Subpopulations Of Peripheral Blood Mononuclear Cells Research Articles

Predicting therapeutic response to fingolimod treatment in multiple sclerosis patients.

Fingolimod, an orally active immunomodulatory drug for relapsing-remitting multiple sclerosis (RRMS), sequesters T cells in lymph nodes through functional antagonism of the sphingosine-1-phosphate receptor, reducing the number of potential autoreactive cells that migrate to the central nervous system. However, not all RRMS patients respond to this therapy. Our aim was to test the hypothesis that by immune-monitoring RRMS patient's leukocyte subpopulations it is possible to find biomarkers associated with clinical response to fingolimod. Prospective study. Analysis of peripheral blood mononuclear cell subpopulations by multiparametric flow cytometry, at baseline and +1, +3, +6, +12months of follow-up in 40 RRMS patients starting fingolimod therapy. Fingolimod treatment induced a severe lymphopenia affecting mainly T and B cells. A relative increase in Treg (memory Treg : 3.8±1.0% baseline vs 8.8±4.4% month +1; activated Treg : 1.5±0.7% baseline vs 3.7±2.1% month +1, P<0.001) as well as transitional B cells (10.5±12.3% baseline vs 18.7±14.6% month +1, P<0.001) was observed. Interestingly, lymphocyte subpopulations were already at baseline significantly different in responder patients. The percentage of recent thymic emigrants (RTE) used to stratify fingolimod responder, and no responder patients was the best biomarker (4.0±1.4% vs 7.4±1.9%, respectively [P<0.001]). The results support that immune-monitoring of lymphocyte subpopulations in peripheral blood is a promising tool to select RRMS candidate for fingolimod treatment.

The influence of stromal cells and tumor-microenvironment-derived cytokines and chemokines on CD3+CD8+ tumor infiltrating lymphocyte subpopulations

ABSTRACT The tumor microenvironment has been identified as a major mediator of immunological processes in solid tumors. In particular, tumor-associated fibroblasts are known to interact with tumor infiltrating immune cells. We describe the influence of fibroblasts and tumor-microenvironment-derived cytokines on the infiltration capacity of CD3+CD8+ cytotoxic T lymphocyte subpopulations using a multicellular 3D co-culture system. 3D tumor microtissues were cultivated using a hanging drop system. Human A549 and Calu-6 cancer cell lines were incubated alone or together with the human fibroblast cell line SV80 for 10 d to form microtissues. On day 10, peripheral blood mononuclear cells (PBMC) were added with or without cytokine stimulation for 24 h. Infiltrating PBMC subpopulations were investigated by flow cytometry. Aggregation of the microtissues and the infiltration of the PBMCs were analyzed by immunohistochemistry, and endogenous cytokine and chemokine expression was analyzed with a multi-cytokine immunoassay. Secretion of chemokines is increased in microtissues consisting of cancer cells and fibroblasts. PBMC infiltrate the whole spheroid in cancer cell monocultures, whereas in co-cultures of cancer cells and fibroblasts, PBMCs are rather localized at the margin. Activated CD69+ and CD49d+ T lymphocytes show an increased microtissue infiltration in the presence of fibroblasts. We demonstrate that the stromal component of cancer microtissues significantly influences immune cell infiltration. The presence of fibroblasts in cancer microtissues induces a shift of T lymphocyte infiltration toward activated T lymphocytes.

Infiltration of lymphocyte subpopulations into cancer microtissues as a tool for the exploration of immunomodulatory agents and biomarkers

IntroductionThe interaction between the immune system and malignant diseases is a proven key target for cancer therapy. We describe an innovative 3D cell culture system comprising both immune and cancer cells to evaluate their interaction and immune cell infiltration to provide an innovative in vitro screening of immunomodulatory agents and biomarkers. Methods3D tumor microtissues were cultivated using a hanging drops system. Human non-small-cell lung cancer cell lines were incubated for 7 days to form microtissues. On day 5, peripheral blood mononuclear cells (PBMC) were added with or without interleukin-2 (IL-2) for 24 or 48h. Viability of cancer cells and the infiltrating PBMC subpopulations were investigated by flow cytometry. Aggregation of tumor cells and PBMC and the infiltration of the PBMC into the tumor microtissues were analyzed by immunohistochemistry. Quantification of infiltration was measured by applying the TissueFAXS system. ResultsImmunohistochemistry revealed PBMC infiltration in all cell lines which increased under IL-2 stimulation. Analysis of infiltrating populations showed both lymphocyte subpopulations and monocytes within the tumor microtissues. In all three co-cultures, CD3+CD8+ and CD3+CD8+CD45R0+CD28+ lymphocytes were increased with IL-2, whereas CD3+CD8+CD45R0-CD28+ PBMCs were decreased with and without IL-2 stimulation. ConclusionIn summary, we present a novel cell culture system to study the interaction between cancer cells and immune cells in 3-dimensional microtissues. In addition, we report for the first time an in vitro infiltration assay based on 3D microtissues. This model has the potential to provide a tool for ex-vivo drug testing and biomarker screening of immunomodulatory agents.

