Unstimulated Peripheral Blood Mononuclear Cells Research Articles

Differential cytokine response of Escherichia coli lipopolysaccharide stimulated peripheral blood mononuclear cells in crossbred cattle, Tharparkar cattle and Murrah buffalo - An in vitro study

Mastitis is a complex disease responsible for huge economic losses to the dairy sector. The causal organisms include a wide variety of micro-organisms including several species of bacteria. Escherichia coli has been identified as one of the most common gram-negative bacteria causing clinical mastitis in cattle. The immune system, of different species and/or breeds, tries to combat these pathogens in an inconsistent manner with differential mode and intensity of immune response, eventually producing contradicting outcomes of this disease. Several reports suggest the existence of variability among different animal breeds/species, resulting in a dissimilar outcome of this disease among them. In order to evaluate the variation among different breeds/species, the present study was undertaken to examine the stimulant effect of E. coli lipopolysaccharide (LPS) on peripheral blood mononuclear cells (PBMCs). The PBMCs were harvested from blood samples of crossbred cattle, Tharparkar cattle and Murrah buffaloes. After 6 h of in vitro stimulation, qRT-PCR was employed to measure the relative mRNA expression levels of CCL5, IL-1β, IL-12β, IFN-γ and IL-10 genes in stimulated and unstimulated PBMCs. The selected genes revealed significant differences in the pattern of innate immune response among crossbred cattle, Tharparkar cattle and Murrah buffaloes. The results clearly indicate the presence of variation in the outcome of immune response even when the immunocytes were stimulated with the same dose of the antigen.

Evidence for a Role of TGF-β-Activated Kinase 1 and MAP3K7 Binding Protein 3 in Peanut-Specific T-Cell Responses

Peanut allergy is considered to be the most common cause for food-induced anaphylaxis. Currently, no approved treatment is available. Avoidance is the only measure to prevent anaphylactic reactions to peanuts. T-helper cells are of special importance for the sensitization process and the maintenance of allergic inflammation. Identifying markers of allergen-specific T-cell responses may help to develop novel treatment approaches. Therefore, we aimed to define new T-cell target genes in Ara h 2-specific T cells and to investigate the possibility of using them as biomarkers of peanut allergy in peripheral blood mononuclear cells (PBMCs). We performed whole mRNA array analysis (whole human genome oligo microarray) of in vitro expanded Ara h 2-specific T cells (CFSE^lowCD3⁺CD4⁺) from 5 peanut-allergic (PA) and 5 non-peanut-sensitized individuals. Expression of selected genes as a result of a two-step bioinformatic approach was confirmed in a second cohort by quantitative PCR. TGF-β- activated kinase 1 and MAP3K7 binding protein 3 (TAB3), calcium/calmodulin-dependent protein kinase type IV (CAMK4) and HemK methyltransferase family member 1 (HEMK1) were significantly upregulated in Ara h 2-specific T cells of PA patients. In addition, the expression of these genes was also assessed in unstimulated PBMCs from a cohort (n = 43) of PA, atopic non-PA, and nonatopic controls. Interestingly, in unstimulated PBMCs, TAB3 expression was significantly downregulated in PA patients compared to atopic non-PA individuals. Thus, TAB3 may play a significant role at the level of T-cell activation and may also be a candidate biomarker for PA.

Intravenous immune globulin affects cytokine production in T lymphocytes and monocytesjmacrophages

