Activation of nuclear factor kappaB in hepatitis C virus infection: implications for pathogenesis and hepatocarcinogenesis.

The hepatitis C virus (HCV) core protein is a multifunctional protein. It may bind to the death domain of tumor necrosis factor receptor 1 (TNFR1) and to the cytoplasmic tail of lymphotoxin-beta receptor, implying that it may be involved in the apoptosis and anti-apoptosis signaling pathways. In vitro studies have been inconclusive regarding its ability to inhibit or enhance TNF-alpha-induced apoptosis. To address this issue, electrophoretic mobility shift assay (EMSA) and immunohistochemical studies were used to show the activation of nuclear factor kappaB (NF-kappaB) in HCV-infected liver tissues and in HCV core-transfected cells. The activation of NF-kappaB was correlated with the apoptosis assays. The results showed that NF-kappaB activation could be shown in HCV-infected livers and HCV core-transfected cells. The data of EMSA correlated with those of immunohistochemical studies, which revealed a higher frequency of NF-kappaB nuclear staining in HCV-infected than in normal livers. NF-kappaB activation conferred resistance to TNF-alpha-induced apoptosis in HCV core-transfected cells. Inhibition of NF-kappaB activation by pyrrolidine dithiocarbamate sensitized them to TNF-alpha-induced apoptosis. These findings suggest that HCV infection may cause anti-apoptosis by activation of NF-kappaB and implicate a mechanism by which HCV may evade the host's immune surveillance leading to viral persistence and possibly to hepatocarcinogenesis.

Hepatitis C virus variants circumventing cytotoxic T lymphocyte activity as a mechanism of chronicity.

BACKGROUND & AIMS: High rate of chronicity after acute hepatitis C virus (HCV) infection cannot be explained in the presence of a multispecific cytotoxic T lymphocyte (CTL) response. The aim of this study was to investigate the effect of virus variants on CTL activity in patients in whom chronicity developed. METHODS: CTL clones specific to a decapeptide epitope derived from hypervariable region 1 were generated from 5 HLA-A2-positive patients with acute hepatitis C by in vitro stimulation with synthetic peptides. The sequential change of this CTL epitope and its influence on the CTL recognition were examined. RESULTS: Virus variants did not appear in 3 patients with recovery, whereas variants with altered peptide ligands capable of antagonizing CTL activity emerged rapidly in the remaining 2 patients in whom chronicity developed. Importantly, these HLA-A2-restricted, hypervariable region 1-specific CTL clones shared the use of T-cell receptor (TCR) genes AV6 and BV17. CONCLUSIONS: These data suggest that there is only a narrow T-cell repertoire responding to a single viral peptide/HLA ligand. The emergence of HCV variants with altered peptide ligands as TCR antagonists accompanied by a limited TCR repertoire may provide a mechanism for HCV chronicity.

Detection of type 2-like T-helper cells in hepatitis C virus infection: implications for hepatitis C virus chronicity.

One striking clinical feature of hepatitis C virus (HCV) infection is that more than 50% of patients with acute hepatitis C will develop chronic infection. To investigate its possible mechanisms, we examined the activation of type 2-like T-helper (Th2-like) cells relating to the development of chronicity. Peripheral blood CD4+ T-cell proliferation and cytokine secretion in response to a panel of recombinant HCV antigens including core (C22), envelope 1 (E1), E2, nonstructural (NS) protein 4 (C100), fusion protein of NS3 and NS4 (C200), and NS5 were assayed in 17 patients with acute hepatitis C. All six patients with self-limited disease had a significant CD4+ T-cell proliferation to C22, E1, C100, C200, and NS5, running parallel with the antigen-stimulated secretion of interleukin (IL)-2 and interferon gamma (IFN-gamma), but not with interleukin (IL)-4 and IL-10, indicating predominant Th1 responses. Among the remaining 11 patients who developed chronicity, 6, 2, and 9 cases showed a specific CD4+ T-cell response to C22, C100, and C200, respectively, and the responses were significantly lower than those of cases with recovery in terms of stimulation index (SI) (P < .05) and of antigen-stimulated IL-2 and IFN-gamma production. Importantly, IL-4 and IL-10 (Th2 responses) were detectable, and C22-specific Th2-like T-cell clones could be generated from patients with chronicity. The data suggested that activation of Th2 responses in acute hepatitis C patients may play a role in the development of chronicity.

