Absence of anti-LKM-1 antibody in hepatitis C viral infection in the United States of America.

Several studies from Europe have observed a relationship between hepatitis C virus infection and anti-liver/kidney microsome-1 (anti-LKM-1) positive chronic hepatitis. It has been suggested that hepatitis C may induce an autoimmune phenomenon that leads to the development of a specific type (type II anti-LKM-1 positive) autoimmune chronic hepatitis. We evaluated 204 sera from patients with well-documented hepatitis C infection from two centres in the United States of America and compared them with sera from 428 French patients from three centres. We evaluated the serological prevalence of anti-smooth muscle antibodies, anti-nuclear antibodies, anti-liver cytosol antibodies, and anti-mitochondrial antibodies subtype anti-M2 in patients with chronic hepatitis C. The two groups were matched in their ages, gender, mode of transmission of hepatitis C infection and severity of liver disease. Anti-LKM-1 was not observed in the patients from the USA at a time when it was noted in 3.7% of French patients. There were no differences, however, in the expression of other auto-antibodies, which were often in low titres. Absence of anti-LKM-1 in USA sera in comparison with French sera suggests that there may be differences in induction of anti-LKM-1 related to environmental and/or host genetic factors, and/or genomic variation in the hepatitis C virus.

Suppressor T-cell deficiency in primary sclerosing cholangitis. Case and family study.

Primary sclerosing cholangitis is considered to be an autoimmune disease of the liver in which there is an association with the HLA phenotypes B8 and DR3 and in which circulating autoantibodies occur. Abnormalities of immune regulation may be present but whether or not they are primary or acquired is not known. This report is of a patient with primary sclerosing cholangitis who was homozygous for HLA B8 DR3, had a circulating antinuclear antibody, and a defect in nonspecific suppressor T-cell activity despite glucocorticosteroid treatment. Nevertheless, family studies revealed no evidence of an immunoregulatory defect in first-degree relatives despite the presence of Raynaud's phenomenon and malignancy in two sisters.

Analysis of T cell receptor beta and gamma genes from peripheral blood, regional lymph node and tumor-infiltrating lymphocyte clones from melanoma patients.

A total of 199 T cell clones from two melanoma patients were derived from progenitor T cells from recurrent melanoma, regional lymph nodes (either involved or uninvolved with malignancy) and peripheral blood by inoculating single cells directly into the wells of microtiter plates before in vitro expansion. The surface marker phenotype of most clones was CD4+CD8-, although some were CD4-CD8+. Genomic DNA prepared from all clones was analyzed by Southern blot hybridization using T cell receptor (TCR) beta and gamma gene probes, seeking clones with identical TCR gene rearrangement patterns as direct evidence for in vivo progenitor T cell clonal amplification. Probing HindIII-digested DNA with TCR beta and TCR gamma probes revealed several clones with identical TCR gene rearrangement patterns. These clones had subsequent probing of BamHI-digested DNA with TCR beta and TCR gamma probes, which showed all but 2 clones to have distinct rearrangement patterns. These analyses provide clear molecular evidence for in vivo polyclonal CD4+ T cell populations in each of several separate immune compartments in these patients.

An immunogenetic study of suppressor cell activity in autoimmune chronic active hepatitis.

Some patients with autoimmune chronic active hepatitis as well as their disease-free first degree relatives show decreased suppressor cell activity of peripheral blood T lymphocytes. Studies were therefore undertaken in families ascertained by the presence of a single chronic active hepatitis patient to determine if this abnormality of immune regulation represents a genetic phenotype simply controlled by a gene or genes at a putative disease susceptibility locus and, further, if this locus showed linkage to either the HLA or the immunoglobulin constant region loci. In addition to determining circulating autoantibody status and genotyping for HLA and immunoglobulin allotypes, suppressor T cells were evaluated by surface markers and by determining their ability to suppress IgG secretion in vitro. The results suggest that immunoregulatory dysfunction in autoimmune chronic active hepatitis is a familial abnormality, but that this abnormality occurs independent of circulating autoantibody status and of the segregation of genes for HLA or immunoglobulin allotypes.

Molecular analyses of in vivo hypoxanthine-guanine phosphoribosyltransferase mutations in human T-lymphocytes: II. Demonstration of a clonal amplification of hprt mutant T-lymphocytes in vivo.