Mononucleated Blood Cell Populations Display Different Abilities To Transmit Prion Disease by the Transfusion Route.

Previous experiments carried out in a sheep scrapie model demonstrated that the transfusion of 200 μl of prion-infected whole blood has an apparent 100% efficacy for disease transmission. These experiments also indicated that, despite the apparent low infectious titer, the intravenous administration of white blood cells (WBC) resulted in efficient disease transmission. In the study presented here, using the same transmissible spongiform encephalopathy (TSE) animal model, our aim was to determine the minimal number of white blood cells and the specific abilities of mononucleated cell populations to transmit scrapie by the transfusion route. Our results confirmed that the transfusion of 100 μl, but not 10 μl, of fresh whole blood collected in asymptomatic scrapie-infected donor sheep can transmit the disease. The data also show that the intravenous administration of 10(5) WBCs is sufficient to cause scrapie in recipient sheep. Cell-sorted CD45R(+) (predominantly B lymphocytes), CD4(+)/CD8(+) (T lymphocytes), and CD14(+) (monocytes/macrophages) blood cell subpopulations all were shown to contain prion infectivity by bioassays in ovine PrP transgenic mice. However, while the intravenous administration of 10(6) CD45(+) or CD4(+)/8(+) living cells was able to transmit the disease, similar numbers of CD14(+) cells failed to infect the recipients. These data support the contention that mononucleated blood cell populations display different abilities to transmit TSE by the transfusion route. They also represent an important input for the risk assessment of blood-borne prion disease transmission and for refining the target performance of leukoreduction processes that currently are applied to mitigate the transmission risk in transfusion medicine. Interindividual variant Creutzfeldt-Jakob disease (vCJD) transmission through blood and blood-derived products is considered a major public health issue in transfusion medicine. Over the last decade, TSE in sheep has emerged as a relevant model for assessing the blood-borne vCJD transmission risk. In this study, using a sheep TSE model, we characterized the ability of different peripheral blood mononucleated cell populations to infect TSE-free recipients by the transfusion route. Our results indicate that as little as 10(5) WBC and 100 μl of blood collected from asymptomatic scrapie infected animals can transmit the disease. They also demonstrate unambiguously that peripheral blood mononuclear cell subpopulations display dramatically different abilities to transmit the disease. These data represent an important input for the risk assessment of blood-borne prion disease transmission and for refining the target performance of leukoreduction processes that currently are applied to mitigate the transmission risk in transfusion medicine.

Association of Elevations of Specific T Cell and Monocyte Subpopulations in Rheumatoid Arthritis With Subclinical Coronary Artery Atherosclerosis.

Coronary artery disease (CAD) is the leading cause of excess deaths in rheumatoid arthritis (RA). However, identification of features denoting patients with a risk of developing CAD is lacking. The composition of circulating peripheral blood mononuclear cell (PBMC) subsets in RA patients differs markedly from that in healthy controls with regard to the extent of T cell activation, with clonal expansion and differentiation to effector memory status, and presence of inflammatory monocytes. In this study, we sought to evaluate whether elevations in these PBMC subpopulations in RA patients could denote those with an increased risk of subclinical CAD, as determined by the presence of coronary artery calcification (CAC). The study cohort comprised 72 patients with RA who underwent cardiac computed tomography to assess CAC. PBMC subsets were determined by multiparameter flow cytometry. Multivariable logistic regression was used to determine the associations between PBMC subpopulations and the presence of CAC. Among the 72 patients with RA, 33% had CAC and exhibited significant increases in the levels of circulating CD4 T cell subsets denoting activation and differentiation to the effector memory phenotypes. Analogous increases in the levels of CD8 T cell subsets, as well as in the CD14(high)CD16+ intermediate monocyte subset, were also present in these patients, as compared to those without CAC. The increases in the CD4 and CD8 T cell subsets were highly intercorrelated, whereas the increases in CD14(high)CD16+ monocytes were independent of elevations in the CD4 T cell subsets. After adjustments for relevant confounders, the levels of CD4+CD56+CD57+ T cells and CD14(high)CD16+ monocytes remained associated with the presence of CAC. These findings indicate that PBMC subsets are markers for the presence of CAC and suggest that mechanisms of atherogenesis in RA may operate in part through the elevations in these subsets, raising further questions about the mechanisms underlying the presence of such alterations in cell composition in patients with RA and the potential for shared etiologic pathways between RA and cardiovascular disease.

