OBJECTIVE: To determine the polymorphism in +252 site of tumor necrosis factor-beta(TNF-beta) gene in patients with or without endometriosis, to evaluate the levels of TNF-alpha and TNF-beta in the serum with or without endometriosis, to explore the relation between polymorphism of TNF-beta gene and the genetic susceptibility of endometriosis, and to explore the pathogenic mechanism of endometriosis at gene level. METHODS: By polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, polymorphism on +252 site of TNF-beta gene was measured in 82 patients with endometriosis (the endometriosis group) and 80 patients without endometriosis (the control group). With the sandwich-enzyme-linked immunosorbent assay (ELISA), the levels of TNF-alpha and TNF-beta in the serum of the two groups were determined. RESULTS: The TNF-beta level in the serum in the endometriosis group with TNF-beta gene +252 site AA genotype significantly increased, compared with GG genotype (t=2.029, P<0.05); while TNF-alpha and TNF-beta level in the serum had no statistical significance in patients with other genotypes in TNF-beta gene +252 site in the endometriosis group and the control group. CONCLUSION TNF-beta gene +252 site AA genotype might be enhance TNF-beta level in the serum of patients with endometriosis.
Adolescent ; Adult ; Endometriosis ; blood ; genetics ; Female ; Humans ; Lymphotoxin-alpha ; blood ; genetics ; Polymorphism, Genetic ; Tumor Necrosis Factor-alpha ; blood ; Young Adult

The localization and functional characteristics of tumor necrosis factor(TNF) beta gene raise the possibility that it may be involved in the susceptibility to autoimmune thyroid diseases. To investigate whether a TNF beta gene polymorphism is associated with autoimmune thyroiditis, we analyzed the TNF beta gene polymorphism with the restriction enzyme NcoI in 48 Korean patients with atrophic autoimmune thyroiditis [23 were found to be thyrotropin binding inhibitor immunoglobulin(TBII) positive, 25 TBII negative], 52 goitrous autoimmune thyroiditis, and 129 healthy controls. Two TNF beta alleles were identified from the restriction fragment length polymorphism studies of amplified genomic DNA. In atrophic autoimmune thyroiditis patients positive for TBII, 7 of 23 patients were homozygous for the TNF beta * 1 allele, 3 were homozygous for the TNF beta * 2 allele, and 13 were TNF beta * 1/2 heterozygous compared to controls(P = 0.20). Also, there were no associations between the TNF beta gene polymorphism and either TBII-negative atrophic autoimmune thyroiditis or goitrous autoimmune thyroiditis. Of the HLA-class II antigens, the frequency of HLA-DR8 was significantly greater among the 23 Korean patients with TBII-positive atrophic autoimmune thyroiditis compared to control subjects (Pc = 0.003). When the HLA-DR8 positive patients with TBII-positive atrophic autoimmune thyroiditis and controls were analyzed separately, the DR8 positive patients with TBII-positive atrophic autoimmune thyroiditis had more homozygotes for the TNF beta * 1 allele(6/12, 50.0%) and no homozygotes for the TNF beta * 2 allele, as compared to the DR8 negative patients with TBII-positive atrophic autoimmune thyroiditis and DR8 positive controls(P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
Alleles ; *Genetic Linkage ; HLA-DR Antigens/*genetics ; Humans ; Korea ; Lymphotoxin-alpha/*genetics ; *Polymorphism, Genetic ; Receptors, Thyrotropin/*immunology ; Thyroiditis, Autoimmune/*genetics

To construct the combined site-directed random mutation library of recombinant human Lymphotoxin (rhLT) for in vitro molecular evolution study, and to study the structure and function relationship. The random point mutations at the sites of 46,106 and 130 were individually generated by overlap PCR amplification with the random nucleotide primers. The three point mutations were combined and cloned into pMD-18T vector to construct the combined mutation library. DNA sequencing was used to evaluate the diversity and randomness of the mutation sites. The combined mutation library was re-engineered, inserted in prokaryotic expression vector pBV220, transformed and expressed in Escherichia coli strain DH5alpha. The biological activity of some of the mutants was tested in 1929 mouse fibroblast cells. As much as 1.5 x 10(5) clones were obtained, which represents 4.5 times of the complete mutation libraries at 99% confidence. Sequencing 50 clones revealed no obvious bias in the nucleotide and amino acid mutations at the sites. Among the 30 expressed samples underwent for the bioassay, 70% (21 samples) were inactive, 23.3% (7 samples) had lower activity than rhLT, the remaining 6.7% (2 samples) had higher activity than rhLT. The combined site-directed random mutation library of rhLT has been constructed successfully. In combination with phase display, the library is ready for in vitro molecular evolution study.
Amino Acid Sequence ; Base Sequence ; Escherichia coli ; genetics ; Evolution, Molecular ; Gene Library ; Humans ; Lymphotoxin-alpha ; genetics ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Recombinant Proteins ; genetics

OBJECTIVETo study the polymorphism of +252 site in intron 1 of tumor necrosis factor (TNF)-beta gene in relation to genetic susceptibility of endometriosis.

METHODSPolymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was employed to detect the polymorphism of +252 site in intron 1 of TNF-beta gene in 82 Chinese Han patients with endometriosis in Guangdong Province and 80 Han patients without endometriosis (control group), and the relation between TNF gene polymorphism and the risk of endometriosis was analyzed.

RESULTSThe +252 site of TNF-beta allele and genotype distribution showed significant difference between endometriosis and control groups (Chi2=6.562, P<0.05; chi2=6.562, P<0.05), and relative risk of endometriosis in relation to allele A was increased by 1.793 fold. The risk of endometriosis was 3.33-fold higher in women of AA genotype than those of GG genotype (Chi2=6.562, P<0.05).

CONCLUSIONSAllele A in TNF-beta gene +252 site can significantly increase the relative risk of endometriosis in women in Guangdong, among which TNF-beta AA genotype might be one of the genetic susceptible factors for endometriosis.

Adult ; Alleles ; China ; Endometriosis ; genetics ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; genetics ; Genotype ; Humans ; Lymphotoxin-alpha ; genetics ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Polymorphism, Restriction Fragment Length

BACKGROUNDWe investigated the possible association of the functional polymorphisms in the tumor necrosis factor (TNF) genes with susceptibility to esophageal squamous cell carcinoma (ESCC) and gastric cardiac adenocarcinoma (GCA).

METHODSThe TNF-alpha-308G/A and TNF-beta+252G/A single nucleotide polymorphisms (SNPs) were genotyped using polymerase-chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, in 555 cancer patients (291 ESCC and 264 GCA) and 437 healthy controls in a high incidence region of North China.

RESULTSAmong healthy controls, frequencies of the TNF-alpha 1/1, 1/2 and 2/2 genotypes were 89.4%, 9.2% and 1.4% respectively, while frequencies of the TNF-beta B1/B1, B1/B2 and B2/B2 genotypes were 12.6%, 32.3% and 55.1%, respectively. No significant difference was found in the overall genotype and allelotype distribution of the TNF-alpha-308G/A and TNF-beta+252G/A SNPs among cancer patients and controls. However, both the B1/B1 genotype and B1/B2 genotype significantly increased the risk of developing ESCC [the age and gender adjusted odds ratio (OR) = 2.04 and 1.91, 95% confidence interval (CI) = 1.04 - 4.43 and 1.14 - 2.60, respectively] and GCA (the age and gender adjusted OR = 2.68 and 2.64, 95% CI = 1.14 - 6.29 and 1.47 - 4.72, respectively) in individuals with negative family history of UGIC, in comparison with the B2/B2 genotype. When the two TNF polymorphisms were combined and analyzed, individuals with the TNF-beta B1/B2 and TNF-alpha 1/2 or 2/2 genotypes significantly reduced the risk of developing ESCC and GCA, in comparison with those harboring the TNF-beta B2/B2 and TNF-alpha 1/1 genotypes (the age and gender adjusted OR = 0.37 and 0.34, 95% CI = 0.15 - 0.92 and 0.13 - 0.90, respectively).

CONCLUSIONSTherefore, the TNF-alpha-308G/A and TNF-beta+252G/A genotyping may be used as a stratification markers to predicate the risk of ESCC and GCA development in North China.

Adenocarcinoma ; genetics ; Adult ; Aged ; Aged, 80 and over ; Carcinoma, Squamous Cell ; genetics ; Esophageal Neoplasms ; genetics ; Female ; Genetic Predisposition to Disease ; Genotype ; Humans ; Lymphotoxin-alpha ; genetics ; Male ; Middle Aged ; Polymorphism, Genetic ; Polymorphism, Single Nucleotide ; Stomach Neoplasms ; genetics ; Tumor Necrosis Factor-alpha ; genetics

BACKGROUNDBacteremia remains a significant cause of morbidity and mortality after kidney transplantation. This study was conducted to investigate whether the polymorphisms of tumor necrosis factor (TNF)-β, interleukin (IL)-1β, and IL-1 receptor antagonist (IL-1ra) gene predicted the susceptibility to bacteremia within the first 6 months after kidney transplantation.

