Blood Mononuclear Cell Cultures Research Articles

Effect of M-CSF on the expression of endothelial progenitor cell markers in blood mononuclear cell culture in coronary heart disease

Aim. To evaluate the nature of changes in the expression of markers of endothelial progenitor cells (VEGFR2, CD34, CD14) and endothelial cells (CD146) in association with the expression of the leukocyte common antigen CD45 in the culture of blood mononuclear cells in the presence of M-CSF in patients with coronary heart disease (CHD) and healthy donors.Materials and methods. The study included 12 patients with CHD with class III–V angina pectoris and 10 healthy donors, from whom 30 ml of venous blood was taken on an empty stomach in the morning and stabilized with heparin. Blood mononuclear cells were isolated by Ficoll density gradient centrifugation (1.077 g / cm3) and subject to immunomagnetic separation using CD14-MicroBeads and CD34-MicroBead Kit (Miltenyi Biotec B.V. & Co. KG, Germany). The resulting CD14+ and CD34+ culture of mononuclear cells was incubated for 6 days in a complete nutrient medium with and without M-CSF 50 ng / ml (Cloud-Clone Corp., USA) with complete replacement of the medium and repeated application of M-CSF on day 3. After 6 days, the proportions of CD45+, CD14+, CD34+, VEGFR2+, and CD146+ cells in the culture were assessed by flow cytometry using CD14-FITC, CD34-PE, VEGFR2-Alexa Fluor 647; CD45-FITC and CD146-PerCP antibodies (BD Biosciences, USA).Results. It was shown that in healthy donors, the proportion of CD146+ cells in the co-culture of blood mononuclear cells with M-CSF exceeded their number in the sample without it, with comparable expression rates of CD45, CD14, and VEGFR2 markers between the control and stimulated cultures. In CHD patients, the number of CD146+ and VEGFR2+ cells did not change when M-CSF was added to the mononuclear cell culture; however, the proportion of CD14+ cells increased and the proportion of CD45+ cells decreased compared to the control sample. The number of CD34+ cells was comparable both between control and stimulated samples, and between the groups of examined individuals. At the same time, in patients with CHD, an increased proportion of VEGFR2+ cells was found in the control and stimulated samples compared to healthy individuals, while an increased proportion of CD14+ cells was detected only in the stimulated culture.Conclusion. The development of CHD disrupts the response of blood mononuclear cells to the effect of M-CSF, increasing the number of CD14+ and reducing the proportion of CD45+ cells in the culture in the absence of stimulating effects on the expression of endothelial cell marker CD146. At the same time, M-CSF does not affect the expression of CD34 and VEGFR2 in endothelial progenitor cells both in patients with CHD and in healthy individuals.

Study on the mechanism of oral administration of tetrandrine during neoadjuvant chemotherapy for colon cancer.

Colon cancer is a digestive tract tumor with one of the highest frequencies worldwide, and with a high fatality rate. The present study aimed to investigate the expression and regulation of inflammatory factors in tumor tissues, monocytes and blood samples in patients with colon cancer (n=46) following treatment with neoadjuvant chemotherapy combined with tetrandrine. All patients underwent tumor resection after neoadjuvant chemotherapy. In the experimental group, 20 cases took tetrandrine during chemotherapy, while in the control group, 26 cases underwent chemotherapy without tetrandrine. Reverse transcription-quantitative PCR and western blotting were performed to detect the mRNA and protein expression levels of TNF-α. ELISA was used to detect the cytokine/chemokine expression levels [IL-15, IL-1β and IL-6, as well as chemokine ligand (CCL)2, CCL5, CCL20, chemokine (C-X-C motif) ligand CXCL1, CXCL2, CXCL3, CXCL5 and CXCL10 in the culture supernatant of colon cancer tissue]. Human blood mononuclear cells were cultured, and cytokine release was determined by ELISA. Cell proliferation ability was assessed using the MTT assay. Compared with the control group, the mRNA and protein expression levels of tumor necrosis factor-α (TNF-α) were downregulated in tumor tissues and serum and the serum levels of IL-15, IL-1β and IL-6 were relatively low in the experimental group. The expression levels of CCL5, CXCL2 and CXCL10 in the supernatant of cancer tissue culture were relatively low, compared with the conditioned medium prepared from tumor tissues of patients not receiving tetrandrine. When the cultured blood mononuclear cells were stimulated by the tissue culture supernatant from the experimental group, less IL-15, IL-1β and IL-6 were released, compared with the medium of tumor tissues of patients not taking tetrandrine. Following stimulation with the tissue culture supernatant from the experimental group, the proliferation ability of HCT116 colon cancer cells significantly declined. During chemotherapy of patients with colon cancer, tetrandrine may inhibit the expression of TNF-α in cancer tissues and blood, reduce the release of inflammatory factors and chemokines and decrease cancer cell proliferation. These findings provide a theoretical basis for the treatment of colon cancer in the clinic.

