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Sample records for mononuclear cells electronic

  1. Mononuclear Cells and Vascular Repair in HHT

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    Calinda eDingenouts

    2015-03-01

    Full Text Available Hereditary hemorrhagic telangiectasia (HHT or Rendu-Osler-Weber disease is a rare genetic vascular disorder known for its endothelial dysplasia causing arteriovenous malformations and severe bleedings. HHT-1 and HHT-2 are the most prevalent variants and are caused by heterozygous mutations in endoglin and ALK1, respectively. An undervalued aspect of the disease is that HHT patients experience persistent inflammation. Although endothelial and mural cells have been the main research focus trying to unravel the mechanism behind the disease, wound healing is a process with a delicate balance between inflammatory and vascular cells. Inflammatory cells are part of the mononuclear cells (MNCs fraction, and can, next to eliciting an immune response, also have angiogenic potential. This biphasic effect of MNC can hold a promising mechanism to further elucidate treatment strategies for HHT patients. Before MNC are able to contribute to repair, they need to home to and retain in ischemic and damaged tissue. Directed migration (homing of mononuclear cells following tissue damage is regulated by the stromal cell derived factor 1 (SDF1. MNCs that express the C-X-C chemokine receptor 4 (CXCR4 migrate towards the tightly regulated gradient of SDF1. This directed migration of monocytes and lymphocytes can be inhibited by dipeptidyl peptidase 4 (DPP4. Interestingly, MNC of HHT patients express elevated levels of DPP4 and show impaired homing towards damaged tissue. Impaired homing capacity of the MNCs might therefore contribute to the impaired angiogenesis and tissue repair observed in HHT patients. This review summarizes recent studies regarding the role of MNCs in the etiology of HHT and vascular repair, and evaluates the efficacy of DPP4 inhibition in tissue integrity and repair.

  2. Cytotoxicity of bovine and porcine collagen membranes in mononuclear cells.

    Science.gov (United States)

    Moura, Camilla Christian Gomes; Soares, Priscilla Barbosa Ferreira; Carneiro, Karine Fernandes; Souza, Maria Aparecida de; Magalhães, Denildo

    2012-01-01

    This study compared the cytotoxicity and the release of nitric oxide induced by collagen membranes in human mononuclear cells. Peripheral blood was collected from each patient and the separation of mononuclear cells was performed by Ficoll. Then, 2x10(5) cells were plated in 48-well culture plates under the membranes in triplicate. The polystyrene surface was used as negative control. Cell viability was assessed by measuring mitochondrial activity (MTT) at 4, 12 and 24 h, with dosage levels of nitrite by the Griess method for the same periods. Data had non-normal distribution and were analyzed by the Kruskal-Wallis test (pporcine membrane induced a higher release of nitrite compared with the control and bovine membrane, respectively (pporcine collagen membrane induces an increased production of proinflammatory mediators by mononuclear cells in the first hours of contact, decreasing with time.

  3. Characteristics of spontaneously proliferating mononuclear cells in rheumatoid arthritis.

    Science.gov (United States)

    Froebel, K; Dickson, R; Lewis, D; Jasani, M K; Sturrock, R D

    1984-10-01

    The phenomenon of increased spontaneous incorporation of 3H-thymidine (3H-TdR) into peripheral blood mononuclear cells in rheumatoid arthritis (RA) has been investigated. The activity was found to be short lived and affected less than 1% of cells. Using a Percoll density gradient we identified two populations of active cells. RA patients with active synovitis and increased 3H-TdR incorporation in the low density population of cells have higher overall 3H-TdR incorporation than normal controls and patients with inactive RA. The low density cell population is enriched for Ia+ cells. The data are consistent with raised spontaneous 3H-TdR incorporation being due to an in-vivo cell mediated immune response.

  4. Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Mononuclear Cells Using Sendai Virus.

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    Soares, Filipa A C; Pedersen, Roger A; Vallier, Ludovic

    2016-01-01

    This protocol describes the efficient isolation of peripheral blood mononuclear cells from circulating blood via density gradient centrifugation and subsequent generation of integration-free human induced pluripotent stem cells. Peripheral blood mononuclear cells are cultured for 9 days to allow expansion of the erythroblast population. The erythroblasts are then used to derive human induced pluripotent stem cells using Sendai viral vectors, each expressing one of the four reprogramming factors Oct4, Sox2, Klf4, and c-Myc.

  5. Fetal hemoglobin accumulation in vitro. Effect of adherent mononuclear cells.

    OpenAIRE

    Javid, J; Pettis, P K

    1983-01-01

    In clonal cultures of erythroid burst-forming units (BFU-E) obtained from blood, the accumulation of fetal and adult hemoglobins (Hb F and Hb A) was measured by radioligand immunoassay. Inclusion of adherent mononuclear cells in the culture promoted a striking increase in the relative amount of Hb F in each of 44 experiments with 14 donors. In two-thirds of the instances, this was accounted for by a selective increase in the absolute amount of Hb F. The differential effect on Hb F and Hb A ac...

  6. Amyotrophic lateral sclerosis multiprotein biomarkers in peripheral blood mononuclear cells.

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    Giovanni Nardo

    Full Text Available BACKGROUND: Amyotrophic lateral sclerosis (ALS is a fatal progressive motor neuron disease, for which there are still no diagnostic/prognostic test and therapy. Specific molecular biomarkers are urgently needed to facilitate clinical studies and speed up the development of effective treatments. METHODOLOGY/PRINCIPAL FINDINGS: We used a two-dimensional difference in gel electrophoresis approach to identify in easily accessible clinical samples, peripheral blood mononuclear cells (PBMC, a panel of protein biomarkers that are closely associated with ALS. Validations and a longitudinal study were performed by immunoassays on a selected number of proteins. The same proteins were also measured in PBMC and spinal cord of a G93A SOD1 transgenic rat model. We identified combinations of protein biomarkers that can distinguish, with high discriminatory power, ALS patients from healthy controls (98%, and from patients with neurological disorders that may resemble ALS (91%, between two levels of disease severity (90%, and a number of translational biomarkers, that link responses between human and animal model. We demonstrated that TDP-43, cyclophilin A and ERp57 associate with disease progression in a longitudinal study. Moreover, the protein profile changes detected in peripheral blood mononuclear cells of ALS patients are suggestive of possible intracellular pathogenic mechanisms such as endoplasmic reticulum stress, nitrative stress, disturbances in redox regulation and RNA processing. CONCLUSIONS/SIGNIFICANCE: Our results indicate that PBMC multiprotein biomarkers could contribute to determine amyotrophic lateral sclerosis diagnosis, differential diagnosis, disease severity and progression, and may help to elucidate pathogenic mechanisms.

  7. Amyotrophic lateral sclerosis multiprotein biomarkers in peripheral blood mononuclear cells.

    Science.gov (United States)

    Nardo, Giovanni; Pozzi, Silvia; Pignataro, Mauro; Lauranzano, Eliana; Spano, Giorgia; Garbelli, Silvia; Mantovani, Stefania; Marinou, Kalliopi; Papetti, Laura; Monteforte, Marta; Torri, Valter; Paris, Luca; Bazzoni, Gianfranco; Lunetta, Christian; Corbo, Massimo; Mora, Gabriele; Bendotti, Caterina; Bonetto, Valentina

    2011-01-01

    Amyotrophic lateral sclerosis (ALS) is a fatal progressive motor neuron disease, for which there are still no diagnostic/prognostic test and therapy. Specific molecular biomarkers are urgently needed to facilitate clinical studies and speed up the development of effective treatments. We used a two-dimensional difference in gel electrophoresis approach to identify in easily accessible clinical samples, peripheral blood mononuclear cells (PBMC), a panel of protein biomarkers that are closely associated with ALS. Validations and a longitudinal study were performed by immunoassays on a selected number of proteins. The same proteins were also measured in PBMC and spinal cord of a G93A SOD1 transgenic rat model. We identified combinations of protein biomarkers that can distinguish, with high discriminatory power, ALS patients from healthy controls (98%), and from patients with neurological disorders that may resemble ALS (91%), between two levels of disease severity (90%), and a number of translational biomarkers, that link responses between human and animal model. We demonstrated that TDP-43, cyclophilin A and ERp57 associate with disease progression in a longitudinal study. Moreover, the protein profile changes detected in peripheral blood mononuclear cells of ALS patients are suggestive of possible intracellular pathogenic mechanisms such as endoplasmic reticulum stress, nitrative stress, disturbances in redox regulation and RNA processing. Our results indicate that PBMC multiprotein biomarkers could contribute to determine amyotrophic lateral sclerosis diagnosis, differential diagnosis, disease severity and progression, and may help to elucidate pathogenic mechanisms.

  8. Autologous Intravenous Mononuclear Stem Cell Therapy in Chronic Ischemic Stroke

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    Bhasin A

    2012-01-01

    Full Text Available Background: The regenerative potential of brain has led to emerging therapies that can cure clinico-motor deficits after neurological diseases. Bone marrow mononuclear cell therapy is a great hope to mankind as these cells are feasible, multipotent and aid in neurofunctional gains in Stroke patients. Aims: This study evaluates safety, feasibility and efficacy of autologous mononuclear (MNC stem cell transplantation in patients with chronic ischemic stroke (CIS using clinical scores and functional imaging (fMRI and DTI. Design: Non randomised controlled observational study Study: Twenty four (n=24 CIS patients were recruited with the inclusion criteria as: 3 months–2years of stroke onset, hand muscle power (MRC grade at least 2; Brunnstrom stage of recovery: II-IV; NIHSS of 4-15, comprehendible. Fugl Meyer, modified Barthel Index (mBI and functional imaging parameters were used for assessment at baseline, 8 weeks and at 24 weeks. Twelve patients were administered with mean 54.6 million cells intravenously followed by 8 weeks of physiotherapy. Twelve patients served as controls. All patients were followed up at 24 weeks. Outcomes: The laboratory and radiological outcome measures were within normal limits in MNC group. Only mBI showed statistically significant improvement at 24 weeks (p<0.05 whereas the mean FM, MRC, Ashworth tone scores in the MNC group were high as compared to control group. There was an increased number of cluster activation of Brodmann areas BA 4, BA 6 post stem cell infusion compared to controls indicating neural plasticity. Cell therapy is safe and feasible which may facilitate restoration of function in CIS.

  9. Immunohistochemical demonstration of lysozyme in normal, reactive and neoplastic cells of the mononuclear phagocyte system.

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    Motoi,Makoto

    1984-04-01

    Full Text Available Using the peroxidase antiperoxidase (PAP method, lysozyme (LZM was shown to exist in normal, reactive and neoplastic cells belonging to the mononuclear phagocyte system (MPS, but was not detected in histiocytosis X cells. Immunostaining for cytoplasmic LZM by the PAP method is useful for identification of mononuclear phagocytes and for diagnosis of the diseases in which these cells participate.

  10. The DNA methylome of human peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Li, Yingrui; Zhu, Jingde; Tian, Geng

    2010-01-01

    DNA methylation plays an important role in biological processes in human health and disease. Recent technological advances allow unbiased whole-genome DNA methylation (methylome) analysis to be carried out on human cells. Using whole-genome bisulfite sequencing at 24.7-fold coverage (12.3-fold per...... strand), we report a comprehensive (92.62%) methylome and analysis of the unique sequences in human peripheral blood mononuclear cells (PBMC) from the same Asian individual whose genome was deciphered in the YH project. PBMC constitute an important source for clinical blood tests world-wide. We found...... that 68.4% of CpG sites and 80% displayed allele-specific expression (ASE). These data demonstrate that ASM is a recurrent phenomenon and is highly correlated with ASE in human PBMCs. Together with recently reported similar studies, our study provides a comprehensive resource for future epigenomic...

  11. Use of cryopreserved peripheral mononuclear blood cells in biomonitoring

    DEFF Research Database (Denmark)

    Risom, Lotte; Knudsen, Lisbeth E.

    1999-01-01

    This study was performed to investigate the effect of storing blood samples by freezing on selected biomarkers and possible implications for biomonitoring. Comparative measurements were performed in order to investigate the use of cryopreserved vs. freshly separated peripheral mononuclear blood c....... We measured the DNA repair activity as dimethylsulfate induced unscheduled DNA synthesis (UDS) in PMBC incubated with either autologous plasma or fetal bovine serum (FBS). Comparison of the hprt mutant frequency by the T cell cloning assay was made in parallel. Finally the content of B....../T-lymphocytes and monocytes was measured in phytohemaglutinin (PHA)-stimulated cultures at different time intervals. The results showed a higher DNA repair activity in cryopreserved samples compared with fresh samples. We also found differences in mutant frequencies with higher values in fresh samples. A significant...

  12. Autologous Bone Marrow Mononuclear Cells Intrathecal Transplantation in Chronic Stroke

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    Alok Sharma

    2014-01-01

    Full Text Available Cell therapy is being widely explored in the management of stroke and has demonstrated great potential. It has been shown to assist in the remodeling of the central nervous system by inducing neurorestorative effect through the process of angiogenesis, neurogenesis, and reduction of glial scar formation. In this study, the effect of intrathecal administration of autologous bone marrow mononuclear cells (BMMNCs is analyzed on the recovery process of patients with chronic stroke. 24 patients diagnosed with chronic stroke were administered cell therapy, followed by multidisciplinary neurorehabilitation. They were assessed on functional independence measure (FIM objectively, along with assessment of standing and walking balance, ambulation, and hand functions. Out of 24 patients, 12 improved in ambulation, 10 in hand functions, 6 in standing balance, and 9 in walking balance. Further factor analysis was done. Patients of the younger groups showed higher percentage of improvement in all the areas. Patients who underwent cell therapy within 2 years after the stroke showed better changes. Ischemic type of stroke had better recovery than the hemorrhagic stroke. This study demonstrates the potential of autologous BMMNCs intrathecal transplantation in improving the prognosis of functional recovery in chronic stage of stroke. Further clinical trials are recommended. This trial is registered with NCT02065778.

  13. Arecoline inhibits endothelial cell growth and migration and the attachment to mononuclear cells

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    Shuei-Kuen Tseng

    2014-09-01

    Conclusion: Arecoline impaired vascular endothelial cells by inhibiting their growth and migration and their adhesion to U937 mononuclear cells. These results reveal that arecoline may contribute to the pathogenesis of oral submucous fibrosis and cardiovascular diseases by affecting endothelial cell function in BQ chewers.

  14. Fish peripheral blood mononuclear cells preparation for future monitoring applications.

    Science.gov (United States)

    Pierrard, Marie-Aline; Roland, Kathleen; Kestemont, Patrick; Dieu, Marc; Raes, Martine; Silvestre, Frédéric

    2012-07-15

    Fish species possess many specific characteristics that support their use in ecotoxicology. Widely used in clinical research, peripheral blood mononuclear cells (PBMCs) can reasonably be exploited as relevant target cells in the assessment of environmental chemical toxicity. The current article focuses on the methods necessary to isolate, characterize, and culture fish PBMCs. These procedures were successfully applied on an endangered species, the European eel (Anguilla anguilla L.), and on an economically important and worldwide exported species, the Asian catfish (Pangasianodon hypophthalmus S.). Proteomic approaches can be useful to screen xenobiotic exposure at the protein expression level, giving the opportunity to develop early warning signals thanks to molecular signatures of toxicity. To date, a major limitation of proteomic analyses is that most protein expression profiles often reveal the same predominant and frequently differentially expressed families of proteins regardless of the experimental stressing conditions. The current study describes a methodology to get a postnuclear fraction of high quality isolated from fish PBMCs in order to perform subsequent subproteomic analyses. Applied on samples from eel, the subproteomic analysis (two-dimensional differential in-gel electrophoresis) allowed the identification by liquid chromatography-tandem mass spectrometry and searches in the full NCBInr (National Center for Biotechnology Information nonredundant) database of 66 proteins representing 36 different proteins validated through Peptide and Protein Prophet of Scaffold software.

  15. The DNA methylome of human peripheral blood mononuclear cells.

    Directory of Open Access Journals (Sweden)

    Yingrui Li

    Full Text Available DNA methylation plays an important role in biological processes in human health and disease. Recent technological advances allow unbiased whole-genome DNA methylation (methylome analysis to be carried out on human cells. Using whole-genome bisulfite sequencing at 24.7-fold coverage (12.3-fold per strand, we report a comprehensive (92.62% methylome and analysis of the unique sequences in human peripheral blood mononuclear cells (PBMC from the same Asian individual whose genome was deciphered in the YH project. PBMC constitute an important source for clinical blood tests world-wide. We found that 68.4% of CpG sites and 80% displayed allele-specific expression (ASE. These data demonstrate that ASM is a recurrent phenomenon and is highly correlated with ASE in human PBMCs. Together with recently reported similar studies, our study provides a comprehensive resource for future epigenomic research and confirms new sequencing technology as a paradigm for large-scale epigenomics studies.

  16. Absence of peripheral blood mononuclear cells priming in hemodialysis patients

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    Santos B.C.

    2003-01-01

    Full Text Available As a consequence of the proinflammatory environment occurring in dialytic patients, cytokine overproduction has been implicated in hemodialysis co-morbidity. However, there are discrepancies among the various studies that have analyzed TNF-alpha synthesis and the presence of peripheral blood mononuclear cell (PBMC priming in this clinical setting. We measured bioactive cytokine by the L929 cell bioassay, and evaluated PBMC TNF-alpha production by 32 hemodialysis patients (HP and 51 controls. No difference in TNF-alpha secretion was observed between controls and HP (859 ± 141 vs 697 ± 130 U/10(6 cells. Lipopolysaccharide (5 µg/ml did not induce any further TNF-alpha release, showing no PBMC priming. Paraformaldehyde-fixed HP PBMC were not cytotoxic to L929 cells, suggesting the absence of membrane-anchored TNF-alpha. Cycloheximide inhibited PBMC cytotoxicity in HP and controls, indicating lack of a PBMC TNF-alpha pool, and dependence on de novo cytokine synthesis. Actinomycin D reduced TNF-alpha production in HP, but had no effect on controls. Therefore, our data imply that TNF-alpha production is an intrinsic activity of normal PBMC and is not altered in HP. Moreover, TNF-alpha is a product of de novo synthesis by PBMC and is not constitutively expressed on HP cell membranes. The effect of actinomycin D suggests a putative tighter control of TNF-alpha mRNA turnover in HP. This increased dependence on TNF-alpha RNA transcription in HP may reflect an adaptive response to hemodialysis stimuli.

  17. MicroRNA Expression in Alzheimer Blood Mononuclear Cells

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    Hyman M. Schipper

    2007-01-01

    Full Text Available Various coding genes representing multiple functional categories are downregulated in blood mononuclear cells (BMC of patients with sporadic Alzheimer disease (AD. Noncoding microRNAs (miRNA regulate gene expression by degrading messages or inhibiting translation. Using BMC as a paradigm for the study of systemic alterations in AD, we investigated whether peripheral miRNA expression is altered in this condition. MicroRNA levels were assessed using the microRNA microarray (MMChip containing 462 human miRNA, and the results validated by real time PCR. Sixteen AD patients and sixteen normal elderly controls (NEC were matched for ethnicity, age, gender and education. The expression of several BMC miRNAs was found to increase in AD relative to NEC levels, and may differ between AD subjects bearing one or two APOE4 alleles. As compared to NEC, miRNAs signifi cantly upregulated in AD subjects and confi rmed by qPCR were miR-34a and 181b. Predicted target genes downregulated in Alzheimer BMC that correlated with the upregulated miRNAs were largely represented in the functional categories of Transcription/Translation and Synaptic Activity. Several miRNAs targeting the same genes were within the functional category of Injury response/Redox homeostasis. Taken together, induction of microRNA expression in BMC may contribute to the aberrant systemic decline in mRNA levels in sporadic AD.

  18. Mechanisms of pancreatic islet cell destruction. Dose-dependent cytotoxic effect of soluble blood mononuclear cell mediators on isolated islets of Langerhans

    DEFF Research Database (Denmark)

    Mandrup-Poulsen, T; Bendtzen, K; Nerup, J

    1986-01-01

    contents of insulin and glucagon in a dose-dependent manner. A maximal effect on islet function was obtained with supernatant concentrations down to 5%. Supernatants of mononuclear cells stimulated with tuberculin were more potent than supernatants produced by lectin stimulation. Culture medium......Supernatants of peripheral blood mononuclear cells from healthy human donors stimulated with recall antigen (purified protein derivative of tuberculin) or lectin (phytohaemagglutinin) markedly inhibited the insulin release from isolated human and rat islets of Langerhans, and decreased rat islet...... reconstituted with tuberculin or phytohaemagglutinin did not impair islet function. Electron microscopy demonstrated that supernatants were cytotoxic to islet cells. The cytotoxic mononuclear cell mediator(s) was non-dialysable, sensitive to heating to 56 degrees C, labile even when stored at -70 degrees C...

  19. Cell type-specific responses of peripheral blood mononuclear cells to silver nanoparticles.

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    Greulich, C; Diendorf, J; Gessmann, J; Simon, T; Habijan, T; Eggeler, G; Schildhauer, T A; Epple, M; Köller, M

    2011-09-01

    Silver nanoparticles (Ag-NP) are increasingly used in biomedical applications because of their remarkable antimicrobial activity. In biomedicine, Ag-NP are coated onto or embedded in wound dressings, surgical instruments and bone substitute biomaterials, such as silver-containing calcium phosphate cements. Free Ag-NP and silver ions are released from these coatings or after the degradation of a biomaterial, and may come into close contact with blood cells. Despite the widespread use of Ag-NP as an antimicrobial agent, there is a serious lack of information on the biological effects of Ag-NP on human blood cells. In this study, the uptake of Ag-NP by peripheral monocytes and lymphocytes (T-cells) was analyzed, and the influence of nanosilver on cell biological functions (proliferation, the expression of adhesion molecules, cytokine release and the generation of reactive oxygen species) was studied. After cell culture in the presence of monodispersed Ag-NP (5-30μgml(-1) silver concentration), agglomerates of nanoparticles were detected within monocytes (CD14+) but not in T-cells (CD3+) by light microscopy, flow cytometry and combined focused ion beam/scanning electron microscopy. The uptake rate of nanoparticles was concentration dependent, and the silver agglomerates were typically found in the cytoplasm. Furthermore, a concentration-dependent activation (e.g. an increased expression of adhesion molecule CD54) of monocytes at Ag-NP concentrations of 10-15μgml(-1) was observed, and cytotoxicity of Ag-NP-treated monocytes was observed at Ag-NP levels of 25μgml(-1) and higher. However, no modulation of T-cell proliferation was observed in the presence of Ag-NP. Taken together, our results provide the first evidence for a cell-type-specific uptake of Ag-NP by peripheral blood mononuclear cells (PBMC) and the resultant cellular responses after exposure.

  20. Transcriptional Profiling of Human Peripheral Blood Mononuclear Cells Exposed to Bacillus anthracis in vitro

    Science.gov (United States)

    2013-03-11

    Bacillus cereus group of bacteria, are attributed to poly- γ-D-glutamate acid (PGA) capsule, lethal toxin (LT) and edema toxin (ET) [10-12]. These toxins...M, Hellman M, Muhie S, et al. (2013) Transcriptional Profiling of Human Peripheral Blood Mononuclear Cells Exposed to Bacillus anthracis in vitro...author and source are credited. Transcriptional Profiling of Human Peripheral Blood Mononuclear Cells Exposed to Bacillus anthracis in vitro Rasha

  1. Naive Treg-like CCR7(+) mononuclear cells indicate unfavorable prognosis in hepatocellular carcinoma.

    Science.gov (United States)

    Shi, Jie-Yi; Duan, Meng; Sun, Qi-Man; Yang, Liuxiao; Wang, Zhi-Chao; Mynbaev, Ospan A; He, Yi-Feng; Wang, Ling-Yan; Zhou, Jian; Tang, Qi-Qun; Cao, Ya; Fan, Jia; Wang, Xiao-Ying; Gao, Qiang

    2016-07-01

    Chemokine receptor-like 1 (CCRL1) has the potential in creating a low level of CCL19 and CCL21 to hinder CCR7(+) cell tracking to tumor tissue. Previously, we found a tumor suppressive role of CCRL1 by impairing CCR7-related chemotaxis of tumor cells in human hepatocellular carcinoma (HCC). Here, we reported a contribution of CCR7(+) mononuclear cells in the tumor microenvironment to the progression of disease. Immunohistochemistry was used to investigate the distribution and clinical significance of CCR7(+) cells in a cohort of 240 HCC patients. Furthermore, the phenotype, composition, and functional status of CCR7(+) cells were determined by flow cytometry, immunofluorescence, and in vitro co-culture assays. We found that CCR7(+) mononuclear cells were dispersed around tumor tissue and negatively related to tumoral expression of CCRL1 (P CCR7(+) mononuclear cells positively correlated with the absence of tumor capsule, vascular invasion, and poor differentiation (P CCR7(+) mononuclear cells was significantly associated with worse survival and increased recurrence. We found that CCR7(+) mononuclear cells featured a naive Treg-like phenotype (CD45RA(+)CD25(+)FOXP3(+)) and possessed tumor-promoting potential by producing TGF-β1. Moreover, CCR7(+) cells were also composed of several immunocytes, a third of which were CD8(+) T cells. CCR7(+) Treg-like cells facilitate tumor growth and indicate unfavorable prognosis in HCC patients, but fortunately, their tracking to tumor tissue is under the control of CCRL1.

  2. Adult peripheral blood mononuclear cells transdifferentiate in vitro and integrate into the retina in vivo.

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    Liu, Qian; Guan, Liping; Huang, Bing; Li, Weihua; Su, Qiao; Yu, Minbin; Xu, Xiaoping; Luo, Ting; Lin, Shaochun; Sun, Xuerong; Chen, Mengfei; Chen, Xigu

    2011-06-01

    Adult peripheral blood-derived cells are able to differentiate into a variety of cell types, including nerve cells, liver-like cells and epithelial cells. However, their differentiation into retina-like cells is controversial. In the present study, transdifferentiation potential of human adult peripheral blood mononuclear cells into retina-like cells and integration into the retina of mice were investigated. Freshly isolated adult peripheral blood mononuclear cells were divided into two groups: cells in group I were cultured in neural stem cell medium, and cells in group II were exposed to conditioned medium from rat retinal tissue culture. After 5 days, several distinct cell morphologies were observed, including standard mononuclear, neurons with one or two axons and elongated glial-like cells. Immunohistochemical analysis of neural stem cell, neuron and retina cell markers demonstrated that cells in both groups were nestin-, MAP2 (microtubule-associated protein)- and GFAP (glial fibrillary acidic protein)-positive. Flow cytometry results suggested a significant increase in nestin-, MAP2- and CD16-positive cells in group I and nestin-, GFAP-, MAP2-, vimentin- and rhodopsin-positive cells in group II. To determine survival, migration and integration in vivo, cell suspensions (containing group I or group II cells) were injected into the vitreous or the peritoneum. Tissue specimens were obtained and immunostained 4 weeks after transplantation. We found that cells delivered by intravitreal injection integrated into the retina. Labelled cells were not detected in the retina of mice receiving differentiated cells by intraperitoneal injection, but cells (groups I and II) were detected in the liver and spleen. Our findings revealed that human adult peripheral blood mononuclear cells could be induced to transdifferentiate into neural precursor cells and retinal progenitor cells in vitro, and the differentiated peripheral blood mononuclear cells can migrate and integrate

  3. Generation of iPS Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors.

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    Su, Ruijun Jeanna; Neises, Amanda; Zhang, Xiao-Bing

    2016-01-01

    Peripheral blood is the easy-to-access, minimally invasive, and the most abundant cell source to use for cell reprogramming. The episomal vector is among the best approaches for generating integration-free induced pluripotent stem (iPS) cells due to its simplicity and affordability. Here we describe the detailed protocol for the efficient generation of integration-free iPS cells from peripheral blood mononuclear cells. With this optimized protocol, one can readily generate hundreds of iPS cell colonies from 1 ml of peripheral blood.

  4. Microarray-based gene expression profiling of peripheral blood mononuclear cells in dairy cows with experimental hypocalcemia and milk fever

    National Research Council Canada - National Science Library

    Sasaki, K; Yamagishi, N; Kizaki, K; Sasaki, K; Devkota, B; Hashizume, K

    2014-01-01

    .... Therefore, peripheral blood mononuclear cells from dairy cows with experimentally induced hypocalcemia or spontaneous milk fever were subjected to oligo-microarray analysis to identify specific biomarker genes...

  5. Studies of biological properties of Uncaria tomentosa extracts on human blood mononuclear cells.

    Science.gov (United States)

    Bors, Milena; Michałowicz, Jaromir; Pilarski, Radosław; Sicińska, Paulina; Gulewicz, Krzysztof; Bukowska, Bożena

    2012-08-01

    Uncaria tomentosa (Willd.) DC is a lignified climbing plant from South and Central America, which (under the name of "vilcacora" or "cat's claw") has become highly popular in many countries due to its proven immunostimmulatory and anti-inflammatory activities and also with respect to its anticancer and antioxidative effects. There are insufficient data on the mechanism of U. tomentosa action on normal blood mononuclear cells. The aim of the study was to analyze the impact of ethanol and aqueous extracts from bark and leaves of Uncaria tomentosa on the structure and function of human mononuclear cells and to find out whether the kind of extractant used modulates biological activity of the extracts studied. Plant material consisted of four different extracts: (1) ethanol extract from leaves, (2) aqueous extract from leaves, (3) ethanol extract from bark and (4) aqueous extract from bark. The effect of these extracts on protein damage as well as on free-radical formation in human peripheral blood mononuclear cells was analyzed. Moreover, changes in viability, size, and granularity as well as apoptotic alterations in human blood mononuclear cells exposed to U. tomentosa extracts were investigated. The oxidative changes were observed in mononuclear blood cells exposed to both ethanol and aqueous extracts obtained from bark and leaves. Moreover, in the cells studied the extracts from U. tomentosa induced apoptosis and a decrease in viability of mononuclear blood cells, with the exception of aqueous extract from leaves. Additionally, no statistically significant changes in the cell size were observed both for aqueous extracts from leaves and bark. Changes in the blood mononuclear cell granularity were observed at 250 μg/mL for all extracts examined. The strongest changes were observed for the ethanol extract of the bark, which increased cell granularity at 50 μg/mL and changed cell size at 100 μg/mL. The conducted research showed differences in biological activity

  6. Conditioning causes an increase in glucose transporter-4 levels in mononuclear cells in sled dogs.

    Science.gov (United States)

    Schnurr, Theresia M; Reynolds, Arleigh J; Gustafson, Sally J; Duffy, Lawrence K; Dunlap, Kriya L

    2014-10-01

    This study was designed to investigate the effects of physical conditioning on the expression of the insulin sensitive glucose transporter-4 protein (GLUT4) on mononuclear cells and HOMA-IR levels in dogs and compared to results reported in human skeletal muscle and the skeletal muscle of rodent models. Blood was sampled from conditioned dogs (n = 8) and sedentary dogs (n = 8). The conditioned dogs were exercised four months prior the experiment and were following a uniform training protocol, whereas the sedentary dogs were not. GLUT4 expression in mononuclear cells and plasma insulin levels were measured using commercially available enzyme-linked immunosorbent assay (ELISA). Blood glucose levels were determined using blood plasma. HOMA-IR was calculated using plasma insulin and blood glucose levels using the linear approximation formula. Our results indicate that the state of conditioning had a significant effect on the GLUT4 expression at the surface of mononuclear cells. HOMA-IR was also affected by conditioning in dogs. GLUT4 levels in mononuclear cells of sled dogs were inversely correlated with the homeostasis model assessment of insulin sensitivity. This study demonstrates that conditioning increases GLUT4 levels in mononuclear cells of sled dogs as it has been previously reported in skeletal muscle. Our results support the potential of white blood cells as a proxy tissue for studying insulin signaling and may lead to development of a minimally invasive and direct marker of insulin resistance. This may be the first report of GLUT4 in mononuclear cells in response to exercise and measured with ELISA.

  7. Immunomodulatory capacity of fungal proteins on the cytokine production of human peripheral blood mononuclear cells

    NARCIS (Netherlands)

    Jeurink, P.V.; Lull Noguera, C.; Savelkoul, H.F.J.; Wichers, H.J.

    2008-01-01

    Immunomodulation by fungal compounds can be determined by the capacity of the compounds to influence the cytokine production by human peripheral blood mononuclear cells (hPBMC). These activities include mitogenicity, stimulation and activation of immune effector cells. Eight mushroom strains (Agaric

  8. Intracoronary infusion of mononuclear cells after PCI-treated myocardial infarction and arrhythmogenesis : is it safe?

    NARCIS (Netherlands)

    Robbers, L. F. H. J.; Nijveldt, R.; Beek, A. M.; Kemme, M. J. B.; Delewi, R.; Hirsch, Alexander; van der Laan, A. M.; van der Vleuten, P. A.; Piek, J. J.; Zijlstra, F.; van Rossum, A. C.

    2012-01-01

    To reduce long-term morbidity after revascularised acute myocardial infarction, different therapeutic strategies have been investigated. Cell therapy with mononuclear cells from bone marrow (BMMC) or peripheral blood (PBMC) has been proposed to attenuate the adverse processes of remodelling and subs

  9. Mononuclear cells in subcutaneous haemorrhage with special consideration of myeloid percursor cells.

    Science.gov (United States)

    Oehmichen, M; Windisch, A; Meissner, C

    2000-10-01

    Various hematogenous markers were used to differentiate and quantify the types of mononuclear cells present in subcutaneous haemorrhages. Fifty samples of subcutaneous bleeding with a survival time of a few minutes to more than 48 hours were studied. The various cell types were detected using the following stains: Naphthol AS-D chloracetate esterase for myeloid cells, including mast cells; (alpha1-antichymotrypsin for monocytes/macrophages; UCHL1 for T-lymphocytes; and L26 for B lymphocytes. The percentage of monocytes/macrophages was found to increase in dependence on survival time, whereas T-lymphocytes declined. Within minutes of injury neutrophilic granulocytes had emigrated into the surrounding tissue and mast cell degranulation had occurred within the haemorrhagic zone. Esterase-positive mononuclear cells, namely metamyelocytes, were detected within minutes after injury and were still present after survival times exceeding 48 hours; however, no dependence on survival time or cause of death was found. Although the increasing number of monocytes/ macrophages and T-lymphocytes was expected, the sometimes high percentage of myeloid precursor cells within the wound were surprising. Possible explanations for this phenomenon are discussed.

  10. The use of the CELLection kit in the isolation of carcinoma cells from mononuclear cell suspensions

    DEFF Research Database (Denmark)

    Werther, K; Normark, M; Hansen, B F;

    2000-01-01

    A study was performed to evaluate in vitro the sensitivity, specificity and variability of a new immunomagnetic microbead isolation technique which provides subsequent immunological staining of captured carcinoma cells. In a mixture of peripheral blood mononuclear cells (PBMCs) and human carcinoma...... cells the epithelial cancer cells were isolated with the Dynal((R)) RAM IgG1 CELLection Kit using Dynabeads M-280 coated with a rat monoclonal antibody (Mab) against mouse IgG1. The rat Mab was biotinylated and attached to Dynabeads via streptavidin and a DNA linker. The anti-epithelial monoclonal mouse...... an average recovery of approximately 60% of a human colon carcinoma cell line HCC-2998 seeded in 5.10(6) PBMCs was obtained, and the recovered cells could subsequently be immunologically stained for the surface antigen CD87 (urokinase plasminogen activator receptor). No positive stained cells were found...

  11. Reduced LAK cytotoxicity of peripheral blood mononuclear cells in patients with bladder cancer

    DEFF Research Database (Denmark)

    Hermann, G G; Petersen, K R; Steven, K

    1990-01-01

    were analyzed using monoclonal antibodies against T cells, natural killer (NK) -cells, monocytes, and activation markers. The cytotoxicities of US-PBMC, PS-PBMC, and LAK cells were all significantly lower in the cancer patients than in the controls (P less than 0.05). The percentages of PBMC positive......The cytotoxicity of unstimulated peripheral blood mononuclear cells (US-PBMC), phytohemagglutinin (PHA)-stimulated PBMC (PS-PBMC) and interleukin-2 (IL-2)-activated PBMC (LAK cells) was assessed in patients with noninvasive and invasive transitional-cell bladder cancer and compared with those...... determined in healthy controls. The differences in the cytotoxicities were correlated with specific changes in the subsets of peripheral blood mononuclear cells (PBMC). PBMC from 37 patients and 13 healthy controls were tested against the bladder cancer cell line T24 in 51Cr-release assays. The PBMC subsets...

  12. A hitchhiker's guide to myeloid cell subsets: practical implementation of a novel mononuclear phagocyte classification system

    Directory of Open Access Journals (Sweden)

    Martin eGuilliams

    2015-08-01

    Full Text Available The classification of mononuclear phagocytes as either dendritic cells or macrophages has been mainly based on morphology, the expression of surface markers and assumed functional specialization. We have recently proposed a novel classification system of mononuclear phagocytes based on their ontogeny. Here we discuss the practical application of such a classification system through a number of prototypical examples we have encountered while hitchhiking from one subset to another, across species and between steady state and inflammatory settings. Finally, we discuss the advantages and drawbacks of such a classification system and propose a number of improvements to move from theoretical concepts to concrete guidelines.

  13. [Production of mature red blood cell by using peripheral blood mononuclear cells].

    Science.gov (United States)

    Jia, Yan-Jun; Liu, Jiang; Zhang, Ke-Ying; Shang, Xiao-Yan; Li, Wei; Wang, Li-Jun; Liu, Na; Wang, Lin; Cui, Shuang; Ni, Lei; Zhao, Bo-Tao; Wang, Dong-Mei; Gao, Song-Ming; Zhang, Zhi-Xin

    2014-10-01

    Most protocols for in vitro producing red blood cells (RBC) use the CD34(+) cells or embryonic stem cells from cord blood, bone marrow or peripheral blood as the start materials. This study was purposed to produce the mature RBC in vitro by using peripheral blood mononuclear cells as start material. The peripheral blood mononuclear cells (PBMNC) were isolated from buffy coat after blood leukapheresis, the mature red blood cells (RBC) were prepared by a 4-step culture protocol. The results showed that after culture by inducing with the different sets of cytokines and supporting by mouse MS-5 cell line, the expansion of PBMNC reached about 1000 folds at the end of the culture. About 90% of cultured RBC were enucleated mature cells which had the comparable morphological characteristics with normal RBC. Colony-forming assays showed that this culture system could stimulate the proliferation of progenitors in PBMNC and differentiate into erythroid cells. The structure and function analysis indicated that the mean cell volume of in vitro cultured RBC was 118 ± 4 fl, which was slight larger than that of normal RBC (80-100 fl); the mean cell hemoglobin was 36 ± 1.2 pg, which was slight higher than that of normal RBC (27-31 pg); the maximal deformation index was 0.46, which approachs level of normal RBC; the glucose-6-phosphate dehydrogenase and pyrurvate kinase levels was consistant with young RBC. It is concluded that PBMNC are feasble, convenient and low-cost source for producing cultured RBC and this culture system is suitable to generate the RBC from PBMNC.

  14. Nuclear thyroid hormone receptor binding in human mononuclear blood cells after goitre resection

    DEFF Research Database (Denmark)

    Kvetny, J; Matzen, L E; Blichert-Toft, M

    1989-01-01

    Nuclear thyroxine and triiodothyronine receptor-binding in human mononuclear blood cells were examined in 14 euthyroid persons prior to and 1, 6, 24 and 53 weeks after goitre resection. One week after resection decreased serum T3 from 1.47 nmol/l to 1.14 nmol/l (P less than 0.05), FT4I from 103 a...

  15. Slc27a2 expression in peripheral blood mononuclear cells as a marker for overweight development

    NARCIS (Netherlands)

    Caimari, A.; Oliver, P.; Rodenburg, W.; Keijer, J.; Palou, A.

    2010-01-01

    Background: Peripheral blood mononuclear cells (PBMC) can be collected easily and repeatedly. Their potential use to reflect the individual's biological status is increasingly explored. Obesity is becoming the most common health problem of the 21st century, being dietary intake an important determin

  16. Age and metabolic risk factors associated with oxidatively damaged DNA in human peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Løhr, Mille; Jensen, Annie; Eriksen, Louise

    2015-01-01

    Aging is associated with oxidative stress-generated damage to DNA and this could be related to metabolic disturbances. This study investigated the association between levels of oxidatively damaged DNA in peripheral blood mononuclear cells (PBMCs) and metabolic risk factors in 1,019 subjects, aged...

  17. Interleukin-8 transcripts in mononuclear cells determine impaired graft function after kidney transplantation

    DEFF Research Database (Denmark)

    Borst, Christoffer; Xia, Shengqiang; Bistrup, Claus

    2015-01-01

    OBJECTIVE: Interleukin-8 (IL-8) has been associated with ischemia reperfusion injury after renal allograft transplantation. Impaired allograft function may cause major impact on patient morbidity and health care costs. We investigated whether transcript levels in mononuclear cells including IL-8 ...

  18. Variation of DNA damage levels in peripheral blood mononuclear cells isolated in different laboratories

    DEFF Research Database (Denmark)

    Godschalk, Roger W L; Ersson, Clara; Stępnik, Maciej

    2014-01-01

    This study investigated the levels of DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG) sensitive sites, as assessed by the comet assay, in peripheral blood mononuclear cells (PBMC) from healthy women from five different countries in Europe. The laboratory in each country (referred ...

  19. Generation of induced pluripotent stem cells with high efficiency from human umbilical cord blood mononuclear cells.

    Science.gov (United States)

    Wang, Juan; Gu, Qi; Hao, Jie; Bai, Donghui; Liu, Lei; Zhao, Xiaoyang; Liu, Zhonghua; Wang, Liu; Zhou, Qi

    2013-10-01

    Human induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine. Generating iPSCs from immunologically immature newborn umbilical cord blood mononuclear cells (UCBMCs) is of great significance. Here we report generation of human iPSCs with great efficiency from UCBMCs using a dox-inducible lentiviral system carrying four Yamanaka factors. We generated these cells by optimizing the existing iPSC induction protocol. The UCBMC-derived iPSCs (UCB-iPSCs) have characteristics that are identical to pluripotent human embryonic stem cells (hESCs). This study highlights the use of UCBMCs to generate highly functional human iPSCs that could accelerate the development of cell-based regenerative therapy for patients suffering from various diseases.

  20. Vigorous, but differential mononuclear cell response of cirrhotic patients to bacterial ligands

    Institute of Scientific and Technical Information of China (English)

    Varenka J Barbero-Becerra; María Concepción Gutiérrez-Ruiz; Carmen Maldonado-Bernal; Félix I Téllez-Avila; Roberto Alfaro-Lara; Florencia Vargas-Vorácková

    2011-01-01

    AIM: To study the role of gram-positive and gram-negative bacteria in the pathogenesis of liver injury, specifically the activation of inflammatory mediators. METHODS: Peripheral blood mononuclear cells of 20 out-patients were studied, 10 of them with cirrhosis. Peripheral blood mononuclear cells were isolated and exposed to lipopolysaccharide or lipoteichoic acid. CD14, Toll-like receptor 2 and 4 expression was determined by flow cytometry, and tumor necrosis factor (TNF) α, interleukin (IL)-1β, IL-6, IL-12 and IL-10 secretion in supernatants was determined by ELISA. RESULTS: Higher CD14, Toll-like receptor 2 and 4 expression was observed in peripheral blood mononuclear cells from cirrhotic patients, (P < 0.01, P < 0.006, P < 0.111) respectively. Lipopolysaccharide and lipoteichoic acid induced a further increase in CD14 expression (P < 0.111 lipopolysaccharide, P < 0.013 lipoteichoic acid), and a decrease in Toll-like receptor 2 (P < 0.008 lipopolysaccharide, P < 0.008 lipoteichoic acid) and Toll-like receptor 4 (P < 0.008 lipopolysaccharide, P < 0.028 lipoteichoic acid) expression. With the exception of TNFα, absolute cytokine secretion of peripheral blood mononuclear cells was lower in cirrhotic patients under nonexposure conditions (P < 0.070 IL-6, P < 0.009 IL-1β, P < 0.022 IL-12). Once exposed to lipopolysaccharide or lipoteichoic acid, absolute cytokine secretion of peripheral blood mononuclear cells was similar in cirrhotic and non-cirrhotic patients, determining a more vigorous response in the former (P < 0.005 TNFα, IL-1β, IL-6, IL-2 and IL-10 lipopolysaccharide; P < 0.037 TNFα; P < 0.006 IL-1β; P < 0.005 IL-6; P < 0.007 IL-12; P < 0.014 IL-10 lipoteichoic acid). Response of peripheral blood mononuclear cells was more intense after lipopolysaccharide than after lipoteichoic acid exposure. CONCLUSION: Peripheral blood mononuclear cells of cirrhotic patients are able to respond to a sudden bacterial ligand exposure, particularly lipopolysaccharide

  1. In vitro transdifferentiation of human peripheral blood mononuclear cells to photoreceptor-like cells

    Directory of Open Access Journals (Sweden)

    Yukari Komuta

    2016-06-01

    Full Text Available Direct reprogramming is a promising, simple and low-cost approach to generate target cells from somatic cells without using induced pluripotent stem cells. Recently, peripheral blood mononuclear cells (PBMCs have attracted considerable attention as a somatic cell source for reprogramming. As a cell source, PBMCs have an advantage over dermal fibroblasts with respect to the ease of collecting tissues. Based on our studies involving generation of photosensitive photoreceptor cells from human iris cells and human dermal fibroblasts by transduction of photoreceptor-related transcription factors via retrovirus vectors, we transduced these transcription factors into PBMCs via Sendai virus vectors. We found that retinal disease-related genes were efficiently detected in CRX-transduced cells, most of which are crucial to photoreceptor functions. In functional studies, a light-induced inward current was detected in some CRX-transduced cells. Moreover, by modification of the culture conditions including additional transduction of RAX1 and NEUROD1, we found a greater variety of retinal disease-related genes than that observed in CRX-transduced PBMCs. These data suggest that CRX acts as a master control gene for reprogramming PBMCs into photoreceptor-like cells and that our induced photoreceptor-like cells might contribute to individualized drug screening and disease modeling of inherited retinal degeneration.

  2. A comparative study of Mono Mac 6 cells, isolated mononuclear cells and Limulus amoebocyte lysate assay in pyrogen testing

    DEFF Research Database (Denmark)

    Moesby, Lise; Jensen, S; Hansen, E W

    1999-01-01

    Pyrogen induced secretion of interleukin 6 (IL-6) in Mono Mac 6 (MM6) cells was measured. The ability of the MM6 cell culture to detect pyrogens was compared to the Limulus amoebocyte lysate (LAL) test and isolated mononuclear cells (MNC). The detection limit of MM6 for lipopolysaccharide (LPS...

  3. A comparative study of Mono Mac 6 cells, isolated mononuclear cells and Limulus amoebocyte lysate assay in pyrogen testing

    DEFF Research Database (Denmark)

    Moesby, Lise; Jensen, S; Hansen, E W;

    1999-01-01

    Pyrogen induced secretion of interleukin 6 (IL-6) in Mono Mac 6 (MM6) cells was measured. The ability of the MM6 cell culture to detect pyrogens was compared to the Limulus amoebocyte lysate (LAL) test and isolated mononuclear cells (MNC). The detection limit of MM6 for lipopolysaccharide (LPS...

  4. Rapid Column-Free Enrichment of Mononuclear Cells from Solid Tissues.

    Science.gov (United States)

    Scoville, Steven D; Keller, Karen A; Cheng, Stephanie; Zhang, Michael; Zhang, Xiaoli; Caligiuri, Michael A; Freud, Aharon G

    2015-07-30

    We have developed a rapid negative selection method to enrich rare mononuclear cells from human tissues. Unwanted and antibody-tethered cells are selectively depleted during a Ficoll separation step, and there is no need for magnetic-based reagents and equipment. The new method is fast, customizable, inexpensive, remarkably efficient, and easy to perform, and per sample the overall cost is less than one-tenth the cost associated with a magnetic column-based method.

  5. Rapid Column-Free Enrichment of Mononuclear Cells from Solid Tissues

    Science.gov (United States)

    Scoville, Steven D.; Keller, Karen A.; Cheng, Stephanie; Zhang, Michael; Zhang, Xiaoli; Caligiuri, Michael A.; Freud, Aharon G.

    2015-01-01

    We have developed a rapid negative selection method to enrich rare mononuclear cells from human tissues. Unwanted and antibody-tethered cells are selectively depleted during a Ficoll separation step, and there is no need for magnetic-based reagents and equipment. The new method is fast, customizable, inexpensive, remarkably efficient, and easy to perform, and per sample the overall cost is less than one-tenth the cost associated with a magnetic column-based method. PMID:26223896

  6. Modulation of adhesion molecules by cholesterol-lowering therapy in mononuclear cells from hypercholesterolemic patients.

    Science.gov (United States)

    Cerda, Alvaro; Rodrigues, Alice Cristina; Alves, Camila; Genvigir, Fabiana Dalla Vecchia; Fajardo, Cristina Moreno; Dorea, Egidio Lima; Gusukuma, Maria Cecilia; Pinto, Gelba Almeida; Hirata, Mario Hiroyuki; Hirata, Rosario Dominguez Crespo

    2015-08-01

    Cholesterol-lowering therapy has been related with several pleiotropic effects including anti-inflammatory action in vascular endothelium; however, their influence on monocyte adhesion molecules is poorly described. To investigate the effect of inhibitors of synthesis (statins) and absorption (ezetimibe) of cholesterol on expression of adhesion molecules L-selectin, PSGL-1, VLA-4, LFA-1, and Mac-1 in mononuclear cells in vivo and in vitro using THP-1 cells. The influence of simvastatin (10 mg/day), ezetimibe (10 mg/day), and their combination (10 mg each/day) on mRNA expression of adhesion molecules was analyzed in peripheral blood mononuclear cells (PBMC) from hypercholesterolemics. The effects of atorvastatin, simvastatin, and ezetimibe on mRNA and protein expression of adhesion molecules were also evaluated in THP-1 cells. Simvastatin/ezetimibe combination, but not the monotherapies, reduced the mRNA expression of the PSGL-1, LFA-1, and Mac-1 genes in PBMC from hypercholesterolemics. Total and LDL cholesterol in serum correlated with PSGL-1 mRNA expression, whereas HDL cholesterol negatively correlated with mRNA levels of L-selectin and VLA-4 genes (P molecules in PBMC from hypercholesterolemics and THP-1 cells. Simvastatin/ezetimibe combination gives more benefits by reducing to a larger extent the expression of adhesion molecules in mononuclear cells. © 2015 John Wiley & Sons Ltd.

  7. Endothelial progenitor cell differentiation using cryopreserved, umbilical cord blood-derived mononuclear cells

    Institute of Scientific and Technical Information of China (English)

    Jun-ho JANG; Hugh C KIM; Sun-kyung KIM; Jeong-eun CHOI; Young-jin KIM; Hyun-woo LEE; Seok-yun KANG; Joon-seong PARK; Jin-hyuk CHOI; Ho-yeong LIM

    2007-01-01

    Aim: To investigate the endothelial differentiation potentiality of umbilical cord blood (UCB), we induced the differentiation of endothelial progenitor cells (EPC)from cryopreserved UCB-derived mononuclear cells (MNC). Methods: MNC from cryopreserved UCB and peripheral blood (PB) were cultured in M199 medium with endothelial cell growth supplements for 14 d. EPC were characterized by RT-PCR,flow cytometry, and immunocytochemistry analysis. The proliferation of differen-tiated EPC was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTI') assay, and vascular endothelial growth factor (VEGF) concentra-tion was measured using an ELISA kit. Characteristics of UCB-derived EPC were compared with those of PB-derived EPC. Results: A number of round-shaped cells were loosely attached to the bottom after 24 h culture, and numerous spindle-shaped cells began to appear from the round-shaped ones on d 7. Those cells expressed endothelial markers such as, Fit-1/VEGFR-1, ecNOS, VE-cadherin, yon Willebrand factor, and secreted VEGF. The patterns of endothelial markers of EPC from PB and UCB did not show striking differences. The results of the prolifera-tion and secretion of VEGF were also similar. Conclusion: We successfully cul-tured UCB cells stored at -196 ℃ into cells with the quality of endothelial cells.Those EPC could be used for angiogenic therapeutics by activating adjacent endothelial cells and enhancing angiogenesis.

  8. Effects of carvedilol on oxidative stress in polymorphonuclear and mononuclear cells in patients with essential hypertension.

    Science.gov (United States)

    Yasunari, Kenichi; Maeda, Kensaku; Nakamura, Munehiro; Watanabe, Takanori; Yoshikawa, Junichi; Asada, Akira

    2004-04-01

    To compare the effects of carvedilol and propranolol on oxidative stress in leukocytes and C-reactive protein levels in patients with hypertension. Sixty hypertensive patients were randomly assigned to carvedilol (20 mg; n = 30) or propranolol (60 mg; n = 30) for 6 months. Thirty normotensive subjects who were given placebo served as controls. Oxidative stress in polymorphonuclear cells and mononuclear cells were measured by gated flow cytometry. C-reactive protein levels were measured by immunonephelometric assay. Oxidative stress in polymorphonuclear cells and mononuclear cells was increased significantly in hypertensive patients compared with in normotensive controls. After 6 months of treatment, carvedilol decreased oxidative stress significantly in polymorphonuclear cells by a mean of 45 arbitrary units (95% confidence interval [CI]: 32 to 59 arbitrary units; P <0.001) and propranolol decreased oxidative stress significantly by 20 arbitrary units (95% CI: 7 to 33 arbitrary units; P <0.003; P = 0.001 for difference between treatments). Carvedilol also decreased oxidative stress significantly in mononuclear cells by 23 arbitrary units (95% CI: 15 to 31 arbitrary units; P <0.001), whereas propranolol decreased oxidative stress by 2 arbitrary units (95% CI: 7 to 12 arbitrary units; P = 0.62; P = 0.002 for difference between treatments). Carvedilol decreased C-reactive protein levels significantly by a median of 0.073 mg/dL (interquartile range, 0.034 to 0.112 mg/dL; P <0.001), whereas propranolol decreased levels by 0.012 mg/dL (interquartile range, 0.009 to 0.032 mg/dL; P = 0.26; P = 0.003 for difference between treatments). These findings suggest that carvedilol inhibits oxidative stress in polymorphonuclear and mononuclear cells, as well as lowers C-reactive protein levels, to a greater extent than does propranolol in hypertensive patients.

  9. Adhesion of subsets of human blood mononuclear cells to porcine endothelial cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Cellular immune response is a major barrier to xenotransplantation, and cell adhesion is the first step in intercellular recognition. Flow-cytometric adhesion assay has been used to investigate the differential adhesions of monocyte (Mo), natural killer cell (NK) and T lymphocyte (T) present within human peripheral blood mononuclear cells (PBMC) to porcine aortic endothelial cells (PAEC), and to demonstrate the effect of human interferon-γ(hIFN-γ) or/and tumor necrosis factor-α (hTNF-α) pretreatment of PAEC on their adhesiveness for different PBMC subsets. The preferential sequence for PBMC subset binding to resting PAEC is Mo, NK and T cells, among which T cells show the slightest adherence; hTNF-α can act across the species, and augment Mo, NK and T cell adhesion ratios by 40%, 110% and 3 times, respectively. These results confirm at the cell level that host Mo and NK cells are major participants in the cellular xenograft rejection, thereby, providing a prerequisite for further studying the human Mo/NK-PAEC interactive mechanisms.

  10. Go it alone: four-electron oxidations by mononuclear non-heme iron enzymes.

    Science.gov (United States)

    Peck, Spencer C; van der Donk, Wilfred A

    2017-04-01

    This review discusses the current mechanistic understanding of a group of mononuclear non-heme iron-dependent enzymes that catalyze four-electron oxidation of their organic substrates without the use of any cofactors or cosubstrates. One set of enzymes acts on α-ketoacid-containing substrates, coupling decarboxylation to oxygen activation. This group includes 4-hydroxyphenylpyruvate dioxygenase, 4-hydroxymandelate synthase, and CloR involved in clorobiocin biosynthesis. A second set of enzymes acts on substrates containing a thiol group that coordinates to the iron. This group is comprised of isopenicillin N synthase, thiol dioxygenases, and enzymes involved in the biosynthesis of ergothioneine and ovothiol. The final group of enzymes includes HEPD and MPnS that both carry out the oxidative cleavage of the carbon-carbon bond of 2-hydroxyethylphosphonate but generate different products. Commonalities amongst many of these enzymes are discussed and include the initial substrate oxidation by a ferric-superoxo-intermediate and a second oxidation by a ferryl species.

  11. Derivation of autism spectrum disorder-specific induced pluripotent stem cells from peripheral blood mononuclear cells.

    Science.gov (United States)

    DeRosa, Brooke A; Van Baaren, Jessica M; Dubey, Gaurav K; Lee, Joycelyn M; Cuccaro, Michael L; Vance, Jeffery M; Pericak-Vance, Margaret A; Dykxhoorn, Derek M

    2012-05-10

    Induced pluripotent stem cells (iPSCs) hold tremendous potential both as a biological tool to uncover the pathophysiology of disease by creating relevant cell models and as a source of stem cells for cell-based therapeutic applications. Typically, iPSCs have been derived by the transgenic overexpression of transcription factors associated with progenitor cell or stem cell function in fibroblasts derived from skin biopsies. However, the need for skin punch biopsies to derive fibroblasts for reprogramming can present a barrier to study participation among certain populations of individuals, including children with autism spectrum disorders (ASDs). In addition, the acquisition of skin punch biopsies in non-clinic settings presents a challenge. One potential mechanism to avoid these limitations would be the use of peripheral blood mononuclear cells (PBMCs) as the source of the cells for reprogramming. In this article we describe, for the first time, the derivation of iPSC lines from PBMCs isolated from the whole blood of autistic children, and their subsequent differentiation in GABAergic neurons.

  12. Protective effects of curcumin on methylglyoxal-induced oxidative DMA damage and cell injury in human mononuclear cells

    Institute of Scientific and Technical Information of China (English)

    Wen-hsiung CHAN; Hsin-jung WU

    2006-01-01

    Aim: To examine the effect of curcumin on oxidative DNA damage and cell apoptosis and injury caused by the reaction of methylglyoxal(MG) with amino acids. Methods: We used DNA strand breaks to examine the effect of curcumin on oxidative DNA damage. In addition, reactive oxygen species(ROS) formation occurs in MG-treated mononuclear cells, so the effect of curcumin on ROS generation was measured using 2',7'-dichlorofluorescin diacetate(DCF-DA) as the detection reagent. Moreover, the impact effects of curcumin on MG-induced cell apoptosis and ROS injury were analyzed by TUNEL and ELISA assay. The collagen I attachment ability of mononuclear cells was examined by trypan blue staining. Results: Our results revealed that curcumin prevented MG/lysine-induced oxidative stress and DNA damage. Curcumin also inhibited MG-induced apoptosis and generation of ROS in mononuclear cells. MG-treated mononuclear cells displayed a lower degree of attachment to collagen (the major component of the vessel wall subendo-thelium), whereas cells pretreated with curcumin before MG treatment exhibited restored affinities for collagen. Conclusion: These results demonstrated that oxidative stress plays a role in MG-induced cell injury and alterations in attachment ability, and that curcumin blocks these effects by virtue of its antioxidant properties.

  13. Effect of malaria components on blood mononuclear cells involved in immune response.

    Science.gov (United States)

    Punsawad, Chuchard

    2013-09-01

    During malaria infection, elevated levels of pro-inflammatory mediators and nitric oxide production have been associated with pathogenesis and disease severity. Previous in vitro and in vivo studies have proposed that both Plasmodium falciparum hemozoin and glycosylphosphatidylinositols are able to modulate blood mononuclear cells, contributing to stimulation of signal transduction and downstream regulation of the NF-κB signaling pathway, and subsequently leading to the production of pro-inflammatory cytokines, chemokines, and nitric oxide. The present review summarizes the published in vitro and in vivo studies that have investigated the mechanism of intracellular signal transduction and activation of the NF-κB signaling pathway in blood mononuclear cells after being inducted by Plasmodium falciparum malaria components. Particular attention is paid to hemozoin and glycosylphosphatidylinositols which reflect the important mechanism of signaling pathways involved in immune response.

  14. Effect of malaria components on blood mononuclear cells involved in immune response

    Institute of Scientific and Technical Information of China (English)

    Chuchard Punsawad

    2013-01-01

    During malaria infection, elevated levels of pro-inflammatory mediators and nitric oxide production have been associated with pathogenesis and disease severity. Previous in vitro and in vivo studies have proposed that both Plasmodium falciparum hemozoin and glycosylphosphatidylinositols are able to modulate blood mononuclear cells, contributing to stimulation of signal transduction and downstream regulation of the NF-κB signaling pathway, and subsequently leading to the production of pro-inflammatory cytokines, chemokines, and nitric oxide. The present review summarizes the published in vitro and in vivo studies that have investigated the mechanism of intracellular signal transduction and activation of the NF-κB signaling pathway in blood mononuclear cells after being inducted by Plasmodium falciparum malaria components. Particular attention is paid to hemozoin and glycosylphosphatidylinositols which reflect the important mechanism of signaling pathways involved in immune response.

  15. Prolactin stimulates integrin-mediated adhesion of circulating mononuclear cells to endothelial cells.

    Science.gov (United States)

    Montes de Oca, Pável; Macotela, Yazmín; Nava, Gabriel; López-Barrera, Fernando; de la Escalera, Gonzalo Martínez; Clapp, Carmen

    2005-05-01

    Attachment of leukocytes to endothelial cells is an essential step for the extravasation and recruitment of cells at sites of inflammation. The pituitary hormone prolactin (PRL) is involved in the inflammatory process. Here, we show that treatment with PRL of human peripheral blood mononuclear cells (PBMC) stimulates their adhesion to human umbilical vein endothelial cells (HUVEC) activated by interleukin-1beta. Stimulation of adhesion by PRL is mediated via integrins leukocyte functional antigen-1 (LFA-1) and very late antigen-4 (VLA-4), because immunoneutralization of both integrins prevents PRL action. Also, PRL promotes the adhesion of PBMC to immobilized intercellular adhesion molecule-1 and fibronectin, ligands for LFA-1 and VLA-4, respectively. Stimulation of integrin-mediated cell adhesion by PRL may involve the activation of chemokine receptors, because PRL upregulates the expression of the G-protein-coupled chemokine receptor CXCR3 in PBMC, and pertussis toxin, a specific G-protein inhibitor, blocks PRL stimulation of PBMC adhesion to HUVEC. In addition, PRL stimulates tyrosine phosphorylation pathways leading to leukocyte adhesion. PRL triggered the tyrosine phosphorylation of Janus kinase-2, of signal transducer and activator of transcription-3 and 5, and of the focal adhesion protein paxillin. Furthermore, genistein, a tyrosine kinase inhibitor, blocked PRL-stimulated adhesion of PBMC and Jurkat T-cells to HUVEC. These results suggest that PRL promotes integrin-mediated leukocyte adhesion to endothelial cells via chemokine receptors and tyrosine phosphorylation signaling pathways.

  16. MHC-unrestricted lysis of MUC1-expressing cells by human peripheral blood mononuclear cells.

    Science.gov (United States)

    Wright, Stephen E; Rewers-Felkins, Kathleen A; Quinlin, Imelda S; Fogler, William E; Phillips, Catherine A; Townsend, Mary; Robinson, William; Philip, Ramila

    2008-01-01

    Many human adenocarcinomas can be killed in vitro by targeted cytotoxic T-lymphocytes (CTL); however, major histocompatibility complex (MHC)-restrictions are typically required. The MUC1 antigen is common in many human adenocarcinomas, and is associated with a variable number of tandem repeats. It has been proposed that antigens with such repeated epitopes may be vulnerable to cytotoxic T-lymphocyte killing without MHC-restriction. Therefore, it is possible that MUC1-expressing malignant cells may be killed by targeted cytotoxic T-lymphocyte in the absence of MHC-restriction. In this study, a human MUC1-expressing murine mammary carcinoma cell line was used to determine if cytotoxic T-lymphocyte killing of MUC1-expressing adenocarcinoma cells requires MHC-restriction. Specifically, MUC1-stimulated human mononuclear cells (M1SMC) were observed to kill human MUC1-transfected, MUC1-expressing murine mammary carcinoma cells, but not the mock-transfected, non-MUC1-expressing murine mammary carcinoma cells. Furthermore, the killing was blocked by antibody to MUC1, indicating MUC1-specific killing. In conclusion, cytotoxic T-lymphocyte killing of MUC1-expressing adenocarcinoma cells can be MHC-unrestricted.

  17. Identification of Suitable Reference Genes for Peripheral Blood Mononuclear Cell Subset Studies in Multiple Sclerosis

    DEFF Research Database (Denmark)

    Oturai, D B; Søndergaard, H B; Börnsen, L;

    2016-01-01

    Quantitative real-time PCR (qPCR) involves the need of a proper standard for normalizing the gene expression data. Different studies have shown the validity of reference genes to vary greatly depending on tissue, cell subsets and experimental context. This study aimed at the identification...... of suitable reference genes for qPCR studies using different peripheral blood cell subsets (whole blood (WB) cells, peripheral blood mononuclear cells (PBMCs) and PBMC subsets (CD4(+) T cells, CD8(+) T cells, NK cells, monocytes, B cells and dendritic cells) from healthy controls (HC), patients with relapsing...... stable combination for analyses of cell subsets between HC and RRMS patients, while the combination of UBC and YWHAZ was superior for analysis of cell subsets between HC, RRMS and RRMS-IFN-β groups. GAPDH was generally unsuitable for blood cell subset studies in multiple sclerosis. In conclusion, we...

  18. Treatment with at Homeopathic Complex Medication Modulates Mononuclear Bone Marrow Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Beatriz Cesar

    2011-01-01

    Full Text Available A homeopathic complex medication (HCM, with immunomodulatory properties, is recommended for patients with depressed immune systems. Previous studies demonstrated that the medication induces an increase in leukocyte number. The bone marrow microenvironment is composed of growth factors, stromal cells, an extracellular matrix and progenitor cells that differentiate into mature blood cells. Mice were our biological model used in this research. We now report in vivo immunophenotyping of total bone marrow cells and ex vivo effects of the medication on mononuclear cell differentiation at different times. Cells were examined by light microscopy and cytokine levels were measured in vitro. After in vivo treatment with HCM, a pool of cells from the new marrow microenvironment was analyzed by flow cytometry to detect any trend in cell alteration. The results showed decreases, mainly, in CD11b and TER-119 markers compared with controls. Mononuclear cells were used to analyze the effects of ex vivo HCM treatment and the number of cells showing ring nuclei, niche cells and activated macrophages increased in culture, even in the absence of macrophage colony-stimulating factor. Cytokines favoring stromal cell survival and differentiation in culture were induced in vitro. Thus, we observe that HCM is immunomodulatory, either alone or in association with other products.

  19. The DNA methylome of human peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Li, Yingrui; Zhu, Jingde; Tian, Geng;

    2010-01-01

    DNA methylation plays an important role in biological processes in human health and disease. Recent technological advances allow unbiased whole-genome DNA methylation (methylome) analysis to be carried out on human cells. Using whole-genome bisulfite sequencing at 24.7-fold coverage (12.3-fold pe...

  20. Intra-arterial Infusion of Autologous Bone Marrow Mononuclear Stem Cells in Subacute Ischemic Stroke Patients.

    Science.gov (United States)

    Ghali, Azza Abass; Yousef, Mohamed Khalil; Ragab, Osama AbdAllah; ElZamarany, Enas Arafa

    2016-01-01

    Based on many preclinical and small clinical trials, stem cells can help stroke patient with the possibility of replacing the cells and supporting the remaining cells. The aim of this study was to evaluate the safety and feasibility of bone marrow mononuclear (BMMN) stem cell transplantation in subacute ischemic stroke patients. Thirty-nine (n = 39) patients with subacute ischemic cerebral infarct due to large artery occlusion in the middle cerebral artery (MCA) territory were recruited. They were distributed into two groups: first group (n = 21) served as an experimental group, which received intra-arterial (IA) mononuclear stem cells (bone marrow-derived mononuclear cell), while the other group (n = 18) served as a control group. All the patients were evaluated clinically by National Institutes of Health Stroke Scale, modified Rankin Scale, Barthel Index, modified and standardized Arabic version of the Comprehensive Aphasia Test, and radiological for 12 months. The stem cell-treated group showed better improvement, but it was not significant when compared with the non-treated group. The volume of infarction changes at the end of the study was non-significant between both the groups. There was no, or minimal, adverse reactions in stem cell-treated group. The study results suggest that autologous BMMN stem cell IA transplantation in subacute MCA ischemic stroke patients is safe with very minimal hazards, but no significant improvement of motor, language disturbance, or infarction volume was detected in stem cell-treated group compared with the non-treated group.

