9th Ljudevit Jurak Symposium on Comparative Pathology

Program | Book of abstracts


PROGRAM

We have a great pleasure of inviting you and your colleagues to this symposium held as a part of a celebration of the 85th anniversrsary of a foundation of °Ljudevit .Jurak” Clinical Department of Pathology (1913 – 1998)


Organizacijski odbor Organizing Committee

Predsjednik/President: Prof. Dr. Mladen Belicza  
“Ljudevit ]urak” Clinical Department of Pathology University Hospital “Sestre milosrdnice”,  Zagreb, Vinogradska 29 Croatia 
Tel/fax: 385-01-3768-302

Dopredsjednik/Vice-president: Prof. Dr. Željko Grabarević 
Department of General Pathology and Pathological Morphology,  Veterinary Faculty,  Zagreb, Heinzelova 55, Croatia 
Tel: 385-01-2390-313, Fax: 385-01-214-697

Tajnici/Secretaries: Prof. dr. Ruža Sabočanec, tel. 385-01-2390-311 
Dr. sci. Božo Krušlin, tel/fax: 385-01-3787-177 or 385-01-3787-244

Scientific Board
Prof. dr. G. Mikutz (Austria) Prof. dr. E Silvestri (Italy)
Prof. dr. Ž. Grabarević (Croatia) Prof. dr. M. Štulhofer (Croatia)
Prof. dr. W Walkiewicz (Poland) Prof. dr. K. Lapis (Hungary)
Prof. dr. H. G. Fassbender (Germany) Prof. dr. M. Bielicza (Croatia)
Prof. dr. K. Čuljak (Croatia) Prof. dr. A. Pirkić (Croatia)
Prof. dr. D. Ferluga (Slovenia) Prof. dr. Z. Biđin (Croatia)
Prof. dr. L. Šenk (Slovenia) Prof. dr. Z. Kusić (Croatia)

Prof. dr. Jasna Talan-Hranilović (Croatia)
Glavna tema simpozija je urološka patologija 
The main topic of the Symposium is Urological Pathology

Mjesto održavanja simpozija/Location of the Symposium 
HOTEL SHERATON, ZAGREB, Kneza Borne 2



  
 

RASPORED PREDAVANJA     PROGRAM

Petak – Friday 
05. 06. 1998.
8,30  Otvaranje skupa Opening ceremony

Ženski gudački kvartet “Dora Pejaćević” (Prim. dr. sci Nada Lang, Doc. dr. sci. Božena ,Šarčević, Dr Blanka Brezovec-Cvetinić, Mr. sci. dr. Mihaela Britvec) 
Izbor iz knjige pjesama “Bijela ptica” patologa dr. Cvjetka Leia 
Female string quartet “Dora Pejaćević” (Prim. dr. sci Nada Lang, Doc. dr. sci. Božena Šarćević, Dr Blanka Brezovec-Cvetinić, Mr. sci. dr. Mihaela Britvec 
A choice of poems from a poem collection “Bijela ptica” by dr. Cvjetko Lež, a pathologist. 

9,00 Dodjela nagrade “Ljudevit Jurak” za doprinos unapređenju komparativne patologije (sponzor Zagrebačka banka) 
The “Ljudevit Jurak” award ceremony, for the contribution to the advance of comparative pathology (Sponsored by Zagrebačka banka) 
9,30 Coffee break Odmor

A. 
Pathological morphology of human and animal diseases 
    Patološka morfologija humanih i animalnih bolesti 
  Chairmen – Predsjedatelji: Mikuz G, Mašera A, Belicza M
10,00 Mikuz G, Rogatsch H (Innsbruck) Memorijalno predavanje Memorial lecture Prostate cancer: Stage prediction by preoperative biopsies.
10,20 Reljić A, Spajić B, Kraljić I (Zagreb)Indications, technique and complications of prostatic needle biopsies.
10.30 Reljić A, Marković-Puač Lj, Spajić B, Tarle M, Čupić H, Kraljić I, Krušlin B, Belicza M (Zagreb)The relationship between clinical and pathohistological fndings in prostatic needle biopsies. 
10.40 Volavšek M, Mašera A, Ovčak Z (Ljubljana) Tumor neoangiogenesis in prostatic carcinoma.
10.50 Glavač D, Mašera A, Ovčak Z (Ljubljana) Molecular heterogeneity in prostate cancer
11.00 Božić Z, Kraus O, Duančić V (Zagreb)A study of transvesical prostatectomy: Retrospective-prospective analysis of 230 Cases.
11.10 Bussani R, Pavletić N, Silvestri F (Trieste)Renal carcinoma: An epidemiological assessment in two periods of time. (Trieste, 1985-86 vs 1994-95). 
 11.20 Bussani R, Pellegrino M, Silvestri F (Trieste)Papillary renal carcinoma (clear cell type) arising in the excretory urothelial district. A very unusual feature. Morphohistopathological findings. 
11.30 Đordević G (Rijeka) Angiogenesis in different types of renal cell carcinoma.
11.40 Kopjar A, Dimanovski J, Stanić G, Krušlin B, Belicza M (Zagreb) Correlation between vimentin expression and nuclear grade in renal cell carcinoma.
11.50 Mašera A, Staniša O, Ovčak Z (Ljubljana) The influence of pH on Ki-67 antigen retrieval in the renal cell carcinoma. 
12.00 Mašera A, Zidar N, Ovčak Z (Ljubljana) Use of immunohistochemistry in the diagnosis of renal angiomyolipoma. 
12.10 Kos M (Zagreb)Congenital malformations of genitourinary system in the I 0-years periods: An autopsy study.
12.20 Ježek D (Zagreb)Mast cell in testicufar biopsies of infertile men. 
12.30 Ježek D (Zagreb) Expression of KiM I P, testosterone and Inhibin in testes of infertile men. 
12.40 Vodovnik A, Mašera A, Ovčak Z (Ljubljana) Immunohistochemistry of spermatocytic seminoma. 
12.50 Lenz D, Čačinović V (Našice) Carcinoma of the penis – A case report. 
Wiener H G, Remkes G W Mian C, Pycha A, Susani M (Vienna)  CYTOLOGY AND QUANTITATIVE CYTOMORPHOLOGY IN NEPHROGENIC ADENOMA
13.00 Discussion and coffee break   Rasprava i odmor 

Chairmen – Predsjedatelji: D. Ferluga, K. Čuljak, Krušlin B
13.30 Talan-Hranilović J, Čupić H, Gnjidić Ž, Hat J (Zagreb) Lymphocytic adenohypophysitis simulating pituitary adenoma. 
13.40 Kalogjera L, Glumbić I, Zurak K, Baudoin T, Rudnički M, Krušlin B (Zagreb) Correlation between eosinophil infiltration and hyperactive response to destilled water chalenge In chronic rhinosinuitis. 
Tomić S, Alujević A, Pešutić-Pisac V Vuković (Split) S FEMALE ADNEXAL TUMOR 0F PROBABLE WOLFFIAN ORIGIN
13.50 Šimec Z, Artuković B, Grabarević Ž (Zagreb) Haemangioendotheliosarcoma of the heart in dog. A case report.
14.00 Gumzej Ž, PirkićA, Bencun-Gumzej P, Klarić P (Zagreb) Cytotopographical expression of ICAM-I and CEA in mucinous ovarian tumors.
14.10 Tomić S, Alujević A, Pešutić-Pisac V, Vuković S (Split) Paraovarial cystic tumor of probable Wollf origin. A case report.
14.20 Alujević A, Janković S, Družijanić N (Split) The malignant mixed tumor of the gallblader (Adenosquamous carcinoma with fibrosarcoma). A case report. 
14.30 Selak I (Sarajevo) Immunological response to epithelial dysplasia in colorectal mucosa.
14.40 Radović S (Sarajevo) Immunological response to epithelial dysplasia in flat mucosa of the colon.
14.50 Discussion and coffee break Rasprava i odmor

Chairmen – Predsjedatelji: Grabarević Ž, Sabočanec R, Gomez-Villamandoz JC 
15.20 Gomez-Villamandoz JC (Cordoba) Pathology and pathogenesis of African swine fever.The role of the monokines. (Invited Lecture)
15.30 Fatma S, Senay B (Ankara) Pathological studies on ovine caseous lymphadenitis in Turkey.
15.40 Gomez-Villamandoz JC (Cordoba) Pathogenesis of thrombocytopenia in classical swine fever Study of the bone marrow and platelets. 
15.50 Gunay A, Vural Atalay S, Nihat T (Arvkara) First report of Peste des Petits Ruminants in lambs inTurkey: Patomorphological and immunohistological feature.
16.00 Modrić Z, Sabočanec R, Turk N, Artuković B, Slunjski A, Čuljak K (Zagreb) Swine leptospirosis caused by Leptospira interrogans (serologic variant Icterohaemorrhagiae)
16.10 Vural Atalay S (Ankara) The comparison of histopathological, immunoperoxidase and immunofluorescence techniques in the diagnosis of rabies in dogs. 
16.20 Carrasco L (Cordoba)Implications of pulmonary intravascular macrophages in pulmonary processes. 
16.30 Ayhan A, Vural Atalay S, Berkin S (Ankara) Incidence and pathological studies on the liver telangiectasis on feedlot cattle.
16.40 Šoštarić B, Lipej Z (Zagreb) The differences in pathological picture in pigs succumbed of hog cholera.
16.50 Juntes P, Lindtner R, Pogačnik M (Ljubljana)Immunohistochemical identification of Leptospira pomona antigen in tissue sections.
17.00 Zrnčić S, Oraić D, Šoštarić B (Zagreb) Pathomorphology of vibriosis in sea bass (Dicentrarchus labrax) of different ages.
17.10 Marinculić A, Božić F, Dominis M, Auslender Ujević V, Grabarević Ž, Duraković E (Zagreb)Immunopathological response againstTrichinella spiralis in mice.
17.20 Discussion and pause   Rasprava i odmor 

B. 
Iatrogenic, experimental and environmental pathology  
Iatrogena, eksperimentalna i patologija okoliša 
Chairmen – Predsjedatelji: Sabolić I, Juntes P, Čurić S
17.40 Curca Dumitru (Bucharest)The influence of thyroid functional modifications on collagen contens of skeletal muscle in the meat chickens.
17.50 Oraić D, Zrnčić S, Šoštarić B (Zagreb) Pathomorphological picture of Myxidium infection in cultivated charp snouted sparus (Puntazzo puntazzo).
18.00 Bassan F, Bergamin C, Bussani R, Pavletic N, Silvestri F (Trieste)Use of LN3 antibody in the analysis of the biological evolution of myocarditis. 
18.10 Bassan F, Zambon S, Bussani R, Pavletic N, Silvestri F (Trieste)Use of microwawe in a histopathology laboratory.
18.20 Baus M, Medjugorac-Popovski M, Sabolić I (Zagreb) Effect of colhicine on redistribution of cell membrane proteins in the rat proximal tubule. 
18.30 Curca Dumitru (Bucharest) Serum cholinesterase activity (PchE) and ascorbinemia calves with neonatal diarrhoea after parenteral administration of ascorbic acid. 
18.40 Međugorac-Popovski M, Herah-Kramberger CM, Sabolić I (Zagreb) Polyclonal antibodies to rat renal cortical endocytic vesicles labe4 proximat tubule cell rnembranes In the rat and human kidney. 
18.50 Herak-Kramberger CM, Thevenod F, Sabolić I (Zagreb and HomburglSaar) Nephrotoxic heavy metals increase the expression of mdrl P-glycoprotein in the rat kidney brush-border membrane. 
19.00 Marinculić A, Kamadan P, Petrinović T, Grabarević Ž, Živičnjak T, Ćurić S, Stojčević D, Džakula N, Beck R, Barbić A, Šantek T, Vukonić L (Zagreb)Clinical and pathofogical study in canine visceral leishmaniasis: natural and experimental infection. 
19.10 Discussion and pause   Rasprava i odmor 
20.00 Congress dinner Kongresna večera

Subota– Saturday 
07. 06. 1997.

C. 
“Herman Jurak” round table on rheumathological diseases 
“Herman Jurak”- okrugli stol o reumatološkim bolestima

Chairmen – Predsjedatelji: Fassbender HG, Đorđević G, Manojlović S

8.30 Nikolić V (Zagreb)Anatomy and biomechanical characteristic of joint.
8.50 Schneider S, Fassbender HG (Mainz) The nosological role of primary necrotizing processes (PNP) in rheumatoid arthritis. 
9.10 Harjaček M, Diaz-Cano S (Zagreb, Muenchen) Signal enhancement method for the digoxigenin-labeled in situ hybridization. 
9.20 Đorđević G (Rijeka) Comparison study of Ag-NOR hierarchy model in clinically confrmed seropositive and seronegative RA patient. 
9.30 Manojlović S, Pećina M, Seiwerth S, Hašpl M (Zagreb) Pathohistological findings in tendons and cartilage in jumpers knee. 
9.40 Discussion and pause  Rasprava i odmor 

D. 
Ethical forum – Forensic medicine: Profession? Science? Politics? 
     Etički forum – Sudska medicina: Struka? Znanost? PoIitika? 
Chairmen – Predsjedatelji: Anđelinović Š, Perić J, Marcikić M 
10,00 Džaja P, Perić J (Zagreb) Animal euthanasia.
10,10 Ugljarević M, Marcikić M, Badžek S (Osijek) Epidemiology of suicide in Osijek county. 
Brkić H. Kaić Z, Keros J, Njemirovskij V (Zagreb)  DETERMINATION OF THE WAR VICTIM’S AGE BY TEETH
10,20 Discussion and coffee break Rasprava i odmor 

E. 
Computer systems and computerisation in pathology  
     Informatički sustavi i kompjutorizacija u patologiji 
Chairmen – Predsjedatelji: Plasaj T, Brezovečki D, Matković M 
11.00 Šimunović D, Belicza M, Lechpammer M,Gladić V, Kusić Z (Zagreb) Thyroid cancer in clinical thyroid diseases register of University Hospital “Sestre milosrdnice”. 
11.10 Cvjetko l, Belicza M, Lechpammer M, Gladić V, Kusić Z (Zagreb) Non-malignant lesions in clinical thyroid diseases register of University Hospital “Sestre milosrdnice”. 
11.20 Kučinar I, Brezovečki D, Matković M, Lechpammer M (Zagreb)Causes of death in autopsies during World War I and 80 years later
11.30 Discussion and coffee break    Rasprava i odmor 

F. 
Stomathological pathology 
     Stomatološka patologija

Chairmen – Predsjedatelji: Pirkić A, Talan-Hranilović J, Kaić Z

11.40 Jukić S, Anić l, Talan-Hranilović J, Najžar-Fleger D (Zagreb)The effects of C02 and Nd-yag lasers on the pulp and periodontal tissue on the dog. 
11.50 Mravak-Stipetić M, Gall-Trošelj K, Pirkić A, Pavelić K, Pavelić J (Zagreb) The lack of p53 and nm23-H I genes deletion in oral lichen planus and leukoplakia.
12.00 Pavlica Z, Juntes P, Pogačnik M (Ljubljana) Defence reaction in a dental pulp after partio-pulpectomy in dog.
12.10 Jukić S, Sisig S, Anić I, Mihelić D, Talan-Hranilović J (Zagreb) The activfty of NADPH – d in chronfcalfy fnflammed dental pulp. 
12.20 Mravak-Stipetić M, Donath K (Zagreb) The Cutting-Grinding technique – a methodological approach and application in dentistry.
12.30 Janjanin N, Manojlović S, Pavelić B, Levanat S (Zagreb) Morphologfcal characteristlcs of odontogenfc cysts. 
12.40 Biočina-Lukenda D, Pirkić A, Cekić-Arambašin A (Zagreb) Expression of c-erB-2 protein in oral lichen planus.
12.50 Kaić Z, Keros-Naglić J, Božićević D (Zagreb) Transcutaneous stfmulatfon analgesia fn stomatofogfcal procedures. 
13.00 Galić .I, Kaić Z, Štefanac-Papić J (Zagreb)Evaluation of four methods for estimating taurodontism. 
13.10 Discussion Rasprava
14.00 Closing ceremony Završetak skupa 

 

BOOK OF ABSTRACTS


Tajnici/Secretaries 
Prof. dr. Ruža Sabočanec

Dr. sci. Božo Krušlin

EDITORS 
Prof.dr.sci. Jasna Talan-Hranilović

Mr.sc. Mirna Lechpammer, dr. med.

