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Cellular and humoral immune responses to vaccines of hepatitis B and rabies as antigens were suppressed by specific inhibitors of cathepsin B, anti-cathepsin B antibody and the specific substrate of cathepsin B. The antigenic peptides of these vaccines are processed by cathepsin B and the fragments are capable of binding with the desetope of MHC class II, beta-chain, because one of the active sites of cathepsin B (14, 15) VN217-222 shares high homology with a part of the desetope, VN57-62, of MHC class II, beta-chain. Rechallenge of the synthesized antigenic peptides of these vaccine molecules shows a strong proliferative response to the splenocyte primed by these vaccines. However, the response to these antigenic peptides was not inhibited by cathepsin B inhibitors. These findings suggest that cathepsin B inhibitors do not inhibit any other processes of immune responses than the proteolytic processing of antigens. Some investigators reported recently that the Ii-chain is degraded by purified cathepsin B in vitro (23-25). However, we showed that the suppression of these immune responses by cathepsin B inhibitors is not due to the inhibition of invariant chain degradation. We found that the invariant chain shares about 40% homology with the cystatin family which are the endogenous inhibitors of cysteine proteases (23, 24). Therefore, the Ii-chain is one of the members of the cystatin superfamily and may participate in the regulation of presentation of antigenic peptides and also antigen processing by cathepsin B.

Autoimmunity against biliary epithelial cells is considered to be involved in the pathogenesis of primary biliary cirrhosis (PBC). However, cytotoxic activity of T lymphocytes against biliary epithelial cells has not previously been examined. This study has demonstrated that spleen-derived T lymphocytes were cytotoxic for autologous biliary epithelial cells in all of five patients with PBC, even though it was only detectable at high effector to target ratios. Such cytotoxicity was not found in non-PBC patients. CD8-positive T lymphocytes were shown to be responsible for the cytotoxicity by negative selection, and its inhibition was dependent on the ratio of cold to hot target cells. These observations may support a current hypothesis that the pathogenesis of PBC is partly due to T cell autoimmunity directed against the bile duct epithelium.

Hepatocellular carcinomas 1 cm in diameter with high or low echogenicity can be detected on ultrasonography and confirmed on fine-needle biopsy, but it is still very difficult to detect small hepatocellular carcinomas with isoechogenicity. In this study, we assessed lymphokine-activated killer cell activity and interferon-gamma production prospectively every 1 to 3 mo for 23 +/- 4 mo (mean +/- 1 S.D.) in 227 patients with cirrhosis. Transient depression of lymphokine-activated killer activity was detected in 43 patients (defective lymphokine-activated killer group), and hepatocellular carcinoma was detected in 24 cases before the end of the 18-mo follow-up. Twenty-one (87.5%) of the 24 hepatocellular carcinoma patients were included in the defective lymphokine-activated killer group. Defective lymphokine-activated killer activity was detected more than 6 mo before detection of a space occupying lesion in the liver or elevation of alpha-fetoprotein level above 400 ng/ml. Serum alpha-fetoprotein level was elevated above 400 ng/ml in only five cases in which hepatocellular carcinoma was detected as a space-occupying lesion. Our results indicate that sequential assessment of lymphokine-activated killer activity may be a predictor of hepatocellular carcinoma and that the depression of immune function in cirrhotic patients is a serious risk factor for hepatocellular carcinoma emergence.

Cellular and humoral immune responses to vaccines of hepatitis B type and rabies were inhibited by specific inhibitors of cathepsin B, specific synthetic substrates of cathepsin B and anti-cathepsin B antibody. Therefore the lysosomal cathepsin B of antigen presenting cells plays an essential role in processing of these antigens for presentation to MHC class II. One of the active sites of cathepsin B, VN217-222 shares highly homologous sequences with a part of the desetope, a binding domain of antigenic peptides, VN57-62 of MHC class II, beta-chain. This evidence suggests that the peptides processed by the substrate specificity of cathepsin B exhibit a common affinity to bind with the desetope of MHC class II, beta-chain.

