Effects Of Tumor Necrosis Factor-alpha Research Articles

Effects of tumor necrosis factor-alpha on porcine oocyte meiosis progression, spindle organization, and chromosome alignment

To evaluate the effects of tumor necrosis factor-alpha (TNF-alpha) on porcine oocyte maturation, spindle dynamics, and chromosome alignment. Controlled, prospective study. University hospital and IVF research laboratory. Ovaries collected from slaughtered prepubertal gilts. Oocyte maturation rate and cytoskeleton distribution. MATERIALS AND METHOD(S): Immature porcine oocytes (GV) were exposed to TNF-alpha at a concentration of 0 (as a control), 1, 5, 10, 100, 200, or 600 ng/mL in M199 medium. Oocytes were cultured for 24 hours to the pre-MI stage or 44 hours to the MII stage. After in vitro maturation for 44 hours, the rates of GV oocytes reaching MII stage were assessed, and MII oocytes were fixed for further examination of the cytoskeleton and the chromosomal distribution. The TNF-alpha concentration at 5 ng/mL decreased the porcine oocyte maturation rate compared with the control after culture for 44 hours, whereas exposure to 10 or 100 ng/mL TNF-alpha resulted in a significant increase in the frequency of defective spindles or abnormal microfilament distribution. Exposed to 200 ng/mL, TNF-alpha caused a significantly higher abnormality rate of chromosome alignment when compared with the controls. Exposure of porcine oocytes to an elevated TNF-alpha concentration clearly caused a reduction in their maturation from GV stage to MII stage and increased the proportion of oocytes with abnormal chromosome alignment and cytoskeleton structure.

Upregulation of immunity-related GTPase (IRG) proteins by TNF-α in murine astrocytes

We examined the effect of tumor necrosis factor-alpha (TNF-α) on murine primary astrocytes. Proteomic analysis demonstrated that four new spots in the TNF-α-treated cells relative to untreated cells. Two of them were identified as Irgb6 and Irgd, members of immunity-related GTPase (IRG) proteins which are the key mediators of interferon-gamma (IFN-γ)-induced resistance of pathogens in numerous cells. Gene expression analysis using RT-PCR showed that TNF-α dose-dependently increased the expression of both proteins. Immunocytochemical analysis showed that TNF-α increased the abundance of both proteins. A subcellular localization study demonstrated that TNF-α induced the partial colocalization of both proteins with the endoplasmic reticulum (ER) and Golgi apparatus, whereas IFN-γ did not induce the colocalization of Irgd protein with the ER and Golgi. Combined stimulation with TNF-α and IFN-γ had a synergistic effect on the expression of Irgb6 and an added effect on the expression of Irgd.

Migration of bone marrow‐derived mesenchymal stem cells induced by tumor necrosis factor‐α and its possible role in wound healing

We aimed to investigate the effect of tumor necrosis factor-alpha (TNF-alpha) on the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in the migration ability of mesenchymal stem cells (MSCs) in the context of wound healing. We also explored the role of p38 mitogen-activated protein kinase and extracellular signal-regulated kinase (ERK) signaling pathways in the migration of MSCs. MSCs were isolated from the bone marrow and cultured. Immunocytochemistry, Western blotting, and reverse transcription-polymerase chain reaction were used to observe the effect of TNF-alpha on the expression of ICAM-1 and VCAM-1 in MSCs. The chemotaxis effect of TNF-alpha on MSCs was investigated by the trans-well system and the inhibition effect of TNF-alpha using its antibody. Western blotting analysis was used to observe the activation of JAK-STAT and mitogen-activated protein kinase signaling pathways, and ERK was inhibited with PD98059 and p38 with SB203580 to observe the effect of TNF-alpha on MSC migration and ICAM-1 expression. The expression of ICAM-1 could be up-regulated by 50 microg/L TNF-alpha (p<0.05), whereas that of VCAM-1 remained unchanged (p>0.05). Also, TNF-alpha showed a chemotaxis effect by enhancing the migration ability of MSCs (p<0.05). TNF-alpha at 50 microg/L increased the expression of phospho-ERK and phospho-p38, and SB203580, but not PD98059, could suppress the chemotaxis effect and up-regulation of ICAM-1 induced by TNF-alpha in MSCs (p<0.05). Thus, TNF-alpha could up-regulate the expression of ICAM-1 in MSCs and enhance the cells' migration ability, and the p38 signaling pathway might be involved in the TNF-alpha-induced migration ability for a role in wound repair and regeneration.

