Presence Of Interleukin Research Articles

The immunomodulatory effects of TNF-α inhibitors on human Th17 cells via RORγt histone acetylation.

The presence of interleukin (IL)-17-related cytokines correlates with rheumatoid arthritis (RA) pathogenesis. Epigenetic modifications, including histone acetylation, regulate gene expression in RA pathogenesis. Tumour necrosis factor-alpha (TNF-α) inhibitors such as etanercept and adalimumab, represent a breakthrough in RA treatment. We aimed to investigate the effects of etanercept and adalimumab on human Th17-polarized cells and the possible intracellular regulators of these effects, including the Th17-specific transcription factors signal transducer, activator of transcription 3 (STAT3), retinoid-related orphan receptor γ-T (RORγt) and epigenetic modification. Human CD4+ T cells from healthy subjects and patients with RA were pretreated with TNF-α inhibitors and then being polarized into IL-17-producing cells. The Th17-related cytokine levels in the culture supernatants were determined with an enzyme-linked immunosorbent assay. Intracellular signalling was investigated by western blot, real-time RT-PCR, and chromatin immunoprecipitation. Th17-polarized cells from patients with RA produced more IL-17A, IL-17F and IL-22 than those from healthy subjects. Etanercept and adalimumab suppressed IL-17A, IL-17F and IL-22 levels in Th17-polarized cells from healthy subjects and patients with RA. Western blot analysis revealed that etanercept and adalimumab decreased mitogen-activated protein kinase-phospho-p38, nuclear factor-κB-phospho-p65, phospho-STAT3 and RORγt levels. Etanercept and adalimumab decreased histone (H)3 and H4 acetylation in the RORγt gene promotor region by decreasing the recruitment of the acetyltransferases p300, CBP and PCAF. The present study broadens our knowledge of the mechanisms underlying the immunomodulatory effects of TNF-α inhibitors in rheumatoid arthritis treatment.

Alloimmune Monitoring after Islet Transplantation: A Prospective Multicenter Assessment of 25 Recipients

Islet transplantation is an effective treatment for selected patients with type 1 diabetes. However, an accurate test still lacks for the early detection of graft rejection. Blood samples were prospectively collected in four university centers (Geneva, Grenoble, Montpellier, and Strasbourg). Peripheral blood mononuclear cells were stimulated with donor splenocytes in the presence of interleukin-2. After 24 h of incubation, interferon- (IFN-) ELISpot analysis was performed. After a total of 5 days of incubation, cell proliferation was assessed by fluorescence-activated cell sorting (FACS) analysis for Ki-67. Immunological events were correlated with adverse metabolic events determined by loss of 1 point of -score and/or an increased insulin intake 10%. Twenty-five patients were analyzed; 14 were recipients of islets alone, and 11 combined with kidney. Overall, 76% (19/25) reached insulin independence at one point during a mean follow-up of 30.7 months. IFN- ELISpot showed no detectable correlation with adverse metabolic events [area under the curve (AUC)=0.57]. Similarly, cell proliferation analysis showed no detectable correlation with adverse metabolic events (CD3+/CD4+ AUC=0.54; CD3+/CD8+ AUC=0.55; CD3/CD56+ AUC=0.50). CD3/CD56+ cell proliferation was significantly higher in patients with combined kidney transplantation versus islet alone (6 months, p=0.010; 12 months, p=0.016; and 24 months, p=0.018). Donor antigen-stimulated IFN- production and cell proliferation do not predict adverse metabolic events after islet transplantation. This suggests that the volume of transplanted islets is too small to produce a detectable systemic immune response and/or that alloimmune rejection is not the sole reason for the loss of islet graft function.

Y-box-binding protein 1 contributes to IL-7-mediated survival signaling in B-cell precursor acute lymphoblastic leukemia.

