Abstracts Immunology, immunopathology and pathology (basic and ...

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Abstracts

Immunology, immunopathology and pathology(basic and clinical)

Nephrology Dialysis Transplantation Vol. 14 n.9 1999

E: Immunolgy, immunopathology andpathology (basic and clinical)

CORRELATION BETWEEN TNM STATUS AND IMMUNOMORPHOLOGYOF RENAL CELL CARCINOMAD Brasanac1, J Markovic-Lipkovski1, GA Müller2, CA Müller3

1Institute of Pathology, University Medical School, Belgrade, Yugoslavia; 2De-partment of Nephrology and Rheumatology, Georg August University, Göttingen,Germany; 3Section of Transplantation, Immunology and Immunohematology,Eberhard-Klaus University, Tübingen, Germany

TNM status is one of the most reliable parameters for tumor prognosis. In thiswork we investigated possible associations between TNM status and variousimmunological characteristics of renal cell carcinoma (RCC).Cryostat sections of 37 RCC (25 clear cell type, 10 granular and 2 chromophobe)were analyzed with indirect immunoperoxidase technique using monoclonalantibodies to: HLA class I (HLA-ABC) and class II (HLA-DR, -DP, -DQ) anti-gens, ICAM-1 (CD54), CD3, CD14, CD4 and CD8 molecules.Comparing to T1/T2 cases, T3/T4 tumors showed higher level of HLA class IIantigens (especially HLA-DQ), more frequently reduced HLA class I level (50%of T3/T4 versus 14% of T1/T2), widespread ICAM-1 expression, prevalence ofT lymphocytes over monocytes among tumor infiltrating mononuclear cellsand CD4/CD8 ratio about one. Similar differences were observed between M1and M0 RCC, with even more pronounced ICAM-1 presence and absence ofCD8+ T lymphocytes domination in all M1 tumors (comparing to 15% of caseswith such domination among M0 tumors). Possible explanations for aforemen-tioned findings could be anergy of T lymphocytes induced by tumor cells due toa lack of necessary co-stimulatory molecules (e.g. B7), blocking activity of shededICAM-1 molecules, or reduced antitumor action of CD8+ (presumably cyto-toxic) T lymphocytes on cells with reduced level of HLA class I antigens.Immunohistochemical analysis of RCC suggests that immune response in T3/T4 and M1 tumors is altered both quantitatively and functionally comparing toT1/T2 and M0 cases.

EFFECT OF PENTOXIFYLLINE ON AUTOIMMUNE GLOMERULONEPHRI-TIS IN BROWN NORWAY RATS.A Wystrychowski1, F Kokot1, H I Trzeciak2 .1 Dept. of Nephrology, Endocrinology and Metabolic Diseases, 2 Dept. ofPharmacology, Silesian University School of Medicine, Katowice, Poland

Pentoxifylline (PTX), a non-selective phosphodiesterase inhibitor, showsimmunosuppressive properties. It reduces the production of TNFα, IL 12 andIFNγ as well as ICAM-1 and IL 2 receptor expression and inhibits proliferationof mesangial cells and formation of collagen.Thus it may be expected that PTX could exert a beneficial effect on glomerulone-phritis. To test this hypothesis we compared the severity of autoimmune glomeru-lonephritis (AGN) induced in male Brown Norway (BN) (230-340 g) rats treatedwith PTX (100 mg/kg b.w., s.c., every 8 hours for 2 weeks) with a control group,which received the same volume of saline. AGN was evoked following a stand-ard protocol (HgCl

2, 1 ml/kg b.w., s.c., every 2nd day for a total of 2 weeks).

After 2 weeks of HgCl2 and PTX treatment (as proteinuria reached a plateau

level) several renal function parameters [plasma urea concentration (Purea

, mmol/l), proteinuria (U

prot, mg/24 h/100 g b.w.), albuminuria (U

alb, mg/24 h/100 g

b.w.), albumin clearance (Calb

, µl/min/100 g b.w.), urine protein-to-creatinineratio (U

prot/1 µl C

cr) and kidney weight (g/100 g b.w.)] were assessed. Two rats

of the control group died in metabolic cages and 5 rats of the PTX group hadcystitis with retention of urine. These animals were excluded from the statisti-cal analysis.group N U

prot U

alb C

alb kidney

wtP

ureaU

Ca µmol/ P

CaU

protg/100g 24h/100g mmol/l /1µl CCr

Control 1 3 6 8 2 5 2 ,17 0 ,85 25 ,4 6 ,2 2 ,12 1 7 1(15) ±31 ±13 ±1,46 ±0,17 ±9,4 ±3,7 ±0,10 ±124

PTX 1 0 21^* 7 * 0,38^ 0,73# 13,8 15,6^ 2,37# 5 2(15) ±19 ±8 ±0,55 ±0,11 ±13,6 ±10,2 ±0,06 ±45

MEAN±SD # p<0,05, * p<0,01, ^ p<0,002 (U Mann-Whitney test) (N)=initial numbers

As seen on the table PTX alleviates the severity of the disease. These findingssuggest that PTX could be of benefit in the treatment of glomerulonephritis.

HEPATITIS C VIRUS (HCV) ASSOCIATEDIMMUNECOMPLEX (IC) GLOMERULAR DISEASE IN PA-TIENTS (PTS) COINFECTED WITH HIVG. Barbiano di Belgiojoso, A. Genderini, N. Landriani, M.T.Barone, D. Scorza, S. Bertoli, M. TrezziRenal Unit, L. Sacco Hospital, Milan, Italy

The presence of HCV infection in pts with HIV coinfection andglomerular involvement has been indicated as responsible ofnephropathy. The association of HCV infection and IC GN inHIV-infected pts has been reviewed. 9 cases have been selected,all submitted to renal biopsy. All patients were white, 8 intrave-nous drug abusers (IDA), one homosexual. Age was between33 and 37 years (mean 34), 4 were males. 7 pts presented withnephrotic syndrome, 2 with proteinuria and microscopichematuria. Low C4 was present in 3 cases, cryoglobulins (cryo)in 1/7 tested cases. At light microscopy 5 pts showedmembranoproliferative GN (among which 3 lupus-like), 2 dif-fuse endocapillary proliferative GN, one IgAGN, oneimmunotactoid GN. At immunofluorescence (performed in allcases) 5 with membranoproliferative GN had intense parietaldeposits of IgG, IgM and C3. Endocapillary forms had promi-nent parietal C3 deposits, IgA-GN mesangial deposits of IgA,IgG and C3. Tactoid GN had IgG deposits in multiple sites,mainly subepithelial. Electron microscopy (performed in all cases)confirmed the presence of large dense deposits in multiple sites,without cryo-type structurated deposits. The clinical and histo-logical aspects of these 9 pts were different from mixed cryoGN, HCV related: no history of purpura or Raynaud phenom-ena, absence of monocytes and structurated deposits, rarity ofC4 reduction and serum cryo.In conclusion, the clear-cut difference between IC GN in HCV-HIVpts and cryo-GN indicates for the former a mechanism otherthan cryo formation, possibly related to HIV viral antigens. Thestrict correlation of HCV with drug abuse suggests that HCVmay represent a marker of such risk group rather than having apathogenetic role.

GLUTATHIONE S-TRANSFERASE ISOENZYME PROFILE INNORMAL HUMAN KIDNEY AND RENAL CELL CARCI-NOMAT Simic1, J Mimic-Oka1, Z Reljic1, D Dragicevic2, P Dragicevic2

1Institute of Biochemistry and 2Institute of Urology, Faculty ofMedicine, University of Belgrade, Yugoslavia.

The exposure of kidney cells to the toxic metabolites of variousorigins, which may have carcinogenic potential, is supposed toplay role in the initiation of renal cell carcinoma. Glutathione S–transferases (GSTs), a family of detoxification enzymes, play acritical role in protecting the kidney by catalyzing conjugation ofdifferent carcinogens with glutathione.GST isoenzyme profile was investigated in normal human kid-ney, different renal tumors and corresponding kidney tissueadjacent to human renal tumors. Enzyme purification was per-formed by using affinity chromatography and isoelectricchromatofocusing.Purification of normal kidneys’ GSTs by affinity chromatogra-phy revealed the presence of two GST fractions: flow–throughtGST (26-28%), with lower affinity for GSH linked to epoxy–activated agarose affinity resin and GST fraction tightly boundto affinity matrix. Further purification of bound GSTs resultedin a rich profile of different GST isoenzymes with balanced ex-pression of both anionic and cationic forms. The results ob-tained suggest efficient cellular anti-carcinogenic potential innormal kidney. However, kidney tissue adjacent to human renaltumors had substantially less flow-through GST fraction (1-4%), whereas renal tumors did not express flow-through GST atall. Isoelectric focussing indicated significantly smaller numberof GST isoenzymes in non-tumor kidney regions when com-pared to normal kidney, with anionic forms being dominant.Isoenzyme profile of renal carcinoma tissue differed significantlyfrom both non-tumor adjacent renal tissue and normal kidney.The GST pattern in renal cell carcinoma showed the predomi-nance of anionic GST forms.Based on the results obtained, it can be concluded that patientswith renal cell carcinoma have defective anti-carcinogenic po-tential due to the qualitative changes in GST expression.

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LIPOPOLYSACCHARIDE-INDUCED CHEMOKINE GENEEXPRES-SION IN MESANGIAL CELLS1H Rim, 2JW Park1Dept. of Internal Medicine, Kosin Univ. Gospel Hospital,Pusan, Korea, 2Dept. of Immunology, Keimyung Univ.,Taegu, Korea

This study was designed to investigate the molecular mecha-nism of chemokine induction by lipopolysaccharide (LPS) in thecourses of infections. Chemokine gene expression was evalu-ated by the RT-PCR assay using RNAs isolated either fromkidneys of LPS-injected mice or from the mesangial cells stimu-lated with LPS. Chemokine biological activity was also demon-strated by the chemotaxis assay. LPS was shown to induce di-rectly IFN-γ inducible protein 10 (IP-10) and monokine inducedby interferon gamma (MIG) through mesangial cells. As the LPSinduction of IL-12 gene expression was shown previously in themesangial cells, chemokine cascade could be induced alsothrough IL-12 pathway. To evaluate this pathway, normal andgamma interferon knock-out (GKO) mice were injected with IL-2 and/or IL-12. MIG and IP-10 gene expression was detected innormal kidney, however, absent in the GKO mouse kidney indi-cating that chemokine induction was mediated throughmesangial IL-12 and IFN- γ. It was also demonstrated that IFN-γ alone induce these chemokines in the mesangial cell culturesystem. Furthermore, sodium salicylate, wortmanin and pip-erazine were added to the mesangial cell culture system to evalu-ate LPS signalling pathway. All of these agents blocked LPSmediated chemokine induction suggesting the activation of nu-clear factor-κ B pathway.It is concluded from this study that mesangial cells are thetarget of LPS in the renal failure resulting from the systemicinfections. LPS induces chemokines directly and/or IL-12 in themesangial cells. Mesangial IL-12 may activate T cells whichinduce also chemokine cascade and inflammation ultimately.

CLINICAL SIGNIFICANCE OF THE RENAL EXPRESSIONOF ICAM-1 IN IgA NEPHROPATHY (IgAN)P Arrizabalaga, C Ascaso, M Solé, X Cuevas1, J Soler2, J Pascual3,A DarnellS. of Nephrology, Pathology, Biostatistical Unit, Hospital Clinic,Barcelona; Hospital of Tarrasa1, Manresa2, Clinica Gerona3. Spain.

Introduction: abnormal ICAM-1 expression on proximal tubuleepithelium is associated with infiltration of T-cells andmacrophages in IgAN (Arch Pathol Lab Med 1998; 122: 817-822).Objective: to analyze the relation between the tubular and inter-stitial expression of ICAM- I and the renal disfunction in theIgAN.Material and methods: in 32 patients with IgAN, 39 ± 16 (x ±SD) years old, protenuria (Pr) 2 ± 1.8 g/24h and creatinine (Cr)in serum 1.76 ± 1.12 mg/dI, we have assessed the tubular andinterstitial expression of intercellular adhesion molecule-1(ICAM-1) with monoclonal antibody CD54 byavidin-biotin-peroxidase and we analyzed their relation withthe Pr and Cr at the moment of renal biopsy and after 2.4 ± 2years. An increase ≥ 50 % over the initial levels was considered asprogressive disease.Results: The ICAM-1+ stain on proximal tubule epithelium wasseen in 13 patients, the median value being 0. 11 ± 0. 18 mm2 /mm2 of tubule. The number of ICAM-1+ interstitial leukocyteswas 234 ± 307 / mm 2 of interstitium. The Pr was 2.7 ± 1.5 g/24 h in the patients with ICAM-1+ tubular expression versus 1.5± 1.8 g/24 h (U=44, p=0.005) in the patients without. Correla-tion was found between the ICAM-1+ tubular expression andthe Pr (r=0.4059, p=0.02). ICAM-1+ interstitial leukocytes were379.3 ± 371 / mm2 in the patients with HBP and 108 ± 164.3 /mm2 in the patients without (U=52, p=0.03). Correlation wasfound between the ICAM-1+ interstitial expression and the Cr(r=0,6343, p<0,001), ICAM-1+ interstitial leukocytes were 516 ±3 60 / mm2 in the patients with increase ≥ 50 % in Cr and 66±87,8 / mm2 in the patients without (U= 16, p=0. 004).Conclusions: the tubular and interstitial ICAM-1+ expressionwould reflect the seventy of the renal disturbance in IgAN.ICAM-1 interstitial more than ICAM-1 tubular can play a roleas marker of progression in this disease.

CAPTOPRIL VERSUS HYDRALAZIN AND MORPHOLOGIC CHANGES INSHR WITH ADRIAMYCIN NEPHROPATHYD.B. Jovanovic1, Lj. Djukanovic1, J. Dimitrijevic1, A. Starevic1, M. Jerkic3, Dj.Jovovic3, J. Varagic3, Z. Dragojlovic2

Clinic of Nephrology1 and Medical Biochemistry2, Clinic Center of Serbia, andInstitute for Medical Research3,Belgrade, Yugoslavia

Antihypertensive therapy has been shown as one of the ways of slowing downthe progression of chronic renal failure (CRF). The most important factor forslowing down CRF progression is to regulate blood pressure independently ofkind of antihypertensive drugs, as shown in our recent study (Clin Nephrol1998;50(6):390-392). The aim of this study was to compare effects of captopril(C) and hydralazin (H) on morphologic changes in spontaneously hypertensiverats (SHR) with adriamycin (ADR) nephropathy.Adult female SHR were divided into four groups: 1. Control group: 12 SHR; 2.ADR group: 27 SHR treated with ADR (2mg/kg i.v. twice in 20 days); 3. ADR-C group: 30 SHR treated with ADR and thereafter with C (60mg/kg/day); 4.ADR-H group: 17 SHR treated with ADR and thereafter with H (6mg/kg/day).Rats were sacrificed at week 18 after second ADR injection and hystologic analy-sis was semiquantitatively performed by calculation of glomerular index, vas-cular index and index of interstitial fibrosis and tubular atrophy.Both antihypertensive agents normalized systemic blood pressure, but failed toprevent proteinuria. H was significantly effective than C in slowing downglomerular changes in ADR SHR (ADR vs ADR-C vs ADR-H vs Control = 5.00vs 3.66 vs 2.23 vs 0.14) (p<0.001). The more severe changes in arterial bloodvessels in week 18 occurred in control group and the difference was significantas compared to all other groups (p<0.001). In ADR-H group changes in arterialblood vessels were significantly lower than in ADR-C group and similar toADR group (2.20 vs 2.90 vs 2.14 vs 3.40). C failed to prevent tubular atrophy inADR SHR, but H reduced tubular changes significantly (2.44 vs 2.20 vs 1.64 vs1.00). H was significantly effective than C in slowing down interstitial changesin ADR SHR (3.22 vs 2.40 vs 1.45 vs 0.14).So, we can conclude that hydralazine reduced morphological changes in ADRSHR more significantly than captopril.