Immune globulin for intravenous use (IVIG) has been used in many inflammatory conditions due to its immunomodulatory potential. The effector mechanisms are incompletely understood. This study dealt with the effects of IVIG on cytokine production in vitro. Cytokine synthesis was identified at the single-cell level using cytokine-specific MAb and indirect immunocytochemical techniques. Peripheral blood mononuclear cells (PBMC) were stimulated for 96 h by immobilized anti-CD3 MAb or by a combination of a protein kinase C activator (PMA) and a calcium ionophore (ionomycin). The addition of IVIG (6 mg/ml) caused a marked inhibition of proliferation and blast transformation despite unaffected cell survival. Anti-CD3-stimulated cultures containing IVIG exhibited a significant inhibition of production of T-cell derived lymphokines IL-2, IL-10, TNF-beta, IFN-gamma and TNF-alpha (made by both monocytes and T cells), while synthesis of the monokine IL-8 was significantly increased. The expression of IL-2 receptors was significantly suppressed. Similar but transient inhibition of most T-cell products (IL-2, IL-3, IL-4, IL-5, IL-10, TNF-beta and GM-CSF) was noted in the PMA/ionomycin-containing cultures. In contrast, no effects were found on IFN-gamma or TNF-alpha production. The superantigen streptococcal pyrogenic exotoxin-A (SPE-A) induced vigorous cell activation and extensive cytokine synthesis. IVIG was added either at the beginning or 24 h after the initiation of cultures in order to elucidate the importance of direct toxin-neutralization. Addition of IVIG from the beginning of cultures induced a strong reduction of blast transformation and an almost complete inhibition of lymphokine production, in particular of IFN-gamma and TNF-beta. Supplementation with IVIG 24 h after initiation of cultures also led to a significant decrease in lymphokine synthesis. Monokine production (IL-1 alpha, IL-1 beta, IL-1ra, IL-6 and IL-8) was either unaffected or even increased. These two facts argue against direct antigen-neutralization as being the only mechanism at work. However, in IVIG-exposed PBMC stimulated with LPS, IL-6 production was significantly reduced. A significant upregulation of IL-1ra was noticed in unstimulated PBMC cultured with IVIG. The results in all the experiments did not indicate a cytotoxic effect by IVIG on cell survival and the production of certain cytokines were unaffected. Instead, the authors believe that the results suggest a previously little examined functional link where the humoral immune response may have direct immunoregulatory effects on the cellular immune system.

2027. Immune Correlates of Protection Against Herpes Zoster (HZ) in People Living with HIV (PLWH)

BackgroundHZ has high morbidity in immune-compromised hosts, including PLWH. Two vaccines against HZ are available, but their efficacy in PLWH is unknown. A surrogate marker of protection against HZ would facilitate efficacy studies of HZ vaccines in PLWH, as compared with HZ clinical outcome studies. To this goal, our study investigated the association of cytotoxic T lymphocytes (CTL) and regulatory T cells (Treg) with incident HZ in PLWH.MethodsThe study used peripheral blood mononuclear cells (PBMC) cryopreserved at mean (SD) of 2.7 (2.5) months before HZ from 31 PLWH cases on ART with plasma vRNA < 200 c/mL, and CD4 counts ≥200 cells/µL. 31 non-HZ controls were matched to cases by CD4 count, age, sex, race, parent study, and ART duration. T cell subsets were measured by flow cytometry after ex vivo VZV- and mock-restimulation and in freshly thawed unstimulated PBMC using the following markers: CD3, CD8, CD25, CD28, CD39, CD57, CD127, CTLA4, FOXP3, IFNg, IL10, KLRG1, LAG3, PD1, perforin, TGFb, TIM3 and TNFa. Data were analyzed by paired t-tests.ResultsAt PBMC collection, PLWH had mean (SD) age 41 (10) years; 16 females; 28 White and 22 Black non-Hispanics; mean (SD) CD4 counts 501 (222) cells/µL; 56 (90%) vRNA < 50 c/mL; median ART duration 50 weeks. In unstimulated PBMC, HZ cases had higher %FOXP3+CD25+(CD4+) Treg compared with controls (P = 0.08). Cases generally had lower responses to ex vivo VZV-restimulation (after subtraction of mock) compared with controls, including %perforin+(CD8+) CTL (P = 0.07) and %IL10+(CD4+) (P = 0.04) and %TGFb+(CD4+) Treg (P = 0.07). %FOXP3+CD25+(CD4+) Treg in unstimulated PBMC did not correlate with VZV-specific %perforin+(CD8+) or %IFNg+(CD4+) effector T cells, but the VZV-specific effector T cell percentages correlated with each other (r = 0.24, P = 0.05).ConclusionAlthough limited by small numbers, this study showed that before development of HZ, PLWH had marginally increased number of Treg in blood and decreased VZV-specific responses. VZV-specific Treg and CTL were equally lower in cases compared with controls, suggesting that a general low VZV-specific T cell responsiveness precedes HZ in PLWH. The lack of association between circulating Treg and VZV-specific T cell responses suggests independent mechanisms of action.Disclosures All authors: No reported disclosures.