Y-box-binding protein 1 (YB-1) is a regulatory protein that is associated with drug resistance and relapse in solid tumors. As YB-1 mediates some of its activity through growth factor receptor signaling dysregulation, the present study compared the expression of YB-1 and interleukin 7 (IL-7) receptor α (IL-7Rα) in pediatric B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) and normal BCP cells. The expression levels of IL-7Rα and YB-1 were higher in relapsed vs. diagnostic samples of primary BCP ALL; however, co-expression was also observed in a minor BCP cell population in samples from healthy donors. Functional crosstalk between YB-1 and IL-7R was detected: Overexpression of YB-1 increased surface levels of IL-7R in B cells, and the stimulation of BCP ALL cell lines and primary samples by IL-7 activated YB-1 by phosphorylation at S102 in a phosphatidylinositol 3-kinase-independent and MEK1/2-dependent manner. Targeted knockdown of YB-1 reduced IL-7-mediated protection against rapamycin, and an inhibitor of MEK1/2 potentiated rapamycin-mediated killing in the presence of IL-7. These data establish a novel link between two well-characterized pro-survival factors in acute leukemia, and suggest that YB-1 inhibition may represent a novel therapeutic strategy for increasing sensitivity to chemotherapy in patients with refractory acute B-cell leukemia.

Th9 and other IL-9-producing cells in allergic asthma.

Allergic asthma is a worldwide increasing chronic disease of the airways which affects more than 300 million people. It is associated with increased IgE, mast cell activation, airway hyperresponsiveness (AHR), mucus overproduction and remodeling of the airways. Previously, this pathological trait has been associated with T helper type 2 (Th2) cells. Recently, different CD4+ T cell subsets (Th17, Th9) as well as cells of innate immunity, like mast cells and innate lymphoid cells type 2 (ILC2s), which are all capable of producing the rediscovered cytokine IL-9, are known to contribute to this disease. Regarding Th9 cells, it is known that naïve T cells develop into IL-9-producing cells in the presence of interleukin-4 (IL-4) and transforming growth factor beta (TGFβ). Downstream of IL-4, several transcription factors like signal transducer and activator of transcription 6 (STAT6), interferon regulatory factor 4 (IRF4), GATA binding protein 3 (GATA3), basic leucine zipper transcription factor, ATF-like (BATF) and nuclear factor of activated T cells (NFAT) are activated. Additionally, the transcription factor PU.1, which is downstream of TGFβ signaling, also seems to be crucial in the development of Th9 cells. IL-9 is a pleiotropic cytokine that influences various distinct functions of different target cells such as T cells, B cells, mast cells and airway epithelial cells by activating STAT1, STAT3 and STAT5. Because of its pleiotropic functions, IL-9 has been demonstrated to be involved in several diseases, such as cancer, autoimmunity and other pathogen-mediated immune-regulated diseases. In this review, we focus on the role of Th9 and IL-9-producing cells in allergic asthma.

T-Cell Therapy Using Interleukin-21-Primed Cytotoxic T-Cell Lymphocytes Combined With Cytotoxic T-Cell Lymphocyte Antigen-4 Blockade Results in Long-Term Cell Persistence and Durable Tumor Regression.

Purpose Peripheral blood-derived antigen-specific cytotoxic T cells (CTLs) provide a readily available source of effector cells that can be administered with minimal toxicity in an outpatient setting. In metastatic melanoma, this approach results in measurable albeit modest clinical responses in patients resistant to conventional therapy. We reasoned that concurrent cytotoxic T-cell lymphocyte antigen-4 (CTLA-4) checkpoint blockade might enhance the antitumor activity of adoptively transferred CTLs. Patients and Methods Autologous MART1-specific CTLs were generated by priming with peptide-pulsed dendritic cells in the presence of interleukin-21 and enriched by peptide-major histocompatibility complex multimer-guided cell sorting. This expeditiously yielded polyclonal CTL lines uniformly expressing markers associated with an enhanced survival potential. In this first-in-human strategy, 10 patients with stage IV melanoma received the MART1-specific CTLs followed by a standard course of anti-CTLA-4 (ipilimumab). Results The toxicity profile of the combined treatment was comparable to that of ipilimumab monotherapy. Evaluation of best responses at 12 weeks yielded two continuous complete remissions, one partial response (PR) using RECIST criteria (two PRs using immune-related response criteria), and three instances of stable disease. Infused CTLs persisted with frequencies up to 2.9% of CD8+ T cells for as long as the patients were monitored (up to 40 weeks). In patients who experienced complete remissions, PRs, or stable disease, the persisting CTLs acquired phenotypic and functional characteristics of long-lived memory cells. Moreover, these patients also developed responses to nontargeted tumor antigens (epitope spreading). Conclusion We demonstrate that combining antigen-specific CTLs with CTLA-4 blockade is safe and produces durable clinical responses, likely reflecting both enhanced activity of transferred cells and improved recruitment of new responses, highlighting the promise of this strategy.