Technical Editor

Dr. sci. Božo Krušlin

Language Editor

Biserka Tomljenović-Belicza

Editors Administrator

Renata Periz

Tiskara 
“Kratis” Zagreb Vrapčanska 15

Design 
Studio grafičkih ideja


A. 
Pathological morphology of human and animal diseases 
Patološka morfologija humanih i animalnih bolesti 



Prostate cancer: Stage prediction by preoperative biopsies 
Mikuz G. Rogatsch H 
Institute of Pathology, Innsbruck, Austria 
The tumor stage and the grade of malignancy are the most important parameters forthe prognostic and therapeutic assessment of the prostate cancer at the moment.To distinguish localized (pT2) from capsule-transgressing (pT3) carcinomas before starting therapy would, therefore, be an important goaLThe clinical methods used for this purpose either have a low accuracy or can only be perFormed with a very expensive equipment available only in few specialized hospitals.Transrectal core needle biopsy is however, perFormed in all patients with elevated PSA and/or other symptoms suspicious for prostate cancer. Several authors tried to determinate the tumors’ stage by morphological and morphometrical analysis of such biopsies. We analyzed 90 prostatectomy specimens and matching needle core biopsies. One half of the patients (N=45) had localized carcinoma (pT2) and the other half of prostatectomy specimens showed an extraprostatic tumor extension (pT3).The Gleason score perFormed on core needle biopsies showed only a minimal difference between the two stages. A simple method for predicting the extension of prostate carcinoma is to count the number of biopsies which contain carcinoma. The method works, however, only if at least 6 biopsies are perFormed. The linear length of carcinoma (in mm) in needle core biopsies can also be used as predictive parameter. In our material pT2 carcinomas showed an average length of 3,4 ~ 2 mm in the biopsies, whereas the average length of carcinomas with extracapsular extension was 20 ± 5 mm (p<0,000 I ).The favorite method used by a number of authors seems to be the measurement of the relative carcinoma volume (vol%) of carcinoma in the core needle biopsies. In our material the mean relative volume of carcinoma tissue in the needle core biopsy of patients with pT2 stage in prostatectomy specimen was 4,9 ± 2,7 vol% pT3 carcinomas in prostatectomy material showed an average relative volume of 30,1 ± 23% in the biopsy (p<0,0001 ). A method previously used in our laboratory was the assessment of the microvessel density in biopsy specimens pT2 carcinomas showed a significantly lower vessel density than pT3. In our opinion the measurement of the relative volume of carcinoma in core needle biopsies fulfills all these criteria in the best possible way. Moreover,the method is not time consuming and does not require special equipment – a pathologist needs no more than a simple ruler to measure the length of the carcinoma as well as of the single biopsy chips.



Indications, technique and complications of prostatic needle biopsies 
Reljić A, Spajić B, Kraljić I 
Department of Urology “Sestre milosrdnice” University Hospital, Zagreb, Croatia 
Prostatic adenocarcinoma is the third most common malignant tumor in Croatian male population and represents a major public health problem. About 85% of prostatic adenocarcinomas are diagnosed in advanced stage.Two years ago, a multidisciplinary approach to the early diagnosis of this disease has been introduced at the Urological Clinic,”Sestre milosrdnice” University Hospital in Zagreb.The aim of this project was to increase the proportion of patients diagnosed in earlier stages of the disease.We present our current protocol for establishing the indication for prostatic needle core biopsy. The experiences on I 34 patients concerning the preoperative treatment, bioptic techniques and complications of the procedure are shown. 
We conclude that prostatic needle core biopsies are necessary for the early diagnosis of prostatic adenocarcinoma.This procedure could be performed in local anesthesia with minimal complications.



The relationship between clinical and pathohistological fndings in prostatic needle biopsies 
Reljić A, Marković-Puač Lj, Spajić B, Tarle M, Čupić H, Kraljić I, Krušlin B, Belicza M 
Departments of Urology, Pathology and Oncology and Nuclear Medicine 
“Sestre milosrdnice” University Hospital, Zagreb, Croatia 
Aim Prostatic needle core biopsy is confirmative method for prostate cancer diagnosis. During a two-year period at Clinic of Urology, University Hospital “Sestre milosrdnice”, 250 patients were examined for prostate carcinoma (PC).The aim of this study was to analyze clinical and pathohistological findings in prostatic needle core byopsies in regard of early diagnosis of the disease. 
Methods The population consisted of ambulatory patients over 50 years of age with lower urinary tract symptoms. All patients were examined according to protocol for early diagnosis of PC. The patients underwent digitorectal examination (DRE) and serum prostate specifc antigen (PSA-Tandem R) measurement. lndication for needle core biopsy were positive DRE and/or PSA value above 4 ng/mL.These. patients underwent transrectal ultrasound examination (TRUS).All visible leasions of peripheral prostatic zone were biopsied and sextant biopsy was taken. If no leasion was detected, random sextant biopsy was perFormed. 
Results  According to above mentioned criteria I 34 of 250 patients underwent biopsy (53,6%). DRE was positive in 67 (50.0%) andTRUS in 70 (52.0%) patients.The PSA level was below 4 ng/mL in 20 of biopsied patients ( I 4.9%), between 4 and I 0 in 66 (49.2%) and above I 0 ng/mL in 48 (35.8%) of patients. Prostatic cancer was found in 39 (29. I %), high-grade PIN (HGP) in I 9 ( I 4. I %) and benign prostatic hyperplasia (BPH) in 76 (56.7%) of patients. PC with low Gleason score (2-4) was in 17 PC (44.0%), with Gleason score 5-7 in 8 patients (20.0%) and with high Gleason score in 14 (36.0%) patients. In cases with well diferentiated cancers PSA was 47.7 ng/ mL (range 5.8-105.0), moderatelly differentiated cancers 68.8 ng/mL (range 17.9-131.0) and in poorly differentiated PC I 8.4 ng/mL (range 0.02-49.8). Average PSA for PC patients was 43.8 ng/mL, for HGP I0.9 ng/mL and 7.6 ng/mL for BPH patients.Volume of the prostate with cancer was roughly 25.0% lower than for BPH. Of 39 detected PC I 2 patients (30.7%) had indication for radical prostatectomy and clinical understaging was 58.3%.There was upgrading of 56.0% of PC in radical prostatectomy specimens comparing with needle core biopsies. 
Sensitivity of DRE was 92.0%, specificity 67.0%, positive predictive value (PPV) and 54.0% and negative predictive value (NPV) 96.0%.Almost identical results were obtained forTRUS. HGP was visualised, by means ofTRUS, in 45.0%. PC was found 5 I .0% and HGP in 10.0% of hypoechoic peripheral zone lesions. For PSA value above 4.0 ng/mL sensitivity was 95.0%, specifoity only I 9.0%, and PPV 32.0%. For PSA value above I 0.0 ng/mL, sensitivity was 69.0%, specificity 78.0%, and PPV 56.0%. 
Conclusions No single test (DRE, PSA,TRUS) had diagnostic capabilities for PC and HGP DRE and PSA should be considered as first line tool for early diagnosis of PC. Although we have biopsied almost every DRE positive and virtually all patients with PSA above I 0 ng/mL,there is enough space for reducing the number of unneccessary biopsies. 



Tumor neoangiogenesis in prostatic carcinoma 
Volavšek M, Mašera A, Ovčak Z 
Institute of Pathology, Medical Faculty, Ljubljana, Slovenia 
Introduction Tumor neoangiogenesis has proven its prognostic value in malignant tumors of different organs. The association with disease progression and metastatic spread has also been shown in prostatic carcinoma after radical prostatectomy. 
Aim The aim of our retrospective study was to disclose its significance in incidental prostatic carcinoma aftertransurethral resection (TURP).We examined all ICP diagnosed at the Institute of Pathology, Medical Faculty in Ljubljana in years 1985-89. 
Methods To determinate how microvessels correlate with the degree of tumor differetiation determined histopathologically and with Gleason score, stage of disease, and the time of survival in at least 6 years after diagnosis, we counted microvessels within the initial carcinoma in 68 patients. Microvessels were higlighted by immunostaining endothelial cells for factorVlll-related antigen and counted in a x200 microscope field (0,8012 mm2) in the most active areas of neovascularisation, Neoangiogenesis and degree of tumor differentiation were then correlated between primary tumors and rebiopsies in I 0 patients. Degree of tumor differentiation was also correlated with the time the disease progression occured. 
Results  Higher microvessel counts were associated with lower degree oftumor differentiation (p=0,005), Gleason score (p=0,001 ), stage of disease in primary tumors (p=0,003), and survival of the patients (p=0,05).The time of disease progression in I 0 rebiopsied patients showed association with lower level of tumor differentiation (p=0,004; 0,006). Microvessel counts were significantly higher in re-biopsies comparing to initial tumors (p=0,006), but no differences could be observed in the degree of differentiation. 
Conclusion  Our results showed that the determination of microvessel density in TURP biopsy specimens of ICP retains its prognostic value already demonstrated in radical prostatectomy specimens of clinical prostatic carcinoma.



A study of transvesical prostatectomy: Retrospective-prospective analysis of 230 Cases 
Božić Z, Kraus O, Duančić V 
Department of Urology “Sestre milosrdnice” University Hospital, Zagreb, Croatia Institute for Cardiovascular Prevention and Rehabilitation, Zagreb, Croatia 
A total of 230 men undergoing transvesical prostatectomy (TP) for benign prostatic hyperplasia (BPH), with glands estimated to weigh more than 60 g, were analyzed retrospectively and followed-up one year afterthe operation.Age, symptoms, test results, histopathological fmdings, postoperative complications and additional operations were evaluated. Reported dysuric symptoms were obstructive in 47,8%, irritative in 26,5%, and combined in 20,4% of the patients. Hematuria was the main symptom in 5,2% of the patients. BPH was histologicaly determined in 87,0%, incidental carcinoma of the prostate in 4,3%, and various types of prostatitis in 8,7% cases. Complications of TP had a lethal outcome in 2,2%, bleeding requiring further surgical procedure in 6, I %, epididymitis in 4,8%,wound infection in 2,6%, urethral stricture in 0,9%, incontinence in 0,9% and postoperative urinary retention without urethral obstruction in 2,2% of the patients,The most frequent reason for reoperation was bleeding in 66,7% cases. Uretheroscopic measurement of the length of the prostatic urethra, measurement ofthe post-micturition residual volumen and intravenous urography cannot be used as a diagnostic criteria in the selection of patients forTP Digital rectal examination of the prostate and cystoscopy retain their diagnostic significance, while ultrasonography offers the best diagnostic possibilites. Significant drawbacks ofTP are high rates of postoperative bleeding and early postoperative lethality.



Renal carcinoma: An epidemiological assessment in two periods of time. (Trieste, 1985-86 vs 1994-95) 
Bussani R, Pavletić N, Silvestri F 
Institute of Pathological Anatomy – University of Trieste, Italy 
Renal cell carcinoma (RCC) is more frequently encountered in older patients and results in significant mortality.The relatively poor outcome may be due to frequent late clinical presentation and resistance of the tumour to the most current forms of adjuvant therapy.The poor prognosis of RCC underlines the importance of determinating the real incidence of the lesion and the necessity for finding some useful prognostic parameters.The purposes of our study are to identify incidence patterns of RCC over two periods of time in the province ofTrieste, and to evaluate the biological evolution of these lesions. One hundred-fourteen new cases of RCC were histologically diagnosed among 29,323 biopsies and 5,656 autopsies performed between I 985-86 at the Institute of Pathological Anatomy of the University ofTrieste. In the period 1994-95, among 36,200 biopsies and 4,600 autopsies, a histological diagnosis of RCC has been made in 99 cases.The average annual incidence was I 3.6 in males and 8.2 in females in the first period oftime, versus I 6.3 and 5.5 in the two sexes respectively in the second period of time. A RCC has been diagnosed only at autopsy in 56 out of the I I 4 cases evidenced between I 985-86 and in 45 out of 99 cases diagnosed between 1994-95. In both periods of time age specific rates of RCC (bioptic and at autopsy diagnosis) increased proportionally with age, reaching the peak incidence in subjects aged 75 and over. On the contrary age specific rates of RCC clinically discovered during life increased only until the 65-74 decade in both periods examined.0ut ofthe 58 patients with RCC diagnosed during life between I 985-86, 42% died from the renal tumour, 4% decreased with the tumour, 27% decreased without signs of the neoplasm and 27% are alive and well. Out of the 54 patients with RCC diagnosed during life between I 994-95, only I 3% died from renal cancer. As for the RCC evidenced at autopsy, about 40% of the patients died from their tumour in both periods of time. Ninety percent of the patients pathologically staged as T I -T2 healed, versus 43% of those stagedT3 and more.The 5-year adjusted survival rate for patients with RCC graded as G I -G2 was 78%, while the survival curves of subjects with RCC graded as G3-G4 was 85% within the fifth year of follow-up.The results from our study indicate that up to today about 50% of RCC are evidenced only at autopsy.Tumour staging seems to be a significant prognostic factor, while nuclear grade was found to be a non important criterion when considering the outcome of patients with RCC.



Papillary renal carcinoma (clear cell type) arising in the excretory urothelial district. A very unusual feature. Morphohistopathological findings 
Bussani R, Pellegrino M, Silvestri F 
Institute of Pathological Anatomy, University of Trieste, Italy 
Clinical features  A 37-year-old man was admitted to the hospital for gross hematuria and frequency The cystoscopy was negative. An abdominal computed tomografic scan discovered a mass arising in the calycopielic district ofthe right kidney. The clinical impression was for a transitional carcinoma ofthe escretory tract. A nefroureterectomy was performed. 
Gross pathology  The kidney weighed 178 grams, and its dimensions were I 2×6.5×3.5 cm; the resected ureter measured 7.5 cm.The intermediate calyx and a part of the renal pelvis were flfed by a white-greyish slightly papillary tumour. Another small roundish white mass was evident in the subcalyceal district, without apparent continuity with the greater lesion: No lesions in the renal cortex and medulla or in the ureteral district were evident.
Microscopic pathology  The tumour was composed of solid sheets of clear cells interspersed with abundant thin-walled blood vessels; sometimes abortive papillae and glomeruloid structures were evident.The tumour cells exhibited diffuse PAS-positivity and reacted with cytokeratin AE I , AE3, vimentin and Ulex-Europeus, whereas there was not positive staining for CEA and S I OO.The tumour macroscopically resembling a transitional carcinoma but characterised by microscopic and immunohistochemical features typical of a clear cell renal carcinoma could be considered as a sort of hamartomatous lesion.



Correlation between vimentin expression and nuclear grade in renal cell carcinoma 
Kopjar A, Dimanovski J, Stanić G, Krušlin B, Belicza M 
Department of Urology University Hospital “Sestre milosrdnice”, Zagreb, Croatia 
Department of Clinical Pathology `Zjudevit Jurak” Clinical Hospital “Sestre milosrdnice”, Zagreb Croatia 
Aim To analyze the correlation between the expression of vimentin and nuclear grade of renal cell carcinoma (RCC). 
Methods  In this study, tumor tissue from 20 patients was analyzed. There were 7 male and I 3 female patients with RCC conf ned to the kidney (pT I , pT2), ranging in age from 36-80 years (mean 60,6 years).Thirteen tumors measured 5 cm in diameter and 7 were <5 cm.Two tumors were papillary and I 8 showed non-papillary pattern. Six tumors were composed of granular cells and I 4 of clear cells. Formalin fixed, paraffin-embedded tumortissue was cut at 5 Nm, deparaffnized, and stained with a monoclonal antibody to vimentin (Dako, Danemark).The intensity of immunostaining was graded semiquantitatively and denoted as (-) for no staining; (+) for weak; (++) for moderate; and (+++) for strong reaction. Nuclear grade was determined according to Fuhrman et al. Am J Surg Pathol I 982;6:655. 
Results There were 4 tumors with nuclear grade I , 3 with grade 2, I I with grade 3, and 2 with nuclear grade 4. Histopathological examination showed vimentin expression in eight cases (40.0%), while I 2 tumors (60.0%) were negative. Positive reaction for vimentin was observed in all four tumors grade I , none of three tumors grade 2, four of I I tumors with grade 3, and in none of 2 tumors with nuclear grade 4. In our study there was no correlation between the expression of vimentin and nuclear grade of RCC. 
Conclusion  Our study shows a lack of correlation between the expression of vimentin and nuclear grade in renal cell carcinomas. Further studies on a larger series of tumors are obviously needed. 



