Cells In Peripheral Blood Mononuclear Cells Research Articles

Effect of polymorphisms in porcine guanylate-binding proteins on host resistance to PRRSV infection in experimentally challenged pigs

Guanylate-binding proteins (GBP1 and GBP5) are known to be important for host resistance against porcine reproductive and respiratory syndrome virus (PRRSV) infection. In this study, the effects of polymorphisms in GBP1 (GBP1E2 and WUR) and GBP5 on host immune responses against PRRSV were investigated to elucidate the mechanisms governing increased resistance to this disease. Seventy-one pigs [pre-genotyped based on three SNP markers (GBP1E2, WUR, and GBP5)] were assigned to homozygous (n = 36) and heterozygous (n = 35) groups and challenged with the JA142 PRRSV strain. Another group of nineteen pigs was kept separately as a negative control group. Serum and peripheral blood mononuclear cells (PBMCs) were collected at 0, 3, 7, 14, 21 and 28 days post-challenge (dpc). Viremia and weight gain were measured in all pigs at each time point, and a flow cytometry analysis of PBMCs was performed to evaluate T cell activation. In addition, 15 pigs (5 pigs per homozygous, heterozygous and negative groups) were sacrificed at 3, 14 and 28 dpc, and the local T cell responses were evaluated in the lungs, bronchoalveolar lavage cells (BALc), lymph nodes and tonsils. The heterozygous pigs showed lower viral loads in the serum and lungs and higher weight gains than the homozygous pigs based on the area under the curve calculation. Consistently, compared with the homozygous pigs, the heterozygous pigs exhibited significantly higher levels of IFN-α in the serum, proliferation of various T cells (γδT, Th1, and Th17) in PBMCs and tissues, and cytotoxic T cells in the lungs and BALc. These results indicate that the higher resistance in the pigs heterozygous for the GBP1E2, WUR and GBP5 markers could be mediated by increased antiviral cytokine (IFN-α) production and T cell activation.

Comparison of peripheral blood B cell subset ratios and B cell-related cytokine levels between ocular and generalized myasthenia gravis

BackgroundThe pathogenesis of myasthenia gravis (MG) depends upon T and B cells, as well as complement and cytokines. The activation of functional subpopulations of T and B cells is the basis of the immune response. However, the activation levels of T and B cell subsets remain unclear in the pathogenesis of MG. Here, we evaluated the proportions of T and B cell subsets and related cytokines in ocular MG (oMG) patients and generalized MG (gMG) patients, and analyzed the potential roles of T and B cell subsets in the pathogenesis of oMG. MethodsIn total, 16 patients with oMG, 31 patients with gMG, and 20 healthy controls (HCs) were included in this study. Peripheral blood mononuclear cells (PBMCs) were separated from venous blood via density centrifugation. The percentages of CD3+CD4+ T cells, CD3+CD8+ T cells, CD4+CD25+CD127- regulatory T cells (Tregs), CD19+CD27+CD38- memory B cells, CD19+CD24hiCD27+ regulatory B cells (Bregs), CD19+CD38+CD138+ plasma cells, and CD19+CD40+ B cells in PBMCs were detected by flow cytometry. The levels of interleukin (IL)-2, IL-4, IL-6, IL-10, IL-17, and interferon (IFN)-γ in serum were detected by enzyme linked immunosorbent assay (ELISA). Differences in T and B cell subsets and related cytokines were compared among the three groups of participants. ResultsThe proportions of CD19+CD27+CD38- memory B cells were significantly higher in the oMG and gMG groups than in the HC group (P = 0.004; P < 0.001), whereas the proportion of CD19+CD27+CD38- memory B cells was significantly lower in the oMG group than in the gMG group (P < 0.001). Moreover, the proportion of CD19+CD24hiCD27+ Bregs was significantly higher in the oMG group than in the gMG and HC groups (P = 0.001). The proportion of CD4+CD25+CD127- Tregs was significantly lower in the gMG group than in the oMG and HC groups (P < 0.001). Finally, the level of serum IL-10 was significantly higher in the oMG group than in the gMG and HC groups (P < 0.05). Compared with the HC group, serum IL-2 levels were significantly increased in the oMG and gMG groups (P = 0.016; P = 0.002). DiscussionThe reduced ratios of Tregs and Bregs may contribute to the progression of oMG to gMG, and the increased proportion of memory B cells may be closely related to the progression of oMG. IL-2 and IL-10 are important in the development of oMG.

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.