Efficient expansion of rare human circulating hematopoietic stem/progenitor cells in steady-state blood using a polypeptide-forming 3D culture

Although widely applied in treating hematopoietic malignancies, transplantation of hematopoietic stem/progenitor cells (HSPCs) is impeded by HSPC shortage. Whether circulating HSPCs (cHSPCs) in steady-state blood could be used as an alternative source remains largely elusive. Here we develop a three-dimensional culture system (3DCS) including arginine, glycine, aspartate, and a series of factors. Fourteen-day culture of peripheral blood mononuclear cells (PBMNCs) in 3DCS led to 125- and 70-fold increase of the frequency and number of CD34+ cells. Further, 3DCS-expanded cHSPCs exhibited the similar reconstitution rate compared to CD34+ HSPCs in bone marrow. Mechanistically, 3DCS fabricated an immunomodulatory niche, secreting cytokines as TNF to support cHSPC survival and proliferation. Finally, 3DCS could also promote the expansion of cHSPCs in patients who failed in HSPC mobilization. Our 3DCS successfully expands rare cHSPCs, providing an alternative source for the HSPC therapy, particularly for the patients/donors who have failed in HSPC mobilization.

Improved harvest and fixation methodology for isolated human peripheral blood mononuclear cells in cytokinesis-block micronucleus assay

Purpose In suspected radiation exposures, cytokinesis-block micronucleus (CBMN) assay is used for biodosimetry by detecting micronuclei (MN) in binucleated (BN) cells in whole blood and isolated peripheral blood mononuclear cell (PBMC) cultures. Standardized harvest protocols for whole blood were published by the International Atomic Energy Agency (IAEA) in 2001 (Technical report no. 405) and 2011 (EPR-Biodosimetry). For isolated PBMC harvest, cytocentrifugation of fresh cells is recommended to preserve cytoplasmic boundaries for MN scoring. However, cytocentrifugation utilizes specialized equipment and long-term cell suspension storage is difficult. In this study, an alternative CBMN harvest protocol is proposed for laboratories interested in culturing PBMCs and storing fixed cells with routine biodosimetry methods. Materials and methods Peripheral blood from 4 males (24, 34, 41, 51 y.o.) and females (26, 37, 44, 56 y.o.) was irradiated with 0 and 2 Gy X-rays. For cells harvested with IAEA 2001 and 2011 protocols, whole blood was used. For cells harvested with our protocol (CRG), isolated PBMCs were used. CRG protocol was validated in DAPI, acridine orange and Giemsa stain, and in three other laboratories. Cytoplasm status, nuclear division index (NDI) and induced MN frequency (MN frequency at 2 Gy – background MN frequency at 0 Gy) (MN/1000 BN) of Giemsa-stained BN cells were compared in IAEA 2001, IAEA 2011, IAEA 2011 + formaldehyde (FA) and CRG protocols. Effects of low and high humidity spreading were evaluated. Results >94% of 1000 BN cells were scorable with clear cytoplasmic boundaries in all donors harvested with CRG protocol. FA addition in IAEA 2011 protocol reduced cell rupture in whole blood cultures, but cell rupture was affected by age, sex and humidity. Almost all cells harvested with IAEA 2001 protocol had cytoplasm loss. PBMCs harvested with CRG protocol stained well in DAPI, acridine orange and Giemsa, and showed high scorable BN frequency in all laboratories. A higher NDI and a lower induced MN frequency were seen in 2 Gy isolated PBMC than whole blood cultures. Conclusion This quick CBMN harvest protocol for isolated PBMCs is a viable alternative to cytocentrifugation, as many scorable BN cells were obtained with routine biodosimetry reagents and equipment. IAEA 2011 + FA protocol should be used to improve CBMN harvest in whole blood cultures. Humidity during spreading should be optimized depending on the harvest protocol. NDI and MN frequency should be separately evaluated for whole blood and isolated PBMC cultures.

Assessment of an Antibody-in-Lymphocyte Supernatant Assay for the Etiological Diagnosis of Pneumococcal Pneumonia in Children.

