TNF Concentrations Research Articles

606 NFKB1 Deficiency Alters Susceptibility to Helicobacter spp. Induced IL-1β Secretion in Bone Marrow Derived Dendritic Cells

Introduction Deletion of specific NF-κB subunits in mice alters the outcome of Helicobacter felis infection. Nfkb1 -/- mice developed more severe gastric atrophy than wild-type (WT) mice 6 weeks after H. felis infection, whereas Nfkb2 -/- mice were protected from this pathology. The mechanisms underlying these outcomes remain unclear, but gastric Il1b transcript abundance was increased in H. felis infected Nfkb1 -/- mice relative to WT, and polymorphisms at the IL1B locus have also been associated with gastric cancer in humans. Il1b transcription is regulated by classical pathway NF-κB signalling. IL-1β secretion also requires the formation of inflammasome complexes, which form following intracellular pathogen recognition, and lead to the autocatalysis of pro-caspase 1 and subsequent cleavage of IL-1β. We hypothesised that inflammasome signalling was altered in mice with abrogated NF-κB signalling and that this influenced H. felis induced pathology. We therefore investigated inflammasome mediated signalling in bone marrow derived dendritic cells (BMDCs) from mice lacking specific NF-κB subunits. Method Cells were harvested from C57BL/6, Nfkb1 -/- , Nfkb2 -/- and c-Rel -/- bone marrow. Dendritic cells were differentiation using 20 ng/ml GM-CSF for 7 days. BMDCs and WT derived gastric epithelial organoids were primed with 20 ng/ml LPS and exposed to 300 μg/ml silica, 5 mM ATP, H. pylori (ATCC 53726) or H. felis (ATCC 49179) (MOI 1:100) with or without a pan-caspase inhibitor (Z-VAD-fmk), an inhibitor of NADPH oxidase (APDC) or 50 mM KCl. Secreted IL-1β and TNF concentrations were measured by ELISA. Results LPS alone induced TNF, but not Il-1β, secretion in all genotypes of BMDCs. Both silica and ATP induced IL-1β secretion in WT BMDCs pre-stimulated with LPS (750 ± 65 and 530 ± 77 pg/ml). BMDCs derived from NF-κB deficient mice secreted similar amounts of Il-1β in response to these stimuli. Inflammasome inhibitors returned IL-1β secretion to unstimulated levels. Gastric epithelial organoid cultures treated with LPS and silica did not secrete either IL-1β or TNF. Following exposure to H. felis or H. pylori , Nfkb1 -/- BMDCs, but not other genotypes, exhibited a 2.6 fold increase in IL-1β secretion compared to untreated cells or cells stimulated with LPS. This was abrogated by Z-VAD-fmk. Conclusion These data identify potent inflammasome activation in NF-κB deficient BMDCs. H. pylori and H. felis also weakly stimulated inflammasome activation in NFkb1-/- BMDCs. This mechanism may contribute to the more severe gastric phenotype that is observed in NFkb1 -/- mice in vivo following H. felis infection. Further studies are required to identify how NF-κB1 deletion influences inflammasome formation, and whether altered IL-1β transcription is involved. Disclosure of interest None Declared.

Cytokine profiles during delivery affect cord blood hematopoietic stem and progenitors cells