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

Proinflammatory Histidyl-Transfer RNA Synthetase-Specific CD4+ T Cells in the Blood and Lungs of Patients With Idiopathic Inflammatory Myopathies.

Autoantibodies targeting histidyl-transfer RNA synthetase (HisRS; anti-Jo-1) are common in the idiopathic inflammatory myopathies (IIMs) and antisynthetase syndrome. This study was undertaken to investigate immunity against HisRS in the blood and lungs of patients with IIM/antisynthetase syndrome. Bronchoalveolar lavage (BAL) fluid, BAL fluid cells, and peripheral blood mononuclear cells (PBMCs) from patients with IIM/antisynthetase syndrome (n = 24) were stimulated with full-length HisRS protein or a HisRS-derived peptide (HisRS11-23 ). BAL fluid and PBMCs from patients with sarcoidosis (n = 7) and healthy subjects (n = 12) were included as controls. The CD4+ T cell response was determined according to levels of CD40L up-regulation and cytokine expression using flow cytometry. Anti-Jo-1 autoantibody responses in the serum and BAL fluid were assessed by enzyme-linked immunosorbent assay. Lung biopsy samples from patients with IIM/antisynthetase syndrome (n = 14) were investigated by immunohistochemistry. In BAL fluid, CD4+ T cells from 3 of 4 patients with IIM/antisynthetase syndrome responded to stimulation with HisRS protein, as measured by the median fold change in CD40L expresssion in stimulated cells compared to unstimulated cells (median fold change 3.6, interquartile range [IQR] 2.7-14.7), and 2 of 3 patients with IIM/antisynthetase syndrome had the highest responses to HisRS11-23 (median fold change 88, IQR 27-149). In PBMCs, CD4+ T cells from 14 of 18 patients with IIM/antisynthetase syndrome responded to HisRS protein (median fold change 7.38, IQR 2.69-31.86; P < 0.001), whereas a HisRS11-23 response was present in 11 of 14 patients with IIM/antisynthetase syndrome (median fold change 3.4, IQR 1.87-10.9; P < 0.001). In the control group, there was a HisRS11-23 response in 3 of 7 patients with sarcoidosis (median fold change 2.09, IQR 1.45-3.29) and in 5 of 12 healthy controls (median fold change 2, IQR 1.89-2.42). CD4+ T cells from patients with IIM/antisynthetase syndrome displayed a pronounced Th1 phenotype in the BAL fluid when compared to the PBMCs (P < 0.001), producing high amounts of interferon-γ and interleukin-2 following stimulation. Anti-Jo-1 autoantibodies were detected in BAL fluid and germinal center (GC)-like structures were seen in the lung biopsy samples from patients with IIM/antisynthetase syndrome. The results of this study demonstrate a pronounced presence of HisRS-reactive CD4+ T cells in PBMCs and BAL fluid cells from patients with IIM/antisynthetase syndrome as compared to patients with sarcoidosis and healthy controls. These findings, combined with the presence of anti-Jo-1 autoantibodies in BAL fluid and GC-like structures in the lungs, suggest that immune activation against HisRS might take place within the lungs of patients with IIM/antisynthetase syndrome.

In vitro and In vivo Susceptibility of Baboons (Papio sp.) to Infection with and Apparent Antibody Reactivity to Simian Betaretrovirus (SRV).

Despite the lack of confirmed reports of an exogenous Simian betaretrovirus (SRV) isolated from baboons (Papio sp.), reports of simian endogenous gammaretrovirus (SERV) in baboons with complete genomes suggest that such viruses may be potentially infectious. In addition, serologic tests have repeatedly demonstrated antibody reactivity to SRV in baboons from multiple colonies. These findings complicate the management and use of such animals for research. To provide further insight into this situation, we performed in vitro and in vivo studies to determine if baboons are or can be infected with SRV. In our initial experiment, we were not able to isolate SRV from 6 seropositive or sero-indeterminate baboons by coculturing their peripheral blood mononuclear cells (PBMC) with macaque PBMC or permissive cell lines. In a subsequent experiment, we found that baboon PBMC infected in vitro with high dose SRV were permissive to virus replication. To test in vivo infectibil- ity, groups of naive baboons were infused intravenously with either (i) the same SRV tissue culture virus stocks used for the in vitro studies, (ii) SRV antibody positive and PCR positive macaque blood, (iii) SRV antibody positive or indeterminate, but PCR negative baboon blood, or (iv) SRV antibody and PCR negative baboon blood. Sustained SRV infection, as defined by reproducible PCR detection and/or antibody seroconversion, was confirmed in 2 of 3 baboons receiving tissue culture virus but not in any recipients of transfused blood from seropositive macaques or baboons. In conclusion, the data indicate that even though baboon cells can be infected experimentally with high doses of tissue culture grown SRV, baboons that are repeatedly SRV antibody positive and PCR negative are unlikely to be infected with exogenous SRV and thus are unlikely to transmit a virus that would threaten the SPF status of captive baboon colonies.

