Ingorn Kimkong

Tumour necrosis factor-alpha gene polymorphisms and susceptibility to oral lichen planus

OBJECTIVE  This study is aimed to investigate the association between OLP susceptibility and clinical type in the Thai population and three polymorphisms within the promoter region of the TNF-α at positions -863, -308 and -238 which have putative functional significances. MATERIALS AND METHODS  Genomic DNA from 75 Thai patients with OLP and 154 healthy controls were genotyped for TNF-α polymorphisms-- -863(rs1800630), -308(rs1800629), and -238(rs361525)--using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS  We found a higher proportion of TNF-alpha-308 AA genotype (high producer genotype) among OLP patients (5/75; 6.67%) when compared to healthy controls (1/154; 0.65%; OR = 10.93; 95% CI = 1.21-251.9). For other polymorphisms (-863 and -238), we did not find any significant association with OLP development; this was also the case with haplotype analysis (-863/-308/-238). CONCLUSION  TNF-α-308AA may play a relevant role in the susceptibility and severity of OLP in the Thai population. However, further investigation of this study is needed.

Association of interferon-gamma gene polymorphisms with susceptibility to oral lichen planus in the Thai population

OBJECTIVE Interferon-gamma (IFN-γ) is highly expressed in oral lichen planus (OLP). The IFN-γ (+874 in intron 1, rs2430561) TT genotype, which has been reported to be associated with high IFN-γ production, was hypothesized to be associated with susceptibility to OLP in the Thai population. DESIGN Genomic DNA samples from 74 OLP and 268 healthy controls were evaluated for IFN-γ polymorphisms by polymerase chain reaction with sequence-specific primers (PCR-SSP) and direct sequencing method. RESULTS The T allele was significantly associated with an increased risk of OLP development as compared to the A allele (OR=1.76, P=0.004; P(c)=0.02). The effect of the T allele was similar to an autosomal recessive disorder; the presence of TT genotype (when compared to AA and AT) conferred an OR of 2.61, P=0.008; P(c)=0.04. CONCLUSIONS We found an association between IFN-γ +874A/T polymorphism and susceptibility to OLP. However, an association study utilising a larger sample size and patients from other races apart from the Asian population should be performed to further verify these findings.

PAAQD: Predicting immunogenicity of MHC class I binding peptides using amino acid pairwise contact potentials and quantum topological molecular similarity descriptors.

Prediction of peptide immunogenicity is a promising approach for novel vaccine discovery. Conventionally, epitope prediction methods have been developed to accelerate the process of vaccine production by searching for candidate peptides from pathogenic proteins. However, recent studies revealed that peptides with high binding affinity to major histocompatibility complex molecules (MHCs) do not always result in high immunogenicity. Therefore, it is promising to predict the peptide immunogenicity rather than epitopes in order to discover new vaccines more effectively. To this end, we developed a novel T-cell reactivity predictor which we call PAAQD. Nonapeptides were encoded numerically, using combining information of amino acid pairwise contact potentials (AAPPs) and quantum topological molecular similarity (QTMS) descriptors. Encoded data were used in the construction of our classification model. Our numerical experiments suggested that the predictive performance of PAAQD is at least comparable with POPISK, one of the pioneering techniques for T-cell reactivity prediction. Also, our experiment suggested that the first and eighth positions of nonapeptides are the most important for immunogenicity and most of the anchor residues in epitope prediction were not important in T-cell reactivity prediction. The R implementation of PAAQD is available at http://pirun.ku.ac.th/~fsciiok/PAAQD.rar.

Association of interferon-alpha gene polymorphisms with chronic hepatitis B virus infection

In this study, the association between the risk of chronic hepatitis B virus infection and the polymorphisms within promoter regions of IFN‐α1 and five genes was explored. This association study was performed on 180 Thai patients with chronic HBV infection [hepatocellular carcinoma (HCC) = 65 and non‐HCC = 115], 173 individuals with self‐limited HBV infection and 140 healthy controls. Our results showed that the A allele of ‐1823G/A SNP within IFNA1 gene was significantly associated with an increased risk of chronic HBV infection as compared to healthy individuals and self‐limited HBV group [OR (95% CI) = 2.20 (1.51–3.19), P = 0.000014 and OR (95% CI) = 1.61 (1.12–2.33), P = 0.0073, respectively]. The effect of A allele was similar to autosomal recessive in which the presence of AA genotype when compared to GG and GA conferred the OR of 2.79 (95% CI = 1.72–4.52, P = 0.0000085). By multifactor dimensionality reduction analysis, we found the interaction between IFNA5 (‐2529) and IFNA1 (‐1823) genes that gave the risk to chronic HBV infection, with the OR (95% CI) of the high‐risk to low‐risk group was 2.79 (1.77–4.40), P < 0.0001. However, further study in functional significance is required.

