Thomas Höhler

Tumor necrosis factor alpha promoter polymorphism at position -238 is associated with chronic active hepatitis C infection

Tumor necrosis factor α (TNF‐α) is involved in the pathogenesis of chronic hepatitis C virus infection. The gene for TNF‐α is encoded in the major histocompatibility locus (MHC). Two polymorphisms at positions ‐308 and ‐238 in the TNF‐α promoter region might influence TNF‐α expression. These promoter polymorphisms have been linked previously to a number of infectious diseases. TNF‐α 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. J. Med. Virol. 54:173–177, 1998.

HLA-DRB1*1301 AND *1302 protect against chronic hepatitis B

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.

Differential genetic determination of immune responsiveness to hepatitis B surface antigen and to hepatitis A virus: a vaccination study in twins

BACKGROUND The course of viral hepatitis is thought to be affected by genetic host variability and, in particular, by genes of the major histocompatibility locus. Hepatitis A and B vaccination is a useful model to study the effect of host factors on the immune response to viral antigens. We aimed to assess the heritability of the HBsAg (anti-HBs) and anti-hepatitis A virus (anti-HAV) immune response and to estimate the effect of the HLA-DRB1 locus and other genetic loci unlinked to HLA. METHODS We did an open prospective study and vaccinated 202 twin pairs with a combined recombinant HBsAg/inactivated hepatitis A vaccine. We measured antibodies to HBsAg and HAV and determined HLA-DRB1* alleles. Heritability was calculated based on variance of antibody response within pairs. Model-fitting analyses were done to analyse genetic and environmental components of vaccine responses. FINDINGS Anti-HBs and anti-HAV showed heritabilities of 0.61 (95% CI 0.41 to 0.81) and 0.36 (-0.02 to 0.73), respectively. For the anti-HBs immune response, 60% of the phenotypic variance was explained by additive genetic and 40% by non-shared environmental effects. The heritability of the HBsAg vaccine response accounted for by the DRB1* locus was estimated to be 0.25, leaving the remaining heritability of 0.36 to other gene loci. INTERPRETATION Genetic factors have a strong effect on the immune response to HBsAg. Although genes encoded within the MHC are important for this immune response, more than half the heritability is determined outside this complex. Identification of these genes will help us to understand regulation of immune responses to viral proteins.

Association of different tumor necrosis factor α promoter allele frequencies with ankylosing spondylitis in HLA-B27 positive individuals

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<0.0004) and the B27-negative (159 wild-type promoter alleles, 39 variant alleles; P< 0.00001) control groups. Positivity for the variant promoter alleles conferred protection and a relative risk of 0.3 to B27-positive individuals. CONCLUSION These data indicate that allelic variations in the TNFalpha promoter influence disease susceptibility in HLA-B27 positive individuals. This protective effect of variant promoter alleles could be related to differences in TNFalpha production or could reflect the association of different B27 haplotypes with ankylosing spondylitis.

MHC class II genes influence the susceptibility to chronic active hepatitis C.

BACKGROUND/AIMS 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. METHODS 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. RESULTS 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; p < 0.001). 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; p < 0.008). 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; p < 0.003). An even stronger protective effect was provided by the presence of DRB1*1301 and DQA1*0103 (relative risk 0.08; p < 0.005). These findings were confirmed in a second group of chronic hepatitis C virus infected patients. CONCLUSIONS 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.

Gemcitabine plus erlotinib followed by capecitabine versus capecitabine plus erlotinib followed by gemcitabine in advanced pancreatic cancer: final results of a randomised phase 3 trial of the ‘Arbeitsgemeinschaft Internistische Onkologie’ (AIO-PK0104)

