key: cord-326785-le2t1l8g
authors: nan
title: Pathological Society of Great Britain and Ireland. 163rd meeting, 3–5 July 1991
date: 2005-06-15
journal: J Pathol
DOI: 10.1002/path.1711640412
sha: 
doc_id: 326785
cord_uid: le2t1l8g

nan

Claire M Allen, D M Hansell. Mary N Sheppard Depanmenls of Diagnosbc Radrology and Lung Palhalogy Royal Bromplon Nahonal Hean and Lung Hosprlal, Sydney Slree:. London SW3 6NP Percutaneous fine needle biopsy 1s an established diagnostic technique for lung lesion^ Afirm diagnOSiSOfbenign versus malignant IS often achieved but hiStologIcal interpretation of small fragments or groups of cells IS difficult Manual cutting (twcut) needles provide asuperior histological specimen but are associated with a high complication rate and have been mainly used for pleural lesions This 1s the first prospective study to assess the feasibility of obtaining histological samplesfrom lung lesionswing apoweredcuning needle (Biopty Gun) We have biopsied 33 patients using Biopty Gun There were no major complications Histological diagnoses were obtained in 30 patients (22 malignant, 8 benign) The malignant lesions identified included 7 nan-small cell carcinomas. 4 adenocarcmamas. 3 squamous cell carcinomas, 3 bronchiolaalveolar cell carcinomas, 2 8-cell lymphomas, 1 small cell carcinoma. 1 atypical camnoid and 1 metastatic breast Carcmoma. The benign 1es1ons included 1 SarcoidosIs. 1 clyptogenic organising pneumonia. 1 Wegener's, 4 resolving pneumonias with Chronic inflammation The rad~OlogiStlS aSSeSSmentaftheamauntoftissueobtainedconelated withgood histology.01 thethreefalsenegativespecimens obtained the radtdogist noted the inadequacy of the samples ~n two cases and the third was a geographic miss Percutaneous biopsy using the Biopty Gun IS a simple and effective means of obtaining good quality histological material from lung parenchyma with a high degree of diagnostic accuracy for bath benign and malignant lesions Inorganic particulate matter in "normal" lung: a study using light microscopy (LM), scanning electron microscopy (SEM), and energy dispersive X-ray analysis (EDXA) London, Onlano. L2partmenI of Palhology 375 Soulh Sireel London Onlano N6A 4G5 Canada h e possible association between inhaled inorganic matter and Some cases of usual inter5tltial flbrOSIS (UIPI has been of longstanding interest to pathologists and cllnoians. Unfortunately. most analytical techniques are impracticable ~n the context of a pathology sewice laboratory In an anempt to find a practicable s01Utm to this problem and to establish a baseline for inorganic particle load in a "normal" population, a study was undertaken using techniqueswhichwauld beavailable ~nmostlargepathologylaboratories Ninecaseswereselectedfram the case(centra1andperipheralparliansaf lower. middleand upperlobesjandexamined with theSEMand EDXAand compared with the LM appearance to determine the panicle type and distrlbutm m a "normal" population. A wide range of inorganic matter was ldentlfied canespanding to SIIICB. aluminium and magnesium s4Cate5, rutile and alumina "like" particles varying from < 1 p to 15p in size In addition. trace elements including zinc. cadmium and increase in number Of particles wasalso noted in areas of fibrosis which were present in two cases (old mflammatory disease) preseumably related to problems in particle clearance The findings of this pilot study suggest that although the SEM and EDXA will likely prove useful tools in the evaluation of lung biopsy soeclmens. the findinq of lnorqanic material in cases of UIP must be interpreted With Caution autopsy SeNICB WhlCh had no known expcsure to lnorganlc dust Sectlons were taken from the nght lung each Pulmonary adenomatosis has been described as adl~tinctive pathologlcal change seen in the lungsolexperimental area Of replacement of normal alveolar lining cells by a taller more glandular type of eplthelwm. usually wlthwt significant cytological atypia. We describe 10 cases I" whlch a smtlar change was seen as an mdental fcndlng ~n resection Specimensfor primary pulmonary adenocaranoma. The lesions (usually multlpleand each 5 mm orless m diameter) were identified in lung parenchymaat a distance from the tumour and consisted of thickened alveolar walls lined by prominent, distinctly atypical cells morphologically Slmllar to type I 1 pneumacytes and cytologically different to the associated turnour Reactive changes 8" lung involved by obstrmtive pneumonitis were not included !n thts Sews All of the associated tumwra were peripheral adenocarcinamas and all showed a pattern of alveolar wall spread at the tumour periphery Clinically 7 of the patients were female and all were smokers or ex-smokers The slgnlflcance of this lesion in the histogenesis of primary pulmonary ademcarcinoma IS. as yet, unclear animals often I" aSSoclatlon wlth exposure to Inhaled camnogens Morphologically the lesion IS a clrcumscrlbed

Departmen1 ofli~slopafhology Sf Richard's Hosprlal. Chrcheslei Wesf Sussex. PO1 9 4SE

A series of 450consecutive personally Conducted autopsies in patlenfsdying Suddenly Outside hospnal and where the death was reported to H M Coroner 15 presented Cot deaths were excluded. In 384 cases the death had been reported because theattendingdoctarwas unwillingtoissueadeathcertlficate. In theother66cases. deathwasnot duetonaturalcausesandfallowedsuicidearan accident Oftheformetgroup, therewere t36Unsuspected malor findings cn 11 8 patients (30.7%). that 1s to say findings which were either the cause Of death or would have led to admission to hospital far assessment and possible treatment 1 they had been discovered in life The largest group were cardiovascular (56) but there were 22 cases of unsuspected malignant disease There were only five malor unsuspected findings in the group of unnatural deaths years). These results highlight the loss of teaching material in the Coroner's system, at a time when hospnal postmortem rates are in universal decline. This matenal would be of value to medical students and to pathologists In traming. and largely comes from cases of lmle or no medico-legal significance. 7%) and these were in Older sublects (mean age 71. A total of 140 postwnortem specimens refened to the Royal Victoria Ho~pdal electron microSCopy unit dunng the years 1984-1988 havebeen reviewed Thkscompnsed3.4Vo of thetotalnumber of sewre-relatedcaaesreferred to theunit.Oulof 14Ocasesreferred. 56wereexaminedandrecorded indetail Theremainderdidnotundergoelectron microscopic examination for various reasons, Such as a concIus~ve diagnosis being reached by light microscopy alone. and semi-thin Sections Showing Severe tissue mtoIysIs. The most common tssues referred for examination were lung, kidney. liver, brain and head The range Of E M. studies carried out included tranSmiSSion electron microscopy ITEM). scanning electron microscopy (SEMI and X-ray micrOanalySiS on SEM. Theelectron micrographs Wererevlewedwlth respect totlssuepresewatlanand th15Wa5cOrrelatedwlth the timeintervalbetweendeathand autopsy. Electron microscopy was considered, an review. to have been diagnostically useful in 46% of cases an which It was deployed

C S Herringtan, A K Graham, K Cooper, J 0 0 McGee

It was Shown prev10usIy that the discrimination of human papillamavirus (HPV) types 6 and 11 by NlSH in archival biopsy matenal requires dlfferent Conditions from those predicted by Conventional solufion kinetic analysis The parameter Tm' (tissue Tm) was defined in order to describe these differences In this study. these pnnciples were extended to the discrimination of HPV 16.31 and 33 in 15 casesol ClN The results of NlSH analysis werecompared with both immunohistochemistry for viral capsid protein and PCR typing These data demonstrate that crosshybndisatm of high nsk viral types occurs ~n clinical lesions under Conventional hybridisation and stnngency WashmgCOndit10nS Thiscross hybridisation isnot due to thepresenceofviralcapsidproteinand 1sm0relikelytO be areflectionoftheendpoint uSedInN1SH.I e thepresenceofvisiblesignal.Practcally.multipleNlSHsignalsduet0 closelyrelatedprobesinarchival materialarenot indicativeofmultipleHPVinfectionunlesstheyarepresent either ~n morphologically discrete areas of the biopsy or their presence has been confirmed by another molecular technique. Motegenerally, thepresenceof asignal in NlSH usingaparticularprobedoes not implythattheidentity of the target nucleic acid IS that of the probe Medical students in the autopsy room Shows that 29 0% thought faces Should be covered during autopsy, and 12 4% thought that genitals Should be covered. about half the group were indifferent to both proposals 35 9% thought the patient's identrty should be concealed from observers Three Students thought there Should be no Conversation at all. 24 0% thought that Conversation Should be limned to procedures and findings and 44 2% thought it Should be limited to professional maners;conversely. 42 1% thought thereshwldbenolimitson thetopicsdiscwaed NearlySO% thoughtstudents Should beencouragedtoassistbutnotpressuredintadoingsa.3.3% thoughtthatallshould becompelledtoassist, but 12 4% thought that students should onlv obsewe. and not be allowed to aSS1s.t. 9 6% of students thought that they should Oni!iObSeNe autopsies on patients they had clerked. whereas 13 4% thought they Should onlfbe on autopsies on patients they had not clerked. Fmally. 31 6% thought that relations Should give spmfic perm1551on before students observed autopsies. whereas 45.4% thought not A quantitative study of the effects of fibroblast growth factor on wound strength and cellularity Fibroblast growih factor (FGF) has potent angiogenic and fibrogenlc effects and IS lmpllcated In the formatlon of granulation tissueand healing Few attempts have been made toquantity theseeffectsmnvo. We havestudled arat skinwound modelusing red cellghostsasan FGFvehicle Tensiometv OfthewOUndSShowed amaxlmaleffectof the FGF after seven days when the wound strength was 50% above that m controls (p < 0 01) This effect had disappeared by fourteen days. Computerized image analys~ using a Joyce Lo& Mini Magiscan measured total nuclear content of areas I" the wounds. permitting a topographic analysis of cellulanty versus distance from the wound centre Cellularlty effects showed adifferent time course from wound strength. a 34% increase at fouldays anda42% decreaseat sevendays. relative tocontrols(bothp < 0.05) Attwelvedaysthecellularityeffectwasstill sQnlflCant at a 31 Vo decrease but by twenw-one days It had dwappeared The results suggest that FGF causes an early transient increase in cellulardy and more rapid increase in wound strength: most of these cells are macrophages and fibroblasts Suggesting a connection between thesetwo ObSeNat10ns. The adhesion molecule$ 81 integrin (CD29). 82 integrin (CDl8). and intercellular adhesion molecule-1 (ICAM-1, CD54) are essential to the intimate co-operation of antigen presenting cells (APCs), T cells and keratinocytes Cyclosponn. Which IS an effective treatment for psonas~s, may cause immUnOSUppreSSiOn by altering antigen presentation We have pertormed a quantitative immunohistochemical assessment of the effect of low dose ~yclosporin on the expression of p1-integnn. p2-integnn and ICAM-f m the epidermis in Chronic plaque psonas1s. Staininglevels werecompared withclinicalresponseasassessed bythePsoriasisAreaSeverihllndex(PAS1 score) pt-lntegnnand CAM expressionon keratioocyteswerenotaltered bytherapy buttherewasasignificantdecrease inthemeanlevelsof P2positivelargedendriticcells(APC5)within theepidermis.BZ-integnn wasnot expressed by keratinacytes There was a strong correlation between p2 expression and PAS1 score after three months on cyclosporln and one month off therapy These results lndlcate that P2 stalnlng on large dendrltlc epidermal cells Previous studies enumerating Silver stained n~c l~l a r organiser regions in problematic cutaneous melanocytic lesions have yielded inconsistent. but generally favourable, resulis. It seems probable that such InconsIstencies. arise largely from differences ~n fixation. staining and counting strategies. Our group, having devised improved methods of AgNOR staining and counting. IS now able to re-examine the potential role 01 AgNORs in borderline lesions Pilot work demonstrated potentially significant differences in AgNOR dispersal between benign and malignant lesions and I" this study bath AgNOR numbers and dispersal patiems have been evaluated A range of melanocytic lesions including banal naev~. dySplaStic naevi. typical melanomas, Spiu naevi, atypical spih le~ions and minimal deviation melanomas were collected Virtualty all of the unusual lesions had been diagnosed by specialists in dermatopathology. Using a combined assessment of AgNOR numbers and dispersal, It proved pOSSible to discriminate borderlip lesions from banal naevl and typical melanomas. Benign borderline lesions -Such as Spit2 naevi ~ possess numerous NOR$ but display tight clustenng in Contrast to malignant melanomas whereNORdiSperSa1 isaprominentfeature DiscriminatInganetypeaf borderlinelesionfromanathercauld not be achieved however, in practice this distinction IS probably less important than assigning a aoIRary specimen to a benign or malignant group A larger prospective trial of AgNORS in melanocytic lesions 6 currently in progress with a mean total exposure of 36 pack-years. Alihough p53 was expressed more commonly in adenocarcinoma (30ROf lo)andsquamouscarc~noma(28~~of29)than~nsmallcelltumours(l0%of2o),thiscouldbeaccountedfor by the smoking history. since patients with non-small cell carcinoma smoked more (a mean af 42 pack-yeas) than those With small cell lesions (mean of 33 pack-years). There was no relationship between p53 expression and SUNlYal.

s. A. sun. J. R. ~a s n e y Depaitmenl ofPathology University of Liverpol, P 0 Box 147, L!vemoOi. L69 3BX Activation of thec-myconcqene With overexpression of itsancopratein product ocurs In bronchlal malignancles ofalltypes, but has beenmost extensivelystudied insmall celicarcmoma, wherensaverexpression~n culturedllnes has beenassociatedwithdevelopmentoffast-growingvanants whlchlosemuchofthelrendocrlnephenohlpeand aner their morphology. It has been suggested that these vanant lines might be the equlvalent of the large cell bronchial endocrine carcinomas sometimes seen I " YIYO, but this is not proven. We have used th0 Myc 1 -9E10 monoclonal antibody and the avidin-biotin technique to study the panem of expression of the p62 oncoprotein product of the c-myc gene in turnour deposits of twelve subjects coming to necropsy with disseminated small cell carcinoma in an attempt to relate 11 l o morphological vanablity and metastatic site. Anhough expression bore no relationship to morphological vanation. it ohen differed markedly horn site to slte. Whereas parts of the primary t m w r strongly overexpressed the protein ~n all but one wbfect, there was considerable vanability between secondary deposits. OOSSiblV mdicatina arelation~hi~ between c-mycex~resslon and propensltvfor metaStaSlS to certain iocationS. Tumour growth rate is a key parameter of neoplastic aggression. and is determined by the balance of cell gain and loss.Apoptos~sisama]ormadeoftumourcellloss, but linleis knownof itsregulation. Dieremesin turnourgrowth conferred by HPVtypeS 16and 16 werestudied in aratfibroblast modelsystem. immortaiisedcells weretransfected with HPV 16 and 18 expression vectors. either alone or With activated c-Ha-rasl. Monoclonal cell lines were established. and their vector DNA Content was confirmed by PCR Tumour Cell 111185 ddfered in their growth properties m wvo and m vilro. HPV 18 containing turnours were larger and Showed less apoptosis than those containing HPV 16, although bothshowed moreapoptasls than thenodulestormed bytheparentfibroblastsalone. In all turnours the presence of ras greatly reduced apoplosis and increased the growth rate. Very similar propetlies wereobswvedin culture, and apoptoticratesshowed astranglnveneconelatlonwlthratesofnet Cellgrowth. HPVS appeared IoStim"latetumOurCei1 apoptasis, butthiswas suppressed byras. melowerapaploslsassoclatedwilh HPV 16 compared with HPV 16 may partly explain the more aggressive phenotype of cervical c a n m containing HPV 16. It hasbecomeapparent thatanumberof maleculesmaybeexpressed byreStingorqu~escentcellsandarelostwith transition into the cell cycle. In addition to being of biological interest, such molecules may prove useful as operatcanal mahers of quiescent cell populationS in histological material and allow the further characterisation of cellular subpopulations. One such molecule IS statin. a 57kD protein previously reported to be expressed only by cells ~n Go. We have shown bylaserconfocalfluarescence microscopythat thestatin antigen is associated with the nuclear envelope with a dstnbution 51m1l ar to that of nuclear lamins. Using a monoclonal antibody (5-44) that recognises slatin we have defined the tissue distribution of statin immunoreactivlty in a range of ContinUOUSly renewing, conditionaily renewing and non-renewing tissues. The distribution Of immunoreactivlty 1s essentially as would be expected of a maher of quiescent ceils. In contrast. in established pancreatic Carcinoma and other epithelial ceil lines. we have found $latin immunoreactivity ~n cycling cells using biotinylated K67 Slatin double labelling techniques. In ConcIusion. statin lmmunoreactivitv in normal tissues Correlates with auiescence but this realtionship IS lost, at least in vifro. 10 Loss of cell-cell and cell-substratum adhesion are imponantfactors during turnourprogression. Tumour promoten are Compounds Which although not carcinogenic themsews increase the frequency of turnour development in animals previously exposed to carcinogens. We have used the turnour promoter TPA on cultured human renal epithelia ~ells10 mimic nwplastlc transformation. Following TPA treatment we haveexamined thedistribution of vlnculin, B1 integrin and actin within thetreatedcells byfluorescencemicroscopy.Treatment of therenal epithelium by TPAcauses arounding up of the cells and a loss of adhesion toeither laminin Or fibronecttn substrata Fiuorescent microscopic examination reveals a progressive loss of reactivity for vinculin and B1 inlegrin within local Conlacts. These changes are accompanied by a redistribution of the actin microfilaments from Orientated bundles of Stress fibres to a circumferential arrangement These Changesofareduction in focal contact componentsand disarganised actin cytoskeietan mimic the changes we have previously described in renal Carcinoma. Glutathione s-transferases are a diverse group Of enzymes with an important role !n the metabolism 01 faelgn compounds including Some carcinogens and Cancer chemotherapeutic agents. Increased expression of GST PI is seen in manyanimal and humancancersandisassociated with resiStanCetocalboplatinandcisplatin in humanlung cancet cell iines. GSTs are involved m steroid hormone transport and metabolism and have a role in the complex metabolic relationships between Settoli ceils and germ cells. We have studied GST isoenzymne expression by an immunohiStochemical method in 16 Stage I teratomata. 16 stage I 1 tetatomata, 6 stage I seminoma, 11 cases of intratubulargerm Cell neaplasia(1TGCN) and a groupof Cryptorchid and normal testes. The Stage I 1 teratomata had beentreated with cisplatin based therapyand bothprimmaryandpost-therapymetaftatictumourtissuewerestudied.

GSTAlphaexpression correlated with morphological evidence of epithelial differentiation in teratomata.Therewas no difference in GST expression between stage I and stage I 1 pnmary testicular IumourS nor between primary testicularand post-therapy metastatic tUmOUrS. GSTexpression did not correlate with survwal. GST Pi was strongly expressed in the neoplastic germ cells Of ITGCN but was weak or negative in normal germ cells. This may be Significant in view 01 the potential for later contralateral turnour development tn patients treated by cisplatin based therapy. In Summary, GSTexpression in testicular germ cell turnours reflectedlheirdifferentiationandappsared to be unrelated l o therapy and subsequent survival. A case illustrating the usefulness of electron-microscopic examination of fine needle aspirates 1s described Fine needleaspiration wasperlormedon asubcutanwusnoduleinthechsstwall ofaneldedyman whowassuspmted tobesuffering from Bronchogeniccaronomaanclinicaland radiologicalfindings.mesmears werenotdiagnan~c. but ~n view of the history af asbestos exposure, the needle washings were submined for eIectrOn-microSCOpic examination, Which showed mesathelial differentiation and a diagnosis of metastatic malignant mesotheiioma was suggested. An autopsy perlormed eight months later confirmed the diagnosis of malignant mesotheboma.