Cellular immune responses in patients with dual infection of hepatitis B and C viruses: dominant role of hepatitis C virus.

Several lines of evidence have suggested that immune mechanisms are involved in the pathogenesis of hepatitis B virus (HBV)- and hepatitis C virus (HCV)-related hepatitis. Study of patients with dual HBV and HCV infection raises the question of which is etiologically more relevant in determining the liver cell damage. To address this issue, proliferation of peripheral blood mononuclear cells (PBMCs) in response to a panel of HBV and HCV antigens was assayed in 13 patients with chronic dual hepatitis B and C, 7 patients with chronic hepatitis B, 7 patients with chronic hepatitis C, and 6 patients with hepatitis B surface antigen (HBsAg) carrier state. Although HBV or HCV hepatitis patients had a significant response to HBV or HCV antigens, respectively, the patients with dual hepatitis B and C exclusively responded to HCV antigens, but not to HBV antigens. One patients who was seropositive for both HBV-DNA and HCV-RNA showed a low response to HBV antigens initially but lost the response 3 months later and became responsive to more HCV antigens. These findings suggest that HCV has a dominant role in the immune response of the patients with dual HBV and HCV infection.

Nutritional, hepatic, and metabolic effects of cachectin/tumor necrosis factor in rats receiving total parenteral nutrition.

BACKGROUND: In orally fed animals, infusion of cachectin/tumor necrosis factor (TNF) caused weight loss and muscular wasting, accompanied by anorexia. Despite muscle wasting, there were gains in weight and protein and DNA contents of the viscera, but no significant metabolic abnormalities. METHODS: To observe the effect of cachectin/TNF on the nutritional-metabolic status, and without the confounding effect of anorexia, cachectin/TNF was infused into rats receiving total parenteral nutrition in sufficient amounts to induce weight gain in controls at the same rate as in orally fed rats. RESULTS: TPN prevented loss of body weight, but cachectin-treated animals had reduced nitrogen retention and carcass weight. By contrast, there were gains in visceral protein levels, which in the liver was due to a marked proliferation of biliary epithelium. In addition, cachectin-treated animals receiving TPN developed hyperglycemia, hyperosmolality, diuresis, and dehydration. They also had azotemia and cholestasis. CONCLUSIONS: In the absence of the effects of anorexia, cachectin reduced nitrogen retention and caused metabolic and multisystem dysfunction, comparable with the effects of clinical sepsis.

Leukocyte CD11b/18 antigen-directed monoclonal antibody improves early survival and decreases hypoxemia in dogs challenged with tumor necrosis factor.

