Separated Peripheral Blood Mononuclear Cells Research Articles

Flow cytometric analysis of immune cell populations in the bronchial and mesenteric lymph nodes of the dromedary camel.

Dromedary camel is an important livestock species with special economic value in arid and semi-arid regions of the world. Given the limited data on detailed immune cell composition and cell marker expression in the dromedary camel lymph node tissue, the present study was undertaken to investigate the immune cell composition of bronchial and mesenteric lymph nodes from healthy dromedary camels using flow cytometry. In this study, we applied flow cytometry and multicolor immuno-fluorescence to phenotype the main populations of immune cells in the bronchial and mesenteric camel lymph nodes and compared them with separated peripheral blood mononuclear cells and granulocytes. We used antibodies to detect several cell surface molecules associated with camel T cells (CD4, WC1), B cells (MHCII, BAQ44A), monocytes/macrophages (CD172a, CD14, CD163), in addition to the pan-leukocyte marker CD45 and the cell adhesion molecules CD44 and CD18. Compared to blood mononuclear cells, camel lymph node cells contained a higher percentage of lymphoid cells with only a minor fraction of myeloid cells. In addition, the lower expression of CD44 and CD18 on lymph node lymphocytes compared to lymphocytes from peripheral blood indicates higher frequency of naïve lymphocytes in the lymph nodes. The frequency of CD4+ T cells, B cells and γδ T cells within camel lymph node lymphocytes compared to blood indicates a similar tissue distribution pattern of lymphocyte subsets in camel and bovine and supports previous reports on the similarity between the camel immune system and the immune system of other ruminants. Lymph node neutrophils were identified as CD45++ CD172a++, CD14+, MHCIIlow, BAQ44A+, CD44++, CD18++ cells. In conclusion, the present study is describing the employment of flow cytometric single-cell analysis and immunostaining for the analysis of the immune cell composition in the camel lymph node.

Label-free microfluidic isolation of functional and viable lymphocytes from peripheral blood mononuclear cells.

The separation of peripheral blood mononuclear cells (PBMCs) into constituent blood cell types is a vital step to obtain immune cells for autologous cell therapies. The ability to separate PBMCs using label-free microfluidic techniques, based on differences in biomechanical properties, can have a number of benefits over other conventional techniques, including lower cost, ease of use, and avoidance of animal-derived labeling antibodies. Here, we report a microfluidic device that uses compressive diagonal ridges to separate PBMCs into highly pure samples of viable and functional lymphocytes. The technique utilizes the differences in the biophysical properties of PBMC sub-populations to direct the lymphocytes and monocytes into separate outlets. The biophysical properties of the monocytes and lymphocytes from healthy donors were first characterized using atomic force microscopy. Lymphocytes were found to be significantly stiffer than monocytes, with a mean cell stiffness of 1495 and 931 Pa, respectively. The differences in biophysical properties resulted in distinct trajectories through the microchannel terminating at different outlets, resulting in a lymphocyte sample with purity and viability both greater than 96% with no effect on the cells' ability to produce interferon gamma, a cytokine crucial for innate and adaptive immunity.

Association between CD137 and ischemia-reperfusion injury in patients with acute ST-segment elevation myocardial infarction

