Tumor Necrosis Factor-α Gene Research Articles

Musculoskeletal Involvement in SSc Is Associated with Worse Scores on Short Form-36 and Scleroderma Health Assessment Questionnaire and Lower Tumor Necrosis Factor-α Gene Expression in Peripheral Blood Mononuclear Cells.

Systemic sclerosis (SSc) is characterized by a wide variety of symptoms and disease manifestations including joint pain, gastrointestinal dysfunction, interstitial lung disease, and cardiomyopathy. Using the Scleroderma Health Assessment Questionnaire (SHAQ) and Short Form-36 (SF-36) we explored how patient-reported physical health, mental health, and functional status related to these clinical characteristics and to cytokine levels utilizing the Hospital for Special Surgery Scleroderma Registry. In a cross-sectional study of 185 patients meeting the 2013 ACR/EULAR criteria for SSc, we compared disease features and patient-reported outcomes (PROs). Interleukin-6 (IL-6), interleukin-1β (IL1β), and tumor necrosis factor-α (TNFα) levels were assessed by luminex and ELISA assays in a subset of 32 patients. The Pearson correlation coefficient, Spearman correlation coefficient, two-sample t test or Wilcoxon rank sum test, ANOVA or Kuskal-Wallis test, and Pearson chi-squared or Fisher's exact test were performed as applicable to detect the association between disease manifestations, PROs, and blood biomarkers. The modified Rodnan skin score (MRSS) was positively correlated with SHAQ scores. Patients who had musculoskeletal involvement scored worse on both the SHAQ and SF-36. Lower levels of TNFα expression in PBMCs were also correlated with musculoskeletal involvement. No other significant correlations were found between clinical factors, PROs, and cytokine data. Musculoskeletal outcomes are a major determinant of quality of life and function in patients with SSc. These results emphasize the importance of musculoskeletal outcomes in clinical studies of SSc.

20-HETE-induced mitochondrial superoxide production and inflammatory phenotype in vascular smooth muscle is prevented by glucose-6-phosphate dehydrogenase inhibition.

20-Hydroxyeicosatetraeonic acid (20-HETE) produced by cytochrome P-450 monooxygenases in NADPH-dependent manner is proinflammatory, and it contributes to the pathogenesis of systemic and pulmonary hypertension. In this study, we tested the hypothesis that inhibition of glucose-6-phosphate dehydrogenase (G6PD), a major source of NADPH in the cell, prevents 20-HETE synthesis and 20-HETE-induced proinflammatory signaling that promotes secretory phenotype of vascular smooth muscle cells. Lipidomic analysis indicated that G6PD inhibition and knockdown decreased 20-HETE levels in pulmonary arteries as well as 20-HETE-induced 1) mitochondrial superoxide production, 2) activation of mitogen-activated protein kinase 1 and 3, 3) phosphorylation of ETS domain-containing protein Elk-1 that activate transcription of tumor necrosis factor-α gene (Tnfa), and 4) expression of tumor necrosis factor-α (TNF-α). Moreover, inhibition of G6PD increased protein kinase G1α activity, which, at least partially, mitigated superoxide production and Elk-1 and TNF-α expression. Additionally, we report here for the first time that 20-HETE repressed miR-143, which suppresses Elk-1 expression, and miR-133a, which is known to suppress synthetic/secretory phenotype of vascular smooth muscle cells. In summary, our findings indicate that 20-HETE elicited mitochondrial superoxide production and promoted secretory phenotype of vascular smooth muscle cells by activating MAPK1-Elk-1, all of which are blocked by inhibition of G6PD.

