Regulation Of TNF-α Research Articles

Protease Inhibitors from Marine Actinobacteria as a Potential Source for Antimalarial Compound

The study was planned to screen the marine actinobacterial extract for the protease inhibitor activity and its anti- Pf activity under in vitro and in vivo conditions. Out of 100 isolates, only 3 isolates exhibited moderate to high protease inhibitor activities on trypsin, chymotrypsin and proteinase K. Based on protease inhibitor activity 3 isolates were chosen for further studies. The potential isolate was characterized by polyphasic approach and identified as Streptomyces sp LK3 (JF710608). The lead compound was identified as peptide from Streptomyces sp LK3. The double-reciprocal plot displayed inhibition mode is non-competitive and it confirms the irreversible nature of protease inhibitor. The peptide from Streptomyces sp LK3 extract showed significant anti plasmodial activity (IC50: 25.78 µg/ml). In in vivo model, the highest level of parasitemia suppression (≈45%) was observed in 600 mg/kg of the peptide. These analyses revealed no significant changes were observed in the spleen and liver tissue during 8 dpi. The results confirmed up-regulation of TGF-β and down regulation of TNF-α in tissue and serum level in PbA infected peptide treated mice compared to PbA infection. The results obtained infer that the peptide possesses anti- Pf activity activity. It suggests that the extracts have novel metabolites and could be considered as a potential source for drug development.

High density lipoproteins improve insulin sensitivity in high-fat diet-fed mice by suppressing hepatic inflammation

Obesity-induced liver inflammation can drive insulin resistance. HDL has anti-inflammatory properties, so we hypothesized that low levels of HDL would perpetuate inflammatory responses in the liver and that HDL treatment would suppress liver inflammation and insulin resistance. The aim of this study was to investigate the effects of lipid-free apoAI on hepatic inflammation and insulin resistance in mice. We also investigated apoAI as a component of reconstituted HDLs (rHDLs) in hepatocytes to confirm results we observed in vivo. To test our hypothesis, C57BL/6 mice were fed a high-fat diet (HFD) for 16 weeks and administered either saline or lipid-free apoAI. Injections of lipid-free apoAI twice a week for 2 or 4 weeks with lipid-free apoAI resulted in: i) improved insulin sensitivity associated with decreased systemic and hepatic inflammation; ii) suppression of hepatic mRNA expression for key transcriptional regulators of lipogenic gene expression; and iii) suppression of nuclear factor κB (NF-κB) activation. Human hepatoma HuH-7 cells exposed to rHDLs showed suppressed TNFα-induced NF-κB activation, correlating with decreased NF-κB target gene expression. We conclude that apoAI suppresses liver inflammation in HFD mice and improves insulin resistance via a mechanism that involves a downregulation of NF-κB activation.

NLRC5 Mediates Cytokine Secretion in RAW264.7 Macrophages and Modulated by the JAK2/STAT3 Pathway

The nucleotide-binding domain leucine-rich repeat proteins (NLRs), a class of innate immune receptors that respond to pathogen attack or cellular stress, have gained increasing attention. NLRC5 is the largest member of NLR family, which has recently been identified as a critical regulator of immune responses. In this study, we explore the role of NLRC5 in cytokine secretion and the role of the JAK2/STAT3 signaling pathway in lipopolysaccharide-induced NLRC5 expression in RAW264.7 cells. We demonstrated that overexpression of NLRC5 results in a downregulation of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) secretion; on the other hand, knockdown of NLRC5 by transfecting siRNA enhanced IL-6 and TNF-α secretion in RAW264.7 cells. These results indicated that NLRC5 plays a negative role in the regulation of IL-6 and TNF-α. Meanwhile, AG490 (a specific inhibitor of the JAK2/STAT3 signaling pathway) and JAK2 siRNA were used to manipulate JAK2/STAT3 activity. Finally, the results showed that AG490 and JAK2 siRNA inhibited NLRC5 expression and the expression levels of p-JAK2 and p-STAT3. We, for the first time, demonstrate that the inhibition of the JAK2/STAT3 signaling pathway results in decrease of NLRC5 expression.

