Proliferation Of Peripheral Blood Mononuclear Cells Research Articles

Bisphenol A induces DNA damage in cells exerting immune surveillance functions at peripheral and central level

Bisphenol A (BPA) is a synthetic xenoestrogen diffused worldwide. Humans are chronically exposed to low doses of BPA from food and drinks, thus BPA accumulates in tissues posing human health risk. In this study, we investigated the effects of BPA on peripheral blood mononuclear cells (PBMC) from human healthy donors, and in glia and microglia of rat offspring at postnatal day 17 (17PND) from pregnant females who received BPA soon after coupling and during lactation and weaning. Results indicated that BPA affected Phytoemagglutinin (PHA) stimulated PBMC proliferation causing an S-phase cell cycle accumulation at nanomolar concentrations while BPA was almost ineffective in resting PBMC. Furthermore, BPA induced chromosome aberrations and the appearance of shattered cells characterized by high number of fragmented and pulverized chromosomes, suggesting that the compound could cause a massive genomic rearrangement by inducing catastrophic events. The BPA-induced DNA damage was observed mainly in TCD4+ and TCD8+ subsets of T lymphocytes and was mediated by the increase of ERK1/2 phosphorylation, p21/Waf1 and PARP1 protein expression. Intriguingly, we observed for the first time that BPA-induced effects were associated to a sex specific modulation of ERα and ERβ in human PBMC. Immunofluorescence analysis of rat hippocampus corroborated in vitro findings showing that BPA induced ɣH2AX phosphorylation in microglia and astrocytosis by decreasing ERα expression within the dentate gyrus. Overall these results suggest that BPA can alter immune surveillance functions at both peripheral and central level with a potential risk for cancer, neuroinflammation and neurodegeneration.

Effect of Ancylostoma ceylanicum hookworm platelet inhibitor on platelet adhesion and peripheral blood mononuclear cell proliferation.

Ancylostoma ceylanicum is a zoonotic parasitic nematode that can cause iron-deficiency anemia and malnutrition in humans. A. ceylanicum hookworm platelet inhibitor (Ace-HPI) can inhibit platelet aggregation in the host to facilitate blood sucking, but whether it possesses platelet adhesion inhibitory activity or immunomodulatory role is yet unknown. To explore the effect of Ace-HPI on platelet adhesion, we expressed the recombinant protein in two competent cells, BL21 (DE3) and Rosetta-gami2 (DE3), and incubated this protein with canine platelets in a 96-well microplate. Ace-HPI was used to stimulate peripheral blood mononuclear cells (PBMC) in vitro to investigate the effect on PBMC proliferation and cytokine expression. Results showed that Ace-HPI expressed in Rosetta-gami2 (DE3) strain was mostly soluble. The inhibitory effect of this protein on platelet adhesion was relatively weak (7-8%). This protein stimulated the proliferation of PBMC and promoted the expression of Treg and Th2 cytokines, such as IL-10 and IL-13. These results lay a foundation for exploring the role of Ace-HPI in hookworm disease pathogenesis and as a candidate molecule for hookworm vaccines.

Determining the immunological characteristics of a novel human monoclonal antibody developed against staphylococcal enterotoxin B

ABSTRACT Staphylococci are the main cause of nosocomial infections globally. The exotoxin staphylococcal enterotoxin B (SEB) produced by methicillin-resistant Staphylococcus aureus is a major cause of pathology after a staphylococcal infection. We previously isolated an anti-SEB human monoclonal antibody designated as M0313. Here we further characterize this antibody in vitro and in vivo. Immunoblotting analysis and ELISA results indicated that M0313 accurately recognized and bound to SEB. Its binding affinity to native SEB was measured at the low nM level. M0313 effectively inhibited SEB from inducing mouse splenic lymphocyte and human peripheral blood mononuclear cell proliferation and cytokine release in cell culture. M0313 also neutralized SEB toxicity in BALB/c female mice. Most importantly, M0313 promoted the survival of mice treated with SEB-expressing bacteria. In-vivo imaging revealed that M0313 treatment significantly reduced the replication of SEB-expressing bacteria in mice. The neutralization capacity of M0313 correlated with its ability to block SEB from binding to major histocompatibility complex II and T-cell receptor by binding to the SEB residues 85–102 and 90–92. Thus, the monoclonal antibody M0313 may be developed into a therapeutic agent.