Overexpression of Regulatory T Cell-Related Markers (FOXP3, CTLA-4 and GITR) by Peripheral Blood Mononuclear Cells from Patients with Breast Cancer

Background:Regulatory T (Treg) cells are immunosuppressor lymphocytes that play a critical role in the establishment and progression of cancers. A number of markers, especially FOXP3, CTLA-4 and GITR influence the function of Treg cells. This investigation aimed to evaluate the expression of a number of important Treg cell-related markers by peripheral blood mononuclear cells (PBMCs) from newly-diagnosed women with breast cancer.Methods:The fresh PBMCs were obtained from 20 women with breast cancer and 20 healthy individuals. The PBMCs from both groups were cultured for 32 hours in the presence or absence of PHA (10 μg/ml). After total RNA extraction from cultured PBMCs, the expression of the FOXP3, CTLA-4 and GITR transcripts was assessed using real time-PCR.Results:The mRNA expression of FOXP3, CTLA-4 and GITR in unstimulated PBMCs from patients with breast cancer were significantly higher than healthy control group (P<0.05, P<0.03 and P<0.04, respectively). Similarly, the expression of FOXP3, CTLA-4 and GITR transcripts in PHA-stimulated PBMCs from patients with breast cancer were significantly increased in comparison with healthy individuals (P<0.01, P<0.005 and P<0.01, respectively).Conclusion:The increased expression of FOXP3, CTLA-4 and GITR represent higher activity of Treg cells in patients with breast cancer that may play an important role in the tumor establishment and development.

In vitro proinflammatory gene expression predicts in vivo telomere shortening: A preliminary study

The chronic psychological stress of caregiving leads to higher risks for many diseases. One of the mechanisms through which caregiving is associated with disease risk is chronic inflammation. Chronic inflammation may accelerate cellular aging via telomere dysfunction and cell senescence, although this has not been examined in human cells from healthy people. We examined peripheral blood mononuclear cells (PBMCs) from 20 healthy mothers of children with autism (caregivers) and 19 mothers of neurotypical children (controls) in an in vitro culture system where PBMCs were stimulated with phytohaemagglutinin (PHA). We measured RNA expression levels of a panel of immune function genes before and after PHA stimulation, as well as telomere length from PBMCs collected from the participants at baseline and 15 months later. Caregivers and controls had similar gene expression profiles in unstimulated PBMCs, but after PHA stimulation, caregivers had increased RNA levels of the master inflammatory regulator NF-κB and its proinflammatory cytokine targets IL-1β, IL-6 and its receptor IL-6R as well as inflammatory chemokines IL-8, CXCL1 and CXCL2. Gene expression analysis suggested caregivers have increased Treg and Th17 T cell differentiation. Additionally, key signaling molecules involved in the upregulation of COX-2, a critical enzyme in the synthesis of the inflammatory mediator prostaglandin, were elevated. When both groups were examined together, higher expression levels of proinflammatory genes were associated with shorter telomere length in PBMCs from blood drawn 15 months later, independent of baseline telomere length. Taken together, these results suggest that chronic stress is associated with an exaggerated inflammatory response in PBMCs, which in turn is associated with shorter telomere length measured from PBMCs collected 15 months later. To our knowledge, this is the first human study that shows increased proinflammatory expression predicts future telomere shortening.