URINARY EXCRETION OF VASCULAR ENDOTHELIALCELL GROWTH FACTOR (VEGF) IN MEMBRANOUSGLOMERULONEPHRITIS (MGN)E. Honkanen, A-M. Teppo, C. Grönhagen-RiskaHelsinki University Central Hospital, Department of Medicine,Division of Nephrology, Helsinki, Finland

VEGF is a dimeric glycoprotein which increases vascular perme-ability, stimulates angiogenesis, and protease activity. It is nor-mally expressed by podocytes and glomerular endothelial cellsbut its role in the pathophysiology of proteinuria and glomeru-lar diseases is unclear.By using a novel sandwich enzyme immunoassay we measuredurinary VEGF excretion in 30 patients with idiopathic MGN, 9with minimal change (MC) glomerulopathy, 8 with necrotizingglomerulonephritis associated with systemic vasculitis (VAS),12 with diabetic nephropathy (DNP), and 33 normal controls.The mean (±SE) urinary VEGF was significantly lower in MGN(16±3 [95%CI 10 to 23] ng/mmol crea) than in the normal con-trols (68±10 [CI 49 to 88] ng/mmol crea; P< 0.0001 ANOVA).In MC and DNP the excretion was unchanged (55±14 [CI 24 to86] and 101±25 [CI 45 to 156] ng/mmol crea, respectively;P=NS) whereas VAS patients had elevated VEGF excretion(184±68 [CI 24 to 344] ng/mmol crea; P<0.01) compared to thenormal controls. In 17 MGN patients followed up for 12 monthsdecreasing proteinuria paralelled increasing urinary VEGF whilepersistent nephrotic syndrome was associated with stable ordecreasing VEGF excretion (D proteinuria vs. D VEGF: r=-0.51,P=0.003).In conclusion, urinary VEGF excretion is decreased in MGN butnot in the other glomerular diseases studied and decreasingclinical activity (proteinuria) is associated with increasing VEGFexcretion. Changes in VEGF excretion may indicate reversiblepodocyte injury in MGN.

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HEMORRHAGIC FEVER WITH RENAL SYNDROMES. Susa, Lj. SusaSerbian Academy of Sciences and Arts, Belgrade, Yugoslavia

Hemorrhagic fever with a renal syndrome (HFRS) is spread overmany countries of the world. In Yugoslavia hemorrhagic feverhas been found and described some time ago. We detected thefirst epidemic in our country in 1961 in a military unit. In ourcountry hemorrhagic fever with renal syndrome which corre-sponds to Corean type of hemorrhagic fever is most frequentlymet. Our to date investigations of the HFRS in 350 ptspathogenesis point out that immunological mechanisms aremoss important in development of this disease. We have notedthe increase of IgG, IgM, IgD and IgA in many of our patients.The level of IgA has increased more quickly than the level of IgG.We have found the highest circulating immune complexes in98,2% of our patients with severe forms of the HFRS, at theacute stage, i.e. in the first 30 days since the beginning of thedisease. After the 8th week all patients had a negative titre. Wealso found immunological disturbances at early stages of dis-ease, especially in the febrile phase. These findings made usaware of the fact that the immunity complexes are being laiddown in the kidneys in the form of deposits which causa kidneyimpairment of varying intensity followed by extensive proteinu-ria of the glomerular type, which indirectly points at variousforms of glomerular lesions. The clinical picture of HFRS is ofvarying severity and depends on many factors, primarily on thedegree of kidney impairment. When renal biopsies were donehistopathological changes were found in all such patients, as-suming various forms of mesangio-proliferative, membrana-proliferative, focal segmental, and membranous glomerulone-phritis with evident interstitial changes in the sense of secondaryinterstitial nephritis. Prevention and treatment should be carriedout on the basis of unique criteria. There is today no specifictreatment of the HFRS. From the point of view of the latestachievements and knowledge about fast diagnosing and use ofmodern technology in the treatment of the HFRS mortality over5% cannot be tolerated.

FLOW CYTOMETRIC DETECTION OF ANTI-ENDOTHELIALCELL ANTIBODIES (AECA) IN RENAL VASCULITIC DIS-EASEP R Evans, S Terry, T Carr, M Rogerson, M Waldron, P BassDepts. of Pathology, Immunology, and Division of Medicineand Paediatric Nephrology, Southampton University Hospital,UK

Endothelial cells are the foremost targets in renal vasculitis. Thepossible pathogenic role of AECA in adult and paediatric renalvasculitis is controversial. Techniques to measure AECA oftenhave lacked standardization. We have used flow cytometry (FC)to detect low levels of antibody directed to the cell surface ofendothelial cell lines to give a more relevant assessment of invitro antibody mediated damage. Eighteen patients with biopsyproven renal vasculitis were studied. Of the adults (N=14) therewere 6 cases of Wegeners Granulomatosis (WG), 5 SystemicLupus Erythematosis (SLE) and 3 Henoch Schonlein Purpuras(HSP); in the paediatric group there were 2 HSP’s, 1 WG and 1SLE. 130 serum samples were examined using flow cytometryand a panel of endothelial, epithelial, hybrid cell lines and renalproximal tubular cells. Carefully screened selected negative con-trol sera were used. AECA were found to be present in signifi-cant amounts in the adult vasculitic group compared to normaladults, particularly in adult WG (p=0.005). AECA in adult SLEwas of borderline significance (p=0.05). The binding intensity ofAECA reflected the clinical course in WG. No antibodies weredetected in the paediatric group. These findings suggest AECAhas a significant pathogenic role in a range of renal vasculitides.

PROTECTIVE MECHANISM OF HEME OXYGENASE(HO)-1 IN HEMIN-INDUCED CELL INJURYT Toma, A Yachie, K Ohta, Y Kasahara, S KoizumiDept. of Pediatr. and Dept. of Lab. Sci., Faculty of Med.,Kanazawa University, Kanazawa, Japan

HO-1 is an inducible form of HO and it is known to protectrenal damage through its anti-oxidant effect. However, pre-cise protective role of HO-1 in stress-induced cell damage islargely unknown. We established a lymphoblastoid cell line(LCL) from a patient with congenital HO-1 deficiency andthe mechanism of cell protection by HO-1 was studied.LCLs were established from the patient and controls byimmortalization with EB virus. Hemin-induced cell injurywas evaluated by a flow cytometry using FITC-conjugatedAnnexin V binding. Apoferritin or bilirubin was added tothe culture at various concentrations. Retrovirus-based HO-1 vector or vehicle control was transfected to HO-1 defi-cient LCL. HO-1 expressions by these cells were detectedby immunoblotting and flow cytometry.HO-1 deficient LCL was extremely sensitive to hemin-in-duced cell injury, whereas LCLs from controls were resist-ant. Apoferritin or bilirubin did not inhibit the cell injury.HO-1 gene transfection resulted in constitutive expressionof HO-1 and nearly complete reversal of the cell death.These results suggest the existence of unknown protectivemechanism in HO-1-mediated protection of the cell injury,in addition to the effects of ferritin or bilirubin. WhetherHO-1 induces a novel cytoprotective molecule, or it plays adirect role in hemin-stimulated cultures, is yet to be evalu-ated.

HEME OXYGENASE (HO)-1 EXPRESSION BY RENAL TUBU-LAR EPITHELIUM IN RENAL DISEASESK Fujimoto, H Kaneda, A Seno, T Toma, K Ohta, Y Kasahara, AYachie, S KoizumiDept. of Pediatr. and Dept. of Lab. Sci., Faculty of Med.,Kanazawa University, Kanazawa, Japan

Tubular epithelial cells are constantly exposed to variousoxidative stresses and are vulnerable to the injury wheneverthese insults are overwhelming. HO-1 protects these cells byneutralizing the noxious agents, including heme proteins. Wecompared HO-1 expression in renal tissue in various renal dis-eases and analyzed the pattern of HO-1 expression in relation toclinical symptoms.Renal biopsy specimens were obtained, dewaxed and stainedfor HO-1 using rabbit anti-human HO-1 antibody. Distributionof HO-1 staining was analyzed and the intensity was gradedinto four different scales.Pathological diagnosis and clinical symptoms, including pro-teinuria and hematuria were compared with the result of immu-nohistochemistry.HO-1 expression was minimum in normal kidney. Regardless ofthe type of renal pathology, HO-1 staining within the proximaltubules tended to be more intense with greater degree ofhematuria. Similar, but less remarkable tendency was observedfor the distal tubules. HO-1 expression was virtually absent inHO-1 deficiency, although gross hemolysis with persistenthematuria was associated with severe injury and atrophy oftubular epithelium.These results indicate that HO-1 expression by tubular epithe-lium may serve as a significant protective barrier in the case ofglomerular injury and subsequent hematuria.

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DETECTION AND PREVENTION OF HEPATITIS C IN ANEPHROLOGY UNIT. A LONG TIME FOLLOW UP.Almroth G, Ekermo B, Svensson G, Månsson A, Widell A,Sweden.

Sera from 21 patients, who had ben in hemodialysis (HD)during 1988-91, known to be positive (or close to the cut offlevel, one) in ELISA, were tested in 1991 for antibodies tohepatitis C virus (anti-HCV) with recombinant immunoblotassay 2 (RIBA 2) and for hepatitis C virus ribonucleic acid(HCV-RNA) with polymerase chain reaction (PCR). RIBAwas positive in 9 cases and indeterminate in 6, while HCV-RNA was present in 12/15 tested sera.In Oct 1991; 9/58 patients (16 %) from our HD unit wereconsidered as positive for PCR or RIBA. By the end of 1996there were 4 out of 37 (11%) HD patients in the unit whowere considered as possible HCV-carriers as determinedby ELISA, RIBA or PCR. The condition in the whole neph-rology unit (HD, peritoneal dialysis, transplanted patients)was also equal between Oct.-91 and the end of 1996 (10 vs 9% possible carriers).Preventive measures may limit the number of carriers in anephrology unit.Indeterminate RIBA-results should be regarded with cau-tion due to the relative immunodeficiency of uremic pa-tients.

INFLUENCE OF HAEMODIALYSIS ON MARKERS OFNEUTROPHILS APOPTOSIS IN VITROE. Majewska*, Z. Sulowska**, Z. Baj*, J. Rysz***, M. Luciak****Department of Pathophysiology and Clinical Immunology,MMA, **Microbiology and Virology Center, Polish Academy ofSciences,***2and Department of Internal Medicine, MMA, Lodz,Poland

Interaction of cell surface with haemodialysis membranes, dia-lysate-derived bacterial contamination, complement activation,IL-1 and TNFα synthesis can influence the neutrophil apoptosis.We tested 12 patients with end-stage renal disease on a long-term haemodialysis (HD) treatment. The blood samples weredrawn before, at 20th min and 4th hr of HD. Using the flowcytometry, we examined the ability of neutrophils to undergoapoptosis by estimating percentage of apoptotic neutrophils(based on Annexin V and propidium iodide binding), apoptosis–related (Fas,p53) and survival–related (bcl-2) proteins expres-sion in the whole blood neutrophils cultured for 18 hrs. All dataare presented as mean fluorescence intensity ± standard error.Evaluation of statistical significance was performed byWilcoxon’s signed rank test and Spearman rank correlation analy-sis. P values ≤ 0.05 were considered significant. The percentageof apoptotic neutrophils in cultured whole blood sampled be-fore HD (61.0±15%) is higher compared with 20th min(42.1±8,3%) and 4th hr of HD (54.8±14,9%). The changes inpercentage of apoptotic neutrophils positively correlate withFas molecule expression. HD does not affect the expression ofp53 and bcl-2 cytoplasmic expression in neutrophils, but wehave revealed the positive and negative correlation between thepercentage of apoptotic neutrophils and p53 and bcl-2 proteinsexpression at each time point, respectively. Presented resultssuggest a transient selected sequestration of potentially apoptoticneutrophils.

GLOMERULAR AND INTERSTITIAL T CELL SUBESTS INPRIMARY CRESCENTIC GLOMERULONEPHRITIS ANDCLASS IV LUPUS NEPHRITISL. Grcevska, M. PolenakovikDepartment of Nephrology, Clinical Center, Skopje, R.Macedonia

A remarkable mononuclear cell infiltrate may be associated withprimary and secondary glomerulonephritides. Class IV lupusnephritis (diffuse proliferation)(LN) may have similar histopatho-logical features as primary crescentic glomerulonephritis(CGN).In the present study monoclonal antibodies to T-cell subsetswere used to analyze interstitial cellular infiltrates andintraglomerular cells in primary CGN and class IV LN. 20 renalbiopsy specimens of primary CGN and 15 of class IV LN wereanalyzed, 10 renal tissues from accidental authopsies wereused as control group. Ten microscopic fields at magnification500 were analyzed for each patient, and the number of positivecells was expressed as M+-SEM. The number of glomerularCD3 cells (3,7+-0,3) was increased in class IV LN, compared toCGN (2,3+-0,4, p<0,01) and controls (0,04+-0,00, p<0,001).Intraglomerular CD4/CD8 ratio was elevated in LN(1,8) com-pared to CGN(1,2 p<0,05) and controls (0,93 p<0,01). The situ-ation was different with the interstitial infiltration, CD3 cellswere increased in CGN (324+-126), compared to LN (282+-69,p<0,05) and controls (3,8+-0,5, p<0,01). But, CD4/CD8 ratiowas higher in LN (2,1) compared to CGN (1,9 p<0,05) andcontrols (1,3 p<0,01). We can conclude that CD3 positiveintraglomerular and interstitial cells were significantly increasedin both CGN and class IV LN, intraglomerular values were higherin LN and interstitial in CGN. Interstitial and intraglomerularCD4/CD8 ratio was also higher.

EXPRESSION AND DISTRIBUTION OF ADHESION MOL-ECULES IN RENAL CELL CARCINOMAG. Basta-Jovanovic, Z. Jovanovic, S. Kovacevic, L. KovacevicInstitute of Pathology, Medical Faculty Belgrade, Yugoslavia

The aim of our study was to analyse the level of expression andthe distribution of different adhesion moleculs in different typesof renal cell carcinoma (RCC), to compaire the expression ofadhesion molecules with the distribution of different compo-nents of extracellular matrix and to compaire adhesion mol-ecules expression with the degree of tumor malignancy. Tumortissue from 14 renal cell carcinomas that were classified accord-ing to nuclear gradus G1-G3 and to cell type: 9 clear cell, 2chromophilic (1 eosinophilic and 1 basophilic), 1 chromophobeand 2 spindle cell. In addition, 5 normal human kidneys wereanalysed as controls. On cryostat sections the monoclonal anti-bodies which defined different adhesion molecules (cadherins E,N, P; integrins alpha 1-6, alphaV; and ICAM-1) were applied byindirect immunoperoxidase method. The results of our investi-gations showed that in renal cell carcinoma there is either ex-pression of N or E cadherins, or their combined expression whichdoes not depend on the cell type of renal cell carcinoma or theirnuclear gradus. Cadherin P was not detected in any investi-gated type of renal cell carcinoma. Expression of alpha1, 2 and4 integrins was variable in different cases. Alpha 3, alpha 5 andalpha V expression was found in almost all our investigatedcases. There was no correlation between alpha1-alpha 5 integrinexpression and the cell type or nuclear gradus of renal cell carci-noma, while alpha V integrin expression was found to be incorrelation with nuclear gradus of renal cell carcinoma. Inten-sive expression of ICAM 1 was found in all cell types of renal cellcarcinoma except in chromophobe type where it was absent.

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Abstracts



INTERSTITIAL MYOFIBROBLASTIC ACTIVITY IN MEMBRANOUS NEPH-ROPATHY (MN) AS A PROGNOSTIC FACTOR AND ITS RELATIONSHIP WITHTHE EXPRESSION OF PDGF AND TGF-β.S Mezzano1, ME Burgos1, A Droguett1, C Aros1, L Ardiles1, I Caorsi1 , J Egido 2.1Div. Nephrology, Univ. Austral, Valdivia, Chile; 2 Fundación Jiménez Díaz,Madrid, España.

The myofibroblast (Mf) is recognized as an important participant in progres-sive renal fibrosis. In order to know whether the presence of Mfs predicts pro-gression of the MN and its relationship with the expression of the profibrogeniccytokines PDGF-BB and TGF-β, we have studied renal biopsies of 25 patientswith idiopathic MN. 13 patients developed progressive disease while 12 showeda non-progressive variant. Mfs were identified by immunohistochemistry (IMH)using monoclonal anti α-SMA antibodies. Detection of PDGF-BB and TGF-β(protein and/or mRNA) was done by IMH and by in situ hybridization (ISH).IMH coupled to ISH was performed to detect simultaneously α-SMA positivecells, and cells expressing TGF-B and PDGF-BB. Monocyte/macrophages wereidentified using monoclonal anti CD68 antibodies.α-SMA interstitial positive cells were mainly detected in patients with a pro-gressive disease (13 out 13 progressive versus 4 out 12 with non-progressive;p=0.0004); the staining distribution was mainly in peritubular andperiglomerular cortical interstitium, with a stronger expression in patientswith a progressive MN. The presence of Mfs was significantly associated withthe tubular interstitial cell infiltration (p=0.02), interstitial fibrosis (p=0.005),and with the severity of proteinuria (p=0.03). In 4 progressive and 3 non-pro-gressive cases, we observed α-SMA positive cells at the glomerular level thatrepresent a phenotypic modulation of activated mesangial cells. Double IMHcoupled with ISH was performed in some progressive cases, observing α-SMAimmunostaining mainly in the peritubular intestitium, and the TGF-B and PDGF-BB mRNA expression in the surrounding tubular epithelial cells. The IMH forCD68 was negative or weak in the majority of cases.On the whole, in patients with MN, the expression of interstitial α-SMA stain-ing indicates myofibroblastic activation and is associated to inflammatory cellinfiltrates and progression of renal disease. The increase in PDGF and TGF-βstaining suggest that they could be involved in the myofibroblast activity. Bycontrast, the glomerular expression of α-SMA, did not correlate with the degreeof glomerulosclerosis. ( Supported by Fondecyt 1970628 ).

