Increase In IL-10 Production Research Articles

Poster 106: Commonly Used Orthopaedic Clinical Therapeutics Affect the Cytokine Activity of Culture-Expanded Mesenchymal Stem Cells

Objectives:Hyaluronic acid (HA), platelet-rich plasma (PRP), and methylprednisolone are widely used for the non-operative management of osteoarthritis (OA). More recently, human bone marrow derived mesenchymal stem cells (BM-MSCs) have garnered significant interest as a non-operative means for symptom modification of OA. Anti-inflammatory cytokine secretion is a major component of proposed BM-MSC action. We sought to describe the effect of HA, PRP, and methylprednisolone on the cytokine profile of BM-MSCs at multiple stages of culture expansion.Methods:Following Institutional Review Board and United States Food and Drug Administration approval, nine BM-MSC cell lines from 4 human donors underwent three stages of culture expansion: passage 2 (P2), passage 3 (P3), and passage 4 (P4). Levels of OA-related cytokines (IL-1β, IL-6, IL-8, IL-10, Stem cell Factor [SCF], Stem Cell Derived Factor-alpha [SDF-α]) were measured using Luminex multiplexing technology. Each of the BM-MSC preparations were evaluated at each passage, 24 hours following exposure to HA, PRP, or methylprednisolone. Paired T-tests and Wilcoxon signed-rank testing were performed as appropriate following assessment of normality. Significance was set at p<0.05.Results:Culture expansion significantly altered cytokine production, however treatment with current OA therapeutics further altered these responses implicating the potential of combined treatment of culture expanded hMSCs and OA therapeutics. Figure 1 demonstrates the behavior of selected cytokines after exposure to HA, PRP or methylprednisolone preparations. Further, we tested different concentrations of each of the therapeutics to determine optimal dosing. Exposure to increasing doses of HA led to reductions in BM-MSC expression of SCF, SDF-α, VEGF, CCL20, and adiponectin (p<0.05 for all). PRP increased IL-1 levels in P2 and P3 (p<0.05 for both), IL-6 in P2 (100 pg/mL vs 5500 pg/mL, p<0.001), and IL-8 in P3 (900 pg/mL vs 3900 pg/mL, p<0.001). PRP did not affect IL-10 expression (p>0.05). Aside from an increase in IL-6 production for P2 (200 pg/mL vs 1500 pg/mL, p<0.05), methylprednisolone did not have significant effects on cytokine expression.Conclusions:Orthopaedic therapeutic adjuvants influence the inflammatory cytokine profile of BM-MSCs at multiple stages of culture expansion. Although further study is needed, these therapeutics have potential to be used in tandem with BM-MSCs to create an optimal cytokine environment for treatment of degenerative joint disease.Figure 1.hMSCs and Osteoarthritis Adjuvants. Human bone marrow derived hMSCs were culture expanded over 4 passages and treated with hyaluronan (HA), platelet rich plasma (PRP) and methylprednisolone (DEPO). Culture expansion significantly altered cytokine production, however treatment with current OA therapeutics further altered these responses implicating the potential of combined treatment of culture expanded hMSCs with OA therapeutics.

The Immunomodulating Effect of Phlorotannins from a Brown Alga, Eisenia nipponica, on Mice Stimulated with Ovalbumin through T Cell Regulation.

The immunomodulating effect of phlorotannin was investigated in mice stimulated by ovalbumin. When analyzing the main components of phlorotannin concentrate (PTC) from Eisenia nipponica, seven phlorotannins [eckol, 6,6'-bieckol, 6,8'-bieckol, 8,8'-bieckol, dieckol, phlorofucofuroeckol (PFF)-A, and PFF-B] were detected. These phlorotannins accounted for approximately 80% of PTC. Oral administration of PTC to mice daily for 21days reduced serum immunoglobulin E (IgE) and total IgG1 levels attributable to Th2 cells. The production of splenic cytokines [interleukin (IL)-10 and transforming growth factor-β1] and Treg cell-mediated expression of forkhead box protein P3 mRNA were significantly increased whereas the production of inflammatory cytokines (interferon-γ, IL-4, IL-5, and IL-17) by Th1, Th2, and Th17 cells was markedly suppressed. IL-21 production and basic leucine zipper ATF-like transcription factor mRNA expression attributable to follicular helper T (Tfh) cells were also suppressed. Flow cytometric analyses demonstrated increased number of Treg cells despite a decrease in the total T cell population. An increase in total B cells was also observed by the flow cytometric analyses in addition to increases in IL-10 production, which activates B cells. In contrast, the significantly suppressed production of inflammatory cytokines and moderate increase in Treg cell subpopulation indicated a direct impact of PTC on inflammatory lymphocytes (Th1, Th2, Th17, and Tfh). Thus, PTC may exert antiallergic effects by immunomodulation of T cells and inactivation of inflammatory lymphocyte.