The influence of pH on Ki-67 antigen retrieval in the renal cell carcinoma 
Mašera A, Staniša O, Ovčak Z 
Institute of Pathology Medical Faculty, Ljubljana, Slovenia 
Introduction Heat based antigen retrieval (HBAR) method using different retrieval solutions is evaluated by immunohistochemical demonstration of Ki-67 antigen in the renal cell carcinoma. In various tissues the optimal conditions forthe antigen retrieval (AR) should be determined by HBAR in solutions with different pH values (1,2). 
Material and methods  Tissue sections from 20 tumor specimens were treated for I 5 min at 97 C in the laboratory microwave oven.Three retrieval solutions were used: 0,05 M citrate buffer pH 6,0; 5mM EDTA/ NaOH pH 8,0 and Target Retrieval Solution (TRS) pH 9,9 DAKO. The automated labeled streptavidin biotin-DAB reaction were performed on the DAKOTechMate 500.Working dilutions ofthe monoclonal antibody MIB-I (DIANOVA) were 1:150, 1:300, 1:600, respectively. 
Results Optimal immunohistochemical reaction was achieved perForming HBAR in citrate buffer with antibody dilution I:150. Using the EDTA/NaOH and TRS the best results were obtained by antibody dilution I :600. Morphometric analysis showed significant higher number of positive nuclei of tumor cells after HBAR in EDTA/NaOH orTRS comparing to the HBAR in citrate buffer. 
Conclusions Ki-67 antigen couldn’t be demonstrated in formalin fixed-parafn embedded specimens,therefore AR method has to be used. In renal cell carcinoma we found the following conditions yielding the best results:HBAR in EDTA/NaOH pH 8,0 andTRS pH 9,9, respectively.The addition of EDTA as a chelating agent for calcium ions enhances the results of HBAR (3). 
Literature 1) Shi S-R et al., J Histochem Cytochem I 997; 45:327. 
2) Taylor CR et al., Hum Pathol I 994; 3:263 
3) Morgan JM et al., J Pathol I 997; I 82:233



Use of immunohistochemistry in the diagnosis of renal angiomyolipoma 
Mašera A, Zidar N, Ovčak Z 
Institute of Pathology, Medical Faculty, Ljubljana, Slovenia 
Introduction  Renal angiomyolipoma (AML) is a rare benign tumor of mixed mesenchimal origin.The diagnosis is based on the recognition of the three components – mature adipose tissue, smooth muscle cells, (which can occasionally be polymorphic) and characteristic blood vessels with thickened walls, but without elastic lamellae. However, all three components are not always present, especially in small biopsy samples. In such cases AML can be misinterpreted for a sarcoma. 
Aim To determine whether immunohistochemistry might be helpful in distinguishing AML from other mesenchymal tumors especially tumors of the smooth muscle origin. 
Methods  Twenty cases ofAML were stained immunohistochemically with antibodies (Ab) against vimentin, smooth muscle actin, desmin, HMB-45 and NKI/C3. Control group consisted of leiomyoma, leiomyosarcoma and lipoma (five cases in each group). 
Results  Afl cases of AML stained diffusely with Ab against vimentin, smooth muscle actin, desmin and NKI/ C3, and only focally with Ab against HMB-45. Leiomyomas and leiomyosarcomas stained with Ab against vimentin, desmin and actin, but not with Ab against NKI/c3 and HMB-45. Lipomas did not stain with any of the Ab used. 
Discussion  The results of our study indicate that immunohistochemistry can be useful in the diagnosis of AML. Staining with anti HMB-45 and NKI/C3 distinguishes AML from benign and malignant smooth muscle cell tumors, and it might be useful in small biopsy samples in which all three components ofAML are not present. Finally, our study showed that NKI/C3 stains smooth muscle cells ofAML diffusely and not only focally as HMB-45 and,therefore, should be regarded as a better markerfor AML than HMB-45. 



Congenital malformations of genitourinary system in the I 0-years periods: An autopsy study 
Kos M, Ilić-Forko D, Babić M, Nola M, Mayer D, Tabain I, Jukić S 
Department of Gynaecological and Perinatal Pathology, Medical School University of Zagreb, Zagreb, Croatia 
Aim To determine the incidence and types of urogenital malformations on our autopsy material in period from I 988- I 997. 
Method  Retrospective analysis of autopsy records and paraff n sections when available. 
Results  7672 autopsies of spontanously or medically aborted fetuses, stillborn and liveborn infants that died in the neonatal period were perFormed at our Department. In 17. I % ( I 32 cases), a malformation of urinary/genital system was diagnosed. Bilateral renal agenesis was diagnosed in I 6.6% (22/ I 32); in 68. I 8% it was isolated, in 3 I .8% combined with other malformations, mostly anal and rectal (57, I 4%). Unilateral renal agenesis was diagnosed in 9.8% ( I 3/ I 32). It was unexpected and isolated in I 5.4% and combined with other malformations in 84.6% cases. Renal hypoplasia was diagnosed in 6.8 I % (9/ I 32) of cases; it was an isolated f nding (except for pulmonary hypoplasia) in I I.I % while it was combined with other malformations in 88.8%.Anomalies of position and form were noted in I 7.4% (23/ I 32); in 73,9% the shape was anomalous (94. I % were kidneys fused at midline) while anomaly of position was diagnosed in 26. I % (6/23). Fused kidneys were an isolated malformation in 30.43% (7/23 cases). Renal cystic disease was found in 23.48% (3 I / I 32); dysplastic kidneys comprised 58.06%;ADPKD 9.67%,ARPKD and renal cortical microcysts 6.45` o each and in I 6. I 3% the type of the cystic disease could not be determined. Renal cystic disease was an isolated f nding in 29.03%, and in 70.96% it was combined with other malformations (in 50% those of CNS). In 9.09% of renal cystic disease combined with other anomalies (2/22), Meckel Gruber syndrome was diagnosed. Prune belly syndrome was diagnosed in 6.8 I % (9/ I 32); pathological changes of ureters (stenosis, duplications, dilatations with consequent hydronephrosis) in 12.9% ( I 7/ I 32) and malformations of the bladder and urogenital sinus 2.3% (3/ I 32) of cases. Isolated anomalies of the genital system were diagnosed in 4.5% (6/ I 32) of the cases. 
Conclusion  The results of this study differ somewhat from the results of other authors, mostly from Western Europe and American continent. It remains to be clarified whetherthe differences are due exclusively to ethnic causes or to different approaches in material evaluation. 



Mast cell in testicufar biopsies of infertile men 
Banek Lj,  Schulze W Goluža T, Hittmair A, Pezerović-Panijan R, Ježek D 
Medical Paculty Uniu of Zagreb, Zagreb, Croatia 
Mast cells of the human testis are occasionally observed within the gonadal interstitium. In some cases of idiopathic male infertility, these cells are known to increase in number. Since there is a lack of data on mast cells in the cases of male infertility, the aim of our study was: a) to characterize these cells according to their location within the testis, b) to determine their number and volume and c) to correlate this data with the status of spermatogenesis (evaluated by the modified )ohnsen score /1-10/).Testicular biopsies (Epon embedded) were divided into the control and infertile group.The blocks were serially cut in order to obtain semithin and ultrathin sections. Semithfn sections were stained with I % toluidine blue and observedlmeasured underthe binocular light microscope (Carl Zeiss).Within the biopsy, mast cells could easily be recognized by their oval (or a little bit elongated) shape and typicai numerous dark-stained granules.The cells were found on two characteristic locations; a) in the vicinity of seminiferous tubules or within the lamina propria itself (“peritubular-” mast cells) and b) in the proximity of blood vessels and Leydig cells of the interstitium (“interstitial” mast cells). Stereological analysis indicated a signifcantly increased number of mast cells in the group of infertile patients when compared to control biopsies.A close association between the low score and the increased number of mast cells was found.The data in our study indicates a significant involvement of mast cells in the male infertility. 
Literature: Hedger MF Rev Reprod (1997), 2:38 Yamamoto M et al. Acta Urol Japon (1 994), 40:541 NagaiT et al. Fertility Sterility (1992), 57: 1331 Hashimoto J et al. Urologia Internat (1988) 43:129 Agarwal S et al. Int J Fertil (1 987) 32:283 Haider SG et al. Andrologia (1 985) 17:532



Expression of KiM I P, testosterone and Inhibin in testes of infertile men 
Ježek D Lebo A, Hittmair A, Rogatsch H, Schulze W 
Medical Faculty Univ, of Zagreb, Zagreb, Croatia 
Within the testis, various cell populations interact by numerous paracrine factors.This is especially true for Sertoli and Leydig cells. (n addition, recent studies on testicular macrophages have shown that these cells, situated within the testis interstitium, could affect testosterone production of Leydig cells. Therefore, the aim of our study was to investigate the expression of KiM I P I (macrophage marker), testosterone and inhibin in testicular biopsies of infertile men.Testicular biopsies were divided into 2 groups: 8 control biopsies and 36 biopsies that displayed various degrees of hypospermatogenesis.Volume of each analyzed testis was determined by ultrasound or orchidometer. Spermatogenesis was evaluated using a modified Johnsen score ( I – I 0) (De Kretser and Holstein, I 976). Cells positive to KiM I P I , testosterone and alfa-subunit of inhibin were measured by the Weibel multipurpose system with 42 test points.Testis of infertile patients displayed a significantly lower testicular volume and score when compared to controls. Morphometric (stereological analysis) showed a significant increase in number and volume of macrophages, testosterone-producing cells (Leydig cails) and inhibin-producing cells (Sertoli and some Leydig cells) per mm3 of the testicular tissue when compared to control values.When the same variables were analyzed per entire organ (absolute number and volume of positive cells identified by immunohistochemistry) no signifcant difference between the two groups could be established (except for number of inhibin-positive Leydig cells which were significantly increased in the group of infertile patients). A significant correlation was found between macrophages, testosterone- and inhibin-producing cells,testis volume and score. Moreover, a positive association between the expression of inhibin, testosterone and macrophages was also found. This data suggests that Sertoli cells, Leydig cells and macrophages could act as a paracrine unit within the testis.



Immunohistochemistry of spermatocytic seminoma 
Vodovnik A, Mašera A, Ovčak Z 
Institute of Pathology, Medical Faculty Ljubljana, Slovenia 
Aim To evaluate a pattern of immunohistochemical reactivity in the spermatocytic seminoma. 
Introduction  Spermatocytic seminoma (SS) is a rare tumor arising in testis only. Immunohistochemistry of SS was rarely investigated so far, exposing discrepancies in cytokeratin and NSE positivity of tumor cells (1). 
Material and methods Three cases of SS were studied. Patients were 51 ,61 , and 63-year-old. In all patients an orchiectomy was perFormed. Immunohistochemical reaction by streptavidin-biotin complex method on alpha-fetoprotein, hCG, wide spectrum keratin, cytokeratins 13, 17, 18, 19 and 20, vimentin, LCA, EMA, CEA, PLAP NSE, S-100 protein, chromogranin A, synaptophysin, Ki-67, and PCNA was performed. 
Results  Tumor cells in all three cases showed positive immunohistochemical reactions on NSE, vimentin, PCNA, and Ki-67. PCNA and Ki-67 immunopositivity was strong to moderate in the nuclei of tumor cells. Cytoplasmic positivity of NSE and vimentin was moderate in all tumor cells. No positive reactions on PLAP and cytokeratins were found. Other immunohistochemical markers revealed negative results as well. 
Conclusions  Recentfy,the PLAP and NSE immunoreactivity in classic seminoma was claimed by some authors as exclusive ( I ). According to our findings, the usefulness of NSE as a discriminator between classic seminoma and SS seems to be questionable. On the ground of wide array of cytokeratins used in our study, we tend to deem these markers as helpful in the differential diagnosis. 
Literature. I . Cummings OW et al., Hum Pathoi 1994; 25:54.



Carcinoma of the penis – A case report 
Lenz D, Čačinović V 
Našice District Hospital, Našice, Hrvatska 
We report a patient 52-years of age who presented with undefined complaints in the region of the glans of the penis. Local finding suggested phymosis. Clinical examination revealed small induration measuring up to I cm while inguinal lymph nodes were enlarged.The patient refused surgery and was seen 8 months later with advanced disease. Total penectomy with inguinal lymphadenectomy was perFormed. Pathohistological analysis revealed a tumor measuring up to 6,5 cm. Histological finding was well-differentiated squamous cell carcinoma infiltrating urethra. The patient is at present alive and without recurrence. 



CYTOLOGY AND QUANTITATIVE CYTOMORPHOLOGY IN NEPHROGENIC ADENOMA 
Wiener H G, Remkes G W Mian C, Pycha A, Susani M 
Departments of Clinical Pathology and Urology Uniu Vienna, Austria 
Predominantly found in transplant patients the incidence of nephrogenic adenomas increases. Although considered a metaplastic lesion of the transitional epithelium, malignant transformation cannot be excluded.The aim of the presented study was to describe the value of cytology and quantitative cytomorphology in the follow up of these lesions. 
Methods  Seven patients with primarily transurethrally resected, histologically proven, nephrogenic adenomas were followed for two years. In summary, I 5 voided urines and bladder washings were available, Cytological diagnosis was done on cytopsin preparations of voided urine and bladder washings. Static image analysis was done on Feuglen stained bladder washings. Based on dual parameter morphometry using the DNA parameter 2cDl and a shape descriptor a risk factor for bladder cancer was estimated. 
Results  Recurrent or persisting lesions were found at five cystoscopic controls of three patients. Cytological evaluation of voided urine reported either cellular features negative for atypia and/or malignancy (n= I 2) orthose summarized as atypia were found (n=3). Static image analysis resulted in estimation of a low (n= I 0) or intermediate (n=2) risk for bladder cancer.Three specimens had to be considered insufficient for image analysis. Recurrent/persisting lesions were removed by transurethral resection and were proven as nephrogenic adenomas. 
Conclusion  The evaluated morphological data stress the benign biological behavior of nephrogenic adenoma and prove cytology and quantitative cytomorphology as most useful parameters in the follow up of these lesions.



Lymphocytic adenohypophysitis simulating pituitary adenoma 
Talan-Hranilović J, Čupić H, Gnjidić Ž, Hat J 
Department of Clinical Patkology `Ljudevit Jurak” “Sestre milosrdnice” University Hospital, Zagreb Clinical Department of Radiology “Sestre milosrdnice” University Hospital, Zagreb Clinical Department of Neurosurgery “Sestre milosrdnice” University Hospital, Zagreb, Croatia 
Lymphocytic adenohypophysitis (LAH) is a rare autoimmune inflammatory process ofthe pituitary gland occurring predominantly during the peripartal period. LAH can cause pituitary expansion and hypopituitarism presenting with features suggestive of a pituitary adenoma.We report a case of a 39-year-old woman who developed bitemporal hemianopsia and artralgic pain in the small joints during the late stage of her second pregnancy. Five months after the delivery visual field defects withdrew, but serum hormone analysis revealed hypothyroidism and increased levels of PRL. Magnetic resonance imaging showed an intrasellar mass with suprasellar extension measuring I 6x I 2mm, that closely resembled pituitary adenoma. A transphenoidal approach was used to remove the patients’ sellar lesion and all of the abnormal tissue was removed. Histopathological examination of the tumor showed an extensive mononuclear infiltration of the adenohypophysis tissue by plasma cells and lymphocytes forming follicles with germinal centers. Neurohypophysis was uninvolved, morphologically normaLThe immunoperoxidase technique showed difFuse presence of immunoreactive prolactin within surviving adenohypophysal cells. Immunohistochemistry revealed CD3 and CD8 positiveT cells at the periphery of lymphatic follicles and in the interfollicular areas. CD 20 positivity was observed in the follicular centers. Diffuse positivity for CD45 was observed throughout the lesion. Our data suggest that the disorder is related to cell-mediated immunity as well as humoral immunity.The question of surgical treatment, of this rare pathological entity, still remains open. 
Literature: 1. Hashimoto K et al. Endocr J ( I 997), 44; 1-1 0 2. IshiharaT et al. Endocr J (1996), 43; 433-40 3. Hartmann I et al. Clin Neuropathol (1 996),15; 234-239 4. Presmann EK et al. J Reprod Med ( I 995), 40; 251-259