CHARACTERIZATION OF RENAL ANDCARDIOVASCULAR APOPTOSIS FOLLOWINGENDOTOXEMIC SHOCK IN RATS.ME Grossman1, AH Campos1 MF Franco2 and N Schor1.Nephrology1 and Pathology2 Divisions, UNIFESP, SP, Brazil.

Apoptosis may be involved in organ dysfunction observedduring endotoxic shock. However, its involvement in renal failureconsequent to sepsis was not clarified. We addressed this questionemploying a model of murine endotoxic shock. Male Wistar ratswere treated with E. coli lipopolysaccharide (LPS, 10 mg/kg ofbody weight; i.p.) and the animals were sacrificed at differenttime intervals. Heart, aorta and kidney apoptosis was evaluatedby means of DNA agarose gel electrophoresis (AGE), Hoechst33342 DNA staining, hematoxilin/eosin (HE), and DNA nickend labeling (TUNEL). Mortality rate among animals treatedfor 24 h was higher than 40%. Neither DNA AGE nor HE stainingwere able to detect apoptosis in survivors after 6, 24 or 48 h inany of the studied organs. In addition, no inflammatory reactionwas disclosed by HE. On the other hand, preliminary data fromHoechst 33342 fluorescent staining showed signs suggestive ofapoptosis in aortic endothelium, myocytes and renal cells after24 h, and to a lesser extent, after 6 h. TUNEL confirmed andextended this findings, showing that renal apoptosis wasconcentrated mainly in the tubular region, sparing relatively theglomeruli, and being present even after 48 h. We conclude that,besides its participation in the cardiovascular compromiseobserved in endotoxic shock, apoptosis may also be involved inthe genesis of sepsis-related renal dysfunction. Support:FAPESP

NO-INDUCED APOPTOSIS AS A MECHANISM OFMESANGIOLYSIS IN ANTI-THY 1.1 NEPHRITIS.Z. Hruby, M. Rosinski, B. Tyran, W. KopelDept. Of Nephrology, Wroclaw University of Medicine, Wroclaw,Poland

Apoptotic cell death has been regarded as a mechanism ofhomeostatic control of mesangial hypercellularity in the prolif-erative phase of anti-Thy 1.1 nephritis in the rat. Nonetheless,since administration of an iNOS inhibitor in the earliest,mesangiolytic phase of nephropathy notably reduced mesangialcell damage, we assessed the extent of NO-linked glomerularapoptosis on days 0-4 after i.v. injection of nephritogenic anti-Thy 1.1 antibody in Wistar rats.On day 2 after induction, the apoptotic index in glomeruli (as-sessed by TUNEL method) reached peak values (0.41+-0.03),accompanied by enhanced generation of nitric oxide in renaltissue (Griess method on slices of renal cortex: 1.83+-0.49 µmol)and high expression of glomerular iNOS mRNA assessed by thein situ RT-PCR. On days 0-3 mesangial cell proliferation wasundetectable, determined by immunostaining for glomerularPCNA. Similarly, we have not revealed any presence of infiltrat-ing macrophages (staining with labeled ED-2 antibody) in ne-phritic glomeruli at this phase of the disease. Administration ofl-NAME, an iNOS inhibitor from day 0 of nephropathy resultedin marked reduction of apoptotic index (day 2, 0.19+-0.05) andno demonstrable production of nitrite in kidney tissueIn conclusion, apoptosis of mesangial cells linked with the au-tologous activity of iNOS, may at least in part, be responsiblefor mesangiolysis at the earliest stage of anti-Thy 1.1 nephritis.

SURFACE MARKER EXPRESSION, IN VITRO LYMPHOCYTESURVIVAL AND SOLUBLE ACTIVATION MARKERS DUR-ING HAEMODIALYSIS SESSIONN. Ursea, L. Petrescu“Dr Carol Davila” Teaching Hospital of Nephrology, Bucharest,Romania

Objective. In order to evaluate the influence of HD session on Tcell and monocyte surface markers expression, lymphocyte acti-vation, and in vitro peripheral blood lymphocyte survival, weinvestigated 20 HD pts [age: 45-55 yrs (51±5.7); HD duration4±1.5yrs.; M=10, F=10; primary renal disease: GN (n=14), IN(n=3) and nephroangiosclerosis (n=3)]; free of intercurrent in-flammatory diseases and without immunosupresive drugs dur-ing last three months. HD (3x4.5hrs/week) was performed withpolysulphone membranes and bicarbonate dialysate. Ten healthy,age and gender-matched controls were used.Methods. Blood samples were drown at the beginning, at 15, 30,60 min and at the end of the HD session. Flow-cytometry wasperformed with CD3/CD4, CD3/CD8, CD11b/CD18 two-colormonoclonal antibodies (MAbs). Peripheral blood lymphocytes(PBL) survival in vitro was assessed by Trypan blue exclusionmethod at 24, 48 and 72 hours of culture. Electrophoresis ofDNA extracted from in vitro cultured PBLs was performed toshow the apoptosis pattern of internucleosomal cleavage.Results. The percentage of CD3/CD4 lymphocytes increasedduring HD session (r=0.902, p<0.05). CD3/CD8 remained con-stant on lymphocytes as well as CD11b/CD18 on monocytes. Invitro survival of PBL decreased faster during the culture time inpatients than in controls (p<0.05 at 48h and 72h) and during theHD session (r=0.995, p<0.001 at 72h), but was similar to con-trols at baseline in the 24 h culture. Cell death was mainlyapoptotic, as suggested by morphological criteria and DNAladder in electrophoresis.Conclusions. HD session had little or no influence on T cell andmonocytes phenotypes, respectively. CD4 positive T cell popu-lation transiently increased during the HD session starting withthe first 15 minutes. PBLs of HD patients seem to be more proneto die in vitro, mostly by apoptosis, but only during the HDsession.

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Abstracts



BLOCKADE OF LFA-ICAM1 INTERACTIONS AND MCP1 DID NOT PRE-VENT THE DEVELOPEMENT OF INTERSTITIAL FIBROSIS IN EXPERI-MENTAL CHRONIC PROLIFERATIVE GLOMERULONEPHRITIS.Segarra A, Arbós MA, Buscà B, Argelaguer X, Alvarez E, Quiles MT, SchwartzS and Piera LL.Research Unit. Nephrology Department. Hospital Vall d’ Hebrón. Barcelona

Aim: To analyze whether the specific blockade of LFA-ICAM interactions andMCP1, reduced interstitial fibrosis and prevented chronic renal failure in anexperimental model of chronic proliferative glomerulonephritis.Methods. Eight weeks after having induced a chronic anti Thy1 proliferativeglomerulonephritis, 60 Wistar male rats weighing 250 g were randomly as-signed to one of six groups. Group 1 received no therapy and served as a control.Groups 2 to 6 were the intervention groups each of them receiving therapy withblocking monoclonal antibodies against both ICAM1 and MCP1 administeredip in increasing doses from week 10 to week 14. Specific antibody binding wasdetermined by flow cytometry. At week 14, all animals were sacrified and theseverity of renal failure, interstitial infiltrate and interstitial fibrosis among thedifferent groups were compared using immunohistochemical techniques andRT PCR for collagen III and TGF beta.Results: At week eight all experimentals groups showed renal failure, proteinu-ria and high blood pressure with no significant differences among them. Duringfollow-up, all animals developed progressive renal failure and increasing pro-teinuria. At week 14, all animals showed advanced renal failure with serumcreatinine levels ranging from 1.9 mg/dL to 4 mg/dL and urinary protein ex-cretion higher than 500 mg/day. Serum creatinine, proteinuria, interstitial in-filtrate, interstitial fibrosis and m RNA TGF beta/B actin and col III/B actinratios were all similar in treated and untreated groups independently of the doseof monoclonal antibody administered.Conclusion. High doses of blocking antibodies against LFA-ICAM1 and MCP1failed to reduce the amount of interstitial mononuclear infiltrate and did notprevent the development of progressive interstitial fibrosis when tested in achronic model of proliferative glomerulonephritis.

COMPARATIVE EFFECTS OF FOUR IMMUNOSUPPRESORDRUGS ON RENAL FUNCTION AND INTERSTITIAL FIBRO-SIS IN THE LATE PHASE OF EXPERIMENTAL CHRONICPROLIFERATIVE NEPHRITIS.Segarra A, Arbós MA, Buscà B, Argelaguer X, Alvarez E, QuilesMT, Schwartz S and Piera LL.Research Unit. Nephrology Department. Hospital Vall d’ Hebrón.Barcelona

Aim: To compare the effect of four immnunossupresor drugs oninterstitial infiltrates, interstial fibrosis and renal function in anexperimental model of chronic proliferative glomerulonephritis.Methods: Eight weeks after having induced a chronic anti Thy1proliferative glomerulonephritis, 50 Wistar male rats weighing225 to 250 g were randomly assigned to one of five groups of 10animals. Group 1 received no therapy and groups 2 to 5 receivedtherapy with prednisone (0,25 mg/day), azathioprine (0,20 mg/day), tacrolimus (0,02 mg/day) and micophenolate (0,01 mg/day) respectively from week 8 to week 16. At week 16, animalswere sacrified and we compared the severity of renal failure,interstitial infiltrate and interstitial fibrosis among the differentgroups using immunohistochemical techniques and RT PCR forcollagen III and TGF beta.Results: At week eight all experimentals groups showed renalfailure, proteinuria and high blood pressure with no significantdifferences among them. During follow-up, groups 1 to 4 showedprogressive renal failure and presistent proteinuria whereas ingroup 5, renal function remained stable. At week 16, serumcreatinines were 2.16 (0.3), 2.3 (0.6), 2.11 (0.4) and 1.98 (0.1)and 1.68 (0.47) respectively (p<0.001) and urinary protein ex-cretion was 0.52 (0.17), 0.82 (0,21), 0.72 (0.22), 0.45 (0.11) and0.21 (0.1) respectively (p<0.001). When compared with the othergroups, group 5 showed significantly lower interstitial fibrosisand lower m RNA TGF beta/B actin and col III/B actin ratios.Conclusions: In the chronic phase of renal interstitial inflamma-tion and fibrosis micophenolate mofetil was the only drug ableto slow down the development of progressive renal failure. Thiseffect was related to a significant reduction of renal expressionof TGF beta and collagen III.

DIFFERENTIAL EXPRESSION AND REGULATION OFENDOTHELIN 1 mRNA AND MDR mRNA IN RENAL TUBULARCELL LINES.F Arrebola, F O’Valle, A Olmo, M Aguilar, B Espigares, M Guillen, DAguilar, RG Del MoralDept. de Anatomía Patológica, Hospital Universitario San Cecilio,18012 Granada, Spain

P-glycoprotein (P-gp) has an important role in protection againstxenobiotics. Endothelin 1 (Et1) is a powerful vasoconstrictor and itsoverexpression in renal cells can be one of the mediators of drug-induced tubular lesions. Transcriptional regulation of the multi-drugresistance (MDR1) and Et1 genes share some activation mechanismsthrough protein kinase C and the AP1 promoter. In this study we showthe different behaviours of MDR and ET1 expression in renal cells invitro, their modulation by immunosuppressive (IS) andchemotherapeutic (CT) drugs, and their relationship with the cell cycle.In vitro assays were done with renal tubule-derived MDCK and LLC-PK1 cell lines maintained in MEM culture medium with 5% IFBS.Pharmacological induction was achieved by incubation for 15 dayswith sublethal doses (IC

30) of the different nephrotoxic drugs (IS and

CT). Levels of expression of the MDR1 and endothelin 1 genes weredetermined by RT-PCR. Flow cytometry was used to measure thefollowing parameters: P-gp expression (JSB-1 clone), percentage ofpositive cells (%POS) and mean specific fluorescence intensity (MSFI).Cell cycle and growth fraction were also determined.Under basal conditions MDR1 mRNA expression was 0.1 ± 0.01 inMDCK and 0.11 ± 0.007 in LLC-PK1; the %POS and the MSFI of P-gpwere also similar in the renal tubule cell lines (%POS MDCK 83.65 ±22.71, LLC-PK1 81.28 ± 20.5; MSFI MDCK 24.8 ± 3.1, LLC-PK1 23.9 ±5.9). Under these conditions, ET1 mRNA expression was greater thanMDR1 mRNA levels (MDCK 0.69 ± 0.01, LLC-PK1 0.51 ± 0.03). Incuba-tion with drugs (IS or CT) increased MSFI of P-gp and MDR1 mRNAexpression in both lines (p < 0.001, ANOVA). The drugs that inducedthe greatest MDR1 mRNA overexpression induced the greatest reduc-tion in ET1 mRNA expression in renal tubule cell lines. RT-PCR stud-ies showed that ET1 and MDR1 mRNA expression were inverselyrelated (Pearson coefficient r= -0.488, p<0.05). P-gp and MDR1 mRNAlevels were directly related to the percentage of cells in S and G

2M

phases (Pearson coefficient r= 0.465 and 0.576, respectively), while ET1mRNA levels showed no statistical correlation.In conclusion, our findings suggest that one of the keys to nephrotox-icity control is the imbalance in MDR1 and ET1 mRNA expression.

COMBINED BLOCKADE OF ANGIOTENSIN II - ENDOTHELIN 1 PATHWAYPRESERVED RENAL FUNCTION AND REDUCED INTERSTITIAL FIBRO-SIS IN AN EXPERIMENTAL MODEL OF CHRONIC PROLIFERATIVEGLOMERULONEPHRITIS.Segarra A, Arbós MA, Buscà B,Argelaguer X, Alvarez E, Griera E, Quiles MT,Schwartz S and Piera LL.Research Unit. Nephrology Department. Hospital Vall d’ Hebrón. Barcelona.

Aim: To analyze the efectiveness of angiotensin II –endothelin 1 pathway block-ade in preventing the progression of interstitial fibrosis in an experimentalmodel of chronic proliferative glomerulonephritis.Methods: Eighty male Wistar rats weighing 225 to 250 g,were used for the experi-ments. Eight weeks after having induced a chronic anti Thy1 proliferativeglomerulonephritis, animals were randomly assigned to one of four groups.One control group (group 0) which received no therapy and three interventiongroups (groups 1,2,3) receiving therapy with the specific endothelin 1 receptorantagonist FR 139137 plus enalaprilate, enalaprilate and FR 139137 respec-tively from week 10 to week 20. At week 20, we compared the severity of renalfailure, interstitial infiltrate and interstitial fibrosis among the different groupsusing immunohistochemical techniques, RT PCR and Northern Blot for colla-gen III, TGF beta, angiotensinogen, ET1 receptor and pre-proendotelin.Results: Eight weeks after the first dose of antithy1, blood pressure increasedsignificantly, all animals developed proteinuria and progresive renal failure.At week 8, serum creatinine, blood pressure and proteinuria were similar in allstudy groups. At the end of follow-up, the three intervention groups showedlower levels of proteinuria, serum creatinine and low blood pressure than con-trol group. There were no differences between groups 2 and 3. When comparedwith groups 2 and 3, blood pressure , urinary protein excretion and serum cre-atinine were all significantly lower in animals treated with both drugs (group1). This later group showed also significantly lower intensity of interstitialfibrosis, and lowerTGF beta, ET1 receptor and col III mRNA expression.Conclusions: Long – term combined blockade of angiotensin II and endotelin Areceptors reduced proteinuria,prevented the progression of interstitial fibrosisand preserved renal function in a model of chronic proliferative glomerulone-phritis. These preventive effects could be attributed to direct actions on intersti-tium but were also related to more effective reduction of blood pressure.

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Abstracts



RELATIONSHIP OF HYALINE ARTERIOPATHY WITH mRNA OF INTRARENAL P-GLYCOPROTEIN AND ENDOTHELINS IN CHRONIC CYCLOSPORIN-INDUCED NE-PHROTOXICITY IN RATSA Olmo, C Ramírez, M Aguilar, F Arrebola, ME Reguero, F Revelles, F O’Valle, RG Del Moral.Dept. de Anatomía Patológica, Hospital Universitario San Cecilio, 18012 Granada, Spain.