Inhibition of JAK3 and PKC via Immunosuppressive Drugs Tofacitinib and Sotrastaurin Inhibits Proliferation of Human B Lymphocytes In Vitro

BackgroundAntibody-mediated response in solid organ transplantation is critical for graft dysfunction and loss. The use of immunosuppressive agents partially inhibits the B-lymphocyte response leading to a risk of acute and chronic antibody-mediated rejection. This study evaluated the impact of JAK3 and PKC inhibitors tofacitinib (Tofa) and sotrastaurin (STN), respectively, on B-cell proliferation, apoptosis, and activation in vitro. MethodsHuman B cells isolated from peripheral blood of healthy volunteers were cocultured with CD40 ligand–transfected fibroblasts as feeder cells in the presence of interleukin (IL) 2, IL-10, and IL-21. The cocultures were treated with immunosuppressants Tofa, STN, and rapamycin (as a control), to analyze the proliferation and apoptosis of B cells by means of Cyquant and flow cytometry, respectively. CD27 and IgG staining were applied to evaluate whether treatments modified the activation of B cells. ResultsTofa and STN were able to inhibit B-cell proliferation to the same extent as rapamycin, without inducing cell apoptosis. After 6 days in coculture with feeder cells, all B cells showed CD27 memory B-cell phenotype. None of the immunosuppressive treatments modified the proportion between class-switched and non–class-switched memory B cells observed in nontreated cultures. The high predominance of CD27+CD24+ phenotype was not modified by any immunosuppressive treatment. ConclusionsOur results show that Tofa and STN can suppress B-cell antibody responses to an extent similar to rapamycin, in vitro; therefore these compounds may be a useful therapy against antibody-mediated rejection in transplantation.

Interleukin 10 promotes immune response by increasing the survival of activated CD8+ T cells in human papillomavirus 16-infected cervical cancer.

Human papillomavirus (HPV)-specific CD8+ T cells are present in HPV-infected cervical cancer patients and have demonstrated potent antitumor properties. However, these cells cannot control tumor progression in most patients. To investigate the underlying mechanisms involved in suppressing or promoting CD8+ T cell functions, we focused on interleukin 10 (IL-10), a pleiotropic cytokine with controversial roles in antitumor immunity. We found that compared to healthy controls, circulating CD8+ T cells in HPV 16-infected cervical cancer patients expressed significantly higher levels of IL-10. Interestingly, these CD8+ T cells from cervical cancer patients, but not those from healthy controls, responded to HPV 16 E6/E7 peptide stimulation by increasing IL-10 expression, demonstrating an antigen-specific IL-10 release. Addition of exogenous IL-10 improved the survival, but did not increase the proliferation, of peptide-stimulated CD8+ T cells. CD8+ T cells cultured in the presence of IL-10 also resulted in significantly higher interferon gamma (IFN-gamma) and granzyme B concentration, primarily due to improved cell survival. In resected cervical tumors, the frequency of tumor-infiltrating IL-10+ CD8+ T cells was positively correlated with the frequency of tumor-infiltrating IFN-gamma+ and granzyme B+ CD8+ T cells. Tumor-associated macrophages were more potent than peripheral blood monocyte-derived macrophages at inducing IL-10 expression in CD8+ T cells, possibly explaining the elevated IL-10+ CD8+ T cell frequency in cervical cancer patients. Together, these results are consistent with an immunostimulatory role of IL-10, which promoted CD8+ T cell response by increasing the survival of activated CD8+ T cells.

Interleukin 6 induces expression of NADPH oxidase 2 in human aortic endothelial cells via long noncoding RNA MALAT1.