The study was aimed to determine the correlations between serum levels of cytokines (GM-CSF, IL-4, IL-10 and TNF) in maternal (MB) and cord blood (CB) and some features of cord blood hematopoietic stem and progenitor cells (CB HSPCs).Study material was MB and concomitant CB samples collected from 98 volunteers at the moment of delivery. The IL-4, IL-10, TNF and GM-CSF concentrations in serum and in supernatants from PMA-stimulated mononuclear cells isolated from both blood types were measured using BD Cytometric Bead Array Flex Set System. CB HSPCs (CD34+CD45low) proportion was also estimated by flow cytometry.The most relevant results concerned the tendency to down regulation of CB HSPCs number with an increase of IL-4, IL-10 and GM-CSF levels, only the TNF concentration seems to have no influence on HSPCs pole size. The strongest positive correlations were found between CD34+CD45low HSPCs number and IL-10 and GM-CSF in MB serum and GM-CSF and TNF from CB supernatants. The strongest negative correlations were found between CD34+CD45low HSPCs number and IL-4 and GM-CSF in CB serum and IL-10 in MB supernatants. Interestingly, we observed ‘opposite correlation’ between serum and supernatant from CB and MB. We concluded that elevated serum levels of IL-4, IL-10 and GM-CSF in CB are indicative of enhanced differentiation of HSPCs and characterize a normal perinatal development. Elevated levels of cytokines seem to stimulate differentiation of HSPCs what is advantageous for neonates during perinatal period.

Immune environment in the knee synovial fluid of patients with spondyloarthritis involves simultaneous high percentages of Treg and Th17 cells and high concentrations of pro-inflammatory cytokines.

Immune environment in the knee synovial fluid of patients with spondyloarthritis involves simultaneous high percentages of Treg and Th17 cells and high concentrations of pro-inflammatory cytokines.

Impact of a Case Series of Corneal Transplant Rejection on the Kinetics of Cytokine Concentrations in Human Tears after Keratoplasty

period, starting after Day 7. The concentrations of IL-2, IL-4, IL-10, TNF, IFN-γ, and IL-17A spiked 24 h at least before rejection (P 10-fold higher in the nonrejection group than in the control and rejection groups. Kinetics analysis showed that IL-6 response was limited to a transient increase during the first 24 h, whereas the other cytokines accumulated throughout the follow-up

Inflammatory Cytokine Profile Associated with Metabolic Syndrome in Adult Patients with Type 1 Diabetes.

Objective. To compare the serum concentration of IL-6, IL-10, TNF, IL-8, resistin, and adiponectin in type 1 diabetic patients with and without metabolic syndrome and to determine the cut-off point of the estimated glucose disposal rate that accurately differentiated these groups. Design. We conducted a cross-sectional evaluation of all patients in our type 1 diabetes clinic from January 2012 to January 2013. Patients were considered to have metabolic syndrome when they fulfilled the joint statement criteria and were evaluated for clinical, biochemical, and immunological features. Methods. We determined serum IL-6, IL-8, IL-10, and TNF with flow cytometry and adiponectin and resistin concentrations with enzyme linked immunosorbent assay in patients with and without metabolic syndrome. We also compared estimated glucose disposal rate between groups. Results. We tested 140 patients. Forty-four percent fulfilled the metabolic syndrome criteria (n = 61), 54% had central obesity, 30% had hypertriglyceridemia, 29% had hypoalphalipoproteinemia, and 19% had hypertension. We observed that resistin concentrations were higher in patients with MS. Conclusion. We found a high prevalence of MS in Mexican patients with T1D. The increased level of resistin may be related to the increased fat mass and could be involved in the development of insulin resistance.

PO-0570 Zymosan But Not Lps, Pam3csk4, Or Flagellin Induces Immune Responses In Monocytes, Dcs, And Monocyte-derived Dcs Of Neonates Comparable To Those Of Adults