New diagnostic tests for the etiology of childhood pneumonia are needed. We evaluated the antibody-in-lymphocyte supernatant (ALS) assay to detect immunoglobulin (Ig) G secretion from ex vivo peripheral blood mononuclear cell (PBMC) culture, as a potential diagnostic test for pneumococcal pneumonia. We enrolled 348 children with pneumonia admitted to Patan Hospital, Kathmandu, Nepal between December 2015 and September 2016. PBMCs sampled from participants were incubated for 48 h before harvesting of cell culture supernatant (ALS). We used a fluorescence-based multiplexed immunoassay to measure the concentration of IgG in ALS against five conserved pneumococcal protein antigens. Of children with pneumonia, 68 had a confirmed etiological diagnosis: 12 children had pneumococcal pneumonia (defined as blood or pleural fluid culture-confirmed; or plasma CRP concentration ≥60 mg/l and nasopharyngeal carriage of serotype 1 pneumococci), and 56 children had non-pneumococcal pneumonia. Children with non-pneumococcal pneumonia had either a bacterial pathogen isolated from blood (six children); or C-reactive protein <60 mg/l, absence of radiographic consolidation and detection of a pathogenic virus by multiplex PCR (respiratory syncytial virus, influenza viruses, or parainfluenza viruses; 23 children). Concentrations of ALS IgG to all five pneumococcal proteins were significantly higher in children with pneumococcal pneumonia than in children with non-pneumococcal pneumonia. The concentration of IgG in ALS to the best-performing antigen discriminated between children with pneumococcal and non-pneumococcal pneumonia with a sensitivity of 1.0 (95% CI 0.73–1.0), specificity of 0.66 (95% CI 0.52–0.78) and area under the receiver-operating characteristic curve (AUROCC) 0.85 (95% CI 0.75–0.94). Children with pneumococcal pneumonia were older than children with non-pneumococcal pneumonia (median 5.6 and 2.0 years, respectively, p < 0.001). When the analysis was limited to children ≥2 years of age, assay of IgG ALS to pneumococcal proteins was unable to discriminate between children with pneumococcal pneumonia and non-pneumococcal pneumonia (AUROCC 0.67, 95% CI 0.47–0.88). This method detected spontaneous secretion of IgG to pneumococcal protein antigens from cultured PBMCs. However, when stratified by age group, assay of IgG in ALS to pneumococcal proteins showed limited utility as a test to discriminate between pneumococcal and non-pneumococcal pneumonia in children.

Prolactin Induces IL-2 Associated TRAIL Expression on Natural Killer Cells from Chronic Hepatitis C Patients In vivo and In vitro.

Natural killer cells (NKC) are a major component of the innate immune response to HCV, mediating their effects through TRAIL and IFN-γ. However, their function is diminished in chronic HCV patients (HCVp). Prolactin is an immunomodulatory hormone capable of activating NKC. The study aims to explore if hyperprolactinemia can activate NKC in HCVp. We treated twelve chronic HCVp (confidence level =95%, power =80%) for 15 days with Levosulpiride plus Cimetidine to induce mild hyperprolactinemia. Before and after treatment, we determined TRAIL and NKG2D expression on peripheral blood NKC, along with cytokine profiles, viral loads and liver function. We also evaluated in vitro effects of prolactin and/or IL-2 on NKC TRAIL or NKG2D expression and IFN-γ levels on cultured blood mononuclear cells from 8 HCVp and 7 healthy controls. The treatment induced mild hyperprolactinemia and increased TRAIL expression on NKC as well as the secretion of IL-1ra, IL-2, PDGF and IFN-γ. Viral loads decreased in six HCVp. IL-2 and TRAIL together explained the viral load decrease. In vitro, prolactin plus IL-2 synergized to increase TRAIL and NKG2D expression on NKC from HCVp but not in controls. Levosulpiride/Cimetidine treatment induced mild hyperprolactinaemia that was associated with NKC activation and Th1-type cytokine profile. Also, an increase in TRAIL and IL-2 was associated with viral load decrease. This treatment could potentially be used to reactivate NKC in HCVp.

MicroRNA profiling of human myeloid angiogenic cells derived from peripheral blood mononuclear cells

Human myeloid angiogenic cells (MACs), also termed early endothelial progenitor cells, play an important role in neovascularization and vascular repair. MicroRNAs (miRNAs) are a class of naturally occurring, noncoding, short (∼22 nucleotides), single-stranded RNAs that regulate gene expression post-transcriptionally. MiRNAs have been shown to regulate MAC function. A miRNA signature of MACs was described approximately a decade ago, and many new miRNAs have been discovered in recent years. In this study, we aimed to provide an up-to-date miRNA signature for human MACs. MACs were obtained by culture of human peripheral blood mononuclear cells in endothelial medium for 7 days. Using qPCR array analysis we identified 72 highly expressed miRNAs (CT value < 30) in human MACs. RT-qPCR quantification of select miRNAs revealed a strong correlation between the CT values detected by the array analysis and RT-qPCR, suggesting the miRNA signature generated by the qPCR array assay is accurate and reliable. Experimentally validated target genes of the 10 most highly expressed miRNAs were retrieved. Only a few of the targets and their respective miRNAs have been studied for their role in MAC biology. Our study therefore provides a valuable repository of miRNAs for future exploration of miRNA function in MACs.