Rapid nitric oxide-dependent effects of tumor necrosis factor-α on suprachiasmatic nuclei neuronal activity

The effect of tumor necrosis factor-alpha (TNF-alpha) on excitability and synaptic function was analyzed in slice preparations of the suprachiasmatic nuclei (SCN), the major mammalian circadian pacemaker. TNF-alpha caused a rapid increase in the spontaneous firing rate in most SCN neurons examined that was paralleled by an increase of inhibitory postsynaptic currents. The nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester abolished these effects. No effect of TNF-alpha was found on miniature synaptic currents. The lack of effect on miniature synaptic currents indicates that TNF-alpha primarily affects neuronal membrane properties to cause the changes in spontaneous firing. TNF-alpha, levels of which show circadian variation in the brain and increase during inflammatory conditions and aging, may thus through nitric oxide induction modulate SCN electrical output to affect downstream circadian rhythms.

Regulation of catabolic gene expression in normal and degenerate human intervertebral disc cells: implications for the pathogenesis of intervertebral disc degeneration

IntroductionThe aim of this study was to compare the effects of tumour necrosis factor-alpha (TNF-α) and interleukin-1-beta (IL-1β) on protease and catabolic cytokine and receptor gene expression in normal and degenerate human nucleus pulposus cells in alginate culture.MethodsCells isolated from normal and degenerate nucleus pulposus regions of human intervertebral discs were cultured in alginate pellets and stimulated by the addition of 10 ng/mL TNF-α or IL-1β for 48 hours prior to RNA extraction. Quantitative real-time polymerase chain reaction was used to assess the effect of TNF-α or IL-β stimulation on the expression of matrix metalloproteinase (MMP)-3, -9 and -13, TNF-α, TNF receptor 1 (TNF-R1), TNF receptor 2 (TNF-R2), IL-1α, IL-1β, IL-1 receptor 1 (IL-1R1) and IL-1 receptor antagonist (IL-1Ra).ResultsMMP-3 and MMP-9 gene expressions were upregulated to a greater level by IL-1β than TNF-α. MMP-13 was upregulated by each cytokine to a similar extent. TNF-α and TNF-R2 expressions were upregulated by both TNF-α and IL-β, whereas TNF-R1 expression was not significantly affected by either cytokine. IL-1β and IL-1Ra expressions were significantly upregulated by TNF-α, whereas IL-1α and IL-1R1 were unchanged.ConclusionsTNF-α does not induce MMP expression to the same degree as stimulation by IL-1β, but it does act to upregulate IL-1β expression as well as TNF-α and TNF-R2. The net result of this would be an increased inflammatory environment and accelerated degradation of the matrix. These results support the hypothesis that, while TNF-α may be an important initiating factor in matrix degeneration, IL-1β plays a greater role in established pathological degradation.

Proinflammatory cytokine and ligands modulate cardiac peroxisome proliferator‐activated receptors