<h3></h3> Increased susceptibility to infection and a tendency toward more severe outcomes than in healthy adults both illustrate the prematurity of neonatal innate immunity mediated via TLRs. However, the details of TLR-mediated neonatal innate immunity are not fully understood. Here, we investigated the differences in TLR-mediated immune responses between the human neonate and adult, focusing on the cytokine profiles of monocytes, dendritic cells (DCs), and monocyte-derived DCs (MoDCs) in cord and adult blood. Purified monocytes, DCs, and MoDCs were stimulated with LPS (TLR4 ligand), Pam3CSK4 (TLR1/2 ligand), flagellin (TLR5 ligand) or zymosan (TLR2 ligand). IL-8, IL-6, and TNF concentrations were analysed in culture supernatants. Compared with the effects in adult blood, LPS-, Pam3CSK4-, and flagellin-stimulated cytokine production in cord blood was weak in monocytes, comparable in DCs, and elevated in MoDCs. In contrast, zymosan stimulation gave comparable cytokine profiles in the monocytes, DCs, and MoDCs of cord and adult blood. The immaturity of TLR-mediated innate immunity in neonates thus depends on monocytes rather than DCs. Zymosan, a cell wall extract from <i>Saccharomyces cervisiae</i>, is known to show vaccine adjuvant activity in adult animal, but the adjuvant activity was unclear in neonatal animal. Our results indicate that zymosan-mediated effective TLR2 signalling in neonates may be useful for developing a neonatal vaccine adjuvant.

135. Comparison of multiplex immunoassays and ELISAs for the determination of circulating levels of inflammatory cytokines

To determine whether multiplex immunoassays yield results that are comparable to those obtained with traditional enzyme-linked immunosorbent assays (ELISAs), cytokine concentrations were determined in 53 EDTA plasma samples from 7 college-age healthy individuals (3 female) undergoing experimental placebo ( n = 4) or endotoxin ( n = 3) exposure; 6–8 blood samples per subject were collected over a 7-h observation period. Plasma was assayed according to the manufacturer’s protocols for IL1-β, IL6, and TNF-α by high sensitivity ELISAs (Quantikine, R& D Systems), and as part of an 11-plex Performance High Sensitivity Human Cytokine multiplex bead-based (Luminex) assay (R& D Systems). When all samples in the placebo and endotoxin conditions were examined together, strong correlations were seen between ELISA and multiplex concentrations of IL1-β ( r = 0.91), IL6 ( r = 0.99), and TNF-α ( r = 0.94; all p values r = 0.89) and IL6 ( r = 0.98; both p values r = −0.28). This difference for TNF was not a function of assay sensitivity, as the calculated TNF concentrations for all placebo samples were within the range of the standard curve on both ELISA and multiplex assays. For IL-1 and IL-6, ELISAs and multiplex immunoassays yield similar results for samples in the physiological circulating range.

IFN-gamma and TNF associated with severe falciparum malaria infection in Saudi pregnant women.

BackgroundTumour necrosis factor (TNF) and interferon gamma (IFN-γ), encoded by TNF-836 C/A (rs 1800630) and IFN-γ -1616 C/T (rs2069705) genes, are key immunological mediators that are believed to both play protective and pathological roles in malaria. The aim of this study was to investigate the relationship between TNF-836 C/A and IFN-γ-1616 C/T polymorphism and susceptibility to severe malaria in pregnant women.MethodsA prospective cohort (cross-sectional) study was conducted in pregnant women attending the out-patient clinic in King Fahad Specialist Hospital in Jazan (KFSHJ), with a clinical diagnosis of malaria. A total of one hundred and eighty six pregnant women were genotyped for single nucleotide polymorphism (SNP) for TNF and IFN-γ using Taqman® MGB Probes. Serum cytokine concentrations were measured by sandwich ELISA method.ResultsA hospital case–control study of severe malaria in a Saudi population identified strong associations with individual single-nucleotide polymorphisms in the TNF and IFN-γ genes, and defined TNF-836 C and IFN-γ-1616 T genotypes and alleles which were statistically significantly associated with severe malaria infection. Furthermore, TNF-836 CC and IFN-γ-1616 TT genotypes were associated with higher serum concentration of TNF and IFN-γ, respectively, and with susceptibility to severe malaria.ConclusionsThis data provides a starting point for functional and genetic analysis of the TNF and IFN-γ genomic region in malaria infection affecting Saudi populations.