Targeting IRAK3 for Degradation to Enhance IL-12 Pro-inflammatory Cytokine Production.

Interleukin-1 receptor-associated kinase 3 (IRAK3) is a pseudokinase mediator in the human inflammatory pathway, and ablation of its function is associated with enhanced antitumor immunity. Traditionally, pseudokinases have eluded "druggability" and have not been considered tractable targets in the pharmaceutical industry. Herein we disclose a CRISPR/Cas9-mediated knockout of IRAK3 in monocyte-derived dendritic cells that results in an increase in IL-12 production upon lipopolysaccharide (LPS) stimulation. Furthermore, we disclose and characterize Degradomer D-1, which displays selective proteasomal degradation of IRAK3 and reproduces the 1L-12p40 increases observed in the CRISPR/Cas9 knockout.

Increased Endogenous Production of Pro‐inflammatory Adipokines by 3T3‐L1 Adipocytes Differentiated with Arachidonic Acid

Inflammatory factors derived from adipose tissue (AT) can set the stage for chronic, low‐grade inflammation. This meta‐inflammation can predispose the individual to further inflammatory‐based disorders and diseases. Polyunsaturated fatty acids (PUFAs) are known to have immune modulating effects, with omega‐6 (n‐6) PUFAs typically being associated with increased production of inflammatory molecules, and n‐3 PUFAs having been shown to be anti‐inflammatory, in both in vivo and in vitro studies. The 3T3‐L1 cell line is an embryonic mouse fibroblast line that can be differentiated into an adipocyte phenotype making it a suitable line for studies on the effects of PUFAs on inflammatory molecule production by AT. The aim of this research was to determine the effects of n‐6 and n‐3 PUFAs on the release of inflammatory adipokines in lipopolysaccharide (LPS)‐challenged and LPS‐unchallenged adipocytes. Fibroblasts were differentiated into adipocytes with the addition of 100 μM of linoleic acid (LA; n‐6), arachidonic acid (ARA; n‐6) or eicosapentaenoic acid (EPA; n‐3) throughout the differentiation period, followed by a 6 h, 1 μg/ml LPS challenge. Cell‐conditioned media was collected before and after the LPS challenge for interleukin (IL)‐6 and prostaglandin E2 (PGE2) quantification via enzyme‐linked immunosorbent assays. Differentiation of adipocytes with n‐6 ARA caused a significant increase in PGE2 levels compared to control cells, or cells differentiated with LA or EPA, both after LPS challenge (two‐way ANOVA; p&lt;0.0001) and before LPS challenge (two‐way ANOVA; p&lt;0.0001). We did not observe differences in PGE2 levels between LPS‐challenged and non‐challenged cells differentiated with ARA (matched pairs t‐test; p=0.42) and EPA (p=0.57), while control (p=0.0005) and LA‐treated cells (p=0.0003) produced higher amounts of PGE2 after LPS stimulation. Interestingly, quantified levels of IL‐6 across treatments did not differ before and after LPS stimulation (matched pairs t‐test; p=0.75), although, as observed with PGE2, there was a significant increase in IL‐6 production by ARA‐differentiated cells compared to control, LPS, LA, and EPA‐treated cells (two‐way ANOVA; p=0.004). In conclusion, use of ARA in the differentiation process of 3T3‐L1 fibroblasts into a final adipocyte phenotype increases their endogenous (non‐LPS stimulated) production of IL‐6 and PGE2.

Influence of polyamines of bacterial origin on the production of key cytokines in the culture of human mononuclear leukocytes

To date, participation of biogenic polyamines has been studied in details, with respect to regulation of microbial gene expression, interrelations between bacteria, development of their persistence state. Opportunity of their use as markers of human pathological conditions is being actively evaluated. The aim of our study was to assess the effect of bacterial diamines, i.e., cadaverine and putrescine, upon production of key cytokines (IFNγ and IL-4) in the culture of human mononuclear leukocytes. We studied leukocytes of peripheral venous blood obtained from 18 healthy male volunteers (mean age 24.0±0.6 years). The leukocytes were isolated by means of gradient centrifugation using a Ficoll-Verografin mixture. For the cultivation of lymphocytes, a micro-method and plastic round-bottom 96-well plates were used. Concanavalin A at a concentration of 5 μg/ml was used as a T cell mitogen. Polyamines were used at final concentrations of 5, 25, 50, 75 and 100 μM/L. The cultivation was carried out in humidified atmosphere with 5% CO2, at 37 °C for 72 hours. At the end of incubation, the culture medium was collected and frozen for subsequent quantitative enzyme immunoassays of cytokine concentrations (IFNγ and IL-4) (Russia). Viable cells were counted using Goryaev chamber after staining with 0.1% trypan blue solution. Statistical analysis was performed using Student’s t test or Mann–Whitney test. Addition of cadaverine at all concentrations reduced IFNγ production in the culture of mitogen-activated cells. When culturing leukocytes supplied with putrescine (5 to 50 μM/L), a dose-dependent decrease of IFNγ was observed. Upon further increase of putrescine concentrations, the IFNγ production is restored to the values of the control samples. Direct toxic effect of polyamines upon the cells was revealed. Both the diamines, at the doses of 50, 75, and 100 μM/L, caused increase of IL-4 production by the mitogen-activated cells. Such changes can be associated both with direct cytotoxic effect of cadaverine and putrescine, being mediated by changes of some metabolic pathways. In addition, the effects of polyamines upon monocytes present in culture can include their anti-inflammatory state, e.c., an increased IL-4 production. In general, cadaverine and putrescine, produced by microorganisms of various taxonomic groups, regulate the effectiveness of compensatory-adaptive reactions that ensure adaptation of microbial populations to changing or unfavorable environmental conditions.