Recent molecular analysis of in vivo-derived hprt mutant T-lymphocytes cloned from human blood show that mutants occurring at the normal frequency (approximately 5 X 10(-6) in healthy young individuals generally represent independent hprt mutations. Here we report that in an individual with a high mutant frequency (86-620 X 10(-6],92% (61/66) of the mutant clones are descendents of an original mature T-cell precursor that has undergone in vivo clonal expansion. Therefore, these mutants could represent as few as one original hprt mutation. If so, correcting for the clonal expansion yields a revised calculated mutant frequency (Mf) value for this individual that is near the normal range. These hprt mutant clones all showed identically rearranged T-cell receptor (TCR) beta and gamma gene patterns by Southern blot analysis. All the clones were surface marker CD4+, showed no obvious chromosomal aberration, and had no detectable hprt gene structural alteration. This TCR-defined T-cell clone appears to have expanded in the blood of the individual over a 6-month period and persists at high levels after nearly 4 years. This finding illustrates the need to analyze mutants from individuals with high mutant frequencies at the molecular level in order to estimate hprt mutation frequency from the calculated hprt mutant frequency. The possibility that spontaneous hprt mutants might arise in vivo preferentially in dividing cells, and implications of this, are discussed.

Immunogenetic studies of autoimmune chronic active hepatitis: HLA, immunoglobulin allotypes and autoantibodies.

The strategy of assigning a surrogate phenotype, defined as the presence of antinuclear and/or antismooth muscle antibodies to disease-free first degree relatives of index cases was used to search for a postulated disease susceptibility gene in autoimmune chronic active hepatitis. In addition to determining circulating autoantibody status, 10 patients, 51 first-degree relatives and 6 spouses of index chronic active hepatitis patients, each ascertained by the single patient, were genotyped for HLA (A, B and DR loci) and immunoglobulin allotype (G1m, G2m, G3m and A2m loci) haplotypes. Among the 10 chronic active hepatitis patients, 6 had HLA haplotypes B8 and DR3, and 3 of these patients had, in addition, the immunoglobulin allotype haplotype Gm a,x;g. Circulating autoantibodies defining the surrogate phenotype was found in 39% of the first-degree relatives. However, segregation analysis offered no support for either single autosomal dominant or recessive inheritance for the autoantibody-positive phenotype. Linkage between the postulated disease susceptibility locus and either the HLA (Chromosome 6) or immunoglobulin (Chromosome 14) locus was excluded by several analyses. Furthermore, logistic regression indicated that neither immunogenetic marker was statistically associated with autoantibody positively in these families. Therefore, despite the occurrence of autoantibody positivity in first-degree relatives of autoimmune chronic active hepatitis patients, we found no evidence that this trait has a simple genetic basis, or that it is an alternative manifestation of a postulated disease susceptibility gene for chronic active hepatitis.

Functional suppressor T cell activity in Crohn's disease and the effects of sulphasalazine.

Suppressor T cell activity was measured in 18 patients with Crohn's disease and 20 controls, using two different functional assays. The effects of sulphasalazine and its metabolites on in vitro suppressor cell activity were also studied. The activity of a Con A-induced suppressor cell system in patients with Crohn's disease did not differ from that of controls, suggesting that the previously reported abnormalities are a secondary phenomenon. Furthermore, the activity of a non-induced suppressor T cell system was also normal in these patients. There was no evidence either in vivo or in vitro to suggest that sulphasalazine exerts its beneficial action by an effect on this aspect of immunoregulation.

Suppressor cell activity in progressive systemic sclerosis.

Prostaglandin-producing suppressor cell activity was significantly increased in patients with progressive systemic sclerosis (p less than .01). No significant differences were found in concanavalin-A induced suppressor T cell activity. Although these results indicate an alteration in immunoregulatory function they may represent a compensatory reaction to a defect in the regulation of fibrogenesis.

Studies on lymphocyte hyporesponsiveness in cirrhosis: the role of increased monocyte suppressor cell activity.

We investigated the possibility that monocyte suppressor cells play a role in the peripheral lymphocyte hyporesponsiveness of chronic liver disease by utilizing assays of monocyte-mediated suppressor activity in 46 patients with chronic liver disease and 46 controls. The percent change achieved by the addition of indomethacin to a PHA-induced proliferative response was significantly increased in patients with cirrhosis compared with controls (p less than 0.001). The increase was seen in cirrhosis regardless of etiology but was not found in patients with chronic hepatitis without cirrhosis. The effects of indomethacin were abolished by monocyte depletion and were greater in autologous serum than in pooled AB serum (p less than 0.02). Monocyte depletion in cirrhotic patients significantly increased the lymphocyte response to PHA (p less than 0.005) but made no significant difference in controls. There was a significant correlation between the indomethacin-induced changes and the changes in lymphocyte response to PHA on monocyte depletion (r = 0.6583, p less than 0.01). Our initial results led us to study the mode of action of the inhibitory effect of cirrhotic serum on lymphocyte response to PHA. Cirrhotic serum significantly reduced the response of normal lymphocytes compared with control serum (p less than 0.02), but the difference was abolished by adding indomethacin and by monocyte depletion. These results suggest that monocyte suppressor cells may play a role in the depressed cellular immunity seen in some patients with cirrhosis. The inhibitory effect of cirrhotic serum appears to be in part monocyte mediated and prostaglandin dependent.

Effects of testosterone, oestradiol and progesterone on immune regulation.

