Influence of the polymorphism of the DUSP14 gene on the expression of immune-related genes and development of pulmonary tuberculosis.

Recently, a genome-wide screening identified a functional single-nucleotide polymorphism in dual-specificity phosphatase 14 gene (DUSP14), which was associated with pulmonary tuberculosis (TB) in a West African study. DUSP14 regulates T-cell proliferation and cytokine production in a negative way via dephosphorylation and inactivation of key signaling molecules. The aim of this study is to further explore the possible significance of the DUSP14 polymorphism. Total RNA was extracted from the whole blood of 109 healthcare workers (HCWs) in Vietnam and subjected to quantitative reverse-transcription PCR for DUSP14 and 20 immune-related genes. DUSP14 rs1051838 was genotyped in 502 new pulmonary TB patients and 506 healthy controls. Among disease-free individuals (HCWs), T-helper type-1 (Th1)-related genes, interferon-gamma receptor 2 (IFNGR2) and signal transducer and activator of transcription-1 (STAT1) mRNA levels significantly increased as the number of A alleles of rs1051838 increased, whereas the DUSP14 mRNA level tended to decrease. The AA genotype was associated with protection against active TB in younger patients (⩽45 years old, OR=0.63, 95% CI 0.44-0.90). Our results suggest that a low-expression genotype of DUSP14 accompanied by high transcript levels of Th1 immune-related genes may confer protection against early TB development.

Association of TLR polymorphisms with development of tuberculosis in Indonesian females.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis and is a major cause of morbidity and mortality worldwide. Many candidate genes have been investigated for a possible association with TB. Toll-like receptors (TLRs) are known to play important roles in human innate immune systems. Polymorphisms in and functions of TLRs have been investigated to identify associations with specific infectious diseases, including TB. Here, we examined whether single-nucleotide polymorphisms (SNPs) in TLRs and genes in TLR signaling were associated with TB susceptibility in Indonesian and Vietnamese populations. A statistically significant association was observed between TB susceptibility in a classified Indonesian female group and rs352139, an SNP located in the intron of TLR9, using the genotype (P = 2.76E-04) and recessive (AA vs AG+GG, P = 2.48E-04, odds ratio = 1.827, 95% confidence interval = 1.321-2.526) models. Meta-analysis of the Indonesian and Vietnamese populations showed that rs352139 was significantly associated with TB in the recessive model. This finding indicated that a TLR9 polymorphism might have an important role in the susceptibility to M. tuberculosis in Asian populations.

HLA-A, -B, -C, -DRB1 and -DQB1 alleles and haplotypes in the Kinh population in Vietnam.

Allele and haplotype frequencies of the human leukocyte antigens (HLA) were studied in the Kinh Vietnamese population. We analyzed 170 unrelated healthy individuals. DNA-based HLA typing was performed using a microsphere-based array genotyping platform with sequence-specific oligonucleotide probes to distinguish HLA-A, -B, -C, -DRB1 and -DQB1 alleles. A total of 21 HLA-A, 37 HLA-B, 18 HLA-C, 25 HLA-DRB1, and 14 HLA-DQB1 alleles were identified. HLA-A*1101, A*2402, A*3303, B*1502, B*4601, Cw*0102, Cw*0702, Cw*0801, DRB1*1202, DQB1*0301, DQB1*0303, and DQB1*0501 were found with frequencies higher than 10%. Two representative haplotypes bearing two to five HLA loci were A*1101-B*1502 and A*3303-B*5801 for HLA-A-B; Cw*0801-B*1502 and Cw*0102-B*4601 for HLA-C-B; B*1502-DRB1*1202 and B*4601-DRB1*0901 for HLA-B-DRB1; DRB1*1202-DQB1*0301 and DRB1*0901-DQB1*0303 for HLA-DRB1-DQB1; A*1101-Cw*0801-B*1502 and A*3303-Cw*0302-B*5801 for HLA-A-C-B; A*1101-B*1502-DRB1*1202 and A*2901-B*0705-DRB1*1001 for HLA-A-B-DRB1, A*1101-Cw*0801-B*1502-DRB1*1202-DQB1*0301 and A*2901-Cw*1505-B*0705-DRB1*1001-DQB1*0501 for HLA-A-C-B-DRB1-DQB1. Allele distribution and haplotype analysis demonstrated that the Vietnamese population shares HLA patterns with southern Chinese, Thai, Javanese and Micronesians, while it also retains unique characteristics.

Coiled-coil domain containing 85B suppresses the beta-catenin activity in a p53-dependent manner.

