CD14 Protein Research Articles

A robust and sensitive flow cytometric method for multiplex RNA in-situ hybridization analysis of blood cells using oligo probes and branched DNA based signal amplification (P3356)

Abstract We present here a novel method for in-situ hybridization (ISH) detection of multiple RNA targets in single cells using standard flow cytometer. This method is based on the use of oligo pairs as probes and bDNA signal amplification for highly sensitive and specific ISH detection of RNA. The oligo pairs are designed to hybridize to specific target sequences and can be readily synthesized. The oligo probes/RNA target complex is detected after signal is generated by bDNA signal amplification. The method is compatible with immunostaining, so simultaneous staining of protein and RNA in single cells is feasible. We demonstrated the co-staining of RNA and surface marker proteins in PBMC, B cells, T cells and leukemic cell lines of U937, K562, Jurkat and M1. In addition, the flow cytometric analysis of cytokine gene induction by lipopolysaccharides/R848 in PBMC was shown by staining with antibody for CD14 protein and probes for RNAs encoding interleukin-1 beta, -6, -8 and tumor necrosis factor-alpha. Taken together, this novel RNA ISH flow cytometry assay offers an invaluable tool for studying immune response, stem cell biology and infectious diseases.

CD14 Protein Acts as an Adaptor Molecule for the Immune Recognition of Salmonella Curli Fibers

Amyloids, protein aggregates with a cross β-sheet structure, contribute to inflammation in debilitating disorders, including Alzheimer's disease. Enteric bacteria also produce amyloids, termed curli, contributing to inflammation during infection. It has been demonstrated that curli and β-amyloid are recognized by the immune system via the Toll-like receptor (TLR) 2/TLR1 complex. Here we investigated the role of CD14 in the immune recognition of bacterial amyloids. We used HeLa 57A cells, a human cervical cancer cell line containing a luciferase reporter gene under the control of an NF-κB promoter. When HeLa 57A cells were transiently transfected with combinations of human expression vectors containing genes for TLR2, TLR1, and CD14, membrane-bound CD14 enhanced NF-κB activation through the TLR2/TLR1 complex stimulated with curli fibers or recombinant CsgA, the curli major subunit. Similarly, soluble CD14 augmented the TLR2/TLR1 response to curli fibers in the absence of membrane-bound CD14. We further revealed that IL-6 and nitric oxide production were significantly higher by wild-type (C57BL/6) bone marrow-derived macrophages compared with TLR2-deficient or CD14-deficient bone marrow-derived macrophages when stimulated with curli fibers, recombinant CsgA, or synthetic CsgA peptide, CsgA-R4-5. Binding assays demonstrated that recombinant TLR2, TLR1, and CD14 bound purified curli fibers. Interestingly, CD14-curli interaction was specific to the fibrillar form of the amyloid, as demonstrated by using synthetic CsgA peptides proficient and deficient in fiber formation, respectively. Activation of the TLR2/TLR1/CD14 trimolecular complex by amyloids provides novel insights for innate immunity with implications for amyloid-associated diseases.

Suppressed Th17 Levels Correlate with Elevated PIAS3, SHP2, and SOCS3 Expression in CD4 T Cells during Acute Simian Immunodeficiency Virus Infection

T helper 17 (Th17) cells play an important role in mucosal immune homeostasis and maintaining the integrity of the mucosal epithelial barrier. Loss of Th17 cells has been extensively documented during human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections. The lack of effective repopulation of Th17 cells has been associated with chronic immune activation mediated by the translocation of microbial products. Using ex vivo analysis of purified peripheral blood CD4 T cells from SIV-infected rhesus macaques, we show that the suppression of interleukin-17 (IL-17) expression correlated with upregulated expression of negative regulatory genes PIAS3, SHP2, and SOCS3 in CD4 T cells. Suppressed Th17 expression was accompanied by elevated levels of soluble CD14 (sCD14) and lipopolysaccharide binding protein (LBP) in the plasma during early stages of infection. Plasma viral loads rather than sCD14 or LBP levels correlated with acute immune activation. Additionally, we observed a significant increase in the expression of CD14 on peripheral blood monocytes that correlated with IL-23 expression and markers of microbial translocation. Taken together, our results provide new insights into the early events associated with acute SIV pathogenesis and suggest additional mechanisms playing a role in suppression of Th17 cells.