We have previously reported depressed gamma-interferon production and depressed lymphokine-activated killer and natural killer activity in patients with relatively large hepatocellular carcinomas. These parameters were normal in cirrhosis. Some evidence had suggested a gamma-interferon production defect as the main cause of depressed lymphokine-activated killer activity in hepatocellular carcinoma, (i.e., gamma-interferon enhances lymphokine-activated killer and natural killer activity and gamma-interferon antibody inhibits lymphokine-activated killer induction). However, we were unable to clinically define the precise mechanism operating here because gamma-interferon production and lymphokine-activated killer activity were both defective in advanced hepatocellular carcinoma. In recent years, it has become possible to detect even small hepatocellular carcinomas on ultrasonography and to confirm them by fine-needle biopsy. In this study, we assessed those immune parameters in 48 patients with hepatocellular carcinomas less than 2 cm in diameter to confirm depressed immune function and to clarify the mechanism of these defects. Lymphokine-activated killer activity was defective in 31 patients (64.6%), whereas gamma-interferon production was defective in only 1 of these patients (2.1%). This observation argues against the hypothesis that defective gamma-interferon production is the primary defect and provides new insight into the mechanism of progression of defective immune function in hepatocellular carcinoma. Thirty-one of the 48 hepatocellular carcinoma patients were treated surgically, and these immune parameters were followed for 6 mo. The recovery of lymphokine-activated killer activity in all hepatocellular carcinoma patients with low lymphokine-activated killer activity suggests the tumor burden as the cause of defective lymphokine-activated killer activity.

In 19 acute hepatitis patients with severe coagulopathy who were fully alert and oriented without any changes of mood or behavior, the P300 latency and the arterial blood ketone body ratio (KBR) were assessed as predictors of fulminant hepatitis. All 5 patients developing fulminant hepatitis had a corrected P300 latency longer than 345 msec and 4 of them had a KBR below 0.6. There was a significant negative correlation between the KBR and the blood ammonia level and between the KBR and the corrected P300 latency, while there was a positive correlation between the blood ammonia level and the corrected P300 latency. These data suggest that hepatic encephalopathy develops when loss of hepatic detoxifying activity allows toxic substances to reach the brain and induce cerebral edema. Our findings also suggest the clinical value of using the P300 latency combined with the KBR as predictors of fulminant hepatitis.

Varicella-zoster virus (VZV) infections are among the most common viral diseases characterized by recurrent episodes alternating with asymptomatic periods. VZV reactivation is believed to be induced by the impairment of the host's cell-mediated immune system; however, the precise mechanisms involved in the latency period and reactivation of herpes viruses in the infected host are not yet fully elucidated. We assessed the immune functions in noncompromised patients with typical herpes zoster to investigate the immunological status during the process of reactivation of VZV. The results indicated depressed immune functions in the early stage of VZV reactivation with gradual improvement during the recovery phase. These findings are in accord with the clinical course of herpes zoster and suggest a possible therapeutic trial.

By Northern blot analysis of total RNA, extracted from normal human and rat Ito cells, either freshly isolated or in culture, recent studies demonstrated the expression of mRNA for collagen type I, III, IV, fibronectin, laminin, transforming growth factor-beta (TGF-beta), hepatocyte growth factor, nuclear retinoic acid receptor-beta and alpha-smooth m muscle actin. Moreover, Ito cells isolated from experimentally induced fibrotic livers exhibited a marked increase in mRNA for collagen type I, associated with a moderate increase in mRNA of collagen type III and TGF-beta. Although endothelial cells from the same liver also showed moderate increase in mRNA for collagen type I alone, hepatocytes showed no changes. From these results it is concluded that Ito cells are the most important cells in hepatic fibrogenesis.

Using dispersion cultures performed in semi-solid medium we demonstrate here that the interaction of T cell antigen receptor molecules with Ia-peptide complexes on the same cell surface results in T cell activation, without the production of lymphokines. This recognition of antigen at the single-cell level, induced a state of anergy which was not due to a decrease of surface T cell antigen receptor/CD3 complexes. The induction of anergy by peptide could not be prevented by the addition of various co-stimulatory signals, including antibodies to CD28, or CD2, high doses of interleukin-2 or phorbol ester.