Inhaled ds RNA and rhinovirus evoke neutrophilic exacerbation and lung expression of thymic stromal lymphopoietin in allergic mice with established experimental asthma

BackgroundRhinovirus infection or dsRNA stimulation increased thymic stromal lymphopoietin (TSLP), an upstream pro-allergic cytokine, in asthmatic bronchial epithelial cells. We hypothesized that dsRNA challenges superimposed on established experimental allergic asthma constitute a useful exacerbation model. We further hypothesized that TSLP is induced at dsRNA- and rhinoviral infection-induced exacerbations.MethodsAllergic mice were challenged with OVA followed by three daily intranasal challenges with dsRNA or saline. Bronchoalveolar lavage fluid (BALF) was analysed for total protein, lactate dehydrogenase (LDH), CXCL1/KC, CCL2/MCP-1 and differential cell counts. Lung tissue histology, neutrophils and TSLP, TNF-α, IFN-β and IFN-λ mRNA were examined. Alternatively, allergen-challenged mice received intranasal rhinovirus-(RV)-1B followed by lung TSLP immunostaining.ResultsIn mice with allergic airway inflammation, dsRNA challenges caused a significant exacerbation increasing lung tissue inflammation score and tissue neutrophilia. Bronchoalveolar lavage fluid neutrophils, total protein, LDH, CXCL1/KC and CCL2/MCP-1 were also increased (P < 0.01), and so were lung tissue expressions of TNF-α, IFN-λ and TSLP (P < 0.01), but IFN-β was not increased. TSLP, IFN-λ and LDH were not increased by allergen or dsRNA challenges alone, but increased exclusively at exacerbations. RV1B infection-induced exacerbation also increased lung tissue TSLP (P < 0.05).ConclusionsdsRNA-induced exacerbation in mice with experimental asthma involved general inflammation, cytokines and interferons, in agreement with previous observations in exacerbating human asthma. Additionally, both dsRNA and RV1B infection increased lung TSLP exclusively at exacerbations. Our data suggest that dsRNA challenges superimposed on allergic inflammation are suited for pharmacological studies of asthma exacerbations including the regulation of lung tissue TSLP, TNF-α, IFN-β and IFN-λ.

Protective effect of iridoid glycosides from Paederia scandens (LOUR.) MERRILL (Rubiaceae) on uric acid nephropathy rats induced by yeast and potassium oxonate

Iridoid glycosides of Paederia scandens (IGPS) are an active component isolated from Chinese herb P. scandens (LOUR.) MERRILL (Rubiaceae). Uric acid nephropathy (UAN) is caused by excessive uric acid, which results in damage of kidney tissue via urate crystals deposition in the kidneys. This study aimed to investigate the protective effects of IGPS on UAN in rats induced by yeast and potassium oxonate. Treatment groups received different doses of IGPS and allopurinol (AP) daily for 35days respectively. The results showed that treatment with IGPS significantly prevented the increases of uric acid in serum and the elevation of systolic blood pressure (SBP), attenuated renal tissue injury, improved renal function and reserved the biological activity of NOS-1. IGPS also inhibited the biological activity of TNF-α and TGF-β1, and suppressed the mRNA expressions of TNF-α and TGF-β1 in renal tissue. Taken together, the present and our previous findings suggest that IGPS exerts protective effects against kidney damage in UAN rats through its uric acid-lowering, anti-inflammatory and immunomodulatory properties. Furthermore, decreasing SBP by up regulation of NOS-1 expression and down regulation of TNF-α and TGF-β1 expression are involved in the effect of IGPS on high uric acid-induced nephropathy.