This study examined the effect of monoclonal antibody (MAb) directed against leukocyte CD11b/18 glycoprotein complex (904MAb) on cardiopulmonary injury induced by tumor necrosis factor (TNF), and death. Eighteen 2-yr-old, purpose-bred beagles with chronic tracheostomies were challenged with TNF (60 micrograms/kg of body weight) intravenously. Nine of 18 animals were treated with 0.5 to 1.0 mg/kg of body weight 904MAb intravenously 45 min before and 12, 36, and 48 h after TNF infusion. Serial femoral and pulmonary arterial catheter hemodynamics, blood gas analysis, and radionuclide cineangiographic left ventricular ejection fractions (EF) were done before and after a fluid challenge. Serial bronchoalveolar lavages (BAL) with cell and protein analysis also were performed using the chronic tracheostomies. Compared with animals given TNF alone, animals treated with 904MAb did not differ in overall survival (TNF alone, 2/9; 904MAb, 3/9); however, the group of animals treated with 904MAb had significantly (p less than 0.01) fewer deaths within the first 30 h of TNF challenge. At 4 h after TNF challenge, all animals had significantly (p less than 0.05) reduced PaO2 after fluid challenge; however, animals given 904MAb (compared with animals given TNF alone) had significantly (p less than 0.05) smaller reductions in PaO2. Throughout the study, animals given 904MAb before TNF or TNF alone had similar changes in cardiac index, mean arterial pressure, EF, and BAL protein and neutrophil concentration. Thus, MAb directed against the leukocyte CD11b/18 glycoprotein complex prolonged survival and reduced the hypoxemia occurring after TNF challenge, but this antibody did not improve overall survival or cardiopulmonary function.(ABSTRACT TRUNCATED AT 250 WORDS)

TNF but not IL-1 in dogs causes lethal lung injury and multiple organ dysfunction similar to human sepsis.

We compared the early and late pulmonary effects of human recombinant tumor necrosis factor (TNF) and interleukin 1 (IL-1) challenges in awake dogs with chronic tracheostomies. Serial blood gas analysis, bronchoalveolar lavage (BAL) with cell and protein analysis, intravascular catheter hemodynamics, and radionuclide left ventricular ejection fractions (LVEF) were determined before and after infusion of TNF (60 micrograms/kg body wt, n = 8), IL-1 (1,000 micrograms/kg body wt, n = 6), or heat-inactivated IL-1 (n = 6, controls). Controls given heat-inactivated IL-1 had no changes (P = NS) in any pulmonary parameter throughout the study. Animals given IL-1 had a transient increase (P less than 0.05) in BAL neutrophil concentration 1 day after infusion but no other changes (P = NS) in pulmonary function throughout the study. Animals given TNF had early (0-4 h) decreases (P less than 0.05) in arterial PO2, increases (P less than 0.05) in physiological shunt fraction and alveolar-to-arterial PO2 gradient, and a high mortality rate (50%). In TNF animals, volume challenges at 4 h were associated (P less than 0.05) with death and noncardiogenic pulmonary edema. In TNF survivors, hypoxemia persisted for 2-3 days and was associated with increases (P less than 0.05) in alveolar protein and neutrophil concentration on days 1 and 3, respectively, which in survivors returned to near normal over 6-21 days. Animals challenged with TNF and not IL-1 had reversible depression of LVEF similar in time course to abnormalities in arterial PO2. In this study, TNF (but not IL-1) challenges were lethal and produced acute pulmonary dysfunction sustained over days (reversible in survivors) that was similar to that seen in human septic shock. The ability of TNF to induce pulmonary injury similar to bacterial shock suggests that TNF is a key mediator of sepsis-induced lung injury. Furthermore, because TNF challenge induced both sustained pulmonary and cardiac injury, TNF may be a common pathway for the multiple organ dysfunction that occurs during septic shock.

Body composition and metabolic rate in rat during a continuous infusion of cachectin.

Changes in brain metabolites, energy balance, resting metabolic rate, body composition, and organ histology were studied over 10 days in control (C), cachectin-infused (CIR), and pair-fed (PFR) (in relation to CIR) rats. The cachectin was continuously infused for the 10 days into the superior vena cava at the rate of approximately 100 micrograms.kg-1.day-1. The brain of the CIR had significantly more tryptophan and 5-hydroxyindole-3-acetic acid than C and PFR. The CIR rats were anorexic, hypermetabolic, relatively hyperglycemic, and had raised blood urea nitrogen with comparable creatinine levels when compared with similarly wasted PFR. They had significant loss of muscle mass, especially in muscles with a predominance of type II fibers. However they gained liver, heart, and lung mass. The loss of muscle mass could be ascribed to dietary deficiency, but the gain in visceral mass was associated with an increase in organ DNA and protein. Histology showed that there was cell proliferation in the liver, heart, and kidneys. The data are consistent with centrally mediated anorexia with nutritionally mediated muscle wasting but with visceral hypermetabolism, protein accumulation, and cell proliferation.