In vitro effect of triamcinolone and platelet-rich plasma on cytokine levels of elbow lateral epicondylitis-derived cells

BackgroundThe pathogenesis and treatment of lateral elbow epicondylitis (LEE) are still controversial. The purpose of the current study was to evaluate the production of inflammatory cytokines by LEE-derived cells and to compare the anti-inflammatory effect of triamcinolone acetonide with platelet-rich plasma (PRP) on cytokines production in primary culture of these cells.MethodsThird passage cells from primary cultures of LEE were assessed for the production of the cytokines IL-1β, IL-6, IL-8, IL-10 and TNF-α by immune-enzymatic assay (ELISA), after the treatment with 1, 10 and 100 μM triamcinolone compared to no treated controls at the time points 6, 12, 18, 24, 48, 72 and 96 h, and to PRP at 48, 72 and 96 h.ResultsThe cytokines IL-6 and IL-8 were produced in high concentrations by LEE cells. One, 10 and 100 μM triamcinolone induced significant decrease in the production of IL-6 and IL-8 at 48, 72 and 96 h, adding the time point 12 h for IL-8. Compared to controls, PRP caused a significant increase in the production of IL-6 and IL-8 and there was a significant increase in IL-10 production with the use of 100 μM triamcinolone at 48 h. The production of IL1-β and TNF-α was very low and did not change when the cultures were treated with triamcinolone or PRP.ConclusionLEE-derived cells produce IL-6 and IL-8, confirming the inflammatory nature of this condition. While triamcinolone inhibited the production of IL-6 and IL-8 by LEE cells, PRP induced an increase in these cytokines compared with controls.

Endothelial NOX5 Expression Modulates Thermogenesis and Lipolysis in Mice Fed with a High-Fat Diet and 3T3-L1 Adipocytes through an Interleukin-6 Dependent Mechanism.

Obesity is a global health issue associated with the development of metabolic syndrome, which correlates with insulin resistance, altered lipid homeostasis, and other pathologies. One of the mechanisms involved in the development of these pathologies is the increased production of reactive oxygen species (ROS). One of the main producers of ROS is the family of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, among which NOX5 is the most recently discovered member. The aim of the present work is to describe the effect of endothelial NOX5 expression on neighboring adipose tissue in obesity conditions by using two systems. An in vivo model based on NOX5 conditional knock-in mice fed with a high-fat diet and an in vitro model developed with 3T3-L1 adipocytes cultured with conditioned media of endothelial NOX5-expressing bEnd.3 cells, previously treated with glucose and palmitic acid. Endothelial NOX5 expression promoted the expression and activation of specific markers of thermogenesis and lipolysis in the mesenteric and epididymal fat of those mice fed with a high-fat diet. Additionally, the activation of these processes was derived from an increase in IL-6 production as a result of NOX5 activity. Accordingly, 3T3-L1 adipocytes treated with conditioned media of endothelial NOX5-expressing cells, presented higher expression of thermogenic and lipolytic genes. Moreover, endothelial NOX5-expressing bEnd.3 cells previously treated with glucose and palmitic acid also showed interleukin (IL-6) production. Finally, it seems that the increase in IL-6 stimulated the activation of markers of thermogenesis and lipolysis through phosphorylation of STAT3 and AMPK, respectively. In conclusion, in response to obesogenic conditions, endothelial NOX5 activity could promote thermogenesis and lipolysis in the adipose tissue by regulating IL-6 production.