Human Lipopolysaccharide-binding Protein (LBP) and CD14 Independently Deliver Triacylated Lipoproteins to Toll-like Receptor 1 (TLR1) and TLR2 and Enhance Formation of the Ternary Signaling Complex

Bacterial lipoproteins are the most potent microbial agonists for the Toll-like receptor 2 (TLR2) subfamily, and this pattern recognition event induces cellular activation, leading to host immune responses. Triacylated bacterial lipoproteins coordinately bind TLR1 and TLR2, resulting in a stable ternary complex that drives intracellular signaling. The sensitivity of TLR-expressing cells to lipoproteins is greatly enhanced by two lipid-binding serum proteins known as lipopolysaccharide-binding protein (LBP) and soluble CD14 (sCD14); however, the physical mechanism that underlies this increased sensitivity is not known. To address this, we measured the ability of LBP and sCD14 to drive ternary complex formation between soluble extracellular domains of TLR1 and TLR2 and a synthetic triacylated lipopeptide agonist. Importantly, addition of substoichiometric amounts of either LBP or sCD14 significantly enhanced formation of a TLR1·TLR2 lipopeptide ternary complex as measured by size exclusion chromatography. However, neither LBP nor sCD14 was physically associated with the final ternary complex. Similar results were obtained using outer surface protein A (OspA), a naturally occurring triacylated lipoprotein agonist from Borrelia burgdorferi. Activation studies revealed that either LBP or sCD14 sensitized TLR-expressing cells to nanogram levels of either the synthetic lipopeptide or OspA lipoprotein agonist. Together, our results show that either LBP or sCD14 can drive ternary complex formation and TLR activation by acting as mobile carriers of triacylated lipopeptides or lipoproteins.

Propofol exerts anti-inflammatory effects in rats with lipopolysaccharide-induced acute lung injury by inhibition of CD14 and TLR4 expression

We investigated the effect of propofol (Prop) administration (10 mg kg-1 h-1, intravenously) on lipopolysaccharide (LPS)-induced acute lung injury and its effect on cluster of differentiation (CD) 14 and Toll-like receptor (TLR) 4 expression in lung tissue of anesthetized, ventilated rats. Twenty-four male Wistar rats were randomly divided into three groups of 8 rats each: control, LPS, and LPS+Prop. Lung injury was assayed via blood gas analysis and lung histology, and tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) levels were determined in bronchoalveolar lavage fluid using ELISA. Real-time polymerase chain reaction was used to detect CD14 and TLR4 mRNA levels, and CD14 and TLR4 protein expression was determined by Western blot. The pathological scores were 1.2 ± 0.9, 3.3 ± 1.1, and 1.9 ± 1.0 for the control, LPS, and LPS+Prop groups, respectively, with statistically significant differences between control and LPS groups (P < 0.05) and between LPS and LPS+Prop groups (P < 0.05). The administration of LPS resulted in a significant increase in TNF-α and IL-1β levels, 7- and 3.5-fold, respectively (P < 0.05), while treatment with propofol partially blunted the secretion of both cytokines (P < 0.05). CD14 and TLR4 mRNA levels were increased in the LPS group (1.48 ± 0.05 and 1.26 ± 0.03, respectively) compared to the control group (1.00 ± 0.20 and 1.00 ± 0.02, respectively; P < 0.05), while propofol treatment blunted this effect (1.16 ± 0.05 and 1.12 ± 0.05, respectively; P < 0.05). Both CD14 and TLR4 protein levels were elevated in the LPS group compared to the control group (P < 0.05), while propofol treatment partially decreased the expression of CD14 and TLR4 protein versus LPS alone (P < 0.05). Our study indicates that propofol prevents lung injury, most likely by inhibition of CD14 and TLR4 expression.

Circulating high-sensitivity C-reactive protein and soluble CD40 ligand are inter-related in a cohort of women with polycystic ovary syndrome

ObjectiveInsulin resistance (IR), the fundamental metabolic alteration in women with polycystic ovary syndrome (PCOS), has recently been reported to represent, in part, a chronic low-grade inflammatory state. This study aimed to assess the inter-relationship between the plasma levels of high-sensitivity C-reactive protein (hs-CRP) and soluble CD40 ligand (sCD40L) – both inflammatory factors – and the pathogenesis of IR among women with PCOS. Study designThis study compared 44 women with PCOS with a group of control women without PCOS (n=39). Participants were matched for age, blood pressure and degree of obesity. Enzyme-linked immunosorbent assay was used to measure the two inflammatory factors. Fasting glucose and insulin were assayed and used to calculate the IR indices. ResultsSignificant elevations in the plasma levels of both inflammatory factors were observed in women with PCOS compared with the control women: 3.17 (2.23–3.89) vs 2.05 (0.93–2.82)mg/l for hs-CRP and 2.46 (1.08–4.16) vs 0.82 (0.43–1.26)ng/ml for sCD40L, respectively. Interestingly, among women with PCOS, the two inflammatory factors were significantly positively correlated with each other (r=0.465, p=0.002), as well as with the IR indices. This association remained significant even after adjustment for body mass index as a measure of obesity. ConclusionIndependent of obesity, women with PCOS suffer low-grade inflammation that is correlated with IR. The observed inter-relationships suggest that CRP in collaboration with the CD40–CD40L system may have a role in the pathogenesis of PCOS.