P-glycoprotein (P-gp) acts physiologically as an efflux pump to expel hydrophobic sub-stances from cells. Our group and others have shown that cyclosporin A (CsA), amongother actions in the kidney, induces P-gp overexpression (Am J Pathol,1995). Endothelin1 (Et1) is widely expressed in the kidney in a variety of physiological or pathologicalsituations, the latter of which can progress to sclerosis. Exposure to cyclosporin A (CsA)of mesangial, endothelial and renal tubule cells induces Et1 overexpression. The patho-physiological function of Et3 in the kidney is currently being debated; Et3 may be involvedin the regulation of water reabsorption through the action of type B tubular receptors.110 male Sprague-Dawley rats fed with a maintenance diet were divided into threegroups: two control groups, one inoculated with 0.9% of sodium chloride (SC) and theother with the solvent used for CsA injection, and an experimental group treated with25 mg CsA per kilogram of body weight per day during 28 or 56 days. We evaluated theexpression levels of P-gp mRNA using the RT-PCR technique. Prepro Et1 and Et3 mRNAwas determined by northern blot (NB).Chronic treatment with CsA induced an increase in the expression of P-gp mRNA in adose and time dependent manner, more evident by 58 days (0.725 vs 0.251, p<0.01,Newman-Keuls test.) (Table). A remarkable finding was that the upregulation of P-gpmRNA was inversely related to the incidence of hyaline arteriopathy (Spearman´s test,r=-0.3819, p<0.01). Prepro Et3 mRNA levels were greatly increased from posttreatmentday 28, whereas prepro Et1 mRNA levels were increased from posttreatment day 56(Table). On day 28 renal lesions clearly correlated with levels of Et3 mRNA. However, onday 56 the key finding was the strong correlation of prepro Et1 mRNA levels in CsAnephrotoxicity with the most important analytical, histological and immunohistochemi-cal alterations.Table 28 Days (x±SD) 56 Days (x±SD) SignificanceGroup: CsA SC CsA SC Two Way

ANOVAP-gp (RT PCR) 0.47±0.33 0.24±0.07 0.72±0.36 0.25±0.22 P<0.01Et1-NB 0.20±0.10 0.23±0.05 0.34±0.09 0.15±0.05 P<0.05Et3-NB 0.48±0.30 0.07±0.11 0.36±0.10 0.10±0.04 P<0.01

The increased expression of P-gp mRNA in CsA-treated compared with untreated ratsand the inverse relation of P-gp mRNA levels with hyaline arteriopathy are in agreementwith previous analytical, histological and immunohistochemical findings, suggesting animportant role for P-gp in the prevention of pharmacological nephrotoxicity by CsAthrough its action as a detoxicant in renal cells. Our results support the hypothesis thatclinical and morphological phenomena related to CsA nephrotoxicity are related to thehypersecretion of endothelins in the progression to CsA-induced interstitial fibrosis. Thechanges are first evident in Et3 expression and angiotensin II accumulation, and are laterreflected in Et1 expression.

PREVALENCE AND DIAGNOSTIC SIGNIFICANCE OF ANTIBODIES TO THEHIGH MOBILITY GROUP NON-HISTONE CHROMOSOMAL PROTEINSHMG1 AND HMG2.Radice A, *Bianchi M E, °Covini G, *Bonaldi T, °Bredi E, Sinico R A and D’AmicoG.Dept of Nephrology S. Carlo Hospital, *Genetics & Biol of MicroorganismsUniversity, °Gen Med Humanitas Inst - Milan, Italy.

Recently, two novel antigens, HMG1 and HMG2 proteins, have been describedas possible targets for ANCA in non vasculitic diseases. However, their preva-lence in different disease groups needs to be ascertained yet. Aim of our workwas to study the prevalence of antibodies to HMG1 and HMG2 in selected cat-egories of ANCA-associated diseases.Anti-HMG1 or HMG2 antibody was assayed by ELISA using recombinantHMG1 or HMG2 from E. Coli as antigen in solid phase.The anti-HMG1 level (mean OD±SD) was significantly increased in lupus erythe-matosus systemic (SLE) (0.653±0.430, p=0.02), ulcerative colitis (UC)(0.668±0.359, p=0.04), autoimmune colangitiis (AC) (0.798±0.414, p=0.03), pri-mary biliary cirrhosis (PBC) (0589±0.334, p=0.0001) and type II autoimmunehepatitis (AH-II) (0.594±0.130, p=0.001) but not in primary sclerosing colangitiis(PSC) (0.692±0.384), type I autoimmune hepatitis (AH-I) (0.718±0.597), ANCA-associated systemic vasculitis MPO/PR3 positive (AASV) (0.525±0.255 and0.331±0.239) in comparison to normal controls (NC) (0.374±0.150).Anti-HMG1 were frequently detected in sera from patients with UC (6/18, 33%,p=0.04), AC (4/7, 57%, p=0.007) and PBC (14/45, 31%, p= 0.04) but were notstatistically significant in SLE (5/17, 29%), PSC (3/7, 43%), AH-1 (3/7, 43%),AH-II (1/8, 12.5%), AASV (6/27, 22%); in normal controls only 1 out of 26 sera(3.8%) were positive.Anti-HMG2 antibody was detected with the same disease distribution but witha lower prevalence. Anti-HMG1 and anti-HMG2 antibodies were detected morefrequently in sera with positivity for P-ANCA by immunofluorescence on etha-nol-fixed granulocytes.In conclusion, our results confirm that HMG1 and HMG2 are new ANCA anti-gens and specific autoantibodies can be found in a variety of ANCA- associateddiseases, ranging from rheumatic to inflammatory bowel and hepatic diseases.This spread distribution decreases their diagnostic significance in comparisonwith other ANCA antigens.

DOES HLA-DR14 ALLELE CONFER SUSCEPTIBILITY TOCRYOGLOBULINEMIC GLOMERULONEPHRITIS?S. Pasquali, W. Mantovani*, A. Zucchelli, S. Casanova**, P. ZucchelliDivisone di Nefrologia e Dialisi Malpighi, Laboratorio Analisi*, Istituto diAnatomia Patologica**, Azienda Ospedaliera S.Orsola-Malpighi, Bologna,Italia.

Although a clear association between hepatitis C virus (HCV) and essentialmixed cryoglobulinemia (EMC) has been established, it is not yet clear whyonly some HCV+ cryoglobulinemic patients develop renal damage.In an attempt to differentiate these 2 subtypes of cryoglobulinemic patients,with and without renal disease, we have studied 23 HCV+ type IIcryoglobulinemic patients (8 males, 15 females, mean age 63.2 years), 16 withand 7 without cryoglobulinemic glomerulonephritis (CGN) followed-up formore than 4 years.The genetic characteristics of the HCV and of the host were analyzed in the 2groups of patients.HCV genotype was identified in 20 patients using type-specific oligonuclectideprobes after hybridization. All the patients were typed for HLA (Classes I andII) by the standard lymphocytotoxicity technique. HLA results were comparedwith normal controls.The genotype distribution in patients with CGN was no different from the onefound in patients without CGN while the presence of HCV subtype 1b correlatedwith the presence of liver damage.Class I HLA-88 was present in 57.1% (4/7) of the patients without renal damagecompared to 10.1% (38/377) of normal controls (p = 0.003, pc = n.s.). Only 1 outof 16 (6.2%) patients with renal damage exhibited this antigen.As regards the expression of class II HLA alleles, a signifiicant association wasfound with HLA-DR14, detected in 5 out of the 16 (31.2%) nephropathic patientscompared to 6.4% (24/377) of the normal controls (p = 0.0038; pc < 0.05). Noneof the patients without renal damage exhibited this antigen.The results suggest that: 1) the genetic heterogeneity of HCV does not influencerenal involvement while it appears to influence liver damage; 2) the develop-ment of CGN requires a genetic predisposition of the host associated with theHLA-DRl4 marker.

MYCOPHENOLATE MOFETIL (MMF) REDUCES APOPTOSIS IN THE RE-NAL ABLATION MODEL IN THE RAT.J Rincón, Y Quiroz, F Romero, G Parra and B Rodriguez-IturbeDept. of Immunobiology, Inbiomed and Renal Service, Hospital Universitario,Universidad del Zulia, Maracaibo, Venezuela..The role of apoptosis in the development of glomerulosclerosis is controversial.Deletion of glomerular cells could be a physiologic response to proliferation or,alternatively, a pathogenetic mechanism, resulting in scarring. Since MMFameliorates the sclerosis and prevents renal failure in the 5/6 nephrectomy (Nx)model (Kidney Int March, 1999) we examined the effects of MMF in the prolif-erative activity (mAb anti-PCNA) and apoptosis (TUNEL method) in this ex-perimental model. We studied 40 male Sprague-Dawley rats after 5/6Nx thatreceived MMF 30 mg.Kg-1.day-1 by gastric gavage (n=20) or vehicle (n=20). Fiveadditional rats were sham operated. Rats were sacrificed 3, 7, 28 and 56 daysafter renal ablation (5 rats from each group at each time interval). Serum creati-nine and proteinuria were determined weekly.Results. MMF treatment prevented the progressive rise in serum creatinine andproteinuria (p<0.001). Eight weeks after surgery, segmental sclerosis was presentin 48.4 ± 8.35% of the glomeruli of vehicle treated rats and in 25 ± 10.5% in theMMF-treated rats (p<0.001). Apoptosis and proliferation are shown in Tables1 and 2.

Table 1. Proliferation (PCNA positive cells/glomerular cross section)3 days 7 days 28 days 56 days

5/6Nx 0.47 ± 0.11 1.16 ± 1.32 1.51 ± 0.58 2.17 ± 1.855/6Nx+MMF 0.40 ± 0.06 0.65 ± 0.48 0.89 ± 0.37 0.60 ± 1.77

Table 2. Apoptosis (TUNEL positive cells/glomerular cross section)3 days 7 days 28 days 56 days

5/6 Nx 0.24 ± 0.20 0.79 ± 0.38 0.43 ± 0.30 0.66 ±5/6 Nx+MMF 0.17 ± 0.10 0.56 ± 0.12 0.39 ± 0.43 0.07 ± 0.08

(All values are mean ± SD). MMF treatment reduces proliferation by about 15%at 3 days to 72% at 56 days and reduces apoptosis by 29% to 5% at the same timeintervals. Decreased proliferation and apoptosis is likely involved in the reduc-tion in glomerulosclerosis resulting from MMF-treatment.

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Abstracts



TGF-α1 AND APOPTOSIS IN GLOMERULONEPHRITISD Goumenos, A Tsamandas*, S Tsakas, F Sotsiou+, D Bonikos*,J Vlachojannis.Departments of Nephrology and Pathology* University of Patras,and Department of Pathology+ Evangelismos Hospital, Greece.

TGF-α1 is considered the most fibrogenic growth factor.Apoptosis, programmed cell death, has been implicated inpathogenesis of wound healing and scarring. Bcl-2 protein pro-longs life of rapidly proliferating cells while bax protein pro-motes apoptosis. The aim of the study was to evaluate thepotential relation among TGF-α1, bcl-2, and bax expression andapoptosis in various types of glomerulonephritis (GN).Renal biopsies were obtained from 40 patients (M/F=25/25,age 50.5±17.6 yr, serum creatinine 1.4±0.2mg%). Streptavidin-biotine technique was employed on paraffin sections using spe-cific antibodies against TGF-α1, bcl-2, and bax protein. Presenceof apoptotic cells was assessed using TUNEL method.Quantitation of immunostained cells was performed usingmorphometric analysis.TGF-α 1 was localized in the cytoplasm of tubular epithelialcells and renal interstitium. Apoptotic nuclei were also detectedin tubular cells and interstitium. Bcl-2 and bax were present intubular epithelial cells. Glomerular bax expression was moreevident than bcl-2. TGF- α1 expression was positively correlatedwith TUNEL(+)cells, and bax expression and reversibly withbcl-2 expression (p<0.05). TUNEL(+)cells were correlated posi-tively with bax and reversibly with bcl-2 expression (p<0.05).TGF α1 and tubular bax expression were correlated with serumcreatinine values (p<0.05)In conclusion, TGF-α1 and bax expression in human GN seem tobe related to enhanced apoptotic process and progressive renaldisease. Bcl-2 expression may provide a survival advantage totubular epithelium.

RENAL AND SYSTEMIC INVOLVEMENT IN MIXEDCRYOGLOBULINEMIAS ASSOCIATED WITH HEPATITIS C VIRUS CHRONICINFECTIOND. Rosca, E. Mota, M. Mota, M. GeorgescuClinical Hospital no. 1 Craiova, Nephrology and Dialysis Deprtment, Facultyof Medicine, Craiova, Romania

In the renal damages associated with hepatitis C virus (HCV) infection are in-volved immune mechanisms, mainly being considered the mixedcryoglobulinemias (MC). MC are type II or III, according with Brouet classifi-cation, in use yet. In the same time, MC are involved in the occurrence of anotherlocal and systemic manifestations.Our goal was to identify the renal involvement and systemic disorders in thechronic infected patients with HCV, with detectable cryoglobulinemia. In thisprospective and retrospective study were included 297 patients with HCV chronicinfection documented by the presence of anti-HCV antibodies (using ELISA II);these patients was tested for cryoglobulis and measured cryocit. All was renaland immunological investigated and some of they benefit by renal or/and skinbiopsy.Cryoglobulins was detected in 115 (38,7%) of the patients chronic infected withHCV, type II in 31 (26,9%) and type III in 84 (73,1%) patients. Chronic liverdisease was documented in 118 patients (39,7%), being present in these situa-tions in the on set of the infection diagnostic. The most frequent features of therenal involvement was proteinuria and microscopic hematuria with nephriticsyndrome in 17 patients, nephrotic syndrome in 3 cases, isolated proteinuriaand/or hematuria in 12 cases, respectively 27,8% with renal involvement. Thesedisorders was detected in only 8 cases in the moment of cryoglobulinemia detec-tion, in the rest of the cases during the time. The evolution to chronic renal failurewas noted in 3 cases, in one of these to the uremia, needing dialysis. Extrarenalinvolvement were palpable purpura in 68 patients (59,1%), arthralgias in 61(53%), peripheral neuropathy in 29 (25,2%) and general features (weakness,increased body temperature, weight loss) in 103 (89,5%). Immune disorderswere: detectable rheumatoid factor in 43 patients (37,4%), decrease of the C4 andC3 serum levels and the increase of liver transaminases in 2/3 of cases, sugges-tive for active liver disease.In conclusion, the renal manifestations in the MC HCV associated are mainly ofglomerular type, rising later in the course of disease, but hepatic involvement ispresent early.

LYMPHOCYTE SUBSETS IN CHLDHOOD IgA NEPHROPA-THY (IgAN)M Ekim1, A Günlemez1, A Ikinciogullari2, N Kara3, E Babacan2, NTümer1.University of Ankara School of Medicine, Pediatric Nephrology1

and Immunology-Allergy2, Social Insurance Hospital PediatricNephrology3, Ankara, Turkey.

The objective of the study is to determine whether or not periph-eral blood (PB) total T, B lymphocytes and their subsets play arole in the pathogenesis of IgAN in childhood. 13 patients (agerange; 9-20 year) and 18 age matched healthy controls (agerange; 7-18 year) were taken into the study. All patients havenormal renal function, blood pressure and no previous therapyhave been applied. Among all, one have persistant microscopichematuria, one have mild proteinuria (<0.5 gr/m2/day), 6 haveboth persistent microscopic hematuria and proteinuria (<0.5gr/m2/day), while 5 have normal urine. Neither macroscopichematuria nor infectious episode have been detected when studywas performed. PB total T lymphocyte (CD3+), B lymphocyte(CD20+),T lymphocyte subsets (CD4+, CD8+) and CD40+ Bcells were analysed using dual color direct immunofluorescencemethod by flowcytometry (Coulter-EPICS XL-MCL). CD3+,CD4+, CD8+, CD20+ and CD40+ measurements were as fallows(mean±SD).

CD3+% CD4+ % CD8+ % CD20+% CD40+%Patients 68.9±5 38.7±9 27.6±7 13±4 19.8±3Controls 67.8±6 38.2±5 25.8±7 13±3 20.9±3

No difference have been detected in PB total T, B lymphocytesand their subset levels in children with IgAN compared to con-trols (p>0.05). Therefore our preliminary results failed to sup-port the major role of celluler immune responce in the pathogenesisof IgAN; however, forworded for further investigations, espe-cially during clinical exacerbation of the diseases.