Human abdominal aortic aneurysm (AAA) is characterized by the induction of intracellular and extracellular inflammatory cytokines and the production of reactive oxygen species (ROS) associated with localized inflammatory responses in the vascular wall. Recent studies have shown that greater circulating levels of the proinflammatory cytokine interleukin-6 (IL-6) are closely associated with AAA presence, suggesting that IL-6 plays an important role in the development of AAA. Previous in vivo studies have indicated that excess activities of NADPH oxidase (NOX), a major oxidase system for ROS production, promote AAA development. Furthermore, long noncoding RNAs (lncRNAs) are involved in the development of AAA. LncRNA MALAT1 has been found closely involved in endothelial cell functions and dysfunctions. In the present study, we explored the effects and the underlying mechanisms of IL-6 and MALAT1 on the expression/activity of NOXs in human aortic endothelial cells (HAOECs). Primary HAOECs with or without overexpression or knockdown of MALTA1 were cultured in the presence of IL-6. We found that IL-6 concentration- and time-dependently elevated the NOX activity as well as the MALAT1 level in HAOECs. Among different NOXs, only NOX2 was induced by IL-6. Overexpression and knockdown of MALAT1 respectively augmented and abolished IL6-induced expression of NOX2, NOX activity/cellular ROS production, and activation of the human NOX2 gene promoter, whereas MALAT1 alone in the absence of IL-6 treatment showed no significant effect. Knockdown of extracellular signal-regulated kinase (ERK) abolished IL6-induced expression of MALAT1. In conclusion, this study provides the first evidence that IL-6 induces expression/activity of NOX2 in HAOECs via inducing MALAT1 by an ERK-dependent mechanism. It adds new insights into the molecular mechanisms underlying AAA development.

Interaction between Treg cells and tumor-associated macrophages in the tumor microenvironment of epithelial ovarian cancer.

Epithelial ovarian cancer(EOC) is the most lethal gynecological malignancy. Inflammatory cells in the EOC microenvironment play a key role in tumor progression. In the present study, we investigated the mechanism of the accumulation of regulatory T cells(Tregs) induced by interleukin-10(IL-10) derived from tumor-associated macrophages(TAMs) in the EOC microenvironment. The frequency of Tregs and TAMs was detected by immunofluorescence in 40EOC tissues and 20benign ovarian tumors, as well as the expression of IL-10 which was assessed by immunohistochemistry. It was found that the frequency of Treg cells and TAMs was significantly higher in the EOC than those in the benign ovarian tumors. The expression of IL-10 was also found to be higher in the EOC than that in the benign tumors. EOC patients with a high frequency of Tregs exhibited a significantly shorter overall survival time compared to those with a low frequency of Tregs. In addition, the expression of IL-10 in ascites and blood serum and the IL-10 released in the co-cultured system supernatants were detected by ELISA. Following CD4+T-cell co-culturing with macrophages and IL-10, it was observed by flow cytometric analysis that the frequency of Treg cells was increased in the presence of IL-10. It was also established that IL-10 released in the co-cultured supernatants was increased. We also detected the mechanism of Treg cells induced by IL-10 invivo. The SKOV3 cell tumor volume and weight were much higher in the presence of IL-10 in a mouse subcutaneous model. These data suggest that IL-10 secreted by TAMs increase the frequency of Treg cells through the activation of Foxp3 during T-cell differentiation and promotes tumor progression.

Clinical-grade regulatory Tcells: Comparative analysis of large-scale expansion conditions.

Recent clinical trials have indicated the high potential of regulatory Tcells (Tregs) in the prevention of acute and chronic graft-versus-host disease (GvHD) after hematopoietic stem cell transplantation, but immune interventions require large numbers of Tregs. With respect to their limited natural occurrence, development and optimization of protocols for large-scale expansion of clinical-grade Tregs are essential if considered for therapeutic use. We compared different clinical-grade large-scale expansion protocols for repetitive transfer of large numbers of Tregs in clinical trials for the prevention of acute and/or chronic GvHD. Donor Tregs were isolated using magnetic-activated cell sorting (MACS) technology with good manufacturing practice-compliant devices. CD8 and CD19 depletion followed by CD25 enrichment resulted in the isolation of CD4+CD25+CD127- Tregs with a mean purity of 77%. Cell populations were expanded exvivo using X-Vivo 15 (±rapamycin), TexMACS (±rapamycin), and CellGro DC (±rapamycin) in the presence of interleukin-2. The highest rates of expansion of clinical-grade Tregs were observed for X-Vivo 15 and CellGro DC without rapamycin in compared with all other expansion media tested. The suppressive capacity of the expanded Treg population was maintained under all conditions investigated. Our data suggest that expansion with CellGro provides data comparable to those obtained with TexMACS or X-Vivo 15 with rapamycin, although all three conditions did not provide the same propagation rate as X-Vivo 15 alone. With respect to functionality, phenotype, and stability, CellGro DC medium represents a reasonable alternative for good manufacturing practice-compatible large-scale exvivo expansion.