Role of main neuroendocrine pathways activated by swim stress on mast cell-dependent peritoneal TNF production after LPS administration in mice

To characterize the effects of swim stress on the early mast cell (MC)-dependent peritoneal production of TNF in response to lipopolysaccharide (LPS) administration in mice, identifying the neuroendocrine mediators involved. Ten to twelve-week-old Swiss Webster, C57BL/6J or c-Kit (Wsh/Wsh) mice were used. Animals were intraperitoneally challenged with LPS at different times after forced swimming (FS) and peak TNF production was determined in peritoneal washes at optimal time after LPS administration. Selective blockage of main neuroendocrine pathways was performed before swim stress. TNF concentrations were determined by ELISA. FS provoked an immediate and transient inhibition of LPS-elicited, MC-dependent TNF accumulation in peritoneum, which lasted around 30min. Suppresive effects of FS were absent on MC-deficient c-Kit (Wsh/Wsh) mice but were recovered after reconstitution with MC. Adrenalectomy or DSP4 administration increased basal ip TNF levels and enhanced LPS-induced TNF release without any effect on stress-induced inhibitory effects, mifepristone did not produce any change on stress-induced inhibition, whereas mecamylamine administration increased basals and attenuated stress effects. Swim stress transiently inhibits the canonical MC-dependent response of TNF production in response to LPS in murine peritoneal cavity with the main participation of the cholinergic anti-inflammatory reflex.

Raised concentrations of lipid peroxidation products (LPO) in pregnant women with impaired glucose tolerance.

Lipid peroxidation (LPO) results from oxidative damage to membrane lipids. Whereas LPO rises in normal pregnancy, the effect of gestational diabetes mellitus (GDM) on this process has not been clearly defined. Fasting blood concentrations of malondialdehyde+4-hydroxyalkenals (MDA+4-HDA), as LPO index, TNFa soluble receptors (sTNF-R1 and sTNF-R2), and soluble adhesion molecules (sICAM-1, sVCAM-1), were measured in 51 women at 28 weeks of gestation. The women were divided according to the results of 50.0 g glucose challenge test (GCT) and 75.0 g oral glucose tolerance test (OGTT): Controls (n=20), normal responses to both GCT and OGTT; Intermediate Group (IG) (n=15), abnormal GCT but normal OGTT; GDM group (n=16), abnormal both GCT and OGTT. Glucose concentrations in women diagnosed with GDM were within the range of impaired glucose tolerance. There were no significant differences in concentrations of either TNF a soluble receptors R1 and R2, or sICAM-1 or sVCAM-1. LPO concentrations [MDA+4-HDA (nmol/mg protein)] were significantly higher in women with GDM than in the other two groups [64.1±24.3 (mean±SD), 39.3±23.1, 47.0±18.1, for GDM, IG and Controls, respectively; p<0.05]. In multivariate analysis, the only significant independent correlation was between LPO level and glucose at 120 minutes of OGTT (rs=0.42; p=0.009). Oxidative damage to membrane lipids is increased in GDM and might result directly from hyperglycaemia. Physiological significance of this phenomenon remains to be elucidated.

AB0101 Articular Adipose Tissue from Osteoarthritis Patients is A Source of Cytokines and Adipokines

BackgroundRecently rheumatoid adipose tissue has been suggested to play an important role in chronic inflammatory joint diseases. Its ability to synthesize cytokines and adipokines has been characterized also in our...

The role of TNF-? in the pathogenesis of multiple myeloma “a study in Iraqi patients”

