Induction Of TNF-α mRNA Research Articles

Characterization of Chemically-Induced Bacterial Ghosts (BGs) Using Sodium Hydroxide-Induced Vibrio parahaemolyticus Ghosts (VPGs)

Acellular bacterial ghosts (BGs) are empty non-living bacterial cell envelopes, commonly generated by controlled expression of the cloned lysis gene E of bacteriophage PhiX174. In this study, Vibrio parahaemolyticus ghosts (VPGs) were generated by chemically-induced lysis and the method is based on minimum inhibitory concentration (MIC) of sodium hydroxide (NaOH), acetic acid, boric acid, citric acid, maleic acid, hydrochloric acid, and sulfuric acid. The MIC values of the respective chemicals were 3.125, 6.25, <50.0, 25.0, 6.25, 1.56, and 0.781 mg/mL. Except for boric acid, the lysis efficiency reached more than 99.99% at 5 min after treatment of all chemicals. Among those chemicals, NaOH-induced VPGs appeared completely DNA-free, which was confirmed by quantitative real-time PCR. Besides, lipopolysaccharides (LPS) extracted from the NaOH-induced VPGs showed no distinctive band on SDS-PAGE gel after silver staining. On the other hand, LPS extracted from wild-type bacterial cells, as well as the organic acids-induced VPGs showed triple major bands and LPS extracted from the inorganic acids-induced VPGs showed double bands. It suggests that some surface structures in LPS of the NaOH-induced VPGs may be lost, weakened, or modified by the MIC of NaOH. Nevertheless, Limulus amoebocyte lysate assay revealed that there is no significant difference in endotoxic activity between the NaOH-induced VPGs and wild-type bacterial cells. Macrophages exposed to the NaOH-induced VPGs at 0.5 × 106 CFU/mL showed cell viability of 97.9%, however, the MIC of NaOH did not reduce the cytotoxic effect of wild-type bacterial cells. Like Escherichia coli LPS, the NaOH-induced VPGs are an excellent activator of pro-inflammatory cytokines (IL-1β and iNOS), anti-inflammatory cytokine (IL-10), and dual activities (IL-6) in the stimulated macrophage cells. On the other hand, the induction of TNF-α mRNA was remarkable in the macrophages exposed with wild-type cells. Scanning electron microscopy showed the formation of trans-membrane lysis tunnel structures in the NaOH-induced VPGs. SDS-PAGE and agarose gel electrophoresis also confirmed that cytoplasmic proteins and genomic DNA released from the VPGs to culture medium through the lysis tunnel structures. Taken together, all these data indicate that the NaOH-induced VPGs show the potency of a safe, economical, and effective inactivated bacterial vaccine candidate.

Animal Models of Acute Photodamage: Comparisons of Anatomic, Cellular and Molecular Responses in C57BL/6J, SKH1 and Balb/c Mice

Human cutaneous photodamage is a major medical problem that includes premature aging and fragility of the skin. Nonxenografted animal models have not been comparatively evaluated for how well they resemble the changes seen in human skin. Here, we sought to identify a suitable mouse model that recapitulates key anatomic, cellular and molecular responses observed in human skin during acute UV exposure. Adult females from three strains of mice, C57BL/6J, SKH1 and Balb/c were exposed to UVB and then evaluated 3 or 20 h after the last irradiation. Skin from UVB-exposed C57BL/6J mice showed features resembling human photodamage, including epidermal thickening, infiltration of the dermis with inflammatory cells, induction of tumor necrosis factor-α (TNF-α) mRNA, accumulation of glycosaminoglycans, particularly hyaluronan in the epidermis and loss of collagen. Hairless SKH1 mouse skin responded similarly, but without any induction of TNF-α mRNA or chondroitin sulfate. Irradiated Balb/c mice were the least similar to humans. Our results in C57BL/6J mice and to a lesser extent in SKH1 mice, show cutaneous responses to a course of UVB-irradiation that mirror those seen in human skin. Proper choice of model is critical for investigating cellular and molecular mechanisms of photodamage and photoaging.

Comparison of Helicobacter pylori and Escherichia coli in induction of TNF-α mRNA from human peripheral blood mononuclear cells

Purpose: To investigate the difference between the abilities of Helicobacter pylori and Escherichia coli to induce expression of TNF-α in human peripheral blood mononuclear cells (PBMC). Materials and Methods:H pylori was isolated from gastric biopsy specimens. The mononuclear cells were isolated from human blood, cultured, and treated with either intact or sonicated E coli or H pylori, and mRNA expression for TNF-α was detected using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Results: TNF-α mRNA expression levels were significantly higher in PBMCs stimulated with E coli compared to those stimulated with H pylori at the same number and identical conditions (P < .001). The results also suggest that sonicated bacteria were significantly (P < .001) less stimulatory for PBMCs than intact bacteria for both E coli and H pylori. Conclusions: The ability of different H pylori strains isolated from biopsy samples to stimulate TNF-α from PBMCs was significantly lower than that of E coli. Sonicated bacteria, as compared to intact bacteria, was a very poor inducer of TNF-α mRNA expression, suggesting that the conformation of lipopolysaccharides (LPS) on the outer leaflet of the outer membrane is not totally conserved in sonicated bacteria.