Encephalopathy and severe coagulopathy in patients with acute hepatitis (AH) are good markers for the diagnosis of fulminant hepatitis (FH), which occurs in only about 1% of AH patients. However, even if patients show severe coagulopathy, it is quite difficult to predict FH before the onset of encephalopathy. The ratio of acetoacetate/beta-hydroxybutyrate in arterial blood (KBR) has been reported to reflect the cellular energy charge level in hepatocytes. In our previous report, KBR was quite low in FH patients and was an excellent marker for predicting the prognosis. KBR of normal subjects is distributed in a range of 1.0-2.1 (1.54 +/- 0.26, mean +/- SD). In this study, we assessed KBR serially in 15 AH patients with severe coagulopathy (hepaplastin test (HPT) < 40%), including seven patients who developed FH, to see if we could predict FH by using KBR as a marker. Seven patients with KBR < 0.6 of long duration (4 days or more) were complicated with hepatic encephalopathy (HE) and it took 3 or more days of KBR below 0.6 before HE appeared. The other eight patients with KBR < 0.6 of short duration (less than 4 days) were not complicated with HE. These data suggest that AH patients with HPT < 40% and a 3-day duration of KBR < 0.6 are at serious risk of FH.

In various organ-specific autoimmune diseases, aberrant expression of major histocompatibility complex class II antigens on each target epithelial cell has been reported. Some researchers have attempted to link this phenomenon to the antigen-presenting capacity and the induction of autoimmunity, whereas others think it might serve as a peripheral mechanism for the induction and the maintenance of self-tolerance in autoreactive T cells. In this study, we showed that intraperitoneal administration of interleukin-2 (1.2 x 10(6) IU/kg) to 4-wk-old male BALB/c mice for 35 consecutive days induced lymphocyte infiltration around bile ducts in the liver and major histocompatibility complex class II expression on biliary epithelial cells, which was immunoelectron microscopically confined to the luminal cell surface. Immunohistochemically, lymphocytes accumulating around bile ducts were mainly T cells, positive for CD3, L3T4 and H-2 class II molecules, and a few of them were positive for Lyt-2 and negative for immunoglobulin. Half of the infiltrates were positive for asialo GM1, and one-third was positive for interferon-gamma. Interferon-gamma-positive, L3T4-positive cells were detected in mirror sections. However, neither the destruction of biliary epithelial cells nor the presence of granulomas was observed. Autoantibodies were serologically undetectable. The existence of interferon-gamma-positive cells in the lesion and the fact that intravenous administration of anti-interferon-gamma twice a week completely inhibited the lymphocyte infiltration and the major histocompatibility complex class II expression on biliary epithelial cells suggested that these changes were induced through endogenous interferon-gamma production.(ABSTRACT TRUNCATED AT 250 WORDS)

The source of activation of T lymphocytes in primary biliary cirrhosis (PBC) is undefined. Hence, T-cell-mediated reactivity against a biliary tract antigenic protein from human bile was studied. The bile protein was fractionated by 30-50% saturated ammonium sulphate and gel-chromatography, and analysed by SDS-PAGE and Western immunoblotting using rabbit antisera to the bile protein. The antisera reacted specifically with human bile duct epithelium. Western blotting of bile proteins showed two major bands, the B1 and B2 antigens. B1 stained for sialoglycoprotein but not lipid, but B2 was negative for both. Cell-mediated reactivity was tested by proliferation of peripheral lymphocytes against B1. Taking the upper limit of the normal range for stimulation indices (S.I.) as less than 1.89 (= mean + 2 SD), a mitogenic response was detected in 14 of 16 patients with PBC (S.I.: 11.7 to 2.3), and in 4 of 15 patients with chronic active hepatitis, but in none of 12 patients with drug-induced intrahepatic cholestasis or obstructive jaundice. The B2 protein was non-stimulatory. Lymphocyte proliferation to B1 in PBC was confined to T cell fractions of peripheral blood leucocytes. There was no cross-antigenicity between B1 and the M2 antigens, according to Western blotting using the rabbit antisera and PBC sera with anti-M2 reactivity. Thus, the B1 biliary protein is a possible source of T cell activation in PBC and hence could be an immunological co-factor in the pathogenesis of this disease.