CIRCULATING SOLUBLE ADHESION MOLECULES INANCA-ASSOCIATED VASCULITIS.J. Ara, A. Saurina, E. Mirapeix, P. Arrizabalaga, R. Rodriguez,R. Abellana*, C. Ascaso*, A. Darnell.Nephrology Service, Biostatistical and Epidemiological Unit*Hospital Clínic. Barcelona. Catalonia. Spain.

The aim of this study was evaluate whether changes in concen-trations of soluble (s) E-selectin, sP-selectin, sL-selectin, sICAM-1 and sVCAM-1 reflect disease activity in patients with ANCA-associated vasculitis. A sandwich ELISA was used to measurethese soluble adhesion molecules in sera from 20 patients withANCA associated vasculitis during active and remission phases(ten patients with Wegener´s granulomatosis and ten patientswith microscopic polyangiitis.At the time of diagnosis, sE-selectin, sICAM-1 and sVCAM-1levels (mean+/-sd) (88+/-42,27 ng/ml, 437,25+/-184,46 ng/ml, 1720,36+/-1174,05 ng/mL, respectively) were significantlyhigher in patients with ANCA associated vasculitis than inhealthy controls (p<0,0001, p=0,002, p=0,001 respectively). sP-selectin values did not differ from those obtained in normaldonors. In contrast, sL-selectin levels (940,8+/-349,09 ng/mL)were significantly lower in patients than those recorded in healthycontrols (p<0,0001). A significant decrease in concentration ofsE-selectin, sP-selectin, sICAM-1, and sVCAM-1 was observedbetween active and remission phases (p<0,0001, p=0,002,p=0,001, p=0,001 respectively). No significant differences wasobserved in sL-selectin levels between active and remissionphases. sL-selectin concentration (802,34+/-306,7 ng/mL) dur-ing the remission phase remained lower than those observed inhealthy controls (p<0,0001). A positive correlation was observedbetween VCAM-1 and creatinine levels during the active phase(0,51,p=0,002). No correlation was observed between other ad-hesion molecules and this variable.The increase in sE-selectin, ICAM-1 and VCAM-1 levels on ac-tive phase and the return to normal values during the remissionphase suggest that the concentration of these adhesion mol-ecules reflect disease activity in patients with ANCA associatedvasculitis.

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Abstracts



RENAL FIBROSIS PRECEDES INFLAMMATION IN A RATMODEL OF OBESITY, A FEATURE AGAINST THE PARA-DIGM OF INFLAMMATION IN NEPHROPATHY.J Chevalier, B Poirier, S Lavaud, C Mandet, MF Bélair, J Bariéty,I Myara.Inserm U430 and C. Bernard Ass., Broussais Hosp., Paris,France.

Macrophages have been proposed as key factor in the process ofrenal injury. We examined the role of inflammation in the onsetand progression of interstitial fibrosis in Zucker obese (fa/fa) ZOrats, which rapidly develop kidney lesions in absence of hyper-tension and hyperglycemia. Lean (Fa/fa) ZL rats act as an usefulinternal control. Type I and III collagens were quantified usingpolarized light and computer-assisted image analyzer. The ki-netics of expression of mRNA encoding matrix components,adhesion molecules, chemokines and growth factors was fol-lowed by reverse transcriptase polymerase chain reaction onpieces of kidney cortex. The presence of synthesized proteins aswell as macrophages/ED

1

+-cells was followed by immunohis-tochemistry. Fibrosis developed in two phases. The first oneoccured as early as 3 months and resulted from a neosynthesisof Type III collagen and fibronectin and a reduction of extracel-lular matrix (ECM) catabolism as shown by an increased ex-pression of the metalloproteinase inhibitor TIMP-1. ECM andTIMP-1 overexpression was probably under the control of TGF-β1 which is synthesized at 3rd-month-onwards in ZO rats. Theseevents occured independently of any macrophage infiltration.The second phase started at 6 month-onwards: interstitial fibro-sis worsened with a large accumulation of Type I collagen. Bycontrast, this process appeared associated with ED1+-cell infil-tration. Thus, in absence of hypertension and hyperglycemia,inflammation cannot explain the onset of interstitial fibrosiswhich develops in young obese Zucker rats. Once the lesions andthe renal impairment were launched, macrophages probablyaggravated the process.

MYOFIBROBLASTIC TRANSDIFFERENTIATION OF TUBULAR CELLS: APOSSIBLE ROLE IN THE PROGRESSIVE RENAL SCLEROSISA Amore, P Cirina, G Conti, and R CoppoNephrology and Dialysis Department, Regina Margherita Children’s Hospital,Torino, Italy

The relentless fibrosclerosis in evolutive nephropathies is characterized by aprogressive expansion of newly formed matrix, in which fibroblastic-like cellsare included. Renal tubular cells (TC) and fibroblasts share a common mesen-chymal origin; the differentiation during the embryogenesis is regulated by thesuppression or transcription of different genes: among those TGFβ3 and type Icollagen are tought to play a role.Aim of this study was to investigate the possibility to instigate in vitro theprogressive fibroblast-like transdifferentiation of TC. Cells were conditionedwith stimuli that in previous studies of our group were able to modulate severalfunctional activities of TC, including Cyclosporin (CyA) (from 250 to 1000 ng/ml), native human serum albumin (HSA) and fatty-acid-free HSA (faf-HSA)(from 2 to 20 mg/ml). As positive controls TC were incubated with 5-10 ng/mlTGFβ3 and 0.1-1 ng/ml type I collagen. We evaluated the following parameters:the phenotype by light microscopy, some specific mesenchymal (vimentin, αactin -αSMA) and epithelial markers (cadherin) by immunoperoxidase andPCR. Moreover, the synthesis of TGFβ (ELISA and western blot) and its specificmRNA (PCR) were detected. The results are reported in the table:

Vimentin E-cadherin α-SMA TGFβ3(pg/ml)

TC basal 10% (trace) 100% (+++) — 31±4.2TC + HSA 10 mg/ml 25% (++) 30% (+) 30% (+) 41±7.8TC+ faf-HSA 10 mg/ml 8% (trace) 97% (+++) — 34±3TC + CyA 500 ng/ml 50% (++) 40% (+) 45% 132±34TC+ TGFβ3 10 ng/ml 55% (+++) 60% (trace) 50% (+++) __

The synthesis of TGFβ3 was confirmed in western blot. In PCR we observed amodulation in the expression of the specific mRNA of the different markersevaluated. A 25-45% of TC treated with HSA and CyA dysplayed a progressivefibroblast phenotype transformation. The transition of TC to a fibroblasticphenotype, detected by light microscopy, correlated with: a) decreased expres-sion of the epithelial markers; b) increased expression of mesenchymal mark-ers; c) synthesis and reorganization of actin fibers.We conclude that different stimuli may elicit in TC a myofibroblastic transfor-mation: this mechanism could be relevant in the progressive sclerosis of neph-ropathies.

IgA NEPHROPATHY: A CLINICOPATHOLOGICAL CORRELATIVE AND FOLLOW-UP STUDY OF 33 CASESS Güçer1, K Tinaztepe1, M Güllülü2, K Dilek2, G Gönüllü2, M Yavuz2, B Can3, C Güven3,M Yurtkuran2,Ped. Nephropathology Unit, Hacettepe Univ. Faculty of Medicine Ankara1, Dept. ofNephrology, Uludag Univ. Faculty of Medicine Bursa2, Dept. of Histology-Embryology,Ankara Univ. Faculty of Medicine, Ankara3, Turkey

Since primary IgA nephropathy(IgAN) presents with a highly variable clinical course andhistopathologic features in individual patients(pts) there have been still ongoing debateson the predictive histologic indicators. In this study, clinicopathological correlations wereinvestigated in 33 pts with IgAN (ages 18-47 yrs, M:16,F:17) at the time of biopsy diagnosisand after a mean follow-up period of 33 months (range:2-120 mos). Treatment consistedof prednisolone (Pred) alone, if unresponsive, Pred+Azathioprine, Pred+ Cyclophospha-mide or Pred+CycA administered for 2-50 mos (mean 20 mos).Two pts received no therapy.Renal biopsies were evaluated by light(LM) and immunofluorescence microscopy (IF) inall and additionally by electron microscopy (EM) in three. Five pts had second renalbiopsies with two yrs. intervals. Hematuria (mainly gross) was present in 92% of the ptsand hypertension in 70%. LM showed histologic sublasses; minimal glomerulopathy (ClassI) (no case), focal glomerulosclerosis type in 19 pts (Class II), focal proliferative GN (ClassIII) in 3, diffuse proliferative GN (Class IV) in 5 and advanced sclerosing GN in 6. Histo-logic scores (HS) [Glomerular(1-6), interstitial (1-13), vascular (0-9) and total (2-32)] weredetermined as previously described and their clinical correlations were analyzed. IF re-vealed heavy mesangial IgA deposition in all with C3(75%), IgM(40%), IgG (14%) in alesser degree and no C1q deposition. EM showed paramesangial deposits with alterationsin GBM. Conclusions of clinicopathological correlations were as follows: 1)There was nocorrelation between hypertension or a high serum IgA level on admission and prognosis(p > 0.05) 2)Clinical improvement was obtained in 16 pts (10 in Class II, two each in otherclasses) while clinical worsening in 7 (4 in Class V, 2 in Class IV and 1 in Class I), unchangedclinical course in (5 in Class II and one in Class I). End-stage renal failure developed in 5pts (3 in Class V and 2 in Class IV) and three were lost to follow-up. 3)Of five pts rebiopsied,two showed a correlative clinical and histologic improvement but the remaining threehad persistent pathologic findings despite clinical improvement. 4)Cases with highglomerular score (≥5) and total HS (≥18) showed a poor clinical course(p<0.05). 5)Therewas no prognostic effect of additional IgG and/or IgM depositions in glomeruli (p > 0.05).6)These preliminary results might indicate the value of rebiopsy studies in determiningthe prognostic indicators in larger series and longer follow-up periods.

THE USE OF DIALYSATE FILTER U 8000 S CANNOT PRE-VENT INDUCTION OF IL-6 PRODUCTION DURING DIALY-SIS WITH CUPROPHAN/HEMOPHAN-MEMBRANES.H-J Guth, S Gruska, H Preez, G Kraatz (intr. by B Osten)Department of Internal Medicine A, Ernst-Moritz-Arndt-Uni-versity, Loefflerstr. 23a, 17489 Greifswald, Germany

Activation of monocytes and other immuno-competent cellsduring hemodialysis is due to contact with membrane surface,blood line tubing systems and endotoxins. The use of cartridges(sodium bicarbonate column) and dialysate filter can reduceendotoxins in dialysate. The kind of membrane can also play animportant role in biocompatibility.Method: 24 patients, undergoing standardized hemodialysisincluding sodium bicarbonate cartridge system withCuprophan/Hemophan (N=12)- and Polyamide (N=12)-mem-branes, were immediately investigated before and after treat-ment using a whole blood stimulation assay for IL-6 (“Dynamix”Il 6 -DIA, Biosource Diagnostics, Germany). Before the nextdialysis treatment all AK 200 hemodialysis machines were con-verted to dialysate filter U 8000 S (all devices from GambroGroup, Lund, Sweden) and the same patients were investigatedagain. In both groups (Cuprophan/Hemophan and Polyamide)baseline concentrations and production capacity for Il 6, ob-tained during dialysis with and without U 800 S filter, werecompared (student’s t-test).Results: In the Polyamide-group the IL-6-concentrations andthe production capacity for IL-6 were not markedly changed (p> 0.05) before and after dialysis with or without dialysate filter.In the Cuprophan/Hemophan-group IL-6-concentrations wereincreased (p = 0.049) and the IL-6-production capacity wassignificantly elevated after dialysis (p = 0.003). The use of U8000 S cannot prevent the induction of IL-6-production capac-ity in peripheral blood mononuclear cells after dialysis (IL-6-baseline p = 0.027; IL-6 stim. p = 0.033).Conclusions: The use of dialysate filter U 8000 S cannot reduceimmune response in Cuprophan/Hemophan dialysis, meas-ured by IL-6-production capacity before and after dialysis.

A26

Abstracts



QUANTITATIVE AND GENOTYPIC ANALYSIS OF THE HEPATITIS C VIRUS(HCV) DETECTED IN LYMPHOCYTES AND MONOCYTES FROM PATIENTSWITH CRYOGLOBULINAEMIC GLOMERULONEPHRITISE. Menegatti#, V. Ghisetti§, D. Rossi*, A. Barbui§, G. Marchiaro§, M. Chiara#,L.M. Sena# and D. Roccatello*.CMID, Ospedale L. Einaudi*, Laboratorio di Microbiologia, OspedaleMolinette§, Dipartimento di Medicina ed Oncologia Sperimentale, Universitàdi Torino#, Italia.

The hepatitis C virus (HCV) RNA is frequently found in sera and is concentratedin crioglobulins from patients with mixed crioglobulinaemia. It is widely ac-cepted that the HCV infection is pathogenetically related to the crioglobulinaemicsyndrome. HCV has a special tropism towards hepatocytes, but also mononu-clear cells of the peripheral blood and bone marrow. Mononuclear cell infectioncould be etiologically relevant to the development of some crioglobulinaemicmanifestation.In the present study, limphocytes and monocytes from 8 patients with glomeru-lonephritis associated with mixed crioglobulinaemia (type II in 6 and type III inother 2 cases) were separately isolated from the peripheral blood by means ofcentrifugation on Ficoll gradients and cell incubation on Petri’s plates (37°C for60 min.)Total RNA was extracted according to Trizol procedure (Life Technologies,Italy). RNA total amount and purity were evaluated by spectrophotometricanalysis. Viral load was determined in cell extracts by RT-PCR (AmplicorMonitor Roche) and results were expressed as genomes/µg total RNA. HCVgenotype was identified in the two cell types and in sera by RT-Nested PCR(Innolipa Immunogenetics, Belgium)Each patient was viremic. Moreover, viral genome was revealed in 6 out of 7monocyte extracts and 4 of 7 lymphocyte extracts. Viral load in monocytes wasfound to be higher than in lymphocytes [ 2.70 (0 - 10.00) vs 0.24 (0 - 1.26) genomes/µg total RNA , p< 0.05)] The same genotype detected in serum was revealed inlymphocytes and monocytes from 6 out of 8 patients. In 2 monocyte extracts,beside the genotype determined in serum (1b), another genotypes was detected(3a and 2a/2c, respectively).These results suggest that HCV has a definite tropism to the peripheral bloodmonocytes, which are more frequently infected (with higher viral load) thanlymphocytes. The occasional presence in mononuclear cells of genotypes otherthan those detected in sera suggests either a co-infection by different viral subtypes(which could use monocyte as a “reservoir”) or a high potential of viral muta-tion.

PHARMACOLOGIC MODULATION OF THE EXPRESSION OF THE CELLCYCLE REGULATORY PROTEINS IN AN EXPERIMENTAL MODEL OFMESANGIAL NEPHRITISE. Menegatti§, M. Chiara§, G. De Rosa#, D. Bellis#, L.M. Sena§ and D. Roccatello*CMID* e Servizio di Anatomia Patologica#, ASL4, Dipartimento di Medicinaed Oncologia Sperimentale dell’Università di Torino§, Italy.

The response of mesangial cells to a phlogistic challenge includes cell prolifera-tion and mesangial matrix expansion. Cell proliferation is a highly regulatedprocess which includes enhancing factor such as cyclins (C) and cyclin-depend-ent kinases (Cdk) and inhibitory proteins, such as p27. Cs and Cdks promote cellentering into G1-phase and progression through cell cycle.Aim of this study was to evaluate the effects, on the cell cycle regulatory system,of the purine analogue roscovitine (R), administered in the florid proliferationphase of an experimental model of mesangial proliferative nephritis induced bythe monoclonal antibody (MoAb) anti Thy-1 antigen.Twenty Wistar male rats (200-220 g body weight) were i.v. given 0.25 µl/kg BWof MoAb anti Thy-1 (OX-7, Cederlane). Three days after nephritis induction, 5rats were given roscovitine (2.12 mg/kg BW, Alexis) suspended in dimethylsul-phoxide (DMSO) and 5 received the veicole alone. Samples of cortical and med-ullary tissues were separately processed by RT-PCR for the gene expression ofcyclins B, D1, D2, D3 and the inhibitory protein p27.The MoAb anti Thy-1 induced, one day after administration, focal aspects ofmesangiolysis and tuft collapse and, after 7 days, a remarkable mesangialhypercellularity in 50 per cent of glomeruli, with aspects of neutrophil exuda-tion.The analysis of the amplification products of the reverse transcribed RNA, sepa-rately extracted from cortical and medullary tissues, showed, at the corticallevel, a pronounced increase in gene expression of cyclin B (involved in the mi-totic process), D1, D2 and, even more, D3 (regulating cell entering into cell cy-cle), and the inhibitory protein p27 one day after the MoAb administration.Cyclin hyperexpression persisted, weakened, at the 7th day.Roscovitine, but not its vehicle, DMSO, markedly reduced cyclin B and D expres-sion and induced, at the cortical level, a remarkable increase in the expressionof the cell proliferation inhibitory protein p27.The present study suggests the possibility to pharmacologically handle the regu-latory system of cell proliferation, in response to a phlogistic challenge in anexperimental model of mesangial glomerulonephritis.