Involvement of Oxidative Stress and the Epidermal Growth Factor Receptor in Diesel Exhaust Particle-Induced Expression of Inflammatory Mediators in Human Mononuclear Cells.

Exposure to diesel exhaust particles (DEPs) has been associated with increased incidence of cardiopulmonary diseases. This study is aimed at examining the proinflammatory effects of DEP on primary human peripheral blood mononuclear cells (PBMC) and the underlying mechanisms using a human mononuclear cell line, THP-1. DEPs were incubated with the PBMC and THP-1 cells for 24 h, respectively. The supernatants were collected and subjected to measurement of proinflammatory mediators including interleukin 8 (IL-8) or tumor necrosis factor α (TNFα) by ELISA. Levels of reactive oxygen species (ROS) were determined using flow cytometry. Phosphorylation of the epidermal growth factor receptor (EGFR) was examined with immunoblotting. Exposure to DEP induced a concentration-dependent increase in the expression of IL-8 and TNFα in the PBMC and THP-1 cells. Further mechanistic studies with THP-1 cells indicated that DEP stimulation increased intracellular levels of ROS, an indicator of oxidative stress, and phosphorylation of the EGFR, indicative of EGFR activation. Pretreatment of THP-1 cells with the antioxidant N-acetyl-L-cysteine (NAC) markedly blunted DEP-induced EGFR phosphorylation, indicating that oxidative stress was involved in DEP-induced EGFR activation. Furthermore, the pretreatment of THP-1 cells with either NAC or a selective EGFR inhibitor significantly blocked DEP-induced IL-8 expression, implying that oxidative stress and subsequent EGFR activation mediated DEP-induced inflammatory response. In summary, DEP stimulation increases the expression of proinflammatory mediators in human mononuclear cells, which is regulated by oxidative stress-EGFR signaling pathway.

Subclinical Epstein-Barr Virus Primary Infection and Lytic Reactivation Induce Thyrotropin Receptor Autoantibodies.

Epstein-Barr virus (EBV) is a herpes virus that mainly infects in B lymphocytes and occasionally reactivates lytically. Most individuals have been infected with EBV primarily in their childhood with no symptoms, and the virus persists latently for life. We have previously reported that EBV-infected B cells with thyrotropin receptor autoantibodies (TRAbs) on their surface [TRAb(+) EBV(+) cells] were present in the peripheral blood mononuclear cells (PBMCs) of healthy adult controls and patients with Graves' disease, and that TRAbs released in the culture medium of PBMCs containing TRAb(+) EBV(+) cells by EBV reactivation. EBV lytic reactivation induced the differentiation of host B cells into plasma cells and antibody production. Various autoantibodies have been detected during the acute phase of infectious mononucleosis (IM) that is the symptomatic primary infection of EBV. Therefore, the autoantibody production may be induced by the asymptomatic primary infection. In this study, we examined the presence of TRAb(+) cells, EBV(+) cells, and TRAb(+) EBV(+) cells in PBMCs from 29 healthy or subclinical children without Graves' disease and one cord blood that were divided into six age groups, and also measured plasma TRAb levels. The results obtained demonstrated that low levels of TRAb production occurred with EBV primary infection and lytic reactivation in children without symptoms of IM. Furthermore, the populations of TRAb(+) cells, EBV(+) cells, and TRAb(+) EBV(+) cells were small in the period of primary infection, but they potentially expand with repeated EBV lytic reactivation. This may partly explain why the onset of Graves' disease often occurs in young adults, but rarely in infancy.

