Subpopulations Of Peripheral Blood Mononuclear Cells Research Articles

Revisiting miR-155 in intervertebral disc degeneration: blood cell signature and local cell-free profiles

We unraveled the expression profiles of coding-noncoding RNAs in intervertebral disc degeneration (IDD). However, it remains elusive regarding miR-155 expression in peripheral blood mononuclear cells (PBMCs) and local extracellular space in IDD. The study aimed for investigating the miR-155 expression of PBMCs, extracellular miRNAs (ex-miRNAs) of human nucleus pulposus (NP) tissues, and morphological changes of cell death in the IDD process. Here, we harvested peripheral blood and NP samples from scoliosis and IDD patients as control and degenerative groups, respectively. Then standard Ficoll density-gradient centrifugation was used to isolate PBMCs. The two subpopulations of PBMCs were divided based on the cellular difference in adherence, i.e., monocytes/ macrophages and lymphocytes. Quantitative real-time PCR was used to evaluate miR-155 relative expression in PBMCs. ex-miRNAs were screened by comparison between GSE19943, GSE63492, and extracellular RNA (exRNA) atlas. The morphological changes of cell death subtypes in IDD were observed in transmission electron microscopy (TEM). Results indicate that the lower expression of miR-155 in PBMCs from IDD patients ascribed to alterations in monocytes/macrophages. Moreover, we identified 594 shared hsa-miRNAs as the intracellular miRNA expression profile and 258 miRNAs as the ex-miRNA expression profile in human NP tissue. miR-155 was amongst intracellular miRNAs in NP. TEM observation presented multiple endocytic secretory vesicles within human NP cells, implicating exosome transporting machinery. Typical necroptosis and pyroptosis were noted in human NP. Collectively, this study reveals miR-155 expression in PBMCs from IDD patients. Moreover, we propose ex-miRNA expression profile and necroptosis/pyroptosis in human NP tissue, shedding novel light on the etiology of IDD.

Expression analysis of miRNAs from the DLK1-DIO3 locus in CD4+ and CD14+ cells in patients with relapsing-remitting multiple sclerosis

To analyze the expression of several previously unstudied miRNAs from the DLK1-DIO3 locus in peripheral blood mononuclear cells (PBMC) and in CD14+ and CD4+ cell subpopulations in patients with relapsing-remitting multiple sclerosis (RRMS) and healthy individuals. MiRNA expression analysis was performed in PBMC, CD14+, and CD4+ cells of 14 patients who did not take immunomodulatory drugs, and 14 individuals without autoimmune and inflammatory diseases by reverse transcription followed by real-time PCR. Expression levels of 10 miRNAs selected based on different criteria were significantly higher in PBMC in men with RRMS compared to healthy men; in women, their expression did not change in a similar comparison. No mass unidirectional changes in the expression of these miRNAs in RRMS men were observed in CD14+ and CD4+ cells. High consistency in the expression of miRNA pairs was also not found. The analysis of PBMC showed the involvement of 10 more miRNAs encoded by genes from the DLK1-DIO3 locus in the development of RRMS. The main observed effect of the increase in the expression levels of these miRNAs in men with RRMS compared with healthy men is not achieved due to the contribution of CD14+ and CD4+ cells. This opens up possibilities for studying the involvement of miRNAs in other PBMC subpopulations in the pathological processes in RRMS.

Potential PD-L1 expressing cytotoxic T-cell immunopathology in Alzheimer disease.

Growing evidence suggested that adaptive immunity involved Alzheimer's disease (AD). Peripheral blood mononuclear cell (PBMC) subpopulations and expression of immune checkpoints such as programmed death-ligand 1(PD-L1) and programmed cell death protein-1 (PD-1) could play roles in Alzheimer disease. The interaction of PD-L1/PD-1 lead PD-1 expressing T cells to attenuate T cell-activating signals. Recent studies had demonstrated the blockade of PD-L1/PD-1 axis modified brain pathology and restored cognitive performance in tauopathy murine model. However similar human experiment suggested controversial results in AD patients. To observe the changes of PBMC subpopulations and PD-L1/PD-1 expression in AD and healthy controls, we utilized flow cytometry, and applied t-distributed stochastic neighbor embedding (t-SNE) algorithm as a new approach to analyze results. AD patients (n=16) with different disease severity (CDR= 1 - 2) and healthy controls (n=16) were enrolled. Expression levels of PD-L1/PD-1 in AD patients and healthy controls were derived by flow cytometric analysis parameters. Moreover, a dimensionality reduction algorithm for visualized t-SNE, was implemented to analyze the PD-L1/PD-1 expressing cell clusters between AD patients and healthy controls. We found no significant PD-1 expression intensity difference between cytotoxic T lymphocytes (CTL) of AD patients and healthy controls. In AD patients, subpopulation of CTL expressing PD-L1 was elevated by 85.53%. Further, PD-L1 expressing CTL clusters between AD patients and healthy controls were compared. We observed that densities of PD-L1high CTL clusters are significantly higher than the healthy controls. Upregulation of PD-L1 and PD-L1 expressing CTL were increased in peripheral blood of AD patients, linking to newly found strong correlation between the level of PD-L1 positive CTL and markers which indicated negative immune climate. Abnormal adaptive immune response of CTL might associate with concurrent viral infection, intracellular bacterial infection, and underlying cancer in AD patients. Our results suggested a novel strategy to prevent AD progression with regulation of PD-L1 expressing CTL, and notation of underlying intracellular infection and malignancy for which could relate to dementia progression.

