Search

OBJECTIVE To investigate the potential association of tumor necrosis factor alpha (TNFalpha) promoter alleles with ankylosing spondylitis. METHODS DNA from 141 HLA-B27 positive Caucasian patients with ankylosing spondylitis and 46 B27-positive and 99 B27-negative healthy Caucasian controls was investigated by polymerase chain reaction amplification of the TNFalpha promoter region and subsequent dot-blot analysis with allele-specific oligonucleotides. RESULTS There was a significant decrease in the promoter alleles TNF-238.2 and TNF-308.2 in the ankylosing spondylitis group (266 wild-type alleles, 16 variant alleles) compared with the B27-positive (75 wildtype promoter alleles, 17 variant alleles; P

Tumor necrosis factor alpha (TNF-alpha) is involved in the pathogenesis of chronic hepatitis C virus infection. The gene for TNF-alpha is encoded in the major histocompatibility locus (MHC). Two polymorphisms at positions -308 and -238 in the TNF-alpha promoter region might influence TNF-alpha expression. These promoter polymorphisms have been linked previously to a number of infectious diseases. TNF-alpha promoter polymorphisms at positions -238 and -308 were studied by DNA sequencing and sequence-specific oligonucleotide hybridization in 82 individuals with chronic hepatitis C and 99 control subjects. Subjects had been HLA class I and class II typed in a previous study. The frequency of the TNF238.2 promoter allele was significantly higher in the hepatitis C group (18.7%) compared to the controls (3.5%; P < 0.0001; pcorr < 0.009). No significant differences in the frequency of the TNF308.2 allele were observed between patients and controls. The increased frequency of the TNF238.2 allele could not be explained by linkage disequilibrium to HLA-B or -DR genes. These findings show an association between the TNF238.2 promoter variant and chronic active hepatitis C. They suggest that this polymorphism or a linked gene may be a host factor contributing to the development of chronic active hepatitis C.

Tumor necrosis factor-α is considered to be one of the important mediators in the pathogenesis of psoriasis. A strong association of juvenile onset psoriasis with the major histocompatibility complex encoded HLA-Cw6 antigen has been reported but it is unclear whether Cw6 itself or a closely linked gene is involved in the pathogenesis. This study has focused on the association of promoter polymorphisms of the major histocompatibility complex encoded tumor necrosis factor-α gene with psoriasis and psoriatic arthritis. Tumor necrosis factor-α promoter polymorphisms were sought by sequence-specific oligonucleotide hybridization and by direct sequencing in Caucasian patients with juvenile onset psoriasis and with psoriatic arthritis and in healthy controls. A mutation at position −238 of the tumor necrosis factor-α promoter was present in 23 of 60 patients (38%; p < 0.0001; Pcorr < 0.008) with juvenile onset psoriasis and in 20 of 62 patients (32%; p < 0.0003; Pcorr < 0.03) with psoriatic arthritis, compared with seven of 99 (7%) Caucasian controls. There was a marked increase of homozygotes for this mutation in the psoriasis group. Another mutation at position −308 was found in similar proportions of patients and controls. Our study shows a strong association of the tumor necrosis factor-α promoter polymorphism at position −238 with psoriasis and psoriatic arthritis. Our findings suggest that this promoter polymorphism itself or a gene in linkage disequilibrium with tumor necrosis factor-α predispose to the development of psoriasis.

Chronic hepatitis C develops in more than 70% of hepatitis C virus infected subjects. Viral factors influence the disease course, but little is known about the importance of host factors.Frequencies of major histocompatibility complex (MHC) class I and class II antigens were analyzed in two groups of patients with chronic hepatitis C virus infection and in control subjects. MHC class I typing was done by standard microlymphocytotoxicity assays. DRB1 and DQA1 genotyping was done by PCR based typing methods.DRB1*0301 was found in 26 of 75 patients with chronic hepatitis C virus infection (34.7%) and in 12 of 101 control subjects (11.9%) (relative risk 3.9; p0.001). Homozygosity for this allele appeared to confer a stronger risk. In contrast, DRB1*1301 was detected in three subjects with persistent infection (4.0%) compared to 21 control subjects (20.8%) (relative risk 0.2; p0.008). This allele was linked with DQA1*0103, which was found in 10 patients (13.3%) compared to 34 control subjects (33.7%) (relative risk 0.31; p0.003). An even stronger protective effect was provided by the presence of DRB1*1301 and DQA1*0103 (relative risk 0.08; p0.005). These findings were confirmed in a second group of chronic hepatitis C virus infected patients.The MHC class II allele DRB1*0301 appears to predispose to progression to chronic active hepatitis C, whereas the class II alleles DRB1*1301 and DQA1*0103 appear to provide protection against chronic active infection with hepatitis C virus.