Analysis of inflammasomes and antiviral sensing components reveals decreased expression of NLRX1 in HIV-positive patients assuming efficient antiretroviral therapy.

Few studies have investigated the importance of different components of the inflammasome system and of innate mitochondrial sensing (IMS) pathways in HIV infection and its treatment. We analysed the expression of several components of the inflammasome and of the IMS in HIV-positive patients taking successful combination antiretroviral therapy (cART). We enrolled 20 HIV-positive patients under cART, who achieved viral suppression since at least 10 months and 20 age and sex-matched healthy donors. By RT-PCR, using peripheral blood mononuclear cells (PBMCs), we quantified the mRNA expression of 16 genes involved in inflammasome activation and regulation (AIM2, NAIP, PYCARD, CASP1, CASP5, NLRP6, NLRP1, NLRP3, TXNIP, BCL2, NLRC4, PANX1, P2RX7, IL-18, IL-1β, SUGT1) and eight genes involved in IMS (MFN2, MFN1, cGAS, RIG-I, MAVS, NLRX1, RAB32, STING). Compared with controls, HIV-positive patients showed significantly lower mRNA levels of the mitochondrial protein NLRX1, which plays a key role in regulating apoptotic cell death; main PBMC subpopulations behave in a similar manner. No differences were observed in the expression of inflammasome components, which however showed complex correlations. The decreased level of NLRX1 in HIV infection could suggest that the virus is able to downregulate mechanisms linked to triggering of cell death in several immune cell types. The fact that HIV-positive patients did not show altered expression of inflammasome components, nor of most genes involved in IMS, suggests that the infection and/or the chronic immune activation does not influence the transcriptional machinery of innate mechanisms able to trigger inflammation at different levels.

Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPT™) and standard density gradient.

BackgroundHigh quality genetic material is an essential pre-requisite when analyzing gene expression using microarray technology. Peripheral blood mononuclear cells (PBMC) are frequently used for genomic analyses, but several factors can affect the integrity of nucleic acids prior to their extraction, including the methods of PBMC collection and isolation. Due to the lack of the relevant data published, we compared the Ficoll-Paque density gradient centrifugation and BD Vacutainer cell preparation tube (CPT) protocols to determine if either method offered a distinct advantage in preparation of PBMC-derived immune cell subsets for their use in gene expression analysis. We evaluated the yield and purity of immune cell subpopulations isolated from PBMC derived by both methods, the quantity and quality of extracted nucleic acids, and compared gene expression in PBMC and individual immune cell types from Ficoll and CPT isolation protocols using Affymetrix microarrays.ResultsThe mean yield and viability of fresh PBMC acquired by the CPT method (1.16 × 106 cells/ml, 93.3 %) were compatible to those obtained with Ficoll (1.34 × 106 cells/ml, 97.2 %). No differences in the mean purity, recovery, and viability of CD19+ (B cells), CD8+ (cytotoxic T cells), CD4+ (helper T cell) and CD14+ (monocytes) positively selected from CPT- or Ficoll-isolated PBMC were found. Similar quantities of high quality RNA and DNA were extracted from PBMC and immune cells obtained by both methods. Finally, the PBMC isolation methods tested did not impact subsequent recovery and purity of individual immune cell subsets and, importantly, their gene expression profiles.ConclusionsOur findings demonstrate that the CPT and Ficoll PBMC isolation protocols do not differ in their ability to purify high quality immune cell subpopulations. Since there was no difference in the gene expression profiles between immune cells obtained by these two methods, the Ficoll isolation can be substituted by the CPT protocol without conceding phenotypic changes of immune cells and compromising the gene expression studies. Given that the CPT protocol is less elaborate, minimizes cells’ handling and processing time, this method offers a significant operating advantage, especially in large-scale clinical studies aiming at dissecting gene expression in PBMC and PBMC-derived immune cell subpopulations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-015-0113-0) contains supplementary material, which is available to authorized users.