Targeting CD47 As a Novel Therapeutic Strategy in Multiple Myeloma

Introduction: CD47 is an immune checkpoint coordinating the innate and adaptive immune systems which allows cancer cells to escape the innate immune response by sending inhibitory signals to macrophages and regulates the adaptive immune anti-tumor response. Hence, CD47 has been identified as a novel therapeutic target in cancers, including multiple myeloma (MM) which is localized mainly in the bone marrow (BM). MM cells rely heavily on the interactions with tumor microenvironment (TME) and immune system. Macrophages are enriched in the BM of MM patients compared to healthy controls, and were demonstrated to support MM growth, in opposition to their intrinsic phagocytic properties. We study the new class of immune checkpoint inhibitor such as anti-CD47 monoclonal antibody (mAb) which enhance MM killing. We tested two anti-CD47 mAbs provided; Vx1000R (blocking only) - blocking CD47/SIRPα binding and Vx1004R (dual function) - blocking CD47/SIRPα binding and inducing cell death activity.The objectives of this study were: (1) to test the expression of CD47 (Vx1000R and Vx1004R binding) to MM cells, stromal cells (MSP-1 and HS5) and peripheral blood mononuclear cell (PBMC) sub-populations; (2) to study anti-CD47 mAbs binding in hypoxic conditions; (3) to investigate the effect of anti-CD47 mAbs on MM cell apoptosis in 2D and the 3D tissue engineered BM (3DTEBM; a scaffold incorporating BM plasma from MM patients, MM cells, and accessory cells) to reproduce the TME; (4) to test the cytotoxicity of Vx1004R on PBMC sub-populations in 3D culture; and (5) to examine the effect of Vx1004R on MM cell phagocytosis ex vivo by macrophages.Methods: We performed meta-analysis of CD47 mRNA expression in dataset available online in healthy, MGUS, smoldering MM and MM subjects. We tested the binding of Vx1000R and Vx1004R mAbs on MM.1S, H929, RPMI8226 and U266 cells and stromal cells (MSP-1 and HS5) in normoxia (21% O2) and hypoxia (1% O2), and in the 3DTEBM at 24h and 72h. We also tested Vx1000R binding to PBMC sub-populations (from 12 MM patients) including CD3 (T cells), CD14 (monocytes/macrophages), CD16 (NK cells, eosinophils, and neutrophils), CD19 (B cells), CD123 (pDC and basophils) and CD138 (MM cells).Next, we examined the cytotoxic activity of CD47 mAbs on MM cells alone and in co-culture with the BM negative fraction (CD138-depleted) and stromal cells (MSP-1) in 2D cultures by performing MTT survival assay, and in 3DTEBM cultures by flow cytometry. The cytotoxicity of Vx1004R was studied on PBMCs incorporated into a similar 3D model and treated with Vx1004R; proliferation and apoptosis of different sub-populations of PBMC were assessed via flow cytometry. Lastly, the effect of Vx1004R on MM cell phagocytosis by macrophages (CD14+; green) was tested after 2h of co-culture with MM cells (pre-labeled with DiD+; red) in the presence or absence of Vx1004R. Phagocytosis was determined as double positive (green+ red+) cells by flow cytometry.Results: The meta-analysis results demonstrated that CD47 mRNA expression increases with tumor progression (compared to early stage disease and healthy subjects). We demonstrated that MM.1S, H929, U266 and RPMI8226 cells and stromal cells (MSP-1 and HS5) express high levels of CD47, which does not alter in hypoxia, and that MM cells increase CD47 expression in 3DTEBM at 72h. CD47 (Vx1000R) is expressed in different sub-populations of PBMCs with the highest expression in MM cells. In addition, we have shown that anti-CD47 dual function mAb inhibits proliferation of MM cells, especially when co-cultured with negative fraction and MSP-1 stroma in 3DTEBM cultures. Moreover, we showed that anti-CD47 mAb induces phagocytosis of MM cells ex vivo by macrophages.Conclusions: CD47 is a promising target candidate for MM treatment, since CD47 mRNA increases with MM progression, and CD47 protein increases in MM cells in the presence of TME in the 3DTEBM, suggesting involvement in pathophysiology of MM. Moreover, anti-CD47 mAb enhances killing of MM cells when co-cultured with TME; however, it does not affect apoptosis of PBMC sub-populations in the 3DTEBM. Finally, anti-CD47 mAb induces phagocytosis of MM cells ex vivo by macrophages. These results suggest that anti-CD47 mAbs are applicable for MM treatment. Further studies are warranted to examine the effect of anti-CD47 mAbs as a novel strategy to inhibit the progression and dissemination of MM in vivo, especially when combined with chemotherapy. DisclosuresVij:Takeda: Honoraria, Research Funding; Jazz: Honoraria; Abbvie: Honoraria; Celgene: Honoraria; Amgen: Honoraria, Research Funding; Janssen: Honoraria; Bristol-Meyers-Squibb: Honoraria; Konypharma: Honoraria. Capoccia:Tioma: Employment. Manning:Tioma: Employment. Azab:Abbvie: Research Funding; Verastem: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner; Selexys: Research Funding; Karyopharm: Research Funding; Cell Works: Research Funding; Cleave Bioscience: Research Funding; Cellatrix LLC: Other: Founder and owner; Glycomimetics: Research Funding; Tioma: Research Funding.