Markers of microbial translocation and risk of AIDS-related lymphoma

Depletion of gut-associated lymphocytes by HIV infection facilitates microbial translocation, which may contribute to non-Hodgkin lymphoma (NHL) risk via chronic immune activation and B-cell hyperstimulation. We therefore examined associations of four microbial translocation markers with subsequent NHL risk in a case-control study nested within four prospective cohort studies of HIV-infected individuals. Prediagnostic blood specimens for 56 NHL cases and 190 controls matched for age, sex, race, specimen type, cohort, and CD4 T-cell count were tested for the endotoxin lipopolysaccharide (LPS), antiendotoxin core antibody (EndoCab), LPS-binding protein (LBP), and soluble CD14 (sCD14). Elevated levels of sCD14 were associated with significantly increased NHL risk [odds ratio (OR) 2.72 (95% confidence interval [95% CI] 1.29-5.76)]. In subgroup analyses, elevated LPS levels were also associated with significantly increased NHL risk [OR 3.24 (95% CI 1.10-9.53)]. EndoCab and LBP levels were not associated with NHL risk. The association of sCD14 and LPS with NHL risk supports an etiologic role for gut microbial translocation in lymphomagenesis among HIV-infected individuals. Additional studies with larger sample sizes are needed to confirm these observations.

Effect of biliary drainage on inducible nitric oxide synthase, CD14 and TGR5 expression in obstructive jaundice rats

To investigate the effect of biliary drainage on inducible nitric oxide synthase (iNOS), CD14 and TGR5 expression in rats with obstructive jaundice (OJ). Male adult Sprague-Dawley rats were randomly assigned to four groups: OJ, sham operation (SH), internal biliary drainage (ID) and external biliary drainage (ED). Rat models were successfully established by two operations and succumbed for extraction of Kupffer cells (KCs) and liver tissue collection on the 8(th) and 15(th) day. KCs were isolated by in situ hepatic perfusion and digested with collagen IV, density gradient centrifuged by percoll reagent and purified by cell culture attachment. The isolated KCs were cultured with the endotoxin lipopolysaccharide (LPS) with and without the addition of ursodeoxycholic acid (UDCA). The expression of iNOS, CD14 and bile acid receptor-TGR5 protein in rat liver tissues was determined by immunohistochemistry. The expression of iNOS and CD14 messenger RNA (mRNA) on the isolated KCs was detected by reverse transcription polymerase chain reaction (PCR) and the TGR5 mRNA level in KCs was measured by real-time quantitative PCR. The iNOS protein was markedly expressed in the liver of OJ rats, but rare expressed in SH rats. After relief of OJ, the iNOS expression was decidedly suppressed in the ID group (ID vs OJ, P < 0.01), but obviously increased in rats of ED (ED vs OJ, P = 0.004). When interfered only with LPS, the expression of iNOS mRNA by KCs was increased in the OJ group compared with the SH group (P = 0.004). After relief of biliary obstruction, the iNOS mRNA expression showed slight changes in the ED group (ED vs OJ, P = 0.71), but dropped in the ID group (ID vs OJ, P = 0.001). Compared with the simple intervention with LPS, the expressions of iNOS mRNA were significantly inhibited in all four groups after interfered with both LPS and UDCA (P < 0.01, respectively). After bile duct ligation, the CD14 protein expression in rat liver was significantly strengthened (OJ vs SH, P < 0.01), but the CD14 mRNA level by KCs was not up-regulated (OJ vs SH, P = 0.822). After relieving the OJ, the expression of CD14 protein was reduced in the ID group (ID vs OJ, P < 0.01), but not reduced in ED group (ED vs OJ, P = 0.591). And then the CD14 mRNA expression was aggravated by ED (ED vs OJ, P < 0.01), but was not significantly different between the ID group and the SH and OJ groups (ID vs SH, P = 0.944; ID vs OJ, P = 0.513, respectively). The expression of TGR5 protein and mRNA increased significantly in OJ rats (OJ vs SH, P = 0.001, respectively). After relief of OJ, ID could reduce the expression of TGR5 protein and mRNA to the levels of SH group (ID vs SH, P = 0.22 and P = 0.354, respectively), but ED could not (ED vs SH, P = 0.001, respectively). ID could be attributed to the regulatory function of activation of KCs and release of inflammatory mediators.