  1. Cytokines profile and peripheral blood mononuclear cells morphology in Rett and autistic patients.

    Science.gov (United States)

    Pecorelli, Alessandra; Cervellati, Franco; Belmonte, Giuseppe; Montagner, Giulia; Waldon, PhiAnh; Hayek, Joussef; Gambari, Roberto; Valacchi, Giuseppe

    2016-01-01

    A potential role for immune dysfunction in autism spectrum disorders (ASD) has been well established. However, immunological features of Rett syndrome (RTT), a genetic neurodevelopmental disorder closely related to autism, have not been well addressed yet. By using multiplex Luminex technology, a panel of 27 cytokines and chemokines was evaluated in serum from 10 RTT patients with confirmed diagnosis of MECP2 mutation (typical RTT), 12 children affected by classic autistic disorder and 8 control subjects. The cytokine/chemokine gene expression was assessed by real time PCR on mRNA of isolated peripheral blood mononuclear cells (PBMCs). Moreover, ultrastructural analysis of PBMCs was performed using transmission electron microscopy (TEM). Significantly higher serum levels of interleukin-8 (IL-8), IL-9, IL-13 were detected in RTT compared to control subjects, and IL-15 shows a trend toward the upregulation in RTT. In addition, IL-1β and VEGF were the only down-regulated cytokines in autistic patients with respect to RTT. No difference in cytokine/chemokine profile between autistic and control groups was detected. These data were also confirmed by ELISA real time PCR. At the ultrastructural level, the most severe morphological abnormalities were observed in mitochondria of both RTT and autistic PBMCs. In conclusion, our study shows a deregulated cytokine/chemokine profile together with morphologically altered immune cells in RTT. Such abnormalities were not quite as evident in autistic subjects. These findings indicate a possible role of immune dysfunction in RTT making the clinical features of this pathology related also to the immunology aspects, suggesting, therefore, novel possible therapeutic interventions for this disorder.

  2. Study of the inhibition by polymorphonuclear leukocytes of TNF-α release from human mononuclear cells and its mechanism

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    @@ The present study was undertaken to investigate the effect of human PMNs on the production of TNF-α by the human peripheral blood mononuclear cells (PBMCs) and to elucidate its tentative mechanism.

  3. Pancreatic Cancer Cell Lines Can Induce Prostaglandin E2 Production from Human Blood Mononuclear Cells

    Directory of Open Access Journals (Sweden)

    Svitlana P. Grekova

    2011-01-01

    Full Text Available Accumulating evidence suggests an important role for cyclooxygenase-2 (COX-2 in the pathogenesis of a wide range of malignancies. The protumorigenic properties of COX-2 are generally thought to be mediated by its product, PGE2, which is shown to promote tumor spread and growth by multiple mechanisms but most importantly through modulation of the local immune response in the tumor. Pancreatic tumor cells produce various amounts of PGE2, some of them being even deficient in COX enzymes or other PGE2 synthases. Here we describe that, beside pancreatic tumor cells or stromal fibroblasts, human peripheral blood mononuclear cells can also produce PGE2 upon coculture with pancreatic cancer cells. Stimulating of cellular cPLA2 within PBMCs by secreted factors, presumably sPLA2, from tumor cells appeared crucial, while the direct contact between PBMCs and PDACs seemed to be dispensable for this effect. Our data is emphasizing the complex interactions participating in the formation of the tolerogenic immune milieu within pancreatic tumors.

  4. Aggressive periodontitis and chronic arthritis: blood mononuclear cell gene expression and plasma protein levels of cytokines and cytokine inhibitors

    DEFF Research Database (Denmark)

    Sørensen, Lars K; Havemose-Poulsen, Anne; Bendtzen, Klaus

    2009-01-01

    BACKGROUND: Cytokines and cytokine inhibitors have been associated with many immunoinflammatory diseases. In the present study, we examined whether peripheral blood mononuclear cell (PBMC) gene expression mirrors the corresponding plasma levels of clinically important pro- and anti-inflammatory c......BACKGROUND: Cytokines and cytokine inhibitors have been associated with many immunoinflammatory diseases. In the present study, we examined whether peripheral blood mononuclear cell (PBMC) gene expression mirrors the corresponding plasma levels of clinically important pro- and anti...

  5. Sunlight-induced DNA damage in human mononuclear cells

    DEFF Research Database (Denmark)

    Møller, Peter; Wallin, Hakan; Holst, Erik

    2002-01-01

    of sunlight was comparable to the interindividual variation, indicating that sunlight exposure and the individual's background were the two most important determinants for the basal level of DNA damage. Influence of other lifestyle factors such as exercise, intake of foods, infections, and age could......In this study of 301 blood samples from 21 subjects, we found markedly higher levels of DNA damage (nonpyrimidine dimer types) in the summer than in the winter detected by single-cell gel electrophoresis. The level of DNA damage was influenced by the average daily influx of sunlight ... to blood sampling. The 3 and 6 day periods before sampling influenced DNA damage the most. The importance of sunlight was further emphasized by a positive association of the DNA damage level to the amount of time the subjects had spent in the sun over a 3 day period prior to the sampling. The effect...

  6. Validation of using gene expression in mononuclear cells as a marker for hepatic cholesterol metabolism

    Directory of Open Access Journals (Sweden)

    Dutta Amrita

    2006-08-01

    Full Text Available Abstract HMG-CoA reductase and the LDL receptor are ubiquitously expressed in major tissues. Since the liver plays a major role in regulating circulating LDL, it is usually of interest to measure the effects of drug or dietary interventions on these proteins in liver. In humans, peripheral blood mononuclear cells have been used as a surrogate for liver to assess regulation of these genes, although there is concern regarding the validity of this approach. The purpose of this study was to evaluate the relationship between liver and mononuclear cell expression of HMG-CoA reductase and the LDL receptor in guinea pigs, a well established model for human cholesterol and lipoprotein metabolism. We extracted RNA from liver and mononuclear cells of guinea pigs from a previous study where the effects of rapamycin, an immunosuppresant drug used for transplant patients, on lipid metabolism were evaluated. Guinea pigs were assigned to three different diets containing the same amount of fat (15 g/100 g and cholesterol (0.08 g/100 g for a period of 3 weeks. The only difference among diets was the concentration of rapamycin: 0, 0.0028 or 0.028 g/100 g. There were no differences in plasma LDL cholesterol (LDL-C among groups. Values were 78.4 ± 14.3, 65.8 ± 17.2 and 68.4 ± 45.4 mg/dL (P > 0.05 for guinea pigs treated with 0, low or high doses of rapamycin, respectively. The mRNA abundance for the LDL receptor and HMG-CoA reductase was measured both in liver (n = 30 and mononuclear cells (n = 22 using reverse transcriptase PCR. In agreement with the finding of no changes in plasma LDL-C, there were also no differences for the expression of HMG-CoA reductase or the LDL receptor among groups. However, a positive correlation was found between liver and mononuclear cells for both HMG-CoA reductase (r = 0.613, P

  7. Electronic Structure and Reactivity of a Well-Defined Mononuclear Complex of Ti(II).

    Science.gov (United States)

    Wijeratne, Gayan B; Zolnhofer, Eva M; Fortier, Skye; Grant, Lauren N; Carroll, Patrick J; Chen, Chun-Hsing; Meyer, Karsten; Krzystek, J; Ozarowski, Andrew; Jackson, Timothy A; Mindiola, Daniel J; Telser, Joshua

    2015-11-02

    A facile and high-yielding protocol to the known Ti(II) complex trans-[(py)4TiCl2] (py = pyridine) has been developed. Its electronic structure has been probed experimentally using magnetic susceptibility, magnetic circular dichroism, and high-frequency and high-field electron paramagnetic resonance spectroscopies in conjunction with ligand-field theory and computational methods (density functional theory and ab initio methods). These studies demonstrated that trans-[(py)4TiCl2] has a (3)Eg ground state (dxy(1)dxz,yz(1) orbital occupancy), which, as a result of spin–orbit coupling, yields a ground-state spinor doublet that is EPR active, a first excited-state doublet at ∼60 cm(–1), and two next excited states at ∼120 cm(–1). Reactivity studies with various unsaturated substrates are also presented in this study, which show that the Ti(II) center allows oxidative addition likely via formation of [Ti(η(2)-R2E2)Cl2(py)n] E = C, N intermediates. A new Ti(IV) compound, mer-[(py)3(η(2)-Ph2C2)TiCl2], was prepared by reaction with Ph2C2, along with the previously reported complex trans-(py)3Ti═NPh(Cl)2, from reaction with Ph2N2. Reaction with Ph2CN2 also yielded a new dinuclear Ti(IV) complex, [(py)2(Cl)2Ti(μ2:η(2)-N2CPh2)2Ti(Cl)2], in which the two Ti(IV) ions are inequivalently coordinated. Reaction with cyclooctatetraene (COT) yielded a new Ti(III) complex, [(py)2Ti(η(8)-COT)Cl], which is a rare example of a mononuclear “piano-stool” titanium complex. The complex trans-[(py)4TiCl2] has thus been shown to be synthetically accessible, have an interesting electronic structure, and be reactive toward oxidation chemistry.

  8. Role of peripheral blood mononuclear cell transportation from mother to baby in HBV intrauterine infection.

    Science.gov (United States)

    Shao, Qingliang; Zhao, Xiaxia; Yao Li, M D

    2013-12-01

    We aimed to investigate the role of peripheral blood mononuclear cell transportation from mother to baby in hepatitis B virus (HBV) intrauterine infection. Thirty HBsAg-positive pregnant women in the second trimester and their aborted fetuses were included in this study. Enzyme-linked-immunosorbent-assay was utilized to detect HBsAg in the peripheral blood of pregnant women and the femoral vein blood of their aborted fetuses. HBV-DNA in serum and peripheral blood mononuclear cells (PBMC) and GSTM1 alleles of pregnant women and their aborted fetuses were detected by nested polymerase chain reaction (PCR) and seminested PCR, respectively. We also examined the location of placenta HBsAg and HBcAb using immunohistochemical staining. The expression of placenta HBV-DNA was detected by in situ hybridization. For the 30 aborted fetuses, the HBV intrauterine infection rate was 43.33%. The HBV-positive rates of HBsAg in peripheral blood, serum, and PBMC were 10% (3/30), 23.33% (7/30), and 33.33% (10/30), respectively. Maternal-fetal PBMC transport was significantly positively correlated with fetal PBMC HBV-DNA (P = 0.004). Meanwhile, the rates of HBV infection gradually decreased from the maternal side to the fetus side of placenta (decidual cells > trophoblastic cells > villous mesenchymal cells > villous capillary endothelial cells). However, no significant correlation between placenta HBV infection and HBV intrauterine infection was observed (P = 0.410). HBV intrauterine infection was primarily due to peripheral blood mononuclear cell maternal-fetal transportation in the second trimester in pregnant women.

  9. Usefulness of liver infiltrating CD86-positive mononuclear cells for diagnosis of autoimmune hepatitis

    Institute of Scientific and Technical Information of China (English)

    Kazutaka Kurokohchi; Shigeki Kuriyama; Tsutomu Masaki; Takashi Himoto; Akihiro Deguchi; Seiji Nakai; Asahiro Morishita; Hirohito Yoneyama; Yasuhiko Kimura; Seishiro Watanabe

    2006-01-01

    AIM: Although the pathogenic mechanism underlying autoimmune hepatitis (AIH) remains unclear, the immune system is thought to be critical for the progression of the disease. Cellular immune responses may be linked to the hepatocellular damage in AIH. Recently, much attention has been focused on the critical functions of costimulatory molecules expressed on mononuclear cells in the generation of effective T cell-mediated immune responses. Analysis of costimulatory molecule expressed on mononuclear cells from the patients with AIH may give us insight into the pathogenic mechanism of hepatocellular damage in AIH.METHODS: Peripheral blood mononuclear cells (PBMC)were taken from the patients with AIH (34 cases) and healthy controls (25 cases). Liver infiltrating mononuclear cells (LIMCs) were taken from the patients with AIH (18 cases), the patient with chronic hepatitis C (CH-C) (13 cases) and the patients with fatty liver (2 cases).Using flow cytometry, the cells were analyzed for the expression of costimulatory molecules, such as CD80,CD86, and CD152 (CTLA-4). The results were compared with clinical data such as the level of gammaglobulin,histological grade, presence or absence of corticosteroids administration and the response to corticosteroids.RESULTS: The levels of CD80+, CD86+ and CD152+PBMC were significantly reduced in the patients with AIH as compared with healthy controls. By contrast,those cells were significantly higher in LTMC than in PBMC of the patients with AIH. Especially, the level of CD86+ LIMC showed a marked increase irrespective of the degree of disease activity in the patients with ATH,although CD86+ cells were rarely present in PBMC. The levels of CD86+ cells were present in significantly higher frequency in patients with AIH than in the patients with CH-C. Furthermore, the patients with AIH with high levels of CD86+ LIMC showed good responses to corticosteroids, whereas 2 cases of AIH with low levels of CD86+ LIMC did not respond well

  10. Age and gender effects on DNA strand break repair in peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Garm, Christian; Moreno-Villanueva, Maria; Bürkle, Alexander;

    2013-01-01

    single-strand breaks (SSBs) and double-strand breaks (DSBs) in human peripheral blood mononuclear cells (PBMCs). Of these lesions, DSBs are the least frequent but the most dangerous for cells. We have measured the level of endogenous SSBs, SSB repair capacity, γ-H2AX response, and DSB repair capacity...... in a study population consisting of 216 individuals from a population-based sample of twins aged 40-77 years. Age in this range did not seem to have any effect on the SSB parameters. However, γ-H2AX response and DSB repair capacity decreased with increasing age, although the associations did not reach...

  11. HCG-Activated Human Peripheral Blood Mononuclear Cells (PBMC) Promote Trophoblast Cell Invasion.

    Science.gov (United States)

    Yu, Nan; Yan, Wenjie; Yin, Tailang; Wang, Yaqin; Guo, Yue; Zhou, Danni; Xu, Mei; Ding, Jinli; Yang, Jing

    2015-01-01

    Successful embryo implantation and placentation depend on appropriate trophoblast invasion into the maternal endometrial stroma. Human chorionic gonadotropin (hCG) is one of the earliest embryo-derived secreted signals in the peripheral blood mononuclear cells (PBMC) that abundantly expresses hCG receptors. The aims of this study were to estimate the effect of human embryo-secreted hCG on PBMC function and investigate the role and underlying mechanisms of activated PBMC in trophoblast invasion. Blood samples were collected from women undergoing benign gynecological surgery during the mid-secretory phase. PBMC were isolated and stimulated with or without hCG for 0 or 24 h. Interleukin-1β (IL-1β) and leukemia inhibitory factor (LIF) expressions in PBMC were detected by enzyme-linked immunosorbent assay and real-time polymerase chain reaction (PCR). The JAR cell line served as a model for trophoblast cells and was divided into four groups: control, hCG only, PBMC only, and PBMC with hCG. JAR cell invasive and proliferative abilities were detected by trans-well and CCK8 assays and matrix metalloproteinase (MMP)-2 (MMP-2), MMP-9, vascular endothelial growth factor (VEGF), tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 expressions in JAR cells were detected by western blotting and real-time PCR analysis. We found that hCG can remarkably promote IL-1β and LIF promotion in PBMC after 24-h culture. PBMC activated by hCG significantly increased the number of invasive JAR cells in an invasion assay without affecting proliferation, and hCG-activated PBMC significantly increased MMP-2, MMP-9, and VEGF and decreased TIMP-1 and TIMP-2 expressions in JAR cells in a dose-dependent manner. This study demonstrated that hCG stimulates cytokine secretion in human PBMC and could stimulate trophoblast invasion.

  12. Circulating angiogenic cells can be derived from cryopreserved peripheral blood mononuclear cells.

    Directory of Open Access Journals (Sweden)

    Tanja Sofrenovic

    Full Text Available BACKGROUND: Cell transplantation for regenerative medicine has become an appealing therapeutic method; however, stem and progenitor cells are not always freshly available. Cryopreservation offers a way to freeze cells as they are generated, for storage and transport until required for therapy. This study was performed to assess the feasibility of cryopreserving peripheral blood mononuclear cells (PBMCs for the subsequent in vitro generation of their derived therapeutic population, circulating angiogenic cells (CACs. METHODS: PBMCs were isolated from healthy human donors. Freshly isolated cells were either analyzed immediately or cryopreserved in media containing 6% plasma serum and 5% dimethyl sulfoxide. PBMCs were thawed after being frozen for 1 (early thaw or 28 (late thaw days and analyzed, or cultured for 4 days to generate CACs. Analysis of the cells consisted of flow cytometry for viability and phenotype, as well as functional assays for their adhesion and migration potential, cytokine secretion, and in vivo angiogenic potential. RESULTS: The viability of PBMCs and CACs as well as their adhesion and migration properties did not differ greatly after cryopreservation. Phenotypic changes did occur in PBMCs and to a lesser extent in CACs after freezing; however the potent CD34(+VEGFR2(+CD133(+ population remained unaffected. The derived CACs, while exhibiting changes in inflammatory cytokine secretion, showed no changes in the secretion of important regenerative and chemotactic cytokines, nor in their ability to restore perfusion in ischemic muscle. CONCLUSION: Overall, it appears that changes do occur in cryopreserved PBMCs and their generated CACs; however, the CD34(+VEGFR2(+CD133(+ progenitor population, the secretion of pro-vasculogenic factors, and the in vivo angiogenic potential of CACs remain unaffected by cryopreservation.

  13. IFN-{gamma} gene expression in pancreatic islet-infiltrating mononuclear cells correlates with autoimmune diabetes in nonobese diabetic mice

    Energy Technology Data Exchange (ETDEWEB)

    Rabinovitch, A.; Suarez-Pinzon, W.L.; Sorensen, O. [Univ. of Alberta, Edmonton (Canada)] [and others

    1995-05-01

    Insulin-dependent diabetes mellitus in nonobese diabetic (NOD) mice results from selective destruction of pancreatic islet {beta}-cells following islet filtration by mononuclear leukocytes. Cytokines produced by islet-infiltrating mononuclear cells may be involved in {beta}-cell destruction. Therefore, we analyzed cytokine mRNA expression, by reverse-transcriptase PCR (RT-PCR) assay, in mononuclear leukocytes isolated from pancreatic islets of four groups of mice: diabetes-prone female NOD mice; female NOD mice protected from diabetes by injection of CFA at an early age; male NOD mice with a low diabetes incidence; and female BALB/c mice that do not develop diabetes. We found that mRNA levels of IL-1{beta}, IL-2, IL-4, IL-10, and IFN-{gamma} in mononuclear cells from islets of diabetes-prone female NOD mice increased progressively as these cells infiltrated the islets from age 5 wk to diabetes onset (>13 wk). However, only IFN-{gamma} mRNA levels were significantly higher in islet mononuclear cells from 12-wk-old diabetes-prone female NOD mice than from less diabetes-prone NOD mice (CFA-treated females, and males) and normal mice (BALB/c). In contrast, IL-4 mRNA levels were lower in islet mononuclear cells from diabetes-prone female NOD mice than from NOD mice with low diabetes incidence (CFA-treated females and males). Splenic cell mRNA levels of IFN-{gamma} and IL-4 were not different in the four groups of mice. These results suggest that islet {beta}-cell destruction and diabetes in female NOD mice are dependent upon intra-islet IFN-{gamma} production by mononuclear cells, and that CFA-treated female NOD mice and male NOD mice may be protected from diabetes development by down-regulation of IFN-{gamma} production in the islets. 56 refs., 4 figs., 3 tabs.

  14. Outcomes of autologous bone marrow mononuclear cells for cerebral palsy: an open label uncontrolled clinical trial.

    Science.gov (United States)

    Nguyen, Liem Thanh; Nguyen, Anh Tuan; Vu, Chinh Duy; Ngo, Doan V; Bui, Anh V

    2017-04-12

    Stem cell therapy has emerged as a promising method for improving motor function of patients with cerebral palsy. The aim of this study is to assess the safety and effectiveness of autologous bone marrow mononuclear stem cell transplantation in patients with cerebral palsy related to oxygen deprivation. An open label uncontrolled clinical trial was carried out at Vinmec International Hospital. The intervention consisted of two administrations of stem cells, the first at baseline and the second 3 months later. Improvement was monitored at 3 months and 6 months after the first administration of stem cells, using the Gross Motor Function Measure (GMFM) and Modified Ashworth Score which measures muscle tone. No severe complications were recorded during the study. After transplantation, 12 patients encountered fever without infections and 9 patients experienced vomiting which was easily managed with medications. Gross motor function was markedly improved 3 months or 6 months after stem cell transplantation than at baseline. The post-transplantation GMFM-88 total score, each of its domains and the GMFM-66 percentile were all significantly higher (p-value  0.05). Autologous bone marrow mononuclear cell transplantation appears to be a safe and effective therapy for patients with cerebral palsy. ClinicalTrials.gov Identifier: NCT02569775 . Retrospectively registered on October 15, 2015.

  15. Proliferation and telomere length in acutely mobilized blood mononuclear cells in HIV infected patients

    DEFF Research Database (Denmark)

    Søndergaard, S R; Essen, M V; Schjerling, P

    2002-01-01

    The aim of the study was to investigate the mobilization of T cells in response to a stressful challenge (adrenalin stimulation), and to access T cells resided in the peripheral lymphoid organs in HIV infected patients. Seventeen patients and eight HIV seronegative controls received an adrenalin...... infusion for 1 h. Blood was sampled before, during and 1 h after adrenalin infusion. Proliferation and mean telomere restriction fragment length (telomeres) of blood mononuclear cells (BMNC) and purified CD8+ and CD4+ cells were investigated at all time points. In patients, the proliferation to pokeweed...... mitogens (PWM) was lower and decreased more during adrenalin infusion. After adrenalin infusion the proliferation to PWM was restored only in the controls. In all subjects telomeres in CD4+ cells declined during adrenalin infusion. Additionally, the patients had shortened telomeres in their CD8+ cells...

  16. Obesity alters the expression profile of clock genes in peripheral blood mononuclear cells

    Science.gov (United States)

    Tahira, Kazunobu; Fukuda, Noboru; Aoyama, Takahiko; Tsunemi, Akiko; Matsumoto, Siroh; Nagura, Chinami; Matsumoto, Taro; Soma, Masayoshi; Shimba, Shigeki; Matsumoto, Yoshiaki

    2011-01-01

    Introduction The aim of this study was to investigate the association between the variation in expression profile of clock genes and obesity using peripheral blood mononuclear (PMN) cells. Material and methods The subjects comprised 10 obese patients and 10 healthy volunteers. Blood was collected at different time-points during the day and levels of blood sugar, IRI, adiponectin and leptin were determined. Peripheral blood mononuclear cells were sampled, and expression levels of brain and muscle Arnt-like protein-1 (BMAL1), Period (PER)1, PER2, Cryptochrome (CRY)1, CRY2, and REV-ERBα mRNA were quantified. Results During the day, the expression levels of BMAL1, CRY1, CRY2 and PER2 genes in PMN cells of the obese group were all significantly higher compared to those in the non-obese group. In addition, expression of BMAL1, CRY1, CRY2 and PER2 genes in PMN cells increased between 12:00 and 21:00 in the obese group. In PMN cells of both groups, PER1 gene expression showed a bimodal pattern, with high expression at 9:00 and 18:00. Conclusions Differences were observed in the expression profile variation of clock genes between the obese and non-obese groups. This study reveals the differences in clock gene expression profiles between obese and non-obese subjects, with evidence for two distinct chronotypes, and suggests a contribution of these chronotypes to fat accumulation in humans. PMID:22328874

  17. Ethanol suppression of peripheral blood mononuclear cell trafficking across brain endothelial cells in immunodeficiency virus infection

    Directory of Open Access Journals (Sweden)

    Lola C Hudson

    2010-01-01

    Full Text Available Lola C Hudson1, Brenda A Colby1, Rick B Meeker21Department of Molecular Biosciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA; 2Department of Neurology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USAAbstract: Earlier studies suggested that the combination of alcohol use and immunodeficiency virus infection resulted in more severe neurologic disease than either condition individually. These deleterious interactions could be due to increased immune cell and virus trafficking or may result from interactions between ethanol and human immunodeficiency virus (HIV-associated toxicity within the brain. To determine the extent to which increased trafficking played a role, we examined the effect of ethanol on the migration of different peripheral blood mononuclear cell (PBMCs subsets across a brain endothelial cell monolayer. We utilized combinations of feline brain endothelial cells with astrocytes, and/or microglia with either acute exposure to 0.08 g/dL ethanol, a combination of ethanol and feline immunodeficiency virus (FIV, or FIV alone. Adherence of PBMCs to endothelium was increased in all combinations of cells with the addition of ethanol. Despite increased PBMC adhesion with ethanol treatment, transmigration of B cells, monocytes, CD4 T cells and CD8 T cells was not increased and was actually decreased in the presence of astrocytes. Expression of three common adhesion molecules, intercellular adhesion molecule-1 (ICAM1, ICAM2, and vascular cell adhesion molecule, was unchanged or slightly decreased by ethanol. This indicated that although adherence is increased by ethanol it is not due to an increased expression of adhesion molecules. RANTES, MIP1α, MIP1β, and MCP-1 mRNA expression was also studied in brain endothelial cells, astrocytes and microglia by reverse transcriptase-polymerase chain reaction. Ethanol treatment of astrocytes resulted in modest changes of

  18. Prevention of diabetic microangiopathy by prophylactic transplant of mobilized peripheral blood mononuclear cells

    Institute of Scientific and Technical Information of China (English)

    Bin ZHOU; Xiao-cang CAO; Zhi-hong FANG; Cui-lin ZHENG; Zhi-bo HAN; He REN; Man-chiu POON; Zhong-chao HAN

    2007-01-01

    Aim: To investigate whether the prophylactic local delivery of mobilized periph-eral blood mononuclear cells (M-PBMNC) could prevent peripheral microangio-pathy in diabetic nude mice. Methods: Diabetic nude mice were induced with intraperitoneal injections of streptozotocin. With the time course of diabetes, we detected the capillary and arteriole density of mice adductor muscles by immuno-histopathy. In situ apoptosis was detected by using TdT-mediated dUTP nick end labeling (TUNEL) methods. M-PBMNC were labeled and locally delivered to the adductor muscles. Mononuclear cells were also isolated and cultured in vitro for the detection and counting of endothelial progenitor cells(EPC). Results: Rarefication of capillaries and arterioles, enhanced apoptosis in adductor muscles,and reduced circulating EPC in diabetic nude mice. Prophylactic local delivery of M-PBMNC halted the progression of microvascular rarefaction in hind-limb skel-etal muscles by inhibiting apoptosis. We detected the survival, migration and incorporation of transplanted M-PBMNC into the murine vasculature in vivo. In addition, more EPC were available from M-PBMNC than non-mobilized cells.Conclusion: These results suggested that the prophylactic local delivery of M-PBMNC may represent a novel approach for the treatment of microvascular complications in diabetics.

  19. High Insulin and Leptin Increase Resistin and Inflammatory Cytokine Production from Human Mononuclear Cells

    Directory of Open Access Journals (Sweden)

    Panayoula C. Tsiotra

    2013-01-01

    Full Text Available Resistin and the proinflammatory cytokines, such as TNF-α, IL-6, and IL-1β, produced by adipocytes, and macrophages, are considered to be important modulators of chronic inflammation contributing to the development of obesity and atherosclerosis. Human monocyte-enriched mononuclear cells, from ten healthy individuals, were exposed to high concentrations of insulin, leptin, and glucose (alone or in combination for 24 hours in vitro. Resistin, TNF-α, IL-6, and IL-1β production was examined and compared to that in untreated cells. High insulin and leptin concentrations significantly upregulated resistin and the cytokines. The subsequent addition of high glucose significantly upregulated resistin and TNF-α mRNA and protein secretion, while it did not have any effect on IL-6 or IL-1β production. By comparison, exposure to dexamethasone reduced TNF-α, IL-6, and IL-1β production, while at this time point it increased resistin protein secretion. These data suggest that the expression of resistin, TNF-α, IL-6, and IL-1β from human mononuclear cells, might be enhanced by the hyperinsulinemia and hyperleptinemia and possibly by the hyperglycemia in metabolic diseases as obesity, type 2 diabetes, and atherosclerosis. Therefore, the above increased production may contribute to detrimental effects of their increased adipocyte-derived circulating levels on systemic inflammation, insulin sensitivity, and endothelial function of these patients.

  20. Beryllium alters lipopolysaccharide-mediated intracellular phosphorylation and cytokine release in human peripheral blood mononuclear cells.

    Science.gov (United States)

    Silva, Shannon; Ganguly, Kumkum; Fresquez, Theresa M; Gupta, Goutam; McCleskey, T Mark; Chaudhary, Anu

    2009-12-01

    Beryllium exposure in susceptible individuals leads to the development of chronic beryllium disease, a lung disorder marked by release of inflammatory cytokine and granuloma formation. We have previously reported that beryllium induces an immune response even in blood mononuclear cells from healthy individuals. In this study, we investigate the effects of beryllium on lipopolysaccharide-mediated cytokine release in blood mononuclear and dendritic cells from healthy individuals. We found that in vitro treatment of beryllium sulfate inhibits the secretion of lipopolysaccharide-mediated interleukin 10, while the release of interleukin 1beta is enhanced. In addition, not all lipopolysaccharide-mediated responses are altered, as interleukin 6 release in unaffected upon beryllium treatment. Beryllium sulfate-treated cells show altered phosphotyrosine levels upon lipopolysaccharide stimulation. Significantly, beryllium inhibits the phosphorylation of signal transducer and activator of transducer 3, induced by lipopolysaccharide. Finally, inhibitors of phosphoinositide-3 kinase mimic the effects of beryllium in inhibition of interleukin 10 release, while they have no effect on interleukin 1beta secretion. This study strongly suggests that prior exposures to beryllium could alter host immune responses to bacterial infections in healthy individuals, by altering intracellular signaling.

  1. The Effects of Royal Jelly on In-Vitro Cytotoxicity of K562 Cells and Peripheral Blood Mononuclear Cells

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    SE Hosseini

    2014-02-01

    Full Text Available Abstract Background & aim: Royal jelly, secreted by worker bees, has different biological activities on cells and tissues. The aim of this study was to evaluate the effects of royal jelly on peripheral blood mononuclear cells and on the tumor category of K562 cell line. Methods: In the present experimental study, three subjects were selected separately with three repetitions. K562 (104 cells and PBMC (105 cells with different concentrations of royal jelly (5, 10, 25, 50 and 100 mg/ml were cultured under standard conditions for 48 and 72 h separately. The fatality rate on PBMC cells and K562 cancer cells was evaluated by using MTT (Tetrazolium Dye-Reduction Assay. The number of viable cells in PBMC that were exposed for 48 hours with Royal Jelly was evaluated by trypan blue staining. Data were analyzed by ANOVA. Results: The royal jelly had no cytotoxicity effect on PBMC cells but at concentration of 50 and 100 mg/mL the cytotoxicity effect were observed on k562 cells whereas, at 10 and 25 mg/ml the number of PBMC viable cells increased. Conclusion: Due to the lack of lethality of royal jelly on PBMC cells and PBMC cell viability and an increase in the fatality rate of cancer cells in the future, royal jelly can be used as a potential candidate for treatment of leukemia. Keywords: Royal jelly, K562, peripheral blood mononuclear cell

  2. Therapeutic angiogenesis in Buerger's disease (thromboangiitis obliterans) patients with critical limb ischemia by autologous transplantation of bone marrow mononuclear cells.

    Science.gov (United States)

    Motukuru, Vishnu; Suresh, Kalkunte R; Vivekanand, Vivekanand; Raj, Sumanth; Girija, K R

    2008-12-01

    Peripheral arterial disease is a significant problem worldwide. In developing countries such as India, the increased incidence of smoking and other forms of nicotine intake has resulted in a large proportion of young individuals with Buerger's disease. The results of surgical and endovascular treatment for this condition have not been very rewarding. Hence, we focused on providing alternative therapies. Neovascularization by autologous bone marrow mononuclear cell transplantation is being tried as an alternative therapeutic option. We have reviewed our series of patients who underwent autologous bone marrow mononuclear cell transplantation during the last 2 years. We enrolled 38 patients who were chosen to undergo autologous bone marrow mononuclear cell transplantation for nonreconstructible Buerger's disease. We injected the bone marrow mononuclear cells into the calf muscles of the affected limbs in 36 patients. We monitored ulcer healing, ankle-brachial index (ABI), and transcutaneous oximetry (TcPo(2)) level. No procedurally related complications occurred, although one injected sample of bone marrow aspirate later revealed infestation with Strongyloides stercoralis. Two patients were seropositive on the Venereal Disease Research Laboratory test and were not injected with the bone marrow mononuclear cells. Three patients (12%) underwent major amputations disease who have critical limb ischemia.

  3. Conversion of mononuclear cells from human umbilical cord blood into hepatocyte-like cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG Fang-ting; FANG Jia-zhi; YU Jie; WAN Hui-juan; YE Jing; LONG Xia; YIN Mei-jun; HUANG Chun-qiao

    2006-01-01

    Objective:To evaluate the differentiation of human umbilical cord blood cells into hepatocyte-like cells. Methods: Mononuclear cells (MNCs) derived from human umbilical cord blood were isolated using Ficoll. The experiment was derived into 3 categories: (1) MNCs co-cultured with 50 mg minced liver tissue separated by a trans-well membrane and then collected at 0 h,24 h,48 h and 72 h; (2) MNCs cultured along supplemented with 100 ml/L FBS, 100 μ/ml penicillin, 100 μg/ml streptomycin, 4. 7 μg/ml linoleic acid, 1×ITS, 10-4 mol/L L-Ascorbic acid 2-P and a combination of FGF4 (100 ng/ml) and HGF (20 ng/Ml). Cells were then collected at 0 d and 16 d to examine the expression profile of hepatocyte correlating markers; (3) 0.2-0.3 ml of MNCs with a cell density of 2×107/ml were transplanted into prepared recipient mice [n= 12, injected with 0.4 ml/kg (20%) CCl4 and 150 ng/kg 5-fluorouracil (5-Fu) prior the transplant 24 h and 48 h, respectively] via injection through tail vein. Mice were sacrificed 4 weeks after transplantation. The hepatocyte correlating mRNAs and proteins were determined by RTPCR, immunohistochemical analysis and immunoflurence technique. Results: (1) After 72 h, a number of glycogen positive stained cells were observed with MNCs co-cultured with damaged mouse liver tissues.The expression of hepatocyte markers, human albumin (ALB), α-fetal protein (AFP) and human GATA4 Mrna and proteins were detected by RT-PCR and immunohistochemistry as well. For the confirmation,the DNA sequencing of PCR products was performed. In control groups, MNCs co-cultured with normal mouse hepatocytes or MNCs cultured alone, all markers remained negative. (2) In growth factor supplemented culture system, MNCs developed into larger volume with richer cytoplasm and binucleation after 16 d. Positive expression of ALB, AFP, CK18 and CK19 Mrna were detected with RT-PCR, and ALB positive staining was observed by immunocytochemistry as well. In contrast, MNCs cultured without

  4. Autologous bone marrow mononuclear cell transplantation in Duchenne muscular dystrophy - a case report.

    Science.gov (United States)

    Sharma, Alok; Sane, Hemangi; Paranjape, Amruta; Bhagawanani, Khushboo; Gokulchandran, Nandini; Badhe, Prerna

    2014-01-01

    Male, 9 FINAL DIAGNOSIS: Duchenne muscular dystrophy Symptoms: Hyporeflexia • hypotonia • weaknes of lower limbs - Clinical Procedure: - Specialty: Neurology. Congenital defects/diseases. Duchenne muscular dystrophy (DMD) is a fatal, genetic, progressive, degenerating muscle disorder. Current treatment options are palliative. Newer options of cellular therapy promise to alter the disease process. Preclinical studies have successfully tested myogenic, neurogenic potential and dystrophin expression of bone marrow mononuclear cells. We treated a 9-year-old boy suffering from DMD with serial autologous bone marrow mononuclear cell transplantations followed by multidisciplinary rehabilitation. Brooke-Vignos score was 10 and he was wheelchair-bound. Over 36 months, gradual progressive improvement was noticed in muscle strength, ambulation with assistive devices, fine motor movements, Brooke-Vignos score, and functional independence measure score. Nine months after the transplantation, electromyography findings showed development of new normal motor unit potentials of the vastus medialis muscle. Magnetic resonance imaging scan of musculoskeletal systems showed no increase in fatty infiltration. This case report provides early investigative findings or the restorative effects of cellular therapy in DMD.

  5. Impact of fexofenadine, osthole and histamine on peripheral blood mononuclear cell proliferation and cytokine secretion.

    Science.gov (United States)

    Karolina Kordulewska, Natalia; Kostyra, Elżbieta; Matysiewicz, Michał; Cieślińska, Anna; Jarmołowska, Beata

    2015-08-15

    This paper compares results of peripheral blood mononuclear cell (PBMC) incubation with fexofenadine (FXF) and osthole. FXF is a third-generation antihistamine drug and osthole is assumed a natural antihistamine alternative. To our best knowledge, this is the first comparative study on FXF, osthole and histamine cytokine secretion and cytotoxicity in PBMC in vitro cultures using cell proliferation ELISA BrdU. The cultures were treated 12, 42, 48 and 72h with FXF and osthole at 150, 300 and 450ng/ml concentrations and histamine at 50, 100 and 200ng/ml. Our study results confirm that FXF, osthole and histamine exert no cytotoxic effect on PBMCs and that IL-6, IL-10 and TNF-α cytokine secretion following osthole cell stimulation was similar to that by FXF stimulation.This confirms our hypothesis that osthole is a natural histamine antagonist, and can therefore be beneficially applied in antihistamine treatment.

  6. PSP activates monocytes in resting human peripheral blood mononuclear cells: immunomodulatory implications for cancer treatment.

    Science.gov (United States)

    Sekhon, Bhagwant Kaur; Sze, Daniel Man-Yuen; Chan, Wing Keung; Fan, Kei; Li, George Qian; Moore, Douglas Edwin; Roubin, Rebecca Heidi

    2013-06-15

    Polysaccharopeptide (PSP), from Coriolus versicolor, has been used as an adjuvant to chemotherapy, and has demonstrated anti-tumor and immunomodulating effects. However its mechanism remains unknown. To elucidate how PSP affects immune populations, we compared PSP treatments both with and without prior incubation in phytohaemagglutinin (PHA) - a process commonly used in immune population experimentation. We first standardised a capillary electrophoresis fingerprinting technique for PSP identification and characterisation. We then established the proliferative capability of PSP on various immune populations in peripheral blood mononuclear cells, using flow cytometry, without prior PHA treatment. It was found that PSP significantly increased the number of monocytes (CD14(+)/CD16(-)) compared to controls without PHA. This increase in monocytes was confirmed using another antibody panel of CD14 and MHCII. In contrast, proliferations of T-cells, NK, and B-cells were not significantly changed by PSP. Thus, stimulating monocyte/macrophage function with PSP could be an effective therapeutic intervention in targeting tumors.

  7. Dietary exposure to benzoxazinoids enhances bacteria-induced monokine responses by peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Damgaard, Dres; Jensen, Bettina Margrethe; Palarasah, Yaseelan

    2015-01-01

    -out, the groups switched diets. Peripheral blood mononuclear cells (PBMCs) were stimulated with Porphyromonas gingivalis, Escherichia coli lipopolysaccharide (LPS), or tetanus toxoid (TT). PBMCs from a healthy donor received the same stimuli in presence of serum from each participant receiving BXs. The production...... of monokines, T-cell cytokines and T-helper cell proliferation were assessed. A 3-wk diet with high BX content enhanced IL-1β responses against LPS and P. gingivalis, as well as TNF-α response against P. gingivalis, after 24 h of stimulation. Moreover, IL-6 was found to be increased after 7 days of stimulation......SCOPE: To examine potentially immunomodulating effects of dietary benzoxazinoids (BXs), present in cereal grains. METHODS AND RESULTS: Nineteen healthy volunteers were randomly distributed into two groups, who received diets with high or low content of BXs for 3 wk. After a week's wash...

  8. Lipopolysaccharide and silica-stimulated mononuclear cell prostaglandin production in ulcerative colitis

    Directory of Open Access Journals (Sweden)

    Neville A. Punchard

    2000-01-01

    Full Text Available Basal, lipopolysaccharide (LPS and silica-stimulated prostaglandin (PG production were compared between peripheral blood mononuclear cells (PBMNC from UC patients and healthy subjects (HS. Basal and LPS-stimulated PBMNC PGI2, but not PGE2, production was greater in UC. LPS stimulated both PGE2 and PGI2 by PBMNC from HS and UC patients. Silica stimulated production of both PGs by cells from HS but only PGE2 by cells from UC patients. The differences in responses to silica and LPS may result from differences in activation of NFκB or, alternatively, prior sensitisation to one of these agents. That PBMNC PGE2 production is not increased in UC, as it is in Crohn’s disease, suggests that there are differences in PBMNC behaviour between these two diseases.

  9. Production of cytokines by mononuclear cells of hypertrophic adenoids in children with otitis media with effusion.

    Science.gov (United States)

    Zelazowska-Rutkowska, Beata; Ilendo, Elzbieta; Skotnicka, Bozena; Wysocka, Jolanta; Kasprzycka, Edwina

    2012-01-01

    Hypertrophic adenoids with otitis media with effusion is a common infectious disease and present a serious otological problem in children. Cytokines, potent inflammatory mediators, play important role in the initiation of immunological response in otitis media. Adenoids excised due to hypertrophy with or without chronic otitis media with effusion were used to isolate mononuclear cells. Secretion of cytokines by non-stimulated and PHA-stimulated cells was determined by specific ELISAs. We found a significant increase in the production of IL-5 and TNF-α secreted by adenoidal cells of children with otitis media with effusion compared to group with hypertrophic adenoids. No differences were found in the secretion of IL-8, IL-6, and IL-10 between these two groups of patients. Our results suggest a difference between the immunological responses in the course of hypertrophic adenoids with otitis media as compared to hypertrophic adenoids.

  10. Transplantation of autologous bone marrow mononuclear cells for patients with lower limb ischemia

    Institute of Scientific and Technical Information of China (English)

    GU Yong-quan; LI Xue-feng; YU Heng-xi; CUI Shi-jun; WANG Zhong-gao; ZHANG Jian; GUO Lian-rui; QI Li-xing; ZHANG Shu-wen; XU Juan; LI Jian-xin; LUO Tao; JI Bing-xin

    2008-01-01

    Background Many treatment options for lower limb ischemia are difficult to apply for the patients with poor arterial outflow or with poor general conditions.The effect of medical treatment alone is far from ideal.especially in patients with diabetic foot.A high level amputation is inevitable in these patients.This study aimed to explore the effect of transplantation of autologous bone marrow mononuclear cells on the treatment of lower limb ischemia and to compare the effect of intra-artedal transplantation with that of intra-muscular transplantation.Methods In this clinical trial,32 patients with lower limb ischemia were divided into two groups.Group 1 (16 patients with 18 affected limbs) received transplantation of autologous bone marrow mononuclear cells by intra-muscular injection into the affected limbs;and group 2(16 patients with 17 affected limbs)received transplantation of autologous bone marrow mononucJear cells by intra-arterial injection into the affected limbs.Rest pain,coldness,ankle/brachial index (ABI),claudication,transcutaneous oxygen pressure(tcPO2)and angiography(15 limbs of 14 patients)were evaluated before and after the mononuclear cell transplantation to determine the effect of the treatment.Results Two patients died from heart failure.The improvement of rest pain was seen in 76.5%(13/17)of group 1 and 93.3%(14/15)of group 2.The improvement of coldness was 100%in both groups.The increase of ABI was 44.4%(8/18)in group 1 and 41.2%(7,17)in group 2.The value of tcPO2 increased to 20 mmHg or more in 20 limbs.Nine of 15 limbs which underwent angiography showed rich collaterals.Limb salvage rate was 83.3%(15,18)in group 1 and 94.1%(16/17)in group 2.There was no statistically significant difference in the effectiveness of the treatment between the two groups.Conclusions Transplantation of autologous bone marrow mononucJear cells is a simple,safe and effective method for the treatment of lower limb ischemia,and the two approaches for the implantation

  11. [Preliminary study on autologous bone marrow mononuclear cells transplantation for lower limb chronic venous ulcer].

    Science.gov (United States)

    Huang, Wen; Wang, Liwei; Tan, Bin; Zhang, Guozhen; Zhao, Yu; Ren, Guosheng

    2011-05-01

    To investigate the effectiveness of autologous bone marrow mononuclear cells transplantation on lower limb chronic venous ulcer. Between May 2009 and September 2010, 17 patients with lower limb chronic venous ulcer were treated with autologous bone marrow mononuclear cells transplantation (transplantation group) and 10 patients treated without cells transplantation served as control group. In the transplantation group, there were 9 males and 8 females with age of (33.3 +/- 6.1) years, including 11 cases of simple great saphenous vein varicosity and 6 cases of chronic venous insufficiency; the area of ulcer was (4.39 +/- 2.46) cm2; and the duration of ulcer ranged from 3 months to 6 years. In the control group, there were 4 males and 6 females with age of (39.2 +/- 10.3) years, including 7 cases of simple great saphenous vein varicosity and 3 cases of chronic venous insufficiency; and the area of ulcer was (5.51 +/- 2.63) cm2; and the duration of ulcer ranged from 3 months to 2 years. All patients in both groups were classified as C6 according to clinical etiology anatomy pathophysiology (CEAP) classification. No significant difference was found in the general data between 2 groups (P > 0.05). The healing process of ulcer was observed. The granulation tissue was harvested for HE staining before operation and at 3 days after operation in the transplantation group. The microvessel density (MVD) and vascular endothelial growth factor (VEGF) expression of ulcer granulation tissue were observed. In the transplantation group, ulcer healing was accelerated; complete healing was observed in 15 cases, partial healing in 1 case, and no healing in 1 case with the median healing time of 22 days. However, in the control group, the healing process was slower; complete healing of ulcer was observed in 7 cases and no healing in 3 cases with the median healing time of 57.5 days. There was significant difference in the healing time between 2 groups (Z = 0.001 4, P = 0.0027). HE

  12. Avidin inhibits PHA-induced human peripheral blood mononuclear cell proliferation

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    Cicia Firakania

    2016-04-01

    Full Text Available Background: Cell proliferation occurs not only in normal but also in cancer cells. Most of cell proliferation inhibition can be done by inhibiting the DNA synthesis, notably by intervening the formation of purine or pyrimidine. In purine de novo synthesis, it was assumed that biotin plays a role as a coenzyme in carboxylation reaction, one of the pivotal steps in the purine de novo pathways. The aim of this study was to see the avidin potency to bind biotin and inhibit mitosis.Methods: Peripheral blood mononuclear cell (PBMC was cultured in RPMI-1640 medium and stimulated by phytohemagglutinin (PHA in the presence or absence of interleukin-2 (IL-2, with or without avidin. The effect of avidin addition was observed at 24, 48, and 72 hours for cell proliferation, viability, and cell cycle. Statistical analysis was done by one-way ANOVA.Results: Avidin inhibited cell proliferation and viability in culture under stimulation by PHA with and without IL-2. Cell cycle analysis showed that avidin arrested the progression of PBMC after 72 hours of culture. Most cells were found in G0/G1 phase.Conclusion: Inhibition of biotin utilization by avidin binding can halt cell proliferation.

  13. Immune effects of cocoa procyanidin oligomers on peripheral blood mononuclear cells.

    Science.gov (United States)

    Kenny, Thomas P; Keen, Carl L; Schmitz, Harold H; Gershwin, M Eric

    2007-02-01

    There has been considerable work on the relationships between nutrition and the immune response, particularly on studies that have focused on adaptive responses. There is increasing recognition of the importance of innate immunity in host protection and initiation of cytokine networks. In this study, we examined the effect of select cocoa flavanols and procyanidins on innate responses in vitro. Peripheral blood mono-nuclear cells (PBMCs), as well as purified monocytes and CD4 and CD8 T cells, were isolated from healthy volunteers and cultured in the presence of cocoa flavanol fractions that differ from another by the degree of flavanol polymerization: short-chain flavanol fraction (SCFF), monomers to pentamers; and long-chain flavanol fraction (LCFF), hexamers to decamers. Parallel investigations were also done with highly purified flavanol monomers and procyanidin dimers. The isolated cells were then challenged with lipopolysaccharide (LPS) with quantitation of activation using CD69 and CD83 expression and analysis of secreted tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, IL-10, and granulocyte macrophage colony-stimulating factor (GM-CSF). The chain length of flavanol fractions had a significant effect on cytokine release from both unstimulated and LPS-stimulated PBMCs. For example, there was a striking increase of LPS-induced synthesis of IL-1beta, IL-6, IL-10, and TNF-alpha in the presence of LCFF. LCFF and SCFF, in the absence of LPS, stimulated the production of GM-CSF. In addition, LCFF and SCFF increased expression of the B cell markers CD69 and CD83. There were also unique differential responses in the mononuclear cell populations studied. We conclude that the oligomers are potent stimulators of both the innate immune system and early events in adaptive immunity.

  14. Human umbilical cord blood mononuclear cell transplantation for delayed encephalopathy after carbon monoxide intoxication

    Directory of Open Access Journals (Sweden)

    Gong D

    2013-08-01

    Full Text Available Dianrong Gong,1 Haiyan Yu,1 Weihua Wang,2 Haixin Yang,1 Fabin Han1,21Department of Neurology, 2Centre for Stem Cells and Regenerative Medicine, Liaocheng People's Hospital, The Affiliated Liaocheng Hospital, Taishan Medical University, Shandong, People's Republic of ChinaAbstract: Stem cell transplantation is one of the potential treatments for neurological disorders. Since human umbilical cord stem cells have been shown to provide neuroprotection and promote neural regeneration, we have attempted to transplant the human umbilical cord blood mononuclear cells (hUCB-MNCs to treat patients with delayed encephalopathy after carbon monoxide intoxication (DEACOI. The hUCB-MNCs were isolated from fresh umbilical cord blood and were given to patients subarachnoidally. Physical examinations, mini-mental state examination scores, and computed tomography scans were used to evaluate the improvement of symptoms, signs, and pathological changes of the patient's brain before and after hUCB-MNC transplantation. A total of 12 patients with DEACOI were treated with hUCB-MNCs in this study. We found that most of the patients have shown significant improvements in movement, behavior, and cognitive function, and improved brain images in 1–4 months from the first transplantation of hUCB-MNCs. None of these patients have been observed to have any severe adverse effects. Our study suggests that the hUCB-MNC transplantation may be a safe and effective treatment for DEACOI. Further studies and clinical trials with more cases, using more systematic scoring methods, are needed to evaluate brain structural and functional improvements in patients with DEACOI after hUCB-MNC therapy.Keywords: human umbilical cord blood mononuclear cells, transplantation, delayed encephalopathy after carbon monoxide intoxication, MMSE

  15. Association of poly(ADP-ribose) polymerase activity in circulating mononuclear cells with myocardial dysfunction in patients with septic shock

    Institute of Scientific and Technical Information of China (English)

    Li Li; Hu Bangchuan; Gong Shijin; Yu Yihua; Dai Haiwen; Yan Jing

    2014-01-01

    Background Severe sepsis and septic shock are the leading causes of morbidity and mortality in hospitalized patients.This study aimed to investigate the association of poly(ADP-ribose) polymerase-1 (PARP-1) activity in circulating mononuclear cells with myocardial dysfunction in patients with septic shock.Methods A total of 64 patients with septic shock were divided into the survival group (n=41) and the nonsurvival group (n=23) according to mortality at 28 days after enrollments.PARP-1 activity in circulating mononuclear cells,brain natriuretic peptide,Acute Physiology and Chronic Health Evaluation Ⅱ score,the cardiac index (CI),the cardiac function index (CFI),global ejection fraction (GEF),and the left ventricular contractility index (dp/dt max) were measured after admission to the intensive care unit.Results PARP-1 activity in circulating mononuclear cells of nonsurvival patients with septic shock was significantly higher than that in survival patients.PARP-1 activity in circulating mononuclear cells was strongly,negatively correlated with the CI,the CFI,GEE and dp/dt max.Multiple Logistic regression analysis showed that PARP-1 activity in circulating mononuclear cells was an independent risk factor of myocardial dysfunction.The optimal cutoff point of PARP-1 activity for predicting 28-day mortality was 942 nmol/L with a sensibility of 78.2% and specificity of 65.1%.Conclusion PARP-1 activity in circulating mononuclear cells is significantly associated with myocardial dysfunction and may have prognostic value in patients with septic shock.

  16. DNA damage in peripheral blood mononuclear cells and neutrophils of dairy cows during the transition period

    Directory of Open Access Journals (Sweden)

    S. Oikawa

    2012-06-01

    Full Text Available This study was designed to investigate the apoptotic process in peripheral blood mononuclear cells (PBMC and polymorphonuclear neutrophil leukocytes (PMN in dairy cattle during the transition period. Blood samples were collected from 4 dairy cattle at 3 weeks before the expected parturition (wk -3, parturition (wk 0 and 3 weeks after parturition (wk +3. The DNA damage of PBMC and PMN was evaluated based on the comet assay using visual scoring (arbitrary units. Undamaged DNA remained within the core (score 0 and the broken DNA migrated from the core towards the anode forming the tail of a comet (scores 1-4. Significantly higher scores in PBMC at wk 0 and wk +3 were observed compared with those in PMN although there were no significant changes of scores in either cell type during the experimental period. It is suggested that the apoptotic rate of PBMC is accelerated compared with that of PMC during the transition period.

  17. Detection of Intracellular Factor VIII Protein in Peripheral Blood Mononuclear Cells by Flow Cytometry

    Directory of Open Access Journals (Sweden)

    Gouri Shankar Pandey

    2013-01-01

    Full Text Available Flow cytometry is widely used in cancer research for diagnosis, detection of minimal residual disease, as well as immune monitoring and profiling following immunotherapy. Detection of specific host proteins for diagnosis predominantly uses quantitative PCR and western blotting assays. In this study, we optimized a flow cytometry-based detection assay for Factor VIII protein in peripheral blood mononuclear cells (PBMCs. An indirect intracellular staining (ICS method was standardized using monoclonal antibodies to different domains of human Factor VIII protein. The FVIII protein expression level was estimated by calculating the mean and median fluorescence intensities (MFI values for each monoclonal antibody. ICS staining of transiently transfected cell lines supported the method's specificity. Intracellular FVIII protein expression was also detected by the monoclonal antibodies used in the study in PBMCs of five blood donors. In summary, our data suggest that intracellular FVIII detection in PBMCs of hemophilia A patients can be a rapid and reliable method to detect intracellular FVIII levels.

  18. A Mononuclear Mn(II) Pseudoclathrochelate Complex Studied by Multi-Frequency Electron-Paramagnetic-Resonance Spectroscopy.

    Science.gov (United States)

    Azarkh, Mykhailo; Penkova, Larysa V; Kats, Svitlana V; Varzatskii, Oleg A; Voloshin, Yan Z; Groenen, Edgar J J

    2014-03-06

    Knowledge of the correlation between structural and spectroscopic properties of transition-metal complexes is essential to deepen the understanding of their role in catalysis, molecular magnetism, and biological inorganic chemistry. It provides topological and, sometimes, functional insight with respect to the active site properties of metalloproteins. The electronic structure of a high-spin mononuclear Mn(II) pseudoclathrochelate complex has been investigated by electron-paramagnetic-resonance (EPR) spectroscopy at 9.5 and 275.7 GHz. A substantial, virtually axial zero-field splitting with D = -9.7 GHz (-0.32 cm(-1)) is found, which is the largest one reported to date for a Mn(II) complex with six nitrogen atoms in the first coordination sphere.

  19. Treatment of refractory cutaneous ulcers with mixed sheets consisting of peripheral blood mononuclear cells and fibroblasts

    Science.gov (United States)

    Ueno, Koji; Takeuchi, Yuriko; Samura, Makoto; Tanaka, Yuya; Nakamura, Tamami; Nishimoto, Arata; Murata, Tomoaki; Hosoyama, Tohru; Hamano, Kimikazu

    2016-01-01

    The purpose of this study was to confirm the therapeutic effects of mixed sheets consisting of peripheral blood mononuclear cells (PBMNCs) and fibroblasts on cutaneous skin ulcers. Vascular endothelial growth factor (VEGF) secretion in mixed cell sheets was much higher than in PBMNCs and fibroblasts. Concerning the mechanism, transforming growth factor beta 1 and platelet-derived growth factor BB secreted from PBMNCs enhanced VEGF production in fibroblasts. In wounds created on the backs of diabetic mice, the therapeutic effect of mixed cell sheets was similar to that of daily treatment with trafermin, a recombinant human basic fibroblast growth factor. Although abnormal granulation tissue and inflammatory cell infiltration were observed in trafermin-treated wounds, the transplantation of mixed cell sheets resulted in the natural anatomy of subcutaneous tissues. The expression patterns of identical wound-healing factors in wounds were different between mixed sheet-transfected and trafermin-treated animals. Because mixed cell sheets transplanted into full-thickness skin defects were eliminated in hosts by day 21 in syngeneic transplantation models, allogeneic transplantation was performed using mice with different genetic backgrounds. The wound-healing rates were similar between the mixed cell sheet and trafermin groups. Our data indicated that mixed cell sheets represent a promising therapeutic material for cutaneous ulcers. PMID:27329845

  20. Productive infection of human peripheral blood mononuclear cells by feline immunodeficiency virus: implications for vector development.

    Science.gov (United States)

    Johnston, J; Power, C

    1999-03-01

    Feline immunodeficiency virus (FIV) is a lentivirus causing immune suppression and neurological disease in cats. Like primate lentiviruses, FIV utilizes the chemokine receptor CXCR4 for infection. In addition, FIV gene expression has been demonstrated in immortalized human cell lines. To investigate the extent and mechanism by which FIV infected primary and immortalized human cell lines, we compared the infectivity of two FIV strains, V1CSF and Petaluma, after cell-free infection. FIV genome was detected in infected human peripheral blood mononuclear cells (PBMC) and macrophages at 21 and 14 days postinfection, respectively. Flow cytometry analysis of FIV-infected human PBMC indicated that antibodies to FIV p24 recognized 12% of the cells. Antibodies binding the CCR3 chemokine receptor maximally inhibited infection of human PBMC by both FIV strains compared to antibodies to CXCR4 or CCR5. Reverse transcriptase levels increased in FIV-infected human PBMC, with detection of viral titers of 10(1.3) to 10(2.1) 50% tissue culture infective doses/10(6) cells depending on the FIV strain examined. Cell death in human PBMC infected with either FIV strain was significantly elevated relative to uninfected control cultures. These findings indicate that FIV can productively infect primary human cell lines and that viral strain specificity should be considered in the development of an FIV vector for gene therapy.

  1. Analysis of quantum dot fluorescence stability in primary blood mononuclear cells.

    Science.gov (United States)

    Summers, Huw D; Holton, Mark D; Rees, Paul; Williams, Paul M; Thornton, Catherine A

    2010-10-01

    A quantitative assessment of fluorescence signal generation and persistence in blood cells, measured at multiple points over a time course, is presented. Quantum dots (QDs) are inorganic fluorophores that are photostable and nonmetabolized and so can provide quantitative measures of cell biology over multiple cell generations. However, if the potential of these nanoparticles for long-term reporting is to be realized, an understanding of the stability of their fluorescence in living cells is essential. CdTe/ZnS and CdSe/ZnS core/shell dots with peak emission wavelengths of 705 nm and 585 nm, respectively, were loaded, via endocytosis into mononuclear cells extracted from primary blood and flow cytometry used to measure the average fluorescence intensity per cell within populations >10⁴. Time-based study showed a saturation-limited uptake of QDs with a characteristic time of 20 min and a maximum fluorescence signal that is linearly proportional to dot solution concentration. The fluorescence signal decreases after attachment and internalization within cells and is accurately described by a biexponential decay with a rapid initial decay followed by a much slower signal loss with characteristic times of 435 and 7,000 min respectively. Comparison with control samples indicates that interaction with the culture media is a major contributory factor to the initial signal decay. These results provide phenomenological descriptions of the evolving QD fluorescence within live cells with associated analytical equations that allow quantitative assessment of QD-based assays.

  2. Medroxyprogesterone acetate increases HIV-1 infection of unstimulated peripheral blood mononuclear cells in vitro.

    Science.gov (United States)

    Sampah, Maame Efua S; Laird, Gregory M; Blankson, Joel N; Siliciano, Robert F; Coleman, Jenell S

    2015-06-19

    Several observational studies suggest that medroxyprogesterone acetate (MPA) injectable contraceptives may increase a woman's risk of sexual HIV-1 acquisition. In-vitro studies are conflicting, mainly due to differences in the type of progestin studied or activation status of the primary cells. We sought to determine whether MPA increases infection of unstimulated peripheral blood mononuclear cells (PBMCs). Freshly isolated PBMCs from normal blood donors were treated with physiologic MPA concentrations ranging from 0.003 to 5 ng/ml and infected with GFP-tagged R5-tropic or X4-tropic HIV-1 pseudoviruses by spinoculation. The infection was limited to a single cycle. Cells were stained with CD3, CD8 and CD14. Infection was quantified as the percentage of GFP cells by flow cytometry. Absolute infection was greater among unstimulated MPA-treated CD3⁺CD8⁻ T cells vs. untreated cells across MPA concentrations of 0.003-3 ng/ml using R5 (P  0.5). The CD3⁺CD8⁻ T-cell population of MPA-treated unstimulated PBMCs were more susceptible to HIV-1 infection than untreated cells. The increased infection was partly due to monocytes and was lost when PBMC were exogenously stimulated. These data provide confirmation of a biological association between MPA exposure and increased susceptibility to HIV-1 infection, particularly among women who inject drugs.

  3. The photodynamic effect of Victoria blue BO on peripheral blood mononuclear and leukemic cells

    Energy Technology Data Exchange (ETDEWEB)

    Fiedorowicz, M. [Hugo Kollatay Univ. of Agriculture, Krakow (Poland); Pituch-Noworolska, A.; Zembala, M. [Polish-American Children`s Hospital, Krakow (Poland). Dept. of Clinical Immunology

    1997-05-01

    The photodynamic effect of Victoria blue BO (VB-BO) and photoirradiation on peripheral blood mononuclear cells was studied. The cells were preincubated with VB-BO followed by photoirradiation and overnight culture. The highest percentage of dead cells (propidium iodide assay in flow cyctometry) was seen in the monocyte population. The lymphocytes showed a lower sensitivity to VB-BO photodynamic action than the monocytes (12% vs 80% of PI-positive cells). The effect of VB-BO and phototreatment on lymphocyte function was studied using a mitogen-induced proliferation assay. A decrease of mitogen response was observed. The VB-BO and photoirradiation were also used on leukemic cells. The leukemic cells from acute myeloid leukemia and B precursors leukemia were sensitive to VB-BO photodynamic action. The high VB-BO sensitivity of monocytes and leukemic cells (myeloid and lymphoid B derived) suggests possible application of VB-BO for selective depletion of monocytes or sensitive leukemic cells. (author).

  4. Do androgen deprivation drugs affect the immune cross-talk between mononuclear and prostate cancer cells?

    Science.gov (United States)

    Salman, Hertzel; Bergman, Michael; Blumberger, Naava; Djaldetti, Meir; Bessler, Hanna

    2014-02-01

    The aim of the study was to examine the effect of androgen deprivation drugs, i.e. leuprolide and bicalutamide on the immune cross-talk between human peripheral blood mononuclear cells (PBMC) and cells from PC-3 and LNCaP human prostate cancer lines. PBMC, PC-3 and LNCaP were separately incubated without and with two androgen-deprivation drugs, i.e. leuprolide and bicalutamide, and the secretion of IL-1β, IL-6, IL-1ra and IL-10 was examined. In addition, the effect of both drugs on the production of those cytokines was carried out after 24 hours incubation of PBMC with both types of cancer cells. Leuprolide or bicalutamide did not affect the production of the cytokines by PBMC or by the prostate cancer cells from the two lines. Incubation of PBMC with PC-3 or LNCaP cells caused increased production of IL-1β, IL-6 and IL-10 as compared with PBMC incubated without malignant cells. While 10(-7) M and 10(-8) M of leuprolide caused a decreased secretion of IL-1β by PBMC previously incubated with prostate cancer cells without the drug, bicalutamide did not affect this PBMC activity at any drug concentration. This observation suggests the existence of an additional mechanism explaining the effect of androgen deprivation therapy in prostate cancer patients.

  5. Binding of toxic-shock-syndrome toxin-1 to human peripheral blood mononuclear cells

    Energy Technology Data Exchange (ETDEWEB)

    Poindexter, N.J.; Schlievert, P.M.

    1987-07-01

    Toxic-shock-syndrome toxin-1 (TSST-1), produced by Staphylococcus aureus and associated with toxic shock syndrome, functions in vitro as both a lymphoproliferative and immunosuppressive protein for human peripheral blood mononuclear cells (PBMs). We analyzed TSST-1-target cell interactions by receptor-ligand binding analyses. In competitive binding experiments, 2 X 10(5) human PBMs or purified cell populations were incubated in the presence of small amounts of (5-50 ng) of /sup 125/I-labeled TSST-1 and increasing amounts of unlabeled TSST-1 (25-10,000 ng). Data were analyzed by the method of Scatchard. Toxin-specific receptors were shown to exist on T lymphocytes within the PBM population. T4+ cells had 27.5 X 10(6) receptors per cell, and T8+ cells had 9 X 10(6) receptors per cell. T4+ and T8+ receptors had dissociation constants of 2.58 X 10(-8) M and 1.8 X 10(-8) M, respectively. These studies confirm earlier work showing that TSST-1 causes the functional activation of a population of T lymphocytes involved in suppression of immunoglobulin responses.

  6. Chemokine receptor expression on the surface of peripheral blood mononuclear cells in Chagas disease.

    Science.gov (United States)

    Talvani, Andre; Rocha, Manoel O C; Ribeiro, Antonio L; Correa-Oliveira, Rodrigo; Teixeira, Mauro M

    2004-01-15

    We evaluated the expression of chemokine receptors (CCR1, CCR2, CCR5, and CXCR4) on the surface of peripheral blood mononuclear cells obtained from patients with chronic chagasic cardiomyopathy (CCC) and noninfected individuals. Only CCR5 and CXCR4 expression was different on the surface of the subsets (CD4, CD8, and CD14) evaluated. Patients with mild CCC had elevated leukocyte expression of CCR5, compared with noninfected individuals or those with severe disease. CXCR4 expression was lower on leukocytes from patients with severe CCC. The differential expression of both receptors on leukocytes of patients with CCC was consistent and clearly correlated with the degree of heart function such that the lower the heart function, the lower the expression of either CCR5 or CXCR4. These results highlight the possible participation of the chemokine system in early forms of chagasic cardiomyopathy and the relevance of heart failure-induced remodeling in modifying immune parameters in infected individuals.

  7. Inverse relationship of tumors and mononuclear cell leukemia infiltration in the lungs of F344 rats

    Energy Technology Data Exchange (ETDEWEB)

    Lundgren, D.L.; Griffith, W.C.; Hahn, F.F.

    1995-12-01

    In 1970 and F344 rat, along with the B6C3F{sub 1} mouse, were selected as the standard rodents for the National Cancer Institute Carcinogenic Bioassay program for studies of potentially carcinogenic chemicals. The F344 rat has also been used in a variety of other carcinogenesis studies, including numerous studies at ITRI. A major concern to be considered in evaluating carcinogenic bioassay studies using the F344 rat is the relatively high background incidence of mononuclear cell leukemia (MCL) (also referred to as large granular lymphocytic leukemia, Fischer rat leukemia, or monocytic leukemia). Incidences of MCL ranging from 10 to 72% in male F344 rats to 6 to 31% in female F344 rats have been reported. Gaining the understanding of the mechanisms involved in the negative correlations noted should enhance our understanding of the mechanisms involved in the development of lung cancer.

  8. Spironolactone induces apoptosis in human mononuclear cells. Association between apoptosis and cytokine suppression

    DEFF Research Database (Denmark)

    Mikkelsen, Martin; Sønder, S U; Nersting, J;

    2006-01-01

    Spironolactone (SPIR) has been described to suppress accumulation of pro-inflammatory cytokines. Here, the suppression of TNF-alpha in lipopolysaccharide (LPS)-stimulated mononuclear cell cultures was confirmed. However, SPIR was also found to induce apoptosis, prompting the investigations...... of a possible association between the two effects: The apoptosis-inducing and the cytokine-suppressive effects of SPIR correlated with regard to the effective concentration range. Also, pre-incubation experiments demonstrated a temporal separation of the two effects of ... preceding apoptosis. An association between the two effects was also seen when testing several SPIR analogues. Contrary to TNF-alpha, the levels of IL-1beta increased in SPIR-treated cultures. However, the amount of IL-1beta in the supernatants depended upon the order of SPIR and LPS addition, as IL-1beta...