Chelidonium majus crude extract induces activation of peripheral blood mononuclear cells and enhances their cytotoxic effect toward HeLa cells

ABSTRACT The aim of the study was to examine the immunomodulatory effect of crude Chelidonium majus L ethanolic extract on ex vivo harvested peripheral blood mononuclear cells (PBMNCs). PBMNCs were isolated by density gradient centrifugation. The PBMNC cytotoxicity assay was performed with HeLa tumor cells as target cells. MTT assay was used to estimate the proliferation effect of extract and cytotoxic efficiency of treated PBMNCs. Flow cytometric analysis was used for immunophenotyping. Treatment induced moderate proliferative response, perturbation in PBMNC ratios, and the emergence of some unconventional subpopulations. The percentage ratio of double positive CD4+ and CD8+ T lymphocytes and monocytes, ratio of T and B lymphocytes expressing CD14, and percentage of NK cells expressing CD57 increased after treatment, indicating activation of PBMNC subpopulations. Cytotoxic activity against HeLa cells was enhanced. Activation of PBMNCs and enhancement of their cytotoxic effect toward HeLa cells indicate the immunostimulatory effect of Ch. majus ethanolic extract.

Assessment of Glial Fibrillary Acidic Protein Binding to the Surface of Leukocytes with Dark‐Field Imaging and Computational Analysis

AbstractThe glial fibrillary acidic protein (GFAP) is widely established as a traumatic brain injury (TBI) biomarker and can be used in early diagnosis. As essential primary human immune cells, leukocytes are recruited to injured cerebral sites during TBI response, where they can interact with and potentially bind to TBI biomarkers. To date, no studies have demonstrated ultra‐low GFAP binding enumeration on leukocytes. Herein, a dark‐field imaging technique coupled with computational analysis is introduced to quantify GFAP bound to peripheral blood mononuclear cells (PBMCs). Dark‐field microscopy (DFM) with a custom‐written image acquisition software is developed for rapid 3D PBMC imaging by utilizing specific antiGFAP monoclonal antibody functionalized gold nanoparticles (antiGFAP‐AuNPs) as contrast‐generating probes. Subsequently, the developed algorithm is utilized in processing thousands of acquired images for rapid visualization and enumeration of bound antiGFAP‐AuNP on each leukocyte. The proposed method is demonstrates the specific binding of GFAP to the surface of PBMCs on a healthy donor blood. Thereafter, subpopulations of PBMCs with antiGFAP‐AuNP binding are identified with the assistance of fluorescence imaging and DFM imaging, paving a new way to understanding the relationship between TBI and leukocyte classes. Hence, this study offers a rapid and ultra‐sensitive strategy for biomarker assessment following TBI.