An 18-year-old male was admitted with headache, nausea, and vomiting. Computed tomography (CT) revealed an enhanced tumor of the pineal region and hydrocephalus. The tumor was partially resected via a parieto-occipital craniectomy. The histological diagnosis was germinoma. No serum tumor markers such as alpha-fetoprotein (AFP) and human chorionic gonadotropin (HCG) were detectable. A ventriculo-peritoneal (V-P) shunt was emplaced and radiation therapy (whole brain 59 Gy) given. The tumor and the hydrocephalus regressed completely and he returned to work. Six years later, he experienced constipation and general fatigue. CT and echotomography of the abdomen showed a large peritoneal tumor and ascites. Laboratory investigation demonstrated serum levels of AFP 7640 ng/ml and HCG 150 IU/l, and high ascitic levels of AFP 12,890 ng/ml and HCG 1030 IU/l. AFP and HCG levels regressed after combined chemotherapy. However, he died due to leukopenia and pneumonia. Autopsy found no metastasis of tumor cells to the central nervous system. The peritoneal cavity contained hemorrhagic fluid and a large tumor 4100 g in weight. The tip of the V-P shunt tube was in front of the tumor. No neoplasm was found in the testis, retroperitoneal cavity, thymus, and other organs. The microscopic appearance of the peritoneal tumor was different to the first pineal tumor. The neoplasm was confirmed as a mixed germ cell tumor with teratoma components and suspected to be a metastasis of the pineal tumor through the V-P shunt system.

Arterial ketone body ratio (AKBR [acetoacetate/beta-hydroxybutyrate]) was measured in nineteen patients under medical supportive therapy for fulminant hepatic failure (FHF), in order to evaluate its predictive value relative to liver transplantation. Of the 19 patients 8 (42%) were salvaged and 11 (58%) died. Seven of 8 survivors showed an increased AKBR over 0.6 at 24-hr after admission, and all of them showed AKBR over 0.8 at 48-hr with subsequent maintenance of the value over 1.0. By contrast, all 11 nonsurvivors demonstrated sustained suppression of AKBR below 0.4 from 24 to 72 hr after admission. AKBR values at 24 and 48 hr showed statistically significant differences between survivors and nonsurvivors. Neither the grade of portal systemic encephalopathy (PSE) nor other conventional laboratory parameters--such as AST, bilirubin, ammonia, prothrombin time, hepaplastin test, fibrinogen, and platelet count--could discriminate between survivors and nonsurvivors by univariate analysis. These results indicate that AKBR can accurately predict the prognosis of FHF at the initial 24-48 hr after admission, and that it can play an important role in setting the indication of FHF for liver transplantation.

Pit cells, which are recovered in low density fraction of lymphocytes derived from the liver (L-Fr.1,2), are recognized as perisinusoidal cells. In the present study, the accumulation of liver-derived low (L-Fr.1,2) and high (L-Fr.3,4) density 51Cr-labeled lymphocytes was assessed in the liver, spleen and peripheral blood of syngeneic BALB/c mice in comparison with those derived from the spleen (S-Fr.1,2 and S-Fr.3,4) and peripheral blood (P-Fr.1,2 and P-Fr.3,4). L-Fr.1,2, L-Fr.3,4 and P-Fr.1,2 accumulated selectively in the liver, whereas P-Fr.3,4 did not accumulate in the liver. L-Fr.3,4 was composed of two major subpopulations; Thy1+CD4+ and Thy1+CD8+. This suggested that L-Fr.3,4 were distinctive from P-Fr.3,4 or L-Fr.1,2 and inhabited the liver. P-Fr.1,2 and S-Fr.1,2 tended to accumulate in the liver and 51Cr labeled P-Fr.1,2 and S-Fr.1,2 were recovered mainly in L-Fr.1,2 L-Fr.3,4, S-Fr.1,2 and P-Fr.3,4 48 h after the transfer of these cells, suggesting that a part of P-Fr.1,2 and S-Fr.1,2 might be in dynamic equilibrium with those of L-Fr.1,2 and could be a part of the source of L-Fr.3,4. Results also suggested that some of L-Fr.1,2 of T cell lineage might change into L-Fr.3,4 in the liver and might be released into the systemic circulation. Thereafter, L-Fr.3,4 might behave as P-Fr.3,4 and no longer show organ-specific accumulation.