Mitogen-activated protein kinases and tumor necrosis factor alpha responses of macrophages infected with Orientia tsutsugamushi

Orientia tsutsugamushi, an obligatory intracellular bacterium, is the causative agent of scrub typhus. The disease is histopathologically characterized by inflammatory manifestations, indicating that orientiae induce mechanisms that amplify the inflammatory response. Here we investigated the transcriptional response of tumor necrosis factor alpha (TNF-α) gene and the host cell responses of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) to O. tsutsugamushi. Induction of the TNF-α gene was not blocked by a eukaryotic protein synthesis inhibitor cycloheximide, suggesting that de novo synthesis of host cell protein is not required for this transcriptional response. The induction of TNF-α mRNA by O. tsutsugamushi was not blocked by the inhibitors of NF-κB activation. Inactivation of Orientia by heat abolished both MAPK activation and TNF-α production, whereas treatment of host cells with cytochalasin D impaired neither NF-κB and MAPK activation nor TNF-α production. In conclusion, our data suggest that O. tsutsugamushi-stimulated TNF-α production and MAPK activation requires cellular attachment by viable orientiae but not bacterial internalization into the host cells.

Induction of Tumor Necrosis Factor-α by UVB: A Role for Reactive Oxygen Intermediates and Eicosanoids¶

UVB irradiation induces nuclear factor–κB (NF-κB) activation, tumor necrosis factor–α (TNF-α) expression and reactive oxygen intermediates (ROI) in keratinocytes. We investigated whether ROI play a role in UVB-induced TNF-α mRNA expression. The antioxidants N-acetyl cysteine, NAC, epigallocathin gallate, EGCG, butylated hydroxyanisole (BHA) and vitamin C could reduce UVB-induced TNF-α mRNA levels to various degrees; vitamin E (α-tocopherol) had no effect. BHA was the most potent inhibitor. The oxidant tertiary butylated hydroperoxide could effectively induce TNF-α mRNA expression. Nordihydroguaiaretic acid (NDGA) and MK-886, inhibitors of lipoxygenase (LOX), and indometacin and quinacrine, inhibitors of cyclooxygenase (COX) and phospholipase A2, respectively, could also reduce UVB-induced TNF-α mRNA expression. Inhibition by NDGA was in concordance with the results for BHA. NDGA, indometacin, quinacrine and BHA could also effectively inhibit the inhibitor of NF-κB degradation, thereby maintaining NF-κB inactivity. In conclusion, we show that ROI are implicated in the induction of TNF-α mRNA by UVB and that not all antioxidants are equally effective inhibitors. COX products and more importantly LOX products, which themselves are products of an oxidative metabolism, are the main ROI implicated in this induction of TNF-α expression by UVB probably via activation of NF-κB.

Mechanisms of Cardiac Depression Caused by Lipoteichoic Acids From Staphylococcus aureus in Isolated Rat Hearts

Lipoteichoic acid (LTA) represents a major virulence factor in gram-positive sepsis. In the present study we perfused isolated rat hearts for 180 minutes with highly purified LTA from Staphylococcus aureus. A progressive decline of left ventricular contractile function paralleled by the expression of myocardial tumor necrosis factor-alpha (TNF-alpha) mRNA and protein as well as the release of TNF-alpha into the perfusate was observed in LTA-perfused hearts. Employment of an anti-TNF-alpha antibody completely prevented the loss in contractile function. When CD14, a prominent pathogen recognition receptor, was blocked by a specific antibody, induction of TNF-alpha mRNA and protein release as well as the associated cardiodepression was diminished in response to LTA. Synthesis of TNF-alpha protein was located to interstitial cells of LTA-challenged hearts as detected by immunohistochemistry. Besides progressive cardiodepression, coronary perfusion pressure (CPP) was moderately increased in LTA-perfused hearts. This was accompanied by the release of thromboxane A2 (TXA2) into the perfusate and the induction of cyclooxygenase (Cox)-2 mRNA and protein in the myocardium. Blocking of TXA2 by the nonspecific Cox inhibitor indomethacin, the thromboxane receptor antagonist daltroban, or the selective Cox-2 inhibitor NS-398 prevented the increase in CPP. LTA causes cardiac depression by activating myocardial TNF-alpha synthesis via CD14 and induces coronary vascular disturbances by activating Cox-2-dependent TXA2 synthesis. These phenomena may contribute to cardiac depression in gram-positive sepsis.

Contributions of angiotensin II and tumor necrosis factor-alpha to the development of renal fibrosis.