Circulating mitochondrial DNA (mtDNA) variants to predict metastatic progression of rectal cancer.

e16132 Background: High rates of systemic failure after curative-intent therapy in locally advanced rectal cancer (LARC) call for new diagnostic tools to improve risk-based treatment stratification. We hypothesized that blood mtDNA polymorphisms, generated by the high mtDNA mutation rate when the mitochondrial biogenesis is mobilized during clonal immune cell activation, correlate with systemic immune control of LARC dissemination and thus can be used as diagnostic marker. Methods: 41 LARC patients given neoadjuvant (chemo)radiotherapy had DNA extracted from whole blood (WB) sampled at the time of diagnosis, for high-throughput full-length mtDNA sequencing followed by annotation of mtDNA variants using the curated reference genome (mitomap.org). The composition of peripheral blood mononuclear cell (PBMC) subpopulations was analyzed by high-dimensional single-cell mass cytometry. Metastatic events were recorded over 24-60 months of follow-up after completion of the multimodal treatment. Results: The total number of WB-mtDNA variants was strikingly different across the study cases but without correlation to patient’s age, body mass index, or metformin use, which may affect mitochondrial function, or to the disease extension at presentation or the local tumor response to the neoadjuvant therapy. All patients had microsatellite-stable tumor. The higher the number of variants in MT-RNR2, encoding one of the two ribosomal subunits of mitochondria, the better distant metastasis-free survival (DMFS). For each patient, two MT-RNR2 sites were homoplasmic for either the normal or a deletion (3105AC > A, 3106CN > C) variant, categorizing the patients into two groups that comprised the same cases for both mtDNA sites. Nine of a total of 10 DMFS events occurred during the 18 first months of follow-up in one of the patient groups ( n = 23). The single event in the other group ( n = 18) appeared in a patient who had declined primary tumor surgery. In the former group, a low number of variants in the ribosomal subunit MT-RNR1 identified all patients who had metastatic progression. These patients had reduced PBMC frequency of CD8+ (cytolytic) T cells that were positive for CXCR4 ( p = 0.001), a chemokine receptor that promotes their homing to tumor sites. Conclusions: The WB-ribosomal mtDNA polymorphisms reflected systemic immune cells endowed with cytolytic and tumor-homing properties. The actual mtDNA variants were single-base alterations and might thus be analyzed by PCR and developed as a feasible assay for metastatic risk assessment in rectal cancer. Clinical trial information: 01816607 .

GRP78 expression in peripheral blood mononuclear cells is a new predictive marker for the benefit of taxanes in breast cancer neoadjuvant treatment

BackgroundBreast cancer treatment is tailored to the specific cancer subtype. Often, systemic treatment is given prior to surgery. Chemotherapy induces significant endoplasmic reticulum (ER) stress-mediated cell death and upregulation of 78-kDa glucose-regulated protein (GRP78). We hypothesized that chemotherapy induces ER stress not only in the tumor tissue but also in immune cells, which may affect the response to anti-cancer treatment.MethodsWe determined the surface expression of GRP78 on 15 different peripheral blood mononuclear cell (PBMC) subpopulations in 20 breast cancer patients at three time points of the neoadjuvant treatment, i.e., at baseline, after anthracycline treatment, and after taxanes treatment. For this purpose, we performed flow cytometric analyses and analyzed the data using ANOVA and the Tukey test. Serum cytokine levels were also evaluated, and their levels were correlated with response to treatment using the t-test after log transformation and Mann-Whitney U Wilcoxon W test.ResultsA significant increase in GRP78 expression in PBMCs was documented during the taxane phase, only in patients who achieved pathological complete response (pCR). GRP78-positive clones correlated with increased serum levels of interferon gamma (IFNγ).ConclusionsThe presence of GRP78-positive clones in certain PBMC subpopulations in pCR patients suggests a dynamic interaction between ER stress and immune responsiveness. The correlation of GRP78-positive clones with increased levels of IFNγ supports the idea that GRP78 expression in PBMCs might serve as a new predictive marker to identify the possible benefits of taxanes in the neoadjuvant setting.

Antitumoral and Immunomodulatory Effect of Mahonia aquifolium Extracts.

The prodrug potential of Mahonia aquifolium, a plant used for centuries in traditional medicine, recently gained visibility in the literature, and the activity of several active compounds isolated from its extracts was studied on biologic systems in vitro and in vivo. Whereas the antioxidative and antitumor activities of M. aquifolium-derived compounds were studied at some extent, there are very few data about their outcome on the immune system and tumor cells. To elucidate the M. aquifolium potential immunomodulatory and antiproliferative effects, the bark, leaf, flower, green fruit, and ripe fruit extracts from the plant were tested on peripheral blood mononuclear cells and tumor cells. The extracts exert fine-tuned control on the immune response, by modulating the CD25 lymphocyte activation pathway, the interleukin-10 signaling, and the tumor necrosis-alpha secretion in four distinct human peripheral blood mononuclear cell (PBMC) subpopulations. M. aquifolium extracts exhibit a moderate cytotoxicity and changes in the signaling pathways linked to cell adhesion, proliferation, migration, and apoptosis of the tumor cells. These results open perspectives to further investigation of the M. aquifolium extract prodrug potential.

