IL-6 Response Research Articles

Sex differences in interleukin-6 stress responses in people with Type 2 diabetes.

People with Type 2 diabetes (T2D) show dysregulated inflammatory responses to acute stress, but the effect of sex on inflammatory responses in T2D remains unclear. The purpose of this study was to investigate differences in interleukin (IL)‐6 stress responses between older men and women with T2D. One hundred and twenty‐one people (76 men; mean age = 64.09, SD = 7.35, 45 women; mean age = 63.20, SD = 6.70) with doctor‐verified T2D took part in this laboratory‐based stress testing study. Participants carried out acute mental stress tasks, and blood was sampled at baseline, immediately poststress, 45 min poststress, and 75 min poststress to detect plasma IL‐6 concentrations. IL‐6 change scores were computed as the difference between the baseline measurement and the three time points poststress. Main effects and interactions were tested using mixed model analysis of covariance. We found a significant main effect of time on IL‐6 levels, and a significant Sex × Time interaction. In adjusted analyses including the three change scores and all the covariates, the significant Sex × Time interaction was maintained; IL‐6 responses were greater in women at 45 and 75 min poststress compared with men, adjusting for age, body mass index, smoking, household income, glycated hemoglobin, oral antidiabetic medication, insulin/other injectable antidiabetic medication, depressive symptoms, and time of day of testing. Different inflammatory stress response pathways are present in men and women with T2D, with women producing larger IL‐6 increases. The long‐term implications of these differences need to be elucidated in future studies.

Interferon-gamma production in Lyme arthritis synovial tissue promotes differentiation of fibroblast-like synoviocytes into immune effector cells.

Lyme arthritis (LA), a late disease manifestation of Borrelia burgdorferi infection, usually resolves with antibiotic therapy. However, some patients develop proliferative synovitis lasting months to several years after spirochetal killing, called postinfectious LA. In this study, we phenotyped haematopoietic and stromal cell populations in the synovial lesion ex vivo and used these findings to generate an in vitro model of LA using patient-derived fibroblast-like synoviocytes (FLS). Ex vivo analysis of synovial tissue revealed high abundance of IFNγ-producing T cells and NK cells. Similar to marked IFNγ responses in tissue, postinfectious LA synovial fluid also had high levels of IFNγ. HLA-DR-positive FLS were present throughout the synovial lesion, particularly in areas of inflammation. FLS stimulated in vitro with B.burgdorferi, which were similar to conditions during infection, expressed 68 genes associated primarily with innate immune activation and neutrophil recruitment. In contrast, FLS stimulated with IFNγ, which were similar to conditions in the postinfectious phase, expressed >2,000 genes associated with pathogen sensing, inflammation, and MHC Class II antigen presentation, similar to the expression profile in postinfectious synovial tissue. Furthermore, costimulation of FLS with B.burgdorferi and IFNγ induced greater expression of IL-6 and other innate immune response proteins and genes than with IFNγ stimulation alone. These results suggest that B.burgdorferi infection, in combination with IFNγ, initiates the differentiation of FLS into a highly inflammatory phenotype. We hypothesise that overexpression of IFNγ by lymphocytes within synovia perpetuates these responses in the postinfectious period, causing proliferative synovitis and stalling appropriate repair of damaged tissue.

Reappraisal of antibodies against Saccharomyces cerevisiae (ASCA) as persistent biomarkers in quiescent Crohn’s disease

A clear correlation exists between microbiota and the dysregulation of the immune response in Inflammatory Bowel Diseases (IBD), which comprise Crohn’s disease (CD) and ulcerative colitis (UC). These unbalanced reactions also involve humoral responses, with antibodies against Saccharomyces cerevisiae. Thus, here we aimed to quantify IgA and IgG specific to S. cerevisiae (ASCA) in quiescent CD and UC, to correlate the production of these antibodies with patient’s inflammatory response and disease clinical presentation. Twenty-nine subjects (16 CD and 13 UC) and 45 healthy controls were enrolled in this study and had plasma samples tested for ASCA and cytokines (IL-2, IL-4, IL-6, IL-10, IFN-γ, TNF-α), besides clinical evaluation. IBD patients had increase IgA and IgG ASCA, especially those with colonic (L2) and fistulizing (B3) CD. Similarly, patients who dropped out the treatment had augmented ASCA, while IgG was reduced in those receiving sulfasalazine treatment. Furthermore, the quiescent CD patients had elevated IL-6 on plasma, especially in the absence of treatment, together with increased counter regulatory response of IL-10. There was a positive correlation between IgA and IgG on CD but not UC, as well as between IgA and TNF in total IBD patients. In addition, the levels of IgG x TNF, IgA x IL-10 and IgG x IL-10 were also correlated in CD, indicating that ASCA production may be influenced by the inflammatory response. Finally, we concluded that ASCA could be pointed as relevant biomarker of CD presentation and residual inflammation, even in clinical remission patients.