T. Dorman, A ti Ismail, B Cunan, M. Leader Pathology Department Hoyal College of Surgeons m Ireland, Dublln many hiStOlogi~allymalignantfeaturessuch as hypercellularltyand high mitOtiCCOUntS, but clinically followa benign course. Demoid tUmOUrS and fibromafoses can be densely cellular but usually quite bland histologically and are associated wdh infiltrative marginsand repeated local recurrences. In addtion irradiated tissue often Contains many cellswithcytologicalfeaturesol malignancy ThiSStUdyexaminesthepIoidyofsuch lesionsbyboth olthecunently availabletechniques using dlsaggregated formalin fixed paraffin embedded tissue Sixteen cases 01 NF, 6 DFSTs. 7

DTs. 29fibromatoses(1ncI~ding palmar, plantar. retrcpentonealand Soh tissue) and 5 miscellaneouscases including 1 leiomyoblastoma, 1 inflammatory pseudosarcoma and plexiform histlocytama. One malignant fibrous histimytoma and t paragangliama Were included as possible poSLIVe Controls. 10 cases containing numerous irradiation fibroblasts were alm analysed All 'pseudosarcwnas' were euploid by both image analysis and flow cytometry The malignant fibrous histiocytoma was aneuploid by image analysis and flow cytometry and the paragangliomawasaneuplaid by imageanalysis and tetraploid by flow cytometv Themndwonsof thisstudv are that pseudoSarcomas are euploid and aneuploidy would appear to be confined to malignant tUmOUrS of mesenchymal ongm. other sites We collected 32 cases to study their morphology and antigenic profile after staining for the epithelial markeffiCAM5.2 flow molecularweight keratins)andlp34 (high molecular wefghtkeratins), thegeneralmelanoma and neural markers S1 W. neurone specific enoIas8 (NSE). protein gene product 9.5 (PGP) and the melanoma S w i f l C marker HMB 45, the neuroepithelia markers Leu 7 and glial fibnllary acidic protein (GFAP), and the intermediate ftlaments vimentln VIM) and nsurofilament (NR The following turnour types emerged: I pure Spindle celltype(N = 5),11,purepolyganalcelltumaun(N = 12)comp0sedofpleomorphiccells(N = 3).undwmcellswith an alveolar IN = 3) or sheet arrangement (N = 6). and 111 mixed pleomorphic spindle and polygonal cell tumouffi (N = 13). Two tumouffi proved to be lymphomas An intraepithelial melanocytic component could be established in 7 cases Within adjacent respiratory or squamous melaplastic epithelium. In 2 cases n e w trunks a~mclated with tumOUr contained increased numbers Of atypical Schwann cells. Consistently expressed were S1W (30130 and colon ovanan tumour antigen (COTA). CSAis a heat stablemucin associated antigen present m normal colonic epithelial cells and IS expressed m greater quantitites tn colon8c adenmarcinomas COTA is a heat Stable antigen present 8 n colanicneoplasiaand mumowwananturnours but not ~nnormalcalonicepithelium 5psmionsfrom to1 primary adenocarcinomas (30 ovanan, 20 Colo-rectal, 9 gastric. 10 breast. 5 Oesophageal. 10 prostatlc. 13 pancreattc, 3endometnal and t gallb1adder)and two adenmarcinomas metastatic tothe liverwere incubated with anti CSA and anti COTA antibodies using the P.A.P technique with positive and negative controls. While anti CSA positivitywasseenin 19d20~0loni~aden0carcin0ma~(9wsakand lO~tr~"g).~twasalsaseen 1n20af30ovanan (13 weekand 7 strong). 6 of 9 gastric (3weakand 3 strong), 6 of 10 breaSt(5 weak and 1 strong). 5 of 5 Oesophageal (4 weak and 1 strong). 5 of 10 prostatic (all weak). 11 of 13 pancreatic (9 weak and 7 strong). 3 Of 3 endometnal (all weak) and the gallbladder ademcarcinoma. Both liver metastatic adenocarcinomaS were negative In additim while anti COTA staining was positive m 16 of 20 colonic adenocarcinomaS (6 weak and 10 strong) and 20 of 30 ovarian adenocarcinomas.~twasalsapositive~n5af 9gastnc(3weakandZstrong).3al lObreast(2weakand 1 StlMg), all 50esophageal(allweak)5of 10pr0~tati~(allweak), 11 0113pa"creatic(lOw8akand t strong),all3endometnal(all weak) and the case of gallbladder endocarcinoma (strons) Both liver metastatic adenocarcinomas were negative. m e C O~C~U S~O~ of this study 1s that anti CSA and anti COTA are not adequately specific in the Identification of a ~olonlc or Ovarian origin af an adenocarcinoma and Cannot reliably be applied to the identification Of a metastatic adenocarcinoma of unknown primary Site 

The early Intfa-epithelial changes of adenocarcinoma of the nose and paranasal Sinuses were sought In the histological sections from 30 high grade adenocarcinomas and 4 cyclindric cell Carcinomas from this region none of which had had previous radiotherapy treatment. 7 of the cases came from wwd-workers. 1 I r m a polishw and 1 from a plasterer Attention was directed to the non-neoplastic epithellm of the Surface and of the sera-muclnous glands. In 14 casesofadenocarcinomaand t of cylindriccell carcinoma surfacechaogeswere detected These took the form of hyperplasm of goblet cells of the rsspiratq epithelwm accompanied by dysplastic changes of other epithelial cdls: m the latter these became enlarged and irregular m Shape with large hyperchromatic nude and prominent nucleoli. lnthsmajorityof IesionsthesechangeswereisolatedIn theepithelium. Changesof thesetypes were never Seen in sefomucinous gland epithelium. In addition similar changes were not noted m the coverlng respiratoryepithelium in 50casesofnasal polyps evenwhen severely inflamed Thesemsmchangesso described Haematoporphynn derivative sensitised tumwr tissue. 1s Underwing evaluaton 4" widesptead and m i s t a n t papillary tumOurSand ,n casesof rvldespreadseveredysplasra/carnnoma,nufu". Arewewof Spallents (21,7m, ages 4 6 8 0 years. pre-and post therapy) has revealed no change ~n histological grade and stage ~n 7 patients, progression to invasion in t case and a redunion to mild urothellal atypla alone in 1 case. Local tissue changes following treatment were limnedto oedema and an acute lntlammatoryreactlon. No metaplastlc. Stromalfibroblast or neNe changes were seen Variations ~n bladdw size. both increased and dlmtnlshed, wwe encountered The histological grading Of TCC following POT Shows little Improvement. but studies are in progress to improve lhght delivery In the bladder, and hence improve treatment outcome.

A Armour. A. S. Jack Bpartment of Pathology, Umverslty of i d s . Leeds. LSZ 9JT Follicular lymphoma IS a disease characterised by widespread lymph node involvement usually at the time of presentation proper tie^ of lymphocyte homing and Circulation appear to be mediated by a variety of cell adhesion molecules. The a m of this study was to compare mrmal nodal lymphocytes with the neoplastic populatlm m folllcularlymphoma. hall caSeSalloftheneaplasticfollicleSexpressed lcaml but thiSYanedfromonlyafewcellsto 70% of cells within a follicle This Dosltlvlty included dendmic retic~l~rn cells. germinal Centre cells and neoplastic lymphocytes. Leu 6 (lymphocyte homing receptor) showed an inverse pattern of expression in all but 3 cases The phenotype (Icaml + L a 87 IS a feature of germinal centres and scanwed paracontcal blasts in reaclive nodes anhough there IS uniformity of expression of lcaml in germinal centres which IS not apparent in any folllcular lyumphoma case. This study showed a loss of lcamt and increased Leu 8 expression by neoplastic lymphocytes within follicles This may relate to the propensity of this disease to spread widely throughout the lymphatic System Activation leads to an alteredfunction and aconcomitant aneration in the chemistry ofthe cell surface, which ~s a site nchincalbohydrate. Rat lymph nodelymphacytes.eitherunactivatedoraclivatedfot5days1namixedlymphocyte reanlon. were treated with biotinylated IeCtinS from a large panel. chosen to probe surface glycans Lmlns were revealed With awdln-phycoerythnn and cell populations were analysed in a fluorescence activated cell-sorter Double-stalnlng with FITC-monoclonal antlbodles defined the functional lymphocyte Subsets. UEA-1 and LTA boundtOnoce11sof anytype. UnactivatedB-cells boundall theremaininglectins.saveMPA. toagreaterextentthan did UnactlvatedT-cellsand B-cell actlvatlon produced no change I" glycan sxpresslon. Unactlvated and actlvatedTcells all expressed a2.6-linked Sialyl residues, but o-2.3-linked sialyl expression was hetrogeneous, did not COneSpOnd toanysubsets,and wa~unchangedonanlvatian.Alargegroupof lectinsshowedlow wno binding to UnactlvatedT-cells, but boundonactivatianexactly In parallel to1L-2 receptorexpressionCTacantigen) Thes~ngle Structure GaWGalNAcal,3Galpl,4GlcNAc-)

GalIGalNAcat ,3Gal~1,4GlcNAc-) R could account for their binding The presence of neoplastic (light chain restricted) B cell follicles In low grade B c d gaStrolnteStlnal (GI) MmPhomaof

Although MALT 1s not present in normal human gastric mucosa, lymphold t,ssue ,* acqulr& ,n response lo

Colonisationof themucosa by Helrcob~cleipyioii. We have investigated the possibility that thisacquiied lymphoid t1SSue IS 01 MALT type WhlCh may povovlde the background I" which lymphoma can arl~e w e examined gast,,c b~0p5~e5fr0m450casesafH~l~~~bacrerassaciatedgastntis.and biopsyandresectionspecimensfram60casesaf t"mours has cOntr'buted to a peKlstence Of Ihe more "Iew arefofl'cle (Fee) gastrlc 8 cell lymphomaof MALT In 175 cases of HeBCobaCIergaStritiS prominent lymphoid follicles were idenfilled In 8aftheee B cell clusters were identified within the gastric epithelium. reminiscent Oltheteatures seen 10 the dome epithelium Of sm11811 inteStinal Peyer'z patch. Thls B cell-eplthelh aSSOclatlOn was not assaclated With the eplthelm cell changes or the glandular destructm seen ~n lymphoeplthellal I~S I O~S 01 MALT lymphomas. In 54/60 cases of gastric MALTlymphoma Helicobaclercould be identified Oltheremaining6cases.5 weiegastrecfamy specimens 8n Which specimen washing may have contributed to the negativefindings. We suggest that gaStrlc MALT 1s acqulred In response to local immunological Stimulation as a result of mucosal coloniSatiOn by Hel~cobaclerpylon, and that the development of MALT lymphoma IS a subsequent event mUCOSa associated hmphold tlSSUe (MALTI has been explained On the bas15 of speclftc colonization at reactlve B lymPhomaSbe Included In lhecatqoryof MALT lymphoma but the frequent presenceotafoll~culapanem in these foltiC1eS by Ihe neaplastlc cantrocyte-like(CCL) It has been low grade Cellthyro1d

We have Ihe and 'nveSflgated the and genotype Of' Of primary low gradeBcelllymphomasofthethyro'd alsodemonstratedfeatures ofMALT1ymphama'ncludtng CCLce115and l y m p h~' t h~' a l l~' o n s . m e appearances and immunoh'Sto'og~ofthefo'l'c'es werethoseofto'l'cularcalon'rat'on described 'nGIMALTMmphomarather thanFCCfoll'cular'~mphoma Thepredomlnant iWg1 ~ane'naffoll~cularcolon~rat~~nconformed tothatdeSlgnated ccLcells show'ng aStnk'ngly h'gh prol'feratton late No evidence Of the '(14 ' ' 1 tranSIoCat'an was found In any On DNA extracted from fresh (n = 1) or paraffln embedded (n = 9) t15sue mese IlndlngS argue against a FCC lineage lor primary thyroid lymphomas and support their 1ncIus1on 8" the MALTcategory We have used a panel of antibode$ to demonstrate Stages of granulocyte maturation by immUnOh~St0ChemiStry I" decalcified. wax-embedded bane marrow trephine biopsies Antibodies reactive with muramidase. u-1 -ant~tryps,n. neutraphil elaslase and CD68 react with early granulocyte precursors. CD15 and calgranulin identity later granulOCyteS. IhdiYldUaI antibodies dlfier rn the populations 01 cells ldentlfled TheantlbcdteS also react With cells of mWlOCyte llneageand provide information concerning theorganlsatlon afmonopolesls about whlCh liffle IS known

In nOrmal marrOw granulopoiesis IS zonal wlth maturation occurring radially around trabeculae and blood vessels This pattern IS exaggerate3 in reactive hyperplasia and chronic granulocytic leukaemia (CGL) There IS marked disruption of this zonal organisation in myeloproliferative and myelodysplastic states, with considerable overlap 01 Patterns between these conditions In chronic mvelomonacvtic leukaemia (CMML) there IS comDlete absence of zonal arrangement of granulopaiesis possibly due to monocytic proliferation obscuring the underlying marrow spaces and that by analogy With CGL CMML represents an exaggeration of this normal panern granulopoletlc Panern We hypotheslse that monopoles15 normally Occurs 8" a randomly dispersed fashion Within As previously Shown by us in animals untreated With Cyclosparin A. the cell birth rate fell from an initial prelmmunisation valueof 32 cellsit 000cellsihour to 18 cells11 000 celldhouran day 2 followed by a rise1044 cellsit 000 cellslhour On day 4 However, in Cyclosporin A treated animals the cell birth rate (1 7 5 ce11s/lOOO cells/hour) was signiticantlydepressed belowthecontrol pre-immunisation levelandremained suppressedupfaday4fallowed by an abrupt rise These results are consisten1 with the hypothesis that T lymphocytes or their pmducts not only drwe The morphological appearancescompnsed lymphoepithelia lesions (In onecase). numerous lymphoid follicles and a diffuse infiltrate of monocytoid cells (centrocyte like cells) There was striking plasma cell differentiation and COlonisation of lymphad folllcks by monacytoid cell?. and neoplastic plasma cells. lmmunahistochemistry convincingly demonstrated heavy and light chain restriction ~n all the biopsies from both the cases The bladder 1s developmentally related to the hind gut and this manifests 8" the variety of metaplastic epithelium seen at this Site Circulating cells. encauntenng the endothelial surface. make contacts 10 an environment rich 8 n glycan. A large panel of biotinylated lectins was used to probe far variations in the glycans expressed on endathelia of artenes. veins, arterioles, venules. capillanes, high endothelia vessels and lymphatics m a range of normal and pathological human tissues Formalin-fixed, paraffln embedded specimens from the files of Manchester Royal Infirmary and ManChester Royal Eye Hospital were used Lectins were revealed with an avidin-peraxidasesystem No differences werefound between arterial. arteriolar, venular, veinous or lymphatic endothelia all expressed abundant complex-type N-linked glycans. of several subtypes. Capillaries were highly variable and Showed heterogeneity ~n their expression Of 1) Outer chain sequences fn N-linked glycansand 2) mucin-type sequences. both between different. normal organs and Within an organ. implying that the surrounding tissue probably had a regulatory effect Where endothelium was reactive, additional aneratiom wete seen and actively growmg endothelium in granulation tissue expressed hqhmannose SIwCtweS. High endolhelial YBSS~IS showed a much lower density and narrower range of glycan expression thandidad~acenlnormalCapillanes,despite theirknownvery highrateofglycansynthesisand secretion The IymphocyteILN-homing receplor(s) would be a component of this restricted porlfolio of glycan Monocyte margination in atherosclerosis associated with immunological injury N. J. Combs, P J Gallagher. P. S Bass

Clinical and experimental evidence indicates that immunological injury IS associated with accelerated atherOSClerOSiS. Allograft recipients may develop accelerated atheroscleross and anlmalsgwen serum Sickness and a high fat diet develop more extensive atherOSCleroSis than controls fed the diet alone We have tested the hypothesis that atherosclerosis ~n these animals IS associated with increased adhesion of monocytes to the aortic endothelium. Chronicswum sickness was induced in genetically hyperlipidaemic rabbits with nativeanionic bovine serum albumin (nBSA) or highly CatiOniSed protein (CBSA) Rabbits given nBSA showed a Spectrum of glomerular endocapillary prollferativechange Thasegiven cBSA developed early membranous glomerulopathy In COntrOlS the numberofmanocytesadherent to theendothelium ranged from 37Y5Isq mm and m animals given SeNm sickness 15-305Isq mm (nBSA)or14~5Isqmm(cBSA) As therewerenosignificant intergroupdifferences theseiesultsdo not support the hypothesis that immunological injury increases monocyte adhesion to the aortic endothelium

Departmen! oIPatholcgy, SOuIhampfon Universily Hospdals Soufhamplon. SO9 4XY Amyloidcan beidentified 1nupta20% of elderty hearts,especiallyin themyocardium oftheatrialappendages In a small prapanionofth%lecasesamylaid~salsapresent inthecardiacvalve~ butisolatedvalvulardepos,tsarerare.

A 73 year old male presented with bilateral leg Weakness and an intradural turnour. He died 3 days after spinal surgeryanddeposltsofaglloblaslomamunlforme wereldentlfledm both thecerebrumand thecord Therewasno hlstory of cardiac disease but all four valves showed translucent verrucous thrkenlngs. These had a uniform eosnophilc hi~lologicalaPpearanCe. stainedwithcongo red and had UltraStNUCtUralfeatUreS of amyloid Amyloid P proteinwas identified immunohistochemically ~n thesedeposits but negative multswere obtained withantibodies t o m . ALand A4 proteins. mwewas no evidenceof cerebrovascular ormyocardial amyloidosis The involvementof all four cardiac valves. the stnking absence of amyloid I" other organs and the a~~o~l a t i o n with a widespread glioblastoma are unusuaI but unexplained features of this case The IMA 1s used as a bypass of narrowed coronary arteries It IS said to be less prone than vein gratk to develop subsequent occlusivedisease. Thisstudy of pairs of IMAsfrom subjects of vario~s ageswas to see if the histological structure ofthevessel might explain immunityto graft disease Sixteen IMAs were fixed in distension byformalln at 150 Cm Of water pressure and secllon~ taken at the level of each nb (first to StXth). All arteries undelwent Similar changes along thelr length. wlth no Slgniflcant difference between left and rlght lntimal changes were minor conssting of lbro-elastic muscular thickening in all age groups and in those who had died from vascular disease TheinternalelaStiC lamellawaswelldefinedat all levels. bUttheexternallamellawasclearlydefinedonlyatthelevel ofthefmhandslxthnbs(I.e. mthedetalartwyJ. Medral thrckne~~decreasedalongthelengthOfthearter~esandInall cases changes from an elastic to a mUSCUlar structure, generally at the level of the fourth or fiflh rib The ratio of medial thickness to numbw of lmdlae in~reases along the artery. notably at its point of change Muscle fibre Orientation changes from inter-mixed cir~ular and longitudinal in the elastic part to predominantly circumferential in the muscular pan. The pronounced StNCtUral differences of the IMA compared to the Similar sired epicardia coronary arteries. which are muscular. may be Of relevance in explaining their markedly different incidence of atheroma.

Naomi Carter, S Variend

Fatty change of the heart IS a pwrly defined pathological entity which in the adult hean can be caused by severe hypoxia nutritional disorders. poisoning by Selected drugs and catecholaminerelease It IS most commonly seen ~n association with coronary artery disease Little data exists with regard to the paediatric hean In 980 pediatric deaths coming to post mortem over a 10 year period. there were 66 cases of myocardial fatty change of varying seventy and distribution detected ~n 0 1 1 Red 0-stained Sections Infection and congenital diSOrdenwereimplicated 1n39deaths Infectionand cangenitaldisordenwereimplicated 1n39deaths. 5910fallcasesoffarmchangeand 2 2% Of all deaths Seven ofthe5e~ases had acombined ~nfecti~u~andcangennalaetiology Othercausesofdeath included turnour, traumaandcomplicationsofbinh 3 6% of casesofSudden Infant DealhSyndrome(S1DS) hada fatty heart Only one case of the total 66 cases showed deflnlte hlstaloglcal evldence of ischaemic myocardial damage. Insome instances, thedegreeaffattychangemayberelatedtathedurationand seventyoftheundertyng condition Someofthesechildren may havean occunnutritional orenlymedisarderwhich ~sexpressedatacellular level ~n the form o! fatty change and that contibutes to their early death.

We havecampared thereparting OftempOralarterybiopsiesbetween 1973-78(135cases)and 198691 (91 cases).