Correlation between eosinophil infiltration and hyperactive response to destilled water chalenge In chronic rhinosinuitis 
Kalogjera L, Glumbić I, Zurak K, Baudoin T, Rudnički M, Krušlin B 
Departments of Otorhinolaryngology and Patholog;y “Sestre milosrdnice” 
University Hospital, Zagreb, Croatia 
It is well documented that non-isotonic aerosols induce hyper-reactive response in nasal mucosa, which can be expressed as the increase in nasal resistance, quantity of nasal secretion or concentration of inflammatory mediators in nasal lavage fluid.The relationship between non specific response to different stimuli and mediators to eosinophil proteins concentration in tissue and nasal lavage was studied on different nasal provocation models, but the relationship between eosinophil inf Itration in sinonasal mucosa and nasal responsiveness to distilled water challenge has not been elucidated. 
This study was designed to analyze the relationship between eosinophil (eo) infiltration in sinus mucosa of the patients suffering from chronic rhinosinuitis and their response to distilled water provocation. 
Material and methods. 
A group of I 5 patients ( I 0 male, 5 female) were provoked with the inhalation of I 0 ccm of destilled waterTheir response to such a provocation was estimated through the succeeding increase in nasal resistance, Nasal resistance was measured by active anterior rhinomanometry with the computer coupled rhinomanometerAtmos PC200 (Atmos, Lenzkirch, BRD). Basal resistance was calculated as the mean ofthree measurements before provocation, and following the provocation with simultaneous inhalation of I 0 ccm of distilled water; 3 measurements were performed within fve minutes.All patients were operated in the period of one month after nasal provocation. Samples of nasal polyps, and mucosa from the ethmoid and maxillary sinuses was fixed in the 10% neutral formalin, embedded in paraffine and stained with hemalaun eosine. Eo and neutrophil leukocyte inf Itration was analyzed in I 0 high power fields (400x) per sample and patients were divided in three groups according to the level of eo infiltration in at least one sample: I .) less than I 0 eo per HPF; 2.) I 0 to 20 eo per HPF ; and 3.) more than 20 eo per HPF Neutrophil infiltration was not recorded in any ofthe samples. Statistical analysis ofthe data was perFormed by using the Mann Whitney Wilcoxon test. 
Results In 6 patients eo infiltration exceedect 20 eo per HPF (group 3.); I patient had eo inf Itration of I 0 to 20 eo per HPF; and 8 patients had less than I 0 eo per HPF. One patient was excluded from the I st group, as he had no evidence of sinus disease and was operated for hypertrophic rhinitis only As the 2nd group was too small for statistical analysis, the patients from the 2nd group were merged with the 3rd group. Mean basal resistance for the 2nd and 3rd group was 0,25 Pa/cm3s- I and for the I st group 0, I 8 Pa/cm3s- I . Mean nasal resistance following provocation was 0,37 Pa/cm3s- ( for the 2nd and 3rd group and 0, I 9 Pa/ cm3s- I for the I st group. Mean increase in 2ndand 3rd group was 34 % and for the I st group 26% (insignificant). Our results confirm previous results of Klementsson ( I 99 I ), which have not demonstrated correlation between ECP levels in nasal lavage and hyper-reactive response to metacholine provocation considering increased secretion to provocation.The basal and provoked resistance’s in 2nd and 3rd group are significantly higher than in the I st group. 
Conclusions Considering these findings, we can conclude that that patients with chronic sinuitis and eo inf Itration in the sinus mucosa of more than 10 eo per HPF have higher nasal resistance than patients with chronic sinuitis without marked eosinophil infiltration, but their responsiveness to distilled water provocation is not significantly higher.



FEMALE ADNEXAL TUMOR 0F PROBABLE WOLFFIAN ORIGIN 
Tomić S, Alujević A, Pešutić-Pisac V Vuković S 
Clinical Hospital Split, Split Croatia 
We report a case of a 39-year old women with adnexal tumor, which arose within the leaves of the broad ligament, connected to the left ovary and the Fallopian tube.The patient remains alive, without evidence of the disease, one year after left salpingoophorectomy, without subsequent adjuvant therapy. Histologically, the tumor cells were arranged in three different patterns: closely packed tubules, solid and sieve like. Cells had uniform round and oval nuclei and mitoses were rare. Immunohistochemical profile of the tumor was similar to that of Wolffian duct remnants. They co-expressed cytokeratin and vimentin and lacked EMA reactivity, in contrast to tumors of M,Ilerian origin which usually express EMA. Although the majority of these tumors are benign, a few cases have shown malignant potentiaLTherefore, such tumors should receive careful follow-up for possible recurrence and /or metastasis. 
Literature: 1) Scully RE. Female adnexal tumor of probable Wolff an origin. Cancer I 973, 3 I :67 I -677. 
2) Daya D. Malignant female adnexal tumor of probableWolffan origin.Arch Path Lab Med I 994, I I 8:3 I 0-3 I 2. 
3) Rahilly MA. Female adnexal tumor of probable Wollfan origin: clinicopathological and immunohistochemical study ofthree cases. Histopathology I 995,26:69-74.



The malignant mixed tumor of the gallblader (Adenosquamous carcinoma with fibrosarcoma). A case report 
Alujević A, Janković S, Družijanić N 
Clinical Hospital Split, Split, Croatia 
A case of adenosquamous carcinoma with fibrosarcoma of the gallblader is described. Adenosquamous carcinoma was characterized by the presence of squamous epithelial and glandular components.Atypical glands were found, among the areas of squamous cell carcinoma, which also contained squamous epithelium with signs of cell atypia.The sarcomatous component was presented by interwoven fascicles of spindle and fusiform, poorly differentiated ceIIs.The clinical and X-ray examinations of the patients revealed a chronic cholecystitis. 
References: I ) Suster S. Adenosquamous carcinoma of the gallblader with spindle cell features. Histopathology I 987, I I :209-2 I 4 
2) Kuaskina G. Adenosquamous cancer of the gallblader. Arkh Patol I 980, 42:57-59



LOCAL IMMUNE RESPONSE IN DYSPLASTIC EPITHELIAL LESIONS IN FLAT BOWEL MUCOSA 
Radović S, Selak I, Bratović I 
Institute of Pathology, Sarajevo University School of Medicine 
Gastroenterology Department, Sarajevo University Hospital Center, Sarajevo, Bosnia and Herzegovina. 
Aim Dysplastic changes of flat bowel mucosa have been investigated immunohistologicaly to determine the cellular immune response.The relationship between the phenotype and localization of the lymphoreticular cells and prognosis of the precancerosis has been assessed. 
Methods  Biopsy specimens from 170 patients were examined: 74 classifed as inflammatory-regenerative and 96 as dysplastfc (38 miid, 28 moderate and 30 severe dysplasia). On paraffin sectfons 3 monoclonal antibodies against various leukocytic antigens were used. 
Using a semiquantitive method the quantity of mononuklear inflammatory cells was defined, their types, distribution, and altered interaction with epithelial cells. 
Results  CD45R0 T lymphocytes were predominant cells in the infiltrates. Less frequent were CD 68 macrophages. CD20 B lymphocytes within the inflammatory cells was generally Iow.The intensity and composition of the local immune reaction increased proportionally with the intensity of epithelial dysplasia.The lymphoreticular cells were accumulated mainly in the lamina propria. Direct contact between cells of the lymphoreticular infiltrate and dysplastic epithelial cells was found rarely. Even where close contact between lymphocytes and macrophages and dysplastic cells was found, no signs of direct lytic effects on the dysplastic cells were observed. It seems that dysplastic epithelial lesions do not provoke a signifcant defence immune response, but the local immune reaction might be a part of etiologically different bowel mucosa inflammation which may lead to lesions. 
Conclusion  Our study proved that specific local immune response in dysplastic epithelial lesions in flat bowel mucosa cannot be used as important prognostic factor.



Pathology and pathogenesis of African swine fever.The role of the monokines 
Gomez-Villamandos J C, Carrasco L, Salguero FJ, Mekonnen T, Ruiz-Villamor E, Bautista MJ, Sierra MA 
Dept. Anatomia Patologica Facultad de Veterinaria Cordoba, Spain 
African swine fever (ASF) is perhaps the most controversial and interesting model of a swine disease with monocyte macrophage as the main target cell for virus replication. The aim of this paper is to show the relationship, in space and time, between macrophage alterations, including pro-inflammatory monokines expression, with vascular lesions, lymphopenia and virus replication in non-MPS cells. A pathogenic mechanism is proposed for the disease. Large White x Landrace cross-bred pigs of both sexses were used for this study The animals were inoculated, by the intramuscular route, with 105 50% HAD50 of the E70 ASF-virus isolate. Tissue samples were routinaly processed for electron microscope. Immunohistochemical study to detect IL-Ia and TNF-a expression was carried out.The pathogenesis of hemorrage is related to the stimulating of endothelial cells and the subsequent loss of the endothelial lining, virus replication takes place in these cells in the f nal stage of the disease. Loss of endothelium allows platelets to adhere to the basement membrane aggravating DIC, which commences prior to the appearance of this lesion. ASF-infected swine suffers severe lymphopenia, that suddenly appears during the middle phase of ASF Our studies have shown that the destruction of lymphocytes in lymphoid organs and in the cellular infiltrate is due to apoptosis. Immunohistochemical study revealed an increased level of IL-Ia and TNF-a expression in these animals. Our finding constitutes the experimental evidence underlying the hypothesis that monokines released by activated macrophages and/or infected macrophages play a major role in the pathogenesis of acute ASFThis study was supported by D.G.E.S. (Spain) grant No. PB95- 158.



Pathological studies on ovine caseous lymphadenitis in Turkey 
Sayin Fatma 
University of Adnan Menderes, Faculty of Veterinary Medicine, Department of Pathology Turkey 
Caseous lymphadenitis caused by Corynebacterium pseudotuberculosis in small ruminants has been reported in many countries of the world (4), includingTurkey For some years, this disease in sheep has become the most important problem of Government farms in Turkey, by reducing both meat and wool production, and by decreasing fertility (1,2,3).This study is aimed to examine the pathology of this topic.The disease was inspected in sheep, belonging to Ceylanpnar farm in southern part ofTurkey, in March 1997. Materials were taken from the lesional regions, and fxed in 10 % neutral formalin solution. After paraffin-embedding, 5mm sections were prepared and stained with hematoxylin- eosin for histological examination. Lesions of caseous lymphadenitis were seen in 23 of 75 clinically suspected animals. Grossly, the typical finding was the abscess formation. They were seen as capsulated nodular lesions in different size and consistency The small, new abscesses were of a pasty consistency while the large, older abscesses were dry and solid.The color of necrotic abscess material was greenish-yellow; there was no odor to them, and cut surFace of some showed a concentric Iamellation.The abscesses in the lung were measured 1-4 cm in diameter, whereas those in liver were 0.5-3 cm, and those in lymph nodes were ranged from 0,5 cm to almost as large as the organ itself. Gross abscesses were seen generally on mandibular (10 cases) and mediastinal (8 cases) lymph nodes, followed by scapular, femoral and bronchial lymph node abscesses in decreasing numbers. Lesions were only seen in lung (7 cases) and liver (2 cases) from the visceral organs. Microscopically, the necrotic areas in the center of the abscess consisted of destroyed neutrophylic leucocytes. It was surrounded by a layer of mainly macrophages and some mononuclear ceIIs.The capsule was made of fibrous tissues. Morphologic and immunohistochemical studies are still going on. 
Literature: 1. Batey, R.G.(1986).AustVet J. 63(4) : 131. 
2. Paton, M.W et all. (1988).AustVet J. 65(4) : 117 
3. Paton, M.VV et all. (1994). AustVet J. 7 1(2) : 47. 4. Pepin, M. et all.(1993). Am J Vet Res. 54(6) : 873.



First report of Peste des Petits Ruminants in lambs inTurkey: Patomorphological and immunohistological feature 
Alcigir, Gunay*; Yural Atalay Sevil*; Toplu, Nihat** 
*University of Ankara, Faculty of Veterinary Medicine, Department of Pathology Ankara, Turkey 
** University of Adnan Menderes, Faculty of Veterinary Medicine, Department of Pathology, Turkey 
Three dead and one live animal were chosen from a flock consisting of 6-7 month old lambs with 210 lambs from the district ofAnkara in 1993 for the determination of an illness showing symptoms of fever, cough, diarrhea and had 30 % mortality. By using pathologic and immunhistologic methods the disease was diagnosed as Peste des Petits Ruminants. Grossly, light necrotic and erosive stomatitis and few erosions in abomasum were identified, 1-2 mm diameter white-gray foci in liver, hyperemia and edema in intestines were seen. In cranial and caudal lobes of lungs there were 2-5 cm diameter red sometimes gray hepatization areas. Histopathological signs were characteristic and there were pathognomic multinuclear syncytial giant cells and intracytoplasmic inclusion bodies in mucosa of tongue and in abomasal glandular cells. Syncytial giant cells with few nuclei were detected in mediastinal and mesenterial lymph nodes. Although there was small and irregular focal necrosis in liver, the main sign was the finding of syncytial giant cells with 3-5 or more nuclei and some intranuclear inclusion bodies. The most evident signs in diagnosis of the disease were observed in lungs. Mainly puruVent, sometimes desquamative catarrhal bronchopneumonia and necrotic areas were apparent. Interalveoler intersititiel pneumonia was present between these areas. Some bronchial epithelial cells were hyperplastic. In addition to these characteristic signs, the diagnosis was supported by the detection of multinuclear syncytial giant cells in alveoler lumina and intracytoplasmic inclusion bodies in bronchi and bronchial glandular cells and sometimes pneumocytes. On the other hand, reddish stained diffuse or granular viral antigens were detected by Avidin-Biotin Complex (ABC) method using rabbit anti-rinderpest hyperimmune serax within the lungs in bronchi and bronchioler epithelial cells, bronchial glandular cells, oral mucosal cells, hepatocyts, particularly reticulum and macrophage cells in lymph nodes. The macroscopic and microscopic structures of the lesions were in accordance with the literature data (1,2,3), except the finding of liver lesions. 



Swine leptospirosis caused by Leptospira interrogans (serologic variant Icterohaemorrhagiae) 
Modrić Z, Sabočanec R, Turk N, Artuković B, Slunjski A, Čuljak K 
Department of General Pathology and Pathological Morphology 
Faculty of Veterinary Medicine of the University of Zagreb, Zagreb, Croatia 
In one of nine piggeries of one pig herd, 18 days after sows farrowed litters some of piglets were severely jaundiced and poor viable were died. Five of this sick piglets had titres for leptospires but twelve sows ofthem had not.Total of fme piglets which were brought to our clinic due to determine diagnosis, had aglutinins for leptospires and pathological changes in one died and two sacrified piglets were severe jaundice, liver and kidney parenchymatous degeneration and splenomegaly. From sacrified piglets kidneys a leptospires serogroup icterohaemorrhagiae were isolated by cultivation in Korthof’s media. 
In two piglets a streptomycin was administered during four days what was resulted with failure in isolation of leptospires from theirs kidneys. 
The obtained facts indicates that the source of infection for piglets in piggeries were very probably rats.



The comparison of histopathological, immunoperoxidase and immunofluorescence techniques in the diagnosis of rabies in dogs 
Vural Atalay Sevil 
University of Ankara, Faculty of Yeterinary Medicine, Department of Pathology Ankara, Turkey 
The effectiveness of the immunoperoxidase staining method for the diagnosis of rabies was investigated in this study, compared to immunofluorescence and histopathological methods.The tissues (cornu ammonis, cerebellum, thalamus, colliculus rostralis, pons cerebri, nucleus caudatus, medulla oblangata, spinal cord, Gasserian ganglion, parotid gland, submandibular salivary gland, adrenal gland, retina, cornea, trachea, stomach, intestine, mouth, nose, ear, and urinary bladder) of 10 rabies dogs were examined.The samples were fixed in 10 % bufferformaline and were blocked in paraffin and sectioned at 4-5 mm.All sections stained with hematoxylin and eosin and Avidin-Biotin Complex (ABC) and indirect immunoflourescent test using rabbit anti-rabies hyperimmune sera*.The nonspecific inflammatory reactions and degenerative changes detected by histopathologic methods regarding rabies were observed in the central nervous system, Gasserian ganglion, parotid gland, adrenal gland, and submandibular salivary gland.The Negri bodies were seen in the cornu ammonis, cerebellum, thalamus, nucleus caudatus, colliculus rostralis, and also, in the Gasserian ganglion. The immunoperoxidase method, showed extra and intra cellular rabies virus antigen and/orthe inclusion bodies in the entire central nervous system and Gasserian ganglion. In addition, immunopositive rabies virus antigens were also identified in retina, submandibular salivary gland, cornea, parotid gland, trachea, stomach, intestine, ear, mouth, nose, urinary bladder, and adrenal gland. The immunofluorescence method, revealed rabies virus antigens in the central nervous system, Gasserian ganglion, retina, parotid gland, ear and mouth. In this study, equally positive results were obtained from the avidin-biotin peroxidase and indirect immunofluorescence method. Although both methods could be used conveniently in formalin fixed and paraffin embedded blocks, the immunoperoxidase method appeared to be more advantageous for use in pathology laboratories since this method permitted usage of high titered sera as well as usage of more simplified microscopes and storage of stained slides for a long time. This immunoperoxidase method has been used forthe first time forthe diagnosis of rabies in our country.The microscopic structures of the lesions were in accordance with the literature data (1,2,3,4). 
Literature: 1. Balachandran,A. et all. (1994).Vet Pathol. 31: 93. 
2. Das, S.K. et all. (1985). J Anim Sci. 55 : 979. 
3. Goldwasser, R.A. et all. (1958). Proc Soc Exp Biol Med. 98 : 219. 
4. Sinchaisri,T et all. (1992). J Vet Med Sci. 54 : 409. *Provided Centre of Expertise for Rabies Agriculture Canada Animal Disease Research Institute Nepean, Ontario. 
1 Brown, C.C et all. (1994).Vet Pathol. 31: 194 
2. Brown, C.C et all. (199 1).Vet Pathol. 28 : 166. 
3. Obi,TU. et all. (1983). ZbI Vet Med B. 30 : 751. Provided Animal Disease Research and Control Institute, Ankara,Turkey.



