Trypanosoma cruzi (T. cruzi) acutely-infected mice. We found increased expression and deposition of TNF-α in the thymus of infected animals compared to controls, accompanied by increased co-localization of fibronectin, a cell migration-related ECM molecule, whose contents in the thymus of infected mice is also augmented. In-vivo studies showed an enhanced export of thymocytes in T. cruzi-infected mice, as ascertained by intrathymic injection of FITC alone or in combination with TNF-α. The increase of immature CD4+CD8+ T cells in secondary lymphoid organs was even more clear-cut when TNF-α was co-injected with FITC. Ex-vivo transmigration assays also revealed higher number of migrating cells when TNF-α was added onto fibronectin lattices, with higher input of all thymocyte subsets, including immature CD4+CD8+. Infected animals also exhibit enhanced levels of expression of both mRNA TNF-α receptors in the CD4+CD8+ subpopulation. Our findings suggest that in T. cruzi acute infection, when TNF-α is complexed with fibronectin, it favours the altered migration of thymocytes, promoting the release of mature and immature T cells to different compartments of the immune system. Conceptually, this work reinforces the notion that thymocyte migration is a multivectorial biological event in health and disease, and that TNF-α is a further player in the process.Previous studies revealed a significant production of inflammatory cytokines together with severe thymic atrophy and thymocyte migratory disturbances during experimental Chagas disease. Migratory activity of thymocytes and mature T cells seem to be finely tuned by cytokines, chemokines and extracellular matrix (ECM) components. Systemic TNF-α is enhanced during infection and appears to be crucial in the response against the parasite. However, it also seems to be involved in disease pathology, since it is implicated in the arrival of T cells to effector sites, including the myocardium. Herein, we analyzed the role of TNF-α in the migratory activity of thymocytes in  Trypanosoma cruzi is the etiologic agent of Chagas disease. Despite substantial progress over the last years in relation to the immunopathology of the disease, the role of the thymus in the course of infection and pathogenesis remains unclear. Severe thymic alterations were observed during experimental Trypanosoma cruzi-acute infection. Thymuses in infected mice develop a progressive atrophy, mainly due to a depletion of CD4+CD8+ double-positive thymocytes +CD8+ cell depletion seems to be linked to a severe stress-triggered apoptosis +CD8+ cells bearing prohibited TCR Vβ segments in secondary lymphoid organs +CD8+ cells +CD8+ cells seems to be influenced by the interaction between fibronectin and the chemokine CXCL12 T. cruzi infection seems to be dual. Control of human and experimental T. cruzi infection is critically dependent on TNF-α activity but its overproduction is detrimental to the host and contributes to disease pathology T. cruzi-triggered thymic atrophy is at least partly dependent on hypothalamus-pituitary-adrenal axis activation, which promoted intrathymic apoptosis of CD4+CD8+ cells by exposure to increased levels of adrenal glucocorticoid hormones The role of tumor necrosis factor-alpha (TNF-α) in T. cruzi-infected mice, TNF-α modulates migratory responses, inducing chemokine expression at systemic level, probably down-regulating CCR5 expression T. cruzi-infected TNF-α receptor double knockout mice (p55−/− plus p75−/−) inflammatory infiltrates in the myocardium are absent T. cruzi infection migration of peripheral T cells is influenced by TNF-α.Processes related to thymocyte migration could be also mediated by TNF-α, but at present, data on a putative role of TNF-α modulating cell migration in the thymus remains lacking. Yet, this hypothesis seems plausible since in T. cruzi infection, systemic or intrathymic production of TNF-α could modulate thymocyte migration, either by itself or in combination with ECM molecules, particularly fibronectin.Under normal conditions, intrathymic cell migration and thymocyte export are complex processes, regulated at least by ECM proteins such as fibronectin and laminin, as well as chemokines T. cruzi infection in mice, we explored herein the role of TNF-α in intrathymic T cell migration, as well as thymocyte export to peripheral lymphoid organs.Taking into account previous results showing disturbances of thymocyte migratory properties during T. cruzi (Clonotype II) were maintained by serial passages in suckling mice. Heparinized blood obtained from infected animals was diluted in saline and washed twice. Live parasites were counted using Neubauer chambers. Mice were infected subcutaneously with 100 viable trypomastigotes. To monitor the systemic repercussion of the acute disease, parasitaemia and the survival time was recorded following infection as described before Male C57BL/6 mice, aging 6–8 weeks, were obtained from the animal facilities at Rosario Medical School and Oswaldo Cruz Foundation. All animal procedures were performed according to protocols for animal care and use, approved by each Institutional Ethical Committee , Resolution N° P-0145-02; and Faculty of Medical Sciences from National University of Rosario, Bioethics and Biosecurity Committees, Resolution N°3740/2009). Trypomastigotes of the Tulahuén strain of 4Cl for red cell lysis.Thymuses, subcutaneous lymph nodes and spleens were removed, minced, washed and resuspended in PBS containing fetal calf serum 5% . Spleen samples were further treated with NH6 cells were resuspended in flow buffer and incubated with a given specific monoclonal antibody for 30 minutes at 4°C in the dark . Once defined the lymphocyte gate, 30,000 events were acquired. Background staining values obtained with fluorochrome matched-conjugate isotype controls were subtracted.For immunostaining, 1.10For recent thymic emigrants (RTEs) determination (see below), cells from mice injected with physiological saline alone were used to define positive FITC labeling in the other groups.In cell migration experiments, migrating cells were also labelled with the appropriate antibodies, fixed with paraformaldehyde 1% and analyzed by flow cytometry. Monoclonal antibodies used to stain cell surface molecules were: PE/anti-CD4, PercP/anti-CD4, PE/anti-CD8, PercP/anti-CD8, APC/anti-CD8, APC/anti-CD3 and PE/anti-TCR (BD Pharmingen). In all cases, dead cells were gated out on the basis of forward- and side-cell scatter. Fluorochrome-labeled isotype-matched negative controls for the specific antibodies were also used (BD Pharmingen). Acquisition of events was done using an FACSAria or FACScalibur flow cytometers (BD Pharmingen). Results were analyzed using the WinMDI 2.8 software .Mice were bled by cardiac puncture at different days post-infection. Blood was centrifuged during 10 min at 1,500 rpm at −4°C. Serum was stored frozen at −20°C until analyzed. At the same time, thymuses were removed, weighted and homogenized in 2 volumes of 300 mmol/L sucrose with protease inhibitors . Homogenates were kept at −80°C until used. TNF-α concentrations were measured in blood and tissue samples by a specific ELISA kit, according to manufacturer's specifications (BD Pharmingen). Detection limit of the technique corresponds to 15.6 pg/ml.In further experiments, intrathymic TNF-α was detected by means of immunoblotting. Lysates of thymuses were prepared in 4 volumes of 300 mmol/L sucrose with protease inhibitors: 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 1 µg/ml aprotinin . Samples (20 µg of protein) were subjected to electrophoresis under reducing conditions on a 15% SDS-PAGE, and transferred onto nitrocellulose membranes. After blocking (5% non fat milk during 2 h at room temperature), blots were incubated overnight at 4°C with rat anti-mouse TNF-α monoclonal antibody (clone MP6-XT3). Membranes were then incubated in blocking buffer with anti-rat Ig G-peroxidase conjugate  and bands were detected by enhanced chemiluminescence kit (Amersham). Autoradiographs were obtained by exposing PVDF membranes to Kodak XAR film, and the bands were quantitated by densitometry, using a Shimadzu CS-9000 apparatus .Thymuses were removed 14 days after infection, embedded in Tissue-Tek  and frozen in liquid nitrogen. Five 5 µm-thick cryostat sections were settled on poly-L-lysine (Sigma)-covered glass slides, acetone fixed and blocked with PBS-BSA 1%. Samples were submitted to specific antibodies as follows: 1∶10 rat anti-mouse TNF-α monoclonal antibody  for 1 hour at room temperature, washed and submitted to appropriate secondary antibody, 1∶400 goat anti-rabbit Alexa 488 and 1∶400 goat anti-rat Alexa 546  respectively. Samples were analyzed by confocal microscopy using a LSM 510 Zeiss device (Germany) and the images obtained were subsequently analyzed using the Image J software .in vivo evaluation of RTEs. FITC efficiently and randomly labels thymocytes, enabling their subsequent identification in the peripheral lymph organs. Intrathymic FITC injections were performed as described by Scollay et al +CD4+CD8+ cells by flow cytometry. Only animals with more than 50% of FITC+ thymocytes were used for subsequent analysis. Peripheral cells were considered RTEs when FITC+ cells could be distinguished from, comparing with animals that only received intrathymic injection of saline. Data were evaluated as absolute numbers of FITC+ cells, and also through an index enabling to estimate the overall rate of emigration (ORE), adjusted to the absolute number of FITC+CD4+CD8+ cells at the periphery, in relation to corresponding secondary lymphoid organ cellularity and the percentage of labelled cells observed in the thymus.Intrathymic injection of fluoresceinisothiocyanate (FITC) allows direct 6 thymocytes in 100 µL of RPMI-1% BSA) were then added in the upper chambers. Migration medium was serum free, so that to avoid serum-derived fibronectin or other soluble cell migration stimuli. After 3 hours of incubation at 37°C in a 5% CO2 humidified atmosphere, migration was defined by counting the cells