MiRNA-181a Regulates Adipogenesis by Targeting Tumor Necrosis Factor-α (TNF-α) in the Porcine Model

Adipogenesis is tightly regulated by altering gene expression, and TNF-α is a multifunctional cytokine that plays an important role in regulating lipogenesis. MicroRNAs are strong post-transcriptional regulators of cell differentiation. In our previous work, we found high expression of miR-181a in a fat-rich pig breed. Using bioinformatic analysis, miR-181a was identified as a potential regulator of TNF-α. Here, we validated TNF-α as the target of miR-181a by a dual luciferase assay. In response to adipogenesis, a mimic or inhibitor was used to overexpress or reduce miR-181a expression in porcine pre-adipocytes, which were then induced into mature adipocytes. Overexpression of miR-181a accelerated accumulation of lipid droplets, increased the amount of triglycerides, and repressed TNF-α protein expression, while the inhibitor had the opposite effect. At the same time, TNF-alpha rescued the increased lipogenesis by miR181a mimics. Additionally, miR-181a suppression decreased the expression of fatty synthesis associated genes PDE3B (phosphodiesterase 3B), LPL (lipoprotein lipase), PPARγ (proliferator-activated receptor-γ), GLUT1(glucose transporter), GLUT4, adiponectin and FASN (fatty acid synthase), as well as key lipolytic genes HSL (hormone-sensitive lipase) and ATGL (adipose triglyceride lipase) as revealed by quantitative real-time PCR. Our study provides the first evidence of the role of miR-181a in adipocyte differentiation by regulation of TNF-α, which may became a new therapeutic target for anti-obesity drugs.

Pulsed Radiofrequency Modulates Pain Regulatory Gene Expression Along the Nociceptive Pathway

Background: Pulsed radiofrequency (PRF) therapy is a clinical treatment utilizing electromagnetic energy aimed to relieve neuropathic pain. This is the first study examining the modulated expression of pain regulatory genes following the induction of the spared nerve injury (SNI) pain model and subsequently treated with PRF therapy. Objectives: The present study investigated the behavioral efficacy of PRF therapy in rats exhibiting sciatic nerve injury and examined gene expression changes in the sciatic nerve, ipsilateral L5 dorsal root ganglia (DRG), and spinal cord. Study Design: A randomized, experimental trial. Setting: Department of Biological Sciences, Illinois State University and Department of Psychology, Illinois Wesleyan University. Methods: An SNI model was used in male Sprague-Dawley rats (weight 260-310 g). A sham surgery was also performed as a control group. After 3 days development of the SNI model, an RF electrode was applied to the sciatic nerve proximal to the site of injury and stimulated for 3 minutes. The response to mechanical stimuli was assessed throughout the duration of the study. Furthermore, changes in gene expression along the nociceptive tract (sciatic nerve, DRG, and spinal cord) were assessed 24 hours post-PRF therapy. Results: It was observed that the mechanical allodynia, induced by SNI model, was reversed to control values within 24 hours post-PRF therapy. Additionally, modulated expression of pain regulatory genes was observed after induction of the SNI model. Following PRF therapy, expression of many of these genes returned to control values (sham) in each of the tissues tested. Increased proinflammatory gene expression, such as TNF-α and IL-6, observed in the sciatic nerve (site of injury) in the SNI group was returned to baseline values following PRF therapy. Up-regulation of GABAB-R1, Na/K ATPase, and 5-HT3r as well as down regulation of TNF-α and IL-6 were also observed in the DRG in the SNI-PRF group relative to the SNI group. Up-regulation of Na/K ATPase and c-Fos was found in the spinal cord following PRF treatment relative to the SNI group. Limitations: Immediate changes in gene expression were observed at 24 hours to better determine the mechanism with no long-term data at this time. Protein expression was not assessed in addition to gene expression changes. Conclusion: These results indicate that the electromagnetic energy applied via PRF therapy influences the reversal of behavioral and molecular effects of hypersensitivity developed from a peripheral nerve injury. Key words: Pulsed radiofrequency, PRF; spared nerve injury, SNI; electromagnetic stimulation; Sprague-Dawley, rat; withdrawal threshold; mechanical allodynia, Von Frey; gene expression; nociceptive pathway; electroneuromodulation; cytokines

Abstract 383: Hypertensive and Renal Injury Responses to Angiotensin II and High Salt Intake in Mice Lacking the Gene for Interleukin-10

