Relationship between precore/basal core promoter mutations of hepatitis B virus and therapeutic effect of peginterferon α-2b,and changes of mutations before and after treatment

AIM:To investigate the relationship between therapeutic effect of peginterferon α-2b (Peg-IFNα-2b) and precore (PC) region G1896A and basal core promoter (BCP) region A1762T/G1764A mutations of hepatitis B virus (HBV), and the changes of the mutations before and after treatment. METHODS:The patients with HBeAg-positive chronic hepatitis B (CHB) (n=69) were treated with Peg-IFNα-2b for 48 weeks and followed up for 24 weeks. The PC and BCP sequences at baseline and the 72th week were determined using polymerase chain reaction (PCR) and direct sequencing. Serum HBsAg, HBeAg, alanine aminotransferase (ALT) and HBV DNA was quantified in the samples taken at baseline (week 0), during the treatment period (weeks 4, 8, 12, 24, 36 and 48), and during follow-up (weeks 60 and 72). RESULTS:Within the total cohort, wild-type (WT) virus was detectable in only 14 patients (20.29%), and mutants were detected in 55 patients (79.71%). The serum HBeAg level in the patients with mutant virus was significantly lower than that in the patients with WT virus (P=0.024). The proportion of WT, PC mutant, BCP mutant and PC+BCP mutant was significantly changed at baseline and week 72 (P=0.004). No significant difference of HBeAg seroconversion and combined response between patients with WT virus or mutants (PC, BCP and PC+BCP) was observed. CONCLUSION:PC and BCP mutations have no effect on the response of HBeAg-positive CHB patients to Peg-IFNα-2b. The proportion of each mutation was significantly changed before and after treatment.     

Study on mutation of mitochondrial gene from rat breast cancer

AIM: To know the variations of the cytochrome b gene in cancer tissue, paracarinoma tissue and normal tissue and to inquire into the relationship between mutations of mitochondrial genome and carcinogenesis. METHODS: Cellular total DNA was extracted.The cytochrome b genes of three tissues were amplifyed with polymerase chain reaction(PCR). PCR products were analysed by DNA auto-sequencing method. RESULTS: The cytochrome b gene of cancer tissue had the C to G mutation at nt 14931, the C to G mutation at nt 15004 and the T to C mutation at nt15435,respectively. The cytochrome b gene of paracarinoma tissue had the A to C mutation at nt 15436. The cytochrome b gene of normal tissue had not mutation. CONCLUSION:Mitochondrial DNA mutations could be the endogenous factors that induce nuclear genome mutation. It could promoto carcinogenesis. The paracarinoma tissue was abnormal in DNA molecular level.     

Paired study on hepatitis B virus S gene mutation in immunoprophylaxis failure to prevent HBV vertical transmission

AIM：To explore the characteristics of hepatitis B virus S gene mutation in the vertical transmission after active and passive vaccination. METHODS：Fifteen cases of immunoprophylaxis failure were enrolled in the study. HBV S gene (including pres-S and S) from the mothers, newborns before active and passive vaccination and 7-month-old infants with immunoprophylaxis failure were detected by PCR amplification. The characteristics of HBV S gene mutation were compared among the 3 groups. RESULTS：The genotype of HBV in the newborns and the infants was the same as that in the mothers. The frequencies of mutation in the 2 fragments of the HBV S gene had no significant difference between the 3 groups. The homology tree model based on HBV S gene was analyzed in the 3 groups, in which every group had their own cluster. There were 15 different mutation sites between 7 pairs of mothers and newborns. There were 3 different mutation sites between 3 pairs of newborns and infants (nt273A→A/G, nt512C→C/T and nt1139C→A), among which the first 2 were located in the S gene region but not in the “a” determinant, and the latter was located in the overlap region of S and X genes. There were 25 different mutation sites between 9 pairs of mothers and infants, but only 1 case had a different mutation site between the mother, newborn and infant. CONCLUSION：The HBV species in newborns and infants with immunoprophylaxis failure were transmitted from the mothers. The mutations in the HBV S gene with immunoprophylaxis failure happened before and after active and passive vaccination, mainly before vaccination. The relationship between HBV S gene mutations and immunoprophylaxis failure should be further explored.     

