OBJECTIVE: To investigate the effects of co-expression of sodium iodide symporter (NIS) reporter gene on the proliferation and cytotoxic activity of chimeric antigen receptor (CAR)-T cells in vitro. METHODS: T cells expressing CD19 CAR (CAR-T cells), NIS reporter gene (NIS-T cells), and both (NIS-CAR-T cells) were prepared by lentiviral infection. The transfection rates of NIS and CAR were determined by flow cytometry, and the cell proliferation rate was assessed using CCK-8 assay at 24, 48 and 72 h of routine cell culture. The T cells were co-cultured with Nalm6 tumor cells at the effector-target ratios of 1∶2, 1∶1, 2∶1 and 4∶1 for 24, 48 and 72 h, and the cytotoxicity of CAR-T cells to the tumor cells was evaluated using lactate dehydrogenase (LDH) assay. ELISA was used to detect the release of IFN-γ and TNF-β in the co-culture supernatant, and the function of NIS was detected with iodine uptake test. RESULTS: The CAR transfection rate was 91.91% in CAR-T cells and 99.41% in NIS-CAR-T cells; the NIS transfection rate was 47.83% in NIS-T cells and 50.24% in NIS- CAR-T cells. No significant difference in the proliferation rate was observed between CAR-T and NIS-CAR-T cells cultured for 24, 48 or 72 h (P> 0.05). In the co-cultures with different effector-target ratios, the tumor cell killing rate was significantly higher in CAR-T group than in NIS-CAR-T group at 24 h (P < 0.05), but no significant difference was observed between the two groups at 48 h or 72 h (P>0.05). Higher IFN-γ and TNF-β release levels were detected in both CAR-T and NIS-CAR-T groups than in the control group (P < 0.05). NIS-T cells and NIS-CAR-T cells showed similar capacity of specific iodine uptake (P>0.05), which was significantly higher than that in the control T cells (P < 0.05). CONCLUSION The co-expression of the NIS reporter gene does not affect CAR expression, proliferation or tumor cell-killing ability of CAR-T cells.
Antineoplastic Agents ; Cell Line, Tumor ; Cell Proliferation ; Iodine ; Lymphotoxin-alpha ; Receptors, Chimeric Antigen ; Symporters ; T-Lymphocytes

OBJECTIVETo investigate the association of TNF-β 252A >G variant with the risk of non-small cell lung cancer (NSCLC).

METHODSTotal 956 patients with NSCLC were recruited between January 2000 and December 2008 at Cancer Hospital, Chinese Academy of Medical Science as the case group, and 994 frequency-matched controls were randomly selected from a pool of cancer-free subjects recruited from a nutritional group. All the participants were unrelated Han Chinese. There were no age, gender restrictions. Smoking status of the subjects was surveyed. Informed consent was obtained and 3 ml peripheral blood was collected from each subject. All samples were genotyped by polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP). The OR and 95%CI were estimated by logistic regression to evaluate the relationship between TNF-β 252 A/G variant and the risk of lung cancer.

RESULTSThe frequencies of TNF-β 252 AA, AG and GG genotype were 30.9% (307/994) , 47.4% (471/994) and 21.7% (216/994) in lung cancer cases and 35.7% (341/956) , 48.1% (460/956) and 16.2% (155/956) in controls. Logistic regression analysis revealed that TNF-β 252 GG genotype contributed to a decreased risk of developing NSCLC (OR = 0.64, 95%CI: 0.49-0.83) compared with AA genotype. When stratified by smoking status, the individuals with 252 GG genotype had a significant increased risk of NSCLC (OR = 1.54, 95%CI:1.00-2.39) among smokers; which was less than those with AA genotype among smokers (OR = 2.88, 95%CI:1.91-2.24). When further stratified by smoking index, individuals with 252 GG genotype had a significant decreased risk of NSCLC among heavy smokers with OR (95%CI) of 2.24 (1.33-3.74), which was less than those with AA genotype (OR = 4.62, 95%CI:2.88-7.41).

CONCLUSIONTNF-β genetic variant may interact with environment factor to contribute to the susceptibility to NSCLC.