ASSOCIATION OF TUMOR ANGIOGENIC CELLS (CD133+/ VEGFA+) AND CIRCULATING CANCER STEM CELLS (CD133+/VEGFR2-) IN ASTROCYTIC GLIOMA PATIENTS

Background: Astrocytic gliomas are the most common primary brain tumors that developed from glial origin. The angiogenic cell population from brain tumor enhances the recruitment of circulating cancer stem cells homing towards tumor site.Objectives: This study aimed to investigate the tumor angiogenic cell population that stained with CD133+ and VEGFA+ markers and its association with circulating cancer stem cell (CD133+/VEGFR2-) population in the peripheral blood mononuclear cells (PBMCs) of astrocytic glioma patients.Methods: A total of 22 astrocytic glioma patients from Hospital Universiti Sains Malaysia who consented to the study were included. Tumors (n=22) were sliced and stained with CD133+ and VEGFA+ angiogenic markers and counter stained with DAPI. The circulating cancer stem cells (CD133+/VEGFR2-) in PBMCs (n=22) were quantified using FACS based on the expression of CD133 and VEGFR2 markers. The paired t-test and Pearson correlation were used for the data analysis.Results: The percentage of angiogenic cell population was significantly higher in brain tumor compared to adjacent normal brain tissue (1.25 ± 0.96% vs. 0.74 ± 0.68%; paired t-test=2.855; df=21, p = 0.009). Positive correlation was found between the angiogenic cells of brain tumor tissue and adjacent normal brain tissue (Pearson correlation, r = 0.53, p = 0.011). Significant positive correlation was found between angiogenic cells in glioma tumor and cancer stem cells in peripheral circulating systems of astrocytic glioma patients (Pearson correlation, r = 0.42, p = 0.049).Conclusion: Angiogenic cells in the brain tumor resident promote the recruitment of circulating cancer stem cells homing to the tumor site and induce the proliferation and growth of the tumor in astrocytic glioma patients.

An increase in circulating B cells and B cell activation markers in peripheral blood is associated with cigarette smoking in a male cohort in Bangladesh

In a cohort of approximately 200 Bangladeshi men, equally divided into smokers and non-smokers and equally divided by exposure to high and low levels of drinking water arsenic, we examined ex vivo a series of immune markers and immune function tests in peripheral blood mononuclear cells (PBMC). These immune parameters included PBMC cell surface markers (CSM) for B, T, monocytes, and NK cells, activated T and B cell markers, cytokine production in vitro, and analysis of CD4 subsets (Th1, Th2, Treg, and Th17 cells). We found that the effects of cigarette smoke were quite different than those associated with arsenic or polycyclic aromatic hydrocarbon (PAH)-DNA adducts. Cigarette smoking was associated with a significant increase in the number of PAH-DNA adducts as well as an increase in urinary levels of 1-hydropxypyrene (1-OHP). After correcting for arsenic exposure and PAH-DNA adducts, we found that cigarette smoking was associated with an increase in the percentage of CD19+ B cells, as well as the percentage of activated B cells (CD19+, HLA-DRbright cells) found in PBMC. These findings demonstrate activation of the immune system during chronic exposure to cigarette smoke, which is a known risk factor for autoimmune diseases.

Effects of Follicular Helper T Cells and Inflammatory Cytokines on Myasthenia Gravis.

Myasthenia gravis (MG) is an autoimmune disorder mediated by antibodies against the acetylcholine receptors (AChR) of the skeletal muscles. An imbalance in various T helper (Th) cells, including Th1, Th2, Th17, Th22 and follicular helper T (TFH) cells, has been found associated with immunological disturbances. In this study, we aim to investigate the role of the Th cells in peripheral blood of MG patients. A total of 33 MG patients and 34 age matched controls were enrolled in this study. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque density gradient centrifugation assay. The proportion of TFH cells in PBMC were analyzed using flow-cytometry assay by determining the levels of cellular markers CD4, CXCR5, CD45RO, CD45RA and ICOS and PD-1. The levels of IFN-γ, IL-4, IL-17 and IL-21 in serum were analyzed by Cytometric Bead Array. The serum IL-22 level was analyzed by ELISA. The frequency of TFH cells in PBMCs was higher than those in healthy subjects and correlated to the severity of MG patients. The levels of pro-inflammatory cytokines IFN-γ, IL-17 and IL-21 were elevated in the serum of MG patients, while there were no significant differences regarding the levels of IL-4 and IL-22 between MG patients and control subjects. Our findings suggest that Th cells and their cytokines balance of MG patients are involved in the clinical condition or severity of MG disease.