Background/Aims: The outcome of acute hepatitis B infection may be influenced by host factors like the major histocompatibility complex (MHC). We have investigated MHC class I and class II antigens in patients with chronic hepatitis B compared to a healthy control population. To confirm the findings of this first study we performed a second study in a group of subjects who had spontaneously recovered from acute hepatitis B infection. Methods: Frequencies of MHC class I and class II antigens were analyzed in patients with chronic hepatitis B virus infection and in control subjects. MHC class I typing was done by standard microlymphocytotoxicity assays. DRB1 and DQA1 genotypes were determined by polymerase chain reaction based typing methods. Results: In the first study the class II allele HLA-DRB1*1301-02 was found in 4 of 70 subjects with chronic hepatitis B virus infection (5.7%) compared to 27 of 101 healthy controls (26.7%, relative risk 0.17; p =0.001; p corr =0.025). This protective effect of the DRB1*1301-02 allele was confirmed in the second study. Eight of 24 patients (33.3%) who cleared hepatitis B virus spontaneously were positive for DRB1*1301-02 (relative risk of developing chronic infection compared to chronic hepatitis B subjects 0.12; p=0.004). Subtyping confirmed that 1301 and 1302 were both decreased in frequency in patients with chronic hepatitis B. Conclusions: The MHC class II allele DRB1*1301-02 is associated with protection from chronic hepatitis B in Caucasian patients.

Liver-kidney microsomal-1 autoantibodies characterize a subgroup of autoimmune chronic active hepatitis. The liver antigen of liver-kidney microsomal-1 antibodies has been identified as cytochrome P450 db1, a microsomal enzyme catalyzing the oxidative metabolism of more than 20 drugs, including debrisoquine, sparteine and bufuralol. A genetic polymorphism (debrisoquin-sparteine polymorphism) is responsible for the lack of P450 db1 protein in the livers of 5% to 10% of Caucasians, leading to impaired drug metabolism and a distinct poor metabolizer phenotype. We investigated whether liver-kidney microsomal-1 positive autoimmune chronic active hepatitis patients express functionally intact P450 db1 in their livers. In four patients with liver-kidney microsomal-1 positive chronic active hepatitis, but not in five patients with various liver-kidney microsomal-1 negative liver diseases, the presence of circulating liver-kidney microsomal-1 antibodies was confirmed by immunofluorescence, radioimmunoassay and immunoblotting analysis using recombinant P450 db1. Moreover, only sera from liver-kidney microsomal-1 positive autoimmune chronic active hepatitis patients strongly inhibited the enzymatic activity of P450 db1 in human liver microsomes in vitro. Immunoblotting detected 50-kd P450 db1 protein in liver biopsy specimens from all patients. The in vivo function of P450 db1 was investigated by determining the metabolic ratio for sparteine and its 2-dehydro and 5-dehydro metabolites in 12-hr urine samples after oral administration of sparteine sulfate. In vivo P450 db1–mediated drug metabolism was of the extensive metabolizer phenotype and did not differ significantly between liver-kidney microsomal-1 positive (metabolic ratio = 1.15 ± 0.32) and liver-kidney microsomal-1 negative (metabolic ratio = 1.18 ± 0.48) patients. Thus patients with liver-kidney microsomal-1 positive chronic active hepatitis express functionally intact P450 db1 in their livers. However, the activity of this enzyme is not significantly diminished in vivo by circulating liver-kidney microsomal-1 autoantibodies that react with the active site of P450 db1 and inhibit its function in vitro. (HEPATOLOGY 1990;12:127–132).

Nonresponsiveness to HBsAg vaccination is observed in 5-10% of vaccine recipients and is possibly caused by a defect in the T helper cell compartment. The immune response to HBsAg is influenced by genes of the major histocompatibility complex. We have investigated MHC class I and class II antigens in 53 adult responders and 73 nonresponders. Results obtained in this first study were tested in a second study with 56 responders and 62 nonresponders from an infant vaccination trial. In addition, the peripheral Vbeta-chain T-cell receptor repertoire was investigated using monoclonal antibodies and flow-cytometry in 26 adult responders and 38 nonresponders. As previously reported, nonresponsiveness to HBsAg vaccination was associated with DRB1*3 and DRB1*7. In addition, DRB1*13 was significantly increased among vaccine responders (35.2% vs 5.4%;p0.0001) suggesting an immune response promoting effect for this allele whereas the closely related allele DRB1*14 was associated with nonresponse in the infant study. There was no evidence for a hole in the T cell receptor Vbeta repertoire. In conclusion, in agreement with results obtained in mice there appears to be a hierarchy of DRB1* genes in the HBsAg immune response. The possible differential association of DRB1*13 and DRB1*14 may allow the identification of differences between responsiveness and nonresponsiveness to a few amino acid differences in the beta1-domain of the class II heterodimer.

