , , ] . older age, severity of illness, lymphocyte count, decreased steroid dose, positive fluid balance, chronic disease or immunosuppression, and nosocomial sepsis were associated with poor icu outcome [ ] . patients who had diarrhoea were more likely to require ventilatory support and icu care [ ] . higher serum sars-cov concentration in the early stage of the disease was a prognostic indicator for later icu admission [ ] . patients presenting with more extensive radiographic involvement also predicted the need for icu care or death [ ] . age alone is a consistent and strong prognostic factor in all series. age-stratified death rates were estimated to be < % in patients below years of age, % between and years, % between and years, and > % in elderly patients over years old [ ] . corresponding estimates in hong kong were % in those below years of age, and % in those over years [ ] . the cause of death in sars is usually progressive respiratory failure with or without concomitant sepsis. sudden cardiac arrest is also possible, and has been hypothesized to be due to hypoxemia (which would worsen during activities including defaecation), direct viral myocardial injury and extreme anxiety, all of which may lead to electrical instability in the myocardium and induction of arrhythmia [ ] . sars is primarily transmitted by direct or indirect contact of mucous membranes (eyes, nose, or mouth) with infectious respiratory droplets or fomites [ , ] . transmission risks increase with duration and proximity of contact. infection control precautions in the icu are shown in appendix [ ] . endotracheal intubation should be considered earlier and in anticipation of impending deterioration, so that ample time is available for preparation. it should be performed by the most skilful airway practitioner in a negative-pressure room behind closed doors. should the operator choose to wear additional personal protective equipment like the airmate hepa powered air purifying respirator system ( m, mn, usa), he/she must be familiar with its mode of operation and the precautions required for gowning and degowning, and must be assisted by a colleague with similar knowledge [ ] . a "modified awake" intubation technique has been suggested as the best possible compromise between patient and operator safety by administration of a combination of midazolam, fentanyl and lidocaine until the patient reaches the desired level of sedation [ ] . the patient is then paralysed after intubation to minimize coughing. alternatively, the "rapid sequence induction" technique with intravenous administration of midazolam and suxamethonium can also minimize patient coughing. it should however be emphasized that, unless there is prior preparation for a surgical airway, neuromuscular paralysis should be avoided in anticipated difficult intubation in order to maintain spontaneous respiration [ ] . both bronchoscopy and niv should be performed in a negative pressure room. although there is widespread fear of infective risk by niv [ , ] , centres with such experience, including ours, have found that its use is safe if the necessary precautions are taken [ , , , ] . finally, strict adherence to infection control measures in the form of strict isolation and effective cohorting, early diagnosis and contact tracing, timely reporting and institution of public health measures, as well as enhancement of environmental ventilation is key components in the effective management of infectious diseases. managing critically ill sars patients is a challenging task. most, if not all, knowledge about the clinical aspects of sars are based on retrospective observational data, and randomized-controlled trials are required for confirmation. physicians and scientists all over the world should collaborate to study this condition which may potentially threaten human existence. summary of probable sars cases with onset of illness from sars reference rd ed. flying publisher the severe acute respiratory syndrome severe acute respiratory syndrome: clinical outcome and prognostic correlates clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study icu management of severe acute respiratory syndrome 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severe acute respiratory syndrome (sars) sars: ventilatory and intensive care a practical approach to airway management in patients with sars infection control precautions in the icu effective staff education in infection control, emphasizing on • precautions to be used in high-risk procedures and alternative procedures to reduce risks • limit opportunities for exposure, e.g., avoid aerosol generating procedures & limit number of health care workers (hcws) present, alternative nursing practices to limit number of hcws exposed to each patient • effective use of time during patient contact • "gowning" and "degowning" without contamination precautions: disposable gloves, gown, cap • eye protection with non-reusable goggles and face-shield • powered air purification respirators (papr) are optional ppe when performing high-risk procedures • pens, paper, personal items and medical records should where feasible, increase to ≥ ach + re-circulate air through hepa filter • preferred: negative pressure isolation rooms with antechambers • a viral/bacterial filter should be placed at the expiratory port of bag-valve mask • place two filters per ventilator: between expiratory port and the ventilator, and another on the exhalation outlet of the ventilator • use closed-system in-line suctioning for endotracheal/tracheostomy tubes • handle contaminated heat and moisture exchangers (hme) and heated humidifiers carefully • scavenger system for exhalation port of ventilator is optional if negative pressure with high air change (> /hour) is achieved • preoxygenate patient and temporarily switch off machine whenever ventilator circuit disconnection is required the authors have declared that no conflict of interest exists. key: cord- - ou ubb authors: weiss, martin m.; weiss, peter d.; mathisen, glenn; guze, phyllis; henderson, donald a.; inglesby, thomas v.; o'toole, tara title: rethinking smallpox date: - - journal: clin infect dis doi: . / sha: doc_id: cord_uid: ou ubb the potential consequences of a competently executed smallpox attack have not been adequately considered by policy makers. the possibility of release of an aerosolized and/or bioengineered virus must be anticipated and planned for. the transmission and infectivity of variola virus are examined. arguments for and against pre-event vaccination are offered. the likely morbidity and mortality that would ensue from implementation of a mass pre-event vaccination program, within reasonable boundaries, are known. the extent of contagion that could result from an aerosolized release of virus is unknown and may have been underestimated. pre-event vaccination of first responders is urged, and voluntary vaccination programs should be offered to the public. two defenses against a vaccine-resistant, engineered variola virus are proposed for consideration. methisazone, an overlooked drug, is reported to be effective for prophylaxis only. the extent of reduction in the incidence of smallpox with use of this agent is uncertain. it is useless for treatment of clinical smallpox. n- respirators (face masks) worn by uninfected members of the public may prevent transmission of the virus. conventional wisdom holds that smallpox presents an unlikely threat to the public health [ ] [ ] [ ] [ ] . that wisdom asserts that the virus is sequestered in secure sites [ ] and that, even if it somehow released, vaccine will abort any potential epidemic [ ] . moreover, conventional wisdom holds that promising drugs are in development to treat smallpox [ ] [ ] [ ] [ ] and that the virus is not highly contagious [ ] . such considerations could prove to be overly optimistic and do not take into account the many uncertainties regarding transmission and infectivity of the smallpox virus. in addition to the possible existence of more-virulent "weaponized" strains, further advances in genetic engineering may permit construction of strains able to evade the current vaccine. australian workers markedly increased mousepox virulence by splicing a mouse il- gene into a laboratory strain [ ] ; similar constructions might be assembled using human smallpox virus (variola major) or another pox virus (e.g., monkeypox virus) and human genes [ ] . this article critically examines some of the current tenets of public health policy and highlights the uncertainty of much of the data. we also examine potential defenses against a release of smallpox virus and make recom-mendations regarding immunization and the development of prophylactic medications. smallpox may be transmitted via respiratory droplets or via fine-particle aerosol. the distinction between the two has critical public health implications. respiratory droplets (i.e., sputum and saliva) have a range likely no more than m (∼ ft) and are therefore a threat only to persons in the immediate vicinity of the affected patient. epidemiological studies support the finding that respiratory droplet spread is the prime route of transmission; the geographic locus of transmission is described as being almost always at the bedside, rather than public areas [ , ] . freefloating aerosolized virions, on the other hand, would have a considerably more extensive range. in , dixon [ ] reviewed the evidence for the alternative mode-aerosolized spread-and concluded that true airborne infection was extremely rare. nonetheless, epidemiological evidence suggests that transmission by means of aerosolized particles may be a real occurrence. in , persons on floors of a german hospital developed smallpox, despite isolation of the coughing, smallpox-infected patient in a single room [ ] . seventeen cases of smallpox developed; none of the patients had direct contact with the initial patient. subsequent "smoke" testing demonstrated air flows consistent with an aerosol spread [ ] . the last recorded death due to smallpox, according to world health organization investigators, was likely associated with virus that had been transmitted by aerosol [ ] . in , janet parker, a medical photographer at the university of birmingham medical school in england, became ill with smallpox and subsequently died. her darkroom was story above and several rooms down the hall from the laboratory of dr. henry bedson, a prominent smallpox researcher. smallpox virus can also be transmitted by fomites, such as clothing and bedding [ ] . laundry workers have developed smallpox. one study found a much higher recovery of smallpox virus from pillows and bedclothes than from air samples of the patient's coughs [ ] . the length of time that these objects remain infectious is unclear, but on the basis of the historical pattern of epidemics, it is likely no more than a few days. current wisdom holds that smallpox, contrary to its popular reputation, is not a highly infectious disease [ , ] . examinations of outbreaks in india and pakistan in the s showed that each case of smallpox gave rise to only new cases during the infectious (dry) season and to new case during the humid season [ ] . such observations-along with the long incubation period of smallpox (mean, - days; range, - days)suggest that there would be adequate time to vaccinate the public and prevent a more widespread outbreak. not revealed in these reports is the extent to which the affected public had already been vaccinated. if the percentage of the population that had been vaccinated was high, the aforementioned findings may merely reflect the population's immune status, rather than a low attack rate. another report placed the vaccination level in india at that time at % [ ] . if accurate, this would support the reason for the low attack rate as being a consequence of a public protected by immunization, rather than due to a virus with low inherent infectivity. indeed, there are data that smallpox is highly contagious. during the period of endemic smallpox, in field studies in africa, % of susceptible contacts became infected [ ] . other sources report attack rates of anywhere from % to % among unvaccinated contacts [ ] . potentially fatal reactions to smallpox vaccination include encephalitis, progressive vaccinia, eczema vaccinatum, and myopericarditis. postvaccinial encephalitis or encephalomyelitis has been reported to occur at an incidence of case per , vaccinations [ ] . in recent data from an ongoing department of defense (dod) study, there was case of encephalitis reported among , vaccinations [ ] ; the patient recovered. there was no evidence by either viral culture or pcr for vaccinia being the etiology. progressive vaccinia (a postvaccination viral dissemination with subsequent shock and localized gangrene) occurs in persons with immunodeficiencies, and eczema vaccinatum (a generalized spread of vaccinia to skin beyond the vaccination site) occurs in persons with atopic dermatitis; neither was reported in the dod study [ ] . fifty cases of contact transfer of vaccinia occurred, primarily in spouses and adult intimate contacts [ ] . the lower-than-expected incidence of adverse events may reflect more-careful screening of vaccination candidates for immunosuppression and eczema (for whom vaccination is contraindicated), the generally healthy status of the population being vaccinated, the previous vaccination in up to two-thirds of vaccine recipients, and covering of the vaccination site, which reduces inadvertent inoculation of contacts. (in previous vaccination campaigns, the vaccination site was left exposed.) an unexpected finding in the dod study above was the occurrence of cases of myopericarditis [ ] [ ] [ ] . there was death among these cases [ ] . other than for that fatality, in all cases for which there was follow-up cardiac testing, there was normalization of electrocardiograms, echocardiograms, exercise testing, and functional status [ ] . there was no increased incidence of coronary events in the dod program [ , ] , but in the much smaller civilian vaccination program (involving , vaccinees), the number of myocardial infarctions observed ( cases) was higher than would have been expected ( cases) [ ] . plaque-purified tissue culture vaccines are in clinical trials and may have a lower incidence of adverse reactions than does the standard calf lymph vaccine [ ] . in addition, attenuated and dna subunit smallpox vaccines are under development [ ] and may prove to be safer for immunocompromised persons. there have been attempts to answer the question of how many deaths would arise from preemptive mass vaccination of the public. depending on the percentage of the population vaccinated, the number of deaths is estimated to be in the range of - [ , , ] . the likely deaths and morbidity that would ensue from a vaccination program must be weighed against the likelihoodand consequences-of a smallpox attack. the conventional wisdom, as noted above, is that smallpox "does not spread rapidly under natural conditions" and, in fact, spreads at a "leisurely" pace [ , p. ]. transmission usually requires "close prolonged contact" for spread [ , p. ]. each case of smallpox "gives rise to (only) about three new cases" [ , p. ]. the long incubation period of - weeks "provides the time to intervene and limit secondary spread" [ , p. ]. we can "readily stop outbreaks within two infective generations (about weeks) after recognition of the initial cases" [ , p. ]. there is a problem in basing public policy on these principles. even if the above is an accurate representation of the contagiousness of smallpox, this paradigm reflects the spread of natural smallpox. unfortunately, any future smallpox epidemic would likely be an unnatural, man-made event. the natural history of an unnatural event may not be natural. a second misconception regards vaccination. contrary to the widely held belief that vaccination is equally successful after implantation of the variola virus, "postexposure vaccination is at best of limited effectiveness" [ , p. ]. the most optimistic report on postexposure vaccination, plotting efficacy against time, utilized a presumed average incubation period. it concluded that postexposure vaccination reduced the clinical case rate by % when administered up to days postexposure [ ] . concerns over the effectiveness of postexposure vaccination have been raised by others [ , ] . bozzette et al. [ ] calculate that there would be , deaths in a "high-impact airport attack," despite the presence of an aggressive postevent immunization program. it could be argued that his calculation may be an underestimation. in their model, bozzette et al. [ ] used a pattern of spread based on outbreaks that occurred after world war ii in a largely smallpox-immune population. vis-à-vis smallpox, the immune status of the older portion of our population is uncertain. it was generally accepted that the immunity provided by vaccination deteriorates with time. two-thirds of persons with smallpox in the s had preexisting vaccination scars [ ] . however, hammarlund et al. [ ] found substantial humoral and or cellular immunity against vaccinia persisting in persons who had been vaccinated - years earlier and cite epidemiological studies that argue for long-term protection. regardless, the immune status of our younger population (i.e., those aged ! years), with regard to smallpox, probably resembles the status of the aztec, inca, and th century american indian populations, rather than that of a vaccinated people. it is possible, therefore, that each index case would give rise to considerably more than just the secondary cases in the outbreaks that occurred after world war ii. as is reported in a consensus statement by smallpox authorities, "a clandestine release of smallpox, even if it infected only - persons to produce the first generation of cases, would spread rapidly in a now highly susceptible population, expanding by a factor or - times or more with each generation of cases" [ , p. ]. this pattern of spread likely occurred in the population of central mexico, which, according to aztec tribute rolls taken before their exposure to smallpox in the early s, was million. the spanish, in , estimated that the population was . million, but other factors, including measles, also probably played a role in the decline [ ] . bozzette et al. [ ] ascribe a mortality rate of . % to the unimmunized population. however, there are data showing a mortality rate of % in an unvaccinated population [ ] . the same long incubation period that some authorities hold to be an advantage in control of the disease [ ] could actually prove to be our achilles' heel. even within the limits of the shortest possible incubation period ( days), high-impact attacks could be repeated-at the same site or at different sites, with no one aware that attacks were taking place. to make a cogent assessment of the consequences of a smallpox attack, several questions must be answered. otherwise, we are engaged in no more than guesswork. the questions are these: ( ) can smallpox virus be aerosolized? ( ) if it can be aerosolized, for how long does it remain viable, and how far can it be carried? ( ) even if it can remain aerosolized and viable for a prolonged period of time, just how infectious is it by this route? smallpox virus can be aerosolized [ ] . however, the current opinion on how long the virus can remain viable in this state is that the viability rapidly decreases after min ("no more than %- % survived" [ , p. ] ), implying that there is nil viability left soon thereafter and, thus, that aerosolization does not represent much of a threat [ ] . unfortunately, closer scrutiny of the science underlying that assertion shows less reason to be sanguine. the great majority of the loss in variola virus viability was already present when first measured min into the study. thereafter, there was but modest further decline over the remaining -min length of the study [ ] . the virus may therefore persist at a relatively stable level of viability for hours. how long the virus can actually remain aerosolized is unknown, as is its infectivity in this mode. if one extrapolates from the results of studies of vaccinia, aerosolized variola virus that is protected from uv light survives for h [ ] . a critical caveat that was not addressed above is that the discussion has been limited to natural smallpox in a natural setting. the soviet union is known to have engaged in an active program to aerosolize bioweapons, including smallpox, for use in bioweapons [ ] . if modified or attached to the appropriate carrier, variola virus could possibly remain suspended and infectious for a considerable period. on the other hand, dissemination of variola virus into the air (e.g., via crop dusters or bomblets) subjects the virus to variables such as uv light, thermal factors, humidity, and wind. the virus might not survive, or it might be dispersed in the atmosphere into such low concentrations that it is no longer infective. because the minimal infective dose has not been determined, the efficacy of such dissemination is unknown. the current bush administration sought widespread preevent vaccination of the public over concern as to whether an effective vaccination program could be implemented after an attack on an unvaccinated public [ ] . the public health com-munity, however, citing safety issues, has opposed immunizing the public [ ] . animal studies demonstrate that cidofovir (vistide; gilead) has activity against poxvirus infections [ ] [ ] [ ] [ ] , but only when it was administered either concurrently or, in one study, within days after the initial challenge with the virus. if its effectiveness extends to humans, this drug would have a prophylactic effect only. it would not be of benefit for treatment of established clinical smallpox. cidofovir has been modified to render the drug bioavailable by the oral route. this modification (adding a lipid tail to produce hexadeclyoxypropyl-cidofvir [hdp-cidofovir]) resulted in a new drug, which, in vitro, is times more effective against variola than is unmodified cidofovir [ ] . methisazone, a thiosemicarbazone, has been reported to be effective for smallpox prophylaxis. a clinical trial in india in the s involving contacts claimed a % reduction in the incidence of the disease ( ) [ , ] . however, p ! . this study has been criticized elsewhere [ ] . treatment and control groups were incompletely randomized, with a possible bias in favor of methisazone. a subsequent fully randomizedbut considerably smaller-trial reported favorable but lessimpressive results (the incidence of smallpox in the control group was almost double that in the methisazone group) [ ] . this finding did not reach statistical significance. methisazone was stated to be effective prophylaxis in the eighth edition (from ) of harrison's principles of internal medicine [ ] , but it is doubtful that many made note of it. by then, smallpox had essentially been eradicated, and there was little reason to pay much attention to the entry. later editions of harrison's virtually eliminated the smallpox chapter, along with discussion of the drug. the agent has since fallen off of our radar screens [ ] . a panel of smallpox authorities assessed methisazone and determined that it had only modest benefit, probably reducing the incidence of smallpox by only %- % [ ] . this reduction should not be dismissed as inconsequential. in the event of a smallpox attack with an engineered virus, even such modest efficacy could prove critical. not addressed, however, is the question of just how effective methisazone would be without coadministration of vaccine. (vaccine could be useless in an attack with a modified virus.) in all of the aforementioned studies, contacts simultaneously received postexposure vaccination and methisazone. one study, on a related poxvirus, suggests an answer to this question. methisazone was investigated as prophylaxis for variola minor (alastrim), where contacts were not vaccinated, and was found to be effective for the prevention of alastrim at a significance level of . [ ] . methisazone is not without side effects. nausea and vomiting have been reported in one-tenth to two-thirds of persons who receive the drug [ , ] . for prophylaxis, the drug must be given within days of the initiation of infection with variola major [ ] . methisazone has a significant weakness: without patent protection, it is essentially an orphan. a pharmaceutical company is unlikely to expend research effort or promotion on such a drug. that weakness, however, is also a strength: in the public domain, it would likely be inexpensive to produce. the smallpox virus is - nm in size. n- respirators, with ulpa (ultra-low penetration air) filters, are . % efficient in filtering particles of у nm in size [ ] . the retail cost of these masks is $ . n- respirators, which are less effective respirators, have been reported to be protective in preventing transmission of severe acute respiratory syndrome coronavirus (size, nm) in health care workers [ , ] , but use of these respirators failed to prevent a cluster of cases in one hospital [ ] . concerns have been raised over leakage around the mask, especially in the absence of fit testing [ ] . nonetheless, these masks, if distributed to the public, could prove to be critical for the control of a smallpox epidemic that was overwhelming our health care system, and they might also prove to be effective in limiting contagion of smaller viruses, such as influenza virus (either natural virus, as in , or engineered virus [ ] ). additionally, an aerosolized smallpox attack would likely paralyze our cities. availability of masks might allow some measure of confidence for essential services to continue. a focus on the hazards of smallpox vaccination without consideration of the potential consequences of a competently executed smallpox attack may lead to skewed analyses and flawed decisions. in particular, the use of a more virulent, "weaponized" strain of smallpox virus could mean that the epidemic would outrun the currently planned postevent vaccination/isolation measures. although conventional wisdom suggests that smallpox, in its natural state, is largely limited to spread via respiratory droplets, concern about the potential for aerosol transmission is real and might be a greater problem in a developed society with large urban populations. despite the potential hazards, we believe that greater efforts should be made to promote pre-event immunization-especially in emergency providers and health care workers. furthermore, consideration should be given to allowing the public voluntary access to the vaccine. with proper informed consent and careful screening to minimize the risk of adverse side effects, such a program could reduce the risk of a runaway epidemic. because of the possibility of an attack involving bioengineered smallpox virus that is resistant to the current vaccine, methisazone should be reexamined, and research should be continued on other antiviral agents. also, an adequate supply of masks 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transmission of severe acute respiratory syndrome (sars) sars among critical care nurses cluster of severe acute respiratory syndrome cases among protected health-care workers-toronto, canada interim domestic guidance on the use of respirators to prevent transmission of sars influenza as a bioweapon active poliovirus baked from scratch a not-so-cheap stunt key: cord- - xn v s authors: rodák, l.; Šmíd, b.; nevoránková, z.; smítalová, r.; valíček, l. title: verification of sensitivity and specificity of group a rotavirus detection in piglets faeces with monoclonal blocking elisa methods date: - - journal: j vet med b infect dis vet public health doi: . /j. - . . .x sha: doc_id: cord_uid: xn v s monoclonal antibodies to group a rotavirus vp protein were prepared and used for verification of three blocking enzyme‐linked immunosorbent assay (elisa) modifications to detect rotavirus a. selected competitive blocking elisa (cb‐elisa) and electron microscopy (em) were used for examination of field faecal samples of piglets affected with diarrhoea. rotavirus was detected in samples ( . %) by cb‐elisa method, whereas in ( . %) samples by em examination. however, of samples positive by em, rotavirus a was detected by cb‐elisa in ( . %) samples; indicating the share of group a rotavirus in all cases of gastroenteritis caused by rotavirus. the sensitivity and specificity of the cb‐elisa was verified both by inclusion of control samples containing transmissible gastroenteritis virus (tgev) and porcine epidemic diarrhoea virus (pedv) in each analysis and by comparative examination of samples with the commercial elisa kit. the cb‐elisa sensitivity was positively affected by examination of samples in the presence of chelating agent. rotaviruses rank among significant causes of gastroenteritis in the majority of vertebrates. data on their prevalence in human populations attest to their significance. a total of - million cases of gastroenteritis caused by rotaviruses with high mortality in children, particularly in developing countries, are annually reported (bajolet and chippaux-hyppolite, ; parashar et al., ) . it follows that rotaviral infections play a significant role in farm animals kept under much worse hygienic conditions. besides rotaviruses, two coronaviruses -transmissible gastroenteritis virus (tgev) and porcine epidemic diarrhoea virus (pedv) -rank among the economically most important viruses causing diarrhoea in pigs. rotaviruses, which are about nm, with doublestranded rna genome, are highly resistant to the conditions of external environment and to both chemical and physical agents (ramos et al., ; fischer et al., ) . they replicate mainly in enterocytes covering small intestinal villi and are spread via the faecal-oral route (benfield et al., ; debouck and pensaert, ; gelberg et al., ) . previously, all rotaviruses were considered antigenically related, containing common vp antigen (group a rotavirus) with mr k. later, rotaviruses without the common antigen were classified as so-called non-group a rotaviruses. seven antigenically distinct groups of rotaviruses (a-g) were described; four (a, b, c and e) of them are pathogenic for pigs. group a rotavirus which is the most often ( . - . %) causal agent of rotaviral gastroenteritis (sigolo de san et al., ; janke et al., ; pongsuwanna et al., ) comprises a series of subgroups and serotypes according to antigenic analysis of vp and vp proteins present in the outer capsid of virion. common group a rotavirus vp antigen is found in the inner capsid of virion, and rotaviral infections caused by that agent may be diagnosed by use of specific vp antibodies regardless of animal species affected. rotavirus detection gives more exact information on the cause of gastroenteritis than determination of antiviral antibodies, which are present almost in all blood serum samples. therefore, both in veterinary and in human medicine, a series of methods are used [latex agglutination, reverse transcription polymerase chain reaction (rt-pcr), enzyme-linked immunosorbent assay (elisa), electron and immune electron microscopy (em) etc.]; some of them are available as commercial diagnostic kits (benfield et al., ; gerna et al., ; al yousif et al., ; raboni et al., ) . the objective of the present study was to compare three modifications of blocking elisa method for the detection of rotavirus a. the results obtained with selected competitive blocking elisa (cb-elisa) method were compared with those obtained by em and by commercial elisa kit. the reference strain of rotavirus a (osu strain, vr- ; serotype g /vp and serotype p /vp ) and three of our own rotavirus a field isolates were propagated in ma- cells grown in minimum essential medium eagle in the presence of trypsin ( lg/ml, t- ; sigma, st louis, mo, usa). the virus was released from the cells - hpi by repeated freezing/thawing. after centrifugation ( · g for min) of the culture medium, the pellet was resuspended in phosphate-buffered saline (pbs) in / of the original volume as crude viral antigen (v-ag). control antigen (c-ag) was prepared similarly from uninfected cells. purified v-ag was prepared by ultracentrifugation of supernatant on a cushion of % sucrose in pbs (optima le- k ultracentrifuge; beckman, palo alto, ca, usa). crude v-ag and c-ag were similarly prepared by propagation of tgev (strain capm v- ; collection of animal pathogenic microorganisms, brno) and pedv (strain cv- ) in porcine kidney (pk- ) and vero cell lines, respectively. they were used for specificity check of all elisa methods used. two hysterectomy-derived, colostrum-deprived, -week-old piglets were kept in sterile conditions, and orally infected with . , tcid of group a rotavirus. seven weeks post infection, piglets were challenged with . . tcid of the virus and killed under total anaesthesia days later. the titre of rotavirus a antibodies in the blood serum obtained (swspos.) was determined by indirect elisa. rotavirusnegative porcine blood serum (swsneg.) was obtained by exsanguination of -week-old uninfected piglet. the immunoglobulin fraction (swigrota) was obtained from positive serum by ion-exchange chromatography and used as a binding antibody in the blocking elisa methods. rota-and coronaviruses were detected by electron microscopic examination of culture media and faecal samples of piglets after negative staining with % ammonium molybdate solution in water, ph . (sˇmı´d et al., ) . inbred mice of the line balb/c were repeatedly immunized with purified rotavirus a. hybridomas were prepared by a standard procedure (galfre`and milstein, ) by fusion of splenic lymphoid cells with cells of the myeloma line sp / . specificity of mab produced by hybridomas was checked by elisa, western blot (wb) and immunoperoxidase (ip) detection of rotavirus a in infected cells. selected hybridomas were used for preparation of ascitic fluids. monoclonal antibodies (mab) purified from ascitic fluids by ion-exchange chromatography were stored at ) °c in % glycerol or used for the preparation of peroxidase conjugates (hrpo-mab-rota). antibodies to swine and mouse immunoglobulins were purified from hyperimmune swine (swamoig) or rabbit (raswig, ramoig) sera by affinity chromatography. these antibodies and mabrota were conjugated with horseradish peroxidase (hrpo, type vi-a; sigma) using the periodate method (boorsma and streefkerk, ) . the stock conjugate solutions were adjusted to mg antibodies/ml. for the ip, wb and elisa they were diluted · to · in pbs containing . % tween and . % lactalbumin hydrolysate (pbst-lah). rotavirus a antibodies in swine and mouse sera, in culture media of hybridomas and in purified mabrota preparations were assayed using the indirect elisa method. the wells of microtitre plates (nunc-immunoplate, polysorp, denmark) pre-coated alternatingly with crude rota a v-ag and c-ag were incubated ( h at °c) with various dilutions of tested samples. after washing the second identical incubation with conjugates to swine or mouse immunoglobulins followed. the reactions were visualized by incubation with chromogen tmb ( , ¢, , ¢-tetramethyl-benzidine; sigma) solution. after min, the reaction was stopped and absorbances were measured spectrophotometrically at nm. control wells filled with the diluent (blank) only, or with control negative or positive sera at the first incubation, were included in each examination. highest samples dilution showing a difference in optical density of at least . (after subtraction of absorbances of blank wells) between the wells with bound v-ag and c-ag were classified as positive. sensitivity and specificity of three variants of a blocking elisa were determined by box titration using faeces of an experimentally infected piglet. the wells of microtitre plates (nunc-immunoplate, maxisorp, denmark) were pre-coated overnight with binding antibodies in carbonate-bicarbonate buffer, ph . ( lg ig/ml; ll/well). during the first incubation ( h at °c), pairs of pre-coated wells were filled with ll mixtures of faeces with swsneg. or swspos. pbst-lah containing . m nacl and mm ethylenediaminetetraacetic acid (ed-ta)aena salt was used for sample dilution at the first incubation. after the second incubation (always h at °c) with ll of detection antibodies (conjugates hrpo-mabrota or hrpo-swigamoig), visualization of the reaction and assessment of absorbances (a) followed, similarly to indirect elisa. between incubations, the wells were rinsed with pbst four times. pairs of wells filled with the diluent (blank) or the mixtures of crude v-ag (rotavirus a, tgev, pedv) and sws neg./pos. during the first incubation were included in each analysis. the samples were regarded as positive if the net absorbance (na), i.e. the difference of average absorbances in the wells incubated with swsneg./pos. was > . , and reactions were blocked by > % in the wells with swspos. blocking percentages were determined using the formula: %b ¼ ) [(a swspos. · ) : (a swsneg. )]. the following variants of the blocking elisa method were investigated: double antibody sandwich elisa (das-elisa): wells precoated with binding mabrota. the first incubation with antigen test samples was performed for h, the second incubation with the detection antibody (hrpo-mabrota). competitive blocking elisa (cb-elisa): wells pre-coated with binding swigrota. after h of incubation with the antigen test samples the wells were supplemented with ll diluent containing lg mabrota/ml, and the incubation continued for another hour. the second incubation was undergone using detection antibody hrpo-swiga-moig. the das-elisa: wells pre-coated with binding swigrota. the first incubation with the antigen test samples was verification of sensitivity and specificity of group a rotavirus detection performed for h and the second incubation was conducted with the detection antibody hrpo-mabrota. commercial kit (ingezim rota das . .rt.k. ; ingenasa; inmunologia y genetica aplicada, s.a.; spain, thereafter das-elisa kit) was used for the detection of rotavirus a in faeces. the kit utilizes biotin-conjugated mab anti-rota a and streptavidin-peroxidase conjugate. examinations were carried out according to the manufacturer's instructions. samples were evaluated as positive at the values of corrected absorbance (ca) > . , dubious at ca ¼ . - . and negative at ca < . (ca ¼ a of the sample tested ) a of negative control antigen). results of the commercial kit examinations were compared with those obtained by em and cb-elisa methods. in total, faecal samples from piglets with diarrhoea were examined. most samples were from piglets younger than days. after delivery to the laboratory, they were diluted in two to three volumes of earle's medium, centrifuged ( min, · g), and the supernatants immediately examined by em. before cb-elisa examination, the samples were kept at ) °c. after verification of the specificity of hybridomas producing mab to rotavirus a, two of them were selected for further use. both mab of the isotype specificity igg a (g /d ) and igg b (b /f ) reacted with the viral protein vp in wb analysis (results not shown). indirect elisa titres of both mab stock solutions containing mg ig/ml reached · . cross-reac-tivity with other viral antigens could not be detected using any of the methods mentioned ( table ) . sensitivity comparison of three variants of the blocking elisa method of rotavirus a detection were performed by box titrations in microtitre plate wells pre-coated with binding antibodies. mixtures containing the faecal sample of an experimentally infected piglet and swspos./swsneg. fivefold diluted - · and - ·, respectively, were examined. the sample of faeces in the entire range of dilutions was assessed as positive by all of the three blocking elisa methods. only slight differences in sensitivity were detected. results obtained with minimal and maximal dilutions of sws only are shown in table . competitive blocking elisa methods proved to be most effective for the determination of coronaviruses (tgev: l. roda´k, b. smid, z. nevorankova, l. valicek and r. smitalova, unpublished data; pedv: l. roda´k, l. valicek, b. smid and z. nevorankova, unpublished data). therefore, cb-elisa method (variant ) was also used for routine rotavirus a detection in field samples; mixtures of faecal samples and swsneg./pos. were examined in working dilutions : and : , respectively. using diluent supplemented with chelating agent in the first incubation, more satisfactory results were obtained in most of samples in comparison with standard diluent (pbst-lah). net absorbance values (na > . ) and %b > was necessary to obtain for positive evaluation of the samples. under these conditions, sample evaluation was highly specific; as established by examination of selected positive and negative samples in eight wells (n ¼ ), determination of mean absorbances (ma) and standard deviations (±sd). calculated coefficients of variation (cv%) were . - . %. only with negative samples giving low absorbance values, the cv% was occasionally higher. sensitivity of the cb-elisa method and das-elisa kit was compared by examination of faecal sample of experimentally infected piglet twofold diluted · to ·. the results document that the highest sample dilution · and · was regarded as positive by das-elisa kit and cb-elisa respectively. cb-elisa absorbances (a) of the sample diluted : were . / . (table ) . it follows that sample with cb-elisa a < . will be probably negative by das-elisa kit. comparative examinations of crude v-ag (rota a, tgev, pedv) proved high specificity of both the methods (table ) . also randomly selected field faecal samples, assessed positively and negatively by em and cb-elisa, were comparatively examined by das-elisa kit (table ) (table ) . polyclonal and mab to group a rotavirus were prepared and used as binding or detection antibodies for sensitivity testing of three elisa variants for rotavirus a demonstration. in similar experiments dealing with tgev and pedv detection (l. roda´k et al., unpublished data), lower sensitivity of das-elisa variants using conjugated mab was proven. therefore, more sensitive variants of cb-elisa, based on the use of unconjugated mab, were selected for routine demonstration of both coronavirus and rotavirus a infections. the use of a uniform methodical procedure thus allows detecting simultaneously all three most important causative agents of viral gastroenteritis. specificity of examination was confirmed by comparative assessment of v-ag (rotavirus a, tgev, pedv) by cb-elisa and by commercial das-elisa kit ( table ). absence of cross-reactivity of cb-elisa with bacterial antigens present in field faecal samples confirmed the results of rotavirus a-negative samples. higher sensitivity of rotavirus a detection by cb-elisa in comparison with commercial das-elisa kit was proven. by examination of positive faecal sample twofold diluted · to ·, at least times higher sensitivity of cb-elisa method was demonstrated (table ) . this was also confirmed by comparative examination of randomly selected field faecal samples. all cb-elisa-negative samples were evaluated negatively by das-elisa kit examination. however, of the remaining cb-elisa-positive samples, only one half of samples were evaluated as positive by commercial das-elisa kit ( table ) . the sensitivity of rotavirus a detection by cb-elisa in comparison with em is also higher. of field faecal samples rotavirus was detected in samples by cb-elisa whereas in samples by em. on the contrary, of em positive samples rotavirus a was detected in ( . %) samples by cb-elisa. the fact that all groups of rotavirus are detected by em, while group a rotavirus only is detected by cb-elisa, can explain this difference. the congruency of em and cb-elisa examinations ( . %) indicates the share of rotavirus a in cases of rotaviral gastroenteritis and correlates with other findings (sigolo de san et al., ; janke et al., ; pongsuwanna et al., ) . the assumption that the remaining seven ( . %) samples likely contain non-group a rotavirus was partly confirmed by demonstration of group c rotavirus electropherotype in one em highly positive sample. therefore, more sensitive rt-pcr techniques will be applied in following experiments. it is well-known that chelating agents destroy the outer capsid of rotavirus and its infectivity (ward and ashley, ; fang et al., ) . therefore, we presumed that their presence in elisa examinations might improve accessibility of antigens localized in the inner capsid of virion, particularly the vp antigen. this was confirmed in our study, and better results were obtained in most samples using solution with chelating agent in comparison with the standard diluent. highly positive values of na and %b were obtained in of cb-elisa-positive faecal samples; it confirms suitability of working dilutions of faeces ( ·) and swsneg./pos. ( ·) selected for routine use. the remaining samples assessed as negative because of the %b < were also positive at higher working dilution. diagnosis of the causal agents of viral gastroenteritis is a basic prerequisite both for introduction of immunoprophylactic measures (schaller et al., ; ebina, ; hodgins et al., ) and confirmation of their effectivity. the use of uniform methods allowing diagnosing simultaneously rotavirus and coronavirus infections could contribute to this goal, as well as introduction of pcr in following experiments. evaluation of a latex agglutination kit (virogen rotatest) for detection of bovine rotavirus in fecal samples rotavirus and other viruses of diarrhea shedding of rotavirus in feces of sows before and after farrowing comparison of a commercial enzyme-linked immunosorbent assay with electron microscopy, fluorescent antibody, and virus isolation for the detection of bovine and porcine rotavirus periodate or glutaraldehyde for preparing peroxidase conjugates? rotavirus excretion in suckling piglets followed under field circumstances prophylaxis of rotavirus gastroenteritis using immunoglobulin purification and characterization of adult diarrhea rotavirus: identification of viral structural proteins rotavirus particles can survive storage in ambient tropical temperatures for more than month preparation of monoclonal antibodies: strategies and procedures the shedding of group a rotavirus antigen in a newly established closed specific pathogen-free swine herd comparative evaluation of a commercial enzyme-linked immunoassay and solid-phase immune electron microscopy for rotavirus detection in stool specimens effects of maternal antibodies on protection and development of antibody responses to human rotavirus in gnotobiotic pigs relative prevalence of typical and atypical strains among rotaviruses from diarrheic piglets in conventional swine herds global illness and death caused by rotavirus disease in children serological and genomic characterization of porcine rotaviruses in thailand: detection of a g porcine rotavirus comparison of latex agglutination with enzyme immunoassay for detection of rotavirus in fecal specimens the stability of porcine rotavirus in feces prevention of human rotavirus-induced diarrhea in gnotobiotic piglets using bovine antibody electron microscopic demonstration of porcine epidemic diarrhea virus in the czech republic incidence of group a and atypical rotaviruses in brazilian pig herds comparative study on the mechanisms of rotavirus inactivation by sodium dodecyl sulfate and ethylenediaminetetraacetate this work was supported by projects qf and mze from ministry of agriculture of the czech republic.the authors wish to thank mrs farnı´kova´, mrs lickova´, ms hoydenova´and ms bacˇinska´for their skilful technical assistance. key: cord- -vbadg ki authors: jeong, jae-ho; kim, gye-yeop; yoon, soon-seek; park, su-jin; kim, you-jung; sung, chang-min; shin, sung-shik; lee, bong-joo; kang, mun-il; park, nam-yong; koh, hong-bum; cho, kyoung-oh title: molecular analysis of s gene of spike glycoprotein of winter dysentery bovine coronavirus circulated in korea during – date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: vbadg ki since the molecular analysis of spike (s) glycoprotein gene of bovine coronavirus (bcov) has been conducted and compared mainly among american and canadian isolates and/or strains, it is unclear whether bcov circulated in the other countries are distinctive in genetic characteristics. in the present study, we analyzed the s glycoprotein gene to characterize winter dysentery (wd) coronavirus strains circulated in korea during – and compared the nucleotide (nt) and deduced amino acid (aa) sequences with the other known bcov. the phylogenetic analysis of the entire s glycoprotein gene revealed that the aa sequences of all korean wd strains were more homologous to each other and were very closely related to respiratory bovine coronavirus (rbcv) strain ok and enteric bovine coronavirus (ebcv) strain ly- , but were distinct from the other known bcovs. based on the phylogenetic analysis of the hypervariable region of the s subunit, all korean wd strains clustered with the respiratory strain ok, bcq and the enteric strain ly- , while the canadian bcq calf diarrhea and wd strains, and the american rbcv lsu, french ebcv f and avirulent vacc, l , and mebus strains clustered on a separate major branch. these data suggest that the wd strains circulated in korea had a genetic property of both rbcv and ebcv and were significantly distinct from the ancestral enteric strain. ). the virion contains five major structural proteins: the nucleocapsid (n) protein, the transmembrane (m) protein, the hemagglutinin/esterase (he) protein, the spike (s) protein, and the small membrane (e) protein (lai and cavanagh, ) . the variation in host range and tissue tropism of coronaviruses is largely attributable to variations in the s glycoprotein (gallagher and buchmeier, ) . the bcov had point mutations or deletions especially in the s subunit of s glycoprotein (chouljenko et al., ; hasoksuz et al., ; gelinas et al., ; rekik and dea, ) . moreover, - /$ -see front matter © elsevier b.v. all rights reserved. doi: . /j.virusres. . . molecular analysis of s gene of bcov has been conducted and compared mainly between american and canadian isolates and/or strains (chouljenko et al., ; hasoksuz et al., ; gelinas et al., ; rekik and dea, ) . in this study, we performed molecular analysis on the s glycoprotein gene to characterize wd coronaviruses circulated in korea during korea during - , compared the nucleotide, and predicted aa sequences with the other known bcov. ten wd strains, kwd -kwd , were originally isolated in the g clone of human rectal tumor cells (hrt- g) from fecal samples of adult dairy or beef cows, with wd in south korea, and identified as a bcov by immunoelectron microscopy, elisa with bcov-specific antisera and monoclonal antibodies, and rt-pcr specific for the part of bcov n gene (jeong et al., ) . rna was extracted from the supernatant fluids of hrt- g cells infected with kwd strains, based on the acid quanidinium-phenol-chloroform rna extraction method as described by cho et al. ( a) . as a negative control, rna was extracted from mock-infected hrt- g cells. the oligonucleotide primers used in the rt-pcr were designed from the published sequence of the s gene of the mebus strain (genbank accession no. u ). the sequence of primers (positions calculated from the start codon of the s gene) are as follows: st upstream primer, -atgtttttgatacttttaatttcc- ( - ); st downstream primer, -acaccagtagatggtgctat- ( - ); nd upstream primer, -gggttacacctct-cacttct- ( - ); nd downstream primer, -gcaggacaagtgcctatacc- ( - ); rd upstream primer, -ctgtccgtgtaaattggatg- ( - ); rd downstream primer, -tgtagagtaatc-cacacgt- ( - ); th upstream primer, -ttc acgacagctgcaaccta- ( - ); th downstream primer, -ccatggtaacaccaatcc ca- ( - ); th upstream primer, -ccctgt-attaggttgtttag- ( - ); th downstream primer, -accactaccagtgaacatcc- ( - ); th upstream primer, -gtgcagaatgct-ccatatggt- ( - ); th downstream primer, -ttagtcgtcatgtgatgttt- ( - ). a one-step rt-pcr assay was performed as described by cho et al. ( a,b) . the rt-pcr products were purified using a genclean ii kit (bio , inc., lajolla, ca) according to the manufacturer's instructions. the dna sequencing was done using an automated dna sequencer (abi system , applied biosystem inc., foster city, ca). using the dna basic module (dnasis max, alameda, ca), nucleotide sequences of our bcov strains were first compared for the s sequence of bcov as follows: the cd strains including mebus (genbank accession no. u ), bcq (genbank accession no. u ), bcq (genbank accession no. u ), bcq (gen-bank accession no. u ), bcq (genbank accession no. u ), and bcq (genbank accession no. aag ); the wd strains including bcq (genbank accession no. aag ) and bcq (genbank accession no. ak ); the rbcv strains including bcq (genbank accession no. aak ), lsu (genbank accession no. aaf ), and ok (genbank accession no. aaf ), and ebcv strains including ly- (genbank accession no. aaf ) and f (genbank accession no. d ). the deduced aa sequences were then assembled and analyzed on the amino acid basic module (dnasis max, alameda, ca). a sequence similarity search was performed for the bcov s glycoprotein using the lalign query program of the genestream network server at institut de génétique humaine. phylogenetic analyses were conducted using phylodraw program at the graphics application laboratory, pusan national university. all of the s genes of the kwd strains contained an orf of nucleotides, encoding a predicted protein of aa residues and having a molecular weight of approximately kda. spike protein further divided it into s and s segments of approximately and kda, respectively, at the cleavage site aa . among all strains analyzed, a total of and polymorphic nucleotides were identified in the s and s subunit of the spike glycoprotein of bcov, respectively, compared with the mebus strain (data not shown). these polymorphisms led to and aa changes at and distinct sites, respectively. we have shown recently that the respiratory and enteric isolates collected concurrently from two different calves in usa were significantly distinct from the ancestral enteric strain, mebus (hasoksuz et al., ) . by drawing comparisons of deduced aa between our strains and other known bcov, the two most similar sequences were those of kwd strain and rbcv lsu strain ( . %) in paired comparisons, and the most distant were those of the kwd strain and the mebus strain ( . %). all the virulent-kwd strains tended to be distant from the ancestral enteric strain, mebus, suggesting that allelic variation resulted in genetic drift over time (hasoksuz et al., ) . based on the total number of aa substitutions, a phylogenetic tree of all s gene sequences was analyzed with prototype strain mebus, rbcv strains lsu and ok, ebcv strains f and ly- , and avirulent ebcv l (fig. ) . the aa sequences of kwd strains were more homologous to each other and were very closely related to the virulent rbcv ok and ebcv ly- strains, among the other known bcov studied in paired comparisons (fig. ) . these data showed that the wd bcovs circulated in korea were genetically similar to both rbcv and ebcv. in addition, the phylogenetic comparison of korean wd strains with other known bcovs, on the sequences of the hypervariable region, revealed that all korean wd strains were also clustered in groups with the virulent rbcv ok and bcq strains, and the virulent ebcv ly- strain (fig. ) . the canadian bcq cd and wd strains, the american rbcv lsu strain, french ebcv f strain and avirulent vacc, l and mebus strains were clustered on a separate major branch (fig. ) . therefore, these results indicated that the bcov strains may be diverging from an enteric tropism to a dual (respiratory and rekik and dea, ) of respiratory (rbcv), enteric (ebcv), calf diarrhea (cd), winter dysentery (wd), and avirulent strains. the cd strains including mebus, bcq , bcq , bcq , bcq , and bcq ; the wd strains including bcq , bcq , and kwd -kwd ; the rbcv strains including bcq , lsu, and ok; ebcv strains including ly- and f . phylogenetic analyses were conducted using phylodraw at the graphics application laboratory, pusan national university. enteric) tropism over time via intermediates (hasoksuz et al., ) . this speculation could be supported by our previous paper (cho et al., a) in which gnotobiotic and colostrumdeprived calves were inoculated with respiratory isolates of bcov shed viruses, both nasally and rectally. this subsequently induced diarrhea. moreover, feedlot cattle, which had shown respiratory disease signs at arrival time, developed diarrhea days after arrival, which peaked at - days post arrival (cho et al., b) . further study will be needed on the differences in pathogenicity, antigenicity, or any other physicochemical characteristics between the kwd strains. in the s glycoprotein, the hypervariable region spanning aa - of the s subunit contains the s b immunoreactive epitope which is the target for virus neutralizing monoclonal antibodies (mabs) (cavanagh, ) . the hypervariable region showed varied antigenicity when reacting with mabs directed against the s glycoprotein. therefore, chouljenko et al. ( ) reported that four rbcv-specific aa substitutions fig. . comparison of the predicted amino acid sequences of the bcov s glycoprotein specified by different strains. light-gray boxes contain aa sites known for respiratory bovine coronavirus-specific (chouljenko et al., ) , dark boxes contain virulent-specific (chouljenko et al., ) , and clear boxes contain korean strains-specific aa changes. at aa , , , and can be predictive and mabsspecific strains for this region may also be distinguished among respiratory, enteric, and vaccine bcov strains. in the present study, three aa sites including aa , , and of rbcv ok and lsu strains were identical in all korean wd strains (fig. ) . in our previous paper (hasoksuz et al., ) , enteric and respiratory bcov pairs collected concurrently from two feedlot calves in usa also had identical aa substitution sites at the same sites. furthermore, aa (n → g) substitution of rbcv ok and lsu strains compared with the mebus strain was also detected in the cd strain bcq (data not shown). since these changes were observed in wd, cd, and rbcv strains, our data does not support the speculation of chouljenko et al. ( ) . in addition, aa and of the s subunit have been reported for ebcv-specific sites, which were observed in ebcv strains f and ly (chouljenko et al., ) . however, aa and of the s subunit of all korean wd strains were identical with those of lsu and ok strains of rbcv, bcq , and bcq of wd isolates, mebus, and l avirulent strains (fig. ) . in particular, aa , reporting its sequence in many isolates and/or strains, are the same as other known bcov including rbcv, cd, wd, and ebcv. these results imply that these aa sites are ebcv f and ly strain-specific, or that there are no ebcv-specific sites in the s gene. the n-terminal region of the s subunit of kwd strains (aa - ; laude et al., ) , being shown to function as a receptor-binding domain in mouse hepatitis virus, had a total of aa changes compared with the mebus strain (data not shown). amino acid substitutions in this region could alter the tropism of the coronavirus. for example, the first aa of the s subunit of the prcv were associated with respiratory tropism and reduced enteropathogenicity (wesley et al., ) . therefore, these aa substitutions in the korean wd strains compared with that of the mebus strain may induce either an alteration of the receptor-binding ability during viral invasion or pathogenicity to cattle. the s protein of bcov has a cleavage site located at aa - (krrsrr) and is divided into the peripheral s glycoprotein from the transmembrane domain of the s glycoprotein (abraham et al., ) . the sequence krrsrr at the predicted proteolytic cleavage site was identified in all kwd strains. interestingly, all kwd strains had an ala to ser change at aa , immediately upstream of the cleavage site. since this change was observed exclusively in the respiratory isolates lsu and ok, it was speculated that the change in aa was a potential marker of respiratory tropism (chouljenko et al., ) . although changes in aa surrounding the cleavage site of the ha molecule of the influenza a virus and sendai virus have been demonstrated to modulate its cleavability (rott et al., ; hsu et al., ) , rekik and dea ( ) reported that this change ( ; a → s) did not appear to modulate cleavability, the rate of viral replication or the type of cytopathic changes induced in hrt- cells for their enteric bcov isolates. in our previous paper (hasoksuz et al., ) , this change ( ; a → s) was observed in both respiratory and enteric strains. taken all together, the aa substitution (a → s) at aa does not appear to be a potential marker of respiratory tropism. of the two cleavage products of s, the s subunit is highly conserved among coronaviruses (gallagher and buchmeier, ) . in the present study, the aa sequences of the s subunit of korean wd strains were comparatively well conserved compared with the s subunit of the mebus strain. the notable aa changes were observed only in the first hydrophobic region of all kwd strains. in this region, there were four aa substitutions (aa , , , and ), resulting in a marked increase of hydrophilicity in comparison with that of the mebus strain (data not shown). interestingly, the other known virulent bcov, including rbcv lsu and ok strains, ebcv ly and f strains revealed an increase in hydrophilicity in the first hydrophobic region compared with avirulent strains vacc, l , and mebus (data not shown). the membrane fusion activity is most likely conferred by the internal hydrophobic sequences within the s subunit (luo and weiss, ; yoo et al., ) . therefore, aa substitutions inducing an increase in hydrophilicity observed in our strains and other virulent bcov may alter the fusion activity or pathogenicity of the viruses. putative virulent-specific sites of bcov were reported to exist at aa sites (six within the s subunit and four within the s subunit) in the s gene through comparison with avirulent and virulent strains (zhang et al., ) . in the present study, the s glycoprotein of all kwd strains contained seven aa substitutions (aa , , , , , , and ) which were common for all virulent strains (fig. ) . this is in agreement with chouljenko et al. ( ) , who identified seven virulent-specific aa changes in the s gene by comparison with the rbcv, ebcv, and vaccine strains. since these virulent-specific sites were observed in all virulent bcov, but not in avirulent bcov, these aa changes may affect the structure and function of the s glycoprotein and alter the pathogenetic potential of these viruses (chouljenko et al., ; zhang et al., ) . deduced sequence of the bovine coronavirus spike protein and identification of the internal proteolytic cleavage site the coronavirus surface glycoprotein detection and isolation of coronavirus from feces of three herds of feedlot cattle during outbreaks of winter dysentery-like disease cross-protection studies between respiratory and calf diarrhea and winter dysentery coronavirus strains in calves and rt-pcr and nested pcr for their detection evaluation of concurrent shedding of bovine coronavirus via the respiratory tract and enteric route in feedlot cattle nucleotide and predicted amino acid sequences of all genes encoded by the genomic portion ( . kb) of respiratory bovine coronaviruses and comparisons among respiratory and enteric coronaviruses bovine coronavirus the genome organization of the nidovirales: similarities and differences between arteri coronavirus spike proteins in viral entry and pathogenesis bovine coronaviruses associated with enteric and respiratory diseases in canadian dairy cattle display different reactivities to anti-he monoclonal antibodies and distinct amino acid changes in their he, s and ns . protein molecular analysis of the s subunit of the spike glycoprotein of respiratory and enteric bovine coronavirus isolates protease activation mutants of sendai viruses: sequence analysis of the mrna of the fusion protein (f) gene and direct identification of the cleavage-activation site detection and isolation of winter dysentery bovine coronavirus circulated in korea during the molecular biology of coronaviruses antibody titers against bovine coronavirus and shedding of the virus via the respiratory tract in feedlot cattle corona and related virus: funtional domains in the spike protein of transmissible gastroenteritis virus roles in cell-to-cell fusion of two conserved hydrophobic regions in the murine coronavirus spike protein comparative sequence analysis of a polymorphic region of the spike glycoprotein s subunit of enteric bovine coronavirus isolates studies on the adaptation of influenza viruses to mdck cells winter dysentery in dairy herds: electron microscopic and serological evidence for an association with coronavirus infection isolation of respiratory bovine coronavirus, other cytocidal viruses, and pasteurella spp. from cattle involved in two natural outbreaks of shipping fever genetic analysis of porcine respiratory coronavirus, an attenuated variant of transmissible gastroenteritis virus the s subunit of the spike glycoprotein of bovine coronavirus mediates membrane fusion in insect cells comparison of the nucleotide and deduced amino acid sequences of the s genes specified by virulent strains of bovine coronaviruses this study was supported by the technology development program for agriculture and forestry, ministry of agriculture and forestry, republic of korea. key: cord- -hvakv t authors: chen, guoqian; chen, da-zhi; li, jianhua; czura, christopher j.; tracey, kevin j.; sama, andrew e.; wang, haichao title: pathogenic role of hmgb in sars? date: - - journal: med hypotheses doi: . /j.mehy. . . sha: doc_id: cord_uid: hvakv t high mobility group box protein (hmgb ) is released by necrotic cells or activated macrophages/monocytes, and functions as a late mediator of lethal systemic and local pulmonary inflammation. passive immunization with anti-hmgb antibodies confers significant protection against lethal endotoxemia, sepsis, and acute lung injury, even when antibodies are administered after the onset of these diseases. in light of observations that three chinese herbal formulations recommended for treatment of severe acute respiratory syndrome (sars) specifically inhibited the release of hmgb from innate immune cells, we hypothesize that hmgb might occupy a pathogenic role in sars by mediating an injurious pulmonary inflammatory response. severe acute respiratory syndrome (sars) has killed more than nine hundreds of people around the world and infected thousands more. it has created international anxiety because of its rapid progression and high mortality. in china, several clinical trials revealed efficacy of traditional chinese herbal remedies in reducing symptoms and mortality of patients with sars [ ] . high mobility group box protein (hmgb , formerly known as hmg- or amphoterin) has recently been identified as a new proinflammatory cytokine and a late mediator of inflammation, sepsis, and acute lung injury. in light of observations that several chinese herbal remedies recommended for treatment of sars specifically inhibited the release of hmgb from activated innate immune cells, we hypothesize that hmgb might occupy a pathogenic role in sars by mediating an injurious pulmonary inflammatory response. hmgb as a proinflammatory cytokine hmgb has been for a long time described as a nuclear dna-binding protein in mammalian cells [ ] . recent evidence has revealed that hmgb can be released by necrotic cells or activated innate immune cells (such as macrophages and monocytes), and functions as a proinflammatory cytokine [ ] [ ] [ ] . in response to exogenous stimuli (such as bacterial endotoxin, lipopolysaccharide, lps) or endogenous cytokines (such as tnf, il- b or ifn-c), cultures of macrophages, monocytes or pituicytes actively release hmgb in a time-and dose-dependent manner [ , , ] . the kinetics of hmgb release from activated macrophages/monocytes is significantly delayed, with hmgb accumulation first detectable at h after stimulation [ , ] . this delayed release of hmgb distinguishes it from tnf and other early mediators of systemic inflammatory responses [ ] . extracellular hmgb is able to stimulate the production of tnf and other proinflammatory cytokines from macrophages, endothelial cells or neutrophils, triggering an inflammatory response [ ] . in murine models of endotoxemia or sepsis, or patients with sepsis or rheumatoid arthritis [ ] [ ] [ ] , serum hmgb levels are significantly elevated [ ] . administration of hmgb via various (e.g., focal, intraperitoneal, intracerebroventricular, and intratracheal) routes induces marked inflammatory responses in vivo [ , , ] . taken together, these studies indicate that accumulation of hmgb can amplify the cytokine cascade, and mediate injurious inflammatory responses. the pathogenic role of hmgb as a late mediator of lethal systemic inflammation has been defined in animal models of endotoxemia and sepsis using anti-hmgb antibodies or inhibitors [ , , ] . anti-hmgb antibodies confer a dose-dependent protection in animal models of sepsis, even when the first dose of anti-hmgb antibodies is delayed for h after the onset of sepsis [ ] . administration of a novel anti-inflammatory agent, ethyl pyruvate, dose-dependently inhibits hmgb release, and confers significant protection against the lethality of sepsis [ ] . in animal models of collagen-induced arthritis, administration of anti-hmgb antibodies also confers significant protection [ ] . thus, therapeutic agents capable of inhibiting hmgb activities (such as anti-hmgb antibodies) or release (such as ethyl pyruvate) may hold potential for the clinical management of sepsis and other inflammation diseases [ ] . note: all herbal ingredients were obtained from ny-tongrentang, inc. (flushing, ny, usa), mixed, and boiled in water for min. the water-soluble fraction (recalibrated to a total volume of ml with sterile water) was cleared by centrifugation ( rpm, min, °c) and filtration (through . lm filter), and the clear water-soluble herbal extract was evaluated for the immunomodulatory properties. intratracheal administration of hmgb to lps-resistant c h/hej mice stimulates lung neutrophil accumulation, and increases pulmonary levels of proinflammatory cytokines such as tnf, il- b, and mip- [ ] . histological examination of lung tissue reveals evidence of an acute diffuse inflammatory response, with accumulation of neutrophils in the interstitial and intraalveolar areas, interstitial edema, and protein exudation into the alveolar space [ ] . in a widely used animal model of acute lung injury induced by intratracheal administration of bacterial endotoxin, administration of anti-hmgb , either before or after endotoxin treatment, significantly decreases endotoxin-induced neutrophil infiltration, and lung edema [ ] . despite the ameliorative effects of anti-hmgb antibodies on the development of lung injury and neutrophil accumulation, this treatment does not affect endotoxin-induced increases in pulmonary levels of tnf, il- b, or mip- , establishing the important role of endogenous hmgb itself as a mediator of acute lung injury [ ] . after the outbreak of the sars in china, the state administration of traditional chinese medicine recommended the use of several traditional chinese herbal remedies for prevention and treatment of sars [ ] . clinical observations suggest that chinese herbal remedies are beneficial in the reduction of (i) fever; (ii) air-space shadowing of the chest radiographs; (iii) the use of corticosteroids; and (iv) the death rate of severe sars patients [ ] . to evaluate the immunomodulatory properties of traditional chinese herbal remedies recommended for sars, we examined their potential effect on the release of several proinflammatory mediators in cultures of macrophages and human peripheral blood mononuclear cells (hupbmcs). three chinese herbal formulations were prepared according to the recipes released by the state administration of traditional chinese medicine [ ] (table ) . at clinically relevant doses ( - ll of extract/ml culture medium), none of these herbal formulations inhibited endotoxin-induced production of tnf or il- b by either macrophages or hupbmcs (data not shown). two out of three herbal remedies significantly attenuated endotoxin-induced production of nitric oxide ( fig. (b)) . surprisingly, all three chinese herbal remedies dose-dependently ( - ll/ml) suppressed endotoxin-induced hmgb release by figure effects of three traditional chinese herbal formulations on endotoxin-induced release of nitric oxide and hmgb . murine macrophage-like raw . cells (panels a, b) or huupbmcs (panel c) were stimulated with lps ( ng/ml) either alone, or in the presence of herbal formulations (f , f , and f ), and levels of hmgb and nitric oxide in the culture medium were determined h later by western blotting analysis or the griess reaction as previously described [ ] . shown in the bar graphs (panels a, b, and c) are means ae sem. of three experiments in duplicates (n ¼ ). student's t-test was performed and a p < : was considered significant (*). shown in the bottom of panel c is a representative western blot. cultures of both macrophages ( fig. (a) ), and hu-pbmcs ( fig. (c) ). an excessive immune response to acute coronavirus infection appears to be the fatal factor in patients who die of sars [ ] [ ] [ ] [ ] . autopsy of sars patients revealed signs of diffuse alveolar damage, airspace edema, and bronchiolar fibrin [ ] [ ] [ ] . our hypothesis is that initial acute coronavirus infection of alveolar endothelial cells or macrophages leads to cell injury via virus-mediated cytolysis, which is accompanied by hmgb release from damaged cells (fig. ) . extracellular hmgb , as a mediator of acute lung injury [ ] , subsequently mediates injurious pulmonary inflammatory responses including neutrophil infiltration, derangement of epithelial barrier, lung edema, and lung injury. collectively, these injurious pulmonary inflammatory responses may consequently lead to respiratory failure, and death. this hypothesis can be tested using anti-hmgb antibodies or hmgb inhibitors (such as ethyl pyruvate) in future studies. for instance, if immunoassays using anti-hmgb antibodies indicate a potential elevation of pulmonary hmgb levels in sars patients, it will support the above hypothesis. similarly, if hmgb -specific antibodies or inhibitors attenuate the progression of sars in animal models or clinical settings, it will support an important role of hmgb in the pathogenesis of sars. figure hypothetical role of hmgb in the pathogenesis of sars. hmgb is released from coronavirus infected cells, and then stimulates excessive inflammatory responses. is traditional chinese medicine useful in the treatment of sars? reversing established sepsis with antagonists of endogenous hmgb hmg- as a late mediator of endotoxin lethality in mice release of chromatin protein hmgb by necrotic cells triggers inflammation hmgb as a late mediator of lethal systemic inflammation proinflammatory cytokines (tumor necrosis factor and interleukin ) stimulate release of high mobility group protein- by pituicytes ifn-gamma induces high mobility group box protein release partly through a tnf-dependent mechanism high mobility group box chromosomal protein : a novel proinflammatory mediator in synovitis high mobility group box chromosomal protein plays a role in the pathogenesis of rheumatoid arthritis as a novel cytokine high mobility group box chromosomal protein , a dna binding cytokine, induces arthritis the cytokine handbook extracellular role of hmgb in inflammation and sepsis hmg- as a mediator of acute lung inflammation ethyl pyruvate prevents lethality in mice with established lethal sepsis and systemic inflammation successful treatment of collagen-induced arthritis in mice and rats by targeting extracellular high mobility group box chromosomal protein activity lipid unites dissipate syndromes of sepsis herbal supplement severe immune response kills sars victims lung pathology of severe acute respiratory syndrome (sars): a study of autopsy cases from singapore the clinical pathology of severe acute respiratory syndrome (sars): a report from china pathological study on severe acute respiratory syndrome we thank drs. ping wang, man yu, and xiaoling qiang for critical reading of the manuscript. this research was supported in part by the north shore-lij research institute, and the national institutes of health (nih, nigms, gm ). key: cord- -tvc eg authors: yang, ming; yu, xin title: china’s rural electricity market—a quantitative analysis date: - - journal: energy doi: . /j.energy. . . sha: doc_id: cord_uid: tvc eg abstract the objective of this paper is to quantify the development of the rural electricity market at county level and below in china. a sectorial energy demand analysis and forecasting model was developed to analyze six chinese provinces with different economic backgrounds. historical data for over years were collected on rural economic development, households, population, per capita income, community infrastructure development, capital investment, electricity consumption, output values in agriculture sector, and township and village enterprises (tves). this paper concludes that by , annual electricity demand will increase at a rate between − . % and . % (depending on the sectors and provinces). it also recommends a preferred order for future rural electricity investment: jiangsu, hebei, henan, shaanxi, liaoning and xinjiang, i.e. from the most to the least developed provinces, if the investment objectives are to find the best market return and the greatest impact on rural market development. over the past few decades, china has successfully implemented economic system reforms in rural areas, and as a result, the rural electricity market is expanding quickly. from january to september , the chinese government invested billion yuan (us$ billion) in urban and rural electricity market development [ ] . its goal was to stimulate domestic demand and push economic development in china, given that about million people ( % of the country's population) are living in rural areas. however, very little of china's rural electricity market is known to the public due to a lack of research and a rapid development of the market. many questions remain unclear, such as ''what are the shares of electricity consumption in rural areas?'', ''how and where will these parameters change in rural china?'', ''how much electricity will a rural household consume?'', ''which part of china will incur the greatest rural electricity expansion by ?'', and ''what is the priority order of investment in rural electricity markets? the objectives of this paper are to establish a model to identify and assess rural electricity market development in china as well as answer the above questions. in this research, we collected a large amount of rural, social and economic data for six provinces in china. we grouped the provinces according to their economic and geographical conditions. jiangsu and liaoning were selected and grouped together because ( ) they both have good economic development conditions in rural areas; and ( ) they are both located in the eastern coastal area. liaoning is in northeast and jiangsu in east china. these two provinces represent china's well-developed areas. hebei and henan, located in central china, are grouped together to represent china's mid-developed provinces. finally, xinjiang and shannxi, located in the western region of china, represent china's least developed provinces. these three groups will also represent six electricity markets that the chinese government plans to establish for power retail competition by . we forecast electricity demand in the rural areas of the six provinces and tried to identify the best investment market in terms of high growth rate of electricity demand and greatest impact on rural electrification and economic development. the paper is comprised of five parts. in section , we present literature reviews on the electricity market. this includes brief assessments of china's electric power sector and provides visions of the rural electricity market that leads to the focus of this study. our model, methodology and approaches are discussed in section . in section , we analyze data, describe variables, and state scenarios used in the study. section , the key section of this paper, presents our results. section shows the weakness and limitations of this research and directs further research in this topic. conclusions and recommendations are presented in section . finally, we have included a summary of electricity market information for the six provinces in appendix a. the information presented in this paper will be useful to policy makers in china and other developing countries when planning for rural electrification and electricity full retail competition. very few articles and documents are available in detailed quantitative analysis for the rural electricity market. historical papers in this field mainly focus on electricity market reform, urban electricity market, and qualitative analysis. yang [ ] presents an international literature review on rural electricity market development with case studies for the usa, thailand and lao people's democratic republic (lao pdr). the paper summarizes the following: investing in rural electricity market in general is not financially viable but economically sound and socially friendly. private sectors have little incentive to participate in rural electricity market development. instead, governments should initiate and/or invest in rural electricity development programs. (ii) the main objectives of the government in rural electricity market development include poverty reduction, rural economic development, sustainable development in rural areas, and mitigating the migration from countryside to city. yang and yu [ ] reviewed and examined the evolution of china's power sector for the period - . the paper covers the areas of institutional development and reform of china's power industry, power shortages in the s, power supply by different resources, capital investment structure, tariff policies, demand side management, energy efficiency policies and practices, laws in the power industry, and environmental impact from power generation. between and , two additional hot topics have been added to china's power literature: ( ) continued reform of the power industry aiming at establishing a full retail market for power sector competition; and ( ) rural electricity market development. yang [ ] reviewed the chinese government strategies during - on power system reform towards a market based power system. the paper summarizes that china's rural market has been initially formed and can provide % of the rural households with electricity by . in , the national government (the state council) issued a national policy document (document no. , ) and outlined four key issues in china's power reform [ ] . rural electricity market development is one of the key issues in china. the chinese government also established ''four principles of facilitations'' to the development of the rural market. these are ( ) facilitating economic development and poverty reduction; ( ) facilitating the merger of urban and rural power systems (any separation is not allowed); ( ) facilitating power supply cost reduction and alleviating farmers' financial burdens; and ( ) facilitating further power system reform in china. china's current electricity situation can be described in three key statements: ( ) short of power supply; ( ) electricity tariff reforming; and ( ) electricity market development. the state electricity regulatory commission said that power supply and demand in was generally balanced. in , the demand exceeded the supply and provinces, autonomous regions and municipalities (including shanxi, jiangsu and shanghai) faced severe power shortages last summer that affected local economic and social development, particularly rural households' daily lives [ ] . the commission attributed the shortages mainly to (i) the lack of electricity market analysis, (ii) incorrect power demand forecasting, (iii) the slow investment in power supply, (iv) climate change (hot summer), and (v) higher production by enterprises after the severe acute respiratory syndrome (sars) outbreak. china's policy makers are pondering responsive market strategies, hoping to accelerate electricity facility construction and balance power supply and demand by ''breaking monopoly and introducing competition'' during the coming years. following the splitting up of the state power corporation at the end of , china plans to create six regional competitive power markets within years in a determined move to break up the traditional province-based electricity monopolies. these comprise: north, northeast, east, west, central and south electricity markets. when established, the markets will be able to sup-port a power plant to sell electricity within the local market as well as export to other markets as long as the transmission line capacities are sufficient. similarly, an end-user will be able to choose a power supplier either inside or outside of his own market system. this is called a ''full retail competition market'' in china. evidently, rural electricity market development plays a very important role in china's power reform. by the end of , china still had about million poor people, most of whom could not access electricity. according to the china statistical press [ ] , one rural family has . people. thus, million rural residents would be equivalent to about . million rural households. if us$ is required today to electrify one rural family [ ] , about us$ . billion is needed. developing the rural electricity market, providing power for these people and raising them above the poverty line by (with limited funds) is a great challenge to the chinese government and the power industry. in the coming decade, china's rural electrification has multiple tasks. the first task should be rural electricity demand forecasting and rural market development planning. the results will assist the government in arranging capital and formulating policies. the second task should be the implementation of institutional reforms to strengthen the rural electrification results. in the long run, the reforms will encourage full retail competition in the rural electricity market. the third task would be the continuation of the ''brightness program'' and the ''developing rural power with wind program''. these programs are very important to electrify remote households with renewable energy technologies, especially in the western region of china. in this study, we focused on electricity market analysis and power demand forecasting for the rural areas-county level and below. a sectorial electricity demand model was developed. on the basis of available data, we divided the rural electricity market into several sectors: township and village enterprises (tves), agriculture and other family business, and households. value added, energy intensity and market share of the economy are the main variables in the study. the model and variables are discussed in detail. the theoretical study of electricity market started in the middle of the th century with the development of computer science. however, progress in theory and practice was slow during the initial period. it was not until the s that the theoretical study of medium-to-long-term electricity demand forecasting began. a series of forecasting methods, such as top-down econometric modeling and bottom-up end-use accounting modeling, was successively developed and widely accepted in the electricity demand forecasting of power systems [ ] . having reviewed the literature of energy modeling, lin made an analysis of energy demand forecasting for china by using an econometric model. the article states that econometric models using gdp and electricity tariff as main driving variables may have at least one disadvantage. since the economic variables employed in the econometric model are likely to be endogenous, estimating electricity demand by a single equation may produce simultaneous bias and lead to unreliable forecasts. there could be many other factors that require proper attention in determining electricity demand in china's rural area. an important one, for example, is the climate. dry climate in a year means that more electricity is needed for irrigation. cold or gloomy days mean that more electricity will be generally used for heating and lighting. electricity demand also varies according to the time of day. therefore, the demand for electricity in rural areas depends on climate changes and peak demands during cold and hot seasons. however, it is difficult to incorporate such climate change factors in an econometric model. for our research, we used seda v , a well-developed mathematical computer model for sectorial energy demand analysis. we developed the first version to analyze the strategies of asian oil importing countries and economies during - [ ] . in the current study, to describe energy consumption in rural households and capture the variable of ''population'', we updated the model with a special module. in this section, we present part of the mathematical equations of seda v . the total electricity demand (ed) in the rural electricity market is the sum of electricity demand for all sectors in a rural area. mathematically, it can be expressed as follows: where ed t is the total electricity demand, ed it is the electricity demand in sectors not including rural households , ed ht is the electricity demand in rural households in period t. eq. ( ) can be further formulated as follows: where va it is the value added by sector i (i ¼ , ,. . .n) at period t (t ¼ , , ,. . .m) (billion yuan); ei it is the electricity intensity in sector i (i ¼ , ,. . .n) at period t (t ¼ , , ,. . .m) (gw h/million yuan); s it is the economic shares of the rural economy in sector i at period t (%); gdp t is the gross domestic product of rural economy at period t (billion yuan). since rural gdp is not available, we use rural economic output in this study; nh t is the number of households at period t (t ¼ , , ,. . .m); eih t is the electricity intensity in rural household at period t (kwh/per household); inh t is the inverse of the number of people per household at period t (household/number of people); pop t is the rural population at period t (million persons). by differentiating eq. ( ), we derived where dec t is the electricity demand change in period t in rural market, p n i¼ ½ei it  gdp t  ds it is the electricity demand change in rural market in period t due to structural effect, p n i¼ ½s it  gdp t  dei it is the electricity demand change in rural market in period t due to sectoral effect, p n i¼ ½ei it  s it  dgdp t is the electricity demand change in rural market in period t due to activity effect, eih t  inh t  dpop t is the electricity demand change in rural households in period t due to population change in rural area, eih t  pop t  dinh t is the electricity demand change in rural households in period t due to the change of number of people per household, in h t  pop t  deih t is the electricity demand change in rural households in period t due to the change of electricity intensity per household. on the basis of historical data and scenarios, we used the above model to analyze the rural electricity market year by year from to . in determining the changes of the parameters (ds it , dei it , etc.), we analyzed the historical values of the variables and used scenario analysis on the basis of china's th five-year economic and social development plan, and interviewed with chinese government officers on social and economic development policies. in the next section, we will discuss the data, variables, and their processes for fitting the model. a panel data set including six provinces over the period from to was constructed from various governmental sources [ ] [ ] [ ] [ ] [ ] [ ] [ ] and on-site interviews with many experts in china. in addition, we projected data for the period - for electricity market analysis. in the following example, we briefly describe the methods used in preparing several historical data in the research. the rural economy was divided into five sectors: tves, agriculture, county level industry (industry), household, and others. given that we only had output data for the county industrial sector in , we estimated industrial outputs below county level and presented the results between and by using provincial gdp for the period. since we did not have any output data for other sectors, we simply assumed growth rates and projected the electricity increase in the period - . in the following, we first analyze historical electricity market. electricity consumption by rural households, rural irrigation, agriculture, and tves in each of the six provinces for the period - is collected to analyze the rural electricity market. on the basis of the research objectives and the available data, we used the primary variables defined in table a . for this study. we collected data of - from the chinese government [ ] . the following list presents the main collected data. (i) yearly national economic growth rate: %. this scenario will guide the assumptions of tve output and other sectors' output in the six provinces. (ii) creating new jobs and transferring rural residents to urban residential areas in years: million. this information tells that rural population might keep decreasing or stable in the future years due to migration. during a field trip to china, the prime author interviewed a number of chinese experts and government officers regarding economic and social development trends for the selected six provinces . the following information was collected from the trip: electricity intensity for rural irrigation will increase slightly after rural electrification. lower electricity prices reduce labors and increase machines in agriculture production. (ii) electricity intensity for tves industry will continue to decrease because tves will change their structure from high electricity intensity industries to low electricity intensity industries. (iii) electricity intensity for ''other sectors'' will decrease slightly because of the electricity conservation effect and increasing outputs from newly developed low electricity intensive enterprises. the number of people in a rural household will slightly decrease at the trend during - due to the government policy of birth control, economic development in rural areas, urbanization and migration. annual rural population growth rate will remain low. (vi) net rural per capita income growth rates in real prices: jiangsu: - %; liaoning: %; henan and hebei: %; shaanxi: - %; xinjiang: - % . (vii) inflation rate may keep between % and %. (viii) the growth rates of tves will be the same as those of individual provincial industries. (ix) the chinese government will continue to invest in rural electrification. during - , the government will invest an additional billion yuan. the government's rural network investment during - ( billion yuan) did not have significant impact on electricity consumption of rural agriculture production because the objective was to retrofit the power network for household electricity supply only. however, reduced tariffs will in some way stimulate electricity consumption in agriculture production. (xi) electricity intensity in tev and other sectors might decrease due to electricity conservation and business structure change. however, it will be at a slower rate than during - because electricity conservation will be more expensive. (xii) rural market reform will continue to aim at full retail competition. in the first section, we mainly considered the historical data of - to set growth rates. in the second and third sections, we took into account both historical trend, future scenarios and findings. in order to keep the article within a reasonable length, we have described the case of jiangsu in detail and combined the discussion and results of the other provinces. table a . in appendix a also presents the projected results of the future electricity market by sector in jiangsu: electricity markets in all sectors will continue to grow; however, growth rates will decrease over the years. in addition to jiangsu, we have quantitatively analyzed electricity markets for liaoning, hebei, henan, shaanxi and xinjiang. the analysis for the five provinces followed the same procedure as that of jiangsu. tables a. electricity market development ranked by growth rates from the highest to lowest are as follows: xinjiang, hebei, jiangsu, shaanxi, henan and liaoning. the fact that xinjiang might have the highest growth rate in electricity demand during - is due to its historically small base of electricity consumption. hebei ranked second in both electricity quantity and growth rate, and jiangsu ranked third in growth rate. this indicates that the electricity market will be reasonably sustainable and stable in both hebei and jiangsu in the near future (see fig. ). table a . and fig. comparatively highlight the analysis results of electricity markets for the six provinces. it is observed that jiangsu, hebei and henan have good rural electricity development potential with both high volumes of electricity demand bases and fast growing rates in the coming - years. among the strengths of the model and the study include conciseness, flexibility, and understandability. the model is developed on the basis of basic theory of energy economics. the study approach can be operated by a professional if he has experience in running computer models and undertaking such energy market analysis assignments. since seda is a bottom-up accounting model, there are a couple of weaknesses or limitations in the research. a bottom-up accounting model requires a large amount of data. if more detailed data is available, the energy sector can be further broken down, and more accurate model results can be achieved. however, data shortage is one of the main limitations in china. household business income and county industry output, for example, are not statistically avail- able. we had to estimate the data. in addition, due to the reform of the statistical system and a change in methodology, some historical data are not consistent throughout the history. for example, the statistical methodology for rural population and economic sectors in liaoning has been changed in the early s. many people living in rural areas have been statistically treated as urban residents. as a result, the rural population according to the chinese official statistical data has been decreasing very quickly. by , there were about . million people in liaoning's rural areas. this figure may not be true. as another example, energy consumption by county small industries has been statistically presented in the industry sector. in recent years, the state power corporation has treated electricity consumption at county level and below as rural electricity consumption. a further limitation of the bottom-up accounting model is that it does not explicitly apply the variables of energy price and income. however, these variables have been acknowledged implicitly in the model. for example, a survey on willingness to pay for electricity at different prices and different income levels has been undertaken in the research and the information has been incorporated in setting the growth rate of household energy consumption. the model can be used to undertake detailed cross-sectorial analysis of similarities and differences among provinces. part of the mathematical formulas of the model for such analysis was stated in yang [ ] . however, conducting such analysis might become the scope of another paper . nevertheless, the main significance of this study is that we used a computer-supported model to analyze the electricity market and forecast electricity demand for the chinese rural areas. the availability of this type of analysis is scarce in the history and literature. the impact of china's rural economic reform on rural sectoral energy consumption-an article in preparation. there is a great electricity market development potential in china's rural areas. even if the chinese rural residents are far above the poverty benchmark, their electricity consumption is very low. for example, in jiangsu-the most developed province among the six ones, the average per capita income of a rural resident was more than yuan/year/capita (us$ ) in . if there are four people in a rural family in jiangsu, the family income will be , yuan/year (us$ /year) . with this income, the family can purchase all the basic family electric appliances such as a tv, a refrigerator, a washing machine in or years. however, our findings show that electricity consumption per family in jiangsu rural area was about . kwh per day. this amount of electricity can only run a refrigerator. it is evident that electricity demand by rural households will be very high. after retrofitting the rural power network (rural electrification) and cutting down the rural electricity prices, electricity consumption in rural households will increase quickly. in the agriculture sector, electricity consumption will also increase during - , even though the sector has declined in electricity consumption in some provinces. after the mass investment in the rural power network since and rural institutional reform, electricity tariffs in china's rural areas have been reduced and this has facilitated electricity substitution for labor and diesel in agriculture production. tves and county industry have been increasing consumers in the rural electricity market. given that tves and county industry will be the most important economic sectors in the rural economy between and , electricity demand will continue to grow. the average annual growth rates of tves between and in the six provinces are in the range from . % in liaoning to . % in xinjiang, while the rates for county industry are between . % in liaoning and . % in jiangsu. electricity demand for other sectors will continue to grow but the shares are not significant in the near future. the reason is that the service industry in rural areas has not been developed well and the shares of electricity demand in total are small, ranging from . % in henan in to . % in xinjiang in (see table ). on the basis of the above electricity market analyses, we would recommend the preferred future rural electricity market investment in the following order: jiangsu, hebei, henan, shaanxi, liaoning and xinjiang. this implies the following order of investment for the development of six national electricity markets: east and south china electricity markets, north and northeast china electricity markets, and central and western electricity markets. china's potential of power blackouts. investment and construction news china's power management china's rural electrification and poverty reduction the reform is not just for reform. china power news china faces power shortage beijing: china statistical publishing house structural change, efficiency improvement versus energy demand forecasting-case study of china's power sector beijing: china statistical publishing house beijing: china statistical publishing house beijing: china statistical publishing house beijing: china statistical publishing house beijing: china statistical publishing house beijing: china statistical publishing house state power cooperation-spc. china rural electrification integration statistical book china people's congress. the planning outline of china's th five-year national economic and social development. a government policy paper approved by the fourth meeting of the ninth people's congress of china acknowledgements are due to the anonymous referees for their excellent comments on the previous versions of the article. the authors are indebted to the editor-in-chief of energy-the international journal for his constant encouragement and advice on refining this article. the key: cord- -ulavigi authors: remington, k. m.; trejo, s. r.; buczynski, g.; li, h.; osheroff, w. p.; brown, j. p.; renfrow, h.; reynolds, r.; pifat, d. y. title: inactivation of west nile virus, vaccinia virus and viral surrogates for relevant and emergent viral pathogens in plasma‐derived products date: - - journal: vox sang doi: . /j. - . . .x sha: doc_id: cord_uid: ulavigi background and objectives human plasma is the source of a wide variety of therapeutic proteins, yet it is also a potential source of viral contamination. recent outbreaks of emergent viral pathogens, such as west nile virus, and the use of live vaccinia virus as a vaccine have prompted a reassessment of the viral safety of plasma‐derived products. the purpose of this study was to evaluate the efficacy of current viral inactivation methods for west nile and vaccinia viruses and to reassess the use of model viruses to predict inactivation of similar viral pathogens. materials and methods virus‐spiked product intermediates were processed using a downscaled representation of various manufacturing procedures. virus infectivity was measured before and after processing to determine virus inactivation. results the results demonstrated effective inactivation of west nile virus, vaccinia virus and a model virus, bovine viral diarrhoea virus, during pasteurization, solvent/detergent treatment and caprylate treatment. caprylate provided rapid and effective inactivation of west nile virus, vaccinia virus, duck hepatitis b virus and sindbis virus. inactivation of west nile virus was similar to that of bovine viral diarrhoea virus. conclusions this study demonstrates that procedures used to inactivate enveloped viruses in manufacturing processes can achieve inactivation of west nile virus and vaccinia virus. in addition, the data support the use of model viruses to predict the inactivation of similar emergent viral pathogens. plasma-derived products, such as clotting factors, intravenous immunoglobulins, alpha -proteinase inhibitor ( α -pi ) and human serum albumin, are routinely used in clinical practice and the safety of these products with respect to transmission of infectious viruses has never been higher. the high margin of viral safety can be attributed to a number of measures that manufacturers have implemented during the last decade. these measures are part of a multifaceted strategy for pathogen safety that includes donor screening, donation testing, virus inactivation or removal measures during manufacture, strict adherence to good manufacturing procedures and post-use surveillance of products. however, recent outbreaks of emergent viruses, such as west nile virus ( wnv ), severe acute respiratory syndrome (sars)-associated coronavirus and monkeypox, have indicated that potential threats to the blood supply exist and have resulted in a re-evaluation of the current pathogen safety strategy for plasma products. although donor deferral and plasma donation screening effectively provide safety for known pathogens, the presence of a new or emergent virus cannot be detected when its existence is not anticipated. consequently, robust viral clearance procedures within manufacturing processes are critical. the capacity of these procedures to remove or inactivate viruses is validated in small-scale studies in which high levels of virus are spiked into a manufacturing intermediate and the reduction of infectious virus is measured. typically, a number of these experiments are performed, each using a different virus that could potentially be present in plasma. some potential contaminants, such as hepatitis c virus (hcv) and parvovirus b , cannot be readily cultured in vitro . for viruses like these, a closely related virus with similar physicochemical properties, and which can be easily propagated in the laboratory, is used [ ] . like hcv, bovine viral diarrhoea virus (bvdv) is a member of the flaviviridae and has been used for many years as a model for hcv. other viruses, such as the flaviviruses yellow fever virus and tick-borne encephalitis virus, as well as the alphaviruses sindbis virus and semliki forest virus, have also been used to model hcv. inactivation/ removal of bvdv and other hcv model viruses by various manufacturing processes has effectively represented hcv clearance, as evidenced by the lack of hcv transmission by products which have claimed significant levels of bvdv clearance. wnv, a mosquito-borne encephalitis virus, is now epidemic in the united states. most wnv-infected individuals have subclinical infections, with a short, but defined, viraemic period. during epidemics of wnv infection, when the prevalence of viraemia within the population is high, the risk of a viraemic blood or plasma donor is also high. in fact, in , wnv was transmitted to transplant recipients from an infected organ donor and to recipients of whole blood and platelets from infected donors [ ] . this resulted in the implementation of a united states food and drug administration (fda)-endorsed wnv screening programme for whole blood. the programme includes donor deferral questions and nucleic acid amplification tests (nats) [ ] [ ] [ ] . the fda and the european agency for the evaluation of medicinal products (emea) concur with manufacturers of plasma products that various viral-inactivation measures used during the processing of these products are sufficient to provide a high degree of safety for this virus [ , ] . the live vaccinia virus ( vv ) vaccine has recently been used as part of the united states national smallpox immunization program. in an interim recommendation, the centers for disease control advised immunization with this vaccine for individuals at risk for monkeypox infection [ ] . the possibility of transmission of vv during whole blood or plasma donations by recently vaccinated individuals has resulted in recommendations by the fda to defer donors who have recently received the smallpox vaccine [ ] . we have recently evaluated the inactivation of two newly emergent potential contamination threats: wnv and vv. vv is a unique enveloped virus, with complex lipid membrane structures [ ] . although some studies have evaluated vv inactivation, it is not always included in the panel of viruses used for virus clearance studies for plasma-derived products. wnv, a member of the flaviviridae, is closely related to bvdv, which has been used for many years as a model for hcv. it would be expected, then, that wnv inactivation would be similar to that of bvdv, and that wnv-inactivation studies could verify the validity of using bvdv as a model for virus-inactivation studies. the virus-inactivation data presented here demonstrate that current virus-inactivation procedures provide inactivation of wnv and vv and support the use of virus-inactivation data from model viruses. manufacturing process intermediates for anti-haemophilic factor (ahf) (koate®-dvi), intravenous immunoglobulin (ivig-s/d when produced using the solvent/detergent process: gamimune®n; ivig-c when produced using the caprylate/ chromatography process: gamunex®), intramuscular immunoglobulin (igim) (baygam®), α -pi (prolastin®) and albumin ( % and %; plasbumin®) were obtained from the bayer healthcare plasma fractionation facility (clayton, nc). human plasma protein solution (hpps) was obtained from the bayer healthcare recombinant factor viii (kogenate®fs) manufacturing facility (berkeley, ca). hpps is used as a component of the production medium for kogenate®fs and is a % human albumin protein solution. for pasteurization studies, an aliquot of stabilized α -pi, hpps, % albumin or % albumin was heated to ± · ° c and then spiked to % (v/v) with wnv, vv or bvdv. the virus spike and product were mixed well and incubated at ± · ° c for h. aliquots were removed at the indicated times, placed on ice and immediately assayed for infectious virus. a separate, unheated, virus-spiked product sample was used to determine the initial virus concentration. the α -pi intermediate, ph · , was stabilized during pasteurization with · m citrate and % sucrose. hpps and albumin were aqueous solutions at ph · ; hpps and % albumin were stabilized with m m n -acetyl-dl -tryptophan and m m sodium caprylate, while % albumin was stabilized with m m n -acetyl-dl -tryptophan and m m sodium caprylate. smallscale pasteurization experiments were performed in - ml volumes. during manufacture, albumin is pasteurized in its final container, and so the volume of the virus experiments was similar to that of production. prolastin pasteurization experiments were approximately · % of production scale and hpps pasteurization experiments were · % of production scale. during the production of ahf, the product intermediate is treated with · % tri-( n -butyl)-phosphate (tnbp)/ % polysorbate (tween ) for h at - ° c. for these studies, a concentrated stock solution of tnbp/ tween was added to aliquots of ahf intermediate solution to · %/ % or · %/ · %. virus was added to the solution to % (v/v). the mixture was incubated at ± · ° c for h, and aliquots were removed for titration at the indicated time-points. virus, added to hank's balanced salt solution (hbss) to % (v/ v), was an untreated control. small-scale experiments were conducted in -ml volumes, approximately · % of production scale. during the production of ivig-s/d or imig, the process intermediate is incubated in · % tnbp/ · % sodium cholate (cholate) for - h at ± ° c. the protein concentration is adjusted to - · % and the ph is adjusted to · - · . for vv-, wnv-or bvdv-inactivation studies, concentrated tnbp/ cholate from a stock solution was added to an aliquot of process intermediate to · %/ · % or to · %/ · % and then spiked to % (v/v) with virus. the mixture was incubated at ± · ° c for h and aliquots were removed at the indicated time-points for quantification of infectious virus. hbss was spiked to % (v/ v) with virus and used as an untreated control. small-scale experiments were conducted in -ml volumes, approximately · % of production scale. the caprylate-inactivation experiments were carried out as described previously [ ] . briefly, caprylate was added to gamunex® intermediate to a specified final concentration. throughout the addition of caprylate, the ph of the process intermediate was maintained at · by adding either · m acetic acid or · m sodium hydroxide. the solution was then spiked to % (v/v) with virus that had been ph-adjusted to · - · and the virus-spiked solution was incubated at ° c for min. aliquots for virus titration were removed from all solutions at the indicated times throughout the incubation. small-scale experiments were conducted in -ml volumes, approximately · % of production scale. although m m caprylate is used in this step in production, the concentrations of caprylate evaluated in these studies ranged from m m to m m . as the gamunex® intermediate contains ≈ m m residual caprylate [ ] , caprylate from a · m stock solution was added to increase caprylate levels to within the desired range. to confirm that the targeted caprylate range was reached during inactivation experiments, the concentration of caprylate in the process intermediate following addition was measured. analytical testing could not be performed on virus-spiked solutions, so mock-spiked solutions, containing % (v/v) virus propagation medium instead of virus, were generated. during the manufacture of gamunex®, the final product at ph · - · is incubated at - ° c for - days as an enveloped virus-inactivation step. for this study, the gamunex® final product that had been adjusted to ph · was spiked to % (v/v) with wnv and incubated at ° c. samples were removed on days , , and , and infectious wnv was quantified. as ph · is above the normal ph range used in production, this represented a 'worst-case' situation. the volumes used for the virus-inactivation experiments were similar to the final container sizes of product. vv (the wr strain) was propagated and assayed at apptec laboratory services (camden, nj). all wnv, except for that used in the gamunex® low-ph incubation study, was also propagated and assayed at apptec laboratory services, where infectious wnv or vv was quantified by making end-point serial log dilutions of the test samples or positive controls in serum-free medium. vv was assayed with a plaque assay using monkey kidney (bs-c- ) indicator cells. wnv (the ny- strain) was assayed with a plaque assay using african green monkey kidney (vero) cells. for both viruses, · -ml aliquots of serial dilutions of samples were plated on multiple wells of six-well plates. virus titres were reported as plaque-forming units (pfu) per ml. wnv (ny- strain), used in the gamunex® low-ph incubation study, was propagated and assayed in vero cells that were obtained from the american type culture collection (atcc; rockville, md). sindbis virus (ar- strain) was propagated and assayed in baby hamster kidney (bhk- ) cells obtained from the atcc. bvdv ( kentucky- strain), obtained from the biological research facility and faculty (ijamsville, md), was propagated in madin-darby bovine kidney ( mdbk) cells and assayed in bovine turbinate ( bt) cells. test samples were neutralized before they were assayed. all in-house viruses were quantified by tissue culture infectious dose ( tcid ) and, for each dilution, - replicates of a · -ml aliquot were plated in each well of -well microtitre plates. cell monolayers were then observed microscopically for the presence of viral infection, as indicated by a viral cytopathic effect (cpe ). each well was scored as positive or negative, and the results were converted into a titre ( log median tcid /ml) by using the method of spearman & karber [ , ] . duck hepatitis b virus ( dhbv ), a hepatitis b virus ( hbv ) surrogate, was obtained from hepadnavirus testing, inc. (palo alto, ca) and consisted of dhbv-containing serum obtained from congenitally infected ducklings. the virus was assayed at apptec laboratory services in primary duck hepatocytes. samples were serially diluted and · ml of each dilution was assayed in quadruplicate in -well plates. ten days following inoculation, monolayers were fixed and wells containing infected hepatocytes were identified using fluorescence microscopy after incubation with a murine monoclonal antibody to the dhbv surface antigen, washing and a subsequent incubation with a fluorescein isothiocyanate (fitc)-conjugated sheep antibody to mouse immunoglobulin g. results were converted into a titre by using the method of spearman & karber [ , ] . for all virus assays, limits of detection were based on the results of cytotoxicity and viral interference experiments, which evaluated whether the product intermediates were toxic to the indicator cells or interfered with the ability of the virus to infect the cells. the data in table show the inactivation of wnv, vv and bvdv in α -pi during a -h pasteurization at ° c. the protein, at a concentration of mg/ml, is in a citrate buffer containing · m citrate and % sucrose for stabilization during heating. vv was reduced by log within h, and by h was undetectable. wnv and bvdv were inactivated to the lower limits of detection within h, and infectious virus was undetectable after h at ° c. a total of ≥ · log , ≥ · log and ≥ · log of wnv, vv and bvdv, respectively, were inactivated. wnv and vv were also inactivated during the pasteurization of hpps (table ). this solution contained mg/ml protein and was stabilized with m m sodium caprylate and m m acetyltryptophan. complete inactivation of wnv was observed after min of heating at ° c, resulting in a total inactivation of ≥ · log . approximately four log of vv was inactivated within min; within h of pasteurization, no infectious vv was detectable. a total inactivation of ≥ · log vv was observed. when % or % albumin was spiked with either wnv or bvdv and pasteurized at °c for h, all infectious virus was inactivated (table ). within min of incubation of % albumin at °c, wnv was near the lower limit of detection and no bvdv could be detected. pasteurization of % albumin inactivated ≥ · log wnv and ≥ · log bvdv. similarly, pasteurization of % albumin resulted in the complete inactivation of both wnv and bvdv within min. a total of ≥ · log wnv and ≥ · log bvdv were cleared. within h of incubation of the ahf intermediate with either · % tnbp/ % tween or · % tnbp/ · % tween , no infectious wnv could be detected (fig. a) . the production operating concentration of · % tnbp/ % tween resulted in ≥ · log inactivation of wnv, and when the concentration was diluted twofold, ≥ · log inactivation was also observed ( table ) . solutions of ahf spiked with bvdv and incubated in · % tnbp/ % tween or · % tnbp/ · % tween resulted in ≥ · log and ≥ · log inactivation, respectively (table ). in contrast, vv was more resistant to inactivation under these conditions. following h of incubation in · % tnbp/ % tween , · log inactivation was achieved ( fig. b, table ). were treated with tri-(nbutyl)-phosphate ( tnbp)/tween and the inactivation of infectious (a) wnv in · % tnbp/ % tween or · % tnbp/ · % tween or (b) vv in · % tnbp/ % tween was determined. for solutions of intravenous immunoglobulin produced using the solvent/detergent process (ivig-s/d), the inactivation of (c) wnv in · % tnbp/ · % cholate or · % tnbp/ · % cholate or (d) vv in · % tnbp/ · % cholate was determined. each wnv point represents the mean of three determinations and each vv point represents a single determination. the dashed lines in panels (b) and (d) represent the limit of detection. overall reduction factors are presented in table . table virus inactivation by tri-(n-butyl)-phosphate (tnbp)/tween in solutions of anti-haemophilic factor (ahf) or tnbp/cholate in intravenous immunoglobulin produced using the solvent/detergent process (ivig-s/d) inactivation step fig. (a) . b from fig. (b) . fig. (c) . e from fig. (d) . for west nile virus (wnv), each value represents the mean of three determinations; for bovine viral diarrhoea virus (bvdv), each value represents the mean of two or three determinations; and for vaccinia virus (vv), the value is the result of a single determination. solvent/detergent solutions were incubated at °c for h. incubation of wnv in an ivig-s/d process intermediate solution, containing either · % tnbp/ · % cholate (manufacturing conditions) or · % tnbp/ · % cholate, resulted in complete inactivation of the virus within min (fig. c) . approximately log of wnv was inactivated (table ) . similarly, in · % tnbp/ · % cholate or · % tnbp/ · % cholate, ≥ · log and ≥ · log bvdv inactivation, respectively, was observed (table ) . unlike the observations with tnbp/tween , complete and rapid inactivation of vv was achieved during the treatment of ivig-s/d with · % tnbp/ · % cholate. within min, > log of inactivation was achieved and, by h, no infectious virus could be detected (fig. d) . a total of ≥ · log of vv was inactivated (table ) . similarly, wnv was readily inactivated when the igim process intermediate was incubated in either · % tnbp/ · % cholate (manufacturing conditions) or · % tnbp/ · % cholate (fig. ) . complete inactivation was observed within min of incubation. total inactivation of ≥ · log and ≥ · log were achieved in · % tnbp/ · % cholate and · % tnbp/ · % cholate, respectively. treatment of a gamunex® intermediate solution with sodium caprylate resulted in the complete inactivation of both wnv and vv within min. in mm caprylate at °c, ≥ · log wnv was inactivated (fig. a, table ). when incubated at °c in mm caprylate, ≥ · log vv was inactivated (fig. b, table ). caprylate has previously been shown to provide robust and effective inactivation of human immunodeficiency virus (hiv), pseudorabies virus (prv) and bvdv [ ] . to further evaluate the efficacy of this innovative method for the inactivation of enveloped virus, additional models for hbv and hcv were used in inactivation studies ( table ). the data demonstrated that no infectious dhbv, an hbv surrogate, could be detected following incubation in mm caprylate. within min, dhbv inactivation was complete (kinetics not shown). another model for hcv -sindbis -was similarly shown to be rapidly and completely inactivated in mm caprylate. inactivation of hiv, prv and bvdv have previously been shown to be robust with respect to caprylate concentration [ ] . robust inactivation of bvdv with respect to protein concentration, ph and incubation temperature have also been demonstrated [ ] . to further investigate the robustness of caprylate inactivation of enveloped viruses, a series of studies were performed to evaluate the robustness of sindbis virus inactivation with respect to caprylate concentration, protein concentration, ph and incubation temperature. these data, shown in table , demonstrate that within the ranges investigated, these operating parameters did not influence sindbis inactivation by caprylate. under all conditions, complete inactivation of sindbis was observed within min (kinetics not shown). incubation of the gamunex® final product (ph · ) at °c, resulted in the effective inactivation of wnv (fig. ) . within fig. kinetics of west nile virus (wnv) inactivation during the treatment of intramuscular immunoglobulin (imig) with · % tri-(n-butyl)-phosphate (tnbp)/ · % cholate or · % tnbp/ · % cholate. each point represents the mean of three determinations. total inactivations of ≥ · log and ≥ · log wnv were achieved in · % tnbp/ · % cholate and · % tnbp/ · % cholate, respectively. table . days of incubation at ph · , wnv was near the lower limit of detection and, by the next time-point ( days), complete inactivation was achieved. a total of ≥ · log inactivation was observed. effective inactivation of wnv, bvdv and vv by pasteurization was demonstrated in a variety of protein solutions, even in the presence of a stabilizer. although albumin, hpps and α -pi utilize different excipients for stabilization (e.g. sucrose, citrate, tryptophan) and were at different protein concentrations, complete inactivation of the viruses was achieved, demonstrating that this technology provides robust inactivation of enveloped viruses. as the complete inactivation of wnv, bvdv and vv was observed in both % and % albumin, similar inactivation of these viruses would be expected for all intermediate concentrations. both wnv and vv have previously been shown to be susceptible to inactivation by heat. complete inactivation of vv was observed during the pasteurization of solutions of immunoglobulin at °c [ , ] , and a recent report has demonstrated the inactivation of wnv during the pasteurization of albumin [ ] . a number of studies, including this one, have demonstrated the inactivation, by pasteurization, of viruses that are closely related to wnv, such as bvdv, tick borne encephalitis virus, yellow fever virus and sindbis virus [ , , ] . the data reported here, which demonstrated a fig. (a) . d from fig. similar inactivation of wnv and bvdv by pasteurization of α -pi and albumin, support the predictive value of inactivation information from model viruses. vv is antigenically similar enough to both variola (smallpox) and monkeypox viruses to be used as a vaccine [ ] . the susceptibilities to pasteurization of monkeypox virus and variola virus, then, are probably similar to that of vv. solvent/detergent treatment is widely used to inactivate lipid-enveloped viruses. treatment of the ahf intermediate with % of the tnbp/tween concentration used in the manufacturing process provided rapid and complete inactivation of wnv. again, these results were very similar to those of the related virus, bvdv, supporting the validity of this virus as a model for wnv. vv, however, was more resistant to inactivation by tnbp/ tween . the production-operating concentration of tnbp/ tween did not achieve complete inactivation of vv, although the virus titre was reduced by over log . the resistance of vv to solvent/detergent has previously been observed [ , ] . vv is unlike other enveloped viruses in that it can be present in two infectious forms, both of which are membranous and one of which is a double membrane with a complex structure [ , ] . in contrast to tnbp/tween , in this study, tnbp/cholate provided effective inactivation of both wnv and vv. treatment of human placental tissue with · % tnbp/ · % cholate at °c resulted in substantial, but incomplete, vv inactivation [ ] . in the current study, tnbp/cholate in a solution of ivig, rather than tissue, was incubated at °c, and complete inactivation of the virus was achieved. cholate may be able to solubilize vv membranes more efficiently than other detergents. thus, these tnbp/cholate data provide evidence of the ability of this method to effectively inactivate enveloped viruses with a wide variety of characteristics. it also suggests that an emergent enveloped virus with characteristics similar to the viruses for which tnbp/cholate data exist will also be susceptible to inactivation by this method. wnv was effectively inactivated during the low-ph incubation step of the gamunex® process, even when the incubation was performed at ph · , above the upper ph limit of the manufacturing range (ph · - · ). the susceptibility of this virus to low-ph-mediated inactivation has previously been demonstrated, and the current data confirm that incubation of this product under conditions of low ph similarly achieve inactivation of the virus [ , ] . it is likely that any manufacturing step that was performed at a low ph would effectively inactivate wnv. vaccinia has also been shown to be susceptible to inactivation at low ph [ ] . caprylate treatment provided rapid and complete inactivation of the wide panel of enveloped viruses that were evaluated. to date, all enveloped viruses evaluated, representing a wide variety of physicochemical characteristics, have been inactivated to the limit of detection within minutes of incubation in caprylate. caprylate inactivation of vv was similar to that of other enveloped viruses, demonstrating that it can be an alternative to solvent/detergent treatment for the inactivation of resistant enveloped viruses. these data also suggest that any emergent enveloped virus with a complex lipid membrane may be similarly susceptible to this means of inactivation. the data presented here support the robust inactivation of enveloped viruses by caprylate. a number of enveloped viruses, representing a variety of virus families, were shown to be completely inactivated within minutes of incubation with caprylate. in addition, data obtained using sindbis virus demonstrated that during the gamunex® manufacturing process, variations in caprylate concentration, protein concentration, ph or incubation temperature would not alter the overall inactivation or the inactivation kinetics of this hcv surrogate. these data also demonstrated the validity of the use of model viruses for inactivation studies. inactivation of wnv by pasteurization, solvent/detergent treatment and caprylate treatment was very similar to that observed with bvdv in this study. the use of surrogate viruses for those viruses that cannot readily cultured in vitro is a widely used strategy that has been advocated by regulatory agencies [ ] . bvdv has been used as a model for hcv for many years, and its ability to predict the inactivation of hcv has been verified by the lack of transmission of this pathogen by blood products subjected to inactivation measures that achieve significant levels of bvdv inactivation. the wnv data presented here support the use of model viruses; wnv inactivation mirrored that of bvdv. the appearance of a novel viral pathogen should not place the safety of plasma-derived products in jeopardy as long as data from viruses with similar physicochemical characteristics support effective inactivation during manufacture. data from studies with a wide panel of model viruses, representing a range of physicochemical properties, should provide a basis for determining whether an emergent pathogen is likely to present a threat to biological products. although data from model viruses was predictive of the safety of plasma-derived products with respect to transmission of wnv, vv or other potential viral contaminants with similar properties, the results of the current study provide definitive confirmation. this is consistent with the conclusion of others [ ] . furthermore, these data justify the model virus approach for assessing the viral safety of these products. cpmp: cpmp note for guidance on virus validation studies: the design, contribution and interpretation of studies validating the inactivation and removal of viruses transfusion transmission of west nile virus: a merging of historical and contemporary perspectives revised recommendations for the assessment of donor suitability and blood and blood product safety in cases of known or suspected west nile virus infection workshop on development of donor screening assays for west nile virus american association of blood banks: recommended guidance for reporting west nile viremic blood donors to state and/or local public health departments and reporting donors who subsequently develop west nile virus illness to blood collection agencies updated interim cdc guidance for use of smallpox vaccine, cidofovir, and vaccinia immune globulin (vig) for prevention and treatment in the setting of an outbreak of monkeypox infections recommendations for deferral of donors and quarantine and retrieval of blood and blood products in recent recipients of smallpox vaccine ( vaccinia virus) and certain contacts of smallpox vaccine recipients assembly of vaccinia virus: role of the intermediate compartment between the endoplasmic reticulum and the golgi stacks evaluation of virus and prion reduction in a new intravenous immunoglobulin manufacturing process properties of a new intravenous immunoglobulin (igiv-c, %) produced by virus inactivation by caprylate and column chromatography biometrie grundzuge biologisch-medizinischer statistik [biometry, the basics of biological and medical statistics joint announcement of the federal health office and the paul ehrlich institute federal office for sera and vaccines: requirements for validation studies to demonstrate the virus safety of drugs derived from human blood or plasma virus reduction in the preparation of intravenous immune globulin: in vitro experiments inactivation and elimination of viruses during the fractionation of an intravenous immunoglobulin preparation: liquid heat treatment and polyethylene glycol fractionation west nile virus and the safety of plasma derivatives: verification of high safety margins, and the validity of predictions based on model virus data virus safety of human immunoglobulins: efficient inactivation of hepatitis c and other human pathogenic viruses by the manufacturing procedure resistance of vaccinia virus to inactivation by solvent/detergent treatment of blood products inactivation and elimination of viruses during preparation of human intravenous immunoglobulin the formation and function of extracellular enveloped vaccinia virus purification of fibroblast growth factor- from human placenta using tri (n-butyl) phosphate and sodium cholate inactivation of west-nile virus during peptic cleavage of horse plasma igg potential contribution of mild pepsin treatments at ph to the viral safety of immunoglobulin products key: cord- -ctx vhi authors: woo, patrick cy; lau, susanna kp; tsoi, hoi-wah; chan, kwok-hung; wong, beatrice hl; che, xiao-yan; tam, victoria kp; tam, sidney cf; cheng, vincent cc; hung, ivan fn; wong, samson sy; zheng, bo-jian; guan, yi; yuen, kwok-yung title: relative rates of non-pneumonic sars coronavirus infection and sars coronavirus pneumonia date: - - journal: lancet doi: . /s - ( ) - sha: doc_id: cord_uid: ctx vhi background: although the genome of severe acute respiratory syndrome coronavirus (sars-cov) has been sequenced and a possible animal reservoir identified, seroprevalence studies and mass screening for detection of subclinical and non-pneumonic infections are still lacking. methods: we cloned and purified the nucleocapsid protein and spike polypeptide of sars-cov and examined their immunogenicity with serum from patients with sars-cov pneumonia. an elisa based on recombinant nucleocapsid protein for igg detection was tested with serum from healthy blood donors who donated years previously and with serum positive for antibodies against sars-cov (by indirect immunofluorescence assay) from patients with sars-cov pneumonia. the seroprevalence of sars-cov was studied with the elisa in healthy blood donors who donated during the sars outbreak in hong kong, non-pneumonic hospital inpatients, and symptom-free health-care workers. all positive samples were confirmed by two separate western-blot assays (with recombinant nucleocapsid protein and recombinant spike polypeptide). findings: western-blot analysis showed that the nucleocapsid protein and spike polypeptide of sars-cov are highly immunogenic. the specificity of the igg antibody test (elisa with positive samples confirmed by the two western-blot assays) was %, and the sensitivity was · %. three of healthy blood donors who donated during the sars outbreak and one of non-pneumonic paediatric inpatients were positive for igg antibodies, confirmed by the two western-blot assays (total, · % of our study population). interpretation: our findings support the existence of subclinical or non-pneumonic sars-cov infections. such infections are more common than sars-cov pneumonia in our locality. background although the genome of severe acute respiratory syndrome coronavirus (sars-cov) has been sequenced and a possible animal reservoir identified, seroprevalence studies and mass screening for detection of subclinical and non-pneumonic infections are still lacking. methods we cloned and purified the nucleocapsid protein and spike polypeptide of sars-cov and examined their immunogenicity with serum from patients with sars-cov pneumonia. an elisa based on recombinant nucleocapsid protein for igg detection was tested with serum from healthy blood donors who donated years previously and with serum positive for antibodies against sars-cov (by indirect immunofluorescence assay) from patients with sars-cov pneumonia. the seroprevalence of sars-cov was studied with the elisa in healthy blood donors who donated during the sars outbreak in hong kong, non-pneumonic hospital inpatients, and symptom-free health-care workers. all positive samples were confirmed by two separate westernblot assays (with recombinant nucleocapsid protein and recombinant spike polypeptide). findings western-blot analysis showed that the nucleocapsid protein and spike polypeptide of sars-cov are highly immunogenic. the specificity of the igg antibody test (elisa with positive samples confirmed by the two western-blot assays) was %, and the sensitivity was · %. three of healthy blood donors who donated during the sars outbreak and one of non-pneumonic paediatric inpatients were positive for igg antibodies, confirmed by the two western-blot assays (total, · % of our study population). severe acute respiratory syndrome (sars) has now affected countries in five continents, with more than cases and more than deaths. a novel virus, the sars coronavirus (sars-cov), is known to be the aetiological agent. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the viral genome has been completely sequenced. , we have also reported the isolation of viruses resembling sars-cov from himalayan palm civets found in a live animal market in the guangdong province of china; this finding implied that animals could be a reservoir of the virus. detection of sars-cov from a non-pneumonic case in singapore (http://www.who.int/csr/don/ _ _ /en/) suggested that non-pneumonic and subclinical sars-cov infections are possible. however, extensive seroprevalence studies and mass screening for detection of subclinical and non-pneumonic infections are still lacking. at present, the most widely used methods for detection of antibodies against sars-cov are indirect immunofluorescence assay and elisa with cell-culture extract. , however, antibody detection by these methods is difficult to standardise and has not been compared with recombinant-antigen-based antibody-detection tests. a recent seroprevalence study, which used the indirect immunofluorescence assay for antibody detection, did not detect sars-cov antibodies in any of health-care workers from a hospital in which a sars outbreak had occurred. an approach of elisa-based antibody-detection tests using recombinant antigens with positive results confirmed by western-blot assays that use two different antigenic proteins offers higher sensitivity, specificity, and reproducibility than indirect immunofluorescence assay and elisa with cell-culture extract and is easier to standardise. [ ] [ ] [ ] [ ] [ ] [ ] in this study, we used a sensitive and specific elisa based on the highly immunogenic nucleocapsid protein of sars-cov and confirmed positive results by western-blot assays with recombinant nucleocapsid protein and recombinant spike polypeptide (another highly immunogenic protein) of sars-cov to examine the seroprevalence of non-pneumonic sars-cov in the general population, non-pneumonic patients in hospital, and health-care workers during the sars epidemic. pellet was resuspended in l dnase-free, rnase-free double-distilled water and was used as the template for rt-pcr. cloning and purification of (his) -tagged recombinant proteins to produce a fusion plasmid of the nucleocapsid protein of the sars-cov for protein purification, primers lpw and lpw (panel) were used to amplify the gene encoding this protein by rt-pcr. the sequence coding for aminoacid residues - of the nucleocapsid protein was amplified and cloned into the bamhi and ecori sites of expression vector pet- b(+) (novagen, madison, wi, usa) in frame and downstream of the series of six histidine residues. the (his) -tagged recombinant nucleocapsid protein was expressed and purified by means of the nickel-loaded hitrap chelating system (amersham pharmacia, piscataway, nj, usa) according to the manufacturer's instructions. roughly mg purified protein was routinely obtained from l escherichia coli carrying the fusion plasmid. for the spike protein of the sars-cov, primers lpw and lpw (panel) were used to amplify the gene encoding aminoacid residues - by rt-pcr. this portion of the spike protein was used because the complete protein could not be expressed in e coli. the pcr product was cloned into the bamhi and kpni sites of vector pqe- (qiagen, hilden, germany). the resulting clone was digested by psti, and the fragment that contained the gene encoding aminoacid residues - of the spike protein was cloned into expression vector pqe- (qiagen, hilden, germany) in frame and downstream of the series of six histidine residues. expression and purification of the (his) -tagged recombinant spike polypeptide were done as described for the nucleocapsid protein. western-blot analysis was done according to our published protocols, with slight modifications. [ ] [ ] [ ] briefly, ng purified (his) -tagged recombinant nucleocapsid protein or (his) -tagged recombinant spike polypeptide was loaded into each well of a sodium dodecyl sulphate % polyacrylamide gel, then electroblotted onto a nitrocellulose membrane (bio-rad, hercules, ca, usa). the blot was cut into strips, and the strips were incubated separately with in dilution of three serum samples, obtained from three patients with sars-cov pneumonia, positive for antibodies against sars-cov detected by our indirect immunofluorescence assay. antigen-antibody interaction was detected with an ecl fluorescence system (amersham life science, buckinghamshire, uk). serum samples from three healthy blood donors were used as controls. assessment of recombinant nucleocapsid protein elisa serum samples from healthy blood donors who donated blood years previously (aged years or older) and patients with pneumonia positive for antibodies against sars-cov detected by our indirect immunofluorescence assay were used for the assessment of the elisa-based igg antibody test. the test was modified from our previous publications. , briefly, each well of a nunc immunoplate (roskilde, denmark) was coated with ng purified (his) -tagged recombinant nucleocapsid protein for h, then blocked in phosphate-buffered saline with % bovine serum albumin. l diluted human serum ( in ) was added to each well of the protein-coated plates, and they were incubated at °c for h. after five washes with washing buffer, l horseradish-peroxidase-conjugated goat antibody to human igg ( in dilution; zymed laboratories inc, south san francisco, ca, usa) was added to each well, and the plates were incubated at °c for h. after a further five washes with washing buffer, l diluted , Ј, , Ј-tetramethylbenzidine (zymed laboratories) was added to each well and incubated at room temperature for min. l · mol/l sulphuric acid was added, and the absorbance at nm of each well was measured. each sample was tested in duplicate, and the mean absorbance for each sample was calculated. the presence of specific antibodies in positive samples was confirmed by retesting of the sample by the elisa based on recombinant nucleocapsid protein and western-blot assays with recombinant nucleocapsid protein and recombinant spike polypeptide separately. using the protocol described above, we tested for the presence of igg antibodies against the nucleocapsid protein in serum samples from healthy blood donors (aged years or older) who donated blood in march-may, ; non-pneumonic paediatric inpatients (aged less than years) and nonpneumonic adult inpatients (aged years or older) admitted to queen mary hospital in may, ; and symptom-free health-care workers. the presence of specific antibodies in all positive samples was confirmed by two separate western-blot assays, one with recombinant nucleocapsid protein and the other with recombinant spike polypeptide as the antigen. the study sponsors had no role in the study design; collection, analysis, or interpretation of data; or the writing of the report. the purified (his) -tagged recombinant nucleocapsid protein and spike polypeptide were separated on denaturing polyacrylamide gels followed by western-blot analysis with serum from three patients with pneumonia positive for antibodies against the sars-cov. prominent immunoreactive protein bands of about kda were visible on the western blots that used either antigen (figure ). these sizes were consistent with the expected size of · kda for the full-length (his) -tagged nucleocapsid protein and · kda for the (his) -tagged spike polypeptide. an elisa-based sars-cov antibody test was developed for the detection of specific antibodies against the (his) -tagged recombinant nucleocapsid protein. box titration was carried out with different dilutions of (his) tagged recombinant nucleocapsid protein coating antigen and pooled serum from three patients with pneumonia positive for antibody against the sars-cov. the results identified ng purified (his) -tagged recombinant nucleocapsid protein per elisa well as the ideal amount for plate coating for igg detection (data not shown). to establish the baseline for the tests, serum samples from healthy blood donors who donated blood years previously were tested in the elisa. for these samples, the mean optical density at nm for igg detection was · (sd · ). an absorbance value of · was selected as the cut-off value (the mean value for the healthy controls plus sd; figure ) . seven of the samples from healthy blood donors had values of more than · in the igg elisa (figure ), but none of them had the specific antibody when tested by the nucleocapsid protein or the spike polypeptide westernblot assay. the specificity of the igg antibody test (elisa confirmed by western-blot assays) was %. the mean value for the samples obtained from the patients with sars-cov pneumonia, positive for igg antibodies against the sars-cov by our indirect immunofluorescence assay, was · (sd · ). samples had optical density of more than · in the igg elisa (figure ). all were confirmed to have the specific antibody by both the nucleocapsid protein and the spike polypeptide western-blot assays. the sensitivity of the igg antibody test, with the immunofluorescence assay as the gold standard, was therefore · %. ( · %) of the healthy blood donors who donated blood in march-may, , eight ( · %) of the non-pneumonic paediatric patients, eight ( · %) of the non-pneumonic adult patients, and one ( %) of the symptom-free health-care workers who had cared for the patients with sars-cov pneumonia were positive for igg antibodies by elisa ( figure ) . however, only three ( · %) healthy blood donors who donated blood in march-may, , and one ( · %) non-pneumonic paediatric patient were confirmed to have specific sars-cov antibodies by both the nucleocapsid protein and spike polypeptide western-blot assays. up to the end of may, patients from a population of about seven million in hong kong ( · %) had developed sars-cov pneumonia, compared with a rate of non-pneumonic sars-cov infections in our study population of about · % (p< · by poisson exact test of equality). previous studies in animal coronaviruses, such as infectious bronchitis virus, have shown that the nucleocapsid protein and spike protein are highly immunogenic, are abundantly expressed during infection, and can be used for serodiagnosis of animal coronavirus infections. [ ] [ ] [ ] in this study, detection of igg antibodies to both proteins was highly sensitive and specific for sars-cov infections. six ( · %) serum samples that were seropositive by the immunofluorescence assay were negative by the elisa. the reason for this discrepancy may be that the nucleocapsid protein did not elicit antibody response in this minority group of patients. conversely, of five patients with sars-cov pneumonia who were seronegative by the immunofluorescence assay but rt-pcr positive for sars-cov, two were seropositive by our elisa, with clearly positive opticaldensity values of · and · and confirmation by western-blot assay (unpublished). furthermore, in four of patients with sars coronavirus pneumonia who had serial serum samples, igg was detected earlier by the elisa than by the immunofluorescence test (unpublished). in another study that used elisa based on cell-culture extract, five of patients with sars-cov pneumonia were positive according to that elisa but negative by indirect immunofluorescence during the time when the elisa titres were low. this finding accords with the results of a previous study on human coronavirus e, which showed that three of serum samples were positive by recombinant-nucleocapsid-protein-based western blot but negative by immunofluorescence, and six of serum samples were positive by immunofluorescence but negative by recombinant-nucleocapsid-protein-based western blot. all these findings show that our elisa may be able to detect additional cases that the immunofluorescence assay has missed. with this potentially more sensitive elisa for igg antibody detection, we assessed the seroprevalence of non-pneumonic sars-cov infections in both the general population and our hospital population; all positive serum samples detected by elisa were confirmed by two separate western-blot assays, with two immunologically unrelated antigens. the spike polypeptide was used in addition to the nucleocapsid protein for western-blot confirmation to eliminate the possibility of cross-reactivity between antibodies against the nucleocapsid proteins of other human coronaviruses and that of sars-cov. however, the aminoacid identities between the nucleocapsid protein of sars-cov and those of the human coronaviruses oc and e are only · % and · %, respectively, and there were no crossreactions between pairs of oc and pairs of e human coronavirus serum samples and the sars-cov. in fact, of the individuals who were igg positive by elisa, ( %) were positive by the nucleocapsidprotein-based western-blot assay, but only four were positive by both the nucleocapsid protein and the spike polypeptide western-blot assays. this apparent high rate of false-positive non-pneumonic cases, as detected by the recombinant nucleocapsid protein elisa, is due to the use of a single antigen for screening asymptomatic or nonpneumonic infections. it is well known that the positive predictive values of serological tests are much affected when the prevalence of the infection is low, especially in clinically incompatible cases. this is the reason why an immunologically unrelated antigen, the spike protein, has to be used for confirmation of the cases detected as "positive" by the nucleocapsid-protein-based assays. cross-reactivity with human coronavirus oc or other sars-cov-like viruses remains an important issue for future studies on sars-cov serology. three of the four individuals with non-pneumonic sars-cov infections were healthy blood donors, and one was a paediatric inpatient. this patient was a -month-old girl who was admitted to hospital in may, , because of fever for days. she was also noted to have had a cough for the previous weeks and repeated vomiting before admission. there had been no breathing difficulty or diarrhoea. the child had no history of direct contact with patients with sars-cov pneumonia. however, a colleague in her mother's workplace had recently been diagnosed as having sars-cov pneumonia. physical examination of the girl revealed only shotty (palpable but too small to be measured) cervical lymph nodes and congested throat. her chest radiograph was normal. apart from mild lymphopenia (lymphocyte count · ϫ /l), her blood results were normal. nasopharyngeal aspirate was negative for influenza viruses, parainfluenza viruses, adenovirus, and respiratory syncytial virus antigens. she was given antipyretic treatment and was discharged the next day. subsequent antibody testing showed that her serum was positive for both igg and igm antibodies against the nucleocapsid protein as well as igg antibodies against the spike polypeptide of sars-cov. the difference between the rate of non-pneumonic sars-cov infections in our study population and the rate of sars in hong kong suggests that non-pneumonic infections are more common than sars-cov pneumonia and may explain cases of sars-cov pneumonia in patients who had no obvious contact with other patients with sars. contributors p c y woo and k y yuen are coprincipal investigators, jointly wrote the report, and coordinated and supervised the study. s k p lau supervised the analysis of all serological data and corrected the report. h w tsoi cloned and purified the nucleocapsid protein and did the serological assays. k h chan supervised the immunofluorescence assay. b h l wong and v k p tam did the serological assays and analysed the data. x y che cloned and purified the spike polypeptide. s c f tam coordinated the collection of clinical specimens. v c c cheng and i f n hung managed the patients' database. s s y wong, b j zheng, and y guan corrected the report. none declared. coronavirus as a possible cause of severe acute respiratory syndrome development of a standard treatment protocol for severe acute respiratory syndrome prospective study of the clinical progression and viral load of sars-associated coronavirus pneumonia in a community outbreak lung pathology of fatal severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome a cluster of cases of severe acute respiratory syndrome in hong kong a major outbreak of severe acute respiratory syndrome in hong kong identification of severe acute respiratory syndrome in canada the genome sequence of the sars-associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome isolation and characterization of viruses related to the sars coronavirus from animals in southern china severe acute respiratory syndromeassociated coronavirus infection mp encodes an abundant and highly antigenic cell wall mannoprotein in the pathogenic fungus penicillium marneffei detection of specific antibodies to an antigenic mannoprotein for diagnosis of penicillium marneffei penicilliosis characterization of afmp : a novel target for serodiagnosis of aspergillosis detection of specific antibodies to an antigenic cell wall galactomannoprotein for serodiagnosis of aspergillus fumigatus aspergillosis enzyme-linked immunosorbent assay for detecting antibodies to borne disease virusspecific proteins an indirect elisa for the detection of antibodies against porcine reproductive and respiratory syndrome virus using recombinant nucleocapsid protein as antigen cloning and characterization of male in burkholderia pseudomallei groel encodes a highly antigenic protein in burkholderia pseudomallei aflmp encodes an antigenic cell wall protein in aspergillus flavus localization of linear b-cell epitopes on infectious bronchitis virus nucleocapsid protein recombinant nucleocapsid protein is potentially an inexpensive, effective serodiagnostic reagent for infectious bronchitis virus an elisa for antibodies against infectious bronchitis virus using an s spike polypeptide detection of human coronavirus e-specific antibodies using recombinant fusion proteins cost-effectiveness of rapid diagnosis of viral respiratory tract infections in pediatric patients this work is partly supported by a research grant council grant (hku / m), the sars research fund, and the university sars donation fund, the university of hong kong. we thank members of the clinical microbiology laboratory, queen mary hospital, and members of the department of medicine, united christian hospital, hong kong for their assistance. key: cord- -iw oobbe authors: wuxing, dai; mingjun, lei; shaoting, wu; zhihao, chen; liang, liang; hujrong, pan; li, qin; shitong, gao; shishan, yuan; renli, zhang title: expression and purification of sars coronavirus membrane protein date: journal: j huazhong univ sci technolog med sci doi: . /bf sha: doc_id: cord_uid: iw oobbe to construct a recombinant plasmid pet a-m, the gene encoding severe acute respiratory syndrome (sars) coronavirus membrane protein was amplified by rt-pcr and cloned into the expression plasmid pet a. results of restriction endonuclease analysis, pcr detection and dna sequencing analysis revealed that the cloned dna sequence was the same as that reported. the recombinants were transformed intoescherichia coli (e. coli) bl (de ) and induced by isopropyl-β-d-thiogalactopyranoside (iptg). the expression of kd ( kd= . ku) protein was detected by sds-page and pured by metal chelated chromatography. results of western-blot showed that this expressed protein could react with antibodies in sera of sars patients during convalescence. this provided the basis for the further study on sars virus vaccine and diagnostic agents. a novel coronavirus (sars-cov) was discovered in association with cases of severe acute respiratory syndrome (sars). the sequence of the complete gnome of sars-cov was determined : ' . it is not closely related to any of the previously characterized coronavirus. the analysis of open reading frames of sars-cov showed that there were similar major structural proteins that may have played important roles in causing the infection. they are spike protein (s protein), nucleocapsid protein (n protein), membrane protein (m protein), and small envelope protein (e protein). to screen and prepare effective sars virus vaccine and diagnostic antigens, we designed a pair of primers to amplify the gene encoding sars coronavirus membrane protein and cloned it into an expression plasmid pet a. sars coronavirus was kindly provided by vice-director he (division of microbiological testing, center for disease control and prevention, shenzhen, china). escherichia coli (e. coli) dh a, bl ( d e ) were kept in our laboratory. vector pmd -t was obtained from the takara biotechnology (dalian) c o . , ltd. expression plasmid pet -a was from emd. bioscience, inc (usa). taq dna polymerase, d n t p , protein marker were purchased from promega company (usa). virus rna purification kit was purchased from qiagen company (germany). rt-pcr kit, t dna ligase, restriction endonuclease s a l i , b a m h i , dna marker were from takara biotechnology co ( d a l i a n ) , dai wuxing, male, born in , associate professor ltd. plasmid dna isolation kit was obtained from shanghai dna biotechnologies co, ltd. isopropyl-~ -d-thiogalactopyranoside ( i p t g ) , sds-na, edta-na , tris base, ampicillin were purchased from shanghai sangon biological engineering &-technology service co, ltd. sheep anti-human igg labeled by horseradish peroxidase was purchased from pierce company ( u s a ) . sera of sars patients during convalescence were offered by shenzhen east lake hospital. preparation and purification of virus rna the preparation and purification of virus rna was performed according to the manual provided by the virus rna purification kit. rt-pcr amplification was performed with the sars-cov genomic rna used as the template by the manual of rt-pcr kit. . /,i cd-na after reverse transcription was used as pcr template. a pair of primers used for the amplification of the whole gene sequences encoding membrane protein were upstream primer: "-gga tcc a t g gca gac aac g g t ac- " and downstream primer: "-gtc gac ctg tac t a g caa agc aat at- ". the reaction was performed with a perkin-elmer thermal cycle. pcr was carried out in txl reaction volume containing pmol of each primer, . m m o l / l of d n t p , . mmol/i. mgcla, iu of taq polymerase and / total volume of x buffer. the cdna was initially denatured for min at ~c and followed by amplification cycles of ~ for s, c for s a n d 'c for s and a min terminal extension at 'c. the pcr products were eleetrophoresed on . ~ agorose gel, got extracted and were cloned into vector pmd -t. the sequence of gene enco-ding m protein was obtained from recombinant plasmid pmd t-m which was digested by b a m h i and s a l i and purified as the target gene fragment. the plasmidpet a was digested by the same enzymes and the larger fragmem was purified as the vector. the larger gene fragment and the vector were joined to construct the plasmid pet a-m. then the plasmid pet a-m was transformed into e. coli dhs~ and the screening of positive clones was performed by colony pcr method. the recombinant plasmid dna was idemified by restriction enzyme digestion and transferred into e. coli bl (i)e ). above constructed plasmid was transformed into the e. ('oil strain, b i . ( i ) e ) , and signal colony was grown in i.b medium with ampieillin ( /,g/ml) and glucose ( m g / m l ) . when the culture was saturated, the culture was transferred to ,b medium at a dilulion of : . the protein expression was induced with . m m o l / i , of ipt(; after a,>, of the cultured medium was around .( . after ipt(; induction at 'c for t h, the cells were harvested by centrifugation. the cell pellet was suspended by x b u f f e r and incubated in a ( water hath for rain. the suspension was centrifuged at () g for rain and the supernatants were collecled for the sds-page analysis. the purification of the m protein the constructed plasmid was transformed into the e. coil strain, bi. ( ) e ) . and signal colony was grown in lb medium. the culture was incubated overnight at ( . then ml of culture was transferred to m[ of i . b m e d i u m al ('. the protein expression was induced with m m o i / l of i p t g after a,; of the cuhured medium was around . . after iptg induction at ( for , h, cells were harvested by centrifngation. the cell pellets were resuspended hy buffer ( mmol t. tris-h('l ph . , mmol/i~ na('l, ~ glycerin). the cells were sonicated in ice bath. l h e lysed ceils was centrifuged at g al { ( for rain and pellets were discarded. then the m protein was purified according to the manual of pet vector dna from n ( ) v a g e and si)s-pac-e was per formed. protein samples were electrophoresed on sds-page and then transferred to nitrocellu lose membrane, which blocked with phosphate buffered saline ( p b s ) , . % non fat-dried milk and . ~ tween . "['he membrane was incubated with mixed sera of i sars patients during conva lescenceat a dilution of : at ( overnight. after being washed by pbst (pbs and . tween ) for times, the blot was incubated with sheep anti-human immunogtohulin ( ( ; ) la beled by h r p a t a dilution of i ' a~ c for h. the blots were then washed and incubated with diaminobenzidine (dab) until a brown precipitate formed in the protein band. the membrane was transferred to the pbs to stop the reaction. the the sequencing result showed that the sequence of m gene from recombinant plasmid pmd t-m was the same as reported in ncbi genbank. the cell lysate supernatants of recombinant e. ('oli bl (de ) were detected by sds-page and the expression of the ki) protein (containing m protein and his tag at carboxyl terminal tail and t tag at nitryl terminal tail), which was the same as expected, was detected ( fig. ). the fused protein containing m protein in the cell lysate supernatants was purified by ni + chelated chromatography ( fig. ). results of western-blot revealed that the recombinant protein could be recognized specifically by the antibodies of m protein in the sera of sars patients, but could not be recognized by the sera of people without sars. it provided the evidence that the recombinant m protein possessed the biological character of membrane protein of coronavirus ( fig. ). coronavirus is enveloped positive-strand rna viruses that contain structural proteins and its genome is about kb e :. the s, m and n proteins have been broadly studied for their important roles in receptor binding and virion budding e . the m protein is the most conversed protein and is the major determinant of virion morphogenesis. it also interacts with the n protein presumably to assemble the nucleocapsid at the surface of the mature virion et?. the m gene encodes a protein of amino acids and its isoelectric point is . . the sequence analysis of amino acids shows that the m protein contains three transmembrane domains but without signal peptide. it has a large carboxyl terminal tail with amino acids, and this tail presumably interacts with the virus nucleocapsid. in this research, we designed a pair of primer according to the whole sequence of m gene in ncbi gen-bank. then the whole sequence of the gene was obtained by r t -p c r and was cloned into expression plasmid pet a. restriction endonuclease analysis, pcr detection and dna sequencing analysis revealed that the gene cloned was identical to the gene sequences from the reported strain. after it was transformed into e. coli bi. ( d e ) and induced by i p t g , western-blot showed that the expressed kd protein which was characterized by sds-page and purified by metal chelated chromatography could react with antibodies in sera of sars patients during convalescence, demonstrating the recombinant protein possessed the biological activity of membrane protein. it provided the evidence that m protein could be employed as antigen to prepare sars virus vaccine and diagnostic agents. but the immunogenicity, the effect of antiinfection and the prospect in clinical application of the recombinant m protein need further research. characterization of a novel coronavirus associated with severe acute respiratory syndrome science the complete sequence of the sars-associated coronavirus the coronaviridae: an introduction the coronavirus spike protein is a class i virus fusion protein: structural characterization of the fusion core complex genetic evidence for a structural interaction between the carboxy termini of the membrane and nucleocapsid protein of mouse hepatitis virus protein interactions during coronavirus assembly characterization of coronavirus m protein and nuclecapsid interaction in infected ceils key: cord- -kira mz authors: strike, philip c.; wardle, jane; steptoe, andrew title: mild acute inflammatory stimulation induces transient negative mood date: - - journal: j psychosom res doi: . /s - ( ) - sha: doc_id: cord_uid: kira mz objective: this study aims to assess the mood changes induced by mild acute inflammatory stimulation (typhoid vaccination). methods: using a double blind study design, healthy volunteers underwent baseline assessments of mood, financial strain and work stress and were randomised to injection of salmonella typhi vaccine or placebo injection. mood, symptoms and body temperature was assessed by a modified version of the profile of mood states at , , , , and h post injection. results: typhoid vaccination induces no increases in physical symptoms or temperature. mood improved over the day in the placebo but not in the vaccine condition. negative changes in mood following injection were correlated with chronic stress (financial strain) in the vaccination condition (r=−. , p<. ). conclusion: a mild acute inflammatory stimulus induces transient negative mood, and responses were modulated by chronic stress. implications for depressed mood in physical illness are discussed. people with infections or inflammation can exhibit profound changes in behaviour. initially, this was thought to be a psychological response to feeling unwell, but more recently the cytokines released as part of the inflammatory response have been implicated. ''cytokine associated sickness behaviour'' is the term used for this collection of symptoms such as malaise, lethargy, anorexia, hyposomnia, inactivity, impaired cognitive function and anhedonia [ , ] . this theory is supported by the fact that the syndrome can be reproduced by infusion of either recombinant cytokines, or molecules that induce cytokine synthesis (e.g., lipopolysaccharide) [ - ] . agents which antagonise cytokines abolish the syndrome [ ] . in animal models, the cytokine interleukin- (il- ) plays a central role, but il- and tumour necrosis factor alpha (tnfa) are also involved. inflammatory responses may also be relevant to depression. it has been known for several years that major depression is frequently associated with activation of the inflammatory response system, involving increased produc-tion of il- , il- receptor antagonist (il- ra), il- and tnfa [ ] , although the evidence relating subclinical depressive symptoms with inflammatory responses is equivocal [ , ] . proinflammatory cytokines affect the brain's metabolism of hxdroxy tryptophan ( -ht) by either directly or indirectly stimulating the enzyme indoleamine , -dioxygenase leading to a peripheral depletion of -ht providing a possible intermediate in the link between inflammation and depressive symptoms [ ] . these inflammatory responses may not be epiphenomena, but actively contribute to depressive symptoms and negative mood states. this has been documented most consistently in studies of mood responses following cytokine-based treatments for cancer. immunotherapy with il- or interferon a causes sadness, loss of interest and other depressive symptoms, and responses correlate with the magnitude of responses of endogenous cytokines [ , ] . depressive symptoms have also been reported following interferon a treatment of hepatitis c [ ] . these effects are rapid, and may be mediated by the effects of cytokines on the hypothalamic -pituitary-adrenocortical (hpa) axis [ ] . there is also evidence that antidepressants have antiinflammatory effects [ ] . taken together, these findings raise the intriguing possibility that depressive symptoms in as disparate illnesses as coronary heart disease, multiple sclerosis and rheumatoid arthritis may be due in part to cytokine activation. the effects of cytokines on mood can be investigated experimentally by administering stimuli that acutely induce inflammatory cytokine release. reichenberg et al. [ ] assessed the effects on mood and cognitive function of salmonella abortus equi endotoxin injection in volunteers. rapid increases of -to -fold in il- and tnfa occurred within - h of administration in endotoxin but not placebo-injected groups. increases in anxiety and depressed mood were reported over the same time-scale in the endotoxin group, and the changes were correlated with the magnitude of cytokine responses. however, a problem with this model is that endotoxin administration leads to fever and malaise. a larger increase in body temperature was recorded by reichenberg et al. [ ] in the endotoxin than placebo groups and although no symptoms of sickness were reported, appetite was substantially inhibited. many participants report flu-like symptoms in response to endotoxin. this makes it difficult to know whether behavioural changes were due to the inflammatory challenge or mild illness induced by the endotoxin itself. in the present study, we used a milder model of experimental inflammation, involving standard typhoid (salmonella typhi) vaccination. this method has been used by vallance et al. to induce inflammatory responses that have been studied in relation to atherogenesis [ ] . increases of four-to sixfold in il- occur, together with il- ra increases of averaging -fold after h [ ] . importantly, body temperature is not affected, and participants do not report any sickness or malaise. the use of vaccine rather than endotoxin may afford a better insight into the changes in mood in response to immune challenge in human healthy volunteers. the first aim of this study was therefore to examine the effects of typhoid vaccination on the subsequent mood of healthy volunteers in a placebo-controlled double blind trial. based on the evidence linking proinflammatory cytokine release with negative moods, we hypothesised that typhoid vaccination would induce a negative mood response. since moods are not constant over the day, changes in mood following vaccination cannot be definitively ascribed to the effects of an intervention in the absence of a comparison group. half the participants were therefore injected with placebo, and the mood changes in this group taken as the reference against which the active vaccination group could be compared. in the light of the kinetics of proinflammatory cytokine responses observed in earlier studies, we hypothesised that compared with placebo, vaccination would induce transient negative moods in the - h following treatment, in the absence of rises in systemic body temperature or symptoms of illness. psychological stress plays an important role in the way the immune system functions [ ] . plasma concentrations of inflammatory cytokines vary with acute stress and with chronic stressors such as care-giving and work strain, and increase with acute stress [ - ] . in a study of experimentally induced upper respiratory infection, background stress was found to correlate positively with il- responses to infection [ ] . it is conceivable that chronic stress might influence cytokine responses to vaccination, and thereby moderate mood responses. the supplementary aim of this study was therefore to assess associations between chronic stress and mood responses to typhoid vaccination. there are many aspects of chronic stress that could potentially be investigated, but in the interests of brevity and appropriateness for the population we limited measurement to two commonly assessed variables-financial strain and job demands. we hypothesised that background chronic stress would modulate cytokine responses, and thereby associations with mood responses in the vaccination but not placebo conditions. twenty-six volunteers ( female, male) between and years old were recruited from university staff. volunteers were all nonsmokers, had no recent illnesses, were on no regular medication, and were not pregnant. none had any history of any mental illness or had received typhoid vaccination within the last months. no volunteers had any allergies or had had any previous problems with vaccinations. all felt well on the morning of the study and had had no stressful events that morning. volunteers refrained from excessive exercise and from any caffeine or alcohol from the day before the study. the study was presented to participants as an examination of immune responses to vaccination (as assessed by saliva samples). ethical approval was given by the university college london medical research ethics committee. two measures of chronic stress were included in this study. the first was the financial strain questionnaire originally developed by pearlin et al. [ ] and utilized in the whitehall ii and other studies [ , ] . eight items (e.g., ''do you have enough money for the kind of clothing you and your family should have?'') were presented, with response options ranging from = no difficulty to = very great difficulty. ratings were summed, so scores could range from to , with higher ratings indicating greater financial strain. the second measure was an index of work demands derived from the demand/control model of work stress, as used in the whitehall ii study [ , ] . four items (e.g., ''do you have to work very intensively?'') were administered, each of which was rated on a four-point scale ranging from = often to = never/almost never. scores were converted to a scale from - , where indicates minimum and maximum demands. mood and symptoms were assessed with a modified version of the profile of mood states (poms) [ ] as described elsewhere [ ] . this consisted of items, each of which was rated on a five-point scale from = not at all to = extremely. six high loading items were taken from the vigour, tension -anxiety, depression -dejection, and confusion scales of the poms, and five items from the fatigue scale. in addition, there were four symptoms (feverish, aching joints, nauseated, and headache), and three filler items. participants were asked to rate how they felt at that moment. body temperature was measured with a sublingual digital thermometer, and blood pressure and heart rate using an electronic sphygmomanometer. all studies began at the same time ( a.m.) to ensure uniformity. the study was performed in a double blind, placebo controlled manner. after initial measurements of temperature, blood pressure, heart rate and mood, randomisation to either vaccine or placebo ( . ml of normal saline) was performed by a coordinator and . mg salmonella typhi vaccine (typhim vi, aventis pasteur msd) or placebo in identical -ml syringes was administered intramuscularly by a qualified nurse or doctor into the nondominant deltoid muscle of the volunteers. both vaccine and placebo were stored in the fridge before administration. there were no complications of vaccination or placebo injection. thereafter administration of questionnaires and clinical observations was performed by researchers blinded to the experimental group of participants. volunteers were assessed at , , , , and h after vaccination. at each of these times, they filled in the modified poms scale and had temperature, blood pressure and pulse measurements. scores for the five poms scales were computed for the baseline sample by summing ratings on individual items. the scores on the fatigue dimension were scaled up to conform with those of the other moods. ratings of the four symptoms were summed to produce a total score. vaccine and placebo groups were compared on baseline mood, symptoms, chronic stress and physiological measures using t tests. changes in symptoms, body temperature, blood pressure and heart rate over the study were analysed with repeated measures analysis of variance with group (vaccine, placebo) as the between-subject factor, and time (base, , , , , and h) as the within-subject factor. to avoid the multiple comparisons when assessing mood responses, we calculated total mood scores as recommended in the poms manual, by summing the negative items (tension, depression, confusion, and fatigue), and subtracting them from the positive vigour score. changes in mood from baseline were then computed and analysed by repeated measures analysis of variance. finally, correlations between chronic stress measures and changes in mood were computed separately for the vaccine and placebo groups. all analyses were carried out using spss v. . results are presented in terms of means fstandard deviation. the background characteristics and baseline measures are summarized in table . there were no significant differences between vaccine and placebo groups in age, financial strain, work demands, or in physiological measures. the mood scores showed a high positive vigour score coupled with low ratings on negative scales, indicating that participants were generally in positive moods. mood scores did not differ significantly between groups, with the exception of fatigue (t = . , p <. ), where ratings were higher in the placebo than vaccine group. however, the scores in both groups were extremely low (possible range - ), so the difference was trivial. none of the participants reported appreciable symptoms preinjection. the mean symptom ratings and body temperature assessed at the seven time points are shown in fig. . there was no significant change over time or difference between groups in either variable. the absence of any rise in body temperature with vaccination indicates that the procedure did not induce even mild illness. the mean symptom scores never exceeded one at any time. since the symptom scores theoretically range from a low of zero to a maximum of , it is evident that neither treatment elicited physical symptoms throughout the study. there were some variations in cardiovascular measures. heart rate showed a significant cubic trend over the study period [ f ( , ) = . , p < . ]. the mean heart rate at baseline ( . f . bpm) decreased to a low of . f . bpm after h. this was followed by a modest rise to . f . bpm after h, with a final fall to similarly, there were no differences between groups in systolic or diastolic pressure over the day. the analysis of changes in total mood score between baseline and later samples show a significant quadratic product moment correlations between mood change and the two background stress variables (financial strain and job demands) were computed separately for the vaccination and placebo groups. no significant effects were found in placebo condition. in the vaccine group, there was a significant negative correlation between change in mood over the first hour following injection and financial strain (r = À . , p < . ). this effect is illustrated in fig. . participants who reported high financial strain showed negative changes in mood over the first hour following vaccination, while positive mood changes were apparent in most participants who reported low financial strain. this suggests that background chronic stress levels may have affected the mood responses to typhoid vaccination. the principal hypothesis tested in this study was that the mild inflammatory response induced by typhoid vaccination would induce transient negative mood. it was necessary to test this hypothesis in a double-blind placebo-controlled fashion, since researchers' and participants' expectations might have influenced the pattern of results. in addition, mood can change over the day irrespective of stimuli such as vaccination, so the effects of this stimulus were tested against the fluctuations observed in the placebo group. mood was assessed using a standard measure, and we analysed the total poms rather than individual scales to provide a global impression of mood change. the results confirmed our expectations about the effects of the inflammatory stimulus. mood improved over the day in the placebo group, reaching a plateau - h postvaccination ( : a.m. to : p.m.), before deteriorating slightly later in the day. the increase in positive mood from the start of the study may have been due partly to time of day, and partly to the relief at having completed the injection. by contrast, mood did not improve over the day in the vaccination condition. the difference between groups was greatest over the first h following vaccination, with some convergence later in the day. by the end of the study ( : p.m.), mood had deteriorated below baseline in the vaccination group, while remaining elevated in the placebo condition. thus, relative to the placebo condition, the vaccination group had a more negative mood following injection. the mood differences were significant but modest. this compares with result of reichenberg et al. [ ] , in which both depression and anxiety increased in absolute terms in response to endotoxin. the difference may be due to the greater intensity of the inflammatory stimulus in this earlier study. in the present study, there was no change in body temperature, no febrile responses, and symptom reports were low and stable. in addition, the study by reichenberg et al. [ ] was more invasive in nature, involving venous cannulation and continuous temperature measurement through a rectal probe. it is notable that anxiety increased in the placebo as well as endotoxin condition in this earlier study. our results suggest that vaccination may be a preferable way of studying the relationship between immune challenges, mood and behavioural responses. a previous study of rubella vaccination has shown increases in depressed mood in a proportion of the young women tested who were initially seronegative [ ] . cytokines were not measured in this study, so it cannot be definitely concluded that mood changes were due to their activation. we are currently carrying out a study to address this issue. however, the time course of responses is consistent with the cytokine changes that would be expected. in previous studies using typhoid vaccination, the maximum rise in il- and il- ra occurred at -to -h postvaccination [ , ] . cytokine levels then fall gradually, but do not reach baseline values even -h postvaccination. these responses parallel the changes in mood observed in the present investigation. the second important finding was that the magnitude of mood change following vaccination was associated with one of the two measures of chronic stress. participants reporting greater financial strain showed larger negative changes in mood in the hour after vaccination. no such association was observed in the placebo group, so it is not a nonspecific response caused by negative affectivity reporting biases. there was also no association with the second measure of chronic stress, job demands. the explanation may be that job demands were relatively low, while financial strain was high on average in this sample compared with other population samples in which the measures have been used. the association with financial strain needs to be confirmed in a larger study involving cytokine measures, so as to discover whether inflammatory or some other responses to vaccine mediate effects. if the association is confirmed, it could be due to one of two processes; either background stress magnifies inflammatory responses to standard challenges such as vaccination thereby increasing the mood change, or it affects the extent of mood change following a fixed inflammatory response. individual differences in mood response to rubella vaccination have also been reported, with greater depressed mood in young women of lower socioeconomic status [ ] . a number of studies of responses to fixed doses of coronavirus or influenza have shown that the likelihood of infection is affected by psychosocial factors such as life stress, negative affect, and social isolation [ , ] . the observation by cohen et al. [ ] that the il- increases following infection with influenza were correlated with psychological stress is consistent with the possibility that the present finding was mediated through individual differences in il- response. studies relating negative moods with inflammation may be relevant to depression in coronary heart disease. inflammation is now thought to be a fundamental factor in the pathophysiology of vascular disease [ ] . acute coronary syndromes are also associated with marked inflammatory responses, with larger increases in il- h followed by raised il- [ ] . the acute increase in il- ra and il- over the first days following hospitalization with unstable angina is associated with adverse clinical outcomes [ ] . depression in the days following admission with acute coronary syndromes has also been shown to predict poor outcome in many studies [ ] , and several mechanisms to explain the association are being investigated [ ] . an additional possibility is that depressed mood is due to inflammation in these patients, and that heightened levels of inflammation underlie the association between depression and adverse cardiac outcomes. we studied a relatively homogeneous group of university workers, the majority of whom were postgraduate academics. they were relatively young, physically fit and active. the study was small, with insufficient numbers to test sex differences in responses. future studies of larger samples will involve cytokine as well as mood measures, to determine whether the magnitude of inflammatory responses is directly associated with mood changes following vaccination. behavioral effects of cytokines cytokine-induced sickness behaviour: mechanisms and implications cytokine-induced sickness behavior: where do we stand? lowered serum dipeptidyl peptidase iv activity is associated with depressive symptoms and cytokine production in cancer patients receiving interleukin- -based immunotherapy acute effects of recombinant human interleukin- on 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and antidepressive treatment cytokine-associated emotional and cognitive disturbances in humans acute systemic inflammation impairs endothelium-dependent dilatation in humans prevention of inflammation-induced endothelial dysfunction. a novel vasculo-protective action of aspirin the effects of psychological stress on humans: increased production of pro-inflammatory cytokines and a th -like response in stress-induced anxiety life stress, mood disturbance, and elevated interleukin- in healthy older women inflammatory cytokines, socioeconomic status, and acute stress responsivity immune dysfunction associated with chronic professional stress in nurses psychological stress, cytokine production, and severity of upper respiratory illness the stress process hardship and depression health inequalities among british civil servants: the whitehall ii study low job control and risk of coronary heart disease in whitehall ii (prospective cohort) study manual for the profile of mood states the effects of exercise training on mental well-being in the normal population: a controlled trial influence of socioeconomic status on behavioral, emotional and cognitive effects of rubella vaccination: a prospective, double blind study prevention of inflammation-induced endothelial dysfunction: a novel vasculo-protective action of aspirin social ties and susceptibility to the common cold psychological stress and susceptibility to the common cold atherosclerosis-an inflammatory disease inflammation and atherosclerosis. circulation increasing levels of interleukin (il)- ra and il- during the first days of hospitalization in unstable angina are associated with increased risk of in-hospital coronary events depression in patients recovering from a myocardial infarction depression as a risk factor for cardiac mortality and morbidity: a review of potential mechanisms this study was supported by the british heart foundation and the medical research council. we are grateful to bev murray, lindsay emmerson and lena brydon for assistance in vaccination. key: cord- -shrmdkco authors: vaqué, josep title: las enseñanzas del síndrome respiratorio agudo grave date: - - journal: gaceta sanitaria doi: . /s - ( ) - sha: doc_id: cord_uid: shrmdkco nan e ntre noviembre de y julio de , merced a los medios de comunicación, la red de internet y las publicaciones médicas, hemos podido seguir de cerca la eclosión, la amplia diseminación y el posterior declive de una enfermedad transmisible emergente, el síndrome respiratorio agudo grave (srag), causada por un coronavirus desconocido hasta hace poco (sars-cov) que, a través una propagación de persona a persona y un fuerte tropismo pulmonar, mostró una notable capacidad patogénica y letalidad. afectó a . pacientes y produjo muertes en países de los continentes, por lo que acertadamente ha sido denominada la primera pandemia del siglo xxi . si en el anestesista y pionero de la epidemiología john snow, mediante el cierre de la llave del suministro de agua, consiguió cortar la epidemia de cólera de londres, de forma parecida -aunque a mucha distancia en el tiempo-, en la primavera e inicios del verano de se ha logrado controlar el srag con el uso de medidas muy clásicas de la salud pública. en este sentido, en el informe sobre la nueva epidemia elaborado por el gobierno de canadá, se expone: «el srag ha sido contenido, al menos temporalmente, no mediante la revolución genómica ni con avanzados productos farmacéuticos, sino con el uso de anticuadas medidas de salud pública, como el lavado de manos, los procedimientos de control de las infecciones, el aislamiento de los casos y el seguimiento y la cuarentena de los contactos» . también, en relación con las medidas que la oms y los países afectados aplicaron con esperanzas de conseguir el control, se ha comentado que los resultados fueron mucho mejores de lo esperado . en realidad, dado el inicial desconocimiento de su etiología, y en especial de sus características epidemiológicas, no se esperaba una evolución tan favorable, y las previsiones iniciales no eran nada optimistas. por ejemplo, burke, en abril de , en una nota editorial titulada «tenemos meses para actuar», publicada en diversos periódicos norteamericanos, razonaba que la epidemia solamente podría erradicarse mediante unos intensos esfuerzos para detectar y aislar todos los casos infectivos, antes de que llegara la nueva temporada invernal y se restableciera la transmisión; partía de su experiencia acerca de la infección por el virus de la inmunodeficiencia humana (vih) cuando, en , al detectar una leve prevalencia de la infección en jóvenes reclutas, se dio cuenta de que su generalización era inevitable . todavía en mayo de , schabas alertaba a la comunidad médica acerca de que «deberíamos acostumbrarnos a vivir con el srag» . afortunadamente, la infección no progresó hasta llegar a constituir un problema generalizado ni permanente, sobre todo debido a las medidas de control adoptadas, a la limitada eficiencia de la transmisibilidad del virus y al posible efecto del cambio estacional cies y permiten la invasión microbiana . también ha evidenciado la necesidad de aumentar la capacidad y la organización de la salud pública para hacer frente a los nuevos eventos. todos estos aspectos no son precisamente nuevos, pues desde el advenimiento de la infección por el vih-sida, cuando se introdujo el concepto de infección emergente, muchas voces e instituciones han proclamado la relevancia de la investigación científica y de la adecuada preparación de los sistemas de salud para afrontar las muy probables nuevas infecciones. la amplia y rápida diseminación del srag ha puesto de manifiesto que hasta ahora todas estas previsiones no han tenido el desarrollo deseado, aunque en los últimos años se ha dispuesto de importantes mecanismos, como la red de alerta global y respuesta de la oms, que ha sido muy efectiva en el srag. la rápida movilización de la oms y de muchas instituciones internacionales frente al srag comportó un impresionante desarrollo de las investigaciones, que en pocos meses condujeron a destacados avances sobre su etiología, diagnóstico, clínica y epidemiología. el conocimiento de sus vías de transmisión y las características de infectividad de los pacientes fue esencial para determinar las medidas de control más efectivas. por otro lado, la respuesta de los países afectados ante la epidemia fue dispar. en china se registró una extensa propagación comunitaria inicial asociada a la precariedad de su sistema sanitario; cuando el gobierno de este país estableció la atención gratuita de todos los afectados y organizó una gran red epidemiológica con numerosos centros para el aislamiento de los casos, la epidemia entró en una fase de control. en singapur y canadá la notable diseminación nosocomial evidenció importantes fallos en la acción diagnóstica, la labor epidemiológica y la aplicación de las medidas de control . las deficiencias de los sistemas de salud frente al srag son objeto en la actualidad de numerosos estudios y publicaciones. tienen especial interés los informes de los gobiernos de canadá y de hong kong , así como el del instituto de medicina de estados unidos . en el informe canadiense se examinan en profundidad las numerosas deficiencias identificadas: falta de capacidad de respuesta asistencial y de salud pública, dificultades en la obtención de resultados de laboratorio, ausencia de protocolos para el intercambio de información entre los diversos niveles gubernamentales, inadecuada capacidad para la investigación epidemiológica de la epidemia, inadecuado control de las infecciones en los hospitales, escasa coordinación entre la salud pública, y los hospitales y la atención primaria, inexistencia de canales de comunicación al público, y muchas otras. como resultado del estudio, en el informe se propone desarrollar una amplia reorganización de las actividades de salud pública en los ámbitos federal y local, así como la reforma de múltiples aspectos de la asistencia sanitaria, como el establecimiento de estándares en los servicios de urgencia de los hospitales. en resumen, en el informe se realiza un amplio repaso de las funciones de la moderna salud pública, con propuestas para su adecuación al entorno del país. seguramente, es el proyecto de cambio y modernización de la salud pública más importante y consistente puesto a punto en los últimos años, y será muy interesante seguir su progresivo desarrollo. la conmoción provocada por el srag en canadá y hong kong ha propiciado el desarrollo de importantes propuestas para reformar sus sistemas de salud pública. muchos países, sin esperar a sufrir una experiencia similar, deberían revisar a fondo su situación y tomar ejemplo de tales propuestas. también, además de esta adecuación interna, la epidemia ha evidenciado la urgente necesidad de disponer en el contexto internacional de potentes organizaciones de vigilancia y respuesta frente a las infecciones emergentes. the severe acute respiratory syndrome learning from sars. renewal of public health in canada. a report of the national advisory committee on sars and public health stray thougts on sars six months to act sars: prudence, not panic ¿volverá el síndrome respiratorio agudo grave? crossing the species barrier -one smal step to man, one giant leap to mankind importancia de la transmisión nosocomial en el síndrome respiratorio agudo grave y su prevención report of the sars expert committee learning from sars. preparing for the next disease outbreak key: cord- - stewshp authors: huang, qing; fu, wei-ling; chen, bing; huang, jun-fu; zhang, xue; xue, qiang title: inactivation of dengue virus by methylene blue/narrow bandwidth light system date: - - journal: journal of photochemistry and photobiology b: biology doi: . /j.jphotobiol. . . sha: doc_id: cord_uid: stewshp abstract peracetic acid was one of the most commonly used disinfectants on solid surfaces in hospitals or public places. however, peracetic acid is an environmental toxin. therefore, safer, alternative disinfectants or disinfectant systems should be developed. because photodynamic virus inactivation with methylene blue (mb)/light system has proven effective in blood banking, mb was selected as a photosensitizing agent, dengue virus as a model virus for enveloped rna viruses, and an in-house fabricated narrow bandwidth light system overlapping the absorption spectrum of mb as the light source. dengue virus was mixed with different concentrations of mb, and illuminated by the narrow bandwidth light system under different illumination distances and times. the amount of dengue virus remaining was evaluated by plaque forming assays. results showed that the concentration of mb working solution, illumination intensity of light source, illumination distance and time were four key factors affecting efficiency of virus inactivation using the mb/narrow bandwidth light system. dengue virus could be completely inactivated at . m in min when mb⩾ . μg/ml. however, when the distance reached . m, only greater concentrations of mb ( . μg/ml) could completely inactivate virus in a reasonably short time ( min), and smaller concentrations of mb ( . μg/ml) could only completely inactivate virus using longer times ( min). the results of this virus inactivation model indicate that our mb/narrow bandwidth light system provides a powerful, easy way to inactivate dengue viruses. effective disinfection systems, tools or reagents have been shown to play an important role in the practice of preventing spread of contagious pathogens (e.g., server acute respiratory syndrome-associated coronavirus). but the use of common disinfectants might result in many by-products with potential genotoxic and/or carcinogenic activity. in a recent study, the micronucleus test in root cells revealed genotoxicity in many samples of water disinfected with sodium hypochlorite, chlorine dioxide, or peracetic acid [ ] . in our practice in china, peracetic acid is the most commonly used disinfectant on solid surfaces in hospitals and public places. because peracetic acid is a stronger oxidizing agent than chlorine or chlorine dioxide, it is much more hazardous for exposure to skin, eyes, the digestive system, or the respiratory system. moreover, the substance is very toxic to aquatic organisms after environmental disposal without any previous treatment. therefore, alternative disinfectants or disinfectant systems should be developed that are safer to the environment. photodynamic technologies (or photochemical methods) have been proven to be appealing methods for virus inactivation in blood banking applications. in these - /$ -see front matter Ó elsevier b.v. all rights reserved. doi: . /j.jphotobiol. . . methods, methylene blue (mb) is the most widely used photosensitizing agent for photodynamic inactivation of viruses. mb (mw: . ), a member of the group of phenothiazine dyes, was shown to inactivate viruses in human plasma on exposure to light [ , ] . photodynamic treatment with mb, which has a high affinity for enveloped rna viruses, can effectively inactivate various enveloped rna viruses, including hiv, hepatitis b virus and hepatitis c virus in plasma, and probably also the non-enveloped parvovirus b [ , ] . in the present study, we selected mb as the photosensitizing agent because it is used clinically and because of its known toxicological properties. we fabricated a narrow bandwidth light system consisting of a light-emitting diode (led) matrix overlapping the maximum absorption wavelength of mb working solution used in this study. dengue virus, an enveloped rna virus, served as the model virus in these experiments. upon photodynamic treatment of dengue virus with the mb/ narrow bandwidth light system, we measured the photodynamic parameters of virus inactivation, and then determined the conditions for effective photodynamic inactivation of dengue virus added to pooled vero cells. stock aqueous solution of mb ( mg/ml) (jichuan, inc., jiangshu, china) was diluted to . lg/ml in water. one milliliter of . lg/ml mb aqueous solution was used to measure its absorption spectrum with a uvspectrophotometer. light-emitting diodes (leds; midpeak bandwidth nm, peak nm) with irradiation spectrum overlapping with the maximum absorbing wavelength of mb was used as a light source. the led matrix was an in-house fabricated light box equipped with · (= ) leds. the illumination intensity and light color were determined with an st- c radiant power meter (photo & electronic factory, beijing, china) and human eyes, respectively. dengue virus was selected as the model virus for evaluating efficiency of photodynamic inactivation with the mb/narrow bandwidth light system. appropriate volumes of stock aqueous solution of mb was added to ml of · pfu/ml dengue virus suspensions in modified eagleÕs minimum essential media (emem) (atcc, usa) supplemented with % fetal bovine serum (fbs) to reach final concentrations of . , . , . or . l g/ml of mb aqueous solution. the mixture of mb/virus system was illuminated from above with the narrow band-width light system for , , , or min at a distance of . , . , . , . , . or . m. after photodynamic inactivation, the virus suspensions were stored at À °c until used as described in the following sections. vero cells were cultured in modified emem supplemented with % fbs, glutamine ( . mg/ml), penicillin ( iu/ml) and streptomycin ( . mg/ml) at °c in a humidified atmosphere of % co . on the day before infection, vero cells were incubated in -well microtitre plates. on the day of infection, the supernatants of cultured infected cells were discarded and the cells were washed once with emem without fbs. mixtures of mb/virus illuminated previously with the narrow bandwidth light system were inoculated into the wells of the microtitre plates containing vero monolayer cells prepared previously. every mixture corresponding to a different concentration of mb working solution was inoculated into wells. inoculums were incubated at °c for h with gentle shaking every - min to allow adsorption of virus by vero cells. after -min intervals during the adsorption, . ml of emem supplemented with % methylcellulose were added to the inoculums, which were then incubated at °c, % co for - days. the plaques were counted after staining with crystal violet. the titration of dengue virus was assessed with plaque-forming units, and the inactivation effects on dengue virus of different concentrations of mb solution were evaluated based on the killing log value (klv). klv was defined as lg n À lg n x , where n is the initial virus titration ( · pfu) and n x is the average titration (pfu) for each concentration of mb working solution which has been added to wells [ ] . three controls were set up. a mixture of mb/virus suspension without illumination served as positive control a, illuminated virus suspension without mb served as positive control b, and dilution solution (i.e., modified emem supplemented with % fbs) without any virus or mb suspension served as negative control. the absorption spectrum of mb was peaked at nm. the leds were selected based on peak absorption wavelength of mb and then fabricated into an led matrix. emission from this narrow bandwidth light system significantly overlapped the mb absorption spectrum, reducing light penetration through the mb aqueous solution. on-axis (forward looking) illumination intensity of this narrow bandwidth light system was mlx at a distance of . m. the emitted color was red as observed by eye. efficiency of virus inactivation of different concentrations of mb working solution was more than % (klv > . ) at different illumination distances for illu-mination time > min. the results indicate that most of the virus is inactivated in the first min (fig. ) . results of dynamic parameters of inactivation showed that . lg/ml mb could not completely kill all virus in any illumination conditions ( fig. (a) ), and . lg/ml mb could only completely kill virus at illumination distance equal to or less than . m when illumination time reached min ( fig. (b) ). however, when mb concentration was equal to or greater than . fig. . photodynamic inactivation of model virus with mb/narrow bandwidth light system working concentrations of mb working solution in a, b, c and d were . , . , . and . l g/ml respectively. in c and d, some of the distance with same inactivation efficiency was combined into one serial in order to give more clear results. the original log value of model virus (i.e., dengue virus) titre was . (i.e., lg · pfu). fifty percent ( %) of klv was . . lg/ml, it could completely kill virus at illumination distance equal to or less than . m in min (fig. (c) and (d)). if the distance was as long as . m, only mb at the highest tested concentration ( . lg/ml) could completely kill viruses, and only when illumination time reached min ( fig. (d) ). however, lower concentrations ( . lg/ml) of mb could completely kill virus after an extended illumination time ( min) (fig. (c) ). the results indicate that mb concentration, illumination time and distance are three key factors affecting efficiency of virus inactivation when the illumination intensity of the light source was held constant. however, because illumination intensity at the target is decreased with distance from the source, our results also indicate that the illumination intensity is the fourth key factor affecting killing efficiency of virus inactivation. moreover, the results also indicate that equivalent inactivation effects could be achieved at lower concentrations of mb and at shorter times and longer distances via increasing illumination intensity of the light source. as a consequence, there were four key factors, which were mb concentration, illumination intensity of light source, illumination time and distance, for our photodynamic virus inactivation instrument. the effects of virus inactivation were increased with the increase of mb concentration, the enhancement of illumination intensity of the light source and the extension of illumination time, as well as the decrease of illumination distance. photodynamic antimicrobial agents based on the well-established phenothiazinium biological stain methylene blue offer a simple method for the inactivation of viruses (e.g., hiv, hepatitis c virus and hepatitis b virus) contained in donated blood and blood products or collections. this method has been widely used in the prevention of transfusion-transmitted diseases in the clinic. moreover, because pathogens of bacterial, yeast and protozoal classes are also susceptible to phenothiaziniums, the mb/light system has also been used in photodynamic antimicrobial chemotherapy [ ] . chemical and biological properties of mb have been thoroughly studied [ , ] . the optical absorption of mb is optimum for the blood banking application, and it is currently being used clinically for photoinactivation of fresh frozen plasma in europe [ ] . mb/lighttreatment has proved to be the most suitable method of virus inactivation since it provides an acceptable compromise between viral safety and impaired materials (e.g., plasma) quality. mb can bind to and enter via the virus membrane, whereupon it intercalates with nucleic acids. upon illumination, it then absorbs visible light energy and becomes activated with generation of highly reactive oxygen species (e.g., singlet oxygen). these disrupt the viral membrane and cause destruction of the nucleic acids, particularly at guanosine residues. the resulting nucleic acid modification can prevent viral replication and induce the inactivation of viruses through both type i and ii pathways [ ] . the mb then reverts, in the presence of oxygen, back to its original state. in traditional photodynamic inactivation of virus, a white light source is commonly used. because the absorption spectrum of mb solution is between nm and nm, it is light in this range that shows reaction activity with mb solution. light of other wavelengths should not inactivate virus with mb molecules. therefore, if a white light source is used, strong illumination intensity is required. however, a high concentration of mb (e.g., lm) and a short distance (e.g., cm) is used in blood banking applications [ ] . because the emission of leds is almost monochromatic and can be characterized by a peak wavelength, light energy of an led light source matching the peak wavelength of the mb photosensitizing agent can be transmitted to mb molecules in a high efficiency manner. upon selecting leds matching the peak absorption of mb, lower illumination intensity was needed to inactivate virus at longer distance (e.g., present studies). compared to a final concentration (i.e., lm) which always works at cm in previous photodynamic inactivation of enveloped rna viruses in plasma, a much lower concentration of mb ( . lg/ml) can completely kill virus at . m in min under our custom-designed narrow bandwidth light system. thus a light source with narrow bandwidth matching the peak absorption of mb is an appealing technology for inactivation of enveloped rna viruses. the results indicated that our mb/narrow bandwidth light system provides a powerful and easy way to inactivate enveloped virus in solution at a longer distance and in a shorter time than with traditional mb/light systems used in blood banking applications. in our mb/narrow bandwidth light system, the four factors affecting inactivation efficiency of mb can be grouped into two types. type i is the property of mb itself, i.e., its final working concentration. type ii is the feature of the narrow bandwidth light system, which includes the illumination intensity, time and distance. increase of mb final concentration and/or illumination intensity enables the narrow bandwidth light system to kill or inactivate enveloped virus at much greater distance in much shorter time. the present studies indicate that the narrow bandwidth light system could be used as an appealing disinfectant tool for inactivation of enveloped virus in environments such as resting or working places where the surfaces were not shaded from the light source. in the european pharmacopoeia, mb is listed as a reagent suitable for external use [ ] . high dose of mb (i.e., . mg/kg) has also been studied in the treatment of human septic shock and no adverse effect was observed [ ] . animal studies examining high-dose mb have also failed to demonstrate significant toxicity [ ] . therefore, our mb/sw-light system can be used continuously for a long time without any harm to humans. mb working concentration and illumination intensity, time and distance are the four key factors affecting the inactivation efficiency of the mb/narrow bandwidth light system. compared to current methods, this mb/narrow bandwidth light system might be a safer and more effective disinfectant tool for inactivation of enveloped rna. methylene blue emem eagleÕs minimum essential media fbs fetal bovine serum leds light-emitting diodes klv killing log value genotoxicity of surface water treated with different disinfectants using in situ plant tests photoinactivation of viruses in human fresh plasma by phenothiazine dyes in combination with visible light no evidence for neoantigens in human plasma after photochemical virus inactivation photodynamic treatment of pooled coumarin plasma for external quality assessment of the prothrombin time virus inactivation of blood products by phenothiazine dyes and light china ministry of health, technical standard for disinfections methylene blue derivatives -suitable photoantimicrobials for blood product disinfection? photo-induced inactivation of viruses: adsorption of methylene blue, thionine, and thiopyronine on qbeta bacteriophage photochemical interactions of methylene blue and analogues with dna and other biological substrates virus inactivation of plasma and its derivatives methylene blue administration in septic shock: a clinical trial methylene blue and erythrocytes in the living animal. contribution to the toxicology of methylene blue and formation of heinz bodies this study was supported by a grant from the scientific technology foundation of chongqing, china (charity no. ). the authors are extremely appreciative of prof. larry baum, department of medicine and therapeutics, the chinese university of hong kong, for his revision and review of this paper. key: cord- -nokybn a authors: zeng, fanya; chow, ken yan ching; hon, chung chau; law, ka man; yip, chi wai; chan, kwok hung; peiris, joseph s.malik; leung, frederick chi ching title: characterization of humoral responses in mice immunized with plasmid dnas encoding sars-cov spike gene fragments date: - - journal: biochem biophys res commun doi: . /j.bbrc. . . sha: doc_id: cord_uid: nokybn a the immunological characteristics of sars-cov spike protein were investigated by administering mice with plasmids encoding various s gene fragments. we showed that the secreting forms of s , s subunits and the n-terminus of s subunit (residues – ) were capable of eliciting sars-cov specific antibodies and the region immediate to n-terminus of matured s protein contained an important immunogenic determinant for elicitation of sars-cov specific antibodies. in addition, mice immunized with plasmids encoding s fragment developed a th -mediated antibody isotype switching. another interesting finding was that mouse antibodies elicited separately by plasmids encoding s and s subunits cooperatively neutralized sars-cov but neither the s nor s specific antibodies did, suggesting the possible role of both s and s subunits in host cell docking and entry. these results provide insights into understanding the immunological characteristics of spike protein and the development of subunit vaccines against sars-cov. severe acute respiratory syndrome (sars) is an infectious viral disease caused by a newly emerged coronavirus (sars-cov) from china guangdong with unknown origin [ ] , with typical corona-like spikes on the surface of the virion [ ] , formed by oligomers of the largest viral glycoprotein, namely the spike protein (s protein) [ ] . the spike protein could be structurally cleaved into n-terminal s and c-terminal s subunits after translation or functionally differentiated into the corresponding domains without cleavage [ ] . the s subunit is the knob region of the spike, involved in viral attachment with the cellular receptors and hence determines the host cell tropism [ ] [ ] [ ] . previous studies in vaccine development against animal coronaviruses suggested that the s subunit contains neutralizing epitopes that conferred protection to animals upon viral challenge [ ] [ ] [ ] [ ] [ ] [ ] . the s subunit is the stem region of the spike and its coiled-coil and transmembrane regions are involved in host cell entry and cell-to-cell fusion [ ] . the s protein of sars-cov is a $ -kda glycoprotein [ ] and the s subunit was defined by the highscore alignment with the amino acid sequences of other coronavirus s proteins ( . % aligned to pfam , conserved domain of coronavirus s glycoprotein). the sars-cov s subunit shares several structural motifs with the well-studied gp protein of hiv- , such as the n-terminal leucine/isoleucine heptad repeat (hr), the c-terminal hr, the loop between two hrs, the transmembrane domain, and the aromatic residue-rich motif [ , ] . however, the sars-cov s has a low sequence homology with the existing animal and human coronavirus s subunits, therefore no protein structure model could be utilized in helping the development of vaccine and drugs. recently, the advances in molecular characterization of sars-cov s have been made. the genetic diversity of the s hypervariable region observed in animal coronaviruses was also reported in sars-cov [ ] . a recent report has identified the ace as a cellular receptor for the virus [ ] and the binding domain has been defined between residues and of the s subunit [ ] . clinical observations in sars patients imply both humoral and cell-mediated immune responses may be needed to prevent sars-cov infection. it was reported that an apparent depletion of t cells occurred in the early infection and a gradual increase to normal level was observed as the patients recovered [ ] . sars-cov specific antibodies were detected at days after the onset of symptoms and kept at high titer for at least months in the recovered patients [ ] . in addition, infusion of convalescent sera to patients had been shown to help disease recovery [sung et al., unpublished data]. the seroconverted individuals with no illness in the epidemic region [ ] and the inability of culturing viruses from rt-pcr positive specimens collected from the patients about days after presentation [ ] implied that the neutralizing antibodies could be stimulated rapidly and might restrict the virus propagation. moreover, in preparation of this paper, gao et al. [ ] reported that the combination delivery of sars-cov s , m, and n genes into macaques using adenoviral vector elicited high titer of neutralizing antibodies, suggesting the potential of using s gene as a vaccine candidate. in the present study, mice were immunized with plasmids encoding various s gene fragments and the humoral responses were investigated. the results showed that antibodies against sars-cov could be induced by both subunits and part of s , and the mouse antisera against s and s could cooperatively neutralize sars-cov. animal, immunization, and serum collection. balb/c mice at the age - week were obtained from the laboratory of animal unit and brought up in animal house of zoology department, the university of hong kong. at day , , and the mice were anesthetized and injected with lg per dose of plasmid in ll phosphate-buffered saline (pbs) through tibialis anterior (ta) muscle on each leg [ ] . in the case of co-administration of two kinds of plasmids in the same mouse, each construct was dissolved in ll pbs and injected into ta muscle of each leg. mouse blood was regularly collected by tail bleeding at day , , , , , , , and , and the sera were kept at ) °c until further use. at day the seroconverted groups were terminally anesthetized and their sera were taken for the assays of virus specific neutralization, antibody isotypes, and geometric titer (gmt). construction of recombinant plasmid. sars-cov cdna was reverse-transcribed from viral rna extract of sars-cov hk- as described previously [ ] . dna fragments covering different regions of the s gene were amplified from the viral cdna with primers at the proper sites. fig. showed a schematic diagram indicating the sizes and the locations of these fragments on s gene. the recombinant s fragments (s r , s r , and s r ) were constructed by joining of short s fragments through overlapping extensions. the cloning sites, bamhi and ecori, start and stop codons were incorporated into pcr fragments by synthesizing on primers when necessary. a kozak sequence was also added to s na and s la ahead of their signal sequence. an expression vector, pci-sp pgh , was reconstructed by adding amino acid residues of pig growth hormone signal peptide sequence (sp pgh ) [ ] at upstream of modified polyclone sites of pci (promega). all the pcr amplified fragments were cloned into pcr . -topo (invitrogen) and sequenced as described [ ] before they were cloned into pci-sp pgh , except s na and s la , which possessed the signal sequence of s gene and hence were inserted to pci. the fragments s nb , s lb , s c , s f , s r , s r , and s r were also cloned into another expression vector pcdna . (+) (invitrogen) without signal sequence. plasmid dna fig. . schematic diagram of full-length sars-cov s protein and its derived fragments used in the study. the full-length s gene is shown immediately under the hydrophobicity profile (upper). signal sequence of s gene is marked as a solid black box and the putative highly hydrophobic regions of s are shown in solid box in light grey color. heptad repeats (hr and hr ) and membrane spanning domain (msd) are indicated as described elsewhere [ , ] . all the fragments including the recombinant fragments were cloned into mammalian expression vector pci-sp pgh , except for s la and s na which were cloned in pci. fragments, s nb , s lb , s c , s f , s r , s r , and s r were inserted into pcdna . in addition. all the constructed plasmids were used for mice experiments. s r - are different recombinants representing the putative hydrophilic regions. used to immunize animals was prepared by plasmid giga kit from qiagen (valencia, ca) according to manufacturer's instructions. expression and western blot of spike protein. to test the expression of s gene and its derived fragments in mammalian cells, all the dna fragments, as shown in fig. , were cloned into pcdna . - Âhis-ires-gfp through bamhi and ecori sites. the vector was modified by adding the Âhis and ires-gfp up-and downstream of the multiple cloning sites of pcdna . (+), respectively. ires-gfp was pcr amplified from the plasmid pbmn-i-gfp, which was a kind gift from dr. g.p. nolan (stanford university school of medicine). the recombinant plasmids were transfected into cos- cells cultured in well microtiter plate in triplicate with dosper liposomal transfection reagent (roche, mannheim, germany) following manufacturer's instruction. the expression was examined by direct observation using fluorescence microscopy. the expression of several selected fragments was further confirmed by western blot. the transfected cos- cells were harvested in - h post-transfection (p.t.). the expressed protein was purified with ni þ -agarose (amersham, uk) from clarified cell lysate and transferred onto pdvf membrane (bio-rad, hercules, ca) by electroblotting after sds-page. monoclonal mouse anti-histag antibody (invitrogen) was utilized to detect expressed protein in western blot. elisa tests of antibody titers. to detect sars-cov specific antibodies in mice immunized with the s gene dna, human anti-sars-cov antibody (igg) detecting elisa kit (beijing huada gbi biotechnology, beijing, china) was used according to manufacturer's instructions, except that anti-human igg was replaced with hrpconjugated goat-anti-mouse igg (h + l) antibody (zymed, south san francisco, ca) as the secondary antibody. according to the manufacturer, inactivated sars-cov viral particles were coated as antigen. mice group and individual were defined as seroconverted if antibody titer was higher than and , respectively. the gmt of the individual serum in the seroconverted groups was tested by end point dilution. sars-cov specific mouse igg and igg a isotypes were detected with the same elisa kit but either hrp-conjugated anti-igg or igg a isotype monoclonal antibody (pharmingen) was used as the secondary antibody. virus neutralization assay. microtiter plate ( -well) containing confluent frhk cells in . ml maintenance medium ( % fcs in mem) was prepared. each of -fold diluted mouse serum ( . ml) starting from in was premixed with . ml of tcid sars-cov (strain hk- ) and incubated at °c for . h. about . ml of the virus-serum mixture was then inoculated in wells seeded with frhk cells in duplicate and further incubated at °c. cytopathic effect (cpe) was examined at and h after. neutralization titer was determined as the highest dilution of serum which gives % cpe of cells. in order to investigate the antigenicity of s protein, especially the s subunit, s gene was segmented into s , different s fragments (s na=b=c , s la=b=c , s c , s - , and s - ), and recombinant s fragments (s r , s r , and s r ). the recombinant s fragments were constructed by joining the various hydrophilic regions of s protein (fig. ) , in an attempt to highly express the possible neutralizing epitopes on the exposing surface of s protein. all of the fragments were inserted in pcdna . - Âhis-ires-gfp, including the full-length s gene, and expressed in cos- cells, as demonstrated by the observed fluores-cence in the transfected cells (data not shown). in such expression system, the green fluorescence protein (gfp) was encoded by the same mrna encoding s gene fragment under the control of cmv promoter, but translated in two independent open reading frames (orf). although the number of fluorescence emitting cells and the density of fluorescence were different among various s fragments transfected cells, it was difficult to establish a connection between gfp expression and antibody titers since the antibody response was not merely influenced by or directly related to the expression of antigen. in addition, the expression of s protein was also confirmed by western blot (fig. ) , and the expression of different s fragments in pcdna . - Âhis-ires-gfp was detected with expected size. however, we failed to detect the expression of s f and other two small fragments, s - and s - , in the same experiment although their expression was proven at mrna level through gfp co-expression. grouped sera were prepared by pooling equal volume of individual serum of the same group. the sars-cov specific antibodies of the grouped sera were detected by elisa and the humoral response profile of the seroconverted groups is shown in fig. . the individual sera in possible seroconverted groups were titrated and are summarized in table . in general, both s and s could elicit sars-cov specific antibodies. the immunogenic importance of the n-terminus of s subunit was revealed by comparing the sars-cov specific antibody titer of mouse groups immunized with plasmids containing s na , s nb , and s nc . these three fragments shared a common region of residues - , but different only in their n-terminus (fig. ) . a higher antibody titer and more seroconverted individuals were observed in s nb group than that of the s na group. the s nc group, however, showed no detectable sars-cov specific antibody response (table ) , although it had the same leader sequence and most of the s amino acid residues as s nb . another series of constructs, s la , s lb , and s lc , showed a similar trend of antibody responses against sars-cov (table ) . groups immunized with pci-sp pgh containing s f , s - , and s - , however, showed no detectable antibody response against the virus, while s c , s r , s r , and s r groups showed a relatively low humoral response (table ) . it was also noted that the groups immunized with pcdna . (without signal peptide) containing the fragments s nb , s lb , s c , s f , s r , s r , and s r showed no detectable antibody response (data not shown) although their expression was confirmed by western blot (fig. ) . the seroconverted sera were further tested for their antibody isotypes of igg and igg a to estimate whether th or th was activated and mediated antibody isotype switching in dna immunized mice. several fragments (s nb , s lb , s c , s r , and s r ) inserted in pci-sp pgh were tested by a combined injection with s lb in pcdna . with no signal peptide attached to investigate if intracellular s could activate th pathway leading to an igg a isotype switching. however, only s nb and s lb groups were seroconverted and hence only the antisera of these two groups were recruited for the study. the result showed that the secreting forms of s nb and s lb could elicit both igg and igg a isotypes. co-administration of an additional nonsecreting s lb to both groups induced a higher igg a/igg ratio in both groups (table ) , although such non-secreting form of s protein seemed to have a negative effect on antibody elicitation, as a lower group's titer and a fewer number of seroconverted mice were observed in group s la /+ and s na /+. antisera from group of s nb , s lb , s c , s , s f , s r , s r , and s r were tested for their neutralizing antibody titer although s c , s r , s r , and s r had only slightly group's antibody and even no detectable antibody response for n, no detectable antibody response/neutralizing activity. n/a, not applicable. a an additional lg pcdna . -s lb dna was delivered in parallel for each mouse in each injection. b by mixing equal volume of the two groups' mouse serum (for neutralizing assay). c titers were measured based on the seroconverted mice. d group sera (s f and control) or combined seroconverted sera in the group (the rest groups) were used. s f in the elisa assay ( table ). the result showed that only the antiserum combined with equal volume of sera from group s lb and s was capable of neutralizing sars-cov. no detectable neutralizing ability was observed by using other serum samples including the mixed s nb and s group serum and the serum elicited by s r dna which covered most hydrophilic regions of s gene (table ) . in the present study, different truncated fragments of sars-cov s gene were cloned into two mammalian expression vectors, with or without secreting signal sequence, and used to immunize mice. the non-secreting form of s protein could not elicit any detectable sars-cov specific antibody response while the secreting forms s , s , and the n-terminus of s (s n ) did. the inability of the non-secreting s protein to elicit sars-cov specific antibody may be explained by the lack of expressed antigens with correct conformation exposed to immune system for post-translational modification including complex folding, glycosylation, and oligomerization, which occurs during protein secretion and sorting. it was widely reported that the expression of viral membrane protein in different cellular locations (e.g., secreted, membrane bound, and non-secreted form) was the immune response of dna immunization. animals vaccinated with dna encoding secreting form of e glycoprotein of classical swine fever virus (csfv) showed both csfv specific antibody response and protection upon viral challenge though the native e was anchor membrane protein ( [ , ] and zeng et al., unpublished data). in addition, a shift in t-helper cell response from th to th was induced by expression of secreting form of hemagglutinin glycoproteins [ ] . the non-secreting form of s protein, however, might be useful in activation of th response, as injection of secreting and non-secreting s into the same mouse enhanced th response, as shown by the raised igg a to igg ratio (table ) . these preliminary data suggest that s may possess epitopes that could activate t-cell responses. the antibody and neutralizing antibody elicited by s and s in the mice experiment, it was demonstrated that sars-cov specific antibodies could be induced by immunization with plasmids encoding s , s and fragment of s subunit. plasmid carrying s nb (residue - ), elicited the highest antibody titer among all the seroconverted groups (table ) . residues near the cleavage site of the signal peptide in matured s were very critical in terms of stimulating sars-cov specific antibodies, as demonstrated by two sets of fragments, s na=b=c and s la=b=c , which were expressed through the same plasmid vector. as indicated in the virus neutralizing assay, none of the antisera from the sars-cov specific seroconverted groups can neutralize the virus. instead, mixed antisera from seroconverted groups immunized with s (s lb ) and s plasmids showed neutralizing activity, although the concentration of the antibodies was diluted one-half as compared to the parent sera. it was also noted that another set of antisera mixed sample from s nb and s showed no observable neutralizing activity as well, although the antibody titer of s nb was higher than that of s lb and the same s serum was used. the obligatory cooperation of s and s specific antibodies in terms of virus neutralization, however, contradicts with previous researches on coronavirus. several authors reported that immunization with recombinant s protein or plasmid encoding the s subunit of infectious bronchitis virus (ibv) could induce protective immune responses [ , , ] , although multiple inoculations were required [ ] . the first paper on immunizing masques with structural genes of sars-cov, however, reported that co-delivery of three viral genes encoding s , nucleocapsid (n), and membrane (m) protein could elicit a high titer of neutralizing antibody and t-cell response [ ] . the authors stated that inoculation of s with n and or m could stimulate a strong antibody and t-cell response, in parallel with the previous experience on developing vaccine against transmissible gastroenteritis coronavirus (tgev) [ ] . from this, together with the experimental data from this report, it can be hypothesized that antibodies against the s glycoprotein are crucial in neutralizing sars-cov but the antibodies against other components of the virus may also play critical roles in virus neutralization. such effects may become significant if the titer of the antibodies, including those against s protein, is low. the putative cooperative role of s specific polyclonal antibody to neutralize the virus may serve as an additional example which directly demonstrates the blockage of virus entry through the binding to the s subunit. further experiments are desirable to define the exact roles s and s specific polyclonal antibodies play in neutralizing sars-cov and their protecting ability against virus challenge. results described here are expected to speed up the design of subunit vaccine and the immunological research against the sars-cov. epidemiology and cause of severe acute 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coronavirus four major antigenic sites of the coronavirus transmissible gastroenteritis virus are located on the amino-terminal half of spike glycoprotein s protection of chickens from infectious bronchitis by in ovo and intramuscular vaccination with a dna vaccine expressing the s glycoprotein induction of protective immunity in chickens vaccinated with infectious bronchitis virus s glycoprotein expressed by a recombinant baculovirus the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex mass spectrometric characterization of proteins from the sars virus: a preliminary report cloaked similarity between hiv- and sars-cov suggests an anti-sars strategy molecular epidemiology of the novel coronavirus that causes severe acute respiratory syndrome (sars) angiotensin-converting enzyme is a functional receptor for the sars coronavirus a -amino-acid fragment of the sars coronavirus s protein efficiently binds angiotensin-converting enzyme measurement of subgroups of peripheral blood t lymphocytes in patients with severe acute respiratory syndrome and its clinical significance profile of specific antibodies to the sars-associated coronavirus prevalence of igg antibody to sars-associated coronavirus in animal traders detection of sars coronavirus in patients with suspected sars, emerg. infect. dis. serial online effects of a sars-associated coronavirus vaccine in monkeys intramuscular and intradermal injection of dna vaccines in mice and primates the complete genome sequence of severe acute respiratory syndrome coronavirus strain hku- (hk- ) porcine growth hormone: molecular cloning of cdna and expression in bacterial and mammalian cells evaluation of genetic vaccine against classical swine fever dna-mediated protection against classical swine fever virus dna immunization: effect of secretion of dna-expressed hemagglutinins on antibody responses cooperation between transmissible gastroenteritis coronavirus (tgev) structural proteins in the in vitro induction of virus-specific antibodies this work was supported by research grant council grant hku / m. key: cord- - id oh n authors: allan, janet; barwick, timi agar; cashman, suzanne; cawley, james f.; day, chris; douglass, chester w.; evans, clyde h.; garr, david r.; maeshiro, rika; mccarthy, robert l.; meyer, susan m.; riegelman, richard; seifer, sarena d.; stanley, joan; swenson, melinda; teitelbaum, howard s.; timothe, peggy; werner, kathryn e.; wood, douglas title: clinical prevention and population health curriculum framework for health professions date: - - journal: american journal of preventive medicine doi: . /j.amepre. . . sha: doc_id: cord_uid: id oh n abstract the clinical prevention and population health curriculum framework is the initial product of the healthy people curriculum task force convened by the association of teachers of preventive medicine and the association of academic health centers. the task force includes representatives of allopathic and osteopathic medicine, nursing and nurse practitioners, dentistry, pharmacy, and physician assistants. the task force aims to accomplish the healthy people goal of increasing the prevention content of clinical health professional education. the curriculum framework provides a structure for organizing curriculum, monitoring curriculum, and communicating within and among professions. the framework contains four components: evidence base for practice, clinical preventive services–health promotion, health systems and health policy, and community aspects of practice. the full framework includes domains. the title “clinical prevention and population health” has been carefully chosen to include both individual- and population-oriented prevention efforts. it is recommended that all participating clinical health professions use this title when referring to this area of curriculum. the task force recommends that each profession systematically determine whether appropriate items in the curriculum framework are included in its standardized examinations for licensure and certification and for program accreditation. i ncreasingly, members of the health professions, policymakers, and the american public understand the importance of health promotion, disease prevention, and population health across a spectrum of issues affecting health, including chronic disease management, emerging infectious diseases, emergency preparedness, disparities in health and healthcare services, and the impact of behavior and lifestyle choices. , increasingly, we realize the inadequacy of diseasebased, episodic, acute care intervention for addressing these issues. nevertheless, a focus on prevention and population health continues to lag behind the emphasis on one-on-one treatment. until prevention is thoroughly integrated into all aspects of our healthcare system, measurable progress addressing these issues will elude us. an essential element of any effort to change a healthcare system must be the education of future clinicians who will practice new approaches in new contexts. thus, an unambiguous emphasis on individual-as well as population-based prevention must be part of clinicians' education. although a few innovative methods to integrate clinical prevention and population health into clinician training have been developed, - no structured, comprehensive curriculum incorporates these topics into most health professionals' education. the goal of implementing such a curriculum is not new. "some years of effort . . .to teach prevention as an integral part of clinical medicine . . .has met with limited success," wrote barker and jonas over decades ago after reviewing the literature and their own experience. despite past limited success, the time may be right for making real progress. the events of fall , the severe acute respiratory syndrome (sars) epidemic, and west nile virus have highlighted the critical role of prevention and public health. the institute of medicine (iom) reports on medical errors and the quality of care highlighted the need to improve patient safety and restructure care systems. , another iom report called for "transforming the content, methods, approaches, and settings used in health professions education" in response to the "changing needs of the population and changing demands of practice." healthy people encouraged the reexamination of clinical education by including an objective "to increase the proportion of schools of medicine, schools of nursing and health professional training schools whose basic curriculum for healthcare providers includes the core competencies in health promotion and disease prevention." traditionally, each health profession designed, developed, and implemented its own curriculum framework. the approach presented here ( ) assumes the need for and value of a common curriculum framework outlining the content that all health professions students should know and skills they all should have; ( ) articulates how to organize such a framework; and ( ) specifies what it should be called. to integrate clinical prevention and population health into clinical practice, this approach assumes the need for effective interprofessional communications and collaboration. a mutually agreed upon curriculum framework articulating key elements of prevention and population health sciences provides not only the common, core subject matter, but also increases the opportunity for education and training in multiprofessional teams. the release of healthy people joined the need for more effective prevention education with the need for greater interprofessional education and practice. although not specifically recommended by healthy people , if a prevention framework is to be widely used, it must be widely accepted across the core health professions-a goal that will be achievable if the framework is developed by leaders from the core clinical professions. the task of developing such a framework provided the impetus for the association of teachers of preventive medicine (atpm) to join with the associa-tion of academic health centers to convene the healthy people curriculum task force, which is composed of a senior academic member and the executive director (or designee) from the following clinical health professional organizations: the task force also includes representation from the student health alliance (a consortium of health profession student organizations) and two resource groups, the association of schools of public health (asph) and community-campus partnerships for health. this unprecedented assemblage of diverse health professions stakeholders developed the clinical prevention and population health curriculum framework (framework) that could be used by students from at least the seven represented health professions. the progenitor for the framework was conceived almost years ago when atpm assembled a group of leaders in prevention to articulate the basic prevention content for a comprehensive medical education curriculum. the resulting inventory of knowledge and skills relating to disease prevention and health promotion provided the guidepost for schools' efforts to broaden prevention training. despite the intent that this document be "shared across many disciplines," it was associated with medical training and rarely used by other health professions. - building on the above inventory, in , the task force developed a preliminary curriculum framework in clinical prevention and population health. widespread web-based review and evaluation of this document were sought from academics, students, practitioners, and through the participating organizations. to encourage each participating clinical health profession to review its curriculum recommendations and/or requirements and consider changes compatible with the framework. the framework allows considerable flexibility for each clinical health profession to determine the depth of curriculum that is recommended, the timing for teaching the material, and the method(s) for delivery. the goal is to provide general recommendations and identify content areas that may require greater emphasis. it is also the intent to point out opportunities for interprofessional education and collaboration. the framework is designed for degree programs rather than postgraduate or residency training, although it is hoped that these programs will build upon the framework. the framework should be viewed as providing the foundation for a curriculum that spans the years of clinical health professional training. the curriculum content will generally need to be incorporated in more than one module or course in a degree program. therefore, a mechanism for integrating this curricular content is important. integration provides the opportunity to stress the interactive or ecologic nature of the factors affecting health and the development and outcome of disease (as stressed in recent iom reports). , the framework also reflects the iom's emphasis on health policy, ethics, and global health as components of public health education. the name "clinical prevention and population health" has been carefully chosen to include both individual-and population-oriented preventive efforts as well as the interactions between them. it is recommended that participating health professions use this title when referring to this area of the curriculum. the framework consists of four components-evidence base of practice, clinical preventive serviceshealth promotion, health systems and health policy, and community aspects of practice-with domains. these components are recommended as a structure for organizing and monitoring curriculum, and communicating within and among disciplines. within each component, the numbered domains are designed to outline content to reflect both individual clinical prevention and population health. the numbered domains allow each profession to identify the content considered relevant to its educational efforts. finally, the listed items in each domain represent examples of the types of materials a particular profession may choose to encourage or require in its curriculum. rates of disease (e.g., incidence, prevalence, case fatality) types of data (e.g., nominal, continuous, qualitative) the following discussion addresses the four components. a full discussion of all elements of the framework, including the domains and general recommendations for implementation is included at www.atpm.org. this full document includes recommendations for timing and coordination of the curriculum, integration of curriculum content, and competency assessment. this component aims to operationalize the background needed to incorporate evidence into practice. the level of quantitative and qualitative analysis and study design understanding needed may vary from one profession to another. however, the numbered domains are designed to identify core competencies that are considered generally applicable for clinical health professional education. this component may be implemented using a variety of educational methods. practice with structured reading of the health research literature will help ensure that students understand the relevance of these concepts. this component aims to ensure that students not simply memorize recommended interventions, but understand the science needed to produce and interpret evidence-based recommendations for an intervention or service. this component is intended to parallel the structure established by the u.s. preventive services task force. the four domains within this component are intentionally structured in parallel, implying a similarity in approach and depth of understanding. each of the four domains could be subsumed into broader clinically oriented education. while it is desirable to connect each domain of this component with other clinically oriented education, it is important that a coherent approach be preserved for teaching the principles of screening, counseling, immunization, and chemoprevention. some clinical health professions may encourage or require knowledge of the content of specific evidencebased recommendations. however, that knowledge is considered separate from accomplishing the aims of this component. a systematic approach to this component has not been part of most clinical health professional curricula. the development of a coherent curriculum that provides a framework for students to use as they experience the u.s. healthcare system is essential to accomplishing the intent of this component. this component may be particularly amenable to interprofessional educational efforts, since the required level of knowledge is not likely to vary by discipline. although ethical responsibilities are included under the health workforce domain, the integration of ethical issues throughout the entire framework is recommended. health policy is not generally required by current accreditation standards or included in most clinical health professions education curricula. nevertheless, the task force overwhelmingly endorsed its inclusion here. the intent is to provide students with a basic understanding of policies that affect their practice and the health of their patients and communities, processes through which policies are developed, and opportunities to participate in policymaking. this component aims to integrate individual clinical prevention with the population health focus of the framework. community is defined broadly to include geographically defined communities as well as those defined by similar demographics, interests, or experiences (e.g., age, employment, diseases, health risks). the need for community-based learning experiences outside traditional institutional settings is fundamental to this component. service learning, communitybased clinical rotations (e.g., public health departments and community health centers), communitybased research, and international health experiences are possible ways to attain this goal. these might include education in the evaluation of web-based information, particularly from sites used by patients; learning firsthand about environmental and occupational exposures; and understanding the structure of international efforts to address current and emerging health problems. the cdc guide to community preventive services can help support the development of prevention education across the health professions. integration of the four components is highly desirable. to integrate the curriculum, one might illustrate the options for intervention from primary prevention through rehabilitation; the level of intervention from the individual to the high-risk group to the general population; or the methods of behavioral intervention, including education, motivation, and training in counseling skills. such integration may require incorporating specific curricular content near the end of the degree program. each clinical profession should address the methods used to evaluate students and to ensure their levels of competency. the task force recommends that each profession systematically determine whether appropriate items in the framework are included as part of its standardized examinations for licensure and certification. the task force encourages clinical health professions to explore creative methods for implementing the framework using the opportunities it provides for interprofessional education. in addition, teaching the content contained in the framework needs to emphasize the involvement of a range of health professionals beyond those represented on the task force. the framework presented here will be used through - , when it will be extensively reviewed and revised. data collection on inclusion of framework content elements in the curricula of each of the seven professions is underway, in order to meet the require-ments of the healthy people mid-course review in . the framework has been endorsed by several professions on the task force, and explorations with their accrediting bodies have begun. to help disseminate and implement the framework, the task force plans to develop an online clinical prevention and population health resource center. a web-based searchable database could eventually provide access to curricula for each of the domains of the framework, and searches would be possible to identify curricula for specific clinical disciplines and for teaching using a variety of formats. linkages to educational consultants and continuing education programs may also be available through the resource center. the task force will examine other options for implementing the framework. for instance, the task force will examine the implications of the iom recommendation that "all undergraduates should have access to education in public health." to facilitate implementation of the framework, graduate-level health professional programs might recommend an undergraduate public health course as part of their preprofessional preparation. the task force succeeded in bringing together a wide spectrum of clinical health profession groups and developed a common framework for organizing, implementing, and monitoring curricula in clinical prevention and population health. continued success will require building on this interprofessional communication and collaboration to develop models for interprofessional education. actual causes of death in the united states mortality and morbidity attributable to use of addictive substances in the united states pew health professions commission. recreating health professional practice for a new century a case-based curriculum for teaching clinical and population-based preventive medicine education for more synergistic practice of medicine and public health the clerkship in public health: a positive experience teaching medical students about epidemiology: utilizing a state health department teaching about the changing u.s. health care system: an innovative clerkship the teaching of preventive medicine in american medical schools, - crossing the quality chasm: a new health system for the twenty-first century health professional education: a bridge to quality academic health centers: leading change in the st century department of health and human services. healthy people inventory of knowledge and skills relating to disease prevention and health promotion prevention education and evaluation in u.s. medical schools prevention for the st century: setting the context through undergraduate medical education who will keep the public healthy? educating public health professionals for the st century improving medical education: enhancing the behavioral and social science content of medical school curricula preventive services task force. guide to clinical preventive services. vols. washington dc: agency for healthcare research and quality advancing the healthy people objectives through community-based education: a curriculum planning guide community preventive services task force. guide to community preventive services. available at: www.phppo.cdc.gov/cdcrecommends key: cord- - k tcgfs authors: burnouf, thierry; griffiths, elwyn; padilla, ana; seddik, salwa; stephano, marco antonio; gutiérrez, josé-maría title: assessment of the viral safety of antivenoms fractionated from equine plasma date: - - journal: biologicals doi: . /j.biologicals. . . sha: doc_id: cord_uid: k tcgfs abstract antivenoms are preparations of intact or fragmented (f(ab′) or fab) immunoglobulin g (igg) used in human medicine to treat the severe envenomings resulting from the bites and stings of various animals, such as snakes, spiders, scorpions, or marine animals, or from the contact with poisonous plants. they are obtained by fractionating plasma collected from immunized horses or, less frequently, sheep. manufacturing processes usually include pepsin digestion at acid ph, papain digestion, ammonium sulphate precipitation, caprylic acid precipitation, heat coagulation and/or chromatography. most production processes do not have deliberately introduced viral inactivation or removal treatments, but antivenoms have never been found to transmit viruses to humans. nevertheless, the recent examples of zoonotic diseases highlight the need to perform a careful assessment of the viral safety of antivenoms. this paper reviews the characteristics of equine viruses of antivenoms and discusses the potential of some manufacturing steps to avoid risks of viral contamination. analysis of production parameters indicate that acid ph treatments and caprylic acid precipitations, which have been validated for the manufacture of some human igg products, appear to provide the best potential for viral inactivation of antivenoms. as many manufacturers of antivenoms located in developing countries lack the resources to conduct formal viral validation studies, it is hoped that this review will help in the scientific understanding of the viral safety factors of antivenoms, in the controlled implementation of the manufacturing steps with expected impact on viral safety, and in the overall reinforcement of good manufacturing practices of these essential therapeutic products. antivenoms are important biopharmaceutical products made from the plasma of immunized horses or sheep. they are used in human medicine to treat the potentially severe pathophysiological complications resulting from the bites and stings from various animals, such as snakes, scorpions, spiders, cnidarians, lepidopterans or fishes, as well as from intoxications with plants [ , ] . envenomings are a serious health problem worldwide and are most particularly dreadful in rural areas of the developing world [ ] , where shortage of antivenom products [ ] and lack of sufficient medical facilities explain numerous fatalities [ , ] . snake bites represent the major cause of envenoming. case fatality associated with some snake bites reach % or more but can be reduced to less than % through antivenom therapy, the only available current treatment [ , e ] . global mortality from snake bites may range from , to , per year, but these figures are likely underestimated [ , ] . viperid snake bite envenoming induces local effects, such as swelling, pain, necrosis, hemorrhage and blistering, often accompanied by secondary infection [ ] . systemic viperid envenoming is characterized by a complex pathophysiological profile including coagulopathy, hemorrhage, hypovolaemia, shock and acute renal failure [ , ] . progressive paralysis may be caused by elapid snake venom neurotoxins, and by some viperid venoms displaying neurotoxic effects. some elapid venoms also induce local necrosis and rhabdomyolysis [ ] . snake bite survivors may have major chronic physical and neurological disability. scorpion stings are the second major cause of human fatalities from envenoming (probably amounting to several hundreds per year). scorpion venoms contain toxins which target sodium, potassium, calcium and chloride channels, causing direct effects and release of neutrotransmitters such as acetylcholine and catecholamines, inducing intense local pain and potentially fatal neurotoxic and hemodynamic disturbances [ ] . the role of antivenom in the treatment of scorpion stings and other arachnids remains controversial but several reports support their manufacture and use, particularly in severe cases [ , ] . fatalities have also occurred from envenoming by jellyfish, and venomous fishes. long-term anecdotal experience supports the beneficial effect of stonefish antivenoms [ ] , but those may need to be given very early to fight a rapid onset of cardiotoxicity. stings by cnidarians, lepidoptera, centipedes and coneshells and bites by spiders, ticks and one genus of octopus, probably account for about deaths per year [ ] . finally, envenomings by massive attacks of africanised bees cause some deaths each year in the americas [ ] . antivenoms are most often made by fractionation of plasma of horses (less frequently sheep) that have been immunized with crude venoms [ ] . pooled hyperimmune plasma is processed to purify the horse immunoglobulin g (igg) fraction that may be subjected to enzymatic treatment to obtain f(ab#) and fab antibody fragments, and caprylate or ammonium sulphate precipitation to improve their purity [ ] . to some extent, some manufacturing steps have features similar to those used to prepare human plasma-derived igg. although there is no known transmission of any infectious agent through antivenoms (albeit under circumstances where rigorous clinical patient follow-up is difficult), theoretical concerns about the possibility of transmission of horse/sheep infectious agents to humans do exist. recent natural transmissions of zoonotic diseases highlight the possible exchanges between humans and the natural reservoirs of biologic agents found in animals [ ] , and the inherent risk of emerging diseases [ ] . examples of such infections originating from animal (or avian) pathogens include human immunodeficiency virus, ebola, hantaan, lassa, nipah viruses and other paramyxoviruses, equine morbilli virus, west nile virus, and probably severe acute respiratory syndrome (sars) coronavirus [ , ] . the parenteral transmission of animal viruses to humans is also possible. infectious sv virus of rhesus monkeys was a contaminant of early polio vaccines which were administered to a large number of people. whether this was the way in which this virus was introduced into the human population is unclear and a controversial issue. the risk of contamination of antivenoms by equine virus is, therefore, of theoretical concern and has been debated at a recent world health organization (who) workshop [ ] . recently, the committee for proprietary medicinal product (cpmp) of the european medicine evaluation agency has published a note for guidance on the manufacture and quality control of animal immunoglobulins and immunsera [ ] . however, most manufacturers of antivenoms are located in the developing world and some of them may not be directly exposed to the state-of-the-art process validation concepts, nor have the financial and logistics capability to perform extensive viral validation studies to assess the viral reduction potential of the process they use. therefore, we found it helpful to examine the viral safety of antivenoms by first reviewing the characteristics of equine viruses. we then evaluated the theoretical ability of manufacturing methods of antivenoms to inactivate or remove those viruses, through a comparison with the well validated technologies used to manufacture human plasma-derived igg preparations. finally, we want to emphasize that applying good manufacturing practices in the production of antivenoms, from the control of the animals to that of all production steps, and ensuring traceability in the whole chain of manufacture, represent the current best investment in the quality and safety of these products. horses can harbour enveloped and non-enveloped viruses. the main structural characteristics of those viruses are presented in table ; epidemiological and clinical data and testing methods are described briefly below. eav is a e nm single-stranded (ss)-rna virus of the arteriviridae family [ ] . eav causes an equine viral arteritis, an endotheliotropic viral disease [ ] . transmission occurs via respiratory and reproductive routes. there is a variety of clinical signs, and strains vary in virulence [ ] , but severe infection can lead to abortions in pregnant mares or neonatal foal death. a one-tube real-time taqman rt-pcr assay was developed for detecting eav. the test was validated using seminal plasma and nasal secretions [ ] . a dna vaccine was shown to induce long-term immunization [ ] . bdv is a e nm ss-rna virus belonging to the new bornaviridae family, order mononegavirales. borna disease, known as 'disease of the head', is a sporadically occurring, progressive viral polioencephalomyelitis that primarily affects horses and sheep. after a few weeks to several months incubation, bdv can cause locomotor and sensory dysfunction followed by paralysis and death. bdv exists world-wide in horses, sheep, cattle, cats, dogs and ostriches and can affect a large number of warm-blooded animal species, including humans [ , ] . the infection can be fatal, but the majority of carriers are asymptomatic [ ] . cross-species transmission of this commensal virus has not been proven, but zoonotic aspects of bdv should be considered [ ] . bdv-specific antibodies and viral rna have been found in humans with various psychiatric disorders. diagnosis can be made serologically, but also by antigen markers in peripheral white blood cells, combined with nucleic acid amplification [ ] . these are closely related e nm ss-rna alphaviruses [arbovirus type a] of the togaviridae family that are transmitted by arthropods (usually mosquitoes). symptoms of eeev infections, an important multisystemic zoonotic disease, include anorexia and colic, changes in sensorium, hyperexcitability, and terminal severe depression. organ coagulative necrosis and cns lesions are observed [ ] . weev is not as neuroinvasive as eeev and the encephalitis caused is not as severe as that caused by eeev. a test to detect eee and wee viral rna has recently been developed [ ] . veev is present in both humans and horses but no evidence of transmission from horses to humans by normal routes of contamination has been found. veev has been identified in pharyngeal secretions and is stable when aerosolized; it has been shown to be stable in dried blood and exudates. vaccines have been developed against those three viruses [ ] ; one possible case of viral transmission in horse has been suspected following vaccination [ ] . ecv is a e nm ss-rna virus of the coronaviridae family. it has been isolated from feces of a diarrhoeic foal, and has close antigenic and/or genetic relationships with mammalian group coronaviruses [ ] . efv, also known as spumavirus, is a e nm ss-rna virus of the retroviridae family. it belongs to the nonpathogenic, complex unconventional retroviruses and has been isolated in nonhuman primates, cattle, cats, and more recently in the blood of horses [ ] . ehvs are large e nm double-stranded (ds) dna gamma herpes viruses. ehv and ehv cause much damage to the horse industry and are ubiquitous in the equine population. they are responsible for life-long latent infections in their hosts even in those with natural or vaccine-induced immunity [ ] . ehv strains are associated with respiratory disease, abortion, and paresis/paralysis, whereas ehv strains induce respiratory disease [ ] . ehv and ehv have a less clear pathogenicity and distribution within the equine population. ehv may have an aetiological role in ocular disease [ ] . the prevalence of ehv in adult horses was found to be up to % in sweden and % in the united kingdom. the prevalence of ehv dna was and % in adult horses in hungary and the united kingdom, respectively [ ] . eiav is a e nm ss-rna lentivirus of the same retroviridae family as hiv. most infected horses are asymptomatic, with life-long latent infection of leucocytes until stressed (e.g. by pregnancy, corticosteroids, surgical operation, disease) or until new virus variants arise. viraemia then increases by -fold. red blood cells become coated by the virus particles and are then lysed by complement, causing jaundice, oedema, hemorrhagic diarrhoea, petechial hemorrhages. eiav can be transmitted from horse to horse by blood. recently a reverse-transcriptase polymerase chain reaction assay (rt-pcr) has been described for quantifying eiav rna in equine plasma [ ] . eiv is a e nm ss-rna virus of the orthomyxoviridae. equine influenza is one of the most economically important contagious respiratory diseases of horses [ ] . hev is a nm ss-rna virus [ ] . the virus is probably transmitted from bats to horses, and causes natural disease in humans and horses. it is the reference virus for a proposed new genus within the paramyxoviridae family, which also includes another newly identified zoonotic bat virus (nipah), and the salem virus. hev has been recognized in australia as a new zoonotic disease of horses since / . this lethal zoonotic viral agent is endemic in certain species of fruit bats (flying-foxes) and over % of them in eastern australia are seropositive [ ] . six species of flying-foxes in papua new guinea have tested positive for antibodies to hev [ ] . hev does not appear to transmit readily between horses under natural and experimental conditions, or from horses to humans [ ] . horses can be infected by oronasal routes and can excrete hev in urine and saliva [ ] . the virus appears to be spread by close contact with body fluids, such as froth from infected lungs [ ] , via nasal discharge, saliva and/or urine [ ] . the most important clinical and pathological manifestation of hev infection in horses and humans is severe interstitial pneumonia caused by viral infection of small blood vessels. from to , epizootic and epidemiological episodes of meningoencephalitis and severe acute respiratory syndromes were reported in australia, malaysia and singapore [ , ] . it may also cause nervous disease. three incidents of hev disease in horses have been recorded in australiadtwo in , which caused the death of two humans and horses and one in , which involved the death of a single horse [ ] . infected horses can develop a severe and often fatal respiratory disease characterized by dyspnoea, vascular endothelial damage and pulmonary oedema. nervous signs may also occur. a number of diagnostic methods have been developed [ ] , based on the examination of blood, lung, kidney, spleen, and, if nervous signs are present, also of the brain. pcr assays have been developed. a rapid and sensitive rt-pcr assay using a fluorogenic (taqman) probe was developed to improve the diagnosis of hev infection [ ] . both are closely related, arthropod-born, e nm ss-rna flaviviruses of the flaviviridae family that may affect the central nervous system of horses [ , ] . evidence of infection with niv was found in the brain of one horse in which inflammation of the meningeal blood vessels occur [ ] . niv emerged in malaysia, spread rapidly through the pig population, and caused the deaths of over people [ ] . salv has been recently identified in horses [ ] . the only known isolate was obtained from a horse that was involved in a disease outbreak of undetermined nature [ ] . vsv is a e nm ss-rna virus of the rhabdoviridae family. it may cause stomatitis in horses, and may represent an emerging equine infectious disease [ ] . an immunoglobulin m (igm) capture enzyme-linked immunosorbent assay (mc-elisa) has been developed for the detection of primary infection of vsv in equine and swine sera [ ] . wnv is a e nm ss-rna arbovirus of the flaviviridae family, first identified in the west nile district of uganda in . wnv is found over a broad geographical range and in a wide diversity of vertebrate host and vector species [ ] . until , the disease was found in african, european, and eurasian countries. more recently, there was an increase in outbreaks of illness due to wnv in animals as well as humans and numerous cases are currently reported in canada and the usa. infected humans may be asymptomatic and the transfusion of donated blood from such individuals has resulted in the infection and death of recipients. this has led to the introduction in of strict regulatory measures in canada and the usa which include the testing by a pcr assay of all blood donations. wnv primarily circulates between birds and mosquitoes, while mammalian infections are incidental. mammal biting mosquitoes become infected when they feed on the blood of an infected bird. once this happens, people, horses and other mammals may get wnv. infected horses are, however, unlikely to serve as important amplifying hosts for wnv in nature [ ] . many wnv-infected horses probably never show symptoms, but a study reported mortality rates close to % [ ] . early symptoms are often indistinguishable from other equine encephalitides including rabies, equine herpesvirus- , equine protozoal myeloencephalitis, and eastern, western, or venezuelan equine encephalomyelitis. a vaccine for horses has been developed, but one horse that received two doses died from the disease [ ] . erav and erbv are picornaviruses reclassified as members of the aphtovirus genus because of resemblance to foot-and-mouth disease virus. they are e nm ss-rna viruses. high neutralizing antibody titers develop and appear to correlate with strong reactivity to vp in western blots [ ] . a new serotype of the genus erbovirus, tentatively named erbv has been identified recently and found in % of horses in australia [ ] . eev is a nm ds-rna virus of the reoviridae family. several serotypes have been identified in southern africa. serotype-specific virus-neutralizing antibody in serum samples from horses suggests a widespread occurrence of infection but there seems to be only a low level of cross-protection in horses to natural reinfection [ ] . erv is a e nm double-stranded rna of the reoviridae. rotaviruses are important pathogens associated with diarrhoeal diseases in almost all species of mammals. a pcr test with a detection limit of approximately . ! tcid per gram faeces, with possible increase in the sensibility by one order of magnitude using nested pcr, was developed, representing a possible diagnostic tool [ ] . antivenoms are produced from the plasma or serum collected from immunized horses or sheep. plasma can be obtained by the centrifugation of whole blood or by apheresis. plasma from several animals is typically pooled into e l batches and subjected to a fractionation process to isolate the igg fraction. following the initial use of crude equine immune serum, still reported to be manufactured by one producer [ ] , various methods of igg purification and refinement have been introduced ( table ). most manufacturers use protocols derived from the basic method described by pope [ , ] , modified by harms [ ] , based on pepsin digestion at low ph, to obtain f(ab#) fragments, followed by ammonium sulphate precipitation of antibody fragments [ , , ] . this basic approach is combined with a number of additional steps, aimed at obtaining a purer [ ] preparation, such as heat coagulation [ ] and ion-exchange chromatography [ , ] . the ph at which pepsin digestion is performed by various laboratories ranges between . and . , at temperatures between and c. pepsin digestion is usually performed in undiluted serum or plasma (protein concentration: e g/l). incubation times range from min to h and with varying pepsin concentrations [ , , , ] . thermocoagulation is used by many laboratories and consists of heating at e c for h [ , , ] although not all methods involving pepsin digestion include heat coagulation [ ] . some fractionation protocols include an additional table steps used for the manufacture of antivenoms screening of production animals for adventitious agents plasma collection (whole blood or apheresis) in bags or bottles plasma thawing at room temperature plasma pooling igg/fragments purification process f(ab#) : b pepsin digestion (acid ph) and ammonium sulphate precipitation, or b pepsin digestion (acid ph) and caprylic acid precipitation fab: ammonium sulphate precipitation and papain digestion at ph e whole igg: b caprylic acid precipitation b ammonium sulphate precipitation igg/fragments concentration: (nh ) so /na so precipitation, ultrafiltration polishing: ion exchange (removes fc and further purifies iggs/ fragments) ultrafiltration sterile filtration aseptic filling storage in the liquid state or lyophilisation acidification step to remove some non-igg globulins ('euglobulins') which are unstable at acid phs [ , ] . one producer includes a bulk pasteurisation step in its antivenom production protocol [ ] . other manufacturers produce whole igg antivenoms using either ammonium sulphate precipitation of igg or caprylic acid precipitation of non-igg proteins [ e ], followed by dialysis or ultrafiltration. recently, a simple, one-step methodology based on caprylic acid precipitation has been described [ ] . the conditions used for caprylic acid fractionation of equine plasma are as follows: plasma ph is adjusted to . and caprylic acid is added directly to this plasma to attain a final concentration of % (v/v). the mixture is stirred during caprylic acid addition and then for one additional hour, after which the precipitate is separated by filtration. caprylic acid is then removed by either dialysis or ultrafiltration and the product is formulated before sterile filtration [ ] . some antivenoms are made of fab antibody fragments, obtained by papain digestion of sheep igg, at neutral ph, after sodium sulphate precipitation of igg [ ] . affinity-chromatography or ion-exchange chromatography steps have been described in the production of some fab antivenoms [ , ] . treatment with b-propiolactone has been evaluated to reduce complement activation by horse plasma-derived products [ ] ; however, it does not appear to be used routinely in laboratories producing antivenoms. in addition to the differences in fractionation protocols, antivenoms made of whole igg, f(ab#) and fab molecules greatly differ in their pharmacokinetic profiles [ , ] . most antivenoms come in liquid presentation, but some are lyophilized. the former need to be stored at e c, since higher temperatures reduce their shelf-life and induce protein denaturation and aggregate formation [ ] . in contrast, lyophilized antivenoms can be stored at room temperature and have a more prolonged shelf-life. most antivenoms contain preservatives to prevent bacterial contamination [ , ] . considerable experience has been gained in recent years on the viral safety of human igg preparations and other human plasma-derived products. historical perspectives on these products help in assessing the viral safety of antivenoms. the safety of human plasma products results from the combination of overlapping strategies that include: (a) selection of donors, (b) viral testing procedures on single donations (e.g. serological tests) and plasma pools (e.g. nucleic acid tests) to exclude donations contaminated by viruses, (c) processing and purification steps to inactivate and remove viruses, and (d) implementation of good manufacturing practices [ ] . whenever feasible, similar approaches should be used for the manufacture of animal plasmaderived products. monitoring the health of animals used for antivenom production is important [ ] . colonies of animals should be kept free of contamination through good husbandry or vaccination, where appropriate. surveillance of the source animals should include screening for pathogens, health monitoring, including routine blood chemistry and haematology tests, and post-mortem examination. ideally, animals should be kept under strictly contained conditions, but this is not readily applicable to horses. however, for instance, animals could be kept free of a particular arthropod-borne virus if they are maintained in an area free of the particular arthropod vector. if a particular pathogen is identified, it should be established whether it is present in plasma. some virological screening methods may be performed, if available and epidemiologically relevant to a particular geographic region, to limit risk of virus presence in the animal herd and/or the plasma pool. to some extent the guidelines from the federation of european laboratory animal science association (felasa), although not referring to horses, may serve as a reference [ e ]. nevertheless, not all of these measures are available in many developing countries nor readily applicable to horses used for antivenom production, and pathogens can escape this surveillance program. this illustrates the need for manufacturing techniques to ensure a sufficient margin of safety with regards to potential infectious agents present in the manufacturing plasma pool. this also strongly emphasizes the crucial importance of ensuring an appropriate traceability system from horse donors to human recipients of antivenoms so that any potential infectious events or risks may be identified in a proper and timely manner and relevant counter-measures taken promptly. confidence in the safety of antivenoms must come from evidence that the manufacturing processes used can reproducibly remove or inactivate high potential levels of viruses. comparison with human plasma fractionation suggests that some of the steps of antivenoms include treatments which may inactivate viruses. human plasma fractionation is a complex integrated manufacturing process from which products like albumin, coagulation factors and igg, are obtained. purification processes combine cryoprecipitation, ethanol fractionation, chromatography, and ultrafiltration [ ] . in addition, various types of dedicated viral reduction treatments have been introduced, most often on purpose, such as chemical treatments by solvent-detergent, caprylic acid precipitation, low-ph incubation, heat treatments in the liquid or in the dry states, and nanofiltration [ ] . among those, caprylic acid and low ph treatments, both of which are commonly used also for the purification of antivenom igg, have been shown to contribute to the viral safety of human plasma igg products as described below. some years ago, unsaturated fatty acids were shown experimentally to inactivate more than log of lipidenveloped viruses (vsv, sindbis virus, hiv) in human plasma protein fractions [ ] . however, specific attention has been given to caprylic acid (also called octanoic acid), as it has been used as precipitating agent for human igg. it has been suggested that the non-ionized form of the caprylic acid disrupts the integrity of the lipid bilayer and membrane associated proteins of enveloped viruses. utilizing the dissociation reaction and varying the concentration of the ionized form of caprylate, a specific amount of the nonionized form of caprylate can be maintained over a wide ph range. treatment of plasma protein solution with caprylate at acidic ph also readily inactivates lipid-enveloped viruses like herpes simplex virus type i, vesicular stomatitis virus, vaccinia virus, and sindbis virus [ ] . detailed validation studies of two caprylic acid treatments of human ig have been published recently. the robustness of a treatment applied to three intravenous immunoglobulin preparations (igg, igm-enriched, and igm preparations) has been investigated using hiv, bvdv, sindbis virus and pseudorabies as model viruses [ ] . the routine treatment conditions for these two igg and this igm preparation are indicated in table . kinetics of inactivation was determined over a period of h of treatment. complete inactivation (corresponding to more than . e . log) is achieved within the first minutes. within a certain range, viral inactivation in the igg product was not affected by ph ( . e . ), temperature ( e c), and protein content ( e g/l). above ph , and most specifically at ph , no bvdv inactivation was found. at a content of caprylic acid of . g/kg or less, inactivation of hiv is significantly reduced. under the conditions applied during manufacture, caprylic acid leads to robust inactivation of lipid-enveloped viruses; ph is a particularly critical parameter and should be less than . these conditions have also been found to inactivate o . log eav, an equine virus used as a model (dichtelmu¨ller, personal communication). another study reported the viral inactivation achieved during caprylic acid precipitation of non-igg proteins from human igg product [ ] . at ph . , c, and in the presence of mm caprylate, r . and r . log of hiv and prv, respectively, were inactivated during the h treatment, but only . log for bvdv was inactivated. at mm caprylate, r . log of bvdv was inactivated within this time period. at ph . , c, mm caprylate, and ph . , c, mm caprylate, complete inactivation of bvdv and of hiv and prv was achieved in less than min. the authors also showed that mm caprylate at ph . and c in supernatant iv- , an intermediate in albumin production, inactivated r . log bvdv almost instantaneously [ ] . the virucidal effect of caprylic acid has also been confirmed in human albumin solution, where it is used as a stabilizer for pasteurisation. elevated temperature and low ph were found to be critical parameters to ensuring significant reduction in virus infectivity. rate and extent of inactivation were sensitive to variations in the caprylate to protein ratio and to changes in ph. in the conditions retained, % w/v protein, mm caprylate, ph . and c, more than log inactivation of the lipid-enveloped viruses tested, including bvdv, was achieved [ ] . it should be kept in mind that treatment of whole plasma or crude fractions, as is the case for equine antivenoms production, may lead to lower rate and kinetics of viral inactivation, due to the high endogenous lipid content, as found in a study that evaluated the virucidal effect of sodium oleate [ ] . finally, one should keep in mind that caprylic acid treatment does not inactivate non-enveloped viruses. several human igg preparations are subjected to an acid ph incubation that was historically introduced to reduce immediate adverse reactions subsequent to intravenous injections. in the late s, acid ph treatment was also shown to contribute greatly to the table comparison of conditions for caprylic acid treatment used for human igg preparations ( ) [ , ] . the treatment of human igg generally consists of incubation at ph , with or without traces of pepsin, at temperatures from to c, using a protein content close to g/l, and for more than h. the treatment is intended to eliminate aggregates. pepsin, when present, is maintained at low concentration to avoid cleavage of the igg molecule. several model enveloped viruses were shown to be inactivated under those conditions [ e ]. more than e log inactivation of hiv and other enveloped viruses used as surrogate models, such as semliki forest virus (sfv), hsv, vsv, and cmv is achieved. by contrast, poliovirus, as other non-enveloped virus, seems more resistant. the presence of trace concentrations of pepsin added to reduce anticomplementary activity during this procedure has been shown to contribute little to virus kill of most processes [ ] . virus inactivation by acid ph and pepsin is influenced by several parameters. hiv, bvdv, sfv, and prv are completely inactivated within mine h at c but inactivation rate and extent is less at lower temperatures. increasing the sucrose content from to % reduced the rate of inactivation of prv but not that of sfv. increasing the nacl content from to mm reduced the rate of inactivation of sfv but that of prv was unchanged. an increase in the igg concentration from to % speeds up the inactivation of prv but decreases that of sfv. therefore, temperature is a major parameter in the virucidal efficacy of ph-pepsin treatment and the impact of solute composition is virus-dependent [ ] . some fractionation protocols used in antivenom production include a low ph treatment to induce the precipitation of 'euglobulins', which are plasma globulins unstable under these conditions. this step was shown to remove between . and . log of eiav, sindbis virus, poliovirus and porcine parvovirus [ ] . virus reduction may occur by co-precipitation with the discarded 'euglobulins' rather than by an inactivation process [ ] . pasteurisation is the treatment of a liquid protein fraction for h at c [ ] and is being used in the production of at least one type of equine-derived antivenom [ ] . experience with human plasma products show that liquid heat treatment may inactivate both enveloped and non-enveloped viruses. pasteurisation of albumin solutions is carried out in the final container in the presence of low concentrations of sodium caprylate alone or with n-acetyl tryptophan. inactivation of sindbis and emc model viruses added to % albumin solution was achieved within min treatment [ ] . however, inactivation of some non-enveloped viruses is less [ ] , and the process appears to be virus-specific. various coagulation factors, protease inhibitors, and intravenous igg are pasteurised during the purification process. most often, pasteurisation is performed in the presence of stabilizers, like amino acids, sugars, or citrate [ , ] , to protect protein functionality, and limit molecular alterations and protein aggregation. protein stabilizers are also known to stabilize viruses, however, further highlighting the need for validating the exact conditions of treatment used. pasteurisation can inactivate viruses of different types, enveloped or nonenveloped, including hiv, hbv, hcv, and hav [ , , , ] . there are few published data on the inactivation of resistant model non-enveloped viruses like porcine parvovirus, sv or reovirus type in plasma products [ , ] . in most instances, inactivation of such viruses is limited to less than e log over the h heating period. heat treatment of whole human plasma at c for h, in the presence of a specific combination of stabilizers, inactivates more than log of hiv, and more than log of enveloped and nonenveloped model viruses [ ] . finally, it was demonstrated that heat treatments like pasteurisation and vapour heat treatment (as well as solvent-detergent and nanofiltration on nm membranes) can inactivate/ remove more than log of wnv [ ] . chromatography is primarily used in human plasma fractionation as a downstream polishing step [ ] . ionexchange chromatography has been described for some antivenom products [ , ] . viral partitioning has been shown to occur during affinity and ion-exchange chromatography, but the mechanism of removal is not easy to predict and control [ ] , making it difficult to consider these as robust viral removal steps. storage at ph . , in the presence of a stabilizer, such as % maltose, for a minimum of days at c, yields aggregate-free and in vitro functionally active human igg preparations [ ] . incubating at ph . for days at c caused a , -fold decrease in bvdv infectivity and complete inactivation of chimpanzee infectious doses per millilitre of hcv [ ] . with the exception of a recent report of a fab ovine antivenom formulated at ph . [ ] , to our knowledge, formulation at low ph is not commonly used for antivenoms but may be considered as a viral safety step, if it does not affect the biological activity, the general safety, and the clinical efficacy of the product. viral validation studies are small-scale experiments designed to provide an estimate of the overall viral reduction level achieved across the manufacturing processes and to identify steps and parameters that are critical for viral inactivation or removal. to perform such studies, as described in the guidelines prepared by the cpmp, production intermediates are voluntarily spiked with known amounts of known viruses, using laboratory-scale mimicking of the production process, and the virus reduction factor is calculated by comparing the infectivity level before and after each individual process step evaluated. when appropriate, a cumulative viral reduction factor can be calculated and provides information on the viral safety margin of processes for a range of model viruses. effective and robust individual steps typically achieve more than log reduction in infectivity under a large range of production parameters. viral validation studies are usually conducted with e model viruses differing in their structural features (presence of an envelope, rna or dna genetic material, size, degree of resistance). ''relevant'' viruses, which are known potential contaminant of the starting plasma, should be used when possible. a list of existing laboratory adapted model viruses that could be used for validation studies of antivenoms manufacturing processes is shown in table . vsv and wnv that may contaminate horses appear of special relevance for process validation of equine products. dedicated viral inactivation and removal production steps should be implemented in a way to ensure the reproducibility in viral reduction and the absence of risks of downstream contamination. examples of approaches used for the manufacture of human plasma products are provided in recent who guidelines [ ] , and a cpmp note for guidance provides recommendation on production of immunsera [ ] . both can be used as reference by manufacturers of antivenoms. viral reduction equipment should have adapted specifications. for instance, equipment for bulk in process virus inactivation, such as the acid ph incubation or the caprylic acid treatment, should ideally be fully enclosed. in addition, vessels should have temperature controls and be designed to avoid ''dead points'' where important parameters such as temperature and ph could not be ensured. similarly, liquid heat treatment or heat shock should be conducted in jacketed tanks with the solution stirred throughout the heat cycle to ensure homogeneity and all points in the tank should be within the specified temperature range. during the qualification phases, the equipment and the required services should be shown to conform to the predefined technical specifications and requirements, and to function within the specified limits. when possible, it is recommended to create a dedicated ''viral safety area'' where production steps are arranged in a clear and logical way, so that recontamination of the virally reduced intermediates is avoided. operating procedures should also be written to reduce the likelihood of crosscontamination. bulk ph or caprylic acid virus inactivation could be carried out in two stages, the first stage being located in a normal production room, followed by a second incubation in another tank located in a segregated, contained area. the duration of the first stage should be such that the majority of virus inactivation (as found during the viral validation studies) has occurred. on completion of the first stage, the product should be aseptically transferred (e.g. through sterile coupling) into the second vessel located in a safety zone for completion of the second stage of viral inactivation. product-contacting-equipment used in the safety area should be dedicated or decontaminated to inactivate any remaining virus. a quality assurance system should ensure that the execution of virus reduction methods conforms to the validated conditions. viral inactivation and removal procedures should be described in approved standard operating procedures (sops) that contain critical process limits for the viral inactivation and removal methods applied to antivenoms. to date there is no documented example of viral transmission to humans from the use of antivenoms, whichever type of product (whole igg, f(ab#) or fab) is manufactured [ ] . considering the difficulty in the containment of large animals like horses, and in an exhaustive viral testing of the starting plasma, the viral safety of antivenoms appears therefore largely dependent upon the capacity of manufacturing processes to reduce infectious agents and on the use of good manufacturing practices, in particular in ensuring production traceability. by contrast to human plasma products, most antivenoms are currently not subjected to purposely introduced viral reduction steps like solvent-detergent, pasteurisation, or nanofiltration [ , , ] . however, a comparison with validated manufacturing processes used for human igg clearly indicates that at least two widely used antivenom production steps, caprylic acid treatment and low-ph incubation, are likely to contribute in a robust manner to viral safety, at least against enveloped viruses. the concentration of caprylic acid ( %) used for antivenoms is higher than that used for human igg (table ) , and the ph of treatment is within the effective range (less than ph ) required for optimal inactivation of enveloped viruses. as the treatment is performed on equine plasma itself, or on crude fractions, the viral kill could be reduced by the presence of lipids. the acid incubation of antivenoms which is performed at a ph ( . e . ), less than that used for human igg, and at a similarly warm temperature ( e c) ( table ) should provide a fast rate of inactivation of lipidenveloped viruses, and possibly also non-enveloped viruses. however, the incubation time ( mine h) is for some processes significantly less than for human igg (typically e h), probably reducing the extent of viral kill achieved. preliminary viral kill data obtained with antivenoms confirm that conditions used for peptic cleavage at low ph of f(ab#) achieve robust inactivation of wnv [ ] . we recommend that further representative viral validation studies using a range of well-established virus models are performed to know the rate and extent of viral kill actually achieved during those production steps. the viruses to use for those validations should be discussed with specialized virologists, and, by analogy with most studies done with human plasma derivatives, might include three lipidenveloped viruses (such as bvdv, vsv, psr, and wnv) and one non-enveloped virus (such as emc, poliovirus, or reovirus ). inactivation data could then be compared to viral kills achieved using similar process steps of human igg products, therefore providing some reference on their robustness and allowing manufacturers and national regulatory authorities to assess scientifically the margin of safety of antivenom products. other manufacturing steps, such as heat coagulation, could be validated as well. formulation of the product at low ph, as done for some human igg preparations, is another potential approach to consider for improved viral safety, but evaluating product stability and efficacy under such conditions would be a pre-requisite. in view of their expected beneficial impact on viral safety, appropriate process implementation of caprylic acid treatments and low-ph incubations should be ensured, as well as measures taken to avoid risks of downstream contamination and, more widely, to respect good manufacturing practices and traceability concepts. such conclusions should also be valid for other animal plasma-derived igg products used in human medicine such as anti-lymphocyte/t-cell, antitoxins, and anti-bacterial or viral agents sera [ ] . snake antivenoms marine antivenoms venomous bites and stings a new pan african polyspecific antivenom developed in response to the antivenom crisis in africa report of a who workshop on the standardization and control of antivenoms venoms, antivenoms and immunotherapy 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within the family paramyxoviridae newly discovered viruses of flying foxes transmission studies of hendra virus (equine morbillivirus) in fruit bats, horses and cats hendra and nipah virus infections the australian paralysis tick may be the missing link in the transmission of hendra virus from bats to horses to humans infection of humans and horses by a newly described morbillivirus hendra virus disease in horses hendra (equine morbillivirus) development of a fluorogenic rt-pcr assay (taqman) for the detection of hendra virus japanese encephalitis in horses in japan japanese b encephalitis in a horse identification and phylogenetic comparison of salem virus, a novel paramyxovirus of horses isolation of salem virus, a novel equine paramyxovirus, and assessment of its etiologic role in a disease outbreak vesicular stomatitis and other vesicular, erosive, and ulcerative diseases of horses development of an immunoglobulin m (igm) capture enzyme-linked immunosorbent assay for detection of equine and swine igm antibodies to vesicular stomatitis virus west nile virus in livestock and wildlife experimental infection of horses with west nile virus west nile virus encephalomyelitis in horses: cases equine rhinitis a virus: structural proteins and immune response equine rhinitis b virus: a new serotype the classification of seven serotypes of equine encephalosis virus and the prevalence of homologous antibody in horses in south africa nested reverse transcriptase-polymerase chain reaction for the detection of group a rotaviruses the action of proteolytic enzymes on the antitoxins and proteins in immune sera. i. true digestion of the proteins the action of proteolytic enzymes on the antitoxins and proteins in immune sera. ii. heat denaturation after partial enzyme action the purification of antitoxic plasmas by enzyme treatment and heat denaturation antivenins in brazil: preparation antivenin production in india preparation of improved f(ab#) antivenoms. an example: new 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virus validation of ph -treated human immunoglobulin products produced by the cohn fractionation process virus inactivation by pepsin treatment at ph of igg solutions: factors affecting the rate of virus inactivation virus inactivation during intravenous immunoglobulin production viral safety of intravenous immunoglobulins g for therapeutic use strategies to produce virus-safe blood derivatives inactivation of viruses in labile blood derivatives. ii. physical methods pasteurization as an efficient method to inactivate blood borne viruses in factor viii concentrates inactivation of hiv, hbv, hcv related viruses and other viruses in human plasma derivatives by pasteurisation a pasteurized therapeutic plasma pasteurization of antihemophilic factor and model virus inactivation studies virucidal heattreatment of single plasma units: a potential approach for developing countries nile virus and the safety of plasma derivatives: verification of high safety margins, and the validity of predictions based on model virus data affinity chromatography in the industrial purification of plasma proteins for therapeutic use chromatographic removal of viruses from plasma derivatives a comparative in vitro study of low ph and enzyme treated immunoglobulin preparation for intravenous use inactivation of hepatitis c virus in low ph intravenous immunoglobulin formulation of a liquid ovine fab-based antivenom for the treatment of envenomation by the nigerian carpet viper (echis ocellatus) world health organization expert committee of biological standardization. guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma products. www.who.int/biologicals-quality% assur-ance% and% safety nanofiltration of plasma-derived biopharmaceutical products inactivation of west-nile virus during peptic cleavage of horse plasma igg we express our sincere thanks to dr. herbert dichtelmu¨ller for his review of the manuscript and expert suggestions. key: cord- -vob bu c authors: tam, theresa w.s; sciberras, jillian e; tamblyn, susan e; king, arlene; robert, yves title: the canadian pandemic influenza plan: an evolution to the approach for national communicable disease emergencies date: - - journal: international congress series doi: . /j.ics. . . sha: doc_id: cord_uid: vob bu c abstract advance planning for a large-scale and widespread health emergency is required to optimize health care delivery during an influenza pandemic. the canadian pandemic influenza plan (cpip) is an example of a successful communicable disease emergency plan that ensures a national, coordinated approach to preparedness, response and recovery activities in the event of an influenza pandemic. the general concepts incorporated into the cpip may be utilised in the contingency planning for a bioterrorism event or other communicable disease emergencies, including: a national, coordinated approach in planning; an emergency management structure to conduct the response; the use of common terminology to facilitate communication and response coordination, and the establishment of specific technical, communications and operational response groups and networks in advance. the multinational outbreak of severe acute respiratory syndrome (sars) in offered the opportunity for the testing of these concepts. the experiences and lessons learnt during the sars response may be utilised to strengthen communicable disease preparedness and response capacity. a future global epidemic or pandemic of influenza is highly likely if not inevitable, but is unpredictable in timing. when a novel influenza virus that is capable of efficient humanto-human transmission and causing high morbidity and mortality emerges, countries will have very little time to carry out the many key activities required to minimise the health, societal and economic impact of a pandemic. with the increasing recognition of the threat posed by the next influenza pandemic internationally, there is an accelerated effort by the world health organization (who) and many countries to develop or strengthen existing pandemic preparedness plans. [ , ] the world is still inadequately prepared for the next pandemic, however, ongoing efforts in preparedness activities and testing of the pandemic plans will continue to strengthen our response networks, processes and infrastructures. at the inception of the canadian pandemic influenza plan (cpip) in , the main focus was on a vaccine strategy. after the hong kong influenza a/h n incident in , the pandemic plan evolved to include a more comprehensive approach, incorporating the following key components: surveillance, vaccine programs, and use of antivirals, health services, emergency services, public health measures and communications. one of the main challenges of pandemic preparedness was to establish the essential close linkages between public health, health care and emergency response sectors. after the terrorist attacks of september , and the subsequent anthrax attacks in the united states, national authorities became acutely aware of the need to strengthen public health infrastructure to respond to health emergencies. in addition to the national pandemic influenza committee (pic), which provides technical advice on influenza pandemics, a national network for health emergency communications and a national forum which integrates public health and emergency measures were formed. the cpip evolved to include three main sections: ( ) preparedness, ( ) response and ( ) recovery, to be consistent with the general principles of emergency response and with the phased approach of the world health organization (who) framework. the general concepts incorporated into the cpip that may be utilised in the contingency planning for other infectious disease emergencies include: a national, coordinated approach to planning; an emergency management structure to coordinate and conduct the response; the need for common terminology (e.g. using the same response phases), and the need to have specific technical, communications and operational response groups and networks formed in advance. the recent national response to an influenza a/h n alert and a multinational outbreak of severe acute respiratory syndrome (sars) offered the opportunity for the testing of these concepts. on february , when the who confirmed a report of two cases of influenza a(h n ) associated with one death in the hong kong special administrative region, health canada activated the pandemic influenza committee and alerted the national surveillance system, ''fluwatch.'' the newly formed national health emergency communication network was also activated and fact sheets on influenza h n were developed. within hours after the first who global alert for atypical pneumonia on march , , health canada received reports of the first cases of sars in canada from provinces that were already on the alert for h n . the pandemic influenza response structures and processes were rapidly and successfully translated to respond to sars. pic was expanded to form the national sars technical advisory group. existing national technical groups (e.g. the canadian public health laboratory network) and pandemic influenza working groups were transformed into sars working groups, with links to the who sars laboratory, clinical and epidemiology networks. although the public health measures for limiting the impact of an influenza pandemic may be different from that for the containment of sars, we are now able to learn from the sars experience the types of measures that have been effective, feasible and acceptable to the public and health care providers. in canada, sars highlighted the deficiencies in the public health infrastructure, policies, procedures and legislation to support urgent public health action. sars reminded us of the need for a clear command structure with dedicated team leadership during a health emergency. health organizations are generally not set up with the command and control structures necessary to respond effectively to a large-scale emergency. jurisdictions that did not have well-developed pandemic influenza plans had to create structures immediately to deal with sars. the province of ontario, with the most involvement with sars, was not able to participate on an ongoing basis in national level technical discussions as all of its experts were contributing to the front line response. a national, technical planning group with members who are able to dedicate their time would be important in future responses. although federal, provincial and territorial public health departments are able to communicate alerts for emerging infectious diseases very effectively through telephone or e-mail systems, there is a need to enhance the delivery of information in a timely manner to front-line health and emergency workers. real time data sharing between hospitals and public health and between different levels of the public health system was a particular challenge, as was timely epidemiological analysis. the development of integrated mechanisms and processes for hospital-and community-based surveillance is required, including the strengthening of hospital surveillance capacity. pre-established data elements and data sharing agreements between local, national and international governments/organisations for emerging infectious diseases will facilitate the determination of key epidemiological parameters at the time of the emergency. the national case definitions designed for monitoring the spread and impact of sars were utilised inappropriately in some situations, e.g. for clinical management. although public health and infection control measures were implemented immediately and effectively, the classification and reporting of cases were delayed as retrospective epidemiological linkage of cases were made. case definitions should be developed and the rationale for any changes to case definitions should be clearly documented. there should be a consistent use of national case definitions and we should strive for international consistency where possible to facilitate international surveillance and communications. there was an unprecedented rapid generation of knowledge on sars through the existing influenza networks and new partnerships. the public health and research laboratories within canada made an immediate and valuable contribution to the international effort of elucidating the etiology of sars, the genetic sequencing of the sars-associated corona virus and the development of new diagnostic tests. however, especially at the provincial laboratory level, the inability to limit testing and to link laboratory data with epidemiological and clinical data resulted in over-testing and the inability to prioritise specimens. the public health measures instituted during sars were effective in containing the epidemic; however, the effectiveness of specific strategies is still being assessed. quarantine and isolation were generally acceptable to the public. cancellation of public gatherings will happen regardless of public health recommendations. in order to implement communitybased public health measures and institution-based infection control measures, the involvement of multiple partners is needed, including key health professional groups, non-governmental organizations, the media, businesses and schools. issues pertaining to international borders, such as travel advice, screening, quarantine and embargo of supplies, arise quickly and must be an integral part of emergency plans. blood safety and supply issues will also arise quickly at the start of any emerging infectious disease emergency. all jurisdictions need to strengthen human resource planning and surge capacity in their health emergency plans. there is a need to enhance our ability to mobilise resources across the country for outbreak investigation and control and to support health care services, including infection control. the sars response highlighted the need for more trained infection control personnel. the focus of hospital infection control resources on the containment of sars may have negative impacts, such as an increase in other nosocomial infections and failure to recognise or report other infections, e.g. tuberculosis. ongoing training of health care workers on the correct method of putting on and removing personal protective equipment is required before and during an epidemic. the negative impact of stringent infection control and other hospital policies on the wellbeing of health care workers, as well as the impact of work exhaustion and post-traumatic stress on responders, must be addressed in emergency plans. since sars was primarily a nosocomial disease in canada, managing hospital triage and patient transfer was essential to the local response. the experience gained in patient triage and in setting up dedicated clinics and other alternate care sites (e.g. screening units established outside hospitals) during the sars response is valuable for pandemic influenza preparedness. the feasibility and effectiveness of using specific hospitals or units within hospitals dedicated to the assessment and management of an emerging infectious disease should be assessed. establishing the security of medical and other supplies, e.g. through stockpiling and multiple suppliers, should be an integral part of logistics planning for health emergencies. preparedness for an influenza pandemic in canada currently includes the security of vaccine supplies and options for stockpiling antiviral drugs. communicating the progress and impact of an epidemic in real time to decision makers and the public is very challenging. considerable resources are required to translate scientific data (especially epidemiological data) into public information. in canada, information on health events is communicated openly and rapidly to the public, experts and health care providers to the media; therefore, public health spokespersons must be proactive in providing the correct information expediently to the public. the first phases of the canadian pandemic influenza plan have been tested and applied to another emerging infectious disease emergency. the plan requires ongoing review and evaluation through emergency exercises and the incorporation of lessons learnt from sars. common approaches to communicable disease emergency plans, with an emphasis on the strengthening of public health infrastructure and infection control in health care facilities, will be the key to future response to emerging infections. world health organization, global agenda on influenza surveillance and control influenza pandemic planning key: cord- -dt m i authors: hellen, christopher u.t.; wimmer, eckard title: viral proteases as targets for chemotherapeutic intervention date: - - journal: curr opin biotechnol doi: . / - ( ) -g sha: doc_id: cord_uid: dt m i many viruses encode proteinases that are essential for infectivity, and are consequently attractive chemotherapeutic targets. the biochemistry and structure of the human immunodeficiency virus proteinase have been characterized extensively, and potent peptide-mimetic inhibitors have been developed. techniques and strategies used to improve the efficiency of these compounds are likely to be applicable to other viral proteinases. many human and animal viruses encode proteinases that play important roles at different stages in the infection cycle, including separation of functionally different domains from a precursor polyprotein (enabling cleavage products to be transported to different cellular compartments) and regulation of a variety of events in viral replication, such as uncoating, activation of replicative enzymes and morphogenesis [ ] . these proteinases are essential for virus infectivity, and have therefore come to be seen as attractive targets for chemotherapeutic intervention, particularly because they have unusual cleavage specificities that differ from those of host proteases. proteinases are encoded by all retroviruses, including hiv and human t-cell leukemia virus and have been identified in a growing number of dna and positivesense rna viruses. these include adenoviruses and herpesviruses [ ", "] as well as picornaviruses, flaviviruses (such as dengue and yellow fever viruses), pestiviruses and the related hepatitis c virus. a number of these viruses cause diseases of medical or veterinary importance that are not amenable to conventional preventive or prophylactic measures, and proteinase inhibition therefore represents a valid alternative therapeutic approach. many viral proteinases have only been identified recently, and characterization is consequently in its early stages. to illustrate potential strategies in the analysis of viral proteinases, and in the design and development of inhibitors we shall therefore focus on the picornavirus and retrovirus proteinases, as they have elicited the greatest academic and industrial interest, and as a result, have been characterized in detail. hiv- has the genetic organization '-gag-pol-env- ' that is typical of retroviruses. the gag and pol genes encode inner structural, and replicative proteins respectively, and are translated as polyproteins that are cleaved at eight sites by the proteinase (pr) ( fig. and table ). these polyproteins are transported to the plasma membrane and cleavage occurs after budding of immature particles, resulting in morphological changes associated with virion maturation. the substrate specificity of hiv- pr is puzzling in that pr catalyzes specific cleavage at a small number of polyprotein sites that show no apparent sequence conservation. analysis of viral and non-viral substrates suggests that no subsite has absolute specificity, and that a combination of moderate interactions may be sufficient to confer catalytic specificity [ "]. heterogeneity in the composition of viral polyprotein cleavage sites probably plays a role in determining the rate, and consequently the order, of cleavage at different sites. a proteinase-deficient hiv mutant produced noninfectious immature virions containing unprocessed polyprotein [ ] , an observation that is crucial to the consideration of pr as a therapeutic target. hiv- pr is a c symmetric homodimeric aspartyl proteinase that consists of two identical -amino-acid subunits. their termini interdigitate at the dimer interface, but otherwise the topology of hiv- pr is similar to that of pepsin-like aspartyl proteinases [ "']. techniques for the large scale purification of recombinant hiv- pr, and for the routine assay of its proteolytic activity are fundamental prerequisites for the development of inhibitors, and various methods have been reported [ "']. the successful design of substratebased inhibitors of other aspartyl proteinases, such as pepsin and renin, suggested the strategy of designing analogous peptide-mimetic inhibitors of hiv- pr by incorporating non-hydrolyzable 'transition-state' mimics into substrate analogues [ "', ", -]. this approach is based on a key step in aspartyl proteinase catalysis: generation of a tetrahedral diol by hydration of a trigonal amide (fig. a) . the high (millimolar) k m values of peptide substrates indicate that potent (i.e. nanomolar) pr inhibitors must incorporate structural features that significantly increase their binding affinity. peptide-based inhibitors have several disadvantages, including vulnerability to degradative enzymes, rapid clearance, and poor oral absorption. these problems are commonly addressed by minimizing size and peptide-like character of promising lead compounds. the first reported pr inhibitor was pepstatin [ ], a diagnostic inhibitor of aspartyl proteinases. this weak inhibitor contains two statine residues that embody transition state analogue i (fig. b ). to identify more potent inhibitors, dreyer et al. [ ] compared the effectiveness as pr inhibitors of five different classes of dipeptide isosteres inserted into a consensus heptapeptide template. heptapeptides (p -p ' or p -p ') are the shortest substrates that are cleaved efficiently by pr. statine-based (i), reduced amide (ii) and phosphinate (iii) transition state analogues exhibited modest potency, but placement of phe-gly hydroxyethylene dipeptide isosteres (iv) into the consensus template yielded compounds that inhibited hiv pr at nanomolar concentrations in vitro and prevented polyprotein processing, virion maturation and viral spread at - btm in cell culture. truncation and extensive structure-activity analysis at the p , pi" and p ' positions led to the identification of highly potent (subnanomolar) pr inhibitors based on dihydroxyethylene (v) [ -] and hydroxyethylene (iv) [ "] isostere transition state analogues. potency was enhanced by incorporating residues that stabilize the extended inhibitor structure, presumably due to optimized hydrogenbonding in the substrate binding cleft. for example, the p ' and p ' residues (leu-phe) can effectively be replaced by various substituted aminobenzocycloalkanes [ " ]. the pi' position can accept side-chains unrelated to natural amino acids, allowing modifications to be made that enhance solubility and thus cell penetration [ ", -] . such substitutions may reduce binding affinity (k i) but the enhanced solubility may nevertheless result in a net increase in antiviral activity. the ability to cleave the amino terminus to proline distinguishes hiv pr from non-viral aspartyl proteinases. hydroxyethylamine (vi) structures that readily accomodate the prolyl imino acid have been incorporated into a number of potent inhibitors [ , ", , " ]. modification of a protected tripeptide incorporating this structure by substituting the imino residue decahydroisoquinoline at the pi' position yielded highly potent inhibitors of pr in vitro and in cell culture, such as the compound ro - [ , " ]. this inhibitor was expected to have considerable selectivity, and indeed it inhibited human aspartyl proteinases such as gastricsin, renin and pepsin by less than % at a concentration of pm. typical ic values for ro - ( - nm) are -fold below its cytotoxic concentration in uninfected host cells. recent reports indicate that a mg oral dose every hours is sufficient to maintain the mean human plasma concentration at about nm. the potency of ro - is strongly dependent on tight binding by the p and particularly the p substituents, whereas binding of a second class of hydroxyethylamine inhibitors (which contain a noncyclic, secondary amine in place of the decahydroisoquinoline residue) [ " ] is less dependent on these interactions, and this second class binds more tightly in the pi"-p ' region. peptide substrates are inherently asymmetric and the pr dimer must therefore lose its perfect c symmetry during catalysis. symmetry is permissible for inhibitors, however, and might even improve binding affinity and selectivity over endogenous aspartyl proteinases. these considerations have led to the design of a series-of diaminoalcohol-and diaminodiolbased inhibitors with c (vii) or pseudo-c symmetry [ , "- " ]. these inhibitors are potent even at subnanomolar concentrations and highly selective in vitro, but most have suffered from poor solubility, leading to modest potency in cell culture. strategies to circumvent this deficiency and thus enhance activity in cell culture have included modification of terminal residues and their linkage groups to increase solubility. these interactions include extensive van der waal's contacts with residues that define the hydrophobic - ' binding pockets, and a hydrogen bonding system that sandwiches the inhibitor strand between the catalytic cleft and the flaps. the hydroxyl groups of type v, vi and vii inhibitors form hydrogen bonds with both catalytic aspartates. significantly, all complexes contain a tetrahedrally coordinated active-site water molecule, which bridges two flap residues and two inhibitor carbonyl groups, prompting suggestions that an improved inhibitor would contain a functional replacement for the water [ ", ] . the similarities in the extended conformation of all inhibitors, as well as in the induced conformational changes that they cause in the enzyme, indicate that it is possible to model and improve peptidic inhibitors on the basis of these known structures. an alternative approach to discover novel templates for the design of non-peptide inhibitors is to search three-dimensional structure databases for molecules with a shape that is complementary to the active-site cleft. to date, this approach has led to identification of the antipsychotic agent haloperidol as a weak pr inhibitor [ ] . the picornaviridae are a family of small icosahedral viruses that includes the etiological agents of several important human and animal diseases. it consists of five genera, including rhinovirus (the common cold virus) and enterovirus (e.g. poliovirus and hepatitis a virus). picornaviruses have a positive-sense monopartite rna genome that encodes a single large polyprotein. it is processed by three different proteolytic activities which can each be regarded as serving a distinct function ( fig. and table ) [ " ]. the initial event in this cascade is cleavage by ap r° at its own amino terminus, separating the p~ structural protein precursor from the nascent polyprotein. secondly, functional proteins are released from the p and p -p (non-structural) protein precursors by cp r° or its precursors. finally, maturation cleavage of the vp capsid protein occurs on encapsidation of viral rna to yield infectious virus particles. in addition to their role in viral replication, the a and c proteinases of poliovirus (and by implication, of other picornaviruses) are responsible for aspects of the dramatic inhibition of host cell rna and protein synthesis that occurs on infection. the a proteinase is involved in degradation of the eukaryotic initiation factor elf~fy, which is correlated with shut-off of cap-dependent translation [ ] , and cp ro inactivates transcription factor iiic, inhibiting polymerase iii transcription [ " ]. sequence alignment and inhibitor studies suggested that both a and c proteinases are related structurally to trypsin-like serine proteinases, with the no- at all eight sites within the polyprotein (fig. ) . sites in other picornaviruses are slightly more heterogenous. poliovirus apro cleaves tyr-gly dipeptides at the p - a junction and within the three-dimensional polymerase, but although all corresponding sites in other picornaviruses have a gly residue at the pi' position, various residues occur at the p position. aliphatic residues occur at the p positions of most ap r° and cp r° sites. mutagenesis and peptide cleavage experiments indicate that cleavage site recognition depends on a minimum substrate length (six residues for cp r°) and the presence of specific residues at positions that differ according to both the virus and the proteinase [ , ", ", "- " ]. there are additional conformational determinants of recognition of cleavage sites within polyproteins, so the large (millimolar) k m values of peptide substrates may reflect their greater conformational freedom. potential peptidemimetic in- hibitors are likely to exhibit similar flexibility, and must therefore be conformationally constrained and incorporate structural features that increase their binding affinity. the lack of absolute specificity at most subsites, and the requirement for peptide substrates to extend to the p position indicates that the substrate binding clefts of cp r° and probably ap r° are capable of extensive hydrogen bond interactions with such inhibitors. however, only a few inhibitors of cp ro have been reported [ ", ] . proteases are encoded by several dna viruses and numerous rna viruses in addition to the picornaviruses and retrovimses discussed above. although they are all potential targets for chemotherapeutic intervention, significant progress in inhibitor development has only been reported for hiv- pr. in the few years since its identification, the structure of pr and numerous inhibitor complexes have been determined, and highly potent peptidemimetic inhibitors have been developed. knowledge of the strategies used in enhancing the potency and specificity of pr inhibitors, and in overcoming the inherent limitations of peptide-based inhibitors is likely to prove invaluable in the development of peptidemimetic inhibitors of other viral proteinases. heinrikson rl: the complexities of aids: an assessment of the hiv protease as a therapeutic target. chem today , : - . an account of the recent progress in substrate-based inhibitor design is complemented by a concise summary of the structure of hiv pr and a clear exposition of its application to structure-based inhibitor design. norbeck : - . the first of" series of disclosures [i "- ", "'] from a group at merck illustrating strategies to selectively modify termini and side-chain residues to increase potency, reduce size and peptidic character, and to increase solubility (and hence antiviral activity) of hydroxyethylene isostere-based inhibitors. this paper describes the effect of modification of the pi', p ' and p ' residues. benzocycloalkyl amines as novel c-termini for hiv- protease inhibitors hiv- protease inhibitors based on hydroxyethylene dipeptide isosteres: an investigation into the role of the pi' side chain on structure-activity systematic modification of the p]' residue of the lead inhibitors described in [ ", "] established that side chains unrelated to natural amino acids are tolerated at this position, permitting substitutions that increase solubility and cell penetration synthesis and antiviral activity of a series of hiv- protease inhibitors with functionality tethered to the p or pi' substituents: x-ray crystal structure assisted design computer-assisted molecular modelling was used to design derivatives of the lead inhibitor l- , [ "] with increased cell penetration and antiviral potency. an x-ray crystal structure of the hydroxyethylamine analogues of the p /p substrate cleavage site are tight-binding inhibitors of the hiv protease effect of hydroxyl group configuration in hydroxyethylamine dipeptide isosteres on hiv protease inhibition. evidence for multiple binding modes in a series of hydroxyethylamine isostere inhibitors, the preferred diastereomeric configuration of an essential hydroxyl group depended on both the length and nature of the peptide framework. this hydroxyl group is hydrogen bonded to the two catalytic asp residues rational design of peptide-based hiv proteinase inhibitors novel binding mode of highly potent hiv-proteinase inhibitors incorporating the (r)-hydroxyethylamine isostere binding of the hydroxyethylamine inhibitor ro - described in na: effects of a specific inhibitor of hiv proteinase (ro - ) on virus maturation in a chronically infected promonocytic cell line (u ) antiviral activity of the potent hiv inhibitor ro - is sufficient to inhibit acute and chronic infections. the low toxicity of this compound renders it a highly promising antiviral agent in aids chemotherapy a series of potent hiv- protease inhibitors containing a hydroxyethyl secondary amine transition state isostere: synthesis, enzyme inhibition, and antiviral activity a novel subclass of potent hydroxyethylamine inhibitors containing a secondary amine isostere in place of the cyclic amine of ro - show differences in structure-activity relationships and in binding mode structure-based c a symmetric inhibitors of tiiv protease design, activity and . a struc-lure of a c symmetric inhibitor complexed to hlv- design of c symmetric inhibitors exploiting the perfect symmetry of hiv pr revealed by x-ray crystallography x-ray crystal structure of the hiv protease complex with l- , , an inhibitor with pseudo-c symmetry an illustration of the application of x-ray crystallography to rational drug design, which in this instance revealed unoptimized hydrogen bonding and several water-mediated pr-inhibitor interactions antiviral and pharmokinetic properties of c symmetric inhibitors of the human immunodeficiency virus type protease this paper illustrates the difficulties in reconciling the competing demands on pr inhibitors for tight hydrophobic interactions with pr subsites, and aqueous solubility required for bioavailability and in vivo efficacy x-ray crystallographic structure of a complex between a synthetic protease of human immunodeficiency virus and a substrate-based hydroxyethyalmine inhibitor structure at . a resolution of chemically synthesized human immunodeficiency virus type protease complexed with a hydroxyethylene-based inhibitor structure-based design of nonpeptide inhibitors specific for the htmmal immunodeficiency virus maturation of poliovirus capsid proteins a concise review of the role of three distinct proteolytic activities in the release of capsid proteins from the poliovims polyprotein and their subsequent assembly into virions poliovirus protease a induces cleavage of eulkaryotic initiation factor f polypeptide p poliovirus proteinase c converts an active form of transcription factor hic to an inactive form: a mechanism for inhibition of host cell polymerase ill transcription by poliovirus the severe inhibition of rna polymerase iii-mediated transcription in polio-infected cells is a result of inactivation of transcription factor iiic by the proteolytic activity of cp r° site-directed mutagenesis of the putative catalytic triad of poliovirus c proteinase site-directed mutagenesis experiments suggest that the catalytic triad of polio cp ro (his , glu , cys ) structurally resembles trypsinlike serine proteinases but differs significantly in its constituent residues analysis of putative active site residues of the poliovirus c protease experiments designed to evaluate two conflicting structural models of polio cp ro suggest that glu is a constituent residue of the catalytic triad whereas asp is involved in polyprotein substrate recognition characterization of poliovirus a proteinase by mutational analysis: residues required for autocatalytic activity are essential for induction of cleavage of eukaryotic initiation factor p polio apro containing a cysl ser substitution within the putative his , asp , cysl catalytic triad retains significant autocatalytic activity expression and characterization of recombinant hepatitis a virus c protease a colorimetric assay was used to characterize cleavage by purified hepatitis a virus cp to putative papainrelated thiol proteases of positive-strand rna viruses. identification of rubi-and aphthovirus proteases and delineation of a novel conserved domain associated with proteases of rubi, c~-and coronaviruses computer-assisted analysis was used to identify the first viral proteirlases that are distantly related to papain-like thiol proteinases role for the p amino acid residue in substrate utilization by the pouovirus cd proteinase ba: a rapid method for determination of endoproteinase substrate specificity: specificity of the c proteinase from hepatitis a virus a novel and rapid technique was used to demonstrate that hepatitis a vires cp ro has a strong preference for smali residues (ala, ser, gly) at the pl' position, but has little specificity at p '. . weidner jr, dunn bm: development of synthetic peptide substrates for the poliovirus c proteinase a high performance liquid chromatography assay reveals that polio cp ro has a strong preference for a proline p ' residue, and a continuous fluorescence assay is reported determinants of substrate recognition by poliovirus a proteinase positions are strict determinants of substrate recognition by polio apro, but p ', pi' and p positions are broadly tolerant of substitution. substrate requirements for cleavage in trans are more stringent than for cleavage in cis hepatitis a virus c proteinase substrate specificity hepatitis a virus cp ro has strong preferences for residues at p and pa positions, and differs in specificity from enteroviral c proteinases structure and stereochemistry of thysananone: a novel human rhinovirus c protease inhibitor from thyanophora penicilloides. telrahedron lett a novel non-peptide (naphthoquinone) inhibitor of rhinovirus c proteinase spiro lndoline beta-lactams, inhibitors of poliovirus and rhinovirus c-proteinases key: cord- -rl yum n authors: wallinga, jacco; teunis, peter title: different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures date: - - journal: am j epidemiol doi: . /aje/kwh sha: doc_id: cord_uid: rl yum n severe acute respiratory syndrome (sars) has been the first severe contagious disease to emerge in the st century. the available epidemic curves for sars show marked differences between the affected regions with respect to the total number of cases and epidemic duration, even for those regions in which outbreaks started almost simultaneously and similar control measures were implemented at the same time. the authors developed a likelihood-based estimation procedure that infers the temporal pattern of effective reproduction numbers from an observed epidemic curve. precise estimates for the effective reproduction numbers were obtained by applying this estimation procedure to available data for sars outbreaks that occurred in hong kong, vietnam, singapore, and canada in . the effective reproduction numbers revealed that epidemics in the various affected regions were characterized by markedly similar disease transmission potentials and similar levels of effectiveness of control measures. in controlling sars outbreaks, timely alerts have been essential: delaying the institution of control measures by week would have nearly tripled the epidemic size and would have increased the expected epidemic duration by weeks. the etiologic agent of the disease, a coronavirus, was identified on april , ( ) . estimates of important epidemiologic measures such as the case-fatality rate and the incubation period were reported in may ( ) ( ) ( ) . despite the rapid progression of understanding of the new disease, differences between the epidemic curves (numbers of probable sars cases by date of symptom onset) have been awaiting further explanation. for the affected regions, these epidemic curves have been publicly available from the end of march onwards ( ) , and they reveal distinct temporal patterns in numbers of sars cases. this is especially remarkable for the epidemics in hong kong, vietnam, singapore, and canada, since these epidemics started almost simultaneously in late february and similar control measures were put in place at almost the same time in march ( figure , parts a-d) . the question arises as to whether the affected regions differed in terms of transmission potential for sars or effectiveness of control measures. in this paper, we interpret the observed epidemic curves with regard to disease transmission potential and effectiveness of control measures, and we compare the epidemiologic profiles of sars outbreaks in hong kong, vietnam, singapore, and canada. the epidemic curve is the number of reported cases by date of symptom onset. for each reported case i, we denote the symptom onset date by t i . for the sars epidemics in hong kong, vietnam, and singapore, we derived the dates of symptom onset from epidemic curves provided by the world health organization ( ) . for canada, we used the epidemic curve provided by health canada ( ) . the generation interval, denoted by τ, is the time from symptom onset in a primary case to symptom onset in a secondary case. sometimes this generation interval is called the serial interval ( ) or generation time ( ) . the generation intervals observed during the sars outbreak in singapore are well described by a weibull distribution with a shape parameter α and a scale parameter β, with values corresponding to a mean generation interval of . days and a standard deviation of . days ( ) . we denote this distribution by τ ∼ w(τ|α,β). the key epidemiologic variable that characterizes the transmission potential of a disease is the basic reproduction number, r , which is defined as the expected number of secondary cases produced by a typical primary case in an entirely susceptible population ( ) ( ) ( ) ( ) . when infection is spreading through a population, it is often more convenient to work with the effective reproduction number, r, which is defined as the actual average number of secondary cases per primary case. the value of r is typically smaller than the value of r , and it reflects the impact of control measures and depletion of susceptible persons during the epidemic. if r exceeds , the number of cases will inevitably increase over time, and a large epidemic is possible. to stop an epidemic, r needs to be persistently reduced to a level below . estimation of r is a simple affair if there is information about who infected whom. then it is possible to construct an infection network ( ) , wherein cases are connected if one person infected the other. estimation of r involves simply counting the number of secondary infections per case. often, estimation of the effective reproduction number is a complicated affair, because only the epidemic curve is observed and there is no information about who infected whom. recent analyses of closely monitored epidemics have shown that it is possible to estimate the probability that one person has infected another if the spatial locations of the infected persons are available ( , ) . however, when only times of symptom onset are available, most investigators resort to approximating r by assuming an exponential increase in the number of cases over time ( , ) or by fitting a specific model that summarizes assumptions about the epidemiology of the disease ( , , ) . such assumptions can be avoided by using a likelihood-based estimation procedure that infers "who infected whom" from the observed dates of symptom onset as provided by the epidemic curve. however, the computational burden of a straightforward numerical evaluation of the likelihood appears to be enormous, since it requires consideration of all possible infection networks, and even for a small outbreak of cases there are almost × possible infection networks (see appendix ). here we show that it is possible to obtain likelihood-based estimates of r while avoiding the computational problems if we use pairs of cases rather than the entire infection network. the relative likelihood p ij that case i has been infected by case j, given their difference in time of symptom onset t it j , can be expressed in terms of the probability distribution for the generation interval. this distribution for the generation interval is available for many infectious diseases, and we denote it by w(τ). the relative likelihood that case i has been infected by case j is then the likelihood that case i has been infected by case j, normalized by the likelihood that case i has been infected by any other case k: the effective reproduction number for case j is the sum over all cases i, weighted by the relative likelihood that case i has been infected by case j: note that we are not making any assumption regarding the distribution of numbers of secondary infections per case (i.e., the p ij are independent in j). additional detail on the derivation of these equations is provided in appendix . the estimation algorithm allows estimation of the effective reproduction numbers for infectious diseases at a finer temporal resolution under more general assumptions than was previously possible. to test how well the estimation procedure approximates the underlying value of an effective reproduction number during a typical sars outbreak, we estimate the effective reproduction numbers from simulated epidemic curves. we have constructed a stochastic, individual-based model that simulates epidemic processes with exactly specified properties. the model allows for a variable effective reproduction number r t as a function of symptom onset data t, and the model parameters are estimated from observations on the sars epidemic in singapore (see appendix ) . applying the estimation procedure to simulated epidemic curves shows that most of the estimates are close to the actual reproduction numbers and that a few estimates based on small outbreak sizes are below the actual values that are used in the simulation model. on average, the estimates tend to be lower than the actual values but deviate less than percent from the actual reproduction numbers. if we account for the effects of incomplete reporting and temporal change in generation interval, the estimates become only slightly less accurate, and on average they deviate less than percent from the actual reproduction numbers (see appendix ) . for hong kong, vietnam, singapore, and canada, we have converted the epidemic curves into the time course of effective reproduction numbers. the results are shown in figure , parts e-h. these four large outbreaks were sparked almost simultaneously by the same index patient. all regions have faced erratic "super-spread events" wherein cases produced more than secondary infections. these "super-spread events" show up in parts e-h of figure as temporary increases in effective reproduction numbers around the symptom onset date of the index case for the "super-spread event." in hong kong, vietnam, and singapore, there were "super-spread events" marking the start of the outbreak. in hong kong, singapore, and canada, there were "super-spread events" after control measures were implemented. during the early phase of the sars epidemic, before the first world health organization global alert was issued on march , , the average effective reproduction numbers were markedly similar across the regions: each case produced approximately three secondary infections (table ) . a value of r slightly higher than is consistent with the observed epidemics in all four regions. around mid-march, control measures were implemented in all regions, and during this period the effective reproduction numbers decreased sharply. for some regions, the effective reproduction numbers continued to decrease at a slow pace, suggesting improvement of control measures while the epidemic was going on. after the first world health organi- ( ). to explore the range of epidemic curves that can result from the same epidemic process, we performed extensive computer simulations using a model of epidemics with characteristics similar to those of sars (see appendix ) . the outcome of , simulations shows a highly variable epidemic size and epidemic duration: the mean epidemic size is cases ( percent confidence interval (ci): , , ), and the mean epidemic duration is days ( percent ci: , ). all observed sizes and durations of sars epidemics are within this very wide range of possible outcomes resulting from chance alone. additionally, we used computer simulations to explore the effect of the timing of implementation of control measures on the epidemic size and duration, in a setting that is typical for the sars outbreaks. the simulation results show a high sensitivity to the timing of implementation of control. on average, a week delay in implementation of control measures results in a . -fold increase in mean epidemic size and a -week extension of the mean epidemic duration. this study showed that there exists a direct relation between the epidemic curve and the time course of the reproduction number r. this relation is determined by the distribution of the generation intervals. the relation can be used to monitor the combined effects of transmission potential and control measures during an epidemic. we have shown that the epidemic curves for sars in hong kong, vietnam, singapore, and canada, though apparently different, are all consistent with a single time course of the effective reproduction numbers for sars. this apparent difference in epidemic curves arises because chance effects, such as the occurrence of a rare "super-spread event," leave a lasting trace on the epidemic curve. in contrast, chance events manifest only as temporary increases in the reproduction number. our analysis of the epidemic curves for sars shows that one should be cautious in taking a smaller epidemic size and a shorter epidemic duration as proof of better infection control. we have presented the relation between observed epidemic curves and inferred reproduction numbers from an infection-network perspective. we are certainly not the first researchers to do so: infection networks have been used extensively in the area of sexually transmitted diseases ( ) , and an infection-network perspective was adopted to analyze a closely observed foot-and-mouth epidemic in great britain ( ) . our contribution is the derivation of likelihood-based estimates of effective reproduction numbers, requiring only the observed time of symptom onset for the observed cases. the use of a likelihood framework provides a set of powerful tools for inference, uncertainty analysis, and model selection ( ) ; the use of only time of symptom onset allows us to apply this method to routinely collected epidemic-curve data. however, estimation of effective reproduction numbers from epidemic curves has an intrinsic limitation that should be kept in mind: the effective reproduction number contains entangled information about the transmission potential (i.e., the basic reproduction number) and the effectiveness of control measures. these two components can be disentangled only when we obtain additional information-for example, about the time of implementation of control measures. moreover, individual contributions to the effective reproduction number are entangled if their date of symptom onset is smaller than the generation interval. this limitation can be overcome if more detailed information on who infected whom is available (see appendix ) . for the sars outbreak in hong kong, it is possible to compare our results with previously published estimates. our estimate of the average effective reproduction number prior to the first global alert (r = . , percent ci: . , . ) is more precise than the estimate obtained by assuming an exponential increase in the number of cases (r = . , percent ci: . , . ) ( ) and more precise than the estimated lower bound excluding "super-spreading events" (r > . ) ( ). our estimate of the average effective reproduction number after the first global alert (r = . , percent ci: . , . ) is much higher than the previously estimated lower bound excluding "super-spread events" (r > . ) ( ). this comparison illustrates that the estimation algorithm presented here allows more precise estimation of the effective reproduction numbers for infectious diseases under more general assumptions than was previously possible. the effectiveness of control measures against sars can be estimated if it is assumed that the sudden decrease in the as the direct threat of a worldwide sars epidemic has waned, the question arises as to how we can use the experience with sars to improve infection control against new infectious diseases. our analysis of the epidemic curves for sars, as reported for hong kong, vietnam, singapore, and canada, shows how crucial the rapid implementation of control measures has been in limiting the impact of the epidemics, both in terms of preventing more casualties and in terms of shortening the period during which stringent infection control measures were in place. a first lesson from the several sars epidemics is that a timely alert against a new infectious disease is most essential. our analysis suggests that the control measures implemented prevented approximately three quarters of all potential secondary infections; this may be insufficient to stop another new infectious disease. a second lesson, then, is that it is crucial to estimate the transmission potential of a new emerging disease as soon as possible and to establish whether additional, more stringent control measures are required. an outbreak of an infectious disease can be described as a directed network in which the nodes represent cases and the directed edges between the nodes represent transmission of infection between cases. we consider an outbreak of n reported cases, of which q cases have contracted infection from outside the population. this leaves nq persons whose primary case is among the reported cases. we label the cases by an index i ∈{ , …, n}. because each case has exactly one primary case, each node in the infection network must have exactly one incoming edge. because a case patient cannot have infected himself or herself, there cannot be any edges from a node to itself. the structure of a network that satisfies these constraints can be uniquely represented by a vector v, of which the ith element v(i) denotes the label of the primary case that has infected the case with label i. we use v(i) = to refer to sources of infection outside the population. we denote the entire set of all infection networks that satisfy the above constraints by v. the number of different network structures in v is (nq) n- , since, for any of the nq nonimported cases, there are n - possible primary cases. note that the set v includes network structures with cycles and that such structures cannot represent transmission between cases. we use a probability model to infer the likelihood that a specific infection network v underlies the observed epidemic curve t. the probability model is built on the assumption that transmission of infection occurred only among the reported n cases. a key element in this model is the probability density function for the generation interval, w(τ|θ). here, τ is the generation interval and θ is a vector of parameters that specify the probability distribution. we require w(τ|θ) = for τ < . all infection networks with cycles have at least one negative generation interval, and these networks are assigned zero probability by this requirement. in the absence of an observed epidemic curve, each infection network is considered equally likely. this is equivalent to requiring independence between unobserved transmission events from case j to case i and from case j to any other case k. henceforth we will refer to this requirement as the "independence condition." in appendix , we simulate epidemic processes that do not meet the above-mentioned technical conditions, and we use these simulations to test for the robustness of the likelihood-based estimation procedure. the probability of observing epidemic curve t, given the parameters θ for the generation interval and v for the infection network, is (a ) because we are interested in the likelihood of sets of infection networks, we sum the likelihood over networks in a set. this requires a "weight function" c(v|θ) for each infection network. the independence condition implies that c(v|θ) is a constant, denoted c. the integrated likelihood over the set of all networks is therefore the integrated likelihood over the set of all infection networks in which case k has been infected by case l is (a ) the relative likelihood that case k has been infected by case l is the average daily reproduction number r t is calculated as the arithmetic mean over r l for all of those cases l who show the first symptoms of illness on day t. when we have observed transmission of infection between some pairs of cases, it is possible to infer both infection network (v) and generation interval (θ) simultaneously. we must consider the likelihood (a ) where (k,l) denotes all pairs of cases for which any case k is known to have been infected by another case l. estimates for both generation interval (θ) and infection network (v) are obtained by maximizing this equation using the expectationmaximization algorithm ( ) . note that the independence condition applies only to the unobserved transmission events. we have constructed a stochastic, individual-based model to generate infection networks that result from epidemic processes with exactly specified properties. we use such simulated infection networks for testing the estimation procedure and for exploring the expected distribution of epidemic size and epidemic duration. the model allows for a variable effective reproduction number r t as a function of symptom onset date t. in the simulations described here, we set the effective reproduction number to a value of r t = for cases with a symptom onset date before the issuance of the first global alert on march , , and to a value of r t = . for cases with a symptom onset date on or after march , . the model draws for each case the number of secondary infections from a negative binomial distribution, which is determined by the mean r and the shape parameter k t = (r t + r t )/σ t . the model uses values of k t = . for cases with a symptom onset date before march , , and k t = . for cases with a symptom onset date on or after march , ; these values correspond to the distribution of the number of secondary infections per case as observed during the severe acute respiratory syndrome (sars) outbreak in singapore ( ). the model draws for each new infection the generation interval from a weibull distribution with a mean and standard deviation of . days and . days, respectively ( ) . each simulation is started by one index case that produces at least secondary cases, which corresponds to a sars outbreak that is started by a so-called "super-spread event," to ensure that the epidemic takes off. we test for accuracy of the proposed estimation procedure by first generating epidemic curves using the stochastic simulation model and then estimating the time course of the reproduction number for the simulated epidemic curves using the proposed method. the estimated reproduction numbers tend to be close to the actual values used in the simulation model, except for simulated epidemic curves with a small number of cases in which the estimated reproduction numbers are well below the actual values (the actual values of the reproduction numbers for the simulated epidemic curves were and . for cases with symptom onset data before and after march , , respectively; the average estimated values were . and . ). the estimation procedure supposes that all infected persons will show overt clinical symptoms and that all cases will be reported, an assumption that might not be correct for an infectious disease like sars. to test for the impact of incomplete reporting on the estimated effective reproduction number, we modified the model such that each infected individual would have a probability of his or her case's being reported of . . the resulting estimates were only slightly less accurate than they were with complete reporting (the actual values of the reproduction numbers for the simulated epidemic curves were and . for cases with symptom onset data before and after march , , respectively; the average estimated values were . and . ). the estimation procedure presented here supposes that the distribution of generation intervals does not change over time. however, for the sars epidemic in singapore, there was a tendency for the generation interval to decrease after control was implemented ( ) . cases with a symptom onset date before and a standard deviation of . days, whereas cases with a symptom onset date on or after march , , must have had a mean generation interval of . days and a standard deviation of . days ( ) . to test for the impact of this temporal change in the duration of the generation interval, we modified the model by using two different weibull distributions, one before march and the other one on or after march , each param-eterized according to the singapore data. the estimates were, on average, slightly lower than they were with use of the same generation interval throughout the epidemic (the actual values of the reproduction numbers of the simulated epidemic curves were and . for cases with symptom onset data before and after march , , respectively; the average estimated values were . and . over simulations). update -sars: chronology of a serial killer a cluster of cases of severe acute respiratory syndrome in hong kong identification of severe acute respiratory syndrome in canada severe acute respiratory syndrome-singapore koch's postulates fulfilled for sars virus epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong transmission dynamics of the etiological agent of sars in hong kong: impact of public health interventions transmission dynamics and control of severe acute respiratory syndrome epidemic curves-severe acute respiratory syndrome (sars) epidemic curve of a sars outbreak in canada the construction and analysis of epidemic trees with reference to the uk foot-and-mouth outbreak infectious diseases of humans: dynamics and control the estimation of the basic reproduction number for infectious diseases mathematical epidemiology of infectious diseases: model building, analysis and interpretation transmission potential of smallpox: estimates based on detailed data from an outbreak transmission intensity and impact of control policies on the foot and mouth epidemic in great britain severe acute respiratory syndrome: temporal stability and geographic variation in casefatality rates and doubling times transmission potential of smallpox in contemporary populations dynamics of measles epidemics: estimating scaling of transmission rates using a time series sir model an assessment of preferential attachment as a mechanism for human sexual network formation maximum likelihood from incomplete data via the em algorithm this article benefited greatly from discussions with members of the world health organization-sars modeling working group (drs. roy anderson, kari auranen, neil ferguson, nigel gay, marc lipsitch, mick roberts, and ping yan) and from comments by drs. hans heesterbeek, nico nagelkerke, and siem heisterkamp. key: cord- -bu j kdr authors: macours, nathalie; poels, jeroen; hens, korneel; francis, carmen; huybrechts, roger title: structure, evolutionary conservation, and functions of angiotensin- and endothelin-converting enzymes date: - - journal: int rev cytol doi: . /s - ( ) - sha: doc_id: cord_uid: bu j kdr angiotensin-converting enzyme, a member of the m metalloprotease family, and endothelin-converting enzyme, a member of the m family, are key components in the regulation of blood pressure and electrolyte balance in mammals. from this point of view, they serve as important drug targets. recently, the involvement of these enzymes in the development of alzheimer's disease was discovered. the existence of homologs of these enzymes in invertebrates indicates that these enzyme systems are highly conserved during evolution. most invertebrates lack a closed circulatory system, which excludes the need for blood pressure regulators. therefore, these organisms represent excellent targets for gaining new insights and revealing additional physiological roles of these important enzymes. this chapter reviews the structural and functional aspects of ace and ece and will particularly focus on these enzyme homologues in invertebrates. of catalysis, classified into five major groups: serine proteases, cysteine proteases, aspartic proteases, metalloproteases, and proteases with an unknown mechanism (barret et al., ) . from these groups, the metalloproteases, with more than families identified to date, are most diverse. in mammalian physiology, the high interest in metalloproteases originates from the fact that they are attractive targets for drug design. in insects, regulatory peptides are the major and most diverse class of signaling molecules. they act as transmitters and modulators in the nervous system and as hormones controlling key physiological processes, behavior, and development. peptidases are recognized as the key components of this peptidergic system, functioning in biosynthesis of active peptides from their precursors and in peptide inactivation. structural analysis of metalloproteases from invertebrate and vertebrate species indicates that these enzymes are very well conserved and arose early during evolution. the evolutionary conservation of universal peptidesignaling mechanisms has led to the general acceptance of the idea that invertebrates provide excellent models for the study of genetics and physiology. the information and knowledge gained by studying invertebrate peptidases will provide novel insights in the process of evolution and may reveal conserved functions of vertebrate peptidases that have remained unknown until now. angiotensin-converting enzyme (ace, dipeptidyl carboxypeptidase i [dcp i], peptidase p, carboxycathepsin, peptidyl-dipeptidase a, peptidyl-dipeptidase i, kininase ii, ec . . . ) was originally isolated in as a ''hypertensin-converting enzyme'' (skeggs et al., ) . classified as a member of the m gluzincin family, ace acts as a zinc-metalloprotease. the general reaction mechanism is identical for all zinc-metalloproteases. coordination of the zinc ion within the catalytic site of metalloproteases is typically avected by an hexxh motif, a third zinc ligand (e) located carboxyl to this motif, and a water molecule. the histidine residues within the active site motif are known to directly coordinate the active site zinc ion, whereas the hexxh glutamate has been shown to coordinate weakly to zinc via the activated molecule, thus facilitating the acid-base catalytic mechanism. the renin-angiotensin system (ras) plays a key role in the regulation of blood pressure and of fluid and electrolyte balance in mammals (inagami, ) . being part of the renin-angiotensin system, ace converts the decapeptide angiotensin i (angi) into the potent vasopressor angiotensin ii (angii). ace also inactivates the vasodilatory peptide bradykinin (bk) . hence, inhibition of ace results in lowering the blood pressure by preventing formation of the hypertensive angii and by inactivation of the hypotensive bk (fig. ) . ace is one of the most studied mammalian peptidases, and in particular for medical applications. ace inhibitors are widely used in treatment of hypertension, diabetic nephropathy, and heart failure (dell'italia et al., ) . the production of captopril, the first clinically used ace inhibitor, represents a classical example of drug design (menard and patchett, ) . vertebrate ace is classified as a type i ecto-enzyme, with the major part of the protein, including the active site, located on the external surface of cells. ace is anchored in the plasma membrane by the c-terminal part of the protein. there is an n-terminal signal peptide present, required for the passage through the endoplasmatic reticulum. two distinct forms of ace are known: a somatic form (sace), which is found in many tissues, and a smaller iso-enzyme, germinal ace (gace, also called testicular ace [tace] ), which is found exclusively in testes, where it has a crucial role in fertility (fig. ). the soluble, circulating form of the sace protein is produced by a ''secretase,'' probably a zinc-metalloprotease, which releases ace from the membrane by cleaving ov the extracellular part . the two forms diver in that the somatic isoform contains two active sites, whereas the germinal form contains only one. sace is composed of two very similar domains (c and n domain), which are both catalytically active (wei et al., ) . tace is, apart from its n-terminal sequence, identical to the c-domain of sace. the close similarity of the n and c domains of mammalian sace and the fact that exons - and - of the human ace gene, which code for the n and c domain, respectively, are very similar in size and have similar codon phases at exon-intron boundaries, strongly indicates that the coding gene arose from duplication of an ancestral ace gene coding for a single domain enzyme (cornell et al., ) . the ancestral copy is predicted to be the c-domain, equivalent to the germinal isoform (lattion et al., ). the fig. schematic representation of human and drosophila ace protein and gene structure. exons are indicated by boxes. exons and corresponding amino acid regions in the protein are colored correspondingly. zinc-binding sites (hexxh) are indicated. human somatic and testicular promoters are indicated by arrows. one-kilobase intronic sequence was omitted at the // signs. two isoforms are transcribed from a single gene, under the control of two distinct promoters. sace is transcribed from a promoter region upstream of the duplication, and tace from a promoter within intron (langford et al., ) . the two domains of sace have relatively broad substrate specificities and share some enzymatic features, but there are several biochemical properties that diverentiate the two active sites . for example, the hematopoietic stem cell regulator, n-acetyl-ser-asp-lys-pro (acsdkp), is an in vivo substrate for the n-domain, but not for the c domain of human ace (azizi et al., ) . the n and c domain can also be diverentiated by their response to chloride ions, with the c domain's specific activity being dependent on chloride ion concentration (wei et al., ) . their inhibitory aynity profiles also diver, illustrated by the existence of an n-domain-specific inhibitor (rxp ) (dive et al., ) . ace acts as a peptidyl-dipeptidase, removing the c-terminal dipeptide from its substrate, but it can also act as an aminopeptidase and as an endopeptidase on peptides that are amidated at the c terminus. to date, only angiotensin i, bradykinin (yang, ) , and the haemoregulatory peptide ac-dskp (azizi et al., ) have been confirmed as in vivo substrates for mammalian ace. the conversion of angi into the vasoconstrictor angii and the degradation of the vasodilator bradykinin reflect the key role of ace as a component of the mammalian ras. in recent years, the ras turned out to be a far more complex regulatory system that involves more angiotensin-derived mediators (angi, angii, angiii, angiv, ang ( - ) ), ang ( - ) ) then initially recognized ( fig. ) . to this day, it includes four identified receptors . in vivo models using ace-deficient mice have greatly advanced the knowledge of the ras and confirmed the role of ace in blood pressure regulation and fertility. knock-out mice all show reduced blood pressure, altered kidney morphology, impaired somatic growth, and male sterility (esther et al., ; eriksson et al., ) . these mice produce normal numbers of sperm that are indistinguishable from wild-type sperm in assays of viability, motility, capacitation, and induction of the acrosome reaction. absence of ace, however, does cause defects in sperm transport in the oviducts and in binding to zonae pellucidae (hagaman et al., ) . knockout of tace, leaving the sace intact, has proven that tace expression is both necessary and suycient for fulfilling the role of ace in male fertility (ramaraj et al., ) . recently, immunocytochemical studies have shown that tace is completely shed from the sperm membrane before ejaculation. this would imply that the defects in sperm transport in the oviducts and in binding to zonae pellucidae after tace knockout are not directly related with tace activity (metayer et al. ) , so the exact role of tace remains to be uncovered. the fact that to date no in vivo substrate has been found for tace is the main bottleneck in tace research. expression of sace, either in renal proximal tubes or in vasculature, is suycient for maintaining normal kidney functions. however, for maintaining blood pressure, sace must be expressed in vascular endothelial cells (kessler et al., ) . expression of tace alone is suycient to restore male fertility in mutant mice without curing other problems of ace À/À mice (ramaraj et al., ) . however, sace cannot substitute for tace, demonstrating that the two isozymes are not interchangeable for fertility functions (kessler et al., ) . yet tace expression in serum of aceÀ/À mice can substitute for sace in maintaining normal renal structure and functions without restoring blood pressure (kessler et al., ) . all this indicates that the two isoforms of ace are indispensable, as suggested by their high degree of evolutionary conservation. also, the specific physiological function of ace requires its expression in the correct tissue. the peptide acsdkp reversibly prevents the recruitment of pluripotent haematopoietic stem cells into the s phase of the cellular cycle by maintaining them into the g o phase. because this peptide has been found to be an in vivo substrate specific for the n domain of sace, it is suggested that ace is implicated in the process of hematopoietic stem cell regulation by permanently degrading this natural circulating inhibitor (azizi et al., ; rousseau et al., ) . however, in a very recent study, mutation of the n domain zinc-binding site in mice, rendering this n domain inactive indeed, resulted in accumulation of acdskp, but no physiological evect was seen (fuchs et al., ) . ace acts as an endopeptidase on the multifunctional neuropeptides substance p and cholecystokinin and may degrade the luteinizing hormone releasing hormone (lh-rh) (skidgel and erdos, ) . its biological significance in this context is not known, and the possibility of functional cross talk between the systems has not been excluded . the broad in vitro specificity (table i ) and the presence of ace in species that lack an identifiable ras shows that the relevance of ace to the animal must be more complex than just its role in the ras. up until now, only one other member of the m family other than ace has been identified. almost years after the discovery of ace itself, two groups (donoghue et al., ; tipnis et al., ) introduced the first homolog of mammalian ace. this protein and its homolog were named ace and aceh, respectively, and were shown to be an essential regulator of heart function (crackower et al., ) . human ace is an -amino acid-long type i integral membrane protein, including a -amino acid n-terminal signal peptide and a putative c-terminal membrane anchor. the extracellular domain shares a % sequence identity with the catalytic domain of sace. it is % identical to human testicular (germinal) ace and also contains a single catalytic site. ace contains six putative n-linked glycosylation sites on the extracellular part (tipnis et al., ) . the c-terminal domain of ace shares a % sequence identity with collectrin (vickers et al., ; zhang et al., ) , a developmentally regulated renal transmembrane glycoprotein. the many similarities in the genomic sequence of ace and ace indicate that there is an evolutionary relationship between these two genes . ace transcripts are detected in heart, lung, kidney, the gastrointestinal tract, and testis (harmer et al., ; komatsu et al., ) . ace is located at the endothelium of coronary and intrarenal vessels and in the renal tubular epithelium. like ace, ace also appears to be susceptible to cleavage and secretion from the cell surface (donoghue et al., ) . however, the sequence identity of the juxtamembrane regions of ace and ace is diverent. the cleavage site present in ace (-ala-arg ser-glu-) is absent in ace . hence, a diverent secretase may be involved in the shedding of ace . ace does not have the same substrate specificity as ace, nor is it inhibited by the typical ace inhibitors such as enalapril, lisinopril, and captropril. ace hydrolyzes angi and angii but cleaves neither bradykinin nor the typical ace substrate hip-his-leu ( fig. ) . to hydrolyze angi and angii, ace acts as a carboxypeptidase, removing one amino acid from the c terminus of the substrate. this is also the case for kinetensin, des-arg bradykinin, and neurotensin (donoghue et al., ; . several other regulatory peptides, including the enkephalins, are not substrates of ace . it seems that ace prefers to cleave a single c-terminal hydrophobic residue from its substrates (table i) and consequently does not hydrolyze bradykinin that contains an arginine at its c terminus. ace is the first mammalian carboxypeptidase identified that contains the hexxh active site. ace also acts on apelin- and apelin- peptides, of which the role is not yet fully understood but involves an evect on hypertension and vasoconstriction . two opioid peptides, dynorphin a( - ) and b-casamorphin, are also ace substrates (eriksson et al., ; vickers et al., ) . these peptides activate g protein-coupled opioid receptors that regulate pain perception and are suggested to negatively avect cardiomyocyte contractility (pugsley, ; ventura et al., ; wenzlav et al., ) . the diverences in substrate specificity between ace and ace have important physiological consequences. whereas ace activity leads to the formation of the vasoconstrictor angii, ace is involved in the formation of vasodilator mediators (danilczyk et al., ) . the targeted disruption of ace in mice results in increased angii levels, impaired cardiac contractility, and up-regulation of hypoxia-induced genes in the heart (crackower et al., ) . renal ace is implicated in diabetic nephropathy. in the diabetic kidney, levels of ace transcription and protein expression are downregulated. it is suggested that this regulation occurs through the actions of ace in the ras (tikellis et al., ) . another, rather unexpected function of ace was recently discovered. it was found to act as a functional receptor for the severe acute respiratory syndrome (sars) coronavirus (cov) (dimitrov, ; li et al., ; xiao et al., ) . sars is the abbreviation for the cov-induced disease that causes sars, with a mortality rate of approximately % (ksiazek et al., ; kuiken et al., ) . the virus associates with cellular receptors to mediate infection of the target cells (gallagher and buchmeier, ; holmes, ) . ace , acting as a cellular receptor, eyciently binds to the s domain of sars-cov, eyciently supporting the replication of sars-cov. adding a soluble form of ace , but not that of ace, blocks the association of sars-cov and its target cell. replication of the virus in cells and the formation of syncitia can be blocked by antibodies against ace , which indicates the importance of ace in the replication of sars-cov (li et al., ) . several attempts to reveal the three-dimensional structure of ace have been made very recently. this provides an insight into the structural variations underlying the diverences in substrate specificity between ace and ace . it seems that these diverences occur in the ligand binding cavity, influencing the binding of the peptide carboxyterminus (guy et al., ) . the crystal structure of ace , native and inhibitor bound, revealed a large inhibitordependent bending movement of the two enzyme domains to one another, positioning important catalytic residues (towler et al., ) . it has been suggested that ridges surrounding the cavity at the top of the molecule serve as a binding region for sars (prabakaran et al., ) . the discovery of this sars-related function of ace , together with the knowledge of its structure, may contribute substantially to the possible treatment of sars patients. lamango and isaac ( ) reported the metabolization of [d-ala , leu ] enkephalin by head membranes of the housefly, musca domestica. this hydrolysis was only partially inhibited by phosphoramidon, an inhibitor of endopeptidase- . . the combination of phosphoramidon and captopril, however, fully inhibited the cleavage of enkephalin, suggesting the presence in this fly of a second enkephalin-metabolizing enzyme in addition to endopeptidase- . . it turned out to be zn -kda-enzyme that was activated by zncl and inhibited by edta as well as the mammalian ace inhibitors captopril, fosinoprilat, and enalaprilat. this represented the discovery of the first invertebrate ace homolog . following the report on this ace-like activity, the first results of putative drosophila melanogaster ace cdna clones were published (cornell et al., ) . since then, ace homologs have been detected in many invertebrate species. cloning or purification of ace from the fruit fly d. melanogaster (cornell et al., ; tatei et al., ) , the cattle tick boophilus microplus (jarmey et al., ) , the leech theromyzon tessalutum (laurent and salzet, ) , the housefly m. domestica (lamango et al., ) , the buvalo fly haematobia irritans exigua (wijvels et al., ) , the mosquito anopheles stephensi , the silk moth bombyx mori , the grey fleshfly neobellieria bullata (vandingenen et al., b) , and the locust locusta migratoria (macours et al., a) have contributed to the molecular, biochemical, and evolutionary characterization of invertebrate aces. the first d. melanogaster ace homolog that was discovered is abbreviated as ance, because ace was already used for acetylcholine esterase in the fruit fly. of the invertebrate aces known today, the two d. melanogaster ace homolog (ance and acer) have been studied in most detail. ance is also known under the name race, as the enzyme was discovered, sequenced, and named by two groups simultaneously (cornell et al., ; tatei et al., ) . nowadays, the focus on ace research is beginning to shift from purification-characterization to a more physiological point of view. considering the fact that most invertebrates have an open circulatory system, there is probably no need for a renin-angiotensin system analogous to that found in vertebrates, at least not for the regulation of blood pressure. hence, understanding the biology of ace homologs in invertebrates may, in due time, provide substantial information on additional roles for ace. in contrast to the occurrence of both a double and a single domain ace in vertebrates, characterization of ace homologs from diverent invertebrates showed that the overall size of these proteins was comparable to the nonglycosylated testicular form of mammalian ace. cloning of the corresponding cdnas confirmed the existence of this single-domain form in insects. until now, there has been no evidence for a functional two-domain form of insect ace. in addition to the absence of a two-domain form, a structural diverence exists between insect ace and the mammalian enzyme, located in the n-and c-terminal ends. cloning of the d. melanogaster ace homologue (ance) revealed that the insect form of ace lacks both the heavily glycosylated n-terminal extension and the c-terminal transmembrane and cytoplasmic regions that are present in mammalian testicular ace. the enzyme contains an n-terminal secretory signal peptide, which indicates that the mature enzyme is being secreted. hence, ance was predicted to code for a soluble secreted protein (fig. ) . confirmation of this prediction came when % of recombinantly expressed ance activity was found in the cell culture medium (cornell et al., ) . although insect ace activity was primarily detected in a membrane preparation from heads of the housefly, m. domestica , this ace protein was later found not to behave as an integral membrane protein. the membrane-associated activity was probably the result of a contamination of the membrane fraction with soluble ace (lamango et al., ) . therefore, the general form of insect ace seems to be a secreted single-domain protein with no recognizable c-terminal membrane anchor. the molecular identification of the ace enzyme in b. microplus, however (jarmey et al., ) , which does posses a c-terminal hydrophobic region, showed that the membrane-bound form of ace is not restricted to vertebrate organisms. also, in the cockroach leucophaea maderae and the noctoid moth lacanobia oleracea, a substantial amount of ace activity was detected in head membranes . the nature of this membrane-associated ace activity in these two organisms is, as yet, unidentified. sequence comparison between human testicular ace and several invertebrate ace homologs showed that the overall sequence identity is approximately % and exceeds % in the regions surrounding the active site. in human ace, and potential n-linked glycosylation sites (asn-xaa-ser/ thr motifs) are present in the somatic isoform and in the germinal isoform, respectively. this contrasts to the situation in invertebrates, where ace homologs have only three or fewer potential glycosylation sites. in d. melanogaster, it was shown that glycosylation of ace is not required for the enzymatic properties. expression of an unglycosylated mutant of ance resulted in the secretion of a catalytically active enzyme, with the same substrate and inhibitor aynity as the wild-type enzyme (williams et al., ) . kim et al. ( ) determined the crystal structure of the d. melanogaster ace homolog ance. ance is composed of a-helices and three antiparallel b-strands. the substrate-binding channel exists as a large continuous internal angiotensin-and endothelin-converting enzymes path that comprises the entire protein molecule. the composition of this substrate-binding site is unusual in that it is composed of two unequal-sized ''chambers'' analogous to a peanut shell. the large chamber, referred to as the n chamber, contains the binding site of the n-terminal sequence of the substrate angiotensin i. the smaller chamber, called the c chamber, will bind the carboxy-dipeptide of the substrate. the size of these chambers is suyciently large for binding short peptide substrates. the bottleneck region connecting these two chambers encloses the zinc ion critical for catalysis and locates the inhibitor binding sites. the opening of these chambers to the exterior appears to be relatively small for the passage of peptide substrates, so flexibility and a so-called ''breathing motion'' are probably required for eycient catalysis. the presence of significant ace activity was reported in extracts of the horse anemone, actinia equine, a member of the cnidarians (coates et al., b) . to this day, no ace peptidase activity has been detected in nematode tissues. however, there is one copy of an ace-like gene (cd d . ) with a recorded expressed sequence tag (est) present in the genome of the nematode caenorhabditis elegans. yet taking a closer look at this ace-like gene reveals that the amino acids crucial for enzyme activity are absent. the sequence lacks all three zinc coordinating his residues, which means that the c. elegans ace homolog does not have a functional zinc-binding domain. therefore, it cannot be classified as a metallopeptidase homolog. studies on this ace-like nonmetallopeptidase, termed acn- , demonstrated that the protein plays an essential role in larval molting and adult morphogenesis. if acn- is indeed evolutionarily related to the ancestral ace, it means that during evolution it lost its peptidase activity but acquired sites for new molecular interactions that have a direct or indirect evect on the molting process in nematodes (brooks et al., ) . this phenomenon can be further expanded to other organisms and enzymes, as in the genome of d. melanogaster and anopheles gambiae, - % of the protease-like genes lack one or more catalytic residues. some of these proteins have probably lost the peptidase activity of their ancestor while acquiring new functions. the analysis of the human ace gene at the structural level forwarded the view that the vertebrate somatic form was transcribed from a tandem duplication (lattion et al., ) . in all vertebrate species examined, from humans at the top down to the electric fish torpedo marmorata (turner et al., ) , the somatic ace enzyme exists as the two-domain form. the analysis of the ace gene of the housefly m. domestica revealed that this organism comprises a smaller form of the enzyme, indicating that this species does not contain the duplicated form of ace. the existence of this single-domain isoform was confirmed when the ance cdna of d. melanogaster was sequenced (cornell et al., ) . all other invertebrate ace enzymes that were discovered later on also consisted of the single-domain isoform. this leads to the view that invertebrate ace represents the ancestral copy of the vertebrate two-domain enzyme and that the duplication event occurred before vertebrate radiation. however, this course of ace evolution started to be questioned when the characterization of d. melanogaster ace homologs began. the high level of homology between ance and acer points toward the existence of an ancestral ace gene. in addition, studying the relationship of ance and acer to the n and c domains of human somatic ace showed that ance was more enzymatically related to the c domain than to the n domain of human ace (williams et al., ) . acer, however, has much less in common with this c domain. the active sites of the n domain of sace and acer share structural features that permit the binding of the rxp inhibitor, which does not bind the c domain of sace. an analysis of the genomic region around the ance gene in d. melanogaster has led to the identification of four additional ace-like genes (isaac et al., ) . from these four, ance , , and seem to be transcribed, because ests were reported for these genes. with the information available today, no est seems to be present for ance . the predicted protein sequence corresponding the ance gene includes a cterminal transmembrane anchor, as is seen in vertebrate ace. ance codes for an ace-like protein with a putative signal peptide, but without a cterminal membrane region. both genes lack crucial active site residues. these two gene predictions are interesting because alternative splicing of the predicted transcripts would lead to a two-domain, membrane-bound ace-like product, as is seen in the vertebrate somatic form (coates et al., a) . taken together, this information indicates that the duplication event that gave rise to the vertebrate tandem protein predates the divergence of the ecdysozoa and deuterostomes, and that the divering activities on the n and c domains have been maintained over a long period of time. insect ace is capable of hydrolyzing the known in vivo substrates of mammalian ace, angiotensin i, bradykinin, and the hemoregulatory peptide ac-dskp. however, there are no reports of insect homologs of these known in vivo substrates, and none of the many insect peptides isolated to date shows any structural resemblance to them. the search for angiotensin i-like peptides in the brain of several insect species could not prove otherwise (isaac et al., b; schoofs et al., ) . in addition, a search in the d. melanogaster genome did not reveal any potential homologs of the precursor proteins of angiotensin i and bradykinin (isaac et al., ) . this is in contrast to the situation in leeches, where several reports on the presence of members of the renin-angiotensin system have been published (laurent and salzet, ; salzet and verger-bocquet, ) . cleavage experiments with ace from m. domestica showed that insect ace exhibits a broad in vitro substrate specificity. the in vitro substrates of mammalian ace, cholecystokinin, enkephalins, substance p, and lh-rh, are successfully cleaved by the endopeptidase activity of musca ace. this shows that insect ace can hydrolyze c-terminally amidated peptides in vitro, functioning as an endopeptidase (isaac et al., a; lamango et al., ) . m. domestica ace is unable to hydrolyze either ccap (crustacean cardioactive peptide) or proctolin. apparently, the penultimate c-terminal prolyl residue of proctolin makes this peptide resistant to ace hydrolysis. the lack of hydrolysis of ccap is likely a result of the presence of a disulfide bridge, which results in a secondary structure that prevents access to the active site. a novel activity of insect ace, namely, prohormone processing, was suggested when it was found to be capable of successfully removing basic dipeptides from locustamyotropin oligopeptides possessing a c-terminal gly-lys-arg and gly-arg-arg extension (isaac et al., b) . many insect peptides are synthesized as inactive prohormones and need posttranslational processing to acquire their biological activity. in several prohormones, the sequence -gly-arg/lys-arg-is an internal consensus recognition sequence for endoproteolysis by prohomone convertases, generating peptide intermediates with a c-terminal -gly-arg/lys-arg-extension. these are substrates for carboxypeptidase e (cpe) that sequentially removes the two basic amino acids from the c terminus, which is followed by amidation of the c terminus (isaac et al., b) . the fact that insect ace is capable of hydrolyzing these partially processed prohormone-like peptides, together with the intracellular colocalization of the enzyme with locustamyotropin peptides, implies a role for ace in the biosynthesis of peptide hormones and neuropeptides . zhu et al. ( ) and coworkers demonstrated that the hexapeptide trypsin modulating oostatic factor from n. bullata (neb-tmof) (bylemans et al., ) is selectively hydrolyzed by ace that is present in substantial amounts in the fly's haemolymph . neb-tmof is capable of regulating vitellogenesis and is thought to be released by the ovaries and transported to the midgut, where it terminates the protein meal-induced trypsin biosynthesis. captopril feeding experiments suggest that neb-tmof represents a prime candidate for being an in vivo substrate for circulating insect ace (vandingenen et al., a) . tmof was also detected in aedes aegypti, probably synthesized by the brain (borovsky and meola, ) . however, this aea-tmof, with six prolines at its c terminus, is not cleaved by neb-ace (k. hens, personal communication) . recently, new substrates were purified from the ovaries of n. bullata. their primary structures were identified as nklkpswqwisl (neb-odaif), nklkpsqwislsd (neb-odaif- - ), nklkpsqwi (neb-odaif- - ), nklkpsq (neb-odaif- - ), slkpsnwltpse (neb-odaif- ), and leqiyhl. these peptides show significant homology to the n-terminal part of fly yolk proteins (hens et al., ; vandingenen et al., b) . kinetic analysis of these peptides with insect ace and human ace shows that the n. bullata ace shares enzymatical properties with the c-domain sace, in addition to features that seem unique to invertebrate ace. characterization of the ance protein demonstrated that this enzyme eyciently hydrolyzes angiotensin i and bradykinin (cornell et al., ; williams et al., ) . ance can operate as an endopeptidase, for instance on tachykinins, substance p, and leucokinins. still, the nonamidated form of a peptide is always preferentially cleaved over the amidated form. furthermore, insect ace preferentially acts as a dipeptidase and shows reduced tripeptidase activity. of the insect peptides tested, locustatachykinin (lom tk- ) proved to be the best substrate for ance. however, the d. melanogaster tachykinins turned out to be poor ance substrates . acer is not capable of cleaving angiotensin i (houard et al., ) , and most peptides tested were hardly degraded by acer . to this day, there are no clues concerning the identity of the endogenous acer substrates. the structural and biochemical similarities between mammalian and invertebrate ace do not necessarily imply a functional conservation as well. although the presence of an invertebrate ras has been reported in a leech that feeds on ducks (laurent et al., ; salzet, a,b, ) , no other components of the ras other than ace have been described in insects or other invertebrates. it is therefore highly improbable that this invertebrate enzyme would physiologically act as a converting enzyme for an angiotensin-like peptide. as already mentioned, functionality of mammalian ace is, however, not restricted to the ras. both its broad substrate specificity and widespread tissue distribution indicate its involvement in several other (but, unfortunately, still poorly described) physiological functions. to fully unravel the in vivo role of ace in insects, information about substrate specificity and tissue and cellular localization of the enzyme, as well as its putative colocalization with its substrates, has to be combined. these data, together with the studies of ace mutants and the evects of ace inhibitors on insect physiology, will eventually lead to the elucidation of the roles of insect ace. today's concept on insect ace functionality focuses on its possible role in reproduction, development, and defense. the discovery of ace in testes of several insect species, including h. irritans exigua (wijvels et al., ) , lymantria dispar (loeb et al., ) , n. bullata, the colorado potato beetle leptinotarsa decemlineata, and the locust l. migratoria schoofs et al., ) , implies that the function of tace may have been conserved during the course of evolution. in l. decemlineata, ace was detected in the germ cells, more particularly in developing spermatids and mature spermatozoa, whereas in l. migratoria, ace was found in peripheral somatic cell bodies of the apical compartment of the testicular follicles and not in the germ cells. in the haematophagus fly h. irritans, expression of testicular ace is induced after a blood meal, indicating a specific role in the maturation of spermatozoa (wijvels et al., ) . the possible importance of testicular ace in reproduction became apparent by the observation that male d. melanogaster transheterozygotes of two mutant ance alleles were found to be sterile (tatei et al., ) , with the infertility linked to the failure of the spermatocytes to develop into active mature spermatozoa. spermatogenesis proceeds normally in the mutants up to the spermatid stage, but these spermatids do not succeed in diverentiating into spermatozoa. this change is accompanied by nuclear scattering and an abnormal morphology of the spermatids. together with the observed expression pattern of ance, the enzyme is believed to be required for spermatid diverentiation through the processing of a regulatory peptide synthesized within the developing cyst (hurst et al., ) . the involvement of ace in the control of spermatogenesis was also demonstrated by studies of the evects of aii and bovine ace on the ability of the testes of l. dispar to synthesize and release ecdysteroids in response to testis ecdysiotropin (te). the evect of te on the initiation and up-regulation of ecdysteroid synthesis could be imitated by both aii and bovine ace, raising the possibility that ace is involved in the production of an aii-like peptide that modulates ecdysteroid synthesis in the testes of insects (loeb et al., ) . in female adults of the mosquito a. stephensi, ace activity increases by a factor . after a blood meal. it is not known where this induced ace is synthesized, but it accumulates in developing ovaries and in mosquito eggs . adding ace inhibitors (captopril and lisinopril) to the blood meal of the mosquito leads to a size reduction of the batch of eggs laid in a dose-dependent manner. the ace inhibitors reduce fecundity by interfering with the transfer of the oocytes along the oviducts (ekbote et al., a) . in the reproductive tissue of the tomato moth lacanobia aleracea, almost all of the ace activity is concentrated in the accessory glands of the male and in the spermatheca and bursa copulatrix of the (virgin) female. these high activities may imply the importance of ace for peptide metabolism in the male and female reproductive tract. changes in the levels of ace activity in the reproductive tissues of males and females during mating, point toward a transfer of ace from male to female during time of copulation (ekbote et al., b) . this male ace donated in the spermatophore might form a hitherto unknown male component of an energy-producing metabolic pathway, as is seen in the serine endopeptidase-dependent proteolytic cascade in the spermatophore of b. mori (aigaki et al., (aigaki et al., , . in the fleshfly n. bullata, feeding of captopril results in an increase of vitellogenin titers in the fly hemolymph. it has been suggested that this evect is mediated through the trypsin-modulating oostatic factor tmof. as already discussed, neb-tmof is an in vitro substrate of insect ace. however, if tmof were to be inactivated by ace, inhibition of ace would lead to an increase of tmof levels in the hemolymph, resulting in a reduced vitellogenin synthesis. therefore, an activating evect of ace on tmof is postulated . the discovery of ace interactive peptides in the ovaries of the fleshfly (vandingenen et al., a) , and the discovery that these peptide substrates originate from yolk gene products, strengthen the idea that ace is a regulator of vitellogenic and developmental processes (hens et al., ; vandingenen et al., b) . these physiological observations, in combination with the discovery of ace protein in the gonads of numerous insects, undoubtedly show that ace is a vital enzyme in insect reproduction. the further elucidation of the full role of ace in this process necessitates including the description of all endogenous substrates. the observation that some mutations in the d. melanogaster ance gene result in death during larval/pupal stages (tatei et al., ) indicates that insect ace is important for normal growth and development. many reports from diverent and independent laboratories have confirmed the role of insect ace in development and metamorphosis. along with the cloning of the first d. melanogaster ace homolog (race, ance), the gene coding for this protein was shown to be a target of the homeobox regulatory gene zerknullt (zen) and decapentaplegic (dpp) (tatei et al., ) . a -bp-long enhancer located in the ance promotor region covers three zen protein binding sites and was shown to mediate selective expression in the amnioserosa. during early development, ance is activated by zen and becomes associated with the diverentiating amnioserosa and with the anterior and posterior midgut, where it persists throughout embryogenesis. in late embryos, ance expression is detected in epithelial cells of the midgut and in the pericardial cells of the heart. ance expression is lost from the presumptive amnioserosa in zen À and dpp À mutants; the expression in the gut stays unavected. in d. melanogaster larvae, high expression of ance is found before and peaks during pupal development, indicating a physiological role for the enzyme during metamorphosis (wilson et al., ) . also, in the tomato moth, l. oleracea, ace activity increases approximately fourfold in the last larval instar and in the early pupal stage (ekbote et al., b) . in insects, each larval molting is preceded by high ecdysteroid titers, and for pupal and adult development, multiple pulses of ecdysteroid hormones are essential. in wing discs of b. mori, the ace gene (bmacer) was found to be directly -hydroxyecdysone ( e) inducible in a dose-dependent manner. because this ecdysteroid-dependency was not found in other tissues examined, transcription of bmacer seems to be organ specific . during the transition from larva to pupa in d. melanogaster, the expression of ance in imaginal disc cells was found to be induced by ecdysteroids. these data point toward a fundamental role for ace during metamorphosis of holometabolous insects. several relationships between ace and metamorphosis are possible: the conversion of precursors into biologically active peptides necessary for metamorphosis, inactivation of signaling peptides, or recycling of amino acids from larval proteins for the synthesis of pupal and adult cuticle/tissues. in a. stephensi, ace expression is induced by a blood meal and ace accumulates in developing ovaries, from which it passes into the mosquito eggs . this indicates a role for ace-generating peptides that are essential for oocyte development and embryogenesis. the characterization of acn- in the nematode c. elegans shows that it is not necessarily the proteolytic function of ace that is involved in developmental processes. the hypodermal expression of acn- is under the control of nuclear hormone receptors that regulate molting in c. elegans. functional acn- knockout by rnai causes morphological defects in larvae and adults. more specifically, acn- was found to be required for larval molting, male tail development, and formation of adult alae (brooks et al., ) . prohormone processing? in n. bullata, l. migratoria, l. decemlineata, l. maderae, the walking stick insect carausius morosus, b. mori, and the caterpillar mamestra brassica, ace immunoreactivity is seen in neuropil regions of the brain. in l. migratoria, l. maderae, b. mori, and m. brassica, ace immunoreactivity is also present in neurosecretory cells of the brain . in l. migratoria, immunoreactive staining can also be traced in the controlateral nervus corporis cardiaca i (ncc i), corpora cardiaca (cc), and suboesophageal ganglion (sog), and in c. morosus, l. decemlineata, and l. maderae, ace immunoreactivity was also detected in the corpora cardiaca (veelaert et al., ) . the presence of ace in neurosecretory cells and neuropil regions of the insect brain points toward a dual role for ace in the insect nervous system . the localization of ace in the neuropil indicates the metabolic inactivation of peptidic neurotransmitters. the presence of peptides from the adipokinetic hormone (akh), kinin, and locustatachykinin family in the central nervous system (cns), as well as the knowledge that these peptides are