Aberrant accumulation of beta-catenin is closely related to carcinogenesis. Mutations in the p53 gene are reported to induce the aberrant accumulation of beta-catenin in the absence of dysfunction in the glycogen synthase kinase 3beta (GSK3beta)-mediated degradation pathway, but the mechanism remains incompletely understood. Here, we show that human coiled-coil domain containing 85B (CCDC85B) is induced by p53 and regulates beta-catenin activity via interaction with the T-cell factor 4 in the nucleus. Moreover, CCDC85B enhances the degradation of beta-catenin and suppresses tumor cell growth. In conclusion, we revealed that CCDC85B-induced degradation of beta-catenin is independent of GSK3beta and other p53-inducible products, Siah-1L, suggesting that CCDC85B constitutes the one of the frameworks of p53-induced multiple regulatory pathways for beta-catenin activity.

Pulmonary Mycobacterium avium complex infection associated with the IVS8-T5 allele of the CFTR gene.

BACKGROUND: The T5 allele in intron 8 (IVS8) on specific haplotype backgrounds (e.g., long TG repeats) causes abnormal splicing in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and is also known to be associated with chronic airway diseases. OBJECTIVE: To investigate the role of CFTR variations for susceptibility to pulmonary Mycobacterium avium complex (MAC) infection. PARTICIPANTS: Three hundred patients with pulmonary MAC infection (72 males, 228 females; mean age at onset 61.6 + or - 12.4 years) took part in this study. Diagnosis of MAC infection was based on American Thoracic Society criteria. Clinical profiles were collected and blood samples were genotyped for TG repeats, poly-T and M470V polymorphisms. RESULTS: We found significantly higher T5 frequency in MAC patients than in healthy controls from our own study (0.035 and 0.005, respectively, P = 0.023) and other reports. Homozygote for the T5 allele was found in two MAC patients. All T5 alleles were associated with longer TG repeats, the TG12 or TG13 allele. Seventeen of the 21 T5 alleles appeared to be associated with the V470 allele. Other polymorphisms did not show any significant differences in frequency. CONCLUSIONS: These findings suggest that the IVS8 5T allele might be involved in susceptibility to pulmonary MAC infection.

Detection of a 5' end subgenome of hepatitis C virus terminating at nucleotide 384 in patients' plasma and liver tissues.

Quadri and Negro [Dig Liver Dis 2001; 33: 480] reported greater distribution of 5' end genomic RNA of hepatitis C virus (HCV) over its 3' end in the liver of patients with recurrent hepatitis C after liver transplantation. We not only confirmed their results by quantifying the 5' end subgenomes in various specimens by using dilution and real-time polymerase chain reaction methods, but also discovered that such subgenomes terminated at nucleotide (nt) 384 of the viral genome or in its immediate upstream. The subgenomes in the plasma uniformly, with a few exceptions, ended at this position, while those in the liver more heterogeneously at various points upstream of nt 384. Subgenome populations ending some points in the downstream of nt 384 were not detected. The amount of the 5' end subgenome, while fluctuating during the clinical course of the patients, exceeded that of the longer sized HCV genomes which included the intact genome, and, when the relative ratio of the 5' end subgenome increased, the amount of longer sized HCV RNA tended to decrease, suggesting a suppressive effect of the 5' end subgenome on viral replication.

Overproduced p73alpha activates a minimal promoter through a mechanism independent of its transcriptional activity.

p73, the gene for a protein related to the tumor suppressor p53, encodes several variants which bear distinct carboxy-terminal structures as a result of alternative splicing. We and others showed that these splicing variants have different transcriptional effects on promoters with a p53-binding consensus sequence (p53BCS). Here we show that when transiently overexpressed, p73alpha but not p73beta activated several minimal promoters without the p53BCS, while p73gamma and p73epsilon activated them to a much lesser extent than p73alpha, and p53 suppressed the promoters without p53BCS as reported previously. Moreover, the results of RNase protection and RNA transfection assays suggested that this activation occurred at the transcriptional level. Deletion analysis of p73alpha revealed that the transactivation domain of p73 was not involved in this activity and the C-terminal region of p73alpha which is a specific structure of this variant was essential, suggesting that this phenomenon occurs independent of the transactivation activity of p73alpha and that the C-terminal extension of p73alpha may affect the basal level of transcription.

Single nucleotide polymorphisms of the mannose-binding lectin are associated with susceptibility to primary biliary cirrhosis.

