Modulation Of Cytokine Production Research Articles

Dydrogesterone and the immunology of pregnancy.

Progesterone is indispensable for the maintenance of pregnancy, both via its endocrine effects and its role in creating a favorable immunological environment for the fetus. This review focuses on the immunological effects of progesterone. Progestogens have been shown to have very interesting effects on cytokine production and decidual natural killer (NK) cell activity. The orally-administered progestogen, dydrogesterone, has the ability to modulate cytokine production patterns in a manner that could be conducive to successful pregnancy. The adverse effects of progesterone deficiency and the beneficial effects of progesterone supplementation in pregnancy pathologies will be discussed.

Interleukin-30 (IL27p28) alleviates experimental sepsis by modulating cytokine profile in NKT cells

Sepsis is an acute systemic inflammatory response to infection associated with high patient mortality (28-40%). We hypothesized that interleukin (IL)-30, a novel cytokine protecting mice against liver injury resulting from inflammation, would generate a protective effect against systemic inflammation and sepsis-induced death. Sepsis was induced by lipopolysaccharide (LPS) or cecal ligation and puncture (CLP). The inhibitory effects of IL-30 on septic inflammation and associated therapeutic effects were determined in wild-type, IL30 (p28)(-/-), IL10(-/-), and CD1d(-/-) mice. Mice treated with pIL30 gene therapy or recombinant IL-30 protein (rIL30) were protected from LPS-induced septic shock or CLP-induced polymicrobial sepsis and showed markedly less liver damage and lymphocyte apoptosis than control septic mice. The resulting reduction in mortality was mediated through attenuation of the systemic pro-inflammatory response and augmentation of bacterial clearance. Mice lacking IL-30 were more sensitive to LPS-induced sepsis. Natural killer-like T cells (NKT) produced much higher levels of IL-10 and lower levels of interferon-gamma and tumor necrosis factor-alpha in IL-30-treated septic mice than in control septic mice. Likewise, deficiency in IL-10 or NKT cells abolished the protective role of IL-30 against sepsis. Furthermore, IL-30 induced IL-10 production in purified and LPS-stimulated NKT cells. Blocking IL-6R or gp130 inhibited IL-30 mediated IL-10 production. IL-30 is important in modulating production of NKT cytokines and subsequent NKT cell-mediated immune regulation of other cells. Therefore, IL-30 has a role in prevention and treatment of sepsis via modulation of cytokine production by NKT.

Putative Roles of SOCS1: SOCS 3 Ratios in Development and Resolution of a Herpesvirus Lesion

This article is the author’s opinion of the effects of the cytokines produced as a result of epithelial cell macrophage interactions during herpes simplex virus type 1(HSV-1) infection. Cell cultures of murine keratinocytes were examined for SOCS1 production following HSV-1 infection and macrophage cell lines were examined for cytokine production following polarization and modification of cytokine production by peptide mimetics of suppressors of the cytokine signaling (SOCS) molecules SOCS1 and SOCS3. It is speculated that the pro inflammatory cytokines produced by M1 polarized cells enhance the inflammatory nature of the lesion produced by HSV-1 and the anti-inflammatory cytokines of the M2 polarized cells promote resolution of the lesion.

Dimethyltryptamine (DMT): a biochemical Swiss Army knife in neuroinflammation and neuroprotection?

Dimethyltryptamine (DMT): a biochemical Swiss Army knife in neuroinflammation and neuroprotection?