Lipopolysaccharide (LPS) segmental lung challenge in nonhuman primates – a model of airway inflammation

We developed a reproducible model of neutrophilic inflammation in cynomolgus monkeys (Macaca fascicularis) in order to study potential novel anti-inflammatory compounds intended for respiratory diseases such as COPD and asthma. Using a pediatric bronchoscope, a baseline bronchoalveolar lavage (BAL) was performed on the left lung prior to LPS segmental challenge to the right lung. At 24, 72 and 168 hours post challenge, BAL fluid (BALF) was collected to assess the level of pulmonary inflammation. At both 24 and 72 hours (Hr) post LPS challenge, total BAL cells were significantly elevated over baseline. BAL cell differentials revealed that mean percent neutrophils significantly increased from baseline (1.3 ±1.6%) to (74.0 ± 21.9% at 24 Hr and 26.6 ± 21.0% at 72 Hr) whereas, BAL macrophage numbers had significantly decreased from baseline (94.4 ± 3.1%) to (31.6 ± 21.2 % at 24 Hr and 64.2 ± 20.1% at 72 Hr) post challenge. In addition to increased inflammation, we also observed a significant increase in BAL cell gene expression of MMP-9 and significant increases in sCD14 and IL-8 from concentrated BALF 24 Hr post challenge. Numbers of infiltrating cells and other inflammatory parameters returned to baseline levels by 168 Hr following LPS challenge. In some studies, intramuscular injections of dexamethasone (Dex [1 mg/kg]) were administered 24 Hr prior and just prior to LPS challenge. Dex did not significantly reduce total BAL inflammation or IL-8 protein, but did significantly reduce percent neutrophils and sCD14 protein within the BALF 24 hours post challenge. Dex also significantly increased percent macrophages 24 Hr post challenge. These data show that segmental challenge with LPS produces an acute pulmonary neutrophilic inflammation, which is reduced by pretreatment with Dex. This reproducible, short-term, large animal model may help us to better understand neutrophillic inflammation pathways contributing to lung injury and host defense.

Emerging Roles of L-Type Voltage-Gated and Other Calcium Channels in T Lymphocytes

In T lymphocytes, calcium ion controls a variety of biological processes including development, survival, proliferation, and effector functions. These distinct and specific roles are regulated by different calcium signals, which are generated by various plasma membrane calcium channels. The repertoire of calcium-conducting proteins in T lymphocytes includes store-operated CRAC channels, transient receptor potential channels, P2X channels, and L-type voltage-gated calcium (Cav1) channels. In this paper, we will focus mainly on the role of the Cav1 channels found expressed by T lymphocytes, where these channels appear to operate in a T cell receptor stimulation-dependent and voltage sensor independent manner. We will review their expression profile at various differentiation stages of CD4 and CD8 T lymphocytes. Then, we will present crucial genetic evidence in favor of a role of these Cav1 channels and related regulatory proteins in both CD4 and CD8 T cell functions such as proliferation, survival, cytokine production, and cytolysis. Finally, we will provide evidence and speculate on how these voltage-gated channels might function in the T lymphocyte, a non-excitable cell.

Establishment of mouse intestinal myofibroblast cell lines

To establish novel intestinal myofibroblast (IMF) cell lines from mouse colonic mucosa and investigate their biological characters. Primary IMFs were isolated from mucosal tissues of mouse colon that was denuded of epithelial cells and smooth muscle layer. For immortalization, primary IMFs were transfected with simian virus 40 large T antigen (designated as LmcMF). We also isolated some primary IMFs that spontaneously became immortalized without transfection (designated as SmcMF). To check immortality and normality of these cells, we examined their proliferative ability and contact inhibition. Moreover, the expression levels of proteins characterizing IMFs [including α-smooth muscle actin (α-SMA), vimentin, desmin, and type I collagen] and proteins associated with the immune response [such as toll-like receptor 4 (TLR-4), CD14, MD2, IκBα, and p-p38] were determined by Western blotting. The localization of several myofibroblast protein markers was also detected by immunofluorescence staining. The cell growth assay results show that both LmcMF and SmcMF cells proliferated logarithmically at least up to passage 20. In addition, the contact inhibition assays show that LmcMF and SmcMF stopped growing after the cells reached confluence. These data suggest that these 2 types of cells were immortalized without losing contact inhibition of growth. Moreover, both LmcMF and SmcMF, like primary IMFs, showed spindle-shaped appearance. The expression levels of key myofibroblast protein markers, including α-SMA, vimentin, and desmin, were also examined by the Western blotting and immunofluorescence analyses. Our results show that these cells were positive for α-SMA and vimentin, but not desmin, as well as that both LmcMF and SmcMF expressed type I collagen at a lower level than primary IMFs. Finally, we investigated the expression level of lipopolysaccharide (LPS) receptor-related proteins, as well as the response of the cells to LPS treatment. We found that the TLR4, CD14, and MD-2 proteins were present in LmcMF and SmcMF, as well as in primary IMFs, and that all these cells responded to LPS. We established 2 novel IMF cell lines from mouse colonic mucosa, namely, LmcMF and SmcMF, both of which were able to respond to LPS.