Peroxisome proliferator-activated receptors (PPAR) mediate inflammatory processes and alter cardiac function. However, it is not clear whether inflammatory cytokines or PPAR ligands regulate PPARs in the cardiomyocytes to modulate cardiac functions. We investigated the effects of tumour necrosis factor-alpha (TNF-alpha) and PPAR ligands on the expression of PPARs in HL-1 cardiomyocytes. HL-1 cardiomyocytes were incubated with and without TNF-alpha (1, 10, 25 and 50 ng mL(-1)) or PPAR ligands (rosiglitazone, pioglitazone and fenofibrate) at concentrations of 0.1, 1 and 10 microM for 24 h. The cells also received SN-50 (NF-kappaB inhibitor, 50 microg mL(-1)), ascorbic acid (100 microM) and coenzyme Q10 (10 microM) alone or combined with TNF-alpha. Using reverse transcriptase-polymerase chain reaction and Western blot, we found that incubation of TNF-alpha (50 ng mL(-1)) for 24 h decreased PPAR-alpha, but increased PPAR-gamma without altering PPAR-delta. These effects were not changed by co-administration of SN-50. However, co-administration of ascorbic acid prevented the effect of TNF-alpha both on PPAR-alpha and PPAR-gamma. Coenzyme Q10 partially attenuated the effect of TNF-alpha on PPAR-gamma but did not alter its effect on PPAR-alpha. The administration of rosiglitazone (10 microM) and pioglitazone (10 microM) for 24 h increased PPAR-gamma mRNA, but did not alter PPAR-alpha or PPAR-delta. Moreover, fenofibrate (0.1, 1 and 10 microM) increased PPAR-gamma without any effects on PPAR-alpha or PPAR-delta. Oxidative stress causes the regulations of PPAR-alpha and PPAR-gamma in the TNF-alpha-treated cardiomyocytes. The up-regulation of PPAR-gamma by PPAR ligands may contribute to their anti-inflammation effects.

Effects of tumor necrosis factor-alpha induced insulin resistance on adipose triglyceride lipase in mice

To investigate the effects of tumor necrosis factor (TNF)-alpha induced insulin resistance (IR) on glucose and lipid metabolism and adipose triglyceride lipase (ATGL). Forty male C57BL/6J mice were randomly divided into 2 equal groups: TNF-alpha group with undergoing intraperitoneal injection of TNF-alpha 6 microg x kg(-1) x d(-1) for 7 days and normal control (NC) group with saline injection. Hyperinsulinemic-euglycemic clamp technique combined with 2-deoxy-[(3)H] glucose as a tracer was used on 20 mice, 10 from each group, to examine the fasting blood glucose (FBG), plasma insulin (INS), total cholesterol (TC), triglyceride (TG), and free fatty acid (FFA). The glucose infusion rate (GIR) was recorded. Other 20 mice, 10 in each group, were killed with their adipose and/or muscle tissues taken out. RT-PCR was used to detect the mRNA expression of ATGL, hormone-sensitive lipase (HSL), carnitine palmitoyl transferase-1 (CPT-1), and peroxisome proliferator activated receptor-gamma (PPARgamma). Western blotting was used to measure the protein expression of ATGL. Muscle glucose uptake (MGU) was measured. After TNF-alpha treatment, the FBG, plasma INS, and FFA were significantly elevated in the TNF group compared with the NC group (all P < 0.05). During the steady-state of clamp test, the plasma INS level of the TNF group was (341.7 +/- 17.7) mU/L, significantly higher than that of the NC group [(84.7 +/- 5.5) mU/L, P < 0.01]. The FFA level of the TNF group was (0.82 +/- 0.03) mmol/L, significantly higher than that of the NC group [(0.43 +/- 0.07) mmol/L, P < 0.01]. The GIR of the TNF group was (39.1 +/- 2.3) mg x kg(-1)x min(-1), significantly lower than that of the NC group [(54.2 +/- 2.2) mg x kg(-1) x min(-1), P < 0.01]. The MGU level of the TNF group was (15.8 +/- 1.7) micromol.100 g(-1) x min(-1), significantly lower than that of the NC group [(20.9 +/- 2.5) micromol.100 g(-1) x min(-1), P < 0.01]. The ATGL mRNA expression level in adipose tissues of the TNF group was (0.85 +/- 0.09), significantly lower than that of the NC group (1.37 +/- 0.12, P < 0.01). The ATGL protein expression level of the TNF group was 0.53 +/- 0.03, significantly lower than that of the NC group (0.65 +/- 0.05, P < 0.05). The PPARgamma mRNA expression level in adipose tissues of the TNF group was 0.83 +/- 0.07, significantly lower than that of the NC group (1.07 +/- 0.07, P < 0.05). In TNF-alpha induced insulin resistance, AGTL may be involved in the pathways of lipid metabolism.