hydrolyzed by ace (lamango et al., ) , indicates that they might be potential in vivo substrates for insect neuronal ace. in neurosecretory cells, ace is likely to be involved in the conversion of prohormones to active peptide hormones that are secreted into the hemolymph by the corpora cardiaca or corpora allata (isaac et al., b) . because insect ace has been shown to exert in vitro prohormone processing activities against the locustamyotropins, and locustamyotropin-containing cells of the locust brain and suboesophageal ganglion also contain ace, this insect enzyme most probably plays a role in the biosynthesis of these hormones (veelaert et al., ) . in contrast to the situation in vertebrates, which have a dual system of immune reaction, namely, an innate and an acquired one, invertebrates only rely on their innate immune responses. this system comprises both humoral and cellular defense responses. well-known humoral responses include the synthesis of a broad spectrum of antimicrobial peptides/proteins (bulet et al., ) , as well as two proteolytic cascades: the blood coagulation system identified in the horseshoe crab (iwanaga, (iwanaga, , and the prophenoloxidase-activating system (pro-po-as) in insects and crustaceans (sö derhäll and cerenius, ) . cellular defenses cover hemocyte-mediated responses like phagocytosis, nodulation, and encapsulation. in insects, a large number of peptides is released into the hemolymph, where they are known to regulate a large diversity of physiological functions. the half-life of these peptides can be regulated by proteases and peptidases present in the hemolymph. the discovery of ace activity in the hemolymph of insects , together with the knowledge of putative prohormone converting activity of ace, indicated the possible involvement of ace in the proteolytic processing of insect antibacterial peptides from their precursors (isaac et al., a) . supportive data concerning this hypothesis came from expression studies in larvae from the sheep blowfly lucilia cuprina and the old screwworm chrysomya bezziana, which are known to secrete or excrete chymotrypsins and trypsins in larval cultures and wound sites. the knowledge that an ace homolog is also secreted or excreted from these larvae indicated a dual role in wound formation. it may degrade chymotryptic and tryptic digestion products from host tissue proteins and may influence the host vascular and inflammatory response by processing or degrading host regulatory peptides (wijvels et al., ) . in the endoparasitic wasp, pimpla hypochondriaca, the female envenomates and oviposits into pupae of some lepidopteran species. this venom was shown to suppress the hemocyte-mediated immune responses of the host by impairing encapsulation and phagocytosis (parkinson et al., ; richards and parkinson, ) . the presence of ace in this venom, together with the presence of antibacterial activity, implies that ace could be one of the enzymes involved in the processing of antibacterial peptides from the venom sac (dani et al., ) . all the mentioned data indeed point toward an involvement of ace in the defense system, but they are still only hypothetical. the first physiological evidence for the existence of a connection between ace and defense came from studies in the locust l. migratoria. locusts that are subjected to an immune challenge through injection of lipopolysaccharides (lps) into the hemocoel showed an increase in ace mrna-expression. comparing the level of ace transcription from lps-treated animals with control animals revealed an approximately -fold increase in ace transcription hours after the lps-injection (macours et al., b) . the exact nature of the ace-defense connection remains to be clarified. in mammals, all blood cells derive from hematopoietic stem cells that diverentiate into diverent lineages. the involvement of ace here resides in the inactivation of ac-sdkp (n-acetyl-seryl-aspartyl-lysyl-proline), a peptide that prevents the recruitment of hematopoietic stem cells (azizi et al., ; baudin, ) . insects also continue to produce hemocytes via a division of stem cells or by continued division of hemocytes in circulation (jones, ; ratclive et al., ) . on the basis of this information, a possible connection between ace and the immune system can be suggested in the replenishment of the hemocytes that are involved in the insect immune response. the possibility of ace being involved in the processing of antimicrobial peptides in locust hemolymph cannot be excluded, but so far, except for lysozyme and coagulogen, no major antibacterial activity could be detected in locust hemolymph (van sambeek and weisner, ). the rise in ace expression that follows lps injection is not likely to result from an involvement of ace in the prophenoloxidase cascade, as this pathway is not activated by injection of lps. however, the immune system of the locust responds to lps injection through the formation of nodules (goldsworthy and chandrakant, ; hovman et al., ) . therefore ace may be required in the process of nodule formation. this speculation is in agreement with the recent findings of goldsworthy and colleagues ( ) , who investigated the evect of lps on the prophenoloxidase cascade and nodule formation in the locust. activation of prophenoloxidase by lps could only be achieved by coinjection with akh, not by the injection of lps alone. when captopril was coinjected, the activation of the ppo cascade remained unavected (with/without akh). however, captopril did inhibit the nodule formation induced by lps (goldsworthy et al., ) . considering these data, a possible role for ace in the immune system of insects may reside in the regulation of nodulation. to date, leeches are the only group of invertebrates in which the existence of a renin-angiotensin system has been reported. in theromyzon tessulatum, a parasite of ducks, an angiotensin ii (aii)-like peptide was isolated (salzet et al., ) . the full sequencing of such aii-like peptide was realized in another leech species, namely erpobdella octoculata . this leech aii peptide divers from the vertebrate aii by a c-terminal amidation. injection of this aii into the leech results in a decrease in mass, reflicting a diuretic evect. also in t. tessulatum, a renin-like peptide was isolated (laurent and salzet, a) and partial peptidic sequences were identified. this enzyme was found capable of releasing ai from an ai-like precursor, angiotensinogen (laurent et al., ) , by cleavage of the leu -leu bond. the mass of this invertebrate renin (ca. kda) was lower than that of vertebrate active renin (ca. kda) but displayed a comparable activity. the leech renin is localized in the excretory and nervous system, which implies the involvement of the leech ras in osmoregulation. the angiotensin-converting enzyme of this leech-ras is a glycosylated membrane-bound enzyme of kda that is released into the hemolymph in a hydrophilic form ( kda) (laurent and salzet, ; vandenbulcke et al., ) . the presence of this ras in coelomocytes of leeches and the involvement of each part of this system in the immune response inhibition indicates the implication of the ras in host-parasite cross-talk (salzet and verger-bocquet, ) . endothelin-converting enzymes (eces) belong to the class of type ii integral membrane zinc metalloproteases named for their ability to hydrolyze big endothelins (big ets) into the smaller vasoactive endothelins. yanagisawa and colleagues ( ) were the first to predict the existence of eces based on the cloning of the prepro-endothelin gene. ece is classified into the neutral endopeptidase or m group of proteins, which contains type ii membrane glycoproteins with zinc peptidase catalytic activity. at present mammalian m family of zinc proteases consists of seven known members: neutral endopeptidase (nep); the endothelin-converting enzymes ece- , ece- , and ece- ; the kell blood group antigen (kell); the phosphate regulating gene (pex); x-converting enzyme (xce); and secreted endopeptidase (sep). each member of this family shows homology to the others, mainly in the c-terminal part of the sequence that is responsible for catalytic activity. in addition, there are structural similarities in that all m members have short intracellular domains. where identified, these enzymes have roles in the processing or metabolism of regulatory peptides and therefore represent potential therapeutic targets. ece- is a membrane-bound protein with a short n-terminal cytoplasmatic tail, a transmembrane hydrophobic domain, and a large extracellular domain, which shares high sequence homology to nep and other members of the metallopeptidase family. ten cysteine residues in the ece sequence are conserved among the other members of the family and are probably involved in disulfide bridges. the ece protein has predicted sites for n glycosylation in the extracellular domain. this is consistent with the observation that ece is a highly glycosylated protein and explains the diverence between the predicted molecular mass and the mass shown on sds-page. purification and cloning studies of rat ece- have shown that it consists as a disulphidelinked dimer, in which cys is solely responsible for the formation of the intermolecular disulphide bond (shimada et al., ) . the large extracellular domain contains the zinc-binding consensus sequence hexxh, in which the glutamic acid (e) is the most important residue for catalytic activity. the two flanking histidine (h) residues act as zinc coordinating amino acids. the third ligand for the zinc atom is e , whereas h is involved in the stabilization of the tetrahedral intermediate during the transition state (shimada et al., ) . four ece- isoforms have been identified in humans and were named ece- a ( amino acids), ece- b ( amino acids), ece- c ( amino acids) and ece- d ( amino acids) (jafri and ergul, ; valdenaire et al., valdenaire et al., , a . the four isoforms are transcribed from a single gene and result from the use of alternate promoters located upstream of specific exons. they only diver in the first half (approximately) of their cytoplasmatic tail and are identical over their remaining sequence, including the enzymatic catalytic site. as a logical consequence, the isoforms display comparable converting activity. the specificity of the isoforms thus lies in their cytosolic tails, which results in a diverent subcellular distribution. whereas ece- a and ece- c are present in the plasma membrane, ece- b and ece- d are retained inside the cell. recently it was shown that ece- isoforms can heterodimerize. it was suggested that heterodimerization of ece- isoforms could constitute a means for regulating their subcellular distribution and, subsequently, their extracellular activity (muller et al., ) . considering the importance of the biosynthesis of both the intracellular and extracellular endothelins, the subcellular distribution of ece isoforms may play a central role in the regulation of the endothelin system. the endothelin system is composed of endothelin (et) peptides and their receptors (fig. a) , which together avect a wide variety of physiological functions and pathological conditions. the discovery of this system started in the mid- s, when an increasing awareness of the role of endothelial cells as active components of the vascular system led to the description of an endothelial cell-derived constricting factor (hickey et al., ) . in , a -amino acid vasoconstricting factor termed ''endothelin'' was isolated from cultured porcine aortic endothelial cells (yanagisawa et al., ) . the expression of et is associated with many pathological processes and with tumor growth. to fulfill their wide spectrum of physiological functions, ets act through autocrine and paracrine mechanisms. their biosynthesis thus requires tight local control. endothelin- (et- ) is a -amino acid peptide with a hydrophobic c terminus. within year of its discovery, two structurally related peptides were identified and termed et- and et- , respectively. the three isoforms are encoded by three diverent genes and show highly conserved sequences in several species. all isoforms are composed of amino acids with two intrachain disulfide bridges. et- exhibits the closest structural similarity to et- , divering by only three amino acid residues, whereas et- divers by six amino acids. et isoforms are widely distributed among various cells and tissues. of the three isoforms, the most widely distributed, best studied, and most potent is et- . et- shares great structural homology to sarafotoxin, a snake (atractapsis engaddensis) venom that induces myocardial infarction by exaggerated contraction of the cardiac vessels and interruption of the blood supply to the heart. ets are produced by a variety of organ and body tissues such as the lung and kidney as well as the brain, pituitary, peripheral endocrine tissues, and placenta. most abundantly, endothelial cells of the vascular endothelium produce et. et binds to two types of receptors (et a and et b ) (arai et al., ; sakurai et al., ) . both contain seven transmembrane domains and activate an overlapping set of g proteins, producing an array of diverent physiological responses (douglas and ohlstein, ) . although structurally highly similar, these receptors possess diverent aynities for the three isoforms of et. like many other regulatory peptides, the ets are maturated through proteolytic processing from larger precursor polypeptides, termed ''prepro-ets.'' this proteolytic cleavage generates an essentially inactive intermediate referred to as big et. the subsequent hydrolysis of big et into the final active product et, and a presumed inactive c-terminal fragment, is catalyzed by ece (fig. a) . in the case of big et- and et- , proteolysis occurs at the trp -val bond. et- is formed through cleavage of the trp -ile bond of big et- (inoue et al., ) (fig. b) . the conversion of big et to et can occur in the extracellular medium and in the secretory pathway; cells thus secrete big ets either alone or together with endothelins . the physiological importance of the cleavage of big et is indicated by a -fold increase in vasoconstrictor potency on cleavage to et. this makes ece an important activating protease in the biosynthetic pathway of et. ece- null mice exhibit a phenotype similar to that of et- -or et a -deficient mice, demonstrating the significance of ece- in generating bio-available et- (yanagisawa et al., ). the endothelin system plays a variety of roles in both normal physiology and pathological conditions in a number of tissues/organs of the body. in blood vessels, et maintains a basal level of vasoconstriction and is involved in the development of hypertension, atherosclerosis, and vasospasm after subarachnoid haemorrhage. in the heart, the endothelin system avects force and rate of contraction and mediates hypertrophy and remodeling in congestive heart failure. in lungs, et regulates the tone of both airways and blood vessels and is involved in the development of pulmonary hypertension. kidney-et regulates water and sodium excretion and acid-base balance, and it participates in the pathophysiology of acute and chronic renal failure. in the brain, the et system modulates cardiorespiratory centers and hormone release (kedzierski and yanagisawa, ) . in ovaries, et is shown to play a role in regulating the female reproductive cycle, where it functions as an important component of the luteolytic cascade (levy et al., ) . egidy and colleagues ( ) suggested that et, as a powerful vasoconstrictor and mitogenic peptide that is produced by many cell lines, might play a role in cancer progression of the colon. studies by johnson and colleagues ( ) revealed that ece- possesses a broad in vitro substrate specificity and is potentially involved in the metabolism of peptides distinct from et. they found that neurotensin, substance p, bradykinin, and the oxidized insulin b chain are in vitro substrates for ece- . given this in vitro substrate specificity, it is likely that ece- is involved in the degradation and processing of many biologically active peptides, both at the cell surface and in the secretory pathway (johnson et al., ) . a second form of ece, ece- , was cloned from bovine adrenal cortex and was reported to possess a % identity with ece- . the main structural features of ece- are conserved in ece- (emoto and yanagisawa ) . both are type ii integral membrane proteins with the hexxh consensus sequence. the cysteine residues conserved between ece- , nep, and kell, are also conserved in ece- , as is the cysteine residue responsible for dimerization of ece- . ece- is also heavily glycosylated, with possible glycosylation signals. although ece- shows specific activity to produce mature et- from big et- , like ece- , the striking diverence between ece- and ece- is the acidic ph optimum of . of ece- , in contrast to the neutral ph optimum of ece- . this indicates that ece- is involved in et synthesis in the intracellular compartments in which the ph is acidic (emoto and yanagisawa, ) . ece- has been reported to be expressed in various organs and tissues including cultured human vascular endothelial cells, uterus, ovary, heart, lung, and liver, indicating various functions for the enzyme. recent targeting studies with mouse ece- revealed that it plays crucial roles in the formation of cardiac outflow structures (yanagisawa et al., ) . four subisoforms of ece- that diver only in their n-terminal cytoplasmatic tails were termed ece- a- , ece- a- , ece- b- and ece- b- , ece- a- and ece- a- are expressed in a variety of tissues including liver, kidney, adrenal gland, testis, and endothelial cells. ece- b- and ece- b- are abundantly expressed in the brain and adrenal gland (ikeda et al., ) . ece- was purified from bovine microsomes. this enzyme was found to be specific for the conversion of big et- to et- and was inhibited by phosphoramidon (hasegawa et al., ) . to the best of our knowledge, this is the only report on an ece- enzyme. neutral endopeptidase (neprilysin, nep), a - -kda plasma membrane protein, is the prototype and best characterized member of the m zinc metallopeptidase family. nep is identical to the neutrophil and cluster-diverentiation antigen cd and is also known as the common acute lymphoblastic leukemia antigen (calla), which is mainly associated with the precursors of b lymphocytes (letarte et al., ) . purification and subsequent cloning of nep revealed it to be a type ii integral membrane protein of approximately residues. neprylisin's extracellular domain, which includes the active site, also contains cysteine residues, of which are involved in highly conserved disulfide bridges that are important in the maintenance of structure and activity of the enzyme (oefner et al., ) . nep comprises three subisoforms that result from alternative splicing in the untranslated region, indicating that these spliceforms do not exhibit functional diverences (ishimaru and shipp, ) . regarding its catalytic activity, nep shares some similarity with the bacterial zinc metalloproteinase thermolysin, and in particular in its substrate specificity (malfroy et al., ) . nep exists as an ectoenzyme that preferentially hydrolyzes extracellular oligopeptides (< kda) on the amino side of hydrophobic residues. nep is the only enzyme in this family of which the x-ray crystallographic structure has been determined. the structure revealed a restricted active site cleft preventing access of large peptides and proteins, explaining its oligopeptidase character (oefner et al., ) . nep is primarily expressed in the kidney, where it has the capacity to cleave and inactivate the natriuretic and vasodilatory peptide (anp), which controls arterial pressure (beaulieu et al., ) . the first physiological function of nep was found in the brain, where nep is located in neuronal cells. there, it controls the half-life of enkephalins and substance p (two neuropeptides involved in pain control) through inactivation (malfroy et al., ) . nep also plays a pivotal role in various cancers, such as prostate cancer and leukemia (letarte et al., ; papandreou et al., ) . a variety of other physiological substrates has subsequently been revealed for nep (turner and tanzawa, ) , although most attention has been focused on the vasodilator anp. the development of selective nep inhibitors, of which the best studied are thiorphan and phosphoramidon, has primarily been driven by their potential as novel cardiovascular agents (roques and beaumont, ) . the medicinal treatment of various azictions with nep inhibitors has become so prevalent that they now constitute a class of drugs used in both gastroenterology and cardiology (tanja et al., ) . there is, however, a potential downside to the clinical use of nep inhibitors, as downregulation of nep levels is seen in various cancers and in the lung, where it potentiates neurogenic inflammatory responses. recent reports that a nep-like enzyme can degrade the neurotoxic b-amyloid peptide involved in alzheimer's disease raises new concern about chronic blockade of nep-like activity in the cns . the kell blood group system is one of the major antigenic systems in human erythrocytes. over diverent blood group antigens have been assigned to this system, and the molecular basis for this polymorphic variation has been shown to be the result of single-base mutations in the kell gene, resulting in the diverent kell phenotypes (lee, ) . the protein is of clinical importance because incompatibility involving kell antigens can cause severe hemolytic reactions to blood transfusions and leads to fetal and neonatal anemia. kell is a -kda type ii erythrocyte membrane glycoprotein that is attached to the cytoskeleton and that comprises a short n-terminal intracellular segment, a single transmembrane sequences and a large, -amino acid extracellular domain. in the c-terminal third of the molecule, kell shares a - % amino acid identity with human nep, ece- , pex, and xce (lee et al., ) . kell has cysteine residues from which the cysteine residues that are preserved in all members of the m family are conserved. one of the cysteine residues, cys , forms a disulfide band with the xk protein, which spans the membrane times (russo et al., ) . the absence of xk, which occurs in the mcleod patients, is correlated with acanthocytic red blood cells and a late-onset form of nerve and muscle disorder . studies using a recombinant, truncated form of kell, lacking the intracellular and transmembrane domain (skell), have shown that kell possesses converting enzyme activity for the substrate big et- . conversion of big et- and big et- is much less eycient. the known substrates for nep are not hydrolyzed by skell. individuals lacking kell do have a normal et- biosynthesis, so the protein probably has other substrates than big et- and other biological functions . the ''phosphate-regulating gene with homology to endopeptidases on the x chromosome,'' pex, was identified from studies of patients with x-linked hypophosphataemic rickets (hyp consortium, ) . hyp is the most common inherited disorder of renal phosphate wasting characterized by hypophosphatemia and defective bone mineralization (scriver and tenenhouse, ) . it is postulated that the substrate of pex is an unidentified peptide hormone (termed ''phosphatin'') that modulates renal tubular phosphate handling. such an activity could involve either the processing of a phosphate-reabsorbing hormone precursor to its active form or the inactivation of a circulating phosphaturetic factor. in situ hybridization revealed the presence of pex in osteoblasts and odontoblasts, implicating the enzyme in the development of bones and teeth (ruchon et al., ) . in addition to these speculations, the physiological function of the pex protein in bone development and its relation to the mechanisms that lead to renal and skeletal abnormalities remain to be defined. cloning of the full-length pex cdna revealed it to be a type ii integral membrane protein with a -residue n-terminal cytoplasmatic tail and a c terminus of amino acid residues in the extracellular compartment. recombinant pex functions as an endopeptidase by hydrolyzing parathyroid-hormone-derived peptides, although it is unlikely that these are physiological substrates, as they do not have the properties of the sought-after phosphatin (lipman et al., ) . a novel member of the metalloprotease family was isolated from mouse embryos in search for an alternative ece enzyme (ikeda et al., ) . this enzyme, called sep (soluble secreted endopeptidase) shares higher structural ( % identity, % similarity) and functional similarities with nep than with eces or other members of this family, indicating that sep and nep may constitute a subfamily within this group of metalloproteases. similar enzymes have subsequently been cloned and characterized from mouse testis (referred to as neprilysin-like ; nl ) (ghaddar et al., ) and rat brain (nep ii) (tanja et al., ) . in vitro alternative splicing of sep yields two isoforms, one membranebound isoform (sep Á ) and another isoform that is secreted into the culture medium (sep). cellular expression of the cdna showed that most of the enzyme is secreted into the culture medium. after translation, both isoforms are inserted into the er as membrane type ii proteins. sep Á then becomes an er resident, whereas sep is proteolytically cleaved and transported to the extracellular compartment (raharjo et al., ) . the presence of a furin cleavage site in the nl sequence indicated that a member of the substilinlike family of convertases is responsible for this secreted form (ghaddar et al., ) . in vitro enzymological analysis shows that sep can eyciently hydrolyze circulating vasoactive peptides, including et- , angiotensin-i, anp, bradykinin, and substance p. the physiological relevance of sep mainly remains to be determined. its presence in testis and brain, however, indicates roles in fertility and neuropeptide inactivation, respectively, as it appears to localize in specific neuronal populations in the cns (tanja et al., ) . x-converting enzyme (xce) got its name from the fact that it is an orphan peptidase for which no substrate has as yet been identified. it was originally identified by homology cloning from a human brain cdna library. it is most abundant in the cns (valdenaire et al., b) . the -amino acidlong xce is more homologous to ece- ( % identity in the last residues) than to nep or any other member of this family. a rat neuronal membrane-bound endopeptidase termed dine (damage-induced neuronal endopeptidase), which is upregulated in response to neuronal injury and that was identified by diverential display pcr, appears to be the rat homolog of human xce (kiryu-seo et al., ) . therefore, it has been suggested that this enzyme may play a role in protecting injured neurons against neuronal death. although the conservation in the xce sequence clearly indicates a peptidase function for xce, its substrate remains to be identified. candidate substrates, for which no cleavage by recombinant xce could be detected, include et- , big et- , calcitonin, bradykinin, enkephalins, and galanin. inactivation of the xce gene in mice resulted in neonatal lethality caused by respiratory failure (schweizer et al., ) , indicating a role in respiration control. involvement of xce in defects in synaptic connections or transmission is a particularly attractive hypothesis because of the specific neuronal expression pattern of xce (in the medulla oblongata), together with its putative metaiopeptidase nature, indicating that one or more neuropeptide transmitters can be considered as potential candidate substrates for this enzyme. for many years, knowledge about the m family of metalloproteases was limited to vertebrates. nowadays, the number of research groups reporting on the presence of these enzymes in nonvertebrate organisms is increasing. this increase began with the notification of an endopeptidase-like activity in several invertebrates and has recently evolved into the direction of molecular identification. early reports described nep-like activity/homologs in invertebrates, but in recent years it has become clear that the presence of true ece activity/homologues in invertebrates also can no longer be denied. the distinction between a nep and ece homolog is not always easily made, as both enzymes share high sequence similarity and similar enzymatic properties. however, putative diverences between the two enzymes have come forward. in vertebrates, nep activity is discriminated from ece activity on the basis of their diverence in susceptibility to the inhibitors thiorphan and phosphoramidon. however, most of the data available on endopeptidase-activity in invertebrates reveal that these inhibitors are not always as eycient as in vertebrates. furthermore, the distinction between these inhibitory profiles does not always seem to be straightforward. because no expression studies with an ece-like protein in invertebrates have as yet been done, at present there is no clarity in this matter. on the molecular level, the distinction between nep and ece is made in the et-binding domain. this sequence, nayy in vertebrate ece, is replaced by a nafy sequence in nep. this knowledge came from mutational studies with vertebrate ece that have shown that the replacement of the first tyrosine by a phenylalanine in ece leads to an -fold reduction of the v max /k m for the conversion of big et- to et- (sansom et al., ) . to date, it is not clear whether this big et substrate of ece is present in invertebrates. the analysis of ets in nonvertebrates is mainly limited to immunocytochemical studies. a small number of studies have indicated the presence of endothelin in invertebrates, using antibodies to vertebrate endothelins (hasegawa and kobayashi, ; kohidai and csaba, ; montuenga et al., ) . neuropeptide-degrading endopeptidase activity was found in synaptic membranes of the locust, schistocerca gregaria. the locust adipokinetic hormone (akh- ) was used as a substrate. it was inactivated by cleavage at the asn-phe bond, resembling a cleavage pattern typical for mammalian endopeptidases. phosphoramidon partially inhibited this endopeptidase activity. this was the first time that the presence of an endopeptidase-like activity in insects was reported. the activity found in this insect species was less susceptible to inhibition by phosphoramidon and thiorpan compared to its mammalian counterparts. this may reflect a diverence in inhibitor binding (isaac, ) . the enzyme responsible for the inactivation of the neurohormone akh is located on the surfaces of tissues; this degradative activity could not be detected in hemolymph (o'shea and rayne, ) . in the mollusc aplysia californica, endopeptidase activity was detected with the use of the substrate leu-enkephalin. this activity could be inhibited by thiorphan and phosphoramidon for approximately % (bawab et al., ; zappulla et al., ) . the presence of an identical endopeptidase-like activity was confirmed by studies in d. melanogaster wilson et al., ) , m. domestica (lamango and isaac, ) , l. dispar (masler et al., ) , l. maderae, and l. migratoria . these researchers studied the cleavage of either akh or locustatachykinin (lomtk)-degradation. in d. melanogaster, the degradation of lomtk- by head membrane fractions yields two main fragments, fygvramide and gpsgfyg. the use of the nep inhibitor phosphoramidon led to % inhibition of the generation of the first fragment, whereas the formation of the second fragment was inhibited by only %. this points toward the presence of at least two distinct nep-like enzymes that are responsible for the degradation of lomtk- , the first resembling nep and the second one resembling ece, which is less susceptible to phosphoramidon . surprisingly, the evect of the inhibitor thiorphan, which allows diverentiation between mammalian nep and ece, was not examined in this study on d. melanogaster endopeptidases. the first evidence of an ece-like activity in insects came from studies in neuronal membranes from the locust l. migratoria, where the conversion of big et- to mature et- was found to be less susceptible to inhibition with thiorphan when compared to phosphoramidon inhibition (macours et al., c) . a second reason for not subscribing this activity to a typical nep enzyme comes from the fact that nep is known to rapidly degrade mature et- at multiple cleavage sites (vijayaraghavan et al., ) . also, the use of a fluorescent substrate highly selective for ece (luciani et al., ) confirmed the presence of ece-activity in the locust. this ece-like activity was found to be very abundant in the reproductive system (ovaries and testes) of the locust, indicating a role in reproduction and development. a lower but substantial amount of activity was found in midgut, brain, and fat body, so multiple functions of this enzyme in insects seem likely. the first invertebrate putative endothelin-converting enzyme homologue was cloned from the coelenterate hydra vulgaris. the coelentrates represent one of the oldest phyla of the animal kingdom. to the best of our knowledge, this is the only invertebrate organism, other than insects, from which an ece homolog has been cloned and reported. this sequence shows the highest percentage of identity ( %) to human ece- and a substantial amount of identity to the other members of the human m family. on the basis of this large sequence homology and the presence of the nayy motif, this molecule was classified as an ece homolog (fig. ) . the cystein that is responsible for the dimerization of the protein in vertebrates is absent in this invertebrate ece homolog, indicating that early forms of this enzyme existed in monomeric form. expression patterns of h. vulgaris ece during head and foot regeneration point toward a role for this enzyme in development. homogenates from this organism also contained immunodetectable levels of big et- and et- . human ets are able to induce contraction in h. vulgaris, indicating the involvement of a h. vulgaris endothelin-like system in contraction of the cytoskeleton (zhang et al., ) . however, human big et could not exert this evect on contraction, indicating that h. vulgaris ece does not properly convert human big ets or that h. vulgaris ece works intracellularly (sarras et al., ) . the unambiguous confirmation of the presence of a functional endothelin-like system in h. vulgaris will have to result from the molecular identification of h. vulgaris et and its precursor big et. sequencing of the entire genome of c. elegans in (the c. elegans sequencing consortium) revealed possible nep/ece-like genes in this organism. intriguingly, one of the c. elegans genes codes for a two-domain product (cf. ace in vertebrates). the temporal and spatial expression of five c. elegans nep/ece-like genes has been studied, revealing all five genes to be expressed in a tissue specific manner, indicating that these genes control diverent physiological processes (turner et al., ) . simultaneously with the detection of ece-like activity in the locust, the first insect ece homolog was cloned (macours et al., c) . the l. migratoria ece (lomece) sequence reveals a putative type ii transmembrane protein of amino acid residues, with a calculated mass of kda deduced from its longest open reading frame. the sequence contains a short n-terminal cytoplasmatic tail of nine amino acids, a hydrophobic stretch representing the putative membrane-spanning domain, and a large extracellular stretch of amino acids containing the characterizing active site hexxh. hence, the overall structure of this insect ece resembles that of mammalian ece. ten potential glycosylation sites are present in lomece, indicating that like vertebrate ece, the insect form is also highly glycosylated. among these glycosylation signals, asn and asn , which are known to be important for the bioactivity of mammalian ece- are conserved (nelboeck et al., ) . the putative zinc-coordinating residue glu , which is located residues downstream from the active site, compares with a -amino acid separation in mammalian ece (shimada et al., ) in contrast to mammalian nep, which possesses a -residue separation (hooper et al., ). the c-terminal cevw sequence, critical for proper enzyme maturation and activity (macleod et al., ) , is present in lomece, as is the endothelinbinding site nayy (fig. ) . the cysteine residue responsible for dimerization of the protein in mammalian organisms (shimada et al., ) is absent in lomece, supporting the hypothesis that early divergent forms of this enzyme may have existed in monomeric form (zhang et al., ) . the need for a dimeric form of ece for enzyme functionality was refuted by studies with solubilized recombinant ece- in which dimerization did not appear to be essential or preferential for full ece activity (korth et al., ) . studies with a monomeric and dimeric from of bovine ece demonstrated that these two forms posses a comparable biochemical profile. the major diverence between the two forms is that the dimeric form is found to be more eycient in catalyzing the conversion of big et- into et- (kulathila et al., ) , sustaining the possibility of other in vivo substrates for the invertebrate ece form. the fact that ece mrna is found in a variety of tissues in the locust is indicative of a major, and most probably diversified, biological role for the enzyme. in the d. melanogaster genome (adams et al., ; flybase consortium, ) , predictions of approximately members of the m family can be made (coates et al., b) . the identification and classification of these peptidase genes was performed with gene prediction strategies, sequence comparison with known genes, and searches for active site regions by use of the merops database (rawlings et al., ) . sequence comparison of these genes with members of the human metalloproteases family identified the cg sequence to posses the highest homology with human ece- (fig. ) . an m homolog that contained all the essential characteristic amino acids necessary for enzymatic activity was cloned from the cns of the slug aplysia californica. this molecule was assigned nep. the sequence contains the nayy sequence in combination with a -amino acid separation ( in vertebrate nep) between the active site and the third zinc-binding ligand, thus combining both ece and nep characteristics (fig. ) . the presence of nep in the central and peripheral nervous system may indicate that it could be a major player in the modulation of synaptic transmission in a. californica and in the metabolism of neuropeptides (zappulla et al., ) . in b. mori, an ecdysteroid-inducible neutral endopeptidase-like gene was cloned from the wing discs (bmnep). its sequence showed % identity with rat-nep. it contained the same zinc-binding motif (heith) as a mammalian nep and contained a -residue separation between the putative third zinc-coordinating ligand glu and the active site (fig. ) . hence, it was classified as a nep homolog. the expression of bmnep is induced by hydroxyecdysone ( e) and is suggested to be a secondary response gene, following the expression of bmacer, which starts within hours after e stimulation. from this point of view, it is suggested that this peptidase is involved in the activation or inactivation of peptides in wing discs, or peptide hormones secreted from diverent organs in the course of metamorphosis (zhao et al., ) . in the d. melanogaster genome, the cg and cg sequences display the highest homology to human nep. the m and m family of metallopeptidases contain both prokaryotic and eukaryotic members. in the genome of the bacteria, gloeobacter violaceus (accession bac ), shewanella oneidensis (accession np_ ), and xanthomonas axonopodis (accession np_ ), the predicted protein sequences of ace homolog resemble the single-domain form of invertebrate ace, with a possible signal sequence and the absence of a transmembrane domain at the c terminus of the enzyme. an invertebrate nep/ece homolog was discovered in the gram-negative bacterium, porphyromonas gingivalis. this is an important pathogen in the manifestation of acute periodontitis. this chronic inflammatory disease is the major cause of tooth loss in adults (havajee and socransky, ; socransky and havajee, ) . the proteases of these bacteria, in addition to their primary function in providing peptides for growth, are thought to be directly involved in tissue invasion and modulation of the host immune system (lamont and jenkinson, ) . the porphyromonas gingivalis pepo sequence (pgpepo) shows % sequence identity with human ece- and . % with human nep. instead of a membrane-bound domain, it contains a putative signal sequence of amino acids at the n-terminal end of the sequence, indicating that the protease could be secreted across the plasma membrane, possibly with pathological consequences as a result. all activityessential amino acids are present in the pgpepo sequence, but the cystein residues that are conserved between all members of the vertebrate nep/ece family are absent. because this also includes the absence of the cystein responsible for dimerization of the ece protein, the bacterial ece homolog exists in monomer form (awano et al., ) . the pgpepo protein displays a substrate specificity similar to mammalian ece. it converts big et- , - , and - into their respective mature endothelin peptides and cleaves substance p, angiotensin i and ii, and neurotensin at multiple sites. urotensin ii, angiotensin iii, and oxytocin are not hydrolyzed by pgpepo . the p. gingivalis bacteria were shown to succeed in invading and multiplication within aortic endothelial cells and are localized in atherosclerotic plaques (deshpande et al., (deshpande et al., - . because several associations between periodontal and coronary artery diseases have been made (beck et al., ) , invasion of these bacteria into cells was suggested to cause a distortion of the finely balanced equilibrium involved in et production. this may provide a possible link between periodontitis and cardiovascular diseases . experiments using a mutant form of bacteria, showing pepo-gene deficiency, showed a decreased invasion of such bacteria in mammalian cells. this indicates that pepo gene is at least involved in the first step of the bacterial cycle; namely, the invasion and lysis of the mammalian cell membrane (ansai et al., ) . in the merops database for peptidases (rawlings et al., ) , a large number of bacteria can be found that all contain at least one m homolog in their genome. also in archeae, m homolog are reported, but the majority of the archeae do not contain an m homolog in their genome. the presence of homolog of these enzymes in prokaryotes could perhaps have resulted from a horizontal exchange of genes between prokaryotes and eukaryotes (froeliger et al., ) . however, because members of the m family are present in eubacteria and some archeabacteria, as well as in eukaryotes, the possibility that an ultimate ancestor of the m family may have been present in the organism that preceded both modern bacteria and eukaryotes cannot be excluded. although the exact evolutionary relationship between eukaryotic and prokaryotic metalloproteases cannot yet be determined, the high level of sequence conservation indicates that these enzymes are under strong evolutionary constraints, thereby protecting structural features essential for enzyme activity. the amyloidogenic pathway of abpp (amyloid beta precursor protein) processing leads to the production of the neurotoxic amyloid b-peptide (ab or ab ). the formation of the ab peptides occurs through the initial cleavage by a b secretase, followed by the action of a g secretase (fig. ) . the pathological accumulation of ab in the brain (formation of plaques) is the primary cause of neuronal cell death and dementia in patients with alzheimer's disease (hardy and higgins, ) . the degree of ab accumulation in the brain is dependent not only on its production but also on its removal. the three zinc-metalloproteases nep, ece, and possibly ace have been put forward as ab degrading enzymes, thus limiting the amount of ab accumulation in the brain. in vitro assays revealed that ace is capable of preventing the aggregation of ab and prevents the deposition of ab onto a synthetic matrix. in addition, it inhibits ab fibril formation and ab-induced cytotoxicity (hu et al., ) . the catabolism of ab by ace occurs through a rather unusual cleavage, between the asp -ser bond of ab. however, infusion of ace inhibitors in the brain of abpp transgenic mice had no evect on the level of ab present in the brain. this raises some questions concerning the significance of ace in the ab catabolism in vivo. this is in contrast to the m metalloproteases, nep and ece, for which the in vivo involvement has been confirmed. in vitro models have characterized ece- as an ab-degrading enzyme that appears to act intracellularly, thereby limiting the amount of ab available for secretion. overexpression of ece- in cells that lack endogenous ece activity results in a pronounced decrease in ab accumulation in the conditioned media (eckman et al., ) . the physiological relevance of ece activity on ab catabolism was supported by the analysis of ab levels in the brain of mice deficient for ece- . here, significant increases in the levels of ab were detected (eckman et al., ) . nep cleaves the ab peptide at five possible positions, with gly -tyr being the initial cleavage site (howell et al., ; shirotani et al., ) . chronic injection of thiorphan in the rat brain leads to an accumulation of ab (iwata et al., ) . in nep-deficient mice, increased levels of ab are detected because of a reduction in the level of exogenous ab catabolism. in addition, areas in the brain of patients with alzheimer's that are enriched in plaques display reduced levels of nep (yasojima et al., a,b) . all this indicates that nep and ece are rate-limiting peptidases participating in ab catabolism (fig. ) . future research may include the upregulation of these metalloproteases as a promising strategy for the treatment and prevention of alzheimer's disease. for many years, the study of m and m metalloproteases in invertebrates lagged behind that in mammals. the first reason was that it was not logical to start searching for peptidic regulators of blood pressure in animals with an open circulatory system. second, many invertebrates are small, and harvesting enough tissue for the purification of enzymes is diycult and time consuming. molecular studies in invertebrates have expanded rapidly with the development of microtechniques that make it much easier to work with these fig. schematic representation of the synthesis of ab from its precursor. the shaded area represents the membrane-bound region. the cleavage of ab by nep, ece, and ace is shown by arrows. small quantities. many of the identified invertebrate genes are also known to be in vertebrate species, where they posses similar characteristics and functions. these observations support the fact that invertebrates provide excellent model systems for the study of genetics. by comparing the human genome with those of other organisms, regions of similarity and divergence can be identified, thereby providing critical clues about the structure and function of human genes. this information may be beneficial for developing better strategies for treating and preventing some human diseases. with each molecule that is sequenced, this approach becomes more powerful. the identification of ace and ece as key components in the regulation of the mammalian blood pressure system made them interesting targets for the development of eycient antihypertensive drugs. however, the large in vitro substrate specificity and the recent discovery of their involvement in alzheimer's disease asks for caution in the use of ace and ece inhibitors as therapeutic drugs. the large cellular and tissue distribution of ace and ece in the human body also points toward the possibility of as-yet-unknown physiological roles of these enzymes. the idea that invertebrate counterparts of these enzymes function as regulators of blood pressure is very unlikely. hence, these organisms can be useful in the clarification of these extra enzymatic functions. future research concerning the roles of these enzymes will have to involve the identification of new substrates and inhibitors. possible substrates can be tested on their in vivo characteristics by colocalization studies and knockout experiments. from the above listed data, it is clear that our knowledge about the occurrence and physiology of the m peptidase family in invertebrates is beginning to take shape, whereas research on the m peptidase family members is still in its infancy. available information on these enzyme families in invertebrates is restricted to ace, nep, and ece. however, the exploration of invertebrate genomes that have already been published indicates that a large number of m and m family members is present. further data mining of invertebrate genomes in combination with the recombinant expression of isolated sequences might lead to the identification of homologs to ace , kell, sep, xce, ece- , and ece- in invertebrates. the genome sequence of drosophila melanogaster a specific endopeptidase baee esterase, in the glandula prostatica of the male reproductive system of the silkworm, bombyx mori auginine carboxypeptidase activity in the male reproductive glands of the silkworm, bombyx mori construction of a pepo gene-deficient mutant of porphyromonas gingivalis: potential role of endopeptidase o in the invasion of host cells cloning and expression of a cdna encoding an endothelin a receptor sequencing, expression and biochemical characterization of the porphyromonas gingivalis pepo gene encoding a protein homologous to human endothelin-converting enzyme angiotensin i-converting enzyme and metabolism of the haematological peptide 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catabolism: roles for neprilysin (nep) and other metallopeptidases? characterization of pgpepo, a bacterial homologue of endothelin-converting enzyme- functional conservation of the active sites of human and drosophila angiotensin i-converting enzyme exploring the caenorhabditis elegans and drosophila melanogaster genomes to understand neuropeptide and peptidase function characterization of putative drosophila angiotensin converting enzyme cdna clones cloning and expression of an evolutionary conserved single-domain angiotensin converting enzyme from drosophila melanogaster peptidyl dipeptidase a: angiotensin iconverting enzyme angiotensinconverting enzyme is an essential regulator of heart function antibacterial and proteolytic activity in venom from the endoparasitic wasp pimpla hypochondriaca (hymenoptera: ichneumonidae) a story of two aces use of angiotensin-converting enzyme inhibitors in patients with diabetes and coronary artery disease invasion strategies of the oral pathogen porphyromonas gingivalis: implications for cardiovascular disease the secret life of ace as a receptor for the sars virus rxp , a phosphinic peptide, is a potent inhibitor of angiotensin i converting enzyme able to diverentiate between its two active sites a novel angiotensin-converting enzyme-related carboxypeptidase (ace ) converts angiotensin i to angiotensin - signal transduction mechanisms mediating vascular actions of endothelin degrading of the alzheimer's amyloid b peptide by endothelin converting enzyme alzheimer's disease b-amyloid peptide is increased in mice deficient in endothelin converting enzyme modulation of human colon tumor-stromal interactions by the endothelin system a mosquito (anopheles stephensi) angiotensin i-converting enzyme (ace) is induced by a blood meal and accumulates in the developing ovary ace inhibitors reduce fecundity in the mosquito angiotensin i-converting enzyme (ace) activity of the tomato moth, lacanobia oleracea: changes in levels of activity during development and after copulation suggest roles during metamorphosis and reproduction endothelin-converting enzyme- is a membranebound, phosphoramidon-sensitive metalloprotease with acidic ph optimum just the beginning: novel functions for angiotensin-converting enzymes mice lacking angiotensin-converting enzyme have low blood pressure, renal pathology, and reduced male fertility the flybase database of the drosophila genome projects and community literature streptococcus parasanguis pepo encodes an endopeptidase with structure and activity similar to those of enzymes that modulate peptide receptor signaling in eukaryotic cells newly recognized physiologic and pathophysiologic actions of the angiotensin-converting enzyme coronavirus spike proteins in viral entry and pathogenesis molecular cloning and biochemical characterization of a new mouse testis soluble zinc metallopeptidase of the neprilysin family the endocrinology of nodule formation in locusts in response to injections of microbial products interactions between the endocrine and immune system in locusts angiotensin-converting enzyme- (ace ): comparative modeling of the active site, specificity requirements, and chloride dependence microbial etiological agents of destructive periodontal diseases angiotensin-converting enzyme and male fertility alzheimer's disease: the amyloid cascade hypothesis quantitative mrna expression profiling of ace , a novel homologue of angiotensin converting enzyme purification of a novel endothelin-converting enzyme specific for big endothelin- immunohistochemical localization of endothelin- in the nervous system of the earthworm eisenia foetida characterization of four substrates emphasizes kinetic similarity between insect and human c-domain angiotensin-converting enzyme characterization of a coronary vasoconstrictor produced by cultured endothelial cells modifications of the hemogram and of haematopoietic tissue of male adults of locusta migratoria (orthoptera) after injection of bacillus thuringiensis sars coronavirus: a new challenge for prevention and therapy membrane protein secretase the drosophila melanogaster-related angiotensin-i-converting enzymes acer and ance. distinct enzymic characteristics and alternative expression during pupal development neutral endopeptidase can hydrolyze betaamyloid( - ) but shows no evect on beta-amyloid precursor protein metabolism angiotensin-converting enzyme degrades alzheimer amyloid beta-peptide (a beta); retards a beta aggregation, deposition, fibril formation; and inhibits cytotoxicity the drosophila angiotensinconverting enzyme homologue ance is required for spermiogenesis a gene (pex) with homologies to endopeptidases is mutated in patients with x-linked hypophosphatemic rickets molecular identification and characterization of novel membrane-bound metalloprotease, the soluble secreted form of which hydrolyzes a variety of vasoactive peptides molecular isolation and characterization of novel four isoforms of ece- the renin-angiotensin system the human preproendothelin- gene. complete nucleotide sequence and regulation of expression neuropeptide degrading activity of locust (schistocerca gregaria) synaptic membranes peptidyl dipeptidase activity in the haemolymph of insects insect angiotensin-converting enzyme: comparative biochemistry and evolution insect angiotensin-converting enzyme. a processing enzyme with broad substrate specificity and a role in reproduction angiotensin-converting enzyme and the metabolism of regulatory peptides in insects inactivation of a tachykinin-related peptide: identification of four neuropeptide-degrading enzymes in neuronal membranes of insects from four diverent orders a novel activity of insect peptidyl-dipeptidase a (angiotensin i-converting enzyme): the hydrolysis of lysyl-arginine and arginyl-arginine from the c-terminus of an insect prohormone peptide conserved roles for peptidases in the processing of invertebrate neuropeptides cleavage of arginylarginine and lysyl-arginine from the c-terminus of pro-hormone peptides by human germinal angiotensin i-converting enzyme (ace) and the c-domain of human somatic ace analysis of the human cd /neutral endopeptidase . promotor region: two separate regulatory elements the limulus clotting reaction the molecular basis of innate immunity in the horseshoe crab metabolic regulation of brain abeta by neprilysin identification of the major abetal- -degrading catabolic pathway in brain parenchyma: suppression leads to biochemical and pathological deposition nuclear localization of endothelin-converting enzyme- : subisoform specificity carboxypeptidase from boophilus microplus: a ''concealed'' antigen with similarity to angiotensin-converting enzyme regulation of hematopoiesis hydrolysis of peptide hormones by endothelin-converting enzyme- . a comparison with neprilysin endothelin system: the double-edged sword in health and disease the germinal isozyme of angiotensin-converting enzyme can substitute for the somatic isozyme in maintaining normal renal structure and functions physiological non-equivalence of the two isoforms of angiotensin-converting enzyme maintenance of normal blood pressure and renal functions are independent evects of angiotensin-converting enzyme crystal structure of drosophila angiotensin i-converting enzyme bound to captopril and lisinopril damage-induced neuronal endopeptidase (dine) is a unique metallopeptidase expressed in response to neuronal damage and activates superoxide scavengers evects of the mammalian vasoconstrictor peptide, endothelin- , on tetrahymena pyriformis gl, and the immunocytological detection of endogenous endothelin-like activity molecular cloning, mrna expression and chromosomal localization of mouse angiotensin-converting enzyme-related carboxypeptidase (mace ) construction, expression and characterization of a soluble form of human endothelinconverting-enzyme- a novel coronavirus associated with severe acute respiratory syndrome expression, purification and characterization of the monomeric and dimeric forms of soluble bovine endothelin converting enzyme- a metabolism of insect neuropeptides: properties of a membrane-bound endopeptidase from heads of musca domestica identification and properties of a peptidyl dipeptidase in the housefly, musca domestica. that resembles mammalian angiotensin-converting enzyme hydrolysis of insect neuropeptides by an angiotensin-converting enzyme from the housefly, musca domestica the endopeptidase activity and the activation by cl-of angiotensin-converting enzyme is evolutionarily conserved: purification and properties of an angiotensin-converting enzyme from the housefly, musca domestica life below the gum line: pathogenic mechanisms of porphyromonas gingivalis. microbiol transgenic mice demonstrate a testis-specific promoter for angiotensin converting enzyme the testicular transcript of the angiotensin-i converting enzyme encodes for the ancestral, non-duplicated form of the enzyme isolation of a renin-like enzyme from the leech theromyzon tessulatum a comparison of the n-terminal sequence of the leech theromyzon tessulatum angiotensin converting-like enzyme with forms of vertebrate angiotensin converting enzymes biochemical properties of the angiotensin-converting-like enzyme from the leech theromyzon tessulatum a comparison of the leech theromyzon tessulatum angiotensin i-like molecule with forms of vertebrate angiotensinogens: a hormonal system conserved in the course of evolution characterization of apelin, the ligand for the apj receptor molecular basis of kell blood group phenotypes proteolytic processing of big endothelin- by the kell blood group protein molecular cloning and primary structure of kell blood group protein common acute lymphocytic leukemia antigen is identical to neutral endopeptidase hormonal regulation and cell-specific expression of endothelin converting enzyme isoforms in bovine ovarian endothelial and steroidogenic cells angiotensin-converting enzyme is a functional receptor for the sars coronavirus cloning of human pex cdna: expression, subcellular localization and endopeptidase activity angiotensin ii and angiotensinconverting enzyme as candidate compounds modulating the evects of testis ecdysiotropin in testes of the gypsy moth, lymantria dispar highly sensitive and selective fluorescence assays for rapid screening of endothelin-converting enzyme inhibitors conserved cysteine and tryptophan residues of the endothelin-converting enzyme- cxaw motif are critical for protein maturation and enzyme activity isolation of angiotensin converting enzyme from testes of locusta migratoria molecular evidence for the expression of angiotensin converting enzyme in hemocytes of locusta migratoria: stimulation by bacterial lipopolysaccharide challenge an endothelin-converting enzyme homologue in the locust, locusta migratoria: functional activity, molecular cloning and tissue distribution molecular cloning and amino acid sequence of human enkephalinase (neutral endopeptidase) high aynity enkephalindegrading peptidase in brain is increased after morphine in vitro metabolism of an insect neuropeptide by neural membrane preparations from lymantria dispar design of angiotensin converting enzyme inhibitors germinal angiotensin iconverting enzyme is totally shed from the rodent sperm membrane during epididymal maturation endothelin-like immunoreactivity in midgut endocrine cells of the desert locust, locusta migratoria heterodimerization of endothelin-converting enzyme- isoforms regulates the subcellular distribution of this metalloprotease glycosylation of asn- and asn- is important for functional expression of endothelin-converting enzyme- structure of human neutral endopeptidase (neprilysin) complexed with phosphoramidon adipokinetic hormones: cell and molecular biology neutral endopeptidase . loss in metastatic human prostate cancer contributes to androgen-independent progression analysis of venom constituents from the parasitoid wasp pimpla hypochondriaca and cloning of a cdna encoding a venom protein a live-cell assay for studying extracellular and intracellular endothelin-converting enzyme activity a model of the ace structure and function as a sars-cov receptor the diverse molecular mechanisms responsible for the actions of opioids on the cardiovascular system isolation and expression of the ecdysteroid-inducible angiotensin-converting enzyme-related gene in wing discs of bombyx mori alternative splicing regulates the endoplasmatic reticulum localization or secretion of soluble secreted endopeptidase invertebrate immunity: basic concepts and recent advances selective restoration of male fertility in mice lacking angiotensin-converting enzymes by sperm-specific expression of the testicular isozyme merops: the protease database kell, kx and the mcleod syndrome venom from the endoparasitic wasp pimpla hypochondriaca adversely avects the morphology, viability, and immune function of hemocytes from larvae of the tomato moth, lacanobia oleracea neutral endopeptidase . inhibitors: from analgesics to antihypertensives the hemoregulatory peptide n-acetyl-ser-asp-lys-pro is a natural and specific substrate of the n-terminal active site of human angiotensin-converting enzyme pex mrna is localized in developing mouse osteoblasts and odontoblasts association of xk and kell blood group proteins cloning of a cdna encoding a non-isopetide-selective subtype of the endothelin receptor elements of angiotensin system are involved in leeches and mollusks immune response modulation structural characterization of a diuretic peptide from the central nervous system of the leech erpobdella octoculata biochemical evidence of angiotensin ii-like peptides and proteins in the brain of the rhynchobdellid leech theromyzon tessulatum molecular modelling and site-directed mutagenesis of the active site of endothelin-converting enzyme structure, expression, and developmental function of early divergent forms of metalloproteinases in hydra immunocytochemical distribution of angiotensin i-converting enzyme-like immunoreactivity in the brain and testis of insects neonatal lethality in mice deficient in xce, a novel member of the endothelinconverting enzyme and neutral endopeptidase family x-linked hypophosphataemia: a homologous phenotype in humans and mice with unusual organ-specific gene dosage cloning and functional expression of human endothelin-converting enzyme cdna rat endothelin-converting enzyme- forms a dimer through cys with a similar catalytic mechanism and a distinct substrate binding mechanism compared with neutral endopeptidase- . neprilysin degrades both amyloid beta peptides - and - most rapidly and eyciently among thiorphan-and phosphoramidon-sensitive endopeptidases peptidyl dipeptidases (ance and acer) of drosophila melanogaster: major diverences in the substrate specificity of two homologs of human angiotensin i-converting enzyme the preparation and function of the hypertensin-converting enzyme novel activity of human angiotensin i converting enzyme: release of the nh -and cooh-terminal tripeptides from the luteinizing hormonereleasing hormone the bacterial etiology of destructive periodontal disease: current concepts role of the prophenoloxidase-activating system in invertebrate immunity a putative novel metalloprotease and its isoforms in cns and testis race: a drosophila homolog of the angiotensin-converting enzyme genome sequence of the nematode c. elegans: a platform for investigating biology characterization of renal angiotensin-converting enzyme in diabetic nephropathy a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase ace structures reveal a large hinge-bending motion important for inhibitor binding and catalysis the angiotensin-converting enzyme gene family: genomics and pharmacology mammalian membrane metallopeptidases purification and characterization of a peptidyl dipeptidase resembling angiotensin converting enzyme from the electric organ of torpedo marmorata the neprilysin (nep) family of zinc metalloendopeptidases: genomics and function aceh/ace is a novel mammalian metallocarboxypeptidase and a homologue of angiotensin-converting enzyme insensitive to ace inhibitors. can a fourth isoform of endothelin-converting enzyme (ece- ) is generated from an additional promoter molecular cloning and characterization xce, a new member of the endothelin-converting enzyme and neutral endopeptidase family, is preferentially expressed in the cns organization of the gene encoding the human endothelin-converting enzyme (ece- ) biochemical identification and ganglionic localization of leech angiotensin-converting enzymes isolation and characterization of an angiotensin converting enzyme substrate from vittelogenic ovaries of neobellieria bullata presence of an angiotensin converting enzyme (ace) interactive factors in ovaries of the grey fleshfly neobellieria bullata captopril, a specific inhibitor of angiotensin converting enzyme, enhances both trypsin and vitellogenin titers in the grey fleshfley successful parasitation of locusts by entomopathogenic nematodes is correlated with inhibition of insect phagocytes immonocytochemical distribution of angiotensin-i converting enzyme in the central nervous system of insects and speculations about its possible function kappa and delta opioid receptor stimulation avects cardiac myocyte function and ca + release from an intracellular pool in myocytes and neurons hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase the hydrolysis of endothelins by neutral endopeptidase the two homologous domains of human angiotensin i-converting enzyme are both catalytically active diminution of contractile response by kappa-opioid receptor agonists in isolated rat ventricular cardiomyocytes is mediated via a pertussis toxin-sensitive g protein cloning and characterisation of angiotensin-converting enzyme from the dipteran species, haematobia irritans exigua, and its expression in the maturing male reproductive system expression of angiotensin-converting enzyme-related carboxydipeptidases in the larvae of four species of fly drosophila melanogaster angiotensin i-converting enzyme expressed in pichia pastoris resembles the c domain of the mammalian homologue and does not require glycosylation for secretion and enzymic activity extracellular peptidases of imaginal discs of drosophila melanogaster the sars-cov s glycoprotein: expression and functional characterization disruption of ece- and ece- reveals a new role for endothelin-converting enzyme in murine cardiac development dual genetic pathways of endothelin-mediated intercellular signaling revealed by targeted disruption of endothelin converting enzyme- gene a novel potent vasoconstrictor peptide produced by vascular endothelial cells a dipeptidyl carboxypeptidase that converts angiotensin i and inactivates bradykinin reduced neprilysin in high plaque areas of alzheimer brain: a possible relationship to deficient degradation of beta-amyloid peptide relationship between beta amyloid peptide generating molecules and neprilysin in alzheimer disease and normal brain cloning and characterization of aplysia neutral endopeptidase, a metallo-endopeptidase involved in the extracellular metabolism of neuropeptides in aplysia californica isolation and expression of an ecdysteroid-inducible neutral endopeptidase . -like gene in wing discs of bombyx mori molecular and functional evidence for early divergence of an endothelin-like system during metazoan evolution: analysis of the cnidarian in vitro degradation of the neb-trypsin modulating oostatic factor (neb-tmof) in gut luminal content and hemolymph of the grey fleshfly, neobellieria bullata key: cord- -o i hy authors: holliday, ian; tam, wai-keung title: e-health in the east asian tigers date: - - journal: int j med inform doi: . /j.ijmedinf. . . sha: doc_id: cord_uid: o i hy objective: the article analyzes e-health progress in east asia's leading tiger economies: japan, hong kong, singapore, south korea and taiwan. it describes five main dimensions of e-health provision in the tigers: policymaking, regulation, provision, funding and physician-patient relations. methods: we conducted a series of fieldwork interviews and analyzed key healthcare websites. results and conclusion: our main finding is that the development of e-health in the region is less advanced than might be expected. our explanation focuses on institutional, cultural and financial factors. the application of information technology (it) to public sector operations sometimes captured in the notion of e-government is starting to have an impact on developed healthcare systems the world over. as time goes by, that impact is expected to become even more pronounced. ''the consensus seems to be that new information technologies will significantly affect almost every aspect of health care,'' wrote blumenthal [ ] . in this article, we examine the progress of e-health in the five leading economies of east asia: japan, hong kong, singapore, south korea and taiwan. each seeks to place itself at the forefront of the information revolution and has high levels of internet access and usage. each also has a sophisticated healthcare system dedicated to securing maximum healthcare benefit at minimal cost. by standard outcome indicators, these systems all have very good records. the tigers, therefore, form a cluster in which e-health might be expected to be notably advanced. however, our finding is that although some progress is being made, it remains limited. it is also variable across the five societies. the article begins by reviewing some of the literature on e-health taking from it a series of critical dimensions and issues. it then briefly analyzes the two relevant contextual aspects of the east asian tigers: their participation in the information age and the nature of their healthcare systems. on these bases, it examines their e-health progress, focusing on the major themes unearthed in the contemporary literature. finding limitations and variations, it concludes by thinking through possible explanatory factors, focusing on institutional, cultural and financial issues. much of the existing e-health literature has been developed in the context of the united states, reflecting both us leadership in the information age and the continuing search for solutions to us healthcare problems. five main themes are prominent. four of the five address distinct dimensions of the broad policy and management framework for healthcare, examining internet impacts on policymaking, regulation, provision and funding. the fifth theme looks inside the healthcare sector and inside the surgery, at the implications of the internet for physician-patient relations. eventually, this may have policy significance, but for now, it is best treated separately. the major argument made about healthcare policymaking is that the us government has been slow to engage with the numerous issues generated by the it revolution [ ] . the core features of that revolution, notably enhanced information flows, increased networking possibilities and novel commercial opportunities, are now well documented [ ] . however, it is said that in us healthcare, most policy actors in both congress and the executive branch continue to focus on pre-information age agendas. although, the bush administration has started to address these concerns, the result remains something of a lack of internet-related policy activity and only a limited number of perspectives on the internet's potential to transform the us healthcare system. clearly structural features of the us system, including fragmentation both of government and of the healthcare sector, play key roles. looking at the narrower sphere of regulation, concerns are expressed about the failure of regulatory agencies to keep pace with internet-related developments. goldsmith notes that the internet generates many potential regulatory problems, ranging from licensing e-health practitioners to monitoring information quality in a virtual world with no boundaries [ ] . fried et al. detail some of the obstacles placed in the way of e-health by existing regulations, holding that individuals and organizations must navigate a maze of rules and codes, old and new, if they wish to implement fresh ideas and approaches [ ] . kassirer's prediction is that the courts will play a role when substandard medical advice provided through web sites or e-mail yields poor medical outcomes. he believes that courts will be especially important when professional advice is given without a direct patient encounter, or when state lines are crossed [ ] . some regulatory issues are us-specific, but many have much wider relevance. partly building on the regulatory theme, analysts have also debated the limitations currently imposed on healthcare provision through the internet. kleinke argues that the internet will not contribute to a solution to the administrative redundancies, economic inefficiencies, and quality problems that have long plagued the us healthcare system. instead, it will exacerbate the cost and utilization problems of a system in which patients demand more, physicians are legally and economically motivated to supply more, and public and private purchasers are expected to pay the bills [ ] . goldsmith holds that the challenges of standardizing coding and formats for clinical information, and protecting patient privacy, will hinder the realization of network computing potentials in healthcare [ ] . the problems to which these and other authors point are structural. economic, organizational, legal, regulatory, and cultural conflicts rooted in the us healthcare system are all barriers to e-healthcare provision. further problems are found in the sphere of healthcare funding. shortliffe criticizes congress for focusing on short-term benefits, arguing that research investment for e-health must be balanced between basic and applied analyses [ ] . robinson examines the effect of distinct forms of capital on the development of the healthcare internet. in the late s, venture capital flooded into the ehealth sector, rising dramatically from us$ million in early to us$ million in late . in the same period, e-health firms went public, raising us$ . billion at their initial public offerings. however, the technology-sector crash in late hit the e-health sector especially hard, prompting an extended period of consolidation between e-health and more conventional firms [ ] . us funding problems thus relate to both the public and the private sectors. finally, analysts have looked inside the surgery at physician-patient relations. existing survey data show that citizens make considerable use of the internet for healthcare information and services, mostly of a generic kind [ ] . indeed, anderson reports that in , % of us adults with internet access did so [ ] . as more patients go online, increasing numbers will turn up in surgeries with internet-fueled questions and concerns. meeting the growing expectations of these individuals will be a significant challenge for physicians [ ] . assessing the likely impact, kassirer argues that the internet will change the physician-patient relationship in unpredictable ways, with some aspects of electronic communication strengthening the bond, and others undermining it [ ] . goldsmith believes patients have most to gain from the emergence of the internet, arguing that it will rebalance the steeply asymmetrical medical knowledge held by patients and physicians [ ] . using information gained through internet searches, patients can now open their dialogues with physicians at a much higher level than before, and thereby gain leverage in the care process. ball and lillis also discuss the potential challenges the internet presents to physicians. as internet searches often generate as many questions as answers, physicians are likely to find themselves under increased workload pressures [ ] . the variable quality of healthcare information accessed through online searches [ ] , a matter that is being actively addressed by bodies such as the health on the net foundation (www.hon.ch) and the internet healthcare coalition [ ] , can only reinforce those pressures. zupko and toth hold that physicians sometimes encounter a form of cultural shock when confronted by well-informed patients [ ] . it is therefore perhaps not surprising that an april survey found that physicians are much more reluctant than patients to use the internet for healthcare interactions. while % of patients wanted to exchange e-mail with their doctors, only about % of doctors actually did so. physician-patient confidentiality, time concerns and increased exposure to malpractice liability were cited as primary reasons for doctors' wariness [ ] . in the face of this mounting speculation and evidence, lumpkin is sanguine, however, contending that the physician-patient encounter is little changed, despite widespread internet usage in healthcare [ ] . though focused on the us, the existing e-health literature generates key themes for an analysis of progress in other parts of the world, including the east asian tigers. however, before exploring those themes, we first present some basic contextual information about our five societies. two features of the east asian tigers are particularly relevant to this analysis: their participation in the information age and the nature of the healthcare systems in which their application of it needs to be assessed. in this section, we examine both features. looking at broad social participation, it was exploited more rapidly in the five east asian tigers than in any other global cluster. for many years, the nielsen//netratings global internet index has ranked all five societies in the top worldwide for personal computer (pc) connections and internet access and usage. the four smaller societies, hong kong, singapore, south korea and taiwan, are particularly advanced. furthermore, the severe acute respiratory syndrome (sars) crisis that hit the region in spring gave a major boost both to internet usage in general, and to e-health in particular [ ] . in hong kong, for instance, the number of active internet users increased by % from february to april , before falling back by % from april to june . the overall increase was %. also at the start of , the time spent online by hong kong people first increased by % and then fell back by %, registering an overall increase of % [ ] . consistent with the image of economic and social dynamism, they have projected for many years now, the east asian tigers are among the most advanced it societies on earth. to some extent, this strong it orientation is the product of developmental state strategies. with the partial exception of hong kong, the east asian tigers have long placed considerable faith in stateled growth strategies. furthermore, for many years they have frequently focused those strategies on it and it-related sectors. in japan, in the s, the fabled ministry of international trade and industry targeted supercomputers and the fifth generation as a major development project [ ] . despite a long period of economic stagnation in the s, the japanese it industry remains a significant global force. in singapore in the s, the state took the lead in nurturing wafer fabrication, the most sophisticated ''front end'' of the semiconductor industry. chartered semiconductor manufacturing, established by the government in , is now the third largest silicon foundry in the world. in south korea, in the early s, the state reorganized the public-sector telecommunications system by closing inefficient firms and allocating profitable segments to major chaebol like samsung and goldstar, enabling them to establish specialized chip businesses. by the early s, samsung had become the world's number one producer of dynamic random access memories for pcs and workstations. in taiwan since the s, the ministry of economic affairs and the state-controlled electronics research service organization have played crucial roles in developing the semiconductor industry. today, it is the fourth largest in the world, and firms within it have entered into strategic alliances with leading industry players in the west [ ] . even in hong kong, where a developmental state took longer to emerge, the government is currently overseeing the construction of a flagship cyberport, intended to host a strategic cluster of companies and professional talents, specializing in it applications, information services and multimedia content creation, and designed to project a hi-tech international digital city image. the east asian tigers are also leading players in the development of e-government. the un/aspa benchmarking survey of all un member states placed singapore at number (a long way behind the us, but only fractionally behind australia and new zealand), south korea at number and japan at number . all three states featured in the top category of high e-government capacity. the survey did not assess hong kong and taiwan, neither of which is a un member state. the report noted that singapore ''demonstrated a balanced and citizen-centric e-government program, while possessing the benefits of a high technological infrastructure and human capital measures''. it held that south korea ''made perhaps the most dramatic advances in its e-government program by successfully implementing several new online transaction features''. it was more critical of japan, arguing that it had ''yet to live up to its rather significant potential''. ''japan's e-government program has not yet reached a comparable level of sophistication as that of the regional leaders due primarily to achieving only a limited interactive presence among national government websites'' [ ] . a january analysis of e-government in east and southeast asia reached similar conclusions, identifying the five tigers as regional leaders [ ] . accenture's survey looked at only two of the five east asian jurisdictions analyzed here. it ranked singapore number in the world after canada, and japan number [ ] . looked at from many different perspectives, then, the east asian tigers are leading participants in the emergent information age. healthcare systems in the tigers share a basic orientation, but are otherwise quite varied. the orientation is best termed productivist, in that in each society social policy has usually been subordinate to economic objectives. while the governments of all five tigers certainly get involved in social policy, they usually do so either for economic reasons or after they have made provision for their various economic goals. the main stimuli to this strong focus on economic development were, in all cases, the devastation brought by the second world war, and the uncertainties of the international order constructed thereafter [ ] . this shared orientation has fed into healthcare policy in three main ways [ ] . in japan and its two former colonies, south korea and taiwan, healthcare was initially left chiefly to the market. only once economic policy was on track and a measure of growth had already been attained, did these societies turn their attention to planning their healthcare systems. in doing so, they concerned themselves chiefly with healthcare finance, creating social insurance systems by gradualist means. now, in all of these societies, the health insurance scheme is universal in aspiration and near universal in fact. across all three societies, healthcare provision remains privatesector-driven, with the state performing a chiefly regulatory role. traditional medicines are significant in all three societies and covered by national health insurance schemes [ ] . however, they are not consistently brought within the planning frame. in hong kong, until the early s, the colonial government took a strictly reactive and incremental approach to healthcare. its major interventions focused on subventing charitable organizations in the healthcare business, though in time, it also built hospitals and delivered care directly through them. throughout, government activity was funded out of general government revenue. the major and to date, only step change came in , with the formation of the hong kong hospital authority (hkha). this imposed state control and state funding on the secondary sector and gave hong kong a miniature version of the british national health service. however, there has never been any attempt to bring primary care within the planned healthcare system. only in , was traditional chinese medicine subjected to anything more than minimal government regulation [ ] . in singapore, the early post-war experience was similar to that of hong kong. here, however, separate initiatives were taken in the spheres of provision and funding. in , much provision was integrated at the secondary care level through creation of the state-run hospital corporation of singapore. this body subsequently sought to drive private-sector disciplines into state provision through ''corporatization''. in , in an attempt to generate integrated pathways of care, it was broken into two territorial clusters focused on the secondary sector but also having primary and tertiary elements. however, as most of the primary sector remained outside the state sector, the extent of integration was limited; in singapore, the state provides % of secondary care but only % of primary care. on the funding side, singapore in created a compulsory savings system, medisave, within the wider central provident fund scheme. it added the insurance schemes medishield and medishield plus in and and created a basic social safety net, medifund, in . these various schemes partially fund secondary care provision. there is also some direct state subsidy. funding of primary care takes place mainly through out-of-pocket expenses. the traditional sector stands outside all state planning and, as in hong kong, has only recently been brought within the regulatory framework. these healthcare systems have enviable records. not only did they make a rapid post-war transition from the contagious disease characteristic of thirdworld countries to the chronic disease characteristic of first-world societies, but also they register very favorable health outcomes as measured by standard input and outcome indicators (table ) . healthcare systems in the east asian tigers thus share a productivist orientation and strong performance. they exhibit varied state roles, with much healthcare activity lying outside the public sector and some of it falling beyond the planning horizon. in japan, south korea and taiwan, state involvement is extensive in finance but limited in provision. in hong kong, the government both funds and directly provides care in the secondary sector, but not elsewhere. in singapore, the state provides a large amount of secondary and some primary care. the funding regime is complex, comprising direct state subsidy, forced individual saving, state-run and private-sector insurance, and out-of-pocket expense. in all five tigers, both the public and private sectors play important roles and face clear incentives to take an interest in harnessing the internet for healthcare gain. note: taiwan data are from . sources: [ ] . against the dual backdrop of sophisticated it societies that make extensive use of the internet and cost-effective healthcare systems driven in variable ways by actors from the public and private sectors, we now turn to a survey of e-health in the east asian tigers. to frame the survey, we begin by providing a brief descriptive overview of the major state-run healthcare websites in the region. we then structure our analysis using the five main analytical spheres that dominate the existing e-health literature: policymaking, regulation, provision, funding and physician-patient relations. all ministries or departments of health in the east asian tigers have their own website. throughout the region, the major quasi-autonomous state agencies, such as the national health insurance agencies in japan, south korea and taiwan, the hkha in hong kong and the two big healthcare clusters in singapore, also have sites. here, we look only at the main government healthcare sites ( table ) . the overall quality is high. all have clickable links to organizational objectives and tasks. most also offer detailed information about subsidiary divisions. all contain links to the government homepage and related healthcare sites so that visitors can conduct further searches and collect additional information. all provide feedback channels. in singapore, the ministry of health (moh) offers online feedback opportunities. in taiwan, citizens can make online appointments with the director of the department of health (doh). in japan, the ministry of health, labour and welfare (mhlw) uses e-mail to solicit however, within this generally strong showing, there are also significant differences, with japan's mhlw and to a lesser extent, hong kong's hwfb lagging behind their regional counterparts in key respects. firstly, the mhlw fails to provide contact details for named officials on its website. this is standard practice in the other four tigers. singapore's moh, for example, gives address, telephone number and e-mail details for key officials. secondly, while healthcare professionals and officials in singapore, south korea and taiwan can communicate with each other through the internet, their counterparts in japan and hong kong cannot. thirdly, the range of options available to users is more restricted in japan and hong kong than in the other three tigers. in south korea, for instance, it has played a role in the surveillance system for communicable disease since . through electronic data interchange and regional database management systems, notifying and reporting systems have been computerized, and an electronic record of all notified and reported cases is kept. using the super-highway communication network, physicians and public health centers can access the notifying and reporting system, disweb, anywhere and anytime through the internet (http://dis.mohw.go.kr). in singapore, the moh site within the government's ecitizen portal enables healthcare professionals to download application forms for license renewal, approval to perform a pregnancy termination, and so on. the policymaking strand of the e-health literature castigates us policymakers for being slow to grasp the potential of the internet. such a charge is less easy to sustain in the east asian tigers, though again experience is variable. singapore and taiwan are the regional leaders. singapore's ecitizen portal addresses many aspects of citizen interaction with government, with healthcare being a prominent theme. the internet is used to reinforce the public health messages that have been disseminated by the singaporean government through other media for many years. behind the scenes, e-mail links pervade the healthcare system and enhance the cohesiveness of policy networks. in a controlled city state, those networks are in any case very tight. in taiwan, the doh in launched an ambitious e-health project, with a timeline stretching to . the health information network that is central to this initiative has a backbone funded by central government and permits local users in both the public and private sectors to participate on a self-paying basis. drawing on us experience, it seeks to promote electronic medical records, based on a smart card system, so that information can flow to all parts of the healthcare sector. a healthcare certification authority, created in , oversees promotion of this initiative. in the other three tigers, progress is less impressive. japan launched an e-japan strategy in january , designed to make it ''the world's most advanced it nation within years'' [ ] . the strategy had an explicit e-government strand. in september , the mhlw followed up by issuing a ''grand design'' for promotion of it in the healthcare sector. the aim was to computerize the entire sector by and to introduce an electronic medical record system covering % of clinics and % of hospitals with plus beds by . progress towards targets appears to be on track. however, japanese performance in the e-health domain is poor by regional standards. hong kong is also quite slow to place healthcare online. the hwfb site contains standard bureaucratic information, such as current policy initiatives and recent speeches, plus public health information that has been developed particularly since the sars crisis. here, the major networking initiative is being taken by the dominant public-sector delivery agency, the hkha. while its primary focus is provision, the networking links being created among hospitals are likely to have policy consequences. as in singapore, e-mail links also bolster ties within policy networks that are already quite cohesive. south korea is making an aggressive attempt to exploit the internet across all areas of government, but in the healthcare sphere, currently remains an average performer. turning to regulation, three main issues are raised in the literature. the first is that e-health generates a number of regulatory problems. the second is that excessive regulation may impede e-health progress. the third is that the courts are likely to have to step in when administrative regulation fails. in the east asian tigers, regulation is clearly a major concern and an evident constraint on ehealth development, often for good reason. one instance is limitations placed on consultations, which in all five tigers quite properly mandate face-toface physician-patient contact before any specific healthcare information or advice can be given. for the foreseeable future, online consultation, though technically feasible, is likely to be restricted by professional concerns. another instance is limitations placed on information sharing and exchange, which in all the tigers are again very properly re- stricted by privacy considerations. however, there is some variation in regional regulatory practice. in singapore, patients requiring repeat prescriptions can place an order online and have the medications delivered to their homes. only after months, do they have to return to the healthcare system to consult a physician. elsewhere, this practice is illegal. in japan, physicians are prohibited from answering specific questions about healthcare or disease by e-mail or telephone. regarding provision, assessments in the us literature are mainly negative. on the one hand, the argument is made that it cannot be expected to solve structural problems in healthcare systems. on the other, barriers even to less ambitious networking initiatives are held to be substantial. these are fair points, but they should not be allowed to obscure the real progress being made by healthcare systems around the world, and in our case in east asia. among the five tigers, taiwan's healthcare websites, both public and private, provide the most comprehensive services to patients. singapore ranks second, and hong kong third. japan and south korea are somewhat behind the regional pace. an overview is given in table . in taiwan, the doh operates a taiwan e-hospital site to provide free online medical advice to patients (http://taiwanedoctor.doh.gov.tw/). currently, medical practitioners and nutritionists from public hospitals form a consulting team to answer questions about specialties. patients seeking general medical advice can send questions to a particular practitioner and receive feedback online or by e-mail. in the private sector, a number of hospitals, such as the chang gung memorial hospital, have online question-and-answer services for patients. the kingnet second opinion webhospital (www.webhospital.org.tw) and the taiwan physician's net (www.doctor.com.tw) are two prominent sites providing free online medical advice to patients. established by kingnet entertainment (www.kingnet.com.tw) in , the webhospital has some voluntary physicians answering questions from the public. the taiwan physician's net brings together about physicians, whose information and advice are posted on the web. apart from getting online medical advice, patients can search for a particular physician and visit his or her office for treatment. in taiwan, patients can also make medical appointments online with many public and private hospitals. looking to the future, the doh is planning to develop a medical information exchange center to promote information sharing and enhance treatment quality. in singapore, health is one of a number of cluster points within the ecitizen site. to date, the internet is mainly used to provide general healthcare information, with the healthcare portal containing comprehensive information about healthcare providers, the healthcare establishment, healthy lifestyles and public health issues such as sars. many searches are possible. the site also allows individuals to submit complaints and feedback. only a few transactions can be undertaken online. as in taiwan, appointments can be made and altered online. through singapore's e-pharmacy services, recurrent prescription items can be ordered online and delivered throughout the island. in one of its two main healthcare clusters, patients can register online and access summary medical records. inside the healthcare system, information flows are starting to change as polyclinics and gps gain access to hospital records online. the likelihood is that enhanced integration of the public and private sectors will result. in hong kong, the hkha, which oversees almost the entire secondary sector, is currently introducing online networking in hospitals. its clinical management system is an integrated clinical workstation giving clinicians access to departmental information and patient records. it will soon develop into a longitudinal electronic patient record within the public hospital system, enabling records to be accessed by many parties simultaneously anywhere, anytime. the system will also actively support clinical decisions by offering alerts, reminders, links to medical knowledge and other aids. it is expected to play an important role in reducing medical errors and improving the quality of patient care. over the next years, the hkha is planning to create a hong kong health information infrastructure, with the aim of networking all healthcare providers in the public, private and social welfare sectors. it also intends to build an electronic medical record for every hong kong resident and provide citizens with an electronic gateway to healthcare information and evidence-based medicine [ ] . these initia-tives are likely to enhance information flows within the public healthcare system. compared with taiwan and singapore, however, hong kong lags behind in developing internet services for patients. individuals cannot register and access summary medical records online. lacking an e-pharmacy service, the hong kong system does not allow recurrent prescription items to be ordered online. japan and south korea are falling behind their regional counterparts in providing online health services to patients. their official health websites do not deliver any electronic service to individual patients. with the exception of initiatives taken by a small number of private hospitals in south korea, like the yonsei eye and ent hospital, neither public nor private hospitals in these two tigers allow patients to register online. however, in , japan's mhlw established telemedicine networks to provide specialized care to people in remote areas. the government will provide us$ million a year to form networks consisting of one large hospital and three clinics working together to supervise patients. each patient will be equipped at home with a computer that can monitor heart rate, blood pressure and other indicators, as well as a phone capable of transmitting video. they will be linked to physicians through an isdn digital phone connection, thus enabling physicians to diagnose illness by electronically transmitted data. from june , the mhlw started to establish such networks a year, so that all districts will have at least one by [ ] . in , south korea's semipublic seoul national university hospital founded ezhospital, which is business-oriented instead of patient-oriented. with three main business elements, education (content services), e-trading and system integration, ezhospital is starting to alter purchasing arrangements for both medical and non-medical supplies. as the south korean system is highly fragmented, the purchasing consortia that can be built through the internet could one day become significant. at present, however, e-purchasing is at an early stage of development. analyses of e-health funding focus on one main issue, the short-termism of us initiatives. in this domain, it is difficult to reach an overall assessment of the tigers' performance. on the one hand, their developmental state orientations make the general climate for it industrial emergence very different from the climate found in the us. in this regard, the tigers look to the long term in a systematic fashion that has no us equivalent. on the other hand, it is hard to find evidence that the tigers are investing heavily in e-health applications. moreover, because the private sector plays such a large regional role in healthcare, as it does in the us, many of the relevant initiatives fall outside the state sector and are hard to capture. there are undoubtedly many small commercial initiatives in east asia as, again, there are in the us. furthermore, like other commercial websites, private healthcare sites throughout the region rely heavily on advertising and sale of products for income. to take just two taiwanese instances, the kingnet webhospital and the taiwan physician's net offer online sales not only of healthrelated products, but also of cinema tickets. the very fragmented nature of private-sector healthcare operations throughout the tigers means that few summary assessments can be made. looking finally at physician-patient relations, the existing literature contains variable forecasts of unpredictable change, little change, and so on. however, there is a clear belief that patients have most to gain from e-health and physicians correspondingly have most to lose. in general, physicians in the tigers have tended to be wary of exploiting the internet for patient interactions. this partly reflects the tight regulatory climate in which many find themselves, with many modes of physician-patient contact outlawed. it may also reflect a certain reluctance on the part of both physicians and patients to engage in the informalities of online contact. until recently, then, the emergence of virtual physician-patient relations was highly limited. since the spring sars crisis, however, the pattern may have started to change. although it is too early to register the longterm impact of the crisis, it is clear that during the sars outbreak, many individuals sought to shift to online interactions with healthcare professionals. the fear of visiting surgeries and, in particular, hospitals that gripped the region in has certainly not disappeared and seems likely to provide a lasting stimulus to virtual delivery of healthcare. furthermore, the generic healthcare information found in great abundance on english-language websites is paralleled on regional websites operating in chinese, japanese and korean. there is also some official encouragement for patients to migrate to e-health. in may , hong-jen chang, ceo and president of taiwan's bureau of national health insurance, argued at an oecd forum that e-health could make a major contribution in informing patients. as evidence, he cited taiwanese experience in confronting hiv/aids and the role of the internet in educating patients about the disease. in the long run, he contended, patients equipped with information gained from online searches ''will translate into quality improvement and efficiency gains for the system'' [ ] . overall, east asian societies retain many traditional features, which generate some resistance to change in established modes of physician-patient contact. nevertheless, there are also factors operating in the opposite direction. one long-term impact of the sars crisis seems likely to be heightened caution about visiting healthcare facilities, for fear of contracting infectious disease, and a consequent boost for e-health. the east asian tigers form the most wired cluster of societies found anywhere in the world. moreover, they have long had a developmentalist orientation that has seen their states become involved in many aspects of economic and social development. in the sphere of e-health, however, their performance is strong at the level of basic web provision, but otherwise not particularly advanced. on the whole, their health ministries or departments have good sites covering all the fundamentals of online provision. outside central government agencies, they often have a wealth of additional sites in the public and private sectors. beyond that, they do not make pioneering use of the internet in healthcare. there are many possible reasons for this slightly disappointing performance, some of which apply to all of the tigers and others which are specific to a particular society. in japan, the structural problems that mired the economy in stagnation for more than a decade from the early s also form part of the explanation for its sluggish e-health performance. a notable feature of the japanese healthcare system is the considerable power of the japan medical association and its extensive links to the liberal democratic party that has governed the country for almost all of the post-war period. in hong kong, the sovereignty transfer was quite disruptive, and only several years on is the political system taking a settled shape on the developmental state model. looking beyond the specific circumstances of individual tigers, however, the major explanatory factors appear to be institutional, cultural and financial. institutionally, east asian healthcare systems tend to be highly fragmented, notably in japan, south korea and taiwan. in consequence, policymakers in healthcare ministries and departments have rather few levers that they can use to direct change. in the e-health sphere, they can quite easily construct official government websites, but generating reform in the wider healthcare system is more difficult and depends on their success in building consortia of interest among many private-sector actors. in part, they seek to do this by offering ring-fenced seed money for specified development projects. in part, they resort to exhortation, calling on all members of society to engage in the project of securing and maintaining regional and/or global leadership in the information age. in these many respects, the east asian tigers have a great deal in common with the us. in the additional domain of culture, they differ from the us. while capitalism is certainly a dynamic force in east asia as in north america, it also co-exists with still vibrant cultural underpinnings. the confucian heritage that characterizes all five east asian tigers has many complex strands. among them is considerable respect for authority, hierarchy, status and so on. in the medical sphere, one consequence is that doctors tend still to be accorded considerable professional status. this may make it difficult for full commercialization to take place and for the market drive that characterizes ehealth in the us to work its way through the system. finally, the financial dimensions of healthcare in the east asian tigers should not be overlooked. these are healthcare systems that deliver the excellent outcomes already mentioned at a fraction of the cost registered in the us and, indeed, in most developed societies. as a proportion of gdp, east asian tigers spend between and % on healthcare, with most coming in at around %. this is far below the us figure of - %, and also below the highincome country standard of almost %. one result of the tigers' success in holding down healthcare costs is that the incentive to experiment with new initiatives is reduced. clearly, there still are some incentives, but they are not as strong as in the us. e-health in the east asian tigers remains at an early stage of development. all have attained a good basic standard, but few are engaged in pathbreaking initiatives. alongside institutional factors that are similar to those found in the us, cultural and financial factors help to explain this rather unsatisfactory level of performance. doctors in a wired world: can professionalism survive connectivity? milbank q the internet promise, the policy reality the information age: economy, society and culture. vol. ii. the power of identity the information age: economy, society and culture. vol. i. the rise of the network society the information age: economy, society and culture. vol. iii. end of millennium how will the internet change our health system? ehealth: technologic revolution meets regulatory constraint patients, physicians and the internet com: the failed promise of the healthcare internet networking health: learning from others, taking the lead financing the healthcare internet rethinking communication in the e-health era consumers of e-health: patterns of use and barriers the 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rhetoric and national inaction high performance, maximum value productivist welfare capitalism: social policy in east asia welfare capitalism in east asia: social policy in the tiger economies traditional medicines in modern societies: an exploration of integrationist options through east asian experience agenda-setting for the regulation of traditional chinese medicine in hong kong networking health: dawning of the e-health era. paper presented to apami-mic conference at the hong kong convention and exhibition centre e-health and the informed patient, paper presented to oecd forum taiwan council of economic planning and development, taiwan statistical data book world development indicators the work described in this article was substantially supported by a grant from the research grants council of the hong kong special administrative region, china [project no. cityu / h]. initial seed funding was provided by the governance in asia research centre, city university of hong kong.we are grateful for the research support we received. we thank academics, officials and practitioners in east asia for talking to us about e-health. the usual disclaimer applies. key: cord- -isff lp authors: han, dong p; kim, hyung g; kim, young b; poon, leo l.m; cho, michael w title: development of a safe neutralization assay for sars-cov and characterization of s-glycoprotein date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: isff lp the etiological agent of severe acute respiratory syndrome (sars) has been identified as a novel coronavirus sars-cov. similar to other coronaviruses, spike (s)-glycoprotein of the virus interacts with a cellular receptor and mediates membrane fusion to allow viral entry into susceptible target cells. accordingly, s-protein plays an important role in virus infection cycle and is the primary target of neutralizing antibodies. to begin to understand its biochemical and immunological properties, we expressed both full-length and ectodomain of the protein in various primate cells. our results show that the protein has an electrophoretic mobility of about – kda. the protein is glycosylated with high mannose and/or hybrid oligosaccharides, which account for approximately kda of the apparent protein mass. the detection of s-protein by immunoassays was difficult using human convalescent sera, suggesting that the protein may not elicit strong humoral immune response in virus-infected patients. we were able to pseudotype murine leukemia virus particles with s-protein and produce sars pseudoviruses. pseudoviruses infected vero e cells in a ph-independent manner and the infection could be specifically inhibited by convalescent sera. consistent with low levels of antibodies against s-protein, neutralizing activity was weak with % neutralization titers ranging between : to : . to facilitate quantifying pseudovirus-infected cells, which are stained blue with x-gal, we devised an automated procedure using an elispot analyzer. the high-throughput capacity of this procedure and the safety of using sars pseudoviruses should make possible large-scale analyses of neutralizing antibody responses against sars-cov. during the first epidemic of severe acute respiratory syndrome (sars), which began in november of in guandong province of the people's republic of china, and lasted for about months, close to people were infected worldwide, among which people died (who, ) . the etiological agent of this atypical respiratory disease has been identified as a novel coronavirus (designated as sars-cov) fouchier et al., ; ksiazek et al., ; peiris et al., ; poutanen et al., ) . with a mortality rate of over %, sars-cov had a major health and socioeconomic impact. fortunately, there have been very few incidences of sars infections during the winter season of [ ] [ ] . however, with multiple modes of virus transmission and a wide range of potential nonhuman reservoirs including wild animals commonly found in markets (e.g., civet cats and raccoon dogs; guan et al., ) as well as domestic cats (martina et al., ) , it is highly likely that a virus of this nature will most certainly resurface in the future. currently, there are no antiviral drugs, immunotherapeutic agents, or vaccines available against the virus. to better control or prevent future epidemics, anti-sars-cov drugs and/or vaccines need to be developed. sars-cov belongs to coronaviridae family. the genomic organization of the virus is similar to that of other coronaviruses with a general order of replicase (rep; orfs- a and b), spike (s)-glycoprotein, envelope (e), membrane protein (m), and nucleocapsid (n) from v to v direction (marra et al., ; rota et al., ) (fig. ) . several openreading frames have also been identified, which may encode additional proteins (marra et al., ; rota et al., ; snijder et al., ) . their functions, however, are not known at the present time. the protein of a major interest as a target of antiviral drug development efforts as well as for developing vaccines is s-glycoprotein. s-protein of coronaviruses, which is thought to function as a trimer (delmas and laude, ) , is responsible for both binding to cellular receptors and inducing membrane fusion for virus entry into target cells (collins et al., ; godet et al., ; kubo et al., ) . mutations in the protein have been shown to alter virulence and cellular tropism (fazakerley et al., ; leparc-goffart et al., ; sanchez et al., ) . taken together, the s-protein plays a critical role in the biology and pathogenesis of coronaviruses. not surprisingly, it is an important target of virus-neutralizing antibodies (chang et al., ; collins et al., ; fleming et al., ; godet et al., ; kant et al., ; kubo et al., kubo et al., , takase-yoden et al., ) . moreover, mice immunized with a recombinant s-protein, or a peptide derived from it, are protected from lethal challenges with murine hepatitis virus (mhv) (daniel and talbot, ; koo et al., ) . s-protein is a type i membrane glycoprotein, which is translated on membrane-bound polysomes, inserted into rough endoplasmic reticulum (rer), cotranslationally glycosylated, and transported to the golgi complex. during the transport, s-proteins are incorporated onto maturing virus particles, which assemble and bud into a compartment that lies between the rer and golgi (lai and holmes, ) . virions are carried from golgi to plasma membrane in secretory vesicles. virions are released from cells when virion-containing vesicles fuse with plasma membrane. excess s-proteins not incorporated onto virus particles are transported to the surface of plasma membrane (lai and holmes, ; tsai et al., ; yamada et al., ) . s-protein of sars-cov is amino acids long (fig. ) . it is predicted to have a amino acid signal peptide at the amino-terminus, a single ectodomain ( amino acids) and a transmembrane region followed by a short cytoplasmic tail ( residues) at the carboxy-terminus (marra et al., ; rota et al., ) . due to low sequence homology between the s-protein of sars-cov and that of the other coronaviruses (marra et al., ; rota et al., ) , the structural and immunogenic properties of sars-cov sprotein must be ascertained experimentally. the cellular receptor for sars-cov has recently been identified to be angiotensin-converting enzyme (ace ; li et al., ) . the molecular interactions between the s-protein and ace are not yet known. better understanding of the interactions could lead to development of virus entry inhibitors. neutralizing antibodies (nabs) play a critical role in protection against a variety of viral diseases. an accurate assessment of nab responses in virus-infected patients is needed to determine immune correlates of protection. it is also an essential and integral part of a vaccine development process. conventional virus-neutralization assays require the use of replication-competent, infectious viruses. evaluating virus-neutralizing activity of a large number of antisera with these assays is undesirable due to safety concerns, especially for a biosafety level (bsl ) pathogen like sars-cov. the same safety concerns have prompted our laboratory to utilize replication-defective pseudoviruses for hiv- neutralization assay (kim et al., ) . in this assay, nonreplicating moloney murine leukemia virus (mulv) particles pseudotyped with hiv- envelope glycoproteins are used (schnierle et al., ) . these pseudoviruses encode a h-galactosidase gene, which allows detection of individual infected cells when stained with x-gal ( -bromo- -chloro- -indolyl-h-d-galactopyranoside). in this study, we report development of a sars-cov pseudovirus neutralization assay, which should be particularly valuable for researchers who may not have easy access to bsl containment facility. additionally, we describe a high-throughput system for quantitative analyses of x-gal stained cells. this assay system should facilitate rapid evaluation of antibody responses to vaccine candidates and/or entry inhibitors against sars-cov. to express sars-cov s-glycoprotein, we initially cloned a dna fragment encoding the protein into pcdna- vector (pcdna-s; fig. b ). to detect s-protein, western blot was performed with convalescent sera from sars-cov-infected patients. however, no clear protein band was detected despite number of attempts. we reasoned that one of the possibilities for the inability to detect the protein is a low level of s-protein expressed from pcdna-s. to increase the amount of sprotein expressed, we subcloned the s gene into phcmv-g vector (burns et al., ) , which expresses high level of vesicular stomatitis virus (vsv) g glycoprotein. although we were able to express higher amount of s-protein (see below), this was not sufficient to detect a clear band on western blots. an alternative explanation is that antibodies against the protein in convalescent sera cannot recognize s-protein subjected to denaturing conditions of sds-page (viz. linear epitopes). however, because results from radioimmunoprecipitation and indirect immunofluorescence assays were also ambiguous, it is most likely that the antibody titer against s-protein is very low in convalescent sera. to further increase protein expression level, s gene was subcloned into a ptm vector (moss et al., ) . with this vector, a protein of interest is under the control of a strong t rna polymerase (t rnap) promoter and the protein is expressed when cells transfected with the plasmid are infected with a recombinant vaccinia virus expressing t rnap (vtf - ; fuerst et al., ) . the presence of encephalomyocarditis virus internal ribosome entry site (ires) at the v end of rna transcripts allows efficient translation of mrna transcribed in cytoplasm. using ptm-s, we were able to detect a faint, but distinct band of approximately - kda by western blot (fig. a , lane ). we also reevaluated pcdna-s as this vector has a dual promoter system (cmv and t promoter). using t promoter, we were able to detect a protein band of a similar size, albeit less clear than using ptm-s (lane ). the lower expression of the protein is likely due to the lack of ires in the pcdna vector. because the calculated molecular weight of s-protein without amino acid signal peptide is about kda, the result suggested posttranslational modification (e.g., glycosylation). to better demonstrate this, we generated another clone (ptm-eshis) that expresses the entire ectodomain of s-protein (amino acids - ) with a six-histidine tag at the carboxy terminus. the ectodomain of s-protein migrated with an approximate molecular weight of kda while its calculated molecular weight is only . kda (fig. b , lane ). to demonstrate that this difference is due to glycosylation, eshis protein was treated with endoglycosidase h (endo-h) or peptide: n-glycosidase f (pngase f). as shown in fig. b (lanes and ), treatment with either glycosidase increased the mobility of the protein to approximately kda. because the mobility of the protein treated with either glycosidases was the same, s-protein is most likely modified with high mannose and/or hybrid, rather than complex, oligosaccharides. while s-glycoprotein of some coronaviruses is cleaved into two subdomains, s and s , the fact that we observed only a single band suggests that sars-cov s-protein functions as a single unit. despite difficulties in detecting s-protein directly by immunoassays, proteins expressed from both pcdna-s and phcmv-s constructs were able to pseudotype mulv particles to produce sars pseudoviruses that could readily infect vero e cells (fig. a) . none of the other cell lines we tested, including hela, a , t, and bs-c- , were susceptible. this is in contrast to vsv-g pseudotyped viruses, which could infect all cell lines (data not shown). the fact that bs-c- cells, which, like vero e cells, are african green monkey kidney cells, were not susceptible was somewhat unexpected. however, when we performed infections with a high multiplicity of infection, we were able to detect infected bs-c- , albeit at a significantly reduced titer ( - -fold compared to vero e cells; data not shown). this result was not too surprising because it has been shown that even t cells, which express small amounts of ace , support some basal level of sars-cov replication . many pseudovirus-infected cells appeared as a doublet, which is the result of a cell division following integration of mulv pseudovirus genome encoding h-galactosidase. these doublets are counted as a single infectious unit. a typical yield of sars pseudoviruses was about  infectious units per milliliter of culture supernatant using phcmv-s, which was about fivefold greater than using pcdna- . this yield is comparable to what we have been able to achieve for hiv- pseudoviruses (between  and  depending on envelopes; kim et al., ) , but lower than vsv-g pseudovirus yield (between  and  depending on target cell lines used). interestingly, sars pseudovirus production was about -fold less efficient when plasmid transfection was performed by calcium phosphate method compared with using cationic lipids (lipofection). this difference, however, was not observed for vsv-g pseudovirus production. an additional difference was that a longer incubation time was needed to achieve peak pseudovirus production for sars-s compared to vsv-g ( vs. days posttransfection, respectively). the reasons for these discordant results are unknown at the present time. cellular entry of coronaviruses can occur either by acidic ph-dependent or -independent pathway (gallagher et al., ; lai and holmes, ; cavanagh, , ; nash and buchmeier, ; payne et al., ) . to investigate whether sars-cov infection requires low ph, we examined sensitivity of pseudovirus infections to lysosomotropic agents chloroquine and nh cl. as expected, infectivity of viruses pseudotyped with vsv-g was reduced by chloroquine and nh cl in a dose-dependent manner and , respectively) . the protein was detected by western blot with anti- Âhis antibody. no band was detected from cells transfected with an empty vector (lane ). acrylamide gradient gel ( - %) was used. ( fig. b and c, respectively) . in contrast, sars pseudovirus infection was virtually unaffected, suggesting that sars-cov infection proceeds in an acidic ph-independent manner. to assess whether sars pseudoviruses we generated could be used to quantify virus-neutralizing antibodies, we examined their susceptibility to convalescent sera from sars-cov-infected patients. as shown in fig. a , sera from two patients were able to specifically neutralize sars pseudoviruses; the same convalescent sera could not neu-tralize hiv- or vsv-g pseudoviruses and no neutralizing activity was observed with a normal serum. to determine neutralizing antibody titers in virus-infected patients, we performed the assay with serially diluted sera from seven patients. as shown in fig. b , antibody levels were quite similar in all patients with % neutralization titer between : and : . although the pseudovirus neutralization assay is sensitive, quantitative, and safe, it has one disadvantage of having to count individual x-gal-stained cells through a microscope. to overcome this problem, we looked into a possibility of automating the data collection procedure using an elispot reader (immunospot analyzer, cellular technology ltd.). although this instrument is commonly used to quantify antigen-specific t cell cytokine responses by counting chromogenic immunospots (e.g., ifn-g), we rationalized that it might be able detect x-gal-stained blue cells. as shown in fig. , there was no problem with using the instrument to count spots at a single-cell resolution and the analysis was highly efficient as the entire -well plate could be processed in less than min. virus-infected cells appearing as doublets did not pose a problem because parameters on the analysis software could be adjusted to count two stained cells adjacent to each other as one. the number of infectious foci counted was quite linear as a function of virus inoculum (fig. c ), validating the methodology. this procedure could be used to quantify other assays based on x-gal staining of cells (e.g., recombinant vaccinia viruses that express h-galactosidase). in this study, we expressed sars-cov s-glycoprotein, which was able to pseudotype mulv particles. sars pseudoviruses were able to efficiently infect vero e cells, which have been shown to support sars-cov infection. the infection did not require low ph, suggesting viral entry is mediated by a direct fusion event between viral and plasma membranes. this result is consistent with a previous report that cell-to-cell fusion mediated by sprotein and its cellular receptor ace occurred at neutral ph (xiao et al., ) . however, our result is in direct disagreement with recently published article by simmons et al. ( ) . there are three major differences in exper-imental procedures between the two studies. first, we pseudotyped mulv particles whereas simmons et al. used hiv- . second, we used an authentic s-glycoprotein whereas they used a c-terminal fusion protein that included a v epitope and polyhistidine tag, which totaled, by our estimation, extra amino acids. whether the discrepant result is due to the use of different s-glycoproteins and/or different virus cores needs to be further investigated. the third difference between the studies is the concentrations of lysosomotropic agents used. while we used nh cl at - am amounts, which are sufficient to inhibit vsv-g-mediated fusion ( fig. ; picard-maureau et al., ) , they used millimolar (mm) amounts. at these concentrations, nh cl could have a secondary effect on sglycoprotein. we were unable to find concentrations of chloroquine used in their study. it is interesting to note that while simmons et al. observed that pseudovirus infections required low ph, s-protein-mediated cell-to-cell fusion did not. the sars pseudoviruses we generated could be specifically inhibited by convalescent sera from sars-cov infected patients, indicating that s-glycoprotein of sars-cov is a target of neutralizing antibodies as it is for other coronaviruses. the major purpose of generating sars pseudoviruses was to devise an assay system to assess virus-neutralizing antibodies safely and rapidly without having to use infectious, replication-competent sars-cov. the results of our study indicate that sars pseudoviruses could be used to evaluate efficacy of various s-glycoprotein-based vaccine candidates to elicit virus-neutralizing antibodies. they could also be used to perform structurefunction analyses of s-glycoprotein. due to a large size of sars-cov genome, it would be difficult to perform such analyses directly in the context of the virus, not to mention potential safety hazards from working with it. in contrast, mutational analyses of the protein could be performed readily using pseudoviruses. our attempt to characterize biochemical and immunological properties of the s-protein was hampered by the fact that antibody titers against the protein in convalescent sera were extremely low; we were able to identify only a faint band on a western blot (with high background) and attempts to detect the protein by immunofluorescence and radioimmunoprecipitation assays were less than successful. in contrast, convalescent sera have been successfully used to detect sars-cov-infected cells by an immunofluorescence assay (hsueh et al., ; peiris et al., ) . together, the available data seem to suggest that s-protein might not be immunogenic, at least compared to other viral proteins. in fact, immunoreactivity analyses of a panel of synthetic peptides derived from s, membrane (m), and nucleocapsid (n) proteins suggested that n protein might be the most immunogenic protein . the nonimmunogenic nature of s-protein might present potential problems in developing a vaccine that can elicit potent neutralizing antibodies against sars-cov. in this regard, it is interesting to note that s-protein is highly glycosylated with potential asparagine-linked glycosylation sites. based on our analyses of the ectodomain of the protein, carbohydrate residues account for approximately kda (based on mobility in sds-page). the glycans were primarily high mannose and/or hybrid type. this, however, needs to be verified using proteins produced from nonvaccinia virus expression system, because the virus infection could possibly affect cellular glycosylation machinery. extensive glycosylation of hiv- envelope glycoprotein has been one of the major obstacles in eliciting good humoral responses against the protein and in developing an effective vaccine against the virus (cho, ) . it remains to be seen whether and to what extent glycans on s-protein affect immunogenic properties of the protein. interestingly, potential glycosylation sites are clustered into three regions of the protein (fig. a) : n-terminal, middle, and c-terminal. it has been shown that individual glycosylation sites on hiv- surface glycoprotein gp may have different functions; while some are important for evading immune responses, others are critical for maintaining proper protein structure necessary to interact with cellular receptors and mediate membrane fusion (ogert et al., ; reitter et al., ) . additional studies are needed to determine whether glycosylation sites in different clusters of s-protein serve different functions. in the absence of an effective vaccine and/or antiviral drugs against sars-cov, early detection of virus-infected patients would be critical for effective containment of future epidemics. quantitative rt-pcr-based diagnostic assays have been described for sars-cov (grant et al., ; lau et al., ; ng et al., ; poon et al., a poon et al., , b tang et al., ; yam et al., ) . despite high sensitivity, their utility has some limitations: (i) the detection rate varies widely between % and % depending on clinical sam-ples and protocols used for the assay; (ii) the window of detectability is limited to early stages of infection; and (iii) the assay is not suitable for routine surveillance. antibodies against sars proteins have been shown to appear as early as days after the onset of illness (hsueh et al., ) . therefore, development of a high-throughput serologybased diagnostics could complement pcr-based assays. in this regard, a virus-neutralization assay could be used as a confirmatory test, which would enhance the accuracy of early diagnosis of sars-cov. because neutralizing antibodies are important for virus clearance, the assay could also be used to assess disease prognosis. in either case, the availability of sars pseudoviruses allows avoiding the use of infectious sars-cov. the overall cloning strategy is shown in fig. b . two parental plasmids encoding a sars-cov s gene (urbani strain), pentr-s and pcr-s, were obtained from the u.s. centers for disease control and prevention. two s-proteinexpressing plasmids (pcdna-s* and pcdna-s) were generated using pcdna- (invitrogen). the s gene in pcdna-s*, which was transferred from pentr-s (bamhi -ecori fragment), lacks the original translation stop codon taa because it was changed to aat of ecori restriction site (gaattc). pcdna-s with a stop codon was constructed by replacing a swai-ecori fragment of pcdna-s* with the same fragment from pcr-s. to generate phcmv-s, a bamhi -ecori fragment from pcdna-s was inserted into a bamhi site of phcmv-g following blunting ends with klenow. to construct ptm-s, a bamhi -xhoi fragment from pcdna-s was cloned into the corresponding sites of ptm-ndei (cho et al., ) . despite the fact that ptm-s has a small open-reading frame that encodes eight amino acids between the internal ribosome entry site of ptm-ndei vector and the s gene, s-protein was efficiently expressed and the plasmid was used as is without further modification. to generate ptm-eshis, v end of the ectodomain was pcr amplified using a sense primer v-gtc gtc aac att caa aaa gaa- v (nts - of s gene) and an antisense primer v-aat gaa gcg gat cccggg tta gtg atg gtg gtg atg atg ttg ctc ata ttt tcc caa- v. base-pairing region (nts - ) is shown in bold and the six histidine residues are italicized. the amplified fragment was digested with swai (nt ) and smai (underlined) and subsequently cloned into ptm-s digested with swai and stui. cell culture, protein expression, and western blots all cell lines, except for vero e , were maintained in dmem supplemented with % fetal bovine serum (fbs), mm l-glutamine, and penicillin -streptomycin antibiotics. vero e cells were maintained in emem with the same supplements plus . mm nonessential amino acids. cells were cultured at jc in % co incubators. to express sprotein, cells were transfected with plasmids by a calcium phosphate precipitation method. briefly, . ml of . m cacl solution containing ag of plasmids was slowly mixed with  hbs ( mm hepes, . mm na hpo , mm nacl, ph . ) and the mixture was added to cells. after an overnight incubation, culture medium was replaced and cells were further incubated for two additional days. for expression from ptm-s and ptm-eshis, transfected cells were infected with vtf - (fuerst et al., ) at a multiplicity of infection of . following days of infection, cells were lysed with a hypotonic cell lysis buffer ( mm tris, ph . , mm nacl, . mm mgcl , % np- ). insoluble cell debris and nuclei were removed by a brief centrifugation in a microfuge. cell lysates were subjected to sds-page and western blot. s-proteins were detected with either a pool of convalescent sera ( : dilution) or anti-his (c-terminal) monoclonal antibody (invitrogen; : dilution) followed by horseradish peroxidase-conjugated goat anti-human or anti-mouse igg antibody (pierce), respectively. protein bands were visualized using supersignal west pico chemiluminescence detection system (pierce). molecular weights of the protein bands were approximated by the mobility of standard molecular weight markers. pseudoviruses were generated as previously described (kim et al., ) . briefly, mulv packaging cell line telceb (schnierle et al., ) was transfected with pcdna-s, phcmv-s, phcmv-g (burns et al., ) , or pltr-gp (hiv- dh ; kim et al., ) using either calcium phosphate precipitation or lipofection (lipofectin; invitrogen) method. two days posttransfection ( days for sars pseudovirus), cell culture medium was harvested and subjected to centrifugation (  g, min) to remove cell debris. supernatant was aliquoted, stored at À jc and used as a virus stock. virus titer was determined in vero e cells for sars-s and vsv-g or in hos-cd -ccr (cheng-mayer et al., ; deng et al., ) for hiv- gp pseudotyped viruses. typically, cells were infected with - infectious units for h. cells were washed with pbs and incubated with a fixative ( % formaldehyde, . % glutaraldehyde in pbs) for min at room temperature. the cells were washed twice with pbs and incubated with a freshly prepared staining solution (pbs containing mm potassium ferricyanide, mm potassium ferrocyanide, mm magnesium chloride, and mg/ml of x-gal) for > h at jc. for routine analyses, x-gal-stained blue cells were manually counted using an inverted microscope. to determine ph-dependency of viral entry, vero e cells were incubated in culture medium containing - am chloroquine for h at jc before adding viruses. vsv-g or sars-s pseudoviruses were allowed to adsorb to cells for h at jc in the absence of chloroquine. following adsorption, virus inoculum was removed, cells were washed, and infection was allowed to proceed for about h in the absence of chloroquine. for nh cl, cells were incubated with - am. due to minimal cytotoxicity, nh cl was present throughout the infection period including h incubation before virus addition. all infections were done in duplicates. neutralization assay was performed as previously described (kim et al., ) using convalescent sera ( - days post-onset of symptoms) obtained from cdc or from patients hospitalized in queen mary hospital, hong kong. approximately - infectious units of pseudoviruses were incubated with serially diluted, heat-inactivated ( jc, min) convalescent or normal sera for h at jc. the mixture was subsequently added to vero e (for sars-s or vsv-g) or hos-cd /ccr (for hiv- gp ) cells. virus infection was allowed to proceed for another h. virus-neutralizing activity was determined relative to no serum control. the general pseudovirus infection procedure is the same as described above. the major difference was that -well plates with a white membrane bottom normally used for elispot assays (plate m ; bd biosciences) were utilized rather than regular tissue culture plates. immunospot analyzer from cellular technology ltd. was used as per manufacturer's recommendations. vesicular stomatitis virus g glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus macrophage tropism of human immunodeficiency virus type and utilization of the cc-ckr coreceptor subunit protein vaccines: theoretical and practical considerations for hiv- membrane rearrangement and vesicle induction by recombinant poliovirus c and bc in human cells monoclonal antibodies to murine hepatitis virus- (strain jhm) define the viral glycoprotein responsible for attachment and cell -cell fusion protection from lethal coronavirus infection by affinity-purified spike glycoprotein of murine hepatitis virus, strain a assembly of coronavirus spike protein into trimers and its role in epitope expression identification of a major co-receptor for primary isolates of hiv- identification of a novel coronavirus in patients with severe acute respiratory syndrome the v a . envelope glycoprotein deletion mutant of mouse hepatitis virus type- is neuroattenuated by its reduced rate of spread in the central nervous system antigenic relationships of murine coronaviruses: analysis using monoclonal antibodies to jhm (mhv- ) virus aetiology: koch's postulates fulfilled for sars virus eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage t rna polymerase alteration of the ph dependence of coronavirus-induced cell fusion: effect of mutations in the spike glycoprotein major receptorbinding and neutralization determinants are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein detection of sars coronavirus in plasma by real-time rt-pcr isolation and characterization of viruses related to the sars coronavirus from animals in southern china microbiologic characteristics, serologic responses, and clinical manifestations in severe acute respiratory syndrome location of antigenic sites defined by neutralizing monoclonal antibodies on the s avian infectious bronchitis virus glycopolypeptide development of a safe and rapid neutralization assay using murine leukemia virus pseudotyped with hiv type envelope glycoprotein lacking the cytoplasmic domain protective immunity against murine hepatitis virus (mhv) induced by intranasal or subcutaneous administration of hybrids of tobacco mosaic virus that carries an mhv epitope a novel coronavirus associated with severe acute respiratory syndrome neutralization and fusion inhibition activities of monoclonal antibodies specific for the s subunit of the spike protein of neurovirulent murine coronavirus jhmv c - variant localization of neutralizing epitopes and the receptor-binding site within the amino-terminal amino acids of the murine coronavirus spike protein coronviridae: the viruses and their replication a real-time pcr for sars-coronavirus incorporating target gene pre-amplification the c mutant of mhv-a is very weakly demyelinating and has five amino acid substitutions restricted to the spike and replicase genes role of ph in syncytium induction and genome uncoating of avian infectious bronchitis coronavirus (ibv) coronavirus ibv-induced membrane fusion occurs at near-neutral ph angiotensin-converting enzyme is a functional receptor for the sars coronavirus virology: sars virus infection of cats and ferrets new mammalian expression vectors entry of mouse hepatitis virus into cells by endosomal and nonendosomal pathways quantitative analysis and prognostic implication of sars coronavirus rna in the plasma and serum of patients with severe acute respiratory syndrome n-linked glycosylation sites adjacent to and within the v / v and the v loops of dualtropic human immunodeficiency virus type isolate dh gp affect coreceptor usage and cellular tropism initial events in bovine coronavirus infection: analysis through immunogold probes and lysosomotropic inhibitors coronavirus as a possible cause of severe acute respiratory syndrome foamy virus envelope glycoprotein-mediated entry involves a ph-dependent fusion process early diagnosis of sars coronavirus infection by real time rt-pcr rapid diagnosis of a coronavirus associated with severe acute respiratory syndrome (sars) identification of severe acute respiratory syndrome in canada a role for carbohydrates in immune evasion in aids characterization of a novel coronavirus associated with severe acute respiratory syndrome targeted recombination demonstrates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence pseudotyping of murine leukemia virus with the envelope glycoproteins of hiv generates a retroviral vector with specificity of infection for cd -expressing cells characterization of severe acute respiratory syndromeassociated coronavirus (sars-cov) spike glycoprotein-mediated viral entry unique and conserved features of genome and proteome of sarscoronavirus, an early split-off from the coronavirus group lineage localization of major neutralizing epitopes on the s polypeptide of the murine coronavirus peplomer glycoprotein interpretation of diagnostic laboratory tests for severe acute respiratory syndrome: the toronto experience a -amino acid stretch in the hypervariable region of the spike protein s subunit is critical for cell fusion activity of mouse hepatitis virus assessment of immunoreactive synthetic peptides from the structural proteins of severe acute respiratory syndrome coronavirus summary of probable sars cases with onset of illness from the sars-cov s glycoprotein: expression and functional characterization evaluation of reverse transcription-pcr assays for rapid diagnosis of severe acute respiratory syndrome associated with a novel coronavirus requirement of proteolytic cleavage of the murine coronavirus mhv- spike protein for fusion activity we are grateful to cdc for providing plasmids encoding sars-cov s gene and convalescent sera, to dr. bernard moss for vtf - , to dr. franc ßois-loïc cosset for telceb cell line, and to drs. jonathan silver and mario skiadopoulos for vero e cells. hos-cd -ccr cell line was obtained from dr. nathaniel landau through the aids research and reference reagent program, division of aids, niaid, nih. we specially thank dr. magdalena tary-lehman for providing assistance with using immuno-spot analyzer. key: cord- - p jk h authors: trampuz, andrej; prabhu, rajesh m.; smith, thomas f.; baddour, larry m. title: avian influenza: a new pandemic threat? date: - - journal: mayo clinic proceedings doi: . / . . sha: doc_id: cord_uid: p jk h in december , the largest outbreak of highly pathogenic avian influenza h n occurred among poultry in asian countries. a limited number of human h n infections have been reported from vietnam and thailand, with a mortality rate approaching %. deaths have occurred in otherwise healthy young individuals, which is reminiscent of the spanish influenza pandemic. the main presenting features were fever, pneumonitis, lymphopenia, and diarrhea. notably, sore throat, conjunctivitis, and coryza were absent. the h n strains are resistant to amantadine and rimantadine but are susceptible to neuraminidase inhibitors, which can be used for treatment and prophylaxis. the widespread epidemic of avian influenza in domestic birds increases the likelihood for mutational events and genetic reassortment. the threat of a future pandemic from avian influenza is real. adequate surveillance, development of vaccines, outbreak preparedness, and pandemic influenza planning are important. this article summarizes the current knowledge on avian influenza, including the virology, epidemiology, diagnosis, and management of this emerging disease. in december , the largest outbreak of highly pathogenic avian influenza h n occurred among poultry in asian countries. a limited number of human h n infections have been reported from vietnam and thailand, with a mortality rate approaching %. deaths have occurred in otherwise healthy young individuals, which is reminiscent of the spanish influenza pandemic. the main presenting features were fever, pneumonitis, lymphopenia, and diarrhea. notably, sore throat, conjunctivitis, and coryza were absent. the h n strains are resistant to amantadine and rimantadine but are susceptible to neu-raminidase inhibitors, which can be used for treatment and prophylaxis. the widespread epidemic of avian influenza in domestic birds increases the likelihood for mutational events and genetic reassortment. the threat of a future pandemic from avian influenza is real. adequate surveillance, development of vaccines, outbreak preparedness, and pandemic influenza planning are important. this article summarizes the current knowledge on avian influenza, including the virology, epidemiology, diagnosis, and management of this emerging disease. s ince mid-december , asian countries-cambodia, china, indonesia, japan, laos, south korea, thailand, and vietnam-have reported outbreaks of highly pathogenic avian influenza caused by the h n strain among poultry. this is the largest outbreak of avian influenza in poultry ever described. most of these countries experienced outbreaks of avian influenza for the first time. during the past months, more than million domestic poultry have died, or they have been culled to contain the epidemic and prevent the potential transmission to humans. despite the widespread infection of avian influenza viruses among poultry in asia, reported infection in humans has been rare to date. as of , only vietnam and thailand have reported influenza h n infection in humans. thirty-two human cases were laboratory confirmed, resulting in deaths, a mortality rate approaching %. however, most of the affected countries have limited capacity for surveillance of human disease. in addition, poultry production contributes substantially to the economies of the affected countries, resulting in considerable political pressure to minimize the extent and seriousness of the epidemic. therefore, the reported human cases of avian influenza may be underestimated. historically, this avian epidemic zoonosis is unprecedented in its scale, geographical distribution, economic losses, challenge for control, and potential public health consequences. the potential development of a human influenza pandemic similar to that of due to spanish influenza is a major concern. two of the key criteria that characterized the pandemic of - have already been fulfilled in the current epidemic: ( ) the ability of the virus to infect humans resulting in high mortality and ( ) a global immunologically naive human population. the third criterion, efficient human-to-human transmission, has thus far not been observed. the adaptation that would result in human-to-human transmission might involve changes in the receptor properties or improved viral replication efficiency. this adaptation might be achieved by mutation of an avian virus genome or by mixing segments of an avian virus with segments from a virus already adapted to humans (genetic reassortment), leading to the emergence of a new influenza subtype with pandemic potential. both events may occur easily. first, influenza viruses mutate frequently, potentially allowing them to change the host receptor specificity from avian to human. second, the segmented viral genome allows the exchange of genes between viruses if they infect the same host cell, which serves as the "mixing vessel." in the current h n outbreak in asia, no genetic reassortment among avian and human influenza viruses has been found; all genes are of avian origin. , however, the widespread epidemic of avian influenza in domestic birds increases the likelihood for mutational events and genetic reassortment. in view of the high mortality observed in the limited number of infections in humans, the prospect of a severe pandemic is of considerable importance. we review current knowledge about avian influenza, including the virology, epidemiology, diagnosis, and management of this emerging disease. for personal use. mass reproduce only with permission from mayo clinic proceedings. influenza viruses type a, b, and c belong to the family of orthomyxoviridae. these enveloped viruses contain a segmented single-stranded rna genome. a key difference among influenza virus types is their host range. type a viruses have been isolated from a wide range of species, including humans, pigs, horses, seals, ferrets, mink, whales, and birds. types b and c viruses infect humans predominantly but have also been isolated from seals and pigs, respectively. influenza a viruses can be divided into subtypes based on surface glycoproteins, hemagglutinin and neuraminidase. to date, different hemagglutinin (h -h ) and neuraminidase (n -n ) proteins have been identified. hemagglutinin facilitates entry of the virus into host cells through its attachment to sialic acid on epithelial cell receptors, promotes membrane fusion, and elicits protective neutralizing antibody response. hemagglutinin is the crucial component of influenza vaccines. neuraminidase has enzyme activity, which cleaves sialic acid on virion proteins, facilitating the release of progeny virions from infected cells. it is an important target for antiviral agents. although all influenza a virus subtypes have been found in birds, only different hemagglutinin (h , h , or h ) and different neuraminidase (n or n ) proteins have circulated widely in humans. the antigenic characteristics of influenza virus change gradually by accumulation of point mutations (antigenic drift) or profoundly by genetic reassortment (antigenic shift) in the genes encoding primarily hemagglutinin and neuraminidase. during antigenic drift, mutations occur because of the lack of the proofreading activity of viral rna polymerases. as a consequence, new antigenic variants emerge constantly, allowing the virus to evade immune recognition and giving rise to annual epidemics. the antigenic drift of influenza viruses requires the replacement of influenza strains used in the vaccine every several years. during antigenic shift, new strains appear to which most humans have no immunity and that have the potential to cause severe global outbreaks of human influenza. these pandemic strains derive from nonhuman viruses by interspecies transmission of the whole virus or by genetic reassortment between avian and human viruses that have infected a single cell. pigs have receptors for both avian and human influenza viruses and have been considered an intermediate host for the reassortment of influenza viruses. reemergence of a previously circulating virus to which most of the population is immunologically naive is another mechanism by which pandemic strains can occur. the nomenclature of influenza viruses includes the type of virus (a, b, or c), host of origin (excluding humans), geographical site of origin, strain number, and year of isolation, followed in parentheses by the antigenic description of the hemagglutinin and neuraminidase glycoproteins, eg, a/chicken/hong kong/ / (h n ). avian influenza viruses do not replicate efficiently in humans, which suggested initially that direct avian-to-human transmission would not occur. the hemagglutinins of avian strains bind preferentially to host receptors terminating in an α( , )-linked sialic acid, whereas human strains bind preferentially to receptors terminating in an α( , )linkage. the predominance of these receptors in different tissues partly reflects the tropism of influenza in different species. receptor specificity and inability of efficient replication in vivo were believed to provide a barrier against human infection by avian influenza virus. high doses of avian influenza virus strains were required for replication in volunteers. the first human infections by avian h n virus in hong kong in showed that receptor specificity was not a definitive host restriction factor. in this epidemic, an intermediate host was not necessary for transmission of avian strains to humans. all birds are thought to be susceptible to infection with avian influenza viruses. wild waterfowl and migrating bird populations provide an extensive natural reservoir for influenza a viruses ( figure ). the virus multiplies in the intestines of these birds, particularly in wild ducks, and they can carry the virus without developing signs of infection, indicating an optimal level of viral adaptation in these hosts. therefore, influenza is not an eradicable disease, and prevention and control of outbreaks are the only realistic goals. unlike mammalian influenza virus strains, avian viruses have shown little antigenic variability during the past decades. the high level of genetic conservation suggests that avian viruses have reached an evolutionary stage in which antigenic changes provide no selective advantage. infected birds excrete large amounts of virus in respiratory secretions, saliva, and feces. in the northern hemisphere, the prevalence of avian influenza peaks between late summer and early winter when the birds leave their breeding grounds and migrate; up to % of these birds may excrete virus. water sources that are contaminated by wild bird droppings and used by domestic poultry are a common and efficient means of viral spread. migratory birds are capable of flying long distances, and those migrating longitudinally appear to play a key role in viral transmission. figure . wild aquatic birds are the main reservoir for influenza a viruses, from which viruses can be transmitted to other hosts such as horses, pigs, poultry, whales, seals, and humans. as indicated by arrows, humans can also be infected by pigs and poultry. the fowl plague was first described in as a disease affecting chickens in italy. the causative agent was isolated from a chicken in , which was the first identified influenza virus (the human influenza virus was not identified until ). domestic poultry flocks are highly vulnerable to avian influenza, particularly birds that are raised outdoors. once introduced into domestic flocks, the virus can spread among farms via contaminated inanimate objects, contributing to the rapid evolution of epidemics. avian influenza in birds is characterized by a wide spectrum of symptoms, ranging from a mild illness to a highly contagious, severe, and rapidly fatal disease with a mortality rate approaching %. the latter syndrome was formerly known as fowl plague; it is now termed highly pathogenic avian influenza and is characterized by coughing, sneezing, excessive lacrimation, cyanosis of the unfeathered skin, edema of the head, ruffled feathers, diarrhea, nervous system disorders, or sudden death without clinical signs. in the past, most outbreaks of highly pathogenic avian influenza in poultry have been due to subtypes h or h . epidemics of avian influenza among poultry have produced serious economic consequences. in addition to a highly pathogenic avian influenza, viruses of low pathogenicity resulting in mild respiratory symptoms and reduced egg production can cause substantial economic losses. in , turkey farms in minnesota were affected by influenza virus h n , resulting in an economic loss of approximately $ million. moreover, avian influenza viruses of low pathogenicity can mutate into highly pathogenic viruses after circulating for several months. during the - epidemic in italy, the h n virus mutated within months to a highly pathogenic form, and more than million birds died or were culled. since , outbreaks of highly pathogenic avian influenza have increased in frequency and severity, and the number of novel strains suggests that the next influenza pandemic is imminent. in , outbreaks of highly pathogenic avian influenza occurred among chickens at farms in delaware, pennsylvania, texas, and maryland. the diseased and exposed birds were killed, and a quarantine zone was implemented around the affected farms. on february , , h n infection was confirmed in dead domestic cats and in a tiger in a zoo in thailand. to date, these animals have not been considered susceptible to influenza viruses. this raises concern about the need for surveillance in animals other than birds. since the late s, live poultry markets have been considered the source of human influenza viruses. these permanent live animal markets (wet markets) are embedded strongly in the food culture of asian countries. close human contact with live animals provides an ideal environment for the zoonotic transfer and evolution of infectious disease agents. the most important control measure for containing the outbreak in hong kong in was rapid destruction of the entire poultry population, proper disposal of carcasses, and rigorous disinfection of farms. to reduce the risk of reemergence of avian influenza, all aquatic birds were removed from retail markets, including ducks, geese, and quails (source of the h n strain). monthly "clean days" were introduced when all markets are emptied and cleaned simultaneously. new regulations mandate quarantine of poultry in designated farms in mainland china for days and testing for h infection before importation to hong kong. other control measures include continuous surveillance of influenza virus strains in humans and in birds, careful protection of cullers through appropriate personal protective equipment, restrictions on the movement of live poultry, and use of the human influenza vaccine to reduce the risk of coinfection in poultry workers and cullers. a global outbreak of influenza usually occurs when a new influenza virus emerges, spreads, and causes disease worldwide. descriptions of widespread and serious epidemics of respiratory disease suggestive of influenza have for personal use. mass reproduce only with permission from mayo clinic proceedings. *many people died within the first few days after infection, nearly half of whom were healthy young adults. †virus is still circulating. ‡illness occurred almost exclusively among persons younger than years. this pandemic did not increase mortality. no. of deaths been recorded for centuries. hippocrates in ancient greece described the first case of influenza-like illness. in the th century, human influenza pandemics emerged with intervals of to years (table ). the spanish influenza of - (h n ) was the most devastating pandemic in recorded human history, resulting in to million deaths worldwide; nearly half of those who died were healthy young adults. this figure is at least double the number of soldiers killed on the battlefields of europe during world war i. other less catastrophic pandemics occurred in (asian influenza), (hong kong influenza), and (russian influenza). the asian (h n ) and hong kong (h n ) pandemic strains were generated by reassortment between human and avian viral genes. the russian influenza virus (h n ) circulated in the s and reemerged in . the illness occurred almost exclusively among persons younger than years. the relatively low mortality rate associated with this pandemic can be attributed to the immunity of older individuals who had antibodies from their previous exposure to nearly identical viruses. to date, human infection with avian influenza viruses has been confirmed on several occasions ( table ). in , the first documented direct transmission of an avian influenza virus to humans occurred in hong kong, when an h n strain caused a severe respiratory disease in previously healthy young adults, of whom died. the outbreak in hong kong was controlled by the destruction of the entire poultry population; within days, more than . million chickens were killed. in , avian influenza h n viruses were isolated for the first time in humans; children were hospitalized with uncomplicated upper respiratory tract infection in hong kong, and both recovered completely. in may and in february and april , the poultry stocks in hong kong were again destroyed when the highly pathogenic h n virus reemerged in flocks. however, no more human cases of h n influenza were identified until february , when cases were confirmed in hong kong residents after travel to china, of whom died. in february , a large outbreak of h n avian influenza occurred in poultry farms in the netherlands. the virus caused mild illness in several farm workers, but a veterinarian who had visited an affected farm died of pneumonia. the virus isolated from the fatal case displayed amino acid substitutions, which possibly contributed to the increased disease severity. all farm workers received mandatory influenza vaccination and prophylaxis with oseltamivir. the h n poultry outbreak subsequently spread to germany and belgium but was eventually controlled by destruction of more than million domestic poultry. in february , another outbreak of h n avian influenza occurred on a poultry farm in british columbia, canada, and resulted in at least humans with infection, mainly conjunctivitis. recently, the crystal structure of the hemagglutinin from the virus was determined, explaining why this virus was able to spread efficiently in the human population despite retainment of the avian receptor-binding site. epidemiological studies suggest that most human h n infections resulted from contact with infected birds or surfaces contaminated with their excretions. however, evidence of limited human-to-human transmission of avian influenza viruses has occurred ( table ) . a case-control study of patients hospitalized for influenza virus h n disease in was conducted in hong kong. exposure to live poultry in the week before the onset of illness was significantly associated with h n disease, whereas traveling, eating, or preparing poultry products and recent exposure to persons with respiratory illness showed no significant association. in a cohort study including hospitals in hong kong at which patients infected with h n strains had been admitted, a significantly higher seropositivity rate for personal use. mass reproduce only with permission from mayo clinic proceedings. for h n was found among exposed health care workers ( . %) than among nonexposed ones ( . %), providing epidemiological evidence of transmission from infected patients to health care workers. in the netherlands in , family members of farm workers were infected during the h n outbreak, demonstrating the ability of human-to-human transmission. the present epidemic of highly pathogenic avian influenza virus in asia is historically unprecedented and extremely challenging. between december and in , sudden death occurred in , chickens at a farm near seoul, south korea; this was later confirmed as being caused by the highly pathogenic avian influenza virus h n . on january , , authorities in vietnam also reported several outbreaks of avian influenza h n at farms in the southern provinces. human cases of h n avian influenza presenting as severe respiratory illness occurred, with a mortality rate of more than %. on january , , the world health organization alerted the global outbreak alert and response network to support health authorities in the epidemiological investigation and containment of human cases. these h n viruses are substantially different from the h n viruses in outbreaks in hong kong in and , indicating that the virus has mutated. on january , , authorities in thailand reported an outbreak of highly pathogenic avian influenza among poultry, with laboratory-confirmed cases of h n infection in humans. japan, the european union, the united states, and other major export markets banned import of poultry products from the affected asian countries. preliminary clinical data from confirmed human cases of h n avian influenza from the current epidemic in vietnam were summarized recently. the mean age of the patients was . years (range, - years); none had any clinically important preexisting medical conditions. of the patients, had a history of direct contact with poultry (chicken or ducks), with a median time before onset of illness of days (range, - days). the main clinical features were fever, shortness of breath, cough, and diarrhea. notably, sore throat, coryza, and conjunctivitis were absent. all patients presented with pronounced lymphopenia (mean lymphocyte count, . × /l) and important chest radiographic abnormalities including diffuse, multifocal, or patchy infiltrates or segmental or lobular consolidation. eight of the patients ( %) died after a mean of days of illness. the clinical features, together with liver dysfunction, renal failure, coagulopathy, and pancytopenia seen in the hong kong h n outbreak, are similar to those seen in patients with severe acute respiratory syndrome. similarly, human infection with avian influenza h n reported in in hong kong presented as an influenzalike illness with pneumonia. reactive hemophagocytic syndrome was the most characteristic pathologic finding and probably contributed to the lymphopenia, liver dysfunction, and abnormal clotting profiles that were observed among these patients. gastrointestinal manifestations, renal failure unrelated to rhabdomyolysis, and pancytopenia were unusually prominent. factors associated with severe disease included older age, delay in hospitalization, lower respiratory tract involvement, and leukopenia or lymphopenia at admission. the preliminary findings suggest that cytokine dysfunction contributes to the severity of for personal use. mass reproduce only with permission from mayo clinic proceedings. testing is indicated for hospitalized patients with radiographically confirmed pneumonia, acute respiratory distress syndrome, or other severe respiratory illness for which an alternate diagnosis has not been established and history of travel to a country with documented h n avian influenza within days of symptom onset testing should be considered on a case-by-case basis in consultation with state and local health departments for hospitalized or ambulatory patients with temperature > °c (> . °f) and one or more of the following: cough, sore throat, shortness of breath and history of contact with domestic poultry (eg, visited a poultry farm, household raising poultry, or bird market) or a known or suspected human case of influenza h n in an h n -affected country within days of symptom onset h n disease. there is no evidence of h n virus replication outside the respiratory tract. viral replication seems to trigger a burst of cytokine production that may ultimately result in multiorgan failure. all patients who present to a health care setting with fever and respiratory symptoms should be questioned regarding their recent travel and exposure history. in patients at risk for avian influenza h n , laboratory testing is indicated ( table ). the optimal specimen for influenza virus detection is a nasopharyngeal aspirate obtained within days of the onset of symptoms; however, nasopharyngeal swabs and other specimens can be used. the h n influenza virus can be detected by rapid antigen tests, virus culture, and reverse transcriptase-polymerase chain reaction (rt-pcr). at least rapid antigen detection assays are commercially available and provide results in to minutes but are not as sensitive or specific as virus culture or rt-pcr. virus isolation in cell cultures enables further antigenic and genetic characterization, drug susceptibility testing, and vaccine preparation. however, cultures take to days and must be performed under biosafety level + laboratory conditions. conventional and real-time pcr assays detect diverse influenza type a viruses, including the avian h n strains. , polymerase chain reaction primers are directed to the conserved matrix gene of the virus, and therefore this assay would be expected to detect recombinant avian strains of the virus. of emphasis, avian influenza virus may be recombinant strains of the virus and may not contain the essential genes for replication in laboratory cultures. rapid antigen detection and rt-pcr can be performed with standard biosafety level laboratory conditions in a class ii biological safety cabinet. serologic diagnosis of influenza is based on the detection of a -fold or greater increase in specific antibody titer in paired serum samples; the first should be collected as soon as possible after onset of illness and the second, to days later. this limits the usefulness of serology in the diagnosis and treatment of acute illness. the current h n strains are resistant to amantadine and rimantadine but are susceptible to neuraminidase inhibitors (oseltamivir and zanamivir). these drugs are effective for prophylaxis and treatment of influenza a virus infection, including the avian influenza h n . however, no efficacy trials have been performed due to the small number of human cases. because a potential vaccine may be in short supply, antiviral drugs may play an important role in reducing the severity and spread of infection during the first stages of a pandemic. for prophylaxis, oseltamivir should be administered to individuals exposed to h n avian influenza within hours. therefore, a global influenza strategy in the st century calls for stockpiling antiviral drugs and drafting plans for rapid distribution. in geographic areas affected by avian influenza, individuals should avoid contact with poultry, and they should perform thorough and frequent hand hygiene using soap and water or alcohol-based hand rubs. the virus is killed by heat ( °c for hours, °c for minutes, or °c for minute) and common disinfectants such as alcohol, bleach, formalin, or iodine compounds. generally, % bleach solution is appropriate for dealing with biohazardous spillage. influenza virus can survive in feces for several months. it can survive in water for up to days at °c, for more than days at °c, and indefinitely in frozen material. therefore, poultry, including the eggs, should be cooked thoroughly. recently, the centers for disease control and prevention issued guidelines for airline flight crews and persons meeting passengers arriving from areas with avian influenza. protection of persons involved in outbreak eradication activities includes strict adherence to hand hygiene practices and the use of appropriate personal protective equipment (gloves, disposable clothing, shoe covers, safety goggles, and particulate respirators). during handling of human specimens, formation of aerosols and droplets should be minimized. isolation precautions identical to those recommended for severe acute respiratory syndrome should be implemented for all hospitalized patients diagnosed as having or being evaluated for avian influenza h n (table ). these precautions should be continued for days after for personal use. mass reproduce only with permission from mayo clinic proceedings. the current inactivated trivalent human influenza vaccine provides no protection against the h and h avian influenza strains. however, an important control measure is providing the seasonal human influenza vaccine to people at risk for avian influenza to reduce the risk of coinfection with avian and human influenza viruses simultaneously and to decrease the possibility of reassortment. the resultant hybrid virus could be highly transmissible among humans. a prototype virus and candidate vaccines have been developed for protection against the h n virus strain that infected people and resulted in death in hong kong in . however, the avian subtypes are rapidly lethal to chicken embryos, and therefore the traditional chicken embryo method is not applicable. alternative means of producing h and h vaccines were exploited, including the plasmid-based reverse genetic technology. a vaccine strain can be created by merging selected hemagglutinin and neuraminidase genes from the target virus with a laboratory virus. the resulting virus is recognized by the human immune system and causes a protective immune response but no disease. however, such vaccines have yet to be studied in clinical trials, and safety testing must be completed. the occurrence of avian influenza h n in humans is another reminder of our vulnerability to an emerging pandemic. several measures can help to minimize the global public health risk. an immediate priority is to halt further spread of epidemics in poultry populations that would reduce the opportunities for human exposure to the virus. clinicians should be cognizant of human influenza h n infection among patients with the appropriate epidemiological exposure, so that patients can be identified quickly and managed appropriately and health care workers can be protected. the reemergence of h n influenza in humans emphasizes the need to develop a vaccine against this virus. for personal use. mass reproduce only with permission from mayo clinic proceedings. available at: www.who.int/csr/disease/avian_influenza/en/. accessibility verified development of a vaccine effective against avian influenza h n infection in humans human influenza a h n virus related to a highly pathogenic avian influenza virus world health organization the pathogenesis of influenza in humans the structure and receptorbinding properties of the influenza hemagglutinin pandemic threat posed by avian influenza a viruses outbreaks of avian influenza a (h n ) in asia and interim recommendations for evaluation and reporting of suspected cases-united states are we ready for pandemic influenza? influenza virus neuraminidase inhibitors realities and enigmas of human viral influenza: pathogenesis, epidemiology and control pandemic influenza: its origin and control influenza type a in humans, mammals and birds: determinants of virus virulence, host-range and interspecies transmission animal influenza virus surveillance influenza: an emerging disease wet markets-a continuing source of severe acute respiratory syndrome and influenza? outbreak of avian influenza a(h n ) virus infection in hong kong in human infection with influenza h n avian influenza a virus (h n ) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome transmission of h n avian influenza a virus to human beings during a large outbreak in commercial poultry farms in the netherlands case-control study of risk factors for avian influenza a (h n ) disease, hong kong risk of influenza a (h n ) infection among poultry workers, hong kong, - cases of influenza a (h n )-thailand world health organization international avian influenza investigative team. avian influenza a (h n ) in patients in vietnam clinical features and rapid viral diagnosis of human disease associated with avian influenza a h n virus pathology of fatal human infection associated with avian influenza a h n virus detection of influenza a viruses from different species by pcr amplification of conserved sequences in the matrix gene multiplex real-time pcr assay for detection of influenza and human respiratory syncytial viruses available at: www.cdc.gov/flu/avian/index.htm. accessibility verified sars: epidemiology, clinical presentation, management, and infection control measures prevention and control of influenza: recommendations of the advisory committee on immunization practices (acip) a. strict hand hygiene before and after all patient contact b. use of gloves and gown for all patient contact c. use of eye protection when within feet ( m) of the patient d. use of shoe covers when entering the isolation room e. placement of the patient in an airborne isolation room key: cord- -sqe h t authors: alfano-sobsey, edith m.; eberhard, mark l.; seed, john r.; weber, david j.; won, kimberly y.; nace, eva k.; moe, christine l. title: human challenge pilot study with cyclospora cayetanensis date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: sqe h t we describe a pilot study that attempted to infect human volunteers with cyclospora cayetanensis. seven healthy volunteers ingested an inoculum of cyclospora oocysts (approximately – , oocysts). the volunteers did not experience symptoms of gastroenteritis, and no oocysts were detected in any stool samples during the weeks volunteers were monitored. highest oocyst counts were purified and concentrated by sucrose and cesium chloride gradients ( ) . see the table for additional inoculum treatment conditions. each candidate inoculum was tested for salmonella spp., shigella spp., campylobacter spp., yersinia spp., mycobacterium spp., escherichia coli o :h , enteroviruses, hepatitis a virus (hav), herpesvirus, cytomegalovirus, coronavirus, astrovirus, rotavirus, norovirus, adenovirus, hiv, clostridium difficile toxin, enterotoxin, and intestinal parasites (data not shown). serum specimens from the donors of cyclospora-positive stools were tested for serum markers of hiv, hav, hepatitis b virus, and taenia solium. if serum from a cyclospora-positive stool donor tested positive for any of the above, oocysts from that person were not used. candidate inocula in which none of these pathogens or toxins were found were used in the human challenge study. this safety-testing protocol was reviewed and approved by both the university of north carolina school of medicine irb and the cdc irb. cell culture infectivity assays and animal models were not available to determine the infectivity or viability of the oocysts. attempts were made to assess the viability of the inoculum by observing sporozoite motility after excystation of the oocysts by different methods. however, these methods did not yield a sufficient number of motile sporozoites to measure viability. the study was conducted at the general clinical research center at the university of north carolina hospital, chapel hill, nc. inclusion and exclusion criteria determined whether a person was eligible to participate in the study. healthy volunteers were recruited from the university of north carolina (unc), chapel hill, nc, and the surrounding community. before enrollment, each candidate received a medical evaluation, and preinoculation serum specimens were collected and archived. the seven study participants comprised four women and three men; three were white, and four were african american. the median age was . after ingesting the inoculum, volunteers were asked to collect all stool specimens for weeks and one specimen a week at weeks , , , and . they were also asked to keep a daily record of physical symptoms and the time of each stool passage. in addition, volunteers provided blood and saliva specimens weekly for weeks and at weeks and postdosing. study outcome measures were a) shedding cyclospora oocysts in stool; b) frequency, weight, color, and consistency of stool; and c) clinical symptoms of gastroenteritis: diarrhea (> stools in hours), nausea, vomiting, abdominal pain, myalgia, headache, fever, chills, or fatigue. stools were examined to detect oocysts at unc chapel hill. all stool specimens were concentrated by using the formalin-ethyl acetate concentration procedure routinely used to examine ova and parasites in stool specimens ( ) . in addition, to increase the sensitivity of detection, all stools from the first weeks postdosing were concentrated by a sucrose floatation procedure ( ) . aliquots of the concentrated sample from each procedure were examined microscopically by using a wet mount preparation ( ) , and % of these concentrates were confirmed by a second laboratory at cdc. criteria to identify cyclospora oocysts were based on size, morphologic characteristics, and ability of the oocysts to autofluoresce under epifluorescence ( ) . inoculum treatment and challenge conditions of this study are described in the table. numbers of stools examined per volunteer ranged from to ; no oocysts were detected in any of the stool samples. volunteer experienced a brief episode of abdominal cramps on day postdosing. this volunteer attributed the symptom to possible dehydration due to strenuous activity performed in the heat that day. volunteer produced four loose stools on day postdosing but reported feeling well. this volunteer also had a mildly elevated leukocyte count ( . x /l; normal range . - . x /l) on day postdosing. however, this volunteer had a mildly elevated baseline leukocyte count ( . x /l) days before dosing. overall, no conclusive evidence based on clinical or parasitologic diagnostic procedures showed that any volunteers became infected. cyclosporiasis continues to be a difficult emerging infectious disease to understand. our results are consistent with other researchers' inability to establish c. cayetanensis infection in a wide variety of animal models ( ). given these results, questions relating to host susceptibility and risk factors for infection with cyclospora, the biology of c. cayetanensis, survival conditions for c. cayetanensis in vitro and in vivo, and factors that allow cyclospora to become infectious in the environment need further study. host susceptibility and risk factors for infection are always a consideration when evaluating host response to pathogen exposure. epidemiologic data suggest that immunity may develop to c. cayetanensis in areas where cyclosporiasis is endemic and that the disease is more severe in naïve populations ( ) . persons affected in foodborne outbreaks of cyclosporiasis in north america were mostly adults who experienced prolonged symptomatic gastroenteritis, and median food-specific attack rates were high ( , ) . in this study, only healthy adult volunteers ( - years of age) were recruited from areas in which cyclosporiasis is not known to be endemic. therefore, although the number of volunteers in this study was small (n = ), epidemiologic data suggest that host susceptibility factors did not substantially contribute to inability of the inocula to cause infection in the volunteers at the doses administered. virulence and characteristics of cyclospora necessary to infect human hosts are unknown. nucleotide sequence variability in the first internal transcribed spacer regions within c. cayetanensis from different geographic origins has been observed and suggests the existence of multiple strains ( , ) . in addition, data from cryptosporidium human volunteer studies demonstrated that the % infectious dose (id ) differed (from to , oocysts), depending on the isolate used in the study ( ) . for these reasons, we attempted to vary the inocula by selecting oocysts from persons in different geographic regions (haiti, missouri, and georgia) and increasing the numbers all oocysts in stool samples in this study were stored in potassium dichromate ( . %), and most of the final inoculum preparations were disinfected with bleach ( . %). cryptosporidium parvum has been stored in . % potassium dichromate (for < weeks to > weeks) and remained infectious in human volunteers, cell culture, and animals ( , ) . also, baboons inoculated with oocysts never exposed to potassium dichromate were not infected with cyclospora (m. eberhard, unpub. data). other parasites of genera related to cyclospora (cryptosporidium, eimeria spp., toxoplasma gondii, sporocysts of sarcocystis spp.) have been shown to resist high levels of bleach ( ) ( ) ( ) . however, the effects of potassium dichromate and bleach on the cyclospora oocysts used in this study are unknown, since methods to evaluate infectivity and viability were not available. naturally occurring cyclospora oocysts may survive for extended periods in the environment, given the marked seasonality of infection in areas where the disease is endemic ( ) . however, many questions remain about the triggers and conditions necessary for cyclospora oocysts to survive and become infectious in the environment. given the results of this study, conditions necessary for cyclospora to become infectious were probably not achieved in preparing and storing the oocysts. future studies are necessary to examine individual and combined effects of temperature, humidity, storage media, and disinfection on the survival, viability, and infectivity of stored cyclospora oocysts. these studies would help determine optimal conditions to stimulate sporulation and maintain infectivity of oocysts in vitro over time. however, such studies will not be possible until suitable cell culture systems or animal host models for cyclosporiasis are developed. in vitro cultivation of cryptosporidium parasites: a guide to laboratory procedures and identification laboratory diagnosis of cyclospora infections attempts to establish experimental cyclospora cayetanensis infection in laboratory animals pathologic and clinical findings in patients with cyclosporiasis and a description of intracellular parasite life-cycle stages cyclospora cayetanensis: a review, focusing on the outbreaks of cyclosporiasis in the s outbreak of cyclosporiasis associated with basil in missouri in intervening transcribed spacer region variability in cyclospora cayetanensis sequence variability in the first internal transcribed spacer region within and among cyclospora species is consistent with polyparasitism virulence of three distinct cryptosporidium parvum isolates for healthy adults the infectivity of cryptosporidium parvum in healthy volunteers influence of temperature on cryptosporidium parvum oocyst infectivity in river water samples as detected by tissue culture assay inactivation of cryptosporidium parvum oocyst infectivity by disinfection and sterilization processes effect of sodium hypochlorite exposure on infectivity of cryptosporidium parvum oocysts for neonatal balb/c mice the toxoplasma gondii oocysts from cat feces we thank all the volunteers in this study; susan pusek for assistance recruiting and caring for study volunteers; and alexandre dasiva, lisa lindesmith, erin-joi collins mcneal, deanne rhodes, douglas wait, and michael arrowood for assistance with laboratory procedures. dr. alfano-sobsey is an epidemiologist and industrial hygiene consultant for the public health regional surveillance team, office of public health preparedness and response, division of public health, north carolina. her professional interests include public health preparedness and emerging infectious diseases. key: cord- -bcyotwkf authors: alkire, sabina; chen, lincoln title: global health and moral values date: - - journal: lancet doi: . /s - ( ) - sha: doc_id: cord_uid: bcyotwkf nan shaped differently depending on the morals espoused. a rights-based or equity approach, for example, would be expected to differ from a utilitarian or humanitarian approach. also, an initiative is only partly justified by its moral expediency. of critical importance are factors shaped by knowledge and by institutional interests. moral soundness about why particular global programmes should be advanced may need to be balanced against the imperative of achieving consensus among people of many different moral views. yet, as we argue in the conclusion, moral clarity-as well as knowledge and institutional interests-can usefully shape what, when, and how health initiatives should best proceed. to stimulate discussion, we have selected four major schools of moral values commonly used to justify global health initiatives: humanitarianism, utilitarianism, equity, and rights. we could have analysed other schools, but these four, we believe, encompass a good range of moral positions. there are, of course, both large and important variations within each school. the appeal for charitable acts to meet pressing humanitarian needs is arguably the most common ethical basis for global health action. humanitarianism can be a form of virtue ethics but it also often a humanistic response to evident social problems. the ethos of humanitarianism is embedded in nearly all religions. in a humanitarian approach, people respond to human suffering and realise human fulfilment by acting in a virtuous manner based on compassion, empathy, or altruism. the virtues might be specific or broad. examples of specific acts are charitable tithing among baptists or zakat (charitable donations) among muslims. broadly proscribed virtues encompass such human qualities as generosity, honesty, trustworthiness, integrity, and fairness. among the wealthy, these virtues might be expressed as philanthropy, which often focuses on health. humanitarianism provides the primary ethical basis of voluntary action undertaken by non-governmental organisations (ngos), and is also an important base of public support for official foreign aid. us president george bush in announcing us$ billion in assistance for hiv/aids control described the pledge as a "work of mercy". public opinion polls in the usa consistently show that alleviating world hunger and providing drinking water are worthy of foreign aid from the usa. emphasis on voluntary generosity and self-expression (rather than on duties or obligations) gives humanitarianism a broad appeal to many social groups, corporations, and governments. contemporary appeals to people's humanitarian impulses focus on the giver: who a person becomes by acting well, and how a person realises a sense of accomplishment or fulfilment. there are dangers that those who are helped can be placed in a dependent position, treated as victims not agents. also, the underlying societal rules and structures that generate the social ills are not necessarily addressed. this approach might be more relevant to humanitarian catastrophes than structural approaches that attempt to correct the root causes of social problems. in a utilitarian framework, the value of health is determined by the subjective utility (happiness, pleasure, or desire satisfaction) that it creates for an individual. across all individuals in a society, the ideal state is one that maximises the aggregate utility. health could be valued because it generates utility directly, or because good health is instrumental to other utilitygenerating states, including opulence, or both. many contemporary health policies are based on a form of utilitarianism in which good health is valued as instrumental to maximising aggregate utility. for example, the who commission on macroeconomics and health calculated the costs and benefits of burdens of disease and argued that investing in health would generate economic growth, thereby enhancing incomes and aggregate utility. the utilitarian approach underscores important interconnections between health and other variables. it can show how improving the health of the deprived can be "in everybody's interest"-including the self-interest of people not inclined to altruism. its difficulties, however, are several. first, the instrumental valuation of health demeans it as an intrinsically valued goal in all societies. second, people's self-assessments do not www.thelancet.com vol september , rights were not granted to include this image in electronic media. please refer to the printed journal. necessarily match their observed health status. for example, the self-reported morbidity rates in the indian state of kerala, where life expectancy is - years, are significantly higher than in bihar, where life expectancy is significantly lower; and self-reported morbidity in the usa is higher still. third, it is rather difficult, even theoretically, to aggregate very different kinds of utility together into a single entity. finally, a utility-maximising approach is not directly sensitive to distributional concerns. equity is a relational concept in which ethical assessments are-at least in part-based on distributional features of one or more variable. fortunately, considerable intellectual work has recently been done on health equity and social justice. , building on the work of political philosopher john rawls, amartya sen has addressed some key features of health equity. first, he poses the question of "equity of what?" should equity be evaluated with reference to health achievement or access to health care? sen argues that equity in health should be assessed in terms of health capabilities and achievements rather than healthcare activities. after all, health care is a human activity; what people actually value is the capability to attain good health. he further notes that equitable social processes should inform evaluations of equity in the health space. in some equity domains, such as gender, completely equal distribution of health achievement could be considered unfair because women-whose lifespan in the absence of gender discrimination exceeds that of men-should, under an equity framework, enjoy longer life expectancies. an equity-based evaluation considers not only allocation of a fixed set of health resources, but also allocation of resources between health and other social objectives. equitable approaches to health have carried considerable power in mobilising support for health components of international development. striking disparities in health achievement and emotively powerful arguments of preventable suffering can animate the public and political leaders. an example is the recent call for public funds to expand antiretroviral treatment to hiv-positive people in poor countries. the disparity between the health of those with access to lifesaving drugs and the avoidable deaths among all others evokes the moral imperative to alleviate preventable human suffering caused by the inequitable access to antiretroviral drug therapy. human rights in health are embedded in several un declarations, and they have deep and wide moral bases. legal formulations were created to specify what was argued in the th century to be an inalienable moral claim grounded in the ontological dignity of human beings. human rights can be described as "things which are owed to man because of the very fact that he is man". some human rights can be expressed in the space of capabilities-for example rights to health, or to inclusion. yet rights also add to the capability perspective by invoking duties and obligations on the part of others. because each human being is recognised as an "end", human rights demand obligatory behaviour on the part of the state, firms, groups, and individuals. obligations may be "perfect" (as enshrined in law) or "imperfect" (a general duty to do what one can to help). calls for a rights-based approach to global health have recently grown. extensions of human rights to children and women both contain references to freedom from preventable suffering and freedom to exercise health choices. , the application of human rights to good health has drawn attention to the duties and obligations that people and institutions have towards human beings, viewed squarely as an "end" worthy of dignity. a human rights approach often assumes some health minimum that all people should be able to realise for human dignity. the challenge is to implement the corresponding "incomplete obligations" among communities, institutions, and states where good health depends upon resources, knowledge, technologies, and social action. these ethical schools do not track precisely to any specific health initiative. none of the schools dominates any specific health action, and several schools are often relevant to any single initiative. at present, whether the by initiative was evaluated according to aggregate utility (increasing the utility of people with hiv/aids) or distributional equity (increasing the numbers of people in developing countries who are given antiretroviral treatment), human rights (for health care), or the need www.thelancet.com vol september , rights were not granted to include this image in electronic media. please refer to the printed journal. for humanitarianism (to alleviate the suffering of those with hiv/aids), in all cases action is morally imperative. ensuring a minimal threshold of health might similarly fit well with humanitarianism and human rights, and equity and justice values will require action on behalf of the most disadvantaged. beyond moral values, the selection of global health initiatives is shaped by other, often implicit but no less valid, factors. among these are knowledge and institutions. to a large degree, ethical assessments will rest not only on the ethical perspective chosen, but also on the information selected for examination. the selection of information is shaped by political and scientific forces as well as by moral theories. paul farmer has written eloquently about the selective scrutiny of information that shapes health action. tuberculosis, especially multidrug-resistant tuberculosis, became recognised as a health crisis when it achieved rapid transmission in new york city. yet tuberculosis-before, during, and after the new york crisis-kills million people annually, most of whom are poor. because of informational selectivity, tuberculosis is a silent crisis among the world's poor, invisible to the rich and powerful. similarly, severe acute respiratory syndrome (sars) achieved front-page news because of its lethal nature and the paralysing effect it had on global commerce. yet, "sars-like" health catastrophes take place daily in thousands of rural villages in low-income countries. these health problems likewise severely affect families and communities, who are invisible to better-off and protected communities. scientific knowledge provides the basis for research and development of health technologies, such as vaccines and drugs. breakthroughs in health research raise moral challenges because they make feasible treatment for conditions that were hitherto incurablefor example, antiretroviral drugs for hiv. morally, there is a big difference between inevitable human calamity and suffering that can be prevented by modern technology. growing knowledge gaps between technological potentiality and health realities present huge ethical challenges. contention is further fuelled if the gap is accentuated by commercial interest. recent debates over affordable access to life-saving antiretroviral drugs have focused on the fairness of international regimes of intellectual property rights that are perceived to favour commerce over human health. global health, like other fields, has a cluster of institutional stakeholders. governments and intergovernmental agencies like the un and the world bank are mandated to play technical, financial, and operational roles. since health is a major component of the global economy, corporations have interests in profits as well as in protecting their public reputations. civil society organisations have many roles, ranging from the direct delivery of services to advocacy on public policies. institutions, like all actors, are endowed with certain capabilities and also seek to advance their bureaucratic, political, and financial agendas. one typical driver of organisational behaviour is to gain command over resources that can translate into more jobs, higher status, and more numerous activities. tracking of financial flows in global health initiatives can help reveal institutional winners and losers. historical studies have examined these institutional motivations in global health. the work of the rockefeller foundation overseas, for example, was often linked to corporate interests and political propagation of capitalism. in an excellent historical analysis of tuberculosis control in mid- th century, sunil amrith postulated that the conduct of tuberculosis programmes was primarily shaped by the state of knowledge and the capabilities of global institutions. field research had shown that directly observed therapy (dots) was highly effective in curing tuberculosis in home-based settings. endowed with new knowledge, yet limited by institutional capacity and scarce funding, who decided to pursue tuberculosis control through vertical programmes involving cadres of specifically tasked field workers rather than attempt to build holistic villagebased primary services. the latter approach would have been far more demanding institutionally and financially. a common usage of moral values is to mobilise public support. sometimes, however, advocates of global health do not give an accurate representation of distinct ethical schools, simply because they want everyone to agree. braveman and groskins, for example, argued that the concepts of equity and rights are essentially identical, and lead to similar strategies. their aim seems to have been advocacy for certain types of health actions rather than for clarification of distinctive moral schools. de cock argued that a public health rather than a human rights approach should frame responses to hiv/aids in africa, but again this analysis is based on a very narrow example of both ethical schools. we argue that clarity in thinking is essential, because different moral schools do indeed raise distinct considerations and it can be useful to evaluate these carefully. at the same time, the urge to seek consensus is also valid, and can be sought without either exaggerating differences, or claiming (inaccurately) that differences between moral schools do not exist. a common usage of moral values is advocacy, often to rich and powerful leaders, institutions, and nation states with the goal of mobilising resources-finance, political will, human motivations-on behalf of particular health action. but here we run into an apparent paradox: how can one use moral values as advocacy tools, when the moral schools are distinct, and when people argue passionately among them? in order to achieve the support, global health programmes also must build consensus among a diverse constituency of resource-holders as to the central value of the initiative. so when it comes to the language of why support for global health is important, we recognise, with cass sunstein, the wisdom of seeking "incompletely theorized agreements" in the moral discourse surrounding global health. in his tanner lectures in human values, sunstein argued that in some cases consensus can be achieved if participants refrain from elaborating their moral positions, because if they scrutinised these positions in depth, consensus could fracture. by contrast, he advocates an approach that "enlists silence, on certain basic questions, as a device for producing convergence despite disagreement, uncertainty, limits of time and capacity, and heterogeneity". sunstein's approach has the advantage of opening space for dialogue, exchange, and discussion, thereby promoting deliberative democracy, political accountability, and reason-giving. incompletely theorised agreements satisfy diverse constituencies who might have very different reasons, including incompatible values, for supporting a particular activity. there is a further point against requiring everyone to agree on only one ethical justification for global health. for not only might different approaches appeal to different groups, different people might also have distinct understandings of what the terms "rights", "equity", or "humanitarianism", actually mean. after all, the support base of global health initiatives is diverse, ranging from heads of state to private-sector executives to religious leaders to activists from ngos to opinion-setters and journalists. it is highly unlikely that these constituencies will share an identical understanding of ethical terms. a global health initiative can receive emphatic support from people who do not necessarily agree on the ethical foundations for their support, and in fact may very clearly disagree with one another as to why a programme should proceed-ie, its ethical or metaphysical justification. advocates of global health initiatives would thus do well to proceed with a general appeal to moral concepts such as social justice and compassion, and this generality belies prudence rather than a lack of moral rigour. yet an eclectic appeal to moral values in order to garner support of global health initiatives is not to imply that distinctions among moral values are trivial. beyond clarifying why an action is important, adopting a particular moral approach can influence health action in other deeply important ways. first is the scope of health action. an example is the programmatic implication of pursuing access to health care versus equitable distribution of health outcomes. in the former case, the programme would invest heavily in building health clinics and outposts, and perhaps in increasing the ratio of medical personnel per citizen. this sounds very appealing until one recognises that a country may have many rural health outposts, and many doctors on salary role, but if these doctors do not turn up to work, and the outposts do not have adequate pharmaceutical supplies, the population's health outcomes might remain very poor. on the contrary, to achieve an equitable distribution of health outcomes it would be necessary to make sure that the investment in health care results in better health across the population. it would also then be necessary to address broader social determinants of health, such as that raised in michael marmot's intriguing research on the under-recognised relation between socioeconomic inequality and health. second, different ethical schools (and different groups within them) may shape how global health programmes are undertaken. charitable acts might treat people as passive recipients of generosity, whereas rights-based approaches would encourage "voice" and participation to strengthen the agency of people for achieving their inherent rights. third, advocacy might use moral values to advance a global health agenda-because they are effective in advancing a global agenda. to mobilise a compassionate response, a picture of a feeble, emaciated, and large-eyed child might be used to stir pity among donors. such advertisements tend to view the poor as helpless victims, rather than people who could be empowered to care for themselves. arguably, much harm has been done by such dehumanising advocacy techniques. yet, it could be argued that such moral approaches are legitimate to use because they are more effective in evoking public support than other moral approaches. when people speak of ethics, the contribution that most readily leaps to mind is motivational: that an appeal to moral values will motivate people to support a set of actions. yet this is only one of the ways in which moral values can support global health initiatives, and is not necessarily the most powerful. discussions on whether to frame the objective of global health initiatives in terms of access to health care, or capabilities for good health, or utility maximisation, help to clarify what global health initiatives seek to accomplish. criteria such as efficiency, or equitable treatment for men and women, clarify which alternative actions to realise similar goals should be selected. consideration of how health activities contribute to or block non-health objectives such as the support of agency, or the rights to self-determination, clarify the importance of how health initiatives are carried forward. thus global health may be far easier to achieve if we pause to follow through different moral analyses and thereby clarify what, which, and how global health initiatives can best proceed. science and the health of the poor speech at the world health assembly health at the world summit on sustainable development philanthropy and health. london: king's college introduction to the principles of morals and legislation. london: the athlone press, . who commission on macroeconomics and health. macroeconomics and health: investing in health for development health: perception vs observation inequality reexamined challenging inequities in health: from ethics to action public health, ethics, and equity why health equity? the rights of man and natural law. london: geoffrey bles, the centenary press human development and human rights: human development report convention on the rights of the child vienna declaration and programme of action informational analysis of moral principles infections and inequalities: the modern plagueupdated edition with new preface rockefeller medicine men: medicine and capitalism in america plague of poverty? the world health organization, tuberculosis and international development, c - poverty, equity, human rights and health: policy and practice shadow on the continent: public health and hiv/aids in africa in the st century legal reasoning and political conflict aristotelian justice and health policy: capability and incompletely theorized agreements is inequality bad for our health key: cord- -yr u qv authors: miesbach, w.; scharrer, i.; asherson, r. a. title: recurrent life-threatening thromboembolism and catastrophic antiphospholipid syndrome in a patient despite sufficient oral anticoagulation date: - - journal: clin rheumatol doi: . /s - - - sha: doc_id: cord_uid: yr u qv we report on a -year old female patient with primary antiphospholipid syndrome (paps) and several thromboembolic events despite stable doses of oral anticoagulation, good patient compliance and maintained inr values of > . over the preceding years the patient had presented a wide spectrum of manifestations of aps, including recurrent venous and arterial thromboses, cardiac, gynecological (hellp syndrome), neurological involvements, livedo reticularis, a mild thrombocytopenia and the most feared manifestation of the catastrophic antiphospholipid syndrome (caps). life-threatening bilateral subdural bleeding occurred while she was anticoagulated. the clinical features appeared to be refractory to oral anticoagulation with phenprocoumon. they were life threatening on each occasion and she developed repetitive episodes of organ damage with cardiac insufficiency (nyha iii), pulmonary hypertension and other residual defects. even during heparinization recurrent thromboembolism supervened as well as livedo reticularis of the extremities. lupus anticoagulants (lac), anticardiolipin (acl) antibodies and anti-β( )-glycoprotein- (β( )gpi) titers were all markedly elevated. this case report shows that recurrent episodes of thrombosis can occur despite seemingly adequate anticoagulation in patients with caps. the antiphospholipid syndrome (aps) is one of the most common causes of acquired thrombophilia and is characterized by arterial and/or venous thrombosis, recurrent pregnancy losses, and the laboratory evidence of antibodies against phospholipids or phospholipidbinding protein cofactors [ ] . aps is considered to be an autoimmune disease with unpredictably occurring episodes of thromboembolism combined with serological demonstration of antiphospholipid antibodies (apl) [ , ] . further manifestations may include thrombocytopenia, valvular heart disease and a variety of neurological and gynecological disorders. the thromboembolic manifestations may be heterogeneous, possibly reflecting the heterogeneity of the apl themselves. common laboratory tests include functional coagulation tests for lupus anticoagulants (lac) and immunological assays for antibodies against cardiolipin (acl) and b -glycoprotein i (b gpi). this syndrome may be 'primary' or may be associated with other diseases, particularly systemic lupus erythematosus (sle). retrospective studies suggest that patients with aps have an increased risk of recurrent thromboembolism [ , , ] , and for this reason it is recommended that they receive oral vitamin k antagonists, such as warfarin, in order to achieve an international normalized ratio (inr) range within a therapeutic level (inr fi ). in most clinical situations, therapeutic doses are sufficient to control this disease and prevent further thromboembolism. the following case study documents a patient who, despite receiving the recommended oral anticoagulant therapy, developed recurring thromboembolic complications. these clinical features were life threatening on each occasion and appeared to be refractory to oral anticoagulation. the diagnosis aps was defined by the international consensus criteria (sapporo criteria) [ ] . the lupus anticoagulants (lac) were measured in platelet-depleted plasma and positive results were established following the guidelines proposed by the subcommittee for standardization of lupus anticoagulants (ssc) of the scientific and standardization committee of the isth [ ] . at our institution, lupus anticoagulants were determined by the kaolin-clotting time [ ] , the textarin/ ecarin time [ ] , a modified dilute russel viper venome time (drvvt) [ ] , and confirmed by the staclot la test [ ] . the detection and quantitative evaluation of circulating antibodies was calculated according to rosner [ ] : (ica=kct ( : mixture of patient/normal plasma)-kct (normal plasma)/ akct (patient plasma). the acl-igg and igm antibodies were determined by solidphase, b -glycoprotein-dependent standardized enzyme-linked immunosorbent assays (elisa) [ ] . an elisa method also measured b -glycoprotein antibodies. the level of protein s activity was low at % (normal value - %). all other tests for thrombophilia (factor v mutation/ activated protein c resistance and factor ii mutation) were negative, and the level of protein c was normal at % (normal value - %). assessment of heparin-induced thrombocytopenia was not performed because the patient's platelets were above /nl throughout her hospital stay. ana, ama, and rheumatoid factor tests were negative. antithrombin, homocysteine levels and lipid profile were normal. liver function tests showed mild elevations of ast and alt ( and u/dl, respectively) and a total protein of . mg/dl with normal albumin. blood protein electrophoresis showed normal values. hepatitis profile revealed that the patient had antibodies against hepatitis b virus. serum cryoglobulins were negative. the patient, a -year-old turkish woman was previously known to suffer from a glucose- -phosphate-dehydrogenase deficiency as well as from a previous hepatitis b infection. at the age of the patient gave birth to a male child in the th week of pregnancy after an emergency cesarean section undertaken because of hellp syndrome. from june to july the patient was admitted to hospital, initially because of nausea and vomiting. examination on entry to hospital showed livedoid discoloration of the third to the fifth toes on the right foot, as well as hepatosplenomegaly. angiography of the inferior vena cava showed an hourglass-shaped stenosis with prestenotic dilatation and retrograde filling of the veins of the liver. a diagnosis of a vena caval thrombosis with budd-chiari syndrome was made (fig. ). the patient was transferred to the thoracic surgery department of the frankfurt university hospital for replacement surgery for the vena cava. anca was negative. phospholipid antibodies and lupus anticoagulants were not determined. on october a pericardial patch operation was carried out in the presence of subtotal membranous inferior vena cava stenosis. two weeks later the patient was readmitted to hospital with tachycardia and dyspnea. a spiral ct scan of the thorax showed the presence on the right side of small peripheral wedge-shaped thickenings, indicating pulmonary emboli. ultrasound of the leg veins showed no evidence of thromboses. a spiral ct scan of the abdomen, using non-homogeneous contrast medium enhancement, showed hepatomegaly. thrombosis with a light restriction of % appeared attached subphrenically to the wall of the vena cava, and this had probably caused the pulmonary embolism. laboratory analysis revealed a ptt of s. lupus anticoagulants and high-titer anticardiolipin antibodies were positive and she was immediately started on oral anticoagulation with phenprocoumon. catastrophic aps: second episode of pulmonary embolization and myocardial infarction twenty months later (from august to september ) she was admitted to intensive care because of the development of a catastrophic aps [ ] . three weeks prior to admission, during a vacation in turkey, she had presented to hospital because of severe dyspnea, tachypnea, tachycardia and fever. her inr on admission was . . lac and acl antibodies were markedly elevated (table ) . she had signs of acute left-ventricular myocardial infarction (elevated values of ck and ck-mb), and chest x-ray now showed bilateral pulmonary infiltrates. sepsis and ards were suspected and intravenous antibiotics were commenced, which partially improved her condition (she became afebrile). cultures from blood, urine and tracheal fluid were negative. because of her serious respiratory condition, she was transferred by plane to the intensive care unit of the frankfurt university hospital for further treatment. a bronchoscopy showed signs of purulent bronchitis, and a ct scan of the lung showed bilateral infiltrates sparing the periphery which were interpreted as ards, as well as a questionable embolus in the right pulmonary artery. there were no signs of coronary artery disease by coronary angiography, and the myocardial infarction was interpreted as caused by coronary embolism. treatment with unfractionated intravenous heparin (aimed to elevate thrombin time to - s) was started. acetylsalicylic acid was not given because of thrombocytopenia ( /nl) and the glucose- -phosphate-dehydrogenase deficiency. although fully anticoagulated with heparin with normal antithrombin levels ( % upon admission), she developed arterial thromboembolic ischemia to the right arm and a toe of the right foot, as well as livedo reticularis (fig. ) . the clinical situation was interpreted as a catastrophic antiphospholipid syndrome (caps), possibly triggered by purulent bronchitis or pneumonia. plasmapheresis (pp) ( -h duration, with l of fresh-frozen plasma) was started and continued for days. this led to a dramatic decrease in acl-igm and anti-b -gp- titers, as well as to substantial clinical improvement. during the course of additional multimodal treatments (intravenous immunoglobulins, steroid therapy) her condition gradually improved; she recovered from shock and became afebrile. a ct scan of the chest revealed marked regression of the bilateral pulmonary infiltrates. bone-marrow histology was normal. five weeks later she was discharged from the hospital with residual signs of skin necrosis, and regular follow-up as well as continued anticoagulation was strongly advised. remarkably, acl-igg and ica and, to a lesser extent, anti-b -gp- titers had again increased when the patient was seen in the outpatient service (day after plasmapheresis initiation). however, there was no clinical evidence of disease. subsequent outpatient observation produced unchanged echocardiogram results, i.e. medium-grade limitation of function of the left ventricle (ef - %) with akinesia of the apex of the heart and the apical septum, as well as anterolateral hypokinesia. the mitral and aortic valves were well separated. there were no indications of right ventricular strain. the right hand showed loss of sensitivity and there was weakness in the median nerve distribution. electromyography studies were, however, normal. the patient in fact had burned her fingers and her hand on several occasions, and also complained of pains in the lower right arm. in spite of many attempts at treatment this residual state remained after the caps. with an inr of . there was once again an emergency readmission to hospital from march to march , with acute dyspnea and right-sided chest pain. again, a suspected acute pulmonary embolism was diagnosed. lac and acl antibodies were markedly elevated. a scintigram/ventilation-perfusion scan showed a subsegmental pulmonary embolism in the lower left in the lingula area, which was confirmed by a ct scan of the thorax. ct scan of the abdomen further showed hepatic venous obstruction with generally thin portal vein and non-homogeneous parenchyma. the vena cava showed perfusion but this was non-homogeneous, especially at the level of the liver. an echocardiogram showed noticeably increased mean pulmonary artery pressure (mpap mmhg). levels of ddimers and troponin t were initially higher, but then decreased in the course of the treatment. after discontinuing treatment with oral anticoagulants, heparinization with unfractioned heparin was introduced while monitoring the thrombin time (thrombin time: - s) methylprednisolone mg daily was also administered. subsequently, oral anticoagulant treatment was recommenced. this was in addition to the administration of clopidogrel. after weeks the patient recovered and could be discharged. running an inr of , the patient was again readmitted to hospital on july with coughing, dyspnea and pronounced weakness, as well as chest pain. lac and acl antibodies were markedly elevated. although an increased troponin test was found, the ck level was not elevated, and thus more invasive investigations were not undertaken. newly appearing subsegmental areas of decreased perfusion, typical of emboli, in the right central fold of the scintigram of pulmonary ventilation-perfusion scans strengthened the suspicions of a further acute pulmonary embolism. an echocardiogram showed the left-ventricular function of % to be decreasing, while the left atrium was distended ( mm). the ventricular septum was dyskinetic. in a duplex ultrasound scan the internal jugular vein was seen to be obstructed and partly recanalized. there was also an obstruction in the left external iliac vein. after discontinuing anticoagulation with phenprocoumon and dosing with unfractioned heparin after thrombin time observation, the patient suffered increasing loss of consciousness accompanied by aphasia days after entering hospital. a ct scan showed bilateral subdural bleeding with compression of the lateral ventricles and displacement of the central line (fig. ) . during transportation for neurosurgical treatment, an increasing pupillary difference with dilated pupils was noticed. on arrival the pupils were dilated and round bilaterally. a left frontal craniotomy was immediately carried out, with relief drainage, and a right frontal drill-hole trepanation with drainage tubes was inserted. during the course of treatment, brainstem reflexes remained constant. ct images that were prepared because of persistent headache showed a left parietal cyst formation, which subsequently dissolved. the oral anticoagulation was changed to warfarin. clopidogrel was discontinued. during the course of further treatments the patient recovered almost completely. she still suffers from disturbances of fine motor function and sensory disorders in the right half of the body. a variety of cardiac, neurologic and gynecologic manifestations are associated with the presence of apl. a particularly serious and often fatal clinical form with a high mortality rate of approximately % despite treatment has been termed the 'catastrophic' antiphospholipid syndrome (caps) (asherson's syndrome) [ , , , ] . these patients present with fulminant thrombotic complications affecting predominantly small vessels in the majority of cases. however, large vessel occlusions, e.g. deep vein thromboses affecting the lower limbs mainly, with complicating pulmonary emboli and cerebral arterial occlusions, do also occur in a minority of patients. despite the increasing understanding of the mechanism and clinical manifestations of aps, thrombotic complications are unpredictable and 'triggering factors' are not identifiable in the majority of cases, although risk factors are now increasingly being identified. these include warfarin withdrawal, inadequate warfarinization, and the administration of certain drugs, particularly oral contraceptives etc. retrospective studies suggest that patients with aps have an increased risk of recurrent thromboembolism [ , , ] . patients with high positive acl antibodies may have more frequent thrombo-occlusive events after cerebral ischemia [ ] . it is therefore recommended that such patients receive oral vitamin k antagonists, such as warfarin, in order to achieve an international normalized ratio (inr) within the recommended therapeutic range. a beneficial role for anticoagulants in decreasing the rate of recurrent thrombosis has been shown in several retrospective studies [ , , ] . oral anticoagulation therapy with warfarin is monitored by the prothrombin time and the international normalized ratio (inr). in general, ther-apy with oral anticoagulants targeted at an inr of . is very effective as it reduces the risk of recurrent thromboembolism by - % [ ] . this approach also holds for patients with the hereditary thrombophilias [ ] . in patients with lupus anticoagulants who have sustained a thromboembolic event the recommended inr for oral anticoagulation is controversial. it has been recommended by some that high-intensity anticoagulation with inrs of . or greater for patients with aps should be administered to prevent further thromboembolic complications [ ] . a comparison of periods with no treatment, aspirin, and several intensities of oral anticoagulation has shown that only high-intensity anticoagulation was associated with a significant reduction of the recurrence rate. with an inr of > , no recurrences were noted by rosove et al. [ ] , whereas others found . events per patientyear during the mean evaluation period of and years [ ] . in contrast, ginsberg et al. saw no recurrent thromboses during conventional-intensity warfarin therapy (inr . - . ) [ ] . data from a recently completed prospective study over a -year follow-up period (warss/apass) has suggested that there is no difference between warfarin (inr between . and . at a suggested target of . ) and aspirin ( mg/day) in preventing recurrent aps manifestations and major bleeding complications [ ] . this is in comparison to a higher rate of recurrence in patients with aps and without any treatment. on the contrary, data from the stroke prevention in reversible ischemia trial (spirit) indicate that high-intensity anticoagulation (inr . - . ) may lead to a significant excess of major bleeding complications [ ] . the role of asa in preventing recurrent pregnancy losses in aps has been well demonstrated [ , ] . there have been recent reports that emphasize the prophylactic role of asa [ , ] , although asa is not administered universally for the primary prevention of thrombosis in apl-positive individuals. recently consensus statements have been published concerning prophylaxis and treatment guidelines of the antiphospholipid syndrome [ , , , , , , ] . in patients with catastrophic aps the most common form of treatment is a combination of anticoagulation, corticosteroids and plasmapheresis [ ] . over a -year period, our patient presented a wide spectrum of manifestations of aps, including recurrent venous and arterial thromboses, cardiac, gynecological (helpp syndrome), neurological involvement, livedo reticularis, a mild thrombocytopenia, and the most feared manifestation of caps. life-threatening bilateral subdural bleeding occurred under anticoagulation. the patient fulfilled the definition of caps according to asherson, with involvement of at least three organs and multiple small vessel occlusions (heart, lung, skin and foot) [ ] and according to the recently proposed classification criteria of caps [ ] . as caps has been associated with preceding infections [ , ] the pulmonary infection leading to an adult respiratory distress syndrome (ards) may have triggered its development. plasmapheresis led to clinical improvement as well as a dramatic decrease of anti-b -gp- titers that has been correlated with successful treatment of caps [ ] after the patient's condition gradually improved owing to the multiple modalities of treatments administered, the acl-igg-titer and ica and, to a lesser extent, anti-b -gp- titers had again increased when she was seen in the outpatient clinic (day after plasmapheresis initiation). there was no clinical evidence of any recurrent complications. the patient developed multiorgan damage with cardiac insufficiency (nyha iii), pulmonary hypertension, and residual defects following recurrent thromboembolic complications. most of these manifestations appeared under anticoagulation with phenprocoumon and an inr > ( table ) . the manifestations of caps, including myocardial infarction caused by arterial thromboembolism, had also occurred in spite of phenprocoumon treatment with an inr of . , a level that is usually deemed sufficient to prevent thrombosis [ ] . however, circulating antibodies have been described which may interfere with correct measurement of the inr [ ] . in patients with aps also, a prolonged prothrombin time may be found as a result of an antibody-mediated decrease in factor ii levels [ ] . even closely monitored anticoagulation using intravenous heparin in an icu setting was unable to prevent ongoing embolism, and the patient suffered from arterial embolism to her right foot while being treated with i.v. heparin with a controlled thrombin time of s (normal range < s). recurrent thrombosis has been observed in patients with caps and individual patients may need repeated plasmapheresis for several years [ ] . however, recurrences of caps are extremely rare. the low protein s level of the patient may have enhanced her susceptibility for thromboembolism, and antibodies against both b -gp- and protein s have been described [ ] . alternatively, the antiphospholipid antibodies may have interfered with protein s measurement. a family protein s deficiency is unlikely, because her family tests were normal. the inhibition of platelet aggregation with clopidrogrel given in addition to the oral anticoagulation was also unable to prevent the ongoing thrombotic process, as recurrent pulmonary emboli and life-threatening cerebral bleeding occurred while she was undergoing this therapy. the major fear of clinicians in advising high-intensity anticoagulation is that bleeding complications will exceed its beneficial effects, and this fear is well founded. the frequent presence of thrombocytopenia or disturbed platelet function in patients with aps may in fact render these patients more vulnerable to this complication. several retrospective studies have reported major bleeding complications in these patients [ , , ] . the risk for severe hemorrhagic complications, notably subdural hematoma, in young aps patients with highintensity oral anticoagulation is certainly increased [ ] . the aps is characterized by the presence of different autoantibodies and is a systemic disease with various immunological abnormalities [ ] . future therapeutic interventions will no doubt concentrate on this aspect, and immunomodulation as well as new treatments for recurrent thrombosis for patients such as ours will undoubtedly become important subjects for future research. because of the rarity of the catastrophic syndrome many newer therapies, e.g. recombinant protein c, have not as yet been documented as being effective in this life-threatening condition. international consensus statement on preliminary classification criteria for definite antiphospholipid syndrome: report of an international workshop etiology and pathogenic mechanism of the anti-phospholipid syndrome unraveled mechanism of autoantibody-mediated thrombosis the antiphospholipid syndrome epidemiology of the antiphospholipid antibody syndrome antiphospholipid syndrome: clinical and immunologic manifestations and patterns of disease expression in a cohort of , patients criteria for the diagnosis of lupus anticoagulants: an update. on behalf of the subcommittee on lupus anticoagulant/antiphospholipid antibody of the scientific and standardisation committee of the isth lupus anticoagulant measurement the textarin/ecarin ratio: a confirmatory test for lupus anticoagulants the use of the dilute russell viper venome time for the diagnosis of lupus anticoagulants detection and quantitative evaluation of lupus circulating anticoagulant activity anticardiolipin antibodies: detection by radioimmunoassay and association with thrombosis in systemic lupus erythematosus combined plasmapharesis and immunosuppression as rescue treatment of a patient with catastrophic antiphospholipid syndrome occurring despite anticoagulation: a case report catastrophic antiphospholipid syndrome. clinical and laboratory features of patients the catastrophic antiphospholipid syndrome pathophysiology of the catastrophic antiphospholipid syndrome (caps) the catastrophic antiphospholipid syndrome -asherson's syndrome igg anticardiolipin antibody titer > gpl and the risk of subsequent thrombo-occlusive events and death. a prospective cohort study antiphospholipid thrombosis: clinical course after the first thrombotic event in patients patients with antiphospholipid antibodies and venous thrombosis should receive long term anticoagulant treatment the management of thrombosis in the antiphospholipid-antibody syndrome genetic susceptibility to venous thrombosis investigation and management of heritable thrombophilia antiphospholipid antibodies and venous thromboembolism tilles bc, the apass study group et al ( ) apl and stroke study (apass) the stroke prevention in reversible ischemia trial (spirit) study group ( ) a randomized trial of anticoagulants versus aspirin after cerebral ischemia of presumed arterial origin randomised controlled trial of aspirin and aspirin plus heparin in pregnant women with recurrent miscarriage associated with phospholipid antibodies antiphospholipid antibody-associated recurrent pregnancy loss: treatment with heparin and low-dose aspirin is superior to low-dose aspirin alone should low-dose aspirin also be a background therapy for all patients with systemic lupus erythematosus (sle) prophylactic antithrombotic therapy for patients with systemic lupus erythematosus with or without antiphospholipid antibodies: do the benefits outweigh the risks? a decision analysis prophylaxis of the antiphospholipid syndrome: a consensus report venous thromboembolism in the antiphospholipid syndrome: management guidelines for secondary prophylaxis stroke and the antiphospholipid syndrome: consensus meeting taormina non-stroke neurological syndromes associated with antiphospholipid antibodies: evaluation of clinical and experimental studies cardiac disease in the antiphospholipid syndrome: recommendations for treatment treatment of pregnant patients with antiphospholipid syndrome catastrophic antiphospholipid syndrome: international consensus statement on classification criteria and treatment guidelines the role of infection in the pathogenesis of catastrophic antiphospholipid syndromemolecular mimicry? therapeutic plasma exchange for the acute management of the catastrophic antiphospholipid syndrome: beta ( )-glycoprotein i antibodies as a marker of response to therapy monitoring warfarin therapy in patients with lupus anticoagulants a mechanism for the hypoprothrombinemia of the acquired hypoprothtrombinemia-lupus anticoagulant syndrome catastrophic antiphospholipid syndrome: response to repeated plasmapheresis over three years dual antibody reactivity to beta -glycoprotein i and protein s: increased association with thrombotic events in the antiphospholipid syndrome hemorrhagic complications of long-term anticoagulant therapy in patients with systemic lupus erythematosus and antiphospholipid syndrome systemic antiphospholipid syndrome key: cord- -jy uvwre authors: yuen, kenneth s.c.; chan, wai-man; fan, dorothy s.p.; chong, kelvin k.l.; sung, joseph j.y.; lam, dennis s.c. title: ocular screening in severe acute respiratory syndrome date: - - journal: am j ophthalmol doi: . /j.ajo. . . sha: doc_id: cord_uid: jy uvwre to investigate the ocular manifestations of patients with severe acute respiratory syndrome (sars) and to monitor the possible ocular complications arising from the treatment regimen with high-dose systemic corticosteroid drugs. design: prospective, observational cohort case series. methods: ninety eyes from patients with the diagnosis of sars during an epidemic outbreak in hong kong were analyzed. relevant medical and ophthalmic histories were taken. ophthalmic examinations, including best-corrected visual acuity, intraocular pressure, slit-lamp, and indirect ophthalmoscopy examination, were performed at baseline and at -month and -month follow-up. setting: faculty practice in university hospital. results: only two patients had mild elevated intraocular pressure at baseline and at subsequent follow-up. there was no loss of visual acuity, cataract progression, or increased cup–disk ratio. fundus examinations were unremarkable in all patients. conclusions: our study did not demonstrate any ocular manifestations in patients with sars. the treatment regimen of high-dose corticosteroid also did not show any significant ocular complications. routine ocular screening of patients with sars for diagnosis or for complications might not be indicated. s evere acute respiratory syndrome (sars) is a newly recognized but highly contagious disease entity that has recently been reported in asia, north america, and europe. , within a short period of time, the outbreak afflicted more than countries and became a global health threat. the infectious agent of this "modern plague" was suspected to be caused by a novel coronavirus strain (sars-cov), and coronavirus has been previously reported to be associated with retinitis and a breakdown of the blood-retinal barrier in animal studies. , the ocular involvements of this novel coronavirus strain, sars-cov, and its disease, sars, in humans were not known. methylprednisolone pulse therapy and high-dose oral prednisolone constituted the major part of the treatment regimen for sars. prolonged use of a corticosteroid drug is associated with glaucoma, cataract, central serous chorioretinopathy, and papilloedema. there is little information in the literature on the significance of routine ocular monitoring during the acute phase in prescribing pulse or high-dose corticosteroid therapy. in march , hong kong was seriously affected by a massive outbreak of sars, and we took that opportunity to conduct a prospective observational study to investigate the probable ocular manifestations arising from sars and the possible short-term complications resulting from the pulse or high-dose corticosteroid therapy. the results are important to physicians and ophthalmologists in determining whether a routine ocular examination should be arranged in suspected cases for diagnosis and in probable or confirmed cases for monitoring of complications. patients with the diagnosis of sars who had been managed in the prince of wales hospital, hong kong, were recruited. we only included cases that were probable and therefore fit the case definition issued by the world health organization. cases were excluded if an alternative diagnosis could fully explain their illness. patients physically unfit for ophthalmic examinations or those who failed to give informed consent were also excluded. the study was approved by the ethics committee of the chinese university of hong kong. relevant medical and ocular histories, including diabetes mellitus, hypertension, glaucoma, high myopia, previous ocular disease, and surgery, were recorded. patients were assessed with a comprehensive ocular examination including best-corrected visual acuity, intraocular pressure (iop) by noncontact tonometer ([nct] xpert noncontact tonometer plus; reichert ophthalmic instruments, new york, new york, usa), slit-lamp, and binocular indirect ophthalmoscopy at baseline and at months and months. use of personal protective equipment recommended by the infectious control unit of the hospital was strictly maintained throughout the examination. a total of patients ( eyes) were recruited; ( . %) were female and were male. their age ranged from to years (mean, years). most patients were recruited during the first week (n ϭ , . %) or second week (n ϭ , . %) of their diseases. four patients had been admitted to the intensive care unit, and of them died during the follow-up, but no postmortem study on the eye was made. the mean Ϯ standard deviation of the iop at baseline was . Ϯ . mm hg. two patients had iop higher than . mm hg. one was . mm hg, and the other was . mm hg. both had a normal visual field with humphrey central - threshold test and had no other glaucomatous features. twenty-seven patients ( %) completed the secondmonth follow-up and patients ( %) completed the third-month follow-up. only patients ( %) were still on prednisolone by the second month (dosage ranged from to mg), and none required any by the third month. no visual acuity loss, cataract progression, or anterior uveitis was observed. fundus examinations were unremarkable in all patients, with particular emphasis on any vitritis, retinitis, vaculitis, central serous chorioretinopathy, and changes at the optic disks. for the patients with mildly elevated iop in both eyes, the iop was persistent elevated at the same level months after corticosteroid cessation. there was no statistically significant difference between iop at baseline and month (t test, p ϭ . ; spss . for windows). at month , when no patient was on corticosteroid, the mean iop was . Ϯ . mm hg. there was only one case with iop greater than . mm hg. this patient had an iop of . mm hg at month and iop of . mm hg at baseline. none of the cases had an iop rise of at least . mm hg compared with the baseline iop. our study did not demonstrate any ocular manifestations resulting from sars or the novel coronavirus. no significant elevation in iop or other corticosteroid-related ocular complications was observed in the short-term follow-up. coronavirus-associated atypical pneumonia is extremely infectious, and the transmission appears to be through direct contact or contact with respiratory droplets in close vicinity to an infected person. health care workers are particularly at risk for infection, as reflected by the numbers of those involved. among the total number of , cases worldwide, , ( %) were health care workers. with the unremarkable ophthalmologic findings of this study, routine ocular screening in patients with sars for diagnosis or for complications may not be worthwhile. p atients with disorders of the cone photoreceptors often have debilitating photophobia, over and above their decreased visual acuity. this prevents them from optimal visual functioning in the daytime and even in ordinary indoor illumination. patients often need to wear a baseball cap or visor in addition to light-absorbing glasses and still find it difficult to function. several investigators have reported improvement in effective vision in cone disorders by using red or grade gray-tinted contact lenses, but these reports have generally been of only one or two severe acute respiratory syndrome (sars)-multi-country outbreak. who-recommended sars cases definition severe acute respiratory syndrome (sars): summary table of sars cases by country on newly discovered coronavirus as the primary cause of severe acute respiratory syndrome the role of apoptosis within the retina of coronavirus-infected mice blood-retinal barrier breakdown in experimental coronavirus retinopathy: association with viral antigen, inflammation, and vegf in sensitive and resistant strains ocular complications of paediatric patients with nephrotic syndrome key: cord- -ei faruy authors: zheng, xiaohong; li, kejun; wang, ruzhu; zhao, liping; xu, lisa x.; chen, yazhu; jin, xinqiao; gu, bo; bai, jingfeng; liu, hongmin; ye, xiaojiang title: experimental investigation of integrated air purifying technology for bioaerosol removal and inactivation in central air-conditioning system date: journal: chin sci bull doi: . /bf sha: doc_id: cord_uid: ei faruy in this research, high voltage static electricity and ultraviolet technologies were integrated to an air purifying device which can be used to trap and kill airborne bacteria and viruses in central air-conditioning systems. an experimental platform was built to mimic the central air system, in which the efficacy of the newly built device was examined. in addition to the standard physical and chemical tests, bacteriophages were used to simulate airborne viruses in the experimental system. the bacteriophage suspension was aerosolized into the air with ultrasonic wave atomization. the result showed that more than % removal efficiency of micro-particles (< micron in diameter) were removed after the device was in operation in a building and more than % of bacteriophages in the experimental system. it is concluded that the integrated air purifier is suitable for controlling air quality and preventing virus transmission through the central air system. since the winter of , severe acute respiratory syndrome (sars) affected nearly countries in all the continents, resulting in deaths by july , as reported by world health organization (who) [ ] , , ) . some research indicated that the sars virus was predominantlyspread by liquid droplets, and/or by direct and indirect close contact [ ] . the central air-conditioning system has been considered as a probable means of sars transmission in hospitals or office buildings. international scientific community has been in the forefront of fighting against sars in discovering the means of sars transmission and searching for effective ways of prevention [ ] . fresh air circulation can dilute the virus concentration inside a room and was found effective in preventing virus spread. but it cannot completely eliminate the sars virus from an air conditioned environment. thus, it is necessary to trap and kill bacteria and viruses in the central air conditioning system. in this work, a central air purifying device was developed by integrating high voltage static electric field and ultraviolet irradiation technologies. an experimental platform simulating central air-conditioning system was built to perform some physical and chemical tests of the device. further, one bacteriophage strain jd-ii- isolated from the environment was used as a viral simulant to examine the efficiency of the device for bioaerosol removal and inactivation. experimental studies ( ) test system. this test system consists of an experimental chamber of m , a test section and an airconditioning section including an ultrasonic humidifier, a finned tube heater, a finned tube heat exchanger and a fan. fig. shows the schematic of the whole system which is completely sealed. air flow is uniform and re-circulated from the chamber to the test section, and then the air-conditioning section. air change per hour (ach) can be adjusted with the speed of the fan. the rest section could be easily replaced by any kind of air purifying device and used to examine the dust-cleaning and bacteria-killing efficiency of the device by circulating micro-particles and bacteriophages in the system. furthermore, the micro-environment (i.e. temperature, relative humidity, etc.) can be adjusted in the system to investigate the climate effect on the virus sensitivity, infectivity, and transmissivity. ( ) integrated air purifier. fig. shows the integrated air purifier installed in the test section. it was built by integrating high voltage static electric field, ultraviolet ray, composite silver electrodes, and an active carbon fiber filter. large particles can be first filtrated by active carbon fibers. small particles and virus aerosols are mainly polarized and trapped by high voltage static electrical field. the silver electric plates of v high voltage together with ultraviolet ray completely kill the trapped bacteria and viruses within minutes. upon binding onto the bacteria cell membrane, silver ion can penetrate and react with sulfhydryl (-sh) to damage the cell synzyme activity, leading to cell death [ , ] . but the silver ion does not react on mammal cells because of the totally different cell membrane structure. the major emission band of the c waveband ultraviolet light is around nm wavelength which is mainly adsorbed by nucleoprotein (> %). when the nucleic acid absorbs high-frequency ultraviolet waves, the chemical bond of the nucleic acid molecule would be damaged, resulting in abnormal decompose or denaturalization to cause death of bacteria and viruses. ( ) bacterophage as a viral simulant for sars and other airborne viruses. bacteriophages are the viruses of bacteria and they have specific host range, one phage strain only infects a limited number of strains in one bacterial species. they do not infect animals or human beings. it is thus safe to use bacteriophage as viral simulant in the place of sars or other severe animal or human viruses [ ] . the amount of the viral simulant particles in the air can be collected and measured for plaque formation units per sample by using a double layer plating technique. four phage strains named jd-ii- , jd-ii- , jd-ii- and jd-ii- were isolated from an aquatic sample by using e. coli mg as the host strain. resistance to environmental extremes including temperature and dryness was tested for all the four phage strains. table shows that the resistance of jd-ii- to both temperature and dryness was superior to sars cov virus. this provides a basis for using this particular phage strain as a viral simulant in place of sars cov and other airborne viruses in the tests for evaluation of bioaerosol removal and inactivation by different types of air purifiers. disinfectant a) the initial bacteriophage titer was pfu/ml. the data were expressed as the percentage of phage particles survived from the treatments. ( ) bioaerosol generating and sampling technique for the viral simulant. it is a key technology in this research to disseminate the bacteriophage stock culture in the air to simulate the virus aerosol caused by actions such as sneezing. ultrasonic humidifier was used to evaporate the phage suspension to create the bioaerosol of the viral simulant. the bacteriophage suspension was aerosolized into the air with ultrasonic wave atomization. aerosolization velocity was measured by quantifying the mass difference of the suspension before and after atomization per unit time. the effects of viral concentration, ion strength and viscosity of the viral suspension on aerosolization speed were also measured in this work. no significant differences were found between the aerosolization speed of pure water and viral suspension containing different ratios of culture medium (between . % %). the aerosolization speed remained constant at . ± . ml/min with all the tested variables. no significant impact on viability of the phage particles was observed with repeated and lengthened treatment under ultrasonic wave in the humidifier. the bacteriophage particles were collected by using a semisolid plate via the impaction of the airflow at m/s in the test tube. gelatin is viscous and becomes liquid after incubation at for min. three kinds of gelatin plates were prepared for testing their sampling efficiency of airborne phage particles. the mp plate contained % of gelatin. ten milliliter % gelatin was added on lb plate to compose glb double layer plate. the gsm double layer plate was constituted with . % agar as bottom layer and % gelatin in sm buffer solution as top. the centimeter diameter plates were arranged at different sites horizontally or tilted toward the incoming airflow to collect phage aerosol. ( ) testing the dust-cleaning efficiency of the integrated air purifier. the particle generation source, such as mosquito-repellent incense, was ignited in the experimental chamber at the beginning of the experiment and closed until the pm concentration measured by the tsi dusttrak monitor reached a preset value. the humidity was controlled around %. the air was circulated through the system and the chamber temperature was controlled by the air-conditioning section. all experiments were performed at a constant air velocity of m/s, a constant temperature of . the deposition of particles onto the inner surfaces of the system were researched by measuring the pm concentration in the center of the experimental test chamber with respect to time, to establish the baseline measurement. then, the pm concentration was measured with the air purifier at v voltage in operation under the same condition. ( ) testing the sterilizing efficiency of the integrated air purifier. the ultrasonic humidifier containing bacteriophage suspension generated homogeneous bacteriophage droplets into recycling airflow. the gsm plates were used to collect bacteriophage particles in the airflow at the beginning and end site in the test tube with airflow at m/s for min. each test repeated times. the plates containing bacteriophage were incubated at for h to melt the gelatin. all the liquid was transferred to a centrifuge tube. ml of the phage liquid was mixed well with . ml of the host bacterial suspension (at od nm = . ). after standing still for min at room temperature, the suspension was mixed with ml melted soft agar and poured quickly onto lb plate. the solidified plates were incubated at overnight. the number of plaques per plate was recorded to calculate phage particles collected. fig. shows that the pm concentration decreases with time or without the air purifier in the experimental chamber. with the air purifier, the pm concentration decreased from . to . mg/m following an exponential decay over one-hour period while it took more than h to reach the same level via natural deposition. to simplify the analysis, it is reasonable to assume that the air is fully mixed and the particulate concentration is uniform inside the test chamber. the air purifier has a filter system capable of trapping and retaining . % to . % of all mono-dispersed particles of micrometers or less in diameter ) when the effect of deposition, resuspension and adsorption with chamber inner surface are considered. the high concentration smoke particle was extraordinary irritant to eyes and nose of tester. the device helped eliminate the irritating smoke and odor in one hour. the pressure drop of the device meets the standard of a high efficiency filter which is around pa at m/s wind velocity measured by an inclined tube micronanometer [ , ] . according to the indoor air quality standard in china gb/t - ) , the ozone in indoor air should be less . mg/m . it is possible that the hydroxyl and negative oxygen ion might be ionized from water vapor in air with the high voltage and ultraviolet ray. the ozone density can be tested with eco ozone monitor. under the situation of using high voltage static electric field and ultraviolet ray, the ozone density fluctuated from . to . mg/m , while the average ozone density is . mg/m using high voltage static field only and . mg/m using ultraviolet ray only. therefore, the air purifier does not introduce the secondary ozone pollution. fig. (a) shows that the plaques formed on a gsm plate were used to sample the airflow containing phage aerosol generated with a source suspension with pfu/ml when the integrated air purifier was turned off. more than plaques were formed in one such plate. shown in fig. (b) , less than plaques were found in one plate when the integrated air purifier was turned on. a % bioaerosol removal efficiency was thus achieved with the integrated air purifier in action. after the treatment of the integrated air purifier, phage concentration in the airflow was reduced to a minimum. it is very important to assure enough accuracy for phage measurement at such a low concentration. in this experiment, removal rate of bacteriophage solely depended on the high voltage static equipment without active carbon fiber film, and no plaque was detectable when the system coupled with the active carbon fiber film. thus, we need to increase the phage titer at the source suspension to ensure accurate detection of airborne phages. fig. shows the phage particles collected from the test tube with the air purifier on or off when the concenrifier was installed in place and turned on. the integrated air purifier reduced airborne viral particles by orders of magnitude. it was demonstrated that the aerosol generation and sampling technology used in this work was able to evaluate the performance of the integrated air purifier effectively in a biologically and statistically sound way. tration of bacteriophage stock culture in the humidifier was at a titer of pfu/ml with one minute sampling time. through the statistic test, the difference of removal efficiency was significant under . % degree of confidence with or without turning the air purifier on. the aerosolization and collection of phage particles were thus reproducible. when the test tube was void of any purifying measures, phage particles collected at the end of the test tube was ( . . ) pfu/plate, the number dropped to ( . . ) pfu/plate if the integrated air pu- the air purifier was also used in the central air-conditioning system of an office building at shanghai jiao tong university and the sixth people's hospital of shanghai. two supply diffusers were disposed on the right side wall and two exhaust grilles were laid on the upper side wall in the test room of the building. the particle removal efficiency was tested in a low initial concentration with one or two air purifiers installed in the exhaust or without fresh air to avoid the environmental effect. fig. shows the total average removal efficiency of . % and thus the bacteria removal efficiency is estimated to be more than % according to the ashrae applications handbook [ ] . fig. . removal rate of particles by the air purifier in the central air-conditioning system in an office building. , natural deposition; , using one hybrid purifier in one supply diffuse; , using two hybrid purifier in two supply diffuse. in addition to particle removal test, airborne bacteria were also sampled in the experimental room with the integrated air purifier. two sampling methods were used to collect the airborne bacteria. depositional sampling was used to sample airborne bacteria by placing culture plates at sites in the room. impaction sampler was used at . l/min speed to measure bacteria in per unit volume of the air. the result showed that the bacteria decreased from . to . cfu/plate, falling by . % after the integrated air purifier was in action for min. the bacteria collected with the impaction sampler showed a decrease by one to two order of magnitude. this demonstrated a significant effect of the integrated air purifier for cleaning the indoor air in a real world building. based upon the integrated technology of high voltage electric field, ultraviolet ray, composite silver material, and activated carbon fibers, an air purifying device has been developed to prevent airborne bacteria and virus spread through central air-conditioning system. an experimental platform mimicking the central air-conditioning system was built to examine the efficacy of the device. in addition to the physical and chemical tests, bacteriophage was successfully used as a viral simulant in the place of sars cov virus to evaluate the viral removal efficiency of the air purifier. it has been demonstrated that the test methods are feasible and that the integrated air purifier has significant removal and inactivation effects both in the laboratory and in real world building. the integrated air purifier holds great promise in prevention of airborne viral transmission and improvement of indoor air quality in the future. emergency measures in hvac systems against sars, heating ventilation & air conditioning & sars overview of disinfection methods of biologic pollution in hvac systems stochastic dynamic model of sars spreading catalytic materials evaluated by adsorption and inactivation of parainfluenza virus cytotoxicity of mammalian cells destruction of living cells by pulsed highvoltage application a continuous treatment system for inactivating microorganisms with pulsed electric fields standard test method for resistance of materials used in protective clothing to penetration by blood-borne pathogens using phi-x bacteriophage penetration as a test system an experiment study of cylinder-shaped hepa filter the theory of air-cleaning technology chapter : health care facilities key: cord- - upcncw authors: zhu, jieqing; xiao, gengfu; xu, yanhui; yuan, fang; zheng, congyi; liu, yueyong; yan, huimin; cole, david k; bell, john i; rao, zihe; tien, po; gao, george f title: following the rule: formation of the -helix bundle of the fusion core from severe acute respiratory syndrome coronavirus spike protein and identification of potent peptide inhibitors date: - - journal: biochem biophys res commun doi: . /j.bbrc. . . sha: doc_id: cord_uid: upcncw severe acute respiratory syndrome (sars) coronavirus (sars-cov) is a newly identified member of family coronaviridae. coronavirus envelope spike protein s is a class i viral fusion protein which is characterized by the existence of two heptad repeat regions (hr and hr ) (forming a complex called fusion core). here we report that by using in vitro bio-engineering techniques, sars-cov hr and hr bind to each other and form a typical -helix bundle. the hr , either as a synthetic peptide or as a gst-fusion polypeptide, is a potent inhibitor of virus entry. the results do show that sars-cov follows the general fusion mechanism of class i viruses and this lays the ground for identification of virus fusion/entry inhibitors for this devastating emerging virus. following the rule: formation of the -helix bundle of the fusion core from severe acute respiratory syndrome coronavirus spike protein and identification of potent peptide inhibitors severe acute respiratory syndrome coronavirus (sars-cov) has been identified as a new distinct pathological entity [ ] [ ] [ ] and the disease infected more than people and killed worldwide, mostly in asia, before it was brought under control in july between the winter and spring in - (who website: www.who.int). it has been shown that the genomic sequence is unrelated to any other known members of family coronaviridae isolated from either humans or animals [ ] [ ] [ ] , thereof designated as a new group, group iv, along with the previous three known groups [ ] [ ] [ ] [ ] [ ] [ ] . members in family coronaviridae are a group of enveloped positive-stranded rna viruses with largest genome among the rna viruses and have - envelope proteins embedded on the surface of the viral envelope lipid membrane [ , ] . the genomic sequencing reveals that, as with other enveloped rna viruses, including the coronaviruses [ ] [ ] [ ] , sars-cov envelope spike (s) protein contains highly conserved heptad repeat regions (hr and hr ), which have been shown as important in virus membrane fusion and successfully used as targets for virus entry/fusion inhibitors in a number of viruses [ ] [ ] [ ] [ ] [ ] [ ] , including a coronavirus, mouse hepatitis virus (mhv) [ ] . the existence of hr regions is also a characteristic of class i viral fusion protein [ ] . currently two classes (classes i and ii) of virus fusion proteins have been classified [ ] . it is generally believed that the envelope protein undergoes a series of conformational changes during the virus fusion process [ , [ ] [ ] [ ] . the hr and hr regions are believed to be important modules/domains in this process and show different conformations in different fusion states [ ] . under the current model, there are at least three conformational states of the envelope fusion protein [ ] . they are pre-fusion native state, prehairpin intermediate state, and post-fusion hairpin state. during these state transitions, the hr and hr are exposed in an intermediate conformational state but bind to each other to form coiled coil structure in an anti-parallel mode in the post-fusion state. therefore, the in vitro introduced hr peptides compete with the endogenous hr counterparts in the intermediate state, preventing the transition into the formation of hr / hr coiled coil bundle, the post-fusion state [ ] . this coiled coil bundle conformation is believed to be important for bringing two lipid membranes (cellular and viral) into proximity with each other allowing the membrane fusion for virus entry. membrane fusion is the key step for enveloped virus infection. the hr / hr coiled coil bundle is called the virus fusion core [ ] . in this structure, as shown by several crystal structures of fusion cores, including hiv, hrsv, influenza virus a, and ebola virus [ ] [ ] [ ] [ ] [ ] [ ] [ ] , three hr bind each other to form a trimeric core whereas three hr surround this core. as both hr and hr are structurally a helical in this fusion core, this structure is also called -helix coiled coil bundle [ ] . to investigate the structural basis of sars-cov fusion and entry and identify new fusion inhibitors, we have carried out the analysis of the sars-cov hr and hr fusion core in this study. both hr and hr have been expressed in escherichia coli and the in vitro assembly of the fusion core was analyzed. the gst fusion or chemical synthetic hr was tested in virus fusion inhibition. the results do show that sars-cov s protein is a typical class i viral fusion protein and hr is a potent fusion inhibitor. this is consistent with the recent observation on another coronavirus, mhv [ ] . virus and cell line. sars-cov virus isolate whu was used in this experiment, which was isolated from a sars patient who died of sars and the virus genome was confirmed by sequencing (genbank accession no. ay ). the virus causes typical cytopathic effect (cpe) on vero e cell monolayer. vero e cell line was obtained from the american type culture collection (atcc) (rockville, md) and maintained in dmem (sigma) containing % fetal bovine serum (fbs) (sigma). gene construction. as shown in fig. , sars-cov s protein is a typical type i membrane protein. the hr and hr regions were predicted using the computer program learncoil-vmf (http://nightingale.lcs.mit.edu/cgi-bin/vmf) [ ] . the hr region covers amino acids - , whereas the hr includes the amino acids - ( fig. ). the polypeptides were then engineered in vitro and expressed in e. coli. the hr was expressed as an n-terminal his-tagged protein as the basic amino acid cluster required for s /s cleavage is not present it is unlikely that sars-cov s protein would be processed further into s and s . however, the positions of the putative s and s are indicated for the convenience to compare with other coronaviruses. "ss" represents signal sequence and "tm" for transmembrane domain. in the lower panel, likelihood of hr and hr predicted by learncoil-vmf program [ ] is shown. in pet a vector (novagen) and hr (two versions) was expressed as a glutathione-s-transferase (gst) fusion protein. a peptide corresponding to amino acids - (hr - ) was also synthesized (sigma-genosys, uk). for construct making, his-hr construct was cloned into pet a vector with bamhi and xhoi, thereof resulting in extra amino acids at the n-terminus of the hr (same strategy as wang et al. [ ] ). gst fusion hr constructs (hr - and hr - ) were cloned into pgex- p- (pharmacia) with unique restriction enzyme sites bamhi and noti (same strategy as yu et al. [ ] ). there were two versions of gst-hr , i.e., gst-hr - and gst-hr - . for the latter, there were extra amino acids after the predicted position at the c-terminus from the s gene (amino acids - ). cloned constructs were verified by direct dna sequencing. protein expression and purification. all the relevant positive expression vectors ( a-hr , gst-hr - , and gst-hr - ) were transformed into e. coli strain bl (de ) competent cells and single colony was inoculated into  yt medium containing lg/ml ampicillin at °c for overnight culture. then the overnight culture was transferred to new  yt medium for large-scale protein production by growing at °c. when the culture density (od ) reached . , the culture was induced with . mm iptg and grown for an additional h at °c before the cells were harvested. the harvested culture was centrifuged and the bacterial cell pellet was re-suspended in pbs and homogenized by sonication. triton x- was then added to a final concentration of %, and the lysate was incubated for min on ice and was subsequently clarified by centrifugation at , g for min at °c. then the supernatant was loaded onto a glutathione-sepharose b column (pharmacia) or ni-chelated sepharose affinity column (pharmacia). the protein-loaded column was then washed with  column volume of pbs. after that, for gstfusion protein, the protein was eluted with mm reduced glutathione. for the gst-removed proteins, the gst- c rhinovirus protease (kindly provided by drs. j. heath and k. hudson) was added into the resin to cleave the gst and the fused target protein and the mixture was incubated with gentle agitation for about h at °c. the target protein was eluted with ml pbs. for the his-tagged hr , the protein was eluted with mm imidazole. hr -hr complex assembly and gel-filtration analysis. equal molar purified his ( a)-hr and gst-removed hr were mixed at room temperature for h. the mixture was then loaded onto a superdex g column (pharmacia). one single peak ( - kda) was detected and the eluted fractions were analyzed on tris-tricine sds-page. gst pull-down experiment. purified gst-hr - was mixed with equal molar a-hr on ice for h and then loaded onto a glutathione-sepharose b column. the column was washed with column volumes of pbs and then eluted with mm reduced glutathione. the existence of both gst-hr - and a-hr in the elution was tested on sds-page. circular dichroism spectroscopy. circular dichroism (cd) spectra were measured on a jasco j- spectrophotometer in pbs. wavelength spectra were recorded at °c using a . cm pathlength cuvette. for the thermodynamic stability, the hr -hr complex protein was measured at nm by monitoring the cd signal in the temperature ranging from to °c with a scan rate of °c per minute. fusion inhibition assay. for the inhibition experiments, vero e cell cultures growing in -well microplates were infected with  tcid /well (tcid was virus titers causing % of cpe on vero e cell monolayer and the tcid used in this experiment was .  /ml). the serial -fold dilutions of polypeptides ( repeats for each dilution) were added at the same time as virus adsorption for h at °c. then the mixtures of virus and the polypeptides were replaced by dmem containing % fbs and continued to cultivate for h until calculating the well numbers of cpe and inhibition of cpe. the ic was calculated according to reed-muench method [ ] . all three polypeptides, a-hr , gst-hr - , and gst-hr - , were expressed in e. coli as soluble pro-teins in this study. as our previous work has shown that the extra amino acids in the a expressed protein in hrsv had no effect on the binding to hr [ ] we carried out all the binding experiments with this a-hr preparation. gst-removed hr - and hr - were both soluble proteins in pbs. when equal molars of a-hr and hr - were mixed a unique complex peak could be detected by gel filtration and the peak molecular weight was estimated approximately as kda (fig. a) . both a-hr and hr were detected in the peak on sds-page ( fig. a) . this matches the calculation of  a-hr and  hr - , implying the formation of -helix bundle complex. we also tested the gst pull-down experiments and showed the tight binding of a-hr to gst-hr - (data not shown). hr and hr complex is a stable a-helix coiled coil cd spectroscopic profile of the hr -hr complex shows a typical a-helix structure, with double minima at and nm (fig. b) . from previous work on other viral fusion core, this structure is coiled coil which is characterized by its extreme stability. thermodynamic measurements of this hr -hr complex showed it remains structured up to °c (fig. c) , indicating its extraordinary stability. in the presence of hr polypeptides (gst-fusion forms or synthetic peptide), we observed an analogous, strong inhibitory effect of sars-cov hr which blocked virus entry (fig. ) . the % effective doses (ic ) for inhibition of the cpe in cultured cells were . - nm for the synthetic hr peptide and . - nm for the gst fusion hr forms (fig. ) . despite our extensive experiments, using both the synthetic and bio-engineered hr s, we did not see any virus-infection inhibition effect from hr (data not shown). this might reflect the difference between hr and hr in the virus entry inhibition as observed in some paramyxoviruses [ , , [ ] [ ] [ ] . the mechanism underlying this difference needs to be further investigated in the future. it is noteworthy that, in a recent study on mhv, only hr inhibition has been observed [ ] . coronavirus entry into the cells starts with viral and cellular membrane fusion which is mediated by the binding of the viral s protein with the cellular receptor/s [ ] . receptor-induced conformational changes in the s protein are crucial in this step [ ] [ ] [ ] . in this study, we have shown that the putative hr and hr of sars-cov bind to each other and form a -a-helix bundle (trimer of hr /hr dimer), a characteristics of class i viral fusion proteins. the importance of the hr regions in mhv fusion has been demonstrated with site-directed mutagenesis study by luo and weiss [ , ] . as the receptor for sars-cov has been identified recently [ ] , receptor-induced conformational changes of s protein should be pursued in the near future, esp. the conformations of hr and hr fusion core in different stages during the fusion process. recently, rottier and co-workers [ ] reported the formation of hr /hr -helix bundle complex of mhv s protein. this and our present result on sars virus are the first evidence for a -helix bundle formation of hr and hr and the hr fusion inhibitor derived from family coronaviridae, implying that coronavirus adopts a similar fusion or entry mechanism to other rna viruses [ ] . taking the fact into account that sars is an acute disease, this kind of peptide inhibitors might act better than that in chronic infection hiv because the therapeutic peptide can be used through intranasal route. the conformational changes of the s protein are very likely to be crucial for coronavirus entry [ ] [ ] [ ] and so this provides a most plausible target for drug design. the strong inhibition of the peptide in the gst ( kda) fusion form indicates that there is significant space for hr to interact with hr in the intermediate state of the s protein during the process of virus fusion. this has also been observed in our previous work on newcastle disease virus and hrsv, members of family paramyxoviridae [ , ] . our results do provide a drug lead for sars-cov and its potential clinical application should be rigorously pursued in the near future. a clear crystal structure of this fusion core of sars-cov would also make a great deal of contribution to our understanding of coronavirus fusion and fusion inhibitor discovery. coronaviridae: the viruses and their replication virus taxonomy: classification and nomenclature of viruses, th report proc. natl. acad. sci. usa proc. natl. acad. sci. usa key: cord- -ot pvtbl authors: chen, f; chan, k. h; jiang, y; kao, r.y.t; lu, h. t; fan, k. w; cheng, v.c.c; tsui, w.h.w; hung, i.f.n; lee, t.s.w; guan, y; peiris, j.s.m; yuen, k. y title: in vitro susceptibility of clinical isolates of sars coronavirus to selected antiviral compounds date: - - journal: journal of clinical virology doi: . /j.jcv. . . sha: doc_id: cord_uid: ot pvtbl abstract effective antiviral agents are urgently needed to combat the possible return of severe acute respiratory syndrome (sars). commercial antiviral agents and pure chemical compounds extracted from traditional chinese medicinal herbs were screened against clinical isolates of sars coronavirus by neutralisation tests with confirmation by plaque reduction assays. interferon-beta- a, leukocytic interferon-alpha, ribavirin, lopinavir, rimantadine, baicalin and glycyrrhizin showed antiviral activity. the two interferons were only active if the cell lines were pre-incubated with the drugs h before viral inoculation. results were confirmed by plaque reduction assays. antiviral activity varied with the use of different cell lines. checkerboard assays for synergy were performed showing combinations of interferon beta- a or leukocytic interferon-alpha with ribavirin are synergistic. since the clinical and toxicity profiles of these agents are well known, they should be considered either singly or in combination for prophylaxis or treatment of sars in randomised placebo controlled trials in future epidemics. although the sars epidemic has been successfully contained with quarantine and infection control measures, the presence of this virus in wild game food animals , stocks in laboratories and possible seasonality of this disease suggest that recurrence of such an epidemic is not unlikely in the coming winters. since all age groups are affected and a high fatality is noted in the elderly and those with co-morbidities (donnelly et al., ) , there is an urgent need to find a cure. prospective clinical and viral load studies in nasopharyngeal secretions from sars patients showed that viral replication peaked at the th day after the onset of symptoms with subsequent clinical deterioration in % of the cases despite a decreasing viral load (peiris et al., ) . therefore the key facet of management should include respiratory support, immuno-modulation in selected cases and early institution of an effective antiviral agent. such an antiviral agent, if given early, may decrease the peak viral load and the associated immuno-dysregulatory damage. at the moment, there are no commercially available antiviral agents tailored-made for sars coronavirus. there is an urgent need to search for an agent with a known in use clinical and toxicity profile so that a randomised placebo control trial can be conducted if the epidemic recurs in one of the coming winters. we report in this study on the in vitro antiviral susceptibility of isolates of sars coronavirus to commercially available antiviral agents and pure chemical compounds including baicalin, glycyrrhizin, and chlorogenic acid extracted from traditional chinese herbs. ten isolates of sars coronavirus from different sars patients who satisfied the revised who criteria for sars are listed in table . the drugs used for antiviral susceptibility specialists to draw up a "technical scheme (tentative) for the prevention and treatment of severe acute respiratory syndrome (sars) using traditional chinese medicine". in the scheme, a recipe "qing fei jie du tang" (soup for clearing the lung and detoxification) was recommended which consists of huang qi (astragalus membranaceus) gm, chai hu (bupleurum chinense) gm, ma huang (ephedra sinica) gm, xing ren (prunus armeniaca) gm, sheng she gao (plaster stone) gm, sheng yi ren (coix lacryma-jobi) gm, gua wei pi (benincasa hispida) gm, jie geng (platycodon grandiflorum) gm, bo he (mentha haplocalyx) gm, huang qin (scutellaria baicalensis) gm, sheng gan cao (glycyrrhiza uralensis) gm, jin yin hua (flos lonicerae) gm, and qing hao (artemisia apiacea) gm. among them, only scutellaria baicalensis, glycyrrhiza uralensis, flos lonicerae and artemisia apiacea have their pure chemically defined ingredients being extracted, purified and documented to have antimicrobial activities. consequently, the main bioactive compounds, namely, baicalin (derived from scutellaria baicalensis), glycyrrhizin (from glycyrrhiza uralensis), chlorogenic acid (from flos lonicerae) and artesunate (from artemisia apiacea) were investigated in the present study. the pharmacological properties of baicalin, glycyrrhizin, and chlorogenic acid are summarized in table . artesunate is not included in this table since it is already well known as an anti-malarial drug in western medicine (price, ) . they were extracted as we have previously reported (lu et al., ) . the concentrations of baicalin, glycyrrhizin, no glycyrrhizin in plasma is found after oral administration of mg glycyrrhizin in healthy persons, presumably glycyrrhizin is metabolized to glycyrrhetinic acid by intestinal bacteria which contain ␤-d-glucuronidase or the amount consumed is too little only traces expected ( mg per person, in human); this may be due also to that the amount consumed is much lower than that for animals standard doses in oral administration in humans ∼ mg baicalin (as tablets); also can be up to ∼ mg baicalin (calculated from herb, assuming g herb used; the herb may contain up to % as baicalin) ∼ mg glycyrrhizin (as tablets) or ∼ mg glycyrrhizin (calculated from the herb assuming that the herb contains . % glycyrrhizin) ∼ mg (calculated from the herb assuming that the herb contains . % chlorogenic acid) serum level (after intravenous administration) chlorogenic acid, and lopinavir in the cell culture system were also monitored by hplc (lu et al., ) whereas the concentration of others were monitored by neutralization assays with the vesicular stomatitis virus indiana strain and a laboratory strain of influenza a h n .the procedures used for in vitro antiviral susceptibility testing are as follows. initial screening of all compounds against the prototype sars coronavirus strain no. was performed in -well microtitre plates seeded with foetal rhesus kidney- cells. two-fold dilutions of antiviral agents starting from more than four times the peak serum concentration after the maximum therapeutic dose to less than one-quarter of the trough serum concentration were tested in quadruplicate against tcid of sars coronavirus. a corresponding set of cell controls with drug but without virus inoculation was used as controls for drug toxicity. the cells were scored for the inhibition of the cytopathic effect (cpe) at and h. those compounds with demonstrable in vitro inhibitory activity were re-assayed against the other nine strains of sars coronavirus collected from different patients from different hospitals of the hong kong special administrative region (hksar). their antiviral activities were also compared in both foetal rhesus kidney- (frhk- ) and vero-e cell lines. those likely to have clinically significant inhibitory activity were tested by the plaque reduction assay. for selected agents with consistent activity in the plaque reduction assay, checkerboard assays for synergy were per-formed for combinations of interferons and ribavirin using the same neutralization test in well microtiter plates seeded with vero cell line. cells were not incubated with the interferons before viral inoculation. vero cells were used instead of vero e and frhk- cell lines because better antiviral effect can be demonstrated in vero but less so in the other two cell lines for ribavirin and the interferons. ten isolates of sars coronavirus from different patients with sars admitted to different hospitals in hk-sar showing seroconversion towards the prototype virus infected frhk- cell line were used in this study (table ) . they were isolated from the lung tissue biopsy (prototype virus, m ), urine, and nasopharyngeal aspirates. initial screening of commercially available antimicrobial agents against the prototype virus grown in frhk- cell line did not reveal inhibitory activities for acyclovir, ganciclovir, cidofovir, foscarnet, interferon-alpha- a, interferon-alpha- b, amantadine, zidovudine, stavudine, nevirapine, abacavir, and ritonavir. inhibitory activities were not detectable for glycyrrhizin, artesunate and chlorogenic acid in frhk- cell line. glycyrrhizin was still included for further testing because this was reported to be active in vero-e cell lines (cinatl et al., a) . further testing by neutralization tests table comparison of antiviral activity of compounds against strains of sars-cov in frhk cell line, against the prototype strains ( ) with the other isolates of sars coronavirus against the active compounds confirmed detectable inhibitory activities for leukocytic interferon-alpha, interferon-beta- a, ribavirin, lopinavir, rimantadine, and baicalin. the range of their effective concentration of compound required to reduce the plaque forming unit by % (ec ) at and h, and their selectivity index are shown in table . when the same neutralization test on these compounds was run in vero-e cell line, rimandatine, glycyrrhizin, leukocytic interferon-alpha and interferon-beta were more active especially at h. moreover, pre-incubation of the cell lines with these two interferons for h before adding the virus markedly enhanced the inhibitory activity by three to over -fold. but ribavirin, lopinavir, and baicalin were less active in the vero-e cell line (table ) . as for the plaque reduction assay, vero cell lines were used instead of vero e or frhk- cell lines because antiviral activity can be demonstrated for most of the agents. only interferon-beta- a, leukocytic interferon-alpha, lopinavir, ribavirin, rimantadine, and baicalin were tested. the ec of interferon-beta- a ( u/ml), leukocytic interferon-alpha ( u/ml), lopinavir ( g/ml), ribavirin ( g/ml), rimantadine ( g/ml), and baicalin ( g/ml) are comparable to the results obtained from cpe assays. tests for synergism between ribavirin and lopinavir have already been reported (chu et al., ) . no synergism could be demonstrated between rimantadine and ribavirin. the most active compounds are the interferons. thus further checkerboard assays were performed with combinations of leukocytic interferon-alpha or interferon-beta- a, and ribavirin. marked synergism was seen at both and h. the combination of leukocytic interferon-alpha ( g/ml) and ribavirin ( g/ml) or interferon-beta- a ( . g/ml) and ribavirin ( g/ml) were shown to be active at h of incubation (table ) . control of sars may be achieved by epidemiological measures, antiviral prophylaxis or treatment, and vaccination. during the last pandemic of sars, the only available means for control were public health measures such as isolation of suspected cases, quarantine of contacts, and personal protective infection control procedures for high-risk individuals such as health care workers. there is an urgent need to find effective antiviral agents with acceptable side effect profiles. in developing countries such as china, commercially available western antiviral medicine is unlikely to be affordable by most people. moreover, the sars mortality of mainland china was only % comparing favourably with the % to % of other areas (who, ) . china is also the only place where traditional chinese medicinal herbs were extensively used for treatment of sars. the development of vaccine will take a much longer time. therefore, we undertook these antiviral susceptibility tests for all commercially available antiviral agents in the hksar and pure chemicals purified from traditional chinese herbs known to have antimicrobial activity. these chosen herbs were included in a standard formula used for the treatment of sars in china and the hksar. only interferon-beta and glycyrrhizin were reported to have significant antiviral activity against sars coronavirus (cinatl et al., a,b) . using the frhk- cell line, we have shown that ribavirin, rimantadine, lopinavir, and baicalin also have detectable antiviral activities. however, like the interferons and glycyrrhizin, their activities tend to decrease with incubation beyond h (table ) . judging from the achievable serum levels with standard oral or parenteral dosing, rimantadine, ribavirin, glycyrrhizin, and even the two interferons are unlikely to have clinically significant in vivo activities. moreover, lopinavir, and rimantadine have a relatively inferior selectivity index of to . upon subsequent testing with vero-e cell line, both leukocytic interferon-alpha and interferon-beta- a were more active and especially after pre-incubation for h before viral inoculation. the findings suggest that prophylaxis with the interferons should be considered. though ribavirin was much less active in the vero cell line, it is highly synergistic with either two interferons. therefore, a combination of ribavirin with either of these two interferons should be considered for the treatment of sars. interferon-gamma was reported not to possess antiviral activity against sars coronavirus (cinatl et al., b) , whereas interferon-beta was confirmed to be active in this study. what is interesting was the demonstration of activity of leukocytic interferon-alpha despite the lack of activity of the recombinant interferon-alpha- a and interferon-alpha- b. this was not unexpected because this preparation of leukocytic interferon-alpha is a multi-subtype natural interferon with predominantly interferon alpha- and alpha- in contrast to the other commercial preparation with a single subtype of recombinant interferon-alpha- . in in vitro studies, different subtypes have been found to have different antiviral activities as well as immunological effects (foster et al., ) . it was also demonstrated that leukocytic interferon-alpha had a superior antiviral effect than that of recombinant interferon on human immunodeficiency virus infection (fan et al., ) . it is important to know that in vitro findings may not correspond with clinical efficacy. despite its in vitro activity, topical or systemic interferon-alpha did not produce a consistent reduction in symptoms or lesion duration of genital herpes (eron et al., ; lebwohl et al., ) . and interferon-alpha was not effective in preventing cmv infections or treating cmv pneumonia in bone marrow transplant patients (meyers et al., ) . despite its broad-spectrum antiviral activities against respiratory viruses in vitro, prophylactic intranasal interferon-alpha is only protective against rhinovirus-induced common cold under natural condition (douglas et al., ) . this was unexpected because intranasal leucocyte or recombinant interferon-alpha protect against experimental human infection by rhinovirus, coronavirus, and respiratory syncytial virus (hayden and gwaltney, ; higgins et al., ; higgins et al., ) . besides the high cost of interferons, the high incidence of fever and flu syndrome of up to % during its initial phase of administration may pose confusions in terms of the response to treatment. the well-known side effect of pancytopenia may also be confused with markers of sars activity such as a decrease in platelets and occasionally neutrophils (raanani and ben-bassat, ) . although interstitial pneumonitis and bronchiolitis obliterans organising pneumonia are rare complications of prolonged use of interferons (karim et al., ; ogata et al., ) , there is always a fear that their proinflammatory effect may worsen the viral pneumonitis caused by sars. as for the less expensive option, baicalin but not glycyrrhizin may be considered if traditional chinese medicine is to be used for antiviral prophylaxis or treatment. the serum level after mg of glycyrrhizin orally was not detectable. even with a mg dose of intravenous administration, the peak serum level is only g/ml which is still below the ec of glycyrrhizin. although an oral dose of . gm of baicalin can only achieve a serum concentration of . g/ml, intravenous administration of a mg dose of baicalin in human can achieve a peak serum concentration of g/ml. thus intravenous baicalin should be con-sidered for treatment in randomised placebo control trials in developing countries where such formulations are available and affordable. baicalin was shown to inhibit hiv- by two mechanisms (kitamura et al., ; li et al., ) . at the level of cellular entry, baicalin can conjugate with selected chemokines such as mip- ␤ and sdf- ␣, and interfere with their capacity to activate cellular receptors ccr and cxcr respectively. these two co-recpetors are essential elements for hiv- infection and therefore baicalin can inhibit env-protein mediated fusion of hiv with cells expressing cd /ccr or cd /cxcr . baicalin has also been known to inhibit hiv- reverse transcriptase probably by interfering with the binding of viral rna to the rt molecule near the active site of the enzyme. in terms of prophylaxis against sars short of an effective vaccine, intranasal leukocytic interferon-alpha or interferon-beta- a are likely to be effective. however the local side effect of nasal irritation can decrease compliance. it could also be considered for randomised placebo-control trials. as for the antiviral treatment of symptomatic sars, it is important to have a rapid and reliable diagnostic test since early institution of antiviral therapy is important to decrease the peak viral load . interferon-beta- a or leukocytic interferon-alpha plus ribavirin appear to be the most effective combination. since interferons may not be effective in inducing an antiviral state in the uninfected host cells during the first h, a combination with a short course of ribavirin appears to be reasonable. this will also reduce the side effects and fluid volume associated with a full course of ribavirin. despite the superiority of interferons in the in vitro assays, there is little clinical data of its use in the treatment of acute viral respiratory infection in human. thus the combination of ribavirin with lopinavir/ritonavir should still be considered since some positive clinical data has already been accumulated in a historical controlled treatment trial (chu et al., ) . glycyrrhizin, an active component of liquorice roots, and replication of sars-associated coronavirus treatment of sars with human interferons the role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong prophylactic efficacy of intranasal alpha -interferon against rhinovirus infections in the family setting therapy of genital herpes with topically applied interferon increased efficacy of human natural interferon alpha (ifn-alpha n ) versus human recombinant ifn-alpha for inhibition of hiv- replication in primary human monocytes different 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human leukocyte interferon for treatment of cytomegalovirus pneumonia after marrow transplantation interferon-related bronchiolitis obliterans organizing pneumonia clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study early diagnosis of sars coronavirus infection by real time rt-pcr artemisinin drugs: novel antimalarial agents immune-mediated complications during interferon therapy in hematological patients world health organization. summary table of sars cases by country we acknowledge research funding from the kai cheong tong sars research fund, the university of hong kong. key: cord- - u cpsc authors: nan title: arboviren—durch arthropoden übertragbare viren: stellungnahmen des arbeitskreises blut des bundesministeriums für gesundheit und soziale sicherung date: journal: bundesgesundheitsblatt gesundheitsforschung gesundheitsschutz doi: . /s - - - sha: doc_id: cord_uid: u cpsc nan unter dem oberbegriff arboviren (arthropod-borne viruses) werden diejenigen viren zusammengefasst, die sich sowohl in arthropoden wie mücken oder zecken als auch in vertebraten (vögeln, säugetieren) vermeh-ren. diese viren können durch arthropoden über biss oder stich bei der blutmahlzeit auf vertebraten übertragen werden. zu den arboviren gruppieren sich heute etwa vertreter verschiedener virusfamilien bzw. virusgenera. in . tabelle sind die für den menschen wichtigsten krankheitserreger, die beteiligten vektoren, die virusreservoire und die geographische verbreitung der erkrankungen zusammengefasst. nicht in allen genera einer virusfamilie findet man für den menschen pathogene erreger, die durch arthropoden übertragen werden. als beispiel sei genannt, dass nur in dreien der insgesamt genera der familie der bunyaviridae virusspezies eingruppiert sind, die durch arthropoden auf den menschen übertragen werden können. hantaviren, die zum genus hantavirus der bunyaviridae gehören, werden dagegen nicht von arthropoden auf den menschen übertragen, sondern durch dessen kontakt mit ausscheidungen der natürlichen wirte, mäuse und ratten. arbeiten mit infektiösen arboviren sind unter erhöhten sicherheitsbedingungen durchzuführen, da diese erreger überwiegend in die risikogruppe bzw. eingruppiert sind [ , ] . in deutschland spielt nach dem heutigen wissensstand nur das virus der frühsommermeningoenzephalitis (fsme), das durch zecken (ixodes ricinus) übertragen wird, epidemiologisch eine wesentliche rolle. die überwiegende anzahl der arboviren gehört zu virusfamilien, die eine lipiddoppelmembran als hüllen aufweisen (umhüll-te viren), d. h., diese viren sind empfindlich gegen lipidlösungsmittel und detergenzien. des weiteren weisen sie eine geringe stabilität gegen hitze auf. eine ausnahme bildet dabei das colorado-tick-fever-virus, das zur familie reoviridae, genus coltivirus, gehört und nicht umhüllt ist (. tabellen und , . abb. ). arthropoden infizieren sich beim saugakt an infizierten vertebraten während der virämischen phase [ ] . in den arthropoden vermehren sich die erreger zuerst im verdauungstrakt, ehe eine ausbreitung im gesamten organismus erfolgt. da die viren im verlauf der infektion auch die speicheldrüsen befallen, können die erreger dann bei folgenden saugakten wieder auf vertebraten übertragen werden. für einige viren, wie etwa west-nil-virus (wnv) und st.-louis-enzephalitis-virus (slev), wurde nachgewiesen, dass die virusvermehrung in den mücken abhängig ist von der durchschnittlichen umgebungstemperatur. je höher die durchschnittstemperatur, desto kürzer die zeitspanne, bis die virusvermehrung in den mücken abgelaufen und virus in hohen titern in der speicheldrüse vorhanden ist [ , ] . inwieweit in deutschland die entsprechenden durchschnittstemperaturen über die notwendigen zeiträume erreicht werden, die eine vermehrung von wnv in mücken ermöglichen, wurde bisher nicht untersucht. in den arthropoden wird eine persistierende infektion etabliert [ ] . zudem wur-virämischen phase können hohe virustiter im blut erreicht werden, die zur infektion von mücken oder zecken ausreichen. dieser zyklus arthropoden-vertebraten bedingt die verbreitung der erreger über größere distanzen, wobei infizierte vögel zu einer überregionalen verbreitung (zugvögel, wanderung von vögeln) und infizierte nager eher zu regional enger umschriebenen endemiegebieten führen [ , ] . wie epidemiologische studien zeigen, ist der mensch nur in einzelnen fällen als virusreservoir anzusehen. ausnah-ten jahreszeit mit verantwortlich. auch bei virusinfektionen, die durch zecken übertragen werden können, spielt die transovarielle Übertragung für die viruszirkulation eine wichtige rolle. zu bemerken ist hierbei, dass zecken im vergleich zu mücken eine erheblich längere lebenszeit haben (jahre im vergleich zu mücken, die wenige wochen bis monate leben) [ , ] . in der regel werden vertebraten mit arboviren nicht persistent infiziert. nach infektion entwickeln sie eine immunantwort und eliminieren den erreger. während der de nachgewiesen, dass die viren auf die eier übertragen werden (vertikale, transovarielle Übertragung). aus infizierten eiern entwickeln sich dann wieder über das larven-und puppenstadium persistent infizierte arthropoden, die die erreger wiederum auf ihre eier übertragen können [ , , , ] . wie der mechanismus der transmission auf die nachkommen genau abläuft, ist bisher nicht geklärt. die transovarielle Übertragung bei den mücken ist für die aufrechterhaltung einer epidemie in trockenzeiten oder in gemäßigten breiten während der kal- wie aus . tabelle zu ersehen, ist der erreger der mitteleuropäischen frühsommermeningoenzephalitis nach dem heutigen kenntnisstand das einzige in deutschland bedeutsame virus, das durch zecken der spezies ixodes ricinus übertragen wird [ ] . infektionen mit anderen erregern können bei reisen in endemiegebiete erfolgen. so werden infektionen mit dem sandfliegenfieber-virus (phlebovirus) z. b. in italien, spanien oder auf dem balkan erworben, dengue-virus-infektionen bei reisen nach südostasien (z. b. thailand) und in die karibik/südamerika oder gelbfieber-virus-infektionen in afrika und südamerika [ , , , ] . man geht davon aus, dass sich etwa % der bevölkerung in deutschen endemiegebieten mit fsmev infiziert haben, wobei z. b. waldarbeiter eine höhere prävalenz aufweisen [ , , ] . eine Übertragung von arboviren über körperflüssigkeiten, wie z. b. blut, ist möglich, spielt jedoch im wesentlichen nur bei den hämorrhagischen fiebern eine rolle (z. b. krim-kongo-hämorrhagisches-fieber, cchf). fälle von transfusionsassoziierten arbovirus-infektionen wurden für wnv in den usa berichtet [ ] , eine dengue-virus-infektion in hongkong [ ] und eine Übertragung von fsme-virus in finnland ( [ ] , vgl. . tabelle ). im jahr wurden in den usa und kanada mittels nukleinsäure-amplifikations-technik (nat) von ca. wnvpositiven blutspendern und von transfusionsassoziierten Übertragungen berichtet. von Übertragungen des fsme-virus durch frische ziegen-bzw. kuhmilch wurde mehrfach berichtet [ ] . inwieweit zugvögel als reservoir von arthropoden-übertragenen virusinfektionen in deutschland dienen können, ist unbekannt. die vermutung liegt jedoch nahe, dass arboviren in einzelfällen durch zugvögel, z. b. aus afrika, dem nahen osten oder südost-europa eingeschleppt werden. infektionen mit sindbis-virus (ockelbo-virus) in südschweden belegen, dass zugvögel das virus aus endemischen gebieten in afrika nach mitteleuropa transportieren können [ ] . die aufrechterhaltung der arbovirus-infektion in arthropodenpopulationen in trockenzeiten und in kälteperioden (winter) wird kontrovers diskutiert. für verschiedene erreger wurde gezeigt, dass einerseits infizierte mücken überwintern können; an- nahme an krankheitsfällen bei menschen in den usa das verhältnis von infektionen zu erkrankungen wird dabei je nach erreger und untersuchtem kollektiv mit : bis . : angegeben. für wnv wird geschätzt, dass weniger als % der infizierten während einer epidemie an einer enzephalitis erkranken [ ] . zurzeit werden untersuchungen durchgeführt, die klären sollen, inwieweit endemische wnv-infektionen in deutschland beim menschen vorkommen. bedingt durch die hohe rate an asymptomatisch oder subklinisch verlaufenden infektionen sind umfangreiche serologische untersuchungen notwendig, um die prävalenz von arbovirus-infektionen abzuschätzen. in einzelnen studien wurde z. b. für fsme gezeigt, dass in deutschen endemiegebieten etwa % der bevölkerung antikörper gegen fsme-virus aufweisen, die nicht auf eine impfung zurückzuführen waren. betrachtet man im vergleich dazu die anzahl der gemeldeten erkrankungen, so ist auch hier davon auszugehen, dass viele fsme-infektionen asymptomatisch oder mit nur leichten krankheitssymptomen verlaufen. nur für einen teil der arboviren sind nachweissysteme kommerziell erhältlich (nachweis für dengue-virus igm, igg, antigen; fsme igg, igm; wnv-pcr, igg und igm). die virämische phase beim menschen ist in der regel kurz und beträgt nur wenige tage. in dieser zeit ist der virusnachweis durch virusisolierung in zellkultur, neugeborenen mäusen oder im bebrüteten hühnerei möglich, wobei sich die wahl des anzuchtmediums nach dem jeweiligen erreger richtet. häufig werden auch insektenzellen zur anzucht von arboviren verwendet. mit auftreten von igm und igg ist der virusnachweis durch anzucht nicht mehr möglich, jedoch bleibt die pcr für einige weitere tage positiv. der nachweis von spezifischem igm bzw. ein mindestens vierfacher titeranstieg in antikörpernachweissystemen (immunfluoreszenz, elisa, neutralisationstest) wird als beweisend für das vorliegen einer frischen infektion ange-sehen. bei der interpretation der ergebnisse ist jedoch zu berücksichtigen, dass kreuzreagierende antikörper auftreten können, hervorgerufen durch impfungen gegen gelbfieber-, fsme-oder japan.-enzephalitis-virus bzw. durch infektionen mit flaviviren. für eine vielzahl dieser viren wurden molekulare nachweissysteme (rt-pcr, realtime pcr) in der literatur beschrieben. verwendet werden dabei für den jeweiligen erreger konservierte genombereiche. eine differenzierung ist durch die wahl geeigneter primer und sonden möglich. die pcr-diagnostik wird in europa in speziallaboratorien durchgeführt (erregerliste und einschlägige adressen bei http:// www.enivd.de). zumindest bei flavivirus-infektionen kann die enge verwandtschaft der viren eine differenzialdiagnose erschweren. durch die auswahl geeigneter primer in der pcr bzw. dem einsatz des neutralisationstestes (z. b. plaquereduktionstest) kann in der regel eine differenzialdiagnose gestellt werden. zur problematik der Übertragung von arboviren hat das scientific committee on medicinal products and medical devices (scmpmd) stellung genommen [ ] . angaben zur prävalenz von arbovirus-infektionen bei blutspendern in deutschland liegen nicht vor. aufgrund der wnv-epidemie in nordamerika und mexiko wurde eine rückstellung für wochen von reiserückkehrern aus diesen endemiegebieten, begrenzt auf das epidemiologisch relevante zeitfenster (vom . . bis zum . . jedes jahres), festgelegt [ ] . darüber hinaus gibt es für die anderen arboviren keine spezifischen ausschlusskriterien. da eine spendertestung auf virusgenom der verschiedenen arboviren findet in deutschland nicht statt. in den usa und kanada wird die spendentestung auf wnv-virusgenom (rt-pcr) wegen der dortigen epidemiologischen lage seit dem . . durchgeführt [ , ] . auf andere erreger wird weltweit nicht getestet. entsprechend . werden spender nach reisen in wnv epidemiegebiete befragt. darüber hinaus findet keine spezielle spenderbefragung im hinblick auf arbovirus-infektionen statt. indirekte ausschlüsse erfolgen durch rückstellung wegen reisen in malariaendemiegebiete und beim auftreten von krankheitssymptomen wie fieber. eine spenderinformation findet nicht statt. bei reiseanamnese werden die spender über eine wnv-spezifische symptomatik informiert (s. . ). es gibt keine untersuchungen zu prävalenz und inzidenz von arbovirus-infektionen in empfängerkollektiven. eine Übertragung von arboviren durch plasmaderivate, die einem virusinaktivierungsverfahren unterzogen wurden, ist unwahrschein-lich, da die relevanten erreger detergenz-, hitze-bzw. säurelabil sind. personen, die in gelbfieber-virus-endemiegebiete reisen, sollten gegen gelbfieber-virus geimpft sein (teilweise für die einreise vorgeschrieben). eine impfung gegen fsme-virus wird für bewohner von endemiegebieten und für reisende dorthin empfohlen. wie die untersuchung von empfängern wnv-kontaminierter blutprodukte gezeigt hat, verlaufen die meisten der infektionen subklinisch, können aber auch zu schweren erkrankungen (enzephalitis) und tod führen. für infektionen mit anderen erregern liegen keine oder nur einzelne fallberichte vor. eine gesicherte therapieempfehlung existiert bisher nicht. eine aktive impfprophylaxe ist möglich für fsme, gelbfieber und japanische enzephalitis. die Übertragung der infektion durch nicht inaktivierte blutkomponenten auf andere personen ist prinzipiell während der virämiephase möglich. Übertragungen wurden außer für wnv (s. auch . ) jedoch nur für erreger viraler hämorrhagischer fieber wie cchf-virus berichtet. aus den vorliegenden berichten kann für deutschland nicht auf ein erhöhtes risiko durch häufige applikation eines produktes geschlossen werden. eine Übertragung kann nur über nicht virusinaktivierte präparate (z. b. erythrozyten, thrombozyten) erfolgen. Über die belastung von plasmapools liegen bisher mit ausnahme von wnv keine angaben vor. die untersuchung der ausgangsmaterialien ist prinzipiell mit molekularen methoden (rt-pcr, taqman-pcr) möglich. während der wnv-epidemie im jahr in den usa wurde in virämischen blutspendern eine durchschnittliche belastung von , · geq/ml und ein maximum von , · geq/ml festgestellt. in den epidemiegebieten in den usa wurde eine prävalenz von in spenden ( , %) gefunden und für die usa insgesamt eine prävalenz von in . spenden ( , %) berichtet. aus diesen daten wurde die mögliche belastung eines plasmapools mit wnv errechnet und eine belastung zwischen , · geq/ml (unter annahme der höchsten prävalenz und belastung der einzelspende) und geq/ml (unter annahme der durchschnittlichen prävalenz von , % und einer durchschnittlichen belastung der einzelspende) angegeben [ ] . diese zahlen sind auf nicht-endemische gebiete, z. b. deutschland, nicht übertragbar. außerhalb eines epidemischen geschehens ist eine sehr geringe prävalenz zu erwarten, die, wenn überhaupt, zu einer weit kleineren belastung von plasmapools führen würde. verschiedene arboviren bzw. vertreter der entsprechenden virusfamilien (z. b. gelbfieber-virus, sindbis-virus, semliki-forest-virus, fsme-virus, wnv, vesikuläres stomatitis-virus) wurden und werden zur validierung von eliminierungsund virusinaktivierungsverfahren bei der produktion von plasmaderivaten (z. b. gerinnungsfaktoren, immunglobuline) eingesetzt. besonders untersucht wurde die wirksamkeit von inaktivierungsverfahren für wnv, und es wurde überzeugend nachgewiesen, dass wnv ebenso inaktiviert wird wie modellviren (z. b. bovines virusdiarrhoe-virus, bvdv), die zum nachweis der hepatitis-c-virus-(hcv-)sicherheit von plasmaderivaten für alle produkte gefordert sind. insgesamt zeigt sich, dass arboviren empfindlich sind gegen hitze und detergenzien und dass sie daher, insbesondere durch diese verfahrensschritte, effizient inaktiviert werden [ , , ]. vertreter der virusfamilien der arboviren werden für die untersuchung der wirksamkeit von inaktivierungsverfahren eingesetzt. die vergleichbarkeit der erregereigenschaften ist insbesondere für wnv gut untersucht. die für alle plasmaderivate geforderte untersuchung mit modellviren für hcv (z. b. bvdv oder sindbis-virus) gibt somit auch aufschluss über die wirksamkeit der verfahrensschritte für die inaktivierung von wnv und anderen arboviren. generell ist das risiko einer transfusionsassoziierten Übertragung von arboviren in deutschland derzeit extrem niedrig. da arbovirus-infektionen häufig asymptomatisch oder subklinisch verlaufen, können nur epidemiologische studien aufschluss über das risiko einer Übertragung solcher erreger geben. das auftreten von fieber und zentralnervösen störungen bis tage nach transfusion von nicht inaktivierten blutprodukten könnte ein hinweis auf eine transfusionsassoziierte arbovirus-infektion sein. die verbreitungsgebiete verschiedener arboviren überschneiden sich teilweise mit malariaendemiegebieten. daher werden durch den ausschluss von reisenden in malariaendemiegebiete auch infektionen mit arboviren erfasst. spezifische ausschlusskriterien gibt es mit ausnahme der rückstellung von reiserückkehrern aus nordamerika und mexiko aufgrund der wnv-epidemie nicht. aufgrund der für deutschland vorliegenden daten zur epidemiologie und dem infektionsverlauf von arbovirus-infektionen sind zurzeit keine über die für wnv getroffenen maßnahmen hinausgehenden reisende in endemiegebiete von arbovirus-infektionen, in denen wie z. b. in den usa hinsichtlich wnv eine akute epidemie abläuft, werden als blutspender für einen angemessenen zeitraum ( wochen) für die gewinnung von zellulären oder nicht inaktivierten blutkomponenten zurückgestellt dieses papier wurde fertig gestellt am . . und vom arbeitskreis blut am . . verabschiedet. es wurde erarbeitet von den mitgliedern der untergruppe "bewertung blut-assoziierter krankheitserreger" des arbeitskreises blut: dr. johannes blümel verordnung zur umsetzung von eg-richtlinien über den schutz der beschäftigten gegen gefährdung durch biologische arbeitsstoffe bei der berufsgenossenschaft der chemischen industrie replication of arboviruses in insect vectors the effect of environmental and physiological conditions of culex tritaeniorhynchus on the pattern of transmission of japanese encephalitis virus effect of rearing temperature on transovarial transmission of st. louis encephalitis virus in mosquitoes mechanisms of arthropod transmission of plant and animal viruses. microbiol growth and transovarial transmission of chandipura virus (rhabdoviridae: vesiculovirus) in phlebotomus papatasi experimental transmission of crimean-congo hemorrhagic fever virus by west african wild ground-feeding birds to hyalomma marginatum rufipes ticks transmission transovarienne des arbovirus par les moustiques potential significance of transovarial transmission in the circulation of tick-borne encephalitis virus experimental transmission of powassan virus (flaviviridae) by ixodes scapularis ticks (acari: ixodidae) migratory birds and spread of west nile virus in the western hemisphere prevalence of antibodies to borrelia burgdorferi and tick-borne encephalitis virus in an endemic region in southern germany arboviruses causing neurological disorders in the central nervous system imported tropical virus infections in germany imported vector-and rodentborne virus infections -an introduction travel-related vector-borne virus infections in germany durch zecken übertragene humanpathogene und bisher als apathogen geltende mikroorganismen in europa. teil i: zecken und viren th international potsdam symposium (ips-v) on tick-borne diseases: tick-borne encephalitis and lyme borreliosis west nile virus screening of blood donations and transfusion-associated transmission -united states local dengue fever cases in tick-borne viral encephalitis in finland. the clinical features of kumlinge disease during - cdc possible west nile virus transmission to an infant through breast-feeding -michigan mosquito-borne viruses in western europe: a review overwintering mechanisms of mosquito-borne arboviruses in temperate climates arboviral ecology and the prospect for overwintering of west nile virus. west nile virus action workshop arboviruses as imported disease agents: the need for increased awareness. arch virol risikogebiete der frühsommer-meningoenzephalitis (fsme) in deutschland west-nil-fieber: erster importierter erkrankungsfall in deutschland transmission of west nile virus from an organ donor to four transplant recipients update: investigations of west nile virus infections in recipients of organ transplantation and blood transfusion update: investigations of west nile virus infections in recipients of organ transplantation and blood transfusion wnv infections in recipients of blood transfusion and organ transplantation investigations of west nile virus infections in recipients of blood transfusions estimated risk of west nile transmission through blood transfusion during an epidemic in queens the impact of arthropod borne diseases (including wnv) on the safety of blood used for transfusion as well as organs used for transplantation in the european community über die zulassung von arzneimitteln/abwehr von arzneimittelrisiken -anordnung des ausschlusses von blutspendern zur verhinderung einer möglichen Übertragung des west-nil-virus durch zelluläre blutprodukte oder gefrorenes frischplasma transfusion transmission of west nile virus: a merging of historical and contemporary perspectives food and drug administration, blood products advisory committee west nile virus and the safety of plasma derivatives: verification of high safety margins, and the validity of predictions based on model virus data cpmp position statement on west nile virus and plasma-derived products tick-borne diseases in transfusion medicine key: cord- -arnuoufn authors: blank, walter a.; henderson, kenneth s.; white, lisa a. title: virus pcr assay panels: an alternative to the mouse antibody production test date: journal: lab anim (ny) doi: . /laban - sha: doc_id: cord_uid: arnuoufn antibody production tests have traditionally been used to test biological materials for viral contamination. now molecular biology techniques have emerged as an alternative. the authors compare map testing with pcr-based detection methods, focusing on differences in animal use, laboratory requirements, sample size, and limits of detection. the transmission of viruses through biological materials is of great concern to many researchers and managers of lab animal facilities. there is ample documentation for the confounding effects of such transmission on research of viral infections in experimental animals (often in the absence of clinical disease). of even greater concern, outbreaks of infection among laboratory workers by zoonotic agents such as lymphotrophic choriomeningitis virus (lcmv) and hantavirus are traceable to contaminated biological materials transplanted into animals. the biosecurity tenets of many research organizations require screening for viral agents of all cell lines, tumors, sera, and other biologicals before implantation or inoculation into animal models. federal guidelines , also dictate that monoclonal antibodies (mabs) and other biotechnology products derived from tissues or cell lines of animal origin are demonstrated to be free of viral contamination before their use for therapeutic or diagnostic purposes. until recently, the mouse antibody production (map) test was the primary method of screening for viruses of murine origin ( table ) , but the application of modern molecular biology methods to this purpose presents certain advantages. in this article, we compare map testing with the polymerase chain reaction (pcr) for the detection of viral agents. although our discussion concentrates on map testing in particular, the concepts presented in this paper are also applicable to rat and hamster antibody production (rap and hap) tests. rowe et al. first developed map testing as a simple and equally sensitive alternative to tissue culture for the detection of polyoma virus, and others have since applied it to simplify the detection of other agents [ ] [ ] [ ] [ ] [ ] . typical testing involves inoculating mice by multiple routes with a test article and holding them in isolation for a minimum of four weeks . researchers then collect serum sized dna molecules may act as templates for further amplification. the result of the process is an exponential increase in the number of target sequences, potentially creating billions of copies (also referred to as 'amplicons') from a single dna template. subsequent detection of the pcr products involves performing gel electrophoresis on an aliquot of the reaction mixture. after staining for visualization, the dna molecules appear as discrete bands separated on the basis of their length, allowing for identification of the pcr product if the target sequence was present in the original sample. because pcr amplifies only dna molecules, one detects viruses with rna samples from the animals and test them for virus-specific antibodies using the enzymelinked immunosorbent (elisa), indirect fluorescent antibody (ifa), or hemagglutination inhibition (hai) assays. detection of virus-specific antibodies indicates the presence of that virus in the test article. the detection of lactate dehydrogenase-elevating virus (ldv), for which an effective antibody test does not exist, consists of screening serum for elevated levels of the enzyme lactate dehydrogenase. the literature contains a number of surveys demonstrating the importance of map testing in the screening for viral agents ( table ) . ldv seems presently to be the most frequent contaminant, although reovirus, lcmv, minute virus of mice (mvm), and mouse hepatitis virus (mhv) are also detected regularly. the general trend during the last three decades seems to be toward fewer cases of contamination of the biological materials tested with the viral agents of interest. this decrease in prevalence coincides with a reduced rate of viral infections in animal colonies, a result of stricter biosecurity measures throughout the laboratory animal community-measures that include map testing of materials. molecular biology methods permit the assaying of test articles directly for the presence of specific nucleic acids from contaminating infectious agents, as opposed to indirectly testing for the elicitation of an immune response. pcr is one of the most powerful and flexible of these molecular techniques, and in recent years it has been applied toward the replacement of map testing [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the basic principle behind pcr is the in vitro amplification of a particular dna sequence (e.g., a viral gene) in a sample to easily detectable levels, using a thermostable dna polymerase enzyme (usually taq polymerase), oligonucleotide primers specific for the target of interest, and a computer-controlled thermocycler that precisely manipulates the reaction temperatures (fig. a) . with each cycle of the pcr, newly synthe- rt-pcr can involve either a two-step reaction (generation of cdna, then transfer of an aliquot to the pcr mixture) or a single step, in which the enzymes required for both reactions are present in the same tube. researchers have adapted numerous variations on the pcr method for the detection of infectious agents, all of which can be performed on dna templates as well as on rna templates after an rt step. a recent development that is particularly useful for diagnostic applications is the fluorogenic ′-nuclease assay , also known as taqman pcr, which makes use of an additional dye-labeled oligonucleotide probe that specifically hybridizes to the pcr product ( fig. b) . as the amplification pro-gresses, the probe binds to the pcr product template and undergoes digestion by the polymerase enzyme, releasing a detectable fluorescent signal that may be read on a fluorometer after completion of the reaction or that can be monitored 'real-time' during the course of the reaction by a specialized thermocycler, permitting quantification of starting templates. product detection by fluorescence permits a very high level of sensitivity, greatly simplifies post-pcr analysis, and lends itself to a -well plate format, allowing the use of semiautomated and automated procedures as throughput needs require. any appropriately equipped laboratory can support the performance of pcr for the detection of murine viruses (see 'laboratory requirements' below), using assays published in the literature or developed on the premises, although several commercial providers of these services exist figure . among the advantages of the use of pcr rather than map testing is the substantial reduction in time needed to complete the test. ( table ). costs will vary not only with providers but also depending on the number of agents of interest for testing. generally, different panels of assays are available depending on customer needs. for instance, charles river laboratories (crl) offers an "essential" panel of viruses and a "comprehensive" panel of viruses, although assays for individual viruses are also available. virus pcr assay panels may also include testing for mycoplasmas, which are common bacterial contaminants of tumor and cell lines , . the steps involved in the map and pcr testing procedures appear in figure . (unless otherwise specified, comparisons between the procedures originate from protocols used at crl.) map test sample preparation consists of homogenization, inactivation by osmotic lysis/freeze-thaw, and dilution as necessary before inoculation into the animals. between and days after inoculation, technicians euthanize for serum collection the animals designated for ldv testing, and they collect serum for viral antibody testing from all other animals no sooner than days after inoculation. for pcr, technicians extract the total nucleic acid (tna; i.e., dna and rna) by chemical lysis of the test articles, followed by binding and washing on miniature columns containing silica resin, and elution from the columns. they can then use the aliquots of the purified tna directly for pcr to detect dna viruses, or they can subject them to rt before pcr for rna viruses. the animal facilities and trained personnel required for map testing are generally already present in organizations doing other types of animal experimentation. because of the potential for infection, it is advisable to conduct map testing in facilities that are separate from areas used to house other animal studies. map testing requires immunocompetent mice free of antibodies against viral pathogens, and one must take additional precautions, such as using sterilized microisolation units and performing all animal manipulation in a biosafety cabinet, to avoid infection by viruses other than those potentially in the test articles and to prevent infection of other animals in the facility. detection of antibodies (or enzyme levels, in the case of ldv) specific to the agents in question requires access to a serological testing laboratory. pcr testing requires personnel trained in molecular biology techniques and specialized equipment, which includes still-air work hoods for sample processing, thermal cyclers, and gel electrophoresis equipment and/or fluorometric detection systems. to prevent false-positive results due to contamination with pcr templates, reagent preparation, sample processing, and pcr amplification/product detection should all take place in separate laboratories. ideally, personnel should have laboratory-specific gowning and equipment, and workflow should proceed from 'clean' to 'dirty' areas to prevent the contamination of reagents and samples with amplicons generated in the amplification area. a primary advantage of using pcr panels is the reduction in the time between submission of the sample and reporting of results. map testing protocols require a minimum of days after inoculation to allow the animals to mount a detectable immune response; specimen preparation and serological testing will contribute additional time to the total required for this method. by comparison, pcr allows the processing of test articles and analysis of results in a period as short as one to two days, although the number of samples being analyzed and resources available may increase turnaround time. generally, pcr results become available in less than one week. standard map testing protocols call for a minimum of . ml of test material, enough to inoculate five mice with . ml intraperitoneally, . ml intranasally, and . ml orally, and to inoculate another five mice similarly with a -fold dilution of the test material. one can dilute smaller amounts of one of the most serious concerns with the use of pcr in a diagnostic capacity is that its high sensitivity makes it susceptible to false-positive results because of contaminating templates. three potential sources of this contamination are other samples, experimental materials such as positive controls, and pcr products generated by previous reactions against the same target sequence . however, adequate precautions and controls can minimize contamination and detect it as such before erroneous results are reported. the most effective method of contamination prevention is separation of tasks (reagent preparation; sample preparation; pcr amplification, and analysis) into different labs and maintenance of strict workflow procedures from 'clean' to 'dirty' areas. there must be no transfer between areas of room-designated equipment (pipettors, sample racks, etc.) and gowning. personnel should only manipulate positive control templates in the amplification lab, and if possible, should prepare them in facilities separate from routine sample extraction. a quality control and tracking program for sample extraction and pcr reagents should also be in place. assigning lot numbers when reagents are prepared and identification of aliquots in use can be very helpful in tracing the source of contamination in the event that it does occur, and personnel should use single-use aliquots whenever possible. to monitor contamination of reagents, personnel should always perform negative control reactions. strongly positive test articles present a potential source of contamination during sample preparation. during the processing steps, it is important to handle samples individually with no more than one tube open whenever possible. still-air hood pcr workstations can help avoid transfer of aerosols between samples. a practice that we recommend is the submission of duplicate samples (e.g., two aliquots of the same test article) for pcr panel testing. only one of the aliquots undergoes initial testing; in the event of a positive result, the untouched 'retain' sample undergoes extraction and retesting by the positive assay (including controls) to confirm the positive result. carryover contamination of pcr products from previous reactions is of particular concern, especially when analyzing pcr products on a gel; one can prevent it by a combination of including the enzyme uracil n-glycosylase (ung) and substituting the nucleotide deoxyuracil (du) for thymine in pcr master mixes . the alternate nucleotide becomes incorporated into all pcr products during the course of the reaction. in subsequent reactions, a brief incubation before pcr allows ung to degrade any dna molecules containing du-that is, any products potentially carried over from earlier reactions-making them unavailable for amplification. 'legitimate' templates containing thymine are unaffected, and the ung is inactivated before pcr begins. serological assays may also give falsepositive results for numerous reasons, including incompletely purified antigens and cross-reactive antibodies . particular difficulties in the interpretation of map test results may arise if antibodies generated against the actual biological material being tested react with the cell culture control materials used in the preparation of elisa antigens. although noninfectious virus particles can elicit an immune response, high anti-body titers are most likely to develop after active viral infections , requiring the presence of adequate numbers of infectious viral particles for map testing to be successful. many viruses can become noninfectious with changes in temperature or ph, possibly resulting in inactivation during storage, transportation, or preparation of the test article. by contrast, pcr testing can detect infectious and noninfectious virus particles alike, provided that the sample has not been mishandled to such an extent that the nucleic acid has degraded. pcr assays may be subject to inhibition by substances present in the test article that are not completely removed during the nucleic acid extraction procedure, leading to false-negative results. it is possible to detect inhibition by performing a separate 'spike' control assay in which limited copy numbers of another template are added along with the extracted sample to the pcr mixture. an independent assay evaluation of this assay with a positive result indicates the absence of pcr inhibitors. degradation of nucleic acids or inefficient recovery during extraction from test articles can also give false-negative results. one can control for this by adding another exogenous template in the early stages of sample preparation, then testing the final tna extract for that template. this nucleic acid recovery control may also act as a spike control for pcr inhibitors, although separation of the two controls can simplify troubleshooting of a failed assay. a summary of the published detection sensitivities of a number of pcr assays, along with some of our own sensitivity data, appears in table . in a direct comparison of the pcr assays listed with map testing, pcr assays were more sensitive than map testing and were equally sensitive. the choice of animals used can influence the sensitivity of map testing itself, because some viruses elicit different levels of antibody response in particular mouse strains , . the limit of detection among pcr assays can vary greatly with the pcr technology, primer design, amplicon size, resource reagents using documented standard operating procedures. appropriate documentation of all data and regular review by a quality assurance/regulatory affairs department are also necessary components of the process. a distinct advantage of pcr panel testing is the replacement of animal-based testing with a series of in vitro assays, consistent with the rs of humane experimental technique (reduction, replacement, and refinement) . crl's standard map testing procedure involves the inoculation of mice with the test article and diluted test article, and another mice with a negative control article. pcr use thus avoids the use of up to animals per test article, along with any associated distress. screening of biological materials for infectious agents before use for in vivo experimentation is an important precaution for research integrity. although the traditional method of accomplishing this has been serologically by the map test, the use of modern molecular biology techniques such as pcr permits such testing to be done much more rapidly with greater sensitivity and high reliability, provided that adequate controls are implemented. cycle parameters, pcr reagents, and nucleic acid extraction procedures. furthermore, comparisons between different pcr assays for the same agent can be difficult to make when based on indirect virus titer methods such as infectivity assays. the sensitivity of these titering methods is highly dependent on a number of variables, including virus strain, host animal or cell line susceptibility, the ratio of infective to noninfective virus particles in the particular virus stock being tested, and culturing techniques. for both map and pcr, assays that are very sensitive for known virus strains may not be capable of detecting divergent or newly emerging strains. such was recently the case with a serological assay for mvm: the mvm elisa did not detect the closely related mouse parvovirus (mpv) because of differences in their respective virus capsid proteins . similarly, pcr primers and probes must be targeted to genetic sequences that are highly conserved among all strains of the agent being tested. agents with divergent sequences between strains or subspecies may require multiple (or degenerate) pcr primers or probes within a reaction mixture, or may require additional assays. for instance, the absence of highly conserved sequences between hantaviruses led us to design separate assays for the hantaan and seoul strains. the widespread use of antibody production tests during the past decades has led to their broad acceptance as the method of choice to detect rodent viruses in biological materials to be used for studies following good laboratory practices (glp), as well as in products for human use such as biotechnology products derived from cell lines or mabs. as such, fda guidelines specifically mention antibody production tests , . one can also use other tests of at least equivalent sensitivity and reliability-including pcr tests-provided that adequate validation of each method is performed with respect to such aspects as sensitivity, selectivity (specificity), reproducibility, and stability . such validation experiments must take place on validated equipment with quality-controlled committee on infectious diseases of mice and rats, institute of laboratory animal resources outbreak of lymphocytic choriomeningitis virus infections in medical center personnel infection of laboratory workers with hantavirus acquired from immunocytomas propagated in laboratory rats points to consider in the manufacture and testing of monoclonal antibody products for human use. fda, docket no. d- international conference on harmonization: guidance on viral safety evaluation of biotechnology products derived from cell lines of human or animal origin commercial production of monoclonal antibodies studies on mouse polyoma virus infection. i. procedures for quantitation and detection of virus a new mouse virus apparently related to the adenovirus group natural history of sendai virus infection in mice an evaluation of the mouse antibody production test for detecting three murine viruses comparison of isolation in cell culture with conventional and modified mouse antibody production tests for detection of murine viruses comparative sensitivity of infectivity assay and mouse antibody production (map) test for detection of mouse thymic virus (mtlv) detection of rodent coronaviruses in tissues and cell cultures by using polymerase chain reaction sequence analysis and molecular detection of mouse hepatitis virus using the polymerase chain reaction detection of rodent parvoviruses by use of fluorogenic nuclease polymerase chain reaction assays comparison of the sensitivity of in vivo antibody production tests with in vitro pcr-based methods to detect infectious contamination of biological materials detection of rodent coronaviruses by use of fluorogenic reverse transcriptasepolymerase chain reaction analysis detection of lymphocytic choriomeningitis virus by use of fluorogenic nuclease reverse transcriptase-polymerase chain reaction analysis detection of sendai virus and pneumonia virus of mice by use of fluorogenic nuclease reverse transcriptase polymerase chain reaction analysis detection of reovirus type by use of fluorogenic nuclease reverse transcriptase polymerase chain reaction the polymerase chain reaction (pcr) reverse transcriptase polymerase chain reaction (rt-pcr) fluorogenic ′-nuclease pcr (real-time pcr) contamination of transplantable tumors, cell lines, and monoclonal antibodies with rodent viruses detection and elimination of contaminating microorganisms in transplantable tumors and cell lines pcr primer: a laboratory manual microbiological assessment of laboratory rats and mice detection of mouse hepatitis virus antibody by protein a-elisa in prevalent inbred strains or outbred stocks of mice effect of mouse strain and age on detection of mouse parvovirus by use of serologic testing and polymerase chain reaction analysis molecular characterization of a newly recognized mouse parvovirus limitations of taqman pcr for detecting divergent viral pathogens illustrated by hepatitis a, b, c, and e viruses and human immunodeficiency virus guidance for industry: bioanalytical method validation the principles of humane experimental technique murine virus contaminants of leukemia viruses and transplantable tumors detection of minute virus of mice using real-time quantitative pcr in assessment of virus clearance during the purification of mammalian cell substrate derived biotherapeutics polymerase chain reaction for detection of rodent parvoviral contamination in cell lines and transplantable tumors a vaccinia virus-vectored hantaan virus vaccine protects hamsters from challenge with hantaan and seoul viruses but not puumala virus modes of seoul virus infections: persistency in newborn rats and transiency in adult rats key: cord- -nno yjae authors: sylvester‐hvid, c.; nielsen, m.; lamberth, k.; røder, g.; justesen, s.; lundegaard, c.; worning, p.; thomadsen, h.; lund, o.; brunak, s.; buus, s. title: sars ctl vaccine candidates; hla supertype‐, genome‐wide scanning and biochemical validation date: - - journal: tissue antigens doi: . /j. - . . .x sha: doc_id: cord_uid: nno yjae abstract: an effective severe acute respiratory syndrome (sars) vaccine is likely to include components that can induce specific cytotoxic t‐lymphocyte (ctl) responses. the specificities of such responses are governed by human leukocyte antigen (hla)‐restricted presentation of sars‐derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information (lauemoller et al., rev immunogenet : : – ). the latter was recently established when a causative coronavirus (sars‐cov) was isolated and full‐length sequenced (marra et al., science : : – ). here, we have combined advanced bioinformatics and high‐throughput immunology to perform an hla supertype‐, genome‐wide scan for sars‐specific ctl epitopes. the scan includes all nine human hla supertypes in total covering > % of all individuals of all major human populations (sette & sidney, immunogenetics : : – ). for each hla supertype, we have selected the top candidates for test in biochemical binding assays. at this time (approximately months after the genome was established), we have tested the majority of the hla supertypes and identified almost potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. eradicated. this would require detection assays that can track the disease and intervention measures that can break the chain of transmission. all of these procedures should be simple, yet effective. unfortunately, no such diagnostic test is currently available, and controlling transmission by containment solely is complicated and extremely costly. further complicating any eradication effort, a nonhuman reservoir appears to exist. thus, a strong case for a sars vaccine can be made. it would be of significant help in any eradication effort and, should that fail, it could protect infected individuals against the disease. the sars-cov infects epithelial cells in the respiratory tract causing interstitial pneumonia ( ) . one would therefore expect that an effective vaccine should induce mucosal immunity such as that effected by secretory immunoglobulin a (iga), which specifically prevents an infectious agent from penetrating the mucosal epithelium, and by cytotoxic t lymphocytes (ctls), which specifically eradicate infected cells ( ) . iga responses are generally considered the major protective mechanism; however, there are examples of ctls, not antibodies, being responsible for early control of mucosal infection ( ) . particularly noteworthy, this is the case for the infectious bronchitis virus of chicks, a prototype of the coronaviridae family, where primary effector cd þ ctls play a critical role in the elimination of virus during acute infection and subsequent control of the infection ( - ). human ctls are specific for peptides presented in the context of human leukocyte antigen (hla) molecules [generically known as ''major histocompatibility complex (mhc) molecules'']. prior to presentation, peptides are generated in the cytosol by limited proteolytic fragmentation of all available protein antigens, translocated to the endoplasmic reticulum, specifically sampled by the mhc molecules and exported to the cell surface, where they await ctl scrutiny. importantly, the hla is extremely polymorphic and the peptide binding specificity varies for the different polymorphic hla molecules ( ). it has, however, been suggested that the majority of all major human populations can be covered with three to nine ''hla supertypes'', where the different members of each supertype bind similar peptides ( ). if one knew exactly how peptides were generated and selected, then genomic/proteomic information could be used to predict the outcome of antigen presentation and forecast immunogenicity. here, we have used advanced immunobioinformatical tools to mimic antigen presentation and, in a highly cost-and timeeffective manner, predicted possible immunogenic epitopes. the complete sars genome/proteome was obtained from gen-bank (nc ) and virtually digested into all unique nonamer peptides ( ) . thus, close to , binding predictions were made for each of the nine hla supertypes. artificial neural networks (anns) were used to predict the binding affinity quantitatively when the corresponding data were available [e.g. for a* ( , ) ]. the performance of the anns is high, as the correlation coefficient between predicted and measured binding is . . the remaining hla bindings were predicted using weight matrices derived from gibbs sampling sequence-weighting methods with pseudocount correction for low counts as well as differential position-specific anchor weighting (nielsen et al. manuscript in preparation). these weight matrices were calculated from available nonamer data from the syfpeithi and mhcpep databases with the peptides clustered into the nine supertypes (a , a , a , a , b , b , b , b , and b ). the positive predictive value of the matrix-driven prediction has been found to be around %, whereas the negative predictive value has been found to be around % (lamberth et al. unpublished observation). proteasomal processing was predicted using netchop . ( ). netchop . has been found to be superior to other proteasomal prediction algorithms ( ) . peptides with a netchop . score below . (i.e. poorly predicted proteasomal processing) were excluded from further analysis. finally, we excluded all peptides that did not represent epitopes conserved in all sars isolates. figure shows a representative example for a member of the hla-a supertype, the hla-a* (this haplotype is particularly common in southeast asia). for each hla supertype, the top-ranking nonamer peptides were synthesized by standard -fluorenylmethyloxycarbonyl (fmoc) chemistry, purified by reversed-phase high-performance liquid chromatography (at least %, usually > % purity) and validated by mass spectrometry. the interaction of these epitope candidates with the appropriate hla was subsequently validated in a biochemical binding assay ( ) . briefly, denatured and purified recombinant hla heavy chains were diluted into a renaturation buffer containing hla light chain, b -microglobulin, and graded concentrations of the peptide to be tested, and incubated at c for h allowing equilibrium to be reached. we have previously demonstrated that denatured hla molecules can de novo fold efficiently, however, only in the presence of appropriate peptide ( ) . the concentration of peptide-hla complexes generated was measured in a quantitative enzyme-linked immunosorbent assay and plotted against the concentration of peptide offered ( ) (fig. ) . because the effective concentration of hla ( - nm) used in these assays is below the equilibrium dissociation constant (k d ) of most high-affinity peptide-hla interactions, the peptide concentration leading to half-saturation of the hla is a reasonable approximation of the affinity of the interaction. an initial screening procedure was employed whereby a single high concentration ( , nm) of peptide was incubated with one or more hla molecules. if no complex formation was found, the peptide was assigned as a non-binder to the hla molecule(s) in question, conversely, if complex formation was found in the initial screening, a full titration of the peptide was performed to determine the affinity of binding. the resulting binding isotherms were analyzed by one-site hyperbola regression (prism graphpad) determining the concentration of hla employed ( - nm, data not shown), the k d of the interaction (table ) and the goodness of the curve fit (r was always > . and in the majority of cases it was > . , data not shown) ( ) . in general, intermediate and high-affinity binders have k d s better (i.e. lower) than nm and nm, respectively, and the higher the affinity, the more likely the peptide is going to be a t-cell epitope ( ) . table summarizes the data for hla-a* and hla-a* . the pep- for the top-ranking peptides, only (or - %) of the combinations involves cross-responses, where a peptide predicted to be a top-ranking binder to one hla molecule turns out to be a binder to a member of another hla supertype, i.e. it supports the contention that hla supertypes effect significant diversification of anti-sars ctl responses. conversely, the overlap between different members of the same hla supertype appears to be extensive. thus, of the peptides predicted to be good binders to a* were found to bind to another member of the a supertype, hla-a* . similarly, nine of the peptides predicted to be good binders to a* were found to bind to hla-a* (table ) once all nine supertypes have been tested, we would project to have found well over different vaccine candidates. these would all have been predicted to be successfully processed by the proteasome and biochemically validated for hla binding. therefore, there should be a high probability that these peptides are indeed presented to ctls. once that occurs, there is an approximately % chance of being able to raise a ctl response ( ) . thus, our data do in all likelihood include some ctl epitopes. to identify these from the > binding peptides, one could search for the corresponding ctl reactivities in peripheral blood of sars survivors using robust and reasonably simple technologies such as interferon-g secretion from stimulated whole blood t cells ( ) peptide binders to hla-a* (top frame) and hla-a* (bottom frame), sorted according to predicted binding strength, were synthesized and the affinities of binding to a* and a* were determined. the peptide sequence is given in single-letter code and the measured binding affinity is given as the kd. in the near future, the genome of any pathogen can be fully sequenced in a matter of days. the bioinformatics tools currently being developed and perfected will be able to use such genomic information to predict immune epitopes computationally, and the corresponding immunological tools will currently be able to validate these predictions in a matter of weeks to months. we predict that epitope identification in the near future will be as fast as a dna sequencing in handling whole organisms. with the dissemination of these tools, one could envision that clinicians and scientists anywhere would be able to analyze pathogens of their interest (or agent of bioterrorism, or tumor cell) for the purpose of fast identification of immunogenic epitopes ( ) . the timeline of the present sars epidemic has demonstrated how fast modern science can identify a pathogen and decipher its genome. using this information in a fast and rational design of vaccines, immunobioinformatics promises to take this development one step further. identifying cytotoxic t cell epitopes from genomic and proteomic information: ''the human mhc project the genome sequence of the sars-associated coronavirus nine major hla class i supertypes account for the vast preponderance of hla-a and -b polymorphism sars coronavirus: a new challenge for prevention and therapy recent advances in mucosal vaccines and adjuvants specific cytotoxic t lymphocytes are involved in in vivo clearance of infectious bronchitis virus cytotoxic t lymphocyte responses to infectious bronchitis virus infection cytotoxic t lymphocytes are critical in the control of infectious bronchitis virus in poultry adoptive transfer of infectious bronchitis virus primed alphabeta t cells bearing cd antigen protects chicks from acute infection memory t cells protect chicks from acute infectious bronchitis virus infection sensitive quantitative predictions of peptide-mhc binding by a ''query by committee'' artificial neural network approach reliable prediction of t-cell epitopes using neural networks with novel sequence representations prediction of proteasome cleavage motifs by neural networks predicting proteasomal cleavage sites: a comparison of available methods establishment of a quantitative elisa-based assay capable of determining peptide-mhc class i interaction efficient assembly of recombinant major histocompatibility complex class i molecules with preformed disulfide bonds the relationship between class i binding affinity and immunogenicity of potential cytotoxic t cell epitopes immunodominance in mhc class i restricted t lymphocyte responses cytokine-based human whole blood assay for the detection of antigen-reactive t cells recombinant polyepitope vaccines for the delivery of multiple cd cytotoxic t cell epitopes delivery of multiple cd cytotoxic t cell epitopes by dna vaccination immunogenicity of a human immunodeficiency virus (hiv) polytope vaccine containing multiple hla a hiv cd (þ) cytotoxic t-cell epitopes an hla-a polyepitope vaccine for melanoma immunotherapy papillomavirus virus-like particles for the delivery of multiple cytotoxic t cell epitopes cytotoxic t cell polyepitope vaccines delivered by iscoms the development of multi-epitope vaccines: epitope identification, vaccine design and clinical evaluation optimization of epitope processing enhances immunogenicity of multiepitope dna vaccines the danish mrc (grant - - ), the th framework programme of the european commission (grant qlgt- - ), the nih (grant ai - ), and the danish national research foundation supported this work. support for this work has been obtained from siga technologies. key: cord- - j y authors: jun, qiao; xian-zhu, xia; song-tao, yang; de-sheng, li; gui-xue, hu; yu-wei, gao; he-ting, sun; zhong-pen, zhao; zhi-jing, xie; fang, yan; wen-qi, he; gen, huang title: serological survey on canine coronavirus antibodies in giant pandas by virus neutralization test date: journal: j for res (harbin) doi: . /bf sha: doc_id: cord_uid: j y in order to survey the infectious situation of canine coronavirus (ccv) in giant panda population, a virus neutralization test detecting specific antibodies against ccv in giant panda’s sera was established by using two-fold dilutions of serum and tcid( ) of the virus. the sera samples of giant pandas, which were gathered from zoos and reserve region of sichuan province, china were detected. the neutralization antibody titer of : was recognized as the positive criterion, sera samples were detected to be positive, and the positive rate was . %. the titers of neutralizing antibody ranged from : to : . it was the first comprehensive investigation on neutralization antibodies against ccv in giant panda population in china. the results of study showed that the infection of ccv in giant panda population was universal, which has posed a threat to the health of giant panda. therefore, it is incumbent on us to study safe and effective vaccines to protect giant panda against ccv infection. canine coronavirus (ccv), a causative agent of enteritis in neonatal dogs, was firstly identified by binn during an epizootic study in germany (binn et al ) . however, since the virus has been demonstrated, canine coronavirus (ccv) appeared to be worldwide in europe, the united states, thailand, and in australia (appel ; kelly et al ; tennant et al ; bandai et al ) . in recent years, it was reported that ccv could infect giant pandas and others precious wild animals (mainka et al ; he et al ; gao et al ; qiao et al ) . in , a strain of ccv was isolated from the liver of an acutely died giant panda deriving from wolong reserve, p. r. china, which further verified that it was a causative agent to giant panda. however, so far, there is no-comprehensive knowledge about the infectious situation of ccv in giant pandas. whether it is necessary to use ccv vaccine to protect this precious animal should be researched, in order to answer these questions, the sera samples of giant panda were collected from zoos and reserve regions in sichuan province, china and used for serological investigation of neutralizing antibodies against ccv. the strain dxmv of ccv was originally isolated from the liver of an acutely died giant panda deriving from wolong reserve, p. r. china. the serum samples of giant panda were gathered from zoos and reserve regions of sichuan province. each serum had a unique number which was donated the corresponding giant pandas. dmem was purchased from gibco company. the -well cell culture plate was obtained from promega company. standard anti-ccv positive serum was prepared by genetic engineer laboratory of pla. negative serum was from institute of virology of jilin province. madin-darby canine kidney (mdck) cell was used for assay of tcid . an ampule of virus was thawed and diluted to -fold serially with virus growth medium (vgm). when containing confluent monolayers mdck cells for inoculation were prepared in -well cell culture plate, the normal medium was removed and - jl dmem was added to wash away fetal bovine serum. then different virus dilutions were transferred to corresponding wells for tjl, absorbed for hours at ~ in a co incubator. inoculums were then removed by using a multi-channel pipette, and ijl of vgm was added to well to incubate for - days. cytopathic effects (cpe) were observed daily under inverted microscope for - days. tcids was calculated by the reed-muench method according to observed results. sera samples were heated to inactivate for min at ~ and then performed serially twofold dilutions. virus was diluted to tcid per #l in vgm and gently mixed with serially diluted serum. and the plates were incubated at ~ for hours, in % co incubator. medium in monolayers mdck cell used for neutralization test was moved and -#l virus-serum mixtures were transferred to each well of the plate. the plates were incubated for h at ~ in a co incubator and then the virus-serum mixture was removed from each well. - jl dmem was added to each well. cpe was observed as described previously. the highest dilution which can completely protect the cell from cpe was taken to be the viral antibody titer. the serum samples of giant pandas were assayed under the preceded conditions of optimization. meanwhile, positive antisera against canine distemper virus, canine adenovirus, canine parainfluenzavirus, rabies virus were also tested referring the above procedures for observing whether the cross reaction would happened. the virus neutralization test (vnt) exhibited higher specificity. positive sera against ccv could effectively inhibit dxmv strain of ccv to infect mdck, while positive antisera against canine distemper virus, canine adenovirus, canine parainfluenzavirus, rabies virus did not exhibit inhibiting activity. neutralization antibodies titers of giant panda's sera were shown in the table . the no. in samples was ccv antibody positive when neutralization antibody titer of : was recognized as the positive criterion. the positive samples were linhai, zhuangzhuang, didi, wumingl, iongxin, chuangchuang, iongfei, liangliang respectively (table ) . noticeably, the serum of chuangchuang exhibited the highest titer for : . canine coronavirus belongs to coronavinises serogroup i, a major antigenic group of coronaviruses and is serologically related to feline infectious peritonitis virus (fipv), feline enteric coronavirus (fecv), transmissible gastroenteritis virus (tgev) and porcine respiratory coronavirus (prcv). these viruses have been distinguished mainly by their host species of origin. it was reported, however, that some strains of ccv can also infect cats, swine and other wildlife animals. when mainka et al. ( ) carried out serological survey of ccv in giant pandas (ailuropoda melanoleuca) in the wolong reserve, china, he found that sera samples in giant panda's sera were ccv antibody positive, which indicated that ccv may infect giant panda and induce neutralizing antibody against ccv. from the virological aspects, generally speaking, animal species susceptibility to different virus has close relationships with the presence or absence of specific receptor in a species. recent studies had indicated that feline aminopeptidase-n on the surface of cell was used for the receptor by ccv. whether feline aminopeptidase-n existed on the giant panda's cell or not was not very clear. since the sera samples of giant pandas detected by mainka were very limited, they did not reflect the infection situation. in our study, sera samples of giant panda were detected by vnt, which indicated that ccv infection was universal in giant panda's populations. since it was very difficult to distinguish ccv infection from other pathogen infection such as cpv- , enteric bacteria, parasites, poisonings and non-infectious causes of diarrhea, laboratory confirmation was necessary in clinic diagnosis (evermann et al ; tennant et al ; keenan et al ; yasoshima et al. ) . at present, diagnostic methods which have been used for the detection of ccv, include electron microscopy (em), isolation on appropriate cell cultures, nested-polymerase chain reaction (n-pcr), enzyme-linked immunosorbent assay (elisa) and so on. of the several methods used for the detection of ccv, em appears to be a valuable diagnostic tool. em has been reported to be more sensitive and useful in virus isolation for detecting both coronaviruses and rotaviruses. however, the frequency of ccv disease has probably been overestimated by diagnostic laboratories when electron microscopy (em) was applied as the principal diagnostic method. for common presence of corona virus-like particles in feces, diagnosis of ccv was difficulties by em and requires confirmation by other tests. immuno-electronmicroscopy with a specific immune serum permits confirmation of the em diagnosis, but it requires specialized laboratories and qualified experts. virus isolation in cell cultures is often used, but it is difficult. recently, an n-pcr assay for the diagnosis of ccv infection has been reported (takeuchi et al ; pratelli et al ; pratelli etal ; rimmelzwaan etal ) . pcr for the diagnosis of ccv infection revealed high specificity and sensitivity, but it also required the qualified experts to perform. the vnt is a sensitive and specific assay to the diagnosis of ccv by detecting specific antibody. this method is not only used for diagnosis, epidemic investigation but also can be developed quickly upon recognition of a novel virus, which is available before suitable purified viral proteins become available for use in other assays. through our serological survey of ccv in giant panda, the results suggested that it is necessary to study effective vaccines to protect giant pandas confronting common infection. although the value of inactivated ccv vaccine for ccv infections in dogs remains controversial, new vaccines such as dna vaccine and recombined adenovirus vector vaccine may be useful to prevent ccv infection in giant panda. these new vaccines are being experimented in our laboratory. virus infections of carnivores i. amsterdam canine coronavirus infections in japan: virological and epidemiological aspects recovery and characterization of a coronavirus from military dogs with diarrhea proceedings of the annual meeting of the us animal health assoc acute hemorrhagic enteritis associated with canine coronavirus and parvovirus infection in a captive coyote population diagnosis of complex infection of canine distemper virus and canine coronavirus to giant panda by multi-pcr mini report on detection of coronavirus in feces of giant panda intestinal infection of neonatal dogs with canine coronavirus - : studies by virologic, histologic, histochemical, and immunofluorescent techniques canine coronavirus infection in the dog following oronasal inoculation serologic survey of giant panda (ailuropoda melanoleuca), and domestic dogs and cats in the wotong reserve diagnosis of canine coronavirus infection using nested-pcr development of a nested pcr assay for the detection of canine c ronavirus cloning and sequence analysis of spike protein gene of canine coronavirus giant panda's isolate the use of enzyme-linked immunosorbent assay systems for serology and antigen detection of parvovirus, coronavirus and rotavirus interactions in dogs in the netherlands electronmicroscope study of experimental enteric infection in neonatal dogs with a canine coronavirus studies on the epizootiology of canine coronavirus feline aminopeptidase n is a receptor for all group i coronaviruses case report on mixed infection of canine parvovirus and canine coronavirus. electron microscopy and recovery of canine coronavirus key: cord- - p ug authors: mcgeer, allison title: let him who desires peace prepare for war: united states hospitals and severe acute respiratory syndrome preparedness date: - - journal: clin infect dis doi: . / sha: doc_id: cord_uid: p ug nan on march , the world health organization (who) first posted a worldwide alert concerning an outbreak of severe acute respiratory syndrome in vietnam, hong kong, and guandong province, china [ ] . in june , the centers for disease control and prevention (cdc) surveyed members of the infectious disease society of america emerging infections network (ein) about sars preparedness in their hospitals. it is a measure of the rapid globalization of both the outbreak and the outbreak response that, ! months after the outbreak was recognized, % of responding members of the ein reported that their hospital had cared for a patient meeting the case definition of sars and that % had plans in place to address sars [ ] . the major characteristics of the plans were remarkably uniform, given the very short time for their development and the rapidly changing data. it is a credit to who, cdc, and the outbreak management teams in each country that as much information flowed as rapidly as it did. the variability in the plans illustrates both the uncertainties inherent in the data available at the time of the survey and the significant challenges in sars preparedness. the most important and most difficult component of sars preparedness programs is the identification of infected patients. sars is a febrile respiratory illness that is often clinically indistinguishable from other causes of fever and pulmonary infiltrates [ ] [ ] [ ] . identification of cases depends on prompt recognition of epidemiological risk and clustered infections. of the ein members responding to the survey in this issue of clinical infectious diseases [ ] , ( %) reported that patients with respiratory symptoms in their emergency department (ed) would be screened for a travel history. routine screening in the ed is a substantial investment for most hospitals and one that some may judge to be of dubious benefit, given that only laboratory-confirmed cases of sars were diagnosed in the united states [ ] . on the other hand, the hospitals of % of respondents that have not implemented screening are dependent on their admitting physicians to consider sars in the differential diagnosis and to order appropriate precautions. this latter system has been shown to repeatedly fail to identify tuberculosis, another cause of acute respiratory disease [ , ] . sars is much more likely than tuberculosis to be transmitted and to result in disease. in the event of another outbreak of sars, systematic screening of ed patients, at least those who are to be admitted to the hospital, should be part of every plan. some aspects of the plans from june will likely now have changed. for instance, the relatively low percentage of plans ( %) that incorporated follow-up of exposed patients and visitors reflected the focus on health care worker infections early in the outbreak. documentation indicating that exposed visitors and patients who became ill were the major source of transmission should result in the incorporation of prompt patient and visitor follow-up into all plans [ , ] . similarly, at the height of the outbreak, uncertainties about transmission led many institutions to impose quarantine on returning travelers (and led some travelers to self-impose quarantine). however, transmission of sars was almost invariably linked to households and hospitals and did not occur before the onset of symptoms [ ] [ ] [ ] . thus, quarantine of travelers is not a necessary measure. recognizing the power of denial, however, some hospitals may continue to require daily checks of returning workers until the full incubation period has passed. as noted by srinivasan et al. [ ] , the survey also highlights more-general issues in infection control in hospitals. it may still be possible to manage sars safely in the significant minority of hospitals ( %) and eds ( %) that lack airborne isolation rooms. however, it is not possible to manage chickenpox, measles, or tuberculosis without appropriate airborne isolation precautions. because % of the responding hospitals admitted a traveler from an area of sars endemicity despite travel restrictions, it seems unlikely that they can avoid caring for all diseases spread by the airborne route. the issue of whether protection from sars requires airborne precautions will continue to generate controversy. a careful assessment of exposures in sars outbreaks, particularly those due to superspreading events and transmission despite compliance with isolation precautions, is needed to determine whether airborne spread occurs [ , [ ] [ ] [ ] . in addition, continued work on the science of health care worker respiratory protection is clearly needed. national standards vary widely in the developed world. in the united states, the standards of the occupational safety and health association recommend annual fit testing for n respirators [ ] . the results of this survey suggest that compliance with this recommendation is the exception rather than the rule. in canada, the canadian standards association, in the absence of data, has made no recommendation for protection against infectious agents [ ] . in some countries of the european union, fit testing is required before use but is not required annually; in others, fit testing of individuals is not currently recommended [ ] . the issue of how best to assure protection for each ward nurse in the middle of a weekend night is real. so is the fear that the logistical problems associated with always having a supply of fit-tested masks for all health care workers will push in-vestigators to downplay the risk of airborne spread. at least issues of importance in hospital preparedness for sars were not touched on in the survey reported by srinivasan et al. [ ] . disaster preparedness plans allow most facilities to contact all staff rapidly at the beginning of an emergency. outbreaks of disease, however, require plans for regular (sometimes twice daily) information updates for hospital staff, patients, visitors, and related medical service professionals. preparedness for sars also requires clear delineation of the roles and responsibilities of hospitals and government agencies in many areas of outbreak management. for instance, it is essential before outbreaks to determine who will set hospital policy (e.g., restrictions on hospital admissions, requirements for managing infected patients, and decisions as to which hospitals will admit patients with sars), who will establish work restriction policies for exposed health care workers, and who is responsible for follow-up of exposed patients, staff, and visitors. a number of ein members surveyed expressed concerns about health care worker compliance with precautions. at least analyses of risks associated with health care worker infection despite the use of precautions now identify that h of infection-control training and confidence that precautions would be protective are associated with substantial reductions in the risk of infection (toronto sars hospital investigation, unpublished data; lau et al. [ ] ). management personnel at all hospitals should now be asking themselves how confident they are that clinical staff clearly understand infectioncontrol precautions and how they can be sure that, if sars should reemerge, all health care workers have the training necessary to comply with precautions. one challenge for hospitals is to maintain the impetus to integrate the rapidly expanding new knowledge about sars into the best prevention programs. a second is to reassess the management of ex-posure to droplet-spread pathogens in hospitals. the cdc is currently recommending that all hospitals consider offering masks to all coughing patients and using droplet precautions (placing patients for whom such precautions are required in a private room, if possible; masking health care workers within m of such patients or upon room entry; and limiting the movement of such patients outside of their room), in addition to standard precautions, for all patients with symptoms of respiratory infections [ ] . although, as srinivasan et al. [ ] suggest, these isolation precautions may have benefits that extend to situations beyond sars outbreaks, it is also true that isolation may have risks, as recently demonstrated by redelmeier et al. [ ] . as life returns to a "new normal" after sars, we urgently need a better understanding of how to minimize the risk of transmission of viral respiratory diseases without creating adverse events associated with additional infection-control precautions. world health organization. cases of severe respiratory illness may spread to hospital staff. released hospital preparedness for severe acute respiratory syndrome in the united states: views from a national survey of infectious diseases consultants discriminative ability of laboratory parameters in severe acute respiratory syndrome (sars) analysis of deaths during the severe acute respiratory syndrome (sars) epidemic in singapore: challenges in determining a sars diagnosis council of state and territorial epidemiologists. sars investigative team, cdc. revised u.s. surveillance case definition for severe acute respiratory syndrome (sars) and update on sars cases-united states and worldwide environmental infection control of tuberculosis nosocomial transmission of multidrug-resistant mycobacterium tuberculosis sars transmission and hospital containment investigation of a nosocomial outbreak of severe acute respiratory syndrome (sars) in toronto, canada. hospital outbreak investigation team epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong clinical features and short-term outcomes of patients with sars in the greater toronto area possible sars coronavirus transmission during cardiopulmonary resuscitation superspreading sars events cluster of sars in medical students exposed to a single patient institute for occupational safety and health. tb respiratory protection program in health care facilities: administrator's guide. september selection, use and care of respirators. document severe acute respiratory syndrome (sars) sars transmission among hospital workers in hong kong public health guidance for community-level preparedness and response to severe acute respiratory syndrome (sars) safety of patients isolated for infection control key: cord- -s qu odd authors: anderson, evan j; weber, stephen g title: rotavirus infection in adults date: - - journal: lancet infect dis doi: . /s - ( ) - sha: doc_id: cord_uid: s qu odd rotavirus has been recognised for years as the most common cause of infectious gastroenteritis in infants and young children. by contrast, the role of rotavirus as a pathogen in adults has long been underappreciated. spread by faecal-oral transmission, rotavirus infection in adults typically manifests with nausea, malaise, headache, abdominal cramping, diarrhoea, and fever. infection can also be symptomless. rotavirus infection in immuno-compromised adults can have a variable course from symptomless to severe and sustained infection. common epidemiological settings for rotavirus infection among adults include endemic disease, epidemic outbreak, travel-related infection, and disease resulting from child-to-adult transmission. limited diagnostic and therapeutic alternatives are available for adults with suspected rotavirus infection. because symptoms are generally self-limiting, supportive care is the rule. clinicians caring for adults with gastroenteritis should consider rotavirus in the differential diagnosis. in this review we intend to familiarise clinicians who primarily provide care for adult patients with the salient features of rotavirus pathophysiology, clinical presentation, epidemiology, treatment, and prevention. infective gastroenteritis causes substantial morbidity and mortality worldwide. although various bacterial species have long been associated with gastrointestinal disease, specific viral causes of these infections were not delineated until the early s. however, with the discovery of norwalk virus in and rotavirus in , the causative agents for most non-bacterial gastroenteritis infections were identified. almost immediately, the spectrum of viruses causing gastrointestinal infection in adults was recognised as differing from that in children. among children younger than years, nearly half of all cases of diarrhoea requiring admission to hospital can be attributed to rotavirus infection. by contrast, among adults most non-bacterial outbreaks of gastroenteritis can be linked to the norwalk-like viruses. the important part played by viral pathogens besides the norwalk-like viruses in adults with gastroenteritis is not yet fully appreciated. specifically, the contribution of pathogens that typically affect children is not recognised by most clinicians who take care of adults. such is the case for adult infections caused by the common paediatric pathogen rotavirus. here we review important features of rotavirus microbiology and pathophysiology, along with relevant clinical and epidemiological features of rotavirus infection. in , bishop and colleagues described unique viral particles obtained from the duodenal mucosa of children with gastroenteritis. viruses with similar morphological appearance had been seen in in the intestinal tissue of mice with diarrhoea. under the electron microscope, the nm diameter viral particles first described in these reports had a wheel-like appearance, prompting the name rotavirus, from the latin rota (figure). rotavirus is a non-enveloped virus now classified within the reoviridae family. segments of doublestranded rna reside within the core. the rna encodes six viral proteins (vp) that make up the viral capsid, and six non-structural proteins (nsp). the core is surrounded by an inner capsid, composed mostly of vp , the primary group antigen, , and includes the epitope detected by most common diagnostic assays. other structural proteins also seem to confer some degree of group specificity. the outer capsid is primarily composed of vp and vp . vp contributes the spoke-like projections to the wheel-shaped appearance of rotavirus. this vp is cleaved by trypsin in vitro to yield vp * and vp *, which appear to play an important part in cellular attachment. the inner and outer capsids give the viral particle the double-layered icosahedral structure visualised on negative-stain electron microscopy. seven distinct groups of rotavirus (named a to g) have been shown to infect various animal species. of these, only groups a, b, and c have been reported as human pathogens. group a is the primary pathogen worldwide and is the group detected by commercially available assays. additional subgroups and serotypes can be identified by further characterisation of vp , vp , and vp antigens. group b seems to be limited to causing epidemic infection in asia and the indian subcontinent, whereas group c rotavirus causes endemic infections that frequently go unrecognised. rotavirus spreads from person to person, mainly by faecaloral transmission. although rotavirus has been detected in urine and upper-respiratory samples, , these body fluids are not believed to be commonly associated with transmission. after ingestion, rotavirus particles are carried to the small intestine where they enter mature enterocytes through either direct entry or calciumdependent endocytosis. after cytolytic replication in the mature enterocytes of the small intestine, new rotavirus particles can infect distal portions of the small intestine or be excreted in the faeces. more than - viral particles per gram of faeces are excreted by children during infection. , the amount of rotavirus excreted by adults might be more variable. in at least one study shedding was - -fold lower in travellers' diarrhoea. symptom-free adults can shed rotavirus in quantities so low as to be undetectable by most routine assays. the mechanism by which rotavirus induces diarrhoea is poorly understood. few investigations have incorporated the study of human mucosal samples. the reports that are available describe various findings: villous shortening, flattening, and atrophy, denudation of microvilli, mitochondrial swelling, distension of the endoplasmic reticulum, depressed disaccharidase concentrations, and infiltration of mononuclear cells. , , additional hypotheses about the pathophysiology of rotavirus gastroenteritis have been generated from animal studies. in one review the diminished ability of the intestinal epithelium to absorb fluid and nutrients, stimulation of the enteric nervous system, and local villous ischaemia and shortening resulting in impaired nutrient absorption were noted. a murine model of rotavirus infection suggests that rotavirus nsp acts as an enterotoxin, potentially by increasing calcium-dependent signalling of chloride secretion. , the diarrhoea induced by rotavirus is unlikely to be completely explained by any one process, rather that several mechanisms contribute simultaneously. these mechanisms are summarised in panel . , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] resolution of rotavirus gastroenteritis depends greatly on the immunological response of the host. in a normal host, rotaviral antigens are transported to peyer's patches, undergo processing by b cells, macrophages, or dendritic cells and are presented to helper t cells. this cascade culminates in stimulation of rotavirus-specific b cell and cytotoxic t-lymphocyte-precursor expansion. bernstein and colleagues noted that stool rotavirus iga concentrations peaked - days after infection and persisted for longer than year, but at declining concentrations. the researchers suggested that serum rotavirus iga is a more consistent marker of rotavirus immunity than other antibody measurements. however, rotavirus-specific iga is frequently undetectable in duodenal fluid or faeces in the first week of infection, although symptoms might resolve within that time. this pattern suggests a mechanism independent of humoral immunity. offit notes that infected mature villous epithelial cells are steadily replaced by less-mature enterocytes, which may be less susceptible to rotavirus invasion. increased peristalsis improves clearance of viral particles and the non-specific activity of interferons can prevent vp translation. de bouissieu has reported that interferon ␣ concentrations correlate with a trend towards shorter duration of diarrhoea among patients who have rotavirus infection. although many physicians presume that rotavirus infection will confer lifelong immunity, multiple investigations show that re-infection can occur. bishop and colleagues noted that infection with rotavirus during the neonatal period did not protect against developing rotavirus infection during the first years of life but did lessen the severity of such infections. in a prospective study of mexican infants followed up from birth, valazquez and colleagues noted that by age years % of infants had experienced a primary rotavirus infection. during the same period, nearly % of the infants experienced a second infection. more than % of the children studied had five or more rotavirus infections during the first years of life. yolken and colleagues had previously noted that by age reduced absorptive surface denudation of microvilli; shortening, flattening, and atrophy of villi; invasion of villi by rotavirus causing ischaemia and shortening , , functionally impaired absorption depressed disaccharidase concentrations; impaired co-transport of glucose and sodium; decreased sodium-potassium atpase activity impairing electrochemical gradient , , , [ ] [ ] [ ] cellular damage impairing absorption mitochondrial swelling; distension of endoplasmic reticulum; mononuclear cell infiltration , , , enterotoxigenic effects of rotavirus protein nsp induces increased intracellular calcium concentrations; in murine models, acts like a toxin to induce diarrhoea , stimulation of enteric nervous system stimulation of intestinal secretion of fluid and electrolytes; stimulation of intestinal motility resulting in decreased intestinal transit time , altered epithelial permeability increased paracellular permeability by weakening tight junctions between cells [ ] [ ] [ ] years, more than % of children had antibodies to two different rotavirus serotypes. therefore, although nearly all adults have antibodies to rotavirus, they might still be susceptible to infection. rotavirus can elude host defences and induce repeat infection through several mechanisms. there are multiple groups, subgroups, and serotypes of rotavirus. initial antibody response to infection is serotype specific, with limited production of cross-reactive antibodies. subsequent rotavirus infections increase antibodies that cross-react with multiple serotypes. additionally, certain elements of the rotavirus-specific immune response are short-lived. rotavirus-specific secretory iga is sometimes not detectable in faeces as early as year after infection. elias reported that rotavirus fluorescent antibody titres peaked in children at age - years but subsequently fell to almost undetectable concentrations in individuals older than years. in a review of multiple studies, jiang and colleagues an appreciation of the typical presentation of rotavirus infection in children is critical to understanding the spectrum of disease among adults. primary infection with rotavirus typically occurs in infants between ages months and years, although infection in neonatal intensive-care units and severe infection in infants younger than months are well documented. , in all age-groups, the classic presentation of rotaviral infection is fever and vomiting for - days, followed by non-bloody diarrhoea. the diarrhoea may be profuse, and - bowel movements per day are common. when examined, the stool from affected patients is generally devoid of faecal leucocytes. especially when associated with vomiting, the diarrhoea of rotavirus infection can precipitate severe and even life-threatening dehydration. infants with repeat rotavirus infections are generally less-severely affected than those with primary disease. among adults, rotavirus infection has been associated with a wide spectrum of disease severity and manifestations. as such, it is difficult to provide a concise description of a typical clinical presentation. nevertheless, prospective studies of voluntary rotavirus ingestion have provided some insight, although the participants in these studies were primarily young healthy adults. data from several such trials are summarised in the table. , [ ] [ ] [ ] for these volunteers, illness most frequently began - days after ingestion and continued for - days. about two-thirds of study participants had an antibody response, with more than half of all participants eventually shedding rotavirus. symptoms were less common than evidence of infection, but most frequently included diarrhoea, fever, headache, malaise, nausea, or cramping. one participant passed stools in day. among the volunteers, rotavirus particles were detectable in the stool from the start of symptom onset and persisted for more than days in some. three studies document rotavirus readministration to volunteers, noting that symptoms and antibody response were much less common. , , in several reports of rotavirus outbreaks among adults similar symptoms have been described. in a prospective study of families with neonates followed up from shortly after birth, only of adults who had serological evidence of rotavirus infection were symptomless. had diarrhoea and had abdominal cramping. none had symptoms that necessitated medical care or absence from work. in a separate study, parents of children with rotavirus gastroenteritis developed serological evidence of infection, but only three had diarrhoea. grimwood and colleagues found, in a study of children with rotavirus in families, that of adult family members exposed to rotavirus developed evidence of infection, and all but four were symptomatic. in a study of college students during a rotavirus outbreak, of the individuals who met the criteria for rotavirus infection, % had diarrhoea, % abdominal pain or discomfort, % loss of appetite, % nausea, and more than % had fatigue, vomiting, headache, chills, subjective or low-grade fever, or myalgia. patients with underlying immunodeficiency are at risk of sustained symptoms and rotavirus dissemination, a phenomenon already recognised among children. this pattern was first described in when two of four children with underlying primary immunodeficiency who had rotavirus infection developed chronic diarrhoea that at least temporarily responded to administration of human milk containing a high titre of rotavirus antibodies. a geriatric patient with impaired naturalkiller-cell activity and impaired cellular and humoral immunity had rotavirus shedding for at least days. gilger and colleagues noted that in four children who had various immune deficits and chronic diarrhoea from rotavirus, rotavirus was identified in the liver and kidney. whether the involvement of liver or kidney was important is unclear. natural infection quantity of cross-reactive neutralising antibody responses against multiple serotypes, - of virus-specific secretory iga at intestinal mucosa surface, , and of virus-specific iga and igg in serum , challenge studies serum serotype specific neutralising antibody; , intestinal neutralising antibody; , serum rotavirus igg, but not necessarily with large doses; [ ] [ ] [ ] and raised prechallenge titres of antibody to specific epitope of rotavirus vp vaccine-induced serum antibodies might be important in protection from natural infection, but interpretation is complicated because of differences in methods of existing studies; and heterotypic antibody response to rotavirus vaccination might be protective against usual infecting serotypes , other investigators have assessed the course of rotavirus infection in patients with malignant disease. a wide spectrum of clinical manifestations and severity of illness have been reported. bolivar screened adults who had various solid tumours and leukaemia, with and without diarrhoea. he noted that two patients had rotavirus infection, both of whom had undergone bone-marrow transplantation and had developed graft-versus-host disease. diarrhoea lasted for days before resolution. in three subsequent studies in bone-marrow-transplant patients results varied. yolken and colleagues prospectively assessed patients undergoing bone-marrow transplantation for infectious gastroenteritis and found that rotavirus occurred less frequently than clostridium difficile or adenovirus, but still occurred in nine of patients. in one patient, adenovirus occurred concomitantly with rotavirus infection. of the eight remaining patients, all developed vomiting, seven had abdominal cramps, six had respiratory illness (infiltrates on chest radiography with appropriate clinical signs and symptoms) and four had diarrhoea. five of the eight rotavirus-infected patients eventually died. troussard and colleagues also prospectively assessed patients undergoing bone-marrow transplantation and noted rotavirus infection in eight of patients. adenovirus occurred concomitantly in two patients. seven of the patients had isolates positive for rotavirus in the winter months, with acute onset, vomiting, and diarrhoea. in a later study of the same topic, rotavirus was noted in four of adult asymptomatic stem-cell-transplant patients followed up prospectively. no patient with gastroenteritis had rotavirus isolated. rotavirus infections in adult patients infected with hiv- frequently present as a chronic diarrhoea with sustained viral shedding in stools. albrecht and colleagues, between and , detailed a retrospective assessment of samples from patients infected with hiv- who had otherwise unexplained diarrhoea. samples from patients without diarrhoea served as controls. samples from nine case patients were positive for rotavirus; two of these samples were rotavirus recurrences months after the initial episode. no symptom-free patients had rotavirus infection. rotavirus was associated with diarrhoea of - weeks' duration in all patients and with abdominal cramping in eight patients. thomas and colleagues looked prospectively at samples from uk hiv- -positive patients with diarrhoea. rotavirus was third in frequency to adenovirus and coronavirus, occurring in ( · %) samples. the median cd count of patients with rotavirus infection was nine. hrdy described five typical settings for rotavirus infections in adults. we propose modification of these classifications to the following: endemic disease, epidemic outbreaks, travelrelated gastroenteritis, and infections transmitted from children to adults. although substantial overlap exists between the groups, our classifications clarify separate risk factors and clinical features. rotavirus infection in children is seasonal, with peak incidence in winter months in temperate climates. iturriza-gomara and colleagues noted that, in the uk between and , notable numbers of infections began in december or january, peaking in march or april and falling to almost zero by july. in the usa, kapikian and colleagues found that rotavirus cause % of diarrhoea cases necessitating admission to hospital in children between november and april, but could not be linked to cases from may to october. in several studies findings suggest that adult disease is not as season-specific as childhood disease. cox ffu=focus forming unit. nr=data not recorded in original paper and taken as not having occurred in calculation of summary percentages. *data included when > % infectious dose ingested (> ffu). † of patients had mild illness (including one patient with no antibody response or shedding). ‡one of four volunteers experienced illness but no specific symptoms were recorded. §summary percentages after rotavirus ingestion calculated only from data from studies in which full clinical syndrome of illness described. all percentages have been rounded to the nearest whole percentage point. adult serum in routine hospital samples are present throughout the year. igm concentrations increased with older age, and antibodies reached % in march and fell to - % during the summer months. cox and medley attributed the high rates to igm persistence, igm crossreactivity, or possibly to non-seasonal high infection rates in adults. other researchers have also found that rates of adult disease do not mirror the winter seasonality of infection in paediatric patients. these studies suggest that endemic disease in adults may not arise solely from unrecognised transmission of rotavirus from children to adults. the contribution of rotavirus as a cause of endemic gastrointestinal disease varies according to geographic distribution and characteristics of patients. in a small prospective study in the uk, rotavirus caused · % of acute diarrhoea in adults admitted to hospital. similarly, % of acute diarrhoea in switzerland, % of infectious diarrhoea pathogens in a swedish clinic for infectious diseases, % of adults with gastroenteritis requiring admission in thailand, - % of adults older than years with gastroenteritis presenting to their family physician in the netherlands, and nearly % of individuals older than years in michigan were due to rotavirus. in studies in other geographic areas even higher rates of infection have been seen. in japan, nakajima and colleagues reported that group a rotavirus had a role in % of patients with diarrhoea. pryor and colleagues noted that rotavirus was second only to campylobacter spp as a cause of diarrhoea among australian adults, accounting for % of all cases. in indonesia, % of patients presenting with diarrhoea had rotavirus-positive stools compared with % of control samples. in a study of mexican adults, % of patients presenting with acute gastroenteritis during winter months were positive for rotavirus. even these results might underestimate the true prevalence of endemic rotavirus infection. group c rotavirus is not routinely detected by commercial assays but it does contribute to endemic rotavirus infection worldwide. in a study in the uk, % of patients were seropositive for group c rotavirus. among adults, clusters of rotavirus infections most frequently occur in communities that are otherwise sheltered from more routine exposure to rotavirus-infected children. one of the largest outbreaks involved nearly people in , in an isolated area of micronesia. since then, other outbreaks have occurred among closed communities, including a finnish military base, an israeli kibbutz, and an isolated south american indian community. outbreaks of rotavirus infection have also occurred in long-term health-care facilities, particularly those with close living quarters; compromised host immunity and multiple comorbid disorders might help facilitate the spread of infection. - cubitt and colleagues described an epidemic of rotavirus among staff and patients in an extended-stay geriatric hospital, in which of residents developed symptoms and seven had confirmed rotavirus infection. halvorsrud and orstavik described an outbreak of cases of acute gastroenteritis among nursing-home patients with identification of rotavirus by comparing acute and convalescent antibody titres. rotavirus has been suggested as the causal pathogen in % of diarrhoea outbreaks in a study of institutions caring for elderly residents. among adults, rotavirus outbreaks are not confined to geriatric populations. group a rotavirus was associated with an outbreak of gastroenteritis among college students in the district of colombia. rotavirus also caused a waterborne outbreak of gastroenteritis in in eagle-vail and avon, co, usa in which severity of symptoms correlated with the amount of tap water consumed. finally, griffin and colleagues screened outbreaks of gastroenteritis in the usa between and and found that rotavirus was implicated in three outbreaks. uniquely affecting asia, group b rotavirus has been associated with outbreaks affecting large numbers of adults in broad geographic distributions of china and india. , rotavirus has been implicated as an important contributor to travellers' diarrhoea among adults, especially among those visiting central america and the caribbean. in a study of travellers returning from jamaica, rotavirus was identified in % of individuals with diarrhoea, making the virus second only to enterotoxigenic escherichia coli as a cause. in two studies of us students travelling in mexico, electron microscopy identified rotavirus in about % of patients who had diarrhoea, compared with % and %, respectively, of symptom-free patients. , in a third study, a substantial rise of antibodies to rotavirus was seen in % of two student groups travelling to mexico. by contrast, only - % seroconverted to norwalk virus. ryder and colleagues found rotavirus in % of panamanian travellers to mexico who had diarrhoea. sheridan and colleagues similarly found that % of us peace corps volunteers and % of panamanian travellers visiting mexico had at least a four-fold increase in rotavirus antibody titres. adult travellers with rotavirus shed - times less rotavirus than do paediatric patients. , although rotavirus can be linked to adult gastroenteritis in each of the other settings, adults who are in contact with children are at particularly high risk of infection. transmission of rotavirus within families from children to parents seems to be a common event. wenman and colleagues showed prospectively that rotavirus infection occurred in of adults caring for children with rotavirus infection. by contrast, only four of adults whose children had no documented rotavirus infection became infected. grimwood and colleagues confirmed this finding in a report that a third of adult family members in new zealand developed evidence of rotavirus infection. the same phenomenon has been seen among parents of more severely ill children. kim and colleagues found evidence of rotavirus infection in % of adult contacts of children who were admitted to hospital with rotavirus, compared with % of adult contacts whose children were not infected. more casual contact might also be sufficient to facilitate rotavirus transmission from children to adults. rodriguez and colleagues reported that nine of adults experienced illness after exposure to children infected with rotavirus in a playgroup. although substantial evidence is lacking, child-to-adult transmission of rotavirus is accepted to occur with some frequency on paediatric wards. many paediatric nurses, medical students, and house officers experience symptoms of gastroenteritis during the winter months when most paediatric rotavirus infections are encountered. von bonsdorff and colleagues described paediatric nurses at several different locations with acute gastroenteritis caused by rotavirus. among seven hospital staff that developed diarrhoea after direct contact with children with diarrhoea staying in hospital, a rise in antibody titres was detected in three. interestingly, in the same study, six of medical students reported gastroenteritis. three of the students had rotavirus particles present on electron microscopy and were noted to be more ill than the parents of the children who were infected. all had diarrhoea for - days and two of the three had low-grade fever and vomiting. another case report supports transmission of rotavirus from children to hospital caretakers. electron microscopy, which permits visualisation of the pathognomonic wheel-like appearance, was initially used for diagnostic purposes, but elisa or eia have become more commonly used. commercial assays are reliable, convenient, and inexpensive, but require at least - virions to generate a positive result. , the false-positive rate of commercial assays is - %. one of the biggest limitations of most commercial assays is that they do not detect non-group-a rotavirus. , other more sensitive and newer methods are being used in research. one such method is pcr, which is up to times more sensitive than immunoassays. in one study using pcr, % of otherwise healthy children shed virus for - days after symptoms developed. although stool cultures are routinely tested for bacterial pathogens, the low frequency of detecting a positive result calls the usefulness of this practice into question. rotavirus infection can occur in a similar number of patients to some bacterial pathogens. sending a sample of rotavirus antigen for testing by elisa or eia could potentially cut costs if by doing so either hospital stay or procedures could be avoided. such a cost-benefit analysis in adult patients has not been published. one limitation is that adults might shed less rotavirus in faeces than do children, further hampering diagnosis. we suggest that obtaining rotavirus antigen testing for patients admitted to hospital with risk factors for rotavirus infection will be cost effective if additional inpatient studies can be avoided. determination of rotavirus infection may also be beneficial if infectious patients can be isolated to prevent nosocomial spread. a positive rotavirus antigen test might also allow physicians to avoid prescribing antibiotics for travel-related rotavirus infections. treatment of rotavirus infections is primarily directed at symptom relief and restoration of normal physiological function. oral rehydration should be attempted initially. in most developing countries, oral rehydration salt solutions are used extensively in children. most adults can be managed by encouraging them to drink fluids. an additional intervention that has been used is administration of lactobacillus spp bacteria to shorten the duration of diarrhoea. , although seldom used in children, codeine, loperamide, and diphenoxylate can help with symptom relief and control of the volume of diarrhoea. bismuth salicylate, in a placebo-controlled double-blinded trial, was efficacious in treating the symptoms of rotavirus diarrhoea. trial use of bismuth salicylate can be considered in adults when other coexistent infectious causes have been ruled out. if symptoms cannot be controlled and the patient becomes dehydrated, administration of intravenous fluids and hospital admission might be necessary. rarely, extraordinary measures have been attempted to help resolve rotavirus infections. for example, human breastmilk has been provided to immunodeficient infected children to help resolve chronic diarrhoea. this option, however, is not practical in adults. several groups report oral administration of human serum immunoglobulins possessing antirotavirus activity to bind free rotavirus antigen. guarino and colleagues noted a mean duration of diarrhoea of h in children who received one oral dose of mg/kg human serum immunoglobulin, compared with h in children who did not. in a study involving three immunocompromised children with chronic rotavirus diarrhoea, oral administration of human serum immunoglobulins (igg mg/kg) cleared rotavirus antigen in all three, but rotavirus antigen recurred in two. among adults, oral immunoglobulin administration of - g daily for days to bone-marrow-transplant recipients has been successful. prevention of rotavirus infection can be facilitated by avoiding exposures and faecal-oral spread. contact with sick children and potentially contaminated food and water should be avoided. since % of rotavirus virions placed on human fingers survive for min, thorough hand washing is critical in prevention. contact isolation for