TUBERCULIN AND ANERGY SKIN TESTING OF PATIENTSRECEIVING LONG-TERM HEMODIALYSISH Taskapan, FS Oymak, A Dogukan, C Utas,Nephrology and Respiratory Disease Dept. of Erciyes Univer-sity Medical Faculty, Kayseri, Turkey

The incidence of tuberculosis (TB) in patients on regularhemodialysis (HD) is higher than that of general population.The tuberculous skin test (TST) is an imperfect test for detectinglatent TB. End stage renal failure is known to be a risk factor forskin test anergy, but the rate of anergy in HD patients is unclear.In this study, the frequency of TB reactions and anergy in 65 HDpatients (Male 35, female 30) were examined. The mean age was38.24±14.90 with range 14 to 70. All patients were tuberculintested using the Mantoux tecnique with 0.1 mL (5 tuberculinunits) of purified protein derivative intradermally injected intothe volar surface of the forearm that did not have the arteriov-enous shunt. Antigen for candida was injected by similar tech-nique, separated by 30 mm of skin. Results were interpreted 48hour later. Anergy was accepted as less than 2mm of indurationto candida antigen. Tuberculin positivity was defined as aninduration of ≥ 10 mm. The duration of HD ranged 4 to 126months with mean 43.12±28.47 months. No reaction to candidaantigens was found in 24 (36.9%) patients. However, one ofthese patients reacted to TST. 14 of 24 patients considered tohave a positive candida antigen test were tuberculin positive. Ofthe 65 participants, 15 (23.1%) were TST positive. There was nosignificant relationship between age, the duration of HD andanergy. Even with a high rate of anergy, TST test appears to beuseful test in HD patients. Anergy testing may be helpful todetermine the predictive value of a negative TST.

EXPRESSION OF METALLOTHIONEIN IN VARIOUSGRADES OF RENAL CELL CARCINOMA. AN IMMUNO-HISTOCHEMICAL STUDY.H. Paraskevakou, S.E. Theocharis, L. Soubassi, A. Athanasiades,P.S. Davaris.Dept of Pathology, University of Athens, Medical School, Ath-ens Greece.

Metallothionein (MTs) are cytosolic proteins rich in cysteine ap-pearing to have a physiological role in the absorption, transportand metabolism of trace elements, mainly of zinc and copper.Recent reports have linked over-expression of cellular MT withthe progression of malignant tumors.The aim of this study was to investigate the possible signifi-cance of MT expression in Renal Cell Carcinoma (RCC) in asso-ciation with the grading of tumors. Forty-five patients (33 M, 12F) who underwent nephrectomy due to RCC consisted the groupof this study. Their age ranged from 36 to 82 years old. Sectionsof paraffin embedded tissues were stained immunohistochemi-cally by the streptavidin-biotin peroxidase technique, using amouse (IgG

1k) monoclonal antibody (Zymed, San Francisco, Calif,

USA) that recognized a common epitope for both MT isoforms(I, II).From 45 tumors examined, 22 (49%) were characterized posi-tive and 23 (51%) negative to MT expression. Specifically MTexpression was detected only in 4 from 24 (17%) cases classifiedas grade I. The frequency became higher in 11 from13 (84%)cases of grade II whilst, all of the 8 (100%) cases of more ana-plastic neoplasms (grades III, IV) showed positivity to MT. Theintensity of staining was graded as mild to intense. MT distribu-tion was observed almost exclusively in the cytoplasm ofneoplastic cells and in the tubular epithelium in normal tissue.In conclusion, the difference in MT expression between grades II,III, IV and I, may indicate that MT overexpression in moreanaplastic RCC is associated with poor prognosis of the dis-ease.

A27

Abstracts



RENAL PROTEINASE-ACTIVATED RECEPTOR-2 (PAR-2) EXPRESSION CORRELATESWITH TUBULOINTERSTITIAL DAMAGE IN IgA NEPHROPATHY (IgAN).G. Grandaliano, R. Monno, E. Ranieri, G. Cerullo, C. D’Altri, L. Gesualdo, F.P. Schena.Div. of Nephrology, Dept of Emergency and Organ Transplantation, University of Bari,Bari, Italy.

Several studies have suggested the involvement of mast cells in the pathogenesis of tissuefibrosis in different organs, although the mechanisms underlying their potential fibrogenicrole is still largely unclear. Tryptase is one of the main components of mast cell secretorygranules and can be released upon cell activation. PAR-2 is a G protein-coupled receptorthat is cleaved and activated by trypsin-like proteolytic enzymes, including tryptase.The aim of the present study was to evaluate mast cell infiltration and PAR-2 gene expres-sion in IgAN renal biopsies and to investigate the potential role of tryptase-PAR-2 inter-action in the pathogenesis of glomerular and interstitial fibrosis. To this purpose the pres-ence of tryptase-positive cells were investigated by histochemistry in 17 biopsies fromIgAN patients and 10 apparently normal portions of kidneys removed for renal carci-noma. PAR-2 gene expression was evaluated in the same tissue samples by RT-PCR,quantified by computerized densitometry and normalized to GAPDH expression. PAR-2gene expression was also evaluated, by RT-PCR, in cultured human mesangial and proxi-mal tubular cells under basal conditions and upon stimulation with IL-1, a pro-inflamma-tory cytokine mainly produced by monocytes. Finally, gene and protein expression of TGF-β, a powerful fibrogenic factor, was evaluated in trypsin-stimulated human mesangialand proximal tubular cells by northern blot and ELISA, respectively.Tryptase-positive cells were not detected either in the glomeruli or in the interstitium ofnormal kidneys, whereas in IgAN biopsies they were present mainly at the interstitial leveland scantly within the glomerular tuft. In normal kidneys, PAR-2 gene expression wasbarely detectable by RT-PCR, whereas in the biopsy samples obtained from IgAN patientsthe mRNA levels for this protease-activated receptor were strikingly increased (PAR-2/GAPDH ratio: Control 0.37+0.06; IgAN 1.6+0.2, p<0.01). Interestingly, PAR-2 gene expres-sion was directly correlated with the extent of interstitial fibrosis (r=0.778, p<0.001) andmononuclear infiltrate (r=0.65, p=0.02). PAR-2 mRNA was expressed by cultured humanmesangial and proximal tubular cells and its expression was induced in a time-dependentmanner by IL-1 in tubular, but not in mesangial cells. Finally, the activation of PAR-2 bytrypsin, in both mesangial and tubular cells, induced a significantly upregulation of TGF-β gene expression with a peak at six hours, followed by an increase of TGF- β proteinsynthesis and release.Our data suggest that tryptase released from mast cells may play a significant role in thepathogenesis of extacellular matrix expansion in IgAN through the interaction with itsspecific cell-surface receptor and the subsequent increase of TGF- β expression

FAS AND BCL-2 EXPRESSION ON LYMPHOCYTE SUBPOPULATIONS INIgA NEPHROPATHYJ Gascó, J Iglesias, R Bernabeu, N Matamoros and X Bestard.Dept. of Nephrology and Immunology. Hospital Son Dureta. Palma de Mallorca.Spain.

Abnormal T and B lymphocyte function and aberrations in lymphocyte mark-ers have been recognized in IgA Nephropathy. We study the peripheral bloodlymphocytes (PBL) spontaneous apoptosis and Fas and Bcl-2 expression inpatients diagnosed by renal biopsy of Idiopathic Mesangial IgA Nephropathy.PBL of 18 p diagnosed of Idiopathic IgA Nephropathy were studied. As normalcontrols, 14 healthy subjects. MoAb used: CD8-PerCP, CD4-PE, CD19-PE andSimultest control from Beckton Dickinson (BD), CD95-FITC (UB-2) by CoulterImmunotech. Two or three colour analysis on a FACScan flow cytometer. For theintracellular Bcl-2 expression, staining of surface antigens was performed first.Whole blood was permeabilized using FACS permeabilizing solution fromBD.Bcl-2 expression was measured using direct staining with Bcl-2 MoAb (FITC-labelled; Dako). PBM were obtained by Ficoll density gradient centrifugationand cultured for 72 hours in complete medium. Apoptoic cells were evaluatedby flow cytometry using 7-aminoactinomycin D (7-AAD, Sigma) as DNAmarker,after staining with surface antigens with diferent monoclonal antibod-ies for specific populations. Data were analyzed using the t-Student test (Bergervs Control patients). 1)CD95 expression on cell subsets. Percentage: CD4+ Tcells, 29+/-10% vs 31+/-8%; CD8+ T cells, 21+/-13% vs 19+/-10%; CD19+,12+/-4% vs 7+/-3% (p=0.004). Mean Fluorescence Intensity (MFI): CD4+ T cells,27+/-6% vs 27+/-3%; CD8+ T cells, 21+/-6% vs 19+/-3%; CD19+, 28+/-8 vs29+/-11. 2)Bcl-2 expression on cell subsets (Berger vs Controls patients). Per-centage: 100% of the cells studied in the diferent subsets were Bcl-2+.MFI:CD14+Tcells, 113+/-29 vs 108+/-14; CD8+ T cells, 95+/-23% vs 109+/-16 ; CD19+,79+/- 18 vs 75 +/-16. 3)Percentage of spontaneous apoptosis on cell subsets:CD4+ T cells, 23+/-7 vs 10+/-6 (p<0.0001); CD8+ T cells, 30+/-13 vs 11+/-6(p<0.0001); CD19+, 41+/-13 vs 35+/-13.Spontaneous apoptosis in peripheric CD4+ and CD8+ T cells from IgA neph-ropathy patients were increased significantly, comparing with controls.Normal levels of spontaneous apoptosis were observed in B lymphocytes(CD19+). Conversely, the expression of CD95 were raised in CD19+ cells. Wehave not found diferences in the Bcl-2 expression between IgA Nephropathypatients and normal controls.

TWO LATENT FORMS OF TGF-b1 ARE EXPRESSED IN THE NORMALHUMAN KIDNEY AND ARE DIFFERENTIALLY UP-REGULATED INGLOMERULONEPHRITIS (GN)Z. I. Niemir, E. Pawliczak, P. Olejniczak, G. Dworacki, M. Kurpisz, R. Waldherr,& S. CzekalskiUniversity of Medical Sciences and Polish Academy of Sciences, Poznan, Po-land, and University of Heidelberg, Germany.

Results concerning the expression of latent TGF-1 forms in glomerular diseasesare rather scarce.We performed an immunocytochemistry study on renal biopsy specimens withfeatures of IgA-GN (n=37), membrano-proliferative GN (MPGN; n=4), idiopathicmembranous GN (IMGN; n=14), focal-segmental glomerulosclerosis (FSGS;n=12), and minimal change disease (MCD; n=11), looking for the expression ofthe large latent TGF-b1 complex (LLTC), small latent TGF-b1 complex (TGF-b1LAP), an active form of TGF-b1, and TGF-b receptor type II (TGF-bRII).Normal human kidney served as control (n=4). The generation of the active formof TGF-b1 in the kidney samples was also analysed by immunoblotting of de-tergent-buffer extracts from these specimens, separated by SDS-PAGE undernon-reducing conditions.Our results show that in the normal kidney LLTC is expressed in mesangialareas, whereas TGF-b1 LAP localises to podocytes. The expression of TGF-bRIIis predominantly noted in association with LLTC. Up-regulation of LLTC isobserved in MCD, early stages of IMGN and FSGS, as well as in IgA-GN withmild mesangial cell proliferation. The expression of LLTC decreases, however,in MPGN, IgA-GN with marked proliferative response of mesangial cells, andin advanced stages of FSGS. The podocyte expression of TGF-b1 LAP increasesparticularly in IgA-GN with intermittent macroerythrocyturia and low-rangeproteinuria, while diminishing in nephrotic patients. The intensity ofimmunostaining for the active form of TGF-b1, confirmed by Western-blot, seemsto correlate with the expression of LLTC.Our results seem to indicate a link between up-regulation of LLTC and the pres-ervation of the glomerular architecture. A negative relationship between thepodocyte expression of TGF-b1 LAP and the glomerular filter permeabilitymay also be suggested.

CYTOKINE INDUCED DIFFERENTIAL EXPRESSION OF ADHESION MOL-ECULES AND HLA-DR IN CULTURED HUMAN GLOMERULAR ENDOT-HELIAL CELLS (HGEC).S Park, WS Yang, SK Lee, JH Kim, HH Jung, H Ahn, JD Lee.Dept. of Internal Medicine, Urology, Biochemistry, University of Ulsan, Seoul,Korea

Glomerular endothelial cells should participate in the process of glomerulardisease by expressions of HLA antigens and adhesion molecules. However, fewhas been known about the regulation of the expression of these molecules in theglomerular endothelial cells. In this study, we investigated the effect of cytokines(IL-1β, TNF-α, IFN-λ) on the expression of ICAM-1, VCAM-1, and HLA-DR incultured HGEC. HGECs were isolated from kidneys resected for renal cell car-cinoma, and confirmed by homogenous positive staining of factor VIII andhomogenous uptake of fluorescent-labeled acetylated LDL (DiI-Ac-LDL). Cel-lular expressions of ICAM-1, VCAM-1, and HLA-DR were quantified by ELISAon fixed adherent cells. The results are as followings; ICAM-1 (n=9)1 VCAM-1 (n=6)1

6h 24h 48h 6h 24h 48hcontrol 3.01±0.192 3.23±0.23 3.39±0.25 1.37±0.22 1.49±0.19 1.60±0.29IL-1β(5ng/ml) 3.74±0.03* 3.73±0.04** 3.75±0.04 2.23±0.21* 2.04±0.31* 1.72±0.29TNF-α(10ng/ml) 3.45±0.03* 3.77±0.03* 3.76±0.03 2.49±0.19* 2.56±0.23* 2.30±0.36*

IFN-σ(20ng/ml) 3.44±0.39* 3.77±0.02* 3.73±0.03 1.34±0.18 1.42±0.14 1.44±0.17 HLA-DR (n=3-4)1

24h 48h 72h 1: each n is the mean of 4-control 0.38±0.05 0.49±0.09 0.45±0.04 8 well experimentIL-1β(5ng/ml) 0.54±0.04 0.65±0.14 0.45±0.06 2: the value of optical denTNF-α(1ng/ml) 0.44±0.05 0.71±0.04 0.47±0.04 sity mean ± standard errorIFN-γ(10ng/ml) 0.76±0.16* 2.11±0.34* 2.40±0.23* *: P<0.05, compared toIFN-γ(20ng/ml) 0.85±0.15* 2.13±0.24* 2.67±0.12* control by paired t test

**: P=0.053

The results showed that 1. ICAM-1 was increased by IL-1β, TNF-α and IFN-γ. 2.VCAM-1 was increased by IL-1β and TNF-α, not by IFN-γ. 3. IFN-γ only increasedexpression of HLA-DR in HGEC. 4. Basal expression of ICAM-1 was higherthan VCAM-1 and HLA-DR in cultured HGEC. 5. The time course of expressionwas different according to adhesion molecule. In conclusion, HGECs expressedadhesion molecules and HLA-DR, which were regulated differentially byinflammatory and immune-regulatory cytokines.

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Abstracts



DETECTION OF SELECTED ENZYMATIC PATTERNS INKIDNEY BIOPSY MATERIAL FROM PATIENTS WITHGLOMERULONEPHRITISM. Gliesing2, U. Ott1, R. Fünfstück1, K.-J. Halbhuber2, J. Schubert3,G. SteinDepts. of Internal Medicine IV1, Anatomy II2 and Urology3,Friedrich-Schiller-University of Jena, Germany

Glomerulonephritis (GN) is not only characterized by changes ofthe glomeruli but also by disturbances of the tubulointerstitium.Enzymatic patterns of tubular cells in kidney biopsy materialfrom 18 pats. with GN were evaluated and compared withnormal renal tissue. The activities of monoamine oxidase(MAOX), cytochrom c oxidase (COX), succinate dehydrogenase(SDH) and glucose-6-phosphatase (G6Pase) were analyzed his-tochemically by lightmicroscopical metal precipitation, in caseof SDH by tetrazolium technique. After quantifying enzymaticactivities by Quantimet 500+ an index for the area of enzymaticreaction to total tubule area in correlation to the mean grey valuewas determined (Tab.).Enzymatic activities:

MAOX COX SDH G6PaseGN 0.2-8.9 0.5-9.8 0.5-6.3 0.1-8.5controls 11.4 15.6 8.1 19.3

No difference between proliferative and non-proliferative GNwas detectable, but the interrelationship between enzyme activi-ties and the degree of tubular damage was significant. SDH wasreduced more focally in contrast to the general reduction ofG6Pase, MAOX and COX. The reduced enzymatic activitiesreflect tubular damage and correlate with proteinuria, espe-cially with a1-microglobulin and albumin excretion.