Increased Levels of Endotoxin Core Antibodies and Decreased Levels of sCD 14 Indicate Chronic Endotoxemia in Coronary Artery Disease (CURES-127)

Introduction: Endotoxin is hypothesized to play an important role in chronic inflammation associated with coronary artery disease (CAD). The activity of the endotoxin is determined by the relative levels of endotoxin core antibodies (EndoCab), LPS binding protein (LBP) and soluble CD14 (sCD14). Towards this end, we estimated EndoCab, LBP and sCD14 levels in subjects with CAD and correlated them with clinical parameters, serum hormones and cytokine levels. Methods: The study subjects were recruited from Chennai Urban Rural Epidemiology Study (CURES). Age and gender matched subjects with (n=46) and without (n=45) CAD were selected from Phase III of the study. The levels of serum lipopolysaccharide (LPS), EndoCab and its translocation markers (sCD14 and LBP) along with insulin, glucagon, adiponectin and leptin and pro-inflammatory cytokines such as TNF-α, IL-6 and IL-1β were measured by immuno-assays. Results: Quartile analysis showed significantly higher percentage of CAD subjects in the highest quartile of EndoCab (>26.6 EMU/ml) (CAD-24% Vs Controls 0%) and LPS (>65.05 EU/ml) (CAD-21% vs. Controls 4%), while a reverse trend was seen for sCD14 (>2198398.7 ng/ml) (CAD-1% vs. Control-24%) (p<0.01). LBP levels were not statistically different between the groups. While EndoCab levels showed positive association with LPS, LBP, sCD14, hsCRP, HOMA-IR and glucagon, sCD14 showed a negative association with most of these markers. Significant increase in the levels of pro inflammatory cytokines TNFα, IL-6 and IL-1β, was also observed in the CAD group when compared to the control. Conclusions: Significantly elevated levels of EndoCab and decreased levels of sCD14 indicate chronic endotoxin exposure in CAD subjects.

Imaging oxytocin × dopamine interactions: an epistasis effect of CD38 and COMT gene variants influences the impact of oxytocin on amygdala activation to social stimuli

Although oxytocin (OT) has become a major target for the investigation of positive social processes, it can be assumed that it exerts its effects in concert with other neurotransmitters. One candidate for such an interaction is dopamine (DA). For both systems, genetic variants have been identified that influence the availability of the particular substance. A variant of the gene coding for the transmembrane protein CD38 (rs3796863), which is engaged in OT secretion, has been associated with OT plasma level. The common catechol-O-methyltransferase (COMT) val158met polymorphism is known to influence COMT activity and therefore the degradation of DA. The present study aimed to investigate OT × DA interactions in the context of an OT challenge study. Hence, we tested the influence of the above mentioned genetic variants and their interaction on the activation of different brain regions (amygdala, VTA, ventral striatum and fusiform gyrus) during the presentation of social stimuli. In a pharmacological cross-over design 55 participants were investigated under OT and placebo (PLA) by means of fMRI. Brain imaging results revealed no significant effects for VTA or ventral striatum. Regarding the fusiform gyrus, we could not find any effects apart from those already described in Sauer et al. (2012). Analyses of amygdala activation resulted in no gene main effect, no gene × substance interaction but a significant gene × gene × substance interaction. While under PLA the effect of CD38 on bilateral amygdala activation to social stimuli was modulated by the COMT genotype, no such epistasis effect was found under OT. Our results provide evidence for an OT × DA interaction during responses to social stimuli. We postulate that the effect of central OT secretion on amygdala response is modulated by the availability of DA. Therefore, for an understanding of the effect of social hormones on social behavior, interactions of OT with other transmitter systems have to be taken into account.

