inhhration in the alveolar capillary, and interstitial space of the lung in the I/R group. ONO- 1714 treatment eftect:vely attenuated these damages, immunohistochemical analysis showed that anti-mtmtyrosine staining in the 1/R group was pusitive in the sinusoidal spaces and hepatocytes m the the liver and alveolar macrophages and interstitial space of the lung, while this staining was absent in ONO-1714 group. CONCLUSION: These data show that NO produced by iNOS contributes to liver damage but had little ettects on lung injury tbllowntg hepatic I/R. C;eneraion of peroxynitrite was suggested to be one of the pathogencsis in this condition and the inhibition of iNOS could be applied for the attenuation of hepatic L/R injury.

$883

Signaling for Hypersensitive Reaction Manuela G Neuman, Neil SheaL Lori H. Shapiro, Izabella M. Malkiewicz, Masud Taeri, Joel Fish, Manuel Gomez, Lawrence Cohen

Hypersensitivity syndrome reactions (HSRs) to anticonvulsants (AC) and sulpbonamides (SMX) are associated with toxicities culminating with toxic epidermal necmlysis (TEN), which has a mortahty of 20-60%, ObJective: l-Validation of an in vitru lymphocyte toxicity assay (LTA) for assessing HSRs to the specific AC or SMX incnmimlted in HSR; 2-identification ot specific detects in innnnno-responses that may establish markers of individuals at increased risk tor HSRs to AC or SMX and 3-Correlation of these markers with an in vitro LTA. Methods: 190 patients with a suspected drug4nduced HSR (94 AC and 96 SMX) and 200 persons that have tolerated these drugs (controls) were tested. These patients had manitested rash fever, hepatic involvement w~thin 8 weeks of exposure to AC (carhamazepine, phenytoin or phenobarbital) or SMX (snlphametaxazole). lndwidual's lymphocytes were exposed in vitro to the incriminated drug in the presence of a microsomal system. The mitocbondnal succinate defiydrogenase activity as a measure of cell viability was expressed as % of cell death. LF?X showing a toxicity higher than 12.5% was considered positive. Differences among groups were determined by the use of confidence intervals and analysts of variance. The Wilcoxon rank sum test was used to compare difterences between groups. Results: 1-A perfect correlation was obsePeed between (he LTA posinve r = 0.89 and the clinical diagnosis in TENs with a posinve predictive vahie to the incritinrcating drug of 96% and a negative predictive ~vtlue of 98%, sensitivity 8995 and speciticity 87%. More importantly some of the patients presented cross-reactivtly to different AC, while others presented a negative LTA to one drug and a positive value to another. Based on the LTA, an ahemative drug may be prescribed, 2-No differences were seen between the control and AC-HSR groups regarding the tumor necrosis factor alpha (TNF-a) serum levels and their mRNA-expression. AC- induced TEN had interleukm (ID 6 significantly lower than in controls and 1L 12 higher demonstrating the involvement of immune system in HSRs, TNF-a serum levels SMX-HSR and the mKNA expression is higher and presents a polymorphism that is dependent on the severity of TEN. IL 6 is siginflcanfly lower than in controls and 1L 8 and IL 12 significantly highenThe LTA technique LS accurate and sensitive. Prediction of a HSR in vitro will prevent drug-induced morbidity and insure a safe administration of therapies. Cytokine and chemokines polymorphism and mitochondrion signal for drug-n:duced HSRs.AA

S884

Propionibacterinm Aches Triggers Inflammatory Responses Via Toll-Like Receptor 2 and Sensitizes for Liver Injury Via Tlr2-independem Pathways Laszlo Romies, Karen Kodys Douglas Golenbc, ck~ Gyongyi Szabo