Peptidoglycan and Lipoteichoic Acid Induces Differential mRNA Response of Immune-Related Genes in PBMC of Crossbred, Tharparkar Cattle and Murrah Buffalo

ABSTRACTSubclinical mastitis, generally caused by Staphylococcus aureus, has a global economic impact all over the world. Hence, it needs to be resolved on higher priority which may be attained via. selection of mastitis resistant animals or inclusion of mastitis resistant trait into herd apart from management care. Diverse hosts with various genetic make-ups encounter pathogens in a diverse manner which in turn leads to contradicting outcome of the disease. Identification of species-wise or breed-wise differential expressed genes in response to S. aureus through relative evaluation of transcripts may be useful for judging the immuno-competency of a species or breed toward mastitis. The present study was undertaken to examine the stimulant effect of S. aureus peptidoglycan (PGN) and lipoteichoic acid (LTA) on Peripheral blood mononuclear cells (PBMC) harvested from blood samples of crossbred cattle, Tharparkar cattle, and Murrah buffaloes. After 6 h of in vitro stimulation qRT-PCR was used to measure the relative mRNA expression of TLR-2, TNF-α, IL-8, IFN-γ and IL-10 genes in stimulated and un-stimulated PBMC. The selected genes revealed significant differences in the pattern of immune response among crossbred cattle, Tharparkar cattle and Murrah buffalo in spite of the same stimulant dose.

Aerobic Training Status and Fatty Acid-Induced hTERT mRNA Expression Following Maximal Exercise

PURPOSE: Telomeres protect the ends of cellular chromosomes from degradation. Although telomere length within immune cells shorten naturally with age, increasing risk of disease and all-cause mortality, physical activity preserves telomere length by increasing mRNA expression of the telomerase component, telomerase reverse transcriptase (hTERT). Therefore, this study examined the influence of aerobic training status on the capacity of peripheral blood mononuclear cells (PBMCs) to express hTERT mRNA following palmitate stimulation. METHODS: PBMCs were isolated from 12 trained (T) and 11 untrained (UT) subjects pre- and post-maximal exercise, and stimulated with or without palmitate (4 hours) to examine changes in hTERT mRNA expression. RESULTS: hTERT mRNA expression remained unaltered following palmitate stimulation in T subjects at rest and in response to maximal exercise. To the contrary, palmitate- induced hTERT mRNA expression was elevated at rest relative to unstimulated PBMCs in UT subjects and following maximal exercise was suppressed relative to unstimulated PBMCs and pre-exercise expression levels (F [1, 81] = 7.874, p = 0.006). In addition, cardiorespiratory fitness (VO2max) was negatively associated with the percent change in hTERT mRNA expression in unstimulated PBMCs (r = -0.496, p = 0.022) and positively associated with the percent change in hTERT mRNA expression following palmitate stimulation (r = 0.468, p = 0.032). CONCLUSION: This data suggests that aerobic training may preserve the capacity of immune cells to protect against inflammatory-induced telomere shortening following acute physiological stress.

Evaluation of the TLR negative regulatory network in CVID patients.

Common variable immunodeficiency (CVID), a clinically symptomatic primary immunodeficiency disease (PID), is characterized by hypogammaglobulinemia leading to recurrent infections and various complications. Recently, some defects in the signaling of TLRs have been identified in CVID patients which led us to investigate the expression of TLR4 and 9 negative regulatory molecules and their upregulation status following their activation. Using TaqMan real-time PCR, SOCS1, TNFAIP3, RFN216, and IRAK-M transcripts among peripheral blood mononuclear cells (PBMCs) were measured with/without TLR4 and 9 activations. TLR4 and 9 were activated by lipopolysaccharide (LPS) and unmethylated CpG-oligodeoxynucleotide (CpG-ODN), respectively. Production of IFN-α and TNF-α cytokines, as a part of the functional response of mentioned TLRs, was also measured using ELISA. Deficient transcripts of IRAK-M and TNFAIP3 in unstimulated PBMCs and lower production of TNF-α and IFN-α after treatments were observed. Upregulation of RFN216 and TNFAIP3 after TLR9 activation was abnormal compared to healthy individuals. Significant correlations were found between abnormal IRAK-M and TNFAIP3 transcripts, and lymphadenopathy and inflammatory scenarios in patients, respectively. It seems that the transcriptional status of some negative regulatory molecules is disturbed in CVID patients, and this could be caused by the underlying pathogenesis of CVID and could involve complications like autoimmunity and inflammatory responses.