TRAF Binding Is Required for a Distinct Subset of In Vivo B Cell Functions of the Oncoprotein LMP1

EBV-encoded latent membrane protein 1 (LMP1) is important for EBV contributions to B cell transformation and many EBV-associated malignancies, as well as EBV-mediated exacerbation of autoimmunity. LMP1 functionally mimics TNF receptor (TNFR) superfamily member CD40, but LMP1 signals and downstream effects are amplified and sustained compared with CD40. CD40 and LMP1 both use TNFR-associated factor (TRAF) adaptor proteins, but in distinct ways. LMP1 functions require TRAFs 3, 5, and 6, which interact with LMP1. However, TRAFs can also contribute to signaling in the absence of direct interactions with cell surface receptors, so we investigated whether their roles in LMP1 in vivo functions require direct association. We show in this study that the LMP1 TRAF binding site was required for LMP1-mediated autoantibody production, the germinal center response to immunization, and optimal production of several isotypes of Ig, but not LMP1-dependent enlargement of secondary lymphoid organs in transgenic mice. Thus, LMP1 in vivo effects can be mediated via both TRAF binding-dependent and -independent pathways. Together with our previous findings, these results indicate that TRAF-dependent receptor functions may not always require TRAF-receptor binding. These data suggest that TRAF-mediated signaling pathways, such as those of LMP1, may be more diverse than previously appreciated. This finding has significant implications for receptor and TRAF-targeted therapies.

Lipopolysaccharide Causes an Increase in Intestinal Tight Junction Permeability in Vitro and in Vivo by Inducing Enterocyte Membrane Expression and Localization of TLR-4 and CD14

Bacterial-derived lipopolysaccharides (LPS) play an essential role in the inflammatory process of inflammatory bowel disease. A defective intestinal tight junction (TJ) barrier is an important pathogenic factor of inflammatory bowel disease and other inflammatory conditions of the gut. Despite its importance in mediating intestinal inflammation, the physiological effects of LPS on the intestinal epithelial barrier remain unclear. The major aims of this study were to determine the effects of physiologically relevant concentrations of LPS (0 to 1 ng/mL) on intestinal barrier function using an invitro (filter-grown Caco-2 monolayers) and an invivo (mouse intestinal perfusion) intestinal epithelial model system. LPS, at physiologically relevant concentrations (0 to 1 ng/mL), in the basolateral compartment produced a time-dependent increase in Caco-2 TJ permeability without inducing cell death. Intraperitoneal injection of LPS (0.1 mg/kg), leading to clinically relevant plasma concentrations, also caused a time-dependent increase in intestinal permeability invivo. The LPS-induced increase in intestinal TJ permeability was mediated by an increase in enterocyte membrane TLR-4 expression and a TLR-4-dependent increase in membrane colocalization of membrane-associated protein CD14. In conclusion, these studies show for the first time that LPS causes an increase in intestinal permeability via an intracellular mechanism involving TLR-4-dependent up-regulation of CD14 membrane expression.

Abstract 9461: Circulating CD14 + CD16 + Monocyte Subsets as Biomarkers for Severity of Coronary Artery Disease in Patients With Stable Angina Pectoris

Background: Circulating monocytes can be divided into two subsets typically identified by the expression of CD14 and CD16. Although previous studies have shown that circulating monocytes contribute to the progression of coronary atherosclerotic lesions, the relationship between the severity of coronary artery disease (CAD) and the two distinct monocyte subsets has not previously been evaluated. We investigated the relationship between monocyte subsets and severity of CAD assessed by coronary angiography (CAG) in patients with stable angina pectoris (SAP). Methods: We enrolled 125 patients who underwent diagnostic CAG. Patients were divided into three groups; those without CAD, those with single-vessel disease (SVD), and those with multiple-vessel disease (MVD), according to diagnostic CAG findings. In addition, severity of CAD was evaluated by Gensini score. Two monocyte subsets (CD14 + CD16 - and CD14 + CD16 + ) were measured by flow cytometry. Results: Circulating CD14 + CD16 + monocytes were more frequently observed in patients with MVD (24.4 [18.5 to 29.8] %) than in those with SVD (vs. 12.8 [10.0 to 16.2] %, p&lt;0.001) or without CAD (vs. 8.9 [7.6 to 10.2] %, p&lt;0.001). High-sensitivity C-reactive protein and soluble CD40 ligand were not significantly different among the three groups. The proportion of CD14 + CD16 + monocytes was positively correlated with Gensini score (r=0.618, p&lt;0.001). Multivariate logistic regression analysis revealed that the preferential increase of CD14 + CD16 + monocytes was an independent predictor of MVD ((odds ratio: 1.475; 95% confidence interval: 1.273 to 1.708, p&lt;0.001). Conclusions: Our results revealed that the increased proportion of CD14 + CD16 + monocytes associated with severity of CAD in patients with SAP. Preferential increase in peripheral CD14 + CD16 + monocytes might closely relate to the pathophysiology of CAD progression.

Mutations of STAT6 DNA Binding Domain Are Characteristic of PMBL and Belong to the Genetic Signature of This Entity.

