Magnitude of activity in chronic hepatitis C is influenced by apoptosis of T cells responsible for hepatitis C virus.

BACKGROUND: The mechanisms of hepatitis C virus (HCV) persistence are unknown, but down-regulation of immune response in a host is likely to play a major role in it. METHODS: To investigate whether T cell apoptosis contributes to such down-regulation, we compared peripheral T cell apoptosis in patients with chronic hepatitis C (CHC) with the serum titre of HCV-RNA, serum alanine aminotransferase (sALT) levels and its change, or peripheral T cell proliferation to the recombinant core antigen of HCV, JCC-1. RESULTS: The percentage of apoptosis in T cells was 0.30 +/- 0.31% (mean +/- SD) in 44 patients with CHC and 0.10 +/- 0.05% in 10 normal volunteers (P < 0.05). In patients with CHC there was no statistical correlation between apoptosis in T cells and sALT levels, titre of HCV-RNA or T cell proliferation to JCC-1 antigen. But, in patients showing relatively more apoptosis in T cells (more than mean + 2SD of apoptosis in T cells from normal volunteers), sALT levels decreased. CONCLUSIONS: Thus, T cell apoptosis in patients with CHC is considered to cause a reduction in sALT, contributing to HCV persistence in patients with CHC.

Unresponsiveness of peripheral T cells induced by apoptotic bodies derived from autologous T cells.

Several reports described the dose-dependent effect of Staphylococcus aureus enterotoxin B (SEB) regarding both levels of apoptosis and anergy of T cells. We investigated here whether T-cell apoptosis induced with SEB causes unresponsiveness of naive T cells. Apoptotic bodies were isolated from human T cells stimulated with antigen-presenting cells (APCs) and SEB by the continuous density gradient centrifugation method. When naive T cells were stimulated with APCs and SEB in the presence of apoptotic bodies, their proliferation was dose dependently suppressed and their TCRs were less downregulated than those of T cells stimulated without apoptotic bodies. Furthermore, those T cells were predisposed not to respond to restimulation with fresh APCs and SEB in the absence of apoptotic bodies. These results, taken together with the observation of tight binding of apoptotic bodies to APCs, imply that T cells stimulated in the presence of apoptotic bodies may undergo unresponsiveness due to interruption of contact with APCs.

An analysis of T cell antigen receptor variable beta genes during the clinical course of patients with chronic hepatitis B.

BACKGROUND: The degree of hepatocyte injury in patients with chronic hepatitis B appears consistent with the number of T cells that respond to hepatitis B virus-related antigens. METHODS: By using a polymerase chain reaction (PCR)-based approach, we monitored a ratio of the T cell antigen receptor (TcR) variable (V) beta gene families against a total TcR V beta gene expression in the peripheral T cells obtained from five patients and four healthy controls. RESULTS: In the healthy controls, there was no significant change in the ratios at an interval of four or eight weeks. In contrast, several TcR V beta families showed the significant changes in the ratios of their gene expression during the follow-up period in all patients. No common highly fluctuated TcR V beta, however, was found among the patients. Furthermore, there was no correlation between their changes and serum levels of alanine aminotransferase. CONCLUSIONS: These findings suggest that the skewing of the TcR family with multiclone is the result of T-cell responses to viral antigens in peripheral blood.

Analysis of human T-cell antigen receptor variable beta gene usage following vaccination with recombinant HBsAg.

We analyzed the TcR Vbeta gene usage before and after vaccination with the hepatitis B vaccine since changes in the TcR Vbeta gene families would be considered to provide preliminary evidence of a mechanism to prevent HBV infection. Six healthy adult volunteers received immunizations. TcR Vbeta usage, T-cell proliferation, and HLA class II alleles were examined in peripheral blood mononuclear cells (PBMC) both before and after vaccination. Furthermore, TcR Vbeta usage in postimmunization PBMC was also compared with PBMC cultured with recombinant HBsAg (rHBsAg). The level of in vitro T-cell proliferation in the presence of rHBsAg increased significantly (P < 0.01) in PBMC isolated after vaccinations. Increases in the different TcR Vbeta genes were also observed in each individual following vaccinations, regardless of the similarity in their HLA alleles. Specific HBV-related antigen-responsive T cells were induced after HB vaccination, without any common restriction for the TcR Vbeta gene families. The mechanism that helps prevent HBV infection was thus found to involve multiclonal alterations in the TcR Vbeta repertoire.

Colonic cancer in a patient with primary sclerosing cholangitis and long-standing ulcerative colitis.