Angiotensin II upregulates tumor necrosis factor-alpha (TNF-alpha) in the rat kidney with unilateral ureteral obstruction (UUO). In a mouse model of UUO, we found that tubulointerstitial fibrosis is blunted when the TNF-alpha receptor, TNFR1, is functionally knocked out. In this study, we used mutant mice with UUO in which the angiotensin II receptor AT(1a) or the TNF-alpha receptors TNFR1 and TNFR2 were knocked out to elucidate interactions between the two systems. The contribution of both systems to renal fibrosis was assessed by treating TNFR1/TNFR2-double knockout (KO) mice with an angiotensin-converting enzyme inhibitor, enalapril. The increased interstitial volume (Vv(int)) in the C57BI/6 wild-type mouse was decreased in the AT(1a) KO from 32.8 +/- 4.0 to 21.0 +/- 3.7% (P < 0.005) or in the TNFR1/TNFR2 KO to 22.3 +/- 2.1% (P < 0.005). The Vv(int) of the TNFR1/TNFR2 KO was further decreased to 15.2 +/- 3.7% (P < 0.01) by enalapril compared with no treatment. The induction of TNF-alpha mRNA and transforming growth factor-beta1 (TGF-beta1) mRNA in the kidney with UUO was significantly blunted in the AT(1a) or TNFR1/TNFR2 KO mice compared with the wild-type mice. Treatment of the TNFR1/TNFR2 KO mouse with enalapril reduced both TNF-alpha and TGF-beta1 mRNA and their proteins to near normal levels. Also, alpha-smooth muscle actin expression and myofibroblast proliferation were significantly inhibited in the AT(1a) or TNFR1/TNFR2 KO mice, and they were further inhibited in enalapril-treated TNFR1/TNFR2 KO mice. Incapacitating the angiotensin II or the TNF-alpha systems individually leads to partial blunting of fibrosis. Incapacitating both systems, by using a combination of genetic and pharmacological means, further inhibited interstitial fibrosis and tubule atrophy in obstructive nephropathy.

Induction of TNF-α mRNA in murine macrophages by virulent and avirulent strains of Salmonella choleraesuisserovar Typhimurium and serovar Choleraesuis

TNF- αmRNA induction in murine macrophages by virulent and avirulent Salmonellastrains was measured in vitroby RT-PCR method. Virulence plasmid-cured strains of S. choleraesuisserovar Typhimurium and serovar Choleraesuis, and rpoS-defective mutant of S. choleraesuisserovar Typhimurium induced significantly higher level of TNF- αmRNA than their parent (virulent) strains in macrophages of C3H/HeN mice. When macrophages of LPS-low responder (C3H/HeJ) mice were used, the difference of the induction level was not observed, indicating that LPS was involved in the enhanced level of TNF- αmRNA induction by avirulent Salmonellastrains. LPSs from virulent and avirulent strains were analysed, but no difference was found for cytokine-inducing activity, and chemical properties. Those results suggested that avirulent Salmonellastrains were damaged more easily, and released more LPS in macrophages to enhance TNF- αinduction.

Overcoming cis-diamminedichloroplatinum (II) resistance of human ovarian tumor cells by combination treatment with cis-diamminedichloroplatinum (II) and tumor necrosis factor-alpha.

Previous studies have demonstrated that tumor cells have different degrees of sensitivity and resistance to various cytotoxic agents. The acquisition of drug resistance is a major concern in cancer treatment. The current study investigates the cytotoxic effect of cis-diamminedichloroplatinum (II) (CDDP) and tumor necrosis factor-alpha (TNF-alpha) used in combination on CDDP-resistant human ovarian tumor cell lines. Cytotoxicity was determined by the microculture tetrazolium dye assay. Synergy was assessed by isobolographic analysis. TNF-mRNA was examined by Northern blot analysis. Treatment of the CDDP-resistant C30 cells with CDDP and TNF-alpha overcame the resistance of C30 cells to CDDP or TNF-alpha. In addition, the combination of CDDP and TNF-alpha resulted in a synergistic effect on the C30-resistant line, the CDDP-sensitive parental cell line A2780, and two freshly derived ovarian carcinoma cell cultures. Treatment of C30 cells with CDDP followed by TNF-alpha showed a synergistic effect, whereas treatment with TNF-alpha followed by CDDP demonstrated a less cytotoxic effect. A possible mechanism of resistance to TNF-alpha in tumor cells is the induction of TNF-alpha mRNA and protein. C30 cells do not produce mRNA constitutively for TNF-alpha; however, treatment of C30 cells with TNF-alpha induces the expression of TNF-alpha mRNA. When CDDP was used in combination with TNF-alpha, the level of TNF-alpha mRNA induced by TNF-alpha was reduced significantly. This study shows that the combination of CDDP and TNF-alpha can overcome the CDDP resistance of tumor cells and that downregulation of TNF-alpha mRNA by CDDP may play a role in the enhanced cytotoxicity seen with the combination of CDDP and TNF-alpha. The synergistic effect obtained with established ovarian tumor cell lines and in short-term cultures of freshly isolated ovarian tumors suggests that combination treatment with TNF-alpha and CDDP may have clinical applications in the treatment of drug-resistant tumors.