Microarray study reveals a transforming growth factor-ß-dependent mechanism of fibrosis in discoid lupus erythematosus.

BACKGROUND: Discoid lupus erythematosus (DLE) is characterized by scarring lesions that develop and perpetuate fibrotic lesions. These are not observed in subacute cutaneous lupus erythematosus (SCLE). The pathophysiological basis of this is currently unknown. OBJECTIVES: To identify contradistinctive signalling pathways and cellular signatures between the two type of lupus, with a focus on the molecular mechanisms leading to fibrosis. METHODS: We conducted a gene expression microarray analysis in lesional and nonlesional skin biopsy specimens of patients with DLE (n = 10) and SCLE (n = 10). Confirmatory reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry were performed on selected transcripts in a new cohort of paraffin-embedded skin biopsies (n = 20). Changes over time of a group of selected inflammatory and fibrotic genes were also evaluated in a second biopsy taken 12 weeks later. In vitro functional studies were performed in primary isolated fibroblasts. RESULTS: Compared with nonlesional skin, DLE samples expressed a distinctive T-cell gene signature. DLE samples displayed a significant CD4 T-cell enrichment with an imbalance towards T helper 1 cytokine predominance and a relative increased forkhead box (FOX)P3 response. RT-qPCR and immunochemical analysis over time showed a progressive increment of fibrotic markers and persistent FOXP3 recruitment. Ex vivo upregulation of SERPINE1, MMP9, TGFBR1, phosphorylated SMAD3 and TGFB1 suggested a transforming growth factor (TGF)-ß-dependent mechanism of fibrosis in DLE, also confirmed by the results observed following in vitro stimulation with TGF-ß. CONCLUSIONS: These results highlight major pathogenic pathways in DLE and provide novel molecular targets for the development of new therapies. The data suggest the existence of a TGF-ß-dependent pathway inducing fibrosis in DLE.

Genetics of Graves' Disease: Special Focus on the Role of TSHR Gene.

As most autoimmune diseases, inherited predisposition to Graves' disease (GD) is polygenic with the main contributory genes being located in the HLA region. Also, as in other autoimmune diseases, family linkage, candidate gene association, and GWAS studies have identified an expanding number of predisposing genes (CTLA4, CD40, PTPN22...) and 2 of them, TG and TSHR, are thyroid specific. In spite of this expanding number of associated genes, it has been estimated that all together they account for only a 20% of the heritability of GD. TSHR is of special interest as it codes for the target of TSHR stimulating antibodies (TSAbs), which are unequivocally pathogenic and an exception in autoimmunity by being stimulating rather than neutral, blocking, or cytotoxic. This is surprising because the generation of stimulating TSHR antibodies by immunisation of laboratory animals has been remarkably difficult, suggesting an underlying mechanism that favours stimulating over neutral or blocking anti-TSHR antibodies must be operating in GD patients. Besides, after HLA, TSHR is the gene most tightly associated to GD. The TSHR polymorphisms conferring susceptibility are located in the unusually large intron 1. Two mechanisms have been already put forward to explain its association with GD. According to one, the risk alleles determine an increase in the expression of TSHR mRNA splice variants that code for a soluble form of the receptor. The wider distribution of soluble TSHR would favour its immunogenicity and the development of an autoimmune response to it. It does not explain why it becomes immunogenic, as immunogenicity and distribution are not necessarily connected, nor why the immune response focus to the production of stimulating antibodies. According to the second mechanism proposed, the risk alleles determine a lower TSHR expression in the thymus and this would favour the escape of more TSHR reactive T cells, that is, central tolerance failure. The unexpected finding that thymocytes express TSHR and that TSAbs stimulate them lead to postulate that this would accelerate their egress from the thymus and a less efficient deletion of the TSHR self-reactive T cells. It can be envisaged that these autoreactive T cells may enhance the production of TSHR-Abs in the germinal centres of the thyroid draining lymph nodes, especially of those capable of further stimulating the egress of autoreactive T cells from the thymus. This mechanism, which does not exclude the former, provides and insight of the way in which TSAbs are favoured over neutral or blocking antibodies. Finally this would explain the frequent finding of thymic hyperplasia in GD patients.

Influence of the dynamics of the hypervariable region 1 of hepatitis C virus (HCV) on the histological severity of HCV recurrence after liver transplantation.