that migrated to the lower chambers containing migration medium alone (RPMI-1% BSA). Migrating cells were ultimately counted, labelled with appropriate antibodies and analyzed by flow cytometry. Differences in intrinsic cytokinesis between displayed thymocytes from infected or noninfected mice driven by TNF-α or fibronectin  were expressed after subtracting the value recorded in wells containing BSA alone. The percentage of each subset was used together with the total cell counting to calculate absolute numbers of each lymphocyte subset from each lymphoid organ. The results are presented in terms of total thymocyte migration as well as the percentages of input seen in each CD4/CD8-defined subpopulation, using the following formula:We evaluated thymocyte migratory response to TNF-α adhered to fibronectin. Briefly, inserts of 5 µm-pore size of transwell plates  were coated with 10 µg/mL of human fibronectin or 10 µg/ml BSA (Sigma) for 1 h at 37°C and then unspecific binding sites were blocked with BSA 10 µg/mL. Various concentrations of recombinant murine TNF-α (25 or 250 pg/mL) were diluted in 70 µL added to upper chamber, allowing adhesion to fibronectin (or BSA) during 15 min at 37°C. Unbound TNF-α was then removed by washing before adding thymic cells. Thymocytes were obtained at 14 days post-infection or from non-infected counterparts. Cells . For qPCR we used approximately 100 ng of cDNA for each sample and SYBR Green Master Mix 2 . cDNA was amplified using specific murine primer sequences: 5′-TCAAAGAGGAGAAGGCTGG-3′ (FP) and 5′-CACCAC AGCATACAGAATCG-3′ (RP) for TNF-R1; (5′ TGTAGCATCCTGGCTATTCC-3′ (FP) and 5′-ATGAAGCAGTTCACCAGTCC-3′ (RP) for TNF-R2 and 5′-GCTACAGCTTCACCACCACAG-3′ (FP) and 5′-GGTCTTTACGGATGTCAACGTC-3′ (reverse) for ACTB (β-actin). All reactions were performed in triplicate. After 45 cycles of amplification, TNF-R1 and TNF-R2 expression in each cell subset was normalized to the housekeeping gene ACTB  Expression of genes coding for both TNF-receptors (TNF-R1 and TNF-R2) was evaluated by real-time PCR. In each experimental condition, thymocytes subsets were FACS isolated from three pooled thymuses stained with anti-CD4 Percp and anti-CD8 APC antibodies, in a MoFlocell sorter . All subsets were enriched to a purity of greater than 98%. After sorting, RNA was extracted using a commercially available kit . Total RNA concentrations were estimated by Ap value was ≤0.05.Differences in quantitative measurements were assessed by the Kruskall-Wallis non parametric analysis of variance and Mann-Whitney U test. Correlations were evaluated by Pearson test. Results were expressed as mean ± standard error (SE) unless otherwise indicated. The GraphPad Instat 4.0 software  was applied for statistical analyses, and differences were considered significant when +CD8+ thymocytes (data not shown) was seen along with infection in C57BL/6 infected mice, thus confirming previous results. These alterations occurred in parallel with a systemic and intrathymic increase in TNF-α contents. TNF-α rising became evident 14 days post-infection in both thymus extracts and serum , we next evaluated whether thymic contents of TNF-α co-localized with fibronectin. Confocal microscopy data showed that co-localization of TNF-α and fibronectin was increased after infection , as well as spleens, were removed and the FITC+ CD4/CD8-defined T cell subsets were ascertained by cytofluorometry  by adjusting the absolute number of FITC+CD4+CD8+ cells in the periphery in relation to the cellularity of a given peripheral lymphoid organ and the percentage of labelled observed in thymus. As seen in +CD4+CD8+ cells compared with FITC alone in both secondary lymphoid organs of infected animals. We did not notice differences in values from FITC+CD4+ RTEs in the spleen after TNF-α intrathymic injection, but, evaluation of ORE values suggest a TNF-α-driven enhancement of FITC+CD4+ and FITC+CD8+ cells in the lymph nodes from infected mice.Taking into account that the absolute numbers of FITCTNF-α lacks chemotactic activity by itself when placed in its soluble form in the bottom well of transwell cell migration chambers. Accordingly, migratory activity of thymocytes through membranes treated with BSA plus TNF-α was similar to that seen on BSA alone (data not shown).versus fibronectin plus TNF 250 pg/mL in both control and infected groups, we found p = 0.06).To evaluate a haptotactic capacity of TNF-α, the recombinant cytokine was placed in the upper chamber 15 min before adding thymocytes. This procedure allows TNF-α to be deposited on the pre-formed fibronectin lattice. As previously reported + cells from infected mice have a significantly higher migratory response than non-infected animals when they migrated through fibronectin alone, or through fibronectin plus 25 pg/mL TNF-α coated wells (group referred as FN+TNF25)  compared with fibronectin alone; suggesting that TNF-α interacts with fibronectin mobilizing thymocytes from infected but not from control animals.For a more precise interpretation of these data, we evaluated the specific migration of CD4/CD8-defined thymocyte subsets (expressed as percentage of input for each subset), taking into account that the relative amounts of distinct CD4/CD8-defined thymocyte subpopulations changed after infection. We noticed that CD4N+TNF25) . FurtherT. cruzi infected mice might be related to the enhanced expression of integrin-type fibronectin receptors VLA-4 and VLA-5 T. cruzi infected mice. By contrast, TNF-R1 or TNF-R2 mRNA were similar in CD4+ or CD8+ subpopulations, independently of whether they derived from infected or control animals. Interestingly, in both conditions, the amounts of TNF-R1 and –R2 transcripts on the DP subset seemed greater than in SP cells, with a higher expression of the isoform 2 of this receptor , as CXCL12, should be informative.Other possibility to explain these data is that Our study is at variance with previous observations, in which TNF-α was proposed as a stop signal for leukocyte migration in vivo and in vitro, immature CD4−CD8− as well as CD4+CD8+ cells displayed a higher migratory response to fibronectin in the presence of TNF-α. These results not only confirm higher fibronectin-driven immature cell migration during the acute phase of infection A relevant aspect derived from our results is that, both per se, enhance fibronectin-triggered migratory response of these cells. In acutely-infected mice, the ultimate migration patterns reveal the abnormal release of immature CD4+CD8+ lymphocytes towards secondary lymphoid organs. Previous work strongly indicates that part of these immature cells released from the thymus in T. cruzi infected animals is potentially autoimmune, since they have bypassed the classic intrathymic negative selection We have previously proposed that thymocyte migration  is a multivectorial event, from the action of various ligand/receptor interactions; each one corresponding to a single migration vector T. cruziIn any case, the release of potentially autoimmune T cells may result in the development of autoimmunity, even in adult individuals. Importantly, recent data suggest that the occurrence of immature T cells in the periphery of the immune system is extended during chronic phase in both mice and patients infected with +CD8+ cell homing to secondary lymphoid organs might be related to a selective expression of fibronectin receptors (VLA-4 and VLA-5) and also with TNF-receptors (TNF-R1 and TNF-R2). Accordingly, during T. cruzi infection, VLA-4 and VLA-5 are enhanced in CD4+CD8+ cells +CD8+ subpopulation, when we observed an increased export of this subset modulated by intrathymic TNF-α contents. Moreover, the relative amount of mRNA TNF-R2 seems to be higher than the TNF-R1 in these cells. This is in keeping with the putative role of TNF-R2 in cell migration events +CD8+ cell migration may be favored by up-regulation of fibronectin and TNF-α receptors.The enhanced CD4T. cruzi, but at the same time are involved in the autoimmune phenomenon seen in chronic Chagas disease T cells are crucial in protective immunity against


The role of tumor necrosis factor alpha (TNF-α) in cancer is complex with both apoptotic and anti-apoptotic roles proposed. However the mechanism is not clear. In the study, we designed to investigate the effect of TNF-α on the activation and expression of nuclear factor kappa B (NF-κB)/p65/SLUG/PUMA/Bcl-2 levels in human lung cancer A549 cell line, and in conditions of TNF-α-induced apoptosis.in vitro effects of siRNA on apoptosis, and sensitivity to 20 ng/ml of TNF-α treatment for 24–48 h.We have engineered three A549 cell lines that were transiently transfected with PUMA siRNA, SLUG siRNA and Bcl-2 siRNA, respectively. We have measured the We found the NF-κB activity and PUMA mRNA/protein was significantly increased after treatment of TNF-α for 24 h in untreated A549 cells, and led to a significant increase in TNF-α-induced apoptosis, no significant increase of SLUG and Bcl-2 level was shown. However, after treatment of TNF-α for 48 h in untreated A549 cells, SLUG and Bcl-2 level was significant increased, and PUMA level was significant decreased, and TNF-α-induced apoptosis was significantly decreased compared to the apoptosis level after treatment of TNF-α for 24 h. Inhibition of the NF-κB activity could effectively decrease the PUMA level and increase the SLUG and Bcl-2 level. PUMA silencing by siRNA led to a significant decrease in TNF-α-induced apoptosis after treatment of TNF-α for 24 h. Bcl-2 and SLUG silencing by siRNA led to a significant increase in TNF-α-induced apoptosis for 48 h. Furthermore, SLUG silencing increased PUMA level and decreased Bcl-2 level.The findings suggested that TNF-α treatment promoted apoptosis via the NF-κB-dependent PUMA pathway. The anti-apoptotic role of TNF-α was via NF-κB-dependent SLUG and Bcl-2 pathway at a later time. TNF-α[GenBank:AAN76506] is a potent pleiotropic cytokine and is a major mediator of inflammation with multiple biological functions . SeveralPUMA is a downstream target of p53 and a BH3-only B-cell