Complexes of DNA with the Antimicrobial Peptide LL37 Augment NK Cell Functions by Inducing Type I Interferon Production from Circulating Monocytes and Plasmacytoid Predendritic Cells.

The cationic antimicrobial peptide, LL37, forms electrostatic complexes with DNA (LL37-DNA), which are potent activators of circulating plasmacytoid predendritic cells (ppDCs) and monocytes. However, the effects of LL37-DNA on other immune cell types, such as NK cells, are poorly characterized. In this study, we show that complexes of human genomic DNA (hgDNA) or synthetic double-stranded oligodeoxynucleotides with LL37 strongly enhance natural cytotoxicity of human peripheral blood mononuclear cells (PBMCs) upon an overnight culture, whereas hgDNA alone has no effect, and LL37 alone is moderately active. LL37-DNA complexes potentiate degranulation of, and interferon (IFN)-γ production by, NK cells upon subsequent encounter of K562 target cells. The complexes do not influence percentages of NK cells among PBMCs or the expression of cytotoxic proteins by NK cells. Using neutralizing anticytokine antibodies and immunomagnetic depletion of different subpopulations of PBMCs, we found that the effect of LL37-DNA on NK cells is indirect and mediated by type I IFNs produced by monocytes and, to a lesser extent, by ppDCs. We discuss possible roles of LL37-DNA complexes in the regulation of NK cell functions and in the treatment of cancer.

A novel subpopulation of peripheral blood mononuclear cells presents in major burn patients

Hypertrophic scars (HTS) are generally believed to result from proliferation and activation of resident connective tissue fibroblasts after burns. To demonstrate a potential role of blood-borne cells, the peripheral blood mononuclear cells (PBMCs) and the effect of PBMCs on dermal fibroblast behavior was investigated.Flow cytometry was used to analyze the surface and intracellular protein expression of PBMCs and fibroblasts. Transwell migration assay, enzyme-linked immunosorbent assay and real-time reverse transcription polymerase chain reaction was performed to assess fibroblast functions.We identified a novel subpopulation of PBMCs in burn patients in vivo that appears at an early stage following major thermal injuries, which primarily express procollagen 1, leukocyte specific protein 1, CD204, toll-like receptor 4 and stromal cell-derived factor 1 (SDF-1) receptor CXCR4. In vitro, the conditioned media from burn patient PBMCs up-regulated the expression of fibrotic growth factors and extracellular matrix molecules, down-regulated antifibrotic factor decorin, enhanced cell chemotaxis and promoted cell differentiation into contractile myofibroblasts in dermal fibroblasts.After thermal injury, this novel subpopulation of PBMCs is systemically triggered and attracted to the wounds under SDF-1/CXCR4 signaling where they appear to modulate the functions of resident connective tissue cells and thus contribute to the development of HTS.

The role of B cells in the early onset of the first demyelinating event of acute optic neuritis.

We evaluated the molecular pathways that operate in the early phase of acute optic neuritis (ON) by studying gene expression profiles of peripheral blood mononuclear cells (PBMCs) subpopulations, including CD19(+) B cells, CD14(+) macrophages, and CD4(+) and CD8(+) T cells. Samples of PBMC subpopulations were obtained from 18 MS patients within 96 hours of the acute onset of the first demyelinating event of ON, and from 14 age- and sex-matched healthy subjects. High throughput gene expression analysis was performed on samples from six ON patients and nine healthy subjects using Affymetrix technology. Data were analyzed using Partek software, and most informative genes (MIGs) were defined as genes with P < 0.01 and fold change > 2.0. Molecular pathways were analyzed by Ingenuity software. Verification of key MIGs was done on samples from five independent ON patients and five healthy subjects by quantitative (Q) RT-PCR and Western blot. Functional assay to test antigen presentation ability of sorted B cells was performed on blood samples from seven additional ON patients. Significantly differentiating gene expression signatures consisting of 467, 55, and 55 MIGs respective of CD19(+), CD14(+), and CD4(+) cells, were identified between ON and healthy subjects. No MIGs were detected for CD8(+) cells. The major involvement of CD19(+) B cells in the early stage of ON was characterized by enrichment of genes involved in activation of immune mechanisms (P = 3.2 × 10(-25) to 2.5 × 10(-3)), including cellular immune response (P = 7.1 × 10(-12)), B-cell cellular growth and proliferation (P = 1.0 × 10(-7)), activation of immune cells trafficking pathways (P = 5.4 × 10(-15) to 2.0 × 10(-3)), and stimulation of antigen presentation (P = 8.9 × 10(-11)). This massive B-cell-restricted initiation of the immune response in the early disease process of ON was followed by low CD14(+) and CD4(+) cells activity and CD8(+) cells anergy. Our findings demonstrate that CD19(+) B cells have a significant role in the pathogenesis of the first demyelinating event of acute ON and suggest their role as a possible target for immunomodulation.