AbstractAbstract 2645Primary mediastinal B-cell lymphoma (PMBL) is a separate entity of aggressive B-cell lymphoma and accounts for about 5% of aggressive lymphomas according to the World Health Organization (WHO) classification. This lymphoma affects young patients and presents as a anterior mediastinal mass consisting of large B-cells that usually express little if any surface or cytoplasmic immunoglobulin and major histocompatibility complex class I and/or class II molecules. PMBL display constitutive activation of janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway, also observed in Hodgkin lymphoma and is characterized by heterozygous missense mutations of STAT6 DNA binding domain that are not observed in diffuse large B-cell lymphoma.To determine whether these mutations could represent a diagnostic or prognostic marker useful in the clinic, we developed a PCR HRM (High Resolution Melting) assay to screen for mutations in STAT6 exon 12, which had been previously identified as a hotspot mutation. We analyzed a series of 80 patients with a mediastinal biopsy and/or histological review of PMBL, enrolled in clinical trials of the GELA (LNH-03) or the GOELAMS (075). DNAs were extracted from FFPE (formalin-fixed and paraffin-embedded) samples with QIAmp DNA mini kit (QIAGEN®) for GELA samples and fully automated Siemens Healthcare Diagnostics proprietary experimental method based on magnetic beads for GOELAMS samples. Sixteen DNAs were not analyzed due to poor amplification in PCR and four DNAs were not analyzed due to other reasons (refused protocol, inclusion at relapse, not included in the tissue microarray/TMA). All cases with abnormal denaturation curves were analyzed by Sanger sequencing. Of the remaining 60 DNA samples, we detected 20 mutated tumors in exon 12 (30%). These mutations were heterozygous single nucleotide variants, often multiple (10 cases, 8 double hit mutations and 2 triple hit mutations) and targeted mostly amino acids 417 (9 hits) and 419 (7 hits). Immunohistochemical analysis of TMA showed that the mutated tumors expressed CD23 (14/20) and MAL proteins (13/15), more frequently than non-mutated ones (10/33, p= 0.009 and 15/27, p=0.049, respectively). Interestingly, these proteins are encoded by genes belonging to the transcriptional signature of PMBL as defined by gene expression profiling in previous studies, There were no significant difference in the clinical presentation between patients with a STAT6 mutation and non-mutated cases regarding age, sex, performance status, Ann Arbor stage, LDH, IPI, treatment response, progression free survival and overall survival. However we noted an absence of elevated β2-microglobulin (1 case elevated/18) and an absence of albumin < 35 g/dl (0 case/8) in the mutated tumors compared with non-mutated tumors (11/31, p= 0.003 and 9/21, p< 0.05, respectively). Finally, we analyzed the correlations of these mutations with cytogenetic abnormalities including CIITA (Class II Transactivator) rearrangement, PD-L1/2 (Programmed death ligand) rearrangement and PD-L1/2 copy number alterations. Indeed, CIITA rearrangements have been described in 38% of PMBL and PD-L1/2 amplification were reported in 63% of PMBL. In this series of patients, CIITA rearrangements were present in 15/49 (31%) and PD-L1/2 low (3–4 copies) and high (>4 copies) level amplification observed in 33/52 (63%) of cases. It is interesting to note that STAT6-mutated cases frequently harbor CIITA rearrangement compared to STAT6-non-mutated cases (9/18 vs 6/31, p= 0.05) as well as PD-L1/2 low level copy number gains (12/19 vs 10/32, p= 0.02), suggesting that the combination of specific genomic alterations cooperate during PMBL oncogenesis.In conclusion, the frequency of this mutation suggests an in vivo selection during PMBL lymphomagenesis. The correlations with the clinico-biological data suggest that STAT6 mutations may be searched for in routine FFPE samples and may help to establish a molecular definition of PMBL that is characterized by alterations of the JAK-STAT pathway. Further investigations are required to identify the mutational mechanisms involved and the oncogenic function associated with these mutations. Disclosures:No relevant conflicts of interest to declare.

Suppression of HIV-1 Infection by APOBEC3 Proteins in Primary Human CD4 + T Cells Is Associated with Inhibition of Processive Reverse Transcription as Well as Excessive Cytidine Deamination

The Vif protein of human immunodeficiency virus type 1 (HIV-1) promotes viral replication by downregulation of the cell-encoded, antiviral APOBEC3 proteins. These proteins exert their suppressive effects through the inhibition of viral reverse transcription as well as the induction of cytidine deamination within nascent viral cDNA. Importantly, these two effects have not been characterized in detail in human CD4(+) T cells, leading to controversies over their possible contributions to viral inhibition in the natural cell targets of HIV-1 replication. Here we use wild-type and Vif-deficient viruses derived from the CD4(+) T cells of multiple donors to examine the consequences of APOBEC3 protein function at natural levels of expression. We demonstrate that APOBEC3 proteins impart a profound deficiency to reverse transcription from the initial stages of cDNA synthesis, as well as excessive cytidine deamination (hypermutation) of the DNAs that are synthesized. Experiments using viruses from transfected cells and a novel method for mapping the 3' termini of cDNAs indicate that the inhibition of reverse transcription is not limited to a few specific sites, arguing that APOBEC3 proteins impede enzymatic processivity. Detailed analyses of mutation spectra in viral cDNA strongly imply that one particular APOBEC3 protein, APOBEC3G, provides the bulk of the antiviral phenotype in CD4(+) T cells, with the effects of APOBEC3F and APOBEC3D being less significant. Taken together, we conclude that the dual mechanisms of action of APOBEC3 proteins combine to deliver more effective restriction of HIV-1 than either function would by itself.