CLL/lymphoma-2 (Bcl-2)[NCBI Reference Sequence:NR073605] family member. It is induced by p53 following exposure to DNA-damaging agents, such as γ-irradiation and commonly used chemotherapeutic drugs . It is aIt has been reported that TNF-α-induced apoptosis is through the NF-κB-dependent PUMA pathway in colon cancer cells . Simultaet al. have shown that TNF-α upregulates SLUG via the NF-κB/HIF1alpha axis, which imparts breast cancer cells with a stem cell-like phenotype  upregulation . In lungLung cancer is the most frequently occurring cancer in the world and causes more deaths in China. Despite advances in treatment modalities including radiation, surgery and chemotherapy, the overall survival in lung cancer remains low. TNF-α has been shown to regulate both apoptotic and anti-apoptotic pathways. In the present study, we investigated the effect of TNF-α on apoptosis in lung cancer A549 cells, and explored its mechanism. We demonstrated that TNF-α treatment promoted apoptosis via the NF-κB-dependent PUMA pathway at an early time. The anti-apoptotic role of TNF-α was via the Bcl-2 upregulation and PUMA downregulation by NF-κB-dependent SLUG upregulation at a later time.2. The inhibitor of nuclear factor kappa-B (IKK), wedelolactone, was obtained from Alexis Biochemical . Recombinant human TNF-α was from R&D Systems . Antibodies for p65/PUMA/SLUG/Bcl-2/goat anti-rabbit IgG-HRP/goat anti-mouse IgG-HRP are from Santa Cruz Biotechnology . Anti-β-actin antibody were purchased from Sigma , and Lamin A antibody from Abcam .The human non-small cell lung cancer (NSCLC) cell line A549 was obtained from the American Type Culture Collection, and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 100 IU/ml penicillin/streptomycin in a 37°C humidified incubator with 5% COTransfection was performed with Lipofectamine 2000  following the manufacturer’s instructions. siRNA transfection was performed 24 h before 20 ng of TNF-α treatment. siRNA duplexes, including PUMA siRNA, SLUG siRNA and Bcl-2 siRNA and the control scrambled siRNA were from Santa Cruz Biotechnology.Cells were plated in 12-well plates at 20 to 30% density 24 h before treatment. Human TNF-α (20 ng) was diluted with appropriate cell culture media. In some experiments, cells were pretreated with 25 μM of the IKK inhibitor, wedelolactone, for 12 h before addition of 20 ng of TNF-α.′-gacacacatacagtgattatt-3′ and 5′-aaacttttcagcttcaatggc-3′; PUMA, 5′-cgacctcaacgcacagtacga-3′, and 5′-aggcacctaattgggctccat-3′; Bcl-2, 5′-cgacgacttctcccgccgctaccgc-3′; reverse, 5′-ccgcatgctggggccgtacagttcc-3′; GAPDH, 5′-acatcgctcagacaccatgg-3′ and 5′-gtagttgaggtcaatgaaggg-3′. The PCR products were fractionated on a 2% agarose gel and visualized after ethidium bromide staining. After 3 minutes at a temperature of 95°C, the experimental reaction consisting of 30 cycles at 95°C for 30 s, 58°C for 60 s, and 72°C for 30 s, and the PCR products were analyzed by gel electrophoresis. The relative expression of mRNA was quantitatively measured using densitometry analysis by gene expression analysis software.Total RNA was extracted from cultured cells with Isogen  according to the manufacturer’s instructions. Random-primer, first strand cDNA was generated from 0.5 μg total RNA with a Takara RNA PCR kit , and the PCR analysis was performed using gene-specific primers and first strand cDNA as the template. The sequence of primers was as follows: SLUG, 5Cytoplasmic and nuclear fractions from cells were prepared using the NE-PER nuclear and cytoplasmic Extraction kit . Cells were lysed with RIPA buffer  containing protease inhibitor cocktail  and 2 mM sodium vanadate. Protein concentration was determined by BCA protein assay reagent kit (Pierce). Equal amounts of cell lysates were separated by 10% SDS-PAGE, electrophoretically transferred to nitrocellulose membrane , immunoblotted with primary antibody against p-p65/P65/SLUG/PUMA/Bcl-2/β-actin. Blots were visualized with anti-rabbit or anti-mouse IgG conjugated with horseradish peroxidase (HRP) and ECL reagents (Pierce).Cell extracts were prepared using a commercially available nuclear extraction kit according to the manufacturer’s protocol (Pierce). Electrophoretic mobility shift assay was performed according to the provided protocol . In brief, a 21-mer oligonucleotide corresponding to the consensus NF-κB site (Promega) was radiolabeled with (γ-32P)-ATP by aT4 kinase reaction. Nucleotides were purified by chromatography through a G-25 spin column  equilibrated in TE buffer. Ten micrograms of nuclear protein extract were incubated with the radiolabeled NF-κB oligonucleotide for 20 minutes at room temperature. For competition studies, a 50-fold molar excess of unlabeled oligonucleotide was added to the reaction (a 21-mer corresponding to the AP-1 consensus sequence (Promega) was used to confirm specificity). DNA protein complexes were separated by electrophoresis through a nondenaturing 4% polyacrylamide gel in 0.5 × TBE at 100 V for 2.5 h. Autoradiographic films were developed after 18 h of exposure to the gels (−20°C).5 cells/ml. After allowing 24 h for cell adherence, cells were transfected and/or treated. Cells were collected by gentle trypsinization, washed in PBS, pelleted by centrifugation and fixed in 70% ethanol. Immediately prior to staining, cells were washed twice in PBS and resuspended in PBS containing RNase (20 μg/ml). Cells were stained with PI  for 10 minutes at room temperature. Samples were analyzed by FACS (FL-3 channel) using a Beckman Coulter Counter Epics XL flow cytometer . For each sample, 50,000 events were collected and stored for subsequent analysis using EXPO software . The percentage of cells in the sub-G0 phase was quantitated as an estimate of cells undergoing apoptosis.To identify the induction of apoptosis, treated cells underwent propidium iodide (PI) staining and FACS as to the manufacture’s instruction. In brief, cells were plated at a density of 1 × 10t-test. A P-value < 0.05 was considered to indicate statistical significance.All statistical analyses were performed using the SPSS10.0 software. The results were presented as means ± SD of three replicate assays. Differences between groups were assessed using analysis of variance (ANOVA) or the Dunnett Treatment with 20 ng/ml of TNF-α significantly stimulated NF-κB transactivation function as determined by the electrophoretic mobility shift assay Figure A. The inTreatment with 20 ng/ml of TNF-α significantly induced Bcl-2 mRNA at 24 h . The results suggest that NF-κB-mediated PUMA induction represents a novel mechanism mediating TNF-α-induced apoptosis.Thirty percent of apoptosis was detected in lung cancer cells following 20 ng/ml of TNF-α treatment for 24 h . Our study demonstrated that SLUG silencing promoted PUMA expression induced by TNF-α  of apoptosis was detected in lung cancer cells following TNF-α treatment at 48 h. This might be due to simultaneous induction of the anti-apoptotic NF-κB targets by TNF-α, such as SLUG and Bcl-2 at 48 h .In addition to PUMA, anti-apoptotic Bcl-2 was also upregulated with TNF-α treatment. Knockdown of Bcl-2 by siRNA also led to a significant increase in TNF-α-induced apoptosis, consistent with the previous finding that Bcl-2 is the major survival factor in lung cancer cells. These results suggest that overexpression of Bcl-2 can compromise TNF-α-induced and PUMA-mediated apoptosis  of apoptosis were detected in A549 cells following TNF-α treatment for 48 h. We found the PUMA mRNA/protein was significantly decreased after TNF-α treatment for 48 h, and the anti-apoptotic proteins Bcl-2 and SLUG, which have been shown to be involved in the control of apoptosis and resistance of various cells to irradiation and chemotherapies was significantly increased -21.et al. had suggested that reducing the expression of SLUG enhanced the sensitivity of neuroblastoma cell lines to imatinib mesylat by attenuating Bcl-2 expression [SLUG, a member of the snail superfamily of zinc finger transcription factors, is the key epithelial-mesenchymal transition (EMT) regulator responsible for conferring acquired resistance to target therapy in lung cancer . Previoupression . SLUG capression . It has pression ,19. Furtpression -22. ThusOur study showed TNF-α treatment for 48 h promoted SLUG and Bcl-2 levels and decreased PUMA levels followed by decreased apoptosis in A549 cells. After siRNA knockdown of SLUG or Bcl-2, apoptosis of TNF-α-induced for 48 h was significantly increased. Otherwise, knockdown of SLUG significantly upregulated the PUMA level and decreased the Bcl-2 level. We therefore suggest that TNF-α treatment for 48 h increased the NF-κB-dependent SLUG upregulation, by which PUMA was inhibited and Bcl-2 level promoted, and then inhibited the apoptosis in A549 cells.We demonstrated that PUMA is a target of NF-κB and a critical mediator of TNF-α-induced apoptosis. At an early time, TNF-α treatment induced apoptosis by PUMA activity. At a later time, TNF-α-induced apoptosis was inhibited by NF-κB-dependent SLUG upregulation, which suppressed PUMA and increased Bcl-2 activity.ANOVA: analysis of variance; Bcl-2: B-cell CLL/lymphoma-2; Bcl-XL: Bcl-extra large; DMEM: Dulbecco’s modified Eagles medium; EMT: epithelial-mesenchymal transition; FBS: fetal bovine serum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HRP: horseradish peroxidase; IKK: inhibitor of nuclear factor kappa-B; MM: multiple myeloma); NF-κB: nuclear factor-kappa B; NSCLC: non-small cell lung cancer; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PI: propidium iodide; PUMA: p53 upregulated modulator of apoptosis; SCF: stem cell factor; TNF-α: tumor necrosis factor alpha.The authors declare that they have no competing interests.YW carried out the studies, carried out the experiment of molecular biology and drafted the manuscript. BY participated in the experiment. XY participated in the sequence alignment. ZW participated in the design of the result and performed the statistical analysis. MW coordinated and helped to draft the manuscript. All authors read and approved the final manuscript.