patients diagnosed with rotavirus infection is necessary, generally for the duration of hospital stay, because of sustained faecal shedding of low concentrations of virus. gloves, gowns, isolation, and rigorous hand washing should be used in the care of individuals infected with rotavirus. sattar and colleagues reported that rotavirus survives best in low humidity on non-porous surfaces at room temperature or cooler. phenolic disinfectants do not inactivate rotavirus; instead hypochlorite or sodium dichloroisocyanurate tablets with a free chlorine concentration of at least parts per million are recommended. a % ethanol solution is also effective in inactivation of rotavirus and can help to prevent environmental spread. rotavirus infection in adults has been successfully prevented by use of a commercially available review disinfectant spray on rotavirus contaminated fomites under experimental conditions. given the substantial disease-related morbidity and mortality associated with rotavirus, the development of an effective vaccine is a priority. although multiple vaccines were under development, the tetravalent rhesus-human reassortant rotavirus vaccine (rrv-tv) seemed to produce the best results. the rrv-tv vaccine prevented about half of rotavirus infections, but was much more effective in preventing severe disease. , shortly after the vaccine was approved, the vaccine adverse-event monitoring system noted by mid- an excess of cases of intussusception among recently vaccinated infants, eventually prompting the vaccine to be withdrawn. two sharply differing perspectives on the risk and benefits of the rrv-tv vaccine and the advisory committee on immunization practices' confirmation of its decision to withdraw its recommendation for the vaccine have been put forward. , other vaccines are under development. vaccines have been primarily developed to attempt to decrease the severity of rotavirus infections in children. although vaccines seem to be fairly safe in adults from the vaccine trials, we are unaware of any plan to consider vaccination in adult patients. vaccination could theoretically be used in adult patients considering travel to central america or the caribbean or among immunocompromised patients to prevent or lessen the severity of rotavirus diarrhoea. despite recognition as an important cause of gastroenteritis in children, rotavirus's role in adult gastroenteritis is underappreciated. immunity to rotavirus is incomplete and most people have multiple infections over their lifetime. adults with rotavirus can be asymptomatic, but the most common symptoms are nausea, malaise, headache, abdominal cramping, diarrhoea, and fever. adults at particular risk of rotavirus infection are travellers, adults exposed to infected children, and immunocompromised people. group a rotavirus, the most common human pathogen, can be diagnosed with many different commercial assays, but all have limited sensitivity. rotavirus testing might be beneficial in certain clinical settings if detecting rotavirus would change management of patients or prevent nosocomial spread. treatment is primarily symptomatic. rotavirus should be considered in the differential diagnosis of adult infectious gastroenteritis. we have no conflicts of interest. we identified sources for this review by searches of medline with use of the key words "rotavirus infection" and "adult." the search strategy and selection criteria included all english and human studies from the year until the present. we further reviewed the abstracts for relevance before inclusion. review of the references of the papers retrieved by the initial search allowed for additional studies to be identified and considered for inclusion. visualization by immune electron microscopy of a -nm particle associated with acute infectious nonbacterial gastroenteritis virus particles in epithelial cells of duodenal mucosa from children with acute non-bacterial gastroenteritis viral gastroenteritis molecular epidemiology of "norwalk-like viruses" in outbreaks of gastroenteritis in the united states epizootic diarrhea of infant mice: identification of the etiologic agent relationship between viruses from acute gastroenteritis of children and newborn calves rotavirus infections: guidelines for treatment and prevention rotaviruses and their replication fields' virology nonenteric sources of rotavirus in acute diarrhea rotavirus infection of the oropharynx and respiratory tract in young children infantile enteritis viruses: morphogenesis and morphology pathogenesis of rotavirus diarrhea rotaviruses of man and animals human rotavirus in an adult population with travelers' diarrhea and its relationship to the location of food consumption excretion of serotype g rotavirus strains by asymptomatic staff: a possible source of nosocomial infection duodenal mucosal damage in infants with gastroenteritis structural and functional abnormalities of the small intestine in infants and young children with rotavirus enteritis role of the enteric nervous system in the fluid and electrolyte secretion of rotavirus diarrhea age-dependent diarrhea induced by a rotaviral nonstructural glycoprotein diarrhea induction by rotavirus nsp in the homologous mouse model system rotavirus infection impairs intestinal brush-border membrane na(+)-solute cotransport activities in young rabbits d-glucose transport in piglet jejunal brush-border membranes: insights from a disease model absence of a campmediated antiabsorptive effect in an undifferentiated jejunal epithelium rotavirus alters paracellular permeability and energy metabolism in caco- cells intestinal permeability assessed with polyethylene glycols in children with diarrhea due to rotavirus and common bacterial pathogens in a developing community rotavirus-induced structural and functional alterations in tight junctions of polarized intestinal caco- cell monolayers host factors associated with protection against rotavirus disease: the skies are clearing induction and persistence of local rotavirus antibodies in relation to serum antibodies rotavirus induces alpha-interferon release in children with gastroenteritis clinical immunity after neonatal rotavirus infection: a prospective longitudinal study in young children rotavirus infections in infants as protection against subsequent infections epidemiology of human rotavirus types and as studied by enzyme-linked immunosorbent assay oral administration of human rotavirus to 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rechallenged with a human rotavirus human rotavirus studies in volunteers: determination of infectious dose and serological response to infection identification of vp epitopes associated with protection against human rotavirus illness or shedding in volunteers homotypic and heterotypic epitope-specific antibody responses in adult and infant rotavirus vaccinees: implications for vaccine development clinical manifestations of rotavirus infection in the neonatal intensive care unit rotavirus gastroenteritis in infants aged - months in melbourne, australia: implications for vaccination comparison of serum and mucosal antibody responses following severe acute rotavirus gastroenteritis in young children orbivirus acute gastroenteritis of infancy rotavirus infection in adults. results of a prospective family study human reovirus-like agent as the major pathogen associated with "winter" gastroenteritis in hospitalized infants and young children spread of rotavirus within families: a community 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evidence for symptomatic and asymptomatic reinfections an outbreak of rotavirus diarrhea among a nonimmune, isolated south american indian community rotavirus infection in a geriatric population an outbreak of rotavirus infection in a long-stay ward of a geriatric hospital epidemic of viral gastroenteritis in an elderly community an outbreak of rotavirus infection in a geriatric hospital outbreaks of astrovirus type and rotavirus gastroenteritis in a geriatric inpatient population an epidemic of rotavirusassociated gastroenteritis in a nursing home for the elderly outbreaks of infectious intestinal disease in residential institutions in england and wales - a community waterborne gastroenteritis outbreak: evidence for rotavirus as the agent outbreaks of adult gastroenteritis traced to a single genotype of rotavirus investigation of an outbreak of adult diarrhea rotavirus in china emergence of adult diarrhoea rotavirus in calcutta, india epidemiology, etiology, and impact of traveler's diarrhea in jamaica rotavirus in travelers' diarrhea: study of an adult student population in mexico human rotavirus in an adult population with travelers' diarrhea and its relationship to the location of food consumption norwalk virus and rotavirus in travellers' diarrhoea in mexico travelers' diarrhea in panamanian tourists in mexico traveler's diarrhea associated with rotavirus infection: analysis of virus-specific immunoglobulin classes human reovirus-like agent infection: occurrence in adult contacts of pediatric patients with gastroenteritis common exposure outbreak of gastroenteritis due to type rotavirus with high secondary attack rate within families rotavirus associated with acute gastroenteritis in adults rotavirus-associated gastroenteritis in two adults probably caused by virus reinfection viral infections of the gastrointestinal tract improved detection of rotavirus shedding by polymerase chain reaction control of outbreaks of viral diarrhoea in hospitals: a practical approach extended excretion of rotavirus after severe diarrhoea in young children oral bacterial therapy reduces the duration of symptoms and of viral excretion in children with mild diarrhea bacteriotherapy with lactobacillus reuteri in rotavirus gastroenteritis rotavirus infections: guidelines for treatment and prevention bismuth subsalicylate in the treatment of acute diarrhea in children: a clinical study oral immunoglobulins for treatment of acute rotaviral gastroenteritis oral administration of human serum immunoglobulin in immunodeficient patients with viral gastroenteritis: a pharmacokinetic and functional analysis severe rotavirus-associated diarrhoea following bone marrow transplantation: treatment with oral immunoglobulin rotavirus survival on human hands and transfer of infectious virus to animate and nonporous inanimate surfaces red book: report of the committee on infectious diseases infection control for hospitalized children institutional outbreaks of rotavirus diarrhoea: potential role of fomites and environmental surfaces as vehicles for virus transmission prevention of surface-to-human transmission of rotaviruses by treatment with disinfectant spray randomised placebo-controlled trial of rhesushuman reassortant rotavirus vaccine for prevention of severe rotavirus gastroenteritis efficacy of the rhesus rotavirus-based quadrivalent vaccine in infants and young children in venezuela reappraisal of the association of intussusception with the licensed live rotavirus vaccine challenges initial conclusions the first rotavirus vaccine and intussusception: epidemiological studies and policy decisions for personal use. only reproduce with permission from the lancet. a -year-old woman presented with a -year history of persistent ulcerous nasal injury that had progressed slowly and without fever, respiratory, or any general symptoms, until the upper lip was affected. a painless ulcero-vegetating injury was seen but the patient was otherwise normal; chest radiographs did not show abnormalities. further examination showed soft tissue attenuation and an ulcered mass lesion in the nasal cavity (figure). the definitive diagnosis was made by isolating mycobacterium tuberculosis from tissue removed during biopsy and antituberculous therapy was started. the lesion had resolved at completion of treatment.tuberculosis of head and neck occurs infrequently and involvement of the nose is rare. granulomatous lesions within the nasal cavity may represent either local disease or a manifestation of a systemic disorder and the differential diagnosis must include tuberculosis. although almost forgotten in industrialised countries, this unusual form of tuberculosis can appear mainly in females and the elderly. it is not thought to be contagious, or to produce noticeable symptoms or physical signs. key: cord- - dlh wg authors: vernet, guy title: molecular diagnostics in virology date: - - journal: j clin virol doi: . /j.jcv. . . sha: doc_id: cord_uid: dlh wg molecular biology has significantly improved diagnosis in the field of clinical virology. virus discovery and rapid implementation of diagnostic tests for newly discovered viruses has strongly beneficiated from the development of molecular techniques. viral load and antiviral resistance or subtyping assays are now part of the biological monitoring of patients chronically infected by human immunodeficiency virus (hiv), hepatitis b virus (hbv), hepatitis c virus (hcv) and cmv. it will be important to add to this panel assays for other viruses of the herpesviridae family. qualitative assays for the detection of blood-borne viruses have increased safety of blood donation and organ transplantation. screening of other blood-borne viruses (parvovirus b , hav), multiplexing of detection and test automation to improve practicability and reduce costs will be the next steps. a major evolution in the near future will be the generalization of nat for the diagnosis of viral etiology in patients, mostly with respiratory, cns or gastro-intestinal diseases. major technical improvements have been made to avoid obstacles that still limit this generalization, i.e. genetic variability of viruses, multiplex detection, contamination risk. commercial offers already exist but menus must be extended to limit the validation and documentation work associated with home-brew assays. real-time amplification has allowed the development of new nat platforms but automation and integration of all steps of the reaction are still required to reduce hands-on-time, time-to-result and costs, and to increase throughput. molecular biology has revolutionized all domains of viruses diagnosis including the rapid identification of emerging or re-emerging viruses, viral safety of blood products or organ transplants and viral disease management. one of the major driving forces for the introduction of molecular techniques in virology has been the absence of easy and performing multiplication techniques similar to those developed for bacteriology. the most striking illustration of the power of molecular techniques concerns blood transmitted viruses-human immunodeficiency virus (hiv), hepatitis b virus (hbv) and hepatitis c virus (hcv) for which spectacular progresses in the detection and treatment of viral diseases have been made following the introduction of qualitative and quantitative nucleic acid tests (nat). the recent discovery of a new human coronavirus responsible for severe acute respiratory syndrome (sars) epidemic is another example. it is obvious that nat will encourage the development of antiviral drugs which, compared to antibiotics, has been delayed partly because performing efficiency assessment techniques were lacking. during the last years, many new human pathogens have been discovered among which eight were viruses with various pathogenicity. molecular techniques have played a central role in their discovery (table ) . the construction of cdna libraries by cloning techniques has been used to identify hcv and hepatitis e virus. representational difference analysis (rda) was successful in identifying human herpes virus (hhv- ), and the hepatic viruses gbv-c and ttv. rda allows the detection of viral sequences by comparing whole nucleic acids sequences in cells from humans or animals before and after infection. reverse-trancription polymerase chain reaction (rt-pcr) with random or degenerate primers has been used to identify human metapneumovirus (hmpv), the virus sen and the coronavirus associated to sars (sars-cov). molecular techniques alone have allowed the characterization of very important human pathogens like hhv- , responsible of kaposi sarcoma or hcv which induce acute or chronic hepatitis, cirrhosis and hepatocarcinoma. however, other viruses detected in a similar way are still waiting for the demonstration of their clinical importance. this illustrates the need to verify koch's postulate and the importance of keeping laboratory competencies for classical virology-tissue culture, electron microscopy and animal experiments-which plays a major role in virus discovery, together with epidemiological studies. large epidemiological studies are also required to assess the clinical interest of new pathogens. molecular diagnostics have been very rapidly implemented in clinical virology laboratories following the discovery of hmpv (van den hoogen et al., ; peiris et al., ; boivin et al., ) and sars-cov (ksiazek et al., ; drosten et al., ; anderson, ) . a prospective study on the prevalence of hmpv could be initiated as early as during the - winter, although the virus has been discovered in only. there has been only a few weeks lag between sequencing sars-cov and availability of the first commercial nat. nat are more and more used to exclude blood donations from patients infected by viruses (allain, ) . hcv and hiv testing has been implemented as part of routine screening in blood banks in and european countries respectively. in france, two commercial offers -procleix (gen-probe, inc. usa) and nuclisens extractor (biomerieux, france) associated with cobas ampliscreen (roche diagnostics, switzerland)-are used to screen donations for hiv and hcv infections. this allowed the reduction of residual risk from in , to in . millions for hiv and in , to in millions for hcv (assal et al., ) . however, there is room from improvement as few contaminated blood units are still missed due to the lack of sensitivity induced by pooling strategies. cost constraints must also be considered if further improvements are to be considered. cost effectiveness of hiv and hcv nat addition to serology testing is already very low: in usa it has been calculated that the cost of each saved life is . - . millions us$ per year (jackson et al., ) . hepatitis b virus screening using molecular biology should also be included as even the most recent antigen assays (hbsag) assays miss infected blood units. as an example, single-sample hbv testing would allow the detection of - additional contaminated units among units tested (biswas et al., ) . monovalent or trivalent assays (hiv, hbv, hcv) are proposed by roche diagnostics (ampli nat) and gen-probe (procleix ultrio) but blood units should not be pooled to provide sufficient sensitivity. procleix ultrio detects single seroconversions days earlier than abbott prism-hbsag but only days in pools of and . days in pools of (cambié, ) . alternatively, an ultracentrifugation step could be introduced following pooling to obtain a higher sensitivity compared to current antigen assays (roth et al., ) . screening for west-nile virus contamination has been implemented in us blood banks. from late june to mid-september , approximately . million donations were screened. twelve hundred eighty-five ( . %) were initially reactive for wnv by using nucleic acid-amplification tests and ( . % of the total donations) are considered presumptive viremic donations (i.e. a donation that is repeatedly reactive by the primary and/or alternate nat assay or a primary nat assay with a very high signal) (cdc, ) .other viruses (parvovirus b , hepatitis a virus) are also transmitted by blood donation and may be part of the screening in a near future. however, nat may not always be the best diagnosis approach and antigen tests or antibody tests may be efficient and less expensive alternatives. automation and integration of nat is necessary to reduce costs and quarantine delays for blood units supply. in this respect, the recent approval by us food and drug administration (fda) of the tigris molecular diagnostic system (gen-probe, san diego, usa) is a major breakthrough as it has been designed to process samples in h. integration and time-to-result are also very important parameters to be considered to insure viral safety of transplant organs, especially lung, heart and liver. it is very important to determine the status of transplants regarding infection by hiv, hbv, hcv and viruses from the herpesviridae family. nat have significantly improved identification of viruses as etiologic agents of human diseases affecting various organs, especially respiratory and gastro-enteric tracts and central nervous system (cns). as a consequence, rapid antiviral treatments can be initiated and considerably reduce morbidity and mortality as, for example, in the case of herpes encephalitis. the increasing number of available antiviral drugs will even accentuate the need for positive viral identification. similarly, unnecessary antibiotic treatments can be avoided or reduced and hospitalisation durations shorten. treatments of chronic viral diseases are very efficiently monitored by viral load assays. however there are still obstacles that prevent a wider dissemination of molecular assays. the extreme genetic variability of some viruses, especially rna viruses (which rna-polymerases have no proofreading activity), often makes their diagnosis difficult. the most striking examples are found in the norovirus family which contains viruses responsible for the vast majority of gastro-intestinal epidemics in adults. hiv diagnosis is also quite difficult to achieve due to its high genetic variability. gardner et al. ( ) have deduced from sequence alignments that a real-time taqman assay should contain not less than nine primer and probe sets to detect with the same sensitivity all hiv strains in a geographically representative panel. to reduce the impact of variability on amplification and detection efficiency, one can use primers and probes with o-methyl bases, degenerate bases or "universal" bases, such as inosine or nebularine. touchdown pcr protocols, in which the annealing temperature slightly decreases during the successive amplification cycles to bracket the melting temperature tm of the reaction, provides sensitivity even when primers have mismatches with target sequences of divergent species in a viral family. finally, degenerate primers or probes, with mixtures of the bases found in sequence databases among various species, may be useful to detect all species of a viral family. it is often desirable to provide the capability for panel detection, i.e. to detect several viruses that can be responsible for a disease. for example, coyle et al. have described at the winter meeting of european society for clinical virology (copenhagen, january ) a molecular viral respiratory strip for the detection of common respiratory viruses. whenever possible, consensus primers able to detect all viruses from a family or genus must be used. there are several examples of such consensus primers for enterovirus (kammerer et al., ) , flavivirus (scaramozzino et al., ) or herpesviridae (tenorio et al., ) . however, their ability to amplify all viruses with the same efficiency must be carefully evaluated. if such an approach is not possible, two different possibilities exist for panel detection: multiplex detection in single tubes or parallel detection in individual tubes. mixing primers in a single amplification tube to achieve multiplex detection of viruses usually results in decreased sensitivity of assays compared to single tests. for example, we have observed, using a dna-microarray assay (see below), that the analytical sensitivity of multiplex rt-pcr detection of six viruses, i.e. influenza a, influenza b, rsv a/b, parainfluenza , and is reduced by a factor of < - logs compared to single detections, depending on the virus. nevertheless, this multiplex assay was able to identify correctly / infections in respiratory specimen (one rsv b infection was misidentified as rsv a; unpublished data). the formation of primer dimers is generally considered as the major cause of sensitivity loss but careful optimisation of all parameters of amplification including primer, enzymes, nucleotides and salts concentrations as well as protocol conditions are required to obtain expected performances. realtime assays (see below) that monitor signal apparition during the amplification step are also limited in their capacity to realize multiplex detection by the number of available wavelengths in existing equipments which currently allows the detection of three viruses only. instead of mixing several pairs of primers in a single tube, nucleic acids purified from the original clinical specimen can be distributed into several tubes for independent amplification and detection. major drawbacks of this approach are the reduction of sensitivity because of lower amounts of nucleic acid available for each individual amplification, higher hands-on times required to manipulate all the different tubes, difficulty to automate the distribution of small volumes of purified nucleic acids without introducing cross-contaminations and higher costs due to the need for enzymes in each tube. internal controls (ic) are important components to monitor each step of the assay from extraction of nucleic acids to detection. because inhibitors of the amplification reaction, which are frequent in some specimen types, will also impact its amplification, the presence of an ic is a strong validation in case of negative result. niesters ( ) has described an original approach for internal control of nat: the use of viral universal controls that can be added to each specimen and be amplified with specific primers, preferably in a multiplex format with primers for the virus to detect. seal herpes virus and phocine distemper virus can be used to control nat for dna and rna viruses respectively. of course, animal viruses which can not infect humans are required. other strategies for ic involve synthetic materials, i.e. plasmids or transcripts which contain sequences able to bind the test primers and a specific probe. clinical virology laboratories have high expectations in term of automation and integration of molecular assays. a major bottleneck in the workflow of these laboratories is at the level of sample preparation. table shows automated systems for nucleic acid purification that are currently commercialised. most of them use the nucleic acid binding properties of silica (boom et al., ) . as many as samples can be handled in a single run on the biorobot (qiagen gmbh, germany). time-to-result and, more important, hands-on-time tend to decrease, with the most recent equipments requiring no longer than min of technician time for more than samples. new labelling technologies that do not need solid-phase separation have allowed the development of real-time molecular assays in which the detection of amplicons is done as soon as they appear during amplification. the most simple real-time detection chemistry uses the sybr green dye which specifically binds during double-stranded dna generated during pcr. several probe technologies (fig. ) have also been designed for real-time assays like taqman ( fig. a ) or molecular beacons (fig. b) in which a quencher molecule is removed from the vicinity of the fluorescent marker upon binding to rna or dna generated during amplification cycles. in the fret technology (fig. c) , two probes, one with a fluorescence donor and one with a fluorescence acceptor molecule are designed to bind adjacent sequences of the amplified material to generate signal (mackay et al., ) . real-time techniques have been designed for pcr or nasba amplification and have several advantages which facilitate automation and reduce time-toresult ( - min) and hands-on-times. they are performed in closed devices which do not need to be opened to transfer amplified material for end-point detection, thus reducing the risk of laboratory or samples cross-contamination. the use of real-time platforms makes the general organisation of molecular biology laboratories easier by reducing the constraints on activities segregation in different rooms to control contaminations. table shows existing real-time automates. the next generation of nat platforms will integrate sample preparation, amplification and detection in a single test device genexpert (cepheid, usa) is the first fully integrated system which allows the detection of bacillus anthracis or group b streptococcus in approximately min directly from clinical specimen. several viral assays based on real-time nasba on this platform will soon be available from biomerieux. several ivd companies offer commercial nat for the diagnosis of viral diseases. besides technical difficulties, another obstacle to the development of molecular assays is the importance of resources needed to optimise, produce and validate home-brew assays and to build up documentation required for qualification of techniques and laboratories. new european community regulations will even increase this need. it is the role of in vitro diagnostic (ivd) companies to provide reagents which are the results of careful optimisation and are produced according to high quality manufacturing procedures. they have clinical and regulatory affairs departments that conduct validation studies and assemble documentation required to get approval of the reagents. however, even for ivd companies, the conception and validation process is time-consuming and timeto-market may be long. this is especially a problem when a diagnostic tool is urgently needed in case of emergence of a new virus. one possibility to reduce time-to-market is to release "research use only" (ruo) assays or assays that have the "ce analytical" approval in europe or the status of "analyte specific reagent" (asr) in the usa. in this case, commercial products that have excellent analytical sensitivity and are manufactured according to quality standards of the ivd industry can be used by clinical virology laboratories, which have the responsibility to validate their use as diagnostic tools and obtain authorization to use them. infections by hiv and hepatitis b and c viruses are usually well diagnosed using serology. only diagnosis of early primo-infections may benefit from nat. platforms like amplicor amplicor from roche diagnostics or easyq from biomerieux that are usually used for viral load measurement during therapy (see below) are also suitable for the early detection of hiv or hcv infections. many other infections and especially acute infections for which igm appear only several days after onset of symptoms cannot be efficiently diagnosed using serology assays. forty to sixty percent of community acquired pneumonia that require hospitalisation have no known aetiology despite intensive investigation and this percentage is even higher when less severe lower respiratory tract infections (lrti) are considered (l. kaiser, personal communication). in a recent study, henrickson et al. ( ) , have shown, using multiplex rt-pcr that % of children hospitalised for lrti are infected with the seven most common respiratory viruses. similarly, a recent survey of encephalitis leading to hospitalisation in the usa from to has revealed that nearly % had no aetiology (khetsuriani et al., ) . absence of specific antiviral treatments for most viruses, which limits prescription of biological tests and weak performances of diagnostic tests based on viral culture or serology are major explanations of this situation. nat, which have been implemented by many large european hospitals, significantly improve viral diagnosis. however, there are several obstacles to the generalization of molecular diagnostics in smaller, decentralized laboratories. a major obstacle is the fact that, except for hiv, hbv, hcv and cmv, virology nat are most often home-brew assays which sometimes suffer from bad performances and poor batch to batch consistency. however, quality of nat is the percentage of false-positive results which reflects laboratory or cross-contaminations and used to be high, dropped to . % (wallace et al., ) . table shows some products currently commercialised by major companies for viruses other than hiv, hbv and hcv, although the list may not be exhaustive. most of them are asr or ruo kits although some are ec marked. the majority of these reagents have been designed to run on realtime platforms. results shown by liolios et al. in illustrate the interest of multiplex detection of respiratory viruses by nat. one hundred forty-three clinical specimen were tested using the hexaplex assay from prodesse inc. (usa) which detects six viruses in a single tube using pcr and detection with microplate capture and peroxidase-labelled probes. samples were detected with the prodesse assay only and not with immunofluorescence or viral culture (table ) . table multiplex nat for the detection of respiratory viruses (hexaplex, prodesse inc.) dna-microarrays or dna-chips are very powerful detection tools that can be combined with amplification techniques to detect viruses or virus variants (reviewed in clewley, ) . wang et al. ( ) have described a microarray spotted with -mer oligonucleotides which represent the five most conserved sequences (more than / bases are conserved among all representative sequences in a virus sequence alignment) of each virus of interest. the chip contains different probes. following pcr amplification of genetic material in the clinical specimen using random primers, hybridisation onto the microarray was able to identify respiratory viruses in the enterovirus, rhinovirus, paramyxovirus, adenovirus and herpesvirus families. however, this technique has not yet been extensively validated for routine diagnosis in a clinical virology laboratory. we have developed assays combining rt-pcr and dna-microarrays for the detection of viruses. the arrays are manufactured using the photolithography in situ synthesis technology (affymetrix, usa) and contain -mer oligonucleotides. sequence signatures are identified using extensive sequence databases because they are conserved in all viruses of a genus or family or in all subtypes or isolates of a virus and are not found in other viruses. for each base of the signature, probes perfectly matching the target and probes with a mismatch at the interrogated position are present on the array. if polymorphisms are present in or near the signature, variant probes may also be present. consensus primers have been designed for enterovirus, flavivirus, herpesviridae, parainfluenza and influenza virus, rsv and adenovirus. they can be combined in multiplex detection pcr or rt-pcr assays for the diagnosis of viral respiratory or cns infections. following amplification, dna is labelled using a newly developed diazomethyl chemistry (laayoun et al., ) . a complete line of instruments (sample preparation, thermocycler, hybridisation station and laser scanner) is available to perform the assay. as described above, a respiratory assay designed to detect six major viruses (parainfluenza , and , rsv a/b, influenza a and b) in a single specimen has demonstrated high clinical sensitivity in preliminary evaluation. fig. illustrates the discriminatory capacity of this technology for cns viruses. assays for the identification of human papilloma virus (hpv) are commercialised by several companies. the detection of a highly pathogenic hpv type has a very high predictive value for cervical carcinoma. however, the number of hpv types that are more or less closely associated to cervical cancer is high. dna-microarrays may thus be appropriate for the multiplex detection of all these types. such an assay has been described by and is distributed by biomedlab co. (south-korea). it is based on a consensus amplification and on -mer probes that are able to detect and discriminate hpv types and has shown an association of . - . % between hpv positivity and lesions of different severity or carcinoma whereas hpv infection was only found in . % of cases when cytology was normal. viral load platforms are available from several ivd companies (versant from bayer diagnostics; cobas ampliprep/ amplicor from roche diagnostics, minimag/nuclisens easyq from biomerieux). significant progress have been made in the ability of most hiv assays to detect all subtypes of hiv- but no commercial assay exist for hiv- . the analytical sensitivity of hbv viral load assays should be increased to reach the same performances as those of hiv assays, especially in the case of infections by variants with low replication competencies. for efficient treatment monitoring of immunosuppressed patients, for example in the case of organ transplantation, viral load assays should also be developed for epstein-barr virus, varicella-zooster virus and hhv . genotyping tests are now commercially available and are part of the biological follow-up of treated patients although home-brew assays are still used in most laboratories (korn et al., ) . hiv and hbv resistance assays as well as hcv subtyping assays are generally based on the sequencing technology (trugene hiv and hbv from bayer healthcare; vi-roseq hiv- from celera diagnostics; genotyping hcv kit from bayer healthcare). hybridisation techniques, such as lipa assays (innogenetics, belgium) are also used. however, the number of probes that can be spotted on nitrocellulose strips is limited and only few polymorphisms can be detected which is convenient for hcv subtyping but does not for resistance tests which are based on the detection of a high number of mutations. in addition, hiv and hbv have highly variable genomes and naturally occurring polymorphisms that are present in the vicinity of resistance mutations may affect the binding efficiency of probes. dna-microarrays are an alternative for resistance or typing reagents for viruses or bacteria . we have developed an assay based on rt-pcr and detection with fig. . biomerieux dna-microarray for the detection of neurotropic viruses. following nucleic acid purification, amplification is performed by pcr using a single touchdown protocol in three tubes, one for herpesviridae (one primer pair for hhv , , , and ), one for enteroviruses (one primer pair for all serotypes) and one for flaviviruses (one primer pair for all viruses). amplification products are combined and labelled using diazomethyl chemistry and hybridised on a dna-microaaray which contains -mer oligonucleotides. two or four probes are used for the detection of each base of sequence signatures determined for each virus. a total of , probes are synthesised on this dna-microarray which has been designed for the simultaneous detection of viruses from the herpesviridae family and from the flavivirus, enterovirus, paramyxovirus, poliomavirus, bunyavirus and orthopoxvirus genus. a: amplicons generated resolved using the bioanalyzer (agilent technologies). b: image of the dna-microarray obtained with a confocal laser reader. c: resolution capability of the array. closely related viruses hybridise with very different efficiency on heterologous probes. high density probe arrays, designed to detect antiretroviral resistance mutations simultaneously in gag cleavage sites, protease, reverse transcriptase, integrase and gp . this assay has been tested on a panel of hiv- patients on a total of relevant codons in comparison with the classic sequence-based method. key resistance mutations were correctly identified in and % of codons in protease and reverse transcriptase, respectively (gonzalez et al., ) . we have also developed a similar assay for the detection of polymorphisms in the complete hbv genome: antiviral resistance mutations in pol gene, vaccine, diagnostic or immunotherapy mutation in s gene and mutations in basic core promoter, pre-core, core, x, pre-s and pre-s regions that may have an impact on disease evolution or treatment efficiency. this assay is currently under evaluation in the frame of hepbvar a european collaborative group for the study of emerging variants of hepatitis b virus. in the coming years, more and more laboratories will offer to clinicians viral diagnosis based on nucleic acid tests. many biological and instrumentation problems that have slowed the generalization of molecular assays have been resolved but several others remain and need to be addressed. major im-provements are expected in the integration and automation of nat diagnostic platforms to reduce hands-on-time, time-toresult and costs and to increase throughput. ivd companies have engaged in development programs to provide clinical virologists with equipments and application menus adapted to their diagnosis needs. technical constraints and recent improvements of molecular assays transfusion risks of yesterday and of today correlation of cervical carcinoma and precancerous lesions with human papillomavirus (hpv) genotypes detected with the hpv dna chip microarray method a novel coronavirus associated with severe acute respiratory syndrome application de la biologie moléculaireà la sécurité virale transfusionnelle: le dépistage génomique viral comparative sensitivity of hbv nats and hbsag assays for detection of acute hbv infection human metapneumovirus infections in hospitalized children rapid and simple method for purification of nucleic acids update: detection of west nile virus in blood donations-united states a role for arrays in clinical virology: fact or fiction identification of a novel coronavirus in patients with severe acute respiratory syndrome limitations of taq-man pcr for detecting divergent viral pathogens illustrated by hepatitis a, b, c, and e viruses and human immunodeficiency virus detection of hiv- antiretroviral resistance mutations with highdensity dna probe arrays national disease burden of respiratory viruses detected in children by polymerase chain reaction the cost-effectiveness of nat for hiv, hcv, and hbv in whole-blood donations nested pcr for specific detection and rapid identification of human picornaviruses burden of encephalitisassociated hospitalizations in the united states quality control trial for human immunodeficiency virus type drug resistance testing using clinical samples reveals problems with detecting minority species and interpretation of test results characterization of a novel coronavirus associated with severe acute respiratory syndrome aryldiazomethanes for universal labeling of nucleic acids and analysis on dna chips comparison of a multiplex reverse transcription-pcr-enzyme hybridization assay with conventional viral culture and immunofluorescence techniques for the detection of seven viral respiratory pathogens real-time pcr in virology clinical virology in real-time children with respiratory disease associated with metapneumovirus in hong kong nat for hbv and anti-hbc testing increase blood safety comparison of flavivirus universal primer pairs and development of a rapid, highly sensitive heminested reverse transcription-pcr assay for detection of flaviviruses targeted to a conserved region of the ns gene sequences a newly discovered human pneumovirus isolated from young children with respiratory tract disease species differentiation and antibiotic susceptibility testing with dna microarrays linkage between the journal and quality control molecular diagnostics (qcmd) microarray-based detection and genotyping of viral pathogens key: cord- -m nbs c authors: yong, voon wee; antel, jack p. title: major histocompatibility complex molecules on glial cells date: - - journal: nan doi: . / - ( ) -n sha: doc_id: cord_uid: m nbs c while glial cells of the central nervous system do not constitutively express class i or ii major histocompatibility complex (mhc) molecules, astrocytes and microglial cells can be induced by a variety of factors to express these antigens. oligodendrocytes have inducible class i but not class ii elements. there are considerable differences in regulation of mhc antigen expression between glial cells from rodent and human brains, both in situ and in vitro. the consequence of glial cell mhc expression for immune interactions in the cns is discussed in the context of glial cell antigen presentation capacity and neural cell susceptibility to cell-mediated immune effector mechanisms. major histocompatibility complex (mhc) antigens comprise a highly polymorphic group of cell surface glycoproteins essential for the process of antigen recognition by t cells . such recognition involves a trimolecular complex of antigen, in the form of a processed peptide, associated with (located in a groove or cleft formed by) an mhc molecule, which is presented to the t-cell receptor of t cells expressing the a and ß chains of the receptor . the mhc recognition process is a requirement for initiation of the immune response through presentation of antigen to t helper (cd +) cells by specialized cells termed antigen-presenting cells, and for either cd + or cd + t cells to carry out effector responses dependent on cell-cell contact, particularly cytotoxic responses directed at specific cell targets . the genes encoding mhc molecules are located on the short arm of chromosome in human (hla region) and on chromosome in mice (h- region) . these genes can be divided into major classes, class i and class ii . among the over human class i genes are the hla-a,b,c, genes (h k,d,l in mouse) that encode a glycoprotein heavy chain that becomes associated with , -microglobulin ; the latter polypeptide is encoded outside the mhc region . the class ii gene region in human (hla-d, corresponding to i-region in mouse) is comprised of at least sub-regions, dp, dq and dr, each of which encodes at least one of the a and one of the ß chains that, together, comprise a class ii mhc molecule . genes outside the mhc region may also contribute to the overall process of immune recognition (minor histocompatibility antigens) . class i mhc glycoproteins are `normally' widely expressed on nucleated cells, except for endogenous cells within the central nervous system (cns) (see discussion below) ; class ii antigens are expressed primarily on cells involved in immune function, such as b cells, cells of the macrophage/monocyte lineage, dendritic cells, activated t cells in humans and specialized phagocytic cells within various tissues, e .g. langerhan's cells in the skin . usually, but not invariably, class i molecules are the mhc recognition molecules for cd + t cells (suppressor/cytotoxic) whereas class ii molecules are recognized by cd + t cells (helper/inducer) . the process of t cell recognition of antigen in the context of mhc molecules is a central means whereby an individual distinguishes self from non-self . lack of class ii mhc expression, as occurs in type ii bare lymphocyte syndrome, results in severe immunodeficiency . at the other extreme, aberrant expression on normally class ii-negative cells carries the risk of inappropriate presentation of self-antigens and is thought to be involved in the pathogenesis of many autoimmune diseases . furthermore, individual susceptibility to a variety of autoimmune diseases in humans has been reported to be influenced by the genetically-determined mhc repertoire, particularly those of the class ii mhc region . ' the cns has traditionally been considered a relatively immunologically privileged site, partly because neural cells were thought not to express mhc antigens, based primarily on early immuno-histochemical analyses of normal tissue in situ . ' the selective advantage to the organism for such non-expression remains unexplained . expression in human cns of mhc antigens, both class i and class ii, appears under pathological conditions, including not only those considered primarily inflammatory disorders ' but also in neurodegenerative diseases where the pathogenesis is not thought to be immunemediated . ' central issues raised by these observations include which cns cells can be induced to express mhc molecules ; what conditions regulate this expression ; and what is the functional significance of such expression with regard to regulation of immune reactivity within the cns and to endogenous cns cells becoming susceptible targets of t cell-mediated immune effector mechanisms . here we focus on mhc antigen expression on filial cells within the cns . we review how both technical and biological variables, including analyses in situ versus in vitro, normal versus pathological tissues, age and species factors, may have contributed to apparently discrepant observations . we present our experience with adult human filial cell cultures obtained from non-inflammatory surgical biopsies and consider the relevance of glial cell expression of mhc with regard to antigen-presentation and target-recognition . in particular we discuss how these may be related with susceptibility to cell-mediated autoimmune diseases of the cns . mhc expression in studies of brain sections human brain sections initial immunohistochemical studies of normal human brain sections did not detect the presence of mhcimmunoreactive cells . ' this has been corroborated by more recent reports . ' in contrast, several studies have described class ii mhc immunoreactivity on neural cells in normal human brain sections . the cell types expressing class ii mhc molecules have been variably identified (either by morphology, ultrastructural features and/or immunohistochemistry) as astrocytes or microglial cells ; ' oligodendrocytes and neurons have consistently been found to be negative for class ii mhc antigen expression in situ . ° the majority of examinations continue to report lack of expression of class i mhc molecules in normal neural tissue . , , although the detection of mhc molecules in normal human brain tissue sections remains equivocal, there is agreement that mhc expression is up-regulated (increased intensity of immunoreactivity and in proportion of immunoreactive cells) in many disease states . these include multiple sclerosis, where inflammatory processes are thought to be involved in the pathogenesis, ' and in conditions where no classical inflammation is apparent, such as neurodegenerative diseases like alzheimer's and brain tumors . [ ] [ ] [ ] the cell types with up-regulated class i or ii mhc expression remain controversial, with suggestions that these are either astrocytes or microglial cells ' or both . the expression of mhc on glial cells has been used as an index that these cells have become `activated' and are participating in the pathological reaction . in human disease states, oligodendrocytes and neurons remain mhc-negative . " °t echnical differences may contribute to discrepant observations using tissue sections . mhc molecules are cell-surface antigens, which can be easily destroyed by inappropriate fixation . prolonged exposure to formalin, used routinely to process postmortem human brain tissue, can mask or destroy the epitope . many studies have used frozen sections and often the morphology of cells was suboptimal, leading to possible misidentification of mhc immunoreactive cells . the choice of fixative (paraformaldehyde or bouin's being preferred) and the duration of fixation have recently been highlighted as important determinants when assessing mhc expression in situ . class ii mhc immunoreactivity can best be detected with fixation intervals of less than h ; staining intensity steadily declined with prolonged fixation until after h, little or no immunoreactivity was observed . under the presumed optimal staining conditions, only microglia expressed class ii mhc in the normal human brain . clinical variables may also influence the detection of mhc antigens in brain sections . pre-mortem intercurrent systemic diseases, particularly infections, can affect class ii mhc expression within the cns (u . traugot and p . lebon, personal communication) . in the presumed postmortem normal brain sections used for mhc detection, it is difficult to exclude the possibility that the tissue donors did not have any underlying pathology, such as subclinical viral infections, that could have modulated mhc antigen expression . a confounding factor in the reports of mhc expression in the cns is species differences . unlike v. w. yong and j. p. antel human brain sections, normal rodent brains in situ do not show immunoreactivity for mhc except on scattered endothelial cells of large blood vessels . whereas class ii mhc-positive astrocytes and microglia are observed in active lesions in multiple sclerosis, astrocytes in the lesions of experimental allergic encephalomyelitis (eae), an animal model of multiple sclerosis, do not appear to express class ii mhc (ia) while microglia do ; - astrocytes and microglia are both class i mhc immunoreactive in eae. in wallerian degeneration following eye enucleation or facial nerve resection, microglia but not astrocytes express la antigens ; , is following facial nerve resection microglia are also immunoreactive for class i mhc . the majority of rat cns cells newly induced to express mhc class ii following days of continuous intravenous administration of gamma-interferon, which is a potent inducer of class ii antigens on astrocytes in vitro (see below), were found to be microglia and not astrocytes . normal mouse oligodendrocytes in situ have been reported to express class ia, although this was not confirmed by others . one immunoelectron microscopic study has described cytoplasmic reactivity for class la in a small proportion in view of the many potential difficulties of experiments in vivo, cultured cells have been used to address the issue of expression of mhc on neural cells . here, live cells can be subjected to immunohistochemical protocols without prior fixation, and the cell type of interest can be reliably identified by using cell typespecific antibodies in double immunofluorescence analyses. bulk isolated cultures also allow for more detailed analyses using molecular biology tools . by immunohistochemical analyses largely performed using cells cultured from neonatal animals, the consensus for rodent cells is that under basal culture conditions class i mhc antigens are expressed on a substantial number of astrocytes , and microglial cells but not oligodendrocytes . , class i can be induced on rodent oligodendrocytes by viral infections and gamma-interferon . , immunoreactive expression of class ii mhc (ia) on rodent astrocytes, microglia or oligodendrocytes in basal culture conditons appears to be absent or negligible . , - analyses at the messenger rna level similarly reveal that cultured astrocytes do not constitutively express mrna for class ii mhc . induction of la on rodent astrocytes occurs after treatment with gamma-interferon, , , , tumor necrosis factor and certain strains of viruses . the administration of gamma interferon in vivo results in the detectable expression of la antigens on rodent astrocytes when isolated and cultured, even when none are detected in vivo . similarly, gammainterferon treatment of mouse or rat microglia in vitro results in la expression ; , neonatal or adult brainderived microglia respond in a similar manner . the gamma-interferon-induced la expression on glial cells seems to be mediated by the protein kinase c signal transduction pathway . in contrast to astrocytes and microglia, rodent oligodendrocytes appear refractory to la expression even when stimulated with gamma-interferon, or supernatants derived from activated mononuclear cells or virus-infected cells . , it is of interest to note that the o- a progenitor cell (which gives rise to the type- astrocytes and oligodendrocytes at least in vitro) and the type- astrocytes can be induced by v. w. yong and j. p. antel gamma-interferon to express la ; oligodendrocytes are refractory . these authors suggest that differentiation of the o- a progenitor cells along the oligodendrocyte pathway is associated with a loss of inducibility of ia antigens . in conjunction with studies of mhc induction, down-regulation of mhc expression has also been examined . for rodent astrocytes, the induction of la by gamma-interferon or tissue necrosis factor is inhibited by adrenaline through ß -adrenergic mechanisms, fi vasoactive intestinal peptides or by transforming growth factor-ß and - . most studies of mhc antigen expression to date, including analyses in situ and in vitro, have used antibodies to recognize mhc determinants . previously, we have observed that cytotoxic t cells could recognize class i mhc on murine astrocytes (i.e . functional recognition), although expression of such molecules was not convincingly demonstrated by antibody immunolabelling . these findings could reflect not only the lack of sensitivity of the immunohistochemical techniques used to detect mhc but also that the determinants recognized by antibody and those recognized by t cells need not be identical . the latter possibility could account at least in part for the failure of some antibodies to mhc molecules to block cytotoxic t cell responses directed against specific cell targets . a much more limited number of studies have explored whether human glial cells in vitro are immunoreactive for mhc antigens . in two studies using autopsy-derived human adult cells, the large proportion (close to % in some cases) of astrocytes and oligodendrocytes expressed class i mhc molecules (hla-a,b,c) whereas a smaller proportion of cultured astrocytes ( - %) from all donors expressed class ii mhc (hla-dr) . of interest, - % of oligodendrocytes from of the donor preparations were immunoreactive for hla-dr . , o in contrast, a study using cells from two autopsy adult samples and another reporting on one autopsy case, did not find hla-dr expression on astrocytes . to circumvent possible post-mortem effects and the variability introduced by pre-mortem intercurrent systemic infections that may affect mhc antigen expression (see above) we have been using glial cells cultured from brain samples surgically resected during treatment for intractable epilepsy . except for the seizures, these subjects were normal . their young age group ( ± years) corresponds to one that is at risk for developing suspected autoimmune diseases such as multiple sclerosis . for class i mhc molecules, close to % of oligodendrocytes, astrocytes and microglia were immunoreactive . _ for hla-dr, virtually all microglia showed positivity ; no oligodendrocytes were found to express hla-dr (figure ) . the analyses of hla-dr expression on biopsy-derived adult human astrocytes showed that this class ii mhc molecule was detected on all human biopsy-derived cultures ; however, different series of human astrocytes expressed hla-dr bearing astrocytes remained relatively constant when analysed at different time points, up to days in vitro . the differential level of expression of hla-dr between human series was not correlated with the age of the human subjects, the extent of gliosis in the resected material or the duration for which the cells have been maintained in culture . when adult human astrocytes were subclassified by morphology (these did not fall neatly into the type- and type- astrocytes characterized in rodent), hla-dr expression was prevalent on process-bearing rather than the flat astrocytes (figure ) , the expression of hla-dr on cultured adult human glial cells in our studies probably represents an induction process in vitro, because freshly isolated cells did not have detectable hla-dr and because the type of serum (example, human ab) and its concentration in the culture medium affected the proportion of astrocytes that expressed hla-dr . it would be of interest to determine whether the individual variability of hla-dr expression on human astrocytes reflects a genetically determined property, as seems to be the case with rodents where inducibility of ia molecules on cultured astrocytes correlates with susceptibility to eae (see below) . in contrast to their human counterparts, rat astrocytes in our hands did not express class ii mhc when isolated and maintained in identical culture conditions to the adult human cells . this result is in agreement with the multitude of reports cited above that rat astrocytes have undetectable levels of la under baseline culture conditions . thus, whereas the expression of mhc on adult human cells probably represents an induction process in vitro, rodent cells are not similarly induced ; this suggests differences in inductive capability, probably at the molecular level, of mhc between species . such a result would reflect the observations in situ (see above) that mhc antigens are more readily detected on human neural cells than those of rodents . in correspondence with rodent cells, class ii mhc expression on human adult astrocytes can be upregulated by treatment with gamma-interferon ; , this effect can be blocked by treatment with betainterferon . more recently, it has been shown that interleukin- can decrease constitutive expression of class ii mhc in a glioblastoma multiforme line . human fetal cells have also been examined for expression of mhc molecules . this literature is small but it seems that both class i and ii mhc can be detected on fetal astrocytes ; ß a small proportion of microglia were immunoreactive for hla-dr (class i was not examined) . oligodendrocytes and neurons in human fetal cultures were negative for mhc expression . summary of tissue culture studies expression of mhc antigens by glial cells is more readily detected in tissue culture studies than in the brain in situ (table ) . although one explanation may be the feasibility of determinations in vitro, it is more likely that the induction of mhc molecules is a result of cell culture . another possibility is that in culture as yet uncharacterized in situ inhibitors of mhc antigen expression are lost . class ii mhc expression on astrocytes and microglia in the human system is observed under baseline culture conditions, unlike in rodent cells (table ) ; we speculate that the molecular factors that control inducible elements of mhc gene expression in the human system are less tightly regulated than those in rodent cells . however, in response to treatment with gamma-interferon, a potent cytokine, induction of mhc seems to be similar across species (table ) . there does not appear to be changes in mhc expression due to age factors, from the limited number of studies currently available, , , significance of mhc expression by glial cells antigen presentation neonatal murine astrocytes and microglia, especially after gamma-interferon treatment, can present antigen, e .g. myelin basic protein, to previously sensitized cd + t cell lines in an mhc class iirestricted manner . , , , astrocytes, even after treatment with gamma-interferon, are, however, unable to induce activation of primary cd + t cells in a mixed lymphocyte reaction ; such responses were achieved by `professional' antigen presenting cells, such as dendritic cells and activated b cells . microglial cells prepared from human brain biopsies are also able to induce a response by previously nonactivated human peripheral blood-derived t cells in a mixed lymphocyte reaction . the capacity of endogenous glial cells to present antigen probably contributes to the observed persistence of activated myelin-sensitized t cells within the cns, as compared to non-neural antigen-reactive t cells . immune-mediated injury to oligodendrocytes or its myelin membrane is postulated to account for the demyelination which characterizes the acquired human disease multiple sclerosis, the induced human disease acute disseminated encephalomyelitis (postvaccination, post-infectious encephalomyelitis) and the induced animal disease eae . in the last, the disease can be passively transferred using myelin antigen-reactive cd + t cells . the mechanisms through which oligodendrocyte/myelin injury occurs are not precisely defined ; mechanisms implicated include direct cd + t cell-mediated injury, cellmediated injury by other t and non-t (macrophage/ microglia) cells recruited to the lesion sites, and injury mediated by soluble factors released by the accumulated inflammatory cells . rat t cells, reactive to myelin basic protein and comprised predominantly of cd + t cells (or cd + t cell hybridomas), have been shown to be cytotoxic to cultured rat oligodendrocytes in a strain-, presumably mhc-, restricted manner . the cytotoxic effect of the cd + t cell lines require the presence of antigen presenting cells, which possibly enhance effector-target interactions ; the effect could not be reproduced by soluble factors . myelin basic protein-reactive cd + -t cells can acquire cytotoxic capability, at least in vitro, and have been shown to lyse cells expressing histocompatible class ii mhc antigens (e.g. b cells transformed with epstein-barr virus) in the presence of myelin basic protein . the target oligodendrocytes did not express class ii mhc although they were induced to express class i antigens when cocultured with t cells . the presumably small number of cd + t cells in the mbp-reactive cd + t cell lines could also have contributed to oligodendrocyte lysis, consistent with our previous observations that allo-reactive cd + -t cells can lyse human oligodendrocytes in vitro in an mhc class i restricted manner . a further potential mechanism whereby cd +-t cells may be cytotoxic to non-mhc class ii expessing cells, such as oligodendrocytes, involves the possibility of their acquiring cytotoxic capability following prolonged activation while losing their mhc restriction ; such cells have been generated in vitro and termed promiscuous killers . we have found that t-a/ß chain-expressing cd + and cd + t cells can induce promiscuous killing of human oligodendrocytes (t.c .g. ruijs, e .a . brown and j .p. antel, unpublished) . the conditions for generating these cells involves maintaining persistent activation with interleukin- ; such conditions in vitro may well parallel those found in vivo in the chronic inflammatory lesions characteristic of multiple sclerosis . the promiscuous cytotoxic effect canot be reproduced with supernatants from such cultures . the classic antigen-restricted t cell expresses the a and ß chain of the t cell receptor, as indicated previously . a t cell population identified by expression of the -y/s chains of the t cell receptor has been observed to accumulate in excess numbers in regions of demyelination in cns brains . these cells can mediate potent cytotoxicity of human oligodendrocytes in vitro . these t-y/s cells are not restricted by either classical class i or ii mhc molecules. the recognition molecule of these cells remains unknown, with much interest focused on heat shock proteins . however, further candidates may be class i-like molecules such as cd or possible equivalents of the mouse qa and tl gene products . a potential link between mhc class ii up-regulation on glia and genetic susceptibility to autoimmune disease was suggested by findings that astrocytes from rodent strains susceptible to eae expressed higher levels of mhc class ii after exposure to gammainterferon in vitro than do astrocytes from resistant strains, although this has recently been disputed . glia from eae susceptible mouse strains are more potent presenters of mpb to sensitized t cells than are glia from eae resistance mice ; no differences were found in this function using spleen cells . susceptibility to immune-mediated encephalomyelitis initiated by jhm-virus infection in rats is a function of inducibility of la (but not class i) antigen on astrocytes . such observations have raised speculations that exposure to cytokines (especially gamma-interferon) or viruses could contribute to cns cells becoming capable of presenting auto-antigen to infiltrating t cells or becoming susceptible targets of such t cells . for human adult astrocyte cultures, it remains to be determined whether the differential expression of hla-dr by various specimens could be correlated with particular hla haplotypes associated with genetic susceptibility to the development of autoimmune diseases of the cns, such as multiple sclerosis (dr locus) . whether the observed deleterious effect of systemically administered gamma-interferon on the course of relapsing multiple sclerosis reflects effects on the cns as well as on the systemic immune system, remains speculative . a novel means of inducing overexpression of mhc molecules in the cns in vivo involves the creation of transgenic animals . a transgenic mouse line created by insertion of a class i mhc gene under control of the myelin basic protein promoter, with resultant restricted expression of the transgene to oligodendrocytes, displayed marked dysmyelination but without inflammation . this model may be more akin to genetic models of impaired myelin production, rather than to immune-mediated myelin destruction as claimed . summary offunctional significance of mhc antigen expression on plia mhc antigen expression on glia has been linked with susceptibility to development of autoimmune cns diseases . the potent inducers of mhc expression on glia include viruses and immune system-derived soluble factors (cytokines), which themselves are the suspected initiators or mediators of such diseases . mhc expression on filial cells is of functional significance with regard to these cells acquiring the capacity to present antigen to t cells and thus promoting (or conversely, potentially inhibiting) immune reactivity, and to these cells themselves becoming susceptible targets of specific immune effector mechanisms . the dynamic expression of mhc molecules within the cns contributes both to the capacity of endogenous glial cells to regulate immune reactivity within this compartment and to such cells becoming susceptible targets of immune effector responses . the functional importance of mhc molecules in antigenspecific t cell responses is well illustrated by the potent effects of systemically administered anti-class ii mhc antibodies, which inhibit the development and disease course of eae . the increasing availability of molecular genetic techniques to manipulate expression of mhc per se within the cns provides new opportunities to assess the role of these molecules in influencing neural-immune interactions . with regard to human immune-mediated cns disease, optimal immunotherapies would be those which are organ-specific, i .e . spare overall systemic immune capability . the apparent effect of biological response modifiers on mhc expression on glial cells implicate v. w. yong and j. p. antel them as potential selective therapies . it still has to be defined whether down-regulation of mhc can be more readily achieved on cns cells with their inducible rather than constitutive expression of mhc, than on systemic antigen-presenting cells that constitutively express them . conversely, in some circumstances, up-regulation may be of advantage, for example to augment filial tumor-directed immune responses . as the molecular mechanisms regulating mhc expression in the cns are further defined, additional means will hopefully become available for therapeutic manipulation of this dynamic system . a molecular basis for mhc class ii-associated autoimmunity the tissue distribution and cellular location of transplantation antigens tissue distribution of la antigens and their expression on lymphocyte subpopulations on the presence of la-positive endothelial cells and astrocytes in multiple sclerosis lesions and its relevance to antigen presentation multiple sclerosis : relevance of class i and class ii mhc-expressing cells to lesion development reactive microglia are positive for hla-dr in the substantia nigra of parkinson's and alzheimer's disease brains detection of hla-dr on microglia in the human brain is a function of both clinical and technical factors expression of class ii major histocompatibility antigens on reactive astrocytes and endothelial cells within the gliosis surrounding metastases and abscesses microglia are the major cell type expressing mhc class ii in human white matter multiple sclerosis : oligodendrocytes in active lesions do not express class ii major histocompatibility complex molecules monoclonal antibody analysis of mhc expression in human brain biopsies : tissue ranging from 'histologically normal' to that showing different levels of glial tumor involvement expression of la molecules by astrocytes during acute experimental allergic encephalomyelitis in the lewis rat the distribution of ia antigen in the lesions of rat acute experimental allergic encephalomyelitis in situ detection of class i and ii major histocompatibility complex antigens in the rat central nervous system during experimental allergic encephalomyelitis . an immunohistochemical study expression of la antigen on perivascular and microglial cells after sublethal and lethal motor neuron injury macrophage function during wallerian degeneration of rat optic nerve : clearance of degenerating myelin and la expression effects of dexamethasone on microglial activation in vivo : selective downregulation of major histocompatibility complex class ii expression in regenerating facial nucleus rat ependyma and microglia cells express class ii mhc antigens after intravenous infusion of recombinant gamma interferon expression and synthesis of murine immune response-associated (ia) antigens by brain cells immune response gene product (ia) on glial and endothelial cells in virus-induced demyelination tumor necrosis factor induces expression of mhc class i antigens on mouse astrocytes astrocytes as antigen-presenting cells . part ii . unlike h- k-dependent cytotoxic t cells . h- ia-restricted t cells are only stimulated in the presence of interferon-y fc receptor density, mhc antigen expression and superoxide production are increased in interferon-gamma-treated microglia isolated from adult rat brain coronavirus infection induces h- antigen expression on oligodendrocytes and astrocytes the expression of mhc antigens on oligodendrocytes : induction of polymorphic h- expression by lymphokines expression of la antigens by cultured astrocytes treated with gamma-interferon inducible expression of h- and ia antigens on brain cells cultured human and rat oligodendrocytes and schwann cells do not have immune response gene associated antigen (ia) on their surface viral particles induce la antigen expression on astrocytes antigen presentation and tumor cytotoxicity by interferon-gamma treated microglial cells induction and regulation of class ii major histocompatibility complex mrna expression in astrocytes by interferongamma and tumor necrosis factor-alpha enhanced interferon-gamma-induced iaantigen expression by filial cells after previous exposure to this cytokine cytokines up-regulate la expression in organotypic cultures of central nervous system tissue regulation of hla class ii molecule expressions by -y-ifn the differentiation of o- a progenitor cells into oligodendrocytes is associated with a loss of inducibility of ia antigens norepinephrine inhibits -y-interferon-induced major histocompatibility class ii (ia) antigen expression on cultured astrocytes via / -adrenergic signal transduction mechanisms transforming growth factors type ßl and suppress rat astrocyte autoantigen presentation and antagonize hyperinduction of class ii major histocompatibility complex antigen expression by interferongamma and tumor necrosis factor-alpha susceptibility of astrocytes to class i mhc antigen-specific cytotoxicity antigen expression by glial cells grown in culture expression of la antigens on the surface of human oligodendrocytes and astrocytes in culture induction of human leukocyte antigen-a,b,c and -dr on cultured human oligodendrocytes and astrocytes by human y-interferon immunohistochemical studies of adult human glial cells human oligodendrocytes are susceptible to cytolysis by major histocompatibility complex class irestricted lymphocytes morphologic heterogeneity of human adult astrocytes in culture : correlation with hla-dr expression expression and modulation of hla-dr on cultured human adult astrocytes interferon-$ impairs induction of hla-dr antigen expression in cultured adult human astrocytes interleukin- ß decreases hla class ii expression on a glioblastoma multiforme cell line differential expression and regulation of major histocompatability complex (mhc) products in neural and glial cells of the human fetal brain human microglial cells : characterization in cerebral tissue and in primary culture, and study of their susceptibility to hiv- infection astrocytes present myelin basic protein to encephalitogenic t cell lines la restricted encephalitogenic t lymphocytes mediating eae lyse autoantigen-presenting astrocytes major histocompatibility complex-expressing nonhematopoietic astroglial cells prime only cd * t lymphocytes : astroglial cells as perpetuators but not initiators of cd ' t cell responses in the central nervous system cytotoxic effect of myelin basic protein-reactive t cells on cultured oligodendrocytes colocalization of lymphocytes bearing ys t-cell receptor and heat shock v. w. yong and j. p. antel protein hsp ' oligodendrocytes in multiple sclerosis the ys t cell receptor and class lb mhc-related proteins : enigmatic molecules of immune recognition hyperinducibility of la antigen on astrocytes correlates with strainspecific susceptibility to experimental autoimmune encephalomyelitis induction of class ii major histocompatibility complex antigens on a population of astrocytes from a mouse strain (balb/c) resistant to experimental allergic encephalomyelitis immunologic differences in murine glial cells and their association with susceptibility to experimental allergic encephalomyelitis inducibility of la antigen on astrocytes by murine coronavirus jhm is rat strain dependent dysmyelination in transgenic mice resulting from expression of class i histocompatability molecules in oligodendrocytes we wish to acknowledge the collaborations of the following individuals : s .u . kim key: cord- -zj gy z authors: schaaf, h.s.; beyers, n.; nel, e.d.; gie, r.p.; donald, p.r. title: seasonal variation in the culture rate of m. tuberculosis in children date: - - journal: tuber lung dis doi: . / - ( ) -x sha: doc_id: cord_uid: zj gy z nan epidemic influenza and of clinical cases of pneumopatia has been determined by means of laboratory assessements (virus isolation, indirect immunofluorescence, specific serologic tests). the type a influenza virus was incriminated in % of the cases and in % of the cases was incriminated the association of influenza virus with parainfluenza viruses, adenoviruses, coronaviruses and s.r. virus. in a small number of cases (children) with severe respiratory diseases, the examination of trachea-bronchic aspirate was positive for bacteria and fungi. the association virus-bacteria was found in % of these cases, confirming the gravity of their evolution and prognostic. further studies, with particular reference to the etiology of severe respiratory diseases, are required, enabling thus a real evaluation of infectious morbidity and an adequate approach of treatment. the mean elisa value in patients was significantly higher as compared to controls (p < .ool). a total of mb strains isolated from different mammalian species: bovines, felines (cats), one camelidae (llama), and seals; and other mb isolates form tb patients, were included in the study. the bp probe hybridized with an unique . kg restriction fragment in mb strains, and either with one fragment in a different position or with more than ohne fragment in other strains-isolated from bovines, llama, cats and tb patients. the restriction fragment patterns of four mb strains from seals were identical and clearly different from all the remaining mb strains. the use of the probe allowed us to confirm transmission of infection in a herd where mb isolates from cows were identical, and different from all other isolates found in that region. most of these argentine mb strains presented only one copy of is , and that limits further use of this element for epidemiological surveillance. enzyme-linked immunoassay (eia) are widely used in clinical practic for antimycobacterial antibody detection still, the development of more eia-variant, based on new more specific micobacteria-derived antigens is never stopped. an antigenic preparat from m. bovis bcg (russia) cell wall was purified by triton x- extraction will subsequent auton sedimentation and ether treatment. this complex antigen was named triton fraction (tf). it contains three serologically active antigenic determinant identical to those of triton fractions separated from virulent micobacterial strains m. bovis and m. humanus. this provides us the opportunity to use it in eia test system for specific antibodies detection in lung tuberculosis patient's sera. with serum sample, deluting : the sensitivity of this method ranges from % to % depending on the form and activity of lung tuberculosis process. the same dilution of sera from the group of healthy blood donors gave % specificity of the test. no more than % false-positive results were obtained while testing : diluted sera from non-tuberculosis lung patients. the method described is used to confirm the diagnosis of tuberculosis in clinical practice as well as for high risk groups detection in mass surveys at epidemic centres. schaaf, h. s., beyers, n., nel, e. d.) conclusion: culture rate of m.tb was significantly higher during spring than during the other seasons. this is supported by the same trend in children with tbm. we speculate that more tb infections occurs during winter which then present with disease in spring. (table ) . drugs involved mostly are h ( %) and s ( %). multidrug resistance ( drugs) (mdr) remains stable since . more than % of mdr are due to h and s resistance. table risk factors for sdr and mdr have been a status as foreign born ( %) and previous treatment ( %). in a great urban population in germany we found increasing and relatively high rates of m.tb. resistance since . the study was conducted on active pulmonary t. b. cases and matched control subjects. all patients hat sputum positive for acid fast bacilli. they were subjected to full clinical examination including chest x-ray. blood samples were taken at admission and at one and two months following the start of anti tb therapy for routine analysis as well as serum ada and a igg. the results of this study revealed that on admission, mean ada level . k . u/l was significantly higher than the control group . & . u/l. the value of ada on admission was significantly higher than at one and two months after treatment. this implies the importance of ada as a predictor for successful treatment. serum igg to a presented significantly higher values after months of treatment than its level on admission. when a cutoff value of units was chosen, . % of patients were found to be positive for a igg on admission, while the figure increased to . % after months of treatment. this may signify a role for a igg on long term follow up as a predictor of immunological response to tb infection. no correlation was found between either ada or a igg and type or extent of radiological findings nor with extent of tuberculin activity. pulmonary tuberculosis (sputum positive) . pulmonary tuberculosis (sputum negative) abdominal tuberculosis milliary tuberculosis renal and urinary track tuberculosis. . skin tuberculosis. . tuberculosis of eye tubercular laryngitis. . genital tuberculosis. . quescent cases normal individuals (monteux positive) normal individuals (monteux negative) key: cord- - la tm authors: hsueh, po-ren; kao, chuan-liang; lee, chun-nan; chen, li-kuan; ho, mei-shang; sia, charles; de fang, xin; lynn, shugene; chang, tseng yuan; liu, shi kau; walfield, alan m.; wang, chang yi title: sars antibody test for serosurveillance date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: la tm a peptide-based enzyme-linked immunosorbent assay (elisa) can be used for retrospective serosurveillance of severe acute respiratory syndrome (sars) by helping identify undetected chains of disease transmission. the assay was developed by epitope mapping, using synthetic peptides from the spike, membrane, and nucleocapsid protein sequences of sars-associated coronavirus. the new peptide elisa consistently detected seroconversion by week of onset of fever, and seropositivity remained through day . specificity was % on normal blood donor samples, on serum samples associated with infection by other pathogens, and on an interference panel. the peptide-based test has advantages of safety, standardization, and automation over previous immunoassays for sars. the assay was used for a retrospective survey of healthy healthcare workers in taiwan who treated sars patients. asymptomatic seroconversions were detected in two hospitals that had nosocomial disease. a peptide-based enzyme-linked immunosorbent assay (elisa) can be used for retrospective serosurveillance of severe acute respiratory syndrome (sars) by helping identify undetected chains of disease transmission. the assay was developed by epitope mapping, using synthetic peptides from the spike, membrane, and nucleocapsid protein sequences of sars-associated coronavirus. the new peptide elisa consistently detected seroconversion by week of onset of fever, and seropositivity remained through day . specificity was % on normal blood donor samples, on serum samples associated with infection by other pathogens, and on an interference panel. the peptide-based test has advantages of safety, standardization, and automation over previous immunoassays for sars. the assay was used for a retrospective survey of healthy healthcare workers in taiwan who treated sars patients. asymptomatic seroconversions were detected in two hospitals that had nosocomial disease. r etrospective surveillance for infection is an important means to screen for and interrupt undetected chains of disease transmission. such surveillance may be key to tracking the severe acute respiratory syndrome-associated coronavirus (sars-cov) because mild and asymptomatic cases of sars-cov infection that do not meet the world health organization's case definition ( ) have been identified by immunoassays ( ) ( ) ( ) , and sars-cov-like viruses have been isolated from wild mammals ( ) . sars-cov may have persisted over the summer in previously affected areas in such difficult-to-recognize reservoirs ( ) . the reemergence of sars in the city of guangzhou of the guangdong province of china in december and january ( ) is evidence that an unknown reservoir exists and signals the need for continued surveillance with laboratory testing. the current laboratory methods for identifying sars are not ideal tools for use in retrospective mass screening. reverse transcription-polymerase chain reaction (rt-pcr) for detecting viral rna is the most sensitive method for early identification of sars. however, viral load rapidly declines beginning or days after disease onset ( ) ( ) ( ) . moreover, rt-pcr requires sophisticated equipment and high laboratory quality-assurance standards ( , ) . identifying seroconversion to sars-cov by immunoassay also is a definitive criterion for laboratory determination of sars ( ) , and seroconversion is the preferred standard for retrospectively detecting sars-cov infection ( ) . sars immunoassays include the enzyme-linked immunosorbent assay (elisa) or western blot with antigen from whole virus or various recombinant proteins, a cumbersome immunofluorescence assay (ifa) using whole virus fixed on glass, and methods to determine neutralizing antibodies ( , ) . immunoglobulin (ig) g to sars-cov, detected by these immunoassays, begins to rise sharply by day after onset of symptoms. virtually all sars patients show virus-specific antibody by week , and anti-sars-cov igg persists through day ( , , ) . although any of these immunoassays can provide a definitive laboratory finding, all but the recombinant tests require biosafety level to contain the virus or are time-consuming to perform, have not been well-standardized, are of unknown specificities, and would be difficult to adapt to large-scale manufacture. improving laboratory methods for the large-scale serologic surveillance of sars, particularly in the presence of other respiratory illnesses, and standardization of diagnostic assays are key priorities for controlling sars ( ) . in this report, a standardized and rapid peptide-based sars elisa is characterized for sensitivity and specificity. beginning in april , delay in recognizing sars cases and in implementing isolation procedures led to several nosocomial clusters of sars-cov transmission in healthcare facilities in taiwan ( , ) . the results from a retrospective serologic survey by the peptide elisa of healthcare workers from facilities affected by nosocomial outbreaks are presented as a working example. we developed an elisa for sars that has synthetic peptide antigens as the solid-phase immunosorbent. over overlapping peptides, deduced from the tor sars-cov genomic sequence ( ) , were synthesized as candidate antigens from the spike (s), membrane (m), and nucleocapsid (n) proteins. candidate immunodominant s, m, and n peptides were selected and refined on the basis of serologic reactivities to a panel of serum samples from patients clinically diagnosed with sars at national taiwan university hospital in taipei and the xiaotangshan sars emergency hospital in beijing ( ) . epitope mapping by serologic validation has been described for the development of peptide-based elisa tests for hiv, hepatitis c virus, and foot-and-mouth disease virus ( ) ( ) ( ) . for the peptide-based sars elisa, wells of microtiter plates were coated with µg/ml of a mixture of the s, m, and n protein-derived peptides, and serologic reactivities were determined by a standard elisa procedure as previously described ( ) , except that the detector was horseradish peroxidase-conjugated goat anti-human igg, and the chromaphore was , ′, , ′tetramethylbenzidine (tmb). in brief, serum samples, including two normal human samples provided as nonreactive controls, were diluted : in phosphate-buffered saline with carrier proteins and preservative. the diluted serum samples were reacted to the peptide-coated microtiter wells for l h at °c. plates were washed times, reacted to the antibody conjugate, again washed times, and reacted to tmb; reactivity was then determined by a . assay results were obtained within h. results were scored on the basis of the signal/cutoff (s/c) ratio, and cutoff absorbance was determined from the mean of the two controls plus standard deviations (sd) from the distribution of normal human samples ( figure ). seroconversion panels were collected as serial serum samples from sars patients at national taiwan university hospital in the course of treatment. these panels tested positive for seroconversion by ifa ( ) and are used here to evaluate analytical sensitivity. a panel of serum samples from convalescent sars patients were provided by the center for disease control, department of health, taiwan, to evaluate diagnostic sensitivity. these serum specimens were confirmed for sars-cov infection clinically by the world health organization diagnostic criteria ( ) and serologically by whole virus-based elisa and ifa; some specimens were also confirmed by rt-pcr ( ) . samples were drawn with appropriate timing for serologic reactivity ( - weeks after onset of symptoms). a panel of , plasma samples collected from random blood donors in florida before was obtained from the gulf coast regional blood center (houston, tx) for specificity evaluation. additional specificity studies were conducted with serum that had serologic reactivities for bloodborne pathogens (hiv- , hiv- , hepatitis c virus, htlv /ii, and syphilis) obtained by united biomedical inc. before from various u.s. sources, an interference panel supplied by boston biomedica inc. (boston, ma) of serum samples with interference substances commonly found in processed clinical samples (edta, acid citrate dextrose, and citrate phosphate dextrose with adenine), and serum supplied by national taiwan university hospital from patients associated with typical and atypical respiratory pathogens other than sars-cov (influenza, rubella, cytomegalovirus, epstein-barr virus, and mycoplasma pneumoniae). serum samples were collected from healthy healthcare workers after interviews to confirm lack of signs and symptoms of sars including fever, respiratory symptoms, and diarrhea. the peptide-based elisa was evaluated for sensitivity to seroconversion on eight seroconversion panels obtained from national taiwan university hospital (figure , table ). in patient , seroconversion was detected by day with an a of . . absorbance remained at > at day . in patient , from whom blood was drawn on days , , , , and (no samples were collected from day to day ), seroconversion was apparent on day after the onset of fever. on day , the a remained > . in five of the other six seroconversion panels, from acute to convalescent phases, seropositivity was observed by days to , and by day in patient , from whom serum had not been collected from days to ( figure ). the peptide-based elisa showed an analytical sensitivity to earliest time of detection by week and for duration of detection beyond day . the seroreactivities of patients to were also evaluated by a standard ifa method ( ) for comparison. seroconversion was detected in all six patients by the ifa method within days of the peptide elisa (data not shown). the diagnostic sensitivity of the peptide elisa was % on a panel of convalescent-phase serum samples from sars patients provided as a reference panel by the center for disease control, department of health, taiwan. these sera, confirmed for sars by diagnostic and serologic criteria, displayed a mean s/c ratio of . (figure ). the specificity of the peptide-based sars elisa was tested on plasma obtained before from , random florida blood donors (gulf coast regional blood center, houston, tx). these normal plasma samples with a presumed zero seroprevalence rate gave a mean a of . ± . (sd). subsequently, the cutoff value for the peptide-based assay was set as the mean a for duplicate nonreactive controls plus . (based on sds from the mean for these , normal plasma samples). the distribution of the s/c ratio for the blood bank samples is plotted in figure with the mean s/c ratio of . . none showed positive reactivity, for a specificity on the normal samples of % ( table ) . the peptide elisa was further evaluated for specificity with a pre- collection of serum samples from patients with seropositivities for bloodborne pathogens such as hiv- , hiv , hepatitis c virus, htlv /ii, and syphilis (various u.s. sources), and with normal serum samples spiked with interference substances heparin, edta, acd, and cpda- (boston biomedica). the samples with seroreactivities for the pathogens all tested negative by the peptide-based sars elisa, as did the sera interference panel ( table ). the peptide elisa was evaluated for specificity on serum samples drawn from patients associated with typical and atypical respiratory pathogens other than sars-cov (national taiwan university hospital). these included samples from ) patients naturally infected with influenza (two sequential bleeds per influenza patient), ) patients with rubella, bacterial agent for atypical pneumonia, and ) pre-and postvaccine blood samples from patients given influenza vaccine. all samples were tested in duplicate. the sitespecific antigens of the peptide sars-cov elisa were free of cross-reactivities to the other respiratory pathogens ( table ) . a prospective study was performed to determine asymptomatic infection among primary healthcare workers in hospitals that treated sars patients. we collected serum samples from healthcare workers without symptoms, not all of whom were in direct contact with sars patients, who agreed to be tested for antibody to sars-cov at ho ping, yang ming, en chu kong, and hsin tai hospitals, approximately weeks after the outbreaks were recognized. ho ping and yang ming hospitals had admitted sars patients before the recognition of sars and before healthcare workers had implemented control measures. subsequently, these facilities experienced transmission to healthcare workers. the en chu kong and hsin tai facilities admitted patients once control measures had been implemented. neither of these hospitals recorded transmission of sars to healthcare workers. elisa detected three cases out of samples from ho ping and one case in blood samples from nursing aides at yang ming. these four positive samples, indicative of asymptomatic infection, were confirmed for seropositivity by ifa. none of the serum samples from the two hospitals without nosocomial infection displayed seroconversion. a convenient elisa to detect igg to sars-cov, based on site-specific synthetic antigens taken from the s, m, and n proteins of the virus, has high specificity. no cross-reactivity was detected in samples associated with common non-coronavirus respiratory pathogens. in addition, the lack of detectable reactivities among the , u.s. blood donors supports a specificity for the assay to distinguish sars-cov infection from infection by other human coronaviruses. the presence of anti-coronavirus antibodies among a u.s. population of this size is strongly anticipated because an incidence as high as % for oc and e respiratory infections has been observed, even among healthy young adults ( ) . the new peptide-based elisa is equivalent in sensitivity to other immunoassays for sars and can be detected after day . the synthetic antigens provide the advantages of high standardization, freedom from biohazard, and ease of scale-up production. moreover, testing by the elisa format can be readily automated for large-scale screening. the highly specific peptide-based sars antibody test is a convenient means to carry out widespread retrospective surveillance, such as that now being proposed for china to trace hotspots of persons carrying antibodies to sars-cov and to track the origins of the disease ( ) . a preliminary survey with the peptide elisa detected asymptomatic clusters of seroconversion among exposed healthcare workers in two taiwan hospitals that also had nosocomial disease. in contrast, no seroconversion was found among the exposed healthcare workers from two hospitals that had no apparent disease transmission to healthcare workers. the finding of asymptomatic seropositive persons indicates that the test will be useful in larger retrospective surveillance studies, which are needed to fully define the epidemiology and spectrum of disease. the protocol and the informed consent documents were approved by the ethics committee of medical research of the biomedical sciences, academia sinica. all participation was voluntary and was documented with written informed consent. dr. hsueh is an associate professor in the departments of laboratory medicine and internal medicine, national taiwan university hospital, national taiwan university college of medicine. his research interests include mechanisms of microbial resistance and molecular epidemiology of emerging pathogens. he is actively involved in a national research program for antimicrobial drug resistance and is a member of the sars research group of national taiwan university college of medicine and national taiwan university hospital. world health organization. case definitions of surveillance of severe acute respiratory syndrome (sars) mild severe acute respiratory syndrome asymptomatic severe acute respiratory syndrome-associated coronavirus infection early indications point to lab infection in new sars case isolation and characterization of viruses related to the sars coronavirus from animals in southern china sars-one year later update : announcement of suspected sars case in southern china; investigation of source of infection for confirmed case begins tomorrow clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study detection of sars coronavirus in plasma by real-time rt-pcr serologic and molecular biologic methods 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envelope protein improved serodiagnosis of hepatitis c virus infection with synthetic peptide antigen from capsid protein differentiation of convalescent animals from those vaccinated against foot-and-mouth disease by a peptide elisa microbiologic characteristics, serologic responses, and clinical manifestations in severe acute respiratory syndrome evidence-based medicine for student health services antibodies to sars-like virus hint at repeated infections key: cord- -avkgldnp authors: perlman, stanley; wu, gregory f. title: selection of and evasion from cytotoxic t cell responses in the central nervous system date: - - journal: adv virus res doi: . /s - ( ) - sha: doc_id: cord_uid: avkgldnp cytotoxic cd t lymphocytes (ctls) are critical for the clearance of noncytopathic viruses from infected cells. this chapter discusses one mechanism used by viruses to persist—namely, the selection of a variant virus in which changes in the sequence of a ctl epitope abrogate recognition. the unique features of cytotoxic cd t cell function in the central nervous system (cns) are discussed. the role of ctl escape mutants in the viral evasion of the immune system and subsequent disease progression in non-cns infections are summarized. the immune response in the cns is similar to the response in extraneural tissue, but several aspects of the activation of the immune response, cellular trafficking, and antigen presentation are unique to the cns. although the cns has classically been considered a site of immune privilege, surveillance of the normal cns by circulating, activated lymphocytes occurs, with a limited number of lymphocytes being present in the normal cns at any given time. in mice infected with mouse hepatitis virus and in some humans persistently infected with human immunodeficiency virus type , hepatitis b virus or hepatitis c virus, ctl escape mutants play an important role in virus amplification and disease progression. the immune response in the cns is similar to the response in extraneural tissue, but several aspects of activation of the immune response, cellular trafficking, and antigen presentation are unique to the cns (reviewed in lassmann, ; lassmann et al., ; matyszak, ; williams and hickey, ) ). the function of cytotoxic cd t cells in the inflamed cns and the major histocompatibility complex (mhc) class i antigen expression in this tissue are most pertinent for the topic of this review and will be preferentially discussed in this section. although the cns has classically been considered a site of immune privilege, surveillance of the normal cns by circulating, activated lymphocytes occurs, with a limited number of lymphocytes being present in the normal cns at any given time (hickey, hsu, and kimura, ) . however, considerable data suggest that the immune response in the cns is initiated not in the parenchyma but, rather, in draining lymph nodes located in the deep cervical tissue (cserr and knopf, ) . dendritic cells, critical for antigen presentation, are virtually absent from the normal cns, suggesting that virus antigen must spread to the draining lymph node tissue before activation can occur (hart and fabre, ; matyszak, ; matyszak and perry, ) . in support of this contention, influenza virus inoculated directly into the cns parenchyma does not induce an immune response until virus spreads to the cerebrospinal fluid (csf) and, soon thereafter, to the draining lymph nodes (stevenson et al., a; stevenson et al., b) . once the immune response has been initiated, breakdown of the blood-brain barrier, presence of plasma proteins, and recruitment of leukocytes characterize the cns inflammatory response. notably, cells with the phenotype of dendritic cells are detected as part of the cellular infiltrate in inflammatory processes and are believed to contribute to sustaining the immune response during chronic inflammation (matyszak, ; matyszak and perry, ) . within the cns, cd and cd t cell effector functions are virtually the same as in peripheral sites of inflammation. cd t cells are responsible for the majority of cytotoxic activity and exhibit three effector mechanisms: perforin/granzyme-mediated killing; fas/fas ligand-mediated killing; and cytokine secretion (liu, young, and young, ; smyth and trapani, ) . in one well-documented example, perforin is critical for the cd t cell response to lcmv, both peripherally and in the cns (kagi et al., ) . fas/fasl interactions have not been documented to be critical for cd t cell effector function in the cns, but fasl expression by infiltrating cd t cells has been impli-cated in oligodendrocyte death in inflammatory settings such as experimental allergic encephalomyelitis (eae) and multiple sclerosis (ms) (d'souza et al., ; sabelko et al., ; waldner et al., ) . secretion of interferon-~/(ifn-~/) and of other cytokines are of critical importance in orchestrating multiple components of the cellular and humoral immune responses. furthermore, in some cases, cytokines are capable of directly inhibiting viral replication. for example, ifn-• appears to be critical for virus clearance from infected oligodendrocytes in mice infected with mouse hepatitis virus (parra et al., ) . in another example, genetic disruption of ifn-~ or its receptor inhibits virus clearance in strains of mice normally resistant to infection with theiler's murine encephalomyelitis virus (tmev) (fiette et al., ) . ctls also release chemokines through granule exocytosis, thereby sustaining the immune response by contributing to the influx of other inflammatory cells (reviewed in baggiolini, dewald, and moser, ; luster, ) . the influx of both antigen-specific and nonspecific cells may contribute to virus clearance but also has the potential for increasing damage to nearby uninfected cells (bystander damage). this mechanism has been postulated to be significant in the pathogenesis of several cns diseases with inflammatory components, including demyelination in animals with virus-induced or autoimmune demyelination (houtman and fleming, b; lassmann, ) . one major difference between the cns and extraneural tissue is found in mhc class i antigen expression. in extraneural tissue, mhc class i antigen is expressed constitutively by most cells. however, in the uninflamed cns, constitutive expression of mhc class i and ii antigen is infrequent and is confined to a subset of endothelial cells (mhc class i) and macrophages/microglia (mhc class ii) (reviewed in lampson, ; shrikant and benveniste, ) . expression of these molecules by astrocytes, oligodendrocytes, or neurons is not believed to occur in the normal cns. however, this conclusion may need to be modified since, in a recent study, the coordinated expression of mhc class i antigen (heavy chain and ~ -microglobulin) and of a t cell receptor component (cd ~) by neurons during development was postulated to be important for synapse formation (corriveau, huh, and shatz, ) . mhc class i and class ii expression are upregulated in the cns in multiple pathological settings with infectious or noninfectious etiologies (also reviewed in lampson, ; shrikant and benveniste, ) . as discussed above, mhc class i expression by antigen-presenting cells resident in the cns is not involved in initiation of the ctl response but is likely to be critical for its perpetuation. expression of mhc class i molecules by infected cells is also required for recognition and lysis by activated antigen-specific cd t cells. the most convincing data show that mhc class i and class ii antigens are upregulated on microglia/macrophages in inflammatory diseases of the cns, including ms and tmev-and mhv-induced demyelinating encephalomyelitis. t cell costimulatory molecules such as b - and b - are also upregulated on these cells and, in some cases, activated microglia/macrophages isolated from the cns can directly stimulate antigen-specific t cells proliferation (bo et al., ; matyszak, ; pope et al., ; xue et al., ) . mhc class i and class ii expression by astrocytes and oligodendrocytes has been demonstrated in vitro, although, in some cases, only after exposure to proinflammatory cytokines, such as interferon- (lampson, ; shrikant and benveniste, ) . whether astrocytes and oligodendrocytes upregulate mhc class i and class ii expression in the inflamed cns remains quite controversial, with expression of these molecules being reported in some studies (lampson, ; shrikant and benveniste, ) . in most recent studies, mhc antigen expression by astrocytes and oligodendrocytes in inflamed tissue was not detected (e.g., pope et al., ; xue et al., ) , but these negative conclusions must be tempered because a biologically significant level of expression might not be detectable with presently available assays. mhc class i antigen upregulation on neurons has not been reported in pathological conditions, although a recent set of elegant studies demonstrated the expression of these molecules following inhibition of electrical activity (neumann et al., ; neumann et al., ) . if verified in the infected cns, these results would suggest that only neurons sufficiently damaged by infection with a virus or another infectious agent would be recognized by antigen-specific ctls. furthermore, this mechanism would minimize destruction of infected, but still functionally active, neurons. although there is no agreement on the specifics, these studies clearly demonstrate that mhc class i and ii antigens are expressed on cns-derived cells in vitro and in vivo during inflammatory processes. as a consequence, they provide a framework for interpreting experiments that describe the selection of ctl escape mutants in the cns of virus-infected mice. occurring outside the cns the first experimental evidence for virus escape from ctl recognition by mutation of a cd t cell epitope was provided by pircher et al., ( ) . in mice that were transgenic for a t cell receptor (tcr) specific for lymphocytic choriomeningitis virus (lcmv), infection with lcmv resulted in the rapid appearance of virus mutated in the target ctl epitope. infection of tcr transgenic mice, but not of immunocompe-tent b mice, with mutant lcmv resulted in a delay in the kinetics of virus clearance. similar results were obtained when wild-type mice were infected with lcmv variants in which some or all of the appropriate ctl epitopes were mutated (lewicki et al., ; moskophidis and zinkernagel, ) . notably, lcmv escape mutants are not generated in immunocompetent mice after infection with wild-type virus. in these mice, ctls recognizing multiple epitopes are detected, suggesting that an immune response directed against several epitopes precluded the selection of ctl escape mutants, at least in settings in which virus clearance was relatively efficient. a subsequent study suggested that ctl escape mutants may emerge and become the predominant virus in human populations in which a single hla allele is overrepresented, and in which an immunodominant epitope is restricted by that allele. the strain of epstein-barr virus (ebv) circulating in humans residing in the coastal regions of papua new guinea contains a mutation in an immunodominant cd t cell epitope that diminished binding to the hla-all molecule (de campos-lima et al., ) . hla-all is present at a high frequency in this population, suggesting that the selection of virus encoding this mutation was immune-driven. however, this conclusion may not be warranted since the same mutation is found in virus circulating in the highland populations of papua new guinea, even though the hla-all allele is not overrepresented in that group (burrows et al., ) . the studies of lcmv-infected mice suggested that efficient virus clearance precluded the selection of ctl escape mutants. conversely, less efficient virus clearance would be predicted to facilitate the emergence of these mutants. therefore, the potential contribution of ctl mutants to pathogenesis was examined next in several persistent infections, including human and nonhuman primates chronically infected with the human immunodeficiency virus (hiv- ), hepatitis b virus (hbv), and hepatitis c virus. the emergence of ctl escape mutants during the course of persistence was documented in each of these infections (franco et al., ; mcmichael and phillips, ; weiner et al., ) . the most convincing data suggesting a role for these variants in disease progression comes from studies in which the emergence of ctl escape mutants are documented early during the infection. selection at early times postinfection is consistent with a role in delaying the elimination of virus from the infected host. in one study, borrow et al. ( ) described an hiv-infected patient in which the initial ctl response was directed at a single hla b -restricted epitope. early in the course of the infection, a mutation in a presumptive anchor residue was detected, leading to a loss of recognition by the patient's ctls. shortly thereafter, the ctl response spread to addi-stanley perlmanandgregoryewu tional, presumably less immunodominant, epitopes. these responses were able to control the virus for a short period, but were, ultimately, inadequate because the patient exhibited a rapid rate of disease progression with a fatal outcome. in another study, mutations resulting in ctl escape were identified in the nefgene in a patient during primary hiv infection. these mutations abrogated recognition by nef-specific ctls harvested from the patient, suggesting that their selection was also immune-driven and that it contributed to virus persistence (price et al., ) . in still another study, ctl escape mutants were identified in hivinfected patients several years after the primary infection, coincident with increased virus replication and decreased cd t cell counts . it is uncertain, in these cases, whether ctl escape caused disease progression or whether it was a consequence of increasingly ineffectual control of virus replication by other components of the immune system, such as antibodies and cd t cells. ctl escape mutants that emerge only after immune surveillance has diminished may still contribute to disease progression in hiv-infected patients, even if they are not the primary cause for the loss of control by the immune system. ctl escape mutants were also selected in an hiv-infected individual treated with ctls directed against a single epitope (koenig et al., ) , thereby showing that, as in lcmvinfected tcr transgenic mice, a strong, monospecific ctl response facilitated their appearance. ctl escape variants are occasionally detected in patients chronically infected with hbv, usually in the context of a narrowly focused, strong immune response (bertoletti et al., a; bertoletti et al., b) . a more common finding in patients infected chronically with hbv is the lack of a significant ctl response (rehermann et al., ) , suggesting that variation in hbv-specific t cell epitopes is important in only a minority of patients. similarly, ctl escape mutants were detected in a chimpanzee chronically infected with hepatitis c virus (weiner et al., ) , but persistence appears to be associated, most commonly, with a weak ctl response (rehermann et al., ) . mutations in an epitope or in its flanking sequences can result in escape from surveillance by cd t cells, by affecting any one of several steps along the pathway used to generate peptides for presentation by mhc class i molecules. antigen processing involves proteolytic cleav-age by proteosomes in the cytoplasm and transport of the resulting peptides to the endoplasmic reticulum for binding to the mhc class i molecule. mutations in sequences flanking a ctl epitope may affect proteolytic cleavage or transport into the endoplasmic reticulum. only a few examples of mutations affecting either of these processing steps have been described (eisenlohr, yewdell, and bennink, ; ossendorp et al., ) . in one example, comparison of the sequence of an immunodominant ctl epitope encoded by two related murine leukemia virus families (akv/mcf and friend/moloney/rauscher [fmr] ) showed that a single amino acid change present in the fmr strains abrogated cd t cell recognition (ossendorp et al., ) . the mutation did not affect binding of peptide to the mhc class i molecule or immunogenicity of a peptide corresponding to the variant epitope. rather, the loss of recognition occurred only after endogenous processing of antigen and resulted from alterations in proteosomal processing, which were shown using purified s proteosomes in vitro. these conclusions assume that processing in vitro mimics what occurs in the cell and that other differences in the amino acid sequence between the two viruses did not contribute to the observed differences. mutations in sequences flanking a ctl epitope are difficult to detect, and therefore may be more important in ctl escape than is appreciated at present. more commonly, mutations in ctl epitopes that affect binding to the mhc molecule, or affect recognition by the tcr, have been identified. some of the mutations described above, in hiv- infected individuals, diminish binding to the mhc class i molecule or result in increased rates of dissociation from the mhc molecule (reviewed in mcmichael and phillips, ) . mutations that impair binding to the mhc molecule are the simplest to interpret and, depending on the diminution of binding, will completely abrogate recognition by epitopespecific cd t cells. mutations that affected binding to tcrs were first reported in studies describing selection of lcmv ctl escape mutants in mice transgenic for an lcmv-specific tcr (pircher et al., ) . peptides mutated in residues that directly contact the tcr have also been described in virus isolated from hiv-infected patients . these mutations may result in partial or complete loss of recognition by autologous ctls. during the course of these studies on viral variation, mutations were identified in viral rna and dna isolated from patients persistently infected with hiv- or hbv that not only diminished recognition by epitope-specific cd t cells, but also inhibited recognition of wild-type epitope if both epitopes were present in the infected cell (bertoletti et al., b; klenerman et al., ) . the mechanism of this phenomenon (tcr antagonism) is not completely understood, but is believed to involve delivery of an altered signal to the cd t cell by the mutated peptide/mhc class i complex, resulting in nonresponsiveness or diminished responsiveness to the target agonist peptide (sette et al., ) . although tcr antagonism has only been demonstrated by using ctl clones, it has also been documented to occur when variant and wild-type epitopes are presented by different cells, a scenario that presumably mimics the situation in the infected host (meier et al., ) . another mechanism similar to antagonism is interference with ctl priming (plebanski et al., ) . in this case, a variant epitope does not inhibit ctl effector function but, rather, the generation of activated antigen-specific ctls from naive precursor cd t cells. this mechanism, identified in humans and mice infected with malaria, has not yet been described in any viral disease. one of the best examples of a pathological setting in which ctl escape mutants contribute to virus persistence and the development of disease is that of mice infected with the neurotropic coronavirus, mouse hepatitis virus--strain jhm (mhv-jhm). selection of escape mutants was not anticipated because immune selective pressure in the cns, a site that is relatively protected from immune surveillance, might be predicted to be less than in the periphery. neurotropic coronaviruses, including mhv-jhm, the a strain of mhv (mhv-a ) and mhv- , cause acute and chronic infections of the cns in susceptible mice and rats (reviewed in houtman and fleming, b; lane and buchmeier, ; stohlman, bergmann, and perlman, ) . mhv-jhm is highly neurovirulent and infection of susceptible mice results in widespread infection of neurons with a rapidly fatal outcome. however, more generally interesting are those model systems in which mhv-jhm and mhv-a are induced to cause either acute or chronic demyelination. general strategies for modifying the acute infection have been recently reviewed (perlman, ; stohlman, bergmann, and perlman, ) , and two general approaches will be briefly described here. in the first, mice are infected with attenuated virus. attenuated virus was cloned from pools of virus harvested from suckling mouse brain (stohlman et al., ) . alternatively, viral mutants were selected by chemical mutagenesis or by treatment with neutralizing antibody directed against the surface (s) glycoprotein (dalziel et al., ; fleming et al., ) . in each case, the variant virus causes acute encephalitis in only a small percentage of mice but is able to persist and cause demyelination in most survivors. in mice infected with these variants, infectious virus is, in general, cleared by - weeks after inoculation. clinical symptoms are most evident within the same time frame. mice that survive the infection slowly recover, although evidence of ongoing demyelination can be detected for several months after the acute infection has resolved. in the second approach, mice are infected with wild-type mhv-jhm and protected from the acute encephalitis by administration of anti-mhv antibody or mhv-specific cd or cd t cells (buchmeier et al., ; stohlman et al., ; yamaguchi et al., ) . this intervention also results in decreased infection of neurons, but does not prevent infection of cells in the white matter or demyelination. virus clearance is enhanced by treatment with protective antibodies or t cells in some studies (buchmeier et al., ; yamaguchi et al., ) , but not all . in a variation of the latter approach, suckling c bl/ (b ) mice are protected from acute encephalitis with nursing by dams previously immunized to mhv-jhm . the suckling mice are protected from acute encephalitis and remain asymptomatic for - weeks. at that time, a variable percentage ( - %) develop histological evidence of demyelination and clinical signs of hind-limb paralysis. immunization of the dams may be accomplished either by active immunization with infectious virus , or by passive infusion of neutralizing antibody (pewe, xue, and perlman, ) , and with similar outcomes. the temporal course of this disease is very different from that observed in other models of mhv-jhm-induced demyelination, because mice are asymptomatic for several weeks before developing disease, and because infectious virus is not cleared. in a series of reports, pewe et al showed that escape from the cytotoxic cd t cell response was a major factor in the disease progression in these mice (pewe et al., ; pewe, xue, and perlman, ; pewe, xue, and perlman, ) . ctl escape mutations were detected in all mice that developed clinical disease several weeks postinfection in this model. in b mice, two cd t cell epitopes, encompassing residues - (s- - ) (h- db-restricted) and - (s- - (h- kb-restricted), of the s glycoprotein are recognized (bergmann et al., ; castro and perlman, ) . these two epitopes are located within a region of the protein that is prone to both mutation and deletion (termed the "hypervariable region") (banner, keck, and lai, ; stanley perlman and gregory f. wu parker, gallagher, and buchmeier, ) . the location of the epitopes within a region that can tolerate mutation, without loss of function, most likely contriljutes to the selection of ctl escape mutants. mutations have only been detected in epitope s- - , the immunodominant epitope of the two , and not in epitope s- - . mutations are not detected in regions of the genome flanking epitope s- - or in most mhv-specific cd t cell epitopes. the one possible exception is that a mutation is detected in a cd epitope encompassing residues - of the s glycoprotein (s- - ) in several mice, although wild-type epitope is also detected in the same animals. the significance of this mutation is not known, because it does not appear to decrease recognition by cd t cells specific for the epitope . mutations have been detected in amino acids at positions - of epitope s- - , with only a single variant generally being detected in any individual infected mouse (pewe, xue, and perlman, ) . a summary of mutations detected thus far is shown in fig. . based on the crystallographic structure of the h- d b molecule and on mutagenesis studies (hudrisier et al., ; young et al., ) , some of these mutations are predicted to affect binding to the mhc class i molecule, whereas others inhibit binding to the tcr of the cd t lymphocyte. mutations in epitope s- - are selected within - days postinfection, a time at which virus is not cleared in other mhv infections (houtman and fleming, a; lin et al., ) . detection of ctl escape mutants at a time before virus clearance is expected to be complete is consistent with a role for these mutants in virus persistence. notably, epitope s- - -specific ctl activity can be detected in the cns as early as days postinfection, prior to the detection of ctl escape mutants (unpublished observations). mutations are also not detected in infected mice with severe combined immunodeficiency (scid mice), suggesting that their selection is cd t cell-driven (pewe, xue, and perlman, ) . in this model, mice that do not develop hind-limb paralysis by - days postinfection remain asymptomatic. mutations are only occasionally detected in asymptomatic mice at - days postinfection suggesting that escape from cd t cell surveillance correlates well with virus amplification in b mice but is not sufficient for the development of clinical disease. as discussed above, the role of ctl escape mutants in viral pathogenesis is controversial. these mutants are not expected to be relevant to pathogenesis if the ctl response is directed at several ctl epitopes, as is believed to occur in many human infections. it has also been postulated that, even if a single epitope is immunodominant, a t cell summary of mutations detected in epitope s- - in virus isolated from mice with hind-limb paralysis. a panel of peptides resulting from single nucleotide changes in the sequence of the wild-type epitope were tested in cytotoxicity assays. only those changes resulting in a -fold decrement in activity are included in the figure. the amino acids listed above the wild-type sequence have been detected in virus isolates, whereas those listed below the wild-type sequence (underlined) have not yet been detected. the l to p change in position results in only a -fold decrease in recognition but has been detected in a minority of cdna clones in single mice with hind-limb paralysis (marked with parentheses). *mhc (m) or tcr (t) contact-based on published reports (hudrisier et al., ; hudrisier et al., ; young et al., ) . response that is comprised of many cd t cell clonotypes would preclude ctl escape, because a single mutation would affect recognition by only a subset of the epitope-specific t cells (ishikawa et al., ) . to determine the biological significance of the mutations that were detected in mhv-infected mice, several additional experiments were performed. first, a panel of variant epitopes were analyzed in cytotoxicity assays, using either lymphocytes derived from the cns of mice with acute encephalitis directly ex vivo or bulk populations of splenocytes cultured in vitro for weeks with peptide s- - pewe et al., ) . this panel comprised all the peptides resulting from single nucleotide changes in epitope positions - and . these assays showed that nearly all of the mutations that were stanley perlman and gregory f. wu selected in vivo resulted in a complete or substantial loss of recognition by either population of lymphocytes. several additional mutations also resulted in a nearly complete loss of recognition by these lymphocytes, but have not yet been detected in vivo (fig. ) strikingly, some mutations in position , a secondary anchor for binding to the mhc molecule, resulted in loss of recognition, but were never selected in infected mice. whether this apparent selectivity reflects a sampling error, or is in fact biologically driven (i.e., results in detrimental changes in rna or protein structure), remains to be determined. the biological significance of these mutations was also addressed by infecting maternal antibody-protected suckling c bl/ mice with virus mutated in epitope s- - . mice infected with this virus do not recognize epitope s- - but still respond to epitope s- - . variant virus-infected mice have significantly greater mortality and morbidity when compared to mice infected with wild-type virus (pewe, xue, and perlman, ) . these results suggest that the cd t cell response to epitope s- - is an important component of the host response to mhv-jhm, and support the idea that escape from this response is beneficial to the virus and results in increased virus replication and disease progression. these results suggest that the ctl response to epitope s- - is monospecific, because single mutations in residues important for binding to either mhc antigen or the tcr result in substantial loss of activity in cytotoxicity assays. the monospecificity of the response is consistent with a monoclonal or an oligoclonal response to the epitope. to directly assess the diversity of the t cell response, tcr v~ element usage and the complexity of the complementarity determining region (cdr ) were determined. the tcr is a heterodimer consisting of an a and a ~ chain. the great diversity in the t cell response results from the large number of different v, d, and j elements in the germ line, coupled with imprecise joining at the v-d and d-j junctions ([ chains) or v-j junction (a chain). the cdr encompasses the junctional region of the a and chains and makes direct contact with the mhc/peptide complex. analysis of a monoclonal or oligoclonal t cell response should reveal usage of only one or a small number of different v~ and va elements and minimal heterogeneity of the cdr . sequence analyses of the cdr were facilitated by the use of soluble mhc/peptide tetramers specific for epitope s- - (tetramer s- ). soluble mhc class i/tetramers were first described by altman et al., and have been shown, in several subsequent studies, to detect antigen-specific t cells with high sensitivity and specificity (e.g., altman et al., ; flynn et al., ; murali-krishna et al., ) . epitope s- - -specific cd t cells were isolated directly from the cns of mice with acute encephalitis by sorting with a fluorescentactivated cell sorter (facs) after staining with anti-cd antibody and tetramer s- . the tetramer s- -positive t cells were analyzed initially for v~ usage. the results showed usage of a large number of different v[~ elements by cd t cells responding to the epitope, with preferential usage of some v~ elements occurring, specifically v~ and v[~ . analysis of individual cdr sequences from a subpopulation of tetramer s- -positive cells, those expressing the v~ element, revealed substantial heterogeneity. the cdr sequences were shown to fit, approximately, a logarithmic distribution (fisher, corbet, and williams, ; taylor, kempton, and woiwod, ) . from this distribution, it was possible to calculate that approximately - epitope s- - -specific cd t cell clonotypes were present in the cns of a mouse with acute encephalitis. similar measurements using lymphocytes harvested from the cns of mice with chronic demyelination revealed that approximately - different clonotypes were present in these animals. previous studies suggested that either , (butz and bevan, ) or (bousso et al., ) precursor cd t cells for any single epitope were present in an individual mouse. our calculation of the number of t cells recognizing epitope s- - agrees more closely with the lower estimate. notably, none of the wild-type epitope s- - rna, or little of it, is present in the cns of mice with chronic demyelination, but cd t cells specific for the epitope are still detectable. whether this represents stimulation by residual wild-type antigen (which may be cleared more slowly than viral rna (knopfet al., ; zhang et al., ) , or by variant sequence remains to be determined. furthermore, many of the [~ chain cdr sequences were detected in more than one mouse. in the case of mice with chronic demyelination, very few cdr sequences were unique to a single animal. thus, unlike other experimental systems (bousso et al., ) , the repertoire of cd t cell tcrs recognizing epitope s- - is not unique to each naive animal but is, in large part, common among all c bl/ mice. this lack of variability may facilitate the emergence of ctl escape mutants in a large fraction of infected mice. these results suggest that ctl escape mutants are critical in virus persistence and in the development of clinical disease in mice infected with mhv-jhm. these results also show that ctl escape mutants are selected in the presence of a polyclonal, but monospecific, cd t cell response. as already mentioned, the importance of ctl escape stanley perlman and gregory f. wu mutants in other infections is much less certain, suggesting that the following features, present in the mhv-jhm-infected cns, must predispose to the selection of these mutants: . ctl escape mutants are observed in mhv-infected rodents only when b mice are inoculated at the suckling stage with virulent wildtype virus and protected from acute disease by nursing by immunized dams. inoculation with virulent mhv-jhm and use of b mice are essential for disease to develop. chronic demyelination does not develop if suckling mice are inoculated with the less neurovirulent a strain of mhv (unpublished observations). in most other models of mhv persistence, older mice are inoculated with attenuated strains. persistence is manifested by the presence of viral rna and ongoing demyelination in the absence of infectious virus (adami et al., ; houtman and fleming, a; rowe et al., ) . ctl escape mutants have not been detected in these models, possibly because clearance of infectious virus is so rapid . a normal immune system is also required, however, because even when clearance is delayed in mice infected with attenuated virus, as occurs in immunodeficient mice, ctl escape mutants are not commonly selected . . infection with wild-type virus at the suckling stage ( - days old) is also crucial in this model. three-week-old b mice inoculated with wild-type virus and protected from acute encephalitis by passive administration of small amounts of neutralizing antibody are protected from acute encephalitis, but they do not develop hind-limb paralysis at later times (sun and perlman, ) . demyelination can be detected in the spinal cords of these mice at months postinfection. these mice have not formally been assessed for the presence of ctl escape mutations at late times after infection, but they clearly do not develop a clinical disease consistent with the selection of ctl escape mutants. these results suggest that inoculation of mice with virulent virus at the suckling stage is essential for the selection of ctl escape mutants or, as a minimum, for their clinical manifestation. this requirement for inoculation of suckling mice may reflect the immaturity of the immune system. a consequence of infecting mice at days of life may be suboptimal clearance of virus. consistent with this possibility, after inoculation at the suckling stage, low levels of infectious virus can be detected at days postinfection in balb/c mice (unpublished observations) . balb/c mice never develop evidence of hind-limb paralysis or other clinical disease at any time and develop completely normally (castro et al., ; . . another consequence of inoculation of mice with an immature immune system may be an aberrantly polarized immune response. a striking feature of the immune response in most c bl/ mice infected with mhv at the suckling stage is the lack of an appreciable antibody response, when they are assayed by elisa or in neutralizing assays (jacobsen and perlman, ; . the cd and cd t cell response in maternal antibody-protected mice that develop chronic demyelination is proinflammatory and readily detected (castro et al., ; . this strongly polarized, cell-mediated immune response in the presence of a defective humoral response may also predispose to the selection of ctl escape mutants. in support of this, balb/c mice inoculated at the suckling stage in the maternal antibody model mount a significant antibody response and do not develop hind-limb paralysis (castro et al., ; . . mhv infection of congenic b .a( r) (kbddl d) mice results in the development of hind-limb paralysis, albeit at a lower frequency than in either b or c bl/ mice (castro et al., ) . two ctl epitopes, s- - and an la-restricted epitope encompassing residues - of the nucleocapsid (n) protein (n- - ) , are recognized in these mice. the immunodominant epitope recognized in b mice, epitope s- - , is not recognized in b .a( r) mice since these mice do not encode the h- d b allele. virus isolated from b .a( r) mice with hind-limb paralysis was evaluated for the presence of mutations in epitopes s- - and n- - , and none were detected. the development of hind-limb paralysis in b .a( r) mice, in the absence of mutations in either ctl epitope, suggests that a factor expressed on the c bl/ background (decreased production of antibody?) may contribute to the increased amount of virus replication observed in these mice. the outgrowth of ctl escape mutants in b mice, in conjunction with this other putative factor, results in a much higher rate of clinical disease than is observed in b .a( r) mice in which ctl escape mutants are not selected. . a ctl response directed at a single immunodominant epitope enhances the likelihood that ctl escape mutants will be selected. in b mice, two mhv-specific epitopes are recognized. cytotoxicity assays using cns-derived lymphocytes from mice with acute encephalitis, directly ex vivo (castro and perlman, ) , and limiting dilution assays using splenocytes harvested from mice intraperitoneally immunized with mhv , suggested, in both cases, that the two epitopes were recognized by similar numbers of cd t cells. more recent measurements, using mhc class i peptide/tetramers to detect antigen-specific cells or methods to detect interferon-~ production after stimulation with peptide, confirmed stanley perlman and gregory f. wu these results (unpublished observations). however, much more peptide s- - is required to sensitize target cells for lysis. thus, the ctl response in these mice may be functionally directed at a single epitope. conversely, a ctl response to a single immunodominant epitope is not sufficient for ctl escape mutants to be selected. the ctl response in balb/c mice is directed at a single epitope (n- - ), yet these mice do not develop hind-limb paralysis in the maternal antibody-protection model (castro et al., ) , and ctl escape mutants are not selected (unpublished observations). the ability of the virus to tolerate mutations in the part of the protein that contains the target cd t cell epitope is also important. epitope s- - is in a region of the s protein prone to deletion and mutation, while the balb/c epitope is located in a highly conserved region of the n protein. as mentioned above, mutations in this epitope are not detected in b .a( r) mice, even though the background of this strain may be favorable for the selection of ctl escape mutants. . mhv-jhm is neurotropic and the infection caused by this agent is confined, for the most part, to the cns. as discussed above, the processes of initiation of an inflammatory response, trafficking to the site of inflammation, and antigen presentation within the cns differ in several aspects from similar processes occurring at extraneural sites. in particular, mhc class i expression is minimal in the normal cns, and while clearly upregulated within the infected cns, determining the cellular sites of expression remains an area of active research. therefore, while it is speculative at present, it is formally possible that infection within the cns results in a modified immune response that increases the likelihood that ctl escape mutants will be selected during the course of an infection. what can be concluded from these studies about the role of ctl escape mutants in the pathogenesis of viral infections in the cns? the following conclusions may also be applicable for ctl escape mutants arising in extraneural tissue. first, ctl escape mutants are not commonly selected in most infections. one explanation for this is that the selection of ctl escape mutants is uncommon if the cd t cell response is directed against several epitopes. however, a cd t cell response directed against one epitope or a few, has been observed in several human and experimental infections (cole, hogg, and woodland, ; flynn et al., ; lehner et al., ; moss et al., ; murali-krishna et al., ; wallace et al., ) --suggesting that an immunodominant response is not unusual. nevertheless, ctl escape mutants are not detected in most experimental settings even when only one epitope is immunodominant, or a few are. second, the recognition of an epitope by a diverse population of cd t cell clonotypes does not prevent emergence of these variants. what does appear to be true, however, is that the diversity of the response matters less than whether the different cd t cell clonotypes recognize the same or different parts of the mhc/peptide complex. a response narrowly focused on one part of the complex will facilitate the selection of ctl escape mutants. the ctl response to epitope s- - is very sensitive to changes in residues and pewe et al., ) , two residues important for binding to the tcr (young et al., ) . strongly focused recognition of central residues of an immunodominant h- kb-restricted epitope, which is recognized in sendai virus-infected b mice, has also been reported (cole, hogg, and woodland, ) . third, in mice infected with mhv and in some humans persistently infected with hiv- , hbv, or hepatitis c virus, ctl escape mutants play an important role in virus amplification and disease progression. in most cases, however, it is likely that there would not have been sufficient time for selection of ctl escape mutants if virus clearance were rapid and complete. thus, selection of ctl escape mutants in the absence of any other factor is probably not sufficient to prevent rapid virus clearance from an infected host. in mhv-infected b mice, their selection appears to be facilitated by a suboptimal humoral immune response. this deficient antibody response may be a consequence of infection of suckling mice in the presence of maternally derived antibody. maternal immunization has been shown to impair immune responses in other infections (wang et al., ) , although it has never been shown to affect selectively humoral, and not cellular, immunity. fourth, an interesting aspect of this model system is the number of questions that it raises about the dynamics of mhv growth and cellular tropism in the cns. unlike the situation in mice chronically infected with tmev, in which macrophages are the main reservoir for virus (lipton, twaddle, and jelachich, ) , no single type of cns cell has been identified as the predominant target in mice persistently infected with mhv. the selection of ctl escape mutants in the cns of mhv-infected mice demonstrates that mhv infection of a cell expressing mhc class i antigen is a critical part of the pathogenic process. astrocytes, macrophages/microglia, and oligodendrocytes are all infected in these animals xue et al., ) . of all of these cells, only macrophages/microglia express detectable levels of mhc class i antigen in mhv-infected mice with chronic demyelination (xue et al., ) . one interpretation of these results is that macrophages/microglia are in fact the primary site of productive virus infection, with replication in oligodendrocytes or astrocytes being of secondary importance or abortive. infection of the latter two cells might be crucial for the development of clinical disease, but might be less important for propagation of the virus. alternatively, astrocytes or oligodendrocytes might express mhc class i antigen, albeit at levels below the detection limits of assays presently available. escape from cd t cell surveillance might occur in these cells, thereby resulting, simultaneously, in increased virus replication and disease progression. infection with mhv affects mhc class i antigen expression by astrocytes in vitro and in newborn mice but this has not been demonstrated in vivo in older mice (correale et al., ; gilmore, correale, and weiner, ; suzumura et al., ; suzumura et al., ) . conversely, proof that astrocytes or oligodendrocytes were the most important sites of mhv-jhm replication and, therefore, the sites where selection of ctl escape mutants occurred, would provide evidence that these cells express mhc class i antigen. evolution of mouse hepatitis virus (mhv) during chronic infection: quasispecies nature of the persisting mhv rna phenotypic analysis of antigen-specific t lymphocytes human chemokines: an update a clustering of rna recombination sites adjacent to a hypervariable region of the peplomer gene of murine coronavirus variability of persisting mhv rna sequences constituting immune and replication-relevant domains the jhm strain of mouse hepatitis virus induces a spike protein-specific 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is virus strain specific late escape from an immunodominant cytotoxic t-lymphocyte response associated with progression to aids demonstration and characterization of ia-positive dendritic cells in the interstitial connective tissues of rat heart and other tissues, but not brain t-lymphocyte entry into the central nervous system dissociation of demyelination and viral clearance in congenitally immunodeficient mice infected with murine coronavirus jhm pathogenesis of mouse hepatitis virusinduced demyelination binding of viral antigens to major histocompatibility complex class i h- d b molecules is controlled by dominant negative elements at peptide non-anchor residues relative implication of peptide residues in binding to major histocompatibility complex class i h- db: application to the design of high-affinity, allele-specific peptides polyclonality and multispecificity of the ctl response to a single viral epitope localization of virus and antibody response in mice infected 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parenchyma without immunologic control the immunogenicity of intracerebral virus infection depends on anatomical site persistent infection by mouse hepatitis virus murine coronaviruses: isolation and characterization of two plaque morphology variants of the jhm neurotropic strain in vivo effects of coronavirus-specific t cell clones: dth inducer cells prevent a lethal infection but do not inhibit virus replication spread of a neurotropic coronavirus to spinal cord white matter via neurons and astrocytes induction of glial cell mhc antigen expression in neurotropic coronavirus infections coronavirus infection induces h- antigen expression on oligodendrocytes and astrocytes diversity statistics and the logseries model fas-and fasl-deficient mice are resistant to induction of autoimmune encephalomyelitis the cytotoxic t-cell response to herpes simplex virus type infection of c bl/ mice is almost entirely directed against a single immunodominant determinant effect of passive immunization or maternally transferred immunity on the antibody response to a genetic vaccine to rabies virus persistent hepatitis c virus infection in a chimpanzee is associated with emergence of a cytotoxic t lymphocyte escape variant traffic of hematogenous cells through the central nervous system antigen specificity of cd t cell response in the central nervous system of mice infected with mouse hepatitis virus depletion of blood-borne macrophages does not reduce demyelination in mice infected with a neurotropic coronavirus protection of mice from a lethal coronavirus infection in the central nervous system by adoptive transfer of virus-specific t cell clones the three-dimensional structure of h- d at . a resolution: implications for antigen-determinant selection directional and compartmentalised drainage of interstitial fluid and cerebrospinal fluid from the rat brain immunology taught by viruses this work was supported in part by grants from the national institutes of health (nih) (ns and ai ) and from the national multiple sclerosis society. g.f.w. was supported in part by a national research service award from the nih. key: cord- -autprmr authors: burrell, louise m.; johnston, colin i.; tikellis, christos; cooper, mark e. title: ace , a new regulator of the renin–angiotensin system date: - - journal: trends in endocrinology & metabolism doi: . /j.tem. . . sha: doc_id: cord_uid: autprmr abstract angiotensin-converting enzyme (ace) is a zinc metalloproteinase and a key regulator of the renin–angiotensin system (ras). ace is a newly described enzyme identified in rodents and humans with a more restricted distribution than ace, and is found mainly in heart and kidney. ace cleaves a single residue from angiotensin i (ang i) to generate ang – , and degrades ang ii, the main effector of the ras, to the vasodilator ang – . the importance of ace in normal physiology and pathophysiological states is largely unknown. ace might act in a counter-regulatory manner to ace, modulating the balance between vasoconstrictors and vasodilators within the heart and kidney, and playing a significant role in regulating cardiovascular and renal function. angiotensin-converting enzyme (ace) is a zinc metalloproteinase and a key regulator of the renin-angiotensin system (ras). ace is a newly described enzyme identified in rodents and humans with a more restricted distribution than ace, and is found mainly in heart and kidney. ace cleaves a single residue from angiotensin i (ang i) to generate ang - , and degrades ang ii, the main effector of the ras, to the vasodilator ang - . the importance of ace in normal physiology and pathophysiological states is largely unknown. ace might act in a counter-regulatory manner to ace, modulating the balance between vasoconstrictors and vasodilators within the heart and kidney, and playing a significant role in regulating cardiovascular and renal function. angiotensin-converting enzyme (ace), a dipeptidyl carboxypeptidase, is a key enzyme in the renin -angiotensin system (ras); it converts the inactive decapeptide, angiotensin i (ang i; or ang - ), to the active octapeptide and potent vasoconstrictor ang ii (or ang - ) (figure ), and inactivates the vasodilator bradykinin [ ] . ang ii is thought to be responsible for most of the physiological and pathophysiological effects of the ras, and inhibitors of ace that reduce the formation of ang ii have been highly successful in the management of hypertension, are standard therapy following myocardial infarction to delay the development of heart failure, and reduce the rate of progression of renal disease [ , ] . recently, however, the classical view of the ras has been challenged by the discovery of the enzyme ace [ , ] , in addition to the increasing awareness that many angiotensin peptides other than ang ii have biological activity and physiological importance [ ] . the reported vasodilatory actions of ang - [ ] , along with the potential involvement of ace in both ang ii degradation and ang - production, add another level of complexity to the ras [ , ] . it is predicted that cardiovascular disease will be the leading cause of death by , and although current approaches to block the ras have been of major benefit in this area, it is now clear that other pathways and enzymes within the ras can modulate its main effector, ang ii. we must question our understanding of the 'classical' ras, and work towards unravelling the complexity of the 'new' ras ( figure ). this should result in alternative strategies for the treatment of cardiovascular disease in the future. ace structure and function ace is the first known human homologue of ace, and was cloned from a human heart failure cdna library [ ] and a human lymphoma cdna library [ ] . analysis of the genomic sequence of ace has revealed that the gene contains exons and maps to chromosomal location xp [ ] . the full-length, human ace cdna predicts a protein of amino acids that has % homology with the n-terminal catalytic domain of ace, and a hydrophobic region near the c-terminus, which probably serves as a membrane anchor. like ace, ace is predicted to have the topology of a type membrane protein, with the catalytic domain on the extracellular surface. unlike somatic ace, ace has only one active enzymatic site and functions as a carboxypeptidase rather figure . the renin -angiotensin system (ras) pathway. schematic diagram of the classical ras (left), showing the main pathway for angiotensin ii (ang ii) generation from ang i via angiotensin-converting enzyme (ace). in the classical view, ang ii mediates all known effects via the at receptor. on the right is the updated view of the ras, showing the role of ace in degrading ang i to ang - , and ang ii to the vasodilator ang - . in this version of the ras, ang ii also mediates effects via the g-protein-coupled at receptor, whereas ang - acts through the mas receptor. than a dipeptidyl carboxypeptidase. thus, ace removes a single c-terminal leu residue from ang i to generate ang - , a peptide with no known function. although ace was described originally for its ability to generate ang - from ang i [ ] , it also degrades ang ii to the biologically active peptide, ang - [ ] ( figure ). indeed, in vitro studies indicate that the catalytic efficiency of ace for ang ii is -fold greater than for ang i [ ] , indicating that the major role for ace is the conversion of ang ii to ang - . the potential role of ang - as a cardioprotective peptide with vasodilator, anti-growth and anti-proliferative actions has been recognized relatively recently [ , ] . taken together, the data suggest that ace might function to limit the vasoconstrictor action of ang ii through its inactivation, in addition to counteracting the actions of ang ii through the formation of the agonist, ang - . recent studies [ ] have identified the g-protein-coupled receptor encoded by the mas protooncogene, mas, as the receptor for ang - [ ] . genetic deletion of the mas receptor abolished the binding of ang - to whole mouse kidney, whereas the functional significance of ang - and its receptor was demonstrated by studies showing that mas-deficient mice could not respond with antidiuresis to ang - after a water load, and that the aortas of mas-deficient mice were unable to relax in response to ang - [ ] . in addition, an unexpected function of ace has recently been identified and characterized; ace is a functional receptor for coronaviruses, including the coronavirus that causes severe acute respiratory syndrome, and is involved in mediating virus entry and cell fusion [ , ] . although not directly relevant to cardiovascular function, this would indicate that the ras including ace has multiple roles in physiology and various pathophysiological states [ ] . ace distribution ace has a much more restricted distribution compared with ace, and in humans ace transcripts have been identified in the heart, kidney and testis [ , ] . like ace, ace is found in endothelial cells, and is present to a lesser degree in vascular smooth muscle cells. in the kidney, ace is found in the proximal tubular epithelium. recently, ace has been identified in the gastrointestinal tract, brain and lung [ ] , suggesting a more ubiquitous distribution than initially thought. the precise physiological function of ace is currently under intense investigation. there has been increasing interest in peptides that are either cleaved or generated by this enzyme. in addition to effects on the angiotensin peptides, ace cleaves the c-terminal residue of the peptides des-arg -bradykinin, neurotensin - and kinetensin [ ] , and hydrolyses apelin- and dynorphin a - with as high a catalytic efficiency as ang ii [ ] . ace has no effect on bradykinin, in contrast to ace, emphasizing the specificity of ace [ , ] . many of the in vitro substrates of ace have actions that are relevant to cardiovascular regulation; apelin is a potent cardiac inotrope [ ] , dynorphin a is an endogenous opioid neuropeptide and des-arg -bradykinin binds to the bradykinin b receptor, which is activated by inflammation and tissue injury [ ] . it is not yet known whether the in vitro substrates of ace are also physiological in vivo substrates, and further studies are needed that address in vivo changes in the levels of putative substrates or products of ace using ace -knockout mice [ ] , ace transgenic animals [ ] and ace agonists, in addition to potent, selective ace inhibitors [ , ] . interestingly, the in vitro enzymatic activity of ace is unaffected by ace inhibitors [ , ] , but there are no data as to the effect of angiotensin receptor blockers on ace activity. in the future, it will be of great interest to assess the in vivo effects of ace inhibitors, particularly in light of the interesting findings with regard to cardiac function, which have been seen in both the ace knockout and transgenic animals [ , ] . it has been hypothesized that ace might protect against increases in blood pressure and that ace deficiency leads to hypertension. in several rat models of experimental hypertension, the gene for ace maps to a defined quantitative trait locus on the x chromosome previously identified as a quantitative locus for blood pressure [ ] . an association between ace and blood pressure has also been reported; in the spontaneously hypertensive rat (shr) and shr stroke prone rats, renal ace levels are reduced compared with normotensive wistar-kyoto rats (wky) [ ] , and we have confirmed that renal ace mrna is reduced in shr compared with wky [ ] . it is not clear whether ace deficiency plays a pathophysiological role to cause hypertension or is simply a consequence of increased blood pressure, and the precise role of ace in hypertension requires clarification. ace and the heart evidence of the biological role of ace in angiotensin peptide degradation comes from studies in ace -knockout mice, which lack ace protein [ ] . these mice develop abnormal heart function, with severely impaired cardiac contractility, and the decrease in function is both sex and time dependent, with more severe abnormalities in male than in female mice, and a more pronounced phenotype in older animals. the importance of ace in cleaving and/or inactivating ang ii is indicated by the increase in plasma, cardiac and kidney ang ii levels in the ace -knockout animals, and confirmed by studies in which genetic ablation of aceon an ace mutant background completely rescued the cardiac phenotype [ ] . hypoxia markers were also upregulated in the heart of the ace -knockout mice, suggesting that loss of ace from the vascular endothelial cells resulted in coronary vessel constriction and reduced oxygen delivery to the myocytes [ ] . certainly, the localization of ace in the rat and human heart to endothelial cells of intramyocardial blood vessels and smooth muscle cells supports a role for ace in the control of local vasodilation. although it was reported that ace protein synthesis was similar in normal hearts and a failing heart from a single patient with idiopathic cardiomyopathy [ ] , in a study of review subjects with idiopathic cardiomyopathy, there was an increase in functional cardiac ace activity assessed by the ex vivo formation of ang - [ ] . to date, there have been no published studies on cardiac ace in the context of ischaemic heart disease. given that marked stimulation of ace and ang ii occurs after myocardial infarction [ ] , ace probably increases after myocardial infarction; this increase would limit the adverse effects of raised cardiac ang ii by increasing levels of the vasodilator ang - . support for this idea comes from studies in the rat myocardial infarction (mi) model, in which the development of heart failure was associated with increased ang - immunoreactivity [ ] . furthermore, infusion of ang - attenuated the development of heart failure after mi, confirming the functional significance of ang - [ ] . it is possible that the relative balance of vasoconstrictor and vasodilator angiotensin peptides is important in the modulation of both haemodynamic and trophic effects of these peptides in the context of ischaemic heart disease. a word of caution is needed with regard to ace and the heart. although the data presented suggest that ace and ang - might have cardioprotective effects after myocardial injury, in transgenic mice with increased cardiac ace expression [ ] , there was a high incidence of sudden death, which correlated with transgene expression levels. electrophysiology revealed severe, progressive conduction and rhythm disturbances, with sustained ventricular tachycardia, that progressed to fibrillation and death. clearly, further studies are needed to determine the functional relevance of ace in the heart. immunohistochemical studies by our group [ ] and others [ ] have shown that in the kidney, both ace and ace protein are localized predominantly to epithelial cells of the distal tubule. the ras has been implicated in the pathogenesis of diabetic complications, in particular diabetic nephropathy [ ] , and ace inhibition provides significant renoprotection. we have recently characterized ace in the kidney of a rodent model of type diabetes mellitus and compared and contrasted it with ace [ ] . previous studies in the kidney using this model of diabetes demonstrated that ace is downregulated in the renal tubules and upregulated in the glomerulus. in a recent study, we found that both ace and ace mrna levels were decreased in diabetic renal tubules by , %. we also found that ace protein synthesis is reduced in the diabetic kidney, and this reduction is prevented by ace inhibitor therapy, suggesting that ace might have a renoprotective role in diabetes [ ] . although there have been no other studies on the expression of ace after renal tissue injury, the synthesis of collectrin, a protein with significant homology to ace is upregulated in a model of progressive renal injury [ ] . the ras has proved to be an important regulator of cardiovascular and renal structure and function, in addition to salt and water balance. blockade of the ras with ace inhibitors or ang ii type receptor antagonists has clearly established its key role in the pathophysiology of an increasing number of diseases, including hypertension, heart failure, ventricular remodelling, renoprotection and diabetic complications. these beneficial results are thought to result from blocking the vasoconstrictor, hypertrophic and proinflammatory actions of ang ii. the discovery of a new enzyme in the ras pathway, ace , which produces the vasodilatory and antihypertrophic peptide ang - , gives rise to the hypothesis that ace provides a counter-regulatory system to ang ii, which might also contribute to the beneficial effects of ras blockade. the identification of ace in the heart and kidney, its modulation in heart failure and diabetes, and the recent description that this enzyme plays a biological role in the generation and degradation of angiotensin peptides provides a rationale for the further exploration of its role in pathophysiological states, including myocardial ischaemia, renal failure, atherosclerosis, and diabetic complications. many questions remain to be answered about ace (box ), in particular the impact of ace on the generation of bioactive peptides, but clarification of the role of ace in health and disease will be assisted by the development of agents that modulate ace activity. the discovery of ace has opened up an exciting new area of cardiovascular and renal physiology, in addition to providing the possibility of identifying novel therapeutic targets. conversion of angiotensin i to angiotensin ii tissue angiotensin converting enzyme in cardiac and vascular hypertrophy, repair, and remodeling tissue angiotensin and pathobiology of vascular disease -a unifying hypothesis a novel angiotensin-converting enzymerelated carboxypeptidase (ace ) converts angiotensin i to angiotensin - a human homolog of angiotensin-converting enzyme -cloning and functional expression as a captopril-insensitive carboxypeptidase counterregulatory actions of angiotensin exploring the structure and function of zinc metallopeptidases: old enzymes and new discoveries the role of ace in cardiovascular physiology hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase vasopeptidase inhibition and ang-( - ) in the spontaneously hypertensive rat angiotensin-( - ) is an endogenous ligand for the g protein-coupled receptor mas angiotensin-converting enzyme is a functional receptor for the sars coronavirus the secret life of ace as a receptor for the sars virus quantitative mrna expression profiling of ace , a novel homologue of angiotensin converting enzyme novel role for the potent endogenous inotrope apelin in human cardiac dysfunction vasoactive potential of the b- bradykinin receptor in normotension and hypertension angiotensin-converting enzyme is an essential regulator of heart function heart block, ventricular tachycardia, and sudden death in ace transgenic mice with downregulated connexins novel peptide inhibitors of angiotensinconverting enzyme substrate-based design of the first class of angiotensin-converting enzyme-related carboxypeptidase (ace ) inhibitors renal ace- expression in development, hypertension and diabetes increased angiotensin-( - )-forming activity in failing human heart ventricles: evidence for upregulation of the angiotensin-converting enzyme homologue ace effects of angiotensin-converting enzyme inhibition on angiotensin and bradykinin peptides in rats with myocardial infarction cardiac angiotensin-( - ) in ischemic cardiomyopathy angiotensin-( - ) attenuates the development of heart failure after myocardial infarction in rats characterization of renal angiotensinconverting enzyme in diabetic nephropathy effects of losartan on renal and cardiovascular outcomes in patients with type diabetes and nephropathy commentary on tikellis. et al. there is more to discover about angiotensin-converting enzyme collectrin, a collecting duct-specific transmembrane glycoprotein, is a novel homolog of ace and is developmentally regulated in embryonic kidneys key: cord- -p oae e authors: o'callaghan, barbara; synguelakis, monique; le gal la salle, gildas; morel, nicolas title: characterization of aminopeptidase n from torpedo marmorata kidney date: - - journal: biol cell doi: . /s - ( ) - sha: doc_id: cord_uid: p oae e a major antigen of the brush border membrane of torpedo marmorata kidney was identified and purified by immunoprecipitation. the sequence of its n terminal amino acids was determined and found to be very similar to that of mammalian aminopeptidase n (ec . . . ). indeed aminopeptidase n activity was efficiently immunoprecipitated by monoclonal antibody k . the purified antigen gives a broad band at kda after sds-gel electrophoresis, which, after treatment by endoglycosidase f, is converted to a thinner band at kda. this antigen is therefore heavily glycosylated. depending on solubilization conditions, both the antigen and peptidase activity were recovered either as a broad peak with a sedimentation coefficient of s ( % chaps) or as a single peak of . s ( % chaps plus . % c( )e( )), showing that torpedo aminopeptidase n behaves as an oligomer stabilized by hydrophobic interactions, easily converted into a kda monomer. the antigen is highly concentrated in the apical membrane of proximal tubule epithelial cells ( gold particles/μm( ) of brush border membrane) whereas no labeling could be detected in other cell types or in other membranes of the same cells (basolatéral membranes, vacuoles or vesicles). monoclonal antibodies prepared here will be useful tools for further functional and structural studies of torpedo kidney aminopeptidase n. aminopeptidase n (ec . . . ) is an abundant membrane bound peptidase of kidney and intestinal microvilli (for reviews, see [ , ] ). it hydrolyses the n-terminal amino acid of short peptides, with preference for amino acids with uncharged side chains [ ] . while it completes digestion of oligopeptides in the intestine [ ] , its function in kidney brush border membrane remains unclear. this enzyme has also been reported in the plasma membrane of various cell types like hepatocytes [ , ] or myeloid cells [ ] . the complete amino acid sequences of human intestinal [ ] and rat kidney [ ] aminopeptidases n have been determined. northern blot analysis revealed the existence of two human rna transcripts, both encoding for the same protein but under the control of different promoters. however, intestinal and kidney epithelial cells express the same mrnas, which are shorter than the transcripts of myeloid or fibroblastic cells [ ] . aminopeptidase n is composed of a single type of subunit, a -kda glycosylated polypeptide (for reviews, see [ , ] ). this subunit possesses a single transmembrane domain near its n terminus while the major part of the enzyme is ectocellular, including the catalytic sites [ , , ] . in the present work, we have characterized the aminopeptidase n from torpedo marmorata kidney and found it to be very similar to the mammalian enzyme. torpedo marmorata were provided by the marine station of areachon (france). crude fractions of kidney membranes were prepared from pooled whole kidneys which had been stored at - °c for seve .r'al days. the tissue was homogenized ( % w/v) in buffer a ( mm naci, mm na phosphate buffer, mm edta, . mm dithiothreitol, % glycerol, final ph . ) to which . mm pmsf was added. the homogenate was centrifuged at gm~ for min. membranes were pelleted ( gm~x for rain) from the low speed supematant. balb/c mice were immunized by three intraperitoneal injections (at -day intervals) of kidney membrane proteins (about /.tg emulsified proteins in freund's adjuvant). three days after a final booster intraperitoneal injection (without adjuvant), immunized spleen cells were fused with the non-secreting myeloma clone p x -ag . cells using polyethylene glycol as the fusing agent [ , ] . hybrids were selected in hypoxanthine, aminopterin and thymidine medium and superuatants from the culture wefts containing hybrid cells were tested for the presence of antibodies binding to the apical membrane of tubular epithelial cells on torpedo kidney frozen sections. clones were selected, some of which were cloned by limiting dilution. crude kidney membranes ( mg proteinhrd) were solubilized in % chaps and . % ci e at °c for min and non-solubilized material was pelleted ( gmx for rain). for screening experiments, proteins (about mg/ml) were biotinylated using immunopure nhs-lc-biotin (pierce, /zg/mg protein) for h, at °c. the reaction was stopped by addition of glycine ( mm final concentration). then, immunoprecipitation was performed in one step: . ml of solubilized proteins ( mg protein/ml), ml of culture supernatant containing monoclonal antibodies, . ml of a suspension of protein a sepharose- b (pharmacia, mg dry powder/ml of mm naci, mm tris buffer ph . ) and /.tl of antimouse igg antibodies raised in rabbit ( mg ig/ml, biosys)were mixed and incubated overnight at °c. beads were recovered by low speed centrifugation ( g for rain) and washed extensively. bound proteins were eluted by resuspension of the beads in p.i sample buffer ( % sds, % fl-mercaptoethanol, m sucrose, mm tris buffer (ph . ), mm bromophenol blue), and boiled for rain. proteins were immunoadsorbed as above and eluted from the protein a beads in i /al sds sample buffer. they were then diluted in volumes of mm na acetate buffer, mm edta, mm fl-mercaptoethanol, mg/ml chaps, final ph . and incubated at °c for h with or without . u endoglycosidase f/n-glycosidase f (boehringer). after concentration under vacuum, samples were resuspended in sds sample buffer and submitted to gel electrophoresis. electrophoresis was performed in - % linear acrylamide gradient gels according to [ ] . proteins in acrylamide gels were either stained with coomassie blue, or electrotransferred onto nitrocellulose as described by [ ] . biotinylated proteins on the blots were indirectly visualized using streptavidin-biotinylated peroxidase complex (amersham). the peroxidase substrates were h and diaminobenzidine. immunoprecipitation was performed as above but starting from mg solubilized kidney membrane proteins, and using ml of culture supernatant (mab k ), / of rabbit antibody to mouse ig ( mg ig/ml, biosys), and ml of a suspension of protein a sepharose- b beads ( mg dry powder/ml). eluted proteins were dialysed against . % sds (overnight) and " . % sds for h (spectrapor ), and concentrated to / under vacuum. electrophoresis was performed as above except that . mm thioglycolate was added to the migration buffer and that a prerun ( min at ma) was performed [ ] . proteins bands were transferred onto 'problott' membranes (applied biosystems) overnight at v in transfer buffer ( mm boric acid, mm tris-base). the kda band was stained by amidoschwartz and cut out. electroblotted proteins were sequenced by dr jp le caer (institut alfred fessard, cnrs, gif-sur-yvette) in a a gaz-liquid protein sequencer (abi) using the problott cart.ridge. phenylthiohydantoin amino acids were analysed on a a pth analyser (abi, foster city, ca). kidney membrane proteins ( mg protein/ml) were solubilized for h at c in one of the following detergent-containing buffers: ) mm naci, mm phosphate buffer, . mm mgci , % glycerol, % chaps; ph . ; ) mm naci, mm phosphate buffer, mm edta, % glycerol, % chaps; ph . ; ) mm naci, mm phosphate buffer, . mm mgci , % glycerol, % chaps, . % c e ; ph . . after eentrifugation ( gmax for h), an aliquot ( or /tl supernatan of solubilized proteins was layered on top of ml sucrose gradient. we used - % linear sucrose gradients in the solubilization buffer but with a -fold lower detergent concentration. proteins markers: alcohol deshydrogenase ( . s, boehringer), catalase ( . s, boehringer), and fl-galactosidase ( s, sigma) were solubilized and submitted to centrifugation in parallel linear sucrose gradients. the kda antigen content in the fractions was estimated after / to / dilution in /.d transfer buffer ( mm tris, . % triton x- , % methanol; ph . ) and blotting onto nitrocellulose, using a dot-blot apparatus. dots were probed with mab ki and antimouse ig antibodies conjugated to peroxidase (institut pasteur productions). intensity of the dot staining was quantified using a scanning densitometer (hoeffer scientific instruments) and compared to that of known amounts of solubilized kidney membrane proteins. enzymatic marker activity was measured as previously described [ , ] . aminopeptidase n activity was followed at nm using l-alanine p-nitroanilide (sigma) as the substrate in a mm tris phosphate buffer (ph . ) [ ] . protein content was determined by the method of lowry [ ] . kidney pieces were fixed immediately after dissection in % paraformaldehyde in mm naci, i mm na phosphate buffer (ph . ) for several hours. they were then incubated in the saline containing increasing sucrose concentrations (up to %) and frozen in isopentane cooled on dry ice. sections ( /.tm thick) were mounted on gelatin coated microscope slides and stored at - °c. primary antibodies (or culture supernatants) were diluted in mm naci, mm tris buffer (ph . ), . % tween and % bovine serum albumin (bsa) and allowed to bind overnight. bound antibodies were visualized using fluorescein conjugated antimouse ig antibodies (institut pasteur productions). kidney pieces were immersed in . % glutaraldehyde in mm naci, mm na phosphate buffer (ph . ), for h at room temperature. tissue pieces were permeabilized by . % saponin in the same buffered saline for rain and preincubated in . by indirect immunofluorescence on frozen kidney sections, clones were selected which produced antibodies that bound to the apical membrane of proximal tubule epithelial cells. each of these culture supernatants was used to immunoprecipitate kidney membrane proteins that had been in some experiments, when kidney membranes were pre-pared in the absence of the protease inhibitor pmsf, an additional kda band was immunoprecipitated by each of the anti- kda antibodies (not shown). this kda can be very easily removed from membranes by a single washing step. most probably, it is a proteolytic fragment of the kda antigen bearing the immunogenic regions but lacking the membrane domains of the protein. the kda antigen was immunoprecipitated by monoclonal antibody k , submitted to gel electrophoresis and electroblotted (see materials and methods). the band at kda was cut out and the n-terminal amino acid sequence was determined using automated edman degradation. starting from about pmol of the kda antigen, pmol pth-aminoacids were obtained in each of the successive cycles. the n-terminal amino acid sequence of the kda antigen (table i) turned out to be very similar to the corresponding sequence of aminopeptidase n (ec . . . ) which has been determined in man [ ] , rat [ ] , rabbit [ ] and pig [ ] . this sequence begins with eight hydrophilic table i . n-terminal amino acid sequence of the kda antigen. " for comparison, the n-terminal amino acid sequences of aminopeptidase n from rat kidney [ ] and from intestines of man [ ] , rabbit [ ] and pig [ ] are shown. ratkidney akg f y i s k s lg i lg illg human intestine ak g f y i s k s lg i lg illg rabbit intestine akg f y i s ka lg i lg fxlg porcintestine akg f y i s l a lg i agxlxv ) . immunopreeipitated proteins were directly submitted to sds gel electrophoresis (-) or submitted to endoglycosidase f/n-glycosidase f action ( . u at °c for h) before electrophoresis. gels were stained with coomassie blue. to check whether the kda antigen was indeed aminopeptidase n, we measured aminopeptidase n activity using alanine p-nitroanilide as the substrate [ ] . ~ln the course of immunoprecipitation experiments, enzyme activity was determined both in non-retained sampies (incubation mixture after removal of immunoadsorbed proteins by low speed centrifugation of protein a beads) and in immunoadsorbed protein samples (washed protein a bead suspensions just before sds elution). a significant proportion ( %) of peptidase activity was immunoprecipitated in a single step by monoclonal antibody k (table ii) . an unrelated monoclonal antibody k [ ] was unable to precipitate any detectable enzyme activity. in the same experiment, the antigen precipitated by antibody k was visualized as a kda band after sds-gel electrophoresis, which was absent from samples incubated with the control antibody k (fig ) . proteins immunoabsorbed by antibodies k or k , and eluted in sds, were treated by endoglycosidase f (see materials and methods). the kda band was converted to a lower molecular mass polypeptide, at kda (fig ) . this demonstrates that torpedo zminopeptidase is heavily glycosylated, deglycosylation reducing its apparent molecular mass by about %. note, for compar-ison, a small effect of endoglycosidase treatment on the heavy immunoglobulin chain migration (at about kda) in both k and k samples. bands at kda most probably correspond to endoglycosidase f ( kda molecular mass). to investigate whether the torpedo kidney aminopeptidase n was a monomer or an oligomer, we studied the velocity sedimentation in sucrose gradients of both the kda antigen and the aminopeptidase n activity, solubilized in nondenaturing conditions. kidhey membranes were treated by % chaps + . % c e . solubilized proteins were sedimented in - % linear sucrose gradients at rpm for h. fractions were collected from the bottom of the gradients and their kda antigen content and peptidase activity were estimated as described in materials and methods. both antigen and activity were recovered as a single peak, with a sedimentation coefficient of . s (fig ) . considering that aminopeptidase n is a hydrophilic membrane protein with a single transmembrane domain [ , ] , we can assume that it binds only a small amount of detergent and behaves as a globular protein in sucrose gra- dients. taking the approximation of martin and ames [ ] , this would give an approximate molecular mass of kda for the kda antigen, which therefore behaves, in these solubilization conditions, as a monomeric protein. then kidney membranes were solubilized by % chaps alone, in the presence of . mm mgc (fig a) or of mm edta ( fig b) . after velocity sedimentation in sucrose gradients containing either mgc or edta, a broad peak of antigen was found, with a sedimentation coefficient around s. in the presence of mgci , the peak of activity was also broad and it did not coincide with that of the antigen ( fig a) : it was shifted to lighter fractions. in addition, a second smaller peak of activity was recovered around . s. in the edta containing gradients, the total peptidase activity was much lower than in the mgc conditions. it corresponds to edta insensitive peptidase activity. its distribution ( fig b) revealed a peak at . s, very similar in amplitude and position to the . s noticed in mgci . some activity was also detected in denser fractions. by difference, it was possible to estimate the distribution of edta sensitive peptidase activity, which is similar to that of the antigen (fig c) . aminopeptidase n has been shown to be a zinc metalloprotein [ , ] and inhibition of its activity by the divalent cations chelator edta was not surprising. when solubilized by chaps alone, torpedo aminopeptidase behaves as a large size oligomer. this oligomer is dissociated in the presence of . % ci eg, and appears therefore to be stabilized by hydrophobic interactions. the presence of dithiothreitol during solubilisation and centrifugation does not modify the sedimentation profile of torpedo aminopeptidase (data not shown), showing that disulfide bonds are not necessary for its oligomerization, as reported for mammalian aminopeptidase [ ] . torpedo kidney organization has been schematically described by gfrard [ ] . a thin tubular segment, with a cili- ated epithelium, connects the glomerule to the proximal convoluted tubule, characterized by its large epithelial cells with their typical brush border apical membrane. the distal tubule follows, with a flat epithelium and epithelial cells devoid of flagella or microvilli. in the large field view presented in figure (upper panel), proximal tubule sections are concentrated on the left whereas distal tubule sections are mainly located on the right. anti- kda antibodies labeled very intensely the apical membrane of proximal tubule epithelial cells. no staining of the basolateral membranes was observed, even at higher magnification (lower panel). distal tubules and glomerules were not labeled. in sections of boundary segments of the proximal tubule, the staining was restricted to the apex of the larger epithelial cells. the distribution of the kda antigen was studied at the subeellular level using monoclonal antibodies k and k (figs and ) . these antibodies were chosen because their binding to kidney membranes adsorbed onto nitrocellulose was not affected by incubation of the blots in % glutaraldehyde (dot blot experiment not shown). binding of the mabs k or k was indirectly visualized using antimouse igg antibodies conjugated to nm gold particles. a general view of the apical portion of a proximal tubule epithelial cell is presented in figure . the apical membrane covers numerous microvilli, about . #m large and several microns long. this membrane is homogeneously decorated with numerous gold particles. in contrast, no labelling was associated to the basolateral membrane, or to the numerous vesicular or vacuolar membranes located under the brush border. in higher magnification views ( fig ) the abundance and selectivity of the kda antigen distribution are demonstrated. the density of gold particles associated to the apical membrane (about particles/btm ) was estimated on membrane profiles perpendicular to the planes of the section. gold particles are located extracellularly, at some distance (about nm) from the membrane, except of course on tangential sections (fig , top panel) . in some sections of proximal tubules, ciliated cells were found intercalated between epithelial cells (fig , lower panel) . no gold particles were associated to their apical membrane, nor to basolateral membranes, in contrast to the microvilli plasma membrane of the adjacent epithelial cells. working on torpedo kidney membranes, we have characterized a major immunogen of the brush border membrane of proximal tubules. in a fusion experiment, we selected hybridoma clones secreting antibodies which bound to these membranes. % of these clones produced antibodies which precipitated the same kda antigen, a protein which was only a minor component of kidney membranes. this antigen was highly purified in a single immunoprecipitation step, followed by gel electrophoresis. this purification allowed us to determine the n-terminal amino acid sequence of the antigen, which turned out to be very similar to that of aminopeptidase n from various mammals including man. the first eight amino acids, which probably correspond to the cytoplasmic domain, are identical in all sequences determined; the following amino acids are hydrophobic and some c o n s e r v a t i v e changes are observed. in addition to the kda antigen, monoclonal antibody k immunoprecipitated efficiently aminopeptidase n activity, showing that the kda antigen carries the activity. mammalian forms of aminopeptidase n are heavily glycosylated membrane proteins composed of a single type of subunit (m r of kda, detergent form [ ] ) which possess a single transmembrane domain near its n terminus (see [ , ] for reviews; [ ] ). the torpedo protein was not extracted from membranes at high ionic strength or by alkaline (ph ) treatment, demonstrating that it is an intrinsic membrane protein. it gave a broad band after sds gel electrophoresis and migrated in these conditions with an apparent molecular mass of kda. when deglycosylated by endoglycosidase f/n-glycosidase f, the antigen gave a thinner band at kda. thus, the torpedo enzyme is also a heavily glycosylated protein; this would explain its high immunogenicity. when solubilized in % chaps alone, torpedo aminopeptidase n was recovered in large protein complexes with a sedimentation coefficient around s. dithiothreitol was unable to dissociate these complexes (not shown), demonstrating that disulfide bonds were not necessary for the oligomerization of the enzyme, as previously reported [ ] . in contrast, low concentrations of ci e were sufficient to dissociate the oligomers showing that they were stabilized by hydrophobie interactions. in some experiments (not shown), when proteases were not inhibited by pmsf, a kda polypeptide was immunoprecipitated in addition to the kda antigen. this polypeptide, which most probably is a proteolytie fragment of aminopeptidase n, could be eliminated by a simple washing of the crude membranes. this shows that it has lost the transmembrane anchoring domain of the molecule and that it does not remain membrane associated through an interaction with an intact aminopeptidase n monomer, as has been reported for proteolytic digests of porcine [ , ] and bovine [ ] aminopeptidase n. in t o r p e d o kidney, a high density of the antigen (about gold particles/pan ) was detected in the brush border membrane in proximal tubule epithelia. no gold particles were associated with the apical membrane of other cells, either ciliated cells in proximal tubules or epithelial cells in distal tubules (not shown). no antigen was detected in other membranes of proximal tubule epithelial cells, neither basolateral membranes nor intracellular membranes such as vacuoles or vesicles membranes were labeled. mammalian peptidase n is concentrated in microvillar apical membranes of both kidney proximal tubules and intestinal epithelia. but in enterocytes, enzyme was found associated to purified basolateral membrane fractions, where its specific activity is times lower than that of the brush border membrane fractions [ ] . similarly, in madin-darby kidney cells transfected with human aminopeptidase n edna, this enzyme was predominantly recovered in the apical membrane but some of it (about %) was associated to basolateral membranes [ ] . it was therefore surprising that we could not detect any enzyme in basolateral membranes of torpedo kidney, especially considering the high density of gold particles associated with the apical membrane and the absence of background staining. sorting of aminopeptidase n has been extensively studied in various cell types: hepatocytes [ ], enterocytes [ , ] and kidney epithelial cells [ ] . in madin-darby canine kidney cells, aminopeptidase n is directly sorted to the apical plasma membrane [ ] . no label was detected in intracellular vesicles in our experiments, suggesting a reduced accessibility of antibodies to these structures. this could also reflect a low turnover of the antigen or possibly that antibodies bind only to mature forms of tl~ enzyme and not to its precursor forms, present in the endoplasmic reticulum and the golgi apparatus [ , ] . the major part of mammalian aminopeptidase n, including the catalytic domain, is located on the extracellular surface of the epithelium [ , ] . this is also the case for the torpedo kidney enzyme. indeed, epitopes labelled by antibodies k and k are exposed in the extracellular medium since gold particles were on the external side of microvilli membranes, located at some distance (about nm) from the membrane. considering the size of gold particles ( rim) and that of immunoglobulins g ( nm), we can estimate that the epitope is about nm outside the plane of the membrane and therefore that a large part of the antigen is localized in the tubular lumen. in conclusion, we have identified the kidney aminopeptidase n from torpedo marmorata. to our knowledge, it is the first characterization of this enzyme from torpedo and from fish in general. monoclonal antibodies prepared here will be useful tools for further functional studies since they bind to the solubilized protein without loss of its enzymatic activity. fine ultrastructural studies will also be possible since these antibodies are able to probe aminopeptidase n in its membrane environment, even after glutaraldehyde fixation of tissues. by its subcellular distribution, its biochemical properties and its n-terminal amino acid sequence, this enzyme closely resembles the mammalian enzymes. biogenesis of the rat hepatocyte plasma membrane in vivo: comparison of the pathways taken by apical and basolateral proteins using subcellular fractionation protein-electroblotting and microsequencing strategies in generating protein data bases from two-dimensional gels rabbit intestinal aminopeptidase n. purification and molecular properties a simple method for polyethylene glycol-promoted hybridization of mouse myeloma cells isolation and characterization of membrane bound arylamidases from human placenta and kidney topology of microvillar membrane hydrolases of kidney and intestine rat intestinal brush border membrane peptidases. i. solubilization, purification and physicochemical properties of five different forms of the enzyme continuous culture of fused cells secreting antibodies of predefined specificity cleavage of structural protein during the assembly of the head of bacteriophage t human myeloid plasma membrane glyeoprotein cd (gpl ) is identical to aminopeptidase n protein measurement with folin phenol reagent the aminopeptidase from hog intestinal brush border a method for determining the sedimentation behavior of enzymes: application to protein mixtures evidence for the transit of aminopeptidase n through the basolateral membrane before it reaches the brush border of enterocytes l'acetylcholinesttrase des organes ectriques des poissons complexes membranalres sorting of endogenous plasma membrane proteins occurs from two sites in cultured human intestinal epithelial cells (caco- ) subcellular fractionation and subeellular localization of aminopeptidase n in rabbit enterocytes immunological identification of a new x m r membranebound protein in torpedo electric organ molecular and cellular basis of digestion complete amino acid sequence of human aminopeptidase n deduced from cloned edna the oligomerie structure of the renal aminopeptidase n from bovine brush border membrane vesicles anchoring and biosynthesis of stalked bursh border membrane proteins: glycosydases and peptidases of erythrocytes and renal tubuli look at ( ) separate promoters control transcription of human aminopeptidase n gene in myeloid and intestinal epithelial cells a human liver aminopeptidase n. the amino acid and carbohydrate contents, and some physical properties of sialie acid containing glycoprotein electrophoretie transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some application on the subunit structure of particulate aminopeptidase from pig kidney amino acid sequence deduced from a edna suggests it encodes the zn-peptidase aminopeptidase n aminopeptidase n is directly sorted to the apical domain in mdck cells human aminopeptidase n is a receptor for human coronavirus e the amino acid sequence of the hydrophobic anchor of rabbit intestinal brush border aminopeptidase n we thank drs jp le caer and j rossier (institut alfred fessard, cnrs, gif-sur-yvette) for amino acid sequencing and dr s o'regan for improving our manuscript. this work was supported by a grant from dret (n ° / ). ° key: cord- -zxx m kz authors: heymann, david l; aylward, r bruce; wolff, christopher title: dangerous pathogens in the laboratory: from smallpox to today's sars setbacks and tomorrow's polio-free world date: - - journal: lancet doi: . /s - ( ) -x sha: doc_id: cord_uid: zxx m kz nan the lancet • vol • may , • www.thelancet.com commentary less than a year after an unprecedented international public-health effort interrupted human-to-human transmission of the coronavirus that causes severe acute respiratory syndrome (sars-cov), some human beings are again infected. sars-cov does not seem to have reentered the human population from the exotic wildanimal markets of china that have preoccupied publichealth officials worldwide, nor from some other source in nature not yet understood. rather, the latest outbreak seems to be from a laboratory source. this scenario is reminiscent of the often forgotten footnote to the smallpox eradication effort when the last human infections did not occur in somalia, the last country with naturally occurring smallpox, but a year later in birmingham, in the uk, originating from a laboratory with inadequate biosafety facilities. auspiciously, the new sars cases are occurring as who's biosafety advisory group prepares to examine the long-term containment of poliovirus stocks, the risks of which will rapidly increase after interruption of transmission and the ending of immunisation with oral poliovirus vaccine. the recent outbreak of nine cases of sars in china, with one death, underlines again the challenges of maintaining appropriate biosafety conditions in laboratories working with dangerous pathogens. in this outbreak, two researchers at the institute for viral disease control and prevention in beijing developed sars in late march and mid-april. all subsequent second-generation and thirdgeneration cases have now been linked to close contact with one of these researchers. investigation of the source of the outbreak, jointly by the chinese ministry of health and who, continues to focus on this virology institute. if the laboratory source is confirmed in china, this will be the third known incident of laboratory-acquired sars-cov infection, the first having occurred at the national university of singapore where a postgraduate developed an illness consistent with sars in late august, . during the investigation that followed, it was concluded that the student most probably acquired the infection in the bsl- laboratory in which he was studying the west nile virus · days before the onset of his illness, which is consistent with the expected incubation period of sars. (biosafety conditions are described as biosafety levels in four categories , with bsl- and bsl- recommended for work with pathogens that cause serious human and animal disease). it seems that transmission occurred as a result of inappropriate laboratory procedures that led to cross-contamination of the west nile virus specimen with sars-cov. no other workers in the laboratory, and none of the medical staff who cared for the student while he was ill, became secondarily infected, nor did household and other contacts. the second reported incident similarly resulted in an isolated case of sars in early december, . it occurred at the institute of preventive medicine, national defence university, taipei, in a laboratory scientist who had been working intensely in a bsl- laboratory, over a long period and for long hours each day. it seems that transmission occurred after exposure to sars-cov from contact with droplets when cleaning the spill of a sars-containing specimen in the laboratory's transport chamber. accidental transmission of a dangerous pathogen from a laboratory can occur when a susceptible and unprotected laboratory worker is exposed to the agent during laboratory procedures. these conditions were met in the smallpox laboratory in birmingham in , and in at least two of the laboratories associated with the recent cases of sars during . if the resulting human infection causes viral shedding, with exposure to susceptible workers in the laboratory, health-care system, or community, an outbreak can result. in the outbreak in brimingham after the smallpox laboratory accident, infection spread from the initial case to a close family member and one other. in the current outbreak of sars, chains of transmission seem to have moved from the laboratory, to a close family member, and to a hospital, from where a nurse who treated the laboratory worker then transmitted infection to five others. proven measures to minimise the risk of reintroducing dangerous pathogens include: limiting the number of sites where they are stored and studied to those that are absolutely necessary; protection of laboratory workers with available vaccines, protective clothing, and safe equipment; closely monitoring illnesses in laboratory workers; and adhering to standard operating procedures. hundreds of years of combined experience in high and maximum containment laboratories have proven these biosafety measures effective if rigorously and faithfully followed-with strict national procedures to verify that appropriate conditions and procedures are maintained. after certification of smallpox eradication, known stocks of variola virus were destroyed or transferred to one of two who reference laboratories where biosafety is periodically verified by the who biosafety advisory group. during the sars outbreak last year, many specimens were obtained from human cases of sars commentary dangerous pathogens in the laboratory: from smallpox to today's sars setbacks and tomorrow's polio-free world and sent to many different national and international laboratories for various studies. in april, , who provided guidelines for handling, packing, and shipping sars specimens, and listed laboratory practices that could safely be done under bsl- and those that required bsl- . these guidelines were reviewed and updated during later who consultations, and laboratory research activities continue at many of these sites. unfortunately, adherence to these guidelines has now failed at two, and possibly three, different laboratories, reaffirming the importance of strong national, and possibly international, monitoring of their implementation. the predictable emergence of new dangerous pathogens in the future further highlights the need for such action. if activities to eradicate poliomyelitis remain on target, interruption of human-to-human transmission will occur sometime during the next months, and the wild poliovirus will be moved from the list of endemic infections to that of dangerous pathogens. that poliovirus reintroduction could occur was seen in when a reference strain of wild-poliovirus type i that is used in the production of inactivated poliovirus vaccine was isolated from a young child being investigated for diarrhoea. the subsequent epidemiological investigation found that the child's father was employed at a production site for inactivated poliovirus vaccine where an accident had occurred. fortunately the child was fully immunised against poliovirus. but the child served as a healthy carrier of poliovirus to the community, although sanitation was adequate and poliovirus vaccination coverage was high enough to prevent an outbreak. a similar poliovirus reintroduction from a laboratory or production facility for poliovirus vaccine to one of the many countries that have indicated their intention to stop poliovirus immunisation after certification of global eradication could result in future outbreaks of poliomyelitis. recognising the risks that would be associated with a poliovirus reintroduction, who and its technical partners in poliomyelitis eradication began the process of establishing a global action-plan for the long-term laboratory containment of wild polioviruses in the mid- s. by , international consensus had been established and the process of surveying and inventorying laboratories for wild poliovirus and infectious or potentially infectious materials began in the three who regions that had interrupted indigenous transmission of wild poliovirus. through the comprehensive surveys of national laboratories that are underway or completed in countries in five continents, over facilities have been inventoried to date. about facilities have reported wild-type poliovirus or potential infectious materials. at the same time, technical and engineering solutions have allowed the continued production of inactivated poliovirus vaccine from wild poliovirus under enhanced bsl- conditions to ensure that such a vaccine is available to the countries that choose to continue routine immunisation against the poliovirus after eradication has been certified globally and the routine use of oral poliovirus vaccine stopped. the current who plan for the period after the global interruption of wild-poliovirus transmission specifies bsl- conditions for wild-poliovirus infectious materials and bsl- for potentially infectious materials in nonvirology laboratories, on the basis of a now outdated assumption of continued universal immunisation. in september, , an expert group recommended that because circulating vaccine-derived polioviruses would, like wild poliovirus, compromise the goal of poliomyelitis eradication, the use of oral poliovirus vaccine for routine immunisation should eventually stop. , this decision, combined with the recognition that many countries plan to forego future routine immunisation with inactivated poliovirus vaccine, has led to the understanding that sabin poliovirus strains will also need to be stored and handled under appropriate biosafety conditions. a biosafety strategy for minimising the risk of a laboratory accident with the sabin poliovirus is under development. in addition, consensus is being sought on the mechanisms and procedures for ensuring that the necessary stockpiles of oral poliovirus vaccine are available should poliovirus be reintroduced into human populations because of a laboratory accident. although an increasing number of pathogens are referred to as dangerous, in reality different pathogens present different laboratory risks. sars-cov seems to represent a high laboratory risk. unlike sars-cov, poliovirus is not efficiently transmitted by droplets from person to person, and a vaccine is available that fully protects laboratory workers from disease and reduces the risk of infection, thereby providing additional assurances against substantial consequences should a laboratory accident occur once routine immunisation with oral poliovirus vaccine has stopped. it is also reassuring that no further accidents have occurred with the smallpox virus stored in two reference laboratories for over years. nevertheless, the recent laboratory accidents with sars-cov are a stark reminder that the security of public-health achievements requires greater investment to ensure that global biosafety standards for dangerous pathogens in laboratories are universally adopted, strictly adhered to, closely monitored, and rigorously enforced. we have no conflict of interest to declare. polio eradication initiative who, geneva , switzerland (e-mail: heymannd@who.int) the basis of five longitudinal studies. the authors conceded that only one of these studies was able to record whether prodromal manifestations of schizophrenia preceded cannabis use. the results of the study indicated that "cannabis users at age years had elevated scores on the schizophrenic symptom scale only if they had reported psychotic symptoms at years", and that people who used cannabis at age years had a higher risk for adult schizophreniform disorder at age years even if psychotic symptoms at age years were controlled for. the researchers concluded that cannabis was a causal factor for psychosis in "vulnerable youths". there is some reason to believe that cannabis contributes to psychosocial problems in adolescents and young adults, and no responsible adult would want young people to take drugs. there is no question that this issue is an important candidate for education and prevention, but there is a fierce debate on the place repressive measures should have in this context. there is little reason to believe that criminalisation has had a strong effect on the extent of cannabis use by young people. moreover, prohibition itself seems to increase the harmfulness of drug use and cause social harm. by stopping all cannabis users from being treated as criminals, i believe this year's change by the british government of its cannabis law (a declassification from class b to c) is a sensible attempt to balance the possible harms caused by cannabis and its prohibition. the concern expressed by peter maguire of the british medical association and others, that "the public might think that reclassification equals safe", is based on the wrong assumption that cannabis became illegal because its use is unsafe and dangerous. many unsafe activities are legal, including skiing downhill, having sex, drinking beer, eating hamburgers, and taking aspirin. cannabis did not become illegal because it was shown to be dangerous but, more likely, because harry anslinger, commissioner of the us bureau of narcotics - , and his colleagues needed a new target and battlefield after the end of alcohol prohibition in . reputed dangers, presented in his statements before the us senate in , were used as a shocking means of manipulation-eg, "a man under the influence of marijuana actually decapitated his best friend; and then, coming out of the effects of the drug, was as horrified as anyone over what he had done." the representative of the american medical association strongly opposed the marijuana tax act of : "to say . . . that the use of the drug should be prevented by a prohibitive tax, loses sight of the fact that future investigation may show that there are substantial medical uses for cannabis." we live in a time in which the unrealistic and unproductive paradigm of complete abstinence from drugs is slowly dissipating. proponents of a drug-free society find this fact hard to accept, and responsible politicians and doctors can find achieving an appropriate position in the debate difficult. however, we must learn to deal with drugs and their possible dangers without fear. i have no conflict of interest to declare. nova-institut gmbh, d- huerth, germany (e-mail: franjo.grotenhermen@nova-institut.de) cannabis can cause anxiety, agitation, and anger among politicians. the consequences of this cannabis-induced psychological distress syndrome (cipds) include overreaction with respect to legislation and politics and a lack of distinction between use and misuse of cannabis. in times of a war against drugs, this distinction might even be regarded as unpatriotic, as irresoluteness in the face of the enemy. one trend associated with cipds involves taking away the driving licence of people who drive and are discovered to have inactive tetrahydrocannabinol metabolites in their urine. in a more severe state of paranoia even medicinal use can be perceived as a threat to society, since it might "destabilize the societal norm that drug use is dangerous", ignoring the fact that many prescription and over-the-counter drugs are potentially harmful. exaggerated laws on cannabis made by anxious individuals could be regarded as a modern version of the generational conflict. rationality and factuality are needed to calm down politicians affected by cipds. that cannabis might cause infertility, cancer, cognitive decline, dependency, traffic accidents, and heart attacks, and that it can lead to the use of more dangerous drugs, are all arguments that have been used to justify the war on cannabis. drugs can be harmful, whether they are legal or illegal, but claims about the dangers of cannabis are often overstated. , one main justification for today's war on cannabis is its possible detrimental effect on the mental health and social wellbeing of adolescents. in this week's lancet, john macleod and colleagues show that the causal relation is less certain than often claimed, and point out several common misunderstandings about the difficulties encountered when studying drug use, such as the limits of confounder adjustment. the results of one often-cited swedish study, for example, indicate a crude odds ratio of · for schizophrenia risk at age years in individuals who used cannabis more than times before age years. this finding suggests cannabis is an important contributor to schizophrenia. after adjustment for several possible confounders, however, the risk decreased to · , a strong indication of residual confounding-ie, the presence of factors that would further reduce the risk if included in the statistical model but that could not be included because of a lack of data. another review details the findings of an investigation into the association between cannabis and psychosis on world health organization. sars in china-updates smallpox and its eradication. geneva: world health organization polio control after certification: major issues outstanding china confirms sars infection in another previously reported case; summary of cases to date. world health organization sars situation update severe acute respiratory syndrome (sars) in singaporeupdate . sars case in singapore linked to accidental laboratory contamination severe acute respiratory syndrome in taiwan, china world health organization. who biosafety guidelines for handling of sars specimens world health organization. who post-outbreak biosafety guidelines for handling of sars-cov specimens and cultures genetic analysis of wild-type poliovirus importation into the netherlands ( - ) • www.thelancet.com commentary poliovirus vaccine production sites be effective for global certification? global action plan for l aboratory containment of wild poliovirus world health organization. guidelines for the safe production and quality control of ipv manufactured from wild poliovirus the role of routine polio immunization in the post-certification era report of an informal consultation on identification and management of vaccine-derived polioviruses key: cord- -ma s hrs authors: madden, david l.; mundon, francis k.; tzan, nancy r.; fuccillo, david a.; dalakas, marinos c.; calabrese, vincent; elizan, tenesita s.; román, gustavo c.; sever, john l. title: antibody to human and simian retrovirus, htlv‐i, htlv‐ii, hiv, stlv‐iii, and srv‐i not increased in patients with multiple sclerosis date: - - journal: ann neurol doi: . /ana. sha: doc_id: cord_uid: ma s hrs we have tested sera from patients with multiple sclerosis, matched controls, and those with other neurological diseases, as well as sera from patients with the acquired immunodeficiency syndrome and controls and patients with tropical spastic paraparesis (tsp) and controls for antibody to human t‐lymphotropic virus type i (htlv‐i), htlv‐ii, human immunodeficiency virus (hiv), simian t‐lymphotropic virus type iii, or simian retrovirus type i by immunofluorescent activity test, and for htlv‐i and hiv by the elisa method. sera from patients with multiple sclerosis and matched controls, and from patients with optic neuritis and parkinson's or other neuromuscular diseases did not have antibody to any of the retroviruses tested. specimens from tsp patients and some controls contained htlv‐i antibody. we conclude from our study that only tsp patients had serological evidence of infection with one of the retroviruses studied. we the none of the elsa hiv od readings on the samples from ms patients, patients with optic neuritis, controls, and patients with other neurological disease were above the cutoff of . . an analysis of the od readings obtained did not indicate that the readings were higher in the ms patients as compared to the controls. seventeen of the tsp patients had htlv-i elisa od readings above the cutoff of . ; patients with other neurological disease also had readings above the cutoff. three of the patients with clinical tsp, with other neurological diseases, and the controls had elisa od readings below the . s annals of neurology supplement to volume , cutoff and were considered negative. none of the htlv-i elisa readings on samples from aids patients or controls or from ms patients, patients with optic neuritis, contro s, and patients with other neurological diseases were above the cutoff of . . analysis of the od readings obtained did not indicate that the readings were higher in the ms patients as compared to the controls. retroviruses have been associated with neurological disease in patients with aids and with tsp. the clinical signs and symptoms of tsp resemble, to some extent, ms. in our study of serum samples collected before hiv infection became prevalent in the united states, we could not demonstrate a serological relationship between the human or simian retroviruses and ms. several explanations for the differences of these findings and those reported by koprowski and colleagues [ are evident. first, it is possible that some patients in key west have htlv-i antibody because it is a tropical island on the edge of the tsp caribbean belt. second, modern society being so mobile, individuals who have lived or traveled to htlv-i-endemic regions may develop antibody, return to more northern latitudes, and later develop ms independent of their htlv-i status. further, it should be expected that ms patients have lifestyles that are similar to the general population. some will develop hiv antibody as a result of homosexual activity, heterosexual transfer, intravenous drug usage, and transfusion. careful questioning may identify these individuals. it has been suggested that the type of test used may influence the results. koprowski [personal communication) believes that there is insufficient antigen on commercial plates to detect the possible cross-reactions with yet unidentified retrovirus. he believes that one must greatly overload the plates with antigen to identlfy the possible cross-reacting antibodies. data from the use of commercial kits indicated that such kits are constructed to be overly sensitive; they identify to % of false positives in order not to miss any true positives. our laboratory has had experience in developing many elisa tests for serological studies. we find it necessary to titrate the antigen and conjugate with positive and negative serum to get the right concentration of each to ensure maximum sensitivity and specificity. altering one parameter, such as increasing antigen concentration, may increase nonspecificity and false-positive reaction. ifa is useful in confirming htlv-i antibody and, by use of proper cell control, in detecting nonspecificity. we conclude from our study that our ms patients did not have serological evidence of retrovirus infection. it is possible for some ms patients to have retrovirus antibody that is not related to ms. during the past years, more than ten different possible agents have been suggested as causes of ms [ . among these are a number of recognized viruses, such as measles, canine distemper, scrapie agent, and coronaviruses, and multiple sclerosis-associated agents of unclear classification, the bone marrow agent, and the chimpanzee agent. the serological evidence for etiological association of a retrovirus with ms seems weaker than that available for measles, rubella, and coronavirus. multiple sclerosis and human t-cell lymphotropic retroviruses search for the cause of multiple sclerosis and other chronic diseases of the central nervous system key: cord- -zaguyxm authors: stephenson, iain; nicholson, karl g; wood, john m; zambon, maria c; katz, jacqueline m title: confronting the avian influenza threat: vaccine development for a potential pandemic date: - - journal: lancet infect dis doi: . /s - ( ) - sha: doc_id: cord_uid: zaguyxm sporadic human infection with avian influenza viruses has raised concern that reassortment between human and avian subtypes could generate viruses of pandemic potential. vaccination is the principal means to combat the impact of influenza. during an influenza pandemic the immune status of the population would differ from that which exists during interpandemic periods. an emerging pandemic virus will create a surge in worldwide vaccine demand and new approaches in immunisation strategies may be needed to ensure optimum protection of unprimed individuals when vaccine antigen may be limited. the manufacture of vaccines from pathogenic avian influenza viruses by traditional methods is not feasible for safety reasons as well as technical issues. strategies adopted to overcome these issues include the use of reverse genetic systems to generate reassortant strains, the use of baculovirusexpressed haemagglutinin or related non-pathogenic avian influenza strains, and the use of adjuvants to enhance immunogenicity. in clinical trials, conventional surfaceantigen influenza virus vaccines produced from avian viruses have proved poorly immunogenic in immunologically naive populations. adjuvanted or whole-virus preparations may improve immunogenicity and allow sparing of antigen. sporadic human infection with avian influenza viruses has raised concern that reassortment between human and avian subtypes could generate viruses of pandemic potential. vaccination is the principal means to combat the impact of influenza. during an influenza pandemic the immune status of the population would differ from that which exists during interpandemic periods. an emerging pandemic virus will create a surge in worldwide vaccine demand and new approaches in immunisation strategies may be needed to ensure optimum protection of unprimed individuals when vaccine antigen may be limited. the manufacture of vaccines from pathogenic avian influenza viruses by traditional methods is not feasible for safety reasons as well as technical issues. strategies adopted to overcome these issues include the use of reverse genetic systems to generate reassortant strains, the use of baculovirusexpressed haemagglutinin or related non-pathogenic avian influenza strains, and the use of adjuvants to enhance immunogenicity. in clinical trials, conventional surfaceantigen influenza virus vaccines produced from avian viruses have proved poorly immunogenic in immunologically naive populations. adjuvanted or whole-virus preparations may improve immunogenicity and allow sparing of antigen. few infectious diseases cause such a huge annual toll of morbidity, mortality, and economic loss as influenza. in addition, influenza can unpredictably emerge to cause pandemics. the spanish pandemic spread around the world within months causing up to million deaths. the transmission of avian influenza h n to at least people during the asian h n epizoonotic period , prompted concerns that the next pandemic is imminent. as highlighted by the recent severe acute respiratory syndrome (sars)coronavirus outbreak, international air travel increases global vulnerability to infectious respiratory pathogens. our ability to combat influenza and its complications depends primarily on vaccination. annual influenza vaccine production is a wellplanned process that takes up to months. current facilities may not be suitable for rapid bulk manufacture of avian influenza virus vaccines in response to a world threat. influenza viruses are enveloped negative-sense rna viruses with a segmented genome belonging to the orthomyxoviridae family. they are classified on the basis of their core proteins into three distinct types: a, b, and c. influenza a viruses infect a range of mammalian and avian species, whereas type b and c are essentially restricted to human beings. influenza a viruses are responsible for annual epidemics and occasional pandemics, whereas influenza b viruses cause outbreaks every - years, but are not associated with pandemics. the main antigenic determinants of influenza a and b viruses are two surface glycoproteins: the neuraminidase and the haemagglutinin, both capable of eliciting immune responses in human beings. the haemagglutinin is involved with receptor binding and membrane fusion. the neuraminidase facilitates cleavage of virus progeny from infected cells, prevents viral aggregation, and aids movement through the mucosal respiratory-tract epithelium. virus strains are classified according to host species of origin, geographic site and year of isolation, serial number, and, for influenza a, by serological properties of subtypes of haemagglutinin and neuraminidase. influenza a h and h subtypes circulating in human beings evolve and undergo antigenic variability continuously. a lack of effective proofreading by the viral rna polymerase leads to a high rate of transcription errors that can result in aminoacid substitutions in surface glycoproteins. virus variants with substitutions in the antibody-binding sites can evade humoral immunity and reinfect individuals. this is termed "antigenic drift". the segmented viral genome allows for a second type of antigenic variation. if two influenza viruses simultaneously infect a host cell, genetic reassortment may generate a novel virus with new surface or internal proteins. pandemic influenza viruses arise by this process of "antigenic shift", when a virus with a new haemagglutinin subtype emerges and spreads efficiently in a naive human population. comparisons of pandemic and interpandemic influenza are shown in table . bird populations. , avian influenza a viruses generally do not cause disease in these natural hosts. the principal site of influenza virus replication in aquatic birds is the gastrointestinal tract resulting in high faecal viral titres and viral transmission in migratory feeding areas. despite the range of virus subtypes, only a few haemagglutinin (h , h , h ) and neuraminidase subtypes (n , n ) have established in human beings and have caused widespread respiratory disease. the haemagglutinin of human influenza viruses preferentially binds to sialic acid receptors containing ␣ , -galactose linkages, whereas avian influenza viruses preferentially bind to those containing ␣ , -galactose linkages. these binding preferences correlate with the predominance of sialic acid ␣ , -galactose linkages on human epithelial cells, and ␣ , -galactose linkages on avian intestinal epithelial cells. [ ] [ ] [ ] although the molecular mechanisms responsible for receptor-binding specificity are poorly defined, it is believed that haemagglutinin of avian origin must acquire human receptor-binding specificity to generate influenza strains capable of sustained human-to-human transmission. site-directed mutagenesis studies have shown that only one or two aminoacid mutations are required for this change. limited passage in human beings of a virus possessing an avian haemagglutinin, such as occurring in asia currently, , may be sufficient to generate such a change. during the th century, an h n virus in , an h n virus in , and an h n virus in caused influenza pandemics. human-avian reassortant viruses seem to have caused the pandemics of and . the h n virus differed by three genes (haemagglutinin, neuraminidase, and the rna polymerase pb ) from the h n virus that infected people between and . the h n virus differed by two genes (haemagglutinin and pb ) from the h n virus that infected people between and . in both cases, the h and h haemagglutinin genes were contributed by avian viruses. sequence analyses of early h and h isolates indicate receptor-binding specificity was altered by a single aminoacid substitution soon after human introduction. since pig trachea contains receptors for both avian and human influenza viruses, and can support replication of viruses of both human and avian origin, it has been suggested that genetic reassortment between avian, swine, and human influenza viruses may occur in pigs, and that they represent a "mixing bowl" for the evolution of human pandemic strains. haemagglutinin and neuraminidase of h n viruses isolated from human beings, poultry, and wild ducks have distinguishable properties. chickens seem to support a separate natural reservoir of influenza viruses, indicating a possible role as intermediate hosts in zoonotic transmission. some avian h viruses established in poultry are capable of two-way transmission between domestic ducks, where they are able to generate multiple reassortants with other cocirculating viruses. these reassortant viruses have haemagglutinin receptor-binding sequences potentially capable of human infection, suggesting that new viruses may emerge directly from the avian pool. the close proximity of people to high concentrations of waterfowl, poultry, and swine in southeast asia, and avian influenza activity, has identified this region as a hypothetical influenza epicentre (figure ). avian influenza viruses generally do not replicate efficiently in human beings, even after experimental infection. before , direct transmission of avian influenza viruses to the human respiratory system was not considered possible. however, it is now recognised that at least some subtypes of avian influenza viruses can replicate within the human respiratory tract. although it is unclear whether the recent reported increase in transmission of avian influenza to people (table ) is the result of heightened surveillance, the geographical expansion of h n poultry outbreaks across asia is an unprecedented and new event. more than confirmed cases of transmission of avian h n virus to human beings has increased the possibility that an avianhuman reassortant virus may emerge to effectively transmit among people. caused by immunity in population result pandemic influenza a antigenic shift: emergence of novel or little or no background immunity high attack rates, excess mortality and re-emerging subtype of influenza a (maybe partial immunity in older morbidity in all age groups people if re-emerging virus) interpandemic influenza antigenic drift: evolution of existing little immunity in infants. partial variable outbreaks or epidemics influenza (a or b) strains immunity in adults by cross-reacting with variable morbidity and antibody to previously seen mortality, usually in elderly and young and related strains (h greatest severity) the first association of avian influenza viruses with respiratory illness in human beings was during when six deaths from human cases of highly pathogenic influenza h n occurred during an outbreak among live-bird markets (table ) . [ ] [ ] [ ] all viral genes were of avian origin, indicating that h n had crossed the species barrier without adaptation or reassortment with human viruses. despite the elimination of ducks, geese, and quail (sources of h n and its donor genes) and cleaning days in the markets, h n viruses have subsequently reemerged in hong kong poultry markets, although hong kong remains free from infection in the h n outbreak across asia. , in late , highly pathogenic h n viruses were isolated from dead waterfowl in hong kong (personal communication, m peiris, university of hong kong), which was notable since h n viruses do not typically cause disease in waterfowl. in february , h n reemerged in hong kong in two family members returning from a trip to china. in , pathogenic h n viruses are causing extensive poultry outbreaks in asia with deaths ( %) of human cases reported in vietnam and thailand. , , in hong kong in , and again in , influenza h n viruses were isolated from children with mild respiratory illnesses. , as with h n , no human-to-human transmission was evident. additional human h infections in china, and detection of antibody to h in human serum samples in china and hong kong suggest that further human h infection has occurred. eurasian h viruses circulating since the late s have been classified into three phylogenetic sub-lineages: g , y (g -like), and y . , those established in live-bird and have also transmitted to swine in hong kong and china. [ ] [ ] [ ] some avian h viruses have acquired receptor-binding characteristics typical of human strains, increasing the potential for reassortment within both human and pig respiratory tracts. in early , outbreaks of highly pathogenic avian influenza h among poultry occurred in the netherlands and extended to belgium and germany. , more than workers involved in control of the outbreak developed viral h n conjunctivitis and a few developed respiratory illness. evidence of limited human-to-human spread, and a fatal respiratory infection, highlighted a significant threat to human health. more recently, cases of avian influenza (h n ) infections have been reported in two cullers during control of an h n poultry outbreak in canada. avian influenza (h n ) seems to have crossed the species barrier from poultry to people for the first time. in egypt in april two infants presenting with mild febrile respiratory symptoms had h n influenza viruses isolated from respiratory samples. as well as h , h , and h viruses, the h subtype has acquired the ability to infect chickens and is rapidly becoming endemic in poultry populations. phylogenetic analyses of h n viruses isolated from wildfowl, showing high nucleotide homology of the internal genes to human h n and h n viruses, suggests these subtypes can transfer genetic material between each other and are potential sources of new strains. h n viruses were responsible for the influenza pandemic and circulated as the only human subtype until when it was replaced by h n subtype. the h haemagglutinin gene, along with the neuraminidase and pb genes, were derived from avian sources. since people born after lack immunity to the h subtype, h viruses pose a pandemic threat to this susceptible population. h viruses continue to circulate in wild ducks, although the conditions required for the re-emergence of human h influenza viruses are unclear. although the virulence of avian influenza viruses has been well studied in avian species, their virulence in mammals is not well understood. infection with avian influenza a viruses in birds causes a wide spectrum of disease ranging from subclinical to overwhelming systemic illness (figure ). both h and h subtypes have the ability to evolve into highly pathogenic forms. although virulence is a polygenic trait, a major contributing factor in birds is the haemagglutinin. cleavage of the haemagglutinin into two subunits is essential for viral infectivity. haemagglutinin from strains of low pathogenicity is cleaved by proteases limited to the respiratory tract of mammalian species and the intestinal tract of avian species. by contrast, haemagglutinin from highly pathogenic viruses can be cleaved by proteases present in a range of tissues, resulting in multi-system infection. structural features at the cleavage site determine the cleavability of the haemagglutinin. the acquisition of multiple basic aminoacids in highly pathogenic h or h haemagglutinin enables cleavage of the protein by these ubiquitous proteases and confers virulence. carbohydrate side chains in the vicinity of the cleavage site may also affect the access of the proteases and hence virulence. other factors in addition to the haemagglutinin cleavage site are likely to contribute to virus pathogenicity in mammals. virulence of mouse-adapted influenza a viruses have been associated with the interferon antagonist properties of the ns protein or the ability of the neuraminidase glycoprotein to sequester circulating plasminogen and promote haemagglutinin cleavage. balb/c mice [ ] [ ] [ ] [ ] and ferrets are useful mammalian hosts for the evaluation of human h n pathogenesis. in these mammalian hosts, the multibasic aminoacid motif in the haemagglutinin is necessary but not sufficient for virulence. , , a single aminoacid substitution in pb is associated with high pathogenicity of human h n viruses in mice although substitutions in other gene products are likely to play a part. in human h n infection, disease progression to respiratory failure is unusually severe, with features of haemophagocytosis, leucopenia, and multiple organ failure. , , in-vitro infection of human macrophages with h n human viruses induces high levels of cytokines compared with some human virus strains. in pig respiratory epithelial cells, the h n human viruses were also shown to be relatively resistant to the inhibitory effects of host antiviral cytokines such as interferons. thus, the severity of h n infection in people is likely to be related to the induction of excessive proinflammatory responses that exacerbate tissue injury. it has been suggested that the nonstructural gene has a role. gene sequence analyses of the pandemic virus, which displayed enhanced virulence in human beings has not yet uncovered molecular determinants previously associated with influenza virus virulence. , the molecular determinants and gene constellations that confer virulence of avian, swine, and human viruses, and the circumstances under which virulent phenotypes emerge remains unclear. understanding the basis of virulence is important for vaccine design, particularly of live vaccines, so that viruses can be attenuated. current inactivated influenza vaccines are produced from virus grown in embryonated hens' eggs, and are of three types: whole-virus, "split-product", or subunit "surface-antigen" formulations. whole-virus vaccines are associated with increased adverse reactions, especially in children, and are little used. most influenza vaccines are split-product vaccines, produced from detergent-treated, highly purified influenza virus, or surface-antigen vaccines containing purified haemagglutinin and neuraminidase. vaccines are usually trivalent, containing g each of two influenza a subtypes (h n and h n ) and one influenza b strain. vaccines elicit a relatively strain-specific humoral response, have reduced efficacy against antigenically drifted viruses, and are ineffective against unrelated strains. the who reviews vaccine composition biannually and updates antigenic content depending on prevalent circulating subtypes to provide antigenically well-matched vaccines. protective efficacy of - % in healthy young adults is obtained when there is a good antigenic match between the vaccine and circulating strains. vaccination of the elderly is associated with - % reductions in hospitalisation for pneumonia and influenza, - % reductions for all respiratory conditions, and - % reductions in all-cause mortality. new influenza vaccines must elicit protective immunity. the haemagglutinin-inhibition test is most commonly used for the detection of antibody to influenza, although single radial haemolysis (srh) may also be used, since both are correlated with immune protection. , in the european union, interpandemic influenza vaccines should fulfil certain criteria, prepared by the committee for proprietary medicinal products (cpmp), which are usually assessed by haemagglutinin-inhibition tests in limited annual clinical studies (panel ). in the event of pandemic influenza, vaccine demand would soar. savings made using monovalent rather than trivalent vaccine ( g haemagglutinin per dose instead of g) would possibly be offset by a two-dose schedule, increased demand, and difficulties with production of egg-grown viruses. new developments include the use of mammalian cell lines to culture influenza virus for vaccines to provide increased flexibility of production at times of heightened demand. immunopotentiating effects of adjuvants and whole-virus vaccine may increase antigenicity, allowing dose content reduction enabling maximum efficient use of limited supplies. the population immune status in a pandemic situation differs from that seen during the interpandemic period. at the onset of the previous pandemics, younger adults were immunologically naive to the new strains, whereas older populations may have been primed by previous infections of related strains that circulated in earlier times. global immune susceptibility to avian influenza subtypes would be expected. the quantity of antigen required to elicit satisfactory immune responses in naive individuals is unclear since few studies have been done after the emergence of a novel virus. current events suggest the urgent need to develop a clearly defined strategy for clinical assessment of safety and immunogenicity of pandemic vaccines. in and , the emergence of influenza a/new jersey/ and a/ussr/ / (h n ) triggered pandemic alerts, and afforded the opportunity for vaccine trials in immunologically naive and primed populations. a series of whole-virus vaccine studies [ ] [ ] [ ] [ ] [ ] reported differences between naive populations (those aged years and not exposed to previous h n strains) and primed populations (older than ). in naive patients, if one dose of vaccine was administered, large doses (in excess of g haemagglutinin) were required to fulfil cpmp criteria. however, if two doses of vaccine were given, lower antigen doses ( g) were needed. whole-virus vaccine was significantly more immunogenic than subunit or split-product vaccines. in primed patients, as is the case during interpandemic periods, no difference in immunogenicity between whole-virus vaccine and subunit or split-product vaccines was reported. however, a consistent finding was that whole-virus vaccine was associated with increased reactogenicity, particularly in children, who developed febrile complications even with low doses. although licensed for use in human influenza vaccines, aluminium salts are rarely used since studies have indicated little clinical benefit. however, encouraging findings were more recently reported in a study of whole-virus a/singapore/ / (h n ) vaccine in immunologically naive people. monovalent alumadjuvanted vaccine containing either · , · , or · g h haemagglutinin per dose was compared with unadjuvanted whole-virus g vaccine. although a single dose of any vaccine was unable to elicit responses associated with protection, a second dose of vaccine boosted responses to mean geometric increase in antibody > · (> in the у years group) number of seroconversions or significant rises in anti-haemagglutinin antibody (ie, four-fold increase in post-vaccination hi titre or % increase in srh zone) should be > % (> % in the у years group) proportion of patients achieving a seroprotective hi titre of у / , or srh titre of > mm post vaccination should be > % (> % in the > years group) rates that fulfilled cpmp licensing criteria across all doses, suggesting that up to an eight-fold reduction in antigen content could be achieved with the addition of alum (figure ). since influenza a viruses possess a segmented genome, simultaneous infection of eggs with two different viruses may result in reassortment of segments to produce a desired vaccine seed strain. the influenza a virus components of annual influenza vaccines are typically derived from egggrown reassortment viruses that have the relevant haemagglutinin and neuraminidase genes of the antigenically relevant strain, and the six remaining gene segments from a/puerto rico/ / (h n ). these pr/ / segments confer high growth properties in eggs favoured for inactivated vaccine production. this process requires large numbers of eggs, and in many companies lacks the flexibility to respond rapidly to a pandemic event. highly pathogenic h and h viruses cannot be grown in large quantities because they are lethal to chicken embryos. such pathogenic strains also impose regulatory and safety issues. as the multibasic sequence cleavage site is believed to contribute to the pathogenesis of human h n infection, vaccine preparation from wild-type h n virus would require heightened biocontainment to protect workers and eliminate the possibility of environmental contamination and infection of susceptible animals. thus, several approaches have been attempted for avian influenza vaccine development. these include: ( ) the production of inactivated vaccine from wildtype virus; ( ) the selection of an antigenically related nonpathogenic vaccine strain; ( ) the use of baculoviruses to express recombinant haemagglutinin; ( ) dna-based vaccines; ( ) the use of plasmid-based reverse genetics systems to construct vaccine seed strains possessing attenuated haemagglutinin; and ( ) plasmid-based reverse genetic systems to construct attenuated donor strain recombinants. reverse genetics is likely to produce the most rapid response in an emerging pandemic. much of the preclinical and clinical development of highly pathogenic avian influenza vaccines has used h virus as a model. after the h n outbreak, inactivated vaccines from wild-type a/hong kong/ / (h n ) virus were prepared in the uk and the netherlands. , whole-virus vaccine was effective in protecting mice against lethal challenge with h n virus. conventional surface-antigen vaccine was poorly immunogenic in chickens and did not protect against lethal dose challenge, although an iscom formulation (antigen as immune-complex stimulators) boosted immune responses and protected against lethal h n challenge. because the use of highly pathogenic strains has safety restrictions, the selection of a surrogate non-pathogenic virus capable of evoking crossreactive immunity to the hong kong h n viruses was investigated. both a/duck/hokkaido/ / (h n ) and a/duck/singapore/ (h n ) have haemagglutinin proteins antigenically similar to a/hong kong/ / (h n ) and were used in experimental vaccines. , although antibody titres to h n induced by a/duck/ singapore (h n ) vaccine were four-fold lower than the homologous strain, inactivated whole-virus vaccine , and alum-adjuvanted subunit vaccine were capable of protecting mice against lethal h n challenge. although attempts to reassort a/duck/singapore (h n ) with a/pr/ / to produce a high-growth virus suitable for vaccine production failed, conventional and mf -adjuvanted a/duck/ singapore/ (h n ) surface-antigen vaccines were clinically assessed in a randomised phase i trial. two doses of · , , or g h haemagglutinin were given weeks apart. antibody responses were measured by haemagglutinininhibition, virus microneutralisation, and srh. although both vaccines were well tolerated, non-adjuvanted vaccine was poorly immunogenic, with only one of ( %) recipients seroconverting by haemagglutinin-inhibition and srh h n , and four ( %) by microneutralisation and srh h n after two g doses. the addition of mf gave significantly higher antibody responses (figure ), and two doses achieved seroconversion rates of / ( %), ( %), ( %), and ( %) by haemagglutinin-inhibition, microneutralisation, srh h n , and srh h n , respectively. antibody titres by srh to h n were about half those to h n , showing the need for close antigenic matching between vaccine and pandemic strains to ensure maximum vaccine efficacy. antibody responses after h n revaccination months later were boosted significantly above those achieved after two doses. it is desirable for vaccines to boost responses after initial priming, since second waves occur - months after the first pandemic wave of infection. one problem in assessing vaccine responses was the insensitivity of the haemagglutinininhibition test, routinely used in assessment of influenza vaccines, for the detection of antibody to h when compared with neutralisation tests. , haemagglutinin-inhibition relies on the ability of antibody to disrupt the haemagglutinin-sialic-acid-receptor binding interaction between virus and erythrocytes. if the erythrocytes used in the test are not optimised for expression of ␣ , galactose linkages that are necessary for avian influenza virus binding, the test is insensitive. however, there are no recognised clinical correlates of immune protection for neutralisation antibody. it was only possible to assess vaccine responses with respect to cpmp licensing criteria after development of a specific srh assay. a modified haemagglutinin-inhibition test, using enzymatically altered turkey erythrocytes or horse erythrocytes, was developed to increase the sensitivity for detecting antibody to avian influenza virus antigens. an alternative to egg-derived vaccines involves the use of haemagglutinin protein expressed in insect cells by recombinant baculovirus. potential difficulties include the use of uncleaved rather than cleaved haemagglutinin and differences in glycosylation in insect cells that may effect immunogenicity. nonetheless, antibody responses to - g doses of recombinant h and h antigens are similar to those induced by licensed vaccines. , baculovirus-derived h and h haemagglutinin vaccines protect against lethal virus challenge in chickens, even when the haemagglutinin sequence homology differs by up to %. however, when clinically assessed in people, a recombinant baculovirus-expressed h vaccine was suboptimal. even after two doses of g, only % subjects seroconverted by microneutralisation, suggesting improvements in immunogenicity are needed. reverse genetics systems can be used to generate attenuated avian influenza viruses, and are likely to prove pivotal in pandemic vaccine development. the appropriate haemagglutinin and neuraminidase genes from a virus can be cloned and, if necessary, the haemagglutinin may be attenuated by removal of the multi-basic cleavage site sequence, and inserted into plasmids. the plasmids are transfected into a cell line together with plasmids encoding the internal genes from the a/pr / virus to generate an appropriate non-pathogenic vaccine seed strain. vaccine candidates expressing the target haemagglutinin from highly pathogenic viruses could potentially be produced within weeks of an emerging event. recombinant h viruses showing desirable properties for h vaccine formulation-including loss of egg lethality, virulence, and infectivity in animal models-have been produced, , although one attenuated recombinant virus showed limited neurovirulence in mice. reverse genetics is thus capable of generating attenuated viruses from pathogenic strains suitable for vaccine production with only limited enhancement of biosecurity measures and using pre-existing equipment and facilities. it should also be possible to prepare panels of reassortant viruses and vaccine seed candidates containing target genes of potential pandemic viruses in advance of any specific threat. however, there are regulatory, safety, and legal problems to overcome before the technology can be used for vaccine development. mammalian cell lines (eg, vero cells) used for transfection must be of certified quality for human vaccine production. viruses generated by reverse genetics may be considered to be "genetically modified organisms", imposing local and national safety regulations regarding research and development. in addition, intellectual property rights on reverse genetics technology are held, and licences may need to be granted for commercial use of vaccines. inactivated influenza vaccines are poor inducers of cytotoxic t-cell (ctl) responses, which aid in recovery from influenza infection. it has been suggested that crossreactive immunity to several influenza virus subtypes could be induced by ctl responses to conserved epitopes in internal proteins. dna vaccines integrate gene sequences for the antigenic protein of interest into bacterial plasmids that are inoculated into the host. the expression of plasmid dna produces the antigen in its natural configuration, which is more likely to stimulate neutralising antibody and undergo hla class i expression inducing ctl responses. h and h haemagglutinin-expressing dna vaccine protects mice and chickens against lethal dose virus challenge. , dna vaccine expressing conserved internal proteins including nucleoprotein give partial protection to mice against h n infection. intradermal dna influenza vaccines are beginning clinical evaluation. iscom-formulated h n vaccine can induce ctl responses and greater longer-lasting antibody responses than conventional vaccine. it offers broad protection against virus challenge with h , h , h , and virulent h viruses in mice. iscom vaccines are tolerated in human beings and induce broad ctl and rapid humoral responses. mucosal delivery of inactivated influenza h n vaccine adjuvanted with modified heat-labile enterotoxin from escherichia coli induces b-cell-dependent heterosubtypic immunity against lethal h n virus challenge in mice. in the absence of an antigenically matched vaccine, alternative vaccine strategies that induce crossreactive immunity by iscom, dna, or mucosal vaccines may provide useful firstline defence against an emerging pandemic strain. since h viruses do not have a multibasic haemagglutinin cleavage site, they show low pathogenicity for avian species and may be grown to high titres in eggs. both g -like (a/hong kong/ / ) and y (g -like; a/hong kong/ / ) h n viruses are capable of human infection. , for an effective h vaccine strategy, an understanding of the relative immunogenicity and crossprotection induced by these lineages is required. although infection of mice with g or g group h viruses did not evoke detectable crossreacting neutralising antibody, they were protected from subsequent rechallenge with the homologous or heterologous virus lineage. whole-virus g h vaccine produced crossreactive antibody responses to both g and g viruses, and protected mice against rechallenge with either virus. by contrast, whole-virus g h vaccine induced homologous antibody titres only and was able to protect against g challenge, but showed reduced protective efficacy against the heterologous g lineage. here, a single dose of h vaccine induced adequate immune responses in mice, by contrast with findings with h n vaccine that required a two-dose schedule to elicit adequate immune responses. , since some y (g -like) h viruses do not grow well in eggs, an a/pr/ / reassortant has been produced. one dose of inactivated g /pr vaccine protected mice against g challenge. two doses of vaccine increased antibody responses capable of protecting mice against both g and g h challenge. whole-virus and subunit a/hong kong/ / (h n ) vaccines were clinically evaluated in the uk. adults were randomly assigned two doses, administered weeks apart, of · , , or g h haemagglutinin content. although well tolerated, whole-virus vaccine was more reactive, in keeping with h n vaccines. there was little detectable crossreactive immune response to an antigenically distinct g h virus (unpublished findings). more than % of the prevaccination serum samples showed reactivity to h n by neutralisation and haemagglutinin-inhibition, suggesting preexisting crossreacting antibody from exposure to earlier haemagglutinins. it is unlikely that h influenza has circulated widely in the uk. further serological testing correlated h reactivity with antibody responses to h , but not h or h haemagglutinin. people with baseline reactivity to h were born before , and thus had been potentially exposed to h during its period of circulation in human beings. subjects were immunologically divided into naive and primed recipients. in truly naive subjects, one dose of either vaccine was poorly immunogenic. although whole-virus vaccine was more immunogenic than subunit vaccine, two doses still left a significant number of vaccinees with serological responses below the protective threshold (table ) . among primed individuals, one dose of either vaccine boosted anti-h responses, fulfilling cpmp criteria. since the second dose was of questionable value, to preserve limited vaccine supplies during the first wave of an emerging pandemic, different schedules in different populations could be considered. a german study among - year-olds reported one dose of g whole-virus vaccine a/hong kong/ / (h n ) capable of fulfilling at least one cpmp criterion and that a second dose of vaccine significantly improved responses. however, age-related responses or the effect of pre-existing reactivity to h n were not analysed. in keeping with the h n experience, alum-adjuvant allowed a reduction in h content to · g per dose while maintaining immunogenicity. intranasally delivered live, attenuated cold-adapted influenza vaccines elicit systemic and local mucosal immune responses and display protective efficacy. attenuated cold-adapted strains are generated by reassortment between a wild-type virus expressing target haemagglutinin and neuraminidase, and a cold-adapted donor such as influenza a/ann arbor/ / (h n ). donor strains are cold adapted, temperature-sensitive, and attenuated. these properties are associated with polygenic mutations. these live attenuated viruses display high levels of phenotypic and genotypic stability and are not transmissible to close seronegative contacts. both attenuated h n and h n /ann arbor cold-adapted recombinant viruses have been generated and are seen to be non-pathogenic in mammalian and chicken models. , concerns over the generation of a reassortant between a live virus vaccine containing an avian influenza virus and a co-infecting human strain, and the possibility of spontaneous genetic change may limit the use of such vaccines in the interpandemic period. while current intramuscular influenza vaccines are effective at inducing relatively strainspecific serum haemagglutination-inhibition igg, they are poor at stimulating secretory iga in nasal wash fluid. , as secretory iga exhibits potential heterotypic crossreactivity to influenza virus strains at the point of entry, , live attenuated virus vaccines may offer wider protection against vaccinedrifted variants that could be advantageous once a pandemic is underway. specific influenza antiviral agents are available for early treatment and prophylaxis of influenza. the adamantanes, amantadine and rimantadine, have been available for more than years and inhibit strains of influenza a including nonhuman subtypes. however, rapid emergence and transmission of drug-resistant virus after treatment may render prophylaxis ineffective. the genetic basis for resistance seems to be single aminoacid substitutions in the viral m ion channel. the h n strains isolated from poultry and human cases in had genotypic changes in the m gene associated with resistance, suggesting these agents would be of little clinical value should these strains become capable of humanto-human transmission. neuraminidase inhibitors, such as zanamivir and oseltamivir, are effective in prevention studies and are highly active against a broad range of influenza a viruses of both human and avian origin, including amantadine-resistant strains. although strains with reduced susceptibility to neuraminidase inhibitors have been isolated after sequential passage of virus in presence of drugs, clinically significant resistant strains have not, as yet, been identified. antiviral drugs may be of benefit in protecting individuals in essential services whilst waiting for an effective vaccine to be prepared; however, supply and cost issues would limit their effect on the course of a pandemic. a sufficiently large supply of antivirals to curb pandemic influenza would require international or need for robust surveillance programmes in human and animal populations and sharing of information between animal and human surveillance systems surveillance information to be open and shared in a timely fashion to assess potential threats potential pandemic strains come from animal reservoir. improved understanding of the antigenic and molecular associations between potential pandemic strains of same subtype improved understanding of immunogenicity against drifted avian influenza strains is required as the ability to generate broad crossprotective immunity is desirable in vaccine candidate. intellectual property rights of attenuated viruses produced by reverse genetics must be addressed in advance because licences for commercial use may be required vaccine virus candidate needs to be able to grow well in eggs (or approved cell culture) to improve ability to respond rapidly to emerging threat improved understanding of virulence determinants in mammalian models to be able to attenuate viruses used for vaccine manufacture safety of virus handling for workers involved in preparation of vaccine to assess and approve mammalian cell lines of human vaccine quality ensure that reagents from animal sources are transmissible spongiform encephalopathies compliant "reverse genetics" generated viruses are labelled as genetically modified organisms-implications for national and local regulatory authorities clinical assessment of vaccines derived by reverse genetics ability to organise antigenicity studies rapidly in response to emerging threat may require prepared approved protocols that can be readily adapted. improvement in assessment of antibody responses to avian influenza vaccines to establish licensing criteria standardisation of assays for detection of neutralising antibody to avian influenza establish correlates of immune protection of neutralising antibody whole-virus vaccine more immunogenic than subunit or split-product vaccine in immunological naive populations (h n , h n ) two doses of vaccine required in immunologically naive populations, the first to prime and the second to boost responses (h n , h n , h n , h n ) in primed populations, a single dose of vaccine can potentially induce responses associated with protection (h n , h n , h n ) addition of adjuvants such as mf and aluminium salts have the potential to significantly enhance immunogenicity and spare antigen use (h n , h n and h n ) avian haemagglutinin (h and to a lesser extent h ) seems to be less immunogenic in people than h and h assessment of antibody responses to avian influenza may require additional serological methods other than the standard haemagglutinininhibition test (h n ) potential crossreactivity with pre-existing antibodies complicates interpretation of immune responses in people (h n ) need to develop understanding of improving vaccine candidates to enhance heterosubtypic crossreactivity and protection need to assess vaccine candidates in advance of pandemic to identify difficulties and establish dosing schedules in different populations national stockpiling before the onset of such an event; this would require considerable expense, and vaccination is likely to remain the principal means of combating pandemic influenza. although pandemic planning and understanding is greater since the first h outbreak in , our ability to respond rapidly remains less than optimal. the asian h n epizoonotic outbreak indicates the urgent need for vaccines against avian influenza viruses. however, regulatory and safety considerations confront their development (panel ). it is necessary to improve our understanding of the virulence determinants in mammalian systems to be able to attenuate viruses to select appropriate and safe vaccine strains that can generate broad crossreactivity. national and international authorities must urgently confront regulatory issues to allow production and clinical assessment of newly generated virus vaccine candidates. there are important observations from our clinical experience with vaccines for pandemic influenza (panel ). despite increased reactogenicity, the greater immunogenicity of whole-virus vaccines could be beneficial in a pandemic. vaccines containing avian h and h haemagglutinin seem to be less immunogenic in human beings than vaccines based on h and h haemagglutinin. whether this is a general event associated with avian subtypes is at present unclear. enhancement with mf or alum salts may provide best antigen use and enhance immunogenicity. overall, so far, clinical trials of avian influenza vaccine candidates have given disappointing results. it remains to be seen how plasmid-derived reverse-genetics 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a virus infection in mice influenza: vaccination and treatment searches of medline, pubmed, current contents, and references from relevant articles, as well as the extensive files of the authors identified data for this review. search terms were "avian influenza", "influenza vaccine'', "pandemic influenza", "h influenza", "h influenza", "h influenza", "influenza vaccines", "pathogenesis" and "virulence". english language articles were reviewed. key: cord- -yq dtf n authors: samaranayake, lakshman p.; peiris, malik title: severe acute respiratory syndrome and dentistry a retrospective view date: - - journal: the journal of the american dental association doi: . /jada.archive. . sha: doc_id: cord_uid: yq dtf n abstract background severe acute respiratory syndrome, or sars, which has created panic in asia and in some parts of north america, is the first epidemic of the new century. although it has been well-contained, sporadic cases continue to emerge. objectives the authors trace the emergence of the sars outbreak from southern china and its spread worldwide, discuss the viral etiology of the infection and its clinical features, and review the infection control guidelines issued during the outbreak by the health authorities in hong kong, the centers for disease control and prevention, the world health organization and the american dental association. they also review the prospects for a new outbreak and preventive measures. overview the disease, which is caused by a novel coronavirus termed the “sars coronavirus,” or sars-cov, essentially spreads through droplet infection and affects people of any age. it has a mortality rate ranging from to percent. a major hallmark of this disease has been the rate at which it has affected health care workers through nosocomial transmission; in some countries, up to one-fourth to one-third of those infected were in this category. however, no dental health care worker has been affected by sars in a nosocomial or dental setting. conclusions and clinical implications researchers believe that a combination of factors, including the universal infection control measures that the dental community has implemented and/or the low degree of viral shedding in the prodromal phase of sars, may have obviated the spread of the disease in dental settings. the dental community should reflect on this outbreak to reinforce the currently applied infection control measures. m icrobial threats continue to emerge, reemerge and persist. some organisms are newly recognized pathogens that have existed for centuries (for example, helicobacter pylori, which causes gastric ulcers). others are old organisms that have learned new tricks (for example, multidrug-resistant tubercle bacilli). a third category consists of totally new organisms. this last group of alarming new infectious agents that are virulent and deadly have emerged in rapid succession during the last few years. some of these, such as the ebola virus infection, are still smoldering in some remote corners of the world, while others, such as the h n (and h n ) influenza a bird flu virus and the west nile virus infections, are emerging in different parts of the world. severe acute respiratory syndrome, or sars, is the latest addition to this deadly assortment of new diseases. in the face of these infectious threats, in particular the pandemic of hiv infection, the dental community has reacted swiftly by adopting standard precautions. dentists follow a uniform infection control protocol to treat all patients, irrespective of their medical histories. however, in the face of a new infection that is considered highly contagious, it is prudent to review infection control procedures. the objective of this article, therefore, is to describe the epidemiology, clinical features, etiology and prevention of sars, as well as to evaluate the current infection control protocols used in dentistry in view of the facts related to the spread of this infection. we also explore the prospects for recrudescence of the disease, its treatment modalities and the promise of a sars vaccine. we do not know with certainty how, where and when the disease now known as sars manifested in humans, although theories abound. in february , the world health organization, or who, coined the term "severe acute respiratory syndrome" for the flulike condition that developed into pneumonia. nonetheless, researchers and clinicians generally believe that the first few cases may have originated in china. these sporadic cases were described sometime in the fall of in the guangdong province in southern china. for decades, the guangdong province had a large concentration of people, pigs and fowl living in close proximity because of mixed farming traditions that date back for centuries. this region also has the dubious distinction of being the deadly source of the asian flu, caused by the h n virus, which killed about million people worldwide in and . in , the avian flu (caused by the h n virus), which killed six people, also originated in the guangdong province. the recent outbreak. the sars outbreak has been identified in more than countries in five continents, affecting more than , people, predominantly in asia (especially china), with mini-outbreaks in north america and a few cases in europe. the disease has led to more than deaths worldwide. clusters of cases are particularly common among close associates of patients and the health care workers who treated them and their household contacts. because of the alarming global spread of the disease, who issued a global alert in march and instituted worldwide surveillance. patient characteristics. most patients identified up to now were previously healthy adults aged through years. a few cases of sars have been reported among children (≤ years of age), in whom the clinical course now is thought to be less aggressive. we provide a summary of the major clinical characteristics of patients with sars, although the information should be considered preliminary because of the broad and nonspecific case definition. clinical features. the incubation period for sars is widely considered to be two to seven days, but occasionally may last up to days. symptomatically, the illness appears to have two phases: an early, prodromal febrile phase and a secondary lower-respiratory phase. in pathological terms, however, it is a triphasic disease with a primary viral replicative phase, a secondary immune hyperactive phase and a pulmonary destructive phase. the disease generally begins with a prodrome of typically high fever (> c) that may be accompanied by chills and rigors. headache, malaise and myalgia also are common. at the onset of the illness, some patients have mild respiratory symptoms. in a few cases, the febrile prodrome may be accompanied by diarrhea, although rash and neurologic or gastrointestinal findings are absent. after three to seven days, the secondary lower-respiratory phase begins with a dry, nonproductive cough or dyspnea that may be accompanied by, or progress to, hypoxemia. in up to one-fifth of the cases, the respiratory illness is severe enough to require intubation and mechanical ventilation. the fatality rate among patients with illness that meets the current who definition for probable and suspected cases of sars ranges from to percent, depending on the age group and possibly other, yet unconfirmed, factors. furthermore, the mortality rate is higher among those with underlying illnesses and among the very elderly. typically, chest radiographs appear normal during the febrile prodrome and, in some patients, throughout the course of the illness. however, in the majority of patients, the respiratory phase is characterized by early focal infiltrates that progress to more generalized, patchy, interstitial infiltrates, sometimes leading to consolidation in the very late stages. in general, in the early phase of the disease, patients may have either a normal or decreased white blood cell count, with a reduction in the absolute lymphocyte count. at the peak of the jada, vol. , september c l i n i c a l p r a c t i c e symptomatically, the illness appears to have two phases: an early, prodromal febrile phase and a secondary lowerrespiratory phase. c l i n i c a l p r a c t i c e nomenon not common among other human coronaviruses. medical workers in hong kong, toronto and germany noted this effect when they inoculated lung tissue of patients into cultured monkey kidney cells. [ ] [ ] [ ] this phenomenon leads to a classic cytopathic effect in which the confluent cell layers in laboratory cell cultures are broken down, causing patchy denudation and detachment of cells. [ ] [ ] [ ] immunofluorescence testing of the infected cells indicated that they were indeed infected with a new form of the coronavirus, which has been termed "sars coronavirus," or sars-cov. furthermore, antibodies to the sars-cov were found almost exclusively in patients with sars during their convalescence, but not in human serum samples from healthy patients or in samples banked before the outbreak, suggesting that the infection is new to humans. until now, human coronaviruses have caused the relatively innocuous common cold. however, coronaviruses that infect other mammals and birds are more virulent. these include avian infectious bronchitis (a major problem in the poultry industry), transmissible gastroenteritis of pigs and feline infectious peritonitis. although there was initial speculation that close contact between poultry and humans in the chinese province of guangdong (where sars is thought to have originated) may have resulted in the virus' crossing the species barrier from poultry to humans, evidence now indicates that the himalayan palm civet cats that are consumed as a delicacy and sold widely in animal markets in china are the source of the infection. however, the sars-cov is not a recombinant of known coronaviruses. analyses of the genetic signatures of the viral strains from different geographic regions indicate that immunological pressure might modulate the evolution of the virus in human population cohorts. , other candidate organisms such as paramyxovirus and chlamydia have been implicated in the disease process, but the consensus is that they play a very small role, if any, in the pathogenesis of sars. general properties of coronaviridae. coronaviridae are a family of rna viruses that have been associated etiologically with respiratory ill-lower respiratory phase, up to one-half of patients exhibit leukopenia and thrombocytopenia or platelet counts at the low end of the normal range ( , to , per microliter). renal function appears to remain normal in the vast majority of patients. the box shows the second interim case definition for sars, provided by the centers for disease control and prevention, or cdc. it is based on clinical, epidemiologic and laboratory criteria. however, in areas such as hong kong, where there has been significant disease activity, the cdc criteria have been amended to include patients who do not respond to appropriate antibiotic therapy for atypical pneumonia caused by conventional agents (such as mycoplasma pneumoniae and chlamydia pneumoniae) and/or are in direct contact with another patient with sars. treatment and prevention. a number of treatment regimens have been explored for sars. these include a variety of antibiotics to presumptively treat known bacterial agents of atypical pneumonia, as well as antiviral agents such as oseltamivir and ribavirin. steroids also have been administered in combination with these antimicrobial agents. however, the most beneficial regimen remains to be determined. until reliable diagnostic tests, an effective vaccine and antiviral drugs are available, control of the epidemic depends on early identification of suspected and probable cases, quarantine of patients (and their close contacts) and effective infection control measures, particularly after patients are admitted to a health care facility. etiology. researchers have confirmed that a new strain of virus belonging to the family coronaviridae is the prime agent of this disease. [ ] [ ] [ ] [ ] although other viruses belonging to paramyxoviruses such as metapneumovirus have been implicated, these appear to play only a secondary role, if any, in the disease process. the coronaviruses-so named for the crown of spikes they carry on their surface -attracted the interest of researchers when they noted that the virus rapidly infected cells in culture, a phe- is the prime agent of severe acute respiratory syndrome. copyright © american dental association. all rights reserved. one or more of the following exposures in the days before the onset of symptoms: dtravel to a foreign or domestic location with documented or suspected recent transmission of sars-cov; dclose contact † with a person with mild-to-moderate or severe respiratory illness and history of travel in the days before onset of symptoms to a foreign or domestic location with documented or suspected recent transmission of sars-cov likely exposure to sars-cov one or more of the following exposures in the days before onset of symptoms: dclose contact with a person with confirmed sars-cov disease; dclose contact with a person with mild-to-moderate or severe respiratory illness for whom a chain of transmission can be linked to a confirmed case of sars-cov disease in the days before onset of symptoms laboratory criteria tests to detect sars-cov are being refined and their performance characteristics assessed; therefore, criteria for laboratory diagnosis of sars-cov are changing. the following are general criteria for laboratory confirmation of sars-cov: ddetection of serum antibody to sars-cov by a test validated by the centers for disease control and prevention, or cdc (for example, enzyme immunoassay); or disolation in cell culture of sars-cov from a clinical specimen; or ddetection of sars-cov rna by a reverse transcriptase polymerase chain reaction test validated by cdc and with subsequent confirmation in a reference laboratory (for example, cdc) dprobable case: meets the clinical criteria for severe respiratory illness of unknown etiology and epidemiologic criteria for exposure; laboratory criteria confirmed or undetermined dsuspect case: meets the clinical criteria for moderate respiratory illness of unknown etiology and epidemiologic criteria for exposure; laboratory criteria confirmed or undetermined a case may be excluded as a suspect or probable sars case if: dan alternative diagnosis can fully explain the illness; dthe case has a convalescent-phase serum sample (that is, obtained > days after symptom onset), which is negative for antibodies to sars-cov; dthe case was reported on the basis of contact with an index case that was subsequently excluded as a case of sars, provided other possible epidemiologic exposure criteria are not present * adapted from the centers for disease control and prevention. † close contact is defined as having cared for or lived with a person who has sars, or having a high likelihood of being in direct contact with respiratory secretions and/or body fluids of a person with sars (during encounters with the patient or through contact with materials contaminated by the patient), either during the period in which the individual was clinically ill or within days of resolution of symptoms. examples of close contact include kissing or embracing, sharing eating or drinking utensils, close conversation (less than feet apart), physical examination, and any other direct physical contact between people. close contact does not include activities such as walking by an individual or sitting across a waiting room or office for a brief time. ness in humans and with other diseases in domestic animals. interestingly, they also are associated to some extent with human diarrheal diseases. structurally, they are to nanometers in diameter, positive-stranded and about kilobases in length. their genome is the largest of all rna viruses, and highfrequency recombination between related coronaviruses leads to the generation of much genetic diversity. the virus has three major proteins. the nucleocapsid protein is enclosed within the viral envelope with the rna in a helical nucleocapsid. the other two proteins are the membrane glycoproteins and the spike glycoprotein. the spike glycoprotein is responsible for the characteristic fringe of crownlike projections. antibodies that elicit spike glycoprotein are thought to confer protection against infection. because the human strains are relatively difficult to culture compared with animal strains, the extent of strain variation in human coronaviruses is unclear. there are three serologically and genetically distinct groups of coronaviruses associated with animal and human disease. in general, they are species-specific, although there are a number of examples of viruses crossing species barriers and establishing themselves in new hosts. , once the host is infected, the virus may produce localized disease that often is restricted to the respiratory epithelium or the gastrointestinal tract, or they may produce disseminated infection causing systemic disease. coronavirus was confirmed as the etiologic agent in sars via serologic techniques demonstrating a rise in antibody titer, its growth in tissue culture, a determination of reverse transcriptase-polymerase chain reaction, or rt-pcr, specific for this virus using molecular genetic techniques, and animal studies. animal studies have helped to satisfy koch's postulates, which are necessary to prove disease causation. these postulates stipulate that to be the causal agent, a pathogen must meet four conditions: it must be found in all cases of the disease, it must be isolated from the host and grown in pure culture, it must reproduce the original disease when introduced into a susceptible host, and it must be found in the experimental host so infected. however, further studies that include control groups are required to determine the role of other agents, if any, in causality or as cofactors for severe disease. virus infectivity and survival. the rapid spread of sars worldwide within a few months points to the contagious nature of the disease. the infectivity during the incubation period is still unclear, and it appears that the risk of transmission during the prodrome is low. in contrast, in coronaviruses that cause the common cold, the viral shedding period usually precedes the onset of clinical symptoms by one to two days, although the peak viral excretion occurs during the symptomatic phase. , , the infectivity of sars during convalescence appears to be low and remains to be determined. some data on the survival and infectivity of the sars coronavirus indicate that, unlike other coronaviruses, it is a rather robust organism that is stable in feces (and urine) at room temperature for at least one to two days. it is more stable (up to four days) in stool from patients with diarrhea (which has a higher ph than does normal stool). however, the virus loses infectivity five minutes after being exposed to commonly used disinfectants and fixatives, including percent formaldehyde, percent hypochlorite, percent ethanol and percent phenol. heat at c kills the sars coronavirus at around , units per minutes (considered to be a quick reduction). spread of the disease. the available epidemiologic data suggest strongly that the main routes of virus spread are droplets, direct contact and fomite (indirect contact) transmission, although airborne transmission has not been ruled out completely. researchers believe that the cause of the large outbreaks among health care workers was the transmission of droplets through aerosol-generating medical procedures, such as the use of nebulizers. , no firm data exist regarding the infectivity of contaminated saliva (as opposed to sputum from the respiratory tract) through the droplet route. in some patients, the infection manifests itself as a mild form of diarrhea, and coronavirus particles have been recovered from fecal matter. hence, it is possible that fecal contamination could lead to the spread of the disease, although more data are needed to confirm this route of transmission. it is interesting that some animal coronaviruses are spread through the fecal-oral route. laboratory diagnosis. the mainstay of the sars diagnosis is its characteristic clinical features mentioned above. however, a number of laboratory tests-including serologic tests, cell culture and molecular diagnostics-can be used to confirm the clinically suspicious or probable cases. , these tests include the following. enzyme-linked immunosorbent assay, or elisa, test. from about days after the onset of clinical signs, elisa tests can be used to detect immunoglobulin, or ig, m and iga antibodies in the serum samples of patients with sars. early antibodies are detected in some patients within two weeks. immunofluorescence assay. sars virusinfected vero cells can be used to detect igm antibodies in serum samples of patients after about day of the onset of the disease. this test is reliable, yet demanding, because the live virus must be grown in cell culture; in addition, subsequent immunofluorescence needs to be demonstrated. cell culture. laboratory workers can detect virus in specimens (for example, respiratory secretions, blood) from patients with sars by infecting cultured vero-e or fetal rhesus kidney , or frhk- , cells. molecular tests. laboratory workers can use pcr assays that detect genetic material of coronavirus in patient specimens (such as respiratory secretions, blood or stool samples). primers that are required for this test now are available widely through various web sites (for example, the cdc, the university of hong kong and the governmental viral unit of hong kong). interpretation of test results. clinicians must exercise caution when interpreting laboratory test results, because the key to diagnosis is clinical evaluation and possible exposure to an infected person. a positive laboratory test result indicates that the patient is, or has been, infected with the sars-cov, while a negative test result does not necessarily rule out sars. , seroconversion of paired serum samples with convalescent serum samples obtained more than days after onset of symptoms is a reliable, sensitive and specific diagnostic method. however, the current diagnostic option of choice for early and rapid diagnosis is rt-pcr detection of virus in respiratory or fecal specimens. this test has low sensitivity, and a negative test result does not exclude the diagnosis. many laboratories are addressing the problem of sensitivity and specificity of the sars diagnostic tests, and it will take some time before a highly sensitive, specific, quick and simple diag-nostic test is available. it is possible that, as is the case with hiv infection, saliva could be used as a diagnostic fluid in this context. many people have been alarmed by the spread of sars in clinical facilities, where a disproportionately large number of health care workers (sometimes up to one-third) have been infected. however, it is reassuring that, to date, there have been no documented cases of sars transmitted in a dental setting. this may be the result of a combination of factors. first, transmission has not been documented during the incubation period before the appearance of febrile symptoms. it is unlikely that patients with sars would visit a dentist for elective treatment while they are in the acute phase of the disease, because of the high fever and other, rather debilitating, attendant symptoms. seto and colleagues conducted a case-control study in which they showed that proper use of standard precautions is adequate to prevent the nosocomial spread of sars in the absence of aerosol-producing procedures. however, as health care providers, dental personnel should be wary of the disease and should know how it is spread, how to identify patients with sars and what modifications need to be made to the practice to prevent transmission of the disease. although sars is well-controlled now, it may emerge insidiously, as has been the case with many other coronavirus infections. we review below the infection control measures that dentists and dental staff members now follow, in light of new epidemiologic data about sars, particularly its spread through aerosols and droplets. our recommendations are based on the recent ada guidelines, the cdc's recent report of recommended infection control practices for dentistry and our own experience in hong kong related to the last outbreak. identification of patients with sars. as health care providers, dentists should be able to identify a suspected case of sars. the cdc's current interim diagnostic criteria for sars are shown in the box. they are subject to change as more is learned about the disease, and should be reviewed periodically by visiting the ada or cdc web sites. to date, there have been no documented cases of severe respiratory syndrome transmitted in a dental setting. as stated above, we doubt that patients with sars who are in the acute febrile phase of the disease will visit a dentist. in the unlikely event that this does occur, the dentist should not treat the patient in the dental office, but should refer him or her to a health care facility as soon as possible for diagnosis and care. dentists also have a duty to report the case to state or local health departments. patient evaluation. as always, dentists should take a thorough medical history from each patient and update it at each recall appointment. the questionnaire used for this purpose may have to be modified to incorporate targeted screening questions regarding sars. although these questions may appear superfluous during the current abeyance of the outbreak, they are important as a guide if there is another outbreak of sars or an outbreak of a similar new disease. these questions may include the following: ddo you have fever? dhave you experienced a recent onset of a respiratory problem, such as a cough or difficulty breathing? dhave you, within the last days (that is, the incubation period for sars), traveled internationally or visited an area where documented or suspected community transmission of sars is occurring? dhave you come into contact with a patient with sars in the past days? in the event that the patient recently has returned from a geographic region with documented or suspected community transmission of sars, the clinician can defer elective treatment until the incubation period is over. dentists can provide emergency treatment, provided they use routine barrier precautions and avoid spatter or aerosol-generating procedures. this emergency treatment should be limited to the control of pain and infection. dentists should not treat patients in the dental office who are suspected of having sars. if a patient replies "yes" to the first two screening questions, the dentist should wear a surgical mask, discuss his or her potential concerns with the patient, call an area medical facility (such as a hospital) and inform the staff that he or she is referring a patient suspected of having sars so that arrangements can be made for transportation and care of the patient. patients with sars need ground emergency medical services. these screening questions should be asked routinely of all patients, because questioning only a select group of patients, for whatever reason, may undermine the early detection of infection and might be construed as a discriminatory practice. clinicians should delay treating convalescing patients for at least one month after they are released from the hospital. convalescing patients are instructed to remain at home for seven days after discharge from the hospital, and during this period they are requested to stay indoors and keep contact with others to a minimum. preprocedural rinsing. a preprocedural antimicrobial mouthrinse (with . to . percent chlorhexidine gluconate) is believed to reduce the number of microbes that are released into the operatory environment. this has been shown in a number of studies in which a longlasting mouthrinse (for example, chlorhexidine gluconate with povidone iodine and essential oils) has reduced the disseminated microbial load during procedures such as ultrasonic scaling. , however, no concrete data show that a preprocedural mouthrinse reduces infection among dental health care workers or patients. a preprocedural rinse would be most useful in situations in which a rubber dam cannot be used, such as when a prophylaxis cup or an ultrasonic scaler is used, and in the absence of assisted, high-volume suction. hand hygiene. microflora on the skin can be divided into two categories: the transient flora colonizing the superficial layers of the skin and mainly acquired through environmental routes, and the residential flora thriving on the deeper layers of the skin and hair follicles. the exogenous, superficial flora are harmful and pathogenic, but are removed easily with clinical hand-washing procedures. by contrast, the endogenous residential flora are almost impossible to remove completely, but are less likely to be associated with infections. the single most important method of preventing transmission of any infectious agent, including the sars coronavirus, is hand washing and appropriate hand care. studies have found that even in critical care units, hand-washing compliance is relatively low, sometimes approaching percent. by contrast, a dramatic reduction in the prevalence of health careassociated infections has been shown when regimented hand hygiene measures were introduced. thus, appropriate hand hygiene is the mainstay of a good dental infection control program. furthermore, recent data indicate that the sars virus, compared with other coronaviruses, is a relatively robust organism and may survive on nonporous surfaces for up to hours. this reinforces the need for good hand hygiene, as well as the importance of thorough surface disinfection. hand hygiene for routine dentistry. for routine dentistry, which entails examinations and nonsurgical procedures, plain soap and water are adequate. recently, the cdc recommended that if the health care worker's hands are not visibly soiled, an alcohol-based hand rub could be used for routine decontamination, because this is as effective as hand washing and also saves time. also, clinicians should decontaminate their hands both before and after removing gloves, because humidity and moisture cause bacteria to multiply rapidly under the glove surface. hand rubs that are based on alcohol alone should not be used owing to their rapid evaporation and lack of residual effect. consequently, hand rubs must be laced with agents such as chlorhexidine, octenidine or triclosan to achieve the needed effect. after using an alcohol-based hand rub, the clinician must dry his or her hands thoroughly before putting on gloves, because any residual alcohol may increase the risk of glove perforation. personal protective equipment. personal protective equipment, or ppe, is designed to protect the skin and mucous membranes of the eyes, nose and mouth from exposure to potentially infectious material. recent experience with the sars coronavirus has shown that vast numbers of health care workers acquired the infection in hospital settings, either as a result of inadequate barrier protection methods or the improper use of these methods. this barrier protection equipment consists of protective eyewear, masks, gloves, face shields and protective overwear. we should note that general work clothes such as uniforms do not protect against a hazard and should not be considered ppe. we describe below the relevant aspects of ppe that pertain to protection against airborne hazards. masks. face masks were first worn by surgeons to minimize postsurgical infection in patients due to microbes that were exhaled or shed by the surgical team. however, the realization that face masks protect the health care worker as well as the patient has led to the routine use of this protective measure in many clinical settings including dentistry. transmission of airborne infection depends on factors such as the virulence of the organism, as well as the number of organisms, transmitted. in the case of coronavirus-induced pneumonia leading to sars, airborne droplet transmission of infection is considered to be the main route of spread. various types of masks and face shields are available. surgical masks usually provide adequate protection in dental care settings, where highly transmissible infectious diseases are not typically encountered. particulate respirators. however, surgical masks are not designed to provide adequate protection against exposure to airborne infectious agents such as tubercle bacilli or droplet nuclei smaller than micrometers. for such purposes, particulate respirators (for example, n- masks) must be used. during the sars outbreak in hong kong, the vast majority of dental practitioners in that country used n masks for routine dentistry. however, these masks are uncomfortable to wear for extended periods because of the difficulty in breathing through a thick impervious fabric, and are not recommended for routine dental office settings. rubber dam isolation. rubber dams help minimize the production of saliva-and bloodcontaminated aerosol or spatter. samaranayake and colleagues reported an up-to- -percent reduction in airborne particles around a -foot diameter of the operational field when a rubber dam was used. a split-dam technique may be used in situations in which gingival areas are involved, such as class v restorations and crownand-bridge preparations. aerosol-generating procedures should be avoided as much as possible if rubber dam isolation is not feasible. some of these procedures include ultrasonic scaling, root-surface débride- ment, and high-or low-speed drilling with water spray. there is no doubt that coronavirus research has gained an unprecedented and urgent momentum owing to the lethality of sars and its nearly worldwide spread within a few months. consequently, laboratories throughout the world are working in unison to provide answers to many unresolved questions, as well as to develop a new preventive vaccine. in dentistry, in particular, a number of questions remain to be resolved, including the following: although the global threat of sars has peaked for the most part, it is helpful to review the response of the community to this novel disease. it is fortunate that sars was not sufficiently infective to cause a repeat of the influenza pandemic that killed millions. even so, we might be able to attribute the relatively low death rate in large part to the worldwide surveillance networks and patient isolation efforts that were introduced rapidly in most countries. in retrospect, an overreaction seems to have been a better option than allowing the disease to run out of control, as was the case with the aids pandemic. culmination of the outbreak. the who lifted all its travel advisories as of june , , and since then, only three new cases of sars have been reported. two of these-one in singapore and the other in taiwan-were acci-dental, laboratory-acquired infections in research technicians working with the organism, while the third patient-from guangdong province in southern china-is thought to have acquired the infection through contact with contaminated rodents. because the initial symptoms of sars mimic those of many variants of atypical pneumonia, a high degree of suspicion by the medical establishment, intense surveillance and immediate quarantine of all close contacts of patients should ward off another, large-scale winter outbreak. if sars does return, its epidemiology may be different from that of the current strain. for instance, the genome of the new sars-cov may differ, and the virus may be more or less infective than the original strain that emerged in . furthermore, we do not know how long the acquired immunity to sars persists. also, will those exposed to the virus be carriers of the disease in the face of a new infection? how many will be silent healthy carriers of the virus? will an emergent strain or strains behave similarly to the older counterpart? we do not have the answers to these questions. mutation of the sars-cov. the reason for the pandemic spread of hiv is its ability to mutate rapidly from one generation to another so it can escape the immune surveillance mechanisms of the host, as well as the prescribed antiviral medications. the sars virus, on the other hand, seems to be remarkably invariant; the genome sequence of isolates from patients in singapore, toronto, china and hong kong has not revealed any changes of major consequence. this does not mean that the sars virus is incapable of mutation; rather, because the virus has encountered little resistance from new human hosts, there is less selective pressure for new mutants to emerge and persist. drugs and vaccines for sars. many researchers are working on potential drugs and vaccines to treat patients with sars. however, their approach has been scattered for the most part, as they screen the multitude of available drugs and compounds for their ability to destroy the sars-cov. thus far, a few have had success. one group reported that the compound glycrrhizin, which is derived from licorice roots, can rid cultured monkey kidney cells of the sars virus. other researchers, using in silico research, have proposed that the newly described proteinase of the sars virus (which converts viral proteins into the active form required for viral replication) could be inhibited by drugs. animal models are essential for experimentation and drug discovery; thus far, the only validated model has been cynomolgus macaque (macaca fascicularis). a smaller and less expensive animal model for sars research has yet to be defined. although vaccines exist for animal coronavirus infections, it may take a few years before a vaccine for sars is developed. it is comforting to note that the existing technology and know-how for animal coronavirus vaccines could be translated directly toward the manufacture of a sars vaccine. furthermore, implicit evidence shows that the vaccine approach to preventing sars is feasible, because patients' conditions appear to improve when they are given hyperimmune serum from recovered patients with sars. sars vaccines could be based on a killed sars virus or on an attenuated virus that is sufficiently potent to replicate itself in humans and initiate a successful antibody response, but not potent enough to cause disease. another option would be to re-engineer a harmless candidate virus to contain genetic sequences of the sars virus. this approach has been used to produce a prototypic vaccine against a coronavirus that causes bronchitis in chickens. should there be a renewed threat of sars, a vaccine would be a mostwelcomed weapon by health care workers. sars is the first readily transmissible infectious disease that the global community has confronted in the new millennium. this, surely, will not be the last contagion that we will encounter. the fact that no dental health care worker or dental patient has thus far contracted sars in a dental setting is a testament to good infection control measures that have been implemented in the vast majority of dental offices. however, the dental community cannot let down its guard, and must be constantly aware of impending infectious threats in various guises, as well as recrudescence of disease, that may challenge the current infection control regimen. i institute of medicine; . . samaranayake lp, peiris js, scully c. ebola virus infection: an overview the ebola virus and the challenges to health research in africa west nile virus: statistics, surveillance, and control. available at infection control for the dental team cumulative number of reported probable cases of severe acute respiratory syndrome (sars) there is nothing permanent except change: the emergence of new virus diseases centers for disease control and prevention. isolation of avian influenza a (h n ) viruses from humans: hong kong epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong clinical presentations and outcome of severe acute respiratory syndrome in children sars: what do we know about the disease updated interim u.s. case definition for severe acute respiratory syndrome (sars) (appendix b ) sars bulletin from hong kong coronavirus as a possible cause of severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome sars-associated coronavirus koch's postulates fulfilled for sars virus coronaviruses of man isolation and characterization of viruses related to the sars coronavirus from animals in southern china identification of severe acute respiratory syndrome in canada comparative full length sequence analysis of sars coronavirus isolates and common mutations associated with putative origins of infection centers for disease control and prevention. interim domestic infection control precautions for aerosol-generating procedures on c l i n i c a l p r a c t i c e patients with severe acute respiratory syndrome (sars) effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars) world health organization. who hospital discharge and followup policy for patients who have been diagnosed with severe acute respiratory syndrome (sars) reduction of viable bacteria in dental aerosols by preprocedural rinsing with an antiseptic mouthrinse reduction of bacteria-containing spray produced during ultrasonic scaling essential microbiology for dentistry how good are hand-washing practices? world health organization. summary on major findings in relation to coronavirus by members of the who multicenter collaborative network on sars aetiology and diagnosis. available at healthcare infection control practices advisory committee, hicpac/shea/apic/idsa hand hygiene task force. guideline for hand hygiene in health-care settings: recommendations of the healthcare infection control practices advisory committee and the hicpac/shea/apic/idsa hand hygiene task force: society for healthcare epidemiology of america/association for professionals in infection control/infectious diseases society of america hospital epidemiology and infection control the integrity of latex gloves in clinical practice the surgical mask unmasked: a review medical progress: how contagious are common respiratory tract infections? evidence of airborne transmission of the severe acute respiratory syndrome virus the efficacy of rubber dam isolation in reducing atmospheric bacterial contamination sars: breaking the chains of transmission chemical modification of glycyrrhzic acid as a route to new bioactive compounds for medicine coronavirus main proteinase ( clpro) structure: basis for design of anti-sars drugs a recombinant fowl adenovirus expressing the s gene of infectious bronchitis virus protects against challenge with infectious bronchitis virus dr. peiris is a professor of virology, faculty of medicine, the university of hong kong, hong kong, and the chief clinical virologist, queen mary hospital, hong kong. key: cord- - d f k authors: mall, sanjay; malcolm east, j.; lee, anthony g. title: transmembrane α helices date: - - journal: curr top membr doi: . /s - ( ) - sha: doc_id: cord_uid: d f k this chapter discusses effects of intrinsic membrane proteins on lipid bilayers and model transmembrane α helices. incorporation of a protein into a lipid bilayer has significant effects on the properties of the bilayer. the rough surface presented by a protein to the surrounding lipid bilayer tends to produce poor packing unless the lipid fatty acyl chains distort to match the surface of the protein. in a liquid crystalline bilayer the lipid fatty acyl chains are disordered, because the chains undergo extensive wobbling fluctuations. the presence of a rigid protein surface reduces the extent of these motional fluctuations. however, the chains tilt and become conformationally disordered to maximize contact with the rough surface of the protein. the net result is that the presence of a protein leads to decreased order for the chains, with a wide range of chain orientations relative to the bilayer normal, but with reduced extent and rate of motion. because of the reduced motion, lipids adjacent to membrane proteins are often referred to as being motionally restricted. it is clear that the reasons for the disorder of the bulk lipids and the disorder of the lipids adjacent to the protein are different; for the bulk phospholipids, the disorder is dynamic, whereas, for the boundary lipids the disorder is static. bilayer of /~, about residues will be required to span the core of the bilayer. the residues in the transmembrane region will be predominantly hydrophobic. however, for membrane proteins such as transporters and ion channels the transmembrane ot helices will also have to contain polar groups; the transmembrane helices will then be amphipathic rather than just hydrophobic. an extreme case could be a transmembrane a helix totally surrounded by other t~ helices in the center of a helical bundle: such a helix would not need to be hydrophobic at all, because it is not in direct contact with the lipid bilayer. however, although small in number, the available high-resolution structures for membrane proteins show no evidence for the presence of such purely hydrophilic transmembrane ot helices. it may be that the process of insertion of membrane proteins into the membrane during biogenesis requires all the transmembrane c~ helices to be relatively hydrophobic. analysis of the compositions of a large number of membrane proteins predicted to contain single transmembrane ot helices has shown that the amino acid composition of a transmembrane ot helix is distinctly different from that of hydrophobic a helices in water-soluble proteins. as expected, hydrophobic residues make up the bulk of the residues, the most common being leu (landolt-marticorena et al., ; wallin et al., ) . amino acids essentially excluded are the basic (arg and lys) and acidic (asp and glu) amino acids and their amide counterparts (asn and gin). transmembrane c~ helices are, however, relatively rich in bulky residues, such as ile, val, and thr, which, in water-soluble proteins, are classed as membrane destabilizers (their bulky side chains interfere sterically with the carbonyl oxygen in the preceding turn of the ot helix and thus destabilize the helical conformation). thus, factors such as residue volume and packing, which are important in determining helix stability in water-soluble proteins, are not so important for transmembrane ct helices, at least for membrane proteins containing single transmembrane c~ helices; effects of large residue volume will, in the membrane, be balanced by the favorable hydrophobic interactions of a large side chain with the fatty acyl chains. in water-soluble proteins, the conformationally flexible gly residue is also classed as a helix breaker, because it is an intrinsically flexible residue with the potential to adopt most of the dihedral angles available in a ramachandran plot. the observation that gly is quite common in transmembrane ot helices suggests that its potential flexibility is constrained in the bilayer environment. because gly possesses the smallest of all the side chains, it may play a role in mediating helix-helix interactions and packing in the membrane. the polar amino acids most commonly found within transmembrane ot helices are cys, thr, and ser. these residues can be stabilized within a hydrophobic environment by hydrogen bonding between their polar side chains and the peptide backbone at positions i - and i - (eilers et al., ) . figure shows the positional preferences of the residues in the transmembrane ot helices of type i membrane proteins with a single transmembrane c~ helix oriented figure positional preferences for amino acids in the transmembrane domains of human type i membrane proteins with single transmembrane a helices. modified from landolt-marticorena et al. ( ) . with its c-terminus on the cytoplasmic side of the membrane (landolt-marticoreno et al., ) . the amino-terminal end of the transmembrane domain contains an lie-rich region followed by a val-enriched region. the carboxyl-terminal half of the transmembrane a helix is leu-rich. ala is found randomly distributed throughout the transmembrane domain. aromatic residues are found located preferentially in the boundary regions, with trp at either end of the transmembrane domain, but with tyr and phe only at the carboxyl-terminal boundary. unlike the other aromatic amino acids, phe, is also found in the hydrophobic segment as well as in the boundary region. the polar regions flanking the transmembrane domain are enriched in arg and lys on the c-terminal side; asn, ser, and pro are enriched in the n-terminal flanking region. the presence of a positively charged c-terminus (cytoplasmic) could play a role in the process of insertion into the membrane, according to the inside positive rule of von heijne ( ) . the presence of particular residues at the n-and c-terminal ends of the helices could also be important in meeting the requirement to "cap" the ends of the ot helices; the initial four --nh and final four --c=o groups of an ot helix have no hydrogen-bonding partners provided by the peptide backbone of the a helix itself, and so suitable hydrogen-bonding partners have to be provided in some other way. one way is to extend the helix by three or four residues at each end with polar residues containing suitable hydrogen-bonding partners such as pro and ash. alternatively, if the hydrophobic, nonpolar residues in the transmembrane c~ helix extend into the headgroup region, hydrogen bonds could form with suitable groups in the glycerol backbone and headgroup regions of the lipid bilayer. either way, if about residues are required to span the hydrophobic core of the bilayer, the total helix length could be up to residues. the result is that there is a degree of indeterminacy in where the ends of transmembrane helices should be drawn; the precise ends of transmembrane ~ helices are often not known. the observed preference for trp and tyr residues for the ends of transmembrane ct helices agrees with measurements of the binding of small peptides at the lipid-water interface, which show that aromatic residues have a preference for the interface (wimley and white, ) . further, a number of small tryptophan analogues have been shown to bind in the glycerol backbone and lipid headgroup region of the bilayer, stabilized partly by location of the aromatic ring in the electrostatically complex environment provided by this region of the bilayer, and partly by exclusion of the fiat, rigid ring system from the hydrocarbon core of the bilayer for entropic reasons (yau et al., ) . thus, although it is agreed that aromatic residues at the ends of transmembrane a helices probably act as "floats" at the interface serving to fix the helix within the lipid bilayer, it is unclear whether the aromatic rings are located in the hydrocarbon or the headgroup region of the bilayer. this uncertainty is also apparent in the crystal structures of a number of membrane proteins. for example, the trp residues in the bacterial potassium channel kcsa (doyle et al., ) are found clustered at the ends of the transmembrane ot helices, forming clear bands on the two sides of the membrane, as shown in fig. . however, the tyr residues clearly form a band on the periplasmic side of the membrane above the band formed by the trp residues. similarly, in the bacterial photosynthetic reaction center (rees etal., ) the majority of the trp residues are found near the periplasmic side of the protein near the ends of the transmembrane c¢ helices, as shown in fig. . however, the band of trp residues is more diffuse than in kcsa, and some trp residues are likely to be located in the hydrocarbon core and some in the headgroup region. the average number of figure the crystal structure of the potassium channel kcsa. a cross section with just two of the four identical subunits is shown. trl o residues are shown in space-fill representation and tyr residues are shown in ball-and-stick representation. two potassium ions in space-fill representation are shown moving through the channel. the separation between the two planes representing the outer edges of the trp residues is /~. (protein data bank [pdb] file ib .) p ¢ figure the structure of the l and m subunits of the photosynthetic reaction center of rhodobacter sphaeroides. trp residues are shown in ball-and-stick representation. an approximate location for the hydrophobic core of the bilayer of thickness a is shown, as defined by the surface covered by detergent. (pdb file aij.) residues in the transmembrane c~ helices of the bacterial photoreaction center is , corresponding to a length of about /~. the stretch of hydrophobic residues in these helices is, however, only about amino acids or about . /~ long (ermler et al., ; michel and deisenhofer, ) . this matches the thickness of the nonpolar region of the complex (about /~) as defined experimentally as the part covered by detergent in the crystal (roth et al., (roth et al., , . detergent is seen to cover some of the trp residues on the periplasmic side of the membrane, but not others (roth et al., ) . the distribution of trp residues on the cytoplasmic side of the complex is much less distinct than on the periplasmic side (fig. ). if the hydrocarbon core of the bilayer around the complex does have a thickness of ]~, then again the trp residues on the cytoplasmic side of the membrane will be located in both the hydrocarbon core and the headgroup regions of the bilayer (fig. ) . in the ca +-atpase of skeletal muscle sarcoplasmic reticulum the situation is more complex, as shown in fig. (toyoshima et al., ) . many of the transmembrane o~ helices extend above the membrane surface to form a central stalk linking the transmembrane region to the cytoplasmic head of the protein. as a consequence some of the helices are very long; helix m , for example, contains residues. a ring of trp residues can be seen on the cytoplasmic side of the membrane helping to define the location of the membrane surface (fig. ) . a lys residue (lys- ) in transmembrane c~ helix m can be seen pointing up from the hydrophobic core of the bilayer like a snorkel. because the cost of burying a charged residue in the hydrophobic core of a bilayer is very high (about kj mo - for a lys residue ; engelman et al., ) , it is likely that the amino group on lys- will be located at the interface; the trp residues in the ca +-atpase will then be located in the headgroup region of the bilayer. the structure of the ca +-atpase is also unusual in that the first transmembrane ot helix contains two polar residues, asp- and arg- , pointing out into the hydrocarbon core; presumably, stacking of asp- against arg- allows formation of an ion pair. the distribution of trp residues on the lumenal face of the ca +-atpase is much more diffuse than on the cytoplasmic side. the hydrophobic thickness of the ca +-atpase could be expected to be about /~, because that is the hydrophobic thickness of a bilayer of di(c : )pc, the phospholipid that supports highest activity for the atpase . however, as shown in fig. , this definition locates the lumenal loops between transmembrane ~ helices m and m i phospholipid designations are pc, ps, and pa for phosphatidylcholine, phosphatidylserine, and phosphatidic acid, respectively. fatty acyl chains are given in the format m:n, where m is the number of carbon atoms and n is the number of double bonds. thus, for example, dioleoylphosphatidylcholine is di(c : )pc. and between m and m within the hydrocarbon core. further, it locates a lys residue (lys- ) totally within the hydrocarbon core, which seems unlikely. the hydrophobic thickness of the bilayer would have to be about a to locate the two interhelical loops and lys- at the lumenal surface (fig. ) ; this is close to the thickness of a bilayer of di(c :i)pc ( a). the crystal structure shown in fig. corresponds to the ca +-bound, e conformation of the atpase. it has been shown that the e conformation of the atpase is favored by di(c : )pc, whereas di(c : )pc favors the other major conformation of the atpase, e (starling et al., ) . thus, it is possible that conformational changes within the transmembrane region of the ca +-atpase lead to changes in the interhelical loops and thus to changes in the effective hydrophobic thickness of the atpase (lee and east, ) . incorporation of a protein into a lipid bilayer can be expected to have significant effects on the properties of the bilayer. the rough surface presented by a protein to the surrounding lipid bilayer will tend to produce poor packing unless the lipid fatty acyl chains distort to match the surface of the protein. in a liquid crystalline bilayer the lipid fatty acyl chains are disordered, because the chains undergo extensive wobbling fluctuations. the presence of a rigid protein surface would be expected to reduce the extent of these motional fluctuations. however, the chains will have to tilt and become conformationally disordered to maximize contact with the rough surface of the protein. the net result is that the presence of a protein will lead to decreased order for the chains, with a wide range of chain orientations relative to the bilayer normal, but with reduced extent and rate of motion. because of the reduced motion, lipids adjacent to membrane proteins are often referred to as being motionally restricted. it is clear, therefore, that the reasons for the disorder of the bulk lipids and the disorder of the lipids adjacent to the protein (the boundary or annular lipids) are different; for the bulk phospholipids the disorder is dynamic, whereas for the boundary lipids the disorder is static. an example is provided by the bacteriorhodopsin trimer, whose crystal structure is unusual in showing a few well-defined lipid molecules (belrhali et al., ; luecke et al., ) . figure shows some of the lipids located at the surface of the trimer. the electron densities for the chains are well defined, but the headgroups are disordered, so that the headgroups could not be identified; the lipids were therefore modeled simply as , -di-o-phytanylsn-propane (belrhali et al., ) . the considerable static disorder of the chains is clear in fig. , the rotational disorder of the chains being necessary to obtain good van der waals contacts with the molecularly rough surface of the bacteriorhodopsin trimer. lipids on the extracellular side of the membrane are better resolved than ~ figure structures of four phospholipid molecules identified in x-ray crystallographic studies of bacteriorhodopsin (belrhali et al., ) . the lipids have been modeled as , -.di-o-phytanyl-snpropane. (pdb file lqhj.) those on the cytoplasmic side; the degree of order of the lipids parallels that of the protein, which is also greater on the extracellular side (grigorieff et al., ) . the average distance between the glycerol backbone oxygens for phospholipids on the two sides of the membrane was . /~ (mitsuoka et al., ) . as expected, this closely matches the hydrophobic length of the transmembrane helices of bacteriorhodopsin; the mean helix length is residues, corresponding to a length of about a. the crystal structure also makes clear the very different conformations adopted by the various lipid molecules located on the surface of the tfimer. for example, one lipid molecule forms a hydrogen bond from its ether oxygens to a tyrosine --oh group at the end of a transmembrane a helix (fig. ; belrhali et al., ; essen et al., ) . the result is that the strength of the interactions of individual boundary lipid molecules with the protein will be different. the disorder of the chains seen in fig. is consistent with the results of molecular dynamics simulations of the bacteriorhodopsin trimer in a bilayer of diphytanyl phosphatidylglycerophosphate (edholm et al., ) . the molecular dynamics simulation agrees with experiment in predicting higher order for both the lipids and the protein on the extracellular side of the membrane; fluctuations in the loops and the ends of helices on the cytoplasmic side of the membrane are greater than on the extracellular side. this is also seen in fluctuations of the lipids, with lipids on the cytoplasmic side of the membrane fluctuating more strongly than those on the extracellular side (edholm et al., ) . the calculated order parameters for the chains are low, mainly due to a static tilt of the chains necessary to allow them to nestle against the rough surface of the protein. the chains in the purple membrane behave more like parts of the protein than parts of a fluid lipid phase, consistent with the idea of boundary lipids (edholm et al., ) . the boundary lipids and the bulk lipids in a membrane can be distinguished experimentally in many systems, because of the static disorder of the boundary lipids and the dynamic disorder of the bulk lipids. static and dynamic disorder give rise to very different electron spin resonance (esr) spectra for spin-labeled lipids, and esr spectra for membrane protein systems usually show two components, one "immobilized," corresponding to boundary lipid, and one relatively mobile, corresponding to bulk lipid (devaux and seigneuret, ; marsh, ) . studies with oriented samples have confirmed a wide range of orientational distributions for the boundary lipid, in contrast to the bulk lipid phase, where motion of the lipid long axis is about the bilayer normal (jost et al., ; pates and marsh, ) . a particularly important feature of a membrane protein as far as the lipid bilayer is concerned is the thickness of the transmembrane region of the protein. the cost of exposing hydrophobic fatty acyl chains or protein residues to water is such that the hydrophobic thickness of the protein should match that of the bilayer. the question then is how the system responds when these do not match. most models of hydrophobic mismatch assume that the lipid chains in the vicinity of the protein adjust their length to that of the protein, with the protein acting as a rigid body. when the thickness of the bilayer is less than the hydrophobic length of the peptide the lipid chains must be stretched. conversely, when the thickness of the bilayer is greater than the hydrophobic length of the peptide the lipid chains must be compressed (fig. ) . stretching the fatty acyl chains will effectively decrease the surface area they occupy in the membrane surface, and, conversely, compressing the chains will increase the effective area occupied in the surface (fig. ) . thus, figure the result of a mismatch between the hydrophobic length of a peptide and the hydrophobic thickness of a lipid bilayer. left: positive hydrophobic mismatch (dp > dl). right: negative mismatch (dp< de). the top shows a "side" view of the chain packing around the peptide and the bottom shows a top view, illustrating the variation in chain cross-sectional area with distance from the peptide. figure based on fattal and ben-shaul ( ) . changes in fatty acyl chain order are linked to changes in average interfacial areas per lipid molecule. a number of terms have been suggested to contribute to the total free energy cost of deforming a lipid bilayer around a protein molecule (fattal and ben-shaul, ; nielsen et al., ): . loss of conformational entropy of the chains imposed by the presence of the rigid protein wall . bilayer compression/expansion energy due to changes in the membrane thickness . surface energy changes due to changes in the area of the bilayer-water interface . splay energy due to changes in the cross-sectional energy available to the chains along their length, resulting from curvature of the monolayer surface near the protein a number of models have been proposed to estimate these terms. fattal and ben-shaul ( ) calculated the total lipid-protein interaction free energy as the sum of chain and headgroup terms. for the chains the loss of conformational entropy imposed by the rigid protein wall was positive (unfavorable) even for perfect hydrophobic matching. the other contribution to the chain term arose from the requirement for hydrophobic matching and the consequent stretching or compression of the chain. the term due to the headgroup region was treated as an interracial free energy, including an attractive term associated with exposure of the hydrocarbon core to the aqueous medium and a repulsive term due to electrostatic and excluded-volume interactions between the headgroups (excluded-volume interactions signify that no two atoms can occupy the same position in space). the resulting profile of energy of interaction as a function of hydrophobic mismatch was fairly symmetrical about the point of zero mismatch. the lipid perturbation energy f (in units of kt per angstrom of protein circumference) calculated by ben-shaul ( ) fits to the equation where dp and dl are the hydrophobic thicknesses of the protein and lipid bilayer, respectively, and the unperturbed bilayer thickness is . /~. the hydrophobic thickness of a bilayer of phosphatidylcholine in the liquid crystalline phase is given by where n is the number of carbon atoms in the fatty acyl chain (lewis and engelman, ; sperotto and mouritsen, ) . a simple, but crude calculation gives an idea of the size of the effect that can be expected from hydrophobic mismatch. it is assumed that the protein is very large and so appears fiat to a lipid molecule. it is also assumed that all the lipid perturbation energy is concentrated in the first shell of lipids around the protein. equation ( ) then shows that if a lipid occupies a of the protein circumference, the lipid-protein interaction energy would change by . kj tool - for a hydrophobic mismatch of a, corresponding to an increase in fatty acyl chain length of carbons, and by . kj mol -z for a hydrophobic mismatch of . a, corresponding to an increase in acyl chain length of carbons. changes in interaction energies of . and . kj mo - correspond to decreases in the lipid-protein binding constant by factors of . and n, respectively. if the change in lipid-protein interaction energy were to propagate out from the protein surface to affect more than the first shell of lipids, effects of hydrophobic mismatch would be reduced. for example, if effects were averaged over three shells of lipids, changes in fatty acyl chain lengths by and carbons from that giving optimal interaction would decrease lipid-protein binding constants by factors of . and , respectively. energies of the magnitude calculated by ben-shaul ( ) are easily sufficient to result in conformational changes on a protein. if a protein conformational change results in a change in the hydrophobic thickness of the protein, the change will result in a deformation of the adjacent bilayer. because the equilibrium constant describing the equilibrium between two conformational states of a protein is determined by the total free energy difference between the two states, the energetic cost of the membrane deformation will contribute toward the equilibrium constant. the approach adopted by nielsen et al. ( ) came to rather similar conclusions. the most important energy terms were found to be the splay energy and the compression-expansion term, the splay energy term being most important close to the protein, with the compression-expansion term being more import'ant further from the protein. even though the bilayer deformation was calculated to extend some a from the protein, most of the deformation was found concentrated in the component immediately adjacent to the protein. an alternative model for mismatch is the mattress model of bloom ( , ) . this again expresses mismatch as two terms. the first is an excess hydrophobic free energy associated with exposing either lipid chains or the protein surface to the aqueous medium. the second is proportional to the contact area between the lipid chains and the hydrophobic surface of the protein. the calculations showed that, for a protein of hydrophobic thickness a, which matches a bilayer ofdi(c : )pc in the liquid crystalline state, the binding constant for di(c : )pc is about a factor of . greater than for a bilayer of di(c : )pc, which will give a bilayer too thick by a (sperotto and mouritsen, ) . thus, effects of mismatch calculated in this way are similar to those calculated using the approach of fattal and ben-shaul ( ) . the importance of hydrophobic matching has been confirmed in a number of experimental studies (dumas et al., ; killian, ) . for example, although bacteriorhodopsin has relatively little effect on the phase transition temperatures of di(c : )pc or di(c : )pc (alonso et al., ) , it increases the transition temperature of di(c : )pc and decreases that of di(c : )pc (piknova etal., ) . this is consistent with hydrophobic matching models; because di(c : )pc gives a too thin bilayer in the liquid crystalline phase, bacteriorhodopsin favors the gel phase, whereas di(c : )pc gives a too thick bilayer in the gel phase, so that bacteriorhodopsin favors the liquid crystalline phase. hydrophobic matching could also be the explanation for the unexpected observation that, in mixtures of di(c : )pc and di(c : )pc at temperatures where the mixture contains both gel and liquid crystalline phases, bacteriorhodopsin partitions equally between the two phases (piknova et al., ; schram and thompson, ) . this contrasts with the observed exclusion of bacteriorhodopsin from gel-phase lipid in mixtures containing single species of phospholipid (alonso et al., ; cherry et al., ) . it has been suggested that this shows that the requirements of hydrophobic matching are of prime importance; the hydrophobic thickness of bacteriorhodopsin is intermediate between the hydrophobic thickness of di(c : )pc in the gel phase and di(c : )pc in the liquid crystalline phase, so that bacteriorhodopsin shows little preference between the two. in mixtures of di(c : )pc and di(c : )pc, where, at low temperatures, two separate gel phases are formed, one enriched in di(c : )pc and one enriched in di(c : )pc, bacteriorhodopsin partitions very strongly into the di(c : )pc-enriched domains; this could be because the hydrophobic thickness of bacteriorhodopsin is better matched to gelstate di(c : )pc than to gel-state di(c : )pc (dumas et al., ) . however, in studies of the ca +-atpase of sarcoplasmic reticulum using either spin-labeled (london and feigenson, ) or brominated phospholipids , strengths of binding of liquid crystalline-phase phospholipids to the atpase were found to be independent of fatty acyl chain length. these results are not consistent with the expectations of hydrophobic matching theory and suggest that, in liquid crystalline bilayers, u-helical membrane proteins are not rigid, but, in fact, can distort to match the thickness of the bilayer. such a distortion could explain why bilayer thickness affects the activity of membrane proteins such as ca +-atpase (lee, ) . the structural distortion could take the form of a change in the tilt of the transmembrane ~ helices with respect to the bilayer normal or could be a change in the packing of the transmembrane c~ helices. an alternative approach to these questions, avoiding the complexity of real membrane proteins, is to use simple model transmembrane ~ helices, which can be synthesized chemically in large quantity. a number of studies have used peptides of the type ac-k -g-ln-k -a-amide (pn) consisting of a long sequence of hydrophobic leu residues capped at both the n-and c-terminal ends with a pair of charged lysine residues. the poly(leu) region forms a maximally stable u helix, particularly in the hydrophobic environment of the lipid bilayer. the charged lys caps were chosen both to anchor the ends of the peptides in the lipid headgroup region and to inhibit the aggregation of the peptides in the membrane. the peptide has been shown to adopt the expected u-helical structure in both liquid crystalline-and gel-phase bilayers (davis et al., ; huschilt et al., ; zhang et al., a) . rates of hydrogen/deuterium exchange for the peptide p in lipid bilayers suggest that at least % of the peptide is in an u-helical conformation in the bilayer, meaning that the whole of the poly(leu) core must be u-helical (zhang et al., b) . rates of hydrogen/deuterium exchange were greater at the n-and c-termini of the peptides than in the middle, suggesting some unraveling of the peptide at its ends (zhang et al, b) . experiments with the peptides of this type suggest that about lipid molecules are required for complete incorporation of the peptide into a bilayer of the appropriate thickness (webb et al., ) . this agrees with estimates from molecular modeling that about - lipid molecules will be required to form a complete bilayer shell around the peptide. at molar ratios of lipid less than this, non-bilayer phases can be induced, particularly when the hydrophobic length of the peptide is less than the hydrophobic thickness of the bilayer and when the peptide contains interfacial aromatic groups (de planque et al., ; killian et al., ; morein et al., ) . effects of single transmembrane u helices on lipid bilayers are likely to be less than those of a protein containing a bundle of transmembrane u helices. the cross-sectional area of a single transmembrane u helix is not much greater than that of a phospholipid molecule in the liquid crystalline phase, so that the hydrophobic surface presented to the lipid molecules is rather small. the structure of the lipid bound to bacteriorhodopsin shown in fig. shows the two chains interacting predominantly with two different transmembrane u helices. this kind of interaction will obviously not be possible with a single transmembrane u helix. less extensive interactions between lipids and single transmembrane u helices than between lipids and membrane proteins is suggested by esr experiments. whereas esr spectra of spin-labeled lipids in the presence of membrane proteins typically show two-component spectra, as described above, esr spectra for lipid bilayers containing the peptide l and for a tryptophan-containing peptide of the type aw (la)nw a are single-component (de planque et al, ; subczynski et al., ) . this means either that the lipid fatty acyl chains are not "immobilized" on the peptide surface or that the rate of exchange between bulk and boundary lipid is fast on the esr time scale (i.e., exchange is faster than s-l). effects of peptides on chain order in di(c : )pc or di(c : )pc measured using deuterium nuclear magnetic resonance (nmr) methods are small in both the liquid crystalline and the gel phases (davis et al., ; de planque et al., de planque et al., , roux et al., ) . thus, addition of a peptide aw (la)tw a to bilayers of di(c : )pc in the liquid crystalline phase resulted in only a . -/~ increase in thickness (de planque et al., ) , whereas about an -/~ increase would be necessary for the bilayer thickness to match the hydrophobic length of the peptide. similarly, nezil and bloom ( ) estimated that the peptide p increased the thickness of a bilayer of (c : ,c : )pc by just . /~, despite the hydrophobic mismatch between the peptide and the lipid bilayer being ca. a. increases in chain order caused by p in (c : ,c : )pc in the liquid crystalline phase were detected using esr, but again the effects were small (subczynski et al., ) . effects of peptides on chain order will depend on the relative hydrophobic length of the peptide compared to the hydrophobic thickness of the bilayer, with long peptides decreasing order and short peptides increasing order, and such effects have been detected using infrared (ir) spectroscopy, but again effects were small (zhang et al., a) . thus it appears that lipids will distort slightly to improve the match between the hydrophobic length of the peptide and the hydrophobic thickness of the bilayer, but the extent of these modifications is very limited and much less than required to produce full matching. a number of studies have been published on the effects of these peptides on the phase transition properties of lipid bilayers. addition of the peptide pi to bilayers of di(ci : )pc both broadens the main gel-to-liquid crystalline phase transition and decreases the enthalpy of the transition (morrow et al., ) . similar effects have been seen on incorporation of membrane proteins such as bacteriorhodopsin and ca +-atpase (alonso et al., ; gomez-fernandez et al., ) . the decrease in enthalpy of the transition has often been taken to mean that the lipids adjacent to the protein (the boundary lipids) are very strongly perturbed by the peptide and so are unable to take part in the normal phase transition: they are effectively withdrawn from the transition. however, deuterium nmr spectra of mixtures of p and di(c : )pc above and below the phase transition are typical of liquid crystalline-and gel-phase lipids, respectively, with no evidence for any "special" lipid unable to take part in the phase transition . similarly, as already described, esr spectra of spin-labeled lipids show the presence of a single type of lipid in the system, not separate bulk and boundary lipids in slow exchange (subczynski et al., ) . thus the peptides (or proteins) do not remove lipid from the main transition, but, rather, perturb the whole lipid bilayer. the peptide decreases the enthalpy difference between the liquid crystalline and gel phases, whereas the lipids in the bilayer remain recognizably liquid crystalline or gel (morrow et al., ) . morrow et al. ( ) showed that mixtures of lipids and peptides can be modeled in terms of regular solution theory (lee, ) . unfortunately, the number of free parameters in fitting to regular solution theory is high, so that little useful information is obtained from the analysis, apart from showing that the data are consistent with regular solution theory. effects of peptides or proteins on phase transition properties have therefore been interpreted qualitatively in terms of a twocomponent model in which one component is more or less unperturbed bulk lipid and the other is highly perturbed boundary lipid, which undergoes a broad phase transition of low enthalpy; this approach works for peptides of the poly(leu) type, but, for some reason, does not work with peptides of the k (la)nk type (zhang et al., a (zhang et al., , . differential scanning calorimetry (dsc) thermograms for mixtures with the poly(leu) peptides have been fitted to two components, attributed to the phase transitions of peptide-free and boundary lipid, respectively. the phase transition temperature for the peptide-free component is slightly less than that for pure lipid; this is probably due to the normal colligative effects that will follow from mixing the "pure" lipid phase with the boundary lipid, the latter acting as an "impurity." the phase transition temperature for the boundary lipid is higher than the bulk transition temperature for short-chain lipids, but is lower for long-chain lipids (zhang et al, a) . the same observation has been made for membrane proteins (dumas et al., ; piknova et al., ) . this is consistent with the idea that short fatty acyl chains have to stretch to match the hydrophobic thickness of the membrane protein, whereas long fatty acyl chains have to compress. however, the experimental changes are much smaller than expected from models ofhydrophobic matching. further insights into how transmembrane ot helices might interact with lipid molecules in a bilayer have come from molecular dynamics simulations. one study was of a transmembrane ot helix of alanine residues in a bilayer of di(c : )pc in the liquid crystalline state (shen et al., ) . the peptide is not an ideal model for a transmembrane ot helix, because it lacks charged groups at each end to interact with the polar headgroups of the phospholipids. nevertheless many features of the simulation are informative. the simulation was started with the peptide as a pure ot helix. the central residues (ala- - ), which interacted just with the lipid fatty acyl chains, remained as a stable t helix. the n-and c-terminal regions of the ot helix, located in the lipid headgroup region, were less stable and fluctuated more, because of transient hydrogen bonding between the peptide bond amide hydrogen and the phosphate or fatty acyl ester oxygen atoms and the water; as a result, the ends of the helices become frayed. the length of the central helical region oscillated slightly about a -a average expected for an ot helix, varying between and a. the helix was tilted up to ° with respect to the bilayer normal. because the helix contains no resides that would make strong contacts in the headgroup region or with water, there is no reason for it not to tilt (shen et al., ) . tilting in fact allows more hydrophobic contact by allowing more of the ala residues to be located in the core of the bilayer. the presence of the peptide had little effect on the calculated properties of the bilayer. the average bilayer thickness was not significantly changed, although the average order parameter for the ch groups in the chains decreased in the presence of the peptide. many different lipid molecules contributed to the immediate surroundings of the peptide (shen et al., ) . even if the fatty acyl chain of a particular lipid was immediately adjacent to the peptide, the headgroup of the lipid could be a substantial distance away. given the diameter of the helix and the size of the phosphate group, a phosphate immediately adjacent to the helix would be between and a away from the center of the helix (shen et al., ) . on average, five lipid molecules having their chains adjacent to the helix also had their headgroups adjacent, using this definition. however, the headgroup, as given by the position of the phosphorus atom, could be up to - a away. since the average distance between lipid headgroups is a, this puts these lipids in the "second" shell around the peptide. other lipids existed between these extremes, suggesting a very diffuse environment around the protein rather than a discrete set of well-ordered shells of lipid. only rarely did an entire lipid molecule pack tightly around the helix. the shell around the helix contains contributions from a large number of lipid molecules, each contributing a small number of atoms (shen et al., ) . thus there is no evidence for a distinct shell of lipids around the peptide and any perturbation of the lipids extends out just a few angstroms. the lack of a clear shell of lipids around the poly(ala) peptide contrasts with the boundary lipids observed for membrane proteins and illustrated for bacteriorhodopsin in figs. and . in part this could be an intrinsic feature of single transmembrane a helices, which will not be able to present a large surface area on which fatty acyl chains could be immobilized. the regular structure of a poly(ala) peptide compared to the rough surface of a typical or-helical peptide might also contribute to the lack of an immobilization shell of lipids. however, a significant factor is also likely to be the lack of polar groups at the ends of the peptide able to interact with the phospholipid headgroups. the importance of polar groups at the ends of the peptides has been shown in a simulation of isolated helices from bacteriorhodopsin (woolf, ) . the simulations show that a small number of the lipids surrounding a helix interact with it much more strongly than other lipids, due to a combination of van der waals (mediated by chains) and charge interactions (mediated by beadgroups) (woolf, ) . a molecular dynamics simulation of the peptide el in a bilayer of di(c : )pc in the liquid crystalline phase showed that the peptide tilted with an average angle of . ° with respect to the bilayer normal, even though the thickness of the hydrophobic region of the bilayer ( a) was a good match to the length of the ~ helix, a (belohorcova et al., ) . a molecular dynamics simulation has been reported for pf coat protein in di(c : )pc (roux and woolf, ) . the coat protein contains an amphipathic a helix on the bilayer surface and a hydrophobic transmembrane a helix. fatty acyl chains next to the transmembrane helix were slightly more ordered than bulk lipids (roux and woolf, ) . similarly, in a simulation of the seventh transmembrane c~ helix of the serotonin ht receptor in di(c : )pc, lipids in contact with the peptide had slightly higher order parameters than bulk lipids (duong et al., ) . the theories described above show that there will be an energetic cost associated with any change in the thickness of a bilayer. this would be reflected in values for the equilibrium constant describing the binding of lipids to the protein. a lipid that can bind to a protein without a change in bilayer thickness would bind more strongly to the protein than one for which binding required a change in bilayer thickness. the strength of interaction between a peptide and a phospholipid in a bilayer can be measured using a fluorescence quenching method (webb et al, ) . peptides used are of the type kkgl wl kka (l ) and kkgl wl kka (l ) containing a central trp residue as a fluorescence reporter group. the peptide is incorporated into bilayers containing the brominated phospholipid dibromostearoylphosphatidylcholine (di(br c : )pc); di(br c : )pc behaves much like a conventional phospholipid with unsaturated fatty acyl chains, because the bulky bromine atoms have effects on lipid packing similar to those of a cis double bond . contact between the bromine atoms in the lipid and the trp residue in the peptide leads to fluorescence quenching. in mixtures of brominated and nonbrominated phospholipids, the degree of quenching of the fluorescence of the tryptophan residue is related to the fraction of the surrounding (boundary) phospholipid molecules that are brominated, and thus to the strength of binding of the nonbrominated lipid to the peptide. an example of the method is shown in fig. . the fluorescence intensity for the peptide l incorporated into bilayers of di(br c : )pc at a molar ratio of lipid to peptide of : is % of that in di(c : )pc, demonstrating highly efficient quenching of the tryptophan by the bromine-containing fatty acyl chains (fig. ) . the fluorescence intensity in mixtures of di(br c : )pc and di(c : )pc decreases with increasing content of di(br c : )pc, reflecting the increasing number of boundary lipids that will be di(br c : )pc. as shown in fig. , fluorescence quenching curves for l in mixtures of di(br c : )pc and (c : )pc show more fluorescence quenching at intermediate mole fractions of di(br c : )pc than in mixtures with di(c : )pc. this shows that di(c : i)pc binds more strongly to the peptide than does di(c : )pc. the results can be analyzed to give relative lipid-binding constants, as described in webb et al. ( ) . these lipid-binding constants for l and l are given in table i. for l strongest binding is seen with di(c : )pc, for which the relative binding constant is about double that for di(c : )pc (table i ). the hydrophobic length of the peptide l is about a, calculated for a stretch of hydrophobic residues in total, with a helix translation of . a per residue. thus, strongest binding is seen when the hydrophobic length of the peptide matches the hydrophobic thickness of the bilayer, as expected from theories of hydrophobic mismatch. however, relative binding constants do not continue to decrease with decreasing chain length from di(c : )pc to di(c : )pc as would have been predicted (table i ). an even larger deviation from theoretical predictions is observed with the short peptide l (table i ). in this case strongest binding is observed with di(c : )pc, with binding decreasing with decreasing chain length to di(c :i)pc, as expected. however, the peptide was found not to incorporate at all into bilayers of di(c :l)pc, instead forming aggregates of peptide separate from the bilayer. ren et al. ( ) obtained very similar results, except that under their conditions, unincorporated peptide bound to the surface of the lipid bilayer, with the long axis of the peptide parallel to the surface. similarly, l was found to be only partly incorporated into ~bilayer hydrophobic thickness d calculated from d = . (n - ), where n is the number of carbon atoms in the fatty acyl chain (sperotto and mouritsen, ) . bestimated hydrophobic length is a for li and a for l . bilayers of di(c : )pc (webb et al., ) . thus, a short peptide cannot incorporate into a too-thick bilayer. it is suggested that a too-thin bilayer can match to a too-long peptide both by a slight stretching of the lipid and by tilting of the long axis of the helix with respect to the bilayer normal so that its effective length across the bilayer is reduced. however, a too-thick bilayer can only match a too-thin peptide by compression of the lipid, which becomes energetically unfavorable when the difference between the bilayer thickness and the peptide length exceeds about /~ (webb et al., ) . possible effects of aromatic residues at the ends of transmembrane ~ helices have been studied using peptides k gfl wl fk a (f l ) and k gyl wl yk a (y li ), in which one leu residue at each end of the poly(leu) stretch is replaced by either a phe or a tyr (mall et al., ) . in contrast to the results with l , peptide f l incorporated fully into bilayers of di(c : )pc, and y li partitioned partially into di(c : )pc. the fluorescence quenching method was again used to obtain binding constants for phosphatidylcholines to the peptides, measured relative to the binding constant for di(c : )pc (table ii) . the effective hydrophobic length of the peptide y li might be expected to be somewhat greater than that of l ; if the peptide is modeled as an c~ helix with the two tyr residues oriented to be roughly parallel to the long axis of the c~ helix, the distance between the two tyr--oh groups is ca. a, about /~ greater than the hydrophobic length of l . the hydrophobic length of y l calculated in this way matches the hydrophobic thickness of a bilayer of di(c : )pc, whereas the relative lipid-binding constants increase from di(c : )pc to di(c : )pc (table ii) . similarly, relative binding constants for f li increase with increasing chain length from di(c :i)pc to di(c : i)pc. the results with y l and f li show that introduction of aromatic "hydrophobic thickness of the bilayer calculated from d = . (n - ), where n is the number of carbon atoms in the fatty acyl chain (sperotto and mouritsen, ). residues at the two ends of the hydrophobic sequence increases the ability of the short peptides to partition into thick lipid bilayers. the observation that the highest relative binding constant is obtained with bilayers considerably thicker than the calculated hydrophobic length of the peptides suggests that the presence of aromatic residues at the ends of the helices could lead to marked thinning of the bilayer around the peptides (mall et al., ) . the chain length dependence of lipid binding to y l is much less marked than for the shorter peptides; the relative binding constant increases from di(c : )pc to di(c :i)pc, but then hardly changes with increasing chain length between di(c :i)pc and di(c :)pc (table ii) . this contrasts with l , which shows markedly stronger interaction with di(c :l)pc than with phospholipids with shorter or longer chains. this again suggests that the introduction of the two tyr residues leads to an increase in the thickness of the bilayer with which optimal interaction of the peptide is observed. interactions between transmembrane c~ helices and the phospholipid headgroups also have to be considered. using the fluorescence quenching method, it was shown that a small number of anionic phospholipid molecules (possibly just one) bound strongly to the peptide l , the remaining molecules binding with an affinity equal to that of phosphatidylcholine. the binding constant for the strongly bound phosphatidic acid molecule relative to phosphatidylcholine in a medium of low ionic strength was . (in mole fraction units), corresponding to a difference in unitary binding energies of - . kj mo - . at ph . , phosphatidic acid bears a single negative charge (cevc, ) . the binding constant for phosphatidic acid changed little with ionic strength, suggesting that the interaction with the positively charged peptide did not follow simply from a high positive potential in the vicinity of the positively charged lys residues on the peptide, increasing the local concentration of anionic phospholipid. the energy of interaction between two ions u is given by where zl and z are the charges on the two ions eo is the permittivity of a vacuum, er is the relative permittivity (dielectric constant) of the medium, and r is the distance between the two ions. assuming a dielectric constant of . (water), we find that an energy of interaction of . kj tool - corresponds to a distance of separation between two monovalent ions of . /~. this therefore suggests that strong interaction requires the anionic headgroup of phosphatidic acid to be in close contact with one of the lys residues on the peptide. once this strong interaction with a single phosphatidic acid molecule has been made, other phosphatidic acid molecules will then interact with el relatively nonspecifically, with a binding constant relative to phosphatidylcholine close to . the relative binding constants for phosphatidylserine were less than for phosphatidic acid and are more sensitive to ionic strength . for phosphatidylserine, the presence of the positively charged ammonium group as well as the negatively charged carboxyl group in the headgroup region may reduce interaction with the positively charged peptide. in contrast to l , the binding constants for anionic phospholipids to l are very similar to those for zwitterionic phospholipids, with a relative binding constant close to . it could be that tilting of l in the bilayer, necessary to match the hydrophobic length of l to the hydrophobic thickness of a bilayer of di(c : )pc, locates the lys residues on the peptide too far from the lipid headgroup region to allow a strong interaction between the anionic phospholipid and the peptide. both phosphatidylserine and phosphatidic acid bind more strongly to the peptides yell and yel than does phosphatidylcholine, the effect of anionic phospholipid decreasing slightly with increasing ionic strength. however, in this case the experiments are consistent with a model in which the binding constants for all the anionic phospholipid molecules binding to the peptide are increased slightly (mall et al., ) . this suggests that the presence of the tyr residues prevents close association of the anionic phospholipid group with the cationic lys residues. these results suggest that the effects of charge on the interactions between phospholipids and transmembrane ot helices will often be rather small and will be strongly dependent on the detailed structure of the peptide and its orientation in the membrane. this picture is consistent with the results of the molecular dynamics simulations of individual ~ helices of bacteriorhodopsin in bilayers of di(ci : )pc, which showed that a small proportion of the lipid molecules interacted with the o~ helices much more strongly than the others, and that these strong interactions were dominated by electrostatic terms rather than van der waals terms (woolf, ) . in general, binding constants for phospholipids to membrane proteins also show relatively little selectivity for anionic phospholipids. for example, binding constants for phosphatidic acid and phosphatidylserine relative to phosphatidylcholine are close to for the caz+-atpase (dalton et al., ) , and binding constants for phosphatidic acid and phosphatidylserine for the (na+-k+)-atpase are about twice those for phosphatidylcholine (esmann and marsh, ) . however, there is evidence for the presence of a small number of "special" anionic phospholipids binding to some membrane proteins, acting as "cofactors." an example is provided by cytochrome c oxidase, whose crystal structure shows the presence of a lipid molecule bound between the transmembrane a helices (iwata et al., ) . interaction between an anionic phospholipid and a binding site on a membrane protein would be specific if strong binding requires close interaction between the anionic headgroup and a positively charged residue on the protein, as suggested by the results presented here. the phase of the phospholipid is important in determining interactions with transmembrane ot helices. as shown in fig. , fluorescence quenching is much more marked in mixtures of di(br c : )pc and di(c : )pc at temperatures where both liquid crystalline and gel phases are present than in mixtures of di(brac : )pc and di(c : )pc (mall et al., ) . thus, l is excluded from regions of lipid in the gel phase and accumulates in regions in the liquid crystalline phase. the binding constants of l| and l z for di(c : )pc in the gel phase relative to di(c :i)pc in the liquid crystalline phase are ca. . (mall et al., ) . this is consistent with the expectation that van der waals contacts between an all-trans fatty acyl chain and the molecularly rough surface of a peptide will be poor; one way that this poor packing can be overcome is by exclusion of the peptide from the gel phase. quenching plots for y l are very similar to those of l (fig. ) , showing that the presence of bulky aromatic residues does not have any significant effect on the selectivity for liquid crystalline-over gel-phase lipid (mall et al., ) . further, since y li shows a preference for longer chain phospholipids than l , y li might have been expected to show a greater preference for gel-phase lipid than l , because phospholipid in the gel phase gives a thicker bilayer than the corresponding lipid in the liquid crystalline phase. because y l and l show equal preferences for liquid crystalline-over gel-phase lipid, any effects of hydrophobic matching between the peptide and the bilayer must be small compared to effects of lipid phase on the interaction energies between the peptides and the lipid (mall et al., ) . preferential partitioning of proteins from domains in the gel phase into domains of liquid crystalline lipid has been demonstrated for a variety of membrane proteins, including bacteriorhodopsin (cherry et al., ) and ca +-atpase kleeman and mcconnell, ) . effects of sphingomyelin at °c are very similar to the effects of gel-phase di(c : )pc ( fig. ; mall et al, ) . mixtures of bovine brain sphingomyelin and di(c : )pc are in a two-phase region at °c, with gel-phase domains enriched in sphingomyelin (untracht and shipley, ) . thus, partitioning of the peptides between gel-and liquid crystalline-phase lipid shows little dependence on the structure of the phospholipid. it has been suggested that plasma membranes of mammalian cells contain domains or "rafts" enriched in sphingomyelin and that particular enzymes, particularly those associated with cell signaling, are concentrated within the rafts (simons and ikonen, ) . the results presented here suggest that membrane proteins containing transmembrane ot helices will tend to be excluded from these rafts, and it may therefore be significant that many of the signaling proteins suggested to be contained within the rafts are anchored to the membrane by glycosylphosphatidylinositol anchors (harder and simons, ) . the presence of cholesterol has a marked effect on incorporation of the peptides into phospholipid bilayers (webb et al., ) . incorporation of cholesterol at a : molar ratio to phospholipid leads to a general reduction in incorporation of the peptides l and l , but superimposed on this effect is a chain length effect. in the presence of cholesterol, the binding constant of p for di(c : )pc relative to di(c : )pc increased from . to about , as expected if the presence of cholesterol increases the effective chain length of the c chain so that it more nearly matches the hydrophobic length of the peptide (fig. ) . consistent with this interpretation, the presence of high molar ratios of cholesterol prevented the incorporation of p into bilayers of di(c :i)pc. nezil and bloom ( ) showed that incorporation of cholesterol at mol% increases bilayer thickness by about ,~. studies with brominated analogues of cholesterol showed that cholesterol binds to the peptides with a binding constant only a factor of about two less strongly than di(c : )pc . this is rather surprising, given the relatively rigid structure of the steroid ring of cholesterol and the molecularly rough surface of the peptide. in other studies, it has been shown that cholesterol binds relatively weakly at the lipid-protein interface of the atpase (simmonds et al., (simmonds et al., , ; comparison with the peptide studies reported here suggests that weak binding of cholesterol to the atpase involves interactions in the lipid headgroup region rather than interactions between the sterol ring and the hydrophobic transmembrane helices. the requirement to match the hydrophobic thickness of a membrane protein to that of the surrounding lipid bilayer could be important in a number of ways. targeting of proteins to their correct final destinations in a cell is essential in maintaining cell integrity. in the bulk flow model, the vast majority of proteins synthesized in the endoplasmic reticulum (er) are believed to leave the er by default and flow along the exocytic pathway until they reach the plasma membrane (nilsson and warren, ) . some proteins, however, have to be retained at particular points along the exocytic pathway. compartmental localization could be achieved in one of two ways. the first involves a retention signal in the protein, which, at the appropriate point in the exocytic pathway, prevents forward movement of the protein by denying it access to budding transport vesicles of the onward pathway. the second involves a retrieval signal, leading to recapture of the protein after it has left the compartment in which it resides. the classical retrieval signal is the kdel sequence found in many er-resident proteins; the situation appears to be different for golgi-resident proteins, where membrane-spanning domains act as retention signals (nilsson and warren, ) . despite the extensive flux of proteins through the golgi, the golgi maintains its own distinctive population of resident proteins. furthermore, the distribution of enzymes within the golgi is organized according to function, so that, for example, the distributions of glycosyltransferases and glycosidases, although overlapping, are distinct (colley, ; roth, ) . many of the proteins in the golgi membrane are predicted to contain a single transmembrane ot helix, oriented with the n-and c-termini on the inner and outer faces of the membrane, respectively. the golgi retention signal in such proteins has been shown to involve the membrane spanning domain (munro, (munro, , nilsson et al., ; swift and machamer, ) . however, the membrane-spanning domains show no sequence homology, and it has not been possible to identify any particular motif leading to retention (bretscher and munro, ; colley et al., ; munro, ) . thus, sialyltransferase remains localized in the golgi even when its -amino-acid transmembrane domain is replaced by leu residues (munro, ) . however, a longer stretch of leu residues did not provide an efficient retention signal (munro, ) . similarly, a -residue insertion into the transmembrane domain of galactosyltransferase reduced its retention in the golgi (masibay et al., ) . the reverse effect has been shown with the influenza virus neuraminidase, which shifted from the plasma membrane to the golgi and er when the number of residues in the transmembrane domain was reduced (sivasubramanian and nayak, ) . the lack of a clear retention motif, together with the inability to saturate the mechanism for golgi retention by overexpression, suggests that retention is not a receptor-mediated event (nilsson and warren, ) . one possible model is then retention by preferential interaction with membranes of optimal thickness (nilsson and warren, ) . both bretscher and munro ( ) and masibay et al. ( ) showed that transmembrane domains of golgi proteins are shorter (average residues) than transmembrane domains of plasma membrane proteins (average residues). it has therefore been suggested that if the golgi membrane is thinner than the plasma membrane, membrane proteins with short transmembrane domains will interact "more strongly" with the lipid bilayer of the golgi than with that of the plasma membrane, leading to retention in the golgi (bretscher and munro, ; masibay et al., ) . the studies with model peptides described above show that a protein containing a transmembrane ot helix with a hydrophobic length greater than the hydrophobic thickness of the golgi membrane will be able to move out of the golgi into the plasma membrane. however, a protein whose transmembrane ot helix has a hydrophobic length less than the hydrophobic thickness of a particular membrane will not be able to enter that membrane, and such a protein would then be retained in the golgi (webb et al., ) . studies of targeting of proteins in yeast are also consistent with a lipid-based model (rayner and pelham, ) . the length of the transmembrane domain is important in targeting with long helices ( residues), ensuring transport to the plasma membrane. however, for proteins with shorter transmembrane domains, the relative hydrophobicity of the transmembrane domain has been suggested to be important as well as its length, this determining targeting to the golgi and the vacuole (rayner and pelham, ) . retention of some membrane proteins in the er could also depend on the length of the transmembrane domain of the protein. an important class of er membrane proteins are those with an n-terminal catalytic domain exposed to the cytoplasm and a c-terminal membrane anchor. such proteins are inserted into the er membrane post-translationally by a signal-recognition-particle-independent pathway. no er retrieval signals have been identified in these proteins. instead, it has been observed that the hydrophobic domain is rather short. for example, cytochrome bs, a protein of this type, has a transmembrane domain containing just hydrophobic amino acid residues (pedrazzini et al., ) . if the length of the hydrophobic stretch is increased to residues, the protein is transported out of the er along the secretory pathway (pedrazzini et al., ) . it could therefore be that matching of the thickness of the lipid bilayer and the transmembrane length of the protein is important in retention in er, as was suggested for the golgi complex. although the length of the transmembrane domain appears to be the most important factor, the structure of the c-terminal, lumenal, region has also been shown to contribute to retention (honsho et al., ) . experiments with another c-terminalanchored protein, the ubiquitin-conjugating enzyme ubc from yeast, suggest that the thickness requirements of the er and golgi membranes may be different, explaining targeting between these two organelles (yang et al., ) . whereas ubc containing the wild-type -residue transmembrane domain targets to the er, increasing the length of the transmembrane domain to residues results in movement to the golgi, and increasing the length further to residues allows movement to the plasma membrane. these experiments show that the length of the transmembrane ot helix is often an important factor in targeting, although it is likely to be only one of a number of important factors. the lengths of the transmembrane ot helices are also likely to be important in the proper function of membrane proteins containing multiple transmembrane ot helices. an example already described is that of the ca +-atpase, which shows highest atpase activity in di(c : )pc and lower activities in bilayers of phospholipids with longer or shorter fatty acyl chains (lee, ) . changes in the atpase underlying these changes in atpase activity are complex (lee, ) , but all must be mediated by the transmembrane ot helices, because these are the parts of the atpase that can "sense" the change in bilayer thickness. in the case of the ca +-atpase it seems that, as described above, the two major conformational states of the atpase (el and e ) have different preferences for bilayer thickness, the e conformation favoring thin bilayers and the e conformation favoring thick bilayers (lee, ) . changing the bilayer thickness could change the tilt of the transmembrane u helices in the ca +-atpase, it could change the packing of the helices, and, possibly, it could lead to changes in the structures of the loops connecting the helices, changing the effective lengths of the helices. all these changes could be linked to changes in the phosphorylation domain of the ca +-atpase, located well above the surface of the membrane. if, as seems likely, the various membranes in a cell have different thicknesses because of their different lipid compositions, the structure of each membrane protein will have evolved to match the thickness of the membrane in 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lengthened membrane anchor escapes from the endoplasmic reticulum and reaches the plasma membrane hydrophobic mismatch and long-range protein/lipid interactions in bacteriorhodopsin/phosphatidylcholine vesicles fluorescence quenching and electron spin resonance study of percolation in a two-phase lipid bilayer containing bacteriorhodopsin transmembrane domain-dependent sorting of proteins to the er and plasma membrane in yeast membrane protein structure and stability: implications of the first crystallographic analyses control of the transmembrane orientation and interhelical interactions within membranes by hydrophobic helix length hydrophobicity of the peptide c=o. • -h--n hydrogen bonded group subcellular organization of glycosylation in mammalian cells detergent structure in crystals of a bacterial photosynthetic reaction centre structure of the detergent phase and proteindetergent interactions in crystals of the wild-type (strain y) rhodobacter sphaeroides photochemical reaction center molecular dynamics of pfl coat protein in a phospholipid bilayer conformational changes of phospholipid headgroups induced by a cationic integral membrane peptide as seen by deuterium magnetic resonance influence of the intrinsic membrane protein bacteriorhodopsin on gel-phase domain topology in two-component phase-separated bilayers transmembrane helix structure, dynamics, and interactions: multi-nanosecond molecular dynamics simulations annular and non-annular binding sites on the (ca + + mg +)-atpase interactions of cholesterol hemisuccinate with phospholipids and (ca +-mg +)-atpase functional rafts in cell membranes mutational analysis of the signal-anchor domain of influenza virus neuraminidase dependence of lipid membrane phase u'ansition temperature on the mismatch of protein and lipid hydrophobic thickness lipid enrichment and selectivity of integral membrane proteins in two-component lipid bilayers characterization of the single ca + binding site on the ca +-atpase reconstituted with short and long chain phosphatidylcholines molecular organization and dynamics of -palmitoyl- -oleoylphosphatidylcholine bilayers containing a transmembrane alpha-helical peptide a golgi retention signal in a membrane-spanning domain of coronavirus e protein crystal structure of the calcium pump of sarcoplasmic reticulum at . /~ resolution molecular interactions between lecithin and sphingomyelin principles of membrane protein assembly and structure architecture of helix bundle membrane proteins: an analysis of cytochrome c oxidase from bovine mitochondria hydrophobic mismatch and the incorporation of peptides into lipid bilayers: a possible mechanism for retention in the golgi experimentally determined bydrophobicity scale for proteins at membrane interfaces molecular dynamics simulations of individual alpha-helices of bacteriorhodopsin in dimyristoylphosphatidylcholine. ii. interaction energy analysis the transmembrane domain of a carboxyl-terminal anchored protein determines localization to the endoplasmic reticulum the preference of tryptophan for membrane interfaces interaction of a peptide model of a hydrophobic transmembrane a-helical segment of a membrane protein with phosphatidylcholine bilayers: differential scanning calorimetric and ftir spectroscopic studies ftir spectroscopic studies of the conformation and amide hydrogen exchange of a peptide model of the hydrophobic transmembrane a-helices of membrane proteins peptide models of helical hydrophobic transmembrane segments of membrane proteins. . differential scanning calorimetric and ftir spectrooscopic studies of the interaction of ac-k -(la) -k -amide with phosphatidylcholine bilayers we thank the biotechnology and biological sciences research council (bbsrc) for financial support of the original studies reported here. key: cord- -etin i y authors: timmins, joanna; ruigrok, rob w.h.; weissenhorn, winfried title: structural studies on the ebola virus matrix protein vp indicate that matrix proteins of enveloped rna viruses are analogues but not homologues date: - - journal: fems microbiology letters doi: . /j.femsle. . . sha: doc_id: cord_uid: etin i y abstract matrix proteins are the driving force of assembly of enveloped viruses. their main function is to interact with and polymerize at cellular membranes and link other viral components to the matrix–membrane complex resulting in individual particle shapes and ensuring the integrity of the viral particle. although matrix proteins of different virus families show functional analogy, they share no sequence or structural homology. their diversity is also evident in that they use a variety of late domain motifs to commit the cellular vacuolar protein sorting machinery to virus budding. here, we discuss the structural and functional aspects of the filovirus matrix protein vp and compare them to other known matrix protein structures from vesicular stomatitis virus, influenza virus and retroviral matrix proteins. enveloped rna viruses from the families of filoviridae, paramyxoviridae, rhabdoviridae and bornaviridae (constituting the order mononegavirales), orthomyxoviridae and retroviridae are a morphologically diverse group of viruses. the viral shape is determined by the matrix protein and the nucleocapsid that are responsible for producing spherical, rod-shaped, bullet-shaped or bacilliform particles. viral matrix proteins generally constitute a major structural protein of the viral particle and are located underneath the viral membrane. during the assembly process in a host cell, matrix proteins self-assemble into higher-order oligomers and/or polymerise at cellular membranes [ , ] . they display a high tendency to aggregate in vitro, which may reflect their ability to self-assemble in vivo. matrix proteins interact with membranes and evidence suggests that they provide a link between the cytoplasmic tails of the glycoproteins and the nucleocapsids that contain the rna genome within the viral particle [ ] . although in some cases matrix protein expression in eukaryotic cells is sufficient to induce virus-like particles (vlps) [ ] [ ] [ ] , the structural requirements for such ordered membrane-associated polymerisation reactions are not well understood and often depend on other viral components such as the glycoprotein [ ] . in addition, cellular factors modulating membrane structures might play a role. consequently, no virus-like particles have yet been produced from soluble matrix proteins and artificial lipid bilayers in vitro. in contrast to segmented and non-segmented negative strand viruses, retroviral matrix proteins are initially part of the gag poly-protein. expression of gag is largely sufficient to perform virus assembly and budding at the plasma membrane which leads to the release of virus-like particles (vlps). in immature viral particles, proteolytic processing generates several distinct protein products, including ma (matrix protein), ca (capsid protein) and nc (nucleocapsid protein) thus producing mature infectious virions. like the matrix protein from negative strand rna viruses ma is associated with the viral membrane and thus performs essentially the same structural function [ ] . lipid rafts are enriched in cholesterol and sphingolipids that can selectively incorporate or exclude proteins and have been first implicated in influenza virus budding [ ] . subsequent studies found the same principle for measles virus, hiv and ebola virus. these studies show that lipid rafts concentrate glycoproteins and matrix proteins thus establishing platforms for efficient assembly and budding [ ] . the first evidence for the involvement of specific gag domains in viral budding came from the work of g€ ottlinger and colleagues who reported that a deletion of the c-terminal region of hiv gag (p protein) caused a significant defect in virus particle release [ ] . electron microscopy studies revealed that these particles failed to pinch off the plasma membrane. subsequent studies then identified a highly conserved pro-thr-ala-pro sequence motif, termed late domain, as playing a crucial role in viral budding [ ] . up to date, several classes of viral late domains have been described, namely p(t/s)ap, yxxl lxxlf and ppxy. they are present alone or in different combinations in the matrix proteins of negative-strand rna viruses and in the gag proteins of retroviruses. in several cases, viral late domains have been shown to be functionally interchangeable, can be positioned at various locations and can act in trans [ ] . recent studies show that the late domains serve as entry points into the vacuolar protein sorting (vps) pathway and connect matrix proteins or gag to cellular factors. the vps machinery was first described in yeast and implicated in membrane protein trafficking from the golgi and plasma membrane via the endosomal system to the lysosome for degradation (for review, see [ ] ). mutation of any of the different yeast vps proteins leads to the formation of enlarged endosomal membrane compartments that cannot mature into multi vesicular bodies (mvb; class e compartment). most of the class e vps proteins exist as soluble proteins or small subcomplexes that are sequentially recruited to the site of mvb formation. initial recognition of the cargo involves class e proteins hrs (vps p), stam, eps (ede p) and clathrin. this leads to the recruitment of escrt- (endosome-associated complexes required for transport), composed of vps p, vps p and vps p to the endosomal membrane where it recognizes ubiquitinated cargo. escrt-i cargo recognition then induces the formation of escrt-ii (vps p, vps p and vps p) and that in turn activates the assembly of escrt-iii multi-protein complexes. finally, another class e vps protein, vps an aaa-type atpase, has been implicated in the disassembly of escrt-iii a necessary step for mvb formation (reviewed by katzmann et al. [ ] ). although the mammalian system is more complex the framework is similar. most of the components have now been described and their interactions have been mapped [ , ] . there are two human class e vps proteins identified that recognize late domains, namely tsg (via p(t/s)ap), which is part of escrt-i and aip- / alix (via lxxlf and yxxl) which in turn interacts with tsg and chmp proteins (escrt-iii), thus providing a link between escrt-i and escrt-iii in retroviral budding [ , ] . it is now recognized that late domain interactions with cellular factors most likely recruit the whole vps machinery to the site of budding. the ppxy motif, the fourth identified late domain has been found in some retroviruses, in rhabdoviruses and filoviruses ( [ ] and references therein). the ppxy motif mediates interactions with proteins that contain ww domains, such as nedd family ubiquitin ligases (e enzyme), which are part of an enzymatic cascade in which single ubiquitin moieties are transferred to lysine residues on the protein substrate. monoubiquitination of matrix proteins is consistent with recent findings that show that ubiquitin plays a role in retrovirus budding [ , ] and that ebola virus vp can be ubiquitinated in vitro [ ] . ubiquitination may thus help to recruit the class e vps components in a similar way as sorting of plasma membrane receptors requires monoubiquitination [ ] . in addition, nedd , which can be also found in lipid rafts [ ] , has been linked genetically to escrt-iii in yeast [ ] . finally, lipids and lipid modifying enzymes, such as endophilins that interact with aip /alix [ ] might play an important role in viral budding [ ] . up to now not all enveloped viruses contain any of the known late domain sequences which could serve as entry points into the vps machinery; however it is likely that they might yet use other short sequence elements which facilitate vps recruitment. in addition this function may not reside within the matrix protein. to date there is still limited structural information on viral matrix proteins as only four different matrix protein structures have been solved, including vp from ebola virus, m from vsv, a fragment of m from influenza virus and a number of retroviral matrix proteins. the ebola virus matrix protein vp is an elongated, two-domain monomeric assembly, composed of two structurally related b-sandwich domains, which are connected by a flexible linker ( fig. (a) ). indeed the unique fold of both domains suggests that the two domains probably arose from a common ancestor by gene duplication [ ] . early work showed that this conformation of vp is metastable, which allows an easy transition into oligomeric ring-like structures in vitro [ , ] . the ring-structures are either octamers or hexamers [ ] (figs. (b) and (c)). in both cases, the nterminal domain of vp constitutes the oligomerisation domain, which forms an anti-parallel dimer and is the building block for oligomerisation [ , ] . the cterminal domains are flexibly attached to the rings and mediate membrane association in vitro and in vivo [ , , ] (figs. (b) and (c)). the octamer was found to bind single stranded rna at the dimer-dimer interface having the sequence -uga- ( fig. (b) ). rna binding creates a new dimer-dimer interface and its binding stabilizes the protein-protein interaction generating the octamers [ ] . based on the crystal structure of the octamer, the complete n-terminal segment from residues to has to unfold in order to create the new ssrna-binding interface. interestingly recent studies suggest that the sds resistant octamer is present in ebola vlps and in virus particles [ ] in contrast to our previous finding which confirmed the presence of rna containing octamers only in infected cells but not in virus particles [ ] . a role for the ring structures in assembly and budding is also evident from the fact that only oligomeric vp interacted with ww from human nedd in vitro via its n-terminal ppxy motif [ ] , an interaction which is important in vivo [ ] . a number of studies also showed the importance of the ptap motif present at the n-terminus for budding [ , ] . this motif binds to the uev domain of tsg independent of its oligomeric state. thus, monomeric vp recruits tsg to the site of budding [ , ] , which in turn might recruit the complete vps machinery for efficient budding as demonstrated in case of hiv- [ ] . the late domain sequences are not present in the crystal structures of vp as they had been removed by proteolysis for efficient crystallisation purposes. note that the c-terminal ends connect to the c-terminal domain, which is missing in the structure. this is also indicated by the flexible attachment of two c-terminal domains (white). (c) full-length vp can be also activated to form hexamers, which is mediated by the nterminal domain. the electron microscopy reconstruction shows that two n-terminal domains assemble to create a ring-structure having threefold-symmetry. like in the octamer structure the c-terminal domains are flexibly attached to the ring structure as indicated by two cterminal domains and by the electron microscopy reconstructions [ ] . the structural studies of ebola virus vp have now firmly established three conformations of ebola virus vp , although their role in assembly and budding or additional functions during the life cycle of ebola virus is far from clear. it is, however, an interesting example of evolution that packs different functional aspects into one relatively small protein probably due to the limiting size of the viral genome. the structure of the matrix protein form vesicular stomatitis virus consists of an n-terminal part composed of a five-stranded anti-parallel b-sheet packed against two b-helices and a small c-terminal part made up by a two-stranded b-sheet and an a-helix, which connects to the n-terminal region via a long linker ( fig. (a) ) [ ] . as with ebola vp , the vesiculovirus matrix protein displays a new fold. no defined oligomeric structures have been described for vsv m, which, however, polymerises in vitro [ ] . for structure determination, vsv m was purified from virus and solubilized by thermolysin cleavage, which removes residues - and - / , thus preventing polymerisation (fig. (a) ). full length vsv m has a high tendency to self-associate into large multimers [ ] , which is sensitive to salt treatment in vitro [ ] . expression of m protein induces the formation and budding of vesicles, which is consistent with its membrane association and polymerisation activities. these particles, however, do not show the characteristic bulletshaped structure of rhabdoviruses indicating that other viral components such as nucleocapsids are responsible for the distinct morphology of vsv [ ] . the structure can be considered to represent the membrane-activated form that, however, no longer binds artificial membranes due to proteolytic removal of residues - / [ ] . it is conceivable that vsv m adopts a different conformation before activation, as cellular expression does not per se lead to polymerisation and membrane binding but produces mostly cytoplasmic m [ , ] . this putative other conformation(s) might then be also responsible for secondary functions of vsv m such as, inhibition of transcription and nucleocytoplasmic transport, nuclear localisation and cell rounding due to the induction of apoptosis [ ] [ ] [ ] [ ] [ ] . vsv m has been also implicated in nucleocapsid condensation [ ] , which may be similar to the function of ebola virus vp in nucleocapsid formation [ ] . in addition, vsv m contains the late domain motifs pppy and psap within the n-terminal residues ( [ ] and references therein), which are missing in the crystal structure. the pppy motif was shown to interact with mouse nedd and proteasome inhibitors reduced virus titers implicating ubiquitin in virus budding [ ] . the question fig. . ribbon diagrams of other known matrix protein structures. the n-and c-terminal ends are indicated. note that the folds of ebola virus vp (fig. (a) ) and the structures shown here are all different. remains whether any conformational diversity of vsv m might contribute to its different functions. the structure of the n-terminal domain of influenza virus m protein has been solved at ph . [ ] and at neutral ph [ ] . both structures are identical and consist of two four-helical bundle subdomains that pack against each other through a hydrophobic interface (fig. (b) ). the fold is again different from other known matrix protein structures. one side of the n-terminal domain of m is strictly positively charged while the opposite one exhibits an all-negative charge [ ] . proteolysis experiments suggest that the c-terminal domain is attached through a flexible linker. established functions of m are membrane association and polymerisation of the nterminal domain and rnp binding of the c-terminal domain [ ] . the n-terminal domain also contains a stretch of basic residues, which have been implicated in nuclear localisation of m and export processes [ , ] . overexpression of m in eukaryotic cells leads to the formation of intracellular tubular structures and the release of virus-like particles (vlps), indicating that m contains all the information for assembly and budding [ ] . vlp formation, however, is also enhanced by coexpression of ha which might augment m membrane association [ , ] . although m has a high tendency to polymerise in vitro, its conformation upon cytoplasmic expression must be postulated to be monomeric as cytoplasmic and nuclear m pools have been reported in addition to membrane associated m [ , ] . the activation process and the structural changes that lead to specific m polymerisation at membranes in vivo have not yet been described. m also exerts a number of additional functions, such as transport of rnp cores out of the nucleus [ ] . no functional late domain sequences have yet been described for m . as the complexity of the mvb machinery provides multiple possible entry points to contribute to its activation for budding processes, it is, however, unlikely that influenza uses another cellular machinery for budding. the crystal structure of the matrix protein from hiv- and siv folds into an arrangement of five a-helices and a three-stranded mixed b-sheet, a fold, which is unique to retroviral matrix proteins [ , ] . helices - pack about a central helix ( ) forming a compact globular domain that is capped by the b-sheet, while helix extends away from the core (fig. (c) ). hiv- ma can form a trimer in solution and can polymerise into a crystalline lattice. the trimers have been suggested to form the building block for the mature ma shell. there, the individual trimers present a largely basic surface on one side that was proposed to interact with the inner membrane of the virus. such an arrangement would pose the myristoylated n-terminal residue that is essential for membrane targeting close to the membrane and the c-terminal helix would point towards the interior of the viral particle [ , ] . in addition to forming a protein shell underneath the viral membrane, ma is also part of the pre-integration reverse transcriptase complex that travels towards the nucleus along microtubules using the kinesin kif- upon viral entry [ ] and seems to be required for nuclear import [ ] . although the sequences of retrovirus matrix proteins differ significantly, the known structures of retroviral matrix proteins (siv; bovine leukaemia virus, htlv-ii; mason-pfizer monkey virus; equine infectious anemia virus) exhibit the same common fold reflecting their evolutionary relationship [ ] . retroviral gag proteins contain either one or two late domain sequences at different locations. hiv- gag has two essential sequence motifs ptap and lxxlf within the c-terminal fragment p while pppy and ptap motifs locate to the c-terminus of ma from htlv-i [ ] . sequence analysis between members of filoviridae (ebola and marburg virus vp ), paramyxoviridae (subfamily paramyxovirinae: sendai virus, sv , measles virus; and subfamily pneumovirinae, human respiratory syncytial virus), rhabdoviridae (vsv; rabies virus) and bornaviridae (borna disease virus) reveals no significant sequence identity (ranging from % to %). the same is true when comparing matrix proteins from the mononegavirales with m from influenza virus and ma from retroviruses, which also underlines their observed structural diversity. in summary, the known matrix protein structures indicate no structural homology between non-segmented negative strand rna viruses such as filoviridae (ebola virus) and rhabdoviridae (vsv) and segmented negative strand rna viruses such as orthomyxoviridae (influenza virus) as well as retroviridae (hiv), although they share common functions. a minimal structural conservation is the presence of late domain sequences that mediate interaction with the class e vps machinery for budding, although this still has to be shown in case of influenza virus m . in addition, there is no striking common motif evident for membrane interaction, a common functional property of all viral matrix proteins. this is in contrast to the functional and structural conservation of the fusion protein subunit of the glycoproteins derived from retroviruses (hiv- ; htlv-i), filoviruses (ebola), paramyxoviruses (sv ) and orthomyxoviruses (influenza virus) which show striking similarities [ ] . they all fold into trimeric rod-like structures composed of a central triple-stranded coiled coil and an outer helical or non-helical layer. this arrangement places the fusion peptide and the transmembrane region at one end of the rod, which facilitates fusion of viral and cellular membranes during viral entry [ ] . the conserved function and conformation of the fusion proteins may therefore be based on a common ancestral viral fusion protein [ ] . the structural dissimilarity of matrix proteins might indicate two different scenarios: (i) matrix proteins from related viruses have evolved from a common ancestor and have changed over time completely as they acquired new functions that helped to adapt to the hosts. such dramatic changes could have been supported by their high mutation rate [ ] . (ii) secondly, matrix proteins have nothing in common with each other as they have been acquired independently during evolution. although there is no doubt that matrix proteins are the major driving forces for assembly and budding there is some evidence that matrix protein-free viruses might have existed as matrix-less measles virus, vsv and rabies virus show infectivity, albeit severely impaired [ ] [ ] [ ] . in addition, other enveloped viruses have solved their envelope acquirement differently, namely flaviviruses and alphaviruses use their glycoprotein to form a protein shell on the outside of the lipid bilayer envelope [ ] . a third class of enveloped viruses, the corona virus uses an integral membrane protein that might function as a matrix protein [ ] . in conclusion, different matrix protein conformations and/or their diverse oligomeric states or polymerisation features are most likely to contribute to the morphological differences between viral families. virus maturation by budding escaping from the cell: assembly and budding of negative-strand rna viruses membrane vesiculation function and exocytosis of wild-type and mutant matrix proteins of vesicular stomatitis virus vesicular release of ebola virus matrix protein vp ebola virus vp -induced particle formation and association with the lipid bilayer intracellular transport of retroviral capsid components influenza viruses select ordered lipid domains during budding from the plasma membrane effect of mutations affecting the p gag protein on human immunodeficiency virus particle release is required for particle production from full-length human immunodeficiency virus type molecular clones expressing protease mechanisms of enveloped rna virus budding receptor downregulation and multivesicular-body sorting the protein network of hiv budding aip /alix is a binding partner for hiv- p and eiav p functioning in virus budding ubiquitin is part of the retrovirus budding machinery a role for ubiquitin ligase recruitment in retrovirus release a ppxy motif within the vp protein of ebola virus interacts physically and functionally with a ubiquitin ligase: implications for filovirus budding raft-partitioning of the ubiquitin ligases cbl and nedd upon ige-triggered cell signaling multivesicular body sorting: ubiquitin ligase rsp is required for the modification and sorting of carboxypeptidase s alix (alg- -interacting protein x), a protein involved in apoptosis, binds to endophilins and induces cytoplasmic vacuolization endophilins interact with moloney murine leukemia virus gag and modulate virion production crystal structure of the matrix protein vp from ebola virus structural characterization and membrane binding properties of the matrix protein vp of ebola virus membrane association induces a conformational change in the ebola virus matrix protein oligomerization and polimerization of the filovirus matrix protein vp the matrix protein vp from ebola virus octamerizes into porelike structures with specific rna binding properties in vivo oligomerization and raft localization of ebola virus protein vp during vesicular budding ebola virus matrix protein vp interaction with human cellular factors tsg and nedd nedd regulates egress of ebola virus-like particles from host cells hiv- and ebola virus encode small peptide motifs that recruit tsg to sites of particle assembly to facilitate egress overlapping motifs (ptap and ppey) within the ebola virus vp protein function independently as late budding domains: involvement of host proteins tsg and vps- crystal structure of vesicular stomatitis virus matrix protein conformational flexibility and polymerization of vesicular stomatitis virus matrix protein solubility of vesicular stomatitis virus m protein in the cytosol of infected cells or isolated from virions aggregation of vsv m protein is reversible and mediated by nucleation sites: implications for viral assembly membrane association of functional vesicular stomatitis virus matrix protein in vivo role of matrix protein in cytopathogenesis of vesicular stomatitis virus the matrix protein of vesicular stomatitis virus inhibits nucleocytoplasmic transport when it is in the nucleus and associated with nuclear pore complexes vesicular stomatitis virus matrix protein inhibits host cell gene expression by targeting the nucleoporin nup complex nuclear localization signals in the matrix protein of vesicular stomatitis virus the cell-rounding activity of the vesicular stomatitis virus matrix protein is due to the induction of cell death role of the vesicular stomatitis virus matrix protein in maintaining the viral nucleocapsid in the condensed form found in native virions the assembly of ebola virus nucleocapsid requires virion-associated proteins and and posttranslational modification of nucleoprotein rhabdoviruses and the cellular ubiquitin-proteasome system: a budding interaction structure of a bifunctional membrane-rna binding protein, influenza virus matrix protein m combined results from solution studies on intact influeenza virus m protein and from a new crystal form of its n-terminal domain show that m is an elongated monomer in vitro dissection of the membrane and rnp binding activities of influenza virus m protein nucleus-targeting domain of the matrix protein (m ) of influenza virus crystal structure of the m protein-binding domain of the influenza a virus nuclear export protein (nep/ns ) influenza virus matrix protein is the major driving force in virus budding influenza virus hemagglutinin and neuraminidase glycoproteins stimulate the membrane association of the matrix protein characterization of the membrane association of the influenza virus matrix protein in living cells nuclear transport of influenza virus ribonucleoproteins: the viral matrix protein (m ) promotes export and inhibits import crystal structures of the trimeric human immunodeficiency virus type matrix protein: implications for membrane association and assembly crystal structure of siv matrix antigen and implications for virus assembly visualization of the intracellular behavior of hiv in living cells two nuclear localization signals in the hiv- matrix protein regulate nuclear import of the hiv- pre-integration complex retroviral matrix proteins: a structural perspective hiv gag mimics the tsg -recruiting activity of the human hrs protein receptor binding and membrane fusion in virus entry: the influenza hemagglutinin structural basis for membrane fusion by enveloped viruses structure of the haemagglutinin-esterase-fusion glycoprotein of influenza c virus rna virus mutations and fitness for survival complementation of m gene mutants of vesicular stomatitis virus by plasmid-derived m protein converts spherical extracellular particles into native bullet shapes a matrix-less measles virus is infectious and elicits extensive cell fusion: consequences for propagation in the brain matrix protein of rabies virus is responsible for the assembly and budding of bullet-shaped particles and interacts with the transmembrane spike glycoprotein g enveloped viruses sars: beginning to understand a new virus we gratefully acknowledge critical comments on the manuscript by dr. stephan becker and apologize to the many colleagues whose work we were unable to cite due to the publishers space limitations. key: cord- -l du authors: puoti, massimo; zanini, barbara; quinzan, gian paolo; ravasio, laura; paraninfo, giuseppe; santantonio, teresa; rollo, adriano; artioli, stefania; maggiolo, franco; zaltron, serena; master hiv/hcv co-infection study group; raise, enzo; mignani, ermenegildo; resta, francesco; verucchi, gabriella; pastore, giuseppe; suter, fredy; carosi, giampiero title: a randomized, controlled trial of triple antiviral therapy as initial treatment of chronic hepatitis c in hiv-infected patients() date: - - journal: j hepatol doi: . /j.jhep. . . sha: doc_id: cord_uid: l du background/aims: interferon and ribavirin combination therapy for chronic hepatitis c induces a low response rate in human immunodeficiency virus (hiv) infected patients. to assess the impact of intensification of interferon administration and of the addition of amantadine on the efficacy and safety of standard anti-hepatitis c virus (hcv) treatment in hiv-infected patients. methods: multicentre, prospective, open-label, randomized, phase iii clinical trial. eighty co-infected patients were randomized to receive ribavirin – mg/day in combination with, group a: interferon alpha a miu thrice weekly; group b: ifnα a miu daily, plus amantadine mg/day; treatment duration was – weeks according to hcv genotype. results: forty-one patients were randomized in group a and in group b. intention-to-treat analysis showed a sustained virological response, defined as hcv-rna negativization, months after stopping treatment in % of patients from group a and % from group b (p> . ). the lack of a -log drop in hcv-rna levels after weeks of treatment showed a % predictive value of lack of sustained response. conclusions: amantadine addition and interferon intensification do not improve the low efficacy of combination of interferon alfa plus ribavirin in hiv/hcv co-infected patients. patients with no early virologic response did not have any probability of sustained response. prevalence of hepatitis c virus (hcv) infection among anti-human immunodeficiency virus (hiv) seropositive patients with a history of intravenous drug use (idu) or transfusion is greater than %. the extensive use of highly active antiretroviral therapy (haart) has dramatically changed the prognosis of hiv infection, prolonging and improving life of anti-hiv seropositives [ ] . on the other hand, mortality and morbidity for liver disease have increased significantly [ ] . consequently, treatment of chronic hepatitis c in co-infected patients has become mandatory. interferon in combination with ribavirin was the gold standard for treatment of chronic hepatitis c in hiv-uninfected patients, inducing a % rate of sustained response [ ] . however, a cumulative sustained virological response (svr) was observed in only % ( % confidence interval (ci), - %) of patients enrolled in four pilot uncontrolled studies aiming to assess the efficacy and tolerability of ribavirin plus interferon alfa administered thrice weekly in hiv/hcv co-infected patients [ - ] . so there is an urgent need for new and more effective treatment schedules for hepatitis c in hiv co-infected patients. among patients treated with interferon alfa three times weekly an intermittent increase of hcv viral load is observed on treatmentfree days [ ] . daily administration of interferon could maintain a sustained antiviral effect on hcv; this schedule is expected to increase the efficacy of interferon alfa . amantadine ( -aminoamantadan) is a tricyclic amine with antiviral activity against toga-, myxo-, arena-, flavi-and coronavirus [ ] . a comparative study recently demonstrated a statistically significant advantage of the addition of amantadine to interferon and ribavirin combination in hiv-uninfected patients with chronic hepatitis c and nonresponders to a previous cycle of interferon monotherapy [ ] . thus addition of amantadine and intensification of interferon schedule with daily administration could be expected to increase the efficacy of standard combination treatment with ribavirin and interferon administered thrice weekly. in order to test this hypothesis we designed a multicentre, randomized controlled trial to compare the efficacy and safety of a new treatment schedule for chronic hepatitis c including ribavirin, amantadine and daily interferon alfa administration with the standard combination treatment. from april to october , hiv/hcv co-infected patients were consecutively enrolled in a multicentre, prospective, open-label, randomized, phase iii clinical trial. the study was conducted by the master hiv/hcv co-infection study group. eligibility criteria included: age between and years; alanine aminotransferase (alt) levels above the upper limit of normal (uln) months before enrolment in the study; detectable plasma hcvrna by qualitative test (polymerase chain reaction (pcr) with amplicor w roche diagnostic system, hoffman-la roche, basel, switzerland); proven hivab seropositivity by elisa confirmed by western blot; stable hiv disease with cd cell count persistently over /ml during the last months; anti-retroviral treatment (art) started at least months before enrolment and demonstrated to be effective or no need for art; exclusion of hepatocellular carcinoma by imaging and alphafetoprotein level lower than ng/ml; willingness not to consume alcohol during the treatment period. exclusion criteria were: reactivity for hepatitis b surface antigen (hbsag), neutropenia (fewer than neutrophils per ml), anaemia (less than g/dl of haemoglobin in women and less than g/dl in men), thrombocytopenia (fewer than , platelets per ml), decompensated liver disease, serum creatinine level more than . times the uln, poorly controlled psychiatric disease, alcohol or drug dependence in the year prior to enrolment; substantial coexisting medical conditions except hiv co-infection, previous treatment with ifn alfa or ribavirin or amantadine; systemic anti-neoplastic or immunomodulatory treatment in the preceding months; current/present hiv-related opportunistic infection or malignancy classified as aids defining events (according to cdc aids surveillance case definitions); concomitant medication with rifampicin and/or rifabutin and/or isoniazid and/or pyrazinamide and/or gancyclovir; evidence of excessive alcohol consumption (. g in males and g in females) and/or illegal substance abuse within the last months; coexisting causes of liver disease; any additional contraindication to any of the drugs used in the study. additional exclusion criteria were pregnancy or lactation, and refusal to practise effective contraception during treatment and follow-up. this randomized controlled clinical trial was conducted at italian centres from april to december . patients were randomly assigned at a : ratio (with a block size of five) to receive: group a: interferon alpha a (ifna) million units (miu) subcutaneously (sc) three times per week plus daily per oral (po) ribavirin (copegus w , roche, mg tablets) or group b: ifna a miu sc daily plus ribavirin (copegus w , roche, mg tablets) and amantadine (mantadan w , boheringer ingelheim, mg tablets) po mg every h. ribavirin was given orally with food at a dose of mg/day (two tablets bid every h) for patients , kg of body weight and mg/day (two tablets in the morning and three in the evening after h) for patients . kg of body weight. treatment duration differed according to hcv genotype: weeks for hcv genotype or , and weeks for hcv genotype or , only if hcvrna was negative at week according to international guidelines [ ] . randomization was centralized in the coordinating centre and stratified according to hcv genotype (genotype or vs. or ). genotype was performed by reverse hybridization assay (inno lipa hcv ii; innogenetics, ghent, belgium). patients were followed up for a treatment-free period of weeks after cessation of therapy. the institutional review boards of the participating centres approved the protocol and all patients provided written informed consent. the study was conducted according to the declaration of helsinki, the applicable regulatory requirements and the ich/cpmp guidelines 'good clinical practice'. clinical examination, laboratory testing (including lactic acid and bicarbonate) and haematological count, including cd cell count, were performed monthly; plasma hivrna was monitored every months using commercially available tests (quantiplex hiv- rna v. . assay chiron corporation emeryville california, usa); hcvrna was measured at time and after , , and weeks by a commercially available secondgeneration rt-pcr test (amplicor monitor version . , roche diagnostic system, pleasanton, ca) according to the manufacturer's instructions. presence of hcvrna in plasma was determined at weeks , and , at the end of treatment and weeks after cessation of therapy using a commercially available second-generation rt-pcr test (amplicor hcv . ; roche diagnostic systems, pleasanton, ca) with a low end detection limit of iu/ml according to the manufacturer's instructions. primary measures of efficacy were end-of-treatment response (eotr) and svr, respectively, defined as hcvrna levels below iu/ml at the end of treatment, and weeks after treatment. measures of safety were: any change in cd cell count, hivrna level, rate of withdrawal for adverse events (ae) or drop-out (do), rate of withdrawal from art or switch of anti-retrovirals. in difficult-to-treat patients (such as 'non-responders' to interferon monotherapy), triple therapy has been proven to increase by at least three times the sustained viral response rate obtained with standard interferon and ribavirin combination [ ] . in order to establish that the svr in the triple therapy arm is at least three times higher than the % sustained response rate observed in hiv-co-infected patients treated with interferon and ribavirin in pilot studies [ ] [ ] [ ] [ ] , it was calculated that at least patients should have been enrolled. a confidence probability of % and a significance level of . were used. intention to treat (itt) and per protocol (pp) analyses were performed. categorical variables were compared using fisher's exact test; distribution of continuous variables was compared using the t-test, mann-whitney two-sample statistic test or wilcoxon ranksum test. relationship between patients' baseline characteristics and svr was examined by univariate logistic regression analysis. to assess the independence of these factors, a multivariate logistic regression analysis was performed with backward selection ðp . : Þ: all p values reported are two-sided, and p was considered significant when , . . statistical analysis was performed using stata software, version . (stata corporation, college station, tx, usa). eighty patients were enrolled in the study; were randomly assigned to group a and to group b; their baseline characteristics are shown in table . no difference was found in demographics or clinical and immunovirological characteristics between the two groups. sixty patients ( %) were taking art, all for more than months according to inclusion criteria, when they started anti-hcv treatment. the rates of hcvrna clearance in both groups are shown in table . itt analysis showed . % eotr, . % in group a and . % in group b. at the end of months, follow-up response rates had decreased by about half: svr was observed in . , % in group a and . % in group b. differences in absolute hcvrna levels or hcvrna change-over baseline between the two groups at any time were not statistically significant. table shows the distribution of some baseline characteristics in patients with and without svr; viroimmunologic and art characteristics were also analyzed, but the tests did not reveal a relationship with svr; genotype or and gamma glutamyl transferase (ggt) baseline level less than . times the upper limit of the normal range were more frequent in patients with svr (p , : fisher's exact test); univariate logistic regression showed their role as predictive factors of svr, with an odds ratio of ( . - . ci %) and . ( . - . ci %), respectively. aspartate aminotransferase (ast) and neutrophil baseline levels were, respectively, lower and higher in patients who obtained an svr (p , : with mann -whitney test). by performing a multivariate logistic regression we found that genotype or and ggt baseline value were independent predictive factors for svr (table ). by week , of the patients ( %) still on active treatment had a virologic response defined as a -log decrease from baseline hcvrna levels or no detectable serum hcvrna (fig. ) . of those with a -week virologic response ( %) subsequently had an svr, dropped out ( because of an adverse event and spontaneously stopped treatment), did have sustained hcvrna negativization, showed a breakthrough after reducing the dose of ribavirin, and patients relapsed after stopping treatment. eighteen out of tested hcvrna negative at weeks. the sustained response rate in this group ( / , %) did not differ significantly from that observed in subjects with detectable hcvrna at weeks ( / ; %). by contrast, of the patients who did not have a -week virologic response dropped out ( because of ae and prematurely stopped treatment), and did not show hcvrna negativization; only two patients showed an etr, but none showed an svr. twenty-five out of patients ( . %) stopped treatment prematurely: patients ( . %) withdrew due to ae and independently stopped therapy: distribution of ae and do was similar in the two treatment groups. pp analysis showed . % of svr in group a and . % in group b: there was not a significant difference between the two treatment arms. the treatment schedule was modified for more than showed an increase in hivrna level at any time during treatment and only of them needed a switch of art due to loss of efficacy. no statistically significant difference was found in cd and hivrna levels between the two treatment groups at any time. eleven patients were undergoing treatment with didanosine, with stavudine, and six with both of them; lactic acidosis was not observed in any patients. the main finding of this study was the very poor rate of svr to combination of standard interferon and ribavirin observed in both treatment arms, with and without amantadine and interferon dose intensification. cumulatively . % of treated patients and . % of those who completed the treatment course cleared hcvrna. this response rate is significantly lower than the % svr rate reported in trials performed on hiv seronegatives [ ] the high withdrawal rate ( %), the large number of subjects requiring adjustment of the treatment schedule ( %) and the low response rate observed in those who completed the treatment schedule suggest that the tolerability and efficacy of interferon alfa and ribavirin in combination are reduced in hiv-co-infected subjects. the proportion of patients who needed interferon dose reduction was significantly higher in group b. we did not find a statistically significant difference in the rates of eotr and svr between the two treatment arms. therefore, the combination of interferon alfa schedule intensification and amantadine addition neither increase the efficacy nor improve the tolerability of combination therapy in co-infected hiv/hcv patients. clinical trials with pegylated interferon formulations [ , ] in combination with ribavirin are ongoing in hiv/hcv-co-infected patients; preliminary results suggest that increased interferon levels induced by pegylated interferons could improve response rates in hiv/hcv-co-infected patients [ , ] . amantadine addition did not improve the efficacy or tolerability of interferon alpha and ribavirin in chronic hcv infection. however, given the low power of this study, additional studies are needed before this drug is discarded from the therapeutic armamentarium for hiv/hcv co-infection. univariate logistic regression analysis of factors predictive of svr did not confirm that of age, sex, degree of fibrosis or baseline hcvrna levels, reported in hiv seronegative patients, but this was probably due to the small size of the sample under study [ ] . the results of our study show that half of the patients with svr had in the past reached a cd cell count of less than /ml; such a low nadir of cd count in the years preceding treatment did not preclude achievement of a svr. these findings, together with the absence of an association between fibrosis score and cd counts and svr, suggest that progression of both hcv and hiv infections does not decrease the response rate to interferon and ribavirin combination in patients without severe immune depletion. we can therefore hypothesize that in patients without a high probability of response and early stage of liver fibrosis a watchful waiting strategy would not reduce the potential efficacy of anti-hcv treatment. this hypothesis needs to be confirmed by larger studies or by a meta-analysis of multiple pilot studies. hcv genotype, early hcvrna clearance and ggt levels were significant predictors of svr in multivariate analysis. the association between hcv genotypes and and a higher rate of svr is well known and has been confirmed in anti-hiv seropositives by pilot studies [ - ] . all but two responders were infected by hcv genotype or and the svr rate in patients infected by genotype or was %. so, taking into account the side effects of treatment, these data suggest a very low cost effectiveness of treatment with standard interferon and ribavirin in hiv seropositives infected by hcv genotype or . the association between earlier hcv clearance and svr suggests that suppression of hcv replication in the early phase of treatment is necessary but not sufficient to induce an svr. high serum ggt in chronic hepatitis c patients was frequently associated with more severe hepatic fibrosis or cirrhosis, or with steatosis, and may in part account for poor response to interferon therapy [ - ] . ggt alteration in hiv seropositive patients is frequent and possibly correlated with one or more of the following factors: hepatic steatosis and/or lipidic dismetabolism, drug-related hepatic toxicity (especially in patients undergoing art), excessive alcoholic consumption, hcv-related hepatic damage (especially genotype ) and immuno-mediated biliary duct damage. although all the included patients denied alcohol abuse, we cannot rule out that high ggt levels were due to undeclared excessive alcohol consumption. body mass index was not significantly associated with treatment response. only one of the svrs showed high ggt at baseline; in this patient ggt normalized the third month after hcvrna negativization. evaluation of art toxicity, careful assessment of alcohol intake and correction of alcohol abuse, diagnosis and treatment of altered lipid metabolism are factors to evaluate before starting treatment for chronic hepatitis c in patients co-infected with hiv. in this study the lack of a -log drop in hcvrna levels after weeks of treatment showed a % negative predictive value of svr. although this data should be interpreted with caution, given the high proportion of dos, this result reinforces/supports the observation of a nearly absolute negative predictive value of the lack of a -week virologic response in hcv non-infected patients treated with pegylated interferons [ , ] . as we enter an era in which hcv treatment is going to be pursued aggressively in hiv/hcv-co-infected persons, more and more emphasis must be placed on identifying at an early stage subjects who will not benefit from an expensive and poorly tolerated therapy. if these results are confirmed by larger studies, anti-hcv treatment could be stopped after weeks, without decreasing the expected rate of svr, in hiv-infected patients who did not show at least a -log drop in hcvrna level. in conclusion, intensification of interferon alpha schedule and amantadine addition do not appear to improve the limited efficacy of standard combination therapy including interferon thrice weekly plus ribavirin for the treatment of chronic hepatitis c in hiv-co-infected patients. the best candidates for anti-hcv treatment are patients infected by hcv genotype or with normal ggt levels. the lack of a -log drop in hcvrna level after weeks of treatment seems to be highly predictive of the poor efficacy of anti-hcv treatment. paolo and ospedale umberto i venezia-mestre) a.poggio, v. mondino (divisione di malattie infettive, ospedale di verbania), m. tinelli, mc. cerri (divisione di malattie infettive ospedale di lodi). mortality for liver disease in patients with hiv infection: a cohort study mortality due to chronic viral liver disease among patients with human immunodeficiency virus randomised trial of interferon a b plu ribavirin for weeks or for weeks vs. interferon a b plus placebo for weeks for treatment of chronic hepatitis c virus chronic hepatitis c in hiv infection: feasibility and sustained efficacy of therapy with interferon alfa- b and ribavirin long term efficacy of combination therapy with interferon alfa b and ribavirin for severe chronic hepatitis c in hiv infected patients interferon and ribavirin combination therapy in chronic hepatitis c in human immunodeficiency virus co-infected patients with congenital coagulation disorders pilot study of interferon alpha high-dose induction therapy in combination with ribavirin for chronic hepatitis c in hivco-infected patients the effects of a high dose, short course of interferon on hepatitis c a controlled trial of amantadine and rimantadine in the prophylaxis of influenza a infection triple antiviral therapy as a new option for patients with interferon non-responsive chronic hepatitis c international consensus conference on hepatitis c. consensus statement introduction to therapy of hepatitis c peginterferon alfa- a plus ribavirin for chronic hepatitis c virus infection peginterferon alfa- b plus ribavirin compared with interferon alfa- b plus ribavirin for initial treatment of chronic hepatitis c: a randomised trial multicenter, randomized trial comparing pegylated interferon alpha- b (peg-ifn) plus ribavirin (rbv) vs. peg-ifn for treatment of hiv/hcv co-infected patients anrs hc -ribavic: a randomised controlled trial of pegylatedinterferona- b plus ribavirin vs interferona- b plus ribavirin as primary treatment of chronic hepatitis c in hiv co-infected patients is an 'À la carte' combination interferon alfa- b plus ribavirin regimen possible for the first line treatment in patients with chronic hepatitis c? retreatment with interferon plus ribavirin of chronic hepatitis c non-responders to interferon monotherapy: a metaanalysis of individual patient data gamma-glutamyl transpeptidase as a response predictor when using alpha-interferon to treat hepatitis c treatment of chronic sporadic-type non-a, non-b hepatitis with lymphoblastoid interferon: gamma gt levels predictive for response steatosis accelerates the progression of liver damage of chronic hepatitis c patients and correlates with specific hcv genotype and visceral obesity the authors wish to thank monica bertoletti and angela braga for their invaluable help and technical assistance in preparing the manuscript. key: cord- -lxlerb authors: lim, w.s; anderson, s.r; read, r.c title: hospital management of adults with severe acute respiratory syndrome (sars) if sars re-emerges—updated february date: - - journal: j infect doi: . /j.jinf. . . sha: doc_id: cord_uid: lxlerb severe acute respiratory syndrome (sars) is a potentially severe and highly infectious disease to which healthcare workers involved in the management of cases are particularly vulnerable. these guidelines briefly summarise optimal and safe practice for clinicians involved in the emergency care of patients with probable or confirmed sars. during severe acute respiratory syndrome caused by a novel coronavirus (sars-cov) emerged as an infectious disease with a significant inhospital mortality and posed a considerable occupational risk for healthcare workers. - the initial sars outbreak ended in july when the world health organisation (who) announced that all known person-to-person transmission of sars-cov had ceased. at the time of preparation of these guidelines, there have been a further two laboratory-acquired cases of sars and further community-acquired cases. these cases emphasise the potential for sars to re-emerge and spread unpredictably. these guidelines document the hospital management of adults with probable or confirmed sars. they are meant only as a brief summary for clinicians. these guidelines do not cover the management in the community of a person under investigation (pui) (see case definitions). guidelines for the management of paediatrics cases have not yet been developed. as more information about sars becomes available, guidance will be appropriately updated. please consult the latest guidance available on the websites of the british thoracic society (http:// www.brit-thoracic.org.uk/) and health protection agency (http://www.hpa.org.uk/infections/ topics_az/sars/menu.htm). the following case definitions (see tables - ) are designed for use during an outbreak of sars, once the re-emergence of sars has been verified by the world health organisation (who). it is anticipated that patients with sars will have a respiratory illness severe enough to warrant hospital admission. management of such cases is covered in sections - . a person with a mild respiratory illness and a potential epidemiological link to sars should be defined as a pui (see table ) and should be assessed in primary care and reviewed within h. a pui does not require routine hospitalisation nor do they require a chest radiograph (cxr) or laboratory investigation for sars cov as part of their assessment. a pui should only be hospitalised if his or her condition deteriorates. the management of such patients is covered in section . if these patients are subsequently found to have radiographic evidence consistent with sars, they should be reclassified as a 'probable sars' case unless an alternative diagnosis is made. a pui should be reported to local health protection units but does not need to be reported to cdsc colindale. please discuss the classification of sars patients with the health protection agency's communicable disease surveillance centre (cdsc) duty doctor (tel.: - - ) and complete a standard sars report form and fax to your local consultant in communicable disease control (ccdc) and cdsc (details at: http://www.hpa.org.uk/infections/ topics_az/sars/forms.htm). † negative antibody test on acute serum followed by positive antibody test on convalescent phase serum tested in parallel or † four-fold or greater rise in antibody titre between the acute and convalescent phase sera tested in parallel (c) virus isolation † isolation in cell culture of sars-cov from any specimen; plus pcr confirmation using a validated method patients are likely to present initially with a clinical picture of pneumonia which may be consistent with sars. therefore, other causes of pneumonia should be considered. confirmation that a patient has sars may occur following further investigation. detailed guidance regarding the infection control issues during hospital management of a patient, or patients, presenting with sars can be found at the hpa website: http://www.hpa.org.uk/infections/ topics_az/sars/hops_infect_cont.htm. briefly, the key recommendations are: (a) give the patient a surgical mask to wear continuously (unless requiring face mask for oxygen confirm the travel history and/or history of contact with a patient with sars. explore other possible causes of pneumonia. assess pneumonia disease severity according to the bts guidelines on the management of community acquired pneumonia (cap) in adults (http:// www.brit-thoracic.org.uk/guide/guidelines.html). in addition, determine whether the patient has any medical history of illness associated with a more severe outcome of sars, i.e. diabetes and cardiopulmonary disease. obtain investigations as listed below (observe high risk infection control measures for all samples). for full details, please see the hpa website at http://www.hpa.org.uk/infections/topics_az/ sars/micro.htm. only send specimens once cdsc have been informed of a case via their standard report form. please observe strict infection control procedures. all specimens should be double bagged and labelled as a biohazard. do not obtain a nasopharyngeal aspirate as this is likely to generate aerosols. admit the patient to a designated isolation unit (see section . ). manage as for severe cap according to bts guidelines. administer fluids and oxygen as required. commence intravenous co-amoxiclav . g tds or cefuroxime . g tds plus erythromycin mg qds or clarithromycin mg bd. please refer to the bts guidelines for alternative recommended regimens. oxygen supplementation should be administered according to standard/local guidelines. however, in order to reduce the risk of aerosol generation and hence spread of infection, high flow oxygen is not recommended, i.e. avoid oxygen flow rates of . l/min. it should be possible to provide - % oxygen supplementation using a standard low flow oxygen system and an air-entrainer together with a ventimask. procedures and practices that promote the generation of aerosols (table ) should be avoided wherever possible to reduce the risk of infection to healthcare workers. , if such procedures need to be performed, e.g. tracheal intubation, it is advised that experienced operators only should undertake these procedures. these should, where possible, be planned and controlled. these procedures should ideally be undertaken in a negative pressure room. only a minimum number of staff should be present and all must wear gowns, gloves, goggles/visors and respirators as described under infection control issues (see section . ). entry and exit from the room should be minimised during the procedure. the use of powered air purifying respirators (paprs) during aerosol generating procedures is not recommended. this is because there are concerns over the removal, disposal, cleaning and decontamination of this equipment, which may increase the potential risk of self-contamination and at this time there is inadequate evidence to determine whether paprs further reduce the transmission of sars. if paprs are used, staff must be properly trained in their safe use. in studies from canada and singapore, approximately % of patients with suspected or probable sars, according to the prevailing who case definition from march to june , required icu admission. , of these patients, - % required mechanical ventilation. average length of icu stay was days. preplanning and early consultation with local critical care providers is recommended. patients who are likely to require intubation, should be identified early and the procedure should be undertaken electively. in order to avoid the use of cpap or niv, early intubation and invasive positive pressure ventilation (ippv) may be required in some patients with impending respiratory failure. the following issues need to be carefully considered: further guidance for the management of critically ill patients is being developed. the use of high-dose steroids has been anecdotally reported to contribute to decrease in fever and need for oxygen supplementation. a study from guangzhou, china has suggested that the early administration of high-dose steroids together with cpap ventilation is associated with a lower mortality. however, these findings are not based on adequately controlled data and there remain concerns regarding the use of high-dose steroids. the use of cpap is certainly no longer recommended (see section . . ). in a retrospective analysis of hong kong patients who had received ribavirin in combination with different steroid regimens, patients who received initial high dose pulsed methylprednisolone intravenously had less oxygen requirement, better radiological improvement and less likelihood to require rescue pulse steroid use than patients who received non-pulse steroid therapy. however, the overall mortality rate, and requirement for mechanical ventilation or admission to the intensive care unit was the same for both regimens. recommendation the current recommendation is to consider moderate doses of steroid (prednisolone - mg/day or iv equivalent) in severely ill patients with sars with increasing oxygen requirements who have a pao , kpa or o sats , % on air. currently there is no convincing evidence that ribavirin alters clinical outcome. in laboratory studies, no in vitro activity against sars-associated coronavirus (sars-cov) has been consistently demonstrated either. in addition, use of ribavirin is associated with significant toxicity including haemolysis (in , %) and decrease in haemoglobin of g/dl or more (in , %). the routine use of ribavirin in patients with sars is not recommended. the antiviral activity of interferons against sars coronavirus has been measured in vitro and interferon beta appears to be particularly active. the world health organisation is currently coordinating plans for clinical trials of interferons in the event of re-emergence of the disease. recommendation none can be given at this time. generate a list of all close contacts. this should be initiated by the attending physician at the time of first contact with the patient. the local hospital infection control and occupational health teams may need to be involved if any healthcare workers are identified as close contacts. record the date on which all close contacts last had contact with the case and inform them about sars. inform the local ccdc/health protection team of any contacts and their details to ensure follow-up. these contacts may continue with everyday activities, as long as they remain well. the local health protection team will contact them on a regular basis to review their health. these contacts should be isolated at home. please refer to the health protection agency's guidelines on voluntary home isolation at http://www.hpa. org.uk/infections/topics_az/sars/homeiso.htm. if a contact becomes unwell within days of their contact with a probable or confirmed sars case they should phone a doctor urgently. for more information please refer to: http://www.hpa.org. uk/infections/topics_az/sars/guidelines.htm. guidelines for the safe discharge of patients recovering from sars have been published by who. please refer to the who website at http:// www.who.int/csr/sars/discharge/en/. briefly, the following criteria should be considered before discharge: (a) afebrile for h (b) resolving cough (c) laboratory tests, if previously abnormal, returning to normal (d) chest x-ray improved. patients should monitor and record their temperature twice daily. if they have an elevated temperature of c or above on two consecutive occasions they should inform (by telephone) the healthcare facility from which they were discharged. patients should remain at home for days after discharge, keeping contact with others at a minimum. this is to reduce the risk of transmission until more is known regarding the potential for continued carriage in convalescent cases. additional home confinement may need to be considered, particularly in patients who are immunosuppressed. inform the local health protection team/ccdc regarding the hospital discharge of patients to ensure follow-up in the community. a standard follow-up form should be completed and faxed to the local ccdc and cdsc on day , day , and/or once the patient is asymptomatic. forms are available from http://www.hpa.org.uk/infections/ topics_az/sars/forms.htm. follow-up post-discharge will be the responsibility of the local infection control team. convalescent serology should be obtained at days after the date of disease onset. puis who have symptoms and signs consistent with a lower respiratory tract infection (lrti) but have a normal cxr do not fulfil the sars case definition (see table ). patients should be discharged and followed up in primary care unless their symptoms or social circumstances warrant continued hospital care. up to % of patients with probable sars may initially present with normal chest radiographs. therefore, puis who need ongoing hospitalisation require careful medical review in the first h following admission. infection control measures as for patients with probable sars should apply (see section . ) until it is clinically clear that the pui does not have probable sars. such patients should be treated as for non-pneumonic lrti. if the patient improves with treatment in the first h following admission, the likelihood of sars is small. infection control measures may be relaxed and the patient discharged if this is clinically appropriate. if the patient does not improve with initial treatment (either no change or deteriorates), a repeat cxr should be obtained. an abnormal cxr with changes consistent with sars would require the patient to be re-classified as having probable sars and be managed accordingly (see section ). if the repeat cxr remains normal, the patient remains a pui. further repeat cxrs may be required at - days intervals depending on clinical circumstances. a pui should be reported to the local health protection unit but does not need to be reported to cdsc colindale. these guidelines were produced as a joint initiative between the british thoracic society, the british infection society and the health protection agency. membership of the guideline development group: lead clinical features and short-term outcomes of patients with sars in the greater toronto area coronavirus as a possible cause of severe acute respiratory syndrome acute respiratory distress syndrome in critically ill patients with severe acute respiratory syndrome critically ill patients with severe acute respiratory syndrome koch's postulates fulfilled for sars virus effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars) clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study identification of severe acute respiratory syndrome in canada a major outbreak of severe acute respiratory syndrome in hong kong bts guidelines for the management of community acquired pneumonia in adults severe acute respiratory syndrome (sars): infection control sars: experience at prince of wales hospital, hong kong anaesthesia and sars development of a standard treatment protocol for severe acute respiratory syndrome description and clinical treatment of an early outbreak of severe acute respiratory syndrome (sars) in guangzhou, pr china high dose pulse versus nonpulse corticosteroid regimens in severe acute respiratory syndrome treatment of sars with human interferons useful contact details: scottish centre for infection and environmental health (scieh) telephone: - - e-mail: jim.mcmenamin@scieh.csa.scot.nhs.uk key: cord- -gvc ij authors: klaunberg, brenda a.; lizak, martin j. title: considerations for setting up a small-animal imaging facility date: journal: lab anim (ny) doi: . /laban - sha: doc_id: cord_uid: gvc ij imaging techniques allow for the conduct of noninvasive, in vivo longitudinal small-animal studies, but also require access to expensive and complex equipment, and personnel who are properly trained in their use. the authors describe their planning and staffing of the nih mouse imaging facility, and highlight important issues to consider when designing a similar facility. state-of-the-art biomedical research often uses rodents and other small animals for disease modeling. a recurring issue for many investigators is the desire to obtain anatomical and physiological information from valuable research animals without sacrificing them. in vivo imaging is a noninvasive way to gain insight into the animal's anatomy and physiology ; however, the unit cost and complexity of many such methods may preclude an investigator's ability to gain access to such devices. location of the imaging equipment in a shared facility can overcome these obstacles. the vision of the nih mouse imaging facility (mif) is to offer various state-of-theart, in vivo small-animal imaging techniques in one facility. the mif is a shared resource for the nih intramural community, and currently has more than active animal protocols. at this time, the mif has three magnetic resonance imaging (mri) scanners, a micro x-ray computed tomography (ct) scanner, two ultrasound scanners, a combined luciferase/gfp imager, and a laser doppler imager. in addition to administrative personnel, the staff consists of a veterinarian, three imaging scientists, an electrical engineer, and three animal technicians. setting up this facility took planning and intellectual contributions from experts in many fields. we provide resources for a wide variety of investigators from many of the various institutes within the nih. this is not a 'how-to' manual, but we will discuss some of the issues that the principal players considered when designing and staffing the mif. a small-animal imaging facility can represent an enormous investment of capital and personnel. to obtain the maximum usefulness and ensure success, a certain amount of planning must take place before an instrument is ordered or facility construction begins. planners should recruit the advice of experts in various imaging fields and involve as many people as necessary to share in the decision-making processes, so that everyone has a voice. knowledge of the needs of the research community is one of the most important priorities in setting up any facility. a consultation with your research community will help to determine which imaging modalities are most needed. it is pointless to include an instrument that no one will use. consultation with experienced operators for each of the imaging modes chosen will elicit useful advice on special needs for each instrument. veterinarians and animal care staff should also have input because they will have a considerable impact on traffic patterns, animal care requirements, and other features. if possible, one should visit other small-animal imaging facilities. what imaging modalities do they have? what problems have they encountered, and how did they solve them? one must consider what types of animals and models could come to the facility for imaging, because these considerations will impact staffing choices, housing availability, and imaging modalities. one should also consider the health status of the animals; a facility that can accommodate immunecompromised animals has more stringent requirements. the biosafety level that the facility will maintain is also a consideration; animals carrying pathogens or treated with radiolabeled agents will require additional restrictions. the mif operates as a clean conventional facility that excludes the following specific pathogens: coronaviruses, pneumovirus of mice (pvm), sendai virus, endoparasites, and ectoparasites. to prevent cross-contamination between rodents, disposable, absorbent material covers all surfaces. all surgical instruments are steam-sterilized for survival procedures, and devices such as nose cones are either placed in cold sterilization or cleaned with a bleach-based disinfectant. planners should decide whether to design and staff the facility so that researchers can be human and animal safety should be the primary consideration when planning an imaging facility design. in addition to excluding specific rodent pathogens, the mif operates at animal biosafety level- (absl- ) [author: edit okay?]