Protective Effect Against Toxoplasmosis in BALB/c Mice Vaccinated With Recombinant Toxoplasma gondii MIF, CDPK3, and 14-3-3 Protein Cocktail Vaccine

Toxoplasma gondii can infect almost all endotherm organisms including humans and cause life-threatening toxoplasmosis in immunocompromised individuals, which leads to serious public health problems. Developing an excellent vaccine against this disease is impending. In present study, we formulated a cocktail protein vaccine including the TgMIF, TgCDPK3, and Tg14-3-3 proteins, which play critical roles in T. gondii infection. The recombinant protein vaccines were constructed and assessed by vaccination in BALB/c mice. We organized the mice in various protein combination groups of vaccines, and all mice were immunized with corresponding proteins at 0, 2, and 4 weeks. The specific protective effects of the vaccines on mice against T. gondii were analyzed by the mensuration of cytokines, serum antibodies, splenocyte proliferation assay, survival time, and parasite cyst burden of mice after the challenge. The study indicated that mice immunized with all three multicomponent proteins vaccine triggered a strong immune response with highest levels of IFN-γ production and IgG antibody compared with the other two protein combinations and controls. Moreover, there was an increase in IL-4 production and antigen-specific lymphocyte proliferation. The parasite cysts were significantly reduced (resulting in an 82.7% reduction), and survival time was longer in immunized mice with three multicomponent proteins compared with the other groups of mice. The enhanced humoral and cell-mediated immunity indicated that the protein cocktail vaccine containing three antigens provided effective protection for mice. These results indicated that recombinant TgMIF, TgCDPK3, and Tg14-3-3 multicomponent proteins were potential candidates for vaccine against toxoplasmosis.

New Insights into the Hypotensins from Tityus serrulatus Venom: Pro-Inflammatory and Vasopeptidases Modulation Activities.

The Tityus serrulatus scorpion is considered the most dangerous of the Brazilian fauna due to the severe clinical manifestations in injured victims. Despite being abundant components of the venom, few linear peptides have been characterized so far, such as hypotensins. In vivo studies have demonstrated that hypotensin I (TsHpt-I) exerts hypotensive activity, with an angiotensin-converting enzyme (ACE)-independent mechanism of action. Since experiments have not yet been carried out to analyze the direct interaction of hypotensins with ACE, and to deepen the knowledge about these peptides, hypotensins I and II (TsHpt-II) were studied regarding their modulatory action over the activities of ACE and neprilysin (NEP), which are the peptidases involved in blood pressure control. Aiming to search for indications of possible pro-inflammatory action, hypotensins were also analyzed for their role in murine macrophage viability, the release of interleukins and phagocytic activity. TsHpt-I and -II were used in kinetic studies with the metallopeptidases ACE and NEP, and both hypotensins were able to increase the activity of ACE. TsHpt-I presented itself as an inhibitor of NEP, whereas TsHpt-II showed weak inhibition of the enzyme. The mechanism of inhibition of TsHpt-I in relation to NEP was defined as non-competitive, with an inhibition constant (Ki) of 4.35 μM. Concerning the analysis of cell viability and modulation of interleukin levels and phagocytic activity, BALB/c mice’s naïve macrophages were used, and an increase in TNF production in the presence of TsHpt-I and -II was observed, as well as an increase in IL-6 production in the presence of TsHpt-II only. Both hypotensins were able to increase the phagocytic activity of murine macrophages in vitro. The difference between TsHpt-I and -II is the residue at position 15, with a glutamine in TsHpt-I and a glutamic acid in TsHpt-II. Despite this, kinetic analyzes and cell assays indicated different actions of TsHpt-I and -II. Taken together, these results suggest a new mechanism for the hypotensive effects of TsHpt-I and -II. Furthermore, the release of some interleukins also suggests a role for these peptides in the venom inflammatory response. Even though these molecules have been well studied, the present results suggest a new mechanism for the hypotensive effects of TsHpt-I

SYNERGISTIC IMMUNOMODULATORY ACTIVITY OF PROBIOTICS BIFIDOBACTERIUM ANIMALIS AND LACTOBACILLUS CASEI IN ENTEROAGGREGATIVE ESCHERICHIA COLI (EAEC)-INFECTED CACO-2 CELLS.

Enteroaggregative Escherichia coli (EAEC) is an E. coli pathotype that presents aggregative adhesion patterns on in vitro cultivated cells, mainly related to persistent diarrhea cases in children. EAEC virulence factors are important for host colonization and pathogeni-city. Intestinal epithelial cells (IECs) recognize pathogen-associated molecular patterns (PAMPs) and initiate an immune response. Several studies using in vivo and in vitro models emphasize the probiotic activity and immunomodulatory capacity of Lactobacillus and Bifidobacterium species. To evaluate the modulation of cytokine production by probiotics Bifidobacterium animalis and Lactobacillus casei in human intestinal Caco-2 cells exposed to different strains of EAEC. Caco-2 cells were incubated with EAEC strains in the presence or absence of probiotics. The production of cytokines IL-8, TNF-α, IL-1β and IL-10 was evaluated in the supernatants by a sandwich enzyme-linked immunosorbent assay (ELISA). Cytokine production did not change when uninfected and EAEC-infected Caco-2 cells were exposed to probiotics separately. All EAEC induced a significant increase in IL-8 production by Caco-2 cells, but the probiotics, even together, could not reduce its production. On the other hand, the synergic activity of probiotic strains significantly increased TNF-α production but decreased the basal production of IL-1ß. Also, probiotics induced a significant increase in the production of the anti-inflammatory cytokine IL-10 during EAEC infection. Our results reinforce the synergistic immunomodulatory activity of probiotics during EAEC infection.