Fluoxetine induces glucose uptake and modifies glucose transporter palmitoylation in human peripheral blood mononuclear cells

ABSTRACTIntroduction: In line with the monoamine hypothesis of major depressive disorder (MDD), the clinical efficacy of the selective serotonin reuptake inhibitor fluoxetine has classically been ascribed to central serotonin enhancing properties. Current research described disturbances in brain energy metabolism in MDD. Additionally, fluoxetine showed beneficial effects in neuropsychiatric disorders associated with central energy imbalance.Areas covered: The effect of in vitro fluoxetine exposure on cellular glucose uptake and cerebral glucose transporter function was assessed in human peripheral blood mononuclear cells (PBMC) and murine neuroblastoma N2a cells. Fluoxetine augmented glucose uptake, measured by utilizing the radionuclide-labled glucose analog [18]F-fluorodeoxyglucose, in PBMC without affecting glucose transporter protein content. Analysis of protein palmitoylation using the acyl-biotinyl exchange method revealed GLUT3 to be palmitoylated in PBMC and N2a cells, while palmitoylation of GLUT1 was detected only in N2a cells. Treatment with fluoxetine significantly increased palmitoylation of GLUT3 in PBMC and strongly induced palmitoylation of GLUT1 in PBMC and N2a cells.Expert opinion: Our findings suggest a novel mechanism exerted by fluoxetine targeting glucose metabolism by regulating glucose transporter palmitoylation. Thus, fluoxetine might evoke its therapeutic effects in neuropsychiatric diseases characterized by disturbances in central energy metabolism at least partly by improving cerebral glucose uptake.

RESVERATROL SIGNIFICANTLY ATTENUATES CELL MEDIATED IMMUNE REJECTION AGAINST XENOGENEIC BOVINE PERICARDIUM

Resveratrol is a major bioactive phenol found in red wine and red grapes that has been shown to reduce T cell activation and proliferation through inhibition of protein kinase B (PKB/Akt) and mTOR pathways. Recent studies have shown that humoral and cell-mediated immune reactions cause structural valve deterioration (SVD) in xenograft valves constructed out of bovine pericardium. Thus, tissue engineered heart valves (TEHV) which involve the process of decellularizing bovine pericardium have been developed to reduce xenoreactive immune response. Our study examines whether resveratrol could further reduce cell-mediated immune rejection against xenograft bovine pericardium thereby serving as a potential adjuvant therapy to TEHV. Human peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Paque gradient centrifugation and resuspended in RPMI 1640 complete medium containing 10% human serum, 2mM L-glutamine, 50 IU penicillin/ml, and 50 ml streptomycin/ml. The cells were further split into a resveratrol-treated group (50μM/ml) and a vehicle-treated group containing ethanol (50μM/ml) and cultured overnight (18hrs) in a 37C 5% CO2 incubator. 2ml of the complete medium from each group containing 1 X 10ˆ6 PBMC cells were then aliquoted into 12-well cell culturing plate with each well containing either native bovine pericardium, decellularized bovine pericardium, allograft pericardium, autograft pericardium, or no pericardium. The exposed culture media were then collected for ELISA analysis after 1 and 3 days. Tumor necrosis factor-a (TNF-a), chiefly produced by macrophages in cell-mediated immunity, were measured. Resveratrol-treated cells showed a significant reduction in all of the tissue exposure types in comparison to vehicle-treated controls at both 1 and 3 days (P < 0.05). Additionally, both vehicle control and resveratrol-treated groups at each time points generated the highest TNF-a concentration with native bovine pericardium exposure followed by the decellularized bovine pericardium, autograft pericardium, allograft pericardium, and no tissue. Resveratrol significantly attenuates cell-mediated immune rejection against xenogenic bovine pericardium exposure to an extent comparable to allograft and autograft human pericardium exposures. Therefore, resveratrol may serve as effective adjuvant therapy in patients receiving TEHV replacements to further decrease the xenogenic immune shown to cause SVD.

Increased Human Interleukin-32 Expression Is Related to Disease Activity of Graves' Disease.