Endogenous interleukin-10 (IL-10) exerts immune down-regulating action on the generation of tumor necrosis factor-alpha (TNF-α). The present study examined the hypothesis that IL-10 plays a protective role in hypertension and renal injury induced by angiotensin II (AngII) and high salt (HS) diet by minimizing TNF-α production. Systemic blood pressure (BP; monitored by implanted radio-telemetry), TNF-α level in plasma and in the kidney (by ELISA) as well as renal injury (glomerulosclerosis, GS by PAS staining and renal interstitial fibrosis, RIF by Trichrome staining) responses to chronic infusion of AngII (400 ng/min; osmotic minipump) for 2 wks were evaluated in wild-type (WT; n=11) and IL-10 gene knockout mice (KO; n=11) which were fed either normal (NS; 0.03% NaCl, n=5) or HS (4% NaCl; n=6) diets. On the last day of the experiment, a 24 hr urine collection was made using metabolic cages prior to sacrificing the mice for the collection of plasma and renal tissue samples. The mean baseline BP in KO was lower (104±3 vs 116±4 mmHg) than that in WT. Increase in BP in AngII+HS treated KO was lower (Δ 20±5 vs Δ 39±2 mmHg) than that in WT but similar in AngII+NS treated KO and WT (Δ 40±3 vs Δ 47±7 mmHg). In AngII+HS treated WT, TNF-α was higher in plasma (69±6 vs 34±4 pg/mL) and in renal tissue (208±15 vs 95±11 pg/mg protein) compared to values in WT treated with AngII+NS. In AngII+HS treated KO, TNF-α was lower in plasma (20±3 vs 180±44 pg/mL) and in renal tissue (205±23 vs 277±23 vs pg/mg protein) compared to values in KO treated with AngII+NS. The urinary nitrate/nitrite excretion rate was higher in AngII+NS (0.56±0.25 vs 0.08±0.01 mM/24 hr) and AngII+HS (1.23±0.12 vs 0.18±0.02 mM/24 hr) treated KO compared to the correspondingly treated WT. The eNOS protein expression was higher in KO treated with AngII+NS (~2 folds) or AngII+HS (~3 folds) compared to those in treated WT. GS (24.6±1.3 vs 13.8±2.1 %) and RIF (10.6±1.1 vs 7.8±0.5 %) changes were greater in AngII+NS treated KO than those in treated WT. However, the changes were minimal in HS treated groups. In conclusion, these data demonstrate that there exists an interaction of IL-10 and eNOS activity in the regulation of TNF-α in the kidney that provides a protective role by minimizing hypertension and renal injury induced by Ang II and HS intake.

Baseline TNFα operational capacity in fetal and maternal circulation prior to the onset of labor: "tuned for different purposes".

In this study, we sought to characterize the tumor necrosis factor α (TNFα) baseline operational capacity in mature fetuses and their mothers prior to the onset of labor. We used an experimental pregnant nonhuman primate model to measure the plasma concentration of TNFα, TNF transmembrane receptor I (TNFRI), and TNFRII with validated enzyme-linked immunosorbent assays. Coefficients of correlations between the maternal and the fetal values and the soluble TNFα, TNFRI, or TNFRII concentrations and ratios were calculated. The TNFα/TNFRI ratio was 3 times lower in fetal circulation than in maternal circulation. No correlations were noted between the maternal and the fetal TNFα, TNFRI, or TNFRII plasma concentrations. These findings suggest that the fetal and maternal baseline circulatory operational capacities of TNFα are independent of each other and tuned differently. This differential regulation of TNFα in fetal and maternal circulation at the end of pregnancy may be guided to protect the fetus from the systemic inflammatory response that is essential for the mechanisms of labor to proceed in the mother.

WW Domain Containing E3 Ubiquitin Protein Ligase 1 (WWP1) Negatively Regulates TLR4-Mediated TNF-α and IL-6 Production by Proteasomal Degradation of TNF Receptor Associated Factor 6 (TRAF6)