Objective: To explore the relationship between the levels of serum soluble CD137 (sCD137) and membrane-bound CD137 (mCD137) and the occurrence of ischemia reperfusion injury (IRI) in patients with acute ST-segment elevation myocardial infarction (STEMI). Methods: This is a cross-sectional study. Consecutive patients with acute STEMI, who underwent emergency percutaneous coronary intervention (PCI) in the Department of Cardiology, Jiangsu University Affiliated Hospital from May 2019 to September 2020, were enrolled. According to the absence or presence of IRI, patients were divided into IRI group and non-IRI group. Clinical data of the two groups were collected and compared. sCD137 level was detected by enzyme linked immunosorbent assay. Ficoll density gradient centrifugation was used to separate peripheral blood mononuclear cells (PBMC) and RNA was extracted, mCD137 mRNA expression level was detected by PCR. Serum sCD137 levels and the mCD137 mRNA levels of PBMC before, after PCI and 24 hours after PCI were compared. The correlation between serum sCD137 level, PBMC mCD137 mRNA level and clinical indicators was observed. The univariate and multivariate logistic binary regression analyses were performed to evaluate the related risk factors of IRI. ROC curve was used to analyze the predictive value of defined parameters for IRI. Results: A total of 112 STEMI patients were enrolled. There were 42 cases (of which 33 were males (78.6%), mean age was (58.6±12.7) years) in non-IRI group and 70 cases(of which 56 were males (80.0%), mean age was (64.5±11.6) years) in IRI group. Compared with the non-IRI group, patients in the IRI group had longer hospital stays, older age, lower rates of obesity, lower systolic and diastolic blood pressure at admission, higher proportion of the the right coronary artery as culprit vessel, lower rate of the use of angiotensin-converting enzyme inhibitor/angiotensin-Ⅱ receptor blocker/angiotensin receptor neprilysin inhibitor, higher levels of urea nitrogen and creatinine, lower glomerular filtration rate, lower triglycerides, higher D-dimer and B-type natriuretic peptidemax, higher proportion of Killip grade Ⅳ and cardiovascular adverse events (all P<0.05). sCD137 levels at the preoperative, postoperative and 24 hours after surgery were significantly higher in the IRI group than in the non-IRI group, while the mRNA levels of CD137 was similar between the two groups. The level of sCD137 in patients after PCI was lower than that before operation, the level of mCD137 mRNA was higher than that before operation (P<0.05). Serum sCD137 levels were positively correlated with hospitalization days, age, B-type natriuretic peptide, creatinine, ischemic time, C Reactive protein (CRP) and CRP/albumin (P<0.05), and negatively correlated with body mass index, glomerular filtration rate and albumin (P<0.05). The mCD137 mRNA expression level of PBMC was positively correlated with hospital stay, age, B-type natriuretic peptide, ischemic time, CRP and CRP/albumin (P<0.05), and negatively correlated with body mass index, glomerular filtration rate, albumin (P<0.05). Multivariate logistic regression analysis showed that higher sCD137 (OR=1.038, 95%CI: 1.009-1.069), aspartate aminotransferase, (OR=1.029, 95%CI: 1.009-1.050) and lower albumin (OR=0.829, 95%CI: 0.703-0.829) before surgery were independent risk factors of IRI (P<0.05). Receiver operating characteristic curve showed that the area under the curve of sCD137 was 0.672 (95%CI: 0.574-0.770, P=0.002) for the prediction of IRI, the best cut-off value was 28.43×10-3 μg/L with sensitivity of 95.2% and specificity of 48.6%. Conclusion: The significantly increased level of sCD137 in acute STEMI patients is positively correlated with reperfusion injury, which is an independent risk factor of IRI and may be related to the prognosis of patients with IRI.

Dietary supplementation of chitosan affects milk performance, markers of inflammatory response and antioxidant status in dairy cows

High-yielding cows produce a lot of free radicals in the body due to high levels of metabolism during lactation, causing oxidative stress and susceptibility to diseases. The influence and mechanism of chitosan (CHI) on antioxidant status and inflammatory response of dairy cows is unclear. This study investigated the effect of dietary supplementation of CHI on promoting milk production and antioxidant status, and reducing markers of inflammatory response of dairy cows. Forty multiple Holstein dairy cows (200 ± 15 days in milk, 34.8 ± 4.95 kg/d of milk yield) were randomly assigned to five treatments (n = 8): control (CON; without CHI addition), supplementation of CHI (mg/kg dietary DMI) with 500 (C500), 1000 (C1000), 1500 (C1500), or 2000 (C2000). The experiment period included 2 weeks for adaptation and 8 weeks for data and sample collection. Blood samples were collected on the 60th day to separate peripheral blood mononuclear cells. The results indicated that supplementation of CHI to the diet linearly increased the milk yield (P < 0.01), energy-corrected milk yield (P = 0.01), milk protein yield (P = 0.03) and lactose yield (P < 0.01) of dairy cows, quadratically increased the proportion of lactose (P = 0.03) and milk urea (P = 0.01), and linearly reduced somatic cell score (P < 0.01) in milk. With the increase of CHI, glutathione peroxidase (P = 0.03) and superoxide dismutase (P = 0.01) activity showed a quadratic increase, while CHI quadratically increased reactive oxygen species (P < 0.01) activity, and linearly reduced malondialdehyde (P = 0.01) and nitric oxide (P < 0.01) content in serum. The supplementation of CHI to the diet suppressed the activity and expression level of gene and protein of inducible nitric oxide synthase, and linearly decreased interleukin-1 content (P = 0.02) and gene expression (P = 0.02). The supplementation of CHI linearly increased the proportion of CD4+ (P < 0.01) and CD3+ (P = 0.03) T lymphocytes. The supplementation of CHI linearly down-regulated the expression of nuclear factor-κBp65 gene (P = 0.01) and phosphorylation level (P = 0.01) in peripheral blood mononuclear cells and linearly inhibited the activity of inducible nitric oxide synthase (P < 0.01) and the production of nitric oxide (P = 0.01). The results suggested that the supplementation of CHI to the diet of dairy cows can improve the milk performance, antioxidant function and reduce pro-inflammatory mediators, which may be related to its inhibition of nuclear factor-κB pathway.