  9. [Research advances on DNA extraction methods from peripheral blood mononuclear cells].

    Science.gov (United States)

    Wang, Xiao-Ying; Yu, Chen-Xi

    2014-10-01

    DNA extraction is a basic technology of molecular biology. The purity and the integrality of DNA structure are necessary for different experiments of gene engineering. As commonly used materials in the clinical detection, the fast, efficient isolation and extraction of genomic DNA from peripheral blood mononuclear cells is very important for the inspection and analysis of clinical blood. At present, there are many methods for extracting DNA, such as phenol-chloroform method, salting out method, centrifugal adsorption column chromatography method (artificial methods), magnetic beads (semi-automatic method) and DNA extraction kit. In this article, a brief review of the principle for existing DNA blood extraction method, the specific steps and the assessment of the specific methods briefly are summarized.

  10. Spironolactone induces apoptosis in human mononuclear cells. Association between apoptosis and cytokine suppression

    DEFF Research Database (Denmark)

    Mikkelsen, Martin; Sønder, S U; Nersting, J;

    2006-01-01

    Spironolactone (SPIR) has been described to suppress accumulation of pro-inflammatory cytokines. Here, the suppression of TNF-alpha in lipopolysaccharide (LPS)-stimulated mononuclear cell cultures was confirmed. However, SPIR was also found to induce apoptosis, prompting the investigations...... of a possible association between the two effects: The apoptosis-inducing and the cytokine-suppressive effects of SPIR correlated with regard to the effective concentration range. Also, pre-incubation experiments demonstrated a temporal separation of the two effects of TNF-alpha suppression...... preceding apoptosis. An association between the two effects was also seen when testing several SPIR analogues. Contrary to TNF-alpha, the levels of IL-1beta increased in SPIR-treated cultures. However, the amount of IL-1beta in the supernatants depended upon the order of SPIR and LPS addition, as IL-1beta...

  11. Activation-induced apoptosis in peripheral blood mononuclear cells during hepatosplenic Schistosoma mansoni infections.

    Science.gov (United States)

    Ghoneim, H M; Demian, S R; Heshmat, M G; Ismail, N S; El-Sayed, Laila H

    2008-01-01

    It is well established that programmed cell death (apoptosis) is an important regulator of host responses during infection with a variety of intra- and extra-cellular pathogens. The present work aimed at assessment of in vitro spontaneous and phytohemagglutinin (PHA)-induced apoptosis in mononuclear cells isolated from patients with hepatosplenic form of S. mansoni infections. Cell death data were correlated to the degree of lymphoproliferative responses to PHA as well as to the serum anti-schistosomal antibody titers. A markedly significant increase in PHA-induced apoptosis in lymphocytes isolated from S. mansoni-infected patients was seen when compared to the corresponding healthy controls. However, a slight difference was recorded between the two studied groups regarding the spontaneous apoptosis. This was accompanied with a significant impairment of in vitro PHA-induced lymphoproliferation of T cells from S. mansoni patients. Data of the present study supports the hypothesis that activation-induced cell death (AICD) is a potentially contributing factor in T helper (Th) cell regulation during chronic stages of schistosomiasis, which represents a critically determinant factor in the host-parasite interaction and might influence the destiny of parasitic infections either towards establishment of chronic infection or towards host death.

  12. Peripheral blood mononuclear cell gene expression in healthy adults rapidly transported to high altitude

    Directory of Open Access Journals (Sweden)

    Herman NM

    2014-12-01

    Full Text Available Nicole M Herman,1 Diane E Grill,2 Paul J Anderson,1 Andrew D Miller,1 Jacob B Johnson,1 Kathy A O’Malley,1 Maile L Ceridon Richert,1 Bruce D Johnson1 1Department of Cardiovascular Diseases, 2Department of Biostatistics, Mayo Clinic Rochester, MN, USA Abstract: Although mechanisms of high altitude illness have been studied extensively, the processes behind the development of these conditions are still unclear. Few genome-wide studies on rapid exposure to high altitude have been performed. Each year, scientists and support workers are transferred by plane from McMurdo Station in Antarctica (sea level to the Amundsen-Scott South Pole Station at 2,835 meters. This uniform and rapid transfer to altitude provides a unique opportunity to study the effects of hypobaric hypoxia on gene expression that may help illustrate the body's adaptations to these conditions. We hypothesized that an extensive number of genes would change with rapid exposure to altitude and further expected that these genes would correspond to inflammatory pathways proposed as a mechanism in development of acute mountain sickness. Peripheral venous blood samples were drawn from 98 healthy subjects at sea level and again on day two at altitude. Microarray analysis was performed on these samples. In total, 1,118 probe sets with significant P-values and fold changes (90% upregulated were identified and entered into MetaCore™ software. Several pathways, including oxidative phosphorylation, cytoskeleton remodeling, and platelet aggregation, were significantly represented by the data set and all were upregulated. Many genes changed expression, and the vast majority of these increased. Increased metabolism in peripheral blood mononuclear cells suggests increased inflammatory activity. Keywords: peripheral blood mononuclear cells, microarray, gene expression, acute mountain sickness

  13. Cord blood versus age 5 mononuclear cell proliferation on IgE and asthma

    Directory of Open Access Journals (Sweden)

    Perera Frederica

    2010-08-01

    Full Text Available Abstract Background Fetal immune responses following exposure of mothers to allergens during pregnancy may influence the subsequent risk of childhood asthma. However, the association of allergen-induced cord blood mononuclear cell (CBMC proliferation and cytokine production with later allergic immune responses and asthma has been controversial. Our objective was to compare indoor allergen-induced CBMC with age 5 peripheral blood mononuclear cell (PBMC proliferation and determine which may be associated with age 5 allergic immune responses and asthma in an inner city cohort. Methods As part of an ongoing cohort study of the Columbia Center for Children's Environmental Health (CCCEH, CBMCs and age 5 PBMCs were cultured with cockroach, mouse, and dust mite protein extracts. CBMC proliferation and cytokine (IL-5 and IFN-γ responses, and age 5 PBMC proliferation responses, were compared to anti-cockroach, anti-mouse, and anti-dust mite IgE levels, wheeze, cough, eczema and asthma. Results Correlations between CBMC and age 5 PBMC proliferation in response to cockroach, mouse, and dust mite antigens were nonsignificant. Cockroach-, mouse-, and dust mite-induced CBMC proliferation and cytokine responses were not associated with allergen-specific IgE at ages 2, 3, and 5, or with asthma and eczema at age 5. However, after adjusting for potential confounders, age 5 cockroach-induced PBMC proliferation was associated with anti-cockroach IgE, total IgE, and asthma (p Conclusion In contrast to allergen-induced CBMC proliferation, age 5 cockroach-induced PBMC proliferation was associated with age 5 specific and total IgE, and asthma, in an inner-city cohort where cockroach allergens are prevalent and exposure can be high.

  14. Repetitive cryotherapy attenuates the in vitro and in vivo mononuclear cell activation response.

    Science.gov (United States)

    Lindsay, Angus; Othman, Mohd Izani; Prebble, Hannah; Davies, Sian; Gieseg, Steven P

    2016-07-01

    What is the central question of this study? Acute and repetitive cryotherapy are routinely used to accelerate postexercise recovery, although the effect on resident immune cells and repetitive exposure has largely been unexplored and neglected. What is the main finding and its importance? Using blood-derived mononuclear cells and semi-professional mixed martial artists, we show that acute and repetitive cryotherapy reduces the in vitro and in vivo T-cell and monocyte activation response whilst remaining independent of the physical performance of elite athletes. We investigated the effect of repetitive cryotherapy on the in vitro (cold exposure) and in vivo (cold water immersion) activation of blood-derived mononuclear cells following high-intensity exercise. Single and repeated cold exposure (5°C) of a mixed cell culture (T cells and monocytes) was investigated using in vitro tissue culture experimentation for total neopterin production (neopterin plus 7,8-dihydroneopterin). Fourteen elite mixed martial art fighters were also randomly assigned to either a cold water immersion (15 min at 10°C) or passive recovery protocol, which they completed three times per week during a 6 week training camp. Urine was collected and analysed for neopterin and total neopterin three times per week, and perceived soreness, fatigue, physical performance (broad jump, push-ups and pull-ups) and training performance were also assessed. Single and repetitive cold exposure significantly (P mixed cell culture, whereas cold water immersion significantly (P < 0.05) attenuated urinary neopterin and total neopterin during the training camp without having any effect on physical performance parameters. Soreness and fatigue showed little variation between the groups, whereas training session performance was significantly (P < 0.05) elevated in the cold water immersion group. The data suggest that acute and repetitive cryotherapy attenuates in vitro T-cell and monocyte activation. This

  15. Characterization of small, mononuclear blood cells from salmon having high phagocytic capacity and ability to differentiate into dendritic like cells.

    Directory of Open Access Journals (Sweden)

    Gyri T Haugland

    Full Text Available Phagocytes are the principal component of the innate immune system, playing a key role in the clearance of foreign particles that include potential pathogens. In vertebrates, both neutrophils and mononuclear cells like monocytes, macrophages and dendritic cells are all professional phagocytes. In teleosts, B-lymphocytes also have potent phagocytic ability. We have isolated a population of small (<5 µm, mononuclear blood cells from Atlantic salmon (Salmo salar L. not previously characterized. In order to identify them, we have performed morphological, gene expression, flow cytometry, cytochemical, ultrastructural and functional analyses. Interestingly, they highly express the gene encoding CD83, the most characteristic cell surface marker for dendritic cells in mammals, and MHC class II limited to professional antigen presenting cells. They did not express genes nor did they have cell markers for B-cells, T-cells, monocytes/macrophages or neutrophils as shown by qRT-PCR, flow cytometry and immunoblotting. A remarkable feature of these cells is their potent phagocytic capacity. Their oxygen-independent killing mechanism, as shown by intense acid phosphatase staining, is supported by lack of respiratory burst and myeloperoxidase activity and the acid phosphatase's sensitivity to tartrate. They show a high level of morphological plasticity, as, upon stimulation with mitogens, they change morphology and obtain branching protrusions similarly to dendritic cells. We suggest, based on our findings, that the small, round cells described here are progenitor cells with potential to differentiate into dendritic like cells, although we can not exclude the possibility that they represent a novel cell type.

  16. Principles of bone marrow processing and progenitor cell/mononuclear cell concentrate collection in a continuous flow blood cell separation system.

    Science.gov (United States)

    Hester, J P; Rondón, G; Huh, Y O; Lauppe, M J; Champlin, R E; Deisseroth, A B

    1995-08-01

    The application of continuous flow apheresis technology to processing bone marrow for collection of the mononuclear progenitor cell population appears to follow the same principles as collection of mononuclear cells from peripheral blood. Unlike peripheral blood, however, where mobilization of cells from extravascular sites during the procedures contributes significantly to the final cell yield, the entire quantity of progenitor cells available for recovery from marrow is present in the original marrow when it is pooled. The process then becomes one of attempting optimal recovery of the cells of interest while excluding contaminating erythrocytes and cells of the myeloid series. This study reports the development of a protocol for recovery of MNC, CD33+, CD34+, and CD34+/DR- cells from harvested marrow for autologous and allogeneic transplants using a continuous flow blood cell separator, the variables influencing the recovery of the cells of interest and the clinical response to infusion of the processed cells.

  17. Generation of Patient-Specific induced Pluripotent Stem Cell from Peripheral Blood Mononuclear Cells by Sendai Reprogramming Vectors.

    Science.gov (United States)

    Quintana-Bustamante, Oscar; Segovia, Jose C

    2016-01-01

    Induced pluripotent stem cells (iPSC) technology has changed preclinical research since their generation was described by Shinya Yamanaka in 2006. iPSCs are derived from somatic cells after being reprogrammed back to an embryonic state by specific combination of reprogramming factors. These reprogrammed cells resemble all the characteristic of embryonic stem cells (ESC). The reprogramming technology is even more valuable to research diseases biology and treatment by opening gene and cell therapies in own patient's iPSC. Patient-specific iPSC can be generated from a large variety of patient cells by any of the myriad of reprogramming platforms described. Here, we describe the generation of patient-specific iPSC from patient peripheral blood mononuclear cells by Sendai Reprogramming vectors.

  18. One-stage focal cartilage defect treatment with bone marrow mononuclear cells and chondrocytes leads to better macroscopic cartilage regeneration compared to microfracture in goats

    NARCIS (Netherlands)

    Bekkers, J.E.J.; Creemers, L.B.; Tsuchida, A.I.; van Rijen, M.H.P.; Custers, R.J.H.; Dhert, W.J.A.; Saris, Daniël B.F.

    2013-01-01

    Objective The combination of chondrocytes and mononuclear fraction (MNF) cells might solve the expansion induced dedifferentiation problem of reimplanted cells in autologous chondrocytes implantation as sufficient cells would be available for direct, one-stage, implantation. Earlier in vitro work

  19. Controlled exposure to diesel exhaust and traffic noise - Effects on oxidative stress and activation in mononuclear blood cells

    DEFF Research Database (Denmark)

    Hemmingsen, Jette Gjerke; Møller, Peter; Jantzen, Kim

    2015-01-01

    unaltered in peripheral blood mononuclear cells (PBMCs). No significant differences in DNA damage levels, measured by the comet assay, were observed after DE exposure, whereas exposure to high noise levels was associated with significantly increased levels of hOGG1-sensitive sites in PBMCs. Urinary levels...

  20. Cpt1a gene expression in peripheral blood mononuclear cells as an early biomarker of diet-related metabolic alterations

    KAUST Repository

    Diaz-Rua, Ruben

    2016-11-23

    Background: Research on biomarkers that provide early information about the development of future metabolic alterations is an emerging discipline. Gene expression analysis in peripheral blood mononuclear cells (PBMC) is a promising tool to identify subjects at risk of developing diet-related diseases.

  1. Quantitative analysis of antiretroviral drugs in lysates of peripheral blood mononuclear cells using MALDI-triple quadrupole mass spectrometry.

    NARCIS (Netherlands)

    Kampen, JJ van; Burgers, P.C.; Gruters, R.A.; Osterhaus, A.D.; Groot, R. de; Luider, T.M.; Volmer, D.A.

    2008-01-01

    We report here on the use of a prototype matrix-assisted laser desorption/ionization (MALDI)-triple quadrupole mass spectrometer for quantitative analysis of six antiretroviral drugs in lysates of peripheral blood mononuclear cells (PBMC). Of the five investigated MALDI matrixes, 2,5-dihydroxybenzoi

  2. Distinct types of fibrocyte can differentiate from mononuclear cells in the presence and absence of serum.

    Directory of Open Access Journals (Sweden)

    S John Curnow

    Full Text Available BACKGROUND: Fibrocytes are bone-marrow derived cells, expressing both haematopoietic and stromal cell markers, which contribute to tissue repair as well as pathological fibrosis. The differentiation of fibrocytes remains poorly characterised and this has limited understanding of their biology and function. In particular two methods are used to generate fibrocytes in vitro that differ fundamentally by the presence or absence of serum. METHODOLOGY/PRINCIPAL FINDINGS: We show here that fibrocytes grown in the absence of serum (SF differentiate more efficiently from peripheral blood mononuclear cells than CD14(+ monocytes, and respond to serum by losing their spindle-shaped fibrocyte morphology. Although fibrocytes generated in the presence of serum (SC express the same range of markers, they differentiate more efficiently from CD14(+ monocytes and do not change their morphology in response to serum. Transcriptional analysis revealed that both types of fibrocyte are distinct from each other, fibroblasts and additional monocyte-derived progeny. The gene pathways that differ significantly between SF and SC fibrocytes include those involved in cell migration, immune responses and response to wounding. CONCLUSIONS/SIGNIFICANCE: These data show that SF and SC fibrocytes are distinct but related cell types, and suggest that they will play different roles during tissue repair and fibrosis where changes in serum proteins may occur.

  3. Autologous bone marrow mononuclear cells in ischemic cerebrovascular accident paves way for neurorestoration: a case report.

    Science.gov (United States)

    Sharma, Alok; Sane, Hemangi; Nagrajan, Anjana; Gokulchandran, Nandini; Badhe, Prerna; Paranjape, Amruta; Biju, Hema

    2014-01-01

    In response to acute ischemic stroke, large numbers of bone marrow stem cells mobilize spontaneously in peripheral blood that home onto the site of ischemia activating the penumbra. But with chronicity, the numbers of mobilized cells decrease, reducing the degree and rate of recovery. Cellular therapy has been explored as a new avenue to restore the repair process in the chronic stage. A 67-year-old Indian male with a chronic right middle cerebral artery ischemic stroke had residual left hemiparesis despite standard management. Recovery was slow and partial resulting in dependence to carry out activities of daily living. Our aim was to enhance the speed of recovery process by providing an increased number of stem cells to the site of injury. We administered autologous bone marrow mononuclear cells intrathecally alongwith rehabilitation and regular follow up. The striking fact was that the hand functions, which are the most challenging deficits, showed significant recovery. Functional Independence Measure scores and quality of life improved. This could be attributed to the neural tissue restoration. We hypothesize that cell therapy may be safe, novel and appealing treatment for chronic ischemic stroke. Further controlled trials are indicated to advance the concept of Neurorestoration.

  4. Autologous Bone Marrow Mononuclear Cells in Ischemic Cerebrovascular Accident Paves Way for Neurorestoration: A Case Report

    Directory of Open Access Journals (Sweden)

    Alok Sharma

    2014-01-01

    Full Text Available In response to acute ischemic stroke, large numbers of bone marrow stem cells mobilize spontaneously in peripheral blood that home onto the site of ischemia activating the penumbra. But with chronicity, the numbers of mobilized cells decrease, reducing the degree and rate of recovery. Cellular therapy has been explored as a new avenue to restore the repair process in the chronic stage. A 67-year-old Indian male with a chronic right middle cerebral artery ischemic stroke had residual left hemiparesis despite standard management. Recovery was slow and partial resulting in dependence to carry out activities of daily living. Our aim was to enhance the speed of recovery process by providing an increased number of stem cells to the site of injury. We administered autologous bone marrow mononuclear cells intrathecally alongwith rehabilitation and regular follow up. The striking fact was that the hand functions, which are the most challenging deficits, showed significant recovery. Functional Independence Measure scores and quality of life improved. This could be attributed to the neural tissue restoration. We hypothesize that cell therapy may be safe, novel and appealing treatment for chronic ischemic stroke. Further controlled trials are indicated to advance the concept of Neurorestoration.

  5. Effect of arsenic, cadmium and lead on the induction of apoptosis of normal human mononuclear cells

    Science.gov (United States)

    DE LA FUENTE, H; PORTALES-PÉREZ, D; BARANDA, L; DÍAZ-BARRIGA, F; SAAVEDRA-ALANÍS, V; LAYSECA, E; GONZÁLEZ-AMARO, R

    2002-01-01

    The aim of this work was to investigate the effect of cadmium, lead and arsenic on the apoptosis of human immune cells. Peripheral blood mononuclear cells (MNC) were incubated with increasing concentrations of these metals and then cellular apoptosis was determined by flow cytometry and by DNA electrophoresis. We found that arsenic induced a significant level of apoptosis at 15 μm after 48h of incubation. Cadmium had a similar effect, but at higher concentrations (65 μm). In addition, cadmium exerted a cytotoxic effect on MNC that seemed to be independent of the induction of apoptosis. In contrast, concentrations of lead as high as 500 μm were nontoxic and did not induce a significant degree of apoptosis. Additional experiments showed that arsenic at concentrations as low as 1·0 μm had a significant pro-apoptotic effect when cells were cultured in the presence of this pollutant for more than 72. Non-T cells were more susceptible than T lymphocytes to the effect of arsenic and cadmium. Interestingly, MNC from children chronically exposed to arsenic showed a high basal rate of apoptosis and a diminished in vitro sensibility to this metalloid. Our results indicate that both arsenic and cadmium are able to induce apoptosis of lymphoid cells, and suggest that this phenomenon may contribute to their immunotoxic effect in vivo. PMID:12100024

  6. Capsaicin Modulates the Immune Cross Talk Between Human Mononuclears and Cells from Two Colon Carcinoma Lines.

    Science.gov (United States)

    Bessler, Hanna; Djaldetti, Meir

    2017-01-01

    Capsaicin, the pungent alkaloid of the chili peppers, has gained a worldwide reputation. In addition to its culinary assets, capsaicin possesses analgesic, anti-inflammatory, antimicrobial, and even carcinopreventive properties. Considering the linkage between chronic inflammation and tumorigenesis, the aim of the study was to evaluate the role of capsaicin in the immune interplay between human peripheral blood mononuclear cells (PBMCs) and HT-29 or RKO cells from human colon carcinoma lines. PBMCs were incubated for 24 hours with either HT-29 or RKO cells and concentrations of capsaicin ranging between 10 and 200 µM. Subsequently, the generation of the following cytokines was examined: tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, interferon (IFN)-γ, IL-1ra, and IL-10. Capsaicin caused a concentration-dependent inhibition of colon cancer cells proliferation but had no effect on PBMC viability. 200 µM of capsaicin suppressed the production of all cytokines tested. At lower concentrations, the secretion of TNF-α, IL-1β, IFN-γ, IL-10, and IL-1ra was inhibited concentration-dependently, whereas that of IL-6 was stimulated. Capsaicin causes a concentration-dependent alteration of the immune balance between PBMC and colon carcinoma cells expressed as an inhibited generation of inflammatory cytokines. These findings indicate the existence of an additional immunomodulatory mechanism by which this alkaloid may prevent tumor development.

  7. Modulation of cytokine release by differentiated CACO-2 cells in a compartmentalized coculture model with mononuclear leucocytes and nonpathogenic bacteria

    DEFF Research Database (Denmark)

    Parlesak, Alexandr; Haller, D.; Brinz, S.

    2004-01-01

    To further investigate the interaction between human mononuclear leucocytes [peripheral blood mononuclear cells (PBMC)] and enterocytes, the effect of a confluent layer of differentiated CACO-2 cells on cytokine kinetics during challenge with bacteria in a compartmentalized coculture model...... cells when leucocytes were stimulated directly with bacteria. This suppression was not paralleled by changes in the production of IL-10, IL-6 and transforming growth factor (TGF)-beta. When the bacteria were applied apically to the CACO-2 cell layer, the production of TNF-alpha, IL-12, IL-1beta, IL-8...... analysis revealed that IL-8 gene expression was equally induced in both CACO-2 and PBMC after apical stimulation with bacteria. Of note, bacteria-stimulated CACO-2 cells produced little or no cytokines in the absence of leucocytes, supporting the concept of leucocyte-epithelial cell cross...

  8. Paracrine Mechanisms of Intravenous Bone Marrow-Derived Mononuclear Stem Cells in Chronic Ischemic Stroke.

    Science.gov (United States)

    Bhasin, Ashu; Srivastava, M V Padma; Mohanty, Sujata; Vivekanandhan, Sivasubramaniam; Sharma, Sakshi; Kumaran, Senthil; Bhatia, Rohit

    2016-01-01

    The emerging role of stem cell technology and transplantation has helped scientists to study their potential role in neural repair and regeneration. The fate of stem cells is determined by their niche, consisting of surrounding cells and the secreted trophic growth factors. This interim report evaluates the safety, feasibility and efficacy (if any) of bone marrow-derived mononuclear stem cells (BM-MNC) in chronic ischemic stroke by studying the release of serum vascular endothelial growth factor (VEGF) and brain-derived neurotrophic growth factor (BDNF). Twenty stroke patients and 20 age-matched healthy controls were recruited with the following inclusion criteria: 3 months to 1.5 years from the index event, Medical Research Council (MRC) grade of hand muscles of at least 2, Brunnstrom stage 2-5, conscious, and comprehendible. They were randomized to one group receiving autologous BM-MNC (mean 60-70 million) and to another group receiving saline infusion (placebo). All patients were administered a neuromotor rehabilitation regime for 8 weeks. Clinical assessments [Fugl Meyer scale (FM), modified Barthel index (mBI), MRC grade, Ashworth tone scale] were carried out and serum VEGF and BDNF levels were assessed at baseline and at 8 weeks. No serious adverse events were observed during the study. There was no statistically significant clinical improvement between the groups (FM: 95% CI 15.2-5.35, p = 0.25; mBI: 95% CI 14.3-4.5, p = 0.31). VEGF and BDNF expression was found to be greater in group 1 compared to group 2 (VEGF: 442.1 vs. 400.3 pg/ml, p = 0.67; BDNF: 21.3 vs. 19.5 ng/ml) without any statistically significant difference. Autologous mononuclear stem cell infusion is safe and tolerable by chronic ischemic stroke patients. The released growth factors (VEGF and BDNF) in the microenvironment could be due to the paracrine hypothesis of stem cell niche and neurorehabilitation regime. © 2016 The Author(s) Published by S. Karger AG, Basel.

  9. Paracrine Mechanisms of Intravenous Bone Marrow-Derived Mononuclear Stem Cells in Chronic Ischemic Stroke

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    Ashu Bhasin

    2016-10-01

    Full Text Available Background: The emerging role of stem cell technology and transplantation has helped scientists to study their potential role in neural repair and regeneration. The fate of stem cells is determined by their niche, consisting of surrounding cells and the secreted trophic growth factors. This interim report evaluates the safety, feasibility and efficacy (if any of bone marrow-derived mononuclear stem cells (BM-MNC in chronic ischemic stroke by studying the release of serum vascular endothelial growth factor (VEGF and brain-derived neurotrophic growth factor (BDNF. Methods: Twenty stroke patients and 20 age-matched healthy controls were recruited with the following inclusion criteria: 3 months to 1.5 years from the index event, Medical Research Council (MRC grade of hand muscles of at least 2, Brunnstrom stage 2-5, conscious, and comprehendible. They were randomized to one group receiving autologous BM-MNC (mean 60-70 million and to another group receiving saline infusion (placebo. All patients were administered a neuromotor rehabilitation regime for 8 weeks. Clinical assessments [Fugl Meyer scale (FM, modified Barthel index (mBI, MRC grade, Ashworth tone scale] were carried out and serum VEGF and BDNF levels were assessed at baseline and at 8 weeks. Results: No serious adverse events were observed during the study. There was no statistically significant clinical improvement between the groups (FM: 95% CI 15.2-5.35, p = 0.25; mBI: 95% CI 14.3-4.5, p = 0.31. VEGF and BDNF expression was found to be greater in group 1 compared to group 2 (VEGF: 442.1 vs. 400.3 pg/ml, p = 0.67; BDNF: 21.3 vs. 19.5 ng/ml without any statistically significant difference. Conclusion: Autologous mononuclear stem cell infusion is safe and tolerable by chronic ischemic stroke patients. The released growth factors (VEGF and BDNF in the microenvironment could be due to the paracrine hypothesis of stem cell niche and neurorehabilitation regime.

  10. Functional and Pharmacological Analysis of Cardiomyocytes Differentiated from Human Peripheral Blood Mononuclear-Derived Pluripotent Stem Cells

    OpenAIRE

    2014-01-01

    Summary Advances in induced pluripotent stem cell (iPSC) technology have set the stage for routine derivation of patient- and disease-specific human iPSC-cardiomyocyte (CM) models for preclinical drug screening and personalized medicine approaches. Peripheral blood mononuclear cells (PBMCs) are an advantageous source of somatic cells because they are easily obtained and readily amenable to transduction. Here, we report that the electrophysiological properties and pharmacological responses of ...

  11. Separation of Escherichia coli bacteria from peripheral blood mononuclear cells using standing surface acoustic waves.

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    Ai, Ye; Sanders, Claire K; Marrone, Babetta L

    2013-10-01

    A microfluidic device was developed to separate heterogeneous particle or cell mixtures in a continuous flow using acoustophoresis. In this device, two identical surface acoustic waves (SAWs) generated by interdigital transducers (IDTs) propagated toward a microchannel, which accordingly built up a standing surface acoustic wave (SSAW) field across the channel. A numerical model, coupling a piezoelectric effect in the solid substrate and acoustic pressure in the fluid, was developed to provide a better understanding of SSAW-based particle manipulation. It was found that the pressure nodes across the channel were individual planes perpendicular to the solid substrate. In the separation experiments, two side sheath flows hydrodynamically focused the injected particle or cell mixtures into a very narrow stream along the centerline. Particles flowing through the SSAW field experienced an acoustic radiation force that highly depends on the particle properties. As a result, dissimilar particles or cells were laterally attracted toward the pressure nodes at different magnitudes, and were eventually switched to different outlets. Two types of fluorescent microspheres with different sizes were successfully separated using the developed device. In addition, Escherichia coli bacteria premixed in peripheral blood mononuclear cells (PBMCs) were also efficiently isolated using the SSAW-base separation technique. Flow cytometric analysis on the collected samples found that the purity of separated E. coli bacteria was 95.65%.

  12. Nipah virus infects specific subsets of porcine peripheral blood mononuclear cells.

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    Beata Stachowiak

    Full Text Available Nipah virus (NiV, a zoonotic paramyxovirus, is highly contagious in swine, and can cause fatal infections in humans following transmission from the swine host. The main viral targets in both species are the respiratory and central nervous systems, with viremia implicated as a mode of dissemination of NiV throughout the host. The presented work focused on the role of peripheral blood mononuclear cells (PBMC in the viremic spread of the virus in the swine host. B lymphocytes, CD4-CD8-, as well as CD4+CD8- T lymphocytes were not permissive to NiV, and expansion of the CD4+CD8- cells early post infection was consistent with functional humoral response to NiV infection observed in swine. In contrast, significant drop in the CD4+CD8- T cell frequency was observed in piglets which succumbed to the experimental infection, supporting the hypothesis that antibody development is the critical component of the protective immune response. Productive viral replication was detected in monocytes, CD6+CD8+ T lymphocytes and NK cells by recovery of infectious virus in the cell supernatants. Virus replication was supported by detection of the structural N and the non-structural C proteins or by detection of genomic RNA increase in the infected cells. Infection of T cells carrying CD6 marker, a strong ligand for the activated leukocyte cell adhesion molecule ALCAM (CD166 highly expressed on the microvascular endothelial cell of the blood-air and the blood-brain barrier may explain NiV preferential tropism for small blood vessels of the lung and brain.

  13. Periurethral muscle-derived mononuclear cell injection improves urethral sphincter restoration in rats.

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    Turco, Marcelo Pitelli; de Souza, Alex Balduino; de Campos Sousa, Isida; Fratini, Paula; Veras, Mariana Matera; Rodrigues, Marcio Nogueira; de Bessa, José; Brolio, Marina Pandolphi; Leite, Katia Ramos Moreira; Bruschini, Homero; Srougi, Miguel; Miglino, Maria Angélica; Gomes, Cristiano Mendes

    2017-03-27

    Investigate the effect of a novel cell-based therapy with skeletal muscle-derived mononuclear cells (SMDMCs) in a rat model of stress urinary incontinence. Male Wistar-Kyoto rats' hind limb muscles were enzymatically dissociated, and SMDMCs were isolated without needing expansion. The cell population was characterized. Twenty female rats underwent urethrolysis. One week later, 10 rats received periurethral injection of 10(6) cells (SMDMC group), and 10 rats received saline injections (Saline group). Ten rats underwent sham surgery (Sham group). Four weeks after injection, animals were euthanized and the urethra was removed. The incorporation of SMDMCs in the female urethra was evaluated with fluorescence in situ hybridization for the detection of Y-chromosomes. Hematoxylin and eosin, Masson's trichrome staining, and immunohistochemistry for actin and myosin were performed. The muscle/connective tissue, actin and myosin ratios were calculated. Morphological evaluation of the urethral diameters and fractional areas of the lumen, mucosa, and muscular layer was performed. SMDMCs population was consistent with the presence of muscle cells, muscle satellite cells, perivascular cells, muscle progenitor cells, and endothelial cells. SMDMCs were incorporated into the urethra. A significant decrease in the muscle/connective tissue ratio was observed in the Saline group compared with the SMDMC and Sham groups. The proportions of actin and myosin were significantly decreased in the Saline group. No differences were observed in the morphometric parameters. SDMSC were incorporated into the rat urethra and promoted histological recovery of the damaged urethral sphincter, resulting in decreased connective tissue deposition and increased muscle content. © 2017 Wiley Periodicals, Inc.

  14. Phenotypes of lung mononuclear phagocytes in HIV seronegative tuberculosis patients: evidence for new recruitment and cell activation

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    José R Lapa e Silva

    1996-06-01

    Full Text Available Mycobacterium tuberculosis preferentially resides in mononuclear phagocytes. The mechanisms by which mononuclear phagocytes keep M. tuberculosis in check or by which the microbe evades control to cause disease remain poorly understood. As an initial effort to delineate these mechanisms, we examined by immunostaining the phenotype of mononuclear phagocytes obtained from lungs of patients with active tuberculosis. From August 1994 to March 1995, consecutive patients who had an abnormal chest X-ray, no demostrable acid-fast bacilli in sputum specimens and required a diagnostic bronchoalveolar lavage (BAL were enrolled. Of the 39 patients enrolled, 21 had microbiologically diagnosed tuberculosis. Thirteen of the 21 tuberculosis patients were either HIV seronegative (n = 12 or had no risk factor for HIV and constituted the tuberculosis group. For comparison, M. tuberculosis negative patients who had BAL samples taken during this time (n = 9 or normal healthy volunteers (n = 3 served as control group. Compared to the control group, the tuberculosis group had significantly higher proportion of cells expressing markers of young monocytes (UCHM1 and RFD7, a marker for phagocytic cells, and increased expression of HLA-DR, a marker of cell activation. In addition, tuberculosis group had significantly higher proportion of cells expressing dendritic cell marker (RFD1 and epithelioid cell marker (RFD9. These data suggest that despite recruitment of monocytes probably from the peripheral blood and local cell activation, host defense of the resident lung cells is insufficient to control M. tuberculosis.

  15. Mononuclear cell therapy reverts cuff-induced thrombosis in apolipoprotein E-deficient mice

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    Lima Leandro C F

    2012-07-01

    Full Text Available Abstract Background Stem/progenitor cell-based therapy has successfully been used as a novel therapeutic strategy for vascular diseases triggered by endothelial dysfunction. The aim of this study was to investigate the effects of mononuclear cell (MNC therapy in situ on carotid cuff-induced occlusive thrombus in the apolipoprotein E knockout (apoE-/- mouse. Methods Spleen-derived MNCs were isolated from green fluorescent protein (GFP-transgenic mice for cell treatment. A cuff-induced thrombus model was produced by placing a nonconstrictive silastic collar around the left common carotid artery in 20-week-old female apoE-/- mice. After 10 days, the cuff was removed, and the animals received in situ MNCs (Cuff-MNC or vehicle (Cuff-Vehicle and were compared with sham-operated animals (Sham. Results The histological analysis showed that the MNC treatment reverted occlusive thrombus formation compared to the vehicle and the vessel lumen area to that observed in the Sham group (MNC, 50 ± 4; Vehicle, 20 ± 4; Sham, 55 ± 2 x103 μm2; p -/- mice. Conclusion In situ short-term MNC therapy was able to revert cuff-induced occlusive thrombi in the carotid arteries of apoE-/- mice, possibly through the homing of EPCs, reduction of oxidative stress and decreased apoptosis.

  16. Hemorrhage activates NF-kappa B in murine lung mononuclear cells in vivo.

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    Shenkar, R; Schwartz, M D; Terada, L S; Repine, J E; McCord, J; Abraham, E

    1996-05-01

    Hemorrhage rapidly increases the expression of proinflammatory and immunoregulatory cytokines in the lungs. Binding elements for the nuclear transcriptional regulatory factors (NF)-kappa B and NF-IL6 (C/EBP beta) are present in the promoter regions of multiple cytokine genes, including those whose expression is increased after blood loss. In the present experiments, we found increased activation in vivo of NF-kappa B in lung mononuclear cells, but not in splenocytes, taken from mice 1 h after hemorrhage. In contrast, hemorrhage did not activate NF-IL6 in lung cells or splenocytes. Inhibition of xanthine oxidase by prior feeding of a tungsten-enriched diet prevented hemorrhage-induced activation in lung cells of NF-kappa B. Incubating splenocytes in vitro with xanthine oxidase activated NF-kappa B but not NF-IL6. Xanthine oxidase-induced activation of NF-kappa B was inhibited by manganese superoxide dismutase, but not by catalase. These results suggest that xanthine oxidase-mediated superoxide anion-dependent activation of NF-kappa B occurs in vivo and in vitro. This mechanism may contribute to increased lung cytokine responses after hemorrhage.

  17. Transplantation of mobilized peripheral blood mononuclear cells for peripheral arterial occlusive disease of the lower extremity

    Institute of Scientific and Technical Information of China (English)

    Xiaofeng YANG; Yanxiang WU; Hongmei WANG; Yifeng XU; Bo XU; Xin LU; Yibin ZANG; Fa WANG; Yue ZHANG

    2006-01-01

    Objectives To assess the clinical efficacy, safety, and feasibility of autologous transplantation of mobilized peripheral blood mononuclear cells (PBMNCs) for patients with peripheral arterial occlusive disease (PAOD) of the lower extremity. Methods A total of 152 patients with PAOD of the lower extremity were enrolled into this non-controlled observational study from November 2003 to March 2006. All patients received subcutaneous injections of recombinant human granulocyte colony-stimulating factor (G-CSF, 450600 μg/day) for 5 days in order to mobilize stem/progenitor cells; their PBMNCs were collected and transplanted by multiple intramuscular injections into ischemic limbs. Patients were followed up for at least 12 weeks. Results At 12 weeks, primarymanifestations,including lower limb pain and coldness, were significantly improved in 137 (90.1%) of the patients; limb ulcers improved or healed in 46 (86.8%) of the 53 patients, while 25 of the 48 (47.9%) patients with limb gangrene remained steady or improved. Ankle-brachial index (ABI) improved in 33 (22%) of the cases, and TcPO2 increased in 45 (30%) of the cases. Angiography before treatment, and at 12 weeks after treatment, was performed in 10 of the patients and showed formation of new collateral vessels. No severe adverse effects or complications specifically related to cell transplantation were observed. Conclusion Autologous transplantation of G-CSF-mobilized PBMNCs might be a safe and effective treatment for lower limb ischemic disorder.(J Geriatr Cardiol 2006; 3:178-80.)

  18. Effect of Static Magnetic Fields on the Peripheral Blood Mononuclear-like Cells

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    Godina-Nava, J. J.; García-Cantú, R.; Cañedo, L.; Rodríguez-Segura, M. A.; Serrano, G.; Alvarado-Alvarez, R.; Toledo-Ramos, F.

    2002-08-01

    In this article the role of static magnetic fields (SMF) in the generation of Ca2+ currents in peripheral blood mononuclear-like cells (PBMLC) is described. Using the sensitivity of Ca2+ channels and pumps to membrane potential and ion concentration we propose a method which uses the conductivity as a dynamical coefficient in the Onsager's reciprocity relations, and the dynamics of the calcium ions described by the electrodiffusion equation deduced by Pelce. The enhanced influx of calcium ion in PBMLC was studied parametrizing the static magnetic fields effects on the conductivity by the coefficients γ, ρ and κ. The parametrization was made according to the symmetry properties of Onsager's reciprocity relations using the most simple expressions. As an example we used available experimental data over chromaffin cell and employing physical considerations concerning to PBMLC, an order of magnitude for the value of ρ ≈ O(-10-5 mol/(Vm2s)), κ = 0, γ ≈ O(-10-3 mol/(VT2m2s) was obtained. The γ parameter was found graphically. With this parametrization, the time to induce calcium current in the cell was always less than the situation without magnetic field application.

  19. Paracrine Effects of Bone Marrow Mononuclear Cells in Survival and Cytokine Expression after 90% Partial Hepatectomy

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    Carlos Oscar Kieling

    2017-01-01

    Full Text Available Acute liver failure is a complex and fatal disease. Cell-based therapies are a promising alternative therapeutic approach for liver failure due to relatively simple technique and lower cost. The use of semipermeable microcapsules has become an interesting tool for evaluating paracrine effects in vivo. In this study, we aimed to assess the paracrine effects of bone marrow mononuclear cells (BMMC encapsulated in sodium alginate to treat acute liver failure in an animal model of 90% partial hepatectomy (90% PH. Encapsulated BMMC were able to increase 10-day survival without enhancing liver regeneration markers. Gene expression of Il-6 and Il-10 in the remnant liver was markedly reduced at 6 h after 90% PH in animals receiving encapsulated BMMC compared to controls. This difference, however, was neither reflected by changes in the number of CD68+ cells nor by serum levels of IL6. On the other hand, treated animals presented increased caspase activity and gene expression in the liver. Taken together, these results suggest that BMMC regulate immune response and promote apoptosis in the liver after 90% PH by paracrine factors. These changes ultimately may be related to the higher survival observed in treated animals, suggesting that BMMC may be a promising alternative to treat acute liver failure.

  20. Metabolic Profiling of Human Peripheral Blood Mononuclear Cells: Influence of Vitamin D Status and Gender

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    Magdalena Stepien

    2014-04-01

    Full Text Available Metabolic profiling of peripheral blood mononuclear cells (PBMC could serve as a less invasive and more direct alternative to tissue biopsies or serum in metabolomic research. We conducted two exploratory independent studies in order to characterise PBMC’s metabolomic profile following short-term vitamin D3 supplementation and to determine gender effects. In the first study, eight healthy males and females aged 40–65 y were randomly selected for profiling of PBMCs after receiving either 15 µg of vitamin D3 or placebo for four weeks. In the second study, twenty younger healthy males and females were studied. Cell metabolites were extracted and deproteinised using methanol/chloroform/water method and analysed by GC-MS. Higher vitamin D status had no effect on the fatty acid profile of PBMCs, but inflammatory biomarkers and adipokines correlated positively with stearic acid levels. In the second study, no gender-specific metabolites were identified. Valine, leucine and aspartic acid were identified as potential BMI-sensitive amino acids. Larger studies are needed to confirm the influence of BMI on these parameters. This work clearly demonstrates the utility of metabolomics profiling of PBMCs and paves the way for future applications of metabolomics in identifying metabolic profiles of blood cells as a measure for dietary intakes or physiological status.

  1. Impact of age on the efficacy of bone marrow mononuclear cell transplantation in experimental stroke

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    Wagner Daniel-Christoph

    2012-08-01

    Full Text Available Abstract Bone marrow-derived mononuclear cells (BM MNC have been effectively used to treat experimental stroke. Most of the preclinical trials have been performed in young and healthy laboratory animals, even though age and hypertension are major risk factors for stroke. To determine the influence of age on the properties of BM MNCs after cerebral ischemia, we compared the efficacy of aged and young BM MNC in an in vitro model of cerebral hypoxia and in an adapted in vivo model of stroke. Human BM MNCs were obtained from healthy young or aged donors and either co-cultured with rat hippocampal slices exposed to oxygen glucose deprivation (OGD, or transplanted intravenously 24 h after permanent middle cerebral artery occlusion in aged (18 months spontaneously hypertensive rats (SHR. Efficacy was examined by quantification of hippocampal cell death, or respectively, by neurofunctional tests and MR investigations. Co-cultivation with young, but not with aged BM MNCs significantly reduced the hippocampal cell death after OGD. Transplantation of both young and old BM MNCs did not reduce functional deficits or ischemic lesion volume after stroke in aged SHR. These results suggest a significant impact of age on the therapeutic efficacy of BM MNCs after cerebral ischemia.

  2. Improved Quality of Life in A Case of Cerebral Palsy after Bone Marrow Mononuclear Cell Transplantation

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    Alok Sharma

    2015-07-01

    Full Text Available Cerebral palsy (CP is a non progressive, demyelinating disorder that affects a child’s development and posture and may be associated with sensation, cognition, communication and perception abnormalities. In CP, cerebral white matter is injured resulting in the loss of oligodendrocytes. This causes damage to the myelin and disruption of nerve conduction. Cell therapy is being explored as an alternate therapeutic strategy as there is no treatment currently available for CP. To study the benefits of this treatment we have administered autologous bone marrow mononuclear cells (BMMNCs to a 12-year-old CP case. He was clinically re-evaluated after six months and found to demonstrate positive clinical and functional outcomes. His trunk strength, upper limb control, hand functions, walking stability, balance, posture and coordination improved. His ability to perform activities of daily living improved. On repeating the Functional Independence Measure (FIM, the score increased from 90 to 113. A repeat positron emission tomography- computed tomography (PET-CT scan of the brain six months after intervention showed progression of the mean standard deviation values towards normalization which correlated to the functional changes. At one year, all clinical improvements have remained. This indicated that cell transplantation may improve quality of life and have a potential for treatment of CP.

  3. Reduction of cytokine release of blood and bronchoalveolar mononuclear cells by ambroxol.

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    Pfeifer, S; Zissel, G; Kienast, K; Müller-Quernheim, J

    1997-03-24

    Ambroxol is a mucolytic agent frequently used in the treatment of chronic bronchitis. It has been reported, following clinical and in-vitro studies, that ambroxol exhibits an anti-inflammatory action. This capability was investigated by activating bronchoalveolar lavage cells and peripheral blood mononuclear cells in-vitro to elicit the release of tumor necrosis factor alpha, interleukin-2 and interferon gamma, whilst simultaneously exposing them to varying pharmacological concentrations of ambroxol (10, 1, and 0.1 microM). After 24 h it was observed that the isolated tissue-culture supernatants showed a dose-dependent reduction in the concentration of the tested cytokines; 10 microM (12 to 37% reduction) and 1 microM to (6 to 27% reduction). At 0.1 microM, a significant reduction could only be observed in the release of interleukin-2 by bronchoalveolar lavage cells. These results demonstrate, that ambroxol exhibits anti-inflammatory actions in concentrations achievable in vivo.

  4. Molecular Imaging of Bone Marrow Mononuclear Cell Survival and Homing in Murine Peripheral Artery Disease

    Science.gov (United States)

    van der Bogt, Koen E.A.; Hellingman, Alwine A.; Lijkwan, Maarten A.; Bos, Ernst-Jan; de Vries, Margreet R.; Fischbein, Michael P.; Quax, Paul H.; Robbins, Robert C.; Hamming, Jaap F.; Wu, Joseph C.

    2013-01-01

    Introduction Bone marrow mononuclear cell (MNC) therapy is a promising treatment for peripheral artery disease (PAD). This study aims to provide insight into cellular kinetics using molecular imaging following different transplantation methods. Methods and Results MNCs were isolated from F6 transgenic mice (FVB background) that express firefly luciferase (Fluc) and green fluorescence protein (GFP). Male FVB and C57Bl6 mice (n=50) underwent femoral artery ligation and were randomized into 4 groups receiving: (1) single intramuscular (i.m.) injection of 2×106 MNC; (2) four weekly i.m. injections of 5×105 MNC; (3) 2×106 MNCs intravenously (i.v.); and (4) PBS. Cellular kinetics, measured by in vivo bioluminescence imaging (BLI), revealed near-complete donor cell death 4 weeks after i.m. transplantation. Following i.v. transplantation, BLI monitored cells homed in on the injured area in the limb, as well as to the liver, spleen, and bone marrow. Ex vivo BLI showed presence of MNCs in the scar tissue and adductor muscle. However, no significant effects on neovascularisation were observed as monitored by Laser-Doppler-Perfusion-Imaging and histology. Conclusion This is one of the first studies to assess kinetics of transplanted MNCs in PAD using in vivo molecular imaging. MNC survival is short lived and MNCs do not significantly stimulate perfusion in this model. PMID:22239892

  5. Allograft inflammatory factor-1 stimulates chemokine production and induces chemotaxis in human peripheral blood mononuclear cells.

    Science.gov (United States)

    Kadoya, Masatoshi; Yamamoto, Aihiro; Hamaguchi, Masahide; Obayashi, Hiroshi; Mizushima, Katsura; Ohta, Mitsuhiro; Seno, Takahiro; Oda, Ryo; Fujiwara, Hiroyoshi; Kohno, Masataka; Kawahito, Yutaka

    2014-06-06

    Allograft inflammatory factor-1 (AIF-1) is expressed by macrophages, fibroblasts, endothelial cells and smooth muscle cells in immune-inflammatory disorders such as systemic sclerosis, rheumatoid arthritis and several vasculopathies. However, its molecular function is not fully understood. In this study, we examined gene expression profiles and induction of chemokines in monocytes treated with recombinant human AIF (rhAIF-1). Using the high-density oligonucleotide microarray technique, we compared mRNA expression profiles of rhAIF-1-stimulated CD14(+) peripheral blood mononuclear cells (CD14(+) PBMCs) derived from healthy volunteers. We demonstrated upregulation of genes for several CC chemokines such as CCL1, CCL2, CCL3, CCL7, and CCL20. Next, using ELISAs, we confirmed that rhAIF-1 promoted the secretion of CCL3/MIP-1α and IL-6 by CD14(+) PBMCs, whereas only small amounts of CCL1, CCL2/MCP-1, CCL7/MCP-3 and CCL20/MIP-3α were secreted. Conditioned media from rhAIF-1stimulated CD14(+) PBMCs resulted in migration of PBMCs. These findings suggest that AIF-1, which induced chemokines and enhanced chemotaxis of monocytes, may represent a molecular target for the therapy of immune-inflammatory disorders. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Improved Quality of Life in A Case of Cerebral Palsy after Bone Marrow Mononuclear Cell Transplantation.

    Science.gov (United States)

    Sharma, Alok; Sane, Hemangi; Kulkarni, Pooja; D'sa, Myola; Gokulchandran, Nandini; Badhe, Prerna

    2015-01-01

    Cerebral palsy (CP) is a non progressive, demyelinating disorder that affects a child's development and posture and may be associated with sensation, cognition, communication and perception abnormalities. In CP, cerebral white matter is injured resulting in the loss of oligodendrocytes. This causes damage to the myelin and disruption of nerve conduction. Cell therapy is being explored as an alternate therapeutic strategy as there is no treatment currently available for CP. To study the benefits of this treatment we have administered autologous bone marrow mononuclear cells (BMMNCs) to a 12-year-old CP case. He was clinically re-evaluated after six months and found to demonstrate positive clinical and functional outcomes. His trunk strength, upper limb control, hand functions, walking stability, balance, posture and coordination improved. His ability to perform activities of daily living improved. On repeating the Functional Independence Measure (FIM), the score increased from 90 to 113. A repeat positron emission tomography-computed tomography (PET-CT) scan of the brain six months after intervention showed progression of the mean standard deviation values towards normalization which correlated to the functional changes. At one year, all clinical improvements have remained. This indicated that cell transplantation may improve quality of life and have a potential for treatment of CP.

  7. Increased peroxisome proliferator-activated receptor-gamma activity reduces imatinib uptake and efficacy in chronic myeloid leukemia mononuclear cells.

    Science.gov (United States)

    Wang, Jueqiong; Lu, Liu; Kok, Chung H; Saunders, Verity A; Goyne, Jarrad M; Dang, Phuong; Leclercq, Tamara M; Hughes, Timothy P; White, Deborah L

    2017-02-02

    Imatinib is actively transported by OCT-1 influx transporter, and low OCT-1 activity in diagnostic chronic myeloid leukemia blood mononuclear cells is significantly associated with poor molecular response to imatinib. Here we report that, in diagnostic chronic myeloid leukemia mononuclear cells and BCR-ABL1+ cell lines, peroxisome proliferator-activated receptor gamma agonists (GW1929, rosiglitazone, pioglitazone) significantly decrease OCT-1 activity; conversely, peroxisome proliferator-activated receptor gamma antagonists (GW9662, T0070907) increase OCT-1 activity. Importantly, these effects can lead to corresponding changes in sensitivity to Bcr-Abl kinase inhibition. Results were confirmed in peroxisome proliferator-activated receptor gamma-transduced K562 cells. Furthermore, we identified a strong negative correlation between OCT-1 activity and peroxisome proliferator-activated receptor gamma transcriptional activity in diagnostic chronic myeloid leukemia patients (n=84; preceptor gamma activation has a negative impact on the intracellular uptake of imatinib and consequent Bcr-Abl kinase inhibition. The inter-patient variability of peroxisome proliferator-activated receptor gamma activation likely accounts for the heterogeneity observed in patient OCT-1 activity at diagnosis. Recently, the peroxisome proliferator-activated receptor gamma agonist pioglitazone was reported to act synergistically with imatinib targeting the residual chronic myeloid leukemia stem cell pool. Our findings suggest that peroxisome proliferator-activated receptor gamma ligands have differential effects on circulating mononuclear cells compared to stem cells. Since the effect of peroxisome proliferator-activated receptor gamma activation on imatinib uptake in mononuclear cells may counteract the clinical benefit of this activation in stem cells, caution should be applied when combining these therapies, especially in patients with high peroxisome proliferator-activated receptor gamma

  8. Infected peripheral blood mononuclear cells transmit latent varicella zoster virus infection to the guinea pig enteric nervous system.

    Science.gov (United States)

    Gan, Lin; Wang, Mingli; Chen, Jason J; Gershon, Michael D; Gershon, Anne A

    2014-10-01

    Latent wild-type (WT) and vaccine (vOka) varicella zoster virus (VZV) are found in the human enteric nervous system (ENS). VZV also infects guinea pig enteric neurons in vitro, establishes latency and can be reactivated. We therefore determined whether lymphocytes infected in vitro with VZV secrete infectious virions and can transfer infection in vivo to the ENS of recipient guinea pigs. T lymphocytes (CD3-immunoreactive) were preferentially infected following co-culture of guinea pig or human peripheral blood mononuclear cells with VZV-infected HELF. VZV proliferated in the infected T cells and expressed immediate early and late VZV genes. Electron microscopy confirmed that VZV-infected T cells produced encapsulated virions. Extracellular virus, however, was pleomorphic, suggesting degradation occurred prior to release, which was confirmed by the failure of VZV-infected T cells to secrete infectious virions. Intravenous injection of WT- or vOka-infected PBMCs, nevertheless, transmitted VZV to recipient animals (guinea pig > human lymphocytes). Two days post-inoculation, lung and liver, but not gut, contained DNA and transcripts encoding ORFs 4, 40, 66 and 67. Twenty-eight days after infection, gut contained DNA and transcripts encoding ORFs 4 and 66 but neither DNA nor transcripts could any longer be found in lung or liver. In situ hybridization revealed VZV DNA in enteric neurons, which also expressed ORF63p (but not ORF68p) immunoreactivity. Observations suggest that VZV infects T cells, which can transfer VZV to and establish latency in enteric neurons in vivo. Guinea pigs may be useful for studies of VZV pathogenesis in the ENS.

  9. Transplantation of mononuclear cells from bone marrow in a rat model of Huntington’s disease

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    Serrano T

    2016-12-01

    Full Text Available Teresa Serrano,1 Paula Pierozan,2 Esteban Alberti,1 Lisette Blanco,1 Karelys de la Cuétara Bernal,1 María E González,1 Nancy Pavón,1 Lourdes Lorigados,1 María A Robinson-Agramonte,1 Jorge A Bergado1 1International Center for Neurological Restoration (CIREN, La Habana, Cuba; 2Department of Biochemistry, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil Abstract: This article investigates the possible effects of transplantation of mononuclear bone marrow cells (mBMCs to ameliorate or prevent the behavioral impairments and the cellular damage observed in a quinolinic acid (QA model of Huntington’s disease. mBMCs were isolated using a standard procedure and implanted within the QA-lesioned striatum. Behavior was explored using motor (beam test and memory (object recognition and Morris water maze tests. Morphology was evaluated using conventional histology (cresyl violet, bisbenzimide (to evaluate cell vitality, and immunohystochemistry to identify neurons or glia. mBMC-transplanted animals showed improvements in motor coordination (beam test. Regarding memory, object recognition was significantly improved in transplanted animals, while spatial memory (Morris water maze test was not severely affected by QA and, therefore, the results after transplantation were significant only in the probe-trial retention test. In samples taken from the animals that participated in the behavioral tests, a preserved morphology of striatal neurons and a reduced glial reaction indicated a possible neuroprotective effect of the transplanted mBMCs. A parallel study confirmed that the transplanted mBMCs have a long survival period (1 year follow-up. The results presented confirm the possibility that mBMC transplantation may be a viable therapeutic option for Huntington’s disease. Keywords: mononuclear bone marrow cells, Huntington’s disease, quinolinic acid, transplant, Fluoro-Jade C

  10. Rac1 regulates the NLRP3 inflammasome which mediates IL-1beta production in Chlamydophila pneumoniae infected human mononuclear cells.

    Directory of Open Access Journals (Sweden)

    Julia Eitel

    Full Text Available Chlamydophila pneumoniae causes acute respiratory tract infections and has been associated with development of asthma and atherosclerosis. The production of IL-1β, a key mediator of acute and chronic inflammation, is regulated on a transcriptional level and additionally on a posttranslational level by inflammasomes. In the present study we show that C. pneumoniae-infected human mononuclear cells produce IL-1β protein depending on an inflammasome consisting of NLRP3, the adapter protein ASC and caspase-1. We further found that the small GTPase Rac1 is activated in C. pneumoniae-infected cells. Importantly, studies with specific inhibitors as well as siRNA show that Rac1 regulates inflammasome activation in C. pneumoniae-infected cells. In conclusion, C. pneumoniae infection of mononuclear cells stimulates IL-1β production dependent on a NLRP3 inflammasome-mediated processing of proIL-1β which is controlled by Rac1.

  11. Autologous transplantation of bone marrow mononuclear cells improved heart function after myocardial infarction

    Institute of Scientific and Technical Information of China (English)

    Guo-sheng LIN; Jing-jun L(U); Xue-jun JIANG; Xiao-yan LI; Geng-shan LI

    2004-01-01

    AIM: To investigate whether autologous transplantation of adult stem cells could improve post-infarcted heart function. METHODS: Bone marrow mononuclear cells (MNCs) were isolated from adult rabbits' tibias after coronary ligation. These cells were exposed to 5-azacytidine 10 μmol/L for 24 h on the third day of culture. After being labeled with bromodeoxyuridine (BrdU), the cells were auto-transplanted into bordering zone of the infarcted area at 2 weeks after injury. The animals were killed at 3 days, 2 weeks, 1 month, and 2 months after transplantation,respectively. The left ventricular functions, capillary density, and cardiac nerve density were measured and the differentiation of the engrafted cells was determined by immunostaining. RESULTS: BrdU-labeled MNCs were well aligned with the host cardiomyocytes. Parts of them were incorporated into capillary and arteriolar vessel walls. In addition to inducing angiogenic ligands (basic fibroblast growth factor, vascular endothelial growth factor) and imflammation cytokines (interleukin 1-β) during the early period of MNCs implantation, MNCs induced 2.0-fold increase in capillary density as well. Moreover, GAP43-positive and TH-positive nerve density were markedly higher in the MNCs-treated groups than that in the non-treated hearts. Left ventricular ejection fraction,LV+dp/dt and LV-dp/dtmax were 47 %, 67 %, and 55 % in MNCs-treated heart respectively, which was higher than that of the control heart, whereas left ventricular end-diastolic volume, left ventricular end-diastolic diameter,and left ventricular end-diastolic pressure were 45 %, 22 %, and 50 % respectively in MNCs-treated heart, which was lower than that of the control heart at 2 months after cell transplantation. CONCLUSION: Autologous transplantation of MNCs induced angiogenesis and nerve sprouting and improved left ventricular diastolic function.

  12. Viability and Functionality of Cryopreserved Peripheral Blood Mononuclear Cells in Pediatric Dengue.

    Science.gov (United States)

    Perdomo-Celis, Federico; Salgado, Doris M; Castañeda, Diana M; Narváez, Carlos F

    2016-05-01

    Cryopreserved peripheral blood mononuclear cells (PBMCs) are widely used in studies of dengue. In this disease, elevated frequency of apoptotic PBMCs has been described, and molecules such as soluble tumor necrosis factor (TNF)-related apoptosis-inducing ligands (sTRAIL) are involved. This effect of dengue may affect the efficiency of PBMC cryopreservation. Here, we evaluate the viability (trypan blue dye exclusion and amine-reactive dye staining) and functionality (frequency of gamma interferon [IFN-γ]-producing T cells after polyclonal stimulation) of fresh and cryopreserved PBMCs from children with dengue (in acute and convalescence phases), children with other febrile illnesses, and healthy children as controls. Plasma sTRAIL levels were also evaluated. The frequencies of nonviable PBMCs detected by the two viability assays were positively correlated (r = 0.74; P dengue, who had a higher frequency of nonviable cells than healthy children and children with other febrile illnesses (P ≤ 0.02), and PBMC viability levels were restored in the convalescent phase. In the acute phase, an increased frequency of CD3(+) CD8(+) amine-positive cells was found before cryopreservation (P = 0.01). Except for B cells in the acute phase, cryopreservation usually did not affect the relative frequencies of viable PBMC subpopulations. Dengue infection reduced the frequency of IFN-γ-producing CD3(+) cells after stimulation compared with healthy controls and convalescent-phase patients (P ≤ 0.003), and plasma sTRAIL correlated with this decreased frequency in dengue (rho = -0.56; P = 0.01). Natural dengue infection in children can affect the viability and functionality of cryopreserved PBMCs.

  13. Aberrant activation of nuclear factor of activated T cell 2 in lamina propria mononuclear cells in ulcerative colitis

    Institute of Scientific and Technical Information of China (English)

    Tsung-Chieh Shih; Sen-Yung Hsieh; Yi-Yueh Hsieh; Tse-Chin Chen; Chien-Yu Yeh; Chun-Jung Lin; Deng-Yn Lin; Cheng-Tang Chiu

    2008-01-01

    AIM: To investigate the role of nuclear factor of activated T cell 2 (NFAT2), the major NFAT protein in peripheral T cells, in sustained T cell activation and intractable inflammation in human ulcerative colitis (UC).METHODS: We used two-dimensional gel-electrophoresis, immunohistochemistry, double immunohistochemical staining, and confocal microscopy to inspect the expression of NFAT2 in 107, 15, 48 and 5 cases of UC, Crohn's disease (CD), non-specific colitis, and 5 healthy individuals, respectively.RESULTS: Up-regulation with profound nucleo-translocation/activation of NFAT2 of lamina propria mononuclear cells (LPMC) of colonic mucosa was found specifically in the affected colonic mucosa from patients with UC, as compared to CD or NC (P < 0.001, Kruskal-Wallis test). Nucleo-translocation/activation of NFAT2 primarily occurred in CD8+T, but was less prominent in CD4+ T cells or CD20+B cells. It was strongly associated with the disease activity, including endoscopic stage (t = 0.2145, P = 0.0281) and histologic grade (t = 0.4167, P < 0.001).CONCLUSION: We disclose for the first time the nucleo-translocation/activatin of NFAT2 in lamina propria mononuclear cells in ulcerative colitis. Activation of NFAT2 was specific for ulcerative colitis and highly associated with disease activity. Since activation of NFAT2 is implicated in an auto-regulatory positive feedback loop of sustained T-cell activation and NFAT proteins play key roles in the calcium/calcineurin signaling pathways, our results not only provide new insights into the mechanism for sustained intractable inflammation, but also suggest the calcium-calcineurin/NFAT pathway as a new therapeutic target for ulcerative colitis.

  14. An active lifestyle induces positive antioxidant enzyme modulation in peripheral blood mononuclear cells of overweight/obese postmenopausal women.

    Science.gov (United States)

    Farinha, Juliano Boufleur; De Carvalho, Nélson Rodrigues; Steckling, Flávia Mariel; De Vargas, Liziane Da Silva; Courtes, Aline Alves; Stefanello, Sílvio Terra; Martins, Caroline Curry; Bresciani, Guilherme; Dos Santos, Daniela Lopes; Soares, Félix Alexandre Antunes

    2015-01-15

    The aim of this study was to investigate the effects of an active lifestyle on mitochondrial functioning, viability, bioenergetics, and redox status markers in peripheral blood mononuclear cells (PBMC) of overweight/ obese postmenopausal women. We performed a cross-sectional study with postmenopausal women aged 45–64 years and body mass index N 25 kg/m2, divided into physically active (n = 23) and sedentary (n = 12) groups. Mitochondria functioning and viability, bioenergetics and redox status parameters were assessed in PBMC with spectrophotometric and fluorometric assays. No differences were found in the enzyme activity of complexes I and II of the electron transport chain (ETC), mitochondrial superoxide dismutase (MnSOD) activity, methyl-tetrazolium reduction levels and reduced glutathione and oxidized glutathione levels between the groups. However, the physically active group presented higher levels of reactive oxygen species (ROS) (P= 0.04) and increased catalase (CAT) (P= 0.029), total (P= 0.011) and cytosolic SOD (CuZnSOD) (P= 0.009) activities. An active lifestyle that includes aerobic exercise for at least 30 min, three times per week may improve antioxidant enzyme activities in PBMC in overweight/obese postmenopausal women, without changes in the activity of the ETC enzymes. However, this low intensity physical activity is not able to induce relevant mitochondrial adaptations.

  15. Effects of Malnutrition on Neutrophil/Mononuclear Cell Apoptotic Functions in Children with Acute Lymphoblastic Leukemia.

    Science.gov (United States)

    Cakir, Fatma Betul; Berrak, Su Gülsün; Aydogan, Gonul; Tulunay, Aysin; Timur, Cetin; Canpolat, Cengiz; Eksioglu Demiralp, Emel

    2017-04-01

    Recent studies claim that apoptosis may explain immune dysfunction observed in malnutrition. The objective of this study was to determine the effect of malnutrition on apoptotic functions of phagocytic cells in acute lymphoblastic leukemia (ALL). Twenty-eight ALL patients (13 with malnutrition) and thirty controls were enrolled. Neutrophil and mononuclear cell apoptosis of ALL patients and the control group were studied on admission before chemotherapy and repeated at a minimum of three months after induction of chemotherapy or when the nutritional status of leukemic children improved. The apoptotic functions of both ALL groups on admission were significantly lower than those of the control group. The apoptotic functions were lower in ALL patients with malnutrition than those in ALL patients without malnutrition, but this was not statistically significant. The repeated apoptotic functions of both ALL groups were increased to similar values with the control group. This increase was found to be statistically significant. The apoptotic functions in ALL patients were not found to be affected by malnutrition. However, after dietary intervention, increased apoptotic functions in both ALL patient groups deserve mentioning. Dietary intervention should always be recommended as malnutrition or cachexia leads to multiple complications. Enhanced apoptosis might originate also from remission state of cancer.

  16. Immunomodulatory activity of Semecarpus anacardium extract in mononuclear cells of normal individuals and rheumatoid arthritis patients.

    Science.gov (United States)

    Singh, Divya; Aggarwal, Amita; Mathias, Amrita; Naik, Sita

    2006-12-06

    Semecarpus anacardium (SA) Linn. (family Anacardiaceae), is a plant well-known for its medicinal value in Ayurveda. The nut extracts of this plant have been traditionally used as antihelminthic, anti-fungal, anti-carcinogenic and in the treatment of nervous debilities and arthritis. In this study we have evaluated crude ethanolic extract of SA nuts for its anti-inflammatory activities in vitro using peripheral blood and synovial fluid mononuclear cells of healthy individuals and rheumatoid arthritis (RA) patients. SA extract inhibited the spontaneous and LPS induced production of proinflammatory cytokines IL-1beta and IL-12p40 but had no effect on TNF-alpha and IL-6 production, both at protein and mRNA level. The crude extract also suppressed LPS induced nuclear translocation of transcription factors, NF-kappaB and AP-1; the inhibition of NF-kappaB was through the inhibition of IkappaBalpha phosphorylation. The extract also suppressed LPS activated nitric oxide production in mouse macrophage cell line, RAW 264.7. Our results for the first time show that SA extract can inhibit proinflammatory cytokine production and demonstrate its mechanism of action.

  17. Shared signatures of social stress and aging in peripheral blood mononuclear cell gene expression profiles.

    Science.gov (United States)

    Snyder-Mackler, Noah; Somel, Mehmet; Tung, Jenny

    2014-10-01

    Chronic social stress is a predictor of both aging-related disease and mortality risk. Hence, chronic stress has been hypothesized to directly exacerbate the process of physiological aging. Here, we evaluated this hypothesis at the level of gene regulation. We compared two data sets of genome-wide gene expression levels in peripheral blood mononuclear cells (PBMCs): one that captured aging effects and another that focused on chronic social stress. Overall, we found that the direction, although not necessarily the magnitude, of significant gene expression changes tends to be shared between the two data sets. This overlap was observable at three levels: (i) individual genes; (ii) general functional categories of genes; and (iii) molecular pathways implicated in aging. However, we also found evidence that heterogeneity in PBMC composition limits the power to detect more extensive similarities, suggesting that our findings reflect an underestimate of the degree to which age and social stress influence gene regulation in parallel. Cell type-specific data on gene regulation will be important to overcome this limitation in the future studies.