Different profile of cytokine production in patients with systemic sclerosis and association with clinical manifestations

Immune dysregulation is a central process in the pathogenesis of systemic sclerosis (SSc). Cytokines produced by lymphocytes and monocytes are important mediators and induce tissue damage, recruit additional inflammatory cells, and promote extracellular matrix production and fibrosis. In the present research, we aimed to study the associations between levels of cytokines in serum and culture supernatants from peripheral blood mononuclear cells (PBMCs) and clinical manifestations in SSc patients. Serum samples were obtained from 56 SSc patients and 56 unrelated age- and gender-matched healthy individuals. Resting and anti-CD3/CD28–stimulated PBMC cultures were obtained from 19 SSc patients and 8 healthy controls. IL-2, IL-4, IL-6, IL-10, IL-17A, TNF, and IFN-γ levels were measured by ELISA or CBA. Serum cytokines, except IL-17A, were below the kit detection limit in most of the patients and controls. In unstimulated PBMC, the production of TNF(p = 0.004), IL-10(p = .048), IL-2(p < 0.001), and IL-6 (p = 0.01) was higher in SSc patients than in healthy controls. After anti-CD3/CD28 stimulation, scleroderma PBMCs had lower concentrations of TNF(p = 0.009), IL-10(p = .018), and IL-2(p = .002) than HC. In unstimulated PBMC, IL-2 concentration was higher in patients with esophageal dysmotility (p = 0.04), and IL-10 levels had a positive correlation with modified Rodnan score (p = 0.03). After anti-CD3/CD28 stimulation, higher levels of IL-2 and IL-4 were observed in SSc patients with lung fibrosis (p = 0.01 and 0.006, respectively), and higher levels of IL-10 (p = 0.04) and IL-4 (p = 0.04) in patients with digital ulcers. In conclusion, SSc patients have a different profile of cytokine production and this was associated with clinical manifestations.

Analysis of cell surface and intranuclear markers on non-stimulated human PBMC using mass cytometry

Mass cytometry is a powerful tool that allows simultaneous analysis of more than 37 markers at the single cell level. Mass cytometry is of particular interest in the identification of a wide variety of cell phenotypes in autoimmune diseases. Moreover, cells can be labelled with palladium isotopes and pooled before staining (barcoding). Nevertheless, immunologists often face an important problem concerning the choice of markers to be included in a panel. This problem arises due to the incompatibility of different buffers used for the fixation and permeabilization of cells with various cell surface epitopes. In this study, we used a panel of 27 markers (19 surface markers and 8 intranuclear markers) to demonstrate disparities in the detection of cell surface antigens when comparing different buffers to stain unstimulated peripheral blood mononuclear cells. These disparities range from mild differences to very important differences in population frequencies depending on the buffers. Finally, we demonstrate the harmful effects of permeabilization prior to barcoding on the detection of some cell surface antigens. Here, we optimize a protocol that is suitable to use when targeting a large panel including both cell surface and intranuclear markers on unstimulated human peripheral blood mononuclear cells.

Polyunsaturated Fatty Acid Biosynthesis Involving Δ8 Desaturation and Differential DNA Methylation of FADS2 Regulates Proliferation of Human Peripheral Blood Mononuclear Cells.

Polyunsaturated fatty acids (PUFAs) are important for immune function. Limited evidence indicates that immune cell activation involves endogenous PUFA synthesis, but this has not been characterised. To address this, we measured metabolism of 18:3n-3 in quiescent and activated peripheral blood mononuclear cells (PBMCs), and in Jurkat T cell leukaemia. PBMCs from men and women (n = 34) were incubated with [1-13C]18:3n-3 with or without Concanavalin A (Con. A). 18:3n-3 conversion was undetectable in unstimulated PBMCs, but up-regulated when stimulated. The main products were 20:3n-3 and 20:4n-3, while 18:4n-3 was undetectable, suggesting initial elongation and Δ8 desaturation. PUFA synthesis was 17.4-fold greater in Jurkat cells than PBMCs. The major products of 18:3n-3 conversion in Jurkat cells were 20:4n-3, 20:5n-3, and 22:5n-3. 13C Enrichment of 18:4n-3 and 20:3n-3 suggests parallel initial elongation and Δ6 desaturation. The FADS2 inhibitor SC26196 reduced PBMC, but not Jurkat cell, proliferation suggesting PUFA synthesis is involved in regulating mitosis in PBMCs. Con. A stimulation increased FADS2, FADS1, ELOVL5 and ELOVL4 mRNA expression in PBMCs. A single transcript corresponding to the major isoform of FADS2, FADS20001, was detected in PBMCs and Jurkat cells. PBMC activation induced hypermethylation of a 470bp region in the FADS2 5′-regulatory sequence. This region was hypomethylated in Jurkat cells compared to quiescent PBMCs. These findings show that PUFA synthesis involving initial elongation and Δ8 desaturation is involved in regulating PBMC proliferation and is regulated via transcription possibly by altered DNA methylation. These processes were dysregulated in Jurkat cells. This has implications for understanding the regulation of mitosis in normal and transformed lymphocytes.