Distinct Immunoregulatory Mechanisms in Mesenchymal Stem Cells: Role of the Cytokine Environment.

Mesenchymal stem cells (MSCs) represent a population of cells which have the ability to regulate reactivity of T and B lymphocytes by multiple mechanisms. The immunoregulatory activities of MSCs are strictly influenced by the cytokine environment. Here we show that two functionally distinct cytokines, interleukin-4 (IL-4) and interferon-γ (IFN-γ), significantly potentiate the ability of MSCs to inhibit IL-10 production by activated regulatory B cells (Bregs). However, MSCs in the presence of IL-4 or IFN-γ inhibit the IL-10 production by different mechanisms. Preincubation of MSCs with IFN-γ led to the suppression, but pretreatment with IL-4 of neither MSCs nor B cells resulted in the suppression of IL-10 production. The search for candidate regulatory molecules expressed in cytokine-treated MSCs revealed different patterns of the gene expression. Pretreatment of MSCs with IFN-γ, but not with IL-4, induced expression of indoleamine-2,3-dioxygenase, cyclooxygenase-2 and programmed cell death-ligand 1. To identify the molecule(s) responsible for the suppression of IL-10 production, we used specific inhibitors of the putative regulatory molecules. We found that indomethacine, an inhibitor of cyclooxygenase-2 (Cox-2) activity, completely abrogated the inhibition of IL-10 production in cultures containing MSCs and IFN-γ, but had no effect on the suppression in cell cultures containing MSCs and IL-4. The results show that MSCs can inhibit the response of B cells to one stimulus by different mechanisms in dependence on the cytokine environment and thus support the idea of the complexity of immunoregulatory action of MSCs.

Generation of clinical-grade CD19-specific CAR-modified CD8+ memory stem cells for the treatment of human B-cell malignancies

AbstractLong-lived, self-renewing, multipotent T memory stem cells (TSCM) can trigger profound and sustained tumor regression but their rareness poses a major hurdle to their clinical application. Presently, clinically compliant procedures to generate relevant numbers of this T-cell population are undefined. Here, we provide a strategy for deriving large numbers of clinical-grade tumor-redirected TSCM starting from naive precursors. CD8+CD62L+CD45RA+ naive T cells enriched by streptamer-based serial-positive selection were activated by CD3/CD28 engagement in the presence of interleukin-7 (IL-7), IL-21, and the glycogen synthase-3β inhibitor TWS119, and genetically engineered to express a CD19-specific chimeric antigen receptor (CD19-CAR). These conditions enabled the generation of CD19-CAR–modified CD8+ TSCM that were phenotypically, functionally, and transcriptomically equivalent to their naturally occurring counterpart. Compared with CD8+ T cells generated with clinical protocols currently under investigation, CD19-CAR–modified CD8+ TSCM exhibited enhanced metabolic fitness and mediated robust, long-lasting antitumor responses against systemic acute lymphoblastic leukemia xenografts. This clinical-grade platform provides the basis for a phase 1 trial evaluating the activity of CD19-CAR–modified CD8+ TSCM in patients with B-cell malignancies refractory to prior allogeneic hematopoietic stem cell transplantation.