CD44+CD24-/low sphere-forming cells of EBV-associated gastric carcinomas show immunosuppressive effects and induce Tregs partially through production of PGE2

EBV-associated gastric carcinoma (EBVaGC) is accompanied by massive lymphocyte infiltration, but therapy resistance and tumor progression still occur in patients with EBVaGC. Cancer stem cells (CSCs) are reported to possess immunomodulatory ability that allows them to resist immune-mediated rejection for many tumor types. However, whether and how CSCs in EBVaGC exhibit immunosuppression has not yet been elucidated. We isolated CSC-like sphere-forming cells (SFCs) from EBVaGC cell line SNU-719 using the cancer sphere method. We validated their CSC-associated properties in the expression of the epithelial-mesenchymal transition (EMT)-related genes, the ability to form colonies, and resistance to chemotherapy drug-induced apoptosis and explored their immunomodulatory ability using the coculture system with PBMC (peripheral blood mononuclear cell). These CSC-like SFCs were CD44+CD24-/low and were more tumorigenic than the parental SNU-719 cells in the xenograft mouse model. Remarkably, in the tumor-PBMC co-culturing experiments, these EBVaGC SFCs demonstrated profound immunosuppression by inhibiting the proliferation of PBMCs and T cell activation as well as inducing the generation of regulatory T cells (Tregs). Furthermore, the induction of Tregs was partially dependent on prostaglandin E2 (PGE2) produced from SFCs. Moreover, the presence of high CD44+CD24-/low cells in tumor tissues predicted a decreased disease-free survival in patients with EBVaGC. Our study collectively confirmed the existence and immune resistance of CSCs in EBVaGC and offers new insights into the development of novel anti-EBVaGC strategies by targeting CSCs.

Priming with inflammatory cytokines is not a prerequisite to increase immune-suppressive effects and responsiveness of equine amniotic mesenchymal stromal cells

BackgroundEquine amniotic mesenchymal stromal cells (AMSCs) and their conditioned medium (CM) were evaluated for their ability to inhibit in vitro proliferation of peripheral blood mononuclear cells (PBMCs) with and without priming. Additionally, AMSC immunogenicity was assessed by expression of MHCI and MHCII and their ability to counteract the in vitro inflammatory process.MethodsHorse PBMC proliferation was induced with phytohemagglutinin. AMSC priming was performed with 10 ng/ml of TNF-α, 100 ng/ml of IFN-γ, and a combination of 5 ng/ml of TNF-α and 50 ng/ml of IFN-γ.The CM generated from naïve unprimed and primed AMSCs was also tested to evaluate its effects on equine endometrial cells in an in vitro inflammatory model induced by LPS. Immunogenicity marker expression (MHCI and II) was evaluated by qRT-PCR and by flow cytometry.ResultsPriming does not increase MHCI and II expression. Furthermore, the inhibition of PBMC proliferation was comparable between naïve and conditioned cells, with the exception of AMSCs primed with both TNF-α and IFN-γ that had a reduced capacity to inhibit T cell proliferation. However, AMSC viability was lower after priming than under other experimental conditions. CM from naïve and primed AMSCs strongly inhibited PBMC proliferation and counteracted the inflammatory process, rescuing about 65% of endometrial cells treated by LPS.ConclusionAMSCs and their CM have a strong capacity to inhibit PBMC proliferation, and priming is not necessary to improve their immunosuppressive activity or reactivity in an inflammatory in vitro model.

Tropomyosin: An Excretory/Secretory Protein from Haemonchus contortus Mediates the Immuno-Suppressive Potential of Goat Peripheral Blood Mononuclear Cells In Vitro.

During host-parasite interactions, binding of excretory/secretory proteins (ESPs) on the host immune cells is considered the fundamental phase for regulation of immune responses. In this study, gene encoding Haemonchus contortus tropomyosin (Hc-TpMy), was successfully cloned and expressed, and the recombinant protein after host cell surface attachment was evaluated for immune functional analysis with goat peripheral blood mononuclear cells (PBMCs) in vitro. The isopropyl-β-D-thiogalactopyranoside (IPTG)-induced recombinant protein was successfully recognized by the sera of rat experimentally infected with rHc-TpMy. The immunofluorescence assay detected attachment of rHc-TpMy on the surface of host PBMCs. Furthermore, immunoregulatory roles of rHc-TpMy on cytokines expression, PBMC proliferation, migration, nitric oxide (NO) production, apoptosis and monocytes phagocytosis were observed. The results showed that expression of IL-4 and IFN-γ cytokines, cell proliferation, NO production and PBMC migration were significantly suppressed by goat PBMCs after co-incubation with rHc-TpMy protein. However, the productions of IL-10, IL-17 and TGF-β1 cytokines, PBMCs apoptosis and monocytes phagocytosis were elevated at dose dependent manner. Our findings indicated that rHc-TpMy is an important ES binding protein exhibit distinct immuno-suppressive roles on goat PBMCs which might be a potential molecular target to control haemonchosis in future.