The overall incidence Of positive biopsies was 24.5% and 26.5% In each period the number of patients with clear clinical evidence of cranial arteritis was 42% but in 1973-78, 58% and in 198691, 66% of these had positive temporal artery biopsies When the histology was reviewed approximately 6% of biopsies in each period had been erroneou~ly reported as healed or atypical artentis. In contrast. a true histological diagnosis of artetitis was missed in~nlya~inglepatient Approximately 18% ofall patientswithaclinicaldiagnosisof giant cellarteritisdevelopedan additional symptom or pathological change associated with Steroid treatment Frequent final clinical diagnoses in patients with negative temporal artery biopsies were transient ischaemic attacks, cerebrovascular accidents. unexplained headache or migraine. polyarteritis or polymyalgia hwmatica These resuns confirm that one third of patients with deflnite clinical evidence of cranial arteritis will have negative biopsies Pathologists continue to misinterpret normal arterial ageing changes as evidence of healed or atypical arteritis

Depanmenf 01 Hslopafhology and Depanmen! 01 Resp8raIoiy Medicne. Sl Banholomew s Hospifal London EClA /BE Adhesion ~fepithelium to extracellularmatrices is mediated partly byafamily of heterodimeric molecules known as megrins We haveexamined theexpressionofthealpha-1 toalpha-6integnn Subunits in~unured human branchia epithelial cells. and in bronchial biops8es from normal subjects and atopic asthamtics. We have also studied the expression of intercellular adhesion molecule-t (ICAM-1, CD54, rhinovirus receptor) Br~nchialepithelia cells from surgical Specimens were grown as explant Cultures on glass covenlips. Bronchial biopsies were taken from right upper and middle lobe carinas lmmunastaining was performed on acetone fixed cells and frozen tissue Sections using alkaline phosphatase and immunoperoxidase techniques All biopsies showed strong positive staining of epithelium for alphe?, alpha~3 and alpha-6 integnns. Stainingforalpha-5 was weak or negativeand epithelium was negative foralpha-1 and alpha-4 except in twoasthmaticswhere itwas weakhi alpha-4 posIttve. In contrast. cultured bronchial epithelial cellswere posItIve for all these mtegrinsexcept alpha-1. Epithelium was positive for CAM-1 in 91 17 asthmatics but negative 10 all Other biopsies. Cultured cells were ~trongly positive for this molecule It 85 concluded that expression of Some adhesion mOleculeS bv bronchial emhelium may vaw ~n relation to the cellular environment and that ths may be imponant tn disease

L%panmen! of Pafhology Univeisrty of Birmingham and Departmenrs o! 'Immunology and 3His!opalho!ogy Eas!

Blood eosinophils are in a relatively inactive State With migration into tissues eosinophils became more activated These activated cells are hypodense compared to most blood eosnoph11s Low affinity receptors for both IgE (FceRII. CD231 and IgG (FcgRIII. CD16) have been documented on activated. hypodense eosinophils This study assessed the expression of these proteins on ttssue eosmophk derived from nasal wlyps The blood and nasal polyps of seven patients undergoing nasal polypectomy were studied Nornodense and hypodense eosinophils were isolated from venous blood by centrifugation an a discontinuous Percoll gradient Percoll wassimilarly used to obtainaneosinophilrichpreparationfromcellsearactedoutafthenasalpolyps Cytospin preparationswetem?.de of these samples Frozen sections of each polyp were also prepared lmmunostaining using an alkaline phosphataseianti-alkalln phosphatase detection system demonstrated that neither blood nor nasal polyp eosinophilsexpressed detectableCD23orCDt6 l t i~p~~~i b l e t h a t e~~i n~p h l l S O f na~alp~lyp~aresimilarto blood eosinophils and are in a relatively inactive State lavage and biopsy studies. the relationships between mucosal inflammation. bronchospasm and bronchial hyperreactivity are unclear Since bronchial Smwth muscle has an essential rolein the pathophydogyof asthma.

we haveexamined theextentto which it ~sin~ol~ed~nallergicinflammation Bi0p~ie~in~l~ding~moothmu5clefr0m 10 asthmatics (age range 19371 who did not use steroids and 4 Controls (age range 2243) were embedded I" Araldite and stainedformast cells ~mOnOCIOnal ant1bodyAAt)and eOSinOphilS(monoclona1 antibody EG2). Mast cell numben In the lamina propria and smooth muscle were similar for both asthmatic and Control Sub e m (mean values.

astham lamina propna 70 81mm2. smooth muscle 72 tImm2, controls 87 5/mm2 and 43 Blmm'respectively) Eosinophil numbers in the lamina propria were increased 10-fold in the asthmatics (p = 0 008) but there was no Significant increase in the number of eosinaphils rn the bronchial smooth muscle EOSinOphil numbers in the asthmatics correlated positively With FEV, We conclude that the role of muco~al inflammation in the pathophysiology Of asthma has yet to be determined We present5 cases ofextra-pulmonary pneumocystosis diagnosed on routinesurgical specimens (two biopsiesof liver. and one each of gastric mucosa. Small intestine and a pen-anal mass). In each case. the histological features were similar to those seen in the lung. and as in Other material from cases of AIDS, munlple pathology was often found Extra-pulmonaly pneumocystasis 1s now being reported from a widerangeof clinical Specialties. One reason forthismcreasemaybe that impraved patient SUIYIY~I with"topicaY inhalatiooalpentamidinetherapyallowsvisceral foci of infection to become clinically apparent This hypothesis IS supported by the finding that 4 ofour5cases were taking nebulised pentamidme Inthesehypersensltivitystatestothatobselved mbronchiectasis (a Chronic Suppurative lung condition not thought to In~olve a hypersensitivity aetiology) and in smokers and non-smokers with no evidence of active pulmonary inflammation Ourresuns havesh0wnthattheB:Tlymphocyteratioisnodifferent inEAAandsarcoidfrOmthat seen ~n bronchiectasis and in normal lungs. We believe that thns IS further evidence to suggest that BAL IS an unrepiesentativetechnlquetorlhestudyof interstitial lung diseasesand that morecansiderationshould begivento the possibility of humoral immune components in the pathogenesis of EA4 determine Its effects upon the number of pulmonary neuroendocrine cells and their peptides. In one experiment. the Concentration of noradrenaline in the lung was estimated by chromatography, and that of the peptides bombesm. neurotensin and caknonin gene-related peptide (CGRP) by radiaimmunoassay. There was significantly Ies5 noradrenaline and bornbesin in the lungs of test rats than in controls but the levels of neurotensin and CGRP were unchanged. In a second experiment, pulmonary neuroendocrlne cells In histological sections were labelled with antiserato bombesin,calc,t~tonin,CGRP andproteingenepmduct9 5[PGP 9 S ) a~d =~" " t~d . T h~, e w =~ nochange in the number of labelled neuroendocrine cells expressed per unit area of lung or per unit length of airway between test and control rats for calcitonln. CGRP or PGP 9.5. Bornbesin-containing cells could not be identified in either

group. An increase ~n pulmonary neuroendocrine cells could not be identified ~n either group. An increase in pulmonary neuroendacrine cells immunoreactive for bomtesin and calcitonin OCCU~S in the early stages of plexOgenicpulmonaryartertopathy in man. Theabsence ofsucha change in mOnOCrOtallneDulmOnaNhYDertenSlOn in the rat suggests that this IS a poor model for the human disease. This preliminary Study was carried Out to assess the quantitative expression of neuroendocrine and mast cells in adun humanlungsofcasesofasbestos-relateddisease.Tencaseseachofasbestasis, pleuralplaques,carcinoma and mesothelioma were studied m comparison with ten normals Wnh no history 01 exposure to asbestos The lung sections were stained lor neuroendocrine markers neurone Specific enolase (NSO and chromagranln. and, chloroacstate esterase and toluidine blue for mast cells. There was a notable variation in the number of neuroendocrine and mast cells between the control and asbestos-related disease group The Variation was also seen between the various asbestos-related diseases. Though not statistically sbgnificant. the trend of the Vanatton indicated that the individual diseases follow a particular pattern a n lhe expresston Of these two cell POPUIPIOIIS. We have studied fibrous turnours of the pleura using morphology. immUnOhiStwhemiSlry and eleclronmaroscopy.

The findings were compared and contrasted with reactive pleural fibrosis and desmoplastlc mesothelioma. The fibroustumwn hadarangeof hiStologicalappearancesand30% weremalignant ~nnature.Theimmunophemtype was uniform and consistent with positive staining for Vimentin and alpha m w t h muscle actin. This was ~n Sharp contrast to findings ~n reactive pleural fibrosis and desmoplastic mesothelioma. UttraStruCtUral appearances of the fibrous tumours of the pleura Were Supportive of a myofibroblastic ongin. We propose that fibrous tumours of the pleura arise from the Submesothelial myofibroblast. The malignant fibrous turnours have a distinct immunohistochemical profile and electron microscopic features to differentiate L from themalignant mesenchymai mesothelioma. A study was undenaken to evaluate the use of immunaperoxidase stains on paraffin embedded tissue lo define the cell type In routine lung cancer preparations. and in particular to identdy a subgroup of turnours showing neuroendocrinedifferentiation. Forty lour consecutivethoracotamycaseswere selected. Following apilot study of 6 cases. to assess digestion times and potentiaily useful antibodies, the remaining cases were processed using a battery of monOClOnal antibodies: cytokeratr IAElfAE3, 348E12). neuron specitic enolase (NSE), chromogranin. and beta 2 microglobulm. In addition to the 3 carcinoid turnours and 1 oat cell Carcinoma in the study 3 large cell Carcinomas and 5 adenmarcinomas demonstrated positive neuroendocrine markers. Uiirastnrcturally, dense core granules could be demonstrated in Only 2/3 of the large cell Carcinomas and in 1/5 adenocarcmomas. The discrepancy between the lmmunOperOxldaSe staining and electron microscopic features likely reflects the heterogeneityafthese turnours. In thisstudy noneof the turnours co-expressed neuroendocrine markersand beta 2 microglabulm. However, the staining panern was inconsistent m the remaining cases High molecular weight cytokeratin (34PE12) was Stronglyposltlve m all casesot squamouscell carcinoma and negativein everyihing else. In summary. monoclonal NSE and chromogranin appeared to provide SuffiCient information to identity neuroendocrinedifterentiatian in thecasesexamined m this Study. High molecularweight cytokeratinwasfound to bea usefuldiscriminaforforsquamouscelicarcinoma. Beta2 microglobulin was negativemall the turnoursshowing neuroendocrine differentiation. butthe inCOnSistenCy Of staining m non neuroendocrinetumaurs. made it less helpful for routine laboratory use

Although endocrine differentiation IS the essence of Small cell Carcinoma of the bronchus, its occurrence m other morphological ("on-small cell) types of bronchial 1Umour (large cell, squamous and adenmarcdnoma) IS welldescribed However, its prevalence m Such tumours IS uncertam, estimates differing from study to Rudy and accordingto how It IS sought. We have examined, byimmunalabellmg, expression offiveendocrine markerproteins (neuron-specific enolase (NSO, protein gene product [PGP) 9.5, the BB isoenzyme of Creatine kinase [CK-BB), SynaptophysinandS-t 00protein)m60 bronchoscopictissue biopsiesof "on-smallcellcarcinomaandasse~ns varlabiltty Within and between tumour deposits In 16 subjects coming to necropsy with disseminated disease

Exactly half of the tissue biopsy specimens immunolabelled for one or more marken; one for four, four for three, twenty lor two and five for one. possibly indicating an element of endocrine dtflerentiation inapparent from their morphology. Expression was even more prevalent amongst the extensively-sampled turnours at necropsy, but Since theinlraductionofthenewGeneralPractnianercontractmApril 1990,lhere hasbeenasignlicant increasein thenurnbeisof skin biopsiesreceivedmHistopathologydepa~ments In ourdepartment there has beenalhreefold increase in numbers of General Practiioner skin biopsies. The aims of this study were to crnlcally appraise these biopsiesand comparethem tosimilarlysized Skin biapsiesreceivedfrom hospital in-patientsviageneraland plastic surgeons for the SIX months prior to and aner 1st April 1990. Data collected included numbers received, ranae of pathological diagnoses, quality 07 information supplied on the request card, accuracy 01 clinical diagnoses. adequacy of excision, age. sex and sites of lesions The resuns showed a Similar range of pathological diagnoses.

The quality of clinical information supplied was comparable in the two groups as was the age and sex of patients. General Practitioner biopsies were less common from the face. Clinical recognition of lesions was somewhat less accurate amongst General Practitioners than amongst Hospital Surgeons Inadequate excision was more common m General Practnioner cases. 5.5% of General Practitioner lesions were found unexpectedly to be premalignant or malignant (eight cases) and all ofthesewere inadequatelyexclsed. Important implicationsemergingfromthisstudy are dlSCUSSed An audit of skin biopsy specimens from general practitioners in Grampian Region: changes in requesting practice and specimen type Assessmen1 ofresaction marginsofsurgicalspecimens~s becming more important m manyfieldsofpathology. We wwe interested in developing a means of assessing surgical margins 10 dermatopatholqy in both conventionally removed skin ellipses and m Skin ellipses removed during M O~S chemasurgery technique Many of the conventionally used markers, such as Indian Ink, Alcian Blue and Tipp-ex Correction fluid are difficult to use I" that they are messytoapply. slow todry and show insufficient Contrast wiih oneanother both lnthegrass specimen and microscopically Weused"Superman'paint,apaintusedforresinandplastermodels,Wh,chcomeSin awlderange of colours. The paint was easyto apply. did not run and dried qwckiy Becauseof the variety of colours avaciablewe were able to apply Contrasting c~l o u n to the vertical and horizontal axes of B X C~S I O~ af skm elhpses removed by MOhSchemosurgery.Thepaint prOYidedagaodmarkergrasslyand wasnot affected byfreezinqthetissue Model paint provides another marker lor surgical BXCIJ~II margins and 1s particularly useful for Moh's chemasurgery where horizontal and vertical axes aremarked inorderto assess theadequacyoftheexcision.The model paint may also be usetul in other branches of surgical pathology where resection margins are Important. In psoriasis there IS altered Bpidermal dmwentiattan and increased epidermal turnover both 01 Which involve changesin intercellularadhesion Aquantnative imm~nOh~st0chernicaicompa~~sonof the expression of the integrin Staining of integrin p SUbunltS 1-4 and e subunlts 1 4 were disclosed usmg an andin-blatln peraxidase techn!que.

Large epidermal dendritic cells (antigen presenting cells) expressed p-2 The psoriatic Skm.showed Increased 6-1 a-3.0-6. and p-2expression. buta-2and~-4showednosignificantdifferencefromnormal Thelncreasedlntegrin expression by keratinocytes seems to be a reflection of awered epidermal differentiation rather than increased keratinoc ?turnover Themcrease in ~-2pasitivedendriticceilscauidbeareflectianofalteredanfigen handkngin psonatic Gin. Interleukm-2 (IL-2). used m the treatment of patients with metastabc disease fallmg to respond to Conventional treatment, can induce regression oftumwrbuik in certain patients However, the systemlc administration ofiL-2 is associated wlth a number of toxic effects, including dermatological compl#catlons. These have been poorly documented We have prospectivehl studied the dermatological reactions 10 5 patlents treated wiih IL-2 for metastats Cdorectal ~arconoma. Pre-and past-treatment biopsies were obtwied where possible, arid sect~ons stained with H 8E. Giemsaand PAS: hesh tissues weresubjected to Immunophenotyplng.There were3 femaleand 2malepatients.with treatmentrour~sranginginnumbwfrom 1-5 Onlyonepatlenthadapastmedlcal hlstoryof any skm complaint (eczema]. Four patlenk suffered a dlffilse eryihematous reaction with mild desquamatm and dryness The other patient developed genwalised erythroderma and an additlonal photosensitivity type reaction.

Histology, anerthe Initialcourse. revealed patchyspmgioS1S. exocylosis and basal layer epidermaldamagewnh a mild perivascular chronic inflammatory cell mfinrate. with SUhseqUent treatment there wasthlckenmg ofthe epiderrnls, plgrnent incontinence. dermal oedema and more marked chronic perivascular cell tnfiltrate. immunohistcchemistry revealed markedchanges inthe expression of CDt , HLADr, CAM-1 and CD25 m the derm15. These Changes were greatly heightsnedwith subsequent liealmsnts. with addnional changes tn other T cetl markers. Clearly IL-2 enhances the parenchymal expression of antibody-dependent and antigen-mdependent accessory molecules which are important in focusing the immune response.

Claire M. Thornton. Maureen Y. Waish

Weexaminedalfprimarycutaneaus malignant melanomas seen rn thmdepartmmtOver a fweyear permd from 1986 to 1990. The total number of tumoum was 354 The number of cases of malignant melanoma both invasive and m M u , increased from 57 cases ~n 1966 to 92 cases In 1990. Superfmal spreading melanoma was the most common type of melanoma, accounting for 60% of the total cases This was followed by the nodular melanoma Whch accountedfor 18%. wlthlentigamallgnamelanomaandacral lent~g~nousmelanomabe~ngtheleast commontypes. Therewasanincreasmg numberoftumourspresentlngwlth aBreslow'sdepthoflessthan t 5mm. thefigurermng from 46% of cases in 1986 lo 64% of cases m 1990 Most cases were stdl of Ciar*e kud ivanhough mcreasing numbersaftumour presented at Clarke levels I and I 1 wlth a corresponding reduction ~n cases presenting at Clarke level V More lesions presented with a flat cross SeCtlonal profile ~n the later years the figures lncreaslng from 30% in 1986 1064% in i w o he numbor of I C S~S showlng surface ulceratan at predentatlon d-reased from 57% In t966to14% In 1990 Themltotlcact~vlty. thedegreeofp~gmentalion. theintensiiyofthe~nnammatorycell infiltrate.

the predominant cell type and the mcldence of vascular lnvaslon Showed no change over the study penod Classification of benign vascular tumours is notoriousiy difliwlt and ciinicopathological correlation is often imprecise. This almost certainly reflects the tendency of pathologists to lump together different lesions under the broad heading 'haemangioma', sometimes with capillarylcavernous subtyping. Twelve cases of a distinctive subset of cavernous haemangiomas, to be known as sinusoidai haemangioma, are presented. These presented in adWs (8 female. 4 male. mean age 49 years. range 2&77). Five arose in the upper limb and five an the trunk (of which two developed in mammary subcutis). All were solitary and presented as a bluish cutanmus swelling up to 3.5 cm in diameter of variable duration. One case was associated with ipsilateral gynaecomastia. Average foiiow-up of 5.6 years has revealed no tendency far local recurrence or metastasis. Histologically these were subCutaneouS/deep dermal lesions with a lobular, sieve-like appearance and focally ill-defined margins. They were composed of dilated. thin-walled interwmmunicating vascular channels with a pseudopapillary architecture. Thrombi were common and two cases showed central infarction. Vascular spaces were lined by monolayered endothelium Which was often plump and hyperchromatic but n d mitotic. Distinction from wnventional cavernous haemangioma and angiosarcoma (particularly in breast lesions) is discussed. Current methods forthe identification of Herpes Simplex Virus (HSVJ may fail to identity the presence ofthe Virus in biopsy or autopsy material. We have investigated 2 autopsy cases and 3 neurosurgical biopsy cases of clinically suspected Herpes Simplex Encephalitis by a nested polymerase chain reaction (PCR). DNA was extracted from routineiyprocessedand paraffin embedded material byproteinase K incubation, phenol chloroform extractionand ethanol precipitation. Nested PCR was performed using known oligonmlaotide primers designed from the HSVtype 1 giywprotein D gene from an area with the lowest homology with HSV type 2. Two of 2 autopsy c a w and 2 of 3 neurosurgical biopsy cases ware PCR positive for HSV. The third neurosurgical biopsy case was not confirmed by PCR as being due to HSV. Such primers lo HSV allow the rapid retraspeaive diagnosis of Herpes Simplex Encephaliis and Should be of value to neurapathologists. Tissueembedded at lowtemperaturein Lowicryl-K4M resin hasbeenshownto besuitableforimmunogoldlabellng of cellular antigens, and to be capable of withstanding the processing required for hybridization of nucleic acid probas.Theaimofthisstudywastoestablish basic wnditionssuitabieforhybridirationof digoxigenin labelled DNA probes to Lowicryl embedded material, both at the light and electron microscope levels. Cultured haemOpoietii cells wereembedded afler brief aldehyde fixation. Digaxigenin labelled Whole human DNA or plasmid pER322 (negative control) probeswereappliedtothinandsem-thinsectionsafterproteolytictilgestion and/or denaturation by heat or alkali. Hybrids were detected in semi-thin sections by standard wlourimetric methods. and in thin sections by immunogold techniques. Elaborate blocking procedures and prolonged washes were found to be unnecessary.