BackgroundToll-like receptors (TLRs) play a pivotal role in the defense against invading pathogens by detecting pathogen-associated molecular patterns (PAMPs). TLR4 recognizes lipopolysaccharides (LPS) in the cell walls of Gram-negative bacteria, resulting in the induction and secretion of proinflammatory cytokines such as TNF-α and IL-6. The WW domain containing E3 ubiquitin protein ligase 1 (WWP1) regulates a variety of cellular biological processes. Here, we investigated whether WWP1 acts as an E3 ubiquitin ligase in TLR-mediated inflammation.Methodology/ResultsKnocking down WWP1 enhanced the TNF-α and IL-6 production induced by LPS, and over-expression of WWP1 inhibited the TNF-α and IL-6 production induced by LPS, but not by TNF-α. WWP1 also inhibited the IκB-α, NF-κB, and MAPK activation stimulated by LPS. Additionally, WWP1 could degrade TRAF6, but not IRAK1, in the proteasome pathway, and knocking down WWP1 reduced the LPS-induced K48-linked, but not K63-linked, polyubiquitination of endogenous TRAF6.Conclusions/SignificanceWe identified WWP1 as an important negative regulator of TLR4-mediated TNF-α and IL-6 production. We also showed that WWP1 functions as an E3 ligase when cells are stimulated with LPS by binding to TRAF6 and promoting K48-linked polyubiquitination. This results in the proteasomal degradation of TRAF6.

Soluble ST2 Does Not Regulate TNF-α and IL-6 Production in Dengue Virus-Infected Human Monocytes

Dengue virus (DENV) produces an acute infection that results in the overproduction of proinflammatory cytokines. Although increased levels of the immunoregulator soluble ST2 (sST2) protein have been reported in the serum of patients with dengue, its importance during DENV infection remains unclear. The purpose of this study was to evaluate the effect of a recombinant human sST2 protein on the production of TNF-α and IL-6 in an in vitro model of DENV infection. Peripheral blood mononuclear cells (PBMCs) were permissive to in vitro DENV infection since viral antigen was detected in CD14+ monocytes by flow cytometry (median, 1%; range, 0–2.2), and in their supernatants TNF-α and IL-6 were detected. However, sST2 protein was not detected. Using multiple staining on infected PBMC we found that only CD14+ cells produced TNF-α and IL-6. Treatment with human recombinant sST2 protein decreased lipopolysaccharide-induced monocyte TNF-α and IL-6 production. However, this effect was not observed when the monocytes were pretreated with sST2 and later infected with DENV-2. These results suggest that sST2 has different roles in the regulation of TNF-α and IL-6 expression in human monocytes stimulated with LPS and DENV-2.

Abstract 551: The Dual Effect of Cigarette Smoke on Macrophage Abca1-dependent Cholesterol Efflux and Protease Production Leads to Decreased Collagen and Increased Necrosis in Plaques of ApoE Deficient Mice

Epidemiological evidence links smoking to an increase in the incidence and severity of cardiovascular disease. However, the molecular mechanisms responsible for susceptibility are unknown. The accumulation of lipids in macrophages induces an inflammatory response, with secretion of cytokines and proteases, and leads to necrosis and loss of collagen content within atherosclerotic lesions. Therefore, the effect of cigarette smoke on lipids in the atherosclerotic process and consequences was examined. Initial studies revealed that cigarette smoke attenuated cholesterol efflux. Subsequently, the end result of cigarette smoke exposure on atherosclerotic lesions in ApoE deficient mice demonstrated a significant increase in necrosis along with a dramatic decrease in the collagen content of lesions in ApoE deficient mice exposed to smoke for 20 weeks. Cigarette smoke extract (CSE) impaired the cholesterol efflux ability for ApoAI and correlated with ABCA1 loss and increased MMPs (9, 12, 13, in human THP-1 cells additionally MMP1 is increased), TLR4/Myd88 and TNFα expression in mouse bone marrow derived macrophages (BMdM) in vitro and in vivo in peritoneal macrophages elicited after 5 days of smoke exposure. Furthermore, by increasing ABCA1 expression with LXR agonist treatment the CSE-induced up regulation of MMPs (MMP-9 and 13) and TNFα was inhibited signifying a correlation between the cigarette smoke induced loss of ABC transporters and MMP induction. Moreover, BMDMs isolated from Cre-LysM-ABCA1 deficient mice displayed an increase in mRNA expression of MMPs (9, 12, 13) at a basal level, which was appreciably augmented with CSE as compared to control macrophages. The LXR induced decrease in MMPs and TNFα expression as well as decreased MMP-9 activity in macrophages was confirmed to be ABCA1-dependent since ABCA1 KO macrophages exposed to CSE did not exhibit this response. These findings establish that the effect of cigarette smoke on macrophage ABCA1-dependent cholesterol efflux impairment leads to both an increase in proteases and inflammatory cytokines and this combination of events is likely synergistic in ultimately generating a more vulnerable plaque through the increase in plaque necrosis and a decrease in collagen content.