Canine Corneal Stromal Cells Have Multipotent Mesenchymal Stromal Cell Properties In Vitro.

The objective of this study was to determine whether corneal stromal cells (CSCs) from the limbal and central corneal stroma in dogs have multipotent mesenchymal stem/stromal cell (MSC) properties, and whether this cell population can be differentiated into keratocyte-like cells (KDCs). Normal, donated, mesocephalic dog corneas were used to isolate CSC in vitro. Immunohistochemistry demonstrated a distinct population of CD90 expressing cells in the anterior stroma throughout the limbal and central cornea. CSC could be cultured from both the limbal and central cornea and the culture kinetics showed a progenitor cell profile. The CSC expressed stem cell markers CD90, CD73, CD105, N-cadherin, and Pax6, while CD34 was negative. Limbal and central CSC differentiated into osteoblasts, chondrocytes, and adipocytes confirming their multipotency. Coculturing allogeneic peripheral blood mononuclear cells (PBMCs) with limbal CSCs did not affect baseline PBMC proliferation indicating a degree of innate immune privilege. Limbal CSC could be differentiated into KDCs that expressed Keratocan, Lumican, and ALDH1A3 and downregulated Pax6 and N-cadherin. In conclusion, canine CSCs have multipotent MSC properties similarly described in humans and could serve as a source of cells for cell therapy and studying corneal diseases.

The anti-inflammatory potential of cefazolin as common gamma chain cytokine inhibitor

A continuing quest for specific inhibitors of proinflammatory cytokines brings promise for effective therapies designed for inflammatory and autoimmune disorders. Cefazolin, a safe, first-generation cephalosporin antibiotic, has been recently shown to specifically interact with interleukin 15 (IL-15) receptor subunit α (IL-15Rα) and to inhibit IL-15-dependent TNF-α and IL-17 synthesis. The aim of this study was to elucidate cefazolin activity against IL-2, IL-4, IL-15 and IL-21, i.e. four cytokines sharing the common cytokine receptor γ chain (γc). In silico, molecular docking unveiled two potential cefazolin binding sites within the IL-2/IL-15Rβ subunit and two within the γc subunit. In vitro, cefazolin decreased proliferation of PBMC (peripheral blood mononuclear cells) following IL-2, IL-4 and IL-15 stimulation, reduced production of IFN-γ, IL-17 and TNF-α in IL-2- and IL-15-treated PBMC and in IL-15 stimulated natural killer (NK) cells, attenuated IL-4-dependent expression of CD11c in monocyte-derived dendritic cells and suppressed phosphorylation of JAK3 in response to IL-2 and IL-15 in PBMC, to IL-4 in TF-1 (erythroleukemic cell line) and to IL-21 in NK-92 (NK cell line). The results of the study suggest that cefazolin may exert inhibitory activity against all of the γc receptor-dependent cytokines, i.e. IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21.

TLR3 activation modulates immunomodulatory properties of human periodontal ligament cells