SECRETION OF PDGF, TNF-α, IL-6 AND IL-8 BY UROEPITHELIAL CELLSSTIMULATED BY E. COLI AND CITROBACTERR Fünfstück1, S Franke1, M Hellberg1, B Knöfel2, E Straube2, J. Hacker3, G Stein1

1Dpt. of Internal Medicine and 2Institute of Medical Microbiology, University ofJena; 3Institute of Molecular Infectional Biology, University of Würzburg; Ger-many

Urinary tract infections induce a local response of mucosal cells and systemicinflammatory reactions including the activation of polymorpho-nuclear cellsand release of various immunoregulatory cytokines, which are important forthe initiation and maintenance of renal inflammation.Cytokine release (PDGF, TNF-α, IL-6, IL-8) of T 24 bladder cells as stimulatedby the noninvasive the E. coli HB 101 (K: 12), the uropathogenic E. coli 0: 18 andthe highly invasive Citrobacter CB 3009 were determined. Analyses were per-formed after incubating 20.000 cells/ml with 20.000-000, bacteria/ml in thesupernatants for 5, 15, 30, 60 and 120 min.Cytokine production was induced by all microorganisms investigated. Theprocesses were time-dependent, with a significant increase in cytokine activityafter 60 min. The release of PDGF and IL-8 was found to be higher due to the E.coli HB 101 than by the other bacteria. Citrobacter CB 3009 induced the highestIL-6 level. A stimulatory effect of TNF-a was detectable 240 min after startingthe experiment.Cytokine levels after 120 min of interaction in FCS (pg/ml)

PDGF IL-6 IL-8 TNF-αtime (min) 120 120 120 240-----------------------------------------------------------------------------CB 3009 176 436 4386 16E. coli 0: 18 118 84 7794 44E. coli HB 101 262 101 8592 33

In all analyses, a significant correlation was found between the timedependentrelease of IL-6 and IL-8 (p<0.05). In case of Citrobacter infection, the PDGF levelsignificantly correlated with IL-6 and IL-8 values (p<0.001).Conclusion: The data demonstrate that urinary tract epithelial cells are able toproduce different cytokines in response to an incubation with E. coli andCitrobacter strains. The virulence of the pathogens does not seem to play a role.This epithelial cell cytokine response can modify immunoregulatory processescaused by an infection responsible for parenchymal injury.

CYTOKINE PROFILES DEFINES DIFERENT RESPONSES TOINTERFERON-α TREATMENT IN PATIENTS WITH CHRONIC HEPATITIS CVIRUS INFECTION (HCV) AND END STAGE RENAL DISEASE (ESRD)G.L. Kissinger, J. Stachowski*, M. Pollok, H. Steffen, K. Jihlke, B. Kneissl, U. Töx,T. Goeser, C.A. Baldamus;*University of Posen, Poland; Medical Clinic IV, University of Cologne, Köln,Germany

Cytotoxic T lymphocytes, natural killer cells (NK) and immunoregulatorycytokines may be important in the clearance of hepatitis C virus (HCV) infectedcells. T-helper type 1 (Th1) cytokines (Interleukin-2, Interferon-γ) are requiredfor antiviral immune response, including cytotoxic T cells and NK activation.In contrary T-helper type 2 (Th2) cytokines (Interleukin-4, Interleukin-10) in-hibit the effective antiviral defense. However there are only a few studies thatspecifically analyzed the levels of immunregulatory cytokines under a specifictherapy in chronic HCV infection and there are no data available of patientswith HCV and ESRD. For these reasons we have studied the effects of Interferon-a(INF-α) monotherapy on Th1 and Th2 cytokine levels in ESRD patients withHCV compared to patients with HCV without ESRD (n=20) and to normal con-trols (n=20).10 consecutive anti-HCV and HCV-RNA positiv patients on maintenancehemodialysis (mean age 45.4±12.1 years) were treated with INF-α2b (5MU 3times/week, INTRON, Essex). Studies were undertaken before, 12 weeks duringand after INF-α therapy.Cytokines were evaluated in culture supernatants of PBMC cultured for 48 hoursin Costar plates. The levels of cytokines were measured by means of a solidphaseenzyme-linkedimmunosorbent-assay (ELISA), specific for human IL-2, IFN-γand IL-4, IL-10 (GenzymeCorporation, Cambridge, USA). Response was de-fined after 12 weeks of therapy (HCVRNA negativ). 4 patients were treatmentresponder. Before therapy all patients had a TH2 pattern compared to normalcontrols. There was no difference between the patients with or without ESRD.INF-α therapy decreased the TH2 cytokines and increased TH1 cytokines. ButNonresponder (NR) had significantly lower TH1 and higher Th2 levels (p<0.01).Our results suggest that persistently high TH2 patterns indicate NR to therapy.Responder showed a stronger switch to TH1 cytokine profiles compared to NR.There was no difference between patients with ESRD and without ESRD. As inpatients without ESRD the stronger modulation towards Th1 pattern may beone mechanism of HCV clearance.

MORPHOLOGICAL INVESTIGATION OF DNA-FRAGMENTATION/APOPTOSIS IN KIDNEY BIOPSIES OF PATIENTS WITH CHRONICGLOMERULONEPHRITIS (GN)Ott, U.1, A. Koscielny1, D. Kinder1, R. Fünfstück1, A. Aschoff2, G. Jirikowski2, G.Stein1

1Dpt. of Internal Medicine IV and 2Dpt. of Anatomy II, Friedrich-Schiller-Uni-versity of Jena, Germany

In many glomerular diseases a hypercellular, proliferative state changes to ahypocellular, sclerotic phase. The mechanism responsible for terminatingglomerular cell proliferation is not clearly understood. It is speculated thatregulation and clearance of cell excess as well as cell proliferation is balancedby apoptotic mechanisms.In renal biopsies of 24 patients with proliferative GN (IgA nephropathy,mesangioproliferative and membranoproliferative GN), 14 patients withnonproliferative GN (focal segmental glomerulosclerosis, minimal change GN,membranous GN) and 7 patients with rapid progressive glomerulonephritis(RPGN), in situ end labeling (ISEL) of fragmented DNA were performed forcharacterizing cells with different degrees of DNA damage probably leading toapoptosis. For ISEL staining of semithin sections bromodesoxyuridine (BrdU)and terminal transferase were used. Incorporated BrdU was visualized by ananti-BrdU antibody and by peroxidase-antiperoxidase. The rate of apoptoticcells could be evaluated by the Quantimet-System (Fa. LEICA).DNA-fragmentation was mainly found in the nuclei of distal tubular epithelialcells, but also in the nuclei of mesangial cells in the glomerulus.DNA-fragmentation in nuclei per 15 000 µm2:

glomerulus tubulusRPGN 29,0+/-4,9 23,6+/-5,6proliferative GN 27,6+/-3,7 17,0+/-6,8non-proliferative GN 19,0+/-4,2 22,0+/-3,4

The difference between proliferative and non-proliferative GN was significant(p < 0,05). The degree of DNA-damage did not correlate with the concentrationof serum creatinine or proteinuria and no correlation was found for urinaryexcretion of albumin, a1-microglobulin and IgG. As one sign of progressivedamage to the cells, DNA-fragmentation could be detected in proliferative GN.In mesangial cells apoptosis is important for the clearance of proliferating cells,but in the distal tubule it could indicate a cell turnover depending on the degreeof inflammation.

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Abstracts



DISTRIBUTION OF TYPE IV COLLAGEN α1 TO α6 CHAINSIN PRIMARY IgA NEPHROPATHY.S Palle1, B Laurent1-2, Y Sado3, C Deprele1-2, JM Koller1-2, F Berthoux1-

2

1Res. Group on Glomerulonephritis and Renal Transplantation(UPRES-EA n°773), 2Dpt. of NDT Univ. of Saint-Etienne, France.3Div. of immunology, Shigei Med. Res. Inst., Okayama, Japan

In several nephropathies, variations in the thickness of glomeru-lar basement membrane (GBM) were generally associated withalterations of extracellular components. Abnormal thinning ofGBM (<275nm in M ; <265nm in F) was found in 40% of patientswith IgA nephropathy (IgAN) leading to hypothesize a defect inthe type IV collagen network.Using immunolabelling technics, we investigated the distribu-tion of the α1 to α6 type IV collagen chains in thin- (n=4) andnormal-GBM (n=4) of IgAN patients as compared with normalkidneys (n=4).No significant changes were observed in α1 to α6 chains betweenIgAN and normal kidneys. We failed to find a significant in-crease of α3 chain despite an increased labelling in IgA patients.On the contrary, a significant increase of α2 chain was evidencedin the GBM of IgAN with normal-GBM; no modifications werefound in the mesangium between the three groups but a signifi-cant increase was also noted in the Bowman’s capsule in bothnormal- and thin-GBM groups. For α4 and α5 chains, a signifi-cant weaker labelling was found in two patients independentlyof thin- or the normal-GBM, while others are comparable tonormal.In conclusion, our study failed to evidence defect or significantalteration in the distribution of collagen IV αchains in IgAN andtherefore Thin-GBM could not be explained by abnormal colla-gen IV network.

ROLE OF INTERLEUKIN-18/ IFN-γ INDUCING FACTOR (IL-18/IGIF) IN IgA NEPHRITISNapodano P, *Libetta C, *Centore F, °Meloni F, Marzorati A,Ferrario F, *Dal Canton A, D’Amico G.Renal Immunopathology Center, Division of Nephrology, S. CarloBorromeo Hospital, Milan and *Departement of Internal Medi-cine and Nephrology and °Istitute of Respiratory diseases,S.Matteo Hospital, Pavia, Italy.

IL-18/IGIF is a recently characterized cytokine, structurally re-lated to IL-1β and produced by monocytes/macrophages, thatinduces IFN-y synthesis by T cells and synergizes with IL-12 inT helper type1 (Th1) cell development, while it inhibits the pro-duction of some anti-inflammatory cytokines, such as IL-10.Moreover, some recent studies have suggested a potential role ofIL-18/IGIF in immunoregulation by mediating immune cell in-filtration into the inflammed tissues and it is already well knownthe important role of leukocyte infiltration in the renal damageof human proliferative nephritis, such as IgA nephritis.In order to investigate the potential role of IL-18/IGIF in IgAnephritis we measured, by ELISA, the serum levels of IL-18/IGIF (IL-18 S) in 6 healthy subjects and, at biopsy time, in 16IgA patients: 9 pts with necrotizing/crescentic IgA nephritis(NC-IgAN) (% of crescents 25.2±10.6; score of interstitial dam-age: 3.2±0.9, range 0-4) and 7 pts with non necrotizing/crescenticIgA nephritis (NNC-IgAN)(score of intersitial damage: 1.7±0.7,range 0-4).IL-18 S in IgA pts were significantly higher than those observedin control subjects (26.2±20.2 vs 4.1±2.5 pg/ml, p<0.01). Moreo-ver, NC-IgAN pts exhibited significantly elevated IL-18 S whencompared with NNC-IgAN pts (28.1±15.4 vs 10.1±4.3 pg/ml,p < 0.02).In conclusion, our results suggest that IL-18/IGIF might play apotential role in the renal damage of IgA nephritis, especially inthose forms characterized by extracapillary proliferation andmarked leukocyte intraglomerular and interstitial infiltration.

THE INFLUENCE OF HEMODIALYSIS ON PERIPHERALBLOOD LYMPHOCYTE APOPTOSIS1J. Rysz, 2E. Majewska, 1J. Mudyna, 1Z. Zbrog, 2Z. Baj, 1M. Luciak,1P. Bartnicki.12nd Dept. of Internal Medicine, WAM and 2Dpt. of Pathophysi-ology, WAM, Lodz, Poland.

The percentage of apoptotic lymphocyte cell was evaluated in10 chronic uremic patients before and after hemodialysis ses-sion and following 24-hr culture. The control group consisted of10 healthy persons. Apoptosis was investigated by means ofAnnexin V-FITC and TUNEL flow cytometry methods. The re-sults are presented as apoptotic cell percentage of the whole cellpopulation (X+SEM):

Healthy Patientsbefore HD after HD

Annexin 2.38±0.69 4.23±2.37 5.43± 8.06TUNEL 10.78 ± 5.90 8.58 ± 0.66 11.85 ± 9.17

24-hr cultureHealthy Patients

before HD after HDAnnexin 8.98±15.3 5.25±2.12a 4.10±2.54a

TUNEL 14.10±24.50 4.65±0.92a 11.03±14.60a

a p<0.05 in relation to healthy persons

It seems that hemodialysis does not immediately numbers ofapoptotic cells. However, following 24-hr culture lymphocytestaken from patients before and after hemodialysis were lower incomparison with those from healthy persons.

PROGNOSIS MARKERS OF TUBULOINTERSTITIAL INJURYIN MESANGIOCAPILLARY GLOMERULONEPHRITIS TYPEIL. Petrica1, M. Raica2, A. Schiller1, S. Velciov1, Gh. Gluhovschi1,V. Trandafirescu1, Gh. Bozdog1, C. Gluhovschi1, F. Bob1

1 - Dpt. of Nephrology, 2 - Dpt. of Histology, County Hospital,University of Medicine and Pharmacy, Timisoara, Romania

A group of 32 patients (p) with mesangiocapillary type Iglomerulonephritis (MCGN) [M-19p (59.37%); F-13p (40.63%);mean age - 42.31 ± 11.28 y] was assessed concerning the rela-tionship between the severity of the tubulointerstitial (TI) le-sions and blood pressure (BP), proteinuria and serum creatinine(SC).All p underwent kidney biopsies which were processed in lightmicroscopy (LM - hematoxylin-eosin, Masson’s trichrome, PAS),immunofluorescence, immunohistochemical (IH) procedureswith monoclonal antibodies [performed with the EPOSsystem-DAKO: antismooth muscle cell actin (a-SMA),anti-desmin (D), anti-cytokeratin (CK), anti-proliferating cellnuclear antigen (PCNA)].Results were analysed by statistical methods (Student’s un-paired ttest, Pearson’s test, Spearman’s rank order test); signifi-cance was considered as P<0.05.The evaluation of the TI lesions in LM revealed in 23 p (71.88%)severe TI injury, which correlated significantly with the intersti-tial IH data: - the extent of the a-SMA positive cells(myofibroblasts) infiltrates (P<0.001), PCNA (P<0.05), D (proxi-mal tubular necroses) (P<0.01), CK (distal tubular necroses)(P<0.001). Proteinuria correlated with a-SMA (P<0.01), PCNA(P<0.05), D (P<0.01), CK (P<0.01); SC with a-SMA (P<0.001),PCNA (P<0.01), D (P<0.01), CK (P<0.05). BP did not correlatewith these parameters.TI lesions in MCGN type I imply important myofibroblasts infil-trates and severe proximal and distal tubular involvement. Thesechanges are consistent with the level of proteinuria and the rateof decline of the renal function.