CORRELATION BETWEEN FRAILTY AND DNA DAMAGE IN HEMATOPOIETIC STEM CELLS: A PILOT STUDY

Frailty is an age-related syndrome characterized by a progressive impairment of multiple physiological systems and leading to poor clinical and functional outcomes. Our aim was to explore the DNA damage, as an effect of increased oxidative stress related to frailty, on peripheral blood mononuclear cells (PBMC) and circulating hematopoietic stem cells (cHSC). According to Fried’s operating definition of frailty, we enrolled three groups of subjects: frail seniors (age >65 years, n=19), fit seniors (>65 years, n= 16) and young controls (age 25-35 years, n=19). We carried out a comprehensive assessment and obtained 3 vials of whole blood for cells and plasma separation. We separated PBMC by Ficoll-Paque and stained with specific conjugated antibodies leucocyte lineage and HSC. We evaluated DNA damage by FACS detection of γ-H2AX in the total PBMC and cHSC subpopulation. We observed an increased percentage of cells, although not significant, with DNA damage in PBMC from frail seniors (0.70%) compared to fit seniors (0.37%) and young controls (0.13%). The percentage of cells with DNA damage in cHSC of frail seniors (2.97%) was higher compared to fit seniors (1.66%, not significant) and young controls (0.46%, statistically significant). Moreover, cHSC present the statistically higher amount of DNA damage, measured as fluorescence intensity, compared to fit seniors and young controls. cHSC from frail seniors show the highest total DNA damage, compared to fit seniors and young controls. This is probably linked to an increase of oxidative stress related to frailty, which we are going to analyze in the near future.

P3688Familial recurrent autoimmune myocarditis associated with a truncating nonsense mutation of the desmoplakin gene

Abstract Background Arrhythmogenic cardiomyopathy (AC) is an important cause of ventricular arrhythmias in children and young adults. AC is associated with mutation of desmosomal proteins, however, cardiac disease penetrance is incomplete and the clinical course varies widely without recognizable exogenous or epi/genetic co-factors. Importantly, DSP mutation carriers may also display entirely non-cardiac e.g. dermatological phenotypes. Methods and results In two brothers with recurrent fulminant myocarditis, mutation screening of 218 cardiomyopathy-related genes identified a truncating mutation Arg1458* of desmoplakin (DSP). DSP immunhistology unexpectedly revealed complete loss (“knockout”) of DSP protein in endomyocardial biopsies (EMBs), but none of the histological anomalies of AC. Criteria for histological diagnosis of myocarditis were not either fulfilled, and cardiac MRI revealed no features associated with AC. Screening for infections was negative, there was no substance abuse, medication or vaccination. Possible disease triggers were competitive sport events. Myosin and troponin I autoantibodies were detected at titers up to 1:320. We used allele-specific RT-PCR to distinguish if the patients' allele classified as “normal” was actually defective due to promotor mutation or epigenetic silencing. RT-PCRs were done on EMBs and peripheral blood mononuclear cells (PBMCs). In a cohort of dilated cardiomyopathy (DCM) patients we were able to detect DSP transcripts in both, PBMC and left-ventricular heart tissue. RNA sequencing of human PBMC subpopulations suggested that DSP transcription may be restricted to certain immune cell subtypes. RT-PCRs revealed that both Arg1458* carriers have a functional second DSP allele, indicating that their “DSP knockout” occurs at the protein level and may be due to protein instability and degradation within desmosomes. We screened additional existing cohorts for such variants and identified stopgain variant Gln307Ter in a 37-yrs-old woman with ARVC. This patient's sister died from heart failure at the age of 39. In a 59-yrs-old female LVNC patient, stopgain variant Y1391X was identified. Here, family history was unclear, her brother probably died from coronary artery disease. In a 71-yrs-old female DCM patient with no family history, stopgain variant Tyr1512Ter was identified. Conclusions The described patients with DSP truncations strongly suggest the existence of additional genetic or exogenous modifiers driving pathogenesis either way. DSP defects may cause recurrent myocarditis, and mutation screening is advisable to enable early detection of high-risk patients with similar phenotypes. Our finding of complete myocardial DSP protein loss emphasizes that DNA sequencing may miss critical molecular disturbances. It is indispensable to also analyze transcriptome and protein level in the tissue actually affected in a patient in order to recognize his/her individual pathogenesis.