Implications of pulmonary intravascular macrophages in pulmonary processes 
Carrasco L, Gomez-Villamandos JC, Bautista MJ, Sanchez-Mascaraque C, Salguero FJ, Millan Y, Quezada M 
Dpto Anatomia y Anatomia Patologica Comparadas, Cordoba, Spain 
In recent years the study of the pulmonary physiology and the pathogeny of pneumonic process of certain species of mammals has suffered important changes, due to the description of a resident population of cells to the Mononuclear Phagocyte System, the pulmonary intravascular macrophages (PIMs), which form a resident population in the pulmonary capillaries of certain animals. The existence of PIMs was first reported in the early 70’s, but they were not studied in any great depth until the late 80’s, when several studies of their features and functional significance were made in goat, bovine, sheep, deer, swine, horse and rabitts.There is evidence that, like macrophage from other sources, PIMs have ample secretory capabilities and influence the pulmonary microvascular physiology through release of proinflamatory mediators that can initiate or enhance acute pulmonary inflamation. However there are few studies that have studied the implications of the PIMs in the development of the acute process that characterized some diseases. From this reason we have analyzed the implications of PIMs in animals experimentaly infected with some viruses, such as African Swine Fever, African Horse Sickness, Rabbit Haemorrhagic Disease; and bacterias as Pasteurella haemolytica and characterized by the establishment of an acute pulmonary process. In these infections we have observed that PIMs sufferer and activation processes, from the first time of infection, were associated with different pulmonary pathological changes, such as neutrof I accumulation in septal capillaries, pulmonary endothelial injury, increased microvascular permeability with a consequent interstitial and alveolar edema and microvascular thrombosis; in African Horse Sckiness and Pastereullosis a concomitant pneumonic proccess was found, characterized by the existence of alveolar macrophages activation and fbrin deposits in alveoli.We can conclude that although the existence of PIMs should be benefical to the animal, since they actively participate in blood clearance, interactions with different antigens, subsequent activation is, also, associated with pulmonary pathological changes. 


Incidence and pathological studies on the liver telangiectasis on feedlot cattle 
Atasever, Ayhan*; Vural Atalay, Sevil**; Berkin, Senay** 
University of Erciyes, Faculty of Veterinary Medicine, Department of Pathology Turkey ** University of Ankara, Faculty of Veterinary Medicine, Department of Pathology, Ankara, Turkey 
The livertelangiectasis is a fairly common lesion offeedlot cattle which causes important economic losses (1).This report deals with the gross and microscopic pathology and incidence of telangiectasis. In this study 3470 feedlot cattle slaughtered in Ankara Meat and Fish Company abbatoir were inspected. Relationship of abscesses to telangiectasis was also examined. All of the tissue samples were collected in a few hours following the slaughter.The samples were fxed in 10% buffer formaline and were blocked in paraffin.All the samples were stained with hematoxylin and eosin, Best, Luna,Toluidine blue, van Gieson and Gram stains.Telangiectasis was observed in 82 of 3470 livers (2.1%). Lesions were mostly localized on the parietal surface of the lobus dexter, and occasionally generalized throughout the organ.They were seen grossly as single or multiple (in 2 cases) reddish-brown foci. Each focus was 1to 3 mm in diameter, occasionally larger. Microscopically, they were generally characterized by distortion and necrosis of hepatocytes with dilatation and congestion in sinusoids.The lesions were observed in 74 livers with normal appearances. Multiple abscesses were observed in 6 livers formed far away from the telangiectasic lesions. Lesions of severe distomatosis were observed in 2 cases. The macroscopic and microscopic structures of the lesions were in accordance with the literature data (1,2,3). No correlation was found between telangiectasis lesions and liver abscesses. This is the first evaluation of this subject in Turkey. 
Literature: 1. Getty, R. (1946).Am J Vet Res. 7:437 
2. Jensen, R. et all.(1982).Am J Vet Res. 43 : 1436 
3. Jensen, R. et all.(1947). J A V M A. 110:256.



The differences in pathological picture in pigs succumbed of hog cholera 
Šoštarić B, Lipej Z 
Croatian Veterinary Institute, Zagreb, Croatia 
Hog cholera recently appeared as a major threat to the European pig industry, whereas it has been present in Croatia, as an endemic disease, during a long period of time. During several decades systematic diagnostic proceeding has been carried out at the Department of Pathology of the CroatianVeterinary Institute; and during that time thousands of animals succumbed due to hog cholera were dissected and pathoanatomicaly evaluated. It has been noted that the pathoanatomical picture had changed during that period, and that lesions earlier considered as pathognomonic are not a useful diagnostic value any more, even more, some of them are not present as a rule, or they are rearly found. Recent pathoanatomical fmdings are more specifically described in this paper. 



Immunohistochemical identification of Leptospira pomona antigen in tissue sections 
Juntes P, Lindtner R, Pogačnik M 
University of Ljubljana, Veterinary Faculty, Ljubljana, Slovenia 
Aim Post-mortem diagnostic of leptospirosis in domestic animals is a difficult task. One way how to do it is by using immunohistochemical procedures (1,2,3). The aim of our study was to produce polyclonal antibodies in rabbits which could be used for immunohistochemical identification of Leptospira interrogans serovar pomona in routinely prepared paraffin embedded tissue sections. 
Methods Rabbits were immunised with non-pathogenic serovars of Leptospira pomona using partly modified previously described procedure (3).The culture for immunisation contained 10 8 Microorganisms and was administered intravenously three times.Animals were bled one week after last application. Sera were tested on smears of Leptospira pomona culture and on formalin fxed paraff n embedded tissue of several animal species DAKO En Vision +(tm) System, was used for the visualization of immunohistochemical reaction, with 3-amino-9-ethycarbazole (AEC) as a substrate. Positive antiserum against Leptospira pomona and positive tissue (a gift from dr. Eugenio Scanziani, Italy) were used as a positive control and incubation without specific antibodies as a negative control. 
Results and conclusions  Antisera that we produced gave a strong and clear immunohistochemical reaction on paraffm tissue sections fixed in buffered formalin.With a very sensitive kit for visualization that we used, we were able to detect leptospiral microorganisms even on material that was not properly fixed. Pattern of reaction products produced on culture smears was granular without recognizable leptospiral morphology. It was confirmed that immunostaining is useful for the detection of Leptospira pomona infections in animal tissues.This procedure is especially valuable in cases when microorganisms in tissue are not viable or microbiological isolation is not possible for other reasons. 
References 1) EllisTM et al.AustrVet J I983;60:364-367; 
2) Poonacha KB et aLVet Pathol I993; 30:362-369; 
3) Scanziani E. ResVet Sci 1991; 50:229-232.



Pathomorphology of vibriosis in sea bass (Dicentrarchus labrax) of different ages 
Zrnčić S, Oraić D, Šoštarić B 
Croatian Veterinary Institute, Zagreb , Croatia 
Vibriosis caused by Listonella (Vibrio) anguillarum is one ofthe most devastating diseases in cage cultivated sea bass (Dicentrarchus labrax) in Croatia.This work derives from our observation of different fish farms alongthe coast systematically monitored forfish diseases. Pathomorphology of this bacterial disease in different ages of sea bass is presented in this paper.The affected fish in all observed cases swam slowly to the water surFace, separated from the lot and were reluctant to eat. Pathoanatomically, fish showed skin decoloration, presence of skin erythema on the f n base, around the mouth, on the vent and in more advanced cases ulcerations were noted as well. Internal examination revealed liver, stomach, intestine, fat tissue, gonads and swim bladder hemorrhages and enlarged spleen.The samples of tissue of all macroscopica!!y changed organs were submitted to histopathological examination and the results are presented in this paper. 



Immunopathological response against Trichinella spiralis in mice 
Marinculić A*, Božić F*, Dominis M**, Auslender Ujević V**, Grabarević Ž*, Duraković E* 
*Veterinary Faculty 
** Pliva 
*** Veterinary Institute, Zagreb 
By the first quantitative phenotypic analysis of intestinal intraepithelial lymphocytes (i-IEL) at the early stages of theTspiralis infection, we found that the kinetic of ((T cells in i-IEL corresponded to the kinetics of worm expulsion after infection of C57BL mice with 200 muscle larvae. Infected mice developed signifcant enteropathy, comprising villus atrophy, crypt hyperplasia, goblet cell hyperplasia and a decrease in i-IEL numbers by 14 days post infection (p.i.), when most of the parasites had been expelled. Most murine i-IEL of the gdT cell lineage tend to be cytolytic when activated.We speculated that gdT cells of i-IEL during the early stages of infection recognize and eliminate damaged epithelial cells generated by parasite antigens, simultaneously accelerating the worm expulsion. However, since the rejection ofT.spiralis worms is dose-dependent effect, to assess whether or not i-IEL, particularly gdT cells, respond comparably to different sized T.spiralis infection, we used C57BL mice, orally infected with 50 or 400 viable T.spiralis larvae. We found that the rapid activation of  gdT cells in i-IEL occured much sooner in mice infected with 400 larvae than in mice infected with 50 worms, as early as 1 day p.i. vs 4 day p.i., respectively In addition we found significant elevations of eosinophiles and mastocytes on day 1 p.i. in mice infected with 400 T.spiralis larvae in comparision with mice infected with 50 larvae. In spite their intrinsic cytolytic activity against transformed cells, no corelation between kinetics of  gdBT cells in i-IEL and T.spiralis worm expulsion was observed in mice infected with 400 or 50 Iarvae.These fndings strongly suggest that bothTspiralis infection levels elicits immunodepression by interFerence with the first line defenceT cell function, therefore influence the outcome of experimental T.spiralis gut infection.



B. 
Iatrogenic, experimental and environmental pathology 
Iatrogena, eksperimentalna i patologija okoliša



The influence of thyroid functional modifications on collagen contens of skeletal muscle in the meat chickens 
Curca Dumitru 
Department of Pathophysiology, Faculty of Veterinary Medicine, Bucharest, Romania 
The collagen contents ofthe adult animal organisms is generally constant, but in some physiological states (parturition) and especially in physiopathological ones, marked modifications take place, both quantitatively and qualitatively. Endocrine glands through the hormones they produce, influence both the quantity and the quality of collagen, thyreoptropine is a stimulator mainly for the hyaluronic acid in the ground substance, while thyroxine stimulates the connective tissue in thyroidectomized animals. Experimental research was conducted on three batches of broilers aged 40 days. Batch I. consisting of eight broilers having a weight of 837.50+/- 13.98 g to be brought into a condition of hyperthyroidism, was administred 2 mgTIROTON/ 5kg of feed, recipe 2 1/1in their combined feed that is about 40mg TIROTON per broiler each day. Batch II. consisting of eight broilers having a weight of 677.50 +/- 20.68 g to be brought into a condition of hypothyroidism, was administred 5 g METILTIOURACIUS kg offeed in their combined feed,which means 100 mg METILTIOURACIL per broiler daily. Batch III was the control. After 42 days of feeding, the broilers were slaughtered with a view to dosing the collagen in their muscular system by means of the indirect biochemical metod which aims at the determination of the hydrowyproline contents according to Neuman and Logans techical indications. Broilers whose feed was supplemented withTIROTON showed a statistically insignificant decrease tendency of collagen in the pectoral muscle (P0.05), while broilers in the batch treated with METILTIOURACIL showed a statistically insignificant increase tendency (P0.05). In the thigh muscles, both batches showed an increase tendency, but statistically insignificant (P0.05).The body weight in both batches was smaller (P<0.01 ) as compared to the controLThe histopathologycal examination ofthe thyroid in the broilers treated withTIROTON revealed the flattening of follicles that are full of colloid. In the broilers treated with METILTIOURACIL, the thyroid follicles are unequal, some of them quite large, the walls become thickened, the colloid sometimes having a haemorrhagic aspect owing to their cystization, which characterises the condition of hypothyroidism. 



Pathomorphological picture of Myxidium infection in cultivated charp snouted sparus (Puntazzo puntazzo) 
Oraić D, Zrnčić S, Šoštarić B 
Croatian Veterinary Institute, Zagreb, Croatia 
Charp snouted sparus (Puntazzo puntazzo) is a native inhabitant of the Adriatic sea.This species has not been cultivated in our fish farms until recently.At a certain fish farm, experimental production of charp snouted sparus started during 1997., using imported fry. Seven weeks old fry was put into growing cages, but after two months high mortalities started. At the beginning, affected fsh swam slowly close to the surface separated from the lot and they were emaciated. Pathoanatomically only slight patchy skin discoloration was observed. By internal examination of recently dead and euthanized moribund fish stomach appeared normal, and was filled with usual amount of ingesta, while immediate proximal anatomical structure of intestine was distended by seromucous transparent content.The intestinal wall was thinner and more translucent then normal. Examination of gut mucosa scrapings using light microscopy (400x) disclosed numerous spores of myxosporean parasite Myxidium leei. Histopathological examination of intestines revealed numerous parasitic structures as well, and therefore confirmed the diagnosis of M. leii.



Use of LN3 antibody in the analysis of the biological evolution of myocarditis 
Bassan F, Bergamin C, Bussani R, Pavletic N, Silvestri F 
Institute of Pathological Anatomy – University of Trieste, Italy 
Myocarditis can def nitively be diagnosed only by endomyocardial biopsy. The traditional histology may be used to: 1) diagnose a “Dallas” myocarditis 2) analyse the myocarditis evolution 3)? evaluate the type and grade of endomyocardial fibrosis 4)? evaluate the myocytes (hypertrophy; atrophy; loss of contractile material, etc.) 5) evaluate the aetiology of some infective myocarditis (bacterial, fungal, protozoal, etc.) What is actually needed is not only an accurate histopathological diagnosis of myocarditis, but also to find some criteria that could be used for identifying the “non Dallas” myocarditis and some prognostic determinants for the patients affected. To assess the overall status of”immunological activation” of the heart we think that the antisera that perform correct immunophenotypical characterisation of inflammatory cardiac infiltrates are: CD45R0, CD45RA, CD4, CD8, CD20, LN3, and CD68-PGM-I. Monoclonal antibodies directed against T or B-cell antigens facilitates identification and quantifcation of inflammatory infiltrates. LN3 (HLA DR-expressing cells) seems to be a particularly useful marker for detection of a generalised autoimmunological process affecting the myocardium. Endomyocardial biopsy (EMB) has been perFormed, at our Cardiology department, on eight-hundred thirty patients with suspected myocardial disease of undef ned aetiology from 1978 to 1996. In 60 patients an active myocarditis has been identifed. The first biopsy showed HLA-DR molecules in all these patients. In the follow-up biopsies LN3 expression in lymphocytes, myocytes and in endothelial cells was evident (43%) only in patients with ongoing myocarditis or in those with dilated cardiomyopathy (DCMP) evolution. A retrospective study evidenced, moreover, LN3 expression in EMB of 10 patients with clinical diagnosis of DCMP and traditional histology positive for”borderline” myocarditis. LN3 may be a diagnostic tool for identification of the “non Dallas” myocarditis and a useful marker in the evaluation of prognostic determinants for patients with myocarditis.