The Effect of Sticopus Hermanii-Hyperbaric Oxygen Therapy to Inflammatory Response of Diabetic Periodontitis

Periodontitis is a major risk factor in the oral cavity in chronic diabetes mellitus. The level of IL-1β as inflammatory cytokine increased in patients with type 2 diabetes and periodontitis, while IL-10 is an anti-inflammatory cytokine, may mediate periodontitis in diabetes. Stichopus hermanii (SH) have been known to have anti-inflammation property. Hyperbaric Oxygen Therapy (HBOT) thas been used as adjuvant therapy in diabetes wound healing. Purpose: To examine the effect of combination SH and HBOT to the expression of inflammatory response of IL-1β and IL-10 on diabetic periodontitis rats. Methods :Thirty male Wistar rats were divided to normal (K-0), diabetic-periodontitis (K-1), and treatment group of SH (K-2), HBOT (K-3), and SH-HBOT (K-4).Experimental diabetic was induced by single dose 65 mg/kg of BW intraperitoneal. The Sticopus hermanii gel 3% by topical application in sulcus gingiva and HBOT 2,4 ATA 3x30 minutes interval 5 minute for 7 days. After 52 days the animals were decapitated and the expression of IL-1β and IL-10 were examined by immunohistochemistry. Data were analyzed with ANOVA and LSD. Results : The expression of IL-1β were raised in K-1 group (10.68±1.50) (p< 0.05), while IL-10 were not raised (8.00 ±1.95) compare to normal group (7.25 ± 0.85) (p>0.05). Treatment with SH in K-2 group resulted in decreased of IL-1β expression (10.00± 1.09) while the expression of IL-10 were raised (8.33 ± 2.60) compare to K-1 (7.25 ± 0.85) (p<0.05). Treatment with HBOT in K-3 group resulted in decreased of IL-10 expression (8.333±0,81) while the expression of IL- 10 were raised (10.67±1.03) compare to K-1 (p<0.05). Treatment with SH-HBOT in K-4 group showed the most decreasing expression of IL-1β (6.50±2.44) and increasing expression of IL-10 (14.83±1.47) compare to K-1 (p<0.05). Conclusions : Combination of SH-HBOT decreased the expression of IL-1β and increased the expression of IL-10 on diabetic periodontitis diabetes rats

The Impact of Resveratrol Supplementation on Inflammation Induced by Acute Exercise in Rats: Il6 Responses to Exercise.

Severe physical activity leads to a sharp increase in free radicals, an oxidative stress, inflammation, and tissue damage. Resveratrol as one of the antioxidants can be effective in preventing the effects of oxidative stress. Therefore, the present study was aimed to evaluate the effect of trans-resveratrol supplementation and training exercise on inflammation-related factors. Sixty-four male Wistar rats were divided into six groups, each group consisting of 16 animals: 1) excursive + trans-resveratrol, 2) exercise group, 3) trans-resveratrol group, and 4) control group. Following the familiarization sessions, a more consistent protocol with an intensity of 65% vo2 max was performed for 12 weeks. Afterward, half of the mice in each group received acute exercise training with an intensity of 70-75% of vo2 max at the age of 20 weeks, until reaching the disability level. Finally, the levels of inflammatory markers were measured using special kits. Our findings depicted that inflammatory factors such as CPR, TNF-α, IL-6, and IL-7 were not affected by endurance protocol (P > 0.05), whereas, they were significantly increased by acute exercise training (P > 0.05). Additionally, we found that RES supplements led to a decrease in CPR and IL-6 levels, while not affecting TNF-α and IL-17 levels. According to available evidence, RES appears to have anti-inflammatory and protective effects during exercise by reducing inflammatory factors. Further studies are required to clarify the role of trans-resveratrol supplementation after exercise training.