Specificnu~l~earsignaiwasseenatbdhthelightandEMisv~iwiththewhaleDNAprobe,revealingdetailsof nuclear DNA distribution not evident in paraffin Sections or cytospun preparations. Non-specific binding and background were minimal. Signal was greatly reduced if the denaturing step was mined, and was slightly increased by protwlytic digestion, though at the expense of cytoplasmic morphological integrity. While the sensitivity of this system is limited by the fact that hybridization occurs only at the suriace of the section, it is a rapid and specific means of nudaic acid detection, and offers the Dossibilitv of accurate iocalization of intracellular human and viral nucleic acid sequences at a fundamental level. of prostatic carcinoma, mouse liver, kidney and gut were used. AcP was demonstrated using an kc-dye cwpling method. The AcP was unaffected by the addition of 10 mM tartrate. although the AcP was known to be 'tartrate Sensitive'. The addition of 100 mM tartrate or 50 mM sodium fluoride weakened but did not eliminate the reaction.

Mouse tissues, prostatic carcinoma and leiomyosarcoma tissues were processed using variausfixatives and embedding procedures. These were tested for AcP and TRAP. The AcP in the muilinucleated giant calk of the leiomyosarcoma survived standard formalin fixation and paraffin wax processing and was tartrate resistant. As expected mouse liver, kidney and gut AcP and prostatic AcP did nd survive most fixatives and embedding procedures. However AcP could be demonstrated in those tissues processed bythe 'AMeX' method. i.e. fixed in acetoneat -2O' C. processed throughmethyi benzoateand xyianeto paraffin wax. TheAcP.thus imalised in paraffin blocks was eliminated by the addition of 10 mM tartrate to the incubating medium. If needed the AMeX pracedure PrRSeNes Some tartrate sensitive AcP in paraffin blocks. AcP that Survives standard fixation and embedding procedures is iikeiy to be tarfrate resistant. showed AcP only in osteoclasts. Various elements ot tissue processing procedures were examined to find the wnditionsnecessarytoachievemaximum AcP localisation. Tissue blockswere pr-SSBdintowaxusingstandard embeddingprocedures. Threefixativeswereused. 10% neutral bufferedfmalin, formalcalcium and ethanol allat +4'Cfor 18 hours.Twodecalcifyingfluids wereemployed, 14%ethylenediamine-tetraaceticacid(EDTA)pH7.2at +4%, ZO' C and37'C.andformicacidIsodium citrateat+4%forW8 hats. Formalinfixed EDTAtreatedtissuaat +4% produced maximum AcP activity. AcP was Shown in osteociasts, Some ostwcytes, chandrocytes. Cement lines., tide mark, periosteaicellsand large macrophage likecells inbona matraw. Formalinfixed tissues decalcified in EDTAatZO'Cand37'C werenegativeforAcPaswerealltissuesfixedinethana1. lnformalcalciumfixedtissueand formic acidlsodium citrate decalcified tissue the AcP reaction was weaker. with some elements, e.g. chondrocvtic AcP missing. All the AcP preserved through paraffin processing was tartrate resistant. It is well recognised that morphology and optical reSOiution are vastly improved with resin as opposed to paraffin embedding of tissue. However, difficulties in producing consistent immunopetoxidase reactions on resin sections have caused many departments to abandon thetechnique. Although antigenicity is preserved the low optical density of Diaminobenzidine (DAE) means that the reaction product is barely visible in thin resin Sections. The aim of this study was to develop a method whereby antibodies wmmonly used on paraffin sections wuld be successfully applied to 1 pm resin sections. Tissues fixed for 18-24 hrs in 10% formol saline were partially dehydrated and infinratedwithLR WhiteResinat4°Cfollowed bypolymerisationat40Cusingacatalyticmethod. 1 pmSectionswere reacted with polyclonai and monocionai antibodms using a standard indirect immunoparoxidase technique. Visualisationwas with a silveramplification SystemforDAE (Amenham) appiiedasthetinalstage. Excellent resuns have been obtained with a rangeof antibodies including S100, von Wiilibrand Factor. immunoglobulins, UCHLl and L26(DAKOLtd.). Usingthistechniqueitisnowpossibletocombinehighresoiutioniightmicroscopywithaprecise immunocytochemicai reaction. The advantages are Obvious and are particularly relevant in the field of lymph node pathology. However, at 15 and 20 Gythe number in muscle dropped Significantly 5.12 and 24 hoursfoilowing treatment, but hadrecoverad to w n t m levels byfivedays. In the lamina propria, AgNOR numbersincreasedinitiailyaflerthe5.10 and 15 Gy treatments but returned to control values by five days. With 20 Gy the AgNOR numbers showed a significant fall 48 hours after irradiation: this decline continued up to five days. It is evident that &NOR numbers within the small intestine are affected following irradiation. The variation in counts is dependent on dose, cell type and time Since irradiation.

Karen M. Britten, W. R. Roche oepartment Of Pathology, Southampton Unwewty General Hosp~lai, Southampton, SO9 4XY in response to ever-increasing demands for immunophendyping in inflammatory disorders, we have developed anernatwefixation and embeddingtechniquesforsmall biopsy specimens. Bronchial biopsieswerefixed in buttered formaiin and processed lor embedding in Araidite or were fixed in acetone containing protease inhibitors and embedded in the water-soiubk resin glycol methacrylate (GMA). GMA allowed for the investigation of a full phenotypic profile akin to that which may be wfwmed in frozen section while yielding far superior morphology and greater numbers of d a n s from Small biopsies. The phenotypic markers included those for T-ceiis (CD3. CD4, CD8,CD25andLFAl),macrophages(CDllC, CD14),mastcelis~B5B6andAAt)andeosinophils(MBP, EGl and EG2). Wehaveaisodemonstratednautrophiieiastase,cytokinesandtheceiiadhesionmolecuiesiCAMl .ELAMand VCAM. Similar high qualty sections were obtained with Araidne but the repertoire of antibodies was restricted to thoseantibodieswhichcannarmailybeappiiedin paraffin. Wesuggestthatforsmaii biopsies whichrequiredetailed immunohistochemistry, such as in the areas of transplantation and mucosa immunology, fixation in acetone at -20% with the inclusion of protease inhibRorS and processing into glvcolmethacwiate with careful temDerature control gives optimum results. These included 10% formaiin at VanOUS temperatures, microwave treatments. Bouin's fluid and a wmmercialiy avaiiabieproduct "Rapid FiX".ThetiSsUeswereroutinelyprocessed. embedded in paranin wax andstained wilhthe haematoxylinandeosin method. An eval"ation~fthefi~alion methodswasaisocarriedoutforimmunocytachemicai Stains. From microscopic examination of the results t was evident that for the haematoxyiin an+eosm stains. 10% formaiinat6O'Cwa~ wnsistentiytheoplimum methodof choice. Farimmunocytochamistryali methodsrasuited in a poor performance using a standard trypsinisetion time. However, an acceptable result was achieved from 10% formalinat60°Canda microwave fixative method. byreducingthetrypsinisation time. This. however, required strict WntrOI. Thus it is possible to fix tissue within one hour. by a method which is cost effective and which can be used within limitations for immunacytochemistry. We set out to study the extent and time dependency of storage related artefacts in cytospin fluid (Shandon). We assessedtheeffectof Stwage in cytospin fluidon the nuciearmarphoiqlyof breast FNAC. 10 FNAspecimsns were coiiected on day 1 and slides were made from each specimen on day 3. day 6 and day 8. These were fixed with a spray fixative. Between each sampk preparation, the cytospin colleaion fluid was kept at 4%. The slides were Fauigen stained and 5 nuclear morphological parameters (Area, Perimeter, Form AR. Farm PE. and Convexity-Concavity) weremeasured using aSeeScanSOiitaire Plus imageanalysis system. There wasasignlticantchange in ail the parametem betwwn day 3 and day 6, and between day 3 and day 8 (p < 0.05). The measurements were repeatableon differentoccasionswithoutsignificant difference in theresuns. These results indicate thatstorage in cytospin fluid significantly aners nuclear morphology. The nuclei become progressively larger and more irregular in shape. il is therefore important to standardize the Storage conditions of FNA specimens. if accurate objective comparisons are required. The identification of macromolecular components of hydrated patholqlicai tissues revealed by low temperature scanningeiectron microscopy (LTSEM) isan emergingfieldof enquiry. In orderthatthetechnicaidetaiisforLTSEM labeilingmaybeestablishedanexperimentaiprotocoi has beendrawninwhichiabeiparticiesofhighatomicnumber are visualized. The experiment comprises a mcdei System in which bovine serum albumen as a known amigen is dissolved in phosphatebutteredsalineand adsorbedontonitroceiiuiosemembtane. Theantigen onthemembrane is subsequently reacted mth rabbt anti-cow antibody and then elther protein A-goid or goat anti-rabbn-gold. Aner treatmentwithadeveiopertoaddaiayerofsiivertothegoidparticies,thepreparationsareanachedtostubs,rapidiy frozen in nitrogen Slush at -21OoC, coated with aluminium and observed on the LTSEM stage at approximately -191°C. BackScaneredeiectronimagingathighacceleratingvoltages(30 kV)isuSBdtodetectsilver-enhancedgoid partic1es.These areciearlyvisuaiized. Thefrozen hydrated preparationsarestabieunderthe described wndtions of LTSEM operation. in Contrast dry, wnventionai. SEM preparations are beam sensitive; their initially observed delicate UitrastNctUre quickly dearades an exeosure to even the moderate I15 kW electron beams emDioved in secondary electron imaging.

A new approach to systematic storage of pathological specimens for low temperature scanning electron microscopy As greater numbers of pathological specimens are stored for wnvenient future imaging by iow temperature scanning electron microscopy (LTSEM). It IS increasingly important to maximise expensive cryostare capacity. A cassenesystem hasthereforebeendeveiopedwhichincreasesthe hoidingcapacityof cryOStoreSbyafactord2.5 over conventional bee storage. Each c-he consists of an aluminium disc 44.5 mm in diameter wilh 6 cylindrical wells10 5mmindiametereveniyspacedand 1.5mmapart. Specimens. mountedan JEOLstubs,areretainedinthe wells by set Screws. Five cassettes fit into each 60 x 60 mm glass storage jar, gwmg a capacny for a 10 Canister cryastore(w4th 4@rs percanister)of 12OOstub-mounted Specimens An addtionai benefit ofthecassettesystem is that each specimen is afforded a mechanically and thermally protected environment. Specimen collection for LTSEM may take place in a distant operating theatre or laboratory. lmmobiiiration of Specimens Whilst they are in transit excludes any possibility of their being damaged during normal handling and improves the prospects for survivalof accidents.Afurther benefitisthataparticuiar mountedspecimencannow bemorespeedily identlledand removed for LTSEM from RS well. Many pathological condtions are charactensed by the presence of cellular degeneration accompanied by cytoskeietai abnormalities. In many such diswders it is not clear whether cytoskeietai abnormalities are pftmary process or are part of a secondary response to cellular insun by other agents or mechanisms. We have used a fibroblast ceii culture mcdei to study the effects of physical distension on the cellular cyioskeielon. inert beads of 2 and4pm were introduced intoceils byallowingendocytosisor bymicroinjection.Thecytoskeietai response tothese beads was studied using immunofiuorescence microscopy. Beads introduced by endocytosis migrated to the prinuciear region and over 46 hours became enmeshed in intermediate filament and mlcrotubuiar aggregates. Actln microfilament organiselionwas not alfected. Incontrast. microinjectionof beadsproduced an immediatecollapseof morofilaments,wiUlvimenBnandtubuiin distribution being preseNed.The immediateresponseto microlnlectlon is similar to the collapse of actin filaments s w n upon thermal stress. This experimental model has shown !hat aggregates of cytoskeietai proteins may be produced within cells as a secondary responseto intraceiiuiar debris, and that microinjection may induce cytoskeietal abnormalnies similar to those seen In thermal Stress. Thedetection of numerical chromosome aberrations m interphasetumourceiis bynonisotopic insitu hybridisation has been previously described but the application d t h s technique to paraffin embedded material has been wmpiicatsd bythe requirementfottissuesectioning with the production of partial nuciei. inthisstudy, the analysis 014-20pm thickparaffin sectionsolconventionally processed CaSki ceilsusing both human papliiomavlrus(HPV1 type l e a d achromosome 11 specificalphoid probe wascompared with resunsoMained usingintactceils.The use of sectioned material did not give signal distributions comparable to those obtained using whale cells. This IS consistent wnh a mathematical model derived for the relationship between Section thickness. nuclear Size and nuclear retention in paranln sections. A method was therefore developed for the extraction and analysis of nuclei fromthick(50pm)paraltinsectionsandappiiedtotheanaiysisof squamouscelicarcinomasofthecervix(n = 11). The number of copies of chromosome 11 varied from 2 to 7 and this variation was apparent both between lesions and between tumour ceiis within the Same ieaon. This IS comparable to resuns obtained wth cervical carcinoma derived csll lines. There was. however, no clear relationship between the presence of HPV sequences and chromosome 11 number. These preliminary results suggest that the postulated loss of host suppression of HPV gene function by deletion of genes on chromosome 11 does not occur through gross chromosomal abnormalities. 

Membranous giomeruionephritls (MGN). an immune-mediated disease, IS a frequent caused renal morbidity in man. Cationic bovine Serum albumin (cBSA), given to NZW rabbits in a chronic serum sickness-type protocol. is knowntoinduceglomerularchangessimilartothe humandisease. We~ssedtheenectofashortcourseofthe immunosuppressivedrug CyAon thedevelopmentof early stage.cBSA-induced MGN. Fourteen male NZWrabbts received an iv immunismg dose of 1 mg cBSA and 1 p g E. coliendotoxin. One week later they commenced daily IY inlectionsaf 25mgcBSAfor21 ~onsec~t~vedays. Three rabbtsweresacftficed atthistime. Sixofthe remaining 11 rabbits were commenced on a short COU~SB of o m CyA. whilst continuing to recewe daily doses of cBSA. The 5 remainingrabbitswsregivencBSAonly.After32consecutivedosesofcBSAthesell anlmaisweresacnficed.Ali3 rabbns given 21 doses cBSA Showed early stage MGN and those given 32 doses of the cationic protein showed a more mature, established disease (thickened glomerular capillary wails wth diffuse. global. granular deposition of igG/C3 and Subepithelial electmn dense deposits). Four of the 6 CyA-treated cBSA rabbns showed a marked reduction in giomerular capillary wail C3 deposition. Three of these 4 rabbns had considerably less severe disease ultrastructuraiiy. These results suggest that CyA may aker the course of cBSA-lnduced MGN.

'oRoartment Of Pathology University of Edmburgh M&~aiSchooi:

Late renal aliogran loss 1s due to arterial intimai proliferation and iumenai narrowing. There are few studies ofthe phenotypes of the intimai cells. We analysed these lesions by light microscopy and immunmyiochemistry using antisera against T-lymphocytes. B-iymphocytes. macmphages, smooth muscle cells, Class ii HLA OR molecules and the proideration antigen PClO 31 vessels were studied from 20 gran nephrectomies resected between 3 and 171 months post-transplantation. We identified an arterial endolheliaiilis, r-rted in cardiac aliografts, but not emphaszed in renal graftrejection. 4 panernsolarterial pathoiogywwerewgnisd: (1) Endotheiialitis in nnteriobuiar arteries without intimal proliferation, (2) Endothelialiis in larger arteries accompanied by intimal prdtferat!on of smooth muscle. (3) '"inactive" lesions with thickened intima (i foam cells) but no endotheliaiitis, and (4) 'Natural" atherosclerosis of larger arteries. The endothellaidis tended to m U r in shmw suMving grafts. The pedominant cell was the macrophage. with fewer T-lymphocytes. PClO was expressed in mononuclear ceiis, smwth muscle cells and endothalid ceiis. pmicuiariy. but not exclusively in younger grafls. We ProposB these ledonsevolve, variably. from an early endotheiiaiitis to late chronic vascular rejmlon or gralt athwmierosis. The predomlnance of the macrophage at ail stages, suggests it plays a significant roia in the evolution of these lesions. The rat IS used to study the response of the renin-angiotensm system m diseases Such as hypertension There are structural differences In the JGA butthere are few comparisons of its response to StimUlalion between species We used renin antisera and an lmmunoperoxidase technique to stain renin-containing cells (RCC) ~n rat (n = I t ) and human kldneys(l5 nephrectomy and 11 autopsycases). Westmulated therenin-angintensin System experimentally by clipping one renal artery (5 rats) and inducing sodium depletion (6 rats) and studied the analogous human diseases-renal artery stenosts (1 0 cases) andAddison'sdissase(6 untreated and 5 treated eases). Wecounted the RCC and plotted their distribution on scatter diagrams. There were some dinerences in distribution between the species but In boththerewasagradient in distributionof RCC which predominated m the superficial renal cortex. In sodium depleted rats. recruitment of RCC in the iuxtamedullary JGAs abolished this gradient. while in some of the animals with renal artery clip hypeitension the normal gradient was reversed, with most RCC in the deep cortex. In bdhu~nreatedAddiSOn'5diseaseand~nrenalanerystanosistherewasanoveralincrease(xS)m RCC butthenormal gradientoftheirdistnbution Withintherenalcortexwasmainlained.Theseresults haveimpl,cationsfortheroleolthe intrarenal renin-anglotensln system in the control of renal haemodynamcs. We have developed a model of adult human prostatic epdhelium that allows arch$tectuml and cytologioai features to be maintained. Epithelial organolds produced by enzymic digestion of human benlgh prostatic hyperplasla tissue weresuspendedm type collagengelandsubcutaneouslyxenogranedintointact malenudemice.ThexenogranIs progressively invaded by mouse stromal cells Thesesurround theepithelial organoids and supportthe reformation of epithelial Structures with a lumen, lined by a mixed epithelial layer. As the lumen forms tall columnar epithelial cells begin to be seen, these progressively express the prostate specific epithelial markers. PSA and PSAP Further the xenograflsexpress appropriatecyiokeratin markers in boththeluminaland basaleplthellal cells. Gelsplaced within a 0.45 pm Millipore chamber, which do not undergo stromal invaston. lose all epithelial organisation with disorganised sheetsand ballsofcellsbeingfound.These cellsdonot express thesecretory markers. In the absence 0lanandr0genic~tim~I~sep,th~l~~l structures wsth a lumen are formed but there are no tall columnar secretory cells and noexpression Ofthesecretory markers. Thismodel hasfurther been investigated todetermine theresponseof human prostatic cells growing In vivo to the antiandrogen Flutamide and to a 4-aza-steroid 5e-reductase inhibitor These observations indicate the essential role of both stromal cells and androgens m dictating functional dlfferentlatnon This model will allow the dissection of the regulatory processes involved in prostatic differentiation. Hepatocyte growth factor (HGF) IS the most Potent known mitogen for adult rat hepatocytes in pimary cuiture and isthoughtto haveanimportant rolelnlivergrowihandrepalr Linle~sknownaboutthemechanismofactionof HGF on hepatocytes. Since the adenylate cyclase system has been implicated in hepalocyie growth control. we examined the role of adenylate cyclase and cyclic AMP WMP) in HGF-stimulated DNA synthesis. Human recombinant HGF (hrHGF. 10 numl) had no elfect on baa1 or stimulated adenylate cyclase activity in membranes prepared from freshly isolated rat hepatocytes. Similarly, hrHGF had noeffect on intracellular CAMPlevels in cultured hepatocytes. Furthermore, agents which increase CAMP Inhibited hrHGF-stimulated DNA synthesis m primary hepatocyte cuI1ures Glucagon had an I . C. , of lo-'; fotskolin (107. IBMX (10"M). 8-broma cAMP (300 pM) and dibutyryl CAMP (300 pM) completely inhiblfed hrHGF-stimulated DNA Synthesvs. From thts. we conciude that adenylate cyclase and cAMP do not have a role m HGF-stimulated DNA synthesis m pnmarycultures oladult rat hepatocytes. The receptor for HGF has recently been identified in tissues Other than the liver, as c-met a protooncogenewith intrinsictyrosinekinaseactivitv. Whether or notc-metactsasthsreceDtorfor HGF ~n heoatocvtes 1s currently under investigation

A novel In wvomodel of intestinal differentiation IS descnbed. Fourteen-day, undifferentiatedfetal rat small intestine. stripped of the major part of 11s mesenchyme. then suspended in a type I collagen gel and renografted ~n a nude mouse. undergoessmall mntestbnal morphogenesis and cytOd$ffeienbat$on. All tourmapr epithelial Imeages. namely Paneth. goblet. columnar and endocrine. are present. Double labelling m sit" hybndlzatlon, employing biotinylated and digoxigenin labelled DNA probes to whole rat DNA and whole mouse DNA, reveals an unusual ]uxtapOSitiOn of species specific stroma. The outer longitudinal Smooth muscle layer, and the major part of the lamina propria. p53isthemost commDnlyalteredgene~n humanturnours. mutati~nleadstothep~oducfionolanabnormalprotein which can be detected by Immunohistology. Such abnormalales are seen in a wide variety of turnours, including colon cancer. Abnormal p53 protein levelshavebeen detewed in 5&55% of sporadiccolorectaltumours. In Order to determine I p53 mutations occur in carcinomas arising from dyspiasla. we have investigated the prevalence of such mutations m colorectal carcinomas from patients Wr)h long-standing ulceratwe colitis 1%). lmmunocytochemicalstalnlng waSpwformedon3freshand32 paralflnembeddedUCcarclnomasand35sporadlc carcinomacontrols matched forsite, stageand grade Six areas Ofdysplasla, (fourassoclatedwlth uCcancers)and 7 sporadic adenomas Were also stained. p53 protein was detected by immunohistochem~stry m the 3 fresh UC cancers and 8/32 (25%) Ofthe paraffin embedded UC cancers usmg the antibodies CMI, Pab 240, Pab 1801 and Pab421. Slmllarresuitswere obtained in thesporadic carCinomasw,th 113fresh cancers positlvefor p53 and 7/32 (22%) of the paraffin embedded Cancers. Two areas of dysplasla which were associated with p53 postlve UC cancer~also Showed positive p53 staining, alongwith an adenoma. Our results indicatethatunlike K-ras mutations, p53 proteinabnormalities Occurat a similarfrequencyln sporadic colorectal carcmomas, carcinomasar8sing !n UC as well as being present in UC dysptasasla. This work suggests that p53 mutations play a mle m the dysplastacarcinoma sequence.