A 27-year-old woman with a 9-year history of ulcerative colitis involving the entire colon was admitted to our hospital in August 1992 because of bloody stools and left lower abdominal pain. She had been treated with sulfasalazine since 1983 and the colitis had been clinically quiescent or mild for 7 years. She had also been diagnosed as having primary sclerosing cholangitis (PSC) 4 years prior to this admission, based on the clinical, laboratory, and cholangiographic findings. A barium enema and colonoscopy showed an irregular mass obstructing the bowel lumen in the distal portion of the descending colon. Biopsy specimens taken from the mass revealed moderately differentiated adenocarcinoma, and a subtotal colectomy was performed. Histologic examination of the mass lesion showed moderately differentiated adenocarcinoma invading the pericolic adipose tissue. She is currently alive 3 years after surgery. PSC has recently been reported as a risk factor for colonic neoplasia in patients with long-standing ulcerative colitis. In Japan, however, colorectal cancer associated with PSC and ulcerative colitis has rarely been reported. The present case suggests that the risk of colonic cancer is higher in patients with ulcerative colitis and PSC than in patients with ulcerative colitis alone.

Apoptosis observed in peripheral T lymphocytes from patients with chronic hepatitis B.

BACKGROUND & AIMS: Activation-induced cell death is involved in regulating peripheral T-cell function. Understanding the kinetics of these T cells is important to elucidate the pathogenesis of chronic hepatitis B, which is mediated by cellular immune mechanisms. METHODS: Subtle apoptotic cells in CD3+ cells were discriminated by flow-cytometric assay using freshly obtained and in vitro recombinant hepatitis B core antigen-stimulated peripheral lymphocytes from patients with chronic hepatitis. RESULTS: The ratio of apoptotic cells in freshly obtained CD3+ cells was significantly higher during the decreasing phase than increasing phase of serum alanine aminotransferase activity in each patient, and apoptosis of CD3+ cells was induced by stimulation with recombinant hepatitis B core antigen. CONCLUSIONS: Activation-induced cell death in peripheral T cells was found in chronic hepatitis B virus infection, similar to some other viral infections. The apoptosis in T cells during the decreasing phase of serum alanine aminotransferase activity results in a vast amount of T-cell deletion that may weaken T-cell function of cytotoxicity over hepatitis B virus-infected hepatocytes. Thus, activation-induced cell death is considered an important modulator in down-regulating the "burst" of responding T cells in patients with chronic hepatitis B.

T cell anergy is programmed early after exposure to bacterial superantigen in vivo.

We have investigated the effects of the protein synthesis inhibitor, cycloheximide (CHX), on the induction of post-thymic T cell tolerance in mice primed with the bacterial superantigen, Staphylococcus aureus enterotoxin B (SEB). A single injection of 1 mg CHX prevented protein synthesis in splenic cells for < 6 h in vivo. The concomitant administration of SEB and CHX prevented induction of SEB-specific anergy, but did not interfere with the deletion of SEB-specific V beta 8+ T cells by activation-induced, programmed cell death. When CHX was given > or = 24 h after SEB administration the expression of anergy was not affected. These findings suggest that anergy and deletion represent independent processes. Furthermore, these observations, together with the fact that SEB retains the potential to induce anergy in specific T cells 8 h after priming in vivo, imply that the determination of alternate fates (anergy or death) occurs at early time points after SEB injection.

Not every superantigen induces tolerance in vivo.

We have distinguished TSST-1 from SEA and SEB with respect to its in vivo effect on T cells, that is, SEA and SEB induce tolerance in treated mice whereas injection of TSST-1 does not result in tolerance. Therefore, previous observations which relate superantigens to the suppression of reactive V beta TCR T cells seem difficult to generalize to all bacterial superantigens. Since the effects of superantigens are beginning to be exploited for immunotherapy, the differences between TSST-1 and SEB in terms of induction of tolerance suggest that all superantigens may not generally be useful in this respect. Each superantigen obviously should be studied in vivo in respect to T cell tolerance induction.

Effects of indomethacin on hepatogenic pulmonary angiodysplasia.

A patient had liver cirrhosis associated with marked hypoxemia. With administration of indomethacin (75 mg/day for six days), PaO2 was elevated up to 50 mm Hg from 44 mm Hg. At that time, dynamic pulmonary perfusion imaging revealed a plateau time course curve of MAA uptake in the lungs, as compared with findings obtained during the state of severe hypoxemia without indomethacin. These observations suggest that part of hepatogenic pulmonary angiodysplasia is a functional vasodilatation that is presumably modulated by vasoactive substances, such as prostaglandins and/or other eicosanoids.

A comparative study of the Ca2+-Mg2+ dependent ATPase from skeletal muscles of young, adult and old rats.