Differences in serum IL-6 response after 1 °C rise in core body temperature in individuals with spinal cord injury and cervical spinal cord injury during local heat stress

Objectives: Passive rise in core body temperature achieved by head-out hot water immersion (HHWI) results in acute increases in serum interleukin (IL)-6 but no change in plasma adrenaline in patients with cervical spinal cord injury (CSCI). The purpose of the present study was to determine the mechanism of heat stress-induced increase in serum IL-6.Setting: A cross-sectional study.Methods: The study subjects were nine with CSCI, ten with thoracic and lumbar spinal cord injury (TLSCI) and eight able-bodied (AB) subjects. Time since injury was 16.1 ± 3.4 years in TLSCI and 16.4 ± 4.1 years in CSCI. Subjects were subjected to lower-body heat stress (LBH) by wearing a hot water-perfused suit until 1 °C increase in core temperature. The levels of serum IL-6, plasma adrenaline, tumour necrosis factor (TNF)-α, C-reactive protein (CRP), and counts of blood cells were measured at normothermia and after LBH.Results: Serum IL-6 concentrations increased significantly immediately after LBH in all the three groups. ΔIL-6% was lower in CSCI subjects compared with AB subjects. Plasma adrenaline concentrations significantly increased after LBH in AB and TLSCI subjects, but did not change throughout the study in CSCI subjects. Cardiac output and heart rate increased at the end of LBH in all three groups.Conclusions: Under a similar increase in core temperature, ΔIL-6% was lower in the CSCI group compared with the AB group. These findings suggest that the observed rise in IL-6 during hyperthermia is mediated, at least in part, by plasma adrenaline.

IL-6 and IL-1β responses to a carbohydrate-electrolyte drink in orienteering athletes

IL-6 and IL-1β responses to a carbohydrate-electrolyte drink in orienteering athletes

Design, synthesis, and biological activity of substituted 2-amino-5-oxo-5H-chromeno[2,3-b]pyridine-3-carboxylic acid derivatives as inhibitors of the inflammatory kinases TBK1 and IKKε for the treatment of obesity.

The non-canonical IκB kinases TANK-binding kinase 1 (TBK1) and inhibitor of nuclear factor kappa-B kinase ε (IKKε) play a key role in insulin-independent pathways that promote energy storage and block adaptive energy expenditure during obesity. Utilizing docking calculations and the x-ray structure of TBK1 bound to amlexanox, an inhibitor of these kinases with modest potency, a series of analogues was synthesized to develop a structure activity relationship (SAR) around the A- and C-rings of the core scaffold. A strategy was developed wherein R7 and R8 A-ring substituents were incorporated late in the synthetic sequence by utilizing palladium-catalyzed cross-coupling reactions on appropriate bromo precursors. Analogues display IC50 values as low as 210 nM and reveal A-ring substituents that enhance selectivity toward either kinase. In cell assays, selected analogues display enhanced phosphorylation of p38 or TBK1 and elicited IL-6 secretion in 3T3-L1 adipocytes better than amlexanox. An analogue bearing a R7 cyclohexyl modification demonstrated robust IL-6 production in 3T3-L1 cells as well as a phosphorylation marker of efficacy and was tested in obese mice where it promoted serum IL-6 response, weight loss, and insulin sensitizing effects comparable to amlexanox. These studies provide impetus to expand the SAR around the amlexanox core toward uncovering analogues with development potential.

Evolution of a maternal immune activation (mIA) model in rats: Early developmental effects