Targeting TNFR2 as a treatment strategy is likely superior to TNFR1 because of its more limited cellular distribution on T cells, subsets of neurons, and a few other cell types, whereas TNFR1 is expressed throughout the body. This review focuses on TNFR2 expression, structure, and signaling; TNFR2 signaling in autoimmune disease; treatment strategies targeting TNFR2 in autoimmunity; and the potential for TNFR2 to facilitate end organ regeneration.The regulatory cytokine tumor necrosis factor (TNF) exerts its effects through two receptors: TNFR1 and TNFR2. Defects in TNFR2 signaling are evident in a variety of autoimmune diseases. One new treatment strategy for autoimmune disease is selective destruction of autoreactive T cells by administration of TNF, TNF inducers, or TNFR2 agonism. A related strategy is to rely on TNFR2 agonism to induce T-regulatory cells (T These fregs) , 3, endoregs) , 5, oligregs) , 7, cardregs) , thymocyregs) , 10, islregs) . Its morregs) , 13.regs is induced upon T-cell receptor activation , certain CNS cells, and endothelial cells, among others. Neither receptor is located on erythrocytes. Typically, cells that express TNFR2 also express TNFR1, with the ratio of expression varying according to cell type and functional role. Because TNFR1 typically signals cell death, while TNFR2 typically signals cell survival, the ratio of their co-expression will shift the balance between cellular survival and apoptosis.TNFR1 and TNFR2 have extracellular, transmembrane, and cytoplasmic components. The extracellular component of both receptors is rich in cysteine, which is characteristic of the TNF superfamily. TNFR1 contains 434 amino acids. Its intracellular region of 221 amino acids contains a death domain that binds TRADD or FADD. In T cells, activation of TRADD or FADD activates the caspases, resulting in apoptosis Figure , 18. The−/− mice homozygous for TNFR2−/− are viable and fertile. They also show normal T-cell development and activity and are resistant to TNF-induced death. The T-cell proliferation responses are diminished and they also show abnormal central nervous system regeneration (JAX Mice Database – 00260).Transgenic mice have been produced to try to understand better the function of TNFR2 . TNFR2−/Tumor necrosis factor, and its signaling through the two receptors, plays several crucial roles during normal development. It shapes the efficacy of the immune system and protects against infectious disease, cancer, and autoimmune disease . Upon reIn the process of developing autoimmunity, abnormal progenitors to T cells and other immune cell types proliferate and begin to mature in the thymus. T-cell education occurs through two parallel pathways for CD4 and CD8 T cells through either HLA class II or HLA class I cell surface structures. For almost all autoimmune disease there is strong genetic linkage to the HLA class II region. This genetic region is rich in immune response genes and contains not only the class II genes themselves but also the HLA class I assembly genes such as the tap transporters (Tap1/Tap2) and proteasome genes (that control self peptide presentation) such as LMP2 (PSMB9), LMP7 (PSMB8), and LMP10 (PSMB10) . During in vitro and in vivo studies, TNF or TNFR2 agonism is associated with pancreatic regeneration , which has been on the market for decades as a vaccine for tuberculosis and as a treatment for bladder cancer. Its chemical equivalent that does not meet FDA’s standards for purity is complete Freund’s adjuvant (CFA). In an early double blinded placebo-controlled Phase I clinical trial, BCG administration produced a transient increase in TNF in the circulation . BCG or Nuclear factor-kB is thwarted from entering the nucleus to transcribe pro-survival genes in autoimmune diseases featuring defects in TNF signaling , 34. InsTumor necrosis factor binds to TNFR1 and TNR2. Another way to make TNF selective for TNFR2 signaling, an effect that could promote tissue regeneration and remove autoimmunity, is to create a TNFR1 antagonist. This strategy would bias TNF to act solely through the TNFR2 receptor. This strategy also appears promising for hepatitis or autoimmunity in murine models . A humanregs co-express CD4+ and the interleukin-2 receptor alpha chain CD25 hi and feature inducible levels of intracellular transcription factor forkhead box P3 (FOXP3). Naturally occurring Tregs appear to express TNFR2 at a higher density than TNFR1 (reg activity: TNFR2 activates and induces proliferation of Tregs (regs (T-regulatory cells are a type of immunosuppressive cell that displays diverse clinical applications in transplantation, allergy, infectious disease, GVHD, autoimmunity, and cancer . Tregs can TNFR1 , 101. Thof Tregs  and TNFRgs (regs .ex vivo or stimulating their production in vivo. In vivo stimulation turns out to be too toxic with standard expansion agents IL-2, anti-CD3, and anti-CD28. These expansion agents can be used to generate large quantities of Tregsex vivo, but the problem is that they produce heterogeneous progeny consisting of mixed CD4+ populations. Heterogeneous progeny carry risk: they are capable of releasing pro-inflammatory cytokines and consist of cell populations with antagonistic properties. Some new approaches are being attempted, including expansion of Tregsin vivo with TL1A-Ig, a naturally occurring TNF receptor superfamily agonist (ex vivo expansion using a newly synthesized TNFR2 monoclonal antibody agonist that produces homogeneous progeny expressing a uniform phenotype of 14 cell surface markers (regs are capable of suppressing CD8+ T cells. In healthy humans, the TNF inducer BCG causes transient expansion of Tregs (reg production in patients with type I diabetes (T-regulatory cells have been proposed to prevent or treat autoimmune disease, but the rate-limiting problem has been obtaining sufficient quantities, whether by generating them  agonist . Additio markers . The TNFof Tregs . In a cldiabetes , which aWhen type 1 diabetes was first reversed in end-stage diabetic mice with boosting of TNF, the research showed an unexpected outcome . The panThe effect of TNF on the pancreas was not the only tissue showing possible regeneration with TNF stimulation. In the field of neuroregeneration, the Ting laboratory showed TNF similarly promoted proliferation of oligodendrocytes progenitors and remyelination . Gradualregs. It holds promise for treating other autoimmune disorders featuring dysregulation of NFkB, which is a key component of the TNFR2 signaling pathway.An overlapping feature across autoimmune disorders is various defects in TNF signaling through its two receptors. TNFR2 is a more attractive molecular target than TNFR1 because of its limited cellular expression. A variety of strategies utilizing TNFR2 agonism can be pursued for treatment of autoimmune disease and also used for regenerative medicine therapies. TNFR2 agonism has been associated with selective death of autoreactive T cells in type 1 diabetes and with induction of TThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The effect of TNF-α and TNF-α antagonists on semen quality in men is controversial. TNF-α levels are usually low in seminal plasma, but they tend to increase in inflammatory and infectious diseases. Etanercept is a highly-specific antagonist of TNF-α. In this report, we describe the development of pregnancy in a couple with a previously infertile husband, who received etanercept for ankylosing spondylitis. TNF-α is a cytokine exerting a wide range of cellular effects. In testis, TNF-α is produced by germ cells and it has a role in the regulation of spermatogenesis. TNF-α leNo studies measuring testicular TNF-α level in patients with ankylosing spondylitis (AS) have been found. We describe pregnancy development after etanercept administration to a husband with AS, in a couple with male factor infertility. It is postulated that increased levels of TNF-α in the testis might have decreased sperm production in this husband. TNF-α blockade by etanercept might have reversed this condition.A 44-year-old man presented with a 20-year history of AS. The patient had morning stiffness of 45-minute duration and advanced deformities: kyphosis, decreased cervical and lumbar spinal mobility, 5 cm of chest-chin distance and 2 cm of chest expansion. He was prescribed a combination of sulphasalazine  and indomethacin , for the first 18-year of the illness. Two years ago, he discontinued this regimen due to active disease and side effects. He then received different analgesic drugs occasionally, during the last two years. On laboratory analysis, the erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) was 53 mm/hour and 2.78 mg/dL  respectively on February 1, 2005.in vitro fertilization in 1996 and 2000, but these attempts were unsuccessful. The husband's sperm concentrations were 2000 per milliliter in 1996 and 15000 per milliliter in 2000. He was receiving sulphasalazine during that time.The patient was married for 20 years. The couple was evaluated for infertility and a diagnosis of “male factor infertility” was confirmed in 1996. They underwent 6 per milliliter concentration, 40% motility, 60% normal morphologic features. The measurements of ESR and CRP level were 35 mm/hour and 2.86 mg/dL respectively on March 16, 2005, and 38 mm/hour and < 0.86 mg/dL respectively on May 3, 2005.The patient was prescribed etanercept 25 mg twice weekly, s.c. On February 16, 2005, the first dose of the agent was injected. His wife, whose last menstruation date was March 5, 2005, had delayed menstruation. On gynecological examination on April 19, 2005 a 6-week gestation was detected. This spontaneous pregnancy developed after etanercept therapy, suggesting the role of etanercept in infertility. Semen analysis done on June 6, 2005 showed a sperm count of 140×10et al. reported that there was no evidence of increased rate of infertility and no association between serum concentration of hormones and ESR.[et al. evaluated testicular function with serum measurements of hormone levels in patients with AS and found abnormal results that correlated with disease activity.[The fertility status and sexual function of patients with AS were explored in several studies and controversial results were obtained.4 Gordon  and ESR. On the oSemen analyses done in 1996 and 2000 revealed sperm concentrations that were in the subfertile ranges. The pati6TNF-α is a potent cytokine produced by neutrophils, activated lymphocytes, natural killer cells, and activated macrophages. In testi1012et al. reported that infliximab infusion did not affect the semen volume, sperm concentration, and forward progression, but it decreased sperm motility and percent of normal oval forms.[The effect of TNF-α and TNF-α antagonists, such as infliximab, on semen quality in men is controversial. A study revealed that TNF-α effectively inhibited apoptosis of human germ cells; but no significant effect of TNF-α on the activation of transcription factor, which is considered to be a mediator of TNF-α -induced survival signals, were observed. Another al forms.et al. incubated sperm suspensions with different doses of TNF-α, TNF-α plus infliximab and only infliximab. Spermatozoa quality declined following incubation with TNF-α in a dose- and time-dependent manner. Sperm motility and membrane integrity were higher in the samples incubated with TNF-α plus infliximab than in the samples treated with TNF-α or infliximab alone. This study demonstrated that exposing spermatozoa to pathological concentrations of TNF-α can result in significant loss of their functional and genomic integrity and that infliximab is capable of reversing the toxic effects induced by TNF-α.[On the other hand, Said by TNF-α.