Objective AIO-PK0104 investigated two treatment strategies in advanced pancreatic cancer (PC): a reference sequence of gemcitabine/erlotinib followed by 2nd-line capecitabine was compared with a reverse experimental sequence of capecitabine/erlotinib followed by gemcitabine. Methods 281 patients with PC were randomly assigned to 1st-line treatment with either gemcitabine plus erlotinib or capecitabine plus erlotinib. In case of treatment failure (eg, disease progression or toxicity), patients were allocated to 2nd-line treatment with the comparator cytostatic drug without erlotinib. The primary study endpoint was time to treatment failure (TTF) after 1st- and 2nd-line therapy (TTF2; non-inferiority design). KRAS exon 2 mutations were analysed in archival tumour tissue from 173 of the randomised patients. Results Of the 274 eligible patients, 43 had locally advanced and 231 had metastatic disease; 140 (51%) received 2nd-line chemotherapy. Median TTF2 was estimated with 4.2 months in both arms; median overall survival was 6.2 months with gemcitabine/erlotinib followed by capecitabine and 6.9 months with capecitabine/erlotinib followed by gemcitabine, respectively (HR 1.02, p=0.90). TTF for 1st-line therapy (TTF1) was significantly prolonged with gemcitabine/erlotinib compared to capecitabine/erlotinib (3.2 vs 2.2 months; HR 0.69, p=0.0034). Skin rash was associated with both TTF2 (rash grade 0/1/2–4:2.9/4.3/6.7 months, p<0.0001) and survival (3.4/7.0/9.6 months, p<0.0001). Each arm showed a safe and manageable toxicity profile during 1st- and 2nd-line therapy. A KRAS wild-type status (52/173 patients, 30%) was associated with an improved overall survival (HR 1.68, p=0.005). Conclusion Both treatment strategies are feasible and demonstrated comparable efficacy; KRAS may serve as biomarker in patients with advanced PC treated with erlotinib. Trial registration number This study was registered at ClinicalTrials.gov, number NCT00440167. Significance of this study What is already known on this subject? Gemcitabine-based chemotherapy remains an international standard of care for patients with non-resectable, advanced pancreatic cancer (PC). Anti-EGFR treatment with the tyrosine kinase inhibitor erlotinib, as well as chemotherapy intensification by application of the FOLFIRINOX regimen, both significantly improved overall survival in randomised phase 3 trials. The optimal (sequential) regimen for the use of gemcitabine, erlotinib and the oral fluoropyrimidine capecitabine remains unclear in advanced PC. Molecular predictors for the efficacy of anti-EGFR treatments in PC have not been defined up to now. What are the new findings? The sequential use of gemcitabine, erlotinib and capecitabine is safe and equally effective in PC; gemcitabine appears to be more effective in 1st- and 2nd-line therapy than capecitabine and therefore remains the preferred combination partner for erlotinib. Skin rash is strongly correlated with efficacy outcome measures in PC patients treated with erlotinib. KRAS wild-type status appears to be associated with improved overall survival in patients treated with erlotinib in this AIO study. Significance of this study How might it impact on clinical practice in the foreseeable future? The benefit of adding erlotinib to chemotherapy is restricted to patients that experience skin rash during treatment; non-rash patients are characterised by a very poor outcome and need to be offered novel treatment strategies. Second-line salvage chemotherapy is effective and safe in selected PC patients. KRAS could serve as the first biomarker for improved survival in erlotinib-treated patients; the predictive value of KRAS for erlotinib efficacy remains to be defined prospectively.

A functional polymorphism in the IL-10 promoter influences the response after vaccination with HBsAg and hepatitis A.

The immune response to hepatitis B surface antigen (HBsAg) is mostly genetically determined. Interleukin 10 (IL‐10) is a central immunoregulatory cytokine with important effects on B‐cells. We have studied the influence of IL‐10 promoter polymorphisms on the immune response to HBsAg and hepatitis A vaccination. We vaccinated 202 twin pairs in an open prospective study with a combined recombinant HBsAg/inactivated hepatitis A vaccine. IL‐10 promoter polymorphisms were investigated in all individuals and their influence on anti‐HBs, and anti‐HAV responsiveness was studied. In the multiple regression analysis accounting for smoking, gender, body mass index and age, the ACC haplotype (−1082, −819 and −592) had a strong influence on anti‐HBs production. Individuals carrying the ACC haplotype had anti‐HBs titres almost twice as high as individuals without this haplotype. In contrast, anti‐HAV production was suppressed by the presence of the −1082A allele in comparison with individuals homozygous for the −1082G allele. The contribution of the shared IL‐10 promoter haplotype accounted for 27% of the genetic influence on anti‐HBs antibody response. In conclusion, genetic variability in the IL‐10 promoter is an important modulator of the immune response against hepatitis viral antigens. (HEPATOLOGY 2005.)