Immunopotentiotor Effect of α-Tocopherol on Cytokine Expression in the Lymphocytes in the Elderly People

Background: Aging is associated with attenuation of immune responses. Studies show that old people are vulnerable to infectious diseases such as influenza. α-Tocopherol as an immunomodulator affects immune responses. In the present study, the effect of α-tocopherol, on lymphocyte responses i.e. interferon-gamma (IFN-γ), Tumor Necrosis Factor-alpha (TNF-α), and nuclear factor-κB (NF-κB) in elderly individuals was evaluated. Materials and Methods: Heparinized blood samples were prepared from 10 elderly individuals (n=10, age &gt;80 years) as the experimental group and 10 young individuals (n= 10, 20-40 years) as the control group. The separated Peripheral Blood Mononuclear Cells (PBMCs) of aged and young individuals were used for treatment with 2 μg/mL of α-tocopherol and 2 μg/mL of Purified Protein Derivative (PPD) after 12 and 24 h incubation period. After isolation of total RNA and synthesize of cDNA, the gene expressions of IFN-γ, TNF-α, and NF-κB were evaluated by real-time PCR method. β-Actin gene was considered as the internal control gene. Results: Results showed that treatment with α-tocopherol increased the IFN-γ expression in old and young lymphocyte groups. The mRNA level of NF-κB increased in the PPD group after 12 h in both old and young groups (p &lt;0.05). There were no alterations in TNF-α expression in both groups. Conclusion: It seems that α-tocopherol is effective in the promotion of cytokine responses in old individuals and may be useful as a supplement for improving the immune system of elderly people.

Separation and Optimisation of a Sucrose Density Gradient Centrifugation Protocol for Isolation of Peripheral Blood Mononuclear Cells (PBMC)

One step centrifugation procedure used commonly for separation of blood cells is the ficoll gradient centrifugation. In this method, after centrifugation, the peripheral blood mononuclear cells (PBMCs) are located on the top of the separation fluid, whereas other blood cells erythrocytes and granulocytes sediment to the bottom. In the present study 75% of lymphocyte suspension could be separated by using a one-step density gradient centrifugation of sodium heparin blood with Sucrose. Sucrose was diluted into different concentrations using miliQ water (10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 100%,). 4 mL of diluted blood was layered on 4 mL of each sucrose solution and centrifuged for 45 minutes at 1000 rpm. Clear separation of PBMCs could be observed in solution with 40% sucrose. The separated PBMCs were analysed in haeme analyser which showed 75% lymphocytes, 23% monocytes and 2% of other cells.

Effects of B7-H3 on T lymphocytes in different activation states

Objective To study the impacts of B7-H3 molecule on the proliferation of T lymphocytes in different activation conditions and on the secretion of relevant cytokines. Methods Peripheral blood samples were collected from healthy subjects to separate peripheral blood mononuclear cells (PBMC) by Ficoll density gradient centrifugation. T lymphocytes were isolated from some of the PBMCs and purified with T Cell Enrichment Kit. PBMC and purified T lymphocytes were activated by anti-CD3/CD28 monoclonal antibodies (McAb) in vitro. Flow cytometry analysis was used to detect the expression of CD28 and CTLA-4 on T lymphocytes at different time points for further analyzing the activation states of T lymphocytes. On this basis, human B7-H3-Fc fusion protein was added into the mixed co-cultivation system on days 0, 1, 2, 3 and 4, and Hu-Fc fusion protein was used as isotype control. CCK-8 method was performed to detect the proliferation of T lymphocytes in each group. ELISA method was used to detect the secretion of cytokines (IL-2, IL-10 and IFN-γ) and to analyze the immune responses induced by stimulating T lymphocytes at different states of activation with B7-H3. Results B7-H3 molecule significantly inhibited the quiescent T lymphocytes from secreting IL-2 and IL-10, but had no significant impact on IFN-γ secretion. Moreover, it significantly promoted the activated T lymphocytes to secret IL-2 and IFN-γ, but had no obvious impact on IL-10 secretion. Results of the cell proliferation assay showed that B7-H3 molecule inhibited the in vitro proliferation of T lymphocytes in the PBMC, but had no obvious impact on purified T lymphocytes. Conclusion The regulatory effects of B7-H3 molecule on the immune functions of T lymphocytes vary with the activation states of T lymphocytes. Key words: B7-H3; T lymphocyte; Co-stimulatory effect

Impaired Immunosuppressive Effect of Bronchoalveolar Mesenchymal Stem Cells in Hypersensitivity Pneumonitis: Preliminary Findings.