  18. Increases of microRNA let-7e in peripheral blood mononuclear cells in Hashimoto's disease.

    Science.gov (United States)

    Kagawa, Tomoya; Watanabe, Mikio; Inoue, Naoya; Otsu, Hiroshi; Saeki, Minori; Katsumata, Yuka; Takuse, Yukina; Iwatani, Yoshinori

    2016-04-25

    MicroRNA (miRNA) is a family of non-coding RNAs that have important roles in various vital functions. It has been reported that let-7e, a miRNA, may be involved in the regulation of interleukin (IL)-10 production. The purpose of this study was to evaluate the role of let-7e as a regulator of IL-10 production in the pathological processes of autoimmune thyroid diseases (AITDs). We evaluated the association between let-7e expression and intracellular expression of IL-10 in the peripheral blood mononuclear cells (PBMCs) collected from 11 healthy volunteers. Then we investigated the expression levels of let-7e in the PBMCs of 50 patients with Graves' disease (GD), 42 patients with Hashimoto's disease (HD) and 28 healthy controls. We found negative correlations between the expression level of let-7e and IL-10 messengerRNA (mRNA) and between the expression level of let-7e and proportion of IL-10(+) cells in stimulated PBMCs from healthy volunteers (r = -0.44, p = 0.0267 and r = -0.49, p = 0.0166, respectively). The expression levels of let-7e were significantly increased in HD patients compared with those in GD patients and healthy volunteers (p = 0.0003 and p = 0.0011, respectively). let-7e may be associated with the pathogenesis of HD through the regulation of intracellular IL-10 expression.

  19. General anesthesia-associatedDNA damage in peripheral blood mononuclear cells of surgical patients

    Institute of Scientific and Technical Information of China (English)

    Wang Haiyan; Zhou Qi; Fu Huo

    2011-01-01

    Objective:To evaluate retrospectively the effect of general anesthesia onDNA damage in the blood mononuclear cells (PBMCs) of surgical patients in order to provide evidence for a better nursing care during the procedure.Methods: Clinical charts of76 patients who underwent operation under general anesthesia and76 healthy control subjects with documented results of DNA damage extent inPBMCs from the single-cell gel electrophoresis(SCGE) or comet assay and serum contents of superoxide dismutase(SOD) and malondialdehyde(MDA)from biochemical analyses were reviewed. The percentage of comet PBMCs and tailDNAand serum contents of SOD and MAD were analyzed by student t-test.Results: Compared with healthy control subjects, generally anesthetized surgical patients had significantly higher % cometPBMCs and % tail DNA(P<0.05) and significantly lower serum concentrations ofSOD (P<0.05) and significantly higher serum concentrations ofMAD (P<0.05). Compared with levels before general anesthesia in surgical patients, % cometPBMCs, % tailDNA, and serum levels ofMADwere significantly higher (P<0.05 or0.01), and serum levels ofSOD were significantly lower (P<0.05), after general anesthesia.Conclusions: General anesthesia during surgery causes a certain degree of hypoxia and PBMC damage. Particular attention should be paid to monitoring and maintenance of blood oxygen saturation in patients undergoing surgery under general anesthesia.

  20. Equine colostral carbohydrates reduce lipopolysaccharide-induced inflammatory responses in equine peripheral blood mononuclear cells.

    Science.gov (United States)

    Vendrig, J C; Coffeng, L E; Fink-Gremmels, J

    2012-12-01

    Increasing evidence suggests that reactions to lipopolysaccharide (LPS), particularly in the gut, can be partly or completely mitigated by colostrum- and milk-derived oligosaccharides. Confirmation of this hypothesis could lead to the development of new therapeutic concepts. To demonstrate the influence of equine colostral carbohydrates on the inflammatory response in an in vitro model with equine peripheral blood mononuclear cells (PBMCs). Carbohydrates were extracted from mare colostrum, and then evaluated for their influence on LPS-induced inflammatory responses in PBMCs isolated from the same mares, mRNA expression of tumour necrosis factor-alpha, interleukin-6 and interleukin-10 was measured as well as the protein levels of tumour necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10). Equine colostral carbohydrates significantly reduced LPS-induced TNF-alpha protein at both times measured and significantly reduced LPS-induced TNF-alpha, IL-6 and IL-10 mRNA expression by PBMCs. Moreover, cell viability significantly increased in the presence of high concentrations of colostral carbohydrates. Carbohydrates derived from equine colostrum reduce LPS-induced inflammatory responses of equine PBMCs. Colostrum and milk-derived carbohydrates are promising candidates for new concepts in preventive and regenerative medicine.

  1. Proteomic analysis of chicken peripheral blood mononuclear cells after infection by Newcastle disease virus.

    Science.gov (United States)

    Deng, Xiaoyu; Cong, Yanlong; Yin, Renfu; Yang, Guilian; Ding, Chan; Yu, Shengqing; Liu, Xiufan; Wang, Chunfeng; Ding, Zhuang

    2014-12-01

    Characteristic clinical manifestations of Newcastle disease include leukopenia and immunosuppression. Peripheral blood mononuclear cells (PBMCs) are the main targets of Newcastle disease virus (NDV) infection. To survey changes in proteomic expression in chicken PBMCs following NDV infection, PBMC proteins from 30 chickens were separated using two- dimensional electrophoresis (2-DE) and subjected to mass spectrometry analysis. Quantitative intensity analysis showed that the expression of 78 proteins increased more than two-fold. Thirty-five proteins exhibited consistent changes in expression and 13 were identified as unique proteins by matrix assisted laser desorption ionization-time of flight mass spectrometer/mass spectrometer including three that were down-regulated and 10 that were up-regulated. These proteins were sorted into five groups based on function: macromolecular biosynthesis, cytoskeleton organization, metabolism, stress responses, and signal transduction. Furthermore, Western blot analysis confirmed the down-regulation of integrin-linked kinase expression and up-regulation of lamin A production. These data provide insight into the in vivo response of target cells to NDV infection at the molecular level. Additionally, results from this study have helped elucidate the molecular pathogenesis of NDV and may facilitate the development of new antiviral therapies as well as innovative diagnostic methods.

  2. A clinical study of autologous bone marrow mononuclear cells for cerebral palsy patients: a new frontier.

    Science.gov (United States)

    Sharma, Alok; Sane, Hemangi; Gokulchandran, Nandini; Kulkarni, Pooja; Gandhi, Sushant; Sundaram, Jyothi; Paranjape, Amruta; Shetty, Akshata; Bhagwanani, Khushboo; Biju, Hema; Badhe, Prerna

    2015-01-01

    Cerebral palsy is a nonprogressive heterogeneous group of neurological disorders with a growing rate of prevalence. Recently, cellular therapy is emerging as a potential novel treatment strategy for cerebral palsy. The various mechanisms by which cellular therapy works include neuroprotection, immunomodulation, neurorestoration, and neurogenesis. We conducted an open label, nonrandomized study on 40 cases of cerebral palsy with an aim of evaluating the benefit of cellular therapy in combination with rehabilitation. These cases were administered autologous bone marrow mononuclear cells intrathecally. The follow-up was carried out at 1 week, 3 months, and 6 months after the intervention. Adverse events of the treatment were also monitored in this duration. Overall, at six months, 95% of patients showed improvements. The study population was further divided into diplegic, quadriplegic, and miscellaneous group of cerebral palsy. On statistical analysis, a significant association was established between the symptomatic improvements and cell therapy in diplegic and quadriplegic cerebral palsy. PET-CT scan done in 6 patients showed metabolic improvements in areas of the brain correlating to clinical improvements. The results of this study demonstrate that cellular therapy may accelerate the development, reduce disability, and improve the quality of life of patients with cerebral palsy.

  3. A Clinical Study of Autologous Bone Marrow Mononuclear Cells for Cerebral Palsy Patients: A New Frontier

    Directory of Open Access Journals (Sweden)

    Alok Sharma

    2015-01-01

    Full Text Available Cerebral palsy is a nonprogressive heterogeneous group of neurological disorders with a growing rate of prevalence. Recently, cellular therapy is emerging as a potential novel treatment strategy for cerebral palsy. The various mechanisms by which cellular therapy works include neuroprotection, immunomodulation, neurorestoration, and neurogenesis. We conducted an open label, nonrandomized study on 40 cases of cerebral palsy with an aim of evaluating the benefit of cellular therapy in combination with rehabilitation. These cases were administered autologous bone marrow mononuclear cells intrathecally. The follow-up was carried out at 1 week, 3 months, and 6 months after the intervention. Adverse events of the treatment were also monitored in this duration. Overall, at six months, 95% of patients showed improvements. The study population was further divided into diplegic, quadriplegic, and miscellaneous group of cerebral palsy. On statistical analysis, a significant association was established between the symptomatic improvements and cell therapy in diplegic and quadriplegic cerebral palsy. PET-CT scan done in 6 patients showed metabolic improvements in areas of the brain correlating to clinical improvements. The results of this study demonstrate that cellular therapy may accelerate the development, reduce disability, and improve the quality of life of patients with cerebral palsy.

  4. Effect of spirulina on the secretion of cytokines from peripheral blood mononuclear cells.

    Science.gov (United States)

    Mao, T K; VAN DE Water, J; Gershwin, M E

    2000-01-01

    ABSTRACT The purpose of this study was to evaluate the immunomodulatory activity of Spirulina, a bluegreen alga used as a food supplement. The effects of Spirulina on the secretion of three cytokines from unstimulated and stimulated human peripheral blood mononuclear cells (PBMC) were examined. In resting PBMC, Spirulina stimulated secretion of interleukin (IL)-1beta, IL-4, and interferon (IFN)-gamma to nearly 2.0, 3.3, and 13.6 times basal levels, respectively. Spirulina induced levels of IFN-gamma (229 +/- 104 pg/ml) that were comparable to those seen after phytohemagglutinin (PHA) stimulation (476 +/- 121 pg/ml). However, it was much less mitogenic than PHA (13.1 +/- 6.9 pg/ml) with respect to the induction of IL-4 secretion (0.34 +/- 0.1 pg/ml). In PHA-stimulated cells, Spirulina enhanced secretion of IL-1beta, IL-4, and IFN-beta by 2.9, 4.0., and 1.6 times, respectively. Although Spirulina stimulates several cytokines, it is clearly more effective in the generation of a Thl-type response. This in vitro study offers additional data for consideration of the potential therapeutic benefits of Spirulina.

  5. Intermittent Hypoxia Alters Gene Expression in Peripheral Blood Mononuclear Cells of Healthy Volunteers.

    Science.gov (United States)

    Polotsky, Vsevolod Y; Bevans-Fonti, Shannon; Grigoryev, Dmitry N; Punjabi, Naresh M

    2015-01-01

    Obstructive sleep apnea is associated with high cardiovascular morbidity and mortality. Intermittent hypoxia of obstructive sleep apnea is implicated in the development and progression of insulin resistance and atherosclerosis, which have been attributed to systemic inflammation. Intermittent hypoxia leads to pro-inflammatory gene up-regulation in cell culture, but the effects of intermittent hypoxia on gene expression in humans have not been elucidated. A cross-over study was performed exposing eight healthy men to intermittent hypoxia or control conditions for five hours with peripheral blood mononuclear cell isolation before and after exposures. Total RNA was isolated followed by gene microarrays and confirmatory real time reverse transcriptase PCR. Intermittent hypoxia led to greater than two fold up-regulation of the pro-inflammatory gene toll receptor 2 (TLR2), which was not increased in the control exposure. We hypothesize that up-regulation of TLR2 by intermittent hypoxia may lead to systemic inflammation, insulin resistance and atherosclerosis in patients with obstructive sleep apnea.

  6. Overexpression of matrix metalloproteinases and their inhibitors in mononuclear inflammatory cells in breast cancer correlates with metastasis-relapse.

    Science.gov (United States)

    González, L O; Pidal, I; Junquera, S; Corte, M D; Vázquez, J; Rodríguez, J C; Lamelas, M L; Merino, A M; García-Muñiz, J L; Vizoso, F J

    2007-10-08

    An immunohistochemical study was performed using tissue microarrays and specific antibodies against matrix metalloproteinase (MMP)-1, -2, -7, -9, -11, -13 and -14, tissular inhibitors of metalloproteinase (TIMP)-1, -2 and -3. More than 2600 determinations on cancer specimens from 131 patients with primary ductal invasive tumours of the breast were performed. To identify specific groups of tumours with distinct expression profiles the data were analysed by unsupervised hierarchical cluster analysis by each cellular type. We did not find well-defined cluster of cases for tumour cells or fibroblastic cells. However, for mononuclear inflammatory cells the dendogram shows a first-order division of the tumours into two distinct MMP/TIMP molecular profiles, designated group 1 (n=89) and group 2 (n=42). Matrix metalloproteinase-7, -9, -11, -13 and -14, and TIMP-1 and -2, were identified as showing significant high expression in group 2 compared with group 1. Multivariate analysis demonstrated that clustering for mononuclear inflammatory cells was the most potent independent factor associated with distant relapse-free survival (group 2: 5.6 (3.5-9.6), P<0.001). We identify a phenotype of mononuclear inflammatory cells infiltrating tumours, which is associated with the development of distant metastasis. Therefore, this finding suggests that these host inflammatory cells could be a possible target for inhibition of metastasis.

  7. The Study of Chlamydia Pneumoniae DNA in the Peripheral Blood Mononuclear Cell of Coronary Heart Disease

    Institute of Scientific and Technical Information of China (English)

    Li Tao; Xu Xiang Guang; Zhang Guo Liang; Fang Weihua

    2004-01-01

    Objectives To detection of chlamydia pneumoniae (Cpn) DNA in the circulating mononuclear cell fractions of coronary heart disease and to investigate the association between infection with chlamydia pneumoniae and coronary heart disease (CHD) and prospectively whether blood -based nested polymerase chain reaction ( nPCR ) is useful in identifying Cpn infection. Methods The peripheral blood mononuclear cell (PBMC) Cpn DNA was examined using nPCR technique and confirmed by electrophoresis in 150 patients with CHD. Select 55 patients with clinical suspected CHD but angiography result are normal as control group (CG). Then we conducted a prospective , randomized, double - blind, placebo -controlled study of 6 months of azithromycin and placebo treatment in CHD group. Patients with Cpn DNA positive were then randomized to receive azithromycin or placebo. After treatment blood sample were collected for repeated measurement . Results Chlamydia pneumoniae DNA was detected in 49(32.7% ) of 150persons with CHD and in 1 ( 1.8% ) of 55 persons with control group,odds ratio 26.2, 95% confidence interva13.52 - 194.98. The positivity rates of nPCR in CHD groups were higher than those in control group. 16 cases (29. 1% ) in latent coronary heart diseases(LCHD) group , 19 cases (39.6%) in unstable angina(UAP) group ,and 14 cases (29.9%) in acute myocardial infarction (AMI)group were Cpn positive by nPCR. There were no significant difference among in AMIUAP and LCHD group. There were significiant difference in Cpn DNA negative rates after the azithromycin and the placebo treatment. Conclusions Chlamydia pneumoniae is present in PBMC of a significant proportion of persons with CHD. The potential role of chlamydia pneumoniae in coronary atherosclerosis may therefore be more related to acceleration of disease or systemic effects by persistent infection than to sudden initiation of progressive coronary artery disease by acute infection. The detection of Cpn DNA in PBMC with nPCR may be

  8. Use of human umbilical cord blood mononuclear cells to prevent perinatal brain injury: a preclinical study.

    Science.gov (United States)

    Dalous, Jérémie; Pansiot, Julien; Pham, Hoa; Chatel, Paul; Nadaradja, Céline; D'Agostino, Irene; Vottier, Gaëlle; Schwendimann, Leslie; Vanneaux, Valérie; Charriaut-Marlangue, Christiane; Titomanlio, Luigi; Gressens, Pierre; Larghero, Jérôme; Baud, Olivier

    2013-01-01

    Cerebral palsy (CP) is the most frequent neurological disorder associated with perinatal injury of the developing brain. Major brain lesions associated with CP are white matter damage (WMD) in preterm infants and cortico-subcortical lesions in term newborns. Cell therapy is considered promising for the repair of brain damage. Human umbilical cord blood mononuclear cells (hUCB-MNCs) are a rich source of various stem cells that could be of interest in repairing perinatal brain damage. Our goal was to investigate the potential of hUCB-MNCs to prevent or repair brain lesions in an animal model of excitotoxic brain injury. We induced neonatal brain lesions using intracranial injections of ibotenate, a glutamate agonist, in 5-day-old rat pups. hUCB-MNCs were injected either intraperitoneally (i.p.) or intravenously (i.v.) soon or 24 h after ibotenate injection, and their neurological effects were assessed using histology and immunohistochemistry. hUCB-MNCs injected i.p. did not reach the systemic circulation but high amounts induced a significant systemic inflammatory response and increased the WMD induced by the excitotoxic insult. This effect was associated with a significant 40% increase in microglial activation around the white matter lesion. hUCB-MNCs injected i.v. soon or 24 h after the excitotoxic insult did not affect lesion size, microglial activation, astroglial cell density, or cell proliferation within the developing white matter or cortical plate at any concentration used. We demonstrated that hUCB-MNCs could not integrate into the developing brain or promote subsequent repair in most conditions tested. We found that the intraperitoneal injection of high amounts of hUCB-MNCs aggravated WMD and was associated with systemic inflammation.

  9. Cytokine production by bone marrow mononuclear cells in inherited bone marrow failure syndromes.

    Science.gov (United States)

    Matsui, Ken; Giri, Neelam; Alter, Blanche P; Pinto, Ligia A

    2013-10-01

    Fanconi anaemia (FA), dyskeratosis congenita (DC), Diamond-Blackfan anaemia (DBA), and Shwachman-Diamond syndrome (SDS) are characterized by the progressive development of bone marrow failure. Overproduction of tumour necrosis factor-α (TNF-α) from activated bone marrow T-cells has been proposed as a mechanism of FA-related aplasia. Whether such overproduction occurs in the other syndromes is unknown. We conducted a comparative study on bone marrow mononuclear cells to examine the cellular subset composition and cytokine production. We found lower proportions of haematopoietic stem cells in FA, DC, and SDS, and a lower proportion of monocytes in FA, DC, and DBA compared with controls. The T- and B-lymphocyte proportions were similar to controls, except for low B-cells in DC. We did not observe overproduction of TNF-α or IFN-γ by T-cells in any patients. Induction levels of TNF-α, interleukin (IL)-6, IL-1β, IL-10, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor in monocytes stimulated with high-dose lipopolysaccharide (LPS) were similar at 4 h but lower at 24 h when compared to controls. Unexpectedly, patient samples showed a trend toward higher cytokine level in response to low-dose (0·001 μg/ml) LPS. Increased sensitivity to LPS may have clinical implications and could contribute to the development of pancytopenia by creating a chronic subclinical inflammatory micro-environment in the bone marrow. © Published 2013. This article is a U.S. Government work and is in the public domain in the U.S.A.

  10. Validation of a quantitative real-time PCR assay for HTLV-1 proviral load in peripheral blood mononuclear cells.

    Science.gov (United States)

    Rosadas, Carolina; Cabral-Castro, Mauro Jorge; Vicente, Ana Carolina Paulo; Peralta, José Mauro; Puccioni-Sohler, Marzia

    2013-11-01

    The objective of this study was to validate a TaqMan real-time PCR assay for HTLV-1 proviral load detection in peripheral blood mononuclear cells. TARL-2 cells were used to generate a standard curve. Peripheral blood mononuclear cell gDNA from 27 seropositive and 23 seronegative samples was analyzed. The sensitivity, specificity, accuracy, precision, dynamic range of the standard curve and qPCR efficiency were evaluated. All of the positive samples amplified the target gene. All of the negative samples amplified only the control gene (β-actin). The assay presented 100% specificity and sensibility. The intra- and inter-assay variability was 2.4% and 2.2%, respectively. The qPCR efficiency, slope and correlation coefficients (r2) were all acceptable. The limit of detection was 1 copy/rxn. This assay can reliably quantify HTLV-1 proviral load.

  11. Peripheral blood mononuclear cell-converted induced pluripotent stem cells (iPSCs) from an early onset Alzheimer's patient.

    Science.gov (United States)

    Lee, Han-Kyu; Morin, Peter; Xia, Weiming

    2016-03-01

    Improvement in transduction efficiency makes it possible to convert blood cells into induced pluripotent stem cells (iPSC). In this study, we generated an iPSC line from peripheral blood mononuclear cells (PBMC) donated by a patient who exhibited memory deficit at age 59; outcome of positron emission tomography scan is consistent with a diagnosis of Alzheimer's disease. Integration-free CytoTune-iPS Sendai Reprogramming factors which include Sendai virus particles of the four Yamanaka factors Oct4, Sox2, Klf4, and c-Myc were introduced to PBMC to convert them to iPSCs without retention of virus. Three germ layer differentiation was induced to demonstrate the pluripotency of these iPSCs. Published by Elsevier B.V.

  12. Peripheral blood mononuclear cell-converted induced pluripotent stem cells (iPSCs from an early onset Alzheimer's patient

    Directory of Open Access Journals (Sweden)

    Han-Kyu Lee

    2016-03-01

    Full Text Available Improvement in transduction efficiency makes it possible to convert blood cells into induced pluripotent stem cells (iPSC. In this study, we generated an iPSC line from peripheral blood mononuclear cells (PBMC donated by a patient who exhibited memory deficit at age 59; outcome of positron emission tomography scan is consistent with a diagnosis of Alzheimer's disease. Integration-free CytoTune-iPS Sendai Reprogramming factors which include Sendai virus particles of the four Yamanaka factors Oct4, Sox2, Klf4, and c-Myc were introduced to PBMC to convert them to iPSCs without retention of virus. Three germ layer differentiation was induced to demonstrate the pluripotency of these iPSCs.

  13. Peripheral Blood Mononuclear Cells Enhance Cartilage Repair in in vivo Osteochondral Defect Model.

    Directory of Open Access Journals (Sweden)

    Niina Hopper

    Full Text Available This study characterized peripheral blood mononuclear cells (PBMC in terms of their potential in cartilage repair and investigated their ability to improve the healing in a pre-clinical large animal model. Human PBMCs were isolated with gradient centrifugation and adherent PBMC's were evaluated for their ability to differentiate into adipogenic, chondrogenic and osteogenic lineages and also for their expression of musculoskeletal genes. The phenotype of the PBMCs was evaluated using Stro-1, CD34, CD44, CD45, CD90, CD106, CD105, CD146 and CD166 cell surface markers. Osteochondral defects were created in the medial femoral condyle (MFC of 24 Welsh mountain sheep and evaluated at a six month time point. Four cell treatment groups were evaluated in combination with collagen-GAG-scaffold: (1 MSC alone; (2 MSCs and PBMCs at a ratio of 20:1; (3 MSCs and PBMC at a ratio of 2:1 and (4 PBMCs alone. Samples from the surgical site were evaluated for mechanical properties, ICRS score and histological repair. Fresh PBMC samples were 90% positive for hematopoietic cell surface markers and negative for the MSC antibody panel (<1%, p = 0.006. However, the adherent PBMC population expressed mesenchymal stem cell markers in hypoxic culture and lacked CD34/45 positive cells (<0.2%. This finding demonstrated that the adherent cells had acquired an MSC-like phenotype and transformed in hypoxia from their original hematopoietic lineage. Four key genes in muskuloskeletal biology were significantly upregulated in adherent PBMCs by hypoxia: BMP2 4.2-fold (p = 0.0007, BMP6 10.7-fold (p = 0.0004, GDF5 2.0-fold (p = 0.002 and COL1 5.0-fold (p = 0.046. The monolayer multilineage analysis confirmed the trilineage mesenchymal potential of the adherent PBMCs. PBMC cell therapy was equally good as bone marrow MSC therapy for defects in the ovine large animal model. Our results show that PBMCs support cartilage healing and oxygen tension of the environment was found to have a key

  14. Effect of dietary fat saturation and cholesterol on low density lipoprotein degradation by mononuclear cells of Cebus monkeys.

    Science.gov (United States)

    Kuo, P C; Rudd, M A; Nicolosi, R; Loscalzo, J

    1989-01-01

    The mechanism by which dietary unsaturated fatty acids lower low density lipoprotein (LDL) cholesterol is unknown. Unsaturated fatty acids incorporated into the cell membrane can increase membrane fluidity and, as a result, dramatically alter membrane-dependent cell functions. Therefore, we examined the effect of long-term dietary consumption of corn oil and coconut oil with and without cholesterol in amounts equivalent to those of a typical Western diet on the degradation of human LDL by peripheral blood mononuclear cells in Cebus albifrons monkeys. Cellular LDL degradation was dramatically enhanced in the mononuclear cells isolated from animals fed corn oil in comparison with those from animals fed coconut oil. The addition of cholesterol to the diets resulted in a slight attenuation of LDL degradation in the corn oil group while no effect was noted in the coconut oil group. Crossover LDL binding and degradation experiments with LDL isolated from animals fed corn oil diets and coconut oil diets demonstrated increased binding and degradation of LDL in mononuclear cells from animals fed corn oil diets. Enhanced mononuclear cell LDL degradation was accompanied by increased cellular cis-unsaturated fatty acyl content, increased membrane fluidity, and decreased plasma cholesterol. Increased cellular cis-unsaturated fatty acyl content with its concomitant increase in membrane fluidity mirrored the dietary lipid profile of the host animal. A linear relationship was observed between cellular LDL degradation and both cellular cis-unsaturated fatty acyl content and membrane fluidity. These observations parallel results noted in whole-animal LDL catabolic studies with these same animals described elsewhere. These data suggest a novel mechanism by which dietary unsaturated fatty acids exert their LDL-lowering effect.

  15. Dynamic changes in the proteome of human peripheral blood mononuclear cells with low dose ionizing radiation.

    Science.gov (United States)

    Nishad, S; Ghosh, Anu

    2016-02-01

    Humans are continually exposed to ionizing radiation from natural as well as anthropogenic sources. Though biological effects of high dose radiation exposures have been well accepted, studies on low-to-moderate dose exposures (in the range of 50-500 mGy) have been strongly debated even as researchers continue to search for elusive 'radiation signatures' in humans. Proteins are considered as dynamic functional players that drive cellular responses. However, there is little proteomic information available in context of human exposure to ionizing radiation. In this study, we determined differential expressed proteins in G0 peripheral blood mononuclear cells (PBMCs) from healthy individuals 1h and 4h after 'ex vivo' exposure with two radiation doses (300 mGy and 1 Gy). Twenty-three proteins were found to be significantly altered in irradiated cells when compared to sham irradiated cells with fold change ± 1.5-fold (p ≤ 0.05), with only three proteins showing ≥ 2.5-fold change, either with dose or with time. Mass spectrometry analyses identified redox sensor protein, chloride intracellular channel protein 1 (CLIC-1), the antioxidant protein, peroxiredoxin-6 and the pro-survival molecular chaperone 78 KDa glucose regulated protein (GRP78) among the 23 modulated proteins. The mean coefficient of variation (CV) for the twenty-three radiation responsive protein spots was found to be 33.7% for 300 mGy and 48.3% for 1 Gy. We thus, conclude that the radiation proteomic response of G0 human PBMCs, which are in the resting stage of the cell cycle, involves moderate upregulation of protective mechanisms, with low inter-individual variability. This study will help further our understanding of cellular effects of low dose acute radiation in humans and contribute toward differential biomarker discovery. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Elevated peripheral blood mononuclear cell-derived superoxide production in healthy young black men.

    Science.gov (United States)

    Deo, Shekhar H; Holwerda, Seth W; Keller, David M; Fadel, Paul J

    2015-03-01

    Several studies have demonstrated that blacks exhibit elevations in systemic oxidative stress. However, the source(s) and mechanism(s) contributing to the elevation in oxidative stress remain unclear. Given that peripheral blood mononuclear cells (PBMCs) can be a major source of NADPH oxidase-derived superoxide production, we tested the hypothesis that young black men demonstrate greater superoxide production and NADPH oxidase expression in PBMCs compared with whites. PBMCs were freshly isolated from whole blood in young normotensive black (n = 18) and white (n = 16) men. Intracellular superoxide production in PBMCs was measured using dihydroethidium fluorescence, protein expression of NADPH oxidase subunits, gp91(phox) (membranous) and p47(phox) (cytosolic) in PBMCs were assessed using Western blot analysis, and plasma protein carbonyls were measured as a marker of systemic oxidative stress. Black men showed elevated intracellular superoxide production (4.3 ± 0.5 vs. 2.0 ± 0.6 relative fluorescence units; black men vs. white men, P superoxide production or NADPH oxidase subunit protein expression. These findings indicate that black men exhibit greater resting PBMC-derived superoxide production and an upregulation of the NADPH oxidase pathway with a possible contribution to increases in systemic oxidative stress.

  17. Changes in transcriptional output of human peripheral blood mononuclear cells following resistance exercise.

    Science.gov (United States)

    Carlson, Lara A; Tighe, S W; Kenefick, R W; Dragon, J; Westcott, N W; Leclair, R J

    2011-12-01

    Various types of exercise alter the population of circulating peripheral blood mononuclear cells (PBMCs) and change their transcriptional output. This work examines changes in PBMC populations and transcription in response to resistance exercise training (RET), and identify key transcriptional changes in PBMCs that may play a role in altering peripheral tissues in response to RET. Ten resistance-trained men (20-24 years), performed an acute bout of RET for ~30 min following a 12 h fast. Venous blood was sampled at rest, immediately following exercise, and at 2 h post-exercise and analyzed for total and differential leukocytes and global gene expression using Affymetrix Genechips. Results showed elevated leukocytes, monocytes, lymphocytes, and lactate values immediately post-exercise (P transcriptional changes in pathways related to immune response, inflammation, and cellular communication. The change in PBMC population (2 h time point) correlated with a dramatic decrease in the expression of CD160, and XCL1, markers of lymphocyte populations. At the 2 h recovery time point upregulation of matrix metalloproteinase 9, orosomucoid 1, dishevelled-associated activator of morphogenesis 2, and arginase 1 suggest an induction in muscle damage and repair during this time frame. These results demonstrate that an acute bout of RET disrupts cellular homeostasis, induces a transient redistribution of certain leukocytes, and results in transcriptional changes in PBMCs translating into systemic changes in response to RET.

  18. Reduced pCREB in Alzheimer's disease prefrontal cortex is reflected in peripheral blood mononuclear cells

    Science.gov (United States)

    Bartolotti, N; Bennett, D A; Lazarov, O

    2016-01-01

    Cyclic-AMP response element-binding protein (CREB) signaling has a critical role in the formation of memories. CREB signaling is dysfunctional in the brains of mouse models of Alzheimer's disease (AD), and evidence suggests that CREB signaling may be disrupted in human AD brains as well. Here, we show that both CREB and its activated form pCREB-Ser133 (pCREB) are reduced in the prefrontal cortex of AD patients. Similarly, the transcription cofactors CREB-binding protein (CBP) and p300 are reduced in the prefrontal cortex of AD patients, indicating additional dysfunction of CREB signaling in AD. Importantly, we show that pCREB expression is reduced in peripheral blood mononuclear cells (PBMC) of AD subjects. In addition, pCREB levels in PBMC positively correlated with pCREB expression in the postmortem brain of persons with AD. These results suggest that pCREB expression in PBMC may be indicative of its expression in the brain, and thus offers the intriguing possibility of pCREB as a biomarker of cognitive function and disease progression in AD. PMID:27480489

  19. Gene expression profiles in peripheral blood mononuclear cells of SARS patients

    Institute of Scientific and Technical Information of China (English)

    Shi-Yan Yu; Yun-Wen Hu; Xiao-Ying Liu; Wei Xiong; Zhi-Tong Zhou; Zheng-Hong Yuan

    2005-01-01

    AIM: To investigate the role of inflammatory and anti-viral genes in the pathogenesis of SARS.METHODS: cDNA microarrays were used to screen the gene expression profiles of peripheral blood mononuclear cells (PBMCs) in two SARS patients (one in the acute severe phase and the other in the convalescent phase)and a healthy donor. In addition, real-time qualitative PCR was also performed to verify the reproducibility of the microarray results. The data were further analyzed.RESULTS: Many inflammatory and anti-viral genes were differentially expressed in SARS patients. Compared to the healthy control or the convalescent case, plenty of pro-inflammatory cytokines such as IL-1, TNF-α, IL-8, and MAPK signaling pathway were significantly upregulated in the acute severe case. However, anti-inflammatory agents such as IL-4 receptor, IL-13 receptor, IL-1Ra,and TNF-α-induced proteins 3 and 6 also increased dramatically in the acute severe case. On the contrary, a lot of IFN-stimulated genes like PKR, GBP-1 and 2, CXCL-10and 11, and JAK/STAT signal pathway were downregulated in the acute severe case compared to the convalescent case.CONCLUSION: Gene expression in SARS patients mirrors a host state of inflammation and anti-viral immunity at the transcription level, and understanding of gene expression profiles may make contribution to further studies of the SARS pathogenesis.

  20. Characterization of Bone Marrow Mononuclear Cells on Biomaterials for Bone Tissue Engineering In Vitro

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    Dirk Henrich

    2015-01-01

    Full Text Available Bone marrow mononuclear cells (BMCs are suitable for bone tissue engineering. Comparative data regarding the needs of BMC for the adhesion on biomaterials and biocompatibility to various biomaterials are lacking to a large extent. Therefore, we evaluated whether a surface coating would enhance BMC adhesion and analyze the biocompatibility of three different kinds of biomaterials. BMCs were purified from human bone marrow aspirate samples. Beta tricalcium phosphate (β-TCP, without coating or coated with fibronectin or human plasma, demineralized bone matrix (DBM, and bovine cancellous bone (BS were assessed. Seeding efficacy on β-TCP was 95% regardless of the surface coating. BMC demonstrated a significantly increased initial adhesion on DBM and β-TCP compared to BS. On day 14, metabolic activity was significantly increased in BMC seeded on DBM in comparison to BMC seeded on BS. Likewise increased VEGF-synthesis was observed on day 2 in BMC seeded on DBM when compared to BMC seeded on BS. The seeding efficacy of BMC on uncoated biomaterials is generally high although there are differences between these biomaterials. Beta-TCP and DBM were similar and both superior to BS, suggesting either as suitable materials for spatial restriction of BMC used for regenerative medicine purposes in vivo.

  1. Response of porcine peripheral blood mononuclear cells to CpG-containing oligodeoxynucleotide

    DEFF Research Database (Denmark)

    Kamstrup, Søren; Verthelyi, D.; Klinman, D.M.

    2001-01-01

    Exposure to bacterial DNA generates a "danger signal" that stimulates cellular elements of the mammalian immune system to proliferate and/or secrete cytokines. Stimulation is critically dependent on hexameric motifs that contain an unmethylated CpG dinucleotide: these are commonly found in bacter......Exposure to bacterial DNA generates a "danger signal" that stimulates cellular elements of the mammalian immune system to proliferate and/or secrete cytokines. Stimulation is critically dependent on hexameric motifs that contain an unmethylated CpG dinucleotide: these are commonly found...... in bacterial but not vertebrate DNA. Different motifs are optimally stimulatory in different species. This work examines whether oligodeoxynucleotides (ODNs) containing CpG motifs stimulate peripheral blood mononuclear cells from pigs. Results show that pigs respond to CpG ODN by proliferating and secreting IL......-6, IL-12 and TNF-alpha. By screening a large panel (>100) of ODNs, the palindromic hexamer 'ATCGAT' was identified as being optimally active in all animals examined (N = 10). These findings are the first to establish the immunostimulatory activity of CpG ODN in pigs, and suggest that the therapeutic...

  2. Flow cytometric probing of mitochondrial function in equine peripheral blood mononuclear cells

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    Coignoul Freddy

    2007-09-01

    Full Text Available Abstract Background The morphopathological picture of a subset of equine myopathies is compatible with a primary mitochondrial disease, but functional confirmation in vivo is still pending. The cationic dye JC-1 exhibits potential-dependent accumulation in mitochondria that is detectable by a fluorescence shift from green to orange. As a consequence, mitochondrial membrane potential can be optically measured by the orange/green fluorescence intensity ratio. A flow cytometric standardized analytic procedure of the mitochondrial function of equine peripheral blood mononuclear cells is proposed along with a critical appraisal of the crucial questions of technical aspects, reproducibility, effect of time elapsed between blood sampling and laboratory processing and reference values. Results The JC-1-associated fluorescence orange and green values and their ratio were proved to be stable over time, independent of age and sex and hypersensitive to intoxication with a mitochondrial potential dissipator. Unless time elapsed between blood sampling and laboratory processing does not exceed 5 hours, the values retrieved remain stable. Reference values for clinically normal horses are given. Conclusion Whenever a quantitative measurement of mitochondrial function in a horse is desired, blood samples should be taken in sodium citrate tubes and kept at room temperature for a maximum of 5 hours before the laboratory procedure detailed here is started. The hope is that this new test may help in confirming, studying and preventing equine myopathies that are currently imputed to mitochondrial dysfunction.

  3. 10th NTES Conference: Nickel and Arsenic Compounds Alter the Epigenome of Peripheral Blood Mononuclear Cells.

    Science.gov (United States)

    Brocato, Jason; Costa, Max

    2015-01-01

    The mechanisms that underlie metal carcinogenesis are the subject of intense investigation; however, data from in vitro and in vivo studies are starting to piece together a story that implicates epigenetics as a key player. Data from our lab has shown that nickel compounds inhibit dioxygenase enzymes by displacing iron in the active site. Arsenic is hypothesized to inhibit these enzymes by diminishing ascorbate levels--an important co-factor for dioxygenases. Inhibition of histone demethylase dioxygenases can increase histone methylation levels, which also may affect gene expression. Recently, our lab conducted a series of investigations in human subjects exposed to high levels of nickel or arsenic compounds. Global levels of histone modifications in peripheral blood mononuclear cells (PBMCs) from exposed subjects were compared to low environmentally exposed controls. Results showed that nickel increased H3K4me3 and decreased H3K9me2 globally. Arsenic increased H3K9me2 and decreased H3K9ac globally. Other histone modifications affected by arsenic were sex-dependent. Nickel affected the expression of 2756 genes in human PBMCs and many of the genes were involved in immune and carcinogenic pathways. This review will describe data from our lab that demonstrates for the first time that nickel and arsenic compounds affect global levels of histone modifications and gene expression in exposed human populations.

  4. Changes in Proteome Profile of Peripheral Blood Mononuclear Cells in Chronic Chagas Disease

    Science.gov (United States)

    Soman, Kizhake V.; Zago, Maria P.; Koo, Sue-Jie; Spratt, Heidi; Stafford, Susan; Blell, Zinzi N.; Gupta, Shivali; Nuñez Burgos, Julio; Barrientos, Natalia; Brasier, Allan R.

    2016-01-01

    Trypanosoma cruzi (Tc) infection causes chagasic cardiomyopathy; however, why 30–40% of the patients develop clinical disease is not known. To discover the pathomechanisms in disease progression, we obtained the proteome signature of peripheral blood mononuclear cells (PBMCs) of normal healthy controls (N/H, n = 30) and subjects that were seropositive for Tc-specific antibodies, but were clinically asymptomatic (C/A, n = 25) or clinically symptomatic (C/S, n = 28) with cardiac involvement and left ventricular dysfunction. Protein samples were labeled with BODIPY FL-maleimide (dynamic range: > 4 orders of magnitude, detection limit: 5 f-mol) and resolved by two-dimensional gel electrophoresis (2D-GE). After normalizing the gel images, protein spots that exhibited differential abundance in any of the two groups were analyzed by mass spectrometry, and searched against UniProt human database for protein identification. We found 213 and 199 protein spots (fold change: |≥ 1.5|, p93% prediction success in classifying infected individuals with no disease and those with cardiac involvement and LV dysfunction. In conclusion, we have identified molecular pathways and a panel of proteins that could aid in detecting seropositive individuals at risk of developing cardiomyopathy. PMID:26919708

  5. Changes in Proteome Profile of Peripheral Blood Mononuclear Cells in Chronic Chagas Disease.

    Directory of Open Access Journals (Sweden)

    Nisha Jain Garg

    2016-02-01

    Full Text Available Trypanosoma cruzi (Tc infection causes chagasic cardiomyopathy; however, why 30-40% of the patients develop clinical disease is not known. To discover the pathomechanisms in disease progression, we obtained the proteome signature of peripheral blood mononuclear cells (PBMCs of normal healthy controls (N/H, n = 30 and subjects that were seropositive for Tc-specific antibodies, but were clinically asymptomatic (C/A, n = 25 or clinically symptomatic (C/S, n = 28 with cardiac involvement and left ventricular dysfunction. Protein samples were labeled with BODIPY FL-maleimide (dynamic range: > 4 orders of magnitude, detection limit: 5 f-mol and resolved by two-dimensional gel electrophoresis (2D-GE. After normalizing the gel images, protein spots that exhibited differential abundance in any of the two groups were analyzed by mass spectrometry, and searched against UniProt human database for protein identification. We found 213 and 199 protein spots (fold change: |≥ 1.5|, p93% prediction success in classifying infected individuals with no disease and those with cardiac involvement and LV dysfunction. In conclusion, we have identified molecular pathways and a panel of proteins that could aid in detecting seropositive individuals at risk of developing cardiomyopathy.

  6. Autologous Bone Marrow Mononuclear Cell Therapy for Autism: An Open Label Proof of Concept Study

    Directory of Open Access Journals (Sweden)

    Alok Sharma

    2013-01-01

    Full Text Available Cellular therapy is an emerging therapeutic modality with a great potential for the treatment of autism. Recent findings show that the major underlying pathogenetic mechanisms of autism are hypoperfusion and immune alterations in the brain. So conceptually, cellular therapy which facilitates counteractive processes of improving perfusion by angiogenesis and balancing inflammation by immune regulation would exhibit beneficial clinical effects in patients with autism. This is an open label proof of concept study of autologous bone marrow mononuclear cells (BMMNCs intrathecal transplantation in 32 patients with autism followed by multidisciplinary therapies. All patients were followed up for 26 months (mean 12.7. Outcome measures used were ISAA, CGI, and FIM/Wee-FIM scales. Positron Emission Tomography-Computed Tomography (PET-CT scan recorded objective changes. Out of 32 patients, a total of 29 (91% patients improved on total ISAA scores and 20 patients (62% showed decreased severity on CGI-I. The difference between pre- and postscores was statistically significant (P<0.001 on Wilcoxon matched-pairs signed rank test. On CGI-II 96% of patients showed global improvement. The efficacy was measured on CGI-III efficacy index. Few adverse events including seizures in three patients were controlled with medications. The encouraging results of this leading clinical study provide future directions for application of cellular therapy in autism.

  7. Bone Marrow Mononuclear Cell Transplantation Restores Inflammatory Balance of Cytokines after ST Segment Elevation Myocardial Infarction.

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    Kirsi Alestalo

    Full Text Available Acute myocardial infarction (AMI launches an inflammatory response and a repair process to compensate cardiac function. During this process, the balance between proinflammatory and anti-inflammatory cytokines is important for optimal cardiac repair. Stem cell transplantation after AMI improves tissue repair and increases the ventricular ejection fraction. Here, we studied in detail the acute effect of bone marrow mononuclear cell (BMMNC transplantation on proinflammatory and anti-inflammatory cytokines in patients with ST segment elevation myocardial infarction (STEMI.Patients with STEMI treated with thrombolysis followed by percutaneous coronary intervention (PCI were randomly assigned to receive either BMMNC or saline as an intracoronary injection. Cardiac function was evaluated by left ventricle angiogram during the PCI and again after 6 months. The concentrations of 27 cytokines were measured from plasma samples up to 4 days after the PCI and the intracoronary injection.Twenty-six patients (control group, n = 12; BMMNC group, n = 14 from the previously reported FINCELL study (n = 80 were included to this study. At day 2, the change in the proinflammatory cytokines correlated with the change in the anti-inflammatory cytokines in both groups (Kendall's tau, control 0.6; BMMNC 0.7. At day 4, the correlation had completely disappeared in the control group but was preserved in the BMMNC group (Kendall's tau, control 0.3; BMMNC 0.7.BMMNC transplantation is associated with preserved balance between pro- and anti-inflammatory cytokines after STEMI in PCI-treated patients. This may partly explain the favorable effect of stem cell transplantation after AMI.

  8. The autologous bone marrow mononuclear cell transplantation by intracoronary route treat patients with severe heart failure after myocardial infarction

    Institute of Scientific and Technical Information of China (English)

    高连如

    2006-01-01

    Objective To investigate the chronic effects of intracoronary autologous bone marrow mononuclear cell (BM-MNCs) transplantation in patients with refractory heart failure (RIHF) after myocardial infarction. Methods Thirty patients with RIHF (LVEF<40%) were enrolled in this nonrandomized study, autologous BM-MNCs (5.0±0.7)×107 were transplanted with via infarct-related coronary artery in 16 patients and 14 patients received

  9. Observation on the safety:clinical trail on intracoronary autologus bone marrow mononuclear cells transplantation for acute myocardiol infarction

    Institute of Scientific and Technical Information of China (English)

    姚康

    2006-01-01

    Objective To investigate the safety of autologous bone marrow mononuclear cell (BM-MNCs) transplantation by intracoronary infusion in patients with acute myocardial infarction (AMI). Methods One hundred and eighty-four patients with AMI treated with percutaneous coronary intervention (PCI) were randomized in a 1:1 way to either intracoronary transplantation of autologous BM-MNCs (n =92) right after PCI or to sodium chloride concluding heparin (controlled, n=92) via a micro

  10. Transplantation of mononuclear cells from human umbilical cord blood promotes functional recovery after traumatic spinal cord injury in Wistar rats

    Energy Technology Data Exchange (ETDEWEB)

    Rodrigues, L.P. [Programa de Pós-Graduação em Neurociências, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil); Iglesias, D. [Laboratório de Hematologia e Células-Tronco, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil); Nicola, F.C. [Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil); Steffens, D. [Laboratório de Hematologia e Células-Tronco, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil); Valentim, L.; Witczak, A.; Zanatta, G. [Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil); Achaval, M. [Departamento de Ciências Morfológicas, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil); Pranke, P. [Laboratório de Hematologia e Células-Tronco, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil); Netto, C.A. [Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil)

    2011-12-23

    Cell transplantation is a promising experimental treatment for spinal cord injury. The aim of the present study was to evaluate the efficacy of mononuclear cells from human umbilical cord blood in promoting functional recovery when transplanted after a contusion spinal cord injury. Female Wistar rats (12 weeks old) were submitted to spinal injury with a MASCIS impactor and divided into 4 groups: control, surgical control, spinal cord injury, and one cell-treated lesion group. Mononuclear cells from umbilical cord blood of human male neonates were transplanted in two experiments: a) 1 h after surgery, into the injury site at a concentration of 5 x 10{sup 6} cells diluted in 10 µL 0.9% NaCl (N = 8-10 per group); b) into the cisterna magna, 9 days after lesion at a concentration of 5 x 10{sup 6} cells diluted in 150 µL 0.9% NaCl (N = 12-14 per group). The transplanted animals were immunosuppressed with cyclosporin-A (10 mg/kg per day). The BBB scale was used to evaluate motor behavior and the injury site was analyzed with immunofluorescent markers to label human transplanted cells, oligodendrocytes, neurons, and astrocytes. Spinal cord injury rats had 25% loss of cord tissue and cell treatment did not affect lesion extension. Transplanted cells survived in the injured area for 6 weeks after the procedure and both transplanted groups showed better motor recovery than the untreated ones (P < 0.05). The transplantation of mononuclear cells from human umbilical cord blood promoted functional recovery with no evidence of cell differentiation.

  11. Transplantation of mononuclear cells from human umbilical cord blood promotes functional recovery after traumatic spinal cord injury in Wistar rats

    Directory of Open Access Journals (Sweden)

    L.P. Rodrigues

    2012-01-01

    Full Text Available Cell transplantation is a promising experimental treatment for spinal cord injury. The aim of the present study was to evaluate the efficacy of mononuclear cells from human umbilical cord blood in promoting functional recovery when transplanted after a contusion spinal cord injury. Female Wistar rats (12 weeks old were submitted to spinal injury with a MASCIS impactor and divided into 4 groups: control, surgical control, spinal cord injury, and one cell-treated lesion group. Mononuclear cells from umbilical cord blood of human male neonates were transplanted in two experiments: a 1 h after surgery, into the injury site at a concentration of 5 x 10(6 cells diluted in 10 µL 0.9% NaCl (N = 8-10 per group; b into the cisterna magna, 9 days after lesion at a concentration of 5 x 10(6 cells diluted in 150 µL 0.9% NaCl (N = 12-14 per group. The transplanted animals were immunosuppressed with cyclosporin-A (10 mg/kg per day. The BBB scale was used to evaluate motor behavior and the injury site was analyzed with immunofluorescent markers to label human transplanted cells, oligodendrocytes, neurons, and astrocytes. Spinal cord injury rats had 25% loss of cord tissue and cell treatment did not affect lesion extension. Transplanted cells survived in the injured area for 6 weeks after the procedure and both transplanted groups showed better motor recovery than the untreated ones (P < 0.05. The transplantation of mononuclear cells from human umbilical cord blood promoted functional recovery with no evidence of cell differentiation.

  12. Responses of synovial fluid and peripheral blood mononuclear cells to bacterial antigens and autologous antigen presenting cells.

    Science.gov (United States)

    Klasen, I S; Melief, M J; Swaak, T J; Severijnen, A J; Hazenberg, M P

    1993-01-01

    The specificity of T cells in the inflamed joints of patients with rheumatoid arthritis (RA) has been the subject of much study. Bacterial antigens are suspect in the aetiology of rheumatic diseases. The responsiveness of the mononuclear cell fraction of peripheral blood and synovial fluid of patients with RA and of patients with rheumatic diseases other than RA to bacterial antigens such as cell wall fragments of the anaerobic intestinal flora, cell wall fragments of Streptococcus pyogenes, intestinal flora derived peptidoglycan polysaccharide complexes, the 65 kilodalton protein of Mycobacterium tuberculosis, and muramyldipeptide was investigated. No significant difference in response was found to all these bacterial antigens in the synovial fluid of patients with RA compared with the responses in patients with other rheumatic diseases. The highest responsiveness in the synovial fluid of the patients with RA was to the streptococcal cell wall fragments and to the 65 kilodalton protein. Higher responses to several bacterial antigens in the synovial fluid of patients with RA were found compared with peripheral blood from the same patient group. The antigen presenting cell population of the synovial fluid in patients with RA and the patients with other rheumatic diseases was found to be stimulatory for autologous peripheral blood T cells even in the absence of antigen. This suggests an important role for the synovial antigen presenting cell in the aetiology of inflammatory joint diseases. PMID:8447692

  13. Characterization of small, mononuclear blood cells from salmon having high phagocytic capacity and ability to differentiate into dendritic like cells.

    Science.gov (United States)

    Haugland, Gyri T; Jordal, Ann-Elise O; Wergeland, Heidrun I

    2012-01-01

    Phagocytes are the principal component of the innate immune system, playing a key role in the clearance of foreign particles that include potential pathogens. In vertebrates, both neutrophils and mononuclear cells like monocytes, macrophages and dendritic cells are all professional phagocytes. In teleosts, B-lymphocytes also have potent phagocytic ability. We have isolated a population of small (neutrophils as shown by qRT-PCR, flow cytometry and immunoblotting. A remarkable feature of these cells is their potent phagocytic capacity. Their oxygen-independent killing mechanism, as shown by intense acid phosphatase staining, is supported by lack of respiratory burst and myeloperoxidase activity and the acid phosphatase's sensitivity to tartrate. They show a high level of morphological plasticity, as, upon stimulation with mitogens, they change morphology and obtain branching protrusions similarly to dendritic cells. We suggest, based on our findings, that the small, round cells described here are progenitor cells with potential to differentiate into dendritic like cells, although we can not exclude the possibility that they represent a novel cell type.

  14. Generation of integration-free human induced pluripotent stem cells from postnatal blood mononuclear cells by plasmid vector expression.

    Science.gov (United States)

    Dowey, Sarah N; Huang, Xiaosong; Chou, Bin-Kuan; Ye, Zhaohui; Cheng, Linzhao

    2012-11-01

    Several human postnatal somatic cell types have been successfully reprogrammed to induced pluripotent stem cells (iPSCs). Blood mononuclear cells (MNCs) offer several advantages compared with other cell types. They are easily isolated from umbilical cord blood (CB) or adult peripheral blood (PB), and can be used fresh or after freezing. A short culture allows for more efficient reprogramming, with iPSC colonies forming from blood MNCs in 14 d, compared with 28 d for age-matched fibroblastic cells. The advantages of briefly cultured blood MNCs may be due to favorable epigenetic profiles and gene expression patterns. Blood cells from adults, especially nonlymphoid cells that are replenished frequently from intermittently activated blood stem cells, are short-lived in vivo and may contain less somatic mutations than skin fibroblasts, which are more exposed to environmental mutagens over time. We describe here a detailed, validated protocol for effective generation of integration-free human iPSCs from blood MNCs by plasmid vectors.

  15. Peripheral blood mononuclear cells: a potential source of homeostatic imbalance markers associated with obesity development.

    Science.gov (United States)

    Oliver, Paula; Reynés, Bàrbara; Caimari, Antoni; Palou, Andreu

    2013-04-01

    Peripheral blood mononuclear cells (PBMC) have a great potential for nutrition and obesity studies. PBMC reflect the nutritional response of key organs involved in energy homeostasis maintenance, which is altered in the obese state. Here, we aimed to determine the usefulness of PBMC as a source of early markers of obesity. To that purpose, we analysed whether PBMC could reflect the insensitivity to changes in feeding conditions associated with obesity during the development of this pathology. Expression of key genes central to energy metabolism was measured by Q-PCR in PBMC samples of normoweight (control) and cafeteria-fed (obese) rats in feeding, fasting and refeeding conditions. Samples were obtained monthly from 2 (beginning of cafeteria diet-feeding) to 6 months of age. In general terms, expression of genes related to fatty acid synthesis (Fasn, Srebp1) and adipogenesis (Pparg) decreased with fasting and increased with refeeding. Conversely, the expression of a key gene regulating beta-oxidation (Cpt1a) and the gene for an orexigenic neuropeptide (Npy)-in accordance with their metabolic role-increased with fasting and decreased with refeeding. This expression pattern disappeared in obese rats, in which insensitivity to feeding conditions was observed after only 1 month of cafeteria diet-feeding. Thus, during development, PBMC accurately reflect nutritional regulation of energy homeostasic genes and the insensitivity to feeding associated with obesity, even in the earlier stages with a low degree of overweight. For this reason, this set of blood cells could constitute a potential source of biomarkers of early homeostatic imbalance which would be useful in nutrition studies that could help prevent the occurrence of obesity.

  16. Selected scorpion toxin exposures induce cytokine release in human peripheral blood mononuclear cells.

    Science.gov (United States)

    Corzo, Gerardo; Espino-Solis, Gerardo Pavel

    2017-03-01

    A cytokine screening on human peripheral blood mononuclear cells (PBMCs) stimulated with selected scorpion toxins (ScTx's) was performed in order to evaluate their effect on human immune cells. The ScTx's chosen for this report were three typical buthid scorpion venom peptides, one with lethal effects on mammals Centruroides suffussus suffusus toxin II (CssII), another, with lethal effects on insects and crustaceans Centruroides noxius toxin 5 (Cn5), and one more without lethal effects Tityus discrepans toxin (Discrepin). A Luminex multiplex analysis was performed in order to determine the amounts chemokines and cytokines IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12-p40, IL-13, interferon alpha (IFN-α), interferon gamma (IFN-γ), tumor necrosis factor alpha TNF-α, and interferon-inducible protein-10 (IP-10) secreted from human PBMCs exposed to these toxins. Although, the ScTx Cn5 is not lethal for mammals, it was able to induce the secretion of cytokines IL-1β, IL-6, and TNF-α, IL-10 and IP-10 in comparison to the lethal CssII, which was able to induce only IP-10 secretion. Discrepin also was able to induce only IP-10. Interestingly, only low amounts of interferons α and β were induced in the presence of the ScTx's assayed. In a synergic experiment, the combination of Discrepin and Cn5 displayed considerable reverse effects on induction of IL-1β, IL-6, IL-10 and TNF-α, but they had a slight synergic effect on IP-10 cytokine production in comparison with the single effect obtained with the Cn5 alone. Thus, the results obtained suggest that the profile of secreted cytokines promoted by ScTx Cn5 is highly related with a cytokine storm event, and also it suggests that the mammalian lethal neurotoxins are not solely responsible of the scorpion envenomation symptomatology.

  17. Chemokine regulation in response to beryllium exposure in human peripheral blood mononuclear and dendritic cells.

    Science.gov (United States)

    Hong-Geller, Elizabeth; Pardington, Paige E; Cary, Robert B; Sauer, Nancy N; Gupta, Goutam

    2006-02-01

    Exposure to beryllium (Be) induces a delayed-type hypersensitivity immune reaction in the lungs of susceptible individuals, which leads to the onset of Be sensitivity and Chronic Beryllium Disease (CBD). Although some mechanistic aspects of CBD have begun to be characterized, very little is known about the molecular mechanisms by which Be activates the host immune response. To gain insight into the cellular response to Be exposure, we have performed global microarray analysis using a mixture of peripheral blood mononuclear and dendritic cells (PBMC/DCs) from a non-CBD source to identify genes that are specifically upregulated in response to BeSO(4) stimulation, compared to a control metal salt, Al(2)(SO(4))(3). We identified a number of upregulated immunomodulatory genes, including several chemokines in the MIP-1 and GRO families. Using PBMC/DCs from three different donors, we demonstrate that BeSO(4) stimulation generally exhibits an increased rate of both chemokine mRNA transcription and release compared to Al(2)(SO(4))(3) exposure, although variations among the individual donors do exist. We show that MIP-1 alpha and MIP-1 beta neutralizing antibodies can partially inhibit the ability of BeSO(4) to stimulate cell migration of PBMC/DCs in vitro. Finally, incubation of PBMC/DCs with BeSO(4) altered the binding of the transcription factor RUNX to the MIP-1 alpha promoter consensus sequence, indicating that Be can regulate chemokine gene activation. Taken together, these results suggest a model in which Be stimulation of PBMC/DCs can modulate the expression and release of different chemokines, leading to the migration of lymphocytes to the lung and the formation of a localized environment for development of Be disease in susceptible individuals.

  18. Controlled meal frequency without caloric restriction alters peripheral blood mononuclear cell cytokine production

    Directory of Open Access Journals (Sweden)

    Longo Dan L

    2011-03-01

    Full Text Available Abstract Background Intermittent fasting (IF improves healthy lifespan in animals by a mechanism involving reduced oxidative damage and increased resistance to stress. However, no studies have evaluated the impact of controlled meal frequency on immune responses in human subjects. Objective A study was conducted to establish the effects of controlled diets with different meal frequencies, but similar daily energy intakes, on cytokine production in healthy male and female subjects. Design In a crossover study design with an intervening washout period, healthy normal weight middle-age male and female subjects (n = 15 were maintained for 2 months on controlled on-site one meal per day (OMD or three meals per day (TMD isocaloric diets. Serum samples and peripheral blood mononuclear cells (PBMCs culture supernatants from subjects were analyzed for the presence of inflammatory markers using a multiplex assay. Results There were no significant differences in the inflammatory markers in the serum of subjects on the OMD or TMD diets. There was an increase in the capacity of PBMCs to produce cytokines in subjects during the first month on the OMD or TMD diets. Lower levels of TNF-α, IL-17, MCP-1 and MIP-1β were produced by PBMCs from subjects on the OMD versus TMD diet. Conclusions PBMCs of subjects on controlled diets exhibit hypersensitivities to cellular stimulation suggesting that stress associated with altered eating behavior might affect cytokine production by immune cells upon stimulation. Moreover, stimulated PBMCs derived from healthy individuals on a reduced meal frequency diet respond with a reduced capability to produce cytokines.

  19. Histamine and histamine-receptor antagonists modify gene expression and biosynthesis of interferon gamma in peripheral human blood mononuclear cells and in CD19-depleted cell subsets

    NARCIS (Netherlands)

    Horváth, B V; Szalai, C; Mándi, Y; László, V; Radvány, Z; Darvas, Z; Falus, A

    1999-01-01

    The effect of histamine and histamine antagonists was examined on gene expression and biosynthesis of bacterial endotoxin (LPS) induced interferon gamma (IFNgamma) both in human peripheral mononuclear cells (PMBC) and in T-cell enriched fractions. We found, that histamine inhibited the LPS induced t

  20. Relationships between human vitality and mitochondrial respiratory parameters, reactive oxygen species production and dNTP levels in peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Maynard, Scott; Keijzers, Guido; Gram, Martin

    2013-01-01

    . Therefore, we measured a number of cellular parameters related to mitochondrial activity in peripheral blood mononuclear cells (PBMCs) isolated from middle-aged men, and tested for association with vitality. These parameters estimate mitochondrial respiration, reactive oxygen species (ROS) production...

  1. Cytotoxic effect of wine polyphenolic extracts and resveratrol against human carcinoma cells and normal peripheral blood mononuclear cells.

    Science.gov (United States)

    Matić, Ivana; Zizak, Zeljko; Simonović, Mladen; Simonović, Branislav; Godevac, Dejan; Savikin, Katarina; Juranić, Zorica

    2010-08-01

    Red and white wine polyphenols have been reported to provide substantial health benefits. In this study, the cytotoxic activity of red and white wine polyphenolic extracts and of resveratrol was evaluated against different cancer cell lines--human cervix adenocarcinoma HeLa, human breast adenocarcinoma MDA-MB-361, and human breast carcinoma MDA-MB-453--and normal human peripheral blood mononuclear cells (PBMCs). Qualitative and quantitative compositions of wine polyphenolic extracts obtained by fractional vacuum distillation of corresponding wines were determined using spectrophotometric methods and high-performance liquid chromatography with diode array detection and liquid chromatography with electrospray ionization-time of flight mass spectrometry analysis. It was demonstrated that wine polyphenolic extracts and resveratrol exerted higher cytotoxic activity against HeLa and MDA-MB-453 cells in comparison to MDA-MB-361 cells and unstimulated and stimulated PBMCs. Furthermore, white wine polyphenolic extract exhibited a significantly higher antiproliferative action on cancer cell lines than red wine extract. The presence of condensed or fragmented nuclei in HeLa cells, pretreated with extract of white wine and stained with a mixture of acridine orange and ethidium bromide, pointed to the morphological signs of apoptosis. In addition, HeLa cells in late stages of apoptosis or secondary necrosis were also observed. Results from our study suggest that polyphenolic extracts from red and white wine may have anticarcinogenic potential.

  2. Characterisation of the Immunomodulatory Effects of Meningococcal Opa Proteins on Human Peripheral Blood Mononuclear Cells and CD4+ T Cells.

    Directory of Open Access Journals (Sweden)

    Claire Jones

    Full Text Available Opa proteins are major surface-expressed proteins located in the Neisseria meningitidis outer membrane, and are potential meningococcal vaccine candidates. Although Opa proteins elicit high levels of bactericidal antibodies following immunisation in mice, progress towards human clinical trials has been delayed due to previous findings that Opa inhibits T cell proliferation in some in vitro assays. However, results from previous studies are conflicting, with different Opa preparations and culture conditions being used. We investigated the effects of various Opa+ and Opa- antigens from N. meningitidis strain H44/76 in a range of in vitro conditions using peripheral blood mononuclear cells (PBMCs and purified CD4+ T cells, measuring T cell proliferation by CFSE dilution using flow cytometry. Wild type recombinant and liposomal Opa proteins inhibited CD4+ T cell proliferation after stimulation with IL-2, anti-CD3 and anti-CD28, and these effects were reduced by mutation of the CEACAM1-binding region of Opa. These effects were not observed in culture with ex vivo PBMCs. Opa+ and Opa- OMVs did not consistently exert a stimulatory or inhibitory effect across different culture conditions. These data do not support a hypothesis that Opa proteins would be inhibitory to T cells if given as a vaccine component, and T cell immune responses to OMV vaccines are unlikely to be significantly affected by the presence of Opa proteins.

  3. microRNA expression profile of peripheral blood mononuclear cells of Klinefelter syndrome.

    Science.gov (United States)

    Sui, Weiguo; Ou, Minglin; Chen, Jiejing; Li, Huan; Lin, Hua; Zhang, Yue; Li, Wuxian; Xue, Wen; Tang, Donge; Gong, Weiwei; Zhang, Ruohan; Li, Fengyan; Dai, Yong

    2012-11-01

    microRNAs are a type of small non-coding RNAs which play important roles in post-transcriptional gene regulation, and the characterization of microRNA expression profiling in peripheral blood mononuclear cells (PBMCs) from patients with Klinefelter syndrome requires further investigation. In this study, PBMCs were obtained from patients with Klinefelter syndrome and normal controls. After preparation of small RNA libraries, the two groups of samples were sequenced simultaneously using next generation high-throughput sequencing technology, and novel and known microRNAs were analyzed. A total of 9,772,392 and 9,717,633 small RNA reads were obtained; 8,014,466 (82.01%) and 8,104,423 (83.40%) genome-matched reads, 64 and 49 novel microRNAs were identified in the library of Klinefelter syndrome and the library of healthy controls, respectively. There were 71 known microRNAs with differential expression levels between the two libraries. Clustering of over-represented gene ontology (GO) classes in predicted targets of novel microRNAs in the Klinefelter syndrome library showed that the most significant GO terms were genes involved in the endomembrane system, nucleotide binding and kinase activity. Our data revealed that there are a large number of microRNAs deregulated in PBMCs taken from patients with Klinefelter syndrome, of which certain novel and known microRNAs may be involved in the pathological process of Klinefelter syndrome. Further studies are necessary to determine the roles of microRNAs in the pathological process of Klinefelter syndrome in the future.

  4. The Gene Expression Patterns of Peripheral Blood Mononuclear Cells in Patients with Systemic Lupus Erythematosus

    Institute of Scientific and Technical Information of China (English)

    LI Shouxin; JIANG Wei; HUANG Rui; WANG Xiaohui; LIU Wen; SHEN Shouyin

    2007-01-01

    This study examined the gene expression patterns of peripheral blood mononuclear cells (PBMCs) in patients with systemic lupus erythematosus (SLE) by using serial analysis of gene expression (SAGE) technology. Following the construction of serial analysis of gene expression (SAGE) library of PBMCs collected from 3 cases of familial SLE patients, a large scale of tag sequencing was performed. The data extracted from sequencing files was analyzed with SAGE 2000 V 4.5 software.The top 30 expressed genes of SLE patients were uploaded to http://david.niaid.nih. gov/david/ease.htm and the functional classification of genes was obtained. The differences among those expressed gene were analyzed by Chi-square tests. The results showed that a total of 1286 unique SAGE tags were identified from 1814 individual SAGE tags. Among the 1286 unique tags, 86.8% had single copy, and only 0.2% tags had more than 20 copies. And 68.4% of the tags matched known expressed sequences, 41.1% of which matched more than one known expressed sequence. About 31.6% of the tags had no match and could represent potentially novel genes. Approximately one third of the top 30 genes were ribosomal protein, and the rest were genes related to metabolism or with unknown functions. Eight tags were found to express differentially in SAGE library of SLE patients. This study draws a profile of gene expression patterns of PBMCs in patients with SLE. Comparison of SAGE database from PBMCs between normal individuals and SLE patients will help us to better understand the pathogenesis of SLE.

  5. Imbalance of Th17 and Treg in peripheral blood mononuclear cells of active tuberculosis patients

    Directory of Open Access Journals (Sweden)

    Jie Luo

    Full Text Available Abstract Objective: Maintaining a right balance between Th17 and Treg might be critical to the immunopathogenesis of active tuberculosis (TB. This study aimed to assess whether the Th17/Treg balance is altered in active TB patients. Methods: 250 study subjects (90 active TB patients, 80 latent TB subjects, and 80 healthy controls were recruited for the study. The expression of Th17 and Treg in peripheral blood mononuclear cells (PBMCs in the 250 subjects was investigated by flow cytometry. Plasma levels of cytokines IL-17 and IL-10, which are related to Th17 and Treg, respectively, were determined by ELISA. Results: The percentages of Th17 and Treg in PBMCs from active TB patients were significantly higher than those from latent TB or control groups (Th17: 4.31 ± 1.35% vs. 1.58 ± 0.71% or 1.15 ± 0.49%, p < 0.05; Treg: 11.44 ± 2.69% vs. 7.54 ± 1.56% or 4.10 ± 0.99%, p < 0.05. The expression of IL-17 and IL-10 was significantly increased in active TB patients in comparison to that in latent TB or control groups (IL-17: 16.85 ± 9.68 vs. 7.23 ± 5.19 or 8.21 ± 5.51 pg/mL, p < 0.05; IL-10: 28.70 ± 11.27 vs. 20.25 ± 8.57 or 13.94 ± 9.00 pg/mL, p < 0.05. Conclusions: Our study demonstrated an altered balance of Treg/Th17 in active TB patients, with higher percentages of Th17 and Treg in PBMCs. Further research on this imbalance may offer a new direction for TB treatment.