Asian Continental Ancestry Group ; Carcinoma, Non-Small-Cell Lung ; Case-Control Studies ; China ; Genetic Predisposition to Disease ; Genotype ; Humans ; Lung Neoplasms ; Lymphotoxin-alpha ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Risk ; Risk Factors ; Smoking

This study was purpose to compare and analyze the chronic lymphocytic leukemia human bone marrow stromal cells (CLL-hBMSC) and normal hBMSC (N-hBMSC) so as to provide theoretical evidence for establishment of CLL-hBMSC interaction model to imitate CLL microenvironment. Mononuclear cells (MNC) were isolated from bone marrow of CLL patients and healthy donors and then were cultured, hBMSC were established by expanding for at least five passages. The mRNA expression of adhesion molecules, such as vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1), was analyzed by real-time PCR. The mRNA and protein expression of lymphotoxin beta receptor (LTβR) were determined by real-time PCR and Western blot, respectively. The individual NF-κB members at protein level of CLL-hBMSC and N-hBMSC were examined by Western blot. The effect of LT&alpha;1β2 on individual NF-κB family members at protein level in CLL-hBMSC and N-hBMSC was also examined by Western blot. The death of CLL cells was determined by flow cytometry with PI staining when cultured with or without CLL-hBMSC and N-hBMSC at different time points. The results showed that the hBMSC could be established successfully from bone marrow of CLL patients, which were similar to N-hBMSC. Adhesion molecules, such as VCAM-1 and ICAM-1, were found to be expressed at similar mRNA levels in CLL-hBMSC and N-hBMSC. LTβR expressions at mRNA and protein levels were comparable between CLL-hBMSC and N-hBMSC. The protein expression of the individual NF-κB family members could be detected in CLL-hBMSC and N-hBMSC with similar expression levels. LT&alpha;1β2 stimulation activated both the classical ( RelA/p50 ) and alternative ( RelB/p52 ) NF-κB complexes in CLL-hBMSC and N-hBMSC. The capacities of CLL-hBMSC and N-hBMSC to protect CLL cell survival were similar. It is concluded that there is no statistical difference between bone marrow from healthy donors and CLL patients in the efficiency of generating of hBMSC. LTβR-NF-κB signaling molecules are expressed and activated on hBMSC with a similar pattern.
Humans ; Intercellular Adhesion Molecule-1 ; metabolism ; Leukemia, Lymphocytic, Chronic, B-Cell ; genetics ; metabolism ; pathology ; Lymphotoxin beta Receptor ; metabolism ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Signal Transduction ; Transcription Factor RelA ; metabolism ; Transcription Factor RelB ; metabolism ; Vascular Cell Adhesion Molecule-1 ; metabolism

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

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

BACKGROUND AND OBJECTIVES: The transplantation of human umbilical cord blood cells (hUCBCs) has been shown to attenuate the unregulated activation of microglia in a rat model of cerebral palsy (CP). To investigate whether hUCBCs transplantation is also anti-inflammatory in humans, we performed a clinical trial in patients with CP. METHODS AND RESULTS: Allogeneic or autologous hUCBCs and erythropoietin (EPO) were intravenously injected into human patients with CP (mean age of approximately 38 weeks), and patients were analyzed for their motor function and social behavior. Blood samples were tested for cytokine levels. The most surprising finding in the study was that the cytokine levels were dependent on the donor cell source (allogeneic or autologous). Interestingly, the allogeneic treatment group demonstrated significantly decreased levels of pro-inflammatory factors, such as IL-1alpha, IL-6, TNF-beta, and RANTES, and showed a statistically significant improvement in motor and social behavior compared to the autologous treatment group. CONCLUSIONS: Given that inflammation plays a pivotal role in CP, our results suggest that allogeneic hUCBCs therapy may be an appropriate strategy for CP treatment. In addition, prior to transplantation, a detailed analysis of the amount of proinflammatory cytokines in cord blood may be needed to avoid exacerbating inflammatory responses.
Animals ; Cerebral Palsy ; Chemokine CCL5 ; Cytokines ; Erythropoietin ; Fetal Blood ; Humans ; Inflammation ; Interleukin-6 ; Lymphotoxin-alpha ; Microglia ; Rats ; Social Behavior ; Tissue Donors ; Transplants ; Umbilical Cord

BACKGROUND AND PURPOSE: Migraine patients are particularly prone to the complication of medication-overuse headache (MOH). Although it has been shown that A allele carriers for the tumor necrosis factor (TNF)-beta gene G252A polymorphism are at high risk of the development of migraine without aura, the relationship between the TNF-beta gene G252A polymorphism and MOH is unknown. We investigated whether the TNF-beta gene G252A polymorphism is involved in the aggravation of migraine by overuse of medications. METHODS: Forty-seven migraine patients (6 males and 41 females; age 36.4+/-10.3 years, mean+/-SD) and 22 MOH patients (1 male and 21 females; age 39.6+/-9.9 years) who had migraine were included in this study. The genotype for the TNF-beta gene G252A polymorphism was determined by polymerase-chain-reaction restriction-fragment-length polymorphism analysis. RESULTS: The distribution of TNF-beta gene G252A genotype frequency differed significantly between migraine and MOH patients (p=0.013). The G/G genotype was carried by 23% of the migraine patients but it was absent in MOH patients. CONCLUSIONS: G/G genotype carriers appear to be less susceptible to the aggravation of migraine by overuse of medications. The G252A TNF-beta gene polymorphism may be one of the factors contributing to the complications of MOH in patients with migraine.
Alleles ; Genotype ; Headache ; Humans ; Lymphotoxin-alpha ; Male ; Migraine Disorders ; Migraine without Aura ; Tumor Necrosis Factor-alpha