Hepatocellular carcinoma (HCC) patients can be divided into two groups according to the degree of lymphokine activated killer (LAK) cell activity; a high LAK activity group (H-LAK-HCC) and a low LAK activity group (L-LAK-HCC). Interferon-gamma (IFN-gamma) production is severely defective in L-LAK-HCC but not defective in H-LAH-HCC. IFN-gamma production is suppressed with the addition of anti-Tac in dose dependent manner, though LAK activity is suppressed only in the presence of high concentration of anti-Tac. LAK activity is suppressed with the addition of anti-IFN-gamma, which is most prominent when the antibody is present during the first 12 hr of incubation. LAK generation is enhanced with the addition of recombinant IFN-gamma, which is most prominent when it is present during the first 12 hr of incubation. However, this enhancing effect is less prominent in L-LAK-HCC as compared to normals, liver cirrhosis, and H-LAK-HCC. This enhancement is regarded to depend on the presence of Leu7+ and Leu11+ subset, as this enhancement is abandoned and IFN-gamma production is inhibited when either of these subsets is deleted. These data suggest that IFN-gamma production and the participation of Leu7+ and Leu11+ subsets is important in sufficient LAK generation, and that poor IFN-gamma production and insufficient response to IFN-gamma may be the cause of severely defective LAK generation in L-LAK-HCC.

We assessed the bilirubin reduction capacity of three different types of devices in vitro: a high-permeable membrane column for double-filtration plasmapheresis (DFP) (Evaflux 2A, Kuraray, Japan), and non-coated charcoal column for hemoperfusion (HP) (N-180, Asahi Medical, Japan), and ion-exchange columns for plasma adsorption (PA) (BR-350, Asahi Medical, Japan, and B-001, Kuraray, Japan). A column for DFP reduced the concentration of low-molecular proteins effectively such as plasma bilirubin and bile acids in an albumin-dependent manner. A charcoal column adsorbed low-molecular substances preferentially. But in these two columns, the loss of fibrinogen is a limiting factor for determining the processing plasma volume. Ion-exchange columns for PA adsorbed bile acids, disconjugated bilirubin, and monoconjugated bilirubin more efficiently compared with delta-bilirubin and unconjugated bilirubin. Pretreatment of the column with heparin reduced the loss of fibrinogen to less than 10%. We applied the BR-350 ion-exchange column in vivo for treatment of three patients with hyperbilirubinemia. After treatment, an alcoholic hepatitis patient with the hepatorenal syndrome (HRS) recovered from acute renal failure. However, in a patient with primary biliary cirrhosis and in a patient with fulminant hepatitis, the decrease of serum bilirubin was transient and no obvious beneficial responses were noted. The capacity and ability of the BR-350 column to adsorb plasma bilirubin was shown sufficient to treat deeply jaundiced patients, because 4 liters of the plasma of a patient with 108 mg/dl of initial total bilirubin concentration was able to be processed continuously without an obvious decrease in bilirubin adsorption capacity.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum inhibition of complement-derived leukocyte chemotaxis was examined in alcoholic liver disease with or without cirrhosis. Chemotactic inhibitory activity (CIA) was detected with higher frequency and degree in alcoholic liver disease, especially with liver cirrhosis compared with normal subjects and non-alcoholic liver cirrhosis. CIA was found in anti-IgA adsorbed fractions in the sera of patients with alcoholic liver cirrhosis. Serum concentrations of IgA1 and IgA2 in alcoholic liver disease were statistically higher than in non-alcoholic liver cirrhosis. However, no correlation between CIA and the concentrations of IgA subclasses was demonstrated in alcoholic liver disease. This serum inhibitor may partly explain the high susceptibility to bacterial infection in alcoholic liver disease.