Levels of inflammatory cytokines from peripheral blood mononuclear cells of children with cow's milk protein allergy.

The aim of the study was to investigate the level of cytokines in cultures of cow's milk protein- stimulated peripheral blood mononuclear cells of patients with cow's milk protein allergy. Eleven children with cow's milk protein allergy and 11 non-allergic controls were studied. Their peripheral blood mononuclear cells were cultured alone and in the presence of cow's milk α-lactalbumin; β-lactoglobulin; αS 1, αS 2, β, and κ-casein fraction mixtures; and a cow's protein mixture from whole milk. Production of cytokines, tumor necrosis factor-α, interleukin-10, and interleukin-12 were determined in culture supernatants. In cow's milk protein-stimulated peripheral blood mononuclear cell cultures of children with cow's milk protein allergy, tumor necrosis factor-α, interleukin-10, and interleukin-12 production was significantly higher than in non-allergic controls (p<0.05). No difference in cytokine production was found between cultures obtained from unstimulated peripheral blood mononuclear cell cultures of both cow's milk protein allergy and non-allergic controls. The findings of this preliminary study align with data from the literature suggesting that the investigation of tumor necrosis factor-α, interleukin-10, and interleukin-12 in cow's milk protein-stimulated peripheral blood mononuclear cell cultures of children may be taken in further consideration to explore whether they might have a predictive role for cow's milk protein allergy. Further studies are therefore needed to extensively investigate this issue.

Non-Viable Lactobacillus Casei Beneficially Modulates Poly I:C Immune Response in Co-Cultures of Human Cells.

Polyinosinic:polycytidylic acid (Poly-IC) has been used as a viral stimulus to mimic in vivo and in vitro infection induced by some viruses. To determine whether non-viable Lactobacillus casei CRL431 (LcM) can modulate the immune response induced by Poly I:C in co-culture models of peripheral blood mononuclear cells (PBMC) and A549 cells. T and NK cell activation was evaluated by flow cytometry and levels of TNF-α, IFN-γ, IL-10, IL-29, and IL-17 by ELISA. Cells in direct contact with A549 (PBMC-A549) and cells with no contact with it (PBMC//A549) were used for this purpose. PBMCs alone and both co-culture systems were stimulated for 24 h with the following stimuli: LPS (10 µg/ml), LcM (106 UFC/ml), Poly I:C (2 µg/ml), Poly I:C+LcM, and LcM (3 h)+Poly I:C. Moreover, unstimulated cells were used as a control. Poly I:C and LcM (3 h)+Poly I:C in PBMC-A549 showed a significant increase in the percentage of CD8+ expression (p<0.05). All stimuli induced significant activation from T CD4+, CD8+ cells compared with unstimulated PBMCs in both co-culture cells system. However, activation percentages were higher in direct co-culture. Poly I:C induced a higher level of pro-inflammatory TNF-α and IFN-γ cytokines as well as IL-17 and IL-29 with lower IL-10 levels in both co-culture systems while LcM induced a beneficial pattern of cytokines that would regulate Poly I:C effect. This in vitro model allowed us to highlight the potential of LcM as a modulator of anti-viral immune response and suggest its potential use in formulations against RNA respiratory viruses.

Genetic Variation at IFNL4 Influences Extrahepatic Interferon-Stimulated Gene Expression in Chronic HCV Patients.