Toll-like receptors (TLR) are a group of receptors that play roles in the innate immune system. Human periodontal ligament cells (hPDL cells) express several TLRs, including TLR3, a nucleotide sensing receptor that recognizes double-stranded RNA from viral infection. However, its role in hPDL cells is unclear. The aim of this study was to investigate the responses of hPDL cells in terms of immunomodulation after TLR3 engagement. HPDL cells were treated with various doses of poly I:C, a TLR3 activator. The expression of interferon-gamma (IFNγ), indoleamine 2,3 dioxygenase (IDO), and human leukocyte antigen G (HLA-G) was determined. Chemical inhibitors and small interfering RNA (siRNA) were used to confirm the role of TLR3. Coculture with human peripheral blood mononuclear cells (PBMCs) with poly I:C-activated hPDL cells was performed. Endosomal TLR3 in hPDL cells was observed by immunocytochemistry. Addition of poly I:C significantly enhanced the expression and secretion of IFNγ, IDO, and HLA-G. Knockdown of TLR3 using siRNA decreased the poly I:C-induced expression of these three molecules. Bafilomycin-A, an inhibitor of auto-phagosome and lysosome fusion, inhibited poly I:C-induced IDO and HLA-G expression, whereas cycloheximide and a TLR3-neutralizing antibody had no effect. In co-culture experiments, poly I:C-activated hPDL cells inhibited PBMCs proliferation and increased mRNA expression of forkhead box P3 (FOXP3), a transcription factor which is a marker of regulatory T cells. Our findings indicated that TLR3 engagement of hPDL cells induced immunosuppressive properties of these cells. Because immunosuppressive properties play an important role in tissue healing and regeneration, activation of TLR3 may help to attenuate tissue destruction by limiting the inflammatory process and perhaps initiate the healing and regeneration process of the periodontium.

Galectin Domain Containing Protein from Haemonchus contortus Modulates the Immune Functions of Goat PBMCs and Regulates CD4+ T-Helper Cells In Vitro.

Galectins are glycan-binding proteins that are widely expressed and distributed in mammalian tissues as well as cells of innate and adaptive immune responses. CD4+ T-helper cells differentiate into effector subsets in response to cytokines. T helper 9 cells are one of the recently described subsets of effector T cells that are relatively new and less studied. In this study, galectin domain containing protein from Haemonchus contortus (Hc-GDC) was cloned, expressed in pET32a, and immunoblotting was performed. Localization of recombinant (r)Hc-GDC on outer and inner surface of H. contortus worm and binding with goat Peripheral Blood Mononuclear cells (PBMCs) were performed using immunofluorescence assay. Moreover, effects of rHc-GDC on proliferation, apoptosis, cell migration, and the nitric oxide production in goat PBMCs were evaluated. Furthermore, modulatory effects of rHc-GDC on production of Th1, Th2, and Th9 cells were evaluated by flowcytometry and on interferon gamma, interleukin (IL)-4 and IL-9 were evaluated by quantitative real-time polymerase chain reaction. The results demonstrated that rHc-GDC was successfully cloned, expressed in expression vector as well as in the gut surface of adult H. contortus worm and successful binding with PBMCs surface were observed. Immunoblotting results revealed that rHc-GDC is an important active protein of H. contortus excretory and secretory products. Moreover, the interaction of rHc-GDC with host cells increased the production of Th2, Th9 cells, IL4, IL-9, PBMC proliferation, nitric oxide, and cell migration. No effects of rHc-GDC were observed on PMBC apoptosis, production of Th1 cells, and secretions of IFN-γ and IL-10 cytokines. These findings indicate that recombinant GDC protein from H. contortus modulates the immune functions of goat PBMCs and has the potential to enhance protective immunity by inducing T helper-9-derived IL-9 in vitro.

Effects of feed restriction and supplementary folic acid and vitamin B12 on immune cell functions and blood cell populations in dairy cows

Cows undergoing a negative energy balance (NEB) often experience a state of immunosuppression and are at greater risk of infectious diseases. The present study aimed to evaluate the impact of a folic acid and vitamin B12 supplement and feed restriction on several immune parameters. Sixteen cows at 45 ± 3 days in milk were assigned to 8 blocks of 2 cows each according to each cow’s milk production in the previous week, and within each block, the cows randomly received weekly intramuscular injections of either saline or 320 mg of folic acid and 10 mg of vitamin B12 for 5 weeks. During week 5, the cows were fed 75% of their ad libitum intake for 4 days. Blood samples were taken before the beginning of the experiment, just before feed restriction and after 3 days of feed restriction, in order to evaluate blood cell populations, the phagocytosis capacity and oxidative burst of polymorphonuclear leukocytes (PMNs), the proliferation of peripheral blood mononuclear cells (PBMCs) and concentrations of non-esterified fatty acids (NEFAs) and β-hydroxybutyrate. The vitamin supplement did not affect any of the tested variables except milk fat and lactose content. Feed restriction reduced milk production and increased the concentration of NEFAs. Feed restriction did not affect blood cell populations but did reduce the percentage of PMN positive for oxidative burst after stimulation with phorbol 12-myristate 13-acetate. The proliferation of PBMCs was reduced when the cell culture medium was supplemented with sera collected during the feed restriction. In conclusion, feed restriction affected the functions of PMN and PBMC and this effect was not prevented by the folic acid and vitamin B12 supplement. These results support the hypothesis that the greater risk of infectious diseases in cows experiencing a NEB is related to impaired immune cell functions by high circulating concentration of NEFAs.