Effects of tumour necrosis factor-alpha (TNF-alpha), IL-1 beta and monocytes on lymphokine-activated killer (LAK) induction from natural killer (NK) cells and T lymphocytes.

Roles of monocytes and cytokines were investigated on LAK induction from T and NK cells. Monocytes augmented more T-LAK induction than did NK-LAK. Expression of IL-1 beta, TNF-alpha and interferon-gamma (IFN-gamma)-mRNA and their cytokine production were superior in NK cells compared with T cells in parallel with their LAK activities. An increase of TNF-alpha, IL-1 beta and IFN-gamma production was induced by co-culturing NK or T cells with autologous monocytes. The augmentation of T cell cytokine production and T-LAK activity by monocytes was more prominent than that of NK cells. TNF-alpha and IL-1 beta were generated 24 h after IL-2 stimulation, and these cytokines were able to almost substitute for monocytes in LAK induction. Conversely, LAK induction was almost completely suppressed by both anti-IL-1 beta and anti-TNF-alpha antibodies, if they were added within 24 h after the start of the LAK induction. IFN-gamma, which was produced at a later stage, scarcely affected LAK induction in spite of the cooperation with TNF-alpha. The results obtained indicate conclusively that the superiority of NK-LAK depends on their superior productivity of both IL-1 beta and TNF-alpha, and that the up-regulation of LAK induction by monocytes is largely due to the enhanced generation of both cytokines.

The effect of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 on chorioamnion secretion of prostaglandins (PG)F2α and E2 in pigs

The aim of the present study was to determine the effect of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) on prostaglandin (PG)F(2 alpha) and PGE(2) secretion as well as cyclooxygenase-2 (COX-2) protein expression in chorioamnion collected on days 25, 30 and 40 of pregnancy in pigs. Fetal membrane slices were incubated for 16 h with TNF-alpha, IL-1 beta, IL-6 (1 or 10 ng/ml of medium) or two combinations of the three cytokines (1 or 10 ng/ml of each cytokine per combination). We demonstrated the stimulatory effect of TNF-alpha, IL-1 beta and/or IL-6 on PGF(2 alpha) and PGE(2) secretion by the porcine fetal membranes. The medium content of these PGs depended on the cytokine type, treatment dose and day of pregnancy. Cytokine stimulation of PGE(2) was more pronounced than that of PGF(2 alpha). In addition, an increase in PGF(2 alpha) and/or PGE(2) secretion was usually associated with an augmentation of COX-2 protein expression. Our results support the notion concerning the possible role of cytokines in modulating production of PGs by fetal membranes during the first trimester of gestation.

Delayed Disruption of Barrier Function in Cultured Human Corneal Epithelial Cells Induced by Tumor Necrosis Factor-α in a Manner Dependent on NF-κB

The corneal epithelium provides a barrier that is both important for corneal homeostasis and dependent on tight junctions (TJs) between adjacent epithelial cells. The authors examined the effects of tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, on barrier function and the expression of TJ proteins in simian virus 40-transformed human corneal epithelial (HCE) cells. The barrier function of cultured HCE cells was evaluated by measurement of transepithelial electrical resistance (TER). The subcellular distribution of the TJ proteins zonula occludens-1 (ZO-1) and occludin and that of the p65 subunit of nuclear factor-kappaB (NF-kappaB) were determined by immunofluorescence staining. The expression of ZO-1 and occludin and the phosphorylation and degradation of the NF-kappaB inhibitory protein IkappaB-alpha were examined by immunoblot analysis. TNF-alpha induced a decrease in the TER of HCE cells in a concentration- and time-dependent manner. It also induced the disappearance of ZO-1 from the interfaces of neighboring HCE cells without affecting the localization of occludin. The abundance of neither ZO-1 nor occludin was affected by TNF-alpha. TNF-alpha induced the phosphorylation and downregulation of IkappaB-alpha and the translocation of the p65 subunit of NF-kappaB to the nucleus. The NF-kappaB inhibitor curcumin blocked the effects of TNF-alpha on TER and the subcellular localization of ZO-1 at late phase. TNF-alpha disrupted the barrier function of HCE cells, apparently by affecting the localization of ZO-1 at TJs in a manner dependent on NF-kappaB at late phase. This action of TNF-alpha may contribute to the loss of corneal epithelial barrier function associated with ocular inflammation.