Although the immunopathogenesis of primary biliary cirrhosis (PBC) remains unknown, familial clustering of patients with PBC suggests an important role for genetic factors. In addition, recent data support the thesis that the mucosal immune response against intraluminal pathogens may be involved with the onset of PBC. Mannose-binding lectin (MBL) is a key factor in innate mucosal defenses and has several key single nucleotide polymorphisms (SNPs). To study whether MBL gene SNPs are associated with susceptibility to PBC, we studied 65 patients with PBC and 218 controls by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequence specific priming-polymerase chain reaction (SSP-PCR) to examine four polymorphic loci: two (H/L and X/Y) within the promoter region and the other two (P/Q and A/B) within exon-1. We also analyzed serum MBL concentrations. Interestingly, the prevalence of haplotype HYPA, leading to hyper-production of MBL, as well as HYPA/HYPA genotype were significantly increased in PBC compared to controls (0.53 vs. 0.44, P=0.031; 33.9%vs. 17.0%, P=0.003, respectively). Furthermore, individuals homozygous for HYPA had a significantly increased risk for PBC (odds ratio (OR)=2.51, 95% confidence interval (CI)=1.34-4.66). Our results demonstrate that the MBL genotype can be significantly associated with increased risk for PBC, and further, that increased production of MBL plays a critical role in immunopathogenesis.

Cytoplasmic localization is important for transcription factor nuclear factor-kappa B activation by hepatitis C virus core protein through its amino terminal region.

We previously reported that hepatitis C virus core protein (core) activates the transcription factor nuclear factor-kappa B (NF-kappa B) when expressed transiently. In the present study, we investigated the relationship between the NF-kappa B activation capacity and subcellular localization of the core. By changing the subcellular localization of the C-terminally truncated core from the nucleus to the cytoplasm, NF-kappa B was activated. In addition, NF-kappa B activity was augmented by forcing the mutated core to move to the endoplasmic reticulum. It was also suggested that the region from aa 21 to 80 of the core is involved in the activation of NF-kappa B.

Regulation of Fas-mediated apoptosis by NF-kappaB activity in human hepatocyte derived cell lines.

Nuclear Factor-kappaB (NF-kappaB) is known to be one of the key regulators of genes involved in survival signaling. The purpose of this study is to investigate the role of NF-kappaB activity in signaling events related to cell survival in hepatocytes, which has been supposed to be one of the most sensitive organs against Fas-induced cytotoxicity. The functions of NF-kappaB activity on Fas-mediated apoptosis in different human cell lines were investigated by a magnetic concentration system for cells with exogenous protein production. We demonstrated that the activation of NF-kappaB was triggered by anti-Fas treatment in hepatocyte derived cell lines, HepG2 and Huh-7 cells. Overexpression of kinase-inactive NF-kappaB-inducing kinase (NIK) and IkappaB kinase (IKK) inhibited the activation of NF-kappaB introduced by anti-Fas treatment in these cells. Notably, inactivation of NF-kappaB by the production of IkappaB-alpha protein made these cells more susceptible to apoptosis induced by Fas stimulation. In contrast, transient expression of NIK showed a suppressive effect on Fas-mediated apoptosis in the same cell lines. These findings suggest the involvement of NF-kappaB in suppression of Fas-mediated apoptosis in human hepatocyte derived cell lines, in which concomitant activation of this transcriptional factor was observed through the stimulation of Fas signaling.

Transcriptional activities of p73 splicing variants are regulated by inter-variant association.

p73 has been identified as a gene that encodes a protein with significant identity with the tumour suppressor p53. The main structural difference between p73 and p53 is the additional C-terminal region of p73. Six isoforms of p73 with differing C-terminal structures, alpha, beta, gamma, delta, epsilon and xi, have been reported. These variants differ in transcriptional activity on p53-responsive promoters. Here we report a possible mechanism of transcriptional activation by p73 splicing variants. C-terminal deletion mutants of p73 alpha showed a significantly higher level of transcriptional activity than wild-type p73 alpha, suggesting that the C-terminal structure of p73 alpha functions to repress the transcriptional activity of p73 alpha. The results of immunoprecipitation assays and two-hybrid assays in mammalian cells showed that the p73 variants interacted with each other, but not with p53. The transcriptional activity of p73 beta was reduced by co-expression with either p73 alpha or p73 epsilon, which bears an identical C-terminal structure to p73 alpha. Co-expression of the C-terminal portion of p73 alpha or p73 epsilon with p73 beta also resulted in reduced transcriptional activity. Moreover, we observed that the level of endogenous p21 protein induced by p73 beta was decreased by co-expression of full-length p73 epsilon or the C-terminal region of p73 alpha or p73 epsilon. These observations suggest that p73-mediated gene expression is regulated by the interactions of p73 splicing variants in the cell.

p73beta, a variant of p73, enhances Wnt/beta-catenin signaling in Saos-2 cells.