MicroRNA-17 Modulates Regulatory T Cell Function by Targeting Co-regulators of the Foxp3 Transcription Factor

Regulatory T (Treg) cells are important in maintaining self-tolerance and immune homeostasis. The Treg cell transcription factor Foxp3 works in concert with other co-regulatory molecules, including Eos, to determine the transcriptional signature and characteristic suppressive phenotype of Treg cells. Here, we report that the inflammatory cytokine interleukin-6 (IL-6) actively repressed Eos expression through microRNA-17 (miR-17). miR-17 expression increased in Treg cells in the presence of IL-6, andits expression negatively correlated with that of Eos. Treg cell suppressive activity was diminished upon overexpression of miR-17 invitro and invivo, which was mitigated upon co-expression of an Eos mutant lacking miR-17 target sites. Also, RNAi of miR-17 resulted in enhanced suppressive activity. Ectopic expression of miR-17 imparted effector-T-cell-like characteristics to Treg cells via the de-repression of genes encoding effector cytokines. Thus, miR-17 provides a potent layer of Treg cell control through targeting Eos and additional Foxp3 co-regulators.

Regulatory T-Cell Plasticity

During the past 2 decades, human and animals studies have clearly documented the crucial role of inflammation in the development and complications of atherosclerosis.1 Both innate immunity and adaptive immunity are involved in this process. The first evidence that suggested a role of adaptive immunity in atherosclerosis was the widespread detection of the major histocompatibility class II in human atherosclerotic plaques and the presence of a large amount of CD3+ lymphocytes in human and mouse atherosclerotic lesions. Most of the T cells in mouse and human atherosclerotic plaques are CD4+ T-helper (Th) cells expressing the αβ T-cell antigen receptor. Among CD4+ T cells, Th1 cells have been shown to exert proatherogenic effects, whereas regulatory T cells (Tregs) display atheroprotective properties. The role of Th2 and Th17 cells is still debated.1 Helper T-cell subsets are defined by the production of cytokines or the expression of characteristic lineage-defining transcription factors. Th1 cells are generated on priming in the presence of interleukin (IL)-12 that promotes the expression of the transcription factor T-bet and stimulates the production of the Th1 prototypical cytokine interferon-γ (IFN-γ).2 Tregs have a central role in the dominant control of immunologic tolerance and maintenance of immune homeostasis. They were first identified in mice and later in humans.3 The transcription factor FoxP3 is essential for the generation and the functions of Tregs.4 FoxP3 deficiency leads to a multiorgan autoimmune disease as can be observed in the scurfy mouse and in human immune dysregulation, polyendocrinopathy, enteropathy X–linked syndrome patients.5 Natural or thymic FoxP3+ Tregs acquire regulatory lineage commitment already on maturation in the thymus,3 whereas adaptive or peripheral FoxP3+ Tregs can be induced from mature CD4+ Th cells in the periphery under the influence of different stimulations, especially transforming …

Regulatory T cells evade restimulation-induced cell death by FOXP3-mediated repression of the SAP promoter.

Abstract An efficient adaptive immune response relies on the rapid clonal expansion of effector T cells, paired with timely disposal of those cells in order to avoid damage to host tissues and maintain immune homeostasis. Restimulation-induced cell death (RICD) is one programmed apoptotic pathway that ensures effector T cell expansion remains in check, triggered by repeated stimulation through the T cell receptor (TCR) in the presence of interleukin-2 (IL-2). CD4+ regulatory T cells (Tregs) require IL-2 and experience frequent TCR stimulation, but are highly resistant to RICD. Although the mechanism remains unclear, previous work has shown that RICD resistance in Tregs is dependent on the forkhead box P3 (FOXP3) transcriptional repressor, the master regulator of Treg function. We have previously shown that T cells from patients with X-linked lymphoproliferative disease (XLP-1), caused by null mutations in the signaling adaptor molecule SLAM-associated protein (SAP), are resistant to RICD. In fact, SAP is required for optimal RICD in effector T cells. We now demonstrate that Tregs express very low levels of SAP compared to conventional T cells (Tconv). Using chromatin immunoprecipitation and reporter assays, we show that FOXP3 reduces SAP expression in Tregs by binding to and repressing the SH2D1A (SAP) promoter. Indeed, knockdown of FOXP3 restores SAP-driven RICD sensitivity in Tregs through a FAS ligand-dependent mechanism. Intriguingly, our preliminary data suggest that FOXP3 also confers RICD resistance in Tconv during initial rounds of activation-induced proliferation. Hence our results provide a mechanism by which FOXP3 confers resistance to RICD to help preserve Tregs and protect early effector Tconv cells.