Polymorphisms at IFNL4 strongly influence spontaneous resolution and interferon therapeutic response in hepatitis C virus (HCV) infection. In chronic HCV, unfavorable alleles are associated with elevated interferon (IFN)-stimulated gene (ISG) expression in the liver, but extrahepatic effects are less well characterized. We used RNA sequencing (RNA-Seq) to examine whether IFNL4 genetic variation (rs368234815) modulates ISG expression in peripheral blood mononuclear cells (PBMC) during chronic HCV infection. ISG expression was elevated in unstimulated PBMC homozygous for the unfavorable ΔG IFNL4 variant; expression following IFN-α stimulation was comparable across genotypes. These findings suggest that lambda interferons may have broader systemic effects during HCV infection.

Interaction of allogeneic adipose tissue-derived stromal cells and unstimulated immune cells in vitro: the impact of cell-to-cell contact and hypoxia in the local milieu.

Multipotent mesenchymal stem cells (MSCs) are an attractive tool for cell therapy and regenerative medicine. Being applied in vivo, allogeneic MSCs are faced with both activated and unstimulated immune cells. The effects of MSCs on activated immune cells are well described and are mainly suppressive. Less is known about the interaction of MSCs with unstimulated immune cells. We evaluated the contribution of tissue-related O2 level ("physiological" hypoxia-5% O2) and cell-to-cell contact to the interaction between allogeneic adipose tissue-derived MSCs (ASCs) and unstimulated peripheral blood mononuclear cells (PBMCs). Under both O2 levels, ASCs affected the immune response by elevating the proportion of CD69+ T cells and modifying the functional activity of unstimulated PBMCs, providing a significant reduction of ROS level and activation of lysosome compartment. "Physiological" hypoxia partially attenuated the ASC modulation of PBMC function, reducing CD69+ cell activation and more significantly supressing ROS. In direct co-culture, the ASC effects were more pronounced. PBMC viability was preferentially maintained, and the lymphocyte subset ratio was altered in favour of B cells. Our findings demonstrate that allogeneic ASCs do not enhance the activation of unstimulated immune cells and can provide supportive functions. The "hypoxic" phenotype of ASCs may be more "desirable" for the interaction with allogeneic immune cells that may be required in cell therapy protocols.

Diagnostic accuracy of a selected signature gene set that discriminates active pulmonary tuberculosis and other pulmonary diseases

We validated the accuracy of host selected signature gene set using unstimulated whole blood (WB), and peripheral blood mononuclear cells (PBMC) in the diagnosis of tuberculosis (TB). The unstimulated WB and PBMC from 1417 individuals with active pulmonary TB patients, other lung diseases and healthy participants were analyzed using real time polymerase chain reaction (RT-PCR). The WB cohort test demonstrates that the combination of GBP5 and KLF2 can differentiate active TB versus HC with sensitivity and specificity of 77.8% and 87.1%, respectively; but most importantly active TB versus OD with sensitivity and specificity of 96.1% and 85.2%, respectively. Again during treatment course, the TB score of GBP5 and KLF2, analytes secretion and clinical parameters were found to be associated in disease progression. In the PBMC cohort test, we found that the only and best discriminatory combination was GBP5, DUSP3 and KLF2 inthe active TB versus HC with a sensitivity and specificity of 76.4% and 85.9%, respectively. Our study reveals that GBP5 and KLF2 may be useful as a diagnostic tool for active TB, also the two-gene set may serve as surrogate biomarkers for monitoring TB therapy.

Quinacrine Suppresses Tumor Necrosis Factor-α and IFN-α in Dermatomyositis andCutaneous Lupus Erythematosus.