Enhanced interleukin-8 production in mononuclear cells in severe pediatric obstructive sleep apnea

BackgroundObstructive sleep apnea (OSA) is a risk factor for cardiovascular disease, metabolic disorders, and cognitive dysfunction. Current thinking links chronic intermittent hypoxia (CIH) with oxidative stress and systemic inflammation. However, the sequence of events leading to the morbidities associated with OSA is poorly understood in children. Monocytes are known to be altered by chronic hypoxia. Thus in this prospective study, we investigated inflammatory cytokine profiles from cultures of peripheral blood mononuclear cells (PBMC) obtained from children with severe OSA and sleep-related CIH.MethodsTen children with OSA (cases) and 5 age-matched children without OSA (controls) were recruited for study. Samples of plasma and PBMC were obtained before and after adenotonsillectomy. The levels of the inflammatory cytokines, interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-12p70, and tumor necrosis factor-α (TNFα), were measured in both plasma and ex vivo culture supernatants of PBMC incubated with lipopolysaccharide (LPS) using the cytometric bead assay.ResultsUpon activation of PBMC by LPS, the levels of IL-8 in the culture supernatants from cases were threefold higher than in controls. The levels of the other cytokines including IL-1β, IL-6, and TNFα, in culture supernatant of PBMC from cases showed no difference from controls; nor were there significant differences in plasma cytokine levels.ConclusionWe speculate that in young children with sleep-related CIH, an enhanced production capacity of IL-8 precedes the development of systemic inflammatory markers. Future work should evaluate IL-8 production capacity as a potential biomarker for OSA severity.

Aberrant DNA methylation of mTOR pathway genes promotes inflammatory activation of immune cells in diabetic kidney disease

DNA methylation has been implicated in the pathogenesis of diabetic kidney disease (DKD), but the underlying mechanisms remain unclear. In this study, we tested the hypothesis that aberrant DNA methylation in peripheral immune cells contributes to DKD progression. We showed that levels of DNA methyltransferase 1 (DNMT1), a key enzyme for DNA methylation, were increased along with inflammatory activity of peripheral blood mononuclear cells in DKD patients. Inhibition of DNMT1 with 5-aza-2'-deoxycytidine (5-Aza) markedly increased the proportion of CD4+CD25+ regulatory T cells in peripheral blood mononuclear cells in culture and in diabetic animals. Adoptive transfer of immune cells from 5-Aza-treated animals showed beneficial effects on the host immune system, resulting in a significant improvement of DKD. Using genome-wide DNA methylation assays, we identified the differentially methylated cytosines in the promoter regions of mammalian target of rapamycin (mTOR) regulators in peripheral blood mononuclear cells of diabetic patients. Further, mRNA arrays confirmed the consistent induction of genes expressed in the mTOR pathway. Importantly, down-regulation of DNMT1 expression via RNA interference resulted in prominent cytosine demethylation of mTOR negative regulators and subsequent decrease of mTOR activity. Lastly, modulation of mTOR resulted in changes in the effect of 5-aza on diabetic immune cells. Thus, up-regulation of DNMT1 in diabetic immune cells induces aberrant cytosine methylation of the upstream regulators of mTOR, leading to pathogenic activation of the mTOR pathway and consequent inflammation in diabetic kidneys. Hence, this study highlights therapeutic potential of targeting epigenetic events in immune system for treating DKD.

Regeneration-associated cells improve recovery from myocardial infarction through enhanced vasculogenesis, anti-inflammation, and cardiomyogenesis.