Clinical observations on differences in the sexual incidence of diseases associated with defects of immune regulation, and of the occasional beneficial effects of pregnancy on disease course suggest that endocrine mechanisms may be important in the immuno-pathogenesis of these disorders. To investigate this possibility the in vitro effects of testosterone, oestradiol and progesterone on selected aspects of immune regulation were studied in normal adults. We observed the effects of these hormones on a mitogen-induced suppressor T-cell system and a monocyte mediated prostaglandin-producing suppressor cell system. The addition of progesterone but not oestradiol or testosterone to the Concanavalin A (Con A) generation of T lymphocyte suppressor cells produced significantly increased suppressor cell activity (P less than 0.005). Pre-incubation of lymphocytes with testosterone but not oestradiol or progesterone in the absence in the absence of Con A resulted in the generation of modest, but highly significant suppressor cell activity (P less than 0.005) No effect on the prostaglandin-producing suppressor cell activity was observed. These findings suggest that certain endocrine changes may alter immunoregulatory function and account for some of the clinical observations previously noted in diseases associated with defects of immune regulation.

Increased suppressor cell activity in inflammatory bowel disease.

We studied the in vitro effect of indomethacin, hydrocortisone, sulphasalazine, and its metabolites sulphapyridine (SP) and 5-amino salicylic acid (5-ASA) on peripheral blood mononuclear cells (PBMC) from 49 patients with inflammatory bowel disease and 34 controls. Indomethacin caused a highly significant increase in the PBMC response to the mitogen PHA-P compared with controls (P less than 0.01), indicating increased activity of a prostaglandin-producing suppressor cell system. On the contrary, sulphasalazine resulted in a reduced response which was significantly greater for the group with inflammatory bowel disease than the control group (P less than 0.05). This reduction was also produced by 5-ASA (P less than 0.05) but not by sulphapyridine. Addition of indomethacin to PBMC incubated with sulphasalazine significantly reduced the effect of sulphasalazine (P less than 0.001). Hydrocortisone resulted in a reduced response which was similar to that of controls and was not altered by the addition of indomethacin. The response to indomethacin, hydrocortisone, sulphasalazine, sulphapyridine, and 5-ASA was not dependent on the HLA type of the patients, disease activity, or drug therapy. The results suggest that increased suppression by a population of prostaglandin-producing suppressor cells plays a role in the immunopathology of inflammatory bowel disease, but that sulphasalazine does not exert its therapeutic effect by acting on this step of the immunoregulatory system. Any trials of indomethacin therapy in inflammatory bowel disease should take into account that, in vitro, sulphasalazine and indomethacin have opposing mechanisms of action in this system.

Immune regulation and HLA types in chronic hepatitis.

Studies were undertaken in 32 patients with hepatitis B-negative or -positive chronic active hepatitis or chronic persistent hepatitis to define the relationship between immunoregulatory activity and the HLA-B8 and B12 phenotypes. Suppressor T-cell activity measured by a concanavalin A-induced suppressor system using allogeneic responder cells was dependent on which individual was selected as a source of responder cells. No differences were noted using isogeneic cells as responders. Suppressor T-cell activity measured by the effect of a noninduced suppressor cell on a mixed leukocyte culture was not different from controls. Increased prostaglandin-producing suppressor cell activity was found in patients with hepatitis B-negative (p less than 0.005) and hepatitis B-positive (p less than 0.05) chronic active activity hepatitis. When results of the suppressor activities were compared among patients with chronic hepatitis dependent on the presence of HLA-B8, B12, or neither of these phenotypes, no significant differences were present. These results provide further evidence of altered immunoregulatory function in patients with chronic active hepatitis, which may reflect increased suppression by a population of prostaglandin-producing suppressor cells. The results do not, however, suggest that a gene coding for altered immune regulation is linked to HLA-B8 or B12.

25-hydroxy vitamin D absorption test in patients with gastrointestinal disorders.

To evaluate the relationships of a 25-hydroxy vitamin D absorption test to intestinal fat absorption and to the absorption of radioactively labeled vitamin D and 25-hydroxy vitamin D, an investigation was undertaken in 19 patients with gastrointestinal disorders. A correlation was noted between the results of the 25-hydroxy vitamin D absorption test and baseline 25-hydroxy vitamin D levels. No correlation was found between results of the 25-hydroxy vitamin D absorption test and quantitative fecal fat excretion. Peak values of the 25-hydroxy vitamin D absorption test correlated with net absorption of 14C vitamin D. No correlation existed between the values obtained in the 25-hydroxy vitamin D absorption test and 3H-25-OH D absorption. These studies indicate that the 25-hydroxy vitamin D absorption test probably does not serve as an effective screening test for intestinal fat malabsorption. The results of the test probably most accurately reflect the body stores of vitamin D at the time of testing, but there appears to be little advantage to performing a 25-hydroxy vitamin D absorption test in lieu of a single determination of the serum level of 25-hydroxy vitamin D as a method of evaluating vitamin D nutritional status.