Vitamin B6 Modifies the Immune Cross-Talk between Mononuclear and Colon Carcinoma Cells

The role of vitamin B6 as a key component in a number of biological events has been well established. Based on the relationship between chronic inflammation and carcinogenesis on the one hand, and the interaction between immune and cancer cells expressed by modulated cytokine production on the other hand, the aim of the present work was to examine the possibility that vitamin B6 affects cancer development by an interference in the cross-talk between human peripheral blood mononuclear cells (PBMC) and those from two colon carcinoma cell lines. Both non-stimulated PBMC and mononuclear cells induced for cytokine production by HT-29 and RKO cells from human colon carcinoma lines were incubated without and with 4, 20 and 100 μg/ml of pyridoxal hydrochloride (vitamin B6) and secretion of TNF-α, IL-1β, IL-6, IFN-γ, IL-10, and IL-1ra was examined. Vit B6 caused a dose-dependent decrease in production of all cytokines examined, except for that of IL-1ra. The results indicate that vitamin B6 exerts an immunomodulatory effect on human PBMC. The finding that production of inflammatory cytokines is more pronounced when PBMC are in contact with malignant cells and markedly inhibited by the vitamin suggests an additional way by which vitamin B6 may exert its carcinopreventive effect.

Modulation of Cytokine Production and Transcription Factors Activities in Human Jurkat T Cells by Thymol and Carvacrol.

Thymol and carvacrol, two main components of thyme, have shown anti-inflammatory effects. The aim of this study was to assess the effects of these components on Jurkat leukemia cells as an in vitro T cell model and their molecular mechanisms of activity. Cells were cultured in the presence of components and subsequently stimulated with phorbol-12-myristate-13-acetate (PMA)/calcium ionophore for evaluating interleukin (IL)-2 and interferon (IFN)-γ production. The activation of T cell transcription factors that included nuclear factors of activated T cells (NFATs), activator protein-1 (AP-1; c-Jun/c-Fos), and nuclear factor (NF)-κB were examined by Western blot analysis. Thymol and carvacrol at 25 µg/ml significantly reduced IL-2 levels from 119.4 ± 8pg/ml in control cells treated only with PMA/Calcium ionophore and the solvent to 66.9 ± 6.4pg/ml (thymol) and 32.3 ± 3.6pg/ml (carvacrol) and IFN-γ from 423.7 ± 19.7pg/ml in control cells to 311.9 ± 11.6pg/ml (thymol) and 293.5 ± 16.7pg/ml (carvacrol). Western blot analyses of nuclear extracts showed that the same concentrations of components significantly reduced NFAT-2 to 44.2 ± 2.7% (thymol) and 91.4 ± 2.3% (carvacrol) of the control (p<0.05), and c-Fos to 31.2 ± 6.2% (thymol) and 27.6 ± 3.1% (carvacrol) of the control (p<0.01). No effects on NFAT-1, c-Jun and phospho-NF-κBp65 levels were observed. Thymol and carvacrol could contribute to modulation of T cell activity by reducing IL-2 and IFN-γ production possibly through down regulation of AP-1 and NFAT-2 transcription factors suggesting their potential usefulness for reduction of T cell overactivity in immune-mediated diseases.

Electric fields are novel determinants of human macrophage functions.

Macrophages are key cells in inflammation and repair, and their activity requires close regulation. The characterization of cues coordinating macrophage function has focused on biologic and soluble mediators, with little known about their responses to physical stimuli, such as the electrical fields that are generated naturally in injured tissue and which accelerate wound healing. To address this gap in understanding, we tested how properties of human monocyte-derived macrophages are regulated by applied electrical fields, similar in strengths to those established naturally. With the use of live-cell video microscopy, we show that macrophage migration is directed anodally by electrical fields as low as 5 mV/mm and is electrical field strength dependent, with effects peaking ∼300 mV/mm. Monocytes, as macrophage precursors, migrate in the opposite, cathodal direction. Strikingly, we show for the first time that electrical fields significantly enhance macrophage phagocytic uptake of a variety of targets, including carboxylate beads, apoptotic neutrophils, and the nominal opportunist pathogen Candida albicans, which engage different classes of surface receptors. These electrical field-induced functional changes are accompanied by clustering of phagocytic receptors, enhanced PI3K and ERK activation, mobilization of intracellular calcium, and actin polarization. Electrical fields also modulate cytokine production selectively and can augment some effects of conventional polarizing stimuli on cytokine secretion. Taken together, electrical signals have been identified as major contributors to the coordination and regulation of important human macrophage functions, including those essential for microbial clearance and healing. Our results open up a new area of research into effects of naturally occurring and clinically applied electrical fields in conditions where macrophage activity is critical.