BackgroundConsidering the impaired function of regenerative cells in myocardial infarction (MI) patients with comorbidities and associated risk factors, cell therapy to enhance the regenerative microenvironment was designed using regeneration-associated cells (RACs), including endothelial progenitor cells (EPCs) and anti-inflammatory cells.MethodsRACs were prepared by quality and quantity control culture of blood mononuclear cells (QQMNCs). Peripheral blood mononuclear cells (PBMNCs) were isolated from Lewis rats and conditioned for 5 days using a medium containing stem cell factors, thrombopoietin, Flt-3 ligand, vascular endothelial growth factor, and interleukin-6 to generate QQMNCs.ResultsA 5.3-fold increase in the definitive colony-forming EPCs and vasculogenic EPCs was observed, in comparison to naïve PBMNCs. QQMNCs were enriched with EPCs (28.9-fold, P<0.0019) and M2 macrophages (160.3-fold, P<0.0002). Genes involved in angiogenesis (angpt1, angpt2, and vegfb), stem/progenitors (c-kit and sca-1), and anti-inflammation (arg-1, erg-2, tgfb, and foxp3) were upregulated in QQMNCs. For in vivo experiments, cells were administered into syngeneic rat models of MI. QQMNC-transplanted group (QQ-Tx) preserved cardiac function and fraction shortening 28 days post-MI in comparison with PBMNCs-transplanted (PB-Tx) (P<0.0001) and Control (P<0.0008) groups. QQ-Tx showed enhanced angiogenesis and reduced interstitial left ventricular fibrosis, along with a decrease in neutrophils and an increase in M2 macrophages in the acute phase of MI. Cell tracing studies revealed that intravenously administered QQMNCs preferentially homed to ischemic tissues via blood circulation. QQ-Tx showed markedly upregulated early cardiac transcriptional cofactors (Nkx2-5, 29.8-fold, and Gata-4, 5.2-fold) as well as c-kit (4.5-fold) while these markers were downregulated in PB-Tx. In QQ-Tx animals, de novo blood vessels formed a “Biological Bypass”, observed macroscopically and microscopically, while PB-Tx and Control-Tx groups showed severe fibrotic adhesion to the surrounding tissues, but no epicardial blood vessels.ConclusionQQMNCs conferred potent angiogenic and anti-inflammatory properties to the regenerative microenvironment, enhancing myocardiogenesis and functional recovery of rat MI hearts.

Divergent Pro-Inflammatory Cytokine Response Induced by GB Virus C/ Hepatitis G virus (GBV-C/HGV) and Torque Teno Virus (TTV) Co-Infections in Hepatitis C Virus (HCV) Infected Thalassemia Patients

Despite common incidence of GB virus C/Hepatitis G virus (GBV-C/HGV) and Torque Teno Virus (TTV) co-infections in subjects with Hepatitis C virus (HCV) infection, pathogenic role of these co-infections in HCV infected subjects has not been clear since studies have mostly been based on liver enzyme profile yielding variable results. Pro-inflammatory cytokines that participate in host immune response against viruses are generated as a consequence of triggering of related genes by Nuclear Factor Kappa B (NF-kB) that is a member of transcription family. Study of three pro-inflammatory cytokines e.g. interleukin 1 beta (IL-1&amp;beta;), Interleukin-6 (IL-6), interleukin-8 (IL-8) in a group of multi-transfused thalassemic subjects in relation to positivity for HCV, HGV and TTV infections showed elevated levels of these cytokines in serum as well as in supernatant of peripheral blood mononuclear cell (PBMCs) cultures in HCV-GBV-C/HGV co-infected subgroup compared to HCV mono-infected subgroup (p &amp;lt;0.05 for comparison of all three cytokines) while HCV-TTV co-infected subgroup showed lowering of these levels compared to HCV mono-infected subgroup (p &amp;lt;0.05 for comparison of all three cytokines). Levels of p65 component of NF-kB i.e.NF-kB p65 in nuclear extracts of lipopolysaccharide stimulated PBMCs correlated positively with the levels of pro-inflammatory cytokines in serum as well as that in supernatants of PBMC cultures in both HCV- GBV-C/HGV co-infected and HCV-TTV co-infected subgroups (p values ranging from &amp;lt;0.001 to 0.004). Levels of NF-kB p65 in nuclear extracts and levels of pro-inflammatory cytokines in serum as well as in supernatants of PBMC culture did not show any alteration compared to thalassemic subjects with HGV or TTV infections alone and healthy non-transfused subjects. Based on the inhibitory role of TTV on activation of NF-kB in TTV-HCV co-infected cases observed in the present study and the reported contribution of NF-kB towards development of hepatocellular carcinoma (HCC) due to establishment of chronicity, it may be worth evaluating if TTV or any component of TTV can be utilized as therapeutic vaccine against development of HCC in HCV infected subjects.