Maternal immune activation (mIA) in rodents is rapidly emerging as a key model for neurodevelopmental disorders such as autism spectrum disorder (ASD) and schizophrenia. Here, we optimise a mIA model in rats, aiming to address certain limitations of current work in this field. Specifically, the lack of clear evidence for methodology chosen, identification of successful induction of mIA in the dams and investigation of male offspring only. We focus on gestational and early juvenile changes in offspring following mIA, as detailed information on these critical early developmental time points is sparse.Following strain (Wistar, Lister Hooded, Sprague Dawley) comparison and selection, and polyriboinosinic-polyribocytidylic acid (poly I:C) dose selection (2.5–15 mg/kg single or once daily for 5 days), mIA was induced in pregnant Wistar rats with 10 mg/kg poly I:C i.p. on gestational day (GD) 15. Early morphometric analysis was conducted in male and female offspring at GD21 and postnatal day (PD) 21, eight dams for each treatment at each time point were used, 32 in total. Subsequent microglia analysis was conducted at PD21 in a small group of offspring.Poly I:C at 10 mg/kg i.p. induced a robust, but variable, plasma IL-6 response 3 h post-injection and reduced body weight at 6 h and 24 h post-injection in two separate cohorts of Wistar rats at GD15. Plasma IL-6 was not elevated at PD21 in offspring or dams. Poly I:C-induced mIA did not affect litter numbers, but resulted in PD21 pup, and GD21 placenta growth restriction. Poly I:C significantly increased microglial activation at PD21 in male hippocampi.We have identified 10 mg/kg poly I:C i.p on GD15 as a robust experimental approach for inducing mIA in Wistar rats and used this to identify early neurodevelopmental changes. This work provides a framework to study the developmental trajectory of disease-relevant, sex-specific phenotypic changes in rats.

Toxoplasma gondii profilin does not stimulate an innate immune response through bovine or human TLR5.

Toxoplasma gondii is responsible for one of the most prevalent infections in people. T. gondii profilin (TgPr) is a protein integral to parasite movement and cellular invasion. Murine TLRs has been described to bind TgPr. Furthermore, more recently, human TLR5 has been described to recognise recombinant TgPr, as well as bacterial flagellin. In addition to infections in humans, T. gondii infects farm animals, but little information is available about its innate recognition. We aimed to investigate whether, similarly to their human orthologue, bovine and porcine TLR5 could also be stimulated by TgPr by using a combination of reporter cell lines expressing full length TLR5 from each species as well as primary cells. Although human and bovine TLR5-transfected cells responded to flagellin, no response was detected upon stimulation with profilin. Furthermore, TgPr failed to elicit IL-6 secretion in human peripheral blood mononuclear cells and CD14+ monocytes. In contrast, exposure of RAW cells, known to express TLR11, to TgPr slightly increased the IL-6 response. Our data cast doubts on the possibility that profilin is a specific ligand for human TLR5 and bovine TLR5. This leaves the immunogenic properties of this potential target antigen (Ag) uncharacterised outside of the murine system.

Remifentanil suppresses increase in interleukin-6 mRNA in the brain by inhibiting cyclic AMP synthesis.

Neuronal inflammation is caused by systemic inflammation and induces cognitive dysfunction. IL-6 plays a crucial role in therapies for neuronal inflammation and cognitive dysfunction. Remifentanil, an ultra-short-acting opioid, controls inflammatory reactions in the periphery, but not in the brain. Therefore, the anti-inflammatory effects of remifentanil in neuronal tissue and the involvement of cAMP in these effects were investigated in the present study. Mice were divided into 4 groups: control, remifentanil, LPS, and LPS + remifentanil. Brain levels of pro-inflammatory cytokine mRNA, and serum levels of corticosterone, catecholamine and IL-6 were measured in the 4 groups. The co-localization of IL-6 and astrocytes in the mouse brain after the LPS injection was validated by immunostaining. LPS and/or remifentanil-induced changes in intracellular cAMP levels in cultured glial cells were measured, and the effects of cAMP on LPS-induced IL-6 mRNA expression levels were evaluated. Remifentanil suppressed increase in IL-6 mRNA levels in the mouse brain, and also inhibited the responses of plasma IL-6, corticosterone, and noradrenaline in an inflammatory state. In the hypothalamus, IL-6 was localized in the median eminence, at which GFAP immunoreactivity was specifically detected. In cultured cells, remifentanil suppressed increase in IL-6 mRNA levels and intracellular cAMP levels after the administration of LPS, and this enhanced IL-6 mRNA expression in response to LPS. Remifentanil suppressed increase in IL-6 mRNA levels in the brain in an inflammatory state, and this effect may be attributed to its direct action on neuronal cells through the inhibition of intracellular cAMP rather than corticosterone.

Characterization of cellular and humoral immune responses to alkaline phosphatase from fertile hydatid cysts in the human peripheral blood.