Galectin Hco-gal-m from Haemonchus contortus modulates goat monocytes and T cell function in different patterns.

BackgroundMonocytes and T cells are two major subpopulations of peripheral blood mononuclear cells (PBMC) and play an essential role in the innate and adaptive immune systems. Different members of the galectin family show multiple and distinct regulatory effects on different cell types. Previous studies have demonstrated that the galectin from Haemonchus contortus (Hco-gal-m) performed immunomodulatory effects on goat PBMC, however, which subpopulation of PBMC is the primary target of Hco-gal-m and whether the immune modulations share the same mechanism remain unclear.MethodsIn this study, the developmental expression of Hco-gal-m was analyzed by RT-PCR and Western blot analysis. The distribution of Hco-gal-m in adult worm was detected by an immunohistochemical test. The binding activity of the recombinant Hco-gal-m (rHco-gal-m) on goat monocytes and T cells were assessed by flow cytometry. The immunomodulatory effects of Hco-gal-m on cytokine secretion, cell activation and apoptosis were observed by co-incubation of rHco-gal-m with goat monocytes and T cells.ResultsHco-gal-m was expressed in L4 as well as adult worms and predominantly localized at the internal surface of the worm guts. rHco-gal-m could bind to both monocytes and T cells. The engagement of rHco-gal-m decreased the production of IL-6, IL-10 and TNF-α in T cells, however, it significantly increased the secretion of IL-10 in monocytes. After rHco-gal-m exposure, the expression of MHC-II on monocytes and that of CD25 on T cells were restricted. Consequently, T cell proliferations were potently inhibited by rHco-gal-m. In addition, rHco-gal-m induced apoptosis in T cells, but not significantly in monocytes.ConclusionsOur results indicated that rHco-gal-m modulated goat monocytes and T cell function in different patterns.Electronic supplementary materialThe online version of this article (doi:10.1186/1756-3305-7-342) contains supplementary material, which is available to authorized users.

BLV-CoCoMo-qPCR: estimation of bovine leukemia virus (BLV) proviral load harbored by lymphocyte subpopulations in BLV-infected cattle at the subclinical stage of enzootic bovine leucosis

Bovine leukemia virus (BLV) is associated with enzootic bovine leukosis (EBL), which is the most common neoplastic disease of cattle. The present study reports the distribution of BLV provirus in peripheral blood mononuclear cell subpopulations isolated from BLV-infected cows at the subclinical stage of EBL as examined by cell sorting and a new quantitative real-time PCR method, BLV-CoCoMo-qPCR, to measure the proviral load of both known and novel BLV variants in BLV-infected animals. Phenotypic characterization of five BLV-infected but clinically normal cattle with a proviral load of > 100 copies per 1 x 105 cells identified a high percentage of CD5+ IgM+ cells (but not CD5-IgM+ B cells, CD4+ T cells, or CD8+T cells). These lymphocyte subpopulations were purified from three cattle by cell sorting or using magnetic beads, and the BLV proviral load was estimated using BLV-CoCoMo-qPCR. The CD5+ IgM+ B cell population in all animals harbored a higher BLV proviral load than the other cell populations. The copy number of proviruses infecting CD5- IgM+ B cells, CD4+ cells, and CD8+ T cells (per 1 ml of blood) was 1/34 to 1/4, 1/22 to 1/3, and 1/31 to 1/3, respectively, compared with that in CD5+ IgM+ B cells. Moreover, the BLV provirus remained integrated into the genomic DNA of CD5+ IgM+ B cells, CD5- IgM+ B cells, CD4+ T cells, and CD8+ T cells, even in BLV-infected cattle with a proviral load of <100 copies per 105 cells.