Threepanernsofstainingareloundin humancolonusingatechniquetodemonstrateO-acetylationofsialomucins (mPASJ. 8% afindivdualsshowunifwmmPAS-positivity. theremaindwareeitherent~~ymPAS-negativewmPASnegativewith occasional positive crypts. We suggestthatthisrepresentspalymorphismof an autosomal gene (0sa) controlling 0-acetylation of sballc acid. isolated crypt-restricted mPAS-posltivky In otherwise negative indivtduais representing somatic mutation of the ma* gene in crypt Stem cells of osa'/osa-individuals. To test this we have Studied colons from 80 patients with rectal carcinoma, half of whom had received 4000cGy radiation 28 days Preoperataely. RadialiOnd~dnotaffecttheprevalenceofthethreepnenotypesbutincreasedthefrequencyofmPASposltlvecryptsinanegativebackground(t6 2~1 0~~~6 . 5~ to*, p <0.05), largelyduetocryptsshowingseclorial mPAS-positivlty (8.2 x lO* YS 0.4 x to4, p < O.C€Ql). consistent with incomplete crypt colonisation by a recently mutated phenotype. The prevalenceofthisosa*/osa~phenot~e(radiated43%, non-radiated 39%) isveqcloseto the predicted heterorygosily rate (40.3%. Hardy-Welnberg Law). Thew resuns suggest that human Colonic crypts are monoclonal with a longer Stem cell cycle than the mouse and that mPAS Staining provides a method for measuring human stem cell mutattonal load. Pouchrris m leo-anal resarvoirs IS a frequent complication of restorative practocolectamy which is associated wtih constderable morbidity. Long term complications of pouchitis are unknown, however patient follow-up wlth sigmoidoscopic surveillanceis mandatory to assessdysplamc or neoplastic changes in resldual rectal mucosa. We examined epithelial cell proliferative activdy in the ileal mucosa of pouch biopsies using the antibody PClO which detects nuclear expression of the 36 kD nuclear protein proliferating cell nuclear antigen (PCNA). Ten patienls with functtoning ,lea-anal pouch reservoirs of at least one year duration were biopsied and assessed hiStOlogiCally for evldence of pouchitis Formalin fixed paraffin embedded biopsies from anterior and posterior pouch wall Were examlned with PClO using the PAP technique. Ten terminal ileum sctions from right hemicokctomy specimens werenormalcontrols. Cryptsin pouch biopsiesand nOrmalcantralshadasimilarPCIOlabelling indexmeanof78K and 82% (mean values 71-81 and 7 W 3 ) respectiveiy The Sides and tips of villi had a Significantly greater PClO labellmg indexinthepouch biopstes(72mean. range59-33). compared withnormaicontrols(t7% mean, range 12-20) These resulk demmstiate an expanded proliferatwe epnhelial compartment in ileal pouch mucwhich in contrast to normal terminal ileum involves villous surfaces. Considering the frequency and long term natllrs 01 pouchitis. thesefindingssupportthe need for continued pathalogical and clinical assessmentof the ileal mucosa in the neo-rectum of these patients.

We, Cui, I C Talbot, J. M. A. Northover

Signiticant alterations in structure. function and gene expression of mltochondna have been reported In cObrectal turnours. but it $5 not known rf these abnormalities are due to mitochondria1 genetic alteration. in this study, total cellular DNA was isolated from 15 l lo rectal Carcinomas, 8 adenomas and their adlacent histologically normal mucosa. These DNA samples were digested separately with 13 different restriction endonucleases. and then analysed by Southern blotting using apurified mtDNAprobe. The restriction fragment pattern oftumourmtDNAwaS comparedta thatofeorrespondingnormalmucosalmtDNA.Theseresultsshowedthattherearenalargedeletions.

insert~ons, or rearrangements $n turnour mtDNA. and no single base changes m the delectable regions In spite of Some polymorphic vanations. Our results suggest that mtDNA changes are unlikely to have a malor role in human colorectal tumoungenesis. hence, alterations in colorectal turnour mitochondria must be dependent upon other mechanisms Crescentic colitis: the clinicopathological spectrum of a distinctive endoscopic feature in the sigmoid colon N. A. Shepherd, S. Gore, S. P. Wilkmson

Oepariments ofNsfopafholcgyand Gastreoterol~y, Gloucestershre Royal Hospml, Great Western Road, Gloucesfw,

Crescentic COIBS describes an endoscopic appearanc0 of the sigmoid colon charactwised by mucOSal swellmg, eqhema and haemorrhagestrictly localised tothecrescentic mucosal folds. In alive yearpericd thisdiagnosiswas made m 34 patrents, representvlg 1 .a% of ail fbeopta endoscopies. There was a male predominance and most Plients were middle-aged or elderly. DwerticulosIs waspresent m most (82%) but theabnomallf~es wareconfined to the CreSCentiC mucosal folds with Sparing of the divenicular onfices. The malorlty of Patients presented with a history of bleeding perano. Histologically there wasaspectrum of changesvaryingfrommlnorvascularcongestian to florid active inflammatory disease with crypt architectural abnormalities mimicking Ulcerative colitis. Three patients presenting with CreSCentc colitis later developed the clinical, endoscopic and histopathological features of distal ulcerative coliitls: two other patients with a history of distal ulcerative colitis were found to have the Charaderlstlc changes 01 crescentic colitis only at endoscopy. Three cases showed the histological features of mucosai prolapse The findings in thisstudy demonstratethat arelativelyspecificendoscopicfeaturemayexhibit a wide spectrum of pathological changes whilst luminal mucosal inflammation of the sigmoid colon, usually in a5sociat1on wiih diverticuioss. may mimic the pathology of chronic inflammatory bowel disease. Asmall propartion of these cases may represenl a Strictly locallsed form of chronic ulcerative colitis. of genome and antigen a n various anatomtcal sees The resuns have imolications lor the mechanism of e n t~ of MV into neurons and for mechanisms of transynaptic viral spread

Neuiopathology, fnstitute of Pathology 

J Chow, J Tobias.' K Co1ston.2T J Chambers

Estrogen IS generally considered to maintain bone mass through suppression of bone resorption We have previously demonstrated that administration of pharmacologic doses of estrogen increased bone formation in ovary-intact rats To assess theeffectsof physiological concentrationsol estrogen on boneformatian. estrogen was administered to ovariectamised rats fi n which bone resorption was suppressed by AHPrBP. Animals receiving exogenous 17p-estradtal (Ed (1 wglkg. t o wgikg and 1 W pgkg daily for 17 days) Showed a dose-dependent increasemtrabecularbonevolumeaft 9%. 25.8% and43.6% respectively, compared wnhthoseratstreated with AHPrBP alone. The increase in bone volume was due to bane formation in E,-treated animals, in Which bone resorption had been almost completely suppressed by AHPrBP Neither ovanectomy. AHPrBP nor E,-treatment had asignificanteffecton thevolumeorrateoflormationofcortical bone Thus. theiocreasedbonerasorptionwhichis aconsequence 01 estrogen-dellclency entralns lncreased bone formation, whlCh masks a Slmunaneous reduction In estrogen-dependent bone formation. It thus appears that estrogen maintains bone volume not only through inhibition of bone resorption. but also through stimulation of bone formation. PGs m bone formation m vmo. which may represent a pathway common to bone anabolism that 8s observed !n response to many e""liO"mental5t1m"ll PGF2, was wlthoul effect We found that nodule mductton by PGS Occutred early the cunures, belore nodules lntegrln expression e n human bone was examkned by immunohistolqlical stalnmg of EDTA-decalcrfec and undecalcllned cryostat sections of fracture-and tumour-associated callus Dbtalned at surgery and neonatal COStochondral )unctions obtained at autopsy. Cases were stained wlth a panel of well-charactsnsed monwlonai antibodies against p1-3. el-5 and uVp3 integrios using ABC peraxidase and indirect immunofluorescence techniques. Osteoclasts stained lor pl , p3. u2 and oVp3 mtegnns. indicating that they express u2p1 NLP-2) and uVp3 (classm vttranectin receptor) Ostmblasts stained lor pl, 04 and u5, and osteocytes stained for p l and 05.

indicating that ostmblasts express n4pl NLP-4) and m5gl NLP-5. classical fibronectin receotorl and that VLP-4 expression 1s reduced or lost during differentiation to astwcytes J Qulnn. N A. Athanasau Nufield Depanment of Paihology and Bactenotogy, Level 4, John RadcOffe Hospital, Oxford, OX3 9DU Osteoclasts are known to effect bone resorption in inflammation and malignancy but whether other cells of the mononuclear phagocyte system. particularly macrophages and macrophage palykaryons, are Similarly capable of pathological bone destruction IS uncertain Macrophages derived from tumours (human lung camnomas. murine MMW-assmated mammary carcinomas) and inflammatory lesions (munne foreign body granubmas) were CuIturedonboneslicesbothIn thepresenceandabsenceaf STZ slromalcells.TheSeneoplastIcand inflammatory ieSiOnS contained a heavy macrophage infiltrate but no giant cells and no calcified tissue. There was superficial roughening ofthebonesurfacebymacrophagesboth InthepresenceandabsenceofST2cellsand.aher t4days. scattered areas of lacunar resorption 8 n co-cultures of macrophages and ST2 cells. Normal pulmonary tissue macrophageSdid not produce resorption lacunaeunder these conditions. The results show that macrophages alone are capable of atype of low-grade boneresorption and that a SUbpOpUWiOn of turnour 01 inflammation-assaoated macrophages following specific interaction With Slromal cells, can differentiate intocellscapable of the special8sed function of high-grade lacunar bone reSOrpt1on Macrophages may thus directly contribute to the mteolysis associated with metastatic turnours and inflammatory lesms in bone Differences in the grade of osteolysis may also account for Clinical differences tn the degrees and rate at which pathological bane resorption occurs 

The George Washmgton University Medtcal Center, Washnqton, 0 C , U S A For decades, new technological advances have been hailedor condemnedas representing the eXtinCliOn 01 cla~s~cal diagnostic surgical pathology, but so far the reports of the death of our specialty have been grossly exaggerated. Indeed, the newtechnologies of the 1990sare seenas aids torathet than replacementsfor careful and intelligent gross and micmscopic examination and interpretation, which havealways remained the 'gold standard" against WniChnewtechniqueSaremeasuted Thedecade will bemarked by anexplosionof pOS5ibilitieSfOrt155Ueas well a5 non-tmue diagnosis, balanced by a Shrinkage in both specimen sizes (already evident in breast pathology) and healthcare budgetS.ThUS. thesurgical pathalogist will haveto becometoanevengreaterextentthecomplete physician. in order to beable to choose wiselyand economically from thediagnostic"menu"availab1e. Thesurgical pathologist w d also have a greater need than heretofore to be a competent cytopathologist as well, as many of his or her cases will also have fine needle aspiration material and a nuclear grading will assume greater significance o n tumor pathology. Finally, the roles of the surgical pathologist ~n both research and patient care (Including direct patteoi contact) will require emphasis m order to attract more medical students into our specialhl. womenorthosewithlowgradedisease.This hasledto theviewthatHPV 161s~nielatedtocer~1c~l cancer Asimple PCR protocol was developed using standards containing 0.1-100 fg Of HPV 16 DNA in 100 ng of normal human placental ONA toestimate levelsaf HPV 16 DNAin smearsor biopsies. The quantltativedistribution of HPV 16 DNA tn normal and abnormal ~ervlcal epithelium was mapped thmughoUt 50 loop biopsy and hysterectomy Specimens using micro-dissectionand histologyof alternate millimeter slices Levels were measured in parallel c~N~c~I smears. Preliminaryresuits show a low lwel of HPV 18 DNA(equiva1ent to one copy per 100 cells) was usual in women with normal smears or low grade abnormalities Replication of HPV 16 DNA to a level of one copy or more per cell was limited to areas of CiN2~3 which may be very Small and to atypical immature metaplasia. The level was reflected ~n the smear Aswitch to a high level of HPV 16 DNA s a biological and potential diagnostic maherfor high grade precancer. Cerv8cal biopsies were stained by the Cracker technique to demonstrate AgNORs. For the purposes of the study, b~opsiesweredividedlntaflVegroups-normal, kallocytosis.CINI.CIN IIandCINIII.UsinganIEMPS/2 hastingan A M S photon framestore a customised image analysls system was created. The image 1s dllated and eroded to close any cavities An outlining procedure locates the AgNORs and records their mid-pants. From this 11st of midpants themeandistanceofeach AgNOR'slhreenearest neighboursIscalculated.Thlsprocedurescanledoutfor several Lelds of view. This study attempts to relate the shape of the associated "mean distance" histograms to the hiStOlOglca1 diagnoses In flRy Cases.

Primary adenocarcinoma of the cervix: a retrospective clinicopathological study of 55 cases R Ananoos, Kamta Nahar.' Alison B,grigg.'S. Roberts? Sergin M. 1sma11 The patientfen healthy with a tendency togain weight being heronlycomplaint. Gynaecological referral revealeda 28 week gestation abdominal mass and bilateral varicose veins. Laparotomy, lee oapharedomy, total abdominal hysterectomy and right salpingo-oophorectomy were carried Out The len ovarian turnour was cyst~c, smoath surfaced. unilocular,cantainedturbid brownnon-greasycontentsand was22cmln diameter.Al0cmsquarearea of lhe lining Showed papillary tutmg. and the remainder was smooth. Hiatology of these papillary areas revealed a papillomatow proliferative squamous neoplasm with keratinization and mild acute inflammatm. wthout evidence of cytologicalatypia.stromal invasionora benigncysticteratoma.Theremainderofthecystshowednat swamous. cuboidal, or low-coIumnar type epithelium with an underlying vaguely endometrial type StrOma in minute focl, with occasional small secondary cysts Thls unusual squamous neoplasm was diagnosed as a proliferative epbdermoid meglomerularperipolarcell isan epithelial cell snuted atthe vascularpole ofthe glomerulus Recently, theexistence of the penpolar cell has been doubted The aims of this study were first, to establish whether the peripolar cell is a m q v e cell type ~n the mammalian kidney. and second, to compare their numbers and morphology in different speces We used scanning electron microscopy to study 1-12 kidneys from each of 11 mammalian species including man. We removed the glomeruli by microdissection and examined a minimum of 20 vascular poles from each kidney Penpolar cells were largest and most numerous m goat and sheep kidneys (1W% and 84% Of glomerul~) meywerescant~estandsmallest 10 humanand bovinekidneys(6 5% and 11 %).Whileaspectsotsome per~polarcells resembled podocytesand otherperipolar cells had features reminiscent of parietal epithelialcells. in each ~pe~ie~wewereabletadistinguishperipolarcellsasaspeciflccellhlpe. Weconcludefirst. that theglomerular peripolar cell 1s a unique cell type ~n the mammalian glomerulus, and second. that their morphology and number Cystlc renal disease occurs in various forms characterised by dilatation of different pans of the nephron. The morphologoal, clinical and genetic features of these diseases IS variable but some animal models have been developed m an attempt to understand the mechanism of cyst formation within the nephron. We describe a pOlyCyBtiC1.510.Ofthe kidneyinthsCEA-N mousew~hanX-linkedrecessive~mmunodetic,entsyndrome There is progressive cystlc dilatation affecting all parts of the nephron. The cyst lining is compoSed of a single l a y m c epithelium with focal nuclear crowding and the formation of micropapillary structures. The Cystlc epithelial cells Show subnuclear vacuolation Focal basement membrane thickening is also a feature. There is no Signdicant inflammatory infiltrate present within these ktdneys. Election rni~roscoptc examination w e a l s that the subnuclear vacdation 1s due to loss of the membrane infoldings at the basal pole of the eplthelial cell wlth flud accumulatlon within the extracellular space. The basement membrane thickening is due to expansion of the lamina dense. The finding of a plycystic kidney lesion on these mice offers an opportunity to investigate the relationship beween the immune system and renal cyst formation. Eqthropomtin. a circulating glywprotem, is the principal humoral regulator of elythropoiesis. It 1s produced in the kidney butthe precisecell oforigin iscontroversial. Erythropoietin participates in a~1~ssicaif88dbackcOntroi system whch attemots to restore Oxvaen deiivew lo the tissues. Normallv erdhrowiatin is resent in the SBNm at picomolar c6mntrations bui revels may h e up to 1001oid duriLg ?&erehkpoxic ;tress. The mechanism linking renal oxygen sensing with erythropoietin synthesis is p o~l y understood but there 1s evidencethat cells of the innsr cortex respond to tissue hypoxia by producing elythropoietln. Eryihropoietin geneexpression in the kidney can be detected by Northern blot anaiysis within one hour of exposure to hypoxie stimulation. The cellular location of erythropaietin m-nger RNAwaSdetected inamurine model byinsdu hybridizationemploying radioiabeiled DNA probes and autoradiography techniques. Tubular cells of the inner renal cortex WBI B identifled as the main site of erVfhropoietin gene transcription. The location oterythropaetm production was confirmed by immunohistochemistry with anti-sera ra4sed to pure recombinam DNA-denvea human elythropoetin. The specific antibodies bound oniy lo tubular cell cytoplasm, confirming the tubular location of erythropoiet,n-producing cells.