Human umbilical vein endothelial cells promote the inhibitory activation of CD4+CD25+Foxp3+ regulatory T cells via PD-L1

BackgroundAtherosclerosis (AS) is a chronic inflammation characterized by massive infiltration of inflammatory cells in arterial wall plaques. Programmed death ligand-1 (PD-L1), a co-stimulatory molecule, plays a vital role in regulating immune responses. We investigated the role and mechanisms of PD-L1 expressed on oxidized low-density lipoprotein (ox-LDL)-impaired human umbilical vein endothelial cells (HUVECs) in promoting activation and cytokine production of CD4+CD25+ forkhead box P3 (FoxP3) regulatory T cells (Tregs). Methods and resultsTregs were incubated alone, with HUVECs or HUVECs pre-stimulated with ox-LDL in the presence of anti-CD3 monoclonal antibodies (mAbs) for 48 h. HUVECs were shown to upregulate the immune phenotypic markers of Tregs, such as glucocorticoid-induced TNF receptor (GITR), cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed cell death-1 protein (PD-1). Moreover, HUVECs modulated cytokine production of Tregs (e.g., interleukin-10 (IL-10) and transforming growth factor-β1 (TGF-β1)). HUVECs treated with anti-PD-L1 mAbs were unable to regulate the surface expression and cytokine production of Tregs. The Transwell culture system suggested that interaction between HUVECs and Tregs via PD-L1 requires cell-to-cell contact. ConclusionExpression of the negative co-stimulatory molecule PD-L1 on HUVECs may upregulate the inhibitory activation and cytokine production of CD4+CD25+Foxp3+ regulatory T cells in AS.

Role of TLR gene expression and cytokine profiling in the immunopathogenesis of viral hepatitis E

BackgroundThe clinical manifestations of Hepatitis E virus (HEV) range from self-limiting acute viral hepatitis (AVH) to acute liver failure (ALF). The varied clinical course is thought to be immune-mediated. Toll-like receptors (TLRs) play a central role in sensing and initiating innate antiviral-response and downstream signaling of TLRs modulates cytokine production, thereby playing an important role in determining the disease course. ObjectivesThe present study was designed to elucidate the role of TLRs and cytokine production in the immunopathogenesis of HEV. Study designPeripheral blood mono-nuclear cells were separated from 50 AVH-HEV, 30 ALF-HEV patients and 50 healthy-controls. One-part of the PBMC was processed for RNA-extraction another pulsed with HEV-ORF2-peptide. Gene-expression levels of TLR (2–4, 7, and 8) were checked using semi-quantitative Real-time-PCR. Cytokine levels were analyzed using Cytokine-Bead-Array. TLR3-silencing experiments were performed and post-silencing cytokine levels were estimated. ResultsTLR3 gene-expression in AVH was significantly higher than ALF (202.4±36.36 Vs 13.71±5.01; p<0.0001). Higher amount of both anti-and pro-inflammatory cytokines; IFNγ, TNF-α, IL10 and TGF-β were detected in the PBMC culture-supernatant of AVH Vs ALF (p<0.0001, p=0.0008, p=0.0002, p<0.0001 respectively). Post-silencing TLR3, significant decrease in IFNγ level was observed in the PBMC culture-supernatant (4.08±1.06 Vs 23.20±12.51; p=N0.0213). ConclusionsTLR3 and IFNγ were found to play an important role in HEV disease pathogenesis. Patients capable of expressing high levels of TLR 3 and robust IFNγ response are able to limit the disease and recover uneventfully; while the patients with lower expression of TLR3 and IFNγ progress to ALF.