The π is missing from the 'Trp in V3 signature pattern 1 could form stabilizing cation- interaction with V2' paragraph of the Results and Discussion. The symbol should appear as follows in the first portion of the paragraph below:Trp in V3 signature pattern 1 could form stabilizing cation-π interaction with V2. We had marked earlier that position 317 (V3 position 22) is usually occupied by an aromatic residue, mostly F and in B’ W. Why should an aromatic amino acid interact with the positively charged region in V2 as suggested by DI ? This interaction could be explained by cation-π interactions between the cationic V2 region and the π-electron system of the aromatic F or W .


Here, we demonstrate that PHF6 is a nucleolus, ribosomal RNA promoter-associated protein. PHF6 directly interacts with UBF through its PHD1 domain and suppresses rRNA transcription by affecting the protein level of UBF. Knockdown of PHF6 impairs cell proliferation and arrests cells at G2/M phase, which is accompanied by increased level of phosphorylated H2AX (γ-H2AX), indicating that PHF6 deficiency leads to the accumulation of DNA damage in the cell. We found that increased DNA damage occurs at rDNA locus in PHF6-deficient cells. This effect could be reversed by knocking-down UBF or over-expressing RNASE1, which removes RNA:DNA hybrids, suggesting that there is a functional link between rRNA synthesis and genomic stability at rDNA locus. Together, these results reveal that the key function of PHF6 is involved in regulating rRNA synthesis, which may contribute to its roles in cell cycle control, genomic maintenance and tumor suppression.Mutation of PHF6, which results in X-linked mental retardation disorder-Börjeson-Forssman-Lehmann syndrome, is also present in about 38% of adult T-cell acute lymphoblastic leukemias and 3% of adult acute myeloid leukemias. However, it remains to be determined exactly how PHF6 acts


Alzheimer’s disease (AD), the most common cause of dementia in the elderly, is characterized by the presence of Aβ plaques, which results in a progressive neuronal degeneration and subsequent cognitive decline. Dendrites and axons in the region surrounding the plaques may become damaged and swollen, forming what are known as dystrophic neurites (DNs). However, the mechanisms by which DNs affect cognitive function in AD patients are not understood.While characterizing the reticulon (RTN)/Nogo family of proteins, which have been shown to negatively regulate BACE1 activity, we found that RTN3, which is primarily neuronally expressed, accumulates in a distinct population of DNs called RTN3 Immunoreactive Dystrophic Neurites (RIDNs) in the brains of AD patients and in APP transgenic mice, an animal model for AD. More importantly, transgenic mice overexpressing RTN3 (Tg-RTN3) produce RIDNs in the hippocampus at a young age, while wild-type mice produce similar RIDNs only at older ages.The importance of RIDNs in relation to cognitive decline is further evidenced by the finding that Tg-RTN3 mice where RIDNs are present show impaired learning and spatial memory, reduced spine density and synaptic plasticity. Molecular characterization of RIDNs indicates that RIDNs contain RTN3 aggregates and inducible expression of RTN3 in a mouse model reveals that RTN3 aggregates are formed irreversibly.Together, our results suggest that inhibition of RTN3 aggregation is a promising approach for improving cognitive dysfunction associated with Alzheimer’s pathology.


N-terminal kinase, SP600125, which is an upstream kinase activating AP-1. Our results thus suggest that TNF-α delays human eosinophil apoptosis via TNF-receptor 1 and the resulting changes in longevity depend on yin-yang balance between activation of NF-κB and AP-1.Eosinophils play a central role in asthma. The present study was performed to investigate the effect of tumour necrosis factor-α (TNF-α) on longevity of isolated human eosinophils. In contrast to Fas, TNF-α inhibited eosinophil apoptosis as evidenced by a combination of flow cytometry, DNA fragmentation assay and morphological analyses. The effect of TNF-α on eosinophil apoptosis was reversed by a TNF-α neutralising antibody. The anti-apoptotic effect of TNF-α was not due to autocrine release of known survival-prolonging cytokines interleukins 3 and 5 or granulocyte-macrophage-colony-stimulating factor as their neutralisation did not affect the effect of TNF-α. The anti-apoptotic signal was mediated mainly by the TNF-receptor 1. TNF-α induced phosphorylation and degradation of IκB and an increase in NF-κB DNA-binding activity. The survival-prolonging effect of TNF-α was reversed by inhibitors of NF-κB pyrrolidinedithiocarbamate and gliotoxin and by an inhibitor of IκB kinase, BMS-345541. TNF-α induced also an increase in AP-1 DNA-binding activity and the antiapoptotic effect of TNF-α was potentiated by inhibitors of AP-1, SR 11302 and tanshinone IIA and by an inhibitor of c-jun- Recombinant human TNF-α and IL-5, anti-human TNF R1 (Tnfrsf1a) and R2 (Tnfrsf1b) neutralising antibodies, recombinant human IL-5, anti-human IL-3 and IL-5 neutralising antibodies and mouse IgG1 isotype control were obtained from R&D Systems Europe . Other reagents were obtained as follows: ammonium pyrrolidinedithiocarbamate (PDTC) and SR 11302 -3-Methyl-7-(4-methylphenyl)-9--2,4,6,8-nonatetraenoic acid) , monoclonal anti-human Fas [CD95] antibody, clone ZB4 , monoclonal anti-human TNF-α antibody , monoclonal anti-human CD95 Fas antibody, clone CH-11 and IgM isotype control , monoclonal anti-human GM-CSF monoclonal antibody , IκBα antibody, actin antibody and horseradish-peroxidase conjugated secondary anti-rabbit antibody , IκBα (phospho S32+S36) antibody , horseradish peroxidase conjugated goat anti-mouse antibody , AP-1 and NF-κB consensus oligonucleotides , PD98059 (2-(2-Amino-3-methoxyphenyl)-4H-1-benzopyran-4-one) , SP600125 (Anthra[1-9-cd]pyrazol-6(2H)-one) , soluble recombinant human APO-1/Fas ligand (RhFasL)  and tanshinone IIA  . Other reagents were obtained as described elsewhere BMS-345541 -1,2-ethanediamine hydrochloride), gliotoxin  was obtained from eosinophilic donors. Donors were either healthy, atopic or asthmatic. They were allowed to use their normal medication . This may affect the basal apoptotic rate of eosinophils as shown previously H-imidazol-4-yl]pyridine), PD98059, SP600125, SR11302 and Tanshinone were dissolved in DMSO. Gliotoxin, methylgliotoxin, BMS-345541, SR11302 and tanshinone IIA were added 60 min before and all other inhibitors 30 min before TNF-α. The pre-incubation times were chosen based on the manufacturer's recommendation or existing literature. The neutralizing antibodies were added 15 min before TNF-α. The cells were incubated under sterile conditions for 40 h for apoptosis assays unless otherwise stated. The final concentrations of ethanol and DMSO were 0.2% and 0.5%, respectively, and this had no effect on the rate of apoptosis in eosinophils (data not shown). However, DMSO may affect the extent of the inhibitory effect of TNF-α on eosinophil apoptosis (in the presence of DMSO (0.5%) the inhibitory effect of TNF-α (10 ng/ml) was 12.7+−2.4% , whereas in the absence of DMSO the inhibitory effect of TNF-α (10 ng/ml) on eosinophil apoptosis was 26.2+−2.4% . However, use of DMSO could not be avoided in the experiments containing pharmacological inhibitors. A similar concentration of ethanol or DMSO was added to the control cultures.Eosinophils were cultured in the absence or presence of TNF-α and the indicated inhibitors. Gliotoxin and methylgliotoxin were dissolved in ethanol. SB203580 (4-[5-(4-Fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1Eosinophil apoptosis was determined by using the relative DNA fragmentation assay in propidium iodide (PI)-stained cells and flow cytometry  as previously described 6/ml) were incubated in RPMI/FCS in the absence and presence of TNF-α (100 ng/ml). The incubations were stopped at the appropriate times (indicated in the text and figure legends) by centrifugation and cytosolic proteins were extracted as previously described Eosinophils (1×10g for 10 s. Nuclei were then resuspended in buffer C and nuclear extracts were obtained as previously described Eosinophils were incubated for the indicated times with TNF-α (100 ng/ml) in the presence or absence of test compounds. Thereafter the cells were rapidly washed with ice-cold PBS and solubilised in 800 µl of hypotonic buffer A GG GAC TTT CCC AGGC-3′5′-AGT TGA G (sense strand). For AP-1 consensus oligonucleotide 5′-d(CGC TTG ATG AGT CAG CCG GAA)-3′ was used. Specificity was determined by the prior addition of a 50-fold excess of unlabeled competitor consensus or oligonucleotide. Binding reactions (3 µg of nuclear extract) and separation of protein/DNA complexes from DNA probe by electrophoresis were carried out as previously described Consensus NF-κB probe containing the decameric NF-κB site (underlined) was Results are expressed as the mean ± standard error of the mean (SEM). Statistical significance was calculated by paired t-test or analysis of variance for repeated measures supported by the Student-Newman-Keuls or Dunnett's test by using Instat software . Differences were considered significant if p<0.05.TNF-α (0.1–10 ng/ml) inhibited apoptosis in a concentration dependent-manner as determined by flow cytometric analysis measuring the relative DNA fragmentation of PI-stained eosinophils . The inhActivation of another member of TNF receptor superfamily, Fas has been previously described to induce apoptosis in human eosinophils Enhancement of eosinophil survival in a co-culture with mast-cells has been attributed to the production of TNF-α and GM-CSF in the culture 1 isotype control (10 µg/ml), TNF-α inhibited