  6. Intravenous autologous bone marrow mononuclear stem cell therapy for ischemic stroke: a multicentric, randomized trial.

    Science.gov (United States)

    Prasad, Kameshwar; Sharma, Alka; Garg, Ajay; Mohanty, Sujata; Bhatnagar, Shinjini; Johri, Sharat; Singh, Kunwar Karni; Nair, Velu; Sarkar, Ravi Shankar; Gorthi, Sankar Prasad; Hassan, Kaukab Maqbool; Prabhakar, Sudesh; Marwaha, Neelam; Khandelwal, Niranjan; Misra, Usha Kant; Kalita, Jayantee; Nityanand, Soniya

    2014-12-01

    Pilot studies have suggested benefit from intravenous administration of bone marrow mononuclear stem cells (BMSCs) in stroke. We explored the efficacy and safety of autologous BMSCs in subacute ischemic stroke. This was a phase II, multicenter, parallel group, randomized trial with blinded outcome assessment that included 120 patients. Patients with subacute ischemic stroke were randomly assigned to the arm that received intravenous infusion of autologous BMSCs or to control arm. Coprimary clinical efficacy outcomes were Barthel Index score and modified Rankin scale at day 180. Secondary outcomes were change in infarct volume, National Institute of Health Stroke Scale (NIHSS) at day 90 and 180. Main safety outcomes were adverse events, any new area of (18)fluorodeoxyglucose positron emission tomography uptake in any body part over 365 days. Fifty-eight patients received a mean of 280.75 million BMSCs at median of 18.5 days after stroke onset. There was no significant difference between BMSCs arm and control arm in the Barthel Index score (63.1 versus 63.6; P=0.92), modified Rankin scale shift analysis (P=0.53) or score >3 (47.5% versus 49.2%; P=0.85), NIHSS score (6.3 versus 7.0; P=0.53), change in infarct volume (-11.1 versus -7.36; P=0.63) at day 180. Adverse events were also similar in the 2 arms, and no patient showed any new area of (18)fluorodeoxyglucose uptake. With the methods used, results of this hitherto first randomized controlled trial indicate that intravenous infusion of BMSCs is safe, but there is no beneficial effect of treatment on stroke outcome. URLs: http://ctri.nic.in/Clinicaltrials and http://www.clinicaltrials.gov. Unique identifiers: CTRI-ROVCTRI/2008/091/0004 and NCT0150177. © 2014 American Heart Association, Inc.

  7. Effects of oral eicosapentaenoic acid versus docosahexaenoic acid on human peripheral blood mononuclear cell gene expression.

    Science.gov (United States)

    Tsunoda, Fumiyoshi; Lamon-Fava, Stefania; Asztalos, Bela F; Iyer, Lakshmanan K; Richardson, Kris; Schaefer, Ernst J

    2015-08-01

    Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have beneficial effects on inflammation and cardiovascular disease (CVD). Our aim was to assess the effect of a six-week supplementation with either olive oil, EPA, or DHA on gene expression in peripheral blood mononuclear cells (PBMC). Subjects were sampled at baseline and six weeks after receiving either: olive oil 6.0 g/day (n = 16), EPA 1.8 g/day (n = 16), or DHA 1.8 g/day (n = 18). PBMC were subjected to gene expression analysis by microarray with key findings confirmed by quantitative real-time polymerase chain reaction (Q-PCR). Plasma phospholipid EPA increased 3 fold in the EPA group, and DHA increased 63% in the DHA group (both p expression in the following pathways: 1) interferon signaling, 2) receptor recognition of bacteria and viruses, 3) G protein signaling, glycolysis and glycolytic shunting, 4) S-adenosyl-l-methionine biosynthesis, and 5) cAMP-mediated signaling including cAMP responsive element protein 1 (CREB1), as well as many other individual genes including hypoxia inducible factor 1, α subunit (HIF1A). The findings for CREB1 and HIF1A were confirmed by Q-PCR analysis. Our data indicate that EPA supplementation was associated with significant effects on gene expression involving the interferon pathway as well as down-regulation of CREB1 and HIF1A, which may relate to its beneficial effect on CVD risk reduction. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  8. Production of nitric oxide by peripheral blood mononuclear cells from the Florida manatee, Trichechus manatus latirostris.

    Science.gov (United States)

    Walsh, Catherine J; Stuckey, Joyce E; Cox, Heather; Smith, Brett; Funke, Christina; Stott, Jeff; Colle, Clarence; Gaspard, Joseph; Manire, Charles A

    2007-08-15

    Florida manatees (Trichechus manatus latirostris) are exposed to many conditions in their habitat that may adversely impact health and impair immune function in this endangered species. In an effort to increase the current knowledge base regarding the manatee immune system, the production of an important reactive nitrogen intermediate, nitric oxide (NO), by manatee peripheral blood mononuclear cells (PBMC) was investigated. PBMC from healthy captive manatees were stimulated with LPS, IFN-gamma, or TNF-alpha, either alone or in various combinations, with NO production assessed after 24, 48, 72, and 96 h of culture. NO production in response to LPS stimulation was significantly greater after 48, 72, or 96 h of culture compared to NO production after 24h of culture. A specific inhibitor of inducible nitric oxide synthase (iNOS), L-NIL (L-N(6)-(1-iminoethyl)lysine), significantly decreased NO production by LPS-stimulated manatee PBMC. Manatee specific oligonucleotide primers for iNOS were designed to measure expression of relative amounts of mRNA in LPS-stimulated manatee PBMC from captive manatees. NO production by PBMC from manatees exposed to red tide toxins was analyzed, with significantly greater NO production by both unstimulated and LPS stimulated PBMC from red tide exposed compared with healthy captive or cold-stress manatees. Free-ranging manatees produced significantly lower amounts of nitric oxide compared to either captive or red tide rescued manatees. Results presented in this paper contribute to the current understanding of manatee immune function and represent the first report of nitric oxide production in the immune system of a marine mammal.

  9. Contribution of mononuclear bone marrow cells to carbon tetrachloride-induced liver fibrosis in rats

    Institute of Scientific and Technical Information of China (English)

    Bao-Qiang Cao; Ji-Zong Lin; Yue-Si Zhong; Shao-Bin Huang; Nan Lin; Zhao-Feng Tang; Rui Chen; Peng Xiang; Rui-Yun Xu

    2007-01-01

    AIM:To study the inhibitory effect of mononuclear bone marrow cell (BMC) transplantation on carbon tetrachloride (CCIt) -induced liver fibrosis in rats.METHODS:Rat liver fibrosis models were induced by CCN and alcohol administration. After 8 wk,twenty rats were randomly allocated into treatment group (n = 10) and control group (n = 10). BMC were infused into the rats in treatment group via the portal vein,while heparinized saline was infused in control group. CCU was hypodermically injected into the rats twice a week for 4 wk. At the end of wk 12,all rats were humanely sacrificed. Liver samples were taken and stained with HE or Masson trichrome. The general conditions,liver fibrSsis (hydroxyproline and collagen fibre) and liver pathological grades in rats were evaluated.± 128.8μg/g in treatment group,and 596.0 ± 341. 8μg/g in control group.The percentage of collagen fibre was 3.75% ± 0.98% in treatment group and 5.02% ± 0.44% in control group.There was a significant difference berween the two groups (P<0.05).Liver pathological grade decreased from grade Ⅳ to grade Ⅲ partially in treatment group (P<0.05) with no obvious improvement in control group (P<0.05).There was a significant difference between treatment group and control group(P<0.05).CONCLUSION: Transplantation of BMC can improve liver fibrosis due to chronic liver injury in rats.

  10. Proteomic methodological recommendations for studies involving human plasma, platelets, and peripheral blood mononuclear cells.

    Science.gov (United States)

    de Roos, Baukje; Duthie, Susan J; Polley, Abigael C J; Mulholland, Francis; Bouwman, Freek G; Heim, Carolin; Rucklidge, Garry J; Johnson, Ian T; Mariman, Edwin C; Daniel, Hannelore; Elliott, Ruan M

    2008-06-01

    This study was designed to develop, optimize and validate protocols for blood processing prior to proteomic analysis of plasma, platelets and peripheral blood mononuclear cells (PBMC) and to determine analytical variation of a single sample of depleted plasma, platelet and PBMC proteins within and between four laboratories each using their own standard operating protocols for 2D gel electrophoresis. Plasma depleted either using the Beckman Coulter IgY-12 proteome partitioning kit or the Amersham albumin and IgG depletion columns gave good quality gels, but reproducibility appeared better with the single-use immuno-affinity column. The use of the Millipore Filter Device for protein concentration gave a 16% ( p appears as a single abundant spot. The average within-laboratory coefficient of variation (CV) for each of the matched spots after automatic matching using either PDQuest or ProteomWeaver software ranged between 18 and 69% for depleted plasma proteins, between 21 and 55% for platelet proteins, and between 22 and 38% for PBMC proteins. Subsequent manual matching improved the CV with on average between 1 and 16%. The average between laboratory CV for each of the matched spots after automatic matching ranged between 4 and 54% for depleted plasma proteins, between 5 and 60% for platelet proteins, and between 18 and 70% for PBMC proteins. This variation must be considered when designing sufficiently powered studies that use proteomics tools for biomarker discovery. The use of tricine in the running buffer for the second dimension appears to enhance the resolution of proteins especially in the high molecular weight range.

  11. Telomerase Activity in Peripheral Blood Mononuclear Cells from Senile Patients with Pneumonia

    Institute of Scientific and Technical Information of China (English)

    LIU Jian; ZHOU Zhen; LIU Xiaoqing

    2006-01-01

    To investigate the changes of the activity of telomerase in peripheral blood mononuclear cells (PBMCs) from senile patients with pneumonia, the telomerase activity was examined before and after the stimulation of phytohemagglutinin-M (PHA-M) in PBMCs from 10 control subjects (group A), 12 non-senile patients with pneumonia (group B) and 9 senile patients with pneumonia (group C). Also observed was the proliferative response of these PBMCs to PHA-M. The results showed that, both with or without the stimulation of PHA-M, the values of telomerase activity in PBMCs from group C patients (A values: pre-stimulation, 0.43±0.04; post-stimulation, 0.63±0.03) were significantly lower than those in PBMCs from both group A patients (A values: prestimulation, 0.65±0.05;post-stimulation, 1.26±0.13;P<0.001, respectively) and group B patients (A values: pre-stimulation, 0.63±0.03; post-stimulation, 0.93±0.03;P<0.05, respectively). The results of MTT test showed that the proliferative activity of PBMCs in group C patients (A value: 0.35±0.03) was also significantly lower than that in group A patients (A value:0. 55±0.04; P<0.05) and group B patients (A value: 0.46±0.03;P<0.05). These results indicate that the telomerase activity decreases in senile patients with pneumonia, which may be one of the mechanisms for the weakened immune function in those patients.

  12. Peripheral blood mononuclear cells HIV DNA levels impact intermittently on neurocognition.

    Directory of Open Access Journals (Sweden)

    Lucette A Cysique

    Full Text Available To determine the contribution of peripheral blood mononuclear cells' (PBMCs HIV DNA levels to HIV-associated dementia (HAD and non-demented HIV-associated neurocognitive disorders (HAND in chronically HIV-infected adults with long-term viral suppression on combined antiretroviral treatment (cART.Eighty adults with chronic HIV infection on cART (>97% with plasma and CSF HIV RNA <50 copies/mL were enrolled into a prospective observational cohort and underwent assessments of neurocognition and pre-morbid cognitive ability at two visits 18 months apart. HIV DNA in PBMCs was measured by real-time PCR at the same time-points.At baseline, 46% had non-demented HAND; 7.5% had HAD. Neurocognitive decline occurred in 14% and was more likely in those with HAD (p<.03. Low pre-morbid cognitive ability was uniquely associated with HAD (p<.05. Log10 HIV DNA copies were stable between study visits (2.26 vs. 2.22 per 106 PBMC. Baseline HIV DNA levels were higher in those with lower pre-morbid cognitive ability (p<.04, and higher in those with no ART treatment during HIV infection 1st year (p = .03. Baseline HIV DNA was not associated with overall neurocognition. However, % ln HIV DNA change was associated with decline in semantic fluency in unadjusted and adjusted analyses (p = .01-.03, and motor-coordination (p = .02-.12 to a lesser extent.PBMC HIV DNA plays a role in HAD pathogenesis, and this is moderated by pre-morbid cognitive ability in the context of long-term viral suppression. While the HIV DNA levels in PBMC are not associated with current non-demented HAND, increasing HIV DNA levels were associated with a decline in neurocognitive functions associated with HAND progression.

  13. Bone marrow mononuclear cell therapy in ischaemic stroke: a systematic review.

    Science.gov (United States)

    Kumar, A; Prasad, M; Jali, V P; Pandit, A K; Misra, S; Kumar, P; Chakravarty, K; Kathuria, P; Gulati, A

    2017-05-01

    Bone marrow mononuclear cell (BM-MNC) therapy has emerged as a potential therapy for the treatment of stroke. We performed a systematic review of published studies using BM-MNC therapy in patients with ischaemic stroke (IS). Literature was searched using MEDLINE, PubMed, EMBASE, Trip Database, Cochrane library and clinicaltrial.gov to identify studies on BM-MNC therapy in IS till June, 2016. Data were extracted independently by two reviewers. STATA version 13 was used for carrying out meta-analysis. We included non-randomized open-label, single-arm and non-randomized comparative studies or randomized controlled trials (RCTs) if BM-MNCs were used to treat patients with IS in any phase after the index stroke. One randomized trial, two non-randomized comparative trials and four single-arm open-label trials (total seven studies) involving 227 subjects (137 patients and 90 controls) were included in the systematic review and meta-analysis. The pooled proportion for favourable clinical outcome (modified Rankin Scale score ≤2) in six studies involving 122 subjects was 29% (95% CI 0.16-0.43) who were exposed to BM-MNCs and pooled proportion for favourable clinical outcome of 69 subjects (taken from two trials) who did not receive BM-MNCs was 20% (95% CI 0.12-0.32). The pooled difference in the safety outcomes was not significant between both the groups. Our systematic review suggests that BM-MNC therapy is safe up to 1 year post-intervention and is feasible; however, its efficacy in the case of IS patients is debatable. Well-designed randomized controlled trials are required to provide more information on the efficacy of BM-MNC transplantation in patients with IS. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. Effects of blood transportation on human peripheral mononuclear cell yield, phenotype and function: implications for immune cell biobanking.

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    Anita Posevitz-Fejfár

    Full Text Available Human biospecimen collection, processing and preservation are rapidly emerging subjects providing essential support to clinical as well as basic researchers. Unlike collection of other biospecimens (e.g. DNA and serum, biobanking of viable immune cells, such as peripheral blood mononuclear cells (PBMC and/or isolated immune cell subsets is still in its infancy. While certain aspects of processing and freezing conditions have been studied in the past years, little is known about the effect of blood transportation on immune cell survival, phenotype and specific functions. However, especially for multicentric and cooperative projects it is vital to precisely know those effects. In this study we investigated the effect of blood shipping and pre-processing delay on immune cell phenotype and function both on cellular and subcellular levels. Peripheral blood was collected from healthy volunteers (n = 9: at a distal location (shipped overnight and in the central laboratory (processed immediately. PBMC were processed in the central laboratory and analyzed post-cryopreservation. We analyzed yield, major immune subset distribution, proliferative capacity of T cells, cytokine pattern and T-cell receptor signal transduction. Results show that overnight transportation of blood samples does not globally compromise T- cell subsets as they largely retain their phenotype and proliferative capacity. However, NK and B cell frequencies, the production of certain PBMC-derived cytokines and IL-6 mediated cytokine signaling pathway are altered due to transportation. Various control experiments have been carried out to compare issues related to shipping versus pre-processing delay on site. Our results suggest the implementation of appropriate controls when using multicenter logistics for blood transportation aiming at subsequent isolation of viable immune cells, e.g. in multicenter clinical trials or studies analyzing immune cells/subsets. One important conclusion might

  15. Are Peripheral Blood Mononuclear Cells Derived from Patients with Certain Myopathies Suitable for Personalized Drug Screening?

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    Andriy V. Shatillo

    2014-12-01

    Full Text Available Background: Limb girdle muscular dystrophies (LGMDs and several other disorders which share their specific phenotype are rare, predominantly hereditary conditions with no curative treatment. Differential diagnosis of these myopathies is quite challenging and expensive in many cases. Therefore, a significant proportion of patients remains undiagnosed and untreated for a long time. At the same time there is a huge amount of drugs and supplements potentially able to modify the course of some of these muscular dystrophies. That is why a simple empirical approach able to define a patient’s reaction to a specific compound seems rational. Because most common basic pathogenetic mechanisms for these quite different disorders increase the vulnerability of muscle cells (or decrease ability for reparation during mechanical stress, we propose a simple, noninvasive and inexpensive approach for individualized drug screening based on the drug’s influence on the mechanical vulnerability of peripheral blood mononuclear cells (PBMC. Methods: PBMC derived from 8 patients with Duchenne muscular dystrophy (DMD, 2 patients with LGMD2A, 1 patient with LGMD2B, 1 with MERRF syndrome, 1 with facioscapulohumeral muscular dystrophy (FSHD and 13 matched control subjects were irradiated by ultrasound in the presence of several compounds (lisinopril, vitamin D3, prednisolon, tocopherol, topiramate, glutargin, α-lipoic acid, essentiale, and physiological solution. Then viability indexes of the samples were detected by citotoxic assays based on vital dye (neutral red and resazurin metabolism. Results: In cytotoxicity tests with active transport of neutral red into PBMC derived from DMD patients, the cells showed signs of destruction at 1.06±0.52 minutes of ultrasounding compared to 1.75±0.6 minutes in control. PBMCs from patients with other myopathies have either normal or decreased resistance to ultrasound. The addition of tocopherol significantly changes the PBMC

  16. Immunostimulatory acivity of Calophyllum brasiliense, Ipomoea pes-caprae and Matayba elaeagnoides demonstrated by human peripheral blood mononuclear cells proliferation.

    Science.gov (United States)

    Philippi, Marina Elisa; Duarte, Bruna Momm; Da Silva, Carolina Vieira; De Souza, Michel Thomaz; Niero, Rivaldo; Cechinel Filho, Valdir; Bueno, Edneia Casagranda

    2010-01-01

    This study evaluates the effect of methanol extracts of three Brazilian medicinal plants on in vitro proliferation of human mononuclear cells. Lymphoproliferation assay was carried out by incubating human peripheral blood mononuclear cells from healthy donors (1 x 10(6) cells/mL) with extracts of Calophyllum brasiliense (roots), Ipomoea pes-caprae (whole plant) and Matayba elaeagnoides (bark), both at 10, 50, 100 and 200 microg/mL, alone or with phytohemagglutinin (PHA, 5 microg/mL), in 96-well microplates at 37 degrees C with 5% CO2, for 72 h. The quantification of cell proliferation assay was performed by blue tetrazolium (MTT) reduction with reading at 540 nm. Cells incubated with only the culture medium were used as negative control for cell proliferation, while the positive control consisted of cells and PHA. The results suggest that the extracts of all three studied plants induce T lymphocyte proliferation. I. pes-caprae showed immunostimulatory activity three times higher than the C. brasiliense extract, while that of the M. elaeagnoides extract was 1.5 times higher. The results demonstrate immunostimulatory effects of these three plants, therefore the continuity of these studies is recommended, in order to determine the active principles.

  17. Rhesus rotavirus VP4 sequence-specific activation of mononuclear cells is associated with cholangiopathy in murine biliary atresia.

    Science.gov (United States)

    Walther, Ashley; Mohanty, Sujit K; Donnelly, Bryan; Coots, Abigail; Lages, Celine S; Lobeck, Inna; Dupree, Phylicia; Meller, Jaroslaw; McNeal, Monica; Sestak, Karol; Tiao, Greg

    2015-09-15

    Biliary atresia (BA), a neonatal obstructive cholangiopathy, remains the most common indication for pediatric liver transplantation in the United States. In the murine model of BA, Rhesus rotavirus (RRV) VP4 surface protein determines biliary duct tropism. In this study, we investigated how VP4 governs induction of murine BA. Newborn mice were injected with 16 strains of rotavirus and observed for clinical symptoms of BA and mortality. Cholangiograms were performed to confirm bile duct obstruction. Livers and bile ducts were harvested 7 days postinfection for virus titers and histology. Flow cytometry assessed mononuclear cell activation in harvested cell populations from the liver. Cytotoxic NK cell activity was determined by the ability of NK cells to kill noninfected cholangiocytes. Of the 16 strains investigated, the 6 with the highest homology to the RRV VP4 (>87%) were capable of infecting bile ducts in vivo. Although the strain Ro1845 replicated to a titer similar to RRV in vivo, it caused no symptoms or mortality. A Ro1845 reassortant containing the RRV VP4 induced all BA symptoms, with a mortality rate of 89%. Flow cytometry revealed that NK cell activation was significantly increased in the disease-inducing strains and these NK cells demonstrated a significantly higher percentage of cytotoxicity against noninfected cholangiocytes. Rotavirus strains with >87% homology to RRV's VP4 were capable of infecting murine bile ducts in vivo. Development of murine BA was mediated by RRV VP4-specific activation of mononuclear cells, independent of viral titers.

  18. Effects of PDE4 inhibitors on lipopolysaccharide-induced priming of superoxide anion production from human mononuclear cells

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    Noëlla Germain

    2001-01-01

    Full Text Available Aims: Phosphodiesterase 4 (PDE4 inhibitors have been described as potent anti-inflammatory compounds, involving an increase in intracellular levels of cyclic 3',5'-adenosine monophosphate (AMP. The aim of this study was to compare the effects of selective PDE4 inhibitors, rolipram and RP 73-401 with the cell permeable analogue of cyclic AMP, dibutyryl-cyclic AMP (db-cAMP and the anti-inflammatory cytokine interleukin-10 (IL-10 on superoxide anion production from peripheral blood mononuclear cells preincubated with lipopolysaccharide (LPS.

  19. Non-major histocompatibility complex-restricted cytotoxic activity of blood mononuclear cells stimulated with secreted mycobacterial proteins and other mycobacterial antigens

    DEFF Research Database (Denmark)

    Ravn, P; Pedersen, B K

    1994-01-01

    Several observations indicate that non-major histocompatibility complex (MHC)-restricted cytotoxicity, mediated for example by natural killer cells and lymphokine-activated killer cells, may serve as an important antimicrobial defense mechanism. The purpose of the present study was to investigate...... the influences of different mycobacterial antigens on non-MHC-restricted cytotoxicity and further to investigate the ways by which various lymphocyte subpopulations contribute to the development of this cytotoxicity. Non-MHC-restricted cytotoxicity was induced following stimulation of mononuclear cells...... the influence of CD4+ cells on the development of non-MHC-restricted cytotoxicity, blood mononuclear cells were depleted of CD4+ cells before antigen stimulation. When mononuclear cells were incubated with purified protein derivative or short-term culture filtrate in the absence of CD4+ cells, cytotoxic...

  20. In vitro expansion of Lin{sup +} and Lin{sup −} mononuclear cells from human peripheral blood

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    Norhaiza, H. Siti; Zarina, Z. A. Intan; Hisham, Z. A. Shahrul [School of Bioscience and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, Selangor (Malaysia); Rohaya, M. A. W. [Department of Orthodontics, Faculty of Dentistry, Universiti Kebangsaan Malaysia, 50300, Kuala Lumpur (Malaysia)

    2013-11-27

    Haematopoietic stem cells (HSCs) are used in the therapy of blood disorders due to the ability of these cells to reconstitute haematopoietic lineage cells when transplanted into myeloablative recipients. However, substantial number of cells is required in order for the reconstitution to take place. Since HSCs present in low frequency, larger number of donor is required to accommodate the demand of transplantable HSCs. Therefore, in vitro expansion of HSCs will have profound impact on clinical purposes. The aim of this study was to expand lineage negative (Lin{sup −}) stem cells from human peripheral blood. Total peripheral blood mononuclear cells (PBMNCs) were fractionated from human blood by density gradient centrifugation. Subsequently, PBMNCs were subjected to magnetic assisted cell sorter (MACS) which depletes lineage positive (Lin{sup +}) mononuclear cells expressing lineage positive markers such as CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a to obtained Lin{sup −} cell population. The ability of Lin{sup +} and Lin{sup −} to survive in vitro was explored by culturing both cell populations in complete medium consisting of Alpha-Minimal Essential Medium (AMEM) +10% (v/v) Newborn Calf Serum (NBCS)+ 2% (v/v) pen/strep. In another experiment, Lin{sup +} and Lin{sup −} were cultured with complete medium supplemented with 10ng/mL of the following growth factors: stem cell factor (SCF), interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF), 2IU/mL of Erythropoietin (Epo) and 20ng/mL of IL-6. Three samples were monitored in static culture for 22 days. The expansion potential was assessed by the number of total viable cells, counted by trypan blue exclusion assay. It was found that Lin{sup +} mononuclear cells were not able to survive either in normal proliferation medium or proliferation medium supplemented with cytokines. Similarly, Lin{sup −} stem cells were not able to survive in proliferation medium however

  1. Production of cytokine and chemokines by human mononuclear cells and whole blood cells after infection with Trypanosoma cruzi

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    Karine Rezende-Oliveira

    2012-02-01

    Full Text Available INTRODUCTION: The innate immune response is the first mechanism of protection against Trypanosoma cruzi, and the interaction of inflammatory cells with parasite molecules may activate this response and modulate the adaptive immune system. This study aimed to analyze the levels of cytokines and chemokines synthesized by the whole blood cells (WBC and peripheral blood mononuclear cells (PBMC of individuals seronegative for Chagas disease after interaction with live T. cruzi trypomastigotes. METHODS: IL-12, IL-10, TNF-α, TGF-β, CCL-5, CCL-2, CCL-3, and CXCL-9 were measured by ELISA. Nitrite was determined by the Griess method. RESULTS: IL-10 was produced at high levels by WBC compared with PBMC, even after incubation with live trypomastigotes. Production of TNF-α by both PBMC and WBC was significantly higher after stimulation with trypomastigotes. Only PBMC produced significantly higher levels of IL-12 after parasite stimulation. Stimulation of cultures with trypomastigotes induced an increase of CXCL-9 levels produced by WBC. Nitrite levels produced by PBMC increased after the addition of parasites to the culture. CONCLUSIONS: Surface molecules of T. cruzi may induce the production of cytokines and chemokines by cells of the innate immune system through the activation of specific receptors not evaluated in this experiment. The ability to induce IL-12 and TNF-α contributes to shift the adaptive response towards a Th1 profile.

  2. p-Cresol affects reactive oxygen species generation, cell cycle arrest, cytotoxicity and inflammation/atherosclerosis-related modulators production in endothelial cells and mononuclear cells.

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    Mei-Chi Chang

    Full Text Available AIMS: Cresols are present in antiseptics, coal tar, some resins, pesticides, and industrial solvents. Cresol intoxication leads to hepatic injury due to coagulopathy as well as disturbance of hepatic circulation in fatal cases. Patients with uremia suffer from cardiovascular complications, such as atherosclerosis, thrombosis, hemolysis, and bleeding, which may be partly due to p-cresol toxicity and its effects on vascular endothelial and mononuclear cells. Given the role of reactive oxygen species (ROS and inflammation in vascular thrombosis, the objective of this study was to evaluate the effect of p-cresol on endothelial and mononuclear cells. METHODS: EA.hy926 (EAHY endothelial cells and U937 cells were exposed to different concentrations of p-cresol. Cytotoxicity was evaluated by 3-(4,5-Dimethylthiazol-2-yl-2,5 -diphenyltetrazolium bromide (MTT assay and trypan blue dye exclusion technique, respectively. Cell cycle distribution was analyzed by propidium iodide flow cytometry. Endothelial cell migration was studied by wound closure assay. ROS level was measured by 2',7'-dichlorofluorescein diacetate (DCF fluorescence flow cytometry. Prostaglandin F2α (PGF2α, plasminogen activator inhibitor-1 (PAI-1, soluble urokinase plasminogen activator receptor (suPAR, and uPA production were determined by Enzyme-linked immunosorbant assay (ELISA. RESULTS: Exposure to 100-500 µM p-cresol decreased EAHY cell number by 30-61%. P-cresol also decreased the viability of U937 mononuclear cells. The inhibition of EAHY and U937 cell growth by p-cresol was related to induction of S-phase cell cycle arrest. Closure of endothelial wounds was inhibited by p-cresol (>100 µM. P-cresol (>50 µM also stimulated ROS production in U937 cells and EAHY cells but to a lesser extent. Moreover, p-cresol markedly stimulated PAI-1 and suPAR, but not PGF2α, and uPA production in EAHY cells. CONCLUSIONS: p-Cresol may contribute to atherosclerosis and thrombosis in patients with

  3. Mesenchymal Stem Cells and Mononuclear Cells From Cord Blood: Cotransplantation Provides a Better Effect in Treating Myocardial Infarction.

    Science.gov (United States)

    Chen, Gecai; Yue, Aihuan; Yu, Hong; Ruan, Zhongbao; Yin, Yigang; Wang, Ruzhu; Ren, Yin; Zhu, Li

    2016-03-01

    The aim of this study was to evaluate the effect of cotransplanting mononuclear cells from cord blood (CB-MNCs) and mesenchymal stem cells (MSCs) as treatment for myocardial infarction (MI). Transplanting CD34+ cells or MSCs separately has been shown effective in treating MI, but the effect of cotransplanting CB-MNCs and MSCs is not clear. In this study, MSCs were separated by their adherence to the tissue culture. The morphology, immunophenotype, and multilineage potential of MSCs were analyzed. CB-MNCs were separated in lymphocyte separation medium 1.077. CD34+ cell count and viability were analyzed by flow cytometry. Infarcted male Sprague-Dawley rats in a specific-pathogen-free grade were divided into four treatment groups randomly: group I, saline; group II, CB-MNCs; group III, MSCs; and group IV, CB-MNCs plus MSCs. The saline, and CB-MNCs and/or MSCs were injected intramyocardially in infarcted rats. Their cardiac function was evaluated by echocardiography. The myocardial capillary density was analyzed by immunohistochemistry. Both cell types induced an improvement in the left ventricular cardiac function and increased tissue cell proliferation in myocardial tissue and neoangiogenesis. However, CB-MNCs plus MSCs were more effective in reducing the infarct size and preventing ventricular remodeling. Scar tissue was reduced significantly in the CB-MNCs plus MSCs group. MSCs facilitate engraftment of CD34+ cells and immunomodulation after allogeneic CD34+ cell transplantation. Cotransplanting MSCs and CB-MNCs might be more effective than transplanting MSCs or CB-MNCs separately for treating MI. This study contributes knowledge toward effective treatment strategies for MI.

  4. Transplanted human umbilical cord blood mononuclear cells improve left ventricular function through angiogenesis in myocardial infarction

    Institute of Scientific and Technical Information of China (English)

    HU Cheng-heng; WU Gui-fu; WANG Xiao-qing; YANG Yan-hua; DU Zhi-min; HE Xiao-hong; XIANG Peng

    2006-01-01

    Background Human umbilical cord blood contains an abundance of immature stem/progenitor cells, which may participate in the repair of hearts that have been damaged by myocardial infarction (MI). This study aimed to evaluate the effects of human umbilical cord blood mononuclear cells (hUCBC) transplantation on cardiac function and left ventricular remodeling in rat model of MI.Methods Forty-five male Wistar rats were randomized into three groups: MI or control group (n=15), MI plus cell transplantation (n=15), and sham group (n=15). Acute myocardial infarction (AMI) was established by ligating the left anterior descending artery, thereafter, hUCBC were implanted into the marginal area of infarcted myocardium. In MI/control group, DMEM was injected instead of hUCBC following the same protocol. Left ventricular function assessment was carried out by echocardiography and invasive hemodynamic measurements one month post MI. All rats were sacrificed for histological and immunochemical examinations.Results The transplanted hUCBC survived and engaged in the process of myocardial repair in the host heart.Echocardiography demonstrated that left ventricular function improved significantly in the rats that underwent cell transplantation. Hemodynamic studies found a significantly decreased left ventricular end-diastolic pressure (LVEDP) [(21.08±8.10) mmHg vs (30.82±9.59) mmHg, P<0.05], increase in +dp/dtmax [(4.29± 1.27)mmHg/ms vs (3.24±0.75) mmHg/ms, P<0.05), and increase in -dp/dtmax [(3.71 ±0.79) mmHg/ms vs (3.00±0.49) mmHg/ms, P<0.05] among MI group with hUCBC transplantation when compared with MI/control group.Masson's trichrome staining revealed that the collagen density in the left ventricle was significantly lower in rats of transplantation group than that in the MI control groups [(6.33±2.69)% vs (11.10±3.75)%, P< 0.01]. Based on immunostaining of α-actin, the numbers of microvessels were significantly (P<0.01) increased at the boundary of

  5. Culture of equine bone marrow mononuclear fraction and adipose tissue-derived stromal vascular fraction cells in different media

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    Gesiane Ribeiro

    2013-12-01

    Full Text Available The objective of this study was to evaluate the culture of equine bone marrow mononuclear fraction and adipose tissue - derived stromal vascular fraction cells in two different cell culture media. Five adult horses were submitted to bone marrow aspiration from the sternum, and then from the adipose tissue of the gluteal region near the base of the tail. Mononuclear fraction and stromal vascular fraction were isolated from the samples and cultivated in DMEM medium supplemented with 10% fetal bovine serum or in AIM-V medium. The cultures were observed once a week with an inverted microscope, to perform a qualitative analysis of the morphology of the cells as well as the general appearance of the cell culture. Colony-forming units (CFU were counted on days 5, 15 and 25 of cell culture. During the first week of culture, differences were observed between the samples from the same source maintained in different culture media. The number of colonies was significantly higher in samples of bone marrow in relation to samples of adipose tissue.

  6. Hemorrhage induces rapid in vivo activation of CREB and NF-kappaB in murine intraparenchymal lung mononuclear cells.

    Science.gov (United States)

    Shenkar, R; Abraham, E

    1997-02-01

    Increased expression of proinflammatory cytokines appears to be an important factor contributing to the development of acute lung injury. In murine models, mRNA levels of proinflammatory and immunoregulatory cytokines, including IL-1alpha, IL-1beta, TGF-beta1, and TNF-alpha, are increased in intraparenchymal lung mononuclear cells 1 h after hemorrhage. Binding elements for the nuclear transcriptional regulatory factors, nuclear factor kappaB (NF-kappaB), CCAAT/enhancer binding protein beta (C/EBPbeta), serum protein 1 (Sp1), activator protein 1 (AP-1), and the cyclic AMP response-element binding protein (CREB) are present in the promoter regions of numerous cytokine genes, including those whose expression is increased after blood loss. To investigate early transcriptional mechanisms which may be involved in regulating pulmonary cytokine expression after hemorrhage, we examined in vivo activation of these five nuclear transcriptional factors among intraparenchymal lung mononuclear cells obtained in the immediate post-hemorrhage period. Activation of NF-kappaB and CREB, but not C/EBPbeta, Sp1, or AP-1, was present in lung mononuclear cells isolated from mice 15 min after hemorrhage. Inhibition of xanthine oxidase by prior feeding with either an allopurinol-supplemented or a tungsten-enriched diet prevented hemorrhage-induced activation of CREB, but not NF-kappaB. These results demonstrate that hemorrhage leads to rapid in vivo activation in the lung of CREB through a xanthine oxidase-dependent mechanism and of NF-kappaB through other pathways, and suggest that the activation of these transcriptional factors may have an important role in regulating pulmonary cytokine expression and the development of acute lung injury after blood loss.

  7. Concise review: bone marrow mononuclear cells for the treatment of ischemic syndromes: medicinal product or cell transplantation?

    Science.gov (United States)

    Cuende, Natividad; Rico, Laura; Herrera, Concha

    2012-05-01

    In November of 2011, the Committee for Advanced Therapies (CAT) of the European Medicines Agency (EMA) published two scientific recommendations regarding the classification of autologous bone marrow-derived mononuclear cells (BM-MNCs) and autologous bone marrow-derived CD133+ cells as advanced therapy medicinal products (ATMPs), specifically tissue-engineered products, when intended for regeneration in ischemic heart tissue on the basis that they are not used for the same essential function (hematological restoration) that they fulfill in the donor. In vitro and in vivo evidence demonstrates that bone marrow cells are physiologically involved in adult neovascularization and tissue repair, making their therapeutic use for these purposes a simple exploitation of their own essential functions. Therefore, from a scientific/legal point of view, nonsubstantially manipulated BM-MNCs and CD133+ cells are not an ATMP, because they have a physiological role in the processes of postnatal neovascularization and, when used therapeutically for vascular restoration in ischemic tissues, they are carrying out one of their essential physiological functions (the legal definition recognizes that cells can have several essential functions). The consequences of classifying BM-MNCs and CD133+ cells as medicinal products instead of cellular transplantation, like bone marrow transplantation, in terms of costs and time for these products to be introduced into clinical practice, make this an issue of crucial importance. Therefore, the recommendations of EMA/CAT could be reviewed in collaboration with scientific societies, in light of organizational and economic consequences as well as scientific knowledge recently acquired about the mechanisms of postnatal neovascularization and the function of bone marrow in the regeneration of remote tissues.

  8. Genotoxicity of alkene epoxides in human peripheral blood mononuclear cells and HL60 leukaemia cells evaluated with the comet assay.

    Science.gov (United States)

    Fabiani, Roberto; Rosignoli, Patrizia; De Bartolomeo, Angelo; Fuccelli, Raffaela; Morozzi, Guido

    2012-08-30

    Volatile organic compounds (VOCs) exert their carcinogenic activity through the production of epoxide metabolites. Because of their high reactivity some epoxides are also produced in the chemical industry for the synthesis of other compounds. Therefore, human exposure to VOCs epoxides does occur and may be an important human health concern. In this study, the in vitro genotoxic potential of epoxides originating from 1,3-butadiene (3,4-epoxy-1-butene: EB; 1,2:3,4-diepoxybutane: DEB), isoprene (3,4-epoxy-2-methyl-1-butene: IO), styrene (styrene-7,8-oxide: SO), propylene (propylene oxide: PO) and 1-butene (1,2-epoxy-butane: BO) in human peripheral blood mononuclear cells (PBMCs) and promyelocytic leukaemia cells (HL60) was measured with the comet assay (single-cell gel electrophoresis, SCGE). The effect of inclusion of foetal calf serum (FCS, 5%) in the cell-culture medium and different durations of exposure (2h, 24h) were also investigated. All epoxides tested produced DNA damage in a concentration range that did not reduce cell viability. HL60 cells were more resistant than PBMCs to the DNA damage induced by the different epoxides. With the exception of IO, the treatment for 24h resulted in an increase of DNA damage. FCS slightly protected PBMCs from the genotoxic effects induced by IO and BO, whilst no such effect was noted for the other compounds. Overall, the dose-dependent effects that were seen allowed us to define a genotoxicity scale for the different epoxides as follows: SO>EB>DEB>IO>PO>BO, which is in partial agreement with the International Agency for Research on Cancer (IARC) classification of the carcinogenic hazards.

  9. Effect of 900 MHz Electromagnetic Radiation on the Induction of ROS in Human Peripheral Blood Mononuclear Cells

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    Mortazavi S.M.J.

    2015-09-01

    Full Text Available Background: Despite numerous studies over a decade, it still remains controversial about the biological effects of RF EMF emitted by mobile phone telephony. Objective: Here we investigated the effect of 900 MHz GSM on the induction of oxidative stress and the level of intracellular reactive oxygen species (ROS in human mononuclear cells, monocytes and lymphocytes as defence system cells. Method: 6 ml Peripheral Blood samples were obtained from 13 healthy volunteers (21-30 year-old. Each sample was devided into 2 groups: one was exposed RF radiation emitted from a mobile phone simulator for 2 hour and the other used as control group which was not exposed to any fields. After that, mononuclear cells were isolated from peripheral blood by density gradient centrifugation in Ficoll-Paque. The intracellular ROS content in monocytes and lymphocytes was measured by the CM-H2DCFDA fluorescence probe using flowcytometry technique. Results: Our results showed significant increase in ROS production after exposure in population rich in monocytes. This effect was not significant in population rich in lymphocytes in comparison with non exposed cells. Conclusion: The results obtained in this study clearly showed the oxidative stress induction capability of RF electromagnetic field in the portion of PBMCs mostly in monocytes, like the case of exposure to micro organisms, although the advantages or disadvantages of this effect should be evaluated.

  10. Evaluation of apoptosis induction in human peripheral blood mononuclear cells and synovial cells in patients with rheumatoid arthritis.

    Science.gov (United States)

    Demian, Soheir R; Abo-Shousha, Seham A; Sultan, Hussein E; Zarka, Wael El

    2005-01-01

    Rheumatoid arthritis (RA) is a chronic inflammatory destructive disease involving the joint and characterized by T-lymphocyte accumulation within the synovial compartment. It is dominated by the presence of macrophages, plasma cells and synovial fibroblasts which are the main pathogenic factors leading to the destruction of bone and cartilage. The survival of these cells may be promoted by inadequate apoptosis leading to synovial hyperplasia. So, the aim of the present study was to evaluate the apoptosis levels before and after induction of apoptosis using anti-Fas mAb, both in peripheral blood (PB) and synovial fluid (SF) infiltrating mononuclear cells (MCs) of patients with RA. CD4+ T cell subsets and cell survival assays were also done to investigate correlations between these parameters. The study was conducted on 15 patients with RA, 10 individual volunteers as a control group and 10 patients with osteoarthritis (OA) as a control group for SF evaluations (have defective Fas expression on their cells). Results of this work revealed that in vitro induction of apoptosis by anti-Fas mAb resulted in increase of: percent (%) reduction of cell viability in PBMCs and SFMCs, % reduction of CD4+ T cell subsets and apoptotic cell % in all studied groups than before induction. The increase in the three parameters is only significant in SF of RA group compared to PB while it is non significant in OA group due to the defective Fas expression on OA cells. Our results also showed a significant positive correlation between CD4+ T cell and viability percentages before induction of apoptosis in SF of RA and between apoptosis levels and CD4+ T cell percentage after induction of apoptosis in the SF of RA group. In conclusion, activated T cells infiltrating SF of RA patients have functional Fas antigen which enable them to undergo in vitro apoptosis using anti-Fas mAb. The cytotoxicity of which is more specific to local lesion such as SF of RA patients suggesting that local

  11. [Effect of airborne fungi on interleukin-4 mRNA expression in the peripheral blood mononuclear cells of rats].

    Science.gov (United States)

    Kan, H; Song, W; Jiang, R

    2001-09-01

    The effect of indoor airborne fungi on interleukin-4 (IL-4) mRNA expression in the peripheral blood mononuclear cells (PBMCs) of rats was studied by inoculating via trachea with fungi suspension. A simplified competitive RT-PCR method was used to measure the IL-4 mRNA expression in plasma. The results showed that interleukin-4 expression was much higher in the affected groups than that in the control one. It suggested that IL-4 might play an important role in the sensitization progress of fungi.

  12. A RNA transcript (Heg) in mononuclear cells is negatively correlated with CD14 mRNA and TSH receptor autoantibodies

    DEFF Research Database (Denmark)

    Habekost, G.; Bratholm, P.; Christensen, Niels Juel

    2008-01-01

    ) patients with early and untreated Graves' disease; and (iii) patients with Graves' disease studied after treatment. In 18 normal subjects and in 20 patients with treated Graves' disease CD14 mRNA was negatively correlated with Heg (P ... receptor autoantibodies were negatively correlated (P ...During a study of gene expression of foxp3 in blood mononuclear cells we observed a DNA product of an unknown RNA fragment. The area of this peak correlated with CD14 mRNA in a small group of subjects. The sequence was localized to chromosome 1. We tested the hypothesis that gene expression...

  13. Microarray profiling of mononuclear peripheral blood cells identifies novel candidate genes related to chemoradiation response in rectal cancer.

    Directory of Open Access Journals (Sweden)

    Pablo Palma

    Full Text Available Preoperative chemoradiation significantly improves oncological outcome in locally advanced rectal cancer. However there is no effective method of predicting tumor response to chemoradiation in these patients. Peripheral blood mononuclear cells have emerged recently as pathology markers of cancer and other diseases, making possible their use as therapy predictors. Furthermore, the importance of the immune response in radiosensivity of solid organs led us to hypothesized that microarray gene expression profiling of peripheral blood mononuclear cells could identify patients with response to chemoradiation in rectal cancer. Thirty five 35 patients with locally advanced rectal cancer were recruited initially to perform the study. Peripheral blood samples were obtained before neaodjuvant treatment. RNA was extracted and purified to obtain cDNA and cRNA for hybridization of microarrays included in Human WG CodeLink bioarrays. Quantitative real time PCR was used to validate microarray experiment data. Results were correlated with pathological response, according to Mandard´s criteria and final UICC Stage (patients with tumor regression grade 1-2 and downstaging being defined as responders and patients with grade 3-5 and no downstaging as non-responders. Twenty seven out of 35 patients were finally included in the study. We performed a multiple t-test using Significance Analysis of Microarrays, to find those genes differing significantly in expression, between responders (n = 11 and non-responders (n = 16 to CRT. The differently expressed genes were: BC 035656.1, CIR, PRDM2, CAPG, FALZ, HLA-DPB2, NUPL2, and ZFP36. The measurement of FALZ (p = 0.029 gene expression level determined by qRT-PCR, showed statistically significant differences between the two groups. Gene expression profiling reveals novel genes in peripheral blood samples of mononuclear cells that could predict responders and non-responders to chemoradiation in patients with

  14. Detection of the covalently closed circular DNA in peripheral blood mononuclear cells of hepatitis B patients and its clinical significance

    Institute of Scientific and Technical Information of China (English)

    朱圣涛

    2014-01-01

    Objective To analyze the correlation between covalently closed circular DNA(ccc DNA)in the peripheral blood mononuclear cells(PBMC)of hepatitis B virus(HBV)-infected patients and serum HBV DNA,hepatitis B surface antigen(HBsA g),hepatitis B e antigen(HBe Ag)and liver histology of hepatitis B patients,and to explore the clinical significance of HBV ccc DNA detection in PBMC.Methods One hundred and eight patients with chronic HBV infection were involved in this

  15. Increased expression of the 20S proteasome in peripheral blood mononuclear cells of type 2 diabetic patients

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Objective To investigate the dynamic expression of the 20S proteasome in peripheral blood mononuclear cells(PBMCs)of type 2 diabetic patients without vascular complications.Methods PBMCs were prepared from 30 type 2 diabetic patients and 30 nondiabetic controls.The general indexes including weight,height and blood pressure were recorded.Fasting plasma glucose,fasting plasma insulin and glycosylated hemoglobin were measured.The protein level of the 20S proteasome was measured by Western blotting.The mRNA exp...

  16. The effects of low-dose methotrexate on thymidylate synthetase activity in human peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Hornung, N; Stengaard-Pedersen, K.; Ehrnrooth, E

    2001-01-01

    ) and dihydrofolate reductase essential for DNA synthesis. This study investigated the effects of low-dose MTX on TS activity and proliferation in human peripheral blood mononuclear cells (PBMC). METHODS: The MTX concentrations in our experiments were chosen according to the plasma concentrations measured in 8 RA......, there was no difference between TS activity measured before and after MTX intake in 6 RA patients on long-term MTX treatment. CONCLUSION: We show that low concentrations of MTX inhibit TS activity in vitro. An in vivo effect cannot, however, be proven given our study design. The role of these in vitro findings...

  17. Effect of Activation-induced Cell Death of Peripheral Blood Mononuclear cells in Patients with Condyloma Acuminatum

    Institute of Scientific and Technical Information of China (English)

    江惟苏; 谭升顺

    2004-01-01

    Objective: To investigate the effect of activation-in-duced cell death (AICD) on cellular immune function in the condyloma acuminatum(CA). Methods: Peripheral blood mononuclear cells(PBMC) were isolated from normal healthy individuals (control group) and patients with CA. PBMC were cultured with PHA-P for 48h in vitro. Apoptosis of the PBMC was detected by flow cytometry. Supernatant cytokines (IL-2 and IL-10) were assayed by ELISA. Results: The rate of PBMC apoptosis in both CA group and control group in fresh PBMC was very low and similar in both groups(P>0.05). The rate of PBMC apoptosis within the CA group was noticeably increased compared to that of the control (P<0.001)after PBMC were cultured for 48h. The level of IL-2 was significantly lower in the CA group than in the control group (P<0.001), The level of IL-10 was significantly higher in the CA group compared to thecontrolgroup(P<0.001). Conclusion: Study results indicate that AICD may affect cellular mediated immune function and play an important role in the pathogenesis of CA.

  18. Reactive oxygen species formation by polymorphonuclear cells and mononuclear cells as a risk factor of cardiovascular diseases.

    Science.gov (United States)

    Yasunari, Kenichi; Watanabe, Takanori; Nakamura, Munehiro

    2006-04-01

    To better identify patients at high risk for cardiovascular events, several markers of risk have been proposed for use in screening. Recently, oxidative stress and inflammation have been evaluated as potential tools for prediction of the risk of cardiovascular events. Among them, we have measured reactive oxygen species (ROS) formation by polymorphonuclear cells (PMNs) and mononuclear cells (MNCs), since they may be a possible link between inflammation and oxidative stress. ROS formation by PMNs and MNCs was measured by a gated flow cytometric assay. Such biotechnological method of measuring ROS formation by PMNs and MNCs will make it possible that we measure vascular oxidative stress and vascular inflammation at the same time from only small amount of blood. We will state in this review that ROS formation by PMNs and MNCs are regulated by different mechanisms, although PMNs and MNCs are circulating in the same blood. Moreover, we will state that ROS formation by PMNs are regulated by blood pressure, Hb A(1C) and oxidided LDL. ROS formation by MNCs are regulated by vascular inflammation, and that ROS formation by MNCs are also related to various cardiovascular risks such as LV mass, norepinephrine, IMT, and nocturnal blood pressure.

  19. Members of the Candida parapsilosis complex and Candida albicans are differentially recognized by human peripheral blood mononuclear cells

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    Eine eEstrada-Mata

    2016-01-01

    Full Text Available The systemic infections caused by members of the Candida parapsilosis complex are currently associated to high mobility and mortality rates, and are considered as relevant as those caused by Candida albicans. Since the fungal cell wall is the first point of contact with the host cells, here we performed a comparison of this organelle in members of the C. parapsilosis complex, and its relevance during interaction with human peripheral blood mononuclear cells. We found that the wall of the C. parapsilosis complex members is similar in composition, but differs to that from C. albicans, with less mannan content and more β-glucan and porosity levels. Furthermore, lectin-based analysis showed increased chitin and β1,3-glucan exposure at the surface of C. parapsilosis sensu lato when compared to C. albicans. Yeast cells of members of the C. parapsilosis complex stimulated more cytokine production by human peripheral blood mononuclear cells than C. albicans cells; and this significantly changed upon removal of O-linked mannans, indicating this wall component plays a significant role in cytokine stimulation by C. parapsilosis sensu lato. When inner wall components were exposed on the wall surface, C. parapsilosis sensu stricto and C. metapsilosis, but not C. orthopsilosis, stimulated higher cytokine production. Moreover, we found a strong dependency on β1,3-glucan recognition for the members of the C. parapsilosis complex, but not for live C. albicans cells; whereas TLR4 was required for TNFα production by the three members of the complex, and stimulation of IL-6 by C. orthopsilosis. Mannose receptor had a significant role during TNF and IL-1β stimulation by members of the complex. Finally, we demonstrated that purified N- and O-mannans from either C. parapsilosis sensu lato or C. albicans are capable to block the recognition of these pathogens by human peripheral blood mononuclear cells. Together; our results suggest that the innate immune

  20. The effect of acute exercise on GLUT4 levels in peripheral blood mononuclear cells of sled dogs.

    Science.gov (United States)

    Schnurr, Theresia M; Reynolds, Arleigh J; Komac, Alyssa M; Duffy, Lawrence K; Dunlap, Kriya L

    2015-07-01

    Using sled dogs as exercise model, our objectives of this study were to 1) assess the effects of one acute bout of high-intensity exercise on surface GLUT4 concentrations on easily accessible peripheral blood mononuclear cells (PBMC) and 2) compare our findings with published research on exercise induced GLUT4 in skeletal muscle. During the exercise bout, dogs ran 5 miles at approximately 90% of VO2 max. PMBC were collected before exercise (baseline), immediately after exercise and after 24h recovery.GLUT4 was measured via ELISA. Acute exercise resulted in a significant increase on surface GLUT4 content on PBMC. GLUT4 was increased significantly immediately after exercise (~ 50%; p0.05). An effect of acute exercise on GLUT4 levels translocated to the cell membrane was observed, with GLUT4 levels not yet returned to baseline after 24h post-exercise. In conclusion, the present investigation demonstrated that acute high-intensity exercise increased GLUT4 content at the surface of PBMC of sled dogs as it has been reported in skeletal muscle in other species. Our findings underline the potential use of peripheral blood mononuclear cell GLUT4 protein content as minimally invasive proxy to investigate relationships between insulin sensitivity, insulin resistance, GLUT4 expression and glucose metabolism.

  1. Sumatriptan increases the proliferation of peripheral blood mononuclear cells from HIV-infected individuals and healthy blood donors in vitro

    DEFF Research Database (Denmark)

    Afzelius, P; Nielsen, Jens Ole

    2000-01-01

    HIV infection is characterized by the loss of CD4+ T cells as well as the loss of T-cell function, leading to severe immunodeficiency. The proliferative capacity of T cells measured in vitro as responses to antigens and mitogens is severely reduced during HIV infection. An increased level...... responsible for regulation of the intracellular levels of cAMP. In a preliminary study sumatriptan increased the proliferative responses of PBMC to a polyclonal activator in vitro in 9 of 10 HIV-seropositive individuals (p=0.007), and in 7 of 9 healthy blood donors (p=0.05). This was probably due...... of the intracellular second messenger adenosine 3',5'-cyclic monophosphate (cAMP) has been shown to cause impaired proliferative capacity of peripheral blood mononuclear cells (PBMC) from HIV-infected individuals in vitro. Sumatriptan, a 5HT1d receptor agonist, inhibits the activity of adenylyl cyclases, the enzymes...

  2. THE IMMUNOMODULATORY EFFECTS OF PROBIOTIC BACTERIA ON PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMCS OF ALLERGIC PATIENTS

    Directory of Open Access Journals (Sweden)

    Somaya M. El Sheikh

    2014-01-01

    Full Text Available Allergic diseases represent major health burden. An allergic reaction is characterized by a disrupted T- helper 1⁄T-helper 2 balance toward a preferential allergen specifically induced TH2 cytokine profile, causing allergic inflammation Probiotic bacteria have various benificial effects in many pathologic situation. Studies have shown that the bacteria present in the intestinal micro flora play a role in the TH1/TH2 balance and its modulation can promote the control of infectious and immune processes. Testing the effects of probiotic bacteria on TH1/TH2 cytokine production by peripheral blood mononuclear cells of allergic patients and control subjects. This study included 24 patients allergic to date pollen and 16 healthy control subjects. PBMC of both groups were separated and cultured for 72 h with date pollen allergen (home-made in the presence or absence of Lactobacillus rhamnosus ATCC 7469 (Living and dead and C- phycocyanin (extracted from Spirulina platensis. The cell culture supernatants were collected to measure Interlukin 4 and Interferon gamma by quantitative ELISA. Incubation of PBMCs of allergic patients with living Lactobacillus rhamnosus ATCC 7469 showed marked reduction in IL4 production (median IL4 concentarion = 3.9 pg. compared to PBMCs callenged with pollen alone (mediam IL4 conentration = 52.6 pg. When PBMC were incubated with living Lactobacillus rhamnosus in absence of allergen significant increase in and IFNγ (median concentration = 42.75 pg. was obtained, compared to PBMC challenged with allergen alone (median = 22.8 pg. When PBMCs incubated with heat killed Lactobacillus rhamnosus either in presence or absence of the offending allergen, marked reduction in IL4 production was obtained (median = 10.6, 3.6 pg respectively compared to PBMC incubated with allergen alone (median = 52.6 pg. When PBMCs incubated with dead Lactobacillus rhamnosus, marked increase in IFNγ production

  3. Dengue viral RNA levels in peripheral blood mononuclear cells are associated with disease severity and preexisting dengue immune status.

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    Anon Srikiatkhachorn

    Full Text Available BACKGROUND: Infection with dengue viruses (DENV causes a wide range of manifestations from asymptomatic infection to a febrile illness called dengue fever (DF, to dengue hemorrhagic fever (DHF. The in vivo targets of DENV and the relation between the viral burden in these cells and disease severity are not known. METHOD: The levels of positive and negative strand viral RNA in peripheral blood monocytes, T/NK cells, and B cells and in plasma of DF and DHF cases were measured by quantitative RT-PCR. RESULTS: Positive strand viral RNA was detected in monocytes, T/NK cells and B cells with the highest amounts found in B cells. Viral RNA levels in CD14+ cells and plasma were significantly higher in DHF compared to DF, and in cases with a secondary infection compared to those undergoing a primary infection. The distribution of viral RNA among cell subpopulations was similar in DF and DHF cases. Small amounts of negative strand RNA were found in a few cases only. The severity of plasma leakage correlated with viral RNA levels in plasma and in CD14+ cells. CONCLUSIONS: B cells were the principal cells containing DENV RNA in peripheral blood, but overall there was little active DENV RNA replication detectable in peripheral blood mononuclear cells (PBMC. Secondary infection and DHF were associated with higher viral burden in PBMC populations, especially CD14+ monocytes, suggesting that viral infection of these cells may be involved in disease pathogenesis.

  4. Increased interleukin-23 receptor+ T cells in peripheral blood mononuclear cells of patients with systemic lupus erythematosus

    Science.gov (United States)

    2010-01-01

    Introduction Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by production of autoantibodies and immune complex deposition in various organs. Aberrations in the T lymphocyte compartment and dysregulated cytokine production are key features of SLE pathogenesis and disease progression. Recently, the role of the interleukin (IL)-17/IL-23 axis in the pathogenesis of SLE has been reported. IL-23 and IL-23R are essential for expansion of pathogenic IL-17-producing T lymphocytes and have been shown to be important in the pathogenesis of lupus in animal models. Methods In this study, the expression of IL-23R and IL-17 in CD4+ and CD8+ T lymphocytes in peripheral blood mononuclear cells (PBMCs) of SLE patients and control subjects were examined by flow cytometry. Twenty-nine SLE patients and 10 control subjects were recruited in this study. Patients were divided into active and inactive groups based on the SLE disease activity index (SLEDAI). As another disease control population, five psoriatic patients were recruited in this study. Results Percentages of both IL23R+ CD4+ and IL-23R+ CD8+ T cell subsets were significantly higher in freshly isolated PBMCs from both groups of SLE patients compared to control subjects (P = 0.0021 and P = 0.0006, respectively). In addition, this difference was maintained after ex vivo stimulation with plate-bound anti-CD3/CD28 antibodies (P = 0.007 and P = 0.0019, respectively). When the fold increase in IL-17+ T cells after ex vivo stimulation for three days was compared between patients and controls, SLE patients exhibited significantly higher increases in CD4+ IL-17+ and CD8+ IL-17+ T cells, suggesting that PBMCs from SLE patients promoted the expansion of IL-17-producing T cells upon stimulation more vigorously than control PBMCs. These trends were not observed in psoriasis patients. The correlations between IL-23R+ T cells and IL-17+ T cells and IL-23R+ CD8+ T cells and SLEDAI scores in patients were also found

  5. Variation in assessment of oxidatively damaged DNA in mononuclear blood cells by the comet assay with visual scoring

    DEFF Research Database (Denmark)

    Forchhammer, Lykke; Bräuner, Elvira Vaclavik; Folkmann, Janne Kjaersgaard;

    2008-01-01

    The comet assay is popular for assessments of genotoxicity, but the comparison of results between studies is challenging because of differences in experimental procedures and reports of DNA damage in different units. We investigated the variation of DNA damage in mononuclear blood cells (MNBCs......) measured by the comet assay with focus on the variation related to alkaline unwinding and electrophoresis time, number of cells scored, as well as the putative benefits of transforming the primary end points to common units by the use of reference standards and calibration curves. Eight experienced......-response relationships of cells exposed to gamma-radiation and it was possible to reduce the variation in oxidized purines in MNBCs from humans by adjusting the level of lesions with protocol-specific calibration curves. However, there was a difference in the level of DNA damage measured by different investigators...

  6. Evaluating the significance of density, localization, and PD-1/PD-L1 immunopositivity of mononuclear cells in the clinical course of lung adenocarcinoma patients with brain metastasis

    DEFF Research Database (Denmark)

    Téglási, Vanda; Reiniger, Lilla; Fabian, Katalin

    2017-01-01

    therapeutic strategies with these agents. The aim of this study was to characterize the distribution of ICs and determine the expression of the checkpoint molecules programmed death protein 1 (PD-1) and its ligand, PD-L1, in brain metastasis of lung adenocarcinoma (LUAD) patients and to analyze......Background. Management of lung cancer patients who suffer from brain metastases represents a major challenge. Considering the promising results with immune checkpoint inhibitor treatment, evaluating the status of immune cell (IC) infiltrates in the prognosis of brain metastasis may lead to better...... their clinicopathological correlations. Methods. We determined the presence of peritumoral mononuclear cells (mononuclear ring) and the density of intratumoral stromal mononuclear cells on brain metastasis tissue sections of 208 LUAD patients. PD-L1/PD-1 expressions were analyzed by immunohistochemistry. Results...

  7. Lethal effect of mononuclear cells derived from human umbilical cord blood differentiating into dendritic cells after in vitro induction of cytokines on neuroblastoma cells

    Institute of Scientific and Technical Information of China (English)

    Zhenghai Qu; Jianxin Zuo; Lirong Sun; Xindong Qu

    2006-01-01

    BACKGROUND: Dendritic cell is the most major antigen presenting cell of organism. It is proved in recent studies that human umbilical cord blood mononuclear cells induced and cultured in vitro by recombinant human granuIocyte-macrophage colony stimulating factor (rhG-MCSF) and recombinant human interleukin-4 (rhlL-4) can generate a great many dendritic cells and promote the lethal effect of T cells on human neuroblastoma, but it is unclear that whether the lethal effect is associated with the most proper concentration of dendritic cells.OBJ ECTIVE: To investigate the lethal effect of human umbilical cord blood mononuclear cells induced in vitro by cytokines differentiating into dendritic cells on human neuroblastoma, and its best concentration range.DESIGN: Open experiment.SETTING: Department of Pediatrics, the Medical School Hospital of Qingdao University.MATERIALS: The study was carried out in the Shandong Provincial Key Laboratory (Laboratory for the Department of Pediatrics of the Medical School Hospital of Qingdao University) during September 2005 to May 2006.Human umbilical cord blood samples were taken from the healthy newborn infants of full-term normal delivery during October to November 2005 in the Medical School Hospital of Qingdao University, and were voluntarily donated by the puerperas. Main instruments: type 3111 CO2 incubator (Forma Scientific, USA), type 550 ELISA Reader (Bio-Rad, USA). Main reagents: neuroblastoma cell line SK-N-SH (Shanghai Institute of Life Science, Chinese Academy of Sciences), RPMI-1640 culture fluid and fetal bovine serum (Hyclone), rhlL-4 (Promega, USA), rhG-MCSF (Harbin Pharmaceutic Group Bioengineering Co. Ltd), rat anti-human CD1a monoclonal antibody and FITC-labeled rabbit anti-rat IgG (Xiehe Stem cell Gene Engineering Co. Ltd).METHODS: ① Human umbilical cord blood mononuclear cells obtained with attachment methods differentiated into human umbilical cord blood dendritic cells, presenting typical morphology of

  8. Lactobacilli, bifidobacteria and E. coli nissle induce pro- and anti-infiammatory cytokines in peripheral blood mononuclear cells

    Institute of Scientific and Technical Information of China (English)

    Ulf Helwig; Stefan Schreiber; Massimo Campieri; Karen M Lammers; Fernando Rizzello; Patricia Brigidi; Verena Rohleder; Elisabetta Caramelli; Paolo Gionchetti; Juergen Schrezenmeir; Ulrich R Foelsch

    2006-01-01

    AIM: To investigate whether the stimulation of peripheral blood mononuclear cells (PBMNC) with the cell debris and cell extraction of different probiotic strains is similar or Species specific.METHODS: Three strains of bifidobacteria, 4 strains of lactobacilli, and E. colinissle were sonicated and centrifuged in order to divide them into cell extract and cell debris. PBMNC were separated by density gradient and incubated for 36 h with either the cell debris or the cell extract of single strains of probiotic bacteria in doses from 102 to 108 CFU/mL. Cell supernatants were taken and interleukin (IL)-10, IL-1β, and tumor necosis factor (TNF)-α were determined by El ISA.RESULTS: Depending on the species super-family, the strains had different stimulation patterns. Except for both L. casei strains, the cell extract of bifidobacteria and lactobacilli had less stimulating capacity than cell debris, whereas the cell extract of E. coli nissle had similar stimulating properties to that of the cell debris of the strain and significantly more stimulating capacity than that of bifidobacteria and lactobacilli. The cell debris of bifidobacteria stimulated more cytokine release than the cell debris of lactobacilli. The cell debris of lactobacilli did not have a stimulating capacity when lower concentrations were used. Neither cell extraction nor cell debris had an inhibitory effect on the production of the tested cytokines by stimulated PBMNC.CONCLUSION: The incubation of probiotic strains,which have been used in clinical trials for inflammatory diseases, with immunocompetent cells leads to different species specific reactions. High IL-10 response to cell debris of bifidobacteria and E. coli nissle can be found. This corresponds to positive effects of bifidobacteria and E.coli nissle in clinical trials for inflammatory bowel disease compared to negative outcomes obtained with lactobacilli.

  9. Mononuclear phagocyte intercellular crosstalk facilitates transmission of cell-targeted nanoformulated antiretroviral drugs to human brain endothelial cells

    Directory of Open Access Journals (Sweden)

    Kanmogne GD

    2012-05-01

    Full Text Available Georgette D Kanmogne1, Sangya Singh1, Upal Roy1, Xinming Liu1, JoEllyn McMillan1, Santhi Gorantla1, Shantanu Balkundi1, Nathan Smith1, Yazen Alnouti2, Nagsen Gautam2, You Zhou3, Larisa Poluektova1, Alexander Kabanov2, Tatiana Bronich2, Howard E Gendelman11Departments of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, 2Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE; 3Center for Biotechnology, University of Nebraska-Lincoln, Lincoln, NE, USAAbstract: Despite the successes of antiretroviral therapy (ART, HIV-associated neurocognitive disorders remain prevalent in infected people. This is due, in part, to incomplete ART penetration across the blood–brain barrier (BBB and lymph nodes and to the establishment of viral sanctuaries within the central nervous system. In efforts to improve ART delivery, our laboratories developed a macrophage-carriage system for nanoformulated crystalline ART (nanoART (atazanavir, ritonavir, indinavir, and efavirenz. We demonstrate that nanoART transfer from mononuclear phagocytes (MP to human brain microvascular endothelial cells (HBMEC can be realized through cell-to-cell contacts, which can facilitate drug passage across the BBB. Coculturing of donor MP containing nanoART with recipient HBMEC facilitates intercellular particle transfer. NanoART uptake was observed in up to 52% of HBMEC with limited cytotoxicity. Folate coating of nanoART increased MP to HBMEC particle transfer by up to 77%. To translate the cell assays into relevant animal models of disease, ritonavir and atazanavir nanoformulations were injected into HIV-1-infected NOD/scid-γcnull mice reconstituted with human peripheral blood lymphocytes. Atazanavir and ritonavir levels in brains of mice treated with folate-coated nanoART were three- to four-fold higher than in mice treated with noncoated particles. This was associated with decreased viral load in the spleen and

  10. Transcriptional profiling of peripheral blood mononuclear cells in pancreatic cancer patients identifies novel genes with potential diagnostic utility.

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    Michael J Baine

    Full Text Available BACKGROUND: It is well known that many malignancies, including pancreatic cancer (PC, possess the ability to evade the immune system by indirectly downregulating the mononuclear cell machinery necessary to launch an effective immune response. This knowledge, in conjunction with the fact that the trancriptome of peripheral blood mononuclear cells has been shown to be altered in the context of many diseases, including renal cell carcinoma, lead us to study if any such alteration in gene expression exists in PC as it may have diagnostic utility. METHODS AND FINDINGS: PBMC samples from 26 PC patients and 33 matched healthy controls were analyzed by whole genome cDNA microarray. Three hundred eighty-three genes were found to be significantly different between PC and healthy controls, with 65 having at least a 1.5 fold change in expression. Pathway analysis revealed that many of these genes fell into pathways responsible for hematopoietic differentiation, cytokine signaling, and natural killer (NK cell and CD8+ T-cell cytotoxic response. Unsupervised hierarchical clustering analysis identified an eight-gene predictor set, consisting of SSBP2, Ube2b-rs1, CA5B, F5, TBC1D8, ANXA3, ARG1, and ADAMTS20, that could distinguish PC patients from healthy controls with an accuracy of 79% in a blinded subset of samples from treatment naïve patients, giving a sensitivity of 83% and a specificity of 75%. CONCLUSIONS: In summary, we report the first in-depth comparison of global gene expression profiles of PBMCs between PC patients and healthy controls. We have also identified a gene predictor set that can potentially be developed further for use in diagnostic algorithms in PC. Future directions of this research should include analysis of PBMC expression profiles in patients with chronic pancreatitis as well as increasing the number of early-stage patients to assess the utility of PBMCs in the early diagnosis of PC.