. some imaging animals may receive agents (chemotherapeutics, infectious organisms) in higher biosafety levels in other facilities. those animals cannot be imaged until they satisfy absl- requirements. planners should determine the need for a preparation room. it is possible to perform simple procedures such as anesthesia induction and tail vein catheter placement adjacent to the imaging instrument. more complicated preparations that require sterile or aseptic procedures will require a dedicated room. in the mif, there is a small preparation area adjacent to each instrument. in addition, we have a preparation room set aside for rodent procedures more complex than anesthesia induction and tail vein intravenous catheter placement. each preparation area is set up with anesthesia and physiological monitoring equipment, as well as a surgical microscope for microsurgery. we use inhalant anesthesia (isofluorane) as much as possible, and each imaging device preparation area is set up identically. all preparation areas have central gas supplies for oxygen, medical air, and nitrogen, as well as a vacuum system for scavenging anesthetic gases. anesthesia and monitoring equipment must comply with magnet safety. anything that enters the scanner room must be nonmagnetic, and any equipment within the fringe field must operate correctly. once there is an understanding of the goals of the research community, the needs of the investigators can determine the imaging techniques to be made available. some techniques, such as optical imaging, are relatively inexpensive in terms of equipment, personnel, and space. individual laboratories may have the financial capability to purchase these types of devices, but more elaborate techniques, such as mri or ct scanning, require a more substantial investment of resources. individual laboratories rarely have the funds to purchase such equipment or the means to maintain them; therefore, mri and ct are usually the core of an imaging facility. please refer to table for a summary of imaging modalities, their applications, and their estimated costs. mri is one of the most powerful noninvasive imaging methods currently available to research. this technology uses radio waves and powerful magnets to generate radiograph-like images of tissue. a strong magnetic field partially aligns the hydrogen atoms on water molecules in the tissue. a radio wave then disturbs the built-up magnetization, and radio waves are in turn emitted as the magnetization returns to its starting place. these radio waves can be detected and used to construct an image (fig. a) . unlike radiographs, the mri patient is not exposed to x-ray radiation, so repeated imaging is not a risky procedure . mri is excellent for imaging different types of soft tissue with high contrast - . researchers can use it for anatomical and functional studies. some applications include tumor growth and treatment, brain function and stroke, and cardiovascular disease. contrast agents, which are drugs that function like histological stains, increase the usefulness of this imaging method. for resource volume , no. lab trained to run the instruments independently, or whether to offer complete service, in which researchers can deliver animals to the facility and then return for images. this choice will have a profound effect on staffing requirements. the mif is designed to be a resource, not a service, so that investigators are encouraged to participate fully. even if the investigator has no desire to learn to run the equipment, it is our requirement that someone on the protocol be present and responsible for the animal during scanning. an important initial decision is whether or not the animals will be housed in the facility. the number of animals housed and the duration of housing will have an impact on space usage. animal welfare guidelines dictate space requirements that must be carefully considered. moreover, one must also consider space for traffic between housing, preparation rooms, and instruments. at the mif, we provide temporary housing (monday-friday) for mice only. we have one -ft animal room with one ventilated rack that can hold shoebox-sized cages. additionally, we have ft of animal husbandry support area for storage and cage washing. animals involved in long-term studies that must come to the mif periodically for imaging are usually returned to a quarantine facility. because of the surrounding magnetic field, an mri system requires a large area (≥ ft ). the magnet itself requires ∼ ft of surrounding clear area. the fringe field is the distance that the surrounding magnetic field extends before it drops to the level of gauss. the fringe field can occupy an area up to ft . the mri suite must have a design that prevents casual visitors from entering the fringe field. a magnetic field > gauss can adversely affect persons with pacemakers or other metallic implants. the distance to the gauss line varies widely with field strength and magnet type. there are magnets available that have a fringe field limited to a foot or less, but these cost more. mr scanners use extremely heavy magnets; therefore, most facilities locate the mr imager on the ground floor. additionally, because the magnets are cooled by cryogens, good ventilation must be present. rapid boil-off of the cryogens could lead to asphyxiation. the instrument manufacturer can provide a detailed description of space requirements and suggested architectural layouts. a facility will need to plan for additional space for the electronics and console for the scanner as well as a preparation area outside the magnetic field. this can amount to another ft . researchers currently consider a -tesla mri system an optimal field for imaging rodents. at the mif we have several bruker avance mri scanners (bruker-biospin, billerica, ma). some other manufacturers of mri consoles are varian (varian medical systems, inc., palo alto, ca), tecmag (tecmag, houston, tx), and mrrs (mr research systems, guildford, uk). a basic setup for the mri includes the superconducting magnet, the imaging console, and several probes for approximately $ , -$ , , , depending on the options. it is ideal to enclose the magnet in a room shielded from radiofrequency, but this construction would generate additional costs. the magnet itself comes with end caps that adequately shield it; however, passing anesthesia and monitoring lines becomes awkward. because of its complexity, the mri requires a scientist with not only the necessary academic qualifications, but also a technical background for operation. there are many mri measurement methods, each with many control variables, designed for specific purposes. the mr operator must be familiar with the basic physics of the mr measurement and the effect of the controls on the image outcome. if new methods are going to be developed or implemented, a postdoctoral-level scientist will be necessary. new methods require sophisticated knowledge of the hardware and computer programming. this individual will be responsible for running the scanner and maintaining the system. mri maintenance requirements include both hardware and software. hardware maintenance includes the associated magnetic field. the magnet requires liquid nitrogen and liquid helium to maintain the superconducting magnetic field. allowing for a safety margin, most magnets need liquid nitrogen once per week and liquid helium approximately twice per year. if the cryogen level is allowed to become too low, the magnetic field can spontaneously quench (i.e., lose its magnetic force). quality assurance includes regular test images of a standard sample. a small loss in performance can result in unusable images. the operating computer and software also require regular maintenance. system software requires periodic updates to install security patches, repair bugs, and add features. these updates are particularly important if the computers are on a network with outside access. despite hardware and software designed to limit external access, new security holes appear on a regular basis. a malicious attack could destroy valuable work and render the instrument useless until the base software can be reinstalled. archiving and removing old data are important parts of computer maintenance. if the number and size of the stored image files grow too large, system performance can slow or even become blocked. a policy for data management can help prevent major problems, but periodic enforcement is still necessary. this policy can range from a simple principle of moving the oldest data first to a more complicated formula based on size and age. at the mif, we encourage everyone to transfer data from the instrument as soon as possible. when data space becomes an issue, the users with the largest amount of data must export or delete files before they can resume scanning. x-ray ct is an imaging method that uses multiple radiographic views of a subject to construct an image (fig. b) . in our system, an x-ray source and detector rotate around the subject degrees while generating a number of projections or views. the mif has a microcat ii ct scanner built by imtek, inc. (knoxville, tn). other manufacturers of the manufacturer can provide exact measurements of the machine footprint. there are several micro-ct manufacturers, and the price ranges from $ , to $ , . the instrument will require one technician for operation and maintenance. this individual should have a sophisticated education in biology or a related field and be comfortable with computers. the ct scanner relies on several computers on a small network. the ct operator will be responsible for maintaining the computer system software, managing the accumulated data, and quality assurance. a good knowledge of anatomy is vital for data interpretation, and the person should be able to interpret radiographs. reporter gene technology has revolutionized the ability to track cell populations in vivo . optical imaging devices, which allow the user to monitor gene expression [author: edit okay?], are easy to operate and have no special environmental requirements. there are several different types of optical imaging. bioluminescence is an enzymatic biochemical process that produces light. the luciferase gene is first inserted into the targeted cell population. image acquisition requires live cells to process the substrate and produce light. advantages of bioluminescence include high sensitivity, and virtually no background noise; disadvantages include the need for cofactors (i.e., oxygen, magnesium, and atp) and expensive substrates (luciferin). fluorescence imaging also requires genetic manipulation; however, the technology does not require a living system to produce an image. fluorescence imaging differs from bioluminescence in that light must be applied to the tissue to generate a signal. one advantage of fluorescence imaging is the commercial availability of numerous markers (gfp, dsred, icg, and cy . ) with no need for substrate or cofactors; a disadvantage is the low signal/noise ratio due to autofluorescence. basic equipment for bioluminescence or fluorescence imaging consists of a light-tight chamber, a sensitive camera (charge-coupled device), and a computer. it is possible for the ambitious investigator to manufacture his or her own imaging device; however, commercial products are available for about $ , . the mif owns a xenogen ivis (xenogen corp., alameda, ca); other manufacturers include eastman kodak (eastman kodak co., scientific imaging systems, rochester, ny), roper scientific (trenton, nj), and advanced research technologies, inc. (saint-laurent, quebec, canada). our imaging device occupies a space measuring ft ; additional benchtop space is necessary for preparing the animal. image acquisition is simple, and it is not necessary to have a dedicated system operator. investigators can easily learn to perform their own imaging study. our system's operating software first acquires a black-and-white photographic image of the subject, then acquires the luminescent image, and finally overlays the image onto the photograph for anatomical signal mapping (fig. ) . both bioluminescence and fluorescence imaging are useful to detect tumor cells, monitor tumor growth, or track cell movement (e.g., metastasis). optical imaging can be highly specific, but it has low spatial resolution. optical data are typically two-dimensional ( d), but d methods are in development. a high throughput of animals is possible because imaging time is resource volume , no. lab micro-ct equipment include ge medical systems (london, ontario, canada), scanco medical (scanco usa, inc., southeastern, pa), and skyscan (aartselaar, belgium). each projection is the equivalent of a radiographic image. a method called 'filtered back projection' permits these projections to be reconstructed into a three-dimensional ( d) data set. a micro-ct scanner for rodents typically uses less x-ray power than a conventional clinical ct scanner, so the effective radiation exposure per scan is reduced. the highest radiation dose we have measured was rad for skin, but typical scans are much less ( rad). the radiation dose is ultimately dependent on the scan parameters (i.e., number of projections, exposure time, x-ray beam strength). ct is excellent for studying the skeletal system, certain internal organs, and fat distribution [ ] [ ] [ ] [ ] . ct can also be used for tumor studies, depending on the location of the tumor. a typical d image with -µm resolution takes about minutes to acquire; higher resolution images may take up to minutes. image reconstruction can take as little as extra minutes or up to hours depending on hardware and software options. as with mri, a contrast agent can be used to enhance the appearance of some organs. in rodents, the half-life of most clinical ct contrast agents is short, so some experimentation is required to find the optimum dose and administration regimen. a micro-ct scanner requires ∼ ft of short, usually from second to minutes. laser doppler is another type of optical imaging that uses a laser light source and the doppler effect to measure capillary blood flow (fig. ) . the equipment consists of a laser source and computer, and costs less than $ , . the footprint of the laser device is small ( ft plus computer), but its sensitivity to movement warrants placement on an antivibration table. this machine is simple to use and is highly sensitive, but it is limited to small vessels within a -mm depth. the mif owns a laser doppler imager from moor instruments, inc. (wilmington, de). another manufacturer is perimed, inc. (north royalton, oh). ultrasound imaging is a rapid, real-time in vivo technique. an ultrasound transducer broadcasts sound waves beyond the audible range into tissue. as the sound waves encounter the interfaces between various types of tissue, they are reflected. the transducer detects the reflected sound waves and uses them to construct an image (fig. ) . the depth of penetration depends on the frequency of the sound wave. the image-processing software produces an image in real time, and the various tissues within the image display different 'echogenic' properties. ultrasound machines used for rodent imaging are similar to those used clinically; however, the larger field of view offered by a clinical scanner is not optimal for imaging smaller subjects. resolution increases (and field of view decreases) as the ultrasound frequency increases; therefore, high-frequency ultrasound is ideal for rodents. ultrasound is excellent for cardiac studies and evaluation of embryonic development , . it may also be used for various organ evaluations, tumors, and guided injections. advantages of ultrasound include rapid image acquisition and ease of use; however, time is required for the operator to develop proficiency in the acquisition techniques and image interpretation. knowledge of anatomy is useful for positioning the transducers, as well as evaluating the resulting images. a well-trained operator should be able to distinguish normal and abnormal anatomy. scattering of the sound waves in the tissue often cause ultrasound images to appear 'noisy' . also, most ultrasound images are d, although some d-capable instruments are available. most ultrasound machines are built on portable carts for easy relocation. the image display is most clearly visible in low ambient light, so this should be a consideration when determining the location for ultrasound imaging. the mif owns two ultrasound machines: a siemens acuson sequoia (siemens medical solutions, malvern, pa) and a visualsonics vevo (visualsonics, inc., toronto, ontario, canada). there are many other ultrasound manufacturers, including philips (philips medical systems, andover, ma) and general electric (ge medical systems, london, ontario, canada). a commercial ultrasound imager can cost about $ , . although one can perform data analysis on the scanner, this does take time away from available scan time. an additional data-processing workstation could cost as much as $ , . the mif does not have a dedicated ultrasonographer at this time. the investigators that use our acuson ultrasound most frequently have experience in its operation and often collaborate with other investigators in need of their expertise. the mif staff is developing proficiency in ultrasonography. positron emission tomography (pet) is an excellent method to perform functional imaging in vivo [ ] [ ] [ ] . uptake of radioactive compounds can demonstrate the presence of tumor, abnormal cell function, or metabolic changes. this imaging technique can generate d data. pet imaging is highly sensitive but suffers from low spatial specificity and needs to be superimposed on an anatomical image for signal location (fig. ) . the footprint of a micro-pet system may only be ft , but it is ideal to locate the micro-pet adjacent to a micro-ct or mri because an anatomical image will be required for co-registration. this allows convenient transport of an anesthetized animal from one machine to another. micro-pet imaging presents its own set of unique considerations. the use of radioactivity dictates its own specifications. the imaging area should be in a location that allows the environment to be properly controlled and appropriate precautions taken.your institution should be able to provide you with a detailed description of the rules and regulations that govern the use of radionuclides. the imaging animals will remain radioactive for some time after imaging, so housing for these 'hot' animals must be considered. the half-lives of radionuclides in the radioactive compounds are typically short (e.g., hours), so housing vice to new users. at the mif, we train investigators to perform simple data analysis. for more complex data analysis, mif staff collaborates with the investigators. this system works well because, having trained an investigator, the technician is freed up for other duties; the investigator can often then pass that training on to new individuals in his or her group as well as other collaborators. once the user base is established, equipment needs are defined, and the facility floor plan is designed, one must consider the staffing needs. a variety of personnel are needed for a successful imaging facility. diversity of experience will enhance the resources as well as expand the knowledge base of the entire personnel staff. certain base criteria must be met for each unique position, but it is our experience that personnel experienced in imaging laboratory animals are difficult to find. many of the necessary technical procedures may be learned on site. we believe that a strong desire to learn may be more important than experience and formal education. it is important to have at least one person on the staff that is skilled in the routine technical procedures and can provide training for new staff members. as with any animal facility, husbandry staff is necessary. ideally, housing for imaging animals should be on site and accessible. the size of the animal housing facility will determine the number of necessary personnel. routine husbandry duties include daily health checks, cage changing, cage washing, room sanitation, sentinel maintenance, quality control, etc. husbandry staff is the foundation of all successful animal research programs. the qualifications of the animal care staff are dependent on how many people will be required to run the facility and in what capacity the people are expected to function. if you can have dedicated husbandry staff, their qualifications will be less stringent, and a person with aalas alat or lat experience would be sufficient. staff members who will be assisting with imaging experiments are expected to work independently, so it is ideal to have personnel with aalas latg certification or equivalent experience. if the technical staff is required to assist with (or perform) husbandry duties, they should understand that it is part of their job description so that no misunderstandings occur later. laboratory animals must be immobilized for imaging procedures. it is important for the laboratory animal technical staff to maintain a working knowledge of anesthesia and physiological monitoring for a variety of species. additionally, basic procedure skills such as venipuncture are required for the administration of imaging contrast agents and other pharmaceuticals. familiarity with aseptic techniques and rodent colony health management techniques will aid in maintaining the level of health standards for the facility. additional useful procedures include intubation of rodents, placement of rodent femoral, jugular, and carotid catheters. the computers acquiring the images and the networks they are connected to can be complicated. many of the personnel operating the imaging devices have sufficient knowledge of computer operation and software programming to repair problems as they occur; however, given the number of computers needed for imaging and personnel support, it is a good idea to have someone dedicated to their maintenance. it is important that these networks are secure and protected from any outside abuse. the mif has shared information technology personnel that maintain computer software security and upgrades. the imaging instruments require regular hardware maintenance as well as periodic repair. the best solution is to have an engineer on the staff to address these issues. it is possible to maintain a service contract with an outside company, but an internal person can aid in quicker diagnosis and repair. the mif has maintenance contract on all equipment, but the level of each maintenance plan varies from a 'parts only' plan to a full-service plan that includes yearly preventative maintenance. the mif is fortunate to have mri scientists who are knowledgable in mri mainte- resource volume , no. march animals can be a temporary situation. the availability of radioactive compounds will have the greatest impact on the success of a micro-pet program. radiolabeled glucose, oxygen, and ammonia are commercially available; however, many studies may need custom-made radioactive compounds. the ideal arrangement is an imaging suite located adjacent to the radiopharmacy that will produce the radionuclides. production of these reagents can represent the largest expense because of the need for highly specific equipment and personnel. manufacturers of micro-pet imagers include general electric and philips medical systems. micro-pet operation will also require a postdoctorate-level scientist to assemble, operate, and maintain the equipment. it is easy to generate an enormous amount of image data in a relatively short amount of time. a single image data set can range from a few kilobytes to over two gigabytes for highresolution three-dimensional data. the amount of image data generated over time can be a problem. mass storage devices must be available for data storage during analysis. if backup and archiving are required, the amount of equipment needed can be considerable. a room will need to be dedicated to computer storage and workstations for post processing images. a minimal room will have space for a rack containing data servers and network equipment. qualified information technology personnel should be able to provide additional advice. in addition to data storage, extra workstations for data processing are almost a necessity. the alternative is allowing data to be analyzed on the acquisition instruments. this reduces the need for extra computers but takes time away from the instruments. the time required for data analysis can be considerable. some consideration must be given to who will analyze data. often, users of the instrument will not have the experience or skill needed to gain the most information from the images. one solution is for image analysis to be part of the imaging service. another approach is to train the investigators to analyze their own data; this reduces the facility burden, but reduces the usefulness of the ser-nance and repair, as well as an electrical engineer who assists with mri and ct maintenance and repair. the imaging facility also needs personnel to attend to nonscientific tasks. administrators must handle daily paperwork such as budget management and supplies, as well as provide other organizational services. these people are critical, because if the facility is successful, none of the scientific personnel will have time to attend to administrative duties. the mif has shared personnel for these tasks. any facility that conducts laboratory animal research should be accessible by authorized personnel only. we cannot overemphasize this because of magnet safety, radiation safety, and animal health. traffic patterns for animals entering the facility may be necessary if the animal health level varies in different areas. the amount of personal protective equipment (ppe) needed will be defined by the types of animals in the facility, as well as the health standard. the minimum ppe for handling animals in the mif includes a disposable lab coat and gloves. because animal studies are ongoing, and we temporarily house mice, personnel must don a lab coat upon entering our facility, regardless of their animal contact. studies with nonhuman primates require a higher level of ppe that includes mucous membrane protection. standard ppe worn around the mri scanners must be nonmagnetic. an institutional animal care and use committee (iacuc) should approve every study conducted within the facility. new investigators may seek the assistance of facility personnel in writing their protocols. this gives the imaging facility personnel a chance to suggest feasible imaging modalities, preparation methods, and anesthesia regimes. the mif has a committee of experienced researchers that review experiments for feasibility, safety, and time requirement. imaging time may be scheduled on a firstcome first-served basis, or otherwise. instruments with short imaging times allow for a larger number of studies in any given period of time. when demands exceed the available scan time, one solution is to assign blocks of time for specific groups. then individuals within the group can work out their own priority for imaging. in our experience, it is easiest to have the equipment operators schedule the investigators. the operators will be most experienced with the requests of the prospective study and can schedule time accordingly. in the mif, magnet scan time during regular working hours is assigned to institutes in blocks; the remaining scan time is assigned on a first-come first-served basis. the other imaging devices require less time per scan, so it has not been necessary to assign blocks of time yet; each investigator is assigned time on a first-come first-served basis. scan time is charged back to the institute by the hour, and the dollar amount is calculated with a budget-driven formula. noninvasive imaging of rodents and other small animals is a powerful tool for biomedical research. setting up an imaging facility is a complex process that involves many decisions affecting everything from available instruments to staff composition. careful planning should help prevent operational snags. the advice of experienced people will be the most valuable asset. it is our opinion that the facility should be designed around a primary mri scanner because mr offers versatility for many applications. acquisition of other devices will be determined by the needs of the research community. growth of the facility is limited only by usage and executive decisions. staff must include a few experienced personnel, but inexperienced eager personnel can be trained to be experts. we have tried to provide an outline of some of the important considerations that went into the creation of the mif. challenges in small animal noninvasive imaging reproductive and teratologic effects of electromagnetic fields mr microscopy and high resolution small animal mri: applications in neuroscience research imaging transgenic animals in vivo imaging of gene and cell therapies electron paramagnetic resonance for small animal imaging applications molecular imaging in small animals-roles for micro-ct the use of microcomputed tomography to study microvasculature in small rodents a review of high-resolution x-ray computed tomography and other imaging modalities for small animal research high resolution x-ray computed tomography: an emerging tool for small animal cancer research advances in in vivo bioluminescence imaging of gene expression advances in ultrasound biomicroscopy noninvasive cardiovascular phenotyping in mice molecular imaging of small animals with dedicated pet tomographs high resolution spect in small animal research radio-imaging in small animals the authors would like to thank stasia anderson of laboratory of diagnostic radiology research, nih, for providing an mr image, dan schimel of the nih mouse imaging facility for providing a ct image, cecilia lo of the laboratory of developmental biology, nhlbi, nih, for providing an ultrasound image, takashimurakami and sam hwang of the dermatology branch, nci, nih, for providing a luciferase image, michael green and the imaging physics laboratory, nih, for providing a pet image, and afonso silva of the laboratory for functional and molecular imaging, ninds, nih, for providing images for this paper. we would also like to thank alan koretsky of the laboratory for functional and molecular imaging for helpful discussions. key: cord- -lsaa nx authors: pearks wilkerson, alison j; teeling, emma c; troyer, jennifer l; bar-gal, gila kahila; roelke, melody; marker, laurie; pecon-slattery, jill; o'brien, stephen j title: coronavirus outbreak in cheetahs: lessons for sars date: - - journal: current biology doi: . /j.cub. . . sha: doc_id: cord_uid: lsaa nx nan comparative insight into the prospects for a coronavirus-based epidemic [ ] . the affected animals died of feline infectious peritonitis (fip), caused by a feline coronavirus (fcov, also called fipv). the presence of a cheetah coronavirus (aju-cov, for acinonyx jubatus coronavirus) was inferred based upon the presence of fipv antibodies and the observation of coronavirus-like particles [ , ] . in domestic cats fcov occurs in two varieties: virulent fipv which causes severe fipv antibody mediated fatal disease in about - % of infected cats, and a subclinical enteric feline coronavirus (fecv) infection. within months of arrival of the two infected cheetahs to winston safari, other cheetahs in the park fell ill. retrospective serum samples tested for antibodies demonstrated that prior to all cheetah serum were negative, but within six months of the sacramento cheetahs' arrival, % of the cheetahs had seroconverted, most with titers > (supplemental figure s ). ninety percent of the cheetahs in the park developed disease symptoms including jaundice, diarrhea, weight loss, gingivitis, hepatic and renal pathology. with a mortality of % within - years, this was the most extreme outbreak of coronavirus in any species recorded. to characterize the genomic disposition of the cheetahs' aju-cov strain, pcr primers based on alignment of seven coronavirus gene segments (pol a, pol b, s, m, n, a/ b, and ′ ′utr), were used to amplify cdna from archived cheetah liver and kidney tissues collected during the winston outbreak. tissues from five different cheetahs were successfully used for amplification of bp of the pol b gene, bp of pol a, bp of n- a, and bp in the ′ ′utr region. phylogenetic analyses of aligned virus genome sequences confirm the monophyly of three previously discovered antigenic groups of coronavirus, plus the divergent sars-cov genome (figure and supplemental data). the cheetah isolates were nested within a group of domestic cat viruses using pol a, n- a and also in a polyphyletic intermix with the ′ ′utr gene segment. the phylogenetic analyses indicate a close similarity of the aju-cov and the fcov strains, suggesting the cheetah virus is closely related to, if not indistinguishable from, domestic cat isolates. the most likely scenario to explain these results is that fcov jumped from the domestic cat into the cheetah. interestingly, the two cheetahs exported from sacramento to winston had visited the u.c. davis veterinary hospital, where many domestic cat fcov isolates were originally isolated, suggesting an opportunity for cross species transmission. second, in cats, cheetahs and humans, the viruses are highly contagious, spreading rapidly through close quarters in weeks, if not days. third, despite these similarities, there is a clear difference in age sensitivity. for sars, there were virtually no childhood cases, and the mortality reaches over % in people over or with pre-existing medical complications [ ] . in cheetahs and domestic cats, mortality is the highest in neonates, infants or subadults; % of cheetah cubs succumbed to the winston outbreak [ ] . fourth, while mortality among humans with sars symptoms and house cats with fcov is low, around - %, cheetahs with aju-cov exhibited the opposite extreme, showing % morbidity and over % mortality. there are two plausible explanations for the cheetah's extreme sensitivity to aju-cov. one is that slight mutational differences transform a relatively benign coronavirus strain to a virulent 'hot' strain. among coronaviruses this scenario has been proposed, but not proven, for the transition of fecv to fipv in cats [ ]. this hypothesis is less likely for the cheetahs, as in the outbreak mentioned above lions became infected concurrently with the cheetahs, but developed no overt symptoms [ ] . also, experimental transmission of the aju-cov to three domestic kittens failed to cause disease [ ] . perhaps also relevant is that when the human sars-cov was induced in domestic cats, they developed no symptoms [ ] . cheetahs are known as the world's fastest land animal but also for their extreme genetic uniformity, a consequence of their escape from extinction some , years ago. remarkably, unrelated cheetahs accept skin grafts from nonrelatives, a characteristic of highly inbred laboratory strains of mice or rats [ ] . the most likely explanation for the high mortality in cheetahs is their genetic uniformity, particularly at immune genes like the mhc. this may have rendered the species susceptible to an emerging virulent strain that had evolved to circumvent the defenses of the first victim. if this hypothesis is correct, the greater genetic diversity of domestic cats and humans may reduce the severity of the epidemic, and also contribute to the occurrence of rare genetically determined sars-cov super-spreaders who can infect with high virulence. this explanation emphasizes the critical role of intrinsic genomic diversity among immune defense genes in any fatal epidemic. as such, the search for explicating genetic susceptibility polymorphisms that may inform prognosis, spread therapy, and prevention of emerging pathogens seems warranted, particularly to anticipate future episodes of the deadly sars coronavirus. revised u.s. surveillance case definition for severe acute respiratory syndrome (sars) and update on sars cases -united states and worldwide sars-looking back over the first days china lags in sharing sars clues officials say isolation and characterization of viruses related to the sars coronavirus from animals in southern china sars virus infection of cats and ferrets genetic basis for species vulnerability in the cheetah prevalence and implications of feline coronavirus infections of captive and freeranging cheetahs (acinonyx jubatus) key: cord- -ipfs hcb authors: mathieu, patricia a.; gómez, karina a.; coutelier, jean-paul; retegui, lilia a. title: sequence similarity and structural homologies are involved in the autoimmune response elicited by mouse hepatitis virus a date: - - journal: j autoimmun doi: . /j.jaut. . . sha: doc_id: cord_uid: ipfs hcb the features of autoantibodies (autoab) to liver fumarylacetoacetate hydrolase (fah) elicited in mice infected with mouse hepatitis virus (mhv) were studied by elisa and western-blot competition assays. all sera tested contained ab to cryptic fah epitopes according with results from western-blot tests, whereas elisa data indicated that some of these same sera did recognize native epitopes of the autoantigen (autoag). such differences were detected in individual sera from various mouse strains, and were ascribed to the fact that proteins insolubilized on solid supports expose a variety of conformational and cryptic antigenic determinants. on the other hand, whereas results from both experimental protocols showed that anti-mhv ab did not cross-react with the soluble autoag, the opposite situation did not show analogous results. thus, binding of autoab to insolubilized fah could be inhibited by mhv depending on the mouse serum or the experimental protocol used. additionally, a set of synthetic homologous peptides from mouse fah and various viral proteins was employed to analyze the ab repertoire of mhv-infected mice. results indicated that two homologous peptides were recognized by most ab: the n-terminal sequences ( – ) from fah and the nucleocapside, both sharing % of identity, and sequence – of the rna polymerase, a peptide showing % of identity with fah – . results indicated that mhv-infection triggers at least three distinct ab populations: anti-mhv, anti-fah and cross-reacting ab. this cross-reaction implies either sequential or conformational epitopes from both the viral proteins and the autoag and may differ between individuals. viruses have been implicated in the generation of autoimmune disorders over the last years [ e ]. it has been proposed that these infectious agents trigger an autoimmune humoral response by diverse mechanisms, including polyclonal b-lymphocyte activation, release of sequestered autoantigens (autoag), antigenic mimicry, modification of self-antigen, epitope spreading of the anti-viral immune response or enhancement of major histocompatibility complex molecule expression [ , , ] . mouse hepatitis viruses (mhv) are known to be lymphotropic and, depending on the viral strain and also on the mouse genetic background, they induce diverse alterations of the immunologic system [ e ]. the mhv strain a (mhv-a ) is a coronavirus that triggers various pathologies in susceptible mice, including hepatitis and thymus involution, igg a-restricted hypergammaglobulinaemia and transient demyelination [ , ] . in a previous paper we reported the presence of autoantibodies (autoab) in sera from various mouse strains after mhv-infection [ ] . the autoab were directed to a -kda protein present in mouse liver and kidney. the autoag was isolated and identified as fumarylacetoacetate hydrolase (fah), a soluble cytosolic enzyme that mediates the hydrolytic formation of fumarate and acetoacetate [ ] . furthermore, during fah purification we found that the autoab detected more weakly another liver protein, which turned to be the enzyme alcohol dehydrogenase (adh). there was no correlation between the igg titers and the presence of autoab to liver fah in cba/ht mice, suggesting that the autoab production was not related to the non-specific polyclonal activation of b-lymphocytes produced after viral infection. additionally, mice immunized with extracts of mouse liver did not develop autoab, thus discarding the release of a self-antigen as the mechanism involved in the autoimmune response elicited by mhv-infection [ ] . since molecular mimicry of viral antigens with self determinants seemed to be the mechanism involved in the mhv-induction of autoab to liver fah, we explored the putative cross-reaction between the enzyme and mhv proteins. elisa and western-blot competition assays, as well as ab reactivity with synthetic peptides from both fah and viral proteins, indicated that the autoab recognized a wide range of cryptic and conformational epitopes of the antigen and that the cross-reaction showed by the anti-mhv ab could be different between individuals. female cba/ht and balb/c mice were bred in isolators at the ludwig institute for cancer research (brussels branch) by dr g. warnier and used for experiments at the age of e weeks. their microbiological status was described previously [ ] . mice were inoculated intraperitoneally with % tissue culture infectious doses (tcid ) of mhv a , grown in nctc cells [ ] . efficiency of mhv infection was checked by testing anti-viral ab by elisa [ ] . the enzyme was prepared as previously described [ ] . briefly, livers from -day-old wistar rats were homogenized in vol (v/w) of chilled . m sucrose, mm tris/hcl buffer containing . mm cacl , u/ml of trypsin inhibitor and mm pmsf, ph . . after centrifugation at , g for min and then at , g for h, ethyl alcohol was added to the supernatant as to obtain a final concentration of % ethanol. this mixture was allowed to stand overnight at (c and then centrifuged at , g for min. the supernatant was mixed with % ethyl alcohol to yield a final alcohol concentration of %. after incubating overnight at (c the precipitated enzyme was packed by centrifugation at , g for min. the pellet was resuspended in mm phosphate buffer ph . and stirred for min. the supernatant fluid was recovered after centrifugation of the suspension at , g for min and solid ammonium sulfate was added so as to obtain a final salt concentration of %. after h at (c and centrifugation at , g for min the pellet was resuspended in mm tris/hcl ph . and dialyzed against the same buffer. an aliquot of this solution was chromatographied in a monoq hr / column (pharmacia lkb biotechnology) equilibrated with mm tris/hcl ph . . proteins were eluted at a flow rate of . ml/min by using a continuous e . m nacl gradient. effluent fractions containing the enzyme were concentrated and then applied to a sephadex g- column ( . ! cm) equilibrated with mm tris/hcl, . m nacl ph . . elution was carried out at a flow rate of ml/h with the buffer cited before and the protein concentration in the effluent fractions was determined at nm. fractions were analysed by western-blot as indicated before [ ] . the nctc adherent cell line derived from normal mouse liver was purchased from the american type culture collection. cells growing in t- bottles were inoculated with mhv a virus at a multiplicity of to tcid /cell. after an adsorption period of h at (c, ml of nctc medium with % fetal calf serum was added to each bottle and incubated at (c. several cycles of freezing and thawing were used to release the virus h after inoculation. the harvested virus was centrifuged at g for min to remove debris and the supernatant was frozen at ÿ (c for storage (mhv stock). the same procedure, but without viral inoculation, was carried out to prepare a control cellular stock (nctc stock). virus titration by endpoint method was performed by inoculating serial dilutions of the mhv stock onto cell monolayers in -multiwell. after h wells with viral cytopathic effect were counted for each dilution and titer was expressed as % tissue infectious doses (tcid ). before using in western-blot and elisa assays the virus was inactivated by incubating the mhv stock h at (c [ ] . protein concentration in both mhv and nctc stocks was determined by lowry et al. [ ] . viral and cellular stocks ( e mg) or purified fah ( e mg) were subjected to % sds-page [ ] and then transferred onto nitrocellulose sheets (amersham, buckingghamshire, uk). after reversible staining with ponceau s to check satisfactory transfer, non-specific ab-binding sites were blocked by incubating the sheets with % non-fat milk in mm tris, . m nacl, . % (v/v) tween , ph . (tbs-m-t) for h at room temperature with shaking. the strips were then incubated overnight at (c with an ab dilution in tbs-m-t. after several washings with tbs containing . % tween , bound ab were revealed with peroxidaselabeled donkey anti-mouse igg diluted : in tbs-m-t (jackson immunoresearch laboratories, inc, west grove, pa, usa) and ecl reagents (amersham, buckingghamshire, uk). the apparent molecular mass (kda) of the detected bands was determined using a wide range protein standard (bdh laboratory supplies poole bh td, uk). to perform competition experiments the diluted ab was incubated overnight at (c with the strips in the presence of different concentrations of competitors, i.e., mhv or nctc stocks, or the purified fah. the intensity of the bands was quantified by densitometric scan of the autoradiograms and the results expressed as percent of control, i.e. band intensity in the absence of competitor. elisa microplates (nunc maxi-sorb) were coated with ml of purified fah ( mg/ml) in . m nahco , ph . , or mhv stock ( mg/ml) in mm glycine, mm nacl, ph . . after overnight incubation at room temperature, the plates were washed with phosphate buffer saline (pbs) containing . % tween (pbs-t) and blocked for h at (c with . m tris, . m nacl, ph . (tms) containing % fetal calf serum (tms-fcs). the plates were then incubated h at (c with the ab diluted in tms-fcs and different concentrations of the competitors. after washing with pbs-t, the bound ab were revealed with peroxidase-labeled donkey igg anti-mouse igg (jackson immunoresearch laboratories, inc., west grove, pa) diluted : in tms-fcs. as a substrate, orthophenylene-diamine-dihydrochloride (opd, sigma chemical co, st. louis, mo, usa) with freshly added h o was used. the reaction was stopped after min by addition of m h so . the absorption was measured by elisa reader (metertech inc., taipei, taiwan) at nm. ten-week-old balb/c mice were immunized subcutaneously on day with mg of fah in ml of saline, emulsified in an equal volume of complete freund's adjuvant (difco laboratories, usa). the animals were boosted on day with the same amount of fah in incomplete freund's adjuvant (difco laboratories, usa) and bled days after the last inoculation. lalign program (http://www.ch.embnet.org/ software/lalign_form.html) using two different algorithms or matrices ( pam .mat, blosum .mat) was utilized to locate multiple matching sub-segments in two protein sequences. sequences of mhv a surface glycoprotein (e ), membrane glycoprotein (e ), nucleocapside (n), rna-direct rna polymerase (rna), hemagglutinin-esterase and kda non-structural protein were aligned with the mouse liver fah amino acid sequence. our minimum criterion for homology was the existence of at least % of sequence identity between fah and each viral protein. homologous peptides ( mers) from fah and viral proteins were synthesized according to the method of geysen et al. [ ] onto activated polyethylene pins, in a standard -well microtiter plate format (mimotopes, san diego, ca, usa). serum reactivity with synthetic peptides was determined by elisa as follows: immobilized pins were blocked for h at room temperature with pbs, ph . , containing % bsa and . % tween . after washing with pbs, ph . , for min at room temperature, pins were incubated overnight at (c in ml of each serum, diluted : in the above-described blocking buffer. pins were then washed four times with pbs, ph . , and incubated for h at room temperature with peroxidase-labeled donkey igg anti-mouse igg diluted : in pbs, ph . , containing % fcs and . % tween . after several washes, the bound ab were detected by incubating the pins for min at room temperature in ml of . mg/ml , #-azino-bis( ethylbenthiazoline- -sulfonic acid) (abts) dissolved in . m na hpo , . m citric acid, ph . , containing . % h o . the absorption was measured by elisa reader at nm. as described previously [ ] , because mouse and rat liver fah share % of sequence identity, we were able to use the purified rat liver enzyme as a substitute of the murine protein. additionally, trace amounts of rat adh co-purified with fah, accounting by about % of total proteins [ ] . thus, for the sake of simplicity hereafter we are going to refer only to ab recognizing rat liver fah as autoag. it was shown that the autoab appeared as soon as days after mhv-infection and persisted up to days post-infection [ , ] . accordingly, ab directed to the different viral proteins could be detected in sera from mhv-infected mice [ ] . under our experimental conditions, after days of mhv-infection only a band corresponding to the mw of the nucleocapside protein (n) was detected in western-blot assays. ab to proteins of mw analogous to surface glycoprotein e (also called spike glycoprotein, peplomer protein or protein s) and membrane glycoprotein e (also termed matrix glycoprotein or protein m) appeared days post-infection and continued to be secreted up to days after the virus inoculation (data not shown). in a previous work we communicated that the autoab occurring in mhv-infected mice were directed to cryptic fah epitopes, since western-blot experiments indicated that simultaneous incubation of sera with either mouse liver extracts or purified rat liver fah did not affect the autoab binding to the insolubilized autoag [ ] . in the present paper we further explored this fact adding competition elisa experiments and testing individual sera from balb/c and cba/ht mouse strains. results from fig. illustrate the different behaviors of the autoab when tested either by competition elisa or by western-blot assays. thus, data from both experimental protocols obtained with a serum from a balb/c mouse indicated that soluble rat liver fah did not alter the binding of autoab to the insolubilized enzyme, corroborating that the autoab were mainly directed to cryptic epitopes of the autoag ( fig. a and b) . however, the soluble enzyme behaved as a good competitor in elisa when a serum from a cba/ht mouse was tested, suggesting the presence of some autoab to native fah epitopes (fig. a) . in contrast, results from western-blot competition experiments performed with this same mouse serum showed that incubation with fah in solution failed to inhibit autoab binding to the insolubilized autoag (fig. b) . results similar to those presented in fig. for the balb/c serum were obtained with three balb/c and one cba/ht sera tested, whereas autoab from one balb/c and two cba/ht sera behaved as that of the cba/ht mouse presented in the same figure. mhv was insolubilized on elisa microplates or nitrocellulose sheets and allowed to react with sera from mhv-infected mice in the presence of different concentrations of soluble rat liver fah. results obtained with sera from five balb/c and three cba/ht mice showed that the enzyme did not compete for ab binding to mhv neither in elisa nor in western-blot competition assays, suggesting that the anti-mhv ab did not recognize the native epitopes exposed in the soluble autoag (see representative results in fig. ) . when the binding of the autoab to insolubilized rat liver fah was tested in the presence of mhv as competitor, three different patterns of autoab reactivity could be distinguished (fig. ) . for instance, when sera from three different cba/ht mice were tested, results indicated that the virus did not inhibit autoab binding to the insolubilized enzyme neither in elisa nor in western-blot experiments (see representative results in fig. a) . conversely, the mhv did compete for autoab binding to the ag in both procedures when a serum from a balb/c mouse was used (fig. b) . lastly, results obtained with four sera from balb/c mice indicated that the virus could behave as a poor competitor in elisa but a very good one in westernblot experiments (see representative results in fig. c ). in no case nctc stock, used as a control, produced any effect (data not shown). various decapeptides displaying at least % identity between the mouse liver fah sequence and the viral surface glycoprotein e , the nucleocapside, the membrane glycoprotein e or rna polymerase were prepared using the pepscan method (fig. ) . as indicated in section , ab binding to the insolubilized peptides was determined by elisa. . . reactivity of ab from mice immunized with rat liver fah sera from balb/c mice immunized with rat liver fah exhibited strong reactivity towards the insolubilized enzyme when tested by elisa, but the anti-fah ab did not recognize mhv proteins neither in elisa nor in western-blot assays (data not shown). results from elisa competition assays showed that soluble rat liver fah strongly inhibited the binding of anti-fah ab to the insolubilized ag, whereas only % of competition was observed in western-blot experiments (fig. ) . on the other hand, the presence of mhv did not affect the ab binding in any procedure (fig. ) . in order to investigate the fine specificity of the anti-fah ab, sera from six mice immunized with the enzyme were tested for their reactivity towards the homologous fah/mhv peptides displayed in fig. . representative results from three different experiments shown in fig. indicated that the individual responses were diverse. however, although a unique pattern of ab reactivity could not be found, anti-fah ab consistently recognized sequences e and e of the enzyme and, depending on the serum tested, bound to various mhv peptides, mainly sequences from e and rna polymerase proteins (fig. ) . as stated before, mice infected with mhv developed autoab to mouse liver and kidney fumarylacetoacetate hydrolase (fah). these ab bound to liver fah from various origins, i.e., the rat, sheep and human enzyme, and also recognized rat liver alcohol dehydrogenase (adh) [ ] . in addition, western-blot competition experiments suggested that the autoab elicited in mhv-infected mice were directed to cryptic epitopes of the ag [ ] . since hypergammaglobulinaemia or tissue damage as mechanisms triggering the mhv-elicited autoimmune process appeared less likely than molecular mimicry, we undertook competition experiments. because individual sera were tested, it could be found that results using elisa and western-blot competition assays were sometimes different, and that such differences did not depend on the mouse strain utilized. thus, whereas western-blot assays indicated that autoab were directed to cryptic fah epitopes in all sera tested, elisa results indicated that some of these same sera did recognize native epitopes of the autoag (fig. ) . these results could be explained taking into consideration that insolubilized proteins expose cryptic epitopes while still retaining some conformational features [ , ] . since proteins submitted to sds-page and then transferred to nitrocellulose should be more denatured than those insolubilized on plastic surface, it is possible that the autoab detected both conformational and cryptic epitopes in elisa and only cryptic determinants in western-blot assays. so, sera showing lack of competition by the soluble autoag in both protocols should have autoab directed mostly to cryptic epitopes of the fah, whereas those displaying dissimilar reactivity may have a mixture of autoab directed to both hidden and conformational antigenic determinants of the enzyme. on the other hand, results from elisa as well as from western-blot competition assays showed that ab to mhv did not cross-react with the soluble autoag (fig. ) . however, results from the opposite situation were not the same. in fact, when mhv stock was used as competitor for binding of autoab to insolubilized fah different patterns of reactivity were obtained. elisa as well as western-blot competition tests showed lack of effect of mhv in solution or, depending on the mouse serum used, both procedures did show an inhibitory activity of the virus (fig. ) . furthermore, some sera displayed different behavior in both procedures, mhv being inhibitory in western-blot experiments but not in elisa assays (fig. ) . optical density ( nm) it is noteworthy to mention than anti-mhv ab elicited in all mice tested were directed to native epitopes of the viral proteins, because mhv strongly inhibited ab binding to the insolubilized virus in elisa as well as in western-blot competition experiments (data not shown). molecular mimicry between viral proteins and self-ag is one of the most probable mechanisms that explain autoimmune responses induced by viral infections [ , ] . murine adenovirus, semliki forest virus, lactate dehydrogenase-elevating virus, herpes simplex virus type- , hepatitis b virus, encephalomyocarditis virus, theiler's murine encephalomyelitis virus, coxsackievirus and cytomegalovirus have been found to mimic physiologically important host proteins [ ] . thus, in order to analyze the ab repertoire of mhv-infected mice a set of mers homologous peptides corresponding to the sequence of mouse fah and viral proteins e , nucleocapside, e and rna polymerase was employed (fig. ) . most ab recognized the n-terminal sequence ( e ) of both fah and the nucleocapside, two peptides that share % of identity. in addition, ab from and days post-infection mice bound to the sequence e of the rna polymerase, a peptide showing % of identity with fah e . thus, recognition of the fah sequence e by ab elicited by mhv infection could be at least partially responsible of the autoimmune response described herein. this observation should also explain the lack of reactivity of anti-fah ab with viral stock. in fact, sera from fahimmunized mice did not react with the n-terminal sequence e of the enzyme, and only showed binding to sequence e or e (figs. and ) . the crystal structure of fah showed that the protein folds into a -residue n-terminal domain of unknown function and a -residue c-terminal domain defined by a novel b-sandwich roll structure [ ] . in that structure, sequence e appears as a coil exposed to the solvent, suggesting that autoab could recognize this structure in the enzyme either insolubilized or in solution. we cannot discard that sequences other than fah e were recognized by the autoab elicited in mhvinfected mice because we did not test peptides spanning the entire fah sequence. moreover, it was reported that similarity of sequences is sometimes not sufficient to mimic epitopes and that structural considerations contribute significantly to the underlying mechanisms of molecular mimicry [ ] . accordingly, results presented in this work indicate that mhv-infection triggers a cross-reaction of either sequential or conformational epitopes from both the viral proteins and the autoag. data also suggest that mhv-infected mice could develop at least three different ab populations: ab directed either to viral proteins or to the autoag, and cross-reacting ab. the observation that immune responses undergo determinant spreading is a major finding shaping current theories regarding autoimmunity and molecular mimicry [ ] . therefore, immune diversification originated from only a single autoreactive determinant, i.e., a common sequence, could probably provide a pathway for the generation of the multifaceted autoimmune response described in this paper. the absence of autoimmune disease in our model remains to be explained. in fact, mice infected with mhv-a develop acute hepatitis but liver regeneration may take place as early as e days after infection [ , ] . additionally, other mhv-a effects, such as thymus involution, the enlargement of the spleen and production of great amounts of igg a, are also transient [ , , ] . thus, in spite of the presence of the autoab reported in this work, no signs of autoimmune hepatitis were evident. our findings remind the response to allo-hppd (allotype of the hepatocyte enzyme -hydroxy-phenyl-pyruvate dioxigenase, alias f liver protein) that has been long studied [ e ]. the mouse f antigen is expressed mainly in the liver ( ÿ m) but leaks into body fluids at a concentration of z ÿ m. it has been proposed that this protein concentration is just sufficient to induce complete tolerance of t cells, whereas b cells are not tolerized [ , ] . although the concentration of liver fah in serum is not known, its similarity in mw and localization with hppd [ , , ] enabled us to speculate that a t cell effect similar to that proposed by n.a. mitchinson and colleagues [ e ] may take place in our model. reactivity of synthetic peptides with sera from mice immunized with rat liver fah. ab binding to the homologous decapeptides showed in fig. was determined by elisa as indicated in section . results are expressed as specific optical density values for ab binding to sequences corresponding to rat liver fah (black bars) or the indicated different mhv proteins (white bars). peptides synthesized on pins were numbered according to fig. . results presented in a, b and c were obtained with individual sera from three different balb/c mice immunized with rat liver fah as indicated in section . the viraleautoimmunity relationship evidence for mimicry by viral antigens in animal models of autoimmune disease including myocarditis mimicking the way to autoimmunity: an evolving theory of sequence and structural homology in vivo polyclonal b-lymphocyte activation elicited by murine viruses the role of infection in the pathogenesis of autoimmune disease viral diseases of the digestive system role of virus receptor-bearing endothelial cells of the bloodebrain barrier in preventing the spread of mouse hepatitis virus-a into the central nervous system morphological analysis of mouse hepatitis virus a -induced pathology with regard to viral receptor expression polyclonal b lymphocyte activation induced by mouse hepatitis virus a infection identification of two liver proteins recognized by autoantibodies elicited in mice infected with mouse hepatitis virus a igg a restriction of murine antibodies elicited by viral infections the coronavirus protein measurements with the folin phenol reagent cleavage of structural proteins during the assembly of the head of bacteriophage t strategies for epitope analysis using peptide synthesis detection of mouse hepatitis virus infection by assay of anti-liver autoantibodies autoantibodies to cryptic epitopes elicited by infection with lactate dehydrogenase-elevating virus binding properties of antibodies to prothrombin and b -glycoprotein i (b -gpi) assayed by elisa and dot blot crystal structure and mechanism of a carbonecarbon bond hydrolase epitope mimics and determinant spreading: pathways to autoimmunity thymus involution induced by mouse hepatitis virus a in balb/c mice self-and allo-specific suppressor t cells evoked by intravenous injection of f protein self-reactive t cell hybridomas and tolerance regulation by non-major histocompatibility complex genes of the allo- -hydroxy-phenylpyruvate dioxygenase (f liver protein) response selection of anti-f protein b-cell repertoires in normal mice the authors are indebted to drs. pierre l. masson (icp, brussels, belgium) and leonor p. roguin (iqui-fib, buenos aires, argentina) for helpful discussions and critical revision of the manuscript. this work was supported by grants from conicet, foncyt and universidad de buenos aires, argentina, and fonds national de la recherche scientifique (fnrs), fonds key: cord- - heg iql authors: armstrong, john; mccrae, malcolm; colman, alan title: expression of coronavirus e and rotavirus vp membrane proteins from synthetic rna date: - - journal: j cell biochem doi: . /jcb. sha: doc_id: cord_uid: heg iql some viruses acquire their envelopes by budding through internal membranes of their host cell. we have expressed the cloned cdna for glycoproteins from two such viruses, the e protein of coronavirus, which buds in the golgi region, and vp protein of rotavirus, which assembles in the endoplasmic reticulum. messenger rna was prepared from both cdnas by using sp polymerase and either translated in vitro or injected into cultured cv cells or xenopus oocytes. in cv cells, the el protein was localised to the golgi region and vp protein to the endoplasmic reticulum. in xenopus oocytes, the e protein acquired post‐translational modifications indistinguishable from the sialylated, o‐linked sugars found on viral protein, while the vp protein acquired endoglycosidase‐h‐sensitive n‐linked sugars, consistent with their localisation to the golgi complex and endoplasmic reticulum, respectively. thus the two proteins provide models with which to study targeting to each of these intracellular compartments. when the rnas were expressed in matured, meiotic oocytes, the vp protein was modified as before, but the e protein was processed to a much lesser extent than in interphase oocytes, consistent with a cessation of vesicular transport during cell division. in the eukaryotic cell, the rough endoplasmic reticulum is the site of synthesis for both secretory proteins and integral proteins of the plasma membrane. to reach the cell surface, both types of protein must traverse the golgi complex. during this transport process, a series of post-translational modifications may occur. since the modifying enzymes are localised along the secretory pathway, the endoplasmic reticulum and golgi complex constitute a series of membrane-limited compartments, each apparently with a distinct complement of proteins [reviewed in . how are these proteins confined to their appropriate destinations, rather than migrating onward to the plasma membrane? we are investigating two viral model proteins, one for the endoplasmic reticulum and one for the golgi complex, with a view to determining the features of each molecule responsible for its correct localisation. rotaviruses are a class of animal viruses which characteristically assemble at the endoplasmic reticulum [ ] . although assembly appears to be a budding process, the matured virion surprisingly lacks a visible envelope . the bovine uk strain encodes two glycoproteins: vp , which forms part of the virion, and vpio, which does not [ , ] . the latter is an integral membrane glycoprotein [ , ] bearing "immature" n-linked sugars which imply a failure to leave the endoplasmic reticulum [ w . by contrast, with coronaviruses the envelope is acquired by budding initially at smooth membranes close to the golgi complex but continuous with the endoplasmic reticulum; as infection progresses budding may also occur in the golgi cisternae or the rough endoplasmic reticulum [ , . the strain mhv-a bears two glycoproteins, the smaller of which, e l , is restricted within the cell and does not appear to reach the plasma membrane except as part of a budded virion , . thus we have adopted the proteins vplo and e l as potential models for the class of membrane proteins which localise within compartments of the secretory pathway. previously we have reported the cloning and sequence analysis of both proteins [ , ] . here we show that both cdnas can be expressed via artificial mrna prepared using sp polymerase [ , . the mrna may be translated either in vitro or after injection into xenopus oocytes or cultured cvi cells. expressed in this way, vplo and e l prove to be valid model proteins for the endoplasmic reticulum and golgi complex, respectively. in addition, we have exploited the fact that xenopus oocytes can be matured into a meiotic state [ , to investigate the transport of the two proteins to their destinations during cell division. all restrictions, ligations, and transformations were carried out according to standard methods . full-length cdna for bovine rotavirus vplo (assembled and kindly provided by h. baybutt) was excised with the enzymes ahaiu and sali, to give a fragment corresponding to nucleotides - [in figure of . this was inserted into the bglii site of the transcription vector psp t [ . e l cdna [ , was excised with aha and fom, representing nucleotides - [in , plus the following bases of the adjacent nucleocapsid gene [ ] and inserted into the bglii site of psp t . both plasmids were linearised with ecori and rna transcribed with sp polymerase in the presence of . mm m g ( ')ppp( ')g exactly as described [ ] . since the vector psp t contains a poly-a tract between the bglii and ecori sites, this method results in the synthesis of capped, polyadenylated mrna in a single reaction. translation in vitro in reticulocyte lysates, injection of xenopus oocytes, immunoprecipitation of proteins, microinjection of cv cells, and immunofluorescence analysis were all performed as before [ ] . for analysis of vplo protein, rabbit antiserum to a fusion protein of bacterial -galactosidase and vpio, whose prepara-tion will be described in detail elsewhere, was used. to detect el, we used a rabbit serum to e l purified by detergent extraction of virus [ ] , kindly provided by s. tooze, embl, heidelberg, who also provided stocks of coronavirus mhv-a and its host, sac-cells. radiolabelled virus was produced by infection of cells at a ratio of pfukell, incubation for hr in methionine-free medium to which was added pci/ml s-methionine (amersham) , and collection of the culture supernatant. endoglycosidase h digestions were carried out as before [ . xenupus oocytes were matured into second meiotic metaphase by incubation in p g / d progesterone as described [ . previously we showed that rna prepared with sp polymerase could be translated in cultured cells, provided it has both a ' cap structure, and a ' poly-a tract added using poly-a polymerase [ ] . e l rna prepared in this way was found to be translated in both cv cells and xenopus oocytes (not shown). we have further simplified this method by using a vector which includes a sequence of a's in the transcribed region [ , eliminating the requirement for a second reaction. rnas prepared by this method for vplo and e l were translated efficiencly in reticulocyte lysates, xenopus oocytes, and cultured cvl cells (figs. , ). rough endoplasmic reticulum; these structures may be cleaved by endoglycosidase h [ ] . vplo protein which had been immunoprecipitated from injected oxcytes was found to be completely sensitive to endoglycosidase h, yielding a species which approximately comigrated with the unprocessed form produced in the reticulocyte lysate (fig. , lanes a-d) . thus the vplo protein is likely to be localised within the endoplasmic reticulum of the oocyte. e l protein was synthesized in oocytes as a spectrum of forms, the most mobile of which comigrated with the unmodified protein from the reticulocyte lysate (fig. , lanes e,f). the oocyte proteins were very similar in both mobilities and relative abundance to the species of e l found in virus particles (fig. , lane ) . these have been shown to contain -linked sugars as their only posttranslational modification [ , ] . since at least the later stages of -linked glycosylation are thought to occur in the golgi complex , most or all of the el protein would appear to have reached this organelle in the oocyte. with both antibodies, no significant labeled proteins were precipitated from oocytes in the absence of the appropriate mrna. sp rnas for vplo and el were microinjected into cvl monkey kidney cells, and the resulting proteins were detected by immunofluorescence. vplo proteins was found in an elaborate pattern around the nucleus and throughout the cytoplasm, characteristic of the rough endoplasmic reticulum (fig. a) . by changing the plane of focus, it became clear that all of the nuclear envelope was labeled (not shown). this was expected as the outer nuclear membrane is continuous with, and usually considered as part of, the endoplasmic reticulum. in contrast, e l protein showed a much more localised fluorescence pattern (fig. b) . labeling was concentrated in a perinuclear area corresponding to the golgi region and coincident with the intracellular pattern shown by fluorescent wheat germ agluttinin, a golgi marker (not shown). the pattern also closely resembled the distribution of e l protein in virally infected cells , . if injected cells were not permeabilised with detergent, no labeling of the cell surface was detected with either protein (not shown). incubation of xenopus oocytes in progesterone causes them to mature from their initial stage, first meiotic prophase, to second meiotic metaphase [ ] . in this state, the golgi apparatus is broken down and protein secretion blocked [ . we investigated the synthesis of vplo and e l in such matured oocytes. vplo was electrophoretically indistinguishable from the form produced in nonmatured oocytes (fig. , lanes a,b) . e l , in contrast, showed a striking reduction in the extent to which it was processed in comparison to nonmatured oocytes (fig. , lanes c,d) , implying that most of the protein is denied access to the modifying enzymes of the golgi complex under these conditions. transcription of rna with sp polymerase has been used as a route for expression of cloned cdnas either in vitro or in the xenopus oocyte [ . if the rna incorporates a ' cap structure and a ' poiy-a tract, it may also be expressed in a b c d fig. . expression of vplo (a,b) and e l (c,d) in normal (a,c) or progesterone-matured (b,d) oocytes. proteins were analysed as for figure , and the fluorograph was exposed for wk. cultured cells [ . by using the vector psp t [ , such rna can be prepared in a single reaction. we assume that the presence of an encoded poly-a sequence in the vector is the critical factor in allowing expression of the rna in cultured cells ; however, the transcribed region also contains ' and ' untranslated regions from a globin cdna, and a sequence of c bases at the extreme ' end [ , and any of these elements may contribute to the stability of the mrna or the efficiency with which it is translated. whichever feature is important, the system provides a quick and versatile approach to the expression of cloned dnas. its principal limitation, at present, is the lack of a technique for the efficient introduction of rna into large populations of cultured cells. it appears that both of the proteins we have studied, vplo and el, are localised in a similar fashion whether they are expressed in isolation in cell types as diverse as oocytes and cvl cells, or in the course of viral infection. thus each is likely to be a legitimate model with which to study targeting of proteins to its respective intracellular destination. in the case of vpio, this is the rough endoplasmic reticulum, as judged by fluorescence microscopy and sensitivity of its n-linked oligosaccharides to endoglycosidase h (figs. , ) . the corresponding protein of rotavirus sall , termed ncvps, bears oligosaccharides of the form man glcnac [lo] . in contrast, the principal location of the e l protein appears to be the golgi complex. as the bulk of the protein made in oocytes has been modified to give forms of similar mobilities to the viral protein (fig. , lanes f,g; see also [ ] ), it has presumably acquired -linked oligosaccharides, an indication of having reached the golgi complex [ ] . most of the oligosaccharides on viral el contain terminal sialic acid [ ] , whose presence is detectable by changes in electrophoretic mobility of the glycoprotein after neuraminidase digestion. however, we have repeatedly failed to observe any effect on mobility of oocyte el by treatment with neuroaminidases from various sources and under various conditions (not shown). the explanation for this is not clear; perhaps the oocyte adds exotic sialic acids which are not susceptible to cleavage. immunofluorescence of cultured cells expressing e l clearly showed a very localised perinuclear distribution (fig. b ) characteristic of the golgi region and similar to the pattern observed during viral infection [ ] . however, not all of the viral protein is restricted to the flattened cisternae of the golgi complex; some at least is found in smooth membranes which are in the same region of the cell but are in fact continuous with the rough endoplasmic reticulum [ , . these membranes are reminiscent of the transitional elements of specialised secretory cells [ ] and are the earliest site of budding during viral infection [ ] . at the resolution of light microscopy it is impossible to say whether the pattern in figure b includes this compartment. it will be of interest to resolve this point by immunoelectron microscopy, which should also allow us to determine how many of the golgi cisternae are labelled. nevertheless, it is probably safe to conclude that the behaviour of the e l protein is largely responsible for determining the intracellular budding site of coronavirus. the localisation of vplo might argue for a similar function in rotavirus morphogenesis. however, this protein's precise role in viral infection remains enigmatic. the other rotavirus glycoprotein, vp , also appears to be restricted to the endoplasmic reticulum when it is expressed in the absence of other viral proteins [ . thus both proteins may be involved in determining the viral assembly site, but the subsequent fate of vplo is unclear, since it is not detectable in purified virions [ ] . perhaps the simplest hypothesis is that the newly budded virus has a lipid envelope including vplo and vp , which is somehow shed at a later stage to leave vp connected directly to components of the capsid [ ] . an aspect of the biogenesis of the endoplasmic reticulum and golgi complex which has recently attracted attention is their fate during cell division. in mammalian cells, both organelles are thought to break up into vesicles which then partition randomly between the daughter cells and fuse together; concomitant with this disruption is a cessation of traffic of secretory and plasma mebmrane proteins to and through the golgi complex [reviewed in . the same phenomena appear to occur in the xenopus oocyte, which has the experimental advantage that its cell cycle state may be manipulated with hormones [mi. the results presented in figure are entirely consistent with this model; vplo is synthesized and processed normally in meiotic oocytes, but e l is processed to a far lesser extent than in interphase oocytes, and thus has probably failed to traverse what is assumed to be the first vesicle-mediated step of membrane traffic, from the endoplasmic reticulum to the golgi complex. it might be of interest to study the vesicles in which e l becomes trapped under these conditions. what features of each protein might be involved in determining its intracellular destination, and what are the mechanisms involved in each case? the two proteins share a superficial topological similarity, in having a very short domain on the lumenal side of the membrane which is n-terminal but does not arise from cleavage of a signal sequence; e has the additional unusual feature of hydrophobic regions large enough to span the membrane three times [ , , , , ] . two mechanisms have been proposed for retention of proteins in the endoplasmic reticulum. one is that a species variously known as bip or grp attaches to improperly folded or assembled molecules and prevents their exit to the golgi complex [ - . the second is that all of the membrane proteins of the endoplasmic reticulum form a continuous interacting network and are unable to diffuse in the lipid bilayer , . in respect of either model, we have never observed any host species which coprecipitates from oocytes with vplo, although admittedly our labelling schedule is perhaps not optimal for detection. (note that the unidentified band in figure , lanes b-d is made only in response to vplo mrna.) neither have we observed such species with our golgi model protein, e l , whose sorting mechanism is even more obscure. however, with the availability of cloned cdna and a reliable expression system, it is now possible to dissect each molecule and identify the characteristics involved in its correct targeting within the cell. molecular cloning: a laboratory manual molecular biology and pathogenesis of coronaviruses this work was supported by grants from the cancer research campaign (to a. colman) and the wellcome trust (to a. colman and m. mccrae). key: cord- - bhnlsgy authors: trifilo, matthew j.; lane, thomas e. title: the cc chemokine ligand regulates cd c(+)cd b(+)cd α(−) dendritic cell maturation and activation following viral infection of the central nervous system: implications for a role in t cell activation date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: bhnlsgy the role of cc chemokine ligand (ccl ) in activation of dendritic cells (dcs) following mouse hepatitis virus (mhv) infection of the central nervous system (cns) was examined. the results indicate that ccl participates in an effective host response to mhv infection by contributing to cd c(+)cd b(+)cd α(−) dc maturation, activation, and migration to cervical lymph nodes (cln). diminished cd α(−) dc activation correlated with reduced ifn-γ expression by virus-specific t cells accompanied by increased il- production suggesting that ccl contributes to an effective host response to viral infection by enhancing the t cell activation potential of dc. the cc chemokine ligand (ccl -macrophage inflammatory protein- a) is capable of activating monocytes and lymphocytes and serves an important role in the initial recruitment of these cells to tissues following microbial infection (cook et al., ; domachowske et al., ) . in support of the importance for ccl in imparting functional signals to t cells are data from our laboratory demonstrating that instillation of mouse hepatitis virus (mhv) into the brains of ccl À/À mice results in an inability to clear virus from the central nervous system (cns) (trifilo et al., ) . mhv-infected ccl À/À mice exhibited a significant reduction in the numbers of infiltrating virus-specific cd + t cells present within the brain indicating that trafficking was impaired. moreover, the ability to produce ifn-g as well as the cytolytic activity of virus-specific cd + t cells was dramatically reduced in the absence of ccl signaling. taken together, these data indicate that ccl signaling significantly enhances the differentiation of primed cd + t cells into effector cells that allows their release from secondary lymphoid organs into circulation and effective migration to the cns. the present study was undertaken to characterize potential mechanisms by which ccl signaling imparts effector function to antigen-specific t cells following mhv infection of the cns. to further understand the relationship between ccl signaling and t cell activation, ccl +/+ and ccl À/À mice were infected with mhv and the presence and activation state of dc-like cells within the brain and draining cervical lymph nodes (cln) determined. our results delineate a ccl -dependent pathway of t cell activation that involves the maturation and activation of a subpopulation of (dendritic cells) dcs (cd c + cd b + cd a À ) within the cns as well as influencing the accumulation of these cells within the cln following mhv infection of the cns. characterization of cd c + cells within the cns following mhv infection of ccl +/+ and ccl À/À mice to characterize the populations of cells present within the cns of mhv-infected mice, cells were harvested at days , , and post-infection (p.i.) and immunophenotyped by flow cytometry. we chose to focus our attention on markers that are associated with professional antigen presenting cells such as dc as recent studies have indicated dc-like cells can be detected within the brains under inflammatory conditions (fisher and reichmann, ; fischer et al., ) . furthermore, we have previously determined that ccl mrna expression is detected within the cns early (b days) and therefore may participate in the appearance of cd c + cells within the brain following mhv infection (trifilo et al., ) . therefore, we sought to characterize the populations of cd c + cells within the brain following mhv infection of ccl +/+ and ccl À/À mice. results in fig. a indicate the frequency of cd c + cells present within the cns of narve ccl +/+ and ccl À/À mice is b %. however, within days following intracranial infection with mhv, there is a marked increase in the frequency of cd c + cells within the brains of both strains of mice (fig. a) . analysis of cd b expression revealed that approximately % of cd c + cells in both ccl +/+ and ccl À/À mice were also cd b + (fig. a) . cd c + cd b + cells isolated from the cns of either ccl +/+ or ccl À/À mice expressed little to no cd a or dec suggesting a phenotype similar to myeloid derived dc (cd c + , cd b + , cd a À , dec À ) ( fig. b ) (anjuere et al., ; henri et al., ) . increased cd a and dec expression on cd c + cd b À cells indicated that the majority of the remaining cd c + cells present within the brain were similar phenotypically to lymphoid derived dc (cd c + , cd b À , cd a + ) (fig. b) (anjuere et al., ; henri et al., ) . although the fig. . analysis of dendritic cells (dcs) within the brain following mhv infection. (a) cells were isolated from the brains of uninfected (narve) or infected (day p.i.) ccl +/+ and ccl À/À mice and stained for cd c + and cd b + expression. gated populations represent cd c + cd b + (upper-right quadrant) or cd c + cd b À (lower-right quadrant) and numbers indicate frequencies of gated cells within the isolated population. (b) cd a and dec expression on cd c + cells. cd c + cd b + cells from either mhv-infected ccl +/+ or ccl À/À mice at day p.i. did not express detectable levels of either cd a or dec while expression of both cd a and dec was readily detectable on cd c + cd b À cells present within the brains of both populations of mice at day p.i. (c) total numbers of cd a + and cd a À dcs within the brains of mhv or sham-infected ccl +/+ and ccl À/À mice at days and p.i. data presented represent an average cell number derived from two separate experiments with a minimum of mice analyzed per experimental group. (d) cd a À cells isolated from brains of ccl +/+ or ccl À/À mice at day p.i. were gated upon and cd , cd , and cd expression was determined by flow cytometry. the mean fluorescence intensity (mfi) for cells obtained from either ccl +/+ or ccl À/À mice is indicated. flow data shown in panels a, b, and d are representative of two separate experiments with a total of mice for each experimental condition. remaining cd c + cd b À cd a À population was not further characterized, it is likely that these cells may be plasmacytoid in origin (cd c + cd b À cd a À b + ) or consist of a yet to be defined population of dc. comparison of the total numbers of cd a + cells within the brains of mhv-infected ccl +/+ and ccl À/À mice revealed no dramatic differences between the two populations of mice at either or days p.i. (fig. c) . in contrast, numbers of cd a À cells were increased by approximately % within the brains of mhv-infected ccl +/+ mice as compared to ccl À/À mice at day p.i. however, by day p.i., there were increased numbers of cd a À cells present in the brains of ccl À/À mice when compared to ccl +/+ mice. in attempt to better evaluate the activation state of cd c + cells within the brains of mhv-infected mice, we next determined the expression levels of co-stimulatory molecules cd (b- . ), cd (b- . ), and cd on cd a À cells within the cns of ccl +/+ and ccl À/À mice. we chose to focus on this subpopulation of dcs in more detail as this clearly was the predominant dc population within the brains of infected mice suggesting a potentially more important role in defense. analysis of cd a À cells isolated from the brains of ccl +/+ mice at day p.i. revealed these cells expressed detectable levels cd , cd , and cd as determined by measuring the mean fluorescence intensity (mfi) (fig. d ). although mhv infection of ccl À/À mice also resulted in enhanced expression of cd on the surface of cd a À cells, the mfi for both cd and cd was dramatically reduced as compared to cd a À cells within the brain of ccl +/+ mice at day p.i. (fig. d ). together, these results indicate that although ccl signaling is not required for the appearance of dc-like cells within the brain, expression of the co-stimulatory molecules cd and cd is muted in the absence of ccl signaling. characterization of cd c + cells within the cln of mhv-infected ccl +/+ and ccl À/À mice following mhv infection of the cns, virus-specific t cells are present within the cln suggesting that the bulk of virus-specific t cells are generated in the periphery (marten et al., ) . therefore, the accumulation of cd c + cells within the cln of infected ccl +/+ and ccl À/À mice was determined. before infection, cd c + cells expressing a myeloid dc phenotype (cd c + cd b + ) and lymphoid dc phenotype (cd c + cd b À ) were present within the cln of both ccl +/+ and ccl À/À mice at an approximate : ratio, respectively ( fig. a) . however, within days of mhv infection of the cns, the frequency of cd c +-cd b + dcs, but not cd c + cd b À dcs, dramatically increased within the cln of ccl +/+ mice ( fig. a) . further analysis revealed that the cd c + cd b + cell population was cd a À dec À while the cd c +-cd b À population was cd a + dec + (fig. b) . although the total number of cd a À cells increased within the cln of ccl À/À mice, there was an approximate -fold reduction in total numbers of cd a À cells as compared to ccl +/+ mice at days and p.i. (fig. c ). similar to the brain, no difference in numbers of cd a + cells within the cln was detected at either days or p.i. (fig. c ). examination of co-stimulatory molecule expression on cd a À dcs present within the cln of ccl +/+ mice correlated with the increased expression of co-stimulatory molecules cd , cd , cd as well as increased mhc i and ii expression when compared to sham-infected mice (figs. d and e) . these data suggest that this population of activated cd a À cells within the cln is able to present antigen and induce t cell differentiation within the cln following mhv infection of the cns. although cd a À dcs isolated from the cln of ccl À/À mice expressed similar levels of cd and cd as compared to ccl +/+ (determined by mfi), expression of cd as well as mhc i and ii were reduced as compared to cd a À cells isolated from mhv-infected ccl +/+ mice (figs. d and e). one mechanism by which dcs influence the t cell response to infection is through the secretion of cytokines that can subsequently polarize the immune response towards either a th or th phenotype depending on the antigenic challenge. to determine if cd a À dcs present within the cln of infected ccl +/+ and ccl À/À mice were capable of secreting either chemokines or cytokines following mhv infection of the cns, these cells were isolated and production determined by elisa. ccl was readily detectable from cd a À dcs obtained from ccl +/+ mice while ccl was not detected in supernatants collected from ccl À/À cd a À dcs (fig. ) . the ccl +/+ cd a À dc population secreted il- p with low-level production of il- ( fig. ) . in contrast, the cd c + cd b + cd a À cells isolated from ccl À/À mice secreted approximately -fold less il- p while il- secretion was increased by -fold as compared to cells from ccl +/+ mice (fig. ) . altered cytokine production in ccl À/À t cells we next evaluated the ability of t cells obtained from the cln of either mhv-infected ccl +/+ or ccl À/À mice to synthesize cytokines following exposure to defined viral antigens. t cells were isolated from the cln of ccl +/+ and ccl À/À mice at days and p.i. following intracranial infection with mhv and stimulated with peptides corresponding to either the immunodominant cd epitope present within the matrix (m) glycoprotein at residues - (m - ) or the immunodominant cd epitope in the surface (s) glycoprotein spanning residues - (s - ) and cytokine production by t cells determined by intracellular cytokine staining (castro and perlman, ; xue et al., ) . the results presented in table indicate similar frequencies of cd + and cd + t cells from ccl +/+ mice produced ifn-g at days and p.i. both cd + and cd + ccl +/+ t cells also secreted il- following specific peptide exposure at day , although expression was limited to the acute stage of mhv infection as the frequency of il- producing cells was reduced at day p.i. (table ). in contrast, the frequency of cd + and cd + t cells isolated from ccl À/À mice secreting ifn-g following peptide stimulation was dramatically reduced. expression of il- by ccl À/À t cells was comparable with ccl +/+ mice at day p.i. however, by day p.i., the frequency of ccl À/À t cells expressing il- remained elevated as compared to ccl +/+ t cells (table ). in addition, only limited frequencies of ccl +/+ cd + and cd + t cells produced il- following mhv infection whereas ccl À/À cd + and cd + t cells displayed an overall increase in the frequency of il- ( table ) . the major findings of this study are (i) mhv infection of the cns results in the appearance of two distinct populations of cd c + cells each expressing markers characteristic of lymphoid (cd c + cd b À cd a + dec + ) and myeloid dendritic cells (cd c + cd b + cd a À dec À ), (ii) the accumulation of cd a À dcs within the draining cln is reduced in the absence of ccl signaling, (iii) expression of co-stimulatory molecules such as cd by cd a À dcs within either the brain and cln of mhv-infected ccl À/À mice is diminished suggesting that ccl signaling enhances expression of these molecules, and (iv) absence of ccl signaling results in the re-direction of the t cell response to viral antigens as determined by cytokine production. these data support and extend recent studies from our laboratory demonstrating an important role for ccl in generating effector anti-viral t cells capable of migrating to the brain in response to viral infection (trifilo et al., ) . i.) ccl +/+ and ccl À/À mice and stained for cd c + and cd b + expression. gated populations represent cd c + cd b + (upper-right quadrant) or cd c + cd b À (lower-right quadrant) and numbers indicate frequencies of gated cells within the isolated population. (b) cd a and dec expression on cd c + cells. cd c + cd b + cells from either ccl +/+ or ccl À/À at day p.i. did not express detectable levels of either cd a or dec while expression of both cd a and dec was readily detectable on cd c + cd b À cells present within the brains of both ccl +/+ and ccl À/À mice at day p.i. (c) total numbers of cd a + and cd a À dcs within the clns of mhv or sham-infected ccl +/+ and ccl À/À mice at days and p.i. data presented represent an average cell number derived from two separate experiments with a minimum of mice analyzed per experimental group. (d) cd a À cells obtained from mhv-infected (day p.i.) or sham mice were gated and the level of cd , cd , and cd expression was determined by flow cytometry. the mfi for cells obtained from either ccl +/+ or ccl À/À mice is indicated. (e) cd a À cells obtained from mhv-infected or sham mice were evaluated for expression of mhc i and ii. the mfi for staining of either mhc i or ii is indicated in the histogram. flow data shown in panels a, b, d, and e are representative of two separate experiments with a total of mice for each experimental condition. the activation of dc and their mobilization to secondary lymphoid organs is thought to be a key step in the initiation of an adaptive immune response (banchereau and steinman, ). recent studies have indicated that following infection of the cns with toxoplasma gondii, cd c + cells are present within the brain and these cells were able to stimulate the proliferation of narve t cells (fisher and reichmann, ) . similarly, our results also indicate an increase in cd c + cells within the cns following viral infection, suggesting that these cells are likely critical for successful t cell priming following migration to draining lymph nodes. whether these cells are present within the cns by differentiation of local antigen presenting cells, or through migration of immature dc has not been determined and is currently under investigation. regardless, our data imply that ccl expression and signaling contributes to the migration of cd a À cd c + cells to secondary lymphoid tissue where they participate in priming of t cells. in support of this, we have shown that ccl is important in arming these cells with the capability to optimally stimulate antigen-specific t cells with the ability to fully differentiate into effector cells (trifilo et al., ) . the data presented in this study support and extend these observations and indicate that these results may be the result of a combination of diminished expression of both mhc class i and ii, reduced expression of cd , as well as a shift in cytokine production by cd a À cells. indeed, cd l:cd mediated interactions between t cells and apc can enhance il- production by dc and blockade of this interaction has been shown to result in reduced autoimmunity by down-regulating th differentiation (cella et al., ; macatonia et al., ) . accumulating evidence indicates that in addition to driving virus-specific t cell proliferation, the activation state of dcs can also directly influence the effector function of t cells through the secretion of proinflammatory cytokines (fischer et al., ; maldonado-lopez et al., pulendran et al., ) . for example, following several viral and bacterial infections, cd a + dcs have been shown to be able to secrete large amounts of the proinflammatory cytokine il- both in vitro and in vivo and this results in a preferential expression of th -associated cytokines, such as ifn-g by responding t cells (aliberti et al., ; maldonado-lopez et al., pulendran et al., ; reis e sousa et al., ) . until recently, the prevailing thought was that cd a + dcs were primarily responsible for production of il- and contributing to a th response. however, recent studies have indicated that cd a À dcs also have the potential for secreting il- and influencing the t cell response (doxsee et al., ) . our studies clearly indicate that cd a À dcs isolated from the draining cln of mhv-infected ccl +/+ mice secrete il- suggesting that these cells help influence a protective th -mediated immune response characterized by the majority of antigen-specific t cells expressing ifn-g rather than il- (table ). in stark contrast is the data indicating that cd a À dcs present in the cln of infected ccl À/À mice predominantly secrete il- and this correlates with limited ifn-g expression and enhanced expression of the th -associated cytokine il- (table ) . therefore, the data indicate that cytokine production, rather than the type of cd c + cell, may control the predominant t cell immune response within the cln. taken together, these data point to an important role in ccl expression in linking innate and adaptive immune responses following viral infection of the cns by contributing to the activation fig. . cytokine and chemokine secretion by cd c + cd b + cd a À cells isolated from mononuclear cells pooled from the cln of mhv-infected ccl +/+ and ccl À/À mice at day p.i. supernatants were analyzed for the production of il- p , il- , and ccl by elisa h following culture. a minimum of three to six mice per group were used for isolation of cells and data presented indicate the average f sd. *p v . . table frequency of cytokine-producing t cells present within draining cervical lymph nodes following mhv infection a day p.i. ifn-g il- il- cd + t cells ccl +/+ f f f data represent two separate experiments with at least three mice per group, n = . data are presented as average f sd. a cytokine expression determined by pooling cells from draining cln of mhv-infected mice at defined times p.i. and pulsing with defined cd + epitope (m - ) and cd + epitope (s - ). b p v . . decreased frequency of ifn-g secreting t cells and increased frequency of il- producing t cells following mhv infection of ccl À/À mice as compared to ccl +/+ mice. c p v . . increased frequency of il- producing t cells in ccl À/À mice as compared to ccl +/+ mice following mhv infection. of dcs through regulating the migration of cells from the cns to lymphoid tissues as well as the expression of both co-stimulatory molecules and cytokine production. however, it is important to note that there is the possibility of other chemokines and chemokine receptors participating in dc responses following viral infection. indeed, the cc chemokine receptor (ccr ) is expressed on professional apc including macrophages and dcs and is thought to contribute to defense following microbial challenge by enhancing recruitment as well as production of antimicrobial products such as tnf-a and no by these cells (luster, ; mccoll, ; serbina et al., ) . in addition, the absence of ccr signaling results in diminished trafficking and accumulation of dendritic cells within secondary lymphoid tissues following antigenic challenge (peters et al., (peters et al., , sato et al., ) . studies are currently in progress to evaluate the contributions of additional chemokine signaling pathways that may also participate in dc activation and migration following coronavirus infection. in conclusion, the studies presented support and extend previous work from our laboratory indicating an important role for chemokines in the migration of t cells into the cns following mhv infection glass et al., ; liu et al., ) . here, we have demonstrated a novel role for the chemokine ccl in enhancing the accumulation and activation of cd a À dcs within secondary lymphoid tissue and this correlates with altered t cell activation and differentiation following viral infection. these results indicate that cd a À dcs likely function in an apc-like role within the cln following mhv infection and that these cells rely upon chemokine instruction to activate t cells. at a more fundamental level, the results presented demonstrate that chemokines serve as critical upstream signals in the innate immune response that later is important with regards to the initiation of a protective adaptive immune responses to viral infection. mhv j . v- was kindly provided by j. fleming (u. wisconsin). ccl À/À and ccl +/+ mice (c bl/ , h- b ) were purchased from jackson laboratories (bar harbor, me). mice were anesthetized by inhalation of methoxyflurane (pitman-moor inc., washington crossing, nj) and injected intracranially (i.c.) with pfu mhvj . v- suspended in al sterile pbs. control (sham) mice were injected with al sterile pbs alone. mononuclear cells were obtained from the brains and cervical lymph nodes [two draining cervical lymph nodes (cln) per mouse] of either ccl +/+ or ccl À/À mice at defined times post-infection (p.i.) using a previously described protocol (trifilo et al., ) . cell surface expression of phenotypic markers was examined using the following reagents for flow cytometric analysis: apcconjugated rat anti-mouse cd ; percp-conjugated rat anti mouse cd (pharmingen, san diego, ca). pe conjugated d b /s - mhc class i tetramer (beckman coulter, san diego, ca) was utilized for identification of cd + t cells specific for viral spike protein antigen (trifilo et al., ) . to determine the presence of dendritic-like cells within the cns and lymph nodes, cells were stained using fitcconjugated rat anti-mouse cd c (serotec, oxford, england) in combination with percp-conjugated rat anti-mouse cd a (pharmingen), rat anti-mouse apc-conjugated cd b (pharmingen), and rat anti-mouse dec (pharmingen). the maturation and activation state of dc were determined using fitc-conjugated rat anti-mouse cd , cd , cd , mhc i, and mhc ii (pharmingen). isotypematched antibodies were used as controls for all staining conditions described. isolation of cd c + cells from the cln ccl +/+ and ccl À/À mice were infected intracranially with pfu of mhv and brains and cln were removed at defined times post-infection for analysis. brain samples were minced and homogenized into a single cell suspension followed by fractionation on a / % percoll gradient at  g for min. for isolation of mononuclear cells from the cln, lymph nodes were homogenized using frosted glass slides and the resulting single cell suspension was treated with sterile h o to lyse red blood cells. due to the low frequency and numbers of cd c + cells within the brain and cln, samples from three to six mice were pooled for each experiment. to enrich for cd c + cd b + cd a À dcs, both brain and cln samples were separately magnetically sorted by negative selection against cd a using macs microbeads coated with anti-cd a (miltenyi biotec, auburn, ca) according to the manufacturer's instructions. cd c + cells were then magnetically selected from the cd a À fraction using macs microbeads coated with anti-cd c (miltenyi). the resulting population was n % pure for cd c + cells that were subsequently determined to be cd b + and cd a À by flow cytometry (data not shown). macs enriched cells were then resuspended in dmem supplemented with % fbs. cytokine and chemokine expression from cd c + cd a À cells freshly prepared cd c + cd a À cells were isolated from the cln from ccl +/+ and ccl À/À mice at day p.i. and seeded into -well plate at a cell density of  cells/ al in dmem containing % fetal bovine serum (fbs, biowhittaker, walkersville, md). following isola-tion, cell viability was n %. after h, supernatants from the samples were collected, and the level of il- , il- , and ccl were determined using quantikine m mouse immunoassays kits (r&d systems, minneapolis, mn) according to manufacturer's specifications. assays had a minimum sensitivity of pg/ml (il- and il- p ) and pg/ml (ccl ). mononuclear cells were obtained from the clns of mhv-infected mice at defined times post-infection and cytokine production by t cells determined by intracellular cytokine staining to defined viral antigens using a previously described protocol (trifilo et al., ) . statistically significant differences between experimental groups was determined by the mann-whitney rank sum test, and p values of v . were considered significant. ccr provides a signal for microbial induced production of il- by cd a+ dendritic cells definition of dendritic cell subpopulations present in the spleen, peyer's patches, lymph nodes, and skin of the mouse dendritic cells and the control of immunity cd + t-cell epitopes within the surface glycoprotein of a neurotropic coronavirus and correlation with pathogenicity ligation of cd on dendritic cells triggers production of high levels of interleukin- and enhances t cell stimulatory capacity: t-t help via apc activation lack of ccr results in increased mortality and impaired leukocyte activation and trafficking following infection of the central system with a neurotropic coronavirus requirement of mip- alpha for an inflammatory response to viral infection the chemokine macrophage-inflammatory protein- alpha and its receptor ccr control pulmonary inflammation and antiviral host defense in paramyxovirus infection the immune response modifier and toll-like receptor agonist s- selectively induces il- and tnf-alpha production in cd c+cd b+cd -dendritic cells brain dendritic cells and macrophages/ microglia in central nervous system inflammation phenotype and functions of brain dendritic cells emerging during chronic infection of mice with toxoplasma gondii reduced macrophage infiltration and demyelination in mice lacking the chemokine receptor ccr following infection with a neurotropic coronavirus the dendritic cell populations of mouse lymph nodes the t cell chemoattractant ifninducible protein is essential in host defense against viral-induced neurologic disease the role of chemokines in linking innate and adaptive immunity dendritic cells produce il- and direct the development of th cells from narve cd + t cells cd alpha(+) and cd alpha(À) subclasses of dendritic cells direct the development of distinct t helper cells in vivo cytokines regulate the capacity of cd alpha(+) and cd alpha(À) dendritic cells to prime th /th cells in vivo kinetics of virus-specific cd +-t-cell expansion and trafficking following central nervous system infection chemokines and dendritic cells: a crucial alliance a mechanism for the impaired ifn-gamma production in c-c chemokine receptor (ccr ) knockout mice: role of ccr in linking the innate and adaptive immune responses chemokine receptor serves an early and essential role in resistance to mycobacterium tuberculosis distinct dendritic cell subsets differentially regulate the class of immune response in vivo in vivo microbial stimulation induces rapid cd ligand-independent production of interleukin by dendritic cells and their redistribution to t cell areas cc chemokine receptor (ccr) is required for langerhans cell migration and localization of t helper cell type (th )-inducing dendritic cells. absence of ccr shifts the leishmania major-resistant phenotype to a susceptible state dominated by th cytokines, b cell outgrowth, and sustained neutrophilic inflammation tnf/inos-producing dendritic cells mediate innate immune defense against bacterial infection the cc chemokine ligand (ccl ) regulates cd + t cell effector function and migration following viral infection identification of a cd + t cell epitope within the m protein of a neurotropic coronavirus this work was supported by national institutes of health grant and national multiple sclerosis society grant -a- to t.e.l. key: cord- -uxxrpfl authors: resta-lenert, silvia title: diarrhea, infectious date: - - journal: encyclopedia of gastroenterology doi: . /b - - - / - sha: doc_id: cord_uid: uxxrpfl nan diarrheal diseases are a major cause of morbidity and mortality around the world, especially in developing countries where children suffer the greatest brunt of infectious diarrhea, malnutrition, and death. annually, approximately million children and infants die worldwide due to diarrheal diseases. in north america, the rate per year is still . diarrheal episodes per child, and in special circumstances (daycare centers, institutions), the incidence is as high as episodes per year. fourteen hospital admissions per children younger than months, per year, result from acute diarrhea. among the adult population, most patients developing acute diarrhea are managed as outpatients or will not seek medical attention. however, . million hospital admissions per year, or . % of all adult hospital admissions annually, are due to diarrhea. in developing countries, inadequate water supply, inef®cient or nonexistent sewage removal systems, chronic malnutrition, and lack of access to oral rehydration are responsible for the high incidence of infectious diarrheal diseases. in the industrialized world, acute diarrhea is still one of the most frequent diagnoses in general practice and children, elderly, and immunocompromised patients are the most vulnerable individuals and account for the majority of these cases. regardless of the etiology, diarrhea is de®ned clinically as the occurrence of three or more episodes of loose stool or any loose stool with blood during a h time period. symptoms lasting less than days represent acute diarrhea, whereas persistent diarrhea lasts more than days but less than weeks, and chronic diarrhea is de®ned by a duration of symptoms greater than weeks. infectious diarrheas are miserable illnesses of overwhelming impact on the general survival of entire populations. throughout history, thousands-strong armies have been defeated by raging diarrheal diseases: from the greeks and macedons under alexander (tucidides), to the romans in the campaigns against the gauls ( julius caesar), to the hundred years war in th century europe, to napoleon, the civil war in america, world war ii, and the vietnam war. scores of previously healthy men suffered and died from the scourge of diarrhea and dysentery in all of these con¯icts. twenty years ago, million to billion episodes of infectious diarrhea and nearly million deaths occurred per year worldwide, primarily in developing countries. ten years later, survival had improved, but the incidence was virtually unchanged despite greater knowledge of the pathophysiology of diarrhea and greater intervention by the world health organization (who). approximately million episodes of acute diarrhea occur in the united states yearly, with an incidence of . to . diarrheal episodes per person-year. medical costs/analyses show that . million americans sought physician care for diarrhea yearly and , required hospitalization. hospitalization and medical costs approached $ million, whereas lost productivity totaled $ million. approximately another million people sought physician care but were not hospitalized. these patients incurred $ million in medical costs and $ billion in loss of work hours. an estimated million cases occurred in people who did not seek physician care, costing nearly $ billion in lost productivity. approximately % of all these cases were presumably of infectious origin. thus, the total cost estimate for diarrheal diseases exceeds $ billion annually in the united states alone. although the elderly have an increased risk for death from diarrhea, death from diarrhea is rare among young children in industrialized countries. in fact, of all pediatric admissions for diarrhea, . % resulted in death, compared with % in patients older than age . increased age was the most important risk factor for death with an odds ratio of . ( % con®dence interval, . to . ) for age or older versus children b years. the national mortality ®gures for the -year period À in the united states show % of diarrheal deaths occurring in individuals older than age . acute infectious diarrhea is transmitted mostly through the fecalÀoral route and by ingestion of contaminated water and food. infection via the fecalÀoral route occurs by direct contact with index cases, especially under conditions of crowding, such as daycare centers or nursing homes. waterborne and foodborne outbreaks are another important source of disease transmission and result from general and/or individual failures in proper standards for the safe handling of foods. in most developing nations, acute diarrhea is endemic due to poor sanitation. furthermore, epidemics of signi®cant proportions often result from natural disasters in areas where water and food supplies are already chronically jeopardized. in some areas of the world, such as asia, africa, and latin america, certain infectious diarrheas (e.g., cholera) have become ongoing pandemics lasting several decades, notwithstanding who efforts at eradication. in most parts of the world, a de®nite seasonality is recognized in the incidence of acute diarrhea. in industrialized nations, the highest incidence of hospital admissions for diarrhea occurs in august and september and in the winter months. in developing nations with warmer climates and endemic conditions, variations in incidence occur from year to year in relation to precipitation indices and crop failures. infectious diarrheas may be classi®ed according to various criteria: duration, underlying mechanism, clinical presentation, etiology, and history. table i summarizes the various criteria for classifying diarrheas in general and infectious diarrheas in particular. in this section, infectious diarrheas are described according to the duration of the main gastrointestinal symptom. acute diarrheas last, by de®nition, less than days and the majority are due to infectious agents. most of these infections are self-limited and generally do not require medical intervention, unless severe dehydration and toxicity develop. however, immunocompromised patients, the elderly, and the very young may develop complications from enteric pathogens that warrant prompt and decisive medical intervention. a list of the major organisms involved in the etiology of acute infectious diarrheas is presented in table ii . not listed is a type of acute enteritis, waterborne and of presumed infectious origin, that has been responsible for several outbreaks of traveler's diarrhea, known as brainerd diarrhea. the etiologic agent of this disease still escapes de®nition. many of the acute infectious diarrheas observed worldwide are diagnosed in the course of local or epidemic outbreaks. three major situations may be encountered: ( ) waterborne infections; ( ) food-borne diarrhea; and ( ) traveler's diarrhea. whereas foodborne diarrhea is often associated with residual microbial toxins, waterborne and traveler's diarrheas are more often caused by active infection via the fecalÀoral route. table iii summarizes the most common causes in these epidemiological situations. a successful enteric pathogen possesses well-developed abilities to colonize, grow, and compete for nutrients in a crowded environment and to interact effectively with the host's enterocytes, inducing changes in the balance between absorption and secretion of water and electrolytes. in most gut infections, a pathogen enters via the oral route and colonizes an area of the ]. in addition to direct effects by microorganisms and their products, enteropathogens induce intestinal damage indirectly via the mucosal in¯ammatory response, which involves secretion of various powerful mediators of secretion and apoptosis. a summary of the current knowledge about the pathogenesis of the most common acute infectious diarrheal syndromes is shown in fig. . on the basis of these three mechanisms, acute infections present as watery, nonin¯ammatory diarrheal syndromes or in¯ammatory diarrheal syndromes. the majority of watery, nonin¯ammatory diarrhea cases are self-limited diseases characterized by low-grade fever, nausea, vomiting, large-volume diarrhea, and the absence of blood and leukocytes in the stools. this presentation is typically reported in patients infected with enterotoxigenic escherichia coli, v. cholerae, clostridial and staphylococcal food poisoning, rotavirus, norwalk virus agent, giardia lamblia, and cryptosporidium. on the other hand, the in¯ammatory diarrheal syndrome is characterized by frequent, small-volume stools that may contain blood and leukocytes, tenesmus, fever, and severe abdominal pain. the most common microorganisms causing this syndrome include salmonella, shigella, campylobacter, enterohemorrhagic e. coli, eiec, clostridium dif®cile, entamoeba histolytica, and yersinia. table iv describes the basic biologic, pathophysiologic, and clinical characteristics pertinent to the most common enteric pathogens. persistent diarrhea is emerging as a major world health problem. children are more likely to develop persistent diarrhea and suffer malnutrition, wasting, and immunocompromise as a consequence. persistent diarrhea is de®ned by looseÀsoft stools occurring at increased frequency and lasting for more than postinfectious persistent diarrhea is a poorly de®ned syndrome that occurs as a sequela of an acute episode with de®nite infectious etiology. patients may develop mild to severe degrees of malabsorption, from lactose intolerance to inability to absorb proteins, fat, and sugars, as well as permanent blunting of villi as assessed by histopathology. the condition is characterized by watery, malodorous stools and progressive wasting. chronic infectious diarrhea occurs mostly in immunocompromised patients. after an acute infectious episode, patients sometimes develop chronic symptoms that are independent of the etiologic agents of acute diarrhea (irritable bowel syndrome with diarrhea, or, occasionally, ulcerative colitis). table vi lists the most common agents isolated from cases of chronic infectious diarrhea. by de®nition, chronic diarrhea lasts more than weeks and patients developing this syndrome quite often are hospitalized and have undergone antibiotic therapy for other reasons. elderly, human immunode-®ciency virus (hiv)/acquired immunode®ciency syndrome (aids), transplant, and cancer patients are easy targets for reinfections or reactivation of only partially subdued infectious organisms. in addition to the causes listed above, bacterial overgrowth can occur in areas of bowel stasis or impaired bowel motility. postsurgery patients, diabetics, posttrauma patients, and intensive care patients are more likely targets of chronic infectious diarrheas from bacterial overgrowth. infectious diarrhea causes high morbidity and mortality among the aging population worldwide. multiorgan complications from an acute episode of infectious diarrhea are also more frequent among the elderly. life expectancy in the united states has risen from an average of years in the th century to years at present. by the year , % of the u.s. population will be older than age . gastrointestinal physiology and gut colonization change constantly with aging and contribute in a signi®cant way to increasing the susceptibility of elderly people to enteric infections. furthermore, the gastric acid barrier in the elderly is impaired. the most frequently isolated organisms and most deadly in elderly patients with diarrhea are c. dif®cile, salmonella, and toxigenic e. coli. these three agents top the list of figure infectious diarrhea: mechanisms of action of major enteric bacteria and viruses. enteric pathogens can induce intestinal injury with consequent diarrhea in three ways: ( ) by producing enterotoxins that interact with receptors located on the gut epithelial cells and evoke anion secretion, such as v. cholera, epec, eaec, stec, c. dif®cile, and s. aureus (a); ( ) by invading the gut epithelium and m cells, thus altering the cell cytoskeleton and activating intracellular pathways through virulence factors. organisms that lead to diarrhea through these mechanisms include eiec, shigella, epec, salmonella, and rotaviruses (b); ( ) by invading mucosal macrophages and inducing in¯ammatory responses leading to intestinal epithelial damage and anion secretion. campylobacter and yersinia use this mechanism (b). outbreaks in long-term and short-term care facilities and salmonella by itself accounts for more than % of cases and more than % of deaths in food-borne outbreaks in nursing homes. more than % of hiv/aids patients in the united states experience infectious diarrhea and this estimate may approach % in developing countries where the hiv epidemic is currently raging unchecked. these patients are more likely to develop persistent or chronic diarrhea after an acute episode because of their impaired immunity, with a signi®cant increase in morbidity and mortality. table vii lists the most common causes of infectious diarrhea in aids patients. the american gastroenterological association (aga) has published a set of general guidelines for the management of chronic diarrhea in aids patients. at least three sets of stool samples should be secured for common enteric bacteria and parasites, including microsporidia, cryptosporidia, and c. dif®cile. febrile patients with diarrhea should have blood cultures for common enteric bacteria. patients with cd lymphocyte counts of cells/mm are at high risk for disseminated mycobacterial infection. the most important ®nding in patients presenting with acute diarrhea is the degree of volume depletion, i.e., dehydration. postural changes in blood pressure are a reliable sign of dehydration. fever, abdominal tenderness, increased bowel sounds, or blood on rectal examination should alert the physician to acute infectious diarrhea. microscopic examination of a stool sample or rectal swab is a traditional and helpful tool in the rapid, bedside investigation of diarrheal illness. the specimen is placed on a glass slide and mixed thoroughly with two drops of methylene blue. the presence of ova, cysts, and/or leukocytes may point directly to a diagnosis. the aga guidelines on managing acute diarrhea indicates empiric antimicrobial therapy in the case of positive fecal leukocytes in a febrile patients. endoscopy has limited utility in the investigation of acute infectious diarrhea and is not cost-effective. it may have a place, however, in cases of persistent or chronic diarrhea. preventative measures against infectious diarrhea must include improvements in sanitation (water supply, sewer systems, housing), education of the general population and, where applicable, vaccination campaigns. unfortunately, no effective vaccines are available for the organisms that cause infectious diarrheas, with the exception of typhoid fever. treatment most acute diarrheal illnesses are self-limited and no speci®c therapy is required. water and electrolyte loss can be prevented or treated with oral¯uidÀelectrolyte solutions. intravenous salineÀglucose solutions are recommended in cases of moderate to severe dehydration. glucose in the intestinal lumen facilitates the absorption of sodium and the cotransport mechanism for these solutes appears to be unhampered by infection with microorganisms or by their toxins. antimotility therapy should be reserved for severe cases and chronic diarrheas and avoided in infants and children. antibiotic or antiviral treatment should be considered in moderate to severe cases in which a microbiological diagnosis is obtained or strongly see also the following articles aids, gastrointestinal manifestations of anti-diarrheal drugs campylobacter cholera cryptosporidium cytomegalovirus diarrhea foodborne diseases food poisoning food safety giardiasis rotavirus salmonella shigella traveler's diarrhea further reading anonymous epidemiology of clostridium dif®cile-associated infections ef®cacy and tolerability of racecadotril in acute diarrhea in children practice guidelines for the management of infectious diarrhea principles and practice of infectious diseases microbes and microbial toxins: paradigms for microbialÀmucosal interactions. viii. pathological consequences of rotaviral infection and its enterotoxin traveler's diarrhea due to intestinal protozoa the role of antibiotics in the treatment of infectious diarrhea infectious diarrhea in children pathogenesis of infectious diarrhea key: cord- - yp wd j authors: may, thomas title: isolation is not the answer date: journal: nature doi: . / a sha: doc_id: cord_uid: yp wd j international scientific collaboration is the best defence against bioterror. the fear of bioterrorism is increasing scientific isolationism in the united states. new restrictions on the publication of sensitive information relevant to biological weapons, on access to 'select' biological agents for research, and on the training of scientists from specified countries are some examples. although restrictions on scientific activities might make sense in the context of nuclear-weapons proliferation, they may end up being counter-productive for the united states' defence against bioterror. biological terrorism poses a unique threat in that the devastation caused by the release of a biological agent is unlikely to be confined to the event itself, but will depend on the ease with which the disease spreads. infectious disease cannot easily be restricted to any location, region or even nation. the world health organization (who) has documented numerous cases of global disease spread, including that of severe acute respiratory syndrome (sars) in . this virus disease originated in southern china and spread to nearly countries, resulting in , infections and deaths over nine months . a bioterror attack would probably be much worse than this natural epidemic, as the infectious agents used and the manner of their release would be designed for maximum effect. if terrorists released a biological agent in a region where quick identification of a disease outbreak was unlikely, they could exploit the ease of international travel to spread the disease to 'targeted'countries once it had established a sufficiently strong foothold to make containment difficult. consequently, attention to the global dimensions of bioterror threats is particularly important, including strengthening international means to identify and contain outbreaks of infectious disease. to date, national-security experts have considered the risk of this sort of attack to be fairly low . it was thought that its indiscriminate nature -placing non-target populations, including the terrorists themselves, in danger -would undermine popular support for the terrorists'agenda.this analysis,however, reflects old expectations of terrorist behaviour based on rational self-interest -rules that simply do not apply to modern terrorists. although many terrorists try to limit casualties for pragmatic reasons, emerging terrorist groups often have radicalized agendas that pay less, if any, concern to public support. in addition, today's terrorists have consistently demonstrated a willingness to die (and to kill innocent civilians) to achieve their goals. in this context,is the united states developing the best strategies for preventing bioterrorism, and for responding to and containing bioterror attacks? homeland defence priorities have emphasized diplomatic,intelligence, law-enforcement (including disruption of terrorists' financial networks) and bordercontrol measures designed to keep potential biological weapons out of the hands of terrorists. these are valid efforts but they do not fully address the international dimensions of modern bioterrorism and the most likely route by which an attack will reach the united states.it is also widely accepted that the 'open' nature of us society creates vulnerabilities to terrorism . but when preparing ourselves for a bioterror attack, an open academic and educational system is one of our most important defensive strengths. new ways of thinking about security are sorely needed. recognition of the true international nature of the bioterror threat should make the united states take a leading role in training foreign scientists, medical professionals and public-health personnel to build a global capacity for identifying and containing disease outbreaks. this must occur at several levels. first, the us centers for disease control and prevention (cdc) in atlanta, georgia, must be better equipped to provide significant support and training to international public-health personnel. although such support has long been a focus of the cdc, the agency does not have the resources to expand these efforts: in the council commentary nature | vol | june | www.nature.com/nature on foreign relations reported that even domestic training is "drastically underfunded". second, efforts at the who to improve infectious-disease surveillance and containment must become priorities for the united states. the unique features of bioterrorism make practical improvements to international healthcare of equal strategic importance to traditional diplomacy. perhaps most important, we must take care to protect the open nature of our academic systems, and to avoid placing undue barriers on the training and education of foreign scientists and medical personnel. although some restriction is necessary, attempts to control scientific expertise must be balanced with the need to promote security through scientific progress, such as the development of new tests and treatments to identify and contain disease outbreaks. some may argue that this will grant terrorists access to sensitive information and expertise, but it does not increase such risks significantly: even with restrictions it is relatively easy to find individuals willing to pursue biological weapons research for the right price. ken alibek, a former leading soviet biowarfare scientist, reported the recruitment of former colleagues by several countries, including iran and north korea. in reality, we cannot control access to biological weapons expertise through control of domestic science alone. in contrast, the dependence of us science on foreign scientists is such that biodefence research will be inhibited if we continue down a road of scientific isolationism. apart from the obvious barriers that restrictions on access to scientific information and tools place on research, restrictions on scientific training for foreign nationals will delay those countries from developing expertise crucial to identifying and containing disease outbreaks -key to any global strategy against bioterrorism. what is required is the proliferation of scientific training worldwide, not scientific isolationism. against all enemies: inside america's war on terror report of an independent task force sponsored by the council on foreign relations international scientific collaboration is the best defence against bioterror. p. martinez monsivais/ap key: cord- - opqbu h authors: leung, h.m.; tan, swee liang; yang, zhen lin title: what has luck got to do with economic development? an interpretation of resurgent asia’s growth experience date: - - journal: j policy model doi: . /j.jpolmod. . . sha: doc_id: cord_uid: opqbu h this paper critically reexamines the belief, currently gathering strength in the literature, that economic development depends on good luck rather than on good policy, and that prometheus is “unchained by chance”. while it is impossible to disprove the role of luck in growth, we argue that luck is endogenous, and good luck is a function of good policy. luck favours those who strive. again contrary to common belief, we show that resurgent asian economies have endured more, not less, than their fair share of economic volatility. they learned their lessons by success and failures, and luck is endogenous through learning-by-investing. in the new millennium, our picture of world economic development is filled with hope and despair. on the one hand, real per capita income in the western developed world has increased faster during the past years than ever before. on the other hand, income distribution across nations is worsening at an alarming rate. uneven development is entrenched, the gulf between rich and poor countries is widening, and rampant poverty continues to haunt millions of people in sub-saharan africa, asia, and latin america. also in the past years, four asian newly industrialised economies-korea, taiwan, hong kong and singapore (nies for short)-have caught the imagination of the world. taken together, the nies have grown at an average rate of . % per year from to , roughly three times the world average. at constant us$ terms, the nies' gross domestic product (gdp) increased by a staggering times from to . singapore, for instance, within a single generation raised her per real capita gdp from % of that of the united states of america in to % in . the nies have presented the world with great hopes and, by the same token, an intellectual puzzle: how did they do it? the nies' growth experience has often been referred to as a "miracle." can poor nations replicate their economic "miracle?" if yes, then how? in life, there are three basic ingredients to good fortune: wisdom, hard work, and luck. luck is important because we live in a world of chance. more precisely, luck can be defined as an unknown and unpredictable phenomenon that causes an event to result one way rather than another. luck can be good or bad, but either way it is characterised by an enigmatic and unknowable volatility. in this regard, two recent papers raise the question of how luck may affect economic development. one is acemoglu and zilibotti ( ) , which argues that "at the early stages of development, the presence of indivisible projects limits the degree of risk spreading (diversification) that the economy can achieve. [. . . ] 'lucky' countries will spend relatively less time in the primitive accumulation stage and develop faster" (p. , italics added). indivisibility is certainly a problem for small countries, and each of the four nies is small. singapore, for instance, is a small city state that deals with indivisibility by targeted-investment policies. largely as a result of her government's economic development policy, . % of her total manufacturing output in came from a single sector of electronic products and components, even though this proportion had fallen from . % in see maddison ( ) , p. . data from world bank's world development indicator cd-rom ( ) and from taiwan statistical yearbook (various years). the total factor productivity debate may be intellectually satisfying, but poor countries in the world are primarily interested in how the nies did it, not whether it was a "miracle" or not. even if singapore had "bought" her success with high saving rates, to many poor nations that surely is a price worth paying for the resulting vast improvement in living standard. it is in this spirit when stiglitz recently wrote that "the total factor productivity debate is much ado about nothing" ( , p. ). . similar stories can be told of taiwan's targeting basic industries such as glass, plastics, steel and cement (see wade, , p. ) , and of korea's targeting shipbuilding, cement, and steel (see amsden, and jones & sekong, ) . applying acemoglu and zilibotti's hypothesis that "chance will always play a key role in his [prometheus'] unchaining" (p. ), these nies must have hit the jackpot by choosing the growth industries. these asian countries could, according to the acemoglu-zilibotti hypothesis, easily have betted on the wrong horse. they have been lucky; things could have been much worse. the other paper is easterly, kremer, pritchett, and summers ( ) , which observes empirically over recent decades that there are (a) low persistence of growth rates, (b) high persistence of country characteristics, and (c) low persistence of shocks such as terms of trade. they conclude that ". . . luck is important relative to policies in determining the long-run path of output" ( , p. ) . not only do easterly and his co-authors assert that luck is important, but that growth policy is not. thus they write, "the finding that much variation in growth rates is due to random shocks should induce caution in attributing high growth rates to good policy (or to a good 'work ethic'). just as a baseball star is dubbed a clutch hitter after a lucky hit, some so-called economic miracles are likely due to random variation" ( , p. , italics added) . the "miracles" they refer to are the four high-flying asian nies, which easterly in another paper predicts "should be heading back toward earth soon" ( , p. ) . sure enough, merely two years after easterly wrote this, all four nies headed "back toward earth" in the form of the asian financial crisis. they did not stay on earth for very long though, and they quickly rebounded to above-average growth rates until, in , another shock hit them (except korea) in the form of a deadly virus called severe acute respiratory syndrome (sars). the asian nies have been lucky, but perhaps their luck is finally running out. because of its inherently unknowable and volatile quality, luck is a concept shrouded by a great deal of ambiguity and obscurity. consequently, we have to be careful in order not to reduce our enquiry to a state of quagmire. luck is often associated with fate, even superstition; a student may think that he did well in an examination paper because he wore his "lucky t-shirt" that day. such prosaicism must be excluded from our discussion. furthermore, any theory based on luck is intellectually distasteful since it can never be falsified. for instance, we can never disprove that the student's t-shirt brought him "luck" that day. by the same token, we can never disprove that asian nies' growth performance was due to "luck" at least in some ways. even though a luck-theory, by its incapacity of being falsified falls within the popperian realm of "non-science," it is still enlightening, meaningful, and indeed crucial to consider how it may help us to understand growth. luck is by definition unknowable. however, we could find out how luck evolves amid economic volatility, and thence infer the role of good luck and good policy in (nies) growth. after all, the history of human civilization is the history of knowing the unknown and of reducing our realm of ignorance, much of which we unwittingly assign to luck. practical policy recommendations are the chief motivation in this paper. the question, "what has luck got to do with growth?" is, for poor countries yearning for economic progress, too important to be ignored. as the only star performers for almost a half century, the nies have carried the hopes of many poor nations in the world. china, india, thailand, malaysia, vietnam and a host of asian countries have begun lately to emulate the four nies with various degrees of success. "how to make a miracle" is without doubt the most encapsulating question today, but much hope will sadly be dashed if the nie miracles are merely freak results from a lucky draw. if the nie's growth really were freak results from a lucky draw, then poor countries should forget about learning from the asian examples, but instead try to improve their fortune through other means. because of the practical importance of the question, "what luck has got to do with development?" it will be worthwhile to examine evidence; doing this will clear our thoughts on the matter. as explained earlier, disproving the luck-hypothesis is an impossible mission; this is not the purpose of this paper. our aim is instead twofold. first, we need to dispel the widespread misconception that the asian nies have had a smooth ride all the way. the fact is the contrary-they have had to endure more than their fair share of volatility along their growth path. second, we argue (in section ) that even luck is endogenous. luck always favours those who work hard, not as an article of faith, but as a consequence of learning-by-doing and learning-by-investing. the asian nie's high degree of volatility was a consequence of their intensive investment policy (see section ); their luck and fortune are spin-offs from their toil and dexterity. our recommendation to poor countries is to formulate good investment and growth policies now, as luck will take the side of those who strive. the plan of the paper is as follows. the next section argues that volatility rises, not falls, with growth. section establishes the positive association between investment shares and volatility. section argues that even luck is endogenous. section concludes. the nies' sustained, robust performance often gives the impression that they have experienced less cycles and less volatility than other countries. for instance, stiglitz ( ) recently notes that "what is remarkable about east asia is not that it experienced a crisis in , but that it had experienced so few crises over the preceding three decades-two of the countries had not had one year of downturn and two had had one year of recession, a better record than any of the supposedly advanced and well-managed organisation for economic co-operation and development (oecd) countries" ( , p. ) . such approbation is not justified. although asian nies did not have a major crisis before , they had to ride through storms and turbulence more severe than other countries at comparable levels of economic development. the purpose of establishing nie's high degree of volatility is not to show that they had been unlucky, but rather to show that their economic "miracle" is a history of struggle, hard work, and learning-by-investing. it is not a whimsy streak of a historical lucky draw. to begin with, we can visualise the cycles by plotting annual gdp changes as a percentage of that year's gdp. fig. a plots the graphs of the asian nies, and fig. b that of four developed countries-australia, sweden, the united kingdom and the united states. australia, sweden, and the uk had similar per capita gdps to the four nies at , while the u.s. is included as another benchmark for comparison. from the figures, we see that what distinguishes the nies is not the absence of fluctuation. both in terms of frequency and magnitude their oscillations are not significantly different from those in the developed countries. the only difference is that the nies fluctuated around a higher mean growth rate than those countries in fig. b. more systematic but conflicting evidence comes from cross-country regressions. kormendi and meguire ( ) and grier and tullock ( ) use crosscountry comparisons, and find that higher standard deviations of output growth are associated with higher mean growth rates. gavin and hausmann ( ) study latin american countries, and find that when other measures of volatility are included, volatility has a positive but insignificant relation with growth rates. contrarily, ramey and ramey ( ) present evidence that volatility and growth rates are negatively linked. marion ( , ) support ramey and ramey's result for a group of developing countries. none of these papers explore in detail the possibility that the volatility facing the rich oecd countries may be of a different category than the volatility facing the poor countries, and again different from those facing the asian nies. we may recall grier and tullock's ( ) warning, that "we do not have a single empirical model of secular growth that applies around the world" (p. ). such warning, however, have not been heeded in the papers just cited. let us examine more carefully ramey and ramey ( ) , who regress the average growth rate of countries (g i ) from to on the standard deviation of growth rate ( i ). using the penn world tables for countries, they report the following regression result where i is a country index, t-statistics are given in the parentheses, and r = . . ramey and ramey find also that this result is robust with the inclusion of four control variables-average investment share of gdp, average population growth rate, initial human capital, and initial per capita gdp. from ( ), ramey and ramey conclude that "volatility has a negative relationship with growth" (p. ). this negative relationship leads to the opinion, prevailing in the literature today, that the fast-growth nies have low volatility. as mentioned in our introduction, this alleged low volatility of nies has led easterly, stiglitz and others to believe that the asian countries have been "lucky". we now set out to show that their perception of the nies' history is erroneous: the fast-growing nies had greater, not smaller, volatility than the advanced oecd countries. in general, volatility rises with growth and not the other way round. since regression ( ) merely tells us the association between growth and volatility rather than causality, and based on our suspicious that (a) the association is non-linear and (b) volatility rises with growth, we put the average - growth rate of country i on the left-hand side and run the following quadratic ordinary least squares (ols) regression i = . ( ) r = . , f = . which is significant at the % level. the data set used is the penn world table (mark . ) with countries. the r-square of . (adjusted r-square . ) is more than twice as large as ramey and ramey's. the quadratic equation gives a distinctly better fit than ramey and ramey's linear specification. using the estimated parameter values from ( ) we plot the resulting u-shaped curve between volatility and growth rate in fig. . the trough of the curve occurs at g = . . volatility rises in both directions from this trough. regressions ( ) and fig. show that three country groups-the slow-growing, the medium-growing, and the fast-growing-have significant similarity within group and differences between groups. many international bodies such as the international monetary fund (imf) also distinguish three country groups-the developing economies (des), the advanced economies (aes), and the asian nies (see table ). table an ols regression using dummy variables will help to identify which country group belongs to which segment of fig. , and lend support to our regression ( ). we extract from the penn world data the countries appearing where, g i = averaged per capita gdp growth rate of country i over - , d j (dummy variables) = if a country belongs to category j, otherwise, j,i = - annual average standard deviation of per capita growth rate of country i belonging to category j, and r = . , f = . which is significant at the % level. three conclusions emerge from ( ). first, the negative relationship between growth rate and volatility holds only for developing economies. second, for the four asian nies, the relationship between growth rate and volatility is positive and statistically highly significant. third, for the advanced economies, the relationship is positive but not significantly different from zero. in short, the u-shaped curve of fig. depicts the behavior of the developing, the advanced, and the asian nies as we move towards the right-hand side along the horizontal axis of average gdp growth rate. let us conjecture for a moment what might have caused this result. consider first of all the downward-sloping portion of the u-shaped curve in fig. . these countries are poor, slow-growing (some have negative growth rates), and technologically backward. many of them rely heavily on agricultural products which have low supply elasticity; agricultural supplies are subject to wide fluctuations because of floods, droughts and other natural disasters. in manufacturing, poor-country workers are unskilled and produce labor intensive, technologically well-diffused commodities. widely available substitutes and intense competition increase demand elasticities for their outputs. the combination of a flat demand curve and a steep supply which shifts easily leads to large swings of sales revenue. the more backward a country is, the more volatility she will face. next we consider the asian nies, which are distinguished by fast growth and intensive investment. in addition, these nations are small in size. as we mentioned earlier, they are restricted by investment indivisibilities and had resorted to industrial targeting policies in order to exploit economies of scale. even though such gambles have paid off in the long run, targeting large-scale investments restricted their economic profile to a limited number of sectors. being small in size, asian nies are driven to rely on export markets located in advanced countries. even though they are catching up with advanced technology, their production remains labor-intensive, and their technology diffused compared to the advanced oecd countries. like the poor countries, the nies face highly elastic demand curves. but unlike the poor countries, the nie's volatility stems mainly from shifts in demand. despite their differences, both the stagnant, less-developed countries and the progressive, asian nies have high volatility. finally we turn to the advanced countries, which typically have moderate growth rates of about - % per year. they possess advanced technologies, but their per capita incomes are now almost indistinguishable from some of the asian nies. table shows that the advanced countries saved less, invested less, and had smaller capital formation than the asian nies. their modest investment means that they take less risk, and that they can hedge their investment better than the asian nies. their high-technology outputs have fewer competitors, greater market power, and less elastic demand curves than the nies'. furthermore, the supply curves of such high-tech outputs do not fluctuate as much as the poor countries' agricultural output, and the demand curves of high-tech commodities do not shift as much as the nies' because of their market power. consequently, the developed countries' gdps are less volatile than the poor, developing countries' and the fast-growing nies'. towards the end of the th century, there were encouraging signs that a group of countries in asia were joining the four nies in their rapid economic growth. as a simple extension we move four other star-performers-malaysia, thailand, indonesia and china-into the asian nie category. repeating the regression ( ) using dummy variables and with eight asian nies gives r = . , f = . which is significant at the % level. in contrast with the findings in ( ), the coefficient for d ae × ae,i in ( ) is now statistically significant. this strengthens our conclusion that the negative growth-volatility relationship only holds for poor, developing countries, and that there is a positive growth-volatility relationship for all the other countries. the positive association just observed raises the prospect that neither growth nor volatility occurs by chance; instead, both are endogenous consequences of economic decisions. we argued earlier that asian nies have resorted to industrial targeting and intensive investment policies, and that their small size forces them to put all their eggs in only a few baskets. we now examine the relationships among volatility, investment intensity and country size. for investment intensity we use investment as a percentage of gdp, and we use log gdp as a proxy for country size. since we are focusing on the positive-sloped segment of the u-shaped curve (see fig. ), only oecd advanced countries and the asian nies (the extended definition, including singapore, hong kong, korea, taiwan, china, pakistan, the philippines, indonesia, thailand and malaysia) are included in the following regression i = . ( . ) + . ( . ) invs i − . (− . ) log gdp i ( ) r = . , f = . which is significant at the % level. the decade-average ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) per capita gdp growth performance was malaysia ( . %), thailand ( . %), indonesia ( . %) and china ( . %). if we exclude the financial crisis and take the average up to , the figures were malaysia ( . %), thailand ( . %), indonesia ( . %) and china ( . %). there is no question that these four countries significantly outperformed the advanced countries and the less-developed countries despite the setback in . growth rates are excluded from this regression to avoid its collinearity with investment share. thus, we confirm that there is a positive and statistically significant association between growth volatility and investment share. the coefficient on economy size is negative, and it is statistically significant at the % level. the smaller the country, the higher the risk from investment indivisibility, and hence the more volatile is its economic growth. we established in earlier sections that asian nies have endured more volatility than other slower-growing nations. some may still believe that the nies had good luck because they stayed afloat despite such adversities. as we argued in the introduction, there is no question that asian nies were lucky. they could have targeted the wrong industries, consumers in their export markets could have found their products not palatable to their tastes, and the nies for many reasons could have foundered instead of soared. in this section, we will give reasons to contend that luck has a habit of favouring those who strive and persevere. as president thomas jefferson once said, "i am a great believer in luck, and i find the harder i work, the more i have of it." in other words, even luck is endogenous; it is a function of cogent policies for growth. luck assumes its preeminence because nature is wrought with chances, crises, and exigencies. in dealing with chances, economists are accustomed to think in terms of risk and uncertainty. a useful conceptual distinction between risk and uncertainty is that by hart ( ) , who defines uncertainty as the reducible component of ignorance about outcomes, whereas risk is the irreducible component. the history of human civilization and industrialisation can be seen as an endless endeavor to reduce our ignorance. this history is one of learning. as we strive, we experiment, make mistakes, learn and sharpen our skills. when we encounter similar circumstances, our performance improves because of what we learned before. analogous to arrow's ( ) hypothesis that production knowledge is enhanced through learning-by-doing, investment knowledge also advances via learning-by-investing. as bernanke ( ) points out, the two key characteristics of all investments are that they are uncertain, and that they are in various degrees irreversible. the difficulty facing poor countries is that they lack investment experience; the irreversibility deters investment decisions since committing to an inferior investment option today means missed opportunities tomorrow. bernanke ( ) also constructs a formal model to show that there is an option value to waiting, and he concludes, ". . . in early stages of the learning process, waiting for information is a valuable activity, and investment tends to be deferred" (p. ). the option value (of not investing) is higher, and investment is deferred for longer, when the uncertainty (our reducible ignorance) attached to the investment is larger. just as learning-by-doing implies increasing returns to production, learning-byinvesting implies increasing returns to investment activities. there is no question that hong kong's li ka shing is a better businessman now than he was twenty years ago, that the korean chaebols have harnessed business expertise unmatched by many backward countries, and that singapore government's economic development board has gathered investment know-how far superior to that possessed by the governments of, say, armenia or the philippines. for any start-up investor the first investment project is inevitably difficult. the second project benefits from the experience of the first, and subsequent decisions are made more accurately and with more conviction as the investor gains from learning-by-investing. thus, bernanke continues, "as knowledge accumulates and priors concentrate, the propensity to invest increases" ( , p. ) . for all less-developed countries, the take-off is the most important and the most difficult part of their industrialisation. making the second million dollars is usually easier than making the first million, not so much because luck favours those who have made the first million, but rather because past experience improves the odds of success in future endeavors. it is important to note that useful experience is gained not only from successful projects; the experience of failure is often a more valuable lesson than success. projects do fail in asian nies. in fact many asian projects fail, and the high degree of nie volatility we observed in sections and testifies to that vividly. the lesson for the poor, undeveloped nations is that the path to growth is bound to be a bumpy ride, but the going can only get better because of learning-by-investing in addition to learning-by-doing. precisely because the beginning is the most difficult, the time to start learning is now. the harder one works, the faster one learns. luck will come to side with those who work hard and those who strive. even luck is endogenous; it is not a faith, but a logical consequence of learning from experience. we conclude therefore that luck indeed has a prominent role to play in economic growth. instead of contrasting "good policy" and "good luck," as easterly et al. ( ) did, good luck should be seen as a natural consequence of good policy. in section we corrected the common misconception that asian nies have maintained high-speed growth with little volatility. we showed, on the contrary, that nies have endured more volatility than the advanced oecd countries. such volatility is, in addition, a necessary part of their learning experience. small country-size and investment indivisibility led asian nies to targeted investment policies, which brought fast growth as well as a high degree of fluctuations. it is precisely from such business ups and downs that these nations learn to sharpen their investment skills; economic growth is not only a process of learning-by-doing, it is also one of learning-by-investing. our conclusion on the role of luck contrasts pointedly with the prevailing belief, as espoused in easterly et al. ( ) and in acemoglu and zilibotti ( ) , that growth is a result of luck, and not a result of good policy. the truth is, even luck is endogenous. poor countries have everything to gain from formulating a progressive, coherent set of growth policies. luck will side with those who strive. this is not a faith; this is part of the human history of learning-by-trying. was prometheus unbound by chance? risk, diversification, and growth policy uncertainty, persistence and growth volatility and investment: interpreting evidence from developing countries like the rest: southeast asia's 'late' industrialisation the economic implications of learning by doing irreversibility, uncertainty, and cyclical investment explaining miracles: growth regressions meet the gang of four good policy or good luck? country growth performance and temporary shocks overcoming volatility in latin-america. inter-american development bank an empirical analysis of cross-national economic growth, - risk, uncertainty, and unprofitability of compounding probabilities government, business, and entrepreneurship in economic development: the korean case macroeconomic determinants of growth: cross-country evidence the world economy: a millennial perspective cross-country evidence on the link between volatility and growth from miracle to crisis to recovery: lessons from four decades of east asian experience governing the market key: cord- -w pewq authors: chou, chih-fong; shen, shuo; tan, yee-joo; fielding, burtram c.; tan, timothy h.p.; fu, jianlin; xu, qiurong; lim, seng gee; hong, wanjin title: a novel cell-based binding assay system reconstituting interaction between sars-cov s protein and its cellular receptor date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: w pewq severe acute respiratory syndrome (sars), a life-threatening disease, is caused by the newly identified virus sars coronavirus (sars-cov). in order to study the spike (s) protein of this highly contagious virus, we established a clonal cell-line, cho-sg, from the chinese hamster ovary cells that stably expresses c-terminally egfp-tagged sars-cov s protein (s-egfp). the ectodomain of the s glycoprotein is localized on the surface of cho-sg cells with n-acetyl-glucosamine-terminated carbohydrate structure. cho-sg cells associated tightly with vero e cells, a sars-cov receptor (ace ) expressing cell-line, and the interaction remained stable under highly stringent condition ( m nacl). this interaction could be blocked by either the serum from a sars convalescent patient or a goat anti-ace antibody, indicating that the interaction is specific. a binding epitope with lesser degree of glycosylation and native conformation was localized by using rabbit anti-sera raised against five denatured recombinant s protein fragments expressed in escherichia coli. one of the sera obtained from the fragment encompassing amino acids - significantly blocked the interaction between cho-sg and vero e cells. the region is useful for studying neutralizing antibodies in future vaccine development. this paper describes an easy and safe cell-based assay suitable for studying the binding between sars-cov s protein and its receptor. severe acute respiratory syndrome (sars), a deadly disease, is caused by a human coronavirus named sars coronavirus (sars-cov). this highly infectious virus killed patients out of probable sars cases from november to july in countries (lingappa et al., http://www.cdc .gov/ncidod/eid/vol no / - .htm) and had a highly disruptive impact on people's lives as well as the economy. sars-cov belongs to the genus of coronavirus in the family of coronaviridae, a family of positive sense rna viruses with large envelopes that propagate in the cytoplasm of host cells and usually cause respiratory diseases in humans and animals. the complete genome of sars-cov has been sequenced from different isolates (marra et al., ; ruan et al., ) and it has a coronavirus genome organization with some unique features but is still distantly related to group coronaviruses (snijder et al., ) . the genome is nearly kb in length and encodes open reading frames (orfs) (marra et al., ; ruan et al., ) including those known coronavirus proteins, such as replicase a and b and four structural proteins, spike (s), envelope (e), membrane (m) and nucleocapsid (n). the rest of the orfs are novel and may encode for proteins with unknown functions. at least two of these proteins have been shown to be expressed during sars-cov infection (tan et al., b; yu et al., ; fielding et al., ) . antibodies against a, also termed as u , were found in the sera of sars patients (tan et al., a; yu et al., ) . aiming to control the sars epidemic, we first focused our studies on the s protein for the purpose of diagnosis, and also studied the interaction between s protein and its receptor for vaccine development. the s protein, a type i transmembrane glycoprotein, locates mainly on the outer envelope surface of sars-cov. its precursor is predicted to be amino acids in length. residues - comprise the putative signal peptide and the ectodomain is from residues to (marra et al., ) . a highly hydrophobic region from residues to is predicted to be the transmembrane domain and the rest (residues - ) are located in the cytoplasm. the s protein of coronavirus plays an important role in the initiation of the viral infection by mediating the attachment of the virus to the cell surface receptors that subsequently induces membrane fusion between the virus and the host. in some coronaviruses, the s protein (group ii mouse hepatitis virus [mhv] and group iii infectious bronchitis virus) is cleaved to generate two distinct functional subunits, s and s , by virus-encoded or host-encoded proteases (ziebuhr et al., ) . s is the nterminal fragment responsible for the attachment to the target cells; whereas, s is required for the fusion (gallagher and buchmeier, ) . the s domain of sars-cov s protein can be easily identified by sequence alignment with other coronavirus proteins but the s domain is not well conserved (spiga et al., ) . although, currently there is no evidence to show that there are cleaved s and s subunits in sars-cov-infected cells, the s equivalent domain has been cloned and demonstrated to bind the sars-cov receptor, ace , of host cells . furthermore, the binding domain of the s protein has been narrowed down to the n-terminal amino acid residues - (babcock et al., ) , - (xiao et al., ) and - (wong et al., ) . in addition to its function in viral entry, the s protein was found to be the major target of the host immune response against coronavirus as it induced neutralizing antibody (taguchi et al., ; kubo et al., ) . chinese hamster ovary (cho) cells are useful hosts to express mammalian genes and the system has been studied extensively to produce recombinant therapeutic glycoproteins. cho cells provide the advantages not only because they can be easily maintained and genetically manipulated, but also because they produce proteins with glycans similar to those native glycoproteins found in humans. in order to avoid the high risks involved in handling live sars-cov and to understand the s protein of this virus, we established a cho clone that expressed a significant amount of the recombinant s proteins on the cell surface and demonstrated that it can be used to study the properties of the sars-cov s protein. the selection of a high yield clone was facilitated by tagging the s protein with egfp at the c-terminus (s-egfp). the green fluorescence was conveniently used to monitor the expression of the protein in living cells and for observing the cell-based binding assay presented in this paper. cell-lines cho-k and vero e were purchased from american type cell collection (manassas, va, usa) and cultured at • c in % co in dmem containing g l − glucose, . mg ml − streptomycin and u penicillin and % fbs (hyclone, utah, usa). to maintain constitutive high level s-egfp expression in cho-sg, the culture medium was supplemented with m znso (tan and hong, ) . sars-cov strain sin (ruan et al., ) , an isolate from a sars patient in singapore was used in this study. the cdna for the cloning of s gene has been previously published (tan et al., a) . the vector pegfp-n as a control for the expression of egfp was purchased from clontech (palo alto, ca, usa). primers for constructing s-egfp from sars-cov strain ( - ) are sarsf ( -gcctcgaggccac-catgtttattttcttattatttcttactc- ) and sar-sr ( -gccccgggatgtgtaatgtaatttgacaccc- ), the s gene without the stop codon was pcr amplified and inserted into the xhoi/xmai sites of pegfp-n for making the fusion gene. the plasmid pegfp-n -s-egfp was further digested with xhoi and noti and then inserted into the xhoi/noti sites of pmmtc vector (tan and hong, ) . the pcr cycles comprised a denaturation step at • c for min, cycles of • c for s, • c for min, • c for min followed by a final elongation step at • c for min. the s-egfp construct was confirmed by dna sequencing. around % confluent cho cells were transfected with s-egfp and pegfp-n using dmrie-c (gibco/brl, gaithesburg, md, usa; brenner et al., ) according to manufacturer's protocol. in general, g ml − dna construct was mixed with g ml − dmrie-c in optimem (gibco/brl). to obtain stable transfectants, cho-sg (s-egfp transfected) and cho-g (pegfp-n transfected) cells were trypsinized h after transfection and then selected with mg ml − g (gibco/brl). the expression of egfp or s-egfp in transfected cells was detected by using a leica dmil-inverted fluorescence microscope fitted with fitc filter. cho-sg cells were lysed with lysis buffer ( % np in pbs, containing g ml − apotinin [sigma, st. louis, mo, usa], mm pmsf [sigma] on ice for h), and the lysate was then separated on % sds page and transferred to nitrocellulose hybond c membrane (amersham-pharmacia biotech, uppsala, sweden). prestained molecular markers were purchased from bio-rad (hercules, ca, usa). the membranes were blocked with % non-fat milk in pbst (pbs containing . % tween- ). antibodies against either gfp (monoclonal, clontech) or s (polyclonal antibod-ies from rabbit; keng et al. (submitted for publication) and horse; shen et al. (submitted for publication)) were used as primary antibody; horseradish peroxidase (hrp)-conjugated sheep anti-mouse (amersham-pharmacia biotech), goat antirabbit (pierce, rockford, usa) or goat anti-horse (bethyl, tx, usa) were used as secondary antibodies and detection was performed with a chemiluminescence kit (pierce). s-egfp was partially purified by using an agarose bound wga (wheat germ agglutinin; vector laboratories, ca, usa) column at • c. cho-sg cell lysate was first loaded onto wga column pre-washed with pbs, then the column was washed again with pbs and subsequently three column volumes of . m n-acetyl-d-glucosamine (glcnac; sigma) in pbs was used to elute the glycoproteins. s-egfp was identified by western blot analysis using rabbit anti-s antibody. non-permeable immunofluorescence staining was used to identify cell surface expression of s-egfp. cho-sg cells were dislodged with . % edta in pbs, and fixed after incubating with primary (patient's serum sample p in tan et al., a) and secondary (rhodamine-conjugated anti-human igg [sigma]) antibody reactions (both antibodies were diluted at : in growth medium and incubated on ice for h), and then loaded onto black teflon memzel slides (merck, darmstadt, germany) and fixed with % paraformaldehyde. the samples were viewed with a zeiss microscope (carl zeiss vision gmbh, hallbergmoss, germany). for all the cell-based binding assays presented in this paper, vero e cells were grown to a confluent layer and the cho-sg and cho-g cells dislodged by . % edta were used as ligands. cho-sg and cho-g cells were resuspended in growth medium and a similar amount of cells were laid over the vero e cell layer at • c for h. after the incubation, the cells were washed once with pbs and then three times with pbs containing nacl of mm, mm, mm, mm, mm or m. to determine whether ace mediates cho-sg and vero e interaction, the vero e cell layer was pre-treated with g ml − of goat anti-ace antibody (r&d systems, mn, usa) in growth medium at • c for h. then the antibody containing medium was removed, followed by incubation with cho-sg cells for two more hours. cells were washed once with pbs and three times with pbs containing mm nacl. to test whether the anti-sera from sars patients or raised against s protein fragments can block the binding between cho-sg and vero e , dislodged cho-sg cells were preincubated with the anti-serum to be tested at • c for h. then the cells were washed with growth medium and laid over the vero e cell layer for two hours. cells were washed once with pbs and then three times with mm nacl. five rabbit anti-s sera were obtained from the immunizations with recombinant s fragments, s (a.a. residues - ), s ( - ), s ( - ), s ( - ) and s ( - ), expressed in escherichia coli (keng et al., submitted for publication) and then sds page separated antigens were purified from gel slices. one mg each of the purified fragments were mixed with equal volume of complete freud's adjuvant (sigma) and used to immunize new zealand white rabbits. all anti-sera used were undiluted including normal human serum and the controls of pre-immune rabbit sera. results of the cell-based binding assay were detected by using a leica dmil-inverted fluorescence microscope fitted with fitc filter. in order to confirm the blocking effect by rabbit anti-s sera, cell lyastes (vero e and cho-sg binding cells) were analyzed by western blot using rabbit anti-s antibody. in order to clone the s portion of s-egfp, we used the viral strain (sin , accession number ay ) (ruan et al., ) isolated from a sars patient in singapore as template for pcr amplification. the primers for amplifying the full-length s gene without the stop codon were designed according to sin sequence (nucleotides to ); the amplified pcr fragment was fused with the egfp gene (fig. a) in pegfp-n . an extra octapeptide (trdppvat) from the egfp vector was generated at the junction, which should have limited effect on the transportation and expression of the fusion protein. for the expression of s-egfp in cho cells, the fusion construct was then cloned into pmmtc (tan and hong, ) . the transmembrane and cytoplasmic domains of the s protein were retained in the construct for proper anchoring and orientation of the ectodomain on the surface of cho cells as well as the intracellular localization of egfp. the expression of s-egfp was detected by using fluorescence microscope. a stable clone, cho-sg (fig. b) , expressing a relatively high level of s-egfp at the cytoplasmic membrane was selected for the study described below. next, we used western blot analysis to confirm the correct expression. s-egfp in the cell lysate of cho-sg (fig. ) was identified by a mouse anti-gfp monoclonal antibody (lane a), and two sera from rabbit (lane b) and horse (lane c) raised against the s protein. all the three antibodies recognized a common band of ∼ kda. the predicted molecular weight (mw) of sars-cov s protein is ∼ - kda (xiao et al., ) ; therefore, the mw of s-egfp is approx- imately - kda with additional kda from the egfp tag (fig. ) . these results concur with the observations from another group (xiao et al., ) that the recombinant s protein was intact in the host cells. additionally, subcellular fractionation (chou and omary, ) together with western blot analysis showed that s-egfp was present mainly in the fraction of plasma membrane (data not shown). the lectin wheat germ agglutinin (wga) is known for its ability to bind the terminal n-acetyl-glucosamine (glc-nac) of glycoproteins. in order to determine the carbohydrate characteristics of the glycoprotein s-egfp, the cell lysate of cho-sg was passed through a wga-agarose column. western blot analysis using rabbit anti-s serum was used to monitor the wga agarose chromatography. fig. shows that nearly all s-egfp was retained on the column (lane f); whereas, it was absent in the flow-through sample (lane e). this result indicates that nearly all s-egfp molecules are glycosylated with glcnac(s) at the end of the oligosaccharide side chains. although fig. b shows that the s-egfp is localized to the plasma membrane, the result did not provide information about its orientation. since the ectodomain of the s protein was expected to be outside the cell, non-permeable ifa was applied to confirm the orientation of the fusion protein. a serum sample (p ) from a convalescent sars patient (tan et al., a) was used as the primary antibody to stain the cho-sg cells followed by staining with a rhodamine-conjugated anti-human igg (fig. ) . the normal human serum was used as the negative control. the result indicates that the s portion of s-egfp was exposed extracellularly of the cho-sg cells. fig. . cho-sg expresses s-egfp at cell surface with expected orientation. non-permeable ifa using the serum (p in tan et al., a ) from a recovered sars patient showed cell surface staining of s-egfp expressing cho-sg cells. the control is normal human serum. the green fluorescence seen in the upper panels was from the egfp tag and the red fluorescence observed in the lower panels was from rhodamine labeled secondary antibody. since ace on the surface of vero e cells is responsible for the binding of and the fusion with sars-cov , we tested the binding activity between cho-sg and vero e cells as well as the binding strength between the ligand (sars-cov s protein on cho surface) and the receptor (ace on vero e ). for the control in the cell-based binding assay, a stable egfp-expressing cho clone (cho-g) was generated by introducing the plasmid pegfpn into cho cells. both cho-g and cho-sg were tested for binding to vero e cell layers and the results are shown in fig. a . the cho-g cells resisted the pbs wash but were detached by the solutions containing supplemented nacl. in contrast, the binding between cho-sg and vero e could resist up to m nacl. we sought to investigate the binding specificity of this interaction by testing whether the serum from a recovered sars patient (p in tan et al., a ) and a goat anti-ace antibody could block the interaction. to test the blocking effect, either cho-sg cells were pre-treated with human sera before incubated with vero e cell layers, or vero e cells were pre-treated with anti-ace antibody before interacting with cho-sg. all panels in fig. b were washed with mm nacl, the controls for the patient's serum was normal human serum, and for anti-ace was pbs. the results showed that both the patient's serum and the anti-ace antibody could block the binding, suggesting that this binding is specifically mediated by the s protein and ace . although, the receptor for sars-cov has been found, it is still important to localize the epitopes that induce neutralizing antibody against the virus, particularly for the purposes of vaccine development. we attempted to localize the epitopes by expressing recombinant s fragments (fig. a ) in e. coli and then immunizing rabbits with the purified fragments. to determine which anti-serum may block the binding, cho-sg cells were pre-treated with the rabbit sera individually before incubating with vero e cell layers. after the incubation, the cells were washed once with pbs and times with mm nacl. the anti-s serum demonstrated the blocking effect ( fig. b and c) indicating that some blocking epitopes are present in s . cho-sg that constitutively expresses a significant amount of s-egfp provides a convenient tool to study the recombinant sars-cov s protein. our results showed that the ectodomain of the s protein on the surface of cho cells mimics the interaction between the virus and vero e cells, presumably through the cellular receptor ace . the cellbased binding assay introduced in this paper should facilitate the screening for drugs, peptides or vaccines that block the entry of sars-cov, minimizing the use of the highly infectious live virus. the clonal cell-line cho-sg may be used for large-scale production to supply s-egfp as a diagnostic marker, and its affinity to wga is a favorable property to assist the purification of s-egfp. although, the expression of s-egfp on cho-sg was significant, self-fusion of cho-sg cells has not been observed in culture, which may be due to the absence or a low level of ace on cho (ovary) cells (donoghue et al., ) . the expressions of recombinant s protein in full-length or fragments had been demonstrated previously xiao et al., ; wong et al., ) by using human kidney cell-lines or t. we also tried two human cell-lines, t and jurkat, to express s-egfp but stable clones were not established due to the undetectable green fluorescence from g resistant colonies. the sars convalescent patient's sera (p and p in tan et al., a) blocked the interaction between cho-sg cells and vero e , confirming the authenticity of this assay. serum sample p does efficiently identify the extracellular expression of the s ectodomain on the cell surface of cho-sg by non-permeable ifa (fig. ) , but with lower potency to block the binding (data not shown in fig. b ). epitopes of sars-cov s protein responsible for the binding to ace was first demonstrated to be in the s region and then narrowed down to the amino acid residues - (babcock et al., ) , - (xiao et al., ) and - (wong et al., ) . the binding and fusion interactions were performed by using expressed soluble s fragments and the cellular receptor. we used the cell-based binding assay to test the blocking effect for five rabbit anti-s sera raised against five recombinant s fragments and three of these fragments, s , s and s (fig. a) covered the a.a. residues from to , which overlapped with the predicted s domain or the s-ace binding region. however, we were only able to identify a significant blocking effect ( fig. b and c) from anti-s (a.a. - ) rabbit serum. in order to rule out non-specific effects from undiluted serum, pre-immune rabbit sera were used as the controls for the binding assay and no blocking effect was ob- fig. . cho-sg and vero e binding assay. in all panels, vero e cells were first cultured to a confluent layer. (a) edta dislodged cho-g (upper panels) and cho-sg (lower panels) were incubated with the vero e cell layers in the cold room for h. cells were washed once with pbs and followed by three washes with pbs containing different amounts of nacl as indicated at the bottom of the figure. (b) the photos shown in the upper panels were taken under tungsten lamp and these in the lower panels were taken under green fluorescence. cho-sg cells were pre-treated with either human normal serum (a) or sars recovered patient's serum (p in tan et al., a) (b) before being added to the vero e cell layer. the cells were washed with mm nacl after the incubation with vero e cells. vero e cultures were pre-treated with goat anti-ace antibody (d) or the pbs control (c) and then incubated with the cho-sg cells followed by mm nacl washes. served. the rabbit sera produced against fragment s (a.a. - ) and s ( - ) also presented some minor blocking effects ( fig. b and c) . rabbit serum anti-s (a.a. - ) could not block the binding (or anti-s with less significant effect) may be due to the antigenicities for those modules requiring higher extent of glycosylation or the native conformation. however, although the region of s contains nine predicted n-glycosylation sites, the rabbit antiserum raised against the denatured fragment produced from e. coli was still able to block the binding. this indicates that there is a non-glycosylated and linear epitope within s that is important for s-ace binding. although the rabbit anti-s serum against s could not block the binding effectively, it was routinely used in a dilution of : as primary anti- fig. . identification of the receptor-binding region of sars-cov s gene. (a) five fragments of sars-cov s protein were expressed in e. coli. rabbits were immunized with these five recombinant s protein fragments. the rabbit anti-sera were tested for blocking cho-sg and vero e cell interaction. (b) the cho-sg cells were first treated with pre-immune serum or one of five rabbit anti-sera described in (a), and then added to the vero e cell layer. after incubation, the cells were washed with mm nacl. the rabbit serum from s had the most significant blocking effect. (c) western blot analysis using rabbit anti-s antibody to show the amount of s-egfp in (b) samples. body for western blot analysis (fig. ) . all sera from either humans or rabbits used in the binding assay were neat; however, diluted sera ( : ) were tested and revealed insignificant blocking effect. our results identified a receptor binding domain of the s protein required lesser extent of glycosylation and native conformation. the blockage of cho-sg cells to vero e cells by anti-ace antibody was nearly complete (fig. b) , indicating that ace is the main receptor on the surface of vero e cells responsible for the binding. however, we cannot exclude the possibilities of other receptors involved in the binding, such as the one closely associated with ace , which may also be blocked by an anti-ace antibody. in patients with sars, atypical pneumonia and diarrhea were common symptoms caused by sars-cov, correspondently the mrna levels of ace were comparably high in gastrointestinal system and lung (harmer et al., ) and the higher ace protein levels have been demonstrated in the epithelia of the lung and small intestine (hamming et al., ) . two human lung cell-lines (mrc and a ) and a human colon cell-line (ht ) were tested for developing the binding assay but results were unexpected due to non-specific binding between cho-sg and these cells. other human cell line of lung or digestive tissues will be examined in the future. ported by the agency for science and technology (a*star) of singapore. amino acids to of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor conserved regulation of the lymphocytespecific expression of lck in the fugu and mammals mitotic arrest with anti-microtubule agents or okadaic acid is associated with increased glycoprotein terminal glcnac's a novel angiotensin-converting enzyme-related carboxypeptidase (ace ) converts angiotensin i to angiotensin - characterization of a unique group-specific protein (u ) of the severe acute respiratory syndrome coronavirus coronavirus spike proteins in viral entry pathogenesis tissuedistribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis quantitative mrna expression profiling of ace , a novel homologue of angiotensin converting enzyme amino acids to in the s domain of sars coronavirus s protein induces neutralizing antibodies: implications for the development of vaccine and anti-viral agent localization of neutralizing epitopes and the receptor-binding site within the amino-terminal amino acids of the murine coronavirus spike protein angiotensin-converting enzyme is a functional receptor for the sars coronavirus comparative full-length genome sequence analysis of sars coronavirus isolates and common mutations associated with putative origins of infection unique and conserved features of genome and proteome of sarscoronavirus, an early split-off from the coronavirus group lineage molecular modelling of s and s subunits of sars coronavirus spike glycoprotein localization of neutralizing epitopes and receptor-binding site in murine coronavirus spike protein gene expression in mammalian cells profile of antibody responses against sars-coronavirus recombinant proteins and their potential use as dianostic markers a novel severe acute respiratory syndrome coronavirus protein, u , is transported to the cell surface and undergoes endocytosis a -amino-acid fragment of the sars coronavirus s protein efficiently binds angiotensin-converting enzyme the sars-cov s glycoprotein: expression and functional characterization identification of a novel protein a from severe acute respiratory syndrome coronavirus virus-encoded proteinases and proteolytic processing in the nidovirales we thank dr. eng eong ooi (environmental health institute, national environmental agency, singapore) for providing inactivated sars rna; drs. yue wang, ding xiang liu, le-ann hwang and wei-ping yu for their precious suggestions; and ms. chay boon loh for her assistance in ifa and the dna sequencing facility in imcb. this project was sup- key: cord- -er ug w authors: maayan-metzger, ayala; itzchak, amir; mazkereth, ram; kuint, jacob title: necrotizing enterocolitis in full-term infants: case–control study and review of the literature date: - - journal: j perinatol doi: . /sj.jp. sha: doc_id: cord_uid: er ug w objective: to examine the increasing number of full-term infants at our hospital exhibiting necrotizing enterocolitis (nec) in order to characterize these cases and to discover common risk factors. methods: medical charts were reviewed for all full-term infants (gestational age > weeks) that were born in our institution during a -year period (from january , to december , ) and that developed definite nec. data regarding the rate of cesarean section (cs) in our institution over the study period and five years prior to the study was also recorded. results: during the years of the study, full-term infants were found to have nec. the incidence of nec in full-term infants increased from . to . per live births in the -year period. mean birth weight was g. all the nec infants except one were delivered by cs, and all of them were fed either with a mixture of breast milk and formula or entirely by formula. seven of the infants ( %) had no major known risk factors predisposing them for nec. mean age of disease onset was very early ( . days) in most of the infants ( infants), and the colon was the main nec site. the short-term outcome was favorable in all but one case, which required explorative laparotomy for intestinal perforation. the number of infants born by cs has been steadily increasing, and was almost three times greater during the study period in comparison to the preceding years. conclusions: the etiology of nec in the full-term population seems to differ from the etiology for the preterm group in its intestinal location and in the timing of its onset. the increase in the rate of cs over the years might be related to the concurrent increase in nec, and this relationship should be further investigated. the most important risk factor for necrotizing enterocolitis (nec) is prematurity. nonetheless, the incidence of nec among the population of very low birth weight (vlbw) infants at our hospital has been decreasing over the years, as has the incidence of mortality and morbidity from this disease. over the past years, however, our hospital has experienced an increase in the incidence of full-term babies exhibiting nec. nec in full-term infants is well documented, accounting for about % of babies with nec; it usually involves babies with known risk factors, such as intrauterine growth retardation (iugr), birth asphyxia, congenital heart disease, gastroschisis, polycythemia, hypoglycemia, sepsis, exchange transfusion, umbilical lines, milk allergy, premature rupture of membranes with and without chorioamnionitis and gestational diabetes. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the purpose of the current study was to characterize this group of full-term infants with nec, to discover whether there are any common known risk factors, and to review the relevant literature. medical charts were reviewed for all full-term infants (gestational age > weeks) that were born in our institution from january , to december , and that developed definite nec. for the purpose of this study, only the files of those babies exhibiting bell stage ii and beyond were reviewed. for those babies, all relevant maternal and infant details were recorded, including details of maternal illnesses and drugs during and prior to the pregnancy, mode of delivery, birth weight, sex, apgar scores, type of feeding prior to disease onset, bilirubin and hematocrit level, blood glucose levels, drugs or procedures administered prior to disease onset, other documented diseases, age at onset of first symptoms, abdominal x-ray interpretations, blood count and culture results, duration of clinical and radiological disease, and early and late complications as evidenced by rehospitalizations in our pediatric department. no significant changes were made in terms of feeding patterns, percent of breast feeding in our nursery ( to %), timing of feeding initiation after birth ( to hours) and type of formula given. no infection outbreaks or change in colonization were recorded during the study time period. there were no changes in obstetrical policies, such as epidural anesthesia or type of medications used (analgetics, antibiotics or other drugs) except for an overall gradual increase in the rate of cs. the incidence of infants who are small for gestational age remained the same in our institution, about %, over the study period. the work-up on each of the infants in the study included complete blood count and blood culture, stool cultures for bacteria (salmonella, shigella, enteropathogenic escherichia coli, yersinia and campylobacter) and viruses (enteroviruses and rota virus), blood electrolytes, and renal and liver function tests. serial abdominal x-rays were taken until complete resolution of clinical and radiological findings. feeding was resumed days after normalization of abdominal x-rays. each infant received intravenous cefotaxim and amikacin for a total of days until cultures came back negative. table shows that over the last years, the incidence of nec among full-term infants at our institution has increased, from . per live births (full-term infants only) in to . per live births in . this increase has been conspicuous mainly over the last years, as shown in the table. the incidence of cs increased from % in to % in (in comparison to to % in the previous years). during the years of the study, full-term infants were found to have nec. in this same period, the rate of nec among vlbw infants born at our hospital decreased from . % in to % in . maternal diseases observed in this group of infants included gestational diabetes, hypothyroidism and turner's syndrome. all but one of the infants with nec were born by cs, six due to nonreassuring fetal heart rate monitoring without apparent birth asphyxia (with one exception). the other seven infants were born by elective cs due to breech presentation, twin pregnancy or previous cs. as shown in table , seven infants had known predisposing risk factors for nec, such as growth retardation, polycythemia, congenital hypothyroidism, birth asphyxia, congenital heart malformation, and one infant with an as yet undiagnosed syndrome. the other seven infants either exhibited no obvious predisposing risk factors or had some common mild conditions, such as hyperbilirubinemia, early asymptomatic hypoglycemia and transient tachypnea. none of these infants was entirely breast-fed. the age of nec onset in most of these babies was only a few days, except for two infants (n & ), with congenital heart disease and undiagnosed syndrome, respectively. nine babies had colon involvement, and in five babies, the radiologist was unable to unconditionally detect the exact location of the nec. one baby was operated on for early perforation. all blood cultures were negative; in one case the urine culture was positive, and in one case clostridium toxin was detected in the stool. all infants were discharged to their homes in good condition. during a -month follow-up period, two infants were re-admitted for obstructive ileus due to late-onset intestinal strictures that required explorative laparotomy with end-to-end anastomosis. the incidence of nec among full-term infants at our hospital has increased markedly over the last years, while the incidence of nec in vlbw infants has gradually decreased. the findings of the current study show that only half of these full-term infants with nec exhibited known predisposing risk factors, including congenital heart disease, undiagnosed syndrome, birth asphyxia, intrauterine growth retardation and congenital hypothyroidism. most of these infants had colon involvement, making this location typical among this group as compared to the preterm group, where the most common site is the jejunum and ileum. the early age at which the first clinical signs became obvious for most of the group is also unique as compared to reports for the preterm group, in whom the mean age of onset is days of age. the incidence of nec in infants with birth weight over g has previously been reported as . per live births, and about to % of all nec cases are term infants. , , , over the last years, a five-fold increase in the number of full-term infants with nec was detected in our population, reaching an incidence of . per live births in , equivalent to % of all nec cases in our institution that year. the clinical presentation of the infants at our hospital was similar to that described for preterm infants, , including abdominal distension, vomiting, bloody stools and septic appearance. we experienced no mortality among our group of patients, whereas the literature describes and % mortality for full-term and preterm babies, respectively. nec appears to occur earlier in full-term infants. in a casecontrol study of full-term infants, wiswell found the median onset of nec to be only days ( developed nec on the first day of life). ruangtrakool studied full-term infants; among them, nec developed . days after birth, in comparison to . days after birth in preterm controls. kabeer also found that nec appeared earlier in a group of full-term infants: at . days versus . days (in preterm infants). in the current study, the group mean age of nec onset was . days among a group of babies; however, for two other babies, one with congenital heart disease and the other with an as yet undiagnosed syndrome, nec appeared much later, suggesting another pathogenesis. the early age of onset may suggest a prenatal ischemic insult in six infants in the study group born by cs due to non-reassuring fetal monitoring, although without postnatal clinical signs of birth asphyxia (except one case). another seven infants were born by elective cs. the possible relationship between increasing rates of cs and early nec in full-term infants may be explained by the fact that sicker infants are saved by cs and by improved neonatal resuscitation. however, the low rate of birth asphyxia in the study group as well as the high rate of elective cs do not support such a hypothesis. so far, cs on its own has not been considered a risk factor for nec by most authors, although uauy et al. did mention cs as a significant risk factor in their group of vlbw infants with nec. recently, minkoff and chervenak discussed a possible mechanism by means of which the anesthesia involved in elective cs causes a possible transient drop of blood pressure, perhaps thus compromising placental blood supply and fetal circulation. in addition, the maternal supine position prior to and during the operation may further reduce fetal circulation and thus interfere with intestinal blood supply. the increasing incidence of cs in our institution may play some role in the higher incidence of nec observed in this study. about % of all elective cs in our institution are under epidural anesthesia capable of compromising splanchnic blood supply to the fetus. this hypothesis should be further investigated by means of fetal doppler blood flow measurements. in the literature, proposed risk factors for nec in term infants include cyanotic heart disease, perinatal asphyxia, hypoglycemia, polycythemia, respiratory distress syndrome, protracted diarrhea, pre-eclampsia, cocaine abuse during pregnancy, cows' milk allergy and anti-c rhesus incompatibility. , , , one case-control study of infants weighing over g at birth who developed nec found a higher frequency of premature rupture of membranes, chorioamnionitis, apgar score less than for the first and fifth minute, respiratory distress syndrome, congenital heart disease, hypoglycemia and exchange transfusion, but no greater frequency of pre-eclampsia, maternal diabetes, maternal drug abuse, meconium-stained amniotic fluid or polycythemia. in that study, only three patients were healthy prior to development of nec. other studies have also confirmed the hypothesis that the occurrence of nec in a full-term infant is usually associated with a predisposing event. out of full-term infants with nec, bolissety found only two that had no prior illness known to be associated with the development of nec. beeby reported eight full-term infants with nec, all with a predisposing event. similarly, wiswell found an unremarkable course prior to the development of nec in only three of full-term infants with nec. our study shows only a % rate of infants with known risk factors for nec. the other seven infants had no known risk factors for nec, but still exhibited some minor medical conditions, such as asymptomatic hypoglycemia, mild tachypnea, hyperbilirubinemia requiring phototherapy, and delivery by cs with or without mild abnormal fetal heart monitoring and with no apparent birth asphyxia. several studies identified a connection between congenital heart disease and nec in term infants. the frequency of this connection ranged from . % to % of term infants with nec: polin f out of , thilo f out of , andrews f out of and bolissety f out of . to the best of our knowledge, only two cases of hypothyroidism and nec have been described so far. the first was the case of a day-old full-term infant with a history of hyperbilirubinemia requiring phototherapy. positive clinical signs included persistent open posterior fontanelle. no thyroid tissue was identified by ultrasound. the infant was treated with intravenous thyroxin. the second patient developed nec at the age of weeks, days after nissen funduplication for severe gastro-esophageal reflux. prior history included hypoxic-ischemic encephalopathy with seizures within the first hours, treated with anticonvulsants. thyroxin was commenced at age days. investigation confirmed thyroid dysmorphogenesis. both infants were diagnosed by the newborn screening test, as was the case presented in our study. a pathogenic relationship between hypothyroidism and nec has been suggested. thyroid hormone deficiency causes decreased gut motility due to peripheral neuropathy of the intestine. in the presence of feeding, this may allow for intestinal bacterial overgrowth, increased gas production and abdominal distension. a second factor relating the hypothyroid state with nec is the hemodynamic effect of thyroid hormone deficiency. in the fetal and perinatal periods, the thyroid hormone is required for normal increase in cardiac output in response to catecholamines. an unsatisfactory increase in cardiac output in response to stress may lead to decreased mesenteric blood flow and bowel ischemia. nec in term infants has also been associated with the repair of gastroschisis and myelomeningocele and lipomyelomeningocele. the pathogenesis is unknown, but several factors appear to play either a primary or a secondary role, including infectious agents and toxins, enteral alimentation, exposure to anesthetic agents and mesenteric ischemia with tissue hypoxia. the development of nec most probably involves multiple factors in the setting of a stressed intestinal system with immature protective mechanisms. about % of nec infants in the study group were receiving all of their meals by mouth prior to the development of nec. it is hypothesized that ( ) incompletely digested formula can provide a substrate for bacterial proliferation and ( ) feeding increases intestinal oxygen demand during nutrient absorption, thus increasing the risk of intestinal tissue hypoxia that can cause mucosal injury and bacterial invasion. most neonatologists increase feeding volumes gradually in preterm infants, although this is usually not the case for full-term infants. human milk appears to be beneficial in preventing nec, most probably by enhancing growth of lactobacilli and by immunologic mechanisms. , the infants in the study group were distinguished by a lack of sufficient breast milk. for many years, mesenteric ischemia was postulated to be the common denominator in nec. cardiovascular stresses such as hypotension, hypothermia, hypoxia, feeding, anemia and umbilical vessel catheterization are all associated with the development of nec. the immature intestines may have reduced ability to regulate blood flow and oxygenation. bacterial colonization of the intestinal tract is a prerequisite for initiation of nec. the occurrence of nec in epidemics suggests that an infectious agent may play a role in the pathogenesis of nec. numerous bacteria and viruses have been isolated in epidemics, including pseudomonas aeruginosa, e. coli, klebsiela pneumonia, clostridium perfringes, butyricum and difficile, enteroviruses, coxsackie b virus, corona virus and rotavirus. nonetheless, most of these isolated organisms are part of the normal intestinal flora. supplementation with bifidobacterium infantis and lactobacillus acidophilus reduced the incidence of nec, probably by preventing the overgrowth of more pathogenic enterobacteria. , , this etiology could not explain the high incidence of nec in the study infants described here. locally produced cytokines may have a detrimental effect during the evolvement of nec. other mechanisms are in place for downward regulation of this proinflammatory activation. a relative deficiency in protective responses may account for the propensity of intestinal injury in premature infants. the concentration of interleukin and (il- , il- ) and tumor-necrosis-factor-alpha (alpha-tnf) is elevated locally. nitric-oxide (no) production by enterocytes is increased and results in enterocyte apoptosis through peroxy-nitrite formation. conversely, no deficiency may also predispose the intestine to nec. moreover, deficiencies in both magnesium and copper can damage the gut's antioxidative defense and produce a pro-nec intestinal cytokine profile. the phospholipid inflammatory mediator, platelet-activating factor, has also been implicated in the pathogenesis of nec. erythropoietin is a trophic hormone that protects against intestinal cell death and was found to have a protective role, as did epidermal growth factor and hepatocyte growth factor. in conclusion, only half of our full-term infants exhibited any major known risk factors that predisposed them to nec. the early onset of nec both with and without any major risk factors might suggest a common mechanism related to a perinatal ischemic insult. such an insult may compromise the intestinal blood supply without our ability at present to detect any sign of it prior to feeding initiation. the increasing rate of cs in our institution, together with the fact that all the study infants except one were born by cs, either elective or urgent, might suggest that cs plays a role in the pathogenesis of nec. the etiology of the increasing number of full-term infants with nec in our institution and the possible role of cs in its pathogenesis deserve further investigation. neonatal-perinatal medicine nelson textbook of pediatrics a regional study of underlying congenital diseases in term neonates with necrotizing enterocolitis necrotizing enterocolitis in term infants necrotizing enterocolitis in fullterm or near-term infants: risk factors necrotizing enterocolitis in the first hours of life necrotizing enterocolitis in term neonates the development of necrotizing enterocolitis following repair of gastroschisis: a surprisingly high incidence neonatal necrotizing enterocolitis. therapeutic decisions based upon clinical staging necrotizing enterocolitis in the full term neonate new concepts in necrotizing enterocolitis necrotizing enterocolitis in full-term infants, a case-control study necrotizing enterocolitis: a comparison between full-term and pre-term neonates neonatal necrotizing enterocolitis. a year review at a country hospital necrotizing enterocokitis in very low birth weight infants: biodemographic and clinical correlates. national institute of child health and human development neonatal research network elective primary cesarean delivery gastro-intestinal complications following neonatal cardiac catheterization risk factors for necrotizing enterocolitis: the influence of gestational age necrotizing enterocolitis and hypothyroidism in a newborn infant: treatment with intravenous l-thyroxin key: cord- -l r f sr authors: lee, chi-wei; tsai, yen-shuo; wong, tai-wai; lau, chor-chiu title: a loophole in international quarantine procedures disclosed during the sars crisis date: - - journal: travel med infect dis doi: . /j.tmaid. . . sha: doc_id: cord_uid: l r f sr this study describes a loophole in the international quarantine system during the recent asian severe acute respiratory syndrome (sars) outbreak. specifically, that of travelers disguising symptoms of respiratory tract infection at international airports, in order to board aircraft to return to their home countries—notwithstanding the infection risks this involves to others. high medical fees for treatment to non-residents in epidemic areas were found to be the main cause for this behaviour. this phenomenon revealed a loophole in the control mechanisms of international quarantine procedures, letting travelers carrying a highly contagious virus slip by undetected and causing possible multi-country outbreaks of communicable diseases. clinical evidence collected from medical records at medical centers can highlight this oversight. from november onwards, the severe acute respiratory syndrome (sars) had spread rapidly via international air travel to at least countries. as of december , , reported cases had numbered with deaths. reasons for its rapid global spread were the highly contagious nature of the virus with its air-borne route of infection, the busy links between affected countries, and probably inadequacies in international quarantine procedures. the increasing volume of international tourism and trade has raised the risks for translocation of exotic diseases. in other words, the increased mobility, mixing and congregation of civilian populations from different nations increase the rate of transmissible diseases. , countries need to cooperate more closely in the future, not only on finding the causes and the management of epidemic outbreaks, but also on preventing the further spread of them. for example, sars had affected people in many areas: in canada (toronto); in china (guangdong, hong kong, shanxi, beijing); in taiwan; in singapore; and in vietnam (hanoi). on march , , the world health organization (who) issued a global alert, . recommending that national authorities implement heightened surveillance for cases of sars. recommendations were aimed at limiting the spread of sars and protecting international air passengers. the screening measures for potential sars symptoms instituted by national health officials and port authorities, included interviews with passengers, as well as the taking of tympanic core body temperatures from boarding and disembarking passengers by way of electronic thermometers and infra-red cameras. national authorities also advised travelers with fever to postpone international travel from sars documented areas. international travelers were educated through the public media on the symptoms of sars and were advised to seek immediate medical attention should such symptoms occur. according to the who report, evidence had indicated that since the start of sars global surveillance at the end of february , a number of suspected and probable cases of sars had departed from affected countries on flights to other countries (http://www.who.int/csr/sars/ archive/ _ _ /en/). local transmission could conceivably have occurred inside the cabin of an aircraft to persons seated close to a sars infected person, by way of droplets discharged through coughs or sneezes. in total, nations ( administrative independent regions, including hong kong) were reported up to april , to be infected by the sars epidemic, in part a consequence of international travel. in fact, the international traveler is an efficient vector for sars as well as new respiratory pathogens yet to emerge. the aim of this study is to describe reasons for a loophole in international quarantine procedures. this study was divided into two parts: information was collected at the international airport of kaohsiung (taiwan), on the physical conditions of passengers who flew from hong kong and landed at kaohsiung from april to , . this information was compiled from questionnaires and brief interviews of arriving passengers. in addition, information was gathered from body temperature measurements performed on each arriving passenger who entered taiwan. records from at the emergency department of e-da hospital, in southern taiwan, of patients suffering from symptoms of respiratory tract infection who had departed recently from sars areas from march , onwards were analyzed. the investigation revealed that during this period a total of passengers were symptomatic during transit on board aircraft, meaning that at least and at most passengers per day were found to be symptomatic during their flight. since the flying time from hong kong to kaohsiung is only to minutes, it is reasonable to suspect that symptomatic passengers were actually aware of their symptoms before boarding the aircraft. thus they fully understood that they were possibly infected with the sars virus when departing from an endemic area and before heading for taiwan, despite the aggressive screening procedures put in place by the hong kong customs and department of health at hong kong's international airport. in order to explain the circumstances for this observed phenomenon and the underlying reasons for such behavior, we proceeded to the second part of our study. here, six patients visiting the emergency department of e-da hospital, from march to , , were found to show symptoms from respiratory tract infections, after they had departed from a sars endemic area and had entered the territory of taiwan by air. as shown in tables and , although none of the six patients were eventually diagnosed wild sars, this observed phenomenon disclosed a very important loophole in the control aspect of international quarantine procedures: the inability to prevent persons with a highly contagious virus from slipping past undetected and thus preventing the further spread of epidemics like sars on international travel routes. all of these patients admitted that extraordinarily high medical fees for non-residents in hong kong, was the major reason for them to hurry back to taiwan, where the cost medical care is significantly lower. in this study, we identified that there were loopholes in the international quarantine system for controlling the international spread of contagious disease like sars, especially when travelers lack a strong motivation to cooperate with national health authorities. this arises particularly when the high medical fees are imposed on non-local residents in endemic areas, were a significant financial burden. furthermore, the emergency room's medical records showed that patients were already aware of their symptoms such as cough or indications of high fever (though not necessarily sars) before they boarded their respective fights. nevertheless, they denied being sick before departure when questioned by health authorities, in full awareness of the infection risks, in order to reach taiwan. since taiwanese residents benefit from very low medical fees in their health care system, in contrast to hong kong's high hospital fees for non-residents. taiwan's medical fees are only v . (nt$ ) per attendance in the emergency room and just % of the total medical expense during the course of admission for in-patient care at a district hospital, with the remaining cost being subsidized by the national health care plan (see table ). tables and show a strong correlation in different medical fees for residents and non-residents both in hong kong and singapore. for example, the admission fee for in-patient care (general acute beds) is v . (hk$ ) per day in hong kong for residents or hong kong identity card holders, while a non-hong kong resident has to pay v . (hk$ , ) per day for the same treatment- times higher. in comparison, all taiwan nationals and residents are covered under the policy of the national health care insurance plan, and thus pay less than v . (nt$ ) per day for in-patient care. this cost differentiation for residents and non-residents in hong kong, is a phenomenon observed almost in every country in the world, with similar examples existing among different member countries in the patients admitted that they were symptomatic before their departure from epidemic areas of sars. european union. this is only a natural human response for a symptomatic traveler to disguise his/ her illness at their point of departure, to flee back to his/her home country for medical care at a significantly lower cost. however, this apparent trivial aspect of human behavior turns out to be a very serious problem in terms of epidemics and quarantine control measurements, where communicable diseases could be introduced into a population by the arrival of outside foreign infectives. citing the example discussed here, the possibility exists that travelers may return home from a foreign trip with an infection acquired abroad. while an experimental model indicates that screening and quarantining of infectives can considerably reduce the infective equilibrium. the egocentric human behavior of certain travelers who break quarantine rules, could be modified by better cooperation between governments. in today's highly mobile society, it is crucial to deter international travelers from spreading contagious diseases during an epidemic and lessons may be learned from the worldwide spread of sars so that precautions can be taken in the policy-making process for the future since a similar tragedy may repeat itself anytime, anywhere in the world. in response to the main issue identified in this report, governments need to set sensible medical fees for the temporary hospitalization of 'aliens' staying in their territories during periods of epidemic outbreaks. this cost could be shared by governments across the globe, with coordination by the who, so as to enforce quarantine measurements more efficiently. the existing cross-border care and international payment coverage policy within the european union can be seen as a good reference base for constructing such a cross-linking system to tackle this emergent problem of international quarantine. this measure may stop people from becoming disease-vectors within their home countries and also to other passengers on the same plane who may carry infection to many different destinations all over the world. thus, it is of importance, that governments in endemic areas publicize such policies, targeting foreigners staying within their boundaries during an outbreak. it is estimated that the sum of inter-governmental medical expenses incurred by infective or potential infective patients hospitalized at sensible cost in the 'host countries', would be substantially lower than the total social costs caused by the spread of communicable diseases, if these infectives were allowed to return to their mother countries. thus, the aim of future research should be focused on the health and safety investment as well as risk control methods. since global surveillance of sars began at the end of february , some evidence has suggested that a number of suspect and probable cases of sars were caused by persons being infected during travel on board aircraft (http://www.wpro.who. int/sars/docs/interimguidelines/part .asp). they were probably seated in close proximity to persons releasing droplets in the air on coughing or sneezing. such cases, gives impetus to more rigorous measures to prevent travel-related spread of sars or other communicable diseases. thus stricter travel enforcement must also be evaluated and added to existing quarantine measurements. this is in addition to the main focus of cross-border care and international payment coverage policies for aliens or temporary 'visitors' in afflicted areas. this sars outbreak may be regarded as a test of whether rigorous contact tracing and other stringent public health measures contained further spread, even though a large numbers of persons may have inadvertently been exposed to the virus. one intervention procedure to control the spread of infectious diseases is to isolate some infectives in order to reduce possible transmissions of the infection to susceptibles. total isolation may have been the first historical known infection control method, since biblical passages refer to the ostracism of lepers, then later in time plague victims were often isolated. the word 'quarantine' meant historically a period of days, the length of time that arriving ships suspected of plague were required to lay in anchor off the harbour before being allowed to dock. this practice started in the th century at mediterranean ports during the outbreak of the bubonic plague. the word quarantine has evolved to signify a forced isolation or a stoppage of interactions with others. over the centuries, quarantine has been used to reduce the transmission of human communicable diseases such as leprosy, plague, cholera, typhus, yellow fever, smallpox, diphtheria, tuberculosis, measles, mumps, ebola and lassa fever. the 'influenza pandemic preparedness plan' developed in the united states of america is an excellent model from which every other country in the world should learn and extrapolate its underlying spirit. this plan has as objectives: to limit the burden of communicable diseases, to minimize social disruption and to reduce economic loss in the future when similar outbreaks of pandemic does occur. early in , epidemiologists had warned the world that should the next pandemic be caused by a virus as deadly as that of influenza pandemic, the potential for disaster would be greater than ever. as the world's population is now more than three times greater than it was in , with nearly half that population residing in urban areas, including hundreds of millions crowded into slums and shanty towns in the developing world. faced with today's highly mobile transportation links, a virulent virus could easily spread around the world in a matter of days. another pandemic would challenge the world's public-health resources as never before. therefore, an effective response to future pandemics of viral infection proportions, such as the influenza pandemic, will demand the full support and complete cooperation of the public. yet the global health community is not prepared for the next viral pandemic, according to klaus stöhr (who, geneva, switzerland), speaking at the international congress on infectious diseases (singapore, march - , ) . furthermore, mankind's history and this case study provides evidence that travelers have contributed significantly to the rapid spread of aids , , influenza , and sars, therefore strict international quarantine enforcement, must be considered for future epidemics. only then, would we be ready to confront similar or even tougher challenges of pandemic outbreaks. in fact, bilateral, as well as regional agreements among different governments on visitors' health care are becoming more common. for example, an extensive list of countries has reciprocal health care agreements with the united kingdom. other non-economic reasons for return home of febrile passengers during the sars epidemic include fear for being infected in the epidemic region, reluctance to be isolated in a foreign country, unfamiliarity with foreign culture, planned travel schedule, etcetera. nevertheless, according to the record of the medical history taken in our emergency department, all six patients admitted that the big gap between medical costs in a foreign country and their mother country was the main reason for disguising their fever on departure. in contrast to sea-voyage, air travel journey is relatively short in duration. infected crew or passengers who travel on board ships would have their diseased status shown clearly during the long sea-voyage, and would have died or being quarantined when they arrived at their destined port. however, due to the relatively short duration of air-travel, the clinical condition of infected passengers on board airplane would not have sufficient time to progress to a serious stage which is obvious enough to detected by the custom at the destined airport. here lies the loophole of international quarantine which would be easily overlooked by airport custom, but not sea-port custom. thus, emergency departments or walk-inclinics are playing the important role in safeguarding the community from imported infectious diseases. emergency physicians should maintain a high level of awareness regarding potential outbreaks of infectious diseases of any kind and play a role in alerting public health authorities to any loopholes in quarantine procedures. the vulnerability of animal and human health to parasites under global change the philippines insurrection and the - cholera epidemic: part i-epidemiological diffusion processes in war the philippines insurrection and the - cholera epidemic: part ii-diffusion patterns in war and peace sars-lessons learned so far healthcare access and mobility between the uk and other european union states: an 'implementation surplus models for transmission of disease with immigration of infectives should i stay or should i go? waiting lists and cross-border care in the netherlands health a review of the application of health economics to health and safety in healthcare effects of quarantine in six endemic models for infectious diseases influenza pandemic planning: review of a collaborative state and national process epidemic and pandemic 'flu ready for the next influenza pandemic? hiv and pandemic influenza virus: two great infectious disease challenges travel and the spread of hiv- genetic variants further development of influenza surveillance in china and global impact on influenza control key: cord- - rjbmsvb authors: ng, m.l.; lee, j.w.m.; leong, m.l.n.; ling, a.-e.; tan, h.-c.; ooi, e.e. title: topographic changes in sars coronavirus–infected cells at late stages of infection date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: rjbmsvb scanning electron and atomic force microscopy was used for the first time to view the maturation of the severe acute respiratory syndrome–associated coronavirus at the cell surface. the surface form of the cells at advanced infection displayed prolific pseudopodia that, in addition to the rest of the plasma membrane, were also active sites of virus release. high magnification of the maturing virus particles showed a rosette appearance with short knoblike spikes under both the scanning electron and atomic force microscopes. the final expulsion step of the maturing virus particles seemed to result in some disruptions to the plasma membrane. the cytoskeletal network along the edge of the infected cells was enhanced and could be involved in transporting and expelling the progeny virus particles. thickening of the actin filaments at the cell edge provided the bending force to extrude the virus particles. scanning electron and atomic force microscopy was used for the first time to view the maturation of the severe acute respiratory syndrome-associated coronavirus at the cell surface. the surface form of the cells at advanced infection displayed prolific pseudopodia that, in addition to the rest of the plasma membrane, were also active sites of virus release. high magnification of the maturing virus particles showed a rosette appearance with short knoblike spikes under both the scanning electron and atomic force microscopes. the final expulsion step of the maturing virus particles seemed to result in some disruptions to the plasma membrane. the cytoskeletal network along the edge of the infected cells was enhanced and could be involved in transporting and expelling the progeny virus particles. thickening of the actin filaments at the cell edge provided the bending force to extrude the virus particles. a new human coronavirus was identified during the recent outbreak of severe acute respiratory syndrome (sars) ( ) ( ) ( ) ( ) . the outbreak started during november in southern china and then spread to hong kong, vietnam, canada, and singapore in early . sequence analyses of various isolates have indicated that the virus is genetically distinct from all known coronaviruses ( ) ( ) ( ) . phylogenetic analysis suggests that the sars-associated coronavirus (sars-cov) does not fit in the three currently known groups of coronaviruses ( , , , ) , which suggests that this is a new virus, not a result of mutation or recombination of known coronaviruses. coronavirus infections are common in both domestic animals and humans ( ) . however, the known human coronaviruses often cause coldlike symptoms, whereas recent infections caused by sars-cov do not. the rate of death for sars infections is %- %, depending on the age of the patients ( ) . sars-cov grows well in vero e cells ( , , ) and enters cells by direct fusion of the virus envelope with the plasma membrane ( ) . the fusion process involving the s glycoprotein is ph independent ( ) . once internalized, the virus core uncoats, revealing flattened, disc-shaped, and electron-dense nucleocapsids described as "doughnutshaped" ( , ) . the uncoated nucleocapsids are found within large, smooth, double-membrane vacuoles together with membrane whorls ( ) . these membrane whorls are postulated to be replication complexes for the virus since they appear very early (within min) after infection. other reports have described double-membrane vesicles as sites of replication for coronavirus (linder strain) ( ), mouse hepatitis virus ( ) , and sars virus ( ) . the latent period observed was - h postinfection ( ). however, a short latent period is common among coronaviruses ( ) . coronavirus infections can be cytocidal for the cells; or, in some cases, persistent infection can result ( ) . the outcome of the infection is dependent on the virus strains and cell types. unlike infection with the hepatitis c virus- e, wherein virus production can continue for weeks without any expression of cytopathic effects ( , ) , infection with sars-cov produces copious progeny virus particles within the first h ( ). the site of assembly of sars-cov was at the golgi complexes, similar to previous reports for other coronaviruses ( ) ( ) ( ) . after assembly, the virus progeny particles are transported in vesicles to the cell periphery for release. the aim of this study was to use scanning electron and atomic force microscopes to investigate changes in the surface topography of sars-cov-infected cells at late infection. the results can assist in further understanding how sars-cov interacts with infected cells at late infection. thus far, replication studies on sars-cov were performed with transmission electron microscopy, which showed detailed intracellular changes during replication in two dimensions. both scanning electron and atomic force microscopy can provide holistic and three-dimensional views as infection progresses. sars-cov ( va ) used for this study was isolated from a sars patient in singapore by the department of pathology, singapore general hospital. the virus was grown in vero e cells (atcc: c ) in the environmental health institute, national environmental agency, singapore. infection of the cells grown on coverslips and subsequent fixation ( % glutaraldehyde) of the infected cells at appropriate times were performed at that institute. the microscopy work on the fixed infected cells was performed at the electron microscopy unit, national university of singapore. vero cells were grown to % confluency on sterile glass coverslips in -well tissue culture plates before infection with µl of sars-cov for h (multiplicity of infection = ). maintenance media supplemented with % fetal calf serum was added to the wells, and the infected cells were incubated in °c incubator with % carbon dioxide. at an appropriate time after infection, the infected cells on the coverslips were fixed with % glutaraldehyde overnight. the coverslips were washed with phosphatebuffered saline before being postfixated in % osmium tetroxide for h. the coverslips were then washed with distilled water and dehydrated through a series of increasing concentration ( %- %) of ethanol. cells on the coverslips were further subjected to critical point drying for . h and left in a °c oven overnight. subsequently, the cells on the coverslips were sputter coated with gold (thickness of nm) and viewed under the xl field emission gun scanning electron microscope (fei company, enidhoven, the netherlands) at kv. infected cells were processed similarly. normally, samples for the atomic force microscopy should be subjected to minimal processing so that the samples are close to their natural condition. however, in view of the pathogenicity of sars-cov, only fixed and gold-coated samples were used for this study. the nanoscope iv multimode atomic force microscope was used (veeco instruments, woodbury, ny). force modulation etched silicon probes were used for imaging (dry tappingmode [veeco]) infected cells. hard tapping using appropriate amplitude setpoints was performed with some samples to show subsurface structures. purified virus fixed in . % glutaraldehyde was put onto a formvar carbon-coated grid and allowed to adsorb for a few minutes before being stained with % phosphotungstenic acid for min. the excess fluid was blotted and the grid left to dry before viewing under cm biotwin tem (fei company, enidhoven, the netherlands). both scanning electron and atomic force microscopy showed that the uninfected vero cells were flat and without prominent form and surface ( figure ). pseudopodia, where present, were not extensive ( figure a and b). in the transmission electron microscopy studies ( , , ) , sars-cov replicated very rapidly and produced large amounts of virus after h of infection. the scanning electron microscopy confirmed that, for some infected cells ( h postinfection), a large quantity of extracellular virus was present (figure a , arrowheads) on the whole cell surface. however, very few virus particles were on the neighboring cell (top right), indicating a nonsynchronous infection. the scanning electron microscopy images showed a holistic view of sars-cov-infected cells compared to ultrathin sections in transmission electron microscopy. another virus-induced change clearly demonstrated by using the scanning electron microscope was the proliferation of pseudopodia on the infected cells and in particular, at the edge of these cells (figure a , arrows compared to figure ). at higher magnification, progeny virus particles protruded at the cell periphery ( figure b , arrow). in the inset (boxed area), a virus particle was seen in the process of extrusion (arrow) after the fusion of the transport vesicle and the plasma membrane. the knoblike spikes surrounding the coronavirus were clearly visible. sars-cov spikes appeared short and stubby ( - nm) when compared to those of other coronaviruses ( nm). this feature gave the virus a rosettelike appearance when viewed under the scanning electron microscope (arrowheads indicate extruded virus particles). the average size of the extracellular virus particles was - nm. the gold sputter coating can also increase in the diameters of the virus particles. from to h after infection, the virus was exported prolifically at the pseudopodia and cell surfaces ( figure a -c, arrows). the surface imaging clearly showed the profuse presence of extracellular virus (arrows). high magnification scanning electron microscopy images of the sars-cov form and structure ( figure c , arrows) appeared to correlate well with those images that used negative staining and tem ( figure c , inset). the knoblike spikes were short and stubby in the negative staining image as well. online figure d (available at http://www.cdc.gov/ncidod/eid/vol no / - -g .htm) shows virus particles were also exported out from the surface of the pseudopodia (arrows). a virus particle in the process of extrusion at the cell plasma membrane was captured with the atomic force microscope at hours after infection. although the proposed mechanism for export of the virus to the extracellular space is through fusion of the transport vesicle membrane at the cell surface, this process seemed to result in localized breaching at the plasma membrane, where the virus extrusion occurred ( figure a, thin arrows) . although fixed and gold-coated samples were used in this study, the atomic force microscope delivered high-resolution images. unfortunately, the knoblike spikes for this virus were not well illustrated in figure a . a threedimensional reconstruction ( figure b ) shows that the virus particle was extruding from a much-thickened cell periphery (arrow). the knoblike structures on the virus surface were further confirmed by atomic force microscopy (online figure c , available at http://www. cdc.gov/ncidod/eid/vol no / - -g .htm). the thickened edges of the infected cells were ruffled and appeared to comprise layers of folded membranes ( figure a and b and online figure c virus particles (arrowheads) could still be exported out of the puffy edge ( figure a, arrows) . a three-dimensional reconstruction ( figure b ) of the height image in figure a shows puffy fronts of the cell edge (arrows) with many virus particles just underneath the surface awaiting extrusion. the large number of progeny virus particles at the cell edge may have resulted in this thickened appearance. virus particles (arrowheads) were pres-ent on other parts of the cell surface as well. thick white arrow shows a clump of virus particles just underneath the plasma membrane. closer examination of the virus-induced changes at the subcellular surfaces of the infected cells, by using the hard tapping mode under the atomic force microscope, showed the involvement of the cell cytoskeleton at late infection. in figure a , gross thickening of the cell skeletal filaments was seen in the cytoplasm (arrowhead) and pseudopodia (arrows). at higher resolution, thickening of the filaments at the edge of cells was obvious ( figure b , arrows). these filaments, which ran parallel to the cell edge, could be the enhanced actin filaments, and together figure . scanning electron microscopy of vero e cells infected with severe acute respiratory syndrome-associated coronavirus at h after infection. a) cell surface is covered with extracellular progeny virus particles, and progeny virus are being extruded from or attached to numerous pseudopodia on infected cell surface (arrows). b) a higher magnification micrograph of the virusclustered pseudopodia (arrows). c) rosettelike appearance of the matured virus particles (arrows). the scanning electron microscopy image complements the form and structure of the virus seen with negative staining (inset) under transmission electron microscopy. d) arrows indicate virus particles being exported from the surfaces of the filopodia. with the accumulated progeny virus particles, could have caused the bulky, puffy-cell periphery. by using transmission electron microscopy, recent studies ( , ) showed the entry events and prolific growth of sars-cov in vero e cells. sars-cov enters the cell by direct fusion and has a latent period of only h. high numbers of progeny virus particles assemble in the swollen golgi sacs before export to the external surface. transmission electron microscopy of ultrathin sections gave good intracellular information but was not able to give a gross morphologic landscape of the infected cells. surface topographic changes induced by sars-cov at maturation and late stages of infections were the focus of this study. the virus-induced modifications at the cell surface or subcellular surface could relate to the eventual destruction of the infected cells as well as shed light on the extrusion mechanism of the progeny virus particles from the cell surface. scanning electron microscopy, an established technique, gives a three-dimensional overview of the virus and the infected cell surfaces. another high-resolution device used in this study is the atomic force microscope. it is also gaining popularity in areas of life science research ( ) ( ) ( ) ( ) ( ) ( ) ( ) . most of these studies were on purified macromolecules. however, the atomic force microscope has also become a virologic standard in recent years ( ) ( ) ( ) ( ) . a recent study on hiv and hiv-infected lymphocytes ( ) demonstrated the strength of this technique for virology. the application of these two selected techniques to study the late sars virus-induced changes in vero cells was rewarding. the sars-cov knobby/rosettelike structures were seen in a three-dimensional form under the scanning electron and atomic microscopy ( figures b, c-online) . the spikes seemed shorter ( - nm) than those of other coronaviruses. at this stage, it is speculative if this could be due to the lack of the hemagglutininesterase protein ( , ) in the spike glycoprotein of this although the scanning electron microscope was able to show virus particles in the process of extruding ( figure b , figure a and b) from the cells, the image derived with the atomic force microscope was superior in resolution. a virus particle was seen pushing out of the cell plas-ma membrane ( figure a ), which resulted in localized loss of membrane integrity at the site. since prolific extrusion of the progeny virus particles occurred at this late stage of infection, the frequent loss of plasma membrane integrity could compromise the physiologic status of the infected cells and lead to cell death. fifteen hours after infection, ruffled, puffy peripheries were visible in infected cells ( figures b, , and ) and not seen in uninfected cells (figure ). this feature was not obvious under the transmission electron microscopy ( ) . subcellular imaging of the thickened edge of the cells showed numerous progeny virus particles awaiting extrusion ( figure b, arrows) . the actin filaments that were parallel to the cell edge appeared to have thickened ( figures a and b compared to figures a and lb) . the enhanced presence of the actin filaments could assist in providing the bending force to expel the progeny virus particles to the exterior. bohn and colleagues ( ) suggested that the forces resulting from the vectorial growth of the actin filaments contributed to membrane bending at the site of virus maturation. actin filaments have also been reported to be directly involved in the budding of both enveloped dna and rna viruses ( ) ( ) ( ) ( ) . in summary, the cellular cytoskeleton network is involved in the sars-cov maturation and possibly replication process. the constant loss of membrane integrity attributable to the prolific progeny virus extrusion resulted in disintegration of infected cells. the microscopy work was supported by the academic research fund, national university of singapore (grant no. r- - - - ). emerging infectious diseases • www.cdc.gov/eid • vol. , no. , november , november topography of sars coronavirus-infected cells dr. ng is an associate professor at the department of microbiology, national university of singapore. her research interests are virology (main focus is on flaviviruses) and microscopy techniques. identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome a major outbreak of severe acute respiratory syndrome in hong kong identification of severe acute respiratory syndrome in canada characterization of a novel coronavirus associated with severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus comparative full length genome sequence analysis of sars coronavirus isolates and common mutations associated with putative origins of infection sars-associated coronavirus coronaviridae and their replication prolific 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studies on cytoskeletons of infected cells actin binding and nucleation by autographa california m nucleopolyhedrovirus role of actin microfilaments in black creek canal virus morphogenesis morphogenesis and release of fowlpox virus actin filaments participate in west nile (sarafend) virus maturation process use of trade names is for identification only and does not imply endorsement by the public health service or by the u.s. department of health and human services purified virus fixed in . % glutaraldehyde was provided by the environmental health institute. key: cord- -cqbayzat authors: rajnarayanan, rajendram v.; dakshanamurthy, sivanesan; pattabiraman, nagarajan title: “teaching old drugs to kill new bugs”: structure-based discovery of anti-sars drugs date: - - journal: biochemical and biophysical research communications doi: . /j.bbrc. . . sha: doc_id: cord_uid: cqbayzat abstract severe acute respiratory syndrome (sars) main protease or c-like protease ( clpro) is essential for the propagation of the coronaviral life cycle and is regarded as one of the main targets for structure-based anti-sars drug design. it is an attractive approach to find new uses for old drugs as they have already been through extensive clinical testing and could easily be accelerated for clinical approval. briefly, we performed virtual screening of a database of small molecules against sars clpro, analyzed inhibitor–protease complexes, and identified several covalent and non-covalent inhibitors. several old drugs that bind to sars clpro active site were selected and in silico derivatized to generate covalent irreversible inhibitors with enhanced affinity. furthermore, we show that pharmacophores derived from clusters of compounds resulting out of virtual screening could be useful probes for future structure–activity relationship studies (sars) and fine-tune the lead molecules identified. severe acute respiratory syndrome (sars) is a lifethreatening viral respiratory illness caused by a new coronavirus (cov). the virus induces symptoms of atypical pneumonia, clinically indistinguishable from similar syndromes and is thought to be of animal origin [ ] . in the course of a few months, sars had spread rapidly from its likely origin in guangdong province, china, to countries. world health organization (who) reported that a total of people worldwide became sick with sars that was accompanied by either pneumonia or respiratory distress syndrome and of these patients died. national laboratories, several biotechnology, and diagnostics firms have joined the global rush to combat the infectious disease [ , ] . the scientific community has already learnt many important lessons from hiv, which could accelerate anti-sars drug/vaccine development. who declared the global outbreak of sars was contained, as no new cases were reported by august . however, the virus is not yet eradicated. the important proteins associated with the sars cov infection include the polymerase, the spike (s) glycoprotein, the envelope (e) protein, the membrane (m) protein, the nucleocapsid (n) protein, and the c-like protease ( clpro) [ ] . sars clpro, which plays pivotal role in the viral replication, is one of the potential targets for structure-based drug design. sars clpro has three domains: i (residues - ), ii ( - ), and iii ( - ). domains i and ii, which contains the active site region, are b-barrel domains and iii is an a-helical domain. sars clpro folds similar to generic serine protease but with a catalytic cys-his dyad playing critical role in the active site. the protease - x/$ -see front matter Ó elsevier inc. all rights reserved. doi: . /j.bbrc. . . reaction is conjured by the active nucleophile cys- and the acid-base catalyst his- . structural conclusions from active site similarity within the coronavirus family and virtual screening on homology models have provided some clues regarding the class of compounds that could interact with sars protease. rhinovirus cpro inhibitors like ag fit into the active site pocket of sars clpro and their derivatives could be potential inhibitors of sars [ ] . the x-ray crystal structure of ag -bound hrv protease formed the basis for early anti-sars drug design (pdb: cqq). further, this is supported by the observation that kz , a derivative of ag , could interact with the active site of the sars protease through six hydrogen bonds [ ] . virtual screening on a d model of the sars clpro against a database of protease inhibitors shows that available protease inhibitors could provide clues toward anti-sars drug design [ ] . molecular dynamics and docking studies using a database of fda approved compounds suggested that l- , a pseudo c symmetric hiv protease inhibitor, fits well into the active site compared to ag [ ] . toney et al. [ ] have reported that sanabandine, a compound from the nci diversity set, could inhibit sars protease. the first crystal structure of the sars clpro was reported in july (pdb: q w) [ ] and a set of crystal structures including sars clpro in complex with a specific hexa-peptide inhibitor have been reported recently (pdb: uj , uk , uk , and uk ) [ ] . bifunctional aryl boronic acid compounds targeting the cluster of serine residues (ser , ser , and ser ) near the active site cavity were found to be effective protease inhibitors [ ] . at this juncture, researchers revisited the old adage ''old drugs for new bugs'' [ , ] and started investigating old drugs that show potential of inhibiting the replication of sars-cov. they have shown that a few old drugs could be used as templates for designing sars clpro inhibitors [ ] . since these old drugs are originally not designed to inhibit sars clpro, they have to be fine-tuned to interact with the new target and ''taught'' how to kill new bugs! the present study employs in silico derivatization as a method to ''teach old drugs to kill new bugs.'' we have designed irreversible covalent inhibitors by selective derivatization of top non-covalent leads, which includes several old drugs especially a class of hiv inhibitors identified from virtual screening. our study has resulted in identification of several peptidomimetics and small molecule candidates as potential non-covalent/covalent inhibitors of sars cl protease. the catalytically active chain a of the sars clpro x-ray crystal structure (pdb id: uk ) without the bound cmk peptide and water molecules was used in the study from the pdb structure. the resultant structure was energy minimized using the discover module of insight ii (accelrys) and used as the initial structure. a database of more than , compounds comprising of protease inhibitors (aspartyl, cysteine, serine, and metallo-proteases), hetatm records extracted from pdb (http://www.rcsb.org/) [ ] , hiv inhibitors (polymerase, integrase, and reverse transcriptase), and a set of thiol reactive compounds filtered from maybridge (http://www.maybridge.com), leadquest (http://www.leadquest.com/), acd laboratories (http:// www.acdlabschem.com), and nci small-molecule databases (http:// dtp.nci.nih.gov) was used in the study. virtual screening of the small molecule inhibitors against sars clpro was performed using the flexx module in sybyl . (tripos) set at default parameters unless otherwise indicated in the text. we utilized multiple well-known scoring functions: a gold-like function [ ] , a dock-like function [ ] , chemscore [ ] , a pmf function [ ] , and flexx [ ] to rank order the complexes resulting from virtual screening. a list of top compounds was selected from each scoring function, generated and purged into a dataset of unique compounds. these compounds were subjected to physicochemical filters and merged into single database of compounds. identified lead molecules were in silico derivatized with suitable thiol reactive warheads. the resultant complexes were subjected to molecular dynamics simulations and energy minimization using discover module of insight ii. molecular dynamics simulations consisted of an initial equilibration of pico seconds (ps) and followed by ps dynamics at k. the final complex structure at the end of the md simulation was subjected to steps of steepest descent energy minimization followed by conjugate gradient energy minimization. for all the above calculations, a distance-dependent dielectric constant and non-bonded distance cutoff of Å were used. molecular graphics images were produced using sybyl . and the ucsf chimera package from the computer graphics laboratory, university of california, san francisco (http:// www.cgl.ucsf.edu/chimera; [ ] ). inhibitor-protease interactions were analyzed using occluded surface (os) [ , ] . designing specific inhibitors to block sars clpro requires a clear understanding of the successful inhibitors designed against class of cysteine proteases. the first set of specific covalent inhibitors for proteases has been designed by adding reactive warheads such as diazo compounds or haloketones to a good substrate of the protease under concern. one of the major disadvantages in using early covalent inhibitors such as haloketones arises due to the inherent reactivity towards non-target molecules. this compromises their stability/ selectivity and in turn makes them unsuitable for in vivo studies. the discovery of e , potent natural epoxysuccinyl inhibitor and michael acceptors such as ag , has shown that lowering the reactivity of the warhead essentially increases the stability, inhibitory potency, and in turn makes them viable for in vivo studies. the entry of ag into clinical trials [ ] [ ] [ ] has rejuvenated the interest in developing irreversible covalent inhibitors for cysteine proteases. essentially the first step is the subsite mapping with a library of peptide substrates [ ] . in lieu of such studies, it is often easier to identify the potential substrate peptide from closer homologues within the family of the protease. a closer examination of the substrate specificity profile of c-like proteases of coronaviruses reveals that p position of the substrate is usually small (gly, ala or ser), a conserved gln at the p position, and the p position of the substrates seems to favor large hydrophobic residues. the side chains of his and phe and the main-chain atoms of met , glu , and his form the s subsite, which confers specificity towards gln. thus, specific covalent inhibitors of sars clpro could be designed by substituting the amino acid at the p position with a thiol specific reactive organic moiety like chloromethyl ketone. the affinity for the peptide with correct pi-pi (where i = , , , etc.) amino acid arrangement has always been the highest. the criteria for any small molecule mimic would be to span the critical length required for the inhibition and make critical interactions with the binding site residues. secondary structure studies using peptide substrates demonstrate that substrates with more b-sheet like structure tend to react fast [ ] . the availability of x-ray crystal structures with bound ligands facilitates computer-assisted design of structural analogues with increased potency. due to the lack of x-ray crystal structures before july , we constructed several homology models based on tgev mpro (pdb id: lvo). though there were significant differences between the homology models and the available crystal structures, by comparison of rms deviations of the binding sites of all the structures alone with respect to that of uk , it could be observed that the differences in the binding site are localized to ÔminorÕ loop reorganization and side chain orientations (fig. a) . our homology homologues ( pa , p s, and p t) and homology models (unb model [ ] and our model) and (ii) sars clpro crystal structures. models were comparable with other models available at that time (fig. b, marked i) . early structures p s [ ] , p t [ ] , and p u [ ] provided structural basis for initial virtual screening efforts. these were based on the assumption that the substrate peptide binds in the normal mode i.e., conserved si-pi and si -pi interactions (where i = , , , etc.). however, in the reported cmk-peptide bound crystal structure, leu-p is partially solvent accessible and does not interact with the s subsite. this results in a shift in subsite interaction; thr-p and asn-p occupy the s and s subsites, respectively. this unusual mode of binding could attribute to lower specificity of the p -amino acid in comparison with other coronaviruses [ ] . the authors have also reported large cooperative movements of the side chains of glu , phe , leu , and tyr , and the n terminus of the partner protomer in the dimer as a function of ph. especially there is a marked difference amongst the structures crystallized at different ph (pdb: uj , uk , uk , and uk ; fig. b , marked ii). our earlier virtual screening studies with homology models of the sars clpro provided clues about potential protease inhibitors. we eliminated molecules with poor scores based on our early virtual screening studies with sars clpro homology models. this not only reduced about % of unwanted molecules but also reduced the computer time taken for virtual screening against the x-ray crystal structure as outlined in materials and methods. virtual screening resulted in unique compounds with small molecule hits containing at least one thiol reactive functional group. these compounds could potentially serve as covalent inhibitors of sars clpro. analysis of the docked complexes reveals that the thiol reactive functional groups are not properly oriented towards catalytic cys in most of the docked complexes. out of the complexes only compounds including kz were oriented properly towards cys but still were - Å away to induce possible nucleophilic attack. reorganization of the ligand fragments to provide proper orientation for protease reaction disrupted critical interactions with the binding site residues. in the process, we learnt that it was easier to modify small molecules that do not contain thiol functionality but bind well to the protease pocket. apart from the potential covalent inhibitors, virtual screening has also resulted in molecules that did not contain any thiol reactive functionality. based on visual examination, we selected small molecules that bind to the substrate-binding pocket by mimicking several critical hydrogen bonding interactions similar to the cmk peptide. the identified non-covalent inhibitors were composed of several popular hiv protease inhibitors (as shown in fig. pharmacophores derived from clusters of compounds resulting from virtual screening form excellent data set for future structure-activity relationship studies (sars). we observed that out of the hiv protease inhibitors shared similar pharmocophoric features (fig. , structure # ) . a d structure-based search of the pharmacophore using crossfire commander v (mdl) resulted in molecules with various functional groups at r - positions. fig. shows the docked pose of successful hits out of the identified compounds in sars clpro-binding site. steric substituents at r - (highlighted in red, fig. ) alter the scores drastically when compared to functional groups at r - positions. molecules with a hydroxyl-or an amino-group at the r position are favored. these molecules could potentially serve directly as non-covalent inhibitors of sars clpro or provide templates for designing covalent inhibitors as described in the following section. we reexamined the set of non-covalent inhibitor bound complexes and observed the class of hiv inhibitors that fit well in sars clpro active site. in an attempt to design irreversible covalent inhibitors, the top candidates from the virtual screening have been subjected to a rule-based secondary screening to select the small molecules that lie within - Å away from the sc of cys- . the resultant candidates were subjected to in silico derivatization and thiol reactive organic warheads had been incorporated at appropriate chemically viable positions as shown in fig. a . the warheads were covalently ligated to sc of cys- and the structures were re-minimized and those with bumps and structural deformity arising out of the new linkage were carefully eliminated by visual examination. in this study, we have incorporated thiol-reactive organic moieties or ''warheads,'' extracted from the covalent inhibitors identified in the study, with both fast and slow reactivity as shown in fig. b . analyses of the inhibitor-bound complexes reveal that covalent inhibitor picks up more interactions compared to its non-covalent analogue. a list of top non-covalent inhibitors selected after secondary rule-based screen with functional groups selected for in silico derivatization is highlighted in red (figs. - ) . graphical illustration of cyclic urea-based non-covalent inhibitor (colored in orange) and its covalent analogue (colored in cornflower blue) bound to sars clpro active site is shown in fig. . visual examination of the interacting residues shows that the covalent inhibitor interacts with the binding site residues much better than the non-covalent analogue. the cmk peptide inhibitor forms six hydrogen bonds with clpro active site residues phe , ser , cys , his , glu , and gln (pdb: uk ). most of the top ranked inhibitors picked in our study form at least hydrogen bonds and the corresponding interacting residues are as follows: thr , asn , phe , asn , gly , ser , cys , his , his , met , glu , and gln . figs. a-c illustrate the docked poses of the cmk peptide, hiv inhibitor (non-covalent inhibitor) and covalent irreversible analogue of the hiv inhibitor, respectively, in sars clpro-binding pocket. occluded surface program (os), a package of programs to calculate the occluded surface and atomic packing of protein structures developed by pattabiraman et al., is used to analyze inhibitor-protease interactions. occluded surface is defined as the molecular surface that is less than . Å from the surface of neighboring non-bonded atoms. that is, if a water molecule cannot fit between two atoms they occlude each other. occluded surface is similar to buried surface but is more sensitive to packing geometry than buried surface using a rolling probe. to calculate occluded surface, normals at the molecular surface are extended outward until they intersect neighboring van der waals surface. the collection of extended normals, and their respective lengths, defines the packing of each atom in a structural model. a combination of occluded surface area and average length of the normals was used to obtain the occluded surface packing (osp) value for each residue and the analysis of inter-chain occluded surface allows a detailed calculation of protein-protein interactions. surfaces of os-identified interacting residues of sars clpro active site are highlighted in figs. a-c. occluded surface scores for each ligand atom with corresponding atoms from the binding site were generated. the os scores averaged per amino acid give a quantitative measure of the protein-inhibitor interactions. the non-covalent inhibitor does not interact with the catalytic residue his . the corresponding covalent analogue interacts with his better than the cmk peptide (fig. d ). both the cmk peptide and the covalent inhibitor bound to cys have higher os score for this residue compared to the corresponding non-covalent analogue (fig. d) . however, the non-covalent inhibitor interacts with the residues glu and gln better than the covalent and the cmk peptide. it is evident that os scores could quantitatively differentiate the interactions of covalent and non-covalent analogue of the hiv inhibitors with binding site residues of sars clpro. we are in the process of in vitro biological testing of top non-covalent inhibitors using cloned sars protease and have initiated the ex silico derivatization of top covalent irreversible inhibitors identified in the study. these results would help focus the substrate-optimization and lead discovery. we have used structure-based screening to identify compounds that bind to the sars clpro-binding site. these molecules could potentially retard the proteolytic action of the sars protease and be used in combination with other anti-viral therapeutics. protease inhibitor design has evolved beyond mere addition of reactive warheads to cognate protease substrates. in silico derivatization of viable functional groups as identified by the secondary rule-based screen enables the old drugs to react with cys by serving as centers of nucleophilic attack. the use of low and high reactive organic warheads provides means to control the reactivity of the inhibitor, its stability, inhibitory potency and in turn helps in designing inhibitors viable for in vivo studies. thus, our strategy not only educates old drugs to kill new bugs but also teaches them to behave according to the needs. occluded surfaces generated scores provide unique and novel method for quantitative comparison of non-covalent and covalent inhibitor bound complexes. unlike with a new drug, old drugs or drugs with minimal modifications do not have to undergo extensive pre-clinical testing to prove their safety, efficacy and have the possibility of gaining accelerated approval by us food and drug administration. our strategy could be extended to identify potent inhibitors and fine-tune old drugs against other disease targets that are cysteine proteases such as cathepsins, caspases, calpains, and papain. severe acute respiratory syndrome (sars) biotech firms jump on sars bandwagon us army joins hunt for sars drug coronavirus main proteinase ( clpro) structure: basis for design of anti-sars drugs binding mechanism of coronavirus main proteinase with ligands and its implication to drug design against sars a d model of sars_cov cl proteinase and its inhibitors design by virtual screening identifying inhibitors of the sars coronavirus proteinase sabadinine: a potential non-peptide anti-severe acute-respiratory-syndrome agent identified using structure-aided design x-ray crystal structure of the sars coronavirus main protease the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor identification of novel inhibitors of the sars coronavirus main protease cl(pro) old drugs for a new bug: influenza, hiv drugs enlisted to fight sars ribavirin in the treatment of sars: a new trick for an old drug old drugs as lead compounds for a new disease? binding analysis of sars coronavirus main proteinase with hiv, psychotic and parasite drugs molecular recognition of receptor sites using a genetic algorithm with a description of desolvation automated docking with grid-based energy evaluation empirical scoring functions: i. the development of a fast empirical scoring function to estimate the binding affinity of ligands in receptor complexes a general and fast scoring function for protein-ligand interactions: a simplified potential approach a fast flexible docking method using an incremental construction algorithm chimera: an extensible molecular modeling application constructed using standard components occluded molecular surface analysis of ligandmacromolecule contacts: application to hiv- protease-inhibitor complexes occluded molecular surface: analysis of protein packing ag- pfizer structure-assisted design of mechanism-based irreversible inhibitors of human rhinovirus c protease with potent antiviral activity against multiple rhinovirus serotypes structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus c protease inhibitors. . incorporation of p lactam moieties as l l-glutamine replacements a combinatorial approach defines specificities of members of the caspase family and granzyme b. functional relationships established for key mediators of apoptosis biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus c-like proteinase homology model of sars-cov mpro protease the protein data bank we acknowledge the national cancer institute (nci) for allocation of computing time and staff support at the advanced biomedical computing center of the national cancer institute, frederick. key: cord- -pbs hy authors: desenclos, jean-claude; van der werf, sylvie; bonmarin, isabelle; levy-bruhl, daniel; yazdanpanah, yazdan; hoen, bruno; emmanuelli, julien; lesens, olivier; dupon, michel; natali, françois; michelet, christian; reynes, jacques; guery, benoit; larsen, christine; semaille, caroline; mouton, yves; christmann, daniel; andré, michel; escriou, nicolas; burguière, anna; manuguerra, jean-claude; coignard, bruno; lepoutre, agnés; meffre, christine; bitar, dounia; decludt, bénédicte; capek, isabelle; antona, denise; che, didier; herida, magid; infuso, andréa; saura, christine; brücker, gilles; hubert, bruno; legoff, dominique; scheidegger, suzanne title: introduction of sars in france, march–april, date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: pbs hy we describe severe acute respiratory syndrome (sars) in france. patients meeting the world health organization definition of a suspected case underwent a clinical, radiologic, and biologic assessment at the closest university-affiliated infectious disease ward. suspected cases were immediately reported to the institut de veille sanitaire. probable case-patients were isolated, their contacts quarantined at home, and were followed for days after exposure. five probable cases occurred from march through april ; four were confirmed as sars coronavirus by reverse transcription–polymerase chain reaction, serologic testing, or both. the index case-patient (patient a), who had worked in the french hospital of hanoi, vietnam, was the most probable source of transmission for the three other confirmed cases; two had been exposed to patient a while on the hanoi-paris flight of march – . timely detection, isolation of probable cases, and quarantine of their contacts appear to have been effective in preventing the secondary spread of sars in france. s evere acute respiratory syndrome (sars) was recently identified as a new clinical entity ( ) . sars likely originated in the guangdong province of people's republic of china ( ) and subsequently spread worldwide as infected persons traveled. during the outbreak, sars was primarily transmitted by person-to-person contact between healthcare workers or household members and ill patients ( ) . community transmission also occurred in several of the most affected areas, and an explosive outbreak from a common source occurred in amoy garden in hong-kong ( ) . as of june , a total of , probable cases and deaths had been reported from countries ( ) . a novel coronavirus has been identified as the cause of sars ( ) ( ) ( ) . based on current knowledge, sars is transmitted from symptomatic patients by close direct or indirect contacts through respiratory droplet secretions ( ) . in specific situations, other modes of transmission, such as airborne spread, may be possible ( ) . the incubation period ranges from to days, allowing sars to spread over long distances by infected persons who travel ( , ) . we describe how sars was introduced in france through a single patient who returned from vietnam on march and present data that suggest transmission from this patient to other passengers may have occurred during his flight back from hanoi to paris. after the world health organization (who) alert on march , , a centralized surveillance system was set up for sars in france ( ) . all persons who returned from an area affected by recent transmission, had been in contact with a probable case during the previous days, and in whom fever was > °c, with cough or difficult breathing, were advised to call the emergency service. these persons were transported to the closest universityaffiliated infectious disease ward or one of the nine infectious disease wards designated as a regional reference center in the french plan of action against bioterrorism, using masks for droplet protection. after performing clinical and introduction of sars in france, march-april, biologic evaluation and chest x-ray, the attending clinician notified the institut de veille sanitaire through a unique telephone number. on the basis of the results of the initial and subsequent evaluations, each notified case was either discharged, kept as a suspect case, or classified as a probable case using the who sars case definition ( , ) . probable and suspected case-patients were kept in isolation until recovery or until the diagnosis was changed, respectively. for this investigation, a probable case of sars was defined as previously described ( ) . for patients who fulfilled the definition of a probable case, respiratory secretion specimens were taken from the nose, throat, or sputum to detect for sars-associated coronovirus (cov) by reverse transcription-polymerase chain reaction (rt-pcr) ( ) at the national reference center for influenza (northern france), institut pasteur, paris. rna extraction and rt-pcr mixes were prepared in designated rooms. rt-pcr procedures included appropriate negative and positive controls in each run: two negative controls for the extraction procedure and one water control and one positive control for each pcr run. two rt-pcr, either both nested or one nested and one real-time, were performed for each sample. real-time rt-pcr, using the sars-cov detection kit from artus (germany), included an internal control that detected pcr inhibitory substances. one-step nested rt-pcr targeting either the bernhard nocht institute (bni) or the centers for disease control and prevention (cdc) fragment of the polymerase gene was used ( , ) . when real-time rt-pcr was performed, which targets the bni fragment, the other rt-pcr was the nested rt-pcr targeting the cdc fragment of the polymerase gene. the real-time and nested rt-pcr, which targeted the bni fragment reliably, detected copies of rna in the assay corresponding to rna molecules per milliliter of specimen. acute and convalescent serum samples were also obtained from probable cases. they were tested for immunoglobulin (ig) g antibodies against the sars-cov using indirect immunofluorescence with vero e cells infected by the sars-cov, negative control vero e cells and fluorescein-labeled goat antihuman igg. results of serologic testing were considered positive either in case of seroconversion or a fourfold increase of observed titers, or if the serum exhibited a titer > . the detection limit of our indirect immunofluorescence assay corresponded to the first dilution used: / . for each probable and confirmed case, information was collected on clinical symptoms, chest x-ray findings, leukocyte counts, illness onset date, demography, all possible contacts with a probable case, and exposures when traveling to affected area (contact with any hospital or place of potential transmission). persons who did not use masks for droplet protection and had contact with a symp-tomatic probable or confirmed case of sars were quarantined at home for days after exposure and contacted daily by telephone. as recommended by who, this follow-up included the passengers who sat within two rows of a sars case-patient on the air france hanoi-paris flight of march and , ( ) . the crew of the air france flight was also followed for days by the air france medical service. during follow-up interviews with the passengers seated close to the index patient (patient a), we obtained a detailed description of his clinical condition, his movements in the aircraft, the contacts he may have had with other persons on board, and the timing of his boarding and deplaning in relation to other passengers, including the stopover in bangkok. passengers on a flight in which a person with a symptomatic probable case had traveled were informed publicly through the media and mail of the potential exposure and advised to call the emergency service phone number to be evaluated and admitted to the closest university-affiliated infectious disease ward if a fever of > °c developed within days of the flight. we estimated the incidence density of sars among passengers who sat within two rows of a case of sars in the af flight of march - by using the total number of person-hours as the denominator. ninety-five percent confidence intervals ( % ci) were calculated by using the exact binomial method ( ) . as of april , a total of suspected cases had been notified to the institut de veille sanitaire and ( . %) met the definition of a probable case of sars. four were men, and their ages were to years. all had fever > °c, four with nonproductive cough and two with dyspnea. none had diarrhea. chest x-rays showed interstitial pneumonia in four patients (bilateral for three) and alveolar consolidation in one. lymphocyte counts were to , /mm . four patients were lymphopenic (< , /mm ); the same four patients also had thrombocytopenia. severe hypoxemia that required mechanical ventilation developed in one patient (the index case, patient a). four patients had been discharged from the hospital within to days after onset, and one died (patient a) from intensive-care complications days after admission. rt-pcr was positive for sars-cov in at least three of the respiratory secretion samples taken on at least different days after onset of symptoms for three of the five patients. acute-phase and convalescent-phase serum samples were obtained for four of the probable cases, and seroconversion to sars-cov occurred in three samples, including samples from the patient for whom rt-pcr was negative (patient d, figure ). however, for patient d, the only respiratory samples available for rt-pcr were taken on day after onset. our subsequent analysis is restricted to the four confirmed cases (patient a to d, figure ). all four cases were related to the outbreak that occurred in the french hospital in hanoi, vietnam ( ). the index patient (patient a), who had worked in this hospital, was the most probable source of secondary transmission to the other three patients. on the basis of information obtained from his colleagues, on march and , he was known to have examined, without respiratory protection from droplet secretions, an ill physician in whom sars subsequently developed. although no precise date of onset is available for patient a, interviews with persons he had met in hanoi during the few days before his departure indicate that symptoms, such as cough and severe fatigue, had developed as early as march . from march to april , three secondary cases occurred (figure seven persons sat within two rows of patient a during the af flight (figure ) , two of whom were medical doctors and did not know him. they indicated that patient a was breathing rapidly (superficial polypnea) and exhibited extreme pallor and pursed lips during the entire flight. he remained calm, had no cough, and left his seat at least twice between bangkok and paris to go to the front lavatory; at each move, he passed through the space between the plane wall and seat k (figure ). during the stopover in bangkok, he disembarked with the passengers on the flight from hanoi to bangkok and then reboarded the plane before the passengers who embarked in bangkok. on landing at charles de gaulle (cdg) paris airport, he disembarked among the last passengers (about passengers left the plane after him) and was cared for by the cdg medical services along with two other physicians who had worked in the french hospital in hanoi and were on the same plane. of the seven passengers who sat within two rows of patient a, sars developed in one (patient b, seat k), which accounted for an incidence density rate of per person hours of exposure ( / hours; % ci . to . ). he reported having handled the same aircraft magazines and using the same lavatory as patient a (wc , figure ). within days of onset and while in hanoi, patient b did not report any contact with the french hospital, other hospitals, or with any sars patients, nor did he stay at the same hotel as patient a. another passenger who sat near patient a ( k) reported a sore throat and a temperature of . °c once during follow-up. the second patient (patient c) sat in seat b. he boarded the plane in bangkok and did not know patient a and did not recall having had any interaction with him during the flight. he used the toilets to the rear behind his seat while patient a used the toilets nearest his seat up front ( figure ) . he was among the first passengers leaving the plane. he did not report any contact with ill persons or hospitals while in thailand. other contacts of patient a included two persons who shared the same car to the hanoi airport, one of whom had met him for hours before departing; two physicians who had worked in the hanoi french hospital and left the plane with him; and four healthcare workers of cdg medical services who cared for him. two taxi drivers (one / hour drive from cdg to his home and one / -hour drive from his home to the infectious disease hospital where he was admitted) were also exposed to patient a, who was then wearing a mask. none of these nine persons had any symptoms during the days after exposure. sars did not develop in any of the unprotected persons who had contact with the three secondary confirmed cases after their onset of fever (duration of contact < / hour to days; < hours for [ . %]). however, a febrile illness for days, with no other symptoms, developed in a household contact of patient d, who had a close unprotected contact with him for about / hour at onset of his symptoms (malaise and fever); a chest x-ray was normal and lymphocyte count was /mm . rt-pcr on nasal and pharyngeal swab was negative for sars-cov. three healthcare workers who cared for patient d and used masks for droplet protection had brief episodes (< hours) of mild fever without any respiratory symptoms and chest x-ray changes. these three episodes were attributed to a common, unidentified, local viral infection. the surveillance system was able to detect the first patient with sars (patient a) and one of his secondary case-patients (patient d). follow-up of passengers seated within two rows of patient a, and the information given to the other passengers of flight af flight allowed patients b and c to be identified. therefore, all casepatients were identified early in the course of the disease and placed under isolation, which contributed to reduction in the risk of secondary transmission and diffusion ( ) . only four of the five probable cases were confirmed either by rt-pcr or serologic testing, although all five met the probable sars case definition. although specific, the sensitivity of the rt-pcr-based detection technique remains to be fully evaluated ( ) . in addition, the time at which respiratory specimens were taken could account for the fact that virus shedding remained undetected for one patient (patient d). of the persons who came into contact with a symptomatic sars patient in france, did not have masks for droplet protection and were exposed, and ( . %) were exposed for a limited amount of time at the onset of illness. no probable case of sars was identified among these persons; a household contact of patient d had a febrile illness (> °c) without any other symptoms and tested negative for the sars-cov by rt-pcr. four contacts of sars cases had an episode of transient, mild or low-grade fever without other signs, including three healthcare workers of the hospital where patient d had been admitted and the passenger seated next to patient a during the af flight. specific antibody testing will be the only way to evaluate if these persons with mild symptoms could have been infected by the sars-cov. since no other exposure could be found within days of onset for patients b and c, their probable source of infection is contact with patient a while in flight, boarding, or disembarking flight af . for patient b, we cannot formally exclude an unrecognized community exposure in hanoi during the days before departure. however, the fact that the sars outbreak was controlled quite rapidly ( ) , without any formal documentation of community transmission, a large unrecognized community transmission most likely did not occur. patient b, in addition to sitting within two rows of patient a, had contact with patient a when he moved to and from the lavatory (at least four close contacts while going and coming at least twice from the lavatory). although a precise date of fever onset is not available for patient a, it appears that he was already symptomatic in the plane and was likely infectious. this finding is based on the following evidence: ) some persons who had met him in hanoi before his departure reported that he had fatigue and fits of cough; ) the passengers closest to him on the plane reported that he was dyspneic; and ) his initial evaluation at admission to hospital on march showed bilateral extended interstitial pneumonia and hypoxemia. the last strongly supports the hypothesis that his illness was ongoing for to days ( , , ) . for patient c, the exact mode of acquisition of sars remains a matter of debate, since he was neither found to have close contact with patient a nor other documented exposure. he had been traveling to thailand, a country where local transmission has never been reported by who ( ). although airborne transmission on the plane cannot be ruled out, a possible hypothesis is an undocumented direct or indirect contact with patient a while boarding or on the plane. our investigation also indicates that the risk for acquiring sars after a contact with a symptomatic case is very heterogeneous, since prolonged contact does not necessarily result in transmission and, conversely, a brief or distant exposure might be sufficient. factors that may explain this observation are the following: ) the virus excretion varies over time, ) the susceptibility to the sars-cov may vary among persons exposed, and ) exposure results in asymptomatic infection. although our study is descriptive and was not designed to evaluate sars control measures, our results support the usefulness of recommendations made to prevent the propagation of sars through air travel (i.e., that persons suspected to have sars should not fly [ ] ). we also believe that timely and sensitive surveillance associated with prompt and strict isolation of cases and quarantine of contacts were effective public health tools to limit the secondary spread of sars in france. a cluster of cases of severe respiratory syndrome in hong kong update: outbreak of severe acute respiratory syndrome-worldwide outbreak of severe acute respiratory syndrome (sars) at amoy gardens, lowloon bay, hong kong. main finding of the investigation world health organization. cumulative number of reported cases of severe acute respiratory syndrome (sars) coronavirus as a possible cause of severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome identification of severe acute respiratory syndrome in canada sars, the internet and the journal epidémie de syndromes respiratoires aigus sévères (sras) world health organization. global surveillance of severe acute respiratory syndrome (sars) sars emergence from uncertainty use of laboratory methods for sars diagnosis world health organization. recommended measures for persons undertaking international travel from areas affected by severe acute respiratory syndrome (sars) biostatistics: a foundation for analysis in the health sciences transmission dynamics of the etiological agent of sars in hong-kong: impact of public health interventions world health organization. cumulative number of reported probable cases of severe acute respiratory syndrome (sars) we thank the following for their contributions: b. basselier, f. lottin, m. allemand, c. aranda, c. saura, p. barboza, c. paquet, i. trema, c. leroy, l. josserand, m. lefort, and b. lina; a. mosnier-mantel, j.m. cohen, the physicians who participated in the "groupes régionaux d'observation de la grippe" network; the physicians of the air france medical service; the clinicians from all infectious disease wards who took care and notified suspect cases of sars to the institut de veille sanitaire; and h.w. doerr for providing the sars coronavirus.all institutions contributed to this study as part of their missions. dr. desenclos is a medical epidemiologist at the institut de veille sanitaire, france and is the head of the department of infectious diseases. his main areas of interest are surveillance of infectious diseases, investigation of outbreaks, and the study of modes of transmission of infectious agents. key: cord- - qoykl authors: shulman-peleg, alexandra; nussinov, ruth; wolfson, haim j. title: recognition of functional sites in protein structures() date: - - journal: j mol biol doi: . /j.jmb. . . sha: doc_id: cord_uid: qoykl recognition of regions on the surface of one protein, that are similar to a binding site of another is crucial for the prediction of molecular interactions and for functional classifications. we first describe a novel method, siteengine, that assumes no sequence or fold similarities and is able to recognize proteins that have similar binding sites and may perform similar functions. we achieve high efficiency and speed by introducing a low-resolution surface representation via chemically important surface points, by hashing triangles of physico-chemical properties and by application of hierarchical scoring schemes for a thorough exploration of global and local similarities. we proceed to rigorously apply this method to functional site recognition in three possible ways: first, we search a given functional site on a large set of complete protein structures. second, a potential functional site on a protein of interest is compared with known binding sites, to recognize similar features. third, a complete protein structure is searched for the presence of an a priori unknown functional site, similar to known sites. our method is robust and efficient enough to allow computationally demanding applications such as the first and the third. from the biological standpoint, the first application may identify secondary binding sites of drugs that may lead to side-effects. the third application finds new potential sites on the protein that may provide targets for drug design. each of the three applications may aid in assigning a function and in classification of binding patterns. we highlight the advantages and disadvantages of each type of search, provide examples of large-scale searches of the entire protein data base and make functional predictions. molecular recognition is one of the central processes in molecular biology. comparison and detection of binding sites is a key step in the prediction of potential interactions. since proteins function by interacting with other molecules, similarity in the binding patterns of proteins is closely related to similarity in their biological functions. there are two potential ways to infer the function of a novel protein. the first is to recognize a sequence or fold similarity with a protein(s) whose function is known. however, a similar fold does not necessarily imply a similar function. for example, proteins with the same fold, like tim barrels, can have multiple functions. on the other hand, proteins with different folds, like subtilisin and trypsin, can share the same function. the alternative approach, implemented in our method, is to investigate the physico-chemical patterns and shape of the protein molecular surface. proteins are assumed to perform similar functions if they share similar binding patterns and recognize similar binding partners, even if they have different sequences and (overall) fold homology. identification of regions on the surface of one protein that resemble a specific binding site of another is especially important for the following three applications. abbreviations used: rmsd, root-mean-square deviation; albp, adipocyte lipid-binding protein; hfabp, heart muscle fatty acid-binding protein; mfb , manduca sexta fatty acid-binding protein; bfabp, brain fatty acid-binding protein; sars, severe acute respiratory syndrome. ( ) functional analysis and classification: recognition of similarity in binding pattern to a well known protein may help in gaining a better understanding of its function and activation mechanism. these are crucial for the development of targeted drug leads like inhibitors. functional annotation of newly determined structures can be a significant contribution to the structural genomics initiative. ( ) potential ligands and ligand fragments: analysis of ligands bound to proteins with similar binding sites may provide hints of chemical groups that can be used to develop a drug for the protein target. the method can be used for lead generation and optimization as well as for de novo drug design. ( ) prediction of side-effects: proteins with similar binding sites may bind the same drug and therefore may potentially cause sideeffects. thorough investigation of such proteins during the drug design process is important for the development of more specific drug leads. other methods that are commonly used for suggestion of new ligands or ligand fragments and for predictions of side-effects are alignment of small molecules , and docking. - these techniques model the interactions of the receptor with specific ligands and therefore do not analyze all potential interactions that a specific binding site may form. this is particularly important, since a single protein-binding site may have several binding patterns. not only can the same binding site bind different ligands with different functional groups, but there is also evidence that at least in some enzymes a single compound can bind in different ways. - a wide variety of methods have been developed for protein structural alignment. most existing methods describe a protein structure by its c a atoms and seek to maximize the overall similarity of the structures. however, when there is no fold similarity between the aligned structures, these methods usually do not provide a biologically significant alignment. analysis of the similarities between binding sites can complement these techniques, ensuring full exploration of available structural data. several methods have been developed to identify specific three-dimensional patterns of amino acid side-chains. artymiuk et al. represented each side-chain by pseudo-atoms and used a subgraph-isomorphism algorithm to identify the spatially conserved patterns. this algorithm (assam) was recently enhanced to include additional constraints such as: the secondary structures, the solvent accessibility and the disulfide bridges. wallace et al. , have introduced "coordinate templates". these allow recognition of the "catalytic triads" that are typical for some of the protein families, like serine proteases, triacylglycerol lipases, ribonucleases and lysozymes. using atomic representation, the geometric hashing technique - was applied to efficiently compare a query protein to the template of the catalytic triad. this algorithm (tess) has been recently updated by jess, which is flexible and unconstrained by the template syntax. binkowski et al. have recently presented an elegant approach to assess the similarity of sequence patterns of surface pockets and voids, which are conveniently organized in castp. jones et al. have reviewed the methods for recognition of functional sites. however, methods that recognize patterns of residues that are conserved in their d positions and in their amino acid identities are not always applicable. there are biological examples of proteins that can bind the same binding partners without sharing any conserved patterns of amino acid residues. , rosen et al. searched for a site on the protein surface that resembles a specific, known active site. the molecular surface was represented using sparse critical points defined by lin et al. , the translation and rotation invariant characteristics of pairs of critical points were used as a key for the geometric hashing procedure. in addition, the reliability of surface comparisons in searches for active sites was examined. it was concluded that although pure geometric surface matching is capable of finding biologically correct solutions, utilizing additional chemical "labeling" information is required to correctly rank and analyze the obtained solutions. kinoshita et al. , performed clique detection on the vertices of the triangulated solventaccessible surface. they constructed a database of binding sites, ef-site, and used a structure of a complete protein structure to search it. however, the number of vertices in their surface representation is too large and it is too sensitive to conformational flexibilities. one of their conclusions was that other representative surface points may be more effective for robust and accurate comparisons. an important contribution was recently published by schmitt et al. they have defined generic pseudocenters that efficiently encode the physicochemical properties important for molecular interaction. each amino acid residue of a protein is represented as a set of such centers. assuming that small molecule binding sites are detected in cavities, they constructed a database of binding sites cavebase, which is integrated with relibase. the clique detection algorithm was used to retrieve cavities that are similar to a specific query cavity. the solutions were ranked according to the similarity of property-based surface patches. here, we present a novel method, siteengine, that is capable of handling large protein structures in a matter of seconds. unlike other methods that use the computationally expensive clique detection algorithm (np-hard), , our heuristic algorithm is based on efficient hashing and matching of triangles of centers of physico-chemical properties. it introduces a low-resolution representation by chemically important surface points and performs fast scoring of all possible solutions, while retaining the correct ones. successive scoring schemes, which are applied to smaller numbers of candidate solutions, perform a thorough exploration of the overall similarity of the surfaces as well as of local shapes of the chemically similar regions. we apply siteengine to a set of biological applications. first, we introduce a benchmark dataset, which is used to construct two surface description databases: one of complete protein structures and the other of binding sites. we compare between various searching applications that can be performed for recognition of functional sites. each application is illustrated by examples of successful recognition of specific types of protein binding sites such as estradiol binding, adenine and atp binding. for each example, we further make some specific predictions by providing a list of proteins recognized to share functional similarities with the query. we provide examples of classification of fatty acid-binding proteins and serine proteases and show the capability of the method to recognize the known similarity of the binding sites as well as of the catalytic residues. at the next stage, we apply siteengine to search a non-redundant dataset of all known protein structures. we describe the binding sites that are recognized to be the most similar to our query binding sites and discuss the quality of the predictions obtained. since siteengine searches a complete structure of each protein in a matter of seconds, we find it to be well suited for such large-scale applications. the method is developed toward the following three search applications: ( ) searching a given functional site on the surfaces of different proteins stored in a database; ( ) comparing a given functional site to a dataset of binding sites; ( ) searching a complete protein structure for the presence of an a priori unknown functional site, similar to known sites. these applications involve two types of comparisons: (i) searching a surface of a complete protein for a given functional site; (ii) comparison between two functional sites. however, from the algorithmic standpoint, the second type of comparison is essentially the same as searching for a given binding site on a protein surface, which is limited to a certain region of interest. therefore, here we describe the algorithmic approach of siteengine for searching a complete protein structure for a region similar to a given binding site. the input to the algorithm consists of a binding site of one protein and of a complete structure of another, where the binding sites are defined by the surface description of the relevant regions. the structure of the complete molecule is searched for the presence of a region, which is similar to the inputbinding site. the output is a transformation that superimposes the input-binding site on the recognized region and a score that measures the similarity between them. the main stages of the algorithm are summarized in figure (a). given the atomic coordinates of a protein struc- recognition of protein functional sites ture, the first step is to calculate the physico-chemical properties of its residues. each amino acid is assigned a set of d points, which are denoted as pseudocenters. each pseudocenter represents an interaction center of one of the following physicochemical properties: hydrogen-bond donor, hydrogen-bond acceptor, mixed donor/acceptor, hydrophobic aliphatic and aromatic(pi) contacts. the rules for the representation of each amino acid as a set of such centers follow schmitt et al. however, unlike their definition, we do not consider a peptide bond as an aromatic property and we do not estimate the directionality of the h-bonding property. figure (a) shows an example of a representation of cavity-flanking residues. in addition, we consider the pseudocenters of h-bonding properties of the side-chains of arg, lys and his to be positively charged, and those of asp and glu to be negatively charged. we have observed that these modifications lead to a slight improvement in experimental results. from the algorithmic standpoint, the similarity of charges is not a prerequisite for matching and is considered only at the scoring stage. a representation by pseudocenters is very efficient and suitable for algorithms like the geometric hashing. however, it is not sufficient for accurate representation and prediction of receptor-ligand interactions, especially in the case of hydrophobic aliphatic and aromatic contacts. therefore, for each pseudocenter, we consider the surface region created by the atoms that contribute to the pseudocenter property. this provides physico-chemical labeling of the surface regions, so that only surface patches with similar properties will be matched. we use a smooth molecular surface as implemented by connolly , and a distance transform grid, as implemented by duhovny et al. for each chemically labeled surface patch we estimate the patch center by a surface point nearest to its center of gravity (see figure (b) and (c)). each patch center is used to estimate the average curvature of its surface patch by calculation of the solid angle shape function. - in this calculation, a sphere of a certain radius is placed at the patch center. the average curvature is approximated by the fraction of the sphere inside the solventexcluded volume of the protein. the radius of the sphere determines the region in which the curvature is approximated. we perform two calculations with different definitions of the radius of the sphere. in the first calculation, we consider a minimum radius sphere bounding the surface patch represented by the patch center. in the second calculation, the radius is user defined (by default, . Å for hydrophobic regions and Å for others). an average of the two values is used to represent the shape of each surface patch. at this stage, we calculate all possible transformations that will superimpose the input-binding site to a similar region of the surface of the other molecule. the algorithm is based on the matching of almost congruent triangles defined by triplets of pseudocenters. figure (d) shows the hashing and matching procedures. each triplet of non-ordered non-colinear pseudocenters of the complete molecule is considered. triplets that form triangles with side lengths within a predefined range are stored in a hash table. a key to the hash table consists of the three parameters of side lengths of a triangle and of an additional physico-chemical index, which encodes the properties of the triangle nodes (see figure (d)). the physico-chemical index is represented by six bits, two for the encoding of the property of each node. this encoding is not unique due to the existence of centers with mixed donor/acceptor property. these can function both as hydrogen-bond donors and acceptors. to overcome this problem, we encode each such node twice, once as a donor and once as an acceptor. each triplet of ordered non-colinear pseudocenters of the query site is considered. triplets that form triangles with side lengths within a predefined range are used to construct a hash key. this key is used to access the hash table and retrieve all congruent triangles of the complete molecule. the construction of the hash table ensures that we will match only triangles with nodes at similar spatial locations and with similar physico-chemical properties. in addition, we require that the values of the shape function of the corresponding nodes of the triangles will be similar up to a user-defined threshold. each pair of matched triangles defines a transformation, which represents a potential solution (superimposition). each candidate transformation is immediately scored by the low-resolution score and only transformations that received a relatively high score are retained. the matching stage of our algorithm performs hashing of geometrical entities in a way similar to well known algorithms such as geometric hashing and pose clustering. these methods select transformations which have received the highest number of votes, e.g. in pose clustering a transformation that was identified by the highest number of matching triangles. the hashing stage of siteengine is extremely efficient, due to the consideration of the physico-chemical properties of the pseudocenters in addition to the geometrical constraints. as a result, we create less false-positive transformations and therefore greatly reduce the overall number of candidate solutions. we can score each candidate transformation and avoid any loss of competitive solutions due to the low number of votes. this approach allows identification of all candidate transformations that consist of at least three matching pseudocenters. the later stages of our scoring scheme will favor solutions with the highest number of matching pseudocenters (see : correspondence score). we implement a hierarchical scoring scheme, detailed in figure (b). the first scheme, which is applied to all potential solutions, is calculated based on a low-resolution representation of the molecules and is therefore highly efficient. as the number of potential solutions is reduced to a smaller subset, the resolution of the molecular representation is increased leading to more precise calculations. the details of the implementation and the default parameters are provided in the supplementary material. the goal of this scoring scheme is to provide the initial ranking of candidate transformations and to filter out biologically unreasonable ones. the main idea is to select a small, chemically meaningful representative set of surface points and use them to efficiently estimate the potential surface similarity of the aligned surface patches. we select these points to be a set of patch centers, i.e. centers of physico-chemical surface patches of the input-binding site. we apply the candidate transformation and consider the local environment to which each patch center is transformed. first, we check whether the given patch centers are transformed to surface regions in the other molecule. second, we check whether the physico-chemical environment to which it is transformed is similar to the one in the original molecule. third, we compare the shape of the region to which it is transformed with the shape measured at the given patch center. similarity in each of these attributes will increase the calculated score. we found it sufficient to consider only the highest-ranking solutions. transformations which superimpose the pseudocenters of the input-binding site so that the root-mean-square deviation (rmsd) between them is lower than a predefined threshold ( Å ) , are considered to belong to the same cluster. for each cluster the best scoring transformation is selected. this scoring scheme is applied to a smaller number of the retained candidate transformations. it can therefore examine them more thoroughly using a higher level of resolution of molecular representation. each candidate transformation is applied to each surface point. then, as in the lowresolution score, we compare the properties of each surface point with the properties of the environment in the other molecule to which this point is transformed. here too, similarity of both chemical and geometrical properties is scored higher than the similarity of only one of these. since the number of considered surface points is much higher, they are divided into different categories by an approach similar to the one described by duhovny et al. , the surface points of the input-binding site are divided into three categories according to their distance from the surface of the molecule on which it is superimposed. each category counts the number of surface points within distance thresholds of Å , Å and Å , respectively. in addition, in each category we calculate the number of points with the same physico-chemical property and charge, and add them to the counter of that category. we calculated a weighted sum of the counters of the three categories. the closer the category is to the surface the higher the weight that it receives. the : correspondence score as described in figure (b), for each retained candidate transformation, we determine a : correspondence (match list) between the sets of pseudocenters of the two molecules. the obtained : correspondence is used for two purposes, to improve each candidate transformation by the least-squares fitting method and to score the similarity of the environments of the corresponding pseudocenters. the match list is defined by calculating the maximum weight matching in a bipartite graph. , the bipartite graph is constructed in the following way. ( ) the nodes of the graph are the pseudocenters of the two molecules. ( ) an edge is added between each pair of pseudocenters that have similar (up to a threshold) spatial locations, physico-chemical properties and shape functions. ( ) each edge is assigned a weight that represents the similarity between the corresponding pseudocenters together with their local environments. it measures the distance, the charge compatibility of the h-bonding properties and the similarity of the local shapes of hydrophobic aliphatic regions. the maximum weight match in this graph provides a : correspondence between subsets of pseudocenters of the two molecules. the obtained match represents a set of pairs of pseudocenters of the two molecules, so that the points of each pair are the most similar in their geometrical and physicochemical properties. at the next stage, we calculate the score of the obtained : correspondence. this score consists of two parts: first, we calculate a score, which estimates the goodness-of-fit between the corresponding pseudocenters of the two molecules. second, for each pair of centers with hydrophobic aliphatic or aromatic properties we perform a more thorough comparison of the corresponding surface patches. there are two factors that we consider to be important in this context: ( ) the size of the overlap region between the patches superimposed by the candidate transformation; and ( ) the shape of the common overlap region. for each potential solution the final score is the combination of all the scores calculated by the algorithm. when performing extensive database searches it is difficult to consider more than one solution for each comparison. in these applications, we select only one solution with the highest value of the final score that maximizes the similarity with the searched pattern. we ignore the other solutions obtained for the same comparison. however, in other applications the number of output solutions is user defined and can be much larger. the overall complexity of our algorithm is dominated by the complexity of the matching and low-resolution scoring stage. the worst case theoretical complexity of an algorithm is oðn m Þ. in practice, this bound is much lower, since there is a limited number of congruent triangles with similar physico-chemical properties. in addition, since we are interested only in triangles that represent potential binding patterns, we limit the side lengths of the considered triangles to be within a limited predefined range. therefore, the practical running times of the method are proportional to oðnm Þ. a sample of the algorithm running times is given in tables - . the time measurements are done on a standard pc workstation ( . ghz xeon processors, gb memory) and do not include the time required for the construction of surfaces and grids, since these can be done in a preprocessing stage. in the section below, we show the experimental results obtained by applying the method to two datasets. first, we introduce a benchmark dataset that is used for a thorough evaluation of the method. we show the usefulness of the method for three types of searching applications as well as for biological classifications. then, we proceed to apply the method to large-scale database searches of the non-redundant dataset constructed from the entire protein data bank (pdb). we analyze and compare the results obtained on the two datasets. a representative protein data set that was constructed to evaluate the performance of the algorithm is detailed in table . two main criteria have motivated the selection of the proteins for the data set. first, we desired to include many structurally diverse proteins that can bind the same ligand. we have selected the adenine-binding proteins as a classical example of such a case. , we included in our data set the proteins used in the study by kuttner et al. thirty-three of these proteins are complexed with atp and with other adenine-containing ligands. other functional families that were included are structurally diverse proteins that can bind estradiol, equilin and retinoic acid. second, our motivation was to include representatives of important and well-studied structural families so that we will be able to check the classification capabilities and the consistency of our method. we have selected seven different protein families: hiv- /hiv- , hiv protease, anhydrase, antibiotics, fatty acid-binding proteins, chorismate mutases and serine proteases. in order to verify the tolerance of the method to local binding site flexibility, we have intentionally included several structures of homologous proteins, that are unbound or complexed with different ligands. the proteins of the data set, listed in table , were preprocessed to construct two types of databases: a list of proteins whose binding sites were recognized to be similar to an adenine-binding site of a camp-dependent protein kinase ( atp) is presented. the proteins are listed in the order of decreasing similarity to the query-binding site. the name of the ligand present in the located binding site is provided. marked by p are the entries that are known to bind adenine. a list of proteins whose binding sites were recognized to be similar to that of a sex hormone-binding globulin ( lhu) is presented. the proteins are listed in the order of decreasing similarity to the query binding site. in all cases, the program has successfully located the bindings sites. the name of the ligand present in the located binding site is provided. marked by p are the entries that are known to bind estradiol. a list of proteins whose binding sites were recognized to be similar to an atp-binding site of "hypothetical" protein mj ( mjh) is presented. the proteins are listed in the order of decreasing similarity to the query binding site. the name of the ligand present in the located binding site is provided. ( ) database of complete protein structures: this database contains the complete protein structures with pre-calculated surfaces. since in many cases the binding site is located between different peptide chains of a protein (e.g. hiv), for each structure we stored all chains whose coordinates appear in the pdb file. ( ) database of protein-binding sites: this database stores only the binding sites extracted from the protein-ligand complexes. each binding site is represented by a surface region around the ligand (surface points of a protein which are closer than . Å to the surface of a ligand). binding sites of ligands that contained less than seven non-hydrogen atoms were not considered. proteins from the data set that have no ligand were not represented in this type of database. the only exception are four proteins from the fatty acid-binding family, for which the binding sites are extracted by comparing to corresponding homologous structures of the dataset. we present three types of searching applications that can be performed on these databases: ( ) application i: searching the database of complete protein structures with a binding site. a query that is used to search the database is a binding site of a specific protein of interest. the search will provide a list of regions from different proteins, that are similar to the query site. ( ) application ii: searching the database of binding sites with a binding site. a known binding site of a protein of interest can be used to search for other binding sites that share the same structural and physicochemical features. ( ) application iii: searching the database of binding sites with a complete protein structure. the query protein structure is searched for regions on its surface that can be similar to known binding sites. whether a certain binding site is used as a query or stored in the database, it is defined exactly in the same manner as described in the previous section. below, we present results obtained by applying our method to each type of application. for each search example, we present a list of solutions that are ranked highest according to the value of the match score (detailed below). these solutions represent the proteins that are recognized to be most similar to the query. below, we describe the calculation of the rmsd, which provides some measurement for the quality of the results obtained. this score represents a portion of the binding pattern of interest found to match during the search. the score is presented as a percentage. we define native_scoreðb; p b Þ as the final score calculated by siteengine when a binding site ðbÞ is searched in its native protein ðp b Þ. since in this case all of the query features are matched, the score represents the maximal possible match ( %). when a binding site ðbÞ is searched in a protein/binding site ðmÞ, the obtained final score of its best solution will be referred to as the search_scoreðm; bÞ. this score will never exceed the native_scoreðb; p b Þ of the same binding site. calculations of the match_score differ according to the type of the search application. in applications i and ii the match_score represents the portion of the query-binding site matched during the search. therefore, it is calculated as a simple normalization of each comparison to a database protein/binding site ðmÞ by the native_score of the query ðbÞ: as a result, this score provides a ranking of the database proteins/binding sites according to the percentage of the query pattern that they match. when the database is searched with a complete protein structure (application iii) the match_score represents how much of the database binding site (b) matches the query protein (m): in this application, this score provides a ranking of the database binding sites, according to the percentage of their features recognized in the query. when the compared proteins share an overall fold, we calculate the rmsd in a manner which is commonly used in unbound docking algorithms. although siteengine aligns binding sites and no ligand information is used, the rmsd deviation calculated between the ligands can provide some insight regarding the results obtained. the rmsd is calculated between the locations of the ligand present in the binding site of the query. it is calculated between two possible locations for this ligand, one obtained when the query-binding site is superimposed by siteengine on the database molecule and the other obtained by aligning the c a atoms of the two molecules. however, when the compared molecules do not have the same overall fold, this calculation cannot be performed. in addition, when the proteins do share the same fold, but manifest high structural variabilities, the alignment between the c a atoms is not straightforward and can be misleading. therefore, this measure is not always applicable and in many cases, instead of providing this value we state "n/a". recognition of adenine-binding sites by searching the database of complete protein structures an adenine-binding site extracted from a campdependent protein kinase ( atp) was used to search the database of complete protein structures. the query-binding site was defined by protein surface points whose distance from an adenine ring of an total the list of the protein structures used for the method verification. atp ligand is under . Å . table presents the highest ranking solutions. the dataset contained five proteins which share the same "protein kinase-like" fold as the query. as expected, all five are recognized as top ranking solutions and the query-binding site is correctly aligned to the binding sites of these proteins. due to the similarity of the fold, we are able to calculate the rmsd between the locations of the ligands obtained by these solutions. as can be seen, in all cases the rmsd is less than . Å . the running times measured in this test case emphasize the ability of the method to handle large protein structures. for example, the longest running time ( . seconds) was observed for a residue molecule ( mu ) represented by pseudocenters. the ability to search the complete surface of a molecule of this size highlights the speed of our method. ranked was a "hypothetical" protein mj . its atpbinding site was correctly recognized when searching for an adenine-binding site. figure presents the alignment obtained between the molecules. as depicted in figure (a) there is no fold similarity between the proteins, however, our method correctly recognizes the similarity between the binding sites. the atp molecules and the complete structure of the camp-dependent protein kinase ( atp) is depicted for illustration only and were not used in the search. figure (b) presents the pseudocenters that are identified to be similar. recognition of estradiol-binding sites by searching the database of complete protein structures the constructed dataset contains proteins that are known to bind estradiol. these proteins belong to four different folds: concanavalin a-like lectins/glucanases ( ), immunoglobulin-like betasandwich ( ), nad(p)-binding rossmann-fold domain ( ), nuclear receptor ligand-binding domain ( ) . the dataset contains a total of seven structures that were complexed with estradiol, while the rest were crystallized with other small molecules. a binding site of a sex hormone-binding globulin ( lhu) was used to search the data set. all data set proteins that are known to bind estradiol are recognized within the best solutions. table presents the highest-ranking solutions. as expected, the top-ranking solution is the correctly recognized binding site of the protein of the sex hormone-binding globulin. figure presents two of the estradiol-binding sites correctly recognized by the algorithm. figure (a) presents an estrogen alpha receptor whose binding site is ranked second and is recognized to be the most similar to the binding site of a sex hormone-binding globulin. figure (b) presents a beta-hydrosteroid dehydrogenase that is ranked th. its binding site is successfully located and is correctly recognized as estradiol binding, but it is identified to be less similar to that of a sex hormone-binding globulin. ranked third and fourth are the binding sites of two proteins which are not known to bind estradiol . highest and lowest-ranking solutions obtained in searching the data set for estradiol binding sites. (a) an estrogen alpha receptor ( qkt), colored cyan, was successfully recognized as estradiol-binding. its binding site, depicted by blue dots, was identified as the most similar to that of a sex hormone-binding globulin ( lhu), depicted by red dots. the ligands from the complexes qkt and lhu are depicted for verification only and are colored in blue and red, respectively. (b) the binding site of a beta hydrosteroid dehydrogenase ( fds), colored cyan, was successfully recognized as estradiol-binding. its binding sites, depicted by blue dots, was ranked and identified as the less similar to that of a sex hormone-binding globulin ( lhu), depicted by red dots. the ligands from the complexes fds and lhu are depicted for verification only and are colored in blue and red, respectively. and are considered to be "false-positive" solutions. figure represents an analysis of a binding site of phoshoinositide -kinase ( e x) that is ranked third. the protein that was used for the alignment is in a complex with atp. however, there is another structure of the same protein in a complex with wortmannin (pdb code e u), which has structural similarity to estradiol. figure (a) -(c) show the similarity between wortmannin (c h o ) and estradiol (c h o ). figure (d) presents the alignment between the surfaces of the binding sites of phoshoinositide -kinase ( e x) and a sex hormone-binding globulin ( lhu). ranked fourth is a binding site of a beta-lactoglobulin complexed with retinoic acid. as in the previous case, the alignment obtained between the binding sites provides a good superimposition between the hydrophobic ligands of estradiol and retinoic acid and places the retinoic acid in the estradiol-binding pocket as would be done with a docking program. , , it is important to note that the binding sites of all estradiol-binding proteins are correctly recognized in spite of the fact that five of them were not complexed with estradiol. some of these ligands are very different from estradiol both in their size and in chemical structure. however, these differences in binding partners as well as the local flexibility that is required to accommodate them did not prevent the successful recognition of the functional similarities made by siteengine. searching the database of binding sites to predict the function of a hypothetical protein a hypothetical protein mj from a hyperthermophile methanococcus jannaschii was crystallized as part of a structural genomics project with the goal of functional recognition. we have extracted its atp-binding site and searched the database of binding sites to recognize those that are most similar to it. table lists the highestranking solutions of this search. all highestranking solutions bind ligands similar to atp. the only exception is an hiv- protease, which was also recognized by schmitt et al. to have a binding niche similar to an atp-binding site of campdependent protein kinase. the measured running times in all comparisons, are less than ten seconds, showing the ability of the method to perform efficient, large-scale database searches. wallace et al. derived d coordinate templates representing the ser-his-asp "catalytic triads" that are typical for some of the protein families, like serine proteases, lipases and lysozymes. these templates were used to classify a representative set of enzymes into four structural groups, up to three subgroups each. rosen et al. selected enzymes to represent this classification. in order to test our ability to recognize the catalytic triads and to classify the protein-binding sites, we have included these protein structures in our data. we have randomly selected three proteins from the three most populated subgroups and used their binding sites to search the data set of complete protein structures. the selected structures were alpha-chymotrypsin ( acb), thermitase ( tec) and serine protease b ( sgb). the protein -protein interfaces of these proteins were defined by the surface points of a protein which are closer than . Å to the surface of their protein binding partner. these were used to search the database of complete protein structures (application i). the results are fully consistent with the classification defined by wallace et al. and members of the same subgroup as the query is always top-ranking. subtilisin-like and trypsin-like folds are the most common examples of proteins with different fold that can perform the same function. proteins of these two folds share the same ser-his-asp catalytic triad and are included in the proteins that represent the classification made by wallace et al. our data set contained proteins of the trypsin-like fold and five proteins of the subtilisin-like. when the database of complete protein structures was searched with the protein -protein interface of thermitase ( tec) the first member of trypsin-like fold ( ela) was ranked . when it was searched with the interface of serine protease b ( sgb) the first member of the subtilisin-like fold ( tec) was ranked . the catalytic residues of histidine and serine, common to the proteins of these different folds, are correctly superimposed by our method with an alignment quite similar to the one presented by schmitt et al. similar to them, the catalytic aspartate residue was not considered in the calculations, since it is not surface exposed. however, the alignment calculated by siteengine, provides a good superimposition of all three residues of the catalytic triad, including the aspartate. figure (a) presents the alignment of two protein-protein complexes obtained during the search with an interface of thermitase ( tec), which is a member of the subtilisin-like fold. ranked , is a member of the trypsin-like fold, b-trypsin ( ptc) complexed with a pancreatic trypsin inhibitor. in spite of the fold differences between these proteins, the similarity in the histidine and serine catalytic residues was correctly recognized. the binding partners of these proteins, although not considered by siteengine, were correctly superimposed by the transformation of the solution. another interesting result, which was obtained by these searches, is the striking similarity of the catalytic histidine residues that was recognized between these proteins and sars-coronavirus main protease. our dataset contained a recently determined structure of sars-coronavirus main protease ( q w), which is related to the severe acute respiratory syndrome (sars) disease. sarscoronavirus main protease, which cleaves the polyproteins of sars-coronavirus, is responsible for the virus replication and therefore for the disease. , the protein of sars-coronavirus main protease ( q w) was ranked when the dataset was searched with the protein-protein interface of thermitase ( tec) and th when searched with serine protease b ( sgb). in contrast to serine proteases which have a ser-his-asp catalytic triad, sars-coronavirus main protease functions through a catalytic dyad, cys-his. our method has successfully detected the spatial similarity between the histidine residues common to all these proteins. figure (b) presents a superimposition of the complexes of tec and sgb on the structure of lq w by the transformation calculated by siteengine. as can be seen, the solution obtained provides a good alignment of the catalytic histidine residues of the three proteins. in addition, the binding partners of these proteins (eglin c from tec and potato inhibi-tor pci- from sgb) are placed in the catalyticbinding site of the sars-coronavirus main protease. searching the database of binding sites with a complete protein structure of a fatty acidbinding protein the goal of this type of database search is to locate potential binding sites of a protein, for which this information is still unavailable. in order to verify our method, we have searched the database of binding sites with a complete structure of a fatty acid-binding protein ( lib). the location of the binding site in this protein is well known and the database of binding sites contained six binding sites that are very similar to it. siteengine was able to correctly select them from the database of binding sites. table presents the highestranking solutions. as can be seen, the six highestranking solutions are the binding sites that are figure . recognition of catalytic residues. (a) when the dataset of complete protein structures was searched with the protein -protein interface of thermitase ( tec, colored green), which has a subtilisin-like fold, ranked was b-trypsin ( ptc, colored orange), which has a trypsin-like fold. the catalytic residues (ordered from right to left) ser , his , asp of tec (colored blue) and ser , his , asp of ptc (colored red) are displayed in ball-andstick. siteengine has recognized the similarity between the residues of serine (right) and histidine (middle). the binding partners pancreatic trypsin inhibitor from ptc and eglin c from tec are colored in red and blue, respectively. no information regarding these binding partners was considered by siteengine. (b) when the dataset of complete protein structures was searched with the protein-protein interfaces of thermitase ( tec) and of serine protease b ( sgb) the structure of sars-coronavirus main protease ( q w), colored blue, was ranked th and th, respectively. the figure presents the catalytic histidine residues (in ball-and-stick) of these proteins, when they are superimposed on q w by the transformation calculated by siteengine. the catalytic his of q w is colored blue, his of tec is green and his of ptc is red. the recognized alignment places the binding partners eglin c of tec (green) and potato inhibitor pci- of sgb (red) in the catalytic site of sars-coronavirus main protease. known to be similar to the query protein. we have calculated the rmsd between the ligands of these proteins when they are superimposed by two transformations: one defined by the superimposition of the c a atoms and the other defined by siteengine. as can be seen, the rmsd in all cases is very low and even existing techniques like docking , consider such transformations to be a success. as detailed below, binding sites of fatty acid-binding proteins that did not receive a high rank are known to exhibit a different binding pattern than the query protein. however, all database binding sites that were extracted from fatty acidbinding proteins were correctly superimposed on the region of the query protein known as its binding site. to visualize these results, figure (a) presents the superimposition of the ligands from these binding sites on the query protein. no ligand information was used during the search and each ligand is superimposed onto the structure of the query protein using the transformation obtained by the alignment between the database-binding site and the query protein. as can be seen, in all cases the ligands are successfully placed in the actual binding site of the query protein. motivated by the previous example, we have applied our program to analyze the function and classify the fatty acid-binding proteins. , for this study, we took all the crystal structures classified by scop as members of the "fatty acid-binding protein-like" family. table lists the pdb codes of these structures and their classification to domains as defined by scop. figure (b) presents the structural alignment between the structures as performed by multiprot. , , as can be seen, the structures and the ligands are very similar; however, the ligand conformations and the binding patterns are very different. we have tested the ability of our program to classify members of this family according to the binding site motifs. we have selected four representative proteins that form the four most highly populated domains of this family: an adipocyte lipid-binding protein ( lid), an intestinal fatty acid-binding protein ( ifb), a cellular retinoic acid-binding protein ( cbs) and a cellular retinol-binding protein ii ( opb). the results of all four searches are summarized in table , where each column ranks the members of the family in decreasing order of similarity to the querybinding sites. the first test was to search the data set with a binding site extracted from the adipocyte lipidbinding protein. according to banaszak et al. adipocyte lipid-binding protein (albp) as well as myelin p (p ), heart muscle fatty acid-binding protein (hfabp) and manduca sexta fatty acidbinding protein (mfb ) interact with their bound table . each ligand is superimposed onto the structure of the query protein using the transformation obtained by the alignment between the database binding site and the query protein. some examples of ligands are depicted to represent the diversity of the binding modes. in green is the palmitic acid molecule from a complex with an ifabp protein ( ifb). in blue is the oleic acid molecule from a complex with the albp ( lid). in red is a retinol molecule from a complex with crabp-ii ( cbs) and in orange is a retinoic acid molecule from a complex with crbpii protein ( opb). in purple is a palmitic acid molecule from mfb protein ( mdc), that has the same binding motif as albp, but exhibits a very high degree of flexibility of the binding site region. fatty acid using the p motif. as observed in table , all members of the albp family and the only structure of myelin p (p ) are top ranking. they are followed by the members of the heart muscle fatty acid-binding protein (hfabp) that have the same binding motif as the querybinding site. members of the brain fatty acidbinding protein (bfabp), share the same "u-shape" fatty acid-binding mode as albp and hfabp and therefore were correctly recognized to be similar to the query. the only member of m. sexta fatty acid-binding protein (mfb ) was ranked . figure depicts the flexibility of the ligands of albp and mfb and provides an explanation for such a low rank. when the data set was searched with the binding sites of the intestinal fatty acid-binding protein ( ifb), the cellular retinol-binding protein ( cbs) and the cellular retinol-binding protein ii ( opb) the results were the same. the proteins were correctly classified and the top-ranking solutions were all the members of the same domain as the query. in this test case the surfaces of complete protein structures were searched for the presence of the binding site of interest. in order to show that the query site was successfully located on the surface of each protein, we present the values of the rmsd which were calculated between the locations of the ligands oleic acid, palmitic acid, retinol and retinoic acid present in the query-binding sites extracted from lid, ifb, lopb, and lcbs, respectively. for each pairwise alignment the rmsd is calculated between the location of the the ranking of the proteins listed in table in the decreasing order of similarity to four different query-binding sites is presented. each entry lists the pdb code and the scop domain and the rmsd between the ligands present in the query-binding sites. the querybinding sites (from lid, ifb, opb and cbs) represent four different binding motifs exhibited by the members of this family. ligand obtained by siteengine and the location of the same ligand obtained by the alignment of the backbones (c a atoms) of the complete structures. although no ligand information was used by siteengine, it can be seen that the rmsd values in most of the cases are very low. the extreme exception is the alignment of the binding site of ifb to the structure of lfo. the structure of the liver fatty acid-binding protein is very different from the rest of the family members due to the fact that more than one fatty acid is bound. the structural alignment by c a atoms leads to an rmsd of . Å between the oleate ligand of lfo and palmitic acid of ifb. siteengine aligns these ligands with an rmsd of only . Å and it correctly detects the primary-binding site of lfo. it must be noted that these results were achieved in spite of the fact that only one best solution was considered for each pairwise alignment. in the absence of an overall fold or sequence similarity between the proteins, assessing the correctness of the obtained results is not straightforward. in these cases, there is no exact definition for the similarity between two binding sites. a query-binding region may contain features that are not essential for the binding, which may differ in proteins with exactly the same function. the absence of an exact definition of the pattern we are looking for makes the evaluation of such partial solutions even more complicated. considering the superimposition between the ligands obtained by an alignment between unrelated proteins can also be misleading. similar binding sites may accommodate ligands that differ in their size and shape and it is not clear what should be the correct superimposition between them. figure (a) presents an alignment between the atp ligands of the hexamerization domain of n-ethylmaleimide sensitive factor ( nsf) and camp-dependent protein kinase ( atp) obtained by the alignment of the corresponding binding sites. as can be seen in the figure, the superimposition of the ligand molecules achieves a good alignment between the ribose parts of the atp molecules while the orientation of the adenine moieties is different. however, when the binding sites are artificially superimposed using the transformation that aligns the adenine moieties, the distance between the phosphate tails of the atp molecules is approximately Å and only six pseudocenters are identified to be similar (as opposed to nine figure . alignment between the ligands induced by the alignment of the binding sites. (a) alignment between the atp ligands of hexamerization domain of n-ethylmaleimide sensitive factor ( nsf) and camp-dependent protein kinase ( atp) obtained by the alignment of the corresponding binding sites. the colored spheres represent the centers of interaction recognized by the program. the coloring of the spheres is as in figure . the spheres of nsf are smaller. it can be seen that the solution provides a good alignment between the ribose parts of the two atp molecules at the expense of the alignment between the adenine moieties. (b) alignment between the nad and atp ligands of lactate dehydrogenase ( ldt) and hypothetical protein mj ( mjh) obtained by the alignment of the corresponding binding sites. the solution provides a good alignment between the ribose parts of the nad and atp ligand molecules. however, the adenine ring of atp is aligned to a nicotinamide ring of nad, and not to its adenine ring as expected. as can be seen, the resulting alignment provides a superimposition of functional groups, that are depicted as balls. moreover, it provides an alignment of two conserved residues his and val of mjh (colored cyan) and his , val of ldt (colored blue) that are located in similar spatial locations. pseudocenters identified by siteengine). therefore, it is not straightforward to identify which solution is the correct one, especially due to the fact that the adenine moiety is known to exhibit two different binding modes. figure (b) presents an alignment between the nad and atp ligands of lactate dehydrogenase ( ldt) and the hypothetical protein mj ( mjh) obtained by the alignment of the corresponding binding sites. once again, the alignment between the binding sites of the proteins provides an alignment between the ribose parts of the nad and atp ligand molecules. however, the adenine ring of the atp is aligned to the nicotinamide ring of nad (and not to an adenine ring as expected). this solution provides the alignment of similar centers of interaction shared by these binding regions. moreover, there are two residues ( mjh, his , val ; ldt, his , val ) that are present in both binding sites and have the same spatial locations as well as identity of the amino acid. since siteengine is a software tool it recognizes regions that maximize the similarity; however, it cannot assess the biological significance of the obtained predictions. these need to be further verified by physical experiments and human expertise. we have applied siteengine to three types of applications. below, we discuss the main advantages and disadvantages of each type (see table ). application i: searching the database of complete protein structures with a binding site this type of database search is the most general and reliable. all of the available information is utilized and we can recognize totally new regions that can function as binding sites. it can be used to suggest a list of proteins that may bind ligands similar to the ligands of the protein of interest and may lead to side-effects. we have illustrated this application by two searches performed with the estradiol-binding site of the sex hormone-binding globulin and with the adeninebinding site of the camp-dependent protein kinase. in both cases, the highest-ranking solutions contained a list of unrelated proteins that can perform the same function as the query site. an additional application of this type of search is the classification of binding patterns. this was illustrated by the examples of serine proteases and fatty acid-binding proteins. application ii: searching the database of binding sites with a binding site constructing a database of binding sites may significantly reduce the time and space required to perform large-scale searches. searches of this type are more focused, since they consider regions that are already known to function as binding sites. less potential solutions are considered, which allows a more careful examination of each. this type of application is limited to the comparison of regions that are already known to serve as binding sites. however, it may be useful in suggesting ligands or ligand fragments for applications such as structure-based drug design. searching with the atp-binding site of the hypothetical protein mj ( mjh) provides an example of how this type of search can assist in the recognition of function and can contribute to structural genomics projects. application iii: searching the database of bindings sites with a complete protein structure the advantage of such an approach is in the recognition of new a priori unknown regions in a protein of interest that can function as binding sites. however, this makes the search extremely unfocused and some of the solutions may align surface regions that have no functional significance. when looking for binding sites which are located in cavities, an alternative strategy may be to extract potential binding pockets, using existing cavity detection methods , - and use them to perform a more focused search. we have illustrated this application by searching the database with a complete structure of a fatty acid-binding protein. siteengine has successfully selected from the database of binding sites those that are known to be similar to the query and suggested a good alignment between them. however, the ranking of binding sites of different size according to their similarity to different regions on the surface of the complete protein is not straightforward. in this work, the ranking was done according to how much of the database-binding site was matched during the search. however, some binding sites received a high rank due to their small size and the fact that some surface patterns have a high probability of appearance on the surface of any protein structure. these considerations must be taken into account when developing more reliable searches of this type. searches of the first type are advantageous over applications ii and iii, since they explore the whole surfaces of complete protein structures. this application is not limited to the set of known binding sites and it can recognize new regions that can function as such. in addition, the construction of a database of binding sites is not straightforward and the results of applications ii and iii are influenced by the selected definition of a binding site. since siteengine is robust enough to search complete protein structures with speed almost equal to comparisons between binding sites, we conclude that, whenever possible, application i is the preferred option. following the successful performance of the siteengine method on our benchmark dataset, we applied it to large-scale searches against a nonredundant dataset constructed from the entire pdb. the evaluation described in the previous section showed that application i is the most general and reliable of all applications. this application searches a database of complete proteins and utilizes all the available information of the proteins structures stored in the pdb. below, we repeat the searches of the previous sections on the non-redundant astral - dataset. this dataset consists of all known protein structures that have less than % sequence identity. following removal of some low-resolution structures that contain only the coordinates of the c a atoms, a total of protein structures were searched by our method. the details regarding the top ranking solutions of all the searches are provided in the supplementary material. the adenine-binding site extracted from campdependent protein kinase ( atp) was used to search the astral database of complete protein structures. as expected, most of the top-ranking solutions are the catalytic sites of other protein kinases. although these proteins have different sequences, they share the same "protein kinaselike" fold. the best solutions are the adenine binding sites of these proteins. in total, there are such sites among the top-ranking solutions. these binding sites were correctly located on the surfaces of these proteins and the performance of siteengine in these cases was similar to the five top-ranking solutions presented in table . it is interesting to note that only five of the recognized adenine-binding sites are complexed with adenine, while the rest are unbound or accommodate other ligands. ranked is the correctly recognized binding site of a replication factor c ( iqp) that accommodates an adp molecule. figure (a) shows the correct recognition of this site and figure (b) presents an additional binding site of d-ala-d-ala ligase ( iow) that accommodates an adp molecule and is ranked . as can be seen the transformation calculated by siteengine provides a perfect alignment between the atp substrate of the query site to the adp of the recognized region. in spite of the difference between the overall structures, these proteins were recognized to have similar shapes of the binding sites and share functional groups located in similar spatial locations. however, the biological "correctness" of many other of top-ranking solutions cannot be verified. some of these were regions of such proteins as photosynthetic reaction centre ( dxr, ranked ), pyruvate phosphate dikinase ( kbl, ranked ) and the gamma subunit of dna polymerase ( jr , ranked ). figure (e) presents one of these solutions that we consider to be a false-positive. the region that is recognized on the surface of photosynthetic reaction centre ( dxr) received a high rank due to the similarity of its hydrophobic patches to the query. some of such false-positive solutions might be filtered out by an additional requirement of the presence of certain features that are required to bind adenine. however, currently there is no automatic way to define such a set of features based on the protein structure alone. the estradiol-binding site of a sex hormonebinding globulin ( lhu) was used to search the recognition of protein functional sites (c) recognized similarity (ranked ) of the estradiol-binding sites of estrogen sulfotransferase ( aqu, blue) and sex hormone-binding globulin ( lhu, yellow). the estradiol ligand of lhu is cyan and that of aqu is purple. (d) recognized similarity (ranked ) of a binding site of a tropinone reductase ( ae ) to an estradiol-binding site of sex hormone-binding globulin ( lhu, depicted as cyan dots). the estradiol ligand of lhu is purple and an estradiol molecule that is placed in the binding site of ae according to its structural homologue ( fds) is shown in green. the two binding sites are recognized to share three residues that have the same spatial location and identity of the amino acid. the residues of ae are blue and those of lhu are red. (e) a false-positive solution that recognizes a similarity astral database of complete protein structures. the two top-ranking solutions are trivial and are the estradiol-binding sites of the query protein (ranked ) and of another sex hormone-binding globulin ( d s, ranked ). in contrast to our benchmark dataset, the non-redundant astral dataset contains almost no proteins complexed with estradiol. although most of the top-ranking regions are indeed binding sites, we do not have the information regarding their ability to bind estradiol. one such case is the cxe (pentaethylene glycol monodecyl ether) binding site of an outer membrane protein nspa ( p t) that is ranked and is illustrated in figure (f). as can be seen, these binding sites have similar surfaces and physico-chemical environments. in total, there are six regions of membrane proteins within the top-ranking solutions. since we do not have the biological expertise to evaluate these results, we consider them to be false-positives. the only dataset protein that is complexed with estradiol is the estrogen sulfotransferase ( aqu), which has a different fold than the query and is ranked . figure (c) presents the obtained superimposition of the estradiol molecules as well as the correctly recognized similarity of the protein regions that accommodate them. ranked is a tropinone reductase ( ae ) that belongs to the same family of tyrosine-dependent oxidoreductases as beta hydrosteroid dehydrogenase ( fds), which is complexed with estradiol (depicted in figure (b) ). the structural alignment of the c a atoms of proteins ae and fds (performed by multiprot , , ), provides a superimposition of the estradiol molecule of fds upon the structure of ae . remarkably, the estradiol molecule is placed on the same region that was recognized by siteengine as the potential estradiol-binding site. figure (d) shows the three residues ( lhu ser , val , leu , and ae ser , val , leu ) that are shared by tropinone reductase ( ae ) and the query. these residues have the same spatial location and identity. as can be seen, the conformations of the estradiol molecules are different. this may be due to a flexible loop (residues - ) at the binding site of ae , which has a different conformation in fds. here, we verify the ability of siteengine to recognize similarities of the catalytic residues of serine proteases, , , , which have become a standard benchmark for evaluation of such methods. in none of its stages did the algorithm consider the information regarding the identity of the amino acid residues. however, the alignment is considered correct only if it superimposes the corresponding catalytic residues. first, the astral dataset was searched with the binding site of thermitase (subtilisin-like fold, tec). the five top-ranking solutions are other proteins of the subtilisin-like fold. ranked seven and eight are the binding sites of members of the trypsin-like fold, which share the same ser-his-asp catalytic triad. the similarity of the corresponding functional groups created by the triads is correctly recognized and the alignments of these solutions are very similar to that presented in figure (a). however, there is one solution that emphasizes a limitation of our method. in addition to the five top-ranking solutions, the astral dataset contained a kexin protein ( ot ), which is also classified as a member of the subtilisin-like fold. the binding site of this protein is correctly located by siteengine and its catalytic triad is correctly matched to the query. however, it is ranked only . this low rank is due to a deviation of almost Å of a flexible loop ( tec, - ) that is present in the binding site. since siteengine does not explicitly address the flexibility of protein molecules, it considers the corresponding loop regions to be unmatched. we proceed to search the astral dataset with a binding site of a member of trypsin-like fold ( sgb). eleven out of top ranking solutions are correctly recognized binding sites of proteins of the trypsin-like fold that share the ser-his-asp catalytic triad. ranked seven is a c cysteine protease ( cqq). this protein is a member of the same scop family as sars-coronavirus ( q w) and the alignment obtained is similar to that presented in figure (b). ranked is the catalytic site of the first member of subtilisin-like fold ( dtw). as before, the corresponding residues of the two catalytic triads are correctly matched by the transformation of siteengine. however, there are two unexpected solutions that received an extremely low rank. one is the epidermolytic (exfoliative) toxin a ( agj), which is ranked . although classified as a member of the trypsinlike fold, it has a different binding site as confirmed by the crystallographic studies. a similar result was obtained for a serine-carboxyl proteinase pscp ( ga ) that is ranked . although its overall structure belongs to a subtilisin-like fold, its binding site is different and it functions through a glu-asp-ser catalytic triad. of the adenine-binding site camp-dependent protein kinase to a region of photosynthetic reaction centre ( dxr). the atp molecule of atp is green. the surfaces of the two binding sites are depicted as dots ( atp, red; dxr, blue) and the functional groups are depicted as spheres. (f) a presumably false-positive solution of similarity of a cxe-binding site of an outer membrane protein nspa ( p t) to an estradiol-binding site of sex hormone-binding globulin ( lhu). the binding sites are depicted as dots ( lhu, red; lp t, blue) and the ligands as sticks ( lhu, green; p t, purple). almost half of the proteins from m. jannaschii, whose structures were determined as a part of the structural genomics project, are classified as functionally unknown "hypothetical" proteins. in this section, we use the astral dataset to show two examples of how siteengine can assist in functional annotation of these proteins. first, we repeat the example of our previous section and use an atp-binding site from mj to search the astral dataset of complete protein structures. as expected, the top-ranking solution is the query-binding site recognized in its native protein. the next two top-ranking solutions correctly recognize the similarity of the query to amp-binding sites of etfp subunits ( o and efv) that belong to the same scop superfamily as the query. figure (a) presents the alignment of the binding site of o to the query. as can be seen, the binding sites are extremely similar and the ligands are perfectly aligned. the recognized similarity of the binding sites in addition to the similarity of the overall structures of these proteins can suggest similarity of their functions. figure (b) presents another example, ranked , where siteengine has recognized a similarity to an adp-binding site of arsenite-translocating atpase figure . (a) the similarity of the functional groups (depicted as spheres) of an amp-binding site of etfp subunit ( o ) to that of the hypothetical protein mj . this alignment is ranked and provides a perfect superimposition of the corresponding substrates. (b) the similarity (ranked ) of an adp-binding site of arsenite-translocating atpase arsa ( ihu) to an atp-binding site of the hypothetical protein mj . although the proteins belong to different overall folds, the similarity of some their secondary structures (e.g. the depicted helices) support the correctness of the obtained solution. (c) the similarity a g protein gialpha ( cip, ranked ) to a bound form of the anp-binding site of the hypothetical protein mj ( mjp). siteengine recognized the similarity of the phosphate-binding regions of these sites. (d) similarity (rank ) of an uma-binding site of udp-n-acetylmuramoyl-l-alanine: d-glutamate (murd) ligase ( uag) to an unbound form of the anp-binding site of the hypothetical protein mj ( b ). the residues shared by these sites are colored blue ( b ) and cyan ( uag). arsa ( ihu). although the alignment of an adp substrate of this protein to the atp of the query is not very good, we have obtained a good alignment of the binding sites as well as of some secondary structure elements. while this protein is classified to a different fold than the query, the similarity, of the secondary structure elements superimposed by the transformation of siteengine suggests that the obtained alignment is correct and can assist in deciphering the function of this protein. another surprising result of this search is the consistency of our presumably false-positive results to what was observed on our benchmark dataset. six out of top-ranking solutions of this search are oxidoreductases. the alignments obtained in these cases are similar to the one presented in figure (b). however, once again, we are unable to confirm the biological correctness of this result. in the next example we applied the method to recognize functional sites similar to an anp-binding site of the hypothetical protein mj with an unknown function. this example is especially challenging, since the binding site of this protein is very unusual. the binding mode of its anp substrate is different from the binding mode in other proteins and its main interaction with the protein is by its phosphate groups, while its adenine ring is exposed and is pointing outwards from the protein surface. here, we have performed two tests, in which the astral dataset was searched with both a bound ( mjp) and an unbound ( b ) form of this protein. as expected the first two solutions are the trivial recognition of the binding sites of the proteins themselves. in both cases, these two solutions were followed by a hypothetical protein yggv ( k k). although extremely similar to the query, this protein is also a part of the structural genomics project and its function is unknown. when searched with a bound form of mj ( mjp), ranked is a binding site of autoinducer- production protein luxs ( j w) and ranked is a tandem phosphatase domain of rptp lar ( lar). ranked is a binding site of adenylate kinase with the substrate-mimicking inhibitor ap a. this inhibitor can be considered as an atp molecule coupled to an amp molecule via the additional phosphate group and its interaction with the protein shows the pathway of phosphoryl transfer. siteengine has recognized a high degree of similarity of the regions that interact with the phosphate groups, which are aligned by the calculated transformation. a similar alignment is also recognized with the gnp-binding site of a g protein gialphal ( cip, ranked ), presented in figure (c). this result seems consistent with the suggestion by hwang et al. that this protein can function similarly to the signal sorters of g-proteins. when searched with an unbound form of mj ( b ), the ranking of the results (other than the top three) is slightly different due to the differences of the surfaces of the bound and unbound forms. ranked is a copper amine oxidase ( ivw), ranked is a tryptophan indol-lyase ( ax ) and ranked is a farnesyl diphosphate synthase ( uby). figure (d) illustrates the result that is ranked , in which we recognize similarity to a udp-n-acetylmuramoyl-l-alanine-d-glutamate (murd) ligase ( uag). the obtained alignment of the binding sites provides a superimposition of the anp substrate of the query to a nucleotide precursor udp-n-acetylmuramoyl-l-alanine (uma). as can be seen, the rings of the two ligands participate in similar aromatic interactions with a phenyl residue that is present in both binding sites. in total, these binding sites share three residues ( b asn , asp , phe , and uag asn , asp , phe ) that have the same spatial location and identity. in this section, we compare the results obtained on the two datasets used in this work. the astral and the benchmark datasets are constructed for different purposes and contain structures with different pdb codes. the benchmark dataset was constructed for the purpose of a thorough evaluation of the method on a set of well-studied examples. consequently, it contains redundancies that are important for the verification of the consistency and for the analysis of falsenegatives. on the other hand, the astral dataset contains no proteins with similar sequences and structures. applying the method to such a representative dataset is important to show the largescale applicability of the method. however, in many cases the proteins of our benchmark dataset were not selected as representative structures and therefore were not included in the astral dataset. one such example is the set of proteins that are complexed with estradiol. there are such proteins in our benchmark dataset and only one in the astral. representatives that are selected for the same protein families do not necessarily bind estradiol, since they may contain mutations that can influence the functional region and can interfere with the binding. this has an impact on the obtained ranking and on our ability to evaluate the results. when searching for regions similar to the adenine-binding site of camp-dependent protein kinase ( atp), the five top-ranking solutions obtained on our benchmark dataset (see table ) are within the ten top-ranking solutions obtained on the astral dataset. the hypothetical protein mj ( mjh), which is ranked eighth on the benchmark dataset is rd on the astral. in both datasets these ranks are within the % of the best solutions †. hexamerization domain ( nsf) is ranked eighth (best %) on the benchmark dataset and its homologue ( d n) that binds adenine is ranked th (best %) on the astral. two additional examples are oxidoreductases, which are represented by the pdb codes b v and le w in the benchmark dataset and by gos and b in the astral. these are ranked th and th (best %) on the benchmark dataset and th (best %) and st (best %), respectively, on the astral. when searching for regions similar to the atpbinding site of the hypothetical protein mj ( mjh) the results are similar to the above. the lactate dehydrogenase ( ldt) that is ranked second on the benchmark dataset (see table ) is represented by li z in the astral and is ranked eighth. another example is the hexamerization domain ( nsf) that is ranked sixth on the benchmark dataset and is within the % of the best solutions. in the astral dataset, the representative structure of the same family ( d n) is ranked th, which is the upper % of the best solutions. another successful example is the members of the nitrogenase iron protein-like family ( a , represented by lihu in the astral dataset) that are ranked seventh on both datasets. in general, there is a similarity of the rankings obtained on the two datasets and results that are within the % of the best solutions of the benchmark dataset are within the % of the best solutions of the astral dataset. recognition of functional sites in protein structures is extremely important for various biological applications, such as prediction of function and ligand binding. we have presented a novel method, siteengine, that in a matter of seconds can search large protein surfaces to recognize such sites and make predictions. we used a benchmark dataset to evaluate the performance of the method for three types of search applications. these experiments have shown that searching the database of complete protein structures is the most general and reliable application. therefore, we have proceeded to use this application to search a nonredundant database constructed from the entire pdb. below, we analyze its main advantages and weaknesses. one of the main advantages of the method is its speed, which is obtained due to the following factors: ( ) introduction of a low-resolution surface representation via chemically important surface points; ( ) hashing and matching triangles of physico-chemical properties; ( ) application of hierarchical scoring schemes for a thorough exploration of global and local similarities. the biological significance of the results obtained by the method is the outcome of the following factors: (a) consideration of both physico-chemical and geometrical properties of a protein molecule; (b) consideration of both discrete (pseudocenters and patch centers) and continuous (surfaces and shapes) representations of the protein molecule; (c) development of a set of scoring schemes that score each type of potential interaction differently according to its main chemical characteristics; (e) scoring each candidate solution, without any specific pre-requirement regarding the size of the matched region. however, siteengine is a software tool and therefore is limited in the quality of its biological predictions. it recognizes geometrically and chemically similar regions that belong to totally unrelated proteins. however, these similarities do not necessarily imply similarity in the binding partners and in the biological functions. siteengine can provide a list of proteins that are most likely to behave similarly to a binding site of interest. however, it cannot assess the biological significance of the recognized similarity. as in many other applications in structural biology, the, major bottleneck of the method is scoring. in the current version of siteengine there is no implicit treatment of electrostatic potentials that have a strong impact on the interaction. addition of such consideration may help to filter the false-positive solutions like the one presented in figure . additional weaknesses of the method are the requirement of high-resolution protein structures and addressing protein molecules as rigid bodies. protein flexibility is addressed only through a set of thresholds that allow a certain variability in the locations. these are definitely insufficient for efficient searches of binding sites that can bind large flexible molecules. other limitations that influence the quality of the results are implied by the screening applications and are general to the problem. one is the absence of a clear definition of what exactly is a functional site and what are the features that define it. when the binding site is defined by its contacts with the smaller ligand, a significant amount of information may be missed. as a result, essential features might be ignored and the extracted pattern might be partially aligned to other functionally different binding sites. there is no simple automatic solution to this problem. one possibility is the construction of a database of consensus binding patterns, common to all proteins with the same function. another problem is assessing the statistical significance of the obtained results. these are strongly influenced by the number of functional sites of the same type present in the searched database. although the astral dataset provides a non-redundant coverage of protein structures, it contains many redundancies of functional sites. in order to provide a truly representative statistical evaluation it is essential to consider a non-redundant dataset of functional sites, the construction of which is a future challenge. in order to efficiently address these problems there is a need for methods for multiple structural alignments between binding sites. in the future, we intend to utilize the insights we have gained in the present method for the development of such algorithms. one fold with many functions: the evolutionary relationships between tim barrel families based on their sequences, structures and functions predicting molecular interactions in silico: i. a guide to pharmacophore identification and its applications for drug design computational methods for the structural alignment of molecules predicting molecular 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epidermolytic toxin a, an atypic serine protease carboxyl proteinase from pseudomonas defines a novel family of subtilisin-like enzymes structure-based identification of a novel ntpase from methanococcus jannaschii high-resolution structures of adenylate kinase from yeast ligated with inhibitor ap a, showing the pathway of phosphoryl transfer ing details of implementation and the top ranking solutions of searches of the astral dataset performed with the binding sites of a c-amp-dependent protein kinase ( atp), a sex hormone-binding globulin ( lhu) we thank maxim shatsky and dina schneidman for useful discussions and for contribution of software to this project. we thank dr shuo liang lin for particularly valuable ideas and for critical reading of the manuscript. we thank drs david zanuy, buyong ma and k. gunasekaran for useful suggestions. this research has been supported, in part, by the "center of excellence in geometric computing and its applications" funded by the israel science foundation (administered by the israel academy of sciences). the research of h.j.w. and a.s.