Beneficial synergistic interactions of TNF-α and IL-6 in C2 skeletal myoblasts—Potential cross-talk with IGF system

The interaction effects of tumour necrosis factor-alpha (TNF-α) and interlukin-6 (IL-6) on skeletal muscle proliferation and differentiation remains controversial. We therefore investigated the potential interactive effects of TNF-α and IL-6 on murine C2 skeletal myoblast survival, differentiation and proliferation. A novel and unexpected positive temporal interaction between TNF-α and IL-6 on cell growth was identified (90%), with maximal beneficial effects obtained in myoblasts treated with TNF-α (10 ng/ml) for 24 h prior to being dosed with IL-6 (2.5 ng/ml) for a further 24 h. This combined treatment significantly (p < 0.05) increased the level of total cellular protein (330%), extracellular signal-regulated kinase (ERK) phosphorylation (55%), and S-phase of cell cycle (2.5-fold), confirming cell growth. The expression of mRNAs of key regulators of muscle mass: insulin-like growth factor binding protein-5, insulin-like growth factor-II (IGF-II), IGF-I receptor (IGF-IR) and IGF-II receptor (IGF-IIR) were also significantly (p < 0.05) increased by 1600-, 1.6-, 27- and 6-fold, respectively, giving an indication of the regulatory mechanisms of this interaction. Moreover, in response to this treatment, the expression level of signal-transducing glycoprotein 130 (gp130) was induced up to 3.5-fold but not after either treatments alone. This may not only explain the beneficial effects of this treatment on skeletal myoblast numbers but also define a functional role of gp130 in skeletal muscle cells. Our data suggest that in the presence of TNF-α/IL-6 functions positively and potentially also cooperatively with the IGF system to achieve the maximal beneficial effect on skeletal myoblast numbers.

Interleukin‐10 inhibits the down‐regulation of ATP binding cassette transporter A1 by tumour necrosis factor‐alpha in THP‐1 macrophage‐derived foam cells

It is suggested that cholesterol efflux mediated by ATP binding cassette transporter A1 (ABCA1) plays an important role in anti-atherogenesis. However, the effects of inflammatory cytokines on ABCA1 expression and cholesterol accumulation in foam cells are little known. This study investigates the effects of tumour necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) on ABCA1 expression and cholesterol content in THP-1 macrophage-derived foam cells. ABCA1mRNA and protein levels were determined by RT-PCR and Western blot, respectively. The total cholesterol content in THP-1 macrophage-derived foam cells was detected by the zymochemistry method. Results revealed that TNF-alpha could increase cholesterol content by down-regulating ABCA1 expression in a time-dependent manner in THP-1 macrophage-derived foam cells, which may contribute to its pro-atherosclerotic effect. In addition IL-10 time-dependently decreased cholesterol accumulation by up-regulating ABCA1 expression and inhibited the down-regulation of ABCA1 by TNF-alpha in THP-1 macrophage-derived foam cells, which may be one of the mechanisms of IL-10 contributing to its anti-atherosclerotic action.

TNF-alpha induces hepatic steatosis in mice by enhancing gene expression of sterol regulatory element binding protein-1c (SREBP-1c).