A novel mannoside-glycocluster adjuvant: Compared in vitro to CpG ODN and MPL and tested in vivo in mouse asthma model

BackgroundAllergen-specific immunotherapy balances the Th2-biased immunity towards Th1 and Treg responses. Adjuvants are used in allergen preparations to intensify the immune responses. The increased prevalence of allergies in developed societies has been associated with decreased microbial load during childhood. This has initiated a search for microbial structures to be used as adjuvants. Our study has shown that a synthetic triacedimannose (TADM) may suppress the Th2-type allergic inflammatory response. The aim of this study was to compare the properties of TADM with capacities of other adjuvants, CpG ODN and MPL, to modulate cytokine production in PBMC and regulate sensitisation in an OVA-sensitised mouse asthma model. MethodsThe effects of TADM were studied in vitro on birch stimulated PBMC cultures of birch allergic rhinitis patients with other known adjuvants. Cytokines in supernatants were measured by Luminex. Effects of TADM were analysed in vivo in a mouse model of OVA-induced allergic asthma by analysing BAL, cytokine mRNA and serum antibodies. ResultsTADM was the only adjuvant that significantly suppressed the production of all birch induced Th2-type cytokines. In a murine model, TADM significantly suppressed the specific IgE production and enhanced IFN-γ production. ConclusionsTADM suppresses the birch allergen induced Th2-type cytokine responses in allergic subjects more efficiently than the two other adjuvants, MPL and CpG ODN. TADM is immunomodulatory also in vivo and decreases the IgE levels and increases the IFN-γ responses in a murine model. These results suggest that TADM may be a promising candidate for novel adjuvants in immunotherapy.

Vpr Enhances Tumor Necrosis Factor Production by HIV-1-Infected T Cells.

The HIV-1 accessory protein Vpr displays different activities potentially impacting viral replication, including the arrest of the cell cycle in the G2 phase and the stimulation of apoptosis and DNA damage response pathways. Vpr also modulates cytokine production by infected cells, but this property remains partly characterized. Here, we investigated the effect of Vpr on the production of the proinflammatory cytokine tumor necrosis factor (TNF). We report that Vpr significantly increases TNF secretion by infected lymphocytes. De novo production of Vpr is required for this effect. Vpr mutants known to be defective for G2 cell cycle arrest induce lower levels of TNF secretion, suggesting a link between these two functions. Silencing experiments and the use of chemical inhibitors further implicated the cellular proteins DDB1 and TAK1 in this activity of Vpr. TNF secreted by HIV-1-infected cells triggers NF-κB activity in bystander cells and allows viral reactivation in a model of latently infected cells. Thus, the stimulation of the proinflammatory pathway by Vpr may impact HIV-1 replication in vivo. The role of the HIV-1 accessory protein Vpr remains only partially characterized. This protein is important for viral pathogenesis in infected individuals but is dispensable for viral replication in most cell culture systems. Some of the functions described for Vpr remain controversial. In particular, it remains unclear whether Vpr promotes or instead prevents proinflammatory and antiviral immune responses. In this report, we show that Vpr promotes the release of TNF, a proinflammatory cytokine associated with rapid disease progression. Using Vpr mutants or inhibiting selected cellular genes, we show that the cellular proteins DDB1 and TAK1 are involved in the release of TNF by HIV-infected cells. This report provides novel insights into how Vpr manipulates TNF production and helps clarify the role of Vpr in innate immune responses and inflammation.

Modulation of Cytokine Production by the Dydrogesterone Metabolite Dihydrodydrogesterone.