-p. is partially supported by the hermann minkowski-minerva center for geometry at tel aviv university. the research of r.n. has been funded in whole or in part with federal funds from the national cancer institute, national institutes of health, under contract number no -co- . the content of this publication does not necessarily reflect the view or policies of the department of health and human services, nor does mention of trade names, commercial products, or organization imply endorsement by the us government. the publisher or recipient acknowledges right of the us government to retain a non-exclusive, royaltyfree license in and to any copyright covering the article. funded, in part, by the nci under contract no -co- . key: cord- -cy y vnt authors: kumar, matam vijay; nagineni, chandrasekharam n; chin, marian s; hooks, john j; detrick, barbara title: innate immunity in the retina: toll-like receptor (tlr) signaling in human retinal pigment epithelial cells date: - - journal: j neuroimmunol doi: . /j.jneuroim. . . sha: doc_id: cord_uid: cy y vnt toll-like receptors (tlrs) are crucial components of innate immunity that participate in host defense against microbial pathogens. we evaluated the expression and function of tlrs in human retinal pigment epithelial (rpe) cells. real time pcr analysis revealed gene expression for tlrs – , , and in rpe cells. tlrs and were the most highly expressed tlrs. protein expression for tlrs , , and was observed on rpe cells and this expression was augmented by treatment with poly i:c or interferon-γ (ifn-γ). tlr is the receptor for dsrna, an intermediate of virus replication. because rpe cells express tlr and are frequently the site of virus replication within the retina, we evaluated tlr signaling. rpe cells treated with poly i:c produced ifn-β but not ifn-α, and this was inhibited by the treatment of rpe cells with anti-tlr antibody. human recombinant ifn-β was shown to be biologically active on rpe cells by inhibiting viral replication. poly i:c treatment of rpe resulted in an increase in the production of il- , il- , mcp- , and sicam- . the presence of tlrs on rpe cells and the resultant tlr signaling in rpe cells suggest that these molecules may play an important role in innate and adaptive immune responses within the retina. toll-like receptors, tlrs, are a family of evolutionary conserved innate immune recognition molecules that recognize molecular patterns associated with microbial pathogens (medzhitov and janeway, ) . they constitute a first line of defense against a variety of pathogens and play a critical role in initiating the innate immune response. tlr recognition of these specific microbial patterns leads to a signal transduction cascade that generates a rapid and robust inflammatory response marked by cellular activation and cytokine release (gordon, ; schnare et al., ) . to date, mammalian tlrs have been identified and each receptor appears to be involved in the recognition of a unique set of microbial patterns (gordon, ; zuany-amorim et al., ) . for example, tlr recognizes various ligands expressed by gram-positive bacteria, whereas tlr engages dsrna and tlr is specific for gram-negative bacteria lipopolysaccharides (lps; alexopoulou et al., ; campos et al., ; johnson et al., ; opitz et al., ) . tlr , on the other hand, recognizes bacterial flagellin, while tlrs and interact with antiviral compounds and tlr binds bacterial dna (bauer et al., ; gewirtz et al., ; ito et al., ) . recently, tlrs were observed to influence the development of the adaptive immune response presumably through the activation of antigen-presenting cells (apc; schjetne et al., ) . the most compelling evidence comes from studies on dendritic cells (banchereau et al., ) . it has previously been shown that multiple tlrs are found on dendritic cells. signaling through these receptors augments antigen presentation by driving cell maturation, upregulating expression of costimulatory molecules on the cell, and inducing cytokines (palucka and banchereau, ) . thus, tlrs may serve as a unique link between innate and adaptive immunity. the retinal pigment epithelium consists of a single layer of cells of neural ectoderm origin, which lie between the photoreceptors of the neural retina and the blood-rich choroid. these vitally important cells phagocytize the shed discs from the photoreceptor outer segments, recycling their components, such as retinoids. additional functions of this monolayer of cells include the transport of nutrients from the choroid into the retina and the transport of waste in the reverse direction. this cell also adsorbs light and provides adhesive properties for the retina (bok, (bok, , . the retinal pigment epithelial (rpe) cell also plays a key role in a variety of retinal pathologic processes. inherited retinal degenerative diseases can be associated with mutations of rpe cellular genes (hamel et al., ; morimura et al., morimura et al., , . moreover, degenerative diseases, such as age-related macular degeneration and diabetic retinopathy, can be associated with early damage to the rpe cell (cai et al., ; lutty et al., ) . in addition, a variety of in vivo and in vitro studies have identified this cell as an ideal target for infectious agents such as cytomegalovirus (cmv), toxoplasma gondii and coronavirus hooks et al., ; nagineni et al., nagineni et al., , . furthermore, this cell is a rich source of cytokines, chemokines, and growth factors that may contribute to or limit pathologic processes (chin et al., ; momma et al., ) . recently, the rpe cell has been shown to play a pivotal role in the immune system. it has been demonstrated that interferon-g (ifn-g) treatment up-regulates the expression of both mhc class-i and -ii molecules on rpe cells (percopo et al., ) . moreover, rpe cells have been reported to act as apc in the retina (percopo et al., ) . hence, rpe cells can incorporate pathogens, produce a variety of cytokines, and present pathogen-derived peptides to sensitized t cells. this finding, that rpe cells function as apc in the retina, extends the activities of this cell beyond its participation as a first line defense cell and underscores its important role in adaptive immunity. therefore, based on these observations, it was of interest to further define the role of this epithelial cell in innate and adaptive immune responses within the retina. to date, there is no information about the presence of tlrs within the retina. the results of this study demonstrate that rpe cells do indeed constitutively express distinct types of tlrs and that their expressions are modulated in the presence of dsrna and cytokines. tlr is a receptor for dsrna, and dsrna is a common replication intermediate for many viruses. moreover, tlr has been described as a specific tlr because it displays the most restricted cellular expression pattern (janssens and beyaert, ) . because tlr gene expression was identified in rpe cells and because the rpe cells are a site of replication for both rna and dna viruses, we further investigated tlr signaling in these cells. our data suggest that the binding of poly i:c, an analog of dsrna, to tlr on human rpe cells resulted in the production of ifn-h and other cytokines, chemokines, and adhesion molecules. thus, tlr signaling within the retina may provide additional protective molecules to mediate viral infections. affinity purified, monoclonal, antihuman tlr and tlr antibodies were purchased from imgenex (san diego, ca) while antihuman tlr (hta ) antibodies were purchased from ebiosciences (san diego, ca). polyclonal antibodies to tlr were obtained from santa cruz biotechnology (santa cruz, ca), lps (salmonella typhosa), poly i:c, and poly di:dc were purchased from sigma (st louis, mo). the recombinant human (ifn-g) and ifn-h were procured from roche molecular biochemicals (indianapolis, in). rna stat- was obtained from tel-test (friendswood, tx). minimal essential media (mem), fetal bovine serum (fbs), penicillin/streptomycin/ fungizone, nonessential amino acids, and normal horse serum (nhs) were purchased from life technologies/gibco (gaithersburg, md). geneamp rna pcr kits and taqman reagents were obtained from perkin elmer (branchburg, nj) . human rpe cell cultures were prepared from donor eyes and grown in mem supplemented with % fbs, nonessential amino acids, and penicillin/streptomycin/fungizone in a % co , humidified jc incubator. characterization of these cells has been described previously (li et al., ; nagineni et al., nagineni et al., , briefly, these cells demonstrated a hexagonal morphology when grown to confluence and formed monolayers with distinct intracellular boundaries. homogeneity of the cultures was established by positive immunostaining with monoclonal antibodies to cytokeratin, an epithelial cell-specific cytoskeletol protein. for the experiments described in this paper, human rpe cultures, at passages to , were used. a human monocyte cell line, u , was grown in an rpmi- medium supplemented with % fbs (atcc, manassas, va). total rna prepared from confluent monolayers of human rpe cells and from suspension cultures of u was used to evaluate the constitutive expression of tlr mrna. to study the effects of tlr activators, human rpe cells were washed with serum-free media (sfm) and incubated in sfm for h in the presence of poly i:c ( ag/ml ), lps ( ag/ml), or ifn-g ( u/ml). total rna was prepared from the cell cultures by using the rna stat- extraction solution. one ag of total rna was used for each rt-pcr reaction. the rt-pcr procedure was performed using an rna pcr kit (perkin-elmer) according to the manufacturer's instructions. pcr products were separated by gel electrophoresis, photographed under uv light, and integrated with an image acquisition system (eagle eye, stratagene, san diego, ca). the following primer pairs were used for the analysis of tlrs, costimulator molecules, and gapdh by rt-pcr: tlr ( bp) v-ctatacaccaagttgt-cagc- v and v-gtctccaactcagtaaggtg- v; tlr ( bp) v-gccaaagtcttgattgattgg- v and v-ttgaagttctccagctcctg- v; tlr ( bp) v-gatctgtctcataatggcttg- v and v-ga-cagattccgaatgcttgtg- v; tlr ( bp) v-tggatacgtttccttataag- v and v-gaaatg-gaggcaccccttc- v; tlr ( bp) v-tagctcc-taatctgatg- v and v-ccatgtgaagtctttgct gc- v; tlr ( bp) v-tctacctgggccaaaact gtt- and v-ggcacatgctgaagagagtta- v; tlr ( bp) v-gccagcgagtctcactgaact- v and v-gccagggcagccaacata- v; tlr ( bp) v-gt ccccacttct ccatg- v and v-gg c aca - (faure et al., ; ito et al., ) , jarrossay et al., ; bauer et al., and tabeta et al., ) . total rna was prepared from quiescent rpe and u by using an rna stat- reagent. quantitative rt-pcr analysis of tlr in rpe and u was performed on an abi prism (applied biosystems, foster city, ca) by using taqman master mix reagent kits according to the manufacturer's instructions. fam-labeled taqman probes and primers for human gapdh and toll-like receptors - (assays-on-demand gene expression products) were obtained from applied biosystems. standard curves were generated to gapdh and tlrs to by ten-fold serial dilutions of rpe and/or u rna. rna samples were analyzed in triplicate under similar conditions as those of the standards in the same -well plates for cycles. fluorescence intensities obtained for the samples were used to calculate relative fluorescence units by normalizing to gapdh fluorescence intensities. results are expressed as relative fluorescence units of tlrs - mrna levels in rpe and u cells. the rpe cells were seeded onto lab-tek tissue culture chamber slides (nalge nunc international, naperville, il). after h, the cells were washed with sfm and stimulated with media, poly i:c ( ag/ml), lps (s. typhosa, ag/ ml), or with ifn-g ( u/ml) for h. the slides were then fixed in equal parts of acetone/methanol and stored at À jc until analyzed. the slides were washed twice with pbs for min and then treated with % nhs in pbs for h at room temperature. the slides were overlaid with primary mouse antihuman tlrs , , or monoclonal antibodies ( ag/ml in pbs with % nhs) or with mouse igg (control) and incubated for -h followed by five washes in pbs containing % nhs. cells were then incubated for h with biotin-labeled horse antimouse igg (h + l; vector laboratories, burlingame, ca). the cells were washed again five times as described previously. fitc-labeled streptavidin ( ag/ml) was added and the cells were incubated for min in the dark (vector laboratories). the slides were then washed twice, mounted, and examined with a fluorescent microscope. . . eia assays for cytokines, chemokines, and adhesion molecules rpe cultures were grown to confluence in -well dishes. cultures were washed with sfm and incubated in the presence of various concentrations of poly i:c or poly di:dc for h at jc. supernatants were collected and stored at À jc until analyzed. the concentration of il- , il- , mcp- , sicam- , ifn-a, and ifn-h in the cell culture supernatant fluids was determined by eia. the assay was performed according to manufacturer's instructions (quantikine eia kits, r&d systems, minneapolis, mn). the data were analyzed using the versamax data analysis program (molecular devices, sunnyvale, ca). results from two representative experiments are presented as the means f s.d. of triplicate cytokine measurements. . . neutralization of poly i:c effects on rpe by tlr antibody rpe cultures were grown to confluence in -well plates in % fbs media. media were removed and replaced with serum free media (sfm). after h, media were removed and replaced with fresh sfm ( ml per well) and polyclonal antibody to tlr ( ug igg/ml). after a -h incubation at jc, poly i:c was added to the wells to obtain a final concentration of or ug/ml. the cultures were further incubated for h and culture supernatants were collected. the levels of secreted ifn-h were determined by eia. rpe cultures were grown to confluence in -well plates. the cultures were washed and incubated for h with serial ten-fold dilutions of recombinant human ifn-h or with media. the monolayers were washed and challenged with approximately plaque-forming units (pfu) of vesicular stomatitis virus. one hour later, the virus inoculum was removed and the cells were washed and refed with ml of mem containing . % methylcellulose and % fbs. after a -h incubation at jc, the overlay medium was removed, the cells were fixed with ethanol and stained with giemsa's solution, and the viral plaques were counted. preliminary studies using rt-pcr analysis indicated that human rpe cells contained detectable amounts of mrna for tlrs , , , , and . u cells are a human monocyte cell line that was used as a control. u cells contained detectable levels of mrna for tlrs , , , , , and . tlr mrna was barely detected in the u cells. in contrast, tlr mrna was highly expressed in human rpe cells. moreover, mrna for two coreceptors for tlrs, cd , and md were detected in human rpe whereas the u cells expressed only cd . in order to more accurately define the constitutive expression of tlrs, we analysed mrna obtained from the two cell types using real time rt-pcr analysis. as shown in table , real time rt-pcr analysis of rpe cells revealed the constitutive expression of varying levels of mrna for tlrs , , , , , , , , and . tlr was not detected and low levels of expression were noted for tlrs , , , and . tlrs and were the most highly expressed tlrs in rpe cells. when compared to u cells, rpe cells contained times more mrna for tlr . we next wanted to determine if poly i:c (dsrna) treatment altered tlr gene expression in rpe cells. the effect of poly i:c treatment on tlrs , , and gene expressions in rpe cells is shown in fig. . nih image analysis of this data indicated that poly i:c treatment increased gene expression of tlr by %, tlr by %, and tlr by %. gene expression for the coreceptors, cd and md , was also evaluated. poly i:c treatment had no effect on cd and md . rpe cells propagated on culture slides were exposed to media alone or media containing poly i:c ( ag/ml) or ifn-g ( u/ml; fig. ). after h, the cells were washed and fixed with equal volumes of acetone and methanol and reacted with normal mouse igg or with antibody directed to tlrs , , or . staining was not observed in cells treated with normal mouse igg (control). the intensity of tlr reactivity was weak on untreated rpe cells, but this was enhanced by pretreatment with poly i:c or ifn-g (data not shown). intensity of tlr reactivity was moderate in untreated rpe cells. this intensity was enhanced with poly i:c ( fig. a and b) or ifn-g. intensity of tlr reactivity was moderate on untreated rpe cells but was augmented following pretreatment with ifn-g ( fig. c and d) or with poly i:c. these data demonstrate that rpe cells express varying amounts of tlrs , , and and that these receptors can be modulated by selected tlr activators. the pattern of staining for tlr and tlr in rpe cells was different. tlr staining appears dispersed throughout the cytoplasm whereas tlr staining appears to be more localized at the cell boarders. tlr has only been detected on a limited number of cell types and recent studies indicate that tlr is a receptor for dsrna produced by viruses. moreover, dsrna binding to tlr on the cell surface results in the production of type- ifns. therefore, we next investigated the possibility that treatment of rpe cells with synthetic dsrna, poly i:c, would induce ifn. rpe cells were treated with varying concentrations of poly i:c or with poly di:dc ( mu/ml). poly di:dc is a synthetic double-stranded polydeoxyinosine/ deoxycytosine (dsdna) and is used as a negative control for poly i:c (dsrna). the cells were incubated for h and supernatant fluids were collected and assayed for ifn-a and ifn-h by eia. as seen in fig. a , poly i:c induced ifn-h in a dose-dependent manner. ifn-a was not detected in these samples or in untreated cells. moreover, cells treated with poly di:dc did not release ifn-a or ifn-h. because we have shown that rpe cells can produce ifnh, we next wanted to determine if ifn-h was biologically active on rpe cells. cell cultures were treated with varying concentrations of recombinant human ifn-h. after incubation for h, cells were challenged with approximately pfu of vsv. virus replication in rpe was evaluated by plaque assay. as seen in fig. b , rpe cells were sensitive to the antiviral action of ifn-h. one unit of ifn-h inhibited vsv by %, whereas and units inhibited virus replication by to %. in order to evaluate the specificity of the poly i:c induction of ifn-h through tlr , we next performed cultures were washed with sfm twice and incubated in the presence of various concentrations of poly i:c or poly di:dc for h. the culture supernatants were collected and analyzed for ifn-a and -h by eia. results from three experiments conducted in duplicate are presented as the means f s.e. (b) human rpe cultures were grown to confluence and were infected with vesicular stomatitis virus (vsv) as described in the materials and methods section. after h, cultures were fixed and stained with giemsa, and the plaques were counted. the data are presented as the means f s.e. of triplicate cultures obtained from a representative experiment. fig. . the effect of anti-tlr antibody on poly i:c-induced ifn-h production in rpe cells. rpe cell cultures grown to confluence were washed with sfm and incubated with media or anti-tlr antibody ( ug/ ml) for h. then, poly i:c was added to the appropriate wells to give a final concentration of or ug/ml. after h of incubation, supernatant fluids were collected and the concentration of ifn-h was determined by eia. results presented were obtained from one representative experiment with quadruplicate samples. antibody inhibition assays. rpe cells were pretreated with anti-tlr antibody for h and were then incubated with poly i:c for h. supernatant fluids were harvested and then analyzed for the presence of ifn-h. as seen in fig. , treatment with or ug of poly i:c induced . f . and . f . units of ifn-h, respectively. pretreatment of the cells with anti-tlr antibody reduced the levels of ifn-h produced to . and . ( p < . ). . . evaluation of poly i:c treatment of rpe cells: cytokines, chemokines, and adhesion molecules we next investigated whether poly i:c treatment of rpe cells resulted in the modification of additional cellular functions such as the production of cytokines, chemokines, and adhesion molecules. rpe cells were incubated with varying concentrations of poly i:c for h. the supernatant fluids were collected and evaluated by eia for il- , il- , mcp- , and sicam- production. as seen in fig. a -d, supernatant fluids from untreated rpe cells did not contain il- or il- and only very low levels of mcp- and sicam- . poly i:c treatment of rpe cells resulted in a dose-dependent enhancement of il- , il- , mcp- , and sicam- . following poly i:c treatment at , , or ag/ml, the concentration of il- increased from to ng/ml, il- increased from to ng/ml, mcp- increased from to ng/ml, and sicam- increased from . to ng/ml. in contrast, treatment of rpe cells under similar conditions with poly di:dc did not enhance the secretion of il- , il- , mcp- , and sicam- (fig. ) . tlrs are critical elements in the host defense against microbial pathogens (takeda et al., ) . in this report, we demonstrate for the first time the presence of tlrs on human rpe cells. real time pcr analysis of tlr gene expression identified tlrs - , and in human rpe cells. furthermore, human rpe cells highly expressed tlr . protein expression for tlrs , and was also observed on rpe cells and this expression was augmented by treatment with poly i:c or ifn-g. because tlr is found on a limited number of cells and was highly expressed in fig. . the effects of poly i:c treatment on the production of cytokines by human rpe cells. rpe cell cultures grown to confluence in -well plates were washed with sfm and incubated without (control) or with sfm containing various concentrations of poly i:c or poly di:dc. after h, the culture supernatants were collected and the concentrations of il- , il- , mcp- , and sicam- were determined by eia. results presented for il- (a), il- (b), mcp- (c), and icam- (d) were obtained from duplicate samples of two representative experiments. rpe cells, we performed studies to analyze signaling through tlr . the interaction of poly i:c with rpe cells resulted in the secretion of ifn-h as well as il- , il- , mcp- , and sicam- . moreover, we show that ifn-h is highly effective in inhibiting virus replication in rpe cells. specificity for tlr signaling was demonstrated by the inhibition of poly i:c induction of ifn-h by pretreatment of rpe cells with anti-tlr antibody. mucosal cells such as gastrointestinal, airway, and urinary epithelial cells are considered as the front line of defense against pathogenic microorganisms (schulz et al., ; hornef et al., ; pitman and blumberg, ; tsuboi et al., ) these cells have developed specific mechanisms for microbial protection that contribute to the innate immune response. recently, it has been demonstrated that these epithelial cells contain several tlrs and respond to microbes by secreting cytokines and chemokines. for example, a murine intestinal epithelial cell line is highly responsive to lps and expresses both tlr and cd . corneal epithelial cells have been reported to express tlr and cd , and the lps treatment of these cells resulted in the secretion of multiple proinflammatory cytokines and chemokines (song et al., ) . like other epithelial cells, the rpe cell can also be considered as a front line defense against invading organisms. it is strategically located between the neural retina and the blood-rich choroid. the rpe forms a barrier, limiting access to photoreceptors and other neuronal cells within the retina. earlier studies by our laboratory and others have revealed that rpe cells can be stimulated to produce a variety of cytokines, chemokines, and adhesion molecules (elner et al., (elner et al., , momma et al., ; nagineni et al., ) . in this report, we show that rpe cells possess a variety of tlrs and the costimulatory molecules, cd and md (tabeta et al., ) . tlr and tlr are the most widely studied members of the tlr family (johnson et al., ; faure et al., ; opitz et al., ) . both of these tlrs were found on the rpe cell. thus, rpe cells may defend against infections by sensing microbial invasion through multiple tlrs and the costimulatory molecules, cd and md . to date, tlr expression has been limited to a small number of cells types. earlier studies demonstrate that tlr is constitutively expressed on intestinal epithelial cells, dendritic cells, and mast cells (cario and podolsky, ; muzio et al., ) . the dendritic cell is a potent antigenpresenting cell that expresses multiple tlrs including tlr (banchereau et al., ; jarrossay et al., ) . several studies suggest that tlr signaling on dendritic cells amplifies antigen presentation by producing proinflammatory cytokines, up-regulating co-stimulatory molecules, such as cd , cd , and cd , and by increasing migration of cells to the lymph node (palucka and banchereau, ) . the dendritic cell and the rpe cell share some common features. both cells express tlr and are apc. the dendritic cell responds to tlr signaling by producing both ifn-a and ifn-h, whereas the rpe cell produces only ifn-h. this difference is probably a reflection of different cellular origins. presently, dendritic cells are considered to be of bone marrow origin while rpe cells are of neural ectoderm origin. additional studies are required to determine if the tlrs on the rpe cell also can augment apc functions. the binding of dsrna or poly i:c, an analog of dsrna, to tlr results in the production of type- ifns and other cytokines and chemokines. specificity for dsrna interactions with tlr was demonstrated by matsumoto et al. ( ) . their studies pointed out that poly i:c-induced ifn-h was suppressed by pretreatment with monoclonal antibody to tlr . additional studies on tlr knockout mice revealed that poly i:c treatment up-regulated the production of type- ifns (ifn-a, ifn-h) in wild-type mice but not in tlr ko mice (alexopoulou et al., ) . in this report, we demonstrate that the poly i:c treatment of human rpe cells results in the production and release of ifn-h and not ifn-a. moreover, these rpe cells are highly sensitive to the antiviral actions of human ifn-h. clearly, dsrna-mediated signaling in rpe cells can have a protective role in viral infections in the retina. in light of this, numerous in vivo and in vitro studies have identified that rpe cells are one of the principle targets for infectious agents, such as cmv, t. gondii, and murine coronaviruses (bodaghi et al., ; detrick et al., ; hooks et al., ; nagineni et al., ) . it is important to point out that additional tlrs may also participate in selected virus infections and further work is needed to better define this interaction (bieback et al., ; compton et al., ; kurt-jones et al., ) . when an infecting agent enters the retina, it is critically important for the host to have a rapid response system to limit damage to nonregenerating retinal cells. the rpe cells are strategically placed to function as protective cells. clearly, the innate immune system composed of tlrs is a primary rapid response system to infection. this study demonstrates that the rpe cell expresses tlrs - , and and therefore can initiate signaling pathways that stimulate host defenses. moreover, these cells respond to tlr stimulation by producing ifnh, il- , il- , mcp- , and sicam- . these cytokines, chemokines, and adhesion molecules together then participate in initiating adaptive immune responses. the demonstration that rpe cells express tlr and release ifnh represents a hitherto unrecognized biological role for the rpe cell. recognition of double-stranded rna and activation of nf-kappab by tolllike receptor dendritic cells: controllers of the immune system and a new promise for immunotherapy human tlr confers responsiveness to bacterial dna via species-specific cpg motif recognition hemagglutinin protein of wild-type measles virus activates toll-like receptor signaling entry of human cytomegalovirus into retinal pigment epithelial and endothelial cells by endocytosis retinal photoreceptor-pigment epithelium interactions. friedenwald lecture the retinal pigment epithelium: a versatile partner in vision oxidative damage and protection of the rpe activation of toll-like receptor- by glycosylphosphatidylinositol anchors from a protozoan parasite differential alteration in intestinal epithelial cell expression of toll-like receptor (tlr ) and tlr in inflammatory bowel disease cyclooxygenase- gene expression and regulation in human retinal pigment epithelial cells human cytomegalovirus activates inflammatory cytokine responses 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pigment epithelial cell autoantibodies are produced during murine coronavirus retinopathy toll-like receptor resides in the golgi apparatus and colocalizes with internalized lipopolysaccharide in intestinal epithelial cells interferonalpha and interleukin- are induced differentially by toll-like receptor ligands in human blood dendritic cell subsets role of toll-like receptors in pathogen recognition specialization and complementarity in microbial molecule recognition by human myeloid and plasmacytoid dendritic cells receptor-mediated monitoring of tissue well-being via detection of soluble heparan sulfate by toll-like receptor pattern recognition receptors tlr and cd mediate response to respiratory syncytial virus interferon-gamma signaling in human retinal pigment epithelial cells mediated by stat , icsbp, and irf- transcription factors changes in choriocapillaris and retinal pigment epithelium in age-related macular degeneration establishment of a monoclonal antibody against human toll-like receptor that blocks double-stranded rna-mediated signaling innate immunity: the virtues of a nonclonal system of recognition differential expression of chemokines by human retinal pigment epithelial cells infected with cytomegalovirus mutations in the rpe gene in patients with autosomal recessive retinitis pigmentosa or leber congenital amaurosis mutations in rgr, encoding a light-sensitive opsin homologue, in patients with retinitis pigmentosa differential expression and regulation of toll-like receptors (tlr) in human leukocytes: selective expression of tlr in dendritic cells synergistic effects of gamma interferon on inflammatory mediators that induce interleukin- gene expression and secretion by human retinal pigment epithelial cells mechanisms of interferon-induced inhibition of toxoplasma gondii replication in human retinal pigment epithelial cells toxoplasma gondii infection induces gene expression and secretion of interleukin (il- ), il- , granulocyte-macrophage colony-stimulating factor, and intercellular adhesion molecule by human retinal pigment epithelial cells transforming growth factorbeta expression in human retinal pigment epithelial cells is enhanced by toxoplasma gondii: a possible role in the immunopathogenesis of retinochoroiditis toll-like receptor- mediates treponema glycolipid and lipoteichoic acid-induced nf-kappab translocation how dendritic cells and microbes interact to elicit or subvert protective immune responses cytokine-mediated activation of a neuronal retinal resident cell provokes antigen presentation first line of defense: the role of the intestinal epithelium as an active component of the mucosal immune system cutting edge: link between innate and adaptive immunity: toll-like receptor internalizes antigen for presentation to cd + t cells and could be an efficient vaccine target toll-like receptors control activation of adaptive immune responses differences in lps-induced activation of bronchial epithelial cells (beas- b) and type ii-like pneumocytes (a- ) the expression of functional lps receptor proteins cd and toll-like receptor in human corneal cells toll-like receptors confer responsiveness to lipopolysaccharide from porphyromonas gingivalis in human gingival fibroblasts toll-like receptors roles of toll-like receptors in c-c chemokine production by renal tubular epithelial cells toll-like receptors as potential therapeutic targets for multiple diseases key: cord- -d h k l authors: nan title: author index, volumes - date: - - journal: vet microbiol doi: . /s - ( ) - sha: doc_id: cord_uid: d h k l nan diaz, a., see alfonso, r., ( ) ( ) dienglewicz, r.l., see shamblin, c.e., ( ) ( ) dizier, i., see thomas, a., ( ) ( ) revised definition of actinobacillus sensu stricto isolated from animals. a review with special emphasis on diagnosis characterisation of bovine strains of pasteurella multocida and comparison with isolates of avian, ovine and porcine origin adhesion of outer membrane proteins containing tandem repeats of anaplasma and ehrlichia species (rickettsiales: anaplasmataceae) to tick cells anaplasma infection in free-ranging iberian red deer in the region of castilla influenza surveillance in birds in italian wetlands ( - ): is there a host restricted circulation of influenza viruses in sympatric ducks and coots? in vitro growth inhibition of major mastitis pathogens by staphylococcus chromogenes originating from teat apices of dairy heifers piscine mycobacteriosis: a literature review covering the agent and the disease it causes in fish and humans first isolation of mycobacterium microti (llama-type) from a dog antimicrobial resistance of commensal escherichia coli from dairy cattle associated with recent multiresistant salmonellosis outbreaks attenuation of a virulent type bovine viral diarrhea virus recombinant lipl antigen-based single serum dilution elisa for detection of canine leptospirosis oxytetracycline as a predisposing condition for chalkbrood in honeybee argentine strain of equine herpesvirus isolated from an aborted foetus shows low virulence in mouse respiratory and abortion models identification of an alkaline ceramidase gene from dermatophilus congolensis evaluation of molecular and immunological techniques for the diagnosis of mammary aspergillosis in ewes differential expression of the msp a gene of anaplasma marginale occurs in bovine erythrocytes and tick cells role of catalase in the virulence of brucella melitensis in pregnant goats characterization of two proteins of staphylococcus aureus isolated from bovine clinical mastitis with homology to glyceraldehyde- -phosphate dehydrogenase the transmission of phocine herpesvirus- in rehabilitating and free-ranging pacific harbor seals (phoca vitulina nanoviruses: genome organisation and protein function transmission of multiple antimicrobial-resistant staphylococcus intermedius between dogs affected by deep pyoderma and their owners genetic characterization of orf viruses isolated from various ruminant species of a zoo efficacy of vaccines against bacterial diseases in swine: what can we expect? correlation between invasion of caco- eukaryotic cells and colonization ability in the chick gut in campylobacter jejuni the clinical expression and emergence of porcine circovirus effect of acidified feed on susceptibility of broiler chickens to intestinal infection by campylobacter and salmonella analysis of differential protein expression in actinobacillus pleuropneumoniae by surface enhanced laser desorption ionisation-proteinchip tm (seldi) technology recombinant major outer membrane protein (momp) of chlamydophila abortus, chlamydophila pecorum, and chlamydia suis as antigens to distinguish chlamydial species-specific antibodies in animal sera occurrence of chlamydiaceae spp. in a wild boar (sus scrofa l.) population in thuringia (germany) multiplex pcr for rapid detection of pseudorabies virus, porcine parvovirus and porcine circoviruses detection of mutations in the gyra gene and class i integron from quinolone-resistant salmonella enterica serovar longitudinal study of interferon-gamma, serum antibody and milk antibody responses in cattle infected with mycobacterium avium subsp pcrbased identification of serotype isolates of actinobacillus pleuropneumoniae biovars i and ii risk assessment of transmission of capsule-deficient routine diagnostics of lawsonia intracellularis performed by pcr, serological and post mortem examination, with special emphasis on sample preparation methods for pcr differentiation of actinobacillus pleuropneumoniae by pcr-rea based on sequence variability of the apxiva gene and by ribotyping enterotoxigenic k þ escherichia coli attachment to host cell receptors inhibited by recombinant pili protein antimicrobial susceptibility of swedish, norwegian and danish isolates of clostridium perfringens from poultry, and distribution of tetracycline resistance genes molecular epidemiology of rabies in botswana: a comparison between antibody typing and nucleotide sequence phylogeny a lawsonia intracellularis transmission study using a pure culture inoculated seeder-pig sentinel model an experimental mouse model of progressive atrophic rhinitis of swine development of maternal antibodies after oral vaccination of young female wild boar against classical swine fever occurrence and characteristics of enterohemorrhagic escherichia coli o in calves associated with diarrhoea resistance of broiler chickens to escherichia coli respiratory tract infection induced by passively transferred egg-yolk antibodies identification of a novel collagen-like protein, sclc, in streptococcus equi using signal sequence phage display protection of pigs from swine dysentery by vaccination with recombinant bmpb, a . kda outer-membrane lipoprotein of brachyspira hyodysenteriae characterization of bohv- ge envelope glycoprotein mimotopes obtained by phage display multiple genetic typing of salmonella enteritidis phage-types , , , and a isolates from animals and humans in the uk pcr detection of a putative n-acetylmuramidase gene from listeria ivanovii facilitates its rapid identification paratuberculosis in farmed and free-living wild ruminants in the czech republic identification of bartonella strains isolated from wild and domestic ruminants by a single-step pcr analysis of the s- s intergenic spacer region erratum to ''identification of bartonella strains isolated from wild and domestic ruminants by a single-step pcr analysis of the s- s intergenic spacer region molecular biology of porcine circovirus: analyses of gene expression and viral replication occurrence, distribution, and role in abortion of coxiella burnetii in sheep and goats in development of a pcr assay for streptococcus iniae based on the lactate oxidase (lcto) gene with potential diagnostic value impact of sawdust and wood shavings in bedding on pig tuberculous lesions in lymph nodes, and is rflp analysis of mycobacterium avium subsp. hominissuis of serotypes and isolated from pigs and environment erratum to ''recent advances in molecular epidemiology and detection of taylorella equigenitalis associated with contagious equine metritis (cem) contagious bovine pleuropneumonia (cbpp) caused by vaccine strain t / of mycoplasma mycoides subsp. mycoides sc mycobacterium nonchromogenicum in nasal mucus from cattle in a herd infected with bovine tuberculosis molecular characterization of porcine tt virus, an orphan virus, in pigs from six different countries comparison of an interferon-g to a phospholipase d enzyme-linked immunosorbent assay for diagnosis of corynebacterium pseudotuberculosis infection in experimentally infected goats intestinal colonisation-inhibition and virulence of salmonella phop, rpos and ompc deletion mutants in chickens genetic typing of bovine viral diarrhoea virus isolates from india detection of specific antibodies against deflagellated salmonella enteritidis and s. enteritidis flicspecific kda polypeptide prevalence and epidemiological features of bovine viral diarrhoea virus infection in lithuania molecular conservation of msp and msp in anaplasma marginale and a. centrale vaccine strain prevalence of bartonella infection in wild african lions (panthera leo) and cheetahs (acinonyx jubatus) detection of carriers of foot-and-mouth disease virus among vaccinated cattle comparable sensitivity and specificity in three commercially available elisas to differentiate between cattle infected with or vaccinated against foot-and-mouth disease virus infection of endothelial cells with anaplasma marginale and a. phagocytophilum evaluation of a lam elisa for diagnosis of paratuberculosis in sheep and goats pulsed-field gel electrophoresis-based subtyping of dna degradation-sensitive salmonella enterica subsp. enterica serovar livingstone and serovar cerro isolates obtained from a chicken layer farm identification and differentiation of avirulent and virulent rhodococcus equi using selective media and colony blotting dna hybridization to determine their concentrations in the environment attaching and effacing escherichia coli isolated from dogs in brazil: characteristics and serotypic relationship to human enteropathogenic serological relationship between cattle exposed to brucella abortus enzyme immunoassay for the diagnosis of brucellosis: chimeric protein a-protein g as a common enzyme labeled detection reagent for sera for different animal species the effect of a killed porcine reproductive and respiratory syndrome virus (prrsv) vaccine treatment on virus shedding in previously prrsv infected pigs chicken anemia virus induced apoptosis: underlying molecular mechanisms phenotypic characterization of brucella strains isolated from livestock in nigeria erratum to ''phenotypic characterization of brucella strains isolated from livestock in nigeria detection of genomic dna of the crayfish plague fungus aphanomyces astaci (oomycete) in clinical samples by pcr haemophilus parasuis: new trends on diagnosis a novel is element, ismpa effect of porcine parvovirus vaccination on the development of pmws in segregated early weaned pigs coinfected with type porcine circovirus and porcine parvovirus fecal shedding of helicobacter spp. by co-housed australian sea lions (neophoca cinerea) and australian fur seals (arctocephalus pusillus doriferus) serotypes and virulence genes of bovine shigatoxigenic escherichia coli (stec) isolated from a feedlot in argentina the high prevalence of helicobacter sp. in porcine pyloric mucosa and its histopathological and molecular characteristics phage types, ribotypes and tetracycline resistance genes of salmonella enterica subsp. enterica serovar typhimurium strains isolated from different origins in italy prevalence and antimicrobial resistance of campylobacter coli isolated from fattening pigs in france viremia and effect of fetal infection with porcine viruses with special reference to porcine circovirus infection higher incidence of malassezia pachydermatis in the eyes of dogs with corneal ulcer than in healthy dogs prevalence and deletion types of the pathogenicity island ett among escherichia coli strains from oedema disease and colibacillosis in pigs safety and efficacy of a modified-live canine coronavirus vaccine in dogs been to the library lately? veterinary microbiology hits a century colostral transmission of maedi visna virus: sites of viral entry in lambs born from experimentally infected ewes interaction between attaching and effacing escherichia coli serotypes o :h and o :k in cell culture antimicrobial susceptibility of mastitis pathogens from first lactation and older cows antimicrobial resistance of salmonella isolated from finishing swine and the environment of alberta swine farms efficacy of live chlamydophila abortus vaccine b in protecting mice placentas and foetuses against strains of chlamydophila pecorum isolated from cases of abortion the effect of mutation on rhodococcus equi virulence plasmid gene expression and mouse virulence comparison of methods for antimicrobial susceptibility testing and mic values for pleuromutilin drugs for brachyspira hyodysenteriae isolated in germany evaluation of variable number tandem repeat (vntr) loci in molecular typing of mycobacterium bovis isolates from ireland physiological and genetic characterisation of some new aphanomyces strains isolated from freshwater crayfish evaluation of three serum i-elisas using monoclonal antibodies and protein g as peroxidase conjugate for the diagnosis of bovine brucellosis influence of sampling time on bacteriological diagnosis of goat intramammary infection phenotypic and genotypic characteristics of staphylococcus aureus isolates from raw bulk-tank milk samples of goats and sheep immunosuppression in postweaning multisystemic wasting syndrome affected pigs pathological findings associated with naturally acquired porcine circovirus type associated disease comparative analysis of marek's disease virus (mdv) glycoprotein-, lytic antigen pp -and transformation antigen meq-encoding genes: association of meq mutations with mdvs of high virulence pathogen carriage by the cat flea ctenocephalides felis (bouche´) in the united kingdom development of a monoclonal antibody based competitive-elisa for detection and titration of antibodies to peste des petits ruminants (ppr) virus influence of porcine intestinal ph and gastric digestion on antigenicity of f fimbriae for oral immunisation inhibition of adhesion of f þ escherichia coli to piglet intestinal villous enterocytes by monoclonal antibody against blood group h- antigen the correlation between salmonella serology and isolation of salmonella in danish pigs at slaughter subviral dnas associated with geminivirus disease complexes infection dynamics of lawsonia intracellularis in pig herds a long-term study in merino sheep experimentally infected with mycobacterium avium subsp. paratuberculosis: clinical disease, faecal culture and immunological studies analysis of variation of bovine viral diarrhoea virus e sequence following transplacental infection of cattle selection of enterococci for potential canine probiotic additives intestinal translocation of streptococcus suis type ef þ in pigs bovine viral diarrhea virus is classified into different subgenotypes depending on the analyzed region within the viral genome genetics and geographical variation of porcine reproductive and respiratory syndrome virus (prrsv) in thailand different approaches to the vaccination of free ranging village chickens against newcastle disease in qwa-qwa the p * adhesin pseudogene of mycoplasma bovis characterization of streptococcus suis serotype isolates from diseased pigs in denmark avian circovirus diseases: lessons for the study of pmws genetic typing of bovine viral diarrhoea virus: most slovenian isolates are of genotypes d and f pseudomonas aeruginosa from canine otitis externa exhibit a quorum sensing deficiency erratum to ''pseudomonas aeruginosa from canine otitis externa exhibit a quorum sensing deficiency antigenic and genotypical characterization of newcastle disease viruses isolated in taiwan between van der meulen characterization of the pcs region of different ehrlichia ruminantium isolates genotypic and phenotypic screening of high and low virulence staphylococcus aureus isolates from rabbits for biofilm formation and mscramms antimicrobial resistance and resistance genes in staphylococcus aureus strains from rabbits conservation of deduced amino acid sequence of fimh among escherichia coli of bovine, porcine and avian disease origin immunoblotting, elisa and culture evidence for chlamydiaceae in sows on belgian farms newly developed primers for the detection of mycobacterium avium subspecies paratuberculosis enhanced efficacy of recombinant brucella abortus rb vaccines against b. melitensis infection in mice conserved regions in the sequence of the f (k ) fimbrial adhesin faeg suggest a donor strand mechanism in f assembly virulenceassociated genes in escherichia coli isolates from poultry with colibacillosis: correction differential clustering of mycoplasma mycoides subsp. mycoides sc strains by pcr-rea of the bgl locus correlation between production of acyl homoserine lactones and proteases in an aeromonas hydrophila aroa live vaccine intrapreputial infection of young bulls with bovine herpesvirus type . (bhv- . ): acute balanoposthitis, latent infection and detection of viral dna in regional neural and non-neural tissues days after experimental reactivation characterisation of a type bovine viral diarrhoea virus isolated from cattle in the uk oxalate degradation by intestinal lactic acid bacteria in dogs and cats comparison of pulsed field gel electrophoresis and repetitive sequence polymerase chain reaction as genotyping methods for detection of genetic diversity and inferring transmission of salmonella excessive porcine circovirus type antibody titres may trigger the development of porcine dermatitis and nephropathy syndrome: a case-control study a specific pcr for the identification of mycoplasma capricolum subsp. capripneumoniae, the causative agent of contagious caprine pleuropneumonia (ccpp) expression of apoptosis-related gene mrnas in feline t-cells infected with feline immunodeficiency virus (fiv) experimental dual infection of pigs with an h n swine influenza virus (a/sw/hok/ / ) and mycoplasma hyopneumoniae the effect of maternal antibodies on the detection of bovine virus diarrhoea virus in peripheral blood samples evaluation of the german cockroach (blattella germanica) as a vector for verotoxigenic escherichia coli f in confined swine production key: cord- -cxq v authors: nitsche, andreas; schweiger, brunhilde; ellerbrok, heinz; niedrig, matthias; pauli, georg title: sars coronavirus detection date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: cxq v we developed a set of three real-time reverse transcription–polymerase chain reaction (pcr) assays that amplify three different regions of the sars-associated coronavirus (sars-cov), can be run in parallel or in a single tube, and can detect < genome equivalents of sars-cov. the assays consider all currently available sars-cov sequences and are optimized for two prominent real-time pcr platforms. including those of coronaviruses, tend to vary over time and with location ( ) ( ) ( ) ( ) . recently, the sequence variations of sars-cov during the first epidemic phases in china in were reported. the neutral mutation rate for sars-cov was almost constant and similar to that of known rna viruses; the s protein, responsible for virus-host receptor recognition, displayed the most extensive amino acid changes ( ) . in addition, the sequence analysis of isolates from recent sars patients in china in has shown that . %- . % of the , bases of s gene, % of bases of m gene, and % of , bases of n gene are isogenous with those submitted to public databases, which date back to the first epidemic in spring ( ) . however, even these minimal changes could render existing pcr assays ineffective should sars-cov reemerge ( ) . to improve the ability to detect sars-cov safely and reduce the risk of eliciting false-negative results caused by genome sequence variations, we established three individual real-time rt-pcr assays. target sequences were chosen by using the following criteria: ) the regions are distributed over the whole genome, including the nonstructural polyprotein a and ab genes and the spike glycoprotein gene (table ) ; ) the regions are highly conserved among the , , and respective sequences available in public sequence databases; ) the regions are suitable for the design of a real-time rt-pcr assay; and ) the designed primers, ′-nuclease probes, and amplicons displayed no considerable homology to other viruses, including human cov oc and e in blast searches (available from http://www.ncbi.nlm.nih.gov/blast/). these assays were based on the fluorogenic oligoprobe chemistry, which uses the ′-exonuclease activity of the dna polymerase to generate a more specific signal than that produced by the use of sybr green i ( ) . the realtime rt-pcr assays were successfully run on the applied biosystems real-time pcr systems (sds and sds ; applied biosystems, foster city, ca) as well as on the roche lightcycler (roche diagnostics gmbh, mannheim, germany). all assays were designed as onestep rt-pcr reactions to be run under identical conditions on the respective pcr platform. this system allowed the simultaneous detection of different sars-cov regions in a single pcr run. moreover, we could combine the three assays in a single tube, which might be important when clinical material is limited. finally, the assays were compared to the ′-nuclease assay published recently ( ) and to a commercially available real-time pcr kit (real-art hpa-coronavirus lc rt pcr reagents, artus gmbh, hamburg, germany). after optimization of primer and ′-nuclease probe concentration and annealing temperature, reaction conditions for our ′-nuclease assay were as follows. for the rt-pcr performed on the applied biosystems platforms, each -µl reaction contained . µl of xquantitect probe rt-pcr master mix (qiagen, hilden, germany), pmol of each primer, pmol of ′-nuclease probe, and . µl of quantitect probe rt mix. rnase-free water was added up to µl, and µl of rna was used. cycling conditions were min at °c for rt reaction, min at °c for inactivation of rt, activation of the taq dna polymerase, and cdna denaturation, followed by cycles of s at °c and s at °c. total running time was min. for lightcycler rt-pcr reactions, each -µl reaction included . µl of . xlightcycler rna master hybridization probes mix (roche diagnostics gmbh), pmol of each primer, pmol of the ′-nuclease probe, and . µl mn (oac) ( mmol/l). rnase-free water was added up to µl, and µl of rna was used. cycling conditions were min at °c for the rt reaction, s at °c for initial denaturation, followed by cycles of s at °c, s at °c, and s at °c. total running time was min. the combined assays were set up by adding all primers and probes in the same concentration; the amount of water was reduced accordingly. protocols are also available from the robert koch-institut homepage (www.rki.de/infekt/sars/pcrprotocol.pdf). the human l gene and the human cyclophilin gene ( ) were amplified under identical reaction conditions as the sars-cov-specific assays on the abi platforms and the lightcycler, respectively, to act as amplification controls. to evaluate the sensitivity of the sars-cov-specific assays, rt-pcrs were performed repeatedly on serial dilutions of rna extracted with the viral rna kit (qiagen) from cultured sars-cov with defined amounts of genome equivalents (ge) by using the international standard of the european network for the diagnostics of imported viral diseases (enivd), distributed through the robert koch-institut (available from: http://www. rki.de/infekt/sars/datasheet.pdf). results are shown in table . detection limits of the three new assays were < ge. comparison of the threshold cycle (c t ) values showed that the new assays were at least as sensitive as the previously described assays ( , ) and the commercially available kit. when we combined the assays in a single tube targeting three different regions on the same rna template, the c t was reduced by to cycles (sds ), which suggests either that sensitivity was unchanged or, when there was an increase, that it was attributable to the combination of all three signals (lightcycler). subsequent agarose gel analysis during optimization steps of the pcr confirmed the simultaneous amplification of the three rt-pcr products (figure, online only; available from: http://www.cdc.gov/ncidod/eid/vol no / - -g.htm). using the single or combined assays, we analyzed bronchoalveolar-lavage fluid samples from suspected sars case-patients and probable sars patients (according to the robert koch-institut case definition, available from http://www.rki.de/infekt/sars/aolg-falldef-arsuu.pdf). all samples were positive for l and cyclophilin control sequences when amplified in parallel. in agreement with the previously published assay results ( ), sars-cov was detectable in three samples from eight probable sars patients, without explicit differences in the c t value of individual assays when the single or combined assays were used. these patients were seropositive and are regarded as confirmed sars patients. respiratory samples and stool samples taken days later from the remaining probable patients as well as the persons with suspected disease were negative by rt-pcr. moreover, these patients remained seronegative and are regarded as unconfirmed sars patients. in addition, serum samples from patients with sars-cov infection obtained - days after disease onset were analyzed. between and , ge/ml of sars-cov-specific rna was detected in of serum samples, even when serum was obtained from patients day after disease onset. (a detailed description of this study will be published later.) none of the assays displayed cross-reactivity to clinical samples containing human cdna from blood; human cov e; influenza viruses a and b; parainfluenzaviruses , , and ; respiratory syncytial virus; rhinoviruses; enteroviruses; adenoviruses - ; human metapneumovirus; mycoplasma pneumoniae; or chlamydia pneumoniae. for these pathogens, we obtained neither a fluorescent signal nor an amplification product in subsequent agarose gel analysis (selection shown in online figure) . although we focused on a one-step rt-pcr to decrease handling and total assay time, the three real-time rt-pcr assays can also be performed as two-step rt-pcr, including a separate cdna synthesis step followed by pcr, and then finally using appropriate ready-to-use master mixes and the same cycling condition, omitting the rt step. the single assays and the combined assay were also used in an external quality assessment to detect sars-cov, organized by the enivd. all assays could detect sars-cov in of samples with virus loads ranging from x to x ge of two isolates of sars-cov per milliliter sample without false-positive or false-negative results. while the application of three single assays to detect sars-cov leads to a higher reliability of negative results, reflecting the negative outcome of three independent amplification reactions, it is a more expensive approach than combining the assays. in conclusion, the real-time rt-pcr assays we describe provide a fast and reliable tool that can complement and improve recently introduced techniques for sars diagnostics. parallel amplification of two human reference genes, l and cyclophilin, confirmed negative results in clinical samples by demonstrating amplifiable rna. the separation of the control reaction was chosen to guarantee the high sensitivity of the sars-cov detection of < ge of sars-cov per reaction. an rt-pcr run is completed in < h, depending on the real-time pcr platform. in cases of small amounts of material or in an emergency situation with a high throughput of samples, the three sars-covspecific assays can be combined into one rt-pcr reaction without loss of sensitivity. furthermore, as the ambiguous diagnostic results in a hospital in canada have recently shown ( ) , targeting three different regions distributed over the whole genome considerably reduces the risk for false-negative results caused by virus sequence modifications. emerging infectious diseases • www.cdc.gov/eid • vol. , no. , july coronavirus as a possible cause of severe acute respiratory syndrome identification of severe acute respiratory syndrome in canada a cluster of cases of severe acute respiratory syndrome in hong kong acute respiratory syndrome. china, hong kong special administrative region of china, and viet nam identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome world health organization. case definition for surveillance of severe acute respiratory syndrome sars rapid diagnosis of a coronavirus associated with severe acute respiratory syndrome (sars) variation of the sequence in the gene encoding for transmembrane protein m of canine coronavirus (ccv) sequence comparison of porcine respiratory coronavirus isolates reveals heterogeneity in the s, , and - genes comparative full-length genome sequence analysis of sars coronavirus isolates and common mutations associated with putative origins of infection the viruses and their replication molecular evolution of the sars coronavirus during the course of the sars epidemic in china people's daily edited by promed-mail wood l, questions about comparative genomics of sars coronavirus isolates a guideline to reference gene selection for quantitative real-time pcr canadian officials watch sars-like mystery bug we thank sabrina wendt for excellent technical assistance, ian m. mackay for critically reading the manuscript, and artus (artus gmbh, hamburg, germany) for kindly providing the real-art hpa-coronavirus lc rt pcr reagents kit.dr. nitsche is a research fellow at the centre for biological safety of the robert koch-institut, berlin. his primary interest is the molecular detection of human pathogens with special focus on emerging viral infections. key: cord- -d loia authors: zhang, xue wu; yap, yee leng title: old drugs as lead compounds for a new disease? binding analysis of sars coronavirus main proteinase with hiv, psychotic and parasite drugs date: - - journal: bioorg med chem doi: . /j.bmc. . . sha: doc_id: cord_uid: d loia the sars-associated coronavirus (sars-cov) main proteinase is a key enzyme in viral polyprotein processing. to allow structure-based design of drugs directed at sars-cov main proteinase, we predicted its binding pockets and affinities with existing hiv, psychotic and parasite drugs (lopinavir, ritonavir, niclosamide and promazine), which show signs of inhibiting the replication of sars-cov. our results suggest that these drugs and another two hiv inhibitors (pnu and uc ) could be used as templates for designing sars-cov proteinase inhibitors. reemergence of severe acute respiratory syndrome (sars) is a distinct possibility. currently neither antiviral therapy nor vaccine is available. viral replicase and protease are preferred targets for the screening and design of antiviral compounds and have been successfully targeted in several viral diseases. the sars-associated coronavirus (sars-cov) main proteinase (mpro or cl pro) plays a key role in proteolytic processing of the replicase polyproteins a and ab, which makes it an attractive target for developing drugs against this new disease. recent report indicated that the proteinase inhibitor kaletra, a mixture of protease inhibitors-lopinavir and ritonavir, approved for treating hiv in , shows signs of effectiveness against the sars virus. in particular, researchers in taiwan discovered that two existing medicines, which have significant effect in inhibiting the replication of sars-cov (http://www. etaiwannews.com/taiwan/ / / / .htm). one is an anti-parasite drug niclosamide, and another is anti-psychotic drug promazine. the purpose of this study is to analyze whether the sars-cov main proteinase could be the target of these existing drugs. we performed in silico binding studies of the drugs using the recently identified crystal structure of mpro, ; to provide information for anti-sars inhibitor design. the atomic coordinates of sars-cov main proteinase were downloaded from protein data bank (pdb id q w). another crystal structure of sars-cov main proteinase is also available (pdb id uj ), the superposition of q w a chain and uj a chain is shown in figure , they overlap very well (rmsd ¼ . ), here we chose q w as docking studies, which was released early. the overall structure of a monomer of sars-cov main proteinase is composed of three domains: domain i (residues - ), domain ii (residues - ) and iii (residues - ), represented by green, pink and white trace in figure . the cleft between domains i and ii is its substrate-binding site. except four drugs (lopinavir, ritonavir, niclosamide and promazine), we also conducted the docking studies of two other molecules, pnu and uc , for their molecular formulas are close to those of niclosamide and promazine, respectively (fig. ) , and they both are the inhibitors of hiv- reverse transcriptase. ; the program hex was employed to conduct the docking of the ligands to the sars-cov main proteinase, its basic approach to parametric functions, which encode both surface shape, electrostatic charge and potential distributions. the surface shape representation uses a novel d surface skin model of protein topology, and a novel soft molecular mechanics energy minimization procedure is used to refine the candidate docking solutions. unlike conventional d fast fourier transform (fft) docking approaches, hex uses spherical polar fourier correlations to accelerate the docking between and times faster than fft docking algorithm. here we used the following parameter set: correlation type ¼ shape + three probes, post-processing ¼ mm minimization, steric scan ¼ (maximum), final search ¼ (maximum), the others are default set. the structural comparison was performed by lga. the visualization of d structure was generated by proteinexplorer (http://www.proteinexplorer.org). figure displays the overall structures of docking for four drugs (lopinavir, ritonavir, niclosamide and promazine) and two inhibitors (pnu and uc ) to sars-cov main proteinase. the binding pockets of these compounds in sars-cov main protease are shown in table , which is defined by those residues that have at least one heavy atom (other than hydrogen) with a distance less than a from a heavy atom of inhibitors, as described by chou et al. the results show that the binding pockets of six compounds can be divided into three classes: ( ) residues - and - for four drugs/inhibitors (lopinavir, niclosamide, promazine and pnu); ( ) residues - and - for uc inhibitor; ( ) residues - and - for drug ritonavir. all these pockets locate in domain i (residues - ), domain ii (residues - ) and a long loop region (residues - ) connecting domains i and ii in sars-cov main proteinase. thus, the four drugs and two inhibitors studied here can basically bind to the active site of sars-cov main proteinase, a cleft between domains i and ii. to estimate the binding affinities of each compound, the inhibitory constant (k i , mole) was calculated from the equation: where dg is the free energy of binding (kj/mol) (here refers to the final docked energy), r is the gas constant . j/k/mol and t is the absolute temperature (at k), as did in jenwitheesuk and samudrala. the results indicate that the inhibitory constants of six compounds are: . · À (lopinavir), . · À (ritonavir), . · À (niclosamide), . · À (promazine), . · À (pnu), . · À (uc ). it is noted that these values are too low, for example, the inhibitory constant of lopinavir was determined as $ À by jenwitheesuk and samudrala. the reason for this difference is that the docked energy value from hex program is a pseudo-energy, which is designed to give reasonably consistent units with conventional energy calculations, not based on experimentally derived parameters, and as a theoretical reference value only when performing the docking algorithm. thus we do not expect these values are the genuine representations of inhibitory constants and we use them primarily for qualitative comparison among the drugs/inhibitors studied here. because the lower the k i is, the greater the binding affinity is, hence hiv drug ritonavir is the compound that bind to the substrate binding site of sars-cov proteinase with the highest binding affinity, followed by hiv inhibitor pnu and anti-parasite drug niclosamide, and uc is the compound with the lowest binding affinity. moreover, the inhibitory constants of ritonavir, pnu, niclosamide, promazine and uc are about À , À , À , À and -fold inhibitory constant of lopinavir, respectively, if we assume that a value of À mol for lopinavir's inhibitory constant is correct, the inhibitory constants of ritonavir, pnu, niclosamide, promazine and uc could be estimated as À , À , À , À and À mol, respectively. the close views of the interactions between sars-cov main proteinase and these drugs/inhibitors are exhibited in figure . the results show that half of lopinavir is left outside the catalytic site (fig. a) , for ritonavir, the thiazole group (p ) and a benzene group (p ) are inserted into s and s specificity pockets, respectively, while another benzene side chain (p ) might be too long to fit the substrate binding pocket perfectly (fig. b) , there is similar situation in the inhibitor ag , which has been experimentally shown to not bind with high affinity to the sars-cov proteinase (http://www.nature.com/ nsu/ / - .html). thus the efficacy of lopinavir/ritonavir could be poor. indeed, consistent with our predictions, experimental observation data indicated that both lopinavir and ritonavir individually have only a weak in vitro activity against sars-cov. however, the addition of lopinavir/ritonavir to ribavirin and corticosteroid treatment regimens appears to reduce incubation and mortality rates, especially when administered early. similarly, the half of niclosamide or promazine is left outside the active site ( fig. c and d) , obviously the propane side chain in promazine is too long. for pnu inhibitor, seems it can basically fit into the active cleft, except the dihydrofuran side chain is a little bit long (fig. e) . finally, the inhibitor uc binds to a position that is slightly away from the active centre (fig. f) , its neopentane or methylfuran side chain is a little long and makes it unable to insert into the active pocket properly. indeed uc is the compound with lowest binding affinity as mentioned above. taken together, our study illustrates that existing drugs/inhibitors may be used as starting points for the discovery of rationally designed anti-sars proteinase drugs. old drugs for a new bug the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor crystal structures of hiv- reverse transcriptase in complex with carboxanilide derivatives evaluation of protein docking predictions using hex . in capri rounds and lga: a method for finding d similarities in protein structures binding mechanism of coronavirus main proteinase with ligands and its implication to drug design against sars improved prediction of hiv- protease-inhibitor binding energies by molecular dynamics simulations identifying inhibitors of the sars coronavirus proteinase coronavirus main proteinase ( clpro) structure: basis for design of anti-sars drugs treatment of severe acute respiratory syndrome with lopinavir/ritonavir: a multicentre retrospective matched cohort study key: cord- -vatfdilt authors: narita, m.; ishii, m. title: encephalomalacic lesions in pigs dually infected with porcine reproductive and respiratory syndrome virus and pseudorabies virus date: - - journal: j comp pathol doi: . /j.jcpa. . . sha: doc_id: cord_uid: vatfdilt four pigs (group ) were infected with an aerosol containing porcine reproductive and respiratory syndrome virus (prrsv) followed days later by pseudorabies virus (prv). three further pigs (group ) received prrsv alone, two (group ) received prv alone, and two (group ) remained as uninfected controls. despite the admittedly small numbers of animals, the experiment appeared to throw light on aspects of synergy. thus, the group pigs showed severe neurological signs characterized by ataxia and muscular tremors. total cell numbers in the bronchoalveolar lavage fluid were increased in all prrsv-infected pigs, and prrsv antigen was detected in the alveolar macrophages. total cell numbers in the cerebrospinal fluid of group pigs were considerably greater than those demonstrated in group , but no prv antigen was found. pigs of groups and showed pulmonary lesions, characterized by interstitial pneumonia and prrsv antigen immunolabelling. non-suppurative encephalitis was found in five of the six pigs of groups and . in particular, one group animal had severe necrotizing encephalitis with intranuclear inclusion bodies and associated immunolabelling of prv antigen. the other three group pigs had prominent malacic lesions, with macrophages. these neuropathological findings strongly suggested that prrsv infection in pigs enhances the severity of brain lesions caused prv. porcine reproductive and respiratory syndrome (prrs) is an arterivirus disease, currently endemic in many swine-producing countries (conzelmann et al., ) . prrs virus (prrsv) primarily infects and destroys alveolar macrophages, which play an important role in pulmonary defence (rossow, ) . prrsv is therefore suspected to have an immunosuppressive effect on pigs. co-infection with prrsv has been reported to increase the severity of disease produced by agents such as porcine respiratory coronavirus, influenza virus, mycobacteria, salmonella choleraesuis and streptococcus suis (groschup et al., ; kawashima et al., ; reeth et al., ; narita et al., ; thacker et al., ; de bruin et al., ; thanawongnuwech et al., ; wills et al., ) . such synergism has been supported by clinical, virological and immunological observations. the immunosuppressive effect of prrsv on the host response to pseudorabies virus (prv) has also been investigated in the respiratory system (shibata et al., ) , but there have been few histopathological studies supporting synergism in dual infections with prrsv and prv, or in dual infection with prrsv and other respiratory viral agents (de bruin et al., ) . the purpose of the present study in admittedly small numbers of specific pathogen-free (spf) pigs was to throw light on the effects (clinical, cytological and pathological) of dual infection with prrsv and prv as compared with single infections. eleven spf pigs aged weeks were used, having been shown to be free from serum antibodies against prv, prrsv and porcine circovirus type . the pigs were divided into four groups (see below), which were housed separately in different blocks to prevent cross-infection. the housing conditions included a filtered air system and regulated temperature. the edrd- strain of prrsv, kindly provided by dr y. murakami of the national institute of animal health, was used after eight passages in swine alveolar macrophages. the ys- strain of prv, also provided by dr y. murakami, was used after three passages in pig kidney cell culture. the infected culture fluids were used as inocula. the pigs were randomly assigned to four groups, as follows: group (pigs - ) were inoculated with prrsv followed by prv; group (pigs - ) received prrsv alone; group (pigs and ) received prv alone; group (pigs and ) were non-infected controls. the inocula were administered as an aerosol produced by a nebulizer, after the animals had been anaesthetized with ketamine hydrochloride ( mg/kg body weight) and xylazine ( mg, intramuscularly). on "day " of the experiment, pigs of groups and received . tcid of prrsv in a dose volume of ml. on day of the experiment, the pigs of groups and received . tcid of prv in a dose volume of ml. the negative control animals of group received ml of non-infected culture medium on day , and ml on day . all animals were killed with an intravenous overdose of pentobarbital sodium on the following days of the experiment: day , pig ; day , pigs , , and ; and day , pigs , , , , and . a csf sample ( ml) was collected by syringe post mortem and placed in a sterile bottle. after removal of the lungs, a cannula ( mm in diameter) was inserted into the right main bronchus, and ml of sterile phosphate-buffered saline (pbs) were introduced into the bronchus and then recovered by suction. counts of nucleated cells per microlitre of csf and balf were made, and . ml of csf and . ml of a in dilution of balf were centrifuged and stained with a diff-quik kit (international reagent corporation, koube, japan). evaluation included a -cell differential count and a morphological description of the cells. the centrifuged csf and balf cells were fixed in cold acetone and stored at c for immunohistochemical examination. specimens from each pig, including parts of the brain (cerebral cortex, frontal lobe, motor area, occipital lobe, corpus striatum, thalamus, colliculus caudalis, cerebellar peduncles, pons, cerebellum and medulla oblongata), spinal cord, trigeminal ganglia, tonsil and lung, were fixed in % neutral buffered formalin. the tissues were embedded in paraffin wax, sectioned ( mm), stained with haematoxylin and eosin (he), and examined by light microscopy. prrsv and prv antigen in the formalin-fixed tissues and cold acetone-fixed csf and balf cells were demonstrated by the streptavidin -biotin (sab) immunoperoxidase (ip) method, with a histofine sab kit (nichirei corp., tokyo, japan). anti-prrsv (chiba - strain) rabbit serum (provided by dr k. kawashima, national institute of animal health, japan) (kawashima et al., ) and anti-prv rabbit serum (provided by dr t. imada, national institute of animal health, japan) (narita et al., (narita et al., , were used as the primary antibodies at dilutions of in and in , respectively. sections were counterstained with methyl green. tissue sections from noninfected control pigs (nos and ) and serum from a non-immunized rabbit were used for control purposes. all four pigs dually infected with prrsv and prv (group ) showed severe neurological signs, characterized by ataxia and muscular tremors, after being infected with prv, and had a severe febrile response (up to c) between days and . the three group pigs, infected with prrsv alone, showed slight respiratory symptoms and had a transient ( -day) febrile response. the two group pigs, infected with prv alone, showed inappetence and pyrexia for a period of days. the two non-infected control pigs (group ) showed no clinical abnormalities during the experimental period. cytology csf cells. the results are shown in table . the total cell number in the csf from pigs infected with prv (groups and ) ranged from to ( £ . )/ ml. the proportions of the various cell types were: neutrophils - %, macrophages - % and lymphocytes - %. the total cell numbers of pigs infected with prrsv alone (group ) and noninfected controls (group ) ranged from to ( £ . )/ml and to ( £ . )/ml, respectively, and no neutrophils or macrophages were found. despite the small numbers of observations, there was an indication that the total number of csf cells may have been considerably higher in group than in group . no prv or prrsv antigen was detected by immunolabelling of the csf cells of any animals. balf cells. the total cell number in the balf from pigs infected with prrsv (groups and ) ranged from to ( £ . )/ml. the proportions of the various cell types were: neutrophils - %, macrophages - %, and lymphocytes - %. the total cell number in pigs infected with prv alone (group ) was to ( £ . )/ml, and in non-infected control pigs (group ) was to ( £ . )/ml. the proportion of neutrophils in pigs infected with prv alone (group ) was - %, that of macrophages was - %, and that of lymphocytes was - %. in non-infected control pigs (group ), the corresponding proportions were as follows: neutrophils - %, macrophages - %, and lymphocytes - %. there was no obvious difference between groups and . prrsv (but not prv) antigen was detected in the balf macrophages of four the seven pigs infected with prrsv (groups and ) (fig. ) . nd, not done. þ, a few immunolabelled cells; , none. see materials and methods for details of inoculation of groups - . the experiment began on "day " and the csf and balf were collected post mortem at the following times: pig (day ); pigs , , and (day ); pigs , , , , and (day ). * £ . for csf, but £ . for balf. macroscopically, all dually infected pigs (group ) showed severe atrophy of the thymus, congestion of the brain, and pneumonia with diffuse tan coloration at the periphery of the lobes. none of the three pigs infected with prrsv alone (group ) or the two pigs infected with prv alone (group ) showed obvious macroscopical lesions, except for slight atrophy of the thymus in the group animals. the two non-infected control pigs (group ) showed no abnormalities. the distribution of microscopical lesions is summarized in table . all dually infected pigs (nos - ) had slight trigeminal ganglioneuritis and slight (pigs - ) to severe (pig ) nonsuppurative encephalitis, consisting of neuronal degeneration and necrosis, neuronophagia, diffuse or focal glial reactions, and perivascular cuffing (fig. ) . the lesions were distributed in the white matter of the lobus frontalis, lobus temporalis and lobus parietalis, but not in the cerebellum or spinal cord. the more severe lesions occurred in the frontalis areas of the cerebral grey matter. some degenerating neuronal and glial cells adjacent to the necrotic areas showed basophilic intranuclear inclusion bodies. three dually infected pigs (nos - ) killed on day of the experiment, had prominent encephalomalacic lesions, localized in table distribution of histopathological lesions in the central nervous system (cns) of pigs infected dually and singly with prrsv and prv encephalitis*/encephalomalacia* in individual pigs of group group group group cns site cerebrum frontalis þþþ/ þ /þþþ þ/þþþ þ/þþþ / / / þ/ / / / temporalis þþ/ þ /þþ þ/þþ þ/þþ / / / / / / / parietalis þ / þ /þ þ/þþ þ/þþ / / / / / / / occipitalis / / / / / / / / / / / hippocampus þ / / / / / / / / / / / thalamus þ / þ / þ / þ/ / / / / / / / cerebellum / / / / / / / / / / / pons þ / þ / þ / þ/ / / / / / / / medulla oblongata þ / þ / þ / þ/ / / / / / / / trigeminal ganglia þ / þ / þ / þ/ / / / / / / / spinal cord / / / / / / / / / / / see materials and methods for details of inoculation of groups - , and for times at which the animals were killed for examination. interstitial pneumonia: moderate (pigs , , , , ), slight (pig ), negative (pigs , , , , ) . tonsillitis: severe (pig ), moderate (pigs , , ), slight (pig ); negative (pigs , , , , , ) . thymic atrophy: severe (pigs - ), slight (pigs , ) , negative (pigs , , , , ). * , negative; þ , slight; þþ, moderate; þþþ, severe. areas near the olfactory bulb and rhinencephalon, including the olfactory tract (fig. ) , olfactory stria, and pyriform cortex. they consisted of cortical cavitation, with macrophages ( fig. ) . pig , infected with prv alone, showed slight perivascular cuffing and glial reaction but no neuronal degeneration or intranuclear inclusion bodies. the four group pigs and two of the three group pigs had slight to moderate interstitial pneumonia (fig. ) , but no necrotizing bronchiolitis. all group pigs had moderate tonsillitis with typical intranuclear inclusion bodies in the degenerating crypt epithelial cells; they also had severe atrophy of the thymus with apoptosis of thymic t lymphocytes (fig. ) . two group pigs also had slight thymic atrophy. such lesions were not observed in the pigs of groups and . the distribution of viral antigens in tissues is summarized in table . in the cerebrum, prv antigen was found in three (nos , and ) of the dually infected pigs. strong immunolabelling was closely associated with the intranuclear inclusion bodies in the neuronal cells of pig , killed on day of the experiment (fig. ) . immunolabelling was observed in a few glial cells of pigs and but not in the macrophages in the encephalomalacic lesions. in the lungs of pigs - , prrsv antigen (but not prv antigen) was detected in macrophages in the alveolar lumina at sites showing interstitial pneumonia (fig. ) . prv antigen was detected in the degenerating epithelial cells of the tonsillar crypts of the four dually infected pigs, and prrsv antigen was detected in the macrophages of two of them (nos and ) and one pig (no. ) that received prrsv alone. neither antigen was found in the thymus of any pig or in any tissue from the two non-infected control pigs. the synergistic effect of dual infection with prrsv and other infectious agents has been investigated previously (groschup et al., ; kawashima et al., ; reeth et al., ; narita et al., narita et al., , thacker et al., ; de bruin et al., ; thanawongnuwech et al., ; wills et al., ) . in this study, pigs infected with prrsv or prv alone did not show severe clinical signs, but there was a febrile response in pigs infected with prv, and there were slight respiratory symptoms in pigs infected with prrsv. the four dually infected pigs (group ), however, showed severe neurological signs characterized by ataxia and muscular tremors. these findings, which resembled those reported by shibata et al. ( ) , suggested that prrsv infection in pigs affects the replication of prv. each of the infectious agents produces characteristic lesions at the various sites of viral replication (narita et al., (narita et al., , (narita et al., , rossow, ) . in the present experiment, seven pigs infected with prrsv (groups and ) had slight interstitial pneumonia but no necrotizing bronchiolitis. these pneumonic lesions were closely associated with the presence of prrsv antigen, and were probably due to prrsv infection since they resembled prrsvassociated lesions described in previous reports (rossow, ; shibata et al., ) . five of six pigs infected with prv (groups and ) had non-suppurative encephalitis and trigeminal ganglioneuritis. one dually infected pig (no. ) had severe necrotizing encephalitis with intranuclear inclusion bodies, while the other three (nos - ) had prominent encephalomalacia, with macrophages. all dually infected pigs had tonsillitis with inclusion bodies and severe atrophy of the thymus. these results strongly suggest that prrsv infection in pigs can increase the severity of brain and tonsillar lesions due to prv replication, and also the severity of lung lesions (shibata et al., ) . moreover, thymic atrophy may be related to dual infection. the total number of csf cells increased -to fold in prv-infected pigs as compared with the numbers in pigs infected with prrsv alone and in the non-infected pigs. moreover, the total number was higher in dually infected pigs that in those infected with prv alone. in prv-infected pigs, neither prv nor prrsv antigen was detected in csf cells. the total cell number in the balf increased by up to -fold in all prrsv-infected pigs, and prrsv (but not prv) antigen was detected in the alveolar macrophages. these results corresponded well with the presence of interstitial pneumonia. thus, the results of csf cell analysis accorded with the severity of the brain lesions in the dually infected pigs, and the results of balf cell analysis with the interstitial pneumonic lesions in prrsv-infected pigs. herpes viruses spread via sensory axons and infect sensory neurons in the ganglia of the peripheral nervous system (cook and stevens, ; narita et al., narita et al., , chowdhury et al., ; yanai et al., ) . in prv infection in pigs, infection spreads from the nasal cavity via the olfactory pathway to produce non-suppurative encephalitis (narita et al., (narita et al., , (narita et al., , (narita et al., , (narita et al., , . in the present experiments, one dually infected pig had severe encephalitis and three had encephalomalacia, characterized by neuronal degeneration with intranuclear inclusion bodies, a diffuse or focal glial reaction, and perivascular cuffing, as well as by cavitation accompanied by large numbers of macrophages. the lesions were mainly confined to the olfactory bulb and rhinencephalon, including the olfactory tract, olfactory stria, and pyriform cortex. the encephalomalacic lesions may therefore have developed from necrotic foci similar to those observed in prv-infected newborn piglets (nunoya et al., ) and in animals with fluctuating temperatures (narita et al., ) . thus, the neuropathological findings strongly suggested that prrsv infection in pigs enhanced the severity of brain lesions caused by prv. this apparent synergy was supported by histopathological findings. neuropathology of bovine herpesvirus type (bhv- ) meningo-encephalitis in a rabbit seizure model molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group pathogenesis of herpetic neuritis and ganglionitis in mice: evidence for intra-axonal transport of infection effects of a porcine reproductive and respiratory syndrome virus infection on the development of the immune response against pseudorabies virus serological studies on the potential synergism of porcine reproductive and respiratory syndrome virus and influenza-, corona-and paramyxoviruses in the induction of respiratory symptoms in swine detection of porcine reproductive and respiratory syndrome virus and mycoplasma hyorhinis antigens in pulmonary lesions of pigs suffering from respiratory distress pseudorabies virus in dexamethasone-treated pigs immunohistological demonstration of spread of aujeszky's disease virus via the olfactory pathway in hpcd pigs immunohistological study of encephalomalacia in pigs infected with aujeszky's disease virus immunohistopathological characterization of pig pneumonia caused by a combined aujeszky's disease virus and actinobacillus pleuropneumoniae infection immunohistochemical characterization of calf pneumonia produced by the combined endobronchial administration of bovine herpesvirus and pasteurella haemolytica immunopathology in aujeszky's disease virusinfected pigs exposed to fluctuating temperatures invasion and spread of equine dual infection of pigs with prrsv and prv herpesvirus in the olfactory pathway of pigs after intranasal inoculation pseudorabies virus infection in piglets acconpanying with the lesion of bilateral encephalomalacia dual infections of feeder pigs with porcine reproductive and respiratory syndrome virus followed by porcine respiratory coronavirus or swine influenza virus: a clinical and virological study porcine reproductive and respiratory syndrome experimental dual infection of specific pathogen-free pigs with porcine reproductive and respiratory syndrome virus and pseudorabies virus mycoplasma hyopneumoniae potentiation of porcine reproductive and respiratory syndrome virus-induced pneumonia pathogenesis of porcine reproductive and respiratory syndrome virus-induced increase in susceptibility to streptococcus suis infection synergism between porcine reproductive and respiratory syndrome virus (prrsv) and salmonella choleraesuis in swine experimental infection with equine herpesvirus (ehv- ) in cats we thank mr m. kobayashi and miss m. shimada for preparing the histological tissue sections, and dr y. ando and mr t. fujisawa for preparing the photographs. key: cord- -hohzjx authors: hamelin, marie-Ève; abed, yacine; boivin, guy title: human metapneumovirus: a new player among respiratory viruses date: - - journal: clin infect dis doi: . / sha: doc_id: cord_uid: hohzjx the human metapneumovirus (hmpv) is a newly described member of the paramyxoviridae family belonging to the metapneumovirus genus. since its initial description in , hmpv has been reported in most parts of the world and isolated from the respiratory tract of subjects from all age groups. despite the fact that prospective and case-control studies have been limited, the epidemiology and clinical manifestations associated with hmpv have been found to be reminiscent of those of the human respiratory syncytial virus, with most severe respiratory tract infections occurring in infants, elderly subjects, and immunocompromised hosts. additional research is needed to define the pathogenesis of this viral infection and the host's specific immune response. part of the pneumovirinae subfamily within the paramyxoviridae family. initial electron microscopic examination of hmpv isolates revealed morphological characteristics consistent with paramyxoviruses. pleomorphic, spherical, and filamentous particles could be observed (figure ) [ ] . spherical enveloped particles vary in size, with a mean diameter of nm. the nucleocapsid has a length varying from ! to ∼ nm and a diameter of nm. hmpv isolates were initially found to grow on tertiary monkey kidney and llc-mk (rhesus monkey kidney) cells, with poor or no viral growth on other cell lines, including madin darby canine kidney, vero (african green monkey kidney), human laryngeal carcinoma (hep- ), human foreskin fibroblast, human rhabdomyosarcoma, transformed human kidney ( ), human lung adenocarcinoma (a- ), and human colon adenocarcinoma (ht- ) [ , ] . in addition, hmpv isolates did not exhibit hemagglutinating activity when tested with human, turkey, chicken, or guinea pig rbcs, which is consistent with other members of the pneumovirinae subfamily [ , ] . intranasal inoculation of ferrets and guinea pigs with hmpv isolates did not cause any clinical symptoms [ ] . also, experimentally infected birds (juvenile turkeys and chickens) did not show clinical signs or virus replication over a -week period. on the other hand, there was evidence of efficient hmpv rep- figure . negative-stain electron micrographs of human metapneumovirus (hmpv). center image shows pleomorphic hmpv particles; note the projections along the periphery of the viruses. upper right and lower left insets show the nucleocapsid and the filamentous rodlike particle, respectively. staining was done with % phosphotungstic acid. bar markers represent nm. from [ ] ; reproduced with permission from the university of chicago press. lication in the respiratory tract of experimentally infected monkeys (cynomolgus macaques) associated with mild upper respiratory tract signs [ ] . thus, in contrast to apv, hmpv is a respiratory pathogen of primates. the hmpv genome consists of a single negative strand of rna of ∼ kb containing genes coding presumably for a nucleoprotein (n), phosphoprotein (p), matrix (m) protein, fusion (f) protein, transcription elongation factor/rna synthesis regulatory factor (m ), small hydrophobic (sh) surface protein, major attachment (g) glycoprotein, and major polymerase (l) subunit, arranged in the order -n-p-m-f-m -sh-g-l- , similar to apv [ , ] . two major differences exist between hmpv and hrsv genomes: the gene order is different, and hmpv does not contain nonstructural genes (figure ) [ ] . the possible relevance of the absence of these viral proteins, which have been associated with ifn antagonism by hrsv [ ] , deserves additional study. for hrsv, the g and f surface proteins are known as the major protective antigens [ ] , and this also needs to be verified for hmpv. phylogenetic analyses have demonstrated that, among members of the pneumovirinae subfamily, hmpv was most closely related to apv serotype c, the avian metapneumovirus that emerged in the united states in the late s. for instance, the amino acid sequence identity between hmpv strains and apv-c and hrsv representatives varies from % to % and from % to %, respectively, when comparing the n, p, m, and f genes [ , ] . the sh and g amino acid sequences of hmpv show important variability and share only % and % identity with their respective apv-c counterparts [ , , ] . it is possible that this important divergence may account for the specific host range of these related metapneumoviruses. genetic analysis on a large number of hmpv isolates has identified major groups and minor genetic clusters within each group [ , ] . however, additional investigations are still required to determine whether these genotypes represent different antigenic groups. recently, the complete nucleotide sequences of hmpv isolates belonging to these major groups were determined [ ] . nucleotide and amino acid sequence identities between the hmpv groups were found to be % and %, respectively, which is similar to what has been reported with hrsv a and b genotypes. since its initial report by the dutch researchers in , hmpv has been found in most parts of the world, with reports from north america (united states and canada), europe (united kingdom, france, germany, italy, spain, and finland), asia (hong kong and japan), and australia (table ). the virus has also been identified in hiv-infected and nonimmunocompromised children from south africa [ ] . the few seroprevalence surveys from the netherlands [ ] , japan [ ] , and israel [ ] have indicated that virtually all children are infected by - years of age. in addition, studies have shown that hmpv is not a new pathogen, with serological evidence of human infection dating from in the netherlands [ ] and viral isolation for the past - years in europe and canada [ , ] . cases of severe hmpv infection in adults [ ] and of reinfection in immunocompromised subjects [ ] suggest that, despite universal infection in childhood, new infections can occur throughout life due to incompletely protective immune responses and/or acquisition of new genotypes. surveys have in- dicated that hmpv has a seasonal distribution overlapping hrsv circulation, with most cases reported during the winter/ early spring months (table ) . although most studies have limited their surveillance to the typical respiratory virus period, our group has shown that % of hmpv isolates recovered in cell culture in quebec, canada, were recovered during the period of december through may [ ] . in addition, we found that outbreaks of hmpv infection tend to peak in early spring over a - week period, slightly later than outbreaks of hrsv infection, which also are more spread out in time [ ] . however, additional studies over multiple years are needed to better define the seasonality of hmpv infection. the role of hmpv as the cause of arti has been evaluated in many studies, mostly using molecular detection methods (table ) . young hospitalized children have been best studied, and hmpv has been generally found in %- % of arti cases in that population. however, in one study from italy, the number of hmpv infections varied widely over a -year period and were associated with %- % of hospitalizations for arti [ ] . the relative role of hmpv in respiratory syndromes of adults has been less studied. in one study from rochester, new york, hmpv was detected by serological tests and/or rt-pcr in . % of young and elderly adults with arti [ ] . interestingly, evidence of hmpv infection was also found by serological testing in almost the same percentage ( . %) in asymptomatic adults, raising the question of the causative role of hmpv in arti in adults. recent studies by our group indicate that hmpv is rarely identified by rt-pcr in npas obtained from asymptomatic young children, with a rate significantly lower than that of subjects with arti (! % vs. %) [ , ] . it is interesting to note that the rates of detection of hmpv have been generally higher in retrospective than prospective studies (table ) , an observation consistent with some selection bias. thus, to better define the role of hmpv in various respiratory conditions, large prospective studies using appropriate controls need to be conducted. in addition, testing of all clinical samples (i.e., not only those samples that are found to be negative for other viruses) must be performed. nevertheless, most studies have so far indicated that hmpv is a frequent cause of severe arti, especially in young children. table summarizes the clinical findings of the first hmpvinfected canadian subjects retrospectively identified through our virology laboratory. symptoms of both upper and lower respiratory tract infections have been associated with hmpv in young children, although most reports are biased towards description of the most severe symptomatology in hospitalized subjects. in young hospitalized children, the clinical features associated with hmpv infections are very similar to those of hrsv [ , [ ] [ ] [ ] . diagnoses of bronchiolitis, with or without pneumonitis, have been most commonly reported. a substantial proportion (up to %) of infected children also has concomitant otitis media [ ] . acute wheezing and asthma exacerbation have been associated with hmpv in some [ , ] but not all [ ] studies. compared with hrsv infections, hmpv cases tend to occur at an older age [ , , ] . the clinical outcome after hrsv infection has been more severe than that after hmpv infection when looking at the proportion of patients with hypoxemia, those with pneumonia, and those admitted to the intensive care unit in studies from canada [ ] and europe [ , ] . in contrast, researchers from hong kong found that hmpv infection was associated with a longer hospital stay and more cases of pneumonia than was hrsv infection [ ] . the latter group reported an estimate of hmpvassociated hospitalizations per , children aged р years. children with underlying medical conditions may have moresevere hmpv disease, leading to hospitalization. in studies from north america, %- % of hmpv cases occurred in children with underlying conditions, such as prematurity, cardiopulmonary problems, and immunosuppression [ , ] . hmpv has been associated with flulike illnesses and colds in healthy adults (table ) [ ] . stockton et al. [ ] identified hmpv in ( . %) of nasal and throat swabs obtained from subjects with flulike illnesses (i.e., patients who tested negative for influenza and hrsv rna) seen by general practitioners in england. eight of the infected patients in that study were adults, and of these patients also had clinical evidence of lower respiratory tract involvement. falsey et al. [ ] found a higher rate of hmpv illness among young adults, although older adults experienced more dyspnea and wheezing than did younger adults, and those with cardiopulmonary conditions were ill for nearly twice as long as younger adults. among retrospectively identified, infected patients aged years who were hospitalized at our institution, developed pneumonitis and died (table ) [ ] . of note, all patients had у underlying illness, such as leukemia/lymphoma and chronic cardiovascular, pulmonary, or neurologic diseases. collectively, available data in-dicate that the clinical presentation of hmpv is very similar to that of hrsv, with more severe outcomes in infants, elderly subjects, and persons with underlying diseases [ ] . the detection of hmpv in special settings merits further discussion. preliminary data suggest that, similar to hrsv, hmpv infection may have a more fulminant course in severely immunocompromised individuals. our group reported the death of a -month-old girl with acute lymphoblastic leukemia due to severe pneumonitis and respiratory insufficiency [ ] . although no autopsy was performed, an npa obtained before death revealed the presence of hmpv only. of interest, this child had presented with a first episode of bronchiolitis caused by hmpv year earlier, and the viral isolates were of different lineages (groups), highlighting the possibility of rapid reinfections in immunocompromised hosts. similarly, hmpv was the sole pathogen identified in the npa obtained from a hematopoietic stem cell transplant recipient who died of progressive respiratory failure after an upper respiratory prodrome [ ] . the presence of a copathogen may also lead to more-severe hmpv infections. a report from england identified hmpv in ( %) of bronchoalveolar fluid specimens obtained from infants with severe hrsv bronchiolitis requiring ventilatory support, raising the possibility that hmpv might be a determinant of hrsv disease severity [ ] . unfortunately, no data regarding dual infections in less severe hrsv disease were available for comparison. in contrast, the rate of bronchopneumonia was not altered by the presence of a second respiratory virus in italian infants with hmpv infection [ ] . finally, the possible synergistic effect between hmpv and a new coronavirus has been recently postulated during outbreaks of severe acute respiratory syndrome (sars) in canada and hong kong [ , ] . in a study of patients with probable sars in hong kong, chan et al. [ ] found evidence of hmpv infections by nested pcr in cell cultures ( %) inoculated with npa samples. in fact, hmpv was more frequently isolated than the sars coronavirus in the latter study. this report is surprising, considering the results of experimental infections in macaques, in which severe multifocal pulmonary consoli-dation was induced by the sars coronavirus only, with no exacerbation after subsequent infection with hmpv [ ] . hmpv growth in cell culture is fastidious; this may be one reason for its late identification. most studies have reported reliable cytopathic effects only in tertiary monkey kidney or llc-mk cells [ , , ] . the cytopathic effect is variable, with some strains inducing hrsv-like syncytia formation and others inducing focal rounding and cell destruction ( figure ) . typically, the cytopathic effect is displayed more than - days after inoculation (mean time was days in our laboratory). confirmation of hmpv cytopathic effect is achieved by rt-pcr testing of infected supernatants in the absence of commercially available antibodies. a recent report by chan et al. [ ] indicated that hmpv could replicate more efficiently in human laryngeal carcinoma (hep- ) cells, despite the absence of cytopathic effect. in that instance, presence of hmpv was confirmed by direct testing of hep- cell culture supernatants by rt-pcr or subsequent passage on llc-mk cells to observe cytopathic effect. because of the unavailability of rapid antigen detection tests and because of the slow and restrictive viral growth, rt-pcr has become the method of choice for the diagnosis of acute hmpv infection. most pcr protocols reported to date have relied on amplification of the l, n, or f gene (reviewed in [ ] ), with primer sequences mainly derived from the prototype strain from the netherlands (genbank accession number af ). because of the existence of hmpv lineages with significant genetic variability within each group, hmpv detection may be underestimated when inadequate primers are selected for pcr amplification. we have recently reported that rt-pcr assays targeting the n and l genes, which code for internal viral proteins, are best suitable for hmpv diagnosis [ ] . rapid and sensitive hmpv assays based on the real-time pcr methodology have recently been described, allowing amplification and detection of this pathogen in р h [ , ] . serological testing only permits a retrospective diagnosis. because infection is almost universal in childhood, a seroconversion or a у -fold increase in antibody titers must be demonstrated to confirm recent infection. the few serological surveys for hmpv were based on an indirect immunofluorescence assay using hmpv-infected cells [ , ] . a home-brew elisa method has also been developed using cell lysates of hmpv [ ] . clearly, simpler elisa tests using viral proteins possibly derived from the main groups are needed to conduct large serological surveys in many parts of the world. no vaccines, chemotherapeutic agents, or antibody preparations are currently approved for the prevention or treatment of hmpv infection. recently, wyde et al. [ ] showed that ribavirin and a polyclonal intravenous immunoglobulin (ivig) preparation had equivalent in vitro activity against hmpv and hrsv. in contrast, no activity against hmpv was conferred by palivizumab, a humanized monoclonal antibody directed against the f protein of hrsv. despite the unavailability of animal studies and the toxicity related to ribavirin administration, the combined use of ivig and ribavirin could be envisaged for treating severe hmpv infection in immunocompromised patients, as has been reported for hrsv infection [ , ] . furthermore, until an effective vaccine is developed, another interesting strategy could consist in the development of hightitered ivig preparations with activity against hmpv. the recent identification of a presumably old viral pathogen is an exciting development in the field of respiratory viruses. available data indicate that hmpv appears to be a significant cause of both upper and lower respiratory tract infections in young children. hmpv reinfections seem to be frequent and could also lead to devastating complications in elderly subjects and immunocompromised hosts, although more studies with adequate control groups are needed to confirm this. overall, the epidemiological and clinical features of hmpv infection appear to be similar to those of hrsv, although differences have been noted. it is important to mention that many fun-damental questions related to viral pathogenesis and the host's specific immune response still remain to be answered. the tucson children's respiratory study ii: lower respiratory tract illness in the first year of life etiology of community-acquired pneumonia: impact of age, comorbidity, and severity prospective comparative study of viral, bacterial and atypical organisms identified in pneumonia and bronchiolitis in hospitalized canadian infants. pediatr a newly discovered human pneumovirus isolated from young children with respiratory tract disease metapneumoviruses in birds and humans characterization of human metapneumoviruses isolated from patients in north america virological features and clinical manifestations associated with human metapneumovirus: a new paramyxovirus responsible for acute respiratory-tract infections in all age groups analysis of the genomic sequence of a human metapneumovirus genetic diversity between human metapneumovirus subgroups cloning and sequencing of the matrix protein (m) gene of turkey rhinotracheitis virus reveal a gene order different from that of respiratory syncytial virus respiratory syncytial virus sequence analysis of the n, p, m and f genes of canadian human metapneumovirus strains subgroup c avian metapneumovirus (mpv) and the recently isolated human mpv exhibit a common organization but have extensive sequence divergence in their putative sh and g genes deduced amino acid sequence of the small hydrophobic protein of us avian pneumovirus has greater identity with that of human metapneumovirus than those of non-us avian pneumoviruses human metapneumovirus-associated lower respiratory tract infections among hospitalized human immunodeficiency virus type (hiv- )-infected and hiv- -uninfected african infants seroprevalence of human metapneumovirus in japan high seroprevalence of human metapneumovirus among young children in israel respiratory tract reinfections by the new human metapneumovirus in an immunocompromised child human metapneumovirus infections in hospitalized children human metapneumovirus associated with respiratory tract infections in a -year study of nasal swabs from infants in italy human metapneumovirus infections in young and elderly adults role of human metapneumovirus and other common respiratory viruses in children's hospitalization for acute respiratory tract infection: a twoyear study using multiplex pcr metapneumovirus and acute wheezing in children presence of the new human metapneumovirus in french children with bronchiolitis high prevalence of human metapneumovirus infection in young children and genetic heterogeneity of the viral isolates children with respiratory disease associated with metapneumovirus in hong kong asthma exacerbations in children associated with rhinovirus but not human metapneumovirus infection prevalence and clinical symptoms of human metapneumovirus infection in hospitalized patients human metapneumovirus infection in the united states: clinical manifestations associated with a newly emerging respiratory infection in children human metapneumovirus as a cause of community-acquired respiratory illness human metapneumovirus infection in the canadian population human metapneumovirus in a haematopoietic stem cell transplant recipient with fatal lower respiratory tract disease human metapneumovirus in severe respiratory syncytial virus bronchiolitis human metapneumovirus detection in patients with severe acute respiratory syndrome identification of severe acute respiratory syndrome in canada koch's postulates fulfilled for sars virus comparative evaluation of real-time pcr assays for detection of the human metapneumovirus molecular assays for detection of human metapneumovirus comparison of the inhibition of human metapneumovirus and respiratory syncytial virus by ribavirin and immune serum globulin in vitro prevention and treatment of respiratory syncytial virus and parainfluenza viruses in immunocompromised patients combination therapy with aerosolized ribavirin and intravenous immunoglobulin for respiratory syncytial virus disease in adult bone marrow transplant recipients evidence of human metapneumovirus in australian children human metapneumovirus and community-acquired respiratory illness in children human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children key: cord- -gl eqlkh authors: murris-espin, m.; prévot, g.; chilon, t. title: par rapport à la vancomycine, le linézolide améliore la guérison et la survie des patients atteints de pneumonias nosocomiales à staphylococcus aureus méthicilline-résistant (samr) r.g. wunderin date: - - journal: revue des maladies respiratoires doi: . /s - ( ) - sha: doc_id: cord_uid: gl eqlkh unknown les pneumonies sont les plus fréquentes des infections nosocomiales [ ] . en unité de soins intensifs, leur mortalité dépasse % [ ] . staphylococcus aureus est un des germes le plus fréquemment isolé, quel que soit le site de l'infection. les souches methicilline-résistantes (samr) représentaient % des isolats de staphylocoques dans l'étude epic (european prevalence of infection in intensive care) [ ] . jusqu'à présent, la vancomycine était le traitement de référence des infections à samr. récemment, une nouvelle molécule, le linézolide, antibiotique de la famille des oxazolidinones a été commercialisé. plusieurs études ont montré une efficacité du linézolide identique à celle de la vancomycine dans les infections nosocomiales à gram positif [ ] [ ] [ ] . il s'agit d'une étude rétrospective à partir des données de deux études prospectives, randomisées, en double aveugle, multicentriques, portant sur patients atteints de pneumonie nosocomiale suspectée à gram positif. chaque patient recevait initialement de l'aztréonam (arrêté si un bacille gram négatif n'était pas isolé), puis, après randomisation, soit du linézolide ( mg x / jour en iv) soit de la vancomycine ( g x /jour) pendant à jours. l'évaluation finale se faisait du e au e jour après la fin du traitement. deux paramètres étaient évalués : la taux de guérison clinique et la survie. sur patients inclus, une pneumonie nosocomiale à staphylocoque doré a été confirmée chez patients dont à samr. le taux de survie globale était non significativement différent entre le groupe linézolide et le groupe vancomycine ( , % vs , %, p = , ) . il en était de même dans le groupe pneumonie à staphylococus aureus ( % vs . %, p = , ). par contre, dans le sous-groupe samr, la survie était meilleure dans le groupe linézolide que dans le groupe vancomycine ( %vs , %, p = , ). dans les pneumonies confirmées à samr par hémoculture ou prélèvement invasif, le bénéfice de survie dans le groupe linézolide était confirmé : % vs %, p = , . après ajustement des variables par analyse de régression logistique, le traitement par linézolide apparaissait comme un facteur de bon pronostic dans la survie des pneumonies nosocomiale (au même titre que des facteurs déjà connus comme un score d'apache ii ≤ , l'absence de pathologie cardiaque et une insuffisance rénale inférieure à µmol/l chez l'homme ( µmol/l chez la femme). en termes de guérison clinique, des résultats identiques étaient retrouvés dans le sous-groupe des pneumonies à samr confirmées par hémoculture ou prélèvement invasif. on connaît la gravité en termes de morbidité et de mortalité des infections nosocomiales, notamment à staphylococcus aureus, et l'intérêt d'un traitement précoce adapté. cette étude, bien que rétrospective, donc imparfaite sur le plan méthodologique, mais reprenant les résultats de deux études méthodologiquement comparables et de qualité, souligne, en termes de survie, l'intérêt d'un traitement empirique précoce par linézolide en cas de pneumonie nosocomiale suspectée à samr. du fait d'une meilleure pénétration tissulaire, des taux intra-pulmonaires constamment supérieurs aux cmi ont été obtenus. bien que relativement bien toléré, plusieurs effets secondaires du linézolide ont été rapportés : thrombocytopénie, diarrhée, céphalées, nausées... [ ], mais la molécule est dénuée de toxicité rénale et un relais per os précoce est possible. le spectre d'efficacité du linézolide inclut presque tous les germes à gram positif dont le samr, les entérocoques vancomycine-résistants, les souches de staphylocoques de sensibilité diminuée aux glycopeptides (gisa), les pneumocoques notamment pénicilline-résistants, ainsi que certains anaérobies dont clostridium perfringens, clostridium difficile, peptostreptococcus spp. et bacteroides fragilis [ ] . on dispose avec cette molécule d'une nouvelle arme vis-à-vis de multiples germes. l'utilisation empirique du linézolide devant une pneumonie nosocomiale conduira inéluctablement à la sélection de souches résistantes. il semble important d'identifier les patients devant rapidement bénéficier d'un tel traitement afin de gagner en termes de survie sans perdre en termes d'écologie bactérienne et de coût [ ]. cette étude a porté sur une cohorte de cas hospitalisés et sur des critères radiologiques et cliniques. les patients remplissaient un questionnaire après confirmation diagnostique. les données étaient centralisées et ont été rétrospectivement analysées. le symptôme le plus fréquemment retrouvé était la fièvre ( %) avec dans à % des cas un syndrome pseudo-grippal. les signes digestifs étaient beaucoup moins fréquents : diarrhée ( %), vomissements ( %), douleurs abdominales ( %). le délai entre l'exposition et le début des symptômes a pu être précisément étudié chez patients uniquement : il était en moyenne de , jours. le délai d'hospitalisation s'est réduit au cours de l'épidémie, passant de , jours début mars à , jours fin avril. le délai moyen entre hospitalisation et décès était de , jours. la durée moyenne d'hospitalisation était de , jours. le taux de mortalité chez les moins de ans a été estimé à , % chez les moins de ans et à , % chez les plus de ans. il est intéressant de noter le raccourcissement dans le délai d'hospitalisation, qui a du conduire à un meilleur contrôle de l'épidémie par un isolement plus précoce des malades. il est possible que la mortalité ait été surestimée car, d'une part, elle ne tenait compte que des patients hospitalisés, donc a priori plus fragiles, et d'autre part, l'étude n'était le reflet que d'une courte période de l'épidémie et ne tenait donc pas compte d'une virulence variable du virus. le taux de mortalité est d'ailleurs variable selon les séries, il varie de , % [ ] à , % [ ] , voire à % à mois [ ] , selon l'âge et les co-morbidités des patients. grâce aux descriptions minutieuses des premiers cas de sras, un tableau clinique a pu rapidement être identifié, permettant l'isolement des cas suspects de sras et probablement de contenir l'épidémie. compte tenu de la mortalité observée, des facteurs pronostiques ont été rapidement isolés, permettant une identification précoce des « sujets à risque ». infections respiratoires a major outbreak of severe acute respiratory syndrome in hong kong clinical features and short-term outcomes of patients with sars in the greater toronto area princess margaret hospital sars study group : outcomes and prognosis factors in patients with severe acute respiratory syndrome in hong kong short term outcome and risk factors for adverse clinical outcomes in adults with severe acute respiratory syndrome (sras) key: cord- -ji jbct authors: morens, david m.; folkers, gregory k.; fauci, anthony s. title: the challenge of emerging and re-emerging infectious diseases date: - - journal: nature doi: . /nature sha: doc_id: cord_uid: ji jbct infectious diseases have for centuries ranked with wars and famine as major challenges to human progress and survival. they remain among the leading causes of death and disability worldwide. against a constant background of established infections, epidemics of new and old infectious diseases periodically emerge, greatly magnifying the global burden of infections. studies of these emerging infections reveal the evolutionary properties of pathogenic microorganisms and the dynamic relationships between microorganisms, their hosts and the environment. merging infections (eis) can be defined as "infections that have newly appeared in a population or have existed previously but are rapidly increasing in incidence or geographic range" . eis have shaped the course of human history and have caused incalculable misery and death. in , a new disease -acquired immune deficiency syndrome (aids) -was first recognized. as a global killer, aids now threatens to surpass the black death of the fourteenth century and the - influenza pandemic, each of which killed at least million people , . of the 'newly emerging' and 're-emerging/resurging' diseases that have followed the appearance of aids (fig. ) , some have been minor curiosities, such as the cases of monkeypox imported into the united states , whereas others, such as severe acute respiratory syndrome (sars), which emerged in the same year , have had a worldwide impact. the anthrax bioterrorist attack in the united states falls into a third category: 'deliberately emerging' diseases. eis can be expected to remain a considerable challenge for the foreseeable future. here we examine the nature and scope of emerging and reemerging microbial threats, and consider methods for their control. we emphasize that emergence results from dynamic interactions between rapidly evolving infectious agents and changes in the environment and in host behaviour that provide such agents with favourable new ecological niches. about million (> %) of million annual deaths worldwide are estimated to be related directly to infectious diseases; this figure does not include the additional millions of deaths that occur as a consequence of past infections (for example, streptococcal rheumatic heart disease), or because of complications associated with chronic infections, such as liver failure and hepatocellular carcinoma in people infected with hepatitis b or c viruses (fig. ) . the burden of morbidity (ill health) and mortality associated with infectious diseases falls most heavily on people in developing countries , and particularly on infants and children (about three million children die each year from malaria and diarrhoeal diseases alone ). in developed nations, infectious disease mortality disproportionately affects indigenous and disadvantaged minorities . eis have been familiar threats since ancient times. they were once identified by terms such as ȕșȓȖș ȝ (loimos) , and later as 'pestilences' , 'pestes' , 'pests' and 'plagues' . many examples can be cited in addition to the black death and the influenza pandemic, such as certain biblical pharaonic plagues and the unidentified plague of athens, which heralded the end of greece's golden age . the age of discovery, starting in the fifteenth century, was a particularly disastrous period with regard to the spread of infectious diseases. importation of smallpox into mexico caused - million deaths in - , effectively ending aztec civilization , . other amerindian and pacific civilizations were destroyed by imported smallpox and measles [ ] [ ] [ ] [ ] [ ] . historians have referred to these events as apocalypses and even as genocide . for centuries, mankind seemed helpless against these sudden epidemics. but the establishment of the germ theory and the identification of specific microbes as the causative agents of a wide variety of infectious diseases [ ] [ ] [ ] led to enormous progress, notably the development of vaccines and ultimately of antimicrobials . in fact, the era of the identification of microbes had barely begun when optimists at the end of the nineteenth century predicted the eradication of infectious diseases . by the s, which witnessed the widespread use of penicillin, the development of polio vaccines and the discovery of drugs for tuberculosis, complacency had set in , and in , the us surgeon general stated that "the war against infectious diseases has been won" . some experts remained sceptical, aware of recurrent lessons from history. they were less persuaded by successes than alarmed by failures such as the lack of progress against infections in the developing world and the global spread of antimicrobial resistance. richard krause, then the director of the us national institute of allergy and infectious diseases, warned in (ref. ) that microbial diversity and evolutionary vigour were still dynamic forces threatening mankind. as krause was completing his book the restless tide , aids -one of history's most devastating pandemics -was already insidiously emerging. the emergence of aids led to renewed appreciation of the inevitability and consequences of the emergence of infectious diseases [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in the past years, some of the factors that resulted in aids have also led to re-emergences of historically important diseases such as cholera, diphtheria, trench fever and plague. many re-emergences have been catalysed by wars, loss of social cohesion, and natural disasters such as earthquakes and floods, indicating the importance not only of microbial and viral factors, but also of social and environmental determinants [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the challenge of emerging and re-emerging infectious diseases the classification of eis as 'newly emerging' , 're-emerging/resurging' or 'deliberately emerging' is useful because the underlying causes of emergence and the optimal prevention or control responses frequently differ between the groups. newly emerging infections are those that have not previously been recognized in man. many diverse factors contribute to their emergences (see box ); these include microbial genetic mutation and viral genetic recombination or reassortment, changes in populations of reservoir hosts or intermediate insect vectors, microbial switching from animal to human hosts, human behavioural changes (notably human movement and urbanization), and environmental factors. these numerous microbial, host and environmental factors interact to create opportunities for infectious agents to evolve into new ecological niches, reach and adapt to new hosts, and spread more easily between them. any discussion of recent eis must begin with the human immunodeficiency virus (hiv) that causes aids. hiv has so far infected more than million people worldwide . before jumping to humans an estimated - years ago , perhaps as a consequence of the consumption of 'bush meat' from non-human primates, hiv- and hiv- had ample opportunity to evolve in hosts that were genetically similar to man (the chimpanzee, pan troglodytes, and the sooty mangabey, cercocebus atys). but hiv/aids might never have emerged had it not been for disruptions in the economic and social infrastructure in post-colonial sub-saharan africa. increased travel, the movement of rural populations to large cities, urban poverty and a weakening of family structure all promoted sexual practices, such as promiscuity and prostitution, that facilitate hiv transmission [ ] [ ] [ ] [ ] . such complex interactions between infectious agents, hosts and the environment are not unique to the epidemiology of hiv/aids. the examples cited below further illustrate how changes in population density, human movements and the environment interact to create ecological niches that facilitate microbial or viral adaptation. some infectious agents that have adapted to non-human hosts can jump to humans but, unlike hiv, are not generally transmitted from person to person, achieving only 'dead end' transmission. infections in animals that are transmitted to humans (zoonoses), and those transmitted from one vertebrate to another by an arthropod vector (vector-borne diseases), have repeatedly been identified as ranking among the most important eis , . examples include the arenavirus haemorrhagic fevers (argentine, bolivian, venezuelan and lassa haemorrhagic fevers) and hantavirus pulmonary syndrome (hps). viruses in these groups have co-evolved with specific rodent species whose contact with humans has increased as a result of modern environmental and human behavioural factors. farming, keeping domestic pets, hunting and camping, deforestation and other types of habitat destruction all create new opportunities for such infectious agents to invade human hosts [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the first epidemic of hps, detected in the southwestern region of the united states in (ref. ) , resulted from population booms of the deer mouse peromyscus maniculatis, in turn caused by climate-related and recurrent proliferation of rodent food sources. increased rodent populations and eventual shortages of food drove expanded deer mouse populations into homes, exposing people to virus-containing droppings. the - malaysian nipah virus epidemic further illustrates the influence of human behaviours and environmental perturbations on newly emerging human infections. pigs crammed together in pens located in or near orchards attracted fruit bats whose normal habitats had been destroyed by deforestation and whose droppings contained the then-unknown paramyxovirus. virus aerosolization caused infection of pigs, with overcrowding leading to explosive transmission rates and ultimately to infections in pig handlers. variant creutzfeldt-jakob disease (vcjd) is another example of a zoonotic disease emerging in humans. vcjd is caused by the humanadapted form of the prion associated with the emerging epizootic (large-scale animal outbreak) of bovine spongiform encephalopathy (bse) figure global examples of emerging and re-emerging infectious diseases, some of which are discussed in the main text. red represents newly emerging diseases; blue, re-emerging/resurging diseases; black, a 'deliberately emerging' disease. adapted, with permission, from ref. . epizootic/vcjd epidemic, primarily affecting great britain, probably resulted from the now-abandoned practice of supplementing cattle feed with the pulverized meat and bones of previously slaughtered cattle. bse itself is suspected to have emerged because of even earlier use of cattle feed containing the agent of sheep scrapie, a prion disease recognized by farmers more than years ago . alarmingly, the new bse prion has become uncharacteristically promiscuous: unlike most known prions, it readily infects multiple species in addition to humans. this suggests the possibility of further emerging diseases associated with prions with currently unknown transmissibility to humans . the recent reports of variant strains of the bse prion suggest that the bse agent could be a more serious threat than other animal prions. infectious agents indirectly transmitted to or between humans by way of human-modified environments account for other emerging zoonoses, as well as certain non-zoonotic diseases, which are discussed below. for example, legionnaires' disease, first identified in , is caused by legionella pneumophila, whose emergence as a human pathogen might not have occurred were it not for the environmental niche provided by air-conditioning systems . campylobacter jejuni and shiga-toxin-producing escherichia coli (e. coli o :h and other agents of haemolytic-uraemic syndrome) infect agricultural animals, gaining access to humans through food, milk, water or direct animal contact. other enteric pathogens, such as the vibrios causing classical cholera (re-emerging; see below) and serogroup o cholera, and the zoonotic protozoa cryptosporidium parvum and cyclospora cayetanensis , seem to have come from environmental or animal organisms that have adapted to human-to-human 'faecal-oral' transmission through water. some eis come from microorganisms that once caused familiar diseases, but which now cause new or previously uncommon diseases. streptococcus pyogenes caused a fatal pandemic of scarlet and puerperal fevers between and (ref. ) . scarlet fever, then the leading cause of death in children, is now rare, but has been largely supplemented by other streptococcal complications such as streptococcal toxic shock syndrome, necrotizing fasciitis and re-emergent rheumatic fever . when new microbes are discovered, their emergences as disease-causing pathogens may be delayed. for example, in , robert koch was unable to show that the newly discovered koch-weeks bacillus caused serious disease. more than a century later, a fatal ei dubbed brazilian purpuric fever was linked to virulent clonal variants of haemophilus influenzae biogroup aegyptius (the koch-weeks bacillus) . although the bases of emergences of new and more severe diseases caused by s. pyogenes and h. influenzae biogroup aegyptius are not fully known, in both cases complex microbial genetic events are suspected. the distinctive clonal variants associated with severe h. influenzae biogroup aegyptius disease have been shown by pcr (polymerase chain reaction)-based subtractive genome hybridization to contain not only a unique plasmid, but also unique chromosomal regions, some of which are encoded by bacteriophages . this research has narrowed the search for virulence determinants to unique proteins, some of which may have been acquired from other organisms by horizontal gene transfer. streptococcus pyogenes has been studied more extensively, but the basis of severe disease emergence seems to be more complex than for h. influenzae biogroup aegyptius. many factors associated with streptococcal virulence have been identified in strains bearing the m surface protein as well as in other m protein strains, among them bacteriophage-encoded superantigen toxins and a protein known as sic (streptococcal inhibitor of complement), which seems to be strongly selected by human host mucosal factors. several lines of evidence suggest that changes in streptococcal virulence reflect genetic changes associated with phage integration, large-scale chromosomal rearrangements and possibly the shuffling of virulence cassettes (clusters of genes responsible for pathogenicity), followed by rapid human spread and immune selection , . infectious agents that are associated with chronic diseases are one of the most challenging categories of newly emerging (or at least newly appreciated) infections. examples include the associations of hepatitis b and c with chronic liver damage and hepatocellular carcinoma, of certain genotypes of human papillomaviruses with cancer of the uterine cervix, of epstein-barr virus with burkitt's lymphoma (largely in africa) and nasopharyngeal carcinoma (in china), of human herpesvirus with kaposi sarcoma, and of helicobacter pylori with gastric ulcers and gastric cancer [ ] [ ] [ ] . some data even suggest infectious aetiologies for cardiovascular disease and diabetes mellitus , major causes of death and disability worldwide. other associations between infectious agents and idiopathic chronic diseases will inevitably be found. re-emerging and resurging infections are those that existed in the past but are now rapidly increasing either in incidence or in geographical or human host range. re-emergence is caused by some of the factors that cause newly emerging infectious diseases, such as microbial evolutionary vigour, zoonotic encounters and environmental encroachment. re-emergences or at least cyclical resurgences of some diseases may also be climate-related -for example, the el niño/southern oscillation (enso) phenomenon is associated with resurgences of cholera and malaria . the impact of both new and re-emerging infectious diseases on human populations is affected by the rate and degree to which they spread across geographical areas, depending on the movement of human hosts or of the vectors or reservoirs of infections. travel has an important role in bringing people into contact with infectious agents . an increase in travel-associated importations of diseases was anticipated as early as , when commercial air travel was still in its infancy . this has since been demonstrated dramatically by an international airline hub-to-hub pandemic spread of acute haemorrhagic conjunctivitis in (ref. ) , by epidemics of meningococcal meningitis associated with the hajj, and more recently by the exportation of epidemic sars (a newly emerging disease) from guangdong province, china, to hong kong, and from there to beijing, hanoi, singapore, toronto and elsewhere (fig. ) . the persistent spread of hiv along air, trucking, drug-trafficking and troop-deployment routes is a deadly variation on this theme [ ] [ ] [ ] . plasmodium falciparum malaria was neglected for several decades, but is now among the most important re-emerging diseases worldwide (fig. ) . years of effective use of dichlorodiphenyltrichloroethane (ddt) led to the abandonment of other mosquito-control programmes, but the insecticide fell into disuse because of mosquito resistance and concerns about the insecticide's potentially harmful effects on humans and wildlife. consequently, malaria has re-emerged, and the situation has been worsened by the development of drug resistance to chloroquine and mefloquine . research efforts focus on the development of vaccines and new drugs, and on re-establishing public health measures such as the use of bed nets. tuberculosis is one of the most deadly re-emerging diseases (fig. ) . the discovery of isoniazid and other drugs initially led to effective tuberculosis cures, empty sanitoria and the dismantling of public health control systems in developed nations. consequently, by the s, when tuberculosis had re-emerged in the era of hiv/aids, local and state health departments in the united states lacked field, laboratory and clinical staff and so had to reinvent tuberculosiscontrol programmes . the remarkable re-emergence of tuberculosis was fuelled by the immune deficiencies of people with aids, which greatly increases the risk of latent mycobacterium tuberculosis infections progressing to active disease, and being transmitted to others. inadequate courses of anti-tuberculosis therapy compound the problem, leading to the emergence and spread of drug-resistant and multidrug-resistant strains , and a need for more expensive treatment strategies such as directly observed therapy. it has been known for over a century that tuberculosis is a disease of poverty, associated with crowding and inadequate hygiene. the continuing expansion of global populations living in poverty makes tuberculosis more difficult to control. . immune deficiency associated with aids, and with chemotherapy for cancer, immune-mediated diseases and transplantation, has contributed to an enormous global increase in the numbers of immunosuppressed people over the past few decades (probably more than % of the world's population), setting the stage for the re-emergence of many opportunistic infections. hiv, which has infected more than million people globally , is the largest single cause of human immune deficiency and markedly increases vulnerability to a wide range of opportunistic pathogens, including pneumocystis carinii, various fungi, tuberculosis, protozoa and herpesviruses . breakthroughs in cancer therapy and in immunosuppressive therapies used to treat immune-mediated diseases and for transplantation , can also leave patients susceptible to opportunistic infections. human organ transplantation adds a further risk of infection with undetected pathogens in donor tissues, and transplantation of animal organs introduces the risk of transmission to humans of animal microbes . the emergence of zoonotic and vector-borne diseases can also be associated with human behaviours and environmental perturbation. although west nile virus is now a major epidemiological concern in the developed world, dengue remains the most significant and widespread flavivirus disease to have emerged globally . a - epidemic in hawaii -fortunately without fatalities -is a reminder that dengue has also re-emerged in locations once considered to be dengue-free. usually transmitted by aedes aegypti mosquitoes, dengue has recently been transmitted by aedes albopictus -a vector switch of potential significance with respect to dengue re-emergence . in the americas, including many us southern states, a. albopictus has been spreading into areas where a. aegypti mosquitoes are not found, and persisting for longer seasonal periods, putting tens of millions more people at risk of dengue infection. dengue re-emergence is further complicated by disturbing increases in a serious and formerly rare form of the disease, dengue haemorrhagic fever (dengue shock syndrome being its highly fatal form). these severe complications are thought to result from the evolution of dengue viruses to escape high insight review articles nature | vol | july | www.nature.com/nature population immunity, seen in increased viral virulence and human immunopathogenesis due to antibody-dependent enhancement of viral infection . cholera is also of interest, not only as an important cause of mortality, but also because of the complexity of factors that determine its re-emergence. both virulent and avirulent strains of these zoonotic bacteria are maintained in the environment and are rapidly evolving in association with phyto-and zooplankton, algae and crustaceans. such environmental strains seem to act as reservoirs for human virulence genes (for example, genes for the phage-encoded cholera toxin and the toxin-coregulated pilus (tcp) factor associated with attachment), and to undergo gene transfer events that lead to new strains containing further virulence gene combinations. these result in periodic cholera emergences that cause epidemics and pandemics . thus, although the disease we know as cholera has appeared to be clinically and epidemiologically stable at least since the third pandemic (in the s), modern evidence suggests that such apparent stability masks aggressive bacterial evolution in complex natural environments. influenza a viruses, which are endemic gastrointestinal viruses of wild waterfowl, have evolved elaborate mechanisms to jump species into domestic fowl, farm animals and humans. periodic gene segment reassortments between human and animal viruses produce important antigenic changes, referred to as 'shifts' . these can lead to deadly pandemics, as occurred in , , and (refs , ) . in intervening years, shifted viruses undergo continual but less dramatic antigenic changes called 'drifts' , which allow them partially to escape human immunity raised by previously circulating influenza viruses. influenza drift is an evolutionary success story for the virus. influenza a has a seemingly inexhaustible repertoire of mutational possibilities at several critical epitopes surrounding the viral haemagglutinin site that attaches to human cells. it remains something of a mystery how zoonotic influenza viruses mix with each other and with human strains to acquire the additional properties of human virulence and human-to-human transmissibility. before , mild cases of human disease associated with avian influenza viruses were occasionally reported . these events have become more frequent, sometimes resulting in severe cases of disease and death. avian influenza has recently made dead-end jumps to humans -for example, the hong kong outbreak of the newly emergent h n influenza, the h n epidemic in the netherlands, the - h n and h n epizootics in asia and elsewhere, and occasional cases of h n disease (fig. ) . meanwhile, back-switches of human h n viruses have emerged in pigs, from which both doubly mixed (pig-human) and triply mixed (pig-human-avian) viruses , have been isolated. such enzootic/zoonotic mixing is suspected to have occurred in the influenza pandemic of - , which was caused by an h n virus with an avian-like receptor-binding site . the predicted virulence genes of this virus are now being sought from -year-old pathology specimens and from frozen corpses . the implications of interspecies genetic mixing for future influenza pandemics are troubling. although much remains speculative about how influenza viruses emerge and spread, it seems clear that the process is driven by prolific and complex viral evolution (genetic reassortment and mutational 'drift'), interspecies mixing and adaptation, and ecological factors that bring humans into contact with animals and each other. by whatever means new influenza virus pandemic strains emerge, they eventually reach a critical threshold of human transmission beyond which epidemic and pandemic spread follows mathematically predictable patterns. the dynamics and determinants of such epidemic development have been studied since the nineteenth century for several infectious diseases. for influenza, both historical and prospective epidemics have been described or predicted using deterministic and stochastic mathematical models, often with surprising accuracy when compared with actual epidemic data. more complicated mathematical models that describe how diseases spread by means other than personto-person aerosol transmission have generally been less successful in describing and predicting epidemics, but have nonetheless been helpful in planning public health responses to epidemics caused by hiv , vcjd and other diseases. mathematical modelling is also used to determine the impact of emerging epidemics. for example, it has been difficult to estimate overall influenza mortality because fatal infections are often neither diagnosed nor accurately recorded in hospital records and death certificates, especially in the elderly. recent epidemiological attempts to obtain improved influenza mortality estimates from seasonal excess mortality data have indicated that influenza mortality may be insight review articles nature | vol | july | www.nature.com/nature greater than was previously suspected, because influenza deaths are frequently coded under seemingly unrelated categories such as cardiovascular diseases. the same approaches also show that other influenza-like deaths may actually be due to other agents, such as respiratory syncytial virus (rsv), a common childhood virus that in the past decade has emerged as a major cause of adult mortality . deliberately emerging microbes are those that have been developed by man, usually for nefarious use. the term 'deliberately emerging' refers to both naturally occurring microbial agents such as anthrax , and to bioengineered microorganisms such as those created by the insertion of genetic virulence factors that produce or exacerbate disease. deliberately emerging microbes include microorganisms or toxins produced in a form that would cause maximal harm because of ease of dissemination, enhanced infectivity or heightened pathogenicity . as concepts, bioterrorism and biowarfare are probably not new. the alleged catapulting of plague-ridden corpses over enemy walls in the siege of caffa (the modern crimean port of feodosia, ukraine) and the dispatch of smallpox-impregnated blankets to indians by british officers in the seven years war ( - ) have frequently been cited as examples of bioterrorism or biowarfare , . two modern attacks have been well documented. in , an oregon religious cult spiked restaurant salad bars with salmonellae in an attempt to sway a local election . a anthrax attack , in which a terrorist mailed anthrax-spore-filled letters to prominent figures, including two us senators, resulted in illness in at least people and the death of five of these individuals. public alarm was elevated by the knowledge that bacillus anthracis is a common and easily obtainable enzootic and soil organism found in laboratories worldwide, and that scientific technology had increased its lethality: the spores had been weaponized by being concentrated, finely milled and packed with a dispersal agent to increase their capacity to disseminate . the united states, the united kingdom, the soviet union and other nations once had sophisticated offensive bioweapons programmes that included the production of weaponized anthrax spores . soviet scientists continued to produce large quantities of organisms adapted for biowarfare and bioterror -among them the agents of smallpox, plague, tularaemia and marburg virus -for several years after their signing of the bioweapons and toxins treaty convention in , which forbade such activities . by , the soviet programme was annually producing , tonnes of weaponized anthrax spores, packing them into warheads and other delivery devices . before the anthrax attacks , the us scientific community had for several years been bolstering its biodefence research capacity. the anthrax attacks greatly accelerated this expansion as part of a national defence plan, which includes efforts to provide a knowledge base for the development of effective countermeasures against agents of bioterror, such as diagnostics, therapeutics and vaccines, and to translate this knowledge into the production and delivery of such measures . bioterror agents have been grouped into three categories of risk . the six category a agents (anthrax, smallpox, plague, tularaemia, viral haemorrhagic fevers and clostridial botulinum toxin) are given top priority because they are highly lethal and readily deployed as weapons. category b and c agents include food-borne and water-borne organisms that incapacitate but usually do not kill. infectious diseases will continue to emerge and re-emerge, leading to unpredictable epidemics and difficult challenges to public health and to microbiology and allied sciences. surveillance and response, the key elements in controlling eis, be they naturally occurring or deliberately engineered, depend on rapid clinical diagnosis and detection and containment in populations and , which along with state and local health departments and other agencies have been making significant strides in national surveillance-response capacity. the enormous influx of us government-funded research resources (largely through the national institutes of health) and public health resources (mainly through the cdc, and state and local public health agencies) in response to the increased threat of a bioterrorist attack will fortify the response capabilities related to all eis. however, it is clear that surveillance and other activities that traditionally fall within the domain of public health are not in themselves sufficient to adequately address the problem of eis. of critical importance are basic, translational and applied research efforts to develop advanced countermeasures such as surveillance tools, diagnostic tests, vaccines and therapeutics . genomics, proteomics and advances in nanotechnology are increasingly being exploited in diagnostic, therapeutic and microbial research applications, and in rational drug and vaccine design. direct and computational structural determination , prediction of protein-protein interactions between microorganisms and drugs, and sophisticated bioinformatics techniques support research in all of the above areas. these technologies have led to numerous advances in real-world utility against eis, most notably in the development of more than antiretroviral drugs that can effectively suppress hiv replication. where they are available and properly used in hiv-infected individuals, these medications have dramatically reduced hiv morbidity and mortality . gene-and protein-based microarrays can be used to detect pathogen signals, to monitor resistance to anti-infective agents, to characterize host gene responses to recent infections, and to facilitate the development of new drugs and vaccines . basic and applied research together have provided promising new vaccine platforms, such as recombinant proteins, immunogenic peptides, naked dna vaccines, viral vectors of extraneous genes encoding immunogenic proteins (including chimaeras), replicons and pseudovirions . many novel vaccine candidates are now being developed against eis such as hiv, ebola virus, west nile virus, dengue, the sars coronavirus, tuberculosis and malaria. of particular note are novel tuberculosis vaccines that recently entered clinical trials -the first time in more than years that new approaches to vaccination for tuberculosis have been assessed in humans . chimaeric flavivirus vaccines for west nile virus, dengue and japanese encephalitis virus are effective in animal models and are in various stages of clinical testing . our growing understanding of the human immune system is also helping to accelerate vaccine development. this is especially true in the case of innate immune responses, which are evolutionarily older, less specific and faster-acting than the adaptive responses that have been the traditional targets of vaccines . as we learn more about innate immunity and its relationship with the adaptive immune system, opportunities to create more effective vaccine adjuvants will emerge. for example, synthetic dna sequences that contain repeated cpg motifs mimic the stimulatory activity that bacterial dna fragments exert on the innate immune system. these sequences show promise as vaccine adjuvants that accelerate and augment immune responses . we can anticipate more progress of this kind as we continue to delineate the complex interactions between innate and adaptive immune responses. the sequencing of the human genome, the genomes of six other animals, including the mouse, and those of microbial vectors and microbes themselves (for example, p. falciparum and its mosquito vector, anopheles gambiae), have elevated microbiology to a wholegenome level. the ability to sequence microbial genomes in a few days or less, and to examine host-vector-microbe interactions at both the genome level and at the tertiary protein structural level, will help us to understand the molecular mechanisms that underlie the pathogenesis of infectious disease and host defences, including resistance and immune evasion. these advances will facilitate the development of new countermeasures. other fertile areas of research include the use of geographical information systems and satellite imaging to support field study and epidemic prevention (for example, predicting hps and rift valley fever epidemics in indigenous areas by satellite imagery of water and vegetation related to animal reservoir and vector prevalence). underlying disease emergence are evolutionary conflicts between rapidly evolving and adapting infectious agents and their slowly evolving hosts. these are fought out in the context of accelerating environmental and human behavioural alterations that provide new ecological niches into which evolving microbes can readily fit. it is essential that broadly based prevention strategies, as well as new and improved countermeasures (that is, surveillance tools, diagnostics, therapeutics and vaccines), be continually tested, refined and upgraded, requiring a strengthened relationship between public health and basic and clinical sciences. the challenge presented by the ongoing conflict between pathogenic microorganisms and man has been well summarized by a noted champion of the war on eis, joshua lederberg: "the future of microbes and mankind will probably unfold as episodes of a suspense thriller that could be entitled our wits versus their genes" . the global scientific and public health communities must confront this reality not only with wit, but also with vision and sustained commitment to meet a perpetual challenge. in figure of this review, the pie chart was drawn incorrectly, with the wedge sizes not in proportion to the total size. the correct figure is shown below. asthma and chronic obstructive pulmonary disease . million factors in the emergence of infectious diseases the wordsworth encyclopedia of plague and pestilence updating the accounts: global mortality of the - "spanish" influenza pandemic centers for disease control and prevention. multistate outbreak of monkeypox -illinois the severe acute respiratory syndrome investigation of bioterrorism-related anthrax, united states, : epidemiologic findings threats to global health and survival: the growing crises of tropical infectious diseases -an "unfinished" agenda emerging infectious diseases among indigenous peoples ǼșȓȖșȕșȍȓȊ -sive, pestis nuperae apud populum londinensem grassantis narratio historica epidemiology of the plague of athens the columbian exchange: biological and cultural consequences of princes and peasants. smallpox in history ch island populations of the pacific the gifts of civilization. germs and genocide in hawai'i agents of apocalypse: epidemic disease in the colonial philippines measles in fiji, : thoughts on the history of emerging infectious diseases die aetiologie der milzbrand-krankheit, begründet auf die entwicklungsgeschichte des bacillus anthracis. beiträge zur biologie der pflanzen spreading germs: diseases, theories, and medical practice in britain the greatest benefit to mankind: a medical history of humanity from antiquity to the present the extermination of infectious diseases the evolution and eradication of infectious diseases infectious diseases: considerations for the st century the restless tide: the persistent challenge of the microbial world (national foundation for infectious diseases committee on emerging microbial threats to health. emerging infections. microbial threats to health in the united states committee on emerging microbial threats to health in the st century. microbial threats to health in the united states: emergence, detection and response emerging and re-emerging infectious diseases: a multidisciplinary perspective emerging and re-emerging infectious diseases infectious history emerging infectious diseases in the st century emerging and re-emerging infectious diseases human frontiers, environments and disease the origins of acquired immune deficiency viruses: where and when? phil population migration and the spread of types and human immunodeficiency viruses the aids knowledge base slowing heterosexual hiv transmission a novel hantavirus associated with an outbreak of fatal respiratory disease in the southwestern united states: evolutionary relationships to known hantaviruses nipah virus: a recently emergent deadly paramyxovirus variant creutzfeldt-jakob disease and the acquired and transmissible spongiform encephalopathies nützliche und auf die erfahrung gegründete einleitung zu der landwirthschaft identification of a second bovine amyloidotic spongiform encephalopathy: molecular similarities with sporadic creutzfeldt-jakob disease scrapie and chronic wasting disease severe streptococcal infections in historical perspective emerging infections brazilian purpuric fever: evolutionary genetic relationships of the case clone of haemophilus influenzae biogroup aegyptius to encapsulated strains of haemophilus influenzae identification and characterization of genomic loci unique to the brazilian purpuric fever clonal group of h. influenzae biogroup aegyptius: functionality explored using meningococcal homology group a streptococcus: allelic variation, population genetics, and host-pathogen interactions genome sequence of a serotype m strain of group a streptococcus: phage-encoded toxins, the high-virulence phenotype, and clone emergence identification of herpesvirus-like dna sequences in aids-associated kaposi's sarcoma microbes and malignancy: infection as a cause of human cancers helicobacter pylori-associated diseases current clinical topics in infectious diseases vol el niño and health epidemiology in relation to air travel acute haemorrhagic conjunctivitis: dealing with a newly emerging disease two worlds of malaria research toward vaccines against malaria the global situation of mdr-tb staphylococcus aureus resistant to vancomycin -united states methicillin (oxacillin)-resistant staphylococcus aureus strains isolated from major food animals and their potential transmission to humans the crisis in antibiotic resistance surveillance for aids-defining opportunistic illnesses, - . mmwr (cdc surveillance summary no. ss- ) infections in patients with cancer undergoing chemotherapy: aetiology, prevention, and treatment impact of current transplantation practices on the changing epidemiology of infections in transplant recipients xenotransplantation: public health risks -patient vs. society in an emerging field the outbreak of west nile virus infection in the new york city area in outbreak of west nile infection west nile virus: epidemiology and ecology in north america dengue and dengue haemorrhagic fever antibody-dependent enhancement of infection and the pathogenesis of viral disease molecular ecology of toxigenic vibrio cholerae the next influenza pandemic: lessons from hong kong a molecular whodunit avian influenza viruses infecting humans structure of the uncleaved human h haemagglutinin from the extinct influenza virus the origin of the pandemic influenza virus: a continuing enigma potential impact of low-efficacy hiv- vaccines in populations with high rates of infection short-term projections for variant creutzfeldt-jakob disease onsets mortality associated with influenza and respiratory syncytial virus in the united states the chilling true story of the largest covert biological weapons program in the world -told from the inside by the man who ran it (random house document zur geschichte des schwarzen todes. mitgetheilt und eingeleitet smallpox and the indians in the american colonies a large community outbreak of salmonellosis caused by intentional contamination of restaurant salad bars biodefence on the research agenda: the world needs new and creative ways to counter bioterrorism threats in bioterrorism: i. cdc category a agents world health organization. consensus document on the epidemiology of severe acute respiratory syndrome (sars) preventing emerging infectious diseases: a strategy for the st century (department of health and human services protein structure prediction and structural genomics guidelines for using antiretroviral agents among hiv-infected adults and adolescents new technology platforms in the development of vaccines for the future the jordan report: th anniversary adjuvants of immunity: harnessing innate immunity to promote adaptive immunity recent advances in the discovery and delivery of vaccine adjuvants the value of complete microbial genome sequencing (you get what you pay for) the authors thank r. m. krause for helpful discussions, and j. weddle for graphic design. the authors declare that they have no competing financial interests.insight review articles key: cord- - s gnxs authors: tee, augustine k.h.; oh, helen m.l.; hui, k.p.; lien, christopher t.c.; narendran, k.; heng, b.h.; ling, a.e. title: atypical sars in geriatric patient date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: s gnxs we describe an atypical presentation of severe acute respiratory syndrome (sars) in a geriatric patient with multiple coexisting conditions. interpretation of radiographic changes was confounded by cardiac failure, with resolution of fever causing delayed diagnosis and a cluster of cases. sars should be considered even if a contact history is unavailable, during an ongoing outbreak. the patient was a -year-old singaporean chinese woman who was a resident of a nursing home. she had a past history of vascular dementia with dysphagia and behavioral abnormalities, ischemic heart disease with atri-al fibrillation, and congestive cardiac failure. in addition, she also suffered from type diabetes mellitus, hypertension, osteoporosis, bilateral osteoarthritis of the knees, and an old traumatic fracture of the left humeral neck. as such, she was fully dependent in her daily activities. she was admitted to the geriatric department of tan tock seng hospital ( ) on march , , for pneumonia and urinary tract infection. these infections responded to a course of intravenous antimicrobial drugs. she also was assessed to have mild dysphagia, which required thickened fluids and blended diet without nasogastric feeding. her chest radiograph before discharge showed persistent bilateral lower zone consolidation (figure ), consistent with bilateral crepitations on auscultation. however, the patient was afebrile and improved functionally to being ambulant with assistance. she was discharged to the nursing home on march . within the next two days, the patient progressively became breathless, with nausea and vomiting. there was no associated cough or diarrhea. she was eventually admitted to the medical department of changi general hospital, a designated non-sars hospital, on march . on admission to the isolation room, she had a maximal tympanic temperature of . °c, with defervescence the next day. she remained afebrile during the remainder of her stay. her blood pressure was / mm hg, pulse rate of beats per minute, and respiratory rate of breaths per minute. her pulse oximetry was % while on l per minute of intranasal oxygen. the jugular venous pressure was not elevated. bilateral basal crepitations were heard on examination. all healthcare workers attending to the patient wore the recommended personal protective equipment, including gown, gloves, and n respirators, each time they entered the isolation room. investigations on admission showed that the patient's hemoglobin was . g/dl, leukocyte count was , /mm ( . % polymorphs, . % lymphocytes), and platelet count was , /mm . the serum urea was . mmol/l; serum potassium, . mmol/l; serum sodium, mmol/l; and creatinine, µmol/l. the liver function tests showed a total bilirubin, . µmol/l; serum albumin, g/l, serum alkaline phosphatase, µ/l; serum alanine transaminase, µ/l; serum aspartate transaminase, µ/l. her creatine kinase was µ/l, and c-reactive protein was elevated at . mg/l. she was diagnosed to have aspiration pneumonia, and intravenous ceftriaxone and metronidazole were prescribed. her chest radiograph showed infiltrates in the right lower zone. her urine, sputum, and blood cultures did not yield any bacterial growth. serologic testing for mycoplasma, legionella, and chlamydia and nasopharyngeal aspirate for common viral antigens were not performed, as clinical suspicion was low. she was subsequently transferred to the geriatric unit. her condition improved, and she was placed in the general ward on march . no protective equipment was used by staff attending her in the general ward. it was ascertained that she was previously admitted to a non-sars ward in tan tock seng hospital. however, on march , the patient became restless and more breathless. a repeat chest radiograph ( figure ) confirmed congestive cardiac failure. her repeat leukocyte count was , /mm ( . % polymorphs, . % lymphocytes), and the platelet count was , /mm . there was mild hyponatremia ( µmol/l) and worsening c-reactive protein levels ( . µg/l) but a stable creatine kinase ( µ/l). intravenous diuretic therapy was instituted, but in view of her poor premorbid functional status, the patient was not intubated or moved to in an intensive care unit. she went into respiratory failure and died on march . death was certified as being caused by pneumonia, with a contributing factor of ischemic heart disease. no autopsy or postmortem specimens were taken. in the week after the patient's death, a cluster of cases of atypical pneumonia surfaced, all of which could be traced to this patient. pneumonia developed in the patient's daughter-in-law, who had visited her in the hospital, and two grandsons living in the same household as the daugh-ter-in-law. another son-in-law, who met this daughter-inlaw during the funeral, also contracted a respiratory illness. a healthcare worker, who was unprotected while caring for the patient, was also admitted to changi general hospital for severe pneumonia. he was later transferred to tan tock seng hospital where he was diagnosed with sars. he required prolonged mechanical ventilation and eventually died of the illness. a female hospital cleaner in changi general hospital, who cleaned the room and tidied the patient's bed in the general ward, became symptomatic days after the patient died. she was admitted to changi general hospital days later and was transferred to tan tock seng hospital the next day. her husband was subsequently admitted to tan tock seng hospital with sars. all cases in the cluster had fever as a presenting complaint. on the basis of epidemiologic data (contact tracing linking her to one of the three original index cases in singapore) ( ) , the index patient's cause of death was determined to be sars (figure ). serologic testing for sars-cov by using enzyme-linked immunosorbent assay (elisa) techniques on various specimens during admission for febrile illness were positive at titers of to , for all patients within the cluster except the patient's daughter-in-law and the healthcare worker from the nursing home. since the issue of a global alert on atypical pneumonia by the world health organization on march , reported cases of sars increased daily and appeared in other countries, including canada, the united states, europe, and africa. the first three cases in singapore were reported on march . these cases were traced to a doctor from guangdong who infected guests at a hong kong hotel ( ) . the clinical features of sars are fairly nonspecific with a body temperature of > °c, occurring in % of patients, being the most sensitive feature in all the case series published thus far, ( ) ( ) ( ) . other symptoms described thus far have included nonproductive cough, dyspnea, malaise, diarrhea, chest pain, headache, myalgia, and vomiting. we describe here a fairly complicated atypical signs and symptoms of sars in an elderly patient. the patient had a fever, which responded to a course of broad-spectrum antimicrobial drugs, thus behaving in a manner not much different from a typical community-acquired pneumonia. the absence of fever during the final course of the patient's hospitalization could have been caused by an altered immune response in the geriatric age group, with a resulting normal leukocyte count. furthermore, prior usage of antimicrobial drugs and possible aspiration from dysphagia may further complicate detection of the disease. the suspicion of sars in this case was thus low before eventual epidemiologic links were established retrospectively. dyspnea is a common symptom reported previously, ranging from % to % of patients. cough has also been noted in % to % of cases in previous studies ( , ) . however the absence of cough, especially in the elderly, could be due to an underlying weak cough reflex. vomiting, though present in our patient, was only accounted for in % of cases in the canadian series ( ) . in a frail older person, this could also be caused by a number of circumstances. our patient had characteristic lymphopenia, which was seen in about % of reported cases. in addition, she also had mild hyponatremia and elevated c-reactive protein. however, thrombocytopenia, elevated transaminases, or raised creatine kinase levels were absent. serial chest radiograph progressed from a predominantly right lower lobe patchy consolidation to a radiographic picture of congestive cardiac failure. reports from sars cases have described mainly basal lung opacities, without any pleural effusion. an underlying poor cardiac function may masquerade the true picture of the air space disease characteristic of sars, especially if the stress of infection decompensates left ventricular ejection fraction. this radi-ologic interpretation could potentially mislead clinicians and lead to more patients, family members, and healthcare workers becoming infected. in addition, a bimodal pattern of time to deterioration of clinical symptoms has been previously reported ( ) . the information currently available on transmission of sars has been attributed to respiratory droplets from close contact which has been defined by who to be having cared for, having lived with, or having direct contact with respiratory secretions or body fluids of a patient known to be a suspected sars case. as the patient lived in a nursing home, the brief social contact during visits by family and friends, may prove sufficient for transmitting the virus. furthermore, the issue of possible coinfection and the influence of coexisting conditions have not been thoroughly investigated, which may change the clinical picture of sars so as to conceal detection. uncharacteristic clinical signs and symptoms, without any travel or contact history, are difficult to recognize. our case serves to highlight atypical signs and symptoms of sars, especially the resolving fever, delay in establishing a positive contact history, and the nonspecific chest radiographic appearance that could be affected by concurrent coexisting conditions, such as cardiac failure. we wish to draw attention to clinicians, so that a high level of suspicion is present as the sars-cov is highly contagious and can cause severe disease. we observed that despite being cared for in the general ward by staff without full personal protective equipment, only one healthcare worker in changi general hospital was infected. this observation supports the hypothesis that the virus may not transmit effectively under certain conditions. nevertheless, late diagnosis may lead to large clusters, as delayed isolation of suspect cases increases the risk of onward transmission in the community ( ) . a positive contact history may not be obvious, particularly in patients with cognitive impairment, until retrospective analysis is done. there is thus a need for continued surveillance of fever and clusters of pneumonia cases to improve the chances of early detection. nonetheless, with the imminent availability of accurate and rapid diagnostic tests, there is hope that the diagnosis of sars can be made with more certainty. this could be further enhanced by a revised case definition. coronavirus as a possible cause of severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome who multicentre collaborative network for sars diagnosis. a multicentre collaboration to investigate the cause of severe acute respiratory syndrome case definitions for surveillance of severe acute respiratory syndrome world health organization a cluster of cases of severe acute respiratory syndrome in hong kong a major outbreak of severe acute respiratory syndrome in hong kong identification of severe acute respiratory syndrome in canada evaluation of who criteria for identifying patients with severe acute respiratory syndrome out of hospital: prospective observational study preventing local transmission of sars: lessons from singapore severe acute respiratory syndrome-singapore severe acute respiratory syndrome (sars) in singapore: clinical features of index patient and initial contacts update: outbreak of severe acute respiratory syndrome-worldwide clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong we thank leo yee sin and mark chen for their input on the case and t. ksiazek for providing the antigen for enzyme-linked immunosorbent assays, according to their method ( ).dr. augustine tee is a member of the royal college of physicians (united kingdom). he is currently working as a registrar in respiratory and critical care medicine at changi general hospital, singapore. key: cord- -adul nzf authors: wu, qingfa; xu, zuyuan; wei, tian; zeng, haipang; li, jingxiang; gang, haixue; sun, min; jiang, fangbo; wang, xiang; dong, wei; yang, ling; wang, jian title: development of taqman rt-nested pcr system for clinical sars-cov detection date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: adul nzf severe acute respiratory syndrome (sars) is an acute newly emerged infectious respiratory illness. the etiologic agent of sars was named ‘sars-associated coronavirus’ (sars-cov) that can be detected with reverse transcription-polymerase chain reaction (rt-pcr) assays. in this study, sets of nested primers covering the sars-cov genome have been screened and showed sufficient sensitivity to detect sars-cov in rna isolated from virus cultured in vero cells. to optimize further the reaction condition of those nested primers sets, seven sets of nested primers have been chosen to compare their reverse transcribed efficiency with specific and random primers, which is useful to combine rt with the first round of pcr into a one-step rt-pcr. based on the sensitivity and simplicity of results, the no. primer set was chosen as the candidate primer set for clinical diagnoses. to specify the amplicon to minimize false positive results, a taqman rt-nested pcr system of no. nested primer set was developed. through investigations on a test panel of whole blood obtained from sars patients and control persons, the specificity and sensitivity of the taqman rt-nested pcr system was found to be and %, respectively, which suggests that the method is a promising one to diagnose sars in early stages. an outbreak of a newly emergent infectious disease referred to as severe acute respiratory syndrome (sars), first identified in guangdong province, china in november , infected as many as individuals and resulted in deaths around the world by july (http://www.who.int/csr/sars/en/). the etiologic agent of sars has been identified as a new coronavirus and named as the sars-associated coronavirus (sars-cov) ksiazek et al., ; drosten et al., ) . sars-cov is an enveloped, positive-strand rna virus. genomes of more than strains of sars-cov have been sequenced (marra et al., ; rota et al., ; ruan et al., ; qin et al., ) . the size of genome is about - kb, which is the longest rna positive strand virus to date. based on the genetic analysis of the genome sequence, the sars-cov has been classified as a distinct group from any previously known coronavirus . the symptoms of sars resemble those of other forms of 'atypical pneumonia' that are usually caused by mycoplasma, chlamydia species, and others. in the absence of effective drugs and vaccines for sars, rapid identification of this disease is of importance for controlling spread. at present, virus isolation by cell culture, elisa, ifa and reverse transcription-polymerase chain reaction (rt-pcr) are the major methods to diagnose sars. elisa and ifa methods depend on the detection of igg and/or igm against sars-cov virus that emerged in the blood during the later phase of infection drosten et al., ) . there is a critical need for a test that can detect sars at early stages of illness (days - ) since persons infected with the sars-cov can infect a large number of individuals easily (rosling and rosling, ) . the detection of viral rna with rt-pcr assays is the most widely used approach for early detection of pathogens poon et al., ) . based on the genomic sequence of bj (accession number: ay ), sets of nested primers covering sars-cov have been synthesized. in this study a methodology for selecting the no. nested primers as the most promising primers for clinical purposes and a taqman rt-nested pcr system using the no. nested primer set are described, and the system was evaluated on a test panel. the primers were designed with the genome of bj strain of sars-cov (ay ) as the target. each set of primers was designed with primer software freely available through internet (http://www.basic.northwestern.edu/bio tools/primer .html). the size of outer amplicon and inner amplicon are - and - bp, respectively. the annealing temperature was restricted at the range of - • c. the primer sequences are summarized in table . these primers were synthesized by a dna biotechnology company (shanghai). the fluoregenic probe for no. nested primer sets -fam-aag gtg aca ctc gct gat gct g-tamra- was provided by sangon biotechology company (shanghai). fam represents the fluorescent reporter, and the tamra quencher is linked with the last nucleotide g. the rna of viruses cultured in vero cell was extracted with the qiaamp viral rna mini kit (qiagen). whole blood samples of patients diagnosed clinically with sars were used in the panel. all patients (female/male = / , age = . ± . ) had high fever, and the day with the highest temperature was regarded as day . in the meantime, whole blood samples of nine control persons were also included in the test panel. the qiaamp rna blood mini kit (qiagen) was used to prepare the whole blood samples of the test panel. according to the manufacture's instructions, rna was eluted in a final volume of l of elution buffer. all the above procedures were performed in biosafety level laboratory. the reverse transcription reaction was carried out separately with a random primer and a specific primer. each reaction mixture includes ng random primers or l of m specific primer, l of total rna and l dntp ( mm). the mixture was heated at • c for min and then placed immediately on ice. the following reagent was then added to the mixture: l of first strand-synthesis buffer ( ×, promega), l of . m dtt (promega), l rnase inhittor ( u/l, promega) and l of superscript ii rnase h-rt ( u/l, invitrogen). the rt reaction was carried out at • c for min, followed by incubation at • c for min, and then finally inactivation of the enzyme at • c for min. the first round pcr was carried out in a l volume containing l of rt product, . m of each primer, unit of taq dna polymerase (promega), . mm dntps, mm tris-hcl (ph . ), mm kcl, . mm mgcl . thermocycling was performed on a thermocyler of mj-research ptc- (mj-research inc.), with initial denaturation for min at • c, followed by cycles of ( • c for s, optimal tm for s, • c for s). the final extension reaction was performed for min at • c to complete all the pcr reactions. the second round of pcr was on undertaken on icycler (bio-rad). to detect the pcr product, sybr green i or taqman probe in the second round reaction was used in the mixture. the reaction volume for the second round of pcr was also l, containing l product of first round pcr reaction, . m of each primer, unit of taq dna polymerase (promega), . mm dntps, mm tris-hcl (ph . ), mm kcl, . mm mgcl and . l of sybr green i or . l of taqman probe ( m). for using the taqman probe in mixture, thermocycling was carried out with initial denaturation for min at • c, followed by cycles of ( • c for s, • c for min). for detection with sybr green i, thermocycling was carried out with initial denaturation for min at • c, followed by cycles of ( • c for s, optimal tm for s, • c for s). a melting curve was completed from to • c, increasing by . • c per cycle, to investigate the amplicons. the sets of nested primers used in the study distributed unevenly in the sars genome, but they spanned all known genes and the two predicted genes identified in the sars-cov genome (fig. ) . to compare the sensitivities of these sets of nested primers, serial -fold di-lution genome cdna of bj that reverse transcribed with random primer was used as the template to carry out the nested pcr. the initial concentration of total rna from virus and vero cells was ng/l, which corresponds to < molecules of sars-cov per microliter. all nested primers sets could detect up to − diluted rna, which corresponds to - copies of sars genome per microliter. for nos. , , and nested primers sets, several copies of sars genome could be detected. although few sporadic signals occurred for diluted templates from − to − , they represent random, unreliable results (fig. ) . to compare the reverse transcribed efficiency of random primer versus specific primer, seven sets of nested primers covering each known gene were chosen for testing. with the -fold diluted bj genome rna series, the reverse transcriptions were carried out separately with a random primer and a specific primer. then, amplification with outer primer on the cdna was performed. the products were monitored with sybr green i that binds with double dna. the results showed that three out of seven sets of nested primers have better efficiency on templates reverse transcribed with the specific primer, and three other sets of primers showed that the reverse transcription with the random primer has higher efficiency than with the specific primer. it is worthy to note that the four sets of nested primers located on the terminus of the genome have higher efficiency when transcribed with the random primer. in contrast, the other three sets of primers targeting the initial region of genome have higher efficiency when transcribed with the specific primer. the outer reverse primer pairs of no. are the most sensitive on cdna reverse transcribed with specific primer. meanwhile, the no. primers can attain the same sensitivity on the cdna reverse transcribed with the random primers (fig. ) . as the no. nested primers set were found to be the most promising primers for clinical diagnosis, a taqman probe targeting the internal region of the no. inner amplicon has been designed to minimize false positive results. the taqman probe and no. nested primers pairs together constitute a taqman rt-nested pcr system. to test the specificity of the taqman rt-nested pcr system, a panel consisting of sets of rna from sars patients' whole blood and sets of rna from control person's whole blood was used. these rna samples were reverse transcribed with the no. outer reverse primer. after the first round of pcr, a second round of pcr was performed and monitored with real time pcr. none of the control samples produced a signal, whereas, of the samples from sars patients produced a detectable signal. the results showed the sensitivity and the lower margin of each bar shows the level of sensitivity of these outer primers. the nos. , , and have higher reverse transcription efficiency when transcribed with specific primer, whereas those primers of nos. , , , and have higher reverse transcription efficiency when transcribed with the random primer. the values on vertical axis represent the diluted concentration of rna compared with that of initial rna ( ng/l). the specificity of the taqman rt-nested pcr system to be and %, respectively (table ) . the most striking features of the sars-associated virus are its spikes on the sphere-like virion, which can be clearly observed under the electron microscope and are the basis of the name 'coronavira'. the spike (s) protein, together with the e and m proteins, constitute the components of the surface of coronaviruses. the replicase (r) protein, which occurs in limited amounts, including a and b is the defined unique non-structural protein responsible for rna replication. according to the northern blot, the subgenomic regions for s, m, and e are transcribed in larger quantities . with the -fold diluted rna as template, the sensitivity of primers targeting the s, m, e, and n proteins is higher than - -fold than those primers targeting the r protein, which suggests that the sensitivity of detection is determined mostly by the quantity of transcripts. nucleic acid amplification techniques combining reverse transcription and the polymerase chain reaction have been applied to the detection of sars. the primer is the primary component necessary for developing rt-nested pcr. the size of outer amplicon was set range from to bp, which were usually sufficiently long to design inner primers with spacing intervals of ∼ bp. compared with longer pcr products, this range and flexibility enables many assays to be produced successfully. for these primers, the reverse transcriptions with random primer and specific primer have different effects. according to the sensitivity and simplicity of our test result, the no. primer appears to be the most promising primers set for clinical diagnosis, which is very useful to simplify the detection process through combining rt and first round of pcr reactions to one-step rt-pcr reaction. because the nested pcr can detect low amounts of virus, it is necessary and important to adopt strict criteria for confirmation of positive results. the product can be confirmed as sars-cov by a number of other methods including taqman, dna chip, pcr-elisa, and sequencing. in this study, the taqman probe was applied to confirm the pcr product. the taqman system was developed by abi and employs a fluoregenic probe-based , exonuclease technology that enables amplification and detection to be carried out simultaneously, eliminating the need for post-pcr analysis (heid et al., ; fortin et al., ) . use of the fluorescent probe in real time pcr provides an additional level of assay specificity. although fluorescence increases in direct proportion to the amount of specific amplicons, the taqman probe was used to specify the product in this study because the fluorescence increases non-linearly in the second round of amplification compared with the original virus rna concentration. to confirm positive amplicon, the pcr procedure should include appropriate negative and positive controls in each run, including negative and positive controls for the extraction procedure and the pcr run. sars-cov has been detected in multiple specimens including extracts of lung, sputum, upper respiratory tract swabs, aspirate, stool, and blood samples via pcr or viral isolation drosten et al., ; poon et al., ) . high concentration of viral rna of up to million molecules per milliliter has been detected in sputum. viral rna has also been detected at extremely low concentrations in plasma during the acute illness phase and in feces during the late convalescent phase, suggesting that the sars-cov may be shed in feces for prolonged periods of time . compared with sputum and feces, blood is used more frequently in clinical practice because it is relatively easy to obtain. in this study, whole blood was therefore used as test sample in order to develop a clinically useful detection method for the early stage of infection. according to the protocol developed in the study, the sensitivity and specificity of the taqman rt-nested pcr system are and %, respectively. negative results cannot exclude the existence of sars-cov in tested samples, which may be attributed to low amounts of rna in the samples. the sensitivity of rt-nested pcr tests for sars depends on the specimen and the time of testing during the course of the illness. rapid diagnosis not only offers considerable benefits when a positive case is quickly identified, but is also equally informative when rapid negative result are obtained for optimal patient management and appropriate therapy. the taqman rt-nested pcr system described here provides a rapid, sensitive, and cost effective approach for the diagnosis of sars-cov infection. identification of a novel coronavirus in patients with severe acute respiratory syndrome use of real-time polymerase chain reaction and molecular beacons for the detection of escherichia coli o :h real time quantitative pcr a novel coronavirus associated with severe acute respiratory syndrome rapid diagnosis of a coronavirus associated with severe acute respiratory syndrome (sars) coronavirus as a possible cause of severe acute respiratory syndrome pneumonia causes panic in guangdong province characterization of a novel coronavirus associated with severe acute respiratory syndrome comparative full-length genome sequence analysis of sars coronavirus isolates and common mutations associated with putative origins of infection the authors would like to acknowledge the specific grant for sars research from the ministry of science and technology of china. key: cord- -u jrbsii authors: yang, gee-gwo; lin, shinn-zont; liao, kuang-wen; lee, jen-jyh; wang, lih-shinn title: sars-associated coronavirus infection in teenagers date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: u jrbsii nan to the editor: a global outbreak of severe acute respiratory syndrome (sars) was reported in march ( ) . most reported cases were in adults. hong kong, however, reported pediatric cases ( ) with less aggressive clinical courses. the disease became endemic in taiwan by the end of april ( ) . hualien city, a geographically secluded city in eastern taiwan, had nine pediatric cases, all mild. the cases occurred in tzu-chi high school, a private boarding school for students to years of age, all of whom live in the same building and eat daily meals together in the school cafeteria. on april , when a student (case-patient ) visited the school nurse on the first day that he had a fever, an infection specialist from affiliated tzu-chi medical center immediately responded. the specialist discovered that this student's close friend in the same class (case-patient ) was already febrile. case-patient , a hong kong resident who leaves taiwan for hong kong every months, had visited hong kong twice in march and april . both students were isolated in the hospital on april . tzu-chi medical center began a search for other febrile students. on april , seven more schoolmates were found to have fever > °c. all were identified on their first day of becoming febrile and were immediately isolated in the hospital. all nine schoolmates underwent chest x-ray examinations and were tested for sars-associated coronavirus (sars -cov) by reverse transcription-polymerase chain reaction (rt-pcr) ( ) and dna sequencing. the tested length for sars-cov was bp in the rna-dependent polymerase region. those teenagers with diarrhea were tested for norovirus in their stool by rt-pcr. for those teenagers with cough, throat swabs were cultured for influenza and parainfluenza virus. to reduce the risk for false-positive pcr results, we followed measures to avoid contamination during specimen handling and processing. two primer sets were used for rt-pcr according to ksiazek ( ) and drosten ( ) . the targets are located in the rna-dependent rna polymerase gene at different regions, which are separated by approximately , bp. the laboratory used in rt-pcr analysis is not involved in viral culture or extraction preparation and is located far away from the laboratory for rna extraction to avoid contamination. negative-control cdna was included in each analysis and confirmed that no contamination had occurred. two operators manipulated rt-pcr analysis for two specimens from the same sample. the specimens were analyzed in different rooms with independent reagents for assurance. realtime rt-pcr instead of nested rt-pcr was used. six schoolmates were positive for sars-cov by rt-pcr, confirmed later by dna sequencing for replicase. the tested dna sequence was > % identical with a published sars-cov sequence. norovirus was identified in one teenager's stool by rt-pcr; this virus belonged to genogroup i by testing partial cdna sequence for capsid protein. the tested length was bp, and the virus was % identical to strain ku agi. culture of a throat swab for influenza and parainfluenza virus did not grow any virus. the initial signs and symptoms of the nine teenagers were self-reported fever ( / , range . °c- . °c), cough ( / ), general malaise ( / ), diarrhea ( / ), rhinorrhea ( / ), headache ( / ), chills ( / ), sore throat ( / ), and myalgia ( / ). cough was productive in three schoolmates and dry in one. chest x-ray results were normal for eight teenagers but showed linear interstitial pneumonia for one teenager. four schoolmates took ribavirin for < days. only the teenager with pneumonia was treated with both ribavirin and clarithromycin, for days. the other four schoolmates did not take medication. all nine schoolmates became afebrile by the third day. seven schoolmates were completely asymptomatic in days. two other schoolmates showed improvement and had normal values of all repeated laboratory tests in days; however, they still had mild coughs on the seventh day, when they were discharged. the one teenager with interstitial pneumonia also had a normal chest x-ray result on the fifth day. all nine teenagers were discharged after week of hospitalization and were continuously isolated in a special dormitory for another weeks. no new cases of fever have occurred in tzu-chi high school in the months since these patients' isolation. case-patient was considered the index patient for sars-cov infection because of his travel history to hong kong. six schoolmates with fever were confirmed by real-time rt-pcr and dna sequences to have sars-cov infection. for students with diarrhea, only one case had coinfection with norovirus. influenza and parainfluenza viral infection was ruled out for students with cough. because the nine ill schoolmates were isolated, no more cases of fever occurred in the school. all epidemiologic, molecular, and clinical studies showed evidence for sars-cov infection. worldwide, sars-cov infection has been clinically severe, characterized by respiratory distress and a % average mortality rate ( ) ( ) ( ) . reported series of sars with high mortality rates have involved mainly adults. theoretically, subclinical or mild illness could be present and easily overlooked, and thus death rates could be overestimated. the schoolmates in our series had mild illnesses and were identified only because of a special situation. on may , , the world health organization (who) estimated that the case-fatality rate for sars ranged from % to %, depending on the age group affected ( ); for teenagers or younger children, the case-fatality ratio was < %. our teenagers with presumed sars-cov infection had very mild courses. this benign course was not related to treatment: only one teenager had a full course of ribavirin treatment, and most of the teenagers had either no specific medications or medications for < days. our preliminary presumption for the benign course was the patients' young ages. the benign course of sars-cov infection in our teenage students supports the who finding of less-severe disease in younger persons. these reasons should be explored more fully and may facilitate the development of more effective treatment and prevention programs in persons of all ages. severe acute respiratory syndrome (sars): multi-country outbreak-updated clinical presentations and outcome of severe acute respiratory syndrome in children control measures for severe acute respiratory syndrome (sars) in taiwan a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome clinical features and short-term outcomes of patients with sars in the greater toronto area severe acute respiratory syndrome (sars) in singapore: clinical features of index patient and initial contacts world health organization. who update , sars case fatality ratio, incubation period we thank chih-bin lin and ai-hsi hsu for caring for the study patients and shih-min hsu for directing nurses of the infection control team for contact tracing and collection of samples.tzu-chi foundation provided both financial and administrative support. and lih-shinn wang* *hualien tzu-chi medical center, hualien, taiwan key: cord- -ska bts authors: roe, m f e; bloxham, d m; white, d k; ross-russell, r i; tasker, r t c; o'donnell, d r title: lymphocyte apoptosis in acute respiratory syncytial virus bronchiolitis date: - - journal: clinical & experimental immunology doi: . /j. - . . .x sha: doc_id: cord_uid: ska bts respiratory syncytial virus (rsv) infection may have an effect on the development of t cell memory responses. rsv bronchiolitis in infants is associated with a transient decline in circulating lymphocytes. we hypothesized that the mechanism underlying this lymphopenia is apoptosis. blood was taken from infants during primary rsv bronchiolitis and three months later. using flow cytometry, we found that absolute numbers of both cd +/cd + t-helper lymphocytes (p = · ) and cd +/cd + cytotoxic lymphocytes (ctl) (p = · ) were significantly reduced during acute infection. up-regulated expression both of fas (p < · ) and tumour necrosis factor-related apoptosis-inducing ligand (trail) receptor (p < · ) was found during acute illness on both cd +/cd + and cd +/cd + lymphocytes, when compared with convalescent samples. expression of fas on cd + lymphocytes was inversely related to cd + number (p = · ). plasma levels of soluble fas ligand (p = · ) and caspase- (p = · ), determined by enzyme-linked immunosorbent assay, were increased during bronchiolitis. plasma interleukin- , a product of caspase- activity, was not raised. taken together, these data suggest that in acute rsv infection, cd + helper lymphocytes and cd + cytotoxic lymphocytes are primed to undergo apoptosis. this is a mechanism through which lymphopenia may occur and t cell memory may be altered. respiratory syncytial virus (rsv), a human paramyxovirus, is the most important cause of viral lower respiratory tract disease in infants and children world-wide. primary rsv bronchiolitis causes substantial morbidity, mortality and cost with hospital admission rates of about · % [ ] . a high proportion of infants will develop recurrent episodes of wheeze, cough and asthma-like symptoms after recovery from bronchiolitis [ ] . interest has therefore focused on the role of the host immune response in the pathogenesis of bronchiolitis and its clinical sequelae. the immunopathogenesis of rsv bronchiolitis plays an important part in the clinical manifestations of the primary infection. evidence from human and mouse studies suggest that lymphocytes clear rsv during infection. children with cell-mediated immunodeficiencies take longer to terminate infection with rsv [ ] . in the mouse model, graham et al . [ ] have studied the effect of depletion of cd + or cd + lymphocytes during acute rsv infection. cd + and cd + lymphocyte subsets are involved in terminating primary infection and if both t lymphocyte subsets are depleted rsv shedding is prolonged. however, depletion of lymphocytes in mice also reduces clinical symptoms and lung injury. research into the increased pathology in recipients of the formalin-inactivated rsv vaccine in the s has implicated aberrant cd lymphocytes as the mediators [ ] . these data suggest that lymphocytes may also play a role in causing lung pathology in rsv infection. previous reviews have commented that the white cell count in rsv bronchiolitis is varied and there is a preponderance of neutrophils [ , ] . the most striking change in circulating white cell count is a reduction in lymphocyte numbers and this effect is more pronounced in sicker children [ ] . it has been suggested that this reduction in lymphocyte count be due to redistribution to the lung [ ] . however, lymphopenia is also known to occur in other viral illnesses, including measles, severe acute respiratory syndrome (sars) and ebola [ ] [ ] [ ] . there are data to suggest that apoptosis of lymphocytes occurs in both these illnesses and may offer a mechanism for this lymphocyte decline. apoptosis is programmed cell death via a number of regulated, energy-dependent pathways. two key apoptotic cascades have so far been identified as important in the removal of mature, circulating lymphocytes; death receptor mediated and mitochondrial-dependent [ ] . cell surface death receptors, including fas (cd ) and tumour necrosis factor-related apoptosis-inducing ligand (trail) receptor, are activated by the binding of specific ligands: fas ligand and trail. both pathways lead to the activation of a cascade of specific proteolytic enzymes, caspases, causing the cell to die. fas ligand may be membrane bound or soluble (sfas) ligand and the soluble form may mediate fas-dependent apoptosis of bystander cells [ ] . the pathways of apoptosis and inflammation are intertwined. caspase- converts the inactive molecules, pro-interleukin- and pro-interleukin- , into the active cytokines, interleukin- (il- ) and interleukin- (il- ), respectively. interleukin- activates the immune system, stimulating chemokine release and the development of a th or th response and il- affects apoptosis through the up-regulation of fas [ ] . in this study of infants admitted to hospital with rsv bronchiolitis we undertook to find evidence to support or refute the hypothesis that circulating cd +/cd + and cd +/cd + lymphocytes may be undergoing apoptosis. our results suggest that rsv disease may be associated with lymphocyte apoptosis and offers a novel mechanism through which rsv may alter longterm immune responses. the study protocol was approved by the addenbrooke's hospital cambridge local and regional ethics committee. infants were included in the study if admitted to hospital with a diagnosis of bronchiolitis at months of age or younger; they were rsv positive by routine nasopharyngeal aspirate immunofluorescence and if parents gave written informed consent. for the purposes of this study, the clinical diagnosis of bronchiolitis also included a requirement for supplemental oxygen to maintain arterial saturations greater than %. all blood samples were taken within h of admission. it is known that parental recollection of day that their child's symptoms began is unreliable and so we chose not state which day of the illness blood was sampled on. however, the day of admission to hospital is usually close to the peak of clinical symptoms and we attempted to obtain blood samples as close to this time as possible. children with a suspected or known immunodeficiency were excluded from the study. parents were asked to return with their child for convalescent blood sampling months following the acute illness. all samples were taken immediately to the laboratory for analysis for processing after being taken. blood samples were heparinized. an edta sample was taken for a full blood count and white cell differential. plasma was separated from the heparinized sample and frozen at - ∞ c. the remaining cellular components were resuspended in phosphatebuffered saline (ph · ) supplemented with · % human albumin (pbsa). lymphocytes were obtained by density gradient separation (histopaque: sigma, poole, uk) and resuspended in pbsa to a concentration of - ¥ /l. flow cytometric acquisition and analysis ¥ lymphocytes were incubated with monoclonal antibodies to cd -ecd (beckman coulter), cd -rpe cy (dako-cytomation, uk), cd -fitc or pe (caltagmedsystems, uk), cd -texas red (caltag), cd -fitc (dakocytomation), and trail r-pe (pharmingen) in four colour combinations. all incubations were in the dark at room temperature for min. the stained cells were then washed twice in pbsa and analysed on a beckman coulter epics xl flow cytometer using system ii software. a standard lymphocyte gate was set using low forward and side scatter properties. the position of the positive regions were set with isotype matched controls. cd expression allowed the extent of red cell contamination in the gated region to be assessed. results were corrected accordingly. results were expressed as a percentage and as the mean fluorescence intensity. flow cytometric data was analysed using winmdi version · software. values are reported as median with interquartile range (iqr) and % confidence limits for the median using box and whisker plots. the data was analysed using mann-whitney test for nonparametric data. a p -value < · was considered to be significant. thirty-two infants ( girls; boys) were enrolled in the study; their characteristics are summarized in table . thirteen ( %) of these children required endotracheal intubation and mechanical *data expressed as median and interquartile range. ventilation for respiratory failure and were admitted to the paediatric intensive care unit (picu) during part of their admission. primary apnoea was not the indication for ventilation in any of these patients. nineteen ( %) children only required paediatric ward admission for supplemental oxygen therapy and fluid management. overall the median (iqr) for the group were · kg ( · - · ) for weight, · weeks ( · - · ) for age at admission and · weeks ( - ) for gestation at birth. in common with previous studies of children those admitted to the picu were younger ( p = · ), weighed less ( p = · ) and had a lower gestational age at birth ( p = · ). all children survived their illnesses and were discharged home. from the acute cohort, ( %) infants returned for a convalescent blood samples at a mean of weeks after the acute illness. in this follow-up cohort, there were girls and boys and of these children had required picu admission. due to the design of the study, the follow-up infants are, on average, months older and correspondingly heavier than the acute group. in one patient from each cohort, a white cell differential count was not obtained. absolute numbers of cd + lymphocytes were reduced in rsv bronchiolitis ( p = · ). both cd +/cd + lymphocytes ( p = · ) and cd +/cd + ( p = · ) lymphocytes were significantly reduced during illness (table ) . in acute illness cd +/cd + lymphocytes had up-regulation of fas (cd ) compared with samples taken at convalescence ( p < · ); both the proportion staining high for fas and the median fluorescent intensity were increased (figs a and a) . cd +/cd + lymphocytes also expressed higher levels of surface fas ( p < · ) compared with convalescent samples (figs b and b) . trail receptor was also up regulated on the cd +/cd + population ( p < · ) and on the cd +/cd + population ( p < · ) (fig. a,b) . to support the hypothesis that increased expression of death receptors on lymphocytes is associated with lymphopenia, we performed a linear regression analysis. levels of fas expression correlated with cd + count ( r = · , p = · ) (fig. ) . plasma levels of sfas ligand were significantly higher in the acute samples compared with the convalescent samples ( p = · ) (fig. ) . total white cell count ( ¥ /l) · ( · - · ) · ( · - · ) · lymphocytes ( ¥ /l) · ( · - · ) · ( · - · ) · cd + t -lymphocytes ( ¥ /l) · ( · - · ) · ( · - · ) · cd + t-helper lymphocytes ( ¥ /l) · ( · - · ) · ( · - · ) · cd + cytotoxic t-lymphocytes ( ¥ /l) · ( · - · ) · ( · - · ) · *data expressed as median and interquartile range. plasma levels of caspase- were significantly higher in the acute samples than in convalescent samples ( p = · ). there was no statistically significant difference between plasma levels of il- from acute and convalescent samples and both medians were within the normal reported range for il- in healthy adults (fig. ) . lymphocyte subsets in acute illness were analysed with respect to age. absolute lymphocyte counts ( r = · , p = · ), cd + ( r = · , p = · ), cd +/cd + ( r = · , p = · ) and cd +/cd + ( r = · , p < · ) counts were all inversely related to age (fig. ) . there was no relationship between age and the same lymphocyte counts in convalescent samples. in this study we have looked for evidence that lymphocyte apoptosis may be a mechanism for rsv-induced lymphopenia. we have found that absolute counts of cd + helper t cells and cd + cytotoxic lymphocytes are reduced in rsv bronchiolitis. we have also demonstrated up-regulated expression of cell surface receptors (fas and trail receptor) on cd + and cd + lymphocytes and increased plasma levels of sfas ligand in the acute illness. increased fas expression correlated with lymphopenia. direct evidence of intravascular apoptosis was not sought since the small quantity of blood that can be obtained from a critically ill baby precluded the use of direct assays such as dna laddering. taken together these findings suggest that death receptor-mediated apoptosis is involved in the reduction of lymphocyte numbers in this disease. our data show that there is an inverse correlation with age suggesting there may be a critical period of infancy during which these effects are most pronounced. we demonstrated elevation of plasma caspase- but not il- in acute illness. il- is important in determining lymphocyte phenotype and has been linked to the modulation of apoptosis. our results for caspase- and il- may also have implications for lymphocyte functioning and the regulation of apoptosis. we have already reported that lymphopenia is more pronounced in sicker children [ ] . de weerd et al . children under the age of years with acute rsv bronchiolitis and showed a significant reduction in cd + cells in all infected infants and reductions of all lymphocyte subsets in those ventilated. their data also found nonsignificant reductions of total lymphocyte, cd + and cd + counts for all rsv positive patients. roman et al . [ ] found nonsignificant reductions in total and all t-lymphocyte subsets. the larger size of our study group and inclusion of more severely affected infants may help to explain our clearer findings. rsv predominantly infects respiratory epithelial cells. it has been shown that in vitro infection of type pneumocytes with rsv leads to apoptosis. increased levels of fas and fas-ligand expression can be demonstrated on infected cells and this is through rsv mediated up-regulation of the nuclear transcription factor nf-il- [ ] . reverse-transcriptase polymerase chain reaction (rt-pcr) has been used to show that rsv viral and mrna can be detected in pbmcs from a small number of infants with acute bronchiolitis [ , ] . however an extensive decrease in the number of lymphocytes is unlikely to be explained by directly infected cells which are at very low levels suggesting bystander apoptosis may be important. in measles, apoptosis of uninfected lymphocytes is also associated with up-regulated cell surface expression of fas and trail-r [ ] . lymphocyte apoptosis occurs in infection with ebola virus [ ] which may be closely genetically linked to rsv [ ] . bystander lymphocyte apoptosis occurs in severe sepsis [ ] . in a mouse model of severe sepsis blocking apoptosis with specific caspases inhibitors is associated with improved outcome. although these models are very different to natural infection, the data from mice suggests possible future clinical therapeutic interventions [ ] . in studies of the coronavirus associated with the severe acute respiratory syndrome (sars), lymphopenia has been described as a hallmark laboratory finding [ ] . the reasons for this lymphopenia are unknown but panesar [ ] has offered the suggestion that the use of steroids or loss of vascular integrity may have contributed. our data would indicate that lymphocyte apoptosis might be a target for future study in this disease [ ] . different subsets of t cells are believed to have different susceptibility to apoptosis. in atopic dermatitis, there is data to suggest that apoptotic cell death may remove th cells allowing th responses to become dominant [ ] . in this way lymphocyte apoptosis may offer a mechanism through which the generation of long-term immune memory may be altered. the debate about the causes of long-term cough and wheeze after bronchiolitis has lead to a polarization of views. children hospitalized with rsv bronchiolitis are at increased risk for allergic sensitization compared with controls [ ] . there is evidence from animal models that rsv can be associated with both the allergic sensitization and the maintenance airways hyperresponsiveness [ , ] . early allergic sensitization has been identified as a key risk factor for persistent asthma but the role of viruses in early life remains controversial [ ] . cell mediated immunity including proliferation, cytotoxicity, memory and cytokine production may all be affected by rsv infection. preston et al . [ ] have shown that t cell proliferative responses may be inhibited by rsv. rsv inhibits cd + t cells infiltrating the lung and the development of cd + t-cell memory [ ] . in the mouse model different proteins of rsv have been shown to prime specific lymphocyte subsets. the surface glycoprotein g is associated with the induction of type t cells, and lung eosinophilia during rsv infection [ , ] . interleukin- is an important early cytokine that, together with il- , regulates interferon-g (ifn-g ) production and promotes the development of the type t-helper response [ ] . in contrast, in the absence of il- , il- may stimulate the development of type immune responses. our data suggest that il- levels are not increased in acute bronchiolitis. legg et al . [ ] have shown that lps stimulated pbmcs from children with acute bronchiolitis have reduced il- mrna expression when compared to those from children with rsv-negative upper respiratory tract infections. in contrast a study of nasal secretions from infants with rsv suggests that il- production by epithelial cells may be elevated [ ] . in summary, our data support the hypothesis that the lymphopenia seen in rsv bronchiolitis is associated with apoptosis. the effect is most pronounced in younger infants suggesting there is an important time in infancy when the immune system may be vulnerable. the reduction in lymphocytes is related to the degree of cellular expression of the receptors for apoptosis. our findings mirror those in severe sepsis where data indicates that inhibition of apoptosis may ameliorate illness. future dissection of this novel mechanism may help to delineate the delayed effects of rsv infection and offer new therapeutic avenues. respiratory syncytial virus-coded 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virus-induced immune suppression interleukin- regulates acute graft-versus-host disease by enhancing fas-mediated donor t cell apoptosis respiratory syncytial virus infection in infants is associated with predominant th- -like response induction of cd (fas) and apoptosis in respiratory epithelial cell cultures following respiratory syncytial virus infection detection of respiratory syncytial virus rna in blood of neonates by polymerase chain reaction respiratory syncytial virus rna in cells from the peripheral blood during acute infection extensive lymphopenia due to apoptosis of uninfected lymphocytes in acute measles patients defective humoral responses and extensive intravascular apoptosis are associated with fatal outcome in ebola virus-infected patients sequence of the major nucleocapsid protein gene of pneumonia virus of mice: sequence comparisons suggest structural homology between nucleocapsid proteins of pneumoviruses, paramyxoviruses, rhabdoviruses and filoviruses sepsis-induced apoptosis causes progressive profound depletion of b and cd + t lymphocytes in humans caspase inhibitors improve survival in sepsis: a critical role of the lymphocyte a major outbreak of severe acute respiratory syndrome in hong kong lymphopenia in sars sars: understanding the coronavirus: apoptosis may explain lymphopenia of sars t helper (th) predominance in atopic diseases is due to preferential apoptosis of circulating memory/effector th cells respiratory syncytial virus bronchiolitis in infancy is an important risk factor for asthma and allergy at age anaphylactic sensitization to aeroantigen during respiratory virus infection respiratory syncytial virus infection prolongs methacholine-induced airway hyperresponsiveness in ovalbumin-sensitized mice viruses and atopic sensitization in the first years of life infectious respiratory syncytial virus (rsv) effectively inhibits the proliferative t cell response to inactivated rsv in vitro respiratory syncytial virus infection suppresses lung cd + t-cell effector activity and peripheral cd + t-cell memory in the respiratory tract distinct types of lung disease caused by functional subsets of antiviral t cells immunity and immunopathology to respiratory syncytial virus. the mouse model regulation of interferon-gamma production by il- and il- type and type cytokine imbalance in acute respiratory syncytial virus bronchiolitis rsv-induced bronchiolitis but not upper respiratory tract infection is accompanied by an increased nasal il- response we thank dr r. patel for help taking some of the blood samples, the nurses and doctors in paediatrics and virology for helping to identify children with rsv bronchiolitis. we are grateful to miss eleanor pinto for assistance in the study and statistical design. we would particularly like to thank all the parents and children who made the study possible. we are grateful to the h.c. roscoe fellowship, british medical association, for the financial support for this project. key: cord- -wu ygt w authors: tambyah, p. a. title: sars: responding to an unknown virus date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: wu ygt w the severe acute respiratory syndrome (sars) is an emerging infection caused by a novel coronavirus which first appeared in southern china at the end of . in early , through a single incident, it spread to hong kong, singapore, canada and vietnam. for busy clinicians in large public hospitals, the response to the virus was initially based on ensuring a high level of protection for staff. however, as the epidemic progressed and more information became available about the virus, procedures were rationalized and the virus is currently under control worldwide. there are, however, numerous unanswered questions concerning super-spreading events, the modes of transmission of the virus and, perhaps most importantly, the rapid detection of the virus early in the course of disease. these issues need to be addressed in case the virus becomes more widespread in the near future. the severe acute respiratory syndrome (sars) is a newly recognized coronavirus infection that emerged in southern china [ ] with subsequent global spread to countries [ ] [ ] [ ] [ ] . in countries where local transmission has occurred, hospitals have been the major foci of infection especially in singapore [ ] and canada [ ] . in february , reports began emerging on promed mail of an outbreak of atypical pneumonia in the guangdong province in southern china [ ] . it is now believed that the first cases of sars occurred in the city of foshan in the guangdong province [ ] . however, throughout november until the late part of february, sars was largely confined to the province of guangdong. the global dissemination of sars is believed to have begun on the ninth floor of hotel m in hong kong, where a physician from guangdong stayed for one night on february [ ] . at least individuals who were staying on the ninth floor of the hotel were subsequently infected with sars, although none of them reportedly had direct contact with the ill physician. the newly infected individuals traveled onward to their homes or next destinations in the usa, canada, singapore, hong kong and ireland sparking off epidemics of varying degrees of severity in each of those countries, mainly in hospitals but also in their respective communities. it is striking to realize that the entire global dissemination of this epidemic can probably be traced to this single event of one overnight hotel stay. it has almost become a cliché to report that the sars epidemic, the first emerging infectious disease of the st century, heralded an unprecedented collaboration between researchers across the globe. within weeks of the first cases, electronic publications reviewed the clinical syndromes [ , ] as well as the characteristics of the virus and methods for its detection by the polymerase chain reaction [ , ] . the genome of various strains of the virus were sequenced, which contributed tremendously to knowledge of its molecular epidemiology [ ] . there was an explosion of reports about the sars coronavirus, with more than a thousand publications available on pubmed by the beginning of march , the first anniversary of the global emergence of the virus. it is beyond the scope of this review to cover all of the virological and clinical information contained in these articles and i would refer the reader to the excellent review by peiris et al. [ ] . the present review focuses on the response to this emerging disease and its evolution in light of increasing information. outstanding issues that remain to be resolved are also highlighted. one of the first unusual aspects of this emerging infection was the recognition that healthcare workers (hcw) were uniquely susceptible to the then unknown etiologic agent. in hong kong, the first clue that a new infection had emerged was a cluster of ill hcws. the same was noted in vietnam and led to the world health organization (who) sending a team to vietnam under the leadership of dr carlo urbani, who later died from the virus he helped to define [ ] . as soon as sars was recognized as a nosocomial infection, guidelines were issued by various authorities including the who it is important to recognize that in the beginning of the sars outbreak there was no information about the agent responsible for the infection or its mode of transmission; hence, there was a tendency to "over-protect." as the epidemic evolved, so did the guidelines, which are constantly being updated and might indeed be out of date by the time this report is read. all guidelines are published on the internet [ - ], and the reader is encouraged to review the websites for the latest information. the physician from guangdong who became the source of the global epidemic through his stay at hotel m was admitted to a hospital in hong kong the day after his arrival in the city. he became progressively more ill and died days later. it is striking that although he was critically ill and intubated on a ventilator in the intensive care unit, only one hcw who attended to him in the emergency department became ill [ ] . the reason for this is likely that hong kong hospitals had been on the alert for highly pathogenic avian influenza. in february, there had been a small cluster of cases of avian influenza in a hong kong family that had traveled to mainland china [ ] . a directive had gone out from the hong kong department of health on february to maintain strict infection control with droplet precautions for all cases of "atypical" community-acquired pneumonia because of concerns that highly pathogenic avian influenza might be easily transmissible from person to person. one of the singaporean women who returned from hotel m was indeed isolated as a possible case of avian influenza in one of singapore's large general hospitals, and no secondary cases resulted from her. because of the concerns for possible avian influenza, or some unknown pathogen with an uncertain mode of transmission, most of the initial strategies devised for the prevention and control of sars were directed against a highly contagious airborne pathogen. in [ ] , previously healthy young people were infected and six died from highly pathogenic avian influenza in hong kong. this mortality rate ( %) is much higher than the normal mortality rate for influenza especially among young healthy individuals [ ] . in response to the outbreak, more than one million chickens were slaughtered and the disease was rapidly brought under control. seroepidemiologic studies of hcws done at the time [ ] demonstrated the efficacy of personal protective equipment (ppe) in preventing transmission and identified the risk associated with close personal contact in addition to the virus's lack of efficient humanto-human transmission capability [ ] . fears of a recurrence of a more virulent or easily transmissible form of avian influenza directed the initial efforts against sars. however, sars possessed an unusual quality in that it seemed to be transmitted in the healthcare setting far more efficiently than in households, where measles, varicella and other airborne viruses usually take rapid hold [ ] . this has yet to be explained completely, but it supports the argument that close contact is the major mode of transmission of the sars virus. fomites have been a cause for concern with the sars coronavirus since the initial global dissemination stemming from individuals in hotel m who had no direct contact with the index case but had stayed in the same corridor and probably had occasion to touch elevator buttons or railings that might have been contaminated with the sars virus. in the outbreak of sars in the amoy gardens apartments in hong kong [ ] , more than individuals who had no known direct contact were infected possibly through the aerosolisation of contaminated sewage. the implications of fomite transmission of sars are considerable and would mandate a much greater degree of environmental cleaning than is currently practiced. however, there are many unanswered questions in this arena. for example, why did the individuals staying on the same hotel floor as the index case in hotel m get infected but none of the staff? [ ] . sars has been convincingly demonstrated to be caused by a coronavirus [ ] . certain other characteristics have been ascertained from the previously known coronaviruses, e and oc , including their ability to survive after drying on inanimate surfaces in the hospital environment as well as differences in the viability of the virus at different conditions of temperature and humidity [ , ] . while the sars coronavirus has a certain amount of homology with the other pathogenic human coronaviruses [ ] , too little is known about its behavior under different environmental and atmospheric conditions to make a definitive statement about the role of the environment in nosocomial transmission. there have been reports of the sars coronavirus persisting for prolonged periods of up to days on environmental surfaces [ ] . survival in stool is reported to be even longer at up to days in alkaline diarrheal stools. this would certainly help explain such circumstances as the hotel m outbreak. the attack rates for sars have generally not been high. in singapore, for example, the index case for the national outbreak was nursed in a general ward by staff who were not wearing protective covering of any kind, and the attack rate was only / doctors, / nurses and / fellow patients in the same ward areas [ ] . again, the distribution of infections suggests that close contact is the most important factor leading to the transmission of this pathogen in hospitals. the widespread emphasis on ppe has been seen by some as placing an undue emphasis on hcw protection without adequately considering the protection of other at-risk individuals, such as other patients in the same area. the use of ppe is also not without its own adverse consequences [ ] as reactions to latex are common among hcws, some with serious consequences. it should also be noted that the use of respirators has been associated with fatal adverse events [ ] . costs are also an issue, and in resource-poor settings -ply cotton masks have been used, which have reportedly been effective in preventing the nosocomial transmission of the virus in at least one large public hospital [ ] . in a case-control study, seto et al. [ ] found that surgical masks were also effective in preventing transmission of sars to hcws, which is in line with our understanding of the epidemiology of the virus. in singapore, one of the successes of our approach to the control of sars was the widespread availability of full ppe for any staff member who requested it. this was even before the widespread dissemination of the virus led to the mandatory use of full ppe in all hospitals, and it provided staff the reassurance that their welfare was a high priority in the midst of an epidemic. there have been concerns that the use of n masks alone might not be adequate for preventing the nosocomial transmission of sars since cases have occurred among "fully protected" hcws [ , ] ; these cases possibly resulted from contact transmission. recognition of the role of contact transmission has led to the inclusion of recommendations for the use of gloves and gowns in all guidelines [ ] [ ] [ ] [ ] [ ] [ ] . while these garments have been shown to be effective in preventing the nosocomial transmission of other respiratory viruses [ ] , few data are available for their efficacy regarding sars. travel restrictions were among the more controversial aspects of the sars epidemic. the economies of most affected countries in east asia were devastated by these travel advisories. while other pathogens have been documented as being transmitted on airplanes, most notably influenza [ ] , the number of individuals infected with sars during air travel was remarkably low. according to the who [ ] , there have been flights carrying symptomatic probable sars patients and of those flights did not result in a single secondary infection. overall, cases of secondary infection resulted from symptomatic individuals. one flight alone, ca , which flew from hong kong to beijing on march, is now known to have accounted for of these cases. olson et al. [ ] reported that one flight with four symptomatic individuals with sars was associated with an attack rate (for confirmed sars) of zero while another flight with a single symptomatic individual was associated with an attack rate of %. the singapore experience [ ] was that three flights with symptomatic sars patients resulted in only one transmission. the overall attack rate for the flights into singapore was thus less than % despite one symptomatic individual being a so-called "superspreader" and another being critically ill at the time of the flight, requiring intubation soon after arrival. interestingly, olson et al. [ ] point out that fully % of the fellow passengers who became infected with sars had no direct contact, as defined by the who, with the index patient on their ill-fated flight. they do not offer any explanation for the differing attack rates, although a careful reader would realize that among the individuals allegedly infected on the flight, ten were traveling together as part of a tour group. also, the flight with the four symptomatic individuals was much shorter than the flight that was associated with widespread transmission. this is supported by icu data from canada [ ] that showed time of exposure to be a major risk factor. overall, however, what these reports demonstrate is that a much more detailed analysis is required in order to truly understand the epidemiology of this unusual virus. during the peak of the sars epidemic in china ( - april ), when the who had travel advisories and alerts in place, there were more than , visitors from china and hong kong [ ] who entered singapore and not a single case of transmission was recorded from any of these individuals. in taiwan, a strict -day quarantine [ ] was placed on all individuals returning from countries that were on the who list of sars-affected countries. a total of , individuals were quarantined, among whom had probable sars and only one of whom had laboratory-confirmed sars. thus, the detection rate was . % for probable sars and . % for laboratoryconfirmed sars. these figures have to be balanced against the costs and psychological impact of quarantine for more than , individuals who were perfectly well. thanks to excellent isolation and case finding with contact tracing, the number of people infected with sars with each successive generation of the outbreak was progressively reaching extinction levels [ ] in the sarsaffected countries. with the re-emergence of sars at the beginning of , drastic travel restrictions have fortunately not been instituted to date since there is no evidence yet of widespread dissemination of the virus across international air routes by travelers. it could also be argued that since we now have much better knowledge of the epidemiology of the virus, travel restrictions might not be necessary the next time around. all of the guidelines agree it would be ideal if patients with sars could be nursed in isolation rooms [ - ]. there are differences, however, in the recommendations for negative pressure with separate ventilation systems, and these perhaps reflect the differences in resources available for healthcare. one drawback of isolation rooms is that unless there are adequate nursing or medical resources, the degree of attention that the patient will receive in a single isolation room is obviously lower than in an open well-ventilated area. patients isolated for infection control purposes are known to be at risk for adverse events in hospital [ ] and this again has to be balanced against the benefits in terms of reduced nosocomial transmission. while all of the available evidence points to droplet and contact transmission, there is a possibility that the virus might be aerosolized during such procedures as high-flow oxygen therapy or possibly via the use of extractor fans, which were blamed for the aerosolisation of contaminated sewage during the amoy gardens outbreak [ ] . therefore, n respirators or higher should be used. this is a cause for concern as in many countries, including singapore, without adequate pre-prepared negative-pressure rooms, powerful extractor fans similar to the ones used in the bathrooms at the amoy gardens apartments are used to create a form of laminar uni-directional airflow. while these may serve to direct the flow of air away from areas of heavy traffic, it is possible that they might be hazardous by facilitating the aerosolisation of infectious droplets. in singapore [ ] and canada [ ] , transmission of the sars virus has been noted in crowded emergency rooms where patients routinely wait for hours for a hospital bed. in singapore, sars was documented as being transmitted to a patient's visitor who was waiting in a corridor during the patient's radiological procedure [ ] , again a common occurrence in many healthcare settings. in our own hospital, the national university hospital, the largest cluster of sars cases occurred in one of our eight-bed wards [ ] where patients are deliberately placed eight to a cubicle in order to support the philosophy of healthcare financing in singapore. the sars outbreak has clearly been a wake-up call for health authorities worldwide [ ] as they try to adjust health systems primarily designed to minimize costs into systems designed to protect staff and patients. the isolation and segregation of patients with suspect and probable sars has been credited with markedly reducing the transmission of the virus [ ] . lipsitch et al. [ ] reported a reduction in the time to isolation of patients with sars as the epidemic progressed with a corresponding decline in the number of secondary cases as knowledge of the virus increased. the majority of individuals with sars have not transmitted the virus to anyone [ ] . while it is tempting to ascribe this to infection control measures, many of these individuals were infected and hospitalized long before the institution of infection control methods. this has given rise to the concept of "super-spreaders." it is known that the presence of common viral upper respiratory tract infections can turn some hcws into so-called "cloud hcws" [ ] . these individuals have been linked with the airborne dispersal of agents that are normally only spread through contact, such as group a streptococci or staphylococcus aureus. the hypothesis is that the presence of upper respiratory tract infections transforms these individuals into efficient transmitters of pathogens through increased coughing, sneezing or nose rubbing. alternatively, airborne dispersal could result from the use of various respiratory therapies. the index patient for the singapore epidemic was not isolated, and hcws, visitors and fellow patients were infected [ ] . the second generation of cases associated with this cluster, before the institution of infection control practices or strict isolation, included only cases. the situation in canada was similar, with cases in the second generation occurring preisolation [ ] . it is quite clear, however, that non-isolated patients are hazardous to staff, visitors and other patients. single non-isolated patients have led to well-documented outbreaks in hospitals in singapore [ ] , taiwan [ ], canada [ ] and hong kong [ ] . the phenomenon of "super-spreaders" has been invoked to explain why so few transmissions resulted from the majority of non-isolated individuals while a few rare cases were associated with the vast majority of transmissions [ ] . the jury is still out as to whether these are indeed individuals who are for some reason more able to transmit infection or whether events, such as the use of high-flow oxygen therapy, are more responsible for what are probably more accurately described as "super-spreading events." until we have more virologic information, we have to assume that all individuals with sars are "super-spreaders" until proven otherwise and we have to take all the necessary precautions. again, because of the concern that an undiagnosed patient might turn out to be a "super-spreader," the threshold to isolation has become progressively lower. initially, hospitals and clinics were using the who case definitions of suspect and probable sars cases to determine which cases to isolate. as we, and others, have pointed out, atypical presentations [ ] are the achilles heel of such a strategy and these have been associated with significant nosocomial transmission. in a very important report from a sars screening clinic, rainer et al. [ ] pointed out that the who criteria, which were actually designed for epidemiologic purposes, while relatively specific, have a sensitivity of only about % in predicting which individuals will turn out to have sars. the implications are that a large number of individuals will need to be isolated and monitored very closely until their clinical course becomes evident. in practice in singapore, this resulted in the conversion of large numbers of hospital wards to isolation facilities, cancellation of elective surgeries and an overall paralysis of the healthcare system. we used a regime of -hourly temperature monitoring without any use at all of antipyretics together with daily serial chest radiographs and blood counts and comprehensive chemistry work ups. with this regime, we found a sensitivity of %, specificity of %, positive predictive value of % and negative predictive value of % for the who criteria at patient presentation [ ] . it is clear that an accurate rapid diagnostic test is urgently needed to allow us to filter out individuals who are at low risk of sars or, better still, at lower risk of transmitting the virus should they not be isolated. current diagnostic tests [ ] , which are based on either molecular methods or serological diagnosis, are severely limited not predominantly by sensitivity or specificity but by the fact that they take awhile to become positive, during which time widespread dissemination of the virus could have occurred from a single non-isolated "super-spreader." fever screening is widely practiced as a sars-prevention measure. there was even a period when the who called for fever screening at airports to prevent the global spread of sars. unfortunately, fever is a non-specific and insensitive screening tool for sars [ ] . atypical presentations of sars without fever have been reported especially in older and immunocompromised patients [ ] . one case is particularly illustrative [ ] . a -year-old man cleared a fever triage area in an emergency room as he was afebrile; he was then admitted to a general ward (not a "fever ward") as a case of heart failure, and he remained afebrile until he developed a lowgrade temperature after being transferred to the medical intensive care unit for progressive shortness of breath. two other patients, one visitor and one nurse who had been in the same emergency department area as this patient were infected with sars. the visitor, a previously healthy -year-old woman, died and her husband and son were subsequently infected. in the brief period the index patient was in the general ward, two other patients and the entire shift of nurses working in the ward at the time, who were only wearing n masks, were infected. by the time the patient became febrile in the intensive care unit, staff were wearing full ppe and no further infections resulted. thus, a single patient who "passed" two strict fever screens managed to be the source of at least nine infections in less than h. this patient was critically ill and died days later; thus, he may have had a very high viral load. this case illustrates the limitations of "cookbook screening" by using fever protocols without paying attention to a careful clinical history and physical examination. in this case, an alert cardiology team who re-did the patient's history and examination and performed a bedside echocardiograph to prove that he was suffering from pneumonia not heart failure made the diagnosis. during the sars outbreak, the inter-hospital transfer of patients in canada [ ] , taiwan [ ] and singapore [ ] was a very efficient means of dissemination of the sars virus. in singapore, on march, the decision was made to close one hospital to new admissions and to concentrate all sars patients there [ ] . this unfortunately led to patients recently discharged from this hospital being shunted to other hospitals and starting off epidemics there. now, in singapore, once a cluster of cases with even a low degree of suspicion is identified, the unit is "locked down" with no admissions, transfers or discharges in order to prevent a recurrence of the former situation. this strategy was also used successfully in vietnam to contain the sars virus, which led to vietnam being the first country to be declared free of local transmission of sars [ ] . while we have learned a tremendous amount in the brief period since sars first emerged in november , there are still a number of unresolved issues for practicing clinicians. the cases of sars in early have quashed hopes that the virus was "put back in the box," and in the formerly affected countries many clinicians are deeply worried about dealing with a devastating resurgence of the virus. i have a personal "wish list" of questions that i would like answered before too long. these include: what are the conditions required for the airborne transmission of the sars coronavirus? when can we be sure that transmission does not occur? when can we get a good rapid diagnostic test that is positive early in the course of the illness? what makes a super-spreading event? is quarantine really necessary? i can only hope that the answers to these and numerous other questions raised by infection control practitioners, hospital epidemiologists, infectious disease clinicians and researchers can be answered before we face the next sars outbreak or something worse! even as this is being written, avian influenza rampages across east asia affecting primarily birds, but also claiming the lives of more than individuals. if this becomes a pandemic form of influenza, sars will pale in comparison. epidemiology and cause of severe acute respiratory syndrome (sars) in people's republic of china a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome a major outbreak of severe acute respiratory syndrome in hong kong identification of severe acute respiratory syndrome in canada clinical features and short-term outcomes of patients with sars in the greater toronto area update: outbreak of severe acute respiratory syndrome-worldwide comparative fulllength genome sequence analysis of sars coronavirus isolates and common mutations associated with putative origins of infection the severe acute respiratory syndrome sars in northern vietnam influenza a h n -hong kong special administrative region clinical features and rapid viral diagnosis of human disease associated with avian influenza a h n virus observations on mortality during the influenza pandemic risk of influenza a (h n ) among healthcare workers exposed to patients with influenza a (h n ), hong kong how contagious are common respiratory infections? 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cloud" healthcare workers update: severe acute respiratory syndrome-taiwan update: severe acute respiratory syndrome-toronto, canada sars experience at prince of wales hospital, hong kong atypical presentations of sars evaluation of who criteria for identifying patients with severe acute respiratory syndrome out of hospital accuracy of who criteria for sars was similar in a non-sars hospital in singapore crouching tiger, hidden dragon: the laboratory diagnosis of severe acute respiratory syndrome atypical sars in a geriatric patient vietnam-sars free key: cord- - hovffso authors: cherry, james d; krogstad, paul title: sars: the first pandemic of the (st) century date: journal: pediatr res doi: . / .pdr. . .fc sha: doc_id: cord_uid: hovffso nan sars (severe acute respiratory syndrome) was a new disease in the fall of , which first occurred in guangdong province, china and spread to countries with cases and fatalities ( ) ( ) ( ) . after an unprecedented global public health effort, the epidemic was controlled within mo of its original occurrence ( ). the scientific effort demonstrated unusual international cooperation and was facilitated by electronic communication. media coverage was incredibly accurate and provided worldwide pictures to augment scientific data. as of march , , there were citations related to sars in the medical literature. of interest, however, is that of these citations only . % are related to pediatric experiences. the purpose of this mini-review is to examine the unique pediatric aspects of sars, to review the epidemiology of the sars-cov in regard to future epidemics, and to use the sars experience as a model for future pandemics. the first train of transmission of sars occurred in fosham city, guangdong province, china ( , ) . during the period from november , , until february , , there were cases reported in guangdong province. sars was spread to hong kong on february , , by a patient from guangdong province who, before his hospitalization, stayed in the metropole hotel in hong kong for d. ten secondary cases occurred in hotel guests, and these infected persons led directly to tertiary cases in two hong kong hospitals and outbreaks in singapore, toronto, and hanoi ( ) . in march , a novel coronavirus (sars-cov) ( fig. ) was isolated from patients with sars and subsequently sequenced ( - ) . this virus was rapidly identified and characterized by a combination of classical virological methods and cutting-edge molecular biology. electron microscopic examination of swabs and sputum specimens from affected patients revealed the presence of viral particles. fortuitously, this newly identified agent replicated in vero cells, in contrast to other human coronaviruses. cytopathic effect was seen by - d after inoculation. in a technological blitzkrieg, the genome was completely and rapidly sequenced by several laboratories. a low-stringency, random primer reverse transcription pcr method was used by drosten et al. ( ) to amplify short fragments of dna using rna recovered from culture supernatants as the template. this method, previously used to identify the human metapneumovirus, was successful again: of fragments identified showed homology to known coronavirus sequences. in a similar approach marra et al. ( ) began by the construction of random primed and oligo-dt primed cdna library, beginning with viral rna recovered from a highly purified virus preparation. these and other molecular tricks of the trade were used to rapidly establish complete sequences of the sars-cov. this was no small feat. coronaviruses have the largest genomes ‫ , ف(‬ bases of positive-sense rna) found in any rna virus. these sequence data not only permitted the rapid development of highly specific diagnostic tests, but also helped in the epidemiologic tracking of the pandemic. moreover, cataloging the genome from human cases assisted in the search for the origin of this disease, when viruses related to the sars-cov were identified in animals [himalayan palm civets (paguma larvata) and raccoon dogs (nyctereutes procyonoides)] in a live animal market in shenzhen, china ( ) . viral genomes from nasal swabs from palm civets were . % homologous to the human sars cov, and represented a distinct phylogenetic group from the human isolates. moreover, early in the epidemic, open reading frame sequences from human isolates were identical to those from palm civets, suggesting animal to human transmission. as the pandemic progressed, most human isolates contained a sars-cov sequence with a deletion of nucleotides in this open reading frame. signature sequences were also identified in the amino acid sequences of the spike protein, which is involved in the attachment of viral particles. it is unclear whether these changes represent adaptive mutation to replication in humans. however, coronaviruses, like other rna viruses, mutate rapidly as a consequence of the error-prone nature of rna polymerases and their characteristically short replicative life cycles. thus, minor (perhaps inconsequential) mutations can emerge rapidly and persist as a founder effect. despite the known high rate of recombination seen with other coronaviruses ( ), there has been no evidence to date that this pandemic reflected the recombination of human and nonhuman coronaviruses, although this predilection could conceivably enhance the diversity of the sars-cov and result in a larger catastrophic pandemic. also of interest, in the investigation in the live animal market, was that of ( %) wild animal traders and of ( %) of those who slaughter the animals had antibody to the animal coronaviruses ( ) . in contrast, only of ( %) vegetable traders in the same market were seropositive, supporting the hypothesis that the sars-cov originated in these animals. the epidemiology of sars is both extremely interesting and frightening ( , , ) . as noted above, sars spread to hong kong on february , . the ten secondary cases associated with the metropole hotel lead to worldwide dissem-ination, which eventually involved countries ( ). how the secondary cases acquired the disease at the metropole hotel is not known. eight of the cases resided on the th floor, which was the same floor as the primary case. since the index patient vomited in the hall of the th floor and this was subsequently cleaned by vacuuming, it is possible that an aerosol was created. of particular interest was the point source outbreak, which involved the amoy gardens housing complex, kowloon bay, hong kong ( , , ) . the primary case in this outbreak was a -y-old man who lived in shenzhen. he had chronic renal disease and he frequently visited his brother in amoy gardens when he made visits to the prince of wales hospital, where his renal disease was being treated. on march and , , he visited his brother who lived in a flat in block e of the housing complex, which had high-rise units (blocks). the patient had diarrhea at the time of his visits and used the toilet in his brother's unit. during the following month, sars cases occurred in amoy gardens, with % occurring in block e residents. the peak of the outbreak occurred on march , , and the block e cases appeared earlier and showed a point-source-type distribution. subsequent study suggested that the secondary cases in block e were due to aerosolization of contaminated sewage through floor vents when toilets were flushed and exhaust fans in bathrooms were switched on. the transmissibility of the virus and human disease is a paradox. on the one hand, in the initial phases of the spread of sars in hong kong, singapore, and toronto, a disproportionate number of health care workers became ill and apparent "superspreader" cases were noted ( - , , , - ) . in contrast, the secondary illness rate in households was only % in a hong kong study and % in a study in singapore ( , ) . these household-contact rates are significantly less than occur with other respiratory infections such as pertussis and measles. with the exception of the amoy gardens outbreak and the secondary cases in the metropole hotel, most other instances of superspreading involved contact by hospital personnel who gave care and had contact with the primary cases without carrying out infection control precautions. sars spread rapidly around the world during march by infected persons who traveled by airplane. surprising, there were relatively few secondary cases acquired by co-airline passengers ( , ) . between february and may , , there were known airline flights with symptomatic probable cases on board ( ). a total of only secondary cases have been linked to probable cases of sars who traveled while symptomatic. however, on one flight from hong kong to beijing with one symptomatic passenger, of the ( %) contacts became ill. the incubation period of sars is - d, with a median of - d ( ). the attack rate in children is reported to be less than that of adults, but when consideration is given to the large number of nosocomial cases in original data sets, it appears that children have similar rates as adults. clinical disease in children is clearly less severe than disease in adults ( , , , , ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . adolescents have illness similar to adults, but the case fatality rate in adolescents is significantly less. although there is evidence that unrecognized infections occur, there is only one reported instance of a possible asymptomatic infection with the sars-cov ( ) . details of clinical illness in children have been presented from studies in hong kong, singapore, toronto, and eastern taiwan. the largest single experience occurred at the princess margaret hospital in hong kong ( ) . the experience there was unique in that ( . %) of the hospitalized children were from the amoy gardens point-source outbreak. in a review of pediatric patients seen in toronto, singapore, and hong kong, the following clinical findings were noted: fever, %; cough, %; rhinorrhea, %; myalgia, %; chills, %; and headache, % ( ) . in patients Ͻ y of age, the most common findings when initially seen were fever and cough, whereas older children (Ն y) also had headache, myalgia, sore throat, chills, and/or rigor. in this study, all cases categorized as probable sars had abnormal chest radiographs or computerized tomography (ct). the most prominent radiographic findings were patchy infiltrates, opacities, and/or areas of consolidation. multifocal lesions were seen in % of the radiographs. hilar adenopathy, extensive pleural effusions, lung abscess, pneumatocele, or pneumothorax were not observed. on ct scan, unifocal or multifocal regions of consolidation and/or ground-glass opacities were observed throughout the lung fields. the most striking laboratory finding is absolute lymphopenia. this occurs in nearly all pediatric patients. in one study, % of children had lymphopenia on presentation, and this frequency rose to % ( ) . the mean value was . Ϯ . ϫ Ϫ /l. other frequently abnormal laboratory findings include thrombocytopenia and elevated lactate dehydrogenase and creatinine phosphokinase. the vast majority of children who were hospitalized with sars were treated with i.v. or orally administered ribavirin. in hong kong, most children were also treated with steroids, whereas in toronto none of the children received steroid treatment ( , , ) . only a small number of children required oxygen supplementation and intensive care and, to our knowledge, no fatalities in children have occurred. the initial diagnosis of sars was based upon clinical and epidemiologic data. the world health organization sars case definition is presented in the . high fever (Ͼ °c) and . cough or breathing difficulty, and . one or more of the following exposures during the d before onset of symptoms: close contact ‡ with a person who is a suspected or probable sars case-patient, history of travel to an area with recent local transmission of sars, residing in an area with recent local transmission of sars. probable case-patient: a suspected case-patient with: . radiographic evidence of infiltrates consistent with pneumonia or respiratory distress syndrome (rds) on chest x-ray or . consistent respiratory illness that is positive for sars coronavirus by one or more assays, or . autopsy findings consistent with the pathology of rds without an identifiable cause. ( ) . laboratory confirmation of a sars-cov infection can be determined by the demonstration of serum antibody by elisa, isolation of the virus from a clinical specimen, or the detection of sars-cov rna by a reverse transcription pcr assay ( , , ) . at the present time, there are two key questions that relate to sars: will the disease reoccur? and, if it does, how should it be treated and can it be contained? this year, two laboratory confirmed cases of sars have been identified in guangong province, china, and no secondary cases have occurred ( ) . severe disease in humans who are infected with animal viruses are an ever-present danger. although perhaps hiv infection is an exception, the only animal viruses that have caused pandemic human disease and continued human to human spread over periods of years are influenza a viruses. other severe diseases acquired from animals such as rabies, lassa fever, and ebola hemorrhagic fever, all of which can be transmitted from person to person, have not resulted in sustained human disease. it would appear that the sars-cov should also be similarly grouped. the sars pandemic of - had two initial events that led to its worldwide dissemination. in retrospect, it seems likely that aerosolization of the virus at the metropole hotel, amoy gardens, and perhaps in some nosocomial situations made the introduction of this virus different from experiences with lassa and ebola viruses. however, concern has to be raised as to the possibility of a future genetic recombinant virus with the sars-cov and a human respiratory cov such as oc or e strains. because live animals and humans have close contact in southern china and infections with human cov are common, dual infection seems quite possible. however, this type of recombination among different groups of cov has, to our knowledge, never been documented. far more likely, however, is the occurrence of a recombinant between a strain of avian influenza (such as h n ) and a circulating human strain (such as h n ). from past experience it appears that an influenza pandemic will occur in the next few years. hopefully, lessons learned from the international response to sars will contribute to its control. the pathophysiologic events in sars are not clear. the illness in adults is biphasic and occasionally triphasic ( , , , - , , ) . the biphasis illness is characterized by an initial febrile period, which is otherwise relatively symptom free, and then a period of respiratory symptoms, chills/rigor, and vomiting and diarrhea. maximum virus shedding occurs during the second phase. about % of adults will have a third phase characterized by acute respiratory distress syndrome. children in general have a single-phase disease and illness in adolescents is biphasic but generally less severe that that seen in older adults ( , , - ) . initial therapy, which was developed on medical services, used both antiviral (ribavirin) and anti-inflammatory (steroids) treatment. the rationale for steroids was based on the perception that severe lung damage was occurring as a result of a "cytokine storm" ( ) . this thought was enhanced by the fact that illness in sars was similar to illnesses in adults due to infection with avian influenza (h n ). laboratory studies with h n virus in tissue culture noted the induction of proinflammatory cytokines ( ) . the most notable laboratory finding in sars is the profound lymphopenia. cui et al. ( ) noted that cd ϩ t cells were reduced in all patients, cd ϩ cells in %, b-lymphocytes in %, and natural killer cells in % of patients. in patients who recover from sars there is a rapid restoration of lymphocytes in peripheral blood ( ) . sars-cov experimental infections in macaques suggest that pulmonary pathology is due to a direct viral effect on type pneumocytes ( ) . at the present, when therapy of pediatric sars patients is considered, it seems clear that there is no indication for routine treatment with steroids inasmuch as children in toronto who did not receive steroids did equally well as those treated in hong kong with steroids. in regard to antiviral therapy, it is disappointing that no laboratory data have become available regarding the evaluation of ribavirin treatment ( ) . at the present time, the use of ribavirin either i.v. or orally is the standard of care. laboratory studies have suggested that pegylated interferon-␣ and interferon-␤ might be useful therapeutic agents ( , ) . an uncontrolled study in adults in toronto suggested that patients treated with interferon alfacon- plus steroids had more rapid recovery than patients treated with steroids alone ( ) . although it seems unlikely to us that pandemic infection with the sars-cov will ever occur, there has been considerable effort to develop a vaccine ( ) . to us, this seems ill advised for two reasons. first, if a reoccurrence of pandemic disease were to occur, it is likely, since its origin will be from an animal, that the new virus will be different from the present human sars-cov. secondly, vaccines against known animal cov have had varied results. of particular concern in this regard is the possibility of enhanced sars rather than protection. this has happened before in humans with other rna viruses (measles and respiratory syncytial virus) and it has happened in the animal setting with a feline cov vaccine (denison mr, personal communication). at the present time, the most important factor in preventing a future epidemic or pandemic of sars, as well as epidemics or pandemics with other new viruses, is sound public health policy and the use of standard infection control procedures. sars gained a foothold because of an unusual event (probable aerosol dissemination), and the failure to recognize the problem and to initially use respiratory isolation procedures, and to use quarantine measures. in the united states in we were lucky because very few of the probable cases were actually infected with the sars-cov. in the spring of , one of us surveyed a number of hospitals, including our own, and found that if a patient with sars were to visit a clinic or emergency room, large numbers of persons would have been exposed before the problem was recognized. a further problem is that all hospitals built during the last y and those being built today do not have the capacity to handle large numbers of patients who require respiratory isolation. world health organization. summary of probable sars cases by onset of illness from severe acute respiratory syndrome (sars) consensus document on the epidemiology of severe acute respiratory syndrome (sars) update: outbreak of severe acute respiratory syndrome-worldwide the severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome characterization of a novel coronavirus associated with severe acute respiratory syndrome sars working group a novel coronavirus associated with severe acute respiratory syndrome the sars coronavirus: a postgenomic era isolation and characterization of viruses related to the sars coronavirus from animals in southern china recombination in large rna viruses: coronaviruses. semin virology outbreak of severe acute respiratory syndrome (sars) at amoy gardens, kowloon bay, hong kong, main findings of the investigation cluster of severe acute respiratory syndrome cases among protected health-care workers-toronto, canada severe acute respiratory syndrome (sars) in singapore: clinical features of index patient and initial contacts a major outbreak of severe acute respiratory syndrome in hong kong secondary household transmission of sars probable secondary infections in households of sars patients in hong kong transmission of severe acute respiratory syndrome on aircraft clinical features and short-term outcomes of patients with sars in the greater toronto area acute respiratory distress syndrome in critically ill patients with severe acute respiratory syndrome hku/uch study group clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study severe acute respiratory syndrome in children infants born to mothers with severe acute respiratory syndrome inflammatory cytokine profile in children with severe acute respiratory syndrome severe acute respiratory syndrome in children: experience in a regional hospital in hong kong other members of the hospital for sick children sars investigation team children hospitalized with severe acute respiratory syndrome-related illness in toronto a young infant with severe acute respiratory syndrome adolescent twin sisters with severe acute respiratory syndrome (sars) severe acute respiratory syndrome (sars): chest radiographic features in children asymptomatic severe acute respiratory syndrome-associated coronavirus infection world health organization case definitions for surveillance of severe acute respiratory syndrome (sars) update: influenza activity-united states, - season detection of sars coronavirus in patients with suspected sars combining clinical and epidemiologic features for early recognition of sars sars-one year later severe acute respiratory syndrome-associated coronavirus in lung tissue induction of proinflammatory cytokines in human macrophages by influenza a (h n ) viruses: a mechanism for the unusual severity of human disease expression of lymphocytes and lymphocyte subsets in patients with severe acute respiratory syndrome significant changes of peripheral t lymphocyte subsets in patients with severe acute respiratory syndrome pegylated interferon-␣ protects type pneumocytes against sars coronavirus infection in macaques antiviral treatment of sars: can we draw any conclusions? treatment of sars with human interferons interferon alfacon- plus corticosteroids in severe acute respiratory syndrome: a preliminary study caution urged on sars vaccines key: cord- - ftnttm authors: gensheimer, k. f title: challenges and opportunities in pandemic influenza planning: lessons learned from recent infectious disease preparedness and response efforts date: - - journal: international congress series doi: . /j.ics. . . sha: doc_id: cord_uid: ftnttm abstract the impact of the next pandemic influenza is likely to be far greater, by orders of magnitude, than most bioterrorism (bt) scenarios. a written pandemic emergency plan and establishment of emergency management teams are critical to mounting a coordinated and effective response to what will be a catastrophic event. members of these teams should include public health, medical, emergency response and public safety officials, organized at each local, state and federal level. the tragic events of september , and the subsequent anthrax attacks have substantially increased funding and support for bioterrorism planning in the united states. thus, public health officials have an unprecedented opportunity to strengthen current systems' planning efforts by promoting dual use bioterrorism/pandemic influenza plans. combining lessons learned from the terrorist incidents, recent preevent smallpox vaccine programs and the history of past influenza pandemics, more effective strategies can be developed. for example, enhanced influenza surveillance systems can provide data that will not only provide early identification of a novel influenza strain, but will provide more timely recognition of other outbreaks of infectious diseases, including public health threats that may initially present as an influenza-like illness (ili). in recent years, we have witnessed emerging and reemerging infectious disease threats that have presented us with challenges similar to those posed by an influenza pandemic. such events highlight the need for advance planning to ensure an optimal response to a health emergency that is certain to be unpredictable, complex, rapidly evolving and accompanied by considerable public alarm. while advance warning for a terrorist attack is unlikely, the warning already exists for a possible new influenza strain, as evidenced by the recent cases of h n in hong kong and the rapid global spread of cases of severe acute respiratory syndrome. influenza is transmitted readily from person to person, and because a novel influenza virus, by definition, is one to which the general population has little to no immunity, an influenza virus with pandemic potential has the potential to cause substantial morbidity, mortality, social disruption, and widespread panic. crosby's [ ] book, ''america's forgotten pandemic: influenza '' notes that more americans died of pandemic influenza than of war-related causalities throughout the entire history of this nation. despite the extent of morbidity experienced, little attention was focused on this catastrophic heath event. in contrast, the tragic events of september , coupled with the use of bacillus anthracis as a bioterrorist weapon of mass destruction, received considerable attention from the media, the public and the political leadership of this country. one of the greatest revelations in the aftermath of these unprecedented events was the realization that public health is a bona fide first responder. in the current era of concern for bioterrorism (bt) disaster-type preparedness, the public health community needs to acknowledge the leadership position it has achieved through these recent events and to accept yet another challenge of thinking broadly and creatively to address the many needs posed by a catastrophic infectious disease disasterbe it an influenza pandemic, a bioterrorism event or an emerging/reemerging infectious disease threat that is yet to be identified. planning for these events can no longer be postponed as advance planning and building of public health infrastructure can make a significant difference in our response. national efforts to prepare for the next influenza pandemic require support and collaboration from multiple partners at the state, local and federal level. establishing relationships with the medical community, law enforcement and public health agencies are not only critical in responding to a potential catastrophic event, but will enhance ongoing everyday work. the recent anthrax events demonstrated that public health's unfamiliarity with the emergency response system's incident command structure impeded investigative efforts. cross-department planning will facilitate a more effective response to pandemic influenza, strengthen ties between public health and emergency response sectors and complement other planning efforts for not only pandemic influenza but for other emergencies including acts of terrorism. a written pandemic emergency plan and an established emergency management team, which includes public health, medical, emergency response and public safety officials, are needed to provide effective leadership, coordination and an effective response to the next influenza pandemic. the planning and public health infrastructure needed to effectively address a bioterrorist event and an influenza pandemic overlap considerably. one such area of overlap is surveillance. global and domestic laboratory and disease surveillance must be strengthened to increase the likelihood of early detection and tracking of pandemic influenza or a bioterrorist event. improvements in state public health laboratory capacity through support of the laboratory response network (lrn) has enhanced rapid testing for influenza. because many potential bioterrorist agents initially cause symptoms that resemble an influenza-like illness (ili), it is critical for every state to have rule-out influenza testing capabilities available on a year-round basis. timely reporting of outbreaks and surveillance for influenza-like illness (ili) are directly relevant to tracking the progression and intensity of influenza activity and may provide an early indication of a bioterrorist event. continued support for the early aberration reporting system (e-ars) and other innovative surveillance strategies will benefit the public during seasonal influenza epidemics, an influenza pandmeic, and any other catastrophic disease event. another critical component of any catastrophic infectious disease plan and response is communication. a key lesson learned from the anthrax attacks was that the public demands up-to-date information on an ongoing basis throughout the emergency. factual information presented by trusted public health officials can assist in minimizing fear and hysteria. most health crises are similar to the recent anthrax attacks, where only a few cases are ultimately diagnosed, but the bulk of the populace seeks information on a rapidly unfolding scenario. demand for factual information will only be heightened for a highly contagious disease entity such as pandemic influenza. data generated as a result of a robust surveillance system can assist public health efforts in minimizing hysteria and preventing the dissemination of misinformation regarding the evolving pandemic: has the novel virus arrived; geographical areas of the country most severely affected; whether disease activity is increasing or decreasing and groups most severely affected. despite the many similarities between pandemic influenza and planning for other catastrophic infectious disease events, including an act of bioterrorism, critical differences do exist. unlike the anthrax events of where there was no forewarning, surveillance should provide days to months of warning for a pandemic, while the pandemic itself will last for several months or years. there will be no ''unaffected'' areas, as the pandemic influenza virus will be present virtually simultaneously in all parts of the country. mutual aid from either the federal government or other regions of the country will be unlikely, as all public health, medical and emergency resources will be dedicated to the disaster at hand locally. support from the federal government will be limited in such a scenario; despite the fact, such assistance usually comes during other states of emergency. resource deficiencies will exist for inpatient/outpatient medical services, biologic products, and key personnel. absenteeism among essential first-line medical and emergency workers will impact services rendered, as no one will be immune to infections from the novel pandemic influenza virus. vaccine will be the primary prevention tool, assuming that vaccine will be able to be developed in a timeframe that will be useful. promoting adult immunization programs, including increasing the use of influenza and pneumococcal vaccine during interpandemic years will also strengthen the public health response. the tragic events of september and the subsequent anthrax attacks and other recent threats posed by severe acute respiratory syndrome and monkey pox have created considerable demand on the medical and public health communities nationwide. as a result, unprecedented resources for enhancing our public health preparedness and response infrastructure at all levels of government have been recently provided to all states by congressional appropriations in the form of bioterrorism cooperative agreements administered by the centers for disease control and prevention (cdc). the request for proposals explicitly note that planning moneys may be used ''. . . to upgrade state and local public health jurisdictions preparedness for and response to bioterrorism, other outbreaks of infectious disease, and other public health threats and emergencies. . .'' [ ] . although these funds will be critical for strengthening this country's preparedness and response to bioterrorism, there exists substantial overlap between the public health infrastructure needed to address bioterrorism-related events and other potential public health threats including pandemic influenza. hence, the current climate presents an opportune time to engage in pandemic preparedness planning. taking advantage of the current funding opportunities will not only optimize our response to such a catastrophe, but will help to limit the total burden of disease in terms of morbidity and mortality, economic loss and social disruption caused by an influenza pandemic. by reflecting upon the lessons learned from the influenza pandemic and recent emerging infectious disease catastrophic events, the public health and medical community can work together to develop an effective preparedness and response plan to strengthen our national readiness to respond to an influenza pandemic as well as to strengthen the health system on which the plan depends. the state and local guidelines developed by the state and federal working group [ ] address the various essential components of an infectious disease catastrophic response: surveillance, communication, emergency preparedness, recommendations for distribution of limited biological products, and infection control/medical management. many lessons were learned through the events of september and the aftermath of the attacks using anthrax through the postal service. public health must assume a leadership position in planning effectively and utilizing newly generated resources to optimally prepare for the next public health catastrophe. like the emerging diseases and recurring disease that have occurred over the past several years, we need to think of bioterrorism and the threat posed by influenza pandemic as an emerging and recurring threat, which will probably continue into the foreseeable future. limiting our planning efforts narrowly focused on bioterrorism will be a lost opportunity. in the highly interconnected and readily traversed 'global village' of our time, one nations' problem soon becomes every nation's problem. . . [ ] america's forgotten pandemic: the influenza of notice of cooperative agreement award: guidance for fiscal year supplemental funds for public health preparedness and response for bio-terrorism (announcement number -emergency supplemental) pandemic influenza: a planning guide for state and local officials ( . ) national academy of sciences, institute of medicine, microbial threats to health: emergence, detection and response we wish to acknowledge the members of the ad hoc influenza pandemic conference planning and steering committee for their continuing dedication and contributions to pandemic planning: lynnette brammer, ron burger, zygmunt dembek, kristine ehresmann, john iskander, deva joseph, donna lazorik, ann moen, mack sewell and gregory wallace. key: cord- -lsuwsm authors: jackson, michael l.; neuzil, kathleen m.; thompson, william w.; shay, david k.; yu, onchee; hanson, christi a.; jackson, lisa a. title: the burden of community-acquired pneumonia in seniors: results of a population-based study date: - - journal: clin infect dis doi: . / sha: doc_id: cord_uid: lsuwsm background. pneumonia is recognized as a leading cause of morbidity in seniors. however, the overall burden of this disease—and, in particular, the contribution of ambulatory cases to that burden—is not well defined. to estimate rates of community-acquired pneumonia and to identify risk factors for this disease, we conducted a large, population-based cohort study of persons aged ⩾ years that included both hospitalizations and outpatient visits for pneumonia. methods. the study population consisted of , seniors enrolled at group health cooperative who were observed over a -year period. pneumonia episodes presumptively identified by international classification of diseases, ninth revision, clinical modification codes assigned to medical encounters were validated by medical record review. characteristics of participants were defined by administrative data sources. results. the overall rate of community-acquired pneumonia ranged from . cases per person-years among persons aged – years to . cases per person-years among those aged ⩾ years. in this population, . % of all pneumonia episodes were treated on an outpatient basis. in multivariate analysis, risk factors for community-acquired pneumonia included age, male sex, chronic obstructive pulmonary disease, asthma, diabetes mellitus, congestive heart failure, and smoking. conclusions. on the basis of these data, we estimate that roughly , cases of community-acquired pneumonia occur annually among seniors in the united states and that ∼ of every persons aged ⩾ years will have a new episode of community-acquired pneumonia each year. the risk of pneumonia increases markedly with age, and pneumonia is a leading cause of death among seniors [ , ] . in the united states, an estimated , hospitalizations for community-acquired pneumonia (cap) [ ] and , deaths attributed to pneumonia and influenza occur among seniors every year [ ] . despite the importance of cap in this population, information on the epidemiology of cap among seniors in the united states is limited. in particular, because many prior studies of pneumonia have included only hospitalized patients, the increasingly important contribution of outpatient disease is not well defined. to better assess the burden of cap in seniors and to iden-bases that record immunizations, medication prescriptions, radiographic test reports, and international classification of diseases, ninth revision, clinical modification (icd- -cm)-coded diagnoses associated with outpatient visits and hospitalizations. each member also has a paper medical record that included copies of hospital admission and discharge summaries and outpatient and emergency department visit notes. hospitalizations for pneumonia among the study population had been identified and validated as part of a prior study of pneumococcal vaccine effectiveness [ ] . in that study, hospitalizations assigned a discharge diagnosis of pneumonia (icd- -cm codes - . ) or streptococcal or pneumococcal bacteremia (icd- -cm codes . , . , . , . , and . ) were selected for chart review. cases of nosocomial pneumonia, defined as cases in which pneumonia symptoms developed after hospitalization or in which the patient had been hospitalized in the previous days, were excluded. a confirmed episode of cap involving hospitalization was defined by an indication that, at the conclusion of their clinical evaluation, the treating physicians considered pneumonia to be the etiology of the presenting illness. episodes of pneumonia treated on an outpatient basis were presumptively defined by an outpatient or emergency department visit with a pneumonia icd- -cm code that was associated with both prescription of antibiotics and obtainment of a chest radiograph within days after the visit. an outpatient episode of cap was defined as confirmed if the chart review indicated that pneumonia was the most likely diagnosis attributed to the illness by the treating physician and if the patient had not been hospitalized in the prior days. definition of baseline covariates. to assess potential risk factors for pneumonia among the study population, a series of baseline covariates was defined from ghc administrative data sources and the regional cancer registry. these variables include ischemic cardiac disease, congestive heart failure, chronic obstructive pulmonary disease (copd), asthma without copd, renal disease, stroke, dementia, lung cancer, serious nonlung cancer, other cancer, diabetes mellitus, receipt of prednisone, receipt of other immunosuppressive medications, pneumonia hospitalization in the year prior to cohort entry, use of home oxygen, and receipt of home health care. the full definitions of these variables are provided in table a in appendix a. smoking status was defined on the basis of data routinely collected at ghc during outpatient visits [ ] . information on smoking status was missing for % of study subjects. these subjects differed in several characteristics from persons with known smoking status. alternate analyses were conducted that excluded persons who were missing smoking information, but because the results did not vary substantially from the results of analyses of the entire study population, the results presented include persons with and persons without smoking data. rates of pneumonia. crude event rates were calculated by dividing the number of cases by the cumulative person-time in each age-and sex-specific stratum. crude event rates were based on all events occurring during the study period and could include multiple events per person. to examine seasonal variation in pneumonia rates, biweekly crude rates were calculated by dividing the number of cases occurring in -week intervals by the cumulative person-time in each interval. we plotted these rates over time and compared the pattern of variation during and outside of influenza seasons with the pattern of proportionate mortality from pneumonia and influenza in the united states, as defined by national vital statistics data [ ] . influenza seasons were defined on the basis of local and national surveillance data [ ] [ ] [ ] [ ] . statistical analysis. differences in rates were assessed using the x test. multivariate cox proportional hazards models [ ] were used to evaluate the association of baseline covariates with the time to first of each outcome event during the study period. additional models were fit to test for the presence of an interaction between age or sex and other covariates with the risk of pneumonia. the attributable risk, attributable risk percentage, population-attributable risk, and population-attributable risk percentage of cap cases associated with smoking were all calculated by comparing the crude rates of cap in current smokers with the rates of cap in former smokers and those who have never smoked using standard formulas [ ] . the cohort consisted of , persons who contributed , person-years of observation during the study period. overall, % of the subjects were male, % were aged - years at baseline, and % were aged - years at baseline (table ). hospitalizations for pneumonia. a total of hospitalizations associated with a pneumonia icd- -cm code among persons were identified. information on ( %) of these events was available for review. of these events, were readmissions for pneumonia and were episodes of nosocomially acquired pneumonia. of the remaining hospitalizations, a clinical diagnosis of cap was confirmed for ( %); cohort members accounted for these cases. an additional confirmed hospitalizations for pneumonia were identified by chart review of hospitalizations associated with a bacteremia icd- -cm code. thus, confirmed capassociated hospitalizations among persons were identified during the study period. a total of episodes of presumptive outpatient pneumonia among persons were identified on the basis of administrative data, and records of ( %) of these events were available for review. of these events, episodes were excluded because a record of care for a respiratory illness was not verified, and were excluded because the respiratory illness was associated with a hospitalization. of the remaining events, a diagnosis of cap was confirmed for ( %), which occurred among cohort members. rates of cap and the percentage of cases resulting in hospitalization increased with age (table ). the rate of pneumonia was consistently higher among men than among women, across all age groups and for both hospitalized patients and outpatients with pneumonia, although the difference was not always statistically significant. overall, . % of cap episodes were treated on an outpatient basis. among persons aged у years, the rate of cap was . events per person-years, which is roughly episode of pneumonia for every persons per year. among persons hospitalized for cap, . % died within days after hospital admission. among outpatients with pneumonia, . % died within days after the first diagnosis. overall, . % of all the deaths in the cohort during the study period occurred within days after a cap diagnosis. we found that the peak rates of all cap (both hospitalizations and outpatient visits for pneumonia) coincided with periods of influenza viral circulation (figure ). in addition, the seasonal pattern of cap in our study population closely mirrored the temporal pattern of the percentage of deaths attributed to pneumonia and influenza nationally. similar seasonal patterns were seen in analyses restricted to either outpatients or hospitalized patients, and the proportion of pneumonia episodes requiring hospitalization did not vary between influenza and noninfluenza periods, either for the cohort overall or by age group (data not shown). the attributable risk for smoking among the study cohort was . cap events per person-years, and the attributable risk percentage for smoking was . %. that is, assuming there is a causal relationship between smoking and cap, we estimate that ∼ % of episodes of pneumonia among smokers were due to smoking. the population-attributable risk for smoking (among the population of persons with smoking data) was . cap events per person-years, and the population-attributable risk percentage for smoking was . %. that is, among the population of persons with smoking data, ∼ % of pneumonia events were due to smoking. there was no significant difference in the attributable risks of smoking across age groups or between men and women. in multivariate analysis, age, male sex, current smoking, diabetes mellitus, congestive heart failure, lung cancer, serious nonlung cancer, copd, asthma without copd, dementia, stroke, receipt of prednisone, use of home oxygen services, greater number of outpatient visits, and hospitalization for pneumonia in the year prior to the study start date were independently associated with risk of all cap (table ) . in general, indicators of chronic illness at baseline were more strongly [ ] [ ] [ ] [ ] . for comparison, the biweekly proportion of all deaths in the united states attributed to pneumonia and influenza (p&i), as reported by national vital statistics data, is also presented [ ] . associated with risk of hospitalization for pneumonia than of outpatient visits for pneumonia. the variable most strongly associated with all cap was copd. there was no significant interaction between age or sex and other covariates on risk of pneumonia. to our knowledge, this is the first contemporary study to assess rates of both hospitalizations and outpatient visits for pneumonia among community-dwelling seniors in the united states, and it provides, to our knowledge, the only populationbased assessment of risk factors for cap among seniors in the united states. our study builds on previous population-based studies of hospitalizations and outpatient visits for cap conducted among communities in finland [ , ] and among the ghc population in the s and early s [ ] , each of which had substantially fewer seniors in their study populations. extrapolating our age-specific rates to the population of seniors in the united states who were not in nursing homes, as reported in census data [ ] , we estimate a disease burden of , cases of cap treated on an outpatient basis and , cases of cap resulting in hospitalization, for a total of , cases of cap. these rates also indicate that ∼ of every persons aged у years will have a new episode of cap each year. our rates of hospitalizations for cap are consistent with those reported by marston et al. [ ] in a centers for disease control and prevention-sponsored prospective study of radi-ographically confirmed hospitalization for cap patients in counties in ohio in . after adjusting for differences in the age distribution of persons aged у years in the study populations, our age-adjusted hospitalization rate of . cases per person-years was nearly identical to the rate of . cases per person-years among community-dwelling seniors reported by marston et al. [ ] . the ohio study did not, however, include an assessment of outpatient visits for cap, which is particularly important, given the trend towards outpatient treatment that has occurred over the past decades. we found that, even among persons aged у years, nearly one-half ( . %) of cap events were treated on an outpatient basis. inclusion of outpatient episodes allows a more comprehensive estimate of the burden of disease and a more accurate assessment of risk factors for cap. persons with chronic conditions who develop cap are more likely to be hospitalized for treatment than are persons without chronic conditions. this means that assessments restricted to hospitalizations for cap will likely overestimate the association of those conditions with the risk of cap. the availability of administrative data on the ghc population allowed us to define the presence of underlying conditions consistently across the study population. because of this, we were able to assess the independent association of those characteristics with risk of pneumonia in multivariate analyses. as previously reported among adults, we found that copd, immunosuppression, smoking, and congestive heart failure were all independently associated with disease risk among sen- iors [ ] [ ] [ ] [ ] [ ] . in addition, we also found that diabetes, lung cancer, serious nonlung cancer, and previous hospitalizations for pneumonia were also risk factors for cap among elderly persons. in this cohort, rates of cap were higher in men than in women, and male sex was a risk factor for cap even after adjusting for age, smoking, and the presence of chronic medical conditions. a survey of hospitalizations associated with a pneumonia icd- -cm code reported in medicare claims [ ] found a significantly higher risk among men than among women, as did a follow-up assessment of the risk of a hospitalization associated with a pneumonia diagnosis among participants of the first national health and nutrition examination survey (nha-nes i) [ ] . neither of these studies assessed whether male sex was associated with pneumonia after adjusting for other risk factors, so it was not clear in these studies whether male sex was simply a marker for a higher prevalence of other risk factors. a population-based study involving elderly persons in finland found that crude cap rates did not differ between men and women and that male sex was not significantly associated with cap after accounting for age and certain chronic medical conditions and risk factors, including asthma, receipt of immunosuppressive therapy, and heart disease [ ] . because our study population was much larger than that of the finnish study, we have more power to detect an independent risk of cap associated with male sex if such a risk truly exists. however, it is possible that the difference in risk we observed could be the result of confounding by factors unmeasured in our population. we found that current smokers were at an increased risk of hospitalization for cap, which is consistent with previous reports. previous case-control studies have shown that smoking is associated with an increased risk of pneumonia [ , ] . one study has reported that, among adults who quit smoking, the excess risk of cap appears to have decreased years after quitting [ ] . we estimate that, among elderly smokers, nearly one-third of pneumonia episodes can be attributed to smoking, suggesting that smoking cessation could potentially reduce the risk of cap among smokers by an important degree. among our study population, the seasonal pattern of pneumonia closely followed the pattern of pneumonia and influenza mortality nationally. correlations between rates of hospitalization for acute respiratory disease and patterns of influenza activity have been reported in adult populations in other geographic areas [ ] [ ] [ ] . the - influenza season is recognized as having been more severe than the several preceding seasons [ ] , and timing of the peak rate of cap for the elderly ghc population in this study period closely matched the peak proportional pneumonia and influenza mortality rates in the united states, occurring in late december and early january . this correlation suggests that data from health management organizations (hmos) on pneumonia-related hospitalizations and outpatient visits could be an additional method for identifying periods of influenza virus circulation or quantifying the severity of influenza seasons. rates of cap in this population were high, especially among some subgroups of subjects, such as those aged у years, and there was close correlation with influenza virus circulation. this is despite the fact that vaccination rates for both influenza and pneumococcal polysaccharide vaccines were high in this population. annual influenza vaccination rates among the study population were %, and % of subjects received pneumococcal vaccine either prior to or during the study period, emphasizing the need for other measures for preventing pneumonia in seniors. the burden of illness could potentially be reduced by the availability of other vaccines, such as pneumococcal conjugate vaccines or vaccines effective against human metapneumovirus or respiratory syncytial virus infection. alternatively, increased emphasis on other strategies to reduce the risk of influenza infection, such as vaccination of caregivers and household contacts, could potentially impact disease risk. one limitation of this study is the possible underascertainment of pneumonia events, because our case finding was primarily restricted to medical encounters assigned a pneumonia icd- -cm code, and so would not capture pneumonia episodes associated exclusively with other diagnosis codes. for outpatient pneumonia, we would also have missed episodes in which a chest radiograph was not ordered. we cannot estimate the extent to which underascertainment may have influenced our results. however, this problem is likely to be more significant for pneumonia treated on an outpatient basis (in which a health care provider may only see a patient early in the course of the illness and assign a diagnosis before the evaluation has been completed) than in cases in the hospital setting (in which the discharge diagnoses are assigned at the end of the hospital stay and should represent a more complete assessment of the illness episode). a second limitation is that the study population includes enrollees of a single health maintenance organization in a single geographic area, and the findings may not be generalizable to the us population as a whole. as noted above, however, our age-standardized rate of cap-related hospitalizations was essentially identical to that observed in the study of cap in ohio [ ] . this similarity, along with the close correspondence between the observed rates over time in our study and the national proportional rates of mortality due to pneumonia and influenza, suggest that our study population is not dissimilar from the elderly population of the united states as a whole. our method for detection of both hospitalizations and outpatient visits for cap among a defined population of hmo enrollees has several advantages for conducting pneumonia surveillance. this type of surveillance can identify high-risk subgroups, can track trends in pneumonia incidence over time, and is suitable for both prospective and retrospective studies. surveillance of this type would be particularly useful for studying the effect of new vaccines or the introduction of new pathogens, such as severe acute respiratory syndrome coronavirus, on rates of pneumonia. incidence of communityacquired pneumonia requiring hospitalization. results of a populationbased active surveillance study in ohio. the community-based pneumonia incidence study group deaths: final data for hospitalized community-acquired pneumonia in the elderly: age-and sex-related patterns of care and outcome in the united states deaths: preliminary data for effectiveness of pneumococcal polysaccharide vaccine in older adults vital statistics mortality data, multiple cause detail update: influenza activity-united states and worldwide, - season, and composition of the - influenza vaccine update: influenza activity-united states and worldwide, - season, and composition of the - influenza vaccine update: influenza activity-united states and worldwide, - season, and composition of the - influenza vaccine update: influenza activity-united states and worldwide, - season, and composition of the - influenza vaccine time-dependent covariates in the cox proportionalhazards regression model epidemiologic methods: studying the occurrence of illness incidence of communityacquired pneumonia in the population of four municipalities in eastern finland clinical efficacy of pneumococcal vaccine in the elderly: a randomized, single-blind populationbased trial rates of pneumonia during influenza epidemics in seattle census briefs: the years of age and older population prospective study of pneumonia hospitalizations and mortality of us older people: the role of chronic conditions, health behaviors, and nutritional status risk factors for community-acquired pneumonia in adults: a population-based case-control study risk factors for community-acquired pneumonia diagnosed upon hospital admission. british thoracic society pneumonia study group risk factors for community-acquired pneumonia diagnosed by general practitioners in the community risk factors for pneumonia in the elderly proportion of community-acquired pneumonia cases attributable to tobacco smoking serious morbidity and mortality associated with influenza epidemics acute respiratory disease hospitalizations as a measure of impact of epidemic influenza influenza-associated morbidity and mortality in young and middle-aged women financial support. vaccine safety datalink contract with the america's health insurance plans, funded by the centers for disease control and prevention.potential conflicts of interest. k.m.n. has received grant support from aventis-pasteur. l.a.j. has received grant support from aventis-pasteur and has served as a consultant to chiron vaccines. all other authors: no conflicts. key: cord- - xhusfth authors: lee‐baggley, dayna; delongis, anita; voorhoeave, paul; greenglass, esther title: coping with the threat of severe acute respiratory syndrome: role of threat appraisals and coping responses in health behaviors date: - - journal: asian j soc psychol doi: . /j. - x. . .x sha: doc_id: cord_uid: xhusfth the present study examines the psychological impact of severe acute respiratory syndrome (sars) by exploring the coping strategies and health behaviors enacted in response to the sars epidemic. hierarchical linear regression indicated that the use of wishful thinking in response to the threat of sars was related to both avoiding public places and avoiding people perceived to be possible carriers of the sars virus, but was not associated with the use of more adaptive health behaviors, such as using disinfectants and hand washing. conversely, those who reported engaging in empathic responding in response to the threat of sars were both less likely to report avoiding people perceived as being at a high risk for sars and more likely to report engaging in effective health behaviors. support seeking was not a significant predictor of the health behaviors examined in the present study. results are discussed in terms of coping with health threats and health promotion. increased understanding of severe acute respiratory syndrome (sars) is necessary for researchers, health-care providers and the general public alike. if we are to prevent the spread of disease and reduce its far-reaching effects, there must be knowledge of not only the virus itself, but also of the social and psychological sequelae of the disease. furthermore, if we are to prevent the spread of disease, study of the impact of sars cannot be limited to those relatively few who have actually contracted sars; it must also include the general public's reactions to the disease. that is, how has the general public reacted psychologically, socially and behaviorally to news of the disease? to what extent does the general public feel threatened by the possibility of the disease spreading, and how do they cope with these fears? do their ways of managing their fear of sars affect their ability to engage in adaptive health behaviors? understanding ways in which the general public's coping with their sars-related fears affects their ability to engage in preventative health behaviors is critical. such knowledge would potentially be applicable, not only in the face of the sars outbreak, but also in general health promotion. one of the most significant effects that sars has had on the general public has been a change in health-related behaviors (bray, ) . these changes were multi-faceted and encompassed everything from frequent hand washing and the use of face masks to complete isolation from the outside world. as evidenced, in part, by the economic fallout of the disease, a common behavior change, particularly in affected areas, was to avoid public venues such as restaurants, marketplaces and airports. although this sort of behavior is undoubtedly associated with decreasing one's risk of exposure to the virus, it was associated with significant economic costs without a correspondingly large reduction in risk (cdc, a; who, a) . another behavioral response to reports of the outbreak was to limit contact with people perceived to be at risk for the virus. this included people who displayed symptoms of the common cold (e.g. coughing or sneezing), workers in the health-care profession, and individuals assumed to have a high likelihood of having sars by virtue of their ethnicity, nationality, or recent travel history. given that the largest outbreak of sars occurred in asian countries, north americans may have grouped those of asian ancestry within this last category. similarly, being canadian (or torontonian in particular) was viewed by some as evidence of being high risk by those outside of canada or north america. avoiding people perceived to be at risk for sars was unlikely to be effective from a health-care perspective, and certainly can be viewed as detrimental from a psychological and social perspective. such behaviors no doubt lead to ostracism of those believed to be at risk. when associated with certain ethnic or racial groups, the obvious result is racism. a third possible behavioral response to the outbreak was to engage in preventative health behaviors as prescribed by the world health organization (who), the center for disease control (cdc) and a variety of other health-care units, hospitals and individual health-care providers (e.g. cdc, a cdc, , b cdc, , c nbc .com, ; who a) . suggestions were widely publicized via television, websites, and lay-oriented news reports indicating that behaviors such as taking more care with cleanliness, using disinfectants, washing hands more regularly, eating well and getting enough sleep were reasonable and useful preventative measures in which to engage. not only were such strategies likely to be helpful in limiting the spread of sars, they also have few negative side effects at both an individual and societal level. although numerous models for the prediction of health behaviors have been put forth (fishbein et al. , ) , few of them consider threats to health as a potentially stressful experience, as is likely to be the case with the sars epidemic. when individuals are exposed to media reports of sars, they may experience an increase in anxiety and threat associated with those reports. how these individuals cope with that threat may either facilitate or inhibit their engagement in the aforementioned health behaviors, which, in turn, may directly alter the risk of disease. examining the role of coping in health behaviors may inform public health efforts to encourage protective health behaviors. models of stress and coping with disease (lazarus & delongis, ; delongis & o'brien, ) provide a useful framework through which to understand the ways in which threats to health, coping and health behaviors are related. these models emphasize the transactional or interactional nature of the stress and coping process and suggest that when people are exposed to stressors, such as news of the sars epidemic, an evaluative thought process is triggered in which they consider whether or not the stressor is a threat to their own well-being. this process in turn prompts an evaluation of options for coping with the perceived stressor. this, then elicits an array of coping behaviors in an attempt to manage various aspects of the stressor. the literature on coping with illness suggests a number of strategies for dealing with illness-related stressors that might meaningfully be applied to the current sars crisis (o'brien & delongis, ) . these include wishful thinking, support seeking and empathic responding. wishful thinking refers to the individual's efforts to cognitively escape from or avoid the situation by wishing, fantasizing or hoping it goes away or is somehow over. wishful thinking has been found to be associated with negative outcomes, such as depression, anxiety, increased stress-related physical symptoms, and poor adjustment to illness (penley et al. , ) . however, we know of no study examining the role of wishful thinking in the implementation of health-related behaviors. support seeking involves efforts to gain emotional, informational or tangible support from others. although there is a strong and consistent association between social relationships and positive health outcomes (coyne & delongis, ; house et al. , ) , there has been little research examining the association of support seeking with engaging in protective health behaviors. finally, empathic responding is a mode of coping that has recently begun to receive attention in the stress literature. individuals engaging in empathic responding try to understand what others are experiencing and offer support and assistance. empathic responding may provide benefit to the recipient of these efforts (o'brien & delongis, ), but, perhaps even more critical, evidence suggests that engaging in empathy and support can provide benefits for the provider as well (brown et al. , ; kramer, ; visitini et al., ) in terms of improved psychological well-being, physical health, and relationship satisfaction. however, we are aware of no research examining the relationship of empathic responding to health behaviors. we sought to examine ways in which the perception of the threat of sars was related to coping and, in turn, how coping was related to health behaviors. understanding the ways in which coping is related to engaging in health behaviors may be critical to encouraging effective health behaviors in the face of health crises such as sars. it was expected that perceived threat of sars would be associated with higher reported frequency of engaging in a variety of coping strategies in an attempt to deal with feelings of threat and fear. specifically, these were expected to include wishful thinking, support seeking and empathic responding. finally, we expected specific coping strategies to be instrumental in facilitating specific health behaviors. that is, given the avoidant nature of wishful thinking as a coping strategy, it seemed reasonable to assume that the use of such a strategy for managing the threat of sars might be associated with avoidance-type health behaviors. here, we expected © blackwell publishing ltd with the asian association of social psychology and the japanese group dynamics association higher levels of wishful thinking to be associated with higher reports of avoiding both public places and individuals perceived to be at risk for sars. finally, empathic responding involves considering the stressful experience not only in terms of one's own feelings and well-being, but also in terms of other's well-being. given the use of empathy, one might be less likely to consider other people as objects to be avoided but rather as people needing and requiring care. without the impulse or need to engage in avoidant behaviors, efforts for engaging in health behaviors may be turned towards more preventative and effective health behaviors such as hand washing. no specific hypotheses were made regarding the relation of social support seeking to health behaviors. although we expected those reporting higher perceived threat of sars to engage in higher levels of support seeking, we had no clear expectations regarding which health behaviors might be facilitated by such a coping strategy given the paucity of previous research. data were collected through an online questionnaire linked to a number of websites, including psychology websites, the laboratory websites of the second and fourth authors and their colleagues, and advertisements on google, a search engine. the google advertisement popped up when individuals searched for information on 'sars'. it asked viewers to complete an online questionnaire about how they were coping with the sars threat. all of the links first took potential participants to an informed consent page. once they indicated they had read the consent form and consented to participate, they were taken to the questionnaire. the questionnaire was written in english. data included here are from questionnaires completed between june and september . sample. seventy-one percent of the sample resided in canada, % of these in toronto. other countries of residence included china, costa rica, germany, hong kong, singapore and the usa. approximately % of the respondents were living in a sars-affected area. fortythree percent reported canada as their country of birth. the majority of the respondents (rs) were under years of age ( %), female ( %) and had over years of education ( %). approximately half the sample was composed of students. other occupations included healthcare workers, engineers, psychologists and office workers. ethnicity was not included due to the difficulties in coming up with a suitable classification system that could be used in a worldwide survey. the sars collaborative research group (including the second and fourth authors) jointly developed the main questionnaire. only those variables related to the current study will be discussed. some of the measures used in the present study were from this original questionnaire (the health behaviors component, demographic information and the support seeking and wishful thinking coping items). other measures (perception of sars threat, empathic coping items, and state anxiety) were added to the questionnaire specifically for use in the study described here. state anxiety. feelings of current anxiety were assessed through an updated version of spielberger et al. 's ( ) measure of state anxiety. respondents were asked to rate their current feelings related to sars on a -point scale ranging from 'not at all' to 'very much so'. the scale consisted of items including such items as 'i feel tense', 'i feel upset', 'i feel nervous'. consistent with previous reports (gaudry et al. , ) internal reliability of the scale in the present study was high (cronbach's alpha = . ). perception of sars threat. participants were asked to rate the extent to which the following statements were true for them at the current moment on a -point scale ranging from 'not at all' to 'a great deal'. the five items were; 'i don't think i could get sars', 'i feel nervous about getting sars', 'sars is threatening my health', 'i don't feel worried about getting sars', 'my daily routine has been disrupted due to thoughts about sars'. internal reliability of the scale was moderate (alpha = . ). ways of coping. items were chosen from the ways of coping questionnaire (folkman et al. , ) that tapped strategies for coping that were applicable to coping with the threat of sars. items from two subscales were included in the present study (wishful thinking and support seeking). in addition, items from the relationship-focused coping scale (empathic responding; o'brien & delongis, ) , which were applicable to the current study, were also included. instructions to respondents for the coping items were 'to what extent have you managed whatever concerns or fears you might have about sars in each of the ways listed below?' wishful thinking. rs were asked the extent to which they had managed their concerns or fears about sars through 'wishing sars would go away or somehow be over with' on a -point scale ranging from 'not at all' to 'a great deal'. support seeking. support seeking was assessed by asking rs to rate the extent to which they had managed whatever concerns or fears they had about sars by 'talking to someone to find out more about sars' and 'talking to someone about how i was feeling about sars' on a -point scale from 'not at all' to 'a great deal'. the reliability of the scale was high (alpha = . ). empathic responding. respondents were asked to report the extent to which they had helped others who might be concerned about getting sars on a -point scale ranging from 'not at all' to 'a great deal'. the four items included: 'tried to understand the other person's concerns about sars', 'tried to understand how the other person felt about sars', 'tried to help the other person(s) by listening to their concerns about sars', and 'tried to help the other person(s) by doing something for them'. consistent with past research (o'brien & delongis, ) , reliability of the scale in the present study was high (alpha = . ). health behaviors. as noted above, items were taken from the web questionnaire on sars developed by members of the sars collaborative research group . instructions to respondents for the health behavior items were 'to avoid getting sars, i have personally…' and specifically for the avoiding people subscale, 'how likely are you to avoid the following people?' avoiding public places. respondents were asked to identify behaviors in which they had engaged to avoid getting sars. the possible behaviors were: avoided travel to sarsinfected areas, avoided eating in restaurants, avoided shaking hands, avoided travel in taxis, avoided travel on subways or commuter trains, avoided eating in food courts/food centers, not gone to work/school, avoided large gatherings of people, avoided particular types of people, and avoided travel by airplane. the number of responses, based on the response alternatives checked, was summed for a final scale. reliability of the scale was moderately high (alpha = . ). avoiding people. respondents were asked to indicate, using a -point scale ranging from 'very unlikely' to 'very likely', the degree to which they avoided people who might be perceived as having a higher risk of having been exposed to the sars virus. the items were: a person you know has just come from an area infected with sars, a person who has a fever, a person who sneezes, a person who looks unwell, a health-care worker, a person who is coughing, a person who you think might possibly be from an area infected with sars, a person who has a family member who has come down with sars, a stranger wearing a surgical/hygiene mask, and a stranger not wearing a surgical/hygiene mask. the final scale consisted of the sum of the checked alternatives. reliability of the scale was high (alpha = . ). taking health precautions. respondents were asked to identify behaviors in which they had engaged to avoid getting sars. these eight health behaviors included: wearing a mask, washing hands more often, taking more care about cleanliness, using disinfectants, eating a balanced diet, exercising regularly, taking an herbal supplement, and making sure they got sufficient sleep. the number of items endorsed was summed into the 'taking precautions' scale. reliability of the scale was moderately high (alpha = . ). table presents descriptive statistics for the study variables. t -tests were conducted examining gender differences in the study variables; the only significant difference to emerge was that women in the study were significantly more likely to report seeking social support than were men ( t ( ) = - . , p < . ). t -tests were also conducted to examine differences between those participants living in sars-affected ( n = ) versus sars-unaffected ( n = ) areas. no significant differences emerged on any of the study variables. the most significant time for the sars outbreak was before mid-july (who, b) . however, given that data collection continued until september, t -tests were conducted examining whether there were significant differences between responses reported before and after mid-july on the study variables. there were no significant differences due to time of data collection (adjusting for the number of tests conducted). table also presents the bivariate correlations among study variables. perception of sars threat was significantly related to reports of both coping (wishful thinking and support seeking) and health behaviors (avoidant behavior, avoiding people perceived to be at risk for sars and taking precautions). as shown in table , rs who reported feeling threatened by sars were also more likely to report wishing sars would go away and to seek support from others to deal with their perceptions of threat. those higher on threat were also more likely to report engaging in avoidant behavior, such as avoiding public places and avoiding people perceived to be at a higher risk of having been exposed to the virus, such as healthcare professionals or people who looked ill. finally, they were more likely to report engaging in such preventative health behaviors as washing their hands and getting sufficient rest. the correlations between coping and health behaviors are presented in table . wishful thinking, support seeking and empathic coping were significantly and moderately intercorrelated ( r 's from . to . ). the intercorrelations among the abbreviated forms of the coping subscales used here are similar in size to those reported in previous studies using the full scales (folkman et al ., ) . as shown in table , rs who tended to wish that sars would go away as a way of dealing with the threat of sars, also tended to report avoiding both public places, such as markets or restaurants and individuals perceived to have a higher likelihood of being exposed to the sars virus. these rs also tended to report engaging in health precautions, such as using disinfectants and taking more care with cleanliness. rs who reported seeking social support in response to the threat of sars were also likely to report avoiding people perceived to be at high risk of sars, such as health-care workers, and engaging in health behaviors, such as eating a balanced diet and exercising regularly. however, these rs were only slightly more likely to report avoiding public places as a response to the sars threat ( r = , p < . ). rs high on empathic responding tended to report avoiding a variety of public places in an effort to avoid exposure to sars. furthermore, those high on empathic responding tended to report engaging in more protective health behaviors, such as hand washing, in their efforts to prevent spread of the disease. however, these bivariate relations are difficult to interpret given the potential confounding of coping and health behaviors with perceptions of the threat of sars and with state anxiety. multivariate analyses were conducted controlling for perception of threat and levels of anxiety, as well as scores on all coping scales, in order to allow a more meaningful picture of the independent associations of each form of coping to each of the three health behaviors examined here. table presents the results of three hierarchical linear regression analyses. these were used to examine the relationship between coping and perceived threat of sars to each of the three types of health behaviors examined in the study, avoiding public places, avoiding people and taking precautions. state anxiety was entered in step of the equation, perception of sars threat in step and the three coping strategies (wishful thinking, support seeking and empathic responding) as a set in step . both wishful thinking ( b = . ), t ( ) = . , p < . and perception of sars threat ( b = . ), t ( ) = . , p < . were significantly positively associated with avoidance of public places even after controlling for state anxiety, support seeking and empathic responding. that is, rs who reported engaging in more wishful thinking to cope with the threat of sars were more likely to report avoiding public places as a way to reduce exposure to the sars virus. sars threat ( b = . ), t ( ) = . , p < . and wishful thinking ( b = . ), t ( ) = . , p < . were significantly associated with a greater likelihood of avoiding people. this held even after controlling for state anxiety, support seeking and empathic responding. empathic responding was associated with significantly lower reports of trying to avoid other people (b = - . ), t( ) = - . , p < . , again controlling for state anxiety, perception of sars threat and the other coping strategies. in other words, rs who reported the use of wishful thinking as a way of coping with the threat of sars were more likely to report avoiding people perceived to be at risk for having sars. however, rs who reported the use of empathic responding were less likely to report engaging in such behavior, once threat of sars and anxiety were controlled. finally, sars threat (b = . ), t( ) = . , p < . and empathic responding (b = . ), t( ) = . , p < . were significantly associated with taking precautions, even after controlling for state anxiety and ways of coping with sars. that is, rs who reported responding to the threat of sars through empathic responding were more likely to report taking health precautions to decrease the risk of getting sars. in sum, multivariate analyses controlling for differences in perceived threat of sars, state anxiety and other ways of coping indicated that both wishful thinking and empathic responding were significantly associated with specific sars-related health behaviors. controlling for differences in perceived threat of sars, state anxiety and other ways of coping, support seeking was not significantly related to the sars-related health behaviors examined in the present study. the current study examined the psychological impact of sars by examining the coping strategies and health behaviors reported in response to the sars outbreak. consistent with the study by tam et al. (this issue) and that by chang and sivam (this issue) , the present findings revealed a link between cognitions and coping responses. our findings suggest that feeling threatened by sars is associated with the use of such coping strategies as wishful thinking and support seeking. it further suggests that patterns of coping with the threat of sars are associated with engaging in specific health-related behaviors intended to reduce the risk of infection. interestingly, this pattern of findings appeared to remain the same regardless of gender, education, and whether the individual was from a sarsaffected area. specifically, those participants who reported engaging in wishful thinking in response to the threat of sars appear more likely to engage in two forms of avoidant health behavior: avoiding public places and avoiding people perceived to be possible carriers of the sars virus. however, those engaging in wishful thinking regarding the virus did not describe themselves as engaging in more of the sort of health behaviors most likely to be viewed as effective by health-care professionals. that is, wishful thinking does not appear to facilitate engaging in critically important health behaviors, such as hand washing and using disinfectants to clean potentially contaminated surfaces. conversely, those who reported the use of empathic responding were not only less likely to report avoiding people who may be perceived as potentially having sars but also more likely to report engaging in precautionary measures and health behaviors likely to be viewed as effective (cdc, a (cdc, , b (cdc, , c who, a) . hence, those who report using empathic responding in response to sars appear to use effective precautionary health behaviors without engaging in avoidant health behaviors that were associated with significant economic and societal costs. consistent with our expectations, it may be that using empathy and perspective-taking to cope with threatening events inhibits a knee jerk reaction to simply avoid anyone who is perceived as being a possible threat. empathic responding may inhibit individuals from viewing the public at large and the places in which they can be found as a primary threat and therefore something to be avoided. as such, health behavior efforts may be geared more toward prevention than avoidance. the present study suggests that those who can respond empathically to others even in the presence of health threats may be better at engaging in productive health behaviors that may contribute to their own and to others' health and well-being. although perception of sars threat was associated with the use of support seeking, this coping strategy was not significantly associated with engaging in the sars-related health behaviors examined in this study, controlling for the other variables examined. there are several possible reasons for this. it may be that we did not have sufficient power to detect an effect given the relatively small sample size. support seeking may also be related to other outcomes not examined in the present study such as mood or feeling comforted, and it may be that any relation between support seeking and health behaviors is indirect via these more direct effects of social support. finally, as is observed in the general literature on social support, the effect of support seeking on outcomes may depend upon the response of others (lehman et al., ) . that is, others may not always respond favorably to requests for support. previous research has indicated that even when support is offered to another, it is not always perceived as helpful. for example, cancer patients report that a sizable proportion of the support that is offered to them fails to provide the comfort or aid that was presumably intended (dakof & taylor, ) . in the context of health behaviors, support may not always be sufficient to promote effective health behaviors. consequently, it may be that we need to examine the interactional patterns between the person eliciting support and the recipient of the support requests if we are to understand the ultimate effects of support seeking. an obvious limitation of the present study is the cross-sectional nature of the data that does not allow the examination of changes over time or processes. the understanding of how individuals cope with sars would be aided by longitudinal data permitting, for example, examination of the endurance of health behavior changes or how fluctuations in perceptions of threat, perhaps related to media reports, are related to changes in health behaviors. furthermore, the sample was collected through the internet using self-report data and thus is subject to the biases and limitations of self-report data and selective sampling due to the use of the internet. benefits of the internet as a collection tool include cost-effectiveness and the enhanced reach of sampling demographics. additionally, there are recommended strategies to avoid such problems as data falsification and data security issues (smith & leigh, ) . however, care must be taken to be aware of the medium's limitations as well. the question of validity is an important one. studies comparing data collected in traditional formats, such as pencil-and-paper (schwarzer et al., ) and telephone survey (chang, ) have found that internet studies have high validity and yield similar results to data collected in more traditional mediums. in fact in some cases (chang, ; murray & fisher, ) , internet surveys have shown higher predictive validity and better psychometric properties than more traditional mediums. however, it is important to note that, as with many mediums, data collected from the internet cannot be assumed to be representative of the general population. according to the american internet user survey, differences include internet users generally being younger, wealthier and more educated than average survey participants (national science foundation, ) . another notable limitation of the present study is that it examined only a few possible coping strategies using brief measures. there may be other coping strategies important to understanding the psychological and behavioral responses to the threat of sars, such as problem-focused coping. additionally, despite examining the role of education, gender and geographic region and failing to find significant differences, our sample is reflective of a well-educated, young, female population mostly residing in canada, which is unlikely to be representative of populations in other countries. given the international nature of sars and its impact, future research should examine a broader sample to see the extent to which the results identified in the present study generalize to other populations. understanding cultural and geographic differences in coping strategies and health behaviors may facilitate designing more effective health promotion campaigns and media messages focusing on those messages with wider applicability across divergent groups. finally, the present study did not examine the impact of coping on close others. for example, network members may both facilitate and hinder health behaviors as well as impact and be impacted by the coping of another. the current study examined the individual in isolation, which does not capture the interpersonal nature of coping in general or of the sars threat in particular. while sars may be transmitted via body fluids person to person, the threat and fear associated with the disease surrounding it travel even faster from person to person. this threat may ultimately be just as damaging to society and individual well-being as the disease itself is to the health of those infected. given the significant relationships that emerged between coping and health behaviors, the present study highlights the importance of considering coping in managing health threats and in encouraging and discouraging various health behaviors (see also chang & sivam, this issue; gan et al., this issue) . the avoidant behaviors reported in the present study (avoiding public places and people perceived to be at a higher risk for having sars) and the study by gan et al. (this issue ) may be associated with significant economic and societal costs to areas affected by sars. for example, the ontario government reported that sars-related costs, including lost revenue related to decreased tourism and commerce, and assistance for individuals, the health-care system and economic recovery totalled $ . billion can © blackwell publishing ltd with the asian association of social psychology and the japanese group dynamics association (ontario government, b) . in communities across canada, community leaders called for awareness programs to address what they saw as a growing aversion towards the asian community (e.g. ontario government, a; rider, ) . understanding that wishful thinking in response to the threat of sars is related to such avoidant behaviors offers the possibility of minimizing the use of this coping strategy and the resultant economic and societal impact this form of coping has had on sars-affected cities around the globe. similarly, the results reported here in relation to avoiding people have significant implications for managing public perceptions of disease threats, particularly when individuals being avoided may belong to a particular ethnic group or groups of health-care professionals. the latter may inadvertently serve to reduce the probability of preventative treatment. having this knowledge can assist public health officials in promoting positive health behaviors while at the same time assisting them in promoting accurate knowledge that will not result in the targeting of particular individuals. notably, our findings suggest methods of coping, such as empathic responding, which might usefully be encouraged via mass media campaigns to both increase effective preventative health behaviors and decrease behaviors that are likely ineffective at best, and potentially damaging to society at worst. it may be important to encourage the use of empathy in coping with health-threatening diseases such as sars in our public health messages given that such coping strategies are not associated with higher feelings of threat and are associated with better health behaviors. given the global impact of sars on health, perceptions of threat, economic functioning, and societal stability, understanding the psychological impact of sars is critical in our attempts to manage the disease and the public reaction to it. knowledge of the psychological reaction to sars may help us create more effective and productive health messages aimed at limiting the detrimental effects such diseases can have on the well-being of society, even among those many who never contract the disease. linda d. cameron (university of auckland), lois c. friedman, mary poon (san francisco general hospital), nicholas difonzo (rochester institute of technology), noelle leonard, prashant bordia (university of queensland), rima styra (university health network), stefano occhipinti (griffith university) and winnie w.s. mak (chinese university of hong kong). . due to the collaborative nature of the questionnaire, there were limitations on the number of items that could be included. given the space restrictions, only one wishful thinking item was included. . while state anxiety may also be viewed as a dependent variable, the current study sought to examine health behaviors as the outcome variable. given this goal, state anxiety was controlled for in the analyses to ensure that the relationships observed between coping and health behaviors were not simply due to their shared variance with general state anxiety. further, in examining the relationship of threat of sars to health behaviors, we wanted to control for the respondent's general level of anxiety, so that we could examine the specific effects of sars-related fears on health behaviors. . we also ran a series of hierarchical linear regression analyses controlling for the effects of education, gender, affected versus non-affected sars region, and the interactions between coping and gender, coping and affected/non-affected areas, and coping and time of responding (e.g. before or after mid-july). the pattern of findings regarding the relationship of coping to health behaviors in these analyses was identical to those reported here. . a regression model was also run with the two items tapping social support (instrumental and emotional) entered separately. the effects of social support on health behaviors remained nonsignificant in these models, regardless of whether the two items were entered together in the same equation or in separate equations. anne marie vartti (finnish national public health institute), arja r aro (erasmus medical center living in the midst of a killer virus providing social support may be more beneficial than receiving it: results from a prospective study of morality hand hygiene in healthcare settings interim guidance on infection control precautions for patients with suspected severe acute respiratory syndrome (sars) and close contacts in households a comparison of samples and response quality obtained from rdd telephone survey methodology and internet survey methodology constant vigilance: heritage values and defensive pessimism in coping with severe acute respiratory syndrome in singapore going beyond social support: the role of social relationships in adaptation victims' perceptions of social support: what is helpful from whom an interpersonal framework for stress and coping: an application to the families of alzheimer's patients factors influencing behavior and behavior change dynamics of a stressful encounter: cognitive appraisal, coping and encounter outcomes flexible coping responses to severe acute respiratory syndromerelated and daily life stressful events validation of the state-trait distinction in anxiety research social relationships and health exapanding the conceptualization of caregiver coping: the importance of relationship-focused coping strategies psychological stress and coping in aging negative and positive life changes following bereavement and their relations to adjustment the internet: a virtually untapped tool for research the application and implications of information technologies in the home: where are the data and what do they say? available at coping with chronic stress: an interpersonal perspective commission urges tolerance and respect during the sars health emergency. news release the association of coping to physical and psychological health outcomes: a meta-analytic review fear of virus fuels racism: ontario must do more to stop return to days of 'yellow peril', asian leaders say assessment of perceived general self-efficacy on the internet: data collection in cyberspace virtual subjects: using the internet as an alternative source of subjects and research environment manual for the state-trait anxiety inventory -form x biases in the perceived prevalence and motives of severe acute respiratory syndrome prevention behaviors among chinese high school students in hong kong psychological stress in nurses' relationships with hiv infected patients: the risk of burnout syndrome frequently asked questions on severe acute respiratory syndrome (sars) this research was supported by a sshrcc grant to the second author and a sshrcc and michael smith doctoral fellowship to the first author. we would like to thank our colleagues in the sars collaborative research group, particularly george bishop, for input regarding the design of this study. key: cord- - qyee authors: mase, m.; tsukamoto, k.; imai, k.; yamaguchi, s. title: phylogenetic analysis of avian infectious bronchitis virus strains isolated in japan date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: qyee to define the origin and evolution of recent avian infectious bronchitis virus (ibv) in japan, a genetic analysis was performed. by phylogenetic analysis based on the s gene including the sequence of the hypervariable regions, ibv isolates in japan were classified into five genetic groups, which included two already-known groups (mass and gray). among them, three major genetic groups were associated with the recent outbreaks of ib in japan. one group is indigenous to japan and could not be placed within the known existing groups in other countries. the remaining two groups, which have emerged recently, are related to isolates in china and taiwan. m. mase et al. post-translational cleavage of two separate polypeptide components, designated s and s [ ] . of these, the s glycoprotein is associated with virus attachment and is a major target of the neutralizing antibodies in chickens, so serotypic evolution in ibv is associated primarily with the sequences of the s glycoprotein [ , ] . hence, the molecular characterization of ibv is based mainly on analysis of the s gene [ , ] . classically, ibv strains have been placed into serologically related groups on the basis of virus neutralization tests (vnt). however, vnts are very laborious and time-consuming. in addition, there is considerable evidence that serologic relationships among ibv isolates, determined by in vitro vnts, have not been reflected by the results of in vivo cross-immunity studies [ ] . moreover, the antigenic variants emerging from wild-type or vaccine viruses by point mutation or rna recombination cannot be detected by vnts [ ] . recently, genetic grouping of ibv on the basis of nucleotide sequences have been applied for virus classification [ , ] . since anyone can apply the genetic information of viruses deposited into the genbank, genetic comparison with other virus strains for epidemiological analysis can be performed quickly. since the first isolation of ibv was recorded in japan in [ ] , the outbreaks have been ongoing. however, the epidemiological analysis of ibv isolates in japan has not been thorough except for a few strains [ , ] . the relationships between japanese ibv isolates and foreign ibv isolates have also remained unknown. we were particularly interested to know whether the current ibv isolates in japan were newly introduced from other countries or whether they arose by mutations of circulating japanese ibv strains. in this study, to define the origin and evolution of recent ibv in japan, we determined the nucleotide sequences of ibv isolated in japan using the reverse transcriptase-polymerase chain reaction (rt-pcr) method coupled with direct sequencing, and analyzed the sequences phylogenetically with viruses isolated in other countries. this information is important for determining strategies to control ib. the ibv isolates employed in this study are listed in table . most of the ibv isolates were obtained from regional laboratories in japan. most of the specimens of ibv were isolated by two or three passages using embryonated specific pathogen-free (spf) eggs. the materials were submitted to our laboratory, and were propagated once in spf eggs. the presence of ibv in the inoculated embryos was initially determined by immunofluorescence assay of allantoic cells using anti-ibv chicken serum and observation of characteristic embryo changes such as dwarfing, stunting or curling, according to the procedures of a previous report [ ] . the following commercial attenuated live vaccine strains in japan were also used in this study: c- , miyazaki, on/ , kita- , ku and tm . they were derived from field cases of ib in japan. viral rna was extracted from infected allantoic fluids using a kit (isogen-ls, nippon gene, tokyo, japan). a reverse transcriptase (rt) reaction was carried out with superscript ii (life technologies, gaithersburg, md, u.s.a.) using random mers. the n-terminus of the s glycoprotein, which includes [ ] : country/region of origin/year of isolation except jp/kh/ strain. jp, japan important structures such as hypervariable regions (hvr) associated with antigenic properties [ , , ] , was selected for this analysis. the following primers were used: ibv-s (forward), agg-aat-ggt-aag-ttr-ctr-gtw-aga-g , and ibv-s (reverse), gcg-cag-tac-crt-tra-yaa-aat-aag-c . (the expected product was about base pairs). these were designed based on a comparison and alignment of the genbank sequences of several known ibv strains. pcr products were purified using the qiaquick pcr purification kit (qiagen, hilden, germany) according to the manufacturer's instructions. purified pcr products were used as a template for sequencing on an applied biosystems s automated dna sequencer using dye terminator cycle sequencing chemistry (perkin-elmer/applied biosystems, foster city, ca, u.s.a.). purified pcr products were sequenced from both directions. the derived nucleotide sequences were analyzed using genetyx-mac ver. . (software development corp., tokyo, japan), and through genbank searches. the phylogenetic analysis with available sequences from genbank was conducted using the clustal [ ] , and the tree was constructed by the neighbor-joining (nj) method [ ] . the expected sizes of dna fragments were successfully amplified by rt-pcr from all the employed ibv samples with our designed primers. the determination of nucleotide sequences of obtained pcr products revealed the diversity of those lengths ( - bp) ( table ) . by phylogenetic analysis, the ibv isolates in japan used in this study were divided into five genetic groups (fig. ). in particular, recent isolates in japan were assigned to three major genetic groups. among them, one group (jp-i) is indigenous to japan and is not present in other countries. the two other groups that have emerged recently (jp-ii and jp-iii) are related to isolates in china and taiwan. some isolates that were mainly isolated in the s, such as jp/ishida/ or jp/nerima/ , and two local attenuated vaccine strains, kita- and ku, were classified into the mass group. another local attenuated vaccine strain, c- , was classified into the jp-i group, and miyazaki and tm strains were classified into the jp-ii group. only one strain (on/ ), which was also a local attenuated vaccine strain, was classified into the gray group. the deduced amino acids from obtained nucleotide sequences of pcr products were aligned with various ibv strains isolated in different geographic regions. the results are shown in fig. . many insertions or deletions of amino acids among ibv strains including the japanese isolates were found in the amplified region. the generated pcr products were employed in restriction endonuclease analysis for the development of a simple and rapid classification of genetic groups. after comparing the obtained sequences in this study, we selected two endonucleases, hae ii and ecor i (takara, tokyo, japan), for this analysis. the restriction profiles fig. . dendrogram of the s glycoprotein of infectious bronchitis viruses. amino acids - (in genbank accession number p ) of the beaudette strain s glycoprotein were used for phylogenetic analysis at the amino acid level. the dendrogram is rooted to murine hepatitis virus ml- strain. horizontal distances are proportional to the minimum number of amino acid differences required to join nodes and sequences of the pcr products of the five genetic groups in this study, as revealed by two restriction endonucleases are shown in table and fig. . each japanese genetic group has a specific profile pattern. the other genetic groups represented, such as connecticut, d , uk/ / , and ark , do not have sites for these two enzymes in their reported sequences. hence, the three major genetic groups in japan were easily differentiated from other genetic groups. recently, novel ibv strains have been proliferating abroad, and genetic analysis of these strains have been mainly based on the s gene [ ] . ibv strains in japan have been considered variants different from foreign ibvs such as massachusetts or connecticut types by vnt or pcr-restriction enzyme fragment length polymorphism based on the s gene [ , , ] . still, it remains unknown whether the current ibv isolates in japan were newly introduced from other countries or whether they arose by mutations of circulating japanese ibv strains. in this study, it was revealed that five genetic groups, which could be differentiated by simple restriction endonuclease analysis, were present in japan. although sequencing is recommended to obtain precise genetic information, restriction endonuclease analysis is simple, easy and convenient for primary characterization in routine diagnosis. among these genetic groups, three major genetic groups were associated with recent outbreaks of ib in japan. a previously published study divided ibv strains into several distinct genetic groupings by analyzing the s gene [ ] . however, most ibv isolates in japan formed genetic groups distinct from these. among three major genetic groups of ibv in japan, one group (jp-i) has not been found in other countries; this group may be indigenous and has been prevalent in japan for a long time, from at least the s. the other two groups that have emerged recently (jp-ii and jp-iii) are related to isolates in china and taiwan. recent ibv isolates in china and taiwan also form distinct genetic groups [ , ] . it is unknown whether these genetic groups originated in japan or in these neighboring countries. interestingly, recently isolated newcastle disease viruses in japan were also closely related, genetically, to those isolated in taiwan and china [ ] . furthermore, it was recently revealed that the coronavirus isolated from pheasants was genetically closely to ibv [ ] . one possibility for the migration of ibv strains into this region is dissemination by some kind of wild birds close to chickens, such as pheasants. the virus neutralization antibodies that form the basis for comparison of ibv isolates are induced largely by the n-terminus in the s glycoprotein [ , ] . in this study, prevalent ibv genetic groups in japan were shown to be completely distinct from other known serotypes from europe and north america by comparing amino acid sequences of the n-terminus in the s glycoprotein. on the basis of the relationships between genotypes and serotypes of ibv [ ] , it was suggested that the serotypes of recent ibv in japan are novel. further analysis would be required to examine the antigenic property of isolates for establishment of a control strategy for ib outbreaks in japan. on the other hand, the variation of ibv may also be attributed to recombination following co-infection of a few distinct strains [ , ] . previously, the kb strain isolated in japan [ ] was revealed to be genetic recombinant [ ] . in japan, in addition to major massachusetts (h strain) and connecticut (l strain) type live vaccines, local attenuated vaccines isolated from outbreaks of ib in japan have been developed and used. they proved to derive from the genetic groups, mass, gray, jp-i or jp-ii, although the genetic backgrounds of these attenuated vaccines have not been clarified. so far, it has been suggested that the recombination events occur between vaccine strains and field strains [ ] . it is unknown whether or not vaccine strains in japan are associated with the emergence of recombinant viruses. to detect recombinant viruses, it would be required to analyze two or more different genetic regions of isolates. further analysis of viruses would clarify whether recombinant variants have become prevalent in japan. all sequences used in this study were sent to ddbj, and their accession numbers areab to ab . completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus coronavirus ibv: structural characterization of the spike protein amino acids within hypervariable region of avian coronavirus ibv (massachusetts serotype) spike glycoprotein are associated with neutralization epitopes infectious bronchitis a nomenclature for avian coronavirus isolates and the question of species status coronaviruses from pheasants (phasianus colchicus) are genetically closely related to coronaviruses of domestic fowl (infectious bronchitis virus) and turkeys use of allantoic cells for the detection of avian infectious bronchitis virus humoral antibody response and assessment of protection following primary vaccination of chicks with maternally derived antibody against avian infectious bronchitis virus serotypes of avian infectious bronchitis virus isolates from field cases in japan cross-immunity in chickens using seven isolates of avian infectious bronchitis virus a novel variant of avian infectious bronchitis virus resulting from recombination among three different strains location of antigenic sites defined by neutralizing monoclonal antibodies on the s avian infectious bronchitis virus glycopolypeptide phylogeny of antigenic variants of avian coronavirus ibv sequence evidence for rna recombination in field isolates of avian coronavirus infectious bronchitis virus differentiation of infectious bronchitis virus serotypes using polymerase chain reaction and restriction fragment length polymorphism analysis typing of field isolates of infectious bronchitis virus based on the sequence of the hypervariable region in the s gene a new typing method for the avian infectious bronchitis virus using polymerase chain reaction and restriction enzyme fragment length polymorphism typing of recent infectious bronchitis virus isolates causing nephritis in chicken phylogenetic analysis of newcastle disease virus genotypes isolated in japan a virus isolated from infectious bronchitis-like diseases of chickens the neighbor-joining method: a new method for reconstructing phylogenetic trees ibv genotypes in japan cloning and sequencing of genes encoding structural proteins of avian infectious bronchitis virus the clustal-x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools evidence of natural recombination within the s gene of infectious bronchitis virus evolutionary implications of genetic variations in the s gene of infectious bronchitis virus relationship between serotypes and genotypes based on the hypervariable region of the s gene of infectious bronchitis virus molecular epidemiology of infectious bronchitis virus isolates from china and southeast asia author's address: dr. masaji mase, department of infectious diseases, national institute of animal health, - - kannondai redaktion: sachsenplatz - , wien, austria. -satz und umbruch we thank drs. t. imada and n. yuasa, national institute of animal health, japan, for valuable discussions and suggestions. we would like to thank the veterinary officials of miyazaki, akita, mie,yamanashi, nagano, shizuoka, shimane, chiba, toyama, aichi, fukui, okayama, kanagawa, osaka, and ibaraki prefectures for their cooperation in the collection of the viral samples. key: cord- -i ow bk authors: cowan, fred m.; broomfield, clarence a.; stojiljkovic, milos p.; smith, william j. title: a review of multi-threat medical countermeasures against chemical warfare and terrorism date: - - journal: mil med doi: . /milmed. . . sha: doc_id: cord_uid: i ow bk the multi-threat medical countermeasure (mtmc) hypothesis has been proposed with the aim of developing a single countermeasure drug with efficacy against different pathologies caused by multiple classes of chemical warfare agents. although sites and mechanisms of action and the pathologies caused by different chemical insults vary, common biochemical signaling pathways, molecular mediators, and cellular processes provide targets for mtmc drugs. this article will review the mtmc hypothesis for blister and nerve agents and will expand the scope of the concept to include other chemicals as well as briefly consider biological agents. the article will also consider how common biochemical signaling pathways, molecular mediators, and cellular processes that contribute to clinical pathologies and syndromes may relate to the toxicity of threat agents. discovery of mtmc provides the opportunity for the integration of diverse researchers and clinicians, and for the exploitation of cutting-edge technologies and drug discovery. the broad-spectrum nature of mtmc can augment military and civil defense to combat chemical warfare and chemical terrorism. t he list of military chemical threat agents has lengthened over the last century. world war i saw the development and deployment of edemagenic and choking agents such as chlorine, phosgene, and cyanide, and blister gases such as sulfur mustard (sm). [ ] [ ] [ ] world war ii cultivated germany's development of nerve gases such as tabun, sarin, and soman, [ ] [ ] [ ] [ ] and this class of agent was used in the to iraq-iran conflict , and in the to terrorist attacks in japan. [ ] [ ] [ ] the cold war yielded more lethality refinements in nerve agents to include development of vx. consistent with past experience, new threat agents with novel mechanisms of toxicity may evolve. countermeasures for defending deployed military personnel against weaponized chemical warfare agents do not seamlessly transition to military force protection and civil defense against chemical terrorism. terrorist threat agents include all classes of chemical warfare agents, toxins, and a broader menu of toxic industrial chemicals. the large number of threats and potential for use of multiple toxic substances in combination can complicate agent identification and medical response. a terrorist assault might more closely resemble the chemical accident at bhopal, india, than a chemical warfare attack. symptoms may be closer to smoke inhalation and acute respiratory distress syndrome (ards) than to the cholinergic crisis caused by nerve agent poisoning. research into medical countermeasures against weaponized battlefield chemical warfare agents has focused on distinct classes of nerve, blister, choking, or edemagenic agents and pathologies specific to these agents, e.g., nerve agent-induced seizure, phosgene-induced ards, and sm blistering. the present challenge of defending against the myriad of threat chemicals and toxins necessitates a more collective and comprehensive strategy that more generally addresses the symptoms and pathology of chemical toxicity rather than a specific chemical mechanism. the multi-threat medical countermeasure (mtmc) hypothesis for chemical toxicity is analogous to the broad-spectrum approach used for protecting against infectious agents authored by dr. ken alibek, former deputy chief of biopreparat, the civilian arm of the former soviet union's biological weapons program. , it may be possible to exploit the fact that the diverse chemistry, sites, and mechanisms of action of toxic chemicals often involve common underlying biochemical signal pathways. these common pathways are key in initiating the cellular processes that contribute to clinical pathologies caused by chemical exposures. clinically defined syndromes or symptoms that represent pathologies of respiratory, neuronal, cardiac, cutaneous, or other systems to include anaphylactic shock, bronchospasm, ards, seizure, excitotoxin-like neuronal degeneration, and blistering are observed after exposure to chemical warfare agents, hazardous chemicals, toxins, and even infectious agents. the mtmc hypothesis suggests that common biochemical pathways contribute to the toxicity that causes the pathologies associated with different classes of toxic chemicals. mtmc drugs that target these key pathways might create a physiological "quiescent" state that protects against various chemical insults. the fact that inflammation causes or contributes to numerous pathologies and that many toxic chemicals provoke an inflammatory response is well established (for review, see refs. [ ] [ ] [ ] . although evidence for a significant role for inflammation in chemical toxicity continues to accumulate, inflammatory response is still often regarded as merely collateral to chemical insult and secondary to toxicity. [ ] [ ] [ ] the evolution over the last decade of the concept that inflammatory response is a key element of chemical toxicity that allows for the development of anti-inflammatory drugs as multi-agent countermeasures is unique to the mtmc hypothesis. [ ] [ ] [ ] the mtmc hypothesis encompasses the interaction of multiple toxic agents and diverse countermeasure drugs on mediators and signaling pathways that cause or sustain inflammation. whether inflamma-tion is a collateral biomarker or causative contributor to chemical toxicity is controversial. however, in either case inflammation can provide a common denominator to facilitate deciphering the relationships between agents, drugs, signaling pathways, and cellular processes pertinent to predicting candidate mtmc drugs. a major tenet of the pharmacology of anti-inflammatory drugs is that drug action can result from the specific inhibition of molecular mediators of inflammation or from modulation of the underlying biochemistry that initiates or sustains an inflammatory response. different inflammatory mediators, pathways, and cell populations can be induced or recruited by different conditions or insults. this multifaceted nature of inflammation may require the use of numerous pharmacologically distinct antiinflammatory drugs to contend with the different manifestations of inflammatory pathology. however, enough uniformity of biochemical pathways of inflammation exists to allow for development of broad-spectrum anti-inflammatory drugs. the biochemical pathways associated with chemical toxicity can involve proteases, inflammatory cytokines such as tumor necrosis factor (tnf), interleukin (il)- , il- , and other molecules such as platelet activating factor (paf), n-methyl-d-aspartate (nmda) glutamate receptors, acetylcholine (ach), substance p, and poly(adp-ribose) polymerase (parp) (for review, see ref. ). these mediators and receptors can influence inflammatory responses associated with cellular processes such as degranulation, apoptosis, and necrosis that contribute to pathologies caused by chemical agents. therefore, many classes of compounds used as countermeasures to chemical warfare agents such as parp inhibitors, proteases inhibitors, adenosine agonists, and nmda receptor antagonist, although not chiefly thought of as anti-inflammatory drugs, have anti-inflammatory pharmacology (table i ) (for review, see ref. ) . the diverse biochemical pathways that influence inflammatory responses may explain why many pathologies associated with intoxication by chemical agents have an inflammatory component. along this line of reasoning, it is not surprising that the majority of countermeasure drugs that have shown efficacy against edemagenic, vesicant, and nerve agents have anti-inflammatory pharmacology (for review, see ref. ) . the presence of inflammatory responses and anti-inflammatory actions of countermeasures implicates inflammation as a cause or a resulting biomarker of the toxicity of these agents. the extent to which common inflammatory mechanisms are coupled to chemical agent toxicity will determine the feasibility of developing anti-inflammatory mtmc. inflammatory response induced by the prototypic edemagenic agent phosgene is the primary cause of the frequently lethal ards. , ards is also associated with pulmonary toxicity of the acetyl-cholinesterase-inhibiting organophosphorus insecticide thionazine, sm, and other toxic chemicals, as well as toxins, infectious-agent pneumonia, shock, sepsis, burns, and other trauma (table ii) . sm dermal-epidermal separation is similar to that caused by proteolysis and certain bullous diseases, and this has fostered the hypotheses that sm vesication involves proteolytic and/or inflammatory responses. [ ] [ ] [ ] [ ] [ ] [ ] experimental evidence has accumulated over the last decade demonstrating sm-increased proteases and the expression of inflammatory enzymes, gene products, mediators, and receptors in tissue or cell cultures. , , [ ] [ ] [ ] [ ] [ ] inflammatory enzymes and cytokines have also been reported in the skin of sm-exposed animals. , cholinergic crisis is the paramount toxicological event in nerve agent intoxication, causing acute toxicity and precipitating seizure and neuronal degeneration. however, along with cholinergic crisis anaphylactoid reactions, pathological proteolytic activity and inflammatory cytokines have also been reported in nerve agent-intoxicated animals. , - the initial observations of anaphylactoid reactions associated with soman poisoning reported by doebler et al. and newball et al. nearly a quarter century ago have recently again been considered by gilat et al. in sarin-intoxicated animals. anaphylactoid reactions, by definition, occur upon initial exposure to a chemical independent of antibody production. anaphylactoid reactions and nerve agent intoxication can generate acute symptoms such as convulsions, bronchoconstriction, respiratory failure, circulatory collapse, and death within a few minutes. , thus, anaphylactoid reactions, , like the more extensively investigated cholinergic crisis and excitotoxin-like neuronal degeneration, are a clinically well-defined and potentially lethal syndrome associated with nerve agent intoxication. mcleod noted neuronal and neurophil degeneration and necrosis associated with soman-induced brain lesions. the pattern of injury was similar to that caused by epilepsy and isch- acute lung/organ dysfunction emic brain injury. some animals also had cardiac lesions characterized by acute necrosis with subsequent mild inflammation and fibrosis. inflammatory cytokines such as tnf, il- , and il- are associated with recruitment of inflammatory cells and cellular processes such as degranulation, apoptosis, and necrosis. inflammatory cytokines are causative factors in phosgeneassociated ards, they contribute to sm injury, and they are implicated in nerve agent seizure and neuronal degeneration. , , , , , , skin from mice sm exposure sites and the brains of soman-intoxicated rats demonstrate increased il- ␤ mrna. , , il- is an inflammatory cytokine that can precipitate other cytokines such as tnf, il- , and il- , and such a cytokine cascade is implicated in sm toxicity. svensson et al. have demonstrated that soman induces the inflammatory cytokine il- ␤ in rat brain, and this activity is highly correlated with convulsions. williams et al. confirmed il- ␤, and further observed an acute and transient upregulation of other inflammatory gene response, i.e., tnf, il- , e-selectin, and intercellular adhesion molecule- caused by exposure to soman. the authors suggest that these molecules may be involved in soman-induced brain injury. eriksson et al. demonstrated that nmda antagonists could suppress excitotoxin-induced il- ␤ mrna in the rat brain. furthermore, vezzani et al. have protected mice against nmda receptor-dependent seizures with soluble il- receptors that specifically antagonize il- activity. the authors suggest that the interaction between il- and il- receptors represents a crucial mechanism of seizure and a target for development of anticonvulsant drugs. the role of inflammatory cytokines in edemagenic, nerve, and blister agent toxicity supports cytokine antagonists as candidate mtmc. anti-inflammatory drugs such as ibuprofen are the drugs of choice in reducing phosgene toxicity. , the majority of drugs that have shown efficacy against sm in animal models have anti-inflammatory pharmacology (for review, see ref. ). our institute has screened more than compounds in the mouse ear vesicant model (mevm) for cutaneous injury; compounds reduced sm histopathology greater than %. - the compounds include seven listed as antiinflammatory drugs, consisting of five capsaicin analogs, a single cyclooxygenase inhibitor, indomethacin, and a calmodulin antagonist, fluphenazine. - the three protease inhibitors and three parp inhibitors included in the list of antivesicant drugs also have the potential to inhibit inflammatory responses. , the remaining six anti-vesicant drugs, sodium -sulfonatopropyl glutathionyl disulfide, % hydrogen peroxide gel, dimercaprol, and three other mercaptopyridines analogs are listed as sm scavengers. , however, mercaptopyridine-like compounds have demonstrated anti-inflammatory pharmacology to include inhibition of the cellular release of the inflammatory cytokine il- . therefore, of the compounds with efficacy against sm toxicity have anti-inflammatory pharmacology, the exceptions being two of the listed sm scavengers. furthermore, antivesicant drugs with anti-inflammatory pharmacology generally demonstrate inhibition of hd-increased il- in human epidermal keratinocyte (hek) cultures (ref. ; fm cowan, ca broomfield, and wj smith, unpublished observations). collectively, the mevm and in vitro sm-increased il- hek assays effectively screen, not just for vesicant countermeasures, but also for anti-inflammatory drugs that inhibit il- . these models further identified drugs such as capsaicin analogs as better antivesicants and anticytokine drugs than major classes of currently available corticosteroids and nonsteroidal anti-inflammatory drugs. the anti-inflammatory action of nerve agent countermeasures to include atropine, carbamates, and benzodiazepines has been extensively reviewed. , , for example, a role for synergy between mediators of anaphylactic response and cholinergic status "hypersensitivity" is accepted for asthma-related lethal bronchoconstriction, and has been implicated for nerve agentinduced lethal bronchoconstriction. , , , kotev has demonstrated that soman-induced bronchial spasm was greatly intensified not only by ach, but also by histamine. hypersensitivity to inhalation of the cholinergic compounds such as methylcholine or the autacoid histamine is a diagnostic test for asthma, and anticholinergics are sometimes used as supplementary treatment. inflammatory, neurological, and tissue pathology pathways involve proteolytic processes. synthesized mediators such as paf and serine proteases such as tryptase and tissue plasminogen activator (t-pa) in addition to autacoids such as histamine are significant factors in inflammatory and anaphylactic responses. , , carbamates can inhibit serine proteases that mediate anaphylaxis and prevent anaphylactic response. the benzodiazepine diazepam is food and drug administration approved for the treatment of seizures associated with nerve agent poisoning. benzodiazepines can, in addition to acting as ␥-aminobutyric acid receptor agonists, also inhibit paf receptors. paf is a dual proinflammatory and neuromessenger phospholipid molecule that participates in pathological phenomena that include protease synthesis, lethal anaphylaxis, vesication and nmda glutamate receptor excitotoxicity, seizure, and neuronal degeneration (for review, see ref. ) . mice are protected from lethal anaphylaxis by serine protease inhibitors or paf antagonists. , the ␥-aminobutyric acid receptor agonist and paf receptor antagonist actions of benzodiazepines might synergistically protect against nerve agent cholinergic and inflammatory toxicities. furthermore, proteolytic and inflammatory pathways are also strongly implicated in nmda receptor-mediated seizure and neuronal degeneration. knockout mice deficient in the protease t-pa were resistant to seizure and neuronal pathology caused by excitotoxins (for review, see ref. ) . furthermore, the synthetic protease inhibitor tpa-stop (american diagnostica, greenwich, ct) suppressed excitotoxin-induced seizure and neuronal degeneration (for review, see ref. ). experimental evidence generated in in vitro and in vivo models of chemical agent toxicity supports the concept of anti-inflammatory mtmc drugs. sm exposure increases the inflammatory il- in hek and human small airway cell (hsac) cultures. , sm-increased il- in hek cultures is a biomarker of dermal inflammatory pathology that is used as an in vitro drug screening and treatment model to complement in vivo studies in the mevm. likewise, sm-increased il- in hsac has recently been developed as an in vitro model for treatment of inhalation toxicity. hsac cultures exposed to to m sm or . to . ppm/min phosgene demonstrated significantly increased il- . a maximum increase of approximately , pg/ml il- in hsac cultures exposed to phosgene or sm was observed. the pattern of il- response, increase to maximum levels followed by inhibition at higher cytotoxic doses, was similar for both agents. ibuprofen ( , , , , and , m) significantly diminished phosgene-increased il- in sac cultures exposed to ppm/min phosgene. furthermore, the doses of ibuprofen that decreased phosgene-increased il- approximately % in hsac ( and m) also inhibited sm-increased il- in hek cultures to about the same extent. , serine protease inhibitors and parp inhibitors can have antiinflammatory pharmacology, and members of these two distinct classes of compounds have shown efficacy for nerve and blister agents in vivo. , , , serine protease inhibitors can prolong the survival of animals intoxicated with the nerve agent soman, and can also protect against vesication caused by the blister agent sm. parp inhibitors can reduce soman-induced neuronal degeneration and sm-induced epidermal necrosis. candidate anti-inflammatory mtmc drugs to include protease inhibitors, paf antagonists, parp inhibitors, nmda receptor antagonist, and adenosine agonists have been suggested (table i) . adenosine receptor agonists illustrate the potential and the complexity of mtmc drugs. van helden et al. used the adenosine a receptor agonist n -cyclopentyl adenosine as a countermeasure to soman poisoning. without any supportive treatment with atropine, oxime, or diazepam, n -cyclopentyl adenosine protected rats from convulsive activity and respiratory distress and improved -hour survival. reduced ach levels in rat brains were reported in this study, and the protection was attributed to cholinergic mechanisms wherein the adenosine a receptor agonists binding to the a adenosine receptor caused inhibition of ach release. however, the cholinesterase-inhibiting nerve agents sarin, tabun, and soman can interact directly with brain a adenosine receptors and may competitively alter the action of these agents at adenosine receptor sites. , adenosine receptors can moderate the activation of other receptors that influence neurotransmitter release and/or synaptic transmission, e.g., ach and nmda receptors. , a , a , and a adenosine receptor agonists can further influence mast cell degranulation, and adenosine-directed treatment modalities have been suggested for asthma. neutrophil infiltration of dermal inflammatory sites, similar to that also observed in sm pathogenesis, was inhibited by administration of adenosine a receptor agonists. , this anti-inflammatory action of adenosine agonists was reversed by an nmda agonist and was mimicked by a glutamate nmda receptor antagonist. hence, in this model, central nmda receptor activity can control peripheral neutrophil accumulation, and adenosine a receptor agonists can influence this nmda receptor excitation-mediated inflam-mation. virag and szabo have further demonstrated that adenosine can inhibit the activation of parp, and they propose that this may affect cell death and inflammation. thus, adenosine agonists can influence multiple inflammatory biochemical pathways associated with nerve and blister agent toxicity and are candidate mtmc (table iii) . the activation of inflammatory responses that contribute to pathology is not restricted to chemical agents. t- toxin, as a major trichothecene mycotoxin, has some radiomimetic properties, and the results of its action, like the blistering induced by sm, can be alleviated by treatment with glucocorticoid hormones. , moreover, paf seems to have an important role in the pathophysiology of t- toxicosis because its selective antagonist bn can prolong survival of rats exposed to lethal doses of this toxin. trichothecene mycotoxin also induce mesenteric and subcutaneous mast cell degranulation and increased secretion of interleukins il- ␤ and il- in vitro. this suggests that superinduction of cytokines might be one of the mechanisms of t- toxin-induced tissue damage. the inflammagenic properties of mycotoxin may further contribute to core pathologies such as ards. likewise, inflammatory cytokines and the complication of ards are associated with the pathology of infectious agents such as anthrax, smallpox, ebola, and, of course, the corona virus infection that causes severe acute respiratory syndrome. [ ] [ ] [ ] the mtmc hypothesis suggests that inflammation is not just a biomarker, but is also a major cause of toxicity for many chemical warfare agents and toxic chemicals. as a counterpoint to the anti-inflammatory mtmc drugs, substances that increase inflammation can synergistically augment chemical toxicity. roth and coworkers (for review, see ref. ) demonstrated that a small dose of the inflammagenic endotoxin lipopolysaccharide (lps), which is without effect by itself, markedly enhances the hepatotoxic effects of aflatoxin b . a similar effect of lps endotoxin occurs with other toxic chemicals and other target organs (for review, see ref. ). stone et al. have reported that lps or the inflammatory cytokines tnf or il- enhanced the cytotoxic effects of the sm stimulant -chloroethyl ethyl sulfide. whether the results of coexposure to an inflammagen such as lps or an intrinsic property of a toxic agent, the inflammatory response can augment sensitivity to chemical toxicities. thus, inflammation associated with chemical toxicity is a target for anti-inflammatory mtmc. the mtmc hypothesis is supported by experimental evidence that distinct classes of drugs such as protease inhibitors and anti-inflammatory parp inhibitors have demonstrated efficacy against the nerve agent soman and the blister agent sm. , , , moreover, t- mycotoxin and sm injury can be alleviated by treatment with glucocorticoid hormones. , by focusing on key biochemical signal pathways and cellular processes that ultimately contribute to pathologies associated with chemical toxicity, the mtmc hypothesis provides the possibility of developing single countermeasure drugs with prophylactic or therapeutic efficacy against distinct pathologies caused by multiple classes of toxic chemical agents. furthermore, the mtmc concept may not be limited to chemical agents. many biological toxins and infectious agents cause inflammatory responses that contribute to pathology. finally, because mtmc addresses clinically recognized pathologies such as ards, collateral improvements in general health care and emergency medicine are possible. once one looks past the diverse chemistry, sites of action, and symptoms, common biochemical pathways may exist for many chemical threat agents. even when threats extend to mixtures of agents and toxic industrial chemicals, or new threat agents evolve, such biochemical signaling pathways may remain more constant and amenable to medical intervention. defining and interdicting these pathways provide strategies for developing civil defense and military preparedness against chemical threats. of perhaps equal significance, the mtmc hypothesis provides an incentive for dialogue and cross-fertilization between diverse chemical defense, academic, clinical, and drug discovery research interests to combat chemical warfare and chemical terrorism. using mtmc to blunt pathological actions of chemical agents is an attractive possibility. a higher form of killing: the secret story of gas and germ warfare chemical and biological weapons: the silent killers organophosphorus compounds as chemical warfare agents organophosphate poisoning: an overview iraq's chemical warfare: case proved analysis of two chemical weapons samples from the iran-iraq war nerve gases as means of chemical terrorism in japan anatomy of aum shinrikyo's organization and terrorist attacks with chemical and biological weapons the first mass chemical terrorism using sarin in matsumoto at war with chemistry putative role of proteolysis and inflammatory response in the toxicity of nerve and blister chemical warfare agents: implications for multi-threat medical countermeasures putative roles of inflammation in the dermatopathology of sulfur mustard a hypothesis for synergistic toxicity of organophosphorus poisoning-induced cholinergic crisis and anaphylactoid reactions efficacy of ibuprofen and pentoxifylline in the treatment of phosgene-induced acute lung injury the temporal profile 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action of -pam and of acetylcholinesterase inhibitors on anaphylactic shock in the rabbit vesicants and nerve agents in chemical warfare. decontamination and treatment strategies for a changed world paf-acether-induced mortality in mice: protection by benzodiazepines neuronal death in the hippocampus is promoted by plasmin-catalyzed degradation of laminin suppression of sulfur mustard-increased il- in human keratinocyte cell cultures by the cox inhibitor indomethacin, the poly(adp-ribose) polymerase (parp) inhibitor -( Ј-bromophenyl)ureidobenzamide and the calmodulin antagonist fluphenazine sulfur mustard-and phosgene-increased il- in human small airway cell cultures: implications for medical countermeasures against inhalation toxicity adp-ribose) polymerase inhibitor, is neuroprotective against soman-induced seizure-related brain damage new generic approach to the treatment of organophosphate poisoning: adenosine receptor mediated inhibition of ach-release binding of some organophosphorus compounds at adenosine receptors in guinea pig brain membranes adenosine a a receptor interactions with receptors for other neurotransmitters and neuromodulators adenosine, mast cells and asthma spinal cord adenosine receptor stimulation in rats inhibits peripheral neutrophil accumulation. the role of n-methyl-daspartate receptors purines inhibit poly(adp-ribose) polymerase activation and modulate oxidant-induced cell death attenuation of general toxic effect of t- toxin in rats treated with various formulations of methylprednisolone potential antiinflammatory treatments against cutaneous sulfur mustard injury using the mouse ear vesicant model protective effect of paf-acether antagonist, bn , in trichothecene toxicosis mesenteric mast cell degranulation in acute t- toxin poisoning cutaneous injury by topical t- toxin: involvement of microvessels and mast cells the effect of t- toxin on il- a and il- secretion in human fetal chondrocytes acute respiratory distress syndrome cutting edge: impairment of dendritic cells and adaptive immunity by ebola and lassa viruses on the role of macrophages in anthrax bacterial lipopolysaccharide exposure alters aflatoxin b( ) hepatotoxicity: benchmark dose analysis for markers of liver injury lipopolysaccharide enhances the cytotoxicity of -chloroethyl ethyl sulfide key: cord- -ho ajsx authors: chen, paul chih‐hsueh; hsiao, cheng‐hsiang title: re: to kf, tong jh, chan pk, et al. tissue and cellular tropism of the coronavirus associated with severe acute respiratory syndrome: an in‐situ hybridization study of fatal cases. j pathol ; : – date: - - journal: j pathol doi: . /path. sha: doc_id: cord_uid: ho ajsx nan we read with great interest the excellent article by to et al, which was published in the february issue of the journal of pathology [ ] . using in situ hybridization (ish), to et al demonstrated the presence of severe acute respiratory syndrome-associated coronavirus (sars-cov) in pneumocytes and in the surface enterocytes of the small intestine. their findings are compatible with those of another study conducted in taiwan [ ] . this observation indicates the cellular tropism of the infectious agent and thus has an impact on our understanding of the disease. supplementing their findings, we report here our recent study, which suggests that the sars-cov may not be exclusively located in pneumocytes, but also in pulmonary macrophages. the lung tissue that we studied was a necropsy specimen provided by one of us (chh). the patient was a -year-old woman who died of sars. histological examination of the lungs revealed diffuse alveolar damage. the sars viral genome had already been verified in the tissue by nested rt-pcr [ ] . the full-length spike protein (s) cdna, membrane protein (m) cdna, and nucleoprotein (n) cdna clones were kindly provided by the national research program for genomic medicine and the genome research center, national yang-ming university, taiwan. these inserts were amplified by pcr and labelled with digoxigenin using the dig-high prime kit (roche, germany). the tissue sections were hybridized with the mixed probes (s, m, and n), ng/µl, at • c overnight. bcip/nbt was employed as a chromogen [ ] . under low magnification, scattered cells containing dark bluish signals, which represented the sars-cov, were visualized ( figure a , nuclear fast red counterstain) within the damaged alveolar spaces, small bronchioles, and even the vascular lumina. high magnification revealed that the virus-carrying cells harbouring the viral particles were frequently multinucleated and laden with anthracotic pigment ( figures b and c : empty arrows indicate anthracotic pigments; arrows indicate virus-carrying multinucleated cells; nuclear fast red counterstain). on the basis of our findings, we believe that some, if not all, positive cells are of the histiophagocytic lineage. first, the co-existence of viral signals and anthracotic pigment in the same cells indicates that those cells are bona-fide macrophages. second, we observed that some virus-harbouring cells were in the vascular lumina. it is unlikely that pneumocytes can be seen in vascular spaces even in a lung undergoing an extensively destructive process. third, the virusharbouring cells were predominantly arranged as single discrete cells interspersed within the parenchyma, a finding also observed in previous reports. we did not identify any adherent virus-positive cells, nor did we discern positive signals in the residual intact pneumocytes attached to the alveolar septa. this feature partly argues for a haematological, rather than an epithelial, origin for the virus-carrying cells. monocytes/histiocytes/macrophages play important roles in the immune response. they produce a plethora of cytokines that have potent regulatory activities in haematopoiesis, inflammation, and diverse immune reactions. in addition to their involvement in cytokine production, they are actively motile cells that respond to chemotactic stimuli and they can phagocytose particulate material and kill micro-organisms [ ] . the mortality of patients with sars has been attributed to a 'cytokine storm' triggered by the host immune response to sars-cov that results in diffuse alveolar damage [ ] . although the underlying mechanism of the 'cytokine storm' has not been clarified, such a hypothesis fits our observation that cells of the histiophagocytic lineage are another carrier of sars-cov. the presence of sars-cov in these cells may induce catastrophic cytokine release. on the other hand, the localization of sars-cov in the cytoplasm of monocytes/histiocytes/macrophages does not justify their role as a host. these cells can phagocytose viral particles, with or without surface virus-specific receptors. also, the presence of many copies of the viral genome does not necessarily indicate viral propagation in these cells. due to the limited availability of sars-covinfected tissue, an ish study on a larger scale would be difficult to perform. nonetheless, this piece of information is critical for future sars investigation-for example, in the search for sars-cov-specific receptors-and also has clinical relevance, such as developing therapeutic agents that target histiocytes. tissue and cellular tropism of the coronavirus associated with severe acute respiratory syndrome: an in-situ hybridization study of fatal cases detection of severe acute respiratory syndrome-associated coronavirus in pneumocytes of the lung detection of epstein-barr virus genome within thymic epithelial tumours in taiwanese patients by nested pcr, pcr in situ hybridization, and rna in situ hybridization normal blood cell the use of corticosteroids in sars. the authors reply this work was supported by grants sars - from taipei veterans general hospital and cstvgh - from the medical research & advancement foundation in memory of dr chi-shuen tsou, taiwan. the research use of the specimen and the procedures followed the ethical standards of the human experimentation committee of the institute. chen and hsiao make an interesting observation on the pathology of severe acute respiratory syndrome (sars). although the nature of the cells with positive in-situ signals has not been confirmed by double immunofluorescence-fluorescence in-situ hybridization (fish) analysis in the letter, we agree that the cells represent macrophages. however, these features were not seen in our limited number of autopsy cases [ ] . in fact, although such positive signals in macrophages were noted in the one case described in their letter, they were not present in the other cases reported by the authors [ ] . thus, this observation appears to be an uncommon event. given the phagocytic nature of macrophages, it is not surprising to see macrophages picking up various cellular and viral components in areas of tissue destruction. nevertheless, there is no convincing evidence that macrophages can be directly infected by sars-cov: the virus has not been isolated or cultured from histiocyte/macrophage-rich organs, including spleen and lymph node, and ultrastructural studies have also failed to detect viral particles in macrophages [ ] [ ] [ ] . furthermore, angiotensinconverting enzyme (ace ), the newly identified functional receptor for sars-cov, is not known to be expressed by macrophages. however, as we suggested, aberrant immune responses contribute significantly to the pulmonary and extra-pulmonary manifestations of key: cord- -srenbxa authors: zhao, jincun; wang, wei; wang, guang-fa; li, yonghua; zhuang, hui; xu, xiaoyuan; ren, furong; zhao, zhendong; gao, xiao-ming title: development and evaluation of an enzyme-linked immunosorbent assay for detection of antibodies against the spike protein of sars-coronavirus date: - - journal: j clin virol doi: . /j.jcv. . . sha: doc_id: cord_uid: srenbxa background: severe acute respiratory syndrome (sars) is caused by infection with sars-associated coronavirus (cov). amino acid residues – of the spike (s) glycoprotein of sars-cov (s - ) contains dominant epitopes for anti-viral antibodies (abs) in patient sera. objectives: to develop and evaluate an elisa system for detection of anti-s abs in patient sera. study design: express recombinant s - in e. coli and evaluate the sensitivity and specificity of an elisa system based on the s - polypeptide. results: the s - -based elisa detected igg abs in out of serum samples from hospitalized patients with probable sars, a result closely correlated with that obtained with a virus-based elisa (r = . , k = . ). differential anti-s igg responses were observed amongst sars patients. some of them produced anti-s abs early during their infection, while others failed to make igg abs against the s - polypeptide. none of the serum samples from healthy blood donors was positive in the s - -based assay. conclusion: the s - -based elisa can detect anti-s igg abs with high sensitivity and specificity. severe acute respiratory syndrome (sars) is caused by sars-associated coronavirus (sars-cov), an enveloped, positive-stranded rna virus of the coronaviridae family (rota et al., ; peiris et al., ) . the genome of sars-cov encodes several structural proteins including the spike (s) glycoprotein, nucleocapsid protein, membrane protein and envelope protein (marra et al., ) . membrane fusion between sars-cov and the host cell is mediated by its s glycoprotein xiao et al., ; dimitrov, ; wong et al., ; wang et al., ; sui et al., ) , which consists of amino acid residues with approximately % homology to that of the other human covs (spiga et al., ; ho et al., ) . the s subunit (amino acids - ) of the s glycoprotein contains a receptor-binding domain and is apparently the main target for neutralizing abs in patient sera (xiao et al., ; sui et al., ; wong et al., ; wang et al., ; babcock et al., ) . western blot (wb) and indirect immunofluorescence assays have been developed for detection of abs against the s protein chang et al., ; wu et al., ; woo et al., ; ho et al., ; he et al., ) . however, so far there is no enzyme-linked immunosorbent assay (elisa) available for easier and more sensitive detection of anti-s abs. our computer-assisted analysis suggested that amino acid residues - of the s glycoprotein (s - ) of sars-cov is largely solvent accessible and likely to contain dominant b cell epitopes. this coincided with recently published results by lu et al. ( ) showing that residues - of the s protein of sars-cov contained dominant epitope(s) for anti-s abs in patient sera, as determined in wb assays. sequences outside the - region were relatively poorly recognized by patient sera . it is also supported by findings of zhou et al. ( ) that residues - of the s protein of sars-cov elicited neutralizing abs against the virus. in this study, the s - fragment was expressed in e. coli and used as ag in an elisa system for detection of anti-s abs. restriction enzymes and t ligase were from invitrogen (usa). a kit for dna extraction and purification was from qiagen (hilden, germany). e. coli strains of bl (oe ) and dh ␣ were obtained from novagen (germany) and invitrogen (usa), respectively. complementary dna encoding full length s of sars-cov was a gift from china cdc. from march , , a major outbreak of sars had taken place in beijing, china. we collected sequential blood samples ( samples in total) from patients (both sexes, - years of age, average . years), admitted to the first affiliated hospital of peking university, beijing, china between th april and th june, . all patients fulfilled the who definition of probable sars (fever • c or higher, cough, new pulmonary infiltrates on chest radiography in the absence of an alternative diagnosis to explain the clinical presentation). the blood samples were processed within h of collection. all patient sera were tested for anti-sars-cov igg abs using an elisa kit produced by huada institute (see below). a total of serum samples from patients, randomly selected from the above patient group, were tested using our s - -based elisa kit. in addition, sera from healthy blood donors (both sexes, - years of age, collected between may and july ) were provided by beijing red cross blood center. a small outbreak of sars took place in april involving nine patients in anhui and beijing, china. sequential serum samples from four of the confirmed patients (second or third generation cases), admitted to ditan hospital between th april and th june , were also included in this study. dna coding for s - of sars cov s protein was pcr amplified using high fidelity taq dna polymerase (takara biotech co. ltd., japan). the sequences of the primers employed in the pcr reaction were s - : cgc gga tcc atg ccc ttt gag aga gac ata tct (forward primer, carrying a bamhi restriction site) and s - : ccc gaa ttc tta aat gtc gca ctc ata aga agt g (reverse primer, carrying an ecori site). the pcr product was gel-purified and cloned into expression vector pet- a (novagen, germany). the resultant construct, pet a-s / , encodes for the s - fragment with a histidine (his) tag ( his) at the n terminus. dna sequence of the insert was determined and compared with the s gene sequence of sars-cov strain urbani (accession number ay ) using dnastar software. pet a-s / was transformed into e. coli bl cells for expansion. a bacterial colony harboring the plasmid was inoculated into yt medium containing kanamycin ( g/ml) and cultured, with continuous shaking, at • c to appropriate density. isopropyl-␤-d-thiogalactopyranoside (iptg) was then added to a final concentration of . mm to induce the expression of the recombinant protein (for further . h). after centrifugation at × g for min, the pellet was resuspended in pbs and subjected to sonication in an ice bath for min. the inclusion bodies were solubilized with m urea, after centrifugation at × g at • c for min, the supernatant was applied to an equilibrated ni column (novagen, germany). after on-column refolding procedure, proteins bound to the column were eluted with mm imidazole in tris-buffered saline (ph . ). the eluted fractions were examined by sds-page and the proteins were either stained with coomassie blue or transferred to nitro-cellulose membrane for wb assays. the nitro-cellulose membranes, on which protein bands had been transferred, were blocked at room temperature for h with block solution ( % non-fat dry milk) and then incubated for h at room temperature with serum sample. after washing in tris-buffered saline (tbs, ph . ) containing . % tween , the membranes were incubated with hrp-labeled goat anti-human igg ab (zhongshan biotech co., china). , -diaminobenzidine tetrahydrochloride (dab, sigma) was used to visualize the reaction. elisa plates (nunc, demark) were coated at • c overnight with recombinant protein ( . pmol/well) in carbonate buffer (ph . ). immediately before use, the coated plates were incubated with blocking solution ( % bsa in pbs) for h at • c and then washed times with pbs containing . % tween (pbs-t). serum samples ( / diluted, l/well) were added in triplicate and incubated for min at • c. after washes with pbs-t, horseradish peroxidase (hrp)-labeled goat anti-human igg ab was added and incubated for h at • c. ortho-phenylenediamine (opd) substrate was used ( l/well) as substrate with m h so solution as stop buffer. the optical density (od) was immediately read at nm. for elisas using the kit produced by huada institute (beijing, china), the manufacturers' instruction was followed. briefly, dilution buffer ( l/well) was added to pre-coated wells followed by l serum and incubated for min at • c. after washes, hrp-labeled detection ab ( / diluted) was dispensed ( l/well) and incubated for min at • c before further washes. substrate buffer containing abts [ , -azino-di-( -ethylbenzo-thiazoline sulfonate)] was then added and allowed to develop for min. after stop buffer was added and the plates were read at nm. all experiments describe here have been repeated at least three times. results obtained using s - recombinant protein-based elisa and sars-cov-specific kit were compared using the correl module of microsoft excel software. the cohen kappa test was performed to analyze the agreement between the results of the two elisa kits using spss software. complementary dna encoding s - of sars-cov was cloned into expression vector pet- a for expression in e. coli bl . the his-tag-containing recombinant protein was purified to more than % homogeneity using a ni column. sds-page analysis of the affinity purified product revealed protein band of expected molecular weight (fig. ) . in wb assays, convalescent serum from a sars patient (pt-lx) and anti-his-tag mab specifically recognized the recombinant s - (fig. ) . in the subsequent study, recombinant s - was employed as coat- ing ag for an elisa kit with hrp-labeled goat-anti-human igg abs as secondary ab. checker-board titration experiments were carried out to optimize the concentration of the coating ag (s - ) and secondary ab (data not shown). a sars-cov-specific elisa kit, developed by huada institute, beijing, china, has been licensed by the ministry for public health of china. it employs the lysate of sars-covinfected vero-e cells as coating ags and has been widely used in china for sars-cov-specific ab testing with reliable results. by using the huada kit, we analyzed sequential serum samples (total sera) from hospitalized patients with probable sars and the results are summarized in table . half of the patients seroconverted for igg abs against sars-cov by day after the onset of illness. nearly % of them produced virus-specific serum abs by days - . sera from three convalescent sars patients and two healthy individuals were serial diluted and tested in the s - -based elisas, which detected anti-s igg abs in a specific and sensitive manner, with the reactivity end point from : to : diluted patient sera (fig. ) . serum samples from healthy blood donors were subsequently screened and none of them was positive (fig. a) , suggesting a high specificity for the s - -based system. when sequential serum samples ( total, / diluted) from patients (randomly selected from the abovementioned patient set) with probable sars were analyzed using the s - -baesd assays, were positive (fig. b) . the huada kit detected anti-viral igg abs in out of the samples (fig. c) , of which were positive in both assays. when the s - -based and virus-based elisa results were plotted against each other, a linear correlation (first degree regression, r = . ) was observed (fig. ) , fig. . sensitivity of the s - -based elisa. elisa plates were coated with recombinant s - . serum samples from convalescent sars patients ( , ♦, ) and healthy individuals ( , ) were serial diluted and dispensed, in triplicates, into the wells. hrp-labeled goat anti-human igg was used as secondary ab with opd as substrate. the results are expressed as absorbance reading at nm wavelength. fig. . comparison of s - -based and virus-based elisa results. the s - -based elisa kit was used to screen serum samples from (a) healthy blood donors and (b) patients (pt to pt , collected between and days from the onset of illness) with probable sars. the serum samples were folds diluted, results are expressed as absorbance reading at nm wavelength. cutoff value was . . elisa results obtained with the huada kit are shown in (c) for comparison. in this case, serum samples were folds diluted, results are expressed as absorbance reading at wavelength of nm. cutoff value was . . each bar represents one serum sample, samples from each patient were group together in the order of collection time (days) after onset of illness. the cohen kappa test also confirmed an agreement between them (κ = . ). time courses of igg responses against the s protein and the whole virus in out of the patients are compared in fig. a and b. sera from patients pt , pt and pt contained high titer anti-s but low titer anti-virus igg abs. in contrast, patient pt was a high responder against the whole virus but low responder against s - . patient pt seroconverted days after the onset of symptoms, suggesting a possible case of sars-cov infection being acquired in the hospital ward. patient pt remained negative in both tests months after the onset of illness and was eventually excluded as a sars patient. both the s - -based and the virus-based elisa kits were employed to screen sera from an additional sars patients infected in april . three of them were high responders against the s protein, while the other one (pt ) responded poorly to s - but strongly to the whole virus ( fig. c and d) . in patient pt , anti-s serum abs appeared earlier than that specific for other viral proteins. specific and sensitive detection methods for anti-viral abs in patient sera are of great value in diagnosis as well as research. virus-based assays have the advantage of being able to detect abs specific for all structural components of the virus. on the other hand, recombinant protein-based tests would allow analysis of anti-viral humoral immunity in much detail. the s protein of sars-cov is heavily glycosylated and contains many disulfide bonds (spiga et al., ; krokhin et al., ; tripet et al., ; ying et al., ) . it is difficult, however, to obtain s glycoprotein expressed in eukaryotic systems, which would otherwise be ideal for detection of anti-s abs in vitro. full-length s protein expressed in prokaryotic systems is often insoluble and therefore unsuitable as coating ag in elisa systems. our on-column refolding procedure allowed correct formation of some of the disulfide bonds in the s - fragment, producing soluble recombinant protein at a reasonably high concentration (data not shown). results reported herein indicate that the s - -based elisa can detect anti-s abs in patient sera with high sensitivity and specificity. it has been suggested that the n-glycans of the s glycoprotein could have a significant effect on its antigenicity, as the presence of n-glycans contributes to the correct folding and biological function of glycoproteins. however, recently published results indicate that bacterial expressed fragment of the s protein (non-glycosylated) could maintain its antigenicity (zhou et al., ; lu et al., ) . in addition, neutralizing abs were successfully raised in mice after immunization using prokaryotically expressed s - of sars-cov (zhou et al., ) . in this study we wound that most sars patients developed strong igg responses against the s glycoprotein of sars-cov. some of them had anti-s abs in significantly higher titer than that against other structural proteins of the virus (e.g. pt and pt ). in this situation, our s protein-based elisa would be more sensitive than the virus-based assays in detecting antiviral abs. there were also cases where the s - was poorly recognized by abs in patient sera (e.g. pt in fig. c and d). it should be emphasized, however, that the s - polypeptide covers less than / of the s protein sequence. a negative result such as this does not rule out possible presence of anti-s abs against epitopes outside the - region of the s protein. moreover, xu et al. ( ) have reported relatively high frequency of variations in s gene of sars-cov. natural variability in the s protein might also affect the validity of the s - -based elisa, although antigenic variation in s - has not yet been fully characterized. amino acids to of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor antibody detection of sars-cov spike and nucleocapsid protein the secret life of ace as a receptor for the sars virus development of a western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome antigenicity and receptor-binding ability of recombinant sars coronavirus spike protein mass spectrometric characterization of proteins from the sars virus: a preliminary report angiotensin-converting enzyme is a functional receptor for the sars coronavirus immunological characterization of the spike protein of sars-coronavirus the genome sequence of the sars-associated coronavirus coronavirus as a possible cause of severe acute respiratory syndrome characterization of a novel coronavirus associated with severe acute respiratory syndrome molecular modeling of s and s subunits of sars coronavirus spike glycoprotein potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s protein that blocks receptor association profiles of antibody responses against severe acute respiratory syndrome coronavirus recombinant proteins and their potential use as diagnostic markers structural characterization of the sars-coronavirus spike s fusion protein core expression cloning of functional receptor used by sars coronavirus a -amino acid fragment of the sars coronavirus s protein efficiently binds angiotensinconverting enzyme relative rates of non-pneumonic sars coronavirus infection and sars coronavirus pneumonia early detection of antibodies against various structural proteins of the sars-associated coronavirus in sars patients the sars-cov s glycoprotein: expression and functional characterization sars-associated coronavirus quasispecies in individual patients proteomic analysis on structural proteins of severe acute respiratory syndrome coronavirus an exposed domain in the severe acute respiratory syndrome coronavirus spike protein induces neutralizing antibodies the first two authors contributed equally to this work. key: cord- - rusr j authors: chan, louis y.; lee, nelson; chan, paul k.s.; wu, alan; rainer, timothy h.; li, philip k.t.; fung, hong; sung, joseph jy title: diagnostic criteria during sars outbreak in hong kong date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: rusr j nan to the editor: a novel coronavirus caused more than , proba-ble cases of severe acute respiratory syndrome (sars) worldwide ( , ) during the outbreak. before the etiologic agent was identified, the diagnosis of sars was made according to a set of clinical-epidemiologic criteria as suggested by the centers for disease control and prevention (cdc) ( - ). these criteria remained important in the initial diagnosis and prompt isolation of patients because the overall sensitivity of initial reverse transcriptase-polymerase chain reaction (rt-pcr) testing for sarsassociated coronavirus (sars cov) rna on upper respiratory specimens ranged from approximately % to % (though sensitivity improved with a second test) ( , ) . in a sars screening clinic at the prince of wales emergency department, the positive predictive value (ppv) of these criteria was estimated to be % ( % ci % to %) ( ) . the relative importance of the clinical versus epidemiologic criteria had not been evaluated. by using paired serologic testing to determine sars-cov infection ( ), we evaluated the relative importance of the clinical-epidemiologic diagnostic criteria during an outbreak. patients with a diagnosis of sars, and who were admitted to one of five regional hospitals in hong kong for isolation and treatment from march to june , , were included in this retrospective analysis. probable sars case-patients were those who met the cdc clinical criteria for severe respiratory illness of unknown etiology ( ), and met the epidemiologic criterion for exposure in either a close or a possible contact. close contact was defined as caring for, living with, or having direct contact with body fluids of a probable sars patient (e.g., working in the same medical ward or staying in the same household) within days of initial symptoms. because hong kong was the documented sars transmission site from february to july , , a modified epidemiologic criterion of possible contact was adopted. possible contact was defined as staying or working in the same hospital compound, or residing in the same building where case clusters of sars had been reported, within days of symptoms onset. laboratory testing of paired immunoglobulin (ig) g antibody to sars-cov was used to determine infection ( ) . positive serologic evidence of infection was defined as a four-fold rise in antibody titer or detection of antibody in convalescentphase serum. seronegativity was defined as absence of antibody in convalescent-phase serum obtained > days after symptom onset ( ). seronegativity in this defined time frame (> days -serum collected before july , , and beyond days) excluded the diagnosis of sars ( ). samples from patients showing nonspecific fluorescent signals were considered negative for sars-cov infection. rt-pcr was performed on clinical specimens (respiratory, fecal) from all patients ( , ( ) ( ) ( ) . demographic and laboratory parameters and history of close contact were compared between the seropositive and seronegative groups. student t test was used to analyze continuous variables. a p value of < . was considered statistically significant. odds ratio (or) and % confidence interval (ci) were calculated for categorical variables. during the study period, patients were hospitalized with probable sars. one hundred patients were excluded because their serologic results were either missing (n = ) or they died before day of illness (no convalescent-phase serum, n = ). three hundred seventy-five patients were included in the analyses; ( . %) patients were serology-positive for sars-cov. two hundred sixty-three of the patients ( . %) had a -fold increase in antibody titers, and of the patients ( . %) had detectable antibody in either acute-or convalescent-phase serum samples (titer - , ). twenty-two patients ( . %) had antibody titer < in their convalescentphase serum samples (median = days; range = - days). no clinical specimens were positive for sars-cov by rt-pcr. thus, the ppv of the clinical-epidemiologic criteria for sars in our cohort was . ( % ci . - . ). the contact history and demographic and laboratory parameters for both seropositive and seronegative groups are depicted in the table. the proportion of patients with a history of close contact was significantly higher in the seropositive group than in the seronegative group ( . % vs . %, or . ; % ci . - . ). only . % of the patients with serologically confirmed results had no close contact history; . % of the seronegative patients were in this category. the ppv of close contact was . ( % ci . - . ), and the ppv of possible contact was . ( % ci . - . ). seropositive patients had a significantly lower lymphocyte count on admission compared to the seronegative patients ( . ± . vs . ± . x /l) (p = . ). the ppvs for possible contact plus lymphopenia < . x /l and < . x /l were . ( % ci . - . ) and . ( % ci . - . ), respectively. seronegative patients were older ( . ± . vs. . ± . years), were less likely to be healthcare workers ( . % vs. . %), had their venue of contact in the community ( . % vs. . %), and had a higher total leukocyte count on admission ( . ± . vs. . ± . x /l). no differences were found in the lactate dehydrogenase, activated partial thromboplastin time, creatinine phosphokinase, and alanine-aminotransferase levels between the two groups. fifteen of the seronegative patients responded to antibiotics ( ); five died of comorbid illnesses (one of carcinoma of lung, one of metastatic carcinoma of prostate, two of chronic pulmonary diseases, and one of congestive heart failure), and two died of bacterial pneumonia. in four patients, bacterial pathogens were identified (one methicillin-resistant staphyl-ococcus aureus, two stenotrophomonas maltophilia, and one pseudomonas aeruginosa). also, ( . %) of the patients had coexisting medical conditions: three had congestive heart failure, four had chronic pulmonary diseases, two had chronic renal failures, two had advanced malignancies, two had diabetes mellitus, and two had parkinson's disease. our findings showed that . % of cases defined as probable sars on the basis of clinical-epidemiologic criteria had no serologic evidence of coronavirus infection. this set of criteria was associated with a ppv as high as . in a local outbreak. the ppv of the cdc epidemiologic criterion of close contact was higher ( . ). the ppv of possible contact was . , but when applied with lymphopenia, the ppv became higher. our analysis illustrated that a history of close contact with patients with sars-cov infection is of major importance when diagnosing such infection. this finding supports the hypothesis that sars-cov is transmitted through respiratory droplets and physical contact with a patient's body fluids. although not specific, lymphopenia and its subsequent progress was highly prevalent among sars patients ( ) ( ) ( ) . clinicians are now advised by the world health organization that hematologic deviations (e.g., lymphopenia) should be considered in sars evaluations ( ). our study was limited by sample size and its retrospective status. nonetheless, we demonstrated the accuracy of diagnostic criteria in an outbreak and the importance of epidemiologic criteria. further studies are needed to evaluate the diagnostic accuracy of these criteria in a nonoutbreak situation when the case prevalence is low. to the editor: malaria continues to cause disease and death in millions of persons living in areas of the world where it is endemic, despite decades of research on vaccines, new drugs, and alternative methods of control. still, by far the most effective method for reducing and controlling the impact of this disease is indoor residual spraying (irs) of insecticides. the most cost-effective and safe insecticide has been, and in many instances still is, dichlorodiphenyltrichloroethane (ddt). this intervention is continually under scrutiny, and we address these issues in this letter. chen and rogan ( ) claim that ddt causes reduced duration of lactation and increased incidence of preterm births, and they posit that ddt used for malaria control would do as much harm as good. the validity of their arguments requires substantial evidence of a causal relationship between ddt and adverse consequences of ddt irs for malaria control. chen and rogan dismiss a field study on births and duration of lactation in south african mothers, some of whom occupied houses sprayed with ddt for malaria control ( ) . however, if claims of large numbers of adverse health effects of ddt irs are correct, then the study should have detected large differences between ddt-exposed and unexposed populations. according to chen and rogan, the median duration of breastfeeding could be as low as - months when mothers are exposed to high levels of ddt. thus, a cross-section of breastfeeding infants in the ddt-exposed population should, on average, have been considerably younger than in the unexposed population. in fact, the average age of breastfeeding infants was slightly greater in the ddtexposed population ( . months versus . months). for both populations, only an insignificant fraction of mothers could not donate milk. furthermore, twice the level of dichlordiphenylethylene (dde, metabolic breakdown product of ddt) that is claimed to cause reduced duration of lactation in humans has no adverse affect on lactation in rats ( ). the authors of the south african study ( ) report no difference in rates of stillbirths between the sprayed and unsprayed areas. the national institute of environmental health sciences study ( ) reported a causal association between ddt and preterm and small-for-gestational-age births but this has not been replicated for african births. the study was not based on a random population of births, and no explanation is offered for including diverse categories of births in the study population. an earlier study in sri lanka presented data on deaths attributed to malaria and to premature births years before ddt was used and years when ddt irs was used in districts ( ) . districts varied greatly in levels of malaria endemicity. after ddt was introduced in , levels of irs in districts were commensurate with levels of endemic malaria. after , malaria deaths declined greatly and the reduction was greatest where ddt usage was highest. during the same period, deaths attributable to premature births increased slightly. inves-tigators attributed this to "improvements in reporting and diagnosis rather than any declines in the health of expectant mothers, which on all other criteria showed improvement." ( ). spearman's correlation analysis for districts shows that the increase in premature birth deaths was slightly greater in areas with less malaria and ddt use. thus, the evidence does not support the idea that the reported increase in premature births was a side effect of ddt use. in any case, the increase in deaths attributable to premature births was orders world health organization. severe acute respiratory syndrome (sars) centers for disease control and prevention. updated interim u.s. case definition for severe acute respiratory syndrome (sars) clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study evaluation of reverse transcription-pcr assays for rapid diagnosis of severe acute respiratory syndrome associated with a novel coronavirus evaluation of who criteria for identifying patients with severe acute respiratory syndrome out of hospital: prospective observational study laboratory diagnosis of sars a major outbreak of severe acute respiratory syndrome in hong kong haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis role of absolute lymphocyte count in the screening of patients with suspected sars key: cord- - muxz c authors: phillips, sally; lavin, roberta title: readiness and response to public health emergencies: help needed now from professional nursing associations date: - - journal: j prof nurs doi: . /j.profnurs. . . sha: doc_id: cord_uid: muxz c nan t he tragedy of september , anthrax attacks, and severe acute respiratory syndrome (sars) and other recent infectious disease outbreaks have heightened our awareness of the need for health care system readiness and response capabilities. at the same time, the economic realities of our modern health care system are reflected in cost-containment strategies toward low-volume inventories, reduced bed availability, downsizing of staff, and a shift to outpatient services (american hospital association, ) . decreased reimbursement structures and workforce shortages have diminished the health care system's ability to meet minimum patient demands, let alone the surge of patients that would be expected in a mass-casualty incident. furthermore, the infrastructure needed for detection and response from the public health sector has been seriously eroded by decades of insufficient funds. agencies within the department of health and human services (hhs) have been working to address readiness and response capabilities, but private organizations and professional associations also have a role to play. in keeping with the public health security and bioterrorism preparedness and response act of , hhs developed a department-wide strategic plan to delineate its priorities. within the plan, the centers for disease control (http://www.bt.cdc.gov) and health resources and services administration (hrsa; http://www.hrsa.gov/bioterrorism. htm) have strategic activities in education, training, licensure, and credentialing for the public health care workforce and for hospital readiness. the agency for healthcare research and quality also has strategic activities related to education and training, as well as uses of information technology and electronic communication networks (phillips, burstin, dillard, & clancy, ; . hhs's working definition of health surge capacity is the ability a health care system has to rapidly expand beyond normal services to meet the increased demand for medical care and public health services that would be required to care for patients in the event of a large-scale public health emergency or disaster. needed resources include beds, personnel to staff the beds, equipment, ability to transport victims and personnel, and the ability to provide ongoing care. all aspects of surge capacity present challenges, but the demand for qualified health care personnel is particularly complex. although nursing is not the only health profession experiencing a workforce shortage, nursing is vital to any largescale demand for care. nationally, there are , , licensed registered nurses, or registered nurses per , people (hrsa, ) . these numbers are insufficient to meet current capacity needs and would be woefully inadequate in the event of a mass-casualty incident. a masscasualty event would require mobilization of additional nurses from outside the affected jurisdiction. such a mobilization, however, would have to overcome issues of credentialing and licensing. when licensed health care clinicians arrived as good samaritans and volunteered after / , hospital administrators turned them away because they did not have the proper credentials. nurses must collaborate and coordinate and train for future crises. issues of competency, standards, and mechanisms for education and training must be approved to certify qualified nurses for mass-casualty events. a major step was taken in march , when the international nursing coalition for mass casualty education (incmce) was founded to ensure a competent nurse workforce in response to mass-casualty incidents. the incmce consists of organizational representatives from schools of nursing, nursing accrediting bodies, nursing specialty organizations, and governmental agencies. in july , incmce developed a set of national, consensus-based, validated competencies for all entry-level nurses (incmce, ) . nurses must also initiate systems that promote their ability to respond in the next crisis. in , president clinton signed presidential decision directive/nsc- (white house, ) , which established a national strategy for ensuring critical infrastructure protection, primarily cybersecurity. in , president bush replaced pdd- with homeland security presidential directive (white house, ) , which identified the roles of the health care and public health sectors. specifically, it charged the sector-specific agencies to "collaborate with appropriate private sector entities and continue to encourage the development of infor- mation sharing and analysis mechanisms" (pdd- ). under the directive, hhs established the healthcare sector coordinating council, which has responsibility for activities such as communicating potential risks, threats, and vulnerabilities to private organizations. nurses make up the largest health profession, yet they lack a mechanism that enables them to gather and disseminate nursing-specific information and communicate potential risks, threats, and vulnerabilities. a coordinating group comprising nurses from university, public health, and response settings, with a secure system that would allow collaboration on issues like identifying and providing a roster of volunteers, would be a good national, consistent approach to identifying and addressing vulnerabilities. this group would provide valuable insight to and receive vital information from the healthcare sector coordinating council and would disseminate that information to nurses throughout the country. nurses would be better prepared and the country would be safer. it is time for nursing to endorse such an entity and become engaged at this level of strategic initiative. by the american hospital association with the support of the office of emergency preparedness, us department of health and human services ahrq's bioterrorism research portfolio: real linkages in real time the agency for healthcare research and quality responds to emerging threats of bioterrorism homeland security presidential directive key: cord- -zzxjv authors: campanacci, valérie; egloff, marie‐pierre; longhi, sonia; ferron, françois; rancurel, corinne; salomoni, aurelia; durousseau, cécile; tocque, fabienne; brémond, nicolas; dobbe, jessika c.; snijder, eric j.; canard, bruno; cambillau, christian title: structural genomics of the sars coronavirus: cloning, expression, crystallization and preliminary crystallographic study of the nsp protein date: - - journal: acta crystallogr d biol crystallogr doi: . /s sha: doc_id: cord_uid: zzxjv the aetiologic agent of the recent epidemics of severe acute respiratory syndrome (sars) is a positive‐stranded rna virus (sars‐cov) belonging to the coronaviridae family and its genome differs substantially from those of other known coronaviruses. sars‐cov is transmissible mainly by the respiratory route and to date there is no vaccine and no prophylactic or therapeutic treatments against this agent. a sars‐cov whole‐genome approach has been developed aimed at determining the crystal structure of all of its proteins or domains. these studies are expected to greatly facilitate drug design. the genomes of coronaviruses are between and . kbp in length, the largest of the known rna viruses, and encode – mature proteins. the functions of many of these polypeptides, including the nsp –nsp replicase‐cleavage products, are still unknown. here, the cloning, escherichia coli expression, purification and crystallization of the sars‐cov nsp protein, the first sars‐cov protein to be crystallized, are reported. nsp crystals diffract to . Å resolution and belong to space group p ( / ) , with unit‐cell parameters a = b = . , c = . Å. with two molecules in the asymmetric unit, the solvent content is % (v (m) = . Å( ) da(− )). the recent epidemics of severe acute respiratory syndrome (sars) represent a real paradigm for emerging viral pathogens, as well as an example of worldwide coordinated efforts to control a serious viral outbreak, a test of the reaction time of the scienti®c community. the ®rst cases of severe acute respiratory syndrome originated from the guangdong province in south east china. the number of cases reported and our current knowledge regarding this illness are still currently evolving, but a number of basic facts have been ®rmly established. the aetiologic agent of sars is a positivestranded rna virus belonging to the coronaviridae family and its genome differs substantially from those of previously identi®ed coronaviruses, including two other human coronaviruses (peiris et al., ; ksiazek et al., ; drosten et al., ; snijder et al., ) . the virus, whose name sars-cov is now currently accepted, is mainly transmitted by the respiratory route. however, evidence for a secondary faeco± oral route of transmission has also been presented. the viral strain probably primarily infected wild animals traded in asian markets and crossed the species barrier to infect humans. there is to date no vaccine and no prophylactic or therapeutic treatments against this agent. a prophylactic treatment would have been useful to combat the epidemics; the only effective measure available to prevent the spread of the virus is to quarantine all persons that have been exposed to sars-cov. the number of antiviral molecules that can be used to treat patients infected by rna viruses is incredibly low. accordingly, it is important to search for ef®cient antiviral drugs for a large number of rna viruses, while giving priority to viruses transmitted by the respiratory route because they have the highest potential for causing pandemic outbreaks. the scienti®c community has reacted promptly and ef®ciently to identify and characterize this new infectious agent, as well as to develop methods for sars-cov detection and containment protocols. in the meantime, a wide effort is being made to design drugs active against sars-cov. ribavirin has been used in the absence of other candidates, but its intrinsic ef®ciency against sars-cov appears to be low (koren et al., ) . to select drugs active against a viral pathogen, one usually relies on screening candidate drugs for their ef®cacy in virusinfected cell cultures and/or animal models. however, during the current research on drugs for treating hepatitis c virus (hcv) infections, a novel and promising approach has been introduced. the rna-dependent rna polymerase of hcv has been puri®ed and crystallized and enzymatic tests have been used to ®nd potent nucleoside and non-nucleoside inhibitors of the virus, the structure±activity relationships of which allow further testing and clinical developments (de francesco et al., ) . this approach is gaining momentum owing to a concomitant increase in the power of new technologies and technological developments. among those, genomics approaches are being conducted to solve the crystal structures of large sets of clinically relevant proteins, which will become the subjects of future structure±function relationship studies. a crystal structure has not yet been determined for any of the predicted mature sars-cov proteins. the crystal structure of the main (or cl) protease of transmissible gastroenteritis virus, a related coronavirus, has been determined and was used to construct a model of the sars-cov cl protease, facilitating future drug design against this important target (anand et al., ) . the putative coronavirus rna-dependent rna polymerase has been puri®ed, but is inactive in vitro (grotzinger et al., ) . in this context, we have developed a sars-cov wholegenome approach aimed at determining the crystal structure of all sars-cov proteins. we anticipate that this will greatly facilitate drug design as well as the study of many other aspects related to the biology of these complex viruses. coronaviruses are enveloped viruses with a single-stranded rna genome of positive polarity (lai & holmes, ) . their genome is between and . kbp in length, the largest of the known rna viruses. like other coronaviruses, the sars-cov genome is known to encode two large replicase polyproteins (the orf a and orf ab proteins), which are processed into a set of mature non-structural proteins (nsps) by internal viral proteases (snijder et al., ) . the functions of many of these products, such as the nsp ±nsp polypeptides produced from the c-terminal domain of the orf aencoded polyprotein, are still unknown. in the related mouse hepatitis virus, which is a group coronavirus, the sars-cov nsp corresponds to a kda cleavage product (p a- ) that is found preferentially in the perinuclear region of infected cells, where it co-localizes with other components of the viral replication complex (bost et al., ) . no clues to the function of the nsp equivalent of any coronavirus have been obtained thus far. here, we report the cloning, expression, puri®cation and crystallization of the sars-cov nsp protein, a -residue protein ( fig. ) , which is the ®rst sars-cov protein to be crystallized. vero cells were infected with sars-cov (frankfurt- strain; ncbi accession no. ay ; drosten et al., ) at a multiplicity of infection of . . at the onset of the cytopathogenic effect (approximately h post-infection), intracellular rna was isolated by cell lysis for min at room temperature with % lithium dodecyl sulfate in let buffer ( mm licl, mm edta, mm tris±hcl ph . ) containing mg ml À of proteinase k. after shearing of the cellular dna using a syringe, lysates were incubated at k for min, extracted with phenol (ph . ) and chloroform and the rna was ethanol-precipitated. cdna was obtained by reverse transcription using primer sav ( h -ggacag-caaccgctggacaatc- h ), complementary to nucleotides ± of the frankfurt- genome, using thermoscript reverse transcriptase (invitrogen). the sars-cov nsp -coding sequence was ampli®ed by pcr from the cdna prepared above using two primers containing the attb sites of the gateway recombination system (invitrogen cultures were grown at k until od reached . and were then stored for h on ice; % ethanol was added for the induction of stress chaperones (gong & shuman, ) . expression was induced by adding mm iptg and cells were incubated for h at k. cells were collected by centrifugation and the bacterial pellets were resuspended and frozen in mm tris±hcl, mm nacl, mm imidazole ph . . cellular suspensions were thawed with . mg ml À lysozyme, . mg ml À dnase and mm mgso and were centrifuged at g. the supernatant was applied onto an ni-af®nity column connected to an fplc system (amersham pharmacia biotech). the protein was eluted with mm tris± hcl, mm nacl, mm imidazole ph . and then applied onto a preparative superdex gel-®ltration column pre-equilibrated in mm tris±hcl, mm nacl ph . . the recombinant protein was characterized by n-terminal sequencing, mass spectroscopy, dynamic light scattering (dls) and circular dichroism (cd). dls was performed with a dynapro microsampler (protein solutions) using a protein solution at . mg ml À in mm tris±hcl, mm nacl ph . . the cd spectrum of the ®nal puri®ed product was recorded between and nm on a jasco j spectrometer using a protein solution at . mg ml À in sodium phosphate buffer ph . containing mm nacl. crystallization screening was performed by vapour diffusion with nanodrops using a cartesian robot as described previously (sulzenbacher et al., ; vincentelli et al., ) . brie¯y, three commercial kits were used: wizard screens and (emerald biostructures), structure screens and and stura footprint screen (molecular dimensions ltd). the crystals were obtained in . m ammonium sulfate, . m phosphate±citrate ph . and with a protein concentration of . mg ml À in the gel-®ltration buffer. the optimization of the crystallogenesis was performed with nanodrops in a twodimensional matrix (lartigue et al., ) with a precipitant range of . ± . m ammonium sulfate and a ph range of . ± . ( . m phosphate±citrate), leading to a crystal size of $   mm (fig. ) . the crystals were cryocooled in a pure solution of silicone oil dc . they were exposed at beamline id -eh , esrf, grenoble using a quantum adsc q r detector. a total of oscillations were recorded with a crystal-to-detector distance of mm and a collection time of s per frame. diffraction data were integrated with denzo (otwinowski & minor, ) and were reduced with scala (collaborative computational project, number , ). we have subcloned sars-cov targets in the gateway system, including full-length proteins and protein domains. to date, constructs have been generated, of which were expressed, were soluble and ®ve were puri®ed. four of them led to small crystals, among which were those of the nsp protein described in this report. expression of selenomethionine-substituted nsp was performed using the method of methionine-biosynthesis pathway inhibition (doublie Â, ) . puri®cation of the selenomethionine protein was performed as described above and crystal optimization is under way. nsp crystals diffract to . a Ê at id -eh (esrf, grenoble). data integration and reduction indicate that they belong to the p space group. r sym is . %, an excellent value considering the redundancy of the data (table ) . re¯ections are observed at multiples of six along the c axis ( l), indicating that the space group is either p or its enantiomorph p . the unit-cell parameters are a = b = . , c = . a Ê , which lead to a v m value of . a Ê da À ( % solvent) with two molecules in the asymmetric unit (matthews, ) . the observed distribution of centric or acentric intensities overlaps with the theoretical curve, an indication that merohedral twinning, a feature that is often observed in trigonal or hexagonal crystals, is not present. sars-cov nsp has been puri®ed to homogeneity in two steps. the identity of the ®nal product has been con®rmed by n-terminal sequencing. the oligomeric status of nsp has been checked using gel ®ltration and dls. the former technique indicates that the protein is monomeric, while the dls analysis is consistent with a monodisperse species with an apparent stokes radius of a Ê and an equivalent mass of kda, which corresponds to a dimer. this discrepancy might be related to the concentration differences between the two techniques. a psi-blast search retrieved seven homologous sequences, all belonging to members of the coronaviridae family. they were aligned using multalign (corpet, ) with standard options. the consensus of the secondary-structure predictions obtained with jpred (cuff et al., ), psi-pred (mcguf®n et al., ) and predict protein (rost, ) converges to a fold of seven -strands. a foldrecognition analysis was performed with the threading programs d-pssm (kelley et al., ) and inbgu (fischer, ) . both programs fail to detect any protein homologue to nsp , but converge to a fold of two seven-stranded -sheets. in agreement, the cd spectrum of puri®ed nsp reveals a structured protein formed by a majority of -strands ( %) and -turns ( %), but which also contains % -helix. random-coil segments account for % of the total. the sarc-cov nsp protein expressed in e. coli was readily crystallized using the nanodrop screening (sulzenbacher et al., ) and optimization (lartigue et al., ) approaches. crystals diffract to . a Ê resolution and are amenable to structure determination using semet substitution and mad methods (hendrickson, ) at synchrotrons. nucleic acids res. , ± . cuff methods enzymol. fields virology acta cryst. d , ± methods enzymol. methods enzymol. acta cryst. d this study was funded by the spine project of the european union th pcrdt (qlrt- - ), by the french genopole programme and by the conseil general of the bouches-du-rhone. we thank h. w. doerr and h. rabenau (institute for medical virology, johan wolfgang goethe university, frankfurt-am-main, germany) for providing us with the virus and p. bredenbeek, s. gorbalenya and w. spaan for technical assistance and helpful discussions/ suggestions. key: cord- -e s el s authors: parashar, umesh d; anderson, larry j title: severe acute respiratory syndrome: review and lessons of the outbreak date: - - journal: int j epidemiol doi: . /ije/dyh sha: doc_id: cord_uid: e s el s nan , the chinese ministry of health notified the world health organization (who) of an outbreak of atypical pneumonia that likely emerged in guandong province, china, in november . during late february to early march , clusters of atypical pneumonia were recognized in vietnam, hong kong, canada, and singapore. [ ] [ ] [ ] [ ] [ ] epidemiological investigations revealed that the index patients for each of these clusters had stayed on the ninth floor of a hotel in hong kong on - february (figure ). further investigation indicated that the likely source of the outbreak was a physician from guandong province (case a) who stayed on the same hotel floor on february. this physician had cared for patients affected by the respiratory disease outbreak and he had been symptomatic with a febrile, respiratory illness since february. this dramatic chain of transmission brought to the world's attention this new respiratory disease, called severe acute respiratory syndrome (sars), and clearly illustrated the potential for sars to spread extensively from a single infected person and to rapidly disseminate globally through air travel. the who issued an historic global alert and, together with its international partners, initiated a rapid and intense response to this global public health emergency. the response led within weeks to the identification of the aetiological agent, sars-associated coronavirus (sars-cov), [ ] [ ] [ ] [ ] and to a series of decisive and effective containment efforts that interrupted the last chain of human transmission in less than months. in this article, we review what has been learned about the aetiological agent and its pathogenesis and pathology, clinical manifestations, epidemiology, and diagnosis plus strategies for control of sars. sars-cov is an enveloped, positive-stranded, rna virus in the coronaviridae family ( figure ). coronaviruses are associated with a variety of enteric and respiratory disease syndromes in several animal species. the two recognized human coronaviruses other than sars-cov, oc , and e, have definitively been associated only with upper respiratory illness. sars-cov was first cultured in vero e cells from autopsy tissue, bronchoalveolar lavage, and nasal swab samples obtained from patients hospitalized in hong kong, canada, germany, and singapore, with identification by electron microscopy and complete genomic sequencing. the aetiological role of sars-cov was confirmed by the replication of an illness similar to that seen among humans in experimentally infected cynomolgus macaques, followed by re-isolation of the virus and detection of a specific immune response in the infected macaques. , the genomic sequence of sars-cov is quite distinct from that of other human and animal coronaviruses, , suggesting that the virus has likely circulated in its natural reservoir for a considerable period of time. the search for the origins of sars-cov and its potential reservoir(s) is ongoing. as many as % of the early sars cases in guandong occurred among people who were involved in animal trade or in food preparation, and people involved in animal trade in guandong were more likely to have antibodies to sars-cov than those who did not trade animals or general population controls. in addition, a coronavirus with % homology with human sars-cov isolates was recovered from civet cats and a racoon dog sold live for food in markets in guangdong. collectively, these observations support the hypothesis that sars-cov was first transmitted from wild animals used for food to humans, with subsequent personto-person transmission. experimental studies have shown that ferrets and domestic cats are also susceptible to infection by sars-cov and that they can efficiently transmit the virus to previously uninfected animals that are housed with them. further clues to the animal reservoir for sars-cov might be provided by studies of wild and domesticated animals from animal markets in guandong and the genomic sequence analysis of any viral isolates obtained from these species as well as from human patients. studies have also demonstrated the presence of antibody to sars-cov or a sars-cov-like virus in a small proportion of healthy adults in hong kong who had sera banked in may , at least years prior to the sars outbreak. this finding suggests that sars may have been transmitted periodically in the past from animals to humans and the virus may have evolved and adapted to human infection and transmission or that chance events resulting in efficient transmission led to the outbreak in . angiotensin-converting enzyme (ace ) was recently identified as a functional sars-cov receptor, and ace is efficiently expressed in lung, heart, kidney, and gastrointestinal tissue in humans. sars-cov has been isolated in cell culture in autopsy specimens from lung, intestinal, and kidney tissue of patients who died of sars. , sars-cov rna can also be detected by reverse transcription-polymerase chain reaction (rt-pcr) assays in the plasma and serum of more than % of patients during the first week of illness. pathological findings in the lungs of sars patients during the first days of illness include pneumocyte proliferation and desquamation, hyaline membrane formation, mixed inflammatory infiltrate, and intra-alveolar oedema. , increased numbers of interstitial and alveolar macrophages, with focal haemophagocytosis in interstitial macrophages, have also been described. in cases of longer duration, diffuse alveolar damage, squamous metaplasia, and multinucleated giant cells, of macrophage origin, are observed. biopsy and autopsy specimens from the colon and terminal ileum of sars patients show relatively normal architecture with no evidence of villous atrophy or inflammatory infiltrates. no distinct histopathological changes have been described in other tissues. the role of cytokines and immunopathogenic mechanisms in sars are being investigated. lymphopaenia is common among patients with sars. while cd t lymphocytes counts are reduced in nearly all sars patients, reductions in circulating levels of cd t lymphocytes, b lymphocytes, and natural killer cells are also common. , cd and cd lymphocyte counts fall early in the course of illness, and low cd and cd levels have been associated with more severe illness. the presence of lymphopaenia and pathological manifestations of diffuse alveolar damage, destruction of the white pulp of the spleen, and haemophagocytosis suggest that proinflammatory cytokines released by stimulated alveolar macrophages may have a major role in the pathogenesis of sars. death from sars usually occurs late in the course of illness (Ͼ week after onset) and has been attributed to adult respiratory distress syndrome, multiorgan failure, thromboembolic complications, secondary infections, and septic shock. coinfection with human metapneumovirus has been reported among patients with laboratory-confirmed sars; however, the role of co-infection in amplifying the pathogenicity of sars-cov is unknown. sars-cov disease has a median incubation period of approximately - days; most patients become ill within - days after exposure. the most common initial symptom is fever, often accompanied by headache, myalgias, malaise, chills, and rigor. respiratory symptoms typically do not begin until - days after onset of fever, but may be present at onset in up to one-third of patients. lower respiratory tract symptoms are common and typically include a non-productive cough with later onset of dyspnoea. upper respiratory symptoms such as rhinorrhoea and sore throat are seen in less than % of patients. diarrhoea has been reported at presentation in approximately % of patients, although this symptom was observed in as many as % of all patients affected by an outbreak at an apartment complex in hong kong that is believed to have resulted from fecal-oral/respiratory transmission of sars-cov. respiratory signs such as rales and rhonchi are present in less than one-third of cases, and their severity often does not correlate with the findings seen on chest radiographs. elderly patients and those with underlying chronic illnesses such as renal failure sometimes present with atypical symptoms, including the absence of fever. in general, sars appears to be milder in children. , haematological abnormalities include lymphopaenia ( - % of patients), thrombocytopaenia ( - %), prolongation of the activated partial thromboplastin time ( - %), and elevated levels of lactate dehydrogenase ( - %), alanine or aspartate aminotransferases ( - %), and creatinine phosphokinase ( - %). none of these laboratory findings can reliably discriminate between sars-cov disease and other atypical pneumonias. chest radiographs may be normal in up to % of patients who are evaluated early in illness, and high-resolution chest computed tomography scans can detect abnormalities in up to two-thirds of patients with normal chest x-rays. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] nearly everyone with sars-cov disease develops radiographic evidence of pneumonia by illness day - . the radiographic abnormalities typically begin as isolated, focal, ground glass opacities predominately in a peripheral location, often in the lower lobes, and progress over several days to focal, multifocal, or diffuse consolidation involving additional lobes or both lungs; mediastinal lymphadenopathy, cavitation, atelectasis, and pleural effusions are uncommon. some - % of patients with sars-cov disease require mechanical ventilation, and about half of these people die despite ventilation therapy. severity of illness and risk of death from sars increases with advancing age and in the presence of certain underlying medical conditions, particularly diabetes mellitus. the overall case-fatality rate of approximately % can increase to Ͼ % in people older than age . during the global epidemic, cases of probable sars with ( . %) deaths were reported in countries. the countries with the greatest number of reported cases included mainland china (n = ), hong kong special administrative region (n = ), taiwan (n = ), singapore (n = ), and canada (n = ). health care facilities were severely affected and transmission in hospitals was a major factor in the amplification of outbreaks. in addition to health care workers, who accounted for % of all cases globally, other patients on the same ward as sars patients and visitors to the hospital were affected. for example, of cases reported from april to june in toronto, canada, % occurred among health care workers, % resulted from exposure during hospitalization, and % occurred among hospital visitors. after health care facilities, households of sars patients were the second most common setting of sars-cov transmission. surveys in hong kong and singapore indicated that - % of people in the households of sars patients may have been secondarily infected. , the risk of transmission was greatest among those involved in direct patient care or other close contact with the patient. transmission to casual and social contacts appears to be uncommon, but secondary cases have been documented after exposures in the workplace and on airplanes and other conveyances. , the epidemiology of sars outbreaks suggests that sars-cov is transmitted primarily through droplets and close personal contact, a conclusion that is supported by the finding that surgical masks confer protection against infection. , studies documenting stability of the virus for days in the environment raise the possibility of fomite transmission, and in a few instances, possible transmission by small-particle aerosols cannot be excluded. in particular, aerosol-generating medical procedures (e.g. endotracheal intubation, bronchoscopy) may be associated with an increased risk of transmission in health care settings. [ ] [ ] [ ] [ ] given that profuse watery diarrhoea is seen in a significant proportion of patients and sars-cov can be shed in large quantities in stool, faeces remain a possible source of virus and fecal-oral or fecal-respiratory spread are the leading hypotheses for a large outbreak affecting more than people at an apartment complex in hong kong. mathematical modelling studies indicate that each sars case infects an average of two to four people. [ ] [ ] [ ] however, some sars patients are very efficient in transmitting sars-cov to susceptible people under certain circumstances, leading to socalled 'super-spreading events'. , [ ] [ ] [ ] [ ] super-spreading events have occurred in a number of settings, including the hotel in hong kong, from which the sars outbreak disseminated globally, and hospitals in many locations. the factors contributing to super-spreading events are not completely understood but may be related to the inherently greater infectiousness of some patients, alternative modes of transmission, or the exposure of large numbers of contacts in an environment conducive to transmission. asymptomatic sars-cov infections have been documented, but they appear to be uncommon and their epidemiological significance in disease transmission has not been established. the risk of transmission from patients appears to be greatest during the second week of illness, which correlates with the timing of peak viral load in respiratory secretions. the timing of peak infectiousness might also explain, in part, the predilection for sars-cov to spread in health care settings, as most patients seek medical care by the end of the first week of illness. although sars-cov rna can be detected in fecal specimens by rt-pcr for more than a month after the onset of illness, no transmission has been documented more than days after the resolution of fever. genome sequencing analyses indicate that sars-cov isolates from the outbreaks in hong kong, vietnam, singapore, toronto, and taiwan are closely related and match the viral isolate obtained from the ill physician from guandong province, supporting the epidemiological conclusion that each of these outbreaks was directly or indirectly linked to the ill physician. on the other hand, sars-cov isolates from guangdong province exhibit greater diversity and other viruses belonging to genetic lineages different from the global outbreak strain were introduced into hong kong prior to march , but these introductions did not lead to large outbreaks. these data raise the question of whether some viral strains may have an inherently greater epidemic potential; this hypothesis needs further evaluation. alternatively, differences in transmission unrelated to virus strain (i.e. super-spreading events) could explain the presence or absence of outbreaks associated with independent human infections. the clinical manifestations of sars-cov disease are not sufficiently distinct from those of other respiratory pathogens to permit a reliable differential diagnosis. however, a combination of clinical and epidemiological features can provide clues to the diagnosis of sars. with no documented person-to-person transmission of sars-cov worldwide, the predilection for sars-cov to cause unusual clusters of pneumonia in health care settings provides a means to focus on surveillance efforts. if the re-emergence of sars-cov is confirmed, then a history of exposure to known sars casepatients or sars-affected areas may be helpful in early recognition of patients. the laboratory diagnosis of sars-cov is based on either detection of viral rna in clinical specimens or the finding of antibodies directed against sars-cov in serum. viral isolation is not recommended for routine diagnosis because of its low sensitivity and the biosafety hazards it poses. available real-time rt-pcr assays for sars-cov are highly specific and can detect as few as - copies of viral rna in clinical specimens, but a low viral load in respiratory and fecal specimens during the first week of illness limits the clinical sensitivity and utility of these assays. limited data suggest that sars-cov rna can be detected in the serum of more than % of sars patients during the first week of illness. because of the potential for contamination, positive rt-pcr results should be confirmed by testing a second specimen in a reference laboratory. the identification of serological antibody to sars-cov remains the reference standard for confirmation of sars-cov infection. however, antibody is usually detectable only after the first week of illness and some patients may not mount an antibody response for up to - days after illness onset. it is important to note that neither a negative rt-pcr test nor a negative test of serum obtained Ͻ days after illness onset can reliably exclude infection with sars-cov. body fluids that should be submitted for testing from patients with suspected sars include respiratory tract secretions, serum, whole blood, and stool. lower respiratory tract specimens (e.g. sputum, bronchial alveolar lavage fluid) appear to have higher yield than upper respiratory tract specimens (e.g. nasal or pharyngeal aspirates or swabs). collection of multiple specimens of different types is likely to increase the overall diagnostic yield, and different specimens will have a greater diagnostic yield at different times in illness (table ) . sars-cov test results should always be considered in the context of clinical and epidemiological findings. in the setting of no sars human-to-human transmission worldwide, the positive predictive value of a sars-cov test is extremely low, and thus any positive laboratory result must be interpreted with extreme caution because of its implications for global public health. diagnostic assays for other respiratory pathogens may be helpful in differentiating sars-cov disease from other illnesses, but sars patients may be simultaneously infected with sars-cov and another respiratory pathogen. factors that should be considered in the evaluation of patients with dual infections include the strength of the epidemiological exposure criteria for sars-cov disease, the specificity of the alternate diagnostic test, and the compatibility of the clinical presentation and course of illness with the alternative diagnosis. in the absence of a vaccine, effective drugs, or natural immunity to sars-cov, the key to controlling sars is the classic public health control strategy of case identification and containment. these measures proved effective and were associated with cessation of transmission throughout the world by july . the key to containment efforts is a surveillance system that provides ready access to timely information on the number of new cases, the likely source of exposure for cases, the number of cases not previously identified as contacts, and the number of contacts with high-risk exposures to known cases (potential prospective cases). since sars anywhere has implications locally, nationally, and globally, it is essential that the health care and public health communities exchange information on individual sars cases and the status of sars transmission in the community and globally. once a potential case of sars is detected, appropriate infection control measures must be implemented immediately to prevent transmission. sars patients often require hospitalization because of the severity of their illness; those with less severe illness are sometimes hospitalized to ensure that strict isolation procedures are followed. in other settings, patients have been managed in residential settings after evaluation of their suitability for this purpose. because the possibility of airborne transmission cannot be excluded, patients who require hospitalization should preferably be admitted to an airborne infection isolation room or specially adapted sars unit or ward where they can be treated safely and appropriately. in some settings, a lack of isolation rooms and/or a need to concentrate infection control efforts and resources may lead to a strategy of grouping patients in individual rooms on the same floor, which proved effective in many settings in the outbreak. health care workers should strictly adhere to use of appropriate personal protective equipment, and the potential for transmission to other non-sars patients and hospital visitors should be minimized through administrative controls. contact tracing, the identification of people potentially exposed to a case of sars, is essential to prevent spread. this provides a means to focus control efforts on people who are at high risk of sars, to identify people early in the course of their illness, and to implement control measures before they can spread the virus to others. contact tracing, evaluation for illness, and monitoring should be an immediate high priority to maximize the chance to rapidly control an outbreak. during the outbreak, quarantine of exposed people was one of the contact management strategies used to prevent inadvertent sars-cov exposures by separating those exposed from the unexposed. this potentially decreased the interval between the onset of symptoms and the institution of control measures. other key components of an effective sars control strategy include the following: ( ) systems for rapid and frequent communication of crucial information about sars; ( ) education and training of public health and health care workers about the clinical and epidemiological aspects of sars, appropriate use and interpretation of laboratory tests, and best practices for effective use of infection control strategies; and ( ) efficient information technology systems that provide a means to link clinical, epidemiological, and laboratory data on sars cases and to disseminate this information locally, nationally, and globally, and systems that allow rapid identification, tracking, evaluation, and monitoring of contacts of sars cases. the emergence of sars-cov dramatically illustrates the potential for a new disease to suddenly appear and spread, leading to widespread health, social, and economic consequences. fortunately, the experience also demonstrates the power of traditional public health measures-including surveillance, infection control, isolation, and quarantine-to contain and control a sars outbreak. it is not possible to predict when and where sars-cov will reappear and whether it will cause similar outbreaks in the future. possible sources of virus for a re-emergence of sars-cov include its original animal reservoir, persistent infection in humans, or the laboratory. the recently detected sars-cov cases in china are hypothesized to represent animal-to-human transmission, , and each of the laboratory-acquired cases in singapore and taiwan highlight the need for stringent biosafety precautions. , to achieve the type of swift and decisive response that is required to control a sars outbreak, we must be prepared and have learned from the many lessons of the sars outbreak. preparation for a response to an outbreak of sars requires co-ordination and co-operation among public health, health care, and other emergency response entities at all levels of government. investments in sars preparedness are likely to yield additional dividends in preparedness to battle other emerging infectious diseases and other threats to public health. epidemiological and aetiological studies of 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systems date: journal: lab anim (ny) doi: . /laban - sha: doc_id: cord_uid: mi saerx housing rodents in ivc racks has many advantages over conventional cages but also presents unique challenges related to health monitoring. the authors review the issues to consider in design of a sentinel program using ivc systems. there exist many methods and strategies for monitoring rodent colonies for microbial agents. several general principles are applicable when developing and assessing the efficacy of a microbiological monitoring program, irrespective of the type of caging used for rodent housing. first and foremost, the monitoring program must specifically target the infectious agents of concern. in particular, there should be consideration of the route and duration of transmission of the infectious agents, the probability that the agents will be found, and the likelihood that the agents will cause disease or affect research results. second, the monitoring program should make the most efficient use of personnel and resources. finally, the outcome of a successful monitoring program should aid research, not hinder it, and should result in animals that are sufficiently pathogen free for research and should facilitate exchange of animals between institutions. rodents in contemporary animal colonies live in several types of caging. the three types of caging systems commonly used are open-top cages, static isolator cages, and individually ventilated cages (ivcs). the type of housing used affects the ease with which infectious agents can be spread between rodents housed in separate cages. rodents housed in open cages and serviced in the open have the highest risk of transmitting infectious agents from cage to cage via aerosols and fomites. this is one reason why this type of housing is being eliminated in many facilities. rodents housed in static isolator cages have a lower risk of infectious-agent transmission than do rodents housed in opentop caging, because the filter lid serves as a physical barrier to infectious-agent transmission, although transmission can occur if filter lids are not properly installed or during cage changes. it has also been shown that the exhaust gases do not actu-ally pass through the filter material in the lid but escape through the gap between the cage and the cage lid . this is the reason that many facilities have instituted the use of ventilated cage change stations for husbandry and experimental manipulations of rodents housed in static isolator cages. ivcs differ from static isolator cages in several ways. each cage receives a supply of hepa filtered air at a rate of between and air changes per hour (ach). this results in lower temperature and humidity as well as lower concentrations of ammonia and carbon dioxide than in static isolator cages . some ivc systems balance the air supply with the air exhaust system, thus permitting control of differential pressure at the cage level. thus each cage, theoretically at least, is its own biocontainment zone. an alternative approach in some ivc systems is to seal the cages to achieve biocontainment (instead of balancing differential pressure). this approach has, however, the obvious disadvantage of being potentially lethal in the event of cage ventilation failure , . rodents housed in ivcs have the lowest risk of cage-to-cage spread of infection [ ] [ ] [ ] [ ] [ ] [ ] (j. schmidt, personal communication). transmission between animals housed in ivc systems generally occurs as a result of dissemination during husbandry procedures or experimental manipulations by investigators, thus justifying the use of ventilated cage change stations for such activities. nevertheless, it is essential to exercise great care to ensure against transmission by fomites on hands or equipment, such as watering valves, particularly when active infections are present , . monitoring methods, such as placement of room sentinels adjacent to the hvac exhaust duct, as used traditionally with mice housed in open-top caging, require reassessment with regard to their efficacy in static isolator cages and in ivc systems. tion of the testing methods (e.g., serology on immunodeficient animals). the use of sentinel mice to monitor a colony decreases the number of mice needed to detect an infection within a colony, because each sentinel can serve in monitoring of many colony mice. there are three types of sentinel animals. first, contact sentinels (i.e., mice housed in the same cage as the colony mice to be monitored) are highly reliable at detecting infectious agents transmitted by all routes (air, feces, urine, wounds, contact, etc.). second, soiled-bedding sentinels (i.e., mice housed on soiled bedding that has been removed from several cages of colony mice) are most effective at detecting agents that are transmitted in feces or urine. third, exhaust air sentinels (i.e., mice that are exposed to rack exhaust air) are effective at detecting agents that are transmitted in respiratory excretions. lipman and homberger provide an overview of the advantages and disadvantages of the three types of sentinels. finally, direct detection of infectious agents present within the environment of a colony is feasible by swabbing such surfaces as cage and cage racks, by taking appropriate samples from rack exhaust air, and so on. several methods exist for testing samples collected from mice or from their environment. traditionally, most viral infections are detected using serological assays. the advantages of serological assays are that they are inexpensive and can provide a historical picture of which agents are present in the colony (i.e., at any time after a mouse becomes seropositive, antibodies to the rel-evant agent can be detected). the primary disadvantage of serological assays is that mice infected with an agent may not become seropositive until or more weeks after the infection's initiation. therefore, in the midst of an outbreak, many mice may become infected and may potentially be transmitting virus to other mice, yet may still be seronegative. additionally, immunocompromised mice, especially those with b-cell deficiencies, may produce few or no antibodies in response to an infection. recently, molecular methods have augmented our arsenal of testing methods. molecular methods, in particular pcr and rt-pcr, detect the nucleic acids present within a bacteria or virus. in general, these methods are highly sensitive and rapid, and can be very effective means to determine which animals are infected with a particular agent. molecular methods are particularly useful during outbreaks, when it is essential to determine rapidly (within a few hours) the location of infected mice within a rack, so as to develop a strategy for containing the spread of the agent. the limitations of molecular methods are that they are relatively expensive, they can yield both false negative and false positive results, their high sensitivity makes them prone to cross-contamination, and many substances found in blood, feces, and other animal tissues can function methods such as exposing sentinels to soiled bedding, as used traditionally with mice housed in static isolator cages, also require reassessment for their applicability in ivc systems. moreover, we should add to our monitoring approach new methods specifically designed to exploit ivc system characteristics by the sampling of air exhausted from the ivc system. there are two ways of monitoring the exhausted air, either from individual cages or from the whole ivc rack. small gauze filters placed on the cage exhaust opening, on the exhaust opening from a zone of the rack (e.g., a row or column of cages), or on the exhaust manifold of the entire rack can permit periodic monitoring of the effluent air. after removal from the rack, molecular pcr-based methods can detect infectious agents on the filters. additionally, there can be continuous monitoring of exhaust air from the ivc rack (or a selected zone of the rack) that is achieved by testing of sentinels housed in a specially designed cage (bioscreen system; biozone inc., fort mill, sc, and biozone ltd., margate, uk) that receives as its air supply a portion of the mixed exhaust air from several cages on the rack, before hepa filtration (fig. ) . the effectiveness of such a system is clearly dependent on the uniformity of ventilation of all cages on the rack so as to be confident that a low incidence of infection will be represented in the exhaust air without regard to the location on the rack of the infected animals . there are many ways of detecting an infectious agent that has been transmitted to one or more rodents housed in ivcs. the most direct method of surveillance is to monitor the colony mice themselves for evidence of an infection. this method seems highly reliable on first consideration, but it presents significant drawbacks. if the animals are properly screened, considering that each cage is essentially a self-contained unit, then it is by far the least cost-effective method, because it requires the largest investment of mice, staff, and testing reagents. moreover, the age, genotype, and experimental use of the animals may interfere with the applica- as inhibitors of the enzymes used in these assays, causing false negative results. finally, the nucleic acids detected may be present in a noninfectious form of the agent, such as in inactivated material present on the surface of cages or change stations. detection of bacteria usually follows culture of fecal, cecal, nasopharyngeal, or other tissue samples on specialized agar plates. specific identification of pathogenic bacteria after culture usually involves a combination of enzymatic and fermentation tests. microscopy is the usual method for visual detection in diagnostic specimens of endoparasites and ectoparasites. there are several infectious agent-specific variables that affect the ease with which an agent can be detected in mice housed in an ivc system. the first influence is the route(s) by which the agent is shed from the animal ( table ). agents that are transmitted only through animal-animal contact, such as mites, are very difficult to detect in mice housed in an ivc system. it is possible to detect these agents directly in colony animals or using contact sentinels. one can effectively detect agents that are transmitted via the fecal-oral route, such as mouse parvovirus (mpv), using contact sentinels, sentinels exposed to soiled bedding, or molecular testing of feces from colony animals, but exhaust air sentinels are not effective at detecting these . in contrast, the use of contact sentinels, exhaust air sentinels, or molecular testing of cage surfaces is effective in detecting agents that are shed in respiratory secretions, such as sendai virus, but sentinels exposed to soiled bedding are ineffective at detecting these . the methods used to sample the cage or rack surfaces for air-borne infectious agents (cage wipe material or protocols, filter material, or placement location) may influence the effectiveness of detecting the agent of interest. for example, our lab detected sendai virus rna on the cages of experimentally infected mice for weeks using calcium alginate swabs wet with saline, whereas another group was able to detect sendai virus rna on the cages of experimentally infected mice for only days using alcohol wipes , . a second major influence that affects the ease with which an infectious agent can be detected in mice housed in an ivc system is the infectious-agent load to which the sentinel mice are exposed. infectiousagent load depends on the duration of shedding, on the concentration of agent shed, and on the stability of the agent after it is shed ( table ) . detection of agents such as murine rotavirus (epizootic diarrhea of infant mice, or edim), which causes an acute infection in adult immuno- erally shed at high concentrations, are much easier to detect using all monitoring methods, but the reliability of detection is still a problem [ ] [ ] [ ] . the stability of the infectious agent in the environment also influences the difficulty of its detection ( table ) . for agents that persist as infectious particles in the environment for only short periods of time, such as most enveloped viruses, detection is more difficult than for agents that are stable as infectious particles for long periods in the environment, such as parvoviruses or pinworm eggs. labile agents can be missed if the timing of exposure by contact, exhaust air, or soiled bedding does not coincide with the period of shedding. even though many enveloped viruses, such as coronaviruses, lose their infectivity upon drying and rupture of the envelope, the nucleic acids inside the viral particles remain detectable . therefore, molecular assays can detect the recent presence of agents even after they lose their infectivity and transmissibility to mice within the colony. for monitoring methods that detect infectious agents in the exhaust air, the size of an agent should theoretically affect its detectability. exhaust air and particulates should carry smaller agents such as viruses more effectively than larger agents such as parasites ( table ) . for example, mpv and helicobacter hepaticus both cause chronic intestinal infections and are shed in feces, yet mpv, a -nm virus, was detectable on filters placed in the rack exhaust air, whereas h. hepaticus, a -to -µm bacterium, was not . there are several system-specific conditions that may influence the efficacy of microbiological monitoring of mice housed in an ivc system. the air change rate that is achievable in an ivc rack is much higher ( - ach) than that achieved in static isolator cages (> ach) . a recent study showed that the higher air change rate in an ivc system compared with static isolator cages resulted in lower relative humidity . furthermore, the mean bedding weight gain per mouse was % greater in static isolator cages than in the ivc system. the less humid cage environment present in the ivc system could result in the dehydration and inactivation of many infectious agents. this effect, together with the dilution of organisms in the exhaust air by the higher ventilation rate, could lead to decreased transmission of agents to colony and sentinel mice. the decreased intercage spread of an infectious agent means that infections are sporadic and confined to just a few cages at a time, and it is therefore essential to use an adequate sample size when monitoring mice housed in ivc systems. several studies have shown that the high airflow achieved in ivc systems results in lower ammonia levels than those seen in static isolator cages at - days after cage change , - . the lower ammonia levels measured in ivcs are probably the direct result of lower relative humidity, because high humidity has been linked to proliferation of urease-positive bacteria that can convert urea to ammonia . furthermore, the type of bedding used in ivc systems can affect the rate of ammonia accumulation, because some bedding types may contain endogenous ureases . the frequency of cage change generally depends on the time required for intracage ammonia concentrations to rise to a level considered irritating to the mucous membranes of husbandry personnel (generally > p.p.m.) . because ammonia levels rise more quickly in static isolator cages, it is generally necessary to change static isolator cages at least once a week, whereas mice housed in ivc systems may need changing only once every weeks. less frequent cage changing decreases demands on personnel time, decreases the quantity of bedding used, increases cage longevity, and may decrease pup mortality , but it can also decrease the efficacy of soiled-bedding monitoring. if husbandry personnel add soiled bedding to sentinel cages only during routine biweekly cage changes, then this reduces the number of times that soiled bedding will be added to the sentinel cage in the ivc system as compared with static isolators, and agents pre-competent mice, with shedding of virus for ∼ days, can be very difficult. direct tests for the agent in the animal or the environment must occur during the short window of shedding and therefore are quite unreliable at detecting a low-level infection within a colony. detection of edim using serology and sentinels exposed to soiled bedding has the limitation that the timing of the soiled-bedding transfer must coincide with the short period of shedding to be effective. in ivc systems, a biweekly cage-changing schedule, with biweekly addition of soiled bedding to sentinel cages, has the disadvantage that the bedding transferred may or may not contain infectious virus. for agents that cause acute infections, methods that monitor the mice continuously over an extended period, such as seroconversion of colony mice, contact sentinels, exhaust air sentinels, or long-term placement of gauze filters in the exhaust air stream, are most effective. frequent monitoring, using sentinels exposed to soiled bedding or shortterm placement of gauze filters in the exhaust air stream, can also be effective. periodic monitoring methods, such as sentinels exposed to soiled bedding, and continuous monitoring methods, such as exhaust air sentinels, are both likely to be effective at detecting agents that cause chronic or persistent infections. the concentration of an excreted infectious agent influences the difficulty of its detection. agents, such as mpv, that cause a chronic infection with low-level fecal shedding, can be difficult to detect. detection of these agents necessitates testing an adequate sample size. a sample size that is too small or a monitoring protocol that samples a nonrepresentative group of cages can result in sporadic detection of the agent within a colony. for example, in a recent study, mice that received a single dose of soiled bedding from a group of cages in which % of the cages housed mpvinfected mice seroconverted, whereas those that received several doses of soiled bedding from a group of cages in which only % of the cages housed mpv-infected mice did not seroconvert . agents, such as mouse hepatitis virus (mhv), that are gen-volume , no. lab animal november sent in the soiled bedding have a longer time in which to become noninfectious before being exposed to susceptible sentinel animals. it will be necessary to optimize soiled-bedding sentinel monitoring protocols used in ivc systems so they can accurately detect agents if they are present. this means following a strict protocol to avoid bias by regularly sampling all cages on the rack, and one should also consider supplemental sampling of soiled bedding between cage changes. both of these have implications for labor and may have the negative effect of increasing the probability of cageto-cage transmission during soiled-bedding collection. the rate at which a particulate accumulates on a filter may also affect the sensitivity of filter-based tests. agents such as mpv are present at low concentrations in exhaust air. recently, we reported that mpv dna was not detectable on filters placed on the rack exhaust filter for h, but mpv dna was detectable on filters placed on the rack exhaust filter for weeks . the positioning of filters within the ivc rack can also influence the chance that an agent will be detected. for example, mpv, mhv, and sendai virus were all detectable on filters placed on the lids of cages housing infected animals for h at days after inoculation, but only mhv and sendai virus were detectable on filters placed on the rack exhaust filter for h at days after inoculation . this indicates that the closer to the source of the infection, where the infectious-agent concentration in the exhaust air is highest, the greater the likelihood of detecting it. it will be necessary to determine for each ivc system the optimal positioning of test filters and the time required to accumulate adequate particulates, which serve as fomites to transport infectious agents through the air, and should consider the ventilation pattern of the system. theoretically, any location within the exhaust system of the ivc rack that a small ( cm ) filter can be temporarily placed can serve as a test site. the type of bedding used in ivc cages could affect the rate of particulate generation and accumulation on filters and therefore could affect air-testing efficacy. aspen chip bedding has been found to be substantially less dusty than wood fiber or straw pelleted bedding . theoretically, the greater the amount of dust generated from bedding, on which agents can be carried through the air, the greater the chance of detecting the agent on a filter. the accuracy of monitoring methods involving exhaust air sampling is dependent on the uniformity of the airflow achieved in the ivc rack and thus on the ivc rack design. when infections are present in only a small number of cages on an ivc rack, as is often the case, the reliability of exhaust air monitoring, whether using gauze filters placed on the rack prefilter or using exhaust air sentinels, is likely to be highly dependent on the uniformity of exhaust airflow within the rack. nonuniform airflow resulting in large variations in exhaust airflow from individual cages may be a cause of inaccurate or unreliable sampling of cages housing infected mice seen in some studies (b. tiemann, personal communication). furthermore, some ivc systems have a single manifold that is used for supply air only, and exhaust air simply escapes from the cage into the room. in such ivc systems, it may be impossible to appropriately sample exhaust air either by using gauze filters or with exhaust air sentinels. one can operate ivc racks using either a positive or negative air pressure differential. under a positive air pressure differential (bioexclusion mode), the supply fan for the rack pushes more air into the rack than is pulled out by the exhaust fan, resulting in a small amount of air leaking out of the cages into the animal room. under a negative air pressure differential (biocontainment mode), the supply fan pushes less air into the rack than is pulled out by the exhaust fan, resulting in a small amount of air being sucked into the cages from the animal room. theoretically, different air pressure differentials might affect the efficacy of air monitoring methods, but in our experience the efficacy of microbiological monitoring was equivalent under positive and negative air pressure differentials . it is important to remember that in an ivc system, the primary means of infectious-disease spread within a rack occurs during husbandry procedures or experimental manipulations. during an outbreak, monitoring exhausted air from individual cages using cage-top gauze filters, or monitoring exhausted air from the entire ivc system using filters placed on the rack exhaust manifold, can be highly efficient in determining the extent of an infection within a rack or within a facility, and in confirming elimination of an agent after such an outbreak. both of these filter-based methods do not require handling of potentially infected mice and can produce results within a few days. this can be very valuable when handling animals for sentinel bleeding or for collection of soiled bedding could lead to spread of the infection. rodent microbiological monitoring continues to be a highly dynamic, nonstandardized, and continually evolving process. monitoring strategies and approaches to diagnostic sample collection are confounded regularly by the convergence of innovative husbandry refinements, new developments in a wide array of caging systems, evolving pathogens, and novel rodent genotypes. in this environment of constant change, and often in the face of relatively meager financial resources for assessment of these mitigating influences, the validation of monitoring approaches has proved to be a challenge. consequently, we recommend a multifaceted approach to monitoring for infectious agents in ivc systems. each monitoring program should consider both infectious agent-related and equipment-related variables. the routes of transmission for the infectious agents to be monitored, the duration and infectiousload shed, and the stability of the agent in the environment should all inform the decision as to which approach to monitoring is appropriate. likewise, the frequency of testing should specifically target the battery of agents to be tested and the type of caging used. it is also essential to understand and consider airflow rates and uni-formity of airflow in each type of ivc system. until techniques and technology, still to be developed, simplify the monitoring process, a fully integrated program of microbiological monitoring should make use of a sensible combination of soiledbedding sentinels, contact sentinels, and, where possible, exhaust air monitoring. even a broad sentinel-based monitoring program, however, is unlikely to be fully reliable and completely sound. consequently, the most comprehensive monitoring program should also include health evaluations of colony rodents that present as clinical cases. received / / ; accepted / / . the international joint meeting twelfth iclas general assembly & conference, seventh felasa symposium assessment of static isolator cages with automatic watering when used with conventional husbandry techniques as a factor in the transmission of mouse hepatitis virus efficacy of three microbiological monitoring methods in a ventilated cage rack rodent quality assurance testing: use of sentinel animal systems detection of sendai virus and pneumonia virus of mice by use of fluorogenic nuclease reverse transcriptase polymerase chain reaction analysis reverse-transcriptase polymerase chain reaction-based diagnosis and molecular characterization of a new coronavirus strain isolator rodent caging systems (state of the art): a critical view the effects of intracage ventilation on microenvironmental conditions in filter-top cages micoenvironment in ventilated animal cages with differing ventilation rates, mice populations and frequency of bedding changes ammonia build-up in animal boxes and its effect on fat tracheal epithelium characterization and qualification of microenvironmental contaminants in isolator cages with a variety of contact beddings american conference of governmental industrial hygienists. threshold limit values for chemical substances and physical agents and biological exposure indices the impact of reduced frequency of cage changes on the health of mice housed in ventilated cages the bedding of laboratory animals as a source of airborne contaminants the influence of filter top caging on the transmission of pinworm infections in mice an evaluation of intra-cage ventilation in three animal caging systems comparison of environment and mice in static and mechanically ventilated cages with different air velocities and ventilation designs failed air supply to individually ventilated caging system causes acute hypoxia and mortality in rats carbon dioxide concentrations in unventilated ivc cages evaluation of isolator caging systems for protection of mice against challenge with mouse hepatitis virus effectiveness of pressurized individually ventilated (piv) cages in reducing transmission of pneumonia virus microbiological validation of the m.i.c.e. caging system performance evaluation of a positive/negative ventilated murine rack system following direct and indirect aerosol exposure to bacillus subtilis spf status of mice can be maintained in spfmice in ventilated cage systems key: cord- - aa pi authors: ho, yu; lin, pi-hsiu; liu, catherine y.y; lee, su-ping; chao, yu-chan title: assembly of human severe acute respiratory syndrome coronavirus-like particles date: - - journal: biochem biophys res commun doi: . /j.bbrc. . . sha: doc_id: cord_uid: aa pi viral particles of human severe acute respiratory syndrome coronavirus (sars cov) consist of three virion structural proteins, including spike protein, membrane protein, and envelope protein. in this report, virus-like particles were assembled in insect cells by the co-infection with recombinant baculoviruses, which separately express one of these three virion proteins. we found that the membrane and envelope proteins are sufficient for the efficient formation of virus-like particles and could be visualized by electron microscopy. sucrose gradient purification followed by western blot analysis and immunogold labeling showed that the spike protein could be incorporated into the virus like particle also. the construction of engineered virus-like particles bearing resemblance to the authentic one is an important step towards the development of an effective vaccine against infection of sars cov. a new and deadly disease, named human severe acute respiratory syndrome (sars), was first recognized in in the asian region, and quickly spread to several locations across the world over the following few months. the causative agent for sars has subsequently been identified as a novel coronavirus [ ] . coronaviruses are among the largest of the enveloped rna viruses. sars-associated coronavirus, sars cov, has a positive-strand rna genome of kb and measures about nm in diameter [ , ] . the viral envelope contains at least three structural membrane proteins, i.e., spike (s), membrane (m), and small membrane or envelope (e) proteins. the s proteins make up the characteristic "club-shaped" or "crownlike" projection of coronaviruses. the s protein is a type i integral membrane glycoprotein, containing a large extracellular domain, a short transmembrane domain, and a small cytoplasmic carboxyl-terminus [ ] , and is responsible for the entry of the virus into host cells. the m protein is the most abundant virion protein, which is a triple-spanning integral membrane protein with a short ectodomain and a large carboxyl-terminal endodomain [ ] . the e protein, the third virion constitution, contains a hydrophobic region flanked by hydrophilic termini [ ] . the sars cov is phylogenetically distinct from all the three coronavirus groups known today [ ] . the receptor binding domain of s of sars cov and the receptor of the host cell have recently been identified [ , ] , which is also different from previous studies for other coronaviruses [ ] [ ] [ ] . thus, although previous studies on coronaviruses showed that possession of the m and e proteins is a minimal requirement for the assembly of viral particles, whereas the s protein is dispensable, but is included when present [ ] [ ] [ ] [ ] , the demonstration that e and m proteins are sufficient for the assembly of sars cov-like particles, and these virus-like particles (vlps) can further incorporate s proteins are still imperative knowledge and technology for rational strategy (ex. vaccine development) to combat this deadly virus. in this communication, we report the formation and isolation of sars cov-like particles through the co-expression of either e and m proteins or e, m, and s proteins using a baculovirusinsect cell system. plasmids and recombinant baculoviruses. the dna sequences coding the s, e, and m proteins of sars cov were obtained from the college of medicine, national taiwan university (genbank accession no. ay ). the coding sequences for the three structural proteins were cloned into a modified transfer plasmid derived from pbacpak (clontech), under the control of a strong viral promoter, polyhedrin promoter. this transfer plasmid also contains a red fluorescent reporter gene, dsred (clontech), driven by a heat-shock promoter. to generate recombinant baculoviruses using autographa californica multiple nucleopolyhedrovirus (acmnpv) viral genome, the individual plasmids containing s, e, and m proteins were co-transfected with linearized viral dna (baculogold, bd biosciences) by using lipofectin (invitrogen) into insect cells, successful recombinants were isolated by the indication of red fluorescence. the recombinant baculoviruses encoding the s, e, and m proteins, assigned as vabhrps, vabhrpe, and vabhrpm, respectively, were obtained by two-or three-round serial dilutions, and all viral stocks were prepared and manipulated according to the standard protocol described by o'reilly et al. [ ] . insect cell culture. the spodoptera frugiperda iplb-sf (sf ) insect cell line was cultured as a monolayer in tnm-fh insect medium, containing % heat-inactivated fetal bovine serum as described previously [ ] . it was used for the propagation and infection of recombinant baculoviruses; titers of viruses were determined by a newly developed quantitative real-time pcr-based method [ ] . isolation of coronavirus-like particles. insect cells were co-infected with vabhrpe and vabhrpm at a multiplicity of infection (moi) of : . at days post-infection (dpi), cells were collected by a cell scraper (costar) and then re-suspended in tris-buffered saline (tbs), containing a cocktail of protease inhibitor ( : dilution, set iii, calbiochem), and lysed by sonication. the post-nuclear supernatant (pns) was obtained by centrifugation at rpm for min, and was then placed on a % (w/w) sucrose cushion for centrifugation at , rpm for h. pellets were washed twice with tbs and resuspended in the same buffer, and then subjected to a - % (w/w) sucrose gradient at , rpm for h. thirty fractions, monitored at a wavelength of nm, were extracted from the centrifuged sucrose gradient using a density gradient fractionation system (isco). the fraction containing vlps was subsequently examined by western blot analysis and by electron microscope as described below. western blot analysis. each fraction of sucrose gradient was analyzed by an % and % of sds-page, respectively; the separated proteins were transferred onto a pvdf membrane (immobilin-p, millipore). the membrane was blocked with % non-fat milk for h and then probed (at a dilution of : ) by antiserum at °c overnight. the antiserum used to probe s protein was developed by immunizing rabbits with the amino acid fragment - of spike protein (a gift from dr. pei-jer chen, graduate school of clinical medicine, college of medicine, national taiwan university), whereas those used to probe e protein were raised by immunizing rabbits with synthetic oligopeptides (lvkptvyv ysrvknl, c-terminal of e protein, genesis biotech). after three washes with . % tween containing phosphate-buffered saline (tpbs), a horseradish peroxidase (hrp)-conjugated goat anti-rabbit igg was added at a dilution ratio of : , incubating at room temperature for h. the blot was then washed with tpbs four times, followed by visualization using chemiluminescent reagent (western lightning, perkin-elmer) and developed on an x-ray film (biomax, kodak). characterization of vlps by electron microscope. ultra-structure of cells co-infected with recombinant baculoviruses was collected, fixed, and then visualized by electron microscopy as described in our previous studies [ ] . to visualize purified vlps using transmission electron microscopy with negative staining, an aliquot of ll of the vlp preparation was loaded onto a carbon-coated grid, letting standstill for min. grids were then stained with % of phosphotungstic acid (pta) for min; after excess pta was drained and the grid was examined directly under electron microscope (em). for immunogold labeling, vlps were loaded onto a collodion-coated em grid for min. after the removal of excess of sample solution by gently blotting with a filter paper at the edge of the grid, an antibody specific against s protein (developed by immunizing rabbits with synthetic peptides corresponding to amino acids - of s protein; img- , imgenex) was added onto the grid and incubated for h at room temperature. grids then underwent s wash six times in sorensen's phosphate buffer at room temperature and were incubated with nm gold conjugated anti-rabbit igg for h. after six s washes in sorensen's phosphate buffer, the samples were stained with % pta for min, then drained, and examined under the em. to express e, m, and s proteins of sars cov in baculovirus-insect system, we first cloned the individual sequences into three different plasmids, under control of a strong viral promoter, polyhedrin promoter (see materials and methods). recombinant baculoviruses encoding e, m, and s protein genes were used to infect sf insect cell, and the expression of each protein was checked by western blot at dpi (data not shown). to study if vlps are able to form in insect cells, we first used vabhrpe and vabhrpm to co-infect sf cells at different moi (i.e., e:m ¼ : - : ), and examined these co-infected cells by electron microscopy (data not shown). we found the formation of vlps was satisfactory at moi of : . fig. a shows a typical electron micrograph of a cell co-infected with recombinant baculoviruses expressing both e and m proteins, in which the boxed region was further magnified in fig. b . the vlps were observed in a large lightly stained vesicle (solid arrow). a higher magnification showed that several heavily stained smaller vesicles with vlps were also found (open arrows) near the lightly stained vesicle. these lightly and darkly stained structures are similar to the electron microscopic studies of a specimen from sars patient [ ] . the distribution of vlps in the baculovirus-insect cell system was also consistent with a previous study demonstrating the assembly of hepatitis c virus-like particles in insect cells [ ] . in figs. c and d, the cytoplasm of a cell is covered with vlps, some of which are arbitrarily indicated by arrowheads. this result suggested that vlp-containing vesicles (open arrow in (b)) were disrupted and vlps were released all over in the cytoplasm in some cells. previously, it was shown that e protein is not essential for the replication of murine coronavirus. however, a virus with e protein mutation gave rise to smaller plaques with at least orders of magnitude lower titer than those typically observed for the wild type virus [ ] . in our study, baculovirus which only expresses m protein was used to infect insect cells. electron microscopic analysis showed that vlps were not found in the infected insect cells (data not shown). this result suggests that e protein is required for the efficient formation of vlp of sars cov, although, we cannot rule out the possibility that vlp may still be formed in the cells with significantly reduced numbers and thus difficult to detect. the s protein is the major antigen determinant site and the protein responsible for the recognition by host cells. in order to determine if s protein could be recruited into vlps for further usage as vaccine or delivery tool to target cells of the sars cov, insect cells were co-infected with vabhrps, vabhrpe, and vabhrpm at an moi of : . : , respectively. after harvesting the infected cells, cell lysate was purified through a sucrose gradient centrifugation to isolate vlps. aliquots of sucrose gradients ( ll each) were fractionated, and the chromatograph was monitored at nm ( fig. a) . the absorbance reached a peak at tube , by which the density was calculated as . mg/ ml, corresponding to % (w/w) of sucrose. the contents of centrifuged fractions were analyzed by % and % sds-page, respectively, and then analyzed by western blot. the presence of s and e proteins was evident in fractions - (fig. b) . these results implicated that vlps contained s protein. to verify the incorporation of s protein onto vlps, fractionated tube was further analyzed by electron microscopy. the isolated vlps were observed by negative staining in electron microscope (fig. ) , whereas the location of s protein was depicted by immunogold labeling using the s protein-specific antibody. as shown in fig. , the vlp, ca. nm in diameter, was specifically labeled with gold particles. our western and electron microscopic analyses accordingly demonstrated that the s protein can be incorporated into the vlp and is located to the periphery of vlp. by western blot analysis, we were able to detect a notable band corresponding to a molecular weight of about kda, which is larger than the predicted molecular weight of s protein amino acid sequence (ca. kda) by kda. the difference could be attributed to the extent of glycosylation since there are about potential n-glycosylated asparagine residues in s protein (data not shown). taking advantage of posttranslational modifications, baculovirus-insect cell sys-tem can be used to generate functional spike proteins, supposedly in an oligomeric form on the surface of the vlps, to raise neutralizing antibodies in the human body to combat sars cov infection. to obtain protective immunity at maximum efficiency, the ideal immunogens used to develop vaccines should elicit immunity, resemble the pathogenic viruses, and be non-pathogenic. vlps closely fit an ideal immunogen. one promising application for the production of non-pathogenic vlps is to act as most effective immunogens to induce protective immunity and to develop immunogen-based assays [ ] [ ] [ ] . another possible application is to define the possible packaging signal of sars cov. the packaging signal of coronavirus rna was previously identified by using defective interfering viral particles [ ] , since the sars-cov is a highly infectious virus, it would be much more feasible to identify this signal by using vlps. moreover, s protein-containing vlps should be able to specifically target to the host cell of the sars cov and serve as a safe and efficient tool to deliver western analysis confirmed the presence of s and e proteins mainly in fractions - . potential therapeutic agents (e.g., sirna) for effective treatment of sars in the future. immunogold labeling of purified vlp. a baculovirus particle (bac) was shown to serve as a negative control. vlp, virus-like particle. characterization of a novel coronavirus associated with severe acute respiratory syndrome assembly of spikes into coronavirus particles is mediated by the carboxy-terminal domain of the spike protein coronavirus particle assembly: primary structure requirements of the membrane protein the small envelope protein e is not essential for murine coronavirus replication a -amino-acid fragment of the sars coronavirus s protein efficiently binds angiotensin-converting enzyme angiotensin-converting enzyme is a functional receptor for the sars coronavirus identification of a receptor-binding domain of the spike glycoprotein of human coronavirus hcov- e recognition of cellular receptors by bovine coronavirus nucleocapsidindependent assembly of coronavirus-like particles by coexpression of viral envelope protein genes the production of recombinant infectious di-particles of a murine coronavirus in the absence of helper virus infectious bronchitis virus e protein is targeted to the golgi complex and directs release of virus-like particles coronavirus pseudoparticles formed with recombinant m and e proteins induce alpha interferon synthesis by leukocytes baculovirus expression vectors: a laboratory manual persistent baculovirus infection results from deletion of the apoptotic suppressor gene p rapid titer determination of baculovirus by quantitative real-time polymerase chain reaction persistent hz- virus infection in insect cells: evidence for insertion of viral dna into host chromosomes and viral infection in a latent status a novel coronavirus associated with severe acute respiratory syndrome hepatitis c virus structural proteins assemble into viruslike particles in insect cells baculovirus expression of chimeric hepatitis b virus core particles with hepatitis e virus epitopes and their use in a hepatitis e immunoassay recombinant hepatitis c virus-like particles expressed by baculovirus: utility in cell-binding and antibody detection assays virus-like particles as immunogens characterization of coronavirus di rna packaging we thank dr. pei-jer chen for providing genes and dr. s.h. yeh for providing antiserum against s protein, which are necessary for these experiments. we also thank drs. michael m. lai and pei-jer chen for their comments. this work was supported by grants from the national science council (nsc - -b- - -y) and academia sinica (imb / and as -ab-imb- ). key: cord- -tq f d authors: weaver, scott c.; barrett, alan d. t. title: transmission cycles, host range, evolution and emergence of arboviral disease date: journal: nat rev microbiol doi: . /nrmicro sha: doc_id: cord_uid: tq f d many pandemics have been attributed to the ability of some rna viruses to change their host range to include humans. here, we review the mechanisms of disease emergence that are related to the host-range specificity of selected mosquito-borne alphaviruses and flaviviruses. we discuss viruses of medical importance, including venezuelan equine and japanese encephalitis viruses, dengue viruses and west nile viruses. rna viruses, including hiv , , dengue virus (denv) , and possibly the severe acute respiratory syndrome (sars) coronavirus [ ] [ ] [ ] , have caused recent pandemics by changing their host range to amplify in humans. mosquito-borne alphaviruses and flaviviruses belong to the arthropod-borne viruses (arboviruses), and both have a positive-sense single-stranded rna genome. most arboviral infections are asymptomatic, or present with an influenza-like illness. however, several mosquito-borne alphaviruses and flaviviruses are important human pathogens that cause central nervous system disease, coma or death (table ) . arboviruses require a host (usually a bird or small mammal) in which they replicate, and a vector, such as a mosquito, for transmission to other organisms. female mosquitoes ingest virus from the blood of an infected animal. on biting another animal the mosquito transfers the virus through saliva into the new host. birds are the most common arbovirus hosts, whereas humans and other animals such as horses are usually dead-end hosts -they do not transmit the virus to others in the 'herd', and cannot function as a reservoir for reinfection of mosquitoes. infection of dead-end hosts can, however, lead to clinical disease (table ) . in this review, we focus on selected viruses such as venezuelan equine and japanese encephalitis viruses (veev and jev, respectively), which cause epidemics by adapting to domestic animals and exploiting them as amplification hosts. we discuss other mosquito-borne viruses including west nile virus (wnv) and denv, which cause neurological disease. we consider the patterns and history of host-and geographical-range alterations that lead to disease emergence, and the experimental model systems that are used to study the evolutionary constraints on arbovirus host-range changes. venezuelan equine encephalitis (vee) was first recognized as a disease of horses, donkeys and mules in south america during the mid- s. the veev genome encodes four non-structural proteins that participate in genome replication and protein processing, and generates a subgenomic mrna ( s), which is translated into three main structural proteins. these structural proteins and the positive sense . -kb rna genome comprise virus particles. recently, interest in veev has been renewed, because it has been developed as an efficient, stable biological weapon that is infectious by aerosol and that is easily produced in large quantities . epidemiology of epizootics. veev was isolated in from a post-mortem brain specimen of a horse with encephalitis , , but it was only in the late s that the veev was identified as a cause of human diseasepresenting as a febrile illness, sometimes accompanied by neurological manifestations and occasional mortality , . until , periodic but unpredictable outbreaks of vee (some involving hundreds of thousands of equine and human cases) were mostly restricted to as well as in mexico and florida. these enzootic strains differed from the strains that were isolated during equine epizootics and epidemics; they were antigenically distinct and generated little or no viraemia in experimentally infected horses. it was therefore thought that enzootic strains were incapable of causing epidemics, although fatal infections documented in panama and mexico established their virulence as human pathogens , . in the late s, antigenic studies shed light on the relationship between epizootic and enzootic veev strains. the strains isolated during major epizootics were antigenically similar, and were classified into subtypes iab and ic. the enzootic strains were antigenically distinct from the epizootic strains and were more diverse. the enzootic strains were grouped into subtypes id-if and ii-vi, and were later classified as several different species , (table ) . as it was initially believed that enzootic veev strains were unrelated to vee outbreaks, because they were incapable of amplification in equines and were never isolated during major epidemics, the source of the subtype iab and ic strains that initiated vee epizootics and epidemics remained unknown . advances in viral molecular genetics, phylogenetic methodology and computational techniques have provided some answers. initially, rna oligonucleotide fingerprinting showed that some enzootic strains in subtype id were more closely related to certain subtype ic epizootic strains than was indicated by their antigenic properties . later, genome sequencing confirmed peru, ecuador, venezuela and colombia (fig. ) . (human cases were only recognized retrospectively for the earliest outbreaks.) widespread and long-lasting vee outbreaks did occur -one epizootic began in el salvador and guatemala in and spread through most of central america and mexico, reaching texas in the united states in . however, after , vee outbreaks and epizootic strains seemed to disappear for years. there were many reasons for this long period of viral inactivity. equine encephalitis has a high mortality rate, and those animals that survive usually produce protective neutralizing antibodies and are immune to reinfection , . insufficient populations of mosquito vectors also limited incidence of the disease. however, in , an outbreak of vee occurred in venezuela ( - venezuelan epizootic/epidemic) , and in , there were epizootics/epidemics in venezuela and colombia , as well as small equine epizootics in southern mexico in and (ref. ) . after identification of veev as a cause of human disease, experimental animal models revealed that equine infection results in a high titre viraemia; the animals therefore serve as highly efficient amplification hosts in the presence of abundant competent mosquito vectors . in agricultural settings, this efficient amplification facilitates human infection. at the same time, viruses with similar antigens, including the vee serocomplex of alphaviruses (fig. ) , were discovered in permanent sylvatic cycles in several tropical and subtropical regions of south and central america, requires knowledge of the host-range changes and their genetic basis. recently, the molecular changes that led to the evolution of epizootic strains from enzootic progenitors have begun to be determined. analysis of related enzootic id and epizootic ic strains from western venezuela facilitated the identification of putative genetic determinants of the epizootic phenotype . also, amino-acid sequences of the envelope glycoprotein revealed a common pattern of positive charge (arginine (arg) or lysine (lys)) substitutions in a region that was probably located on the surface of the viral particle, which might encode the antigenic determinants , . preliminary studies in which these mutations were introduced into the envelope protein of an enzootic id strain indicate that an arg or lys residue at amino-acid position is the main determinant of the equine viraemia phenotype. interestingly, these mutations also change the serotype from id to iab/ic, explaining the correlation between veev serotype and epizootic potential. this single amino-acid substitution, which has a major effect on the vertebrate host range, has resulted in the repeated occurrence of vee emergence over the past years. this period correlates with the introduction of equines into the new world from europe, and the establishment of equine populations that are large enough to mediate efficient amplification. rna viruses, including alphaviruses, have very high mutation frequencies because their error-prone rnadependent rna polymerases lack proof-reading ability . estimates of mutation frequency for a closely related alphavirus, eastern equine encephalitis virus (eeev), are approximately - substitutions per nucleotide. this indicates that the single mutation leading to the lys or arg substitution at position in the envelope glycoprotein e occurs regularly in nature, because veev populations in mosquitoes and vertebrate reservoir hosts usually exceed plaque-forming units (pfu) ml - . an interesting exception to the association between equine veev amplification and epizootic transmission is the recent mechanism of equine disease emergence in mexico . although phylogenetic studies support the recent acquisition of the equine virulence phenotype by local, enzootic subtype ie strains , , the link between neuroinvasion and viraemia -which occurs in all other epizootic strains (subtypes iab and ic)is broken in the mexican strains isolated from encephalitic horses in and . these veev strains cause equine encephalitis in the absence of high-titre viraemia, which indicates increased neurotropism, as high plasma levels of circulating veev are usually necessary for cerebral invasion . the lack of equine amplification by these strains probably explains their failure to spread to the united states and central america, despite similar ecological conditions and a geographical position in the path of the widespread - subtype iab outbreak. other epizootic amplification hosts are being sought in mexico to explain the sudden emergence of vee in , but selection by deforestation for adaption to an alternative, non-sylvatic vector may also be involved . this similarity . phylogenetic studies concluded that the enzootic subtype id strains were ancestors of the epizootic viruses -now known to have evolved independently on at least three occasions (fig. ) . this led to the prediction, in , that additional epizootics would follow despite a -year hiatus . in the case of the - venezuelan outbreak, and both mexican outbreaks, phylogenetic studies identified sympatric, closely related enzootic strains that seemed to represent the epizootic progenitors. the venezuelan/ colombian isolates were almost identical to strains isolated in the same regions during a - epizootic/epidemic, suggesting either release from a laboratory source or unprecedented genetic stability in a cryptic, natural transmission cycle . enzootic and epizootic strains of veev use different vertebrate hosts and mosquito vectors (box ) . therefore, understanding mechanisms of vee emergence by antigenic shifts and the acquisition of the equine viraemia phenotype sympatric having overlapping geographical distributions. - , , , , subtype iab - subtype iab , - , , , - , subtype iab - - , indicate that viral adaptation to epizootic mosquito vectors might also be important for vee emergence , . recently, these findings were extended to the subtype id enzootic viruses (putative epizootic progenitors), supporting the role of adaptation to mosquito vectors in vee emergence , . interestingly, the same genetic determinant, the e -spike envelope glycoprotein, seems to regulate adaptation to both mosquito and equine hosts . studies are now underway to determine if the same amino-acid substitutions mediate both host-range changes. veev emergence -unanswered questions. there are many important unanswered questions about veev emergence. why has only one of the closely related enzootic veev lineages (fig. ) apparently generated all epizootic iab and ic strains responsible for major outbreaks? do slight differences in the gene and protein profiles of other enzootic veev lineages prevent adaptation to equine hosts and/or epizootic vectors by altering the effect of minor genetic mutations? as equine populations decline in latin america, and human and domestic animal populations increase, will the scale and frequency of vee epizootics decrease? can humans or domestic animals such as cattle replace equines as amplification hosts? humans can sustain levels of viraemia similar to those of equines, but are probably mosquito host range and emergence. as well as adaptation to equine amplification,veev emergence from enzootic progenitors requires an alteration in the mosquitovector host range. all seven of the vector species identified in enzootic veev cycles are members of the culex (melanoconion) subgenus of mosquitoes, and all of these mosquitoes are also members of the spissipes section (comprising species) of this subgenus . the ecological and physiological properties that facilitate efficient transmission of enzootic veev by these mosquitoes are unknown, but might include their relative population stability, as they inhabit areas around permanent water sources. however, during outbreaks, floodwater mosquitoes of genera such as ochlerotatus (formerly named aedes) and psorophora -which show large seasonal changes in population density -are the main transmission vectors of epizootic veev. outbreaks often occur in desert-like habitats -such as the guajira peninsula of venezuela and colombia -at times of unusually heavy rain. similar climatic events might contribute to the emergence of other arboviruses such as rift valley fever virus, which, unlike veev, is efficiently maintained by transovarial transmission . studies showing that ochlerotatus taeniorhynchus (probably the most important epizootic vector in coastal outbreaks) has a higher oral susceptibility to epizootic (subtype iab) compared with enzootic (subtype ie) veev zoonotic pathogens or diseases that normally circulate among nonhuman animals but that can be transmitted to humans. figure) . veev adaptation to domestic amplification hosts (equines) requires mutations in the e gene that encodes the envelope glycoprotein. these mutations increase the level of host viraemia, and have occurred at least three times during the past century. this change is accompanied by an alteration in the veev serotype to iab or ic. adaptation to epizootic mosquito vectors also seems to accompany some veev outbreaks. dengue viruses have emerged as the most important human arboviral pathogens by altering their host range, from non-human primate enzootic reservoirs to humans, in an ecologically and separately evolving urban endemic transmission cycle (see figure) . the four dengue serotypes all emerged independently as human pathogens hundreds to thousands of years ago, when human populations had attained sufficient density to enable continuous horizontal transmission, probably supplemented by vertical transmission in mosquitoes. ochlerotatus and psorophora spp. (veev), culex tritaeniorhynchus, culex spp. human amplification (for example, urban dengue virus,yfv, chikungunya) spillover from enzootic cycle (for example, wnv, sylvatic dengue virus, yfv, veev) amplification in domestic animals (for example, epizootic veev, jev) (weev) habitats. the adaptation of these viruses to equine hosts would have profound public and veterinary health implications. the recombinant ancestors of weev are eeev-and sindbis-like alphaviruses, both of which use avian enzootic hosts. however, eeev, but not sindbis, causes equine and human encephalitis, indicating that the non-structural and/or capsid-protein genes or cis-acting rna sequences are important determinants of this pathogenic phenotype . although jev is an arbovirus, it is a member of the genus flavivirus, family flaviviridae. the genome of jev is similar to that of veev, as it comprises a singlestranded, positive-sense rna molecule, approximately , nucleotides in length. however, in contrast with alphaviruses, the flavivirus genome has no ′ poly(a) tail. structural proteins, including a single envelope protein, are encoded by the ′ quarter of the genome. japanese encephalitis -the disease. although japanese encephalitis (je) has been recognized as a disease since the s (ref. ), the jev was first isolated in in tokyo, japan, from the brain of a fatal human exposed to mosquito vectors less frequently than domestic animals , . cattle, dogs and other domestic animals are susceptible to infection with veev, but levels of viraemias are low, and adaptation would probably be required for amplification in these hosts. during , epidemic vee occurred in regions of venezuela lacking equine populations, but with abundant goats, sheep and people, implicating humans or other animals as amplification hosts (g. medina and n. perez, personal communication). can epizootic veev strains persist in nature in the absence of efficient equine amplification and transmission? is epizootic adaptation to equine and mosquito hosts species-specific, resulting in a loss of fitness for the sylvatic hosts that are required for permanent viral circulation? further research is needed to predict the future impact of vee, and to design effective measures to prevent and control natural emergence events and possible terrorist introductions. there are other interesting questions about eeev and western equine encephalitis virus (weev). unlike veev, these viruses do not amplify efficiently in equines (they produce low levels of viraemia), and epizootics involve avian hosts and usually occur only in close proximity to their enzootic swamp (eeev) or agricultural the level of viraemia is too low to infect mosquitoes. similarly, an unusually wide range of animals, including birds, dogs, bats and snakes are dead-end hosts that are unable to infect mosquitoes. pigs and birds are the major amplifying hosts of jev, although infection usually does not produce clinical disease. these animals do, however, develop high-titre viraemias, which provide an excellent source of infection for mosquitoes. in parts of asia where pigs are often kept adjacent to human dwellings, these animals are an important source of viral amplification and significantly enhance human exposure and infection. also, as most domestic pigs are slaughtered by months of age, annual birth cohorts provide a population of animals that are susceptible to infection . as jev does occur in areas of asia where pig populations are low, they are not essential hosts for the enzootic transmission cycle. interestingly, in central java, where there are few pigs, it seems that cattle, which are normally considered a dead-end host, might be involved in the natural transmission cycle of jev . migratory birds allow jev to travel large distances. unlike veev, there case of encephalitis . jev is the largest worldwide cause of epidemic encephalitis. the virus causes epidemics of paediatric encephalitis, mainly in india, korea, china, south-east asia and indonesia. owing to its large geographical range, more than two billion people are at risk of infection, and case-fatality rates often exceed %. approximately , cases occur each year, of which , are fatal; importantly, up to % of those who survive the disease suffer from neurological sequelae that last from months to years. the ratio of apparent to inapparent (asymptomatic) infection varies from in to in , depending on the geographical area. for example, in northern thailand, cases of encephalitis caused by jev have been diagnosed each year since the late s, with most cases occurring during the rainy season in june, july and august. the fatality rate of virologically confirmed cases is %, and, in thailand, about in humans that are infected with jev develop encephalitis. similar to veev, jev is also a veterinary problem and horses can succumb to encephalitis. however, unlike veev, equines are dead-end hosts for jev, as ie-pa -menaii ie-gu - u ie-gu - u ie-mx -oax ie-mx - ie-mx -oax ie-mx - - id- proven. the differences in jev genotypes might have arisen in response to geographical variation in mosquito vectors or amplifying hosts. the relevance to human disease of the genotypic and antigenic differences among jev strains is unknown. early genetic studies used rna oligonucleotide fingerprinting to identify genetic differences; however, determination of the nucleotide sequence of the first jev genome in (strain jaoars , genotype iii) facilitated comprehensive genetic studies. representative genomic sequences of the four genotypes have been determined, and results are consistent irrespective of the technique used (t mapping, partial or complete genome sequencing). the genotypes show different levels of geographical clustering (fig. ) . genotype i is found in australia, japan, korea, northern thailand and cambodia; genotype ii is found in australia, southern thailand, malaysia, sarawak and indonesia; genotype iii is found throughout asia and genotype iv is found only in indonesia. initial studies suggested that there were genetic differences between strains associated with endemic (round the year) disease in tropical regions of asia such as malaysia, indonesia and the philippines, compared with epidemic (summer-only) disease in temperate regions of asia (for example, japan, taiwan, korea and china). however, this has not been confirmed, and differences in endemic and epidemic disease are probably due to climatic variation. je is an emerging disease, and its geographical distribution is increasing. australia is the latest country in which jev has been isolated, and the virus was probably introduced by viraemic migratory birds or wind-borne infected mosquitoes. most phylogenetic studies of the genus flavivirus imply that the flaviviruses originated in africa and then spread to other continents . phylogenetic studies of jev reveal that genotype iv is most divergent, with up to % nucleotide and . % amino-acid variation compared with genotype i, whereas the other three genotypes differ by no more than % at the nucleotide and . % at the aminoacid levels . the phylogeny of jev (fig. ) indicates that genotype iv, which is found only in indonesia, is ancestral. genotypes ii and iii have also been found, sometimes concurrently, in the indonesia/malaysia region. introduction of jev into badu island in the torres straits, australia, in involved genotype ii, whereas the subsequent introduction of jev into northern australia in involved genotype i. genotype iii is most commonly isolated, and is found throughout asia (but not australia). similarly, genotype iii was the only genotype found in japan until , when genotype i was first isolated. overall, these results indicate that jev originated in the indonesia/malaysia region and subsequently spread to surrounding areas and countries. the rate of evolution of jev indicates that it is a relatively 'young' virus, with genotype iv diverging from the progenitor ancestral virus is no evidence that enzootic jev requires adaptation (mutation and selection for replication in pigs or birds) to initiate amplification in these epizootic amplification hosts. the principal vectors of jev are culex tritaeniorhynchus mosquitoes, and maximum virus isolation rates from mosquitoes occur during late july, concurrent with human and equine epidemics. in malaysia, both culex gelidus and c. tritaeniorhynchus are important vectors. other mosquito vectors include members of the aedes, anopheles, mansonia and armigeres species. antigenicity and genotype of jev. studies have indicated that there are antigenic and genetic differences among jev strains, with at least four genotypes and five antigenic subtypes [ ] [ ] [ ] . these genotypes and subtypes might correspond, although this has not been experimentally proteins (part a in figure) . during infection, two mrnas are synthesized -a full length mrna that forms the viral genome and a smaller mrna that is used to produce virion (part b, right) shows the rna genome in the centre surrounded by copies of the capsid protein, and the lipid-bilayer envelope, which is derived from the host-cell plasma membrane. the capsid protein is arranged into hexons and pentons on a t = lattice, which complements the organization of the envelope glycoproteins. the e envelope glycoproteins are parallel to the envelope and the e envelope glycoproteins form spikes on the surface. the e protein is believed to be the site of mutations that alter the host range of veev for equines and mosquitoes to mediate epizootic emergence, probably through cellular-receptor interactions. the nucleocapsid (part c) has capsid proteins arranged with t = icosahedral symmetry. the figure was reproduced with permission from ref . it is thought that migratory birds might be important in the emergence of jev. the asiatic cattle egret (bubulcus ibis coromandus) is an important amplifying host, and its geographical range increased in asia during the nineteenth century because of changes in agricultural practices . this coincided with the evolution and spread of recent jev genotypes. it is also tempting to hypothesize that the second world war had an important role in jev emergence, because isolation of jev strains had been restricted to japan prior to this conflict. some important questions remain. why is genotype iv restricted to the indonesian/malaysian region, whereas recent genotypes are geographically more widespread? genotype iv has only been identified once (in ), from five mosquito isolates, and its ability to cause human disease is unproven. although it is thought that genotype iv existed prior to , this has not been confirmed. the sequence of the structural protein genes of a strain (muar) from malaysia indicates that muar is ancestral to genotype iv and is a fifth genotype (fig. b) source of the virus, because humans only develop a low-titre viraemia and are therefore considered deadend hosts. bioterrorism is also an unlikely source, as the epicentre of the outbreak was in the queen's district of new york city, and the virus was transmitted by mosquitoes. one explanation is that wnv was introduced to new york by a viraemic animal that was subsequently bitten by a mosquito, which then spread the disease to humans. many exotic animal species, particularly birds, are imported into the united states through new york, and it is possible that an imported viraemic bird initiated transmission. alternatively, an infected migratory bird might have carried the virus to the united states. normally, migratory birds fly in a north-south direction, rather than in an east-west direction. the north-south route would imply that the virus entered the united states from central/south america. this seems unlikely, as wnv was only found in the southern united states at least one year after it was discovered in new york. another possibility is the east-west route. although this might also seem unlikely, vagrant racing-pigeons from europe occasionally appear in the united states. recently, the uk media reported that a homing pigeon called 'billy' was released in france en route to britain, but instead flew to new york . finally, an infected mosquito that was transported aboard an aircraft could have introduced wnv to the united states. aircraft departing from the middle east to the united states are not routinely treated with insecticide, and mosquitoes often enter aeroplanes -attracted by humans or bright light. phylogenetic studies using both complete and partial genome sequences have shown that the wnv strain found in new york in is most closely related to a israeli strain isolated from a goose, which indicates that the virus was recently introduced into the united states. this conclusion was supported by serological studies that showed a lack of anti-wnv antibodies in individuals living in new york prior to (ref. ). initial studies revealed that the nucleotide sequences of strains found on the eastern seaboard of the united states were similar to the prototype 'new york ' (ny ) strain isolated from a flamingo in the bronx zoo, and indicated that the ny strain spread westward in - . recent studies have shown that a genetic variant termed 'north america' has arisen since and has replaced ny as the dominant genotype present in north america . the 'north america' genotype only differs by . % from ny , but has characteristic nucleotide and amino-acid substitutions. other genetic variants have been identified, including a south-east coastal texas genotype that differs by . % from ny and by . % from 'north america', and a mexican genotype that differs by . % from ny (ref. ). overall, these data might indicate a lack of darwinian selection in the evolution of a particular genetic variant, and support genetic drift of the virus during its dissemination across north america. consistent with this hypothesis, the southeast coastal texas genotype was only isolated in from the texas-louisiana border, and might have become extinct. to date, there is no evidence for selection of any phenotypic differences among north american strains, and it remains to be seen if wnv will evolve towards reduced virulence as it adapts to new hosts in the western-hemisphere ecosystem. there are many hypotheses about the mechanism of wnv emergence in the united states. it is unlikely that viraemic human travellers are a each year . in urban settings, denvs are transmitted among human hosts by the peridomestic mosquito vectors aedes aegypti and aedes albopictus. however, studies of dengue virus ecology in sylvatic habitats of west africa and malaysia , have identified transmission cycles involving non-human primates as reservoir hosts and arboreal, tree-hole dwelling aedes (stegomyia) spp. as vectors. efficient inter-human denv transmission probably requires a human population of , to million people, a feature of urban civilizations that did not exist until about , years ago, and therefore the sylvatic cycle is probably ancestral . endemic/epidemic denv is therefore thought to have evolved in africa or asia from sylvatic viral forms. initial phylogenetic studies of both endemic/ epidemic and sylvatic strains of denv- showed evolutionary divergence of these ecologically distinct forms . recent studies indicate that epidemic/endemic forms of denv- , - and - , which now use humans as reservoir hosts, evolved independently from sylvatic progenitors in the past , years, accompanied by host-range changes from non-human primates to humans, and from arboreal aedes spp. to a. aegypti and a. albopictus vectors , (fig. ) . the highly efficient peridomestic transmission cycle -which is now independent of the ancestral sylvatic cycles -benefits greatly from the ecology of a. aegypti. this species lays its eggs in waterstorage containers and in refuse, it readily enters human habitations and it often takes several blood meals during each reproductive cycle for both egg production and nutrition. once infected, this competent vector transmits the virus to many human hosts , . interestingly, as with sylvatic denv, a. aegypti originated from another tree-hole-dwelling aedes mosquito which is found in sylvatic african habitats (aedes aegypti formosus) . however, the sylvatic african forms of denv do not use a. aegypti formosus as a principal vector, and this subspecies is relatively refractory to infection . yellow fever virus also uses a. aegypti aegypti as its main vector during african urban epidemics, but this cycle seems to be temporary and cannot be detected during inter-epidemic periods. it is not known why yfv has not adapted to a permanent human-a. aegypti endemic cycle, which would have devastating public health implications. the adaptation of rna viruses to new hosts is generally host-specific . the evolutionary hypothesis therefore predicts that a. aegypti and a. albopictus should have increased susceptibility for endemic/ epidemic denv compared with its sylvatic progenitors. recent experimental infection studies of populations of a. aegypti and a. albopictus support this hypothesis. when fed blood meals containing equivalent viral titres, the endemic/epidemic strains of denv- consistently infect a higher proportion of mosquitoes than sylvatic strains . if similar adaptation to human hosts occurred during denv evolution, differences in pathogenicity might be expected between the endemic and sylvatic denv strains. studies are required in regions of africa and asia, where humans are exposed to sylvatic infection, to confirm these differences. genetic north america. however, differences in surveillance and the high incidence of human infection through blood transfusion, mother-to-foetus transmission, transmission in breast milk and by organ transplantation have caused major public health concerns [ ] [ ] [ ] [ ] [ ] [ ] [ ] . an important question that remains unanswered is how wnv will adapt to new ecosystems in the new world. there are two main hypotheses based on comparisons with other members of the jev group members. first, wnv could become enzootic and endemic, similar to st louis encephalitis virus (slev), and cause limited human disease through spillover. alternatively, the virus could become epidemic, similar to asian strains of jev, and could cause annual outbreaks affecting large numbers of humans and animals. the 'explosive' nature of the north-american wnv epidemic as well as the equine and avian epizootics might reflect highly efficient enzootic amplification and avian virulence. north-american birds have probably not developed resistance to wnv because of low levels of viral exposure. virulence in the avian host might subsequently decline because of the strong selection for resistance to wnv resulting from high bird-mortality rates. if resistance is accompanied by a decline in viraemia levels, enzootic amplification could subside. then, spillover to humans and equines would diminish. the transport of wnv into latin america by migratory birds raises important questions about pre-existing human and avian immunity to other flaviviruses such as slev, denv, yfv and rocio virus. cross-reactive immunity could protect against wnv infection and/or disease, or could increase pathogenesis through immune enhancement, which is thought to contribute to the aetiology of dengue hemorrhagic fever. it is still not known if wnv and south american flaviviruses can share the same hosts and ecosystems (similar to wnv and slev in the united states). it is also not known how wnv will spread and cause human disease in the presence of flavivirus antibodies. competition between wnv and slev for avian hosts and mosquito vectors might be expected to result in competitive exclusion of one of these viruses . however, these viruses coexist in the southern united states where they seem to use the same avian hosts and vectors. low rates of immunity to slev in birds and low rates of infection in c. quinquefasciatus might indicate that host resources are not limiting factors, which could allow for indefinite coexistence. denvs (serotypes - ) are the most important arbovirus human pathogens, and are also unusual as they use humans as reservoir hosts (see further information in the online links box). during the past years, the prevalence of dengue fever, as well as lifethreatening dengue haemorrhagic fever and shock syndromes, has increased exponentially, with approximately , million people (two-fifths of the world's population) at risk, and about million cases recorded peridomestic in, and around, human habitations. cycles that rely on humans as exclusive reservoir hosts. (the smallpox virus has been eradicated from natural transmission by smallpox vaccine and, similarly, poliovirus is nearly eliminated worldwide.) however, any eradication programme would rely on an inability of the sylvatic strains to re-emerge -currently the likelihood of this is not known. studies are required to quantify the number of mutations leading to vector adaptation during emergence of urban dengue from ancestral sylvatic cycles. this will allow assessment of the frequency with which arboviruses can undergo host adaptation. these studies are important because denv vaccines are currently being developed that could eradicate urban malaysia -p - malaysia -p - malaysia -p - thailand -tc philippines - philippines - philippines -h- -p thailand -d sri lanka -s malaysia -p - indonesia - indonesia - tahiti - results . another arbovirus, vesicular stomatitis virus (vsv), can also adapt simultaneously to sandfly and vertebrate host cells, and the mutation rate of the virus in specialized compared to alternating-host passages is the same. this contradicts the hypothesis that arboviruses are constrained in their evolutionary potential . surprisingly, vsv populations that are specialized for replication in vertebrate cells showed fitnessincreases for replication in sandfly cells, indicating a lack of specificity in some adaptation events. further study of these model systems is needed to determine if alternating passages generate mutations that increase fitness in both cell types, or if they generate a polymorphic population with host-cell-specific adaptive mutations. recent in vivo experiments have produced results that contradict the cell-culture experimental models of arbovirus evolution. when veev was placed into a laboratory transmission cycle -consisting of ten cycles in hamsters alternating with ten cycles in a. aegypti mosquitoes -genetic stability of the virus isolates was observed, and virus fitness was either reduced or unchanged. however, specialization for replication in the hamster host resulted in rapid increases in virus fitness with many mutations, consistent with virus adaptation and evolutionary constraints imposed by the alternating-host cycle of arboviruses . cell-culture models probably do not fully reflect in vivo conditions, as artefacts (such as selection for virus binding to heparin sulphate) might distort results , . the development of experimental systems to study mechanisms of replication of these important human and animal pathogens in different hosts should reveal how these rna viruses emerge to cause disease in humans. although it is still unclear if there are common mechanisms of emergence of arboviruses, studies using molecular genetics and viral ecology should enable researchers to predict emergent strains. furthermore, common determinants of emergence could be used to develop rationally designed antiviral strategies. although host-range changes have occurred throughout arbovirus evolution, with important consequences for human health, the ease and frequency with which these events occur is unknown. viruses that have obligate alternating replication cycles in taxonomically disparate hosts -vertebrate and arthropod -should be compromised because they are 'generalists', owing to their fitness in both hosts. other viruses that use a single host as their main reservoir might be able to adapt more readily to related hosts. this might account for the slow rates of evolution of most arboviruses (approximately substitutions per nucleotide per year) compared with many single-host rna viruses (approximately - substitutions per nucleotide per year) . experimental cell-culture model systems have been developed to test this hypothesis, but results are only partially supportive. in vitro transmission cycles of eeev have been established by serial passage in vertebrate (baby-hamster kidney), or mosquito (a. albopictus c / ) cells . eeev has also been introduced into an alternating-host cycle of both cell types to mimic the natural transmission cycle. as predicted, virus-fitness increases occurred in both hosts following specialization. viruses that had specialized to one host type showed a decline in fitness in the alternate cells, as well as in comparable vertebrate or mosquito cells, consistent with specificity of adaptation during specialization. surprisingly, the alternating-host-cell cycles resulted in simultaneous adaptation to both cell lines, with increased levels of fitness comparable to those associated with host specialization, contradicting the hypothesis that arboviruses are limited in their adaptation potential by the alternating-host transmission cycle. however, specialization resulted in a larger number of mutations than alternating-host passages, consistent with the reduced rates of nucleotide-sequence changes typical of arboviruses. alternating passage of eeev in chicken and mosquito cell lines produced similar origin of hiv- in the chimpanzee pan troglodytes troglodytes timing the ancestor of the hiv- pandemic strains evolutionary relationships of endemic/epidemic and sylvatic dengue viruses this paper demonstrates the convergent evolution of endemic denv virus strains from three of the four serotpes of sylvatic progenitors, and places a time frame on urban dengue emergence that is congruent with historical and epidemiological predictions the global emergence/resurgence of arboviral diseases as public health problems the genome sequence of the sarsassociated coronavirus isolation and characterization of viruses related to the sars coronavirus from animals in southern china characterization of a novel coronavirus associated with severe acute respiratory syndrome agents: a brief synopsis the causative agent of infectious equine encephalomyelitis in venezuela venezuelan equine encephalomyelitis venezuelan equine encephalitis the arboviruses: epidemiology and ecology experimental infection of horses with enzootic and epizootic strains of venezuelan equine encephalomyelitis virus virulence and viremia characteristics of epizootic subtype ic venezuelan equine encephalitis viruses and closely related enzootic subtype id strains emergence of a new epidemic/epizootic venezuelan equine encephalitis virus in south america re-emergence of epidemic venezuelan equine encephalomyelitis in south america. vee study group association of venezuelan equine encephalitis virus subtype ie with two equine epizootics in mexico el virus de la encephalitis equina de venezuela como determinante de infecciones en humanos, descripción de un caso fatal ocurrido en jaltipán veracruz en venezuelan equine encephalitis this landmark paper established the antigenic relationships of enzootic and epizootic strains of veevs and provided the framework for future genetic studies that identified the origins of outbreaks genetic variation of venezuelan equine encephalitis virus strains of the id variety in colombia genetic evidence that epizootic venezuelan equine encephalitis (vee) viruses may have evolved from enzootic vee subtype i-d virus this paper provides the first phylogenetic evidence that epizootic strains of veev have evolved repeatedly from enzootic subtype id progenitors potential sources of the venezuelan equine encephalitis subtype ic epidemic genetic and phenotypic changes accompanying the emergence of epizootic subtype ic venezuelan equine encephalitis viruses from an enzootic subtype id progenitor variants of venezuelan equine encephalitis virus that resist neutralization define a domain of the e glycoprotein positively charged amino acid substitutions in the e envelope glycoprotein are associated with the emergence of venezuelan equine encephalitis virus origin and evolution of viruses geographic distribution of venezuelan equine encephalitis virus subtype ie genotypes in central america and mexico equine amplification and virulence of subtype ie venezuelan equine encephalitis viruses isolated during the and mexican epizootics venezuelan equine encephalitis emergence: enhanced vector infection from a single amino acid substitution in the envelope glycoprotein natural enzootic vectors of venezuelan equine encephalitis virus climate and satellite indicators to forecast rift valley fever epidemics in kenya vector competence of mosquitoes as a marker to distinguish central american and mexican epizootic from enzootic strains of venezuelan encephalitis virus vector infection determinants of venezuelan equine encephalitis virus reside within the e envelope glycoprotein virological and serological studies of venezuelan equine encephalomyelitis in humans the encyclopedia of arthropod-transmitted infections the encyclopedia of arthropod-transmitted infections western equine encephalitis virus is a recombinant virus epidemiology of japanese encephalitis isolation of japanese epidemic encephalitis from mosquitoes caught in nature this paper is one of a series of ten papers that describes the ecology 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the northeastern united states the epidemic of west nile virus in the united states genetic variation among geographically distinct west nile virus isolates collected in the united states during west nile virus in mexico: evidence of widespread circulation since hopelessly lost, the homing pigeon whose crosschannel hop took him to new york. daily mail west nile virus and wildlife health west nile virus: emerging threat to public health and animal health the role of birds in the ecology of west nile virus in europe and africa the ecology and epidemiology of west nile virus in africa detection of west nile virus in blood donations -united states west nile virus encephalitis transmission of west nile virus through blood transfusion in the united states in transmission of west nile virus from an organ donor to four transplant recipients west nile virus west nile virus infection transmitted by blood transfusion the struggle for existence dengue and dengue haemorrhagic fever amplification of 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slow rates of evolution in new world alphaviruses genetic and fitness changes accompanying adaptation of an arbovirus to vertebrate and invertebrate cells differential evolution of eastern equine encephalitis virus populations in response to host cell type lack of evolutionary stasis during alternating replication of an arbovirus in insect and mammalian cells genetic analysis of epizootic venezuelan equine encephalitis virus emergence mechanisms. thesis binding of sindbis virus to cell surface heparan sulfate adaptation of sindbis virus to bhk cells selects for use of heparan sulfate as an attachment receptor genetic determinants of venezuelan equine encephalitis emergence venezuelan equine encephalitis the authors declare no competing financial interests. key: cord- -krb eidw authors: shen, s; law, y.c; liu, d.x title: a single amino acid mutation in the spike protein of coronavirus infectious bronchitis virus hampers its maturation and incorporation into virions at the nonpermissive temperature date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: krb eidw the spike (s) glycoprotein of coronavirus is responsible for receptor binding and membrane fusion. a number of variants with deletions and mutations in the s protein have been isolated from naturally and persistently infected animals and tissue cultures. here, we report the emergence and isolation of two temperature sensitive (ts) mutants and a revertant in the process of cold-adaptation of coronavirus infectious bronchitis virus (ibv) to a monkey kidney cell line. the complete sequences of wild type (wt) virus, two ts mutants, and the revertant were compared and variations linked to phenotypes were mapped. a single amino acid reversion (l( )-to-q) in the s protein is sufficient to abrogate the ts phenotype. interestingly, unlike wt virus, the revertant grows well at and below °c, the permissive temperature, as it carries other mutations in multiple genes that might be associated with the cold-adaptation phenotype. if the two ts mutants were allowed to enter cells at °c, the s protein was synthesized, core-glycosylated and at least partially modified at °c. however, compared with wt virus and the revertant, no infectious particles of these ts mutants were assembled and released from the ts mutant-infected cells at °c. evidence presented demonstrated that the q( )-to-l( ) mutation, located at a highly conserved domain of the s subunit, might hamper processing of the s protein to a matured -kda, endo-glycosidase h-resistant glycoprotein and the translocation of the protein to the cell surface. consequently, some essential functions of the s protein, including mediation of cell-to-cell fusion and its incorporation into virions, were completely abolished. the coronaviridae family contains causative agents of a wide spectrum of diseases affecting humans, mammals, and birds. it is the etiologic agent of severe acute respiratory syndrome (drosten et al., ; fouchier et al., ) . the molecular basis of host specificity and tissue or cell tropism of coronavirus partially resides in the specific interaction between the surface spike protein and cellular receptor(s). this interaction would determine the outcome of infection: whether it is acute or chronic and persistent (rowe et al., a (rowe et al., , b , which organs or tissues are targeted, what symptoms are shown (ballesteros et al., ; leparc-goffart et al., ; navas et al., ) , and even whether host specificity is changed (kuo et al., ) . coronaviruses are a group of enveloped rna viruses with positive-sense, single-stranded genomes of to kb that are packaged into helical structures by the nucleocapsid protein (n). the viruses acquire their envelopes by budding of the structural components into the intermediate compartment (ic) between the endoplasmic reticulum (er) and the golgi apparatus (krijnse-locker et al., ; lai and cavanagh, ; tooze et al., ) . the membrane glycoprotein (m) and the envelope protein (e) mediate the formation of the envelope, as they are involved in the induction of virion assembly and incorporation of other structural components into virus particles (vennema et al., ) . the spike (s) glycoproteins incorporate into the envelope (de haan et al., ) in the form of homooligomeric, nm projections, giving the distinctive morphology of the coronavirus virion. some coronaviruses also contain another envelope protein, the hemagglutinin esterase (he). the largest structural s protein is a type i integral membrane glycoprotein and spans the viral envelope once. in some cases, the s protein is proteolytically cleaved into the n-terminal s and the c-terminal s subunits of equal size by a host proteinase . the s and s subunits are noncovalently associated to each other. a hydrophobic transmembrane domain (tm) of s anchors on the envelope with a short cytoplasmic-tail inside. one leucine-zipper (heptad repeats) domain overlaps with the tm domain and another extends outside the envelope, folding into a-helices and coiled-coil structures involved in oligomerization (de groot et al., ) . it was found that the tm domain and endodomain mediated the incorporation of s into virions but the details of the s/m interaction remain to be elucidated. although s protein is dispensable for the formation of virus-like-particles (vlps), its incorporation into virion is critical to the assembly of infectious virus particles. the s protein is cotranslationally n-glycosylated in the er and also forms stable complex with m in the pre-golgi membrane (holmes et al., ; opstelten et al., ; sturman et al., ) . it was then transported to the golgi apparatus where its high mannose side chains are subsequently trimmed and further modified. the matured s protein is an endo-glycosidase-h-resistant (endo-h), -kda form (luytjes et al., ) . at the trans-golgi, the matured virions are encapsulated into vesicles of the constitutive secretory pathway and released from infected cells. a portion of s protein might be transported to the plasma membrane and responsible for the cell-to-cell membrane fusion, resulting in the formation of typical syncycia and spread of virus infection to neighboring cells. the tm, cysrich and leucine-zipper domains of s and the disparate region of s are associated with the membrane fusion activity of the protein (chang et al., ; gallagher et al., ; krueger et al., ; luo et al., ; tsai et al., ) . cleavage of the s protein enhances the membrane fusion, though not necessarily required. the s protein contains receptor-binding domains in the s subunit (kubo et al., ; godet et al., ; saeki et al., ; suzuki and taguchi, ) . upon binding of the s protein to the receptor(s), conformation changes induce the virus -cell membrane fusion and subsequently unloading of the ribonucleocapsid inside the cell. it has been demonstrated that the cell-to-cell spread of mhv infection does not need the presence of the receptor, but the s protein is required to mediate the cell-to-cell fusion (gallagher et al., ; nash and buchmeier, ) . for coronaviruses, however, the detail of molecular mechanisms of membrane fusion, especially those linked to the s region, is yet to be fully elucidated. in this study, ts mutants were generated by growing the beaudette strain of ibv at progressively lower temperatures from to jc in vero cells. two ts mutants were isolated from passages grown at jc (ts ) and jc (ts ). a revertant was also obtained by growing the ts mutant at the nonpermissive temperature ( jc). sequence comparison revealed that mutations in the s subunit were responsible for the ts phenotype. comparative studies of viral protein synthesis and growth properties of the wt, ts mutant, and revertant viruses demonstrated that the ts mutants were extremely unstable at the nonpermissive temperature. if they were allowed to enter cells by absorption for h at the permissive temperature ( jc), the s protein would be core-glycosylated and partially modified at the nonpermissive temperature, but was unable to assemble into virions. the membrane fusion activities of the ts mutants were totally abolished in virus-infected cells and in cells overexpressing the mutant s protein at the nonpermissive temperature. the beaudette strain of ibv, grown in vero cells at jc, was plaque-purified and was initially adapted to grow at jc. after passages, the virus was subsequently adapted to grow at progressively lower temperatures by culturing at jc for passages, jc for passages, jc for passages, jc for passages, jc for passages, and jc for passages. when the virus was initially adapted to a lower temperature, no cpe was observed within - h postinfection. apparent cpe appeared usually after to passages. the virus was continuously passaged at the same temperature until cpe became visible within - h postinfection, and then shifted to a lower temperature. plaque assays of different passages were performed at both and jc. it was found that viruses from early passages grown at jc could form plaques at both temperatures at similar levels. but passages grown at jc produced much fewer plaques at jc than at jc, suggesting the emergence of ts mutants during cold-adaptation. these ts mutants dominated quickly at jc. two ts mutants, designated ts and ts , were plaque-purified from passage grown at jc and passage grown at jc, respectively. plaque assays showed that both ts and ts formed plaques only at the permissive temperature, but not at all at the nonpermissive temperature. by growing the ts mutant at jc overnight and then at jc for days, a revertant, rev- , was obtained which produced plaques at both and jc at a similar level ( table ). the titers of rev- were much higher than that of the wild-type virus wt (isolated from passage at jc) at jc, as it was derived from the cold-adapted virus (table ) . to map the defects responsible for the ts phenotype described above, the complete nucleotide sequences of wt , ts , ts , and rev- were determined using rt-pcr products and cdna clones from each virus. comparison of the sequence of wt with ts revealed point mutations, a -base-deletion, a -base-, -base-and a -base-insertion (table ). these alterations caused amino acid substitutions in eight mature viral proteins, a single amino acid insertion in the a protein, a truncated b (compared with early passages of wt virus) caused by a single base insertion in the b of ts , and a -amino acid insertion in the n protein. in ts , similar point mutations, deletion and insertion were found ( table ). the most different feature of the two ts mutant is the insertion in the b gene. in ts , a single a insertion was found at a poly-a stretch at positions to , causing frameshift of the b gene and resulting in a c-terminally truncated b protein (shen and liu, ) . however, a three-a-insertion at the same poly-a stretch was found in ts , which does not affect the open reading frame of the b gene. comparison of the sequence of rev- with the two ts mutants showed only one amino acid change (q -to-l ) in the s protein and one amino acid deletion in the nonstructural protein a (i ), which exists in both ts and ts but not in rev- and wt virus. the genotype of the rev- at these two positions (q and i ) are therefore the same as that of wt , but different from the two ts mutants. as both wt virus and the revertant could form plaques at and jc, these two mutations might be responsible for the ts phenotype. a blast search of genbank clearly showed that the amino acid at position of the s protein was a conserved glutamine. the leu residue in the two ts mutants is located immediately downstream of a hypervariable region and the upstream of a conserved region of the s subunit. one additional mutation, d -n , was found in ts only, and an i -m mutation in the two ts mutants and rev- . after mapping the potential determinants responsible for the ts phenotype, we would like to explore the mechanisms by which these mutations affect the propagation of the ts mutants at the nonpermissive temperature. synthesis of the s protein in cells infected with wt virus, ts mutants, and revertant was analyzed. two sets of experiments were carried out. first, vero cells were infected with wt , ts , ts , and rev- at and jc, respectively, and lysates were immunoprecipitated with anti-ibv antibodies. it was obvious that synthesis of the ts mutant s intergenic region between m and a , table titers of wild type, mutant and revertant viruses at jc and jc (pfu/ml) titer jc jc wt .  .  ts .  ts .  rev- .  .  protein was detectable at jc but not at jc (fig. a , lanes , , , and ) under these conditions. in contrast, expression of the s protein was observed in cells infected with wt and rev- at both temperatures ( fig. a , lanes , , , and ), though the level of the proteins detected in cells infected with wt was much lower at jc. this was consistent with the lower titers of wt virus at jc. in the second set of experiments shown in fig. b , cells were absorbed with each virus at jc for h and one of the duplicates was shifted to jc. under these conditions, the two ts mutant s proteins were synthesized at both and jc (fig. b, lanes , , , and ), like those of the wt virus and revertant (fig. b , lanes , , and ). these results indicated that the viral structural protein could be synthesized at nonpermissive temperature if the mutants were allowed to enter cells. the viral subgenomic rna and one of the processed proteins, the c-like proteinase, were also detected in the same ways (data not shown), and the results were consistent with those described above. comparison of the nucleotide sequences and the s protein synthesis of wt, ts mutants and revertant indicate that the single amino acid mutation (q -l ) in the s protein may be responsible for the temperature sensitivity of the mutant virus. this possibility was studied by biochemical and functional characterization of the s protein from the revertant and a ts mutant. the s gene of rev- and ts was cloned under the control of a t promoter to investigate whether membranefusion activity was affected by the mutations in the s protein. cells were infected with recombinant vaccinia/t virus and were transfected with plasmids containing the s gene from either ts or rev- . the expression of the s proteins was analyzed by western blotting using anti-ibv antibodies. as shown in fig. a , the s protein of both revertant and ts mutant was expressed at and jc (lanes , , , and ), though the s protein of ts was expressed at a relatively lower level than the rev- at jc (compare lanes and ). the fusion activity of the s protein derived from the revertant and ts , respectively, was then examined. as shown in fig. b , membrane fusion was observed in cells expressing the s gene of ts at jc (panel c) but not at jc (panel f), at h posttransfection. in contrast, membrane fusion was observed in cells expressing the s gene of revertant at both and jc (panels b and e). interestingly, membrane fusion appeared at h posttransfection, after the cells expressing the s gene of ts were shifted from to jc at h posttransfection (panel i). the recombinant s genes of the ts mutant and revertant were transfected into cells in mm dishes at jc and one of the duplicates was shifted to jc at h posttransfection. radiolabeled proteins were immunoprecipitated and treated with endo-h at jc. as shown in fig. a , the ts mutant s protein, synthesized at jc, was sensitive to endo-h digestion, as no -kda form was observed (lane ). in contrast, a portion of the ts mutant s protein, synthesized at jc (lanes ), was resistant to endo-h digestion. the revertant s protein, synthesized at both and jc, was also resistant to endo-h digestion (lanes and ). these a b fig. . analysis of the expression of the s protein from wt (wt ), ts mutants (ts and ) and revertant (rev- ) viruses. (a) cells were infected with each virus at jc and jc as indicated on the top for h and were maintained at the same temperatures. (b) two dishes of cells were infected with each virus at jc for h. one of the duplicates was maintained at jc (lanes , , , and ) and the other one was shifted to jc (lanes , , , and ). radiolabeled cell lysates were immunoprecipitated with anti-ibv antibodies. the proteins were separated on . % polyacrylamide gels and detected by autoradiography. numbers on the left indicate molecular mass in kilodalton and the position of the s protein is indicated on the right. results indicate that the ts mutant s protein synthesized at jc was not a mature form of the glycoprotein. in addition, a novel glycosylated form of the s protein, migrating between the endo-h treated -kda and the matured -kda forms, was observed. it might represent trimming of an initial, core-glycosylated form of the s protein in the er and the cis-golgi. interestingly, this band was also observed in ts -infected cells (fig. b, lane ) , when the incubation time at jc was extended before the culture was shifted to jc. this band is much stronger than the -kda form (lane ), giving direct evidence that the mutations in the s protein of ts dramatically hampered the maturation process of the s glycoprotein. compared with ts , the s protein of ts contains another amino acid substitution, d -n (table ) , which is not present in the s protein of rev- and wt . as this band was not observed in ts -infected cells (data not shown), it is not clear at this point if this additional mutation may also play a role in the maturation of the s protein. it was also noted that, at jc for all viruses, the s and s subunits were hardly detected in cell lysates but abundant in the supernatants (fig. b) . at the nonpermissive temperature, the s protein was not cleaved at all (lanes and ). these results may suggest that only the mature form of the s glycoprotein could be cleaved into s and s and fig. . membrane fusion activity of the s protein expressed from wt and ts . (a) vero cells were transfected with plasmids without insert (lanes and ) or with the s gene from the wt (lanes and ) and ts (lanes and ), and were cultured at jc (lanes , , and ) or jc (lanes , , and ). the expression of the s protein was examined in western blot using anti-ibv antibodies, and h-tubulin was immunostained as loading controls. (b) the membranefusion activity of the s protein from the wt (panels b, e, and h) and ts mutant (panels c, f, and i) at jc (panels b and c) and jc (panels c and f) days post-transfection was compared. panels h and i show induction of membrane fusion on cells transfected with the wt and ts by shifting cells shown in panels e and f to jc for day. panels a, d, and g show cells transfected with empty plasmids. reinforce the conclusion that the majority of the s protein of the ts mutants synthesized at the nonpermissive temperature may represent a pre-mature form of the s glycoprotein. to detect whether the ts mutant s protein would be transported to the plasma membrane at the nonpermissive temperature, the s protein was transiently expressed in vero cells using the vaccinia virus-t expression system. the transfected cells were treated with cycloheximide at . h posttransfection for min to inhibit further protein synthesis. the cells were then incubated at and jc, respectively. the subcellular localization of the s protein was analyzed by indirect immunofluorescent staining at , , , and h postinfection, respectively. the confocal microscopy images of transfected cells are shown in fig. . plasma membrane staining of the ts mutant s protein was not observed at any time points posttransfection at jc, but was clearly seen at jc at and h postinfection (fig. ) . the revertant s protein was expressed on the cell surface at both temperatures at and h posttransfection. these results may explain the lack of membrane fusion activity of the ts mutant s protein in both infected and transfected cells. endo-h treatment of the s protein from rev- and ts . the s protein derived from rev- (lanes , , , and ) and ts (lanes , , , and ) were expressed in vero cells at jc (lanes , , , and ) and jc (lanes , , , and ), using a t -vaccinia expression system. the radiolabeled proteins were immunoprecipitated with anti-ibv antibodies, the eluted proteins were endo-h-(lanes , , , and ) or mock-treated (lanes , , , and ) and analyzed by sds-page. (b) detection of the less matured s protein from ts at jc and the defect in cleavage of the mutant s protein. cells were infected with ts and rev- for h at jc, one of the duplicates was maintained at jc (lanes , , , and ), and the other one was shifted to jc (lanes , , , and ). cells were radiolabeled and viral proteins were immunoprecipitated with anti-ibv antibodies and analyzed by sds-page. lanes , , , and refer to viral products detected from cell lysates and lanes , , , and refer to viral proteins detected from virus particles released to the cultured media. quantitative analysis of surface expression of the wild type and ts mutant s protein was carried out by immunofluorescent staining with anti-s protein antiserum and the positive staining cells were sorted by flow cytometry. as shown in fig. , . % of nonpermeabilizing (panel a) and . % of permeabilizing (panel f) cells expressing the empty plasmid showed background staining. when the cells were incubated at the permissive temperature, . % ( . - . ) of hela cells expressing the wt s protein (panel b) and . % ( . - . ) of cells expressing the ts mutant s protein (panel c) displayed surface staining. after permeabilizing with . % saponin, . % ( . - . ) of cells expressing the wt s protein (panel g) and . % ( . - . ) of cells expressing the ts mutant s protein (panel h) showed positive staining. when the cells were incubated at the nonpermissive temperature, . % ( . - . ) of cells expressing the wt s protein (panel d) and . % ( . - . ) of cells expressing the ts mutant s protein (panel e) exhibited surface staining. after permeabilizing with . % saponin, . % ( - . ) of cells expressing the wt s protein (panel i) and . % ( . - . ) of cells expressing the ts mutant s protein (panel j) showed positive staining. these results confirm that the ts mutant s protein could not be efficiently translocated to the cell surface at the nonpermissive temperature. to investigate whether the s protein of ts mutant was assembled into virion or not, ts and rev- -infected cells were radiolabeled and the virus particles were purified through sucrose gradients twice. immunoprecipitation of the purified virions using anti-ibv antibodies showed that the s protein of ts was not detected at jc (fig. , lane ), but was observed at jc (fig. , lane ) . the s protein was detected at both temperatures for rev- (fig. , lanes and ) . these results render support that the spike protein of the ts mutant may not be assembled into virus particles at jc. coronavirus s protein is responsible for receptor binding and membrane fusion. it also induces neutralizing antibodies and bears determinants for virulence. a considerable diversity in s protein among coronaviruses exists, which contributes to host specificity, cell and organ tropisms, and pathogenesis. characterization of mutants with point or deletion mutations in the s protein has helped to establish links between the variations and the functions or altered antigenicity and virulence of viruses. through sequence analysis of wt virus, two ts mutants, a revertant and different passages of a cold-adapted ibv, data presented in this study not only mapped the defects of the ts mutants to the s gene, but also revealed the molecular events occurred during evolution of the ibv s gene. compared with the parental wt virus, mutations that are identical in the two ts mutants and the revertant might be associated with the cold-adaptation phenotype, while those identical only in the two mutants but different from the revertant and wt virus were considered to be linked to the ts phenotype. according to this criterion, the q -to-l mutation in the s protein that exists in the two ts mutants, and interestingly, occurred at a highly conserved domain among ibv viruses immediately downstream a variable domain in the s subunit may be responsible for the ts phenotype. the mutants with this mutation accumulated and became dominant under selective pressures, in this case, changes in the hosts and temperatures. the i -m mutation emerged earlier than the q -l mutation, which among variations in other gene products may be associated with cold-adaptation, suggesting potential segregation of mutations responsible for either cold-adaptation or ts phenotypes. further confirmation of the possibility that the q -to-l mutation may cause the ts phenotypic changes is currently being carried out by introducing the mutation to an infectious ibv clones and isolation of a recombinant virus containing this mutation only. the s protein is co-translationally n-glycosylated in the er, oligomerized (luytjes et al., ) if folded properly, and associated with the m protein in pre-golgi membrane (opstelten et al., ) . one of the essential steps in nlinked glycosylation is transfer of a preformed, -coreunit-oligosaccharide to a specific asn residue in the sequence asn-x-ser/thr (where x is any residue except pro, asp, or glu). the oligosaccharide chain is donated by dolichol-pyrophosphate-oligosaccharide to the protein chain in the er. it is trimmed down in the er and cis-golgi, and different external sugars are then added to the trimmed chain in the medial-and trans-golgi. glycoproteins with high mannose oligosaccharides in the er and cis-golgi remain sensitive to endo-h. they become endo-h resistant after being processed by the medial-and trans-golgi resident enzymes to glycoproteins with complex oligosaccharides. the acquisition of endo-h resistance is therefore an indication that viral n-linked glycoproteins are properly processed and transported to the golgi apparatus (luo and weiss, ; luo et al., ; luytjes et al., ) . examination of the amino acid sequence of ibv s protein showed that there are and potential glycosylation sites in the s and s subunits, respectively. the calculated molecular weight of the s protein is kda. if all sites are used, the initially transferred oligosaccharides account for kda. in cells overexpressing s protein, core-glycosylation of the s protein in the er resulted in a -kda product as expected. trimming of the s glycoprotein in the er and cis-golgi led to a partially processed -kda form, larger than the endo-h-treated -kda form which only has one sugar residue left at each site. both the trimmed -and core-glycosylated -kda forms were sensitive to endo-h digestion. at the permissive temperature, further modification of the mutant s protein in the medial-and trans-golgi by addition of sugars to the trimmed chains resulted in the maturation of s, which co-migrates with the core-glycosylated -kda form but is resistant to endo-h digestion. at the nonpermissive temperature, however, no mature form of the fig. . analysis of the structural proteins on purified ts and rev- virions. immunoprecipitations were performed using virions, purified from ts -(lanes and ) and rev- -infected (lanes and ) vero cells at jc (lanes and ) and jc (lanes and ) . the viral proteins were separated on . % polyacrylamide gels and detected by autoradiography. numbers on the left indicate molecular masses in kilodalton and the positions of the three structural proteins are indicated on the right. mutant s protein was produced. in virus-infected cells, the endo-h sensitive, -kda form of ts was abundant at the nonpermissive temperature, clearly indicating that the mutant s protein was trimmed in the er and cis-golgi but was not transported to the trans-golgi. furthermore, the mutant s protein was not cleaved into s and s at the nonpermissive temperature, which usually occurs in the trans-golgi shortly before the release of virions from cells (frana et al., ) . as only spikeless virions were produced, these results demonstrated that the core-glycosylated or the trimmed forms of s were unable to incorporate into virions. in addition, the mutant s protein fails to induce cell -cell fusion at the nonpermissive temperature. as the q -to-l mutation is not in the fusogenic domain of s, it renders support to the conclusion that the mutation causes the defect in its modification and intracellular transport, instead of having direct impact on the fusion process. taken together, these studies clearly indicate that the mutant s protein could not reach the site where the s protein is incorporated into virions. the reasons for the retention of the mutant s protein in the er and cis-golgi are yet to be explored. previous studies show that the retention of monomeric s protein in the er is due to misfolding induced by disruption of disulfide bonds (opstelten et al., ) , and also caused by lack of oligomerization of endo-h-sensitive s protein of ts mutants though the locations were not determined (luytjes et al., ) . it is not clear whether mutations in ibv s protein have an effect on its folding or oligomerization. nevertheless, in both circumstances, the intracellular transport, modification of the s protein and its incorporation into virion would have been affected. studies with some of the other enveloped viruses demonstrated that molecular chaperones (like calnexin and calreticulin) in the er transiently and specifically interact with partially trimmed, monoglycosylated form of viral n-linked glycoproteins (tatu and helenius, ) . for instance, calnexin interacts with the influenza hemagglutinin and vesicular stomatitis virus g protein (hebert et al., ; hammond and helenius, ) . these and other proteins could facilitate proper folding and ensure quality control for viral glycoproteins. proteins that fail to fold and oligomerize are prevented from transport and ultimately degraded. with the ts mutants, it is possible to explore the interaction of the s protein with molecular chaperones and folding enzymes. this approach would provide new insights into the maturation and assembly of the coronavirus s protein. the ts mutant quickly lost its infectivity at the nonpermissive temperature. this low thermostability indicated that even if the mutant s protein was synthesized, fully modified and assembled into virions at the permissive temperature, the resulting particles were relatively unstable and lost infectivity at the nonpermissive temperature. it suggests that the q residue in the conserved s domain of ibv may also play an essential role in maintaining the thermal stability of the spike in the virion, resembling the s residue in the s region of an mhv ts mutant (ricard et al., ) . in addition, if cells were incubated with the ts mutants at the nonpermissive temperature, viral proteins were hardly detected, indicating that at the nonpermissive temperature, the ts mutant s protein may be unable to either bind efficiently to the receptor or induce virus -cell membrane fusion in contrast to the s protein of the revertant and wt virus. this observation suggests that the mutation in the s protein might induce conformation changes of the spikes on virions at the nonpermissive temperature and hamper either process. vero cells were maintained in complete dmem medium (gibco brl), supplemented with newborn calf serum ( %), streptomycin ( ag) and penicillin ( units/ml). the beaudette stain of ibv was purchased from atcc and propagated in chicken embryonated eggs for three passages. the virus was then adapted to grow and passage on vero cells for times at jc. in this study, the viruses were further passaged on vero cells at progressively lower temperatures for passages (at , , , , , , and jc for , , , , , , and passages) . virus stocks were used for plaque assays, plaque-to-plaque purification and viral rna extraction as described previously (shen et al., ) . the wild-type virus, wt , was plaque-purified from passage grown at jc, while ts mutants, ts and ts , were purified from passage grown at jc and passage grown at jc, respectively. the revertant, rev- , was plaque-purified from cells infected with ts for h at jc and then shifted to jc for days. recombinant vaccinia/t (v/t ) virus was propagated and was tittered on vero cells. virus stocks were kept at À jc until use. confluent monolayers of vero cells were infected with viruses at a multiplicity of infection (moi) of . . after h absorption, the viruses were radiolabeled by replacing medium with methionine-free dmem supplemented with aci/ml [ s]-methionine. after -h incubation at temperatures appropriate for each virus, cells were harvested and virus stock was prepared by freezing and thawing three times. cell debris was removed by centrifugation at rpm for min (beckmen, . ). the supernatant was centrifuged through a % sucrose cushion and the resulting pellet was resuspended in tne buffer ( mm tris -hcl, ph . , mm nacl, mm edta). the viruses were further purified by centrifugation through a - % su-crose gradient in tne buffer at , rpm (beckmen, sw ) twice. fractions containing the virus were pooled together and used for protein analysis. viral structural proteins were labeled with [ s]-methionine, immunoprecipitated and separated on . % polyacrylamide gels as described previously (liu et al., ) . the anti-ibv serum was raised in rabbit against purified ibv virions and was used in immunoprecipitation and western blot as previously described (liu and inglis, ) . a portion of cell lysates was immunoprecipitated with anti-ibv serum. the pellets were washed three times with standard ripa buffer, dissolved in al of digestion buffer ( mm tris, ph . , . % sds) and incubated at jc for min. ten microliters of the supernatants were mixed with al of digestion buffer with or without endo-h ( . mu, boehringer mannheim) and incubated for h at jc. confluent monolayers of vero cells were infected with recombinant vaccinia/t viruses at a moi of . . after h absorption, cells (  ) were trypsinized, centrifuged at g and resuspended in ml of pbs. the cells ( . ml) were mixed with ag plasmid and electroporated (easyject, equibio) at v in a . -cm cuvette (biorad). the cells were then plated in a -mm dish containing ml of dmem with % of newborn calf serum and incubated at temperatures indicated. viral rna was extracted from the purified viruses using the rneasy mini kit (qiagen) according to the manufacturer's instructions. reverse transcription and polymerase chain reaction (rt-pcr) were performed using the expand reverse transcription and high fidelity pcr kits (boehringer mannheim). annealing and extension times of pcr were optimized for amplification of pcr products with different sizes using different primers. more than specific primers were used for amplification, sequencing and cloning. automated sequencing was carried out using pcr products or cdna clones and specific primers as previously described (shen et al., ) . sequence analysis was carried out using the gcg and blast suite of programs as described previously (shen and liu, ) . hela cells were infected with vaccinia/t virus, transfected with constructs encoding wt and ts mutant s protein using qiagen effectene transfection reagent, and incubated at the permissive and nonpermissive temperatures, respectively, for h. cells were harvested, washed once with pbs, resuspended in blocking buffer ( % fbs and % bsa in pbs), incubated on ice for min. a half of the cells were permeabilized with . % saponin in facs washing buffer ( . % fbs and . % sodium azide in pbs) incubated for min at room temperature, and stained with : diluted primary antiserum, the rabbit anti-ibv s protein. the other half was stained directly with the same primary antibody. cells were then washed two times with the facs washing buffer, stained with : diluted fitc conjugated swine anti-rabbit antibody (dako). after washing two times with the facs washing buffer, cells were then fixed with % ice cold paraformaldehyde and analyzed by flow cytometry. two amino acid changes at the n-terminus of transmissible gastroenteritis coronavirus spike protein result in the loss of enteric tropism coronavirus-induced membrane fusion requires the cysteine-rich domain in the spike protein evidence for a coiled-coil structure in the spike of coronaviruses mapping of the coronavirus membrane protein domains involved in interaction with the spike protein identification of a novel coronavirus in patients with severe acute respiratory syndrome aetiology: koch's postulates fulfilled for sars virus proteolytic cleavage of the e glycoprotein of murine coronavirus: host-dependent differences in proteolytic cleavage and cell fusion alteration of the ph dependence of coronavirus-induced cell fusion: effect of mutations in the spike glycoprotein cell receptor-independent infection by a neurotropic murine coronavirus major receptorbinding and neutralization determinants are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein folding of vsv g protein: sequential interaction with bip and calnexin calnexin and calreticulin promote folding, delay oligomerization and suppress degradation of influenza hemagglutinin in microsomes tunicamycin resistant glycosylation of coronavirus glycoprotein: demonstration of a novel type of viral glycoprotein characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the rer to the golgi complex requires only one vesicular transport step variations in disparate regions of the murine coronavirus spike protein impact the initiation of membrane fusion localization of neutralizing epitopes and the receptor-binding site within the amino-terminal amino acids of the murine coronavirus spike protein retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier the molecular biology of coronaviruses altered pathogenesis of a mutant of the murine coronavirus mhv-a is associated with a q l amino acid substitution in the spike protein association of the infectious bronchitis virus c protein with the virion envelope proteolytic mapping of the coronavirus infectious virus b polyprotein: evidence for the presence of four cleavage sites of the c-like proteinase and identification of two novel cleavage products roles in cell-to-cell fusion of two conserved hydrophobic regions in the murine coronavirus spike protein amino acid substitutions within the leucine zipper domain of the murine coronavirus spike protein cause defects in oligomerization and the ability to induce cell-tocell fusion characterization of two temperature-sensitive mutants of coronavirus mouse hepatitis virus strain a with maturation defects in the spike protein spike glycoprotein-mediated fusion in biliary glycoprotein-independent cell-associated spread of mouse hepatitis virus infection murine coronavirus spike protein determines the ability of the virus to replicate in the liver and cause hepatitis disulfide bonds in folding and transport of mouse hepatitis coronavirus glycoproteins envelope glycoprotein interactions in coronavirus assembly a conditional-lethal murine coronavirus mutant that fails to incorporate the spike glycoprotein into assembled virions evolution of mouse hepatitis virus: detection and characterization of spike deletion variants during persistent infection generation of coronavirus spike deletion variants by high-frequency recombination at regions of predicted rna secondary structure identification of spike protein residues of murine coronavirus responsible for receptor-binding activity by use of soluble receptor-resistant mutants determination of the complete nucleotide sequence of a vaccine strain of porcine reproductive and respiratory syndrome virus and identification of the nsp gene with an unique insertion emergence of a coronavirus infectious bronchitis virus mutant with a truncated b gene: functional characterization of the b protein in pathogenesis and replication rearrangement of the vp gene of a group a rotavirus in combination with a point mutation affecting trimer stability sequence analysis and in vitro expression of genes and of an ovine group b rotavirus isolate, kb : evidence for a non-defective, c-terminally truncated nsp and a phosphorylated nsp proteolytic cleavage of the e glycoprotein of murine coronavirus: activation of cell-fusing activity of virions by trypsin and separation of two different k cleavage fragments analysis of the receptor-binding site of murine coronavirus spike protein interactions between newly synthesized glycoproteins, calnexin and a network of resident chaperones in the endoplasmic reticulum replication of coronavirus mhv-a in sac-cells: determination of the first site of budding of progeny virions a -amino acid stretch in the hypervariable region of the spike protein s subunit is critical for cell fusion activity of mouse hepatitis virus nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes this work was supported by the biomedical research council, agency for science technology and research, singapore. key: cord- -g zynbul authors: person, bobbie; sy, francisco; holton, kelly; govert, barbara; liang, arthur; garza, brenda; gould, deborah; hickson, meredith; mcdonald, marian; meijer, cecilia; smith, julia; veto, liza; williams, walter; zauderer, laura title: fear and stigma: the epidemic within the sars outbreak date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: g zynbul because of their evolving nature and inherent scientific uncertainties, outbreaks of emerging infectious diseases can be associated with considerable fear in the general public or in specific communities, especially when illness and deaths are substantial. mitigating fear and discrimination directed toward persons infected with, and affected by, infectious disease can be important in controlling transmission. persons who are feared and stigmatized may delay seeking care and remain in the community undetected. this article outlines efforts to rapidly assess, monitor, and address fears associated with the severe acute respiratory syndrome (sars) epidemic in the united states. although fear, stigmatization, and discrimination were not widespread in the general public, asian-american communities were particularly affected. p ublic health strategies that deal with rapidly evolving disease outbreaks of new and emerging infectious diseases require a delicate balance between protecting the public's health and initiating exclusionary practices and treatments that can lead to fear and stigmatization of, and discrimination against, specific populations. the outbreak of severe acute respiratory syndrome (sars) illustrates these difficulties. sars spontaneously appeared in the southern province of guangdong, people's republic of china, in november ( , ) . by july , the epidemic, had spread to more than countries with , cumulative probable cases and deaths and was identified as a global threat to health ( ) . in the united states, cases were reported with classified as probable sars; no deaths occurred ( ) . as with many disease outbreaks, scientific information and data related to the disease changed almost hourly, as public health scientists and practitioners responded to the worldwide outbreak, which was coupled with widespread fear ( , ) . while persons, agencies, and governments sought to identify modes of transmission, strategies for disease containment, and treatment for sars, fear spread unchecked throughout the global community. fear of sars arose from the underlying anxiety about a disease with an unknown cause and possible fatal outcome ( ) . stigmatization of potential sars patients emerged early in the outbreak, as global media reported dramatic stories from asia in print media, television, and the internet. headlines from the english-language press heightened the fear. "concern is mounting over the continuing spread of the deadly sars virus. some experts say it could have a similar impact to the flu epidemic that killed million-or the current world hiv crisis," wrote the british broadcasting corporation from london, england ( ) . "china has threatened to execute or jail for life anyone who deliberately spreads the killer sars virus," stated the cable news network from beijing, china ( ) . studies have shown that during serious disease outbreaks, when the general public requires immediate information, a subgroup of the population that is at potentially greater risk of experiencing fear, stigmatization, and discrimination will need special attention from public health professionals ( ) ( ) ( ) . the recent sars outbreak was a classic example of such an outbreak. fear is further fueled when infection control techniques and restrictive practices such as quarantine and isolation are employed to protect the public's health ( , ) . while exclusionary practices based upon the best available scientific evidence may be scientifically and ethically sound for one population, those same practices may not be sound for all populations ( , ) . during the sars outbreak, some persons became fearful or suspicious of all people who fear and stigma: the epidemic within the sars outbreak looked asian, regardless of their nationality or actual risk factors for sars, and expected them to be quarantined. some americans did not understand that quarantine and isolation practices appropriate for sars-affected areas in asia, where community transmission was a concern, were practices that were not appropriate in the united states where the disease was not community acquired. for example, some persons, who had recently traveled to areas where sars was spreading, isolated themselves, even though they had no symptoms and had not been exposed to someone with sars. fear of being socially marginalized and stigmatized as a result of a disease outbreak may cause people to deny early clinical symptoms and may contribute to their failure to seek timely medical care ( ) . such fear can ultimately increase stigmatization when cases are identified at a later date ( ) . stigmatization associated with discrimination often has social and economic ramifications that intensify internalized stigmatization and feelings of fear ( ) . containing fear, which is integral to the public health management of a new and emerging disease such as sars, is best accomplished by a behavioral strategy that addresses the needs of a segment of the population at risk of becoming stigmatized and discriminated against. this strategy works best as a complement to a larger public health education and communication campaign. typically during outbreaks, initial risk communication is targeted to frontline public health professionals through vehicles such as the morbidity and mortality weekly report. initial communication provides information on case definitions and laboratory-testing strategies, as well as interim guidelines for infection control and other critical issues. communication strategies for the general public most frequently involve television sound bites, press conferences with dignitaries and health officials, and targeted release of information to mass media outlets such as newspapers and internet sites ( ) . although these risk communication activities are critical for keeping the general public informed during an outbreak, they can fail to meet the personal needs of the affected population and the general public. during the first week of april , the national center for infectious diseases (ncid) at the centers for disease control and prevention (cdc) formed a -member, multidisciplinary ncid/sars community outreach team as part of its emergency response to the global sars outbreak. while other ncid/cdc response teams dealt with laboratory investigations, surveillance, communica-tion, and clinical infection control practices, the community outreach team worked to implement rapid public health strategies to document, monitor, and assist in ameliorating specific problems associated with fear, stigmatization, and discrimination attributed to the sars outbreak in the united states. in creating a rapid public health intervention to mitigate behaviors and practices associated with sars-related fear, the team recognized the need to address the experiences of persons at greatest risk for experiencing sars-related fear, stigma, and discrimination. the team monitored stigmatizing ideas and behaviors in the general population and the media, particularly toward asian americans, who were disproportionately reporting fear, stigmatization, and discrimination compared to the general public. the team began working with asian-american communities to develop a culturally tailored intervention that ) promoted community understanding of the facts related to the transmission and prevention of sars; ) contributed to the strengthening of community resiliency and capacity to mitigate fear, stigmatization, and discrimination; and ) encouraged appropriate health-seeking behaviors for those who may have been exposed to sars and were experiencing early symptoms. the team also worked to dispel myths; keep the general public better informed; prevent discrimination against sars-affected communities; and provide guidance for institutions, agencies, and organizations hosting international visitors from sars-affected countries. during the first weeks of april , the ncid/sars community outreach team conducted a rapid situational analysis to determine the impact of sars-related fear, stigmatization, and discrimination within the asian-american community in the united states. the team carried out the following activities: ) facilitated group discussions with key opinion leaders within the asian community in the united states; ) collected and monitored the cdc public response service data; ) collected and monitored asian-language newspapers, internet sites, and other information sources; ) reviewed polling data and other communication information; ) conducted community visits, panel discussions, and media interviews; ) solicited information from state and regional minority health liaisons nationwide; ) developed ongoing relationships with the asian-american communities; particularly in major metropolitan areas throughout the united states; and ) determined new datagathering strategies as needed. the team conducted group interviews through teleconferences with national, state, and local influential leaders in the asian-american community throughout the united states. the team also conducted group interviews with chambers of commerce and trade association members, school officials and representatives, state public health department staff, academicians at universities, mental health professionals, and others. the teleconferences the team conducted reached more than persons who represented more than agencies, organizations, and communities. the goals of the group interviews were the following: ) determine the impact of sars-related fear on the asian community; ) document examples of fear, stigmatization, and discrimination; ) determine strategies for identifying and reaching "hidden populations"; ) develop partnerships with leaders and community members of the affected populations; ) determine the needs of affected populations; and ) respond appropriately to those needs through a targeted intervention with activities and asian-language materials. five major recommendations were derived from the facilitated group discussions with key informants: ) develop simple, tailored sars prevention messages; ) develop sars information materials in various asian languages; ) disseminate sars information through multiple and culturally appropriate channels, including (but not limited to) community visits, town hall meetings, and health education and communication channels to complement mass media messages; ) establish partnerships with local asian-american community-based organizations to educate the community; and ) ensure that cdc would continue to provide leadership and coordination in preventing and controlling sars. the relationships developed during these group discussions allowed team members to monitor and document ongoing stigmatizing situations related to the disease outbreak in real time and to deal more effectively with intentional and unintentional discrimination. cdc operates the public response service (cdc prs) under contract with the american social health association. this contract provides hotline service to the general public requesting information via telephone and email about bioterrorism and other disease emergencies, including sars. the ncid/sars community outreach team worked with the cdc prs to track a daily sample of incoming sars-related calls, specifically noting questions associated with fear, stigmatization, and discrimination directed toward the asian-american community. this system allowed the team to help determine specific answers to frequently asked questions for hotline staff and to develop simple, prerecorded asian-language messages. passive data collection of sars fear-related concerns began on april , . during may , , sars-related calls were received; , ( . %) of these calls were pas-sively sampled. of these sampled calls, an average of % of callers expressed concerns related to fear, stigmatization, and discrimination. a caller could express more than one concern. major concerns included the following: fear of buying asian merchandise ( calls); working with asians ( calls); living near asians ( calls); going to school with asians ( calls); and more generic issus such as being on a cruise ship or airplane ( calls); and church, school, or workplace issues ( calls). most sars calls related to transmission, symptoms, and treatment of disease and travel advisories. one critical component of the team's activities was determining where members of the asian-american community were getting sars-related information. team members monitored english-language and asian-language electronic, print, and television media coverage and informal chat rooms in the united states and other countries to stay abreast of changing information about the nature of the sars outbreak that could influence fear, stigmatization, and discrimination. the assessment showed that many people within the asian-american community were getting information from asian-language newspapers, television, and internet sites directly from china, hong kong, taiwan, and other asian areas-usually hours ahead of information providers in the united states. the information provided by these asian-language sources was often inconsistent with newspaper, television, and internet coverage in the united states, thus creating fear and suspicion that the united states government might not be telling the truth about the outbreak in this country. independent contentanalysis research conducted by intertrend communications (san francisco, ca) compared four of the most popular chinese language newspapers in the united states with two popular national mainstream english-language newspapers from march to april , ( ) . intertrend data showed that ) chinese-language newspapers were more likely to highlight sars news related to the chinese community in the united states or from china more prominently than mainstream englishlanguage newspapers; and ) chinese-language newspapers were more likely to have articles on sars, including featured in-depth articles, than mainstream english-language u.s. national newspapers ( ). these findings supported the team's initial assessment (based on an informal convenience sample of asian-language papers). general email inquiries sent to the cdc communications center and information from public health professionals, health providers, and community members led the team to sars-related internet sites that contained rumors and inaccurate information, which added to general misunderstanding, confusion, and fear. even legitimate public health internet sites from different parts of the world provided disparate information as the outbreak unfolded, furthering uncertainty and fear in the united states. the team also monitored internet sites that supported community fears as they promoted home remedies, medicinal cures, and inappropriate and unnecessary protective equipment. monitoring the information sources of the affected population was a critical activity, allowing the team to separate fact from fiction with accuracy and timeliness and address salient issues and concerns during community visits. based on its rapid situational assessment, the team was able to develop interventions to assist in mitigating fear, stigmatization, and discrimination. team members carried out the following activities: ) advised other sars emergency response teams on how to minimize the risk of stigmatizing groups in their own communications by focusing messages on the virus and the relevant behavioral risk factors; ) assisted with developing culturally tailored health education materials; and ) conducted community visits, panel discussions, and media interviews to positively influence negative behaviors occurring in communities. these visits and other contacts with the asian-american community allowed cdc to develop ongoing relationships and helped the team determine new data-gathering strategies. during a disease outbreak, information changes rapidly as scientific evidence is collected and analyzed. vital components of the team's activities were prioritizing and translating existing information and guidance documents and developing health education materials to address the specific needs of the asian-american community. an inhouse translation service did not exist, and the rapidly evolving scientific evidence challenged the turnaround time for developing, translating, and disseminating information. the team worked to identify priority documents for translation and to ensure asian-language translation for web and print products tailored to the asian-american community. to ensure accurate translations, cdc contracted with professional translation services and had all documents back-translated. web-based information on sars included documents in traditional chinese, simplified chinese, korean, vietnamese, and japanese, as well as french and spanish. the team also created brief, recorded educational hotline messages in chinese and vietnamese. the main messages for people in the united states were the following: ) the risk of sars is low; ) severe cases of sars have been uncommon, and there have been no deaths in the united states; ) methods for disease prevention in the general public are like those of other viral diseases; and ) although no evidence of community spread currently exists, continued vigilance, aggressive case management, and infection control are needed. team members conducted field visits to asian communities in boston; new york city; oakland, california; san francisco; washington, d.c.; edison, new jersey; and los angeles to respond to the direct needs of the communities and gather information. the team met with community leaders, toured the communities, informally gathered further information, and gave community sars presentations in seven cities, reaching approximately persons. through community visits, the team was able to ) provide the latest in evidence-based information on sars with asian-language education materials; ) dispel misconceptions, myths, and rumors; ) act as a catalyst for bringing together a broad spectrum of organizations and persons in the community to create local networks to promote community resiliency; and ) provide credibility and reassurance to those who felt vulnerable. speakers also presented a public health model for mitigating fear, stigmatization, and discrimination that could be instituted by public health officials, clinicians, and community members. through open discussion sessions and informal information gathering in the community, the team found that sars-related stigmatization was occurring more frequently within the asian community than from outsiders directed toward the asian community. the team also found that those persons with sars-like symptoms who used traditional herbal physicians and pharmacies were less likely to be referred to, or seek out, public health officials, suggesting that further research into strategies to reach this population is needed. conducting community visits also showed that cdc was responding to the needs of the community at risk for sars-related fear, stigmatization, and discrimination and was modeling positive behaviors to the public. other infectious disease epidemics have been associated with specific ethnic groups. fear, stigmatization, and discrimination plagued russian jewish immigrants when the outbreaks of typhus fever and cholera in new york city were traced to russian jewish immigrants from eastern europe ( ) . in the spring of , the chinatown community in san francisco was faced with extreme discrimination due to an outbreak of bubonic plague, the "black death," attributed to rats transported on a ship from hong kong ( ) . in an outbreak of hantavirus infection in the four corners area (where the borders of four states-arizona, new mexico, utah, and colorado-meet) of the united states was initially referred to by reporters as a navajo disease, which led to severe fear, stigmatization, and discrimination of native americans in the region ( ) . previous scientific studies have shown that fear associated with stigmatization and discrimination has negatively affected public health efforts with chronic conditions and diseases such as mental illness, hiv/aids, tuberculosis, leprosy, and epilepsy ( ) ( ) ( ) ( ) ( ) . more recently, stigmatization associated with fear and the aids epidemic negatively influenced voluntary testing, counseling, and treatment of those infected with the disease ( ) . health providers have also seen reluctance by recent refugees and immigrants to get tested and treated for tuberculosis because of possible social stigmatization ( ) . the potential of being labeled at-risk for having or transmitting a stigmatizing condition such as sars creates fear and anxiety, and an entire population of people can be at risk for becoming stigmatized in society ( ) . protecting the health of the public while preventing stigmatization of segments of the population during a rapidly evolving disease outbreak is complex. the team's experience during the recent sars outbreak demanded anticipatory insight, perceptive planning, and a rapid response to a targeted audience with specific cultural perspectives and influences. it also required us to recognize the distinctive features of sars in a medical, social, and cultural context. weiss states, "preventing fear and stigmatization depends on controlling or treating the target health problem, countering tendencies of those who stigmatize others, and supporting those who are stigmatized through emotional support and social policies" ( ) . the data collected during the rapid situational assessment were critical in guiding activities of the team. both the data and the data collection process assisted the team in establishing interpersonal relationships with community leaders, determining priority needs, identifying responsible intervention strategies, and developing effective communication channels. the team was able to better understand community perceptions and attitudes by identifying the communities' trusted sources of information. when conducting community visits, the team was able to address discordant information, myths, and rumors; provide simple asian-language messages and materials; and act as a catalyst to build community resiliency and prepare for the possibility of future emerging diseases. the team was also able to keep cdc/ncid leaders informed and to intervene when they identified discriminatory policies, practices, and actions that were inconsistent with evidence-based public health recommendations and guidelines. quelling fear-driven stigmatization and discrimination during the sars outbreak required tailored intervention strategies carried out by the sars community outreach team. these activities complemented traditional risk com-munication for the general public. to be effective, behavioral intervention approaches, messages, and materials had to be salient for the affected population, in this case asian-american communities within the united states. further, these interventions aimed at promoting an accurate understanding of the epidemic both in the general population and within the affected community, that is, the dynamic nature of the outbreak and its cause, treatment options, and prevention strategies. through interpersonal connections, the team members worked to promote reassurance and enhance community resiliency. public health professionals must understand the necessary balance needed to protect the public's health with appropriate exclusionary practices, while at the same time preventing fear, stigmatization, and discrimination of specific segments of the population. as we prepare for the next new or reemerging disease outbreak, we should also be preparing to deal with the fear epidemic that will likely accompany it. by developing effective behavioral and health education strategies and providing timely attention to the special needs of affected populations, we can ensure that, no matter what the infectious disease, we can limit the associated epidemic of fear and stigmatization. update: severe acute respiratory syndrome-worldwide and united states pneumonia causes panic in guangdong province managing sars amidst uncertainty fear is spreading faster than sars-and so it should. peter sandman column [serial online contagion, defect: issues in the anthropology of public health. stigma and global health: developing a research agenda sars: is global panic justified? bbc news uk edition death for sars spreaders: china east european jewish immigrants and the new york city epidemics of in search of equality: the chinese struggle against discrimination in nineteenth-century america hantavirus infection-southwestern united states: interim recommendations for risk reduction stigma interventions and research for international health. stigma and global health: developing a research agenda centers for disease control and prevention. use of quarantine to prevent transmission of severe acute respiratory syndrome-taiwan agency for toxic substances and disease registry. a primer on health risk communication principles and practices. the agency on stigma and its public health implications. stigma and global health: developing a research agenda report: sars article content analysis, comparison between chinese newspapers and mainstream newspapers in los angeles subjective experiences of stigma. a focus group study of schizophrenic patients, their relatives and mental health professionals thinking about aids and stigma: a psychologist's perspective leprosy: urgent need to end stigma and isolation tuberculosis beliefs among recent vietnamese refugees in new york state stigma in the lives of adolescents with epilepsy: a review of the literature critical delays in hiv testing and care: the potential role of stigma when tuberculosis treatment fails. a social behavioral account of patient adherence stigma: notes on the management of spoiled identity we thank the following cdc staff members who volunteered their time to translate critical information into multiple languages during the sars outbreak: feng chai, rachanee cheingsong, feng xiang gao, wenlin huang, han li, wenkai li, xiaofang li, timothy lim, gang liu, yuko mizuno, christine huong montgomery, xuanthao ngo, doan quang, yang xia, and yingtao zhou.ms. person is a senior behavioral scientist in the national center for infectious diseases, centers for disease control and prevention. her research interests focus on health and behavior, with a specific interest in cross-cultural behavioral interventions for the prevention and control of infectious disease. key: cord- -tg jkss authors: wang, haibin; mao, yuanli; ju, liancai; zhang, jing; liu, zhiguo; zhou, xianzhi; li, qinghong; wang, yuedong; kim, sunghee; zhang, lurong title: detection and monitoring of sars coronavirus in the plasma and peripheral blood lymphocytes of patients with severe acute respiratory syndrome date: - - journal: clin chem doi: . /clinchem. . sha: doc_id: cord_uid: tg jkss nan severe acute respiratory syndrome (sars) is a newly emerged infectious disease, and a novel sars-associated coronavirus (cov) has been identified as a causative agent ( ) ( ) ( ) . reliable and sensitive determination of the sars cov load would aid in the early identification of infected individuals, provide guidance for treatment (especially the use of steroid hormones and antiviral agents), and aid in monitoring of a patient's clinical course and outcome. among the available tests, viral gene amplification by reverse transcription-pcr (rt-pcr) provides a relatively rapid and specific test for the diagnosis of individuals showing sars-associated symptoms ( , ) . rt-pcr was successfully used to detect sars cov in nasopharyngeal aspirates, nasopharyngeal swabs, throat swabs, and broncheoalveolar lavage of sars patients ( , ) . however, because the composition of these samples varies with time and among individuals, they are unlikely to serve as a standardized sample source for quantification, comparison, or monitoring of sars cov infection. to meet clinical needs, some improved rt-pcr methods have been developed, and plasma has been used as a sample source ( - ) . the consistency of plasma composition makes it a good sample source for monitoring the cov load. cov-enriched samples are critical for achieving a high detection rate. current methods have a limited detection window, largely because they do not fully utilize the cov viral rna in the sample. because of its high false-clinical chemistry , no. , negative rate, the current method is unable to definitively rule out sars within days after onset ( ) . an increase in viral rna input could enhance the chance of detecting cov ( ) . the input to pcr can be increased by reducing the loss of cov during sample preparation ( , ) or by increasing the amount of prepared cov rna and cdna product used in the final pcr amplification. a high input of cov sample may increase the detection rate. guided by this concept, we made the following modifications to a previously reported real-time quantitative rt-pcr for cov ( ): (a) instead of using a rna capture column, we used trizol to extract total rna to increase the yield of cov from samples; (b) instead of using - % of the total obtained cov rna for the reverse transcription step, we dried the whole trizol-extracted cov rna sample, dis- solved it in the reverse transcription reaction mixture, and used it for the reverse transcription step; (c) instead of taking % of the reverse transcription product for pcr amplification, we used % of the reverse transcription product. in addition, to ensure efficient nested annealing of pcr primers, we performed reverse transcription with the cov sequence-specific primer that extends nucleotides beyond the pcr primer sequence. by making these modifications, we were able to achieve a detection rate of % as determined by testing of samples from sars patients admitted to our hospital and diagnosed according to the who definition for sars during the outbreak from march to june . among them, were male and were female, ages - years, with a mean age of years. the patients were aware of and willing to participate in this test. for a more detailed description of the patients and methods, please see the data supplement that accompanies the online version of this technical brief at http://www.clinchem.org/content/vol /issue /. in addition, there were no false positives detected by our method, as assessed with samples from patients infected with other viruses. furthermore, when plasma from a sars patient was tested four times, it yielded a mean of cov load of copies/ . ml of plasma with a an interassay cv of %. our main goal in establishing this method was to monitor cov load during the clinical course. we first examined the cov load in sars patients at different sars stages, classified into three groups: group , days - after fever onset (n ϭ patients); group , days - after fever onset (n ϭ patients); and group , days - after fever onset (n ϭ patients). the data (fig. a) indicated that the mean cov copy number in patients during days - after fever onset (group ) was / . ml of plasma and that of ( %) patients had a cov load substantially Ͼ copies, which could be easily detected by our modified method. in patients beyond days after onset (groups and ), the mean cov copy number decreased dramatically, to ϳ in . ml of plasma. these data imply the following. (a) the peak shedding of cov corresponds to the peak course of sars, when the virus has the highest transmission potential, consistent with the epidemiologic data. (b) the residual sars cov may persist in a patient's circulation for a relatively long time without obvious effects on the host. the pathophysiologic significance of a detectable residual cov load lasting up to - months is not clear, and attention should be paid to this phenomenon. (c) in the development of sars tests, it is important to take into account the timing of sample collection in the evaluation of the sensitivity and detection rate. (d) finally, if a procedure cannot detect abundant cov in the first-week sample, then it is unlikely to be effective for early diagnosis, monitoring of the therapeutic effect, or detecting subclinical sars cov infection. our improved method can detect the presence of a few copies of cov and has a high detection rate for first-week samples. notably, the number of cov copies detected in acute-phase patients (days - after fever onset) by our method is much higher than the values reported elsewhere ( ) . this may attributable to differences in the severity of patients' infections and to differences in sample input at three steps: cov mrna extraction, cov cdna production, and pcr amplification. a small difference in sample input at each step could lead to a large difference in the final cov copy number because of amplification of enzyme reactions. we next tested whether our improved method could be used to monitor changes in cov load in individual sars patients. the method could detect the cov load during the sars course, as demonstrated in fig. b , representative data from the patients tested. whereas patient a had a sharp decrease in cov titer, patient b maintained a high cov titer, reflecting individual differences in sars course. such dynamic changes in cov concentrations in a patient's circulation provide guidance for therapeutic interventions, especially the adjustment of doses of prednisolone and ribavirin. another way to increase cov sample input to enhance cov detection is to use a sample that is originally enriched in cov. because the sars cov is an rna virus, it targets cells and uses the cellular machinery to replicate itself. in addition to the epithelial cells lining the respiratory tract, which are targets of cov, the lymphocytes are also highly likely to be targeted by cov because lymphopenia occurs in almost all sars patients ( ) ( ) ( ) . we therefore separated lymphocytes from ml of blood with ficoll, extracted the total rna from ϫ of the lymphocytes, and used the recovered rna in our modified quantitative method for cov. although lymphocytes and plasma from healthy individuals and patients with influenza or other viral infections (e.g., mumps and rubella) had no detectable cov, a high concentration of cov was found in ϫ lymphocytes from patients tested - days after fever onset and patients who were recovering from sars, which was one to four orders of magnitude higher than the concentration measured in . ml of plasma from the same patients (fig. , c and d) . this finding provides evidence that lymphocytes are a target or reservoir for sars cov and that they are a better sample source than plasma for detecting sars cov. in conclusion, we believe that the use of lymphocytes, which are highly enriched in cov, and of a standardized simple ficoll separation make the proposed method suitable for detecting and monitoring sars-cov. diagnosis of iron deficiency (id) or functional iron deficiency (fid) is particularly challenging in patients with acute or chronic inflammatory conditions because most biochemical markers for iron metabolism are affected by the acute-phase response (apr) ( ). the hemoglobin content of reticulocytes (chr) is an early and sensitive indicator of fid ( ) . recently, we presented a novel approach to provide insights into the diagnosis of fid in apr by use of the chr ( ). id and fid were defined as a chr Ͻ pg based on the distribution of chr and biochemical markers of iron status in healthy controls. when a chr Ͻ pg was used for identification of id and fid in anemic patients, the values of ferritin, soluble transferrin receptor (stfr), and the stfr-f index (stfr/ log ferritin) ( ) performed significantly better in patients without apr [based on a c-reactive protein (crp) cutoff of mg/l]. in id combined with inflammation, the cutoff value for the stfr-f index was . , and in simple id, it was . . a diagnostic plot was developed that combined chr and stfr-f index, which allowed identification of four major categories of id: (a) iron repletion, normal erythropoiesis; (b) patients with reduced iron supply, but not yet in an iron-deficient erythropoietic state; (c) depletion of storage and functional iron with decreased hemoglobinization of erythrocytes, classic id; and (d) fid in an iron-replete state, with decreased hemoglobinization of erythrocytes. the plot provided a useful approach to the diagnosis of iron-deficient states. to date, the measurement of reticulocyte hemoglobin has been restricted to the analyzers of a single manufacturer. now a second manufacturer has produced what would appear to be a comparable index, the so-called ret-y ( , ) generated by the sysmex xe- analyzer. the ret-y is the mean value of the forward-scatteredlight histogram within the reticulocyte population. a corresponding value, the rbc-y, is the mean value of the forward-scattered-light histogram within the mature erythrocyte population. preliminary studies (see below) have demonstrated a good correlation between rbc-y and mean cell hemoglobin (mch), better, in fact, than with mean cell volume (mcv). a mathematical transformation applied to rbc-y can therefore produce a hemoglobin equivalent for erythrocytes (rbc-h e ) expressed in picograms. applying the same transformation to the ret-y gives a reticulocyte hemoglobin equivalent expressed in picograms. an appropriate name for this index would be reticulocyte hemoglobin equivalent (ret-h e ). the objectives of this study were twofold: (a) to establish the diagnostic equivalence of the ret-y (ret-h e ) with the chr; and (b) to describe its clinical assessment in the diagnostic plot combining the ret-y with the stfr-f index as a tool for the diagnosis and therapeutic monitoring of iron-restricted erythropoiesis. during a -month period, we studied adult anemic patients ( men and women) with hemoglobin Ͻ g/l for men and Ͻ g/l for women. specimens were collected for complete blood cell count, chr, ret-y, stfr, ferritin, and crp within h of admission. the patient diagnoses included cancer-related anemias, anemias of end-stage renal failure, anemias of inflammatory disorders, anemias of pregnancy, and anemias of heterogeneous origin. blood counts were performed with the advia (bayer diagnostics) and sysmex xe- (sysmex corporation) automated hematology analyzers. in the reticulocyte channel of the sysmex xe- , the sample, stained by a polymethine dye specific for rna/dna, is analyzed by flow cytometry by use of a semiconductor laser. a two-dimensional distribution of forward-scattered light and fluorescence is presented as a scattergram indicating mature erythrocytes and reticulocytes. ret-y is the mean value of the forward-scattered-light histogram of the imatinib compared with interferon and low-dose ara-c for newly-diagnosed chronic phase chronic myeloid leukemia imatinib induces durable hematologic and cytogenetic responses in patients with accelerated phase chronic myeloid leukemia: results of a phase study imatinib induces hematologic and cytogenetic responses in patients with chronic myelogenous leukemia in myeloid blast crisis: results of a phase ii study hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia resistance in the land of molecular cancer therapeutics clinical resistance to sti- cancer therapy caused by bcr-abl gene mutation or amplification multiple bcr-abl kinase domain mutations confer polyclonal resistance to the tyrosine kinase inhibitor imatinib (sti ) in chronic phase and blast crisis chronic myeloid leukemia ph(ϩ) acute lymphoblastic leukemia resistant to the tyrosine kinase inhibitor sti has a unique bcr-abl gene mutation high frequency of point mutations clustered within the adenosine triphosphatebinding region of bcr/abl in patients with chronic myeloid leukemia or ph-positive acute lymphoblastic leukemia who develop imatinib (sti ) resistance bcr-abl gene mutations in relation to clinical resistance of philadelphia-chromosome-positive leukaemia to sti : a prospective study molecular and chromosomal mechanisms of resistance to imatinib (sti ) therapy several types of mutations of the abl gene can be found in chronic myeloid leukemia patients resistant to sti , and they can pre-exist to the onset of treatment roots of clinical resistance to sti- cancer therapy denaturing high-performance liquid chromatography: a review characterizing mutations in samples with low-level mosaicism by collection and analysis of dhplc fractionated heteroduplexes temperature modulation of dhplc analysis for detection of coexisting constitutional and mosaic sequence variants in tsc a mutation conferring resistance to imatinib at the time of diagnosis of chronic myelogenous leukemia presence of the bcr-abl mutation glu lys prior to sti (imatinib) treatment in patients with phϩ acute lymphoblastic leukemia mechanisms of autoinhibition and sti- / imatinib resistance revealed by mutagenesis of. bcr-abl clinical resistance to the kinase inhibitor sti in chronic myeloid leukemia by mutation of tyr- in the abl kinase domain p-loop roots of clinical resistance to sti- cancer therapy sensitive and quantitative detection of mutations associated with clinical resistance to sti- an endonuclease/ ligase based mutation scanning method especially suited for analysis of neoplastic tissue a novel coronavirus associated with severe acute respiratory syndrome characterization of a novel coronavirus associated with severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus world health organization multicentre collaborative network for severe acute respiratory syndrome diagnosis. a multicentre collaboration to investigate the cause of severe acute respiratory syndrome a cluster of cases of severe acute respiratory syndrome in hong kong serial analysis of the plasma concentration of sars coronavirus rna in pediatric patients with severe acute respiratory syndrome quantitative analysis and prognostic implication of sars coronavirus rna in the plasma and serum of patients with severe acute respiratory syndrome detection of sars coronavirus in plasma by real-time rt-pcr the big question now: will it be back? early diagnosis of sars coronavirus infection by real time rt-pcr lymphopenia in sars haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis sars-coronavirus replicates in mononuclear cells of peripheral blood (pbmcs) from sars patients key: cord- -n epxwn authors: nan title: ecr – final programme: scientific and educational exhibits date: journal: nan doi: . /s - - - sha: doc_id: cord_uid: n epxwn nan contrast enhanced ultrasonography (ceus) of pancreatic masses m. d'onofrio, s. vasori, u. rozzanigo, s. caffarri, n. faccioli, c. procacci; verona/it purpose: to evaluate the role of ceus in the study of solid and cystic masses of the pancreas. materials and methods: masses of the pancreas, found on conventional us, were studied with ceus, by using sonovue (bracco) on a sequoia (acuson). a dynamic observation from the early contrastographic phase (during arterial enhancement), to the late contrastographic phase (after venous enhancement) was possible. all the lesions were cytologically/histologically proved. the results were compared with those obtained at spiral ct and/or mri. seven distinct enhancement patterns were observed: ) rapid intense enhancement in the early contrastographic phases with microbubble entrapment in the late phase [ neuroendocrine tumors, metastases from hypervascular tumors, poorly differentiated carcinomas]; ) moderate enhancement in the early contrastographic phase, resulting hypoechoic in the late phase [ poorly differentiated carcinomas, acinar cell carcinoma, neuroendocrine tumors]; ) capsular enhancement in the earliest phases [ solid and papillary epithelial neoplasm; neuroendocrine tumor]; ) no enhancement [ ductal adenocarcinomas and neuroendocrine tumor]; ) progressive enhancement [ solid and papillary epithelial neoplasm]; ) "parenchymographic" enhancement [ inflammatory masses]; ) parietal nodules and septas enhancement [ cystic tumors] . results: ceus correctly characterized / ( %) solid pancreatic tumors. in particular all ( / ) the ductal adenocarcinomas were correctly diagnosed. small nodules and septas of pancreatic cystic tumors, not visible at conventional us, were detected at ceus. conclusions: ceus can demostrate macrocirculation and microcirculation of solid pancreatic masses and can improve the characterization of cystic pancreatic lesions. is dual-phase spiral ct adequate for the confirmation of resectability and preoperative staging of pancreatic carcinoma? s. mylona, l. thanos, a. pagonas, v. kalioras, s. lyra, n. batakis; athens/gr purpose: to present the value of dual-phase spiral ct for the confirmation of resectability and pre-operative staging of pancreatic carcinoma. materials and methods: in a period of years patients with pancreatic carcinoma underwent dual-phase spiral ct for preoperative staging. ct scans after iv administration of contrast material with an electronic injector were obtained in the arterial (scan delay = sec) and in the portal venous phase (scan delay = sec) under a protocol with a total volume of ml, flow rate ml/sec. we have correlated the ct findings with surgical-pathologo-anatomic findings. results: dual-phase spiral ct was positive for pancreatic carcinoma in patients ( . %). in patients it was false-positive ( . %). in ( . %) out of patients it showed hepatic metastases, in ( . %) out of patients it showed lymph node enlargement, in ( %) out of patients vascular invasion, and in ( . %) out of patients peritoneal metastases. the accuracy of the method was up to %. conclusion: dual-phase spiral ct is a safe, noninvasive, and accurate method to investigate pancreatic carcinoma and should be considered as the standard preoperative method for assessing lesion resectability. assessment of resectability of pancreatic head cancer with ct imaging g.g. karmazanovsky, v.a. kubyshkin, a.v. kotchatkov; moscow/ru purpose: to investigate the value of preoperative ct in assessment of resectability of pancreatic head cancer. methods and materials: eighty-nine patients ( males and females; mean age ) with pancreatic head adenocarcinoma were investigated with spiral ct with bolus intravenous contrast enhancement. all of the patients were operated on. whipple procedures were performed on patients and palliative operations were performed on patients. results: tumors of patients were considered unresectable. patients (group ) had tumors that were encircling superior mesenteric or portal veins ( pts), superior mesenteric ( pts) or hepatic arteries ( pts) completely. no fat layer was identifiable between the tumors and the vessels. patients (group ) had ct appearances of tumors that partially (less than / of the circumference and in less than mm) circumscribed the vessels. tumors of all group patients were not resectable with a negative margin. among group , four patients ( . %) had unresectable tumors; six patients ( . %) had tumors that were difficult to dissect from the vessels; patients ( . %) had no evidence of vascular involvement and in fact went through curative surgery. conclusion: the ct findings predicted correctly surgical findings in of unresectable tumors ( . %). when the tumor partially circumscribed the vessels ct was not to be relied upon in predicting whether or not the tumor was fixed against the vessels. contrast-specific ultrasound (cs-us) imaging of the spleen: a pictorial essay of traumatic and nontraumatic disorders a. nunziata , o. catalano , m. mattace raso , i. matarazzo , a. siani ; naples/it, pozzuoli/it learning objective: to illustrate the appearance of several splenic lesions as shown during real-time, contrast-specific, harmonic imaging. background: contrast-enhanced, low-mechanical index us is a new technique allowing continuous, real-time assessment of splenic abnormalities. in our institution we use the technology named contrast tuned imaging (esaote, italy) and we employ a second-generation, sulfur-exafluoride based, microbubble contrast agent (sonovue-bracco, italy). the contrast agent, at a volume of . or . ml, in injected through a peripheral vein and a g needle, using a three-way stopcock and a -ml normal saline flushing. imaging findings: a wide spectrum of splenic disorders is depicted: injury, spontaneous hematoma, abscess, capillary and cavernous hemangioma, lymphomatous infiltration, metastasis, splenomegaly, and accessory spleen. several potential pitfalls are also shown. conclusion: contrast-specific us is being used with increasing frequency in the evaluation of the spleen. this is a superficial organ, with an almost homogeneous echotexture and with a dense and persistent contrast-enhancement; these characteristics make the spleen an optimal organ to be studied with contrast-specific us. radiologists should be aware of the typical and atypical appearance of splenic disorders as shown at contrast-specific us imaging. the spleen: normal variants and pathologies (imaging review) s. vessal, a. anbarasu, s.b. rai, w. shatwell, r. ramachandra; birmingham/uk learning objectives: to familiarise with the normal anatomy of the spleen. to further demonstrate common variations and splenic pathologies encountered in clinical practice. background: the spleen is the largest single mass of lymphoid tissue seen in the body. we familiarise with its normal image appearance using ct, uss and mri. the pattern of contrast enhancement of the spleen during the arterial and venous phases in ct can mimic disease by forming pseudomasses. normal variants such as splenunculus, splenic clefts/lobulations, liver wrap-around, wandering spleen as well as congenital heterotaxic syndromes will be discussed. a spectrum of splenic pathologies are demonstrated and the list of differential diagnoses that should be considered when seeing splenomegaly, cystic lesions, solid lesions and splenic calcification. examples of splenic trauma -laceration, subcapsular and intraparencymal haematoma will be discussed and complications such as splenosis demonstrated. image findings: images of the spleen on plain radiography, ct, mri, uss and radionuclide imaging from our institution are demonstrated along with imaging protocols. conclusion: there are several normal variants of the spleen which should be readily recognised. congenital abnormalities of the spleen such as polysplenia are rare, this should not be misdiagnosed as splenosis. it is useful to assess the presence and extent of splenomegaly. further classification of parenchymal lesions into cystic or solid can be made. solid lesions can be further subclassified into those with calcification. this poster demonstrates some splenic pathologies, a clear list of differential diagnosis is essential in interpreting any splenic lesion. non-traumatic acute splenic lesions: role of ct f. garibaldi, y. hetmaniak, b. gallix, j.-m. bruel; montpellier/fr learning objectives: -to illustrate a pictorial review of various acute splenic pathologies of non traumatic origin. -to learn characteristic imaging features of spontaneous spleen rupture or splenic infarction. -understand the pivotal role of abdominal ct when acute splenic pathology is suspected. background: splenic pathology is rarely suspected in patients with acute pain of the left upper abdominal quadrant. ct imaging is a well established tool for splenic injury of traumatic origin but ct features of non traumatic acute splenic pathology are less known. the purpose of this exhibit is to review the main causes of non traumatic acute splenic lesions and to describe their specific ct patterns. b d e f a g procedures: material consisted of patients with acute non traumatic splenic pathology who underwent multiphase contrast enhanced ct examination of the abdomen. the medical report of these patients were reviewed. cases were categorized according to the presence of the main ct patterns we found: spontaneous rupture, ischemic lesion, abscess or pseudo cyst or complicated tumoral involvement. conclusion: helical ct, in association with patients history and biology results is accurate to characterize the severity and the causes of acute non traumatic splenic injury. the new ultrasound who-classification for diagnosis, staging and follow-up of patients with cystic echinococcosis (ce) w.p. hosch, g.w. kauffmann, t. junghanss; heidelberg/de learning objects: the new who-classification provides, for the first time, a standardized sonomorphological staging of echinococcal cysts. we demonstrate the clinical value of this classification with examples from our cohort of patients. specific signs supporting the diagnosis of ce, and criteria for assessing cyst viability are presented. this allows staging of cysts relevant for treatment decisions (surgery, percutaneous drainage (pair), albendazole or "watch&wait") and treatment monitoring. background: ultrasonography plays a central role for diagnosis and follow-up of abdominal ce due to problems regarding sensitivity and specificity of clinical symptoms and serological tests. this technique allows the staging of cysts with respect to the involution process (spontaneous and treatment induced). the treatment options of ce have broadened in recent years with percutaneous drainage (pair), medical treatment and "watch&wait" as preferable strategies over surgery for specific cyst stages. in a standardized classification has been published by who allowing inter-observer comparison of treatment decisions and follow-up. procedure details: patients with a follow-up of up to years have been seen in our referral centre for ce since . apart from patients with pulmonary lesions and patient with a spinal manifestation, all other patients had abdominal cysts ( hepatic, spleen, kidney, peritoneal). we evaluated the value of the who-classification for diagnosis, therapeutic decision making and follow-up. conclusion: on the basis of the who-classification ultrasonography is not only an excellent tool for the primary diagnosis of ce but also very valuable for clinical decision making and monitoring treatment response. the spectrum of manifestations of metastatic malignant melanoma c.s. ng, v. kundra, m.j. jacobson, j. szklaruk, d.g. bedi; houston, tx/us learning objectives: . to gain an appreciation of, and be alert in clinical practice to, the wide range of sites, both common and uncommon, of metastases from malignant melanoma, including the lymphatic, central nervous, and musculoskeletal systems, thorax, abdomen and pelvis. . to become familiar with the radiologic appearances of metastatic melanoma on ct, mr, and ultrasound. background: malignant melanoma is a tumor that is increasing in incidence throughout the world, with a widely varying prognosis and distinctly capricious behavior. if, and when, the tumor metastasizes it can spread in an expected manner; however, frequently both the timing and sites of dissemination can be entirely unpredictable. imaging findings: this exhibit will present the wide range of manifestations of metastatic melanoma throughout the body, including the lymphatic, central nervous, and musculoskeletal systems, chest, abdomen and pelvis, and soft tissues, as identified by the major cross-sectional modalities of ct, mr, and ultrasound. the hypervascular and hemorrhagic tendency of the tumor, and its typical mr signal characteristics, will be illustrated. the exhibit will demonstrate the typical sites of metastases, namely lymphatics, brain, lungs, and liver; but will also illustrate the wide spectrum of more unusual sites of dissemination, including meninges, pleura, pancreas, gallbladder, gastrointestinal tract, genitourinary tract, peritoneal cavity, skeletal muscle and bone. the illustrative examples will be correlated with the tnm staging system. conclusions: metastatic malignant melanoma has an extremely wide spectrum of manifestations. practicing radiologists should be alert to these manifestations. malignancies in the aids affected population other neoplasms such as cervical cancer, hodgkin's disease and others have been observed with an increased incidence, probably because of the longer survival of aids patients. the aim of our exhibit is to describe ct and mr features of aids related neoplasms. procedure details: we reviewed ctand mr findings of aids patients examined between - , amongst which demonstrated pathologically confirmed neoplasms. ct's were performed by using spiral (plus, plus , siemens, erlangen, germany), and multislice ct equipment (lightspeed ge medical systems, milwaukee, usa); mr examinations were performed by using a . unit (vision; siemens medical systems, erlangen, germany) . we demonstrated nhls, hodgkin's disease, ks, cervical cancers, leukaemias, testicular, larynx, lung, breast, esophagus, stomach, liver, kidney, and adrenal carcinomas, intrahepatic multinodular cholangiocarcinoma and stromal tumors of the gastrointestinal tract. / nhl showed extranodal involvement to the liver ( / ), brain ( / ), lung ( / ), pleura ( primary effusion lymphomas with lymphomatous cells in the pleural effusion and no identifiable tumor mass) and gastrointestinal tract ( / ). ks involved the gastrointestinal tract ( ), lung ( ), liver ( ), larynx ( ), muscle ( ) , adrenal gland ( ), spleen ( ), pancreas ( ) . conclusion: ct and mr proved useful in the evaluation of the variegate pathologic findings of aids related neoplasms. abdominal imaging of post transplant lymphoproliferative disorders (ptld) k. burney, i. lyburn, r. hopkins; cheltenham/uk learning objectives: this exhibit will review the pathogenesis, staging and abdominal imaging findings of post-transplant lymphoproliferative disorders (ptld) seen in cardiac, lung, renal and liver transplant organ recipients. background: post-transplant lymphoproliferative disorders (ptld) are a complication of immunosuppression in solid organ transplant recipients. we reviewed the imaging of patients with a histologically proven diagnosis of ptld treated at our sub-regional oncology unit over the past years. conclusion: ptld is estimated to occur in between - % of patients. clinical manifestations are variable ranging from focal disease with graft dysfunction to systemic illness. ptld may be unsuspected or more extensive than expected and imaging plays an important role in diagnosis, staging and follow-up. ct (and to a lesser extent us) is the imaging modality of choice. does clinical examination of polytrauma guide properly ct evaluation? a. kalai, y.k. maratos, t. loeb, o. clément, g. frija; paris/fr learning objectives: to know the new definition of polytrauma patient based on the risk factor. to know that a whole body scanner is indicated in polytrauma patient. to know the ct protocol in polytrauma. background: the evaluation of the polytrauma patient is still most of time based on regional ct oriented with the clinical finding. based on our experience in patients this exhibit purposes a systematic evaluation with whole body scanner. major findings and pitfalls are illustrated. procedure details: whole body scanner includes: axial head ct, cervical spine helical ct with sagittal and coronal recontructions, helical ct with contrast iv with an arterial phase on the thorax and portal phase an the abdomen and pelvis, sagittal reconstruction on the dorsolombar spine. the severity of the lesions after the clinical evaluation were compared to the iss (index severity score) after the whole-body scan. % of the patients had at least one missed lesion when compared to clinical assessment .the average iss of these patients was higher and there was a surmortality. conclusion: whole-body scanning is highly recommended for polytrauma victims in order to enable prompt and early treatment of lesions which might have been missed at the initial clinical evaluation. radiological exploration of the embalmed cadaver of eva peron c. gotta, a. buzzi; buenos aires/ar learning objectives: to exhibit for the first time a medical document of eva perón's embalmed cadaver. background: eva perón was juan domingo peron's wife (he was president in argentina between argentina between and argentina between , and between argentina between and . for her political and social action she is known worldwide. she died of a cervix cancer in . when she died, she weighed less than kg, so many people doubt that her embalmed cadaver (small and consumed) belonged to her. the cadaver was embalmed by dr pedro ara, professor of anatomy at the school of medicine of the university of cordoba. he was born in zaragoza, spain, and he was established in argentina in the decade of . professor ara carried out excellent work with eva peron's cadaver. contrary to the classical technique, eva peron's embalmed body conserves its viscera. imaging findings: when the régime was overthrown in eva peron's cadaver was deposited in the general confederation of work ("confederacion general del trabajo", cgt). the new authorities doubted of the authenticity of the cadaver. radiological exams were made by dr guido gotta (father of one of the authors), who confirmed that the body corresponded to a human being, and that there was evidence of neoplasia (abdominal nodules, bone metastasis). conclusions: besides its medical use, thanks to its capacity to examine the interior of bodies, since their discovery x-rays were used for other purposes. in this case, they were used to determine the authenticity of eva peron's embalmed cadaver. imaging of complications of drug abuse s. van background: the material is culled from our databank of the emergency department of our institution. the age of the patients in our series ranged between and -years-old. imaging findings: complications of drug abuse in the drug addict will be classified according to their pathogenic mechanism or by their way of administration. pathologically, complications are usually due to their vasoconstrictive and thrombogenic effect on different organs, including the brain (stroke), heart (myocardial ischemia), the gastro-intestinal system (ischemic colitis), the nose (nasal septum perforation due to cocaine snorting). a direct toxic effect of drug metabolites is another pathogenic mechanism resulting in tissue necrosis, as typically seen in liver cell necrosis due to ecstacy. the route of administration (intravenously, orally, inhalation or smoking) may also reflect the radiological presentation. local abscess formation, septic thrombophlebitis, septic pulmonary emboli or bloodborn infections result from intravenous access, whereas inhalation and smoking may result in pneumothorax, pneumomediastinum and epidural pneumatosis. indirect complications, such as an increased risk for traffic accidents can be attributed to the additive effect of a cocktail of drugs and/or alcohol. complications related to smuggling are due to accidental rupture of packed drugs in the gastrointestinal lumen. the radiologist should be aware of the possible causal relationship with drug abuse, especially when those complications are encountered in a young population. abdominal background: there is a broad spectrum of neurogenic tumors that involve the abdomen. in this exhibit, we will describe the variable radiologic findings of abdominal neurogenic tumors. this study included patients with abdominal neurogenic tumors which were confirmed histologically by surgical resection (n = ) or us guided biopsy (n = ). two radiologists reviewed radiologic findings retrospectively and correlated with pathologic results. imaging findings: we categorized them into groups by cell of origin; nerve sheath origin (schwannoma in , neurofibroma in , plexiform neurofibromatosis in , malignant peripheral nerve sheath tumor in ), paraganglionic cell origin (paraganglioma in , pheochromocytoma in ), and ganglion cell origin (ganglioneuroma in , ganglioneurofibroma in , ganglioneuroblastoma in , neuroblastoma in ). conclusion: although abdominal neurogenic tumors show a broad spectrum, some neurogenic tumors revealed characteristic radiologic findings and correlated well with pathologic findings. familiarity with various neurogenic tumors in the abdomen is helpful in the approach of the diagnosis. purpose: to illustrate the broad spectrum of imaging findings (ct, us, urography and barium contrast studies) of patients with proven abdominal tuberculosis. we review between january and july , the typical and atypical imaging findings of patients that had a final diagnosis of genitourinary ( ), adrenal ( ), bowel ( ), peritoneal ( ), and hepatosplenic ( ) tuberculosis. results: most patients had been at increased risk because of drug abuse, alcoholism, aids and steroid therapy. radiological findings included low density lymph nodes, splenomegaly, hepatomegaly, intrasplenic and intrahepatic masses, ascities, diffusely thickened and enhanced peritoneum, mottled low density masses in the omentum, pleural effusion, thickening of the bowel, adrenal enlargement, atrophy and calcification, fallopian tube fibrosis, uterine atrophy and adhesions, ureteral and bladder fibrosis, and different patterns of renal hydronephrosis and calcification. conclusion: patients with aids had more severe form of involvement than those who did not have aids. sonography was the only imaging modality to demostrate septations within the tuberculous ascites, but showed the fewest morphologic details in renal tuberculosis. ct is the imaging modality that best determined the extent of intrabdominal tuberculosis. recognition of these manifestations should help optimize the correct diagnosis and management of tuberculosis in order to obtain a favorable outcome, biopsy is mandatory in almost all cases. predictive to develop a predictive model for the presence of esophageal varices (ev) in patients with hepatic cirrhosis (hc). this model would limit the endoscopic screening to the group of patients with high risk of bleeding from ev. methods and materials: consecutive patients with hc were included in this prospective study ( men, women; mean age . ). physical and laboratory examinations, upper gastrointestinal endoscopy and doppler ultrasonography were recorded. patients were divided in two groups: ) no ev or small ev, ) medium or large ev. the distribution of clinical, biochemical, and doppler ultrasonographic variables were compared in each group. the u statistic of mann-whitney was performed for the quantitative variables, whereas the chi-square or the fisher's exact tests were used for the qualitative ones. variables were also included in a multivariate stepwise logistic regression model. a roc-analysis was performed. results: in the stepwise logistic regression, presence of medium or large ev was independently predicted by splenomegaly, decreased platelet count, hcv infection and a decrease of the maximum portal flow velocity (mpv). the discriminating ability of the prediction rule was high, with an area under the roc curve of . ( % ci: . - . ). conclusion: patients with hc should be screened by upper gastrointestinal endoscopy only if splenomegaly, low platelet count, infection by hcv and decreased mpv are present, since these parameters confidently predict the existence of medium or large ev. premier experience of multiphasic contrast material-enhanced dynamic study on . tesla magnetic resonance (mr) imaging t.-j. hsieh, g.-c. liu; kaohsiung/tw purpose: to evaluate the accuracy and sensitivity of a dynamic contrast material-enhanced three-dimensional ( d) volumetric breath-hold hepatic magnetic resonance (mr) imaging examination on a . tesla machine. methods and materials: . tesla mr imaging with serial multiphasic contrast material-enhanced dynamic study was performed in patients with hepatocellular carcinoma (hcc). all patients had pathologic comfirmation by fine needle biopsy and followed angiographies were performed in . months. each test result was interpreted independently by two radiologists. separate reading sessions were performed for images from the multi-phases. sensitivity and positive predictive values were calculated for each reading session. results: one hundred twenty-nine early-enhancing lesions were detected in the patients on the . tesla mr images. the followed angiographies showed hypervascular tumor stains. all the lesions showed in the mr images were identified in the angiographies. the breath-hold dynamic examination on . telsa showed a overall sensitivity of . %. conclusion: multiphasic contrast material-enhanced dynamic study on . tesla magnetic resonance (mr) imaging . tesla mr imaging is insensitive for the diagnosis of hccs. role the insulinomas are rare functional pancreatic neuroendocrine tumours. the most common clinical presentations include hypoglycaemia (< . mmol/l), confusion, and loss of consciousness. diagnosis is difficult, and performed by computed tomography, magnetic resonance or selective angiography of the pancreatic arteries by digital subtraction angiography (dsa). the aim of the study was to analyse head-to-head the diagnostic value of pancreatic dsa, computed tomography and magnetic resonance in patients ( female, male; ± years) with insulinoma. the diagnosis was established by pathohistology after the surgical treatment in all patients. the tumours were localised in the pancreatic body in / patients ( %), in the pancreatic tail in / patients ( %) and in / patients ( %) purpose: to estimate variants in the anatomic structure of the gallbladder, the biliary and pancreatic ducts and to develop a technique of research. material and methods: patients underwent complex mri using a . t unit. t w haste, t w turboflash, including fs, and mrcp (single-slice turbose and multi-slice haste in different orientations) were acquired. patients also examined with ercp. results: in % of patients the merger of the hepatic lobar bile ducts was extrahepatic. % revealed an additional right hepatic duct, more than half running into the common bile duct (cbd) low. the low fusion of the cystic duct and cbd was revealed in % of the patients, and its left arrangement in %. anomalies in the form of the gallbladder are revealed in % and additional pancreatic duct in % of patients. in % patients the main pancreatic duct and cbd had a common ampoule, in more than % of cases they enter the duodenum parallel, in % cases separately. it is expedient to study the mutual relationships of ducts, parenchyma and vessels with application of t -w haste. general submission about bile and pancreatic ducts and gallbladder allows receiving single-slice tur-bose. for better analysis of details of anatomic structure it is necessary to study of source images multi-slice haste in different orientations. the results of research especially are useful for planning of operative treatment. conclusions: complex mri, especially reception of t -w images and realisation of mrcp, is an excellent tool for revealing variants in anatomic structure of the gallbladder and ductal system. the clinical utility of diffusion imaging out of the central nervous system y. murakami , k. imoto , n. aito , t. sakamoto , m. yamasaki , a. furukawa , r. ito , k. murata ; kohka/jp, shiga/jp the clinical utility of diffusion imaging out of the central nervous system is not yet clear. the purpose of this prospective study is to determine the role and the clinical usefulness of diffusion-weighted mr imaging combined with conventional mr imaging for the detection of visceral disease without of the central nervous system. material and methods: one hundred and thirty-four cases who were referred to our hospital for the examination of visceral disease without the central nervous system (including cases with neoplasms and cases with inflammatory disease, upper abdomen; , pelvis; , other organ; ) were investigated. mr imaging ( . t) was performed using a diffusion-weighted single-shot spin-echo echoplanar sequence, t -weighted fast spin-echo sequence, and t -weighted spin-echo sequence. an experienced reviewer evaluated the diffusion-weighted mr images for the presence of a high-signal-intensity lesion and compared with other mr sequences. imaging findings were correlated with findings from histological specimen in patients and with findings from clinical follow-up examination in other patients. results: diffusion-weighted mr imaging depicted high signal intensity lesions, including cases with malignancy and five cases with inflammation, and a case of castleman's disease. in three patients with lymphomas, high signal intensity lesions reflected the activity of the disease process. there was no high signal intensity lesion in cases with eight adnexal diseases. conclusion: diffusion-weighted mr imaging out of the central nervous system may possibly be useful for the extent or the activity of malignant or inflammatory disease. evaluation of computed tomography for assessment in lipodystrophy syndrome j.a.g.s. gallego, f.m.a.i. ardoy, s.p.u. padilla, f.g.r. gutierrez; elche/es purpose: lipodystrophy syndrome (peripheral fat loss and/or visceral fat accumulation) is a main problem in hiv-patients treated with antiretroviral therapy. the diagnosis is based on physical examination and/or standardized questionaires so there is a need for quantitative diagnostic tools. the aim of this study is to evaluate computed tomography (ct) to measure fat distribution in hiv patients. methods and materials: hiv-patients ( men) were included. lipodystrophy was diagnosed clinically on . subcutaneous limb adipose tissue area and their percentage were measured by a single slice at mid thigh and mid arm by ct. the subcutaneous (sat), visceral (vat), and total (tat) abdominal adipose tissue as b d e f a g well as the vat/tat, sat/tat and vat/sat ratios were calculated by a single slice at umbilical level. results: vat/tat and vat/sat ratios were significantly greater in lipodystrophic than in non-lipodystrophic patients: . vs . , p < . and . vs . , p < . respectively in men. . vs . p < . and . vs . p < . in female patients. sat/tat and mid thigh fat tissue percentage were significantly lower in lipodystrophic patients: . vs . p < . and . vs . p < . respectively in men. . vs . p < . and . vs . p < . in female patients. no significant differences were found at mid arm. a vat/sat ratio > . for men and > . for women had a sensitivity of % and % respectively and a specificity of %, to diagnose lipodystrophy syndrome. conclusion: computed tomography may be a useful technique to quantify lipodystrophy in hiv-infected patients. withdrawn by authors ultrasonographic assessment of body fat distribution in type ii diabetes mellitus of insulin resistant type and its relation to anthropometric measurements n. previous studies have shown that in subjects with type ii diabetes mellitus of insulin resistant type there is a relation between cardiovascular diseases and obesity, insulin resistance and body fat distribution. in these patients body fat distribution assessed with dexa and antrophometric measurements were correlated with ultrasonographic fat assessments. materials and methods: subjects ( male and female with mean age of . ± . ) in whom type ii diabetes mellitus of insulin resistant type was diagnosed with homa (homeostasis model assessment) were enrolled in the study. their weights, heights, abdominal diameters, and gluteal diameters were recorded. body mass index (bmi, kg/m²) and the ratio of abdominal diameter to gluteal diameter (a/g) were calculated. subcutaneous (fatsc), preperitoneal (fatpp), and visceral (fatv) fat thicknesses were ultrasonographically assessed. results: there was a significant correlation between bmi and fatsc or fatv (p < . ); and between total fat ratio and fatsc and fatv (p < . and p < . , respectively). there was no relation between a/g and fatsc, fatpp and fatv. conclusion: ultrasonographically assessed subcutaneous and visceral fat thicknesses in type ii diabetes mellitus patients of insulin resistant type may be used as prognostic factors of cardiovascular diseases. withdrawn by authors c- mri of the upper abdomen using a new free-breathing navigator triggered t -weighted tse-sequence (pace-tse) c. klessen, p. asbach, r. kirsch, b. hamm, m. taupitz; berlin/de purpose: to evaluate image quality of a new free-breathing navigator triggered t -tse sequence (pace = prospective acquisition correction) for magnetic resonance imaging of the upper abdomen in comparison to a established standard breath-hold t -weighted sequence. materials and methods: prospective evaluation of consecutive patients with various pathologies referred to our clinic for mri of the upper abdomen. all patients were examined at . t (siemens magnetom quantum) using the following sequences: pace-tse (work-in-progress sequence, x matrix, fov mm, axial slice orientation, tr ms, te ms, turbo factor , slices, mm slice thickness, n = excitations), breath-hold haste sequence ( x matrix, axial slice orientation, tr ∞, te ms, slices, mm slice thickness, n = excitation). three independent abdominal mri experienced radiologists reviewed all images and evaluated motion artifacts, liver-spleen contrast, depiction of intrahepatic vessels, depiction of the pancreas and the adrenal glands and overall image quality on a -point scale. in addition, to directly evaluate the influence of the respiratory trigger technique, in patients the t -tse sequence was repeated with the respiratory trigger function switched off. results: mean examination time of the pace-tse was . minutes. the depiction of anatomical details and contrast was significantly better (p < . ) using the pace-tse sequence compared to the standard haste sequence. the pace-t -tse sequence has the potential to significantly im-prove recognition of anatomical details and contrast compared to standard breathhold haste imaging. reactive lymph node hyperplasia: a diagnostic problem in the staging of hodgkin's disease (hd) l. ilic-todoric, b. lukac; belgrade/yu purpose: therapeutic approach to hd requires determination of the disease extent to supra/infradiaphragmatic lymph nodes (ln) . a problem appears when ln are of borderline size or only slightly enlarged. should such a finding on ct be considered as reactive hyperplasia or a pathological finding? the purpose of this report is to solve this dilemma. methods and materials: infradiaphragmatic spiral ct scan was performed in patients with histologically confirmed supradiaphragmatic hd. bipedal lymphography represented the gold standard for final staging. taking reactive hyperplasia into consideration, we estimated and statistically analysed ct's ability to differentiate benign and malignant conditions, and its value for staging. results: lymphography showed infradiaphragmatic disease in % of patients, % of whom had enlarged ln, while in % ln were of normal size or smaller. of the total number of patients, reactive hyperplasia and lymphographically normal findings were found in % and % of patients, respectively. ct detected enlargement in % of patients, and in % of these this finding concurred with the lymphographic one. ct could neither detect affected normal/reduced ln, nor differentiate reactive from malignant hyperplasia. postlymphographic restaging of the disease showed that % of patients would be understaged and % overstaged on the basis of ct finding only. conclusion: reactive hyperplasia represents a risk of up/downstaging if staging relies on ct only. errors can be partially assigned to the inadequate interpretation of the ct findings, which cannot be taken for granted when ln are of borderline sizes. in such cases additional diagnostic procedures are necessary. purpose: to compare the diagnostic accuracy of mr (magnetic resonance) images with msct (multislice ct) for preoperative t-staging in patients suffering from cardiac cancer. materials and methods: mr and msct were performed in cases of cardiac cancer diagnosed by biopsy prior to operation. mr and msct scans were performed on two different days. mr sequences included fset w, fset w, fse t w with fat suppression and dynamic enhanced fspgr with fat suppression; msct was applied with dynamic triphasic contrast enhancement. all these findings were prospectively analyzed by two doctors separately and correlated with the surgery pathological findings. statistic work was performed with spss. results: according to histopathologic staging, the accuracy for t -staging detected with mr and msct were % and % respectively, % and % for t staging, % and % for t -staging, % and % for t -staging. compared with msct, dynamic enhanced and delayed mr with fat suppression was superior for revealing the involvement of esophagus and aorta, early stage of invasiveness and providing more evidence in t from t or t from t staging (p < . ). among these mr plain scan sequences, t w with fat suppression was outstanding in depicting the gross features of the tumor, presence of ulceration, and adjacent lymph node swelling. diagnostic accuracy determined by roc analysis was marginally higher with mr scan than with msct scan in t-staging of cardiac cancer. conclusion: mr is superior in t-staging of cardiac cancer and can be used to optimize the therapeutic strategy, thus avoiding unnecessary operation. multidetector ct and virtual endoscopy of the esophagus s. mazzeo, d. caramella, e. neri, p. giusti, c. cappelli, r. bertini, a. belcari, c. bartolozzi; pisa/it purpose: to assess the diagnostic capabilities of multidetector ct in various esophageal pathological conditions. methods and materials: thirty-three patients underwent a multidetector ct study after esophageal distension by means of effervescent powder administered after induction of hypotonia. all acquired images were post-processed with d and d software tools. the ct data was compared with the results of conventional radi-ology ( ), endoscopy ( ), or surgery ( ) . follow-up ranged between and months. results: esophageal distension in the upper and middle third was classified as "good" in / cases ( %); in the lower third, esophageal distension was "good" in / cases ( %). final diagnoses were: leiomyoma ( ), squamocellular carcinoma ( ), adenocarcinoma ( ), esophageal infiltration by thyroid cancer ( ), benign polyposis ( ), chronic esophagitis ( ), post-sclerotherapy stenosis ( ) , no abnormalities ( ). when good distension was achieved, the thickness of unaffected esophageal wall was < mm (range between . and . mm, mean . mm). pathological wall thickening was observed in / cases ( %) with values ranging between . and mm (mean . mm). spiral ct demonstrated true positive cases and true negative cases. there were false negative cases and false positive case. sensitivity was %, specificity %, diagnostic accuracy %, positive predictive value %, negative predictive value %. conclusions: evaluation of the esophagus with multidetector ct proved to be a promising and easy to use technique, allowing panoramic exploration, virtual endoluminal visualization, accurate longitudinal and axial evaluation, and simultaneous evaluation of t and n parameters. (ibd) . to describe the technique of barium tagged dark-lumen mrc. to outline the advantages, disadvantages and limits of the method. background: conventional colonoscopy (cc) is the most widely used method for the assessment of the extension and the activity of ibd. however, cc is an invasive technique and requires long-lasting bowel preparation. furthermore, not infrequently the examination of the entire colon and terminal ileum wouldn't be possible due to severe pain or fecal residue. these disadvantages are eliminated with barium tagged dark-lumen mrc which makes this examination more tolerable and acceptable by patients. imaging findings: ten patients who present with symptoms of ibd underwent cc first and subsequently mrc. for fecal tagging, barium sulphate-containing contrast agent is given with meals, beginning hours before the examination. for mrc the colon was filled with ml of diluted barium. after intravenous administration of . mmol/kg gd-dtpa, coronal t -weighted three-dimensional gradient-echo sequence is performed. additional fat-saturated axial two dimensional gradient-echo and coronal true-fisp sequences are obtained. colon segments are interpreted seperately (rectum, descending, transverse, ascending colon, caecum) when assesing the extent of the disease. bowel wall thickening and congestion of marginal vascularity are considered as signs of the disease. out of sixty segments, of them had colonoscopic correlation. of these in ( %) the extent of disease correlated with cc. non-correlation of findings of mrc and cc was detected in ( %) patients. conclusion: barium tagged dark lumen mrc can be used for the assesment of the extent of ibd. evaluation of ct gastric virtual endoscope (ve) findings: comparison with real gastric fiberscope (gf) lesions k. inamoto, k. kouzai, k. hamada; osaka/jp learning objectives: to check ve findings compared with gf lesions in cases. to learn about images of ve and to consider possibilities to use ve clinically. background: gf is an invasive examination for patients. if ve can be used for checking pathologies of gastric diseases it will be beneficial to detect malignant changes of the stomach in their early stage. procedure details: md-ct images were obtained on the same day as gf with somaton sensation , siemens, erlangen, germany. thin slice ct images with mm thickness were reconstructed to ve images by surface rendering and volume rendering with advantage workstation . _ . with volume analysis voxtool . . e, ge, milwaukee, wi.usa. results indicated that ve images succeeded in showing subtle alteration of mucosal folds. gastric cancer, polyp, ulcer, erosion and gastritis were clearly visualized. by comparison studies between ve and gf, a sensitivity . %, specificity . %, positive predictive value . %, negative predictive value . % and accuracy . % were calculated. conclusion: d md-ct examination of the stomach is noninvasive except for radiation exposure. it will be expected as a new technique of the stomach examinations. gastrointestinal stromal tumor: current concepts and imaging characteristics j.h. lee, k. kim, c. park, w. jeong, j. lee, e. kang, i. cha, h. seol; seoul/kr learning objects: to provide an in-depth review of gastrointestinal stromal tumors (gist's) with their epidemiology, histopathology, clinical manifestations and the most up-to-date therapy. to describe radiologic features of gist's. to discuss differential diagnosis of gist's throughout the abdomen. background: gastrointestinal stromal tumors (gist's) are unique neoplasms that occur throughout the gastrointestinal tract, mesentery, omentum, and retroperitoneum. they are the most common mesenchymal neoplasm of the gastroin-testinal tract and are defined by their expression of kit (cd ), a tyrosine kinase growth factor receptor. the expression of kit is important to distinguish gist's from other mesenchymal neoplasms such as leiomyomas, leiomyosarcomas, schwannomas, and neurofibromas and to determine the appropriateness of kit-inhibitor therapy. imaging features of gist's vary depending on tumor size and organ of origin. since most gist's arise within the muscle layer of the gastrointestinal tract, they most commonly have an exophytic growth pattern and manifest as dominant masses outside the organ of origin. procedure details: the purpose of this exhibit is to provide an in-depth review of gist's with their epidemiology, histopathology, clinical menifestation and differential diagnosis. this exhibit will also reflect our experience in the radiologic evaluation of patients with gist's, with emphasis on the typical findings present on small bowel series, ultrasonography and ct (including d images using multidetector ct). examples of unusual gist's and pathologic correlation as well as the most up-to-date therapy will be included. conclusion: after interacting with this exhibit the radiologist will have an enhanced understanding of gist's including histopathology and radiologic features. imaging of jejunal and ileal primary tumors with pathologic correlation b. rodríguez-vigil, m. lamas, t. berrocal, j. fernández, v. tarín, a. alvarez; madrid/es learning objectives: to illustrate the spectrum of usual and unusual primary neoplasms involving the jejunum and ileum. to evaluate the efficacy of barium studies, us, ct and mr imaging in the management of these tumors. to understand the radiologic findings on the basis of pathologic correlation. background: preoperative diagnosis of jejunal and ileal neoplasms can be a challenge for both clinicians and radiologists. symptoms are nonspecific and endoscopy is commonly unsatisfactory. since early diagnosis is crucial for prompt therapy, imaging plays an essential role. tumors develop in all histologic components of the small intestine including epithelial cells, lymphoid tissues, lymphatics, vessels, nerves, and muscle. imaging findings: we retrospectively reviewed the imaging findings of jejunal and ileal neoplasms from our digestive pathology database. findings were correlated with pathology in all cases. specific neoplasms addressed include benign (adenoma, leiomyoma, lipoma, familial polyposis, hemangioma, lymphangioma and fibroma); and malignant neoplasms (adenocarcinoma, carcinoid tumor, lymphoma, leiomyosarcoma, direct extension to the small bowel from extraintestinal tumors, and metastasic lesions). enteroclysis is the primary and most effective radiologic modality in the evaluation of these neoplasms. ct should be the complementary radiologic modality and used for staging. mri may help in specific cases. pitfalls, diagnostic difficulties and differential diagnoses are emphasised. conclusions: evaluation of patients with small bowel neoplasms frequently requires multiple imaging modalities for diagnosis and treatment planning. because of the characteristic imaging appearances of many of these tumors, this exhibit will help the radiologist in training to better understand and recognise jejunal and ileal neoplasms. imaging findings of sigmoid diverticulitis: a pictorial essay of anatomy, pathology, differential diagnosis and radiologic treatment options m.c. freund, j. bodner, e.m. gassner, m. rieger, a. gschwendtner, w.r. jaschke; innsbruck/at learning objectives: to understand the anatomy and pathology of sigmoid diverticula and of the mesosigmoid. to be familiar with the typical findings of different imaging modalities in sigmoid diverticulitis and its complications and differential diagnosis. to be familiar with percutaneous and endovascular radiologic treatment options including abcess drainage and embolization for diverticular bleeding. background: sigmoid diverticulosis and diverticulitis represents a commonly encountered disease in western countries. an exact diagnosis is a prerequisite for further therapy. imaging findings: different imaging modalities are proposed for detection of self-limiting or complicated sigmoid diverticulitis e.g. abdominal radiography, contrast enema, ultrasound and even catheter angiography. during the last years ct has evolved to be a primary diagnostic imaging modality in patients with left lower abdominal pain. this pictorial essay will demonstrate the relevant anatomy of the sigmoid, mesosigmoid, and vascular supply as well as the typical pathology of the diverticula and its inflammatory complications including diverticulitis, peridiverticulitis, perforation, contained abcess in the mesosigmoid or rarely free intraperitoneal perforation with peritonitis, as well as fistula formation, obstruc- the learning objectives: to depict the ct imaging spectrum of the faeces sign, which is a consequence of small bowel occlusion, and to illustrate its causes. background: abdominal ct scan has been shown to be the most valuable technique, in confirming the diagnosis of small bowel occlusion and to depict the site, level and cause of occlusion. very few studies have focused on the contents of the bowel. particulate faeces-like material mixed with gas bubbles within the lumen of a dilated small bowel loop is always a pathological condition. it is the result of a delayed intestinal transit. to access prevalence, characteristics of this ct sign, and differential diagnosis, we have reviewed ct scans of small bowel occlusion over a period of two years. imaging findings: our analysis suggests that the small bowel faeces sign is probably more frequent than the previous reported studies, in demonstrating cases of small bowel faeces sign mainly observed in the ileal loops, and pointing to the site of occlusion. faeces sign appeared as tubular gas bubbles mixed with particulate matter with no capsule in dilated segments of small bowel loop, due to adhesions, closed loop, and tumours (adenocarcinoma, carcinoid). differential diagnosis of two phytobezoars is also illustrated and discussed. conclusion: small bowel faeces sign may not be confused with colonic segments leading to a failure to recognize the exact level of obstruction. moreover, it represents a sentinel sign in determining the site of occlusion and consequently the cause of the obstruction. imaging appearances and predictors of disease response in metastatic gastrointestinal stromal tumours (gist) treated with imatinib s. cox, b. lanka, m.b. taylor; manchester/uk learning objectives: to evaluate changes in radiological appearances in patients with metastatic gist treated with imatinib. to highlight any specific imaging features that can be used to predict disease relapse. background: imatinib (glivec, sti- ) is a kit tyrosine-kinase inhibitor drug, recently developed and found to be effective in the treatment of metastatic gist. we reviewed the pre-and post-treatment imaging findings on ct in ten patients. the duration of treatment varied from two to forty one months. the pre-treatment, eight and sixteen weeks scans were reviewed, along with the scans at the time of any relapse. the sites of metastases, size of lesions and cystic change were assessed. imaging findings: metastases were present in the liver and peritoneum in seven patients each; mesentery and retroperitoneum in four and two patients respectively; splenic, renal and subcutaneous in one patient each. one patient had progressive disease at eight weeks. nine patients responded to treatment. the maximum reduction in size of the metastasis was at eight weeks. most metastases underwent cystic change with treatment, particularly in the liver and peritoneum. two patients are known to have relapsed at fourteen and fifteen months. in both cases, cystic metastases developed more solid components prior to disease progression as indicated by an increase in the tumour volume. conclusion: ninety percent of the patients responded to treatment. cystic change in the liver and peritoneal metastases correlates with disease response to imatinib. an increase in solid components may be a useful predictor of disease relapse. ct appearances of neoplasms of the small bowel as a cause of obstruction s. sadiq, d. markham; swansea/uk learning objectives: this poster presents a pictorial review of the ct appearances of primary and secondary tumours of the small bowel in patients presenting with obstruction, including neoplasms that are the lead point of an intussusception. background: patients presenting with small bowel obstruction in two district general hospitals over the last twelve months and who were subsequently investigated by ct were selected. the imaging features of those with a neoplastic aetiology were reviewed. image findings: the imaging reflects recent studies examining the incidence of primary small bowel tumours. adenocarcinoma, non-hodgkins lymphoma, carcinoid and leiomyosarcoma comprise the majority. secondary tumour deposits also lead to small bowel obstruction. we demonstrate those that metastasize intraperitoneally (such as ovarian carcinoma), haematogenously (such as melanoma), or by local tumour extension (such as liposarcoma and bladder carcinoma). conclusion: it is well established that ct is a valuable diagnostic procedure in patients with acute small bowel obstruction. it is not only useful in distinguishing obstruction from ileus, but frequently establishes the cause of obstruction. small bowel neoplasms are rare but have a high mortality since tumour-related symptoms occur late and are non-specific. endoscopy is not feasible in most cases, as enteroscopy is limited to specialist centres. small bowel contrast studies do not reach the high level of accuracy obtained in the evaluation of the upper and lower gastrointestinal tract. ct not only allows tumour detection, but demonstrates possible complications and offers the possibility of pre-operative staging in the acute or elective situation. ct in adult intussusception: radiologic-pathologic correlation c. triantopoulou, p. maniatis, i. siafas, n. giannakou, c. avgerinos, c. dervenis, j. papailiou; athens/gr learning objectives: review the ct characteristics of bowel intussusception in adults through a wide variety of cases. emphasize the ability of ct imaging in narrowing the diagnostic possibilities and defining the cause of intussusception. stress the need for familiarization with the different aspects of this rare entity. background: intussusception is a relatively common cause of intestinal obstruction in children but is a rare clinical entity in adults. however, there is a lead point in % of adult cases. cases of adult intussusception were retrospectively reviewed. all ct examinations were conducted on a spiral unit with administration of both oral and intravenous contrast agent, using slice thickness of - mm anda pitch of . site, level, cause and degree of obstruction as well as signs of threatened bowel viability were evaluated and correlated with surgical findings and pathology examination. imaging findings: cases of ileocolic intussusception and of ileoileal were revealed. in patients findings included: cases of polypoid lesions, malignant neoplasms, lipoma, inflammatory diseases and in adhesions due to preceding surgery. in the other three cases no underlying pathology was proven so they were characterized as idiopathic conditions. all neoplastic lesions were correctly depicted before surgery while in the case of lipoma a specific diagnosis was made. ct is a powerful tool in adult intussusception, providing valuable information regarding the involved enteric segments, the underlying pathology and possible local complications, thus assisting in preoperative planning. diagnosis of perforation of stomach or duodenum on multislice computed tomography j.-i. nishimura, t. ohkoshi, y. yamamoto, t. inoue; yokohama/jp learning objectives: a review to determine the variety and relative conspicuity of findings in patients with perforation of the stomach or duodenum. background: multislice computed tomography (ct) gives us detailed information as thin-slice sagittal, coronal, and -dimensional images rapidly. a small volume of free air can be detected, and the perforation holes were predicted in some cases. during a -year period patients with perforation of the stomach or duodenum underwent preoperative ct scans. there were men and women averaging . ± . ( to ) years old. all patients underwent to surgical treatment, and all sites of perforation were confirmed. imaging findings: levels of perforation were the antrum of stomach (n = ), body of stomach (n = ), duodenal bulb (n = ), and descending limb of duodenum (n = ). ct images showed local extraluminal gas near the perforation sites in patients ( %), local ascites near the perforation sites in patients ( %), edematous wall of perforation sites in patient ( %), dirty increased density in the fat tissue in patients ( %), and a perforation hole in patients ( %). diffuse extraluminal gas and ascites were showed in , and patients respectively. in of patients ( %), we could predict the level of perforation. conclusion: multislice ct is a valuable method in the diagnosis of perforation in stomach or duodenum. the diagnosis can be established rapidly with high sensitivity. defecographic imaging of anorectal sphincter dyssynergia syndromes: our experience in subjects a. nunziata , a. salzano , p. de feo , a. de rosa , f. de rosa , m. covello ; naples/it, frattamaggiore/it, napoli/it learning objective: anorectal sphincter dysfunction can be responsible for evacuation difficulties and constipation; in addition, pelvic floor disorders can be associated. by using defecography associated with videoproctography, the evacuation time and the sphincters' opening time can be assessed, obtaining further information about the physiopathology of rectal empting and anal sphincter function. the aim of this study is to have functional radiological parameters of anal activity to detect anorectal sphincter dyssynergia syndrome. background: on a consecutive series of patients suffering from constipation we assessed by defecography combined with videoproctography cases of sphincter dyssynergia syndrome ( males and females with mean age of years, range of - years). all patients underwent clinical examination and endoscopy; patients were investigated by anorectal manometry. imaging findings: in all patients defecography with videoproctography provided a sphinter opening delay > s (normal values: - s) and increased evacuation time > s (normal values: - s). manometry revealed anal hypertonia in cases. conclusion: defecography associated with videoproctography is an accurate, simple and non-invasive method to approach anosphincteral pathology, permtting study of this particular type of terminal constipation, which is difficult to demonstrate using other techniques. background: gist are rare mesenchymal tumors of the abdomen, for which an effective, molecular-targeted therapy with imatinib mesylate has been recently developed. imaging findings: pts with advanced gist ( / with liver lesions and / with peritoneal involvement) undergoing therapy with imatinib mesylate. all pts had a baseline mr, and then at , , , months during treatment. pts responsive to therapy ( / ) showed: a) tumor decrease in size (with an in-crease in tumor volume at months from treatment start in some cases ( %) due to necrosis or bleeding): b) hypointensity of the lesions on t w images (lesion was hyperintense in rare case due to bleeding) and hyperintensity of the lesions on t w images (correlating with the amount of degenerative tissue or necrosis): c) hypovascularization of the lesions on contrast-enhanced t w images in comparison to pre-treatment assessment, due to the presence of degenerative tissue or necrosis: e) evidence of peritoneal fluid in all pts at the first months, reabsorbed in the following months. conclusions: mri is useful to identify tumor response to imatinib mesylate in advanced gist as from the early months of therapy with the following indicators of treatment activity: a) size of lesions; b) signal intensity; c) vascularization; d) amount of degenerative tissue or necrosis; e) presence of peritoneal fluid. crohn's disease: spectrum of findings on helical contrast enhanced ct enterography a.j. madureira, r. cunha, p. varzim, f. magro, i. ramos; porto/pt learning objectives: to illustrate the spectrum of imaging findings of smallbowel crohn's disease at contrast enhanced spiral ct after oral hyperhydratation with isotonic solution (ct enterography). background: spiral ct has become one of the most valuable methods in the assessment of parietal inflammatory changes and extraluminal involvement in small-bowel crohn's disease. the accuracy of this technique depends on the presence of well distended bowel loops. procedure details: after oral hyperhydration with ml of isotonic polyethylene glycol electrolyte-balanced solution, contrast enhanced ct of the abdomen and pelvis was performed with a collimation of mm and mm, respectively. the following ct findings were evaluated: wall thickening, parietal contrast enhancement, extraparietal anomalies (lymph nodes, vessels and mesenteric fat), and presence of inflammatory complications (abscesses, fistulas). the spectrum of findings of small-bowel crohn's disease is thoroughly illustrated and discussed based on our experience with over patients. conclusion: this method provides important information about the extent and activity of small-bowel crohn's disease and its extraluminal involvement and is a potential alternative to small-bowel ct enteroclysis. bowel intussusception in adults: an imaging review h.d. roach, j.m. hanson, r. bagree, c. evans, a. yong; cardiff/uk learning objectives:to outline the mechanism and causes of intussusception in the adult population. to provide a review of the imaging findings in intussusception using several imaging modalities. background: bowel intussusception is more commonly associated with the paediatric population, but can occur in adults, affecting virtually any part of the gastrointestinal tract. in adults there is more often an underlying structural lesion, which acts as a lead point. imaging findings: bowel intussusception has a classical appearance on barium studies and cross-sectional imaging and occasionally a lead point can be identified. due to the underlying structural causes, surgery is more often required in adults than children, resulting in the availability of histopathologic specimens for correlation. however within the small bowel even in adults it has been shown that the majority of cases are self-limiting. in this exhibit, we review the imaging findings of intussusceptions affecting various sites in the gastrointestinal tract using different modalities. conclusion: bowel intussusception is less common in the adult population, but is often associated with underlying structural lesions. the imaging findings are reviewed here. can double contrast barium enema exams incidentally detect terminal ileum tumoral lesions? c. gotta, s. merola, e. sanabria, m. landi, s. ballester, c.a. dominguez; buenos aires/ar learning objectives: to describe a number of radiological signs, found in barium enema double contrast exams in patients, that are specific or suspicious of terminal ileum tumoral lesions. background: since there have been few reports about radiological signs in double contrast barium enema exams that can help in the diagnosis of terminal ileum pathology. some of them use glucagon to improve the reflux of contrast material through the ileo-cecal valve in order to allow the visualization of the reviewing the available literature, there are no reports which describe specific radiological signs on this matter. procedure details: a total of cases of tumoral terminal ileum pathology were found. all lesions were seen for the first time at the double contrast barium enema, and lately examined and characterized by the follow through or small bowel enema. in most of them abdominal computed tomography was performed. cases had histological confirmation based on surgical specimens. of the remaining cases, were lipomas with typical densitometric values diagnosed by computed tomography, thus these patients did not undergo surgery. the other lesions described as polipoid did not have histological confirmation as the patients refused surgical resection. conclusion: double contrast barium enema can detect the presence of terminal ileum tumoral pathology even when not yet clinically suspected. there are a few radiological signs which allow the radiologist to recognize and diagnose it. background: magnetic resonance is a powerful diagnostic tool for abdominal imaging, particularly for extra-visceral tissue depiction. newer pulse sequences, such as single shot fast spin echo (ssfse) and steady state free precession (ssfp) with a water solution infusion via naso-jejunal tube can accurately depict the bowel lumen in a fashion similar to conventional double contrast enema. furthermore d and d fast t weighted sequences with iv contrast administration allows the evaluation of wall involvement. imaging findings: findings are summarized into lumen abnormalities, wall involvement and extra-luminal alterations. corresponding dcsbe are shown and compared. signs of disease activity at mre plus conventional mri are stressed. contribution of different pulse sequences is evaluated. conclusions: mre plus conventional mri is feasible in the diagnosis, staging and follow-up of crohn's disease. subtle mucosal lesions are still poorly depicted, compared to dcsbe. extra-luminal involvement is superbly showed, particularly by new ssfp sequences. disease activity signs seem to be the added value of magnetic resonance in crohn's disease assessment. diagnosis and staging of colorectal carcinomas using multislice ct colonography (msctc): pictorial essay with pathologic correlation r. iannaccone, a. laghi, c. catalano, a. lamazza, f. mangiapane, s. trenna, d. marin, a. schillaci, r. passariello; rome/it learning objectives: to assess the role of msctc in the detection and staging of colorectal carcinoma. background: patients affected by colorectal carcinoma underwent colonoscopy and msctc on the same day. patients free of colonic lesions were included as a control group. ct parameters were: collimation, x mm; effective mas, - ; and kvp, . msct colonography was performed before (prone position) and after (supine position) intravenous administration of contrast material with a -sec delay-time. msctc images were interpreted by two radiologists on a workstation. the results of msct colonography were correlated with colonoscopy and with the results of the histopathologic examination performed on the resected surgical specimen. intra-operative ultrasonography was used as the reference standard for the presence of liver metastases. sensitivity and specificity for tumor detection and staging accuracy (according to the tnm classification) of msctc were calculated. imaging findings: surgery revealed carcinomas ( primary and synchronous lesions) in patients. colonoscopy failed to visualize the entire colon in patients due to obstructive neoplasms and missed all synchronous carcinomas. msctc provided a complete evaluation of the colon in all cases, correctly identified all primary and synchronous carcinomas ( / ; sensitivity, %), and yielded three false-positive diagnoses (specificity, . %). msctc also detected polyps ( true-positives and three false-positives). overall, msctc correcly staged of primary tumors (overall tnm staging accuracy, . %). conclusion: msctc can be proposed as a comprehensive diagnostic test for the pre-operative evaluation of patients with known, or strongly suspected, colorectal carcinoma. gastrointestinal to present the characteristic features of malignant gist and correlated these with histopathologic findings. background: in our pathologic database from to , we found patients with gist and retrospectively reviewed their radiologic findings. thirty one ct, four us, and four barium studies were included. imaging findings: involved organs were as follows; in the small bowel ( %), in the stomach ( %), five in the rectum ( %), two in the mesentry ( %) and one in the colon ( %). on histochemical analysis, eight were classified as benign ( %), six as borderline ( %) , and as malignant ( %). mean tumor size was . cm ( . cm- cm): benign (mean; . cm, range; . cm- cm): borderline (mean; cm, range; cm- . cm): malignant (mean; . cm, range; cm- cm). multiplicity was demonstrated in one benign and one malignant tumour. six cases showed microscopic metastasis to the surrounding lymph nodes without evidence on radiologic imaging. ct features were relatively smooth margination and homogeneous enhancement of smaller tumors. poor margination with surrounding infiltration, central necrosis or ulceration was seen in larger tumors. no evidence of intestinal obstruction, peritoneal seeding or ascites was demonstated. barium examination showed a well demarcated, smooth marginated tumor with normal overlying mucosa. some ulceration was seen in larger masses (bull's-eye sign). us features were welldemarcated homogeneous hypoechogenicity of small masses, hyperechoic foci of ulcerated masses. the characteristic radiologic findings of immunohistochemically kitpositive malignant gist were large tumor size, poor margination, central necrosis or ulceration. the clinical significance of each feature and differential points from benign gist or other conditions will be discussed. learning objectives: to illustrate ct findings of common and uncommon duodenal diseases with pathologic correlation. to demonstrate a comprehensive ct anatomy of the duodenum and adjacent organs. to describe the pitfalls on ct interpretation of duodenal diseases. background: the duodenum may be involved by secondary process as well as primary pathology because it is close to adjacent organs including the stomach, pancreas, liver, gallbladder, hepatic flexure of the colon, aorta, ivc, right kidney and adrenal. duodenal pathology can be overlooked on ct interpretation but understanding of ct anatomy of the duodenum and adjacent organs may be helpful in the diagnosis of various duodenal pathology. procedure details: helical ct during the portal phase with water as an oral contrast agent was performed for better detail of duodenal lesions. we reviewed ct findings with pathologic correlation in patients with various duodenal diseases. duodenal diseases were classified as follows; congenital (duplication, malrotation, diverticulum, and annular pancreas), trauma (intramural hematoma, iatrogenic perforation by endoscopy), inflammatory (perforated peptic ulcer, secondary process by pancreatitis or cholecystitis), primary neoplasms (lipoma, adenoma, adenocarcinoma, lymphoma, and gastrointestinal stromal tumors), secondary invasion of adjacent neoplasms (pancreas, stomach, colon, and gallbladder), bezoar, and postsurgical afferent-loop syndrome. conclusion: helical ct during the portal phase with water as an oral contrast agent provided a comprehensive view of the duodenum and adjacent organs. understanding of ct anatomy and the pitfalls on ct interpretation was helpful in the diagnosis of various duodenal diseases. to investigate utility of mr volumetry for resectable rectal cancer before and after preoperative concurrent chemoradiotherapy. methods: patients with respectable locally advanced rectal cancer underwent preoperative concurrent chemoradiotherapy. among them, patients ( men and women; median age years; range, - years) who had mri before, and month after concurrent chemoradiotherapy were enrolled into this study. radiation of gy/ fractions was delivered to the pelvis, followed by a . gy/ fractions boost to the primary tumor. chemotherapy was administered concurrently with radiotherapy and consisted of cycles of -fu ( mg/m / day) and leucovorin ( mg/m /day) for five days during the first and fifth weeks of radiotherapy. surgery was performed - weeks after the completion of chemoradiation. mr volumetric examinations for tumor volume before and after chemoradiotherapy were perfomed in workstation software using t -weighted axial images. results: according to dworak grade, grade was present in . % ( / ), grade in . % ( / ), grade and in . % ( / ), respectively. primary tumor downstaging occurred in . % ( / ). the mean tumor volume was . cm ( . - . cm ) in mr volumetry before chemoradiotherapy and . cm ( - . cm ) in after chemoradiotherapy. when the tumor volume reduction rate was correlated with the dworak grade, mean tumor volume reduction rate was . % ( . - %) in grade , . % ( - . %) in grade and . % ( . - %) in grade and . the tumor volume reduction rates demonstrated statistically significant differences among dworak grades. conclusion: in mr volumetric evaluation for rectal cancer before and after preoperative chemoradiotherapy, tumor volume reduction rate is well correlated with histologic reduction grade. functional the rectum was insufflated with air. we used t -weighted haste sequences at rest, squeeze and strain, in the axial and sagittal planes. we also performed a dynamic study using a t -weighted haste modified sequence, acquiring a single mid-sagittal slice, repeated every second for seconds, starting with the patient at rest then in squeezing and finally in straining. mr findings were compared with evacuation proctography, considered as gold standard. rectocele and pelvic floor descensus were graded either with the standard reference values, and with modified values obtained from normal volunteers. results: mri vs evacuation proctography identified / rectoceles, / anorectal junction descents, / rectal prolapses, and / diskinetic pubo-rectalis syndromes. mri detected additional findings in % of patients ( enteroceles, uterine descents, cystoceles, perianal pathologies-abscesses, anal fistulas). mri, using specific reference values provided % sensitivity, % accuracy, % npv for the anorectal junction descent, while using conventional values provided % sensitivity, % accuracy and % npv. conclusions: defeco-mri requires a short examination time, is well accepted and gives reliable information for the diagnosis of posterior pelvic disorders. specific reference values should be used to correct the bias of the supine position. correlation between mr findings and pathology in evaluation of the affected bowel wall in crohn's disease f. maccioni, m. colaiacomo, a. bruni, a. cocco, s. parlanti, f. siliquini, m. marini, a. marcheggiano; rome/it purpose: to correlate mr findings with the corresponding pathologic specimens obtained at the level of the pathologic wall in patients with crohn's disease (cd). method and materials: patients with known cd underwent mri after oral administration of a superparamagnetic contrast, using t -weighted plain and fatsuppressed haste sequences, and gd-enhanced t -weighted flash sequences on axial planes. degree and pattern of wall signal, wall thickness and abnormalities of perivisceral fat were evaluated. disease activity was evaluated using standard clinical scores. endoscopic or surgical pathologic specimens were obtained in all patients (gs) . mr findings were inter-correlated and correlated with pathologic findings (gs) either prospectively or retrospectively, with a sideby-side analysis of results. results: high wall gd-enhancement and high t wall signal were observed in patients with active disease and diffuse transmural inflammatory cells infiltrates at gs (n. ). in patients with active disease a layered pattern of gd-enhancement was observed, associated with either submucosal oedema or fibrosis at gs (n. ); in this group of patients, however, t -weighted images showed high or low wall signal in presence of oedema or fibrosis, respectively. in patients with non active disease poor gd-enhancement and low t wall signal were observed and preoperative evaluation of early gastric cancer for laparoscopic gastrectomy by d-ct angiography using -row multislice ct angiography s. kumano , h. miki , t. mochizuki ; onsen-gun/jp, ehime/jp purpose: the purpose of this study was to determine the efficacy of d-ct angiography ( d-cta) using -row multislice ct (msct) in the pre-operative simulation of laparoscopic gastrectomy for early gastric cancer. method and materials: twenty patients with early gastric cancer underwent ct examination using -row msct ( . -mm x ). ml/kg of mgi/ml contrast material was intravenously injected at a rate of ml/sec. timing for the arterial phase scan was determined by using a test bolus injection. portal phase scan was performed s after the start of bolus injection. for d-cta, ct images of . mm thickness were reconstructed at . mm intervals. with a computer workstation, d-cta was produced using volume rendering and maximum intensity projection techniques. results: d-cta demonstrated the left gastric artery (lga), right gastric artery (rga) and left gastric vein (lgv) in all cases. d-cta also demonstrated the lga arising from the celiac trunk in cases and from common hepatic artery (cha) in one case; the left hepatic artery (lha) from the lga in two cases; an accessory lga from lha in one case; and the rga from the proper hepatic artery in cases, from the cha in five cases, and from the lha in one case. regarding the venous system, d-cta demonstrated lgv flowing into portal vein in cases, splenic vein in cases, and the junction of portal and splenic veins in one case. conclusion: -row msct d-cta clearly demonstrated the vascular anatomy in all cases that is necessary for laparoscopic gastrectomy, and was thought to be useful for the preoperative simulation of laparoscopic gastrectomy. douglas pouch hernias: evaluation with entero-colpo-defecography m. estienne , m. moretti , m. valle , p. meinero , o. brunetti ; lavagna/it, santa margherita ligure/it purpose: to assess the usefulness of entero-colpo-defecography (ecd) in the evaluation of douglas pouch hernias and to improve the technique by a simple and non-invasive method. methods and materials: female patients (mean age: years) with clinical symptoms of obstructed defecation underwent ecd. our standard technique includes: ml of diluted barium suspension given orally at least ½ hours before examination; a small gauze opacified with iodamide placed into the vagina; rectal filling with ml of thick barium paste. static and spot lateral radiographs of the pelvis are obtained at rest, during squeeze and push, and during and after evacuation. if widening of the rectovaginal space (rvs) is observed during the dynamic phase, in the absence of an enterocele, a barium sigmoid enema is performed and further lateral films are obtained at maximum straining. results: widening of rvs was observed during ecd dynamic phase in of the patients ( %). thirteen of these patients had small bowel loops prolapsing into rvs (enterocele). for the three remaining patients, in which the widening of rvs could not be explained by enterocele, we performed a sigma barium enema demonstrating the presence of a sigmoidocele. we did not find a peritoneocele without bowel contents. conclusions: douglas pouch herniations may account for symptoms of obstructed defecation. defecography alone is not able to evaluate the hernia contents and peritoneography is an invasive technique. visualization of douglas pouch hernias is improved by the use of our ecd technique allowing a correct identification of enteroceles and sigmoidoceles. were prospectively studied by mdctc and compared with colonoscopy. all patients were referred to ct immediately after colonoscopy. ct was performed with x . mm, kv and mas in both supine and prone positions after colonic air insufflation. this method was well tolerated for all patients. independently, two experienced radiologists interpreted axial, sagittal and coronal images and d endoluminal reconstructions (virtual colonoscopy). in cases of disagreement, the diagnosis was established by consensus. sensitivity, specificity, predictive values and kappa test were analysed. both methods were compared with chi-squared test. results: in our series, the prevalence of colorectal polyps was %. when polyps were mm in size or greater mdctc had a % sensitivity, % specificity, % pvp and % pvn. the agreement between colonoscopy and mdct was observed in %. no statistical differences were found between these methods. conclusion: ct colonography is emerging as a potential alternative to conventional colonoscopy in the screening of colorectal cancer. row mdctc represents a reliable imaging method to detection of colorectal polyps with a high accuracy, particularly when the polyp size is mm or greater. this suggests an improvement over single slice ct. a. kalogera-fountzila, c. kouskouras, a. lefkopoulos, n. fotiadis, d. karanikolas, i. tsifountoudis, a.s. dimitriadis; thessaloniki/gr purpose: to prospectively evaluate the characteristic spiral ct features of intestinal ischemia and necrosis in patients with acute abdomen. materials and methods: twenty patients, aged - (median ), were examined in a three year period, using helical ct without oral contrast administration and after intravenous injection of contrast medium. the clinical manifestations consisted of acute abdomen and/ or sepsis in all patients. the diagnosis of bowel ischemia was confirmed with surgery, where colonic and/ or small bowel resection was performed in patients within hours. one patient with the presence of portal gas was found to be normal at surgery and one patient died before surgery and had a confirmatory autopsy. results: the causes of critical intestinal ischemia were diverse, including superior mesenteric artery thromboembolism in ( %) patients, inflammation in ( %), intussusception in ( %), volvulus in ( %), adhesions in ( %), hiv infection in ( %) and unknown etiology in ( %). spiral ct identified necrosis of colon and small bowel in ( %) patients, necrosis of small bowel only in ( %), and of colon in ( %). suggestive findings included intramural air in ( %) patients, thickening of the bowel wall in ( %), thinning in ( %), double or concentric ring sign in ( %) and bowel dilatation in ( %). presence of increased density of mesenteric fat was noted in ( %), mesenteric vein dilatation in ( %), free intraperitoneal air in ( %) and peritoneal fluid or blood in ( %). conclusion: spiral ct can accurately demonstrate signs of bowel ischemia and is often helpful in determining the primary cause of ischemia or necrosis. a comparison of single and multi row ct colonography in the detection of colorectal polyps p. vagli, e. neri, s. picchietti, g. naldini, m. rossi, c. bartolozzi; pisa/it purpose: to compare the diagnostic accuracy of single and multi row ct colonography (ctc) for detection of colorectal polyps. materials and methods: between and , seventy-two consecutive patients with a moderate to high risk of developing colorectal cancer underwent ctc followed by complete cc within hours. ctc was obtained by means of single row ct (n = ) and multi-row ct (n = ). sensitivity and specificity of single and multi-row ctc for detection of colorectal polyps was determined by means of lesion location in four colonic segments (cecum-ascending c-a, transverse t, descending d, sigmoid-rectum s-r). results: all colonic segments were completely visualised in each patient both by single and multi row ctc study. ctc identified polyps (< mm) in pa-tients (c-a n = , t n = , d n = , s-r n = ) , and polyps ( - mm) in patients (c-a n = , t n = , d n = , s-r n = ) . ctc generated false positives (fp) for polyps smaller than mm and fp for polyps - mm. false negatives (fn) were for polyps < mm, and for polyps - mm. sensitivity of single and multirow ctc for detecting polyps were % and % respectively for polyps < mm, and % and % for larger polyps - mm (p < . ). overall sensitivity and specificity of ctc for small polyps (< mm) was % and % respectively. overall sensitivity and specificity increases for larger polyps ( - mm) at % and % respectively. conclusion: multi-row ctc demonstrated a higher diagnostic accuracy than single-row ct in detection of colorectal polyps. paring the two ct techniques, considering patient discomfort and respiratory artifacts. results were compared with double-contrast small bowel enema and evaluated by two experienced radiologists. the integrated diagnosis showed negative patients, with signs suggestive for crohn's disease, with reflux ileitis, with ischemic ileitis and with non-specific ileitis. ct detected positive loops (sensitivity %, specificity %, diagnostic accuracy %); conventional radiology showed positive loops (sensitivity %, specificity %, diagnostic accuracy %). ct-enteroclysis false positives were due to poor bowel distention and false negatives to proximal bowel localization of disease; conventional study false negative cases were due to unseparable loops. mild respiratory artifacts were present in % ( of patients) of sdct examinations vs % ( of patients) of mdct examinations. conclusions: mdct-enteroclysis in comparison with sdct-enteroclysis reduces scanning time, respiratory artifacts and patient discomfort, allows a better spatial resolution, more evident on mpr, and improves pathological pattern representation. detection and staging of gastric lesions combining axial ct and virtual endoscopy images s. visconti, v. panebianco, m. celestre, t. celano, d. tancredi, f. iafrate, r. passariello; rome/it purposes: the aim of our study was to develop a virtual endoscopy examination protocol for the stomach after co distention, and to identify agc lesions according to borrmann's classification based on tumor morphology. methods and materials: virtual endoscopy of the stomach was performed in patients with gastric lesions. the stomach was distended with g of top effervescent granules used for double contrast upper gi studies (duogas, bracco), after intramuscular injection of mg of scopolamine butylbromide. all patients underwent a spiral ct volume zoom examination ( / / mm/ mm/ mm/ .l sec kv/mas/slice-coll./slice-width/feed-rot/rot.time). in cases spiral ct was repeated in a prone position because of a limited gastric distension by air. real time endoscopy images were reconstructed using a volume rendering technique with a dedicated workstation and specific software (vitrea . -vital images), and evaluated combined with axial images by two radiologists. one week later a fiberoptic gastroscopy was performed in all patients. results: image quality was graded as optimal in cases. in cases the stomach was not distended. virtual endoscopy correctly showed gastric lesions (ulcerative lesions n = , vegetating lesions n = , benign lesions n = ). we found a good correlation with surgical specimens in cases. the actual role of virtual gastroscopy combining axial ct and ve images is to evaluate the locoregional and distant extension (perigastric tumor involvement, distant metastasis) of tumors. it's potential roles are replacing barium studies and examining patients with surgical gastric exclusion. traumatic bowel perforation after blunt abdominal trauma: analysis of the ct findings according to the perforation site and the elapsed time since the trauma h. kim, h. shin, s. park, s. park, h. kim, w. bae, i. kim; cheonan/kr purpose: to evaluate the efficacy of ct in predicting the perforation site and the differential findings according to the elapsed time from the trauma in patients with bowel perforation after blunt abdominal trauma. methods and materials: abdominal ct's of patients with bowel perforation after blunt abdominal trauma confirmed by surgery were retrospectively analyzed without knowledge of the surgical results. the interval from the trauma to ct was to hours (mean, hours). the ct findings of traumatic bowel perforation including extraluminal air collection, segmental bowel wall thickening, localized inter-mesenteric fluid, bowel wall discontinuity, and mesenteric hematoma were analyzed. the predicted perforation sites were classified as: duodenum, jejunum, jejuno-ileal junction, ileum, and colon. based on the mean elapsed time ( hours), the patients were divided into the early lapse group (n = ) and the late lapse group (n = ), and the differential ct findings were analyzed. results: diagnostic accuracy of predicting the perforation site was: duodenum ( %), jejunum ( %), jejuno-ileal junction ( %), ileum ( %), and colon ( %). extraluminal air collection was more frequently seen in the late lapse group ( %) than in the early lapse group ( %) (p < . ), whereas other ct findings showed no statistically significant difference in the incidence. contrast-enhanced magnetic resonance imaging of the terminal ileum in children with crohn's disease p. paolantonio, a. laghi, o. borrelli, c. miglio, m. celestre, d. marin, s. cucchiara, r. passariello; rome/it introduction: to evaluate the diagnostic value of gadolinium enhanced mri with polyethylene glycol solution as oral contrast agent (ce-peg-mri) in revealing inflammation of the distal ileum in children with crohn's disease (cd) and in differentiating them from other inflammatory bowel diseases. subjects and methods: seventy-five consecutive paediatric patients referred for suspected cd underwent ileo-colonoscopy with biopsy and ce-peg-mri. the distal ileal mucosa was assessed by endoscopic and histological scores. the cd activity was measured by means of paediatric crohn's disease activity index (pcdai). results: active cd was diagnosed in cases, active ulcerative colitis (uc) in , spondyloarthropathy and indeterminate ileo-colitis in . in all cd patients ce-peg-mri showed a high concordance with endoscopy and histology. of the uc patients the ce-pegmri was negative in and showed mild parietal contrast enhancement of the terminal ileum in of the patients with backwash ileitis. among the group of spa patients, ce-peg-mri was negative in and revealed mild parietal contrast enhancement in . no increase in wall thickness in any of the uc and spa patients was demonstrated. sensitivity and specificity of ce-peg-mri were % and %, respectively. the test optimally correlated with endoscopic and histological scores (r: . ; r: . , respectively) as well as with the pcdai in the cd patient group (r: . ). conclusions: high correlation of ce-peg-mri with ileal endoscopy, histology and pcdai makes this test of great interest as a tool for monitoring the clinical course and the effect of therapy in cd patients. small bowel sonography in celiac disease after oral administration of sonographic contrast medium p. mirk, r. foschi, i. de vitis, l. guidi, a. vecchioli-scaldazza, p. marano; rome/it purpose: to assess the usefulness of sonography in celiac disease after small bowel loop filling by sonographic contrast medium. materials and methods: consecutive patients with clinical suspicion or diagnosis of celiac disease were prospectively examined by us before and after ( '- ') drinking of - ml of an isosmotic water solution of polyethilene glycol. patients had celiac disease (positive histology or aga or ema antibodies); patients had previous celiac disease but were clinically negative, or had other benign disorders, and were considered as normal. results: after contrast administration intestinal loops were better or slightly better shown in / and / patients ( . % and . %), or unchanged in / ( . %). among celiac patients, observed intestinal abnormalities were: segmental dyskinesia; transient intussusception; reduced number of jejunal folds; increased number of ileal folds; hypotonic dilated fluid-filled loops; increased intestinal motility. extra-intestinal abnormalities in celiac patients were: spleen size reduction; mesenteric lymphadenopathy; liver steatosis; free peritoneal fluid. celiac patients had no detactable abnormalities (false negatives); there were no false positives. overall accuracy was . % (sensitivity: . %; specificity: %; ppv %; npv . %) conclusions: small bowel filling by sonographic contrast medium improves us evaluation of wall thickness, mucosal fold pattern, and intestinal motility. transient intussusception was the single most diagnostic sign, whereas other features were less specific and of limited value unless found in combination. preoperative mri and transrectal us in the staging, and post-irradiation therapy restaging, of rectal cancer i. miucin-vukadinovic; sremska kamenica/yu purpose: the goal of the study was to determinate and compare the value of mri and transrectal us (trus) in the staging, and post-irradiation therapy restaging, of rectal cancer. this study was designed to evaluate the down-staging effect of preoperative radiation. method and materials: patients (range, - ) with biopsy-proven rectal can-cer underwent imaging using a . t scanner and transrectal us ( . or mhz transducer) before, and after radiation therapy ( gy, a daily fracton of gy, days a week).tumors were staged according to the tnm staging system. two observers independently scored the tumor stage. the standard of reference was histopathology (after surgical resection). results: all patients underwent radiotherapy before surgery for rectal carcinoma. before radiotherapy mr examination detected . % t (b ), . % t (b ), . % t n (c ) and . % t / n (c ) tumors. down-staging of one or more t stages occurred in . % with tumor regression in . %. the mri tumor stage agreed with the histological stage in % of patients. trus correctly staged % of lesions. t category was correctly restaged after preoperative radiation in only . %. conclusion: preoperative radiotherapy is becoming the standard of care for resectable, locally advanced adenocarcinoma of the rectum. mri and trus enable selection of appropriate patients for preoperative radiotherapy. preoperative trus can establish the depth of penetration of a tumor and showed less sensitivity for lymph node staging. mri showed better diagnostic accuracy (with tendency for overstaging) then trus for interpretation of complete pelvic status in the pre-and post-irradiation period. echographic efficacy evaluation of gastric ulcer treatment s. pimanau, n. mikhailava; vitebsk/by purpose: the objective of this study was to investigate the possibilities of noninvasive abdominal echography in efficacy evaluation of chronic peptic ulcer pharmacotherapy. method and materials: patients with gastric ulcers in the exacerbation phase were examined before the course of pharmacotherapy and after it. ultrasound investigation was carried out after administration of - ml deaerated liquid. on echography a chronic gastric ulcer was visualized in cases. in the acute exacerbation phase the peptic ulcer was deep, filled with echogenic contents, the ulcer edge being sharp and overhanging. periulcerous infiltration was hypoechogenic, - mm long, - mm thick, and its area made up about - mm . the peripheral edge of periulcerous infiltration seemed sharp. in the course of effective treatment both ulcer dimensions and periulcerous infiltration area reduced on echograms. periulcerous infiltration reduction speed made up about mm /day. the reparative process was also accompanied by ulcer flattening. in cicatrization the ulcer was not visualized on echograms, a hypoechogenic stripe - mm thick generally remaining. in the absence of reparation, the echographic picture of the ulcer and periulcerous infiltration didn't change essentially. parallel endoscopic study of the stomach revealed either coincidence with echographic findings of qualitative characteristics of the reparative process positive dynamics or its absence. in cases the results of ultrasound and endoscopic investigations didn't coincide; echograms showed slight infiltration with a central small echogenic area at the site of ulcer regarded as ulcer reparation, while endoscopy revealed a red scar. conclusion: thus, abdominal ultrasound investigation of the stomach enables reliable evaluation of gastric ulcer dynamics in the course of conservative treatment. comparison of multi-slice ct-colonography in ultra-low-dose technique with high resolution video-colonoscopy in the detection of colorectal polyps m. cohnen, c. vogt, k. andersen, a. saleh, a. beck, s. vom dahl, v. aurich, d. häussinger, u. moedder; düsseldorf/de purpose: to prospectively compare msct-colonography using an ultra-low-dose technique (uld-msctc) with high-resolution video-colonoscopy (hr-vc) as the standard technique for detection of colorectal cancer and polyps. patients and methods: patients underwent msct-colonography (uld-msctc) after standardized oral bowel cleansing immediately before video-colonoscopy (hr-vc). patients were scanned with an ultra-low-dose msctcolonography protocol ( mas, ctdiw eff: . mgy). after noise reduction using an mathematical algorithm by dedicated software, uld-msct-colonographic images were analyzed by a team of two readers in a blinded fashion and the results were compared with the results of hr-vc. results: lesions were detected by hr-vc in patients. sensitivities for detection of polyps < mm, - mm, and > mm in size were % ( of polyps), % ( of polyps) and % ( of polyps) respectively. all colorectal tumors ( of , %) were prospectively diagnosed. the sensitivity to detect flat lesions was % ( of flat lesions). the overall specificity was calculated at %. the calculated effective dose ranged between . and . msv. conclusions: uld-msctc has an excellent sensitivity and specificity for the detection of colorectal lesions > mm despite a significant reduction in radiation exposure. defecographic the pelvic floor in straining state descended an average of . cm from the inferior margin of the ischial tuberosity, its broad range of position from - . cm to . cm implying a wide variation of anorectal angle and perineal descent. mild degree rectocele with less than cm of depth was found in out of cases. rectal intussusception was noted in six and rectal incontinence was seen in one. formation of rectocele and intussusception during defecation was common in asymptomatic young volunteers. the wide range of defecographic measurements warrants the necessity of other complementary studies on anorectal function to improve the diagnostic accuracy. therefore, the interpretation of defecographic findings should be made with caution and should not be used as the sole criteria for selection of a treatment modality. right-side colonic diverticulitis: sonographic and ct findings (differential diagnosis) e. blanc, m.j. martínez, t. ripollés, m. agramunt, c. soto, r. pastor; valencia/es purpose: to describe the sonographic and ct features in right-side colonic diverticulitis and to differentiate this entity from other right-side acute abdominal conditions. methods and materials: patients with a final diagnosis of diverticulitis of the ascending colon or cecum were retrospectively reviewed. sonography was performed in all patients and ct in . the following findings were evaluated: the presence of an inflamed right colonic diverticulum, pericolonic infiltration, focal colonic wall thickening and the identification of a normal appendix. the diagnosis was confirmed by surgery, clinical course, or barium enema. results: sonography and ct showed pericolonic inflammation and focal colonic wall thickening in all cases. the inflamed diverticulum was seen with sonography and ct in ( %) and ( %) patients respectively. the normal appendix was identified with sonography in ( %) and with ct in ( %) cases. the initial diagnosis was: right-side colonic diverticulitis (n = ), acute appendicitis (n = ), perforated colonic carcinoma (n = ) and an inflammatory mass (n = ). conclusions: right-sided colonic diverticulitis is a condition that can clinically mimic other causes of right abdominal pain like acute appendicitis, perforated colonic carcinoma, salpingitis, ileocecal inflammatory pathology, epiploic appendicitis and omental infartion. in this series the most common sonographic and ct findings included the presence of a right colonic diverticulum with thickening of the adjacent pericolonic wall and pericolonic infiltration. both ultrasound and ct can be extremely useful in the early diagnosis of this entity avoiding unnecessary surgery procedures, in a primarily benign and self-limiting condition that can be treated conservatively. an accurate scanning technique for detecting recurrent rectal cancer: enhancement of early-phase dynamic helical ct j. tanaka, s. tsukuda, a. heshiki; iruma-gun/jp purpose: to evaluate the usefulness of dynamic enhancement of helical ct in the detection of local recurrence of resected rectal cancer. in patients with a history of curatively resected t or t rectal cancer, follow-up plain ct indicated that they had a loco-regional recurrent tumor; consequently, they underwent follow-up pelvic helical ct providing accurate early-phase contrast enhancement in the lower pelvic region. the patients were divided into four groups according to grade of contrast enhancement of the suspected mass-like lesion before and after contrast enhancement, and correlation between groups and results obtained by biopsy and/or surgery was assessed. if no correlation was found, patients were followed up for a period lasting from days to two years. as of june , results had been obtained for patients; and correlation between these results and grouping by contrast enhancement ratio was analyzed. results: all patients in the highest-grade group (n = ) had local recurrence. when only the highest-grade group was considered positive, there were no false positives and only one false negative. conclusion: our results suggest that early-phase contrast enhancement is useful for accurately detecting recurrent rectal cancer. this can be easily achieved using smartprep function, and should be used to distinguish recurrent tumors from post-surgical scar tissue, because these two tissue types do not exhibit similar enhancement patterns. staging of gastric carcinoma by mr imaging in vitro c. sato , s. naganawa , h. kumada , t. miura , t. ishigaki ; nagoya/jp, toyohashi/jp purpose: to evaluate the accuracy of cancerous invasion to gastric wall with magnetic resonance imaging in vitro. twelve specimens of gastric carcinoma were examined with a . t mr using a small loop surface coil. they were fixed in formalin within days prior to imaging. the field of view was mm, a matrix size was x , and the section thickness was mm. the t -and t -weighted images were obtained. two radiologists evaluated the mr images independently, and consensus was obtained if there were any discrepancies between the two. findings on mr images were compared with histopathologic findings. results: mr images depicted the normal gastric wall as consisting of the layers clearly. cancerous invasion was detected in of specimens with mucosal invasion, with submucosal invasion, with the invasion to muscularis propria, with subserosal invasion, and the rest that extended into the serosa. the mr imaging-determined grade correlated with the histopathologic findings for of tumors. the overall accuracy was . %. conclusion: mr imaging has an accurate diagnostic capabilities for the evaluation of the cancerous invasion to the gastric wall in vitro. the results of the present study may encourage the further development of an endoscopic surface coil. role to investigate the value of mri in the overall assessment of crohn's disease (disease activity, disease extension, complications). method and materials: consecutive patients with crohn's disease underwent mri in the context of a clinical evaluation including biochemistry, endoscopy, histology and radiology. all patients underwent mri after oral administration of a superparamagnetic contrast agent, using standard sequences (haste t weighted, with and without fat-suppression and gd-enhanced t -weighted flash). two independent radiologists evaluated mr images on printed films. disease length, presence of strictures, fistulae, abscesses, phlegmons and any other abdominal complication were evaluated at the level of each segment. gold standard (gs) for morphological assessment were: barium studies, ct or us, and surgery when performed. at the level of the affected segments bowel wall thickness, t wall gd-enhancement, t wall signal, t fibro-fatty proliferation signal were evaluated and graded ( - ) to assess disease activity. gs for disease activity were endoscopy, biological activity and cdai. results: mri detected % of overall disease length, % of fistulae, % of strictures; adhesions were overestimated. in % of patients mri showed complications requiring surgery ( hydronephrosis, abscesses, phlegmons, pancreatic duct stone, enterovesical fistula, ovarian involvements). the following mri findings were statistically correlated with the clinical and biological signs of active disease: bowel wall thickness (r = . ), wall gd-enhancement (r = . ), t wall signal (r = . ), t fibro-fatty proliferation signal (r = . ). learning objectives: to recognize usual and unusual radiologic findings of pseudomyxoma peritonei with pathologic correlation. background: pseudomyxoma peritonei is a rare neoplastic condition in which gelatinous intraperitoneal fluid collections associated with mucinous disseminated implants are observed. although a ruptured appendiceal mucocele is the most common cause, pathologies arising from other origins may cause this condition with various imaging manifestations. imaging findings: the usual imaging findings were of mucinous, fluid-like material spread over the peritoneal cavity with organ scalloping observed in the liver, splenic and mesenteric margins, with central displacement of small intestinal loops. within the material, cyst-like round structures may be observed and clarified by the septal enhancement on contrast-enhanced images. punctate, annular, or curvilinear calcifications may be disseminated. dropping appendiceal mucocele or a rare urachal tumor located in the female pelvis may simulate an ovarian tumor and be misdiagnosed as gynaecological disease. demonstration of the continuity to the cecum or urachus was the diagnostic clue. localized disease in the female pelvis may simulate ovarian carcinomatous peritonitis, and scalloping of the uterine margin may suggest this condition. because synchronous mucinous tumors of the ovary and the appendix may occur, careful observation to detect the slight irregularity of the omentum adjacent to the cecum is necessary to avoid overlooking an occult primary appendiceal lesion. conclusion: to recognize various imaging findings is important to make the correct preoperative diagnosis. in particular occult appendiceal lesions must not be overlooked. multiplanar observation on ct and mri was important to recognize the complex organic-pathologic relationships to identify the primary neoplasm. retroperitoneal cystic masses: ct findings d. yang, h. kim, j. kang, t. seo, h. kim, s. kim, j. kim; incheon/kr learning objectives: to illustrate the ct findings of the different types of retroperitoneal cystic masses. background: retroperitoneal cystic masses, which arise within the retroperitoneal space but outside the major organs of that compartment, are uncommon. however, the widespread use of computed tomography (ct) for evaluating abdominal and retroperitoneal diseases has increased the detection rate of retroperitoneal cystic lesions. in this exhibit, we conduct a literature review and present the ct findings of our own patients to illustrate the ct appearance of different types of retroperitoneal cystic masses. imaging findings: a variety of ct features were identified in various retroperitoneal cystic masses. the disease entities include cystic lymphangioma, retroperitoneal mucinous cystadenoma, müllerian cyst, cystic change of paraganglioma, epidermoid cyst, cystic teratoma, tailgut cyst, mucocele of appendix, perianal mucinous adenocarcinoma, cystic change of leiomyosarcoma after chemotherapy, pancreatic and nonpancreatic pseudocyst, lymphocele, urinoma, and hematoma. conclusion: knowledge of ct findings of various retroperitoneal cystic masses may be helpful for differential diagnosis. learning objective: our purpose is to describe the ct and mr findings of cystic dystrophy of the duodenal and gastric wall in heterotopic pancreas (cdhp) which allows an accurate diagnosis of this pathology usually associated with chronic pancreatitis. background: cystic dystrophy in heterotopic pancreas is characterized by the development of cysts in heterotopic pancreatic tissue localized in the duodenal or gastric wall. cyst formation is related to cystic dilatation of an anomalous duct bordered by pancreatic excretory epithelium, or to pseudocysts caused by pancreatitis of the heterotopic pancreas. we reviewed ct and mr findings in five patients with cystic dystrophy in heterotopic pancreas (cdhp). they underwent spiral ct (n = ) and mr/ cholangio-mr (n = ). diagnosis was confirmed by endoscopic us in all cases. three cases were pathologically proven after surgery. imaging findings: the ct and mri findings of cdhp consists of cysts, (normally multiple), located in the inner wall of the second part of the duodenum and less frequently in the stomach. the duodenal or gastric walls affected are thickened and inflammatory changes are observed around the cysts. signs of chronic pancreatitis are frequently seen. mr/ cholangio-mr shows high intensity lesions corresponding to the cysts. conclusion: cdhp presents some ct and mr features which permit an accurate and reliable diagnosis. when a cystic lesion is found in the duodenal or gastric wall cdhp should be included in the differential diagnosis with other cystic masses such tumors or abcesses. , and thanks to its favorable influence on serum glucose and lipid profiles, this technique is the preferred treatment for patients with severe type i diabetes mellitus and end-stage renal disease (simultaneous pancreatic-kidney transplantation) . therefore an understanding of the anatomic configuration and the spectrum of postsurgical complications is needed. imaging findings: we retrospectively reviewed computed tomographic scans (ct), ultrasounds (us) and doppler studies of patients who had undergone pancreatic transplantation using portal-enteric drainage in our hospital between january and july . we review the usefulness, limitations and potential pitfalls of ct and us. acute postoperative complications, including acute rejection, transplant pancreatitis, peripancreatic collections and abscess, pseudocysts, vascular complications (thrombosis, pseudoaneurysm) and exocrine leaks, are depicted. furthermore, ct-guided percutaneous biopsy and drainage is a safe and an alternative method for obtaining tissue from the transplanted pancreas with graft dysfuntion, and collections. conclusion: knowledge of normal anatomic configuration and the radiologic appearance of normal pancreatic transplant will allow proper interpretation and early treatment of the spectrum of portsurgical complications in this kind of pancreatic allograft. the more common current gastric bypass procedures include the roux-en-y procedure and bilio-pancreatic diversion with duodenal switch procedure. the more typical complications of these surgeries include anastomotic leaks and stenoses, both of which have been reported in the current literature. more atypical complications, however, have not been comprehensively demonstrated. imaging findings: gastric bypass/gastric stapling is a common surgical procedure at our institution. we have encountered several atypical complications, documented with flouroscopic studies using water soluble contrast. we offer five unusual complications which include the following: internal herniation through the small bowel mesentery, internal herniation through the transverse mesocolon, external herniation through the abdominal wall incision, enterocutaneous fistulas, roux-en-y configuration with anti-peristaltic inversion of the gastroenteric roux limb, and incorrect anastomosis of the roux limb with the excluded stomach (resulting in a roux-en-o configuration). conclusion: our findings expand those of the current literature to include the more infrequent complications of gastric bypass surgery. in the fluoroscopic evaluation of post-operative gastric bypass patients, a thorough understanding of expected post-operative bowel configuration is essential. in addition to assessing anatomical abnormalities, it is equally important to assess for abnormalities of motility. in this study, we performed a retrospective review of the medical records and imaging studies of patients with proven fmf, diagnosed between - . imaging findings: the commonest clinical manifestation were recurrent peritoneal attacks with abdominal pain ( . %) and fever ( . %). abdominal imaging findings included ileus (n = ), focal peritonitis (n = ), ascitis (n = ), splenomegaly (n = ), and hepatomegaly (n = ). one patient developed fatal peritoneal mesothelioma, and . % of patients developed amyloidosis with sonographic findings of renal parenchymal disease or cardiomyopathy. arthritis was second in frequency occurring in . % of patients. radiographs were normal (n = ) or showed joint effusion and soft tissue swelling (n = ) due to synovitis. two patients developed seronegative destructive arthropathy. pleuritis was encountered in . %. polyarteritis nodosa (pan) was present in patients, multiple sclerosis in one, and autoimmune hemolytic anemia in one patient. conclusion: fmf predominantly involves abdominal viscera but can affect other organs. the majority of patients have nonspecific imaging findings and the radiologic diagnosis is rarely considered. amyloidosis, mesothelioma, and destructive arthropathy are potential serious complications of fmf. polyarteritis nodosa, multiple sclerosis, and autoimmune hemolytic anemia are rare associations or coincidence with fmf. mucosal associated lymphoid tissue lymphomas: imaging evaluation with pathologic correlation d. voultsinou, t. gerukis, n. staurogianni, k. anastasiadou, a. anagnostopoulos, p. palladas; thessaloniki/gr learning objectives: to identify the radiological features of extra nodal non hodgkin malt lymphoma. to illustrate the spectrum of appearance of the disease in its various locations in the human body. to list the various imaging strategies. to discuss the differential diagnosis. background: mucosal associated lymphoid tissue (malt) lymphoma arises in extranodal mucosal lymphoid tissue and has only been recognized recently. it affects several extranodal structures such as the stomach, the lung, the eye and the salivary glands. it is generally low grade and has an indolent course, as it remains long confined to the initial site. in this exhibit we describe and illustrate the most common radiological patterns of malt lymphoma. imaging findings: we retrospectively reviewed clinical and radiographic records in cases with confirmed low-grade gastric malt lymphoma. the study group consisted of men and women (mean age years). the predisposing factors of malt lymphoma, number and location of lesions, and course of the disease were evaluated. we determined the most probable diagnosis on the basis of the radiologic findings and correlated them to pathologic and immunohistochemical findings of endoscopy, biopsy and bone marrow examination. out of patients presented with malt lymphoma of the stomach, in the parotid gland, in the lung, in the large intestine (sigmoid colon, rectum), in ileocecal valve and in the orbit rectal malt lymphoma coexisted in one patient, and parotid gland and lung lymphoma in patients. all patients were treated with chemotherapy. the disease resolved in patients. the correlation of imaging, endoscopic and pathologic findings significantly contributes to diagnosis and follow-up of malt lymphomas, early diagnosis of which results in complete regression. to describe a new approach to ct colonography for detecting colo-rectal polyps with multidetector row ct (mdct). to display the results and to present the benefits provided by this technique. background: virtual dissection‚ (gems) is an original application to display the entire inner surface of the colon without the need for navigation. the method consists of displaying the straightened and flattened colon using surface rendering. we performed virtual colonoscopies with a ge lightspeed mdct. the acquisition parameter and each step of the imaging procedure will be described. procedure details: all patients underwent ct colonography in both supine and prone positions, the following parameters were used: kvp; - mas; . mm collimation; . -mm reconstruction intervals and rotation time, . - . sec. the colon was assimilated to a cylinder manually defined. time to display the straightened and flattened colon using surface rendering was about minutes. normal aspect, polyps and artifacts were displayed. polyp detection was faster and easier with this method compared to axial images and the usual virtual endoscopy mode with forward and reverse viewing fly-through volume rendered movies. conclusion: this method makes it possible to stretch the colon virtually and to cut it along its axis similar to a real dissection on the pathologist's ( ) and luschka duct ( ). ce-mrcp succesfully revealed the origin of the leak in patients. conclusion: contrast enhanced mr cholangiography with iv mangafodipir trisodium can accurately diagnose the presence and location of bile duct leaks in patients who have undergone laparoscopic cholecystectomy. internal hernia of the anterior part of the abdomen: imaging in the multidetector-row ct era n. hongo, h. mori, s. matsumoto, y. okino, a. adachi, a. kaku; oita/jp purpose: internal hernia of the anterior part of the abdomen (ihapa) is difficult to diagnose preoperatively because of its rarity and lack of imaging landmarks. the purpose of this study is to clarify the ct features of ihapa by ct, especially with multi-detector row ct (mdct). methods and materials: the ct scans in patients with surgically confirmed ihapa through a transverse mesoclolon (n = ) or an omentum (n = ) were retrospectively reviewed; furthermore, the transverse mesocolic type was classified into intramesenteric type (n = ) and transmesenteric type (n = ). special attention was paid to the herniated sac-like appearance, hernial orifice, running course of the omental fat and transverse colon. results: the closed loop of the small bowel was observed in all cases. a sac-like appearance, enclosed by transverse colon was identified in both cases with intramesenteric type, but was not observed in other types of ihapa. fatty continuation between the hernial orifice and omentum was identified in two out of four cases with omental type of ihapa. furthermore, the transverse colon passed above to the hernial orifice in all cases with omental type, while this finding was not observed in any of the transverse mesocolic type. conclusion: the relationship between the hernial orifice and transverse colon, and sac-like appearance enclosed by transverse colon are the diagnostic key of ihapa on ct. particularly, mdct would become feasible to diagnose the ihapa preoperatively, even though it lacks of vascular landmarks. contrast enhanced mri using oral peg solution in the follow-up of patients with known crohn's disease m. scettro, a. grasso, f. monetti, g. rescinito, g. rollandi, c. neumaier; genoa/it purpose: aim of our study was to analyze, in the context of an extensive evaluation, crohn's disease activity index (cdai), and conventional enteroclysis, the value of mri to identify disease extension and complications in patients with known disease. material and methods: abdominal mri of patients in a period of months with a diagnosis of crohn's disease were analyzed by two experienced gastrointestinal radiologists, in consensus, looking for any intestinal and extra-intestinal abnormality. mri studies were performed on a . t (intera nt, philips) with a phased array coil. mri was performed using a polyethylene glycol (peg) solution b d e f a g as oral contrast agent to distend the small bowel (ce-peg-mri), using standard sequences (ss-tse t and tfe t with fat saturation after gd-dtpa and buscopan mg i.v.) . results: mr imaging had an overall sensitivity of % and a specificity of % for active disease. bowel wall enhancement (ratio of signal intensity of abnormal to normal bowel > . : ), bowel wall thickening greater than / mm and increased mesenteric vascularity were useful in identifying active disease. the experience shows that the method is complementary to conventional enteroclysis in the detection of superficial and transmural abnormalities in patients with crohn's disease. in addition, ce-peg-mri can provide excellent information concerning mesenteric involvement, disease activity, and complications of crohn's disease. finally mri may become a gold standard in the identification of recurrant disease. withdrawn by authors the number of x-ray based examinations (using a contrast medium) of the oesophagus, stomach, and large bowel, decreased by %, % and %, respectively. x-ray based examination of the small bowel and plain radiography of the abdomen remained unchanged. endoscopies of oesophagus / stomach and the large bowel increased by nearly %. x-ray based examination of the biliary tract almost disappeared with us, ercp and mrcp taking over. the annual collective effective dose for the all gi tract examinations was reduced by %, from . mansv in to . mansv in . the shift in modalities used for diagnostic imaging of the gastrointestinal tract from x-ray examination to us, endoscopies and mri resulted in a significant reduction in exposure to ionising radiation of patient, and need to be considered when discussing further development and structure of diagnostic imaging in general. perfusional enhanced ultrasound in the diagnostic evaluation of sicca syndrome g. argalia, d. salera, g. giuseppetti; ancona/it purpose: sicca syndrome is a relatively common disorder especially among older women. it may be caused by an autoimmune disorder like sjogren's syndrome (ss) or by several other illnesses. our purpose is to evaluate the diagnostic possibilities of contrast enhanced ultrasound in the characterization of sicca syndrome. we studied consecutive patients with sicca syndrome, affected by ss ( with primary sjogren's syndrome, with secondary sjogren's syndrome due to connetivitis) and patients with a sicca syndrome not related to ss. all patients were selected according to the european community study group diagnostic criteria for sjogren's syndrome (the gold-standard of the study) and underwent contrast enhanced ultrasound examination with timeintensity curve analysis obtained before and during saliva stimulus, salivary glands scintigraphy and labial gland biopsy. results: ss patients had an echo contrast enhancement before and during saliva stimulus significantly lower (p < . ) than the non ss ones. the patients with primary ss showed a significantly lower enhancement during saliva stimulus than the secondary ss patients (p < . ); no statistically different enhancement was seen in basic conditions (p = . ). in the study group ( subjects with sicca syndrome) the enhanced ultrasound showed a sensitivity of . %, a specificity of % and an accuracy of . % on the diagnosis of ss. our experience confirmed the good diagnostic accuracy of salivary glands scintigraphy and labial biopsy. the preliminary results show this method is useful in the functional study of parotid glands in the characterization of sicca syndrome. fusion imaging of d angiographies of arteries and veins around the stomach by multiphase fusion technique under a single-breath hold using row multidetector row ct: its usefulness for preoperative simulation and intraoperative navigation of laparoscopy-assisted gastrectomy m. matsuki, h. kani, i. narabayashi; takatsuki/jp purpose: gastric arteries and veins can vary between patients; therefore, it requires much time to ligate arteries, and veins can be damaged during regional lymph node excision under laparoscopy-assisted gastrectomy (lag). we evaluate the efficacy of fusion imaging of three-dimensional angiographies of arteries and veins around the stomach by the multiphase fusion technique under singlebreath hold using row multidetector row ct (mdct) in the preoperative simulation and intraoperative navigation of lag. methods and materials: contrast-enhanced ct scan using das mdct was performed on patients before lag. images at both arterial and venous phases were obtained under a single-breath hold. three-dimensional ct angiographies at the arterial and venous phases were reconstructed using the volume-rendering technique and then fused. ) the detectability of the left gastric artery (lga), right gastric artery (rga), left gastric coronary vein (lcv), right gastric vein (rgv) and helens' gastrocolic trunk (gct) on the multiphase fusion images was evaluated in comparison with the surgical findings. ) in intraoperative navigation, the clinical usefulness of multiphase fusion imaging was evaluated. results: . in nine of ten patients ( %), the multiphase fusion images could demonstrate clearly, simultaneously and three-dimensionally the lga, rga, lcv and gct without a respiratory gap. . we could arrange and rotate a multiphase fusion image to correspond to the operative view, which was very useful in the intraoperative navigation of lag. the multiphase fusion imaging is considered to be very useful in the preoperative simulation and intraoperative navigation of lag. three-dimensional endosonographic guidance of needle positioning in interstitial brachytherapy a.f. christensen, m.b. nielsen, s.a. engelholm; copenhagen/dk interstitial brachytherapy is used in the treatment of anal carcinoma, so far without image guidance during needle positioning.the aim of the study was to describe a procedure for optimizing needle positioning guided by three-dimensional ( -d) endosonography. twelve patients who received external radiation therapy for anal carcinoma were referred for interstitial brachytherapy under -d endosonographic guidance. the procedure was initiated by anal endosonography performed with a mhz rotating endoprobe. cross-sectional images of the anal sphincters were stored on a -d system during retraction of the endoprobe. afterwards, any projection could be reconstructed. from this scanning the optimal positioning of the needles was determined. the needles containing radioactive isotopes were inserted through holes in an externally fixated disc. repeated endosonography assured that optimal tumor coverage was obtained by adjusting the number, loading or position of the needles. in all patients endosonography was able to visualize the extent of the tumor and the position of each needle so that both the distance from needle tip to upper tumor border, as well as the distance from the anal orifice to the lower tumor border could be determined. in patients endosonography showed a, at least clock-positions larger circumferential tumor size than manual examination. this made an increase in number of needles necessary. in patients endosonography influenced the extent of loading of the needles. in patients endosonography led to a change in positioning of the needles. -d endosonography guidance of interstitial brachytherapy in anal carcinoma seems possible, which may influence the radiation coverage. to determine ct criteria that allows accurate classification of interstitial hernias. methods and materials: ct scans of patients with surgically proved spigelian hernia (n = ) or interparietal inguinal hernia (n = ) have been retrospectively reviewed. ct assessment included: ) assessment of the hernial sac (anatomic situation, extension, content) and ) assessment of the fascial defect (anatomic situation, relationship to the inferior epigastric vessels). results: in all cases (n = ), the hernial sac was located between the internal and external oblique muscles. the fascial defect was located at the spigelian aponeurosis, above the inferior epigastric vessels in the seven cases of spigelian hernia, and at the internal inguinal ring, lateral to the inferior epigastric vessels in the six cases of interparietal inguinal hernia. conclusion: assessing only the hernial sac does not allow accurate classification of interstitial hernias. diagnosis is established by precise analysis of the anatomic situation of the fascial defect. the role of magnetic resonance cholangiography in patients with biliaryenteric anastomosis y.a. akhmetov; almaty/kz purpose: the study was aimed at investigating the role of mr-cholangiography (mrc) in the examination of patients treated with biliary-enteric anastomosis. methods and materials: mrc was performed in patients ( female and male, mean age: . years) undergoing biliary-enteric anastomoses ( hepatico-jejunostomies and choledocho-duodenostomies). mrc was performed with a non-breath-hold d turbo spin echo sequence (tr = msec, te = msec, etl = ) with an acquisition time from min to min. patients subsequently underwent percutaneous transhepatic cholangiography (ptc) in order to confirm the mrc findings and to perform a therapeutical procedure. patients with choledochoduodenostomy were examined with ercp. the remaining patients, with no evident symptoms or signs of bile duct dilation, were examined during their surgical follow-up without the performance of any invasive procedure. the degree of bile duct dilation was correctly evaluated with good panomaric assessment of the ducts and site of anastomosis in all patients. both dilated and non-dilated bile ducts were well depicted. mrc correctly showed stenosis of anastomosis in of patients, - mm stones in of patients and bile ducts irregularities in of patients with cholangitis. conclusion: mrc is a safe, noninvasive technique in the study of biliary-enteric anastomoses and can be used to the screening of symptomatic patients. mrcp images may serve as a guide for planning of interventional procedures. cystic duct imaging using both magnetic resonance cholangiography (mrc) and d learning objectives: to describe the indications and diagnostic limits of each procedure, to present the steps taken to perform each procedure as well as the possible complications, and potential pitfalls. background: the early diagnosis of breast cancer is a challenge in breast imaging work-up. to detect malignancy in non-palpable breast lesions without unnecessary open biopsies, percutaneous biopsy of the suspect lesion should be undertaken. percutaneous breast biopsy can be performed under sonographic or x-ray guidance. whatever method is used, certain principles apply. exhibit details: the exhibit will be divided into subsections focusing on the indications and diagnostic limits of each procedure, the necessary equipment for each method and the steps taken to perform each procedure through sample cases. we will focus on practical issues such as needle size, site of entry, number of tissue probes that we need to take according to the lesion type. also the avoidance of complications, and other useful tips will be presented. a special reference will be made to the histological findings that require surgical excision and the potential pitfalls in case of false negative samples. we will also present a short time follow-up diagram for the histologically confirmed benign lesions. conclusions: whenever a non-palpable breast lesion is considered for biopsy, either x-ray or ultrasound guided procedures are performed. each method has its indications, advantages and disadvantages, and the examiner should be familiar with those factors for proper procedure selection. the use of carbon marking after stereotactic -gauge vacuum-assisted breast biopsy and during advanced breast results: eleven cases were considered fn ( %). a) four cases were found after -g core biopsies: two architectural distorsions initially diagnosed as radial scars proved to be malignant after surgery, and one case of microcalcifications, first diagnosed as adenosis, underwent month follow-up corresponding to a ductal carcinoma in situ. also a ductal carcinoma in situ was found inside a fibroadenoma (first diagnosed as fibroadenoma). b) also seven cases were found after vacuum-assisted biopsies, all of them microcalcifications that were confirmed in the radiograph of the specimen: five cases were initially diagnosed as atipical ductal hyperplasia and corresponded to four ductal carcinomas in situ, and one infiltrating ductal carcinoma; the remaining two cases were first reported as ductal ectasia and interstitial microcalcifications, becoming two ductal carcinomas in situ (one of them was diagnosed months later after core biopsy). the average diameter of the lesion was . mm. none of the lesions were removed completely. conclusion: results such as radial scar, atypical ductal hyperplasia, interstitial microcalcifications and ductal ectasia may occult a carcinoma after needle core biopsies. the percutaneous triple sample (fnac, core biopsy, and cylinder smears) in breast cancer diagnosis: results in patients i. vizcaino, v. torres, s. picó, e. blanc, c. soto, s. isarria; valencia/es purpose: to evaluate a combined method to sample tumoral tissue in breast cancer. a prospective study using at the same time fnac, core biopsy and cylinder smears was performed in patients. we used a g needle ( - passes) and a thin-wall g needle ( - cylinders, mm/ cm sized). smears of core biopsy cylinders were systematically prepared to cytological examination. the lesions were targeted using palpation, ultrasound, coordinates plate, and a stereotactic device depending on the lesion. all patients had breast cancer. the results were evaluated regarding the sensitivity to breast cancer diagnosis. the overall sensitivity of three-way diagnosis was % (the false negative case was a non-palpable dcis that showed necrosis in cylinder and no tumoral cells in both, fnac and cylinder smears). the sensitivity of fnac was the same as core biopsy: %. the sensitivity of cylinder smears was %. benign tissue in cylinders with malignant cells in cylinders smears was seen in cases ( dcis, necrotic medullar carcinoma, intracystic carcinoma and idc). fibrous tissue was found in the cylinders of these cases. the combination of fnac, core biopsy and cylinder smears show a high sensitivity to breast cancer diagnosis. cylinder smears must be obtained systematically in core biopsy in order to prevent the false negative results. weak cohesion of tumoral cells can explain this phenomenon. purpose: mammographic follow-up of patients after conservative treatment for breast cancer by screen-film mammography is difficult and sometimes impossible due to the post-radiation and surgical changes. digital mammography (dm), with post-processing tools, might facilitate the reading. the purpose of this presentation is to evaluate the accuracy of dm and to describe the post-therapeutic changes in these patients. materials and methods: dm (senograph d, gems®) was performed in patients and months after conservative treatment (surgery and radiotherapy) for breast cancer. mammograms were indepedently read by two senior radiologists. both radiologists performed screen-reading, using commercially available post processing tools (magnification, zoom, contrast inversion, thickness compensation). they evaluated the overall image quality. the following features were also analysed: breast density (according to birads classification), skin thickening, parenchymal changes with glandular oedema ( grades), scar and microcalcifications. results: all dm were considered as of diagnostic quality by both radiologists. importance of glandular oedema was correlated to breast density (birads , n = ; oedema , n = ), (birads , n = ; oedema , n = ). analysis of skin and dermal thickness was facilitated by the algorithm of thickness compensation. visibility of scars (n = ) and microcalcifications (birads , n = ; bi-rads - - , n = ) was improved by digital magnification and digital contrast variations. conclusion: digital mammography due to the post processing tools seems to facilitate dramatically reading of mammography in patients with previous conservative treatment for breast cancer, and helps to delineate post-therapeutic changes. the conclusions: it appears that the achievable image quality of a system not only depends on detector characteristics but also on available combinations of target, filter and tube voltage. therefore image quality comparisons show different results using optimized spectra for each digital system, compared to using the same spectrum for all systems. to show a short way to present breast mr dynamic imaging obtained using -mm isotropic d acquisition and single-voxel proton spectroscopy. background: during a standard breast mr exam, including a precontrast shorttau inversion recovery (stir) or fast spin-echo t -weighted fat-sat and a dynamic gd-enhanced d sequence, at least native and subtracted images are produced. moreover, maximum intensity projections (mips) of a subtracted phase and dynamic curves must be shown. if performed, proton spectra could be given with localizing images, too. procedure details: seventy breast mr exams were performed at our department as follows: -slice axial stir; t -weighted d gradient-echo coronal -mm partitions ( x -mm field-of-view; x matrix; -mm voxel) with . mmol/kg gd-chelate and -s time resolution ( postcontrast phases), giving native and subtracted for a total of images; single-voxel spinecho (te ms) proton spectroscopy. we present firstly an axial mip of the first subtracted sequence similar to cranio-caudal x-ray mammography (xm) views, two lateral mips similar to lateral degrees xm views, and a coronal mip. then, only selected images are presented: stir, precontrast t -weighted, and postcontrast subtracted images (morphology); percent enhancement-to-time curves for regions of interest (dynamics); proton spectra with/without choline peak at . - . ppm and lipids peaks (metabolism). representative cases will be shown in correlation xm, ultrasound, and pathologic proof. conclusion: breast mr high-resolution imaging and proton spectroscopy can be effectively summarized in no more than images ( x -in-one films), % of the acquired/subtracted images. mr guided interventional procedures in breast pathology management i. herraiz, l. concepción, j. ballesteros, a. fernández-moscoso, j. gallego, m. garcía-franco; alicante/es; presented by s. lopez-celanda; alicante/es learning objectives: to descibe mri-guided core biopsy of mri-suspected lesion, hook wire placement or clip marking for excisional biopsy of non-palpable, mri-only visualized lesions. procedure details: all procedures were performed on a . t philips gyroscan intera unit. standard ce breast mri imaging protocol included a dynamic volumetric t -weighted acquisition prior and after gd-dtpa injection employing a bilateral surface coil. localization was achieved either by employing a stereotatic device or by triangulation methodology with external markers with a flexible unilateral surface coil. imaging acquisition was tailored to each case. titanium clips (inrad®) or nitinol (somatex®). g titanium coaxial needles (somatex®) and g automatic biopsy gun were employed (tsk acecut®,). our series includes patients with mri suspected lesions. reevaluation of them with conventional methods (mammography and sonography) allowed location of half of them resulting finally in a total of mri-guided interventional procedures. this includes mri-guided core biopsies in lesions measuring - mm, mriguided presurgical hook wire localization and clip placements to assist location of a mri visible single lesion. pathologic evaluation resulted in cases of infiltrative ductal carcinoma and benign lesions. the only biopsy false negative result was in the smallest lesion ( mm). conclusions: all mri-suspected lesions must be reevaluated by conventional methods. in mri-only visible lesions we favor clip marking or hook wire location if size is smaller than mm. in lesions highly suspicious for malignancy additional clip marking during core biopsy procedure is recommended. background: accuracy of pre-treatment diagnosis of breast carcinoma extension is essential in order to plan the most effective therapeutic approach. extension of carcinomas can be difficult to interpret on d images, especially if their distribution is segmental or irregular. also, additional multifocal or multicentric foci can be problematic to localize in respect to the main tumour and the anatomy of the remaining breast. it is furthermore important for breast surgeons to apprehend the extent of the disease in radiological images in order to plan the surgical approach. procedure details: t -weighted flash d pre-and post-contrast images were obtained in patients diagnosed with breast cancer on core biopsy. analysis of images was done with a dedicated software for breast mri (asymedâ). parametric images for maximum enhancement speed, maximum enhancement, multiplanar and d reconstructions, speed of enhancement curves and enhancement curves are analysed in each patient. d images were viewed as video clips with a degree rotation around both breasts. maximum tumor diameter was measured in all images and distribution was reported following surgical anatomy. all shown cases have been histopathologically proven in respect to size and extension. conclusion: besides analysis of morphology and speed of enhancement curves, maximum enhancement speed parametric d images stand as a useful approach to tumor extension, giving additional information and an overall view of distribution of the disease in respect to breast anatomy. background: the rate of breast cancer recurrence is - % per year. follow-up of patients after conservative surgery includes a periodic clinical examination and a mammography every months during the first years and every year thereafter. post-treatment changes may mimic or obscure recurrent cancer. diagnostic problems are encountered with conventional imaging and fine-needle aspiration cytology. increased vascularity and vascular permeability occurs in recent scars and inflammation; however majority of scars months after surgery do not enhance significantly at mrm while all malignancies do. imaging findings: in our series all cases of proved recurrence showed over % maximum signal intensity enhancement at minute and plateau or washout dynamic curve at mrm. only two cases of scar showed more than % maximum signal intensity enhancement at minute and the curve in all benign cases was gradual. the quantitative data should be always correlated with the morphology of enhancement curve. irregular shape or margins and inhomogeneous enhancement proved to be important criteria for recurrence assessment. conclusion: mrm appears a valuable tool to differentiate post-treatment changes from recurrent carcinoma and to guide the pathological confirmation. its high negative predictive value may have an impact on follow-up of treated breasts. the correlations between enhancement parameters and histopathological findings were analyzed using stepwise multiple regression analysis, student's-t test and spearman moments correlation coefficients. results: significant correlations were determined between the presence of lymph node metastasis and tumor size (r = . , p < . ), contour characteristics (r = . , p < . ), and edge characteristics (r = . , p < . ). a highly significant correlation was found between histological grades and qualitative enhancement patterns (r = . , p < . ). statistically significant differences were found between the groups with and without lymph node metastasis regarding enhancement in the st minute (p < . ) and tic slope (p < . ). a significant difference was found between the histological grades i and iii regarding all quantitative enhancement parameters, whereas no difference was found between the grades i-ii, and ii-iii. conclusion: dce-mri helps to predict prognostic factors of breast cancer by revealing qualitative and quantitative enhancement features of the primary tumor. additional morphological factors further improve our ability to predict lymphatic metastasis. we measured the number of vessels, the major systolic velocity (ps), the end diastolic velocity (ed), the resistance index (ri) and the glosing index (pi) appearing in all the slices we took. we noted the maximum and minimum values of all the above parameters that appeared in the same mass in different slices and calculated the differences. results: for the benign tumors the mean values for the differences between the maximum and the minimum number of vessels was (diffves) . (p < . ), the diffps was . cm²/sec (p < . ), the diffed was . cm²/sec (p < . ), the diffri was . (p < , ) and diffpi was . reliability of us-diagnosis of metastatically affected regional lymph nodes in the presence of female breast cancer e. shevchenko, a. zubarev; moscow/ru purpose: analysis of false-positive and false-negative results of us in diagnosis of metastatic lymph nodes (l.n.) in breast cancer. materials and methods: patients with breast cancer (stages t n -t n ) were examined on hdi atl philips with morphological verification. patients ( . %) had metastatic deposition in regional l.n. results: precision of us-diagnosis of unchanged l.n. was equal to . , specificity - . %, sensitivity - . %. false-negative results were found in cases ( . %). in b-mode the oval shape remained unchanged in all cases, cortical layer and core were differentiated. thickness of cortex: . - . cm, node size: . x . cm- . x . cm. in cases blood flow was located in portal region, cases - longitudinal vessel in center of the l.n. sizes of the l.n. with micrometastases were up to . cm. we had false-positive results in patients ( . %). round shape was found in cases, oval -in , in patients l.n. joined together in irregularly shaped node. in cases there was uneven thickening of cortex in one pole of node with size fluctuation. differentiation of cortex and core was absent in cases and echogenity was diffusely lowered. subcapsular vessels were located in cases, aberrant vessels -in , distortion of central vessel -in , local absence of perfusion -in . false-positive cases corresponded to sinus-histiocytosis, hyperplasia of lymphoid tissue. conclusion: only use of the full complex of us-symptoms of metastatically affected l.n. lets us improve the precision of diagnosis. inter-and intraobserver agreement in breast ultrasonography: significance of internal echoes at image interpretation j. tsutsumi, k. shimamoto, m. ikeda, a. sawaki, h. satake; nagoya/jp purpose: to evaluate the significance of internal echoes among diagnostic us criteria for breast lesions. materials and methods: breast masses ( benign, malignant) were interpreted by radiologists and radiological technologists using a four-point rating scale, and the kappa statistics were employed for analying interobserver agreement. to assess reproducibility in judgments, double reading was done in cases, and a total of cases were interpreted for each observer. us criteria included shape, border, boundary echoes, internal echoes (homogeneity and echo level), posterior echoes, lateral shadow, gland surface, depth-width ratio (d/w) and total impression in differentiating benign from malignant lesions. to evaluate the significance of internal echoes at us image interpretation, us images were modified to make the internal echoes invisible by painting with black color using the adobe photoshop. : roc analysis showed a significant difference in diagnostic performance between the original us images and modified us images (p < . ). the kappa value of posterior echoes was highest whereas that of boundary echoes showed the lowest value in both the original and modified us images. inter-observer agreement in shape, border, and category in the original images was significantly higher than that in the modified images (p < . ). reproducibility in boundary echoes and gland surface was fair, and that in posterior echoes and the d/w was good. can a mammogram dictate to you the diagnosis? a review of characteristic mammographic findings a.n. chalazonitis , j. tzovara , t. vrakatselis ; athens/gr, ioannina/gr learning objectives: participants will obtain an approach to various mammographic patterns and improve their diagnostic skills in breast imaging. background: mammography is considered by far the single most important imaging modality for breast diseases. except from radiologists with vast experience, many other physicians without specific knowledge are unable to discriminate normal from abnormal findings in the mammograms. imaging findings: the main aim of this exhibit is to provide a pictorial review of characteristic radiological findings in different mammograms. various examples due to different causes are illustrated and mammographic findings, as well as differential and final diagnosis are also discussed. all cases have been selected by reviewing our hospital teaching files. conclusion: our exhibit will allow participants to challenge their skills in the detection of both normal and abnormal mammographic signs. breast imaging and histopathologic correlation of fibroadenomas in patients with transplanted kidney: different features from usual fibroadenoma e. son , e.-k. kim , k. oh ; sungnam/kr, seoul/kr learning objectives: to analyze the imaging and histopathologic features of fibroadenomas in kidney transplanted patients and to compare findings of fibroadenomas in kidney transplanted patients with those in a control group. background: from to , , patients underwent renal transplantation at our institute. all patients received immunosuppressive therapy with cyclosporin a and steroid after renal allograft. we examined ten female patients who were diagnosed with fibroadenomas during chemotherapy, and compared the data obtained with that of fibroadenomas in the normal population. procedure details: twenty-two fibroadenomas developed in ten patients. eight of the patients had multiple fibroadenomas and had bilateral fibroadenomas. the mean diameter of the fibroadenomas was . ± . cm. by mammography the lesions were spherical in shape and had a well circumscribed margin with a high density mass. none of the lesions showed calcification or spiculation. sonographic findings of the masses showed relatively high echogenecity with higher l/t ratios than benign masses, however, features of homogeneous internal echo and well circumscribed margin were consistent with benign. conclusion: the fibroadenomas that developed in patients with renal transplantation showed a tendency to be multiple, bilateral and larger than those in the control group. these fibroadenomas also exhibited a more rapid growth, a more spherical shape and an unusually high internal echo and higher l/t ratio than usual fibroadenomas. to present mammographic and ultrasonographic (us) findings in various types of reconstruction using autogenous myocutaneous flap after mastectomy or breast conserving operation. background: breast reconstruction is an integral part of a woman's breast cancer management and yields positive psychological benefits for the patient. autologous tissue reconstruction has gained in popularity in part owing to adverse publicity regarding prosthetic implant safety. it can alter breast anatomy and architecture drastically. in this exhibit, we describe mammographic and us findings in the various types of reconstruction using autogenous myocutaneous flap after mastectomy or breast conserving operation. procedure details: mammography and ultrasonography obtained in patients who had undergone reconstruction mammoplasty using the autogenous myocutaneous flap procedure were reviewed to facilitate recognition of both normal and abnormal postoperative appearances of the various types of reconstruction using autogenous myocutaneous flap after mastectomy or breast conserving operation. normal mammographic and us findings include predominance of fatty appearance, surgical clips, and surgical scars. abnormal mammographic and us findings include fat necrosis, calcifications, and locally recurrent carcinoma. us findings of fat necrosis were cystic, complex and solid appearing masses with circumscribed or ill-defined margins in peripheral portions of flap. us finding of locally recurrent carcinoma was ill-defined heterogeneous hypoechoic lesions in reconstructed breast, similar to those of primary breast cancer. conclusions: breast reconstruction using autogenous myocutaneous flap has increased in popularity with various methods. mammography and sonography facilitated excellent visualization of normal and abnormal findings of various reconstructed breasts using autogenous myocutaneous flap. extramammary pathology mimicking breast disease e. rabanal, r. rosell, j. salvia, r. garcia; sabadell/es learning objectives: to present the imaging findings in cases suspected initially of breast disease which turned out to have extramammary pathology. background: we reviewed our records for the last ten years. during that time, , mammary studies were performed. all cases with extramammary pathology were selected. they were classified into two broad categories: those which had abnormal physical findings (palpable mass or skin thickening) and those with abnormal findings in the mammogram (calcifications, nodules and thickened lymphatics). imaging findings: cases were classified as follows: microcalcifications secondary to filariasis. we will see more of these cases due to increased immigration. it's important to know this entity to make the correct diagnosis and avoid unnecessary examination or biopsy. uniform skin thickening and/or oedema, secondary to systemic disease (sclerodermia), congestive heart failure or lymphoedema. palpable skin nodules (infected sebaceous cysts), simulating mammary abscesses. chest wall disease: empyema necessitatis draining into the breast. extramammary (chest wall) lipomas and dercum's disease. neurofibromas in von recklinghausen's disease. the imaging findings of these processes will be reviewed and their salient features described in order to suggest the correct diagnosis. conclusion: it is important to know the features of extramammary disease in order to make the correct diagnosis, avoiding further examinations and unnecessary invasive procedures. the reality of microcalcifications in breast tissues shown by synchrotron radiation imaging j. okamoto, y. kanemaki, k. imamura, n. ehara, y. inada, i. maeda, k. miyamoto, y. nakajima, m. fukuda; kawasaki/jp purpose: the aim of this study was to approach the reality of microcalcifications of benign and malignant diseases using synchrotron radiation imaging, and to make a comparison with conventional x-ray images. materials and methods: surgical and biopsied specimens fixed in wax blocks were imaged using synchrotron radiation (sr), and conventional mammography unit (conv) as well. studied were patients ( benign, malignant). sr imaging was performed at spring- in japan in refraction-enhancement mode. conv images were evaluated visually using a magnifying glass. sr images were inspected on a monitor without magnification. morphology of microcalcification was categorized to groups: small round, amorphous (am), and pleomorphic (pl). results: , calcifications were observed in conv images and , calcifications in sr images. , of , ( %) were invisible in conv images. sr imaging revealed that great numbers of specks were recognized collectively as a single calcification in conv images, and of , calcifications in conv images were found to consist of multiple specks. the incidence was especially higher in am and pl types ( / and / , respectively). comparing malignant with benign diseases, there was a striking contrast in am-type calcifications; majority of am-type calcifications in malignant diseases were found as collectives of fine specks ( / ), to the contrary / in benign (p < . ). conclusion: synchrotron radiation imaging showed that majority of amorphous and pleomorphic type calcifications, especially of amorphous type of malignant diseases, in conventional images were essentially collectives of multiple specks. psychological perceptions of women due to mammographic follow-up b. barreau , r. gilles , s. tastet results: women answered the questionnaire. all women were satisfied with the mammography and the medical staff except that the mammography was painful in cases. for women, the period between mammographic follow-up was too long. in women, the quality of life was altered. communications of the experience to a relative was frequent ( / cases) but satisfactory in only cases. for women, medical information was considered relevant. the median of the scale of stress was ( - ). there is two pickaxes, one at , the second at . "low-stressed" women could have an avoidance coping. "high-stressed" women could use a helplessness-hopelessness coping strategy. conclusion: women reported relevant medical information and comprehension of the short follow-up -mammography. they were reassured by the medical care, but the evaluation of the scale of stress was high, probably due to the diagnosis. mammographic density changes in postmenopausal hormone therapy: effects of various agents using a new scoring system s. orguc, c. göktan, g.y. ovali; manisa/tr purpose: to evaluate the changes of mammographic breast density due to postmenopausal hormone replacement with various agents using a new scale and to compare the new scoring system with the classical methods. materials and methods: women who received hormone replacement therapy in celal bayar university hospital between - were examined with baseline and follow-up mammograms. the effects of various hormone regimens, selected according to the gynecological status, were evaluated by two radiologists. wolfe classification and a new scoring system were used to assess the change of mammographic density. the new scale divides each breast into ten wedge shaped slices on the cc positioned mammograms. change of density is scored according to the number of slices, which have an increase of density in comparison with the baseline mammograms. the results were statistically evaluated to compare the effects of various hormone regimens using a control group of patients who did not receive any hormonal therapy. statistical analysis was also carried out to determine the effect of using different scales. results: tibolone effected breast density is less than preparations containing oestrogen. degree of mammographic change differs depending on the scale used. the new scale is more sensitive in depicting changes of breast density. conclusion: preparations containing oestrogen effect the mammographic density more than tibolone regimens. the new scaling system is highly efficient and objective in determining the changes of breast density. to evaluate sensitivity and specificity of positron emission tomography (pet) and -f-fluorodeoxyglucose ( f-fdg) in breast cancer diagnosis and to assess tumor dissemination. materials and methods: patients were examined: without mammary disease, with fibrous cystic mastopathy, and with breast cancer, which was hystologically proven. pet-scans (ecat exact ) in "whole body" mode were performed min after intra-venous injection of - mbq f-fdg. the t/ nt ratio for radiopharmaceuticals was evaluated. results: in all patients with malignant tumors the t/nt ratio for pet- fdg was higher than . (m ± m = . ± . ). it was shown that pet has a high diagnostic accuracy in breast cancer detection with sensitivity % and specificity %. pet scanning in the "whole body" mode was allowed to assess dissemination of tumor process with high accuracy. pet f-fdg sensitivity was around . % to % in diagnosis of regional and distant metastases. it was related to metastases localisation. conclusion: pet with f-fdg has high diagnostic accuracy in diagnosis of breast carcinoma and staging of tumor extent. results women aged between - were invited in three rounds in the mentioned period of time. bilateral mammography with double projection was performed, and read in a double blind manner, according to the strict, prescribed methods. if needed, additional mammographic x-ray, us, fnab and core biopsy was performed. results: the acceptance rate was low, around . % ( women were screened during five years). we called back around % of the screened patients for additional x-ray examination ( . %) or for us ( . %). we performed fnab in % of all cases ( women). cases were proven benign. the overall rate of hystopathologicly verified invasive cancers per women were ( patients). we could detect an increasing rate of small cancers and better lymph node state by the time, because around % of the examined women were already screened by us before, during the -year-period. number of interval carcinomas was nine ( . %). conclusions: comparing to european standards our screening program was the same, but the acceptance rate was lover. the high non-acceptance rates can be explained mainly by the cross-screening possibilities of budapest, hungary and because the high risk population is not informed well enough. however, due to the mental hygenic program running in hungary, we could detect an increased number of inquisitive women. tubular carcinoma of the breast: clinical, mammographic and ultrasonographic findings f. zandrino , m. calabrese , l.e. bacigalupo , f. musante ; alessandria/it, genova/it purpose: tubular carcinoma is a well differentiated invasive adenocarcinoma, with a prevalence ranging from to % of all carcinomas of the breast. the purpose of this presentation is to analyse its clinical, mammographic, and ultrasonographic features, with histopathological correlation. materials and methods: in a retrospective review of consecutive histologically proven carcinomas of the breast, pure (tubular component of at least %) tubular carcinomas were found ( . %) in women (age ± yrs). results: three lesions were palpable. on mammography, five were not detectable; the remaining presented as nodules with spiculated ( lesions) or irregular ( ) margins, spiculated nodules with microcalcifications ( ), distorsion ( ), or asymmetric density ( ). on us, lesion was not detectable; the remaining presented as hypoechoic lesions with irregular ( ) or well defined ( ) borders. for tumours, diagnosis of carcinoma was made with cytology; in cases core biopsy: in the first a complex sclerosing lesion with atypical cells was suggested, in the second differential diagnosis between tubular carcinoma and sclerosing adenosis was proposed. lesion size was . ± . mm. only in patient were metastatic axillary nodes found. conclusion: in our series, tubular carcinoma presented mainly as a non-palpable, small-sized lesion, with non-specific mammographic patterns and a hypoechoic ultrasonographic appearance. lymph node metastases are rare, as previously reported in the literature. dose distribution in tangential irradiation for breast h. ohtani, s. usui, y. jincho; tokyo/jp purpose: skin injury, lung disease and conservation radiation therapy for breast is reported. in a previous investigation, local management and survival rates were reported on the face of treatment effect's records, but the real irradiation doesn't become clear. this report is to measure the dose distribution of the effect on the critical organ by tangential irradiation of the left breast. materials and methods: measurement was performed with a semiconductor detector. irradiation was performed with x-ray beams at degrees. irradiation field sizes were established × cm to investigate influence by field sizes. irradiation was done on the wedge of degrees. results: dose equivalent was accurately measured with semiconductor detectors. from the results, breasts, thyroid gland, heart, both lung fields, kidney, ovary, and uterine doses become clear. the dose distribution at tangential irradiation of left breast was measured. consequently, the breast in which a radiation injury tends to be encountered, lung field, and the dose of important internal organs could be obtained. the necessity to confirm the dose distribution in the body for tangential irradiation in breast preservation treatment, refers to a radiation injury, and this study estimates these doses. the to assess the influence of days of repetitive hyperbaric simulations on the form and structure of single-lumen silicone gel-filled implants. materials and methods: new implants from different manufacturers and removed implants were submitted for simulated dives in a hyperbaric chamber, with an average of dives a day and with a maximal depth of m. a standard x-ray and mri were performed before, after and days of repetitive diving, and days after the experiment. mri was performed using a . -t superconductive magnet (symphony-, siemensag, erlangen, germany) with a body and spine coil, and a gradient echo t -w sequence ( d flash), slice thickness mm. the prostheses were checked for bubble formation, volume changes, integrity and morphological appearance. results: there were no significant changes in form, nor in shell integrity. after days of repetitive diving, there were some tiny bubbles in the implants, confirmed by x-ray and mri. after days, there were significant bubbles in of the implants (increase in implant volume up to %); in the other there was a slight increase in number and volume of bubbles. there was no significant change in the bubbles days after the last dive. the significant bubble formation in some single-lumen silicone gelfilled implants after days of repetitive diving, raises concern about the influence of repetitive stress on the lifespan of implants in correlation with the number and depth of dives. resection of non-palpable breast cancer using d imaging created by multislice helical ct: a new interventional technique to design resection lines in breast preserving surgery k. oda, t. kubota, h. satake, a. sawaki, t. ishigaki, y. nimura; nagoya/jp purpose: we report a new method for an adequate segmental resection of nonpalpable breast cancer employing an interventional technique using three-dimensional images of contrast-enhanced ct. materials and methods: eight patients with non-palpable breast cancer (dcis , invasive carcinoma ) were included in this study. diagnosis of carcinoma was made by cytology, but the extent of the lesion could not be clearly shown by us. one hour before the operation, contrast-enhanced helical ct was performed using a toshiba aquilion (toshiba corp. tokyo) with the patient in the supine position, following the insertion of vats (video assisted thoracoscopic surgery) markers (hakko, japan) under us guidance. ct scan was performed seconds after the injection of contrast medium. the detector row configuration was x -mm. maximum intensity projection (mip) was performed on three-dimensional reconstruction ( d-ct). using three-dimensional images of ct, anatomical relationship of vats markers, nipple and carcinoma demonstrated as a well-enhanced focal-clumped lesion or segmental lesion was evaluated and lines of resection with to cm surgical margin were designed. curative resection was confirmed in x-ray films of resected specimens based on the relationship between the lesion and the vats markers in preoperative three-dimensional ct images. results: in all cases, adequate segmental resection with cancer free surgical margins was accomplished and the cosmetic results of the surgery were satisfactory. conclusion: three-dimensional images of contrast-enhanced ct with this interventional technique is a useful method to design resection lines for non-palpable mammary carcinomas. european breast screening performance: does case volume matter? h.j. scott , a.g. gale , d. wooding , d. walter ; derby/uk, augsburg/de purpose: u.k. breast screening radiologists typically read over , screening cases per annum, whereas in europe this figure is much lower as in many countries national breast screening programs are in their infancy. the performs scheme in the u.k. permits radiologists annual self-assessment of their filmreader skills. as part of the eu funded european breast cancer network a number of german radiologists have now read the current performs assessment set. we investigated whether real-life case volume affects reading performance by the comparison of matched groups of radiologists from these two countries. we analysed the data from current sets of difficult recent screening cases. for each case individuals identified which key mammographic features were present, whether the case was abnormal and should be recalled or not. for this analysis the participants were matched on age, gender, film-reading protocols and years of experience. assessment of case volume was elicited by questionnaire data. the radiologists were compared on several key performance measures; cancers detected, correct recall and correct return to screen, signal detection performance statistics and real-life screening practice. results: it was found that whilst the performance of the german radiologists on the current test sets was excellent (correct cancer detection rate > %) on average they performed less well than their uk counterparts. we argue that this is closely related to the volume of cases read per annum by individuals within each country, and theoretically their performance will be equivalent as their case volume increases. the cytogenetic method of dosimetric control for screening mammography v. demin, e. djomina; kiev/ua purpose: it is advisable to study a degree of risk due to the unfavourable consequences of irradiation of the breast with screening mammography in ukraine after the chernobyls disaster. the correlation of cytological (cytogenetic) and physical findings were accomplished. the test-tubes with donor human blood were placed on upper and lower surfaces of breast of tissue-equivalent aldersons phan-tom. the test-culture of lymphocytes of human peripheral blood were used for biological dosimetry. physical dosimetry are realised with the thermoluminescent system alnor. the chromosome aberrations are becoming + . % on the upper surface of breast (normal control level is %) on mammography in two projections (upper-lower and lateral), the fragments of chromosome type are %, radiation markers -dicentric chromosome are . %; equivalent dose is . %. conclusion: there is a risk of radiation induced genetic lesions in breast tissue after mammography. instability genome is a basic radiation carcinogenesis. this is very real for women in ukraine after the chernobyls disaster. efficiency of the multidetector row ct in the diagnosis of breast cancer and evaluation of intraductal spreading h. kani, m. matsuki, i. narabayashi; takatsuki/jp purpose: the purpose is to analyse the efficiencies of multidetector row ct (mdct) in the diagnosis of breast cancer and evaluation of intraductal spreading. materials and methods: pathologically diagnosed breast lesions from patients were examined by mdct. the lesions included the following: invasive carcinomas, noninvasive ductal carcinomas, fibroadenomas, papillomas and other benign lesions. non-contrast ct and contrast-enhanced ct scans at , and sec after the start of enhancement were performed using row mdct. the imaging was performed under the following conditions: . -sec gantry rotation speed, . helical pitch, mm slice thickness and reconstruction intervals of mm. the items examined were as follows: ) in time-enhancement patterns during four phases, the early enhancement and plateau patterns were defined as a malignant tumor and gradual enhancement pattern was defined as a benign lesion. its ability in the diagnosis of malignant tumor was evaluated in the comparison with histopathologic findings. ) the abnormal enhancement continuing to the tumor at sec after the start of enhancement was estimated as intraductal spreading of breast cancer. the detectability of intraductal spreading was evaluated by the comparison with histopathologic findings. results: . the sensitivity, specificity and accuracy of the diagnosis of malignant tumor were . %, . % and . %, respectively. . the sensitivity, specificity and accuracy of the detactability of intraductal spreading of breast cancer were . %, % and %, respectively. conclusion: mdct is very useful for the diagnosis and preoperative estimation of breast cancer. an exploratory study about mammographic practice (acr from bi-rads) three months after a medical continuing education on breast cancer b. barreau , s. tastet , m. deghaye , v. picot , i. brault , p. marelle , l. ceugnart , d. aucant , s. haber ; paris/fr, bordeaux/fr objectives: evaluation of practices, three months after a medical continuing education, when abnormalities acr (probably benign) are detected on mammography. materials and methods: a questionnaire ( items) about these practices was sent to radiologists three months after medical continuing education (may-december ) . the answers were analysed with chi-square test. results: radiologists completed the questionnaire; radiologists practiced breast screening; used the bi-rads classification; radiologists followed-up the abnormality with a short interval, made a biopsy and overclassed the image; radiologists asked for advice from other radiologists, from an expert, submitted to a multidisciplinary committe and asked for a second reading. a short interval-follow-up disturbed radiologists about an eventual unfavourable evolution of the abnormality and radiologists about legal problems; had difficulty in explaining to patients the short interval-followup. the median of the scale ( to ) of the radiologists perceived stress is . conclusion: these french radiologists have consistent practices with the bi-rads classification and the anaes recommendations. they may have difficulties in explaining the procedure to patients. the radiologist's ethic is based on the "primum non nocere". c b d e f a g histopathological perspective on the first german mammography screening project: a two year review g. gohla , u. sauer , p. hanisch , w. boecker , h. junkermann , u. bonk ; bremen/de, münster/de purpose: the aim of this investigation was to critically review the role of the histopathologist in a mammography screening project. materials and methods: the first model mammography screening project in germany has been running in bremen for the last two years. all women between and years of age have been offered mammography and any suspect findings were investigated by core biopsy. these cases were reviewed in the pathology dept. of the university münster and discussed at a weekly multidisciplinary meeting. lesions were classified using the point b classification scale as recommended by the european union. results: in the first two years of the screening project, a total of women had mammography. suspicious areas were detected and core biopsies were obtained from of these women. % were classified b (malignant) % of these cases were classified b , (regular breast tissue, benign) and % of these cases were classified b and b (atypia probably benign, suspicious of malignancy). in % of all cases there was agreement amongst the reviewing histopathologists, but in % there was no interobserver consensus. these disagreements centered on flat epithelial atypia, atypically hyperplasia and papillary lesions. the consensus amongst experienced breast histopathologists is satisfactory in clearcut cases, but it should be improved in less well classified lesions. these lesions are more likely to be seen in a screening situation and in core biopsies. in this area it is certainly advisable to cooperate closely with the clinician and radiologist. usefulness of multi-slice ct for nonpalpable breast lesions with microcalcifications: correlation with mammographic findings h. satake, a. sawaki, k. shimamoto, s. ishigaki, k. oda, t. imai, t. ishigaki; nagoya/jp purpose: to evaluate the usefulness of multi-slice ct for nonpalpable breast lesions with microcalcifications, compared with mammographic findings. materials and methods: cases with nonpalpable microcalcification of the breast recommended for biopsy on mmg. they included malignant lesions (ductal carcinoma in situ, n = ; ductal carcinoma in situ with microinvasion, n = ) and benign lesions (mastopathy, n = ; intraductal papilloma, n = ; other, n = ). on multi-slice ct images, the presence of focal or segmental enhancement in the region consistent with microcalfications was regarded as positive diagnosis for malignancy. all mammographic and ct findings were assessed with a consensus between two radiologists. results: cases of benign lesions pathologically were assessed as "suspicious malignant" on mmg, while, in three of them, focal or segmental enhancement appeared on multi-slice ct. although seven cases of breast cancer were assessed as "probably benign" on mmg, six cases acquired focal or segmental enhancement on ct. for cancer diagnosis, the sensitivity of mmg and multi-slice ct were . % and . %, specificity was . % and . %, and negative predict value were . % and . %, respectively. breast conservation surgery with simulation using d ct images was performed successfully in cases of breast cancer. the sensitivity and the negative predict value of multi-slice ct for nonpalpable breast microcalcifications were superior to those of mmg. when biopsy is recommended according to mmg, additional examination of multi-slice ct enables the indication for biopsy to be more optimized and is useful for planning the treatment. the value of magnetic resonance imaging in the assessment of adult patients with corrected transposition of the great arteries s. shine, e. kavanagh, c. we fully illustrate the morphology, contrast enhancement and mobility of myxomas in rare locations such as the right ventricle and unusual attachment to the tricuspid valve. background: although benign myxoma is the most common primary cardiac tumor ( %), it remains a rare finding with an incidence of . per million population per year in the western world. myxomas are found in the left ( %) or right ( %) atrium and seldom in the ventricles. these pedunculated tumors typically arise from the atrial septum near the fossa ovalis and much less frequently from the mitral valve. intracardiac obstruction and systemic emboli are the major complications. imaging findings: myxomas in an uncommon cardiac location should be differentiated from other primary (malignant) and secondary (thrombus; metastasis) cardiac tumors. mri allows visualization of the narrow pedicle, its attachment and the changing tumor shape and mobility during the cardiac cycle, unlike most malignant processes. in contrast to most thrombi, myxomas have low signal intensity on cine gradient-echo images and show contrast enhancement. the water-rich myxomatous stroma, fibrous stroma, calcifications and hemorrhage account for the heterogeneous t -weighted se images. increased interstitial space in the myxoid region and inflammatory zones enhance after intravenous gd-dpta on t -weighted se images. lobular tumor surface and higher volume increase the pre-and postoperative risk for systemic emboli. furthermore, the exact location on mri is most helpful in planning the surgical access. conclusion: myxomas in uncommon locations can be differentiated from other cardiac tumors by evaluating tumor morphology, mobility and enhancement. how to study with cmr a patient submitted to ross procedure: a step by step follow-up protocol r. ribes , a. luna , j. vida , p. caro ; cordoba/es, jaen/es, cadiz/es learning objectives: to describe an adequate protocol to study patients submitted to ross procedure with mr. to identify and quantify postsurgical complications in this group of patients with mri. b d e f a g background: ross procedure is widely accepted as one of the best methods for aortic valve replacement, especially in children and young adults. ultrasound is usually used in their follow-up, but tit offers a limited evaluation of the right outflow tract, being important to discard homograft stenosis. mri overcomes the limitations of ultrasound and may replace the number of digital subtraction angiograms traditionally performed in the follow-up of these patients. procedure details: we describe step by step our mr protocol, to study patients submitted tothe ross operation. examples of the mr features of common presentations and complications are shown from our series of patients. our protocol consists of the following steps: • black-blood coronal and transverse turbo spin echo t -weighted images. • cinemr gradient-echo sequences allow functional qualitative assessment of themotion of valves and chambers, the detection of either valvular regurgitation orstenosis, and areas of altered myocardial contractility or relaxation. we perform gre-cinemr sequences in left two chamber, right two chamber, fourchamber, short axis (at several levels from the apex to the base), left ventricular outflow tract, right ventricular outflow tract, and aortic rootviews. • phase contrast images are performed to obtain quantitative data about magnitude anddirection of blood flow through the right and left ventricular outflow tracts. conclusions: cmr allows an accurate evaluation of patients submitted to ross procedure and identification of the postsurgical complications. the causes of focal outpouching of the left ventricle are as follows: ) true aneurysm; a chronic complication of myocardial infarction, which contains the endocardium, epicardium, and thinned fibrous tissue replacing the myocardium, ) pseudoaneurysm; a consequence of rupture of the ventricular free wall due to acute infarction and confined by the pericardium, ) diverticulum; a congenital outpouching of ventricular wall including all of three layers, and ) hibernating myocardium due to chronic ischemia with paradoxical outpouching at systole. mr imaging is useful in differentiating these causes by using variable imaging sequences to evaluate the myocardial contractility, perfusion and viability. in this exhibit, we will show the usefulness of mr imaging to differentiate these pathologies using various mr imaging sequences. imaging methods and findings: cardiac mr imaging methods include; ) cine imaging for wall motion analysis, ) first pass myocardial perfusion imaging, and ) delayed enhancement imaging. images were obtained along the short axis and horizontal long axis of the heart. by analyzing the shape, contractility, motion, perfusion, and contrast enhancement, it is possible to differentiate the causes of the outpouching lesion of the left ventricle. conclusion: cardiac mr imaging is useful in differentiating various causes of focal outpouching of the left ventricle. features of cardiac disease demonstrated on ct pulmonary angiography s. conclusion: ctpa is an increasingly frequently used investigation for the detection of pulmonary embolism. most patients investigated have pathology other than pe as a cause of their symptoms. frequently information about the heart is yielded that provides important clues to determine the cause for the presenting symptoms and signs or reveals co-existing pathology. with this investigation being used more frequently in daily practise in many patients with unexplained breathlessness or chest pain it is important to have a clear understanding of the features of cardiac disease as seen on a ctpa. mri has been done twice - - days and - months after surgery. mri was performed with a . t mr imager using ecg-gated t -se (or t -tse), bright-blood gre-cine-technique and phase-contrast mri. results: pts (of ) had a positive dynamic study of heart chambers dimensions and volumes in early postoperative stage. in the area of prostheses signal of low intensity was defined in all the patients (artifacts). narrow flow of low signal intensity along the prostheses leaflets corresponded to normal function of prosthesis ( of valves). pts had signs of mechanical valve dysfunction ( mitral paravalvular leaks, -aortic prosthetic valve stenosis). mri data were in agreement with the results of echocardiography. mri was able to diagnose such postoperative complications as atelectasis ( ), mediastinal haematoma ( ), pleuritis ( ). conclusion: mri could give an objective information about heart morphology (chamber dimensions, hypertrophy degree) and mechanic valve functioning (normal and abnormal flow) in patients after surgery. it can be safely performed in patients with artificial heart valves. negative calcium scoring: can it rule out severe stenosis? p.m. carrascosa , c. capuñay , p. garcia merletti , p. johnson , s. chandra , r. pissinis , j. carrascosa ; buenos aires/ar, cleveland, oh/us objective: to determine the presence of stenosis greater than % (in at least one coronary vessel) proved by digital angiography (da) in patients with a negative calcium score and correlate findings with ct angiography. methods: patients were evaluated. cts were performed with a -row ct scanner. the calcium score was calculated according to the agatston method. a calcified plaque was defined as a lesion of at least two adjacent pixels with signal intensity above hu. the second enhanced acquisition was performed following the administration of ml of non-ionic contrast with x mm collimation and . mm slice increment. das were performed on a digital angiographer. meas- car car car cardiac diac diac diac diac urements were done by means of qca. stenosis was considered positive if it was greater or equal than %. results: there were patients with high grade calcium score. eleven had positive da for at least coronary artery and in cases the da did not show severe stenosis. there were patients with negative calcium scores (grade ), had negative da findings, and had positive da findings. in these patients, the contrast ct acquisition revealed that the cause of the stenosis was due to soft plaques. conclusion: high grade calcium scores predict severe stenosis. negative results do not exclude them. in this study the % of the patients had negative calcium scores but significant stenosis due to soft plaques. a contrast ct acquisition instead of the calcium score will be necessary to rule out all the severe stenosis. positron emission tomography and n-ammonia application to evaluate myocardial blood flow in the asymptomatic patients with coronary artery calcification d.v. ryzkhova, i.e. itskovich, l.a. tyutin, l.a. kofal; st. petersburg/ru the purpose of this study was to assess myocardial blood flow (mbf) in the asymptomatic patients with coronary artery calcification (cac). methods: symptom-free patients with cac were included in study. all of them hadn't any symptoms of cad (chest pain, severe arrhythmia, heart failure) and myocardial infarction history. the patients with significant myocardial hypertrophy weren't included. the myocardial blood flow at rest (mbf rest) and during dipyridamole test (mbf stress) was evaluated by n-ammonia dynamic pet ("ecat exact ", siemens). the coronary calcium score (ccs) was measured by msct coronaroangiography ("somatom volume zoom", siemens) using the agatston method. results: myocardial perfusion abnormalities during stress test were revealed in patients with cac, there were patients with silent ischemia ( st group). we observed normal myocardial perfusion at rest and during stress test in the remaining patients ( nd group). the mean values of total ccs were less than units and close between the both group ( . ± . units vs. . ± . units, p = ns). mbf-values at rest didn't differ between the both patients' groups ( . ± . ml/ g/min vs. . ± . ml/ g/min, p = ns). mbf stress mean values were significantly decreased in the st group ( . ± . ml/ g/ min vs. . ± . ml/ g/min, p < . ). the ccs less than units isn't a reliable marker of flow-unlimiting stenoses in asymptomatic patients.soft lipid-laden plaques is a cause of myocardial ischemia in symptom-free patients with cac and low ccs. these patients should undergo stress studies to detect silent ischemia. detection and assessment of myocardial inflammation and fibrosis by echo-densitometry and ce-mri in patients with mild and medium myocarditis: validation by wbc-mibi spect m. deryugin, i. itskovitch, v. soukhov, s. boytsov, a. svistov; st.petersburg/ru background: echo-densitometry and ce-mri are novel methods for assessment of inflammatory myocardial infiltration (imi) and myocardial fibrosis (mf). the purpose of the current study was to validate these methods in patients with mild/medium myocarditis using wbc-mibi spect. methods: ce-mri was performed in patients using t -weighted turboflash inversion recovery. inflammation was evidenced by use of threshold method (signal intensity > + . sd). left ventricle echo-densitometry performed in long-axis views from left parasternal border at es/ed with following percentage cyclic variation indexes (cvi) calculation. spect was performed - hrs p.i. of mbq mtc-wbc and min p.i. of mtc-mibi. results: all patients were divided into groups based on scintigraphic data: first group - patients with diffuse leukocytes uptake that considered as imi; in second group there were patients with local leukocytes uptake and abnormal mp (imi+mf); third group consisted of patients with only mf. control group patients (n = ) had normal mp and no leukocytes uptake. cvi was ± % in controls; . ± % in st ; - . ± % in nd and - ± % in rd group. ce-mri was able to detect imi in % of patients. regression analysis revealed good correlation between imi by ce-mri and by wbc-spect (r = . , p < . ). furthermore, good correlation was found between cvi and mtc-mibi uptake (r = . , p < . ) both for imi and mf detection. conclusion: ce-mri enables exact detection of imi as compared to wbc-spect. the results of videodensitometry studies demonstrated that different types of imi were associated with definite cvi percentage, while cvi became negative with mf appearance. one-year ct evaluation of pulmonary veins following percutaneous cryoablation in patients treated for atrial fibrillation b. ghaye , d. szapiro , c. background: pulmonary vein isolation (pvi), using radiofrequency energy, for treatment of atrial fibrillation (af) has been associated with complications including pulmonary vein (pv) stenosis or thrombosis. the purpose of this study was to prospectively evaluate the pv morphology following cryoablation. method: patients underwent percutaneous pvi in academic centers. contrast-enhanced, single-slice or multi-slice spiral ct ( - . mm thick slices) was obtained before, and months following pvi. all examinations were read blinded to the location(s) of ablation. pvs were evaluated quantitatively and qualitatively: the diameter at ostium and at cm from ostium were measured. the presence and location of luminal irregularity or thrombosis was also assessed. the aims of this study were comparison of the local myocardial contraction between the myocardium with de and without, and evaluation of myocardial de's prognostic significance in patients with congestive heart failure due to nonischaemic dcm. materials and methods: delayed enhanced mri and myocardial tagged images were taken in patients ( ± yrs, male) referred for congestive heart failure due to nonischaemic dilated cardiomyopathy, whose left ventricular (lv) end-diastolic volume index > ml/m and lv ejection fraction < %. the patients were followed for a mean of ± months. results: myocardial de was present in patients ( %). in these patients, the average amount of myocardial de was ± % of lv myocardium ( - % of lv). myocardial axial thickening was ± % in the segments with myocardial de, and ± % without (p = . ). myocardial circumferential shortening was ± % in the segments with myocardial de, and ± % in without (p = . electron beam ct (ebct) in the detection of abnormal myocardial contrast enhancement in patients with recent and chronic myocardial infarctions t. vesselova, v. sinitsyn, s. ternovoy; moscow/ru purpose: of the study was to determine the occurrence of low-density areas in the myocardium of the left ventricle (lv) in patients with myocardial infarction (mi) seen during non-invasive ebct coronary angiography. material and methods: patients ( . ± . years old) with ischemic heart disease and mi were included in the study. in patients with first myocardial infarction (mi) ebct was performed during first weeks after coronary event. in patients ebct was done months after an mi. ebct angiography and venticulography were done with imatron c- imager. results: low-density areas in lv myocadium were seen on ebct images in all patients with mi. in % of patients these findings were accompanied by myocardial thinning and in % cases intracavitary thrombi were present. in . % patients with transmural mi low-density myocardial regions were visualized on non-contrast images. in . % patients with mi subendocardial low-density zones were seen. results of ebct were in agreement with myocardial scintigraphy. characteristics of hypodense areas (hu values) in patients with recent and chronic mi were similar. calcifications and significant stenoses of coronary arteries were found in all patients with old mi, the mean (sd) agatston score was . ± . . in patients with subacute mi the mean agatston score was . ± . (ns). four patients from this group had coronary score = . conclusion: during ebct coronary angiography most of the low-density areas corresponded to sites of previous myocardial infarctions. some of these areas may correspond to hypo-perfused areas of ischemic, but viable myocardium. progression of coronary artery disease in relation to coronary stenosis morphology: quantitative coronary angiography analysis j. saponjski, m. ostojic, b. beleslin, v. vukcevic, m. nedeljkovic, s. stojkovic, a. dordjevic/dikic, i. nedeljkovic; belgrade/yu objective: the aim of this study was to analyze progression and regression of coronary stenosis in relation to stenosis morphology. the study group consisted of patients ( male, female; mean age ± years). coronary stenoses were analyzed by quantitative coronary arteriography including percent diameter stenosis (%ds) and changes in obstruction diameter between two arteriographies. according to the ambrose angiographic lesion morphology groups were identified: group i (n = )-simple concentric lesion morphology, group iia (n = ) -simple eccentric lesion morphology, group iib (n = ) -complex lesion morphology, and group iii (n = ) with complex and multiple coronary stenoses. the follow-up period between first and second arteriography were similar for all groups (i, ± months; iia, ± months; iib, ± months; iii, ± months; p = ns). results: progression of coronary artery disease was identified in %, %, % and % of lesions in ambrose groups i, iia, iib, and iii, respectively. on the contrary, regression of coronary stenosis was documented in %, %, % and % of lesions in groups i, iia, iib, and iii respectively. however, comparing the lesions with non-significant and significant stenosis (cut-off point % ds), progression of coronary stenosis was higher for the lesions with ds < % for all groups (except group iii where all the lesions were with ds > %), reaching statistical significance in group i (i, + . ± . vs - . ± . mm, p < . ; iia, . ± . vs . ± . mm, p = ns; . ± . vs . ± . mm, p = ns). conclusion: coronary lesion morphology of simple type was associated with lower rate of coronary stenosis progression. noninvasive the aim of this study was to assess coronary microcirculatory function in postmenopausal women. methods: we measured myocardial blood flow (mbf) with a n-ammonia pet scan at rest, during cold pressor testing (cpt) for an indirect measurement of endothelium-dependent vasomotion, and during dipyridamole hyperemia (endothelium-independent vasomotion) in postmenopausal women without coronary artery disease. young healthy women served as controls. results: rest-mbf and hyperemic-mbf did not differ between the young and the postmenopausal women (rest: . ± . vs. . ± ,. and dipyridamole: . ± . vs. . ± . ml/ g/min; ns). coronary flow reserve was similar between the two groups (the young women: . ± . , the postmenopausal women: . ± . ; ns). cpt induced a similar increase of rate-pressure product in the young and the postmenopausal women ( ± vs. ± beats/min/mmhg), cpt-mbf was significantly decreased in the postmenopausal women ( . ± . ml/ g/min) compared with the young women ( . ± . ml/ g/ min; p < . ). conclusion: pet with n-ammonia during cpt is a noninvasive imaging technique to detect mbf abnormalities, which associated with endothelial dysfunction in postmenopausal women. contrast-enhanced magnetic resonance imaging at true end-diastole to quantify reproducible transmural extent of myocardial hyper-enhancement y.