A preliminary evaluation of the effects of opioids on innate and adaptive human in vitro immune function

BackgroundStudies have demonstrated that whereas some opioids have little effect on immunity (eg, buprenorphine), others can be immunosuppressive (eg, morphine) or immunostimulatory (eg, tramadol). However, a variety of approaches have...

Detection of hepatitis C virus (HCV) negative strand RNA and NS3 protein in peripheral blood mononuclear cells (PBMC): CD3+, CD14+ and CD19+

BackgroundAlthough hepatitis C virus (HCV) is primarily hepatotropic, markers of HCV replication were detected in peripheral blood mononuclear cells (PBMC) as well as in ex vivo collected tissues and organs. Specific strains of HCV were found to be capable to infect cells of the immune system: T and B cells and monocytes/macrophages as well as cell lines in vitro. The direct invasion of cells of the immune system by the virus may be responsible for extrahepatic consequences of HCV infection: cryoglobulinemia and non-Hodgkin’s lymphoma.The aim of the present study was to determine the prevalence of markers of HCV infection: negative strand HCV RNA and non-structural NS3 protein in PBMC subpopulations: CD3+, CD14+ and CD19+. The presence of virus and the proportion of affected cells within a particular PBMC fraction could indicate a principal target cell susceptible for HCV.MethodsPBMC samples were collected from 26 treatment-free patients chronically infected with HCV. PBMC subpopulations: CD3+, CD14+, CD19+ were obtained using positive magnetic separation. The presence of negative strand RNA HCV and viral NS3 protein were analyzed by strand-specific RT-PCR and NS3 immunocytochemistry staining.ResultsNegative strand HCV RNA was detectable in 7/26 (27%), whereas NS3 protein in 15/26 (57.6%) of PBMC samples. At least one replication marker was found in 13/26 (50%) of CD3+ cells then in 8/26 (30.8%) of CD14+ and CD19+ cells. The highest percentage of cells harboring viral markers in single specimen was also observed in CD3+ (2.4%), then in CD19+ (1.2%), and much lower in CD14+ (0.4%) cells.ConclusionsOur results indicate that CD3+ cells are a dominant site for extrahepatic HCV replication, although other PBMC subpopulations may also support virus replication.

Differential microRNA expression in blood in multiple sclerosis

Background: microRNAs (miRNAs) regulate the expression of the genome at the post-transcriptional level. They play a role in autoimmunity and inflammation, and show potential for use as therapeutic targets in many diseases. With the recent detection of miRNAs in body fluids, the possibility for using miRNAs as diagnostic biomarkers has emerged. Objective: We assessed whether miRNAs contribute to the altered immune activation state in relapsing–remitting multiple sclerosis (RRMS) patients and investigated the possible use of miRNAs as diagnostic biomarkers in multiple sclerosis (MS). Methods: We performed global miRNA expression profiling analysis in peripheral blood mononuclear cells (PBMCs) and selected miRNAs were measured in plasma. We detected expression of miRNAs by real-time qPCR and compared results with cytokines related to inflammation and disease activity. Selected miRNAs were analyzed in PBMC subpopulations, after isolating them by magnetic bead separation. Results: We found that among validated miRNAs, let-7d correlated with the pro-inflammatory cytokine interleukin-1B. The miR-145 was 3-fold up-regulated in MS patients; its possible use as a diagnostic biomarker in PBMCs, plasma and serum was confirmed by ROC-curve analysis (Area under the curve (AUC) 0.785, p = 0.0004; 0.785, p = 0.004; 0.981, P < 0.0001, respectively). Conclusions: RRMS patients in remission had altered expression of miRNAs. We validated miR-145 as a potential diagnostic biomarker for the diagnosis of MS in blood, plasma and serum.