Association of Tumor Necrosis Factor-α (TNFα) Gene Polymorphisms with HLA Class II Alleles in HIV/AIDS Patients

Aim of Study: To identify the factors of molecular genetic risks during the development of infection in HIV, based on the TNFα cytokine gene polymorphism in combination with HLA DRB1/DQA1/DQB1 genes, as well as to analyse their possible association with the progress of the disease. 185 HIV infected patients and 173 individuals control group have been analysed. The DNA was extracted from peripheral blood, by using QiagenQIAamp DNA kit reagents. The quality and quantity of DNA was checked by using Qubit ® fluorometer HLA typing for HLA DRB1/DQB1/DQA1* was performed by RT-PCR with sequence-specific primers (SSO). TNFα gene G–238A and G–308A polymorphic variant incidence was determined by RT-PCR analysis. Original Research Article Eglite et al.; BJMMR, 13(12): 1-10, 2016; Article no.BJMMR.23065 2 Results: We have detected TNFα gene allele 308A in 11% HIV infected patients, whereas in control group this allele have been detected only in 4% patients. Although the incidence of the TNFα gene –238A allele was twice as high in the control group (6%) as in the HIV infected patients (3%), it did not prove to be statistically valid (p = 0.253). The incidence analysis of three-locus haplotypes DRB1-DQB1-DQA1 – in TNFα position-238A/G -308A/G showed that haplotypes 01:01/05:01/01:01-TNFα-238(GA)/308(GG) and 01:01/03:02/03:01 TNFα-238(AA)/308(GG) are more frequent in the control group in comparison to the groups of infected patients. This means that these haplotypes have a protective function, which significantly affects the progress of infection. The association of 15:01/05:01/01:01 -TNFα-238(GG)/308(GG) and 03:01/05:01/01:01TNFα238(GG)/308(GА) genotypes indicates a high risk of developing a fulminant infection. The genetic factors of AIDS-related complex of syndromes development are associated not only with the HLA complex class II alleles, but also with the SNP polymorphism in the promoter region of cytokine genes.

Association of tumor necrosis factor-α gene polymorphism with osteoarticular tuberculosis prognosis in a Hebei population.

This study investigated the association of tumor necrosis factor-α (TNF-α)-308, -238, and -863 polymorphisms with osteoarticular tuberculosis (OA-TB) prognosis in a Hebei population. Genomic DNA was extracted from venous blood samples of 120 OA-TB patients and 100 healthy volunteers. TNF-α-308, -238, and -863 were analyzed by PCR-restriction fragment length polymorphism; genotype and allele frequencies were calculated. Serum TNF-α level was significantly higher in OA-TB patients (283.16 ± 51.68 ng/L) than in control (122.54 ± 54.65 ng/L; P < 0.05). Higher frequency of TNF-α-308 GG genotype in healthy volunteers (91.0%) than in OA-TB patients (79.2%) indicated that it was a protective factor against OA-TB (OR = 0.405, 95%CI = 0.147-0.657, P = 0.007). Higher frequencies of TNF-α-308 GA genotype and TNF-α-308 allele (A) in OA-TB patients (20.8 and 10.4%, respectively) than in healthy volunteers (8.0 and 5.0%, respectively) indicated an association with increased risk of OA-TB (OR = 3.112, 95%CI = 1.520-6.343, P = 0.003; OR = 3.109, 95%CI = 1.676-6.538, P = 0.006; respectively). Haplotype association analysis of TNF-α polymorphisms (-308/-238/-863) showed a higher frequency of TNF-α AGA in OA-TB patients (12.1%) than in healthy volunteers (3.5%), indicating that it was a risk factor for OA-TB (OR = 4.201, 95%CI = 1.80-9.91, P = 0.010). TNF-α-308 G/A and TNF-α AGA (-308/-238/-863) were associated with a predisposition to OA-TB, which could aid clinical detection, prevention, and prognosis of OA-TB.

Associations between the tumor necrosis factor-α gene and interleukin-10 gene polymorphisms and risk of alcoholic liver disease: A meta-analysis