Bronchoalveolar mesenchymal stem cells (MSCs) play an important role in the maintenance of lung integrity. Therapeutic application of bone marrow-derived MSCs reduced chronic bronchial inflammation in idiopathic pulmonary fibrosis, and improved the ratio of survivors in sepsis with pneumonia. This study investigated the effect of MSCs from bronchoalveolar lavage fluid (BALF) of hypersensitivity pneumonitis (HP) on T-cell function under in vitro conditions. Bronchoalveolar MSCs were obtained via bronchoscopy with BAL from children with severe subacute HP. As control, BALF MSCs were assessed from children without any inflammatory lung disease. Isolated MSCs were characterized via immunophenotyping by flow cytometry and confocal laser scanning microscopy. HP-derived and healthy separated peripheral blood mononuclear cells (PBMCs) were stimulated by 5 µg/mL phytohemagglutinin in the presence of HP-derived or control MSCs in 5-day cultures. Proliferation and activation of T-cells were characterized by the mean fluorescence intensity (MFI) of 5,6-carboxyfluorescein-diacetat succinimidyl ester (CFSE) and CD25, CD69 as well as HLA-DR surface positivities, respectively. HP-derived MSCs showed significantly lower level of CD73, CD90, and CD105 expression compared to control MSCs in both flow cytometric and confocal microscopic experiments. MSCs from HP did not reduce T-cell proliferation based on CFSE MFI values, while the level of CD25 expression on both control and HP-derived CD4+ and CD8+ T-cells was significantly reduced by normal MSCs, while HP-derived MSCs did not have any significant effect. The level of other activation markers was not markedly modulated by MSCs. BALF MSCs from HP are unable to downregulate the proliferation and activation of T-cells that may support the development of recurrent intrapulmonary inflammation in HP. © 2016 International Clinical Cytometry Society.

Comparative Study between Polymerase Chain Reaction and Different Rapid Tests for Detection of Mycobacterium Infection.

The objective of this work is to reach a sensitive and rapid method for the laboratory diagnosis of active tuberculosis disease in patients and latent TB infection in household contacts and healthy community controls. We recruited 86 subjects divided into 3 groups: Thirty six patients with suspected active TB (Twenty four suspected pulmonary TB cased and 12 suspected extrapulmonary TB cases). 28 household contacts of confirmed pulmonary TB patients and 22 healthy community controls. Methods: For diagnosis of active tuberculosis the following tests were evaluated: Ziehl-Neelsen (Z-N) staining and culture on Lownestein-Jensen (L-j), tuberculin skin test (TST), nested polymerase chain reaction (PCR) assay on separated peripheral blood mononuclear cells (PBMCs), antibody detection by Immunchromatographic test (ICT), and JFN assay by QuantiFERON TB Gold In-Tube method for diagnosis of latent tuberculosic the following tests were evaluated: TST and Quantiferon TB Gold (In-tube method). The results showed that in pulmonary TB group, TST was the most sensitive test (92.3%), while QFT-Gold IT plus ZN was the most sensitive combined test (100%). In extrapulmonary TB group, TST, nested PCR and QFT-Gold IT were the most sensitive single tests (75%) while QFT-Gold IT plus ZN was the most sensitive combines test (100%). For diagnosis of LTBI: QFT-Gold IT was as sensitive as the TST. TST was 54.5% specific, while QFT Gold-IT was 94.5% specific TST showed that the exposure in the contacts group increased the risk of infection in the contacts group 0.9 times more than the control group (OR, 0,9m 95% Cl, 0.25-3.22; P=0.85). by QFT-Gold IT, exposure increased the risk of infection in the contacts group 8.4 times more than the control group (OR, 8.4; 95% Cl, 0.9-195.63; P=0.028). In household contacts group the concordance between TST and QFT-Gold IT was poor (64.3%, with a k value of 0.230.18). In conclusion, using QFT-Gold IT plus ZN staining is very helpful in the diagnosis of active TB disease. For diagnosis of LTBl. QuantiFERON-TB Gold In-Tube method is more helpful than Tuberculin skin test.

Molecular mechanisms of HBeAg in persistent HBV infection.