Relationship between HBV genotype,PreS/S gene mutation and immunoprophylaxis failure to prevent HBV mother-to-child transmission

AIM：To investigate the relationship between hepatitis B virus (HBV) genotype, PreS/S gene mutation and immunoprophylaxis failure to prevent HBV mother-to-child transmission.
METHODS：Pregnant women with positive HBV surface antigen (HBsAg) and HBV DNA≥1×1010 IU/L were divided into case group (15 cases) and control group (45 cases) according to their neonates with immunoprophylaxis failure or not. The genotypes of HBV and the mutation rate and mutational hot spots in PreS/S gene were detected by PCR amplification technique in the two groups.
RESULTS：(1) Genotypes B and C of HBV were detected in both case and control groups, and the majority of HBV genotype was B in the two groups. Genotype distribution difference between case and control groups was not statistically significant (P>0.05). (2) There was no significant difference in the mutation rate of PreS/S gene between case and control groups (P>0.05). The mutation rates of PreS/S gene between genotypes B and C were significantly different (P<0.05), but when the HBV genotype was the same, the mutation rate of PreS/S gene had no significant difference between case and control groups. Homology tree model based on PreS2/S gene formed genotype B and genotype C clusters, and in each cluster, the sequences of case and control groups did not formed smaller different clusters further. (3) 529G-A, 530A-G, 826A-G1 and 166het-dupC were hot spots of mutation in PreS2/S gene and were found in 4 cases in case group, respectively. A530T (1 case), A530G (2 cases), T531C (3 cases) were found in control group.
CONCLUSION：(1) The mutation rates of PreS/S gene are different in various genotypes. (2) The mutation in PreS/S gene of HBV is prevalent, but not all of the mutations are related to immunoprophylaxis failure to prevent HBV mother-to-child transmission. To find mutational hot spots which are related to immunoprophylaxis failure is more important.     

Mitochondrial DNA 8 point mutations in patients with type II diabetes mellitus

AIM: To explore the relationship between various mitochondrial (mt) DNA tRNA Leu (UUR) and ND1 gene mutations and type 2 diabetes mellitus (T2DM) among Chinese in Hubei Province. METHODS: PCR restriction fragment length polymorphism (PCR-RFLP) analysis was used to screen point mutations of mtDNA (3 243, 3 256, 3 290, 3 316, 3 394, 3 421, 3 426, 3 460, 3 593) in 174 T2DM and 207 healthy controls. Then, DNA sequencing, reverse dot blot hybridization and Genchip were used to compare and confirm mutations. All mutations were analyzed by DNASTAR and Antherprot softwares. RESULTS: In diabetic group, there were 5 carriers (2.9%) of 3 316 G→A (Ala→Thr) mutation, 4 (2.3%) of 3 394 T→C (Tyr→His) mutation, 1 (0.6%) of 3 593 T→C(Val→Ala) mutation, and 1 (0.6%) of 3 618 T→C(Phe→Phe) mutation. Among 3 316 (G→A) mutations , there were more than 1 point mutations in 2 cases, one accompanied with 3 256 C→T(Arg→Arg) and 3 688 G→C (Ala→Pro) mutations, another accompanied with 3 606 A→G(Leu→Leu) mutation. 3 606 (A→G), 3 618 (T→C) and 3 688 (G→C) were novel mutations, GenBank accession number is DQ092356. In controls, only 3 316 (G→A) mutation was found in 1 subject (0.5%). There was significant difference between two groups for 3 394 (T→C) mutation frequencies (P<0.05). CONCLUSION: mtDNA ND1 gene mutations at np 3 316 and np 3 688, np 3 394 might contribute to the pathogenesis of T2DM with other genetic and environmental factors.     

Association between IL-6-572C/G as well as IFNAR1-168G/C and hepatitis B virus infection in populations of Dai and Han ethnicities in Yunnan Province

ATM: To explore the association between IL-6-572C/G (rs1800796) as well as interferon alpha receptor 1 (IFNAR1)-168G/C (rs2257167) and prognosis after hepatitis B virus (HBV) infection in populations of Dai and Han ethnicities in Yunnan Province. METHODS: The blood samples were collected from Dai people and Han people, each nation including 100 healthy controls and 200 infected individuals (100 spontaneous recovery individuals and 100 chronic patients). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing were used to identify the gene type. RESULTS: In Dai people, no significant difference was found between genetic polymorphism of -572C/G and prognosis after HBV infection. The differences of C and G alleles between spontaneous recovery group and chronic hepatitis B group, and healthy controls and HBV infection group were not statistically significant. Meanwhile, GG and CG genotypes were a vital protective factor for the person who developed into a chronic heptatitis B patient under the G allele dominance mode (GG+CG/CC) (P<0.05). In Han people, no statistically significance for IL-6-572C/G genotype and allele distribution in each group comparisons had been found, as well as the C allele recessive mode and C allele dominance mode. For the above 4 indicators, no statistically significant difference of IFNAR1-168C/G in Dai and Han people had been found.CONCLUSION: The GG+CG genotype of IL-6-572C/G may be a protective factor for the HBV-infected Dai people to develop into chronic hepatitis B patients. However, there is no significant association between the IFNAR1-168G/C polymorphism and prognosis after HBV infection in the 2 ethnicities.     