During recent years, there has been an increasing interest in the investigation of the cytokines roles in pathogenesis of cancer, thus the study aimed at evaluating the level of tumor necrosis factor-alpha(TNF-?) in sera of Iraqi multiple myeloma (MM) patients. Beta 2-microglobulion (?2-m) was assessed to determine if there was any association between this cytokine and the level of ?2- m, as the latter is related to the stage of the disease. In addition, the age and gender were also taken into consideration. Furthermore, we investigated the relationship between IgG and TNF-? in sera of patients. 49 Iraqi patients (27 males and 22 females).The patients were also divided into two groups: the first group included (17) patients who were recently diagnosed and not received any treatment at the time of collecting samples while the second group included (32) patients who received treatment. A further group was also investigated which included (12) apparently healthy individuals (9 males and 3 females), who were regarded as a control group. Serum TNF-? and ?2- m were determined using enzyme-linked immunosorbent assay (ELISA).while the concentration of IgG was measured by radial immune diffusion plates The study reached to the following results: TNF-? levels were not significantly elevated in the patients with MM compared to control group (5.98 ± 8.47 SD vs. 4.85 ± 12.1 SD) and no significant differences (P &gt; 0.05) were observed in the mean (6.02 ±8.1SD vs 5.9 ±9.4SD) concentration of TNF-? in patients with MM who received treatment, when compared with those who did not take the treatment .In addition, there are positive significant correlations between TNF-? and ? 2 Microglobulin (r = 0.316, P = 0 .027), and no relationship between IgG and TNF-? (r = - 0.032, P = 0.829).Furthermore, the study observed that, there was no correlation between TNF-? on the one hand and factors of age and gender on the other hand.

The role of TNF-? in the pathogenesis of multiple myeloma “a study in Iraqi patients”

During recent years, there has been an increasing interest in the investigation of the cytokines roles in pathogenesis of cancer, thus the study aimed at evaluating the level of tumor necrosis factor-alpha(TNF-?) in sera of Iraqi multiple myeloma (MM) patients. Beta 2-microglobulion (?2-m) was assessed to determine if there was any association between this cytokine and the level of ?2- m, as the latter is related to the stage of the disease. In addition, the age and gender were also taken into consideration. Furthermore, we investigated the relationship between IgG and TNF-? in sera of patients. 49 Iraqi patients (27 males and 22 females).The patients were also divided into two groups: the first group included (17) patients who were recently diagnosed and not received any treatment at the time of collecting samples while the second group included (32) patients who received treatment. A further group was also investigated which included (12) apparently healthy individuals (9 males and 3 females), who were regarded as a control group. Serum TNF-? and ?2- m were determined using enzyme-linked immunosorbent assay (ELISA).while the concentration of IgG was measured by radial immune diffusion plates The study reached to the following results: TNF-? levels were not significantly elevated in the patients with MM compared to control group (5.98 ± 8.47 SD vs. 4.85 ± 12.1 SD) and no significant differences (P &gt; 0.05) were observed in the mean (6.02 ±8.1SD vs 5.9 ±9.4SD) concentration of TNF-? in patients with MM who received treatment, when compared with those who did not take the treatment .In addition, there are positive significant correlations between TNF-? and ? 2 Microglobulin (r = 0.316, P = 0 .027), and no relationship between IgG and TNF-? (r = - 0.032, P = 0.829).Furthermore, the study observed that, there was no correlation between TNF-? on the one hand and factors of age and gender on the other hand.

P-0205 - Cytokines, Depression and Anxiety in Colorectal Cancer Patients in Different Stages of the Antitumor Therapy