  11. Hepatitis B virus (HBV) variants fluctuate in paired plasma and peripheral blood mononuclear cells among patient cohorts during different chronic hepatitis B (CHB) disease phases.

    Science.gov (United States)

    Coffin, C S; Osiowy, C; Gao, S; Nishikawa, S; van der Meer, F; van Marle, G

    2015-04-01

    Hepatitis B virus is classically considered a hepatotropic virus but also infects peripheral blood mononuclear cells. Chronic hepatitis B has different disease phases modulated by host immunity. We compared HBV variability, drug resistance and immune escape mutations in the overlapping HBV polymerase/surface gene in plasma and peripheral blood mononuclear cells in different disease phases. Plasma and peripheral blood mononuclear cells were isolated from 22 treatment naïve patient cohorts (five inactive, six immune-active, nine HBeAg negative and two immune-tolerant). HBV was genotyped via line probe assay, hepatitis B surface antigen titres were determined by an in-house immunoassay, and HBV DNA was quantified by kinetic PCR. The HBV polymerase/surface region, including full genome in some, was PCR-amplified and cloned, and ~20 clones/sample were sequenced. The sequences were subjected to various mutational and phylogenetic analyses. Clonal sequencing showed that only three of 22 patients had identical HBV genotype profiles in both sites. In immune-active chronic hepatitis B, viral diversity in plasma was higher compared with peripheral blood mononuclear cells. Mutations at residues, in a minority of clones, associated with drug resistance, and/or immune escape were found in both compartments but were more common in plasma. Immune escape mutations were more often observed in the peripheral blood mononuclear cells of immune-active CHB carriers, compared with other disease phases. During all CHB disease phases, differences exist between HBV variants found in peripheral blood mononuclear cells and plasma. Moreover, these data indicate that HBV evolution occurs in a compartment and disease phase-specific fashion.

  12. Feeding conditions control the expression of genes involved in sterol metabolism in peripheral blood mononuclear cells of normoweight and diet-induced (cafeteria) obese rats

    NARCIS (Netherlands)

    Caimari, A.; Oliver, P.; Rodenburg, W.; Keijer, J.; Palou, A.

    2010-01-01

    Peripheral blood mononuclear cells (PBMC) are easily obtainable cells from blood whose gene expression profiles have been proven to be highly robust in distinguishing a disease state from healthy state. Sterol metabolism is of physiological importance, and although its nutritional response in liver

  13. Feeding conditions control the expression of genes involved in sterol metabolism in peripheral blood mononuclear cells of normoweight and diet-induced (cafeteria) obese rats

    NARCIS (Netherlands)

    Caimari, A.; Oliver, P.; Rodenburg, W.; Keijer, J.; Palou, A.

    2010-01-01

    Peripheral blood mononuclear cells (PBMC) are easily obtainable cells from blood whose gene expression profiles have been proven to be highly robust in distinguishing a disease state from healthy state. Sterol metabolism is of physiological importance, and although its nutritional response in liver

  14. Generation of human iPSC line GRX-MCiPS4F-A2 from adult peripheral blood mononuclear cells (PBMCs with Spanish genetic background

    Directory of Open Access Journals (Sweden)

    Sonia Cabrera

    2015-09-01

    Full Text Available We have generated iPSCs from peripheral blood mononuclear cells (PBMCs of a healthy man using heat sensitive and non-integrative Sendai virus containing Sox2, Oct3/4, c-Myc and Klf4. Human GRX-MCiPS4F-A2 cell line was established and characterized through this study.

  15. Antiepileptic and neuroprotective effects of human umbilical cord blood mononuclear cells in a pilocarpine-induced epilepsy model.

    Science.gov (United States)

    Costa-Ferro, Zaquer Suzana Munhoz; de Borba Cunha, Fernanda; de Freitas Souza, Bruno Solano; Leal, Marcos Maurício Tosta; da Silva, Adelson Alves; de Bellis Kühn, Telma Ingrid Borges; Forte, Andresa; Sekiya, Eliseo Joji; Soares, Milena Botelho Pereira; Dos Santos, Ricardo Ribeiro

    2014-03-01

    Status epilepticus (SE) is a condition of persistent seizure that leads to brain damage and, frequently, to the establishment of chronic epilepsy. Cord blood is an important source of adult stem cells for the treatment of neurological disorders. The present study aimed to evaluate the effects of human umbilical cord blood mononuclear cells (HUCBC) transplanted into rats after induction of SE by the administration of lithium and pilocarpine chloride. Transplantation of HUCBC into epileptic rats protected against neuronal loss in the hippocampal subfields CA1, CA3 and in the hilus of the dentate gyrus, up to 300 days after SE induction. Moreover, transplanted rats had reduced frequency and duration of spontaneous recurrent seizures (SRS) 15, 120 and 300 days after the SE. Our study shows that HUCBC provide prominent antiepileptic and neuroprotective effects in the experimental model of epilepsy and reinforces that early interventions can protect the brain against the establishment of epilepsy.

  16. Human peripheral blood mononuclear cells exhibit heterogeneous CD52 expression levels and show differential sensitivity to alemtuzumab mediated cytolysis.

    Directory of Open Access Journals (Sweden)

    Sambasiva P Rao

    Full Text Available Alemtuzumab is a monoclonal antibody that targets cell surface CD52 and is effective in depleting lymphocytes by cytolytic effects in vivo. Although the cytolytic effects of alemtuzumab are dependent on the density of CD52 antigen on cells, there is scant information regarding the expression levels of CD52 on different cell types. In this study, CD52 expression was assessed on phenotypically distinct subsets of lymphoid and myeloid cells in peripheral blood mononuclear cells (PBMCs from normal donors. Results demonstrate that subsets of PBMCs express differing levels of CD52. Quantitative analysis showed that memory B cells and myeloid dendritic cells (mDCs display the highest number while natural killer (NK cells, plasmacytoid dendritic cells (pDCs and basophils have the lowest number of CD52 molecules per cell amongst lymphoid and myeloid cell populations respectively. Results of complement dependent cytolysis (CDC studies indicated that alemtuzumab mediated profound cytolytic effects on B and T cells with minimal effect on NK cells, basophils and pDCs, correlating with the density of CD52 on these cells. Interestingly, despite high CD52 levels, mDCs and monocytes were less susceptible to alemtuzumab-mediated CDC indicating that antigen density alone does not define susceptibility. Additional studies indicated that higher expression levels of complement inhibitory proteins (CIPs on these cells partially contributes to their resistance to alemtuzumab mediated CDC. These results indicate that alemtuzumab is most effective in depleting cells of the adaptive immune system while leaving innate immune cells relatively intact.

  17. Critical role of fractalkine (CX3CL1) in cigarette smoke-induced mononuclear cell adhesion to the arterial endothelium.

    Science.gov (United States)

    Rius, Cristina; Company, Chantal; Piqueras, Laura; Cerdá-Nicolás, Jose Miguel; González, Cruz; Servera, Emilio; Ludwig, Andreas; Morcillo, Esteban J; Sanz, Maria-Jesus

    2013-02-01

    Cigarette smoking is an important risk factor for the development of cardiovascular disease, yet the pathways through which this may operate are poorly understood. Therefore, the mechanism underlying cigarette smoke (CS)-induced arterial endothelial dysfunction and the potential link with fractalkine/CX(3)CL1 upregulation were investigated. Stimulation of human arterial umbilical endothelial cells (HUAECs) with pathophysiological concentrations of CS extract (1% CSE) increased CX(3)CL1 expression. Neutralisation of CX(3)CL1 activity under dynamic flow conditions significantly inhibited CSE-induced mononuclear cell adhesion to HUAECs (67%). The use of small interfering RNA (siRNA) revealed that nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 5 (Nox5) but not Nox2 or Nox4 is the main NADPH isoform involved in CSE-induced CX(3)CL1 upregulation and mononuclear cell arrest. Knock down of HUAEC tumour necrosis factor α expression with siRNA or pharmacological inhibition of p38 mitogen-activated protein kinase and nuclear factor κB also abolished these responses. Interestingly, circulating monocytes and lymphocytes from patients with chronic obstructive pulmonary disease (COPD) (n=29) versus age-matched controls (n=23) showed CX(3)CR1overexpression. Furthermore, CX(3)CL1 neutralisation dramatically diminished their enhanced adhesiveness to CSE-stimulated HUAECs. Finally, when animals were exposed for 3 days to CS, a mild inflammatory response in the lung was observed which was accompanied by enhanced CX(3)CL1 expression in the cremasteric arterioles, an organ distant from the lung. CS exposure resulted in increased leukocyte-arteriolar endothelial cell adhesion which was significantly reduced (51%) in animals lacking CX(3)CL1 receptor (CX(3)CR1). These results suggest that CS induces functional CX(3)CL1 expression in arterial endothelium and leukocytes from patients with COPD show increased CX(3)CL1-dependent adhesiveness. Therefore, targeting the CX(3)CL1

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

    OpenAIRE

    2013-01-01

    Background Although hepatitis C virus (HCV) is primarily hepatotropic, markers of HCV replication were detected in peripheral blood mononuclear cells (PBMC) as well as in ex vivo collected tissues and organs. Specific strains of HCV were found to be capable to infect cells of the immune system: T and B cells and monocytes/macrophages as well as cell lines in vitro. The direct invasion of cells of the immune system by the virus may be responsible for extrahepatic consequences of HCV infection:...

  19. Optimization of lentiviral vector transduction into peripheral blood mononuclear cells in combination with the fibronectin fragment CH-296 stimulation.

    Science.gov (United States)

    Chono, Hideto; Goto, Yumi; Yamakawa, Satoko; Tanaka, Shinya; Tosaka, Yasuhiro; Nukaya, Ikuei; Mineno, Junichi

    2011-03-01

    Large scale T-cell expansion and efficient gene transduction are required for adoptive T-cell gene therapy. Based on our previous observations, human peripheral blood mononuclear cells (PBMCs) can be expanded efficiently while conserving a naïve phenotype by stimulating with both recombinant human fibronectin fragment (CH-296) and anti-CD3 monoclonal antibodies. In this article, we explored the possibility of using this co-stimulation method to generate engineered T cells using lentiviral vector. Human PBMCs were stimulated with anti-CD3 together with immobilized CH-296 or anti-CD28 antibody as well as anti-CD3/anti-CD28 conjugated beads and transduced with lentiviral vector simultaneously. Co-stimulation with CH-296 gave superior transduction efficiency than with anti-CD28. Next, PBMCs were stimulated and transduced with anti-CD3/CH-296 or with anti-CD3/CD28 beads. T-cell expansion, gene transfer efficiencies and immunophenotypes were analysed. Stimulation with anti-CD3/CH-296 resulted in more than 10-times higher cell expansion and higher gene transfer efficiency with conservation of the naïve phenotype compared with anti-CD3/CD28 stimulation method. Thus, lentiviral transduction with anti-CD3/CH-296 co-stimulation is an efficient way to generate large numbers of genetically modified T cells and may be suitable for many gene therapy protocols that use adoptive T-cell transfer therapy.

  20. Study on the relationship between level of CD58 expression in peripheral blood mononuclear cell and severity of HBV infection

    Institute of Scientific and Technical Information of China (English)

    XIE Ming; WANG Xiang-ling; JI Yu-qiang; LI Jie; MENG Zhao-jun; SHI Lin; YUAN Yu-kang

    2005-01-01

    Background As one of the intercellular adhesion molecules, CD58 plays important roles in promotion of the adhesion between T cells and target cells, hyperplasia, activation of T cells and natural killer cells, and balance between Th1 and Th2. We studied the relationship between the levels of CD58 expression in peripheral blood mononuclear cells (PBMCs) and severity of HBV infection. Methods The levels of CD58 mRNA in PBMCs were detected using quantitative reverse transcription PCR. The percentage of CD58 positive cells was detected by flow cytometry in patients and healthy controls. Results The levels of CD58 mRNA and the percentage of CD58 positive cells in patients infected with HBV were significantly higher than that in the control. Based on severity of HBV infection, the patients were classified into four groups. The expression of CD58 increased significantly in an order from mild chronic, moderate chronic, severe chronic to severe hepatitis groups. The levels of CD58 mRNA and the percentage of CD58 positive cells in PBMCs from patients with HBV infection were both positively correlated with serum levels of ALT and AST.Conclusion The level of CD58 expression is related with the severity of HBV infection and the degree of liver damage.

  1. Use of autologous bone marrow mononuclear cells and cultured bone marrow stromal cells in dogs with orthopaedic lesions.

    Science.gov (United States)

    Crovace, A; Favia, A; Lacitignola, L; Di Comite, M S; Staffieri, F; Francioso, E

    2008-09-01

    The aim of the study is to evaluate the clinical application in veterinary orthopedics of bone marrow mononuclear cells (BMMNCs) and cultured bone marrow stromal cells (cBMSCs) for the treatment of some orthopaedic lesions in the dog. The authors carried out a clinical study on 14 dogs of different breed, age and size with the following lesions: 1 bone cyst of the glenoid rime; 2 nonunion of the tibia; 3 nonunion of the femur; 2 lengthening of the radius; 1 large bone defect of the distal radius;1 nonunion with carpus valgus; 4 Legg-Calvé-Perthés disease. In 9 cases the BMMCNs were used in combination with a three dimensional resorbable osteogenic scaffold the chemical composition and size of which facilitates the ingrowth of bone. In these cases the BMMNCs were suspended in an adequate amount of fibrin glue and then distribuited uniformly on a Tricalcium-Phosphate (TCP) scaffold onto which were also added some drops of thrombin. In 1 case of nonunion of the tibia and in 3 cases of Legg-Calvè-Perthés (LCP) disease the cultured BMSCs were used instead because of the small size of the dogs and of the little amount of aspirated bone marrow. X-ray examinations were performed immediately after the surgery. Clinical, ultrasounds and X-ray examinations were performed after 20 days and then every month. Until now the treated dogs have shown very good clinical and X-ray results. One of the objectives of the study was to use the BMMNCs in clinical application in orthopaedic lesions in the dog. The advantages of using the cells immediately after the bone marrow is collected, are that the surgery can be performed the same day, the cells do not need to be expanded in vitro, they preserve their osteogenic potential to form bone and promote the proper integration of the implant with the bone and lastly, the technique is easier and the costs are lower.

  2. Electron Transfer Pathways in Cell

    OpenAIRE

    Yan Liu

    2012-01-01

    Analysis of the electron salvation process data indicates that the electron transfer between the electron donor and acceptor is hindered by the electron salvation process. It is proposed that the electron transfer in the cell environment must be assisted by intermediate messenger called the “transport protein”.

  3. RESULTS OF INTRAMYOCARDIAL ADMINISTRATION OF A MONONUCLEAR FRACTION OF AUTOLOGOUS BONE MARROW CELLS IN CHD PATIENTS WITH CONCOMITANT CARDIAINSUFFICIENCY

    Directory of Open Access Journals (Sweden)

    A. M. Cherniavsky

    2013-01-01

    Full Text Available Aim. Evaluation of long-term results of drug therapy and intramyocardial administration of a mononuclear fraction of bone marrow cells in CHD patients with chronic cardiac insufficiency. Materials and methods. 109 patients were randomized into two groups by using an envelope method. Intramyocardial administration of a mononuclear fraction of autologous bone marrow cells and cardiac insufficiency therapy were performed for the 1st group (n = 55, while the 2nd group (n = 54 received drug therapy only. All patients underwent clinical examination at admission and at 6 and 12 months after the onset of the study. Results. In the 1st group the angina functional class was reliably lowered (from 3.3 ± 0.2 at the onset of the study down to 2.5 ± 0.1 after 12 months. The distance covered during a 6-minute walk test increased from the initial 185 ± 39 meters up to 359 ± 69 me- ters by the end of the 12th month. The angina class decreased from 3.1 ± 0.4 at the onset of the study down to 1.6 ± 0.4 by the end of the 12th month. Minnesota Life Quality Index reduced from 65.3 ± 21 points down to 22.4 ± 6 points in the first group, while in the control one it decreased down to 59.9 ± 16 points. On the contrary, cardiac insufficiency in patients of the second group tended to continually progress: from NYHA FC 3.5 ± 0.1 at the beginning of the study up to 3.9 ± 0.1 in the course of 12-month observation. The angina class remained the same (3.5 ± 0.5 at the beginning and 3.5 ± 0.4 after 12 months respectively. Conclusion. Intramyocardial implantation of a mononuclear fraction of autologous bone marrow cells is a safe method that contributes to the improvement of the left ventricular function, clinical data and prognosis. 

  4. Plasmodium and mononuclear phagocytes.

    Science.gov (United States)

    Mac-Daniel, Laura; Ménard, Robert

    2015-01-01

    Plasmodium, the causative agent of malaria, initially multiplies inside liver cells and then in successive cycles inside erythrocytes, causing the symptoms of the disease. In this review, we discuss interactions between the extracellular and intracellular forms of the Plasmodium parasite and innate immune cells in the mammalian host, with a special emphasis on mononuclear phagocytes. We overview here what is known about the innate immune cells that interact with parasites, mechanisms used by the parasite to evade them, and the protective or detrimental contribution of these interactions on parasite progression through its life cycle and pathology in the host.

  5. Non-Anticoagulant Fractions of Enoxaparin Suppress Inflammatory Cytokine Release from Peripheral Blood Mononuclear Cells of Allergic Asthmatic Individuals.

    Directory of Open Access Journals (Sweden)

    Madhur D Shastri

    Full Text Available Enoxaparin, a low-molecular-weight heparin, is known to possess anti-inflammatory properties. However, its clinical exploitation as an anti-inflammatory agent is hampered by its anticoagulant effect and the associated risk of bleeding.The aim of the current study was to examine the ability of non-anticoagulant fractions of enoxaparin to inhibit the release of key inflammatory cytokines in primed peripheral blood mononuclear cells derived from allergic mild asthmatics.Peripheral blood mononuclear cells from allergic asthmatics were activated with phytohaemag glutinin (PHA, concanavalin-A (ConA or phorbol 12-myristate 13-acetate (PMA in the presence or absence of enoxaparin fractions before cytokine levels were quantified using specific cytokine bead arrays. Together with nuclear magnetic resonance analysis,time-dependent and target-specific effects of enoxaparin fractions were used to elucidate structural determinants for their anti-inflammatory effect and gain mechanistic insights into their anti-inflammatory activity.Two non-anticoagulant fractions of enoxaparin were identified that significantly inhibited T-cell activation. A disaccharide fraction of enoxaparin inhibited the release of IL-4, IL-5, IL-13 and TNF-α by more than 57% while a tetrasaccharide fraction was found to inhibit the release of tested cytokines by more than 68%. Our data suggest that the observed response is likely to be due to an interaction of 6-O-sulfated tetrasaccharide with cellular receptor(s.The two identified anti-inflammatory fractions lacked anticoagulant activity and are therefore not associated with risk of bleeding. The findings highlight the potential therapeutic use of enoxaparin-derived fractions, in particular tetrasaccharide, in patients with chronic inflammatory disorders.

  6. Is the regulation of the electronic properties of organic molecules by polynuclear superhalogens more effective than that by mononuclear superhalogens? A high-level ab initio case study.

    Science.gov (United States)

    Li, Miao-Miao; Li, Jin-Feng; Bai, Hongcun; Sun, Yin-Yin; Li, Jian-Li; Yin, Bing

    2015-08-21

    The regulation of the electronic properties of organic molecules induced by polynuclear superhalogens is theoretically explored here for sixteen composite structures. It is clearly indicated by the higher vertical electron detachment energy (VDE) that polynuclear superhalogens are more effective in regulating the electronic properties than mononuclear structures. However, this enhanced regulation is not only determined by superhalogens themselves but also related to the distribution of the extra electron of the final composites. The composites, in which the extra electron is mainly aggregated into the superhalogen moiety, will possess higher VDE values, as reported in the case of C1', 7.12 eV at the CCSD(T) level. This is probably due to the fact that, compared with organic molecules, superhalogens possess stronger attraction towards the extra electron and thus should lead to lower energies of the extra electrons and to higher VDE values eventually. Compared with CCSD(T), the Outer Valence Green's Function (OVGF) method fails completely for composite structures containing Cl atoms, while MP2 results are generally consistent in terms of the relative order of VDEs. Actually if the extra electron distribution of the systems could be approximated by the HOMO, the results at the OVGF level will be consistent with the CCSD(T) results. Conversely, the difference in VDEs between OVGF and CCSD(T) is significantly large. Besides superhalogen properties, the structures, relative stabilities and thermodynamic stabilities with respect to various fragmentation channels were also investigated for all the composite structures.

  7. Transplanted bone marrow mononuclear cells and MSCs impart clinical benefit to children with osteogenesis imperfecta through different mechanisms.

    Science.gov (United States)

    Otsuru, Satoru; Gordon, Patricia L; Shimono, Kengo; Jethva, Reena; Marino, Roberta; Phillips, Charlotte L; Hofmann, Ted J; Veronesi, Elena; Dominici, Massimo; Iwamoto, Masahiro; Horwitz, Edwin M

    2012-08-30

    Transplantation of whole bone marrow (BMT) as well as ex vivo-expanded mesenchymal stromal cells (MSCs) leads to striking clinical benefits in children with osteogenesis imperfecta (OI); however, the underlying mechanism of these cell therapies has not been elucidated. Here, we show that non-(plastic)-adherent bone marrow cells (NABMCs) are more potent osteoprogenitors than MSCs in mice. Translating these findings to the clinic, a T cell-depleted marrow mononuclear cell boost (> 99.99% NABMC) given to children with OI who had previously undergone BMT resulted in marked growth acceleration in a subset of patients, unambiguously indicating the therapeutic potential of bone marrow cells for these patients. Then, in a murine model of OI, we demonstrated that as the donor NABMCs differentiate to osteoblasts, they contribute normal collagen to the bone matrix. In contrast, MSCs do not substantially engraft in bone, but secrete a soluble mediator that indirectly stimulates growth, data which provide the underlying mechanism of our prior clinical trial of MSC therapy for children with OI. Collectively, our data indicate that both NABMCs and MSCs constitute effective cell therapy for OI, but exert their clinical impact by different, complementary mechanisms. The study is registered at www.clinicaltrials.gov as NCT00187018.

  8. Bacillus Calmette-Guérin enhances production and secretion of type IV collagenases in peripheral blood mononuclear cells.

    Science.gov (United States)

    Kageyama, Y; Kawakami, S; Fujii, Y; Kihara, K; Oshima, H

    1997-03-01

    Intravesical administration of bacillus Calmette-Guérin (BCG) is an effective and widely accepted treatment for superficial bladder cancer. Rapid progression of the disease after BCG therapy, however, has been reported in some cases refractory to the treatment. We examined whether BCG treatment and coexistence of peripheral blood mononuclear cells (PBMCs) alter the invasive potential of bladder cancer cells. Production and secretion of two type IV collagenases, matrix metalloproteinase (MMP) 2 and MMP 9, by PBMCs from five healthy donors or bladder cancer cells (T24, JTC 30, and JTC 32) were evaluated by gelatin zymography, western blot analysis, and northern blot analysis. Invasion of bladder cancer cells was also examined using reconstituted basement membrane (Matrigel). BCG (5, 50, and 500 micrograms/ml) had no effect on secretion of MMP 2 and MMP 9 by bladder cancer cells, but increased the production and secretion of MMP 9 by PBMCs in a dose-dependent manner. The coexistence of PBMCs increased invasion of T24 cells and BCG further enhanced the invasion. Thus, BCG promotes invasion of bladder cancer cells under certain conditions. An increase in the secretion of MMP 9 by PBMCs may account in part for the effect.

  9. In vitro effects of monophthalates on cytokine expression in the monocytic cell line THP-1 and in peripheral blood mononuclear cells from allergic and non-allergic donors

    DEFF Research Database (Denmark)

    Glue, C; Millner, A; Bødtger, Uffe;

    2002-01-01

    was to investigate the in vitro effect of metabolites of phthalate plastisizers, such as whether an adjuvant effect is paralleled by changes of the cytokine expression in the monocytic cell line THP-1 and in peripheral blood mononuclear cells (PBMCs) from allergics and non-allergics. The toxicity monitored by cell...... determined using Quantitative Competitive RT-PCR. PBMCs from allergics and non-allergics were incubated with monophthalate 220 microg/ml) for up to 48 h and cytokine expression (IL-4, IL-5, IFN-gamma) was measured using real-time PCR. The cytotoxic level of monophthalates is 20-200 microg/ml, depending...

  10. CDNA microarray analysis of gene expression patterns in blood mononuclear cells of SLA-DRB1-defined Yorkshire pigs.

    Science.gov (United States)

    Nino-Soto, M I; Jozani, R J; Bridle, B; Mallard, B A

    2008-01-01

    Three lines of commercialYorkshire pigs with defined SLA-DRB1 alleles were developed at the University of Guelph for xenotransplantation and immune response studies. Two of the SLA-DRB1 alleles have been previously reported (SLA-DRB1*0502 and *0701), whereas the third one is a new allele. The influence of defined SLA-DRB1 alleles on transcriptional patterns of immune-related genes in blood mononuclear cells (BMCs) of pigs was explored using cDNA microarray. Microarray analysis showed significant differential expression of inflammatory genes in association with the various SLA-DRB1 alleles. A better understanding of the association between SLA genotypes and gene activity can increase the knowledge of the function of these molecules, as well as define new strategies to control animal health and optimize animal production.

  11. Reduced heat shock response in human mononuclear cells during aging and its association with polymorphisms in HSP70 genes

    DEFF Research Database (Denmark)

    Singh, Ripudaman; Kølvraa, Steen; Bross, Peter

    2006-01-01

    Age-dependent changes in heat shock response (HSR) were studied in mononuclear cells (monocytes and lymphocytes) collected from young (mean age = 22.6 +/- 1.7 years) and middle-aged (mean age = 56.3 +/- 4.7 years) subjects after 1 hour of heat shock at 42 degrees C. Genotype-specific HSR...... was measured by genotyping the subjects for 3 single nucleotide polymorphisms, HSPA1A(A-110C), HSPA1B(A1267G), and HSPA1L(T2437C), 1 each in the 3 HSP70 genes. A significant age-related decrease in the induction of Hsp70 occurred after heat shock in both monocytes and lymphocytes. The noninducible...

  12. Transplantation of autologous peripheral blood mononuclear cells in the subarachnoid space for amyotrophic lateral sclerosis: a safety analysis of 14 patients.

    Science.gov (United States)

    Li, Xiao-Yan; Liang, Zhan-Hua; Han, Chao; Wei, Wen-Juan; Song, Chun-Li; Zhou, Li-Na; Liu, Yang; Li, Ying; Ji, Xiao-Fei; Liu, Jing

    2017-03-01

    There is a small amount of clinical data regarding the safety and feasibility of autologous peripheral blood mononuclear cell transplantation into the subarachnoid space for the treatment of amyotrophic lateral sclerosis. The objectives of this retrospective study were to assess the safety and efficacy of peripheral blood mononuclear cell transplantation in 14 amyotrophic lateral sclerosis patients to provide more objective data for future clinical trials. After stem cell mobilization and collection, autologous peripheral blood mononuclear cells (1 × 10(9)) were isolated and directly transplanted into the subarachnoid space of amyotrophic lateral sclerosis patients. The primary outcome measure was incidence of adverse events. Secondary outcome measures were electromyography 1 week before operation and 4 weeks after operation, Functional Independence Measurement, Berg Balance Scale, and Dysarthria Assessment Scale 1 week preoperatively and 1, 2, 4 and 12 weeks postoperatively. There was no immediate or delayed transplant-related cytotoxicity. The number of leukocytes, serum alanine aminotransferase and creatinine levels, and body temperature were within the normal ranges. Radiographic evaluation showed no serious transplant-related adverse events. Muscle strength grade, results of Functional Independence Measurement, Berg Balance Scale, and Dysarthria Assessment Scale were not significantly different before and after treatment. These findings suggest that peripheral blood mononuclear cell transplantation into the subarachnoid space for the treatment of amyotrophic lateral sclerosis is safe, but its therapeutic effect is not remarkable. Thus, a large-sample investigation is needed to assess its efficacy further.

  13. Flow cytometry analysis of hormone receptors on human peripheral blood mononuclear cells to identify stress-induced neuroendocrine effects

    Science.gov (United States)

    Meehan, R. T.

    1986-01-01

    Understanding the role of circulating peptide hormones in the pathogenesis of space-flight induced disorders would be greatly facilitated by a method which monitors chronic levels of hormones and their effects upon in vivo cell physiology. Single and simultaneous multiparameter flow cytometry analysis was employed to identify subpopulations of mononuclear cells bearing receptors for ACTH, Endorphin, and Somatomedin-C using monoclonal antibodies and monospecific antisera with indirect immunofluorescence. Blood samples were obtained from normal donors and subjects participating in decompression chamber studies (acute stress), medical student academic examination (chronic stress), and a drug study (Dexamethasone). Preliminary results indicate most ACTH and Endorphin receptor positive cells are monocytes and B-cells, exhibit little diurnal variation but the relative percentages of receptor positive cells are influenced by exposure to various stressors and ACTH inhibition. This study demonstrates the capability of flow cytometry analysis to study cell surface hormone receptor regulation which should allow insight into neuroendocrine modulation of the immune and other cellular systems during exposure to stress or microgravity.

  14. The role of Card9 overexpression in peripheral blood mononuclear cells from patients with aseptic acute pancreatitis.

    Science.gov (United States)

    Yang, Zhi-wen; Weng, Cheng-zhao; Wang, Jing; Xu, Ping

    2016-03-01

    Activated mononuclear cells are an early event in the course of severe acute pancreatitis (SAP). To date, the molecular mechanism triggering peripheral blood mononuclear cells (PBMCs) is poorly understood. The aim of this paper was to determine the potential role of Card9 in SAP. We collected data from 72 subjects between January 2013 and June 2014. Subsequently, PBMCs were isolated on day 1, 3 and 5 of pancreatitis. Immunofluorescence staining, quantitative real-time PCR, Western blotting, immunoprecipitation and ELISA were used to determine the role of Card9 in SAP. Microbial culture showed that SAP patients at the early period did not develop any bacteria and fungi infection. Card9 expression in SAP patients was higher than that in mild acute pancreatitis and volunteer healthy controls, up to the peak on day 1. The monocyte-derived cytokines interleukin (IL)-17, IL-1β, IL-6 and tumour necrosis factor-α mediated by the induction of Card9 markedly increased in SAP patients compared with the control group. Furthermore, the inducible formation of Card9-Bcl10 complex was found in PBMCs, which may be involved in nuclear factor kappa B (NF-κB) and p38 activation in SAP. Receiver operating characteristic curve indicated that Card9 levels had a high sensitivity of 87.5% and specificity of 67.7%, showing the close correlation with SAP patients. Card9 overexpression was firstly found in aseptic SAP, which may be played an important role in NF-κB and p38 activation in PBMCs. It also provided the new insights into therapeutic interventions by targeting monocytes activation in SAP patients.

  15. Assessment of the cytokine profile in peripheral blood mononuclear cells of naturally Sarcoptes scabiei var. canis infested dogs.

    Science.gov (United States)

    Singh, Shanker K; Dimri, Umesh; Sharma, Bhaskar; Saxena, Meeta; Kumari, Priyambada

    2014-12-15

    The mechanism of cytokine secretion from T lymphocytes plays an important role in the immune response of dogs and parasitic skin infestations. Assessment of the cytokine profile of naturally S. scabiei var. canis infested dogs could augment understanding of the pathobiology of canine sarcoptic mange. Therefore, the present study examined the cytokines in peripheral blood mononuclear cells of dogs suffering from sarcoptic mange. Thirteen dogs naturally infected with sarcoptic mange participated in the study. The dogs were found positive for S. scabiei var. canis mites in skin scraping examinations and revealed at least three clinical inclusion criteria. Another five clinically healthy dogs were kept as healthy controls. Peripheral blood mononuclear cells were isolated from heparinized blood samples and used for extraction of mRNA. Further, cDNA was synthesized by using 1 mg of mRNA by reverse transcription using oligonucleotide primers. Relative levels of cytokine expression were compared with normalized glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts. The levels of interleukin-4, interleukin-5 and transforming growth factor beta (TGF-β) mRNA expression in dogs with sarcoptic mange were significantly higher (P ≤ 0.01), whereas the level of tumor necrosis factor alpha (TNF-α) was significantly lower (P ≤ 0.01) in comparison with the healthy dogs. No remarkable difference was seen for interleukin-2 mRNA expression between these animals. An overproduction IL-4 and IL-5 might be involved in immuno-pathogenesis of canine sarcoptic mange. S. scabiei var. canis mites possibly induce an overproduction of TGF-β and reduced expression of TNF-α and thus could be conferring the immune suppression of infested dogs.

  16. Relationship between the expression of Toll-like receptor 2 and 4 in mononuclear cells and postoperative acute lung injury in orthotopic liver transplantation

    Institute of Scientific and Technical Information of China (English)

    CHI Xin-jin; CAI Jun; LUO Chen-fang; CHENG Nan; HEI Zi-qing; LI Shang-rong; LUO Gang-jian

    2009-01-01

    Background The aim of this study was to investigate the potential relationship between the dynamic expression of Toll-like receptor 2 and 4 (TLR2/4) in peripheral blood mononuclear cells as well as changes in serum concentration of inflammatory factors and acute lung injury (ALl) in patients after orthotopic liver transplantation (OLT).Methods The peripheral blood samples of 27 patients (23 men and 4 women with ASA Ⅲ to Ⅳ) who received OLT were collected for measurement of TLR2/4 at T1 (after induction of anesthesia), T2 (25 minutes after anhepatic phase), T3 (3 hours after graft reperfusion) and T4 (24 hours after graft reperfusion). The expression of TLR2/4 in mononuclear cells was measured by flow cytometry. The serum concentrations of tumor necrosis factor (TNF)-a, intedeukin (IL)-113 and IL-8 were measured by enzyme-linked irnmunosorbent assay (ELISA). Twenty-seven patients were assigned to ALI group (n=9) and non-ALI group (n=18) according to the diagnostic criteria of ALI. The expression of TLR2/4 in the ALl group or non-ALI group was analyzed.Results Compared to the non-ALI group, the volumes of blood loss, ascites, total output and transfused red blood cells were higher in the ALI group, and the anhepallc phase lasted longer (P0.05). The expression of TLR2/4 in mononuclear cells increased significantly at T3 and 14 in the ALI group (P<0.05, P<0.01). A positive correlation was noted between the expression of TLR4 in mononuclear cells and the serum concentrations of TNF-α, IL-1β (P=0.041, P=0.046) in the ALl group. In the non-ALI group, statistical results showed that the expression level of TLR2/4 in mononuclear cells was not significantly different during the peri-operative period of OLT (besides TLR4 expression at T4). Compared to the non-ALI group, the increasing amplitude of TLR2/4 expression in mononuclear cells was more significant in the ALI group. The patients whose TLR2/4 expression in mononuclear cells exceeded that at T1 by one time were

  17. Influenza a virus induces an immediate cytotoxic activity in all major subsets of peripheral blood mononuclear cells.

    Directory of Open Access Journals (Sweden)

    Sanda Sturlan

    Full Text Available BACKGROUND: A replication defective influenza A vaccine virus (delNS1 virus was developed. Its attenuation is due to potent stimulation of the innate immune system by the virus. Since the innate immune system can also target cancer cells, we reasoned that delNS1 virus induced immune-stimulation should also lead to the induction of innate cytotoxic effects towards cancer cells. METHODOLOGY/PRINCIPAL FINDINGS: Peripheral blood mononuclear cells (PBMCs, isolated CD56+, CD3+, CD14+ and CD19+ subsets and different combinations of the above subsets were stimulated by delNS1, wild type (wt virus or heat inactivated virus and co-cultured with tumor cell lines in the presence or absence of antibodies against the interferon system. Stimulation of PBMCs by the delNS1 virus effectively induced cytotoxicity against different cancer cell lines. Surprisingly, virus induced cytotoxicity was exerted by all major subtypes of PBMCs including CD56+, CD3+, CD14+ and CD19+ cells. Virus induced cytotoxicity in CD3+, CD14+ and CD19+ cells was dependent on virus replication, whereas virus induced cytotoxicity in CD56+ cells was only dependent on the binding of the virus. Virus induced cytotoxicity of isolated cell cultures of CD14+, CD19+ or CD56+ cells could be partially blocked by antibodies against type I and type II (IFN interferon. In contrast, virus induced cytotoxicity in the complete PBMC preparation could not be inhibited by blocking type I or type II IFN, indicating a redundant system of activation in whole blood. CONCLUSIONS/SIGNIFICANCE: Our data suggest that apart from their well known specialized functions all main subsets of peripheral blood cells also initially exert a cytotoxic effect upon virus stimulation. This closely links the innate immune system to the adaptive immune response and renders delNS1 virus a potential therapeutic tool for viro-immunotherapy of cancer.

  18. Long-term three-dimensional perfusion culture of human adult bone marrow mononuclear cells in bioreactors.

    Science.gov (United States)

    Schmelzer, Eva; Finoli, Anthony; Nettleship, Ian; Gerlach, Jörg C

    2015-04-01

    The construction and long-term maintenance of three-dimensional in vitro bone marrow models is of great interest but still quite challenging. Here we describe the use of a multi-compartment hollow-fiber membrane based three-dimensional perfusion bioreactor for long-term culture of whole human bone marrow mononuclear cells. We also investigated bioreactors with incorporated open-porous foamed hydroxyapatite scaffolds, mimicking the in vivo bone matrix. Cells in bioreactors with and without scaffolds were cultured to 6 weeks and compared to Petri dish controls. Cells were analyzed for gene expression, surface markers by flow cytometry, metabolic activity, hematopoietic potential, viability, and attachment by immunocytochemistry. Cells in bioreactors were metabolic active during long-term culture. The percentages of hematopoietic stem cell and mature endothelial cell fractions were maintained in bioreactors. The expression of most of the analyzed genes stabilized and increased after long-term culture of 6 weeks. Compared to Petri dish culture controls, bioreactor perfusion culture improved in both the short and long-term, the colony formation unit capacity of hematopoietic progenitors. Cells attached to the ample surface area provided by hydroxyapatite scaffolds. The implementation of a hydroxyapatite scaffold did not influence colony formation capacity, percentages of cell type specific fractions, gene expression, cell viability or metabolic turnover when compared to control cells cultured in bioreactors without scaffolds. In conclusion, three-dimensional perfusion bioreactor culture enables long-term maintenance of primary human bone marrow cells, with hydroxyapatite scaffolds providing an in vivo-like scaffold for three-dimensional culture. © 2015 Wiley Periodicals, Inc.

  19. Peripheral blood mononuclear cells as a model to study the response of energy homeostasis-related genes to acute changes in feeding conditions

    NARCIS (Netherlands)

    Caimari, A.; Oliver, P.; Keijer, J.; Palou, A.

    2010-01-01

    Peripheral blood mononuclear cells (PBMCs) are readily accessible biological material and a potential tissue source to discover novel biomarkers of response to environmental exposures including nutrition. We analyzed whether PBMCs could reflect molecular changes that take place in response to differ

  20. Peripheral blood mononuclear cells as a model to study the response of energy homeostasis-related genes to acute changes in feeding conditions

    NARCIS (Netherlands)

    Caimari, A.; Oliver, P.; Keijer, J.; Palou, A.

    2010-01-01

    Peripheral blood mononuclear cells (PBMCs) are readily accessible biological material and a potential tissue source to discover novel biomarkers of response to environmental exposures including nutrition. We analyzed whether PBMCs could reflect molecular changes that take place in response to differ

  1. Upregulation of human immunodeficiency virus (HIV) replication by CD4 cross-linking in peripheral blood mononuclear cells of HIV-infected adults.

    Science.gov (United States)

    Than, S; Oyaizu, N; Tetali, S; Romano, J; Kaplan, M; Pahwa, S

    1997-08-01

    This study was conducted with peripheral blood mononuclear cells from 67 human immunodeficiency virus (HIV)-infected adults. It supports the hypothesis that cross-linking of CD4 molecules by HIV gp120 can result in HIV upregulation and spread of infection. Underlying mechanisms include activation of latent infection by factors in addition to, or other than, tumor necrosis factor alpha.

  2. Association between HLA-DR2 and production of tumour necrosis factor alpha and interleukin 1 by mononuclear cells activated by lipopolysaccharide

    DEFF Research Database (Denmark)

    Bendtzen, K; Morling, N; Fomsgaard, A

    1988-01-01

    The production of tumour necrosis factor (TNF) and interleukin 1 (IL-1) by lipopolysaccharide-activated mononuclear cells from 39 healthy donors was studied in vitro by bioassay and ELISA. The donors were typed for HLA-A, -B, -C, -DR, and -DP antigens. There was no detectable production of TNF beta...

  3. Differential mRNA expression and production of interleukin-4 and interferon-gamma in stimulated peripheral blood mononuclear cells of house-dust mite-allergic patients

    NARCIS (Netherlands)

    Laan, M.P.; Baert, M.R.M.; Vredendaal, A.E.C.M.; Savelkoul, H.F.J.

    1998-01-01

    Summary : Optimal culture conditions were established for the analysis of interleukin-4 (IL-4) and interfe-ron-gamma (IFN- ) mRNA expression and protein production, as well as proliferative capacity of peripheral blood mononuclear cells (PBMC). These culture conditions permitted the analysis of diff

  4. Peripheral blood mononuclear cells as a source to detect markers of homeostatic alterations caused by the intake of diets with an unbalanced macronutrient composition

    NARCIS (Netherlands)

    Diaz-Rua, R.; Keijer, J.; Caimari, A.; Schothorst, van E.M.; Oliver, P.; Palou, A.

    2015-01-01

    Peripheral blood mononuclear cells (PBMC) are accessible in humans and their gene expression pattern was shown to reflect overall physiological response of the body to a specific stimulus, such as diet. We aimed to study the impact of sustained intake (4 months) of diets with an unbalanced

  5. TSH, thyroid hormones and nuclear-binding of T3 in mononuclear blood cells from obese and non-obese women

    DEFF Research Database (Denmark)

    Matzen, L E; Kvetny, J; Pedersen, K K

    1989-01-01

    The specific nuclear-binding of T3 (NBT3) in mononuclear blood cells, and the concentrations of TSH, thyroid hormones, and binding proteins were measured after overnight fasting in 12 obese and in 14 non-obese women, none of the subjects were taking any medicine. The concentrations of TSH and free...

  6. Mycobacterium tuberculosis-specific immunospot assay of pleural exudate mononuclear cells is useful for the exclusion of tuberculous pleuritis in patients with lupus pleuritis.

    Science.gov (United States)

    Takase, K; Ohno, S; Ideguchi, H; Murakami, S; Takeno, M; Ishigatsubo, Y

    2009-02-01

    It is often difficult to make a diagnosis of pleuritis associated with rheumatic diseases because of lack of specific diagnostic tools. We report a patient with lupus pleuritis from which tuberculous pleuritis was distinguished by Mycobacterium tuberculosis-specific enzyme-linked immunospot assay of pleural exudate mononuclear cells. After the diagnosis of lupus pleuritis, the patient was successfully treated with prednisolone.

  7. Interactions of allogeneic human mononuclear cells in the two-way mixed leucocyte culture (MLC): influence of cell numbers, subpopulations and cyclosporin

    Science.gov (United States)

    Sato, T; Deiwick, A; Raddatz, G; Koyama, K; Schlitt, H J

    1999-01-01

    With organ allografts considerable numbers of donor-type mononuclear cells are transferred to the recipient, leading to bilateral immunological interactions between donor and recipient lymphocytes. To study such bilateral immune reactions in detail, human two-way MLC were performed. In this model proliferation kinetics, patterns of activation, and survival of the two populations were analysed, and the relevance of initial cell subset composition, relative cell numbers, and the effect of immunosuppression on this co-culture were evaluated. It could be demonstrated that with an initial 50:50 ratio of two populations of allogeneic cells one population dominated after 21 days of co-culture in 78 out of 80 combinations (97%) tested; the other population decreased markedly after an initially stable phase of 6–7 days. With unequal starting conditions the larger population dominated when resting cells were used, but small populations of preactivated cells or separated CD8+ cells could also dominate. Depletion of CD16+ natural killer (NK) cells and of CD2− cells (B cell and monocytes) had no effect on domination. Addition of cyclosporin delayed or blocked the domination process while addition of IL-2 accelerated it. Disappearance of one population was associated with detection of apoptotic cells. The findings indicate that co-cultures of allogeneic mononuclear cells are generally not stable for more than 1 week, but lead to active elimination of one population. CD8+ cells and particularly preactivated cells seem to play the most important role in that process, while NK cells are of less importance. Cyclosporin can prolong survival of allogeneic cells in co-culture. These observations suggest that under the conditions of clinical organ transplantation even small amounts of immunocompetent donor cells transferred by the graft may persist for some time and may, thereby, have the chance to exert immunomodulatory functions. PMID:9933457

  8. Bisphenol A and its analogs induce morphological and biochemical alterations in human peripheral blood mononuclear cells (in vitro study).

    Science.gov (United States)

    Michałowicz, Jaromir; Mokra, Katarzyna; Bąk, Agata

    2015-10-01

    Few studies have addressed the cellular effects of bisphenol S (BPS) and bisphenol AF (BPAF) on cells, and no study has been conducted to analyze the mechanism of action of bisphenols in blood cells. In this study, the effect of bisphenol A (BPA), bisphenol F (BPF), BPS and BPAF on human peripheral blood mononuclear cells (PBMCs) was analyzed. It was shown that BPA, BPF and BPAF in particular, decreased cell viability, which was associated with depletion of intracellular ATP level and alterations in PBMCs size and granulation. Bisphenols enhanced ROS (including OH˙) formation, which led to damage to lipids and proteins in PBMCs. The most significant alterations in ROS level were induced by BPF, and particularly BPAF. Moreover, it was shown that BPAF most strongly provoked lipid peroxidation, while BPA and BPS caused the greatest damage to proteins. It may be concluded that BPA and its analogs were capable of inducing oxidative stress and damage in PBMCs in the concentrations ranging from 0.06 to 0.5 μM (0.02-0.1 μg/ml), which may be present in human blood as a result of environmental exposure. Although, most of bisphenols studied decreased cell viability, size and ATP level at higher concentrations, BPAF exhibited its cytotoxic potential at low concentrations ranging from 0.3 to 3 μM (0.1-1.0 μg/ml) that may correspond to concentrations in humans following occupational exposure.

  9. Synovial fluid mononuclear cells provide an environment for long-term survival of antibody-secreting cells and promote the spontaneous production of anti-citrullinated protein antibodies.

    Science.gov (United States)

    Kerkman, Priscilla F; Kempers, Ayla C; van der Voort, Ellen I H; van Oosterhout, Maikel; Huizinga, Tom W J; Toes, René E M; Scherer, Hans U

    2016-12-01

    In rheumatoid arthritis (RA), observations point to a crucial role for (autoreactive) B cells in disease pathogenesis. Here, we studied whether cells from the synovial environment impact on the longevity of autoreactive B cell responses against citrullinated antigens. Synovial fluid mononuclear cells and peripheral blood mononuclear cells (SFMC/PBMC) were obtained from patients with established RA and assessed for the presence of B cell subpopulations. Cells spontaneously secreting anti-citrullinated protein antibodies (ACPA-IgG) directly ex vivo were detected by antigen-specific Enzyme-Linked ImmunoSpot (ELISpot) assay. SFMC and PBMC were cultured to assess the degree of spontaneous ACPA-IgG secretion. Cells surviving for several weeks were characterised by carboxyfluorescein succinimidyl ester (CFSE) labelling and Ki-67 staining. Cells spontaneously secreting ACPA-IgG were readily detectable in peripheral blood and synovial fluid (SF) of patients with ACPA-positive RA. SFMC showed an up to 200-fold increase in ex vivo ACPA-IgG secretion compared with PBMC despite lower numbers of B cells in SFMC. ELISpot confirmed the presence of spontaneously ACPA-IgG-secreting cells, accounting for up to 50% (median 12%) of all IgG-secreting cells in SF. ACPA-IgG secretion was remarkably stable in SFMC cultures, maintained upon depletion of the CD20(+) B cell compartment and detectable for several months. CFSE labelling and Ki-67 staining confirmed the long-term survival of non-dividing plasma cells (PCs). This study demonstrates a high frequency of differentiated, spontaneously ACPA-IgG-secreting cells in SF. These cells are supported by SFMC for prolonged survival and autoantibody secretion, demonstrating that the synovial compartment is equipped to function as inflammatory niche for PC survival. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  10. Intravenous Autologous Bone Marrow Mononuclear Cell Transplantation for Stroke: Phase1/2a Clinical Trial in a Homogeneous Group of Stroke Patients.

    Science.gov (United States)

    Taguchi, Akihiko; Sakai, Chiaki; Soma, Toshihiro; Kasahara, Yukiko; Stern, David M; Kajimoto, Katsufumi; Ihara, Masafumi; Daimon, Takashi; Yamahara, Kenichi; Doi, Kaori; Kohara, Nobuo; Nishimura, Hiroyuki; Matsuyama, Tomohiro; Naritomi, Hiroaki; Sakai, Nobuyuki; Nagatsuka, Kazuyuki

    2015-10-01

    The goal of this clinical trial was to assess the feasibility and safety of transplanting autologous bone marrow mononuclear cells into patients suffering severe embolic stroke. Major inclusion criteria included patients with cerebral embolism, age 20-75 years, National Institute of Health Stroke Scale (NIHSS) score displaying improvement of ≤ 5 points during the first 7 days after stroke, and NIHSS score of ≥ 10 on day 7 after stroke. Bone marrow aspiration (25 or 50 mL; N = 6 patients in each case) was performed 7-10 days poststroke, and bone marrow mononuclear cells were administrated intravenously. Mean total transplanted cell numbers were 2.5 × 10(8) and 3.4 × 10(8) cells in the lower and higher dose groups, respectively. No apparent adverse effects of administering bone marrow cells were observed. Compared with the lower dose, patients receiving the higher dose of bone marrow cells displayed a trend toward improved neurologic outcomes. Compared with 1 month after treatment, patients receiving cell therapy displayed a trend toward improved cerebral blood flow and metabolic rate of oxygen consumption 6 months after treatment. In comparison with historical controls, patients receiving cell therapy had significantly better neurologic outcomes. Our results indicated that intravenous transplantation of autologous bone marrow mononuclear cells is safe and feasible. Positive results and trends favoring neurologic recovery and improvement in cerebral blood flow and metabolism by cell therapy underscore the relevance of larger scale randomized controlled trials using this approach.

  11. Increased SOCS expression in peripheral blood mononuclear cells of end stage renal disease patients is related to inflammation and dialysis modality.

    Science.gov (United States)

    Rastmanesh, M Mehdi; Braam, Branko; Joles, Jaap A; Boer, Peter; Bluyssen, Hans A R

    2009-01-05

    Inflammation is a characteristic of cardiovascular disease and is increased in end-stage renal disease. Suppressors of cytokine signaling (SOCS) inhibit and reflect activation of intracellular inflammatory pathways. We hypothesized that SOCS expression in peripheral blood mononuclear cells of end stage renal disease patients is increased. Whether SOCS expression in peripheral blood mononuclear cells is related to inflammation, dialysis, and dialysis modality was investigated. Monocytes and lymphocytes were isolated from peripheral blood mononuclear cells of patients not on dialysis (n=8), on peritoneal dialysis (n=8), on hemodialysis (n=14) and of healthy control (n=15) subjects. SOCS expression was assessed by real-time quantitative PCR and plasma cytokines by ELISA. In end stage renal disease patients monocyte SOCS1, and lymphocyte SOCS1 and cytokine-inducible SH2 containing protein-1 (CIS-1) gene expression were increased along with increased plasma levels of interleukin (IL)-6, tumor necrosis factor (TNF)alpha ,and C-reactive protein (CRP). Monocyte SOCS1 correlated with IL-6. Lymphocyte CIS-1 was increased in non-dialysis and peritoneal dialysis but not in hemodialysis patients. Lymphocyte CIS-1 in peritoneal dialysis patients correlated with plasma TNFalpha. Despite the relatively low number of patients studied we observed increased expression of SOCS1 in both monocytes and lymphocytes, and of CIS-1 solely in lymphocytes of the patients. SOCS expression was related to increased systemic inflammation, illustrated by a significant correlation between monocyte SOCS1 and plasma IL-6. SOCS expression in peripheral blood mononuclear cells was also affected by hemodialysis, indicated by increased lymphocyte CIS-1 in non-dialysis and peritoneal dialysis but not in hemodialysis patients. We suggest that increased SOCS expression in peripheral blood mononuclear cells of end stage renal disease patients reflects whether and to which extent systemic inflammation

  12. Functional and Pharmacological Analysis of Cardiomyocytes Differentiated from Human Peripheral Blood Mononuclear-Derived Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Michael Riedel

    2014-07-01

    Full Text Available Advances in induced pluripotent stem cell (iPSC technology have set the stage for routine derivation of patient- and disease-specific human iPSC-cardiomyocyte (CM models for preclinical drug screening and personalized medicine approaches. Peripheral blood mononuclear cells (PBMCs are an advantageous source of somatic cells because they are easily obtained and readily amenable to transduction. Here, we report that the electrophysiological properties and pharmacological responses of PBMC-derived iPSC CM are generally similar to those of iPSC CM derived from other somatic cells, using patch-clamp, calcium transient, and multielectrode array (MEA analyses. Distinct iPSC lines derived from a single patient display similar electrophysiological features and pharmacological responses. Finally, we demonstrate that human iPSC CMs undergo acute changes in calcium-handling properties and gene expression in response to rapid electrical stimulation, laying the foundation for an in-vitro-tachypacing model system for the study of human tachyarrhythmias.

  13. Intravenous transplantation of bone marrow-derived mononuclear cells prevents memory impairment in transgenic mouse models of Alzheimer's disease.

    Science.gov (United States)

    Kanamaru, Takuya; Kamimura, Naomi; Yokota, Takashi; Nishimaki, Kiyomi; Iuchi, Katsuya; Lee, Hyunjin; Takami, Shinya; Akashiba, Hiroki; Shitaka, Yoshitsugu; Ueda, Masayuki; Katsura, Ken-Ichiro; Kimura, Kazumi; Ohta, Shigeo

    2015-04-24

    Stem cell transplantation therapy is currently in clinical trials for the treatment of ischemic stroke, and several beneficial aspects have been reported. Similarly, in Alzheimer's disease (AD), stem cell therapy is expected to provide an efficient therapeutic approach. Indeed, the intracerebral transplantation of stem cells reduced amyloid-β (Aβ) deposition and rescued memory deficits in AD model mice. Here, we show that intravenous transplantation of bone marrow-derived mononuclear cells (BMMCs) improves cognitive function in two different AD mouse models, DAL and APP mice, and prevents neurodegeneration. GFP-positive BMMCs were isolated from tibiae and femurs of 4-week-old mice and then transplanted intravenously into DAL and APP mice. Transplantation of BMMCs suppressed neuronal loss and restored memory impairment of DAL mice to almost the same level as in wild-type mice. Transplantation of BMMCs to APP mice reduced Aβ deposition in the brain. APP mice treated with BMMCs performed significantly better on behavioral tests than vehicle-injected mice. Moreover, the effects were observed even with transplantation after the onset of cognitive impairment in DAL mice. Together, our results indicate that intravenous transplantation of BMMCs has preventive effects against the cognitive decline in AD model mice and suggest a potential therapeutic effect of BMMC transplantation therapy. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Intestinal mononuclear cells primed by systemic interleukin-12 display long-term ability to aggravate colitis in mice.

    Science.gov (United States)

    Pedrotti, Luciano P; Sena, Angela A; Rodriguez Galán, María Cecilia; Cejas, Hugo; Correa, Silvia G

    2017-03-01

    To address whether the burst of systemic interleukin-12 (IL-12) influences intestinal inflammation elicited by luminal stimuli, we induced IL-12 release by cDNA injection in C57BL/6 mice and simultaneously started dextran sulphate sodium administration. The sequence of the inflammatory response triggered by IL-12 release was characterized by assessing myeloperoxidase activity and histological damage in colon samples on days 1, 3, 5 and 7 after colitis induction. To evaluate the persistence of IL-12 priming, colitis was induced in mice 7 or 60 days after cDNA injection. Under IL-12 influence, the development of acute colitis presented a faster and selective infiltration of inflammatory mononuclear cells in the lamina propria. Recruitment was driven by systemic cytokines rather than luminal antigens. Interestingly, when colitis was triggered 7 or 60 days after the cytokine storm, cells maintained the ability to worsen clinical signs of intestinal inflammation. Together, a systemic IL-12 burst effectively primed intestinal cells that became more prone to develop inflammatory responses. Activation was long-lasting because intestinal cells maintained their inflammatory potential and their ability to aggravate colitis. © 2016 John Wiley & Sons Ltd.

  15. Evaluation of annexin V and Calcein-AM as markers of mononuclear cell apoptosis during human immunodeficiency virus infection

    Directory of Open Access Journals (Sweden)

    Priscila F.R. Palma

    2008-04-01

    Full Text Available Evaluation of apoptosis by flow cytometry is generally accomplished by methods that use annexin V-FITC as vital dye, which access phosphatidylserine exposed on the external membrane at the beginning of this process. In addition, the concomitant use of propidium iodide makes possible to verify the characteristic nuclear alterations in the late stages of apoptosis, as a consequence of the increase in membrane permeability. On the other hand, the use of calcein-AM in association with ethidium homodimer (EthD-1 allows the evaluation of cell apoptosis through detection of esterase activity and cellular membrane physical and chemical alterations. The aim of this study was to compare the sensibility of calcein-AM and EthD-1 with annexin V-FITC and propidium iodide for early apoptosis evaluation in peripheral blood mononuclear cell culture, obtained from HIV-infected patients. Apoptosis and cellular viability were detected and quantified by flow cytometry after 24 and 48 hours incubation times. Our results showed that calcein-AM/EthD-1 was more sensitive for apoptotic cell quantification in both incubation times than annexin V-FITC/propidium iodide (mean of 46.95% ± 3.56, p < 0.0001, for 24 hours and mean of 37.67% ± 2.47, p < 0.0014 for 48 hours, besides allowing to clearly define viable, apoptotic and dead cell populations.

  16. Functional and pharmacological analysis of cardiomyocytes differentiated from human peripheral blood mononuclear-derived pluripotent stem cells.

    Science.gov (United States)

    Riedel, Michael; Jou, Chuanchau J; Lai, Shuping; Lux, Robert L; Moreno, Alonso P; Spitzer, Kenneth W; Christians, Elizabeth; Tristani-Firouzi, Martin; Benjamin, Ivor J

    2014-07-08

    Advances in induced pluripotent stem cell (iPSC) technology have set the stage for routine derivation of patient- and disease-specific human iPSC-cardiomyocyte (CM) models for preclinical drug screening and personalized medicine approaches. Peripheral blood mononuclear cells (PBMCs) are an advantageous source of somatic cells because they are easily obtained and readily amenable to transduction. Here, we report that the electrophysiological properties and pharmacological responses of PBMC-derived iPSC CM are generally similar to those of iPSC CM derived from other somatic cells, using patch-clamp, calcium transient, and multielectrode array (MEA) analyses. Distinct iPSC lines derived from a single patient display similar electrophysiological features and pharmacological responses. Finally, we demonstrate that human iPSC CMs undergo acute changes in calcium-handling properties and gene expression in response to rapid electrical stimulation, laying the foundation for an in-vitro-tachypacing model system for the study of human tachyarrhythmias.

  17. Bisphenol A and its analogs exhibit different apoptotic potential in peripheral blood mononuclear cells (in vitro study).

    Science.gov (United States)

    Mokra, Katarzyna; Kocia, Magdalena; Michałowicz, Jaromir

    2015-10-01

    There are only a few studies that have assessed the effect of bisphenol A (BPA) on human blood cells and no study has been conducted to analyze the impact of BPA analogs on human leucocytes. In this study, we have investigated the effect of BPA and its analogs like bisphenol F (BPF), bisphenol S (BPS) and bisphenol AF (BPAF) on apoptosis induction in human peripheral blood mononuclear cells (PBMCs). In order to clarify the mechanism of bisphenols-induced programmed cell death, changes in various signaling molecules of this process have been assessed. We observed an increase in cytosolic calcium ions (Ca(2+)) level and reduction of transmembrane mitochondrial potential (ΔΨm) in PBMCs incubated with all compounds examined, and particularly BPA and BPAF. All compounds studied changed PBMCs membrane permeability, activated caspase-8, -9, -3 and induced PARP-1 cleavage and chromatin condensation, which confirmed that they were capable of inducing apoptosis both via intrinsic and extrinsic pathway. Moreover, we have found that modus operandi of bisphenols studied was different. We noticed that BPAF and BPS caused mainly necrotic and apoptotic changes, respectively, whereas BPA induced comparable apoptotic and necrotic effects in the incubated cells.

  18. Butachlor induced dissipation of mitochondrial membrane potential, oxidative DNA damage and necrosis in human peripheral blood mononuclear cells.

    Science.gov (United States)

    Dwivedi, Sourabh; Saquib, Quaiser; Al-Khedhairy, Abdulaziz A; Musarrat, Javed

    2012-12-01

    Butachlor is a systemic herbicide widely applied on rice, tea, wheat, beans and other crops; however, it concurrently exerts toxic effects on beneficial organisms like earthworms, aquatic invertebrates and other non-target animals including humans. Owing to the associated risk to humans, this chloroacetanilide class of herbicide was investigated with the aim to assess its potential for the (i) interaction with DNA, (ii) mitochondria membrane damage and DNA strand breaks and (iii) cell cycle arrest and necrosis in butachlor treated human peripheral blood mononuclear (PBMN) cells. Fluorescence quenching data revealed the binding constant (Ka=1.2×10(4)M(-1)) and binding capacity (n=1.02) of butachlor with ctDNA. The oxidative potential of butachlor was ascertained based on its capacity of inducing reactive oxygen species (ROS) and substantial amounts of promutagenic 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) adducts in DNA. Also, the discernible butachlor dose-dependent reduction in fluorescence intensity of a cationic dye rhodamine (Rh-123) and increased fluorescence intensity of 2',7'-dichlorodihydro fluorescein diacetate (DCFH-DA) in treated cells signifies decreased mitochondrial membrane potential (ΔΨm) due to intracellular ROS generation. The comet data revealed significantly greater Olive tail moment (OTM) values in butachlor treated PBMN cells vs untreated and DMSO controls. Treatment of cultured PBMN cells for 24h resulted in significantly increased number of binucleated micronucleated (BNMN) cells with a dose dependent reduction in the nuclear division index (NDI). The flow cytometry analysis of annexin V(-)/7-AAD(+) stained cells demonstrated substantial reduction in live population due to complete loss of cell membrane integrity. Overall the data suggested the formation of butachlor-DNA complex, as an initiating event in butachlor-induced DNA damage. The results elucidated the oxidative role of butachlor in intracellular ROS production, and

  19. The application of human umbilical cord blood mononuclear cells in the management of deep partial thickness burn

    Directory of Open Access Journals (Sweden)

    Yefta Moenadjat

    2013-05-01

    Full Text Available Background: Wound healing in burn is a complex process and early complete wound closure still enfaces many problems. Application of stem cells is found to be the future method of wound healing. Among the available sources of allogenic stem cells, umbilical cord blood is quite easy to be obtained, has less ethical issue, and contain multipotent stem cells, which are characterized by low immunogenicity. The study aims to evaluate the potential of human umbilical cord blood mononuclear cells (hUCBMNCs treatment in the management of deep partial thickness burns. Methods: Twenty patients with deep partial thickness burns were treated with topical application of 2 x 107 hUCBMNCs and silver sulfadiazine (SSD cream on the comparable wound size in the other sites. The treatments were applied for six times in every two consecutive days. Wound surface area was measured with Visitrak® on day 0, 7, and 11. Pain intensity was evaluated using Wong Baker’s faces scale on each wound dressing change. Histology examination was performed in some samples of collected skin biopsy of the newly re-epithelialized area of hUCBMNCs and SSD-treated wound at the end of treatment. HLA typing is used to evaluate the issue of safety. Wilcoxon signed rank test was used to compare the rate of wound healing. Results: Sixteen patients of hUCBMNCs-treated showed a significant wound closure in faster than SSD-treated; measured on day 7 (p = 0.041 and day 11 (p = 0.021. Number of patients with reduced pain intensity, from approximately scale 3 to 1/0 on day 7 and 11, were higher in hUCBMNCs-treated compared to SSD-treated wound. In spite of the HLA-mismatch, no allergic reaction, rejection, and infection found on hUCBMNCs-treated wound suggested the safety of this therapy. Histology examination found the formation of dermal-epidermal junction and rete ridges equal to the normal skin on hUCBMNCs-treated wounds. Conclusion: hUCBMNCs are effective and safe to promote re

  20. Effects of active bufadienolide compounds on human cancer cells and CD4+CD25+Foxp3+ regulatory T cells in mitogen-activated human peripheral blood mononuclear cells.

    Science.gov (United States)

    Yuan, Bo; He, Jing; Kisoh, Keishi; Hayashi, Hideki; Tanaka, Sachiko; Si, Nan; Zhao, Hai-Yu; Hirano, Toshihiko; Bian, Baolin; Takagi, Norio

    2016-09-01

    The growth inhibitory effects of bufadienolide compounds were investigated in two intractable cancer cells, a human glioblastoma cell line U-87 and a pancreatic cancer cell line SW1990. Among four bufadienolide compounds, a dose-dependent cytotoxicity was observed in these cancer cells after treatment with gamabufotalin and arenobufagin. The IC50 values of the two compounds were 3-5 times higher in normal peripheral blood mononuclear cells (PBMCs) than these values for both cancer cell lines. However, similar phenomena were not observed for two other bufadienolide compounds, telocinobufagin and bufalin. These results thus suggest that gamabufotalin and arenobufagin possess selective cytotoxic activity against tumor cells rather than normal cells. Moreover, a clear dose-dependent lactate dehydrogenase (LDH) release, a well-known hallmark of necrosis, was observed in both cancer cells treated with gamabufotalin, suggesting that gamabufotalin-mediated cell death is predominantly associated with a necrosis-like phenotype. Of most importance, treatment with as little as 8 ng/ml of gamabufotalin, even an almost non-toxic concentration to PBMCs, efficiently downregulated the percentages of CD4+CD25+Foxp3+ regulator T (Treg) cells in mitogen-activated PBMCs. Given that Treg cells play a critical role in tumor immunotolerance by suppressing antitumor immunity, these results suggest that gamabufotalin may serve as a promising candidate, as an adjuvant therapeutic agent by manipulating Treg cells to enhance the efficacy of conventional anticancer drugs and lessen their side-effects. These findings provide insights into the clinical application of gamabufotalin for cancer patients with glioblastoma/pancreatic cancer based on its cytocidal effect against tumor cells as well as its depletion of Treg cells.

  1. Neuroprotective effects of intravenous transplantation of bone marrow mononuclear cells from 5-fluorouracil pre-treated rats on ischemic stroke.

    Science.gov (United States)

    Li, Y; Mao, W W; Zhang, C G; Wan, L; Jing, C H; Hua, X M; Li, S T; Cheng, J

    2016-03-15

    Our previous findings showed bone marrow mononuclear cells (BMMNCs) from 5- fluorouracil (5-FU) pre-treated rats (named BMRMNCs) had a better therapeutic efficacy in ischemia/reperfusion rats as compared to BMMNCs from untreated rats. This study was undertaken to explore the potential mechanisms underlying the neuroprotective effects of BMRMNCs in middle cerebral artery occlusion (MCAO) rat model. Rats were intravenously pre-treated with 5-FU and BMRMNCs were collected at different time points. The contents of growth factors in the supernatant and CXCR4 expression were detected by ELISA and flow cytometry, respectively. MCAO was introduced to rats, and BMMNCs and BMRMNCs collected at 7 days after 5-FU pre-treatment were independently transplanted via the tail vein 24h later. The neurological function was evaluated before cell transplantation and at 24h, 7d and 14d after cell transplantation. Rats were sacrificed at 14d after cell transplantation, the brains were collected for TTC staining, infarct volume detection, NISSL staining, counting of viable cells in the CA1 region, and observation of transplanted cells. BMRMNCs had elevated expressions of growth factors as well as CXCR4 expression. Our results confirmed the better therapeutic effects of BMRMNCs in MCAO rats, demonstrated by reduction in infarct volume, improvement of neurological function and more viable cells in the hippocampus. In addition, more transplanted cells were found after BMRMNCs transplantation at 7 days and 14 days although there was no marked difference at 14 days. These findings indicate that BMRMNCs transplantation may protect ischemic stroke, at least partially, via increasing the secretion of growth factors and migration to the injured site.