Hepatitis C virus infection in an area hyperendemic for hepatitis B and chronic liver disease: the Taiwan experience.

To assess the contribution of hepatitis C virus (HCV) in liver disease in Taiwan, antibody to HCV (anti-HCV) was studied by radioimmunoassay in 392 patients with chronic liver disease and in 440 healthy adults and 444 subjects at risk. The anti-HCV prevalence was 0.95% in 420 volunteer blood donors, 90% in 100 hemophiliacs, and 81% in 58 parenteral drug abusers. Anti-HCV was present in 6 (7.7%) of 78 hepatitis B surface antigen (HBsAg)-positive and 28 (65%) of 43 HBsAg-negative patients with chronic hepatitis, 3 (10%) of 31 HBsAg-positive and 13 (43%) of 30 HBsAg-negative cirrhotics, and 7 (17%) of 42 HBsAg-positive and 15 (63%) of 24 HBsAg-negative patients with hepatocellular carcinoma (HCC). An outbreak of non-A, non-B hepatitis revealed 18% of 57 patients to be positive for anti-HCV, and in 29 patients with posttransfusion hepatitis prospectively followed, 7 (24%) developed anti-HCV. Thus, HCV infection appears to play a relatively minor role in HBsAg-positive liver disease in Taiwan but is strongly associated with HBsAg-negative chronic liver disease and HCC. The infection is extremely common in hemophiliacs and parenteral drug abusers.

Survival of primates in LD100 septic shock following therapy with antibody to tumor necrosis factor (TNF alpha).

The purpose of this study was to determine the efficacy of treatment with anti-TNF monoclonal antibody in preventing the deleterious effects of sepsis in a nonhuman primate. Experiments were carried out on anesthetized baboons intravenously infused with a lethal dose of Escherichia coli (E. coli). Twelve baboons (six control and six experimental) received 2 hr infusions of E. coli. The experimental group was administered a bolus of anti-TNF antibody, 15 mg/kg, 30 min after beginning the E. coli infusion. Control baboons lived an average of 19 hr (12-34 hr). All antibody-treated baboons survived more than 7 days with a significantly improved quality of life compared to the control group. Although some adverse changes occurred during the monitoring period in surviving baboons, they maintained nearly normal arterial pressures, and serum urea nitrogen and creatinine concentrations. The severe histopathologic changes in lungs, liver, adrenals, kidneys, and spleen documented at death in baboons receiving E. coli only were absent after 7 days in baboons given E. coli and early post-treatment with antibody to TNF.

Antibodies to cachectin/tumor necrosis factor reduce interleukin 1 beta and interleukin 6 appearance during lethal bacteremia.

Cytokines secreted in response to invading micro-organisms are important mediators of detrimental hemodynamic and metabolic changes in the host. To test whether cachectin/TNF plays a role in triggering release of other cytokines in the setting of infection, anesthetized baboons were passively immunized against systemic cachectin/TNF before infusion of a LD100 dose of live Escherichia coli. Bacteremia led to significant increases in circulating levels of cachectin/TNF, IL-1 beta, and IL-6. Although bacterial endotoxin/lipopolysaccharide is a potent stimulus for the synthesis and release of IL-1 and IL-6 in vitro, specific neutralization of cachectin/TNF in vivo with mAb pretreatment significantly attenuated both the IL-1 beta and the IL-6 responses despite fulminant overwhelming bacteremia. These data suggest that cachectin/TNF is essential for the initiation or amplification of IL-1 and IL-6 release during lethal gram-negative septic shock syndrome.