Tissue-regenerative potential of the secretome of \u03b3-irradiated peripheral blood mononuclear cells is mediated via TNFRSF1B-induced necroptosis

Peripheral blood mononuclear cells (PBMCs) have been shown to produce and release a plethora of pro-angiogenetic factors in response to γ-irradiation, partially accounting for their tissue-regenerative capacity. Here, we investigated whether a certain cell subtype of PBMCs is responsible for this effect, and whether the type of cell death affects the pro-angiogenic potential of bioactive molecules released by γ-irradiated PBMCs. PBMCs and PBMC subpopulations, including CD4+ and CD8+ T cells, B cells, monocytes, and natural killer cells, were isolated and subjected to high-dose γ-irradiation. Transcriptome analysis revealed subpopulation-specific responses to γ-irradiation with distinct activation of pro-angiogenic pathways, cytokine production, and death receptor signalling. Analysis of the proteins released showed that interactions of the subsets are important for the generation of a pro-angiogenic secretome. This result was confirmed at the functional level by the finding that the secretome of γ-irradiated PBMCs displayed higher pro-angiogenic activity in an aortic ring assay. Scanning electron microscopy and image stream analysis of γ-irradiated PBMCs revealed distinct morphological changes, indicative for apoptotic and necroptotic cell death. While inhibition of apoptosis had no effect on the pro-angiogenic activity of the secretome, inhibiting necroptosis in stressed PBMCs abolished blood vessel sprouting. Mechanistically, we identified tumor necrosis factor (TNF) receptor superfamily member 1B as the main driver of necroptosis in response to γ-irradiation in PBMCs, which was most likely mediated via membrane-bound TNF-α. In conclusion, our study demonstrates that the pro-angiogenic activity of the secretome of γ-irradiated PBMCs requires interplay of different PBMC subpopulations. Furthermore, we show that TNF-dependent necroptosis is an indispensable molecular process for conferring tissue-regenerative activity and for the pro-angiogenic potential of the PBMC secretome. These findings contribute to a better understanding of secretome-based therapies in regenerative medicine.

Abstract # 3100 Targeted modulation of neurotransmitter receptors on specific leukocyte subpopulations in rheumatoid arthritis

Background and Aim Current treatment strategies for rheumatoid arthritis (RA) including treat-to-target approaches are only partly successful. Therefore, new treatment options are still needed. There is some evidence for a role for dopamine (DA) in the pathogenesis of RA. Moreover, dopaminergic receptors (DR) are expressed on immune cells. The aim of this study was to further investigate the dopaminergic pathway in RA. Methods The expression of DRs (DRD1-5) was examined via flow cytometry in peripheral blood mononuclear cells (PBMC) of patients with RA (n = 5) and of healthy controls (HC, n = 5). In vitro experiments with DR-agonists were performed to determine the effects of DR-stimulation on immune cells. Results All investigated PBMC subpopulations expressed DRs, with the highest expression levels in NK cells and monocytes. Expression of DRD5 tended to be higher in RA NK cells, and DRD1 was upregulated in RA B-cells compared to HC. The activation marker CD69 was detected in all investigated PBMC subpopulations after 6 h and 22 h of stimulation with DR-agonists. Conclusion Our results show that DRs are present on immune cells and their expression seems to be modulated on defined subpopulations during RA. B cells, which are critical in RA, and NK cells appear attractive in context of dopaminergic treatment approaches. Further experiments are ongoing to better define the role of DA on peripheral immune cells during the course of the disease.

Effects of ruminal pH on gene expression in the rumen epithelium, peripheral blood mononuclear cell subpopulations, and blood metabolites from Holstein calves during weaning transition.

We investigated the relationships between ruminal pH, gene expression in the rumen epithelium (RE), peripheral blood mononuclear cell subpopulations, and blood metabolites in Holstein calves during weaning transition. Calves (Weaning group, n=7) were assigned to one of two groups, and fed calf starter with forage (Forage group, n=3) or without forage (Starter group, n=4). Ruminal pH was measured continuously. Samples were collected at −1, 0, 1, and 3 weeks (blood and rumen fluid) or 3 weeks (rumen epithelium) after weaning. In the Weaning group, ruminal pH increased, and several blood metabolites increased (blood urea nitrogen [BUN], beta-hydroxybutyrate [BHB], and gamma-glutamyl transferase [GGT]) or decreased (total cholesterol [T-CHO] and phospholipid) after weaning. Ruminal pH was positively correlated with CD8+CD45R− cell populations and blood metabolites (BUN, glucose, and BHB) and negatively correlated with GGT activity. The 24 hr mean ruminal pH was higher in the Forage group during weaning transition, and toll-like receptor 4 mediated signaling pathway was activated in the Starter group at 3 weeks post-weaning. The number of CD8+CD45R− cells tended to be higher, and several blood metabolites (glucose, triglycerides, T-CHO, and phospholipid) were higher in the Forage group after weaning. Calves with higher ruminal pH also showed a greater energy metabolism status simultaneously with lesser hepatic disturbance enzymes in the peripheral blood. The results of our study indicate that serum GGT activity may be a plausible biomarker for predicting ruminal acidosis in Holstein calves during weaning transition.