Recently, abnormal expression of interleukin-32 (IL-32) has been involved in various inflammatory or autoimmune diseases, but the level of IL-32 expression in Graves' disease (GD) is still unknown. This study is aimed to explore the human IL-32 expression in GD and the association of IL-32 expression with the disease activity of GD. A total of 125 GD patients and 97 normal controls (NC) were recruited in this study. We examined IL-32 mRNA level in peripheral blood mononuclear cells (PBMCs) of 43 GD patients and 41 controls using real-time polymerase chain reaction (RT-PCR). Serum IL-32 level of 40 GD patients and 34 controls was measured by enzyme linked immunosorbent assay (ELISA). In another cohort including 42 GD patients and 22 controls, we detected the percentages of IL-32α+ cells, CD4+IL-32α+T cells, and CD4−IL-32α+ cells in PBMCs by flow cytometry. In GD patients, IL-32 mRNA expression was dramatically higher than that in controls (P < 0.001) and positively associated with FT3 (P = 0.036, r = 0.321). Subgroup analysis revealed that IL-32 mRNA level was elevated in both newly onset GD and refractory GD group (P < 0.01, P < 0.001, respectively) compared with controls. Furthermore, in refractory GD group, the IL-32 mRNA expression also positively correlated with FT3 (P = 0.019, r = 0.560). In addition, serum IL-32 level was notably higher in GD patients than that of controls (P < 0.01). Subgroup analysis also indicated that serum IL-32 level in both newly onset GD and refractory GD group was higher in comparison with controls (P = 0.015, P = 0.023, respectively) and serum IL-32 level in refractory GD patients positively correlated with TRAb (P = 0.043, r = 0.481). The percentages of IL-32α+ cells, CD4+IL-32α+ T cells, and CD4−IL-32α+ cells were all significantly enhanced in GD patients compared with controls (P = 0.005, P = 0.017, P = 0.016, respectively). IL-32 and IL-32α+ cells may be associated with the pathogenesis of GD. IL-32 may become a promising target for the treatment of GD.

Mycobacterium avium sp. paratuberculosis (MAP) induces IL-17a production in bovine peripheral blood mononuclear cells (PBMCs) and enhances IL-23R expression in-vivo and in-vitro

Johne’s disease (JD) is a chronic inflammatory gastrointestinal disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Control of JD is difficult largely due to insensitive diagnostic tools, a long subclinical stage of infection, and lack of effective vaccines. Correlates of protection are lacking in model systems of JD and the sources of inflammation due to JD are not well characterized. Commonly studied immune responses, such as the Th1/Th2 paradigm, do not adequately explain host responses to MAP. A potential role for non-classical immune responses to MAP, such as that mediated by Th17 cells, has been suggested. Indeed, MAP antigens induce mRNAs encoding the cytokines IL-23 and IL-17a in bovine peripheral blood mononuclear cells (PBMCs). IL-23 and IL-17a production have both been associated with Th17-like immune responses. Th17 cells are also defined by surface expression of the IL-23 receptor (IL-23R). To determine the relative prevalence of potential Th17 cells in PBMCs from MAP test positive and MAP test negative cows, PBMCs were isolated and analyzed by immunostaining and flow cytometry. Fresh PBMCs from MAP test positive cows (n = 12) contained a significantly higher proportion of IL-23R positive cells in populations of CD4+, CD8+, and Yδ + T cells than in cells from MAP test negative cows (n = 12; p < 0.05). Treatment with MAP antigens increased the percentage of all T cell subsets with surface expression of IL-23R when compared to untreated (n = 12; p < 0.05) cells. ELISA results for IL-17a secretion revealed a higher concentration of IL-17a secreted from PBMCs treated with MAP antigen (n = 20) than from PBMCs not treated with MAP antigens (n = 20) (p < 0.001), regardless of the JD test status of source cows. Also, we observed a moderate negative correlation between JD diagnostic scores for JD + cows and plasma IL-17a concentration (n = 42; r = −0.437; p-value < 0.004). Plasma with low and mid JD- scores (n = 31; n = 9; 0.1 ≤ X < 0.3) had significantly more IL-17a when compared to plasma with high JD- scores (n = 10; 0.3 ≤ X < 0.46; p-values < 0.05). Similarly, plasma with low JD + score values (0.55 ≤ X < 1.0; n = 9) had significantly more IL-17a when compared to plasma with high JD + score values (X ≥ 2.0; n = 21; p < 0.05). Overall, plasma from JD + cows (0.55 < X ≤ 2.86; n = 41) had significantly less IL-17a than plasma from JD- cows (0 < X ≤ 0.46; n = 70). Our data suggests that Th17-like cells may indeed play a role in early immune responses to MAP infection and development or control of JD.