Health impact of the commercially cultivated mushroom Agaricus bisporus and wild-growing mushroom Ganoderma resinaceum - a comparative overview

The health promoting effects of hot water extracts obtained from fruiting bodies of the commercially cultivated mushroom Agaricus bisporus (AbHW) and the wild-growing mushroom Ganoderma resinaceum (GrHW) originating from northern Serbia are presented in this research. These abilities were compared in vitro by the prevention of lipid peroxidation (LPx) in a linoleic acid model system, inhibition of the angiotension converting I enzyme (ACE) that could help in the maintenance of a normal blood pressure level and strengthening the ability of the central cholinergic neuron by inhibiting the activity of acetylcholinesterase (AChE). Cytotoxic activities were observed towards selected human malignant (HeLa and K562) cell lines and normal- -human peripheral blood mononuclear cells (PBMC). GrHW contains higher phenolics (5.9 g (100 g)-1), inhibition of LPx (EC50 = 1.07 mg mL-1), ACE (IC50 = 0.54 mg mL-1) and AChE (IC50 = 0.37 mg mL-1), and exhibited a significant selectivity in the antitumour action against HeLa (IC50 = 0.14 mg mL-1) and K562 (IC50 = 0.11 mg mL-1) cells. AbHW contained higher total protein (6.4 g (100 g)-1), carbohydrate (75.4 g (100 g)-1) and ?-glucan (55.1 g (100 g)-1) contents and induced significant proliferation of healthy PBMC from 152?116 % in the concentration range of 0.047?0.187 mg mL-1. The difference in the biological activity of the extracts provides guidance on their use as functional food.

Rice bran hydrolysates induce immunomodulatory effects by suppression of chemotaxis, and modulation of cytokine release and cell-mediated cytotoxicity

Objective: To evaluate the immunomodulatory effects of rice bran hydrolysates on cultured immune cells and their underlying mechanism. Methods: Rice bran hydrolysates were prepared from pigmented rice (Oryza sativa L.) by hydrothermolysis and protease digestion. Rice bran hydrolysates were assayed for phenolic content and antioxidant activity. Cell proliferation of Jurkat, THP-1 and peripheral blood mononuclear cells (PBMC) was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Chemotaxis was evaluated by transwell chamber methods. Immunoadherence of THP-1 was performed on cultured human umbilical vein endothelial cells (HUVEC). Cytokine released from PBMC was measured by ELISA assay kits. Lymphocyte-mediated cytotoxicity was carried out on KKU-452 cells. Proteins associated with immunomodulation were analyzed by Western immunoblotting assay. Results: Rice bran hydrolysates were rich in phenolic compounds, such as ferulic acid, catechin, quercetin, and quercetin glycosides. Rice bran hydrolysates suppressed phytohemagglutinin (PHA)- stimulated proliferation of PBMC and Jurkat cells, chemotaxis of Jurkat and THP-1 cells, and immunoadherence of THP-1 on HUVEC cultured cells. The cellular mechanism of rice bran hydrolysates involved the activation of AMPK as well as suppression of mTOR, NF-κB and VCAM-1. Rice bran hydrolysates potentiated PBMC on the PHA-stimulated release of IL-2, TNF-α, and IL-4, and enhanced PHA-induced non-MHC-restricted cytotoxicity on KKU-452 cancer cells. Conclusions: The immunomodulatory effect of phytochemicals derived from rice bran hydrolysates suggests its therapeutic potential for further investigation.