-j. kim, b. choi, k. choe; seoul/kr purpose: to determine feasibility of contrast-enhanced mri (ce-mri) at true enddiastole (ed) free from limitation of time for inversion-recovery and trigger window for quantifying transmural extent of infarction. methods and materials: mri was performed in patients with myocardial infarction. cine imaging and ce-mri with same registered slices in short axis were performed. to allow true ed ce-mri, ecg synchronization should use two rrintervals for one acquisition of a segment of k-space by setting the heart rate to half that of the true heart rate. trigger delay time was adjusted to the rr-interval for imaging at ed and to the sum of rr-interval plus the time between r-wave and the end-systole (es) determined in cine images for imaging at es. results: wall thicknesses of the ed and the es ce-mri were greater than those of the ed and the es cine images ( . ± . mm > . ± . mm, . ± . mm > . ± . mm respectively). subendocardial hyperenhancement was detected in patients. among them, systolic wall thickening was observed with cine imaging in patients and the transmural extent of hyperenhancement measured on ed ce-mri decreased by - % on es ce-mri proportional to the degree of systolic thickening of the epicardially nonenhanced myocardium. transmural hyperenhancement in patients showed no difference in thickness between enddiastole and end-systole. conclusion: ce-mri at ed is constantly possible by using two rr-interval per acquisition of a segment of k-space and useful to avoid the variation in infart sizing with irreproducible cardiac phase other than end-systole in case of subendocardial infarction. comparison of computed tomography (ct) and magnetic resonance (mr) for screening of pulmonary veins (pvs) complications of cryotherapy for percutaneous ablation of atrial fibrillation (af) b. ghaye , d. szapiro , l.-m. rodriguez , c. timmermans , r.f. dondelinger ; liège/be, maastricht/nl purpose: to prospectively compare ct and mr for evaluation of potential complications, including stenosis or thrombosis of pvs, of cryoablation for af. methods and materials: patients underwent percutaneous cryo-ablation of af. cardiac spiral ct and mr were performed before the procedure, at hours, and at months following af cryo-ablation. ct was performed with spiral ct (pq , phillips, eindhoven, the netherlands) using mm thick slices reconstructed every mm, -s rotation time, ma and kv per rotation following iv peripheral injection of ml of % iodinated cm. mr was performed on a . t symphony va system (siemens, erlangen, germany) using a multi-slice t -weighted dark-blood turbo-spin-echo pulse sequence and bright-blood cine trufisp sequence. all ct and mr examinations were quantitatively and qualitatively read blinded to the location(s) of ablation. results: for ct, the mean diameters at la-pv junction and at cm from ostium were . ( . ( . - - ) . associated cardiac and extracardiac anomalies were evaluated on fetal echocardiography. postnatal diagnosis or autopsy were correlated with fetal echocardiography. results: in fetuses with right aortic arch, combined congenital cardiac diseases were as follows, tof ( ), vsd( ), and dorv with pulmonary stenosis ( ) . associated extracardiac anomalies were oligohydramnios( ), arthrygryposis( ), splenic cyst ( ), and shortening of long bones( ). on postnatal echocardiography, previously non-diagnosed vsd were found on two neonates and tga without vsd in one neonate. overall incidence of combined cardiac defects is about . %. conclusion: right aortic arch associates variable cardiac defects and extracardiac anomalies in about half of fetuses. prenatal diagnosis of right aortic arch must be followed further echocardiography and targeted fetal sonography for exact diagnosis. potential myocardial iron content evaluation by mr in β β β β β-thalassemia major patients treated by deferoxamine or deferipron m. galia, m. midiri, t. bartolotta, a. maggio, r. lagalla; palermo/it purpose: to evaluate the usefulness of mri to assess myocardial iron content in patients with β-thalassemia major treated by deferoxamine b mesylate (df) or deferiprone (l ) chelation therapy. method and materials: consecutive patients with β-thalassemia major ( treated by df and by l ) underwent, at enrollment and after one year treatment, heart mri ( . t unit) with an electrocardiogram-triggered gradient-echo t weighted sequence. liver mri was obtained at the same time in all patients. measurements of the heart to muscle signal intensity ratio (hsirs) were compared between the two groups and with laboratory parameters, liver iron concentration (lic) and liver to muscle signal intensity ratio (lsirs). results: hsirs were significantly increased in df (t = - . ; p = < . ) and l group (t = - . ; p = < . ) after one year treatment. no statistically significant difference in the values of hsirs was present between the two groups at the beginning (p = . ; t = . ) and after one year treatment (p = . ; t = . ). hsirs were inversely correlated to lic (r = . ; p = < . ) but not to ferritin levels (r = . ; p = . ). a positive correlation was found between the variation of hsirs and that of lsirs (r = . ; p = < . ), and a mild correlation (r = . ; p = < . ) was found between the γ-glutamyltransferase levels and the hsirs values. conclusion: mri is usefull to detect hsirs variations during iron chelation therapy. moreover it is non-invasive and easily repeatable. application of -iodine labelled -iodinepentadecanoic acid for detection of myocardial viability in patients with chd and resting regional lv dysfunction v. soukhov, i. savicheva, a. partsernyak, a. svistov; st.petersburg/ru purpose of this study was to assess -iodine labeled -iodinepentadecanoic acid ( i-pda) -synthetic radiolabeled free fatty acid with the potence for detection of myocardial viability. methods: patients ( male and female, mean age ± yrs) who had history of myocardial infarction and resting regional left ventricular dysfunction were examined. blood supply, regional myocardial oxidative metabolism and regional metabolic substrate utilization were assessed by spect at - and - min after i.v. injection of mbq with i-pda. echocardiography with low doses of dobutamine was used for measuring inotropic reserve. results: early i-pda spect demonstrated high specificity in diagnostics of perfusion defects (up to %). the delayed images showed foci of remaining higher activity in regions of ischemic, but viable myocardium. of a total of myocardial segments had abnormal resting wall motion. in of them no oxidative metabolism and inotropic reserve were detected, that indicated scarred myocardium. of them were considered to be viable as they demonstrated both oxidative metabolism and inotropic reserve. conclusion: i-pda as an agent for simultaneous perfusion and oxidative metabolism evaluation may be used for identifying myocardial viability. diagnostic value of this method in combination with low doses of dobutamine echocardiography even in the cases of severe myocardial metabolic damage is nearly equal to pet with fdg and should be applied for detection of viable myocardium. comparison of mri and nuclear medicine in detection and analysis of left ventricular infarction r.m. macmillan , t. vakhtangadze , m.r. rees ; philadelphia, pa/us, bristol/uk aims: to compare standard spect perfusion imaging with last contrast enhanced mri for the detection of infracted myocardium. materials and methods: patients ( female, male) from two centres with proven myocardial infarction by ecg, clinical and echo criteria underwent stress/ rest tc sestamibi gated spect scanning with a dual headed gamma camera and late contract enhanced mri on identical . tesla scanners in each centre using a protocol which imaged minutes after injection of . mmol/kg iv gadolinium. analysis of segments used a segment system, with a total of segments analysed in both imaging methods. results: segments demonstrated scars on mri and segments demonstrated scars on isotope imaging. comparison of isotope imaging to mri showed that segments who had scars on isotope scanning failed to show on mri whilst scars on mri were not demonstrated on nuclear imaging. the majority of these scars were small or sub-endocardial. in some cases these scars had islands of functioning myocardium within them. some scars seen as completely non perfused defects on nuclear imaging were demonstrated to be partial thickness scars on mri. there was no significant difference in the findings from each centre indicating reproducibility and transferability of technique and findings. mri was able to detect smaller scars than nuclear imaging and viable myocardium in proximity to scar tissue. prenatal diagnostics of obstructive heart lesions g. spanovic , i. jovanovic , v. parezanovic , a. zvezdin ; novi sad/yu, belgrade/yu purpose: aim of prenatal diagnostics of obstructive heart lesion is to make right decision about the way leading further pregnancy, and decision about the time, place and way of labor. sensitivity and sensibility of fetal echocardiography are very high -between and %. methods and materials: fetuses were examined in period of seven years. fetuses with the diagnosis of some obstructive lesions of the heart were investigated. control group was made of fetuses with normal echocardiograms divided into four groups according to gestational weeks. echocardiographic techniques used: m-mode, d and doppler techniques. results: examination were made between and weeks of gestation. all cardiac structures show linear growth with progression of pregnancy. both halfs of the fetal heart are the similar size. obstructive lesions of the left heart and lesions of the right heart were diagnosed. there is statistically significant difference in size of the left and right structures in fetuses with obstructive lesions according to each other and also the same structure of the normal fetal heart. conclusions: obstructive lesions of the left heart were diagnosed much more frequently ( %) than the obstructive lesions of the right heart ( %) in the examination material. left heart hypoplastic syndrome was dominated lesion- % of obstructive lesions of the left heart and % of all diagnosed obstructive lesions. difficult obstructive lesions are progressive, have poor prognosis and present one of the indications for abortion. background: encouraged by beautiful cardiac multislice ct (msct) images presented at radiology and cardiology meetings, and by the favorable preliminary reports on high diagnostic value, more and more radiologists consider starting up their own cardiac msct program. the quality of the examinations initially obtained then, however, often does not live up to the high expectations. procedure details: a common cause for insufficent image quality is the poor attention to detail during image acquisition. in fact cardiac msct depends on the skill and dedication of the ct-technician. as an international training center for cardiac msct, we have noticed some common mistakes likely made by less experienced operators. generally, the basic procedures issues tend to go wrong: e.g., positioning the patient, placing the ecg-electrodes, choosing appropriate filtering, applying basic cardiac anatomy and physiology knowledge, etc. we will provide a step-by-step "how-to" pictorial with tips and tricks for successful cardiac ct imaging, illustrated by many examples of "right and wrong". issues addressed include handling of patient and workflow, data acquisition, and standardized image post-processing. conclusion: a step by step approach to cardiac msct scanning prevents scan failure. tomographic imaging of sinus venosus defects with multidetector ct angiography (mdcta) and magnetic resonance imaging-angiography (mri-mra) j.c. hellinger, a. napoli, f. chan; stanford, ca/us learning objectives: . describe tomographic imaging techniques for assessment of sinus venosus defects (svd). . illustrate intra and extracardiac anomalies during mdcta and mri-mra for svd. background: svd with associated partial anomalous pulmonary venous return (papvr), accounts for - % of congenital heart anomalies. clinical presentation is nonspecific and thus, diagnosis relies on imaging. transesophageal echocardiography (tee) and digital subtraction angiography (dsa) have been established as standard for this purpose. however, both are invasive and not without risk. mdcta and mri-mra are alternative noninvasive means to image these patients. mri offers the additional benefit to assess shunt ratios with cine phase contrast (pc) sequences. in this exhibit, we highlight our experience with mdc-ta and mri-mra in exams having surgically proven svd-papvr. procedure details: five exams were performed on or channel mdct. contrast was injected at cc/sec, employing bolus trigger technique. two of the -mdct were retrospectively ecg-gated with . mm thickness. non-gated studies were acquired at . mm, with a pitch of . - . and rotation time of . - . seconds. sequences employed in the four mri-mra exams included ecggated t , balanced steady state free precession, pc, and d-spgr angiography. d post-processing employed volume rendering and maximum intensity projection techniques. conclusion: anatomic defect size and location; number of abnormal veins) and dynamic evaluation for svd-papvr corroborated tee, dsa, and/or surgical findings. we identified on all mdcta, a jet of contrast crossing the defect into the left atrium (ct shunt sign). our preliminary experience indicates tomographic imaging is a reliable noninvasive alternative to tee and dsa for assessment of svd-papvr. to sensitize radiologists to the application of reformatting and d-reconstuctions in cardiac ct which depict the coronary arteries in a manner consistent with standard catheter angiography in routine clinical practice. to demonstrate a step by step approach to acquistion, optimized reconstruction techniques, and read-out algorithms in the evaluation of the coronary vessels. background: recent advances in the temporal and spatial resolution of mdct have opened the door to the use of ct in routine cardiac diagnostics, which is evolving rapidly as a noninvasive method. ct coronary angiography has compared favorably with conventional catheter coronary examinations, however new reconstruction and visualization methods are continuously being developed and are often lacking in meeting the demands of the routine clinical setting. it is crucial that radiologists become attuned to the standard approach to the coronary angiogram used in clinical cardiology so that advantage is not lost in the competition to provide services. procedure details: in this educational module, we present a step by step assessment of coronary vessels based on d-reconstructions of isotropic mdct datasets with special consideration to projections and visualization techniques which correspond with the standards of clinical cardiology. optimization of time consuming d renderings of ecg triggered datasets as well as read-out algorithms are demonstrated in examples for normal coronary vessels in addition to representative cases of coronary artery pathology. the resulting effective temporal resolution is - msec (depending on heart rate and reconstruction algorithm). larger volumes can be studied with isotropic spatial resolution and higher temporal resolution. the capability to scan larger volumes increases the feasibility and the accuracy of this technique in the visualisation of coronary artery bypass grafts (cabg). the scan protocol to evaluate cabgs with -row msct is described. different types of conduits (arterial and/ or venous), artefacts (surgical clips) and distal anastomoses of the graft are illustrated with dsa correlation. furthermore, atherosclerotic graft's disease (stenoses and occlusion) as well as native coronary artery disease is displayed with dsa. conclusion: ecg-gated -row multislice ct coronary angiography with . s gantry rotation time provide improved image quality in the evaluation of cabg. significance objective: to compare the msct and pet results in studying the condition of myocardial blood supply and to decide advisability of using them together. materials and methods: patients with suspected diagnosis of coronary artery disease were examined by msct and pet. msct data was obtained using siemens volume zoom ct scanner. retrospectively ecg-gating image reconstruction was conducted. pet was performed on ecat-exact- (siemens) with n ammonia at rest and during pharmacological test with vasodilatators (adenosine, dipyridamole). results: the first group comprised ( . %) patients without stenoses in coronary arteries according to the results of msct. pet also didn't detect reduction in perfusion of myocardial ischemic genesis in these patients. the second group included ( . %) patients with low-grade (< %) stenosis of coronary arteries. in these patients pet at rest and during stress didn't reveal a reduction in perfusion of myocardium. invasive coronarography was not performed for the and groups. the third group included ( . %) patients with different grades of stenosis in coronary arteries, according to the msct. there was reduction in perfusion in the corresponding myocardial segments revealed during at rest and/ or stress-pet. there was invasive coronarography done for these patients, which confirmed the existence of stenosis. methods and materials: ten volunteers (m:f = : , mean age ) without ischemic heart disease and consecutive patients (m:f = : , mean age ) with ischemic chest pain underwent ct coronary angiography (sensation , siemens, germany). reconstruction was done around - ms from r peak and multiplanar reformation and volume rendering were included. myocardial bridge was determined by demonstration of tunneled segment dip into myocardium. invasive coronary angiography was performed in patients. results: twelve tunneled segments in lad were detected in two volunteers ( %) (n = ) and patients ( . %) (n = ). nine were located in middle lad ( in volunteers, in patients) and in distal lad (in patients). with to mm in length, to . mm in greatest depth. the cross-sectional areas of tunneled segments were % smaller than those of the just proximal and % smaller than those of just distal to the tunneled segments. calcification in lad was detected in volunteer and patients with myocardial bridge but not in tunneled segment itself. significant stenosis was detected in proximal lad of two patients and mild stenosis in one patient which were confirmed by invasive coronary angiography. five patients showed only myocardial bridging in lad without coronary arterial stenosis. conclusion: msct directly visualizes tunneled segment within myocardium and gives useful information about myocardial bridging and neighboring coronary arteries. multi-detector row ct for the assessment of atrial septal defect and pulmonary venous drainage in adults g. morgan-hughes, a. marshall, c. roobottom; plymouth/uk purpose: to evaluate retrospectively ecg-gated multi-detector row computed tomography (ct) for the full evaluation of secundum and sinus venosus atrial septal defects (asd), in adults, in comparison to trans-esophageal echocardiography (toe). materials and methods: twelve patients undergoing toe for further assessment of presumed asd, as a prelude to definitive treatment, also underwent retrospectively ecg-gated multi-detector row ct. the multi-detector ct scans were compared to toe for defect and pulmonary vein visualisation. secundum asd sizing; measurements, before possible device closure, were compared, and virtual angioscopy of the right atrium was evaluated for direct, three-dimensional defect visualisation. results: larger secundum asds (greater than . mm) were well visualised with multi-detector row ct, but smaller defects and sinus venosus defects were not. for maximal secundum defect size there was a mean difference between the techniques of . mm (limits of agreement minus . to plus . mm). overall there was moderate agreement for the defect and rim sizing measurements. pulmonary venous drainage was universally fully and correctly evaluated with multidetector ct, which compared favorably to toe. virtual angioscopic visualisation of secundum defects was possible in patients, but the images produced were considered of limited clinical value. conclusions: retrospectively ecg-gated multi-detector row ct offers alternative imaging of asd in adults, allowing visualisation of significant secundum defects, pulmonary venous drainage and defect sizing. further investigation, to establish the accuracy of defect and rim sizing measurements and to re-explore direct defect visualisation with advanced post-processing tools, is required. detection of necrotic myocardial segments using multi-slice computed tomography: comparison with nuclear medicine p. . mm slice width, . mm slice increment and ml of non-ionic contrast ( mgi/ml) administered with a delay scan of seconds. nm and cts datasets were reoriented perpendicular to the left ventricle long axis. segmental analysis was performed on segments within the short axis planes ( basal, mid-ventricular and apical segments). a positive nm finding was defined as a perfusion defect within a segment at both rest and stress. a positive ct finding was defined as a hypo-intensive region within a segment. statistical analysis was performed using a % confidence interval calculated by the exact binomial method. results: segments were evaluated on both nm and msct. nm detected necrotic segments whereas msct detected . there were true-positive, true-negative, false-positive and false-negatives findings. the sensitivity was . %, specificity . %, positive predictive value . % and negative predictive value . %. conclusion: msct showed high sensitivity and specificity for the detection of myocardial necrotic segments compared with nm. evaluation of myocardial bridging by multislice computed tomography p.m. carrascosa , c. capuñay , m. vembar , p. johnson , r. pissinis , j. carrascosa ; buenos aires/ar, cleveland, oh/us to determine the usefulness of multiphase reprocessing msct coronary angiography in the identification of myocardial bridging (mb) and the detection of related ischemia. methods: patients were studied. cts were performed in a -row ct scanner with x mm collimation, . mm slice increment and ml of non-ionic contrast. ten patients with mb diagnosed with msct were evaluated with nuclear medicine (nm). rest and stress studies were performed with mci technetium m sestamibi. segmental analysis were performed on segments within the short axis plane ( basal, mid-ventricular and apical) in both methods. positives nm findings were defined as a perfusion defect within a segment at both rest and stress. positives ct findings as hypo-intense region within a segment. results: all the mbs were placed in the lad. da detected cases, whereas msct only . the sensitivity was . % and the specificity . %. in cases the mb of the lad had greater diameter in the % phase and they were compressed in the % phase. six cases showed ischemia in the nm studies. there were true-negatives, true-positives and false-negatives findings. the sensitivity was . % and specificity %, positive predictive value was % and the negative predictive value was %. conclusion: msct can detect the presence of mb in coronary arteries. it is essential for this identification a multiphase reprocessing of the ct data and can also detect the presence of myocardial perfusion defects. to predict the local, gonadal, thyroidal and eye dose with and without different protection devices in retrospective-gated multi slice ct of the heart. material and methods: radiation dose was measured with an alderson phantom and a digital dose meter with and w/o an infradiaphragmatic lead drape, a cervical lead protection and an eye shield. measurements were done with the ionization chamber placed over the heart, uterus, thyroid gland and eyes. helical ct of the heart was performed with an aquilion msct (toshiba). the dose was determined with slice collimation and pitch between and . using constant voltages and current-time-products for retrospective gated ct. results: local dose over the heart ranged from to µsv. gonadal dose was determined with to µsv w/o lead protection and to µsv using a circular infradiaphragmatic lead drape, meaning a gonadal dose reduction of up to %. with use of protection devices the dose to the thyroids could be reduced by . % and % in terms of using an eye shield. conclusion: radiation causing gonadal dose in cardiac ct is predominantly extracorporal radiation and only in a lesser degree due to scatter radiation from inside the body. an effective dose reduction of % can be achieved by consequent use of a circular lead drape closely adapted to the inferior helical range. the use of organ adapted shields covering the thyroids or eyes mainly results in a decrease of the surface dose and is recommended as well for all patients receiving cardiac ct. images were acquired using a -row spiral ct scanner with . s gantry rotation time. in all patients detailed clinical, ecg, us and scintigrafic (when available) data were collected in order to identify the presence of mi. when a patient with mi was found, mdcta images were analyzed on both axial and multiplanar reconstruction in order to confirm the presence of an infarcted area. results: our study population included patients who previously underwent coronary percutaneous transluminal angioplasty (pta) and stent placement (a total number of stents) and patients with previous by-pass surgery. after clinical assessment, mi was found in patient ( septal, apical, anterior wall of left ventricle, infero-septal). mdct was able to detect the presence of mi in / cases ( . %), showing a regional hypoattenuated area and wall thinning. in all cases, localization and extention of ischemic area were perfectly correlated with clinical known data. in two patients with sub-endocardial involvement, mi could was not detectable with mdct. in one patient the presence of motion artefacts affected image quality and myocardial scar wasn't visualized. the presence of mi is usually well depicted with retrospective ecg gated mdct. mdct coronary angiography: optimization of the image reconstruction phase y. nagatani, r. takazakura, n. nitta, m. takahashi, k. murata; otsu/jp purpose: to investigate the relationship between the heart rate and the optimal image reconstruction phase in mdct coronary angiography. material and method: patients underwent cardiac mdct ( das) (aquilion , toshiba, japan) to obtain coronary angiography. scan data were subsequently reconstructed at different kinds of phases in ecg wave. these methods can be classified as follows according to the used wave in ecg; ).the center of the reconstruction phase is positioned at the peak of t-wave (method t-p), the end of t-wave (method t-e) and the midpoint of descending curve of t-wave (method tm). ).the end of the reconstruction phase is positioned at the peak of p-wave (method p). ).the center of the reconstruction phase is positioned using nterval (method r , , , , , ) . all patients were classified into groups according to their heart rates (hr) (group ; hr < (n = ), group ; < hr; < (n = ), group ; hr < (n = )). two radiologists assessed image quality in blind fashion and scored as follows ( :good, :fairly, : bad). bonferroni/dunn test was performed for statistical analysis. results: in group , significantly better image quality was achieved in method p, rr , rr than other methods. among them, highest image quality was obtained by method p. the best result was obtained by method p in group and tm in group . conclusion: optimal image reconstruction phase in mdct coronary angiography shifts from late diastolic phase to late systolic phase as heart rate increases. assessment of aorto-coronary bypass grafts patency by detector rows computed tomography g. two out of five tumors (right atrium lipoma and aortic valve fibroelastoma) were discovered in a routine msct study of coronary arteries and left ventricle myxoma in an elective coronarography. atypical left ventricle myxoma with mitral stenosis and epicardial cyst were primary visualized in transoesophageal echocardiography. results: both methods allows to evaluate tumors size, localization and structure. the biggest advantage of echocardiography was aknowledged possibility of evaluation of lesions mobility while the biggest advantage of msct was possibility of both d and d imaging in arbitrary planes using multiplanar reformations with evaluation of tumor relations towards adjacent tissues and simultaneous visualisation of coronary arteries. conclusions: msct and echocardiography (tte and tee) are complementary methods in diagnosis of primary cardiac tumors. usefulness of -slice multidetector row computed tomography (msct) in assessment of dilated cardiomyopathy s. hosoi, t. mochizuki, t. haraikawa, j. funada; ehime/jp purpose: the purpose of this study is to show the potential benefits of -slice msct for the diagnosis of dcm. we studied consecutive patients, including dcm, coronary artery disease (cad), and patients with non-cardiac disease as a normal control. dcm was diagnosed on the basis of the clinical data by cardiologists. the msct system used in this study was a lightspeed (ge medical systems) with available gantry rotation speeds of . sec. all cardiac images reconstructed were divided into phase data sets in a r-r interval on ecg. end-diastolic (ed) and end-systolic (es) phase could be visually extracted, and the lv wall thickness and volume was measured. moreover, the segmental lv wall motion was evaluated by the cardiac movie image which was reconstructed using the phase data. results: image quality of mdct were considered sufficient for this analysis. concerning about the rate of coronary artery stenosis, dcm group were significantly less than cad group (p < . ). in dcm group, both lv wall thickness of ed and es were smaller than other groups (p < . ). lv volume between dcm and cad group had no significant difference. lv wall motion in dcm group had a tendency to be diffusely hypokinetic. conclusion: using -slice msct, lv function and coronary artery stenosis was able to be evaluated and distinguished the features of dcm and cad. this study revealed a potential ability of mdct as a useful method for the diagnosis of dcm. background: a variety of pulmonary complications occur in bone marrow transplant (bmt) recipients and are a major cause of morbidity and death. imaging findings: hrct is useful in detection of pulmonary abnormalities, but these findings are generally non-specific. these complications, which reflect the immunologic status of the patients, occur in three phases. this pattern can be used to interpret ct scans. the neutropenic phase (up to weeks after bmt) is characterized by fungal infections, alveolar haemorrhage, pulmonary oedema, and drug reactions. at ct, fungal infections like angioinvasive aspergillosis appear as nodules surrounded by a halo of ground-glass attenuation; alveolar haemorrhage and drug reactions, as bilateral areas of ground-glass attenuation or consolidation. the second phase ( weeks to days after bmt) is dominated by cytomegalovirus pneumonia, which appears as multiple small nodules with areas of consolidation or ground-glass attenuation, and pneumocystis carinii pneumonia again mostly as ground-glass attenuation. the late phase (more than days after bmt) is characterized by bronchiolitis obliterans revealing bronchial dilatation and a mosaic pattern of attenuation, bronchiolitis obliterans with organizing pneumonia (boop), the ct findings usually being patchy consolidation or ground-glass attenuation and chronic graft-versus-host disease. conclusion: if ct findings are considered in relation to the time elapsed after bmt, diagnostic options can be narrowed sufficiently to enable accurate diagnosis. small pulmonary nodules: an easy approach to the differential diagnosis j. rimola, x. gallardo, e. castañer, j.m. mata, a.m. quiles, p. bermúdez; sabadell/es learning objectives: to know the anatomy of the secondary pulmonary lobule, which is essential to the interpretation of high-resolution ct (hrct). to learn an easy way to approach the differential diagnosis of small pulmonary nodules. background: a wide variety of pathologies may present with small pulmonary nodules, which are defined as rounded opacities smaller than cm in diameter. differences in appearance, distribution and attenuation of nodules provide useful clues for correct diagnosis. helical ct and hrct enable correct identification and characterization of small nodules. imaging findings: we illustrate different examples of multinodular lung diseases in an easy-to-understand way. a simple algorithm is used to help in the differential diagnosis. we classify nodules according to distribution (centrilobular, perilymphatic or random) and show the most representative cases of each pattern. we present a wide variety of entities, including: infections such as tuberculosis (endobronchial spread or miliary tb), nontuberculous mycobacteria, fungus; sarcoidosis; tumors (lymphangitic carcinoma, hematogenous metastases); silicosis and coal worker's pneumoconiosis; hypersensitivity pneumonitis; cryptogenic organizing pneumonia; histiocytosis; etc. conclusion: an easy way to assess multiple nodular opacities in the lung is useful to reach an accurate diagnosis. differential diagnosis of unilateral hyperlucent lung m. nishino , k. hayakawa , h. hatabu ; boston, ma/us, kyoto/jp learning objectives: to review differential diagnosis of unilateral hyperlucent lung, understand pathological mechanisms causing the findings, and to be familiar with the imaging findings and clinical manifestations characteristic in each condition. background: unilateral hyperlucent lung on chest radiograph is seen with various pathological conditions. the conditions include both acute and chronic processes, and thus need accurate and prompt diagnostic evaluation. the findings on chest radiograph can provide clues to differentiate various causes of this condition and help to achieve proper management. imaging findings: we present cases of unilateral hyperlucent lung, emphasizing specific imaging findings along with characteristic clinical manifestations. the cases include proximal interruption of pulmonary artery, pulmonary hypoplasia, swyer-james syndrome, congenital lobar emphysema, foreign body aspiration, and bochdalek hernia. findings on chest ct which can confirm or support the diagnosis are also reviewed and discussed. conclusion: familiarity with the characteristic imaging findings of unilateral hyperlucent lung is essential for radiologists to make accurate diagnosis for appropriate patient management. multislice ct of primary and multisystemic pulmonary vasculitis: a patternbased approach to differential diagnosis k. marten , m. prokop , p. schnyder , e.j. rummeny , c. engelke ; munich/de, utrecht/nl, lausanne/ch . clinical and radiologic features of these tumors are often indistinguishable from those of malignant tumors. however recognition and early diagnosis of these lesions may allow for conservative treatment and excellent patient outcome. the aim of this study is to illustrate the ct findings of variable benign tumors of tracheobronchial tree and correlate with the pathologic findings. imaging findings: this study included patients with benign tracheobronchial tumors which were confirmed histologically by surgical resection (n = ) or bronchoscopic biopsy (n = ). the tumors were classified the anatomic locations of the lesions into trachea (n = ), bronchus intermedius (n = ), lobar bronchus (n = ), and segmental bronchus (n = ). pathologic diagnoses were hamartoma (n = ), leiomyoma (n = ), lipoma (n = ), schwannoma (n = ), amyloidoma (n = ), inflammatory polyp (n = ), and bronchial adenoma (n = ). ct demonstrated all tumors confined in the tracheobronchial lumen. of bronchial tumors occluded the bronchial lumen completely, resulting in distal parenchymal collapse. to enhance the limitations of chest x-ray and axial chest ct slices in estimating the volume of pleural effusions. this interactive presentation will allow the reader to test himself, comparing his own subjective estimation to the real volume computed from d shaded surface displays (ssd). to outline the usefulness and ease of post-processing with spiral ct to obtain a true assessment of the pleural fluid volume. background: assessment of the volume of pleural effusions is very imprecise both on chest x-ray and axial chest ct slices. the amount of fluid may be required by clinicians for therapeutic purposes. d ssd of the effusion provides rapid computation of its volume. in this exhibit we will recall the physiopathologic mechanisms involving the preferential locations of pleural effusions. we will show pleural effusions of various volumes on chest ct along with the corresponding chest x-rays and the precise volume of the effusion computed from d reconstruction. procedure details: various cases of free or collected pleural effusions on spiral chest ct (ct twin and mx idt), are presented, including coronal and sagittal reformations. ct images stand along with the corresponding chest x-ray, obtained in the upright and supine position, and with the computed volume of the effusion using d reconstruction. conclusion: pleural volumetry is readily available using spiral ct data recontruction. it should be more often used, to assess the amount of fluid to withdraw with thoracocentesis, before drainage or for monitoring in intensive care unit patients. lung consolidations in various clinical entities: clinico-radiologic correlation presented in an interactive cd-rom in html format s. mylona, s. tandeles, l. thanos, s. lyra, p. ellinas, n. mpatakis; athens/gr learning objectives: to present lung consolidations as found in ct and plain radiographs in various clinical entities. to give a differential diagnosis based on clinical and radiologic features. background: radiologists often encounter lung consolidations in plain radiographs and lung ct. there is a broad spectrum of clinical entities, which either present as lung consolidations or involve lung consolidations. clinical information and radiologic features help to approach the right diagnosis. procedure details: cases are presented in a question and answer format using html resources in an interactive cd-rom. reference is made to proper diagnostic work up and to other relevant clinical information. histopathological confirmation is presented where it is needed. conclusion: radiologists must be trained to deal with patients presenting with lung consolidations using clinical and radiological features. hrct patterns of cardiogenic pulmonary edema e. chambrier, s. zafatayeff, y. badachi, p.a. grenier, c. beigelman-aubry; paris/fr learning objectives: to be familiar with typical and atypical hrct appearances of pulmonary edema based on patients. to emphasize the role of hrct in suggesting this diagnosis in the setting of acute dyspnea, especially if pulmonary embolism is suspected. background: pulmonary edema, a frequent cause of acute dyspnea, can be clinically difficult to detect. hrct is crucial in suggesting this diagnosis, particularly when pulmonary embolism is suspected. actually, bolus intravenous injection of iodinated contrast in case of pulmonary edema may worsen heart failure and lead to patient death. imaging findings: the hrct signs of pulmonary edema are usually divided into interstitial and alveolar patterns, and both can be associated. classic signs of interstitial edema, which accounts for % of cases, are peribronchial and peribronchovascular thickening, septal lines, ground glass opacity, pleural effusion, cardiac enlargement and pulmonary veins dilatation. alveolar edema is characterized by bilateral, patchy or coalescent areas of airspace consolidation, predominantly basal and perihilar. butterfly pattern is found in % of cases. atypical patterns are due to preexisting lung or heart disease, such as right upper lobe edema related to mitral regurgitation, or heterogeneous distribution of edema due to preexisting lesions of emphysema or constrictive bronchiolitis. highly selected cases will be used to illustrate typical and atypical patterns. the varied hrct appearances of cardiogenic pulmonary edema have to be recognized in order to delay the performance of angioct of pulmonary arteries if pulmonary embolism is suspected. smoking-related interstitial lung diseases: radiologic-pathologic correlation a. hidalgo, t. franquet, a. giménez, r. pineda, m. madrid; barcelona/es learning objectives: . to review the radiological findings of smoking related interstitial lung diseases and their pathologic correlations. . to provide some radiological clues for the correct diagnosis. background: interstitial lung disease (ild) represent a heterogeneous group of lung disorders of known or unknown cause. recently, it has been appreciated that cigarette smoking is related to the development of several ilds including desquamative interstitial pneumonia (dip), respiratory bronchiolitis-associated interstitial lung disease (ribld), pulmonary langerhans'cell histiocitosis (plch) and idiopathic pulmonary fibrosis (ipf). procedure details: we reviewed the plain radiograph, ct, and hrct findings of four intersitital lung disorders that have been linked to smoking. several examples of the radiological forms of srild and their differential diagnosis are provided. we will also show how the radiologists can get the diagnosis through some clinical and radiological clues. the link between several interstitial lung disease and smoking has been recently indicated. radiologists have to be aware of these entities and to suggest a diagnosis as accurate as possible by the knowledge of clinical context and the radiological features. the spectrum of ct appearances in pulmonary amyloidosis a. aylwin, p. gishen, a. nicholson, s. copley; london/uk learning objectives: to review the ct appearances of pulmonary amyloidosis. background: amyloidosis has three main histopathological subgroups, with typical imaging characteristics: ) tracheobronchial disease with plaques, nodules or circumferential thickening of the trachea or segmental airways; ) nodular parenchymal disease with single or multiple nodules demonstrating sharp lobulated margins, spiculated masses simulating tumours, and a rare form associated with sjögren's syndrome and lymphocytic interstitial pneumonitis (nodules and lung cysts); and ) diffuse alveolar septal disease showing ground glass opacification, interlobular septal thickening, and traction bronchiectasis on hrct. it is often possible to divide cases into localized amyloidosis with tracheobronchial deposition or nodular disease, or the systemic type with diffuse parenchymal disease or lymphadenopathy on the imaging findings, but cases demonstrating overlapping features are not uncommon. procedure details: the ct appearances of cases of biopsy-proven pulmonary amyloidosis will be demonstrated. the spectrum of ct appearances of pulmonary amyloidosis of both localised and systemic types will be demonstrated. the ct features of localized and systemic disease may overlap. thoracic conclusion: incidence and imaging features of these complications will be illustrated. paraquat poisoning of the lung: a study of patients with paraquat intoxication for years a. ichinose , k. kimura , m. tsuboi , h. saito , t. ishibashi , s. takahashi ; miyagi/jp, akita/jp learning objectives: to analyze patients who had taken paraquat intoxication and to compare those with % paraquat intoxication to those with % paraquat-diquat intoxication, especially with respect to the change of their chest x-rays and ct. background: paraquat is a widely used herbicide that has toxic effects on the lungs, liver, and kidneys. paraquat has been known to induce acute pulmonary fibrosis, causing progressive respiratory failure and death. in japan, % paraquatdiquat product has been used since , in place of % paraquat product,. however, there is no report that compares the influence these products have on the lung. procedure details: there were patients from whom chest x-rays were taken, and of those ( . %) had abnormal findings, which were composed of ground-grass patterns, reticulogranular patterns, pulmonary edemas, cardiomegalies and pulmonary congestions, pneumothoraxs, and mediastinal emphysema. pulmonary edema was found only in the patients with % paraquat intoxication; in all cases, the plasma paraquat concentration was much higher than others. all reticulogranular patterns were found a few days later, and those in the patients with % paraquat-diquat intoxication were potentially reversible. conclusion: % paraquat product has more influence on the lung than % paraquat-diquat product. pulmonary fibrosis caused by % paraquat-diquat product is potentially reversible. thoracic parenchymal abnormalities, enlarged lymph nodes, pleural effusion, and pleural thickening were evaluated. in patients who underwent surgical biopsy or autopsy, the ct-pathological correlation was performed with the actual specimens. imaging findings: on the ct scans, abnormal findings were seen in patients ( . %). the ct findings consisted of ground-glass attenuation (n = ), centrilobular nodules (n = ), thickening of bronchovascular bundles (n = ), and consolidation (n = ). these abnormalities were predominantly seen in the peripheral lung parenchyma (n = ). pathologically, these findings corresponded with atypical lymphocytes infiltration along the interstitium and the alveolar spaces. pleural effusion and enlarged lymph nodes were found in patients and patients, respectively. conclusion: the ct findings in patients with adult t-cell leukemia/lymphoma consisted mainly of ground-glass attenuation, centrilobular nodules, and thickening of the bronchovascular bundles in the peripheral lung. these ct findings, though non-specific, are considered as suggestive of thoracic involvement in patients with adult t-cell leukemia/lymphoma. high resolution ct imaging of the lung for patients with primary sjögren's syndrome c. lohrmann, m. uhl, k. warnatz, n. ghanem, e. kotter, o. schaefer, m. langer; freiburg/de purpose: to assess pulmonary abnormalities in patients with primary sjögren's syndrome using high-resolution computed tomography (hrct). the hrct scans of patients over a ten-year-period with diagnosis of primary sjögren's syndrome were retrospectively reviewed regarding the presence, extension and distribution of pathological findings. results: patients ( . %) showed pathological findings and in ( . %) the hrct scan was normal. a predominance of abnormalities in the lower lobes and subpleural areas was detected. bronchiectasis, thin-walled cysts and small pulmonary nodules in patients ( . %), ground-glass attenuation and emphysema in patients ( . %), interlobar-septal thickening in patients ( . %), honeycombing in patients ( . %), bronchial wall thickening and tree-in-bud pattern in patients ( . %), mosaic perfusion in patients ( . %), and architectural distorsion in patients ( . %). airspace consolidation, air trapping, large nodules ( - mm) and masses (> mm), mediastinal lymph node enlargement (> mm) and free pleural fluid were seen each in patient ( . %). in of patients ( %) with thin-walled cysts areas of ground-glass attenuation were detected. conclusion: hrct seems to be contributive to the characterization of the wide variety of lung abnormalities in primary sjögren's syndrome. airway disease alone or in association with the presence of varying degrees of interstitial disease represent the main findings in accordance with earlier reports. unexpectedly, almost half of the patients have thin-walled cysts on the hrct scans, which etiology is unclear but could be associated with areas of ground-glass attenuation indicating lymphocytic interstitial pneumonia. investigation of the bulky hilum: how accurate are radiologists at detecting hilar pathology on the chest radiograph? s. desigan, d. murray; london/uk purpose: to determine the accuracy of radiologists in detecting hilar pathology on chest radiographs. materials and methods: a retrospective study was performed, analysing the chest radiograph and computed tomography (ct) findings of patients, all of whom had initially been referred for thoracic ct as part of the investigation for a "bulky hilum" as detected on chest radiography. radiologists, comprising consultants and specialist registrar trainees who were blinded to the initial ct findings, were asked to assess the chest radiographs for the presence or absence of a hilar mass and their recommendations for clinical follow-up in each case were recorded. they were then scored for accuracy as compared to the thoracic ct results. the results showed marked variation between observers in the two groups. the participants had an accuracy of %, sensitivity of % and specificity of %. positive predictive values of registrars and consultants were % and % respectively. negative predictive values were % for registrars and % for consultants.the group as a whole detected only out of hilar masses confirmed by ct. the wide range of responses demonstrates the very subjective nature of interpretation of the lung hila on chest radiographs, and the difficulty involved in correctly identifying or excluding hilar masses on chest x-rays. one of the most useful aspects of this study has been to provide feedback to individuals for educational purposes and personal audit. b d e f a g m. paslawski, w. krupski, k. krzyzanowski, j. zlomaniec; lublin/pl purpose: the aim of the study is to evaluate the diagnostic value of hrct in revealing small bronchiectases and bronchiolectases in patients with previously diagnosed pneumoconiosis. the study comprises a group of patients, men and women, aged between and years, with pneumoconiosis in which hrct was performed. hrct was performed in all patients in the prone position, from the level of the apices to the level of the diaphragm. the collimation of the scans was mm, with cm intervals between sections. the hrct examinations were assessed retrospectively by two radiologists, and the presence and character of bronchiectases were noted. results: bronchiectases were seen in patients ( %). in of them the "signet-ring sign" and "tram track sign" were seen, with lack of normal tapering. bronchi were visible in peripheral lung areas, within cm from the parietal pleura in patients. patients had varicose bronchiectases with the typical "string of pearls" appearance. in patients, cystic bronchiectases were seen, presenting with the picture of "cluster of grapes". conclusions: bronchiectases is very often seen in patients with pneumoconiosis, especially in advanced stages. hrct is the modality of choice to diagnose the presence of small bronchiectases and bronchiolectases. typical hrct patterns include "signet-ring sign", "tram track sign", lack of normal tapering, and visible bronchi in peripheral lung areas, within cm from the parietal pleura. in varicose bronchiectases, the "string of pearls" appearance, and in cystic bronchiectasis, the "cluster of grapes" appearance are typical. introspective background: spns occur on in chest radiographs with % being due to lung cancer. conventional imaging is fairly effective in determining the nature of the spn, although - % remain indeterminate following ct scan. the spn will often have needle biopsy either via bronchoscopy or percutaneously which have a recognised complication rate of - %. depreotide is a tc- m labeled analogue of somatostatin with affinity to receptors types , and . various tumours including lung cancer, breast cancer, lymphoma and neuroendocrine tumours express these receptors. we undertook a retrospective review of one years' referrals for depreotide imaging comparing the results with subsequent biopsy or surgical histology where available. additionally we assessed if the referring clinical question had been answered. results: depreotide studies performed, with showing increased uptake within the spn. patients ( %) with positive studies had had a previous indeterminate lung biopsy thus enabling planning of further biopsy or wedge resection with frozen section. depreotide and histology agreed in all patients who had subsequent histology of the spn ( malignant, benign). further biopsy was avoided in out of patients with negative studies. specific answers were given to the clinical referring question in cases. conclusion: depreotide effectively determined the nature of the spns with ability to avoid biopsies in the % of patients with benign disease and to prompt further invasive investigation following an indeterminate biopsy. pleural invagination in progressive massive fibrosis (pmf) in silicosis and mixed dust pneumoconiosis h. arakawa, k. honma, h. shida, h. mori, y. saito; tochigi/jp purpose: invagination of the pleura often accompanies progressive massive fibrosis (pmf) in pneumoconiotic lung. to elucidate the relation between pmf and the pleura covering them, we conducted a retrospective study comparing ct and autopsy findings in silicosis and mixed dust pneumoconiosis (mdp). the study group was an autopsy series of consecutive patients with complicated silicosis or mdp. ct images were examined to determine the location, shape, composition and size of pmf, distance between the lesion and the covering pleura, presence of pleural thickening, a band-like structure between the lesion and pleura, and pleural effusion. autopsy data were reviewed for the presence of pleural thickening and invagination and were compared with the ct findings. results: we identified pmf lesions. pleural invagination was found in ( . %) pmf and was always associated with pathologically determined pleural thickening. ct images showed pleural thickening in ( . %) and invagination in ( . %) lesions. lesions were associated with ipsilateral pleural effusion (p < . ). statistical analysis revealed pleural invagination was associated with pleural thickening (p < . ) and proximity of the lesion to the pleura (p < . ) but not with radiographic profusion of pneumoconiotic opacities, or the shape, composition, size of pmf (p > . ). pmf were radiographically identical to rounded atelectasis. conclusions: fibrous thickening of the pleura and invagination into the adjacent pmf are not uncommon in advanced silicosis and mdp. some lesions resemble rounded atelectasis. ct is beneficial in assessing these pathologic features. imaging the aim of this teaching exhibit is to illustrate a spectrum of usual and unusual cystic diseases involving the lung, and to evaluate the efficacy of plain radiographs and computed tomography (ct) imaging in the diagnosis and management of these conditions, with emphasis in high resolution ct (hrct). we retrospectively reviewed the imaging findings of patients with diffuse cystic lesions involving the lung from our data base of thoracic pathology. a cystic lesion was defined as an air-containing, well-defined space, variable in size. the pertinent embryology and pathologic basis of the radiographic findings are discussed. pitfalls, diagnostic difficulties and differential diagnoses are emphasised. results: specific topics addressed include diffuse cystic diseases of the infancy (bronchopulmonary dysplasia, pulmonary interstitial emphysema, types i, ii, and iii congenital cystic adenomatoid malformation, chronic eosinophilic pneumonia, and pneumatoceles), and diffuse cystic diseases of the adulthood such as honeycomb lung (secondary pulmonary fibrosis), langerhans cell histiocytosis, pulmonary lymphangioleiomyomatosis, tuberous sclerosis, and lymphoid interstitial pneumonia. we also include pulmonary diseases simulating cysts such as congenital diaphragmatic hernias, diffuse bronchiectasis, and emphysema. conclusions: hrct is an adequate imaging modality to assess diffuse cystic disease of the lung, allowing an excellent visualisation of the cysts as well as other secondary findings (distribution, size, location, and wall thickness). because many of these disorders have characteristic imaging appearances, this exhibit will help practising radiologist to better understand, recognise and differentiate cystic diseases involving the lung. beware the normal chest x-ray in suspected lung cancer: a pictorial review of the potential pitfalls a.k. basu, k. jeyapalan, j. entwisle, r. bhatt; leicester/uk purpose: to demonstrate that a standard pa chest x-ray can be falsely reassuring in patients where there is a high index of clinical suspicion for lung cancer. we reviewed the patients with suspected lung cancer whose chest x-rays were radiologically normal and had further ct cross sectional imaging. emphasis was placed on the hidden review areas on a standard pa chest x-ray, e.g. the mediastinum, the apices, behind the diaphragm and behind the heart. we present a pictorial review of these hidden areas and illustrate the potential lesions not seen on a "normal" chest x-ray. results: ct can often demonstrate lesions not visible on chest x-ray. conclusion: a normal pa chest x-ray may be falsely reassuring in patients suspected of having lung cancer. therefore in patients with a high index of clinical suspicion a ct should be carried out despite the normal pa chest x-ray with careful review of the hidden review areas. should a chest radiograph be requested in all cases of febrile neutropenia admissions? a study in a university hospital a.v. acharya, a.k. burnett; cardiff/uk purpose: to evaluate the clinical utility of obtaining chest radiographs, in the absence of chest symptomatology, in patients admitted with acute neutropenic sepsis. methods: prospective study of in patients with febrile neutropenia admitted to the haematology ward was carried out in this university hospital over a period of months. symptoms & signs, chest x-ray findings and changes in management because of these findings were assessed in these patients. we also conducted a questionnaire survey of haematologists of south wales regarding obtaining chest radiographs in these patients. results: chest radiographs were obtained in . % of the patients at the time of diagnosis of febrile neutropenia but only . % of the patients had chest signs/ symptoms. if chest signs/symptoms were present, . % had abnormal chest x-rays and this influenced change in clinical management. in the group where chest signs/symptoms were absent, radiographs were normal and no change in management resulted from a chest radiograph. the survey of haematologists suggested that majority of them ( %) requested chest radiographs in acute neutropenic sepsis irrespective of presence or absence of chest signs or symptoms. in neutropenic patients with no chest signs/symptoms, a chest radiograph on admission is not likely to be abnormal and is unlikely to change the management. on the basis of our study we suggest that the chest radiographs in these patients in the absence of clinical signs or symptoms, are of little value. this would however not exclude the possibility that other imaging approaches would be of clinical value. low to evaluate the usefulness of low dose thin-section ct by multidetector-row ct (mdct) for lung cancer screening in both the determination of malignancy or benignancy and the differential diagnosis. methods and materials: cases with pathologically proved solitary pulmonary nodules, which sizes were less than cm in diameter, were entered into this study. after the routine full dose thin-section ct ( . mm collimation, fov cm, pitch : , kvp, -ma/rotation, . second/rotation, high-spatial frequency algorithm), additional two kinds of low dose helical thin-section ct ( . mm collimation, pitch : , -(in initial cases), -(all cases), -(in remaining cases) ma/rotation) were scanned using a mdct scanner (lightspeed qxi, ge, milwaukee, wi). using roc analysis, an observer performance study, in which three observers indicated the confidence level for the determination of malignancy or benignancy for each nodule, was done. in addition, three observers recorded the final diagnosis of each case. accuracies of the final diagnosis were compared by mcnemer's test. in the determination of malignancy or benignancy, there was no significant difference among these four kinds of thin-section ct (full dose, -, -, and -ma/rotation; mean az = . , . , . , and . , respectively). accuracy of the final diagnosis by low dose thin-section ct ( -, -, and -ma/ rotation; %, %, and %, respectively) was significantly lower than that of full dose thin-section ct ( %) (p < . ). conclusion: efficacy of low dose thin-section ct by mdct is comparative to that of full dose thin-section ct in determining whether small nodules are malignant or not. lung cancer screening using low-dose spiral ct: preliminary results in asymptomatic smokers s. giunta, m. crecco, f. facciolo, l. carpanese, v. cilenti, m. caterino, p. visca, m. mottolese; rome/it purpose: to evaluate the feasibility of lung cancer screening using low-dose spiral ct and to present initial baseline data from a non randomized screening trial using low-dose spiral ct. methods and materials: asymptomatic smoker volunteers, aged years or older who had smoked packs-year or more, underwent baseline low-dose multislice spiral ct (volume zoom siemens) of the chest without contrast material enhancement following the elcap protocol. annual repeat screening was performed on participants. lesions up to mm were deemed non suspicious and low-dose ct was repeated after months. results: at baseline, non calcified nodules were identified in participants. nodules were larger than mm. biopsy of lesions revealed nsclc in cases ( adenocarcinoma, squamous cell carcinoma, large cell carcinoma, adenoid cystic carcinoma). of these were stage i ( . %); stage ii ( %); stage iii ( %) and one stage iv ( . %). were benign lesions ( hamartomas, lymph node, fibrosis). in cases the pet was negative, in cases the nodules resolved after antibiotics; participants refused biopsy. conclusion: low-dose spiral ct seems to be a promising method for screening lung cancer. our preliminary cancer screening results with low-dose spiral ct, demonstrated a prevalence of asymptomatic cancers in . % of a smoking population including a high proportion of early tumor stages. pathological mismatched findings of specimens between ct guided biopsy and resection in noguchi's classifications of small peripheral lung adenocarcinoma s. yoshida, h. ue, s. itou, y. murata; nankoku/jp purpose: we sometimes experience mismatched pathological findings of specimens between preoperative ct guided biopsy and resection in the noguchi's classification of small peripheral lung adenocarcinoma. in this study we discuss the mismatched pathological findings in each case and explored the possibility of reducing the incidence of mismatch. we selected cases, in which preoperative ct guided biopsy and consecutive resection were performed. tumors were peripheral lung adenocarcinoma of cm or less in diameter. the final diagnosis by resected specimen of these cases were type b, type c and type f peripheral adenocarcinoma. we compared hrct findings of them to identify the ct density in each tumor. results: though the final pathological findings show type c tumors, the specimen by ct guided biopsy show only type a or b tumors. further more, though the final pathological findings show type f, not having a replacement growth pattern, the specimen also show type a or b, having a pure replacement growth pattern. conclusion: pathological mismatched findings between ct guided biopsy and resection were observed. additional ct image comparison between mediastinal and lung window settings gave us probable information about the tumors having replacement growth patterns or not. before performing ct guided biopsies, image recognition about the solid density helped us choose the most suitable biopsy site. with more thorough information derived from hrct and biopsy on the types of tumors patients have, physicians can offer patients more accurate prognosis. purpose: to analyze high-resolution ct (hrct) findings in asthmatics and control subjects, and to evaluate the relationship between hrct findings and pulmonary function tests in asthmatic subjects. to try to evaluate the correlation between the bronchial wall thickening (bwt) with pathologic findings and reflect the thickness of epithelial basement membrane on basis of the hrct findings. materials and methods: using hrct, the author analyzed the ratio of bwt, the frequency of bronchial dilatation, the extent of air trapping and centrilobular nodules. in addition, the author assessed hrct findings of asthmatic patients for correlation with pulmonary function tests and then obtained bronchoscopic biopsy at the second divisional branch of patient among the asthmatics. the author measured the thickness of the epithelial layer and basement membrane after h-e stain and then statistically compared with bwt on hrct. results: among the total asthmatics, bwt on hrct showed significant correlation with fev (p = . ) and tlc (p = . ). pefr (p = . ) shows significant correlation with the air trapping on hrct statistically. among the patient of asthmatics, bwt on hrct shows statistically significant correlation with basement membrane thickness on pathology, but not with epithelial layer thickness on pathology (p = . , r = . ). the ratio of bwt between asthmatics and control subjects are significantly different on hrct. the grade of bwt shows significant different fev and tlc statistically, and that of extent of air trapping reveal significant different pefr statistically. the bwt on hrct reflect the thickness of epithelial basement membrane. computer-aided diagnosis: a shape classification of pulmonary nodules imaged on high-resolution ct s. iwano, t. nakamura, t. ishigaki; nagoya/jp purpose: the purpose of this study was to decide whether the computer image analysis could classify shape of pulmonary nodules like radiologists. materials and methods: based on the high-resolution ct (hrct) findings, two radiologists classified pulmonary nodules ( - mm in diameter) into types of shape: round (n = ), lobulated (n = ), polygonal (n = ), tentacle (n = ), spiculated (n = ), ragged (n = ), and irregular (n = ). on the other hand, an image processing computer software extracted several quantitative measures of the shape of each nodule from dicom data of hrct images. to identify the contour of the pulmonary nodules, - hu was used as the predefined threshold values. these quantitative measures were contrasted to the classification by radiologists. results: as quantifiable features to characterize nodule shape, the circularity (= π*area/ perimeter ) and the second moment were suitable. the combination of the circularity and the second moment could classify round, tentacle, spiculated, ragged, and irregular nodules. however, it was difficult to separate lobulated from polygonal nodules. conclusion: computer image analysis of hrct could classify round, tentacle, spiculated, ragged, and irregular pulmonary nodules like radiologists. this method may be useful for the differentiation between malignant and benign nodules. hrct of the lungs in cystic fibrosis: more sensitive marker of cf lung disease than lft m. polakovic, h. kayserová, p. boruta; bratislava/sk purpose: our study was aimed at determining the usefulness of hrct in evaluating pathomorphological changes of lung parenchyma, the reversibility of these changes, and correlation of these changes to pulmonary function and mutations in cftr gene. two hrct examinations of the chest were performed on cf patients ( males, females) with lft (maximal interval: two weeks) in the period of to . follow-up studies were evaluated by a modified bhalla scoring system and compared with initial studies. lung function testing was performed on a mir spiro-bank, using ers (zapletal) predicted values. in group with df /df genotype ( pts) the first examination mean score reached . (rl)/ . (ll), second . (rl)/ . (ll). lung function tests were: first examination -fvc %, fev %, mef % and second one fvc %, fev %, mef %. for groups with df /other genotype ( pts) and other/other genotype ( pts) similar results were obtained. conclusion: hrct is a more sensitive method of evaluating pathologic process in lungs of cf patients than lft. lft is a more complex examination, and the results of pulmonary functions are modified by the physical status of the cf patient (nutrition, strength of muscle, acute exacerbation) and his/her ability to cooperate with spirometry (age). severe mutations of cftr gene in genotype are accompanied by an increasingly high hrct score in spite of therapy applied and stable lung functions. high resolution computed tomography in the diagnosis of bronchiolitis obliterans syndrome after lung transplantation a.e. berstad, t.m. aaløkken, a. kolbenstvedt, Ø. bjørtuft; oslo/no purpose: to evaluate the clinical value of high resolution computed tomography (hrct) of patients after lung transplantation. materials and methods: hrct with insp-and expiratory scans was performed in patients ( women, single-and double lung-, including two heartlung transplanted) who had survived for months or more. the median followup time was months (range to months). air trapping was evaluated on expiratory scans constructed from two short spiral scans in two levels with minimum intensity projection (minip)-reconstruction. the degree of air trapping was classified on a to scale according to the affected surface area. a score of or more was defined as significant air trapping. results: patients developed the bronchiolitis obliterans syndrome (bos) as defined by reduction of pulmonary function test results to less than % of the best postoperative values after a median time of months. of these had an air trapping score of or more. among patients without bos (n = ), patients showed significant air trapping. the sensitivity, specificity and accuracy of a score of or more in the diagnosis of bos were , and % respectively. bron-chiectasis developed more frequently in bos than in non-bos patients ( and %, respectively). conclusions: hrct, including expiratory scans with minip-reconstruction, or development of bronchiectasis was of limited accuracy in diagnosing bos after lung transplantation. hrct indices and pulmonary function tests in patients with chronic bronchial asthma c. kalogeropoulou, d. kalampoka, p. zampakis, i. tsota, k. spyropoulos, t. petsas; patras/gr purpose: to quantitatively evaluate the presence and extent of low attenuation areas on chest high-resolution ct (hrct) in patients with chronic bronchial asthma and correlate such findings with the pulmonary function tests (pft). we examined patients (m: and f: , age . ± . yrs) with chronic bronchial asthma. all patients underwent inspiratory and expiratory hrct as well as forced spirometry, maximum voluntary ventilation, somatic plethysmography and cardiopulmonary exercise test. hrct images were evaluated for the presence and distribution of low attenuation areas. objective quantification of low attenuation areas was obtained by determining the relative area of pixels representing lung tissue with a density below - hu (pixel index). the resulting pixel index was determined for a single slice at the upper middle and lower zone. results: pulmonary function parameters correlated well with pixel index during expiratory scan. there was significant correlation between the upper part of the lung and forced vital capacity (fvc),%fvc, forced expiratory volume in sec (fev ), maximum voluntary ventilation (mvv) and total lung capacity (tlc). the middle part of the lung correlated with fvc, fev , fef - and resistance. the lower part of the lung was also correlated with fev , fef - , residual volume (rv) and total resistance. pixel index during inspiratory hrct scans did not correlate with pulmonary function tests. conclusions: expiratory hrct in asthmatics correlates well with pulmonary functions, which suggests that in asthmatics substantial gas trapping may occur during expiration because of severe airway narrowing and/or airway closure. thoracic imaging during treatment with pergolide r. dore, a. gervasio, m. braschi, v. vespro, g. meloni, c. nascimbene; pavia/it purpose: pergolide is a dopaminergic agonist widely used in the treatment of parkinson's disease. toxicity has been described in the form of fibrosis, inflammation of the pleura, retroperitoneal fibrosis, constrictive pericarditis and inflammatory lung infiltrates. we analyzed thoracic abnormalities in patients who have been taking pergolide long term and the possible differential diagnosis. hospital, "s.matteo" irccs, a group of neurological patients treated with pergolide underwent chest ct, because of clinical and chest x-ray abnormalities. we scanned each patients before and after intravenous injection of contrast medium; the lung parenchyma was evaluated with high resolution algorithm; three patients with pulmonary opacities also underwent fnab. results: all patients had round atelectasis associated either with pleural plaques of vary entity or with atypical and reactive pleural effusion. the thickest pleural plaques were located in the costovertebral angles, followed by the diaphragmatic pleura without effusion. in all the cases the pleural plaques in the parietal side were definetly irregular, linear in the lung side. association with lung interstitial septal thickening was inconstant. conclusions: although our cases are few in number and need further validation, pleural involvement, either fibrotic or inflammatory, always seem to be associated with pulmonary abnormalities. ct pleural signs are non specific; differential diagnosis may include tumors, asbestos exposure and inflammatory disease; however the association with relevant lung round atelectasis is significant. these ct findings must be held in mind and thought to be due to toxicity in patients treated with pergolide. the chest radiological appearances of severe acute respiratory syndrome methods and materials: patients with sars confirmed clinically had serial chest plain films, of whom also underwent chest ct scans. the radiological appearances and evolution patterns of sars were analyzed retrospectively. based on the extent and duration of the lesions on the chest plain films, the patients were classified into three types: simple type (type i) in which the lesions were limited, progressive type (type ii) in which more than three lungs fields were involved, and prolonged type (type iii) in which the lesions remained for more than six weeks. the follow-up chest plain films and ct scans were analyzed comparatively in patients of this group discharged from the hospital after clinical cure. results: in cases, on the chest radiography, cases showed predominately pulmonary parenchymal lesions in the form of ground-glass opacity and consolidation, cases showed predominately interstitial infiltration and cases showed both parenchymal and interstitial lesions. the radiological appearances were nonspecific with good patient prognosis in type i (n = ), and the radiological appearances were relative specific with high patient fatality rate ( %) in type ii (n = ). all of the patients ( %) in type iii (n = ) had residual lesions. ct was superior to the chest plain film on detecting residual lesions. the radiological appearances of sars were relatively specific. the exact diagnosis of sars must combine radiological appearances with the clinical, epidemiological and laboratory findings. hrct to identify hrct features that indicated predisposition to potentially fatal asthma and to evaluate serial follow-up hrct scans of patients with nearfatal asthma (nfa). abnormalities of the large airways (bronchial wall thickness) and small airways (centrilobular thickening and air trapping) were measured semi-quantitatively on hrct scans of non-nfa, nfa, and control subjects. in addition, these abnormalities were re-evaluated after intensive and relatively long-term (> -month) treatment with inhaled steroids. results: centrilobular thickening was observed in % of mild asthma cases, in % of moderate to severe asthma cases, and in % of nfa cases. centrilobular thickening, but neither bronchial wall thickness nor the area of air trapping, was significantly increased in nfa, as compared with mild or moderate to severe asthma (p < . , respectively). in the non-nfa and nfa patients who underwent follow-up hrct scans, only bronchial wall thickness was decreased significantly in the nfa cases (p < . ), while bronchial wall thickness and centrilobular thickening were significantly decreased in the non-nfa cases. these small airway abnormalities were partially reversible in the both groups. residual centrilobular thickening after long-term steroid treatment was significantly higher in nfa than non-nfa patients. the results of our study indicate that extensive small airway abnormalities may be associated with nfa, and that these abnormalities are partially reversible after the successful control of asthma symptoms. a. kalogera-fountzila, a. haritanti-kouridou, a. lefkopoulos, g. sevas, a. tzinas, a. sarafopoulos, a.s. dimitriadis; thessaloniki/gr purpose: we prospectively studied aids and non-aids immunocompromised patients with lung infection in order to evaluate the hrct findings, to characterize the infection and seek for differences in appearance of pcp in the two groups. methods and materials: hiv positive patients were referred to the special infectious diseases unit and non-aids immunocompromised patients were referred to the oncology unit of our hospital with a diagnosis of clinically suspected pulmonary infection. all patients were evaluated with hrct ( . mm collimation, mm interval, kvp, mas, and -s scan time). all non-aids immunocompromised patients had bronchoalveolar lavage, sputum culture, or ct guided biopsy as well as did of the hiv-positive patients which revealed pcp. in the rest of the patients pcp was confirmed after successful anti-pcp treatment. the data were calculated using the chi square test. results: all patients in both groups had the typical findings of pcp, such as presence of ground glass appearance (patchy or diffuse), consolidation (focal or diffuse), bronchiectasis or bronchioloectasies, septal thickening, cysts and nodules. the incidence of consolidation, especially diffuse, was a significantly higher finding in non-aids patients (p = . ). on the contrary, presence of ground glass in a central/perihilar distribution (p = . ), septal thickening (p = . ) and bronchioloectasis (p = . ) was significantly higher in aids patients. conclusion: hrct findings of pcp in non-aids immunocompromised patients are more aggressive, with diffuse consolidation as the cardinal sign and are associated with a more advanced disease. primary and secondary lung malignancies treated with percutaneous radiofrequency ablation: to describe the important ct findings to evaluate for therapeutic efficacy on follow-up ct after rf ablation for the lung cancer. the study group included lung cancers and metastatic nodules in patients. all patients underwent follow-up helical ct examinations immediately, one month later, and then every three months after percutaneous rf ablation. two reviewers interpreted the ct findings with consensus. the serial changes in the enhancement pattern, size, peripheral groundglass opacity, and other findings in the treated area were assessed on the follow-up ct. sensitivity, specificity, and the positive predictive value for evaluating the complete ablation using enhancement pattern on immediate follow-up ct were determined using the chi-square test. in the completely ablated group (n = ), the ablated lesions demonstrated absolutely no contrast enhancement on follow-up ct and the mean percentage of ablated lesion size decreased at , , , , and months ( . %, . %, . %, and . %, respectively) compared with immediate follow-up ct. in the partially ablated group (n = ), the ablated lesions demonstrated varying patterns of enhancement and the mean percentage of ablated lesion size showed a gradual increase after the -month follow-up ct scans. enveloped ground glass opacity surrounding the tumor was seen in ( . %) of lesions on immediate follow-up ct. conclusion: of the ct findings of lung malignancy after rf therapy, the enhancement pattern and the change in size of the ablated lesion are the most important factors in determining whether complete ablation can be achieved. (ip) is an uncommon benign lesion characterized by an irregular growth of inflammatory cells. the purpose of this study was to evaluate ct findings of the thoracic inflammatory pseudotumor. methods and materials: chest ct findings of pathologically proven seven thoracic ip were reviewed retrospectively. mean age of the patients was years old ( - years). pre-and post-contrast enhancement ct were performed in all patients. location and contour with margin of the lesion, pattern of contrast enhancement, calcification in the lesion, lymphadenopathy, associated parenchymal or pleural lesion were reviewed. clinical findings of the patients were also evaluated. results: six were in the lung parenchyma (five in peripheral, one in central; four in upper lobe, two in lower lobe), and one was in the anterior mediastinum. average size of the lesions was . cm ( - cm). of the six parenchymal lesions, three revealed as ill-defined mass-like consolidation, two as well defined masses, and one as speculated mass. one mediastinal mass showed ill-defined margin. all lesions showed heterogeneous contrast enhancement. three had eccentric calcification. none had satellite nodule. four cases had adjacent pleural thickening or effusion. lymphadenopathy was noted in one patient. they complained of mild fever (n = ), hemoptysis (n = ), coughing (n = ), weight loss (n = ). four patients had past history of pulmonary tuberculosis. conclusion: thoracic ip showed variable ct findings. however, when dealing with a peripheral pulmonary mass with heterogeneous contrast enhancement without remarkable lymphadenopathy, the radiologists should always include ip among the entities of the differential diagnosis. early to highlight the potential benefit of including fdg-pet in an early lung cancer detection protocol. materials and methods: from september to october , asymptomatic smokers (mean age . years, sd . , mean tobacco consumption . pack-years) were studied with ldct. repeat short-term high-resolution ct follow-up was done for non-calcified pulmonary nodules (ncpn) < mm to exclude growth. ncpn mm or more were considered as potentially malignant and complementary fdg-pet scanning was recommended. biopsy was recommended for ncpn found to be positive on the fdg-pet. results: ncpn were found in ( %) asymptomatic subjects. the diameter of the largest ncpn was: mm or less in ( . %) participants, > -< mm in ( . %) and mm or more in ( %). subjects underwent fdg-pet exam ( positive, negative). among the fdg-pet positive ncpn were resected (lung cancer), underwent fine needle aspiration cytology (fnac) (non-specific / necrosis). the remaining subject refused further work-up. ct follow-up was decided for the other ncpn. results will be updated after inclusion of more participants. this ldsct based screening programme demonstrated a prevalence of lung cancers in a population of ( . %) asymptomatic subjects at high risk, all with surgical stage t n m . addition of fdg-pet to the protocol may reduce the number of invasive procedures for histologically benign nodules. technical aspects postprocessing procedures after mdct for airway and lung diseases a.-l. brun, c. beigelman-aubry, y. badachi, p.a. grenier; paris/fr learning objectives: to be familiar with the postprocessing procedures after multi-detector row ct (mdct) which can optimize the assessment of airway and lung disease. background: axial slices are considered as the gold standard for analysis of airways and lung diseases. since the era of mdct, excellent reformations, and d reconstructions have become available and frequently used in case of airway disease. infiltrative and cystic lung diseases may also benefit from these techniques, provided that they are optimally used. procedure details: all d and d reconstruction techniques available were retrospectively applied on mdct volume data set from patients with airway and/or lung disease, using various parameters. optimized multiplanar volumetric reconstruction and d ct bronchography are particularly well adapted for assessing tracheobronchial stenosis and bronchiectasis. maximum intensity projection is recommended for assessing small nodular and mosaic perfusion patterns. minimum intensity projection is helpful: ) to detect and assess ground glass opacity; ) to visualize the airways within any lung parenchymal lesions, helping guide selective bronchial aspiration and bronchoalveolar lavage; and ) to depict bronchial adenolectasia in patients with copd or bronchiectasis. virtual endoscopy is particularly helpful in depicting tiny nodulation of the tracheobronchial mucosa in granulomatous and malignant diseases. highly selected cases will be used to illustrate the most striking additional information provided by postprocessing techniques in airway disease, infectious lung disease, and diffuse infiltrative lung disease. postprocessing procedures in addition to axial slices allow an excellent assessment of tracheobronchial and parenchymatous diseases, particularly concerning their depiction, characterization and extent assessment. volume rendering: less-time consuming and accuracy technique in diagnosis of rib fractures e. chavarri, c. trinidad, e. guerra, g. fernandez, c. delgado, p. moreno de la santa; vigo/es learning objectives: to asses the useful of volume rendering d images (vr) in the evaluation of rib fractures using a -slice multidetector ct. to compare vr and conventional images in determining the number and location of fractures. to describe the advantages of vr over conventional images. background: multislice ct has improved the resolution in evaluation of traumatic patients because it permits thinner slices and multiplanar reconstructions with near isotropic voxel. however, the number of images to be evaluated is greater and the diagnosis of rib fractures remains difficult due to the obliquity of costal arches in the axial view. we evaluated chest ct examinations in patients with rib fractures. in this exhibit, we describe ct technique, reconstruction parameters and the advantages of vr over conventional images to ensure number and location of rib fractures. procedure details: a -slice multidetector ct was performed using the following parameters: mas, kv, x . mm collimation, during a single breath hold. images were reconstructed with mm thickness, % overlapping and soft tissue kernel filter (b ). two experimented radiologists analysed axial, multiplanar and vr images. the number of fractures, location and part of arch fractured (anterior, medium and posterior) was described. conclusion: volume rendering is more accurate than conventional images in determining the presence and location of rib fractures, particularly when arches are fractured in more than one site. also, it is a less time consuming technique with better diagnosis confidence. we recommend its use in a routine practice for chest-ct in traumatic patients. c b d e f a g volume rendering d bronchography imaging of the airways with multidetector row ct c. trinidad, g.c. fernandez, m. rodriguez-castilla, c. martinez, e. guerra, f. tardáguila; vigo/es learning objectives: to describe the technique of d volume rendering bronchography (vr-b) with multidetector row ct. to evaluate the usefulness of vr-b of the tracheobronchial tree in clinical practices comparing with axial and multiplanar reconstructions. background: multidetector row ct allows superior imaging resolution in axial and multiplanar images, and also contributes to advances in d-volume rendering that can now be feasible in clinical routine practice. in this exhibit, we will describe the ct technique and vr-b parameters to obtain good quality d images for visualization of central and small airways. in addition, we will evaluate the role of this technique in clinical practice and in different pathologies of the tracheobronchial tree. procedure details: patients with airways pathology due to different etiologies were prospectively studied with -row multidetector ct. images were acquired at end-inspiration during a single breath-hold with x . collimation detector and reconstructed with - mm collimation, . - mm increment (overlapping %) and soft tissue kernel filter (b ). vr-b images were showed using - to - uh window with, - to - uh window level, % opacity and unshaped. conclusion: good quality vr-b images are feasible in daily practice. in patients with benign stenosis of the central airways additional information in assessing length and degree is provided. this technique improves confidence diagnosis in extrinsic compression by malignancies. however, no additional benefit in the evaluation of endobronchial lesions and bronchiectasis is shown. clinical procedure details: all scans covered from the vocal cords to the mainstem bronchi. ct parameters included a slice thickness of mm, a reconstruction interval of mm, a pitch of . mm, and a table speed of mm per rotation. data were obtained during a single-breath-hold acquisition in a craniocaudal direction. twodimensional reformation images including multiplanar reconstructions (mpr), multiplanar volume reconstructions (mpvr), and three-dimensional reconstructions (external and internal rendering) were obtained at a workstation (vitrea ; vital imaging). the main applications of msct in airways disease as well as its limitations and pitfalls are emphasized. specific topics addressed include congenital airways anomalies (anomalous origin of bronchi, vascular rings, tracheomalacia, bronchopleural fistula), extrinsic airways compression, tracheal and bronchial wall infiltration (bronchogenic carcinoma, angiosarcoma), intraluminal airways masses (carcinoid tumor), inflammatory diseases and posttraumatic and iatrogenic airways injuries. conclusion: msct provides very high-quality image postprocessing (multiplanar and three-dimensional images), overcoming limitations inherent to sdct. additional benefits include preprocedural planning for bronchoscopy and surgery, and a more comprehensive spatial anatomy for both radiologists and referring physicians. ultrasound: guided aspiration of supraclavicular lymph nodes in patients with suspected lung cancer m. kumaran, r. benamore, r. vaidhyanath, j. entwisle; leicester/uk learning objectives: to illustrate the utility of ultrasound-guided supraclavicular lymph node aspiration in the cytological diagnosis of lung carcinoma. we describe our preliminary experience in patients over the last months. background: lung carcinoma is the leading cause of cancer death in the uk. histological diagnosis traditionally requires invasive techniques to obtain tissue, such as bronchoscopy, mediastinoscopy or image-guided lung biopsy. we have introduced this technique in patients who have at least n disease on staging ct. if positive, this helps both to stage the patient and provide a cytological diagnosis. it may prevent the need for more invasive techniques, such as percutaneous lung biopsy or mediastinoscopy. procedure details: all patients were scanned and nodes over mm in size were aspirated under ultrasound guidance, using a gauge green needle. a capillary "coring" technique was employed, without suction and aspiration. the samples were then expelled from the needle into cytospin fluid, for cytological examination. the procedure could be performed without local anaesthetic and as an out patient. of the patients examined, had significant lymphadenopathy. of these, had positive malignant cytology, although had indeterminate cell type. samples showed no evidence of malignancy and pet confirmed no evidence of supraclavicular nodal involvement in case. conclusion: this is a promising non-invasive technique in the staging and diagnosis of patients with lung cancer. simulated low radiation dose ct angiography of pulmonary arteries d. tack , v. de maertelaer , c. suess , p. muller , p. scillia , w. petit , p. gevenois ; baudour/be, brussels/be, forchheim/de, charleroi/be purpose: to compare standard dose and simulated low-dose ct angiography of pulmonary arteries (ctpa). raw data of ctpa acquired in consecutive patients with pulmonary emboli (pe) were included. ct acquisitions (volume zoom, siemens) were: x mm, kv, effective mas. from the native raw data, four sets of simulated raw data were generated with , , and effective mas, respectively. from each raw data set, ct slices were reconstructed with . mm thickness and . mm increment, and with a soft tissue kernel. three readers who were blinded to the patients names and to the simulated mas levels analyzed the ct series twice, in random order. pe in central pulmonary arteries (pa) and segmental pa were graded with a four-point scale. agreements (kappa statistics) and readers performances using mas as the gold standard were calculated. results: all pe seen at mas were depicted at all lower radiation dose levels. intraobserver agreements were independent from the radiation dose but strongly depended on the reader's experience. agreements were good for central pe but only moderate for peripheral pa branches. accuracy of low-and lower dose ctpa ranged from to %, and were independent from the radiation dose (p < . ) but not on the reader. conclusion: reducing the radiation dose during ctpa is feasible with high accuracy, and may have less influence on ctpa interpretation than may have changing the reader or repeating a reading. receiver operator characteristic analysis of observer performance in virtual bronchoscopy p. maniatis, c. triantopoulou, h. labrakis, k. malagari, i. tsalafoutas, i. siafas, j. papailiou, d. kelekis; athens/gr purpose: to evaluate the observer performance with the combined interpretation on a workstation of vb, axial, coronal and sagittal images against hard-copy films of thin section ct in low grade bronchial abnormalities using receiver operating characteristic (roc) curves. patients and methods: bronchial sections in patients were evaluated in a prospective observer study using spiral ct data. exclusion criteria included occlusions, high-grade bronchial stenoses (> %) and tracheal lesions. all patients underwent a spiral ct acquisition with mm collimation. two reconstruction intervals were used: mm for virtual bronchoscopy and mm for thin section ct. both examinations were reviewed by two independent radiologists. the findings were correlated with those of flexible bronchoscopy and the statistical analysis was done with receiver operating characteristic curves. results: a total of lesions were identified by flexible bronchoscopy. areas under curves for virtual bronchoscopy and multiplanar reconstructions were . and . for the two observers respectively, while areas . and . were provided from thin axial ct images (by the two observers respectively). these results differed at statistical significant level (p < . for the first observer and p < . for the second observer). the combination of multiplanar reconstructions and virtual bronchoscopy in simultaneous viewing on a workstation is particularly useful for the evaluation of low grade bronchial abnormalities at statistical significant level when compared to thin section axial ct images alone. b d e f a g optimization of voltage combination for chest radiography in a new dualenergy subtraction system n. bandai , h. tagashira , m. yoshimoto , t. kiriyama , t. maruyama ; onsen-gun/jp, matsuyama/jp the new dual-energy subtraction system allows the fast ( millisecond) acquisition of two, high and low voltage images using csi:tl amorphous silicon flat-panel technology. the purpose of this study was to determine the best combination of the voltage for the soft tissue of the chest radiography. the dual-energy subtraction imaging increases the sensitivity and specificity of the pulmonary nodule detection in the chest radiography by reducing the contrast of overlying the bone structures. the range of the high voltage is from kvp to kvp and the low voltage is from kvp to kvp, respectively. the physical factor such as contrast, density and graininess and the anatomical factor such as visibility of the vessel were evaluated by five radiologists using the normalized-rank approach. all rank orders were transformed to the distance scale by the statistical method. the ranking in the low voltage was better in the order corresponding to kvp, kvp and kvp (p < . ). the ranking in the high voltage showed better in the order corresponding to kvp, kvp, kvp and kvp. there were significant differences (p < . ) except the distance scale between kvp and kvp. the best combination for the soft tissue of the chest in a new dualenergy subtraction system was kvp (high voltage) and kvp (low voltage). spiral low-dose ct of the chest: a "one-stop-shop" modality for early detection of the leading causes of death g. bastarrika, o. cosín, j.c. pueyo, Á. alonso, d. cano, j.j. zulueta; pamplona/es purpose: spiral low-dose ct (ldct) can identify very small lung cancers in high-risk individuals. the purpose of this study is to assess feasibility of quantifying coronary calcium using non ecg-gated ldct exams performed in individuals participating in an early lung cancer detection programme (elcdp). a comparative study was performed in asymptomatic smokers included in an elcdp ( male, female; mean age . years; sd . ). subjects consecutively underwent a ldct of the chest and an ecg-gated cardiac ct with a four-row msct scanner (volume zoom, siemens, erlangen, germany). standard parameters were employed: ldct: kv, mas, mm slice, mm coll., mm table feed, . mm rec. interval; ecg-gated cardiac ct: kv, mas, mm slice, . mm coll., . mm table feed, . mm rec. interval. retrospective reconstruction using the raw data from the ldct was performed reproducing the specific ecg-gated ct parameters. intraclass correlation coefficient (icc), paired-sample t-test and bland and altman plot were employed for the statistical analysis. the paired sample t-test showed there was not a systematic bias in the measures of calcium (p = . ). the icc for the overall calcium score and for the different main coronary arteries showed and excellent agreement (overall icc . , % ci . - . ). conclusion: a clear correlation when evaluating quantification of coronary calcium with both ct techniques is observed. a single ldct exam may be useful for early detection of leading causes of death: lung cancer and coronary artery disease. dual energy in digital chest x-ray: material and methods: patients scheduled to undergo routine chest ld-msct were examined either without ( mas, kv, . mm slice width fused to mm effective width, spiral acquisition, n = ) or with ecg-triggering ( mas, . mm slice width, axial incremental acquisition, n = , age-, sex-and bmi-matched) on a philips mx -row msct scanner. three independent radiologists scored axial slices at four different levels (carina, apical, mid-cardiac, basal), using a four-point scale for discrimination of pulmonary structures in the hilar region, the lung core and the subpleural region. mediastinal structures were scored on a three point scale. scores were compared using wilcoxon's test. results: for the more experienced ct radiologist, delineation of pulmonary structures did not improve significantly with ecg-triggering (p = . ), while improvement in the delineation of mediastinal structures was highly significant (p = . ). for the less experienced ct radiologist, there were no significant differences between ld-mscts with and without ecg-triggering for either pulmonary or mediastinal structures (p = . resp. p = . ). although not formally evaluated in this study, both radiologists reported a higher quality of sagittal and coronal mprs from non-triggered data due to lower susceptibility to breathing artifact. conclusion: results indicate that ecg-triggering improves delineation of moving structures (heart, mediastinum) in ld-msct of the chest. ecg-triggering improves delineation of pulmonary structures only in paracardial lung tissue. results: time-distance curves of the breathing-cycle using mri correlated highly significantly with spirometry (p < . ). vc calculated by the model was similar to vc measured in spirometry ( . l vs . l). tvc correlated highly significant with spirometry (p < . ). vertical parameters had a more profound influence on tvc change than horizontal parameters. conclusions: dynamic mri is a simple non-invasive method to evaluate local chest wall motion and respiratory mechanics. it widens the repertoire of tools for lung examination with a high temporal resolution. influence the aim of this study was to investigate influence of reconstruction filters on image quality in ct chest exams. methods: patients were examined with standard chest scanning protocol. after scanning, images were reconstructed with smoothing filter. both set of images were filmed under the same conditions. in blinded assessment, three radiologists independently rated films for overall image quality and chest anatomic details ( -unacceptable, -substandard, -acceptable, -above average, and -superior). for all exams and reconstruction filters image noise were measured in relation to the descending thoracic aorta. results: for all three readers overall image quality was higher for standard filter. image quality for standard protocol was . and for images reconstructed with smoothing filter . . average image noise for images reconstructed with standard filter was . and for images reconstructed with smoothing filter the noise was . . all three radiologists rated greater chest anatomic details for standard protocol images. for both protocols the best-rated detail was that of the trachea and main bronchi ( . standard, . smoothing), and the worst was that of the esophagus ( . standard, . smoothing). the greatest difference between rates of the two protocols was for paratracheal tissue ( . standard, . smoothing) and the smallest for carina and lymph node area ( . standard, . smoothing). conclusion: standard filter is better in visualization of all anatomic detail in general chest exams including mediastinal structure and lymph node area. greater image noise for image reconstructed with standard filters does not influence visualization of anatomic detail. usefulness new quality assurance systems for the evaluation of detectability of lung nodules on the digital chest radiograph y. fujiwara, k. higashimura, h. kimura, h. itoh; fukui/jp purpose: the goal of this study is to develop a new quality assurance system for evaluating detectability of lung nodule of the digital chest radiograph. method and materials: first, we made an oval shaped phantom, which simulated lung tissue such as muscle, fat, and pulmonary field. then several nodules with variable ct values were inserted into the center of the phantom. second, we took images of the phantom using ct and digital radiography (dr) with a flat panel detector.the contrast of the nodules relative to the pulmonary field were measured both on chest ct and dr. the relationship between contrast from ct and dr was analyzed in regression using rd order polynomial. finally, the same analysis was also performed on nodules from clinical cases with lung cancer (n = ), and clinical data was fitted into the same polynomial line. results: all nodules with the ct value less than in the difference between nodules and lung field could not be detected on dr. the relationship between the contrast from ct and dr in clinical cases was also regressed with the same rd order polynomial line from the phantom data. the current result is very useful for the evaluation of the detectability on chest dr. when one want to check it using variable nodules with known ct values, if the contrast of the nodules on dr is not regressed on above line, the assurance of dr might be problematic and/or dr system may need to be reconditioned. b d e f a g pulmonary thromboembolism: acute versus chronic (pictorial review) a. oikonomou , c.j. dennie , j.m. seely , f.r. matzinger , f.d. rubens , p.k. prassopoulos ; alexandroupolis/gr, ottawa, on/ca learning objectives: to present the spectrum of imaging findings that allow the differentiation of acute pulmonary embolism (ape) from chronic thromboembolic pulmonary artery hypertension (cteph) based on chest radiography and helical computed tomography (ct). background: patients with a prompt diagnosis of ape may recover completely following anticoagulation therapy. in a minority, pulmonary emboli do not completely resolve leading to organization, artery obstruction and ultimately to cteph. although the evolution process of ape to cteph has not been fully elucidated, the distinction of the two entities is critical since the treatment of ape is conservative, while cteph warrants pulmonary thromboendarterectomy. imaging findings: radiographic findings of ape are nonspecific and include atelectasis, peripheral consolidation and pleural effusion. localized lung oligemia with associated dilatation of a central artery is extremely rare. right ventricular and central pulmonary enlargement, patchy oligemia and peripheral scars may be seen in cteph. the cardinal signs of ape on ct angiography are central or completely occluding arterial filling defects, which may be associated with peripheral airspace disease and pleural effusion. eccentric filling defects adherent to the vessel wall, irregular intimal thickening, abrupt vessel narrowing or tortuosity are features of cteph. dilatation of central arteries, abrupt vessel cut-off and peripheral scars are more common in cteph than in ape but arterial webs, bands and mural calcifications are only observed in cteph. if arterial findings suggestive of cteph are present, the addition of mosaic oligemia on hrct is pathognomonic of cteph. conclusion: ct allows differentiation of ape from cteph. pulmonary hypertension: ct of the chest in pulmonary veno-occlusive disease a. resten, s. maître, m. humbert, f. capron, g. simonneau, d. musset; clamart/fr purpose: pulmonary veno-occlusive disease is a rare cause of pulmonary hypertension and often difficult to distinguish from severe primary pulmonary hypertension. unfortunately, medical treatment of the primary pulmonary hypertension by prostacyclin (pgi ) can be fatal in veno-occlusive disease and an early pre-therapeutic diagnosis of this uncommon condition is critical. so, the aim of the study was to evaluate ct of the chest as a non invasive approach of this disease. we reviewed cross-referenced records from to in our departments of radiology and pathology and identified patients with an initial pre-therapeutic ct and a pathologically confirmed pulmonary venoocclusive disease. ct scans were compared with the ct scans of consecutive patients with a pathologically confirmed primary pulmonary hypertension. results: ground glass opacities were significantly more frequent in pulmonary veno-occlusive disease (p = . ), abundant, with a random repartition, and a preferentially centrilobular distribution (p = . ). sub-pleural septal lines, and adenopathy were also significantly more frequent (p < . ). the association of these three findings appeared to be quite pathognomonic of pulmonary venoocclusive disease as the cause of pulmonary hypertension (specificity = %) with a % sensitivity. conclusion: on the initial pre-therapeutic chest ct, association of ground glass opacities (particularly with a centrilobular distribution), septal lines, and adenopathy are indicative of pulmonary veno-occlusive disease in patients displaying pulmonary hypertension. caution must be taken before initiating vasodilatator therapy in the presence of such radiological abnormalities. . x . mm ) was performed during and after a single injection of . mmol/kg body weight of gadomer. image data was compared to pre-embolism gd-dtpa-enhanced mri and post-embolism thin-section multi-slice ct (n = ). snr measurements were performed in pulmonary arteries and lung. results: one animal died after induction of pe. in all other animals, perfusion mri and mra could be acquired after single injection of gadomer. at perfusion mri, pe could be detected by typical wedge-shaped perfusion defects. while the visualization of central pe at mra correlated well with ct, peripheral pe were only visualized by ct. gadomer achieved a higher peak snr of the lungs compared to gd-dtpa ( ± vs. ± ). conclusion: contrast-enhanced d perfusion mri and mra allow the assessment of pe using a single injection of the blood pool contrast agent gadomer. c b d e f a g detection of pulmonary emboli with thin slice ( mm) maximum intensity projection reconstructions of multi-detector ct pulmonary angiography s.k. venkatesh, s. wang, p. goh; singapore/sg purpose: to compare thin-slice ( -mm) maximum intensity projection (mip) reconstructions with mm axial scans for detection of acute pulmonary emboli. materials and methods: multi-detector ct pulmonary angiography ( x mm, . pitch; . sec) was performed in patients. from each raw data set, axial mm and mm scans were reconstructed. thin-slice ( mm) mips in axial, coronal and rotational planes were reconstructed from mm axial data using a d workstation. the data sets were reviewed for evidence of embolus on a per vessel basis by radiologists with discrepancies resolved by consensus. emboli demonstrated on the axial mm images were taken as standard for comparison. results: pulmonary emboli were detected in vessels (mean of . emboli per patient) with . % in subsegmental arteries. the thin-slice mip images demonstrated more emboli as compared to axial mm images. axial and rotational mm mip images detected most of the emboli ( . % and %). significant advantage of axial and rotational mm mips over axial mm images was seen at subsegmental artery level ( . % and . % versus . %) (p < . ). among the mips, axial mips detected the most number of emboli in upper and middle lobes, whereas the rotational mip detected slightly higher emboli in the lower lobes. conclusions: thin slice mips are superior to axial mm for pulmonary embolus detection. the advantage of mips is most significant at the level of subsegmental artery level and beyond. we recommend routine use of axial mm mips in the detection of pulmonary embolus. primitive angiosarcomas of the pulmonary arteries: difficulties in ct diagnosis r. dore, m. mantelli, v. vespro, d. savulescu, g. digiulio; pavia/it purpose: primitive angiosarcomas of the pulmonary arteries are very uncommon tumors. the radiologist should consider them in the differential diagnosis with thromboembolic disease, in patients with clinical suspicion of pulmonary embolism, as early surgical treatment may be beneficial. between and , at our hospital, "s.matteo" irccs, we identified three cases of primitive angiosarcomas of the pulmonary arteries, subsequently confirmed at surgical examination, on chest cts of patients with clinical suspicion of thromboembolism. we used a single-slice spiral ct scanner ( sec., - mm collimation), with iv contrast injection at high flow rate. both pulmonary parenchyma and vasculature were evaluated, along with the enhancement pattern of the endoluminal lesions. results: in all cases, the left main pulmonary artery was affected (the distal segment in one case and proximal segments in two). in one patient, the right main pulmonary artery was also involved. all the vascular lesions were occlusive, without anterograde flow and extravasal diffusion. in two patients, contrast enhancement of the endoluminal mass was better detected in the delayed scan. in one patient, contrast enhancement had a periferical distribution. in all the patients, there were no parenchymal signs of thromboembolic disease. conclusions: primitive angiosarcomas of the pulmonary arteries are associated with occlusion of the main pulmonary artery and lack of parenchymal signs. as the differential diagnosis between massive thromboembolic disease and primitive angiosarcoma may be difficult with monophasic ct scan, the evaluation of the contrast enhancement of intraarterial lesion in the delayed phase becomes fundamental. purpose: to demonstrate the imaging features of chronic thromboembolic pulmonary hypertension (ctph) on chest ct scans and to evaluate the correlation between the extent of mosaic pattern of lung attenuation and hemodynamic measurements. methods and materials: chest ct scans of patients with ctph were reviewed to assess vascular and parenchymal changes. in all patients diagnosis was confirmed by surgery. the diameter of the main pulmonary artery (pa) and the ratio of the diameters of the main pulmonary artery to the ascending aorta (rpa) were correlated with mean pulmorary artery pressure and pulmonary vascular resistance. mosaic attenuation was quantitated by adding the number of abnormal lobes (including lingula, score - ). this score was correlated with the hemodynamic measurements. results were analysed by regression analysis and spearman's correlation. results: thrombi were observed in the central pulmonary arteries in % of cases. in all ct scans a mosaic pattern of lung attenuation was found. scars related to the pleural surface were identified in % of cases, bronchial artery collaterals in %. ct scans showed dilated pa in % and rpa ≥ in %. mean pulmonary artery pressure correlated more strongly with rpa than with diameter of pa. the score of mosaic attenuation showed a strong correlation with pulmonary vascular resistance (r = . ; p < . ). to be able to recall the most common ct and mr intrathoracic manifestations in patients with breast cancer. . to be able to describe the radiologic features of this entity. . to be able to recall helpful clues for narrowing the differential diagnosis and appropriately directing patient management decisions. background: breast cancer is a common cancer in women and is second only to lung cancer as a cause of cancer-related deaths in women. the thorax is a common site of metastasic disease and complications related to surgery, chemotherapy, radiotherapy and autologous bone marrow transplantation. imaging findings: the purpose of this exhibit is to review the ct and mr findings of the various intrathoracic manifestations occurring in patients with breast cancer. these include complications related to direct extension of tumor, to metastatic spread, and complications secondary to treatment. the complications can be divided into those that affect the chest wall (chest wall invasion, chest wall metastases, axillary and internal mammary lymphadenopathy, local recurrence, postradiotherapy sternal and costal tumors, surgical complications), mediastinal and hilar manifestations (lymphadenopathy, abscess formation, lymphatic obstruction), pulmonary manifestations (lymphangitic carcinomatosis, pulmonary metastases, endobronchial metastases, neoplastic emboli, lung infections related to immunosuppressive therapy, obliterative bronchiolitis post bone marrow transplantation) and pleural manifestations (pleural effusion, pleural metastases). conclusions: knowledge of the full spectrum of radiologic manifestations of the thoracic complications of breast cancer is useful in diagnosis and in preventing diagnostic errors. bronchogenic cysts: range of radiological appearances f. almolani, p.m. logan; dublin/ie learning objective: to review and illustrate the range of plain film, ct and mri appearances of bronchogenic cysts. background: bronchogenic cysts are one of the congenital cystic lesions in the chest. it has variable appearances on imaging and overlapping features with other mediastinal lesions. it is often identified incidentally. imaging findings: we reviewed the imaging on proven bronchogenic cysts. plain radiographs and ct images were available for all cases. mri images were available in cases. most of bronchogenic cysts have a classic mediastinal location and well defined margin on plain films, with homogeneous low density appearance on ct. however they can be found within the lung parenchyma, be cystic or cavitary, have thick walls and be seen to expand. on mri, fluid levels can be demonstrated. occasionally, they can be multiple or co-exist with significant unrelated pathology. conclusion: bronchogenic cysts have a wide range of imaging appearances and familiarization with these appearances is essential for appropriate inclusion of the diagnosis within a list of differential diagnoses. imaging appearances of mediastinal cystic lesions s.p. prabhu, k. burney, p. goddard; bristol/uk learning objectives: to illustrate the radiological appearances of various mediastinal cystic lesions using various imaging modalities. background: cysts of the mediastinum constitute a small but important diagnostic group, representing to % of all primary mediastinal tumors. mediastinal cysts can be classified based on their aetiology, and include bronchogenic, oesophageal duplication cysts of foregut origin, mesothelial derived pericardial and pleural cysts, cystic thymic lesions, and other miscellaneous cysts. neuroenteric cysts may develop by abnormal septation of the embryogenic germ cell layer, which closely associate with the vertebral column. in addition, mesothelial cysts, including pericardial and pleural cysts, and thymic cysts also occur in the mediastinum, as well as other rare cysts. imaging findings: characteristic location and internal architecture of different mediastinal cystic lesions are used to aid the clinician in formulating a diagnostic plan. plain chest radiographs can identify mediastinal cysts in a large proportion of cases. ct usually reveals well-circumscribed rounded masses of water density or a little higher and is useful in delineating the size, shape, and extent of the mediastinal masses. characteristic fluid signal on mri performed in selected patients differentiates the benign cystic lesion from solid mediastinal masses. mri has therefore succeeded in providing valuable pre-operative specific diagnostic confirmation in regard to mediastinal cysts in selected cases. in this pictorial review, we review the radiological appearances on plain radiographs and cross-sectional imaging for the entire spectrum of cystic lesions of the mediastinum. non-infectious radiographic manifestations of bone marrow transplantation: a pictorial review d. beckett, j. oliff; birmingham/uk learning objectives: to illustrate the radiological manifestations following stem cell transplantation for haematological malignancy. to outline the incidence, clinical presentation and role of imaging within this patient sub-group. background: bone marrow transplantation (bmt) is a critical therapeutic intervention for a variety of pathological conditions. the complications of bmt include chemotherapy and radiation toxicity, graft versus host disease, recurrent malignancy and miscellaneous conditions. complications of allogenic bmt manifest in a variety of clinical settings affecting all organ systems. plain radiography, contrast enhanced studies, sonography, ct, mri and interventional techniques are essential in diagnosing these complications and evaluating their response to therapy. imaging findings: pulmonary manifestations include drug and radiation toxicity, bronchiolitis obliterans, bronchiolitis obliterans organizing pneumonia, lymphocytic interstitial pneumonitis and diffuse alveolar haemorrhage. gastrointestinal presentation with veno-occlusive disease, typhlitis and pneumatosis intestinalis are also reviewed, as are central nervous, musculoskeletal and cardiovascular system manifestations. the spectrum of imaging findings following bmt is diverse. a firm understanding of the clinical presentations and incidence in combination with typical radiological findings is essential in the successful management of this patient sub-group. thoracic manifestations of chronic renal failure a.m. barnacle, p.g. gishen, s.j. copley; london/uk learning objectives: to review the radiological spectrum of thoracic manifestations of chronic renal failure, including the more unusual imaging appearances. background: the effects of chronic uraemia are evident in a multitude of organ systems, including the pulmonary, cardiovascular and musculoskeletal systems. imaging findings: the poster illustrates common thoracic manifestations such as pulmonary oedema and uraemic pericarditis. more unusual manifestations of chronic renal failure such as metastatic pulmonary calcification, renal osteodystrophy, amyloidosis and peritoneal dialysis fluid collection within the mediastinum are highlighted. plain radiographic and ct imaging findings of both these common and more unusual pulmonary manifestations are reviewed. the study illustrates the diversity of thoracic manifestations encountered in chronic renal failure patients and the less well recognised sequelae that should not be overlooked when imaging such patients. background: while the availability of bone marrow transplantation is improving and increasing numbers of adults and children are undergoing the procedure for the treatment of a wide variety of malignant tumours and haematological disorders, it remains a procedure which carries with it a very significant complication risk. despite prophylactic therapy, patients remain at particularly high risk for bacterial, viral and fungal infection in the post-procedure time period. infectious complications are diagnosed and monitored using plain radiography, ultrasound, ct, mri, image-guided biopsy and less often, nuclear medicine. other commonly encountered complications requiring radiological investigation include hepatobiliary complications, veno-occlusive complications and graft-versus-host disease. imaging findings: this educational exhibit demonstrates the radiological appearances of the spectrum of common and unusual complications encountered by patients who have undergone bone marrow transplantation, as depicted using a wide variety of imaging modalities. conclusion: while bone marrow transplantation is a life saving procedure it still retains a high degree of morbidity and unfortunately a wide range of complications can arise following the procedure. the role of the radiologist in using a number of imaging modalities to accurately diagnose and follow these complications remains an important one. air-space pattern lung metastasis l. herraiz, a. cima fernandez, s. dieguez, m. grande, m. sanchez nistal; madrid/es learning objectives: to identify and describe the imaging findings on air-space pattern in lung metastasis. background: lepidic pattern growth in lung metastasis is an uncommon manifestation of disseminated malignancies, mainly described in gastrointestinal adenocarcinomas. on ct, differentiation from other lung diseases as multifocal pneumonia, bronchiolitis obliterans organizing pneumonia (boop) and brochioloalveolar carcinoma (bac) may be difficult, and diagnosis of bac cannot be made with confidence if a primitive extrapulmonay adenocarcinoma has not been ruled out. imaging findings: we reviewed x-ray films and ct studies of patients with proven air-space pattern lung metastasis. metastasis was classified as air-space disease when it showed one or more of the following ct findings: air-space nodules, including cavitating and pseudocavitating nodules; parenchymal consolidation containing air bronchogram and focal or diffuse ground-glass opacities. the primary tumors were colorectal adenocarcinoma in patients; pancreatic adenocarcinoma in ; adenocarcinoma of the ampulla of vater in and infiltrating duct breast carcinoma in . in patient the diagnosis of pulmonary disease preceded the discovery of primary malignancy and was synchronous in other patients. cavitating nodules were seen in patients; parenchymal consolidation in ; ground-glass opacities were seen in ; pseudocavitated nodules in other patients; solid nodules in ; nodules with air bronchogram in two; mass with air bronchogram in and lymphangitic carcinomatosis in patient. conclusions: air-space pattern is an uncommon manifestation of lung metastasis. differentiation from boop, multifocal pneumonia and bronchioloalveolar carcinoma can be difficult because this can be the onset of the disease. imaging of chest wall disorders j. lee, k.-n. lee, s. kim, j. lee, m. roh; busan/kr purpose: to illustrate the radiologic features of a wide spectrum of chest wall disorders according to anatomic location. we have retrospectively reviewed the radiologic findings of patients with chest wall lesions pathologically proven by biopsy or surgery between january and july . we excluded cases of pleural effusion or thickening, chest wall invasion by lung cancer or mediastinal malignant tumor, and secondary metastatic lesions. all patients had plain films and spiral ct scan with intravenous contrast enhancement. several patients underwent supplementary mr with gadolinium enhancement. results: pleural lesions include empyema, tuberculous empyema, empyema neccessitatis, localized fibrous tumor of pleura, malignant mesenchymal cell tu-mor, malignant mesothelioma, pleuroblastoma, fibrosarcoma, angiosarcoma and asbestos-related pleural plaques. muscle and subcutaneous fat are involved in poland's syndrome, inflammation, abscess, tuberculous abscess, empyema neccessitatis, post-operative hematoma, epidermal cyst, intramuscular hemangioma, elastofibroma dorsi, pectoralis muscle fibromatosis, neurofibromatosis, malignant schwannoma, muscle involving nhl, subcutaneous t-cell lymphoma and extraskeletal ewing's sarcoma. rib lesions include chronic inflammation, osteochondroma, enchondroma, eosinophilic granuloma, and plasmacytoma. sternal lesions include chronic inflammation and pigeon breast. a case of chondrosarcoma of the t vertebral body is also included. a wide of spectrum of disease processes affect the chest wall. the ct and mr imaging has enabled precise localization and differential diagnosis of chest wall lesions. therefore, familiarity with these radiologic features will make accurate diagnosis possible and and allow for optimal patient treatment. standardized in all patients ml contrast-agent was injected with a flow-rate of . ml/sec, scan delay was sec. results: in ( %) of patients chest injuries were detected. the following posttraumatic thoracic injuries were revealed: in patients ( %) emphysema, in ( %) hematoma of chest wall; in patients ( . %) fracture of the thoracic spine, ( . %) fractures of the ribs, ( . %) of clavicle, ( . %) of sternum, ( . %) of scapula; ( %) patients had a pneumothorax, ( . %) a hematothorax, ( . %) a tension pneumothorax; patients ( . %) had a lung contusion, ( . %) a lung laceration; patients ( %) had a pneumomediastinum; ( . %) a rupture of thoracic aorta; ( . %) rupture of a supraaortal ar tery and ( . %) a rupture of diaphragm. no injuries of tracheobronchial system, esophagus or the heart were detected. conclusion: a standardized msct examination protocol represents the preferred screening method in the evaluation of patients who have sustained chest trauma. purpose:to find out important ct findings for the prediction of prognosis of thymoma with a survival analysis. materials and methods: patients with thymoma, who underwent surgical resection, were retrospectively analyzed. clinical data were collected from chart review. preoperative plain and contrast enhanced ct was performed in all patients. two independent chest radiologists evaluated ct images paying special attention to diameter, contour, shape, cystic or necrotic portion, calcification, mediastinal fat obliteration, invasion of adjacent mediastinal structure, thickening of adjacent pleura, infiltration of adjacent lung, infiltration of adjacent chest wall, pleural effusion, pericardial effusion, mediastinal lymphadenopathy, enhancement pattern, and degree of enhancement. kaplan-meier survival curves were generated. influence on survival for each ct finding was analyzed by logrank test. results: cystic or necrotic portion, calcification, mediastinal fat obliteration, invasion of adjacent mediastinal structure, thickening of adjacent pleura, infiltration of adjacent lung, infiltration of adjacent chest wall, pleural effusion, pericardial effusion, mediastinal lymphadenopathy, homogeneous enhancement pattern and high degree of enhancement were seen in %, %, %, %, %, %, %, %, %, %, % and % of patients respectively. the ct finding that were most predictive of survival was calcification (logrank test: p = . ). cystic or necrotic portion (p = . ), infiltration of adjacent chest wall (p = . ), and pleural effusion (p = . ) were independent predictors of long-term survival. there were no differences in survival for the other findings. conclusion: calcification, cystic or necrotic portion, infiltration of adjacent chest wall, or pleural effusion on ct suggest poor prognosis of thymoma. b d e f a g low attenuation splenic lesions found at chest ct: differential diagnosis and investigation a.k. choudhary, e.l. senior, n.j. screaton; cambridge/uk learning objectives: . to illustrate the causes of low attenuation splenic lesions. . to discuss clinical and imaging features which aid diagnosis in patients with known or suspected malignancy. background: abnormalities of the spleen are commonly encountered during ct of the thorax and abdomen in patients with suspected thoracic disease, posing diagnostic difficulty particularly in patients with otherwise operable malignancy. we present a pictorial review of low attenuation splenic lesions in patients with suspected thoracic disease. the most common causes are benign (including pseudocysts caused by trauma, infection or infarction), infections (mycobacterial, fungal, hydatid), sarcoidosis, haemangiomata, and lymphangiomatosis. malignant causes include lymphoma and metastases (lung, breast, melanoma, stomach, liver, colon). in patients with known malignancy, splenic metastases are rare in the absence of widespread metastatic disease. although the morphological pattern on ct may suggest malignancy (multiplicity of lesions, irregular margins, enhancement and splenomegaly), other imaging studies may be complementary. on ultrasound focal anechoic lesions are typical of benign cysts while multifocal or diffuse solid lesions are suggestive of malignancy. on mri heterogeneous lesion signal is suggestive of malignancy. in cases where morphological imaging is non-specific, fdg-pet may play an important role. the accuracy of splenic biopsy in indeterminate lesions approaches % but significant complications occur in . %. conclusion: isolated low attenuation splenic lesions must not be considered synonymous with metastases even in patients with known cancer. awareness of the differential diagnosis and further imaging permit accurate diagnosis and management. evaluation . whole-body msct scan were performed using the following protocol: ) native head and neck: mm slice thickness, um mode, reconstruction . mm; ) administration of - ml nonionic contrast media using automatic injector at flow rate of ml/sec and smart prep feature; ) thorax, abdomen and pelvis scan: . mm slice thickness, us mode, reconstruction . mm; ) scan time under minute; ) images were used for mpr and d-vr. results: in management of polytrauma patients, time is of the essence. in high-risk patients who were selected for msct examination following injuries were found: cranial (sub and epidural hematoma, contusion, fracture etc); thoracic (hematothorax, lung contusion, pneumothorax etc); abdominal (contusion and hematoma of parenchymal organ, retroperitoneal bleeding); aortic (rupture, dissection, periaortal hematoma); fractures (mostly ribs - [ %] and pelvis - [ %]) conclusion: in our hospital msct represents the major diagnostic tool in the management of high-risk polytrauma patients, reducing time spent outside of emergency unit. it is a fast and reliable method for diagnosis of skeletal trauma, parenchymal organs and aortic injuries. the radiologist becomes important member of the trauma team in management of acute polytrauma patients. learning objectives: to illustrate the fundamentals of digital imaging, the basic operations of image display and manipulation, the principles of image compression and filtering. backgound: radiological scanners generate digital images and image interpretation is performed using dedicated workstations. the knowledge of digital imaging, image compression and filtering is crucial to understand the new technologies and the post processing operations. procedure details: the structure of a digital image, the encoding algorithm, the operations for image display and manipulation, and the compression algorithms are described. a phantom and clinical cases are used to show the influence of these aspects on the quality and information of the images. conclusion: several aspects affect the information and the quality of digital images. the basic criteria for assessing image quality are defined. web-based radiology-teaching library for board examination of radiologists with object-oriented database system n. we have developed a web-based radiology-teaching library for a board examination of radiologists in japan. this library receives imaging cases from all of japan via the internet. the web-based radiology-teaching library is developed with an object-oriented database, and it is readily available with a simple operation to radiologists. the submission process makes full use of the internet. the reviewers, who are expert radiologists, carefully review the submitted cases. when the reviewed case is sent back to the submitter with the reviewers' comments, the submitter should correct their text and images according to the reviewers' comments in order to obtain the reviewers' approval. the computer assists these processes automatically, and it enables submitters and reviewers to do collaborative work in registering cases without temporal or spatial restriction. finally, approved cases can be registered to our library. results: up to august , our radiology-teaching library has collected imaging cases in all subspecialties. in these cases, cases have been registered. in august , the board examination of radiologists in japan has been performed with selected cases from this library in all subspecialties. conclusion: our library is readily available to radiologists who do not have much computer knowledge. our web-based radiology-teaching library will be useful for the education of all radiologists. pacs: ergonomic considerations c.l. arnoldus, c.j. zylak, m. flynn; detroit, mi/us learning objectives: ergonomic issues associated with picture archivial and communication systems (pacs) should be addressed during the early phase of incorporation in the radiology department. this reviews how a large tertiary care teaching hospital addressed these issues. background: existing computer and alternator workstations in the radiology department were observed for six months. the radiologists were surveyed on workstation issues related to space, noise, lighting, and repetitive motion. a pacs ergonomic work group consisting of radiologists, physicists, and administrators accepted the task of addressing these issues and recommending solutions. procedure details: on non-ergonomic workstations, repetitive strain occurred. of the forty-one radiologists on staff, four who used computer workstations extensively had elbow and shoulder tendinitis from extended periods of reach while manipulating images with the mouse. carpal tunnel symptoms, neck strain from periods of flexion or extension, and eye fatigue were also experienced. alternators placed near monitors reflected light and ghost images on monitor screens. eye level evaluation of films was not possible with fixed monitor height. crowded workstations and alternators were observed. based on the findings, adjustable workstations with articulating keyboards, mousing surfaces with gel wrist rests, flat screen liquid crystal display (lcd) monitors, and multi-adjustable posture support chairs were evaluated. placement of view boxes and ambient lighting issues were addressed. adjustable height workstations for small group consultation and conferencing will be strategically located. voice amplification is being considered. conclusion: ergonomic issues should be identified early before pacs transition to allow a workplace design to maximize workflow and minimize repetitive strain. withdrawn by authors in any single patient we use the same position during each scan to get proper images coregistration. during body scans patients were instructed to breath freely, but not deeply, and not to move. we used a dedicated system for image fusion that combines volumetric information from pet, ct and mri to get proper coregistration in all cases. conclusion: metabolic and anatomical information can be combined precisely to asses anatomic locations of lesions and artifacts with proper coregistration obtained with separate scanners using dedicated software. background: current limitations with regard to medical image quality control and consistency have been discussed previously. we have recently suggested proposals to several american industrial and scientific bodies aimed to improve image quality performance criteria for both clinical and diagnostic images. although some regional and national guidelines and regulations exist, practical universal standards have not yet been achieved. procedure details: this exhibit shows how international radiology and technical societies, regulatory groups, and standards agencies may collaborate in order to improve medical image quality performance and standards, including but not limited to ( ) performance criteria for softcopy and hardcopy, ( ) extending communication and control standards for the performance of output devices, and ( ) initiation and support for research into the relationship between display physical metrics and diagnostic performance, to assess clinical consequences of variations in image quality metrics, and to define what constitutes image quality from a diagnostic perspective. the exhibit has presented some initial steps that will improve the consistency and reproducibility of medical images in electronic imaging systems. the international medical community has an opportunity to build upon the work of many regional and national efforts in a collaborative effort to improve medical image quality with anticipated benefits to patient care, medical practitioners, and the medical industry. b d e f a g modeling and simulation of the blood flow using multi-slice ct: application of computational fluid dynamics s. yamamoto , s. hamada , t. johkoh , s. murai , s. yoneyama , s. maruyama , n. tomiyama , m. komizu , h. nakamura ; osaka/jp, tokyo/jp learning objectives: to understand the prediction based medicine model using computer fluid dynamics (cfd). to support the informed consent of the patient using four-dimensional display. to describe the technical procedure of ct scanning for blood flow analysis. background: aortic aneurysms or dissecting aneurysms are serious and lifethreatening conditions that requires early diagnosis and prompt surgical treatment. it is difficult to directly measure the blood flow and pressure without invasive examination. computer simulation is one of the solutions for non-invasive diagnosis, or for comparison of pre-and post-operative measurement of the blood flow and pressure. in this exhibit we will describe the application of cfd based on four-dimensional vessel reconstructions from multi-slice ct data. procedure details: the volume data ( . - mm slice thickness) was acquired from half-second multi-slice ct (aquilion, toshiba, tokyo, japan). segmented vessels were converted surface display by marchine cubes method. input parameters of blood were applied as an ideal incompressible fluid. steady and transient analysis of cfd was performed using commercially available software (acusolve, acusim software, mountain view, ca) by a finite element method. original program modules for image processing of both vector and medical (di-com) data was implemented on a commercially available workstation (amira . , tgs, san diego, ca). variable dynamic display of both steam line of blood and blood pressure was superimposed on the three-dimensional ct data (multi planer reformat, maximum intensity projection and volume rendering etc) conclusion: modeling and simulation applications to the blood flow could enable us to build rapid prototypes of the models for quick visual validations. using remote asp and internet- for integrating digital radiological images with a regionalinformation system in a new population-based purchasing system for public healthcare provision o. barbero , j. guanyabens , c. rúbies , j. fernández , j. maideu , i. cerdà ; sabadell/es, vic/es to learn about the improvements in health care management due to the implementation of several integrated his/ris and pacs in a regional area.to learn how new technologies like java, internet , asp and remote image archival can be used to improve health care management and provision. background: a pilot project to evaluate the possibilities of digital radiology in the improvement of health care is underway within the framework of the catalan health service's strategic plan to establish a system in the county of osona(*), not far from barcelona (spain). the sharing of information forms the backbone of the project, the aim being to enable all authorized healthcare personnel to have -hour-a-day access to all digital information and images from examinations performed in all healthcare centers in the county, whether private or public. procedure details: for the evaluation and analysis: . improvements brought about in healthcare services; . organizational impact; . cost-benefit analysis; . elements to take into account for the system's implementation and extension. from the technological point of view: . remote asp archive; . high-speed internet- links; . information systems integration based on web, java, xml and dicom. the analysis and evaluation of the results of this innovative project and health care provision strategy will be presented in ecr. (*) the health care ministry will pay a fixed amount of money per person and per year to all the health care providers of the area in exchange for public health care services. a new parameter enhancing breast cancer detection in computer aided diagnosis of x-ray mammograms k. murase , n. tanki , m. iinuma , h. kubota , m. nagao ; osaka/jp, matsuyama/jp purpose: to introduce a new parameter enhancing breast cancer detection in computer aided diagnosis (cad) of x-ray mammograms. we used the database of mammograms generated by japan society of radiological technology. this includes patients with benign masses and patients with malignant masses. the age ranged from to years ( . ± . years). the new parameter called -dimensional fractal dimension ( d-fd) was obtained from the slope of the graph with the logarithm of the cutoff value on the horizontal axis and that of the number of pixels with a gray level above the cutoff value on the vertical axis. in addition to d-fd, the conventional image features such as eccentricity (e) and curvature (c) were extracted. the conventional fractal dimension (c-fd) was also calculated using the boxcounting method. the artificial neural networks were used as a classification algorithm, and a receiver operating characteristic (roc) curve and the area under the roc curve (az) were calculated to evaluate the diagnostic accuracy in the task of differentiating between benign and malignant masses. results: when using e, c, c-fd and age as inputs, the az value was . ± . . on the other hand, when d-fd was added to inputs in addition to the above parameters, the az value was significantly improved to be . ± . . the d-fd is a new parameter which can enhance breast cancer detection in cad of mammograms, because it is effective for discriminating between benign and malignant masses. web computed radiography: a solution for x-ray examinations in africa d. soft tissue tumor distributed decision support system m. robles, l. marti-bonmati, j. garcía-gómez, c. vidal; valencia/es purpose: to provide distributed decision support services in soft tissue tumor (stt) to assist the radiologist. previous studies have shown the capability of pattern recognition techniques to classify between benign/malignant character and different histological groups. methods and materials: radiologist access to the stt distributed decision support services is achieved using a graphical interface, with four windows that offers the main system functionalities areas: . access to local or distributed stt registers that contain the features to study. new data can be imported from microsoft access formatted files or other databases. . statistical analysis that provides the graphical representation and reports of statistical and evaluation information like probability distributions per class, correlation studies, roc curves. . graphical representation of stt registers provides graphical representation of stt features from the protocol, using pca-transformation, or feature selection. . automatic classification access to stt classifiers distributed web-services, developed with pattern recognition technology. results: a location independent and multi-platform system has been developed to provided decision support services for radiologists dealing with stt diagnosis. the system architecture considers the access from hospitals using local or distributed data and connections to clinical decision support servers. the nodes maintenance and upgrade are automatically controlled by the architecture. conclusion: this system helps radiologists with novel and powerful methods in stt diagnosis and characterization. it provides access to distributed data, statistical analysis, graphical representation and pattern recognition classification. grid technology will incorporate more facility, security and powered communications to the stt distributed decision support services. integration of broadband teleradiology into picture archiving and communication system (pacs) y. zhao, k. chen, z. yao, d. wu, z. pan, b. guo; shanghai/cn purpose: the availability of broadband wide-area networks (wans) facilitates a range of new applications in healthcare. teleradiology is increasingly integrated into picture archiving and communication system (pacs). this paper introduces a broadband telemedicine system for transmitting interactive audio and video signals, and high-quality medical images directly acquired from a hospital-wide pacs. methods: a high-performance telemedicine system was developed over the existing metropolitan broadband multimedia network in shanghai to exploit the possibilities offered by internet protocol (ip) over synchronous digital hierarchy (sdh) technology to support real-time tele-consultation for medical imaging. a trial session was held with two channels of fiber-optic connection transmitting real-time duplex audio/video signals and high-quality dicom images between rui jin hospital and shanghai international convention center, kilometers apart. the system enabled direct retrieval of dicom images from the pacs, and allowed to use interactive telediagnosis tools for image visualization and processing. results: during the teleradiology session, dicom images archived at the pacs were readily accessed without any loss of information. a number of functionalities of visualization software were utilized and tested, combined with real-time videoconference session. the system was proven robust, efficient and providing for high performance. conclusion: the broadband telemedicine system under study has given us a useful insight about the potential of applying broadband technology in healthcare, especially the performance of videoconferences with telediagnosis of radiological images. with the close integration of teleradiology into a hospital-wide pacs, it yields sufficient sound and image quality for remote educational and clinical collaboration. performance assessment of a wavelet contrast enhancement method in dense parenchyma based on simulated lesions s. skiadopoulos, l. costaridou, p. sakellaropoulos, c. kalogeropoulou, e. likaki, g. panayiotakis; patras/gr purpose: evaluation of an adaptive wavelet-based contrast enhancement method in dense parenchyma based on simulated radiopaque lesions. methods and materials: sixty normal mammographic images of dense breasts were selected from the ddsm database. simulated radiopaque lesions of known image characteristics, such as low contrast ( . %- %) and unsharp contour ( %- %), were generated and superimposed in dense breast areas. this resulted in a case sample that is difficult to achieve and verify with real lesions. all images with simulated lesions were processed by the wavelet-based method. evaluation was carried out by an observer performance comparative study between the wavelet-processed images and original ones. the sample was presented to three experienced radiologists, classifying each simulated lesion with respect to contour type, using a five-point rating scale. a binormal roc curve was individually fitted to the scores of each observer with a maximum-likelihood procedure and the area under the roc curve (a z ) as well as the % confidence interval were calculated, for each technique. results: combining observers' responses, the a z values are . and . and their corresponding confidence intervals are ( . , . ) and ( . , ) for wavelet method and original one, respectively. this difference in a z values is statistically significant (students' t-test, p < . ), indicating the effectiveness of the wavelet method. conclusion: improved contour characterization of low contrast simulated lesions sample is achieved with the use of the wavelet-based contrast enhancement method. use of simulated lesions sample servers for verification purposes, required to assess the performance of various digital image post-processing techniques. estimating the arterial compliance in early stages of atherosclerosis: evaluation of a new index on the curve-fitted doppler sonograms n. erdogan, s. kara, f. dirgenali, m. okandan; kayseri/tr purpose: to develop a computational method to define a new index to estimate the arterial compliance in early stages of atherosclerosis. the doppler data was obtained from a nonstenotic arterial site (carotid artery) in patients with femoropopliteal atherosclerosis (n = ) and healthy volunteers (n = ) using the audio output of a commercial color doppler ultrasound unit. the signals were transferred to a personal computer and two dimensional sonograms were plotted by fast fourier transformation. smoothing and curve-fitting operation is performed on the sonograms. a moving average method was used for smoothing and interpolant-cubic spline was processed to make curve-fitting. the time interval between the first transmitted peak (t ) and the second compliance peak (t ) in the arterial waveform was evaluated as a new index. our preliminary data showed no significant difference between healthy subjects and the patients with regard to the acceleration, resistivity and pulsatility indices, although a significant difference exists for the new index ( . ± . msec for patients vs. . ± . msec for healthy people). the proposed new index on the the curve-fitted fast fourier transformation sonograms seems to be promising in assessing the cardiovascular risk by detecting the degree of compliance in atherosclerotic vessel walls before the overt clinical symptoms develop. however, its efficacy and prognostic implications need to be confirmed by large scale studies. the contribution of virtual endoscopy (ve), in the imaging of the upper airways and tracheobronchial tree a. morichovitou, a. manolitsas, s. stratilati, i. tsitouridis; thessaloniki/gr purpose: the purpose of our study is to demonstrate the contribution of virtual endoscopy in the imaging of the upper airways and tracheobronchial tree. we studied cases, ( male and female, aged to years old, mean yrs), with tracheobronchial diseases. the virtual endoscopy images were compared with those of actual endoscopy in patients. the examinations were performed using a picker pq spiral ct scanner and a picker voxel q visualization workstation. thickness: mm, pitch: . , overlap: % ( mm). results: ct virtual endoscopy depicted the anatomical structure of nasopharynx, larynx, trachea, and main bronchi in all patients as the actual endoscopy. the results show that virtual endoscopy can study the tracheobronchial tree as far as the segmental bronchi, and sometimes the subsegmental bronchi (in patients). in addition, it can evaluate the extraluminal location of the lesions. lesions in patients were shown by virtual endoscopy ( occlusions, stenosis, compressions) and in patients the examinations were negative. the findings were always in agreement with those of the actual endoscopy and surgical findings. we studied the results of the two methods and describe the advantages and disadvantages of each of them. conclusion: virtual endoscopy can be used in the primary evaluation of the patient before actual endoscopy is performed. it can be used for preparing, guiding and controlling actual endoscopy or in the postoperative follow-up when immediate biopsy is not necessary. pancreatic duct imaging using the curved thin-slab minimum intensity projection method t. ogura , t. ikari , t. tachikawa , a. kamei , k. takano , s. asahara , n. fujita , m. shimizu ; maebashi/jp, tokyo/jp purpose: the recent wide use of curved mpr was applied for pancreatic duct imaging, but curved mpr requires a great deal of skill for the trace of narrowed and tortuous pancreatic duct. mis-tracing on pancreatic duct has led to various artificial images such as false pancreatic stenosis. so novel curved thin-slab minimum intensity projection methods have been employed to obtain correct pancreatic duct image. the purpose of this study is to explain the protocol, reconstruction method, and describe the potential advantages and various problems of this method in the diagnosis of pancreatic diseases. materials and methods: pancreatic duct image was reconstructed from three dimensional volume data using multidetector-row ct (qx/i, gems) scans. axial images were taken with . -mm collimation, a table speed of . mm/rot and . mm image reconstruction intervals. image processing was performed with the virtual place (m.i.lab). results: this method was successful in depicting a wide range of tortuous pancreatic ducts. also the narrowed pancreatic duct is easily visualized, and even branch ducts. the characteristic of this method is that we can easily make a pancreatic duct image quickly and with little dependence on expert technique. in abdominal studies, this method enabled the visualization of the narrow, bending pancreatic duct, reducing false pancreatic stenosis, and also could be applied for screening examination for anomalous union of pancreaticbiliary ductal system in the future. evaluation of the optimal scanning protocol for ct virtual endoscopy using a -slice scanner w. wojciechowski , w. de wever , a. urbanik , g. marchal , r. chrzan , j. kozub ; krakow/pl, leuven/be purpose: the objective of this study was evaluation of optimal scanning protocol for ct virtual endoscopy using a -slice scanner in a phantom study. methods and materials: using the mdct scanner somatom sensation (siemens, germany), ct-images of a phantom were performed, with diferent combinations of slice-thickness ( . , , , , mm), pitch ( . , , . ), slice-collimation ( x . and x . ) and reconstruction increment ( % and %). the phantom consists of simulated round lesions of different size (diameter between and mm) placed into a thin-walled plastic tube of -cm diameter. quantitative analysis was assessed by evaluation of changes in size and shape of the lesions and calculation of sensitivities. qualitative analysis was perfomed by evaluation of lesion's blurring of stairstep artifacts and rippling artifacts. results: overall sensitivity was % with slice thickness . , and mm and not dependent on pitch. the smallest lesions (diameter mm) were not detected with slice-thickness of and mm. image quality of endoluminal images was graded as optimal for slice-thickness . mm and mm. there was no statistical significant difference in quality using a different pitch in these images. however mild rippling artifacts were documented together with an increase in pitch value. conclusion: slice-collimation and slice-thickness are the most important parameters in selection of on optimal scan-protocol for ve: slice-collimation of x . and slice-thickness of mm seem to be optimal. pitch and increment are less important, although values of and % seem to be optimal. selection of optimal parameters is also depended on lesion size. automatic methods and materials: fully automatic and manual methods were developed and compared using in vivo studies. this tool allowed automatic determination of a region of interest (roi) with background correction. by means of this technique, a roi was set, corrections performed and calculation of different parameters (flow, stroke volume and velocity) and flow curves made. this tool was designed using matlab . (mathwork, inc, natick, usa) with a friendly graphical user interface. comparisons were performed by means of intraclass correlations (icc). this model was validated in patients, men and women, age range: - years-old. mr examinations were performed on a . -t system (gyroscan nt intera, the netherlands) with a phase contrast sequence. image parameters were as follow x matrix; fov, mm; mm slice thickness; tr msec, te , excitations, flip angle ° and cardiac phases with use of peripheral retrospective gating. the automatic method performed significantly better than the manual analysis, icc of . - . and . - . , respectively. at the same time partial volume effect caused by inclusion of stationary tissue was corrected. conclusion: automatic setting of roi flow measurements at the cerebral aqueduct is feasible with an adequate reproducibility. it makes the measurements independent of the radiologist, generalizing the calculus of flow parameters. virtual duodenoscopy on the basis of high-resolution mrcp data for the simulation of ercp procedures t. yamagishi , k.h. höhne , k. abe ; tokyo/jp, hamburg/de purpose: ercp has been regarded as one of the most difficult endoscopic procedures for a trainee because of a high possibility of technical failures. therefore, it is considered useful to prepare a rehearsal of ercp. we present a virtual duodenoscopy for practical ercp simulation. the hr-mrcp data sets are imported into the voxel-man program (institute of medical informatics, university hospital hamburg-eppendorf, germany). an interpolated movie of the duodenal inner surface is computed by tracing where the endoscopic tip goes through to the duodenal papilla following the actual ercp procedure. three orthogonal radiological planes of the source mrcp are integrated into the movies, which are showing the conic extent of the endoscopic viewing field. the usefulness of making a prior simulation for a successful ercp was evaluated. the post-processed endoscopic view is precise and realistic enough to execute a practical simulation of ercp, following the actual procedures step by step, e.g., which angle and position of the endoscopic tip is best for the cannula insertion into the common bile duct. the movie created can indicate an ideal approach to the duodenal orifice in exactly the same way performed by ercp experts. the radiological planes are useful as external images like x-ray fluoroscopy. a practical simulation of ercp can be performed by a combination of hr-mrcp data and the voxel-man interactive visualization program. the presented method can provide useful information prior to actual ercp. to review the physics of the interaction between the us beam, the "bubble" and the corresponding us image of the liver and portal system. to estimate the degree of portal hypertension through better visualization of collateral pathways using usca. background: a short historical review of the evolution of usca is followed by a detailed presentation of the physics and technology of recent usca. the interaction between the "bubbles" and the us beam is explained with analytical drawings and real-time us videos. procedure details: the technique and the protocol of the hemodynamic examination of the hepatic veins and the portal system are explained. a detailed anatomical review of the portal venous system is presented with special attention to the main collateral pathways present in cases of portal hypertension. the findings of imaging methods (us, ct, mr) as well as endoscopic methods are presented and their limitations are underlined. new non-invasive hemodynamic us imaging of the main collateral pathways (gastric and esophageal varices, splenorenal, splenogastric, retroperitoneal anastomoses, recanalization of the paraumbilical vein) are described and presented in images and videos after administration of second generation usca. c b d e f a g the benefits as well as the limitations of usca, including those caused by artifacts, are thoroughly discussed so that an unbiased conclusion on new diagnostic possibilities in the study of the portal hypertension can be drawn. clinical impact of liver specific contrast agents in liver imaging a. ba-ssalamah, m. uffmann, s. mehrain, s. schweighofer, w. schima; vienna/at learning objectives: to illustrate the usefulness of liver specific contrast agents in identifying morphological and functional features of focal liver lesions. to educate radiologists about new mr liver specific contrast agents and mr imaging techniques. to demonstrate the value of the combined use of liver specific and non liver specific contrast agents as a problem solving tool. background: liver specific contrast agents used in combination with non-specific gadolinium chelates may serve as a problem solving tool in the diagnosis of focal liver lesions. hepatic focal lesions with atypical imaging appearances usually need further assessment, namely biopsy, particularly in patients with history of malignant disease. liver specific contrast agents offer new opportunities for liver imaging and provide information about the functional and morphological features of focal liver lesion. currently a variety of contrast agents, subdivided in different categories are available for clinical use in mri of the liver. procedure details: in this exhibition, we describe the use of reticuloendothelial system-specific iron oxide particulate agents, hepatocyte-selective agents, nonspecific gadolinum chelates and the combined use of these contrast agents for a large variety of liver diseases.we distinguish between specific imaging characteristics for various liver diseases and nonspecific imaging characteristics that may be found in both malignant and benign focal liver diseases. the combined use of liver specific and non liver specific contrast agents is a valuable tool for the diagnostic work up of special focal liver lesions and may replace the invasive biopsy in the vast majority of cases. doppler rescue: a review of the use of ultrasound microbubble contrast agents in the visualisation of the large vessels k. satchithananda, m.e.k. sellars, s.m. ryan, p.s. sidhu; london/uk learning objectives: understanding of the types of ultrasound contrast agents available and nature of enhancement. understand when these agents could be used to improve diagnostic images and cost effectiveness. an appreciation of the clincial appearances of "doppler rescue" in different areas of vascular imaging. background: ultrasound (us) assessment of the vascular system remains an accurate imaging modality despite the advancement of magnetic resonance angiography. us allows real-time, multi-planar and safe assessment of the vasculature which is convenient to both operator and patient. limitations of us, es-pecially in the vascular system are well documented. full diagnostic doppler assessment is not always possible for a variety of reasons. color doppler assessment greatly improves vascular imaging and recent technical advances have further improved the capabilities of doppler us. a further important development is the introduction of microbubble contrast, which allows previously non-diagnostic studies to become interpretable and avoids further imaging: termed "doppler rescue". image findings: the use of microbubble contrast will be illustrated in the cerebral circulation using trans-cranial us, the extra-cranial carotid system, the liver vasculature, renal artery and the peripheral arterial system. conclusion: both color doppler enhancement and low mechanical index b-mode non-linear imaging will be demonstrated. one learning objectives: initial work-up of trauma patients is time-critical. diagnostic demands require different examination techniques including unenhanced and several contrast enhanced scans for musculoskeletal, angiographic and parenchymal diagnosis. -channel mdr-ct overcomes previous technical limitations, which used to require thickening of slices or segmenting for whole body coverage. we therefore developed a multi-phase contrast protocol allowing acquisition of musculoskeletal, angiographic and parenchymal organ information in a single scan. ct technique and contrast protocol, advantages and limitations of this procedure are described and illustrated. procedure details: consecutive trauma patients were examined on a channel mdr-ct (philips, mx idt). after exclusion of cerebral hemorrhage application of iodine contrast media ( mg iodine/ml, imeron , altana, germany) was started with a bolus of ml at a flow of . ml/sec, followed by a pause of sec. subsequently, a second ml contrast bolus ( . ml/sec) is followed by a saline chaser. the ct-scan is initiated with a delay of sec using a collimation of x . mm, slice thickness of mm and an increment of mm. results: this protocol provides excellent diagnostic image quality covering all issues of trauma work-up. within a table time of less then minutes all relevant pathologies were assessed as proved by further clinical follow-up. all fractures including two petrosal ones were detected. parenchymal pathology included lacerations of liver, spleen, and kidneys. all arterial and venous hemorrhages were diagnosed without the need of additional diagnostic work-up. intravenous, oral and, rectal contrast media for abdominal spiral ct examinations: when to use, how much, how fast, and when to scan a.n. chalazonitis , j. tzovara ; athens/gr, ioannina/gr leaning objectives: to optimise the choice of contrast media initiation parameters for abdominal single slice spiral ct examinations, tailored to each specific organ and to the most common clinical conditions. background: introduction of multi-slice spiral ct permitted greater flexibility in abdominal examinations. however single slice spiral ct still remains a powerful tool in routine imaging practice. procedure details: the aim of this exhibit is to describe lesion or organ behaviour before and/or after contrast material initiation in order to help the participant to understand why, when and how these agents should be administrated for optimal abdominal single slice spiral ct evaluation. contrast volume, flow rate and scan delay before initiation, are discussed. the influence of patient weight and circulatory parameters such as cardiac output and inflow obstructions on contrast enhancement will be also analysed. special techniques in order to achieve optimum gi opacification will be also discussed. purpose: to compare hepatic signal intensity change using superparamagnetic iron oxide (spio)-enhanced mr imaging with scintigraphy using technetium- mgalactosyl human serum albmin ( mtc-gsa) and a visual grading score, in patients with hepatic dysfunction. methods and materials: forty-six patients with hepatic dysfunction who underwent spio-enhanced mr imaging and scintigraphy with mtc-gsa were examined prospectively. in terms of clinical cirrhosis severity, patients were classified into four group; patients were classified as non-cirrhotic, were classified as group a, as group b, and as group c (child-pugh classification). on mr imaging, t -fast field echo (t -ffe) images were obtained before and after the administration of spio. to assess the effect of spio, post contrast relative reduction in signal-to noise ratio (reduction-snr) was calculated. the findings of mtc-gsa-scintigraphy were classified into types according to visual grading score. the clinical cirrhosis severity, reduction-snr, and visual grading score were analyzed with nonparametric methods. the clinical cirrhosis severity was positively correlated with visual grading score (spearman rank test, p < . ). there were significant differences between the clinical cirrhosis severity and the reduction-snr on t -ffe and between the visual grading score and the reduction-snr (kruskall-wallis test, p < . , all comparisons). the index of blood clearance (hh ) was positively correlated and the receptor index (lhl ) was negatively correlated with the reduction-snr on t -ffe (spearman rank test, p < . , all comparisons). conclusion: to evaluate the reduction-snr on spio-mr imaging is helpful in predicting not only kupffer cell function but also hepatic function as assessed by mtc-gsa-scintigraphy. are efficient contrast agents available for use with ultra-high field mr imaging? r.n. muller, p.a. rinck, l. vanderelst, a. roch; mons/be purpose: molecular mr imaging requires specific markers exhibiting high relaxivities at ultra-high fields ( - tesla). although there are theories on the design of optimal paramagnetic centers, actual relaxivities are still below expectations. methods and materials: measurements of relaxivites s - mm - ) to establish nuclear magnetic relaxation dispersion profiles of existing and possible future contrast agents were performed on two field cycling relaxometers and on a . t mr research machine. results: at high field, r and r of small gadolinium complexes are low and decrease; high-field relaxivities can be increased through reduction of molecular motion. to some extent, it is possible to increase the r of small dysprosium complexes. nmrd profiles of superparamagnetic particles reveal a high r /r ratio at ultra-high fields. however, one particle of nm contains around , iron ions, whereas one macromolecular complex contains one or a few gadolinium ions. assuming equivalent relaxivites, the gd complex should carry approximately , ions to present, per molecule, the same relaxivity as the magnetic particle which leads to a molecular weight increase of , . conclusion: for small complexes of dy, r can be modestly increased at high fields. both r and r of gd complexes can be enhanced at high fields by an increase of the molecular size, however, only r keeps increasing. superparamagnetic particles seem clearly more efficient. this fact will be of paramount importance in the context of molecular imaging. they will act as negative agents. results: the contrast ratios of rat abdominal aorta and portal vein on the first to fifth contrast imaging were higher for nms than those for gd-dtpa with every injection doses. the peak contrast ratios of aorta and portal vein for nms were . - . and . - . times as high as those for gd-dtpa. the renal excretion of nms was as fast as gd-dtpa. conclusion: nms showed stronger contrast enhancement for the rat vascular system, especially the portal vein and provided mr angiograms of higher image quality when compared with gd-dtpa at the same injection dose. d-fspgr imaging was performed before and after , , , , min. delineation of popliteal lymph node (pln) and lymph duct (ld) (extremity region, iliac, para aorti, thoracic duct) was examined.on the evaluation of ld, visual evaluation ("good visualization": ++, "visualization": +, "poor visualization": ±, "no visualization": -) was carried out. on pln, snr was calculated as follows: snr = si (pln)/sd (noise). results: in gd-dtpa ( , , ), gd-eob ( , ) and gd-bopta ( , , ), grading of ld from lower extremity region to para-aortic region was "good visualization" or "visualization". in gd-dtpa ( ), grade of ld from lower extremity region to para-aortic region was "poor visualization". on the other hand, using gd-eob ( ), gd-bopta ( ), we could see ld from lower extremity region to iliac region.we found only ld of lower extremity region in gd-dtpa-dea ( , ).these grades were "good visualization" or "visualization". each concentration besides gd-dtpa ( ), contrast enhancement of plns were very well. the pln almost showed peak value after enhancement for most cases ( / ) in five minutes. we saw pln very well after enhancement minutes later, and, at the time of gd-dtpa ( ), gd-eob-dtpa ( , ), gd-bopta ( , , ), gd-dtpa-doa ( , ), snr value was more than . conclusion: using hepatobiliary agents and extracellular agent we were able to do good mr lymphography. optimized iv contrast administration protocols for diagnostic pet/ct imaging t. beyer, g. antoch, s. rosenbaum, l. freudenberg, t. fehlings, j. stattaus; essen/de purpose: we compared different iv-contrast injection protocols for pet/ct-exams (dual-slice) to assess their efficacy in avoiding high-density artifacts on ct and subsequent false positives on corrected pet. methods: four groups of pet/ct-referrals ( mbq fdg at min pi) each were investigated with/-out iodinated contrast ( mg/ml) application: (a) without, (b) multi-phase / ml ( / . ml - ) cranio-caudally with s delay, (c) / / ml ( / / . mls - ) cranio-caudally with s delay, and (d) / ml ( / . mls - ) caudo-cranially with s delay. ct-based attenuation correction (ac) of the pet was applied routinely. ct image quality (iq) was graded (very good), (good), or (insufficient) by three radiologists. image quality and artifacts were reported for head/neck, thorax, and abdomen. two nuclear medicine physicians reviewed the ac-pet images for potential artifacts from iv-contrast without knowledge of the ct, and reported lesion-to-background (l/b) values for potentially related uptake on pet. all reviewers were blinded to the contrast protocol and patient names. results: average ct iq was poorest ( . ) for protocol a across all regions, but improved to . (b), . (c), and . (d). high-density (> hu) ct artifacts were introduced only in the upper thorax in % (b) and % (c) of the patients. mean l/b in corresponding artificial lesions was . and . , respectively, thus resembling false positives. protocol d resulted in artifact-free images. pet/ct iq increased in the presence of iv-contrast agents. highdensity artifacts from bolus injection may lead to false positives on ac-pet. the artifacts can be avoided by alternative contrast administration protocols (e.g. d). contrast enhanced ultrasound in the evaluation of hepatic metastases treated using radiofrequency ablation: hours, and months follow-up assessment g.a. vorkas, i.a. chryssogonidis, c.a. papadopoulos, e. syndouka, k. lytras, l. grassos; thessaloniki/gr purpose: this study evaluates the importance of periodic follow-ups in the assessment of hepatic metastases treated with radiofrequency ablation (rf) using contrast-enhanced sonography. materials and methods: hepatic metastases in selected patients were included in our study. all lesions were studied with ce-us using real-time, continuous scanning with esaote esatune equipment with the cnti software, enabling non-destructive imaging at low mi, following intravenous bolus administration of . ml of a second-generation agent (sonovue-bracco). the enhancement patterns of lesions were assessed during the arterial, portal and late phases. the examination was conducted pre-ablation, hours, month and months after thermoablation. purpose: an up-to-date study showed that for high-risk patients the nonionic, dimeric radiographic contrast medium (rcm) iodixanol had less nephrotoxicity than the nonionic, monomeric rcm. the lower incidence of secondary effects is often attributed to the lower osmolality of the dimeric rcm. to compare the effect of dimeric iotrolan to monomeric iohexol and iomeprol independently of their osmolality, we have modelled the vascular activity of the rcm. in an organ bath, isolated segments of porcine renal arteries, uncontracted or precontracted by micromol/l phenylephrine, were incubated with increasing concentrations of iotrolan- , iohexol- , iomeprol- and mannitol solution iso-osmolal to the rcm. results: expressed in terms of concentration of iodine in the buffer as well as in terms of concentration of the rcm-molecule in the buffer, iotrolan induced a significantly lower relaxation than the monomeric rcm (p < . ). there was no significant difference between the two monomeric rcms iohexol- and iomeprol- (p > . ). the iso-osmolal formulation iotrolan- induced a smaller relaxation than the iso-osmolal formulation of iomeprol- (p < . ). precontracted segments were strongly relaxed by all the rcms as compared to mannitol (p < . ). iotrolan had no significant effect on the basal tone of the vessel whereas iohexol and iomeprol induced a small relaxation effect. conclusion: independently of its osmolality and dilution effect, dimeric iotrolan causes less relaxation of isolated, precontracted segments of porcine renal arteries than monomeric iohexol and iomeprol. this effect can be attributed to the different biological properties of the monomeric and dimeric rcm-molecules. ultrasound contrast agent in the study of traumatic splenic rupture g. caruso , g. salvaggio , g. bellissima , v. ricotta , g. ascenti , r. lagalla ; palermo/it, messina/it purpose: to evaluate the role of ultrasound contrast agent (uca) in the identification of traumatic splenic lesion. materials and methods: from may to july , consecutive patients were admitted to the emergency department for blunt abdominal trauma. ultrasound examinations were performed with an atl-hdi machine with a - mhz convex trasducer capable of fundamental and harmonic imaging. patients were studied before and after uca ( . ml of sonovue®). multi-detector computed tomography was used as the gold standard. all examinations were performed within hours. results: ninety seconds after uca administration, homogeneous contrast enhancement of the whole of the splenic parenchyma was observed in patients. in four patients, no contrast enhancement was observed in a subcapsular splenic area: inferior splenic pole (n = ) and mid-portion of the spleen (n = ). multidetector ct performed in the portal venous phase confirmed traumatic splenic rupture in four patients, and showed an unremarkable spleen in the remaining patients. conclusion: ultrasound after sonovue administration is useful in showing traumatic splenic rupture. contrast-enhanced, wide-band phase-inversion harmonic power doppler imaging of hepatocellular carcinoma j.r. janica; bialystok/pl purpose: to determine whether examination of hepatocellular carcinoma by wideband, pulse inversion sonography offers excellent accuracy in depiction of specific characteristics of these pathologies. b d e f a g eighteen patients were examined. the presence of hcc was confirmed by ultrasound-guided biopsy or surgical resection. all patients, prior to enhanced sonography, had undergone b-mode gray scale sonography, color doppler, and power doppler examinations. after injection of . g of levovist intravenously, analysis of contrast agent arrival was performed by wide-band, pulse inversion power doppler sonography. the b-mode gray scale sonography, color and power doppler sonography were non-specific for hcc in cases in our examination. however, based on the wide-band, phase inversion power doppler sonography findings, all patients with hcc were diagnosed. all typical anatomical features of hcc as chaotic appearance with irregular paths, breaks in calibre and arteriovenous shunts were clearly visible. in cases, computed tomography had failed to disclose pathology while pulse inversion sonographic images were completely suggestive, which was later finally confirmed by histologic examination. conclusions: our data demonstrates the usefulness of wide-band, phase inversion power doppler sonography in the diagnosis of hepatocellular carcinoma by visualizing all characteristic anatomical details. characterization of small liver lesions with contrast enhanced harmonic sonography (ceus) in patients with chronic liver disease p. cabassa, r. taranto human comparative study on zinc and copper excretion via urine after administration of magnetic resonance imaging contrast agents j. kimura, t. ishiguchi, j. matsuda, a. nakamura, s. kamei, k. ohno, k. murata; aichi/jp purpose: to evaluate the in vivo kinetic stability of magnetic resonance (mr) imaging contrast agents, excretion of zinc and copper via urine were studied for three gadolinium (gd) chelate complexes. methods and materials: urine samples were taken before, three hours, and six hours after intravenous administration of gd-dtpa-bma, gd-dtpa, and gd-dota at . mmol/kg to each of five patients who underwent contrast-enhanced mr imaging. five patients who had non-contrast mr imaging were evaluated as controls. urine was assayed for quantitative analysis of zinc and copper using atomic absorption analysis. results: gd-dtpa-bma caused the highest increase in zinc excretion among the three agents, ± µg at hours and ± µg at to hours. gd-dota did not cause a significant increase in zinc excretion, ± µg at hours and ± µg at to hours. gd-dtpa caused moderate increase in zinc excretion, ± µg at hours and ± µg at to hours. excretion of copper did not show a significant difference between the three agents. the difference in zinc excretion among the mr contrast agents reflects in vivo transmetallation of the gd chelate complexes and correlates with the respective stability of the contrast agent. gd-dota was found to be the most kinetically inert among the three agents tested. effect of contrast material pushed with saline solution using a dual injector on enhancement of abdominal aorta, portal vein, and liver parenchyma in multidetector row ct of the liver f. tatsugami low-high and high-low biphasic injection forms in ct examinations of the upper abdomen l. martí-bonmatí, e. tobarra, e. arana, s. costa; valencia/es purpose: our objective was to analyze the influence of different biphasic injection rate protocols in abdominal ct. a double-blind, randomized, parallel group study was designed and conducted in patients without differences regarding gender, age and weight. all of them were studied with the same ct helical protocol. patients were randomly distributed into three groups: a) monophasic ( ml at . ml/s); b) low-high biphasic ( ml, the first ml at a rate of ml/s and the other at . ml/s); and c) high-low biphasic ( ml, the first ml at a rate of . ml/s and the other at ml/s). all the patients were injected, through an antebrachial route, with mg i/ml non-ionic contrast media. all ct scans were obtained at the portal phase with a fixed delay time of seconds. contrast enhancement was evaluated by attenuation coefficients measurements at liver, inferior cava and portal veins, renal cortex, superior aorta and aortic bifurcation. results: the biphasic protocols obtained statistically higher enhancement at the aortic bifurcation (anova, p = . ). however, there was no other statistically significant difference (anova, p > . ) among the three protocols at the different levels. although non-significative, enhancements following monophasic protocol were always higher than those obtained with biphasic protocols, with the exception of aortic bifurcation. conclusion: monophasic injection of contrast agents on helical ct of the upper abdomen gives higher enhancement of parenchymal and venous structures. no significant difference was observed between low-high and high-low biphasic protocols. quantitative analysis showed a statistically significant difference between si of myometrium on plain and sinerem-enhanced mr images assessed by means of t-test. c/n between lesion and normal myometrium significantly increased following sinerem administration. in four patients sinerem-enhanced images provided additional information leading to more accurate t staging. conclusions: intravenous injection of sinerem provides a significant decrease of si of normal myometrium with higher c/n between neoplastic lesion and normal myometrium. sinerem-enhanced mri provides better evaluation of myometrial neoplastic invasion increasing the conspicuity of neoplastic lesions in patients with endometrial and cervical carcinoma. further studies are necessary in order to realize the physiologic mechanism of myometrial uptake of sinerem. purpose: verification of radiological methods with histology has always been difficult due to poor correlation of slice thickness, orientation and confinement to two dimensions. a method for a quantitative three-dimensional reslicing of histological sections is introduced (patent pending epa . ) permitting exact matching of slice geometry. this will enable proper correlation with radiological data. hacatras tumor (mouse) were examined by acquiring dynamic t -weighted mr images and mr microangiography. the tumors were subsequently serially sectioned ( ìm slice thickness) and vessels stained with immuno-fluorescence markers. slices were digitally captured with a microscope, assembled in the sectioning plane and co-registered in the vertical axis. a ray-tracing algorithm performed three-dimensional visualization allowing the virtual re-slicing of the histological sections to create thick-slices ( . mm) in accordance with mri slice geometry. thick-slices were processed as parameter-maps of marker volume density, giving information beyond the area density signal of immuno-fluorescence images. results: d-reconstructions of immuno-fluorescence images displayed diffuse angioarchitecture which is typical for malignant tumors. resliced images revealed the exact architecture of vessel structures seen on mra. also, large single vessels could be clearly delineated on reconstructions. parameter maps of vessel density in virtual thick slices matched well with mr-parameter maps. the quantitative d approach to histology essentially improves correlation of histological and radiological data due to proper matching of slice geometry. as this new method can be used with any histological stain, it provides a tool for verification of new molecular imaging techniques by means of histology. monitoring a human fibrosarcoma (ht ) was implanted in nude mice and grown up to a size of - mm. tumor bearing animals were intravenously injected with a vascular targeting agent (vta) inducing selective thrombosis of tumor neovasculature (treatment group) or saline (controls) respectively. mri ( . t) was performed using an ultrasmall superparamagnetic iron oxide agent (uspio, shu c, schering®) hours after initiation of treatment. iron oxide-induced change in r * (∆r *) was measured using a t w dual echo-epi sequence. ∆r * values of tumor tissue were calibrated by ∆r * of muscle for determination of the vascular volume fraction (vvf). parametric ∆r *-maps were calculated for visualization of tumor perfusion patterns. correlative immunhistochemistry was performed for assessment of the treatment effects in both groups. results: ∆r *-maps revealed a clear reduction of tumor perfusion in treated animals compared to controls. anti-angiogenic tumor treatment resulted in an approximate % decrease of iron oxide induced susceptibility effects. vvf was significantly reduced after injection of the thrombogenic peptide ( the in vivo detection of arthritis by autofluorescence using an antigen-induced arthritis (aia) model was investigated. material and methods: for autofluorescence investigations of joints, a mobile fluorescence-detector was designed consisting of a lens/mirror system attached to a conventional spectrofluorometer and of optimized fiber optic cables leading to and from the site of investigation. measurements were performed in arthritic and healthy mice. aia-and healthy mice were used for histological examinations. results: at the exudative stage (day ) of aia, a decrease of fluorescence signal intensities (arbitrary units, a.u.) for excitation wavelengths of nm (emission at λ = - nm; mean signal intensity . ± . a.u.) and excitation at nm (emission at λ = - nm; mean signal intensity . ± . a.u) were observed. signals increased on day (maximum of cellular infiltration; mean signal intensity . ± . for λ exc = nm and . ± . for λ exc = nm). chronic inflammation (day and ) led to a signal decrease again. signal intensities of nad(p)h differed significantly (p ≤ . ) from controls at days and . conclusion: arthritis influences autofluorescence signals in-vivo. the detected excitation/emission pairs can be assigned to collagen/elastin and nad(p)h. autofluorescence of nad(p)h can be a helpful tool for detecting arthritis noninvasive and in real-time. postpartum uterus: what is normal? what is pathological? evaluation with ultrasound and ct i. escape, j. martinez, f. bastart, l. ortega, c. solduga; barcelona/es learning objectives: to know the normal findings, mean diameters and evolutive changes in postpartum uterus and uterine cavity, in early, middle and late puerperium with ultrasound and ct. to differentiate them from pathological conditions. background: the majority of postpartum patients undergo an unremarkable clinical course. a small percentage of patients develop serious complications in the abdomen and pelvis. endometritis, the most common cause of fever in the postpartum period, complicates - % of vaginal deliveries and up to % of cesarean sections. post partum hemorrhage is most often caused by uterine atony and retained products of conception, and complicates - % of vaginal deliveries. imaging findings: in early puerperium, uterus is most often retroverted and empty with fluid and debris in the cervical area. intrauterine cavity increases on days - post partum with fluid and debris in the whole cavity and it may be normal to see an intracavitary echogenic mass. ap diameter of the uterus diminishes progressively in - weeks. endometrial gas is occasionaly visualized after normal vaginal delivery in the immediate puerperium and disappears within - weeks. it can be difficult to differentiate from endometritis, which can progress to pelvic inflamatory disease. endometrial stripe decreases during puerperium; if it remains thickened, complications such as retained products of conception or hypotonic uterus may be suspected. doppler us can be useful for differentiating between them. we describe these features with ultrasound and ct. conclusion: it is important to recognize normal postpartum uteral findings in order to differentiate them from pathological conditions. pelvic floor dysfunction: interest of mr imaging of ortheses and prostheses j. villeval , m. de graef , r. guillon , c. courtieu , a. maubon , j. rouanet ; montpellier/fr, limoges/fr learning objectives: to display the mri appearance in static and dynamic conditions of intra-vaginal device and post surgical procedures used for the treatment of pelvic floor dysfunction. background: we retrospectively analysed mr exams performed before and after surgical treatment and mr exams without and with intra vaginal device of patients with pelvic floor dysfunction: urinary incontinence (tension-free vaginal tape n = , artificial urinary sphincter n = , intra vaginal device n = ) and pelvic prolapse (intra vaginal device n = , promontofixation n = , prosthetic b d e f a g plaques permacol and polypropylène in recto-vaginal and uretro-vaginal wall n = ). mri consisted of t turbo spin echo, t turbo spin echo and dynamic single shot sequences. imaging findings: we will show the mri appearances of the devices in static and dynamic conditions. prosthetic plaques and tension-free vaginal tape appear as low signal intensity structures which are difficult to visualize compared to promontofixation, artificial urinary sphincter and intra vaginal device. mr imaging represents a good functional post operative evaluation for prosthetic plaques and intra-vaginal device in pelvic prolapse with dynamic studies especially in cases of suboptimal post treatment outcome. conclusion: a radiologist involved in pelvic mri should be aware of the various surgical treatments of pelvic floor dysfunction in order to avoid misinterpretation and evaluate their functional results. mri allows detection of complications for a better collaboration between radiologist and pelvic floor surgeon. pathologies of the uterine endometrial cavity: usual and unusual manifestations and pitfalls on magnetic resonance imaging m. takeuchi background: the endometrial cavity may demonstrate a spectrum of imaging manifestations ranging from normal, to that of a reactive, inflammatory, and benign and malignant neoplastic cause. imaging findings: thickened endometrium or endometrial mass included benign endometrial hyperplasia or polyps, endometrial carcinomas or carcinosarcomas, and gestational trophoblastic diseases. hyperintensity similar to the endometrium suggested rather benign hyperplastic lesions. hyperplasia or polyps associated with adjuvant tamoxifen therapy tended to be large masses with cystic changes and prominent stromal proliferation. myometrial thinning suggested malignancy but was occasionally overestimated because of the fragility of the myometrium with adenomyosis. submucosal leiomyoma with edematous change may mimic endometrial mass, and demonstration of the continuity to the myometrium was the clue to its myometrial origin. uterine sarcomas may involve endometrial cavity, and endometrial stromal sarcomas often showed endometrial masses with characteristic myometrial invasion. adenomyomatous endometrial polyp may simulate endometrial stromal sarcoma due to arborescent smooth muscle components which resemble the preserved bundles of myometrium within the tumor on t -weighted images. fluid collection in the cavity was well visualized on mri to distinguish between hydro-, pyo-, and hematometra. conclusion: to recognize various imaging findings of the uterine endometrial cavity; it is important to make a correct preoperative diagnosis to avoid unnecessary or excessive surgical intervention and to preserve the fertility of the patients. learning objectives: to demonstrate wide variations of the fluid collection in the female pelvis with pathophysiologic correlation and evaluate the diagnostic clues to the differential diagnosis. background: there are various situations with fluid collection in the female pelvis. although physiologic ascites is often observed in the reproductive era, other pathophysiologic fluid collection with both benign and malignant conditions may occur. imaging findings: mucinous ascites with organ scalloping in pseudomyxoma peritonei is characteristic. hemorrhagic ascites, which shows high attenuation on computed tomography (ct) and is well visualized on magnetic resonance imaging (mri) may be observed with malignant tumors, adnexal torsion or ruptured endometriosis. lactate detection on magnetic resonance spectroscopy (mrs) may suggest its malignant condition. non-neoplastic diseases such as ovarian hyperstimulation syndrome, pelvic inflammatory diseases, endometriosis, adnexal torsion and massive ovarian edema may cause accompanying fluid collection with adnexal enlargement. benign tumors may cause ascites, such as meigs' and pseudo-meigs' syndrome, and malignant tumors may cause carcinomatous peritonitis with fluid collection. ascites in postmenopausal woman is an unnatural finding and may suggest adhesion due to old inflammation or surgical procedure, or existence of hidden neoplastic disease. ascites in a tumor-bearing woman may suggest an advanced stage of disease with occult tumor implants. localized fluid collection like peritoneal retention cyst is observed in adhesive pelvis after surgery, inflammation or endometriosis with characteristic contour shape. conclusion: to evaluate the nature or biochemical components of ascitic fluid by mri and mrs may be helpful for the differential diagnosis. recurrent ovarian cancer: patterns and spectrum of imaging findings m. moon, s. kim, j. cho, y. lee; seoul/kr learning objectives: to illustrate the spectrum of imaging findings of the recurrent ovarian cancer. background: ovarian cancer is the most common cause of death among the gynecological malignancies. the treatment of ovarian cancer has traditionally been initial surgical staging and cytoreduction, followed by adjuvant chemotherapy. then, a second look operation is performed to reassess the tumor status after first line chemotherapy. once pathological complete response is defined, the management of ovarian cancer depends on non-invasive methods such as serology and cross sectional imaging. on the follow-up evaluation, identifying patients with minimal recurrent disease is important as it provides the best chance for complete clinical response and long-term survival following second-line chemotherapy. imaging findings: recurrent ovarian cancer usually manifests as local recurrence, peritoneal seeding and nodal recurrence. vaginal stump recurrence is the most common manifestation of the local recurrence. peritoneal seeding presents as ascites, peritoneal enhancement, peritoneal nodularity, and/or mesenteric infiltration on cross sectional imaging. nodal recurrence is most commonly seen in the external iliac group, which is not the presumed site based upon anatomic studies. although hematogenous dissemination is less frequently seen in the recurrent ovarian cancer, hepatic or pleuropulmonary involvement is a relatively common manifestation of hematogenous spread. the unusual manifestations of hematogenous dissemination include metastases in the extrahepatic abdominal solid organs, bone, central nervous system, and abdominal wall involving subcutaneous fat or muscle. conclusion: familiarity with the patterns and spectrum of imaging findings of recurrent ovarian cancer will facilitate accurate diagnosis and prompt treatment. ing of cervical carcinoma. the particular advantages of mri over ct include its multiplanar imaging capability and its superior depiction of soft tissue planes. accurate staging of cervical carcinoma at presentation is essential from the point of view of prognosis and also for management planning, as patients who present with stage iia or lower grade carcinoma proceed to surgery, and those with higher grade tumours are treated primarily with radiotherapy. recurrent disease is also best depicted using mri. patients can present with or develop a wide variety of complications as a result of their tumour and mri can be of particular advantage in the investigation of local complications. imaging findings: this educational exhibit illustrates the mr imaging findings of cervical carcinoma of all grades. we also demonstrate the mr imaging features of a wide variety of local and distant complications, including local invasion, fistula formation, small bowel obstruction and obstructive hydronephrosis. conclusion: mri is the imaging modality of choice for the diagnosis and staging of primary cervical carcinoma. it is also of benefit for the investigation of disease related complications and for the investigation of recurrent disease. the most common is serous papillary carcinoma. these tumors can show totally solid appearance, but mainly solid mass with some cystic portions is more common. they have unique imaging findings especially on mr in that papillary masses with branching pattern are frequently accompanied by peritoneal seeding masses. serous surface papillary carcinoma of the ovary, a distinct subtype of these tumors, is mainly located on the surface of the ovary. sometimes normal ovaries can be found inside papillary masses on mr, which is a very unique and striking appearance. brenner tumor and endometrioid carcinoma are another subtypes of epithelial tumors that can show solid appearance. extensive calcifications can be found in some solid brenner tumors. endometrioid carcinomas are sometimes accompanied by endometrial pathologies including endometrial carcinoma. conclusion: knowing these characteristics can help make correct diagnosis on ct and mr, especially differential diagnosis with metastatic tumors. wide spectrum of uterine leiomyomas mr imaging: radiologic and pathologic correlation h.k. kim; seoul/kr learning objective: to provide an in-depth review of uterine leiomyomas with their epidemiology, histopathology, clinical manifestations and the most up-todate therapy. to describe radiologic feature of usual and unusual appearance of uterine leiomyomas on mr image and histopathologic correlation. to discuss differential diagnosis of uterine leiomyomas from gynecologic and non-gynecologic disorder. background: leiomyomas are the most common uterine neoplasm and are composed of smooth muscle with varying amounts of fibrous connective tissue. imaging features of uterine leiomyomas vary depending on tumor size, location, degeneration and other histologic findings, and specific types of unusual leiomyomas. uterine leiomyomas are classified as submucosal, intramural or subserosal. the common types of degeneration are hyaline (> % of cases), cystic, myxoid, red and hemorrhage. degenerated leiomyomas have variable appearances on t -weighted and contrast-enhanced images. specific types of unusual leiomyomas include lipoleiomyoma and myxoid leiomyoma, which have characteristic mr findings enough to differentiate from other gynecologic and nongynecologic disease. the differential diagnosis of leiomyomas includes adenomyosis, solid adnexal mass, focal myometrial contraction, and uterine leiomyosarcoma. the purpose of this exhibit is to provide in-depth review of epidemiology, histopatholoy, clinical manifestations and differential diagnosis. this exihibit will also reflect our experience in radiologic evaluation of patients with leiomyomas, with emphasis on the typical and atypical findings on pelvic mri. examples of unusual uterine leiomyomas, pathologic correlation as well as the most up-to-date therapy will be included. after interacting with this exhibit the radiologist will have enhanced understanding of uterine leiomyomas including histopathology and radiologic features. experience of mri in women with suspected and known endometriosis: a pictorial review r. benamore, l. grosvenor, a. liddicoat; leicestershire/uk learning objectives: review of imaging features in women with endometriosis since introduction of a . t magnetic resonance (mr) scanner. women were referred when they did not wish to undergo laparoscopy or where surgical intervention and visualisation was difficult. background: endometriosis affects women during the reproductive years between and , with an estimated prevalence of - %. presentation range from pain (symptoms and severity do not correlate) to asymptomatic (diagnosed during infertility investigations). endometriosis (functional glands and stroma outside normal endometrium) is characterised by deposits, endometriomas (cysts) and adhesions, commonly ovarian but is documented to involve any site within the peritoneum and extraperitoneum. mr is noninvasive with high sensitivity and specificity, small foci appearing hyperintense on t and variable signal on t weighted images. endometriomas (> cm) appear hyperintense on t and hypointensity on t , with hyperintense foci on t dependant on age of haemorrhage. fat saturation sequences are essential to improve lesion conspicuity on t . we reviewed all women referred for investigation of suspected endometriosis, and for evaluation of disease extent and sites (notably recto-vaginal pouch) in known cases of endometriosis. procedure details: sequences used were axial t , sagittal t , axial oblique fat saturation (tirm) and narrow section coronal t imaging. mr identified endometriosis involving ovaries, bilateral thick walled and unilateral endometriotic cysts, extra-ovarian disease within peritoneum and soft tissue. fibroids and simple ovarian cysts were common, with evidence of concurrent endometriosis. conclusion: mr detected small foci of endometriosis outside of the ovaries. there was a high incidence of other gynaecological pathologies allowing early review and treatment. d transvaginal sonography in uterine leiomyomas with hysteroscopic and pathomorphological correlation v. gazhonova, k. sokolskaya, t. kurganskaya, a. zubarev; moscow/ru purpose: to determine the possibilities of d transvaginal (tv) sonography in preoperative assessment of uterine leiomyomas and to correlate the findings with hysteroscopic, laparoscopic and macroscopic data. method and materials: women with symptomatic uterine leiomyomas were evaluated before myomectomy and hysterectomy. multiplanar images of the uterus were created in d tvs. preoperative location of the leiomyomas, and interactions with uterine cavity, cervix and iliac vessels were assessed in each case. us results were compared with hysteroscopy in pts, laparoscopy in , open surgery in and pathomorphology data in pts. results: surgical management was changed after d tvs examination in cases. d coronal images allowed better location of the submucous and centripetal leiomyomas especially in multiple closely lying fibromyomas. d tvs results correlated with hysteroscopy in % ( / ) of cases vs. % ( / ). d power doppler tvs provided precise evaluation of the pedunculated submucous and subserous leiomyomas thus enabling adequate myomectomy. in comparison to macroscopic data of the uterus, d tvs were superior to d tvs in differentiation of the type of leiomyomas in % vs. % of cases, and in assessment of the interactions with the cervix in % vs. %. multiplanar reconstruction of the coronal planes on d tvs improved determination of the uterine cavity interactions with large subserous-interstitial leiomyomas before laparoscopic myomectomy. conclusion: d tvs is a useful complement to d tvs in preoperative assessment of leiomyomas. accurate evaluation of the leiomyomas on d mpr can decrease the possibilities of intraoperative hemorrhage and uterine trauma. b d e f a g treatment of the symptomatic large and multiple uterine fibroids by uterine artery embolization s. speca, a.m. costantini, c. di stasi, g. tropeano, v. summaria, p. marano; rome/it purpose: to evaluate the potential usefulness of uterine artery embolization as an alternative to traditional surgery (hysterectomy or myomectomy) for the treatment of symptomatic large or multiple fibroids. material and methods: premenopausal women, aged - years (mean age . ), who presented with menorrhagia, pelvic pain, and/or mass-related symptoms due to large (< or = cm) or multiple fibroids (> or = cm) and were unwilling to undergo surgery or had an increased surgical risk, underwent bilateral uterine artery embolization. before embolization all pts underwent to a complete clinical examination, a basal fsh and estradiol dosage, a vaginal and ureteral tampon, a papanicolau test, a trans abdominal and trans vaginal ultrasonography and color doppler flowmetry. for embolization we used polyvinyl alcool particles, embosphere or spongostan. detailed clinical and ultrasound follow-up were obtained at regular intervals ( - months). the procedure was technically successful in / patients. a mean clinical follow-up of months for pain, mass-related symptoms and menorrhagia was done. % of the patients treated reported a marked to complete symptomatic improvement. only patient experienced no changes in her symptoms. nobody had immediate or late complications. a median reduction of % in the uterine volume and a median decrease of % in the dominant fibroid volume were observed in patients who had follow-up ultrasounds for up to months. conclusions: in our experience artery uterine embolization may provide an important therapeutic alternative for women with symptomatic large or multiple fibroids who desire to avoid surgery. how to conduct quantitative evaluation of uterine neoplasms using gddynamic contrast-enhanced mri k. hayasaka, y. tanaka materials and methods: women with histopathologically proved cervical carcinoma underwent preoperative t -weighted fast spin-echo, dynamic imaging using gradient-echo and postcontrast t -weighted spin-echo mr imaging with a phased-array surface coil. the axial plans in each sequence were reviewed at separate sessions by three radiologists blind to the histopathologic data. results: for the conspicuity of tumor, dynamic and t -weighted images showed high detectability more than postcontrast t -weighted images. most appropriate enhancing time in dynamic study was seconds. in assessing parametrial invasion, the accuracy of t -weighted, dynamic and contrast-enhanced t -weighted mr imaging was . %, . % and %, respectively; no statistically significant difference was observed. conclusion: dynamic imaging is useful in assessing tumor visualization, and the most adequate enhancing time in dynamic study is seconds. but in diagnosis of parametrial invasion, the addition of dynamic mr images does not improve the accuracy compared with t -weighted images alone. ( ), on clinically progressive disease ( ) or patient death ( ). patients had undergone previous surgery ( radical hysterectomy, trachelectomy). patients had non-surgical treatment (chemo and/or radiotherapy). a recurrent mass was identified in patients (mean size cm, . - cm). on t w, the recurrence was hyperintense in %. in the post-surgical group, the recurrence was in the vaginal stump in patients ( %) with parametrial involvement in ( %). in the non-surgical group, the recurrence was in the cervix in patients ( %) with involvement of vagina in % and uterus in %. uterosacral ligament and/ or pelvic sidewall involvement was present in patients ( %). bladder invasion was suspected in % and rectal in %. nodal enlargement was seen in %. bone metastases were present in patients while had dxt changes ( sacral insufficiency fractures). recurrence in the pelvic muscles was also seen. conclusion: recurrent disease in patients with cervical carcinoma usually involves the cervix or vagina, however can present with varied manifestations. knowledge of the site and pattern of disease recurrence can help in early and accurate detection of recurrence. hysterosalpingography: is still useful in the diagnosis of peritubal pathology? s. deftereos, j. manavis, g. alexiadis, g. kafetzis, p. prassopoulos; alexandroupolis/gr purpose: to reevaluate the role of hysterosalpingography in the diagnosis of adhesions related to infertility and in patients managment. materials and method: consecutive patients with more than two years infertility underwent hysterosalpingography (hsg), followed by laparoscopy. patients with infertility related to uterine abnormality were not included. diagnosis of peritubal adhesions was based on the presense of the following six imaging findings: convoluted tubes, vertical tubes, loculation of contrast medium in peritoneum, halo effect, ampullary dilatation or fixed laterodeviation of the uterus. results: in total, tubes were delinated. no abnormality was detected in tubes on both hsg and laparoscopy. laparoscopy disclosed adhesions in tubes. diagnosis of adhesions was made in tubes ( correct, false positive), when the presence of one abnormal sign on hsg was considered as criterion; and in , when two or more signs are present ( correct, false positive). ten live births five to eight months after hysterosalpingography occured. conclusions: accurate diagnosis of adhesions on hsg requires the presence of at least two abnormal signs. the six-month interval between hsg and laparoscopy might be shortened when adhesive peritubal involvement is radiologically suspected. laparoscopy is less indicated when no abnormality is detected on hsg. withdrawn to illustrate ct-urographic patterns of neoplasia of the collecting system. background: conventional excretory urography and conventional computed tomography (ct) were considered the standard techniques used to examine patients with urinary tract neoplasia. multi-detector row ct offers high speed of acquisition and high resolution images, allowing axial and d urographic acquisitions. imaging findings: pts with transitional cell carcinomas were analysed. all patients underwent unenhanced and post-contrast ct axial scans, followed by a urographic acquisition in the excretory phase ( mm collimation, . mm reconstruction interval). d reconstructions of excretory phase images were created with on an independent workstation using a mip algorithm. neoplastic lesions were visualised as solid papillomas in / cases, as wall thickening of the collecting system in / cases and as solid tissue in the renal sinus in / case. lesions were more evident on the source axial images of the excretory phase urographic acquisition. in the post-contrast images, the lesions resulted quite vascularized. urinary tract dilation was evaluated in / patients. dilation was better evaluated on the d mip ct-urographic images in the case of normal renal excretory function and on the source axial images of urographic acquisition in the case of functionally excluded kidneys. conclusion: ct-urographic axial and d images combined with conventional ct imaging allowed good evaluation of urinary tract neoplasia. the more frequent pattern was the wall thickening of the collecting system ( % cases), associated to urinary tract dilation ( % cases). renal background: lc is a minimally invasive surgical technique for patients with small renal masses; mr provides an effective tool for imaging follow-up of renal lesions treated with lc. methods and materials: pts with renal masses underwent renal lc. all patients underwent follow-up mri hrs after surgery, and at , , and months. mr examinations were performed using gre t w, tse t w and ce fs-gre t w sequences. two radiologists reviewed mr images for i) signal intensity, ii) size, iii) vascularization, and iv) perinephric changes after treatment. imaging findings: a) t w images showed isointense cryolesions, and cryolesions isointense to renal parenchyma with hypo-or hyper-intense foci. on t w images all cryolesions were hypo-intense with iso-or hyper-intense foci; b) hrs after treatment all cryolesions were more than cm larger than the original masses; cryolesions decreased in size of an average of % at month, % at , % at , and % at months; c) ce-fs-gre t w images showed complete ischemia of cryolesions in cases; d) cases showed a perinephric haematoma at , and months. the more significant mr pattern in the follow-up of renal lesions treated with lc were the decrease in size of the cryolesions over time and the complete ischemia of the cryolesions. ( ), benign obstruction ( ), not obstructed ( ) and transplanted kidneys ( ). procedure details: we used ultrasound-guidance in cases and ct-fluoroscopy-guidance in cases. the technique was standard seldinger. most patients received -or f. nephrostomy catheters. we had access failures, usually in patients with nondilated systems. we encountered major complications (bleeding and two big urinomas) and only minor complications. catheter dislodgement was relatively frequent ( cases). conclusion: urinary diversion at renal level is necessary in the following clinical situations: as short-term palliation in a terminal case, as a temporary measure prior to definitive diversion, to relieve obstruction where immediate surgical intervention is not feasible and for prolonged drainage where surgical intervention is not indicated. since its first description in , percutaneous nephrostomy has developed into a technique that is now routinely used for a wide range of clinical applications. imaging of angiomyolipoma focused on the clinical issues k.-s. cho, y. jung, s. kim, j. kim; seoul/kr learning objectives: to discuss the role and findings of us, ct and mri in the detection and diagnosis of angiomyolipoma. to discuss the complication of angiomyolipoma and confusion in differentiation from other malignant neoplasm. to discuss the clinical issues of angiomyolipoma including the natural history concerning growth, efficacy of biopsy for diagnosis, and association with tuberous sclerosis. background: angiomyolipoma is the most common benign neoplasm of the kidney and consists of mature adipose tissue, smooth muscle and thick-walled blood vessels derived from perivascular epithelioid cells. although most angiomyolipomas are asymptomatic and cause no problem, some cases raise important clinical issues. ( ) angiomyolipomas may be complicated by risk of hemorrhage and cause confusion in differentiation from malignant neoplasm when they contain minimal amount of fat. ( ) angiomyolipomas are often related with the stigmata of tuberous sclerosis. ( ) the natural history of this benign neoplasm has been controversial, with regard to growth on follow-up images. ( ) the efficacy of biopsy for diagnosis is controversial when image findings are indeterminate. procedure details: in this illustration, we evaluate the role and findings of ultrasonography, computed tomography and magnetic resonance imaging in the detection and diagnosis of angiomyolipoma. discussion will be focused on the important clinical issues. conclusion: multi-detector row ct is the most accurate diagnostic modality for evaluation of angiomyolipoma especially in cases with minimal amount of fat in tumor or associated with complication. mr can differentiate minimal fat angiomyolipoma from small renal cell carcinoma. characterization to understand how to further characterize incidentally detected renal masses found on ct by using the appropriate modality with optimal techniques. backgrounds: due to advances in ct technology and its wide spread use for imaging a variety of abdominal conditions, small renal masses are being incidentally detected more frequently on ct. since at least half of renal cell cancers are detected incidentally by imaging, the cost and time for further imaging of small renal masses may increase. in addition, some renal cell cancers may be cystic and it is important to have a strategy for dealing with cystic renal lesions that do not meet the ct criteria for simple cysts. imaging findings: we will discuss and show a variety of small incidental renal masses detected on unenhanced and single phase contrast-enhanced ct. topics discussed will include the following: ct criteria for distinguishing lesions that do not need further imaging for characterization from lesions that require further imaging for characterization; technical considerations affecting imaging findings; updated bosniak classification for cystic lesions; pitfalls in interpretation; and an algorithmic approach for "indeterminate" masses by using ultrasound, dedicated renal ct, mri, or percutaneous biopsy. the majority of small renal masses incidentally found on ct can be characterized or managed through an appropriate diagnostic pathway. color-duplex imaging (cdi) and magnetic resonance angiography (mra) in reno-vascular disease l. olivetti , g. rozzi , p. pecchini , p. ravani , l. grazioli , e. botturi , i. laparoscopic cryoablation of small renal cell carcinoma: medium term outcome. g. cardone, p. mangili, a. cestari, g. balconi; milan/it purpose: to determine safety and efficacy of laparoscopic cryoablation in the management of small renal cell carcinoma and to assess its medium term outcome. methods and materials: patients underwent laparoscopic cryoablation of tumors between july and june . all treatments were delivered under laparoscopic us guidance. patients were followed up clinically, biochemically and by mr imaging hrs after surgery, and subsequently at , , , , and months. results: hrs after treatment all cryolesions were more than cm larger than the original masses; cryolesions decreased in size by an average of % at month, % at , % at and % at months following cryoablation. early post-procedure mr images showed complete ischemia of all cryolesions. follow-up (mean months) revealed no evidence of recurrence in / patients. one patient showed local recurrence at months, one patient demonstrated an metachronous nodule in the same kidney at months and another patient showed a pancreatic metastatic nodule at months. no significant rise in creatinine was noted post-procedurally. after surgery, retroperitoneal effusion was found in all cases; / cases showed an intralesional haematoma, / cases showed low signal intensity foci due to haemostatic material and / cases showed a perilesional haematoma at and month follow-up. conclusions: our medium term experience suggests that laparoscopic cryoablation is a safe, well tolerated and minimally invasive therapy for small renal cell carcinoma, and mr is an effective imaging technique in the follow-up of renal lesions treated with laparoscopic cryoablation. results: echographic findings postbiopsy were normal, or with loss of corticomedullary differentiation, enlargement and gross edema. clinical indication in transplanted kidneys was deranged renal function, and in native kidneys, nephrotic syndrome. acute rejection was the most frequent diagnosis in transplanted, and focal glomerulosclerosis in non transplanted kidneys. we observed cases ( % of all procedures) of postbiopsy complications ( haematomas, perirenal collections, decreased vascularization, arterio-venous fistulaes and deranged renal function), but none of them were significant. of them ( . %) were in native kidneys, and the remaining cases ( . %) were in renal allografts. we show an alternative way to perform the procedure in order to minimize patient risks and maximize results. we conclude that complications may be more frequent in renal allografts than in native kidneys. the complex preoperative assessment of patients with renal masses with complex mri-study i. platitsyn, e. zaytseva, a. zubarev, v.v. gazhonova; moscow/ru purpose: to study possibilities of complex mri-study in patients with renal masses in preoperative planning of renal surgery. material and methods: patients with renal masses detected by us were evaluated with complex mri-study. complex mri-study included mri before and after ce, d ce-mra and d ce-mru using fast d mra gre sequence were performed with . t system (magnetom harmony, siemens). dsa was used as diagnostic correlation as a gold standard. the obtained mr-data were compared with the results of surgical operation and histopathology. the renal tumors were identified with mri-study in patients; patients had cystic lesions. hypervascular masses were found in patients, hypovascular masses in patients. in one cases of cystic lesion detected with mri-study the patient had hypovascular cysto-solid masses. next, renal vascular variants were revealed: unilateral ara were detected with d mra in cases, with dsa in cases; bilateral ara -in and cases (consequently); triple unilateral ara -in and cases (consequently); multiple ara -in ( arteries) and ( arteries) cases (consequently); the sensitivity of d ce-mra was %. d ce-mru provided high-quality images of the urinary tract in all cases. mrstudy showed an almost complete correlation with results of operative treatment, histopathology and dsa. conclusion: complex mri-study allows noninvasive preoperative assessment of renal tumors localization, morphology and vascularisation, renal arterial system, renal parenchyma, urinary tract and perirenal region. it is valuable for planning of renal surgery. in all cases, the d free-breathing sequence was superior to the breathhold rare sequences in terms of spatial resolution, with equivalent contrast, and without significant motion artifacts. as a result, the level and length of stenoses were always better demonstrated with the d sequence than with the breathhold rare sequences. mip reconstructions allowed analysis of the entire urinary tract with different projections. the duration of the d sequence ( minutes) was comparable to the overall duration of the rare sequence, which requires several slice positioning to explore the entire urinary tract. less t -weighted sequences (haste and trufisp) were superior to d sequences to analyse the cause of obstruction, as they visualised the ureteric wall and the adjacent structures. the d turbo spin echo sequence is robust and especially useful in case of tortuous ureters. this sequence has now replaced the rare sequences in our protocol. haste and trufisp sequences remain useful to analyse the ureteric wall and the adjacent structures. prenatal ultrasonographic findings of multicystic dysplastic kidney: emphasis on cyst distribution m. moon, j. cho, m. song, y. lee; seoul/kr purpose: we retrospectively analyzed multicystic dysplastic kidneys to evaluate the prenatal sonographic characteristics, emphasizing the distribution of cysts. a total of cases was included in this study. the sonographic assessment included the site of the involved kidney, the size of the multicystic dysplastic and the contralateral normal kidney, the distribution of cysts, and associated anomalies. according to the distribution of cysts, multicystic dysplastic kidneys were categorized as subcapsular and random distribution, and interobserver agreement was determined using cross table analysis. the largest longitudinal diameters of the multicystic and the contralateral normal kidney were measured and data were plotted on the normal reference chart. results: multicystic dysplastic kidney was left sided in . %, right sided in . % and bilateral in . %. subcapsular distribution of cysts was observed in . % (n = ) for radiologist , . % (n = ) for radiologist . interobserver agreement was excellent (k = . ). the longitudinal diameter of the multicystic dysplastic kidney was above the % in % and that of the contralateral normal kidney was normal in %. there were major anomalies in cases and fetal karyotyping was offered in cases including cases with associated major anomalies. the results were always normal. conclusion: subcapsular distribution of cysts in multicystic dysplastic kidney is more common than random distribution, so the distribution of cysts may be helpful in the prenatal diagnosis of multicystic dysplastic kidney. tubular extraction rate of mag in patients with impaired renal function s. beatovic, e. jakšic, r. han; belgrade/yu purpose: to analyze whether clearance of mercaptoacetyltriglycine (mag ), which is equal to its tubular extraction rate (ter) could serve as a sensitive parameter of renal function impairment. methods and materials: investigation was carried out in patients, who were divided into nine groups, according to the diagnosis and the degree of renal failure. dynamic renal scintigtraphy was performed minutes after i.v. injection of - mbq of mag . ter was determined by single-sample, volume distribution method using the standard solution of labeled mag . results: results of ter were correlated with blood urea nitrogen (bun), serum creatinine (cr) and creatinine clearance (ccr). significant linear correlation between ter and ccr was found (r = . ; p < . ). correlation between ter/ bun and ter/cr was exponential (r = - . and r = - . , respectively; p < . ). conclusion: analysis of our results show that ter is a more sensitive parameter than ccr, cr and bun, especially in mildly deteriorated function. in advanced renal failure, the sensitivity of ter is similar to the sensitivity of bun and cr. daily we found some significant correlation between pulsatility indices (pi), resistance indices (ri) and renal volume (trv), with creatinine blood level (cr) in the patients with normal evolution, if we correlated the parameters using -day data. there was significant correlation between ri and cr in patients with nf ( days, p = . ). in the patients with atn there was significant correlation between trv and cr ( days, p = . ). significant correlation between trv and cr was found in rd evolution ( days, p = . ) and between pi and cr ( days, p = . ) in patients with rejection. these results confirm the findings obtained by dissection in five cadavers. the analysis of ct scans of retro-extraperitoneal pelvic processes demonstrates the inferior extension of the renal fasciae to the pelvis. these fascial planes serve as pathway for the spread of retro-extraperitoneal processes from the abdomen to the pelvis and vice versa. one can so explain, for example, the occurrence of an abcess in the prevesical space in case of sigmoid diverticulitis. to investigate the effect of alpha-blockers on prostatic blood flow and by means of power doppler image quantification. materials and methods: patients suffering from lower urinary tract symptoms (luts) were treated with alpha-blocker for weeks. transrectal power doppler ultrasound (trpdus) of the periurethral prostate as well as comparative cystometry were performed before and after therapy. for trpdus an acuson sequoia (acuson, mountainview, usa) fitted with a high frequency transrectal probe (ec -c ) was used at standardized machine settings. color pixel density (cpd) was calculated with computer assistance from transrectal power doppler images using scion image image analysis software. cpd and standard urodynamic parameters were recorded in each run at filling volumes of ml, ml and maximum cystometric capacity. in presence of nacl, mean cpd rose by % at ml and by % at full distension, whereas with kcl filling, mean cpd rose by % at ml and by % at full bladder capacity. after therapy, mean cpd was significantly increased at empty bladder by % compared to mean cpd before therapy. during nacl filling, mean cpd rose by % at ml and by % at full distension, while in the presence of kc , mean cpd rose by % at ml and by % at full bladder capacity. conclusions: using trpdus and cpd, relative changes of periurethral prostatic blood flow could be quantified. alpha-blocker lead to a significant increase of prostatic blood flow. these results may explain the therapeutic effects of alphablockers on luts. superselective results: using grey-scale trus, prostate tumors were hypoechoic before treatment. using d pd trus, tumors ( %) were hypervascular before treatment. during treatment the tumors' echogenicity increased gradually, focal fibrosis was detected; the degree of vascularity decreased gradually. in months the fibrosis was not revealed in ( . %) tumors and the hypervascularity was revealed in ( . %) patients that correlated with negative clinical response to hormonal treatment in patients. conclusion: d trus is an informative method in the assessment of the response to hormonal treatment of pc. methods: seven patients with adenocarcinoma of the prostate were scanned supine on a . t system. prior to imrt and during the -week treatment course, mm transverse, coronal and sagital haste-t -weighted images of the pelvis were acquired weekly. the bladder and rectal volumes, position of the prostatic margins, and center of prostate (cop) relative to the bony pelvis were measured. results: all pre-treatment positions were within a . mm range from the ontreatment mean position in each patient. the cop variability in the ap, cc and ml directions were . , . and . mm, respectively. the largest prostatic margin variability was . (posterior) and . mm (cranial and caudal). beyond a rectal volume of . , a strong correlation was found between rectal volume and anterior cop movement (p = - . ). a weak correlation (p = - . ) was found between bladder volume and cranial cop movement. beyond . ml, the mean variability of the cranial and caudal margins increased up to . mm, between and ml . mm, and above ml . mm. the pre-treatment prostate positions were representative of ontreatment positions. a . mm ctv expansion in any direction was sufficient to ascertain % coverage of the ctv within the ptv, assuming a rectal volume < . ml (rectal suppository) and bladder volume around ml (voiding followed by drinking ml of water to keep small bowel away from treatment field). local results: there was no significant difference between the number and severity of complications in the group with local anesthesia compared to the group without anesthesia. there were no significant side effects caused by the infusion of the anesthesic drug. however the infusion of the drug may cause a degradation of the trus image. air bubbles are sometimes infused with the drug causing a "fuzzy" image, which results in a degree of difficulty in guiding the needle. a thorough trus examination of the prostate prior to the anesthesia helps the radiologist in planning the biopsies in advance and thus guiding the needle to the appropriate area, even through a "fuzzy" image. conclusion: us-guided local anesthesia, before trus biopsy, is useful for assuring patient cooperation during this often painful procedure and essential if trus findings call for a large (more than ) number of biopsies. conclusion: conventional mr images combined with pyelo-urographic techniques allowed a good evaluation of urinary tract neoplasia. more frequent patterns were solid papillomas or wall thickening of the collecting system ( % cases), associated with collecting system dilatation ( % cases). there is a "difficult zone" located - cm below the iliac vessels. examination with mild compression is used to solve this problem. color doppler sonography is valuable to distinguish the ureter from vessels. conclusion: abdominal sonography is a cheap and informative method in the diagnosis of ureteral disorders and may replace traditional technologies. trauma of the urinary system: spectrum of findings at helical ct a.j. madureira, c. tavares, l. melao, i. ramos; porto/pt learning objectives: to review and illustrate the spectrum of imaging findings associated with blunt and penetrating trauma to the urinary system on helical ct. to discuss the importance of an appropriate technique and the major pitfalls encountered. background: injury of the urinary system is a common complication of major abdominal trauma. helical ct is a powerful imaging modality in the evaluation of patients with suspected injuries of the adrenal gland, kidney, ureter and bladder. the renal injury ct classification system is valuable and clinically important as it serves as a guide for patient management. procedure details: the authors present a comprehensive review of the imaging findings in common and rare traumatic lesions of the adrenal gland, kidney, ureter and bladder based on the experience of a level trauma center. the indications for helical ct are discussed and a special emphasis is placed in the use of an appropriate technique and discussion of the major pitfalls that may be encountered. conclusion: helical ct is a useful modality in the diagnosis, grading and followup of patients with trauma to the urinary system. helical ct protocols should be optimized and adapted to the clinical situation in question. characterization learning objectives: this poster will present the anatomical waxes dedicated to urogenital anatomy which are displayed at the museum "la specola" of florence. background: teaching gross human anatomy is traditionally based on demonstration and study of the different organs and systems directly on corpses. however, availability of specimens for this purpose can be difficult for a variety of problems and, at present, anatomical models made of plastic material and electronic files are used in most schools of medicine. these problems have been always present in the history of medicine. to overcome this, in , a special laboratory was established at the "imperial regio museo di fisica e storia naturale" of florence to create a collection of anatomical waxes, copied from anatomic dissections, to enable three-dimensional anatomic studies with schematic drawings and captions indicating anatomical details. imaging findings: from to , the laboratory prepared a large number of models, and the collection in florence consists of about pieces. in the collection there are over preparations, showing the complete urogenital system in the male and the female. special models have been created to demon-strate sectional anatomy, the internal structures of the kidney, the relationships of the prostate with the bladder base and the seminal vesicles, the penis, the vagina and the uterine cavity. many waxes depict the gravid uterus and the foetus, with its vasculature and its relations with placental vessels. conclusion: this allows the collection to be considered as the first d "textbook" of anatomy in medical history. characterisation of adnexal masses with magnetic resonance imaging a. saini; london/uk learning objectives and background: the accurate assessment of adnexal masses remains a challenge. to determine key differentiating features of adnexal masses, we retrospectively reviewed the magnetic resonance (mr) characteristics in patients and correlated their appearances with the findings at histopathology. imaging findings: benign solid lesions were characterised by fat, haemorrhage or fibrous components. mature teratomas (n = ) possessed high fat content. haemorrhage was a predominant feature of endometriomas (n = ) but also seen in some malignant tumours. fibromas (n = ), cystadenofibromas (n = ), benign brenner tumours (n = ) and fibrothecomas (n = ) that have a similar fibrous component shared a distinctive short t relaxation. benign cystic lesions comprised serous or mucinous cystadenomas (n = ), were recognised as thin walled uni-or multilocular cysts. borderline (n = ) and malignant epithelial tumours (n = ) were predominantly cystic and distinguished by papillary projections and the secondary features of malignancy, such as ascites, rather than their tissue signal intensity. malignant solid primary ovarian masses were less common. clear cell carcinomas (n = ) were recognised by a predominantly solid mass with more modest cystic elements. granulosa cell tumours (n = ) had variable amounts of cystic change and intratumoural haemorrhage. immature teratomas (n = ) contained circumscribed foci of variable signal-intensities. inflammatory masses (n = ) also presented as complex adnexal cysts. in these cases, the clinical history and examination was key in making the diagnosis. the ability to manipulate tissue contrast with mr imaging makes it an invaluable tool in the assessment of complex adnexal masses enabling characterisation and identification of features associated with less common pathology. to discuss the imaging features so that when this tumor is encountered the diagnosis may be considered. to correlate the pathologic characteristics with the imaging findings. to discuss the key elements to be included in the differential diagnosis and to provide examples of them. background: urachal adenocarcinoma is an extremely rare malignant neoplasm that develops from embryonic remnants and has a variable clinical course. clinical, pathologic and imaging findings were retrospectively evaluated for patients with urachal adenocarcinoma referred from centers. imaging studies (ct, n = ; mri, n = ) were reviewed for morphologic features, such as tumor size, homogeneity, margins and degree of enhancement. imaging findings: the mean tumor size was . cm. the tumors were heterogeneous in / patients, with irregular margins in / , calcifications in / and strongly enhanced in / . all tumors extended anteriorly from the bladder dome; endoscopy was normal in all cases. enlarged lymph nodes were visible in / patients. histologically proven invasion of the bladder dome was detected in / patients and invasion of the anterior abdominal wall in . d reconstructions in the sagittal plane (n = ) evoked a urachal mass and assessed its relationship with the urinary bladder, umbilicus and rectus muscles. all tumors were resected. conclusion: multimodality imaging is essential to obtain an early and accurate diagnosis of urachal adenocarcinoma. multiplanar ct and mr give the best assessment of the extent of tumor invasion. reduction of ionising radiation exposure to patients due to new imaging technology for medical diagnostics of the urinary tract: a retrospective study covering years in a norwegian referral hospital a. nyquist , i. børretzen , h. olerud , b. bjørnarå , t. gudmundsen ; drammen/no, oslo/no purpose: to examine possible changes in ionising radiation doses to patients (collective effective dose) undergoing diagnostic imaging procedures of the urinary tract over the last years in view of shift in modalities from conventional x-ray examination to ultrasonography (us). retrospective study of all patient files for the period from to . the number of plain radiographs, intravenous pyelography (ivp), ultrasonography (us), computer tomography (ct) and magnetic resonance imaging (mri) examinations were registered. for each type of examination the mean effective radiation dose were obtained from the norwegian radiation protection authority (nrpa) as published in . the number of plain radiographs of the lower abdomen covering the urinary tract increased by approximately %, ivps were reduced by %, and ct examination decreased by %. mri examinations did not play any significant role in examining the urinary tract. us of the urinary tract increased from in to in . this shift in modalities from totally x-ray based procedures, to nearly exclusively us often supplemented with one plain radiograph, caused a decrease in the annual collective effective dose for this group of patients with % from . mansv in to mansv in . the shift in modalities used for diagnostic imaging of the urinary tract from conventional x-ray to almost exclusive use of us, resulted in a significant reduction in exposure to ionising radiation of patients, and need to be considered when discussing further development and structure of diagnostic imaging. comparison of the adrenal vein sampling value between primary aldosteronoma and nonfunctioning adrenal adenoma: evaluation with receiver operating characteristic ( , voiding cystourethrography, urethroscopy and biopsy. combined retrograde and voiding sug with saline and echocontrast media levovist® were used in pts with urethral obliteration and chronic suprapubic catheters. the transperineal or transrectal ultrasound was performed to evaluate anterior and posterior urethra respectively. the images obtained allowed detailed morphology assessment of the urethra at different levels. results: compared with rug, the sug more accurately measured stricture length and diameter that was confirmed by measurements at optical urethrotomy ( pts) and urethroplasty ( pts). the introduction of contrast agents enhanced the visibility of the flow in the most strictured segment (less than . mm), which was diagnosed by rug as urethral obliteration in pts. the ce sug accurately diagnosed diverticula ( ), false passages ( ), which were confirmed by urethroscopy in all pts. conclusion: our preliminary results show, that the combined retrograde and the voiding power doppler sonourethrography with ultrasound contrast media might be an effective support in diagnostics and in the planning of treatment of patients with complex strictures and urethral obliteration. ce sug is an informative method in the differential diagnostics of patients with urethral obliteration and complex strictures. differentiation of adrenal adenomas from metastases with unenhanced ct using a scoring system h. gufler , g. eichner , a. results: attenuation values on unenhanced ct were significantly lower for adenomas than for metastases ( . ± hu versus . ± . hu, p < . ). the combined score parameter including all ct criteria showed the largest area under the roc curve. the highest predictive power suggested by the model was calculated with a cut-off point at . for benign lesions with a sensitivity of . and a specificity of . . at . points the scoring system yielded a sensitivity of . % and a specificity of . %. conclusion: compared to densitometry alone, diagnostic accuracy in the differentiation between adrenal adenomas and metastases is improved by including all ct criteria in the evaluation. the presented scoring system is simple but efficient and easy to use in the clinical routine. eswl of the unusually located stones in pediatric patients z. siric, m. radovanovic, a. slavkovic; nis/yu aim: to show the possibilities of lithotripsy in the treatment of bladder and urethral stones in pediatric patients. in a twelve year period, we treated pediatric patients, aged from months to years, by eswl. in children, stones were located in the urinary bladder, and in the urethra in another . all of them were male patients; we decided to use eswl instead of invasive methods of treatment. eswl procedures were performed under general anesthesia in and under sedo-analgesia in patient. during treatment, the patients were placed in the prone position (patients with bladder stones) or in a modified supine position (perineal approach for urethral stones). an average shock waves were applied per patient with a maximal energy of . kv. average duration of treatment was minutes. results: successful fragmentation was achieved during the first session in patients. in patient with a urethral stone, the fragmentation was not satisfactory and the treatment was repeated days later. complete elimination of stone fragments was achieved during the first days in the patients with successful first treatment, but was not achieved at all in the patient with repeated treatment. cystoscopy was performed and the stone was found within a urethral diverticulum. there were no complications in our patient group. conclusion: eswl in our experience seems to be useful as a non-invasive method in the treatment of stones with in unusual locations. it could be recommended in male patients without anatomical variations of bladder and urethra. methods: this is a retrospective study of helical ct of patients that showed iliopsoas abnormalities ( neoplasms, abscesses, hematomas, cases of atrophy and of calcifications). the study group included men and women, - years old (mean years). all studies were obtained over a -year period. final diagnosis were correlated with different ct features to determine findings that could be used to differentiate these abnormalities. these included enlargement of the iliopsoas muscle, irregular margins and fat infiltration, attenuation of the lesion, calcification, bone fracture and associated adenopathy. result: psoas pathology is more frequent in men (p < . ). low attenuation and heterogeneity are the most reliable ct features of iliopsoas abscesses (p < . ). the sensitivity and specificity of low attenuation for the diagnosis of abscesses are % and % respectively. the specificity of heterogeneity for the diagnosis of abscesses is %. bone fracture is useful for the diagnosis of hematoma (p < . ). bone fracture is % sensitive and % specific for diagnosis of hematoma. conclusions: low attenuation, heterogeneity and bone fracture are helical computed tomography features useful for differentiating iliopsoas pathology. results: all masses were classified correctly by both analysis. the mean percentage of signal intensity on subtraction images were ± (means ± sd) and nonadenomas ± (means ± sd), respectively. the difference between two groups was statistically significant (t test, p = . ). our results show that chemical shift subtraction imaging is an objective technique that allows the adrenal adenomas to be differentiated from the other tumors. the objectivity of the technique may help the unexperienced radiologists to improve their interpretation. patient ( %) had uretheral dilatation. the mru visualized the dilatation and determined the obstruction level in cases ( %). the excretory urography detected uretheral dilatation and determined the obstruction level in cases ( %). the mru detects with great accuracy the dilatation and also, obstruction level in patients with obstructive uropathy and could be used as an alternative to excretory urography in specific cases. oro objective: many congenital dysplasias of the osseous labyrinth have been identified. differentiation of these dysplasias is essential for patient management. the purpose of this study is to describe the imaging findings which can be found in anomaly of the inner ear. we retrospectively reviewed imaging findings of children with congenital sensory neural hearing loss ( male, female, mean age . years, age range - years) who had ct (n = ) and mr imaging (n = ). ct was performed on a ge hispeed/i scanner (general electric medical systems, milwaukee, wis, usa). . mm thick direct coronal axial sections were obtained with bone algorithms. mr was performed on a ge signa mr/i with temporal t fse thin section mm scan and routine brain axial, coronal, flair, fse t , se t , coronal fse t , sagittal se t weighted images. results: there were cochlear aplasia with large vestibule (n = ), cochlear hypoplasia (n = ), mondini malformation with large vestibular aqueducts (n = ), mondini dysplasia with large vestibule (n = ), large vestibular aqueduct and endolymphatic sac (n = ), small internal auditory canal (n = ) and large vestibule (n = ). four cases were involved unilaterally. seven cases had combined deformities. five cases had cochlear implant. conclusions: both ct and mr can be used to look at inner ear malformations, but often both techniques are complementary. ct is preferred when associated middle or external ear malformations must be excluded. mri is preferred when subtle changes in the membranous labyrinth or abnormalities of the nerves in the internal auditory canal must be visualized. purpose: benign paroxysmal positional vertigo (bppv) is probably the most common cause of vertigo and the most common peripher vestibular disorder. bppv has been postulated to be the result of freely floating debris of degenerative otoconia in the endolymph of a semicircular canal (sc), canalolithiasis or debris that becomes adherent to the cupula of the semicircular canal. the key in determining the diagnosis of bppv is still examination performing positioning manovers and the use of frenzel glasses. differential diagnosis can be difficult. the aim of this study was therefore to provide a new diagnostic approach by d-nmr technique. our special interest has been if there was a structural change in the sc or the cupula, which could be identified in patients with incurable vertigo compared to those with self-limited disease. methods and materials: we investigated normal and bppv subjects using a . t ge mri with d data set: tr , te , mm slices, x matrix, x fov, . nex. results: all bppv patients had pathological filling defect of the sc before therapy which must be considered as an anomaly canalolithiasis. we postulate there must be structural changes in its lumen which makes the endothelium adhesive which is recognizable in hr- d-mr as a filling defect. conclusion: a significant improvement in quality of high-resolution mri of the inner ear using ciss could be obtained at . t despite potential drawbacks. to obtain at . t, the same snr would require approximately double measuring time, optionally increasing the risk for an accidental head movement which may prove detrimental at high resolution. ( ), dysplasia of the vestibule with or without abnormality of the lateral scc ( ), mondini-deformation ( ), cochlear dysplasia and absence of cochlear nerve ( ) and dysplasia of the modiolus ( ). one patient showed labyrinth hemorrhage at the time of mri. ten cases presented typical clinical features of a progressive hearing loss. in patients a constantly moderate or profound sensorineural hearing loss was present since early childhood. one patient was deaf. one patient suffered from a pendred syndrome. conclusion: a large vestibular aqueduct is a common finding in patients with suspected inner ear malformation. nevertheless, especially in isolated lva, it is sometimes missed in routine work. even if there is no therapy, an early diagnosis is needed to explain to the patient, the cause of hearing loss, the prognosis and behaviour which can possibly delay progression. color digital radiography density measurements in differentiation of periapical granulomas and radicular cysts i.k. rozylo-kalinowska; lublin/pl purpose: the differentiation of periapical granulomas and radicular cysts is crucial for decision on conservative or surgical treatment and therefore influences the success and long-term results of the treatment. although radiograms are a valuable diagnostic tool, relying solely on the evaluation of these images may lead to mistakes in the choice of treatment. the aim of the study was determination of possibile applications of digital radiography density measurements in differentiation of periapical granulomas and radicular cysts of inflammatory origin. the material consisted of digital periapical radiograms obtained using digora, rvg and dixi digital radiography systems in patients aged to , divided into two groups: granulomas and radicular cysts. by means of digora . software, maximum and minimum densities were measured along a line and the difference between these densities was calculated. the largest dimensions of the lesions were measured both perpendicular and parallel to the root canal axis. results: the differences between the results for granulomas and cysts were statistically significant. when the difference of densities exceeded . , the lesion was a cyst and when it was below . , a granuloma was diagnosed. combined application of two criteria, namely the calculated difference between densities and the largest dimension, increased diagnostic possibilities of radiological differentiation of granulomas and radicular cysts. conclusion: it was proved that to some extent it was possible to differentiate granulomas and radicular cysts by means of digital radiography software. ameloblastomas d e f a g x-rays, ct and mri. the use of d-ct and dentascan proved especially helpful in determining tumor dimensions. histological examination of the lesions showed that were of the follicular type, presented as mural ameloblastomas, were squamous type, plexiform and was a malignant ameloblastoma. results: in patient the extension of disease was proven inoperable. in patients various types of mandibulectomies along with immediate reconstruction were performed. in cases of mural ameloblastomas local excision only was performed, whereas in patients the mandibular defect was reconstructed with a titanium plate. conclusion: in large monolocular lesions treatment can be conservative surgery, whereas in large multilocular lesions radical surgical treatment that includes segmental ostectomies with immediate reconstruction of either vascularized or autogenous bone grafting is the treatment of choice and produces the best post operative results. the interpretation of the d reconstruction images and the dentascan are of great importance when such an operation is decided upon. differential growth in vestibular schwannoma g.c. bockeler, v. nandapalan, t. lesser, h. lewis-jones; liverpool/uk purpose: mri plays a pivotal role in the management of patients with vestibular schwannoma (vs), yet there are no universally agreed guidelines on the use of this expensive investigation. this study analyses the site, size and morphology of vs referred for possible intervention. where clinically a wait-and-watch approach was chosen, growth of vs was determined on subsequent imaging. methods and material: mr images of patients with unilateral vs referred to a tertiary centre were classified according to the site of vs with respect to the internal acoustic canal. a variety of measurements were obtained to determine the most sensitive method of measurement. growth was expressed as absolute and relative change in the direction of the largest increase in size. results: vs showed growth in % of patients, % remained static and % regressed. growth was most frequent in vs with an intra-and extracanalicular component whereas purely intracanalicular vs showed no growth. the initial size did not predict growth. growth rates were largest in predominantly extracanalicular vs. conclusion: our findings confirm previous findings of infrequent and smaller growth in purely intracanalicular vs and more frequent and larger growth in predominantly extracanalicular vs. an incremental delay in follow-up imaging of the former vs appears justifiable in the absence of new symptoms. conversely most vs require close monitoring as even small changes in size may lead to symptoms necessitating invasive treatment. sonographic sialadenitis/sialolithiasis: ultrasound is accurate in the assessment of acute and chronic inflammation and abscess formation. it has replaced sialography in many centres for evaluating suspected stone disease and duct dilatation. granulomatous disease: sarcoidosis and sjögren's syndrome may both involve the submandibular glands. ultrasound is able to delineate the phases of disease progression in sjögren's and is also used in lymphoma surveillance in these patients. other lesions of the submandibular space: including adenopathy, cystic hygroma, brachial cleft cyst, ranula and lipoma may mimic submandibular gland lesions and are readily identified sonographically. conclusion: ultrasound represents a safe, widely available and accurate means of assessing the submandibular space and is able to delineate and characterize lesions. ultrasound can be used to guide aspiration or biopsy and reduce the need for surgical excision. ultrasound-guided core biopsy of the parotid gland: results of patients k.k. lewis , m. williams , d.c. howlett , g. manjaly , a.b. moody , n. violaris ; eastbourne/uk, brighton/uk purpose: to evaluate the role of ultrasound-guided biopsy in the management of palpable parotid lesions. materials and methods: one hundred patients were included in this prospective study over a -year period. initial ultrasound examination was performed using a - mhz high frequency transducer. guided biopsies were taken under local anaesthesia (single operator) with a spring loaded variable throw ( mm/ mm) biopsy gun using or g needles with an average of passes per patient. results: in patients, biopsy revealed benign neoplasms, malignant lesions (including primary and secondary lesions as well as lymphomas) and other pathologies including sarcoidosis, tuberculosis, reactive lymphadenopathy, sjogren's and actinomycosis. only lymphoma patients needed surgery for further histological grading. diagnostic accuracy was % for benign versus malignant pathology in the patients who underwent subsequent surgery. there was % correlation between biopsy and operative histology with only two non-correlates. these involved misdiagnosis of squamous cell carcinoma and mucoepidermoid carcinoma. these would have required further histochemical staining for differentiation. surgery was avoided in forty-six patients after biopsy diagnosis. there were no immediate complications of biopsy. conclusion: ultrasound guided biopsy gives % benign versus malignant pathological differentiation. it also has % correlation with operative histology. correct pre-operative diagnosis influences the decision to undertake conservative or radical surgery and unnecessary surgery can be avoided. a core of tissue allows histopathological analysis where assessment of tissue architecture, tumour grading and immunochemical staining is possible. this is invaluable for accurate diagnosis. contrast enhancement of the cochlear aqueduct in mr imaging: its frequency and clinical significance t. nakamura , s. naganawa , h. fukatsu , y. sakurai , i. aoki , a. ninomiya , t. nakashima , t. ishigaki ; nagoya/jp, tochigi/jp purpose: there have been no previous reports on contrast enhancement of the cochlear aqueduct in magnetic resonance (mr) imaging. the purpose of the present study was to evaluate the frequency and significance of this finding. methods and materials: thirty-one patients ( men and women; age range - years) with otologic symptoms (sudden sensorineural hearing loss, vertigo or tinnitus) were examined using contrast-enhanced imaging on a . t mr scanner. the normal ear served as the control. two radiologists evaluated contrast enhancement in the area of the cochlear aqueduct. result: forty-eight of ears ( . %) showed contrast enhancement of the cochlear aqueduct, but no significant differences in the frequency of contrast enhancement were observed between patients with and patients without vertigo, tinnitus, sensorineural hearing loss, cerebellopontine angle tumors or a highriding jugular bulb. in addition, no gender or age-related differences were noted. conculusion: contrast enhancement of the cochlear aqueduct was frequently observed, but the frequency of enhancement in symptomatic ears was not significantly higher than in control ears. the results of this study may prove helpful in avoiding unnecessary examinations and potential diagnostic confusion. high-resolution mr imaging of basaliomas of the facial soft tissue with a microscopy coil h. gufler, c. zörb, w.s. rau; giessen/de purpose: to correlate the tumor extension measured on high-resolution mr images with histology. patients and methods: five patients with basaliomas of the facial soft tissue were examined before surgery with high-resolution mri using a . tesla system with a mm microscopy coil. axial t -and t -weighted tse images were obtained from all patients before gd-dtpa application and additional axial and/ or sagittal t -weighted sequences with and without fat suppression after cm application (fov mm, matrix x , slice thickness . mm, acquisition time - minutes). qualitative analysis of mr images was performed by two radiologists who measured the extension of the tumor and decided whether bone erosion or infiltration of the orbit or of the nasal cartilage was present. results: t -weighted unenhanced axial images proved to be the most useful sequences in predicting the extension of the basaliomas. for the evaluation of b d e f a g orbital invasion, additional sagittal t -weighted unenhanced images were helpful. the extension of the lesions correlated well with the results on histology. in one case, however, nasal cartilage infiltration was not correctly diagnosed on the basis of high resolution mr. conclusion: high-resolution mr imaging using a microscopy coil is a promising method to predict the exact extension of basaliomas of the facial soft tissue. three orbit mri of the superior oblique muscles to measure the asymmetry in patients with iv nerve paresis m.a. eleta, f. shokida, f. seclen, j. gabriel, c. zanchez, f.a. eleta; buenos aires/ar purpose: to evaluate the section of the superior oblique muscles (so) in patients with unilateral congenital or acquired pareses to detect asymmetry and the relationship between the degree of vertical deviation and mri measurements. method and materials: from march to september , patients underwent orbits mri. seventeen patients had unilateral so muscle paresis: congenital and acquired. fifteen patients had normal so. the area of the superior oblique muscle in the section of maximun width was obtained in primary gaze position, supra and infraversion. asymmetry of the superior oblique muscles was defined arbitrarily as the difference between both so in the same patient and the measurement was compared with the values obtained in the normal group of patients. results: mean maximal difference was . ± . mm (p < . ) between the healthy and paretic eye. in the normal group of patients it was . ± . mm (p < . ). eleven out of the patients with congenital paresis showed asymmetry of the superior oblique ( . %). asymmetry determination of the superior oblique muscles disclosed . % sensitivity and % specificity. the relationship between vertical deviation and the maximal difference between the section of the normal versus the paretic eye was p > . . conclusion: imaging of the superior oblique muscles in patients with congenital iv nerve paresis showed significant asymmetry in the section area with regard to the contralateral muscle. however, no correlation was found between the degree of vertical deviation and interocular asymmetry. high resolution gray scale and color doppler imaging was performed in more than patients using a high frequency linear array transducer. various planes of imaging of the eye correlating with drawings of normal anatomy are shown. views are used to illustrate the complex anatomical relationships. an overview of the normal anatomy and pathologic conditions is presented with clinical correlation. results: evaluation with high resolution gray-scale and color doppler ultrasound can often determine diagnosis without further imaging procedures. us is sometimes insufficient to evaluating a deep structures of the orbit. paradigmatic us images from different pathologic entities of the eye (for instance: intraocular foreign bodies, haematoma, cataract, vitreal pathology and ablation of the retina etc.) are shown. conclusion: adequate knowledge of the anatomy of the eye and orbit is needed to correctly limit the differential diagnostic possibility of pathology of the eye and orbit. us is an available method for an accurate diagnosis of the eye with ct, and preferably mr, being reserved for cases which require additional information about deep structure, or those with in which there is discrepancy between the sonographic and clinical diagnosis. nasopharyngeal angiofibroma: the role of angiography in diagnosis and treatment a. szymanska, r. pietura, a. drelich-zbroja, m. szymanski, m. szczerbo-trojanowska; lublin/pl background: nasopharyngeal angiofibroma (na) is a rare, benign, unencapsulated tumour affecting adolescent males. surgery and radiotherapy are main treatment modalities. we evaluated typical angiography findings of na, its role in defining tumour blood supply and treatment planning. material and methods: forty patients with jna ( males and females aged to ) underwent angiography. we assessed tumour vascular composition, its location in relation to the maxillary artery (ma), feeding vessels and feasibility of preoperative embolization. relationship between stage of tumour, presence of intracranial extension and internal carotid artery (ica) blood supply were statistically evaluated. results: on angiograms, all tumours presented intensive inhomogenous blush. . % of tumours were supplied by ma, % by ascending pharyngeal and . % by the facial artery. in % of tumours, feeding vessels originated from the ica and in ( . %) case from the vertebral artery. the relationship between ica blood supply and tumour stage was statistically significant. ica blood supply had no correlation with the presence of tumour intracranial extension. two patients with abundant ica supply were disqualified from surgery and underwent irradiation. in patients with previous external carotid atery ligation, preoperative embolization of vascular recurrent tumour was not feasible. in % of cases ma was displaced by tumour lateral extension. conclusions: angiography shows typical features of na (nasopharyngeal, highly vascular tumour supplied by ma), confirms diagnosis of this tumour and enables preoperative embolization. analysis of tumour blood supply is useful in determining best therapeutic approach. angiography visualises displaced ma, which helps the surgeon identify and ligate it during tumour removal. is an increased capsular width a reliable indirect indicator of temporomandibular joint effusion? f. tognini , d. manfredini , v. zampa , g. tognini , m. bosco ; pisa/it, parma/it purpose: to establish the most accurate cut-off value of ultrasonographic (us) capsular width which consents to discriminate between temporomandibular joints (tmj) with and without magnetic resonance (mr) effusion. the study group consisted of patients who sought treatment for temporomandibular disorders (tmd). all the tmjs (n = ) were evaluated in order to detect the presence of effusion by means of us and mr. ultrasonographic examination allowed measurement of the capsular width, in the sagittal-oblique scans, as the distance between the hyperechoic lines representing condylar surface and the glenoid fossa. after capsular width was measured, roc curve analysis was performed to establish the most accurate cut-off value to discriminate between joints with and without mr effusion. results: diagnostic accuracy of us to predict mr evidence of tmj effusion was good (area under the roc curve = . ). us sensitivity was high for values lower than the cut-off value of . mm (true positive rate (tpr) = . %; false positive rate (fpr) = . %), while specificity was high for values higher than the cut-off value of . mm (tpr = . %; fpr = . %). conclusion: ultrasonography proved to be accurate in detecting the presence of effusion in the temporomandibular joint. analysis of receiver operating characteristic curve seems to reveal that the critical area of capsular width indicating effusion is that around the value of mm. patients were referred for ct angiography of carotid arteries in our institution. of these patients, were found to have carotid body tumors and were included in the study. all tumors were confirmed with several imaging modalities (us, msct, mr, digital subtraction angiography and somatostatin scintigraphy) and with histologic examination performed on the resected surgical specimen. imaging findings: the us (including power-and color-doppler scanning), msct angiography, mr angiography, digital subtraction angiography and somatostatin scintigraphy findings of carotid body tumors were reviewed. tumors were unilateral in eight cases and bilateral in the remaining two patients. all lesions were well-defined (ranging in size from . to . cm) and located within the carotid bifurcation, causing splaying of the carotid branches. all tumors were highly vascularized (mainly by branches of the external carotid artery) and no sign of malignancy was identified. after pre-operative embolization, all lesions were surgically removed with no complications and were confirmed to be carotid body tumors at histology. the diagnostic possibility of a carotid body tumor has to be considered when a solid mass is detected within the carotid bifurcation. imaging studies are essential to differentiate carotid body tumors from other masses of the neck (lymphadenopathy, tumors, cysts, etc). imaging background: evaluation of cervical spaces is an important procedure for patients with cervical pathology because it assesses the prognosis of the patients and helps to select adequate treatment. ultrasound, computed tomography and magnetic resonance play an important role in the imaging diagnosis of this area. however, before examination of pathology, a clear understanding of the anatomy of cervical spaces is essential. the neck is divided into eight regions, enabling the radiologist to examine all areas of the neck in a systematic way in order not to miss a lesion. procedure details: we have reviewed all the normal studies of the neck that were performed during the last year using at least one of the three techniques. we selected the most illustrated cases to describe the anatomy of the cervical spaces. the neck is a complex anatomic area. this education exhibit helps the radiologist to have a better knowledge of imaging of the cervical spaces. preoperatively, the thyroid nodules were assesed in terms of number, compressibility, texture, complexity, margins, volume, vascularity, interactions with great vessels and the presence of thyroid, parathyroid and cervical lymph nodes. d usa multiplanar reconstructions (mpr) reslicing thyroid nodules for virtual surgery were created. us results were compared with intra-operative findings and final pathomorphology. results: fifty-three patients had operations ( lobectomies, thyreoidectomies including with lympadenectomies and thyroid resections). six cancers, adenomas, colloid nodules, goiters and with thyroiditis were found. demonstration of the nodules in three orthogonal planes by d usa and assessment of the nodule's character and vascularity helped to change surgical management in cases; in cases post puncture morphological diagnosis and in cases operation was rejected. d usa was superior to conventional d in differential diagnosis of thyroid nodules. the preliminary decision to perform a lobectomy or a total thyroidectomy was based on variety of factors, many of which were delineated by detailed ultrasound examination. preoperative evaluation of thyroid vascular anatomy and mpr of the nodules were of value for changing surgical management for thyroid resections and in rejecting the necessity of operation. conclusions: d usa is a useful complement to conventional ultrasound that allows precise preoperative evaluation of thyroid nodules which influences surgical management. the value of mr imaging and mr angiography in the differential diagnosis of carotid space tumors p. virtual laryngoscopy: comparison with fiberoptic laryngoscopical findings z. celej , a. wygoda , c. przeorek , w. przeorek , j. baron , a. siemianowicz ; gliwice/pl, katowice/pl purpose: to evaluate the concordance between virtual and direct laryngoscopy in the estimation of laryngeal carcinoma staging. material and methods: multislice ct and direct laryngoscopy were used to examine patients with laryngeal carcinoma. ct data were obtained on a sixteen row detector ( . mm slice thickness, pitch factor ) during free breathing and "e" phonation both before and after contrast enhancement. the patients were examined before radiotherapy and surgical treatment. postprocessing was performed using multiplanar rendering (mpr) and virtual laryngoscopy (vl) as a surface rendering algorithm with boundary density of - ,- and - hu. all ct examination results (mpr and vl) were compared with fiberoscopy. virtual images created at boundary density of - hu revealed better concordance to direct laryngoscopy. every case of laryngeal carcinoma, even plain infiltration at t stage, was correctly diagnosed by virtual endoscopy. the disorder of vocal cord function was better demonstrated in fiberoscopy. virtual laryngoscopy is complementary to fiberoptic endoscopy and should be combined with axial slices and mpr images for the estimation of laryngeal carcinoma staging. ultrasound characteristics and histopathologic correlation in primary thyroid carcinomas c. to describe the technique and problems related to spect/ pet/ct/us image fusion in the neck region and to demonstrate the clinical relevance of the information gained by multimodality imaging on a series of patients with neck tumors, lymph node metastasis, graves disease and chronic ent inflammations. background: small tumors or subtle changes in inflammatory diseases cannot be detected in only one imaging series. multimodality imaging allows for a combination of functional and anatomical information in one dataset and therefore gives more detailed informations on distinct changes. procedure details: for reproducible immobilization of the patient, an individual mold of the patient's head, neck and shoulder was produced using the bodyfix vacuum device (medical intelligence, schwabmünchen, germany). up to modality-specific markers were reproducibly attached to the skin and to the mold. the data-sets were transferred to the treon stealthstation navigation system (medtronic inc.) and image fusion was performed based on the markers and anatomical landmarks. image fusion allowed precise correlation of suspicious tracer-enhancements to tumorous contrast-enhanced nodules on the ct images. in patients with graves disease and ent inflammations focal disease in homogeneous tissues was uncovered due to increased tracer uptake. us images were used for screening and to depict vascularisation. the presented method aids in the detection of tumors and lymph node metastasis in the neck area, influencing the surgical strategy in most patients. in patients with graves disease and chronic inflammations the real amount of inflammation can be determined. lemierre's syndrome (necrobacillosis): the imaging findings s. powell, m. powell, d. bakshi, j. evans; liverpool/uk learning objectives: the purpose of our poster is to demonstrate the imaging findings in the rare and interesting lemierre's syndrome (necrobacillosis), to heighten clinincal suspicion and therefore allow more rapid diagnosis. background: lemierre's syndrome is the triad of pharyngitis, internal jugular vein thrombosis and pulmonary infection. the causative organism is fusobacterium necrophorum. this is a commonly fatal disease requiring both the radiologist and clinician to keep an index of suspicion. the diagnostic findings include pharyngitis with direct local invasion into the lateral pharyngeal space and thrombophlebitis of the internal jugular vein. metastatic infection is common and the usual site is pulmonary ( %). imaging findings: the imaging findings include pharyngitis with direct spread of infection into the lateral pharyngeal spaces. internal jugular vein thrombosis and pulmonary infiltration leading to cavitation are the next most common findings.other findings include pleural effusions, spinal and brain abscesses, septic arthritis and ivc thrombosis. a variety of imaging modalities are employed to assess the patient with disseminated fusobacterial infection. conclusion: our poster aims to provide a comprehensive collection of imaging findings in lemierre's syndrome. lymph node staging in head and neck cancer: a pictorial review s. connolly, h. lewis-jones, r. hanlon; liverpool/uk learning objective: the purpose of this poster is to describe and show the anatomical location of the lymph node levels as described by the american joint committee on cancer. it will also cover some of the difficult diagnostic areas involved in reporting head and neck staging images. background: as a radiological department attached to a large head and neck unit, we have wide experience of head and neck cancer. the extent and level of cervical node involvement is probably the most important prognostic factor for patients with a primary squamous cell carcinoma. node level has also been shown to be a highly significant predictor of survival. procedure details: using anatomical drawings and cross sectional images of pathology that we have encountered in our department, we will show examples of nodes in the described levels. the pictorial review will include examples of normal reactive nodes as well as typical examples of extracapsular spread, squamous cell cancer metastatic nodes and lymphoma. we will also depict some of the more unusual cases we have experienced, such as histologically proven recurrent metastatic nodes, which on imaging grounds appeared benign and cystic, along with some examples of intraparotid nodal disease. the pictorial review will allow greater understanding of the staging process of head and neck cancer and its implication on patient prognosis and survival. ten patients with squamous cell carcinoma of the neck (t - , n - ) planned for primary radiochemotherapy were examined at several time points in the course of the weeks therapy (before, after beginning, and at and weeks). the examinations were performed with aplio us-system (toshiba) with . mhz broadband transducer. pd and adf (either d and d) were applied pre and post application of ultrasound contrast agent (intravenous bolus of . ml sonovue). the methods were compared by visual assessment. results: both ce adf and pd are sufficient to depict blood supply. while pd delivers better b-mode information, adf allows continuous examination due to the lower mi thus giving more time to acquire d data set and determining signal intensity time curve after one bolus application of contrast. adf showed less artifacts concerning blooming and cluttering in number and extent in almost all cases. in cases, we were able to demonstrate a perfusion after weeks of therapy but in it was only possible using ce techniques. conclusion: although pd delivers more b-mode information adf is the method of choice since it has the same sensitivity for detecting blood vessels but allows continuous examination and is less susceptible for artifacts. conclusion: percutaneous islet cell transplantation appears to be a simple and clinical promising radiological intervention and an alternative to surgical pancreas transplantation in patients with type diabetes refractory to insulin therapy. imaging findings and procedure details: diagnostic imaging and interventional minimally invasive percutaneous procedures in disk diseases are reviewed through an interactive hypertext-based teaching file with images and movie demonstrations. ct, mr imaging and discogram findings in disk pathology will be described including degenerative disk diseases, spondylosis, disk herniations, traumatic disk diseases, septic diskitis, and postoperative disk diseases. techniques, indications, contraindications, complications and results of percutaneous minimally invasive procedures of intervertebral disk diseases will be described and illustrated. these procedures include percutaneous periradicular and epidural steroids injection, percutaneous diskogram, diskal biopsy and drainage, percutaneous radiofrequency and laser nucleotomy. conclusion: for disk disease with appropriate indications, minimally invasive interventional radiologic procedures are able to relieve pain and to minimize the risk of disability. colonic stents in acute malignant colorectal obstruction: cases p. bermudez bencerrey, j. falco fages, j. perendreu sans, j. fortuño andrés, m. alcantara moral, d. gil bello; sabadell/es learning objectives: to illustrate our experience with colonic stents in the treatment of acute obstruction from colorectal cancer. to describe the results and complications of the procedure based on a series of patients. background: colorectal cancer is an important health problem with a high morbidity and mortality rate. between - % of patients with colon cancer present with acute obstruction. % of acute obstructions are in the left colon and rectum, and only % of left sided carcinoma colonic obstructions can be treated with intraoperative lavage and subtotal colectomy. the rest of the patients need a temporary or, more probably, permanent colostomy with an important impact on quality of life. in this exhibit we will describe the use of colonic stents as a temporary treatment prior to elective surgery of left sided colon and rectal cancer and as a palliative treatment in the unresectable colon cancer. between july and august , patients with acute colonic obstruction were treated with colonic stents in our department. the procedure, indications, contraindications, and the results are described and illustrated. procedure details: plain abdominal radiography and abdominal ct was performed to all patients prior to the procedure. two different types of colon stents were used: enteral wallstent (boston scientific) and hanarostent (mitech co., ltd.) conclusion: clinical resolution of the obstruction was achieved in %. anastomosis was successfully performed in % of the patients undergoing surgery. major complications developed in % ( perforations, obstructions and migrations). palliative treatment was effective in all cases. saline injection of solid tumours prior to radiofrequency ablation: our experience a.m. camenzuli, a. attard, j.c. evans; liverpool/uk learning objectives: . to outline the procedure we have adopted to instil saline into the tumour. . to describe the appearances of complete necrosis on the inital, early and late follow-up scans. . to describe the complications and patterns of recurrence of tumours after radiofrequency ablation. background: the injection of saline to potentiate the effects of radiofrequency ablation in solid organ tumours in experimental models is well described in the literature. we have injected saline prior to ablation into primary hepatocellular carcinomas, colorectal metastases to the liver and primary renal cell carcinomas. procedure and iimaging details: using either ultrasound guidance or ct and under general anaesthesia, we lace the tumour with ml of % saline via a coaxial needle. a radioablation probe is then placed along the needle -the latter is then removed prior to applying the rf pulse. we routinely ablate the track along which the probe has travelled to avoid seeding. routine follow-up with a dual phase abdominal ct scan is done at month and months. the appearances of ablated tumours, recurrences and complications are reviewed in this presentation. conclusion: saline infiltration is a safe and effective way of potentiating radiofrequency ablation of solid tumours in the liver and kidneys. percutaneous background: renal abscess is a collection of purulent material confined to the renal parenchyma. it is often life threatening leading to septicemia and is difficult to manage only with the use of antibiotics especially in immuno-compromised patients. ct is currently the most accurate modality in detecting and following renal abscesses. treatment with ct-guided percutaneous drainage and appropriate antibiotics can be very useful. in a period of two years we treated patients with renal abscesses. we demonstrate the technique and discuss the indications and complications of ct-guided percutaneous drainage of renal abscesses. the procedure is performed with the patient in prone position under local anesthesia supplemented with minimal sedation. initially a gauge chiba needle is passed into the renal abscess under ct guidance. on confirmation of an abscess we use a pigtail trocar catheter of to f for the drainage. conclusion: ct-guided percutaneous drainage is an effective and well-tolerated method for the therapeutic management of renal abscesses. the imaging features and percutaneous drainage of mediastinal abscesses secondary to oropharyngeal infection s. sadiq, s. owen, r. evans; swansea/uk learning objectives: oropharyngeal infection, such as dental abscess and acute pharyngitis, are recognised but rare causes of mediastinal abscess formation. we highlight the use of multimodality imaging and percutaneous image guided drainage. the hospital records of four such patients, including casenotes, microbiology data and imaging were reviewed. the clinical courses were followed in order to identify the causative pathogen and illustrate the imaging features and subsequent radiological interventional management. the patients presented over a two-year period. the age range of these patients was sixteen to forty seven. the primary aetiology in three patients was a dental abscess. the fourth patient had an acute pharyngitis. image findings: a combination of ultrasound (us), computed tomography (ct) and magnetic resonance imaging (mri) of the neck and thorax was used to define the anatomical compartments involved. radiological placement of an fpigtail catheter using us or ct avoided a major surgical procedure in all patients. the common pathogen isolated was streptococcus viridens. patients were treated with the appropriate antibiotics and all made an uneventful recovery. conclusion: an understanding of the anatomical spaces of the neck and their communication with the mediastinum is demonstrated and we recommend that in this condition, which has a high mortality when managed surgically, interventional radiology is an effective treatment option with a low complication rate. complications occurring after drainage are not uncommon and add to the morbidity of the procedure. the authors display their experience in handling complications (remedial procedures with appropriate images) that occurred in patients with malignant biliary obstruction, in whom ptbd and/or stenting was performed over a -year-period. the authors will also list precautions to be observed to prevent such complications. procedure details: severe hemorrhage was treated by catheter repositioning, angiographic embolization of pseudoaneurysm and coil embolization of a portal vein branch rent via a ptbd tract. cholangitis and bilomas required placement of additional drainage catheters. a migrated plastic stent was repositioned with vascular snare. a ring biliary catheter that broke during procedure, the proximal part lying in peritoneal cavity, was removed by image guided forceps, followed by replacement. blocked plastic stents were removed endoscopically or via the percutaneous route by canulation with a guidewire followed by replacement. ring biliary catheter and additional stents plastic/metallic stents were used for blocked metallic stents. pericatheter leaks responded to flushing, wire passage or larger catheter exchange. conclusion: it is important to be aware of complications that occur during ptbd and stenting, and how to deal with them before embarking on a full time biliary decompression program. intellectual property: development and protection c. cook , m. rees ; weston-super-mare/uk, bristol/uk learning objective: to discuss the different methods of intellectual property (ip) protection. to review the methods of interventional radiology device product development. background: there are many ways of protecting intellectual property. we discuss each of these in turn. we discuss the differences between the methods used in the united states and the european union. we also discuss the methods of product development. details: trademark, copyright, patents are all reviewed. we discuss the need for patent attorneys and specialist ip management units. we describe the different types and levels of protection available. we also discuss specific means by which an idea can be developed, then protected. we look at the methods available to link with development companies, leading to manufacture. all the principles are specifically discussed with respect to the development of a interventional radiological device. there are numerous ways of protecting ip, and we describe each of these in turn. in addition we look at the means of developing and ultimately manufacturing a radiological interventional device. purpose: ethanol injection (pei) and radiofrequency (rf) are the percutaneous radical therapies most frequently employed in europe to treat liver tumors in non surgical patients. we present our experience in these techniques over the last years at the hospital clinic in barcelona. since multisession pei was applied in patients with hepatocellular carcinoma (hcc) (uninodular ≤ cm or multinodular ≤ cm). rf was performed in patients since : hcc, metastasis and others. ct and us (with contrast agents in the later years) were used for efficacy assessment. results: pei: initial complete response (cr) = . %. extra and intratumoral recurrence = and %. one, and year survival rates in child a patients = , and % and in child b patients = , and % (mean follow-up = months). rf: cr in hcc patients = %. extra and intratumoral recurrence = and %. one, and years survival rates = , and % (mean follow-up = months). in patients with metastasis cr = %, with a recurrence rate = % (mean follow-up = months). major complications, that includes hemoperitoneum, liver abscess, needle-tract seeding and cardiovascular death are . % in pei and . % in rf. conclusion: pei and rf are effective local curative methods in liver tumors, with more frequent major complications in the rf group. and excellent ( - ). the efficacy was also evaluated with the days until the patients unable to move started to walk again after pvp. results: pvp was technically successful in all patients with three cases of minimal complications. the mean vas score for patients was . before pvp compared with . after the procedure. on the degree of improvement, the groups of ineffective, poor, effective and excellent consisted of . , . , . and . % of patients, respectively. patients ( . %) could walk the next day after pvp and the mean value until ambulation was . days. conclusion: pvp is a safe and effective treatment for relieving severe pain of osteoporotic vertebral compression fractures, leading to avoid various complications associated with prolonged immobilization in aged patients. effect of vessel size on creation of pulmonary radiofrequency lesions in sheep: assessment of the "heat sink" effect k. steinke , k.s. haghighi , k.k. hazratwala , d.l. morris ; basle/ch, sydney/au purpose: to evaluate the effect of vessel size on radiofrequency lesion creation in the lung with respect to potential for perfusion-mediated heat sink effect and for vascular and bronchial injury. methodology: radiofrequency lesions targeted to tissue encompassing a variety of vessels were created in vivo in the lung of cross-bred sheep. the access was either open after thoracotomy along the th intercostal space, or percutaneous. acute, subacute and chronic changes have been investigated, with immediate, hours and days sacrifice of the sheep post ablation. macroscopic and histopathologic analysis of the vessels and bronchi was performed. the degree of vascular and bronchial injury and viability of perivascular pneumocytes were recorded. results: heat sink effect, macroscopically indicated by invagination of the lung tissue between the vessel and the radiofrequency lesion, was not observed in vessels with a diameter less than mm. half of the vessels mm or smaller were thrombosed, while the arteries of the same size looked to be patent. macroscopically no vessel wall injury could be detected. all vessels mm and above showed a certain extent of invagination, no clots were visible. occasionally within the bronchial lumen small amounts of dark solid material was seen, macroscopically believed to be consolidated blood-tinged sputum. the histopathological results are expected soon. conclusion: as already shown for liver vessels, lung vessels seem to also have a perfusion-related heat sink effect, consistently seen in vessels above mm in diameter. peritoneal seeding of hepatocellular carcinoma after radiofrequency ablation: post-rf bleeding was detected in cases. two of these patients had hepatic arterial embolization performed due to severe bleeding. in terms of seeding pattern, massive perihepatic and ometal masses were seen in cases and multiple small peritoneal nodules were noted in a further cases. other cases showed small single mesenteric mass and gastrohepatic ligament thickening. the rate of peritoneal tumor seeding after rf ablation of hcc was . %. the capsular attachment, tumor bulging and post-rf bleeding seems to be risk factor of peritoneal seeding after rf ablation of hcc. r. marcello, l. broglia, m. castellana, g. gasparini, m. castrucci; rome/it purpose: the aim of our study was the evaluation of the spio contrast-enhanced mr pattern of malignant focal liver lesions treated with percutaneous radiofrequency ablation. methods and materials: focal liver lesions were evaluated with magnetic resonance ( . tesla philips, the netherlands), with spio (resovist schering, germany) contrast media after rf percutaneous ablation. lesions ranged in size from . cm. to . cm. mr examination sequences protocol was as follows: tfe t w and ffe dynamic study before contrast media injection and after and sec spio iv administration; tse t w and spir tse t w before contrast media injection and after - min after spio injection. all patients underwent mr imaging evaluation after days, days and months the procedure was carried out. the signal intensity appearance was examined in order to identify size, signs of absence, residual or recurrence of neoplastic tissue. the gold-standard was clinical and ct imaging follow-up. results: spio enhanced sequences were superior in conspicuity, presence or absence and residual or recurrence of tumoral tissue. in % of lesions a homogeneous pattern was observed within the lesion with a hyperintense peripheral rim; in % of cases inhomogeneity of lesions was observed in examinations performed at days. residual tumor was depicted at days examination as presence of hyperintense foci localized within the treated area. in this preliminary study spio appeared to be an efficacious contrast agent for detection and follow-up of percutaneous rf ablated focal hepatic lesions, especially on t w and the sec t w dynamic mr study. radiofrequency ablation of hepatic dome lesions g. papaioannou, l. thanos, a. nikita, e. alexopoulou, d. loggitsi, d.a. kelekis; athens/gr purpose: to present the additional difficulties and technical peculiarities of radiofrequency ablation (rf) for hepatic dome lesions. materials and methods: patients with focal subphrenic hepatic lesions ( hccs, metastases) underwent percutaneous ct-guided rf ablation. electrodes with or active tips were advanced under ct-guidance. although technically difficult due to the great needle angulation and penetration of a long hepatic portion, transhepatic route was followed in all cases. transpulmonary penetration was considered high-risk due to the large diameter of the electrode. results: all patients experienced mild discomfort during the procedure. three complications were noted: small subcapsular hematomas and pleural effusion, which were treated conservatively. % of the hccs and . % of the metastatic lesions showed signs of complete necrosis immediately post procedure. residual non-necrotic tumour was present in cases and repetition of the procedure was required. tumour recurrence was demonstrated in patients ( . %) on -year follow-up control and was treated with a new session of rf ablation. overall, rf sessions were performed. conclusion: hepatic dome lesions can be treated successfully with rf ablation. careful planning and technical skill are required due to their location. results: no treatment complications were noted. the patients recovered well post injection. ct performed in both children after three months showed ossification of the lytic lesions, allowing the cervical braces to be removed. conclusions: to our knowledge no prior experience in endosteal cervical steroid injection has been reported. these two cases confirm the improvement of osteolytic lesions in children with lch after treatment with endosteal infusion of steroids. stabilisation of the cervical spine was particularly beneficial, as the children were no longer in pain and removed the need for cervical braces three months after the treatment. the influence of radiofrequency ablation on hepatic vessels in porcine liver k. satoh, k. nakamura, m. hamuro, y. sakai, n. nishida, y. inoue, r. yamada; osaka/jp purpose: the objective of this study was to clarify the influence of radiofrequency ablation on hepatic blood vessels. hepatic rfa was performed on swine (mean body weight: kg). the livers were removed either immediately following ablation, week or weeks after ablation. the patency of vessel was determined by ct, and the extent of endothelial disorder with diameter less than . mm was determined histopathologicaly. the portal vein patency rate of immediately, or weeks after rfa were: . ± . %, . ± . % (less than . mm), . ± . %, . ± . % ( . mm and more). hepatic vein patency rate were: . ± . %, . ± . % (less than . mm), . ± . %, . ± . % ( . mm and more). the patency rate was higher for larger vessel diameters. the patency rate of the portal and hepatic veins with diameters less than . mm was found to decrease with time. at weeks after ablation, the patency rate for vessels with diameters of . mm and over were significantly higher (p < . ) compared to those of diameters less than . mm. endothelialization were found in patent vessels from week to weeks after ablation. conclusion: endothelization was confirmed from week to weeks after rfa in patent vessels. blood vessels with diameters of . mm and over maintain high patency even after rfa. it is therefore possible to maintain blood flow in the distal area of hepatic parenchyma following ablation for hepatic tumors, thus rfa has a limited impact on hepatic function. in three cases, a successful second trial after an initial failure was performed. the procedure was performed by means of a transhepatic (n = ) or a transperitonea l (n = ) access route. during procedure, the seldinger technique was used (transhepatic approach, n = , transperitoneal approach, n = ) in patients, while in the remaining patients the procedure were performed using a trocar technique with a transperitoneal approach. chiba needles ( g, mdtech, denmark, n = ), jelco needles ( g, johnson & johnson, usa, n = ) and secalon catheters ( g, ohmeda, uk, n = ) were used for initial puncture of gb. only minor complications such as abdominal pain (n = ), hemorrhage (n = ), referred pain (n = ) occurred in cases ( %). abdominal pain is more frequent in transhepatic approach ( %) than in transperitoneal approach ( %), but not statistically significant (p = . ). overall complication rate is lower in transperitoneal approach ( %) than in transhepatic approach ( %)(p = . ). according to puncture needle, there is no significant difference in complication rate between the chiba needle ( %) and the g needle (jelco needle and secalon catheter, %). conclusion: percutaneous cholecystostomy has proved to be safe and effective treatment for patients with acute cholecystitis. t.v. bartolotta, t. angileri, m. midiri, f. sorrentino, g. sparacia, a. carcione; palermo/it purpose: to evaluate the ct role in treatment of lumbosciatalgia through chemonucleolysis with oxygen-ozone (o -o ) mixture with periradicular and periganglionic technique. materials and methods: from january to june , patients ( women, men; age range: - years) with lumbosciatalgia, underwent intradiscal, periradicular and periganglionic treatment with o -o mixture, corticosteroids and anaesthetics. the analysis of the results was made by visual analogue scale (vas) day before treatment (t ) and, respectively, weeks (t ), months (t ), months (t ) later. all the procedure was performed in a step-wise fashion and controlled by ct scans: from positioning the needle in the centre of the nucleus polposus to mixture administration within the nucleus and in periradicular and periganglionic sites. results: in / ( . %) patients a partial remission of the complaints just after the first treatment (vas at t ) was observed; vas was at t and at the last evaluation, six months later (t ), it was . in / ( . %) patients vas was at t and another treatment was performed, with an improvement of vas at t and t respectively of and in / cases. the remaining / patients did not show any improvement even after the second treatment and vas ranged from to at t . conclusions: chemonucleolysis with o -o mixture with periradicular and periganglionic technique under ct guidance is a reliable and competitive method in treatment of lumbosciatalgia. multiple in patients with malignant biliary obstruction of the hilum and unavailable bilateral access, drainage of biliary tree was obtained with one percutaneous access through which multiple stent deployment was performed with a cross-positioning technique. the first self-expandable endoprosthesis was positioned in the common bile duct. thereafter a balloon catheter was inserted along a previously introduced guide wire to dilate the mesh of the stent. after having removed the catheter, a second stent was implanted through the dilated wall of the first stent, across the hilum. overall stents have been cross-implanted. in patients stents have been inserted, in patient stents, in another patient cross-positioning of stents was required. results: no relevant complications have occurred. patient survival and stent patency rates were comparable with those of multiple-access stenting technique. the procedure is generally more protracted implying an increased radiation exposure for both the patient and the operator. conclusion: biliary stenting with one percutaneous access and cross-positioning technique is a useful procedure in all cases in which bilateral access in not possible. it reduces the risks connected to multiple passages of liver parenchyma though at the expense of a longer time of exposure for both the patient and the radiologist. results: in ( %) patients pain relief was complete within one to two days after injection. one ( %) patient with multilevel hemangioma failed to respond. we did not observe any complications. conclusion: percutaneous vertebroplasty is a promising therapy for patients with painful vertebral hemangiomas. this method is effective, with low risk of complications, and may be appliciable to asymptomatic cases as a preventive treatment. percutaneous a retrospective analysis was performed of percutaneous nephrostomies in patients ( male, female; age range: days- years). twenty patients had bilateral nephrostomies and patients were recatheterized. the procedures were performed with sonographic and fluoroscopic guidance under sedation or general anesthesia. the catheters ( . - fr) were placed either with a seldinger technique or an accustick introduction system. results: percutaneous nephrostmy catheters were placed due to up junction obstruction in patients, uv junction obstruction in patients, posterior urethral valve in patients, neurogenic bladder in patients, congenital anomalies in patients, trauma in patients, stones in patients, tumor in patient and renal insufficiency in patients. pyonephrosis was detected in patients. nine dislodged catheters were replaced percutaneously. all procedures were carried out successfully. no procedure related mortality was seen. one patient arrested right after the procedure because of methemoglobinemia due to local anesthetic and treated with methylene blue. no other major complications were detected. catheter dislodgement and infection were detected in and patients, respectively. duration of catheterization was between and days. all the catheters were removed after medical, surgical or percutaneous treatments. conclusion: image guided percutaneous nephrostomy in pediatric population is a safe and effective procedure with low mortality, morbidity and high success rates. percutaneous ct-guided facet screw fixation in lumbar spines: technical aspect h.y. kang, s.h. lee; seoul korea/kr purpose: to report our experience of the technique of percutaneous ct-guided facet screw fixation of cases in lumbar spines. the ct gantry has an angle tilt limitation of approximately . degrees, but the z-axis (coronal) angle of facet joints is about - degrees. thus under local anesthesia, positioning was head first, prone with cephalad elevation of upper body to compensate for the facet angle. after selecting the route of the procedure, the guide wire and fixed screws were inserted, after measurement of the size of fitting screw. this procedure was performed in patients, with combination of anterior lumbar interbody fusion (alif, patients) and cases of discitis ( patients). results: all procedures of facet screw fixation with alif were performed successfully, there were no definite malpositioning of screw or segmental instability on postoperative images at one month follow-up. in cases of discitis, the procedure were performed also successfully, but with more slightly decreased disc height on image at one month follow-up. the controlateral translaminar facet screw fixations were performed at the levels of l - and l - , and ipsilateral transfacet screw fixations were done at l -s level. conclusion: percutaneous ct-guided facet screw fixation in lumbar spines was a safe and reliable procedure for a method of spinal fusion. minimally invasive management of benign biliary diseases a. doros, v. weszelits, a. peter, a. nemeth; budapest/hu purpose: report our experience in treating percutaneously benign biliary strictures, fistulas, post-operative leakages, residual stones/sludge, bilomas and abscesses. in the last five years, patients ( women, men, - years) with benign biliary diseases were referred to our interventional radiology unit. twenty patients underwent laparoscopic or open cholecystectomy, six had residual stones, three had biliary fistulas. twenty patients had hepatico-or duodeno-jejunostomy stenoses, four had cholangitis-induced liver abscesses, and three had chronic pancreatitis. results: forty percutaneous biliary interventions were performed with good longterm results. strictures were dilated times. six metallic stents were placed into three patients. two plastic stents were placed in one patient with a rendez-vous maneuver. two fistulas were treated with drainage and glue. five stones were removed percutaneously with balloons or dormia baskets. one patient with chronic pancreatitis was treated with a metallic stent. another patient underwent insertion of three metallic stents for intrahepatic stenosis. of the four abscesses, two were surgically and one was percutaneously drained. one patient had two small abscesses treated with iv antibiotics. conclusions: percutaneous management is useful in selected benign biliary diseases. dilatation, stone extraction, and abscess drainage can be performed successfully. in difficult cases combined surgical, endoscopic and percutaneous approach must be chosen. role of aspiration-lavage in the treatment of abscesses in the gallbladder bed after laparoscopic cholecystectomy a.b. stoian, z.a. sparchez, r. badea; cluj napoca/ro purpose: the evaluation of the efficacy of ultrasound guided aspiration followed by lavage with antibiotics in the treatment of gallbladder bed abscesses after laparoscopic cholecystectomy. materials and method: patients with gallbladder bed abscesses were treated percutaneously between - . the abscesses were located in gallbladder bed with extension into subhepatic area % ( / ). the volume of aspirated pus was - ml (average ml). the first step of the treatment was the abscess puncture by a transhepatic % ( / ) or another approach % ( / ) using an g needle ( . mm). after pus aspiration, the cavity was washed - times with a metronidazol + ampicillin solution, the amount of antibiotics once injected being half the amount of the aspirated pus. oral or iv antibiotics (corresponding to antibiogram) were administrated for - days. in the case of fluid reaccumulation the treatment was repeated. background: ptpe is a reliable tool to induce liver regeneration of the nonembolized lobe. this facilitates extended right resection in e.g. klatskin tumor, hcc or liver metasases. between and we performed ptpe. in three cases we noted a failure in regeneration of segment ii + iii but a growth of segment iv after right lobe embolization. in portal vein ligations which regularly spare segment iv the regenerative outcome was significantly reduced compared to ptpe including segment iv. procedure details: in / patients we use a subxiphoidal access to puncture the left portal vein under us-guidance, otherwise a transhepatic right intercostal access. via f guiding catheters a . f microcatheter is placed in each segmental branch in order to apply - ml histoacryl-lipiodol solution in total. segment iv branches are difficult to reach because of the configuration of the rex recessus anatomy. since the left lateral segments lie adjacent, the emolization of segment iv is critical and has to be performed with the utmost care to prevent spill over of embolzation material. conclusion: segment iv turnes out to be the key for optimal portal vein embolization results. although bearing the risk of embolization material displacement to segment ii and iii the better results clearly suggest application of this technique. . palliation of duodenal obstruction via trans-oral route (n = ) and trans-gastric route (n = ). procedural details and results: for group and , and the trans-oral route in group , the technique is essentially an extension to the well established technique of oesophageal stenting of malignancy (except longer wires and catheters are used when more distal stenting is required). as more distal lesions are tackled, the intrinsic difficulty of manipulating long wires and catheters increases and gives way to the more direct transgastric route approach of group . this is also an extension to the well established technique of radiological insertion of gastrostomy using t-bar fixation. failure of insertion occurred in patients in the duodenal obstruction group via the trans-oral route. there were no immediate or delayed complications and no re-interventions were required. five patients ( %) returned to normal diet, patients managed semi-solid diet ( %) and patients ( %) had no significant improvement post stent insertion respectively. the mean survival for groups ( ) and ( ) (i.e. primary carcinoma) was days (range to days). conclusion: radiological placement of gastroduodenal stents is effective for palliation of upper gi malignancies. impact of multi-detector-row-ct ( x) prior to angiographic intervention m.s. juchems, s. pauls, h.j. brambs, a.j. aschoff; ulm/de learning objectives: to demonstrate the additional impact of contrast enhanced multi-detector-row-ct angiography (mdr-cta) compared to digital subtraction angiography (dsa) for planning of angiographic intervention. backround: in selected cases a pre-interventional cta can be useful to facilitate angiographic intervention. we selected four patients with different disease entities (pulmonary av malformation; bleeding caused by hepatic aneurysmosis and from renal pseudoaneurysm after partial renal resection; hepatocellular carcinoma (hcc) prior to transarterial chemoembolization (tace) with aberrant arteries) from our collective who underwent cta prior to angiographic intervention. procedure details: all ct scans were performed using a channel multi-detector-row-ct (philips mx idt). both multiplanar reconstructions (mpr) and slab maximum intensity projections (slab mip) were performed. after cta, patients underwent angiographic intervention (coil embolisation in the first three, tace in the last case). mdr-cta can demonstrate the exact extent and localize multiple vascular malformations, as demonstrated in the first case. in addition, mdr-cta can not only find the cause of hemorrhage but also demonstrate the exact localization of the specific vascular pathology ( nd and rd case). these findings facilitate the intervention, resulting in decreased in three children, a renal artery stenosis between and % due to fibromuscular dysplasia was discovered. in two of these three, the stenosis was unilateral and in one bilateral. in the remaining child, a long stenosis of unknown etiology was discovered. procedure details: in all patients, ptra was performed after having exactly defined the morphology and the hemodynamics of the renal artery stenoses. in three children we have achieved very good. results: ptra was successful resulting in an increased diameter of the arterial lumen. ptra was repeated and unsuccessful in one child with a severe stenosis and an emphasised "recoil" phenomenon. autotransplantation was therefore performed. reference is made to current controversies on this issue. background: endoluminal repair is an acceptable treatment modality for abdominal aneurysms. implant selection is based on the aneurysm's neck anatomy, resulting in two main implant groups; those fixed above the level of the renal arteries, and those fixed below this level. each group presents advantages and disadvantages that should be considered in the selection process. type i endoleaks are the most common complications derived from implant selection. procedure details: in order to select the implant, the patient should undergo a contrast-enhanced abdominal ct scan with coronal reconstructions to image in detail the abdominal aorta, and measure the size of the aneurysm, the distance from the renal arteries to the upper border of the aneurysm, and several other measurements needed. alternatives are abdominal aortic dsa and mra. the two main features that will determine the fixation site are the neck's shape and distance. sealing is always infrarenal. suprarenal devices provide better fixation, although some series report small renal parenchyma infarcts, due to emboli. infrarenal devices are easier to handle, provide for small corrections one placed, but cannot be placed in tortuous necks. conclusions: two main implant groups are used in endoluminal abdominal aneurysm repair, the suprarenal and infrarenal fixation devices. proper patient selection is needed to decide which device is to be used, in order to minimize post-procedural complications. in-vivo transcatheter implantation of a new aortic stent-valve for the temporary relief of acute aortic insufficiency m. hashimoto , t. kaminou , p. haage , y. ohuchi , k. nakamura , k. sugiura , y. noishiki , r.w. günther , t. ogawa ; yonago/jp, aachen/de, yokohama/jp purpose: acute aortic regurgitation is a life-threatening condition, particularly when associated with type a aortic dissection or aortic ectasia. the aim of this animal study was the evaluation of a novel temporary stent-valve as a potential temporizing measure to prevent progression to sudden death until conventional surgical repair can be performed. an umbrella-shaped highly porous polyester mesh valve was placed in the subcoronary portion with the attached metallic stent fixed in the ascending aorta. the prosthesis (stent-valve) was implanted in pigs via the right carotid artery through an fr introducer. the efficacy of the valve was assessed by aortography, pressure measurement and electrocardiography. hour after the implantation, the animals were killed and the stent-valve was subjected to macroscopic examination. results: a severe peri-prosthetic leak and cardiac arrest due to the deep implantation of the stent-valve were seen in case. although, a few premature ventricular complexes were seen in and slight blood stagnation in the valsalva sinus was seen in two, excellent performance of the mesh valve was demonstrated in of cases. there was no significant thrombus formation inside the umbrella valve. the presented new temporary stent-valve shows promise for possible future clinical application. further studies are necessary to assess valve thrombogenicity and coronary flow. percutaneous ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) with hepatic posttransplantation biliary complications who required treatment with interventional radiology techniques. were pediatric patients and were adults. diagnostic percutaneous transhepatic cholangiography (ptc) was performed in all patients, followed by placement of an internal-external biliary drain in the same procedure. in patients presenting with stenosis in the area of the anastomosis, dilatation with balloon angioplasty was also carried out. in patients with biliary calculi, lithotomy was performed followed by expulsion of the calculi to the digestive tract. one patient presented with a massive biliary leak that did not respond to percutaneous drainage and required implantation of a metal prosthesis. results: the transhepatic approach was used for the diagnostic study, for locating the cause of the complication and to carry out the therapeutic maneuvers required for its resolution. in the patients studied, the initial outcome of percutaneous treatment was satisfactory ( % technical success) and in of them ( %) it was the definitive therapeutic measure. among the remaining patients, underwent surgical re-anastomosis and re-transplantation. drainage placement was beneficial in all patients, allowing elective surgical treatment once the infectious-obstructive complication was resolved. conclusion: interventional radiology plays an important role in the therapeutic management of hepatic post-transplantation biliary complications. ptc allows precise early diagnosis of the complication. the interventional technique reduces the need for reoperation, thereby reducing associated morbidity and mortality. a comparative study of transjugular intrahepatic portosystemic shunt and transcatheter sclerotherapy for gastric varices t. ninoi , k. nakamura , n. nishida , y. sakai , t. kitayama , m. hamuro , r. yamada , y. inoue , t. kaminou ; osaka/jp, yonago/jp purpose: the rupture of gastric varices (gv) is a serious complication in patients with portal hypertension. transjugular intrahepatic portosystemic shunt (tips) and transcatheter sclerotherapy (ts) are effective treatments of gv. the effects on the portal pressure completely differ in tips and ts. however, to our knowledge, no comparative study of the two treatments for gv has been reported. the purpose of this study was to compare tips with ts on gv bleeding and survival. materials and methods: a total of cirrhotic patients with gv underwent endovascular treatment. of the patients, without hepatocellular caricinoma were enrolled in this study; patients were treated by tips, and patients by ts. gv bleeding and survival rates were compared between the tips and ts groups. multivariate analysis was used to identify the prognostic factors for gv bleeding and survival. results: the cumulative gv bleeding rate at year was % in the tips group, and % in the ts group (p < . ). the prognostic factor associated with gv bleeding was the treatment method. the cumulative survival rates at , , and years, were %, %, and %, in the tips group, and %, %, and %, in the ts group, respectively (p < . ). the prognostic factors for survival were the treatment method and the child-pugh classification. conclusion: ts may be more useful for gv than tips in consideration of gv bleeding and survival. results: tpsae was technically feasible in all patients without procedure-related complications. despite significant higher injury grade ( . vs ), higher rate of active contrast extravasation ( % vs %) and higher amount of hemoperitone-um on ct, secondary bleeding rate requiring splenectomy was lower in the tp-sae group ( . % vs %). conclusion: tpsae is a feasible and safe procedure in the treatment of splenic injury. tpsae allows a non-operative management despite a selection of severe splenic injuries with one of the lowest reported secondary splenectomy rates. recanalization of acute vertebro-basilar occlusion by a coronary waterjet thrombectomy device t.e. mayer, g.f. hamann, h. brueckmann; munich/de purpose: with a death rate of more than %, vertebrobasilar thromboembolism still remains a dangerous disease. this high rate can be mainly attributed to the overall failure of recanalization of %, depending on the extensiveness of the clot. this is especially in multisegmental occlusions, where the recanalization rate is very low and mortality is about %. we investigated the feasibility of using a coronary mechanical device based on the bernoulli effect (angiojet) for rapid recanalization. methods: ten patients with acute vertebrobasilar occlusion were included in the pilot study. inclusion depended on the presence of multisegmental intracranial ( patients) or any extracranial occlusions and coma lasting no more than hours. local intra-arterial fibrinolysis had failed in two patients before the angiojet was used, and thrombolysis was administered to two patients afterwards (rt-pa). five patients underwent additional intracranial balloon or stent angioplasty, and five received platelet inhibition by tirofiban. results: treament was primarily done with the angiojet and succeeded in complete recanalization in % of the patients ( % of the vessel segments). addition of fibrinolysis resulted in success in % of patients and segments. three hemorrhages were associated with additional thrombolysis, tirofiban, or both. seventy percent of the patients survived, and % had moderate to excellent outcome. the use of the coronary angiojet in the vertebrobasilar system is feasible. a success rate of % recanalization was achieved in a pilot study. compared to historical data of multisegmental thrombosis, the death rate was cut in half and good neurological outcomes increased. experience of transradial and transulnar abdominal angiography and intervention t. sato, m. kajikawa; hiroshima/jp purpose: in coronary angiography and angioplasty, transradial and transulnar approach has currently been accepted as an alternative catheterization method to transfemoral or transbrachial approach. the purpose of this study was to report practical and clinical applicability of these methods in abdominal angiography and intervention. methods and materials: between february and august , abdominal examinations were tried transradially or transulnarly in cases. cases were for liver or gall bladder tumor, cases were for other abdominal tumors, cases were due to gastrointestinal bleeding, cases were for renal or superior mesenteric arterial thrombosis and case was for ulcerative colitis. examinations were with further intervention. cases were examined twice, cases were examined times, case was examined times, cases were examined times, case were examined times and cases were examined times during this period, transradially. results: radial puncture was failed in examinations ( . %) and changed to brachial puncture. in cases with weak radial pulsation, ulnar puncture was tried. in cases of hepatocellurar carcinoma, superselective catheterization was also available and transarterial chemoembolization was done. in cases of abdominal branch aneurysm, successful coil embolization was done in cases and a femoral approach was needed on another occasion for complete embolization in one case. no puncture site hematoma was seen. radial arterial stenosis was found in cases at the next examination. no cerebrovascular problems were noted. conclusion: transradial and transulnar approach are alternative catheterization methods even in abdominal angiography and intervention. pre-and post-operative ct scans before discharge, at , , months and biannually thereafter were performed; pla and pima were evaluated in each followup scan and the number of pla was counted.these data were correlated with the frequency of el-ii. pre-and postoperative volumes of the aneurysmal sacs were measured from ct data. results: median follow-up was months (range - months).in total ct scans were performed and analyzed. six months after stent-graft implantation pima and/or at least pla were seen in patients ( . %); patients ( . % of the study cohort) had an el-ii. no el-ii was seen in patients without pla/ pima. three groups were identified and compared: patients without pla/pima/ el-ii and patients with pla/pima with/without el-ii. a tendency to minor mean volume loss in patients with pla/pima and a significant trend to volume increase in patients with el-ii in later follow-ups was seen. conclusion: there is a high incidence of pla/pima in patients after evr. persistent lumbar and inferior mesenteric artery perfusion is associated with significantly higher el-ii rates after evr of aaa. significant differences in volume changes in later follow-ups were seen between patients with or without el-ii. chemoembolization of hepatocellular carcinoma in candidates for orthotopic liver transplantation: histopathologic findings s. terraz, p. majno, l. rubbia, p.-a. schneider, c.d. becker; geneva/ch purpose: to evaluate the effects of selective transarterial chemoembolization (s-tace) in patients with suspected hepatocellular carcinoma (hcc) before orthotopic liver transplantation (olt). materials and methods: candidates for olt, with viral or alcoholic cirrhosis and a total of nodules suspected to be hcc ( - nodules of . - . cm), underwent s-tace with doxorubicine/lipiodol in - sessions. catheter position was segmental or subsegmental in % and unilobar in %. particles were added unless complete stasis was observed. the delay between s-tace and olt was ( - ) days. histologic findings of the explanted livers was correlated with all clinical, biologic and radiologic parameters. results: based on the final diagnosis, nodules were hcc, five were regenerative nodules and one was focal nodular hyperplasia. we observed total or neartotal necrosis (> %) in ( . %) hcc nodules and no dissemination was present. twenty-three hcc ( . %) nodules were partially necrotic; in of these, capsular and/or vascular invasion were present. one patient developed a pseudoaneurysm at the puncture site; no other complication was observed. conclusion: s-tace leads to complete necrosis in the majority of hcc nodules. olt candidates with a long waiting period should be followed by repeated imaging studies, in order to be able to prevent tumor regrowth or spread by repeating s-tace when necessary. kasabach merritt syndrome: treatment with transarterial embolization k.s. malagari, d. antonopoulos, d. loggitsi, d. danassi, a. rigopoulou, a. sissopoulos, a. nikita, g. papaioannou, d. kelekis; athens/gr aim: kasabach merritt syndrome is a rare life threatening condition characterized by consumption coagulopathy, most often associated with liver hemangiomas. we report on the value of transarterial embolization in the treatment of this condition. material and methods: two patients are included in the study (mean age yrs, range - ). both were women. kasabach merrit was the first manifestation of the liver lesion in one patient and a complication of a known cavernous liver hemangioma in the second. hct values were % and %, plt , and , / mm , fibrinogen and mg/dl respectively. spiral ct of the liver showed typical hemangiomas with a lace-like enhancing pattern (mean diameters and cm respectively). patients were infused with platelets, fresh plasma, cryoprecipitates, aprotinin and antithrombin prior to embolization. one patient required also units of packed red blood cells. selective hepatic angiography was performed prior to embolization, followed by superselective injection of the feeding vessel(s). microspheres of - microns were injected under fluoroscopic guidance until cessation of flow. the procedure was uneventful in both patients. partial restoration of plt cell count was observed from the following day and within a week fibrinogen levels and plt levels were completely restored in one patient and partially to the other. conclusions: transarterial embolization may be life saving in kasabach merritt since it can safely control coagulation abnormalities and allows planning for more definitive treatment including surgical resection or liver transplantation for non operable hemangiomas. in vivo characteristics of microspheres injected into the hepatic arteries of rabbits: angiographic and microscopic comparison of embosphere and beadblock m. takahashi, y. saida; tsukuba/jp purpose: the authors presently utilize non-resorbable microspheres (ms) for the transarterial embolization (tae) of hepatocellular carcinoma (hcc). to correlate their clinical features and in vivo characteristics, two different types of industrially-produced ms, injected into the hepatic arteries of rabbits, were microscopically compared. eight japanese-white rabbits were divided into four groups. hepatic arteries were selectively catheterized by the femoral approach. in each group, lipiodol, embosphere ( - and - µ) or beadblock ( - µ) were injected. ms were prepared as a ml suspension with contrast material, in which ml ms were contained. animals were sacrificed immediately after the injection. the liver was excised with ligation of all major vessels. sections were made in the radiate plane along with hepatic arteries. results: lipiodol spread into the hepatic sinusoid and reached the central veins. they were found also in the peribiliary plexus (pbp). ms reached maximally glisson's sheath but was not found around the pbp nor the sinusoids. embosphere occluded the artery after an injection of some milliliters. microscopically they were often clustered and compressed but their spherical shape was maintained. smaller embosphere penetrated deeper than larger one. beadblock once occluded the artery with a smaller amount of suspension than embosphere. after a few minutes, however, the arterial flow resumed and additional injections were possible. microscopically they formed casts and tend to reach deeper tissue than embosphere of the same size. conclusion: embosphere is elastic but robust, while beadblock is plastic, forming casts in the vessel. these characteristics explain much of their features at the time of injection. the at another patient hours after the implantation the rupture of common femoral artery aneurysm was stated. there were also observed certain post-hospitalization complications which included: a) clotting of stent-graft leg in cases b) leak type i which demanded implantation of additional element of stent-graft in one case c) small leaks type i in cases and type ii in cases which did not demand any further intervention-the type ii leaks were observed in cases months after the operation d) the increase of parietal circular thrombus inside the main part of the stent-graft in two cases e) complaints connected with the internal iliac artery occlusion were declared by patients. the catheter has four microsurgical blades that cut directly into the stenotic plaque during inflation. recently they were proposed for the use in peripheral arterial system using a balloon diameter raging from up to . mm. material and methods: seven consecutive patients (mean age years) were treated with cutting balloon pta (cb-pta) from june until september . all of them had severe multiple, calcified stenosis of the femoro-popliteal artery, maximum cm in length, never treated before. all patients had important associated risk factors such as insulin-dependent diabetes mellitus, hyperlipoproteinemia and smoking. results: cb-pta was technically successful in all patients, with lumen diameter improvement at least of % or residual stenosis less than %; moreover we never observed major dissection of the intimal plaque and we never required stent placement. conclusion: cb-pta in lower limb calcified stenoses is a promising technique that seems to be superior to pta alone, without requiring stenting in femoropopliteal arteries. aneurysm diameter in all patients were > cm (average diameter were . cm, range, . - . cm). the mean follow-up was months. average hospital stay after the intervention was - days. results: the stent-grafts were successfully deployed in all patients. control spiral ct hours after the procedure revealed endoleak in patients ( . %). spiral ct follow-up on th day following the intervention showed that in patients endoleak spontaneously stopped and in one patient small endoleak was seen, but it also stopped within a month following the intervention. in two patients ( . %) acute thrombosis of iliac graft extensions was diagnosed a month after the intervention. in both cases successful endovascular thrombolysis was performed with good clinical outcome. in one patient ( %), progression of atherosclerotic disease resulted in migration of the stent-graft two years after the placement of the graft. reintervention was done with successful deployment of additional aortic extension of the stent-graft. average reduction of aneurysm diameter was . mm (range, - mm). conclusion: endovascular treatment of aaa with talent stent-graft is a safe and effective procedure. in patients with well defined indication it is safe alternative to open surgery procedure. direct thrombin injection for non-femoral pseudoaneurysms r. corso, m. solcia, m. castoldi, r. vercelli, m. intotero, a. rampoldi; milan/it purpose: percutaneous thrombin injection under sonographic guidance has been shown to be an effective and well tolerated treatment for a iatrogenic femoral pseudoaneurysms. for these reasons some authors suggest thrombin injection as alternative treatment also in other body districts. we illustrate our experience, techniques and results in the ablation of pseudoaneurysms of different etiologies and locations whit direct thrombin injection. materials and methods: from june we treated six patients, age range - years, affected with pseudoaneurysms developed after iatrogenic trauma in three patients (arising from radial, posterior tibial and thoraco-acromial arteries), post-pancreatitis (splenic and superior mesenteric arteries) in two patients and in one patient after spontaneous rupture of renal angiomyolipoma in tuberous sclerosis. two patients were anticoagulated. the diameter of pseudoaneurysms varied between . - . cm. after disinfection of the entry site, we slowly injected bovine thrombin ( u/ml of solution) percutaneously under ultrasound guidance in four cases, while in two cases we injected directly the thrombin in the pseudoaneurysm cavity with a fluoroscopically guided endovascular angiographic catheter. results: all interventions were successful and showed no immediate or late complications. doses of thrombin needed varied between - units ( . - ml of solution). a complete thrombosis of the pseudoaneurysm sac was accomplished within minutes of thrombin injection. no recurrence was seen at the follow-up. conclusion: in selected cases, direct thrombin injection may be useful in treating non-femoral pseudoaneurysms resulting in rapid and persistent occlusion. in our limited experience it represents a safe and effective minimally invasive technique and an alternative to surgical repair. the purpose: bronchopulmonary sequestration is a combined lung and vascular anomaly witch is usually surgically treated. in those cases witch the anomaly is mostly vascular, coil embolization is a safe alternative to surgery. method and materials: coil embolization of intrapulmonary sequestration was performed in three adult patients, two of them admitted with hemoptysis and the third one with low left thoracic pain and fever. the initial diagnosis was made by ct and confirmed with angiography. we embolised the feeding arteries using coils (from to mm, cook). angiography after embolization indicated almost complete occlusion and all patients were discharged the next day. results: there were no immediate nor long term complications. follow-up ct was performed six months and one year later, at witch time there was complete occlusion of the anomalous artery. all patients are asymptomatic at present. conclusion: arterial embolization of intrapulmonary sequestration with a predominant vascular component, is a less invasive method of occluding the anomalous artery in selected patients and is a safe alternative method to surgery. emergency endovascular repair of ruptured hepatic artery pseudoaneurysm with coronary stent-graft: two cases a. park; pusan/kr purpose: to report two cases of successful emergent endovascular treatment of ruptured pseudoaneurysm from the hepatic artery using balloon expandable coronary stent-grafts. materials and methods: case . a -year-old male underwent whipple's operation for a cholangiocarcinoma in the distal common bile duct, but his postoperative course was complicated by serious blood loss from a ruptured pseudoaneurysm in the common hepatic artery. using premounted coronary stent-grafts (jostent, jomed, germany), the neck of the aneurysm was completely excluded. case . a -year-old male underwent surgical treatment of a klatskin's tumor and hepaticojejunostomy. four weeks later, an episode of gastrointestinal bleeding prompted celiac angiography. active bleeding from a small pseudoaneurysm in the right hepatic artery was controlled with endovascular deployment of a premounted coronary stent-graft (jostent, jomed, germany). the final angiogram demostrated a total exclusion of the aneurysm with preservation of the arterial lumen. results: transcatheter embolization using a variety of embolizing materials has been considered the treatment of choice for true or false hepatic artery aneurysms. however stent exclusion of the hepatic artery bleeding in an emergent situation has been reported only in few cases. for patients with a compromised blood supply due to portal vein occlusion or exclusion of important collateral pathways by surgical operation, a stent-graft may become the procedure of choice. in emergent situations of hepatic artery bleeding, endovascular implantation of a stent-graft may be an alternative procedure to transcatheter embolization or surgical repair. materials and biomaterials for interventional radiology t. hasebe , a. shimada , t. suzuki , y. matsuoka , h. yoshimura , s. kuribayashi ; tokyo/jp, sagamihara/jp, hiyoshi/jp learning objectives: . to describe the most often used interventional devices (id) benefiting from the recent intensive research activity to improve their functionality or their biocompatibility. . to provide an update on the current status of new materials and biomaterials available in the field of vascular and non-vascular interventional radiology, including our original experimental data . to discuss the importance and the safety issues related to the use of biomaterials and coating materials concerning id. background: in the past decade, interventional radiology has allowed percutaneous access devices and therapeutic implants to be more reliable and effectively used due to the availabiliity of new materials. currently, there are several passive or active stent coatings commercially available with good results in preliminary studies. these materials more often result from industrial research and less frequently from medical research. procedure details: in this paper, we discuss biomaterials currently available for use, together with new biomaterials for the vascular or non-vascular id, such as, for example, the drug-eluting stents, coils for embolization, and biliary/ureteral catheters or stents. finally, we discuss the future of biomaterial use in interventional radiology with the original data in our laboratory (e.g., antithrombogenic and biocompatible stent coating: diamond-like carbon). conclusion: biocompatible issues are very critical for id in order to coexist with living tissue without causing harm. considering the recent advances in new materials with biological effects, we believe the biomaterials will soon constitute a new core of interventional radiology. the radiologists should establish new research domains and complete new and effective products. clinical usefulness of a unified multidetector-ct we can always generate a control image during intervention without repositioning the patient. we have performed highly integrated imaging diagnosis and interventional therapy with this system in more than patients. procedure details: angiography assisted multidetector-ct was performed for the diagnosis and the target therapy of hepatic, pancreatic, adrenal and renal tumors and uterine myomas. in addition, multidetector-ct assisted angiography and interventional therapy was performed for vascular recanalization, such as aortic and peripheral vascular stent therapy; for endovascular treatment of aneurysms and for percutaneous therapy of varices, with a variety of three-dimensional reformatting techniques. we also performed several interventional procedures, such as percutaneous biopsy, biliary and abscess drainage, with this system. conclusion: we introduce our highly integrated intervetional ct system with technical merits to clinical use. background: ct is the imaging method of choice to evaluate hemodynamically stable patients with significant blunt abdominopelvic trauma. contrast enhancement is essential for the diagnosis of many visceral injuries in these patients. it has been demonstrated that contrast-enhanced ct is valuable in the detection of active extravasation of contrast material. the detection of contrast extravasation is critical because it indicates an ongoing, potentially life-threatening hemorrhage. imaging findings: the purpose of this exhibit is to review our experience with active contrast extravasation in patients with blunt trauma to the abdomen and pelvis. findings of contrast-enhanced ct are correlated with those of conventional angiography. the role of angiography and transarterial embolization in patients with active extravasation on contrast-enhanced ct will be discussed. conclusion: contrast extravasation on ct can be used to localize anatomic sites of hemorrhage and to guide immediate angiographic or surgical intervention in patients with abdominopelvic trauma. state-of-the-art ct enables a more detection of contrast extravasation than is possible with conventional and single detector helical ct. for the presented in vitro study, peas (mean diameter . mm) were embedded within a gel-phantom in order to simulate small target lesions. positional data of the patient and of the needle guide mounted to the robot arm were acquired via an optical tracking system (ndi polaris). a preliminary ct calibration procedure allowed assignation of d coordinates to each d pixel of the particular ct scan for fast and accurate intervention planning. the best needle trajectory was defined graphically by simply selecting skin entry point and target point -appropriate position and angulation of the robotic arm were calculated and sent to the robot controller. after confirmation of the planning, the robot moved to the start position using different modes of motion. the coaxial biopsy was performed manually using a -gauge puncture needle, combined with an -gauge biopsy needle and an automated biopsy device. the complete intervention could be monitored and documented by means of superimposed information on the actual ct scan. to prove efficacy of biopsy the length of the harvested specimen, as well as the distance between actual needle tract and centre of the lesion was measured. results: biopsy specimens were successfully obtained from all targets with only one needle pass necessary. radial distance between needle trajectory and centre of the target was surpassing clinical requirements. conclusions: robotic assisted biopsies with high accuracy were feasible using ct guidance. a new laser guidance device for ultrasound guided percutaneous biopsy a.e. samir, j. koukounaras, p. u, a. moorhouse, m. brooks; melbourne/au introduction: ultrasound guided needle placement is usually performed by inserting a needle along a path in the plane of the ultrasound beam. this permits visualisation of the needle in "real time", allowing accurate needle placement. a novel laser guidance device that facilitates needle placement during ultrasound guided biopsy is presented. methods: a laser module was constructed by mounting a laser diode in a nylon tube. a lens was used to transform the pencil laser beam into a linear alignment beam. the laser module was mounted on an ultrasound probe using a customdesigned clip that consisted of two polycarbonate half shells. the linear laser beam was calibrated to be coplanar to the ultrasound beam. no probe modifications were necessary. fourteen biopsy operators performed eight free hand and eight laser guided biopsies each of capsicum-stuffed olives suspended in gelatine phantoms. endpoints measured were: ( ) time to biopsy, ( ) sample quality and ( ) perceived usefulness. the laser beam was visible on the surface of needles ranging from to gauge in size. when the needle was inserted colinear to the laser beam it was visible on the ultrasound image. a significant difference in procedure time or sample quality was not observed. however, % of operators stated that laser guidance had a positive effect on the procedure, and % said that they would use it in the future. conclusion: a probe-mounted laser guidance device is perceived to facilitate accurate ultrasound guided needle insertion by the majority of operators in a pilot study. planning of the intervention is on basis of ct-imaging data sets acquired immediately before an intervention. spatial relation between imaging space and targeting device is either established by using of an optical tracker system or via robot registration based on a ct data set. after graphically selection of the target point and manual pre-positioning of the device, correct needle angulation will be set automatically by the system. during the intervention, the robot kinematic holds the needle guide in a defined position/ orientation to the patient's body -needle insertion will be performed manually by the physician. results: the developed system is easy-to-use and does not interfere to the clinical work-flow. system accuracy and clinical relevance currently is being tested in a series of in vitro tests. the designed and realized prototype of a modular automated needle guide allows positioning of a biopsy needle based on an intra-operatively planning with good cost/benefit ratio. effect of radiotherapy and ischemia on rat liver z. vigvary, p. kupcsulik, a. szijartó, o. hahn; budapest/hu aim: the aim is to create a model of a liver resection combined with intraoperative irradiation for micrometastases. this combined ischemic-reperfusion and irradiation injury of the liver has not yet been investigated. methods: normothermic segmental liver ischemia were created on - g male wistar rats. rats were divided into four groups for , , and minutes of ischemia. hepatic microcirculation was studied by laser doppler flowmeter. histological alterations were followed. i, ii, iv lobes were exposed to , and gy of cs- ã-radiation. alp, alt, ast, ldh, bilirubin and tnf-á tests were performed. results: reperfusion was assesed by post-ischemic flux plato maximum (pm), by area under the curve (rt) and by the time to maximum flux level (rmi). flowmetery showed a nonlinear fashion of reperfusion according to ischemic period. after and minutes ischemia the rate of the reperfusion is almost the same. minutes of ischemia represents an intermediate lesion in term of recovery. after minutes, minirapid deterioration of flux (below % of control) was observed. survival is rather determined by the length of the ischemic period, than the irradiation dose. low dose irradiation ( gy) with short-term ischemia ( min) did not result in increased liver enzymes and resulted only in minimal histological changes. the group of gy irradiation with normotherm, short-term ischemia seems to be tolerable for the tumor-free liver. conclusion: liver toleratse irradiation injury suprisingly well and preconditioning promotes restitution of liver circulation after ischemic injury. purpose: the aim of the study was to assess radiation exposure to the radiologist incurred with ct fluoroscopy. the spectrum of indications and radiation exposures for the radiologist were assessed for an -month period. scatter exposures were measured with and without radiation protection devices: without and with placement of a lead drape on the patient, with and without use of thin rubber radiation protection gloves. in addition, scatter radiation was determined for a combination of lead drape and radiation protection gloves. results: there is a wide variety for the use of ct fluoroscopy ranging from diagnostic biopsy procedures to therapeutic interventions such as radiofrequency ablation of liver metastases and ct fluoroscopy-guided osteosynthesis of fractures. scatter exposure rates to the hand of the radiologist ranged from to micro sv per case without use of a lead drape and radiation protection gloves. the lead drape reduced the scatter exposure to the hand of the radiologist by percent. radiation protection gloves reduced scatter radiation by percent. the combination of both radiation protection devices was most effective in decreasing the dose by percent. conclusion: ct fluoroscopy is a useful targeting method with a wide variety for interventional procedures. however, significant radiation exposures may occur. therefore, the radiologists should be aware of different techniques of ct fluoroscopy guidance and the methods to reduce scatter radiation. purpose: workforce planning, deskilling scenarios and risk containment are generating a need for interventional training processes remote from patients. fixed models lack ultimate realism, and animal models lack pathology with political problems in the uk. virtual environments for interventional needle puncture training require accurate haptics, based on force relationships between tools and tissues, and visualization. to achieve this demands advancements in tissue segmentation, assignment of tissue properties and generation of synthetic images from abnormal patient anatomy and pathology. these challenges require wideranging expertise and will probably take over a decade to achieve fully. no existing simulator model covers interventional radiological needle access procedures (eg: targeted biopsy, nephrostomy, biliary and abscess drainage). thus a substantive collaboration is essential to develop appropriate virtual training environments. internet search highlighted key development areas and, together with conventional networking, relevant departments and individuals were located with major involvement in medical simulations. results: the craive collaboration was established and comprised interventional radiologists, computer scientists, physicists, clinical engineers and psychologists. the key challenges of interventional procedures have driven specific proposals for accurate representation of haptics, from measured in vivo forces, within imaging-derived, immersive, simulated environments. conclusion: achieving effective simulator modeling for training and rehearsal requires research advances and further development in the underlying technologies. while currently beyond the scope of any research group or individual company, a substantive new collaboration, craive, aims to develop interventional radiological simulator models, initially based on a spectrum of interventional needle access procedures. and % (on low dose mode) reduction. image quality measurements were consistently superior ( % higher resolution in fluoroscopy and % in dsa) with significantly more low contrast iodine targets and moving wires detected and a larger dynamic range or working thickness range. the results of our first clinical investigations give further evidence for superior image quality. conclusions: on all modes, dose monitoring and image quality of the digital flat panel system compared favorably to the conventional image intensifier system. the results of our first clinical investigations on image quality based on our routine daily practice will be presented. differential diagnosis of femoral head bone marrow edema pattern i. kalaitzoglou , p. papadopoulou , a.s. dimitriadis ; thessaloniki/gr, kavala/gr learning objectives: to present the imaging features which help in the differential diagnosis of the femoral head "bone marrow edema pattern". background: bone marrow edema of the femoral head is a non-specific mri finding associated with various painful conditions of the hip like osteonecrosis, idiopathic transient osteoporosis, transient bone marrow edema syndrome, migratory osteoporosis and stress fracture. a total number of approximately hip mri examinations were performed in three different diagnostic centers from january to july . bone marrow edema was present in cases. procedure details: mri units used ranged from . to . tesla and the examination protocols included t se and t fse fat-sat or stir axial and coronal images, while sagittal images and contrast administration were reserved for specific cases. patients with typical findings leading to the diagnosis of osteonecrosis or fracture were excluded. the remaining patients had non-specific bone marrow edema of the femoral head which was demonstrated as diffuse, ill defined, low signal intensity on t w images, high on t w and stir images, and extended to the femoral neck region. in half of the patients follow -up examinations were also available. we selected and present the imaging criteria, at presentation and follow -up, which can lead to an early specific diagnosis. conclusion: bone marrow edema of the hip has to be correctly interpreted in order to avoid overtreatment in transient and self-limiting conditions. the presence of subtle subchondral lesions and the temporal evolution are usually key to diagnosis. periosteal reactions: the spectrum of radiological appearances with histopathologic correlation i. pinilla, d. bernabéu, c. martín-hervás, a. alvarez, a. bravo, f. lópez-barea; madrid/es learning objectives: the aim of this exhibit is to illustrate the spectrum of imaging features of periosteal reactions with plain radiography, computed tomography (ct) and magnetic resonance imaging (mri) with emphasis in pathologic correlation. imaging findings: patients with histologically proved diagnosis of benign and malignant lesions in our institution between january and december were identified from the pathology data base. imaging findings with plain radiography, ct and mri were retrospectively reviewed and correlated with key pathologic features. the various patterns of periosteal reaction are arranged into the following categories: continuous (smooth, lobulated, and ridged shells, solid, single lamella, laminated or "onion skin", and parallel spiculated or "hair on end" reactions), interrupted (buttres, codman triangle, lamellated, and spiculated reactions), and complex (divergent spiculated or "sunburst", and combined reactions). the most representative cases of each form of periosteal reaction are displayed. specific topics addressed include benign and borderline bone tumors (osteoid osteoma, osteoblastoma, periosteal chondroma, aneurysmal bone cyst, giant cell tumor, langerhans cell histiocytosis, and adamantinoma), primary malignant bone tumors (osteosarcoma, chondrosarcoma, ewing's sarcoma and non-hodgkin's lymphoma), skeletal metastases, and non-neoplastic conditions such as osteomyelitis and myositis ossificans. the changes in the periosteum are common and important imaging features of bone diseases. recognizing and understanding the different radiological patterns and underlying histopathologic changes help the radiologist to narrow the differential diagnosis. this teaching exhibit gives an overview of the wide spectrum of radiological appearances of periosteal reactions together with histopathologic correlation. in mr images, spin-echo (se) t -weighted images (t -wi) and t -wi were obtained and gd-dtpa contrast enhanced images were obtained in cases. in these cases, the location, the radiographic findings, signal and enhanced pattern on mri were analyzed. imaging findings: in cases, the tumor located in epiphysis ( . %), in cases in apophysis ( . %), in cases in apo-metaphysis ( . %), in cases in epimetaphysis ( . %). in all cases, the tumor was depicted as radiolucent area and in cases, the sclerotic margin was recognized. in all cases, the tumor was revealed as iso-intensity comparing with surrounding muscle on t -wi. on t wi, in cases, the tumor revealed as iso-intensity with bone marrow and in the other cases, it revealed as low intensity. on t -wi, in cases, high intensity area was included which corresponds to the cystic degeneration. in all cases, the tumor was moderately enhanced on contrast enhanced t -wi. in cases, t -t -prolonged lesion which is considered to be the reactive inflammatory change were shown in the bone marrow around the tumor. conclusion: to analyze the image characteristics of chondroblastoma is important to approach the exact diagnosis of bone tumor. chondrosarcomas background: parosteal osteosarcomas represent only - % of all osteosarcomas, and have better prognosis than the classical osteosarcoma.they are frequently low-grade tumors, which may rarely dedifferentiate into high grade sarcomas. imaging findings: mr images performed on - . t mr system of histologically proven cases of parosteal osteosarcoma were retrospectively analyzed for the bones involved, location and the relationship between the signal intensity and the grade of the tumor.there were females and males. ages ranged from to (mean . ). most commonly affected bone was femur, on its distal end, posterior aspect ( ). histological grading was described as follows:grade i: hypocellular stroma with subtle atypia; grade ii: mild increase in stromal cellularity and more prominent cytologic atypia, grade iii: marked pleomorphism with high cytologic atypia and mitotic rate, also called dedifferentiated type. regarding the described grading above, of our cases were grade i (low), were grade ii (intermediate) and were grade iii (high). on mri, all of the parosteal osteosarcoma cases were of hypointense signal intensity on t -weighted images and were hyperintense on t -weighted images with no exception. on gadoliniumenhanced images, heterogeneous contrast enhancement was observed. conclusion: in our study, mri signal intensity was the same in all of the cases; hypointense on t -weighted images and hyperintense on t -weighted images; regardless of the grade of the tumor. giant cell tumor of knee: imaging findings of the most common site v. zampa, s. giusti, c. spinelli, s. ortori, c. bartolozzi; pisa/it learning objectves: . to describe imaging findings useful for an effective diagnosis . to discuss the main differential diagnosis with giant cell tumor (gct) of knee. background: gct is a relatively common skeletal tumor. it is a benign but locally aggressive and destructive lesion composed of primitive histiocytes and large, multinucleated giant cells. the most frequent symptom is pain, local swelling, limited range of motion and pathologic fracture ( %). location of gct and age of patients suggest the diagnosis. it usually affects long tubular bones and, in particular around the knee, the most common site is distal femoral and proximal tibia ( and % of all tgc). the main differential diagnoses include osteosarcoma (teleangectatic or fibrogenic), malignant fibrous istiocytoma, chondromyxoid fibroma and aneurysmal bone cysts. treatment of choice is surgical resection but recurrence is frequent. imaging findings: on radiography gct appears as an eccentric and geographic bone lysis, associated with a narrow zone of transition and lacking surrounding sclerosis. ct is superior to conventional radiography in outlining the extent of the tumor and in demonstrating cortical invasion but mr is currently the best imaging modality in determining extra-osseous extent, articular surface involvement and tumor recurrence with optimal definition of either osseous sclerosis or pseudocapsule. the signal characteristics of the lesion on the different mr sequences will be described to find out if they can help in differential diagnosis. conclusion: radiologic appearances of gct is important in allowing prospective diagnosis, guiding therapy, and facilitating early detection of recurrence. superscans: a pictorial review s.a. o'keeffe, t. geoghegan, w.c. torreggiani; dublin/ie learning objectives: the objective of this poster presentation is to pictorially describe the causes, findings and interpretative pathways of patients undergoing radionuclide bone scintigraphy resulting in a superscan. background: a superscan is a radionuclide bone scan which demonstrates markedly increased, homogenous skeletal radioisotope uptake relative to soft tissues and is usually associated with absent or faint renal images. a retrospective analysis of all whole body radionuclide bone scans performed over a fiveyear period was performed to identify the presence of superscans. analysis was based on review of reports identified on a computerised radiology information system (ris). images were reviewed in association with other relevant imaging and in select cases, with laboratory data and clinical notes. a variety of cases were then selected for this pictorial review. imaging findings: a total of whole body bone scans were performed in our institution in the last five years. superscans were selected for inclusion in this pictorial review. the vast majority of superscans identified were related to metastatic disease of which prostate cancer dominated. superscans were also identified secondary to hyperparathroidism, metabolic bone disease including osteomalacia and pagets disease. conclusions: in this pictorial review, we describe the patho-physiological pathways resulting in the appearances of a superscan. in addition, we pictorially demonstrate a variety of causes resulting in superscan. finally, interpretative pathways of analysis and helpful signs are suggested. usefulness of d reformations in the assesment of spondylolisthesis and spondylolisis of the lumbar spine e. vilar bonacasa, p. sanchez santos, j. torres nuez, g. martinez sanz; teruel/es learning objectives: to obtain images that provide a full and direct view of the pars interarticularis in order to determine the isthmal affectation in lumbar spondylolisthesis. background: nine patients with spondylolisthesis of the lumbar spine among sent to our department for ct study during the last year were performed a volumetric acquisition from l or l to s instead of the conventional axial scanning. later, a d image was obtained and modified in the work station, to achieve a view of the pars interarticularis from the vertebral canal. procedure details: using this method, we obtained images wich allowed us to asses the isthmal affectation in a direct way in all patients. moreover, it permits the study of the repercussion of vertebral bodies slide over the vertebral canal and the intervertebral foramina. conclusion: convencional ct has some limitations to determine exactly the type and degree of isthmal affectation in spondylolisthesis as well as its repercussion on intervertebral foramina. helical ct, using volumetric acquisition with three-dimensional reformations can resolve those limitations. morphology in these cases, the improvement of the skeletal pain were scored in five-levels by patients' interview from = marked improved to = worse and the duration of the symptom's improvement was monitored. results: a total of taes (mean . times) were performed. all procedures were technically successful. skeletal pain was relieved in patients ( %) between = slightly and = markedly improvement. in of patients with malignant lesions, the duration of pain relief ranged from one to months (mean months). no serious complications were observed except for a case with transient neurogenic bladder. conclusion: tae using sap-ms was a safe and effective treatment for the pain control with long duration of unresectable bone tumor. bone marrow edema at mri: diagnostic clue to the underlying cause of knee pain a. blanco, m. martinez, e. parlorio, m.a. corral, s. torres; murcia/es purpose: bone marrow edema (bme) is usually associated with traumatic injuries of the knee but there are other conditions, often unsuspected, that can cause this pattern. our purpose was to evaluate the patterns of distribution of bme in patients with knee pain and try to correlate them with their possible underlying cause. we retrospectively reviewed mri examinations of patients referred for knee pain, with or without history of trauma, during a month period. the examinations were performed in a . t unit using our standard protocol which included fluid-sensitive sequences. in some cases intravenous gadolinium was used. results: patients ( . %) showed bme with variable distribution, resulting from: acute trauma ( ), stress fracture ( ), degenerative disease ( ), spontaneous osteonecrosis ( ) and osteochondritis dissecans ( ) of the femoral condyle, soft-tissue infection ( ), excesive lateral pressure syndrome ( ), iliotibial-band friction syndrome ( ), transient bme ( ), tumor ( ), and nonspecific ( ). in many cases associated with trauma, the distribution of edema allowed to explain the mechanism of injury and predict with accuracy the soft-tissue abnormalities that may be present. conclusion: bme patterns at mri can help to establish the possible cause of knee pain. when there is a history of acute trauma, edema shows typical features that can give the clue to a possible mechanism of injury. on the other hand, if clinical data is not clear, the distribution of edema can orientate to other unsuspected pathologic conditions. appropriate recognition is necessary for an accurate diagnostic and therapeutic orientation. percutaneous percutaneous thermoablations was performed with radionics cool tip rf generator system by using probes with or cm unprotected tip applied for minutes in the center of the lesion. the procedure consisted of localizing the osteoid osteoma on ct imaging and positioning within the lesion a kirschner wire, rigged on a hand-drill device. once the nidus was engaged we proceeded to exchange the thread of kirschner for a rf probe and emit the radio waves. results: at a mean follow-up of months the treatment has been considered effective in % of the cases. in four patients pain has subsequently recurred; one of these cases has been treated for a second time with success. in two cases procedural complications of minor degree, represented by limited cutaneous burns have occurred. conclusion: in our experience ct-guided percutaneous radiofrequency treatment of osteoid osteoma proved to be effective in % of the cases. deprived of severe complications it may represent an uncomplicated, minimally invasive, rapidly effective procedure, alternative to the surgical treatment. order to obtain the load-to-fracture, the specimens were tested destructively at side impact. results: in the standard regions of interest, correlations between fracture load and bmd (bmc) ranged from r = . to r = . , p < . (r = . to . , p < . ). for the newly defined roi in the upper neck of the proximal femur, significantly higher correlations between bmd, bmc and fracture load (r = . and . , respectively; p < . ) were obtained. only a weak statistical relationship between fracture load and fnal was found (r = . , p < . ). normalization of fracture load with respect to fnal of the specimens did not significantly improve results. the mechanical competence of the hip highly depends on the bmd and bmc of the proximal femur. of all the regions of interest analysed in this study a newly defined roi in the upper neck showed the strongest correlation with fracture load. under in-vitro conditions, only a weak association between mechanical strength and fnal was observed. or secondary (n = ) diagnostics were analysed visually. the final diagnosis based on histology, imaging and follow-up. results: histologically the primary lesions were es (n = ), pnet (n = ) and osteomyelitis (n = ). the sensitivity and specificity of fdg-pet for disease (presence of et, and/or its metastases) were and % respectively. fdg-pet and/ or other imaging modalities detected altogether focal lesions. sensitivity and specificity regarding focal lesions were and %. this lower sensitivity is due to small lesions. wbs detected all primary bone lesions present at wbs investigation, but only of bone metastases in four patients, while fdg-pet detected both the primaries and metastases. conclusion: fdg-pet investigation is a valuable method in case of et-s. concerning the detection of bone metastases of et-s fdg-pet is superior to wbs, while for the depiction of small lesions mainly represented by pulmonary metastases fdg-pet is less sensitive than helical ct. the determination of the role of whole body fdg-pet in the diagnostic algorithm needs further investigations. the role of an optimized mr protocol in the detection of osteomyelitis neighbouring metallic bone implants g. michailidis , n. economopoulos , c. kontopoulou , s. argentos , n.l. kelekis ; piraeus/gr, athens/gr purpose: osteomyelitis presents as a complication in a percentage of patients after metallic bone-implant placement. early and accurate detection is crucial for proper treatment. conventional mr images are usually severely degraded from intense artifacts, rendering interpretation often impossible. we report our results using an optimized mr imaging protocol in detecting foci of osteomyelitis near metallic bone implants. materials and methods: during the last year we examined patients ( men and women, age range - ) with metallic bone implants and clinical suspicion of osteomyelitis. the mr protocol consisted of t -weighted stir and single shot turbo spin echo sequences. for t and t contrast-enhanced images we employed turbo spin echo sequences with slice thickness of mm, echo train length > , minimum echo spacing and minimum te. diagnosis of osteomyelitis was based on the detection of one or more of the following findings: subperiosteal fluid, bone sequestrum, fistula and abscess. results: all mr examinations were of diagnostic quality with minimal artifacts present. in patients one or more of the above mentioned findings were present and were diagnosed with osteomyelitis. in the remaining patients no findings suggestive of osteomyelitis were observed. confirmation was obtained by followup and clinical outcome. conclusion: with the use of an optimized scanning protocol mri may overcome its limitations caused by metallic artifacts and is able to detect foci of osteomyelitis in patients with metallic bone implants. primary methods and materials: patients were included, aged to years old. flat bones (pelvis = , sternum = ) were involved as well as metaphysis of tibia (n = ) and femur (n = ). pathologic diagnosis was obtained in all cases. results: in long bones, tumor originated from the medulla, excentered and metaphyseal in location. in all cases, agressive patterns were present such as osteolysis, cortical break and tumoral extension in surrounding soft tissues, lack of peritumoral reactional osteocondensation. periosteal reaction was present in one case. no calcification was noted. on mr imaging, tumors demonstrated intermediate signal intensity on the t -weighted images with heterogeneous enhancement following gadolinium injection. pathological findings included smooth muscle cells with interlacing fascicles of spindle cells, without osteoid production or cartilagineous matrix. despite adequate surgical resection, metatastatic diseases appeared in cases. conclusion: primary leiomyosarcoma of bone is a rare tumor which must be included in the diagnostic possibilities in case of agressive purely osteolytic lesion. results: patients included females and males (aged from to years) with pain in extremities. osteogenic sarcoma was diagnosed in patients, ewing sarcoma - , chondrosarcoma - , bone lymphoma - . in cases on plain film bone damage wasn't detected. on bone scintigraphy, the majority of lesions showed greater than mild uptake, in cases showed no uptake. at mr imaging isointense or low t w signal and increased t w signal was present in all cases. specific cellular constituents (e.g. fibrous, chondroid, blastic or teleangiectatic components), can modify signal characteristics. lesions that are primarily blastic, and, therefore, sclerotic on plain film radiographs, demonstrate low signal intensity on both t -and t -weighted sequences. edema characterized by indistinct margins, is a poor indicator of malignancy. conclusion: study shows that there is overlap in the radiological appearances of primary malignant bone tumors especially in the early stages. magnetic resonance imaging is shown to be a useful and sensitive modality in the detection and evaluation of bone tumors. mr imaging accurately assesses soft tissue and synovial involvement, as well as marrow infiltration, which may be radiographically silent. in patients with extremity pain mr imaging is necessary and mri data wasn't correlated with conventional radiology. all of them were underwent lateral flexion-extension radiography and lumbar mr imaging. differences of interspinous distances at flexion-extension radiography were analyzed at upper, lower, and spondylolisthetic segments. the mr features of the interspinous ligament were divided into groups according to their signal intensities: group , with inflammatory type presented as high signal intensity at t -weighted image and low signal intensity at t -weighted image, and group , fibrotic or fatty type. the incidence of the interspinous ligament with the inflammatory type was analyzed on the spondylolytic segment and the upper and lower segment without spondylolysis, respectively. results: spondylolysis at l , l and l levels were , and , respectively. of patients, the inflammatory type of the interspinous ligament was ( %) on the spondylolisthetic segment and ( %) on the upper segment without spondylolysis (p < . ). the mean difference of interspinous distance on the spondylolisthetic segment and the upper segment were . mm and . mm, respectively (p < . ). in patients of isthmic spondylolisthesis, the upper segment without spondylolysis showed more inflammatory type of the interspinous ligament on mr imaging than spondylolisthetic segment probably due to interspinous hypermobility. advantages of multislice ct in the diagnosis of traumatic osseous disease c. remaining cases, to assess the articular surface involvement and the real extension of the fracture. the isotropic mpr reconstructions carried out with the multislice ct scanners allow us to scan the patient in only one position, usually the axial plane, saving time and using less radiation dose. the mpr reformats takes an important role in the final diagnosis of many cases. the consideration of positioning in radiography for the waist of the scaphoid bone fractures s. matsubara; hiroshima/jp purpose: the most common type of scaphoid bone fracture is the transverse fracture of the waist (central / ). however, traditional positioning in radiography frequently fails to visualize bone fracture line. this may be caused by the fact that x-rays cannot be irradiated perpendicularly onto the scaphoid long axis. in this study, we searched optimal limb position for visualizing the waist of the scaphoid bone. method: in volunteers, the scaphoid bone was taken in the ulnoradial direction at °ulnar deviation (group a) and ° ulnar deviation with the fist clenched (group b). images measured the angle between the cassette plane and long axis of the scaphoid (c-s) and the angle between the cassette plane and the volar tilt (c-v). result: the c-s angle in group a was . ± . ° and in group b was . ± . °. the c-v angle was . ± . °. we devised new positioning that lift distal side of forearm upward at about ° in group a, at about ° in group b. these positioning make the long axis parallel to the cassette plane. especially, the later view was able to visualize the ulna-scaphoid joint more clearly without overlap of radius and scaphoid bones as compared to traditional method. conclusion: in fractures of the scaphoid, in which early diagnosis is critical, optimization of first-choice x-ray imaging is a key factor, and an additional image involving a ° scaphoid waist view will be useful. background: mri imaging is a very powerful tool in the evaluation of the acl, with higher sensitivity than the clinical examination of the knee. some of its lesions are well known and easily identifiable, but others require careful interpretation and familiarity with the spectrum of acl pathology. over knee mri examinations performed in three different diagnostic centers from january to july were reviewed retrospectively. acl lesions were found in approximately knees. procedure details: mri units ranged from . to . tesla, and various imaging protocols were used, with t and t coronal and sagittal images obtained in all cases. traumatic tears were the most common lesions identified. tears were classified as acute or chronic, complete or partial. we illustrate and discuss the primary and secondary findings of acl tears, as well as the imaging criteria in the evaluation of the postoperative acl. we also present fractures of the anterior tibial spine with acl avulsion, acl ganglion cysts and mucoid degeneration, which are less commonly seen. in the examination of the acl pathology the radiologist should evaluate multiple signs and be aware of some less common pathologies in order to effectively contribute to the clinical management. ultrasound and x-ray findings in painful shoulder involvement in rheumatoid arthritis l. rutkauskiene; vilnius/lt learning objectives: to illustrate and compare conventional x-ray and ultrasonography diagnostical possibilities of painful shoulder in ra. background: up to percent of rheumatoid patients have shoulder pain. a rational decision on the treatment of shoulder disease in rheumatoid arthritis depends on an accurate assessment of its cause and the extent of rheumatoid involvement. imaging details: a group of ra patients, who reported a pain in the arm and/ or limitation of the active movement, were examined first with conventional anterioposterior and obligue -outlet -view with - angulation x-ray of both shoulders. the most common findings were: joint space narrowing, erosions, subchondral cysts, irregularities of greater tuberosity, deformity and flattening, superior migration of humeral head, erosions in acromioclavicular joint, sometimes soft tissues swelling. ultrasonography study was performed with a . results: clinical signs and symptoms (pain and swelling) due to foot involvement were present in ( %) of the patients while frequency of involvement was ( %) with mr imaging assessment. the most common involved anatomical region was the hindfoot ( %) following by midfoot ( %) and ankle ( %). the mr imaging findings were bone erosions, achilles tendinitis, para-articular enthesophyte, joint effusion, plantar fasciitis, joint space narrowing, subchondral sclerosis, soft tissue edema, bone marrow edema, enthesopathy of the achilles attachment, plantar fascia attachment, and plantar ligament, subchondral edema, retrocalcaneal bursitis, subchondral cysts, subchondral fissures, and bone ankylosis. conclusion: mr imaging may detect bone, soft tissue, cartilage, tendon, and joint abnormalities in as patients, even if as patients did not have clinical signs and symptoms of foot involvement. this imaging modality may be of importance especially early diagnosis of inflammatory changes in the foot. additionally, familiarity of the typical mr appearances of foot involvement in as patients may be helpful in narrowing the wide differential diagnosis of spondyloarthropathies. mr handle meniscal tear using the following criteria: a) bow tie sign; b) double posterior cruciate ligament sign; c) coronal truncation sign; and d) displacement of the free meniscal fragment. results: in / cases a bucket-handle meniscal tear was identified. in of these latter an infero-medial displacement of a free fragment of the medial meniscus was identified. in all cases a lesion of the posterior horn involving the inferior articular surface was observed on axial plane. on coronal plane was observed the truncated triangle aspect of the peripheral, not displaced, meniscal portion. on all planes the displaced free meniscal fragment was identifiable as a little hypointense formation, medially to proximal tibial extremity, inferiorly to meniscal plane and deep to tibial collateral ligament. in all cases diagnosis was confirmed by arthroscopy. conclusions: mri allowed the detection of the infero-medial displacement of a free meniscal fragment thus helping the arthroscopic approach. high-resolution mri of ulnotriquetral ligament injury and its anatomical variations with a microscopy coil t. ueno , h. yoshioka , t. tanaka , y. kujiraoka , m. shindou , k. ooyama , y. saida ; tsukuba-shi, ibaraki/jp, boston, ma/us purpose: to demonstrate ulnotriquetral ligament (utl) injury and its anatomical variations using a microscopy coil. materials and methods: patients ( males and females with age ranging - year-old), who suffered ulnar side wrist pain and suspected triangular fibrocartilage complex injuries, were investigated in . t mri system with a microscopic coil ( -mm in diameter). axial and coronal t *-weighted images, sagittal and coronal proton density-weighted images, coronal stir images were obtained with a -mm fov and a - . mm of slice thickness for all patients. result: utl was identified in patients and its injuries were found in patients. regarding anatomical variations, in proximal side, utl always connected to volar radiocarpal ligament and disc proper. however, there were many variations of the attachment to triquetrum bone. in patients, bands attached to triquetrum at different site: one attached to the distal aspect of triquetrum, and another attached to a notch which was found at volar aspect of triquetrum. in patients, one attached to proximal side of the notch. patients showed separate utl bands. conclusion: high-resolution mri with a microscopy coil is a promising method to demonstrate utl injury. it would be useful to know normal anatomical variations for understanding the mechanism of utl injury. pictorial review of diagnostic imaging techniques in hip arthroplasty follow-up j. rimola, p. melloni, r. valls, p. bermúdez, d. gil, a. massuet; sabadell/es objective: to evaluate the spectrum of findings for plain-film radiography and other imaging techniques in patients with early or late complications of hip prostheses. we describe the usefulness of each imaging technique in follow-up. background: over the last years more than hip replacements have been performed in our hospital annually. all patients are followed up immediately after surgery, at months and then yearly or when necessary, using plain films and sometimes sonography (us) and/or computerized tomography (ct). us and ct are used to confirm or rule out osteointegration of the prosthesis and complications such as periprosthetic abscess and/or hematoma. imaging findings: we analyzed radiological features of the normal and pathological evolution of hip prostheses. we describe early complications (affecting . % of patients), such as infection, cement extrusion, and periprosthetic fracture, as well as late complications (affecting . % of patients), including aseptic loosening, osteolytic lesions, heterotopic calcifications, migration of the acetabular component, femoral diaphysis fracture, and dislocation of the prosthesis. conclusion: plain films are essential for evaluation and detection of hip arthroplasties. us is usually used to guide percutaneous aspiration of soft tissue. ct enables correct assessment of osteointegration of hip prostheses. results: with wrist arthroscopy as the standard of reference, average sensitivity and specificity of non-enhanced mri, for tears of the tfcc were . and . respectively and . / . / for arthroscopy. conclusion: mr imaging-plays a major role in evaluation and management of traumatic wrist injuries, improves clinicians diagnostic accuracy and reduces the need for arthroscopy. these preliminary results illustrate the ability of mri to assess the integrity of the tfcc and suggests its use as the first imaging method following plain radiography in the evaluation of patients with chronic posttraumatic pain on the ulnar side of the wrist. wrist arthroscopy is a more valuable technique in determining the location, size, and extent of ligament injuries within the wrist. high patients ( women, men; mean age: years) referred from e.r. for a suspect traumatic dislocation of the a-c joint. all patients underwent plain x-ray examination (conventional projections integrated by "outlet" view and stress projections) and hrus scan (longitudinal and axial scan planes of interested ligaments) of both the interested and contralateral a-c joint. ultrasonographic criteria of lesion were considered the absent recognition of the ligament and strong echostructural inhomogeneity a-c joint. measures of a-c and coraco-clavicular (c-c) distances were drawn by means of both methods. dislocation's degree was defined according with rockwood's classification. results: in / cases of indirect trauma, diastasis of the a-c joint ( - mm), was observed, whereas c-c joint was not involved and c-c ligaments were normal. in / patients with direct trauma, distracting lesion of the a-c ligament was observed in cases, and involvement of both a-c and c-c ligaments with diastase of both joints was observed in the remaining cases. a complete correspondence of the two methods was observed. conclusion: hrus enables early anatomical-structural changes detection and lesion's degrees assessment in acute traumatic lesions of the a-c joint, and could be considered more than complementary to x-ray exam in the emergency clinical setting, mantainig its role in the follow-up. ligaments and ac joint soft-tissue morphology, acromioclavicular distance and coracoclavicular distance were evaluated. sonographic findings were correlated with the results of conventional radiographic examination, which was performed in all cases with anteroposterior projections, taken with and without weights, and with "outlet" projection. sonographically, all patients presented lesion of ac ligament with high amount of sero-hemorrhagic fluid inside and outside the joint. in / patients, which had indirect trauma, we observed ac joint dislocation with ac ligament lesion that was not associated to involvement of cc joint and ligament. in / patients, which had direct trauma, we observed lesions of ac and cc legaments with associated joints dislocations. conclusions: ultrasonography, if performed with appropriate technique and adequate transducer, is accurate in the evaluation of acute traumatic ac joint lesion and dislocation, integrating and correlating well with the informations offered by conventional radiography. study of the mr imaging of the posterolateral structures of the normal knee c. yu, k. li; beijing/cn puropse: through the study of the mri appearance and localization of the posterolateral structures (pls), to provide a practicable method for intactly displaying them. methods: thirty tibial bone specimens were observed to establish the bony landmark for localizing the knee. in cadaver knees, the angles between lateral tibial plateau and the long axis of the individual structure of pls were measured. then we can determine the scan methods of the oblique mr images based on above results. the routine and oblique scans of t wi were performed in normal knees. the display effect and appearance of the pls were observed in mri. the lateral tibial plateau is a stable bony landmark for measuring and localizing of the knee. in the pls, the fibular collateral ligament can be intactly displayed on ° posterior coronal oblique images in cases ( %). the popliteus can be better seen on either ° posterior coronal oblique in cases ( %) or ° medial sagittal oblique planes in cases ( %). the popliteofibular ligament can be intactly appreciated on both ° posterior coronal oblique in cases ( %) and ° lateral sagittal oblique images in cases ( %). although the arcuate ligament and the fabellofibular ligament can occasionally be seen on routine and oblique images, their presence rate is low. the oblique mr imaging can intactly display the main structures of pls, and can be an useful tool in diagnosing these structures' injuries. patients are also asked for the presence of their prolonged physical activity in their life. results: patients had type ii, patients had type i and of them showed type iii patellar shapes, out of . patients had patella alta whereas all others stayed in normal range as patellar position. in patients' case no other abnormalities were seen apart from the signs of iliotibial band friction syndrome and three of them had shown type iii patellar shape. conclusions: no apparent correlation was found between iliotibial band friction syndrome and patellar position. as it is much more rare in the normal population wiberg type iii patella showed considerably high prevalance in our study. shoulder to assess by mri the potential benefit and efficacy of selective meniscectomy combined with radiofrequency ablation in cystic degeneration of the lateral meniscus treatment. materials and methods: sixty-one patients entered this study. patients underwent arthroscopic selective meniscectomy and patients underwent arthroscopic selective meniscectomy and radiofrequency treatment (arthocare, sunnyvale, ca) of the meniscal remnant. all of them were submitted three months later to mri and clinical evaluation. in case mri examination was performed before and after the surgical treatment. mri examination was performed using a dedicated . t (artoscan esaote italy) and a . t superconductive unit (ge signa horizon usa) employing se t -w, se t -w and ge t -w sequences on axial and longitudinal scan planes. in case arthro-rm was also performed. clinical evaluation included physical examination and questionnaire. results: clinical evaluation did not show statistically significant discordance between the two groups. mri follow-up after months demonstrated a very clear decrease of the degenerative spots inside the meniscal remnant in the group with combined treatment respect to the group treated only with selective meniscectomy. conclusion: in conclusion our experience considers mri the method of choice in the evaluation of post surgical treatment of degenerative cystic diseases of the lateral meniscus of the knee. high-resolution ultrasonography of the extrinsic carpal ligaments: examination technique and performance d.v. guntern, n. favarger, p. schnyder, n.h. theumann; lausanne/ch purpose: to develop an examination technique to visualize the extrinsic carpal ligaments by ultrasonography and to assess the performance of high resolution ultrasonography in the visualization of different parts of these ligaments. the extrinsic carpal ligaments of wrists of healthy volunteers were examined with a mhz linear array transducer by two muskuloskeletal radiologists in consensus. an examination technique was developed for each extrinsic carpal ligament (radioscaphocapitate, radiolunotriquetral, palmar scaphotriquetral, dorsal scaphotriquetral, palmar ulnotriquetral, dorsal ulnotriquetral, dorsal radiotriquetral and radial collateral ligaments). delineation of the proximal attachment, the body and the distal attachment of each ligament was evaluated as good, poor or absent. results: with a profound knowledge of the anatomy, especially of the osseous landmarks, an easy and fast examination of the extrinsic carpal ligaments by high resolution ultrasound is possible. the more superficial ligaments such as the dorsal scaphotriquetral and the dorsal radiotriquetral ligaments are well visualized by high resolution ultrasound whereas ligaments in a deeper position, such as the proximal and distal insertion of the palmar scaphotriquetral and the distal insertion of the radioscaphocapitate are less well visualized. high resolution ultrasonography is an accurate method to visualize the extrinsic ligaments of the wrist. direct mr arthrography of the glenohumeral joint with capsular distension using a posterior approach r. drescher, j. ludwig, f. rubenthaler, o. köster, g. schmid; bochum/de purpose: evaluation of the clinical use of direct mr arthrography of the glenohumeral joint with maximum distension of the joint capsule in patients with glenohumeral instability using a posterior approach. patients and methods: direct mr arthrography of the shoulder joint was performed on a . t system in patients ( women, men, mean age years). right and left shoulders were evaluated. all patients had anterior or bidirectional instability ( posttraumatic, constitutional). using a fluoroscopically guided posterior approach, ml iopamidol m and ml . % mepivacaine were injected, followed by a % dilution of dimeglumine gadopentetate in . % sodium chloride until full capsular stretching was achieved. mr sequences were t -weighted spin-echo axial and oblique-coronal, fat-saturated t d axial and oblique-coronal, and axial t flash- d. results: arthrography was possible in all patients. the total amount of intraarticularly injected fluid was - ml (mean ml). no complications occurred. mr imaging showed significant capsule distention in patients ( %) and ventral capsule defects in patients ( %). labral lesions ( anterior, posterior), bicipital tendon lesions ( complete, partial ruptures) and partial ruptures of the rotator cuff were noted. patients underwent surgery. regarding labral lesions, sensitivity of mri was . %, specificity was %, and diagnostic accuracy was . % compared with surgical results. conclusions: glenohumeral direct mr arthrography allows accurate detection of labral and tendinous lesions. maximum capsular distension allowed preoperative evaluation of capsular laxity as a source of shoulder instability. anterior talofibular ligament injury: evaluation with mr imaging j. ishida , j. yoshigi , h. okizuka , k. ooae , m. takao , t. yoshizako , k. izaki , n. uchida , h. kitagaki ; izumo/jp, himeji/jp purpose: inversion stress is most frequent injury of all ankles. atfl is the weakest of lateral collateral ligaments and first to rapture. the purpose of this study is to evaluate the usefulness of high-resolution mri for diagnosis of atfl disruption divided into acute and chronic phase. method and materials: patients suspected atfl disruption underwent mri ( . t). cases were in acute phase (< days) and cases were in chronic phase (> month). axial spin-echo (se) t -wi (tr/te = / , fov cm, mm/ . mm, x , nex,) and axial fast se t -wi (tr/te = / , etl , fov cm, mm/ . mm, x , nex,) were obtained with an extremity coil. atfl disruption was diagnosed when either finding is positive:discontinuity, irregularity, non-visualization of aftl, and high signal intensity lesion in atfl on t -wi. results: arthroscopy confirmed atfl disruption in cases in acute phase and b d e f a g cases in chronic cases. in acute phase, sensitivity was %, specificity was %, and accuracy was %. only false negative was seen. in chronic phase, sensitivity was %, specificity was %, and accuracy was %. / was false negative. conclusion: in acute phase, mri was accurate for evaluation of aftl disruption. however, in chronic phase, mri was less accurate because granulomatous tissue after disruption cannot be detected with mri. ct arthrography of the shoulder a. gligorievski, z. temelkovski; skopje/mk purpose: to present ct arthrography (cta) as a new method in our practice in order to point out its advantages and to compare the results with conventional arthrography and mr arthrography. we describe the technique of the examination in detail, performed at the institute of radiology in cooperation with an orthopaedic surgeon and radiologist that form the diagnostic-therapy team. cta performed with patients, aged to , men and women. conventional arthrography was performed in and mra in patients. results: patients presented with lesion of the glenoid labrum, with rotator cuff lesion, with a frozen shoulder, with a hill-sachs lesion, with biceps brachi tendon subluxation and case with slap lesion. we report our first experiences and the results gained are compared to similar recent studies as well as to the conventional radiologic techniques. cta is a method of choice in case of frozen shoulder and glenohumeral instability, but mra is better in case of rotator cuff lesion. we can conclude that cta is a sophisticated method of extreme diagnostic value in the evaluation of the humeral joint especially in case of glenohumeral instability. the invasiveness of the method is small, no complications have been noticed and compared to mr it is significantly cheaper and more available for the medical centers equipped with a ct scanner. mr imaging of the metatarsophalangeal joint with standard mr imaging: evaluation of six patients with clinical suspicion of plantar plate disruption p. hauser; lausanne/ch purpose: to report the mr imaging findings of painful injured metatarsophalangeal joints (mtpj) in case of clinical suspicion of plantar plate (pp) tear. a series of patients with clinical suspicion of pp rupture of first (mtpj ) or second (mtpj ) ray underwent mr studies with . tesla units with use of an extremity coil. the patients lay on their back with the ankle in plantar flexion and the toes in extended position. the standard mr imaging protocol performed in all specimens consisted of t weighted spin echo sequences in sagittal and coronal plan and t fse fat satured sequences in sagittal, coronal and transverse plan. the examinations were interpreted separately by two musculoskeletal radiologists assessing whether or not a pp rupture and associated lesions were present. after the imaging study all the patients underwent surgical revision in a mean delay of one month. results: tears of the pp were visualized in cases, degenerative lesions of the pp were visualized in two cases and two other cases showed no lesion. the mr imaging results concerning pp rupture although allowed depiction of several associated lesions including injuries of the extensor hood, the collateral ligament complex, ostechondral lesions and joint deformities. conclusion: although surgical treatment is generally the rule for hallux valgus deformities, surgical repair is also often required in cases of traumatic injuries of the mtp joints. the force of accord between radiologists and surgical observation of pp ruptures is good. mrt monitoring of autologous hyaline cartilage grafts c. müller, e. grönewäller, c.d. claussen, f. schick; tübingen/de purpose: seven different mr sequences were compared to localize cartilage defects in knee and to evaluate the stage of maturation of the hyaline cartilage graft. methods: mrt was performed in patients aged - years with autologous transplantation of a hyaline cartilage tissue graft after a knee trauma. examination dates were: before transplantation to localize the defect, and weeks, , and months after transplantation to control the morphology and the maturation of the autologous graft. standard t -and proton-density weighted turbo spin echo (tse) sequences, t -weighted spin echo (se) sequences, gradient echo (gre) sequences with and without magnetization transfer (mt) pulses as well as experimental diffusion weighted gre sequences (psif) were used. results: for depiction of the graft short time after surgery t -weighted tsesequences showed the best results, to identify potential secondary damages before transplantation pd weighted sequences were superior. and months after transplantation spoiled d-gre-sequences displayed the graft in high spatial resolution. from the diffusion measurements no statistically significant discrimination between cartilage and graft was obtained at any time, whereas, different images from gre-sequences with and without mt pulse provided high contrast between cartilage and surrounding tissue. the quantification of the mt effect showed an assimilation of the graft to the original cartilage within months. conclusion: mrt should be performed before every transplantation because cartilage defects and secondary damages can be detected clearly. after transplantation mrt can easily identify the graft and detect hypertrophy or debonding in early stage. quantitative macroradiographic study with fractal signature analysis measures bone loss in the rheumatoid arthritis hand l. disini , m. foster , j. buckland-wright ; london/uk, loughborough/uk purpose: fractal signature analysis (fsa), a computerised method of textural analysis, permits the separate measurement of changes in vertical and horizontal trabeculae based on the fractal dimension over a range of trabecular widths (fractal signature). we determined whether the fsa of high definition macroradiographs (x magnification) quantified differences in trabecular organisation at sites of osteopenia and erosion formation in the rheumatoid arthritis (ra) hand. methods: sixty-seven ra patients had macroradiographs of the left hand. the distal radius was scored and grouped from very mild (ra ) to moderate (ra ) disease. macroradiographs were digitised and fsa of horizontal and vertical trabecular organisation were measured in the radius at sites of osteopenia, erosion formation and a mid-shaft site. the fractal signatures were compared to healthy non-arthritic subjects using the t-test. results: compared to the non-arthritics, fsa at the distal radius in groups ra to ra measured a significant reduction (p < . ) in fractal signature at the osteopenic and mid-shaft sites, affecting small to large sized vertical trabeculae, and at the erosion site, involving small and medium sized horizontal and vertical trabeculae. the reduction was smallest in ra and greatest in ra . conclusion: fsa quantified the trabecular bone loss in the distal radius of ra patients of increasing radiographic severity based on a proportional reduction in fractal signature. disease related bone loss involved varying trabecular widths with vertical trabeculae at the osteopenic site and both vertical and horizontal trabeculae at the erosion site. potentially, fsa can distinguish changes consequent to bone-mediated ra drug treatment. the results: the value of d ct reconstructions in external projections was the highest in revealing of the osteophytes of anterior margins of vertebral bodies; the anterior osteophytes were found in ( . %) patients on d ct images. the d ct reconstructions cut off in sagittal plane along the spinous process were the best imaging modality in evaluation of the osteophytes of the posterior margin of the vertebral body, which were seen in cases ( %) and in evaluation of narrowing of the intervertebral foramens ( cases - %). conclusions: d ct reconstructions are valuable in determining the localization and the extent of the cervical spine osteophytes. they are the imaging modality of choice in assessment of the degenerative narrowing of the intervertebral foramens. d ct reconstructions should be the integral part of ct examination of patients with the neck pain. purpose: the aim of our study was to determine the incidence of jspa in croatian population in the last years in relation to other rheumatic diseases and the presence of hla -b in children with jspa. as the cause of jspa is still unknown, the strong association with hla -b suggests genetically determined mechanism. methods and materials: the patients from to years of age diagnosed during the period - at department of paediatric rheumatology, university hospital zagreb following strict criteria of european spondyloarthropathy study group. we identified newly diagnosed patients with rheumatic disease using hospital and outpatient records. results: were identified as having jspa ( . %) amongst , patients with rheumatic disorders. sex ratio was females against males, mean age of . years. patients had undifferentiated spondyloarthropathy ( . %), patients had reiter's syndrome ( . %), had arthritis of inflammatory bowel disease, i.e. crohn's ( . %), two patients had psoriatic arthritis ( . %), and two had ankylosing spondylitis ( . %). ocular complications were present in patients ( . %), enthesitis in ( . %), sacroileitis in ( . %) and peripheral arthritis in all patients ( %), diagnosed radiologicaly. hla -b was present in patients ( . %). conclusion: our epidemiological study showed similar incidence of jspa among other rheumatic diseases in childhood and adolescents ( . %) compared to results of american, canadian and british studies in which this incidence is . - . %. however, the presence of hla -b antigen is lower ( . %). postprocessing optimization of computed tomography data of acetabular fractures v. moschenko, t. shamova, a. sklyarenko, i. bobkova; kharkov/ua purpose: to study diagnostic opportunities of postprocessing programs -multiplanar (mpr) and d shaded surface display (ssd) reconstruction of ct data on acetabular fractures. methods and materials: patients aged - with acetabular fractures in various terms since the moment of trauma (from days to months) were examined. plain radiographs and ct with the subsequent application of programs mpr and ssd were conducted. the acetabular fractures classification by judet -letournel was used. results: use of postprocessing programs allowed in ( . %) cases to specify a type of fracture and character of deformation of acetabulum, therefore in ( . %) -the originally prospective conservative treatment was replaced by operative. considering complexity of choice certain reformations mpr and ssd for analysis and documenting of views, we used standard projections: for mpr - ) along anterior column, ) along posterior column, ) along both columns; for ssd - ) anteroposterior view of the pelvis; ) direct projection (lateral view) of the acetabulum; ) obturator oblique view of the pelvis; ) iliac oblique view of the pelvis. in ( . %) cases use of only these projections was enough to establish the type of acetabular fracture. conclusion: use of mpr and ssd in ct data postprocessing allows establishing the type of acetabular fractures that defines a choice of treatment tactics. our standard protocol of mpr and ssd reduces analysis time of research results and creates conditions for uniform perception of injuries by all participants of diagnostic process. conclusion: routine x-ray projections prove insufficient to establish a diagnosis in / th of the cases. confusion is caused by overlaying of fracture edges and pelvic bone in the direction of x-ray beams. the latter makes hardly visible the small posterior fragment, too. therefore, ct scanning is required for the diagnostic protocol of displaced femoral head fractures. is learning objectives: to describe us and mr imaging findings of nerve infiltration by extrinsic masses, including lipomas, hemangiomas, non-hodgkin lymphomas and ganglion cysts. to learn to differentiate between these pathologic conditions. to emphasize the role of us and mr imaging in the management of these lesions to further delineate the nature and extent of the process. background: in addition to peripheral nerve sheath tumors (e.g. neurofibromas, schwannomas and malignant peripheral nerve sheath tumors), there are softtissue masses, such as hemangiomas, lymphomas and ganglion cysts, which do not originate from the nerve itself but may occasionally infold among the nerve fascicles and expand into the neural tissue. the intraneural growth of these masses is rare, may cause nerve dysfunction and local symptoms and should not be confused as the more common nerve sheath tumors. imaging findings: - mhz us and . t mr imaging findings for a series of eight retrospectively collected cases of masses infiltrating nerves are illustrated with emphasis about the different pathogenetic mechanisms of intraneural infiltration. hemangiomas involved the median and ulnar nerves, ganglion cysts the peroneal nerve, non-hodgkin lymphomas the sciatic and median nerves. correlation of imaging findings with gross surgical views is provided in / cases. conclusion: high-resolution us and mr imaging can detect nerve infiltration by extrinsic masses. knowledge and recognition of the characteristic imaging features can aid in the proper diagnosis and treatment of these lesions. mri of lipomatous soft tissue tumors: radiologic pathologic correlation m. pilavaki, d. chourmouzi, g. boulogianni, a. drevelegas; thessaloniki/gr purpose: to study the mr imaging findings for each type of lipomatous soft tissue tumors and to correlate them with the pathological findings. we studied retrospectively the mr examinations of patients with histological proven lipomatous soft tissue tumors. the histological types of the tumors were: lipomas, fibrolipoma, lipoblastoma, lipomatosis and liposarcomas ( well-differentiated, myxoid, and mixed type) t -, t -, and fat-suppressed and t -weighted images after administration of gadopentetate dimeglumine were obtained. the signal intensity on different sequences, the internal characteristics, the presence of septa and other nonadipose component, revealed on the mr images and the degree of contrast enhancement of septa and nonadipose component were evaluated. results: from the group of benign lipomatous lesions lesions were without a recognizable nonadipose component. lesions had thin septa without contrast enhancement. in case of fibrolipoma areas of nonadipose component correlated with fibrous tissue. in the group of well-differentiated liposarcomas had thin septa and thick septa with mild contrast enhancement. the remaining liposarcomas had thick septa with of them having a nodular and globular non-fatty component with enhancement after the administration of paramagnetic agent. conclusion: statistically significant imaging features favoring a diagnosis of liposarcoma included the presence of thick septa, enhancement of septa and the presence of nodular or patchy nonadipose components. imaging features suggesting benignancy included thin septa with no enhancement relative to muscle and no recognizable nonadipose component. guidelines for diagnosis of soft tissue lipomatous masses (stlm) g. chave, d. ranchere-vince, p. meeus, p. thiesse; lyon/fr learning objectives: to clarify the steps of pretreatment diagnostic procedures in case of soft tissue lipomatous masses (stlm). to describe the mri findings for lipomas and liposarcomas. to develop the principles of percutaneous biopsy. background: differential diagnosis between lipomas and liposarcomas, especially atypical lipomas is not always easy. despite similarities, differential diagnosis can be reliably established employing image parameters. nevertheless, the staging process of stlm culminates in biopsies, allowing to manage the proper treatment. biopsies can be performed percutaneously using imaging guidance. an open biopsy with the resection of the entire mass is disputed when the stlm is composed essentially of fat on mri imaging to avoid the misdiagnosis of benign lipoma. each procedure must be carefully planned: an inadequate biopsy may fail to allow proper diagnosis, have a negative impact on survival, and ultimately necessitate an amputation to accomplish adequate margins of resection. procedure details: after the description of characteristics and different histological subtypes of lipomas and liposarcomas, we will describe the mri findings for each diagnosis, according to a review of the litterature by illustrating with mri imaging of patients followed in our center. we will develop the principles of percutaneous biopsies according to litterature and our experience. conclusion: accurate diagnosis of stlm is based on three factors: clinical presentation, radiological features and pathologic evaluation. the importance of careful planning and performance of biopsies cannot be overemphasized because errors may have a negative impact on survival and impede proper diagnosis. high background: clinical assessment of extensor tendon injuries is not straightforward because of the peculiar anatomy of these tendons made of several interconnecting layers and different attachment sites in the fingers. the development of very high-frequency "footprint" transducers has improved the ability of us to scan the extensor tendon complex in the fingers, allowing accurate depiction of the central and lateral slips of the extensor tendon, and the sagittal and lateral bands of the extensor hood. imaging findings: paradigmatic - mhz and - mhz us images from a series of retrospectively collected cases with a variety of extensor tendon injuries in the fingers are illustrated, including partial and complete rupture of the central slip (boutonnière deformity), tears of the lateral slip, avulsion injury of the terminal tendon (mallet finger), injury of the sagittal band over the metacarpophalangeal joint with tendon instability (boxer knuckle). dynamic us is essential to diagnose injuries of the extensor hood mechanism. correlations of us findings with schematic drawings and the results from . t mr imaging are also provided. conclusion: high-resolution us is promising for evaluating the extensor tendons complex in the fingers. it can identify and characterize a variety of abnormalities. by providing unique information on these tendons, us has potential for major impact on treatment planning. high-resolution us of tenosynovitis in the wrist and hand a. picotti , l.e. bacigalupo , r. podestà , g. succio , f. pugliese , s. bianchi , c. martinoli ; siena/it, genoa/it, geneva/ch learning objectives: to describe the spectrum of tenosynovitis of flexor and extensor tendons in the wrist and hand by means of high-resolution us. to correlate us findings with clinical features and mr imaging. to emphasize the role of us in the management of these lesions to further delineate the nature and extent of the process. background: tenosynovitis of wrist and hand tendons are common and account for a high percentage of orthopaedic consultations. they can be related to local causes, particularly overuse due to sport or occupational activities or may be the result of systemic musculoskeletal disorders. imaging findings: paradigmatic - mhz and - mhz us images from a series of retrospectively collected cases with a variety of tenosynovitis in the wrist and hand are illustrated with emphasis on the pathogenetic mechanism of tendon disease, including de quervain disease, intersection syndrome, extensor pollicis longus, extensor carpi ulnaris, flexor carpi radialis and flexor digitorum tenosynovitis. the main findings in infectious and hypertrophic tenosynovitis are also described and a spectrum of unusual causes producing them is reported, including foreign bodies and conflict with fracture residuals and osteosynthesis material. conclusion: due to its widespread availability, low cost and high spatial resolution, high-resolution us is an excellent tool for investigating a variety of tenosynovitis of the wrist and hand. background: the patients with inflammatory myopathies (inclusion-body myositis (ibm) = , polymyositis (pm) = and dermatomyositis (dm) = ) underwent whole-body mr muscle screening examination. mr examination is performed at six different levels: neck, shoulder, abdomen, pelvic girdle, and upper and lower leg. t -and t -weighted images with and without fat suppression were performed for each level. signal intensity of muscles, volume and the symmetry of involvement were quantified in individual muscles on each side. imaging findings: ibm, dm and pm are rare diseases with clinical and histopathological similarities. ibm usually does not respond to immunosuppressive therapy. therefore, it is necessary to distinguish these entities correctly. ibm is characterized by more frequent fatty infiltration, atrophy, inflammation, a distal predominance, and anterior group involvement. the finding of selective involvement of the medial gastrocnemius and of the vasti with sparing of rectus femoris is typical for sporadic ibm. the selective involvement of flexor digitorum profundus is also highly suggestive for ibm. t , t and fat-sat images contain different but complimentary information. pm and dm predominantly affect muscle groups of the shoulder girdle and the thighs. a newly introduced mr technique provides an excellent overview of all diseased muscles in the body. moreover, it helps us to distinguish ibm from other iims. mri may help establish the diagnosis, suggest appropriate sites for biopsy, and enable assessment of disease progress or regression over time. background: elastofibroma dorsi is a rare, typically slow-growing, often bilateral, para-neoplastic formation. it's composed by fibro-elastic and adipose tissue and is selectively localized along the postero-lateral side of thoracic wall, under the scapula. the certain diagnosis is cyto-histological. mri was the hitherto elective diagnosis. all cases of elastofibroma in our series were studied with us; some of them also with ct and mri; in a retrospective analysis us allowed to suspect presence of elastofibroma in all patients: of them underwent surgery that confirmed the diagnosis. procedure details: in our series the us examination, performed with . - mhz linear probe, showed solid masses, sometimes in both sides, with badly visible margins, hypoechoic with inside hyperechoic stripes, obliquous from the transducer plan. conclusions: in all cases us and clinical findings allowed the diagnosis of elastofibroma dorsi. the mri, performed in patients that underwent surgery, confirmed the us findings. the remaining patients are undergoing follow-up with us. so, us findings, within site, slow growth and possible presence of bilateral masses, allow the diagnosis of elastofibroma dorsi, avoiding costly mri. a comparison between ultrasound, echo-color-doppler, and mri in the evaluation of achilles enthesopathy in psoriasis: preliminary results f. maggi, f. di gregorio, c. de simone, c. pagliarello, m. politi; rome/it purpose: psoriasis is often associated with achilles enthesopathy. this study compares the efficacy of ultrasound, echo-color-doppler and mri in the evaluation of early signs of achilles enthesopathy in psoriatic patients. methods and materials: consecutive psoriatic patients, men, women, mean age years (age range - ), mean pasi score , entered the study. all patients underwent sonography of the achilles tendon and peritendinous structures with aplio (toshiba) equipped with a linear multifrequence ( - mhz) transducer; the study was completed with color-doppler analysis. an mri study, before and after gadolinium, was then performed using e-scan xq (esaote), . tesla superconducting magnet. axial and sagittal t and t weighted, sagittal ge and stir sequences were obtained. after gadolinium, axial and sagittal t weighted sequences were obtained. results: the more frequent us findings were microcalcifications inside the achilles tendon ( % of cases) and signs of bursitis ( %), while uncommon there were degenerative lesions and signs of peritendinitis. completion with echo-color-doppler did not add significant data to us study. mri imaging showed degenerative lesions and peritendinitis as well as us, but was not able to identify any microcalcification. all the alterations were identified in the basal study; administration of gadolinium did not allow any additional pathological finding. conclusion: our preliminary results show that mri, especially with low magnetic field, is not sensitive compared to us in detecting early changes of achilles enthesopathy in psoriatic patients, while echo-color-doppler and enhanced mri sequences had limited utility in diagnostic imaging of early stages of this pathology. characterization high resolution us and mri in the evaluation of plantar fibromatosis i. gallesio, s. parodi, r. pastorino, d. schettini, m. falchi, e. silvestri, g. garlaschi; genoa/it purpose: plantar fibromatosis is a benign fibroproliferative disorder of the plantar fascia clinically presenting subcutaneous nodules in the foot sole. this study aimed to evaluate the application potential of us and mri performance in detection of these lesions. material and method: patient with palpable nodules suspected plantar fibromatosis were evaluated. the us examination was performed by means of a high resolution transducer through tranverse and longitudinal scans along with evaluation of intra-nodular vascularization by power-doppler. mri was performed by means of a . t unit with axial and sagittal planes. t weighted mri images after administration of iv contrast medium were acquired from two patients. results: nodules were, overall, detected, most of them located in the medial plantar fascia. all nodules were oval-shaped, hypoechoic at us and hypointense at mri. considering number and size of nodules, us and mri overlapped (were strikingly similar). moreover, nodules showed hypervascularization at power-doppler, confirmed at mri with paramagnetic contrast medium. this pattern might suggest a different pathoanatomical condition of these lesions, as hypervascularization is a sign of an early stage lesion, featuring a large fibrovascular proliferation. no correlation was found between vascularization degree and symptoms the latter resulting from the neurovascular structures disorder. conclusions: us associated with power-doppler is the first choice method in the evaluation of plantar fibromatosis, able to detect number, size and activity pattern of nodules, leading to a pre-surgery diagnosis. in severe plantar fibromatosis mr is to be considered a complementary method in evaluation of the extent. purpose: conventional mri is a well-accepted technique in evaluating soft tissue tumors. however it is not specific in differentiating benign and malignant lesions. in this study the differential value of the dynamic contrast enhanced mri (dce-mri) was investigated. methods and materials: turbo flash dce-mri was performed on subjects ( - yrs) with soft tissue tumors. enhancement in the st min (e ), nd min (e ), maximum enhancement (emax), and time-signal intensity curve (tic) slopes were calculated. discriminant analyses were performed to reveal parametric differences of the benign and malignant lesions. results: histopathological diagnosis of benign (n = ) tumors were hemangioma (n = ), neurogenic tumor (n = ) lipoma (n = ) and desmoid (n = ), whereas malignant lesions (n = ) were classified as liposarcoma (n = ), osteosarcoma with soft tissue component (n = ), malignant fibrous histiocytoma (n = ), synovial sarcoma (n = ) and giant cell tumor (n = ). for malignant lesions mean values for e , e and emax were %, %, and %, respectively. mean tic slope was . . for benign lesions the above mentioned values were %, %, %, and . , respectively. anova based discriminant analysis was correctly classified % of the lesions to benign and malignant groups using e , % of the lesions using e or emax, and % of the lesions using tic slope. the combination of the above mentioned parameters had resulted a % accuracy (p < . ). conclusion: dce-mri parameters are the surrogate markers of tumoral microcirculation and tissue perfusion. these parameters may be used as key factors when deciding proper treatment alternatives and to reveal malignant transformations. the relation of flexor retinaculum laxity with age, gender, and hand dominance t. altinok, h.m. karakas; malatya/tr purpose: carpal tunnel syndrome (cts) is an entrapment neuropathy involving the median nerve within its fibroosseous tunnel at the wrist. one of the main radiologic parameters in diagnosing cts is the palmar displacement (pd) of the flexor retinaculum in this study, possible physiologic factors that may affect the palmar displacement and decrease the sensitivity of this criterion were investigated. materials and methods: normal wrists of male and female with ages between and (mean: . ; sd: . ) were ultrasonographically investigated with - mhz linear transducer. pd values obtained were correlated with with age, gender, and hand dominance was analyzed. results: there was no significant difference between different genders and between dominant and non-dominant hands regarding palmar displacement. when considering all subjects this parameter was varied between . and . mm (mean: . ; sd: . ). palmar displacement exhibited a very strong correlation with age (p < . ; r = . and r = . , for dominant and non-dominant hand, respectively). when considering all subjects, linear regression between both factors was found to be . (p < . ). the sensitivity of pd in diagnosing carpal tunnel syndrome was variously reported to be lower than other radiologic criteria. we have found this relative insensitivity to be originated from the age dependent laxity. the cut-off value of . mm given in the relevant literature must therefore be considered normal for the paients older than years of age. the use of the normalization data presented may help to improve the sensitivity of the palmar displacement. vascular soft tissue tumor discrimination with mr image findings by pattern recognition techniques c. vidal, j. garcía-gómez, l. marti-bonmati, m. robles; valencia/es purpose: to discriminate within soft tissue tumors (stt) those with a vascular origin from other histological groups by using pattern recognition techniques with mr image findings. a total of consecutive patient with confirmed musculoskeletal stt from different european centres were examined with mr. t and t -weighted fat-suppression or stir images were obtained in all subjects. the recorded variables were: age, clinical presentation, localization, size, shape, mr signal intensity, margins, homogeneity, edema, t -hyperintense tracts, mul-tiplicity, target appearance, muscular atrophy, intratumoral hemorrhage, calcification, intratumoral fat, fibrosis, fascial relationship, bone alterations, vessels. the k-nearest neighbour (knn), support vector machine (svm) and decision trees (dt) were used to classify vascular from cystic, fatty, nervous, synovial and fibrous origin. the sample was splitted with % of cases used as test. results: efficacy of vascular in front of each histological group using knn was high (nervous . , cystic . , fibrous . , fatty . and synovial). vascular vs. all other stt gave an efficiency of . with dt, . with svm and . with knn. conclusion: discrimination of vascular from nervous and cystic origin was easier than from fatty, synovial and fibrous stt. however, the overall values were very high. the computer assisted diagnosis in the discrimination of the vascular histological group from the others in stt diagnosis is possible by using pattern recognition techniques. spring ligament chronic injury on mr imaging a. iovane, m. midiri, m. galia, t. bartolotta, r. lagalla; palermo/it purpose: to evaluate the usefulness of mr to depict spring ligament (sl) and to visualize its chronic abnormalities even when associated to injury of the posterior tibial tendon (ptt). materials and methods: mr images from patients ( female and male; age range - years), with clinical suspect of injury of the ptt, were retrospectively examined for sl normal appearance and abnormalities. all exams were performed by a . t mr using t weighted spin-echo and t weighted fast spinecho sequences on the axial plane. asymptomatic control patients ( female and male; age range - years), were examined in the same interval time using t weighted spin-echo and t weighted fast spin-echo sequences on an oblique plane ( °), parallel to sl. we evaluated if sl was totally or partially visualized, sl thickness and signal intensity. results: in all control patients sl was completely visualized, - mm thick and homogenously hypointense. / patients had injury of ptt. among these patients sl was visualized in its medial portion in patients of which had chronic abnormalities of ls. at standard mr examination was not possible to visualize the inferior portion of sl in almost all patients. conclusions: mr seems to be an important diagnostic technique to evaluate sl abnormalities. the high association of sl lesions with ptt injury has to be known and considered when studying patients with proved advanced ptt injury in order to plan correctly the mr study to sl whole examination. primitive muscular hydatidosis: a pictural review of cases a. konan, h. rajhi, n. mnif, m. karray, m. zlitni, r. hamza; tunis/tn purpose: soft tissue involvement by hydatid disease remains unusual even in endemic areas. in this report our aims are to review the imaging findings of muscular hydatidosis and underline possible peculiarity. we review retrospectively patients ( men, women) who underwent surgery for primitive muscular hydatid disease. imaging technique used included ultrasonography (n = ), ct scan (n = ) and magnetic resonance imaging (n = ). results: ultrasonography found multiple anechoic and hypoechoic cystic lesions (n = ), necrozing tumor-like lesion (n = ). ct scan showed evidence of multivesicular cysts in all cases. mri showed typical liquid lesions (n = ), heterogenous signal due to scraps (n = ) and evaluate accurately the extent of involvement. conclusion: in our experience multivesicular cysts is the main lesion showed in primitive hydatidosis soft tissue. ultrasound is still in routine relevant to detect muscular hydatidosis; ct scan and mri confirme the diagnosis and are helpful to demonstrate the relation between cysts and adjacent structures and evaluate extent of involvement before surgical management. results: mri showed degenerative signs. in % disc enhancement was peripheral, parallel to the end plates.the disk enhancement was at l -l in % and l -s in %. it was found in % of end plates inflammation and in % of fatty changes. it was found in % of stage ii and iiia normal or earlier degenerative stages, % of stage iiib and % of iv stage latter degenerative stages. biopsies were performed avoiding disc infection. of the mri of patients with ankylosing spondylitis showed large contrast enhancement parallel to the end plates without end plates inflammation. mri of operated patients showed linear uptake parallel to end plates associated with enhancement in the posterior part of operated disc. mri of patient with infectious spondylitis showed central disk enhancement with end plates inflammation in % and vertebral abscess in %. conclusion: disc enhancement parallel to end plate is a disc degenerative sign in operated and non-operated disc. it may be present in inflammatory conditions as ankylosing spondylitis. central disk enhancement appears to be an infection sign. effectiveness of us-guided core needle biopsy in the diagnosis of musculoskeletal lesions i. lecumberri, j.l. del cura, o. gorriño, i. lópez, a. legorburu, d. grande; bilbao/es purpose: to evaluate effectiveness and accuracy of ultrasound (us) guided core needle biopsy in musculoskeletal lesions. material and methods: us guided core needle biopsies were performed in patients with musculoskeletal lesions imaged by us. all lesions were tumors on us exam, except two that appeared as ill-defined alterations in soft tissues. twelve were arising from or involving bone. location of the lesions was axial in and in extremities in . results of core biopsy were correlated with the final diagnosis, that was based on the exam of surgical specimen in patients who were operated on, and in clinical course in the rest. results: no complications occurred. core biopsy was considered diagnostic in cases. fifty-two lesions were finally diagnosed as malignant and were benign. in cases final diagnosis was different from that of core biopsy. two lesions with non-diagnostic result were finally diagnosed as malignant and another one as benign. five lesions, benign in core biopsy, were diagnosed as a different benign entity after surgery. three lesions, considered benign after core biopsy, were finally diagnosed as malignant. us-guided core needle biopsy achieved % sensibility, % specificity, % positive predictive value and % negative predictive value to detect malignancy in musculoskeletal lesions. in us-visible musculoskeletal lesions, us-guided core biopsy can establish a diagnosis with great accuracy and frequently can avoid other explorations. thus, it should be considered a stable technique in the management of musculoskeletal pathology. extra-abdominal desmoid tumours in the post-partum period: diagnosis and staging with ultrasonography and mri l. dogliotti , m. bazzocchi , n. gandolfo , f. pugliese , f. prefumo , g. serafini ; pietra ligure/it, genoa/it purpose: the extra-abdominal desmoid tumour (eadt) is a rare disease more common in young females. this study describes the ultrasonographic features of eadts, and compares them with mri in the diagnosis and staging of the disease. a total of lesions of the anterior abdominal wall and perineum have been assessed with ultrasonography and mri. six of them were subsequently diagnosed as eadts at histology. of these, were located in the anterior abdominal wall and one, the largest, was embedded within the perineal muscles. all patients were female and reported being pregnant in the year preceding the diagnosis. three had delivered by caesarean section. the ultrasound examination were performed with a - mhz multifrequency transducer. mri imaging employed tse-t w sequences with and without gadolinium, and tse-t w sequences with and without fat suppression. results: in the cases of eadt ultrasonography showed solid homogeneous masses, slightly hypoechogenic compared to the adjacent fibromuscular structures. in the anterior abdominal wall cases, the margins were clearly identified on a transversal plane but spiculated and irregular along the longitudinal plane in out cases. the large perineal lesion (diameter cm) appeared dyshomogenous, but margins were regular and clearly identifiable. at color doppler exami-nation, all lesions showed a rich vascularisation. mri confirmed the fibrous nature of the lesions and helped in the differential diagnosis with endometriosis. conclusion: eadts appear as solid, homogeneous, clearly delimited lesion at ultrasonography. mri is useful for excluding endometriosis in case of dyshomogeneous lesions. retrospective analysis of ultrasound guided core biopsy in the diagnosis of soft tissue masses f.j. perks, i. beggs; edinburgh/uk purpose: to assess the accuracy of ultrasound guided core biopsy of soft tissue masses. we reviewed soft tissue biopsies in consecutive patients. diagnoses were compared to the post excision diagnosis in patients. all core biopsies were performed by the same radiologist using ultrasound guidance. biopsies were performed as outpatient procedures. results: biopsies repeated. ( %) biopsies diagnostic of a wide range of benign and malignant disease. of ( %) biopsies accurately representative of post excision histology. biopsies diagnosed disease that did not warrant excision. false negative biopsies when sampling liposarcoma, lymphoma, chondrosarcoma and metastasis. overall sensitivity and specificity was . % and % respectively. there were no complications. the purpose of this exhibit is to describe the imaging features of sapho in adults. this retrospective study included patients ( women, men). all imaging procedures, radiographs, ct, mri and scintigraphy, were reviewed. background: sapho (acronym for synovitis, acne, pustulosis, hyperostosis and osteitis) is a syndrome of obscure cause, commonly considered to represent a seronegative spondylarthropathy, affecting especially young adults and children and leading to osteoarticular and skin manifestations. osteosclerosis, cortical thickening, enlargement of bone, arthritis are the predominent radiologic pattern, but periostitis and soft tissue swelling also may be seen and these findings can simulate osteomyelitis, sarcoma, lymphoma. imaging findings: of the patients, topographic localizations were the upper anterior chest wall ( %), spine ( %), periphral bones and joints ( %), pelvis ( %), mandible ( . %). the main radiologic findings correlated with hyperostosis, osteitis and synovitis. the radiologist plays a major role in the diagnosis: this exibit reviews the radiologic appearance of this syndrome and illustrates difficulties in differential diagnosis. erdheim-chester disease: detection and staging with multimodality imaging a. miquel , e. dion , c. graef , y. menu , p. grenier , j.