Previous studies have shown that dydrogesterone, the orally administered progestogen, upregulates the production of Th2 cytokines and downregulates Th1 cytokine production. This study was designed to ascertain whether dihydrodydrogesterone (DHD), the major metabolite of dydrogesterone, is similarly capable of modulating cytokine production by peripheral blood mononuclear cells (PBMC) from women with a history of unexplained recurrent miscarriage. Mitogen-stimulated PBMC from women with unexplained recurrent miscarriage were exposed to progesterone or dydrogesterone or DHD, and the levels of pro-inflammatory (IFN-γ, TNF-α) and anti-inflammatory (IL-4, IL-10, IL-13) cytokines were estimated by ELISA. To ascertain whether DHD mediates its effects via the progesterone receptor, RU486, a progesterone agonist, was added to cultures along with mitogen and DHD. The metabolite DHD, like its parent molecule dydrogesterone, suppresses the production of the pro-inflammatory cytokines IFN- γ and TNF-α and upregulates the production of the anti-inflammatory cytokine IL-4. The progesterone antagonist RU486 reverses the effect of DHD, suggesting that this molecule mediates its cytokine-modulating effect via the progesterone receptor. Dihydrodydrogesterone retains the immunomodulatory effects of the progestogen dydrogesterone by bringing about a shift in cytokine production profiles that might be conducive to the success of pregnancy.

CXCL8 and CCL20 Enhance Osteoclastogenesis via Modulation of Cytokine Production by Human Primary Osteoblasts.

Generalized osteoporosis is common in patients with inflammatory diseases, possibly because of circulating inflammatory factors that affect osteoblast and osteoclast formation and activity. Serum levels of the inflammatory factors CXCL8 and CCL20 are elevated in rheumatoid arthritis, but whether these factors affect bone metabolism is unknown. We hypothesized that CXCL8 and CCL20 decrease osteoblast proliferation and differentiation, and enhance osteoblast-mediated osteoclast formation and activity. Human primary osteoblasts were cultured with or without CXCL8 (2–200 pg/ml) or CCL20 (5–500 pg/ml) for 14 days. Osteoblast proliferation and gene expression of matrix proteins and cytokines were analyzed. Osteoclast precursors were cultured with CXCL8 (200 pg/ml) and CCL20 (500 pg/ml), or with conditioned medium (CM) from CXCL8 and CCL20-treated osteoblasts with or without IL-6 inhibitor. After 3 weeks osteoclast formation and activity were determined. CXCL8 (200 pg/ml) and CCL20 (500 pg/ml) enhanced mRNA expression of KI67 (2.5–2.7-fold), ALP (1.6–1.7-fold), and IL-6 protein production (1.3–1.6-fold) by osteoblasts. CXCL8-CM enhanced the number of osteoclasts with 3–5 nuclei (1.7-fold), and with >5 nuclei (3-fold). CCL20-CM enhanced the number of osteoclasts with 3–5 nuclei (1.3-fold), and with >5 nuclei (2.8-fold). IL-6 inhibition reduced the stimulatory effect of CXCL8-CM and CCL20-CM on formation of osteoclasts. In conclusion, CXCL8 and CCL20 did not decrease osteoblast proliferation or gene expression of matrix proteins. CXCL8 and CCL20 did not directly affect osteoclastogenesis. However, CXCL8 and CCL20 enhanced osteoblast-mediated osteoclastogenesis, partly via IL-6 production, suggesting that CXCL8 and CCL20 may contribute to osteoporosis in rheumatoid arthritis by affecting bone cell communication.

Combined immunomodulator and antimicrobial therapy eliminates polymicrobial sepsis and modulates cytokine production in combined injured mice

Purpose: A combination therapy for combined injury (CI) using a non-specific immunomodulator, synthetic trehalose dicorynomycolate and monophosphoryl lipid A (STDCM-MPL), was evaluated to augment oral antimicrobial agents, levofloxacin (LVX) and amoxicillin (AMX), to eliminate endogenous sepsis and modulate cytokine production.Materials and methods: Female B6D2F1/J mice received 9.75 Gy cobalt-60 gamma-radiation and wound. Bacteria were isolated and identified in three tissues. Incidence of bacteria and cytokines were compared between treatment groups.Results: Results demonstrated that the lethal dose for 50% at 30 days (LD50/30) of B6D2F1/J mice was 9.42 Gy. Antimicrobial therapy increased survival in radiation-injured (RI) mice. Combination therapy increased survival after RI and extended survival time but did not increase survival after CI. Sepsis began five days earlier in CI mice than RI mice with Gram-negative species predominating early and Gram-positive species increasing later. LVX plus AMX eliminated sepsis in CI and RI mice. STDCM-MPL eliminated Gram-positive bacteria in CI and most RI mice but not Gram-negative. Treatments significantly modulated 12 cytokines tested, which pertain to wound healing or elimination of infection.Conclusions: Combination therapy eliminates infection and prolongs survival time but does not assure CI mouse survival, suggesting that additional treatment for proliferative-cell recovery is required.