Ten patients with hepatocellular carcinoma, three of whom had pulmonary metastasis, were treated with adoptive immunotherapy using autologous lymphokine-activated killer cells plus recombinant interleukin 2. Patients received 15 micrograms per day of recombinant interleukin 2 consecutively (for 14 to 64 days), from Day 7 prior to the first leukapheresis, and received 10(9) to 10(10) lymphokine-activated killer cells once or twice per week intravenously; the lymphokine-activated killer cells had been generated from mononuclear cells obtained through leukapheresis. Preadministration of recombinant interleukin 2 prior to the first leukapheresis resulted in a remarkable increase of lymphokine-activated killer activity in seven of nine cases in whom lymphokine-activated killer activity had been poorly inducible even at high concentrations of recombinant interleukin 2. At the end of the treatment, liver tumor regression (34 and 63%, respectively, of two-dimensional size) was observed in two of two patients with a solitary tumor; no increase of liver tumor size was observed in seven patients with massive or multiple tumors, and no changes in the size or number of pulmonary metastatic tumors in any patients were observed. More than a 35% decrease in serum alpha-fetoprotein level was noted in four of nine alpha-fetoprotein-positive patients. However, Child's grades, performance status and lymphokine-activated killer activity on entry into the study could not be used as parameters to predict therapy responsiveness. Neither serious side effects nor significant changes of serum bilirubin, ALT and creatinine were noted. Thus, this treatment seems to be well tolerated even in advanced hepatocellular carcinoma with poor liver function reserve, and tumor regression could be expected in small-burden hepatocellular carcinoma. The assessment of the therapeutic effects and application in hepatocellular carcinoma awaits the development of this trial.

Cytotoxic T lymphocyte (CTL) activity against HBs antigen (HBsAg)-coated autologous mononuclear cells as target cells was examined in hepatitis B virus infection. Target cells were prepared by coincubation of mononuclear cells with purified HBsAg in 0.5 mM CrCl3. The distribution and uniformity amounts of HBsAg on target cells was shown by immune fluorescence and by analyzing the fluorescence intensity with a fluorescent activated cell sorter. CTL activity was detected in 7 of 9 patients with acute hepatitis type B, in 4 of 11 chronic active hepatitis type B, in none of 8 healthy HBsAg carriers, and in none of 22 healthy volunteers. The antigen specificity of the cytotoxicity was confirmed by a blocking experiment with purified HBsAg and by cold target inhibition with HBsAg or bovine serum albumin (irrelevant antigen) coupled cold target cells. CTL lysed HBsAg coupled allogeneic target cells that shared HLA-A or B locus antigens. This finding suggests that HLA restriction may be involved in the killing mechanism. This HBsAg specific CTL clone may participate in the immunopathogenesis of hepatitis B virus infection.

Lymphokine-activated killer activity and natural killer activity in hepatocellular carcinoma patients were assessed. Maximum lymphokine-activated killer activity was induced at 3 to 5 days of incubation, and lymphokine-activated killer activity tended to increase in a manner dose dependent of recombinant interleukin-2. However, the maximum increase of lymphokine-activated killer activity in hepatocellular carcinoma was not as high as that of normal subjects or liver cirrhosis patients. Lymphokine-activated killer activity was impaired in hepatocellular carcinoma as compared to that in normal subjects. Hepatocellular carcinoma seemed to consist of two groups: i.e. a high-lymphokine-activated killer activity group and a low-lymphokine-activated killer activity group. Reduction of natural killer activity was also observed in hepatocellular carcinoma as compared with that in normal subjects and patients with liver cirrhosis. No correlation could be demonstrated between natural killer activity and lymphokine-activated killer activity in normal subjects, liver cirrhosis patients and hepatocellular carcinoma patients. With regard to the presence of HBsAg or alpha-fetoprotein concentration in the sera, there was no significant difference in natural killer and lymphokine-activated killer activity in hepatocellular carcinoma patients. Patients with a small mass lesion showed a low lymphokine-activated killer activity, and depressed lymphokine-activated killer activity was not necessarily related to tumor size. In comparison with the high-lymphokine-activated killer group, the low-lymphokine-activated killer group showed a significant decrease in gamma-interferon production and a preserved function of indocyanine green clearance.