Cellulose nanocrystals induce a dose-dependent effect on cytotoxicity and proliferative activity of human peripheral blood mononuclear cells

<p>Introduction. Cellulose nanocrystals (CNCs) as attractive natural materials, have numerous applications in the biomedical field. Their unique biomechanical characteristics, surface chemistry, low cost and sustainable nature make them an engaging alternative to conventional materials and potentiate their use as progressive material. Therefore, it is critical to evaluate the cytocompatibility and immunomodulatory properties of nCNCs, which have not been completely explored. The objective of this study was to examine a dose-dependent effect of native (n)CNCs on cytotoxicity and proliferative activity of human peripheral blood mononuclear cells (PBMNCs) in vitro. Methods. PBMNCs, obtained from the healthy blood donors, were cultivated with nCNCs. Cell viability was analyzed by flow cytometry assay, while proliferative activity was determined by MTT, [3H]-thymidine uptake assay and detection of IL-2 production. Results. The cytotoxicity results suggested that no concentration of nCNCs (50-400 mg/ml) affected necrosis of PBMNCs, whereas apoptosis was induced by the highest concentration of nCNCs compared to control (p<0.05). Unexpectedly, the highest concentration of nCNCs increased the metabolic activity of PHA-stimulated cells compared to control (p<0.05). In contrast to these findings, lower concentrations of nCNCs (50 mg/ml and 100 mg/ml) stimulated proliferation of PBMNCs (p<0.05 and p<0.001). It was followed by increased production of IL-2 (100 mg/ml) (p<0.001). Conclusion. The results suggest that non-cytotoxic concentrations of nCNCs modulate the proliferative activity of human PBMNCs, a phenomenon which has not been published up to now and which is relevant for further studies.</p>

Design, creation and in vitro testing of a reduced immunogenicity humanized anti-CD25 monoclonal antibody that retains functional activity

Humanized and fully human sequence-derived therapeutic antibodies retain the capacity to induce anti-drug antibodies. Daclizumab (humanized version of the murine anti-Tac antibody; E.HAT) was selected for a proof of concept application of engineering approaches to reduce potential immunogenicity due to its demonstrated immunogenicity in the clinic. Reduced immunogenicity variants of E.HAT were created by identifying and modifying a CD4+ T cell epitope region in the VH region. Variant epitope region peptides were selected for their reduced capacity to induce CD4+ T cell proliferative responses in vitro. Variant antibody molecules were created, and CD25 affinity and potency were similar to the unmodified parent antibody. Fab fragments from the variant antibodies induced a lower frequency and magnitude of responses in human peripheral blood mononuclear cells proliferation tests. By the empirical selection of two amino acid mutations, fully functional humanized E.HAT antibodies with reduced potential to induce immune responses in vitro were created.

Vitamin K2 immunosuppressive effect on pediatric patients with atopic dermatitis.

Over 20 kinds of steroids, tacrolimus ointments, and cyclosporine capsules are usually recommended for the treatment of atopic dermatitis (AD), depending on the symptoms of patients. However, several side effects sometimes occur with the extensive use of these agents for the treatment of pediatric AD patients. The purpose of this study was to explore whether vitamin K2 could be a new immunosuppressive candidate for pediatric patients with AD. The immunosuppressive efficacy of vitamin K2 was evaluated through a cell-culture procedure using mitogen-activated peripheral blood mononuclear cells (PBMCs) obtained from pediatric AD patients. The mean (SD) IC50 value of vitamin K2 for the proliferation of concanavalin A-activated PBMCs was 15.37 (30.05)μmol/L, while the value for tacrolimus was 0.10 (0.28) ng/mL (0.12 (0.35)nmol/L). There was a significant correlation between the IC50 values for vitamin K2 and those for tacrolimus (P = 0.0001, r = 0.8871). However, there was no significant correlation between the IC50 values of vitamin K2 and those of cyclosporine A or methylprednisolone. A significant correlation between the IC50 values of vitamin K2 or tacrolimus and blood eosinophil counts (P = 0.0099, r = 0.7086 and P = 0.0032, r = 0.7722, respectively) was observed. Vitamin K2 -inhibited T-cell mitogen stimulated proliferation of PBMCs from pediatric AD patients in a dose-dependent manner. The PBMCs from pediatric AD patients were more sensitive to the immunosuppressive efficacy of vitamin K2 than the PBMCs from healthy subjects. The individual immunosuppressive pharmacological efficacy of vitamin K2 and of tacrolimus could be inferred from the blood eosinophil count of pediatric AD patients.