The aim of this study is to investigate the T-lymphocyte subpopulation and expression of programmed cell death-1 (PD-1), toll-like receptor (TLR)3, TLR4, and interferon (INF)-γ to illustrate the relationship between hepatitisBe antigen (HBeAg) and persistent hepatitisB virus (HBV) infection. Blood was taken from normal subjects into anticoagulation tubes to separate peripheral blood mononuclear cells (PBMCs). The PBMCs were divided into four groups and cultured with various concentrations of HBeAg for 72h. Changes in the T-cell subset were analyzed through cell counting by flow cytometry, and expression of TLR3, TLR4, and PD-1 was assessed by flow cytometry and Western blot. The concentration of IFN-γ was analyzed using enzyme-linked immunospot (ELISPOT) experiments. PBMCs were stimulated with various concentrations of HBeAg for 72h and assayed by flow cytometry to determine CD4+and CD8+cell counts. The relative frequencies of CD4+and CD8+subpopulations and the CD4+/CD8+ratio decreased compared with the control group, and T-cell impairment was significantly associated with higher HBeAg load. TLR3, TLR4, and PD-1 protein expression was assessed using flow cytometry and Western blotting. Expression of TLR3, TLR4, and PD-1 increased with increasing concentration of HBeAg. ELISPOT experiments were used to determine the concentration of IFN-γ. IFN-γ production in treatment groups was lower than in the control group. Comparing IFN-γ production in treatment groups, IFN-γ production in PBMCs stimulated with high dose of HBeAg was lower than for those stimulated with low-dose HBeAg. HBeAg can inhibit proliferation of lymphocytes, increase TLR3, TLR4, and PD-1 expression, and decrease IFN-γ production. This may be one of the molecular mechanisms of HBV immune tolerance.

Roles of 1,25(OH)2D3 and Vitamin D Receptor in the Pathogenesis of Rheumatoid Arthritis and Systemic Lupus Erythematosus by Regulating the Activation of CD4+ T Cells and the PKCδ/ERK Signaling Pathway

Background/Aims: The study aims to elucidate the roles of 1,25(OH)₂D₃ and vitamin D receptor (VDR) in the pathogenesis of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) by regulating the activation of CD4⁺ T cells and the PKCδ/ERK signaling pathway. Methods: From January 2013 to December 2015, a total of 130 SLE patients, 137 RA patients and 130 healthy controls were selected in this study. Serum levels of 1,25(OH)₂D₃ and VDR mRNA expression were detected by ELISA and real-time fluorescence quantitative PCR (RT-qPCR). Density gradient centrifugation was performed to separate peripheral blood mononuclear cells (PBMCs). CD4⁺ T cells were separated using magnetic activated cell sorting (MACS). CD4⁺T cells in logarithmic growth phase were collected and assigned into 9 groups: the normal control group, the normal negative control (NC) group, the VDR siRNA group, the RA control group, the RA NC group, the VDR over-expressed RA group, the SLE control group, the SLE NC group, and the VDR over-expressed SLE group. The mRNA and protein expressions of VDR, PKCδ, ERK1/2, CD11a, CD70 and CD40L were detected by RT-qPCR and Western blotting. Bisulfite genomic sequencing was conducted to monitor the methylation status of CD11a, CD70 and CD40L. Results: Compared with healthy controls, serum 1,25(OH)₂D₃ level and VDR mRNA expression in peripheral blood were decreased in SLE patients and RA patients. With the increase of concentrations of 1,25(OH)₂D₃ treatment, the VDR mRNA expression and DNA methylation levels of CD11a, CD70 and CD40L were declined, while the expressions of PKCδ, ERK1/2, CD11a, CD70 and CD40L were elevated in SLE, RA and normal CD4⁺T cells. Compared with the SLE contro, RA control, SLE NC and RA NC groups, the expressions of PKCδ, ERK1/2, CD11a, CD70 and CD40L decreased but DNA methylation levels of CD11a, CD70 and CD40L increased in the VDR over-expressed SLE group and VDR over-expressed RA group. However, compared with the normal control and normal NC groups, the expressions of PKCδ, ERK1/2, CD11a, CD70 and CD40L increased, but DNA methylation levels of CD11a, CD70 and CD40L decreased in the VDR siRNA group. Compared with the normal control group, the expressions of PKCδ, ERK1/2, CD11a, CD70 and CD40L increased, but DNA methylation levels of CD11a, CD70 and CD40L decreased in the SLE control and RA control groups. Conclusion: Our study provide evidence that 1,25(OH)₂D₃ and VDR could inhibit the activation of CD4⁺ T cells and suppress the immune response of SLE and RA through inhibiting PKCδ/ERK pathway and promoting DNA methylation of CD11a, CD70 and CD40L.