Relationship between RUNX3 gene 364 locus C→T mutation and gastric cancer in Chinese

AIM: To study the frequency difference of RUNX3 gene 364 locus C→T mutation between normal people(controls) and gastric cancer (GC) patients, and mutation in gastric mucosa of subjects with H.pylori infection. METHODS: Genomic DNA was extracted from peripheral blood and gastric mucosal biopsy specimens of normal people and GC patients in lower or higher prevalence region. Gene mutation was analyzed by PCR-RFLP. RESULTS: The frequency of RUNX3 T/T genotype was no significant difference between controls and GC in lower (χ2=0.57, P>0.05) or higher prevalence region (χ2=0.16, P>0.05). A higher mutation rate in mucosal tissue infected with H.pylori was not discovered. CONCLUSION: RUNX3 gene C364T mutation may be not a genetic susceptibility to GC in Chinese. The mutation is impossibly involved in the pathway of H.pylori infection resulting in gastric carcinoma.     

Association between asthenospermia and mtDNA mutations in ND3 and ND4L genes

AIM: To explore the molecular mechanism of asthenospermia(AST) by preliminary screening of nucleotide sequences from the ND3 and ND4L genes of mitochondrial DNA(mtDNA). METHODS: Samples from 50 AST patients and 42 age-matched normal controls were collected according to the WHO criteria. Density gradient centrifugation was applied to separate spermatozoa with different vigor. The ND3 and ND4L genes of mtDNA were amplified and sequenced directly from the extracted genomic DNA from AST patients and normal controls. The sequences were compared with revised Cambridge Reference Sequence(rCRS) to analyze the variants. RESULTS: A total of 22 nucleotide variations were found in ND3 and ND4L genes of mtDNA in asthenospermia group and control group. G10320A, A10398G and T10609C were missense mutations, while A10157G and A10313C were the reported for the first time in this study. Haplotype N in patients with AST(33/50) was higher than that in control group(14/42, P<0.05), and haplotype R9 in patients with AST(15/50) was also higher than that in control group(4/42, P<0.05) through genetic testing of ND3 gene. Rates of sperm progressive motility of haplotype F1, F2 and R9 were significantly lower than those of haplotype M and M rest. Two haplotype differences, haplotype M and N, were found in the same AST patient’s spermatozoas which had different vigor. Haplotype M had stronger vigor, while haplotype N had lower vigor. By sequencing ND3 gene of mtDNA from 50 AST patients, we detected G10310A heteroplasmic mutation in 2 specimens of asthenospermia with poor and moderate motility spermatozoa, respectively. No mutation occurred in good motility spermatozoa. CONCLUSION: Haplotype of mitochondrial may have some correlation with sperm motility. The nt10398G-10400T polymorphisms may have benefit for sperm motility, whereas the mutation in nt10310A may impair sperm motility.     

Application of PCR-HRM in the study of relationship between leptin gene promoter polymorphisms and liver cirrhosis