Association of IFI200 Gene Polymorphisms with Susceptibility to Systemic Lupus Erythematosus

To the Editor: The MNDA (myeloid nuclear differentiation antigen), IFIX (interferon-inducible protein X), IFI16 (interferon-inducible protein 16), and AIM2 (absent in melanoma 2) are a group of interferon (IFN)-inducible genes whose products belong to the gene family denoted hematopoietic interferon-inducible protein with 200 amino acid repeat (IFI200). These genes map on chromosome 1q21–23, which is the major susceptibility locus of systemic lupus erythematosus (SLE). They are proposed as new candidate genes for SLE susceptibility for several reasons: (1) genetic mapping from a murine model of lupus1,2; (2) upregulation of all 4 genes in patients with SLE3; (3) the role of IFI16 as autoantigen in patients with SLE4,5; (4) the ability of IFI16 to bind single-strand DNA in a process of DNA repair6; and (5) the discovery of AIM2 as an intracellular DNA sensor leading to inflammation and apoptosis7. Therefore, IFI200 might be associated with abnormal inflammation and loss of tolerance to dsDNA observed in patients with SLE. We recruited 200 SLE patients (194 women, 6 men; mean age 36.21 ± SD 10.76 yrs) from King Chulalongkorn Memorial Hospital, Bangkok, each having at least 4 of the American College of Rheumatology revised criteria for SLE8, and 200 ethnically matched healthy volunteer blood donors from the Thai Red Cross Society (147 women, 53 men; mean age 23 ± SD 12.3 yrs)9. The study was approved by the ethics committee of the Faculty of Medicine, Chulalongkorn University, and all subjects gave their informed consent. Within 60 minutes after blood draw, peripheral blood mononuclear cells (PBMC) from 15 healthy donors containing different IFI16 genotypes were isolated by Ficoll-Hypaque gradient (Robbins Scientific, Sunnyvale, CA, USA) and resuspended in RPMI-1640 medium (Sigma, New York, NY, USA) with 10% … Address correspondence to Dr. Hirankarn; E-mail: fmednpt{at}md.chula.ac.th

EpicCapo: epitope prediction using combined information of amino acid pairwise contact potentials and HLA-peptide contact site information

BackgroundEpitope identification is an essential step toward synthetic vaccine development since epitopes play an important role in activating immune response. Classical experimental approaches are laborious and time-consuming, and therefore computational methods for generating epitope candidates have been actively studied. Most of these methods, however, are based on sophisticated nonlinear techniques for achieving higher predictive performance. The use of these techniques tend to diminish their interpretability with respect to binding potential: that is, they do not provide much insight into binding mechanisms.ResultsWe have developed a novel epitope prediction method named EpicCapo and its variants, EpicCapo+ and EpicCapo+REF. Nonapeptides were encoded numerically using a novel peptide-encoding scheme for machine learning algorithms by utilizing 40 amino acid pairwise contact potentials (referred to as AAPPs throughout this paper). The predictive performances of EpicCapo+ and EpicCapo+REF outperformed other state-of-the-art methods without losing interpretability. Interestingly, the most informative AAPPs estimated by our study were those developed by Micheletti and Simons while previous studies utilized two AAPPs developed by Miyazawa & Jernigan and Betancourt & Thirumalai. In addition, we found that all amino acid positions in nonapeptides could effect on performances of the predictive models including non-anchor positions. Finally, EpicCapo+REF was applied to identify candidates of promiscuous epitopes. As a result, 67.1% of the predicted nonapeptides epitopes were consistent with preceding studies based on immunological experiments.ConclusionsOur method achieved high performance in testing with benchmark datasets. In addition, our study identified a number of candidates of promiscuous CTL epitopes consistent with previously reported immunological experiments. We speculate that our techniques may be useful in the development of new vaccines. The R implementation of EpicCapo+REF is available athttp://pirun.ku.ac.th/~fsciiok/EpicCapoREF.zip. Datasets are available athttp://pirun.ku.ac.th/~fsciiok/Datasets.zip.

Increased ATG5-ATG12 in hepatitis B virus-associated hepatocellular carcinoma and their role in apoptosis

AIM To investigate autophagy-related genes, particularly ATG12, in apoptosis and cell cycle in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) and non-HBV-HCC cell lines. METHODS The expression of autophagy-related genes in HBV-associated hepatocellular carcinoma and non-HBV-HCC cell lines and human liver tissues was examined by quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) and western blotting. The silencing of target genes was used to examine the function of various genes in apoptosis and cell cycle progression. RESULTS The expression of autophagy related genes ATG5, ATG12, ATG9A and ATG4B expression was analyzed in HepG2.2.15 cells and compared with HepG2 and THLE cells. We found that ATG5 and ATG12 mRNA expression was significantly increased in HepG2.2.15 cells compared to HepG2 cells (P < 0.005). Moreover, ATG5-ATG12 protein levels were increased in tumor liver tissues compared to adjacent non-tumor tissues mainly from HCC patients with HBV infection. We also analyzed the function of ATG12 in cell apoptosis and cell cycle progression. The percentage of apoptotic cells increased by 11.4% in ATG12-silenced HepG2.2.15 cells (P < 0.005) but did not change in ATG12-silenced HepG2 cells under starvation with Earle’s balanced salt solution. However, the combination blockade of Notch signaling and ATG12 decreased the apoptotic rate of HepG2.2.15 cells from 55.6% to 50.4% (P < 0.05). CONCLUSION ATG12 is important for HBV-associated apoptosis and a potential drug target for HBV-HCC. Combination inhibition of ATG12/Notch signaling had no additional effect on HepG2.2.15 apoptosis.