Dual-specificity phosphatase 3 knockout female mice are resistant to LPS and to polymicrobial induced septic shock in TNF dependent manner. (P1222)

Abstract We report the generation of dual-specificity phosphatase 3 (DUSP3) deficient mice. These mice develop normally and do not exhibit any spontaneous phenotype. However, VHR-/- females, but not males, are resistant to LPS- and to polymicrobial infection-induced septic shock. After LPS injection, while VHR-/- males and VHR+/+ mice of both genders, displayed an increased serum levels of TNF-α and IFNγ, the levels of these cytokines remained significantly low in the VHR-/- females. In vitro experiments using peritoneal macrophages showed the same results suggesting that the systemic cytokines profiles observed are macrophages-dependent. Adoptive transfer of VHR-/- females bone marrow to irradiated VHR+/+ female mice, but not to VHR-/- or VHR+/+ males, protected them from death after administration of LPS. Interestingly, VHR-/- females were sensitive to TNF-α- induced lethality. We also report that the decrease of TNF-α production observed in VHR-/- female’s macrophages after LPS activation was associated with a decreased ERK1/2, but not MEK1/2, activation. Interestingly, pervanadate (PTP pan inhibitor) treatment prior to LPS activation restored ERK1/2 activation in the VHR-deficient macrophages, suggesting that VHR is targeting one of the ERK1/2 PTPs or DUSPs. These results, together with our observation that DUSP3 is the most highly expressed phosphatase in macrophages, suggest a key non-redundant role of VHR as positive regulator of TNF-α in innate immune response in females.

Regulation of TNF‐α with a focus on rheumatoid arthritis

Cytokines and chemokines represent two important groups of proteins that control the human immune system. Dysregulation of the network in which these immunomodulators function can result in uncontrolled inflammation, leading to various diseases including rheumatoid arthritis (RA), characterized by chronic inflammation and bone erosion. Potential triggers of RA include autoantibodies, cytokines and chemokines. The tight regulation of cytokine and chemokine production, and biological activity is important. Tumor necrosis factor-α (TNF-α) is abundantly present in RA patients' serum and the arthritic synovium. This review, therefore, discusses first the role and regulation of the major proinflammatory cytokine TNF-α, in particular the regulation of TNF-α production, post-translational processing and signaling of TNF-α and its receptors. Owing to the important role of TNF-α in RA, the TNF-α-producing cells and the dynamics of its expression, the direct and indirect action of this cytokine and possible biological therapy for RA are described.

Genetic polymorphisms of tumour necrosis factor alpha (TNF‐α) promoter gene and response to TNF‐α inhibitors in Spanish patients with inflammatory bowel disease

Tumour necrosis factor alpha (TNF-α) has an important role in inflammatory response. Alterations in the regulation of TNF-α have been implicated in a variety of inflammatory disorders, including Inflammatory bowel disease (IBD). Indeed, a common treatment for IBD is the use of TNF-α inhibitors. Polymorphisms in the TNF-α promoter region are known to affect the level of gene expression. Our aim was to investigate the influence of these single nucleotide polymorphisms (SNPs) in TNF-α promoter gene play in the risk of IBD in a Spanish population and their individual response to anti-TNF-α treatment. DNA samples from patients with IBD and controls were screened for TNF-α -238G/A (rs361525) and -308G/A (rs1800629) SNPs by PCR-SSOP using a microbeads luminex assay and compared with response to TNF-α inhibitors. There were not statistical differences in -238G/A and -308G/A allele and genotype frequencies between patients. However, we found an increased frequency of -308A allele and -308GA genotype in these nonresponders patients to TNF-α inhibitors with respect to responders patients (Pc<0.05). This -308GA genotype has been classified as high producer of this cytokine. This fact could actually be interesting to explain the different response of patients with IBD with respect to TNF-α inhibitors. TNF-α promoter gene polymorphism does not seem to play a role in IBD susceptibility, but particular TNF-α genotypes may be involved in the different responses to TNF-α inhibitor treatment in Spanish patients with IBD.