C4A deficiency and nonresponse to hepatitis B vaccination

BACKGROUND/AIMS Hepatitis B vaccination failure has been linked to the presence of certain human leukocyte antigen class II alleles. However, the functional background of these associations has remained unclear. Complement component C 4 is encoded within the major histocompatibility complex and is essential for classical pathway activation. METHODS Healthy individuals (n=4269) were vaccinated in a prospective trial with Engerix B. Nonresponse was classified as anti-HBs<10 U/l after the last vaccination. Seventy-three nonresponders (NR) (1.7%) were identified. For comparison 53 responders (R) (anti-HBs>10 IU/l) were drawn randomly from the same cohort. C4 allotyping was carried out by high-voltage agarose gel electrophoresis and C4alpha-chain typing using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. C4 gene deletions (C4Del) were studied by Southern blot. RESULTS C4AQ0 alleles were observed in 45/73 (62%) NR compared to 17/53 (32%) R (P=0.001). C4ADel was observed in 24/73 (33%) NR and in 6/52 (12%) R (P=0.006). C4AQO alleles were present in 21/49 (43%) NR without C4Del compared to 10/46 (22%) in R without C4Del (P=0.031). In a logistic regression with DRB1*0301, DRB1*07, DRB1*1301 and C4AQ0 all except for DRB1*0301 showed a significant association. CONCLUSIONS C4AQ0 shows a DRB1*0301 independent association with vaccine failure. C4AQ0 alleles probably contribute to inefficient complement activation and failure of B cells to secrete anti-HBs.

IL10.G Microsatellites Mark Promoter Haplotypes Associated With Protection Against the Development of Reactive Arthritis in Finnish Patients

OBJECTIVE To investigate the association of microsatellites and single-nucleotide promoter polymorphisms (SNPs) in the gene for the cytokine interleukin-10 (IL-10) with susceptibility to and outcome of reactive arthritis (ReA). METHODS From genomic DNA, IL-10 microsatellites G and R and IL-10 promoter polymorphisms at positions -1087 and -524 were typed by polymerase chain reaction, automated fragment length analysis, and restriction fragment digestion in 85 Finnish patients with ReA and 62 HLA-B27-positive Finnish controls. ReA patients had been followed up for 20 years. Genotypes and haplotypes of IL-10 were correlated with distinct features of the disease course, such as triggering agent, chronic arthritis, development of ankylosing spondylitis, and other chronic features. RESULTS There was a significant decrease in the promoter alleles G12 (allele frequency 0.206 versus 0.033; corrected P < 0.001, odds ratio 0.14) and G10 (0.183 versus 0.092; P < 0.05, odds ratio 0.44) in the ReA group compared with the HLA-B27-positive controls. Chronic arthritis developed significantly more frequently in the B27-positive subjects than in the B27-negative subjects (P < 0.05) as well as in patients with [corrected] the IL10.G8 allele. No associations were observed for either SNP or for the IL10.R microsatellite polymorphism. CONCLUSION IL10.G12 and G10 microsatellite alleles show a strong protective effect against the development of ReA in Finnish subjects. Since these polymorphic markers themselves do not have direct functional implications, they most likely mark promoter haplotypes with distinct functional properties, suggesting that differential production of IL-10 is an important susceptibility factor for the development of ReA.

The Influence of Major Histocompatibility Complex Class II Genes and T-Cell Vβ Repertoire on Response to Immunization with HBsAg

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%;p < 0.0001) 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.