apoptosis . In the presence of anti-TNF-R1 antibody (10 µg/ml) TNF-α did not anymore inhibit apoptosis . In contrast, in the presence of anti-TNF-R2 antibody (10 µg/ml) TNF-α reduced the percentage of apoptotic cells  similarly to that seen in the presence of isotype control. Thus, the anti-apoptotic effect of TNF-α in human eosinophils seems to be mainly due to binding of TNF-α to the TNF-R1.Human eosinophils express both TNF receptors 1  and 2  Previously, TNF-α has been reported to activate p38 mitogen-activated protein kinase in eosinophils In resting cells NF-κB is localized to the cytoplasm because of binding to inhibitory protein IκB. Upon activation NF-κB-inducing kinase (NIK) is activated, which in turn activates a complex of specific IκB kinases (IKKs) resulting in IκB phosphorylation. Phosphorylation of IκB leads to a rapid ubiquitination which makes it a substrate for the proteasome To examine the functional role of NF-κB activation in TNF-α-driven eosinophil survival, a pharmacological approach was adopted. Eosinophil apoptosis was assessed in cultures supplemented with inhibitors of this transcription factor. Ammonium pyrroledithiocarbamate (PDTC) and gliotoxin were used as well characterised inhibitors of NF-κB The finding that PDTC not only reverses, but may even enhance eosinophil apoptosis in TNF-α stimulated eosinophils  and thatActivation of c-jun-N-terminal kinase (JNK) is known to precede the activation of AP-1. If AP-1 activation is “pro-apoptotic” in TNF-α stimulated cells, blocking JNK– AP-1 pathway by an inhibitor of JNK should potentiate the inhibitory effect of TNF-α on eosinophil apoptosis. In fact, SP600125, an inhibitor of JNK, amplified the inhibitory effect of TNF-α on eosinophil apoptosis . To furtIn this study we have shown that in contrast to Fas, TNF-α enhanced survival of human eosinophils by inhibiting apoptosis and that the effects of TNF-α on human eosinophil survival seem to be mediated mainly via TNF-R1 and a balance between activation of NF-κB and AP-1.In addition to the agonistic monoclonal Fas antibody, the naturally occurring agonist FasL produced an increase in the rate of eosinophil apoptosis confirming the previous reports TNF-α has previously been reported to enhance eosinophil survival Previously, both TNF-R1 and TNF-R2 have been reported to be involved in the survival-prolonging action of TNF-α in human eosinophils yin-yang balance between NF-κB and JNK-AP-1 pathways which determines whether the eosinophil survives or undergoes apoptosis.While much has been learned about the mechanism, by which various members of the TNF superfamily signal for apoptosis, proliferation and survival, much remains unknown. For example, the reasons why TNF and its family members activate both apoptosis and anti-apoptotic pathways simultaneously and why almost all members of the TNF family induce apoptosis of some cells and proliferation of others remains to be elucidated in vivo. Furthermore, TNF-α not only modulates eosinophil survival but is able to activate them as well as several other cell types Delayed eosinophil apoptosis is considered as a pathogenic mechanism in eosinophilic diseases Figure S1Specificity of TNF-α -induced NF-κB DNA binding in human eosinophils. Incubations were terminated at the indicated time-points after addition of TNF-α (100 ng/ml). NF-κB DNA-binding activity was analyzed by electrophoretic mobility shift assay. Nuclear extracts were incubated with 50-fold excess of either the specific unlabeled NF-κB consensus probe or with the nonspecific competitor (5′-CGC TTG AGT CAG CCG GAA-3′) prior to the addition of the labeled NF-κB probe. Arrowheads indicate the different specific bands found in each experiment.(TIF)Click here for additional data file.


Some of the rheumatoid arthritis (RA) risk allele variants were related to tumor necrosis factor (TNF) signaling pathways which raised the hypothesis that they might influence the response to anti-TNF drugs.HLA-DRB1 and RA risk alleles specifically selected for their relevance on RA biologic pathways with the response to anti-TNF treatment in a Southern European population using a nationwide register.The primary aim of our work was to investigate potential association between the We evaluated 383 RA patients for associations between anti-TNF treatment response assessed by an absolute change in DAS28 at six months as the primary outcome, and seven single nucleotide polymorphisms (SNP). We also studied the same association taking the proportion of EULAR good responders and non responders at six months as the outcome. Univariate and multivariate linear and logistic regression analyses were performed, adjusting for clinical variables that influenced treatment response.TRAF1/C5 region was associated with a poor anti-TNF treatment response either in linear  -0.43, -0.06; p-value 0.009) and in logistic univariate (odds ratio (OR) 0.61; CI 0.41, 0.92; p-value 0.018) and multivariate regression analyses.The minor allele (G), which is the risk allele for RA susceptibility, rs3761847 SNP in the HLA-DRB1 or the other allele variants with the response to anti-TNF treatment were not observed.Associations between TRAF1/C5 risk RA locus influenced the anti-TNF treatment response in the Southern European population assessed in this study. Additional studies in other populations are necessary to confirm the relevance of this finding.The rs3761847


In this study, we aimed to investigate the association of six well-characterized polymorphisms in tumor necrosis factor alpha and beta (TNF-α and TNF-β) genes with the risk for sarcoidosis via a comprehensive meta-analysis.-307A , -1031C , -863A , -238A , and -857T  alleles, but a significant association for TNF-β 252A allele . Under a random-effects allelic model, there was marginally significant increased risk of sarcoidosis for -307A allele among Caucasians  but not among Asians . There was a low probability of publication bias as reflected by the fail-safe number.The electronic MEDLINE (Ovid) and PubMed databases covering the period from the earliest possible year to June 2013 were searched. Total 13 qualified articles including 1584 patients with sarcoidosis and 2636 controls were recruited. The data were analyzed by RevMan software, and risk estimates were expressed as odds ratios (ORs) and 95% confidence intervals (95% CIs). Analyses of the full data set failed to identify any significant association of TNF-α gene This meta-analysis extended previous findings on the association between the TNF-α and TNF-β genetic polymorphisms and sarcoidosis, by showing that the TNF-β gene A252G polymorphism might be a potential risk factor for the development of sarcoidosis. Sarcoidosis is a multiorgan inflammatory disorder with unknown etiology; it is characterized by the accumulated activation of the CD4+ T lymphocytes and macrophages at disease sites and by the formation of noncaseating epithelioid cell granulomas . The onsEpidemiological data supported that the increased production of tumor necrosis factor (TNF) family members may have physiological implications on the development of sarcoidosis . TNF famMeta-analysis of observational studies poses particular challenges due to the inherent biases and differences in study designs. In this context, we carried out this meta-analysis in accordance with the standards set forth by the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guideline (please see Checklist S1) .TNFA or TNF-β or lymphotoxin-α or LTA” and “sarcoidosis”, combined with “gene or variant or polymorphism or allele”. The research was also supplemented by reviews of reference lists, hand-searching of relevant journals, and correspondence with authors. If multiple publications were available from the same study group, the most complete and the recent results were abstracted. Search results were limited to articles published in English language and studies performed in human subjects.The electronic Medline (Ovid) and PubMed databases covering the period from the earliest possible year to June 2013 were searched for potentially eligible publications. The key words used for the search were “TNF-α or Our analyses were restricted to articles that fulfilled the following inclusion criteria : 1) if they investigated the association between genetic polymorphisms in TNF-α or TNF-β gene and sarcoidosis among unrelated subjects; 2) if they had genotypes of examined polymorphisms tested by validated sample size; 3) if they were on a case-control study design; 4) if they provided sufficient information on the genotypes or alleles of examined polymorphisms to allow an estimation of odds ratio (OR) and its corresponding 95% confidence interval (95% CI).Articles were excluded (one was sufficient) if they investigated the progression, severity, phenotype modification, response to treatment or survival, as well as if they were conference abstracts, case reports/series, editorials, review articles, or the non-English articles.Data were extracted from qualified articles independently by two investigators (YF and WN) according to a standardized Excel template . The following data were collected from each study: first author, year of publication, ethnicity, baseline characteristics of the study population, total number of cases and controls, and genotype or allele distributions in cases and controls. Information on the Hardy-Weinberg equilibrium test was also tracked or calculated if unavailable. After data extraction, discrepancies were adjudicated by discussion and a consensus was reached.http://ims.cochrane.org/revman/download. Hardy-Weinberg equilibrium was assessed with the Pearson χ2 test or the Fisher’s exact test .The meta-analysis was calculated using the Review Manager version 5.0.19 