Ethanolic extract of the Goldenseal, Hydrastis canadensis, has demonstrable chemopreventive effects on HeLa cells in vitro: Drug–DNA interaction with calf thymus DNA as target

This study tested chemotherapeutic potential of Hydrastis canadensis (HC) extract in HeLa cells in vitro, with emphasis on its drug–DNA interaction and apoptosis induction ability. Nuclear uptake of HC by DAPI, Ao/Eb staining and internucleosomal DNA damage by comet assay was studied through fluorescence microscopy. Possible changes in MMP and apoptotic signalling events were critically analyzed. Cell cycle progression studied through FACS and fragmented DNA through “TUNEL” assay were critically analyzed. RT-PCR studies were conducted for analyzing Cyt-C and Bax translocation in mitochondrial and cytosolic extracts, and Caspase 3 in whole cell lysate. Role of p53-mediated regulation of NF-κβ and TNF-α was elucidated by Western blot analysis. Data of CD and Tm profile of CT-DNA were analyzed. Overall results indicated anti-cancer potential of HC through its ability to induce apoptosis, and interaction with CT-DNA that changed structural conformation of DNA, proving HC to be a promising candidate for chemoprevention.

IL-15 that a regulator of TNF-α in patients with diabetes mellitus type 2

Diabetes mellitus type 2 (DM2), obesity, and the metabolic syndrome are considered that a low-grade chronic inflammatory condition. Although synthesis of different pro- and anti-inflammatory cytokines has been implicated in this process, this low-grade inflammatory condition is characterized especially by an increase in Tumor Necrosis Factor alpha (TNF-α), a cytokine that has been associated to the development of insulin resistance and muscle wasting in the diabetic patients. In consequence, mechanisms implicated in attenuating the deleterious effects of TNF-α are activated. One of these mechanisms may involve interleukin 15 (IL-15), which has proven effect on intermediate metabolisms, regulating blood glucose and lipid levels, and diminishing proteolysis in striated muscle. This suggests that the induction of endogenous IL-15 production by a simple event as physical activity may aid in decreasing or even inhibiting the negative effects of TNF-α in patient with DM2 or obesity, in which a low grade chronic inflammatory condition is present.

Effect of high‐fat diet (HFD) on blood pressure and placental levels of tumor necrosis factor (TNF)‐α and soluble fms‐like tyrosine kinase (sFlt)‐1 in pregnant rats

During preeclampsia (PE), placental ischemia increases circulating levels of inflammatory and anti‐angiogenic factors that ultimately leads to hypertension and proteinuria. While obesity is a leading risk factor for developing PE, the mechanisms whereby obesity increases this risk are unclear. Our aims were to examine the effects of HFD on cardiovascular and metabolic systems of normal pregnant rats. Twelve‐week‐old Sprague‐Dawley female rats were fed a normal diet (ND, 13% fat kcal; n=12) or HFD (40% fat kcal). After 9 weeks, rats receiving HFD were grouped in obese prone (OP, weight gain=78.6±2.2 g; n=10) and obese resistant (OR, wg=57.4±2.0 g; n=11) (vs. ND wg=53.6±3.2 g; P&lt;0.05). Rats were then allowed to breed, and mean arterial pressure (MAP) was measured by carotid catheters and tissues were harvested at gestational day 19. Body weight/litter size and visceral fat mass were similar in ND, OR and OP (32.0±2.6 vs. 32.7±2.6 vs. 30.0±1.4 g and 10.9±0.6 vs. 12.2±0.6 vs. 13.2±1.0 g; P&gt;;0.05). MAP was also similar in ND, OR and OP (112±7 vs. 108±3 vs. 110±4 mmHg; P&gt;;0.05). Placental levels of TNFα and sFlt1 were comparable between ND, OR and OP (1.0±0.1 vs. 1.5±0.2 vs. 1.2±0.1 ng/μg and 0.8±0.1 vs.0.9±0.1 vs. 0.9±0.1 pg/μg; P&gt;;0.05). In summary, while HFD increases body weight in OP virgin rats, HFD in OP pregnant rats had no effect on blood pressure regulation or placental levels of TNFα and sFlt1. Funding: HL51971 and HL105324.