Abstract C056: MIV-818 stimulates an antitumor immune response in vitro and enhances the effects of pembrolizumab

Abstract Purpose: Investigation of immune-modulating activity of MIV-818. Background: MIV-818, a phosphoramidate prodrug of troxacitabine was designed to target the active metabolite troxacitabine-triphosphate to the liver while minimizing systemic exposure. MIV-818 is being developed for the treatment of cancer in the liver, and is currently being evaluated in a phase 1/2 study. Preclinical studies have demonstrated that some DNA damaging agents may further enhance antitumor immune responses in addition to direct cytotoxic effects. We therefore investigated whether MIV-818 induces similar immunomodulating effects as single agent or in combination with pembrolizumab. Methods: Immune-mediated tumor cell killing was determined by co-culturing labelled SKOV3 cancer cells with peripheral blood mononuclear cells (PBMC) and quantified over time using the IncuCyte ZOOM system. Effects on inflamed host tumor microenvironments (TMEs) were investigated by cytokine profiling co-cultures of activated PBMCs, primary fibroblasts or endothelial cells and HT29 colon adenocarcinoma cells. Effects on Staphylococcal enterotoxin B (SEB)-stimulated PBMC were determined by quantifying thymidine incorporation, and cytokine expression in supernatants. Results: Co-incubation of SKOV3 and PBM cells with MIV-818 induced a substantially higher level of apoptosis in the cancer cells (74±25%) compared to co-incubation in the absence of MIV-818 (37±23%). MIV-818 inhibited the proliferation of SKOV-3 cells in the absence PBMCs, but with no significant induction of apoptosis (IC50 7.5nM; ≤5% apoptosis). In a more complex model of the immune TME, MIV-818 treatment impacted inflammatory and angiogenesis-related responses in both the stromal and vascular co-culture systems, increasing IFNγ, IL-10, IL-17A, and TNFα in a manner comparable to that of pembrolizumab alone while also inhibiting IL-6. Combining MIV-818 with pembrolizumab further enhanced an immune-stimulatory effect by increasing IL-10, IL-17A and IFNy, while also modulating immune-related (GranB, IL-2, IL-6), and inflammation-related cytokines (VCAM-1, IP-10, TNFα). MIV-818 demonstrated dose-dependent inhibitory effect on proliferation of SEB-stimulated PBMCs. Despite this growth inhibitory effect, it led to a strong, dose-dependent, increase in IL-2 levels as a single agent which was further enhanced in combination with pembrolizumab. Conclusions: MIV-818 exerts favorable immunomodulating effects in vitro by increasing immune cytokine levels and the PBMC-mediated killing of cancer cells. These effects are further enhanced in combination with pembrolizumab and support future combination of MIV-818 with PD-(L)1 inhibitors in patients with HCC and other liver cancers. The effects of MIV-818 on tumor immune activation markers are being assessed the ongoing phase 1/2 clinical trial. Citation Format: Fredrik G Oberg, Karin Tunblad, Ana Slipicevic, Richard Bethell, Mark R Albertella. MIV-818 stimulates an antitumor immune response in vitro and enhances the effects of pembrolizumab [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C056. doi:10.1158/1535-7163.TARG-19-C056

MiR-98 Modulates Cytokine Production from Human PBMCs in Systemic Lupus Erythematosus by Targeting IL-6 mRNA.

Objective There is evidence that interleukin-6 (IL-6) upregulation plays a critical role in immunopathology of systemic lupus erythematosus (SLE). MicroRNA- (miRNA-) 98 was predicted to bind with the 3′-untranslated region (3′-UTR) of IL-6 gene. We hypothesized miR-98 through its regulation of IL-6 gene expression to influence cytokine production from peripheral blood mononuclear cells (PBMCs) in SLE. Methods The expression of miR-98 and IL-6 mRNA in the PBMCs of 41 SLE patients and 20 healthy controls (HC) was detected by quantitative reverse transcription PCR (qRT-PCR). The correlations between miR-98 expression and clinical features were evaluated. Luciferase reporter assay was performed to identify miR-98 targets. miR-98 mimics, miR-98 inhibitor, and IL-6 overexpression vector were generated. Cell viability of PBMCs was assessed using MTT assay. Gene expression and protein level were determined by qRT-PCR and Western blotting. TNF-α, IL-8, IL-1β, and IL-10 levels in cultured supernatants were quantified using ELISA. Results The expression of miR-98 was downregulated in PBMCs of SLE patients, and its expression is negatively associated with IL-6 levels. miR-98 expression was correlated with disease activity, lupus nephritis, and anti-dsDNA antibody. IL-6 mRNA was a target gene of miR-98. IL-6 overexpression promoted the proliferation of PBMCs and increased the levels of TNF-α, IL-8, IL-1β, and IL-10. Those effects were further enhanced by miR-98 inhibitor, while were suppressed by miR-98 mimics. miR-98 regulated the levels of STAT3 phosphorylation via its target gene IL-6. Conclusion The current study revealed that miR-98 could ameliorate STAT3-mediated cell proliferation and inflammatory cytokine production via its target gene IL-6 in patients with SLE. These results suggest that miR-98 might serve as a potential target for SLE treatment and other IL-6-mediated diseases.