The critical roles of tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) in the pathogenesis of alcoholic liver diseases (ALD) suggest that functional variations in the TNF-α (TNFA) and IL-10 genes may be related to individual susceptibility to ALD. As available studies examining the associations between TNFA or IL-10 polymorphisms and ALD risk have yielded conflicting results, a meta-analysis was conducted to clarify the potential relation between TNFA and IL-10 polymorphisms and the risk of ALD. A comprehensive literature search was conducted to identify relevant studies. Pooled odds ratios and 95% confidence intervals were calculated using a random-effects model. The heterogeneity between studies was assessed using the Cochran's Q statistic and the I(2) statistic. Publication bias was assessed using funnel plots and the Egger's regression test. A total of 17studies and 12studies were identified and included in the meta-analysis of the associations between TNFA polymorphisms and ALD risk, and IL-10 polymorphisms and ALD risk, respectively. The pooled results showed that the "A" allele of the TNFA-238G>A polymorphism was significantly associated with an increased risk of ALD. Significant differences in the allele and genotype distributions of the IL-10-1082A>G polymorphism were detected in the comparison between ALD patients and healthy controls, but not when comparing ALD patients and alcohol dependent individuals without ALD. No significant associations between other polymorphic loci and ALD risks were detected. The TNFA-238G>A polymorphism was significantly associated with ALD risk, while the TNFA-308G>A polymorphism and IL-10 polymorphisms (-1082A>G and -592C>A) may not be associated with the individual susceptibility to ALD. The impact of combined TNFA and IL-10 polymorphisms on individual susceptibility to ALD needs to be investigated in future studies.

Tumor necrosis factor-alpha (− 308G/A, + 488G/A, − 857C/T and -1031 T/C) gene polymorphisms and risk of ischemic stroke in north Indian population: A hospital based case–control study

BackgroundGenetic factors may play a role in the susceptibility of Ischemic stroke (IS). Previous studies have shown that Tumour necrosis factor-α (TNF-α) gene polymorphisms were associated with the risk of IS in multiple ethnicities. The present case–control study tested the hypothesis that genetic polymorphisms of the TNF-α gene may affect the risk of IS in North Indian population. We investigated the association of four single nucleotide polymorphisms (−308G/A, +488G/A, −857C/T and -1031T/C) within TNF-α gene promoter and their haplotypes with the risk of IS. MethodsIS was classified using the Trial of Org 10,172 in Acute Stroke Treatment (TOAST) classification. Genotyping was performed for 250 IS patients and 250 age- and sex-matched IS free controls by using SNaPshot technique. Multivariate logistic regression was used to control the confounding effects of demographic and risk factor variables. Haplotype analyses were done by using PHASE software and Linkage disequilibrium (LD) analyses were done by using Haploview version 4.2 software. ResultsAn independent association between TNF-α +488G/A (OR=2.59; 95%CI 1.46 to 4.60; p=0.001) and -857C/T (OR=1.77; 95%CI 1.01 to 3.11; p<0.04) and risk of IS was observed under dominant model. However, no significant association between -308G/A and -1031T/C gene polymorphisms and risk of IS was observed. Haplotype analysis showed that A308-G488-C857-T1031 haplotypes were significantly associated with the increased risk of IS [OR=1.66; 95%CI 1.02 to 2.71; p=0.003]. Strong linkage disequilibrium (LD) was observed for +488G/A and -857C/T (D’=0.41, r2=0.004). ConclusionsTwo SNPs (+488G/A and -857C/T) of TNF-α gene and their haplotypes are significantly associated with the risk of IS in the population enrolled from North India. Our findings indicate that polymorphisms and haplotypes of TNF-α gene may be used as a genetic marker for identifying individuals at increased risk for developing IS.

Heat shock protein 70-2 and tumor necrosis factor-α gene polymorphisms in Chinese children with Henoch-Schönlein purpura.

Henoch-Schönlein purpura (HSP) or IgA-associated vasculitis is related to immune disturbances. Polymorphisms of the heat shock protein 70-2 gene (HSP70-2) and the tumor necrosis factor-a gene (TNF-α) are known to be associated with immune diseases. The purpose of this study was to investigate the likely association of HSP70-2 (+1267A/G) and TNF-α (+308A/G) gene polymorphisms with HSP in children. The polymerase chain reaction restriction fragment length polymorphism method was used to detect the HSP70-2 and TNF-α polymorphisms in 205 cases of children with HSP and 53 controls; and the association of these polymorphisms with HSP and HSP nephritis (HSPN) was analyzed. The G/G genotypic frequencies at the +1267A/G position of HSP70-2 in the HSP group (22.9%) were significantly higher than those in the healthy control group (9.4%) (χ(2)=4.764, P<0.05). The frequencies of the A/A, A/G and G/G genotypes of HSP70-2 in patients in the nephritis-free group and the HSPN group showed no statistically significant difference. The A/A genotype frequency at the +308G/A position of TNF-α in the HSP group was 8.3%, which was higher than that in the control group (χ(2)=6.447, P<0.05). The A allele frequency of TNF-α in the HSP group was higher than that in the control group, with a statistically significant difference (χ(2)=7.241, P<0.05). The HSP70-2 (+1267A/G) and TNF-α (+308G/A) gene polymorphisms were associated with HSP in children. The G/G homozygosity of HSP70-2 and the A/A homozygosity of TNF-α may be genetic predisposing factors for HSP.