Mesenchymal stem cells and Interleukin-6 attenuate liver fibrosis in mice.

BackgroundMesenchymal stem cell (MSC) transplantation has emerged as a promising therapy for liver fibrosis. Issues concerning poor MSC survival and engraftment in the fibrotic liver still persist and warrant development of a strategy to increase MSC potency for liver repair. The present study was designed to examine a synergistic role for Interleukin-6 (IL-6) and MSCs therapy in the recovery of carbon tetrachloride (CCl4) induced injured hepatocytes in vitro and in vivo.MethodsInjury was induced through 3 mM and 5 mM CCl4 treatment of cultured hepatocytes while fibrotic mouse model was established by injecting 0.5 ml/kg CCl4 followed by treatment with IL-6 and MSCs. Effect of MSCs and IL-6 treatment on injured hepatocytes was determined by lactate dehydrogenase release, RT-PCR for (Bax, Bcl-xl, Caspase3, Cytokeratin 8, NFκB, TNF-α) and annexin V apoptotic detection. Analysis of MSC and IL-6 treatment on liver fibrosis was measured by histopathology, PAS, TUNEL and Sirius red staining, RT-PCR, and liver function tests for Bilirubin and Alkaline Phosphatase (ALP).ResultsA significant reduction in LDH release and apoptosis was observed in hepatocytes treated with a combination of MSCs and IL-6 concomitant with upregulation of anti-apoptotic gene Bcl-xl expression and down regulation of bax, caspase3, NFκB and TNF-α. Adoptive transfer of MSCs in fibrotic liver pretreated with IL-6 resulted increased MSCs homing and reduced fibrosis and apoptosis. Hepatic functional assessment demonstrated reduced serum levels of Bilirubin and ALP.ConclusionPretreatment of fibrotic liver with IL-6 improves hepatic microenvironment and primes it for MSC transplantation leading to enhanced reduction of liver injury after fibrosis. Synergistic effect of IL-6 and MSCs seems a favored therapeutic option in attenuation of liver apoptosis and fibrosis accompanied by improved liver function.

Glutamine and alanine-induced differential expression of intracellular IL-6, IL-8, and TNF-α in LPS-stimulated monocytes in human whole-blood

To investigate the effects of the commonly-used immunomodulators l-glutamine, l-alanine, and the combination of both l-alanyl-l-glutamine (Dipeptamin®) on intracellular expression of IL-6, IL-8, and TNF-α during endotoxemia, lipopolysaccharide (LPS)-stimulated human monocytes in a whole blood system were investigated by flow cytometry.Whole blood of twenty-seven healthy volunteers was stimulated with LPS and incubated with three different amino acid solutions (1. l-glutamine, 2. l-alanine, 3. l-alanyl-l-glutamine, each concentration 2mM, 5mM, incubation time 3h). CD14+ monocytes were phenotyped in whole-blood and intracellular expression of cytokines was assessed by flow cytometry.Our investigations showed for the first time in whole blood probes, imitating best physiologically present cellular interactions, that l-glutamine caused a dose-independent inhibitory effect on IL-6 and TNF-α production in human monocytes stimulated with LPS. However, l-alanine had contrary effects on IL-6 expression, significantly upregulating expression of IL-6 in LPS-treated monocytes. The impact of l-alanine on the expression of TNF-α was comparable with glutamine. Neither amino acid was able to affect IL-8 production in LPS-stimulated monocytes. The combination of both did not influence significantly IL-6 and IL-8 expression in monocytes during endotoxemia, however strongly reduced TNF-α production.For the regulation of TNF-α, l-glutamine, l-alanine and the combination of both show a congruent and exponentiated downregulating effect during endotoxemia, for the modulation of IL-6, l-glutamine and l-alanine featured opposite regulation leading to a canceling impact of each other when recombining both amino acids.