We investigated the effect of tumor necrosis factor-alpha (TNF-alpha), a member of the proinflammatory cytokine family, on steatosis of the mouse liver by analyzing morphological changes and hepatic triglyceride content in response to TNF-alpha. We also examined expression of the sterol regulatory element binding protein-1c gene. Intraperitoneal injection of TNF-alpha acutely and dramatically accelerated the accumulation of fat in the liver, as evidenced by histological analysis and hepatic triglyceride content. This treatment increased liver weight, increased serum levels of free fatty acids, and increased fatty acid synthase and sterol regulatory element binding protein-1c mRNA expression. Furthermore, intraperitoneal injection of lipopolysaccaride (LPS) to induce TNF-alpha expression also accelerated hepatic fat accumulation. Pretreatment with anti-TNF-alpha antibody attenuated the development of LPS-induced fatty change in the liver. Antibody pretreatment not only decreased sterol regulatory element binding protein-1c expression in LPS-treated mice but also attenuated the expression of suppressors of cytokine signaling-3 mRNA. This study suggests that TNF-alpha, acting downstream of LPS, increases intrahepatic fat deposition by affecting hepatic lipogenetic metabolism involving sterol regulatory element binding protein-1c.

Cytokine upregulation of proteinase-activated-receptors 2 and 4 expression mediated by p38 MAP kinase and inhibitory kappa B kinase beta in human endothelial cells.

Up-regulation of proteinase-activated receptor-2 (PAR2) is a factor in a number of disease states and we have therefore examined the signalling pathways involved in the expression of the receptor. We investigated the effects of tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), trypsin and the PAR2 activating peptide, 2-furoyl(2f)-LIGKV-OH on both mRNA and functional expression of PAR2 in human umbilical vein endothelial cells (HUVECs). The effect of specific chemical inhibitors and dominant negative adenovirus constructs of the mitogen-activated protein kinase (MAPK) cascade and the nuclear factor kappa B (NF-kappaB) signalling pathway was assessed. Methods included semi-quantitative and quantitative RT-PCR, [(3)H]inositol phosphate (IP) accumulation and Ca(2+)-dependent fluorescence. The above agonists induced both mRNA and functional expression of PAR2; PAR4 mRNA, but not that for PAR1 or PAR-3, also increased following TNFalpha treatment. Inhibition of p38 MAP kinase reduced PAR2 and PAR4 expression, whilst inhibition of MEK1/ERK/JNK was without effect. A similar dependency upon p38 MAP kinase was observed for the expression of PAR4. TNFalpha -induced enhancement of PAR2 stimulated [(3)H]-inositol phosphate accumulation (IP) and Ca(2+) signalling was abolished following SB203580 pre-treatment. Infection with adenovirus encoding dominant-negative IKKbeta (Ad.IKKbeta(+/-)) and to a lesser extent dominant-negative IKKalpha (Ad.IKKalpha(+/-)), substantially reduced both control and IL-1beta- induced expression of both PAR2 and PAR4 mRNA and enhancement of PAR2-stimulated IP accumulation and Ca(2+) mobilisation. These data reveal for the first time the signalling events involved in the upregulation of both PAR2 and PAR4 during pro-inflammatory challenge.

Changes of Src-suppressed C kinase substrate expression in cytokine induced reactive C6 glioma cells

To investigate effect of tumor necrosis factor-alpha (TNF-alpha) on the Src-suppressed C kinase substrate (SSeCKS) in C6 glioma cells. Cultured C6 glioma cells were randomly divided into two groups. In time-dependent group, cells were cultured with TNF-alpha (2 ng/mL) for 0 h, 1 h, 3 h, 6 h, 12 or 24 h, respectively; in dose-dependent group, cells were cultured with TNF-alpha (0 ng/mL, 0.02 ng/mL, 0.2 ng/mL, or 2 ng/mL) for 6 h. The expression of SSeCKS was detected by Realtime PCR and Western blot analysis, and immunocytochemistry was used to investigate SSeCKS's subcellular localization. TNF-alpha induced rapid phosphorylations of protein kinase C (PKC) substrates in C6 glioma cells, and upregulated SSeCKS expression in a time and concentration dependent manner. Immunocytochemistry suggested that SSeCKS was localized in the cyroplasm and the leading end of podosomal extensions in control groups, while TNF-alpha induced translocation of SSeCKS perinuclear. This effect could be partly reversed by PKC inhibitor Ro-31-8220. TNF-alpha activates PKC and upregulates SSeCKS expression in C6 glioma cells. These effects are associated with PKC activity, suggesting that SSeCKS plays a role in response to glia activation in PKC mediated pathway.