Severe reversible acute renal failure and IgA nephropathy R Jackson, k L. C. McLay Unrvemty Diws~lon of Pathology, Giasgow Ropl Infirmary, Castle Sireel. Glasgow, G4 OSF Whilst progressive, irreverable renel failure is well known in the evolution of a stgnificant proportion of cases of IgA nephropathy, disease-related. reuerstbie, severe deteiioretion in renei function 1s a less well recognized phenomenon. Relatively few cases aredocumented in the literatureand these have invariably been associated with m-iw haematuna. Renal failure has been largely attributed to a combination of tubular obstruction by red -11 cast~andacutetubularn%crosisthaughttabetheresunof adirecttubulo-toxiceffectof released haemoglobin. In a review of 122 cases of I@ nephropathy we have encountered two cases of this type. The clinical. histological.

immunohistochemical and ultrastructural data relating to these cases are presented. On the basis of this evidence and an analysisof the relevant literature. 11 isconcludedthat the accepted pathogenetic hypothesisdnot provide an entirelysatisfactaryexpianationof the phenomenon. itisfurlhersuggested that immunologically mediated injury dfrected at the glameruiar vascular pole and possibly the extraglamerular mesangium could compromise thetubular blood supply and might also lead to haemorrhage into the distal tubule at the macuia densa. Scanning electron microscopy (SEMI when combined with electron microprobe analysis (MPA) 1s an expeditious and accurate method of identitying Substances deposited in tissues. We report a case in which these techniques were used to study crystals in a transplanted kidney. The patient presented at age three with Vitamin D resistant rickets and Fanconi syndrome on the basis of cystlnosis. He gradually went into renal failure and received a kidney transplantfrom hismother. itfunctionedwsiifarafewmonths butthen deterioratedandwas removedone year after transpiantation.Afewcrystalswereob~ed by light microscopy ma biopsytakenattwomonths. Arepeat biopsy at four months was examined by SEM and contained the typical hexagonal CryyStaIs of cystine. The s o i i i Sulphur peak obtained with MPA confirmed their elemental CompoJltiOn. The hydrophilic nature of the Cystlne prevented their identificetion within routine sectionsfor transmission electron microscopy (TEM). The hexagonal o~tline of aystaiswas confined to interstiitat macrophages rather than tubular arglomeruiar ceiis. 10 pm frozen sections mounted on carbn pianchets diowed vlsualisation and analysis of the crystal deposits by SEM. The use of appropriate processing methods may be crucial m identifying crystal dspasition within pathological specimens. During the 10 year period 7981-90.1 1 new cases of dense deposit disease (m~~b,a"op,olif~=ti"~ giomerulonephritis type Ill were diagnow in Nonhern Ireland. Two further known patients developed recurrent disease in renal transplants. The mean age of onset was 14 years (range eight-22 years) with five males and eight females affected. Renal function was impaired in 38% patients at presentation which ranged from nephrotic syndrome (55%). nephrilidnephrotc syndrome (15%) and macroscopic haematura With mild protelnuria (15%) lo mild protemuria f microsopic haematuna (15%). Serum C 3 was universally low. Haw of initial biopsies showed cr-entsusuailyaiatedwith rapidansefrenaifailure. 38% ofpatientsrequired diaiysisatorwithin twomonths of presentation. A further 15% d patmnts developed renal failure within three pars of disease onset of the six patientstranaplanted haw havedaveioped rmrrent diseasin their grafts Six patients havenot required dialysisat ameanfoilaw upoffour yearr(ranse0neloeightyears). Mhaugh reialweiyuncommon, dense deposit disease isan important causeof renal failure in children and adolescents. We have confirmed that this disease has a poor outlook in terms of renal function with a tendency towards recurrence in grafts A review of 73 renal biopsies from patients with diabetes mellitus. [1981] [1982] [1983] [1984] [1985] [1986] [1987] [1988] [1989] [1990] . showed 45 with diabetic giomeruionephropathyalone, 25 with other renal diseaseand3 with both diabetic giomerulonephropathyand other renal disease. Eight with the histologiwl appearances of diabetic nephropathy without known diabetes were alsn identified. Clinical details were available on 68 ofthe 73 patients. These showed that in type 1 (insuiin dependent) diabetes (24). most (19) Showed diabetic giomeruionephropathy, 2 Showed both diabetic glome~lon~phropathy and another diagoaas, and 3 showed aniy another diagnosis in type 2 (non insuiin dependent) diabetes (44). 23 Showed diabetic glomerulonephropathy with 20 showing another renal diagnosis without diabetic giomBrUlOnephrOpathy, and 1 With both diabetic glomeruionephropathy and another diagnosis. 01 6 patients investigated with nodular glomerulosclerosis resembling that Seen in diabetes but not known to be diabetic at the time of biopsy, 5 Showed abnormal glucose metabolism on investigation and t showed a paraprotein. This review ShowSthatinpatientswithciinicairenaldiseaseanddiabetesmeliitus,therenaldiseasecannotalways beassumed to be diabetic nephropathy especially ~n type 2 diabetes where a wide range of other renal disease can occur

Histological findings of diabetic nephropathy may occur in patients before diabetes is diagnosed cllnlwiiy.

A J. Hawie, R. L. Bryan

In a biopsyof arenaltransplant. we notedan artmalabnormalitythat wasunrecorded in standard texts. In an arcuate artery. theiewastheappearanceafformation ofanewartery inside theoid, with layersof muscleandelastic fibres that in places resembled an internal elastic lamina. separated from the original internal elastic lamina by loose connective tissue. in a Systematic study of 156 consecutive transpiant biopsies and 17 consecut~ve transplant nephrectomies. another six examples of this abnormality were found. These SIX and the index specimen showed changes cons~stent with chronic Vascular rejection. In a Systematic study of 28 consecutive nan-transplant renal biopsies showing interstitial nephritis. another example ofthis lesion was found, in a kidney showing chronic interstitial nephntis. Thischange isprobablyavmantfom of muscularisation ofthsarteriai intima, isseen in chronlc renal damage, and occurs in transplanted and native kidneys. Localised myloidasis ofthe lower genito-unnarytractIsararedisease. Few studleshaveanempted tocharacterise theamyloidtypeusing immunOhiStochemicalstains. Wereportaseriesofnin~casssinvoivingthebladder(7), lower Ureter (1)and penile urethra(1). Thesecomprised7 malesand Pfemaies with an age ranged 5&79years. Nonsof the patients had evidence of systemic amyioidoss. Three patients had a past history of lower genito-urinq tract infection and of these, one had repeated instrumentation. One patient had prostatic carcmma The cmmonast presenting wmpiaint washaematuriaand the mostfavouredsurgicaldiagnosiswasa neoplasm. Fowpat#ents had repeat biapsiesfor persistent orrecurtentamyloidosiswith atimeintmal ofupto2t yearsfram initial presentation. bght microscopy showed amyloid deposmon throughoutthebiopsies, in lamina propria. muscle. adiposetissueand vessel wails. with a variable giant cell reaction and lymphoplasmacytic infillrate. An ABC-immunaperoxidase techniquewas usedwith antibodmsto Pcamponent, serum amyioidAprote8n prealbummandkappaand lambda light cham in an attempt to classify the amyloid type. This was found to b e d non-PA. non-prealbumm type. A negative or equivocal reaction was seen for kappa and lambda light chains However, Such antibodies may not necessarily be immunoreactive with light chains or fragments of light chains m amyloid deposits. features of bile reflux type gastntis: glandular distmion (branching, "carkscrewmg"). nuclear regeneratwn, intramucosal smooth musclefibresandpaucityof inflammation. HelicObacterpylanarganiams werenoted Bileacid Concentration #n gastrc aspirates collected by endoscopy at the time of biopsy was estimated using an optical densitymethod.GI23casesshowingglandulardistorti~7 hadraisedgasttic bileacidconcentratiOn.M18cases with mahednuclearregeneration6hadraised bileacidsandof 11 case~withinbamucosalsmoothmuxlefibres4 had raised bile acids Nine of 12 cases with raised bile acids Showed a chronic inflammatory response The hiStOlogical features of bile reflux type gastritis which correlate best with estimation of bile m gastric juice are glandular branching, "corkscrewing" and intramucosal smooth muscle fibres whereas pauclty of ChrOnlC inflammation does not H pylon was identified in 8 of 12 cases with raised bile acid. AIMS To compare the bacterial flora of normal and inflamed appendices. and to conelate this with various histological features. TO establish the incidence of Yeninla infection ~n acute appendicihs !n Southampton. METHODS Resectedappendicesweresentheshta histopathologyandal Cm portionwasremovedandcultured for aerobic and anaerobic organisma. The remainder was fixed ~n 1 0% buffered formalin and pocessed for rWtine histology RESULTS: 38 appendices Showed acute ap+endicitis, and 30 were normal. HISTOLOGY There were Statistically Significant differences between the two groups for the p s e n c e o f faecollfhs (p < 0.01). fibrosis (P < 0.01) and prominent follicles (p < 0 05) -all were more common ~n the normal appendices. MICROBIOLOGY NO Yer~inia was cultured in enher group. There were Statistically significant differences in the number in each group which grew anaerobes and StreptocOCCI. Anaerobes were more common in the normal group (p < 0.01). and streptococci morecommon in inflamedappendaes(p< 0 05) CONCLUSIONS. 1.Thereisanaiiwed bactenalflora in acute appendicitis. 2 Yersinia does not contnbute to acute appendicitis in Southampton. 3 Faecoliths. fibrosis and prominent follicles are significantly more common in normal appendces

Le antigen (using CAt9-9) a i d type 2 H antigen using a specific moncbnal antiserum. We f w n d a widespread distribution oftype 1 structures in both benign and malignant epnhelium Oftheextrahepatic biliarytract and ampulla of Vater. Type 2 H antigen expression was seen in benign epnhelium and in non-papillary tumoun and ampullary carinomas. lmmunohistochemical detection of tyW 1 blood Qrou0 antlQenS does not aDpear to be OfPrwnOStlC the majority of cases bearclose relationship to human ulcerativecolitisclinically, endoscopcallyarid I" re-nseto treatment One hundred postmortem IiYerswerestudiedfromconontoptamanns with totalseversul~ataecolitis who were pathogen free and had a histological picture resembling human uIcerallve cdhs Only 39 IIVwere normal In 20 there was a mild penportal chronic inflammation. 16 had extens~ve steatosts, 4 had an appearance reSembling Chronic active hepatitis and ~n 4 the histology resembled that of sclerosng CholangitJa. Other hepatic pathologies were s e n in Smaller numbers. These changes parallel the Itver disease Seen In association wlfh "ICBratiVBCOlitiSInman. Webelievetheconon tOptamannprovidesthefirst modelof l i~e r d i~~~~e m u l~e r a t i v e~~l n to allow sturty of the pathogenesis of the enralntestinal manifestations of ulcerative colitis. r-rvolr and Ilm-anal anastomosis. Pre-and pan-surgical specimens were studied and compared using routine histological and histochemicaltechniqm Themalontyofcasssexhibitedan lncreasem chronc lnflammatlon wlth 1 1 n 1 0 or noacute inflammation.The levelsof chronic innammation were found to bemost severe I" those cases which ~~fferedhompouchiti~. Morphomstncalanalysisrevealedan~ncreaseincrypt depthtovillousheightration(CDVH) ~n 88% of cases. The CDVH in the pouchitis cases was greater than 8 n the Other specimens studied The resuns of m u m histochemical analysis did not show any charactenstic changes occumng. in the cases studied. Lectm histochemistry demonstrated an increase an supranuclear staining of the pouches with DBA. SEA, WFA and WA. which IS similar to that fWnd In poximal colon. Staining with PSA, LCA. U f 3 and LTA revealed charactenstic changes in binding panem Th-may mdjcale changes m tucosylation of certain cellular canponents. Certain changes in lectin binding were found to be more sjgnificant in the pouchitis group of cases. The changes in the ra~ewoir mUcOSa are most Irkely to be an adaptive response to the new mtra-lummal environment wlth the acqmdfon of cmain mime ChaiaCteriStiCS. Thereare alsosp~ciftc changes which occur to a greatsrdegree m the cases with wuchits, these may occur as a result of or lead to the occunence of puchms. These resuns may be Cryostat sctionshomten patientswith ileal pouches lor extensive colitis, ten distal ulcerative ~olitics and five normal mall intestines were assessed immunohistochemically for macrophages (Leu M5 antibody). RFDl gnterdigitating antigen presenting cells). and RFDT (resting macrophages)antigencantainingcellsIn lminapropna. Result~areexpreSSBdasape~=~~t=g~~f thetotal. Normal small intestine contained a mean 7?4 6% and 8% (ranges 3-9.2-1 1, and 2-13) Leu M5. RFDl and RFDT positive ~ell~re~pecl~~elywh~chcontrastswiththoseh~ghervaluesinpouchbiapsies-86%,30%and88%(ranges71-94, 21-52and 7694)respectively. Distal ulcerative coilticscontalned increased macrophagenumbers(43.43 and27% for the three antibodies) but have a predominance of the RFDI positive population in Contrast to pouchitis patients in whomthe RFD7 positive cells predominate Thesefindings demonswate remarkably high macrophagenumbersin pouchitis, howeverthe RFD7 positive sub-populations predominance suggests an aetiologv other than Ulcerative colitis for muchitis.

Margaret Balsltis. Yah Mahida

The wide family of cell adhesion molecules (including the subgroups of the immunoglobulin supergene family. the integnn receptors andtheselectins) are involved In controlling interactions between endothella cells and leucacytes !n inflammatory states. This IS partly by their influence on adhesion and migration Of leucacytes to and through endothellurn. Using monoclonal antibodies, we have compared expr~ss~on of the three cell adhesion molecules ICAM-t (interCeIIUlar adhesion molecule-1). ELAM-t lendothelial leucocyte adhesion molecule-t and VCAM-1 (vascular cell adhesion molecule-t) ~n normal colonic mucosa with muwSa from cases of inflammatory bowel disease. Wehavefoundexpressianafthese threemoleculesto beincreased~ninflammatarybaweldiseaseandthis change involvesendothelial cells as well asleucocytes We will dixUSSthBrelationship between adhesion mOlecUle expression and dlsease actlvlty and pOsslble therapeutic Implications Granulomatous enterocolitis associated with therapeutic irradiation 0.6. Mangham. K M Newbold, S Dover The Universty of Bmmgham Department of Palhology, SCi3oOl of M&mI Science The Medrcal Schwl, Edggbaslon.

Chronic radiation-induced entero~olitis 1s a weli recognised Complication of radiotherapy for mtra-abdominal or pelvbcmalignancy. The pathological features Includepwforatlon. fistula formation. segmental necros~s. and StnCtUre formation. The histologicalfeaturesareessentially lxhaamic in nature. consequent upon the characteristic vascular damage. TWO case5 of Inadtation-induced granulomatous enterocolltis are described ~n which non-caseating epithelcoid granulomas were present ~n the bowel well. To our knowledge. granulomas have not been previously &="bed in radiation coldisor any other formof Ixhaemic bowel disease Thsgranulomaswere largely confined to the mmosz-associated lymphoid tissue and the draining lymph nodes. Naked submucosal granulomas were, however atso present. This distribution is similar to that seen m Crohn's disease and tuberculosis. NO evidence of these, or any othersystem!c ~r a n " l~r n a t~~~~~n d i t l~n~ was present These cases suppon theview that granulomas in bowel disease are secondary to an tmpaired muwsal bamer to antigens and highlight the non-specifiaty of granulomas m the diaynoscs of inflammatozy bowel diseases 

Between 1 and 5% of Crohn's disease patients have involvement of the upper gaStrOinteStlna1 tract The identification of granulomata IS normally required to make a specific diagnosts but this may be dinicult In Small endoscopic biopsies. We present t o cases where there was a strong suggestion of upper gastrOlnteStinal Crohn's disease with a definitive diagnosis established On more distal InVOIVBment. Four of the biopsies contained granulomata. the remaindershowedeitherpatchy Inflammation, local ulceration orvillo~s dtJtmtlon only In aneffort to establish whether Crohn's disease biopsies contained specific macrophage or lymphoid populations we applied the~onoclonal antibodies. MT1 (CD43). UCHLl (CD45RO)). muramidass. MAC 387, alpha-1 antltwsin and KPt (CDg8) using immunohistochemistry. Theresuns were compared with 6 casesot "On-Specific duodenitis Orlelunltis Crypt-restricted loss of G6PD activity has been used to quantify carcinogen-induced somatic mutation of the X- Samples of peripheral nerve (sciatic nerve) of mice from varylng age groups Were examlned over the past 8 years. Development olage~related penpheral neivefibredegeneraflon was Observed among mlce over 18 months age. The spontaneous peripheral nauropathy was characterized by Walierlan type degeneratlon. Teased nerve flbres from mice of 2 years old Showed evidence of swelling of the myelin Sheath. fragmentation d myelin into myelin balls or ovoids, with areas of segmental demyellnatlon. Light microscopic examination Of H 8 E Stained sections revealed axonal degeneration, and myelin fragmentation. with vawolation of nerve fibres. On ultrastructural studies. there was evidence of axa-myelin degeneration. Axxoplasm showing dark stained bodles suggemng of degenerate mitochondria The myelin sheath a l~o showed disorganization and fragmentation 01 myelin, sometimes with whorl lormatron rithcm the cyroplasm of schwann cetk, wtth evidence of autophagocytosis by schwann ceiis. in some inslancestherawere sgnsofproliferation at schwann cells. Nervefibredegeneration affected both non-myelinated and myelmated fibres Generally, therewereapparentlygreaternumbers 0fdegeneratenan-myelinated nerrefibres than myelmated nerve fibres. Furthermore the numben Of degenerate myelinat& nerve fibres were less than in seontaneous peripheral neuopathy in ageing rats (personal ObSeNat1on) SWntaneOuS peripheral neuropathy of sciat,cneNesmainlyappearedin miceaged 18 manlhsormore, 1s5eenonlvvelyr2rely in mlceof 6 months. Younger mice did not Show any evidence of this change Age-related penpheral nelimpathy IS not usually accornpan~~d by any Clln,cal 5,gns Neur~.9fhology. Uhversrty ofIowa, Iowa. U S A V~scuiarendothellalcclls(rn)conslitufet~elntertace betweenthe bloodstreamand thetcsske5and pertoormsevetal key roles ~n the development 01 immune and inflammatory responses Endothellal Cell5 from the braln display slgnlflcantly different momhoiogy from Other En. having ilght ]unctlOns between them and 2 pauClty at micropinacytolic ves~cles Dunng inflammatory conditions of the CNS interaction between mlammataly cells and RBB En 8s an initial important event. It 1s known that expression of several adhesion mOleCUleS on b r m En is upregulated !n intlammatorycondtti~ns and cytokine-induction Ofthese molecules has been investigated However an ~mparlanl mechanism of adheson molecule induction and hence modulation of tmflammatq cell/En binding might beviral infect8onofbrainEn Wehaveexamined theabilityofmeaslesvirus(Human2)andHerpes 1 simplex virus to infect cerebral endothelial cells. Adhesion of syngenelc splenocytes to both vlrally-infected and mock infected cells was determined using a chromiumS1 assay. By 48h of infection with measles YINS, cytopathic effect was eadent. and splenocyte adherence was increased to a mean of 138% of the control mock-infected Aner 24h mock-infecled Expression of the adhesion molecules MECA-325 and MALA-Lon virally-infected cells was determined Possible mechanisms for enhancement of adhesion will be discussed and subsequent implications of virus inlectiun of cerebral EN ~n relation to homing of inflammatory cells into the brain. UltraStrUCtUlal DbseNamns were made on the mechanism and route of Innanmatory cell diapedesis through cerebral vessel walls in an experimental model of can~ned~stempervlrusencephalamyel~t~s ~n the hamster Migrating monocyies and lymphacyiesextended pseudopodiawhich contacted, Indented and adhwedtoendothelwm. They lhen invaded the endnthel12l cells. becoming enveloped by endothelial cytoplasmic processes Which re-establishec the continuity of the vascular lining as the migrating cell passed through Although mlgratlng cells were frequently seen close to intei endotreiml junctions. they Were ever Seen wlthln Iprctlons. or between adlacent endothelial cellsand therewas noev!denceof opening of mterendothelial tlghtpnmons Aherpasslng through the endothel~al layer. cells squeezed through small pores (migration porest in the t h r sub-endothellal basal lamina me present study confirms and extends previous observations on the operation u;#thr the CNS of a trans-endothel~al.

paraiunclianal route for d8apedeSiS of mflammatary cells.