software available at website I2) was used to quantify the presence of between-study heterogeneity with a statistical significance of 0.1. In this study, we applied the random-effects model for all comparisons because it accommodates the possibility that the underlying effect differs across studies. For practical use, the random-effects model is more conservative and has a wider 95% CI than the fixed-effects model.The inconsistency index  with the significance set at 0.05 for each meta-comparison. Specifically, if the calculated Nfs value was smaller than the number of studies observed, the meta-analysis results might have publication bias. We calculated the Nfs0.05 according to the formula Nfs0.05 = (ΣZ/1.64) 2 - k, where k is the number of articles included.Publication bias was assessed by the fail-safe number , -1031C , -863A , -238A , and -857T  alleles in association with sarcoidosis. In contrast, relative to the TNF-β 252A allele, the risk of having sarcoidosis was 1.65 times  for the TNF-β 252G allele. -307A allele among Caucasians  but not among Asians   .Nfs values were calculated to assess the potential existence of publication bias. At a significant level of 0.05, the Nfs0.05 values were consistently greater than the number of studies included in this meta-analysis for all polymorphisms under investigation.The In this study, we sought to investigate the association of the TNF-α and TNF-β genetic polymorphisms with sarcoidosis risk from English journals by conducting a meta-analysis of 13 articles and 4100 subjects. The principal finding of this study was that the TNF-β gene A252G polymorphism might be a potential risk factor for the development of sarcoidosis. Moreover, subgroup analysis indicated a contributory role of the TNF-α gene G-307A polymorphism in Caucasians. To the authors’ knowledge, this is the first meta-analysis to date investigating the association of the TNF-α gene T-1031C, C-863A, C-857T polymorphisms and the TNF-β gene A252G polymorphism with sarcoidosis.-307A allele frequencies across ethnic populations in this meta-analysis (data not shown). As such, it is necessary to construct a database of susceptible genes and polymorphisms implicated in sarcoidosis in each racial or ethnic group.In 2007, Medica and coworkers have meta-analyzed the TNF-α gene G-307A polymorphism in association with sarcoidosis, and found that this polymorphism was linked to the occurrence of sarcoidosis [Extending previous findings, we provided convincing evidence on the contributory effect of the TNF-β gene A252G polymorphism in the development of sarcoidosis. Although only three articles were available for this association, considering the estimated risk estimate (OR=1.65), it seems unlikely that our results could be explained by confounding. In addition, there was a low probability of publication bias for this comparison as reflected by the fail-safe number. It is reasonable to speculate that, the TNF-β gene A252G polymorphism, if involved, might be implicated in the pathogenesis of sarcoidosis by increasing the production of TNF-β. Large epidemiological and clinical studies are required to fully answer this speculation.Meta-analysis is recognized as a powerful tool to summarize results of individual studies; however, it is important to recognize certain limitations. First, all qualified studies were retrospective in design, which precludes further comments on causality. Second, albeit a low probability of publication bias in this meta-analysis, potential selection bias cannot be ruled out, because we only retrieved articles published in English language. Third, we selected only five polymorphisms from TNF-α and TNF-β genes, and did not cover other sarcoidosis-susceptibility genes, such as annexin A11 gene  and butyTaken together, this meta-analysis extended previous findings on the association between the TNF-α and TNF-β genetic polymorphisms and sarcoidosis, by showing that the TNF-β gene A252G polymorphism might be a potential risk factor for the development of sarcoidosis. Nevertheless, despite the small sample sizes involved, this meta-analysis provides an anchoring point for better understanding of the pathogenesis of sarcoidosis. For practical reasons, we hope that this study will not remain just another endpoint of research instead of a beginning to establish the background data for further investigation on mechanisms of the TNF-α and TNF-β genes in sarcoidosis.Checklist S1PRISMA checklist.(DOC)Click here for additional data file.


TNF-α is one of the most important cytokine involved in JIA pathogenesis. Several single nucleotide polymorphisms (SNPs) have been identified within the region of the TNF-α gene but only a very small minority have proven functional consequences and have been associated with susceptibility to JIA. An association between some TNF-α SNPs and adult rheumatoid arthritis (RA) susceptibility, severity and clinical response to anti-TNF-α treatment has been reported. The most frenquetly studied TNF-α SNP is located at −308 position, where a substitution of the G allele with the rare A allele has been found. The presence of the allele −308A is associated to JIA and to a poor prognosis. Besides, the −308G genotype has been associated with a better response to anti-TNF-α therapy in JIA patients, confirming adult data. Psoriatic and oligoarticular arthritis are significantly associated to the −238 SNP only in some works. Studies considering other SNPs are conflicting and inconclusive. Large scale studies are required to define the contribution of TNF-α gene products to disease pathogenesis and anti-TNF-α therapeutic efficacy in JIA.Whether tumor necrosis factor alpha (TNF- It. Itα gen stimuli .α levels as showed by in vitro and in vivo studies. Differences in cytokine production may be partly attributed to the presence of single nucleotide polymorphisms (SNP) within its corresponding gene. At least 12 SNP have been identified in the TNF-α locus, some of which have also been shown to influence the rate of transcription and production of TNF-α cytokine [Genetic factors may affect TNF-cytokine , 50.α polymorphism is the −308A/G, also known as TNF 1/2  [in vivo/vitro analysis [α production [α production and autoimmune disease. This genetic propensity to produce elevated TNF-α levels, due to the presence of the −308A polymorphism, may alter the course of an immune response [In vitro studies using different techniques  failed to demonstrate function in vitro for the −308 TNF-α SNP [In vivo studies have demonstrated that the −308A TNF-α allele had higher transcriptional activity compared with the −308G allele [The most commonly studied TNF-analysis . The preoduction . Severaloduction , 53–55. response , 55, 56.NF-α SNP , 54, 57.G allele , 58. HowG allele , 60. OthG allele .α repressor site, but it has shown contradicting function [α production with respect to the −238G allele [Another possible functional promoter SNP is the −238G/A  that is located within the TNF-G allele , but thiG allele , 61–64. P = 0.002) in a monocyte cell line [The rare −376G/A  is a binding site for the transcriptional factor OCT-1 . OCT-1 sell line , 66.α [Studies regarding the −863C/A  and −857C/T genotypes  showed that the rarer A and T alleles provide increased promoter activity and high production of TNF-α .Many other promoter variants have been described, as +489, +386, −1301, −857, −419, −376, and −244, but these SNPs are rare, particularly in Caucasian, with conflicting and inconsistent data , 53.α protein and its receptors in the pathogenesis of JIA has been suggested by many studies [α plays a key function in the initial and prolonged inflammation and in joint destruction, controlling the production of interleukin 1 (IL-1) and other proinflammatory cytokines including interleukin-6 (IL-6) and interleukin-8 (IL-8) [α mediates joint inflammation and destruction by inducing the synthesis and release of inflammatory metalloproteinases, prostaglandins, and nitric oxide in a variety of cell types, as well as inhibiting the production of matrix components [α cytotoxic effect on synovial cells. The role of TNF-α in JIA is suggested by the findings of high TNF-α levels in the synovial fluids of these patients [α and developing a polyarthritis [α biologic drugs in arthritis patients [The involvement of TNF- studies , 23, 33.8 (IL-8) . TNF-α mmponents . Althougpatients , from strthritis , and fropatients –73. P = 0.005) but not in the Czech JIA subjects; the authors suggested a possible ethnic allele distribution. Besides, in both JIA cohorts, the distribution of genotypes was not significantly different among different JIA subsets [In order to better understand the genetic background of JIA and the role of cytokine SNPs in this disease, several studies have been carried out recently see . The rol subsets . So, most JIA studies are in accord with adult RA results, demonstrating a direct involvement of this polymorphism in the severity of arthritis , 76–78.Some authors demonstrated that the −238G/A allele has a significant association with JIA , particuα production [Other SNPs have also been investigated. A study by Date et al. demonstrated that the −863A, −1013C, and −857T alleles were significantly higher in systemic JIA patients with respect to healthy controls. This association was not found in oligoarticular and polyarticular juvenile arthritis subsets. Moreover, the −857T allele seems to enhance the effect of DRB1*0405/DQB1*0401 haplotype in predisposing the development of systemic JIA. Indeed the author suggests that this polymorphism is associated with higher TNF-oduction . Studiesoduction , 80 but oduction , 74. Theoduction ; Oen et oduction .