METHODSSubjects comprised 82 infected kidney transplant recipients and 60 non-infected kidney transplant recipients. Bacteremia was diagnosed in 16 of the 82 infected recipients. Genomic DNA from these 142 kidney transplant recipients was extracted from peripheral blood leukocytes. Regions containing the NcoI polymorphic site at position +252 of TNF-β gene and the AvaI polymorphic site at position -511 of IL-1β gene were amplified by polymerase chain reaction (PCR) and subsequently digested with NcoI and AvaI restriction enzymes, respectively. The polymorphic regions within intron 2 of IL-1ra gene containing variable numbers of a tandem repeat (VNTR) of 86 base pairs were amplified by PCR.

RESULTSGenotypic and allelic frequencies were similar between infected recipients and non-infected ones. Individual locus analysis showed that recipient TNF-β and IL-1ra gene polymorphisms were not associated with the presence of bacteremia (P = 0.684 and P = 0.567, respectively). However, genotype analysis revealed that recipient IL-1β-511CC genotype was strongly associated with susceptibility to develop bacteremia (P = 0.003). Recipient IL-1β-511CC genotype (odds ratio 5.242, 95% confidence intervals 1.645-16.706, P = 0.005) independently predicted the risk for bacteremia within the first 6 months after kidney transplantation.

CONCLUSIONSThese findings indicate a critical role of IL-1β gene polymorphisms in susceptibility to bacteremia after kidney transplantation, which may be useful to screen for patients at higher risk for post-transplant bacteremias. Thus, the identified individuals can benefit from preventive treatment and a less potent immunosuppressive regimen.

Adolescent ; Adult ; Bacteremia ; genetics ; Female ; Genotype ; Humans ; Interleukin 1 Receptor Antagonist Protein ; genetics ; Interleukin-1 ; genetics ; Kidney Transplantation ; Lymphotoxin-alpha ; genetics ; Male ; Middle Aged ; Multigene Family ; genetics ; Polymorphism, Genetic ; genetics ; Young Adult

OBJECTIVETo investigate the single nucleotide polymorphisms (SNPs) at interleukin 6 (IL-6)-174 and TNF-β NcoI in Chinese Han children in Guangzhou, China and to provide basic information for study on the association between IL-6-174 and TNF-β NcoI polymorphisms and systemic inflammatory response syndrome (SIRS).

METHODSAllele-specific polymerase chain reaction and polymerase chain reaction-restriction fragment length polymorphism were used to determine the SNPs at IL-6-174 and TNF-β NcoI in 481 children selected from the Han population in Guangzhou in 2012. Genotype analysis and comparison with other populations were made with reference to relevant literature.

RESULTSChinese Han children in Guangzhou had only GG genotype at IL-6-174, and the SNP at this locus was rare or not seen in the Han population in Guangzhou. At TNF-β NcoI, the frequencies of TNF-β 1*1, TNF-β 1*2, and TNF-β 2*2 genotypes were 24.7%, 49.7%, and 25.6%, respectively. The sample distribution was in accordance with Hardy-Weinberg equilibrium. The TNF-β 1 allele frequency was significantly higher in Guangzhou Han population than in European and American white population (P<0.05).

CONCLUSIONSTNF-β NcoI SNP is prevalent in the Han population in Guangzhou, and the distribution of alleles is significantly different from that in the white population. The sample from an Hardy-Weinberg equilibrium population can be further used for study on the association between TNF-β NcoI SNP and SIRS in Chinese Han children in Guangzhou. IL-6-174 SNP is rare or not seen in the Han population in Guangzhou, so SNP at this locus cannot be selected for disease association analysis.

Asian Continental Ancestry Group ; genetics ; Child, Preschool ; China ; ethnology ; Deoxyribonucleases, Type II Site-Specific ; metabolism ; Female ; Gene Frequency ; Humans ; Interleukin-6 ; genetics ; Lymphotoxin-alpha ; genetics ; Male ; Polymorphism, Single Nucleotide ; Systemic Inflammatory Response Syndrome ; genetics

OBJECTIVETo investigate the association between tumor necrosis factor-beta (TNF-beta) gene polymorphisms and coronary heart disease (CHD).

METHODSPolymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequence specific primer-PCR (PCR-SSP) were used for the detection of TNF-beta genotype in 210 patients with CHD and 186 healthy controls. The serum TNF-beta levels were measured by enzyme-linked immunosorbent assay (ELISA).