Lenalidomide reduces microglial activation and behavioral deficits in a transgenic model of Parkinson's disease.

BackgroundParkinson’s disease (PD) is one of the most common causes of dementia and motor deficits in the elderly. PD is characterized by the abnormal accumulation of the synaptic protein alpha-synuclein (α-syn) and degeneration of dopaminergic neurons in substantia nigra, which leads to neurodegeneration and neuroinflammation. Currently, there are no disease modifying alternatives for PD; however, targeting neuroinflammation might be a viable option for reducing motor deficits and neurodegeneration. Lenalidomide is a thalidomide derivative designed for reduced toxicity and increased immunomodulatory properties. Lenalidomide has shown protective effects in an animal model of amyotrophic lateral sclerosis, and its mechanism of action involves modulation of cytokine production and inhibition of NF-κB signaling.MethodsIn order to assess the effect of lenalidomide in an animal model of PD, mThy1-α-syn transgenic mice were treated with lenalidomide or the parent molecule thalidomide at 100 mg/kg for 4 weeks.ResultsLenalidomide reduced motor behavioral deficits and ameliorated dopaminergic fiber loss in the striatum. This protective action was accompanied by a reduction in microgliosis both in striatum and hippocampus. Central expression of pro-inflammatory cytokines was diminished in lenalidomide-treated transgenic animals, together with reduction in NF-κB activation.ConclusionThese results support the therapeutic potential of lenalidomide for reducing maladaptive neuroinflammation in PD and related neuropathologies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-015-0320-x) contains supplementary material, which is available to authorized users.

Butyrate Alters mRNA and Protein Levels of Tumorigenic Chemokines in U937 Leukemic Cells

Butyrate (NaB) is a HDAC inhibitor implicated as a potential therapy for cancer. Due to its ability to promote gamma globin, low concentrations (&lt;1.5 mM) are used for treatment of hemoglobin disorders. Conversely, high concentrations (&gt;1.5 mM) activate apoptosis in several cancer cell lines. Low concentrations of NaB influence signaling pathways that modulate cytokine production. However, it is not known whether high concentrations alter these pathways thereby regulating cytokine expression in cancer cells and altering the microenvironment, which was the purpose of this study. We exposed leukemia cells to high concentrations of NaB and analyzed for apoptosis, cytokine mRNA and protein levels, and chemotaxis. Our results show NaB induced activation of caspase‐3, but significantly decreased chemokine CCL2 and CCL5 mRNA and protein levels. Moreover, monocyte migration towards culture media from NaB‐treated cells significantly decreased. To elucidate the signaling mechanism, we investigated if NaB alters activation of AKT, MAPK, and Jak proteins. NaB induced p38, but suppressed ERK and AKT. NaB did not influence JNK or Jak proteins. Pretreatment with a p38 inhibitor abolished NaB‐induced reduction in CCL5; however, this was not observed in CCL2. This data suggests that while promoting apoptosis, NaB influences the cancer microenvironment by decreasing chemokine production via the MAPK pathway. These effects may influence the biology of normal and cancer cells thereby altering the status of the patient receiving NaB therapy. Ms Pulliam is supported by NHLBI T32 Pre‐doctoral training grant 2 T32 HL007735‐16 to Dr. Adunyah and CTSA award UL1TR000445 from the NCATS. Dr. Adunyah is supported by NCI U54 grant 5 U54 CA163069‐02 and NIMHD MeTRC grant 8 U54 MD007593‐04.