Tolblike r~ceptor 2 (TLR2), a pattern recognition receptor, recognizes gram-positive bacteria and lipoproteins Propionibacterium acnes (P. acnes), a gram-posinve hacterium, is a macro- phage and Thl-celI act:valor that primes the liver to endotoxin induced injury modeling fulminant hepatitis We and others recently showed that P. aches mediates cell activation v~a TLR2. Thus, tha aim of this study was to investigate the role of TLR2 in P. aches induced priming of the liver to LPS-induced injury" in vivo. METHODS: 6-8 week old C57BU6 (WT; 3/group) or TLR2 deficient (-/-) mice were chal- lenged with heat-killed P acnes (1 rag, Lp.) or the TLR2 ligands, PGN and/or LTA (each 5ug/g bw. i.p., Staph, A.), stimulated with IPS (05 mg/g b.w., E. coil 011liB4) 7 days later and sacrificed at various tin:epoints. Serum TNFa, iL-12(p70), 11_-6 and 1FN"/(ELISA), liver IL-12p40, iFNy, 1L-lot, 1E-113, ll.-1Ra, IL-IO and IL-6 RNA (RNase protection assay) levels, aud tiver hlstopatbology (H&E) were assessed RESULTS: P. aches induced NF-KB activation in CHO cells expressing human TLR2/CD14 but not in CHO TLR4/CD14 cells. TLR2qned:ated cell activation by P. aches (10-100 lag/ ml) was turther suggested by up-regulation of [L-8 product:on (p<.O004) in TLR2, but not in I LR4,~%fD-2 transtected H}:!K cells. However, investigation of the TLR2-mediated pathways revealed that P. acnes induced granuloma tbrmation as well as sensitization for LPS-induced liver injury both in WT and in TLR2 -/- mice. P. aches augmented IpS-indnced serum TNFc~, IL-6 and IFN% but not IL-12 levels; liver cytokine RNA levels were increased both in WI' and TLR2*/- mice. Finally, unlike P acnes, selective TLR2 ligands (PGN and/or LTA) failed to sensitize for LPS induced mira T evidenced by the lack of serum cytokine increase or liver granulomas CONCLUSIONS: Our data demc, nstrate that P. acnes induces activation of inflammatory pathways via TLR2. However selective activation via TLR2 is not sufficient to substitllte for the liver-sensitizing effects of P. aches The observation that liver sensitization by P. aches occured m the ab~nce of TLR2 expression suggest involvement of mechanism(s) other than TLR2-mediated pathways pnming of the liver by P. acnes.

Serum cytokine levels induced by P. acnes or PGN+LTA preVeatnlmnt tilen LPS challenge ( ~ p r ~ J

WT I P. a~es TLR2 4- I P.acnes ~ I PGN+LTA TNFa 37 2,3 39 12 13 ~0.5 IL4 16122 12211 265.3 IFNy 22,8 6,8 72 32 1,48 0,1 ik-l~'~?O) 18-516 677,9 361,8

$885

Propionibaeterium Aches Sensitizes The Liver for Toll-Like Receptor 4, Not for Tlr2 Mediated Injury Involving Up-Regulation of The Tlr4/Md-2 Receptor Complex Laszlo Romics, Karen Kodys, Weibo Li, Angela Dolganiuc, Gyongyi Szabo

Propioinbacterium aches (P.acnes) is a non-specific macrophage activator that has been shown to prime the liver to lipopolysaccharide (LPS) induced injury via induction of INF,, IL- 12 and IL- 18 resulting in recruitment of inflammatory cells in the liver modeling fulminant hepatitis. IPS (stock LPS, sipS) used in these models has been shown to activate Toll-like receptor (TLR)4 via LPS as well as TLR2 due to the lipopeptide contamination in sIPS. The LPS signaling complex includes multiple molecules, particularly TLR4 and MD2. The purpose of this study was to investigate whether P. aches sensitized the liver for TLR2 or TLR4 mediated injury. METHODS: Human TLR2 and TLR4/MD-2 transfected HEK 293 cells were activated with stock (TLR4 and TLR2 ligand) or purified LPS (pipS; TLR4 ligand) (E.Coli 0111:B4; 0.1- I ~g/ml) and PGN + LTA (TLR2 figands) (StapK A.; 0.1-1 ~g/ml) overnight and lL-8 production (EL1SA) was measured. 6-8 week old female C57BL/6 mice (3 animals/group) were challenged with 1 mg heat-killed P. aches (1000 lag, i.p.), one week later animals were stimulated with sipS (0.5 ~g/g b.w.,), pipS (0.1 ~g/g b.w.), or PGN+LTA (5 ~g/g b.w. each). Serum TNFa, total IL-12, IL-6 and IFN3' (ELISA), liver inflammatory cytokine RNA (RNase protection assay) levels, liver histopathology (H&E) and liver TLR4, TLR2, MD-2 and MyD88 protein levels were assessed (Western blot). RESULTS: In vitro, sipS (TLR4 and 2 figand) and PGN (TLR2 figand), not pipS (TLR4 ligand), triggered IL-8 production in HEK/TLR2 cells while pipS and s ips induced 11.-8 in HEK/TLR4/MD2 cells. In vivo, one week P. acnes treatment primed the liver for injury' induced by sipSeand pipS as reflected by increased serum TNFex, IL-6, total IL-12 and IFN~I protein and increased RNA levels for 1L-12p35, 1L-12p40, 1L-lc~, 1L-l~3, IL-1Ra, Ib 18 and IL-6 in the liver. In contrast, P. aches priming failed to sensitize the liver for TLR2- mediated injury (PGN + LTA). Interestingly, after one week of P. aches treatment both TLR4 and TLR2 protein levels were upregulated in the liver. In addition, the TLR4 co-receptor, MD-2, and not MyD88, the downstream adaptor molecule common to TLR2 and 4, was increased in the P. aches primed lwer. CONCLUSIONS: Our data define that P. acnes sensitizes the liver for TLR4- and not for TLR2-mediated injury that involves up-regulation of the TLR4/MD-2 complex. Our results also suggest that unlike TLR4 activation, TLR2 upregulation do not trigger fiver injury'.