Hydatid cyst, the larval stage of the tapeworm Echinococcus granulosus and a causative agent of cystic echinococcosis, possesses a vast number of antigenic peptides that are constantly presented in the host immune system during infection. Here, we sought to provide more information about the cellular/humoral components engaged in the peripheral immune reactions to the fertile-cyst-derived Echinococcus alkaline phosphatase (E.ALP) in human hosts. Lymphoproliferative and cytokine responses after recall of E.ALP suggested the presence of specific immune reactions against the antigen. Interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin (IL)-10 had the highest fold increase over the spontaneous levels in response to hydatid crude antigen (HCA). Recall of E.ALP, as well as its encounter, boosted IFN-γ, TNF-α, IL-2, and IL-6 responses in peripheral blood mononuclear cells cultures (PBMCs). The HCA-driven levels of all the cytokines in the culture supernatants of normal PBMCs were higher than those measured after E.ALP encounter. Immunoglobulin G (IgG)-profile in response to HCA showed the dominance of specific IgG1, IgG2, and IgG4 antibodies, but relatively lower affinity of IgG3 to this antigen. IgG1 and IgG3 were the only isotypes detected in serum responses to E.ALP. Our findings suggested that E.ALP contributes to the early phase of immune responses to the parasite, likely by induction of proinflammatory profiles and clonal expansion of high-affinity IgG1- and IgG3-secreting plasma cells, suggesting the value of E.ALP as a candidate to develop novel therapeutic and immunization strategies.

Ginsenoside Rg1 enhanced immune responses to infectious bursal disease vaccine in chickens with oxidative stress induced by cyclophosphamide.

This study was designed to evaluate the effect of oral administration of ginsenoside Rg1 on oxidative stress induced by cyclophosphamide in chickens. Ninety-six chickens were randomly divided into 4 groups, each consisting of 24 birds. Groups 2 and 3 received intramuscular injection of cyclophosphamide at 100 mg/kg body weight for 3 d to induce oxidative stress and immune suppression. Groups 1 and 4 were injected with saline in the same way as groups 2 and 3. Then chickens in group 3 were orally administrated Rg1 of 1 mg/kg body weight in drinking water for 7 d. After that, groups 1 to 3 were orally vaccinated with attenuated infectious bursal disease vaccine (Strain B87). Blood samples were collected for determination of infectious bursal disease virus-specific antibodies, cytokines, and oxidative parameters. Splenocytes were prepared for lymphocyte proliferation assay. The results showed that oral administration of ginsenoside Rg1 significantly enhanced specific antibody, IFN-γ, and IL-6 responses, and lymphocyte proliferation induced by concanavalin A and lipopolysaccharide in chickens injected with cyclophosphamide. Antioxidant activity of ginsenoside Rg1 was also observed in chickens by increased total antioxidant capacity, total superoxide dismutase, catalase, glutathione peroxidase, glutathione, ascorbic acid, and α-tocopherol, as well as decreased malondialdehyde and protein carbonyl. Therefore, oral administration of Rg1 was shown to improve the immune responses to infectious bursal disease vaccine in chickens suffering from oxidative stress.

CD8+ T cells promote cytokine responses to stress

Psychological stress is known to have profound effects on immune function and to promote inflammatory conditions. Elevated circulating levels of cytokines associated with stress are known to increase the risk to several diseases, but little is known about this mechanism. This study assessed the role of T cells on cytokine levels after exposure to stress in the learned helplessness paradigm. Adoptive transfer of CD4+ T cells into Rag2−/− mice did not change cytokine levels to stress while CD8+ T cells resulted in an increase in TNF-α, IL-6 and IFN-γ in stressed Rag2−/− mice. Moreover, depletion of CD8+ T cells in WT mice abolished these cytokine responses to stress. Corticosterone and behavioral stress responsiveness was impaired in Rag2−/− mice reconstituted with CD8+ T cells. Notably, depletion of these cells in WT mice had no effect on behavior or corticosterone levels. Exposure to stress did not change the expression of canonical markers of T cell activation including CD62L and CD44 or modified intracellular cytokine content, suggesting that they are not the main producers of circulating cytokines in response to stress. These results show that CD8+ T cells promote TNF-α, IL-6 and IFN-γ responses to stress, possibly by stimulating non-lymphoid cells.

Murine macrophage TLR2-FcγR synergy via FcγR licensing of IL-6 cytokine mRNA ribosome binding and translation.