RESULTSThe frequencies of CC, CA and AA genotypes of C804A in patients and controls were 25.7% and 37.1%, 49.5% and 45.7%, 24.8% and 17.2%, respectively; there were statistically significant differences in the distributions of the genotypes (P<0.05) and the allele frequencies (P<0.05) between the two groups; the risk of suffering from CHD in those of AA and CA genotypes was 1.704 times that in those of CC genotype (OR=1.704, 95%CI: 1.109-2.617). However, there was no significant difference in the distribution of the genotype of G252A between the patients and controls, though significant difference was seen between the subgroups of the CHD group. The serum TNF-beta and high sensitive C-reactive protein (hsCRP) levels of the patients were significantly higher than those of the controls (P<0.05); however, there were no significant differences in regard to different TNF-beta genotypes among the patients and controls respectively.

CONCLUSIONThe single nucleotide polymorphism (SNP) at position 804 in the exon 3 of TNF-beta gene is associated with CHD and the allele A may be a risk factor for CHD in Chinese. The polymorphism of G252A may not play an important role in the pathogenesis of CHD.

Aged ; Asian Continental Ancestry Group ; Coronary Disease ; genetics ; Exons ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; Genotype ; Humans ; Introns ; Lymphotoxin-alpha ; genetics ; Male ; Middle Aged ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single Nucleotide

OBJECTIVETo investigate the changes of inflammation-related cytokine expression profiles in human periodontal ligament fibroblasts (hPDLF) under mechanical stress.

METHODSThe periodontal ligament attached to the mid-third part of the fresh root of young premolars extracted for orthodontic treatment was scalped and removed. hPDLFs were cultured by the method of digesting by I-type collagenase combined with tissue adhering, and then isolated and purified by cells passages. hPDLFs were then divided into two groups, group with mechanical force and group without mechanical force and then cultured for 24 h. Employing cytokine-microarray analysis to assess, in a comprehensive manner compared to the hPDLFs culture system without a static force. The quantity of different cytokine-related genes in hPDLFs was analyzed by means of quantitative with the special primers of up-and down-regulated genes. The mRNA of inflammation-related cytokines was examined by real-time PCR, and the expression of the cytokines in hPDLFs detected by cytokine flowcytomix assay.

RESULTSThe relative expression of interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)-&alpha;, TNF-β and interferon (IFN)-γ mRNA in the hPDLFs with 24 h persistent-pressure (0.3633 ± 0.0874, 0.4200 ± 0.0285, 0.1697 ± 0.0284, 0.0983 ± 0.0131, 0.2840 ± 0.0676 and 3.1067 ± 0.2857) was significantly higher than the group without mechanical force (0.1173 ± 0.0176, 0.1691 ± 0.0174, 0.0117 ± 0.0021, 0.0243 ± 0.0050, 0.0000 ± 0.0000 and 0.1433 ± 0.0125), P < 0.05. The cell culture supernatant cytokines expression of IL-1β, IL-6, IL-8, TNF-&alpha;, TNF-β and IFN-γ after 48 h cultured [(18.21 ± 1.01), (1634.11 ± 472.41), (1461.47 ± 50.53), (20.71 ± 2.52), (884.11 ± 118.86) and (1461.47 ± 333.37) ng/L] was significantly higher than the group without mechanical force [(5.32 ± 4.97), (373.56 ± 155.92), (679.11 ± 141.42), (4.32 ± 0.73), (3.56 ± 0.92) and (204.11 ± 35.36) ng/L], P < 0.05. The relative mRNA and protein expression of IL-2, IL-4, IL-5, IL-10 and IL-12 showed no significant difference between the both groups.

CONCLUSIONSPersistent static mechanical force could regulate the expression of some inflammation-related cytokines. These up-regulated cytokines may be invloved in remodeling of hPDLFs, bone resorption and periodontal microenvironment.

Bicuspid ; Cells, Cultured ; Cytokines ; genetics ; metabolism ; Fibroblasts ; cytology ; metabolism ; Humans ; Interferon-gamma ; genetics ; metabolism ; Interleukin-1beta ; genetics ; metabolism ; Interleukin-6 ; genetics ; metabolism ; Interleukin-8 ; genetics ; metabolism ; Lymphotoxin-alpha ; genetics ; metabolism ; Periodontal Ligament ; cytology ; metabolism ; RNA, Messenger ; metabolism ; Stress, Mechanical ; Tumor Necrosis Factor-alpha ; genetics ; metabolism