Mo1739 LPS Stimulated Whole Blood Cytokine Production and the Association With Disease Characteristics in Patients With Quiescent Crohn's Disease

Introduction: Individual cytokine production is for 60-70% under genetic control, but levels of secreted cytokines after stimulation with lipopolysaccharide (LPS) vary widely between individuals. In various fields of inflammatory diseases the variation in LPS stimulated cytokine production has been linked with disease behavior. So far, studies in patients with Crohn's disease (CD) used single nucleotide polymorphisms (SNPs) as surrogate marker to identify patients with a high innate cytokine production, but it may be more accurate to measure cytokine production itself for this purpose. Our aim was to measure levels of tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, IL-6 and IL-10 after whole blood stimulation with LPS and correlate this with historical disease characteristics of patients with CD who are currently in remission. Methods: Only CD patients in remission and not on immunosuppressants or biologicals, were eligible for participation as drugs or disease activity can modulate cytokine production. Remission was defined as Harvey-Bradshaw Index <5, with normal C-reactive protein (<10 mg/L) and leukocyte levels. Whole blood samples were incubated with E.coli O55:B5 LPS (1 ng/ml) for 4 hours (TNF-α) or 24 hours (IL-1β, IL6 and IL-10). Cytokine levels were determined in supernatants with specific enzyme-linked immunosorbent assays (ELISAs). Non-parametric statistics were used to test correlations between cytokine levels and historical disease characteristics. Results: Sixty-nine patients (50 female / 19 male) were included. Mean disease duration at inclusion was 24.1 (SD= 11.5) years and the average age was 51.2 (SD=12.3) years. No relation was found between disease localization (ileal vs. colon vs. ileocolonic) or disease behavior (presence of fistulas or strictures) and levels of TNF-α, IL-1β, IL-6 and IL-10. Additional analyses were performed to compare mild and severe disease based on phenotype (ileal disease without fistulas versus ileocolonic disease with fistulas), number of bowel resections and percentage of years in which the patient received immunosuppressants or biologicals. A significant difference (p= 0.04) for IL-6 was found between patients with a single (median 2258 pg/ml, interquartile range [1630-2726]) and those with multiple resections (2816 pg/ml [1851-3531]) (see Table). Conclusion: In conclusion, our results show a relation between IL-6 levels and the number of bowel resections. Altogether, the use of LPS stimulated cytokine production as a biomarker to predict disease behavior in CD seems limited. Relation between cytokine production and disease severity.

Linezolid, vancomycin and daptomycin modulate cytokine production, Toll-like receptors and phagocytosis in a human in vitro model of sepsis.

Conventional antibiotics exhibit immunomodulatory properties beneficial in the treatment of sepsis. Antibiotic-resistant Gram-positive bacteria have become a problem in sepsis therapy, giving rise to increased use of last-resort antibiotics; for example, linezolid (LIN), vancomycin (VAN) and daptomycin (DAP). As the immunomodulatory properties of these antibiotics in treating sepsis are unknown, this study examined the effect of VAN, LIN and DAP on the immune response under sepsis-like conditions in vitro. Lipopolysaccharide (LPS)-activated THP-1 monocytes were incubated with LIN, VAN or DAP. Gene expression of cytokines (TNFα, IL-1β, IL-6, IL-10) and Toll-like receptors (TLR1, 2, 4, 6, 7 and 9) was monitored and phagocytosis was determined following coincubation with E. coli. The antibiotics differentially modulated the gene expression of the investigated cytokines. While LIN and VAN upregulated the expression of all TLRs, DAP downregulated mRNA levels of TLR1, TLR2 and TLR6, which recognize pathogen-associated molecular patterns from Gram-positive bacteria. In addition, LIN inhibited, whereas VAN promoted the phagocytic activity of monocytes. Our results suggest that LIN and VAN possess pro-inflammatory properties, whereas DAP might reduce the immune response to Gram-positive bacteria in sepsis. Furthermore, VAN might be beneficial in the prevention of Gram-negative infections by increasing the phagocytosis of E. coli.