Macrophages (MØs) are sentinels of the immune system that use pattern recognition receptors such as Toll-like receptors (TLR) to detect invading pathogens and immune receptors such as FcγR to sense the host’s immune state. Crosstalk between these two signaling pathways allows the MØ to tailor the cell’s overall response to prevailing conditions. However, the molecular mechanisms underlying TLR-FcγR crosstalk are only partially understood. Therefore, we employed an immunologically-relevant MØ stimulus, an inactivated gram-negative bacterium that bears TLR2 agonists but no TLR4 agonist (iBTLR2) opsonized with a monoclonal antibody (mAb-iBTLR2), as a tool to study FcγR regulation of TLR2-driven production of IL-6, a key inflammatory cytokine. We chose this particular agonist as an investigational tool because MØ production of any detectable IL-6 in response to mAb-iBTLR2 requires both TLR2 and FcγR signaling, making it an excellent system for the study of receptor synergy. Using genetic, pharmacological and immunological approaches, we demonstrate that the murine MØ IL-6 response to mAb-iBTLR2 requires activation of both the TLR/NF-κB and FcγR/ITAM signaling pathways. mAb-iBTLR2 engagement of TLR2 drives NF-κB activation and up-regulation of IL-6 mRNA but fails to result in IL-6 cytokine production/release. Here, Src family kinase-driven FcγR ITAM signaling is necessary to enable IL-6 mRNA incorporation into polysomes and translation. These results reveal a novel mechanism by which FcγR ITAM signaling synergizes with TLR signaling, by “licensing” cytokine mRNA ribosome binding/translation to drive a strong murine MØ cytokine response.

Perceived discrimination is associated with the inflammatory response to acute laboratory stress in women at risk for cardiovascular disease

Cardiovascular disease (CVD) is the leading cause of death in the United States and exacts a disproportionate toll on minorities. Growing evidence demonstrates that perceived discrimination is a significant contributing factor to psychological distress, chronic low-grade inflammation, and cardiovascular health. However, little is known regarding the extent to which perceived discrimination contributes to the inflammatory response to acute stress. Therefore, the purpose of this study was to examine the influence of perceived discrimination on the inflammatory response to a laboratory acute stress paradigm in women at risk for CVD. A cross-sectional sample of 99 postmenopausal women (50 African American and 49 non-Hispanic White) (mean age 60.2 years) with at least two risk factors for CVD underwent the Trier Social Stress Test (TSST). Subjects completed the Detroit Area Study Discrimination Scale (DAS-DS) Everyday Discrimination subscale and provided blood and saliva samples prior to the TSST and every 15 min up to 90 min post-TSST to measure a pro-inflammatory cytokine, interleukin-6 (IL-6). Perceived discrimination was significantly associated with the salivary IL-6 response to the TSST (b = 0.49, SE = 0.13, p = <0.001) controlling for age, race, marital status, household income, BMI, statin use, childhood maltreatment, depressive symptoms, and subjective social status. Women who reported higher levels of perceived discrimination had higher levels of salivary IL-6 at baseline and following the TSST as compared to women who reported lower levels of perceived discrimination. Results suggest that higher levels of perceived discrimination, regardless of race and socioeconomic status, may heighten levels of inflammation, prior to and following an acute stress exposure. The circulating Il-6 response was associated with BMI only and did not correlate with salivary IL-6. These data suggest that perceived discrimination may contribute to the salivary-IL-6 acute stress response. However, more research is needed to help clarify the complex relationships among stress and salivary proinflammatory cytokines.

Distinct Phenotypes of Islet Antigen-Specific CD4+ T Cells among the Three Subtypes of Type 1 Diabetes