α in the inflammatory process makes this cytokine an excellent therapeutic target [α promoter and some reports have shown that production of TNF-α is influenced by these SNPs, an association has been suggested between some TNF-α promoter SNPs, JIA subtypes, and clinical response to biologic therapy [The central role of TNF-c target . Germlinc target , 83. Bioc target , 84; howc target , 85. As  therapy , 14, 50.α receptor combined with the Fc portion of the human immunoglobulin molecule, is the first TNF-α antagonist approved for the use in JIA and to date it has proven to be highly efficacious in children with polyarticular JIA [α antibody, not formally approved for JIA patients but commonly used in selected cases [α SNPs on the anti TNF-α effects in paediatric arthritis population.Etanercept, a fusion protein of extracellular domain of the TNF-ular JIA . Other ied cases . Three sα polymorphisms in the etanercept therapy response, Schmeling and Horneff (see α SNPs considered (−238A/G and −308A/G) and clinical response to anti-TNF-α [α SNP on the metalloproteinase-9 (MMP-9) levels and on the clinical response to etanercept in 66 polyarticular JIA children. They found that patients with the −308GG genotype achieved a clinical response more significant than those with the −308AA genotype (P = 0.035) and that MMP-9 levels in patients with the genotype −308GG were significantly decreased after 1 year of treatment with etanercept [To test the influence of TNF-neff see  studied ti-TNF-α . More reanercept .α therapy showed some important evidence (see α drugs with respect to 676TG [P > 0.05) [α drugs. In adult cohorts, Mugnier et al. [α SNP influences the response to infliximab therapy in RA patients. According to these authors, patients with −308GG genotype were better infliximab responders and they concluded that this genetic evaluation can be useful for predicting infliximab therapy response. Balog et al. considered the influence of TNF-α gene −308G/A polymorphism on therapeutic efficacy of infliximab in patients with RA and Crohn's disease. Most of nonresponders carried the TNF-α A allele [α bioactivity resulted was higher in −308A/A or A/G patients than in G/G subjects [Major information can be obtained from studies performed in adults affected by rheumatoid arthritis (RA). It is known that RA and JIA are two distinct entities whereas they have several common characteristics . Works ience see . A reporto 676TG . Besides > 0.05) , 90–92.  > 0.05) . Similarr et al.  tested iA allele . Other sA allele , 96, 97.A allele , 98–101.subjects .α SNPs: −308A/G, −238A/G e, −857C/T. At evaluation after 12 weeks of therapy no association between the three TNF-α SNPs and clinical response was noted. However, the GGC haplotype  in a homozygous form presented significant association with lower clinical response in patients on adalimumab plus methotrexate treatment [Another study in RA patients considered 152 patients subdivided in 3 groups of treatment: adalimumab plus methotrexate, adalimumab plus other DMARDs, and adalimumab alone. The authors studied 3 TNF-reatment . α inhibitors and in 2009 the US Food and Drug Administration (FDA) placed a black box warning for these drugs, as result of the identification of 48 malignancies cases occurring in children exposed to anti TNF-α biologic drugs [In the biologic era, several studies reported an elevated rate of malignancy in JIA patient treated with TNF-ic drugs . Studies in children exposed to biologic drugs reported an increased risk of lymphoma and other cancers with respect to healthy population –107. NevIndeed, two paediatric studies in JIA patient never treated with biologic drugs reported a 2- to 3-fold increased risk of cancer, and in particular 4-fold increased risk of lymphoproliferative disease , 109. AlA very recent work of Nordstrom et al. considering a cohort of biologics-naïve patients diagnosed with JIA between 1998 and 2007, matched with a non JIA cohort, found that the JIA incidence rates of cancer were significantly higher in JIA with respect controls . However, they found a nearly 3-fold increased risk of cancer in biologics-naïve JIA patients . In these studies, it is difficult to evaluate the real risk of cancer because of the potential risk of malignancy associated with underlying illnesses and the use of concomitant immunosuppressants; a clear causal relationship could not be established and the findings suggest an elevated underlying risk of cancer in this disease population independently from biologic therapy. Paediatric studies confirmed the more consistent data reported in biologic treated adult patients. Wolfe and Michaud found about 3-fold increased risk for lymphoma in 18.572 RA biologic drug treated patients with respect to controls. However, the authors referred that increased lymphoma rates observed with anti-TNF therapy may reflect channeling bias, whereby patients with the highest risk of lymphoma preferentially receive anti-TNF therapy and consequently the data are insufficient to establish a causal relationship between RA treatments and the development of lymphoma . The samThe Italian LOHREN registry reported data in contrast with the previous studies. This work considered 1114 RA patients treated with anti-TNF agents after failing to respond to traditional DMARDs, over an average observational period of 23.32 months. Comparison with the general population showed that the overall cancer risk was similar, but the risk of lymphoma was about five times higher in the RA patients treated with a biological agent . Howeverα biologic drugs naïve have a significantly higher risk of malignancy compared with general population [The reported studies seem to indicate that not the biologic drug use but the prolonged inflammatory state itself due to the autoimmune disease that can be responsible for the increased risk of cancer , 118. Inpulation .α levels and cancer development [α on tumoral cell [At this regard, several studies showed an association between elevated circulating TNF-elopment , 121 stielopment , causingelopment  and promelopment . This daral cell , an indiral cell  and a syral cell , 128.α polymorphisms and development of different types of cancer. The +488A and the −857T polymorphisms have been associated to bladder cancer [Moreover, a relationship was found between some TNF-r cancer . The +48r cancer , 131. Thr cancer  and the r cancer . The −30r cancer , breast r cancer , and livr cancer –136 and r cancer .Although some biologics seem to have a high association with certain cancer compared to control, there is no consistency of data. Genetic pathways themselves can be also related to higher tumor development risk. So caution is needed in interpreting the data and more research is needing.α gene polymorphisms in the pathogenesis of JIA is still incomplete. Numerous studies have focused on understanding the contribution of TNF-α polymorphisms in the RA and JIA pathogenesis. The actual knowledge regarding the role of TNF-α polymorphisms have shown an association with higher or lower levels of circulating TNF-α, aggressive or mild disease and poor or good prognosis related to the response to anti TNF-α treatment. The TNF-α SNP is located at −308 position, where the presence of rare A allele was associated with a major gene expression, high level of TNF-α expression, and more aggressive JIA phenotypes [The most studied TNF-enotypes , 43, 74 enotypes , 74. Sevenotypes . The preenotypes , dermatoenotypes , 140, inenotypes , 142, anThis −308 SNP was found mostly in Caucasian population and represents an important risk factor for JIA appearance in this population. α gene polymorphism has been associated with higher TNF-α production and more aggressive JIA phenotypes [Also the −238A TNF-enotypes , 74, 75.enotypes –65, 74.Several other polymorphisms have been identified but their frequency, their pathogenic role, and their influence on biologic drug response have been poorly characterized. α blocking agents are among the most effective therapies for JIA but unfortunately not all patients have a good response. Actually, the reasons for the interindividual variability in the response to anti-TNF-α therapy are unclear, although it is supposed that the genetic background might play a role. Considering the increasingly wide range of biologicas available for AIG and the cost of these therapies, there is an increasing need to predict responsiveness to identify patients more suitable to the therapy, to define the timing of treatment, and to avoid complications.Results of the studies are mostly conflicting. This is likely related to several elements: the complex pathogenesis of JIA, involving different cytokine genes and non HLA genes, largely still not well defined, and also environmental factors that are, in the major part actually unknown. Anyway the identification of singular patient genetic pattern can change the medical therapeutic approach. TNF-α as candidate genes potentially involved in the modulation of clinical response to anti TNF-α blocking agents. Many studies demonstrated that patients with the −308GG genotype are better responders to anti TNF-α therapy. Results of studies in RA patients indicate TNF-α treatment response also in JIA [Now, conflicting results have been found in the few paediatric studies. Indeed only some authors showed that the −308G/G genotype is associated with a better anti-TNF-o in JIA , 85. Otho in JIA , therefoα and consequently on the severity of JIA and the response to anti-TNF-α treatment. Further and larger studies are needed to investigate the influence of TNF-α polymorphisms on the treatment response to individualize the management of the disease.To conclude, data gathered so far indicate a possible influence of the −308 SNP promoter position on the production of TNF-