  2. Effect of bovine lactoferrin on functions of activated feline peripheral blood mononuclear cells during chronic feline immunodeficiency virus infection.

    Science.gov (United States)

    Kobayashi, Saori; Sato, Reeko; Aoki, Takako; Omoe, Katsuhiko; Inanami, Osamu; Hankanga, Careen; Yamada, Yuichi; Tomizawa, Nobuyuki; Yasuda, Jun; Sasaki, Juso

    2008-05-01

    Feline immunodeficiency virus (FIV) infection is characterized by chronic overactivation of immune and inflammatory system, resulting in anergic state and dysfunction of immune cells. Lactoferrin (LF), a glycoprotein present in exocrine secretions and neutrophils, plays an important role in host defense system. Our previous study showed that oral administration of bovine LF (bLF) suppressed oral inflammation, improved the clinical symptoms and decreased serum gamma-globulin as a marker of inflammation in FIV-infected cats with intractable stomatitis. The anti-inflammatory effect was partly involved in regulation of neutrophil function by bLF. In this study, to clarify the relationship between anti-inflammatory effects of bLF and peripheral blood mononuclear cells (PBMC), we examined the effect of bLF on proliferation, cell cycle progression and cytokine expression in mitogen-activated PBMC. MTT [3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide] assay showed that bLF inhibited the concanavalin A (ConA)-induced cell proliferation in FIV-infected cats with the asymptomatic carrier and AIDS-related complex (ARC) phase. Bovine LF restored ConA-induced cell cycle progression and resulted in suppression of the induced apoptosis in feline PBMC. Real-time RT-PCR showed that bLF suppressed ConA-induced expression of interferon-gamma and interleukin-2 in cells of the ARC group regardless of the time of its addition to the medium. These results suggest the hypothesis that therapy with bLF may have the potential to improve and protect functions of overactivated lymphocytes by modulating the cell proliferation, cell cycle and cytokines expression in cats in terminal stage of FIV infection.

  3. Concentration of bone marrow mononuclear cells for in vitro treatment and AB0-incompatible transplantation: a rapid and reproducible procedure using the haemonetics V50 cell separator.

    Science.gov (United States)

    Wiesneth, M; Hertenstein, B; Koerner, K; Heimpel, H; Heit, W

    1988-01-01

    Forty-nine allogeneic and 14 autologous bone marrow grafts were processed with the Haemonetics V50 cell separator (Haemonetics Corp., Braintree, USA) for in vitro treatment with antibodies and cryopreservation respectively. The concentration of hemopoietic progenitor cells was performed without any sedimentation or density gradient agents. The recovery is given in percent (mean +/- sd) of the original marrow values: mononuclear cells (MNC) 74 +/- 10%, polymorphonuclear cells (PMC) 48 +/- 17%, red blood cells (RBC) 12 +/- 5%, granulocyte/monocyte progenitors (CFU-GM) 83 +/- 36%, and erythroid progenitors (BFU-E) 78 +/- 38%. The recovery of nucleated cells (NC) was 90 +/- 13% and the viability 82 +/- 11% after cryopreservation. The technique described provides a simple, rapid and efficient preparation of large bone marrow volumes for in vitro treatment and AB0-incompatible transplantation.

  4. Infection of chicken bone marrow mononuclear cells with subgroup J avian leukosis virus inhibits dendritic cell differentiation and alters cytokine expression.

    Science.gov (United States)

    Liu, Di; Qiu, Qianqian; Zhang, Xu; Dai, Manman; Qin, Jianru; Hao, Jianjong; Liao, Ming; Cao, Weisheng

    2016-10-01

    Subgroup J avian leukosis virus (ALV-J) is an oncogenic retrovirus known to induce tumor formation and immunosuppression in infected chickens. One of the organs susceptible to ALV-J is the bone marrow, from which specialized antigen-presenting cells named dendritic cells (BM-DCs) are derived. Notably, these cells possess the unique ability to induce primary immune responses. In the present study, a method of cultivating and purifying DCs from chicken bone marrow in vitro was established to investigate the effects of ALV-J infection on BM-DC differentiation or generation. The results indicated that ALV-J not only infects the chicken bone marrow mononuclear cells but also appears to inhibit the differentiation and maturation of BM-DCs and to trigger apoptosis. Moreover, substantial reductions in the mRNA expression of TLR1, TLR2, TLR3, MHCI, and MHCII and in cytokine production were detected in the surviving BM-DCs following ALV-J infection. These findings indicate that ALV-J infection disrupts the process of bone marrow mononuclear cell differentiation into BM-DCs likely via altered antigen presentation, resulting in a downstream immune response in affected chickens.

  5. Global gene expression analysis of peripheral blood mononuclear cells in rhesus monkey infants with CA16 infection-induced HFMD.

    Science.gov (United States)

    Song, Jie; Hu, Yajie; Hu, Yunguang; Wang, Jingjing; Zhang, Xiaolong; Wang, Lichun; Guo, Lei; Wang, Yancui; Ning, Ruotong; Liao, Yun; Zhang, Ying; Zheng, Huiwen; Shi, Haijing; He, Zhanlong; Li, Qihan; Liu, Longding

    2016-03-02

    Coxsackievirus A16 (CA16) is a dominant pathogen that results in hand, foot, and mouth disease and causes outbreaks worldwide, particularly in the Asia-Pacific region. However, the underlying molecular mechanisms remain unclear. Our previous study has demonstrated that the basic CA16 pathogenic process was successfully mimicked in rhesus monkey infant. The present study focused on the global gene expression changes in peripheral blood mononuclear cells of rhesus monkey infants with hand, foot, and mouth disease induced by CA16 infection at different time points. Genome-wide expression analysis was performed with Agilent whole-genome microarrays and established bioinformatics tools. Nine hundred and forty-eight significant differentially expressed genes that were associated with 5 gene ontology categories, including cell communication, cell cycle, immune system process, regulation of transcription and metabolic process were identified. Subsequently, the mapping of genes related to the immune system process by PANTHER pathway analysis revealed the predominance of inflammation mediated by chemokine and cytokine signaling pathways and the interleukin signaling pathway. Ultimately, co-expressed genes and their networks were analyzed. The results revealed the gene expression profile of the immune system in response to CA16 in rhesus monkey infants and suggested that such an immune response was generated as a result of the positive mobilization of the immune system. This initial microarray study will provide insights into the molecular mechanism of CA16 infection and will facilitate the identification of biomarkers for the evaluation of vaccines against this virus.

  6. Effects of aspirin-triggered resolvin D1 on peripheral blood mononuclear cells from patients with Chagas' heart disease.

    Science.gov (United States)

    Ogata, Haline; Teixeira, Maxelle Martins; Sousa, Rodrigo Cunha de; Silva, Marcos Vinícius da; Correia, Dalmo; Rodrigues Junior, Virmondes; Levy, Bruce David; Rogério, Alexandre de Paula

    2016-04-15

    Chagas disease is caused by Trypanosoma cruzi (T. cruzi). In some patients with Chagas disease, symptoms progress to chronic chagasic cardiomyopathy. Endogenously, inflammation is resolved in the presence of lipid mediators such as aspirin-triggered RvD1 (AT-RvD1) which has anti-inflammatory and pro-resolution effects. Here, we demonstrated, for the first time, the effects of AT-RvD1 on T. cruzi antigen-stimulated peripheral blood mononuclear cells (PBMCs) from patients with Chagas heart disease. The levels of IFN-γ, TNF-α, IL-10, and IL-13 increased in PBMCs from cardiac-form Chagas patients in stage B1 (patients with fewer heart abnormalities) stimulated with T. cruzi antigen compared to those in non-stimulated PBMCs. AT-RvD1 reduced the IFN-γ concentrations in PBMCs from patients with Chagas disease stimulated with T. cruzi antigen compared to stimulated with T. cruzi antigen cells. AT-RvD1 treatment resulted in no observable changes in TNF-α, IL-10, and IL-13 levels. AT-RvD1 significantly decreased the percentage of necrotic cells and caused a significant reduction in the proliferation rate of T. cruzi antigen-stimulated PBMCs from patients with Chagas disease. These findings demonstrate that AT-RvD1 modulates the immune response in Chagas disease patients and might have potential to be used as an alternative approach for slowing the development of further heart damage.

  7. Effect of thermal stress on expression profile of apoptosis related genes in peripheral blood mononuclear cells of transition Sahiwal cow

    Science.gov (United States)

    Somal, A; Aggarwal, A; Upadhyay, R.C

    2015-01-01

    The study was conducted to evaluate the effect of thermal stress on expression profile of genes related to apoptosis in peripartum Sahiwal cows. For this, twelve pregnant dry Sahiwal cows were selected from Livestock Research Centre at National Dairy Research Institute, Karnal. The cows were divided into two groups consisting of six Sahiwal cows each. Cows of group I calved during thermoneutral temperature conditions (THI=67.3) and cows of group II calved in summer season (THI=79.9). Blood samples were collected on -15, 0 and +15 days with respect to calving where day ‘0’ represents the day of calving. The peripheral blood mononuclear cells (PBMC) were separated and total RNA was isolated for the BCL-2 (B-Cell Lymphoma-2), BAX (BCL-2 antagonist killer-1), BAK (Bcl-2-associated X protein), CASP-3 (cysteine-aspartic proteases-3) and P53 (tumour protien-53) mRNAs expression. It was found that there was up regulation of CASP-3 on the day of calving during both temperature conditions. Comparison between the two temperature conditions showed that expression of CASP-3, BCL-2, BAK, P53 and ratio of BAX/BCL-2 in PBMC increased during summer as compared to thermoneutral condition suggesting the susceptibility of these cells to apoptosis. Based on the above findings it can be concluded that during calving PBMC are more susceptible to apoptosis, and summer being more stressful potentiates the apoptosis of PBMC in Sahiwal cows. PMID:27175165

  8. Senescence-Related Changes in Gene Expression of Peripheral Blood Mononuclear Cells from Octo/Nonagenarians Compared to Their Offspring

    Directory of Open Access Journals (Sweden)

    Amirah Abdul Rahman

    2013-01-01

    Full Text Available Mechanisms determining both functional rate of decline and the time of onset in aging remain elusive. Studies of the aging process especially those involving the comparison of long-lived individuals and young controls are fairly limited. Therefore, this research aims to determine the differential gene expression profile in related individuals from villages in Pahang, Malaysia. Genome-wide microarray analysis of 18 samples of peripheral blood mononuclear cells (PBMCs from two groups: octo/nonagenarians (80–99 years old and their offspring (50.2 ± 4.0 years old revealed that 477 transcripts were age-induced and 335 transcripts were age-repressed with fold changes ≥1.2 in octo/nonagenarians compared to offspring. Interestingly, changes in gene expression were associated with increased capacity for apoptosis (BAK1, cell cycle regulation (CDKN1B, metabolic process (LRPAP1, insulin action (IGF2R, and increased immune and inflammatory response (IL27RA, whereas response to stress (HSPA8, damage stimulus (XRCC6, and chromatin remodelling (TINF2 pathways were downregulated in octo/nonagenarians. These results suggested that systemic telomere maintenance, metabolism, cell signalling, and redox regulation may be important for individuals to maintain their healthy state with advancing age and that these processes play an important role in the determination of the healthy life-span.

  9. Phenotypic characterization of mononuclear cells and class II antigen expression in angular cheilitis infected by Candida albicans or Staphylococcus aureus.

    Science.gov (United States)

    Ohman, S C; Jontell, M; Jonsson, R

    1989-04-01

    In the present study we characterized the phenotypes of infiltrating mononuclear cells in angular cheilitis lesions to further explore the pathogenesis of this disorder. Frozen sections from lesions infected by Candida albicans and/or Staphylococcus aureus were subjected to immunohistochemical analysis utilizing monoclonal antibodies directed to subsets of T-lymphocytes, B-lymphocytes, and macrophages. In addition, the expression of Class II antigens (HLA-DP, -DQ, -DR), the interleukin 2- and transferrin-receptors was studied on resident and infiltrating cells. An intense infiltration of T-lymphocytes was accompanied by expression of Class II antigens on the epidermal keratinocytes in lesion infected by Candida albicans. The Staphylococcus aureus infected lesions displayed a diffuse infiltration of T-lymphocytes but virtually no expression of Class II antigen by epidermal keratinocytes. These observations suggest that the cell-mediated arm of the immune system is involved in the inflammatory reaction of lesions infected by Candida albicans. In addition, the present study confirms that epidermal expression of Class II antigens is closely related to the type and magnitude of the infiltrating T-lymphocyte. Finally, these findings indicate that the type of inflammatory reaction in angular cheilitis is primarily dependent on the isolated microorganism, although the clinical pictures of the disorder are virtually identical.

  10. Rapid separation of mononuclear hodgkin from multinuclear reed-sternberg cells.

    Science.gov (United States)

    Kongruttanachok, Narisorn; Cayre, Yvon E; Knecht, Hans; Mai, Sabine

    2014-03-01

    We describe a method to isolate small mononucleated Hodgkin (H) cells from multinucleated Reed Sternberg (RS) cells of Hodgkin lymphoma using the ScreenCell filter device. This filtration-based approach lends itself to future clinical applications in that it enables the separation of H and RS cells from lymph node biopsies, bone marrow aspirates, pleural effusions, and blood, including the isolation of monoclonal Hodgkin precursor cells from the blood.

  11. Study of the inhibition by polymorphonuclear leukocytes of TNF-α release from human mononuclear cells and its mechanism

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The present study was undertaken to investigate the effect of human PMNs on the production of TNF-α by the human peripheral blood mononuclear cells (PBMCs) and to elucidate its tentative mechanism. Human PMNs and PBMCs were isolated from the venous blood of healthy donors by dextran sedimentation and density gradient centrifugation. In the presence of lipopolysaccharide (LPS), PMNs and PBMCs were cocultured at the ratio of 2:1 for 20 h and the concentration of TNF-α in the supernatant was measured by enzyme-linked immunosorbent assay. The binding rate of monocytes with the fluorescein isothiocyanate-labeled LPS (FITC-LPS) and the mean surface fluorescence intensity of monocytes were analyzed by flow cytometry. Results showed that PMNs were capable of inhibiting the TNF-α release from PBMCs (P<0.05). PMNs suppressed the TNF-α release from PBMCs by 45% on average when PMNs and PBMCs cocultured at the ratio of 2:1. Paraformaldehyde-fixed PMNs still demonstrated the same inhibition (P<0.05),which proved that the inhibition was dependent on cell-to-cell contact and suggested that effector molecules responsible for this effect existed on the cell surface of PMNs. In the presence of PMNs, the binding rate of monocytes with the FITC-LPS and the mean surface fluorescence intensity of monocytes were not affected compared with PBMCs alone (P>0.05). As incubation time was prolonged, the binding of FITC-LPS to monocytes increased (P<0.05). Thus PMNs did not block the binding of LPS with monocytes. It was concluded that PMNs suppressed the TNF-α release from PBMCs via cell-to-cell interaction. In a cell-contact dependent manner, PMNs might interfere with the signal transduction pathway through which LPS activated PBMCs, thus attenuating the response of PBMCs to LPS and downregulating the TNF-α release.

  12. Peripheral blood mononuclear cell proliferation and cytokine production in sheep as affected by cortisol level and duration of stress.

    Science.gov (United States)

    Ciliberti, M G; Albenzio, M; Inghese, C; Santillo, A; Marino, R; Sevi, A; Caroprese, M

    2017-01-01

    A large number of studies recognize glucocorticoids (Gc) as suppressors of inflammation; Gc exert an important role in coordinating the magnitude and duration of host immune responses. In the present in vitro investigation, we tested incremental levels of cortisol to verify the immunosuppressive or immunopermissive role of cortisol in sheep peripheral blood mononuclear cells (PBMC) after acute and chronic stress. Phytohemagglutinin (PHA)-stimulated PBMC were cultured for 24h and 96h at 37°C with 5% of CO2 and varying cortisol levels: 10 ng/mL (baseline), 100 ng/mL (physiological poststressor), and 1,000 ng/mL [hyperactivated hypothalamic-pituitary-adrenal (HPA) axis]. The cell-free supernatants were collected for determination of IL-6, IL-1β, and IL-10 by ELISA, and the bromodeoxyuridine assay was performed on cells. Physiological cortisol concentration negatively affected the levels of IL-6 secreted by PBMC, resulting in increased cell proliferation after acute stress (24h of incubation). However, physiological cortisol concentration exhibited a reduction in cell proliferation induced by increased levels of IL-6 secreted by PBMC during chronic stress (96h of incubation). The cortisol concentration representing a hyperactivated HPA axis led to a reduction in cell proliferation after acute stress, which was probably induced by the elevated IL-10 production. Our results demonstrate that in sheep the effect of Gc on the immune system was related to the magnitude and the duration of stress. In particular, cortisol levels higher than physiological concentrations suppressed cell proliferation soon after acute stress. Instead, the physiological poststressor concentration of cortisol affected the immune responses in a bidirectional manner depending on the duration of the stressor. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  13. A flow cytometry technique to study intracellular signals NF-κB and STAT3 in peripheral blood mononuclear cells

    Directory of Open Access Journals (Sweden)

    Chavarin Patricia

    2007-07-01

    Full Text Available Abstract Background Cytokines have essential roles on intercellular communications and are effective in using a variety of intracellular pathways. Among this multitude of signalling pathways, the NF-κB (nuclear factor kappaB and STAT (signal transducer and activator of transcription families are among the most frequently investigated because of their importance. Indeed, they have important role in innate and adaptive immunity. Current techniques to study NF-κB and STAT rely on specific ELISAs, Western Blots and – most recently described – flow cytometry; so far, investigation of such signalling pathways are most commonly performed on homogeneous cells after purification. Results The present investigation aimed at developing a flow cytometry technique to study transcription factors in various cellular types such as mixtures of B-cells, T-lymphocytes and monocytes/macrophages stimulated in steady state conditions (in other words, as peripheral blood mononuclear cells. To achieve this goal, a two step procedure was carried out; the first one consisted of stimulating PBMCs with IL1β, sCD40L and/or IL10 in such a manner that optimal stimulus was found for each cell subset (and subsequent signal transduction, therefore screened by specific ELISA; the second step consisted of assessing confirmation and fine delineation of technical conditions by specific Western-Blotting for either NF-κB or STAT products. We then went on to sensitize the detection technique for mixed cells using 4 color flow cytometry. Conclusion In response to IL1β, or IL10, the levels of phosphorylated NF-κB and STAT3 – respectively – increased significantly for all the studied cell types. In contrast, B-cells and monocytes/macrophages – but, interestingly, not T-lymphocytes (in the context of PBMCs – responded significantly to sCD40L by increasing phosphorylated NF-κB.

  14. Structural, vibrational, and electronic properties of an uncoordinated pseudoephedrine derivative and its mononuclear and trinuclear copper(II)-coordinated compounds: A combined theoretical and experimental study

    Science.gov (United States)

    Valencia, Israel; Ávila-Torres, Yenny; Barba-Behrens, Norah; Garzón, Ignacio L.

    2014-11-01

    Multicopper oxidases are fundamental in a variety of biological processes in bacteria, fungi and vertebrates. The catalytic center in these enzymes is formed basically by three copper ions, bridged by oxygen bonds. In order to get insights into the reactivity of these complex systems, biomimetic compounds are usually synthesized. Accordingly, in this work, we studied structural, vibrational, and electronic properties of an uncoordinated pseudoephedrine derivative, as well as its corresponding mononuclear and trinuclear copper(II)-coordinated complexes by means of density functional theory. The calculations are compared with experimental results using measurements of the infrared spectra. It is obtained that the molecular configuration of the pseudoephedrine amino-alcohol derivative is stabilized by hydrogen bonding Osbnd H⋯N and by Csbnd H⋯π interactions that are not present in the mononuclear and trinuclear compounds. The coordination compounds show octahedral and square pyramid geometries, respectively, which are slightly distorted by Jahn-Teller effects. The analysis of their theoretical and experimental IR spectra reveals signals related with hydrogen bonding as well as metal-ligand vibrational modes. Regarding the electronic structure, the density of states was calculated in order to analyze the atomic orbital contributions present in these compounds. This analysis would provide useful insights about the optical behavior, for example, in the visible region of the spectrum of the coordinated compounds. At these energies, the optical absorption would be influenced by the orbital interaction of the Cu2+d orbitals with sp ones of the ligand, reflecting a decrease of the HOMO-LUMO gap of the organic ligand due to the presence of the copper(II) ions.

  15. The Role of Amnion Membrane-Derived Mesenchymal Stem Cells on Differentiation and Expansion of Natural Killer Cell Progenitors Originated From Umbilical Cord Blood Mononuclear Cells

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    Ahmadi

    2015-11-01

    Full Text Available Background Natural killer (NK cells are members of the innate immune system. Their unique properties, including recognition of viral infected and tumor cells without major histocompatibility complex (MHC restriction or prior sensitization, make them a suitable choice for immunotherapy. Low numbers of NK cells in circulating blood is the most important obstacle for this goal. Objectives The aim of this study was to make an optimum in vitro condition to proliferate and differentiate cord blood (CB-NK cell progenitors to mature NK cells, which can be used for cell therapy. Materials and Methods In our study, CB-Mononuclear Cells’ (MNCs CD3+ lymphocytes were positive depleted using immunomagnetic microbeads. This CD3-depleted (CD3-dep CB - MNCs compartment was used for in vitro expansion with or without a layer of amnion membrane mesenchymal stem cells (MSCs in combination with cytokines that are essential for NK cells expansion (IL-2, IL-3, IL-15, and FLT3 ligand. The expansion period lasted for one week. On day seven, immunophenotype and fold expansion of differentiated cells were measured. Results Combination of cytokines and MSC layer yielded significant fold expansion in comparison with cytokines without feeder conditions (day 7: 5.2 ± 1.12 and 2 ± 0.78, respectively, P < 0.05. CD3-/CD56+ cells percentage increased during the culture period in MSCs/with cytokine and cytokine/without feeder, respectively (day 0: 4.4 ± 0.42% and day 7: 22.9 ± 3.6% and 13.9 ± 1.92 % for MSC/with cytokine and cytokine without feeder, respectively. Conclusions Our results suggested that CB-NK cells progenitors could proliferate and differentiate on feeder layer of amnion membrane MSCs in combination with specific cytokines to produce NK cells for immunotherapy.

  16. Physalin F, a seco-steroid from Physalis angulata L., has immunosuppressive activity in peripheral blood mononuclear cells from patients with HTLV1-associated myelopathy.

    Science.gov (United States)

    Pinto, Lorena A; Meira, Cássio S; Villarreal, Cristiane F; Vannier-Santos, Marcos A; de Souza, Claudia V C; Ribeiro, Ivone M; Tomassini, Therezinha C B; Galvão-Castro, Bernardo; Soares, Milena B P; Grassi, Maria F R

    2016-04-01

    Human T-lymphotropic virus type 1 (HTLV-1) induces a strong activation of the immune system, especially in individuals with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Physalin F is a secosteroid with potent anti-inflammatory and immunomodulatory activities. The present study aimed to investigate the effects of physalin F on peripheral blood mononuclear cells (PBMC) of HAM/TSP subjects. A concentration-dependent inhibition of spontaneous proliferation of PBMC from HAM/TSP subjects was observed in the presence of physalin F, as evaluated by (3)H-thymidine uptake. The IC50 for physalin F was 0.97 ± 0.11 μM. Flow cytometry analysis using Cytometric Bead Array (CBA) showed that physalin F (10 μM) significantly reduced the levels of IL-2, IL-6, IL-10, TNF-α and IFN-γ, but not IL-17A, in supernatants of PBMC cultures. Next, apoptosis induction was addressed by using flow cytometry to evaluate annexin V expression. Treatment with physalin F (10 μM) increased the apoptotic population of PBMC in HAM/TSP subjects. Transmission electron microscopy analysis of PBMC showed that physalin F induced ultrastructural changes, such as pyknotic nuclei, damaged mitochondria, enhanced autophagic vacuole formation, and the presence of myelin-like figures. In conclusion, physalin F induces apoptosis of PBMC, decreasing the spontaneous proliferation and cytokine production caused by HTLV-1 infection.

  17. Inflammatory cytokines in vitro production are associated with Ala16Val superoxide dismutase gene polymorphism of peripheral blood mononuclear cells.

    Science.gov (United States)

    Montano, Marco Aurélio Echart; da Cruz, Ivana Beatrice Mânica; Duarte, Marta Maria Medeiros Frescura; Krewer, Cristina da Costa; da Rocha, Maria Izabel de Ugalde Marques; Mânica-Cattani, Maria Fernanda; Soares, Felix Alexandre Antunes; Rosa, Guilherme; Maris, Angélica Francesca; Battiston, Francielle Garghetti; Trott, Alexis; Lera, Juan Pablo Barrio

    2012-10-01

    Obesity is considered a chronic low-grade inflammatory state associated with a chronic oxidative stress caused by superoxide production (O(2)(-)). The superoxide dismutase manganese dependent (SOD2) catalyzes O(2)(-) in H(2)O(2) into mitochondria and is encoded by a single gene that presents a common polymorphism that results in the replacement of alanine (A) with a valine (V) in the 16 codon. This polymorphism has been implicated in a decreased efficiency of SOD2 transport into targeted mitochondria in V allele carriers. Previous studies described an association between VV genotype and metabolic diseases, including obesity and diabetes. However, the causal mechanisms to explain this association need to be more elucidated. We postulated that the polymorphism could influence the inflammatory response. To test our hypothesis, we evaluated the in vitro cytokines production by human peripheral blood mononuclear cells (PBMCs) carrier's different Ala16Val-SOD2 genotypes (IL-1, IL-6, IL-10, TNF-α, IFN-γ). Additionally, we evaluated if the culture medium glucose, enriched insulin, could influence the cytokine production. Higher levels of proinflammatory cytokines were observed in VV-PBMCs when compared to AA-PBMCs. However, the culture medium glucose and enriched insulin did not affect cytokine production. The results suggest that Ala16Val-SOD2 gene polymorphism could trigger the PBMCs proinflammatory cytokines level. However, discerning if a similar mechanism occurs in fat cells is an open question.

  18. In vitro effects of two extracts and two pure alkaloid preparations of Uncaria tomentosa on peripheral blood mononuclear cells.

    Science.gov (United States)

    Winkler, C; Wirleitner, B; Schroecksnadel, K; Schennach, H; Mur, E; Fuchs, D

    2004-03-01

    In the traditional Peruvian medicine, hot aqueous extracts of Uncaria tomentosa have been used for the treatment of a wide range of health problems, particularly digestive complaints and arthritis. Some of the beneficial effects observed in patients suggest an immunomodulatory capacity of Uncaria tomentosa extracts. In this study, the effects of two extracts and two mixtures of tetracyclic and pentacyclic oxindole alkaloids of Uncaria tomentosa were investigated in freshly isolated human peripheral blood mononuclear cells (PBMC) stimulated with the mitogens phytohaemagglutinin (PHA) and concanavalin A (Con A) in vitro. Neopterin production and tryptophan degradation were monitored in culture supernatants to determine the effects of the test substances on immunobiochemical pathways induced by interferon-gamma. Compared to unstimulated cells PHA and Con A increased the production of neopterin and degradation of tryptophan (p < 0.01). HCl and ethanol extracts and mixtures of alkaloids of Uncaria tomentosa inhibited both effects in a dose-dependent manner, the lowest effective concentrations of the extracts were 500 - 1000 microg/mL and of the alkaloid mixtures 100 - 175 microg/mL (p < 0.05 and < 0.01). With the highest concentrations of extracts and mixtures complete suppression of mitogen-induced neopterin production and tryptophan degradation was observed. These data demonstrate that Uncaria tomentosa extracts and mixtures of alkaloids modulate the immunobiochemical pathways induced by interferon-gamma. The findings imply a potential application of the extracts as immunoregulators and would be in line with observations in patients using these extracts.

  19. DNA damage and methylation induced by glyphosate in human peripheral blood mononuclear cells (in vitro study).

    Science.gov (United States)

    Kwiatkowska, Marta; Reszka, Edyta; Woźniak, Katarzyna; Jabłońska, Ewa; Michałowicz, Jaromir; Bukowska, Bożena

    2017-07-01

    Glyphosate is a very important herbicide that is widely used in the agriculture, and thus the exposure of humans to this substance and its metabolites has been noted. The purpose of this study was to assess DNA damage (determination of single and double strand-breaks by the comet assay) as well as to evaluate DNA methylation (global DNA methylation and methylation of p16 (CDKN2A) and p53 (TP53) promoter regions) in human peripheral blood mononuclear cells (PBMCs) exposed to glyphosate. PBMCs were incubated with the compound studied at concentrations ranging from 0.1 to 10 mM for 24 h. The study has shown that glyphosate induced DNA lesions, which were effectively repaired. However, PBMCs were unable to repair completely DNA damage induced by glyphosate. We also observed a decrease in global DNA methylation level at 0.25 mM of glyphosate. Glyphosate at 0.25 mM and 0.5 mM increased p53 promoter methylation, while it did not induce statistically significant changes in methylation of p16 promoter. To sum up, we have shown for the first time that glyphosate (at high concentrations from 0.5 to 10 mM) may induce DNA damage in leucocytes such as PBMCs and cause DNA methylation in human cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Paracrine Factors from Irradiated Peripheral Blood Mononuclear Cells Improve Skin Regeneration and Angiogenesis in a Porcine Burn Model.

    Science.gov (United States)

    Hacker, Stefan; Mittermayr, Rainer; Nickl, Stefanie; Haider, Thomas; Lebherz-Eichinger, Diana; Beer, Lucian; Mitterbauer, Andreas; Leiss, Harald; Zimmermann, Matthias; Schweiger, Thomas; Keibl, Claudia; Hofbauer, Helmut; Gabriel, Christian; Pavone-Gyöngyösi, Mariann; Redl, Heinz; Tschachler, Erwin; Mildner, Michael; Ankersmit, Hendrik Jan

    2016-04-29

    Burn wounds pose a serious threat to patients and often require surgical treatment. Skin grafting aims to achieve wound closure but requires a well-vascularized wound bed. The secretome of peripheral blood mononuclear cells (PBMCs) has been shown to improve wound healing and angiogenesis. We hypothesized that topical application of the PBMC secretome would improve the quality of regenerating skin, increase angiogenesis, and reduce scar formation after burn injury and skin grafting in a porcine model. Full-thickness burn injuries were created on the back of female pigs. Necrotic areas were excised and the wounds were covered with split-thickness mesh skin grafts. Wounds were treated repeatedly with either the secretome of cultured PBMCs (Sec(PBMC)), apoptotic PBMCs (Apo-Sec(PBMC)), or controls. The wounds treated with Apo-Sec(PBMC) had an increased epidermal thickness, higher number of rete ridges, and more advanced epidermal differentiation than controls. The samples treated with Apo-Sec(PBMC) had a two-fold increase in CD31+ cells, indicating more angiogenesis. These data suggest that the repeated application of Apo-Sec(PBMC) significantly improves epidermal thickness, angiogenesis, and skin quality in a porcine model of burn injury and skin grafting.

  1. Time-Course Study of the Transcriptome of Peripheral Blood Mononuclear Cells (PBMCs) from Sheep Infected with Fasciola hepatica

    Science.gov (United States)

    Scheerlinck, Jean-Pierre; Ansell, Brendan R. E.; Hall, Ross S.; Gasser, Robin B.; Jex, Aaron R.

    2016-01-01

    Fasciola hepatica is a parasitic trematode that infects a wide range of mammalian hosts, including livestock and humans, in temperate and tropical regions globally. This trematode causes the disease fascioliasis, which consists of an acute phase (≤ 12 weeks) during which juvenile parasites migrate through the host liver tissues, and a chronic phase (> 12 weeks) following the establishment of adult parasites in the liver bile ducts. Few studies have explored the progression of the host response over the course of Fasciola infection in the same animals. In this study, we characterized transcriptomic changes in peripheral blood mononuclear cells (PBMCs) collected from sheep at three time points over the first eight weeks of infection relative to uninfected controls. In total, 183 and 76 genes were found to be differentially transcribed at two and eight weeks post-infection respectively. Functional and pathway analysis of differentially transcribed genes revealed changes related to T-cell activation that may underpin a Th2-biased immune response against this parasite. This first insight into the dynamics of host responses during the early stages of infection improves the understanding of the pathogenesis of acute fascioliasis, informs vaccine development and presents a set of PBMC markers with diagnostic potential. PMID:27438474

  2. Prion protein expression and processing in human mononuclear cells: the impact of the codon 129 prion gene polymorphism.

    Directory of Open Access Journals (Sweden)

    Christiane Segarra

    Full Text Available BACKGROUND: So far, all clinical cases of new variant Creutzfeldt-Jakob disease (vCJD, thought to result from the Bovine Spongiform Encephalopathy (BSE prion agent, have shown Methionine-Methionine (M/M homozygosity at the M129V polymorphism of the PRNP gene. Although established, this relationship is still not understood. In both vCJD and experimental BSE models prion agents do reach the bloodstream, raising concerns regarding disease transmission through blood transfusion. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the impact of the M129V polymorphism on the expression and processing of the prion protein in human peripheral blood mononuclear cells (PBMCs from three blood donor populations with Methionine-Methionine (M/M, Valine-Valine (V/V and M/V genotypes. Using real-time PCR, ELISA and immunoblot assays we were unable to find differences in prion protein expression and processing relating to the M129V polymorphism. CONCLUSIONS/SIGNIFICANCE: These results suggest that in PBMCs, the M129V PrP polymorphism has no significant impact on PrP expression, processing and the apparent glycoform distribution. Prion propagation should be investigated further in other cell types or tissues.

  3. Optimal Thawing of Cryopreserved Peripheral Blood Mononuclear Cells for Use in High-Throughput Human Immune Monitoring Studies

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    Ramu A. Subbramanian

    2012-07-01

    Full Text Available Cryopreserved peripheral blood mononuclear cells (PBMC constitute an important component of immune monitoring studies as they allow for efficient batch- testing of samples as well as for the validation and extension of original studies in the future. In this study, we systematically test the permutations of PBMC thawing practices commonly employed in the field and identify conditions that are high and low risk for the viability of PBMC and their functionality in downstream ELISPOT assays. The study identifies the addition of ice-chilled washing media to thawed cells at the same temperature as being a high risk practice, as it yields significantly lower viability and functionality of recovered PBMC when compared to warming the cryovials to 37 °C and adding a warm washing medium. We found thawed PBMC in cryovials could be kept up to 30 minutes at 37 °C in the presence of DMSO before commencement of washing, which surprisingly identifies exposure to DMSO as a low risk step during the thawing process. This latter finding is of considerable practical relevance since it permits batch-thawing of PBMC in high-throughput immune monitoring environments.

  4. Antigenotoxic potential of aqueous extracts from the chanterelle mushroom, Cantharellus cibarius (higher Basidiomycetes), on human mononuclear cell cultures.

    Science.gov (United States)

    Mendez-Espinoza, Claudia; Garcia-Nieto, Edelmira; Esquivel, Adriana Montoya; Gonzalez, Monica Montiel; Bautista, Efrain Velasco; Ezquerro, Carmen Calderon; Santacruz, Libertad Juarez

    2013-01-01

    Cantharellus cibarius is one of the most important wild, edible, and ectomycorrhizal mushrooms growing at La Malinche National Park, Tlaxcala, Mexico; therefore, the assessment of its biological properties is of great interest to know its potential as an alternative treatment to chemopreventive strategies when it is consumed as part of a diet. Comet assay was used to evaluate the antigenotoxic properties of several concentrations of aqueous extracts (0.0125, 0.025, 0.05, 0.1, and 0.2% w/v) prepared at room temperature (22 ± 2°C). As a test system we used human mononuclear cells exposed to methyl methanesulphonate (MMS) in vitro according to 3 different protocols: previous, simultaneous, and posterior. Previous (0.0125%) and simultaneous (0.1%) treatments resulted in the highest inhibitory efficiency. In the former, the cells assessed showed a tail length of 94.9 ± 64 µm; in the latter, the tails measured 106.2 ± 40 µm. Resulting percentages of reduction in damage were 236% and 196.1%, respectively. We did not obtain a dose-dependent response. The mean tail length for each protocol (previous, 133.1 ± 80 µm; simultaneous, 127.8 ± 57 µm; posterior, 146.3 ± 74 µm) was statistically significant with regard to the positive control (MMS).

  5. The effect of cocoa procyanidins on the transcription and secretion of interleukin 1 beta in peripheral blood mononuclear cells.

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    Mao, T K; Powell, J; Van de Water, J; Keen, C L; Schmitz, H H; Hammerstone, J F; Gershwin, M E

    2000-03-03

    Recent data has demonstrated that cacao liquor polyphenols (procyanidins) have antioxidant activity, inhibit mRNA expression of interleukin-2 and are potent inhibitors of acute inflammation. Given the widespread ingestion of cocoa in many cultures, we investigated whether cocoa, in its isolated procyanidin fractions (monomer through decamer), would modulate synthesis of the pro-inflammatory cytokine, interleukin-1 beta. Both resting and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) were investigated at the levels of transcription and protein secretion. Individual cocoa fractions were shown to augment constitutive IL-1 beta gene expression, although values varied between subjects. Interestingly, the smaller fractions of cocoa (monomer-tetramer) consistently reduced IL-1 beta expression of PHA-stimulated cells by 1-15%, while the larger oligomers (pentamer-decamer) increased expression by 4-52%. These data, observed at the transcription level, were reflected in protein levels in PHA-induced PBMC. The presence or absence of PHA did not alter the effects of the cocoa procyanidins with the exception of the pentamer. This study offers additional data for the consideration of the health-benefits of dietary polyphenols from a wide variety of foods, including those benefits associated specifically with cocoa and chocolate consumption.

  6. Characterization of surface interleukin-2 receptor expression on gated populations of peripheral blood mononuclear cells from manatees, Trichechus manatus latirostris.

    Science.gov (United States)

    Sweat, J M; Johnson, C M; Marikar, Y; Gibbs, E P

    2005-12-15

    An in vitro system to determine surface interleukin-2 receptor (IL-2R) expression on mitogen-stimulated peripheral blood mononuclear cells (PBMC) from free-ranging manatees, Trichechus manatus latirostris was developed. Human recombinant IL-2, conjugated with a fluorescein dye was used in conjunction with flow cytometric analysis to determine changes in surface expression of IL-2R at sequential times over a 48-h period of in vitro stimulation. Surface expression of IL-2R was detected on manatee PBMC, which also cross-reacted with an anti-feline pan T-cell marker. An expression index (EI) was calculated by comparing mitogen-activated and non-activated PBMC. Based on side- and forward-scatter properties, flow cytometric analysis showed an increase in the number of larger, more granular "lymphoblasts" following concanavalin A (Con A) stimulation. The appearance of lymphoblasts was correlated with an increase in their surface expression of IL-2 receptors. Surface IL-2R expression, in Con A-stimulated PBMC, was detected at 16 h, peaked at 24-36 h, and began to decrease by 48 h. Characterization of the IL-2R expression should provide additional information on the health status of manatees, and the effect of their sub lethal exposure to brevetoxin.

  7. Assessment of 188Re marked anti MHC class Ⅱ antibody by peripheral blood mononuclear cells stimulated by donor alloantigen

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    DING Guo-ping; CAO Li-ping; LIU Jie; LIU Da-ren; QUE Ri-sheng; ZHU Lin-hua; ZHOU Yi-ming; MAO Ke-jie; HU Jun-an

    2011-01-01

    Background Previous studies showed that anti MHC-Ⅱ monoclone antibody (MAb) only had partial inhibiting effect of alloreactive mixed lymphocyte reaction (MLR) in vitro and it was unsteady and non-persistent. The aim of this research was to determine whether radioactive isotope 188Re marked MHC-Ⅱ antibody could benefit the allograft acceptance in transplantation as compared to normal MHC-Ⅱ antibody.Methods 188Re was incorporated to 2E9/13F(ab')2 which is against swine MHC class Ⅱ antigen (MAb-188Re). Porcine peripheral blood mononuclear (PBMC) cells were examined for proliferation and cytokine mRNA expression after stimulation with MHC-Ⅱ MAb or MAb-188Re.Results The proliferative response of recipient PBMCs in mixed lymphocyte reaction (MLR) to donor alloantigen showed that the stimulation index of MAb-188Re group was significantly lower than the MHC-Ⅱ MAb group and control (P<0.05). mRNA expression of interleukin 2, interferon Y and tumor necrosis factor α (type 1 cytokines) was lower in MAb-188Re group than the MHC-Ⅱ MAb group, while interleukin 10 (type 2 cytokines) was higher in MAb-188Re group in the first 24 hours.Conclusion MAb-188Re could help the graft acceptance by inhibiting T cell proliferation, lowering the expression of type 1 cytokines and elevating the type 2 cytokines produced by PBMC.

  8. Preventive activity of olive oil phenolic compounds on alkene epoxides induced oxidative DNA damage on human peripheral blood mononuclear cells.

    Science.gov (United States)

    Fuccelli, Raffaela; Sepporta, Maria Vittoria; Rosignoli, Patrizia; Morozzi, Guido; Servili, Maurizio; Fabiani, Roberto

    2014-01-01

    The aim of this study was to investigate the ability of epoxides of styrene (styrene-7,8-oxide; SO) and 1,3-butadiene (3,4-epoxy-1-butene; 1,2:3,4:-diepoxybutane) to cause oxidative stress and oxidative DNA damage on human peripheral blood mononuclear cells (PBMCs) and whether a complex mixture of olive oil phenols (OOPE) could prevent these effects. The DNA damage was measured by the single-cell gel electrophoresis (SCGE; comet assay). We found that the DNA damage induced by alkene epoxides could be prevented by N-acetyl-cysteine (10 mM) and catalase (100 U/ml). Alkene epoxides caused a significant (P DNA glycosylase (FPG)- and Endonuclease III (ENDO III)-sensitive sites in PBMCs, demonstrating the presence of oxidized bases. OOPE (1 μg of total phenols/ml) was able to prevent the alkene epoxide induced DNA damage both after 2 and 24 h of incubation. In addition, OOPE completely inhibited the SO-induced intracellular peroxide accumulation in PBMCs and prevented the oxidative DNA damage induced by SO, as evidenced by the disappearance of both FPG- and ENDO III-sensitive sites. This is the first study demonstrating the ability of OOPE to prevent the DNA damage induced by alkene epoxides providing additional information about the chemopreventive properties of olive oil.

  9. Peripheral blood mononuclear cells as a potential source of biomarkers to test the efficacy of weight-loss strategies.

    Science.gov (United States)

    Reynés, Bàrbara; Díaz-Rúa, Rubén; Cifre, Margalida; Oliver, Paula; Palou, Andreu

    2015-01-01

    Peripheral blood mononuclear cells (PBMC) constitute an easily obtainable blood cell fraction useful in nutrition and obesity studies. Our aim was to study the potential use of PBMC to reflect metabolic recovery associated with weight loss in rats. By real-time PCR, the fasting response of key energy homeostatic genes in PBMC samples of control and cafeteria-obese rats and of rats fed a control diet after the intake of a cafeteria diet (post-cafeteria model) was analyzed. Fasting caused decreased mRNA expression of lipogenic (Fasn and Srebp1a) and adipogenic (Pparγ) genes in PBMC, whereas it increased the expression of the key beta-oxidation gene Cpt1a and the orexigenic gene Npy. Fasting response of the genes studied was impaired in cafeteria-obese animals but was recovered in post-cafeteria rats, which showed a significant body weight decrease and normalization of adipose and metabolic parameters. Npy expression analyzed in PBMC has been revealed to be especially useful as a marker of fasting sensitivity, as its fasting response is not affected by the age of the animals and it is recovered even after shorter time of exposure to a balanced diet. PBMC reflect homeostatic balance recovery associated with weight loss in obese animals, when reverting from a hyperlipidic to a control balanced diet. © 2014 The Obesity Society.

  10. Intrauterine insemination of cultured peripheral blood mononuclear cells prior to embryo transfer improves clinical outcome for patients with repeated implantation failures.

    Science.gov (United States)

    Madkour, Aicha; Bouamoud, Nouzha; Louanjli, Noureddine; Kaarouch, Ismail; Copin, Henri; Benkhalifa, Moncef; Sefrioui, Omar

    2016-02-01

    Implantation failure is a major limiting factor in assisted reproduction improvement. Dysfunction of embryo-maternal immuno-tolerance pathways may be responsible for repeated implantation failures. This fact is supported by immunotropic theory stipulating that maternal immune cells, essentially uterine CD56+ natural killer cells, are determinants of implantation success. In order to test this hypothesis, we applied endometrium immuno-modulation prior to fresh embryo transfer for patients with repeated implantation failures. Peripheral blood mononuclear cells were isolated from repeated implantation failure patients undergoing assisted reproductive technology cycles. On the day of ovulation induction, cells were isolated and then cultured for 3 days and transferred into the endometrium cavity prior to fresh embryo transfer. This immunotherapy was performed on 27 patients with repeated implantation failures and compared with another 27 patients who served as controls. Implantation and clinical pregnancy were increased significantly in the peripheral blood mononuclear cell test versus control (21.54, 44.44 vs. 8.62, 14.81%). This finding suggests a clear role for endometrium immuno-modulation and the inflammation process in implantation success. Our study showed the feasibility of intrauterine administration of autologous peripheral blood mononuclear cells as an effective therapy to improve clinical outcomes for patients with repeated implantation failures and who are undergoing in vitro fertilization cycles.

  11. In Vitro Infection of Human Peripheral Blood Mononuclear Cells by GB Virus C/Hepatitis G Virus

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    Fogeda, Marta; Navas, Sonia; Martín, Julio; Casqueiro, Mercedes; Rodríguez, Elena; Arocena, Carlos; Carreño, Vicente

    1999-01-01

    GB virus C (GBV-C), also known as hepatitis G virus, is a recently discovered flavivirus-like RNA agent with unclear pathogenic implications. To investigate whether human peripheral blood mononuclear cells (PBMC) are susceptible to in vitro GBV-C infection, we have incubated PBMC from four healthy blood donors with a human GBV-C RNA-positive serum. By means of (i) strand-specific reverse transcription-PCR, cloning, and sequencing; (ii) sucrose ultracentrifugation and RNase sensitivity assays; (iii) fluorescent in situ hybridization; and (iv) Western blot analysis, it has been demonstrated that GBV-C is able to infect in vitro cells and replicate for as long as 30 days under the conditions developed in our cell culture system. The concentration of GBV-C RNA increased during the second and third weeks of culture. The titers of the genomic strand were 10 times higher than the titers of the antigenomic strand. In addition, the same predominant GBV-C sequence was found in all PBMC cultures and in the in vivo-GBV-C-infected PBMC isolated from the donor of the inoculum. GBV-C-specific fluorescent in situ hybridization signals were confined to the cytoplasm of cells at different times during the culture period. Finally, evidence obtained by sucrose ultracentrifugation, RNase sensitivity assays, and Western blot analysis of the culture supernatants suggests that viral particles are released from in vitro-GBV-C-infected PBMC. In conclusion, our study has demonstrated, for the first time, GBV-C replication in human lymphoid cells under experimental in vitro infection conditions. PMID:10196301

  12. Modulation of transglutaminase activity in mononuclear phagocytes and macrophage-like tumor cell lines by differentiation agents

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    Goldman, R.

    1987-01-01

    The effect of glucocorticosteroids, retinoids, 1,25-dihydroxyvitamin D/sub 3/ (1,25(OH)/sub 2/D/sub 3/) and the tumor promoter phorbol myristate acetate (TPA) on the expression of transglutaminase activity in in vitro differentiating bone marrow-derived mouse and rat mononuclear phagocytes (BMDMP) and mouse and human myeloid leukemia cell lines was assessed. Dexamethasone was found to induce an increase of about 100% in transglutaminase activity in mouse and rat BMDMP. The effect was time- and dose-dependent, and specific for steroids with glucocorticoid activity. Retinoic acid (RA) suppressed transglutaminase activity in mouse BMDMP and enhanced it in rat BMDMP. In murine and human myeloid leukemia cell lines, dexamethasone enhanced transglutaminase activity to a varying degree, RA suppressed it in P388D1 cells and enhanced it in the other cell lines. 1,25(OH)/sub 2/D/sub 3/ induced a rather small augmentation of enzyme expression, whereas TPA suppressed enzyme expression (70-100%). The species-specific differences previously observed by the authors for the effect of RA, dexamethasone and 1,25(OH)/sub 2/D/sub 3/ on the formation of BMDMP from mouse and rat bone marrow progenitor cells are now shown to extend also to effects on expression of transglutaminase activity. From a mechanistic point of view it is of interest that dexamethasone uniformly enhanced transglutaminase activity, whereas TPA suppressed it. The data suggest that modulation of transglutaminase activity by the four agents occurs via disparate mechanisms.

  13. Centella asiatica modulates cancer cachexia associated inflammatory cytokines and cell death in leukaemic THP-1 cells and peripheral blood mononuclear cells (PBMC's).

    Science.gov (United States)

    Naidoo, Dhaneshree Bestinee; Chuturgoon, Anil Amichund; Phulukdaree, Alisa; Guruprasad, Kanive Parashiva; Satyamoorthy, Kapaettu; Sewram, Vikash

    2017-08-01

    Cancer cachexia is associated with increased pro-inflammatory cytokine levels. Centella asiatica (C. asiatica) possesses antioxidant, anti-inflammatory and anti-tumour potential. We investigated the modulation of antioxidants, cytokines and cell death by C. asiatica ethanolic leaf extract (CLE) in leukaemic THP-1 cells and normal peripheral blood mononuclear cells (PBMC's). Cytotoxcity of CLE was determined at 24 and 72 h (h). Oxidant scavenging activity of CLE was evaluated using the 2, 2-diphenyl-1 picrylhydrazyl (DPPH) assay. Glutathione (GSH) levels, caspase (-8, -9, -3/7) activities and adenosine triphosphate (ATP) levels (Luminometry) were then assayed. The levels of tumour necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1β and IL-10 were also assessed using enzyme-linked immunosorbant assay. CLE decreased PBMC viability between 33.25-74.55% (24 h: 0.2-0.8 mg/ml CLE and 72 h: 0.4-0.8 mg/ml CLE) and THP-1 viability by 28.404% (72 h: 0.8 mg/ml CLE) (p cells, CLE (0.2-0.8 mg/ml) decreased IL-1β and IL-6 whereas increased IL-10 levels (p cell lines, CLE (0.05-0.2 mg/ml, 24 and 72 h) increased GSH concentrations (p cells (p cell lines (p cells, CLE proved to effectively modulate antioxidant activity, inflammatory cytokines and cell death. In THP-1 cells, CLE decreased pro-inflammatory cytokine levels whereas it increased anti-inflammatory cytokine levels which may alleviate cancer cachexia.

  14. Mononuclear Phagocyte-Derived Microparticulate Caspase-1 Induces Pulmonary Vascular Endothelial Cell Injury.

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    Srabani Mitra

    Full Text Available Lung endothelial cell apoptosis and injury occurs throughout all stages of acute lung injury (ALI/ARDS and impacts disease progression. Lung endothelial injury has traditionally been focused on the role of neutrophil trafficking to lung vascular integrin receptors induced by proinflammatory cytokine expression. Although much is known about the pathogenesis of cell injury and death in ALI/ARDS, gaps remain in our knowledge; as a result of which there is currently no effective pharmacologic therapy. Enzymes known as caspases are essential for completion of the apoptotic program and secretion of pro-inflammatory cytokines. We hypothesized that caspase-1 may serve as a key regulator of human pulmonary microvascular endothelial cell (HPMVEC apoptosis in ALI/ARDS. Our recent experiments confirm that microparticles released from stimulated monocytic cells (THP1 induce lung endothelial cell apoptosis. Microparticles pretreated with the caspase-1 inhibitor, YVAD, or pan-caspase inhibitor, ZVAD, were unable to induce cell death of HPMVEC, suggesting the role of caspase-1 or its substrate in the induction of HPMVEC cell death. Neither un-induced microparticles (control nor direct treatment with LPS induced apoptosis of HPMVEC. Further experiments showed that caspase-1 uptake into HPMVEC and the induction of HPMVEC apoptosis was facilitated by caspase-1 interactions with microparticulate vesicles. Altering vesicle integrity completely abrogated apoptosis of HPMVEC suggesting an encapsulation requirement for target cell uptake of active caspase-1. Taken together, we confirm that microparticle centered caspase-1 can play a regulator role in endothelial cell injury.

  15. IMPROVING P-gp EXPRESSION IN HUMAN MONONUCLEAR CELLS IN VITRO TRANSFECTED BY MULTIDRUG RESISTANCE-1 mRNA

    Institute of Scientific and Technical Information of China (English)

    Yang Xiang; Lei Li; Fang Tian; Xiu-yu Yang

    2005-01-01

    Objective To evaluate the expression and function activity of P-glycoprotein (P-gp) in human mononuclear cells (MNCs) in vitro transfected by multidrug resistance-1 (MDR1) mRNA.Methods Two MDR1 cDNA vectors, pT7TS_MDR1 and pGEM5Zf(+)_MDR1, were constructed and transcripted in vitro. Vector pGEM5Zf(+)_MDR1 only contained the coding region ofmdrl cDNA, and pT7TS_MDR1 also included Xeponus β-globin 5' and 3' untranslated region. MNCs were prepared from peripheral blood of parvicellular lung cancer patient. The two human mdrl mRNAs were then transferred into human MNCs in vitro by DOTAP. And the expression efficiency and pump functon of P-gp were measured with flow cytometry.Results Expression of P-gp significantly elevated in both transferred cells compared with untransferred cells (P< 0.01).And pT7TS_MDR1 showed higher capability in elevating the expression of P-gp than pGEM5Zf(+)_MDR1 (P < 0.01). The P-gp function was elevated in both pT7TS_MDR1 and pGEM5Zf(+)_MDR1 groups. The survival ratio of MNCs in erythrocyte-lysis-solution (ELS, 86.07%) and lymphocyte-isolation-solution (LIS, 83.67%) had no significant difference.The CD34+ cells content of the MNCs used for transfection was 2.65% and 1.01% in ELS and LIS group, respectively (P <0.01).Conclusions It is a feasible approach to improve P-gp expression in human MNCs by transfection of MDR-1 mRNA.And the ELS may be more suitable for purifing MNCs for mRNA transfection than LIS.

  16. Male microchimerism at high levels in peripheral blood mononuclear cells from women with end stage renal disease before kidney transplantation.

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    Laetitia Albano

    Full Text Available Patients with end stage renal diseases (ESRD are generally tested for donor chimerism after kidney transplantation for tolerance mechanism purposes. But, to our knowledge, no data are available on natural and/or iatrogenic microchimerism (Mc, deriving from pregnancy and/or blood transfusion, acquired prior to transplantation. In this context, we tested the prevalence of male Mc using a real time PCR assay for DYS14, a Y-chromosome specific sequence, in peripheral blood mononuclear cells (PBMC from 55 women with ESRD, prior to their first kidney transplantation, and compared them with results from 82 healthy women. Male Mc was also quantified in 5 native kidney biopsies obtained two to four years prior to blood testing and in PBMC from 8 women collected after female kidney transplantation, several years after the initial blood testing. Women with ESRD showed statistically higher frequencies (62% and quantities (98 genome equivalent cells per million of host cells, gEq/M of male Mc in their PBMC than healthy women (16% and 0.3 gEq/M, p<0.00001 and p = 0.0005 respectively. Male Mc was increased in women with ESRD whether they had or not a history of male pregnancy and/or of blood transfusion. Three out of five renal biopsies obtained a few years prior to the blood test also contained Mc, but no correlation could be established between earlier Mc in a kidney and later presence in PBMC. Finally, several years after female kidney transplantation, male Mc was totally cleared from PBMC in all women tested but one. This intriguing and striking initial result of natural and iatrogenic male Mc persistence in peripheral blood from women with ESRD raises several hypotheses for the possible role of these cells in renal diseases. Further studies are needed to elucidate mechanisms of recruitment and persistence of Mc in women with ESRD.

  17. Male microchimerism at high levels in peripheral blood mononuclear cells from women with end stage renal disease before kidney transplantation.

    Science.gov (United States)

    Albano, Laetitia; Rak, Justyna M; Azzouz, Doua F; Cassuto-Viguier, Elisabeth; Gugenheim, Jean; Lambert, Nathalie C

    2012-01-01

    Patients with end stage renal diseases (ESRD) are generally tested for donor chimerism after kidney transplantation for tolerance mechanism purposes. But, to our knowledge, no data are available on natural and/or iatrogenic microchimerism (Mc), deriving from pregnancy and/or blood transfusion, acquired prior to transplantation. In this context, we tested the prevalence of male Mc using a real time PCR assay for DYS14, a Y-chromosome specific sequence, in peripheral blood mononuclear cells (PBMC) from 55 women with ESRD, prior to their first kidney transplantation, and compared them with results from 82 healthy women. Male Mc was also quantified in 5 native kidney biopsies obtained two to four years prior to blood testing and in PBMC from 8 women collected after female kidney transplantation, several years after the initial blood testing. Women with ESRD showed statistically higher frequencies (62%) and quantities (98 genome equivalent cells per million of host cells, gEq/M) of male Mc in their PBMC than healthy women (16% and 0.3 gEq/M, ppregnancy and/or of blood transfusion. Three out of five renal biopsies obtained a few years prior to the blood test also contained Mc, but no correlation could be established between earlier Mc in a kidney and later presence in PBMC. Finally, several years after female kidney transplantation, male Mc was totally cleared from PBMC in all women tested but one. This intriguing and striking initial result of natural and iatrogenic male Mc persistence in peripheral blood from women with ESRD raises several hypotheses for the possible role of these cells in renal diseases. Further studies are needed to elucidate mechanisms of recruitment and persistence of Mc in women with ESRD.

  18. Photoacoustic detection of hemozoin in human mononuclear cells as an early indicator of malaria infection

    Science.gov (United States)

    Custer, Jonathan R.; Kariuki, Michael; Beerntsen, Brenda T.; Viator, John A.

    2010-02-01

    Malaria is a blood borne infection affecting hundreds of millions of people worldwide2. The parasites reproduce within the blood cells, eventually causing their death and lysis. This process releases the parasites into the blood, continuing the cycle of infection. Usually, malaria is diagnosed only after a patient presents symptoms, including high fever, nausea, and, in advanced cases, coma and death. While invading the bloodstream of a host, malaria parasites convert hemoglobin into an insoluble crystal, known as hemozoin. These crystals, approximately several hundred nanometers in size, are contained within red blood cells and white blood cells that ingest free hemozoin in the blood. Thus, infected red blood cells and white blood cells contain a unique optical absorber that can be detected in blood samples using static photoacoustic detection methods. We separated the white blood cells from malaria infected blood and tested it in a photoacoustic set up using a tunable laser system consisting of an optical parametric oscillator pumped by an Nd:YAG laser with pulse duration of 5 ns. Our threshold of detection was 10 infected white blood cells per microliter, which is more sensitive than current diagnosis methods using microscopic analysis of blood.

  19. Procalcitonin neutralizes bacterial LPS and reduces LPS-induced cytokine release in human peripheral blood mononuclear cells

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    Matera Giovanni

    2012-05-01

    Full Text Available Abstract Background Procalcitonin (PCT is a polypeptide with several cationic aminoacids in its chemical structure and it is a well known marker of sepsis. It is now emerging that PCT might exhibit some anti-inflammatory effects. The present study, based on the evaluation of the in vitro interaction between PCT and bacterial lipopolisaccharide (LPS, reports new data supporting the interesting and potentially useful anti-inflammatory activity of PCT. Results PCT significantly decreased (p Salmonella typhimurium (rough chemotype and Escherichia coli (smooth chemotype. Subsequently, the in vitro effects of PCT on LPS-induced cytokine release were studied in human peripheral blood mononuclear cells (PBMC. When LPS was pre-incubated for 30 minutes with different concentrations of PCT, the release of interleukin-10 (IL-10 and tumor necrosis factor alpha (TNFα by PBMC decreased in a concentration-dependent manner after 24 hours for IL-10 and 4 hours for TNFα. The release of monocyte chemotactic protein-1 (MCP-1 exhibited a drastic reduction at 4 hours for all the PCT concentrations assessed, whereas such decrease was concentration-dependent after 24 hours. Conclusions This study provides the first evidence of the capability of PCT to directly neutralize bacterial LPS, thus leading to a reduction of its major inflammatory mediators.

  20. Use of accelerator mass spectrometry to measure the pharmacokinetics and peripheral blood mononuclear cell concentrations of zidovudine.

    Science.gov (United States)

    Vuong, Le T; Ruckle, Jon L; Blood, Arlin B; Reid, Michael J; Wasnich, Richard D; Synal, Hans-Arno; Dueker, Stephen R

    2008-07-01

    The remarkable sensitivity of accelerator mass spectrometry (AMS) is finding many new applications in pharmacology. In this study AMS was used to measure [(14)C]-Zidovudine (ZDV) concentrations at the drug's site of action (peripheral blood mononuclear cells, PBMCs) following a dose of 520 ng (less than one-millionth of the standard daily dose) to a healthy volunteer. In addition, the pharmacokinetics of this microdose were determined and compared to previously published parameters for therapeutic doses. Microdose ZDV pharmacokinetic parameters fell within reported 95% confidence intervals or standard deviations of most previously published values for therapeutic doses. Blood, urine, stool, saliva, and isolated PBMCs were collected periodically through 96 h postdose and analyzed for ZDV and metabolite concentrations. The results showed that ZDV is rapidly absorbed and eliminated, has one major metabolite, and is sequestered in PBMCs. (14)C mass balance assessments indicated a significant portion of ZDV remained after 96 h with a much prolonged elimination half-life. Results of this study demonstrate the usefulness of microdosing and AMS as a tool for studying the pharmacokinetic characteristics, including PBMC concentrations, of ZDV and underscore the value of AMS as a tool with which to perform pharmacokinetic and mass balance studies using trace amounts of radiolabeled compound.

  1. Gene expression changes in peripheral mononuclear cells from schizophrenic patients treated with a combination of antipsychotic with fluvoxamine.

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    Chertkow, Yael; Weinreb, Orly; Youdim, Moussa B H; Silver, Henry

    2007-10-01

    Antipsychotic treatment combined with Selective Serotonin Reuptake Inhibitor (SSRI) antidepressant can improve negative symptoms in schizophrenic patients that are unresponsive to antipsychotic drugs alone. The mechanism of this therapeutic effect is not clear. The current study examined molecular changes induced by the combined treatment in human peripheral mononuclear cells (PMC) in order to get insight into its mechanism of action. Gene expression profile of PMC from antipsychotic-treated patients was examined before addition of the SSRI fluvoxamine, and 3 and 6 weeks after. Gene expression patterns screened with a cDNA array, comprising 1176 genes, revealed homologous changes in a range of transcripts related to G-protein coupled receptors (GPCR). Genes related to GPCR-family were assayed using customized cDNA array and the results verified by real-time RT-PCR. The mRNA expression of chemokine receptors, IL8RA and CCR1, and of RGS7 was significantly down-regulated following fluvoxamine augmentation. The clinical assessments showed improvement in negative symptoms following the combined treatment. The transcriptional analysis suggests that the therapeutic mechanism of the combined antipsychotic-fluvoxamine treatment may involve genes associated with G-protein coupled receptors (GPCR). Our findings suggest that gene expression changes in PMC may be useful in investigating the mechanism of drug action in schizophrenia.

  2. Interleukin-10 inhibits the production of inflammatory cytokines by antigen-stimulated mononuclear cells from asthmatic patients

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    Toshiya Takahashi

    2000-01-01

    Full Text Available Bronchial asthma, characterized by chronic airway inflammation, involves many inflammatory cytokines. Interleukin (IL-10 is a potent inhibitor of cytokine synthesis. Thus, the effects of IL-10 were examined on the production of granulocyte-macrophage colony stimulating factor (GM-CSF, IL-5, IL-1 β, IL-2 and interferon (IFN-γ by antigen (Dermatophagoides farinae, Df- stimulated mononuclear cells obtained from asthmatic patients who were sensitized with the antigen and from healthy subjects in vitro. Production of IL-5 and IL-2 was enhanced by Df antigen in the asthmatic subjects, but not in the healthy controls. In contrast, levels of GM-CSF, IFN-γ and IL-1 β production were enhanced by the antigen in both groups. Exogenous IL-10 (10 ng/mL inhibited the production of GM-CSF, IFN-γ and IL-iβ induced by Df antigen in both groups and also inhibited the production of IL-5 and IL-2 induced by the antigen in the asthmatics subjects. The inhibition of GM-CSF production by IL-10 was stronger than that by IL-4. These results indicated that the responsiveness to the inhibitory effect of IL-10 on the production of inflammatory cytokines is not abrogated in asthmatic patients and that IL-10 may be useful in the treatment of bronchial asthma.

  3. Potential antidepressant and resilience mechanism revealed by metabolomic study on peripheral blood mononuclear cells of stress resilient rats.

    Science.gov (United States)

    Li, Juan; Zhang, Shu-Xiao; Wang, Wei; Cheng, Ke; Guo, Hua; Rao, Cheng-Long; Yang, De-Yu; He, Yong; Zou, De-Zhi; Han, Yu; Zhao, Li-Bo; Li, Peng-Fei; Xie, Peng

    2017-03-01

    Resilience is an active coping response to stress, which plays a very important role in major depressive disorder study. The molecular mechanisms underlying such resilience are poorly understood. Peripheral blood mononuclear cells (PBMCs) were promising objects in unveiling the underlying pathogenesis of resilience. Hereby we carried out successive study on PBMCs metabolomics in resilient rats of chronic unpredictable mild stress (CUMS) model. A gas chromatography-mass spectrometry (GC-MS) metabolomic approach coupled with principal component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA) was used to detect differential metabolites in PBMCs of resilient rats. Ingenuity Pathways Analysis (IPA) was applied for pathway analysis. A set of differential metabolites including Malic acid, Ornithine, l-Lysine, Stigmasterol, Oleic acid, γ-Tocopherol, Adenosine and N-acetyl-d-glucosamine were significantly altered in resilient rats, meanwhile promoting antidepressant research. As revealed by IPA that aberrant energy metabolism, HIFα signaling, neurotransmitter, O-GlcNAcylation and cAMP signaling cascade in peripheral might be evolved in the pathogenesis of coping mechanism. The GC-MS based metabolomics may contribute to better understanding of resilience, as well as shedding light on antidepressant discovery. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Quantification of HIV-1 proviral DNA from peripheral blood mononuclear cells using a high throughput four-competitor competitive PCR.

    Science.gov (United States)

    Comandini, U V; Sönnerborg, A; Vahlne, A; Yun, Z

    1997-12-01

    A multiple competitor PCR (mcPCR) was developed to quantify HIV-1 proviral DNA from peripheral blood mononuclear cells (PBMC). DNA extracted from a mixture of HIV infected PBMC and four size-mutated DNA competitors were co-amplified. The Cy5-fluorescence labelled PCR products were denatured by heating, separated using an automated DNA sequencer and quantified by a fragment analysis computer software. An internal standard was generated by plotting the peak areas of the four competitors against their inputs. Based on the internal standard, HIV sample DNA was quantified by extrapolating the corresponding signal. The linear range of the mcPCR was three log wide and the quantitation limit was about 20 copies of HIV DNA/10(6) PBMC. Using the mcPCR, HIV DNA was quantified from 14 long-term non progressors (LTNP) and 14 patients with advanced disease. A significantly lower copy number of HIV DNA was obtained in the LTNP (p = 0.018). These data suggest that the mcPCR is sensitive, reliable and especially useful for HIV DNA quantification of a large number of clinical samples.

  5. Medroxyprogesterone acetate alters Mycobacterium bovis BCG-induced cytokine production in peripheral blood mononuclear cells of contraceptive users.