Type 1 diabetes (T1D) is classified into three subtypes in Japan based on the manner of disease progression: acute onset (AT1D), slowly progressive (SP1D), and fulminant (FT1D). While cellular immunity is considered to play a major role in pancreatic β cell destruction in T1D, the phenotypes of autoreactive T cells in each subtype remain largely unknown. In this study, we performed an integrated assay that permits the determination of ex-vivo cellular immune responses and T cell repertoires specific for islet antigens. Briefly, peripheral blood mononuclear cells obtained from 20 AT1D, 17 SP1D, 18 FT1D patients, and 17 nondiabetics (ND) were stimulated for 48 h with GAD65, preproinsulin (PPI), IGRP, and ZnT8 peptide clusters, after which cytokine/chemokine secretion was measured (ex-vivo assay). Subsequently, peptide-reactive T cells were further expanded with IL-2 to identify CD4+ T cell subsets based on intracellular cytokine staining (ICS assay). In the ex-vivo assay, GAD65-specific IL-6 and IP-10 responses, and PPI-specific IP-10 responses were significantly upregulated in AT1D as compared to ND. Furthermore, GAD65-specific IL-1β and G-CSF, and PPI-specific G-CSF responses were significantly upregulated in FT1D when compared to ND. The ICS assay further revealed that GAD65- and PPI-specific CD4+ T cells were skewed towards Th1 cells in AT1D (P = 0.007 and P = 0.004, respectively), whereas GAD65- and IGRP-specific Th2 cells were more prevalent in SP1D (P = 0.01 and P = 0.03, respectively) than in ND. Interestingly, GAD65-specific Th1 cells were more abundant in SP1D with HLA-DR9 than in those without DR9 (P = 0.03). Of note, FT1D displayed significantly less Tr1 cells specific for all four antigens and GAD65-specific Th2 cells than ND. In conclusion, the phenotypes of islet antigen-specific CD4+ T cells differ among the three T1D subtypes. These distinct phenotypes of pathogenic T cells may be associated with the manner of progression of β cell destruction. Disclosure D. Chujo: Research Support; Self; Mitsubishi Tanabe Pharma Corporation, Sanofi K.K., Kyowa Hakko Kirin Co., Ltd.. A. Kawabe: None. N. Takahashi: None. M. Matsushita: None. C. Tsutsumi: Research Support; Self; MSD K.K.. F. Haseda: None. A. Imagawa: Research Support; Self; Kowa Pharmaceutical, Sanwa Kagaku Kenkyusho Co., Ltd., Ono Pharmaceutical Co., Ltd., Sanofi K.K., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Novo Nordisk A/S, Eli Lilly and Company, MSD K.K., Mitsubishi Tanabe Pharma Corporation, Shionogi &amp; Co., Ltd., Nippon Boehringer Ingelheim Co. Ltd.. Speaker's Bureau; Self; Sanofi K.K., Novo Nordisk A/S, Mitsubishi Tanabe Pharma Corporation, Daiichi Sankyo Company, Limited, Nipro, Eli Lilly and Company, AstraZeneca, Nippon Boehringer Ingelheim Co. Ltd., Taisho Pharmaceutical Co., Ltd., Ono Pharmaceutical Co., Ltd., Sanwa Kagaku Kenkyusho Co., Ltd., Terumo Medical Corporation. T. Hanafusa: Speaker's Bureau; Self; Novo Nordisk Inc.. K. Yagi: None. M. Shimoda: None. K. Ueki: Speaker's Bureau; Self; Daiichi Sankyo Company, Limited, Eli Lilly and Company, Boehringer Ingelheim Pharmaceuticals, Inc., MSD K.K., Novo Nordisk Inc., Mitsubishi Tanabe Pharma Corporation, Kyowa Hakko Kirin Co., Ltd., Takeda. Research Support; Self; Takeda, Astellas, Novo Nordisk Inc.. H. Kajio: None.

THE EFFECTS OF THE RESISTANCE TRAINING ON SERUM CORTISOL, IL-6, IL-8, AND TNF- α

Objective: The purpose of this study is to investigate effects of resistance training on IL-6, IL-8, TNF-α, blood hematocrit and cortisol levels. &#x0D; Materials and Methods: Thirteen players from Manisa Celal Bayar University soccer team and 14 sedentary male students were used as the study subjects. The subjects, whose average age was 18-24 years old, were healthy and free of any cardiovascular diseases. Anthropometrics measurements and blood samples were obtained from all the subjects. Blood samples were obtained basal sample (before the exercise), immediately after the training program (post-exercise), and 2 hours after the training program (2h post-exercise). All subjects participated in the training program in which intensity prescribed individually in 10 different exercises; seated leg press, knee extension, knee flexion, chest press, chest flys, lat pull down, shoulder press, triceps extension, biceps curl and sit-ups. Three sets of intensity of the each exercise were arranged according to pyramidal system at 70-80 % of the each participant. The volume of resistance training was 50-60 minutes. &#x0D; Results: Post exercise IL-6 (p= 0,05) and IL-8 (p= 0,04) concentration of athletes were statistically lower compared to that of sedentary group. Furthermore, serum cortisol concentrations were found to be decreased in both study groups in post exercise and 2h post exercise samples compared to basal values (p&lt;0,05). Post exercise IL-8 (p=0,04) and TNF-α (p=0,04) values of sedentary group increased significantly compared to values at 2h post exercise.&#x0D; Conclusion: IL-6, IL-8, and TNF-α responses to resistance training vary depending on the recruitment of different muscle fiber types by the trained individuals during the resistance training and the recovery of glycogen storage, which is found to be different from that of sedentary individuals.