Proinflammatory Cytokines Regulate Cementogenic Differentiation of Periodontal Ligament Cells by Wnt/Ca(2+) Signaling Pathway.

Periodontal inflammation can inhibit cell differentiation of periodontal ligament cells (PDLCs), resulting in decreased bone/cementum regeneration ability. The Wnt signaling pathway, including canonical Wnt/β-catenin signaling and noncanonical Wnt/Ca(2+) signaling, plays essential roles in cell proliferation and differentiation during tooth development. However, little is still known whether noncanonical Wnt/Ca(2+) signaling cascade could regulate cementogenic/osteogenic differentiation capability of PDLCs within an inflammatory environment. Therefore, in this study, human PDLCs (hPDLCs) and their cementogenic differentiation potential were investigated in the presence of cytokines. The data demonstrated that both cytokines interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) inhibited cell proliferation, relative alkaline phosphatase activity, bone/cementum-related gene/protein expression, and canonical Wnt pathway-related gene/protein expression in hPDLCs. Interestingly, both cytokines upregulated the noncanonical Wnt/Ca(2+) signaling-related gene and protein expression in hPDLCs. When the Wnt/Ca(2+) pathway was blocked by Ca(2+)/calmodulin-dependent protein kinase II inhibitor KN93, even in the presence of IL-6 and TNF-α, cementogenesis could be stimulated in hPDLCs. Our data indicate that the Wnt/Ca(2+) pathway plays an inhibitory role on PDLC cementogenic differentiation in inflammatory microenvironments. Therefore, targeting the Wnt/Ca(2+) pathway may provide a novel therapeutic approach to improve periodontal regeneration for periodontal diseases.

Effects of interleukin 6 and tumor necrosis factor-α on the proliferation of porcine theca interna cells: Possible role of these cytokines in the pathogenesis of polycystic ovary syndrome

ObjectiveWe studied the effects of interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) on the proliferation of porcine theca interna (TI) cells and further elucidated the roles of IL-6 and TNF-α in the pathogenesis of polycystic ovary syndrome. Materials and MethodsTI cells were treated with 10 pg/mL, 100 pg/mL, and 1000 pg/mL IL-6 or TNF-α. TI cell proliferation was then examined by carboxyfluorescein diacetate succinimidyl ester labeling and flow cytometry. ResultsCell proliferation was not significantly different in TI cells cultured in medium alone (control) or in the presence of IL-6. At 72 hours of treatment, the mean fluorescence intensity was significantly lower in TI cells treated with 100 pg/mL and 1000 pg/mL TNF-α than in the control (p < 0.05). ConclusionTNF-α, but not IL-6, was able to promote TI cell proliferation. Our results suggest that TNF-α might play a role in hyperandrogenism, cortex thickness, and the increased ovary volume observed in polycystic ovaries.

0180 : Role of non-muscular myosin light chain kinase (nmMLCK) in the inflammation associated with a model of intermittent hypoxia

Obstructive Sleep Apnea Syndrome (OSAS) is defined by repetitive obstructions of upper airway during sleep inducing intermittent decrease in oxygen saturation or intermittent hypoxia (IH). IH alters endothelial function favoring inflammation and could accelerate atherosclerosis-induced cardiovascular diseases. A protein that may play a role in this process is the nonmuscular myosin light chain kinase (nmMLCK), protein involved in the regulation of endothelial permeability. The aim of this study was to analyze the implication of nmMLCK in inflammation induced by IH on endothelial cells. Human aortic endothelial cells (HAoECs) were exposed to 6h of IH, including 30min of hypoxia (O2 5%) followed by 30min of normoxia (O2 21%), in absence or presence of ML-7 (5μM), a nmMLCK inhibitor, and in absence or presence of interleukin-6 (IL-6, 40ng/ml), an inflammatory cytokine found in OSAS patients. After stimulation, we investigated inflammatory process by evaluating p65-NF-κB pathway and the release of several inflammatory cytokines by HAoECs. Activation of p65-NF-κB inflammatory pathway is increased in response to IH, as well as secretion of certain inflammatory cytokines such as IL-6 and CXCL-1 in HAoECs. While nmMLCK inhibition by ML-7 did not prevent p65-NF-κB activation, it partially decreased IL-6 and CXCL-1 secretion induced by IH. Moreover, to mimic the pro-inflammatory environment already established in OSAS patients, we costimulated HAoECs with IL-6 and IH. Under these conditions, IL-6 alone increased activation of STAT3 but did not potentiate the effects of IH regarding p65-NF-κB activation and cytokine secretion. These results suggest that nmMLCK may participate to the IH-induced inflammatory process in endothelial cells by modulating IH-induced cytokine secretion. Furthermore, IH is sufficient to induce inflammatory process independently of the proinflammatory environment. The author hereby declares no conflict of interest