$886

Continuous Immune Tolerance on Liver Injury ls Induced by Repeated Intravenous Injection with Concanavalin a in Mice Yosui Tamaki, Kimihide Nakamuna, Shiro Yokohama, Satoshi Okamoto, Masashi Yoneda, Mitutaka Okada, Taku ko, Kazunobu Aso, lsao Makino

It is reported that repeated challenge with foreign antigens, such as bacterial toxins and toxic drugs, induces immune tolerant state, resulting in a reduction of the tissue injury and inflammation. The intravenous injection of concanavafin A (Con A) activates T cells and induces cytokme dependent liver injury in mice. However, the gradual decline of liver injw was shown by repeated challenge with Con A. Purpose: To investigate how long the immune tolerance on liver injury is maintained after the cessation of repeated Con A challenge and whether pro- and anti-inflammatory" cytokines are involved in this tolerant state. Methods: Female Balb/c mice (7 weeks old) were intravenously injected with Con A (20 mg/kg) or saline vehicle once a week for six weeks. Ten, 17 and 24 days after the cessation of CoaA or saline injection, mice were intravenously re-injected with Con A (20 mg/kg), and plasma ACT level was determined enz)'maticafly 8 h after the East Con A injection. Plasma TNF-a, IFN-~/and IL-t0 levels were determined by ELISA 0, 2, 4 and 8 h after the last ConA injection. Results: The inhibition of plasma ALT level 8 h after Con A injection was maintained until 24 days after the cessation of repeated Con A challenge but not after saline (mean +- SE, KU/L: vehicle 2179 + - 413; Con A 79 + - 24 at 10 days, 6830 +- 1178; 67 +- 12 at 17 days, 6288 + - 1566; 187 + - 144 at 24 days, n = 5-8), Furthermore, mice at 17 da)~ after the cessation of repeated injection with Con A, plasma TNF-ct and 1FN-~/levels at 2, 4 and 8 h after the last Con A injection were significantly decreased compared with that of repeated vehicle treatment. By contrast, plasma IDIO at 2 and 4 h after the last Con A injection significantly increased by repeated injection with Con A at 17 days (mean +- SE pg/ml: vehicle 141 + - 29; Con A 1572 + - 220 at 2 h, 148 + - 32; 3113 +- 665 at 4 ll~ n = 5-8). Conclusion: These findings suggest that repeated intravenous injection with Con A induces continuous immune tolerance on T-cell-mediated liver injury by the inhibition of proinflammatory cytokines and the induction of anti-inflammatory cytokine prodactiorl in mice.

$887

Effect of a 72-kDa Heat Shock Protein against Endotoxemia in Cirrhotic Rats Ken-lchiro Mikami, Takashi Goto, Kouichi Minra, Shigetoshi Ohshima. Kazuo Yoneyama, Jiun-Guey Lin, Daisnke Watanabe, Ei Kataoka, Daisuke Segawa, Michiro Otaka, Sun:io Watanabe

Background & Aim: In liver cirrhosis, endotoxemia is a common complication with high mortality, It has been reported that 72-kDa heat shock protein (HSP72) protected non- cirrhotic rats against endotoxemia. However, its cytoprotective elfect against endotoxemia in cirrhotic rats has not yet been studied. In this study, we investigated the cytoprotective effect of HSP72 on lipopolysaccharide (ipS)-induced liver injury in cirrhotic rats, Methods: Liver cirrhosis was produced by 8-week intraperitoneal injection of carbon tetrachloride (CCI4) in male Spragne-Dawley rats. One week after the cessation of CC14 administration, the heat shock group (group HS) was exposed to hyperthermia (42.5 degrees C, 15rain). The control group (group C) received no pretreatment except anesthesia. After 48-haUl recovery, IPS (10mg~g) was administered intraperitoneally, Expression of HSP72 in the

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