Effects of Tumour Necrosis Factor-α on Human Sperm Motility and Apoptosis

The aim of this study was to evaluate the effects of tumour necrosis factor-alpha (TNF-alpha) on sperm motility, mitochondrial membrane potential (DeltaPsi), phosphatidylserine (PS) externalization, sperm chromatin packaging quality, and DNA fragmentation. Motile spermatozoa, obtained from 10 normozoospermic men, were incubated with increasing concentrations of TNF-alpha and analyzed 1, 3, 6, and 24 h after incubation by flow cytometry. TNF-alpha decreased total motility 24 h after incubation at 10 ng/mL and progressive motility 3 h after incubation. Accordingly, TNF-alpha reduced sperm DeltaPsi in a concentration- and time-dependent manner. TNF-alpha increased the percentage of spermatozoa with PS externalization from the concentration of 1 ng/mL 1 h after incubation. TNF-alpha produced sperm chromatin and DNA damage in a concentration- and time-dependent manner. In conclusion, these findings may explain the reduction of fertility, secondary to upregulated production of TNF-alpha, in men with urogenital infections.

Lactobacillusplantarum inhibits epithelial barrier dysfunction and interleukin-8 secretion induced by tumor necrosis factor-α

To determine whether Lactobacillus plantarum can modify the deleterious effects of tumor necrosis factor-alpha (TNF-alpha) on intestinal epithelial cells. Caco-2 cells were incubated with TNF-alpha alone or in the presence of L. plantarum. Transepithelial electrical resistance was used to measure epithelial barrier function. Interleukin 8 (IL-8) secretion by intestinal epithelial cells was measured using an ELISA. Cellular lysate proteins were immunoblotted using the anti-extracellular regulated kinase (ERK), anti-phospho-ERK and anti-IkappaB-alpha. A TNF-alpha-induced decrease in transepithelial electrical resistance was inhibited by L. plantarum. TNF-alpha-induced IL-8 secretion was reduced by L. plantarum. L. plantarum inhibited the activation of ERK and the degradation of IkappaB-alpha in TNF-alpha-treated Caco-2 cells. Induction of epithelial barrier dysfunction and IL-8 secretion by TNF-alpha is inhibited by L. plantarum. Probiotics may preserve epithelial barrier function and inhibit the inflammatory response by altering the signal transduction pathway.

Retinoic acid inhibits tumor necrosis factor-alpha induced injury in human lung epithelial cells

To study the effect of tumor necrosis factor-alpha (TNF-alpha) on the proliferation and apoptosis of type II lung alveolar epithelial cells and the regulation of retinoic acid (RA) to this effect. Human type II lung alveolar epithelial cells of the line A569 were cultured and divided into 2 groups: control group (cultured in culture fluid only), TNF-group (cultured in the culture fluid with TNF-alpha 10 micromol/L for 24 or 46 h respectively), RA group (cultured in culture fluid without RA 1 microg/L), and TNF-alpha plus RA group (cultured in culture fluid with TNF-alpha 10 micromol/L + RA 1 microg/L). MTT method was used to test the proliferation of the A549 cells. The cell apoptosis was detected by flow cytometric assay. The proliferation rates of A549 cells treated with TNF-alpha of the concentrations of 0, 0.1, 1, 5, and 10 microg/L were 95.0%, 90.0%, 79.6%, 72.4%, and 59.6% after 24 h (F = 18.04, P < 0.001), and were 93.2%, 82.7%, 61.5%, 50.3%, and 44.7% after 48 h (F = 40.61, P < 0.0001). The inhibition effect on the proliferation of A549 cells treated with 10 microg/L TNF-alpha for 24 h could be reversed, however, the inhibition effect on the proliferation of A549 cells treated with 10 microg/L TNF-a for 48h could not be reversed. RA alone did not significantly promote the proliferation of the A549 cells, but weakened the inhibitory effect of TNF-alpha on the proliferation of the A549 cells. The apoptotic rate of the A549 cells treated with TNF-alpha for 12 h, 24 h and 48 h respectively were 14.3% +/- 3.2%, 18.6% +/- 2.9%, and 43.4% +/- 3.5% respectively, all significantly higher than those of the control group (6.3% +/- 1.2%, 8.2% +/- 1.3%, and 26.1% +/- 2.5% respectively, all P < 0.01), and the apoptotic rate of the A549 cells treated with both TNF-alpha and RA for 12 h, 24 h, and 48h were 4.8% +/- 1.1%, 5.2% +/- 1.3%, and 16.4% +/- 2.3% respectively, all significantly lower than those of the TNF-alpha group (all P < 0.01), and the apoptotic rates of the A549 cells treated with both TNF-alpha and RA for 24 h and 48 h respectively were both significantly lower than those of the control group (both P < 0.05). RA relieves the injury of alveolar epithelial cells and protects the pulmonary surfactant by inhibiting the destruction of TNF-alpha to type II lung alveolar epithelial cells.