Use of microwawe in a histopathology laboratory 
Bassan F, Zambon S, Bussani R, Pavletic N, Silvestri F 
Institute of Pathological Anatomy – University of Trieste, Italy 
Microwaves are very much in use for performing special staining and are also being used for other procedures in Histotechnology. The microwave electrical field that is produced creates friction as it passes through dipolar molecules causing them to reverse themselves 2450 million time/sec. The use of the microwave was firstly introduced in immunohistochemistry applications, but only recently its use extended to the field of histochemistry. Microwaves have been a great property of reinstating immunogenetic cells to sever every cross-linking formed by fixative and proteins (NH2 -COOH). Histochemistry is able to reduce greatly the time of tissue incubation in staining procedures and especially when silver-nitrate impregnation-method is concerned. We found particulatly staining techniques for polysaccharides, neutral mucosubstances and basement membranes demonstration using PAS reaction, for connective and muscle tissue and glial fbres with Azan Mallory techniques. The best result that we got in this field has been the silver-nitrate impregnation for Borrealiae checking in paraffine-embedded tissue sections.We elaborate a special method that clearly underlines Spirochaetes reducing to a minimum the background staining.



Effect of colhicine on redistribution of cell membrane proteins in the rat proximal tubule 
Baus M, Medjugorac-Popovski M, Sabolić I 
Institute for Medical Research and Occupational Health, Zagreb, Croatia 
Epithelial cells along the mammalian nephron and in other tissues establish, maintain, and modify their plasma membrane composition by the process of membrane recycling (Brown D and Stow JL, Physiol. Rev. 1996, 76:245). In general,the recycling of various membrane components between the plasma membrane and intracellular organelles by means of endo- and exocytosis depends on intact microtubules (MT). However, the details of the recycling mechanism for specific plasma membrane proteins are poorly understood.ln the renal proximal tubule (PT) cells,the rate ofthe MTdependent recycling of a particular protein may be related to its localization in the specifc plasma membrane domain (brush-bordervs. basolateral) orto its association with the membrane, e.g. whether a protein is an endo-,transmembrane-, or ecto-protein. Another discriminating factor in this recycling may be the role the specific protein plays in acute or chronic adaptations to the tubular load with the filtered substrate. In this work, we used an MT depolimerizing agent, colchicine, to study the distribution of Na/K-ATPase [a basolateral (BLM) transmembrane protein], vacuolar proton-ATPase [V ATPase; a brush-border membrane (BBM) endo-protein], sodium-phosphate co-transportertype 2 (NaPi-2; a BBM transmembrane protein), and cell adhesion molecule CAM-105 (a BBM ecto-protein) in the rat kidney PT cells. Rats were injected i.p. with a single dose of colchicine (0.35 mg/ 100 g B.M.) or phosphate-buffered saline (controls).Twelve hours later, the animals were anesthetized and kidneys were fixed by perfusion in vivo. An indirect immunofluorescence cytochemistry in 4 (m thick cryosections of the kidney cortex was used to label specific membrane proteins in the PT cell plasma membrane domains and intracellular vesicles. A pattern of staining with the monoclonal and polyclonal antibodies to the specifc membrane proteins, followed by the fluorescein- or CY3-labeled secondary antibodies, showed in colchicine-treated rats: a) disappearance of MT in the PT cells, b) marked loss of the 31 kDaV ATPase sub-unit and NaPi-2 from the BBM, and their redistribution in numerous membrane vesicles randomly scattered in the cell cytoplasm, and c) unchanged distribution of the CAM- 105 and Na/K-ATPase in the BBM and BLM, respectively. We conclude that the abundance of V ATPase and NaPi-2 in the BBM, e.g. transporters whose concentration in the PT cell membrane is known to adapt quickly to acute changes in acid and phosphate loads in the PT, respectively, are very sensitive to MT depolimerization.The slowly-recycling membrane proteins, such as the Na/ K-ATPase in the BLM, and the CAM- 105 in the BBM, are not significantly affected by MT disruption. 



Serum cholinesterase activity (PchE) and ascorbinemia calves with neonatal diarrhoea after parenteral administration of ascorbic acid 
Curca Dumitru 
Department of Pathophysiology, Faculty of Veterinary Medicine, Bucharest Romania 
Comprehensive studies made on ascorbic acid’s (AA) multiple biochemical function have shown that the demand for this vitamin, to get healthy calves with optimum perFormances, is signifcantly higher than the quantity needed to prevent some clinical deficiencies, being noticed an obvious difference between the minimum and optimun needs. Synthesis and using of AA is feed-back regulated, being influenced by living conditions as food, climate, biologic agents action, to which the calves functional peculiarities, like the hormonal and metabolic characteristics are added. A study was made of the total blood level of AA by the Roc and Kuether method, and of PChE activity by the Ellman et al. method, in calves with moderate diarrhoea, after intravenous administration of AA 1g/day for 4 successive days, as compared with untreated calves and with clinically healthy congeners.A statistically significant decrease of ascorbinemia (P<0.05) and PChE activity (p<0.0 1) was found in calves with moderate diarrhoea aged 4 to 14 days as compared to congeners clinically healthy calves, whereas the hematocrit did not show statistically signifcant modifications (P0.05). After the daily intravenous administration of 1g AA for 4 successive days an increase of ascorbinemia to 3.289 +/- 0,533 mg/dl blood was noted in calves with neonatal diarrhoea,the difference being statistically nonsignificant (P0.05) versus nontreated clinically healthy calves. After treatment, the ascorbinemia level was restored, but the PChE activity remained low, 176.475 +/- f 6.01 mU/ml blood serum,the difference being neatly significant (P<0.01) as compared to nontreated clinically healthy calves. Nevertheless, there was an improvement of appetite, a restoration of the habitus, although no correlation was noted with the increase of the PChE activity underthe conditions of statistically nonsignifcant (P0.05) modifications ofthe hematocrit. The results also reveal the existence of a relatively wide variation of AA and PChE activity in calves, depending on age, state of health and parenteral administration of AA.



Polyclonal antibodies to rat renal cortical endocytic vesicles labe4 proximat tubule cell rnembranes In the rat and human kidney 
Međugorac-Popovski M, Herak-Kramberger CM, Sabolić I 
Institute for Medical Research and Occupational Health, Zagreb, Croatia 
Proteins that are f Itered in the renal proximal tubule (PT) are reabsorbed by endocytosis, a process which occurs via specialized intracellular organelles – endocytic vesicles (EV, endosomes). Due to imperFect methods, EV are hard to isolate without a significant contamination with other (intra) cellular membranes.The aim of this study was to produce specific polyclonal antibodies to the antigens in endosomal preparations that could be used either in immunoisolation experiments to get cleaner preparations of EV and other organelles or/and for specific labeling of the cell plasma membrane (PM) domains and intracellular structures in animal and human kidney. Endosomes were isolated from the rat kidney cortex homogenate by the established method (Sabolia I. and Burckhardt G., Methods Enzymol. 1990, 191:505), and characterized functionally and enzymatically. The final vesicle preparations exhibited high activity of the baf lomycin-sensitive proton-pump (V ATPase) and minimal contamination with the brush-border (BBM) and basolateral membranes (BLM). (mmune sera to the EV preparations were raised in rabbits. As found by indirect immunofluorescence cytochemistry in 4 (m thick cryosections of the rat kidney cortex, the immune sera brightly stained the BBM and various intracellular organelles in the PT cells. In immunoblotting experiments,the sera strongly labeled 4 high (Mr 55-276 kDa) and 5 low molecular mass (16-36 kDa) protein bands. However, none of the bands were specific for EV.An antibody to the 104 kDa protein band was purifed by immunoadsorption. This antibody strongly stained the BBM and subapical vesicles in PT cells of the rat and human kidney cortex. In the human kidney cortex, it also stained the BLM of some collecting duct ceIIs.Thus, common antigenic domains exist in the cell membrane proteins in rat and human kidney tubules. Conclusion: Polyclonal antibodies to the rat renal EV failed to specifically label endosomes and, therefore, could not be used for better purification of these organelles. The antibodies, however, might be used in cell biology studies of the common membrane antigens along the rat and human nephron.



Nephrotoxic heavy metals increase the expression of mdrl P-glycoprotein in the rat kidney brush-border membrane 
Herak-Kramberger CM, Thevenod F*, Sabolić I 
Institute for Medical Research and Occupational Health, Zagreb, Croatia 
*II Physiologisches Institut, University of Saarland, Homburg/Saar, Germany 
The multidrug resistance P-glycoprotein (mdr 1) is an 150- 170 kDa membrane protein that catalyzes the ATP-dependent efflux of hydrophobic and potentially cytotoxic compounds from the cells and, thus, it may represent an important cellular mechanism of detoxication. In various epithelia, including the kidney proximal tubule (PT), the protein is expressed on the apical cell membrane. An altered expression of mdrl is commonly seen in tumors treated with chemotherapy or in some cultured cells exposed to environmental stress or cytotoxic heavy metals (1). Using a monoclonal anti-mdrl antibody (C2 19), we studied the expression of mdr 1protein in PT cells by indirect immunofluorescence in 4 (m thick frozen sections of the fixed kidney cortex and by immunoblotting in isolated renal cortical brush-border membranes (BBM) from control rats and from the rats treated in vivo with various heavy metals. Rats were treated with daily s.c. injections of chloride salts of Cd, Hg, Cu, Zn, Mn, Ca, Mg, AI, La (2 mg/kg B.M.) or Pb (5 mg/kg B.M.) for 14 days, or with a single i.p. injection of cis-Pt (5 mg/kg B.M. 5 days before sacrifce). Control rats received an equivalent amount of saline for a comparable time period.A relative amount of mdrl in BBM was correlated to the metal concentration in the renal cortical tissue measured by atomic absorption spectrometry. ln control rats,avery lowtissue concentration ofthe metals was detected and a heterogeneous expression of mdr 1in BBM along the PT was found (S 1<S2<S3). In metal-treated rats, the concentration of metals in the renal cortical tissue increased between 7-(Mn) and 3000-fold (Cd). In rats treated with Cu, Zn, Mn, Ca, Mg,AI, and La, the abundance of mdr 1in the cortical BBM was not different from that in controls. On the contrary, in rats treated with the well-known nephrotoxic heavy metals, such as cis-Pt, Pb, Cd, and Hg, the mdr 1expression in the cortical BBM increased 3-, 6-, 15-, and 18-fold, respectively. Conclusion: nephrotoxic heavy metals (Cd, Hg, Pb and cis-Pt) increase the expression of mdrl in the rat kidney cortex BBM in vivo.The increased expression of mdr 1may represent a part of a specific cytoprotective response of the PT cells to oxidative stress induced by nephrotoxic heavy metals. Functioning as a lipid-translocase (2), mdrl could assist in removal of toxic lipid peroxides generated in the presence of heavy metals by translocating them across the apical membrane into the PT lumen. 
References: 1. Gottesman MM and Pastan I, Annu. Rev. Biochem. 1993, 62:385 
2.Van Helvoort A et al., Cell 1996, 87:507 



Clinical and pathofogical study in canine visceral leishmaniasis: natural and experimental infection 
Marinculić A, Kamadan P, Petrinović T, Grabarević Ž, Živičnjak T, Ćurić S, Stojčević D, Džakula N, Beck R, Barbić A, Šantek T, Vukonić L 
Veterinary Faculty, Zagreb, Croatia 
Canine leishmaniasis is a severe, often fatal, systemic disease of the dog, caused by the protozoan parasite Leishmania infantum. The disease is endemic in Mediterranean countries including Southern Croatia. It is well known that the dog, apart from suffering the disease itself, is the principal reservoir of the disease. In the dog as in other hosts leishmaniads multiply inside macrophages causing chronic inflammatory processes .The purpose ofthe study reported here was to compare the fndings of the clinical aspects of the disease and the clinical pathology in naturally and experimentally infected dogs with a field strain of  Leishmania infantum. The special aim was also to define the virulence and pathogenicity of the same strain in dogs. In order to def ne the clinical aspect of the natural infection, three dogs were hospitalized and thoroughly examined during the period of 1month or up to the death. Among naturally infected dogs, different dermatologic patterns were found like symetric alopecia, scaling and ulcerative dermatosis beside cachexia and parakeratosis. According to the experimental design, a group of six dogs was inoculated with parasites and the infection lasted for 120 days. Samples were obtained from dogs prior to infection and at weekly intervals for complete blood count and serum chemical analyses. Significant changes in rectal temperatures and body weights were not observed between infected and control animals over a 12 week period. All dogs developed a persistent infection in the period of eight weeks The systemic infection was defined by the parasitological findings of parasites in lymph node biopsies. Infected dogs, also developed signifcantly altered hematological values consisting of mild anemia and moderate leukopenia. Beside mentioned, other clinical signs were not found until the end of the experimental period.The lack of full-blown (eishmaniasis in the infected dogs is probably related to the chronic nature of the disease in which long incubation periods are ratherthe rule. It is not clearwhy in some dogs, clinical signs ofthe disease are poorly manifested and in other dogs, the disease is fatal. 



C. 
“Herman Jurak” round table on rheumathological diseases 
“Herman Jurak”- okrugli stol o reumatološkim bolestima



Anatomy and biomechanical characteristic of joint 
Nikolić V 
Department of Anatomy, Medical Faculty Zagreb, Croatia 
Diarthroses or synovial joints are non continuous and cavitated articulations with a fine capillary cleft between articular surfaces (synovial cavity) which contains synovial fluid – synovia.The synovial fluid is produced by the synovial membrane that lines the fibrous membrane and whole articular cavity other than articular surfaces of the joint. Bones are linked by the fibrous capsule and covered by articular cartilage.

The synovial membrane 
The synovial membrane lines nonarticular surfaces in synovial joints (fibrous capsules, intracapsular nonarticural bony surfaces, intracapsular ligaments and tendons), bursae and tendon sheaths were movements occur between contacting surfaces.The synovial membrane ceases at the articular cartilage margin. Macroscopically it is smooth, pink and shining with small synovial villi, folds and fringes on the internal surface. In some joints accumulation of adipose tissue in the synovial membrane occurs in the form of articular fat pads.The synovial membrane varies in local structure. It has an interlacing discontinuous cellular layer with A and B -type synoviocytes one to three cells deep, embedded in thin amorphous fibre free intercellular matrix.The functions of the cells are: 1. Removal of debris from articular cavity (mainly by type A cells). 2. Synthesis of the hyaluronic acid and glycosaminoglycans, or proteoglycan – like material lubricin (which acts as a lubricant of the articular cartilage) of the synovial fluid, and some of the constituents of the extracellular matrix . 3. Some synoviocytes produce antigens. Migrating synoviocytes carry ingested material to the lymphatics of the synovial subintima. Division rate of synoviocytes increases in response to acute trauma or haemarthrosis.

The synovial fluid 
Occupies and can be found in synovial joints, bursae and tendon vaginas. It is clear, viscous and slightly alkaline with a small population of mixed ceIIs.Volume of synovial fluid in normal joints is Iow.The composition of synovial fluid is similar to a dialysate of blood plasma containing: proteins about 0.9 mg/ 100 ml, mucin, mostly hyaluronate and glycosaminoglycan. Proteins are partly free and partly bound to glycosamonoglycans and hyaluronate, 10 to 30% derive from blood plasma. The origins of some other proteins are probably B type cells of synoviaf intima. A healthy human joint contains approximately 1to 5 ml of synovial fluid. 
Functions of synovial fluid are: 
1.To provide a liquid environment forjoint surfaces, internal fluid compartment and hydrodynamics of cartifage and menisci. 
2. Nutrition of articular cartilage cells, discs and menisci, acting as a medium for osmosis between the joint and capillary network. 
3. Lubrication and reductions of shear and erosion. 
Synovial fluid has non-Newtonian properties, i. e.The fluid is highly viscous with low rates of shear; with increased rates of shearthe viscosity nonlinearly decreases, meaning that in slow movements weight bearing capacity would be maximal. The product of viscosity and shear rate is almost constant, and also the weight bearing capacity. Synovial fluid obtained from degenerative joints show reduced non-Newtonian flow properties compared to fluid from healthy joints.