Sarcoplasmic reticulum (SR) vesicles isolated from skeletal muscle of Sprague-Dawley rats ranging in age from 4 months to 28 months were studied and compared. A marked decline, with age, was observed in the amount of (total) SR proteins isolated per gram of muscle tissue used. This decline is in line with the known loss of muscle fiber mass and size with advancing age; however, whether the magnitudes of these two effects are indeed identical, remains to be studied. In contrast, no analogous age-related change was detected in the amount of SR protein per unit mass of rat cardiac muscle. The calcium contents, per mg protein, in SR vesicles isolated from rats of all age groups studied did not differ significantly, and represented only a small fraction of the total capacity of the vesicles for this cation. This capacity was found to decline at old age and this effect, combined with the age-related decrease in the concentration of SR proteins in the tissue, indicate a significant decline in calcium sequestration ability in old muscle. Both basal (Ca2+ independent) and calcium stimulated ATPase activities were found not to be affected by age. In contrast, the efficiency of Ca2+ transport across the SR membrane, as reflected by the number of calcium ions pumped into the vesicles per ATP cleaved, declined from a value of 0.37 at 3-4 months to 0.15 at 24 months. This change may represent an age-related reduction in the fraction of coupled SR vesicles, possibly due to alterations in the membrane. SR vesicle preparations from both young and old rats displayed strongly biphasic inactivation kinetics when incubated at 37 degrees C. This may reflect the heterogeneity of muscles in the tissue used, or be due to the presence of a mixture of coupled and uncoupled vesicles in the SR preparations. The rate of the first step in the ATPase inactivation, in which about 75% of the activity is lost, was found to be affected by age, the old SR vesicles being markedly more labile than their young counterparts. In contrast, no difference was detected between the inactivation kinetics of young and old ATPase proteins dissolved in Triton X-100 and the inactivation was monophasic down to less than 6% of the original activity. These results indicate that the age-related modifications in the stability of the SR calcium pump system involve the membrane but not the ATPase protein. The inactivation of the SR ATPase is believed to proceed via dissociation of the dimeric enzyme to (unstable) subunits. It is therefore likely that changes in the SR membrane in old muscle render the ATPase more dissociable.

Detection of pulmonary telangiectasia using dynamic pulmonary perfusion imaging in patients with liver cirrhosis.

Two cases of liver cirrhosis associated with marked hypoxemia are presented. Chest radiographs and cardiopulmonary function showed no abnormalities, except for the low diffusion capacity of carbon monoxide and slight elevation of the shunt ratio (20 and 6.2%, respectively), as estimated under conditions of 100% oxygen inhalation. Pulmonary perfusion imaging with Tc-99m macroaggregated albumin (MAA) revealed a significant radioisotope uptake in the lungs, brain, spleen, and both kidneys. Shunt ratios, estimated by the quantitative radionuclide method, were 60 and 68%, respectively. Dynamic pulmonary perfusion imaging revealed a gradual reduction in uptake in all areas of both lungs. The discrepancy of the shunt ratio between the two methods results from an abnormal dilatation of alveolar capillaries. The gradual reduction of radioactivity in areas of the lungs is caused by the passage of MAA particles through widened pulmonary capillaries.

Immunological analysis and characterization of lymphocyte subsets in specimens of human hepatocellular carcinomas and metastatic liver cancers.

The distribution and number of CD2 (Coulter T11)+ cells, CD16 (Leu 11b)+ cells, Leu 7+ cells, CD8 (OKT 8)+ cells, CD11 (Leu 15)+ cells, CD4 (Leu 3a + 3b)+ cells and Leu 10+ or Leu 14+ cells in the liver of patients with hepatocellular carcinoma (HCC) and metastatic liver cancer (MLC) were investigated using monoclonal antibodies and immunohistological methods. In the majority of those with HCC and MLC, CD8 (OKT 8)+, Leu 7+ and CD16 (Leu 11b)+ cells were present both in the tumor and non-tumor tissues. The CD8 (OKT 8)+ cells were more numerous than Leu 7+ and CD16 (Leu 11b)+ cells. No significant difference was observed in the distribution and number of Leu 7+ and CD16 (Leu 11b)+ cells, in any area, in both groups. The number of CD8 (OKT 8)+ cells predominated in the non-tumor area, in both groups. CD11 (Leu 15)+ cells and CD8 (OKT 8)+ cells were present in the ratio of 1:3 or 1:4. The number of CD4 (Leu 3a + 3b)+ cells was less than that of CD8 (OKT 8)+ cells in both groups, especially in the tumor area. A few Leu 10+ or Leu 14+ cells were present in all areas, in both groups. In most cases of MLC, the CD8 (OKT 8)+ cells were absent in the tumor area. There was no correlation between the distribution and number of these cells and anti-tumor chemotherapy or non-specific immunotherapy.