Rituximab impacts Th17 cells and related cytokines in patients with diffuse large B-cell lymphoma in vitro

Objective To explore the impact of rituximab on Th17 cells and related cytokines in patients with diffuse large B-cell lymphoma (DLBCL) in vitro and its significance. Methods 20 cases of DLBCL untreated patients and 20 healthy subjects were enrolled in the name of DLBCL group and health control group, respectively. 4 peripheral blood samples were collected from every case to separate peripheral blood mononuclear cells (PBMCs), which were assigned to 4 subgroups according to different culture conditions: blank subgroup(subgroup A), rituximab subgroup (subgroup B), rituximab and serum subgroup (subgroup C) and polarization subgroup (subgroup D) (added IL-6 and TGF-β). After cultured in vitro, the percentage of Th17 cells in each subgroup was tested by flow cytometry, and the cytokine IL-17 in the above-mentioned culture fluid was measured by enzyme-linked immunosorbent assay (ELISA). Results In health control group, the percentage of Th17 cells and the level of IL-17 in subgroup D [(17.12 ± 4.90) % and (45.735±10.012) pg/ml] were significantly higher than those in subgroup A, B, C (P 0.05). The percentage of Th17 cells and the level of IL-17 in the DLBCL subgroup A were significantly lower than those in health control subgroup A [(0.69±0.24) % and (6.012±1.312) pg/ml vs (2.43± 0.61) % and (8.217±1.681) pg/ml (P< 0.05)]. In DLBCL group, after cultured with rituximab, the percentages of Th17 cells in subgroup B, C, D were (2.34±0.48) %, (2.31±0.53) % and(16.92±4.81) %, and the levels of IL-17 were (7.944 ± 1.538) pg/ml, (7.957 ± 1.533) pg/ml and (44.417 ± 9.881) pg/ml, respectively, which were all significantly higher than those in subgroup A. Besides, the percentage of Th17 cells and the level of IL-17 in DLBCL subgroup D were significantly higher than those in subgroup B, C (P < 0.05), while there was no difference between subgroup B and subgroup C. Conclusion Experiments in vitro confirmed that the percentage of Th17 cells in PBMCs of DLBCL patients was lower than that in healthy persons, and rituximab could elevate the percentage of Th17 cells in PBMCs of DLBCL patients. Key words: Lymphoma, large B-cell, diffuse; Rituximab; Th17 cells; Interleukin-17; In vitro experiment

The role of mononuclear cell tissue factor and inflammatory cytokines in patients with chronic thromboembolic pulmonary hypertension.

Thrombosis and inflammation are two major factors underlying chronic thromboembolic pulmonary hypertension (CTEPH). Tissue factor (TF), C-reactive protein (CRP), tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein 1 (MCP-1) may play critical roles in the process of CTEPH thrombosis and pulmonary vascular remodeling. Ten patients with a confirmed diagnosis of CTEPH, 20 patients with acute pulmonary thromboembolism and 15 patients with other types of pulmonary hypertension were enrolled in this study, along with 20 healthy subjects as the control group. The immunoturbidimetric method was used to determine the plasma content of CRP. The plasma levels of TNF-α, MCP-1, and TF antigen were measured by an enzyme-linked immunosorbent assay, and TF activity was measured by the chromogenic substrate method. Percoll density gradient centrifugation was used to separate peripheral blood mononuclear cells from plasma. The level of monocyte TF mRNA was examined by reverse transcriptase-polymerase chain reaction. The correlations between all indices described above were analyzed. In CTEPH patients, the expression of CRP, TNF-α, and MCP-1 was significantly higher than that in controls (P < 0.05). The levels of TF activity, TF antigen, and TF mRNA in monocyte cells were increased in CTEPH patients when compared with control subjects, but only the TF antigen and TF mRNA levels were significantly different (P < 0.05). In CTEPH patients, levels of CRP, MCP-1, and TNF-α significantly correlated with the level of TF antigen in plasma. TF gene expression was increased in patients with CTEPH, suggesting that blood-borne TF mainly comes from mononuclear cells. TF expression significantly correlated with levels of CRP, TNF-α and MCP-1. These factors may play an important role in the development of CTEPH via the inflammation–coagulation–thrombosis cycle.

Expression profile of cold shock protein genes in goats (Capra hircus ) during different seasons

Summary The study was conducted to demonstrate expression profile of cold shock protein genes; cold inducible RNA binding protein (CIRP) and RNA binding motif protein 3 (RBM3) in goats ( Capra hircus ) during different seasons to check the gene expression in different thermal conditions. Blood samples were collected from healthy goats of tropical and temperate region during peak winter, moderate season and peak summer. Goats were divided in three (n=6 in each group) age groups- (I) up to 2 years, (II) 2 to 5 years and (III) above 5 years. RNA isolation was done from separated peripheral blood mononuclear cells (PBMCs) by Trizol reagent. Real-time PCR was applied to investigate mRNA expression of examined factors. The mRNA expression of CIRP and RBM3 was higher in all age groups during peak winter season as compared to peak summer season in both tropical and temperate region goats. To conclude, our results demonstrated expression of CIRP and RBM3 mRNAs in caprine PBMCs and increased expression of these genes during winter season could possibly play a significant role to ameliorate deleterious effects of cold stress to maintain cellular homeostasis in goats.