Autophagy machinery impaired interferon signalling pathways to benefit hepatitis B virus replication.

BACKGROUND Autophagy-related genes ATG4B, ATG7, and ATG12 have been identified to play a critical role in viral replication. However, these genes have yet to be identified in hepatitis B virus (HBV). OBJECTIVE To characterise the role of ATG4B, ATG7, and ATG12 genes in HBV infection. METHODS The mRNA expression was examined by quantitative real-time RT-PCR and Western blotting. Short hairpin RNA (shRNA) of the target gene was used to examine the function of the gene in HBV replication. Evaluation of HBV DNA level was performed by real-time PCR. RESULTS Our findings revealed that ATG12 gene expression was significantly up-regulated (p < 0.005), whereas ATG7 gene expression was down-regulated (p < 0.0001) in HepG2.2.15 cells when compared to HepG2 cells. However, no significant difference in mRNA level of ATG4B was observed. These results were consistent with protein level findings. Moreover, we analysed the function of ATG12 in HBV replication by using ATG12 shRNA and evaluated HBV DNA level. We found that the amount of HBV was decreased in ATG12-knockdown HepG2.2.15 cells when compared to control HepG2.2.15 cells (P < 0.05). The mRNA expression of interferon-alpha (IFN-α), interferon-beta (IFN-β), and interferon-inducible genes (IFI) was also investigated. Our results showed that the expression of IFN-α, IFN-β, and IFI27 genes were increased in ATG12-knockdown cells but not in Mx1 gene when compared to control cells (p < 0.005, p < 0.0001 and p < 0.005, respectively). CONCLUSION These autophagy-related genes, ATG12 may play a role in HBV replication via impairing IFN pathway. However, the biological significance of other autophagic genes such as ATG7 warrants further study.

Gene polymorphisms of interleukin 28B and the risk to chronic hepatitis B virus infection in Thai

In this study, we aimed to evaluate the effect of two single nucleotide polymorphisms (SNPs) of interleukin 28B (IL28B) (rs12979860C/T and rs8099917G/T) on chronic hepatitis B virus (CHB) infection in Thai population. We studied 375 subjects: 83 CHB with hepatocellular carcinoma (HCC) patients, 128 CHB without HCC and 164 individuals with self-limited HBV infection, by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and TaqMan allelic discrimination assay. Results revealed significant risk of IL28B rs8099917T allele associated with CHB without HCC compared with self-limited HBV [odds ratio (OR) (95% confidence interval (CI)) = 2.56 (1.08-6.28), P = 0.019]. The effect of this T allele was similar to that of an autosomal recessive gene in the presence of TT genotype compared with GG and GT genotype [OR (95% CI) = 2.70 (1.11-6.77), P = 0.016, P (logistic regression) = 0.048]. The two locus haplotype analysis of the two IL28B SNP loci did not show any association with CHB (P > 0.05). In conclusion, these results suggested a IL28B rs8099917T allele predispose for susceptibility to chronic HBV infection but not leading to HCC in Thai population.

Association of IFNAR2 and IL10RB genes in chronic hepatitis B virus infection.

In this study, we investigated the effects of two functional polymorphisms, type I interferon receptor 2 gene (IFNAR2)-F8S and interleukin-10 receptor subunit beta gene (IL10RB)-K47E, on chronic hepatitis B virus (HBV) infection. We included 227 Thai patients with chronic HBV infection [100 with hepatocellular carcinoma (HCC) and 127 non-HCC], 170 individuals with self-limited HBV infection and 150 healthy controls. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to analyze these two single nucleotide polymorphisms (SNPs). In this study, the C allele of IFNAR2-F8S was found to be significantly increased in chronic HBV patients when compared with healthy controls [odds ratio, OR (95% confidence interval, CI)= 3.31 (2.11-5.21), P = 6.214 × 10(-9) and corrected P-value, P(c)= 1.864 × 10(-8)]. The effect of this allele was similar to that of an autosomal dominant gene in the presence of CC and CT genotype, when compared to TT with an OR of 4.02 (P = 4.631 × 10(-9) and P(c)= 1.389 × 10(-8)). Furthermore, AA genotype of IL10RB-K47E was found to be significantly decreased in chronic HBV patients compared with individuals with self-limited HBV infection (P = 0.006, P(c)=  0.018 and OR = 0.45). For haplotype analysis, we found CA and CG haplotypes were associated with susceptibility to chronic HBV (P = 0.014, OR = 6.84 and P = 0.002, OR = 3.75, respectively) when compared with healthy individuals. This study suggests that IFNAR2-F8S polymorphisms might be involved in the susceptibility to chronic HBV infection. Moreover, AA genotype of IL10RB-K47E may provide a protective effect in this disease. However, an association study using a larger sample size should be performed to confirm these findings.