Recurrence of hepatitis C virus (HCV) infection after liver transplantation is almost universal and usually leads to chronic hepatitis with different degrees of severity. The pathogenic mechanisms underlying the variable outcome of HCV infection recurrence are not well defined, but recent data suggest that the dynamics of HCV quasispecies may be involved. HCV quasispecies evolution was traced by longitudinal single strand conformation polymorphism, direct sequencing, and cloning analyses of pre- and post-transplant HCV-1b isolates from patients with histologically severe (seven cases) or mild or moderate (nine cases) HCV infection recurrence. Differences between the two groups of patients that concerned the level of viremia or the degree of HCV quasispecies complexity and diversity were not observed at any of the three time points analyzed. However, emergence of nucleotide and amino acid changes during the 12 months follow-up was significantly more frequent in patients with mild or moderate than in those with severe HCV infection recurrence. The ratio of non-synonymous to synonymous nucleotide substitutions 12 months after transplantation was also greater in the former, suggesting that the HVR1 of HCV is under stronger selective pressure in these subjects. These findings suggest that the degree of amino acid diversification in the HVR1 of HCV, which probably reflects the strength of immune pressure on HCV, is inversely related to the histological severity of HCV infection recurrence.

Influence of the genetic heterogeneity of the ISDR and PePHD regions of hepatitis C virus on the response to interferon therapy in chronic hepatitis C.

Two genomic regions of hepatitis C virus (HCV), the interferon sensitivity-determining region (ISDR) of the non-structural 5A gene (NS5A) and the protein kinase-RNA activated (PKR)-eukariotic transcription factor (eIF2-alpha) phosphorylation homology domain (PePHD) of the structural E2 gene, interact in vitro with the interferon-inducible cellular PKR protein kinase. Mutations within these regions might, therefore, influence the response to interferon therapy. Viral load at baseline and sequence heterogeneity of HCV in NS5A and E2 regions was studied in 74 HCV-1b and in 12 HCV-3a infected patients with chronic hepatitis C who were treated with interferon. As previously reported by us, in a smaller series of patients in which the ISDR region was analyzed [Saiz et al. (1998) Journal Infectious Diseases 177:839-847], in the present study a low viral load and a high number of amino acid mutations within the ISDR, but not within the PePHD region, were significantly associated with long-term response to interferon among HCV-1b infected patients. No relationship between these viral features and response to therapy was disclosed in patients infected with HCV-3a.

High amino acid variability within the NS5A of hepatitis C virus (HCV) is associated with hepatocellular carcinoma in patients with HCV-1b-related cirrhosis.

Interferon therapy may decrease the risk of hepatocellular carcinoma in patients with hepatitis C virus (HCV)-related liver cirrhosis. Interaction of the cellular protein kinase PKR with the PKR-binding domain (PKR-bd) of HCV-NS5A protein may affect cellular growth control and viral resistance to interferon therapy. Mutations within the PKR-bd, which comprises the interferon sensitivity determining region (ISDR), have been associated with interferon sensitivity. To determine whether or not there is an association between HCV heterogeneity and the presence of hepatocellular carcinoma, HCV-1b genomic regions were amplified and directly sequenced from serum samples obtained from 82 patients with liver cirrhosis, 53 with, and 29 without hepatocellular carcinoma. None of them had received antiviral therapy. When compared with the deduced consensus sequence, the median number of amino acid changes in the PKR-bd was higher among samples from patients with (4.22) than from those without hepatocellular carcinoma (1.62; P <.001), and isolates with 3 or more amino acid changes were significantly more common among the former (60%) than among the later (6%, P <.001). No such differences were observed in other viral regions, including Core, E2-HVR-1, E2-PePHD, NS3, and the 5' and 3' PKR-bd flanking regions. In addition, amino acid variation in viral regions other than HVR-1 did not accumulate over time in the analyzed sequential serum samples obtained from patients with or without hepatocellular carcinoma. Therefore, a mutated HCV-PKR-bd phenotype is very common in cirrhotic patients with hepatocellular carcinoma.

Genetic evolution of GB virus C/hepatitis G virus (GBV-C/HGV) under interferon pressure.