Introduction: Colorectal cancer (CRC) is a leading cause of cancer mortality in the world. Depression and anxiety are the most prevalent psychological disorders in patients with CRC. This study aimed to investigate whether serum cytokine levels correlate with anxiety and depression in colorectal cancer patients in different stages of the antitumor therapy. Methods: A total of 100 subjects were divided in 5 groups. Group 1: Healthy volunteers free of any psychiatric or immune system disease (n = 20); Group 2: Patients with colorectal cancer who did not undergo surgical resection of tumor (n = 20); Group 3: Patients who underwent surgical resection and who did not start adjuvant therapy (n = 20); Group 4: Patients undergoing chemotherapy (for about 3 months) (n = 20); Group 5: Patients who have completed adjuvant chemotherapy regimen (about 6 months after the end of treatment) (n = 20). Depression and anxiety were analyzed using the Hospital Anxiety and Depression Scale (HADS) and serum levels of IL-1, IL-6, IL-8, IL-10, IL-12, TNF-&agr; and TGF-&bgr; were measured by CBA. Results: Clinically relevant levels of anxiety and/or depression were found in all CRC patients in different stages of the antitumor therapy. Serum levels of the proinflammatory cytokines IL- 1, IL- 6, IL- 8 and TNF- &agr; were increased in CRC patients in pre and postoperative stages. However, these levels were reduced during and after chemotherapy. Furthermore, we found decreased levels of IL-10 in serum of patients in CRC patients in pre and postoperative stage. By analyzing the correlation between HADS scores and serum cytokine levels, we observed a positive association of anxiety and/or depression with concentrations of IL-1, IL-6, IL-8 and TNF-&agr; and negative compared to levels serum IL-10 in different stages of the antitumor therapy. Conclusion: These results indicate a link between serum levels of pro-inflammatory cytokines, anxiety and depression in CRC patients, suggesting that cytokines are involved in the pathophysiology of these comorbidities.

Low-level laser therapy attenuates the myeloperoxidase activity and inflammatory mediator generation in lung inflammation induced by gut ischemia and reperfusion: a dose-response study.

Intestinal ischemia and reperfusion (i-I/R) is an insult associated with acute respiratory distress syndrome (ARDS). Herein we evaluate the dose-response effect of low-level laser therapy (LLLT) on lung inflammation induced by i-I/R. Mice were subjected to mesenteric artery occlusion (45 min) and killed after clamp release and intestinal reperfusion (2h). Increasing doses (1, 3, 5 and 7,5 J/cm(2)) of laser irradiation (660 nm) was carried out on the mice skin over the upper bronchus for 5 min after initiating reperfusion. Neutrophils activation was determined by myeloperoxidase (MPO) activity. The mRNA expression and protein concentration of inflammatory mediators IL-1β, IL-6, TNF and IL-10 in lung were measured by RT-PCR and ELISA, respectively. With exception of 1J/cm(2), LLLT reduced MPO activity as well as IL-1β levels in the lungs from inflamed mice. LLLT was also markedly effective in reducing both IL-6 and TNF expression and levels in the lungs from mice submitted to i-I/R in all laser doses studied. Otherwise, LLLT significantly increased the protein levels of IL-10 in inflamed mice by i-I/R; however only in the dose of 1J/cm(2). We conclude that the LLLT is able to control the neutrophils activation and proinflammatorycytokines release into the lungs in a model of i-I/R in mice.

Posttranscriptional regulation of tristetraprolin and tumor necrosis factor through a two‐compartment model (902.12)

TNF is a pro‐inflammatory cytokine that influences a broad range of immunological responses, including chronic inflammation of the gastrointestinal tract. Prolonged inflammation of gastrointestinal tract is linked with inflammatory bowel disease. TNF biosynthesis is regulated both at transcription and posttranscriptional levels. However, the stimulation‐induced increase in translation rate is much larger. This might indicate the possibility of a posttranscriptional regulatory mechanism. How, during basal conditions, is the free concentration of TNF tightly regulated at low levels in cytosol? The stability and translational efficiency of TNF transcript are regulated by an AU‐rich element (ARE) in the 3′‐UTR of messenger RNA. A transacting protein, TTP, binds to ARE and enhances the mRNA turnover. Here, we examine a proposal that TNF homeostasis is regulated by a two‐compartment interaction loop between TTP and TNF. We propose a computational framework of this regulatory loop by modeling the shuttling of TTP between nuclear and cytosolic compartments. This two‐compartment regulatory loop between TTP and TNF is based on activation, deactivation, and translocation/re‐translocation cycle of TTP protein. The mutual interaction of these two proteins regulates the biosynthesis response of both TTP and TNF during basal and inflammatory conditions.