-d. laredo ; le kremlin-bicêtre/fr, paris/fr learning objectives: to illustrate the clinical, radiological and histological spectrum of findings in erdheim-chester disease. to be able to recognise the disease in a patient with elementary symptoms. to describe the imaging modalities that are necessary for a precise staging of the disease. background: erdheim-chester disease is a multisystemic histiocytosis associated with characteristic skeletal abnormalities. bone (bone pain), skin (xanthomas), orbits (exophthalmos), pituitary gland (diabetes insipitus), retroperitoneum (hydronephrosis) and lungs may be affected by the disease. the relashionship of the disease with langerhans cell histiocytosis is still unclear. seventeen patients with erdheim-chester disease were evaluated between and , using conventional x-rays, us, ct, bone scintigraphy and mri. radiological-pathological correlations were available in cases. imaging findings: osteosclerosis of long bones in lower limbs is the most common feature ( / cases) and should be a trigger for the diagnosis. mri demonstrates replacement of the high signal of the bone marrow by a decreased signal on t and t -weighted images. atypical osseous manifestations include involvement of ribs, skull and pelvis; focal lytic lesions are seldom encountered ( / ). visceral involvement may affect pituitary gland, orbits, pericardium, lungs, kidneys and retroperitoneum. pathologic study shows accumulation of lipid-laden histiocytes and fibrosis. conclusion: bone lesions are rather specific and should suggest the diagnosis of erdheim-chester disease. detection of visceral involvement, sometimes asymptomatic, requires imaging of brain, lung and retroperitoneum. histological analysis is necessary to assess the diagnosis and differentiate the disease from other cases of histiocytosis, especially langerhans cell histiocytosis. the learning objective: we aim to highlight the manifestations of myeloma using referrals from such specialities as haematology, orthopaedics and general medicine. the classical appearances of myeloma are presented with aids to enable the radiologist confirm the diagnosis and differentiate from other pathologies of similar appearance. background: multiple myeloma is the commonest primary malignant neoplasm and therefore as pathology the radiologist should be mindful of its many appearances. imaging findings: through a pictorial review we show not only the classical appearances of myelomatosis on plain film and bone scans but we also present its' appearance on ct scan and mri. the limitations and benefits of each modality are discussed. furthermore we highlight the recent findings which show that not only is mri better at staging multiple myeloma but compared to the dated "durie and salmon" criteria it negates the need for repeat blood and urine samples. conclusion: multiple myeloma may not always present with classical appearances and therefore it is paramount that a radiologist be aware of some of the unusual forms. through an assortment of imaging modalities we present not only the characteristic appearance but also atypical varieties. vertebral development: normal and abnormal aspects-interactive a. marinescu, a. pavel, h.c. ionescu, g. iana; bucharest/ro learning objectives: the program is conceived as an interactive lesson for those who wants to easy understand the vertebral development and also the pathology of an inadequate development. it is addressed to any radiologist in training. background: the vertebral development consists in three stages: mesenchymal, cartilaginous and bony. during this development appear many centers of the future vertebrae (mesenchymal, chondrifications and ossifications centers).o:p> procedure details: using a graphic presentation (originally and schematically) of the vertebrae for any stage and a color convention (the mesenchimal stage is represented in orange, the cartilaginous stage in blue and the third -the bony stage in pink); you can follow all the steps of the vertebral development. the program is interactive, meaning that you can follow the graphic presentation of the steps of the vertebral development, but also: • if you want to know more about the elements on the graphics, you can use the left button of the mouse clicking on the red sign. • before "details" and you will get the information and explanations. then, you can come back to the slide with the marks ü "details". • you can see, for each type of pathology which can occur in different steps of vertebral development, a graphic presentation and radiography (you press the left button of the mouse on the red sign before "pathology". conclusion: all the radiology trainees in our department have been satisfied with the program, instead of reading some arid pages for this topic. nevoid background: nevoid basal cell carcinoma syndrome (nbccs) is a rare autosomal dominant inherited disorder with complete penetrance but variable expressivity. although many radiologists are familiar with typical findings (e.g. major criteria) of this disease there are also findings that are only rarely demonstrable (e.g. minor criteria). imaging findings: we present nbccs-findings of patients. they include numerous pathologies such as multiple cutaneous basal cell carcinomas, odontogenic jaw cysts, ectopic calcifications of the falx cerebri as well as palmar or plantar pits. characteristic images of the patients faces as well as bifid or fused ribs, vertebral anomalies, bridging of the sella turcica, flame shaped pseudocysts of the hands, lymphomesenteric cysts as well as increased frequency of tumors particularly medulloblastomas and ovarian fibromas that are also very important to recognise will be shown. the radiological findings are specific for this disease and may confirm the diagnosis in combination with the clinical presentation. furthermore early diagnosis as well as periodical follow-up examinations is very important since the nevi after puberty turn out to be aggressive. imaging with pathological correlation of sacral and presacral lesions s.a. o'keeffe, t. geoghegan, w.c. torreggiani; dublin/ie learning objectives: the objective of this poster presentation is to pictorially review the imaging of a series of patients with sacral and presacral lesions and to correlate these with pathology. background: presacral tumours are extremely rare accounting for less than in , hospital admissions. there are many pathological causes as this is an anatomical area of embryologic fusion. a review of reports on the radiology information system (ris) was undertaken to identify those patients who had pre- in this pictorial review, we describe the anatomical contents of the presacral space and correlate this with the possible pathological causes of a lesion in this area. in addition, the imaging and pathology of a selection of sacral and presacral tumours is presented and methods of analysis of imaging are suggested. diagnosis background: chordomas are very rare tumors, for which standard medical therapies are lacking. following detection of pdgf-r expression in tumor samples, it was decided to treat some patients with advanced chordoma with imatinib mesylate on an individual basis. these patients were treated at the istituto nazionale tumori, milano, italy, as from august . imaging findings: five pts with advanced disease ( with sacral, one with clivus chordomas) undergoing imatinib mesylate were evaluated with mri at baseline, and then every months. four pts showed hints of tumor response. these were: i) minor and slow decrease in size in the pt with the longest follow-up, following a transient initial increase in size, which was seen in all pts; ii) hypodensity of lesions on t w images and hyperintensity on t w images; iii) hypovasculature of lesions on contrast-enhanced t w images in comparison to baseline assessment. conclusion: actual responsiveness of chordomas to molecular-targeted therapy with imatinib mesylate is left to be determined. however, preliminary observations suggest that mri may be useful to assess tumor response. signs of response may be subtle, likely depending on changes in tumor tissue rather than (or before) decrease in size. groin pain in athletes: mri findings f. idoate saralegui; pamplona/es learning objectives: this exhibit reviews the rmi findings in athletes with groin pain. background: groin pain is a common complaint in athletes, particularly prevalent among soccer players. this condition is frequently multifactorial, and the differential diagnosis can cover a rather broad area of entities, mainly involving adductor muscle and tendinous group, pubic bone even inguinal hernia. between january and august , athletes with groin pain were examined at our institution. the mri findings of the possible causes of groin pain in athletes are described and illustrated. imaging findings: the examinations were performed in a tesla imaging system (magnetom impact expert, siemens) with a surface coil. t -weighted spin echo and t -weighted fat suppressed turbo spin echo images were obtained in axial plane; stir a t -weighted turbo spin echo sequences were performed in coronal plane. the pathological findings observed were: osteitis pubis, stress fracture of pubic bone, muscle tears (adductor, obturator, rectus abdominis, pectineus, iliopsoas, gracilis and rectus femoris groups), abdomino fascial abnormalities, inguinal hernia, bursitis and inflammation conditions of the spermatic cord. conclusion: mri depicts adequately the numerous pathologic entities related to groin pain in athletes. infection background: prosthetic, graft or catheter infection, irrespective of location, invariably equates with a poor outcome. modern aseptic technique, in addition to the use of antibiotic-coated prostheses, have dramatically reduced the incidence of such infection. aggressive antibiotic suppression is effective in fewer than % of cases, the remainder requiring removal of the infected prosthesis, with associated morbidity, and occasionally, mortality. clinical signs and symptoms are frequently non-specific, including lethargy, pyrexia and anorexia. it is for this reason that quite often the task of locating a causative factor relies upon the expertise of the radiologist. imaging: using a pictorial review we depict the numerous pitfalls in the radiological diagnosis of prosthetic and endovascular graft infections. in addition, a guide to radiological imaging of less frequently encountered infections, such as those of breast and penile prostheses, ventriculo-peritoneal shunts, intra-ocular lens, cardiac pacemakers and valves, will be provided. the limitations and benefits of conventional radiography, digital subtraction angiography, computed tomography, ultrasound, magnetic resonance imaging and radio-isotope scintigraphy with respect to the site of infection will be explored in turn. the radiological diagnosis of infection of synthetic materials is notoriously difficult. the use of these materials for therapeutic, cosmetic and palliative purposes is ever-increasing. it is therefore of vital importance that the radiologist be aware of the advantages and disadvantages of available imaging modalities in arriving at a confident, swift diagnosis. a new method for radio frequency of lumbar disc in treatment of chronic low back pain n. azulay , m. forgerit , a. moumouh , e. alava , p. vandermarq , j.-p. tasu ; poitiers/fr, niort/fr, barcelona/es purpose: treatment of chronic low back pains remains a challenge for physicians. recently, radio-frequency heating of inter vertebral disc has been proposed, as an alternative to surgery. we propose here a new method of discal radiofrequency in which heated water is used to spread the thermal energy. materials and methods: seventeen patients were included prospectively according to the criteria used in previous intra discal radiofrequency studies. the catheter is placed in the nucleus and heated water is used as thermal conductor to burn the annulus nociceptors. intra discal pressure and impedance measurements were used to control the energy delivered during the procedure. clinical results were evaluated by the oswestry score. the treatment was scored as a success with at least a % pain reduction on global perceived effect on oswestry score. the global oswestry before treatment was . points and points b d e f a g after corresponding to a . % improvement. on the patients, were successfully treated ( %). we have shown that injection of heated water is sufficient to spread thermal energy to the anular nociceptor. this method is technically more simple than previous techniques using anular needle, avoiding navigation of the catheter through the anulus. ultra low dose ct in navigated closed reduction and percutaneous screw fixation of pelvic ring fractures h.g. staedele, r. huegli, s. meckel, a.l. jacob, h. roser, p. messmer, j. roth; basle/ch purpose: to reduce radiation doses in image-guided pelvic closed reduction and percutaneous fixation crpf without compromising guidance quality and procedural outcome. prospective study including patients with pelvic ring fractures treated with ct-guided crpf. patients received screws which were navigated over guide pins. imaging modality used was a single slice scanner (general electric, usa). tube current was reduced by % with every step starting with routinely used scan parameters for diagnostic and interventional procedures ( ma). if the image quality was good the next control scan was performed with % reduction of ma again. results: patients with fractures underwent crpf. were males, females, with a mean age of years (range . - . ). dose reduction was possible in all patients in to steps ending up in ma (n = ), ma (n = ) and ma (n = ) respectively. ma is the lowest current provided by the ct. number of misplaced screws was . number of additional correction of guide pin was . number of fallbacks to the next dose level was . conclusion: by lowering scan doses no relevant morphological information needed for safe guidance of instruments and implants was lost. scan protocols can be changed to lower ma values without compromising the safety of crpf. immediate procedural outcome was not affected. withdrawn by authors pelvic painful syndrome in athletes: diagnostic imaging evaluation l. zugaro, f. iannessi, a. catalucci, a. barile, c. masciocchi; l'aquila/it purpose: to describe the imaging features of the anterior and posterior painful syndrome in athletes. materials and methods: athletes ( males, female) suffering from pelvic pain (groin pain = and hamstring syndrome = ) underwent mri after plain film and ultrasound preliminary evaluation. in patients ct evaluation was also performed. mr examination was performed using a . t unit (ge medical system). proton density, t weighted and t weighted sequences on axial and coronal planes were employed. in one case mri was also obtained after surgical treatment. in all cases mri demonstrated pathological findings: anterior painful syndromes were related to osteitis pubis, bursitis, inguinal hernia, post-traumatic muscular lesions and stress fractures. hamstring syndrome was related to insertional pathology of hamstring tendons, inflammation disease of regional bursae and fibrotic changes of fatty tissue surrounding the ischial tuberosity. conclusion: anterior and posterior pubic painful syndromes are painful conditions often affecting athletes. mri provides a correct evaluation of these syndromes enabling an adequate therapy. the value of qualitative and semiquantitative ultrasonographic findings in the differential diagnosis of superficial lymph node enlargements o.o. okten , n. tuncbilek , h.m. karakas ; edirne/tr, malatya/tr purpose: the value of the gray-scale (gsusg), color doppler (dusg), and power doppler (pdusg) ultrasonography in the differential diagnosis of superficial lymph node enlargements were evaluated. the study group was consisted of males and females ( - yrs old, mean age . yrs) in whom physical examination had revealed superficial lymph node enlargement(s). all patients were investigated with power doppler usg (sonoline elegra advanced, siemens, germany) using . mhz linear transducer. longitudinal/transverse dimensions (l/t), edge characteristics and hilar echogenecity of lymph nodes were evaluated with gsusg. pulsatility (pi) and resistivity (ri) indexes were calculated using cdusg. vascular pattern described as hilar or non-hilar were determined by pdusg. above parameters were analyzed to determine their differential diagnostic values using anova based discriminant analysis. results: when clinical data and histopathological findings were combined, subjects were diagnosed as lymphoma, and cases were classified as lymphadenitis. multiple comparison analysis of qualitative gsusg and pdusg features consisting of edge characteristics, hilar echogenecity and vascular pattern was able to differentiate lypmhomatous lymph nodes and lymphadenitis with % accuracy (p < . ). in contrary, semiquantitative features (ri, pi and l/t) were not able to classify lymph nodes ( % accuracy, p < . ). conclusion: semiquantitative rdus parameters are not valuable in the differential diagnosis of superficial lymph node pathologies. qualitative usg and pdusg features, on the other hand, may be used as an alternative work-up to cytological studies in patients in whom diagnostic surgical procedures cannot be performed. modern imaging in langerhans cell histiocytosis a. geoffray, k. oudjhane, l. lau, s. weitzmann; toronto, on/ca purpose: to review imaging features of pediatric langerhans cell histiocytosis (lch) with particular attention to cross-sectional imaging and aggressive cases. we reviewed retrospectively the imaging database of all the cases treated in our institution for lch during a years period (january to december ). there were patients, boys, girls, with an age range of weeks to years. results: patients had no imaging anomalies and were excluded. among the with anomalies, had a solitary bone lesion (skull , orbit , spine , iliac , long bones ), had multiple bone lesions associated with other organ involvement in (lung , liver and spleen , thymus ), patients had no bone lesions ( cervical adenopathy, abdominal involvement, abdominal and lung, pituitary gland, middle ears), patient had lch associated with neuroblastoma, and presented with lymphadenopathy, mediastinal and sacrococcygeal masses. imaging modalities were conventional radiography in %, nuclear scans %, ct %, mr %, us %. conclusion: lch in children is characterized by a wide spectrum of organ involvement. cross sectional imaging is useful in assessing the diagnosis and managing the most aggressive cases. can calcaneal spur help in the evaluation of a painful heel? n. sabir, s. demirlenk, b. yagci, n. karabulut; denizli/tr purpose: to determine the value of presence of calcaneal spur in evaluating painful heels. materials and methods: heels of female and male patients with a mean age of . ± . years and mean body mass index (bmi) of . ± . complaining of bilateral (n = ) and unilateral (n = ) heel pain were studied. lateral radiographs of the symptomatic and asymptomatic heels were obtained to assess presence of calcaneal spur. mr imaging of the symptomatic heels was performed using . t superconductive system. the images were taken and reviewed for presence of plantar fasciitis (pf). heel pad thickness was also measured. results: calcaneal spur was seen in of ( %) symptomatic and in of ( . %) of asymptomatic heels. mr has revealed pf in of ( . %) painful heels and calcaneal spur was present in of the cases with pf. calcaneal spur was significantly correlated with weight (p < . ), bmi (p < . ), age (p = . ), heel pain duration (p = . ) and pf (p < . ). no correlation was seen between calcaneal spur and both height and heel fat pad. conclusion: although calcaneal spur is not the cause of pain, it may provide an objective assessment of the changes due to chronic repeatitive trauma to plantar fascia secondary to obesity or age. on the other hand foot radiography should be part of the initial diagnostic methods as it helps in excluding different causes of heel pain. purpose: the positive areas in scintigraphy of sentinel lymph nodes are sometimes difficult to be anatomically identified. we have examined the usefulness of bone scintigraphy performed together with lymphoscintigraphy for identification of the anatomical positions of the positive area in lymphoscintigraphy. materials and methods: sixteen patients, cases of malignant melanoma and cases of squamous cell carcinoma were examined by lymphoscintigraphy. nine patients, cases of malignant melanoma and cases of squamous cell carcinoma, were examined by bone scintigraphy together with lymphoscintigraphy. the patients were divided into three groups. in the first group, lymphoscintigraphy was performed alone. in the second group, the patients were first injected intravenously with tc- mhmdp or tc- mmdp and then lymphoscintigraphy was performed after intradermal administration of tc- m sulphur colloid. three hours after injection of tc- mhmdp or tc- mmdp, bone scintigraphy were taken. in the third group, lymphoscintigraphy was performed minutes after bone scintigraphy. result: anatomical identification of the positive areas in lymphoscintigraphy was difficult when used alone, whereas their identification became easier when used together with bone scintigraphy. when bone scintigraphy was performed after lymphoscintigraphy, evaluation of lesions in the bones near the original focus was sometimes not easy. however, evaluation of lymphoscintigraphy was not affected by prior performance of bone scintigraphy. conclusion: identification of the anatomical positions of the positive areas in lymphoscintigraphy was easier when used together with bone scintigraphy than in lymphoscintigraphy alone. in cases of the joint use, lymphoscintigraphy should be operated after bone scintigraphy. ultrasonographic detection of iatrogenic foreign bodies in an in vitro study i.k. rozylo-kalinowska, l. barczewski, e. szczepanik, r. lenard; lublin/pl purpose: sometimes foreign bodies being parts of surgical appliances remain in soft tissues as a result of iatrogenic faults. their presence is dectected clinically or radiologically, when the objects are radiopaque. nevertheless, radiolucent or faintly radiopaque objects often are a diagnostic challenge as their presence is difficult to diagnose on the basis of conventional radiograms. the purpose of the study was application of ultrasonography for detection of radiolucent and faintly radiopaque foreign bodies in an in vitro model. examples of radiolucent and poorly radiopaque foreign bodies such as plastic fragments of syringes, intravenous catheter, different cotton and gauze swabs, were embedded in containers filled with gelatine, which served as a model of soft tissues as well as played the role of a stand-off pad. the ultrasound scanning was performed by means of atl hdi machine with a high-frequency broadband linear transducer. results. there was determined the possibility of detection of various types of foreign bodies. there were described ultrasonographic image patterns of different objects. image findings: all types of used materials were well visible and hyperechoic in ultrasound image. plastic objects caused appearance of reverberation artifacts. the swabs produced characteristic patterns of internal structure. variations in ultrasonographic image patterns of foreign bodies studied in vitro allowed differentiation of various types of the examined materials. conclusion: ultrasonography may become a useful, widely available and costeffective tool in detection and localisation of foreign bodies of iatrogenic origin that otherwise remain undetected. spectrum of mr findings of herniation pit of the femoral neck m. ferrer, r. dominguez, m. romera, e. castella, x. merino, l. casas; barcelona/es background and purpose: herniation pit of the femoral neck has been considered a normal variant. this lesion is located in the superior aspect of the femoral neck, below the anterior cortex of the femur. the aim of this study was to describe the spectrum of magnetic resonance imaging (mri) findings associated with herniation pit of the femoral neck. material and methods: we retrospectively reviewed the mr studies in patients with hip pain performed in our hospital over the last years. a total of patients presented herniation pit. the features analyzed included: type of herniation pit (unilateral/bilateral), iliofemoral ligament thickening and asymmetry, iliopsoas muscle/femoral head relationship, acetabular coverage angle, cervicodiaphisary angle, femoral anteversion angle, and associated imaging findings (synovitis). results: in patients with hip pain and herniation pit, the following data were found: no other apparent cause of the pain ( %), iliofemoral ligament thickening ( %), decreased acetabular coverage angle ( %), altered cervicodiaphisary angle ( %). the elevated incidence of alterations in acetabular coverage and cervicodiaphisary angle in herniation pit, together with other alterations in stabilizing elements, lead us to suggest that, rather than a separate entity, herniation pit can be considered a component of the spectrum of adult hip dysplasia. b d e f a g high resolution ultrasound of the peripheral nerves of the lower limb: technique of examination and normal us appearance x. montet , s. bianchi , c. martinoli , j. fasel ; geneva/ch, genoa/it objective: to present the basic normal us appearance of peripheral nerves evaluated in vitro and in vivo. to describe the examination technique, the normal us findings and the main anatomic variations of the nerves of the lower limb. materials and methods: us images were obtained with commercially available equipment utilizing two linear probes ( - , - mhz). the normal us appearance of a peripheral nerve was evaluated by in vitro examination a human sciatic nerve. sonograms were then obtained in normal volunteers and correlated with mri and images from anatomic textbook. results: the in vitro us appearance of the a normal peripheral nerve correlates well with the internal nerve structure. fascicles appear as hypoechoic bundles embedded within the hyperechoic connective tissue. the normal anatomy of the sciatic, peroneal, tibial nerve is first briefly described. then for each nerve the scanning technique is presented with emphasis on patient position, probe orientation and dynamic maneuvers. to improve the understanding of us images, correlative drawings showing the adjacent anatomic structures and diagrams showing the position of the probe are presented. high frequency transducers allow a detailed analysis of even smaller peripheral nerves. we present a practical manner to realize a us examination of the nerves of the upper extremity as well as us images of normal nerves and of their main anatomic variants. withdrawn by authors radiologic and sonographic examinations in patients with heterotopic ossifications j. nagy; budapest/hu purpose: to evaluate the diagnostic capability of radiologic and ultrasonographic examinations in patients (pts) with heterotopic ossifications (ho). during the last four years we have examined pts ( women, men; age: / - / years) in case of the clinical suspicion of ho. dynamic conventional radiologic and ultrasonographic ( . mhz linear transducer) examinations were performed. results: in the background of ho, spinal cord injury ( pts, %), brain injury ( pts, %), limb damage ( pts, %), orthopedic surgery ( pts, %) or limb amputation ( pts, %) were found. the most frequent localizations of ho were as follows: one side ( pts) or on either of the sides of the hip ( pts), elbow ( pts), knee ( pts), hip + knee ( pts) or rare localizations (around the femoral stump) ( pts). more than one localization was found in half of the pts. when the clinical suspicion of ho was not proved by radiology, dynamic ultrasonographic examinations were positive in each cases ( pts, %). conclusions: ( ) ho can occur in more than one localization after cns or limb injuries, ( ) the most frequent localization was around one or both hip joints, ( ) sonographic examinations showed earlier positivity compared to conventional radiology, ( ) rare localizations of the ho can induce differential diagnostic problems in clinical practice. ultrasonographic experiences of spine-related lesions in patients with back pain: correlation with mri w. jin; incheon/kr purpose: multiple paraspinal lesions or spine-related lesions have been overlooked on transabdominal ultrasonograhic examination. therefore, we correlated transabdominal ultrasonographic findings with mri findings in spine-related lesions. materials and methods: patients were underwent transabdominal ultrasonographic examinations for routine check prior to surgery. they were men and women aged to (average, ) years. all patients also were checked with mri. in transabdominal ultrasonographic examination, compression of abdominal wall and inner contents were done. after this, we observed closely the contour of body, the height of body, disc height, and paraspinal lesion. results: in patients, there were ) compression fractures or burst fractures; patients, ) spondylitis; patients, ) bone tumorous condition; patients, ) abscess formations in paraspinal muscle; patients, ) ankylosing spondylitis; patient, and ) disc herniation; patients. in these patients, transabdominal ultrasonographic findings were relatively correlated with mr findings, but lesions in posterior and middle elements of spine could not be evaluated with transabdominal ultrasonographic approach. conclusion: despite of many limitations of transabdominal ultrasonographic examination for detection of spine and paraspinal lesion, when spine and paraspinal structures or lesion are visible on transabdominal ultrasonography, ultrasonography suggested to be a screening method in patients with back pain. in particular, ultrasonography was superior to mri in detecting the minimal change of anterior disc, prediscal space, or anterior longitudinal ligament of spine. creation of a radiological musculoskeletal database (db) for statistic referrement, differential-diagnosis and didactic use (stuart project) s. trupiani, e. baldan, r. stramare, c. saccavini, l. bacarini, g. feltrin; padua/it purpose: to create a radiological db, with normal and pathologic cases, to be used for statistic endings, differential-diagnosis and for consultation. the db is available to everyone who as investigation or consultation needs or else for cases submission; for this reason is necessary to follow some fixed criteria unless the case is not accepted. the submission modalities have a starting schedule that contains: patient's anagraphic data, documentation source, sequence of technical-methodological procedures used, a description of every semeiological aspect contained in every single image, gamuts criteria-based diagnosis and final diagnosis. the cases are resumed by acr code. the definitive diagnosis, that contain a synthesis of anatomical-clinical and diagnostic aspects about the pathology observed, is open to critical observations by everyone that is consulting the db. every db consulter is able to introduce his opinion in a special window. the case report ends with a bibliographic correlation that contains the latest literature articles about the argument. the db contents can be utilized as a cases archive for clinical research (for this aspect, very important is the acr code), or as a support in the diagnostic report making, or as a didactical archive. moreover the db users can exchange their opinions, modify or add informations for every case by the web. ment was present in cases ( . %) and discal involvement was present in case ( . %). patients had soft tissue involvement and abscess was present in patients. all cases showed vertebral and discal enhancement. patients showed diffuse involvement of vertebral corpus and patients showed endplate involvement. cases showed both vertebral corpus and endplate involvement. intervertebral space narrowing was present in cases, height loss in vertebral body was observed in cases and cases showed end plate deformities. we detected epidural extension in cases, posterior longitudinal ligament (pll) elevation in , spinal cord compression in and root compression in cases. conclusion: spondylodiscitis is a rare complication of brucellosis and it can cause permanent neurologic deficits and spinal deformities. mri is a very sensitive and non invasive imaging technique which must be firstly preferred for the early diagnosis of spondylodiscitis. imaging findings: a total of aneurysms in patients were evaluated. all were proved at surgery. five small aneurysms were not depicted at d-cta-ssd (three were less than mm, two were less than mm). but only three small aneurysms were missed in d-cta-vrt (two were less than mm, one was less than mm). in ( %) of the patients, d-cta-vrt gave additional informations: orientation (n = ), defining exact neck anatomy (n = ), incorporation of vessels into aneurysm (n = ), lobulation (n = ), and blebs of aneurysms (n = ). conclusion: d-cta-vrt is superior to d-cta-ssd in the detection of very small aneurysms and evaluation of complex anatomy of intracranial aneurysms. gadolinium enhanced ultra-fast mra: a primary diagnostic tool in the recognition and follow-up of dural sinus thrombosis s. gaudino, t. tartaglione, f. molinari, v. valentini, c. colosimo; rome/it dural sinus thromboses (dst) are not extremely rare, they still account for % of ictus in young adults. they need an accurate diagnosis at the onset of symptoms, and during the follow-up, to reduce the risks related to prolonged thrombolytic therapy. digital subtraction angiography (dsa) and, in the last years, phase contrast mr angiography (pc mra) with conventional mr sequences have become the methods of choice for dst diagnosis. our purpose was to evaluate the usefulness of gadolinium-enhanced ultrafast mra (ge-uf mra), routinely employed in supra-aortic vessel evaluation, in the diagnosis and follow-up of dst. materials and methods: we retrospectively evaluated patients with diagnosis of dst, studied using . t magnet, conventional se, tse and ge-uf mra sequences. we could compare ge-uf mra images with dsa in patients and pc mra in patients. results: in all dsa controlled patients, we found a complete agreement between ge-uf mra and dsa concerning location and extent of dst. in all patients with both ge-uf and pc mra, the overall dst diagnosis was identical, even if the diagnosis of minimal dural sinus involvement was easily and definitively assessed with uf-ge mra, which allows a superior diagnosis confidence. conclusions: our preliminary experience suggests that ge-uf mra is a very fast modality, is easily obtained and analyzed and has a superior diagnostic confidence compared to pc mra. in our opinion ge-uf mra should be proposed as the primary diagnostic tool in the recognition and follow-up of dst. imaging imaging appearances: the images were obtained using unenhanced ct and routine mri sequences for brain imaging (ie sagittal t , axial t and coronal proton density weighted sequences). fat suppression sequences were sometimes used for further clarification. the characteristic ct and mri appearances of intracranial fat and the anatomical location of the lesion allow the provision of a narrow differential diagnosis using routine imaging protocols. this facilitates decisions on the need for further imaging modalities. background: vascular dementia represents a very heterogeneous diagnostic category. utilizing the ninds-airen criteria and a centralized imaging rater to determine eligibility for enrollment in recent large-scale clinical trials testing new treatments for vascular dementia has provided increased consistency in the diagnosis of vad. the diagnostic criteria for probable vascular dementia include the development of dementia temporally related to stroke with neuroimaging confirmation. the criteria include a table listing brain imaging lesions associated with vascular dementia. in this study, patients have been screened for the presence of cerebro-vascular disease using brain ct or mri. imaging findings: the wide spectrum of neuroradiological features that are associated with vad may result from cerebral small-vessel disease with extensive leukoencephalopathy or lacunes (basal ganglia or frontal white matter), or may be the consequence of single strategically located infarcts or multiple infarcts in large-vessel territories. it may also be the consequence of global cerebral hypoperfusion, intracerebral hemorrhage or other mechanisms such as genetically determined arteriopathies. conclusion: neuroimaging is required for confirmation of cerebrovascular disease in vad and provides information about the topography and severity of vascular lesions. neuroimaging may also assist with the differential diagnosis of dementia associated with normal pressure hydrocephalus, chronic subdural hematoma, arteriovenous malformation or tumoral diseases. imaging spectrum of cerebral complications after cardiac surgery e. testempassi, g. katsou, v. vantali, m. kolios, d. exarchos, d. chondros; athens/gr learning objectives: to present a systematic approach to imaging findings of cerebral complications caused by cardiac surgery. to review and understand the mechanisms and pathophysiology of cerebral involvement after cardiac surgery. background: a wide spectrum of neurologic complications may occur after cardiac surgery, such as motor, sensory or visual disturbances, stupor or depression of consciousness. the role of imaging in differentiating a variety of conditions that may cause the above neurologic symptoms is very important. morphology and patterns of cerebral lesions also enables an assessment of the pathophysiology and hemodynamics of brain complications after cardiac surgery. we retrospectively reviewed the brain ct images of symptomatic patients, who previously had cardiac surgery performed. imaging findings: a variety of common and uncommon findings on ct and mr images of neurologic complications after cardiac surgery are illustrated. acute infarctions involving a vascular territory, hemorrhagic infarctions, multiple infarctions, watershed infarctions, bilateral infarctions, braistem and cerebellar infarctions, uncal herniation, brain edema and inflammatory processes are depicted. preoperative cerebral ct or mr images are helpful in diagnosing new lesions in patients with old infarctions, lacuna or ischemic leucoencephalopathy. the radiologist must be familiar with the imaging appearance of complications that affect the brain after cardiac surgery. imaging findings of an ethmoidal dural arteriovenous fistula with venous drainage in the vein of galen v.t. skiadas, a. prasouli, p. mitseas, g. kottas, s. laxanis; athens/gr learning objectives: to illustrate the mri, mra and angiographic findings of an ethmoidal dural arteriovenous fistula (avf) in the anterior cranial fossa. background: ethmoidal avfs are rare vascular anomalies. only cases have been presented since , when they were first described by lepoire. they can be either congenital or acquired lesions. ethmoidal arteries are the main feeding vessels. the vast majority drain to the superior sagittal sinus and to the cavernous sinus. the presenting symptom is intracranial hemorrhage, mostly subarachnoid or intracerebral. their propensity for hemorrhage justifies immediate surgical removal of the lesion. imaging findings: an mri scan was performed demonstrating an abnormal region of signal void, suggesting a vascular anomaly involving the base of the left frontal lobe. for further evaluation, the patient underwent an mra examination. a vascular linear lesion, probably a vein, at the base of the left frontal lobe and enlargement mainly of the left, as well as of the right ocular artery was discovered. a digital subtraction angiography (dsa) was performed and an edaf on the left ciribriform plate supplied by bilateral ethmoidal arteries was revealed. it drained via a dilated abnormal vein to the left vein of galen. so far, only one other case has ever been reported with venous drainage to the vein of galen. conclusion: specific mri and mra imaging findings should arouse the suspicion of an edaf and dsa can effectively demonstrate the nidus of the lesion, as well as the feeding and draining vessels and confirm the diagnosis. functional based on the hypothesis of focal cortical inhibition, low-frequency transcranial magnetic stimulation (tms) was used, resulting in a slight reduction of hallucinations in some patients. in this case study, functional magnetic resonance imaging was employed to image blood oxygenation level dependent (bold) effect changes in the temporal lobes under tms-therapy. methods: in a curative attempt, a -year old schizophrenic patient (dsm-iv) with medication resistent acoustic hallucinations was treated with low-frequency tms (fstim = hz) over a four week period. fmri was performed in a . t clinical scanner using the standard head coil and a ge-epi sequence. the tmseffects were detected based on the event related bold-fmri of auditory hallucination and auditory hallucinations rating scale. fmri was performed prior to and after the tms series to visualize possible cortical activation changes in the stimulated area. data analyses were performed with spm (http:// fil.ion.ucl.ac.uk.spm). results: after the third week, the patient presented a reduced frequency of acoustic hallucinations of approx. %, while the loudness of the hallucinations remained unchanged over the weeks of stimulation. fmri demonstrated a bold effect activation reduction after tms in speech related areas, which exceeded the local stimulation area. discussion: fmri data revealed an activation reduction in temporal and temporo-parietal areas after tms corresponding to the clinical recovery. the combination of tms and functional imaging is promising, allowing an insight into neuro-biological mechanisms during tms intervention, which may help to improve treatment success. went immediate hyperbaric oxygen (hbo) therapy and the initial result was excellent. he was discharged with subtle weakness in the right lower extremity. the second woman didn't undergo hbo therapy and was discharged with left hemiparesis. imaging findings: mr imaging with diffusion-weighted images (dwis) and adc maps were performed at admission. the first man showed high signal intensities bilaterally of the perirolandic cortex and occipital cortex on t -weighted images (t wis) and dwis. these areas showed dark signal intensities on adc maps, suggesting cytotoxic edema. the second woman showed high signal intensities bilaterally of the perirolandic cortex, bilateral globus pallidus and left occipital periventricular white matter on t wis and dwis. these areas also showed dark signal intensities on adc maps. conclusion: mr imaging findings are similar to co poisoning in that cytotoxic edema involves the globus pallidus bilaterally. but we experienced some different mr imaging finding to co poisoning in that cytotoxic edema involves the perirolandic area bilaterally, which can be seen in profound perinatal asphyxia in term infants. computed tomography angiography ( volumic acquisition of the brain with multislice detector ct at arterial phase is analyzed systematically with d maximum intensity projection (mip). in most cases, volume rendering reconstructions are also performed. all patients with negative cta have a conventional cerebral angiography to visualize entire intracranial circulation. cta and conventional cerebral angiography are performed by senior neuroradiologists. imaging findings: multislice detector cta explores all the locations of intracranial aneurysms. cta is a quick and reliable technique for the detection and therapy planning of intracranial aneurysms. rarely, cta depicts arteriovenous malformations. in some cases, hemorrhage suggests cerebral venous thrombosis that is also diagnosed on cta. we present selected cases emphasizing the utility and the limits of the technique. conclusion: cta is a non invasive, reliable tool to explore patients with non traumatic intracranial hemorrhage. the use of cerebral cta by expert neuroradiologists allows a significant reduction of the number of emergency diagnostic angiography required. brain activations during bi-script reading in chinese: a direct comparison of alphabetic and non-alphabetic reading using functional magnetic resonance imaging (fmri) y. li , x. feng , y. chen , w. tang ; shanghai/cn, oxford/uk learning objectives: a direct comparison between reading alphabetic and nonalphabetic scripts was done using fmri during bi-scripts reading of chinese characters and its alphabetic sound symbols known as pin yin. background: six mandarin speakers and skilled readers of chinese participated in this study. all the subjects were right handed. experiments were performed on ge mri scanner at the huashang hospital. sixteen slices covering the whole brain were collected with blood oxygenation level dependent (bold) sequence. the anatomical images were obtained in the transverse plane during the same scan session with d fspgr sequence after the functional measurements. the four types of stimuli were distributed equally in two main conditions i.e. chinese and pinyin. in each condition, half stimuli were words and half were non-words. data analysis was carried out using feat, the fmrib easy analysis tool, an extension of medx. imaging findings: the activations in the right inferior frontal gyrus, the left fusiform gyrus, the bilateral cuneus and the bilateral superior frontal gyrus were stronger for chinese character reading than for pinyin reading. the activations in the bilateral supramarginal gyus, the bilateral superior parietal gyrus and the bilateral middle frontal gyrus, however, were stronger for pinyin reading than for chinese character reading. different parts of the middle temporal gyrus were also activated for reading these two scripts. the low hemispheric lateralisation for both chinese character and pinyin reading indicate an important role for both hemispheres in reading. conclusion: fmri is one of the best methods of illustrating difference of brain activations during alphabetic and non-alphabetic reading. mri of the brain in inherited neurometabolic disorders: a pictorial review s.p. prabhu, s.a. barnard, n. stoodley, s.a. renowden; bristol/uk learning objectives: to illustrate typical mri appearances of the brain of patients with inborn errors of metabolism and toxic inherited white matter disease. a simple algorithm to unravel the possible cause of imaging findings is incorporated as part of this presentation. background: the diagnosis of a neurometabolic disease is usually suggested by clinical history and physical findings and is confirmed by appropriate special studies, which may include neuroradiological investigations. when the patient is referred with a nonspecific diagnosis, such as delayed development, the aim is to suggest the possibility of a neurometabolic disorder and initiate further evaluation including possible therapy and genetic counselling. on occasion, routine neuroradiological studies may incidentally produce results suggestive of a neurometabolic disorder. imaging findings: disorders including glutaric aciduria, pyruvate dehydrogenase deficiency, leigh's encephalopathy, maple syrup urine disease, methylmalonic aciduria, adrenoleukodystrophy, metachromatic leukodystrophy, krabbe's leukodystrophy and pelizaeus-merzbacher disease are illustrated. a pattern approach based on which part of the brain is affected is used to provide an aid to the diagnosis. conclusion: magnetic resonance imaging plays an important role in the identification, localisation and characterisation of underlying white matter abnormalities in affected patients and is also extensively used to monitor the natural progression of such disorders and the response to therapy. background: to compare d-dsa with d-dsa and d multi-detector ct angiography (cta) with vrt in the detection and evaluation of intracranial aneurysms. imaging findings: a total of aneurysms were evaluated. seven very small aneurysms (diameter less than mm) and two small aneurysms (less than mm) that were not depicted at conventional d-dsa and d-cta-vrt were depicted at d-dsa and all were proved at surgery. in ( %) of the patients, d-dsa gave additional informations: orientation (n = ), defining exact neck anatomy (n = ), incorporation of vessels into aneurysm (n = ), lobulation (n = ) and blebs of aneurysms (n = ). conclusion: d-dsa is superior to d-dsa and d cta-vrt in the detection of very small aneurysms and evaluation of complex anatomy of intracranial aneurysms. low background: dwi is a useful diagnostic tool that improves our background understanding to pathology. the b-value choice has a significant repercussion on signal-to-noise ratio, dwi contrast and t shine-through phenomenon. we have created a straightforward animated microsoft power point presentation showing diffusion physical principles and its imaging consequences. the dwi studies from normal subjects and patients (including brain tumours, encephalitis, demyelinating and cerebrovascular diseases) were further analyzed. all studies were b d e f a g carried out with three different b-value ( , , s/m ). exponential images and apparent diffusion coefficient (adc) maps were subsequently computed. in normal individuals, the signal and adc in the hemispheric white matter, basal ganglia and brain cortex were measured. lesions' signal with different bvalues, and their contrast rate on computed maps, were subjectively compared. imaging findings: when we visualised some acute lesions, higher b-values improved contrast compared with lower b-values. other lesions, like tumours, showed mixed dwi intensities and their contrast also improved with high b-value images. exponential and adc maps were useful for discarding t shine-through effects, moreover with low b-values, but contrast rate was significantly dismissed. conclusion: high b-value dwi provides a better contrast and a reduced t shinethrough phenomenon but it does not display more lesions than low b-value dwi. as computed maps add useful information to that provided by dwi, it seems that the b-value choice depends on each group preferences. utility of mr spectroscopy, diffusion and perfusion mr imaging in the evaluation of brain lesions in aids patients l. . diffusion weighted imaging were performed using a diffusion gradient of sec/mm applied in three axis planes. mr perfusion was performed using tr ms and te ms, following bolus infusion of intravenous contrast material, obtaining relative cerebral blood volume maps (rcbv). to determine metabolites a mr spectroscopy (mrs) was performed with single-voxel press- sequence. we classified the brain lesions in two majors groups: one with brain lesions that cause mass effect (n = ) and the other with infiltrating white matter lesions (n = ). three patients did not have any lesion. mass effect lesions could be inflammatory/infectious diseases: toxoplasmosis (n = ), tuberculosis (n = ), cryptococcosis (n = ) and neoplasic lesions: lymphoma (n = ). as infiltrating white matter lesions, we included progressive multifocal leukoencephalopathy (n = ), hiv encephalopathy (n = ), cmv infection (n = ) and hsv infection (n = ). brain infarct (n = ) is an important condition in aids patients and is more prevalent than in the general population. the mrs, diffusion and perfusion mri have been shown to be extremely useful in characterizing and making the correct diagnosis of brain lesions in aids patients. "string-knot shape" enhancement: a presenting feature of cerebral sparganosis in mri c. shuguang; shanghai/cn learning objectives: to describe the mri character of cerebral sparganosis. background: the cases of cerebral sparganosis have shown a trend of increased frequency with the change of eating habits, especially eating under-cooked products of snake or frog. there is sparse literature available which illustrates nonspecific findings of cerebral sparganosis in mri including nodular lesions, white matter degenerations or haemorrhages. mr imaging of six patients with cerebral sparganosis were reviewed retrospectively in our exhibit. imaging findings: all six cases of cerebral sparganosis showed a single lesion. the location of lesions were superficial with three in the parietal lobe, one in the frontal lobe, one in the temporal lobe and one in the posterior part of brain stem. five lesions were irregular in shape and about cm diameter with moderate hypointensity on t wi and hyperintensity on t wi. a presenting feature of the parasitic granuloma was a "string-knot shape" enhancement, which was demonstrated in cases. one case showed a cystic lesion with peripheral enhancement. there was slight hyperintensity surrounding the nodular lesions which corresponded to edema. no white matter degenerations or hemorrhages were revealed which had been described by previous studies. diagnosis of sparganosis was made on the basis of characteristic enhancement in mri for cases and one was diagnosed as parasitic infectious disease. during surgery, live larvas of sparganum were successfully removed in five cases with "string-knot shape" enhancement lesions. in the case with a cystic lesion, only fragments of worm body were obtained. conclusion: "string-knot shape" enhancement is a characteristic mri finding which has a high value in diagnosis of cerebral sparganosis. cystic lesions of the brain: a pictorial essay with mr imaging s. cakirer, m. basak, e. serin, i. ince, m. birinci; istanbul/tr learning objectives: to illustrate the spectrum of intracranial cystic lesions. to clarify the underlying reasons for specific mr appearances. to define the role of additional mr sequences for differential diagnosis of cystic lesions with similar appearances. background: intracranial cystic lesions are common in radiological practice. the differential diagnosis of intracranial cystic lesions includes a wide spectrum of diseases and is much easier with the use of standard and advanced mr imaging techniques including diffusion and perfusion-weighted studies and mr spectroscopy. between january and august , patients with intracranial cystic lesions were studied in our institute. mr imaging characteristics of the lesions, the underlying reasons for the specific mr appearances and the role of additional mr sequences for the differential diagnosis were defined following the categorization of the lesions. imaging findings: intracranial cystic lesions can be categorized into five groups. they include cystic components of primary and secondary neoplasms (glioblastoma multiforme, pilocytic astrocytoma, pleomorphic xanthroastrocytoma, ependymoma, ganglioglioma, central neurocytoma, hemangioblastoma, macroadenoma, craniopharyngioma, metastases), benign developmental cysts (arachnoid cyst, colloid cyst, epidermoid cyst, dermoid cyst, pineal cyst, rathke cleft cyst, choroid plexus cyst), post-infectious and inflammatory cysts (abscess, parasitic cysts including cystisercosis and hydatid cyst, multiple sclerosis, adem), post-traumatic or post-infarct cysts (porencephalic cyst, encephalomalacic cyst, leptomeningeal cyst) and normal variant cysts (enlarged tumefactive perivascular spaces, cavum septum pellucidum, cavum vergae). the underlying reasons for mr appearances of cystic lesions, role of additional sequences for their diagnosis and differential diagnoses between lesions with similar appearances have been evaluated. cta was performed on a single-slice spiral ct scanner (hispeed lxi, ge) with slice thickness mm, reconstruction interval . mm and pitch . an area from base of the skull to above the clinoid processes was covered. a non-ionic contrast medium was injected in cubital vein ( mgi/ml, ml/ s, ml) and scanning started after delay of - s. corresponding d image reconstructions (maximum intensity projection (mip) and volume rendering (vr)) were produced at accompanying workstation (aw . , ge). conclusion: intracranial aneurysms were visualized in of patients, but only were further evaluated. although intra-arterial dsa is still regarded as "gold standard" in diagnostic evaluation of the sah, cta can serve as guidance for intervention. proton mr spectroscopy of the brain: patterns and pitfalls in diagnosis of brain tumors c.c.t. lim, h. parmar, h. yin, v.g.e. chua; singapore/sg learning objectives: to review typical spectral patterns of brain tumours and non-neoplastic lesions and to understand the potential mimics and pitfalls in using proton magnetic resonance spectroscopy (mrs). background: mrs provides metabolic information that is independent of conventional mr imaging. mrs has been applied to distinguish brain tumors from other diseases and is available on many clinical scanners. post-processing of single-voxel and multi-voxel studies may be presented either as metabolic spectra or parametric maps in the latter. imaging findings: in brain tumours, there is elevation of choline (a marker of cell membrane turnover) and decreased n-acetyl aspartate (a neuronal marker) and creatine (an energy substance). lactate and mobile lipids are sometimes present. conversely, non-neoplastic lesions typically do not show increased choline. lactate is sometimes detected in ischemia, abscess and demyelination. toxoplasmosis and tuberculomas typically show a dominant lipid peak. bacterial breakdown products including acetate, alanine and succinate are detected in abscesses. occasionally, increased cellular infiltrates and reactive astrogliosis may result in elevated choline levels, mimicking tumour. conclusion: with increasing clinical application of mrs, radiologists should be familiar with common spectral patterns of brain disease as well as the potential pitfalls in interpretation. decreased signal intensity on t -weighted mri in the basal ganglia in patients with white matter disorders p. arguis, s. capurro, j. berenguer, t. pujol, m. olondo, j.m. mercader; barcelona/es learning objectives: to describe some aspects of brain iron metabolism which can be studied by mri. to learn how decreased signal intensity on t -w images in the basal ganglia can be seen in several white matter disorders. background: the iron is taken up by capillary endothelial cells in basal ganglia and then is transported axonally to projection sites where it is used in local metabolism, there the iron is stored in oligodendroglia cells. interruption of normal axonal transportation of iron caused by white matter abnormality might lead to increased accumulation of iron at the basal ganglia since it could still be taken up but not be transported. it is generally accepted that there is a correlation between the accumulation of iron in the extrapyramidal system and the degree of hypointensity observed at this site on t -weighted images. imaging findings: low signal intensity on t -w images were observed in the basal ganglia in patients with different white matter disorders (congenital, multiple sclerosis, postradiation toxicity, ischemia, aids-related and tumoral). mr studies were performed with a . t scanner. conclusion: decreased signal intensity on t -w images in the basal ganglia can be seen in several white matter disorders. typical background: wernicke encephalopathy (we) is a severe neurologic disorder caused by thiamine deficiency related to malnutrition or malabsorption of b vitamin that occurs mainly, but not exclusively, in alcoholic patients. we reviewed the risk factors as well as the usual and unusual clinico-radiological features including two cases in non-alcoholic patients (one with gastroesophageal reflux and other with pyloric stenosis secondary to biliogastric reflux). a third of patients with acute we present with the classic clinical triad: opthalmoplegia, ataxia and confusion. other initial manifestations are hypothermia, hypotension, coma, nutritional polyneuropathy, cardiovascular involvement. early diagnosis is crucial because of non-specific clinical findings, a high mortality rate but a possible complete recovery with immediate thiamine replacement. imaging findings: typical mr findings include symmetrical signal changes in medial thalami, tectum and periaqueductal region. in the acute stage, enhancement after contrast injection may be visible in these regions as well as in the mamillary bodies, but at chronic stages atrophy is the main finding. atypical features described include signal changes in the cerebellum, dentate nuclei, pons, red nuclei, basal ganglia and central and precentral sulci. massive haemorrhages are rare whereas microscopic foci are frequent. differential diagnosis includes de/dysmyelinating disorders, infarctions and creutzfeldt-jakob variant but symmetrical distribution of findings rules out most of them. conclusion: we is a reversible, severe disorder and a high index of suspicion is recommended to diagnose it, even in non-alcoholic patients. to determine whether functional mr imaging sequences (diffusion tensor, tractography, perfusion, spectroscopy, blood oxygenation level dependent-bold), can help add new information to the pre-surgery approach to brain tumors. to determine the efficiency and efficacy of these news tools in evaluating brain tumors. during the period of months, mr exams of patients with brain tumors ( men, women; mean age . years) were performed in a . t clinical scanner (siemens, germany) , using mr clinical standard protocol and functional sequences (diffusion tensor imaging, perfusion, spectroscopy and bold sequence). all the patients had their histopathology diagnosis confirmed by brain biopsy and surgery; glioblastoma multiforme (n = ), anaplastic astrocytoma (n = ), low grade glioma (n = ), gliomatosis cerebri (n = ) and metastases (n = ). high grade glioma and metastases had hyperperfusion, high picks of choline and low n-acetylaspartate (naa). low grade glioma and gliomatosis cerebri had low relative cerebral blood volume, low naa, high choline and myoinositol. diffusion tensor imaging showed alterations of the main tracts within and nearest the high grade tumors. low grade tumors had main tracts dislocated but not interrupted. the white mater tract were preserved in gliomatosis cerebri. bold sequence identified the sensory-motor and language activating areas. mr imaging has been used to evaluate brain tumors. with new neuroimaging advanced tools, we can do this analysis in a more accurate and precise way and it takes less time than it was thought to. it is a huge contribution to the evaluation and management of patients, providing a type of diagnosis close to histopathology diagnosis. individual approach to recanalization treatment of acute ischemic brain contrast-enhanced mra were obtained three to five days after the insult. we retrospectively reviewed the mr findings and clinical courses of patients with anterior circulation territorial infarction. those with ica and mca lesions were divided into six and five groups respectively, according to the level and mechanism of the occlusion. pwi findings can be another factor in the management planning. here, we will present representative cases of each group and discuss the management with thorough consideration of various factors, such as pattern and type of arterial occlusion, the extent of dwi/pwi mismatch, the duration of the ischemia, age of the patient, the severity of neurologic deficits, or use of other anticoagulants, etc. we believe that mr imaging is very helpful in the evaluation and management planning of patients with hyperacute stroke. the treatment should be individualized to enhance the effectiveness and safety of the various treatment modalities. similarities and differences in functional connectivity of the prefrontal cortex: task in episodic memory (em), task-and item-related processes have been postulated to contribute to asymmetries in cortical involvement with the prefrontal cortex (pfc) being of particular interest. this study demonstrates task-and itemrelated differences in functional connectivity of pfc in verbal vs non-verbal em. methods and materials: twelve healthy volunteers performed visual non-verbal (line drawings of known objects) and verbal (abstract words) em tasks undergoing o- -butanol-pet to measure regional cerebral blood flow. using correlation analyses, functional connectivity of item-related (verbal vs non-verbal) processes during episodic encoding and retrieval was assessed. results: across all tasks/items we observed strong prefrontal interactions regarding the dorsolateral (dl) and frontopolar (fp) pfc. during encoding we found bilateral but left dominant pfc interactions with any material, right dlpfc showed no interactions during the abstract word condition. during retrieval of pictures we found an increase of right fppfc interactions and a symmetrical increase of dlpfc interactions. during retrieval of abstract words we observed an increase of left fppfc interactions, while right dlpfc showed no interactions. memory performance was higher for pictures vs words. conclusion: firstly, our results support the idea of a common bilateral pfc involvement across different stimuli and mnemonic operations. secondly, item-related processes point to a left lateralized pfc involvement for words and a bilateral pfc involvement for pictures. these data support the "dual coding" hypothesis, introduced by paivio (paivio, ; paivio ) , postulating a double (verbal and non-verbal) processing pathway for pictoral stimuli, hence a supplementary mnemonic strategy to increase memory performance. perfusion weighted dynamic susceptibility ( method: fifteen patients with unilateral - % carotid artery stenosis were studied with dsa and perfusion-weighted dynamic susceptibility (dsc) mri and were compared to age/sex matched controls. regional cerebral blood volume (rcbv) and mean transit time (mtt) values were calculated in the middle cerebral artery (mca) and borderzone (bz) territories. all patients underwent tea within one week from mri and were re-examined one month after surgery. results: there was no significant difference in rcbv and mtt values between the hemispheres in the symptomatic patients. there was a significant difference in mtt values in the borderzones between patients and controls. after tea we found a decrease of mtt of both hemispheres in bz territories while the remaining hemodynamic parameters persisted unchanged. the decrease in mtt values after tea lead these values to be similar to those of normal subjects. the results of our study suggest that there is an adequate compensation of unilateral stenosis when it is less than %. a hemodynamic compensation mechanism between the two hemispheres, particularly for the distal border zone territories that tend to have a slower mtt with respect to the control subjects before tea, and the restoring of mtt values in the bz after surgery, seems to be demonstrated as well. parkinson's disease: pre-and post-apomorphine perfusion evaluation with dynamic susceptibility contrast (dsc) mri f. gaudiello, s. marziali, a. ludovici, e. ferone, r. floris, g. simonetti; rome/it purpose: our aim was to study whether dsc-mri perfusion method may detect an altered pattern of regional cerebral blood flow (rcbf) in parkinson's disease (pd) patients in comparison to normal subjects and whether this altered pattern may be normalised by apomorphine. material and methods: twenty subjects affected by idiopathic pd according with the brain bank criteria were enrolled for this study. nineteen normal subjects were included as controls. ten of them performed a retest procedure. pd patients, after at least days of therapy withdrawal, were submitted to perfusion dsc-mri. sixteen of them were retested after apomorphine injection at least hours. relative regional blood flow was evaluated by using regions of interest (roi) of pixels manually placed in different region of basal ganglia. results: pd patients showed a significant inter-hemispheric asymmetry due to a higher perfusion in the more affected side (< . ), while normal subjects did not. pd exhibited an abnormal "asymmetry index" in the measured nuclei. dsc-mri performed after subcutaneous apomorphine injection did not show any significant asymmetry in pd patients. retest in normal subjects did not show any significant variation. conclusion: dsc-mri of basal ganglia confirms the asymmetry observed in pet studies of pd, suggesting this method as a promising technique in neurodegenerative diseases. b d e f a g steeper (r = . , p < . ) comparing to wm (r = . , p < . ) and almost negatively linear. conclusion: our study shows that ageing effects on metabolite concentrations are more pronounced in the gm regions of the brain. determination of absolute metabolite concentrations, rather than use of ratios only, is essential for characterizing age-related changes in brain metabolites. diagnosis of cerebral vascular malformations with ct angiography in patients with subarachnoid hemorrhage s. stathopoulou , c. kokkinis , n. makris , a. petinelli , k. vassiou , m. vlychou , p.j. papadaki , g.m. zavras , j.b. fezoulidis ; athens/gr, larissa/gr purpose: to evaluate the diagnostic accuracy of spiral ct angiography in patients with subarachnoid hemorrhage due to vascular malformations. methods and material: one hundred and fifty-eight patients suffering from subarachnoid hemorrhage (sah) underwent ct angiography (cta) as well as intraarterial digital subtraction angiography (ia-dsa). cta was performed using spiral ct with ml of intravenous contrast at ml/sec, after a delay of sec. slice thickness . mm and a reconstruction index of . was used. axial slices and multi-planar reformatting (mpr), maximum intensity projection (mip) and d surface shaded display (ssd) reconstructions were reviewed. ia-dsa followed in all patients. there was comparative study between two methods. results: twelve cases of vascular malformations were diagnosed by cta and these comprised arteriovenous malformations and venous angiomas. in cases, the cause of sah was vascular malformation and in cases was an incidental finding. in comparison with ia-dsa, cta missed only a small dural arteriovenous malformation but diagnosed a small venous angioma missed by ia-dsa. these two malformations appeared without hemorrhage. conclusion: cta is a useful non-invasive method in detection of cerebral vascular malformations causing sah, with similar results to ia-dsa. even though ia-dsa offers more anatomical detail in the evaluation of supplying and draining vessels, cta can be a useful diagnostic tool as the tridimensional aspect offers additional information in the therapeutic planning of these vascular malformations. material and methods: ct angiography (cta) was performed using spiral ct with ml of intravenous contrast at ml/sec, after a delay of sec. a slice thickness of . mm and a reconstruction index of . were used. axial slices and multi-planar reformatting (mpr), maximum intensity projection (mip) and surface shaded display (ssd) reconstructions were studied to evaluate aneurysm characteristics. then the image data was analyzed using volumetric d rendering technique to succeed endoscoping imaging. this technique extracts ct numbers in the boundary region between the vessel wall and contrast media within the vascular lumen. results: forty cases of cerebral aneurysms were detected by cta. with the technique of ct virtual angioscopy, we assured an excellent visualization of the inner contours of the vessels including the neck and the dome of the aneurysm (fly through). also d morphology of the internal structure of the aneurysm was demonstrated (fly around). the size of the sac (true lumen) and the neck were exactly estimated. blood vessels flowing into and out of aneurysms were visualized. in patients with ruptured aneurysms that were operated, virtual angioscopy showed the site of rupture, wich was confirmated by operation. conclusion: virtual angioscopy following spiral cta is a very useful technique supplementary to cta in non-invasive evaluation of cerebral aneurysms and selection of the most appropriate therapeutic modality. serial mri in adult-onset rasmussen's encephalitis reveals two main radiographic and clinical variants n. danchaivijitr, r. nicholas, t. smith, i. hart; liverpool/uk purpose: rasmussen's encephalitis (re) is a rare autoimmune disorder that classically affects one cerebral hemisphere. it is characterized by intractable epilepsy with progressive neurological deterioration and usually presents in childhood. adult onset re was believed to have a more benign clinical course and imaging features are less well described. here, we characterize the mri brain findings of a series of adult onset re patients and correlate with the clinical progression. we retrospectively reviewed the serial mri brain scans of patients with pathologically confirmed adult onset re ( scans in total). five patients had complete series of annual mri studies from their onset. clinical data including neurological signs, cognitive deficits and functional disability were recorded prospectively. results: we recognized patterns of imaging findings; patients had focal increase signal on t w in subcortical white matter of the temporal lobe. later, these were followed by ipsilateral hemispheric atrophy. these patients had a rapidly progressive clinical course in which neurological deficits occurred within months after onset. the second group ( patients) had initial focal cortical atrophy followed later by diffuse atrophy of affected hemisphere without evidence of high signal changes. in addition, bilateral involvement and caudate atrophy were more frequent than childhood variant. conclusion: adult re can be difficult to diagnose because the early mri findings can be subtle and physicians may be unaware of the possible presentation of one hemisphere atrophy without inflammation. pattern of initial mri features may help to predict clinical course of the disease. radiofrequency neurotomy for the treatment of headache associated with the third occipital nerve g.b. marshall, c. siwak, b. frizzell; calgary, ab/ca purpose: to assess the efficacy of a fluoroscopically-guided neurotomy technique in patients with cervicogenic headache associated with the third occipital nerve. methods: retrospective analysis of patients ( women and men) who underwent third occipital neurotomy for cervicogenic headache. these patients had failed conservative treatment and were selected based on response to facet joint injection and medial branch block. neurotomy procedures were completed by two radiologists. a chart review of pre-and post-procedural pain levels was undertaken in addition to follow-up patient interview to determine whether they had improvement in their activities of daily living and recreation. results: all patients reported a reduction in the frequency of their headaches. pain levels reported on a visual analog scale dropped from a pre-procedural mean of . / to a post-procedural mean of . / . the mean duration of pain relief was . months, with of the patients describing ongoing relief of symptoms. all patients reported restoration of activities of daily living, albeit for varying lengths of time. no significant complications were encountered. this radiofrequency neurotomy technique may be a useful means of providing symptomatic relief from cervicogenic headache associated with the third occipital nerve. the study justifies proceeding to prospective evaluation of the technique. functional magnetic resonance imaging (fmri) study of memory functions in elderly and young healthy controls and alzheimer's disease (ad) patients a. urbanik, m. binder, b. sobiecka, j. kozub, m. kuniecki; krakow/pl purpose: the aim of the study was to assess differences in the neural correlates of nonverbal memory functions in pathological and normal ageing. methods: thirty-six volunteers were examined in a . t mr scanner. three groups of twelve subjects were examined: young and elderly healthy controls, and patients with probable ad diagnosis. subjects were required to memorize complex geometrical figures. after the scanning session, they were all asked to redraw these figures. their performance was scored. image data were analysed with spm statistical package. results: both control groups copied drawings more accurately than the ad group. in all groups, the most prominent differences were seen in frontal regions and the occipital lobes, extending to the temporal. for each group this pattern was dissimilar. for the ad group moderate activation in the occipital lobe was observed, but no activation in frontal lobes. in turn, the elderly group revealed quite weak activation in occipital lobes and strong bilateral activation in the frontal lobes. finally, in the young adults group there was very prominent activation in occipital lobes, as well as in the frontal lobes, with apparent left-hemispheric dominance. we have succeeded in revealing differential patterns of brain activation in the studied groups during nonverbal memory encoding. our results suggest that a successful encoding requires an involvement of frontal lobes, that are probably responsible for the strategic aspects of memory functions. as was shown in the elderly control group, frontal lobes can compensate for the deteriorated visual memory which is known to decline over time. the special features of cerebral hemodynamics were studied in patients with chiari malformation (cm) with the help of magnetic resonance angiography (mra) and transcranial dopplerography (tcd). mra data analysis ( ) revealed the frequent presence of vertebrobasilar system (vbs) maldevelopment and embryonic type of circle of willis. tcd data analysis ( ) discovered vertebrobasilar insufficiency and bilateral blood flow increase in the intracranial part of the internal carotid arteries giving evidence of the compensated blood flow from the carotid system to the vbs. % of patients with cm and communicating hydrocephalus had tcd signs of intracranial hypertension. the most significant changes in cerebral hemodynamics were found in patients with marked pathology and older than years. conclusions: . changes of cerebral hemodynamics in patients with chiari malformation are conditioned by a complex of various causes; vertebrobasilar system underdevelopment, extravasal compression of the vbs arteries, rombencephalon hernia (cm) compensated blood flow from internal carotid system to vbs and the presence of intracranial hypertension. . the role of vascular factors in pathogenesis of the clinical signs of chiari malformation is confirmed. acute disorders of cerebral ischemic circulation in vbs and syncopal conditions were mainly found in patients with the nd / rd degree chiari malformation above years of age, that correlates with the greatest changes of cerebral hemodynamics by transcranial dopplerography findings. routine mr imaging in parkinsonian syndromes m. cosottini, r. ceravolo, g. lazzarotti, u. bonuccelli, m. michelassi, c. bartolozzi; pisa/it purpose: to assess the usefulness of routine mri in the differential diagnosis of parkinsonian syndromes such as multiple system atrophy type p or c (msa p and msa c), supranuclear palsy (psp), and corticobasal degeneration (cbd) from idiopathic parkinson disease (pd). we retrospectively evaluated patients with extrapyramidal signs and symptoms that had been followed for five years until a definitive clinical diagnosis. ninety five patients had pd, msa p, msa c, psp, cbd. mr examinations were obtained with . and . t equipment in cases and cases, respectively. axial dp-t and sagittal t -weighted images were evaluated considering the most frequently abnormal findings reported in parkinsonian syndromes. pd mr examination was supposed to be normal. results: using the reported radiological criteria, imaging by mri revealed a sensitivity and specificity of % and % in psp, % and % in msa p, % and % in msa c, % and % in cbd. a correct diagnosis of atypical parkinsonian syndrome was made in % of patients with extrapyramidal signs and symptoms. conclusions: several features on brain mr imaging may improve the diagnosis of atypical parkinsonian syndromes and pd in a substantial proportion of patients. brain with done by pc mra and the intracranial circle was evaluated with d tof mra. dsa was then performed to confirm the grading of stenosis and to depict the presence of collateral intracranial compensatory circles. patients were divided into two groups on the basis of the presence (group ) or absence (group ) of a regional or hemispheric perfusion defect detectable at visual inspection of mtt map. results: in group (six patients) there was a prolonged mtt, a reduced cbf and an increased cbv in two cases. a poor depiction of mca with mra and a significant reduction of mbf in the mca homolateral to ica stenosis ( ml/ min), as well as an angiographic impaired willisian compensation with a recruitment of vessels arising from the external carotid artery and from superficial collateral supply was detected in group . conclusions: pwi reveals oligemic abnormalities in only some patients with ica stenosis. increased mtt and reduced cbf are associated with an impaired willisian collateral supply. probably, bolus dispersion may influence the pwi-detected oligemia in ica stenosis. specificity results: statistical differences were found between the dementias group (ad and vd) and the non-demented group (depression and mci). the ratio choline/ creatine (cho/cr), myoinositol/creatine (mi/cr) and n-acetylaspartate/creatine (naa/cr) showed differences between the studied pathologies, only in ppgm, without significant differences in rtl. naa/mi and mi/cr distinguished between ad and the other pathologies, in ppgm, with the best area under the roc curve for naa/mi, showing the higher sensitivity ( %) and specificity ( %) for the diagnosis of the ad. conclusion: h mrs is a non-invasive tool for the study of ci, showing biochemical information to differentiate ad, vd, depression and mci. the metabolical alterations observed contribute to the differential diagnosis with a high sensitivity and specificity in pathologies with ci, in particular for the diagnosis of ad. value of transcranial doppler (tcd) measurements and "hyperdense middle cerebral artery" sign on ct for acute ischemic stroke prognosis y.t. efendiev, l.b. khalilova; baku/az methods: ct examination was carried out on the first day of stroke for patients (age range to ), who had ischemic infarcts in the middle cerebral artery (mca) territory and repeated on the rd , th and th days. "hyperdense middle cerebral artery" sign reflecting active intraluminal thrombosis was found in patients. the same patients had tcd on the st day. results: symmetric blood flow velocity (bfv) in both mcas was revealed in % of the patients. in this group, clinical course was rather auspicious in more than % of patients. infarcts area in these patents had limited, cortical ( %) and subcortical ( %) localization. no lethal outcome was noted. asymmetric blood flow with decreased mca bfv by more than % on the affected side was noted in % of the patients. in this group, the infarctions had cortico-subcortical localization on final ct. the clinical course was more severe and complicated than in the first group.one lethal case occurred. in %, tcd demonstrated signs of hyperperfusion of hemodynamics with increased of mca bfv by more than %, on the affected side, combined with high peripheral resistance index. infarcts had mainly deep basal location, and lethal cases occurred. later ct detected hemorrhagic transformation of necrotic area ( cases, %). conclusion: "hyperdense middle cerebral artery" sign on ct is significant for early diagnosis of stroke, whereas the tcd measurements in mca is important to predict the clinical course and possible impairment at the early stage of the disease. b d e f a g imaging of brain parenchymal and leptomeningeal metastases from lung cancer: significance of contrast enhancement mri m.p.i.m. jakimovska, t. markoski, m. grunevski, s. jakimovska, s. jovanoska; skopje/mk purpose: to estimate significance of iv application of paramagnetic contrast in mr imaging in diagnosis of parenchymal brain metastases, especially leptomeningeal carcinomatosis seeding from lung cancer. next purpose is to compare sensitivity between ct and mr imaging. material and methods: thirty-six patients with lung cancer were evaluated radiographicaly to determine the present of metastases in cns. we used ct as a screening tool and mr after i.v. application of gddtpa. results: our series with patients included or % with non small-cell lung cancer and patients or % with small-cell lung cancer. three patients ( %) of with small-cell lung cancer had occult metastatic disease to the brain. eight patients ( %) of with non small-cell lung cancer had metastatic disease to the brain parenchyma. in this series, leptomeningeal metastases were not found. ct scans were performed in all patients, mri was performed in cases, some with i.v. application of gddtpa, and in patients brain metastatic disease was found. post contrast mr imaging presents leptomeningeal carcinomatosis as thin linear enhancement along the cortical surface, which follows the gyral pattern of the cortex. conclusion: all patients with lung cancer have to be investigated for brain metastases because of a great percentage (about %) of occult metastatic disease. contrast enhanced mr of the brain is a superior method compared to ct because of excellent contrast resolution, is safer and has multiplaner abilities. to demonstrate meningeal seeding, contrast enhanced mri is the modality of choice. evaluation of the uselfulness of high b-value diffusion-weighted mr imaging in patients with acute cerebral infarction h. hirota , e. shimosegawa , k. takahashi , m. mineta , t. yamada , w. yamamoto , k. nagasawa , h. sato , t. aburano ; asahikawa/jp, akita/jp, iowa, ia/us purpose: we occasionally encounter cases without distinct abnormal findings on conventional diffusion-weighted mr imaging despite there being obvious clinical findings of acute cerebral infarction. our purpose was to evaluate positive predictive value of high b-value diffusion-weighted mr imaging in subjects with acute cerebral infarction. we prospectively assessed subjects with acute cerebral infarction within hours from the onset. all examinations were performed with . t unit using single-shot echo planar diffusion-weighed mr imaging at three different b values of , and s/mm . seven days later from the onset, we re-examined the subjects with t weighted image for confirming the final diagnosis. four experienced neuroradiologists assessed each diffusionweighted images for the presence of cerebral infarction and then compared the findings of each images with those of t weighted images. we divided the findings of each diffusion-weighted images into five categories by location of abnormal signal intensities and calculated true positive rate in each categories. purpose: arterial visualization of intracranial d-cta is difficult in the area of the skull base. this is due to the close proximity of the internal carotid artery and skull base and the fact that they both have almost the same ct values. accordingly, subtraction artifacts due to misregistration are unavoidable using the conventional subtraction method. even a slight difference of the start position in data between pre-and post-contrast ct may lead to the production of considerable artifacts. nowadays, subtraction of two sets of volume data has become possible on a workstation. we assessed whether this technique was useful in intracranial d-cta. methods and materials: subtraction was performed in both phantom and patients and the accuracy was tested. results: precise measurement was shown to be possible in the phantom experiment. following this, several clinical cases were applied. conclusion: subtraction is effective due to being almost free of misregistration. does an interpolated high resolution matrix improve the detection of brain hyperintensities on flair images in multiple sclerosis? f. sardanelli, a. fausto, g. spadaccini, b. cotticelli; milan/it purpose: to test whether an interpolated high resolution matrix improves the detection of brain hyperintensities on flair images in multiple sclerosis (ms). materials and methods: seven ms patients ( male, females, - years old, median ) underwent brain mr at . t using a fast-flair para-axial sequence (tr/te/ti = / / ms; fov mm; no-gap -mm slices; matrix x ; pixel size = . x . mm; time ' "). the sequence was repeated changing only from the standard matrix to an interpolated matrix with a pixel size of . x . mm. the two sequences were read on a remote console in a random blinded fashion, using a one-in-one format and free windowing, grading them as certain (c) or probable (p); for confluent plaques, only the lesions with more than half the contour detectable were counted. wilcoxon test was used. results: a total of hyperintensities (c/p = / ) with and of ( / ) with were counted. they ranged in total - (median = ) with and - ( ) with ; c hyperintensities - ( ) and - ( ); p hyperintensities - ( ) and - ( ), respectively. data were similar in / patients; in the remaining patient (female, years old) total hyperintensities were ( ) and ( ), with a c/p of / and / , respectively. in spite of this outlier, no significant overall difference was found between and for total, c, and p hyperintensities (p > . ). conclusion: interpolated matrices do not significantly improve the detection of brain hyperintensities on flair images in ms. a method for reducing radiation exposure in cerebral perfusion study using multi-detector row ct t. nanjo , k. murase , y. sugawara , m. hirata , t. mochizuki ; osaka/jp, onsen-gun/jp purpose: radiation exposure during ct perfusion study is a serious problem. the purpose of this study was to devise a method for reducing radiation exposure in cerebral perfusion studies using multi-detector row ct. methods and materials: nine patients ( males and females; mean age . years) participated in this study. following a standard protocol for ct perfusion study, continuous (cine) scans ( . - . sec/rotation x sec) consisted of four -mm-thick contiguous slices performed after an injection of iodinated contrast material ( - ml) using a multi-detector row ct scanner (light speed qx/i, ge). new image data were generated by thinning out the original images thus acquired. the thinned-out images were interpolated by linear or cubic interpolation. the functional images of perfusion parameters such as cerebral blood flow (cbf), cerebral blood volume (cbv) and mean transit time (mtt) were generated from these images by applying deconvolution analysis based on singular value decomposition pixel by pixel. we calculated the correlation coefficients between the perfusion parameters obtained from original and thinned-out images for regions of interest in the grey and white matter. results: when using continuous images with a scan time of . - . sec and the thinned-out images with a scan interval of . - . sec, the radiation exposure could be reduced to . - . %, with the correlation coefficients of cbf, cbv and mtt being kept greater than . . conclusion: this method can reduce radiation exposure while keeping the accuracy of perfusion parameters equivalent to that obtained from original images. dynamic scanning. the data acquisition conditions (analysis algorithm, analysis matrix, contrast medium injection rate, image reconstruction algorithm and exposure dose) were optimized while maintaining high quantitative analysis capabilities. cerebral blood flow was then measured in volunteers in their s, in their s, in their s, in their s, in their s, and in their s. none of the subjects had cerebral blood flow disorders. results: the use of optimized data acquisition conditions provided high reproducibility while permitting the exposure dose to be reduced by % or more. a decrease in cerebral blood flow with advancing age was confirmed in ct dynamic perfusion studies. the use of optimal data acquisition conditions allows the exposure dose to be reduced and provides high reproducibility while permitting acceptable quantitative cerebral blood flow analysis. this has made it possible to determine age-specific normal values for cerebral blood flow in dynamic ct perfusion examinations. it is expected that dynamic ct perfusion analysis will prove clinically useful for the assessment of diffuse cerebrovascular diseases and chronic-stage blood flow disorders, as well as for the diagnosis of acute-stage cerebral infarction. diffusion weighted imaging (dwi) epi on low field mr system ( . t): comparison with . t and preliminary report on clinical use in brain ischemia p. purpose: this study assessed the diagnostic value of low field dwi in brain stroke. the secondary goal was to compare apparent diffusion coefficient (adc) values obtained in high and low systems. methods: dwi were performed in groups of volunteers, on . t and . t systems. dw images at b = and mm /s were recorded. adc values from regions of interest (roi) were compared. forty patients with acute symptoms of cns stroke were examined by ct and mr, - h after symptoms onset. dwi-epi images were obtained. follow-up mr weeks later were set as a reference. results: statistical analysis showed significant differences of adc values dependent on roi location within the brain hemisphere. there was no difference between right and left hemisphere. adc values for low and high field systems were similar. in patients, ischemic lesions were found in the first examination. follow-up confirmed infarcts. four patients out of with negative dwi revealed infarction in follow-up mr. sensitivity, specificity, and diagnostic accuracy were . %, %, and %, respectively. conclusions: low field systems are capable of diffusion weighted imaging. for the same b value, adc values at . t and . t are similar. in clinical practice dwi on low field mr systems can be used as a fast and reliable tool for detection of acute ischemic lesions in the brain. advantages of a d multislab t turbo spin echo magnetic resonance (tse mr) sequence for high resolution brain imaging b. it is feasible to scan the whole brain with t contrast in mm³ isotropic resolution with high snr and cnr using a multislab d tse sequence. postoperative multidetector-row ct angiography ( ( ), hemimegalencephaly( ), bilateral perisylvian syndrome( ), lissencephaly( ) and focal cortical dysplasia( ). all were the subject of conventional brain mr imaging studies using the mp-rage (magnetization-prepared rapid gradient-echo) sequence and the resulting -d data sets were processed on a commercially available workstation. abnormal gyral configurations were reviewed. results: abnormal gyral patterns were seen of patients. the configuration and orientation of affected gyri were clearly evaluated in the brain surface-rendering image. in two cases of the schizencephaly (involved the frontal lobe), there were wheel shaped broad gyral pattern of the superior, middle, and inferior frontal lobes. in two cases of the hemimegalencephaly, there were thick gyral pattern and enlarged ipsilateral brain. in two cases of the lissencephaly, the frontoparietal cortex was not delineated and showed a markedly thick and smooth gyral pattern. in two cases of the bilateral perisylvian syndrome, there were opened insular cortex and a thickened gyral pattern. in the focal cortical dysplasia, there were irregular serrated or thick and enlarged gyri. in patients with cerebral cortical dysplasia, brain surface-rendering mr imaging detects a detail gyral pattern and involvement site of abnormal gyri. it can create difficulties in the differential diagnosis and can therefore cause problems in choosing the appropriate therapy. this is especially true in cases with alzheimer's versus lewy-body disease ((dlb), which require different therapeutical approach. we applied h-mrs to assist the diagnostic process and to look for specific differences in the metabolic content of the brain tissue in these two entities. methods: h-mrswas performed using . t scanner in t -weighted images in orthogonal planes in elderly subjects with consensus criteria dlb (n = ), nincds-adrda ad (n = ), and normal control subjects (n = ). h-mrs was performed with single-voxel (svs) technique using steam sequence (tr ms, te ms). voxel was positioned in the temporal, occipital lobe and centrum semiovale. results: in both alzheimer's and lewy-body group, h-mrs showed changes in the metabolite pattern in all voxel locations, with the most distinct expression within temporal lobes. naa and creatine peaks were lowered in both patients groups, comparing to the controls. in spectra of lewy-body patients myoinosytol peak remained unchanged as opposite to the alzheimer's where it was increased. conclusions: h-mrs can be an additional, very important tool in dementia studies and differential diagnosis of this group of diseases. in dlb patients h-mrs is feasible but there are difficulties in scanning patients in the later stages of the illness due to tremor and the degree of brain atrophy. the activation tasks consisted of left finger movement, sensory stimulation, listening to comprehension (for sensory language area), word generation (for motor language area) and working memory. the reference function was boxcar waveform. activation maps were thresholded at uncorrected p = . . the thresholded activation maps were placed into talarach's space. results: cerebellar activation was observed in the motor, the word generation and the working memory tasks. in the hot sensory and listening comprehension tasks, there was not any visible activation in the cerebellum. left and right posterior cerebellar (declive) activation was observed in the left motor task. ipsilateral cerebellar activation was more prominent than contralateral cerebellar activation during the left motor task. left posterior cerebellar (declive) activation was observed in the working memory task. left posterior cerebellar (declive) and both anterior cerebellar (culmen) activations were noted in the word generation task. conclusion: cerebellar activation was observed in the afferent actions of the brain including motor, motor language and working memory tasks. there was no activation of the cerebellum in the efferent action of the brain including sensory and sensory language tasks. the cerebellum is involved in a variety of functional tasks including motor, word generation and working memory. the brain smell centres: comparison of localisation and activation in male and female subjects using functional mr imaging (fmri) m.d. marchwicka-wasiak, b. goraj; lodz/pl the study was conducted in order to determine and to compare the location and activation of smell brain centres in females and males brains using olfactory nerve-mediated (geraniol) and combined olfactory and trigeminal nervemediated (patchouli) stimulants. methods: ten normal volunteers (five women and five men), right-handed, nonsmokers, without any cns diseases were examined to determine the activated cortex areas during stimulation by geraniol and patchouli. mr brain scans were obtained using a . t clinical scanner, with the head-neck coil. the imaging was performed in each subject using se and epi sequences with a blood-oxygenlevel-dependent (bold) effect. the individual inhaled odorized air during the seconds period and alternating room air over the same period. the mean pixel intensity of activated images was substracted from the mean pixel intensity of preactivated images. results: the olfactory system-mediated stimuli (geraniol) evoked bilateral activation of female brain smell centres compared to right hemisphere centre activation in male brains. the exposure to the olfactory and trigeminal nerve-mediated stimuli (patchouli) showed more activated regions in both sexes than to the olfactory nerve-mediated stimuli. conclusion: fmri proved to be a useful method to compare the location and activation of male and female brain smell centres. we compared the group of psp patients with that of control subjects and found a highly significant difference in antero-posterior midbrain diameters (mann-whitney test: p < . ), absolute midbrain volumes (p < . ) and naa/ cr ratios (p < . ), as well as a significant difference in total intracranial volume-normalized midbrain volumes (p < . ). we also found out a significant correlation between naa/cr ratios and antero-posterior midbrain diameters in psp patients (spearman's rank correlation test: rs = . , p < . ). the aim of this study was to compare the diagnostic accuracy of t fast spin echo (t fse), fluid attenuated inversion recovery (flair) and inversion recovery (ir), in evaluating hippocampal sclerosis and to determine a costeffective protocol for this disease process. in this prospective study, patients (aged months- years) with drug-resistant temporal lobe epilepsy (based on clinical symptomatology and electroencephalographic (eeg) registrations) were evaluated by mri with a . tesla unit. the protocol consists of axial t and pd/t se, and flair, t fse and ir in a coronal plain perpendicular to the long axis of the hippocampus. whenever necessary, we applied t + gd se in axial and coronal planes. films were interpreted by two experienced neuroradiologists. sequences were statistically analyzed in pairs using wilcoxon sign ranks test. results: there were patients ( . %) with mr and eeg findings suggestive of hippocampal sclerosis. eight of them underwent therapeutic surgery and there was histopathologic confirmation of the diagnosis. flair detected hyperintensity of the hippocampus in / ( %) and was much more sensitive than t fse ( / p = . ) and pd/t ( / p = . ). reduced hippocampal size ( / ), atrophy of hippocampus ( / ) and enlarged temporal horn ( / ) were better detected with ir than t ( / p = . ). conclusion: a mr protocol using thin coronal sections angled perpendicular to the hippocampus with flair and ir sequences is cost-effective for mesial temporal sclerosis because it is highly sensitive, inexpensive, quick and easily applicable in most units. reversibility of the brain white matter changes in adults with late diagnosed and treated phenylketonuria m. gizewska, l. cyrylowski, d. koziarska, p. nowacki, a. walecka, m. walczak; szczecin/pl purpose: phenylketonuria (pku) is an autosomal recessive disorder related to absent or reduced activity of phenylalanine hydroxylase what leads to increased plasma phenylalanine (phe) concentration. because screening the whole newborn population for pku in poland began in the late s, many pku adults have been diagnosed and treated very late. patterns and extent of the brain white matter changes in these patients is described. in this paper, late diagnosed and treated adults with pku, including males and females, is presented. the mean age at diagnosis was . y (range - ). microcephaly was present in , seizures in , aggressive behavior in ; all but one had speech difficulties, including total mutism in . mri of the head was performed on . t unit with fse and flair sequences without contrast medium administration. results: mri of the head detected white matter changes, hyperintense on t weighted images, in all but one patients. all but one of them had changes in parietal and/or occipital regions. the treatment with low-phe has been introduced in with improvement in all of them. the follow-up mri were performed after mean . months, revealing decreased severity of the white matter changes in all, from mean (according to cleary's scale) on the initial mri to . on the follow-up. our results indicate the possibility of white matter changes reversibility in late diagnosed and treated pku adults. conclusion: dwi is thought to be useful in the diagnosis of cns infections. in our experience, dwi may allow the differentiation of brain abscess from necrotic or cystic brain tumor. dwi is also a useful tool to differentiate subdural and epidural empyemas. detection . coronal views were performed for flair imaging, and coronal and axial views for dp and t -weighted imaging. images were reviewed by two seniors radiologists in consensus for localisation of the signal abnormalities. results: lesions of the olfactory system were observed in patients ( %). signal abnormalities were localised in the temporal lobe in cases ( % with cases of unilateral lesions and cases of bilateral lesions) and in the orbito frontal cortex in cases ( % with cases of bilateral lesions and cases of unilateral lesions). with flair, dp, and t weighted imaging, lesions was detected respectively in %, % and %. olfactory bulb signal abnormalities were diagnosed in cases ( % with cases of unilateral lesions and cases of bilateral lesions). for this localization, the accuracy of flair, dp and t sequences was respectively %, %, %. in post-traumatic anosmia, coronal flair and coronal pd imaging is required to detect respectively signal abnormalities in the parenchyma and in the olfactory bulbs. t weighting imaging seems to be inferior. cerebral hydatid disease: ct and mr imaging findings y. bükte, s. kemaloglu, h. nazaroglu, a. uyar, c. akgül, m. simsek; diyarbakir/tr purpose: cerebral hydatid disease is very rare, representing only % of all cerebral space occupying lesions, even in the countries where the disease is endemic. the aim of this study was to describe the discriminative computed tomography (ct) and magnetic resonance (mr) imaging features of cerebral hydatid disease. we retrospectively reviewed the imaging findings of patients with pathologically confirmed cerebral hydatid disease over a period of years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . among these patients, there were cases of echinococ-b d e f a g cus granulosis and case of echinococcus multilocularis (alveolaris). they were male ( . %) and female patients ( . %), ages ranging from to years with an average age of . years. results: common ct and mr imaging findings of e. granulosis lesions were well-defined, smooth thin-walled, round or spherical, homogeneous cystic lesions with no contrast enhancement, no calcification and no surrounding edema. the lesion seen in e. multilocularis showed a well-defined multiseptated mass consisting of solid and cystic components with calcifications of solid portions. cystic lesions with surrounding hyperintense area of perifocal edema, complete and incomplete rim of contrast enhancement were seen in two patients and were labeled as complicated and infected cysts. conclusion: although the cystic cerebral hydatid disease could be equally well demonstrated on ct and mr examinations, ct is superior in detecting calcification of the cyst, when present. mr is better in demonstrating cyst capsule, detecting multiplicity and defining the anatomic relationship of the lesion with the adjacent structures and helps in surgical planning. ct it may be used to identify regional differences within a patient. qualitatively, single scan cbv maps were noisier than maps using the dynamic method. conclusion: cbv estimates from a single contrast-enhanced scan may identify abnormal areas. however, differentiation between normal and abnormal regions is not as pronounced as when using quantitative, dynamic methods. virtual endoscopic imaging of cerebral ct angiography (veccta) in selecting patients for neurosurgical intervention or endovascular treatment r. prpic-vuckovic, m. rados, m. batinica, j. papa, m. seronja-kuhar; zagreb/hr purpose: to assess the usefulness of virtual endoscopic cerebral ct angiography data analysis in patients with aneurysm rupture before making a decision between neurosurgical intervention or endovascular treatment. materials and methods: during a one-year period, mscta was performed in patients after a conventional ct proven subarachnoid hemorrhage. images were acquired using msct lightspeed ultra, ge. helical data acquisition followed after rapid i.v. administration of contrast material ( . ml/sec), with - sec delay. data was postprocessed on ge advantage workstation ultra v . using reconstruction protocols mip (maximum intensity projection), ssd (shaded surface display), vr (volume rendering) and ve (virtual endoscopy). results: detailed analysis of aneurysmal neck, dome, vessel of origin and surrounding vascular or bone structures was acquired by ssd, mip or by high-accuracy vr protocol. modified surface rendering and ve protocol were used for endoscopic mode viewing to visualize neck shape, accessibility from the parent vessel, presence of thrombotic clots or calcifications inside the aneurysm and vessel of origin, as well as to define orifices of branches arising from the dome. conclusion: accurate characterization of morphology of the aneurysm, especially depiction of orifices and aneurysmal neck are essential for choice between neurosurgical intervention or endovascular treatment. when endovascular treatment is an option, it is useful to know angulation between parent vessel and neck and real volume of aneurysmal dome for proper estimation of coil size and shape. vr and ve imaging of the aneurysmal sack and surrounding anatomy may provide additional useful information for endovascular treatment or neurosurgical intervention. we have demonstrated the existence of non-monoexponential diffusion in the human csc that can be approximated by fast and slow diffusion components. our results are within the limits of human brain and full dti data for the rat spinal cord. the aim of this study is to review the embryology and the development of this structure. to describe the normal ct and mr appearances and to show the neuroradiological findings of pathology that can effect the craniocervical junction. background: the craniocervical junction is an anatomical crossroads with characteristic pathologic entities, mainly malformative and traumatic in origin. we illustrate the normal anatomy of the craniocervical junction and a wide spectrum of pathological conditions involving this structure through a selection of cases from a retrospective review of the ct, mr and plain-film examinations carried out in our centre over the last five years. imaging findings: normal anatomy and a wide spectrum of pathologies affecting the craniocervical junction are illustrated by ct and mr examinations. we group the findings according to etiology: malformation, trauma, infection, inflammatory and tumor, placing emphasis on the radiological features and the differential diagnosis. conclusion: ct depicts the bony structures of the craniocervical junction superbly while mri shows the neurological components better, enabling excellent characterization of lesions and recognition of all potential types of disorders that need to be recognized by general as well as neuroradiologists. the spectrum of t hyperintensities of the sellar region f. with the introduction of high field strength it is expected to overcome the limited signal to noise ratio and subsequently decreased spatial resolution in routinely used standard field strength . - . tesla mr scanners. this would have clinical impact in the preoperative work up of patients with sellar processes. imaging findings: in this exhibit we present a large variety of sellar diseases using tesla mr imaging with emphasis on detection of microadenomas and visualization of anatomic structures. furthermore, the differentiation between infiltration or just compression of the medial wall of the cavernous sinus in case of macroadenoma is illustrated. the excellent visualization of normal anatomy and tumor topography makes this technique a very valuable tool for planning neurosurgical operations. cns manifestations following stem cell transplantation: a pictorial review d. beckett, a. banerjee, j. oliff; birmingham/uk learning objectives: to illustrate the spectrum of radiological manifestations in the central nervous system and head and neck following stem cell transplantation for haematological malignancy. to outline the advantages and limits of plain film radiography, cross-sectional imaging, interventional radiology and radionuclide imaging. background: bone marrow transplantation (bmt) is a critical therapeutic intervention for a variety of pathological conditions. the complications of bmt include chemotherapy and radiation toxicity, graft versus host disease, recurrent malignancy, infection and miscellaneous conditions. complications of allogenic bmt manifest in a variety of clinical settings. the neurological system is commonly affected. in this presentation we will review the incidence, clinical presentation and the radiographic findings of cns involvement following stem cell transplantation. plain radiography, contrast material enhanced studies, sonography, ct, mri and interventional techniques are essential in diagnosing these complications and evaluating their response to therapy. imaging findings: findings may be broadly classified into either infective, cerebrovascular or treatment induced. common infective manifestations include human herpes virus , invasive mucormycosis and central nervous system tuberculosis. classical image findings of haemorrhage and infarction are seen in addition to graft versus host disease cerebrovascular angiitis. infarcts, diffuse white matter disease and cortical atrophy are seen following total body irradiation. the imaging findings of posterior leukoencephalopathy in cyclosporin toxicity is illustrated. the spectrum of imaging findings in the central nervous system is diverse. a firm understanding of the clinical presentations and incidence in combination with typical radiological findings is essential in the successful management of this patient sub-group. b d e f a g background: diffusion weighted imaging has been widely used in stroke patients to detect early acute cerebral infarction and find out ischemic penumbra. however, we often unexpectedly encounter restricted diffusion on dwi of nonvascular origin mimicking hyperacute or acute infarction in clinical practice.we reviewed nonvascular diseases with restricted diffusion on dwi for past three years by retrospective selection. imaging findings: the characteristic findings mimicking hyperacute or acute infarction are high signal intensity and low apparent diffusion coefficient values, mainly distributed in the white matter and incompatible with arterial territory with no stenoocclusive lesion on mr angiography or conventional angiography, and are reversible. these findings may increase possibility of nonvascular diseases such as infection, inflammation (viral or bacterial encephalitis), hemorrhage, leukodystrophy, toxic (anti-cancer drugs, co poisoning), metabolic origin and tumors. conclusion: we present various diseases, under certain clinical settings, and systematically discuss the mechanism of restricted diffusion in a limited number of specific diseases. background: in patients with vitamin b deficiency, symptoms related to the spinal cord may often help to find the diagnosis. however, in patients with atypical symptoms mri shows typical findings often diagnostic for scd. beneath a general discussion, we present cases of scd in whom mri findings were leading to the diagnosis of b -deficiency. patient presented with tingling in her extremities. neurologic examination showed a subacute loss of proprioception and profound loss of vibration sense. vitamin b serum levels were normal. t w-mri showed increased signal in the posterior columns of the spinal cord. dwi showed disturbance of diffusion in the same location. patient , who practiced a vegan alimentation, was admitted with incomplete paraplegia with signs of spasticity. vibration sense and proprioception were impaired as well. mri revealed the typical findings of severely increased signal within the posterior columns of the spinal cord. both patients presented in very early stages of the disease. in summary, the mri findings showed typical signal hyperintensities in t w images in the posterior columns of the cervical and/or thoracic myelon-segments. conclusion: even in patients with subtle atypical symptoms, mri with its typical findings often allows the diagnosis of scd even in the rare case of normal levels of vitamin b . fiber tracking and tensor metrics in diffusion tensor mr imaging c.c.t. lim, h. yin, m. xu, f. hui; singapore/sg learning objectives: to review anatomical patterns and distribution of white matter tracts in the brain and to understand the potential uses of diffusion tensor imaging (dti), including pathological disruption and destruction of fiber tracts. background: dti is a new mr imaging technique that non-invasively visualizes white matter fiber tracts and may have potential to assess demyelination and other brain disorders. using single shot echo planar imaging (tr - /te - ms, mm section thickness, b value - s/mm in - non-collinear directions at . and t) and offline post-processing of diffusion tensor metrics (fractional anisotropy, mean diffusivity and principal eigenvector) and fiber connectivity images (using fast marching tractography) could be studied. imaging findings: in normal patients, the fractional anisotropy (fa) maps clearly visualized white matter tracts as areas of high fa compared to gray matter. the corticospinal tracts, corpus callosum and optic radiations could be consistently identified. abnormalities such as brain neoplasm, cerebral infarction and multiple sclerosis lesions showed decreased fractional anisotropy and elevated mean diffusivity. principal eigenvector maps of water diffusion showed deviation in the direction of the white matter tracts surrounding benign brain neoplasms and malformations. conclusion: quantitative in vivo information on water diffusivity, degree of directionality and directional vector in space, that are not available on conventional mr imaging, can be measured in dti studies. with a potential new clinical application of dti, radiologists should be familiar with anatomy and pathology of the white matter fiber tracts. neuroimaging of background: multiple gliomas are rare findings with variable incidence ( . to %). they can by classified according to: a) time of presentation as early = synchronous (at initial diagnosis) or late = metachronous (diagnosed during the treatment); b) the imaging characteristics as multicentric (arise independently in different sites, there is absence of macroscopic connections or way of dissemination) or multifocal (they spread from primary tumor to other areas in the brain, with evidence of routes of dissemination). we reviewed documentations of patients with diagnosis of glial tumor who grade iii-iv who underwent surgery between january and december . in all patients radiological and histological diagnosis was performed and cases were established as multiple glioma. imaging fingings: most of the patients had one bigger lesion and or smaller ones. the lesions were usually hyperintense on t -weighted images and hypoor isointense on t wi with strong postcontrast enhancement. most of them had moderate edema. we recognized multicentric and multifocal gliomas, were synchronous and one metachronous. conclusion: multiple gliomas are often radiologically reported as metastasis, sometimes as lymphomas or infectious diseases. the differential diagnosis of multiple masses in the brain is very important for therapeutic consequences and multiple glioma should considered because of suggested increasing frequency of this entity. if strong postcontrast enhancement and moderate edema around the lesions are observed, this diagnosis should be suggested. role background: gks is a method of treatment allowing the delivery of a conformal radiation dose to a stereotactically defined target. the significant development of interest in the role of gks as an alternative or adjunct to the neurosurgical management of many brain disorders, requires accurate and reliable methods of acquisition of image modalities for the stereotactic planning. imaging and procedures: between december and august , we treated patients for a large variety of indications (metastases %, vascular malformations %, trigeminal neuralgia %, pituitary adenoma %, primary brain tumor %, other tumors %, vestibular schwannoma %, meningioma %, other functional < %). all patients had an mri combined with ct in stereotactic conditions. some patients also benefited from stereotactic dsa (n = ) or pet (n = ). in all cases, mr was used to delineate a target before radiosurgery. ct was always used as a control for mr distorsion. dsa was used in combination with angiomr for all arteriovenous malformations. when the tumor was ill-defined, we anticipated some limitation of target definition (infiltrative lesions, recurrence of metastases, pituitary adenomas) and also used stereotactic pet. we present a pictorial review illustrating the role of neuroimaging for appropriate targeting in gks. these images are also critical for the evaluation of the potential risks and complications. the complementary information provided by the combination of various imaging modalities allows appropriate and accurate delineation of a target volume before stereotactic irradiation. to familiarize non-greek radiologists with the definition of greek eponyms and to emphasize on those terms and words that have been derived from greek mythology. background: medical eponyms serve as mnemonics, add color to medical writing and are useful shorthands in medical communications. a long catalogue with the most representative greek medical terms is listed. the exact definition and meaning of each word is explained and a short narrative of the relative myth is presented for the neuroanatomical terms which gained their name from greek mythology. conclusion: accurate knowledge of the meaning of these words will help the radiologist to remember the terms and make their learning fun. preoperative to present the potential of diffusion tensor imaging (dti) and colored coded fractional anisotropy (fa) maps in identifying the anatomy of white matter tracts for pre-operative planning. materials and methods: dti data were acquired using dw-se-epi sequences and a . t mr-unit, with the following parameters: tr - , - slices, mm slice thickness, . mm intersclice gap, x matrix and gradients encoding in directions, b- sec/mm . data were acquired in normal subjects and patients with brain tumors, avm's or seizure foci being considered for surgery. fa images and corresponding colored coded maps were generated. also, fse t or flair images were acquired in identical locations for comparative use. results: major white matter tracts (intrahemispheric, interhemispheric, and brain stem) were readily visualized, including the following: superior longitudinal fasciculuus, superior fronto-occipital fasciculus, inferior longitudinal fasciculus, inferior fronto-occipital fasciculus, uncinate fasciculus, corpus callosum, cingulum, optic radiations, anterior commissure, corona radiata, internal capsule, pyramidal tracts, medial lemniscus, medial longitudinal fasciculus, cerebellar peduncles and gracile and cuneate fasciculi. the spatial relationships of these tracts and resectable lesions were analysed, with particular attention to the risk of a postoperative deficit. all results were correlated with the pre-and post-operative deficits. conclusion: white matter anatomy can now be imaged using dti/fa and access to pre-operative eloquent white matter mapping has great potential to improve the outcomes of neuro-surgical candidates. the future management of neuro-surgical patients will be impacted by future refinements and developments of this technique. to present a retrospective analysis of our experience in the endovascular coil trapping for ruptured vertebral artery (va) dissecting aneurysms. materials and methods: we treated patients who presented with acute subarachnoid hemorrhage due to rupture of a vertebral artery dissecting aneurysm. embolization of the entire segment of the dissected site with detachable platinum coils (internal trapping) was performed in all patients. in one patient with posterior inferior cerebellar artery (pica)-involved va dissecting aneurysm, internal trapping of the aneurysm followed by occipital artery-pica bypass was performed by a neurosurgeon. results: coil embolization was successful in all patients. there were no procedure-related complications. there was no sign of infarction in the pica territory in all patients. there was no recurrent bleeding or ischemic symptoms during the follow-up periods. follow-up angiography showed complete occlusion of the aneurysm without recurrence in all patients. conclusion: endovascular coil embolization is a safe and highly effective treatment of ruptured va dissecting aneurysms. high-grade stenoses of the carotid artery were treated with stent implantation. dw mr imaging of the brain was performed a day before and after the endovascular treatment. all patients underwent neurological examination before and after the procedure. results: dw mr images showed a total of new hyperintense lesions in ( %) of the procedures performed. eight lesions were ipsilateral to the treated vessel and lesions, in patients, were contralateral. these patients with contralateral lesions also had new ipsilateral lesions. the lesions were located in parietal lobe (n = ), periventricular (n = ), occipital lobe (n = ), frontal lobe (n = ) and corona radiata (n = ). all lesions measured < mm in diameter. none of the patients had neurological symptoms. results: angiographic occlusion was complete in ( %) and ( %) aneurysms in groups a and b, respectively. mean percentage of volumic occlusion in these groups was . and . %, respectively. perioperative morbidity and mortality rates were and %, respectively in group a, and were respectively similar in group b. no significant difference between the two groups was observed. however, percentage of volumic occlusion correlated with sac-to-neck ratio smaller than . (p = . ) and with sac size (p = . ), except when or more threedimensional coils per aneurysms were used (p = . ). the better the percentage of volumic occlusion, the better the percentage of angiographic occlusion (p = . ). percentage of volumic occlusion was an independent predictor of angiographic complete occlusion (p = . ). wfns grade was an independent predictor of perioperative mortality (p = . ). the use of three-dimensional coils improved the coil-packing and angiographic and volumic occlusion of aneurysms with a neck greater than mm at the time of treatment, provided the sac-to-neck ratio was . or greater and the largest number of three-dimensional coils were positioned first. minimum apparent diffusion coefficient: a quantitative parameter for prediction of malignancy of gliomas preoperatively x. yun; sendai/jp introduction: in gliomas, choline signal (cho) correlates with total membrane metabolism, apparent diffusion coefficient (adc) inversely with cell density, cho*meanadc with each cell's metabolism. we examined which parameter is the most reliable in prediction of malignancy of gliomas. methods: twenty-three pathologically proven glioblastomas (gbm) and anaplastic gliomas (ag) were retrospectively evaluated with preoperative mri with mr spectroscopy (mrs) and dwi. ki index was counted to evaluate pathological malignancy. to evaluate clinical malignancy, we selected cases in which at least enhancing lesions were removed and classified into progressed and stable groups. the minimum adc (madc) of gbm was shown to be significantly lower than ag (p = . ). the highest value of madc in gbm was . , and the lowest in ag was . . using this threshold, subjects were divided into groups; a: gbm with madc < . (n = ); b:gbm and ag with . < madc < . (n = ), c: ag with madc < . (n = ). six cases in group a were classified as progression ( pathologically proven recurrence, two dissemination) and all cases in group c were stable. six cases in group b showed progres-sion ( recurrence, dissemination). fisher test showed significance (p = . ). in group b, the madc of progressive gbms was significantly lower than stable gbms (p = . ). the ncho and ncho*meanadc showed no difference between gbm and ag (p = . , p = . ). inverse correlation was found between ki and madc (p = . ), no correlation was found between ki and ncho (p = . ) and meanadc*ncho (p = . ). the madc suggestive of cell density was the most reliable in prediction of malignancy of gliomas preoperatively. usefulness methods: forty-one cases of skull base lesions ( meningiomas; petroclival, clinoidal, olfactory, in foramen magnum, spheno-petro-clival, in the planum sphenoidale, in posterior pyramid and in pca; acoustic schwannomas, epidermoids, pituitary adenomas, craniopharyngiomas, posterior fossa aneurysms, trigeminal schwannoma, dermoid and juvenile angiofibroma) have been evaluated. data was collected, fused, integrated and reconstructed by a dedicated stealthstation system for neuronavigation. ct images were acquired on axial non-overlapping slices, - mm thick; mri images were obtained with . t device, same field of view and thickness. results: neuronavigation was possible in all cases and was successfully applied in pre-operative planning and during surgical procedures. we calculated some data to check the efficacy of the method, having a mean fiducial error of . mm and an accuracy value at cm of depth . mm. discussion and conclusion: imaging fusion for pre and intra-operative neuronavigation provided, in all cases, great advantages in the choice of the best approach, placing of bone flap, early identification of deep normal or distorted anatomic or pathologic structures and their eventual encasement or involvement by the pathologic primary process, correct definition of tumor boundaries and meningeal implant and relationship with functional areas. neuronavigation appeared ideal for skull base meningiomas making surgical maneouvres safer, more effective and less invasive. a study of mr diffusion changes in normal appearing white matter (nawm) of patients with relapsing remitting multiple sclerosis (ms) f. gaudiello, s. marziali, a. ludovici, e. ferone, r. floris, g. simonetti; rome/it background and purpose: the aim of our study was to monitor water diffusion changes over time in nawm of patients with relapsing-remitting ms and to monitor water diffusion changes in patients with different disability status (edss) and with different clinical status (poussè). methods: we selected subjects who had completed a month follow-up. the mr scans were obtained at baseline, at months and at months. two regions of interest (roi) were placed in each hemisphere in the nawm in each mr study and the apparent diffusion coefficient (adc) value were calculated in patients and in healthy controls. results: tadc in nawm in patients was significantly higher then in controls (controls mean tadc ± sd = . ± . mm /sec x - ; patients mean tadc ± sd = . ± . mm /sec x - ; p < . ). in nawm in patients with higher edss, tadc was significantly higher then in patients with lower edss (patients with higher edss: mean tadc ± sd = . ± . mm /sec x - ; patients with lower edss: mean tadc ± sd = . ± . mm /sec x - ; p < . ). tadc in nawm in patients with poussè was significantly higher then in patients without poussè (patients with poussè mean tadc ± sd = . ± . mm /sec x - ; patients without poussè mean tadc ± sd = . ± . mm /sec x - ; p < . ). purpose: evaluation of uveal melanoma imaging characteristics in ultrasound, computed tomography and magnetic resonance imaging and correlation of those findings suggestive of melanoma. material and methods: eleven patients ( men, women, age - years) with possible diagnosis of uveal melanoma were evaluated. the patients underwent ophthalmoscopic and ultrasonographic examination followed by ct and mr examination. ct imaging was performed with mm axial pre-and post-contrast and coronal post-contrast sections. axial and coronal mm t and t images were obtained, followed by post-contrast t images in axial and coronal direction. additional t fat-sat post contrast images were also obtained depending on the relative position of the melanoma in order to evaluate possible extra-orbital extension. results: of the melanomas, six were located in the left globe, ranging from mm to mm in size and most commonly on the lateral wall. the tumors appeared as hyperechoic lesions on us with a reflectivity range of - %, suggestive of melanoma. retinal detachment was detected in four patients. on ct images the melanomas appeared as homogenous slightly enhancing lesions, while in two patients, the tumor was less obvious due to the retinal detachment. on mr images the tumors appeared as homogenous hyperintense lesions on t wi in five patients. characteristic signal void on t wi due to melanin deposition was noted in three patients. conclusion: ultrasonography appears to be a valuable tool in tissue differentiation of uveal melanoma although magnetic resonance, and to a lesser degree, computed tomography are more specific in evaluating the extra-orbital extension. purpose: gadobenate dimeglumine (gd-bopta: t relaxivity in blood = . mmol - s - ) was evaluated to determine whether the high relaxivity offers advantages over other agents for cns imaging. methods: preliminary studies evaluated gd-bopta at cumulative doses up to . mmol/kg to ascertain the optimal dose for mri of cerebral metastases. two phase iii controlled trials were conducted to compare gd-bopta at cumulative doses up to . mmol/kg with gadodiamide (gd-dtpa-bma) at cumulative dose up to . mmol/kg. two intra-individual, blinded, fully randomized comparative studies were conducted in patients with cerebral gliomas or metastases to determine differences in quantitative (tumor contrast, cnr ratios) and qualitative (lesion contrast, delineation, internal morphology, structure) enhancement parameters versus gadopentetate dimeglumine (gd-dtpa) and gadoteric acid (gd-dota) at a dose of . mmol/kg. in patients with metastases a dose of . mmol/kg gd-bopta proved sufficient for most situations while a cumulative . mmol/kg dose provided additional information in certain cases. comparative studies revealed that . mmol/ kg gd-bopta offers improved performance compared to . mmol/kg gd-dtpa-bma and that cumulative . mmol/kg gd-bopta is equivalent to cumulative . mmol/kg gd-dtpa-bma. the intra-individual comparative studies revealed superior qualitative enhancement performance for gd-bopta compared to both gd-dtpa and gd-dota for all evaluations. the subjective assessments were confirmed by the objective measurements of signal intensity and cnr which were significantly higher for gd-bopta (p < . ). discussion: gd-bopta is a safe and valuable contrast agent for the assessment of cns neoplasms offering statistically significant advantages over gd-dtpa, gd-dota and gd-dtpa-bma for brain lesion enhancement. the in cases the examinations were performed before the treatment, in patients as an assessment of drug treatment. in cases the efficacy of stereotactic effect (se) was assessed on the suitable target structures. results: a metabolism decrease (md) in the right caudate head (ch) was noted in two patients before drug treatment. in two patients hypermetabolism was observed in the anterior cingulate (ac). follow-up examinations revealed a md in the ac and an metabolism increase (mi) in the right ch in these patients. in one case an mi in both dorsal thalami (dt) was observed. a md in both dt before drug treatment was seen in two cases. a bilateral mi was noted in dt by followup examinations in these patients. in all cases of se, it revealed a md in suitable structures. in cases, a md was observed in ac and both the ch. in two cases, a md was seen in the ac, in a bilateral md in the dt. changes of glucose metabolic rate were associated with severity of clinical manifestations in all cases. this data proves participation of limbicostriatal system in forming aod. our results demonstrate the possibility to use f-fdg pet for aod diagnosis and estimation of treatment efficiency. b d e f a g magneting resonance imaging in patients with intractable partial seizures: a preoperative assessment a. lefkopoulos, a. kalogera-fountzila, c. kouskouras, n. fotiadis, g. spanos, e. papadopoulou, a.s. dimitriadis; thessaloniki/gr purpose: the aim of this study was to assess the prevalence of detected structural abnormalities in medically intractable epilepsy, especially those who are amenable to a surgical therapy, and also to evaluate the sensitivity of different dedicated mr sequences. materials and methods: one hundred and twenty patients (aged months- years) with medically intractable partial seizures underwent an mri examination in a . t unit, with t and pd/t se in an axial plane and fluid attenuated inversion recovery (flair), t fse and inversion recovery (ir) in a plane perpendicular to the long axis of the hippocampus. whenever necessary, we applied t se + gd dtpa in axial and coronal planes. results: seventy-nine of the patients had a pathological scan ( . %). abnormalities were hippocampal sclerosis (hs) in patients ( %), cerebral tumors in patients ( %), vascular malformations in patients ( . %), cortical infarcts in patients ( . %), cerebral infections in patients ( . %) and patients with developmental disorders ( cortical dysplasias, pachygyrias, polymicrogyrias, subcortical heterotopias and patient with tuberous sclerosis). all patients with cerebral tumors and eight patients with hs were operated and there was a pathologic confirmation of the diagnosis. patients with vascular malformation underwent a therapeutic embolization. flair had higher sensitivity in detecting hippocampal sclerosis and cerebral infarcts while ir was particularly useful in detecting developmental disorders. conclusions: mr imaging, with an epilepsy dedicated protocol, plays a cardinal role in locating and characterizing anatomic epileptogenic foci in patients with refractory epilepsy and helps in defining the type of treatment. the purpose: the aim of this study was to evaluate the possibilities of combined pet/ct imaging in detecting glioblastomas and in estimation of efficiency of their surgical treatment and radiotherapy. materials and methods: seven patients ( men, women, age between - years) with biopsy-proven glioblastomas were scanned before and/or after treatment by pet/ct (biograph, siemens). a dose of to mbq fluorine- fluorodeoxyglucose (fdg) was administered for each pet scan with an acquisition time of minutes for bed and interactive reconstruction. acquisition time for ct was seconds, bringing the total acquisition of combined pet/ct study to minutes. results: three patients with high-grade astrocytomas demonstrated hypometabolic fdg nodules without contrast enhancement at ct scan before surgery. two patients showed nodules with hypermetabolism of fdg (suv - ) and intensive enhancement of contrast medium due to the recurrence of tumor after combined therapy. in one patient, after radiation therapy, the region of treatment had decreased uptake of fdg and increased enhancement of contrast medium at ct because of radionecrosis. conclusion: it was confirmed that combined pet/ct study could effectively determine whether a lesion was malignant and provide evidence of tumor recurrence. the collection of prominent medullary (white matter) veins appears like a "medusa head" on mr angiographic images and gadolinium-enhanced d-tof sequences; this sign is considered pathognomonic. conclusion: mri is of great value in the accurate identification and morphologic specification of vascular abnormalities. traditionally, these cases required invasive diagnostic imaging. va correct identification is very important because this anomaly is asymptomatic but the ablation can induce venous infarctions. abdominal imaging findings: gastrointestinal tract (gi), hepatobiliary system and the pancreas are involved in cf. the gi complications include obstructive (distal intestinal obstruction syndrome, intussusceptions, fibrosing colonopathy, post surgical adhesions) and non-obstructive conditions (pneumotosis intestinalis, gastroesophageal reflux, duodenitis, and rectal prolapse). liver pathology includes steatosis, cirrhosis, and portal hypertension. the pathological mechanism of cirrhosis may be due to obstruction of the biliary ductules with thickened secretions and secondary inflammation producing fibrosis. biliary tract abnormalities include gallstones, micro gallbladder, and sludge. excessive loss of faecal bile acids due to pancreatic insufficiency is associated with increased lithogenicity of the bile. the pancreatic abnormalities include fat infiltration, fibrosis, atrophy and occasionally cysts and calcification. the primary problem is the plugging of the intralobular ductules by inspissated viscid mucus. conclusion: abdominal symptoms in patients with cf may be due to the various causes related to cf or may be due to coincidental pathology e.g. appendicitis, renal disease. the radiologist should keep an open mind and not restrict his thought process only to the conditions related to cf. neck masses in infants and children: a pictorial review j.r.a. turkington, a. paterson, l.e. sweeney, g. thornbury; belfast/uk learning objectives: this poster presents a pictorial review of pediatric neck masses and their imaging features. particular emphasis is applied to the anatomical site of the mass to aid in differential diagnosis. background: infants and children with neck masses frequently present to the radiologist for further evaluation. the most common aetiologies include congenital lesions and their complications, lymphadenopathy and malignant masses. the role of the radiologist is to differentiate between these conditions using im-aging modalities such as ultrasound and colour doppler, ct and mri. where appropriate, the radiologist will also stage lesions for management purposes, and aid in guiding aspiration or biopsy. imaging findings: the radiological appearance of congenital lesions such as thyroglossal duct cysts, branchial cleft cysts, cystic hygromas, haemangiomas, dermoid cysts and sternomastoid "tumours" are discussed. cervical lymphadenitis and abscesses, and neoplastic masses such as neurogenic tumours, rhabdomyosarcoma, neuroblastoma and lymphoma are also described. lastly, lesions of the thyroid and salivary glands are evaluated. it must be emphasised that the radiological findings should always be interpreted in conjunction with the patient's age, the clinical history and the findings on physical examination. conclusion: neck masses are common in children and a frequent cause of attendance at a&e departments. identifying the anatomical site and radiological appearance aids the differential diagnosis. conclusion: a typical osteoarticular lesion in a typical site can alert you to the diagnosis of sapho, but an unusual site or a primary presentation with no skin lesions will make diagnosis harder or there may be misdiagnosis. children may be seen in a number of specialties, which can lead to unnecessary delays in diagnosis and possible incorrect treatments. this makes the radiologist pivotal in the diagnosis of sapho, although the differential diagnosis should always be considered. to demonstrate the problems which can be caused when trauma x-ray films are incorrectly marked when using the red dot system. background: many radiographs are returned to accident and emergency departments without formal radiological reports, and the red dot system, whereby radiographers attach a red dot to radiographs which they suspect to be showing a fracture, is a widely practised system, used throughout the national health service. it is acknowledged that whilst this system can be beneficial to accident and emergency departments it is not without its problems. procedure findings: our poster examines the problems which can arise in children where normal variants are often highlighted as "fractures" by radiographers. most of the common misinterpretations occur in the appendicular skeleton, e.g. normal variants of epiphyseal ossification or accessory epiphyses, especially of the metacarpal and metatarsal bones. other common "errors" include the pisiform, or os trigonum and accessory ossification centres for the medial and lateral malleoli. in the axial skeleton, vascular markings in the skull, facial bone sutures and end plate ossification centres for the vertebral bodies are some of the commoner areas of concern. conclusion: issues are raised for both accident and emergency departments and for radiology departments if these "errors" are not minimised, including patient complaints of over-diagnosis and potentially unnecessary treatment and follow-up visits. the radiology of pediatric extracorporeal membrane oxygenation: normal appearances and complications a.m. barnacle, m.p. hiorns; london/uk learning objectives: to demonstrate the normal appearances of the pediatric chest during extracorporeal membrane oxygenation (ecmo). the spectrum of radiological abnormalities arising from the complications of ecmo is illustrated, including cannula position and anticoagulation complications. background: ecmo is a means of cardiopulmonary bypass therapy used in patients with cardiorespiratory failure who are unresponsive to conventional intensive care interventions. there is a range of indications for ecmo therapy in the pediatric population, which include congenital diaphragmatic hernia and cardiac disease. there are a number of complications associated with the use of ecmo, many of which are related to the high levels of anticoagulation required during therapy. radiological assessment prior to the commencement of treatment and sequential imaging during therapy are mandatory, and appropriate imaging protocols are discussed. imaging findings: the normal radiological appearances of the chest during ecmo therapy are demonstrated, including the correct positions of both va and vv ecmo cannulae. several technical points are highlighted, to avoid the misinterpretation of certain radiographic appearances. the radiological manifestations of a wide range of complications are illustrated. these include intracranial haemorrhage and extra-axial collection, haemothorax, peritoneal collections and vascular complications. inappropriate cannula positions are also reviewed. the radiologist must be familiar with both the normal and abnormal imaging appearances during ecmo therapy and should be aware of the range of potential complications. the importance of sequential imaging is highlighted and appropriate imaging protocols discussed. acute disseminated encephalomyelitis or multiple sclerosis: what tips the balance? s. avula, a. ghatak, n. wright, r. appleton; liverpool/uk learning objectives: to identify the imaging features and clinical parameters which help to distinguish between acute disseminated encephalomyelitis (adem) and multiple sclerosis (ms) in children. background: both adem and ms are disseminated inflammatory disorders affecting the central nervous system. distinction between the two is difficult, especially at the initial presentation when the prognosis and risk of relapse is an important issue. this exhibit will highlight the salient distinguishing features stated in the literature and illustrate cases of adem and ms which presented over a three year period. procedure details: literature review of childhood adem and ms was carried out and a retrospective review of cases presenting over a three year period with a subsequent diagnosis of adem or ms was performed. there is a considerable overlap between the imaging and clinical features of adem and ms. though there are a few features that predominate in either condition, the temporal progression of the disease with presence of new lesions with or without relapse of symptoms as noted in ms, is the most important factor in distinguishing the two. mri in assessment of fetal renal anomalies j.l. hughes, w. jan; london/uk introduction: in patients with renal anomalies causing severe oligohydramnios, the lack of fluid around the fetus significantly degrades the ultrasound image, making assessment of the fetal abdomen difficult. the presence of renal anomalies has great postnatal significance and successful antenatal counselling depends on the availability of the most complete information. the advent of fast mr sequences has allowed the acquisition of high signal to noise ratio images of the fetus even in the absence of amniotic fluid. we discuss the technique of mr of the fetal renal tract. we describe the normal appearances fetal kidneys on mr and present data on illustrative cases where mr provided additional information over and above a detailed ultrasound examination. methods: t w single-shot rapid acquisition with relaxation enhancement (rare) and t w d fast low angle shot (flash) were obtained of the fetal kidneys in the three orthogonal planes. all images were acquired without fetal sedation with the mother in the supine position within minutes. comparison ultrasound and mr images are presented. results: mri is able to diagnose and assess a variety of renal anomalies such as renal aplasia, dysplastic renal tissue, polycystic kidney disease, congenital hydronephrosis, and ectopic renal tissue. conclusions: fetal mr offers further information over ultrasound examination and hence allows appropriate ante-natal counselling. pediatric urethral anomalies a. sharma, s. maroo; glasgow/uk imaging evaluation of neonatal cystic pelvic masses v.v.t. mascarenhas, a. duarte, l. lobo, j. fonseca-santos; lisbon/pt learning objectives: to describe imaging features of cystic pelvic masses (cpm) in the newborn. to outline main differential diagnosic pitfalls. to emphasise the role of imaging evaluation in treatment and follow-up orientation. background: neonatal cpm are among the most common abdominal masses in the neonate and usually first suspected during fetal ultrasonography. a more precise diagnosis in the neonatal period is essential to an adequate orientation in treatment and follow-up. on this regard, imaging evaluation, particularly us and mri as the main techniques, undertake a pivotal role. we describe diagnostic imaging features, differential diagnosis limitations and a systematic diagnostic approach to cpm among selected cases in our department. imaging findings: we reviewed the referred cases based on sex, location, relation to adjacent anatomic structures and morphological features. main diagnostic entities considered were pre-sacral (sacrococcygeal teratoma and myelomeningocele) and non pre-sacral masses (genitourinary related namely hydrocolpos, ovarian cyst, bladder diverticulum; digestive tract related e.g. duplication cyst; others, particularly lymphangioma). morphological imaging findings according to their uni-or multilocularity, simple or complex components and the presence of specific findings were considered. ultrasound was the first and sometimes the only imaging modality performed. selected cases were further evaluated with other imaging modalities such as conventional contrast studies (e.g. cystography) or mri. conclusion: although there is a wide range of entities presenting as a cpm in the neonate, a systematic imaging approach allows a more precise diagnosis or at least a considerable narrowing of diagnostic possibilities in the majority of cases, which is essential for therapeutic orientation. normal sonographic appearance of kidneys and adrenals in neonates and young infants: unique distinctive features m. vakaki, g. pitsoulakis, h. manoli, c. koumanidou; athens/gr learning objectives: to familiarize residents in radiology or pediatric radiology with the sonographic characteristics of neonatal and infantile kidneys and adrenals. backround: the kidneys are the most commonly sonographically examined abdominal organs during the first months of life. the adrenals, due to their position adjacent to kidneys, are almost always clearly visualized during the renal sonogram. their sonographic appearance is quite different from that in older children and adults and often causes misleading incorrect diagnoses. imaging findings: examples of various normal sonographic findings of the kidneys and adrenals in neonates and young infants are presented and embryologically, anatomically or functionally explained. they include the hyperechogenicity of the renal cortex, the "enlarged" hypoechoic pyramids, the fetal lobulation, the inter-renicular septum, the transient neonatal renal medullary hyperechogenicity, the papillary projections in the pyelocalyceal system, the renal pyelectasis, the large and hyperemic neonatal adrenals and even more. these sonographic interpretations are often misdiagnosed as abnormal conditions, which are also provided. conclusion: this exhibit will allow an interesting way for young radiologists and pediatric radiologists to learn the common, but also the rare sonographic appearances of neonatal and infantile kidneys and adrenals. this knowledge is essential in order to prevent misdiagnoses and needless further investigation. background: difficulties with blind corticosteroid injection of the subtalar joint are commonly encountered in children with jra. ultrasound (us) guidance can make the puncture more accurate by directly showing the needle tip within the joint. procedure details: twelve consecutive patients with jia who had clinical signs of active arthritis and intraarticular fluid or synovial proliferation in the subtalar joint detected at us received either a blinded injection (n = ) or a us-guided (n = ) injection with free-hand technique using high resolution ( - mhz) us. patients were assessed at hours, months and months of follow-up to compare the level of clinical response and the frequency of complications between the two treatment groups. conclusion: although significant improvement was documented in both groups of patients with respect to baseline, those injected under us-guidance had a lower clinical dysfunction score at hrs and months follow-up. no major complications were noted. our preliminary results indicate that intraarticular corticosteroid injection of the subtalar joint in children with jia is more accurate and successful when performed under us guidance. background: doppler sonography is a well-established technique to evaluate a number of pediatric abdominal conditions, but the field of diagnostic applications is now on the brink of major changes. many contrast-specific modalities have been developed in recent years by academic researchers, ultrasound scanner manufacturers, and pharmaceutical companies. our aim is to update the diagnostic value of conventional doppler modalities, and to analyse potentially useful applications and indications for the more recently developed techniques. imaging findings: specific entities will be used to illustrate clinical principles that can be applied to multiple clinical situations. topics addressed include: vascular patency (arterial or venous thrombosis or stenosis, biliary atresia, liver and bowel transplantation, and surgical shunts); anomalous vessels and tubular structures (dilation of the biliary tree vs. blood vessels, pulmonary sequestration, varicocele); and evaluation of hyperemic and ischemic lesions (appendicitis, intussusception, pyelonephritis, orchitis / testicular torsion). conclusion: b-mode sonography, as well as doppler techniques, have a key role in pediatric diagnosis for several reasons (non-invasivity, lack of ionizing radiation, sonographic access is better than in adults, no need for anesthesia or sedation). understanding the physical principles that govern doppler imaging is essential for its successful clinical application. symptomatic cases: n = . mean onsd right eye = . mm (sd = . ), mean onsd left eye = . mm (sd = . ). a wilcoxon signed ranks test showed no significant difference between these results. using the largest reading for each eye when well and symptomatic: mean asymptomatic onsd = . mm (sd = . , n = ), mean symptomatic onsd = mm (sd = . , n = ). a wilcoxon signed ranks test showed a significant difference in these results (p = . ). conclusion: onsd increases in acutely raised icp. our results suggest that children with spina bifida and shunted hydrocephalus cannot be assumed to have onsd within the normal range even when clinically well. in the long term we aim to compile new onsd reference ranges for children with shunted hydrocephalus. withdrawn by authors hyperechoic foci in the kidney: an incidental sonographic finding in neonates and infants m.a. pourbagher, a. pourbagher, f. tiker, z. koc, e.a. niron; adana/tr purpose: the purpose of this study was to determine the incidence and significance of non-shadowing hyperechoic foci in the kidney and to evaluate metabolic disturbances in these patients. in a period of years abdominal ultrasonography (us) was performed as a screening procedure in neonates and infants. a followup us was performed in of ( female and males) patients with hyperechoic foci in the kidney approximately - months after the initial examination. all patients underwent metabolic evaluation, including serum electrolyte measurement and -hour urine collection. results: non-shadowing hyperechoic foci were detected in ( %) patients ( kidneys). the size of foci were - mm (mean mm). the foci were located in the lower pole calix in %, upper pole calix in % and middle zone calix in % kidneys. sixty kidneys had single, had and had or more foci. at - months follow-up the clearance for foci was %. size and site of foci has no relation with foci clearance. urinalysis showed elevated urinary calcium in and hematuria in patients. in patients foci were passed without intervention, and uric acid was the dominant foci composition in both patients. serum electrolyte measurement were normal in all patients. the incidence of hyperechoic foci in our study was %. that is usually discovered incidentally during sonography of the abdomen and generally resolve spontaneously. follow-up of the patients is advocated with us and urinalysis. germinal matrix morphology in antenatal ventricular dilatation k.m. koprivsek , t. vanderheyden , s. counsell , j. allsop , a. stonebrige-foster , s. kumar , n. fisk , m.a. rutherford ; sremska kamenica/yu, london/uk purpose: the germinal matrix (gm), a densely cellular zone lining the lateral ventricles, and give rise to the developing cortex. the mri appearance of gm during fetal development has been well-documented, but data about gm morphology in pathological conditions is sparse. antenatal ventricular dilation (vd) is associated with significant neurological morbidity. we used antenatal magnetic resonance imaging (mri) to measure the gm in fetuses with and without vd. methods: retrospective analyses were conducted of fetal mr images acquired over a -year period, using fast t weighted sequences. in fetuses (normal ventricles in , vd in cases), we evaluated the signal characteristics and the maximum germinal matrix diameter (gmd) at three sites: the anterior and posterior ventricular horns and the caudothalamic notch (ctn). between-group comparisons of gmd in normal and fetuses with vd were made using a students t-test. results: gm had the same low signal intensity regardless of ga and/or ventricular volume. none of the infants with vd had evidence of gm haemorrhage. the mean gmd in controls showed a linear decrease with brain maturation. in fetuses up to weeks of ga with normal ventricles the gmd was greatest at the ctn. the similar pattern of changes in gmd was measured in vd group, where the mean gmd at the tcn were slightly, but not significantly lower then in controls (t = . : p > ( . )). conclusion: gm diameter decreased with increasing gestational age. we did not detect any significant difference in gm morphology in the group of fetuses with antenatal vd. contrast enhanced color doppler ultrasonography in diagnosis of scrotal lesions in children u. zaleska-dorobisz, e. czapiga, k. moron; wroclaw/pl purpose: to investigate the usefulness of ultrasound with contrast agent levovist-schering (usca) for the detection scrotal lesions in children. between - , boys with scrotal masses were examinated. mean age was . years and ranged from - . diagnostic assessment included in all cases conventional us, color doppler and power doopler before and after the adminstration of contrast agent keeping the same methodology in every examined patient. focal testicular lesions (cystic and solid tumor) and extratesticular structures were documented regarding size, localization, range, vascularity and ultrasound diagnosis. in testicular tumors, the degree of tumor vascularity was subjectively graded from to iv. the ultrasound diagnoses were compared to the final diagnoses based on clinical tests, surgical and histological examination. results: testicular and extratesticular tumors showed more enhancement than adjacent testicular parenchyma, seen between to s (mean s) and to s (mean s) after injection. the duration of enhancement was to s (mean s). cross sections and adjacent vessels were only seen in: teratoma, embryonal carcinoma and yolk sac tumor. conclusions: usca is an important and significant aid in the diagnosis of scrotal diseases, in the assessmentof of degree of vascular change and is predictive of malignancy. mr features of the preterm children with athetotic cerebral palsy k. hayakawa, t. kanda, y. yamori; kyoto/jp purpose: the recent development of the neonatal intensive care medicine has caused not only a dramatic decrease in the incidence of kernicterus, but also marked improvement in the survival of premature babies. consequently, the incidence of pure athetotic cerebral palsy has decreased, whereas the incidence of spastic cerebral palsy associated with athetosis, termed mixed cerebral palsy, has increased. the purpose of this study is to evaluate the brain mr features of this condition and to assess the frequency of basal ganglia injury. the subjects consisted of boys and six girls diagnosed with mixed cerebral palsy. their gestational age range from to weeks (average = weeks) and the birth weight ranged from g to g (average = g). mr brain scans were assessed with particular attention to the basal ganglia. imaging of pancreatic disorders in pediatric patients y. herrero, t. berrocal, c. prieto, j. fernández, m. parrón, r.r. lemos; madrid/es purpose: the aim of this teaching exhibit is to illustrate a wide spectrum of usual and unusual disorders involving the pancreas in children, and to evaluate the efficacy of ultrasound (us), duplex and color doppler sonography, endoscopic retrograde cholangiopancreatography (ercp), computed tomography (ct) and magnetic resonance (mr) imaging in the diagnosis and management of these conditions. methods and materials: the imaging findings of patients with disorders involving the pancreas were reviewed from our database of pediatric pancreatic pathology. ultrasound was performed in all cases, while ct, and mri were performed in all tumors and whenever us and doppler images were not conclusive. ercp was used to study the pancreatic ducts. the embryologic and pathologic bases of the imaging findings are discussed in appropriate cases. the key findings at each imaging modality are shown and compared with the underlying pathologic features when available. pitfalls, diagnostic difficulties and differential diagnoses are emphasized. results: specific topics addressed include congenital anomalies (pancreas divisum, annular pancreas, ectopic pancreas, pancreatic cysts, cystic fibrosis, shwachman-diamond syndrome, von hippel-lindau syndrome, and nesidioblastosis); acute and chronic pancreatitis; pancreatic cyst and pseudocysts; primary neoplasms (insulinoma, cystadenoma, and papillary cystic carcinoma); metastasis of burkitt lymphoma; and miscellaneous (hemochromatosis, cushing's disease, steroid and cytostatic therapy, and parenteral nutrition). conclusion: evaluation of patients with pancreatic disorders frequently requires multiple imaging modalities for diagnosis and planning surgical correction. because many of these disorders have characteristic imaging appearances, this exhibit will help practicing radiologist to better understand and recognize pathologic processes affecting the pancreas. imaging congenital vertebral disorders in pediatric patients m. fernández -velilla, t. berrocal, a. royo, a. fernández, j. gutierrez, n. gómez-león; madrid/es purpose: to illustrate a wide spectrum of usual, unusual and exceptional congenital anomalies involving the vertebrae, and to evaluate the efficacy of plain radiographs, computed tomography (ct) and magnetic resonance (mr) imaging in the management of these conditions. methods and materials: we retrospectively reviewed the imaging findings of patients with congenital abnormalities involving the vertebrae from our database of spinal pathology. abnormalities were divided into isolated vertebral anomalies and anomalies associated with systemic diseases or syndromes. plain radiographs were performed in all patients. ct or mr were performed whenever plain radiographs were inconclusive. the embryology and pathologic basis of the radiographic findings are discussed. pitfalls, diagnostic difficulties and differential diagnoses are emphasized. results: specific topics addressed include anomalies in number (vertebral agenesis, sacral agenesis, omovertebral bone); anomalies in size and shape (vertebral and sacral hypoplasia, hemivertebra, occipitalization of c , os odontoideum, lumbarization, sacralization); anomalies of fusion (blocked vertebra, klippel-feil abnormality, spina bifida, butterfly vertebra, coronal cleft vertebra); anomalies of alignment (spondylolisthesis, spondilolysis, scoliosis); neural arch defects (absence of the pedicle and lamina); caudal spinal anomalies with anorectal and urogenital malformations (terminal mielocystocele, caudal regression syndrome, vacter association, oeis complex, currarino triad); and skeletal dysplasias (achondroplasia, thanatophoric dwarfism, chondrodysplasia punctata, spondyloepiphyseal dysplasia, and mucopolysaccharidoses). conclusion: evaluation of patients with vertebral abnormalities frequently requires multiple imaging modalities for diagnosis and planning treatment. because many of these disorders have characteristic imaging appearances, this exhibit will help trainee radiologist to better understand and recognize pathologic processes affecting the vertebral spine. defecography in chronically constipated children a.d. yang, m.-t. cheng, k. lee; changhua/tw purpose: to evaluate the defecographic findings in children suffering from chronic constipation. between may to august , we performed a prospective study of children ( boys, girls) with an age range of - years old who fit the criteria for chronic constipation in our institution. defecography was performed using flouroscopy with radiographs were taken during different phases of defecation; the whole study was video recorded. results: of the children studied ( ) children were found to have abnormal defecographic findings: partial relaxation to non-relaxation of the puborectalis muscle on defecation was found in patients; anterior rectocele in , posterior rectocele in , rectal intussusception in , mucosal prolapse in , rectal prolapse in ; sigmoidoceles in : grade i ( ), grade ii ( ), grade iii ( ). conclusion: . the defecographic findings that are seen in adults could also be found in children. . defecographic evaluation could be a potentially valuable and simple radiologic study which could be used in the evaluation of children suffering from chronic constipation. evaluation of the usefulness of d ct reconstructions of multifragmented fractures in children m. wozniak, e. dybiec, p. wieczorek; lublin/pl purpose: the aim of the study was the evaluation of the usefulness of d ct reconstructions in the imaging of post-traumatic alterations of the bones in children. the analysis was based on three dimensional ct reconstructions of the fractured bones. there were cases of extensive, complex bone fractures, when evaluation based on conventional radiographs was insufficient. all examinations were performed on a shimadzu sct tx scanner. surface and volume rendering were employed in the processing. the results of the study show that d reconstructions allow very precise evaluation of post-traumatic alterations, especially in the bones of complex anatomical structures. they enable the imaging of small fracture fissures invisible on conventional radiographs and ct. in the cases of bone indentations d reconstructions allow very detailed evaluation of the depth of indented fragments, their displacement and rotation. results: ct examinations were performed over the nine month study period. examinations using tube current of more than % greater than protocol were noted. these reduced from % of examinations prior to the introduction of the protocol to % following its introduction. reductions were most noticeable in helical examinations of thorax, abdomen and pelvis. conclusion: weight related protocols reduce the tube current used and thus the radiation dose in a large percentage of paediatric ct examinations. this has proved to be an effective method of reducing dose both in this study and in previous studies. bone sarcomas as a second malignancy in children c. renal dysplasia and intrarenal reflux in boys with posterior urethral valves: high resolution sonographic findings m. vakaki, g. pitsoulakis, h. manoli, c. koumanidou; athens/gr purpose: voiding cystourethrography (vcug) is considered the gold standard for the diagnosis of the posterior urethral valves (puv) and the accompanying vesicoureteral reflux (vur) in boys. however, its contribution to the characterization of intrarenal pathology, which is considered to be of great prognostic value, is minimal. it is only with recently introduced high resolution ultrasonographic equipment that details of disturbed renal architecture can be visualized. materials and methods: infant boys with puv, aged day to months were examined sonographically with - mhz convex and - mhz linear transducers. the presence of puv was confirmed with vcug in all cases. results: apart from the classic sonographic findings of puv, which were also demonstrated, findings specific to the kidney included: a) bilaterally increased renal parenchymal echogenicity and loss of corticomedullary differentiation ( / ), which with b) small cortical cysts ( / ) imply renal dysplastic lesions, c) increased medullary echogenicity, cupping of dilated calyces, which is attributed to intrarenal reflux ( / ), d) hypoechoic lines pointing towards rounded calyces, corresponding to the pelvotubular backflow of urine ( / ), e) small subcapsular urinomas ( / ), f) urinoma and urine ascites ( / ) and g) intrarenal abscesses ( / ) and fungal balls in the collecting system ( / ), due to post-surgical candidiasis of the urinary tract. conclusion: ultrasonography with high-resolution transducers is able to visualize underlying renal pathology and to image the parenchymal architecture, contributing not only to the diagnosis but also to the definition of the prognosis in boys with puv. meningiomas within the vertebral column are even rarer. we present the imaging characteristics of meningiomas ( intracranial and intracanal) in girls and boys, aged . - years (mean age . years). these meningiomas were diagnosed during year period ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . none of the studied children had previously been treated with radiotherapy. both children with vertebral meningioma suffered from neurofibromatosis. the imaging studies included brain ct ( / ) and mr of the brain ( / ) and vertebral column ( / ) before and after intravenous administration of gd-dtpa. the histologic examination after the surgical tumor resection showed meningioma of the transitional type in children and co-existing malignant elements in the th child. following tumor resection, children were free of symptoms and no other treatment was required. in contrast, the patient with the meningioma with malignant elements was treated with chemotherapy and radiotherapy, after subtotal tumor resection. conclusion: imaging of meningiomas (intracranial and intracanal) provides essential information for surgical planning, demonstrating the extraparenchymal nature of the neoplasm, its location, its dimensions, possible calcifications, as well as its effects on adjacent anatomic structures. rare hepatic vascular anomalies in pediatric patients z. harkanyi, g. balazs, g. tasnadi; budapest/hu purpose: to present a selection of rare cases of hepatic vascular anomalies in pediatric patients. us, helical ct and mr were used in the diagnosis and in the follow-up of these lesions. various venous anomalies were detected including intrahepatic arterio-portal and porto-hepatic shunts. a case of porto-caval shunt was also identified. the most frequent anomaly was portal cavernoma due to portal vein thrombosis. one case of caval web has been followed up for years. another case of budd-chiari syndrome has been followed up for two years. several cases of hepatic and portal vein anatomic variations are also documented. d and color doppler us was used as the first line examination in the diagnosis and follow-up different congenital hepatic vascular anomalies. helical ct, cta and, more recently, mra has been used for further analysis of these lesions. conclusions: us is still the first method for the evaluation of the pediatric liver and the hepatic vessels. color doppler is the modality of choice for functional evaluation of the vascular anomalies and is also useful in the follow-up of the patients. contrast-enhanced mra and cta is helpful in selected cases to demonstrate the extent of the lesions and other associated anomalies, such as development of systemic collaterals. acute [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , (n = ) children with septic sacroiliitis and/or acute osteomyelitis of the pelvic bones were imaged by our radiology department. other laboratory tests were already performed and in ( / ) young patients, material from needle aspiration had been sent for cytological examination. the imaging initial approach included plain film study and triple-phase tc radiophosphate scan. in addition, ( / ) patients underwent mri examination and ( / ) children had ct. the laboratory results confirmed the diagnosis of osteomyelitis and/or that of septic sacroiliitis. the retrospective study of the imaging examination results indicated the superiority of scintigraphy and of mri in the diagnosis of these entities. plain film radiographic signs of disease were observed only in patients ( / ) though the bone scintiscan was positive in all patients ( / ). the ct examination was positive in young patients ( / ) while mri's sensitivity was % ( / ). at the end of therapy all patients recovered completely. conclusion: a bone scan is warranted in every case of clinically suspected osteomyelitis and/or septic sacroiliitis in which the radiographs are unrevealing. however, mri is a useful adjunct in the evaluation of the bone edema, the detection and localization of abscess and in the demonstration of the soft tissue extent of the infectious process. the results: hrct showed that / of the cases had bronchiectasis, with cystic fibrosis as the underlying cause in cases. cases had bronchiolitis obliterans and cases had macleod's syndrome. cases with aspiration pneumonia had gastroesophageal reflux, and a intrabrochial foreign body was detected in one case. one case had bronchopulmonary dysplasia, and had tb lymphadenitis compressing bronchi. chest radiography revealed findings of bronchiectasis only in of the cases. hyperaeration was detected in the chest radiographs of only cases out of , whose hrct revealed air trapping. hrct showed peribronchial thickening in cases, however, chest radiography showed the same finding in patients. hrct made it possible to observe linear densities caused by linear atelectasis / scars in cases. hrct showed the underlying pathology and consequent pulmonary lesions in out of cases, and linear densities in . conclusion: hrct has a significant role in detecting the underlying abnormalities and consequent lesions in children with recurrent pulmonary infections. compared to chest radiography, hrct gives much more information. analysis of doppler signals: application for detection of fetal movements a. kribeche, s. benderbous, m. berson, d. kouame, l. pourcelot; tours/fr the objective is to develop and validate a new doppler ultrasound system allowing the imaging of fetal movements in real time. we describe the prototype of new ultra-sound sensors and present the results obtained in pregnant women. the apparatus enabling us to acquire the signals coming from fetal activity consists of ultrasonic sensors, a doppler module and a computer provided with a chart of acquisition of the signals. after suitable recording and signal processing, fetal heart rhythm (fhr) and the fetal breathing movements (fbm) are extracted. study of the doppler signals after fast fourier transform enables us to characterize the fetal movements in term of the amplitude, displacement and frequency. estimate of displacement is obtained by calculating the phase of the signal resulting from the analytical filtering of the doppler signal and to separate the direction from the doppler signals. results: twenty recordings were carried out on four women in their last quarter of pregnancy. during the last quarter of pregnancy, the fhr is approximately beats.min - . however, the fhr that we detected is about beats.min - . we are thus in the presence of the fetal heart rhythm. conclusion: this is the first time that parameters giving the fetal movements are extracted. as a first approach, the fft gives satisfactory results. assesment of skeletal age with hand and wrist sonography: could a standardised method replace radiography? s. megremis, g. cavallo, m. michalakou, e. kehagias, n. segkos, e. agianniotakis, e. sfakianaki; iraklion/gr purpose: to investigate the capability of hand and wrist sonography in skeletal age assessment, and to propose a technique that could be commonly used. materials and methods: forty-two children, aged months to years, underwent a left hand radiograph for skeletal evaluation. they were also sonographically examined on the same hand, with focus on distal epiphyses of radius and ulna, carpal ossification centers, adductor sesamoid bones of metacarpophalangeal joint of the wrist and the cartilage of the third's finger distal phalange. scanning and measurements were obtained in both transverse and longitudinal planes, and the images were digitally recorded for further evaluation. results: all areas of interest were identified both in the left hand radiograph as well as in the sonographic examination, for each individual. furthermore by dorsal and palmar scanning of the carpal ossification centers, accurate mapping and measurements were feasible. gaining experience, and developing a common procedure, we were able to progressively minimize most technical difficulties. conclusion: hand and wrist sonography promises an accurate, safe and cost effective assessment of skeletal age. detailed knowledge of hand and wrist anatomy, and familiarization with a standardized method, as the one we propose, in acquiring and interpreting images is indispensable. in addition, the designation of sonographic charts for the evaluation of skeletal age could be possible in the future. the role of hepatobiliary scintigraphy in the differential diagnosis of neonatal hepatitis, intrahepatic cholestasis, and biliary atresia e. palócz, t. györke, k. bártfai, l. balogh, l. szönyi, i. máttyus, É. kis, t. verebély, e.k. makó; budapest/hu purpose: biliary atresia (ba), intrahepatic cholestasis (ihc) and neonatal hepatitis (nh) are the main causes of neonatal direct hyperbilirubinaemia (ndh). the distinction of ba needing early surgical intervention from nh and ihc syndromes is essential, but difficult. the impact of hepatobiliary scintigraphy (hsc) in the differential diagnosis of ndh was evaluated. methods and materials: hscs of patients (age: days- months) with ndh were included in our retrospective study of a five years period. after administration of . mbq/kg (min.: mbq) mtc-brida serial abdominal images were performed up to hours. the scintigraphic diagnoses (sd) of ba, ihc, nh and indeterminate (ind) were defined based on visual scoring of liver uptake, intrahepatic radiopharmaceutical transport and bowel activity. sds were compared with clinical diagnoses (cd), which were based on laboratory, histological or operative findings and/or clinical follow-up. results: the cds were: ba, ihc, and nh. the sds were: ba, ihc, nh, ind were correct in and false in cases, while the cd in the ind cases was ba. regarding the shortage of bowel activity as a basis for sd of ba the sensitivity and specificity of hsc in the diagnosis of biliary atresia were and % respectively. conclusion: our results suggest that hepatobiliary scintigraphy is a useful tool in distinguishing ba requiring urgent surgical intervention from other kinds of pathologic conditions causing ndh, but that it is unreliable in further narrowing of the differential diagnosis. various radiologic findings of wilms' tumor y.-w. kim , w. kim , i.-o. kim , k. yeon ; busan/kr, seoul/kr purpose: wilms' tumor is the most common pediatric renal mass. although it usually manifests as a solid intrarenal mass with a pseudocapsule and distortion of the renal parenchyma and collecting system, some atypical radiologic findings may produce diagnostic confusion. the purpose of our exhibit is to illustrate various radiologic findings of wilms' tumor. we retrospectively reviewed radiologic findings of cases, which were pathologically proven from to (the age of patients; between months and years). results: this exhibit includes: ) typical intrarenal wilms' tumor (n = ), ) wilms' tumor with vascular invasion (n = ; renal vein , ivc , right atrium ), ) wilms' tumor with perirenal extension (n = ; psoas & ln , perirenal hematoma , subcapsular hematoma ), ) wilms' tumor with extension into the urinary tract (n = ; renal pelvis , ureter & bladder ), ) wilms' tumor with metastasis (n = ; lung , bone , pleura , mediastinum , scrotum ), ) bilateral wilms' tumor (n = ), ) wilms' tumor in a horseshoe kidney (n = ), ) atypical wilms' tumor (teratoid wilms'; n = , cystic wilms'; n = , extrarenal wilms'; n = , intrapelvic wilms'; n = ). the knowledge of these various radiologic findings of wilms' tumor can be helpful in the diagnosis and management of the tumor. extracorporeal shock wave lithotripsy monotherapy for pediatric urinary tract calculi z. siric, a. slavkovic, m. radovanovic, m. mrvic; nis/yu purpose: to show the safety and efficacy of extracorporeal shock wave lithotripsy (eswl) for the urolithiasis in the pediatric age group and to evaluate the complications encountered after the treatment. material and methods: children (age range months to years) with stones underwent eswl. kub were taken on the day after treatment. if stonefree status was achieved, the patient was followed with ultrasonography and urinalysis every six months. if fragments < mm were present, follow-up was repeated every months. results: stone size ranged from . to . cm. kidneys, ureters and bladers underwent eswl sessions. retreatment was required in % patients. the mean number of sessions per child was . . mean fluoroscopy time per session was . min. the overall success rate was %, but the stone -free status was achieved in % of children. the composition of the stones were struvite and calcium oxalate in the majority of the patients. auxiliary procedures (jj stents) were used in % children. transient hematuria, ecchymoses and colic p p p p pedia edia edia edia ediatric tric tric tric tric were the complications encountered after treatment in % of children. perirenal haematoma was identified in one patient. ( %) children developed obstruction and required nephrostomy. conclusions: eswl is non-invasive procedure suitable for stones in all locations in the pediatric patients. it may be the first-line treatment for all pediatric urinary tract calculi, although the long-term effects of lithotripsy on the kidney are unknown. imaging to meckel's diverticulum (n: ), small bowel tumour (n: ), duplication enteric cyst (n: ), postoperative (n: ) and bowel wall hemorrhage (as in henoch-schonlein purpura (n: ). the enema failed to reduce the intussusception in all cases. all children underwent definitive surgery rather than inappropriate treatment by attempted radiological reduction. conclusion: abdominal sonography is the most efficient examination for the diagnosis of intestinal intussusception. various pathological lead points in intussusception can now be defined by ultrasound. secondary intussusception treatment is surgical. diagnostic value of diffusion weighted mr imaging in pediatric cerebral neurological diseases y. oksuzler, h. cakmakci, s. kurul, m. oksuzler, e. dirik; izmir/tr purpose: to detect the diagnostic value of diffusion weighted (dw) magnetic resonance (mr) imaging in different diseases involving the cerebral white and gray matter and to compare the diffusion properties with age matched normal children. conventional and dw mr imaging was performed in children with neurologic disorders, and normal control subjects. neurological disorders included neurodegenerative brain diseases (n: ), anoxic encephalopathy (n: ), acute disseminated encephalomyelitis (adem) (n: ), encephalitis (n: ), subacute sclerosing panencephalitis (sspe)(n: ). apparent diffusion coefficients (adc) were measured from the brain lesions and normal appearing white and gray matter areas in study group. normal appearing areas were also measured in the control group. results: adc values obtained from the normal subjects were similar to values described in the literature. adc values of study groups were significantly different from the control subjects. adc values for the neurodegenerative disease group, anoxic encephalopathy group, adem group, encephalitis, sspe group, were respectively between . x - - . x - cm /s, . x - - . x - cm /s, . x - - . x - cm /s, . x - - . x - , . x - - . x - cm /s. conclusion: although this study is limited due to population size and disease heterogeneity dw mri provides useful and complementary information regarding the degree of involvement in different pediatric neurological disorders. clinical presentation and mri findings in non-accidental trauma in children under two years of age p.c. sundgren , m. petrou , j. jennings , j. ksar , j. smythe , b. foerster , p. maly , p. eldevik ; ann arbor, mi/us, lund/se purpose: to describe the clinical presentation and illustrate the mri findings in suspected non-accidental brain trauma in children under two years of age. we reviewed the hospital charts and the mr reports of patients examined due to clinical suspicion of non-accidental brain trauma. we noted the clinical presentation and the mri findings in each case. both the radiological diagnoses and the final diagnoses were extracted and compared, and the impact of the mri examination on the clinical course of the patient was evaluated. results: subdural hematoma was the most common finding seen in of the patients, petechial hemorrhages and brain edema was other common findings in these patients. discussion/conclusion: non-accidental brain trauma is not uncommon and in most cases ct of the brain is the method of evaluation. mri is only performed in a limited number of cases, for example, when dating the abnormalities is crucial, and to evaluate for additional findings of axonal shearing injury. examples of this will be illustrated. congenital hip dysplasia: mri after reduction of the femoral head e. svedström; turku/fi purpose: in the treatment of developmental dysplasia of the hip, maintenance of the correct position of the reduced femoral head is very important. many modalities such as conventional x-ray images, tomography, ct and us have been used in the evaluation of the femoral head position after reduction. these techniques either use ionizating radiation or require an experienced examiner. they might even be subject to technical limitations or sometimes be inaccurate. this poster describes the use of mri in the assessment of femoral head position after closed or open reduction. mri studies with an open-configured . t mr-imager were performed on four children. some of these children ( - months of age) were studied multiple times during the follow-up. no sedation was used. coronal and axial sequences were used. position of the femoral head, appearance of the limbus and formation of the pseudoacetabulum were noted. results: all mri studies were successfully performed without any sedation. the plaster did not reduce image quality in any of the studied hips. in four still-dislocated hips mri showed correct diagnosis. in two of these hips an inverted limbus could correctly demonstrated. conclusions: accurate diagnosis is very important after reduction of the femoral head. these children commonly require multiple imaging studies during followup. therefore, mri and us, which do not use ionizating radiation, are the preferred modalities. after reduction, mri findings are easily reproduced and are accurate. even other findings like inverted limbus in interposition and formation of pseudoacetabulum can be demonstrated. b d e f a g practical experience with the application of the "digital addendum of the european protocol for quality assurance in mammography" h. bosmans , f. rogge , a.-k. carton , k. young , r. van engen , m. thijssen , g. marchal ; leuven/be, guildford/uk, nijmegen/nl learning objectives: to teach different aspects of the digital addendum (da) to the european protocol for quality control in mammography: • the purpose of the tests as described in the document. • the test methods. • the applicability of the test methods and the discussion of the first measurement results. background: digital mammography is a new imaging modality for mammographic applications. in order to assure sufficiently high quality standards, new test protocols had to be developed. a group of european medical physicists has cooperated under the coordination of the european breast cancer network and euref. the first publication is expected for october . the (pre-released) version (aug. ), work in progress, has been evaluated on different full field digital mammography (ffdm) systems and is the basis of the present teaching session. procedure details: following systems were included: senographe d (ge), senoscan (fischer), ma (fuji), ac (fuji), embrace cr (agfa) and embrace dr (agfa). the tests as described in the protocol included direct performance tests, contrast detail analysis, patient dose measurements and physical measurements such as mtf and dqe. for these last measurements, acceptable and achievable levels have not yet been defined. the help of a service engineer was necessary in most systems to get access to raw data. the tests as proposed in the protocol could then be performed. contrast detail analysis is the most critical part and should be automated to reduce interobserver variability. patient doses were comparable or lower than doses in conventional mammography. the acquisition of mtf and dqe requires a further standardization of the method. the digital addendum in its current phase was applicable to all systems tested so far. practical experience is growing and will add to the applicability and value of the document. anisotropic resolution in helical cone-beam ct reconstruction d. heuscher , s. utrup , f. noo ; highland heights, oh/us, salt lake city, ut/us learning objectives: to describe how the different aspects of helical cone-beam reconstruction affect spatial resolution in all three dimensions. to demonstrate that depending on the radial and angular position of a voxel, this resolution can be anisotropic. to describe appropriate filtering and interpolation steps that can be taken to reduce these effects. background: a number of methods have been described recently that provide either exact or near-exact reconstructions of helical cone-beam scan data. in this exhibit, we will describe key aspects of the implementation of these reconstruction algorithms that affect spatial resolution. we will show how the anisotropy of in-plane resolution can be related to the corresponding effect in single-slice d reconstructions. alternative filtering and interpolation steps will be suggested. procedure details: fwhm slice-sensitivity profile and in-plane resolution measurements were performed on helical reconstructions of a phantom consisting of seventeen . mm diameter spherical beads located at , , and mm radii. the scans used an mx -idt x . mm detector configuration with a . pitch factor. single-slice d reconstructions were performed on the same phantom using both fan and parallel convolution-backprojection algorithms to produce comparative in-plane measurements. alternative hi-order interpolation and voxel-dependent filters were investigated to produce nearly isotropic resolution. conclusion: significant spatial variation in resolution can be observed in exact or near-exact helical cone-beam reconstructions. a more uniform and isotropic result can be achieved by using hi-order interpolation to produce parallel projection data along with longitudinal voxel-dependent filtering. comparison in pet/ct studies iv contrast agents increased the suv of the mediastinum to . ( . ) and the liver to . ( . ). these increases in suv were coincident with changes in ct density from hu to hu (mediastinum) and from hu to hu (liver). suv values in the colon wall seem unaffected by positive oral contrast compared to pet: . ( . ) vs . ( . ). conclusions: physiologic suv from pet/ct appear somewhat higher than suv from standard pet when the ct is acquired with standard volumes of iv contrast media. adventures in non medical radiography a.a. alhajeri, d. mckenna, b. tuohy, c. roche, p. mccarthy; galway/ie learning objectives: to demonstrate the non-radiological use of familiar imaging modalities and show how far we have come since the first x-rays of mrs. roentgen's hand. background: today, x-rays technology has a wide application and is presently in use in fields as diverse as engineering, art, forensic pathology and security. procedure details: through a pictorial review we show how x-rays have a role in non-medical applications. we demonstrate how law enforcement agencies have adapted x-ray technology to screen luggage at airports, sometimes even using ct. furthermore newer technology in use in europe has been developed, termed "back scatter", which makes possible the detection of illegal immigrants and contraband material concealed within trucks. the characterisation of the bony skeleton with the aid of x-rays is not only helpful in identification but sometimes can also indicate to the forensic pathologist the mode of death. industrial engineers are able to evaluate the integrity of a structure with the aid of x-rays. examples include the examination of airplane engines and pipes for hairline fractures, unidentifiable to the unaided eye. in the art world, x-rays have been used to detect forgeries and even to create original art works. conclusions: this poster reaffirms the benefits of different imaging modalities showing how technology developed for medical imaging has been adapted for use in non-medical areas. radiation-induced temporary hair loss after cerebral perfusion studies with multi-detector ct y. imanishi, a. fukui, h. niimi, s. nakaji, k. nozaki, y. furuya, m. uzura, s. hashizume, y. nakajima; kawasaki/jp learning objectives: learning presence of complications by a cumulative or multiplier effect of radiation exposure from multiple diagnostic techniques. background: as various imaging technologies become increasingly advanced, they make it possible to get various kinds of information and large amount of imaging data. in some imaging technologies, the radiation dose increases with the ability to obtain better images or more detailed information. imaging findings: we encountered cases of temporary bandage-shaped hair loss, which was caused by perfusion studies of the head by multi-detector computed tomography (mdct) for evaluation of blood flow in the brain. in all of the , digital subtraction angiography of the head was performed within a period of the ct perfusion studies. this suggested the possibility that radiation exposure from angiography performed in serial examinations, combined with the perfusion studies effectiveness of a new dual-energy subtraction system using csi:tl amorphous silicon flat-panel technology h. tagashira, n. bandai, m. yoshimoto, y. yasuhara; ehime/jp learning objectives: to describe the theory of the new dual-energy subtraction system. to demonstrate the system with chest radiology cases. background: lung cancer is the current leading cause of cancer death and continues to be increasing. chest radiography has been shown to have relatively low sensitivity and specificity for the detection of the pulmonary nodules. this new dual-energy subtraction system allows the fast ( millisecond) acquisition of two, high and low voltage images and provides soft tissue and bone structure image of the chest with relatively low dose. procedure details: the radiologists' performance for reporting chest radiographs was evaluated by use of receiver operating characteristic (roc) analysis. the performance of radiologists was improved significantly when the dual-energy subtraction image was used (p < . ). conclusion: diagnostic accuracy will be improved by using this dual-energy subtraction system. longitudinal filters for uniform cone-beam resolution d. heuscher, s. utrup; highland heights, oh/us purpose: this presentation demonstrates the non-uniform longitudinal resolution inherent in today's cone-beam reconstructions and how, through the appropriate use of longitudinal filtering, improvements in image quality can be achieved. simulations and scans for acquisition geometries of or more detector rows were evaluated. a circular half-scan (hs) and a helical scan (pitch factor . ) were performed on an mx idt using a x. mm detector configuration. a phantom consisting of steel beads was used to measure longitudinal resolution at angles and radii. simulations were performed using the same phantom geometry, including a x. mm helical scan. a voxel-dependent filter was used to achieve uniform longitudinal resolution and compared to projectionbased filtering. both real and simulated thorax phantoms were used to evaluate the effects on image quality. results: resolution measurements demonstrate an angular variation in longitudinal resolution for large-pitch helical and circular hs cone-beam scans. the fwhm ssp measurements varied by over % and % at and mm radii respectively. reconstructions with projection-based filters did not reduce this variation. similar results were observed for helical scans with the larger cone angle ( x . mm detectors), wherein the resolution pattern varied at half the frequency along the longitudinal direction. applying voxel-dependent longitudinal filtration achieved uniform resolution, demonstrating improved image quality in both real and simulated scans. conclusion: there is an inherent non-uniformity of longitudinal resolution for cone-beam scans, particularly for large-pitch helical and circular half-scans. uniform resolution can be achieved using voxel-dependent filters. potential of phase contrast in x-ray imaging p. monnin , r. meuli , j. hoszowska , j.-f. valley , f.r. verdun ; lausanne/ch, grenoble/fr purpose: one of the limitations of conventional radiology is that small or weakly absorbing structures cannot be detected on images. coherent properties of an x-ray beam produce phase contrast images where a change of the refraction index of tissues translates into an edge enhancement phenomena on the images that allows the detection of weakly contrasted structures. this study shows the feasibility of implementing the in-line holography principle with an aim to imaging very small structures. material and methods: phase-contrast images were obtained using synchrotron radiations at esrf (grenoble, france). images of to micrometer nylon wires, immersed in different media, were recorded with a ccd camera ( . micrometers pixel size) in different imaging conditions. images were compared with the results obtained by means of fresnel diffraction theory. the coherent x-ray beam and the defocusing distance between the structures to be imaged and the detector produced phase contrast images of the wires. the comparison of the recorded images with the predicted image formation from the fresnel diffraction theory revealed the performance of this technique in terms of sensitivity, geometrical and energy requirements. images of a biological sample (i.e. pork kidney, cam's) were also produced where very fine low contrast structures, unnoticed in pure absorption contrast, appeared. the potential of the technique in neo-vascular process imaging will also be discussed. the potential of in-line holography techniques to improve image contrast of biological tissues has been demonstrated. experimental the aim of the present study was to evaluate y al o :ce (yag:ce) phosphor for radiographic and mammographic imaging. yag:ce, which has never been previously used in diagnostic radiology, emits green light with very short decay time ( ns). it is expected to match the spectral sensitivities of most photodetectors. radiographic test screens of various coating thickness ( - mg/cm ) were prepared in laboratory employing yag:ce phosphor in powder form with . µm median grain size. luminescence emission efficiency (emitted light over x-ray exposure) and light emission spectrum were experimentally evaluated as a function of x-ray tube voltage employing both tungsten and molybdenum target x-ray tubes. in addition, the spectral compatibility of yag:ce emission spectrum with orthochromatic films, photocathodes, ccd arrays and amorphous silicon photodiodes, used in both conventional and digital radiography, was determined. results were compared with other phosphor materials (gd -o- s:tb, csi:tl) currently employed in medical imaging. results: maximum yag:ce emission efficiency was observed for mg/cm screen at - kv using tungsten target x-ray tube. emission spectra peaked at nm. the spectral compatibility with amorphous silicon photodiodes ( . ) and ccds ( . ) was found to be very high, better than the corresponding compatibility of csi:tl, as mostly used in digital radiography detectors currently. conclusion: considering the yag:ce performance, its short emission decay time and its spectral compatibility with amorphous silicon detectors and ccds, this phosphor material is appropriate for use with digital radiography detectors. b d e f a g reduction of eddy current artifacts in echo planar diffusion tensor imaging with a stimulated echo preparation g. steidle, f. schick; tübingen/de purpose: diffusion-tensor-imaging (dti) using se-epi in tissue with short t values, such as kidney or skeletal muscle, suffers from a poor snr for higher bvalues. using a stimulated-echo diffusion preparation, sufficiently high b-values can be obtained by increasing the mixing time (tm) rather than te. a diffusionweighted stimulated-echo epi sequence is proposed which minimizes the influence of eddy currents (ec) without any increase in the minimum te or acquisition time. methods and materials: assuming a monoexponential decay for the ec with time constant λ, gradient pulses can be implemented within the tm interval such that eddy current effects proportional to exp (-λ x t) will vanish. in-vivo dti measurements were performed in human calf musculature on a . t siemens sonata unit. measurement parameters were te = ms, tm = ms, tr = ms, b = resp. s/mm , matrix x , fov mm, nex = , slice thickness mm. diffusion weighting was applied in six directions in order to calculate the diffusion tensor. results: diffusion images were acquired with and without ec correction. images without ec correction showed obvious scaling and shearing distorsions whereas in images obtained with ec correction no significant distorsions were observed. conclusion: diffusion-weighted mr imaging using epi is very sensitive to image distorsions caused by eddy currents. inserting additional gradient waveforms in a stimulated-echo epi sequence drastically reduces these artifacts and allows a calculation of the diffusion tensor in various tissues with low t /t ratios without significant misregistration. rf-induced artifacts due to aneurysm clips in mrt at . t u.a. lauer, h. graf, f. schick, c.d. claussen; tübingen/de purpose: metallic implants such as aneurysm clips may cause enlarged artifacts at higher magnetic field strengths, not only due to susceptibility effects. aneurysm clips made of ti-alloy were investigated with special respect to mr-imaging results and possible rf-induced artifacts. materials and methods: aneurysm clips were measured in gd-dtpa doped water at . t and . t with adapted se-sequences. additionally, two models made of copper were investigated at . t. to separate rf induced effects, the transmitter voltage was bisected stepwise to verify the rf-induced deviation of the local flip angle near to the implant. this was also done with d-tse sequences. results: in comparison to . t, more pronounced hyperintense zones were found at . t. they were independent of the exchange of phase-and frequency-encoding direction (swap) and became more pronounced at reduced transmitter voltage. comparative investigation of two cu-models and one selected ti clip at reduced transmitter voltage revealed rf induced effects depending on inductive and capacitive structures of the clip. conclusion: metallic implants have to be considered carefully at . t due to the electrical conductivity. rf-induced artifacts can appear not only for longish objects but also on smaller devices. in the case of aneurysm clips, a slight rfinduced effect could be detected. potential patient endangering due to a local enhancement of the secific absorption rate (sar) can be estimated as negligible. optimum is currently recommended as a consistent image display function. however, while the cielab function is still in use in europe, comparative evaluation of these two functions for optimum display function has not yet been reported. the purpose of the present study was to identify which of gsdf and cielab is the optimum display function for thoracic ct imaging by means of visual contrast analysis (vci), i.e. the ratio of luminance contrast to ct value contrast. methods and materials: monochromatic m lcd monitor and photometer of luminance calibration tool kit (data ray corp, westminster, usa) were used. the luminance contrast of this monitor was measured by a photometer displaying gsdf and cielab. luminance change and digital driving level (ddl) that corresponded to ct value (hu) were tabulated. ct value was represented by window setting of thoracic ct imaging, i.e. for mediastinum (ww: ~ , wl: ) and lung (ww: ~ , wl:- ) using gsdf and cielab. the ratio of luminance contrast to ct value contrast was calculated as visual contrast index (vci). the vci of gsdf and cielab was evaluated using t-test. result: using t-test, vic of cielab was significantly higher than that of gsdf in window setting of thoracic ct imaging (p < . ). based on visual contrast index analysis, cielab was more useful than gsdf as a monitor display function of thoracic ct imaging. we suggest that cielab is the optimum monitor display function of thoracic ct imaging because it intrinsically has excellent luminance contrast characteristics. follow-up of patients subjected to high skin radiation dose as a consequence of repeated interventional cardiology procedures r. padovani, g. bernardi, e. quai, m. signor, h. toh; udine/it purpose: as part of a quality assurance program, to retrospectively evaluate cumulative skin dose (csd) received by patients subjected to repeated interventional coronary procedures and to detect radiation induced skin injuries in patients who have received the highest dose. materials and methods: from april to february , cardiac procedures, coronary angiography (ca) and percutaneous transluminal coronary angioplasty (ptca) have been performed on patients. mean dap was . gycm . patients with a cumulative dose-area product (dap) higher than gycm have been extracted. they were patients ( . %), female and male, mean age years, who underwent a mean number of . procedures resulting in a mean cumulative dap of gycm and a maximum value of gycm . from dap, csd has been estimated adopting . and . - cm - as conversion factors for ca and ptca, respectively. clinical follow-up has been performed adopting the lent-soma methodology and scale. results: the highest csd estimated was . gy and only patients ( %) exceed a skin dose of gy. no skin injuries were detected at clinical follow-up. these results allow to estimate a frequency for skin injuries of less than . - in patients undergoing repeated cardiac procedures. the results of this study implies that the frequency of skin injuries may be very low when a quality assurance programme, including regular patient dose monitoring, periodic evaluation of procedure protocol, quality control of equipment and operator training, is established. ultrasound purpose: ultrasound is the most frequently used diagnostic imaging modality. the main problem of ultrasound, however, is interobserver variability. the intention of the development of ultrasound computer tomography was to increase the reliability of ultrasound imaging and to supply standardized images similar to other cross section imaging procedures. methods and materials: instead of a manually-controlled linear transducer array, we used ultrasound computer tomography (usct) to image a volume directly. a few thousand ultrasound transducers were arranged in a cylindrical array around a tank containing the object to be examined coupled by water. every single transducer was small enough to emit an almost spherical sound wave. while one transducer was transmitting all other transducers worked as receivers simultaneously. afterwards a different transducer emitted the next pulse. for volume reconstruction each transmitted, scattered and reflected signal was used. in an experimental set-up a phantom containing nylon filaments, straws, and a cadaveric forearm were investigated. results: nylon filaments with a diameter of . mm were visualized. the skin and soft tissue of the cadaveric forearm were clearly detectable but the bone structures could not be delineated sufficiently. usct supplied reproducible image sequences. conclusion: usct promises to supply standardized high quality three-dimensional images without ultrasound shadowing artifacts and without interobserver variability. furthermore, the tissue is not deformed, in principle offering multimodal image fusion with other cross section imaging modalities. prior to clinical set up extended development especially of large transducer element tubes and improvement of software is necessary. multicentre assessment of a complexity index for ptca procedure a. peterzol , e. quai , r. padovani , g. bernardi , j. kotre , a. dowling ; udine/it, newcastle/uk, dublin/ie purpose: multicentre assessment of a procedure-severity-based complexity index (ci) for the introduction of reference levels (rls) in percutaneous transluminal coronary angioplasties (ptca) as a function of procedure complexity. materials and methods: , and ptcas performed at three different centres were investigated. multiple linear stepwise regression analysis, including clinical, anatomic, and technical factors, was performed in order to obtain fluoroscopy time (ft) predictors. based on the regression coefficients of the emerging model a scoring system was defined, and a ci was obtained by adding up the single scores for each individual procedure. since a good correlation (r = . ; p < . ) was found between the dose-area product (dap) and the resulting ci, the latter was used to classify dose values into three groups. ci groups were determined by means of an anova test: different sets of groups were explored until the matrix of pairwise comparison probabilities reached a minimum value (p < . ). the resulting dap and ft third quartiles corresponding to each ci group are suggested as preliminary rls in ptca as a function of procedure complexity. results: ptca preliminary rls: (i) for dap values: , and gycm , and (ii) for ft: , and min, respectively for the three ci groups: low, medium and high complex procedures. the possibility to introduce rls as a function of procedure complexity, as confirmed by this multicentre study, can improve the process of patient dose optimisation in interventional cardiology. investigation of the radiation detection properties of lu sio :ce and gd o s scintillators for applications in medical imaging d. nikolopoulos, p. liaparinos, d. margetis, d. linardatos, p. mellisaropoulos, k. kourkoutas, g. panayiotakis, d. cavouras, i. kandarakis; athens/gr purpose: the aim of this study was to use monte marlo techniques to examine the detection efficiency of three ce-doped fast emitting scintillators, yap (yalo : ce), lso (lu sio :ce) and gos (gd o s:pr, ce, f) when used with photon energies and coating thicknesses employed in medical imaging. a general monte carlo code has been generated for tracking photons in the energy range covering all medical imaging applications (diagnostic radiology, nuclear medicine). simulated scintillator materials were selected to be in blocks and arrays of dimensions equal to those usually employed in medical imaging. the results for the yap scintillator, showed that the fraction of the incident photon energy which was totally absorbed decreases very rapidly for energies higher than kev (e.g. from % at kev to % at kev for . mm coating thickness). lso was better than yap, its absorption efficiency being significantly higher (p < . ) at - kev (mammography), - kev (x-ray imaging), kev (gamma ray imaging) and at kev (positron imaging). gos was also found to exhibit significant detection efficiency in the whole energy range examined (e.g. from % at kev to % at kev for . mm coating thickness), however its applications are limited due to its longer decay time. conclusion: lso was found to be of adequate efficiency for most imaging applications, while yap could be considered most suitable for x-ray mammography applications. analyses of physiological kinetic functions in dynamic chest radiography by the use of time series spectroscopy y. tsuchiya , y. kodera , m. tsuchiya , a. fukui , s. itou , y. machida ; shizuoka/jp, nagoya/jp purpose: the purpose of this study was to distinguish between movement of diaphragm and heart wall, and to evaluate the kinetic function of these structures in dynamic chest radiography with computer aided diagnoses. we obtained a dynamic chest radiograph of one healthy volunteer during respiration to assess the distinction between structures and another radiograph of a patient during a left ventriculogram to evaluate heartbeat calculation using an image intensifier system. we performed fft and band pass filter processing to obtain an intensity curve of the spectrum of dynamic chest radiography and detected the "change-point" of the phase by using characteristic analysis. fifty-five kinds of band pass filters were used to compare "detection rate of change-point", "matching rate of heartbeat" and "heartbeat error". results: movement of diaphragm and heart wall could be extracted selectively by the use of an extreme low pass filter ( . ~ . cycle/mm) and middle frequency pass filter ( . ~ . cycle/mm). we obtained % of matching rate, when detection rate was % with these two kinds of filters. there was correlation significantly between them (r = . , p < . ). improvement of detection rate decreased heartbeat error. but there was no significant difference between them by the non-repeated measures anova (p = . ). conclusion: our method was effective in analysing the dynamic chest radiograph in order to evaluate precise kinetic function, when suitable filters were used. therefore it is expected to apply for computer-aided diagnoses. reduction of ionising radiation exposure in patients as a consequence of new imaging technology for medical diagnostics of the spine: a retrospective study covering years in a norwegian referral hospital a. nyquist , h. olerud , b. bjørnarå , l. borgen , t. gudmundsen ; drammen/no, oslo/no purpose: to examine possible changes in ionising radiation doses to patients (collective effective dose) undergoing diagnostic imaging procedures for spinal problems over the last years in view of the shift in modalities from conventional x-ray examination and myelography to computed tomography (ct) and later on to magnetic resonance imaging (mri). retrospective study of all patient files for the period from to . the main modality groups for each of the three parts of the spine (cervical, thoracal, lumbar) were: conventional radiographs, myelography, ct, and mri. for each type of examination the mean effective radiation dose were obtained from the norwegian radiation protection authority (nrpa) as published in . the number of conventional x-ray examinations performed was nearly unchanged. use of myelography reduced by %, %, and % for the cervical, thoracal and lumbar regions, respectively. ct usage increased steadily, remained nearly unchanged, and was reduced by nearly % for the cervical, thoracal, and lumbar regions, respectively. the annual collective effective dose from examinations the spine was reduced by % in our hospital from . mansv in to . mansv in . the shift in modalities from conventional x-ray and myelography to ct and mri resulted in a significant reduction in patient exposure to ionising radiation. the results may be explained by our intended policy to select mri insted of ct whenever possible, and is an overture to the discussion of the further development and structure of diagnostic imaging in general, and for the spine in particular. dose evaluation for clinical proton beam with standard dosimetry h. ohtani , t. hiraoka , h. saitoh , t. irifune , t. katoh ; tokyo/jp, chiba/jp purpose: the standard dosimetry was revised on august in japan; it had calling standard dosimetry of absorbed dose in external beam radiotherapy (standard dosimetry ). the purpose of standard dosimetry are the establishment of traceability for dose of radiotherapy and quality assurance / quality control, according as we measure the dose of high-energy radiation using the standardized international method. we explained the dosimetry of absorbed dose with the standard dosimetry for proton beams and the depth dose was measured with the reference ionization chamber and the parallel plate ionization chamber. the maximum energy of the proton beam used in this study was mev. electric separation induced radicals were calibrated for squarer field of × cm with some ionization chambers. the absorbed dose to water calibration factor is obtained by the reference ionization chamber calibrated co gamma rays. the remainder range in water calibrated the beam quality conversion factor for proton beam. using these coefficients, the absorbed dose was calibrated. the absorbed dose used the parallel plate ionization chamber was relatively decided by the results for the reference ionization chamber. conclusion: the absorbed dose for proton beam was calibrated at each depth. dosimetry in water was recommended on the standard dosimetry . however, it is difficult that we have the waterproof dosimeter. we suggested that the method and problem for proton dosimetry using the standard dosimetry . b d e f a g kinetic analysis of the temporomandibular joint with computer-aided detection system n. bandai , s. sanada , k. ueki , s. funabasama , s. tsuduki , y. otani , t. matsui ; onsen-gun/jp, kanazawa/jp, otsu/jp purpose: the purpose of this study was to develop a method of kinetic radiography and a computer-aided detection (cad) system for quantitative evaluation of the temporomandibular joint (tmj). methods and materials: dynamic images of the tmj were obtained from one healthy volunteer by digital fluoroscopy in the lateral view on the right and left sides. the accumulated image subtraction technique extracted the condyle in each image. a sequential similarity detection algorithm (ssda) was employed to trace the movement path and the velocity of the condyle. results: there were no significant differences between by manual and computer analysis. the shape of the path of the right condyle was smoother than that of the left condyle. the size of the maximum vertical and horizontal movements of the condyle were . ± . mm and . ± . mm, respectively. the velocity of the movement of the condyle was higher in the area close to the articular eminence than in any other area during the opening and closing of the mouth. conclusion: our cad system will contribute to the kinetic analysis of the tmj for screening, follow-up study, and informed consent, providing speed, quantitation, and cost-effectiveness. assessment of fetal dose in x-ray examinations of pregnant patients a.j. servomaa , a. kettunen ; helsinki/fi, oulu/fi purpose: when a pregnant woman undergoes examination of the lower abdomen or pelvic area, the fetus is directly irradiated. determination of the absorbed dose to the fetus is therefore of interest as a basis for risk estimates. in finland, the practice with regard to both estimation of the dose to the fetus and recording of the examination in patient records varies considerably. the fetal dose calculated by means of various dose calculation methods are compared and the results presented. methods and materials: using the "fetdose" and "pcxmc" programs, the normalized fetal dose was calculated for abdominal and pelvic examinations of various radiation qualities, esd and conceptus depths. the doses obtained were compared against those reported in the literature. the calculated nuds (normalized uterus doses) obtained for various parameters by means of various calculation methods agree, with reasonable accuracy. the nud occurring in a pelvic ap examination ( kv, mmal filtration, fsd cm) is about . for a gestational age of - weeks and for a fetal depth of cm. the dose to the ovaries differs slightly from the dose occurring to the uterus during the same examination. detailed data about the nuds in various common radiography examinations are presented. the results of calculations obtained with different programs show the same nud with reasonable accuracy. normalized fetal doses and radiation risks corresponding to the european esd recommendations in conventional radiography examinations may help the radiological staff to assess the significance of the exposure to the fetus. withdrawn by authors quality advances via noise reduction in laser imaging t.r. lindquist; oakdale, mn/us purpose: noise sources in medical imaging and printing can limit diagnostic accuracy but are often not assessed quantitatively. a method is described for objectively quantifying noise by means of a film digitizer and analysis software. a second method, based on human perception of simulated nodules on films with varying amounts of noise, is also described. in the first method, a film digitizer scans a print of a constant gray level image. the scanned digital image file is then processed by software that: ( ) extracts and averages density data from narrow horizontal and vertical strips, and ( ) performs one-dimensional fast fourier transform (fft) processing to yield a noise power spectrum (nps) for each horizontal or vertical strip. in the second method, a computer program is used to synthesize a test image with very low contrast dots ("nodules") superimposed on a gradually changing background. in timed experiments, prints of these images are placed on a light box, and an observer is asked to mark the locations where the dots are believed to be present, after which the true and false positives are counted. results: prints were made on a variety of medical imagers, using film with a variety of quality levels. prints made with the latest laser imager and film yielded lower measured noise and higher perception scores than prints made with prior technology. conclusion: measurable progress has been made in reducing the noise level of medical imaging printers. this is further demonstrated by improved perception of subtle details. estimation of maximum skin dose and average lung dose in coronary angiography procedures a. karambatsakidou, p. tornvall, n. saleh, p.-o. löfberg, a. fransson; stockholm/se purpose: a comparison of phantom and patient based techniques to estimate the maximum skin dose (mesd) and average lung dose from coronary angiography procedure is described. conversion factors for the dose to the skin and lung, normalized to the dose-area-product (dap), have previously been obtained from measurements using an alderson phantom. in this study, measurements on patients using slow radiographic film and diodes were used in combination with the software winods to evaluate the impact of different operators, and of variations in patient size, on these factors. the transmission ion chamber integrated into the angiographic unit was used to measure dap. the conversion factor for mesd from the phantom simulation was reported as . msv/gycm . the corresponding value for measurements on patients in this study was . ± . msv/gycm ( sd; patients). no significant difference in conversion factor between the operators was found. the lung dose varied with the body mass index (bmi). in slightly overweight patients (bmi: - ), the lung dose was about % lower than in normal sized patients (bmi: - ) for the same dap-value, while for overweight patients (bmi > ) this difference increased to %. conclusion: measurements on phantoms and on patients yield similar conversion factors for mesd in coronary angiography procedures. the bmi-value seems to be a robust indicator of the variation in dose to internal organs from such procedures. with film/diode measurements, the differences in techniques (geometry; projections) between operators can be easily verified, and as such be integrated into the training of new angiography operators. radiation doses in a newly installed flat-panel digital system in interventional cardiology department v. tsapaki, s. kottou, n. kollaros, p. dafnomili, v. neofotistou; athens/gr purpose:the purpose of the study was to investigate the radiation doses of the newly installed flat-panel fluoroscopy (fpf) system in an interventional cardiology (ic) department and to examine possible methods of technique optimization regarding the new digital system. materials and methods: fifty coronary angiographies (cas) and percutaneous transluminal coronary angioplasties (ptcas) were investigated. patient data were: sex, age, weight, height, dose area product (dap), fluoroscopy time (t) and total number of frames (f). further more, dose rates in all fluoroscopic and cine modes were measured. image quality was assessed using a dedicated test tool. results: median values of dap, t and f were: . gycm , . min and in ca and . gycm , . min, in ptca. our results are comparable with those found in the recent literature. regarding the technical characteristics of the digital system, the high contrast resolution (hcr) is not affected by fluoroscopic mode, whereas low contrast resolution (lcr) is slightly decreased in the lowest mode. the results of the study concerning the fpf system lead to the conclusion that the lowest fluoroscopic mode and the lowest frame rate should be used routinely for dose optimization and that the new digital technology has comparable radiation dose to the conventional technology. purpose: an earlier study using the mte scanner enabled us to register in vivo wave-like activity of the skin electrical landscape (sel), which is supposedly macroscopic manifestation of intercellular communication. the present study of the chaotic sel activity was done to try to find out distinctive dynamic features between melanoma and adjacent tissues. methods and materials: mte is a new functional, non-invasive digital imaging modality, which enables dynamic visualization of the skin and underlying tissues in terms of spectral electroimpedance and electropotential parameters, with high spatial and temporal resolution (~ . mm). non-thermal mm-emf ( min) and weak mf (< . t) were used as test influences. initial and induced sel dynamics have been graphically and statistically analyzed. specifically, the throughimage-sequence analysis resulted in detailed dispersion maps of the scan-area. the sels of (i) healthy and allergic subjects, and (ii) those with cutaneous melanoma were investigated. results: primary melanoma manifested not only as a sharp electro-abnormality, but also as a distinctly stable/hyposensitive zone (sigma = . - . %) in all measurement parameters.this hyposensitive zone(s) was surrounded by islet areas of marked hypersensitivity (sigma is up to %). dispersion range of the more distant environment had intermediate values. in cases of allergy, strongly pronounced dependence of the mm-emf response upon time of investigation was observed at the same subject, i.e.: from sharply marked reactions at the stage of sensitization to practically indistinguishable ones during remission. in healthy subjects, in most cases, significant trigger reversible changes of histograms were registered in response to both tests. intracranial elastance analysis using mri in normal-pressure hydrocephalus t. miyati , m. mase , m. onoguchi , h. kasai , m. hara , k. yamada , y. shibamoto , s. matsunaga , t. kasuga ; kanazawa/jp, nagoya/jp purpose: in order to assess the state and dynamics of the intracranial system in normal-pressure hydrocephalus (nph), intracranial elastance (ice) was measured with magnetic resonance imaging (mri). the ice index, which is the ratio of pressure to volume change, was obtained with alperin's method; intracranial volume and pressure changes during the cardiac cycle were calculated from the net transcranial blood and cerebrospinal fluid (csf) flow measured with phase-contrast (pc) cine mri. ice indices were determined in patients (n = ) with nph, brain atrophy or asymptomatic ventricular dilation (vd), and in healthy volunteers (control group; n = ). concurrently, a time constant of the integral of the csf response function, which represents the intracranial mechanical property was determined with pc cine mri. the changes in ice indices were also analyzed after intravenous injection of acetazolamide. these values were then compared with the volume-pressure response (vpr) during the shunt operations. results: the ice index in the nph group was significantly higher than in the control or vd group, but no difference was found between the control and vd groups. the ice index increased in all groups after an acetazolamide injection because of an increase in cerebral blood volume. there were significant correlations between the ice index and the time constant. a positive correlation was noted between the ice index and the vpr. conclusion: ice analysis measured by pc cine mri makes it possible to noninvasively obtain a detailed assessment of intracranial state and dynamics in the nph and to assist in diagnosis of the nph. quantification of magnetization transfer by sampling the transient signal using mt-prepared single-shot epi g. helms , g.e. hagberg , c.d. claussen , f. schick ; tübingen/de, rome/it purpose: using trains of mt-pulse and epi readout, the transition to steady state can be measured with higher accuracy than the steady state. a novel quantification method based on sampling transitions at different repetition periods (pr) is introduced. gaussian mt-pulses of . ms duration and flip angle of degrees were repetitively applied at khz offset at pr = , , , , ms. an axial slice through the centrum semiovale of the brain was measured by single-shot se-epi ( ms te, mm thickness, cm fov, x matrix). in brain tissue, the signal dependence was described by analogy to progressive partial saturation, where the direct saturation is increased by pr-dependent mtcontribution proportional to macromolecular content and differential saturation. macromolecular content, kinetic and relaxation parameters and the saturation were estimated by a global non-linear least-square fit of the signal equation for rois in cortical grey matter (gm) and central white matter (wm). results: the macromolecular content was ( . ± . )% in wm and ( . ± . )% in gm, the apparent relaxation rates ( . ± . ) /s and ( . ± . ) /s. the apparent transfer rates were ( . ± . ) /s and ( . ± . ) /s. macromolecular saturation of a single mt-pulse was ( . ± . )% in wm and ( . ± . ) in gm; the direct effect on water less than %. signal correction for csf contributions was necessary. conclusion: quantitative mt-mapping of the whole brain may become feasible in vivo at higher field strengths by means of permutation of the slice order. b d e f a g the concept of radiography in health sciences: a concept analysis s. sorppanen , a. servomaa ; oulu/fi, helsinki/fi learning objectives: to clarify the content and uses of the concept of radiography in health sciences and to compare it to the concept of radiography in physics and technology. background: in the field of radiography, theoretical research related to concepts has so far being rare. nevertheless, the concept development is an important part in constructing theories and clarifying the identity of a discipline. in finland, the discipline of radiography started in at the university of oulu, and since then it has offered radiographers academic education in their own field. for being able to build its own knowledge base and theories to be used in developing the profession and education of radiographer, and for being able to stand for its position as an independent academic discipline, the young discipline of radiography needs systematic conceptual clarification and development. procedure details: the concept of radiography was analysed using so-called evolutionary method of concept analysis (presented by rodgers). the contents and uses of the concept were studied by determining the concept's characteristics and by observing these characteristics in the cross-disciplinary comparison. the data consisted of literature and internet pages, and were analysed using deductive-inductive content analysis. conclusion: the concept of "radiography" in health sciences can be determined as "dual, dynamic, social and situation-related expertise of radiographers in the use of radiation, which is based on a versatile synthesis". it seems to be wide and complex in nature, and radiographer-centered. the content and the use of the concept varies according to the discipline. measurement of distribution of horizontal dose amount to chest x-ray by gafchromic film k. nishikiori , m. miyazawa , a. otonashi ; habikino/jp, itabashiku/jp, suita/jp learning objectives: to illustrate horizontal dose distributions of human diagnostic level in the chest phantom by gafchromic film and glass dosimeters. to point out the characteristics of horizontal dose distributions by materials in ten shots and in fluoroscopy. background: in general, we use an ionization chamber dosimeter or tld to measure dose distribution of diagnostic level. however tld can not indicate horizontal and consecutive dose distributions, because it has only scattered dose data. consequently, we used gafchromic film to obtain a horizontal consecutive dose distribution of human diagnostic level. imaging findings: . first, we placed a gafchromic film and glass dosimeters in the specific place of the chest phantom. then we irradiated the chest phantom with diagnostic level x-rays. the dose distribution which was acquired by the film and glass dosimeters was then analyzed. . next, we examined the result of fluoroscopy. . the results of the first method were compared with the result of the fluoroscopy and the difference between the distributions in (gafchromic film and glass dosimeters) and (fluoroscopy) examined. the characteristic dose distributions of the incidence dose in diagnostic level were confirmed by both horizontal dose distributions. . horizontal dose distribution which was specific and consecutive data could be obtained from the gafchromic film. . horizontal dose distribution which was specific and scattered data could be acquired by glass dosimeters. we obtained almost the same as results in fluoroscopy. horizontal dose distribution of fluoroscopy showed a sharp reduction on the subcutaneous tissue of the chest phantom. barium enemas: are radiographers up to it? p. vora, a. chapman; leeds/uk aim: to determine the types and rates of complications encountered by radiographers when performing double contrast barium enemas. a similar complication rate to those encountered by radiologists will reassure those who have adopted this new skill mix and encourage its further development whereas an increase in complications may point to areas where radiographer training needs to be improved. materials and methods: questionnaires were posted to radiographers who had in the last years attended one of the biannual leeds barium enema training courses. results: of questionnaires posted ( . %) were returned completed. approximately, , barium enema examinations had been performed. radiographers reported complications, including intraperitoneal and extraperitoneal perforations. there were five deaths (mortality rate - in , ). the five deaths included two of the twenty-four ( %) perforations, two of the forty-five ( %) cardiac events and one cva that occurred during an examination. conclusions: radiographers have been regularly performing dcbes for almost a decade. the mortality rate for radiographer performed double contrast barium enemas is similar to that reported for radiologists. a slightly higher than expected rate of perforation is noted in this study and so this is an area where radiographer training should be targeted. the level and incidence of the fear in patients subjected to radiological examinations e. czekajska-chehab, m. makara-studzinska, a. koczynasz, i. nowak, a. drop; lublin/pl purpose: analysis of incidence and intensity of fear in patients subjected to diagnostic procedures. methods: an anonymous survey concerning the occurrence and level of fear connected with radiological examinations was conducted among individuals: patients attending for various contrast examinations and women before mammography, using the questionnaire prepared by the authors. the level of fear was evaluated according to the adopted scale in relation to the obtainable maximum values. results: out of individuals ( %) reported their fears before planned radiological examinations. the frequency of fear was extremely high for mammography ( %) and lower for contrast procedures ( %). the strongest fear was associated with the uncertainty of diagnosis and length of waiting for results ( - %). the fears of patients before contrast examinations were mostly related to results and harmful effects of radiation. the fears concerning allergic reactions were reported by almost a half of patients before ct, urography and barium enema. in patients before contrast procedures the highest fear level was related to the uncertainty of results ( - %). the lower level ( %) was associated only with urography. the fears about the examination itself, allergic reactions or harmful effects of radiation showed similar intensity ( - %), irrespective of the kind of examination. conclusions: fears before radiological examinations are common and concern over a half of patients subjected to contrast procedures and almost all of those before mammography. the most common causes of fear include: uncertainty about diagnosis, exposure to harmful effects of radiation and length of waiting for results. audit of adequacy of inpatient ct requests: implications and consequences w. chooi, p. heath, m.j. bull; sheffield/uk purpose: vetting of ct requests ensure that the examination is justified in accordance to local guidelines and also allow the radiologist to select the most appropriate ct protocol. however the provision of poor clinical information severely limits this step. this audit aims to determine the prevalence of inadequate clinical details in ct request forms and also identify trends that result in poor referring technique. methods and materials: for a two week period all inpatient electronically generated ct request were prospectively monitored. outpatient & out-of-hour requests were excluded. results: out of a total of requests, were successfully audited ( %). the types of examinations included ct head ( %), abdomen ( %), thorax ( %), abdomen and thorax ( %), and musculoskeletal ( %). problems were encountered with requests ( %). the most common problem encountered was that of inadequate or misleading information (n = ). others issues included better alternative examination (n = ), undisclosed renal impairment (n = ), failed examinations due to agitated patients (n = ), ward cancellations (n = ), requests without contact details (n = ), and wrong ward details (n = ). as a result of these requests were not performed. the majority of these requests are completed by junior doctors. conclusion: vetting of ct requests is an essential first step towards a successful ct examination. the provision of inadequate clinical information is prevalent and junior doctors require closer supervision when completing ct requests. to determinate whether use of high iodine concentration contrast medium offers more complete information. diagnosis and follow-up of aortic dissection with multislice computed tomography y. herrero, m. martí, j. echeveste, m. fernández-velilla, g. garzón, n. gómez-león; madrid/es purpose: to assess the utility of multislice computed tomography (msct) in the diagnosis and follow-up of aortic dissection. materials and methods: seventy-one patients referred to rule out aortic dissection underwent msct following a standardised protocol. the volume of interest was the entire aortoiliac system, ml of non-ionic iodinated contrast were administered at a flow rate of ml/sec. a collimation of mm, table speed of mm per rotation, pitch of and reconstruction interval of mm were used. multiplanar (mpr) and three-dimensional reconstructions (external rendering) were obtained in a workstation (vitrea ; vital images plymouth, minn). aortic dissection was diagnosed when an intimal flap separating the true and false lumen was observed. patients diagnosed with aortic dissection underwent an endovascular repair (stentgraft placement). follow-up ct examinations after endovascular repair were acquired with the same msct protocol, after performing a basal nonenhaced study. complete closure of the entry site, and both stent-graft and visceral arterial branches patency were considered as adequate stent-graft function signs. results: nine of the patients were diagnosed with aortic dissection (six acute and three chronic dissections), two of which involved the ascending aorta and seven the descending thoracic aorta. follow-up demonstrated in all cases a residual abdominal aortic dissection with progressive decrease in size of the false lumen. conclusion: msct determined the exact anatomy of the dissection flap, the location of the entry and reentry sites, and the major branch vessels that were supplied by the true lumen. endovascular stent-graft placement for treatment of thoracic aortic aneurysms m. fernández -velilla, g. garzón, i. acitores, f. ibáñez, l. riera; madrid/es learning objectives: to assess the safety and efficacy of endovascular stentgraft placement for repairing descending thoracic aortic aneurysms in high-risk patients. to analyse the major associated complications. to show our experience in the management of these patients over a six-year period. background: by helping patients avoid open surgery, endovascular aneurysm repair minimises the perioperative derangement of pulmonary, renal, cardiac, and gastrointestinal function. the greatest potential benefit is in high-risk patients who have large aneurysms and who are poorly suited to any of the current alternatives. procedure details: endovascular aneurysm repair was performed in highrisk patients who could not undergo conventional repair. the feasibility of endovascular treatment and the prosthesis' size was determined based on preoperative spiral ct and intraoperative angiography findings. stent-grafts were surgically inserted through exposed femoral arteries with fluoroscopic guidance. the anaesthetic technique was either epidural or general. endovascular grafts were constructed with gianturco stents and polytetrafluoruroethylene (ptfe) graft. stent-grafts were placed in disecting, atherosclerotic, five traumatic, and one post-coarctation descending thoracic aortic aneurysm. % of them were chronic. we analysed the results obtained in both stable patients, in which an elective treatment was performed of sacular, fusiform, traumatic and chronic dissection aneurysms, and in emergency patients (intramural bruise, penetrating ulcer, and aortic dissection). conclusion: endovascular treatment of thoracic aortic aneurysms offers considerable benefits. long-term follow-up is needed to ascertain the durability of the procedure especially in dissecting aneurysms. an interdisciplinary and experienced team is needed to manage endovascular and surgical treatment. radiologic appearances of thoracic outlet syndrome: evaluated by pat optimized d contrast enhanced mr angiography and intraarterial dsa Ö. Özsarlak, o. d'archambeau, f. delrue, p.m. parizel; antwerp/be learning objectives: thoracic outlet syndrome: pat optimized contrast enhanced (ce) mra and intraarterial digital subtraction angiography. background: seventeen patients clinically suspected of having a vascular thoracic outlet syndrome were retrospectively reviewed. the patient population consists of women, and men with an age range between and . in eight patients, ce-mra was performed in . t mr system. gre flash d mr angiography with care bolus technique was used with a pat acceleration factor of two. the remaining nine patients were examined by intraarterial, and intravenous dsa of the upper extremity. all examinations, including mra were performed both in neutral and in adson positions with abduction of arms. imaging findings: none of the patients showed abnormality during neutral position. examinations repeated during adson position revealed subclavian artery lesions in patients: occlusions, high-grade stenoses, and mild-grade stenoses. in patients, the lesion was located on the left side, in patients on the right side, and remaining patients' lesions were bilateral. conclusion: a review of the functional anatomy, clinical symptoms, ce-mra and dsa findings are presented with illustrative cases. the advantages of patoptimized ce-mra are highlighted. mri evaluation of patients with peripheral vascular malformations g. calabrese, g. falda, g. lucchini, a. cazzulani; garbagnate milanese/it learning objectives: to define a mri protocol to study patients with angiodysplasia in order to evaluate soft tissues and bone involvement and to demontrate the appearance of the vascular malformations. background: imaging studies must show all the abnormal vessels and provide information on the degree of surrounding tissue involvement. in this exibit we describe the mri findings in patients with angiodysplasia of the lower or upper limbs. procedure details: t weighted fse sequences and t weighted fe sequences in the sagital and coronal planes were acquired. a sagital t fse sequence b d e f a g with echos and fat-saturation was used to demonstrate vessels with slow flow. dsa and dsv were performed to proceed to embolization or operation. t images gave good morphological demonstration of muscle involvement. on t fe images the presence of small vessels within the subcutaneous fat or within the bones was easily shown. images acquired with the echos t sequence were processed with the m.i.p. function to show the abnormal vessels; this sequence was especially useful in the venous malformations and in some cases the draining veins were also identified. conclusion: mri is the technique of choice to demonstrate the anatomic extent of the lesions and their relationship to the adjacent soft tissues. even if mra is improving from the technical point of view, still it remains quite complex and time consuming. in our protocol a simple t sequence showed to be a good method to demonstrate abnormal vessel with slow flow. occlusion background: thrombosis of the ivc and its tributaries is most commonly associated with malignancy, resulting from direct invasion by carcinoma of the kidney, liver or adrenal gland. thrombosis of the ivc secondary to benign causes is less common. predisposing factors include pregnancy, connective tissues disorders and coagulation abnormalities. recognition of caval thrombosis on ultrasound may be difficult. however, depiction of the ivc on both contrast-enhanced computerised tomography and mri is more accurate. although non-contrast mri is useful for demonstrating ivc occlusion, contrast-enhanced mr venography offers the opportunity for rapid evaluation in a single breath-hold. imaging findings: the normal appearance of the ivc as demonstrated by mri is illustrated, along with examples of ivc occlusion associated with both benign and malignant pathology. simple rules for differentiating bland versus tumour thrombus are presented and illustrated. a method for optimising the timing of data acquisition for ce-mra is presented. the merits of "indirect" versus "direct" ivc mr venography are also discussed. finally, artefacts that may mimic ivc thrombus are described. conclusion: mri/mra accurately evaluates the ivc. the multiplanar imaging capabilities, lack of ionising radiation, coupled with the added benefit gained from the use of intravenous gadolinium based contrast agents in mra, makes mri the modality of choice in evaluating patients with suspected ivc occlusion. the swollen leg: the various etiology and its ct venography findings w. jun, w. lee, j. chung, j. park; seoul/kr learning objectives: to illustrate the various causes of swollen legs. to recognize the characteristic imaging findings of various causes of swollen legs. to evaluate the accuracy of ct venography in the differential diagnosis of the etiology in patients with swollen legs. background: the swollen leg is a common clinical problem that complicates many medial and surgical disorders. clinical applications of computed tomography (ct) venography have increased with the improved technology of multidetector ct systems. ct venography showed the diagnosis of dvt and other soft tissue disease in patients with swollen leg. for the past two years, ct angiography of lower limbs was performed in patient (age - years) with swollen legs. the various causes and imaging findings of swollen legs are described and illustrated. imaging findings: the characteristic imaging findings of acute deep vein thrombosis (dvt) are a definite intraluminal filling defect, venous distension and muscle compartment enlargement. findings of chronic dvt are small, thick-walled, poorly enhancing veins and presence of collaterals. findings of lymphedema are skin thickening, subcutaneous tissue thickening, fat infiltration: lines parallel to skin and perpendicular to skin. findings of cellulitis including necrotizing fasciitis, are fascial thickening, fat infiltration, focal fluid collection, soft -tissue gas and muscle involvement. conclusion: ct venography showed promise for the diagnosis of dvt and other soft tissue disease in patients with swollen legs. ct venography is the modality of choice when clinical and other imaging findings are indeterminate. the aorta revisited: spectrum of findings on helical ct a.j. madureira, j.p. jesus, j. loureiro, i. ramos; porto/pt learning objectives: to review the imaging findings of the anatomical variants and disease processes that may affect the aorta from the aortic valve to the bifurcation. to describe the pitfalls that may be encountered. background: helical ct is a frequently used method in the evaluation of diseases affecting the aorta. it provides invaluable information in the assessment of acute aortic syndromes and is often employed in diagnosis and follow-up of dissection, aneurysm and bypass surgery. procedure details: the imaging findings of the diseases that may affect the aorta are presented based on the experience of our tertiary care hospital and level trauma center. the anatomical variations of the aortic arch vessels, acute aortic syndromes, aneurysmal disease and aortic bypass surgery are discussed and illustrated thoroughly. conclusion: helical ct is a firstline imaging method in the evaluation of aortic disease due to it's widespread availability, good spatial resolution and speed. learning objectives: to illustrate the mr findings that differentiate severe carotid artery stenosis with a tiny residual distal lumen from patients with total carotid artery occlusion. background: due to the small but substantial stroke risk in patients undergoing digital subtraction angiography, contrast enhanced magnetic resonance angiography (m.r.a.) has become the standard for evaluation of carotid occlusive disease in many centres. m.r.a. is highly accurate in differentiating significant stenosis ( %- %) that requires carotid endarterectomy, from insignificant (< %) disease. m.r.a. is also highly accurate in differentiating severe stenosis from occlusion. however, demonstration of a string sign depends on careful interrogation of the source images as the residual patent lumen may not be visualised on standard maximum intensity projections. given the clinical importance of differentiating between these two groups, we present and contrast the imaging findings of patients with significant carotid stenosis and those with total carotid artery occlusion. imaging findings: we describe the varied findings in both patients with total carotid artery occlusion and those with severe stenosis. we present a simple algorithm for rapid evaluation of patients referred for differentiation of severe stenosis from occlusion. background: anomalies of ivc and renal veins occur with a prevalence of . % to . %, and are easily identified on ct, frequently incidentally. if unidentified, they can be misinterpreted as pathology (adenomegaly, retroperitoneal masses, normal venous structures and/or collaterals during acquired obstruction) or can lead to morbidity during radiologic and surgical procedures (positioning ivc filters, varicocoele sclerotherapy, renal venous sampling, shunt placement for portal hypertension, liver or kidney transplantation, ivc ligation for thromboembolic disease, abdominal aortic aneurysm repair). we describe the ct findings in cases of anomalies of the ivc and the lrv. imaging findings: female and male patients, aged - years, were studied. we found anomalies of the suprarenal segment (interrupted ivc with azygos continuation), anomalies of the renal segment ( circumaortic lrv, retroarotic lrv), and anomalies of the infrarrenal segment ( retrocaval ureter, transposition of the ivc, and duplications of the ivc). one patient with agenesis of the ivc with azygos continuation had profound venous thrombosis, and another had bronchiectasis. in any other case the anomaly was responsible for the patient's symptoms. results: mean size of the ascending aorta in these patients at initial presentation was . cm (range . to . cm). dissection of the ascending aorta occurred in %. median size of ascending aorta at time of dissection was . cm. all patients underwent surgery for their aneurysms. the postoperative mortality was . %. we have compared the two groups of marfan's syndrome patients whom had dissections: with an aortic-root diameter from . to . cm ( %) and with an aortic-root diameter from . to . cm ( %). there is correlation between the aortic-root diameter and the probability of aortic dissection. analysis to isolate risk factors for dissection revealed that size larger than . cm increased the probability by %. the incidence of dissection increased with aneurysm size and the radiological criteria for surgical intervention in marfan's syndrome are aneurysm diameter of . cm. maximal information about size and dissection of aneurysms in marfan's syndrome patients are obtained from analysis of angiography data. an evaluation of internal carotid artery and cerebral blood flow volume using color duplex sonography in patients with vertebral artery hypoplasia m. acar, b. degirmenci, a. yucel, r. albayrak; afyon/tr purpose: vertebral artery hypoplasia (vah) may be defined as va flow volume below ~ to ml/min using color duplex sonography. the aim of this study was to evaluate the effect of va hypoplasia on internal carotid artery (ica) flow volume and cerebral blood flow (cbf) volume. materials and methods: in this study, patients with va hypoplasia and control subjects with normal va flow volume were enrolled. the patients and control subjects were - years of age. the ica and va flow volumes were determined by using color duplex sonography. cerebral blood flow volume was calculated as sum of flow volumes in the ica and va of both sides. the ica and cbf volumes of patients with va hypoplasia were compared with control group flow volumes. results: in va hypoplasia and control group, there were no significant differences between ica flow volumes of right and left sides ( ± versus ± ml/ min; p = . on the right side, ± versus ± ml/min; p = . on the left side) or sum of flow volumes of both ica ( ± versus ± ml/ min; p = . ). however cbf volume tended to be lower in patients with va hypoplasia than control group. conclusion: evaluation of cbf using color duplex sonography is noninvasive and easily applicable to all patients and provides valuable diagnostic data. to the best of our knowledge, this is the first study demonstrating lower sbfv and normal ica flow volume in patients with va hypoplasia. the floating layer: a new sonographic sign of ruptured abdominal aortic aneurysm a. nunziata , o. catalano , m. mattace raso , f. sandomenico , a. siani ; naples/it, pozzuoli/it purpose: in patients with ruptured abdominal aortic aneurysm we have occasionally seen a floating layer, similar to the intimal flap recognizable in patients with aortic dissection but slightly thicker. we reviewed our series to assess the overall prevalence, specificity and diagnostic significance of this finding. materials and methods: in a year period (from january to december ) we have evaluated with us, consecutive patients with abdominal aortic aneurysm (larger axial diameter of the aorta > mm). twenty-nine aneurysms were proven to be ruptured (sonographic and/or ct diagnosis) and were surgically confirmed. the remaining patients were not symptomatic and were proven to have a non-ruptured aneurysm at follow-up. results: a floating membrane was found in out of patients with ruptured aneurysm ( %) and in none of the subjects with non-ruptured aneurysm. the image appeared as a thick layer floating within the aneurysm lumen, clearly detached from a more or less eccentrically placed luminal thrombosis (present in all cases), with a paradoxical movement unrelated to overall vessel pulsation. the floating layer is an uncommon but specific sonographic sign of abdominal aortic aneurysm rupture. recognizing this findings should lead to prompt surgical treatment, without need for further imaging (contrast-enhanced ct). color duplex measurement of cerebral blood flow volume in patients with polycystic ovary syndrome m. acar, a.s. cevrioglu, b. degirmenci, a. yucel, r. albayrak, m. yilmzer; afyon/tr purpose: polycystic ovary syndrome (pcos), one of the commonest endocrinopathies in the human, is known to be associated with risk factors for cerebrovascular disease. we want to determined whether there is a change in cerebral blood flow volume in pcos. we reported women with pcos and healthy women with similar body mass index (bmi). both groups of women were aged to years. we detected internal carotid (ica) and vertebral artery (va) flow volume using color duplex sonography. cerebral blood flow (cbf) volume was determined as the sum of flow volumes of internal carotid and vertebral arteries of both sides. results: there were no significant differences between patients with pcos and control group in cbf volume or the bilateral sum of flow volumes in the ica and va. but bilateral mean velocity of va was significantly lower in patients with pcos compared to control group. correlation between age of patients and mean velocity of va and ica were assessed with pearson correlation analysis. in control group, mean velocity of va and ica was inversely correlated with age (r = . , p = . versus r = . , p = . ). however no correlation was found for patients with pcos. conclusion: this is the first study demonstrating normal cbf volume using duplex sonography in pcos. it is known that the increased risk factors for cerebrovascular disease does not increase stroke mortality in pcos. normal cbf volume and absence of decrease in va and ica mean velocity by aging may be protective mechanisms. thoracic aortic calcification in the elderly g.s. karlsdottir, t. aspelund, s. sigurdsson, g. eiriksdottir, l. launer, t. harris, v. gudnason, r. detrano; kopavogur/is purpose: though demographic associates of coronary and abdominal aortic calcification in elderly persons are known, similar information about the thoracic aorta is not available. we developed a protocol for scanning the thoracic aorta from the aortic bulb to the descending aorta at the level of the cardiac apex. methods and materials: computed tomographic images were acquired with a siemens sensation scanner, using sequential mode and prospective ecg gating. the ct images were processed using calcium scoring software for coronary arteries, which is modified in order to label areas and quantify calcium in the thoracic aorta. the thoracic aorta is divided into three segments: arch, ascending, and descending. these segments are labeled and calcium within them quantified and results expressed in agatston scores. results: agatston scores were acquired from individuals ( men, women) with an age range of - (mean ) years. there was a % increase ( %ci: %- %) in the total agatston score for every years of age for men, and % ( %ci: %- %) for women. there was no significant difference between men and women in the age group - and, men in the age group + had significantly lower scores than women. conclusion: a robust method for quantifying calcium in the thoracic aorta has been established. age and gender distributions of thoracic aortic calcification in the elderly show strong age dependance for both genders but significantly stronger for women than for men. b d e f a g materials and methods: ct scans of fourty patients (mean age years, female: previous aortic repair (n = ), atherosclerosis (n = ), trauma (n = ), other (n = )) with aortic or supraaortic emergency were reviewed for aortic rupture (ar), aortic or supraaortic fistula (af), covered aortic perforation (cap) with transection (at), dissection (ad), or ruptured ulcer (rau), and rapid aortic aneurysm expansion (raae). morphological findings were correlated by dsa, surgery or post-mortem. results: there were ar, af ( tracheobronchial, oesophageal, duodenal), cap ( at, ad, rau) and raae. cta was diagnostic in all cases except five af: four were diagnosed by dsa, one at post-mortem. in cases, cta correctly determined the decision of patient management. one separate vertebral artery origin was missed. two patients died before surgery. patients underwent emergency repair ( surgical, endovascular); two were observed. five thoracic af were treated by endovascular repair, two by surgery, one infradiaphragmatic af by surgery. conclusion: cta is valuable for diagnosing aortic and supraaortic emergencies and determining their treatment. however, in suspected af complementation by dsa is usually required. low materials and methods: seventy-five patients with peripheral arterial disease, divided into three groups of , underwent mdcta. mdcta scanning protocol was: collimation x . mm, thickness mm, gantry rotation time . s, kv , delay time s. the only variable among the three groups was mas, respectively , and , with a resultant ctdiw of . , and . mgy. all patients underwent dsa within hours from cta. the volume of contrast medium ( . gi/ kg of iomeprol mgi/ml), injected at . ml/s, was flushed with ml of saline. d real time interaction approach on a dedicated workstation was performed in all cases, with different reconstructions algorithms. image quality and degree of stenosis were independently evaluated by blinded readers. results: regarding image quality no significant difference was found among three protocols. slight artefacts related to beam hardening were present in the pelvis with the lower mas protocol without impairment of diagnostic quality. interobserver agreement was substantial to almost perfect in all cases. the sensitivity, specificity and accuracy resulted of , and %, independent of the mas value. conclusions: low-dose mdcta provides qualitatively acceptable images. it also appears accurate in the assessment of patients with peripheral arterial disease providing results comparable to higher dose protocols. image noise on native lowest dose protocol can be reduced or eliminated by thickening mip or mpr images. diseases had lymphadema, ( . %) had superficial venous thrombosis, ( . %) had popliteal vein or artery aneurysm. nine ( . %) limbs had pseudoaneurysms ( femoral and located at the tibial-peroneal trunk), ( . %) had avf ( detected to be femoral and at the level of the external iliac artery). four ( . %) had either abscess, or enlarged lymph nodes in the groin, compressing the veins, and ( . %) had some kind of musculoskeletal problem (muscular tear). conclusion: complete venous evaluation with duplex imaging can be very helpful in the determination of the underlying cause of leg swelling. dvt is the commonest. sonographic assessment of intima-media thickness (imt) and plaque morphology of the carotid system: a comparative study a.a. kotis , p. brestas , l. guindaglia , g. chatzakis , k. papanastasiou ; rhodes/gr, athens/gr purpose: to investigate the potential association of increase in imt with carotid artery disease extension, plaque morphology and formation of carotid bifurcation stenosis. materials and methods: patients females and males referred for color duplex ultrasound were included. risk factors for cerebrovascular disease were recorded. measurements of common carotid (cca) imt adjacent to the bulb and carotid bifurcation plaque characterization and classification were realized. coefficients of variation and k-statistics were used for assessment of variability in imt measurements and plaque characterization respectively. controversy cases underwent shu a contrast agent injection. results: significantly higher imt values were measured a) in men than in women (p < . ) b) on the left side compared with the right (p < . ). mean imt measurements showed positive linear relationship with age. we identified plaques in of carotid bulbs and stenosis (> %) in of them. echogenic us plaque appearance ( / ) predominated in comparison with echolucent ( / ) and relationship of lumen stenosis with the presence of echolucent plaques (p < . ) was found. multiple regression model revealed significantly higher imt values in patients with plaques at any side (p = . ), with more evident difference in those with stenosis (p < . ). the magnitude of plaques correlated significantly with imt. there was no relationship between plaque echogenity and imt (p > . ). we realize the existence of correlation between increase in imt in cca and extent of carotid bifurcation atherosclerotic disease. color doppler ultrasonography is accurate and reproducible for assessment of imt and detection of carotid plaque morphology and evolution. to emphasize the role of mr angiography in the evaluation of renovascular disease, and to discuss the performances, advantages and limitations of different mr angiography techniques. materials and methods: more than mr angiograms of renal arteries were reviewed for the presence of any anatomic variations, the number of renal arteries, the presence of renal artery stenosis, occlusion, or other arterial diseases together with the limitations, artifacts and pitfalls of the technique. more than examinations were performed after applying parallel acquisition technique with acceleration factors up to , using a -channel phased array body coil. influence of pat on slice thickness, image matrix, total examination time, and image quality are discussed. results: phase contrast acquisitions are extremely helpful in evaluating flow patterns in the renal arteries, and offer unique information about the flow velocity patterns. pat using sense reconstruction algorithm leads faster d mra acquisitions. this results in, not only shortening the examination time, but also shortening the breath-hold time, and providing better image quality due to decreased motion artifacts. pat-optimized d contrast enhanced mr angiography is unique among noninvasive imaging modalities in that it offers a comprehensive d evaluation of renal anatomy, and renal vascular territories in a very short acquisition time. the main challenge in the evaluation of renovascular hypertension is the detection of hemodynamically significant renal artery stenosis. a combination of different techniques (phase contrast and contrast-enhanced mra) yields the best results, comprising both morphologic and functional information. possibilities results: pathologic changes in the visceral arteries were revealed in cases, including findings with various grades of stenosis of the coeliac trunk, with stenosis of the sma, -occlusion of the splanchnic arteries. causes of visceral ischemia were: atherosclerosis (the main cause), found in patients, aorto-arteriitis -in , coarctation of abdominal aorta -in , extravasal compression-in . high correlation between conventional angiography and ce-mra was determined in patients. the functional phase-contrast mra was done in patients with stenoses of small and middle degree; it gave an opportunity to acquire information on flow parameters and to elaborate the grade of stenotic changes. the ce-mra with pc-mra gives a chance to determine the grade of stenosis with high accuracy, visualize colateral vessels, to get information on flow parameters in damaged splanchnic arteries. b d e f a g imaging findings: all degrees of local vascular damage were seen. various imaging modalities may be employed in characterising such lesions, including ct, mri and conventional angiography and venography. an addict may injure any vessel during injection and the superficially placed femoral vessels are particularly susceptible to damage. septic vascular complication is common. arterial injury resulting in haematoma formation, arterial dissection and thrombosis, arteriovenous fistula and mycotic aneurysm formation is shown. a case of torrential haemorrhage from a ruptured pseudo-aneurysm treated with coil embolisation is demonstrated. deleterious effects secondary to the injected substance, including distal ischaemia and tissue necrosis, are demonstrated. venous thrombosis is common and can be shown by multiple imaging modalities. infected venous pseudo-aneurysms with secondary septic embolisation may occur. conclusion: it is important for radiologists to become familiar with the appearance of vascular complications occurring in the setting of injection drug use. early imaging to define disease extent and possible complication is important in this difficult patient group. carotid intima-media thickness ( the aim of the study was to investigate the probable association of atherosclerotic disease with endothelial cell adhesion molecule icam- , vcam- and e-selectin levels. carotid imt was measured by high resolution ultrasonography of the common carotid arteries in predialysis patients with esrd, patients on chronic haemodialysis (hd) and controls. compared with controls, esrd and hd patients had increased imt (p < . and p < . respectively), as well as serum icam- (p < . and p < . respectively) and vcam- (p < . and p < . respectively) levels. imt was correlated with age in controls, esrd and hd patients (p = . , p = . and p = . respectively). no correlation was found between imt and other cardiovascular (cv) risk factors such as gender, lipid levels, mean arterial blood pressure, ca x p products or hd duration in hd patients. in univariate analyses, imt was correlated with history of cv events, logcrp and icam- both in esrd (p = . , p = . and p = . respectively) and hd patients (p = . , p = . and p = . respectively). moreover, compared with patients with normal imt, esrd and hd patients with increased imt (> . mm) had increased icam- (p = . and p = . respectively). multivariate analyses showed that icam- was a strong independent correlate of imt values both in esrd and hd patients (p = . and p = . respectively). vcam- and e-selectin levels failed to be a significant contributor to imt. in conclusion, chronic uraemia is associated with advanced atherosclerosis. in patients with esrd, both on conservative treatment and on hd, icam- levels are potentially a useful surrogate marker of the extent of atherosclerosis. evaluation of tumor microvessels using monochromatic x-ray after administration of angiogenesis inhibitor t. yamashita, s. imai, n. maehara, a. yamamoto, m. kobatake, y. kajihara; kurashiki/jp purpose: to quantitatively evaluate changes in tumor microvessels using synchrotron radiation in vivo after fr- administration as an angiogenesis inhibitor. the fr- agent (using propylene glycol as a solvent) was continuously infused subcutaneously to rabbits with vx carcinoma implantation at auricles. the rabbits were divided into six groups: control- and control- received only propylene glycol for three or seven days, fr- - and fr- - received mg/day/kg of fr- for three or seven days, fr- - and fr- - received mg/day/kg for three or seven days. microangiograms using a synchrotron radiation system were obtained to evaluate the tumor microvessels and tumor size. results: compared to control- group, fr-x- groups showed no significant tumor size difference (fr: diameter = . ± . mm, volume = . ± . mm , control- : . ± . mm, . ± . mm ), but microangiologically fewer vessels were observed. fr-x- groups presented significant difference (fr: diameter = . ± . mm, volume = . ± . mm , control- : . ± . mm, . ± . mm ), and many irregular vessels, findings similar to control- . there was no significant difference in tumor size and microangiographic findings between fr- -x and fr- -x. conclusion: our study demonstrates the application of new technique to examine antiangiogenic effects in vivo. the growth of the vx carcinoma were sup-pressed remarkably in the fr- -day-groups, and the microangiographic findings were similar to those of control- . the synchrotron radiation microangiogram is a useful tool to evaluate antiangiogenic agent. early atherosclerotic changes in hiv positive patients treated with protease inhibitors m. cristofaro, n. petrosillo, l. rovighi, c. bibbolino; rome/it purpose: antiretroviral therapies with protease inhibitors (pi) produce derangement of lipid metabolism with possible occurrence of early atherosclerotic changes. aim of this study is to evaluate onset of early atherosclerotic changes of epiaortic vessels in hiv positive patients, treated with pi. purpose: hematoporphyrin (hp) accumulates in arteriosclerosis selectively and shows specific fluorescence spectrum change in accordance with the stages of the sclerosis, and this fact is under discussion as to its availability to diagnosis and therapy. we paid our attention to the fact that hp accumulates in the interface of swollen collagen type iv and lipid. we assumed such a model of atheroma, and we examined the possibility of a photodynamic therapy for arteriosclerosis using collagen type iv and phosphatidyl choline liposome. material and method: we added hp to collagen type iv, then the liposome containing cholesterol to the mixture, and examined the agglutination state of the protein-lipid complex. afterwards, we irradiated that complex with led pulse of nm for one hour, and examined scattered spectroscopy, interference spectroscopy using nm semiconductor laser and high sensitivity ccd in order to observe the hp and the agglutination state of complex, and, then, analyzed the alteration of protein-lipid complex in terms of fourier analysis. result: hp gave influence to the various state of assembly of protein-lipid complex. in addition, protein-lipid integration state was changed by the irradiation of light pulse and the change of scattered light that signifies the state of dispersion was observed. conclusion: this study suggested that the molecule composition of the proteinlipid complex in the plaque was changed by the irradiation of light pulse after the addition of hp, and that the photo stimulation could contribute to the decomposition of the cumulated lipid and the stabilization of plaque. depiction of arterial branches in the abdomen using multidetector-row ct n. terayama , o. matsui , k. ueda , t. kobayashi contrast medium ( mgi) was administered intravenously at a rate of ml/sec. parameters of data acquisition were as follows: a slice thickness of . mm, reconstruction intervals of . mm, a pitch of . , and fov of cm. axial images were transferred to advantage workstation . (ge medical systems) and multi-projection volume reconstruction (mpvr) images were obtained. if the origin of each arterial branch and the route until the target organ was depicted, we regarded that the branch was visible. results: coeliac, superior and inferior mesenteric, renal, intercostal, lumbar, right and left hepatic, and anterior and posterior pancreaticoduodenal arteries were visible in all cases. cystic, right gastric and dorsal pancreatic arteries were visible in cases ( %), in cases ( %) and in cases ( %), respectively testicular arteries were visible in cases ( %), and in cases ( %), respectively. conclusion: reformation images of mdct can visualize thin arterial branches in the abdomen experimental lung injury prior to the ct examinations. in addition, segmental and subsegmental pulmonary embolism was artificially induced in four healthy pigs. scanning was performed using a -slice msct protocol using contour finding and adaptive thresholding, both plain and enhanced ct volumes were separated into lung parenchyma, vascular structures and airways. the high contrast information was used to locally match and align sub-volumes. the matched parenchymal volumes were low pass filtered, subtracted and normalized to the enhancement level in a major vessel. results: scanning was successfully performed during a total ~ s scanning time in all pigs. post-processing was technically feasible, even in underlying experimental lung injury. in pe, filling defects corresponded to areas of decreased enhancement. conclusion: it is expected that this imaging technique will increase the sensitivity and specificity of msct determination of arterial input function for measurement of brain perfusion index with tc- m compounds using a solid-state gamma camera y. uenishi, k. murase, m. nagayoshi, m. kawamata, m. takasawa, n. oku, a. takahashi, j. hatazawa; osaka/jp purpose: cerebral blood flow (cbf) can be non-invasively quantified using the brain perfusion index (bpi) determined from radionuclide angiography (ra). for measurement of bpi, accurate determination of arterial input function (aif) is necessary. we have developed a method for the automatic extraction of aif using fuzzy clustering and a solid-state gamma camera (digirad). methods and materials: ra was performed using tc- m hmpao for sec with a digirad placed on the heart and a -head spect (spect h). for comparison, the data were also acquired using a conventional gamma camera (rc- i). the aifs were obtained using fuzzy clustering (aif a ) and manual drawing of a region of interest (roi) (aif m ) from the region of aortic arch. the rois were also drawn over the left and right cerebral hemispheres. the bpi values were calculated using spectral analysis (bpi a : bpi obtained using aif a , bpi m : bpi using aif m ). the deadtimes of digirad and rc- i were measured using the reference source method. results: the mean and sd of bpi m became larger with increasing roi size, while the bpi a was almost constant regardless of roi size. the sd of bpi a was smaller than that of bpi m . the deadtime of digirad ( . ± . µsec) was much less than that of rc- i ( . ± . µsec). furthermore, the deadtime of digirad was almost constant regardless of the volume of phantoms, while that of rc- i increased with increasing volume. the proposed method appears promising for calculation of the bpi, because it allows automatic and objective determination of aif. spectroscopic imaging of radiation-induced effects in the brain after brachytherapy g. bajzik , j. julow , i. repa ; kaposvar/hu, budapest/hu purpose: the effect of brachytherapy, characterized by rapid dose fall off from the radiation source, is different compared to external radiation. we used proton mr spectroscopic imaging to investigate brain tissue response to brachytherapy. methods and materials: twenty glioma patients treated with surgery and/or brachytherapy were studied with mr imaging (t weighted images and post contrast t weighted images) and d proton mr spectroscopy (tr: ms, te: ms, voxel size . cm ). brachytherapy resulted in central necrotic zone, surrounded by an enhancing rim (demarcation zone) and an outer edematous zone on mr images. in each patient sample, spectra of the three zones and the normal appearing white matter were analyzed. results: in demarcation zone, choline/creatine (cho/crea) ratio ( . ± . vs. . ± . , p = . , two-tailed unpaired student's test) and choline/n-acetylaspartate (cho/naa) ratio ( . ± . vs. . ± . , p = . ) was increased compared to normal white matter. in edematous zone cho/crea ratio was increased ( . ± . vs. . ± . , p = . ), cho/naa ratio was not different ( . ± . vs. . ± . , p = . ) compared to normal white matter. in demarcation zone we found elevated cho/crea ratio ( . ± . vs . ± . , p = . ) and cho/naa ratio ( . ± . vs . ± . , p = . ) compared to edematous zone. in the central necrotic zone we could only detect lipid and/or lactate peaks. conclusions: mr spectroscopy demonstrates significant spectral differences in the brain following brachytherapy. tumor recurrence can be suspected in cases where focal choline accumulation is detected. does ct-perfusion imaging based on parametric deconvolution permit prediction of infarct volume in acute stroke? g. bohner , a. förschler , c. zimmer , r. klingebiel ; berlin/de, leipzig/de objectives: suitability of quantitative ct-perfusion (ctp) imaging for assessing the early as well as final infarct volume in acute stroke patients. material and methods: twenty-two patients with acute cerebral ischemia underwent ctp on admission. parameter maps of cerebral blood perfusion (cbp), cerebral blood volume (cbv) and mean transit time (mtt) were generated using a parametric deconvolution algorithm. additional diffusion weighted imaging (dwi) was performed in seven cases. volume of diffusion abnormality was compared with the infarct volumes as assessed by two blinded neuroradiologists for each of the perfusion maps. in addition, final infarct volumes were determined on the basis of follow-up studies. results: mean volumes of ischemia as assessed by cbp, cbv and mtt were . , . and . ml respectively. amongst the patients in whom dwi was performed, six showed perfusions deficits in all perfusion images. in one patient, who had no dwi abnormality, perfusion deficits only were found on cbp and mtt images. mean volume of dwi changes was . ml. the most significant correlation was found between the extent of cbv reduction and dwi infarct volume (r = . , p < . ). mean final infarct volume was . ml for all patients. cbv reduction showed the best correlation (r = . , p < . ) with final infarct volume. conclusion: our findings suggest that cbv maps based on the ctp technique applied in this study permit prediction of infarct volume in early stroke, so ctp would deliver information about the ischemic penumbra. hepatic encephalopathy in alcoholic and non-alcoholic subjects: correlation of magnetization transfer ratio, apparent diffusion coefficient and critical flicker frequency m. cohnen, g. kircheis, h.-j. wittsack, f. miese, f. wenserski, a. saleh, d. häussinger, u. moedder; düsseldorf/de purpose: to correlate quantitative changes of water content in brain parenchyma using magnetization transfer imaging (mti) with different stages of hepatic encephalopathy (he). materials and methods: forty-three patients with hepatic encephalopathy and age-matched control subjects underwent mr imaging ( . t vision, siemens) including mti, mr-spectroscopy (mrs) and diffusion weighted imaging (dwi). liver cirrhosis was due to alcohol in patients whereas non-alcoholic disease was found in . patients underwent assessment of critical flicker frequency (cff) as test for he with a frequency under hz considered pathologic. magnetiza-tion transfer ratios (mtr) and apparent diffusion coefficient (adc) values were determined in basal ganglia, posterior white matter and pons using roi-measurements. single-voxel mrs was performed using a steam sequence (te = ms, tr = . s) assessing basal ganglia and posterior white matter. results: mrs showed typical findings with decreased myo-inositol combined with increased glutamine/glutamate with increasing degree of he indicated by a reduced cff. compared to controls, mtr showed a significant decrease both in the basal ganglia as well as in the posterior white matter. in alcoholic subjects, this was independent from the degree of he, whereas in posthepatitic cirrhosis a correlation was found. a non-significant increase in adc was noted with a decrease of cff in both groups. conclusion: the results may indicate an increase of free protons in the brain parenchyma of patients with he possibly supporting the hypothesis of a lowgrade cerebral edema. the differences between alcoholic and non-alcoholic subjects may hint at a general brain damage due to alcohol independent of hepatic failure. assessment of cerebral blood flow values obtained from dynamic perfusion computed tomography: a comparison with positron emission tomography h. yokote, k. nakayama, t. okamura, n. tsuyuguchi, k. kitamura, n. ozawa, s. minamikawa, t. tashiro, y. inoue; osaka/jp purpose: among imaging modalities measuring regional cerebral blood flow (cbf), dynamic perfusion computed tomography (ct) studies are easy to perform and readily available without specialized equipment. however, quantitative studies comparing perfusion ct with positron emission tomography (pet) have been few. we assessed the correlation of cbf values obtained from perfusion ct and pet. methods and materials: eleven patients with various cerebrovascular diseases underwent perfusion ct and pet. the perfusion ct studies were performed with a four-channel multi-detector row ct scanner. four contiguous -mm ct sections were cine-scanned during a ml bolus of intravenous contrast medium at a rate of ml/s, with a total acquisition time of s. cbf maps were obtained from perfusion ct data by central volume principle and deconvolution method. on cbf maps of both perfusion ct and pet, multiple regions of interest (roi) were drawn independently, including frontal, temporal, parieto-occipital territories, caudate nucleus, thalamus and white matter in both hemispheres. rois were visually matched by agreement of three radiologists. mean cbf values of multiple rois with both ct and pet were compared through linear regression analysis. results: correlation coefficients for individual analysis ranged from . to . , overall . . linear regression analysis showed moderate correlation between perfusion ct and pet values (slope = . , intercept = . ). conclusions: our study has shown moderate correlation of cbf values between perfusion ct and pet. despite the various alterations of cerebral blood flow in our cases, perfusion ct studies were validated to be an easy-to-perform imaging technique to assess cbf values. ageing of the cerebrum: assesment with quantitative proton mr spectroscopy j. ostojic, r. semnic, d. kozic, d. bogdanovic, m. prvulovic; sremska kamenica/yu introduction: aging is recognized to originate from a diversity of mechanisms that blur the limits between normal and pathologic processes. the purpose of this study was to determine the early effect of normal aging on the regional distribution of brain metabolite concentrations, including n-acetylaspartate (naa), choline (cho) and creatine (cr). methods: seventy nine volunteers, ages to years, were examined by localized single-voxel proton mr spectroscopy at . t using press sequence.voxels x x cm were placed in the middle white matter (wm) of the centrum semiovale in the left hemisphere and in the occipital gray matter (gm) of posterior parietal lobes. the ratio of naa/cho were calculated, as well as absolute concentrations of naa, cr and cho.quantification was performed using method of external standard. results: absolute concentrations (mm/kg) of cho increased with age both in wm (r = . , p < . ) and in gm (r = . , p < . ). cr concentrations were much higher in gm than in wm and significantly higher in the old than young subjets (r = . , p < . ). the change of wm cr with age failed to meet p < . criteria. naa concentration was higher in gm than in wm and did not differ between young and old subjects. the age dependency of gm naa/cho ratio was technical inadequacies of plain films in an emergency department b. rajashanker, f. jabeen, r.w. whitehouse; manchester/uk purpose: . to assess the diagnostic quality of radiographs performed in the emergency department in a major referral hospital. . to determine the type and number of inadequacy encountered most often. materials and methods: two specialist registrars (trainees) in radiology, reviewed plain films performed in a busy emergency department. films were examined for inadequacies, which impair the diagnostic quality of the examination. despite artefacts or exposure problems, if a reasonable diagnostic conclusion could be made, the film was considered to be adequate. if a film was found to be inadequate, clinical details (that could have contributed to the poor quality) were looked at, and taken into consideration. artefacts obscuring the area of interest, inadequate collimation, incomplete examinations and improper exposure factors were considered as inadequate. the number and the type of inadequacy, the type of examination were noted. results: . the study showed that about % of the total films performed were inadequate. there are no national figures we could find to compare our results with to evaluate the performances on our emergency department. . the type of inadequacy encountered most often was the presence of jewellery obscuring the area of interest ( %), followed by artefacts caused by clothing ( %). chest radiograph was the most frequently encountered inadequate examinations ( %). conclusion: national guidelines regarding target quality radiographs that need to be achieved in institutions need to be published. the inadequacies were pointed out for better performance in future. drug courier pays a high price for easy money b. szabó, z. mocsári, s.o. farkas, p. magyar, Á. takács, k. karlinger, e.k. makó; budapest/hu purpose: the aim of this study is to show the most common methods for examining drug couriers and their results. method: native abdomen ct scans and native abdomen x-rays and us examination were performed on probably positive patients. in the year until now we examined patients. four positive cases were found. one of them after the ct examination died in the hospital, because leakage of a capsule. conclusion: swallowers before traveling swallow - cm long, cm diameter capsules, which they hope to retrieve after traveling. stuffers insert drug filled corpus alienums into the rectum or vagina. the danger of this type of drug smuggling is the braking or bursting of the capsules, leading to potential fatal drug overdose. steps to european union from europe: reforms of education of hungarian radiographers a. maléta, z. mocsári, s.o. farkas, Á. mester, k. karlinger, e.k. makó; budapest/hu purpose: the aim of this presentation is to show a short summary of the education of hungarian radiographers currently and in the future. method: assessment based on the bologna contract new education system with the cooperation between the society of hungarian radiographers and hungarian ministry of education according to the european directives. results: the base of the modular education system is already laid down. the construction and edition of the new scientific books and the preparation of the teachers and professors has already commenced. conclusion: since the hungarian educational system of radiographers has a history of nearly ninety years we need to use the lessonst of the past into the new system. since the result of the reforms requires a long time, well-organized feedback mechanisms is needed to ensure the effectiveness of the reforms. origin of military radiology: first use of x-rays in ottoman empire a. tunaci, n. yildirim, y.i. ulman, e. yekeler, h. genchellac, m. tunaci, g. acunas; istanbul/tr x-ray usage as a diagnostic device was realized in turkey first time at the military medical school in by esad feyzi, who was an intern and physics assistant. this new invention attracted attention so much that dr. salih and rifat osman also joined him and they started to work in a small unit, where they practiced radiographic examination.the following year, yildiz portable military hospital was reserved for wounded patients from the battlefield of the ottoman-greek war. this mobile hospital, which was activated in , was exported from germany and comprised duquer system pavilions that were reassembled within hours. they took away the x-ray equipment from the military medical school to yildiz and began to produce images of bullet and shrapnel pieces from the patients sent from the battlefield. kuttner and nasse from german red cross came to istanbul in . they were surprised to see that this new invention was already being used there. on the greek side english physicians used two x-ray devices that were transferred from england to pire harbour on may . therefore the ottoman-greek war took its place in medical literature as the first war that radiographic images were taken, first by the turkish and soon after by german and english physicians. references stated that english physicians took x-ray images of wounded greek soldiers and there is no record about greek physicians taking part in this work. turkish physicians continued to work on radiographic images during the war which they had started shortly after the discovery of x-rays. to demonstrate how to perform a numeric flow simulation in patient-specific vessels based on isotropic ctdatasets. to illustrate the possibilities of this promising tool on clinical cases. background: cfd was introduced recently to investigate the behaviour of blood flow. different studies determined cfd as a valuable method to demonstrate the flow and pressure. most studies use simplified standard models for the simulations. this exhibit explains methodological aspects of cfd and how numeric flow simulations are performed in patient-specific vessels based on high-resoloution ct datasets. possible applications of cfd in the cardiovascular field are discussed. procedure details: isotropic ct-angiography data are used to segment the vessels. after generation of a surface mesh using the marching cube algorithm, the model is transferred into a volume mesh and the different entities and walls were defined. specific parameters as velocity at the inlet boundary, flow parameters, and material specific parameters have to be assigned in a next step.the simulation itself provides detailed information about flow-behaviour and pressure. this helps to identify possible zones for development of artherosclerosis or, especially in aneurysms, locations for a possible rupture. furthermore, blood flow can be quantified and e.g. effects of stent-graft implantations or the benefit of a coronary bypass can be demonstrated. conclusion: cfd in combination with patient-specific patient-data is a promising method to visualise flow-effects. in the near future cfd will also gain increased importance for pre-interventional planning of vascular procedures. background: evaluation of the central veins is easily achieved with contrastenhanced mr angiography. "indirect" ce-mrv, a technique where the veins are evaluated during the "venous" phase has been largely replaced by "direct" ce-mrv, a technique whereby dilute contrast agent is injected into the upper extremity veins and imaging is performed during the first-pass. imaging findings: in order to overcome t *-induced susceptibility-effect commercial strength contrast-agent is diluted with saline. in our institution we use a % solution ( cc of contrast agent in cc saline). - cc of contrast is injected over the duration of the breath-hold scan (tr/te/flip msec/ msec/ deg, matrix x , fov - mm x - mm. - slices, mm interpolated to mm). mask subtraction of a pre-contrast scan is performed. the spectrum of findings in normal and diseased veins is illustrated. examples of thrombosis, stenosis and occlusion of the central veins will be illustrated. common pit-falls and artefacts will also be emphasised. conclusion: "direct" ce-mrv is an effective modality for evaluating the central veins. a simple robust method is presented, along with examples of the appearance in normal and pathological states. multidetector row ct angiography of the lower extremities for the assessment of traumatic vascular injury e.e. williamson, j.c. hellinger, a. napoli, g.d. rubin, d. fleischmann; stanford, ca/us learning objectives: . to describe the ct angiographic (cta) technique used in the evaluation of traumatic lower extremity (le) vascular injury. . to illustrate the spectrum of vascular and nonvascular pathology found during le-cta in the setting of trauma. background: digital subtraction angiography (dsa) has long been considered the imaging standard for traumatic vascular injury. cta is a more readily available, noninvasive imaging alternative which is less expensive, less resource intensive and can be used to assess bone and soft tissue injuries without the need for additional imaging tests. in this exhibit we highlight our experience with cta in trauma patients.procedure details: all examinations were acquired using or -channel multidetector-row ct (mdct). contrast media was administered at - cc/sec using bolus-tracking technique. thin section images were obtained from one vascular segment above the level of trauma through to the foot with collimation of . and pitch of . - . . table feed and gantry rotation speed were adjusted to optimize scan duration to match the peripheral delivery of iv contrast. all studies were reformatted in -dimensions using volume rendering, maximum intensity projection and curved planar reformation techniques. conclusion: the spectrum of direct vascular injuries included large and small vessel occlusions, pseudoaneursyms, active extravasation, and vasospasm. nonvascular pathology with indirect effects on the vascular system included complex fractures, open soft tissue wounds, hematomas, and joint effusions. ct angiography of the extremities is a promising technique in the evaluation of traumatic le arterial injury with the ability to assess nonvascular injuries without the need for additional studies. there is a wide range of imaging techniques available for mrv evaluation of the central chest veins, including conventional non-contrast time of flight imaging and contrast enhanced methods such as the newer vespa (venous enhanced subtracted peak arterial) sequences. imaging findings: examples of common central venous pathologies will be illustrated from our database of over patients, who underwent mr at . t using various sequences including non contrast time-of-flight, and post gadolinium true fisp, t -weighted vibe and d flash sequences. typical scan parameters included matrix x , fov - x - mm, - slices, mm interpolated to mm. common pathologies demonstrated included thrombo-embolic disease, central venous obstruction due to local tumour extension, congenital and acquired venous stenoses, av fistulas and infected venous pseudo-aneurysms. conclusion: mrv is a robust, highly accurate and non-invasive technique for assessment of central venous pathology. accuracy of automated centerline approximation algorithms for lower extremity vessels in a cta phantom a. la cruz , m. straka , a. köchl , m. Šrámek , e. gröller , d. fleischmann ; vienna/at, stanford, ca/us purpose: the accurate determination of the central vessel axis is a prerequisite for automated visualization (curved planar reformation) and quantitation. the purpose of this work was to assess the accuracy of different algorithms for automated centerline detection in a phantom simulating the peripheral arterial tree. materials and methods: six algorithms were used to determine the centerline of a synthetic peripheral arterial vessel (aorto-to-pedal arteries, diameter - . mm) dataset ( x x , voxel size . x . x . mm). they are ray-casting/ thresholding (rct), ray-casting/maximum gradient (rcmg), block matching (bm), fitting to ellipse (fe), center of gravity (cog), and randomized hough transform (rht). gaussian noise with a sigma: , and was used to observe the accuracy of the method under noise influence. the accuracy of automatic centerline determination was quantified by measuring the error-distance between the derived centerlines, and the known centerline course of the synthetic dataset. results: bm demonstrated unacceptable performance in large vessels (> mm) when the shift used was less than voxels. rcmg demonstrated a greater error (mean of the error . mm) in large diameter (> mm) vessels than in small diameter (< mm) vessels (mean of the error . mm). because rht and fe use canny edge detector preprocessing, both are sensitive to noise. cog and rct keep the mean of the error-distance significantly smaller ( . mm and . mm respectively) than all other algorithms. conclusion: cog and rct algorithms provide the most efficient centerline approximation over a wide range of vessel diameters. detection of the adamkiewicz artery by subtraction magnetic resonance angiography h. hyodoh, r. shirase, h. akiba, m. tamakawa, k. hyodoh, k. aratani, n. kawaharada, k. morishita, m. hareyama; sapporo/jp purpose: to assess the ability of magnetic resonance angiography (mra) to depict the adamkiewicz artery. materials and methods: fifty-one patients with thoracoabdominal disease underwent mra of the bypass graft region. subtraction maximum intensity projection (mip) images and cine-mode displays were produced to identify the adamkiewicz artery, its side of origin and branching level, and the drainage vein. for statistical analysis, signal intensity ratio (snr) and contrast noise ratio (cnr) were also calculated. results: in ( . %) of the patients, at least one adamkiewicz artery was seen to arise from an intercostal artery. two adamkiewicz arteries were identified in ( . %) of the patients. spinal drainage veins were visualized in ( . %) of the patients, a single drainage vein in of the ( . % of the total patient group) and two drainage veins in the remaining ( . % of the total patient group). in of the patients, both the adamkiewicz artery and the drainage vein were detected during the early phase. conclusion: subtraction mra depicts the adamkiewicz artery in a high percentage of patients. -row multislice computed tomography of pulmonary veins: assessment before and after cryothermal energy ablation r. maksimovic , f. cademartiri , m. scholten , l. jordaens , p.m.t. pattynama ; belgrade/yu, rotterdam/nl purpose: electrical isolation of the pulmonary veins (pvs) with radiofrequency ablation in treatment of patients with paroxysmal atrial fibrillation is associated with pv stenosis. cryothermal ablation (ca) is a new promising method in treatment of these patients. although initial experience of ca showed no associated pv stenosis, results at follow-up have not been reported. the aim of the study was to assess pvs for the presence of stenosis three months after ca by contrast enhanced angiography on -row detectors multislice computed tomography (msct). materials and methods: twenty four patients (mean age . ± . years, males) with symptomatic atrial fibrillation underwent ca in pvs. all patients underwent complete clinical work-up, electrophysiological study, ultrasonography and msct angiography before ca. msct pulmonary angiography was performed three months after the procedure to evaluate ostium and the proximal mm of all pvs. results: dimensions of the treated pvs remained unchanged: the coronal ostial diameter was . ± . preprocedural vs. . ± . mm at follow-up, p < . , ratio of coronal and axial diameter . ± . vs. . ± . , p < . , respectively and the coronal diameter of the proximal mm from the ostium . ± . mm vs. . ± . mm, p < . , respectively. conclusions: ca of pvs in atrial fibrillation has not been associated with stenosis at the orifice and proximal mm of the pvs after three months follow-up. msct pulmonary venography is a reliable, noninvasive method for assessment of pvs in a three-dimensional manner prior to ablative treatment and during the follow-up period. magnetic resonance venography without contrast media for patients with lower extremity varicose veins j. koizumi , m. wada , t. horie , e. kimura , i. muro , k. myojin , t. niibori , y. imai ; isehara/jp, wako/jp purpose: to compare three different sequences of magnetic resonance (mr) venography without contrast media for patients with varicose veins below the knee. materials and methods: for patients with lower extremity varicose veins, mr-venography using time-of-flight (tof), fat suppressed t -weighted turbo spin echo (tse) and balanced turbo field echo (btfe) were compared.the original images on . tesla gyroscan (philips) were transferred to a workstation (m quadra, ziosoft inc.) and d images were reconstructed using maximum intensity projection (mip), multiplaner reformation (mpr), and volume rendering (vr) techniques. for analysis, the veins were divided into superficial, deep venous systems, perforating and varicose veins, and were evaluated using three points scale by a radiologist and a surgeon.results: superficial, deep venous systems and perforating veins were best visualized on btfe (p < . ), while varicose veins were equally visualized on tse and btfe. tof provided poor visibility except in popliteal veins. coexisting arterial system seen on btfe could be discriminated from the deep venous system especially on axial reconstructed images. the varicose veins connected with the superficial venous systems and perforating veins on the surface of the muscle were easily recognized on vr only using btfe. conclusions: mr-venography using btfe displays the best images of the whole venous system in patients with varicose veins below the knee. magnetic resonance angiography in potential live renal donors: a joint radiological and surgical audit a. mizzi, g. roditi, m. subramaniam; glasgow/uk purpose: to assess the impact of joint surgical and radiological audit on the accuracy of mra reports in the evaluation of potential live renal donors. materials and methods: we analysed the case records of live renal donors who underwent gadolinium enhanced mra as part of pre-operative evaluation to assess renal vasculature between august and july . in cases of discrepancy between mra reports and surgical findings, studies were retrieved to the mri workstation and subjected to detailed joint clinical and radiological review. scan quality was assessed and sources of discrepancy were identified. results: there were donors, men and women. reported mra findings were fully confirmed at surgery in of cases. these scans were not analysed further. in donors, the findings at surgery were discrepant with the radiological reports. there were cases of "missed" early branches and cases of "missed" accessory arteries. in the first year of audit there were discrepant cases out of ( %), all of which were radiological reporting errors. the number of discrepant cases in the second year dropped to out of cases ( %). neither of these was a radiological reporting error. there was one "missed" early renal artery branch in the third year of audit, which was visualised on mra review. conclusion: the accuracy of preoperative mra in potential live renal donors is high. radiological reporting of mra examinations is improved through careful clinical feedback, audit and interdisciplinary co-operation. d contrast-enhanced mr portography to scan the entire volume of upper abdomen with maximal spatial resolution using parallel acquisition techniques: preliminary experience d. horák, m. herman, j. bucil, j. klein, j. nekula; olomouc/cz purpose: to assess the feasibility and to evaluate the benefit of a d contrastenhanced (ce) mr portography (mrp) covering the entire volume of upper abdomen with maximal spatial resolution using grappa (generalized autocalibrating partially parallel acqusitions). materials and methods: patients were examined before or after surgical creation of porto-systemic shunt. t -weighted d angiographic sequence with d centric reordering optimized using a grappa factor of was performed at a . t scanner (siemens symphony maestro class) in three phases after administration of - ml of . -molar or ml of -molar gadolinium-dtpa followed by ml saline solution into the canula in antecubital vein. anatomical postcontrast t -weighted d spoiled gre with fat saturation was added. parameters of hires d-ce-mra sequence: in-plane resolution = . x . mm, number of partitions = , slice = . mm, partial-fourier = / , fov = , tr/ te/fa = . / . / , ta = sec. results: large evaluated volume hires mrp sequence is suitable for detailed postprocessing (subvolume mips) in any plane, imaging borderline vessels like recanalized umbilical vein or retroperitoneal collaterals, usually best seen in portal phase. robust protocol can be very easily adapted for uncooperative patients covering the same evaluated volume. functional information (flow direction) can be added with phase contrast mra flow quantification sequence. conclusion: our preliminary results show that the application of grappa allows to capture the whole portal vascular system without missing any important collateral with high spatial resolution in cooperative patients and with sufficient spatial resolution in non-cooperative ones. it's possible to image the flow in surgically placed porto-systemic shunts. gadobenate dimeglumine (multihance®) in contrast-enhanced mr angiography m. kirchin , r. la ferla , m. daprà , g. pirovano , a. spinazzi ; milan/it, princeton, nj/us purpose: gadobenate dimeglumine (gd-bopta) has high t relaxivity in blood (r = . °mm- s - ) compared to other gadolinium agents. this review is aimed at defining the usefulness of gd-bopta for ce-mra. methods: preliminary intraindividual, crossover studies were conducted in healthy volunteers to compare gd-bopta and gd-dtpa at the same dose and injection rate for mra of the abdominal aorta and run-off vasculature. subsequently, phase ii multicenter trials were performed in the renal/abdominal arteries (n = patients), the pelvic arteries (n = patients) and the carotid arteries (n = patients) to establish the optimum dose of gd-bopta for ce-mra. standard unenhanced and contrast-enhanced sequences were employed and images were evaluated by independent, off-site blinded radiologists on the basis of segmental and overall diagnostic quality scores per patient and dose. the crossover studies demonstrated significantly greater signal intensity enhancement after gd-bopta compared to gd-dtpa both in the abdominal aorta and run-off vasculature. the phase ii studies revealed dose-related trends for the contrast-enhanced images in each territory and good correlation between readers. the greatest increase in diagnostic quality from unenhanced mra to ce-mra occurred at . mmol/kg bw while a higher dose of . mmol/kg bw provided little or no additional benefit. ce-mra led to an increased number of patients with lesions detected and increased reviewer confidence for lesion characterization. conclusions: ce-mra with gd-bopta is more efficacious than unenhanced mra and appears superior to ce-mra with conventional gadolinium chelates. the optimum dose for ce-mra of vascular disease is indicated to be . mmol/ kg bw. influence of ct scanner and observer on volume measurement: five glass phantoms filled with diluted contrast material underwent one single-detector row ct (sdct) and two multi-detector row ct (mdct) examinations. for each ct scanner the clinical protocol for ct angiography of the abdominal aorta was used. in a first step the resulting volumes were measured using a special software with automatic outline detection without need of manual segmentation and thus without intra-and interobserver variability to evaluate differences between the ct scanners. in a second step the volumes were measured by three observers using a standard software for manual outline detection to assess inter-and intraobserver variability. results: there was no statistically significant difference between the ct scanners with the corresponding standard protocols (p = . ). inter-and intraobserver variability was . and . ml and lead to significant difference in volume measurement between the observers (p = . ). conclusion: volume measurement does not seem to be influenced by the used ct scanner. the interobserver variability is significant, but slight. consequently, changes of the volume of abdominal aortic aneurysms in follow-up ct angiography after endovascular repair can be considered as true change and not as scanner related. -slice multi-detector ct angiography in the assessment of stenoocclusive disease of the carotid artery bifurcation and vertebral artery origin s. lee, h. roh, h. byun; seoul/kr purpose: to determine the accuracy of -slice multi-detector ct angiography ( -mdcta) in the assessment of steno-occlusive disease of the carotid artery bifurcation and vertebral artery origin. materials and methods: in patients, carotid arteries and vertebral arteries were evaluated with -mdcta and conventional digital subtraction angi-ography (dsa). standardized -mdcta acquired from the circle of willis to the aortic arch. -mdcta data were displayed with maximum intensity projection (mip) for carotid and vertebral arteries and with volume rendering (vr) for carotid artery. the percent diameter stenoses of the carotid artery bifurcation and vertebral artery origin were measured separately on mip, and/or vr ct images and dsa. the carotid stenosis was graded as no, mild (< %), moderate ( - %), severe (> %), and occlusion with nascet criteria. the vertebral artery stenosis was also assessed with the same five-grade scale. the results of -mdcta were correlated with the gold standard of dsa. results: all carotid and vertebral arteries were assessable on mip and/or vr ct images. correlation between stenosis measured with -mdcta and that measured with dsa were good (pearson correlation: on carotid mip images, c = . , < . ; on carotid vr images, c = . , < . ; on vertebral origin mip images, c = . , < . ). the aggrement rate of stenosis grading between -mdcta and dsa was also high ( . % on carotid mip; . % on carotid vr images; . % on vertebral origin mip images). conclusion: -mdcta has a high accuracy for the evaluation of carotid and vertebral artery stenosis and provides multiplanar information preoperatively. does high iodine concentration contrast agent improve arterial enhancement in mdct angiography of the run off vessels? comparison of three different iodine concentration contrast agents f. venditti, c. catalano, m. francone, f. fraioli, a. napoli, v. votta, r. passariello; rome/it purpose: to evaluate whether a high iodine concentration contrast agent may determine a greater arterial contrast enhancement in the visualization of aortoiliac and peripheral vessels. materials and methods: sixty-six patients, randomly divided into two groups, a and b, were studied with a multi detector-row ct (mdct) scanner. both groups were divided in three subgroups (a = , a = and a = mgi/ml; b = , b = and b = mgi/ml) in which different iodine concentrations were injected. in group a variable flow rates were used. in group b the flow rate was kept constant at . ml/s. quantitative and qualitative analyses were performed at predefined fixed vascular segments. results: in both groups a greater density value was achieved using the high concentration contrast agent, in particular at the level of the superficial femoral artery for subgroup a and at the level of the popliteal artery in subgroup b . for all vascular segments the best enhancement was visualized for group b . no statistical significant differences were achieved between the two different groups in the visualization of sub-popliteal vessels. conclusion: our study demonstrates that the use of a high iodine concentration contrast agent increases arterial enhancement of the peripheral arterial tree using mdct scanner. multidetector ct angiography in arterial occlusive disease of the lower extremities: a step forward in vascular diagnostic procedures? m. maggi, s. alborino, e. paci; ancona/it purpose: multidetector computer tomography (mdct) angiography is a new available technique for studying arterial disease of the lower limbs. in this study we report our experience with mdct angiography in evaluating atherosclerotic lesions of peripheral arteries. materials and methods: patients ( / male/female, age ± , in and in rutherford category) were investigated by mdct between april and march , patients underwent digital subtraction angiography (dsa) of the lower extremities within days from mdct angiography. we used a - channels ge medical system ct-scanner, needle-cannula, flow rate ml/ sec, ml iodinated contrast medium / mgi/ml, bolus tracking program (smart prep). mip and volume rendering reformations were used. dsa and mdct angiograms were blindly evaluated by two vascular radiologists according to the following parameters: stenosis (lumen reduction - %), occlusion (> %), absence of lesion (< %) in arterial segments, for each patient. therapeutical implications of mdct angiography were also evaluated. results: concordance between mdct angiography and dsa was . % in detecting and . % in excluding lesions, . % in defining stenosis and . % for occlusions. in / patients percutaneous approach was modified according to ct images. conclusions: mdct angiography could be considered as the first step in diagnostic procedures of peripheral arterial disease and could also modify therapeutical strategy and vascular radiologist's performance. further studies are needed value of long-term routine ultrasound assessment of hemodialysis fistulae in comparison with clinical parameters a. wojciechowski, m. golebiowski, m. grzeszczyk, w. kania; warsaw/pl purpose: the aim of the study was to correlate the results of clinical and ultrasonographic long term follow-up of hemodialysis fistulae. the utility of routine ultrasound examination was discussed. materials and methods: patients underwent doppler ultrasound evaluation of dialysis fistulae. patients were randomly assigned for evaluation. in addition routine tests for blood cell count, hematocrit, kt/v, urr and blood pressure in the venous line were available in all patients. all patients had native fistulae. a single unit (atl ultramark hdi) was used for ultrasound examinations. results: results of laboratory and clinical parameters were abnormal in almost % of patients. low levels of hematocrit (< %) were observed in . %, albumin (< . g/l) in . %, elevated pressure in venous line (> mmhg) in . %. abnormal kt/v (< . ) and urr (< %) in . and . % respectively. us revealed: pseudoanerysms in . % of patients, hematomas adjacent to the fistula . %, arterial steal syndrome in . %, stenosis in . % and mural thrombi in . %. in . % of patients brachial artery blood flow was less than ml/min and in % of cases dialysis fistula flow was greater than ml/min. conclusion: only . % patients had no abnormal either clinical, morphological nor flow parameters. . % of patients had abnormal clinical and us morphological parameters. colour doppler ultrasound is more sensitive than clinical or laboratory methods in detection of hemodialysis fistula dysfunction. it is strongly recommended to start regular ultrasound examinations of all dialysed patients. material and methods: patients with brescia-cimino-shunts were examined by independent examiners with i. a. dsa and vascular ultrasound. all patients had a shunt-volume of less than ml/min., an angiographical stenosis of the anastomosis of at least % or changes of the venous portion of the shunt were examined. studies were performed with the aid of a multifrequency ultrasound probe ( - mhz, logic , , ge) using b-mode, color coded duplex (ccd) and b-flow, first of all in the brightness modification. results: in the group of anastomotic stenosis measurements of intrastenotic diameter gave for i. a. dsa values of (average: . mm), for b-mode (average: . mm), for ccd (average: . mm), for brightness mode of b-flow (average: . mm) and for b-flow with b-mode information values of (average: . mm). the best agreement with dsa was achieved by brightness mode of b-flow. while in b-flow in cases of anastomotic lesion or venous stenotic lesions, hypoechogenic vessel wall alterations were detected, they were not seen in b-mode in any case and not reliably in ccd due to blooming artifacts. in cases of venous aneurysm and vascular elongation a flow detection free of artifacts was only demonstrated by b-flow. conclusion: ultrasound b-flow detects anastomotic stenosis of hemodialysis fistula better than other ultrasound modes. the visualization of hypoechogenic vessel wall alterations and the degree of stenosis is also better appreciated by this mode. ultrasonographic assessment of internal mammary artery in the screening of radiotherapy induced coronary artery disease n. tuncbilek , o.o. okten , h.m. karakas ; edirne/tr, malatya/tr purpose: radiotherapeutically treated breast carcinoma patients are prone to the development of iatrogenic fibrosis and plaque formation in coronary vasculature. in this group of patients, early changes in the diameter and the flow characteristics of internal mammary artery, hypothesised as the surrogate markers of the coronary artery disease, were ultrasonographically (usg) investigated. the study group was consisted of breast cancer patients with ages between and (mean age . yrs) and age-matched control subjects. all patients were radiotherapeutically treated to month ago. internal mammary arteries were ultrasonographically investigated with . mhz linear transducer in longitudinal planes. diameters of the arteries were measured in m-mode; resistivity indices (ri), pulsatility indices (pi), peak systolic and end diastolic values were measured with color doppler usg. mean values of the above mentioned parameters were statistically compared among patient and subject groups. results: mean diameter of the internal mammary arteries were found to be . mm in patients and . mm in control subjects. there was a statistically significant difference between two groups regarding the stated parameter (p < . ). in contrary, the difference of ri, pi, peak systolic and end diastolic values were not statistically significant in two groups studied. conclusion: shortly after radiotherapy the diameter of the internal mammary artery significantly decreases but its flow spectrum does not change. ultrasonographically performed simple diametric measurements may be used as rapid and noninvasive screening tests to predict the development of coronary artery disease. assessment and follow-up of endovascular repair of aortic aneurysm with multislice computed tomography m. fernández -velilla, m. martí, y. herrero, j. echeveste, g. garzón, n. gómez-león; madrid/es purpose: to evaluate the usefulness of multislice computed tomography (msct) after placement of endovascular stent-grafts for treatment of aortic aneurysms. matrials and methods: seventy-six patients underwent msct angiography following treatment of thoracic or abdominal aortoiliac aneurysm with endoluminal stent-grafts. msct was performed from the supracoeliac aorta to the femoral artery bifurcation in patients with aneurysm affecting the abdominal aorta (n = ), and from the thoracic inlet to the femoral bifurcation in patients with thoracic aneurysm (n = ). the standardized protocol consisted of a nonenhanced ct ( mm collimation, pitch of , reconstruction interval of mm, table speed of mm per rotation and a single-breath-hold acquisition), followed by a contrast-enhanced ct. all images were postprocessed in a workstation (vitrea ; vital images, plymouth, minn). the image postprocessing included multiplanar reformats, mip, shaded-surface display, and volume rendering. the studies were assessed for stenosis, thrombosis, perigraft leakage, migration, angulation of graft, and enlarging sac size without visible endoleak (endotension). follow-up ct was performed prior to discharge and at six and twelve months after intervention. results: after stent-graft placement in the patients, ct demonstrated graft permeability in ( %), perigraft leaks in ( %), angulation of the graft in ( %) and endotension in ( . %). conclusion: msct was able to study the entire aorta with a maximal and homogeneus opacification. the msct technique allowed fast scanning, great anatomic coverage during a single breath hold, few motion artefacts, and high spatial resolution in the longitudinal plane, improving the diagnostic accuracy of the examinations. evaluation of vertebral artery using color duplex sonography: comparison of vertebral artery velocity and flow volume measurements for diagnosis of vertebrobasilar insufficiency m. acar, b. degirmenci, a. yucel, r. albayrak; afyon/tr purpose: the aim of this study was to compare the measurements of vertebral artery (va) flow velocity and flow volume for diagnosis of vertebrobasilar insufficiency. matrials and methods: we examined patients referred for evaluation of vertebrobasilar insufficiency. net va flow volume and mean systolic flow velocity were determined by using color duplex sonography. we grouped the patients into three: group consisted of patients with severely damped va flow volume (< ml/min), group moderately damped ( - ml/min) and group , normal (> ml/min). the mean systolic flow velocities in each group were compared by one-way anova. results: damped va flow volume was determined in of patients, of these patients had severely damped, had moderately damped, had normal va flow volume. mean va systolic flow velocities of group , and were ± , ± and ± cm/sec, respectively. mean va systolic flow velocity in group was significantly lower than that of group (p = . ). however there were no significant differences between va systolic flow velocities in group and group (p = . ). conclusion: according to our findings, measurement of va velocity is enough for diagnosis of vertebrobasilar insufficiency in patients with severely damped va flow volume but may not always be helpful in differentiation of moderately damped flow volume from normal. therefore we conclude that for the diagnosis of vertebrobasilar insufficiency, measurement of volume in addition to velocity is necessary in detection of moderately damped va flow volumes. rotational angiography of femoropopliteal arteries after percutaneous transluminal angioplasty (pta): preliminary results f. pozzi-mucelli, m. belgrano, g. tona, r. pozzi-mucelli; trieste/it purpose: to report our experience in rotational angiography (ra) after pta of the femoropopliteal arteries and to compare this modality to standard anteroposterior (ap) projection. materials and methods: forty patients underwent pta of the femoropopliteal arteries in the last months. twentysix were stenoses (lenght: - cm) and occlusions (lenght: - ). in all cases pta was successful and the result was checked with ap projection and with an ra acquisition. the procedure were performed on the philips integris allura system providing the ra tool. results: ra after pta was successful in all cases. in % of cases there was no difference between the static ap projection and ra, whereas in % the static acquisition, compared to ra, underestimated the degree of residual stenosis by - %. in about % of these cases a disagreement on the presence and extent of subintimal flaps between static and rotational acquisition was identified. in another % of cases there was substantial agreement on the residual stenosis but not on the pta-induced dissection which was always better identified on ra. conclusions: ra allowed optimal evaluation of all cases improving the information on the conventional technique based on one or two projections. in our experience this information modified our therapeutic approach leading to repeated balloon angioplasty or stenting in % of cases. all the diagnosis were confirmed by digital angiography or surgical treatment. in the first group (endoluminal repair), the complications were: a) periprosthetic hematoma (n = ); b) neointinal hyperplasia (n = ); c) partial stent-graft thrombosis (n = ); d) total stent-graft thrombosis of an iliac branch (n = ); e) endoleak type ii (n = ); f) endoleak type iii (n = ), separation of the stents' components (n = ). in the second group (surgical repair) we observed: a) periprosthetic infections (n = ); b) supraprosthetic aneurysm (n = ); c) pseudoaneurysm (n = ); d) aortoenteric fistula (n = ). conclusion: cta is accurate, fast and minimally invasive imaging method in the evaluation of complications after abdominal aortic aneurism repair. interobserver agreement in the ct evaluation of carotid artery stenosis p.m. carrascosa , f. meli , c. capuñay , t. sampere , e. martin lopez , d. smith , s. chandra , j. carrascosa ; buenos aires/ar, cleveland, oh/us objective: to determine the usefulness of ct angiography (cta) in the detection and quantification of carotid stenosis in comparison with da. materials and methods: thirty-eight carotid arteries were evaluated in patients with transient ischemic attacks. ctas were performed with a helical ct scanner (pq ; picker). images were evaluated by two radiologists, who were blinded to the percentage of stenosis determined by da. the stenosis were considered positive if they were ≥ %. different methods of stenosis quantification (nascet(n), esct (e) and area quantification (a)) were performed on the same vessel to determine the sensitivity and specificity of each one in comparison with da. interobserver variability of the three methods was evaluated. statistical analysis: kappa coefficient was performed to determine the interobserver variability. results: observer : sensitivity: n: %, e: % and a: % and specificity: %, . % and . % for each method, respectively. observer = sensitivity: n: %, e: % and a: % and specificity: %, . % and % for each method, respectively. kappa: . , and . for n, e and a respectively. conclusion: cta showed high s and sp in the carotid stenosis quantification, specially with the nascet method. emergency multislice ct angiography (mscta) in patients with suspected aortic dissection r. stern padovan, m. lusic, b. oberman, k. potocki; zagreb/hr purpose: the purpose of the study was to evaluate positive msct-angiography findings in patients with spontaneus-nontraumatic aortic dissection. mscta is a noninvasive, time-saving radiological procedure for patients with aortic dissection because they need prompt and accurate diagnosis and treatment. aortic dissection is defined as a separation of the inner aortic wall layers. it is divided by stanford classification into type a that involves the ascendent aorta and type b involves distal to the origin of the left subclavian artery. materials and methods: mscta was performed in patients ( ( . %) female and ( . %) male age - ) with clinically suspected aortic dissection over a period of months on lightspeed ultra slices, ge. slice thickness was . mm with % overlap of reformatted images. we injected - ml of nonionic contrast material at a flow rate of ml/sec with smart prep feature. mip and mpr were usually used. results: aortic dissection was found in ( . %) patients. ( . %) had acute aortic dissection and ( . %) chronic type. acute aortic dissection stanford type a was found in ( %) and type b in ( %). in patients we did not find aortic dissection but other ct findings could explain the symptoms: aa, rupture of aa, mesenteric ischemia, pericardial effusion. conclusion: mscta is noninvasive, rapid and relatively comfortable radiological procedure for patients with suspected acute aortic dissection in cardiovascular emergency. this technique enables correct and detailed diagnostic information and obviates catheter aortography. diagnosis of aortic and supraaortic emergency with ct angiography: how does aortic morphology influence the choice of treatment? c. engelke , k. marten , a. chavan , j.f. reidy , a.-m. belli , e.j. rummeny ; munich/de, hannover/de, london/uk objective: ct angiography (cta) is the imaging method of choice for diagnosis of aortic and supraaortic emergency. in this retrospective evaluation of patients with aortic or supraaortic emergency undergoing intial cta at four european centres diagnosis and determination of treatment are reviewed. the assessment of diagnostic value of vmax, vmin, pi, ri measurements in the diagnosis of coeliac trunk stenosis before and after levovist injection -roc curves analysis a. drelich-zbroja, t. jargiello, w. krzyzanowski, p. kurczab, m. szczerbo-trojanowska; lublin/pl purpose: to assess the diagnostic value of vmax, vmin, pi, ri measurements in the diagnosis of coeliac trunk stenosis before and after levovist injection. in patients doppler examination of coeliac trunk before and after levovist injection was performed measuring vmax, vmin, pi, ri. in conventional doppler and after levovist administration stenoses were diagnosed. number of diagnosed stenoses in relation to number of normal coeliac trunks was representative enough to perform the statistical analysis using roc curves. results: . the areas under curves (aucs) for vmax, vmin, pi, ri, before and after levovist. a. before levovist: aucvmax = . *, aucvmin = . *, aucpi = . ns , aucri = . ns . a. after levovist: aucvmax = . *, aucvmin = . *, aucpi = . ns , aucri = . ns . *p < . when compared with auc = . ; ns-not significant when compared with auc = . . the comparison of aucs for vmax and vmin before and after levovist. aucvmax after levovist -aucvmax before levovist = . ns , aucvmin after levovist -aucvmin before levovist = . ns , aucvmax before levovist -aucvmin before levovist = . *, aucvmax after levovist -aucvmin after levovist = . *. *p < . ; ns-difference not significant conclusions: only vmax and vmin are useful in the diagnosis of coeliac trunk stenosis. the diagnostic value of vmax is significantly higher than diagnostic value of vmin. the use of levovist has no an effect on diagnostic value of vmax and vmin. the effect of body mass index on the ultrasonographic measurements of the portal venous system n. tuncbilek , o.o. okten , s. guldiken , h.m. karakas ; edirne/tr, malatya/tr purpose: the effect of body mass index (bmi) on portal venous diameter and pulsatility index was investigated. the study group consisted of obese and control subjects with normal bmi. inferior vena cava (vci) and portal vein (pv) diameters and their pulsatility indices (pi) were prospectively evaluated with . mhz convex transducer. diametric measurements were employed in m-mode imaging. the difference between obese and normal subjects regarding above mentioned parameters and their correlations with pi are investigated. results: bmi varies between . and . kg/m in control, and between . and . kg/m in obese subjects. in control subjects, mean pi is . for pv and . for vci. in obese patients, these values are . and . , respectively. pi for pv significantly differs between two groups (p < . ) whereas vascular diameters for vci and pv, and pi for vci are not statistically different. in both groups there is a negative correlation between pi and bmi (for control subjects: r = - . , p < . ; for obese subjects r = - . , p < . ). conclusion: pi of the portal venous system is regarded as the surrogate marker of right cardiac function. however factors such as inspiratory and expiratory intraabdominal pressure changes are known to affect this parameter. when considering pi as a marker of right cardiac function, attention must be paid to the relation of pi and bmi, proved by this study to be one of these factors. significance of patent pyloric branch in hepatic arterial infusion chemotherapy s. yoshioka, y. yasuhara, t. murakami, s. kumano, t. mochizuki; ehime/jp purpose: to investigate the clinical significance of the patent pyloric branches after right gastric artery (rga) embolization for arterial infusion chemotherapy. materials and methods: sixty-six patients who received catheter placement in the hepatic artery for arterial infusion chemotherapy were retrospectively recruited from the pool of clinical records between and . two interventional radiologists reviewed the digital subtraction angiographies (dsas) and clinical records of these patients. they assessed the following items by consensus: ) location of the rga, ) frequency of the patent pyloric branchs after coil embolizaton of the rga, ) outcome of arterial infusion chemotherapy through the catheter. results: the location of the rga could be determined in patients. rga-coil embolization was performed in patients. in patients, fine pyloric branchs were patent after embolization. they were too fine to be embolized by the catheter technique. in of these patients, the catheter was successfully placed to avoid the drug infusion into the pyloric branch. the clinical outcome of the other patients were as follows: one patient received the arterial infusion chemotherapy without any complication. one patient experienced transient abdominal pain in the course of arterial infusion chemotherapy. two patients received replacement of the catheter because of abdominal complication. the last patient received surgical ligation of the rga because of reopening of the rga. conclusion: the frequency of abdominal complication in arterial infusion chemotherapy was not negligible in the cases with patent pyloric branchs after rga embolization. erdheim-chester disease: imaging features c. graef, e. dion, p. cluzel, r. renard-penna, j. haroche, b. wechsler, c. beigelman-aubry, p.a. grenier; paris/fr learning objectives: to be familiar with the radiologic features of erdheim-chester disease, a rare form of non-langerhans cell histiocytosis of unknown etiology. background: the disease is characterized by tissue infiltration with lipid-laden foamy histiocytes, polymorphic granuloma of inflammatory cells and collagen fibrosis. the patients have a mosaic of infiltrative involvement of various organs. bone involvement is almost always present but often clinically asymptomatic. the prognosis depends on the severity of cardiovascular, renal or cerebral involvement. imaging features of histologically proven cases were reviewed. imaging features: radiological bone involvement was present in all cases, characterized by bilateral and symmetrical spongy bone densification, cortical thickening and periostosis of long bones from the metaphysis to the diaphysis associated with scintigraphic tracer uptake. renal and perirenal involvement seen on ct scans consisted of bilateral and symmetrical perirenal tissue infiltration (hairy kidney) (n = ), extended to the adrenal fossae, the renal sinuses, and the proximal ureters. cardiovascular involvement consisted in ) thoracic and abdominal circumferential periaortic tissue infiltration (coated aorta) with extension to the coronary, supraaortic and pulmonary arteries, and abdominal branches, ) stenosis of renal or mesenteric arteries (n = ), ) involvement of pericardium (n = ) and myocardial mass (n = ). tissue infiltration involved pituitary in one case and orbits in another one. conclusion: a diagnosis of erdheim-chester disease should be considered in patients who demonstrate bilateral and symmetric densification of the long bones with increased uptake on scintigraphy in association with perirenal fat infiltration and/or a periaortic fibrosis. human nk- cells were labeled with ferumoxides and ferucarbotran using simple incubation, lipofection and electroporation techniques. incubation times were varied from - hours and added contrast agent concentrations were varied from - microgram fe/ cells. pellets of labeled cells and non-labeled controls were evaluated by mr imaging. the cellular iron oxide uptake was proven by prussian blue stains and spectrometry as a standard of reference. in addition, cell viability was tested with the trypan blue exclusion test. differences between labeled cells and non-labeled controls were tested for significance using the t-test. results: nk- cells could be labeled with ferucarbotran and ferumoxides by lipofection and electroporation, but not simple incubation. lipofection had to be performed for h, but resulted in only minor, not significant impairment of cell viablility ( - %) compared to controls ( - %), while electroporation caused cell labeling within seconds, but reduced the viablility of the cells significantly ( - %), thereby necessitating additional cell culture time for cell recovery. the intracellular iron oxide uptake was proven histologically and quantified by spectrometry. ferucarbotran and ferumoxides-labeled cells could be depicted by a significant signal decline on t *weighted mr images. conclusion: human nk- cells can be labeled with ferucarbotran and ferumoxides by lipofection and electroporation. optimized labeling techniques allow a subsequent cell depiction with a standard . t mr scanner. which temporal frame rate is necessary in quantitative dynamic ³he-mri? c. heussel , a. dahmen , f. lehmann , m. salerno , k.k. gast , h.-u. kauczor , j.p.i. mugler , e.e. de lange , w. schreiber ; mainz/de, charlottesville, va/us purpose: dynamic ³he-mri permits regional analysis of distribution ventilatory kinetics. dedicated post-processing offers image-based lung function parameters. sliding window reconstruction of interleaved-spiral acquisitions provides high temporal rates, but do functional parameter values depend on the temporal frame rate? methods and material: single-slice coronal dynamic ³he-mri was acquired continuously for s during free respiration. image reconstruction was performed using sliding window technique with . ms data sampling-time (repetition-time ms). post-processing was performed using a self-written software with linear motion correction for calculation of: rise time (tr, interval of lung-signal from % to % of peak signal), delay- time (td , interval of trachea-signal % and lung-signal %), amplitude (peak lung-signal), and peak flow (pf, maximum signal slope). analysis was done for rois using a temporal resolution of ms, ms, and ms ( nd , th , and th image). this study included exemplary patients (asbestosis, x asthma, copd) so far. results: post-processing of the data-sets lasted to mins at an actual pc ( . ghz p , gb ram). tr ranged from to ms (median ms), td ranged from to ms (median ms), amplitude ranged from to a.u. (median a.u.), pf ranged from to a.u. (median a.u.). no relevant difference was detected in the evaluation of different temporal frame rates for all patients or within an individual patient. conclusion: since these preliminary data suggest a limited effect of high temporal frame rates, improvement of spatial rather than temporal resolution might be the emphasis in the future. to determine the compensatory mechanisms involved in recovery of motor function following resection of the supplementary motor area (sma) and their relation to the clinical characteristics of recovery. subjects and methods: thirteen patients referred for surgery of low-grade gliomas located in the sma were compared to nine healthy controls using fmri before and after surgery during self-paced movements of both hands, successively. activation within regions of interest (rois) (primary sensorimotor cortex (smc), premotor cortex (pmc), sma) were compared and tested for correlation with anatomical characteristics of the tumor and resection, and clinical data. interaction analysis between rois was performed using partial correlation. results: tumor growth induced preoperative underactivity in the adjacent sma and overactivity in the opposite sma. postoperative recovery was associated with recruitment of the lateral premotor cortex in the healthy hemisphere. in the affected hemisphere, pre-and postoperative correlations between sma, pmc and smc were decreased. in the healthy hemisphere, postoperative correlations between pmc and sma or smc were increased. shortened onset and duration of recovery was associated with preoperative changes in sma activation. conclusions: these findings suggest that tumors induced a dysfunction in activation and connectivity of the ipsilateral sma, which was partially compensated by a recruitment of the sma in the healthy hemisphere. this preoperative remodeling of sma activity did not prevent the occurrence of the postoperative deficit but was associated with shortened recovery. sma resection was compensated by the recruitment of an ipsilateral premotor circuitry. the use of internet filters to build a radiology education and teaching resource p. davison, j. revell, m.r. rees; bristol/uk purpose: to develop a resource for radiology undergraduate and postgraduate education from a series of research projects dealing with internet filter aided search. supported by a research and education grant from the ear. materials and methods: two independent research projects into internet search were examined for their application to radiology education and these methods were compared to conventional image search protocols. this experience was translated into constructive advice for undergraduate students undertaking special study modules in radiology. the two internet filters developed were designed to improve accuracy of text search and to improve the search results from image search enquiries in radiology and medical subjects. a second iteration of the image search filter was developed to produce a web crawler that selected images from previously carried out text searches. the text and image based filters were tested with other search engines over a preset number of criteria. results: both the text based search facility and the image based facility showed advantages when compared to conventional search methods. the image search facility was successful in handling medical searches compared to common search engines and the web-crawler was efficient at producing a large number of images from selected sites. these methods were used to develop medical students skills in the use of the internet and has been used in the construction of radiology image based special study projects. conclusion: web based filter programmes and education in search techniques can result in improving the efficiency of internet based material for radiology education. single breath-hold subtraction: novel approach to diagnosing pulmonary embolism by multi-slice ct (work-in-progress) j.e. wildberger , a.h. mahnken , h. ditt , m.u. niethammer , e. spüntrup , e. klotz , r.w. günther ; aachen/de, forchheim/de purpose: in the work-up of pulmonary embolism (pe), ct allows for direct visualization of emboli. in addition, perfusion defects permit direct assessment of the extent of pe, using color-coded lung densitometry. this new image processing technique has been deployed for a -slice multi-slice ct (msct) system. for further improvement, a subtraction technique within a single breath-hold will be mandatory. materials and methods: three healthy pigs underwent lung lavage to induce c- assessment of the angioarchitecture and hemodynamic characteristics of cerebral avms by contrast enhanced static and non enhanced dynamic mr angiography m. essig; heidelberg/de purpose: the aim was to improve the definition of the angioarchitectural components of the avm based on a dynamic mra (dmra) and a high resolution d multi-bolus and multi-phasic contrast-enhanced mra (ce-mra). both techniques will be compared with d-tof-mra and transfemoral high resolution plain film angiography performed for treatment planning. materials and methods: dmra will be performed by tracking a blood bolus through a vessel structure using the spin labeling technique star (signal targeting with alternating radiofrequency sequences). time-resolved ce-mra will be acquired with an ultrashort d fast low-angle shot (flash) sequence (tr/te . / . msec) using asymmetric k-space sampling in readout, phase-encoding, and partition directions. results: all used sequences were successful in the assessment of patients' cerebral avms. the integration of acquired images into the treatment planning protocol was possible for all modalities. the different avm compartments, feeding arteries, avm nidus, and draining veins were detected easily and best on the dmra. the method also allowed to hemodynamically assess the malformations: small avms generally showed shorter shunt times, however, a short shunt-time was associated with a higher risk of bleeding. ce-mra also proved to be superior than tof-mra in the assessment of the angioarchitecture, however, the time resolution of the dynamic varient was too slow to achieve a substantial hemodynamic characterisation. discussion: dmra and ce-mra are better suited than tof-mra to assess avm angioarchitecture. the differentiation of different compartments of the malformation and the hemodynamic assessment is best seen with dmra. diagnostic investigations in relation to determination of paramagnetic centres and free radicals in the model of testicular tumor metastases m. kekelidze, f. todua; tbilisi/ge purpose: to assess the possible connections between diagnostic imaging (spiral ct, mri) of retroperitoneal metastases of testicular tumor and the detection of paramagnetic centres and free radicals in the resected lymph node tissue after retroperitoneal lymph node dissection (rplnd). materials and methods: patients with stage i morphologically confirmed nonseminomatous testicular tumors were subjected to rplnd. diagnostic imaging included abdominal spiral ct with iv contrast and mri. specific tumor markers (afp, cgt) were detected. highly sensitive ( - spin/gas) electron paramagnetic resonance (epr) of resected lymph node tissue was performed to detect the presence of paramagnetic centres and free radicals (fe + , mn + , n ) signals. ct or mri scans were judged as positive or negative for rp metastases on the basis of size, contrast enhancement criteria and compared with morphological and epr data. results: in patients estimated as n on ct and mri the increased signals of free radicals were revealed in tissues. among patients (n ) with resected lymph node tissue, showed low signals of fe + mn + n and material (morphologically confirmed as metastatic) high signals of free radicals, while the increased presence of paramagnetic centers were not detected in these tissues. spiral ct and conventional mri fail to detect micrometastases in cases. there exists real correlation (p < . ) between the increased signals of free radicals (fe + , mn + , n ) and low stage metastatic lesions of rp lymph nodes, which can reflect the connection between particular molecular events and malignant degeneration. diagnostic accuracy of gadolinium-enhanced mr-angiography vs. contrastenhanced helical ct in the diagnosis of pulmonary embolism in the presence of lung infiltrate: an animal study t. franquet, s.e. kalloger, a. oikonomou, s.l. macdonald, e.m. baile, r.j. mayo; barcelona/es purpose: to compare gadolinium-enhanced mr angiography with contrast-enhanced spiral ct for the detection of small pulmonary emboli in pigs with and without pneumonia. materials and methods: ten female juvenile pigs were anaesthetized, intubated and ventilated. sub-segmental sized pulmonary emboli were introduced by injecting . mm methacrylate beads into the external jugular vein. lung infiltrates were simulated by intra-bronchial injection of human plasma. animals were imaged in the supine position at suspended inspiration with cm h peep. contrast media was injected via brachial vein. dual gradient (twinspeed) . t mr images were acquired using dimensional spoiled gradient-echo time of flight sequence; tr . msec, te . msec, * flip angle, mm section thickness, cm fov, x matrix, one average. two mra sets, pulmonary arterial and venous, were acquired in seconds. contrast enhancement was provided by ml of gd dimeglumine followed by ml of normal saline, injected at ml/sec. non-contrast and contrast enhanced ct images were obtained using an track spiral ct; . mm detector aperture, ml contrast at ml/sec. subtraction perfusion images were calculated by subtracting contrast enhanced from non-contrast ct images. after imaging the pig was euthanized and a methacrylate cast of the pulmonary vasculature was obtained and used as a "goldstandard". cta, cta with subtraction perfusion images and mr images were independently interpreted by two observers in a blinded fashion. results: no significant difference (p > . ) was seen between cta, cta with subtraction perfusion images and mr for mean sensitivity ( %, % and %) or mean specificity ( %, % and %), respectively. no significant difference (p > . ) was seen in sensitivity or specificity between pigs with and without infiltrates. a prospective power analysis based upon this preliminary data indicates that the addition of two more readers (n = ) would yield significant differences (p£ . ) between cta and cta with subtraction perfusion images compared to mra for sensitivity and specificity. conclusion: preliminary results indicate that cta and cta with subtraction perfusion images provide improved detection of sub-segmental sized pe in comparison to gadolinium-enhanced mra.