AIM: To analyze the feasibility of PCR-high-resolution melting curve analysis (HRM) for detecting the site mutations of C2549 and G2548 in leptin gene promoter from the patients with liver cirrhosis, and to explore the relevance between mutant genotypes and physiological and biochemical indexes in liver cirrhosis patients. METHODS: Compared with the method of PCR-restriction fragment length polymorphism (RFLP), the present research used the method of PCR-HRM to analyze the site mutations of C2549 and G2548 in leptin gene promoter in control group (n=100) and liver cirrhosis group (n=100). The physiological and biochemical indexes of the patients were also detected and compared. RESULTS: Leptin gene promoter polymorphism was detected using PCR-HRM with effectiveness, high flux and accuracy. Preliminary results showed that the main mutation of the patients with liver cirrhosis was in C2549 site, but not found in G2548 site. Leptin, free leptin index (FLI), fasting insulin (FINS) and insulin resistance index estimated by homeostatic model assessment (HOMA-IR) in liver cirrhosis group were higher than those in control group. Insulin sensitivity index (ISI) and soluble leptin receptor (sOB-R) in liver cirrhosis group were lower than those in control group with significant difference except leptin level. Meanwhile, FLI showed positively correlated with FINS and HOMA-IR (r=0.45, r=0.53, P<0.05), and negatively with ISI (r=-0.34, P<0.05). In the patients with liver cirrhosis, C2549A heterozygous mutation was predominant. The indexes of HOMI-IR, leptin, sOB-R and FLI of C2549A homozygotes and heterozygotes were higher than those of the wildtypes, which showed significant difference except leptin and sOB-R levels (P<0.05). CONCLUSION: PCR-HRM can be more accurate for identifying leptin promoter polymorphism. The increase in the frequency of C2549A mutation may be closely related with liver cirrhosis. Existence of hyperinsulinemia and insulin resistance may be correlated with leptin level in the patients with liver cirrhosis.     

Effect of hepatitis virus B protein on the functions of PBMCs isolated from patients with chronic HBV infection

AIM: To study the effect of hepatitis virus B proteins on peripheral blood mononuclear cells (PBMCs) from patients among various types of chronic hepatitis B virus (HBV) infection.METHODS: 80 patients of various types of chronic HBV infection were observed, including 40 HBeAg positive with abnormal alanine aminotransferase (ALT) (A group), 20 HBeAg positive with persistent normal ALT(B group), 20 HBeAg and HBV-DNA negative with persistent normal ALT level(C group). IL-10, IFN-γ in CD8+CD28+T cells, after stimulation with PHA, HBeAg and HBcAg for 48 h, were inspected respectively in PBMCs.RESULTS: IFN-γ was significantly lower in HBeAg positive patients. IL-10 was significantly higher in HBeAg positive with normal ALT. CD8+CD28+T were significantly lower than others. CONCLUSION: In HBeAg positive group, secretion of cytotoxic T lymphocyte (CTL) and Th1 type cellular immunologic reaction is decreased, Th2 type cellular immunologic reaction is enhanced.     

Relationship of subclasses of serum HDL and Apo A-Ⅰ gene polymorphism in hyperlipidemia

AIM: To investigate apolipoprotein A-Ⅰ gene (Apo A-Ⅰ) polymorphism and its relationship with serum HDL subclasses in patients with hyperlipidemia (HL). METHODS: Apo A-Ⅰ genotype was assayed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The subclasses of serum HDL in 118 patients with hyperlipidemia and 109 healthy subjects were determined by two-dimensional gel electrophoresis conjunction with immunodetection method. RESULTS: Both in HL group and the control group, G/G and C/C genotypes were the most frequent at －78 bp and ＋83 bp of Apo A-Ⅰ gene, respectively. The frequency of rare A allele at －78 bp in HL group was significantly higher than that in control group. In HL group, subjects with G/A mutation had higher serum levels of TG, Apo C-Ⅲ, pre β1-HDL and HDL3a, and lower levels of HDL2a and HDL2b compared to the subjects with G/G genotype. CONCLUSION: The G/A transition in the －78 bp position of the Apo A-Ⅰ gene promoter in patients with hyperlipidemia is associated with HDL subclasses. There is a general shift toward smaller sized HDL, which, in turn, indicates that HDL maturation might be abnormal.     

Role of hepatitis B virus in intrahepatic TGF-β1/Smads signaling pathway

AIM:To explore the effects of hepatitis B virus (HBV) on intrahepatic expression of transforming growth factor β1（TGF-β1） and Smads. METHODS:The expression of intrahepatic TGF-β1, HBsAg and HBcAg in control group and chronic hepatitis B (CHB) group was detected by immunohistochemical method.The serum HBV DNA content was determined by real-time PCR. The role of HBV in the expression of TGF-β1, Smad3 and Smad7 in human hepatic stellate cell line LX-2 in vitro was observed by cell culture and Western blotting. RESULTS:The average score of intrahepatic TGF-β1 expression in CHB group was higher than that in control group. With the increase in serum HBV DNA content, intrahepatic TGF-β1 expression was also enhanced. In the HBcAg positive hepatic tissue, there was higher TGF-β1 expression than that in the liver tissue of HBcAg negative. Compared with control group and HBV+anti-TGF-β1 group, HBV caused increased expression of TGF-β1 and Smad3 in HBV group in vitro. No difference of Smad7 protein among control group, HBV group and HBV+anti-TGF-β1 group was observed. CONCLUSION: The expression of intrahepatic TGF-β1 is related to serum HBV DNA and hepatocellular HBcAg in the patients with CHB. HBV-induced liver fibrosis mainly relies on positive regulatory mechanisms of Smad3,and the negative regulation by Smad7 almost does not function.     