T Helper Cell Cytokine Profiles After Endurance Exercise

Endurance exercise can cause immunosuppression and increase the risk of upper respiratory illness. The present study examined changes in the secretion of T helper (Th) cell cytokines after endurance exercise. Ten highly trained road cyclists [mean±SEM: age 24.2±1.7 years; height 1.82±0.02 m; body mass 73.8±2.0 kg; peak oxygen uptake 65.9±2.3 mL/(kg•min)] performed 2 h of cycling exercise at 90% of the second ventilatory threshold. Peripheral blood mononuclear cells were isolated and stimulated with phytohemagglutinin. Plasma cortisol concentrations and the concentration of Th1/Th2/Th17 cell cytokines were examined. Data were analyzed using both traditional statistics and magnitude-based inferences. Results revealed a significant decrease in plasma cortisol at 4-24 h postexercise compared with pre-exercise values. Qualitative analysis revealed postexercise changes in concentrations of plasma cortisol, IL-2, TNF, IL-4, IL-6, IL-10, and IL-17A compared with pre-exercise values. A Th1/Th2 shift was evident immediately postexercise. Furthermore, for multiple cytokines, including IL-2 and TNF (Th1), IL-6 and IL-10 (Th2), and IL-17 (Th17), no meaningful change in concentration occurred until more than 4 h postexercise, highlighting the duration of exercise-induced changes in immune function. These results demonstrate the importance of considering "clinically" significant versus statistically significant changes in immune cell function after exercise.

High-Throughput Analysis of NF-κB Dynamics in Single Cells Reveals Basal Nuclear Localization of NF-κB and Spontaneous Activation of Oscillations

NF-κB is a transcription factor that upon activation undergoes cycles of cytoplasmic-to-nuclear and nuclear-to-cytoplasmic transport, giving rise to so called “oscillations”. In turn, oscillations tune the transcriptional output. Since a detailed understanding of oscillations requires a systems biology approach, we developed a method to acquire and analyze large volumes of data on NF-κB dynamics in single cells. We measured the time evolution of the nuclear to total ratio of GFP-p65 in knock-in mouse embryonic fibroblasts using time-lapse imaging. We automatically produced a precise segmentation of nucleus and cytoplasm based on an accurate estimation of the signal and image background. Finally, we defined a set of quantifiers that describe the oscillatory dynamics, which are internally normalized and can be used to compare data recorded by different labs. Using our method, we analyzed NF-κB dynamics in over 2000 cells exposed to different concentrations of TNF- α α. We reproduced known features of the NF-κB system, such as the heterogeneity of the response in the cell population upon stimulation and we confirmed that a fraction of the responding cells does not oscillate. We also unveiled important features: the second and third oscillatory peaks were often comparable to the first one, a basal amount of nuclear NF-κB could be detected in unstimulated cells, and at any time a small fraction of unstimulated cells showed spontaneous random activation of the NF-κB system. Our work lays the ground for systematic, high-throughput, and unbiased analysis of the dynamics of transcription factors that can shuttle between the nucleus and other cell compartments.