Hepatocellular carcinoma is one of the most common human cancers with an annual incidence of about 1,000,000 cases worldwide. Although hepatocellular carcinoma is predominant in hepatitis B virus endemic areas, it has also become a major problem in Europe, Japan and North America in close association with the increasing incidence of hepatitis C virus infection. The pathogenetic role of hepatitis C virus infection in the development of HBsAg-negative hepatocellular carcinoma needs to be clarified. In this paper the case of a 66-year-old HBsAg-negative and anti-HCV positive female who developed hepatocellular carcinoma in a cirrhotic liver is reported. After 1 year of follow up, urgent laparotomy had to be performed due to highly differentiated ovarian metastases of the hepatocellular carcinoma. Plus- and minus-stranded HCV-RNA was detected by reverse transcription and "nested" polymerase chain reaction in both the patient's serum and in the metastatic ovarian tissue.

Several concepts concerning the pathogenicity of antineutrophil cytoplasmic antibodies (ANCA) exist, but till now only sparse data about ANCA-endothelial interactions are available. In this study we have investigated the expression of proteinase 3 (PR-3) in human umbilical endothelial cells (HEC) using purified anti-PR3 antibodies (C-ANCA) of patients with Wegener’s granulomatosis (WG) and monoclonal antibodies to PR-3 (human and murine) as probes. Performing cytoELISAs, laser scanning microscopy and Western blot we were able to show that treatment of HEC with IL-1-alpha led to an increased PR-3 expression in the cytoplasm and to a transient translocation into the EC-membrane. Representing an important missing link of ANCA-endothelial interactions, our data give a hint at a possible direct pathogenicity of anti-PR-3 antibodies in WG and other vasculitides.

Avarol is a cytostatic and anti-human immunodeficiency virus (HIV) agent. In this study, the avarol caused induction of gamma-interferon (IFN-gamma) in buffy coat cells (human peripheral blood lymphocytes) is demonstrated by immunological and molecular biological techniques. IFN-gamma production was detected after a 24-hr incubation period with avarol; maximal production was obtained after 5 days in the presence of the optimal avarol concentration of 0.75 microgram/ml. Blotting experiments using human IFN-gamma cDNA and beta-actin cDNA containing plasmids showed that in the absence of avarol no IFN-gamma transcripts were present in lymphocytes. Already after a 24-hr incubation with avarol, IFN-gamma gene induction was detected, and maximal induction was found after a 5-day incubation period. The enhanced IFN-gamma production seems to be caused by a change at the transcriptional and/or post-transcriptional level, but not during subsequent nucleocytoplasmic transport of mRNA. This molecular event is specific, at least in relation to the expression of the beta-actin gene. Our studies demonstrate that avarol displays, besides its potential anti-tumor and anti-HIV activity, a potential immunomodulating effect.

The influence of the immunomodulator bestatin on the expression of terminal deoxynucleotidyl transferase and of DNA polymerase alpha and beta in Molt-4 cells has been studied. Bestatin was found to stimulate cell growth within the range of 0.3-33 microM, while concentrations higher than 300 microM were inhibitory during an incubation period of 48 h. The cell surface bound microsomal leucine aminopeptidase (bestatin receptor) activity decreased gradually during incubation at concentrations of bestatin above 3 microM. This effect was also observed after incubation with amastatin, but not with leupeptin or tunicamycin. Determinations of the activities of DNA synthesizing enzymes from bestatin-treated Molt-4 cells revealed a direct correlation between the decrease of the surface bound microsomal leucine aminopeptidase activity and the increase of the terminal deoxynucleotidyl transferase and DNA polymerase alpha activity; the DNA polymerase beta activity remained unchanged. From these experiments it is hypothesized that bestatin might cause a promoting effect on the differentiation processes of precursor T cells in vivo.

HTLV-IIIB-infected H9 cells are shown to contain a high level of the natural UAG suppressor glutamine tRNA(UmUG Gln); this tRNA has been demonstrated to be required for the synthesis of Moloney murine leukemia virus (Mo-MuLV)-encoded protease. After cultivation of HTLV-IIIB-infected H9 cells with Avarol at a concentration (1 microgram/ml), previously found to protect the cells against the cytopathic effects of HTLV-III, an almost complete inhibition of the synthesis of the tRNA(UmUG Gln) was observed. Moreover, we obtained some evidence that the processing of the HTLV-III precursor protein p53 to p24 is inhibited by Avarol in infected cells, suggesting that the compound interferes with the expression of the viral protease gene.