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• SEROPREVALENCE OF ANTI HE'/IN A WESTERN COUNTRY

G.Macedo, T.Pinto, JA Sarmento, AMH Vale, T.Ribeiro, Gastrenterology Unit, Hosp. S. Jo&o, Porto, Portugal

Introduction: Emmigration and Immigration flows to and from subtropical countries (Brazil, Venezuela, Portuguese-speaking african countries), where some cases of Hepatitis E viral (HEV) infection have occursd, question whether this infection can spread into western countries. Besides entsral route, the finding of HEV in blood, suggests the possibility of parenteral exposure in these areas. In developing countries, it could be wise to screen HEV in blood donors.

Aim: To evaluate the prevalence of antiHEV in the serum of asymptomatic volunteer blood donors (AVBD) and in chronic liver disease patients.

Material and methods: We have analysed 50 blood samples from AVBD without HBV or HCV markers and normal aminotransferass (mean age, 36 years old) and 103 samples from chronic liver disease patients (HBV or HCV infected, or alcoholic liver disease). All samples were tested with HEY EIA (Abbott Diagnostic), using recombinant antigens from open reading frames 2 and 3 (SG3 and 8-5), coding respectively, non structural and structural proteins from 3 urma virus. This teat detects only IgG antibodies.

Results: AntiHEV was detected in 2/50 samples (4%) from AVBD and 7/103 (6,8%) from liver disease patients. The values of optic density/cutoff) found in AVBD samples were 1,1 and 1,5, and in the patient group, varied between 1,3 and 2,3. Five of the positive samples in this group, belong to cirrhotic patients, with hypergammaglobuline- mia.

Conclusions: 1. The prevalence of anti HEV tested in HEV EIA Abbott, was 4% in AVBD and 6,8% in chronic liver disease patients, 2. All positive samples showed values of OD/cutoff that were not over two times the cutoff point which seems to demonstrate that these values should be confirmed by a supplemental assay.

• THIRD GENERATION TESTS FOR HCV: WHAT ADDITIONAL INSIGHTS?

G.Macedo, T.Pintc, B.Juatiga, Gastrenterology Unit, Hosp. S. Jo~o and Hematology Department, Hosp. S t°. Antdnio, Porto, Portuga~

Aim: To evaluate the performance of an anti HCV test, Elisa third generation test, in 3 different groups of asymptomatic volunteer blood donors (AVBD), with s second generation HCV test, negative or doubtful.

Material and methods: Samples from AVBD with negative or doubtful values for 2 nd generation Elisa (Ortho), were evaluated with a 3 ra generation Elisa (Ortho) test, using specific core antigens, NS3/NS4 and NS5. The samples were collected from 22 HBs Ag positive AVBD (group A), from 107 with aminotransferases (ALT) elevation (group B) and from 31 with borderline values in EIA-2 anti HCV (group C), in at least 2 out of 3 tests (optic density/cutoff in between 0,8 and 1,2). All EIA 2 positive samples had a Riba 3 (Ortho) test, and in the 3 groups, the markers of hepatitis B viral infection were searched.

Results'. In group A, one sample (1/22, 4,5%) was EIA 3 antiHCV reactive. In group B (ALE between 70-466 u/I, x:106 _+ 48,6) one sample (1/107, 0,94%) was EIA3 reactive. In 4/107, the HBC antibody was found isolated, and in 11/107, the anticore and antibody against surface antigen were associated (total: 15/107, 14%). Both EIA3 rscativs samples from these A and B groups were not positive in Riba 3. From group C, all EIA2 values between 0,8-1,2 (x:0,96 -+ 0,13) were negative in EIA3 (31/31, 100%). No sample was positive, isolately, to NS5 band.

Conclusions: 1. Being more specific (28/31, 90,3%; EIA3/Riba3 negative samples) the third generation test did not detect 2 indeterminate and 1 reactive sample in Ribs 3. This fact supports the existence of false negative results (less sensibility) even in third generation tests. 2. It seems to be critically important to perform a clinical and serological evaluation of high ALT indviduals, as the prevalence of viral markers in this set was only t4%.