Distribution characteristics of polymorphisms of interleukin-22 gene in Guangxi population and their association with serum lipid levels

AIM: To investigate the distribution characteristics of interleukin-22 (IL-22) gene rs2227485C/T and rs2227491A/G polymorphisms in Guangxi people and the distribution differences with other ethnic groups, and to explore the difference levels of common lipid indexes in different genotypes. METHODS: SNaPshot technique and DNA sequencing were used in 280 Guangxi persons to examine IL-22 genotypes and to analyzed the distribution frequencies of allele and genotype in these sites. The distribution frequencies in different sexes, and the differences between groups and diffe-rence levels of common lipid indexes in different genotypes were analyzed statistically. RESULTS: Three genotypes of CC, CT and TT were found in rs2227485C/T with the frequency distribution of 17.1%, 49.3% and 33.6%, respectively. No significant difference between different sexes of each genotype and allele frequency in the Guangxi population was observed (P>0.05). Compared with the distribution frequencies of genotype and allele in HapMap-TSI, HapMap-HCB, HapMap-JPT and HapMap-MEX, those in Guangxi population showed statistically significant differences (P<0.05). Three genotypes of AA, AG and GG were found in rs2227491A/G with the frequency distribution of 16.1%, 52.8% and 31.1%, respectively. There was no significant difference between different sexes of each genotype and allele frequency in the Guangxi population (P>0.05). The significant differences of genotype frequencies among Guangxi population, HapMap-TSI, HapMap-JPT and HapMap-MEX were detected (P<0.05). Compared with the other 4 populations, allele frequencies in Guangxi population had significant difference (P <0.05). There were significant differences in the levels of HDL-C and LDL-C among the 3 genotypes of rs2227491A/G. The level of HDL-C had difference between AG/AA genotype and GG genotype. In addition, the level of LDL-C had difference between AG/GG genotype and AA genotype (P<0.05). CONCLUSION: rs2227485C/T and rs2227491A/G polymorphisms of IL-22 gene have differences in different populations. The rs2227491A/G polymorphism may be associated with serum lipid levels.     

One novel mutation and copy number variation of NF1 gene in Chinese patients with type 1 neurofibromatosis

AIM: To investigate the mutations and the copy number variation of neurofibromatosis 1(NF1) gene in 2 sporadic patients with type 1 neurofibromatosis in China. METHODS: All coding exons and exon-intron boundaries of NF1 were amplified by PCR. The PCR products were sequenced. The DNA samples from 50 normal subjects were also sequenced for control. Multiplex ligation-dependent probe amplification (MLPA) was also employed to detect the copy number variation of NF1 gene in these patients. Long range PCR was used for the identification of the breakpoint in the large deletion of the gene. RESULTS: The novel mutation, c. 6345_6346 ins G (p. Leu2116Alafs*4), was detected in patient S736. This mutation was absent in her parents and the controls, indicating a de novo mutation. It caused open reading frame shifting, introducing a premature stop codon and resulting in the truncation of the 721 amino acids at the C terminus of the wild-type protein. This truncation cut off part of the armadillo (ARM)-type fold domain in the wild-type protein. A 1.3~1.9 Mb deletion of the gene was also detected in the other patient S743. The deletion spanned the whole NF1 gene and part of the flanking regions in both ends, but the breaking point was still unknown. CONCLUSION: We have identified a novel mutation of NF1, c.6345_6346 ins G (p.Leu2116Alafs*4). We also first report the copy number variation of NF1 gene in Chinese patients. The investigation will be helpful for the molecular diagnosis and understanding the pathogenesis of the disease.     