Endotoxin and tumor necrosis factor challenges in dogs simulate the cardiovascular profile of human septic shock.

Survivors of both human and animal bacterial shock develop a characteristic pattern of progressive changes in cardiovascular function over a period of 7-10 d. In this present study, we examined whether endotoxin (a product of Gram-negative bacteria) or TNF (a cytokine released from macrophages) could reproduce the same complex cardiovascular changes observed in septic shock over a period of 7-10 d. To test this hypothesis, we implanted a thrombin-fibrin clot containing purified endotoxin from E. coli into the peritoneal cavity of eight dogs, and infused TNF into eight different dogs. Over the next 10 d, serial simultaneous heart scans and thermodilution cardiac outputs were performed in these awake nonsedated animals. By day 2 after challenge with either endotoxin or TNF, animals developed a decrease (p less than 0.05) in both mean arterial pressure and left ventricular ejection fraction. With fluid resuscitation, animals manifested left ventricular dilatation (increased [p less than 0.05] end diastolic volume index), increased or normal cardiac index, and decreased or normal systemic vascular resistance index. In surviving animals, these changes returned to normal with 7-10 d. The time course of these changes was concordant (p less than 0.05) with that previously described in a canine model of septic shock using viable bacteria. During the 10-d study, control animals receiving sterile clots or heat-inactivated TNF had not significant changes in hemodynamics. The results from this canine model demonstrate that either endotoxin or TNF alone can produce many of the same hemodynamic abnormalities seen in human septic shock and in a canine septic shock model induced by live bacteria. These findings support the hypothesis that the action of endogenous mediators (TNF) responding to bacterial products (endotoxin) is the common pathway that produces the serial cardiovascular changes found in septic shock.

Cachectin/tumor necrosis factor induces cachexia, anemia, and inflammation.

Cachexia is a potentially lethal syndrome of unknown etiology characterized by anorexia, weight loss, and protein wasting that frequently complicates the treatment of chronic inflammation and cancer. Cachectin/TNF was isolated during the search for a humoral mediator of cachexia and found to stimulate the breakdown of energy stores from adipocytes and myocytes in vitro, but the chronic effects of the monokine in vivo are not known. Sublethal doses of recombinant human cachectin administered twice daily for 7-10 d caused cachexia in rats, as evidenced by reduced food intake, weight loss, and depletion of whole-body lipid and protein stores. Significant anemia is also observed and found to be the result of decreased red blood cell mass, not expanded plasma volume. Leukocytosis and histopathological evidence of tissue injury and inflammation are observed in several organs, including omentum, liver, spleen, and heart. These data suggests that the exposure of the normal host to cachectin is capable of inducing a pathophysiological syndrome of cachexia, anemia, and inflammation similar to that observed during inflammatory states or malignancy.

Anti-cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteraemia.

Bacterial infection of the mammalian bloodstream can lead to overwhelming sepsis, a potentially fatal syndrome of irreversible cardiovascular collapse (shock) and critical organ failure. Cachectin, also known as tumour necrosis factor, is a macrophage-derived peptide hormone released in response to bacterial lipopolysaccharide, and it has been implicated as a principal mediator of endotoxic shock, although its function in bacterial sepsis is not known. Anaesthetized baboons were passively immunized against endogenous cachectin and subsequently infused with an LD100 dose of live Escherichia coli. Control animals (not immunized against cachectin) developed hypotension followed by lethal renal and pulmonary failure. Neutralizing monoclonal anti-cachectin antibody fragments (F(ab')2) administered to baboons only one hour before bacterial challenge protected against shock, but did not prevent critical organ failure. Complete protection against shock, vital organ dysfunction, persistent stress hormone release and death was conferred by administration of antibodies 2 h before bacterial infusion. These results indicate that cachectin is a mediator of fatal bacteraemic shock, and suggest that antibodies against cachectin offer a potential therapy of life-threatening infection.