Reversal of age-related effects in rat muscle phosphoglycerate kinase.

Rat muscle phosphoglycerate kinase is one of several enzymes in which age-related effects have been identified. Thus, samples of this enzyme isolated from old rats display a greatly increased heat stability as compared with enzyme isolated from young animals. Previous studies detected no differences in the sequence of amino acids or in the net charge between the young and old forms of the enzyme and it was concluded that the age-related structural modifications are purely conformational. The present study was conducted with the aim of critically testing this hypothesis. To this end, samples of phosphoglycerate kinase purified from skeletal muscle of young and old rats were unfolded by an 18-hr incubation in a 2 M guanidine hydrochloride solution at 4 degrees C, a treatment that results in extensive loss of the three-dimensional structure of the enzyme. A complete reactivation of both enzymes was achieved by dilution of the unfolded enzyme solutions into a large excess of denaturant-free buffer followed by 4 hr of incubation at 25 degrees C. The reactivation kinetics of the unfolded young and old enzymes were practically identical and the refolded products, compared using heat-inactivation kinetics as a sensitive probe, were found to be identical. Moreover, their heat inactivation coincided with that of young untreated phosphoglycerate kinase. These results demonstrate the reversibility of age-related effects at the molecular level and provide strong support for the hypothesis that the modifications in phosphoglycerate kinase in old muscle are purely conformational and, hence, clearly postsynthetic.

Immunohistochemical characterization of inflammatory infiltrates at the site of bile duct injury in primary biliary cirrhosis.

Surgically obtained liver specimens from four patients with primary biliary cirrhosis (PBC), Stages I-II, were studied immunohistochemically using a broad panel of monoclonal antibodies. At the site of bile-duct injury (CNSDC), the inflammatory cells were recognized to be Coulter T-11+ (directed against all T-cells) and OKT-8+ (directed against cytotoxic/suppressor: C/S T-cells) cells. The MHC-class I antigen (i.e. HLA-A, B, C) was expressed weakly in the cytoplasm of the minority of damaged bile-duct epithelial cells, and the MHC-class II antigen (i.e. HLA-DR) was not expressed. Thus, OKT-8+ cells may play an important role in the immunologically mediated destruction of ductular epithelium in PBC. Strong MHC-antigen expression and OKT-8+ cell infiltration in destructive bile-duct lesions were not simultaneously observed. In the portal lymphocyte-rich areas, OKT-4+ (directed against helper/inducer: H/I T-cells) predominated over OKT-8+ cells. B-lymphocytes were present predominantly in the peripheral zones of the lymphoid aggregates. The distribution of helper/inducer T-cells and B-lymphocytes indicates that they may play a role in the induction of immunoglobulin in the lymphocyte-rich areas. The inflammatory mononuclear cells within the granuloma observed in one patient were Coulter T-11+, OKT-8+ and Leu-3a+3b+ (directed against helper/inducer T-cells) cells.

Steroidogenic and morphological characteristics of granulosa and thecal compartments of the differentiating rabbit corpus luteum in culture.

On the day after ovulation, the thecal tissue and associated mural granulosa lutein cells of the rabbit corpus luteum were separated from the granulosa lutein 'core' by dissection and these tissues were cultured separately or together (whole corpus luteum) in defined medium for 10 days on stainless-steel grids. The medium was changed completely every 24 h. Replicate tissues were cultured with testosterone (10 ng/ml), but no other hormones were added to the medium. Progesterone production increased during the first 2 days of culture for whole corpus luteum, granulosa lutein cells and the thecal compartment which also included granulosa lutein cells. After 3 days, the production of progesterone declined gradually, but was still detectable on Day 10. The production of the metabolite, 20 alpha-dihydroprogesterone, by whole corpus luteum was equal to or greater than that of progesterone. Without the addition of testosterone, the granulosa lutein cells produced little (10 pg/culture) oestradiol during 1 day of culture, but the thecal compartment and whole corpus luteum each produced about 100 pg/culture on Day 1 and declining quantities over the next 2 days. In the presence of testosterone added to the medium, the formation of oestradiol was greatly increased for all tissues for 5-6 days of culture, after which time oestradiol was no longer detectable with or without testosterone in medium. Transmission electron microscopy of cells after 10-12 days of culture revealed fine structure that is characteristic of luteal cells, including abundant smooth endoplasmic reticulum, lipid droplets, and junctions between the luteal cells. The corpus luteum in culture resembles the corpus luteum in situ in that steroidogenesis and differentiation can proceed for a period after ovulation without extrinsic hormonal stimulation.