Evaluation of Anti-Aging Activity of the Biofield Energy Treated Novel Test Formulation Using SIRT1 and Telomerase Activity in in Vitro Model

Telomerase and SIRT1 (member of the sirtuin protein family) along with the lifestyle and diet are the major determinants of aging and its associated diseases such as cancer and cardiovascular disorders. The study objective was to investigate the effect of Consciousness Energy Healing based novel test formulation in pre-adipocytes (3T3-L1) and human peripheral blood mononuclear cells (PBMCs) for anti-aging activity using SIRT1 and telomerase assay. The test formulation was divided into two parts. One portion was denoted as the untreated test item without any Biofield Energy Treatment, while the other portion was defined as the Biofield Energy Healing Treatment, which received the Biofield Energy Healing Treatment by a renowned Biofield Energy Healer, Mahendra Kumar Trivedi. The cell viability using MTT assay showed that the cell viability of 3T3-L1 and PBMCs cells was more than 70% indicating a safe and nontoxic profile. The experimental data in PBMCs cells showed that the Biofield Energy Treated Test formulation showed a significant improved telomerase activity by 39.25%, 20.86%, and 17.95% at concentrations 0.01, 5, and 100 µg/mL, respectively as compared with the untreated test formulation group. These results indicate that the Biofield Energy Healing Treatment would be the significant approach to prevent aging-related disorders such as decline cardiovascular diseases, osteoporosis, dementia, osteoarthritis, Alzheimer’s, hypertension, cancer, Parkinson's Disease, Chronic Obstructive Pulmonary Disease (COPD), Stress, Asthma, cataract, age-related macular degeneration (AMD), hearing loss and metabolic disorders.

Chitosan gold nanoparticles induce different ROS-dependent cell death modalities in leukemic cells.

BackgroundNanotechnology proposes the use of gold nanoparticles (AuNPs) for drug delivery, diagnosis, and treatment of cancer. Leukemia is a type of hematopoietic cancer that results from the malignant transformation of white blood cells. Chitosan-coated AuNPs (CH-AuNPs) are cell death inductors in HeLa and MCF-7 cancer cells without affecting peripheral blood mononuclear cells (PBMC). Considering the selectivity and versatile cytotoxicity of CH-AuNPs, we evaluated whether their selectivity is due to the cell lineage or the characteristics of the cancer cells, by assessing its cytotoxicity in leukemic cells. Moreover, we further examined the cell death mechanism and assessed the implication of nuclear damage, autophagosome formation, and the cell death mechanism induced in leukemic cells.Materials and methodsWe synthesized CH-AuNPs by chemical methods and analyzed their cell death capacity in a T-acute lymphocytic leukemia cell line (CEM), in a chronic myeloid leukemia cell line (K562), and in healthy cells from the same lineage (PBMC and bone marrow, BM, cells). Then, we assessed ROS generation and mitochondrial and nuclear damage. Finally, we evaluated whether cell death occurred by autophagy, apoptosis, or necroptosis, and the role of ROS in this mechanism.ResultsWe found that CH-AuNPs did not affect PBMC and BM cells, whereas they are cytotoxic in a dose-dependent manner in leukemic cells. ROS production leads to mitochondrial and nuclear damage, and cell death. We found that CH-AuNPs induce apoptosis in CEM and necroptosis in K562, both undergoing autophagy as a pro-survival mechanism.ConclusionCH-AuNPs are selective cell death inductors in hematologic cancer cells, without affecting their healthy counterparts. Cell death induced by CH-AuNPs is independent of the cancer cell type; however, its mechanism is different depending on the type of leukemic cells.

Hetero-Polysaccharides Secreted from Dunaliella salina Exhibit Immunomodulatory Activity Against Peripheral Blood Mononuclear Cells and RAW 264.7 Macrophages.

Several species of microalgae have been known to produce exopolysaccharides (EPS) with potential immune activity. In the present investigation, ethyl acetate fraction of crude EPS secreted by Dunaliella salina was explored for immunomodulatory activity against peripheral blood mononuclear cells (PBMC) and RAW 264.7 macrophages. Effect of EPS on cell growth and cytokines production were measured using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and ELISA respectively. Griess reagent was used for measuring the nitric oxide production in RAW 264.7 macrophages. FTIR analysis and mass spectroscopy were carried out for the characterization. Ethyl acetate fraction exhibited dose dependent increase in proliferative index and cytokines production (IFN-γ, TNF-α, TGF-β). At low concentration (250 and 500µg/mL), it showed growth inhibition and at higher concentration (1000 and 1500µg/mL), it enhanced the cell growth. Interestingly, the pronounced increased TNF-α production was observed in ethyl acetate fraction treated PBMC cells at higher concentration (750 and 1000µg/mL) indicating the immunostimulatory effect. In RAW cells, concentration dependent diminished cell growth (IC50 = 691µg/mL) and nitric oxide production (IC50 = 630µg/mL) was observed. FTIR analysis showed the presence of polysaccharides due to the detection of hydroxyl (-OH), Carbonyl (C-O) and alkyl (C-H) groups. Mass spectroscopy results revealed ethyl acetate fraction as penta-saccharide (m/z = 887.56 and 886.54) which are confirmed to be hetero-polysaccharides consisting of hexoses and pentoses along with association of ions. These results suggest that penta-saccharide (ethyl acetate fraction) isolated from D. salina may have the potential to be used for therapeutic purpose as immunomodulatory agent.