Increased Prevalence of Methanosphaera stadtmanae in Inflammatory Bowel Diseases

BackgroundThe gut microbiota is associated with the modulation of mucosal immunity and the etiology of inflammatory bowel diseases (IBD). Previous studies focused on the impact of bacterial species on IBD but seldom suspected archaea, which can be a major constituent of intestinal microbiota, to be implicated in the diseases. Recent evidence supports that two main archaeal species found in the digestive system of humans, Methanobrevibacter smithii (MBS) and Methanosphaera stadtmanae (MSS) can have differential immunogenic properties in lungs of mice; with MSS but not MBS being a strong inducer of the inflammatory response. We thus aimed at documenting the immunogenic potential of MBS and MSS in humans and to explore their association with IBD.MethodsTo validate the immunogenicity of MBS and MSS in humans, peripheral blood mononuclear cells from healthy subjects were stimulated with these two microorganisms and the production of inflammatory cytokine TNF was measured by ELISA. To verify MBS and MSS prevalence in IBD, stool samples from 29 healthy control subjects and 29 patients suffering from IBD were collected for DNA extraction. Plasma was also collected from these subjects to measure antigen-specific IgGs by ELISA. Quantitative PCR was used for bacteria, methanogens, MBS and MSS quantification.ResultsMononuclear cells stimulated with MSS produced higher concentrations of TNF (39.5 ng/ml) compared to MBS stimulation (9.1 ng/ml). Bacterial concentrations and frequency of MBS-containing stools were similar in both groups. However, the number of stool samples positive for the inflammatory archaea MSS was higher in patients than in controls (47% vs 20%). Importantly, only IBD patients developed a significant anti-MSS IgG response.ConclusionThe prevalence of MSS is increased in IBD patients and is associated with an antigen-specific IgG response.

DOP060 CD62L (L-selectin) shedding for assessment of functional blockade of TNF alpha in anti-TNF treated inflammatory bowel disease patients: clinical feasibility and perspectives

Background Tumor necrosis factor (TNF) inhibition is central to the therapy of inflammatory bowel diseases (IBD). However, loss of response (LOR) is frequent and additional tests to help decision making with costly anti-TNF Therapy are needed. Methods Consecutive IBD Patients receiving anti-TNF therapy (Infliximab (IFX) or Adalimumab (after IFX LOR) from Bern University Hospital were identified and followed prospectively. Patient whole blood was stimulated with a dose-titration of two triggers of TLR receptors human: TNF and LPS. Median fluorescence intensity of CD62L on the surface of granulocytes was quantified by surface staining with specific antibodies (CD33, CD62L) and flow cytometry and logistic curves to these data permits the calculation of EC50 or the half maximal effective concentration TNF concentration to induce shedding [1]. A shift in the concentration were CD62L shedding occurred was seen before and after the anti-TNF agent administraion which permits to predict the response to the drug. This predicted response was correlated to the clinical evolution of the patients in order to analyze the ability of this test to identify LOR to IFX. Results We collected prospective clinical data and blood samples, before and after anti-TNF agent administration, on 33 IBD patients, 25 Crohn's disease and 8 ulcerative colitis patients (45% females) between June 2012 and November 2013. The assay showed a functional blockade of IFX (PFR) for 22 patients (17 CD and 5 UC) whereas 11 (8 CD and 3 UC) had no functional response (NR) to IFX. Clinical characteristics (e.g. diagnosis, disease location, smoking status, BMI and number of infusions) were no significantly different between predicted PFR and NR. Among the 22 Patients with PRF, only 1 patient was a clinical non responder (LOR to IFX), based on clinical prospective evaluation by IBD gastroenterologists (PJ, AM), and among the 11 predicted NR, 3 had no clinical LOR. Sensitivity of this test was 95% and specificity 73% and AUC adjusted for age and gender was 0.81 (Figure 1). During follow up (median 10 mo, 3–15) 8 “hard” outcomes occured (3 medic. flares, 4 resections and 1 new fistula) 2 in the PFR and 6 in the NR group (25% vs. 75%; p < 0.01). Correlation with clinical response is presented in Figure 2. Figure 1. Figure 2. Correlation clinical response - log EC50 changes: 1 No, 2 partial, 3 complete clinical response. Conclusion CD62L (L-Selectin) shedding is the first validated test of functional blockade of TNF alpha in anti-TNF treated IBD patients and will be a useful tool to guide medical decision on the use of anti-TNF agents. Comparative studies with ATI and trough level of IFX are ongoing. 1. Nicola Patuto, Emma Slack, Frank Seibold and Andrew J. Macpherson, (2011), Quantitating Anti-TNF Functionality to Inform Dosing and Choice of Therapy, Gastroenterology, 140 (5, Suppl. I), S689.