The epidemiology and clinical features of chronic GBV-C/HGV infection have largely been explored, but there is little information about the mechanisms enabling GBV-C/HGV to cause persistent infection. Since analysis of the genomic variation of GBV-C/HGV under interferon pressure might provide some insight into this issue, we analyzed the nucleotide sequence variation of the 5'NC and NS3 regions in GBV-C/HGV isolates obtained sequentially from seven patients co-infected with HCV and treated with interferon. A reduction of GBV-C/HGV-RNA serum level below the detection limit of the RT-PCR assay was observed during treatment in all patients, but upon interferon withdrawal, viral RNA remained undetectable in only two patients. Among the five patients who did not clear GBV-C/HGV-RNA, viral strains emerging after treatment were identical to those present at baseline in three cases. In a further case, in whom GBV-C/HGV-RNA re-emerged during therapy (breakthrough episode), several mutations appeared in relapse samples. In the remaining patient, with a mixed infection before therapy, only one of the two GBV-C/HGV strains present at baseline was detected upon treatment withdrawal. These data raise the possibility that positive selection may act over GBV-C/HGV genome during interferon therapy, and contribute to persistence of infection with this virus.

Prevalence and route of transmission of infection with a novel DNA virus (TTV), hepatitis C virus, and hepatitis G virus in patients infected with HIV.

OBJECTIVES: To evaluate the prevalence, route of transmission and clinical significance that current co-infection with TT virus (TTV), hepatitis C virus (HCV), and hepatitis G virus (HGV) have in HIV-1-infected patients. DESIGN: Presence of TTV, HCV, and HGV was analyzed in plasma samples from 160 HIV-1-infected patients with parenteral (38 intravenous drug users [IVDUs] and 41 patients with hemophilia) or sexual (39 homosexuals and 42 heterosexuals) risk of exposure, and in 168 volunteer blood donors. Alanine aminotransferase (ALT) levels and CD4+ counts were also analyzed. METHODS: HCV and HGV RNA were detected by specific reverse transcriptase (RT) nested polymerase chain reaction (PCR) and TTV DNA by specific heminested PCR. RESULTS: TTV DNA was detected in 39% of the patients and in 14% of the volunteer blood donors. HCV and HGV infections were detected in 42% and in 14% of the patients, and in 0% and 3% of the blood donors, respectively. Prevalences of TTV and HCV infection were higher among patients with parenteral (62% and 68%) than in those with sexual (17% and 16%) risk of exposure. A higher prevalence of TTV infection (but not of HCV or HGV infection) was observed among patients with hemophilia (76%) than IVDUs (47%), and among homosexuals (26%) than among heterosexuals (10%). Abnormal ALT levels were related with the presence of HCV infection, independently of the detection of TTV DNA. TTV infection did not seem to alter the levels of CD4+ T cells. CONCLUSIONS: Prevalence of current TTV infection is high among HIV-infected patients with parenteral risk of exposure, but TTV is also transmitted through sexual routes; detection of TTV does not seem to influence the clinical or immune status of HIV-infected patients.

Infection with a novel human DNA virus (TTV) has no pathogenic significance in patients with liver diseases.

BACKGROUND/AIMS: A recently identified DNA virus, termed TT virus (TTV), has been associated with post-transfusional hepatitis, and a high prevalence of TTV infection in patients with acute or chronic liver disease of unknown etiology has been reported from Japan, but few data are available about TTV infection in other countries. METHODS: Using hemi-nested-PCR amplification to detect TTV-DNA sequences in serum, we investigated TTV infection in blood donors and in patients with liver diseases of varied etiology. RESULTS: The prevalence of TTV infection was 13.7% in blood donors (23/168), 18.6% in chronic hepatitis C (19/102), 28.6% in chronic hepatitis B (16/56), 29.9% in hepatocellular carcinoma (20/67), 9.1% in cryptogenic chronic liver disease (2/22) and 39.6% in fulminant hepatitis (19/48). The prevalence of TTV infection in patients with virus-induced or idiopathic fulminant hepatitis was similar. Comparison of TTV-infected and non-infected patients did not reveal significant differences concerning demographic, epidemiological or histopathological features. In patients with hepatitis C, response to interferon therapy was not related to TTV infection. Phylogenetic analysis of TTV isolates showed that at least three different types of TTV are present in Spain. CONCLUSIONS: Our data suggest that TTV infection is frequent among blood donors and patients with acute liver disease. However, pathogenic effects associated with TTV infection were not observed.

Molecular evidence of mother-to-infant transmission of hepatitis G virus among women without known risk factors for parenteral infections.

Hepatitis G virus (HGV) RNA was detected in 18 of 133 pregnant women from Tanzania without known risk factors for HGV infection and in 7 of 18 children born to HGV RNA-positive mothers. Molecular evidence of mother-to-infant transmission was obtained only for three of seven children. HGV RNA was also detected in 4 of 42 children born to non-HGV-infected women. Thus, mechanisms other than materno-filial may play an important role in HGV transmission during early childhood.