1-ГЕРМАТРАНОЛ-ГИДРАТ – АКТИВАТОР ТРИПТОФАНИЛ-тРНК-СИНТЕТАЗЫ

The effect of 1-germatranol hydrate, compared to trekrezan, on protein synthesis and the related systems regulating the development, differentiation, regeneration and maintenance of both cellular and tissue homeostasis was studied in vitro using the human peripheral blood mononuclear cells (PBMC) culture. The activating effect of 1-germatranol hydrate on the total activity of tryptophanyl-tRNA synthetase, one of the key enzymes in the extraribosomal stage of the tryptophanyl-tRNA synthetase protein synthesis, is shown. A 15 μg / ml dose of 1-germatranol hydrate increases the expression of tryptophanyl-tRNA synthetase by 33-45% more than a 15 μg / ml dose of trecrezan. By stimulating the biosynthesis of immunoglobulins, germatranol hydrate exhibits an actoprotective effect, thus accelerat-ing the formation of both forms of aminoacyl tRNA synthetase. Activated tryptophanyl-tRNA synthetase, suppressing pathological processes of abnormal aggressive proliferation of blood vessels, stimulates the restorative physiological development of blood vessels. The data obtained suggest that 1-germatranol hydrate can be used as a pharmaceutical exhibiting immunomodulatory and adaptogenic action.

A Sensitive Method for Detecting Peptide-specific CD4+ T Cell Responses in Peripheral Blood from Patients with Myasthenia Gravis.

Myasthenia gravis (MG) is an autoimmune neurological disorder typified by skeletal muscle fatigue and most often production of autoantibodies against the nicotinic acetylcholine receptor (AChR). The present study was undertaken to assess the extent of AChR-peptide recognition in MG patients using co-culturing (DC:TC) of autologous monocyte-derived dendritic cells (moDCs) and highly enriched CD4+ T cells from the blood as compared to the traditional whole peripheral blood mononuclear cell (PBMC) cultures. We found that the DC:TC cultures were highly superior to the PBMC cultures for detection of reactivity toward HLA-DQ/DR-restricted AChR-peptides. In fact, whereas DC:TC cultures identified recognition in all MG patients the PBMC cultures failed to detect responsiveness in around 40% of the patients. Furthermore, reactivity to multiple peptides was evident in DC:TC cultures, while PBMC cultures mostly exhibited reactivity to a single peptide. No healthy control (HC) CD4+ T cells responded to the peptides in either culture system. Interestingly, whereas spontaneous production of IFNγ and IL-17 was observed in the DC:TC cultures from MG patients, recall responses to peptides enhanced IL-10 production in 9/13 MG patients, while little increase in IFNγ and IL-17 was seen. HCs did not produce cytokines to peptide stimulations. We conclude that the DC: TC culture system is significantly more sensitive and better identifies the extent of responsiveness in MG patients to AChR-peptides than traditional PBMC cultures.

Possible Immunomodulating Effect of Retinol on Cytokines Secretion in Patients with Recurrent Furunculosis

Recurrent furunculosis is an infection of hair follicles which results in formation of abscesses. Previous studies showed that the pathogenesis of the disease may include an immune-mediated component as the proliferative response of peripheral blood lymphocytes to staphylococcal antigen is depressed. The aim of our study was to evaluate cytokines concentration in the plasma of patients with recurrent furunculosis and to determine whether retinol affects the secretion of those cytokines in patients with recurrent furunculosis and healthy subjects. Blood samples were taken from 15 patients with recurrent furunculosis and 15 age-matched healthy subjects. A quantitative determination of selected cytokines (IL-17, 13, 2, 10, 4, IFN-γ, TNF-α) was performed in the plasma at baseline and after 72-h culture of peripheral blood mononuclear cells with and without retinol in both groups. In the plasma of patients with recurrent furunculosis, concentration of IL-10, 2, and TNF-α was significantly higher, whereas IL-13 significantly lower when compared with healthy subjects. After retinol stimulation, the concentration of IL-17 and IFN-γ increased significantly in both groups. Secretion of anti-inflammatory cytokines, especially IL-10 (p < 0.002) and 13 (p < 0.01), achieved lower levels in recurrent furunculosis samples than in those of healthy controls. Network of cytokines differs in patients with recurrent furunculosis from healthy subjects. Retinol stimulation affects secretion of both pro-inflammatory and anti-inflammatory cytokines. Further studies are recommended for better understanding the pathomechanism of recurrent furunculosis and potential clinical use of retinol in patients affected by recurrent furunculosis.

RT-LAMP assay: an alternative approach for profiling of bovine heat shock protein 70 gene in PBMC cultured model.