Construction and identification of an RNA interference lentiviral vector targeting the mouse TNF-α gene.

The aim of this study was to construct RNA interference (RNAi) lentiviral vector particles targeting the mouse tumor necrosis factor-α (TNF-α) gene. Three types of small interfering RNA (siRNA) targeting the mouse TNF-α gene were designed, synthesized and transfected into RAW264.7 cells. Screening was performed to identify the siRNA sequence exhibiting the highest inhibition efficiency; based on this, recombinant lentiviral plasmids were constructed and co-transfected into 293T cells with packaging plasmids for the production of lentiviral particles. The screening results showed that the TNF-α mRNA expression levels of the three siRNA groups were significantly lower than those of the negative control group, with the highest inhibition rate in the siRNA2 group (83.09%). Similarly, the expression levels of TNF-α protein in the three siRNA groups were significantly lower than those of the negative control group, and the highest inhibition rate was found in the siRNA2 group (51.16%). The mRNA expression of interleukin (IL)-1β and IL-6 showed no significant difference among the siRNA groups and the negative control. The recombinant lentiviral shuttle plasmid was constructed, and electrophoresis revealed the polymerase chain reaction product to be 343 bp, while that of the empty vector was 306 bp; DNA sequencing showed partial insertion. The virus titer was calculated to be 2×106 TU/µl. In conclusion, RNAi lentiviral vector particles targeting the mouse TNF-α gene were successfully obtained in the present study. This method may be used to produce lentiviral vector for the in vivo study of RNAi gene therapy targeting TNF-α.

Advances in molecular genetics of exfoliation syndrome

Exfoliation syndrome (XFS), one of the most common causes of glaucoma, represents an age related, complex, multifactorial and late-onset disease worldwide. The etiopathogenesis involves both genetic and environmental factor. However, the exact etiopathogenesis of XFS is still unclear. The purpose of this review was to discuss the recent research progress of the molecular genetics of XFS. Some candidate genes linked to XFS include lysyl oxidase-like 1 (LOXL1) gene, clusterin (CLU)gene, contactin associated protein-like 2 (CNTNAP2) gene, apolipoprotein E (ApoE) gene, matrix metallo proteinases (MMPs) gene, glutathione S-transferase (GST) gene, transforming growth factor-β1(TGF-β1) gene, tumor necrosis factor-α (TNF-α)gene and so on. These genes may be modifying genes for the development of XFS. Key words: Exfoliation syndrome; Molecular genetics; Gene

Systematic Review by Multivariate Meta-analyses on the Possible Role of Tumor Necrosis Factor-α Gene Polymorphisms in Association with Ischemic Stroke.

A number of studies have investigated the association between tumor necrosis factor (TNF)-α gene polymorphisms and ischemic stroke susceptibility. However, results of different individual studies are often inconsistent. To provide a more robust evaluation of the association between polymorphisms of the TNF-α gene and ischemic stroke risk, we performed a systematic review with multivariate meta-analyses. PubMed, Embase, CNKI, and WanFang databases were searched up to December 20, 2014. Two reviewers independently extracted information and assessed quality of included studies after all the eligible studies were identified. Afterward, multivariate meta-analyses were performed using Stata 13. The estimation of polymorphisms and disease risk was presented by odds ratios (ORs) and corresponding 95 % confidence intervals (CIs). Forty-nine eligible case-control studies from 25 articles that explored the association between 10 TNF-α polymorphisms and ischemic stroke were indentified from aforementioned databases. The results of multivariate meta-analysis showed a significant association between -238G/A polymorphism (4760 patients and 4389 controls) and ischemic stroke risk in heterozygotes compared with wild genotype (AG vs. GG: OR 1.44, 95 % CI 1.11-1.87; AA vs. GG: OR 1.98, 95 % CI 0.73-5.40). No significant association of -308G/A, -857C/T, and -1031T/C polymorphisms was observed. The results of stratification analyses of -238G/A polymorphism showed that the AG genotype only increased the risk of ischemic stroke in Asians compared to GG genotype. No additional significant association was observed in this study. In conclusion, the present systematic review and meta-analysis support a prominent role of the TNF-α -238G/A polymorphism in the risk of ischemic stroke in Asian adults only, but do not support the role of -308G/A, -857C/T, -1031T/C, -244G/A, -367G/A, -646G/A, -806C/T, -863C/A, and +448G/A in the risk of ischemic stroke. The current evidence warrants further studies with high quality and large sample size to confirm.