Estimation of bovine leukemia virus (BLV) proviral load harbored by lymphocyte subpopulations in BLV-infected cattle at the subclinical stage of enzootic bovine leucosis using BLV-CoCoMo-qPCR

BackgroundBovine leukemia virus (BLV) is associated with enzootic bovine leukosis (EBL), which is the most common neoplastic disease of cattle. BLV infection may remain clinically silent at the aleukemic (AL) stage, cause persistent lymphocytosis (PL), or, more rarely, B cell lymphoma. BLV has been identified in B cells, CD2+ T cells, CD3+ T cells, CD4+ T cells, CD8+ T cells, γ/δ T cells, monocytes, and granulocytes in infected cattle that do not have tumors, although the most consistently infected cell is the CD5+ B cell. The mechanism by which BLV causes uncontrolled CD5+ B cell proliferation is unknown. Recently, we developed a new quantitative real-time polymerase chain reaction (PCR) method, BLV-CoCoMo-qPCR, which enabled us to demonstrate that the proviral load correlates not only with BLV infection, as assessed by syncytium formation, but also with BLV disease progression. The present study reports the distribution of BLV provirus in peripheral blood mononuclear cell subpopulations isolated from BLV-infected cows at the subclinical stage of EBL as examined by cell sorting and BLV-CoCoMo-qPCR.ResultsPhenotypic characterization of five BLV-infected but clinically normal cattle with a proviral load of > 100 copies per 1 × 105 cells identified a high percentage of CD5+ IgM+ cells (but not CD5- IgM+ B cells, CD4+ T cells, or CD8+T cells). These lymphocyte subpopulations were purified from three out of five cattle by cell sorting or using magnetic beads, and the BLV proviral load was estimated using BLV-CoCoMo-qPCR. The CD5+ IgM+ B cell population in all animals harbored a higher BLV proviral load than the other cell populations. The copy number of proviruses infecting CD5- IgM+ B cells, CD4+ cells, and CD8+ T cells (per 1 ml of blood) was 1/34 to 1/4, 1/22 to 1/3, and 1/31 to 1/3, respectively, compared with that in CD5+ IgM+ B cells. Moreover, the BLV provirus remained integrated into the genomic DNA of CD5+ IgM+ B cells, CD5- IgM+ B cells, CD4+ T cells, and CD8+ T cells, even in BLV-infected cattle with a proviral load of <100 copies per 105 cells.ConclusionsThe results of the recent study showed that, although CD5+ IgM+ B cells were the main cell type targeted in BLV-infected but clinically normal cattle, CD5- IgM+ B cells, CD4+ cells, and CD8+ T cells were infected to a greater extent than previously thought.

Quantitative flow cytometric analysis of expression of tumor necrosis factor receptor types I and II on mononuclear cells

Background: Tumor necrosis factor (TNF)-α is an inflammatory cytokine, the biological effects of which are mediated by the interaction with specific membrane-bound receptors. To assess TNF-α receptor (TNFR) expression, it is important to estimate both the number of cells that carry these receptors and the number of receptors per cell, because the cell fate depends on the balance between TNFRI and TNFRII signaling. Objective: The aim of the present study was to develop an optimized protocol to estimate the level of expression of membrane-bound TNFRI and TNFRII, using QuantiBRITE PE calibration beads. Materials and methods: The percentage of cells that expressed membrane-bound TNFRI and TNFRII and the mean number of receptors per cell were determined by flow cytometry using PE-labeled antibodies against TNFR. To create a calibration curve and convert cell fluorescence intensity values to absolute numbers of receptors, we used QuantiBRITE PE beads. Results: CD19+ B lymphocytes had the least percentage of cells expressing TNFRI and the greatest number of receptor molecules per cell, whereas CD3+ T lymphocytes had the greatest percentage of cells expressing TNFRII and the lowest density of these receptors. We also established that stimulation of peripheral blood mononuclear cells (PBMCs) with the lipopolysaccharide (LPS) significantly increased the number of TNFRI and TNFRII on CD14+ monocytes. Conclusion: Application of the protocol-identified differences in the percentage of cells that expressed TNFRs, as well as the absolute number of receptors per cell, among different subpopulations of PBMCs, and between PBMCs cultured with and without LPS.

AngiomiR-126 expression and secretion from circulating CD34+ and CD14+ PBMCs: role for proangiogenic effects and alterations in type 2 diabetics