Analysis of IL-6 and IL-1β release in cryopreserved pooled human whole blood stimulated with endotoxin.

To overcome the lack of availability of fresh human whole blood for pyrogen detection, we explored the feasibility of utilizing cryopreserved pooled human blood to detect the responses of the pro-inflammatory cytokines IL-6 and IL-1β to LPS. Whole blood was obtained from five donors and incubated with LPS. The quantities of pro-inflammatory cytokines were measured using ELISA, and the results were compared among the samples. After the blood was cryopreserved with Dimethyl sulfoxide (DMSO) (10% v/v) and stored for 4 mo at –196℃, the detection limits of the IL-6/IL-1β responses to LPS were 0.2/0.4 endotoxin units (EU)/ml, respectively, and IL-6/IL-1β release increased in response to LPS in a dose-dependent manner. When these experiments were performed in three separate laboratories, the within-laboratory reproducibility of the IL-6/IL-1β responses was 100%/86.7%, 93.3%/100%, and 86.7%/80%, and the inter-laboratory reproducibility was 92.9%/85.7%, 64.3%/63.6%, and 57.1%/66.7%, respectively. The sensitivity (the probability of correctly classifying positive samples) and specificity (the probability of correctly classifying negative samples) of the IL-6/IL-1β tests were 81.7%/82.5% and 100%/100%, respectively. The results of this study suggest that cryopreserved pooled blood is a convenient and viable alternative for evaluating in vitro pyrogenicity. Additionally, maintaining cryopreserved pooled blood promotes safety for the user because it is released only after pretesting for infection parameters and has lower variation than fresh donations from a variety of donors.

Interplay of strain and race/ethnicity in the innate immune response to M. tuberculosis.

BackgroundThe roles of host and pathogen factors in determining innate immune responses to M. tuberculosis are not fully understood. In this study, we examined host macrophage immune responses of 3 race/ethnic groups to 3 genetically and geographically diverse M. tuberculosis lineages.MethodsMonocyte-derived macrophages from healthy Filipinos, Chinese and non-Hispanic White study participants (approximately 45 individuals/group) were challenged with M. tuberculosis whole cell lysates of clinical strains Beijing HN878 (lineage 2), Manila T31 (lineage 1), CDC1551 (lineage 4), the reference strain H37Rv (lineage 4), as well as with Toll-like receptor 2 agonist lipoteichoic acid (TLR2/LTA) and TLR4 agonist lipopolysaccharide (TLR4/LPS). Following overnight incubation, multiplex assays for nine cytokines: IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12p70, IFNγ, TNFα, and GM-CSF, were batch applied to supernatants.ResultsFilipino macrophages produced less IL-1, IL-6, and more IL-8, compared to macrophages from Chinese and Whites. Race/ethnicity had only subtle effects or no impact on the levels of IL-10, IL-12p70, TNFα and GM-CSF. In response to the Toll-like receptor 2 agonist lipoteichoic acid (TLR2/LTA), Filipino macrophages again had lower IL-1 and IL-6 responses and a higher IL-8 response, compared to Chinese and Whites. The TLR2/LTA-stimulated Filipino macrophages also produced lower amounts of IL-10, TNFα and GM-CSF. Race/ethnicity had no impact on IL-12p70 levels released in response to TLR2/LTA. The responses to TLR4 agonist lipopolysaccharide (TLR4/LPS) were similar to the TLR2/LTA responses, for IL-1, IL-6, IL-8, and IL-10. However, TLR4/LPS triggered the release of less IL-12p70 from Filipino macrophages, and less TNFα from White macrophages.ConclusionsBoth host race/ethnicity and pathogen strain influence the innate immune response. Such variation may have implications for the development of new tools across TB therapeutics, immunodiagnostics and vaccines.