Tumour Necrosis Factor-α Gene Polymorphism Is Associated with Metastasis in Patients with Triple Negative Breast Cancer.

Tumour necrosis factor-α (TNF-α) is critical in the regulation of inflammation and tumour progression. TNF-α-308G > A is associated with constitutively elevated TNF-α expression. The purpose of this study was to assess the association between TNF-α-308G > A and breast cancer (BC) risk by subtype and the connection between genotypes and clinical features of BC. A total of 768 patients and 565 controls were enrolled in this study, and genotypes were detected using the TaqMan assay. No effect on susceptibility for any BC subtype was found for the TNF-α-308 polymorphism in our study or in the pooled meta-analysis. This polymorphism was shown to be associated with age at menarche in all BC and in progesterone receptor-negative BC. Interestingly, triple negative breast cancer (TNBC) patients with TNF-α-308A had an increased risk of distant tumour metastasis (OR = 3.80, 95% CI: 1.31–11.02, P = 0.009). Multi-regression analysis showed that TNF-α-308A was also a risk factor for distant tumour metastasis after adjustment for tumour size and lymph node metastasis status (OR = 6.26, 95% CI: 1.88–20.87, P = 0.003). These findings indicate that TNF-α might play a distinct role in the progression of TNBC, especially in distant tumour metastasis of TNBC.

TNF-a (-238G/A and -308G/A) gene polymorphisms may not contribute to the risk of ischemic stroke

Background: The common and major pathological change in ischemic stroke is atherosclerosis in the artery. Tumor necrosis factor-a (TNF-a) is closely related to the pathogenesis of atherosclerosis. The aim of our study was to investigate whether TNF-a gene variants (-238G/A and -308G/A) are associated with ischemic stroke. Methods: A total of 619 ischemic stroke patients and 612 controls were recruited to estimate the frequencies of two TNF-a (-238G/A and -308G/A) single nucleotide polymorphisms using a Sequenom MassARRAY time-of-flight mass spectrometer. The association between TNF-a gene polymorphisms and ischemic stroke risk was evaluated by computing the odds ratio (OR) and 95% Confidence Interval with multivariate unconditional logistic regression analyses. Results: The OR results indicated that no significant associations were found between TNF-a gene (-238G/A and -308G/A) polymorphisms and the risk of ischemic stroke using five genetic models, including the allele model (A vs. G), co-dominant model 1 (GA vs. GG), co-dominant model 2 (AA vs. GG), the dominant model (AA+GA vs. GG), and the recessive model (GG+GA vs. AA). Conclusions: The TNF-a (-238G/A and -308G/A) gene polymorphisms may not be a susceptible predictor of ischemic stroke in Chinese populations.