Antimalarials are used to treat dermatomyositis (DM) and cutaneous lupus erythematosus (CLE). Although hydroxychloroquine (HCQ) is frequently used, addition of quinacrine (QC) has shown additional clinical effects when combined with HCQ. To quantify the effects of HCQ versus QC in suppressing secretion of tumor necrosis factor-α (TNF-α) and IFN-α from the peripheral blood mononuclear cells of DM and CLE patients, lipopolysaccharide-stimulated and control peripheral blood mononuclear cells from DM and CLE patients and control subjects were analyzed for the effect of HCQ and QC on TNF-α and IFN-α production using ELISA testing. Flow cytometry showed the effects of these therapies on intracellular TNF-α inmyeloid dendritic cells and monocytes of DM patients and control subjects. QC significantly suppressed TNF-α relative to HCQ from unstimulated and lipopolysaccharide-stimulated peripheral blood mononuclear cells of DM and CLE patients (P < 0.0001). It suppressed IFN-α as significantly as HCQ from cytosine phosphodiester guanine-stimulated peripheral blood mononuclear cells of DM and CLE patients (P < 0.0001). Flow cytometry showed that QCsignificantly suppressed intracellular expression of TNF-α from the lipopolysaccharide-stimulated myeloid dendritic cells and monocytes of DM patients (P-values ≤ 0.0008). In conclusion, QC likely has a different mechanism of action than HCQ, given the broader inhibition of proinflammatory cytokines, including both TNF-α and IFN-α.

Age-dependent alterations in serum cytokines, peripheral blood mononuclear cell cytokine production, natural killer cell activity, and prostaglandin F2α.

This study aimed to determine age-dependent alterations in serum cytokines, peripheral blood mononuclear cell (PBMC) cytokine production, natural killer (NK) cell activity, and urinary 8-epi-prostaglandin F2α (PGF2α). Nine hundred eighty-seven healthy and nonobese subjects were divided into five age groups: 20-34 (group 1), 35-44 (group 2), 45-54 (group 3), 55-64 (group 4), and 65-80 (group 5) years of age. After adjusting for BMI, sex, and smoking and drinking status, serum interferon (IFN)-γ levels decreased in groups 3, 4, and 5 compared with those in groups 1 and 2. Production of IFN-γ by unstimulated PBMCs was lower in the older groups (groups 4 and 5) than in the younger groups (groups 1 and 3). Serum interleukin (IL)-12 was lower in group 5 than in groups 1 and 2. In contrast, both serum and PBMC IL-6 were higher in group 5 than in groups 1, 2, and 3. Urinary 8-epi-PGF2α increased in group 3 compared with that in group 1 and further increased in group 5. Multiple linear regression analysis revealed that serum IFN-γ levels were negatively affected by age, and NK cell activity at a ratio of E:T=5:1 was positively affected by PBMC IFN-γ. This study shows the age-related reductions in serum and PBMC IFN-γ and serum IL-12 and age-related increases in serum and PBMC IL-6 and oxidative stress in healthy nonobese subjects. Additionally, circulating IL-6 levels may be a better marker of the chronic low-grade inflammatory activity associated with aging than systemic levels of high-sensitivity C-reactive protein, TNF-α, and IL-1β.

A Novel Single-Cell FISH-Flow Assay Identifies Effector Memory CD4+ T cells as a Major Niche for HIV-1 Transcription in HIV-Infected Patients.

ABSTRACTCells that actively transcribe HIV-1 have been defined as the “active viral reservoir” in HIV-infected individuals. However, important technical limitations have precluded the characterization of this specific viral reservoir during both treated and untreated HIV-1 infections. Here, we used a novel single-cell RNA fluorescence in situ hybridization-flow cytometry (FISH-flow) assay that requires only 15 million unfractionated peripheral blood mononuclear cells (PBMCs) to characterize the specific cell subpopulations that transcribe HIV RNA in different subsets of CD4+ T cells. In samples from treated and untreated HIV-infected patients, effector memory CD4+ T cells were the main cell population supporting HIV RNA transcription. The number of cells expressing HIV correlated with the plasma viral load, intracellular HIV RNA, and proviral DNA quantified by conventional methods and inversely correlated with the CD4+ T cell count and the CD4/CD8 ratio. We also found that after ex vivo infection of unstimulated PBMCs, HIV-infected T cells upregulated the expression of CD32. In addition, this new methodology detected increased numbers of primary cells expressing viral transcripts and proteins after ex vivo viral reactivation with latency reversal agents. This RNA FISH-flow technique allows the identification of the specific cell subpopulations that support viral transcription in HIV-1-infected individuals and has the potential to provide important information on the mechanisms of viral pathogenesis, HIV persistence, and viral reactivation.