FLOW CYTOMETRIC LYMPHOCYTE SUBSET ANALYSIS USING MATERIAL FROM FROZEN WHOLE BLOOD

Multicenter immune monitoring programs commonly rely on storing and shipping cryopreserved peripheral blood mononuclear cells (PBMC), isolated from whole blood before freezing. However, under many conditions in the field, facilities to separate PBMC are absent. Here, we investigate the feasibility of using whole blood (WB) frozen at −80°C as a source of viable lymphocytes for use in immunological studies. We compare the percentage of CD4 and CD8 T lymphocytes and their subsets from frozen WB with results from cryopreserved PBMC in five random healthy blood donors (three female, two male). We report that CD4 and CD8 values in lymphocytes from WB frozen up to 120 days were very similar to those of PBMC frozen up to 10 days. These data suggest that within the limits of parameters investigated in this study, contrary to our original assumptions, whole blood frozen at −80°C may in fact be an appropriate source of viable lymphocytes for T cell enumeration assays in immunological and epidemiological studies.

Evaluation of a robotic system for the recovery of peripheral blood mononuclear cells

Investigations into immune responses are often based upon recovery of peripheral blood mononuclear cells (PBMC). To this purpose, the recovery of PBMC by gradient centrifugation is labour-intensive and requires a reasonable level of skill by the laboratory technician. Thus, we set out to determine whether laboratory automation equipment could be used for the recovery of PBMC from blood samples of horses, pigs and cattle, based on the Ficoll-Paque gradient centrifugation technique. Mixing of blood samples with PBS, layering of diluted blood onto Ficoll-Paque gradients, recovery of separated PBMC in RPMI 1640 medium were performed using an automated robotic system, the SBF200 (AM Robotic Systems, Warrington, UK) under laminar air flow conditions. Tubes were tagged with bar codes and manually placed after gradient centrifugation into a tube reader to measure the volume and position of the PBMC layer. The results of the automated procedure compared very well to those of the manual one in terms of percent cell recovery, sterility and cell viability. Also, a high throughput of samples could be implemented: with the integration of cell counting it should be possible for 96 blood samples to be processed, including the production of aliquots, by one person in a day.

The peripheral blood mononuclear cell microRNA signature of coronary artery disease

Background MicroRNAs are being used in the oncology field to characterize tumors and predict the survival of cancer patients. Here, we explored the potential of microRNAs as biomarkers for coronary artery disease (CAD) and acute coronary syndromes. Methods and results Using real-time PCR-based profiling, we determined the microRNA signature of peripheral blood mononuclear cells (PBMCs) from stable and unstable CAD patients and unaffected controls. 129 of 157 microRNAs measured were expressed by PBMCs and low variability between separate PBMC pools was observed. The presence of CAD in general coincided with a marked 5-fold increase ( P < 0.001) in the relative expression level of miR-135a, while the expression of miR-147 was 4-fold decreased ( P < 0.05) in PBMCs from CAD patients as compared to controls, resulting in a 19-fold higher miR-135a/miR-147 ratio ( P < 0.001) in CAD. MicroRNA/target gene/biological function linkage analysis suggested that the change in PBMC microRNA signature in CAD patients is probably associated with a change in intracellular cadherin/Wnt signaling. Interestingly, unstable angina pectoris patients could be discriminated from stable patients based upon their relatively high expression level of a cluster of three microRNAs including miR-134, miR-198, and miR-370, suggesting that the microRNA signatures can be used to identify patients at risk for acute coronary syndromes. Conclusions The present study is the first to show that microRNA signatures can possibly be utilized to identify patients exhibiting atherosclerotic CAD in general and those at risk for acute coronary syndromes. Our findings highlight the importance of microRNAs signatures as novel tool to predict clinical disease outcomes.