Expression of hepatitis B virus core gene in Pichia pastoris

AIM: To study the expression of hepatitis B virus core gene in Pichia pastoris and to obtain high-level expressed recombinant HBcAg with good immunoreactivity and high specificity. METHODS: HBV core gene was amplified by PCR from plasmid pHBV1 which contained HBV whole DNA sequence. The PCR product was cloned into pGEM-T vector by TA cloning strategy. After confirmed by DNA sequence analysis, the gene of interest was inserted into the yeast expression vector pPIC9. The recombinant plasmid pPIC9-cAg was constructed and transformed into GS115 by electroporation. The recombinant yeast GS115 was induced by 0.5% methanol. The expressed product was analysed by SDS-PAGE,Western blot and ELISA. RESULTS: The restriction analysis and DNA sequence analysis proved that HBV core gene had already been cloned to yeast expression plasmid pPIC9. The expressed HBcAg existed in SDS-PAGE. Good immunoreactivity and high specificity of the recombinant HBcAg have been proved by ELISA and Western blot. The titre of the recombinant HBcAg in the cell lysate was 1∶12 800. CONCLUSION: The recombinant plasmid pPIC9-cAg was successfully constructed. The recombinant HBcAg with good immunoreactivity and high specificity was successfully expressed in Pichia pastoris expression system and can be applied to further developing HBcAb immunoassay.     

Role of multidrug resistance gene 1 polymorphism in prognosis of hepatocellular carcinoma patients treated with liver transplantation

AIM: To evaluate the relationship between three multidrug resistance gene 1 (MDR1) polymorphisms (C1236T, G2677A/T, C3435T) and the prognosis of hepatocellular carcinoma (HCC) in Chinese liver transplantation (LT) patients.METHODS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was applied to determine the genotypes of MDR1 gene in 50 HCC patients treated with LT. The tumor-free survival and overall survival were compared among these patients according to the polymorphisms of MDR1 by Kaplan-Meier method, multivariate regression analysis was also performed.RESULTS: No significant association was found between C1236T, G2677T, C3435T and prognosis of these patients. But interestingly, 2677A carrier group had significantly higher tumor-free survival rate than 2677A noncarrier group (P<0.05). The multivariate regression analysis revealed that 2677A carrier genotype was one of the independent factors for predicting tumor-free survival (RR=0.143, P<0.01).CONCLUSION: MDR1 2677A carrier genotype is correlated with the tumor-free survival. MDR1 2677A carrier genotype may be a useful independent prognostic factor in HCC patients treated with LT.     

Association of OPG gene single nucleotide polymorphisms with susceptibility to rheumatoid arthritis in Chinese Han population

AIM：To investigate the association of osteoprotegerin (OPG) gene single nucleotide polymorphisms (SNPs), 163A/G (rs3102735) and 245T/G (rs3134069), with susceptibility to rheumatoid arthritis (RA) in Chinese Han population. METHODS：A total of 205 patients with RA and 171 healthy control subjects were enrolled into this study. Genotyping was performed by polymerase chain reaction-based restriction fragment length polymorphism and subsequently confirmed by DNA sequencing. Odds ratio (OR) and 95% confidence intervals (CI) were calculated for the risk genotypes and alleles. RESULTS：OPG gene polymorphisms 163A/G and 245T/G were conformed to the Hardy-Weinberg equilibrium. The statistical differences in the genotypes of AA, AG and GG at 163A/G locus were found in RA and controls. The G allele was associated with an increased risk of RA, with OR of 1.219 (95％ CI: 1.066~2.339). No significant difference was observed between RA group and control group with respect to genotypic and allelic frequencies of OPG gene 245T/G (P>0.05）. CONCLUSION：The OPG gene 163A/G SNP may be associated with RA susceptibility, and G allele may be the risk factor for developing RA.     

CD4+CD25+FOXP3+ Tregs and HBV-specific CTLs in peripheral blood from chronic hepatitis B patients

AIM: To investigate the roles of CD4⁺CD25⁺FOXP3⁺ regulatory T cells (Tregs) and HBV-specific cytotoxic T-lymphocytes (CTLs) in peripheral blood from the patients with chronic hepatitis B (CHB).METHODS: Peripheral blood mononuclear cells from 28 patients with CHB and 15 healthy controls were analyzed for Treg frequency using flow cytometry and for HBV-specific CTLs using enzyme-linked immunospot assay (ELISPOT).The clinical data of HBV-infected patients were considered.RESULTS: The frequency of CD4⁺CD25⁺FOXP3⁺Tregs was higher in the patients with CHB than that in the patients of healthy controls (3.14%±0.97% vs 1.95%±0.68%, P<0.05), and a positive correlation was found between Tregs and the DNA levels of HBV (r=0.831, P<0.01).HBV-specific CTLs were detected by ELISPOT in CHB patients and a negative correlation was observed between Tregs and CTLs (r=-0.540, P<0.01).CONCLUSION: Peripheral blood CD4⁺CD25⁺FOXP3⁺ Tregs in CHB patients are increased and closely correlated with the DNA replication of HBV and CTLs, suggesting that the clearance of HBV can be influenced by the inhibition of cellular immunoreaction through Tregs.     