The Wnt/beta-catenin pathway and p53 are very common targets for genetic alterations in colorectal cancer, and relationships between them have been reported. Here, we describe the relation between Wnt/beta-catenin signaling and the p53-related gene p73. p73, but not p53, activated a promoter containing the Tcf-binding sequence in Saos-2 cells, and the degree of activation was positively correlated with that on a p53-responsive promoter. Moreover, p73beta enhanced Wnt/beta-catenin signaling synergistically with Wnt-3a or exogenously expressed beta-catenin, unlike p53, and the enhancement was not caused by the accumulation of beta-catenin. These results show that the effects of p73 on Wnt/beta-catenin signaling differ from those of p53.

Hepatitis C virus (HCV) genotype 1b sequences from fifteen patients with hepatocellular carcinoma: the 'progression score' revisited.

The genome of hepatitis C virus (HCV) associated with hepatocellular carcinoma (HCC) may have some characteristics which would barely be found in those of HCV from asymptomatic carriers (ASC). We analyzed 15 HCC patients who were infected with HCV genotype 1b (HCV-1b) for complete nucleotide sequences of the viral genomes. Of the 15 isolates, three were sequenced up to the first nucleotide of the 5'UTR, and six were sequenced to encompass the X-tail at the 3' end: sequencing of at least three-quarters of the 5'UTR and entire polyprotein-ORF was accomplished in all 15 isolates. Analyses of these sequences together with those reported previously by Nagayama et al. [Hepatology; 31 (2000) 745] suggested that nine residues (nt 119 of 5'UTR and aa 90, 434, 938, 962, 1176, 1412, 2143, and 2774 of polyprotein) might be useful to discriminate HCC-type sequences from ASC-type ones. The 'progression score' was 1.4+/-0.9 in ASC versus 3.7+/-1.5 in HCC (P=3.87E-07) when calculated with the Nagayama et al.'s seven residues, but was 1.4+/-0.6 versus 4.6+/-1.9 (P=1.33E-09) with our nine residues: a greater difference between HCC and ASC was achieved in the latter system. Further analyses, by increasing the sample size and/or by extending the comparison to include entire 5'UTR and 3'UTR/X-tail, may thus contribute to define the 'progression score' more appropriately.

Hepatitis C virus core protein enhances the activation of the transcription factor, Elk1, in response to mitogenic stimuli.

Mitogen-activated protein kinase (MAPK) pathways play key roles in cell proliferation, transformation of mammalian cells, and the stress response. We and other investigators showed that hepatitis C virus (HCV) core protein has an oncogenic potential, but its mechanism has remained unknown. We previously demonstrated that the MAPK-extra-cellular signal-regulated kinase (ERK) kinase (MEK)-ERK pathway and its downstream target, the serum response element (SRE), is activated in BALB/3T3 cells producing HCV core protein. To elucidate the precise mechanism by which HCV core protein activates the MEK-ERK pathway, we transiently expressed HCV core protein in several cell lines and studied the signal transduction of the pathway, using Gal4-Elk1 luciferase assay, in vitro kinas assay of MAPK, and Western blotting analysis. We discovered that, in the presence of mitogenic signal, HCV core protein enhanced Elk1 activation working downstream of MEK without affecting ERK activity and Elk1 phosphorylation. Our data suggest that HCV core protein may activate Elk1 through a pathway alternative to the typical phosphorylation cascade. These findings might give new insights into the role of HCV in hepatocarcinogenesis.

Genetic polymorphism of the MxA gene promoter and interferon responsiveness of hepatitis C patients: revisited by analyzing two SNP sites (-123 and -88) in vivo and in vitro.

We have previously reported a single nucleotide polymorphism (SNP) at nucleotide (nt) position -88 (G or T) within an interferon-stimulated response element-like sequence in the promoter region of the MxA gene, which correlated with responsiveness of hepatitis C patients to interferon. Upstream of it, we then identified another SNP (C or A at nt -123) and investigated whether this SNP also correlates with interferon responsiveness. The two SNPs showed a high linkage to each other: all the individuals having G at -88 had C at -123, and 73% of those having T at -88 had A at -123. As was expected from this observation, the SNP at -123 also exhibited a correlation with interferon responsiveness (C/C homozygotes were more frequent among nonresponders than among responders: 65% of 107 vs. 40% of 52, p = 0.0028). These in vivo data from patients were further supported by results from in vitro experiments. The MxA promoter sequence with A at -123 and T at -88 showed about 4-fold higher activity of upregulating the downstream reporter gene than that with C at -123 and G at -88, in a luciferase reporter assay.