The purpose of this study is to develop a novel Reverse Transcriptase Loop-mediated isothermal amplification (RT-LAMP) based assay for in vitro profiling of heat shock protein 70 (Hsp70) in bovine peripheral blood mononuclear cell (PBMC) culture model utilizing the absorbance level of magnesium pyrophosphate-a by-product of LAMP reaction. A set of bovine Hsp70 specific RT-LAMP primers were designed to detect the differential absorbance level of magnesium pyrophosphate by-product which signifies the degree of Hsp70 amplification from cDNA of thermally induced cultured cells at different recovery periods. The study revealed significant (P < 0.05) correlation between absorbance level and the fold change of Hsp70 transcripts at different kinetic intervals of heat stress recovery in bovine PBMC cell culture models. RT-LAMP based absorbance assay can be used as an indicator to measure the degree of bovine Hsp70 transcripts produced during thermal stress and can be used as an alternative to the traditional Real time PCR assay. Developed RT-LAMP assay can be used as a cost-effective method for profiling of bovine HSP70 gene.

Abstract B095: Multiplexed detection of RNA and proteins on the nCounter® platform with low sample input protocol

Abstract As our understanding of the immune responses to cancer continues to grow, the need to extract greater amounts of information from ever smaller sample sizes increases. One of the biggest challenges facing the field is to develop a comprehensive understanding of how the immune system responds to a tumor, and multi-omic profiling (DNA, RNA, and protein) is crucial to building a holistic model of tumor immunity. The NanoString nCounter platform has become an important tool in quantifying transcriptional responses from a wide variety of sample types by enabling direct digital counting of up to 800 targets from a single sample using nucleic acid probes that directly hybridize to the RNA sequence of interest and then are quantified with optical barcoding technology. The platform has now been extended to permit quantification of proteins at the same time using primary antibodies conjugated to DNA oligos that hybridize with the barcodes. We have developed new technology that enables characterization of up to 30 proteins and 770 RNA transcripts in key immuno-oncology pathways from a very low amount of starting material - as few as 50,000 cells. NanoString has previously developed the nCounter Vantage RNA:Protein Immune Profiling Panel which allows digital counting of extracellular proteins which facilitates quantitation of multiple immune cell populations and provides information about their activation status. We have now applied this technology to detect intracellular and secreted proteins as well in the new nCounter Vantage RNA:Protein Immune Signaling panel. This panel is able to detect key transcription factors, signaling molecules, and secreted proteins from peripheral blood mononuclear cells (PBMC), dissociated cells, or cell culture. Additionally, we have recently developed a universal cell capture technology that utilizes anti-β2M antibody coupled to magnetic beads to pull down nucleated cells from a large starting volume. As proof of concept, PBMC from a healthy donor were treated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, TNFα, or IFNγ and RNA and protein were characterized with both the Immune Profiling and Immune Signaling panels. nCounter protein detection compared favorably when validated with flow cytometry, and the additional information imparted by simultaneous profiling of the RNA transcriptome enabled mapping of signaling pathways activated by treatment. Furthermore, RNA and protein counts from the low input protocol were representative of counts obtained from higher cell inputs, indicating that no loss or skewing of data resulted from reducing the starting material. This advance in multi-analyte, multiplexed digital molecular profiling will accelerate immuno-oncology research by reducing sample size requirements and may enable the discovery and development of novel immunotherapies and their associated companion diagnostics. Citation Format: Sarah Warren, Gary Geiss, Brian Burditt, Qian Mei, Alan Huang, Maribeth Eagen, Eduardo Ignacio, Dwayne Dunaway, Lucas Dennis, Joseph Beechem. Multiplexed detection of RNA and proteins on the nCounter® platform with low sample input protocol [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B095.

Рівні інтерлейкіну-4 та інтерлейкіну-17 (IL-4, IL-17) крові у пацієнток зі звичним невиношуванням вагітності, яка настала у циклі екстракорпорального запліднення

The objective: was to investigate the levels of cytokines IL-4 and IL-17 in serum and conditioned medium cultures of blood mononuclear cells (MNC) and evaluation association between their products and miscarriage, which occurred in IVF cycles. Patients and methods. We observed 240 patients with recurrent miscarriage, came in IVF cycles, and 100 apparently healthy fertile women in the control group. The concentrations of IL-4 and IL-17 in serum and conditioned medium of MNC cultures were determined. Results. The levels of IL-4 in the serum and conditioned medium in spontaneous and stimulated mitogen secretion was not significantly different from those in the control group, whereas IL-17 levels were higher than those in the control group serum, in conditioned media of stimulated and non-stimulated MNCs. Conclusion. Disregulation of activity of circulating blood mononuclear cells in women with recurrent miscarriage that followed IVF, is accompanied by increased secretion of IL-17 and almost constant production of IL-4 on the back of high stimulation index of production of these cytokines. Key words: in vitro fertilization, miscarriage, interleukin-4, interleukin-17, serum stimulated and non-stimulated mononuclear blood.