Relationship between SNP of TNFR gene and incidence/severity of pneumonia

AIM：To analyze the relationship between the single nucleotide polymorphism (SNP) of tumor necrosis factor receptor (TNFR) gene and the incidence and severity of pneumonia. METHODS：Total 132 Chinese individuals were enrolled in this study. There were 66 patients with pneumonia and 66 healthy subjects. The SNPs of TNFR gene including TNFR1+36A/G, TNFR1-609G/T, TNFR2+676T/G, TNFR2+1663T/G, TNFR2 +1668A/G and TNFR2 +1690C/T were genotyped by polymerase chain reaction-restriction fragment length polymorphism or gene sequencing for all subjects. Polymorphisms affecting pneumonia incidence and severity were calculated by SPSS. RESULTS：The frequencies of TNFR1-609G and T alleles in pneumonia patients were 40.9% and 59.1%, while those in healthy subjects were 53.8% and 46.2%. The frequency of TNFR1-609T in pneumonia patients was higher than that in healthy subjects (P<0.05). Besides, the frequencies of TNFR1-609G and T alleles in severe pneumonia patients were 25.0% and 75.0%, while those were 46.0% and 54.0% in non-severe pneumonia patients. The frequencies of TNFR2 +1690C and T alleles in severe pneumonia patients were 81.1% and 18.9%, while those were 61.0% and 39.0% in non-severe pneumonia patients. The frequencies of TNFR1-609T and TNFR2 +1690C in severity pneumonia subjects were higher than those in mild subjects (P<0.05). CONCLUSION：It appears that TNFR1-609T is associated with high incidence of pneumonia. TNFR1-609T and TNFR2+1690C are the risk factors of severity in pneumonia in Chinese.     

Association of IFN-γ and IL-4 gene polymorphisms with childhood bronchial asthmatic susceptibility and the levels of plasma IFN-γ,IL-4 and IgE

AIM: To investigate the gene polymorphisms of interferon-γ(IFN-γ) and interleukin-4(IL-4) and the association with asthmatic susceptibility and the levels of plasma IFN-γ, IL-4 and IgE of asthmatic children. METHODS: 100 asthmatic children and 122 control children were enrolled the study. The genotypes of IFN-γ gene-179G/T polymorphism, IL-4 gene-33C/T and-589C/T polymorphisms were tested by PCR-RFLP.The genotype of IFN-γ gene +874A/T polymorphism was tested by AS-PCR.The CA repeat polymorphism of IFN-γ gene was detected by capillary electrophoresis technique.The levels of serum IFN-γ, IL-4 and IgE were measured by ELISA. RESULTS: 100 asthmatic children and 122 control children were all GG homozygotes at -179 locus of IFN-γ gene.-179 locus of IFN-γ gene has no mutation. The genotypes and allele frequency of IFN-γ gene +874A/T and CA repeat polymorphisms showed no significant difference between asthmatic children and the control(P>0.05). An association was revealed between IFN-γ gene +874A/T polymorphism and the level of plasma IFN-γ.The level of IFN-γ was lower in AA genotype than in AT genotype(P<0.05). The genotypes and allele frequency of IL-4 gene -33C/T and -589C/T polymorphisms showed significant difference between asthmatic children and the control(P<0.05).The levels of plasma IL-4 and IgE were higher in TT genotype at -33 locus and -589 locus than those in CT genotype, but only -33C/T polymorphism was associated with the level of plasma IL-4(P<0.05). CONCLUSION: The IFN-γ gene +874A/T and CA repeat polymorphisms were not correlated with asthmatic susceptibility, but there is significant correlation between the level of IFN-γ and +874A/T polymorphism. TT genotype of IL-4 gene -33 locus and -589 locus maybe the susceptible genotype of asthma in children, and the -33 locus polymorphism is associated with the level of IL-4.