Frequency of bovine viral diarrhea virus detected in subpopulations of peripheral blood mononuclear cells in persistently infected animals and health outcome

Bovine viral diarrhea viruses (BVDV) cause acute and persistent infections. Acute infection results in generalized immunosuppression characterized by a decrease in circulating lymphocytes as a result of depletion of CD4+ and CD8 + T cell populations. Persistent infection with BVDV is the result of immune tolerance and is generally not associated with lymphocytopenia. The health outcome of persistently infected (PI) calves varies widely; some die of mucosal disease, some succumb to ill thrift and others appear normal and survive to adulthood. Detection of BVDV at the single lymphoid cell level is important to the study of subpopulations of peripheral blood mononuclear cells (PBMC) during BVDV infections, however there are few methods available for the detection and quantification of BVDV at this level. To circumvent this difficulty, a novel flow cytometry-based PrimeFlow RNA assay using in-situ detection of BVDV was developed. This assay was used to evaluate differences in viral distribution within subpopulations of PBMC over time in PI calves carrying one of two different species of BVDV (type 1 and type 2). Calves were sampled at 3 different time points approximately one month apart. During the course of the study, a subset of the calves died from ill thrift. Mucosal disease was not indicated in any of the deaths. Using RNA probes specific for the BVDV Npro-Erns coding regions for each respective virus, BVDV RNA was detected in all PBMC of PI that appeared clinically healthy. Calves that succumbed to ill thrift were found to have no or little virus in T cells. The clearance of virus from T cells suggests a breakdown in immune tolerance in these calves. This is the first report of a pattern observed in the viral load in the T cell subpopulations and survival in PI calves.

A Co-culture Model of PBMC and Stem Cell Derived Human Nasal Epithelium Reveals Rapid Activation of NK and Innate T Cells Upon Influenza A Virus Infection of the Nasal Epithelium.

Background: We established an in vitro co-culture model involving H3N2-infection of human nasal epithelium with peripheral blood mononuclear cells (PBMC) to investigate their cross-talk during early H3N2 infection.Methods: Nasal epithelium was differentiated from human nasal epithelial stem/progenitor cells and cultured wtih fresh human PBMC. PBMC and supernatants were harvested after 24 and 48 h of co-culture with H3N2-infected nasal epithelium. We used flow cytometry and Luminex to characterize PBMC subpopulations, their activation and secretion of cytokine and chemokines.Results: H3N2 infection of the nasal epithelium associated with significant increase in interferons (IFN-α, IFN-γ, IL-29), pro-inflammatory cytokines (TNF-α, BDNF, IL-3) and viral-associated chemokines (IP-10, MCP-3, I-TAC, MIG), detectable already after 24 h. This translates into rapid activation of monocytes, NK-cells and innate T-cells (MAIT and γδ T cells), evident with CD38+ and/or CD69+ upregulation.Conclusions: This system may contribute to in vitro mechanistic immunological studies bridging systemic models and possibly enable the development of targeted immunomodulatory therapies.

Abstract 16945: Identification of Peripheral Blood Mononuclear Cell Subpopulations in Chronic Left Ventricular Dysfunction Patients With Improved Functional Outcomes: A Biorepository Evaluation From the CCTRN FOCUS Trial