Impaired immunomodulatory ability of type 2 diabetic adipose-derived mesenchymal stem cells in regulation of inflammatory condition in mixed leukocyte reaction.

The immunomodulatory properties of type 2 diabetic patients' adipose-derived mesenchymal stem cells (D-ASCs) has not been extensively studied. In this study, we compared the immunomodulatory properties of D-ASCs and non-diabetic subjects mesenchymal stem cells (ND-ASCs) in co-culture with mixed leukocyte reaction (MLR). ASCs were isolated from adipose tissue samples of type 2 diabetic and non-diabetic subjects (age: 40-55). D-ASCs and ND-ASCs were co-cultured with two-way MLR. Peripheral blood mononuclear cells (PBMCs) proliferation ratio, protein levels of IFN-γ and IL-10, mRNA expression of COX-2, TNF-α, TGF-β1 and IL-6 genes in MLR, D-ASCs and ND-ASCs co-cultures were assessed using XTT, ELISA and Real-time qRT-PCR, respectively. PBMCs proliferation on days 2 and 4 of D-ASCs co-culture was higher than ND-ASCs co-culture of the same days (p < 0.001). IFN-γ level decreased on day 4 compared to day 2 of ND-ASCs co-culture, but its level had not changed in D-ASCs co-culture. COX-2 expression on days 2 and 4 of D-ASCs co-culture was lower than ND-ASCs co-culture of the same days (p < 0.05). The expression of TNF-α and IL-6 on days 2 and 4 of D-ASCs co-culture were higher than ND-ASCs co-culture of the same days (p < 0.001). TGF-β1 on day 4 of ND-ASCs co-culture showed a slightly higher expression than D-ASCs co-culture of the same day. Lower suppression of PBMCs proliferation, declined expression of anti-inflammatory and upregulated expression of pro-inflammatory factors in D-ASCs co-culture, indicated an impairment of these cells in modulation of the inflammatory condition.

In vitro peripheral blood mononuclear cell proliferation, cytokine secretion and oxidative stress modulation by pyrido[2,3-d] pyrimidines

The aim of this work was to study the in vitro effects of new pyrido[2,3-d] pyrimidines, especially 4-(N-phenylethylamino)-5,7-diphenylpyrido[2,3-d] pyrimidine (3a) and 4-(N-benzylamino)-5-(4-chlorophenyl)-7-(3-methoxyphenyl) pyrido[2,3-d]-pyrimidine (3b), on the proliferative responses of human peripheral blood mononuclear cells, cytokine secretion, ATP levels and intracellular redox status. Peripheral blood mononuclear cells were isolated using differential centrifugation on a density gradient of Histopaque. These cells were cultured with the mitogen concanavalin A (Con A) and with different concentrations of the compounds 3a and 3b (1–100 µM). Proliferation (MTT assay), IL-2, INFγ and IL-4 (Elisa kits), ATP levels, cytotoxic effect (micronucleus test) and oxidative markers (intracellular glutathione, superoxide dismutase, catalase, hydroperoxide and carbonyl protein contents) were investigated after 48-h incubation. Our results showed that 3a was immunostimulant at low concentrations but immunosuppressive at high concentrations (50, 100 µM) with a modulation of cytokine secretion. 3b was immunostimulant in a dose-dependent manner. 3a induced an increase in intracellular ATP levels at 1–25 µM but a decrease at 50–100 µM. In the presence of 3b, cell ATP contents increased with 3b concentrations. Peripheral blood mononuclear cell redox markers were consistent with the presence of an oxidative stress induced by high concentrations of 3a and 3b, with a significant increase in the micronucleus frequency in lymphocytes, suggesting cytotoxic effects. However, 3a at low concentrations (1–25 µM) tended to activate antioxidant defense in cells. In conclusion, 3a and 3b modulate cell-mediated immunity, cell energy and redox status at low concentrations.