Ireland

Falbology Royal viclona Hospital, Bcl/asl HI 12 6BL lrrlnrld A SeWICe for the blochemtcal dlagnosts Of lysosamd 8torPge diseases has now been o n operalaon m BetlaSt forslx y n a r~ It currently lids assays available for the measurement 01 16 lysasamal acid hydrolases in plasma. Serum. lkwcocvtes. Cultured sk8nfibroblaSts. amnioticfluld and cultured amniatlccellstordlagnos~s Of 20 lysosomalstorage diaurders Solar 250 patients have been referred from throughout Ireland wtth 53 having a positive diagnosis of these. 18 were diagnosed as being hamozygous for a Specific lysosomal enzyme deficiency, 6 were identified as having multiple enzyme acl~ciencies (mucolipidosis Type 1111-cell disease) and 29 had heterozygote (carrier) enzyme IBVPIS 01 t t v latier. 28 w~i e either parents (obligate hetemrygotes) or siblings of homozygotes and one was a heterozyquti' lor X~lirlhrll recessively inherited Fabry's disease. In addttlon. prenatal diagnosis has been performed on 11 motherswithafam,Iy hi~toryOfI-celldiseaseand/orHurler'ssyndmme.Oneofthesepmvedto bepositivefor Huller 9 Tht' resuIIc 01 the biochemical Investigations of these cases are presented. 

High levels of circulating immunoglobulins are common ~n liver dlsBase. In alcohol8c hver disease depos1s of 8mmunaglobullnA(lgA) have been found in the liver and therenal mesanglum, and Werepart acaseof a3Oyearoldfemale with a2year history of cardiomyopathy and progressivemuscle weakness anerthe birth of her baby Shewas Subsequentlyflnedwith apacemaker. Clin~cally, therewas sevweweaknessof neck flexors with proximodistal weakness tn both arms and mild weakness of htp flexors. The most stnking weaknesswasin herbreathmg muscles Therewas no ptosisorfaaal weakness Rellexeswere symmetrical and her plantarswere flexor HerCKwas normal. Aquadriceps musclebiopsy revealed abnormal vanatcon o n fibre diameters affecting bothflbrelypes. Occasional pink hyalme ~nclus~ons which stainedforacid phosphataseand Wlth PAS wwe seen I~I both fibre types Electron microscopy showed these i n~l~s i o n~ to cwtsist of aggregates 01 10 nm diameter filamentaenmeshedwithinacentral coreofdenseamorphousmatenal. Inotherareastheamorphausmatsnal layas inegutar patches within !he sarcoplasrn mainly at the level of the "z" tine causing disintegration of the sacomere Immunoelection microscopy wing collmdai gold showed that the denseamorphous material reacted slrongiyw<th desmiri antisera and CaLld therefore represent a phosphorylated form of the pratein. Tubular AggrCgatesITAs] haYebeendescr,bedInassociat,on witha widevarietyofdisorders wherethey most Onen occurin type %fibres Recentlytwolam~I~eshavebeenrepoRed with muscularweaknessandTAsasthedom~nant biopsylinding Wereporla thtrdfamilyol whatseemsla beadistinctcondition Twopatients afalherandda-ohter presented with slowly progressive p.oxima1 weakness. limitation of eye movements and Aciiilles t e o d a r.antractufes Creatine kina-was S t 0 times normal By light mcroscopy, basophllic accmUlationS were ~e e n in both fibre types Clecson m!croscopy revealed that these COnslSted 01 tightly packed parallel tubular a m p (TAs) These varied somewhat m theiiulliast,wct~ral appearance and were therefore clawfled accordingly Thisreport lhrows fuRner Bwdexe m Support of a OtStmCt CllntcOpathOlogiCai enttry. The c-myconcaprotein (p62) IS a DNA-bmdmg protem. increased expression of which IS assmated wth shartenmg of the GoG, phaseof the cell cycle. Quantitahon of p62 m breast cancers may have prognoshc and therapeutc Implicatrons. Flow cyiometnc quantitatton (FCMO) of p62 has been shown to correlate wxth certatn parameters m breast carcinoma. However, thsre are factors which we felt could potenttally confound this FCMQ m foimalln flxed paraffinembeded(FFPE)tissues. Bothnuclear(N)andcyt~plsmic(C)express~oncanoccur. dependlngonthetlssue fixative used. Cytoplasmic stripping and exposure of nuclei to the digestwe enzyme, trypsin, occw dwng the preparation of the nuclear suspension from FFPE tissue. This study documents c-myc oncoprotein stainlog I" 5* tissue Senion of 242 p m a r y breast Carcinomas: turnour cells, adlacent benign epithelium and stromal cells were assessed. The monccional antibody OM-1 1-908 (CRB) was employed using the ABC immunocytochemical technique Thi~antibodyrecognisesp62 andacyiaplasm1~40 kD protein The resultsoftrla trypsmsatm of tissue SBCtionSpriOrtOStsiningwerealsonoted. POSItIVBStainingwaSseenIn90Y~oftumaun. both NandCstainmg belng present In 65% Benign epithelium, many endotheiial, fibroblast and lymphocyte nuclel showed posltlvlty Trypsinisatwn ledto lossof Staining 4ntensity.Thusin FFPE breastcawnomas. ~, " g l e p~~~m = t =~F C M Q~f OM-1 1 -908anti-C-mycantiobody Isconfounded by3 facts((i) much extratumoural c-mycexpresslon cccurs, (11) cytoplasmic stripping may iead to OncoprotRin loss and (111) breast oancercells,Iisinducedaver20-fald by phorbolssten.Amphiregulin bindstotheEGFreceptotandcan be stimulatoryorinhibitorydependingan theceil line. ltal~oblndsto~tsown receptor, pOssiblyerbB-3.Weevaluated expression of amphiregulin in 52 primary breast Cancers using immunocyiochemistry. 60% of turnours Showed staining using amonoclonal antibody toamphireguiin which WaSdlffUSeOrfoCal in tumourcells.Amphiregulin was not expressed in Stroma and much less often in normal breast ep!thellum than m tumours. Immunohistochemistry waspositiveincases withthe highest mRNAexprsssion(concordance80%)asmeasuredusingacDNAprobeand qualitativenortherblotsanddat blotsanaBplatecounter All caseswereassayedforEGFraod ER. 14 ER'EGFr-.

12 ER* EGFr*. 11 ER-EGFr-. 14 ER-EGFr'. There was no association of amphtregulln expresston wlth other receptors. These results suggest amphiregulm may be an Independant factor regulating growth of a Substantla proportion of breast cancers. In h a s t cancer, overexpression of the c-erbB-2 oncogene is associated with poorer prognosis, and in a prwious study we found it to be associated with lack of response to endocrine therapy Bolus doxorubicin with infmons of ifosfamiddmesna appears to be a very active chemotherapy combination for advanced breast cancer in young women. Thirty-nine women (median age = 41 yrs) with rapid recurrence of breast cancer (median disease free interval = 15 mth) were treated with doxarubicin and Ifosfamlde/mesna. and c-erbB-2 expression in their pnmary tumours (determined immunohistochsmically) related to treatment response. The tUmOUrS of 11 patlents Strongly expressed c-erbB-2 protein and 9 of these patients had objective responses to chemotherapy In 29 patlents with turnourseitherweakly expressingc-erbB-2(n = 6)otshowingnostaining(n = 23)therewereobject,veresponses,n 21. These data indicate that c-erbB-2 expression ~n primary breast Carclnoma, while associated with resistance to hormonetherapy. daesnot preclude responsetosystemic chemotherapy. and sugge~taposs~blerolefarc-erbB-2 Status in directing treatment strategies for individual breast cancer patients. pmicularly adpvant therapies ~n early stage disease. 

Transforming growth factor IS a muitifuncyional regulatory protein which can either stimulate or inhibit cell proliferation. can inhibit immune responses and can enhance both angiogenesis and the formation of connective tlssue.TGFpmRNAhas beendetectedin breaStcancercellIines, andtheprotein hasbe~nfoundinthemediafrom Several Such lines. In view Of the prominent stromal reaction seen in many breast cancen. TGFP may have a srgnificant role in tumour development and Subsequent behawour. TGFB, protein expression has been examined ~mm~noh~StDchemically using a specific polyclonal antiserum in 75 formalin fixed. paraffin embedded invaswe and in s,fu breast carcinomas Occasional Staining Of normal acinar cells was seen. 20% of invawe cancers had prominent staining of cells for TGFB. often with associated stromal staining. 30% showed occasional cellular and stromal staining. and staining of Stroma only wasobserved in 17%. Theremainder werenegative Staining of ins it^ carcinomas was less with Only 12% having prominent staining and 60% being negative. There was no conelatkon with type or grade of tumour. or oestrogen receptor status but all carcinomas with prominent reactivity had metastasised to nodes. This along with the differences between lnvasive and m srtu carcinomas suggests that TGFP may be a determining factor for invasion and metastasis. Depanment OfPathoIwy, Univers,ty ofshemeld, POBoxSQ6, Shefiela S102UL

Chromatin changes ~n neoplasticcells are well recagnised Quantitation of nuclear chromatin distcibuhon is dcfficult becauseaf thevanetyof parameterswhich couldpossibly bemeasured in thisstmiyamethodof quantttatmg the distribution of DNA was developed for a Seescan Solitaire Plus Image Analysts System. The parameters Homogeneity, Condensation and Clumping were Calculated by segmenting the nucleus grey Scale intensity into three levels, BLACK, GREY and WHITE, byamethod of adaptivethresholding.Theseleveis represent the darkwand lighter regions of chromatin distribution. The relative pr~portlons and positions of each of these regions were calculated by viewing the nucleus through a 4 pixel wide computer generated "mesh" The system was used to compare 22 benign and 28 malignant Feulgen stained breast FNAC specimens. The Homogeneily and Clumping Parameters Were Significantly different between the two groups (p < 0 05). but complete discrimination was not possible In order to test the valtdity of the system. 50 spicimens were graded subp3wely (1 to 3) and compared

With the image analysis results. There was 80% correlation for Homogeneity and 70% lor Clumping. indicating the difference in chromatin distribution 1s real and not a result of system error The role of nuclear DNA analysis. oestrogen receptor and thymidine kinase (TK) activity as prognostic indicators in human breast cancer was assessed. These parameten were investigated in relation to traditional pmgnostlcators. theoveralloblectivewastodeveiopaprognosticindexthat canpredlctforsuNlVal.Tumourmatenalfmm 136 breast cancer patients was anaiysed The nuclear DNA content and oestrogen receptor Status were determined by Static cylophotometry and a multipoint titration assay respectively thymldlne klnase actlvlty was assessed using ATP or CTP as a phosphate donor. Patients were followed-up for a median of 31 months. The Cox proprtlonal hazards model wasused todetermine which parameterscould predict survival.Pathologicalgrade, tumours12e. tOtalTKand CTPJATPratiowerefo~ndto beindependent predictonof survival.Aprognosticscore wasderivedforeachpatlent and these were assigned to one of three prognostic groups. Using these prognostic groups the rlsk of reduced SUNWPI could be predicted in 77.78% of patients. Inconcluslon, the assesment of pathological grade, turnow S m and thymidine kinase activity provides a reliable means of predicting prognosis year periodarereviewed.The median follow-up was78 months. On the baSiso1 margln contour, degreeofstmmal overgrowth and atypla, mitotic activity. nuclear pleomorphism and the presence of speclallsed stroma, 27 benlgn and5malignant phyllodestumounwereidentified Turnoursoccurredin womenof msdlanage52 years whomost often presented with a palpable, Occasionally painful mass With a medlan diameter of 5 cm EXclSon (1 4) or smple mastectomy(l7) werethemorefrequentformsof therapy. LOcalrecurrenceswereexperlenced by Gpatlentswhose pnmary tumours although classified as benign contained areas of stromal overgrowth and specnaltsed stroma: all had been primarily treated by a histologically inadequate local e~~i~i~n~r~~b c~t a n e o~~ mastectomy (l).These local recunenceswere successfully controlled by local surgery (4)and mastectomy(2) Mf~vemal~gnant tumours treated by mastectomy, none has recurred locally or developed metastases. Flve mcompletely emsed turnours whlch showed neither evidence of stromal overgrowth nor speciallsed stroma remaln disease free at 7 years. It 1s concluded that histologically malignant phyllodes tumoun are unllkely to develop distant dlsease I treated appropriately andthatstromal overgrowth may indicatethe possibilityof recurrence ~n inCompietelyexcisedbenign lesions. Several recent Studies have evaluated the incidence in primary breast carcinoma of "micrornetastases" ~n multiple Sections of axillary lymph nodes. HOWBYB~, the significance of these findings ~n terms of prognosis and SUNW remainsunclear. Inanattempt toelaborateon this, wereviewedthecasesof60patlentsdlagnoSedas havlng'bodenegative'' breast Carcinomaon routine histologyand in whom therewasafollow-upperladof at least fwe years. The anginal diagnostic H 8 E slides were reviewed in conjunction With eptthellal immunohistochemical pr-aratlons UsmgCEA,EMA. HMFG, andKeratin, andappropriateclinicalfollow-updatawasaccumulated.Thestudycasesfell into two categories, those which retained a 'node-negatlve' status after revew, including immunohistachemical appraisal (63%). and those which demonstrated individual or grouped malignant epithelial cells (37%) ~n the peripheral or deeperainuses of the lymph node. of the former group. all pattents but one werealive With noevidence of recurrent breast CarCinoma five yean after mastectomy. Of the Second cohort. posnttve far "micrometastases". 64% showed nodiseaserecurrence. while36% had gross metatatic d i s e a s e o r h a d w c c~m b t o thecarcmoma within 1hefiveyearpenod.Thefindingsdescnbed.aIthough basedon low patient numbenln thisprelimmarysludy.

indicate the appropriateness of a large multicentre SUNW to evaluate completely the prognostic significance of these '"msrometastases".

Sadhna Bhatnagar. J. McClure. D A. Kalbhen

The injection of sodium iodoacetate (0.6 mg) into the avian knee joint on two consecutive days induces changes which macmSCopiCally and radiologically resemble osteoarthrosis. Knee-pnts from groups of SIX hens were studied atregularinterval~upt025 weeksalterinjection by histological and histochemlcel methods. Characteristlcles~ons were identified and assessadon asemi-quantiative basisand indwdual Ision profilesacrossthetime course of the expenment were constructed. During the first five weeksthe obvious changes wwe chondrocyte loss and depleted matrix staining. Chondrone formation was also noted. Fmm SIX weeks there was also tibravascular proliferation at the chondro-asseousiunction wim modelling activity and new bone fwmation. in addition near the insenion of ligaments there was chondrocyte differentiation and proliferation. The amount of cartilage fibrillation and flsswng increase3 during the expenment aithough there were 3so areas of apparent resurfacing. The decreased matrlx staining noted early in the expenmen1 stabilised and many lacunar spaces are occupied by chondmcytes towards the end. Subchondral bone hyperplasia and osteaphyte formation are prominent persisting features. Such sequentialcharacterisation may be usefulin a-ssinglhee(fectsofput=~~vetherapeuticcompounds onthe model. Rodentachilles tendonsweiesubiected to midpointtenotomy and allowed to recover for various timesin sllu before the operaled tissue was mmved, placed into a Millipore dinusion chamber and insened inhapRntoneally into syngeneic hosts. Diffusion chambers were removed at weekly intervals such that the toM time post-operation :I.e. time 10 sRu plus time wnhin the diffusion chamber) was up to 8 weeks. Histological examination demonstrated that ectopiccartilage wasprodwed withinthediffusion chamber byatotal of 4 weeks but only if thefirSt 2 weeks were recovery In situ. Wnh increasing time. calcified cartilage, mtteoid and pnmary spongmsa bone was obseived Evtdence suggests that the camlage forms via direct tendon metaplasia and that the bone may form as a result of dinerentialive changes within hypertrophic chondrocytes. tlbla and fibula appearto be directly exposed to tha surface of the medial femoral condyle and inner ridge of the lateral femoral condyle. The exposed load-bearing area of the femur and tibia contain demonstrable amyloid around chondrocytes and in the matrix below the surface. These areas are also covered with the densest hyalmd fibro-cartilage. Amyloid p r e e u~r s from h e synovlum may penehatethe surface Ofthe cartilage under pressure or precursm from cartilage matrix or chondrocytes may be converted into amyloid and are napped below the surface. The inpction of sodium todoacetate into avian knee iolnts is followed by the development of an osteoarthrotic pmess To studytheeftectal sodium lodoacetateon chondrocytemetabalism m wire, ChickStmal chondrocytes grown I~th~ee-dimensionalcollagen gels wereexpmedtothe compound. Sterna from Chick embryos(l7-18 days

In om) were digested with collagenase releasing the chondrocytes which were then cultured in collagen gels.

W i u m iodoacetate was added to 7-day old CUltUreS at concentrations of 0.5. 1 and 2 wg/rnl. (0 25,0.5 and 1 .O Y lodM)tor3 hours. Theeftectswere assessed lor up to21 days by: 1) Staining wnhfluorescein diacetate Which pmducesfiuorescenceiniive. metabolisingceilsonly.2)Thelevelofproteoglycansinthemedium wasestimated by complexing with dimethylmethylene blue and measuring the increase in absorbance at 530 nm. 3) Histologycultures were fixed in glutaraldehyde, embedded in Paraffin, sectioned. and starned wm alcoholic toluidine blue. alcaan blueandpicmsinusred.Treatmentwlthiodoacetateproducedadoserelateddecreaseincellnumbersand intheamount of proteoglycmsreleased into themedium (lessthan 50% at2pg/ml).The histolagyshawed thatthe treated cells were smaller and produced less matrix. These results show that sadium iodoacetate reduces cell number and interferes wqth the manufacture of matrix components.

Instftute of Pathology, Ro9I Vcfona Hosprtal, Belfast. 8712 68L. Irelend Theoblectivesof this study were tocorrelatecertain clmcal features of ependymoma. (i e. age, sex, anatomcai slte. turnour Size. meningeal dissemination and raised intracranial pressure with certain pathological features. 1. e DNA content, mnotic rate. immunocytochsmistry and ultrastwctural features) with prognoss A total 01 35 cases was available for clinical pathological assessment

UniVeWty Of LiVerpoOl, P 0 BOX 147, LiverpoOi 169 3BX and 'Sari Andres Unrvemty

It IS not surprising that wmal exposure to high altitude Causes Increased activity of the hypothalamic-pltultaryadrenocortical axis. but fflidence suggests that continued exposure to hypoxla leads It to perstst long after homeoStas1sis restored and acclimatization achieved

Their pltuatary glands were not sgnlflcantly different insizefromthoseof thesea-levelsubpcts, but c~ntainedlargerpopulat~onsofcort~cotrophs~n termsof the total population of their anterior lobes (25.6 vs 19.4%: P < 0 001) These fmdlngs are in keeping with those of previous studies of the hypothalamic-pituitary-=d~=~~cottical axis in man and animais exposed to prolonged hypoxia at natural high altitude or In the laboratory. They Suggest that greater amounts of ACTH might be necessary to sus1ain normal adrenocoltid function under such circumstances and that adrenocortical hyperplasla

Bone marrow wnsists of a haamopolstic system whch IS nn intimate association with aftbrous stramal network.adun mammalian bonemanowstromais knownto have both chondmgenicand ostecgeniccapabilitiesinvlvoand stromal tissue has been shown to tom fibroblastic Wlonies capable of giving nse to bone in vitro. However, the mqbility of manow stroma to form Cartilage in who has not been addressed When grown in identical conditions to mammalian stroma. mechanically dissociated single cell suspensions of chick embryo bane manow hom 1 >21 days in ovo and from 3 day hatchlings readily lorm bath fibroblastic and cartilage colonies in vitro. Cells within the cartilage colonies are polygonai In morphology, are separated by a relradile exhaceIIuIar matrix and Svnthesise catilage specific pmeogiycans and collagens. as shown histochemicelly, biochemically and immunocytochemically. In Contrast, adult chicken bone manow does not form cartilage. instead, the cells appear osteoblast-like and synthesise coilagens typical of bone.