In vivo assessment of the therapeutic effects of lyophilized leech saliva extract from (Huridinaria manillensis) on LNCaP tumor xenograft model in nude mice.

271 Background: Ancient traditional physicians from many countries used leeching to treat a wide range of diseases for thousands of years. A large number of peptides and proteins have been identified and characterized in leech saliva extract (LSE), including anti-thrombotic agents, cancer metastasis inhibitors and anti-microbials Currently, leech therapy is established as an important tool in microsurgery and reconstructive operations having demonstrated superior clinical outcomes for the optimal salvage of grafted tissues. Methods: In the current study, we have determined the in vivo efficacy of LSE from (Huridinaria manillensis) on castration resistant LNCaP xenograft mouse model. Mice were divided into three groups of six, mice were subcutaneously injected with either LSE (5 mg/kg), docetaxel (10 mg/kg), or vehicle once a week. PSA and tumor volume were measured weekly. After four weeks of treatment, mice were euthanized, tumors and organs were collected for transcriptome and immunohistochemical (IHC) analysis. Results: There was a significant decrease in the tumor volume and PSA with either docetaxel (10mg/kg) or LSE (5 mg/kg) treated groups compared to the control. While there was no significant difference between the anti-tumor activity of docetaxel (10mg/kg) and LSE (5 mg/kg). IHC showed significant increase in caspase-3 and significant decrease in Ki-67 and PCNA expression in the LSE treated mice compared to the control group. Interestingly, transcriptome analysis of tumor samples showed that LSE modulated cytokine production, monocyte adhesion, steroidogenesis, and P38 MAPK signaling pathways. Conclusions: LSE has significant anti-tumor activity in LNCaP tumor xenograft models with no apparent side effects. This can be attributed, at least partly, to its inhibition of cellular proliferation, induction of apoptosis, modulation of immunity and steroidogenesis.

Regulatory T cell levels and cytokine production in active non-infectious uveitis: in-vitro effects of pharmacological treatment.

The aim of this study was to quantify the proportion of regulatory T cells (Treg ) and cytokine expression by peripheral blood mononuclear cells (PBMCs) in patients with active non-infectious uveitis, and to evaluate the effect of in-vitro treatment with infliximab, dexamethasone and cyclosporin A on Treg levels and cytokine production in PBMCs from uveitis patients and healthy subjects. We included a group of 21 patients with active non-infectious uveitis and 18 age-matched healthy subjects. The proportion of forkhead box protein 3 (FoxP3)(+) Treg cells and intracellular tumour necrosis factor (TNF)-α expression in CD4(+) T cells was determined by flow cytometry. PBMCs were also either rested or activated with anti-CD3/anti-CD28 and cultured in the presence or absence of dexamethasone, cyclosporin A and infliximab. Supernatants of cultured PBMCs were collected and TNF-α, interleukin (IL)-10, IL-17 and interferon (IFN)-γ levels were measured by enzyme-linked immunosorbent assay (ELISA). No significant differences were observed in nTreg levels between uveitis patients and healthy subjects. However, PBMCs from uveitis patients produced significantly higher amounts of TNF-α and lower amounts of IL-10. Dexamethasone treatment in vitro significantly reduced FoxP3(+) Treg levels in PBMCs from both healthy subjects and uveitis patients, and all tested drugs significantly reduced TNF-α production in PBMCs. Dexamethasone and cyclosporin A significantly reduced IL-17 and IFN-γ production in PBMCs and dexamethasone up-regulated IL-10 production in activated PBMCs from healthy subjects. Our results suggest that PBMCs from patients with uveitis express more TNF-α and less IL-10 than healthy subjects, and this is independent of FoxP3(+) Treg levels. Treatment with infliximab, dexamethasone and cyclosporin A in vitro modulates cytokine production, but does not increase the proportion of FoxP3(+) Treg cells.