Elevated levels of arginase activity are related to inflammation in patients with COPD exacerbation

IntroductionWithin the pathogenesis of the chronic obstructive pulmonary disease (COPD) there are interactions between different inflammatory mediators that are enhanced during an exacerbation. Arginase is present in bronchial epithelial cells, endothelial, fibroblasts and alveolar macrophages, which make it a probable key enzyme in the regulation of inflammation and remodelling. We aimed to find a potential relationship between arginase activity, inflammatory mediators in COPD patients in stable phase and during exacerbations.MethodsWe performed a prospective, observational study of cases and controls, with 4 study groups (healthy controls, stable COPD, COPD during an exacerbation and COPD 3 months after exacerbation). We measured arginase, inflammation markers (IL-6, IL-8, TNF-∝, IFN-γ and C reactive protein), and mediators of immunity: neutrophils, monocytes, total TCD3 + lymphocytes (CD3ζ), CD4 + T cells, CD8 + T cells, NK cells.ResultsA total of 49 subjects were recruited, average age of 69.73 years (59.18% male). Arginase activity is elevated during an exacerbation of COPD, and this rise is related to an increase in IL-6 production. The levels of IL-6 and IL-8 remained elevated in patients with COPD at 3 months after hospital exacerbation.We did not find a clear relationship between arginase activity, immunity or with the degree of obstruction in COPD patients.ConclusionsArginase activity is elevated during an exacerbation of COPD, and it could be related to an increase in the production of IL-6. Levels of IL-6, IL-8, and arginase activity remain elevated in patients with COPD at 3 months after hospital exacerbation. Arginase activity could contribute to the development of COPD.

Effects of cigarette smoke extract on bronchial epithelial cells stimulated with Cryptococcus neoformans.

In the airways, the adhesion of Cryptococcus neoformans with airway epithelial cells is crucial for the establishment of cryptococcosis. Tobacco smoke is considered a risk factor for cryptococcosis. Here, we evaluated the effects of cigarette smoke extract (CSE) on human bronchial epithelial cells (BEAS-2B) stimulated with C. neoformans. Multiplicities of infection (MOIs) of 1-100 of C. neoformans per cell led to increased IL-8 production and no cytotoxic effects when compared to those of controls. C. neoformans (MOI 100) also significantly increased the concentration of IL-6. In cells stimulated with CSE doses (1.0, 2.5 and 5.0%) from one or five cigarettes, increased IL-1β production was observed only in doses from one (1.0%) and five (2.5%) cigarettes when compared to that of controls. However, only 1.0% CSE failed to show cytotoxic effects. In addition, CSE significantly increased the concentration of IL-8. Cells stimulated with both CSE and C. neoformans demonstrated a reduction in IL-6/STAT3 signalling compared to that in cells stimulated by C. neoformans. In addition, a significant increase in IL-10 production was also observed. No alterations in NF-kB or ICAM-1 expression were observed among the groups. The combination of CSE and C. neoformans favoured the increase of fungal numbers and extracellular adhering of C. neoformans on BEAS-2B cells. In addition, the internalization of C. neoformans on BEAS-2B cells was reduced after CSE stimulation. In conclusion, the association of CSE and C. neoformans induced an anti-inflammatory effect in bronchial epithelial cells, which might favour the development of C. neoformans infection in the airways.

Physico-chemical properties of Silicea terra 200cH and activity on macrophages in vitro

Introduction Silicea terra 200cH can modulate macrophage activity in mice inducing resolution of chronic wounds, whose mechanisms are unknown. Herein, we describe the relation between the activity of Silicea terra 200cH in vitro, on macrophages infected with Calmette-Guérin Bacilli (BCG) - a microorganism involved in chronic granuloma lesions - and physico-chemical properties of the remedy. Objective Establish the relation between physico-chemical properties and biological activity of Silicea terra 200cH in vitro. Methods BCG-infected RAW 264.7 macrophages were analyzed considering their capacity of bacilli internalization, lysosome activity, hydrogen peroxide (H2O2) and cytokines production, using a CBA (BD) kit. The vehicle (succussed water) was used as control. The electrical conductivity and the presence of micro particles in the remedy were also studied. Results Silicea terra 200cH showed significant reduction of hydrogen peroxide production (p?0.001), higher lysosome activity (p?0.001) and increase of IL-10 production (p?0.05), an anti-inflammatory cytokine, in relation to the control. The electrical conductivity of Silicea terra 200cH was statistically higher in relation to pure water (p