Patients with BRCA mutated ovarian cancer may have fewer circulating MDSC and more peripheral CD8+ T cells compared with women with BRCA wild-type disease during the early disease course.

Immunosuppressive myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) are associated with immunologic tolerance and poor prognosis in ovarian cancer (OvCa). We hypothesized that women with germline BRCA1 and BRCA2 mutation-associated (gBRCAm) OvCa would have fewer circulating immunosuppressive immune cells compared to those with BRCA wild-type (BRCAwt) disease during their early disease course (<5 years post-diagnosis) where gBRCAm is a favorable prognostic factor. We collected and viably froze peripheral blood mononuclear cells (PBMCs) from patients with recurrent OvCa olaparib clinical trials (NCT01445418/NCT01237067). Immune subset analyses were performed using flow cytometry for Tregs, exhausted CD8+ T cells, monocytes and MDSCs. Functional marker expression, including cytotoxic T lymphocyte-associated protein 4 (CTLA-4), T cell immunoglobulin and mucin domain 3 (TIM-3) and programmed cell death protein 1 (PD-1) was evaluated. Data were analyzed using FlowJo. Pretreatment PBMCs were collected from 41 patients (16 gBRCAm/25 BRCAwt). The percentage of MDSCs among viable CD45+ PBMC was lower in gBRCAm OvCa compared with BRCAwt OvCa (median 0.565 vs. 0.93%, P=0.0086) but this difference was not seen in those women >5 years post-diagnosis. CD8+ T cells among viable CD45+ PBMCs and CTLA-4+/CD8+ T cells were higher in gBRCAm carriers than patients with BRCAwt, in particular for those <5 years post-diagnosis (median 20.4 vs. 9.78%, P=0.031 and median MFI 0.19 vs. 0.22, P=0.0074, respectively). TIM-3 expression on Tregs was associated with poor progression-free survival, independent of gBRCAm status (P<0.001). Our pilot data suggested that patients with gBRCAm OvCa may have fewer circulating MDSCs but higher CD8+ T cells in PBMCs during their early disease course. This may contribute to the observed survival benefit for these women in their first post-diagnosis decade.

Autophagy protects peripheral blood mononuclear cells against inflammation, oxidative and nitrosative stress in diabetic dyslipidemia

BackgroundType 2 diabetes mellitus (T2DM) results in severe oxidative and nitrosative stress and inflammation when associated with hyperlipidemia. In this study, we have attempted to explore the role of autophagy in T2DM subjects with or without dyslipidemia. MethodsExperiments were carried out in isolated Peripheral blood mononuclear cells (PBMC) from study subjects and insulin resistant HepG2 cells utilizing flow cytometry, confocal microscopy and molecular biology techniques like western blotting, immunofluorescence and real time PCR. ResultsIn case of T2DM with dyslipidemia, higher population of autophagy positive cell was detected compared to T2DM which may have been originated due to higher stress. Flow cytometric data indicated autophagy to be triggered by both oxidative and nitrosative stress in PBMC of diabetic dyslipidemic patients, which is a novel finding of our work. Expression of LC3 puncta, a hallmark of autophagy was observed at periphery of PBMC and Hep G2 cells in case of diabetic dyslipidemic condition. Increased expression of ATG5, LC3B and Beclin1 supports the autophagic pathway in both PBMC and Hep G2 cells. Upon blocking autophagy by 3-methyl adenine (3MA), the apoptotic cell population increased significantly. Autophagy was also been evidenced to control oxidative stress mediated up-regulation of inflammatory markers like IL-6, TNF-α. ConclusionInduction of autophagy emerged to be a protective mechanism for the diabetic cells coupled with dyslipidemia. Not only Reactive oxygen species, but also reactive nitrogen species was involved in autophagy induction process. Moreover inhibition study documented autophagy to have a protective role in pro-inflammatory responses. Thus, enhancing autophagic activity may be an efficient mechanism leading to new therapeutic strategy to restore the glycemic regulation.