BACKGROUND: The tumor necrosis factor (TNF) is believed to play an important role in the pathophysiology of osteoarthritis (OA). Evidence shows that genetic polymorphisms make substantial contributions to the etiology of OA. METHODS: We investigated the genotypes TNF-alpha and TNF-beta in 301 OA patients and 291 healthy subjects as controls. We employed a polymerase chain reaction-restriction fragment length polymorphism and a polymerase chain reaction-single strand conformation polymorphism assay to identify the genotypes TNFA -G308A and TNFB +G252A, respectively. RESULTS: For TNFA -G308A, the percentages of genotypes GG, AG, and AA were 26.3% (79/301), 62.5% (188/301), and 11.3% (34/301) in OA patients and 88.7% (258/291), 11.3% (33/291), and 0% (0/291) in controls. For TNFB +G252A, the percentages of genotypes GG, AG, and AA were 15.3% (46/301), 41.9% (126/301), and 42.9% (129/301) in OA patients and 12% (35/291), 52.6% (153/291), and 35.4% (103/291) in controls. There were significant differences in genotypes and alleles of TNFA -308 between OA patients and controls (p<0.0001) and in alleles of TNFB +252 (p=0.0325). The risk of OA was significantly higher for carriers of the TNFA -308A allele and the TNFB +252 AA homozygote (p=0.0224). CONCLUSIONS: The results suggest close relationships between TNFA -G308A and TNFB +G252A polymorphisms and individual susceptibility to OA in the Korean population.
Alleles ; Genetic Predisposition to Disease ; Genotype ; Homozygote ; Humans ; Lymphotoxin-alpha ; Osteoarthritis ; Polymorphism, Genetic ; Tumor Necrosis Factor-alpha

OBJECTIVES: Proinflammatory process has been implicated as an underlying mechanism of bipolar disorder and schizophrenia. Previous studies have suggested a possible role of lymphotoxin alpha (LTA) gene in the development of schizophrenia and have prompted further investigation in bipolar patients. Association of the LTA +252A/G polymorphism with susceptibility to bipolar I disorder itself as well as with vulnerability among a subset of psychotic bipolar patients were tested. METHODS: DNA extraction was done by a standard method and genotyping was carried out by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method in 114 Korean patients with bipolar I disorder and 202 healthy controls. SPSS v18.0 was used for statistical analysis. Comparisons of the genotype and allele distributions in LTA +252A/G polymorphism were made using a chi-square test. The genotype and allele associations were also evaluated using odds ratio (OR) and 95% confidence interval (CI). Statistical significance was accepted when p was < 0.05. RESULTS: No significant association was found between the LTA +252A/G polymorphism and bipolar disorder. However, LTA +252G allele was present with significantly higher frequency among bipolar patients with psychotic features compared to those without (chi2 = 4.69, p = 0.034, OR = 2.495, 95% CI = 1.069-5.827). CONCLUSION: The results suggest that the allele LTA +252G of the polymorphism may be associated with the psychotic subset of bipolar disorder but not with bipolar I disorder itself. Adequately powered subsequent studies should be conducted.
Alleles ; Bipolar Disorder ; DNA ; Genotype ; Humans ; Lymphotoxin-alpha ; Odds Ratio ; Schizophrenia

OBJECTIVE: To analyze Th1 and Th2 immune balance related cytokines and clinical significance in obstructive sleep apnea hypopnea syndrome children without allergic rhinitis and asthma. METHOD: Collected 91 cases of obstructive sleep apnea hypopnea syndrome children with obstructive level data, and measured the serum Th1 cytokine TNF-beta and IFN-gamma, Th2 cytokines IL-4 and IL-5 levels. One hundred and five normal children were enrolled for the same detection of serum cytokines. RESULT: Non-allergic rhinitis and asthma children serum levels of IFN-gamma was lower than control group children, the difference was statistically significant (P < 0. 01). Other cytokines (TNF-beta, IL-4 and IL-5) were no significant difference with the control group. CONCLUSION Th1 and Th2 immune response was imbalance in non-allergic rhinitis and asthma obstructive sleep apnea hypopnea syndrome children. The decline in Th1 cell-mediated protective immune response cells may cause disease.
Asthma ; Case-Control Studies ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Interferon-gamma ; blood ; Interleukin-4 ; blood ; Interleukin-5 ; blood ; Lymphotoxin-alpha ; blood ; Male ; Rhinitis, Allergic ; Rhinitis, Allergic, Perennial ; Sleep Apnea, Obstructive ; blood ; Th1 Cells ; metabolism ; Th2 Cells ; metabolism