Articular cartilage 
The articulating bone surfaces are covered with 1to 5 mm thick articular cartilage. Articular cartilage’s primary functions are to distribute joint loads over a wide area decreasing the stresses on the contacting joint surfaces and to allow movement with minimal friction and wear. 
Composition and structure of articular cartilage: 
Articular cartilage is a special variety of hyaline cartilage and consists of two compartments: cellular (chondrocytes) and extracellular (matrix), but is an isolated tissue, devoid of blood and lymph vessels, and nerves. 
Cells in articular cartilage – chondrocytes take less than 10% of the tissue’s volume with different zonal arrangement according to layer of depth. Chondrocytes produce, secrete and maintain the organic components of extracellular compartment, the organic matrix. 
The organic matrix is composed of a dense network (mesh) of type 11 collagen f brils (10 to 30% by wet weight) enmeshed with proteoglycans molecules (3 to 10% by wet weight) remaining 60 to 87% is water, inorganic salts and small amounts of other matrix proteins and lipids. 
Coilagen has a high level of structural organization in articular cartilage, inhomogeneously, but having a layered organization. Tropocollagen molecules polymerise into collagen fbrils having a diameter from 25 to 200 nm according to different investigation. The collagen fibre has tensile stiffness and strength as a basic and most important mechanical property, but is weak and buckles with compression. Articular cartilage is anisotropic, its mechanics and material properties differ with the direction of mechanical loading in relation to the collagen fibers arrangements within the planes parallel to the joint surface; the number and density variations in collagen fibers cross-links as well with variation in collagen, proteoglycans and synovial fluid interactions. Proteoglycans (PG) in cartilage are large protein-polysaccharide molecules that exist either as monomers or as aggregates. PG monomers consist of an 200 nm long protein core to which about 150 glycosaminoglycan chains – GAG (keratin sulfate and chondroitin sulfate – polymer chains of specific repeating disaccharide units) are covalently attached. The distribution of GAGs along the protein core is heterogeneous but in arrangement like a bottle brush structure. PG aggregates are formed from up to 150 PG monomers attached to a central hyaluronate core. Hyaluronic acid core molecule is to 4 (m long filament of nonsulfated disaccharide chain. Proteoglycan aggregation promotes immobilization of proteoglycans to the collagen meshwork and structural rigidity ofthe extracellular matrix. 
Water, containing free cations, mostly sodium and calcium, is the most abundant component of articular cartilage. Concentration of water decrease with the depth of articular cartilage from 80% on surface to 65% in the deep zone. It permits difFusion of nutritiens metabolites between chondrocytes and the surrounding synovial fluid. Only a small percentage of the water is intracellular, 30% is associated with the collagen fibers and 70% occupies the intermolecular space and is free to move. This movement is important for controlling cartilage mechanical behavior and joint lubrication.

Biomehanical behaviour of articular cartilage 
Articular cartilage may be considered as a porous permeable medium (organic solid matrix: collagen fibers and PG mesh) filled with fluid (synovial fluid).The contact areas in human joints vary according to joint localization in range of few square centimeters and loading forces from 0 to several times of body weight. Mechanical behavior of articular cartilage is viscoelastic and is a combination of the response of viscous fluid and elastic solid. Two fundamental responses of viscoelastic material are creep and stress relaxation. With a constant load a viscoelastic solid as articular cartilage responds with a rapid initial deformation followed by a slow time-dependent progressively increasing deformation – creep, until an equilibrium is reached.When articular cartilage is subjected to the action of constant deformation (strain) high initial stress is followed by a slow, progressively decreasing of stress (stress relaxation). The compressive viscoelastic behavior of articular cartilage is due primarily to the flow of interstitial fluid, and in shear is due to the motion of long polymer chains – collagen and PGs. During creep the load applied to the surFace is balanced by the compressive stress developed within the collagen-PG solid complex and frictional drag generated by the flow of interstitial fluid during exudation. Creep ceases when the compressive stress within the solid matrix is sufficient to balance load alone and no fluid flow and deformation equilibrium is reached. LubricationThere are several models proposed for lubrication in joints: 1) Fluid film lubrication model- bearing surfaces are separated by a layer of lubricant. It effect depends on the reological properties of the fluid. The load on the bearing surfaces is supported by the pressure in this fluid. 2) Elastohydrodynamic model and boundary lubrication in which properties ofthe solid surfaces are combined with those of a thin monolayer of lubricant. Molecules adsorbed on both bearing articular surfaces. Thickness of these two layers ranged from 1 to 100 nm (Mow and all 1989, Swann 1985) 3) Weeping lubrication model in which the articular cartilage is considered as porous, deformable fluid filled material and, under load, the surfaces were lubricate by a film offluid expressed from the pores.4) Boosted lubrication model in which the compression of the articular cartilage traps fluid in irregularities of articular surfaces. Increasing compression push small molecules through pores into the cartilage and the synovial fluid on the surfaces becoming enriched in hyaluronates, more viscous and than more effective lubricant. Diarthroidal joint surfaces are composed of porous, permeable fluid filed cartilage that can imbibe and exude a lubricant fluid (Mow et al. 1989.) Loading and bearing, static and dynamic conditions are extremely variably suggesting that a mode of lubrication must be a combination of several mechanisms depending on biomechanical demands. 
Friction Friction is defined as the resistance to motion between two bodies in contact and is defined by coefficient of friction (. Frictional force (F) is directly proportional to applied load (W) F = (/W. Coefficient of friction for articular cartilage in synovial joints was measured between 0.002 and 0.06 (for steel on steel (is 0.6 to 0.8, Ice on ice 0. 1) but is increased with magnitude of the load and time of application of the load, and is lower when synovial fluid was used as lubricant than with saline. Wear of articular surfaces According to Maw and al, wear is the removal of material from solid surfaces by mechanical action.There are two components of wear: 1) InterFacial wear due to the interaction of bearing surfaces coming into direct contact with no lubricant film separating them.There are two ways of this type of wear: a) Adhesive wear – surface fragments adhere to each other and are torn from the surFace during sliding. b) Abrasion occurs when a softer material is scraped by a harder one usually by an opposite bearing surFace or by loose particles between the bearing. Both adhesive and abrasive wear may take place in a degenerated joint. Structurally impaired and defected articular cartilage becomes more permeable and softer. 
Lubricant film may leak away through the cartilage and this loss of lubricant results in direct contact between the asperities of articular cartilage and exacerbate the removal of cartilage fragments. 2) Fatigue wear results from the accumulation of microscopic damages within the articular cartilage and subchondral bone under repetitive or cyclically application of load. Failure may occur over a short period with repeated high load or over a long period with repetition of low load. (Armstrog and Mow)

References: Armstrong CG, Mow VC:Variations in the intrinsic mechanical properties of human articular cartilage with age, degeneration and water content. J Bone Jt Surg 64A, 88-95, 1982. Armstrong CG, MowVC: Biomechanics of normal and osteoarthrotic articular cartilage. In: Clinical trends in orthopaedics. ED:Wilson PD,Jr, Straub LR,Thieme – Stratton, NewYork 1982. FungYC: Biomechanics, Mechanical properties of living tissues, SpringerVerlag, NewYork 1981. Junqueira LC, Carneiro J, Kelley RO: Osnove histologije, Školska knjiga Zagreb, 1995. MowVC, Proctor ChS, Kelly MA: Biomechanics of articular cartilage. In: Nordin M, Frankel VH: Basic biomechanics of the musculosceletal system., Lea & Febiger, Philadelphia, London, 1989. Mow VC, Zhu W, Ratcliffe A: Structure and function of articular cartilage and meniscus. In: MowVC, HayesWC: Basic Orthopaedic Biomechanics, Raven Press, NewYork. 199 1. MowVC, Soslowsky LJ: Friction, lubrication and wear of diathrodial joints. In: MowVC and Hayes WC: Basic Orthopaedic Biomechanics. Raven Press, NewYork, 1991. NikolićV., Hudec M: Principi i elementi biomehanike, Školska knjiga, Zagreb, 1988. Swann DA, et al.: The molecular structure and lubricating activity of lubricin from bovine and human synovial fluids. Biochem J 225: 195 201, 1985.Williams PL,Warwick R, Dyson M, Bannister LH: Gray’s Anatomy. 37th Ed. Churchill Livingstone, Edinburgh, London, Melbourne, New York 1989.



The nosological role of primary necrotizing processes (PNP) in rheumatoid arthritis 
Schneider S, Fassbender HG 
Zentrum für Rheuma-Pathologie (WHO Centre), Mainz, FRG 
The profile of RA is characterized by three different components: I. by immunologic inflammation 
2. by an oncologic destruction process 
3. by primary necrotizing processes (PNP) 
The nosologic significance of PNP hitherto was estimated too low. PNP only occur in patients suffering from seropositive RA. The following structures can be affected: Tendon, joint capsule, myocard, artery wall, eye and lung. The tissue destruction is caused by proteases, released by previously proliferated fibroblasts. While inflammation and destruction are fimited to joints and tendon sheaths, PNP are able to manifest at many sites of the organisms, sometimes fatally, and, under certain circumstances, they can dramatically influence the nosological picture of RA.



Signal enhancement method for the digoxigenin-labeled in situ hybridization 
Harjaček M, Diaz-Cano S 
Department of Pediatrics, University Hospital Rebro, Zagreb, Croatia Institute for Pathology, GSF Forschugscentrum, Neuherberg, Germany 
In situ hybridization (ISH) in tissue sections has a wide range of applications in biomedical research. However, ISH still remains a time consuming procedure, and in cases where expression of target mRNA is presumably low, positive results using a standard NBT/BICP chromogen are often difficult to interpret. We report a novel, simple, rapid and particularly sensitive method for detection of the low copy target mRNA. Our new signal detection method is based on the utilization of biotinylated, monoclonal anti-digoxin-Ab which cross-reacts with digoxigenin and has not been previously used for ISH hybridization. We chose to study cytokine expression in various tissues, in particular IL-4 which is known to have smail transcripts (-650 bps) and is found only in a small subpopulation of activated T ceIIs. The low number of expected positive cells coupled with small transcripts should ensure the development of highly sensitive methodology. As a positive control for the high copy target mRNA, a riboprobe for oncogene c-myc was also employed. Our results show that this method is very sensitive, reliable and quick, and offers some unique features that were not available with standard color detection methods used previously for the digoxigenin-labeled ISH.



D. 
Ethical forum – Forensic medicine: Profession? Science? Politics? 
Etički forum – Sudska medicina: Struka? Znanost? PoIitika?



Animal euthanasia 
Džaja P, Perić J 
Zavod za sudsko i upravno veterinarstvo, Veterinarski fakultet u Zagebu 
Euthanasia in veterine represents human and painless killing of animals. It should be performed particulary due to economic and medical reasons. We stress the importance of euthanasia that sholud be performed according to the Iaw. Therefore in this paper we stressed legislature that determine the perFormance of euthanasia. In the majority of cases the owners should decide about euthanasia of their animals, except in those cases that are regulated by law.



Epidemiology of suicide in Osijek county 
Ugljarević M, Marcikić M, Badžek S 
Department of Forensic Medicine and Pathology University Hospital Osijek, Osijek, Croatia 
We choose to study suicide in Osijek Country focusing on trends and levels based on demographic characteristics. Compilation of most descriptive statistics was performed by a standard statistical analysis computer package. There were 280 reported suicide cases, among Osijek Country residents, from 1986. to 1995. The average annual rate was 15,9/100 000 residents from 1986. to 1990., and 18,9/100 000 residents in the second time period from 1991. to 1995. Male suicides contributed to the increased rate of suicide after 1990. There was a fairly uniform spread of suicide over the months of the year, with slight increase in spring and fall, and low figure for January. The suicide rate for male was the highest for the age group of 25 to 29 year olds and 60-64 year olds. Among women suicide rate was the highest for the age group of 60 to 64 year olds. Married and retired committed the largest proportion of suicides. Firearms were preferred by men and poisons by women. There was a significant rise in the suicide rate by firearms after 1990. Depression associated with physical incapacitation and intolerable loneliness were common reasons for suicide among elderly men. 



DETERMINATION OF THE WAR VICTIM’S AGE BY TEETH 
Brkić H. Kaić Z, Keros J, Njemirovskij V 
Department of Dental Anthropology, School of Dental Medicine Zagreb, Croatia 
In this paper authors present the the dental age determination methods of the bodies exhumed from the mass graves in Croatia. The analysis of teeth is found to be one of the main methods in the identification process in the following ways: 
1. Dental identification achieved by comparison with antemortem data; 
2. Identification achieved through an interdisciplinary approach; 
3. Identification achieved by the analysis of the DNA isolated from the dental tissues cells. 
When the interdisciplinary approach is used, involving forensic scientists from different disciplines, the odontologist determinates the age and sex of the body based on the analysis of teeth and corresponding bones structures. Dental age has been determined through clinical and x-ray analysis based on: a) chronology of the teeth eruption; b) quantity of mineralization of hard dental tissues; c) changes on dental tissues acquired during Iifetime.The results show that the determination of dental age is a very reliable method that contributes to faster and precise identifications of the numerous victims of war. 
Literature: Brkić H et al., Dental Identification of War Victims from Petrinja in Croatia. International Journal of  Legal Medicine 1997: 110:47. 
Galić J et al., Dentition Based Postmortem Age Identification. Acta Stomatologica Croatica 1996:29: 197. 
Brkić H et al., Dental Identification. Hrvatski  Stomatološki Vijesnik 1997:5:4. 
Gustafson G Age Detrminations on Teeth. Journal of American Dental association 1950:4 1:45.



E. 
Computer systems and computerisation in pathology 
     Informatički sustavi i kompjutorizacija u patologiji 



Thyroid cancer in clinical thyroid diseases register of University Hospital “Sestre milosrdnice” 
Šimunović D, Belicza M, Lechpammer M, Gladić V, Kusić Z 
“Ljudevit Jurak” Clinical Department of Pathology, “Sestre milosrdnice” University Hospital, Zagreb, Croatia 
The aim of our research is to make an analysis of the hospital population through the period of eighteen years (1980- 1997) and to calculate representation of the thyroid cancer in that sample. We were interested in histological representation, sex distribution with female to male ratio (F/ M), average age at diagnosis, presence of metastasis at diagnosis and the size of the tumor. Following the representation of the follicular carcinoma through the years we wanted to discover possible abnormalities of the daily iodine uptake at the examined population. Šeparović et al. claim that in areas without endemic goiter representation of the follicular carcinoma among malignant tumors is 5 to 15% while in the areas without an endemic goiter representation is up to 40%. At the same time we have started creating the computer register supplemented with a clinical data for further following of the thyroid cancer. Our intention was to add part of the database in “Thanatos” computer system that relates to malignant diseases in the Department of Clinical Pathology “Ljudevit Jurak” to the hospital’s Central Register for Cancer. Biopsy materials were acquired after partial or total thyreoidectomias of the patients that were worked on in “Sestre milosrdnice” University hospital. Materials were embedded in paraffin wax, cut in slices 5 (m thick, colored with hemalaun-eosin and analyzed with microscope. Pathology specialists analyzed all materials. During the eighteen year period (1980- 1997) at the Department of clinical pathology “Ljudevit Jurak”, University hospital “Sestre milosrdnice” in Zagreb, there was a total of 196 878 biopsy findings. We have separated pathohistological diagnosis of the thyroid cancer. With a total of 379 thyroid cancer biopsy findings. We have found 219 (57,8% of all cancer) papillary carcinoma, 77 (20,3%) follicular carcinoma, 29 (7,7%) medullar carcinoma, 30 (7,9%) anaplastic carcinoma and on category other 24 (6,3%) of all thyroid cancer. Findings by other categories of our interest were: papillary carcinoma had female to male ratio 3,38:1, metastasis in 26% an average size 1,78 cm (0,73-3,83). Female to male ratio in follicular carcinoma was 2,21:1, metastasis in 9,1% and average size 2,86 cm (1,5-5). Medullar carcinomas average size was 2,29 cm (0,5-4), metastasis in 5 1,7 % and F/M ratio 1,42: 1. F/M ratio for anaplastic carcinoma was 1,73:1, average size 2,75 cm (1,5-5) and presence of metastasis in 33,3%. Average age for papillary carcinoma was 45,4 years, for follicular cancer 51,1yr., medullar carcinoma 50,7 yr, and anaplastic carcinoma 55,6 yr. Among all thyroid cancers, there has been one difference observed. Average age of the patients with papillary carcinoma (45,4 yr.) is greaterthan quoted in Iiterature. The representation ofthe follicular carcinoma among malignant tumors according to values quoted in literature is 5- 15 % in areas without an endemic goiter. The representation in our sample is 20,3% and it is in correlation with the presence ofthe endemic goiter in Croatia.0ur result suggests further research. Other results are in correlation with the literature. 
Literature: 1 Belgian registry ofthyroid cancer Preliminary epidemiological characteristics revealed by retrospective study (1988- 1993). Meurisse M et al., Ann Endocrinol (Paris) 1997. Delisle MJ et al., Ann Endocrinol (Paris) 1997; 1: 4 13. Park SH et al., Jpn J Clin Oncol 1988; 4: 297