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Abstracts



THE COMPARISON OF ANTIBODY RESPONSE TO INFLUENZA VACCINATION INCAPD, HD AND RENAL Tx PATIENTSC. Cavdar, M. Sayan, A. Sifil, C. Artuk, N. Yýlmaz, S. Kavukcu, H. Bahar, T. CamsarýDokuz Eylül University Hospital & Ankara Refik Saydam Hýfzýssýhha Instýtute - Türkiye

The immune system in CAPD, hemodialysis (HD) and renal transplanted (Tx) patientshave been supressed. Generally these patients give weakly antibody (Ab) response tovaccination in comparison to healthy adults.The aim of this study is to detect Ab response to influenza vaccine as an indicator of thelevel of immunity in CAPD, HD and Tx patients and then to investigate the differenceof these groups. In this study 48 patients including 17 Tx, 16 CAPD and 15 HD werevaccinated. 10 healthy adults were incorporated to this study as control group. DuringOctober - November 1997, a purified, split-virus, commercial trivalent influenza vaccine(VAXIGRIP - Pasteur Merieux Connaught) was given to both patients and control groupin the dosage of 0.5 mL one times to deltoid muscle intramuscularly. Vaccine contained 15µg of each hemagglutinin of A/Johannesburg/82/96(H1N1),A/Nachang/933/95(H3N2)and B/Harbin/07/94 strains. Serum samples were collected before and 1 monthafter vaccination for Ab determinations. Determination of Ab response was performedwith hemagglutination-inhibition test (HAI). Wilcoxon paired t test and Mann - WhitneyU test were used for statistical analysis. p < 0.05 was accepted as statistically significant.All of the results related with study groups are shown in Table 1.Table 1: The Results of the Study GroupsAb Response Tx CAPD HD ControlB (Before) 32,35±4,97 27,50±4,33 34,00±5,84 46,00±7,92B (After) 96,47±34,82* 62,67±8,92* 74,67±12,72* 88,00±16,65*∆B 2,88±0,52 2,53±0,29 1,93±0,20 2,00±0,26H1N1 (Before) 30,59±9,01 25,00±5,70 19,67±4,59 28,00±6,63H1N1 (After) 75,29±17,62* 60,00±12,19* 40,67±5,89* 102,00±28,67*∆H1N1 3,06±0,51 3,67±1,07 2,23±0,30 3,80±0,76H3N2 (Before) 38,82±4,61 36,25±7,0 32,00±6,91 48,00±13,73H3N2 (After) 67,06±12,33*+ 58,67±12,07*+ 75,33±15,18*+ 204,00±61,81*∆H3N2 1,68±0,25+ 2,30±0,51 2,32±0,52 5,00±1,46* Ab levels were increased statistically significant after influenza vaccine+ Ab response against to H

3N

2 was significantly lower in Tx, CAPD and HD

∆B, ∆H1N1, ∆ H3N

2: The magnitude of Ab rising

In conclusion, Ab levels after vaccination in Tx, CAPD and HD patients increased statis-tically significant and there were no differences between them ; this resulted in increasingthe proportion of patients having protective Ab titration however these proportions werefound lower than those of control group. Although the ratio of patients having protectiveAb titration with vaccination increased in Tx, CAPD and HD patients, the magnitude ofAb rising against to H3N2 and the levels at first month were detected as lower than thatof control group. Tx, CAPD and HD patients should be vaccinated every year because ofmedical complications caused by influenza infection. Trial of high dose vaccination protocolsmay be useful to increase Ab response.

THE BOOSTER PHENOMENON IN TUBERCULIN SKIN TESTING (TST) OFHEMODIALYSIS (HD) PATIENTS AND THE RELATIONSHIP WITH SOLU-BLE INTERLEUKIN 2 RECEPTOR (sIL-2R) LEVELSA. Akçay, Y. Erdem, B. Altun, S. Ulusoy, C. Usalan, Ü. Yasavul, Ç. Turgan, S.Çaglar and S. KirazliHacettepe University School of Medicine, Ankara-Turkey

The booster effect is defined as an initially negative TST in a patient which isthen boosted to a positive test by the testing procedure itself. The significance ofthe booster effect in sequential testing of this patients has not been determined.It is suggested that elevated serum sIL-2R levels may decrease the bioavailabilityof IL-2 for T cell response to antigenic stimuli in HD. We examined the frequencyof the booster effect and anergy, and the relationship with serum sIL-2R levelsin HD patients.Fifty three HD patients, aged 20-72yr, M:F 35:18 were studied. Ten (18.8%) pa-tients had a history of active tuberculosis. Skin tests were placed by the Mantouxmethod, using 0.1 mL of tuberculin (5 TU) and candidin control antigen (10.000BE/ml). Patients with <10 mm induration to the initial TST had a repeat tuber-culin test 7 days later. Nineteen (35.8%) of 53 patients demonstrated a signifi-cant tuberculin reaction (>10 mm) on the initial TST, and 10 (18.8%) additionalpatients showed a significant reaction (booster effect) to a second TST (p<0.001).All the patients with a history of tuberculosis had a strongly positive tuberculinreaction (>15 mm) on the initial TST. Ten (18.8%) of 53 patients did not responseto either candidin or two-step TST, and this patients were considered completelyanergic. Serum sIL-2R levels were measured in 10 healthy subjects (age 22-62,5M, 5F), 10 anergic (age 24-56, 4M, 6F) and 10 non-anergic (age 28-61, 6M, 4F)HD patients. The mean values of serum sIL-2R were significantly increased inthe patients as compared to healthy subjects (12.65±5.64 vs. 4.13±0.76 pg/mL,p<0.001). The levels of serum sIL-2R were significantly higher in the anergicgroup than in the non-anergic group (17.98±5.69 vs. 7.72±1.24 pg/mL, p<0.005).Our findings suggest that the booster effect is not uncommon phenomenon inend-stage renal disease and the elevation of serum sIL-2R levels may play animportant role in the pathophysiological mechanisms of cutaneous anergy inuremic patients.

INTERFERON-Α (IFN) THERAPY IN HEMODIALYSIS PATIENTS (HD) WITHASSOCIATED HEPATITIS C VIRUS INFECTION (HCV): INFLUENCE ON THE TH1AND TH2 BALANCE USING INTRACELLULAR CYTOKINES ANALYSISStachowski J., Kissinger G.L., Steffen M., Pollok M., Jilke K., Kneissel B., Eilers R., BaldamusC.A.Medical Clinic IV, University of Cologne, Germany & Clinic of Pediatrics Poznan, Poland

Th1 activity correlates with the expression of IFN-γ and IL-2 whereas Th2 with IL-4 andIL-10 production. Th1 cells promote delayed type hypersensitivity reaction including cellmediated cytotoxicity, whereas Th2 exert suppressive effects and induce immune toler-ance. The clearance of HCV infected cells depends on an adequate cytotoxic activity (Tand NK cells) and Th1/Th2 balance. Influence of INF therapy on Th1/Th2 balance inHD patients with HCV infection is not well known. The study was undertaken in HDpatients (n=15) with associated HCV infection before and during IFN-therapy (5MU 3x /week for 3 month and 3MU 3x/week for 9 month) therapy in patients non-respondingto IFN. Non-response (NR; n=7) was defined after the first 3 month of ineffective IFN-therapy (HCV-RNA+). FACS technique was used in analysis of intracellular cytokines inperipheral blood lymphocytes (PBL). Cytokine expression in activated CD4+ lymphocyteswas estimated by using 3-colour fluorescence. Activated PBL were stained with anti-CD4(Cy-Chrome), fixed, permeabilized (saponine) and then labelled with anti-IL-2 and anti-IFN-γ (FITC) or anti-IL-10 and anti-IL-4 (PE). Results are presented as % of cytokineCD4+cells. Data of NR to IFN and subsequent responders (R) or NR to the long-lasting (9month) IFN-therapy were compared to healthy donors (controls). *p<0.05; +p<0.01;#p<0.001 (Table)

Th1 Th2

IL-2 IFN-γ IL-4 IL-10

IFN-α-therapy NR

Before ↓20+ ↓21+ ↓22+ ↓24+ 33 ↑34* ↑40* 30

5 MU ↑43# ↑43# ↑47# ↑49# ↓12* ↓10* ↓15* ↓12*

3 MU ↑42* ↑42# ↑45* ↑45* 24 18 31 25

Direct after IFN-α NR R NR R NR R NR R

↑48# ↑↑62# ↑52# ↑↑↑68 ↓↓12# ↓↓↓8# ↓14* ↓↓↓9#

3 month later ↓21+ ↑30# ↓21+ ↑33+ 31 24 ↑41* 28The patients with double NR to IFN after 3 month and subsequent 9 month therapyrevealed very low activity of Th1 cells (Th1<<<Th2) before the therapy: in the course ofIFN therapy the Th1/Th2 balance was moved into Th1 activity. In NR the reversed Th1/Th2 (Th1>Th2) activity in the course of long-term IFN-therapy was significantly lowercomparing to R to IFN therapy (Th1>>>Th2). Conclusion: Long-lasting IFN therapy isuseful in order to eliminate HCV in HD patients but the high Th1 activity is only observedduring the therapy.

IMMUNOLOGICAL CHANGES IN ANCA POSITIVE NE-PHROPATHYR. Vladimirova, V. Draganov, B. Kenarova, K. Plochev, R.PenkovMilitary Medical Academy, Sofia, Bulgaria

The aim of the study is to assess the parameters of humoraland cellular immunity and levels of some cytokines in se-rum and urine of 11 patients with clinicolaboratoryconstelation of systemic vasculitis with nephropathy. Pa-tients: Included in the study were 3 patients with poliarteritisnodosa, 3 with SchonleinHenoch nephropathy, 3 withpulmo-renal syndrome of Goodpasture, 2 with other formsof systemic vasculitis, and 10 healthy persons as controls.Methods: The parameters of cellular immunity were deter-mined through the flowcytometry method. The titers ofantineurophil cytoplasmic antibodies (ANCA) were meas-ured through indirect immunofluorescense and the levelsof IL-2, IL6, and INF-gamma were measured through theELISA method. Results: Total T-cell population and T-helpersin ANCA positive patients were significantly increased incontras to T-suppresors and B-cells, which were decreased.The levels of IL-2 and INF-gamma were significantly in-creased, especially in the urine of the patients as comparedto the controls, but there was no difference in IL-6 levels.Conclusion: Our finding in this survey supports the possi-bility of Th1 way of immune caskade playing important ormaintanance of glomerular barrier dysfunction.

A31

Abstracts



IMPACT OF HEPATITIS B AND/OR C ON THE OCCURRENCE OF NEPH-ROPATHY IN PATIENTS WITH HEPATO-SPLENIC SCHISTOSOMIASISH El-Ghoneimy*, ZA Shaker #, MG Saadi *, I Shaltout*, A El-Shamaa*, A El-Bassiouni#, KA Maher#, MM Kamel#.Departments of int. medicine and nephrology*, and clinical pathology #, facultyof medicine, Cairo University, Egypt.

Nephropathy has been proved to occur with infestation by Schistosoma man-soni, particularly following porto-systemic shunting. The effect of associatedinfection with viral hepatitis B and/or C on the tendency of occurrence of thisnephropathy has not been studied.Forty eight patients with Schistosoma mansoni hepatosplenomegaly (HSS) werestudied, including 13 with -ve HCV antibodies and HBsAg (gp I), 11 with +veHBsAg (gp II), 12 with +ve HCV (gp III), and 12 with +ve HCV and HBsAg (gpIV). In addition 24 were included as controls 12 with -ve virology and 12 with+ve HCV.The assessment included clinical data, laboratory investigations for the urine,stools, liver and kidney functions, and abdominal ultrasonography. Upper en-doscopy and occasionally a liver biopsy were done for the patients. The immu-nological assessment included testing for circulating Schistosomal antigens(CSA), circulating immune complexes (CICs), complement C3, C4 and C3d.Microalbuminuria was tested as an indicator of nephropathy.CSA and CICs were significantly higher in all groups than reference values ofcontrols. CICs were highest significantly in group II 68.714 ± 36.142 mg/ml p<0.01 and group IV 79.458 ± 32.806 mg/ml p< 0.001. C3 and C4 were signifi-cantly lower in the patients groups than in controls, and C3d was +ve in mostpatients being more in groups II and IV, 72.72% and 75% respectively.Microalbuminuria was present in a greater percentage (25%) and higher value(72.8 ± 25.67 mg/ml in patients of group IV than the other groups.The risk of developing nephropathy in HSS patients is increased by concomitantHCV or HBV infections, particularly when both are present together in thesepatients.

EVIDENCE THAT AN ENHANCED TH2, TRANSFORMING GROWTH FAC-TOR BETA-1 (TGF-β1) AND TUMOR NECROSIS FACTOR α (TNF-α) IMMUNERESPONSE IS PRESENT IN PATIENTS WITH CHRONIC HEPATITIS C VIRUS(HCV) INFECTION.Garinis G1,2, Spanakis N1,2, Ioannidis T1, Theodorou V1, Manolis E 1,4, ValisD 1,2, Karameris A31. MEDICANALYSIS Institute of Molecular Biology Applications, Athens,Greece. 2. HYGEIA Therapeutic Center, Unit of Nephrology, Athens, Greece. 3.401 General Army Hospital, Dept. of Pathology, Athens, Greece. 4. University ofAthens, School of Nursing, Dept. of Anatomy, Histology and Embryology, Ath-ens, Greece

Objective. In this study, we report serum circulating levels of IL-1β, 2, 4, 6, TNF-αand TGF-β1 in two different HCV related groups: HCV (+) haemodialysis (HD)patients and HCV (+) non-HD patients. Both groups were subcategorized in a)patients with active chronic hepatitis and b) HCV carriers without liver dam-age. Results were compared with HCV viraemia profiles and liver biochemis-try parameters. Methods. IL-2, 4, 6, 1β, TNF-α and TGF-β1 serum levels weredetermined in 92 individuals: 52 HCV (+) individuals and 40 controls: [20 HCV(-) HD patients and 20 healthy volunteers (HCV (-), non-HD)]. Patients weredivided into the following four groups: 1.HCV (+) HD active chronic hepatitispatients (n=10), 2. HCV (+) HD carriers with no liver damage (n=12), 3. HCV (+)non-HD active chronic hepatitis patients (n=11), 4. HCV (+) non-HD carrierswith no liver damage (n=12). Results. IL-2, 4, 6, TNF-α and TGF-β1 but not IL-1βserum levels (P>0.05) were significantly higher (<0,03) in HD active chronicHCV patients versus HD HCV carriers with no liver damage: [100.1±26.30,113.4±36.8, 135±33.64, 81.4±76.12, 193.4±60.3, 48.8±19.2 vs. 69.45±22.49,76.90±19, 82.81±10.39, 145.18±26.8, 104.18±48.71, 35±12.107 pg/mL] and innon HD active chronic HCV patients vs. non HD HCV carriers with no liverdamage [87.92±36, 98.08±35.65, 11.25±33.4, 203±69.4, 142±28.49, 11.46±3.44vs. 33.61±15.93, 40.83±11.73, 45.41±19, 92,66±26, 51.25±17.58, 13.30±5.08 pg/mL]. Elevated serum levels of both IL-4 and 6 were greater than IL-2 cytokinein all patient groups versus controls. A substantial number of HD and non-HDpatients (n=5) with chronic hepatitis C were shown to have normal or nearnormal alanine aminotransferase/ aspartate aminotransferase/γ-glutamyltransferase (ALT/AST/γ-GT) values. No correlation was observedbetween cytokines serum concentrations and HCV viraemia (>0.05). Conclu-sion. Our results suggest that irrespective of HCV viraemia and liver enzymelevels an enhanced Th2, TGF-β1 and TNF-α immune response is present in chronicHCV infection.

1,25-(OH)2VIT.D

3 INHIBITS iNOS mRNA EXPRESSION AND

NO PRODUCTION FROM LPS-STIMULATED RAW 264.7JM Chang, SJ Hwang, JC Tsai, JH Tsai, YH LaiDivision of Nephrology, Kaohsiung Medical College, Kaohsiung,Taiwan.

The activity of inducible nitric oxide synthase (iNOS) ofmacrophages is greatly enhanced during infection. 1-α-hydroxy-lase activity and 1,25-(OH)

2Vit.D

3 (Vit.D) synthesis are also in-

creased. The increase of Vit.D may in some occasion inducehypercalcemia and lead to clinical illness. The significance of theincreased amount of Vit.D is, however, not clear. The aim of thisstudy is to clarify the effect of Vit.D on the iNOS mRNA expres-sion and the nitric oxide (NO) production.Mouse macrophage-like cell line, RAW 264.7, was used in thisstudy. They were cultured in DMEM/F12 supplemented with5% FCS. 24 hours before study, the medium was changed toDMEM/F12 and charcoal-treated bovine serum in order to re-move the influence of Vit.D in the serum. After quiescent, vari-ous doses of endotoxin — E coli lipopolysaccharide (LPS,O127:B8) — and Vit.D were added to RAW 264.7 for 24 hours.NO gas production into the medium was measured by NOdetector, ISO-NO MARK II (World Precision Instruments), andcells were collected to assess the expression of iNOS mRNA,using GAPDH as internal control.After total extraction of RNA, RT-PCR was used to assess iNOSmRNA expression. Vit.D, ≥10-10 M, showed an inhibitory effecton iNOS mRNA expression and the inhibition was dose-de-pendent. The inhibition even became evident from ≥10-11 M in thelower dose of LPS stimulation. The change of NO gas produc-tion generally followed that of iNOS mRNA. NO productionwas gradually decreased by the increase of Vit.D and the de-crease became significant when Vit.D concentration was ≥10-11

M. Under very low dose of LPS stimulation, as low as 10-12 MVit.D was able to decrease NO production.Our study revealed the inhibitory role of Vit.D on the iNOSexpression and NO production from LPS-stimulatedmacrophages. Vit.D may be one of the rescue systems to salvagethe cell from the oxidation injury due to the abrupt productionof NO during infection.

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