13 Tumor necrosis factor-α gene expression before and after exercise in middle-aged and older horses

s / Journal of Equine Veterinary Science 35 (2015) 383e391 388 13 Tumor necrosis factor-a gene expression before and after exercise in middle-aged and older horses M.M. Darby*, S.R. Malone , D.W. Horohov , and D.E. Chappell 1 Morehead State University, Morehead, KY, USA; University of Kentucky, Lexington, KY, USA Tumor necrosis factor-a (TNF-a) is a pro-inflammatory cytokine that is expressed at different levels depending on the age of the horse. Age influences the animal's ability to launch an appropriate immune response and as horses age they exhibit a lower inflammatory response. Twenty-six adult horses (n 1⁄4 8 mares; n 1⁄4 18 geldings) were used to test the hypothesis that older horses would have higher pre-exercise TNF-a values and lower postexercise values than middle-aged horses. Horses were used in a University riding program andwere ridden for approximately one hour undergoing exercise of varying intensity. Blood samples (~4 mL) were collected via venipuncture into PAXgene tubes before exercise (pre) and 3 h after exercise (post). Blood samples were analyzed using real-time polymerase chain reaction (PCR) with equine-specific primers. Statistical significance was set at P < 0.05 and analysis was done using an ANOVA. Horses were split into age groups, middle-aged (<15 years; n 1⁄4 13; mean age 10 years; range 7e14 years) and older (1⁄4 15 years; n 1⁄4 13; mean age 17.6 years; range 15e21 years). The results are displayed as the mean ± SEM. Older horses had similar (P 1⁄4 0.24) pre-exercise TNF-a mRNA expression as middle-aged horses (middle-aged 0.075 ± 0.06; older 0.18 ± 0.07). Post-exercise there was no significant difference (P 1⁄4 0.119) between groups for TNF-a mRNA expression (middle aged 0.32 ± 0.08; older 0.14 ± 0.07). The magnitude of the response to exercise was similar in both groups of horses. Older horses had slightly, but not significantly, higher TNF-a mRNA expression pre-exercise and this could be expected due to chronic inflammation or underlying conditions. Future studies may focus on a more diverse age population with a wider gap betweenmiddle-aged and older horses andmore control over underlying medical conditions.

Isolation of human genomic DNA from archival dried blood spots for neonatal disease screening and its application to methylation detection

Objective To establish an effective DNA isolation method for neonatal disease screening, so as to explore its application to the methylation detection. Methods The 20 dried blood spots samples were randomly divided into 2 groups according to the gender: the traditional method group (n=10) and the improved kit method group(n=10). The DNA quality was evaluated based on its concentration, integrity and whether it could be used in polymerase chain reaction (PCR). These DNA samples with or without bisulfite treatment were used as template in the methylation-specific polymerase chain reaction (MSP). The methylation levels of Leptin and tumor necrosis factor-α (TNF-α) gene promoter region were detected. Results DNA concentration of the improved kit method [(5.70±0.81)mg/L] was significantly higher than that of the traditional method[(3.50±0.45) mg/L](t=2.79, P<0.05), and biochemical analyzer analysis showed a better DNA integrity.Agarose gel electrophoresis revealed that 18S gene fragment could be successfully amplified by PCR method, suggesting its potential application to PCR study.MSP results showed diffe-rent DNA methylation levels of Leptin and TNF-α genes promoter regions from various samples. Conclusions The improved kit method can effectively extract DNA from dried blood spots samples, and these DNA can be used in methy-lation research.The study can provide a new research direction and technical method to reveal the pathogenesis of disease from the perspective of DNA methylation. Key words: Archival dried blood spot; Isolation of human genomic DNA; Methylation; Methylation-specific polymerase chain reaction

Boldenone-induced apoptotic, structural, and functional alterations in the liver of rabbits

Boldenone undecylenate (BOL) is an anabolic androgenic steroid used in livestock to improve growth and food conversion. This study investigated the actions of BOL on structure and functions of rabbit liver as well as the effects of its withdrawal. Eighteen mature male New Zealand rabbits were divided into 2 groups: Control group (n=6) were injected with 0.25 mL corn oil/kg body weight (BW), while BOL group (n=12) received 3 intramuscular injections, 2 wk apart, of BOL (4.5 mg/kg BW). Animals were scarified 1 d after last injection except for 6 rabbits from BOL group that served as the BOL-withdrawal group (4 wk after the 3rd injection). Intramuscular injection of BOL increased ( P <0.05) malondialdehyde (MDA) level, but markedly lowered activities of superoxide dismutase (SOD) and catalase (CAT) and reduced glutathione (GSH) concentration compared to both control and BOL-withdrawal groups. Treatment with BOL significantly ( P <0.05) increased serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) compared to the control group. BOL injection caused different histopathological alterations and apoptosis in liver, but these changes were less evident in the BOL-withdrawal group. Expression of p53 and tumour necrosis factor-α (TNF-α) genes was up regulated in BOL compared to control group, while the expressions of p53 and TNF-α were down regulated in BOL-withdrawal group in comparison with BOL group. In conclusion, BOL injection induced structural and functional changes in the liver of rabbits, increasing oxidative stress and mediators of apoptosis such as ROS, p53 and TNF-α. All these parameters returned to near the control values after withdrawal.