Vitamin D status and its modulatory effect on interferon gamma and interleukin-10 production by peripheral blood mononuclear cells in culture

ObjectivesWe aimed to investigate the influence of vitamin D on the production of the pro-inflammatory cytokine, interferon gamma (IFN-γ), and the anti-inflammatory cytokine, interleukin-10 (IL-10), in peripheral blood mononuclear cell (PBMC) cultures. MethodsThirty healthy subjects were investigated. Serum levels of calcium, 25(OH) D, and parathyroid hormone (PTH) were assessed. PBMCs were activated in-vitro by phytohemagglutinin (PHA) in the presence and absence of vitamin D3 and then levels of IFN-γ and IL-10 were determined in culture supernatant using enzyme immunoassay. ResultsSerum calcium levels were significantly lower in the vitamin D deficient group while serum PTH levels were significantly higher in the vitamin D deficient group. PTH levels were inversely correlated to both calcium and 25(OH) D levels. In culture, vitamin D inhibited IFN-γ production and increased IL-10 production by PBMCs. Serum vitamin D status had no influence on the amount of cytokine produced in culture. ConclusionsThis study demonstrates that vitamin D modulates IFN-γ and IL-10 production and provides a rationale for evaluating vitamin D as an immunomodulatory agent.

Coenzyme Q10 Supplementation Prevents Iron Overload While Improving Glycaemic Control and Antioxidant Protection in Insulin-Resistant Psammomys obesus.

This study investigated the anti-diabetic preventive activity of coenzyme Q10 (CoQ10) in a murine model of diet-induced insulin resistance (IR), Psammomys obesus (Po). IR was induced by feeding a standard laboratory diet (SD). CoQ10 oil suspension was orally administered at 10mg/kg body weight (BW)/day along with SD for 9months. Anthropometric parameters, namely, total body weight gain (BWG) and the relative weight of white adipose tissue (WAT) were determined. Blood glucose, insulin, quantitative insulin sensitivity check index (QUICKI), total antioxidant status (TAS), iron, malondialdehyde (MDA) and nitrite (NO2 (-)) were evaluated. NO2 (-) level was also assessed in peripheral blood mononuclear cells (PBMCs) culture supernatants. Our results show that CoQ10 supplementation significantly improved blood glucose, insulin, QUICKI, TAS, iron and MDA, but influenced neither NO2 (-) levels nor the anthropometric parameters. These findings support the hypothesis that CoQ10 would exert an anti-diabetic activity by improving both glycaemic control and antioxidant protection. The most marked effect of CoQ10 observed in this study concerns the regulation of iron levels, which may carry significant preventive importance.

Propagation of Influenza Virus in Lymphocytes Determine by Antiviral Effects of Honey, Ginger and Garlic Decoction

Three influenza A virus isolates, one from a human, one from a chicken, and one from a wild bird dropping, were used to infect human peripheral blood mononuclear cells (PBMCs) in culture. The influenza strains were H1N, H5/H7-N1/N2 (mixture) and H9N, respectively and were assessed in vitro in human PBMCs in the presence of Phytohemagglutinin (PHA). Viral replication was estimated by visual cytopathic effect (CPE). H1N2 CPE included budding of lymphocytes, fusion of infected cells with neighboring lymphocytes, and syncytial formation. H5/H7- N1/N2 and H9N2 each caused CPE that included bulging of cells with large vacuoles. The supernatant of infected lymphocyte culture was used to infect MDCK cell lines. Influenza viral RNA was detected in extracts of MDCK cell lines and from lymphocytes infected with each of the three virus strains as judged by RT-PCR, confirming that all three isolates were able to replicate in human lymphocytes in vitro. The antiviral activity of a mixture of honey, ginger, and garlic (HGG) extracts, an over the counter drug commonly used in Pakistan to treat patients with influenza virus, was compared to the antiviral drug amantadine for its ability to decrease replication of the H1N2 strain in human PBMCs in vitro. HGG significantly inhibited H1N2 virus growth as judged by CPE, haemagglutination assays, and qRT-PCR. Interestingly, HGG also appeared to promote proliferation of human lymphocytes. These finding suggest that a mixture of crude extracts from honey, ginger, and garlic may be clinically useful against influenza virus.