AbstractSeveral peripheral blood mononuclear cell (PBMC)–derived cell populations can promote angiogenesis, and differences in CD34+ or CD14+ surface expression have been used to separate PBMC subpopulations in this respect. AngiomiRs, microRNAs regulating angiogenesis, are key regulators of angiogenic processes. The present study examines differential angiomiR expression/secretion from CD34+/CD14+, CD34+/CD14−, CD34−/CD14+, and CD34−/CD14− PBMC subsets and their relevance for different proangiogenic properties. Notably, both circulating human CD34+/14+ and CD34+/14− PBMC subsets and their supernatants exerted more potent proangiogenic effects compared with CD34− PBMC subsets. MiR-126 was identified as most differentially expressed angiomiR in CD34+ compared with CD34− PBMC subsets, determined by miR-array and RT-PCR validation. Modulation of miR-126 by anti–miR-126 or miR-mimic-126 treatment resulted in significant loss or increase of proangiogenic effects of CD34+ PBMCs. MiR-126 levels in supernatants of CD34+ PBMC subsets were substantially higher compared with CD34− PBMC subsets. MiR-126 was secreted in microvesicles/exosomes, and inhibition of their release impaired CD34+ PBMCs proangiogenic effects. Notably, high-glucose treatment or diabetes reduced miR-126 levels of CD34+ PBMCs, associated with impaired proangiogenic properties that could be rescued by miR-mimic-126 treatment. The present findings provide a novel molecular mechanism underlying increased proangiogenic effects of CD34+ PBMCs, that is, angiomiR-126 expression/secretion. Moreover, an alteration of angiomiR-126 expression in CD34+ PBMCs in diabetes provides a novel pathway causing impaired proangiogenic effects.

HIV Type 1 Infection Up-Regulates TLR2 and TLR4 Expression and Function in Vivo and in Vitro

Toll-like receptors (TLRs) play a critical role in innate immunity against pathogens. Their stimulation induces the activation of NF-κB, an important inducer of HIV-1 replication. In recent years, an increasing number of studies using several cells types from HIV-infected patients indicate that TLRs play a key role in regulating the expression of proinflammatory cytokines and viral pathogenesis. In the present study, the effect of HIV-1 stimulation of monocyte-derived macrophage (MDM) and peripheral blood mononuclear cell (PBMC) subpopulations from healthy donors on the expression and functions of TLR2 and TLR4 was examined. In addition, and to complete the in vitro study, the expression pattern of TLR2 and TLR4 in 49 HIV-1-infected patients, classified according to viral load and the use of HAART, was determined and compared with 25 healthy subjects. An increase of TLR expression and production of proinflammatory cytokines were observed in MDMs and PBMCs infected with HIV-1 in vitro and in response to TLR stimulation, compared to the mock. In addition, an association between TLR expression and up-regulation of CD80 in plasmacytoid dendritic cells (pDCs) was observed. The ex vivo analysis indicated increased expression of TLR2 and TLR4 in myeloid dendritic cells (mDCs), but only of TLR2 in monocytes obtained from HIV-1-infected patients, compared to healthy subjects. Remarkably, the expression was higher in cells from patients who do not use HAART. In monocytes, there was a positive correlation between both TLRs and viral load, but not CD4(+) T cell numbers. Together, our in vitro and ex vivo results suggest that TLR expression and function can be up-regulated in response to HIV-1 infection and could affect the inflammatory response. We propose that modulation of TLRs represents a mechanism to promote HIV-1 replication or AIDS progression in HIV-1-infected patients.

Size dependent induction of proinflammatory cytokines and cytotoxicity of particulate beta-tricalciumphosphate in vitro

Cellular responses to particulate calcium phosphate ceramics can lead to inflammatory reactions under certain conditions that depend on particle composition, size and morphology. In this context, the potential influence of varying sizes of particulate beta-tricalciumphosphate (beta-TCP) on the induction of inflammation and cytotoxicity remains to be determined. The present work investigates the effects of beta-TCP particles of five different sizes (1, 3, 13, 32 and 40 μm) on human peripheral blood mononuclear cells (PBMC) in vitro concerning the release of TNF-alpha, IL-1beta and IL-8 after six and 18 h of incubation (ELISA) as well as intracellular TNF-alpha, IFN-gamma, IL-1alpha, IL-1beta and IL-8 levels within distinct PBMC subpopulations after 12 h (FACS). Potential cytotoxic effects were determined by assaying lactate dehydrogenase (LDH) and morphological analyses (electron microscopy). Beta-TCP 1 μm did not induce any cytokine after 6 h but slightly increases TNF-alpha, IL-1beta and IL-8 release after 18 h. Larger particles (32 and 40 μm) consistently caused higher levels of cytokine release by increasing the fraction of cytokine producing monocytes. They also caused higher levels of LDH release as did smaller, phagocytosable particles. These data suggest a less inflammatory and cytotoxic profile of beta-TCP devices with a smaller primary particle size when compared to larger particles.