A. M. Fianagan T. J. Chambers &wtmment of H~slopatholosy. Sl George's Hosptal Medlcel Sch-I, London. SW17 ORE Osteaclastshavebeensuccessfullygeneratedinculturesofmurinehaemopoisticcells. ItWouldbeUseful ifasmila, model were available to analyse the mechanisms of regulatlm of human Mteaclast formaim IR normal and pathological states. Although the osteoclast IS present in reverai neoplastic conditions. It is not known whether It forms part of other haemowietic malignancies, lor example, polycythaemia rubra "era or chrmic lymphatic leuhemia. We used strategies based on our experience with murine osteoclastogenesis. However, we have been unabletogeneratefunctional human OSteoclastScapableoIresorbingbone m vrtro. Large numbersofmultinucleate cells developed in these cultures mese cells did not show a typical panem of reactivity with asteociast-specific manoclonal antibodies, nor did they bind '251sCT but rath@r possessed an antigenic profile characteristic of macmphage polykaryons. It IS pecullarthat human tissue fails to support osteoclast generation stnce cells of the other haemopoietic lineages were consistently generated in our cuitures. In murine cunuras it is known that a particular shomal cell type IS requtred for osteoclastagen@sis; it is possible therefore that this cell population IS SpaMin adult humantissue.miswouldreflecttheiownumberofosteoc1asts presentinhuman adults comparedto mice Elucidation of Conditionssuitableforthe generation Mthehuman osleoclastm wtrow~ll helDUs understand the mechanisms by which it IS regulated in health and disease spaceof one year. The other case 1s B middie-agedwoman who &=being affected bythe disease far sevaral years developed separate SarcOmas In each lower limb dunng a nine month pwiod. Each tumour was treated bv Pamal amputation but she died from widespread metastases within one year of the first amputationThe distribution of proline-4-hydroxylase in a range of human tissues shown by a monoclonal antibody, FIB 585 S. Smlth, P. Revell aepariment of Mobd Anatomy and Bone and h m f Research Unrt, The London HospW Medcal Cdl€ge, London. E l FIB 505. a commercially available monoclonal antibady to the B Subunit at the enzyme proIine-4-hydroxyla~ invalved~ntheproductionofcallagen, wasappl'edtaacetoneBxedcryostat sect~onsofawiderangeoftissues. In hver, hspatocytesshowedvarying degrsesof labelling with the antibody, along with spindle shapedcells(SSCs) in associated connective tissue In both tonsil and lymph node. SSCs in the Connective tissue were Melled with the antibody along With a number of other cells (lymphocytes) within the germinal centres. In addition to this. perivascularcellsand cells intheconnectivetissue iayerimmediatelybelowtheepithelium also labeliedin sections of tonsil. In skin, very few cells in the dermis were positive for pm11ne-4-hydroxylase. as were also only a small numbetof epidermal cells. Afew chondrocyteswere marked bythisantibody in artlcularcartilageand intervertebral disc,along withswneostwcytesin bone.ManycellsInthepleuraandtheepimysiumof normal~keletalmusclew~e posotive. There was no staining at all in specimens of kidney. In addition to these 'normar tissues, examples of a seminoma, a breast carcinoma and a thymoma were examined. Each of the turnours showed FIB 585 labeliiw of SSC~withinthestromalt~ssue.Theseresultsshowthat FIB5B5sele~tivelylabelsSSCs.whlCharapresumedto be f8brobiasts. in a wide rangEd1 both normal and pathological tissues. which produce and maintaln the collagen matnx. Proime-4-hydoxylase IS an enzyme involved in the pmduction of collagen and demonshahle in the rER of fibroblasts. It may therefore be cmsldered as a possible matker for fibroblasts in tissue sections. We have applied the mouse moncclonal antlbdy FIB 505, raised against the $ subunn of prol1ne-4-hydroxylase. to alcohol fixed paramn embeddedsections and acetonefixedcryostat sect~onsolnormal (n = 4) and meumatoid (n = 30) synovia. Synoviocytes labelled very strongly, but the distribution vaned between samples. Insome, mostsynoviocytes weremahed, whilein others ~nlythedeepiayerofcells wasmitive. It isnot known whether thts dillerence ~n prdine-4-hydroxylase expression is due to the severity and duration Of disease. drug treatment or other factors. It IS of interest, however, that FIB 505 labelling was not wnflned to the type B (fibroblast-llke) synoviocytes and that the more supficlal type A synoviocytes also contained proline-4hydroxylase. Spindleshapsdeells I" thesublntlmaleonnectiva tissue were labelledma vanablemanner. InaddltlM to these findings for fibroblastic cells. a small cap of FIB 505 posnive cells was seen around lymphoidfollicles. sometimes polansed towards the synovial surface. This enzyme expression by lymphocytes is under further Invsstlgatlo". Vitronectin 1s an adhesive glycoprotein which shares several functional similarities with fibronectin. It is a major component of extracellular matnx and plays a role in cell-matrix mteradians, monocyte function. and the coagulation and wmplement systems. We have employed a monoclonal antibody to assess the distribution of vironectin in frozen synovial biopsies from patients with rheumatoid arthntis, ostwnhntis. ankylosing Spmdyiitis andtraumatic non-inflammato~ control^ (49 cases). lmmunoreactive VltroneCtin was identified in the synovium. Similarto the panem of immunOreactiYefibronectin, itwas located in adensefibrillarpanem surrounding cellsof the synovial lining layer. Vitronectin was also associated with fibres throughoutthe sub-intimal Connective tissues. being most prolific in fibrotic areas. Vltronectin was identified in the sub-endothellal layen of blood vessels. but unlike fibronecttn 11 was not associated with basement membranes. Similar distributlon patterns wme observed in all biopsies studied Thelocalisation ofvitronectitin in synovial tissues suggestsapossibleroie inanachmmtofcellsto the extracellular matrix and that 11 may be important in the pathophysiology of inflammation and repair Manolayercultured articularchondrocytesare known to rapidly losethelrexpressionof collagen typeil.The purpose of this study was to compare the expression of various collagen types in twoand three-dimensional Cultures 01 articular chandracytes. In addition. the expression of S-1W protein and its alpha and beta subunits was studied in monolayer cuI1ures. Bavine articular chondrocytes were isolated by collagenase digestion from ankle joints and cuhured in monolayerandspheroid culturesin DMEMwim 10% FCS lmmunocytochemical studieswere performed using the indirect peroxidase technique an monolayers affer methanollethanol (1 1 vlv) fixation and on frozen Sections of Spheroids. The reaction was scored semi-quantitatively as negative (-), weakly (+) moderately (++) Ontha basisofthenumbersafcasesrefsiredtoustottheidentificalionofjointcry~talsintissuesectians, webelieve that calcium pyraphosphatedihydrate(CPPD) deposnion disease isbeing underdiagnosed byhistopathologists. A crltical light microscopic reviewof 18cases. all with thediagnosisconfimed bymicroanalysis(energydispersiveXrayspectroscopyln thescanning electron microscope, infrared spectroscapywboth) revealed adistinctwefeathery or br"Shlikeappearanceinal1suchdeposits.Thisfeaturewasapparentat lowpower, whileconvincingvisualisation of CryStalSwithinthedepoSitswasdiff~cuneven withanoil immerSionobiective.Thesignof birehingeneeof CPPD crystals is more dificult to demonnrate I" tissue sections than synoviai fluids. due to IactoIs Such as stain. fragmentation and heaping up of crystals, buf it can be more readily assessed in unstained sections or following microincineration of the section. In six of our c a m the deposits were exclusively within bone: demonstration depended on relatiYeunderdecalcification: deposits in this position have not previously been recognised. This study thus provides new information an the histological identdication of CPPD deposns in tisues and implies heterogeneity in the pathogenesis of such deposits. In human knee osteoarthrosis (OA) overt unicornpartmental disease is frequently accompanied by a macroscopically normal second cornpartmsm. However. there are a number of light micmscopical changes within the latter which might amount to an "early osteoarthrosis". These are chondrocyte pmliferation and chondrone formation. decreased proteoglycan staining and disruption of the chandra-osseous interface with duplication of the tidemark and resorption ofthe cartilage by chondmclasts. Tidemark duplication IS also a feature of established OA re~resenti~gadistu~anceDfcabif~cat~oninrone5ofarticularcartilage.SlnceSlWp~~t~i~~~~ knowncalciumion transponmoleculs.altsrationm~~sstainingpanernand~ntens~tym~grn beanlndexotearlyo~. sl~~protemstamng was studied In the tiblai plateau cartdage of 31 cases: 12 with unicornpartmental OA. 11 with bicompartmental OA and8 Controls. lntheianerall chondrocytesgavepositivemembrana and cytoplasmic staining (especially in zones 2 e n d 3 ) , d~u s e w e a k m a t~~c~l s t~~~~~g~~~~~e l , s t~o~g p a r i c e l l~i a~a~d~~t~~-t~~t o~~l s t~i~~~g~~~o~~s 4 a~d 5 .

In overt and early OA the Orderly malrlcal panem was disrupted With prominent patchy staining around chondrocyte clusters. The significance of the change in the matncal distribution ~n OA IS unknown and unlikely lo be related to a disturbance of calcification in zone 5. Road, London. WCl m e histogenesis at Alveolar son part sarcoma (ASPS) IS still unsenled: nonchromaffin paraganglioma. malignant granular cell myoblastoma, neuml tumour, myogenic tumour. habdomyosarcoma and renin-producing tumoui theories have been proposed lmmunohistochemical studies have yielded discrepant results We recentlyobsewed that the diastase resistant crystalline material in ASPS was strikingly reactive for vimentin by the immunoperoxidase method on paraffh sections 01 one case. We then studied paraffin sections of seven other cases from our files between 1980 and 1991 ~ by routine light microscopy, immunohistochemistry and electron microscopy All eight cases were reactlve for vimentin. Which highlighted the crystalline materlal as In the first case. Vanabie numbers of immunoreactlve cells for desmin were found in three. and smooth muscle specific actin in four. All cases Showed some reactivity for neuron Specific enolase. and seven of the eight cases reacted for S-100 protein. The clinical and pathological lea lure^ of 20 cases of synoviai Sarcoma have been reviewed. These tumours are aggressive with a Poor prognosis. typically affecting young aduns with a long history before presentation and detinltive diagnosis. A high index of suspicion IS required If the diagnosis IS not to be missed Several features, including age, size. site. histological type (monophasicibiphasic), mitotic actlvity and necms~s have been investigated withaview toestablishing possible prOgnosticindicatorS. The bestguideto prognosisis assessment 01 mitotic actlvity. Size and histological type dld not affen the outcome. in this study the effecl of subcapsular OrChideCtOmy on Skeletal metastases from prostatic carcinoma was Studied using bone histomorphomatnc paramsten. Twenty-eight patients with bone metastases were studied immediately before and for Seven months after orchidectomy. Tetracycline labelled bone biopsies were taken from metastases and turnour free areas at the beginning and end of study. Sixteen of the 28 patients also underwent o~teocla~t Inhibition tor SIX months using Disodium Pamidronate (30 mg I.V. weekly for 4 weeks then anemale weekly for five months) to enable differentiation of the skeletal response to castration. Histamarphametnc anslysi~ 01 tumour frse bonerevealed adrapin overall bonevolume. Histological analysisofmetastasesshawedadecrease m osteoblastic activity but widespread osteoclast mediated ostealysis. Tumour regression and manow recolonisation were present in most C~S B S but malignant foci remained in 56% of repeat metastatic biopsies, inducing a typical. lacalised disruption of bone metabolism. It is Concluded that orchidectomy causes ostmblastic regression blrt induces increased OStBoClaSt mediated bone destruction, which is most pronounced within metastases. Although tumour regression and marrow recolonisation usually occur within metastases, active turnour foci anen perstsf. ( 11) 

Chairman S. G. Silverberg, Washington

Theeffectsafoncogeneson thedeveloping nelvoussystem havebeenstudiedinaneuraltransplant system ~ntats, taking advantage of the extraordinary capacity of fetal CNS to differentiate in and fully integrate with, the adult host brain. Gene transfer tnto tetal brain cells was mediated by In viiro infection with replication-defsctwe retroviral vectors. Fetal rat brain suspensions were then stermtaxically injected Into the caudoputamen of adult F344 rats.Animals carrying transplants exposed In vim lo the polyoma medium Tantigen developed endothellal hemangiomas in the graft which often led to fatal cerebral hemorrhage within 13-50 days alter transplantatm lntroductm of the viral SIC gene caused astrocytic and mesenchymai tumors affer latency periods of 2 4 months.Following infection of fetal donor cells with a vector encoding the V-myc oncogene led to the development of only a single embryonal CNS tumor whereas exposure to V-Ha-ras and v-myc resulted in the rapbd induction of multiple malignant neoplasms. When injected intracerebrallyinto newborn ratsin vivo, complementation of theseongogenes led 10 the development of malignant hemangioendotheliomas, undinerentiated neural tumors andlor leukmia.Oncogene transferthus constitutes a challenging new model to assess the effects of translorm~ng genes on the developing nervous system.

Since the presentahon of molecular genetic evidence Supporting the Knudson hypothesis m the eariy 1980s there hasbeen increasing interestinthe ideathatasimilartwo-hit mechanism mayaperateln sporadadlccancers Therens now evidencBthatlassofonealleleandretentionottheother~nmutated/wildfarmaccuninsparad~ccalono. lung. renal and breast cancers In breast Cancer lossof heterorygoslty(L0H) has been demanstratedfarseveral alleleson chromosomes 1 l p 13q and an the Short arm of chromosome 17. LOH has also been demonstrated on other chromosomes Thegeneral viewisthat LOH indicatesthatasuppressorgene may bepresentattheseloct. Hawevet In interpreting LOHthere 1sdifficulty indetermining when LOH becomessignificant because Itsincidencevariesfrom 1%700/0 depending on the locus examined. In this presentation, knowledge about LOH on chromosome 11 p (and 11 q where a new deletion has been recently discovered), and 17p will be presented and the pathqlenetic Significanceand ciinicsl relevanceexamined. lnthe lattercontext anewrapid methodfordetemlnmg alleljc dosage will a l x be described which does not requlre RFLP analysis.

Expelt systems -1.e. computer software that can function as cansuhant, decision support system, or process contr~llerhas undergone a rapid development durlng the past decade. Applications to histopathology have 

Gnaw Some 2,OCQdiffuse maiignantmesotheliomas(0MM) occurannuailymN. Americaat thepresenttime.Cumulat~~ely, substantial numbersot lesser-knownfarms of meSothelia1 turnourare also seen, including serous paptllary tumoun of the peritoneum, well differentiatedpapillarymesothellomas and benign cystic mesotheliomas, but incidencedata are not available.The relativeiyfrequent atypical reactive hyperplmasof w o s a membrane add further vanstytothe range of mesothellai lesions which may present diagnostic problems. The expenence of a U.S.-Canadian MeSOtheliomaPanel Suggests that DMM vs. metastatic CarcinOmacOntinUeS to bethe commonest mesatheliamarelateddiagnostieehaliengeandthatthedifficunyandalwayssolved bytheappiicationofspecialstains Almostas common are le~ions in Which the ditferential diagnosiscenters on DMM us. a reactive process (atypicai mesotheliai hyperplasia, fibrous pleurisy) Within the laner group It IS particulady difflcun lo obtain a strong consensus opinion, andfoilaw-uphasconfirmedthemaiorityopinioninonly69%ofcases. SarcomatousDMMvs.sarcomaNOSisaiso a notable area of difficuny. Most mesothelial lesions tn North Amenca. including DMM. are reviewed within a Short time of biopsy by a pathologist in a major teaching centre, only a Small number (5-10%) being subpct to further scrUtiny by a panel or ~n the coune of epidemiological or clinical studies However, many cases resurface later ~n a legal Sening when the material and clinl~al information 1s often available. The original diagnosis of the hosoital pathologist 1s usually endaned by the courts.

Sharon W. Weiss

LlpoSarcoma is one of the most common forms of son tissue Sarcoma and may be divided into several subwes well-differentiated. myxoid. round Cali, phmwpha, and dedifferentiated. This presentation will discuss the 1) diagnosticcnteriaof lipblasts2) d8agnosisand behaviorof myxoidround cell iiposarcoma andthe3) behavior and incidence of "dedifferentiation" dwell-differentiated Ihposarcoma. The diagnosis of lipsarcoma depends in part On the identdication of lipoblasts or pnmitive fat ceils. Since iipoblast-l!ke cdls may be seen m a variety Of conditions apart from iiposarcoma, strict diagnostic C!i tma must be applied in identlfving these ceiis. These cells contain a hyperchromatic. indented or scalloped, eCmtnc nucleus set in a cytoplasm containing one or more lipid-nch vacudes. These cells must also occur in an appropriate histaiqic background. Vanous lesions Which may contain Ihpoblast-like cells and which may. therefore, mimic liposarcoma include fat necrosis, fat atrophy, silicone granuloma, signet nng carcinomas or melanomas, and a variety of malignant tumors with fixation artifact. Myxold androundcell lipsarcomarepresentthemost commanfarmofsarcomaaccuningmearlytamidadunlifeandare commoniy located #n the region of the Mlgh and popitteal fossa. Although the designation of "myxold and "raund CelY Suggest two separateturnortypes. they represnt ends d a common h~stOloglCSpBCtNm. Myxaid liposeicoma represents the well differentiated end of the spenrum. whereas round cell represents the pcarly differentiated end.However, transitional or mixed forms ex~st. accounting for confusion as to how such tumors should be classlfled Mymd liposarcomas are characterized by nodules Of bland stellate or rounded cells set 8 n hyaiuronic aod-rich strama. An intricate plexiform vasculature and numerous lipblasts are easily VdentAed. Wlth progression to round celliiposarcomathecellsbecomelarger,moreatypicalandthestromalessmyxaid. LipOblaStSaremoredlfticultto find and a plexiform vasculature 1s less apparent. Recent work by Evans Suggests that eValUatlOn of myxoidlfound cell Ihpasarcomashould includethe percentage of round cell component wdhin atumorsincethisdirectclh/ mtluences prognosis. Our policy IS to carefully sampk such tmors, submining t section for each centimeter In greatest diameter of the tumor. A rough estimate of the percentage of a round cell component 1s made. We w a r d tumors havinglessthan 1010faroundcellcomponentasGradeI.Thosehav,ngbetween 1&25% roundcelicomDonent are considered Grade 11. whereas those with more than 25% are considered Grade 111. Well dmerentiated liposarcoma IS one of the most common Sarcomas accuning m late adult life. Characteristically affecting the deep muScleSaftheextremities, theretropentonealspace. and the groin, this tumor ischaractenzed by varying amounts of mature fat interspersed with fibrous bands, atypical hyperchromaric spmdledceiis, and lipoblasts. These lesions are considered low grade Sarcomas having a high rate of local recurrence but no abilny to metastaslze. Recently Evans and Ammi eial Suggested that well-differentiated liposarcomas occurring m the wbcutaneous tissue and muxles of the extremity cause so hnle morbidity that the use of the term "atypical lipma" Should be used rather than Ihposarcoma. in contrast. well-differentiated liposarcomas in the retropentoneum cause significant morbidihl and pose a significant risk of death from local disease. Thus. nearly all pathologists agree that the term "weii differentiated lipasarcoma" Should be retained for these tumors ~n the retroperitoneum. However. none of these recent studies have followed a large number of these tumors for a proianged period at time m order to assess the long term behavior and specihcalty risk that such 1es1ons may progresswith time to a higher grade lesions (1.9 dedifferentiate). We have recently completed a follow-up study of 98 cases of well ditferentiated IipoSarcoma accurringmthemusc~oftheextremity,retropntoneum,andgroin. Inalllmatlonsthenskof local recurrence was high. However,dedifferentiationoccurred~n 33% of casesandwasnotrestnnedtotumors~nanyparticularlacatlon. butcouldbe beStconelatedwiththedurationofthetumor. withmostcasesoccun~ngaflerl0Mmoreyean Thus. dedifferentiation IS not a site-dependent phenomenon, as has previously been suggested. but rather a time dependentphenomenon.Anhough thesedatado notneee~~arily indicatethatwe Should abandon theterm"atwca1 itpoma" they do indicate the need tor proianged foilow-up of patients wlth wei&-dlfferent,ated DDosarcOma and the small but deflnne risk of dedifferentiation as a long term complication of the disease.