Increased Vascular Inflammation in Early Menopausal Women Is Associated With Hot Flush Severity

Context: Menopause has been related to an increased atherosclerotic risk. Presence and severity of hot flushes in menopausal women have been associated with impaired endothelial function and advanced subclinical atherosclerosis. Objective: The objective of the study was to evaluate the effect of menopausal transition on vascular inflammation indices and investigate the association of hot flush severity with these indices in early menopausal women. Design, Setting, and Participants: This was a cross-sectional study that included 120 early menopausal women (age range 42–55 yr, <3 yr in menopause) recruited from the menopause outpatient clinic of an academic hospital and 24 age-matched premenopausal women (controls). Main Outcomes: Serum high-sensitivity C-reactive protein, P-selectin, and soluble CD40 ligand (sCD40L) levels were measured. Results: P-selectin and sCD40L were increased in early menopausal compared with control women (P = 0.006 and P = 0.02 respectively), whereas high-sensitivity C-reactive pr...

Modulation of IL-10-Specific B Cell MicroRNAs by Latent Membrane Protein 1 and Epstein-Barr Virus

Post-Transplant Lymphoproliferative Disorder (PTLD) is a potentially fatal side effect of the immunosuppressive agents used to prevent allograft rejection. PTLD is associated with Epstein-Barr Virus (EBV) infection and can result in malignant B cell lymphoma. Latent membrane protein 1 (LMP1), a viral mimic of the B cell costimulatory protein CD40, is the major oncogene of EBV and can usurp host cell pathways to promote cell survival. LMP1 activates the Akt/PI3K signaling pathway to drive IL-10 production, which is essential for the autonomous proliferation of EBV+ B cell lymphomas. MicroRNAs are small, non-coding RNAs that post-transcriptionally regulate gene expression to control cellular events such as differentiation, proliferation, and cell survival. As aberrant expression of microRNAs is found in a variety of human malignancies, our goal was to determine if microRNAs participate in LMP1-induced IL-10 production in PTLD-derived cell lymphomas. To first establish that EBV infection can modulate host cell microRNA, we used Taqman microRNA qPCR arrays to detect the microRNA expression profiles of six B cell tumor lines derived from patients with PTLD, two B cell lines generated by infection with the B95.8 strain of EBV obtained from a patient with mononucleosis, and one EBV-negative B cell line. Expression data was then normalized internally and to the EBV-negative B cell line. Our analysis showed that compared to the EBV-negative B cell line, B cells infected with the benign B95.8 strain of EBV had the altered expression (2-fold) of 294 microRNAs while the PTLD-derived EBV+ B cell lines had the altered expression of 224 microRNAs. Twenty-eight microRNAs were altered in both cases of EBV infection. To examine the specific role of LMP1 in host cell microRNA expression, we used an inducible system of LMP1 activation and expressed different LMP1 molecules in an EBV negative B cell lymphoma line. Activation of the B95.8 form of LMP1 modulated 62 cellular microRNAs and 142 microRNAs were modulated by all five of the PTLD-derived LMP1 molecules. Notably, only 28 microRNAs were modulated by both benign and tumor LMP1. The microRNAs aberrantly regulated by either EBV infection or LMP1 activation were next analyzed with target prediction software for potential binding to the 3″UTR of IL-10. MicroRNA-21, -106A, -106B, -194, -340, -374 and -374-5p fit these criteria and were tested for direct binding to the 3″UTR of IL-10 in luciferase reporter assays. The overexpression of microRNA-106A, -106B, -194, -374, -374-5p each negatively regulated the IL-10 3″UTR. Conversely, microRNA-21 overexpression positively modulated the 3″UTR of IL-10 while microRNA-340 overexpression had no effect. Together, these findings indicate that EBV infection and LMP1 activation induce host cell microRNAs that regulate production of the autocrine growth factor IL-10 in EBV+ B cell lymphomas. Thus, cellular microRNAs are a potential target for inhibiting the growth of PTLD-associated B cell lymphomas.