Lenalidomide Enhances Anti-BCMA Chimeric Antigen Receptor T Cell Function Against Multiple Myeloma

The development of chimeric antigen receptor (CAR) T cells that target antigens expressed on the surface of malignant plasma cells is a promising new treatment approach for relapsed refractory multiple myeloma patients. Although early clinical trials investigating single agent anti-B cell maturation antigen (BCMA) CAR-T cells achieved clinical responses in myeloma, additional combinatorial therapies may increase the effectiveness of this treatment. Lenalidomide, a small molecule immunomodulatory agent approved for use in multiple myeloma, can have both direct tumoricidal and T cell modulatory effects. Recent publications have highlighted the ability of lenalidomide to enhance T cell and CAR-T function in preclinical models.The immunomodulatory effects of lenalidomide were investigated in combination with an autologous cellular drug product transduced to express a BCMA-specific CAR with 4-1BB costimulatory and CD3 zeta endodomains. The intrinsic effects mediated by lenalidomide on CAR-T cell function were evaluated using antigen-coated beads labeled with varying concentrations of BCMA, allowing investigation into how stimulus strength and lenalidomide concentration influence CAR-T cell functionality. Lenalidomide increased CAR-T cell cytokine production across stimulation conditions and CAR-T cells exposed to low levels of antigen demonstrated the most marked increase in cytokine production and activation with the addition of lenalidomide in culture. To further investigate the effect of lenalidomide upon CAR-T cell stimulation, RNA-seq and ATAC-seq molecular analysis of CAR-T cells was carried out after 24 hours or 7 days of stimulation on BCMA coated beads with or without lenalidomide. Differential expression and accessibility results demonstrated an enriched involvement of immune synapse-associated genes, cytokine signaling, and T cell activation pathways consistent with the functional gains measured in the presence of lenalidomide.The effect of lenalidomide on CAR-T cell cytolytic activity, cytokine production, and proliferation was further evaluated in co-culture with multiple myeloma cell lines displaying a range of BCMA expression. The addition of lenalidomide during co-culture increased CAR-T cell effector cytokine production and cytolytic activity. Lenalidomide also increased CAR-T cell performance over 4 weeks in a repeated serial stimulation of BCMA CAR-T cells with MM1.S cells, an assay designed to predict CAR-T drug product fitness. The lenalidomide-associated increase in persistence and proliferation correlated with an increase in IL-2 production and activation marker expression at multiple time points following repeated stimulation. Next, to measure effector function after chronic stimulation, CAR-T cells were cultured for 7 days with BCMA beads, debeaded, and then cultured with multiple myeloma cell lines. Pre-stimulation on BCMA beads decreased CAR-T cell cytolytic activity and cytokine production in this assay. The addition of lenalidomide during CAR-T cell pre-stimulation increased activation marker expression, decreased PD-1 expression, and prevented the decrease in cytolytic activity relative to vehicle pre-stimulated CAR-T cells.To evaluate the in vivo activity of daily administration of lenalidomide in combination with BCMA CAR-T cells, NSG mice were injected intravenously with OPM2 multiple myeloma cells. Two dosing strategies were evaluated to determine the effect of dosing lenalidomide concurrently with CAR-T administration or two weeks following CAR-T administration. Both lenalidomide dosing schedules enhanced the function of sub-curative doses of CAR-T cells, as demonstrated by decreased tumor burden and increased animal survival.Overall, the addition of lenalidomide to BCMA CAR-T cells increased CAR-T function including activation, proliferation, cytolytic activity, and cytokine production in a concentration-dependent manner. The lenalidomide-induced increase in CAR-T function may be advantageous in the myeloma tumor microenvironment as a variety of factors can limit CAR-T efficacy including CAR-T exhaustion and low antigen density expressed on the tumor. Taken together, these data suggest that lenalidomide may potentially increase CAR-T cell activity upon encounter with surface expressed BCMA to enhance an anti-tumor response. DisclosuresWorks:Juno Therapeutics: Employment, Equity Ownership. Soni:Juno Therapeutics: Employment, Equity Ownership. Hauskins:Juno Therapeutics: Employment, Equity Ownership. Sierra:Juno Therapeutics: Employment, Equity Ownership. Baturevych:Juno Therapeutics: Employment, Equity Ownership. Jones:Juno Therapeutics: Employment, Equity Ownership. Curtis:Juno Therapeutics: Employment, Equity Ownership. Johnstone:Juno Therapeutics: Employment, Equity Ownership. Kugler:Juno Therapeutics: Employment, Equity Ownership. Jiang:Juno Therapeutics: Employment, Equity Ownership. Hause:Juno Therapeutics: Employment, Equity Ownership. Sather:Juno Therapeutics: Employment, Equity Ownership. Salmon:Juno Therapeutics: Employment, Equity Ownership. Ports:Juno Therapeutics: Employment, Equity Ownership.