Effect of Leukapheresis on Gene Expression Profiles of Donor’s Peripheral Blood Mononuclear Cells

Leukapheresis has commonly been used to obtain the cell products intended for clinical cell therapy. Hypocalcemia related to citrate toxicity and some circulatory effects such as hypovolemia and hypotension are well-known complications of leukapheresis. In this study, we analyzed the gene expression profiles of peripheral blood mononuclear cells (PBMCs) obtained before and after leukapheresis to determine if the hemodynamic changes can affect the gene expression profiles of leukocytes. PBMCs were isolated from EDTA blood from 5 healthy donors collected before and immediately after apheresis. RNA was isolated, amplified, and analyzed using a cDNA microarray with 17,500 genes. Hierarchical clustering analysis was performed to evaluate the differences of gene expression profiling. Hierarchical clustering separated PBMCs from different donors with each other, but did not separate PBMCs collected before and after leukapheresis. Comparison of gene expression by PBMCs collected before and after leukapheresis found only 25 genes were differentially expressed (15 were up-regulated and 10 were down-regulated after leukapheresis) (F-test, P<0.005). Stress induced apoptosis-related genes, ANXA3, DEDD, and ATXN2L, and cytokine-related genes, IL13RA1 and IK, which were also related to stress, were up-regulated after leukapheresis. Genes involved in DNA and protein binding, such as CLSTN3, LRBA, SATB2, and HSPA8, were down-regulated. Leukapheresis had little effect on gene expression of PBMCs. Some genes showing differences between before and after leukapheresis were mainly involved in stress-related reactions.

Systematic Genomic Screen for Tyrosine Kinase Mutations in CLL.

Genomic screens have identified activating somatic mutations in tyrosine kinases in a spectrum of acute and chronic hematologic malignancies. These mutations lead to increased cellular proliferation and/or impairment of apoptosis. Chronic lymphocytic leukemia is a common chronic leukemia of adults that is thought to result at least in part from failure of apoptosis. Recent data suggest that CLL cell turnover is higher than previously thought and may correlate with disease progression, suggesting that somatic mutations that activate signal transduction may contribute to CLL pathogenesis and/or progression. The purpose of this study was to look systematically for activating mutations in protein tyrosine kinases in CLL using high-throughput DNA resequencing. 95 CLL patients were selected to encompass the range of prognostic groups defined by somatic hypermutation of the immunoglobulin heavy chain gene, ZAP-70 status and FISH cytogenetics. DNA was isolated from Ficoll separated peripheral blood mononuclear cells, and germline DNA was obtained from buccal swabs and/or saliva samples. 70 tyrosine kinases were selected for resequencing based on their involvement in lymphocyte biology, known altered gene expression in CLL, or location at a site of chromosomal gain, loss or mutation in CLL. All coding exons of ZAP-70 were included. Nested M13 tailed primers were designed to amplify and sequence the exons encoding the activation loops and JM domains. Bidirectional sequence analysis was performed using Mutation Surveyor version 2.28 (SoftGenetics). Candidate mutations were reamplified and sequenced from the original CLL DNA sample for verification; in parallel, the same exon was amplified and resequenced from the paired germline sample to assess whether non-synonymous mutations not known to be SNPs were present in the germline. A total of 178 exons were sequenced per sample. Analysis of 6.21 megabases of sequence data identified nine novel non-synonymous alterations in seven different genes; at least seven of these nine alterations are present in the germline, with the other two presently undergoing analysis. Although these non-synonymous alleles may have functional relevance to the pathogenesis of CLL, these data indicate that a significant proportion of non-synonymous alleles identified in high-throughput genomic screens are non-synonymous polymorphisms. These findings further suggest that the rate of acquired somatic mutation in the tyrosine kinome in CLL is low, and demonstrate the importance of matched germline DNA to allow assessment of candidate non-synonymous mutations in human cancer samples.

A possible link between sinusitis and lower airway hypersensitivity: the role of Staphylococcal enterotoxin B

Background and aimsThe prevalence of asthma has been keeping arising with unknown etiology. The cumulative evidence indicates that chronic rhinosinusitis (CRS) closely relates to asthma, but the detailed mechanisms remain unclear. The present study aimed to take insight into the role of Staphylococcus enterotoxin B (SEB) in a possible association between CRS and asthma.Methods38 patients with both CRS and asthma underwent functional endoscopic sinus surgery. Serum specific IgE and cytokines, clinical symptoms of CRS and asthma were evaluated before and after the surgery. Peripheral blood mononuclear cells (PBMCs) were separated from the patients and cultured. Th2 response of the cultured PBMCs in the presence or absence of specific antigens and SEB was evaluated.ResultsBesides the improvement of CRS symptoms, amelioration of asthma was also observed in the patients with both CRS and asthma after the sinus surgery. The preoperatively elevated Th2 cytokines, IL-4 and IL-5, normalized postoperatively. Th2 response was generated with separated PBMCs in the presence of specific antigens. SEB was required for maintaining Th2 response in these separated PBMCs.ConclusionThe present results indicate that a possible link exists between CRS and lower airway hypersensitivity. Sinusitis derived SEB may play a role in sustaining Th2 responses in the low airway hypersensitivity related to sinusitis.