Effects of mutation in hepatitis C virus nonstructural protein 5A on interferon resistance mediated by inhibition of PKR kinase activity in mammalian cells.

The IFN-induced double-stranded RNA (dsRNA)-activated protein kinase PKR is one of the key molecules in the antiviral effects of IFN. To clarify the effects of hepatitis C virus nonstructural protein 5A (NS5A) on antiviral activity of IFN, in particular on PKR kinase activity, in mammalian cells, we established inducible NS5A-expressing cell lines derived from human osteosarcoma (Saos-2). The cells expressing NS5A derived from an IFN-resistant clone (NS5A-lb) that interacted with endogenous PKR in vitro, showed a suppressive effect on IFN function as determined by interference with vesicular stomatitis virus (VSV) infection, whereas NS5A (NS5A-2a) from an IFN-sensitive clone did not block the antiviral effect of IFN. A mutant with deletion of the IFN sensitivity determining region (ISDR) in NS5A-1b (NS5A-AISDR) also interacted with PKR and suppressed its activity in vitro. However, neither NS5A-2a nor the C-terminal truncated mutant of NS5A-1b (NS5A-deltaC) blocked PKR activity. These observations confirmed the previous report that the inhibitory effect of NS5A on IFN activity is mediated at least in part by the repression of PKR. In addition, we showed that IFN sensitivity was determined not only by the ISDR but that the involvement of the C-terminal region of NS5A-1b is important for the suppression of PKR activity.

Full or near full length nucleotide sequences of TT virus variants (Types SANBAN and YONBAN) and the TT virus-like mini virus.

TT virus (TTV) is a common virus and consists of many genotypes and variants. In addition, there exists a virus which both differs greatly from and retains a considerable resemblance to TTV, such as the TTV-like mini virus (TLMV) as we reported previously. Here we report the near full length genomic sequences of 4 isolates of a new variant of TTV (designated YONBAN) along with the full length sequences of 2 isolates of the TTV-SANBAN lineage and 7 isolates of the TLMV species derived from human sera. The TTV-YONBAN sequences showed only about 50% identity at the nucleotide level to those of the prototype TTV (TA278) and to SANBAN, and even less to TLMV. Moreover, the ORF1 of YONBAN lacked the ATG initiation codon which is shared by all the TTV and TLMV isolates so far identified in humans; instead, YONBAN had a Kozak's rule-compatible ACG codon as the candidate initiation site for the ORF1 translation. Nevertheless, the overall genetic structure and the conserved amino acid motifs within the ORF1 and the ORF2 were well shared among the prototype TTV strains, the SANBAN and YONBAN variants, and TLMV. The most conserved nucleotide sequence was found in the noncoding region just upstream from the ORF2, allowing construction of a phylogenetic tree which implied that the TTV genotypes and variants, the TLMV, and chicken anemia virus could be coclassified under a superfamily for which we proposed the name of 'Paracircoviridae' in our previous report.

Identification of a single nucleotide polymorphism in the MxA gene promoter (G/T at nt -88) correlated with the response of hepatitis C patients to interferon.

The interferon (IFN)-inducible MxA protein is known to play an important role in the host defense against certain viruses. We aimed to see if any genetic polymorphism in the promoter region of the MxA gene is associated with the IFN responsiveness of hepatitis C virus (HCV)-infected patients. Initially we sequenced the promoter region of the MxA gene in 12 subjects and found a polymorphic site. We then constructed a specific PCR-RFLP system for this site and subjected 63 samples from chronic hepatitis C patients who were nonresponders (NR) to IFN therapy to it, 52 with sustained response (SR), and 42 healthy controls. Subjects were all Japanese, and unrelated. A single nucleotide polymorphism (SNP) was identified in the MxA promoter region: G/T alleles at nt position -88. Interestingly, this SNP was involved in a genetic element highly homologous to the IFN-stimulated response element consensus sequence, and the G-to-T change there makes this homology a little greater. The rate of G.G homozygosity was 31% in the SR patients, significantly lower than in the NR patients (62%, p = 0.0009), while that of healthy controls was between the two groups (48%). Differences in HCV genotypes did not influence this result. Based on these findings, we propose that the SNP of the MxA promoter at nt -88 identified in this study affects the expression of MxA protein, and may thus be associated with the response of HCV patients to IFN.