Characterization of βN-Octadecanoyl-5-hydroxytryptamide Anti-Inflammatory Effect.

Background: N-octadecanoyl-5-hydroxytryptamide (C18-5HT) is an amide that can be obtained by the coupling of serotonin and octadecanoic acid. This study aims to characterize the in vivo and in vitro anti-inflammatory activity of C18-5HT. Methods: A subcutaneous air pouch model (SAP) was used. The exudates were collected from SAP after carrageenan injection to assess cell migration and inflammatory mediators production. RAW 264.7 cells were used for in vitro assays. Results: C18-5HT significantly inhibited leukocyte migration into the SAP as well as nitric oxide (NO) and cytokines production and protein extravasation. We also observed an reduction in some cytokines and an increase in IL-10 production. Assays conducted with RAW 264.7 cells indicated that C18-5HT inhibited NO and cytokine produced. Conclusions: Taken together, our data suggest that C18-5HT presents a significant effect in different cell types (leukocytes collected from exudate, mainly polumorphonuclear leukocytes and cell culture macrophages) and is a promising compound for further studies for the development of a new anti-inflammatory drug.

P2x7 Receptor Signaling Blockade Reduces Lung Inflammation and Necrosis During Severe Experimental Tuberculosis.

The risk of developing severe forms of tuberculosis has increased by the acquired immunodeficiency syndrome (AIDS) epidemic, lack of effective drugs to eliminate latent infection and the emergence of drug-resistant mycobacterial strains. Excessive inflammatory response and tissue damage associated with severe tuberculosis contribute to poor outcome of the disease. Our previous studies using mice deficient in the ATP-gated ionotropic P2X7 receptor suggested this molecule as a promising target for host-directed therapy in severe pulmonary tuberculosis. In this study, we assessed the effects of P2X7 pharmacological blockade on disease severity. First, we observed an increase in P2RX7 gene expression in the peripheral blood of tuberculosis patients compared to healthy donors. Lung leukocytes of mice infected with hypervirulent mycobacteria also showed increased expression of the P2X7 receptor. P2X7 blockade in mice with advanced tuberculosis recapitulated in many aspects the disease in P2X7-deficient mice. P2X7-directed therapy reduced body weight loss and the development of inflammatory and necrotic lung lesions, as well as delayed mycobacterial growth. Lower TNF-α production by lung cells and a substantial reduction in the lung GR-1+ myeloid cell population were observed after P2X7 inhibition. The effector CD4+ T cell population also decreased, but IFN-γ production by lung cells increased. The presence of a large population with characteristics of myeloid dendritic cells, as well as the increase in IL-6 production by lung cells, also indicate a qualitative improvement in the pulmonary immune response due to P2X7 inhibition. These findings support the use of drugs that target the P2X7 receptor as a therapeutic strategy to improve the outcome of pulmonary tuberculosis.

Antimicrobial Triclosan (TCS) Increases Production of Interleukin 6 (IL‐6) in Human Immune Cells

Triclosan (TCS) is an antimicrobial compound that is widely used in personal hygiene products such as mouthwash and toothpaste. TCS can be ingested or absorbed through the skin and has been found in human blood, breast milk, and urine. Interleukin (IL)-6 is an important pro-inflammatory mediator produced by lymphocytes, monocytes, and other human cells, which regulates cell growth, tissue repair, and immune function. Increased levels have been associated with a number of diseases including rheumatoid arthritis and certain cancers. In a previous study we showed that TCS at concentrations between 0.05-5 µM increased the secretion of IL-6 from immune cells within 24 h. Here we will examine whether the increase in IL-6 secretion stimulated by TCS is due to an increase in the cellular production of IL-6 or only due to release of existing stores of the cytokine. Human peripheral blood mononuclear cells (PBMCs) were exposed to TCS at concentrations of 0-5 µM. The cellular production (combination of secreted and intracellular levels) of IL-6 was measured at 10 minutes, 30 minutes, 6 hours, and 24 hours. Secreted levels were measured in supernatants from exposed cells using enzyme-linked immunosorbent assay (ELISA) and intracellular levels were measured by lysing the exposed- cell pellet followed by Western Blot. Results indicate the production of IL-6 was increased by exposure to one or more concentration of TCS at each length of exposure. The greatest increase in IL-6 production was seen at 6 h, where all TCS exposures caused quite substantial increases in IL-6 production in all donors. The magnitude of these increases varied dependent on the donor. These results indicate that TCS has the capacity to increase cellular production of the important pro-inflammatory cytokine IL-6 from immune cells. TCS-induced fluctuations of IL-6 secretion may serve to produce unwarranted inflammation and the pathologies associated with it.