Background: Although cell therapy could potentially slow or reverse progression of chronic ischemic heart failure (HF), clinical trials of bone marrow-derived mononuclear cells (BM-MNC) have met with minimal success. It is unknown why some patients improve (i.e. improvers) and others do not (i.e. non-improvers). B cell numbers, in fact, have been associated with improvement after acute myocardial infarction (MI). We hypothesized that improvers have a higher percentage of B cells in peripheral blood (PB) vs. non-improvers after study treatment. Methods: We used flow cytometry to evaluate temporal changes in the frequency of PB subpopulations after BM-MNC or placebo therapy in HF patients enrolled in the FOCUS-CCTRN trial. PB was collected at day 0 (baseline) and days 1, 30, 90, and 180 from consented participants. Cohort 1 (n=17) patients, who improved in left ventricular ejection fraction (LVEF), left ventricular end-systolic volume (ESV), and maximal oxygen consumption (VO 2 max) during the 6-month course, were compared to patients who declined in the above three outcomes, cohort 2 (n=11). Linear mixed model was used to determine the changes over time in each cell population. The difference between each time point to day 0 was calculated as linear contrast. Results: Cohort 1 had a higher frequency of B cells (CD45+CD19+) at day 0 (15.24±7.55 vs. 8.95±3.49, p=0.002), day 1 (14.67±5.53 vs. 8.78±4.51, p=0.003), day 90 (14.98±6.64 vs. 8.49±4.33, p=0.006) and day 180 (13.44±7.85 vs. 8.07±4.34, p=0.033) and regulatory B cells (CD19+CD11B+) at day 0 (2.06±1.20 vs. 1.09±0.58, p=0.011) and day 90 (2.49±1.50 vs. 1.32±0.62, p=0.016) when compared to cohort 2. Both cohorts had an increase in CD11B+ at day 1 when compared to baseline (91.53±3.43 vs. 93.09±3.69, p=0.020) (92.01±3.63 vs. 94.60±8.70, p=0.005), which remained higher in cohort 2 until day 30 (92.01±3.63 vs. 93.92±1.91, p=0.020). Conclusion: A higher frequency of B cells and subsets was observed in PB of patients who improved independent of treatment. These data support prior findings of the importance of B cells in recovery after MI and suggest an important relationship between the humoral immune system and HF.

Aberrant expression of the innate restriction factor bone marrow stromal antigen-2 in primary Sjögren's syndrome

ObjectiveThe objective of this study was to analyze bone marrow stromal antigen-2 (BST-2) levels in labial glands, total peripheral blood mononuclear cells (PBMCs) and PBMC subpopulations from primary Sjögren's syndrome (pSS) patients and determine the correlation between BST-2 expression and clinical characteristics. Materials and methodsPBMC subsets were positively separated using magnetic microbeads. BST-2 mRNA levels in labial glands, total PBMCs and PBMC subsets of 30 pSS and 30 healthy control (HC) subjects were investigated using real-time polymerase chain reaction (RT-PCR). Distribution of BST-2-positive cells in the labial glands was assessed by immunohistochemistry. ResultsBST-2 was significantly increased in pSS labial glands and was positively correlated with the VAS value for parotid gland swelling and rheumatoid factor and β2-microglobulin serum levels. BST-2 levels were statistically different between pSS patients with positive and negative expression of anti-SSA antibody. Positive focal infiltrating lymphocytes and adjacent ductal epithelial cells were observed in labial glands from pSS patients, while there were a few scattered positive ductal epithelial cells in controls. BST-2 was also up-regulated in CD19+ B cells and the remaining CD4-CD8-CD19- PBMCs. ConclusionBST-2 was aberrantly expressed in pSS patients, and expression in labial glands was positively correlated with important clinical characteristics; thus, it may be a potential biomarker of pSS activity.

Molecular profiling of regulatory T cells in pulmonary sarcoidosis

BackgroundSarcoidosis is characterized by exaggerated immune response to unknown agent and can affect different organs. One of the main players in the pathology of the disease are regulatory T cells (Tregs), however, up to date the mechanisms of the possible molecular alterations of this particular cell subset are not known. MethodsIn the current study we looked for the global transcriptomic changes of miRNAs, using predefined array, and mRNAs (RNA seq analysis) of Tregs of patients with the most predominant form of the disease - acute pulmonary sarcoidosis (PS). For this purpose sorted CD4+/CD25+/CD127- Tregs from peripheral blood (PB) and CD4+/CD25 + Tregs from bronchoalveolar lavage (BAL) were used. ResultsMiRNA analysis revealed that Tregs isolated from PB and BAL display significantly different miRNA profile, suggesting an important role of the pulmonary microenvironment in creating these changes. Among disease-related miRNAs of PB Tregs we identified miR-155 and miR-223. Moreover, looking at the global transcriptome of PB Tregs, we recognized alterations in TLR-2 signaling pathway and in the downstream of NF-κB apoptosis and proliferation signals. However, induction of TLR-2 expression was found not only in Tregs, but also in the heterogeneous population of peripheral blood mononuclear cells (PBMC) as well as two PBMC subpopulations (CD4+/CD25-and CD4-/CD25-) of patients with PS. This indicates that activation of TLR signaling pathway in sarcoidosis does not occur only in Tregs. ConclusionOur findings offer a deeper insight into the molecular mechanisms of Tregs reduced suppression and increased apoptosis in patients with PS. Based on the current results, future studies should focus on possible therapeutic effect of TLR-2 signaling inhibition.

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.