EFFECTS OF ANTI-LEUKOCYTE ADHESION MOLECULE ANTIBODIES, NITRIC OXIDE SYNTHETASE INHIBITOR AND STEROID ON ENDOTOXIN SHOCK. T.Maeda, S.Marubayashi, Y. Ohshiro, K. Sugino, K. Dohi, S. Koyama*, K. Yamada*. The 2 n d Depa r tmen t of Surgery a n d Depar tment of Biochemistry*, Hiroshima Univers i ty School of Medicine

The objective of this s t u d y is to compare the the rapeu t i c effects of ant i - leukocyte adhes ion molecule an t ibodies (mAbs) , nitric oxide syn t he t a se inhibitor (m0nomethyl -L-arg in ine(NMiA)) a n d methy lp redn i so lone (MP) on exper imenta l endotoxin shock in mice. LPS(3Omg/ kg) was admin i s t e r ed to ICR mice intraperi toneal ly. S imul taneous ly , l m g / k g of mAbs, S - 2 0 m g / k g of NMLA or 3 0 m g / k g of MP was admin i s t e red in t ravenous ly . In the placebo group, phospha t e buf fe red sal ine (PBS) was admin i s te red . In the placebo group, the survival rate at 48 h o u r s was 3 8 % ( 2 1 / 5 5 ) . An t i -C Dl l a , CD18, anti-LECAM- 1 or MP increased the survival ra te to 70%(7/10) , 62% ( 8 / 1 3 ) , 64%(7 /11) or 100%( 11/11) , respec t ive ly . Anti-CD1 lb , ICAM-I or NMLA showed no s ignif icant effect. In the placebo group, p l a sma level o f n i t ra te a n d nitrite e levated f rom 20pM to 260vM at 4 h o u r s a n d 1000HM at 1 6 h o u r s after LPS adminis t ra t ion . NMLA comple te ly restricted nitric oxide p roduc t i on at 4 hours . MP part ia l ly restricted it up to 1 6 h o u r s . In the placebo group, l iver MDA level e levated f rom 0.5 to 2 . 4 6 n m o l e s / m g prot. at 4hours . Anti- CD18 or MP reduced the level to 1.80 or 1 .41 , respect ively. NMLA showed no effect on the level. The mAbs a n d MP are t hough t to be useful as therapeut ic r eagen t s for endotoxin shock. Nitric oxide seems to have no con t r ibu t ion to liver in ju ry u n d e r endotoxemia.

• ETHANOL INHIBITS CALCIUM-INDEPENDENT PHOSPHOLIPASE A 2 IN RAT LIVER. B Ma~enheim, P Stal*, R Hulterantz*, A Musat. Dept. of Medicine, University of Wisconsin, Madison, WI, and Karolznska Instltute, Karollnska Hospltal, Stockholm, Sweden.

The mechanisms of ethanol induced liver injury are only partially understood. There is evidence that chronic ethanol ingestion increases the activity of calcium-dependent phospholipase A 2 (PLA2) , and since these enzymes are important participants in inflammation, cell injury, and remodeling of membrane phospholipids, they may help mediate ethanol induced hepatic injury. The aim of this study was to characterize the activity of calcium-independent liver PLA 2 in chronic ethanol ingestion. We pair-fed rats a liquid alcohol diet according to Lieber and DeCarli for four weeks. There was no difference in serum aminotransferase levels between control and ethanol treated rats, and light and electron microscopic examination of liver tissue from alcohol-treated rats showed only steatosis. For determination of PLA2 activities we applied a novel methodology (Anal Bioehem 1994;217:210-19) which is based on quantitation of major molecular species of l-acyl-lysophosphatidylcholine, generated by the action of PLA 2 on the endogenous pool of phospholipids in cell membranes. We incubated lyophilized liver tissue in TRIS buffer at 37°C for 30 minutes with I mM CaCI 2 or 4 mM EGTA/EDTA, respectively, l-Stearoyl-(S-LPC), l-palmitoyl-(P-LPC), and l-oleoyl-lysophosphatidylcholine (O-LPC) were quantitated by reversed-phase HPLC and mass detection. Incubation of liver tissue from ethanol treated rats in the absence of calcium demonstrated a seven fold and a three fold lower rate of accumulation of S-LPC and P-LPC, respectively as compared to controls. S-LPC: 8.34±2.38 vs 1.11±1.74 (p<O.05); P-LPC: 11.53±1.85 vs 3.54±1.79 (p<0.02); O-LPC: 5.23±0.59 vs 3.4±0.73 (p<0.4) nmol/g dry tissue/min (mean±SEM). There was no statistically significant difference in the calcium dependent PLA 2 activity between controls and alcohol-treated rats. Thus, our study demonstrates that chronic ethanol ingestion inhibits calcium-independent PLA 2 in the liver, an effect which may be important in ethanol induced liver cell injury.