No association between pyrite content and lung cell responses to coal particles

There has been an increase in the identification of cases of coal workers’ pneumoconiosis (CWP) in recent years around the world. While there are a range of possible explanations for this, studies have implicated the pyrite content of coal as a key determinant of CWP risk. However, experimental studies to support this link are limited. The aim of this study was to assess the association between the pyrite content, and subsequent release of bioavailable iron, in coal particles and the response of lung cells involved in the pathogenesis of CWP (epithelial cells, macrophages and fibroblasts). Using real-world Australian coal samples, we found no evidence of an association between the pyrite content of the coal and the magnitude of the detrimental cell response. We did find evidence of an increase in IL-8 production by epithelial cells with increasing bioavailable iron (p = 0.01), however, this was not linked to the pyrite content of the coal (p = 0.75) and we did not see any evidence of a positive association in the other cell types. Given the lack of association between the pyrite content of real-world coal particles and lung cell cytotoxicity (epithelial cells and macrophages), inflammatory cytokine production (epithelial cells, macrophages and fibroblasts), and cell proliferation (fibroblasts) our data do not support the use of coal pyrite content as a predictor of CWP risk.

Relationship of cytokine levels with neuropsychological parameters in patients with vibration disease

Introduction. The results were obtained using the method of solid-phase enzyme immunoassay and neuropsychological examination of patients with vibration disease caused by the combined effect of local and general vibration. The aim of the study was to evaluate the relationship between the content of pro-and anti-inflammatory cytokines and changes in neuropsychological parameters in patients with vibration disease associated with combined exposure to local and general vibration. Materials and methods. Examined men were divided into groups: first group (n=28) - with vibration disease of the second degree, due to the combined effect of local and general vibration and the second (n=16) - the comparison group. There are methods of statistical analysis with the determination of the Shapiro-Wilk W-test, the Mann-Whitney U-test, and the Spearman correlation analysis. Results. In patients with vibration disease when compared to second group was higher values characterizing the analytic-synthetic (p=0.002) and conceptual thinking (p=0.016), short (verbal) (p=0.002), hand-shaped (p=0.002) and long (p=0.041) memory, dynamic (p=0.001) and constructive (p=0.001) praxis, visual gnosis (p=0,042), impressive speech (p=0.041), as well as index test, MMSE (p=0.003). Dysregulation in the immune system manifests in the form of a statistically significant hyperproduction of IL-8 and IFN-γ diagnosed in first group patients when compared with group II at p&lt;0.05. There were statistically significant direct correlations between the concentration of IL-4 and the indicator of conceptual thinking, an indicator that characterizes finger gnosis (p=0.021; 0.025, respectively). Statistically significant dependences indicate that the increase in tests on the arithmetic triple score, the selection of opposites in the active plan, showing the specified finger on the sample and the name, an explanation of the logical-grammatical constructions, indicating a decrease in the function of analytical and synthetic thinking, conceptual thinking, finger gnosis, impressive speech, this is due to an increase in production of IL-8, IFN-γ (p=0,013; 0,044; 0,025; 0,008; 0,048; 0,046; 0,028; 0,018 respectively). Conclusions. The decrease in indicators that characterize analytical-synthetic, conceptual thinking, short-term (auditory-speech), long-term, operational visual memory, dynamic and constructive praxis, visual gnosis, and impressive speech is accompanied by hyperproduction of IL-8, IFN-γ. This fact may characterize the activation of the neuroinflammatory process as an important determinant of cognitive decline under the combined influence of local and general vibration. The established relationship between increased levels of IL-4 and indicators of conceptual thinking, visual gnosis, probably indicates the importance of more pronounced cytotoxic reactions in the violation of the mechanisms of visual perception and thought process in patients with vibration disease.

Campylobacter coli strains from Brazil can invade phagocytic and epithelial cells and induce IL-8 secretion.

Campylobacter spp. have been a predominant cause of bacterial foodborne gastroenteritis worldwide, causing substantial costs to public healthcare systems. This study aimed to assess the invasion and pro-inflammatory cytokine production capacity of Campylobacter coli strains isolated in Brazil. A total of 50 C. coli isolated from different sources in Brazil were analyzed for their capacity of invasion in Caco-2 and U-937 cell lines. The production of pro-inflammatory cytokines was quantitatively measured in response to C. coli. All the strains studied showed invasion percentage ≥ 40% in polarized Caco-2 cells. In U-937 cells assay, 35 of 50 C. coli strains studied showed invasion percentage ≥ 50%. A significant increase in IL-8 production by infected U-937 cells was observed for 17.5% of the C. coli isolates. The high percentages of invasion in Caco-2 and U-937 cells observed for all studied strains, plus the increased production of IL-8 by U-937 cells against some strains, highlighted the pathogenic potential of the C. coli studied and bring extremely relevant data since it has never been reported for strains isolated in Brazil and there are a few data for C. coli in the literature.