Relationship of the genomic complexity of hepatitis C virus with liver disease severity and response to interferon in patients with chronic HCV genotype 1b infection [correction of interferon].

In patients with chronic hepatitis C, the influence of the genetic heterogeneity of the hepatitis C virus (HCV) on the progression of liver disease and on the responsiveness to interferon therapy is a matter of controversy. In this study we evaluated the genetic complexity of HCV by single-strand conformation polymorphism (SSCP) analysis of amplicons from the hypervariable region 1 (HVR1) in 168 patients with chronic genotype 1b HCV infection, of whom 122 received a single course of interferon therapy (3 MU, three times weekly for 6 months). No correlation was observed between the degree of genetic complexity of HCV (indicated by the number of bands in the SSCP assay) and patient age, serum alanine aminotransferase activity, or serum HCV-RNA concentration, measured by competitive polymerase chain reaction. HCV genomic complexity was not related to gender nor to the presumed source of infection. The number of SSCP bands detected in serum samples from patients with chronic hepatitis, either mild (8.1 +/- 3.9), moderate (8.0 +/- 3.3), or severe (9.2 +/- 3.3), and in patients with liver cirrhosis, either compensated (8.0 +/- 2.9), decompensated (6.3 +/- 2.9), or with superimposed hepatocellular carcinoma (9.5 +/- 2.9), was similar. The number of SSCP bands detected in patients with sustained response (7.5 +/- 3. 9), transient response (8.3 +/- 2.9), or no response (8.2 +/- 3.6) to interferon administration was similar as well. These observations suggest that the genetic complexity of hypervariable region (HVR1) of HCV, as estimated by SSCP analysis, is not related to the severity of liver injury nor to the type of response to interferon therapy. Thus, information offered by SSCP analysis of HVR1 of HCV in chronic HCV genotype 1b infection does not appear to be useful in the clinical management of these patients. (HEPATOLOGY 1999;29:897-903.)

Genetic evolution of hepatitis G virus in chronically infected individual patients.

Comparative sequence analysis of different isolates of hepatitis G virus (HGV) has demonstrated significant intersubject genetic heterogeneity, but few data on intrasubject genetic evolution have been reported. To further investigate the genetic diversification of the HGV genome, 36 plasma samples from eleven patients chronically infected with HGV serially obtained 2-4 years apart were analysed. We determined the viral nucleotide sequence of the 5' non-coding (NC) and the NS3 regions by directly sequencing the RT-PCR amplified products obtained from the viral RNAs. Intrasubject sequence variation was found to be 1.3-2.4 x 10(-3) base substitutions per genome site per year within the 5' NC region and 1.3-9.4 x 10(-3) base substitutions per genome site per year within the NS3 region. Depending on the genomic region analysed (i.e. 5' NC or NS3 region), pairwise comparisons and phylogenetic reconstructions showed that intersubject genetic distances were 17.5- to 20.8-fold greater than intrasubject ones. Overall, the evolution rate of HGV in the regions analysed is not significantly different from that found in hepatitis C virus.

Prevalence and genotypes of GB virus C/hepatitis G virus (GBV-C/HGV) and hepatitis C virus among patients infected with human immunodeficiency virus: evidence of GBV-C/HGV sexual transmission.

The development of new antiretroviral agents may improve survival of HIV-infected individuals, and therefore chronic viral hepatitis may become more relevant in these patients. The presence of GBV-C/HGV and hepatitis C virus (HCV) RNA were investigated by reverse transcriptase-nested polymerase chain reaction in plasma from 168 Spanish HIV-infected patients belonging to four different risk groups: intravenous drug users (IVDUs), hemophiliacs, homosexuals, and heterosexuals. GBV-C/HGV-RNA and HCV-RNA were detected in 18% and 43% of the patients, respectively. The prevalence of current infection with these viruses was notably high, 19% for GBV-C/HGV and 69% for HCV, among individuals with parenteral risk of infection (intravenous drug abusers and hemophiliacs), but sexual transmission with GBV-C/HGV was also suggested because 16.5% of patients with sexual risk, either homosexual or heterosexual, had GBV-C/HGV-RNA in plasma. Although investigation of GBV-C/HGV-RNA possibly underestimates the actual prevalence of infection with GBV-C/HGV, the above data suggest that sexual contact may play a relevant role in the spread of this virus. Phylogenetic analysis showed no evidence for clustering of NS3 sequences into different genotypes or subtypes of GBV-C/HGV.