Non-malignant lesions in clinical thyroid diseases register of University Hospital “Sestre milosrdnice” 
Cvjetko l, Belicza M, Lechpammer M, Gladić V, Kusić Z 
“Ljudevit Jurak” Clinical Department of Pathology “Sestre milosrdnice” University Hospital, Zagreb, Croatia 
Recent nationwide study showed that 8- 15% of Croatian population has endemic goiter. The aim of our retrospective study was to make an analysis of the hospital population during the eighteen year period from 1980 to 1997. We have calculated incidence, sex distribution, and time of the diagnosis for each of the non-malignant thyroid Iesion. The analysis was done using the “Thanatos” computer system developed at the “Sestre milosrdnice” University Hospital. “Thanatos” is a computer data base of the biopsy findings together with the clinical data. There were total 3552 non-malignant thyroid lesions during that period.The most common was goiter: 2098 cases or 59,1%.  Among those there were 256 men, and 1842 women with female to male ratio (F:M) 7,2:1. Average age (AA) of the men was 49,7 yr., and AA of the women was 46,8 yr.There were 979 adenomas (27,6%): 138 among men (AA=46,7yr), and 841 among women (AA=44,5yr.) with F:M=6, 1: 1. There were 247 cases (7%) of the Basedow disease: 23 males (AA=46,4yr.), and 224 females (AA=37yr) with F:M=9,7:1. Hashimoto disease was found in 156 cases (4,4%): 8 men (AA=5 1,6yr.), and 148 women (AA=49,8yr.) with F:M= 18,5: 1. Hyperplasia was the diagnosis in 64 cases (1,8%): 6 men (AA=35,2yr.), and 58 women (AA=35,3yr.) with F:M=9,6: 1. We have also found five women with the fibroses thyreoiditis (AA=52yr). More then half or 59, 1% of the non-malignant slesions goes to goiter. All non-malignant thyroid lesions are more common in women, especially Hashimoto disease (F:M= 18,51). Most of the non-malignant thyroid lesions occur during the fourth and fifth decade of life. Hyperplasia is being diagnosed at the average age of 35,2 yr. in men and 35,3 in women. Basedow disease in women is being diagnosed at the average age of 37yr 
LITERATURE: 1. Kusić Z Endocrinol Invest 1996; 19:204-9. 2. Žurić M et al. Krušlin B, Robić M. Book of abstracts. The eight “Ljudevit Jurak” international symposium on comparative pathology. Zagreb, 1997; 33. 3. Park SH et al. Jpn J Clin Oncol 1988; 18:297-302. 4. O’Hanlon DM et al. Ir Med J 1997; 90: 70.



Causes of death in autopsies during World War 1 and 80 years later 
Kučinar I, Brezovečki D, Matković M, Lechpammer M 
Department of Clinical Pathology Ljudevit Jurak Clinical Hospital “Sestre milosrdnice” 
The main purpose of this study was to investigate major differences and changes in the causes of death of patients submitted after their death to our pathologic department but with the time distance of  80 years. For that purpose we analysed causes of death recorded from September 1916. to October 1918. in the autopsy reports of the Pathology Unit of Zagreb’s Public Health Institutions (Prosektura javnih zdravstvenih zavoda grada Zagreba), today’s “Ljudevit Jurak” Clinical Department of Pathology. Obtained data were compared with the causes of death recorded in the archives of the same institution from January 1995. to December 1997. Memorising and processing of pathological diagnoses was provided by the “Thanatos” computer system for clinical pathology. 
Comparison of pathologic data originating from 3 war years during World War I, and 3 years after Croatian war for independence imposes by itself some significant differences. First, all patients from the 1st investigated period were male – soldiers died at one of 7 military hospitals existing in Zagreb at that time. Due to that, 65.5% of them where younger than 49 years, while 29.8% were younger than 29 years of age. Only 2.8% were older than 50 years at the time of death. In contrast, patients from the 2nd investigated period were citizens of Zagreb of both sexes died at one of 12 clinical Departments of the Sestre milosrdnice University Hospital. Their average age at the time of death was 67 years. 
However, comparison of these two undoubtedly very different time periods offers some very intriguing differences in causes of deaths. Among 1118 deaths in the 1st period it was established that infectious diseases were the main cause of death (60.5%), followed by injuries (12.8%) and respiratory diseases (10,1%). In the 2nd observed period total of 3538 deaths was recorded. The main causes of death were diseases of cardiovascular system (23,45%), followed by respiratory causes (16,6%). It is interesting that remaining 59,95% of deaths is distributed in smaller ratios on wide variety of causes of death ranging from injuries and malignancies to paediatric deaths.



F. 
Stomathological pathology 
Stomatološka patologija



The effects of C02 and Nd-yag lasers on the pulp and periodontal tissue on the dog 
Jukić S, Anić l, Talan-Hranilović J 
Department of Dental Pathology, School of Dentistry Zagreb, Croatia 
“Ljudevit Jurak” Clinical Department of Pathology, “Sestre milosrdnice” University Hospita~ Zagreb, Croatia 
C02 and Nd:YAG lasers are most commonly used in medicine and dentistry. The aim of this study was to evaluate the effect of these two lasers on dental pulp and peridontal tissue. Mixed breed dogs weight 25 kg were used for experiments. The animals were anesthetized with pentobarbitural. Pulp chambers of third premolars and molars were opened and pulp tissues were removed from it with sharp spoon. The remainder pulp tissue at the root canal opening of right upper jaws was lased with C02 laser (2 W / 5 tps / 10 ms). The teeth in the left sides of the jaws were lased with Nd:YAG laser (2W / 20 pps). First and second premolars served as positive and negative controls.The teeth were temporary filled with policarboxylat cement and procedure was repeated 15 days later on teeth of lower jaws. Peridontal tissue was exposed by intentional penetration od the pulp chamber floor with steel bur. After hemostasis and drying the furcation region of upper right molars was irradiated with C02 laser (2 W / 5 tps / 10 ms) for 3 s. The left side of the jaws was lased with Nd:YAG laser (2 W / 20 pps) for the same period of time. The same treatment was repeated on teeth of lower jawes for 15 days. The animals were sacrified by an overdose of pentobarbital 30 days after treating lower jaws and tissue was processed for histopathological analysis. The most frequent finding in the pulp tissue in case of using both lasers and regardless oftime oftreatment,was necrosis.A strong chronical inflammatory reaction without dentin bridge formation over canal of rices was found.Appearance of more carbonized tissue and changes in deeper portions of pulp tissue was found in case of Nd:YAG laser irradiation. Fluency that is used in laser soft tissue surgery causes superficial necrosis, inflammatory cells’ infiltration and in deeper portions osseous transformation. Inflammatory resorption of cement and cellular infltration of peridontal ligament is often found beneath superficial wound layer. Nd:YAG laser in case of peridontal tissue, also penetrates more and causes sever changes of tissue. 45 days after treatment with C02 laser, first signs of osseous healing response appeared. In contrast, peridontal tissue lased with Nd:YAG laser did not show any sign of healing.



Lack of p53 and nm23-H I genes deletion in oral lichen planus and leukoplakia 
Mravak-Stipetić M, Gall-Trošelj K, Pirkić A, Pavelić K, Pavelić J 
School of Dentistry University of Zagreb, Zagreb, Croatia 
Department of Molecular Medicine Institute “Ruder Bošković”, Zagreb, Croatia 
“Ljudevit Jurak” Clinical Department of Pathology “Sestre milosrdnice” University Hospital, Zagreb, Croatia 
In our attempt to identify genetic changes involved in epithelial cell proliferation, in certain cases as an earliest step in malignant epithelial neoplasm development, we examined p53(PCR/RFLP) and nm23-H 1(PCR/LOH) allelic deletions in oral benign , potentially malignant epithelial lesions. The study group comprised of 25 benign epithelial lesions, clinically diagnosed as lichen planus (N= 17) and leukoplakia (N=8). Two reccurent aphtous ulcers as well as one specimen diagnosed a benign migratory glossitis were also included in the study. Among 21 samples analyzed for exon 4 (p53 gene) LOH, only 6 (28,5%) were informative with no presence of loss of either allele. Of 23 samples tested for LOH at intron 6 of p53 gene, 8 (42, 1%) were informative, again with no presence of LOH. The frequency of alleles was 73,4 and 26,6% for alleles with and without a specific restriction site. For nm 23 H- 1gene the analysis was perFormed on total of 24 cases. Of them 16 (16,6%) were informative, however, non sample exhibited LOH at his locus. In conclusion, whereas the presence of gross gene alterations (LOH) would have been definitive evidence for the p53 and/or nm 23 involvement in hyperproliferation process, the absence of LOH does not exclude the presence of either smaller mutations, or disregulation of normal gene, or disfunction at the level of wild type protein. Alternatively, p 53 and nm 23-H I have no relation to oral lesion formation, and can not be considered as an early step in benign maybe neoplastic, tissue transformation.



Defence reaction in a dental pulp after partio-pulpectomy in dog 
Pavlica Z, Juntes P, Pogačnik M 
University of Ljubljana, Veterinary Faculty, Ljubljana, Slovenia 
Aim The aim of our work was to study the reactions of a dental pulp in dogs after radical procedures on teeth. A partial vital pulpectomy was used as a model. 
Material and methods  A class V cavity was prepared on upper third right and left incisors and the dental pulp was exposed.The same sterile bur was used to create a 2 to 3 mm partial pulpectomy.The partial pulpectomy wound was treated with calcium hydroxide and covered with cement and left for standard restoration with composite (1,2,3,4,5). Partial pulpectomy was performed on 33 teeth. On the 60th day the test teeth were extracted under anesthesia and prepared for histology. Paraffin blocks of the decalcifed teeth were oriented to permit buco-lingual sectioning of the teeth. Eight Nm thick paraffin sections were stained with hematoxylin-eosin and examined forthe type and degree of pulp inflammation (hyperaemia, pulpitis, necrosis or gangrene). Degenerative changes of the dental pulp (vacuolation, calcification, amyloid or hyaline changes) were also determined. 
Results and conclusions   Inflammation was seen in one incisor only. A hard continuous barrier with well formed tertiary dentine and dentinal tubules was seen in all teeth and in all samples it was formed laterally in a continuous barrier initially. The tertiary dentine differed from the normal dentine in having fewer tubules, which were strongly curved. Partial pulpectomy of the dental pulp produced better quality, uninterupted dentinal bridges in several dogs, when compared to the less radical methods like direct pulp capping, which we have studied earlier. On the basis of this results, the radical method of partial pulpectomy was recommended as the most suitable method for retaining teeth vitality in dogs.

References 1) Ten Cate AR. J Dent Res 1985; 64:549-5 1; 
2) Ivanović V et al. Oral Surg 1989; 67:684-8; 
3) Schroder U. J Dent Res 1985; 64: 5 1-8; 
4) Matsuzaki K et al. Bull Tokyo Dent Coll 1990; 3 1:9- 15; 
5) Cullum DR et al. Oral Surg Pathol 1985; 60:218-23.



The activity of NADPH – d in chronfcalfy fnflammed dental pulp 
Jukić S, Sisig S, Anić I, Mihelić D, Talan-Hranilović J 
Department of Dental Pathology School of Dentistry, Zagreb, Croatia 
“Ljudevit Jurak” Clinical Deparpment of Pathology “Sestre milosrdnice” University Hospital, Zagreb, Croatia 
NADPH – diaphorase serves as an electron donor for nitric oxide (NO) synthase that converts L- arginin in nitric oxide and citrulin. Recently, it has been found that nitric oxide may act as a neurotransmitter. This study was undertaken to determine the activity of NADPH – diaphorase in human dental pulp with chronical pulpitis and in health controLThirty human dental pulps were collected out of the routine dental procedure from carious profunda, nonperiodontaly involved teeth, treatment planed for endodontic therapy. Health controls were obtained from premolars or third molars extracted due to orthodontic therapy. Enzyme-activity of human NADPH-diaphorase was determined by the Pearse et al. (1968) method. High NADPH-d activities were obtained in pulps with chronical pulpitis.



Transcutaneous stfmulatfon analgesia fn stomatofogfcal procedures 
Kaić Z, Keros-Naglić J, Božićević D 
Department of Dental Antropology, School of Dental Medicine, Zagreb, Croatia 
Pain Control Centre, Neurosurgical Clinic, School of Medicine, University ofZagreb, Zagreb, Croatia 
The aim of this study was to evafuate the efficiency of transcutaneous electrostimulation analgesia (TEA) in different stomatological procedures.The study comprised 180 patients. In 90 of them the stomatological procedure was carried out under TEA, while the other 90 patients were controls. We used three points corresponding to the traditional Chinese procedure (Lf-4, LI-20, ST 6) and WQ I 0-A pulse generator with frequency of 30 and 50 Hz. Data processing yielded the following results: the stomatological procedure is less painful when TEA is used.There is no statisticaLly significant difference of the dental procedure regardless to the use of TEA with respect to age and sex. The statistically significant difference has been found between gingival painfulness at the beginning of procedure, than 15 and 25 minutes after in patients under TEA (x2=93,48, df=4, p<0,05). It has also been found that 58, or 64% of the patients who sustained TEA in a satisfactory manner, would agree with procedure again. ThereforeTEA may be considered as an adequate model in study of analgetic value of TEA in condition of acute pain. Although TEA has no absolute advantage over traditional pain control procedures, it surely has its indication in patients suffering from drug allergies, chronic or malignant diseases,AIDS and other progressive destructive conditions, who cannot be treated with traditional pharmacological analgesics.

Literature: 1) Turk DC et al. Handbook of pain assessment. NewYork, Guilford Press, 1992: 15. 
2) Lowe NK et al. Confirming the theoretical structure of the McGill Pain Questionnaire in acute clinical pain. Pain, 199 1;46. 
3) Frucht s et al. Muskelentspannung durch transkutane Elektrostimulation (TENS) bei Bruxismus. Munchen Fortschr Kieferortop. 1995;56. 
4) Mumford JM et aLToothache and related pain. Edinburgh, Churchill-Livingstone, 1976; 118. 
5) Tabu HA et al. Studies of acupunccture for operative dentistry. JAD, 1997;95:555. 
6) Martin RW et al. Dental dolorometry for human pain research: methods and apparatus. Pain, 1979; 6:349.



Evaluation of four methods for estimating taurodontism 
Galić J, Kaić Z, Štefanac-Papić J 
Department of Dental Anthropology, School of Dental Medicine, University of Zagreb, Zagreb, Croatia 
Taurodontism (T) is defined as an apical extension ofthe pulp chamberwhich results in shortened root. The aims of this study were to evaluate four methods of “T” estimation and determination of its concordance.The number of 221 lower molars were traced from the orthopantomograms of 87 males 24-3 1yr of age. Seven variables were measured on the tracings:Variable 1) lowest point of the roof – highest point of the floor of the pulp chamber, 2) lowest point of the roof of the pulp chamber – apex of the longest root, 3) baseline connecting the two cementoenamel junctions (neck) . highest point of the floor of the pulp chamber, 4) neck – deepest pit of the occlusal surFace, 5) neck – root bifurcation, 6) root bifurcation – deepest pit of the occlusal surface, 7) root bifurcation – apex of the longest root. Each tooth was assessed twice by the same author (J.G.) within 2-4 days. T was estimated according to the methods proposed by Shifman (1),Tulensalo (2),Jorgenson (3) and Seow (4).The value ofVariable 3 proposed by Shifman was adjusted for magnification on the orthopantomograms while the index proposed by Seow was adjusted for the second and third molars. Intraexaminer reliability in reproducing the same classification was, on average, 96,84%, the highest for Shifman’s method (99,10%) and the (owest for the Seo~n%s method (94,57%).Table 1 shows frequencies of taurodontism found and methods used.Table 1The concordance in diagnosingT was the highest for Shifman’s and Jorgenson’s method (94, 1%) and the lowest fofTulensalo’s and Seow’s method (83,8%). According to Shifman’s criterion only two molars were classified as taurodontic and those were the only teeth thet were classified the same by all four methods.Thus, the method for estimatingT described by Shifman proved to be the most reliable for use in later epidemiological investigations.

References: 1) Shifman A. et al. Community Dent Oral Epidemiol 1978; 26:200. 
2)TulensaloT et al. Community Dent Oral Epidemiol 1989; 17:258. 
3) Jorgenson R.J. et al. J Cran Genet Dev Biol 1982; 2: 125. 
4) Seow W K. et al. Pediatric Dentistry 1989; 11(3):2 14