Macrophage–sensory neuronal interaction in HIV-1 gp120-induced neurotoxicity

BackgroundHuman immunodeficiency virus (HIV)-associated sensory neuropathy (SN) is the most frequent neurological complication of HIV disease. Among the probable mechanisms underlying HIV-SN are neurotoxicity induced by the HIV glycoprotein gp120 and antiretroviral therapies (ART). Since HIV-SN prevalence remains high in patients who have not been exposed to toxic ART drugs, here we focused on gp120-mediated mechanisms underlying HIV-SN.MethodsWe hypothesized that a direct gp120–sensory neurone interaction is not the cause of neurite degeneration; rather, an indirect interaction of gp120 with sensory neurones involving macrophages underlies axonal degeneration. Rat dorsal root ganglion (DRG) cultures were used to assess gp120 neurotoxicity. Rat bone marrow-derived macrophage (BMDM) cultures and qPCR array were used to assess gp120-associated gene expression changes.Resultsgp120 induced significant, but latent onset, neurite degeneration until 24 h after application. gp120–neurone interaction occurred within 1 h of application in <10% of DRG neurones, despite neurite degeneration having a global effect. Application of culture media from gp120-exposed BMDMs induced a significant reduction in DRG neurite outgrowth. Furthermore, gp120 significantly increased the expression of 25 cytokine-related genes in primary BMDMs, some of which have been implicated in other painful polyneuropathies. The C–C chemokine receptor type 5 (CCR5) antagonist, maraviroc, concentration-dependently inhibited gp120-induced tumour necrosis factor-α gene expression, indicating that these effects occurred via gp120 activation of CCR5.ConclusionsOur findings highlight macrophages in the pathogenesis of HIV-SN and upstream modulation of macrophage response as a promising therapeutic strategy.

Oral administration of melatonin modulates the expression of tumor necrosis factor-α (TNF-α) gene in irradiated rat cervical spinal cord

AimWe aimed to determine the changes in TNF-α expression and Malondialdehyde (MDA) level in a short time after irradiation. Furthermore, we evaluated the effect of melatonin on the modulation of TNF-α gene expression. BackgroundThe radio-sensitivity of the cervical spinal cord limits the dose of radiation which can be delivered to tumors in the neck region. There is increasing evidence that TNF-α has a role in the development of the acute phase of spinal cord injury. Materials/MethodsFour groups of rats were investigated. Group 1 (vehicle treatment) served as the control. Group 2 (radiation) was treated with the vehicle, and 30min later, the rats were exposed to radiation. Group 3 (radiation+melatonin) was given an oral administration of melatonin (100mg/kg body weight) and 30min later exposed to radiation in the same manner as in group 2. Group 4 (melatonin-only) was also given an oral administration of melatonin (100mg/kg body weight). 5mg/kg of melatonin was administered daily to rats in groups 3 and 4, and the vehicle was administered daily to rats in groups 1 and 2. ResultsThree weeks after irradiation, TNF-α gene up-regulated almost 5 fold in the irradiated group compared to the normal group. TNF-α gene expression in the melatonin pretreatment group, compared to the radiation group, was significantly down-regulated 3 weeks after irradiation (p<0.05). MDA levels increased after irradiation and then significantly decreased under melatonin treatment. ConclusionWe suggest that inhibition of TNF-α expression by oral administration of melatonin may be a therapeutic option for preventing radiation-induced spinal cord injury.