Trafficking of Shiga toxin/Shiga-like toxin-1 in human glomerular microvascular endothelial cells and human mesangial cells

This study has determined the intracellular transport route of Shiga-like toxin (Stx) and the highly related Shiga toxin in human glomerular microvascular endothelial cells (GMVECs) and mesangial cells. In addition, the effect of tumor necrosis factor-alpha (TNF-alpha), which contributes to the pathogenesis of hemolytic-uremic syndrome, was evaluated more profound. Establishing the transport route will provide better understanding of the cytotoxic effect of Stx on renal cells. For our studies, we used receptor-binding B-subunit (StxB), which is identical between Shiga toxin and Stx-1. The transport route of StxB was studied by immunofluorescence microscopy and biochemical assays that allow quantitative analysis of retrograde transport from plasma membrane to Golgi apparatus and endoplasmic reticulum (ER). In both cell types, StxB was detergent-resistant membrane associated and followed the retrograde route. TNF-alpha upregulated Gb3 expression in mesangial cells and GMVECs, without affecting the efficiency of StxB transport to the ER. In conclusion, our study shows that in human GMVECs and mesangial cells, StxB follows the retrograde route to the Golgi apparatus and the ER. TNF-alpha treatment increases the amount of cell-associated StxB, but not retrograde transport as such, making it likely that the strong TNF-alpha-induced sensitization of mesangial cells and GMVECs for the toxic action of Stx is not due to a direct effect on the intracellular trafficking of the toxin.

Therapeutic effectiveness of orally administered transgenic low‐alkaloid tobacco expressing human interleukin‐10 in a mouse model of colitis

Inflammatory bowel disease (IBD) represents a spectrum of diseases in which inflammation leads to acute and chronic gut injury. It is a growing health issue for which no cure exists. The pathogenesis is multifactorial with links to infectious and environmental events that trigger disease in genetically predisposed individuals. Treatment of the two major forms of IBD, Crohn's disease and ulcerative colitis, involves the reduction of inflammation with toxic immunosuppressive drugs or blocking of the pro-inflammatory effects of tumour necrosis factor-alpha (TNF-alpha) with antibodies. Here, we show that the oral administration of transgenic low-alkaloid tobacco expressing the contra-inflammatory cytokine human interleukin-10 (hIL-10) reduces the severity of colitis by down-regulating TNF-alpha expression directly at the sites of inflammation in IBD-susceptible IL-10(-/-) mice. hIL-10 expressed in plants is biologically active and displays resistance to gastrointestinal degradation. Dietary supplementation with plant tissue delivering up to 9 microg of hIL-10 daily for 4 weeks was well tolerated by treated mice. Gut histology was significantly improved relative to controls (P = 0.002), and was correlated with a decrease in small bowel TNF-alpha mRNA levels and an increase in IL-2 and IL-1beta mRNA levels. Transgenic plants expressing IL-10 to directly attenuate TNF-alpha expression at sites of inflammation in the gut may become a useful new approach in the luminal therapy of IBD.