Decreased numbers and impaired function of dendritic cells in patients with hepatitis B-related acute-on-chronic liver failure

Objective To investigate the frequencies of circulating dendritic cell (DC) subsets and the function of monocyte-derived dendritic cells in patients with hepatitis B-related acute-on-chronic liver failure. Methods Peripheral blood was collected from hepatitis B-related acute-on-chronic liver failure patients (ACLF, n=40) and chronic hepatitis B (CHB, n=40) as well as normal controls (NCs, n=20). Circulating myeloid dendritic cell (Mdc) and plasmic dendritic cell (pDC) frequencies in peripheral blood mononuclear cells (PBMC) were analyzed by flow cytometric analysis. Purified monocytes were isolated by combination of Histopaque-1.077 and CD14 Microbeads. Monocyte-derived dendritic cells (MoDCs) generated in vitro in the presence of interleukin (IL)-4 and granulocyte macrophage colony-stimulating factor upon activation by poly I∶C. Costimulatory molecule expression and allostimulatory mixed lymphocyte reaction (AMLR) of MoDCs were detected in patients with hepatitis B-related ACLF. Results The number of circulating mDC decreased only in patients with hepatitis B-related ACLF compared with that in normal controls. However, pDC numbers decreased in both CHB and ACLF patients. We observed a further decrease the pDC numbers in ACLF compared to CHB patients without statistical significance (P>0.05). MoDC from ACLF patients showed lower expression of costimulatory molecules CD80, CD86 and the mature marker CD83, as well as MHC Ⅱ molecule (HLA-DR) compared to CHB and NC group. Interestingly, MoDC impaired allostimulatory mixed lymphocyte reaction from ACLF patients compared to those in CHB patients and NCs. Conclusions Patients with hepatitis B-related ACLF have a significantly lower expression of surface markers and impaired AMLR of MoDC, as well as decreased number of circulating mDC and pDC, which may be partially related to HBV disease progression in these patients. Key words: Liver failure, acute/ME; Dendritic cells/ME

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive expression of families of related receptors, a broad range of related ligands and the difficulty of dealing with primary cultures of specialized endothelial cells. Here we describe a bioassay for the detection of ligands to the vascular endothelial growth factor receptor-2 (VEGFR-2), a key transducer of signals that promote angiogenesis and lymphangiogenesis. A cDNA encoding a fusion of the extracellular (ligand-binding) region of VEGFR-2 with the transmembrane and cytoplasmic regions of the erythropoietin receptor (EpoR) is expressed in the factor-dependent cell line Ba/F3. This cell line grows in the presence of interleukin-3 (IL-3) and withdrawal of this factor results in death of the cells within 24 hr. Expression of the VEGFR-2/EpoR receptor fusion provides an alternative mechanism to promote survival and potentially proliferation of stably transfected Ba/F3 cells in the presence of a ligand capable of binding and cross-linking the extracellular portion of the fusion protein (i.e., one that can cross-link the VEGFR-2 extracellular region). The assay can be performed in two ways: a semi-quantitative approach in which small volumes of ligand and cells permit a rapid result in 24 hr, and a quantitative approach involving surrogate markers of a viable cell number. The assay is relatively easy to perform, is highly responsive to known VEGFR-2 ligands and can accommodate extracellular inhibitors of VEGFR-2 signaling such as monoclonal antibodies to the receptor or ligands, and soluble ligand traps.