Antioxidant and anti-inflammatory activities of 3,5-dicaffeoylquinic acid isolated from Ligularia fischeri leaves

A potent anti-inflammatory compound was isolated and identified from leaves of Ligularia fischeri. The ethyl acetate fraction was used for purification of the compound and yielded 3,5-dicaffeoylquinic acid (DCQA). The identity of the compound was confirmed using 1H, 13C NMR, and MS analysis. DCQA showed strong DPPH (IC50=4.26 μg/mL), ABTS (TEAC value=0.9974) radical scavenging, and FRAP activities (=3.84 mmole of Trolox equivalent/g). DCQA effectively inhibited production of NO in RAW 264.7 cells induced using lipopolysaccharide (LPS) in a concentration dependent manner (50–250 μg/mL). DCQA significantly suppressed (p<0.05) up-regulation of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and tumor necrosis factor-α (TNF-α) gene expressions. The isolated DCQA can be useful for development of effective anti-inflammatory agents.

Establishment of a mouse model to express bovine CD14 short hairpin RNA.

BackgroundCluster of differentiation 14 (CD14) functions as a co-receptor for Toll-like receptor (TLR)-4 and myeloid differentiation factor (MD)-2 in detecting bacterial lipopolysaccharide. Together, these complexes promote the phagocytosis and digestion of Gram-negative bacteria, and initiate immune responses. To date, much of our understanding of CD14 function during Gram-negative bacterial inflammation comes from studies on mouse knockout models and cell transfection. To identify the effect of CD14 knockdown in this process in large livestock animals, we established a mouse model expressing bovine CD14 short hairpin (sh) RNA. shRNA fragments targeting bovine CD14 were screened by co-transfection in HEK 293 cells, and the most effective CD14 shRNA fragment was cloned into the eukaryotic expression vector pSilencer4.1-CD14 shRNA-IRES (internal ribosome entry site) and transferred into mouse zygotes by pronuclear microinjection to obtain transgenic mice. Expression of the enhanced green fluorescent protein (EGFP) reporter and genes related to the TLR4 signaling pathway was detected by immunohistochemistry (IHC) and quantitative polymerase chain reaction (PCR), respectively.ResultsOne effective shRNA fragment (shRNA-674) targeting bovine CD14 was obtained, the sequence of which was shown to be conserved between cows, buffalos, sheep, and humans. Thirty-seven founder pups were obtained by pronuclear microinjection, of which three were positive for the transgene. In the F1 generation, 11 of 33 mice (33%) were positive for the transgene as detected by PCR. IHC analysis detected exogenous EGFP expression in the liver, kidney, and spleen of transgenic F1 mice, indicating that they were chimeric. The expression of endogenous CD14 mRNA in the heart, liver, spleen, lung, and kidney of transgenic F1 mice was decreased 8-, 3-, 19.5-, 6-, and 11-fold, respectively. The expression patterns of endogenous MD-2, TLR4, interleukin-6 and tumor necrosis factor-α genes in transgenic mice also varied.ConclusionsThis study confirms that transgenic mice expressing bovine CD14 shRNA can be generated by pronuclear microinjection, and demonstrates inhibited endogenous mouse CD14 expression that alters gene expression related to the TLR4 signaling pathway.

Change in soluble epoxide hydrolase (sEH) during cisplatin-induced acute renal failure in mice.

Cisplatin is one of the most effective chemotherapeutic agents against various types of cancers; however, it is also associated with nephrotoxicity. Recently, it was reported that inflammatory mechanisms play a key role in the development of nephrotoxicity. Epoxyeicosatrienoic acids (EETs) have an anti-inflammatory effect and are metabolized by soluble epoxide hydrolase (sEH: encoded by EPHX2 gene). Here, we determined the change in sEH activity and EPHX2 expression in renal tissue associated with the development of cisplatin-induced nephrotoxicity. Cisplatin administration decreased hydrolase activity accompanied by down-regulation of sEH and EPHX2 expression. The down-regulation occurred prior to the elevation of blood urea nitrogen (BUN) and tumor necrosis factor-α (TNF-α) gene expression or at treatment with low dose cisplatin. In addition, a negative correlation was found between EPHX2 expression and renal thiobarbituric acid reactive substance (TBARS), and edaravone, a radical scavenger, administration did not down-regulate expression of this gene. The results of this study suggest that cisplatin decreased sEH activity through the down-regulation of sEH and EPHX2 expression, and this down-regulation was involved in a negative feedback loop to protect renal tissue from further damage. Thus, sEH is a potential therapeutic target of cisplatin-induced nephrotoxicity.