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    Léanie Kleynhans

    Full Text Available Most individuals latently infected with Mycobacterium tuberculosis (M.tb contain the infection by a balance of effector and regulatory immune responses. This balance can be influenced by steroid hormones such as glucocorticoids. The widely used contraceptive medroxyprogesterone acetate (MPA possesses glucocorticoid activity. We investigated the effect of this hormone on immune responses to BCG in household contacts of active TB patients. Multiplex bead array analysis revealed that MPA demonstrated both glucocorticoid and progestogenic properties at saturating and pharmacological concentrations in peripheral blood mononuclear cells (PBMCs and suppressed antigen specific cytokine production. Furthermore we showed that PBMCs from women using MPA produced significantly lower levels of IL-1α, IL-12p40, IL-10, IL-13 and G-CSF in response to BCG which corresponded with lower numbers of circulating monocytes observed in these women. Our research study is the first to show that MPA impacts on infections outside the genital tract due to a systemic effect on immune function. Therefore MPA use could alter susceptibility to TB, TB disease severity as well as change the efficacy of new BCG-based vaccines, especially prime-boost vaccine strategies which may be administered to adult or adolescent women in the future.

  6. Porcine blood mononuclear cell cytokine responses to PAMP molecules: comparison of mRNA and protein production

    DEFF Research Database (Denmark)

    Sørensen, Nanna Skall; Skovgaard, Kerstin; Heegaard, Peter M. H.

    2011-01-01

    -α and IL-12 p40, and PGN, LPS and Pam3Cys inducing varying amounts of IL-12 p40, IL-1β, TNF-α, IL-6 and IL-10. Surprisingly, the ssRNA-mimic poly-U induced IL-6 and IL-1β only. Using CpG, PGN and LPS, the kinetics of cytokine production measured as mRNA (reverse transcription (RT)-qPCR) and protein (ELISA...... the induction of IFN-α, IL-12 p40, IL-1β, TNF-α, IL-6 and IL-10 by PAMP-molecules [CpG oligonucleotide D19 (CpG), peptidoglycan (PGN), lipopolysaccharide (LPS), Pam3Cys and poly-U] in porcine blood mononuclear cells (BMC) within a 24h period. As expected, cytokine responses were PAMP-specific, CpG inducing IFN......), respectively, correlated well, mRNA responses preceding protein responses. With the exception of IL-1β and IL-6, mRNA-responses were transient, whereas protein responses, except for TNF-α, followed saturation kinetics. Remarkably, LPS-induced TNF-α mRNA was not followed by a protein response. These results...

  7. Mechanism and role of MCP-1 upregulation upon chikungunya virus infection in human peripheral blood mononuclear cells

    Science.gov (United States)

    Ruiz Silva, Mariana; van der Ende-Metselaar, Heidi; Mulder, H. Lie; Smit, Jolanda M.; Rodenhuis-Zybert, Izabela A.

    2016-01-01

    Monocyte chemoattractant protein-1 (MCP-1/CCL2)-mediated migration of monocytes is essential for immunological surveillance of tissues. During chikungunya virus (CHIKV) infection however, excessive production of MCP-1 has been linked to disease pathogenesis. High MCP-1 serum levels are detected during the viremic phase of CHIKV infection and correlate with the virus titre. In vitro CHIKV infection was also shown to stimulate MCP-1 production in whole blood; yet the role and the mechanism of MCP-1 production upon infection of human peripheral blood mononuclear cells remain unknown. Here we found that active CHIKV infection stimulated production of MCP-1 in monocytes. Importantly however, we found that communication with other leukocytes is crucial to yield MCP-1 by monocytes upon CHIKV infection. Indeed, blocking interferon-α/β receptor or the JAK1/JAK2 signalling downstream of the receptor abolished CHIKV-mediated MCP-1 production. Additionally, we show that despite the apparent correlation between IFN type I, CHIKV replication and MCP-1, modulating the levels of the chemokine did not influence CHIKV infection. In summary, our data disclose the complexity of MCP-1 regulation upon CHIKV infection and point to a crucial role of IFNβ in the chemokine secretion. We propose that balance between these soluble factors is imperative for an appropriate host response to CHIKV infection. PMID:27558873

  8. Adenosine A2A Receptor and IL-10 in Peripheral Blood Mononuclear Cells of Patients with Mild Cognitive Impairment

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    Beatrice Arosio

    2011-01-01

    Full Text Available Adenosine suppresses immune responses through the A2A receptor (A2AR. This study investigated the interleukin 10 (IL-10 genetic profile and the expression of A2AR in peripheral blood mononuclear cells (PBMCs of patients with mild cognitive impairment (MCI, Alzheimer disease (AD, and age-matched controls to verify, if they may help distinguish different forms of cognitive decline. We analyzed the IL-10 genotype and the expression of A2AR in 41 subjects with AD, 10 with amnestic MCI (a-MCI, 49 with multiple cognitive domain MCI (mcd-MCI, and 46 controls. There was a significant linear increase in A2AR mRNA levels and A2AR density from mcd-MCI to a-MCI, with intermediate levels being found in AD. The IL-10 AA genotype frequency was 67% in a-MCI, 46% in AD, 35% in mcd-MCI, and 20% in controls. These data suggest that the assessment of the IL-10 genotype and the expression of A2AR in PBMCs may be a valuable means of differentiating between a-MCI and mcd-MCI.

  9. Differential expression analysis of miRNA in peripheral blood mononuclear cells of patients with non-segmental vitiligo.

    Science.gov (United States)

    Wang, Yi; Wang, Keyu; Liang, Jianhua; Yang, Hong; Dang, Ningning; Yang, Xi; Kong, Yi

    2015-02-01

    Vitiligo is a common depigmentary skin disease that may follow a pattern of multifactorial inheritance. The essential factors of its immunopathogenesis is thought to be the selective destruction of melanocytes. As a new class of microregulators of gene expression, miRNA have been reported to play vital roles in autoimmune diseases, metabolic diseases and cancer. This study sought to characterize the different miRNA expression pattern in the peripheral blood mononuclear cells (PBMC) of patients with non-segmental vitiligo (NSV) and healthy individuals and to examine their direct responses to thymosin α1 (Tα1) treatment. The miRNA expression profile in the PBMC of patients with NSV was analyzed using Exiqon's miRCURY LNA microRNA Array. The differentially expressed miRNA were validated by real-time quantitative polymerase chain reaction. We found that the expression levels of miR-224-3p and miR-4712-3p were upregulated, and miR-3940-5p was downregulated in the PBMC. The common clinical immune modulator Tα1 changed the miRNA expression profile. Our analysis showed that differentially expressed miRNA were associated with the mechanism of immune imbalance of vitiligo and that Tα1 could play an important role in changing the expression of these miRNA in the PBMC of patients with NSV. This study provided further evidence that miRNA may serve as novel drug targets for vitiligo therapeutic evaluation.

  10. Interleukin-6 infusion during human endotoxaemia inhibits in vitro release of the urokinase receptor from peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Ostrowski, S R; Plomgaard, P; Fischer, C P

    2005-01-01

    Leucocyte expression of the urokinase receptor [urokinase-type plasminogen activator receptor (uPAR)] is regulated by inflammatory mediators. This study investigated the in vivo effect of endotoxin, interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha on uPAR-release in vivo and in vitro...... in humans. Healthy subjects received intravenous endotoxin injection [high-dose, 2 ng/kg (n=8) and low-dose, 0.06 ng/kg (n=7)], coadministration of 0.06 ng/kg endotoxin and 3 h recombinant human (rh)IL-6 infusion (n=7) or 3 h infusion of rhIL-6 (n=6), rhTNF-alpha (n=6) or NaCl (n=5). Soluble uPAR (su......PAR) was measured by enzyme-linked immunosorbent assay in plasma and supernatants from unstimulated and phytohaemagglutinin and lipopolysaccharide-stimulated peripheral blood mononuclear cell (PBMC) cultures incubated for 24 h. The spontaneous and stimulated uPAR-release from PBMC cultures was enhanced 5 h after...

  11. Gene expression profiling in human peripheral blood mononuclear cells using high-density filter-based cDNA microarrays.

    Science.gov (United States)

    Walker, J; Rigley, K

    2000-05-26

    Microarray technology has provided the ability to analyse the expression profiles for thousands of genes in parallel. The need for highly specialised equipment to use certain types of microarrays has restricted the application of this technology to a small number of dedicated laboratories. High-density filter-based cDNA microarrays provide a low-cost option for performing high-throughput gene expression analysis. We have used a model system in which filter-based cDNA microarrays representing over 4000 known human genes were used to monitor the kinetics of gene expression in human peripheral blood mononuclear cells (PBMCs) stimulated with phytohaemagluttinin (PHA). Using software-based cluster analysis, we identified 104 genes that altered in expression levels in response to PHA stimulation of PBMCs and showed that there was a considerable overlap between genes with similar temporal expression profiles and similar functional roles. Comparison of microarray quantitation with quantitative PCR showed almost identical expression profiles for a number of genes. Coupled with the fact that our findings are in agreement with a large number of independent observations, we conclude that the use of filter-based cDNA microarrays is a valid and accurate method for high-throughput gene expression profiling.

  12. The Markers of Glutamate Metabolism in Peripheral Blood Mononuclear Cells and Neurological Complications in Lung Cancer Patients

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    Slawomir Michalak

    2016-01-01

    Full Text Available Objective. To evaluate the involvement of glutamate metabolism in peripheral blood mononuclear cells (PBMC in the development of neurological complications in lung cancer and during chemotherapy. Methods. The prospective study included 221 lung cancer patients treated with chemotherapeutics. Neurological status and cognitive functions were evaluated at baseline and after 6-month follow-up. Glutamate level, the activities of glutaminase- (GLS- glutamate synthetizing enzyme, glutamate dehydrogenase (GDH, and glutamate decarboxylase catalyzing glutamate degradation were analyzed in PBMC and in sera of lung cancer patients by means of spectrophotometric and colorimetric methods. Results. Chemotherapy of lung neoplasms induced increase of glutamate content in PBMC and its concentration in serum increased the activity of GDH in PBMC and decreased activity of glutaminase in PBMC. The changes in glutamate metabolism markers were associated with initial manifestation of neurological deficit in lung cancer patients and with new symptoms, which appear as a complication of chemotherapy. Moreover, the analyzed parameters of glutamate control correlated with a spectrum of cognitive functions measures in lung cancer patients. Conclusion. We have demonstrated dysregulation in glutamate and glutamate metabolism controlling enzymes as promising indicators of risk for chemotherapy-induced neurological complications in lung cancer patients with particular emphasis on cognitive impairment.

  13. A clinical study of the effectiveness of oral glutamine supplementation during total parenteral nutrition: influence on mesenteric mononuclear cells.

    Science.gov (United States)

    Aosasa, S; Mochizuki, H; Yamamoto, T; Ono, S; Ichikura, T

    1999-01-01

    Bacterial translocation (BT) is a well-known insult during total parenteral nutrition (TPN) and a high incidence of morbidity has been reported in septic patients receiving TPN. Inflammatory cytokines were shown to play an important role in the pathogenesis of critical complications following sepsis. Previous studies have indicated that supplementation of TPN with glutamine is effective in preventing BT in animals, but its effectiveness in humans is unclear. The aim of this study was to determine the effectiveness of oral glutamine supplementation to patients receiving TPN in suppressing cytokine production of mesenteric blood mononuclear cells (M-MNC). Fifteen colorectal cancer patients were divided into 3 groups according to preoperative nutrition management. (1) TPN group: TPN with conventional glutamine-free amino acid solution. (2) Gln group: TPN with oral glutamine supplementation of 30 g/d. (3) CONTROL GROUP: oral intake of normal food. M-MNC were obtained immediately after laparotomy and tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) production of M-MNC was evaluated with or without lipopolysaccharide (LPS) stimulation. TNF-alpha and IL-10 production by LPS-stimulated M-MNC was increased in the TPN group and suppressed in the Gln group. In conclusion, oral glutamine supplementation to patients with TPN was shown to be effective for the prevention of M-MNC activation to avoid excessive production of cytokines.

  14. Hepatitis G Viral RNA Co-infection in Plasma and Peripheral Blood Mononuclear Cells in Patients with Hepatitis C

    Institute of Scientific and Technical Information of China (English)

    LI Shuli; ZENG Linglan; LUO Duande; LIU Wei; GUO Jingsong; YANG Xiaoming

    2001-01-01

    The incidence of the co-infection of hepatitis G virus (HGV) and hepatitis C virus(HCV) and its clinical implication was investigated and the difference in the positive rate of HGV RNA and HCV RNA between plasma and peripheral blood mononuclear cells (PBMCs) observed. By using reverse transcriptase polymerase chain reaction (RT-PCR) assay, HCV-RNA and HGV-RNA in plasma and PBMCs of 72 patients with hepatitis C was detected. It was showed that HGV RNA was positive in plasma of 11 patients, in PBMCs of 15 patients, and simultaneously in both of plasma and PBMCs of 10 patients with the co-infection rate being 22.2 %. Nine patients were both HGV RNA and HCV RNA positive in plasma, 11 patients were both HGV RNA and HCV RNA positive in PBMC, and 6 patients were both HGV RNA and HCV RNA positive in both plasma and PBMC with the positive rate being 12.4 %, 15.3 % and 8.3 % respectively. The positive rate of both HGV RNA and HCV RNA in PBMCs was higher than in plasma. It was concluded that the HGV co-infection rate in the patients with hepatitis C was 22. 2 %. Simultaneous examination of plasma and PBMC can improve clinically detectable rate.

  15. Peripheral blood mononuclear cell traffic plays a crucial role in mother-to-infant transmission of hepatitis B virus.

    Science.gov (United States)

    Xu, Yuan-Yong; Liu, Hui-Hui; Zhong, Yan-Wei; Liu, Chang; Wang, Yong; Jia, Lei-Li; Qiao, Fei; Li, Xin-Xin; Zhang, Chuan-Fu; Li, Shen-Long; Li, Peng; Song, Hong-Bin; Li, Qiao

    2015-01-01

    The role of peripheral blood mononuclear cells (PBMCs) in HBV intrauterine infection is not fully defined. Particularly the origin of PBMCs in HBV-infected neonates remains to be addressed. We carried out a population-based nested case-control study by enrolling 312 HBsAg-positive mothers and their babies. PBMC HBV DNA as well as serum HBsAg and HBV DNA was tested in cohort entry samples. Totally, 45.5% (142/312) of the newborns were found to be infected with HBV in perinatal transmission. 119 mother-infant pairs were identified to be different in the genetic profile of maternal and fetal PBMCs by AS-PCR and hemi-nested PCR. Among them, 57.1% (68/119) of the maternal PBMCs in index cases were positive for HBV DNA while 83.8% (57/68) of the HBV DNA positive maternal PBMCs passed the placental barrier and entered the fetus. Furthermore, maternal PBMC HBV infection was significantly associated with newborn infants HBV infection. PBMC traffic from mother to fetus resulted in a 9.5-fold increased risk of HBV infection in PBMC HBV DNA positive newborn infants. These data indicate that maternal PBMCs infected with HBV contribute to HBV intrauterine infection of newborn infants via PBMC traffic from mother to fetus.

  16. Complex Effects of Vitamin E and Vitamin C Supplementation on in Vitro Neonatal Mononuclear Cell Responses to Allergens

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    Robert N. Barker

    2013-08-01

    Full Text Available Low maternal dietary vitamin E (but not vitamin C intake during pregnancy has been associated with increased in vitro cord blood mononuclear cell (CBMC proliferative responses, childhood wheezing and asthma. We investigated whether these associations reflect direct effects of vitamin E by investigating the effects of supplementing CBMC cultures with physiological concentrations of vitamin E. CBMC from seventy neonates were cultured supplemented with either nothing, α-tocopherol or ascorbic acid. Proliferative, IFN-γ, IL-4, IL-10 and TGF-β responses were measured. In general, vitamin E supplementation was associated with a trend for reduced proliferative responses after stimulation with antigens and house dust mite, and with increased proliferation after stimulation with timothy grass allergen. There was a trend for CBMC cultures to exhibit decreased secretion of IFN-γ, IL-10 and IL-4. Supplementation with vitamin C had no effect on CBMC proliferation, but increased IFN-γ and IL-4 production, and decreased IL-10 production. In conclusion, in vitro vitamin E and C supplementation of CBMC modifies neonatal immune function, but not in a manner predicted by observational epidemiological studies. The observed associations between vitamin E and childhood respiratory disease are complex, and the nature and form of nutritional intervention need to be carefully considered before inclusion in trials.

  17. Relationship between leptin concentration and body fat with peripheral blood mononuclear cells cytokines among obese and overweight adults.

    Science.gov (United States)

    Zarrati, M; Salehi, E; Razmpoosh, E; Shoormasti, R S; Hosseinzadeh-Attar, M J; Shidfar, F

    2017-02-01

    Overweight and obesity has been suggested to be well correlated with altered levels of pro-inflammatory cytokines. The purpose of this study is to assess the relationship of body fat mass (BFM), body fat percentage (BFP) and leptin levels with peripheral blood mononuclear cells (PBMCs) cytokines among obese and overweight adults. Eighty-two overweight and obese individuals were divided into two BMI-category groups (BMI leptin and anthropometric indices were measured and PBMCs were cultured. Mean of leptin concentrations were 23.14 ± 4.07 and 28.25 ± 4.35 pg/ml among individuals with BMI leptin (P leptin (P Leptin had a positive significant correlation with IFN-γ (p = 0.03, r = 0.251). Regarding these results, we proved that BFP, BFM and leptin levels have significant correlations with some PBMC cytokines. Focusing on such strategies may lead to promises for alleviating obesity and its co-morbidities.

  18. Diminished production of TWEAK by the peripheral blood mononuclear cells is associated with vascular involvement in patients with systemic sclerosis.

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    Otylia Kowal-Bielecka

    2010-02-01

    Full Text Available Widespread vasculopathy and profound fibrosis are key features of the pathogenesis of systemic sclerosis (SSc. We hypothesized that the TNF-like weak inducer of apoptosis (TWEAK, a recently recognized multifunctional cytokine which regulates angiogenesis and tissue remodeling, may play a role in the development of SSc. The production of TWEAK by the peripheral blood mononuclear cells (PBMC was investigated, by means of ELISA, in 24 SSc patients and 14 healthy subjects. Moreover, production of TWEAK was correlated with clinical features of SSc. PBMC were isolated using density gradient centrifugation on Histopaque and were cultured in FCS supplemented RPMI medium at 37 degrees C under 5% CO2. Production of TWEAK by PBMC was significantly diminished in patients with more severe microvascular damage, as indicated by the presence of "active" capillaroscopic pattern, compared with SSc patients with less pronounced microangiopathy ("slow" pattern, and healthy subjects. Moreover production of TWEAK correlated inversely with duration of Raynaud's phenomenon. PBMC from patients with scleroderma-related interstitial lung disease tended to produce lower amounts of TWEAK compared with SSc patients without lung involvement but the difference was not significant. The results of our study suggest that diminished production of TWEAK might play a role in the pathogenesis of vascular injury in SSc patients. Whether TWEAK may represent a new therapeutic target in SSc requires further studies.

  19. Peripheral blood mononuclear cells from field cattle immune to bovine viral diarrhea virus (BVDV) are permissive in vitro to BVDV.

    Science.gov (United States)

    Gupta, V; Mishra, N; Pateriya, A; Behera, S P; Rajukumar, K

    2014-01-01

    The aim of this study was to determine the in vitro permissivity of peripheral blood mononuclear cells (PBMCs) from bovine viral diarrhea virus (BVDV)-immune field cattle to homologous and heterologous BVDVs. PBMCs from seventeen BVDV-naïve and sixteen BVDV-immune animals were infected with noncytopathic BVDV-1 or BVDV-2. The immune status of cattle was indicated by the presence of virus neutralizing antibodies, while viral load of PBMCs was determined by real-time RT-PCR. The results revealed that the PBMCs from naïve or immune animals were permissive to either BVDV-1 or BVDV-2, but the viral load was significantly higher for the naïve than for the immune animals. Furthermore, the load of homologous virus in PBMCs from immune animals was lower than that of heterologous virus. Our results provide evidence that the PBMCs from BVDV-immune cattle in field are susceptible to reinfection with homologous or heterologous BVDV, albeit to a lower extent in the former case.

  20. Delineation of canine parvovirus T cell epitopes with peripheral blood mononuclear cells and T cell clones from immunized dogs.

    NARCIS (Netherlands)

    G.F. Rimmelzwaan (Guus); M.C.M. Poelen (Martien); R.H. Meloen; J. Carlson; F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert)

    1990-01-01

    textabstractThree synthetic peptides derived from the amino acid sequence of VP2 of canine parvovirus (CPV) which were recently shown to represent three distinct T cell epitopes for BALB/c mice could prime BALB/c mice for a CPV-specific proliferative T cell response upon immunization. Proliferative

  1. Profile of serum alkaline phosphatase after inoculation of mononuclear cells and bone morphogenetic protein in the repair of osteochondral defects in rabbits

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    Luiz Augusto de Souza

    2011-12-01

    Full Text Available In this study, serum alkaline phosphatase activity was measured in response to the repair of osteochondral defects in twenty-four New Zealand rabbits. The animals were divided into three groups: a control (GC, those treated with bone marrow mononuclear cells (GCM and those that received mononuclear cells with autologous bone morphogenetic protein (BMP + GCM. After exposing the trochlear groove of the left stifle joint, a wedge-shaped segment was removed. Later, the defect was filled with an osteochondral autograft preserved in 98% glycerin. For the GC group, only the bone graft was performed. For the GCM, in addition to the graft, 2x106 seed mononuclear cells were implanted. For the GCM + BMP, the same number of cells, associated with 1μg of bone morphogenetic protein, were intraarticularly administered. The osteoblastic response was measured by analyzing the serum alkaline phosphatase on day 0 (preoperative 3, 15, 30, and 45 after surgery, and by radiographic examinations. Analysis of variance in randomized blocks, factorial and Tukey’s test (p = 0.05 were made. The overall mean GCM was superior to the other groups and the highest rates were among the 15th and 45th days postoperatively. The discrepancy in values between individuals of the same group casts doubts on the veracity of the test.

  2. Co-stimulation of cultured peripheral blood mononuclear cells from intrinsic asthmatics with exogenous recombinant IL-6 produce high levels of IL-4-dependent IgE.

    Science.gov (United States)

    Sánchez-Guerrero, I M; Herrero, N; Muro, M; Vegara, R P; Campos, M; García-Alonso, A M; Alvarez, M R

    1997-09-01

    Asthma is an inflammatory airway disorder, traditionally subdivided into extrinsic, immunoglobulin E (IgE)-mediated, and intrinsic asthma of unknown aetiology. IgE synthesis requires contact between T- and B-cells and a signal provided by interleukin (IL)-4, which can be modulated by IL-6. The objective of this study was to evaluate the effects of IL-4 and IL-6 on total IgE synthesis by peripheral blood mononuclear cells from intrinsic and extrinsic asthmatics. Peripheral blood mononuclear cells from intrinsic and extrinsic asthmatic patients and from healthy subjects were cultured and stimulated with pokeweed mitogen, recombinant IL-4 and IL-6. The IgE level in serum and supernatants was measured by an enzyme-linked immunoassay. Serum IgE was significantly lower in intrinsic asthma than in extrinsic asthma, but significantly higher than in control subjects. IgE production by cultured mononuclear cells from extrinsic asthmatics was not modified after exogenous IL-4 and IL-6 addition. However, intrinsic asthmatics showed enhancement of IgE synthesis in response to IL-4 stimulation, reaching a threefold increase of the spontaneous IgE values, when simultaneous recombinant IL-4 plus IL-6 stimulus was used. Our results indicate that exogenous recombinant interleukin-6 can significantly upregulate the interleukin-4-dependent immunoglobulin E synthesis in intrinsic asthma. This suggests that immunoglobulin E could also play a role in the pathogenesis of intrinsic asthma, in which an interleukin-6 threshold would be critical.

  3. Autologous bone marrow mononuclear cell transplantation in patients with decompensated alcoholic liver disease: a randomized controlled trial.

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    Laurent Spahr

    Full Text Available OBJECTIVE: Impaired liver regeneration is associated with a poor outcome in patients with decompensated alcoholic liver disease (ALD. We assessed whether autologous bone marrow mononuclear cell transplantation (BMMCT improved liver function in decompensated ALD. DESIGN: 58 patients (mean age 54 yrs; mean MELD score 19, all with cirrhosis, 81% with alcoholic steatohepatitis at baseline liver biopsy were randomized early after hospital admission to standard medical therapy (SMT alone (n = 30, including steroids in patients with a Maddrey's score ≥32, or combined with G-CSF injections and autologous BMMCT into the hepatic artery (n = 28. Bone marrow cells were harvested, isolated and reinfused the same day. The primary endpoint was a ≥3 points decrease in the MELD score at 3 months, corresponding to a clinically relevant improvement in liver function. Liver biopsy was repeated at week 4 to assess changes in Ki67+/CK7+ hepatic progenitor cells (HPC compartment. RESULTS: Both study groups were comparable at baseline. After 3 months, 2 and 4 patients died in the BMMCT and SMT groups, respectively. Adverse events were equally distributed between groups. Moderate alcohol relapse occurred in 31% of patients. The MELD score improved in parallel in both groups during follow-up with 18 patients (64% from the BMMCT group and 18 patients (53% from the SMT group reaching the primary endpoint (p = 0.43 (OR 1.6, CI 0.49-5.4 in an intention to treat analysis. Comparing liver biopsy at 4 weeks to baseline, steatosis improved (p<0.001, and proliferating HPC tended to decrease in both groups (-35 and -33%, respectively. CONCLUSION: Autologous BMMCT, compared to SMT is a safe procedure but did not result in an expanded HPC compartment or improved liver function. These data suggest either insufficient regenerative stimulation after BMMCT or resistance to liver regenerative drive in patients with decompensated alcoholic cirrhosis. TRIAL REGISTRATION

  4. Clinical Symptoms in Fibromyalgia Are Better Associated to Lipid Peroxidation Levels in Blood Mononuclear Cells Rather than in Plasma

    Science.gov (United States)

    Cano-García, Francisco J.; De Miguel, Manuel; Carrión, Angel M.; Navas, Plácido; Sánchez Alcázar, José A.

    2011-01-01

    Background We examined lipid peroxidation (LPO) in blood mononuclear cells (BMCs) and plasma, as a marker of oxidative damage, and its association to clinical symptoms in Fibromyalgia (FM) patients. Methods We conducted a case–control and correlational study comparing 65 patients and 45 healthy controls. Clinical parameters were evaluated using the Fibromyalgia Impact Questionnaire (FIQ), visual analogues scales (VAS), and the Beck Depression Inventory (BDI). Oxidative stress was determined by measuring LPO in BMCs and plasma. Results We found increased LPO levels in BMCs and plasma from FM patients as compared to normal control (P<0.001). A significant correlation between LPO in BMCs and clinical parameters was observed (r = 0.584, P<0.001 for VAS; r = 0.823, P<0.001 for FIQ total score; and r = 0.875, P<0.01 for depression in the BDI). We also found a positive correlation between LPO in plasma and clinical symptoms (r = 0.452, P<0.001 for VAS; r = 0.578, P<0.001 for FIQ total score; and r = 0.579, P<0.001 for depression in the BDI). Partial correlation analysis controlling for age and BMI, and sex, showed that both LPO in cells and plasma were independently associated to clinical symptoms. However, LPO in cells, but not LPO in plasma, was independently associated to clinical symptoms when controlling for depression (BDI scores). Discussion The results of this study suggest a role for oxidative stress in the pathophysiology of fibromyalgia and that LPO in BMCs rather than LPO in plasma is better associated to clinical symptoms in FM. PMID:22046409

  5. Mycolactone diffuses from Mycobacterium ulcerans-infected tissues and targets mononuclear cells in peripheral blood and lymphoid organs.

    Directory of Open Access Journals (Sweden)

    Hui Hong

    Full Text Available BACKGROUND: Buruli ulcer (BU is a progressive disease of subcutaneous tissues caused by Mycobacterium ulcerans. The pathology of BU lesions is associated with the local production of a diffusible substance, mycolactone, with cytocidal and immunosuppressive properties. The defective inflammatory responses in BU lesions reflect these biological properties of the toxin. However, whether mycolactone diffuses from infected tissues and suppresses IFN-gamma responses in BU patients remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Here we have investigated the pharmacodistribution of mycolactone following injection in animal models by tracing a radiolabeled form of the toxin, and by directly quantifying mycolactone in lipid extracts from internal organs and cell subpopulations. We show that subcutaneously delivered mycolactone diffused into mouse peripheral blood and accumulated in internal organs with a particular tropism for the spleen. When mice were infected subcutaneously with M. ulcerans, this led to a comparable pattern of distribution of mycolactone. No evidence that mycolactone circulated in blood serum during infection could be demonstrated. However, structurally intact toxin was identified in the mononuclear cells of blood, lymph nodes and spleen several weeks before ulcerative lesions appear. Importantly, diffusion of mycolactone into the blood of M. ulcerans-infected mice coincided with alterations in the functions of circulating lymphocytes. CONCLUSION: In addition to providing the first evidence that mycolactone diffuses beyond the site of M. ulcerans infection, our results support the hypothesis that the toxin exerts immunosuppressive effects at the systemic level. Furthermore, they suggest that assays based on mycolactone detection in circulating blood cells may be considered for diagnostic tests of early disease.

  6. Biological quality control for extracorporeal photochemotherapy: Assessing mononuclear cell apoptosis levels in ECP bags of chronic GvHD patients.

    Science.gov (United States)

    Taverna, Francesca; Coluccia, Paola; Arienti, Flavio; Birolini, Annalisa; Terranova, Laura; Mazzocchi, Arabella; Rini, Francesca; Mariani, Luigi; Melani, Cecilia; Ravagnani, Fernando

    2015-06-01

    Extracorporeal photochemotherapy (ECP) is a treatment approved by the FDA for cutaneous T-cell lymphoma, and it is currently used off-label for graft-versus-host disease (GvHD) and other conditions. In agreement with good practices for the therapeutic use of human cells, quality control has to be performed to validate the ECP procedure with the off-line technique. Since no gold-standard biological test is available, we assessed the apoptosis generated in the ECP bag using a flow cytometric analysis. Thirty-one ECP procedures performed on 13 patients with chronic GvHD were studied by monitoring the induction of mononuclear cell (MNC) apoptosis using annexin V/propidium iodide double staining; residual lymphocyte proliferation to standard mitogens was also measured in 17 of the procedures. The kinetics of apoptosis was analyzed at different times in MNCs untreated or treated with 8-methoxy-psoralen plus ultraviolet A; the variation (ΔAPOPTOSIS ) after 24 h revealed the efficacy of the treatment. In 88.6% of the 31 ECP procedures, ΔAPOPTOSIS was >15% (the "alerting" threshold for ΔAPOPTOSIS was set at 15% on the basis of our data); in the remainder (19.4%), the increment in apoptosis was lower. In four procedures, the proliferation assay was useful for assessing the effect of ECP on the apheretic bag. In conclusion, both flow cytometric assays enabled a biologically significant result to be obtained. In our opinion, the apoptosis test-being faster and easier than the proliferation test-could be a reliable way to validate ECP procedures. © 2014 Wiley Periodicals, Inc.

  7. DDE and PCB 153 independently induce aryl hydrocarbon receptor (AhR) expression in peripheral blood mononuclear cells.

    Science.gov (United States)

    Gaspar-Ramírez, Octavio; Pérez-Vázquez, Francisco J; Salgado-Bustamante, Mariana; González-Amaro, Roberto; Hernandez-Castro, Berenice; Pérez-Maldonado, Ivan N

    2015-01-01

    Recent studies have demonstrated that compounds inducing pro-inflammatory cytokines enhance AhR expression. The aim of this study was 2-fold: (1) to determine if two pro-inflammatory compounds, dichlorodiphenyldichloroethylene (DDE) and 2,2',4,4',5,5'-hexa-chlorobiphenyl (PCB 153), independently affect AhR gene expression in peripheral blood mononuclear cells (PBMC); and (2) if affected, to determine whether the mechanism involved was due to AhR activation or to a pro-inflammatory effect of the chemicals. PBMC isolated from healthy individuals were incubated in the presence of DDE (10 µg/ml) and PCB 153 (20 ng/ml) over time and AhR and CYP1A1 expression was assessed with a real-time PCR technique. The results indicated there was over-expression of the AhR mRNA in PBMC when the cells were treated with DDE and PCB 153. No changes in expression levels of CYP1A1 mRNA were found. Importantly, when the cells were exposed to DDE and PCB 153 in the presence of an antagonist of tumor necrosis factor (TNF)-α, the over-expression of AhR was abolished; as expected, the expression of CYP1A1 was unaffected. In conclusion, these studies demonstrated for the first time an increment of AhR expression "in vitro" in PBMC treated with two pro-inflammatory environmental pollutants, DDE and PCB153. Moreover, the over-expression of AhR was dependent of TNFα induced by DDE and PCB 153 and was independent of AhR activation.

  8. Application of the comet assay for investigation of oxidative DNA damage in equine peripheral blood mononuclear cells.

    Science.gov (United States)

    Marlin, David J; Johnson, Lucy; Kingston, Demelza A; Smith, Nicola C; Deaton, Chris M; Mann, Sarah; Heaton, Paul; Van Vugt, Fenneke; Saunders, Kelly; Kydd, Julia; Harris, Pat A

    2004-08-01

    Oxidative stress occurs when antioxidant defense mechanisms are overwhelmed by free radicals and may lead to DNA damage, which has been implicated in processes such as aging and diseases such as cancer. The two main techniques presently used to quantify DNA damage are measurement of 8-hydroxydeoxyguanosine and the Comet assay (also known as single-cell gel electrophoresis). The aim of this study was to apply the comet assay to equine peripheral blood mononuclear cells (PBMCs) and identify two conditions in which we hypothesized that oxidative DNA damage would be increased in PBMCs: aging and equine recurrent airway obstruction (RAO, a condition similar to human asthma). The images obtained were similar to those previously published for humans, cats, and dogs. The optimum concentration of H(2)O(2) to estimate susceptibility to exogenous damage was 50 microM. Mean intraassay coefficients of variation were 4.7 and 9.7% for endogenous and exogenous tail-DNA quantities, respectively, and 7.3 and 8.3%, respectively, for interassay coefficients. There was no significant difference in either endogenous or exogenous percentages of tail DNA for samples collected from six ponies on three consecutive days. There was no significant difference in endogenous, exogenous, or exogenous (corrected for endogenous) oxidative DNA damage between mature and aged ponies. However, young pony foals had significantly less endogenous DNA damage than mature or aged ponies (P < 0.05). RAO-affected horses without airway inflammation (i.e., in clinical remission) had significantly greater endogenous damage compared with non-RAO-affected control animals (P = 0.009). There was a significant correlation between endogenous percentage of tail DNA in PBMCs and red blood cell hemolysate glutathione concentration (r = 0.720; P < 0.001). In conclusion, the comet assay appears to be suitable for investigating DNA damage in equine PBMCs.

  9. Mapping Variation in Cellular and Transcriptional Response to 1,25-Dihydroxyvitamin D3 in Peripheral Blood Mononuclear Cells.

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    Silvia N Kariuki

    Full Text Available The active hormonal form of vitamin D, 1,25-dihydroxyvitamin D (1,25D is an important modulator of the immune system, inhibiting cellular proliferation and regulating transcription of immune response genes. In order to characterize the genetic basis of variation in the immunomodulatory effects of 1,25D, we mapped quantitative traits of 1,25D response at both the cellular and the transcriptional level. We carried out a genome-wide association scan of percent inhibition of cell proliferation (Imax induced by 1,25D treatment of peripheral blood mononuclear cells from 88 healthy African-American individuals. Two genome-wide significant variants were identified: rs1893662 in a gene desert on chromosome 18 (p = 2.32 x 10-8 and rs6451692 on chromosome 5 (p = 2.55 x 10-8, which may influence the anti-proliferative activity of 1,25D by regulating the expression of nearby genes such as the chemokine gene, CCL28, and the translation initiation gene, PAIP1. We also identified 8 expression quantitative trait loci at a FDR<0.10 for transcriptional response to 1,25D treatment, which include the transcriptional regulator ets variant 3-like (ETV3L and EH-domain containing 4 (EHD4. In addition, we identified response eQTLs in vitamin D receptor binding sites near genes differentially expressed in response to 1,25D, such as FERM Domain Containing 6 (FRMD6, which plays a critical role in regulating both cell proliferation and apoptosis. Combining information from the GWAS of Imax and the response eQTL mapping enabled identification of putative Imax-associated candidate genes such as PAIP1 and the transcriptional repressor gene ZNF649. Overall, the variants identified in this study are strong candidates for immune traits and diseases linked to vitamin D, such as multiple sclerosis.

  10. Opposing effects of low molecular weight heparins on the release of inflammatory cytokines from peripheral blood mononuclear cells of asthmatics.

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    Madhur D Shastri

    Full Text Available T-cell-mediated inflammatory cytokines, such as interleukin (IL-4, IL-5, IL-13 and tumor necrosis factor-alpha (TNF-α, play an important role in the initiation and progression of inflammatory airways diseases. Low-molecular-weight heparins (LMWHs, widely used anticoagulants, possess anti-inflammatory properties making them potential treatment options for inflammatory diseases, including asthma. In the current study, we investigated the modulating effects of two LMWHs (enoxaparin and dalteparin on the release of cytokines from stimulated peripheral blood mononuclear cells (PBMCs of asthmatic subjects to identify the specific components responsible for the effects.PBMCs from asthmatic subjects (consist of ~75% of T-cells were isolated from blood taken from ten asthmatic subjects. The PBMCs were pre-treated in the presence or absence of different concentrations of LMWHs, and were then stimulated by phytohaemagglutinin for the release of IL-4, IL-5, IL-13 and TNF-α. LMWHs were completely or selectively desulfated and their anticoagulant effect, as well as the ability to modulate cytokine release, was determined. LMWHs were chromatographically fractionated and each fraction was tested for molecular weight determination along with an assessment of anticoagulant potency and effect on cytokine release.Enoxaparin inhibited cytokine release by more than 48%, whereas dalteparin increased their release by more than 25%. The observed anti-inflammatory effects of enoxaparin were independent of their anticoagulant activities. Smaller fractions, in particular dp4 (four saccharide units, were responsible for the inhibitory effect of enoxaparin. Whereas, the larger fractions, in particular dp22 (twenty two saccharide units, were associated with the stimulatory effect of dalteparin.Enoxaparin and dalteparin demonstrated opposing effects on inflammatory markers. These observed effects could be due to the presence of structurally different components in the two

  11. Can Melatonin Act as an Antioxidant in Hydrogen Peroxide-Induced Oxidative Stress Model in Human Peripheral Blood Mononuclear Cells?

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    Solaleh Emamgholipour

    2016-01-01

    Full Text Available Purpose. We aimed to investigate the possible effects of melatonin on gene expressions and activities of MnSOD and catalase under conditions of oxidative stress induced by hydrogen peroxide (H2O2 in peripheral blood mononuclear cells (PBMCs. Materials and Methods. PBMCs were isolated from healthy subjects and treated as follows: (1 control (only with 0.1% DMSO for 12 h; (2 melatonin (1 mM for 12 h; (3 H2O2 (250 μM for 2 h; (4 H2O2 (250 μM for 2 h following 10 h pretreatment with melatonin (1 mM. The gene expression was evaluated by real-time PCR. MnSOD and catalase activities in PBMCs were determined by colorimetric assays. Results. Pretreatment of PBMCs with melatonin significantly augmented expression and activity of MnSOD which were diminished by H2O2. Melatonin treatment of PBMCs caused a significant upregulation of catalase by almost 2-fold in comparison with untreated cells. However, activity and expression of catalase increased by 1.5-fold in PBMCs under H2O2-induced oxidative stress compared with untreated cell. Moreover, pretreatment of PBMCs with melatonin resulted in a significant 1.8-fold increase in catalase expression compared to PBMCs treated only with H2O2. Conclusion. It seems that melatonin could prevent from undesirable impacts of H2O2-induced oxidative stress on MnSOD downregulation. Moreover, melatonin could promote inductive effect of H2O2 on catalase mRNA expression.

  12. Downregulation of TIM-3 mRNA expression in peripheral blood mononuclear cells from patients with systemic lupus erythematosus

    Energy Technology Data Exchange (ETDEWEB)

    Cai, X.Z. [Central Laboratory, First Affiliated Hospital, China Medical University, Shenyang (China); Department of Immunology, College of Basic Medical Sciences, China Medical University, Shenyang (China); Huang, W.Y.; Qiao, Y.; Chen, Y.; Du, S.Y.; Chen, D.; Yu, S. [Central Laboratory, First Affiliated Hospital, China Medical University, Shenyang (China); Liu, N. [Department of Nephrology, First Affiliated Hospital, China Medical University, Shenyang (China); Dou, L.Y. [Central Laboratory, First Affiliated Hospital, China Medical University, Shenyang (China); Jiang, Y. [Central Laboratory, First Affiliated Hospital, China Medical University, Shenyang (China); Department of Immunology, College of Basic Medical Sciences, China Medical University, Shenyang (China); Department of Dermatology, First Affiliated Hospital, China Medical University, Shenyang (China)

    2014-10-17

    The T-cell immunoglobulin and mucin domain (TIM) family is associated with autoimmune diseases, but its expression level in the immune cells of systemic lupus erythematosus (SLE) patients is not known. The aim of this study was to investigate whether the expression of TIM-3 mRNA is associated with pathogenesis of SLE. Quantitative real-time reverse transcription-polymerase chain reaction analysis (qRT-PCR) was used to determine TIM-1, TIM-3, and TIM-4 mRNA expression in peripheral blood mononuclear cells (PBMCs) from 132 patients with SLE and 62 healthy controls. The PBMC surface protein expression of TIMs in PBMCs from 20 SLE patients and 15 healthy controls was assayed by flow cytometry. Only TIM-3 mRNA expression decreased significantly in SLE patients compared with healthy controls (P<0.001). No significant differences in TIM family protein expression were observed in leukocytes from SLE patients and healthy controls (P>0.05). SLE patients with lupus nephritis (LN) had a significantly lower expression of TIM-3 mRNA than those without LN (P=0.001). There was no significant difference in the expression of TIM-3 mRNA within different classes of LN (P>0.05). Correlation of TIM-3 mRNA expression with serum IgA was highly significant (r=0.425, P=0.004), but was weakly correlated with total serum protein (r{sub s}=0.283, P=0.049) and serum albumin (r{sub s}=0.297, P=0.047). TIM-3 mRNA expression was weakly correlated with the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI; r{sub s}=-0.272, P=0.032). Our results suggest that below-normal expression of TIM-3 mRNA in PBMC may be involved in the pathogenesis of SLE.

  13. Inflammatory cytokine detection in adenotonsill and peripheral blood mononuclear cells- culture in adenotonsillectomy patients: a comparative study

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    Farhadi M

    2013-04-01

    Full Text Available Background: Tonsils and adenoid hypertrophy is a major respiratory symptom in children which is partly due to recruitment of inflammatory cells in upper airway lymph nodes as a result of the effects of synthesis and release of different inflammatory cytokines. It seems that infections play role in concert with these cytokines leading to tonsilar hypertrophy and other pathologic consequences. It is proposed that cellular infiltrate of tonsils and adenoids may secrete different quantities of these cytokines compared with peripheral blood mononuclear cells (PBMC cultures.Methods: Among patients who were admitted for adenotonsillectomy to the ENT ward, 37 patients, under 1-12 years old patients with fulfill criteria selected to include the study. Excised adenoid and tonsils cultured and inflammatory cytokines Interferon-γ (INF-γ, Interlukine-1 (IL-1, IL-6, IL-8 and tumor necrosis factor-α (TNF-α measured in cellular culture supernatant. The same cytokines measured in PBMC cultures.Results: The data shows that there is a significant difference between IFN-γ and IL-8 amounts in adenoid tissue culture supernatant and PBMC culture of our patients. Furth-ermore, the amounts of IFN-γ, IL-1 and IL-8 showed considerable difference between tonsilar tissue culture supernatant and PBMC culture of these patients. Although there is a significant correlation between IL-6 amounts in tissue culture supernatant and PBMC culture (P=0.02, the respective data for TNF is only almost significant.Conclusion: Inflammatory cytokines may have significant role in the early provoke of inflammation occurred in hypertrophied tonsils and adenoid. The majority of these cyt-okines increase the expression of adhesion molecules on epithelial cells and influence the recruitment of leucocytes and inflamed tonsils. On the other hand lack of sufficient cytokine release may lead to persistent infections and may cause chronic inflammation and hypertrophied tissue.

  14. Members of the Candida parapsilosis Complex and Candida albicans are Differentially Recognized by Human Peripheral Blood Mononuclear Cells.

    Science.gov (United States)

    Estrada-Mata, Eine; Navarro-Arias, María J; Pérez-García, Luis A; Mellado-Mojica, Erika; López, Mercedes G; Csonka, Katalin; Gacser, Attila; Mora-Montes, Héctor M

    2015-01-01

    The systemic infections caused by members of the Candida parapsilosis complex are currently associated to high morbility and mortality rates, and are considered as relevant as those caused by Candida albicans. Since the fungal cell wall is the first point of contact with the host cells, here we performed a comparison of this organelle in members of the C. parapsilosis complex, and its relevance during interaction with human peripheral blood mononuclear cells (PBMCs). We found that the wall of the C. parapsilosis complex members is similar in composition, but differs to that from C. albicans, with less mannan content and more β-glucan and porosity levels. Furthermore, lectin-based analysis showed increased chitin and β1,3-glucan exposure at the surface of C. parapsilosis sensu lato when compared to C. albicans. Yeast cells of members of the C. parapsilosis complex stimulated more cytokine production by human PBMCs than C. albicans cells; and this significantly changed upon removal of O-linked mannans, indicating this wall component plays a significant role in cytokine stimulation by C. parapsilosis sensu lato. When inner wall components were exposed on the wall surface, C. parapsilosis sensu stricto and C. metapsilosis, but not C. orthopsilosis, stimulated higher cytokine production. Moreover, we found a strong dependency on β1,3-glucan recognition for the members of the C. parapsilosis complex, but not for live C. albicans cells; whereas TLR4 was required for TNFα production by the three members of the complex, and stimulation of IL-6 by C. orthopsilosis. Mannose receptor had a significant role during TNFα and IL-1β stimulation by members of the complex. Finally, we demonstrated that purified N- and O-mannans from either C. parapsilosis sensu lato or C. albicans are capable to block the recognition of these pathogens by human PBMCs. Together; our results suggest that the innate immune recognition of the members of the C. parapsilosis complex is differential

  15. Combination of autologous bone marrow mesenchymal stem cells and cord blood mononuclear cells in the treatment of chronic thoracic spinal cord injury in 27 cases

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    Lian-zhong WANG

    2012-08-01

    Full Text Available Objective To investigate and evaluate therapeutic effects of transplantation of autologous bone marrow mesenchymal stem cells in conjunction with cord blood mononuclear cells for late thoracic spinal cord injury. Methods Data from 27 patients with late thoracic spinal cord injury who received transplantation of autologous bone marrow mesenchymal stem cells in conjunction with cord blood mononuclear cells in Neurosurgery Department of 463rd Hospital of PLA between July 2006 and July 2008 were collected and analyzed. The full treatment course consisted of 4 consecutive injections at one week apart. Indicators for evaluation followed that of the American Spiral Injury Association (ASIA Impairment Scale (AIS grade, ASIA motor and sensory scores, ASIA visual analog score, and the Ashworth score. The follow-up period was 6 months. Evaluations were made 6 weeks and 6 months after the treatment. Results Improvement from AIS A to AIS B was found in 4 patients. In one patient, improvement from AIS A to AIS C and in one patient from AIS B to AIS C was found 6 weeks after the treatment. The AIS improvement rate was 22.2%. In one patient improvement from AIS A to AIS B was found after 6 months. The overall AIS improvement rate was 25.9%. ASIA baseline motor scores of lower extremties were 0.5±1.5, 1.7±2.9, 3.1±3.6 before the treatment, 6 weeks and 6 months after the treatment, respectively, and showed a statistically significant improvement (P < 0.05. ASIA sensory scores including light touch and pinprick were 66.6±13.7 and 67.0±13.6 respectively before treatment, and they became 68.8±14.4, 68.4±14.7 and 70.5±14.4, 70.2±14.4 six weeks and six months after the treatment. The changes were statistically significant (P < 0.05; Modified Ashworth Scale scores were 1.8±1.5, 1.6±1.2,1.1±0.8 respectively at baseline, 6 weeks and 6months after the treatment, and showed a statistically significant descending trend (P < 0.05. Conclusion Transplantation of

  16. Evaluating the effect of four extracts of avocado fruit on esophageal squamous carcinoma and colon adenocarcinoma cell lines in comparison with peripheral blood mononuclear cells.

    Science.gov (United States)

    Vahedi Larijani, Laleh; Ghasemi, Maryam; AbedianKenari, Saeid; Naghshvar, Farshad

    2014-01-01

    Most patients with gastrointestinal cancers refer to the health centers at advanced stages of the disease and conventional treatments are not significantly effective for these patients. Therefore, using modern therapeutic approaches with lower toxicity bring higher chance for successful treatment and reduced adverse effects in such patients. The aim of this study is to evaluate the effect of avocado fruit extracts on inhibition of the growth of cancer cells in comparison with normal cells. In an experimental study, ethanol, chloroform, ethyl acetate, and petroleum extracts of avocado (Persea americana) fruit were prepared. Then, the effects if the extracts on the growth of esophageal squamous cell carcinoma and colon adenocarcinoma cell lines were evaluated in comparison with the control group using the MTT test in the cell culture medium. Effects of the four extracts of avocado fruit on three cells lines of peripheral blood mononuclear cells, esophageal squamous cell carcinoma, and colon adenocarcinoma were tested. The results showed that avocado fruit extract is effective in inhibition of cancer cell growth in comparison with normal cells (PAvocado fruit is rich in phytochemicals, which play an important role in inhibition of growth of cancer cells. The current study for the first time demonstrates the anti-cancer effect of avocado fruit extracts on two cancers common in Iran. Therefore, it is suggested that the fruit extracts can be considered as appropriate complementary treatments in treatment of esophageal and colon cancers.

  17. Evaluating the effect of four extracts of avocado fruit on esophageal squamous carcinoma and colon adenocarcinoma cell lines in comparison with peripheral blood mononuclear cells.

    Directory of Open Access Journals (Sweden)

    Laleh Vahedi Larijani

    2014-03-01

    Full Text Available Most patients with gastrointestinal cancers refer to the health centers at advanced stages of the disease and conventional treatments are not significantly effective for these patients. Therefore, using modern therapeutic approaches with lower toxicity bring higher chance for successful treatment and reduced adverse effects in such patients. The aim of this study is to evaluate the effect of avocado fruit extracts on inhibition of the growth of cancer cells in comparison with normal cells. In an experimental study, ethanol, chloroform, ethyl acetate, and petroleum extracts of avocado (Persea americana fruit were prepared. Then, the effects if the extracts on the growth of esophageal squamous cell carcinoma and colon adenocarcinoma cell lines were evaluated in comparison with the control group using the MTT test in the cell culture medium. Effects of the four extracts of avocado fruit on three cells lines of peripheral blood mononuclear cells, esophageal squamous cell carcinoma, and colon adenocarcinoma were tested. The results showed that avocado fruit extract is effective in inhibition of cancer cell growth in comparison with normal cells (P<0.05. Avocado fruit is rich in phytochemicals, which play an important role in inhibition of growth of cancer cells. The current study for the first time demonstrates the anti-cancer effect of avocado fruit extracts on two cancers common in Iran. Therefore, it is suggested that the fruit extracts can be considered as appropriate complementary treatments in treatment of esophageal and colon cancers.

  18. Effective use of frozen donor peripheral blood mononuclear cells for human immunodeficiency virus type 1 isolation from vertically infected pediatric patients.

    Science.gov (United States)

    Paul, M O; Tetali, S; Pahwa, S

    1994-05-01

    In this study, we examined variables related to human immunodeficiency virus (HIV) isolation utilizing samples from 51 HIV-infected (153 plasma and 122 peripheral blood mononuclear cell [PBMC] samples) and 57 uninfected (182 plasma and 163 PBMC samples) infants. Our chief observation was that cryopreservation of donor PBMCs does not significantly alter their sensitivity or specificity for isolation of HIV from patient PBMCs or plasma.

  19. TSH, thyroid hormones and nuclear-binding of T3 in mononuclear blood cells from obese and non-obese women

    DEFF Research Database (Denmark)

    Matzen, L E; Kvetny, J; Pedersen, K K

    1989-01-01

    The specific nuclear-binding of T3 (NBT3) in mononuclear blood cells, and the concentrations of TSH, thyroid hormones, and binding proteins were measured after overnight fasting in 12 obese and in 14 non-obese women, none of the subjects were taking any medicine. The concentrations of TSH and fre...... concentration and was not caused by a primary tissue resistance. The higher TSH and TT3 in the obese women could be caused by a greater caloric intake....

  20. Unique and differential protein signatures within the mononuclear cells of HIV-1 and HCV mono-infected and co-infected patients

    OpenAIRE

    Boukli Nawal M; Shetty Vivekananda; Cubano Luis; Ricaurte Martha; Coelho-dos-Reis Jordana; Nickens Zacharie; Shah Punit; Talal Andrew H; Philip Ramila; Jain Pooja

    2012-01-01

    Abstract Background Pathogenesis of liver damage in patients with HIV and HCV co-infection is complex and multifactorial. Although global awareness regarding HIV-1/HCV co-infection is increasing little is known about the pathophysiology that mediates the rapid progression to hepatic disease in the co-infected individuals. Results In this study, we investigated the proteome profiles of peripheral blood mononuclear cells from HIV-1 mono-, HCV mono-, and HIV-1/HCV co-infected patients. The resul...

  1. Carbohydrate restriction and dietary cholesterol modulate the expression of HMG-CoA reductase and the LDL receptor in mononuclear cells from adult men

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    Volek Jeff S

    2007-11-01

    Full Text Available Abstract The liver is responsible for controlling cholesterol homeostasis in the body. HMG-CoA reductase and the LDL receptor (LDL-r are involved in this regulation and are also ubiquitously expressed in all major tissues. We have previously shown in guinea pigs that there is a correlation in gene expression of HMG-CoA reductase and the LDL-r between liver and mononuclear cells. The present study evaluated human mononuclear cells as a surrogate for hepatic expression of these genes. The purpose was to evaluate the effect of dietary carbohydrate restriction with low and high cholesterol content on HMG-CoA reductase and LDL-r mRNA expression in mononuclear cells. All subjects were counseled to consume a carbohydrate restricted diet with 10–15% energy from carbohydrate, 30–35% energy from protein and 55–60% energy from fat. Subjects were randomly assigned to either EGG (640 mg/d additional dietary cholesterol or SUB groups [equivalent amount of egg substitute (0 dietary cholesterol contributions per day] for 12 weeks. At the end of the intervention, there were no changes in plasma total or LDL cholesterol (LDL-C compared to baseline (P > 0.10 or differences in plasma total or LDL-C between groups. The mRNA abundance for HMG-CoA reductase and LDL-r were measured in mononuclear cells using real time PCR. The EGG group showed a significant decrease in HMG-CoA reductase mRNA (1.98 ± 1.26 to 1.32 ± 0.92 arbitrary units P

  2. The Expression of Caspases Is Enhanced in Peripheral Blood Mononuclear Cells of Autism Spectrum Disorder Patients

    Science.gov (United States)

    Siniscalco, Dario; Sapone, Anna; Giordano, Catia; Cirillo, Alessandra; de Novellis, Vito; de Magistris, Laura; Rossi, Francesco; Fasano, Alessio; Maione, Sabatino; Antonucci, Nicola

    2012-01-01

    Autism and autism spectrum disorders (ASDs) are heterogeneous complex neuro-developmental disorders characterized by dysfunctions in social interaction and communication skills. Their pathogenesis has been linked to interactions between genes and environmental factors. Consistent with the evidence of certain similarities between immune cells and…

  3. The Expression of Caspases Is Enhanced in Peripheral Blood Mononuclear Cells of Autism Spectrum Disorder Patients

    Science.gov (United States)

    Siniscalco, Dario; Sapone, Anna; Giordano, Catia; Cirillo, Alessandra; de Novellis, Vito; de Magistris, Laura; Rossi, Francesco; Fasano, Alessio; Maione, Sabatino; Antonucci, Nicola

    2012-01-01

    Autism and autism spectrum disorders (ASDs) are heterogeneous complex neuro-developmental disorders characterized by dysfunctions in social interaction and communication skills. Their pathogenesis has been linked to interactions between genes and environmental factors. Consistent with the evidence of certain similarities between immune cells and…

  4. A new portable device for automatic controlled-gradient cryopreservation of blood mononuclear cells

    DEFF Research Database (Denmark)

    Hviid, L; Albeck, G; Hansen, B

    1993-01-01

    , which has proved suitable for field conditions. We report here the development and testing of a similar micro-controller regulated device, allowing unattended and automatic controlled-gradient cell freezing. The equipment exploits the temperature gradient present between the liquid N2 surface...

  5. Hepatitis C virus replicates in peripheral blood mononuclear cells of patients with occult hepatitis C virus infection

    Science.gov (United States)

    Castillo, I; Rodríguez-Iñigo, E; Bartolomé, J; de Lucas, S; Ortíz-Movilla, N; López-Alcorocho, J M; Pardo, M; Carreño, V

    2005-01-01

    Background: Occult hepatitis C virus (HCV) infection is characterised by the presence of HCV-RNA in the liver in the absence of anti-HCV, and serum viral RNA. Up to 70% of these patients also have HCV-RNA in peripheral blood mononuclear cells (PBMC) but it is not known if HCV is replicating in these cells. Aim: We studied possible HCV replication in PBMC of 18 patients with an occult HCV infection who were selected on the basis of HCV-RNA positivity in PBMC. Methods: Detection of HCV-RNA positive and negative strands in PBMC was done by strand specific reverse transcriptase-polymerase chain reaction (RT-PCR) and by in situ hybridisation. Results: The presence of HCV-RNA positive strand in PBMC was confirmed in all patients by strand specific RT-PCR and by in situ hybridisation. Mean percentage of PBMC which had the HCV-RNA positive strand was 3.3% (95% confidence interval (CI) 2.1–4.4) The HCV-RNA negative strand was found in the PBMC of 11/18 (61%) patients by strand specific RT-PCR and confirmed by in situ hybridisation, and the percentage of PBMC harbouring the HCV-RNA negative strand was 3.1% (95% CI 0.8–5.5). There was a significant correlation (p = 0.001, r = 0.84) between the percentage of PBMC with the HCV-RNA positive strand and that of PBMC with the HCV-RNA negative strand. Conclusion: HCV replicates in the PBMC of patients with occult HCV infection and thus, although these patients do not have serum HCV-RNA, they could be potentially infectious. PMID:15831916

  6. Gene network analysis of bone marrow mononuclear cells reveals activation of multiple kinase pathways in human systemic lupus erythematosus.

    Directory of Open Access Journals (Sweden)

    Magdalene Nakou

    Full Text Available BACKGROUND: Gene profiling studies provide important information for key molecules relevant to a disease but are less informative of protein-protein interactions, post-translational modifications and regulation by targeted subcellular localization. Integration of genomic data and construction of functional gene networks may provide additional insights into complex diseases such as systemic lupus erythematosus (SLE. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed gene expression microarray data of bone marrow mononuclear cells (BMMCs from 20 SLE patients (11 with active disease and 10 controls. Gene networks were constructed using the bioinformatic tool Ingenuity Gene Network Analysis. In SLE patients, comparative analysis of BMMCs genes revealed a network with 19 central nodes as major gene regulators including ERK, JNK, and p38 MAP kinases, insulin, Ca(2+ and STAT3. Comparison between active versus inactive SLE identified 30 central nodes associated with immune response, protein synthesis, and post-transcriptional modification. A high degree of identity between networks in active SLE and non-Hodgkin's lymphoma (NHL patients was found, with overlapping central nodes including kinases (MAPK, ERK, JNK, PKC, transcription factors (NF-kappaB, STAT3, and insulin. In validation studies, western blot analysis in splenic B cells from 5-month-old NZB/NZW F1 lupus mice showed activation of STAT3, ITGB2, HSPB1, ERK, JNK, p38, and p32 kinases, and downregulation of FOXO3 and VDR compared to normal C57Bl/6 mice. CONCLUSIONS/SIGNIFICANCE: Gene network analysis of lupus BMMCs identified central gene regulators implicated in disease pathogenesis which could represent targets of novel therapies in human SLE. The high similarity between active SLE and NHL networks provides a molecular basis for the reported association of the former with lymphoid malignancies.

  7. Effects of estradiol and progesterone on the proinflammatory cytokine production by mononuclear cells from patients with chronic hepatitis C

    Institute of Scientific and Technical Information of China (English)

    Ying Yuan; Katsuyoshi Tamaki; Masayuki Shono; Tetsuji Takayama; Ichiro Shimizu; Mi Shen; Eriko Aoyagi; Hidetaka Takenaka; Tatuzo Itagaki; Marl Urata; Katsutaka Sannomiya; Nao Kohno

    2008-01-01

    AIM: To investigate the effects of estradiol (E2) and progesterone on the unstimulated and oxidative stressstimulated production of tumor necrosis factor (TNF)-α,interleukin (IL)-1β, IL-8, and macrophage chemotactic protein (ICP)-1 by peripheral blood mononuclear cells (PBMCs) from patients with chronic hepatitis C and healthy controls.METHODS: The PBMCs were separated from agematched 72 males and 71 females with and without chronic hepatitis C, who were divided into two groups based on a mean menopausal age of 50 years. Oxidative stress was induced by hydrogen peroxide in the cells incubated in serum-free media. CytokJnes in the culture supernatant were measured by an enzyme-linked immunosorbent assay.RESULTS: The highest levels of the spontaneous production of TNF-α, IL-1β, IL-8, and MCP-1 by the unstimulated PBMCs were in the older male patients with chronic hepatitis C and the lowest levels were in the premenopausal female-healthy controls. E2 inhibited the cytokine production by the unstimulated PBMCs from the older male and post-menopausal female patients, which was further stimulated by progesterone. The exposure to hydrogen peroxide in the PBMCs from the younger male and pre-menopausal female healthy subjects induced the production of cytokines. The change rates of the hydrogen peroxide-stimulated cytokine production were suppressed by E2 and enhanced by progesterone.CONCLUSION: These findings suggest that E2 may play a favorable role in the course of persistent liver injury by preventing the accumulation of monocytes-macrophages and by inhibiting proinflammatory cytokine production,whereas progesterone may counteract the favorable E2 effects.

  8. RNA-Seq analysis of peripheral blood mononuclear cells reveals unique transcriptional signatures associated with disease progression in dengue patients.

    Science.gov (United States)

    Banerjee, Arup; Shukla, Shweta; Pandey, Abhay Deep; Goswami, Saptamita; Bandyopadhyay, Bhaswati; Ramachandran, Vishnampettai; Das, Shukla; Malhotra, Arjun; Agarwal, Amitesh; Adhikari, Srima; Rahman, Mehebubar; Chatterjee, Shatakshee; Bhattacharya, Nemai; Basu, Nandita; Pandey, Priyanka; Sood, Vikas; Vrati, Sudhanshu

    2017-08-01

    Patients infected with Dengue virus usually present a mild, self-limiting febrile dengue infection (DI) that occasionally leads to a potentially lethal complication, called the severe dengue (DS). The ability to identify the prognostic markers of DS could allow an improved disease intervention and management. To identify the transcriptional signatures associated with the dengue disease progression, we carried out the high-throughput sequencing of the RNA isolated from the peripheral blood mononuclear cells (PBMCs) of the dengue patients of varying severity and compared with that in the patients with other febrile illnesses (OFIs) or the healthy controls. The transcriptional signatures that discriminated the DS patients from OFI and DI patients were broadly related to the pathways involving glycine, serine, and threonine metabolisms, extracellular matrix organization, ubiquitination, and cytokines and inflammatory response. Several upregulated genes in the inflammatory process (MPO, DEFA4, ELANE, AUZ1, CTSG, OLFM4, SLC16A14, and CRISP3) that were associated with the dengue disease progression are known to facilitate leukocyte-mediated migration, and neutrophil activation and degranulation process. High activity of MPO and ELANE in the plasma samples of the follow-up and recovered dengue patients, as well as and the presence of a larger amount of cell-free dsDNA in the DS patients, suggested an association of neutrophil-mediated immunity with dengue disease progression. Careful monitoring of some of these gene transcripts, and control of the activity of proteins encoded by them, may have a great translational significance for the prognosis and management of the dengue patients. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. High-throughput sequencing of microRNAs in peripheral blood mononuclear cells: identification of potential weight loss biomarkers.

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    Fermín I Milagro

    Full Text Available INTRODUCTION: MicroRNAs (miRNAs are being increasingly studied in relation to energy metabolism and body composition homeostasis. Indeed, the quantitative analysis of miRNAs expression in different adiposity conditions may contribute to understand the intimate mechanisms participating in body weight control and to find new biomarkers with diagnostic or prognostic value in obesity management. OBJECTIVE: The aim of this study was the search for miRNAs in blood cells whose expression could be used as prognostic biomarkers of weight loss. METHODS: Ten Caucasian obese women were selected among the participants in a weight-loss trial that consisted in following an energy-restricted treatment. Weight loss was considered unsuccessful when 5% (responders. At baseline, total miRNA isolated from peripheral blood mononuclear cells (PBMC was sequenced with SOLiD v4. The miRNA sequencing data were validated by RT-PCR. RESULTS: Differential baseline expression of several miRNAs was found between responders and non-responders. Two miRNAs were up-regulated in the non-responder group (mir-935 and mir-4772 and three others were down-regulated (mir-223, mir-224 and mir-376b. Both mir-935 and mir-4772 showed relevant associations with the magnitude of weight loss, although the expression of other transcripts (mir-874, mir-199b, mir-766, mir-589 and mir-148b also correlated with weight loss. CONCLUSIONS: This research addresses the use of high-throughput sequencing technologies in the search for miRNA expression biomarkers in obesity, by determining the miRNA transcriptome of PBMC. Basal expression of different miRNAs, particularly mir-935 and mir-4772, could be prognostic biomarkers and may forecast the response to a hypocaloric diet.

  10. Malachite green toxicity assessed on Asian catfish primary cultures of peripheral blood mononuclear cells by a proteomic analysis.

    Science.gov (United States)

    Pierrard, Marie-Aline; Kestemont, Patrick; Delaive, Edouard; Dieu, Marc; Raes, Martine; Silvestre, Frédéric

    2012-06-15

    The potential genotoxic and carcinogenic properties reported for malachite green (MG) and the frequent detection of MG residues in fish and fish products, despite the ban of MG, have recently generated great concern. Additional toxicological data are required for a better understanding of the mechanism of action and a more comprehensive risk assessment for the exposure of fish to this fungicide. To date, the use of fish peripheral blood mononuclear cells (PBMCs) has not been exploited as a tool in the assessment of the toxicity of chemicals. However, PBMCs are exposed to toxicants and can be easily collected by blood sampling. The present study aims at better understanding the effects of MG by a proteomic analysis of primary cultured PBMC from the Asian catfish, Pangasianodon hypophthalmus, exposed to MG. The two lowest concentrations of 1 and 10 ppb were selected based on the MTS (water soluble tetrazolium salts) cytotoxicity test. Using a proteomic analysis (2D-DIGE), we showed that 109 proteins displayed significant changes in abundance in PBMC exposed during 48 h to MG. Most of these proteins were successfully identified by nano LC-MS/MS and validated through the Peptide and Protein Prophet of Scaffold™ software, but only 19 different proteins were considered corresponding to a single identification per spot. Our data suggest that low concentrations of MG could affect the mitochondrial metabolic functions, impair some signal transduction cascades and normal cell division, stimulate DNA repair and disorganize the cytoskeleton. Altogether, these results confirm that the mitochondrion is a target of MG toxicity. Further studies on the identified proteins are needed to better understand the mechanisms of MG toxicity in fish produced for human consumption.

  11. Pro-inflammatory action of MIF in acute myocardial infarction via activation of peripheral blood mononuclear cells.

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    David A White

    Full Text Available OBJECTIVES: Macrophage migration inhibitory factor (MIF, a pro-inflammatory cytokine, has been implicated in the pathogenesis of multiple inflammatory disorders. We determined changes in circulating MIF levels, explored the cellular source of MIF, and studied the role of MIF in mediating inflammatory responses following acute myocardial infarction (MI. METHODS AND RESULTS: We recruited 15 patients with MI, 10 patients with stable angina and 10 healthy volunteers and measured temporal changes of MIF in plasma. Expression of MIF, matrix metalloproteinase-9 (MMP-9 and interleukin-6 (IL-6 in cultured peripheral blood mononuclear cells (PBMCs and the media were measured by ELISA or real-time PCR. Compared to controls, plasma levels of MIF and IL-6 were significantly elevated at admission and 72 h post-MI. In contrast, expression of MIF, MMP-9 and IL-6 by PBMCs from MI patients was unchanged at admission, but significantly increased at 72 h. Addition of MIF activated cultured PBMCs by upregulating expression of inflammatory molecules and also synergistically enhanced stimulatory action of IL-1β which were inhibited by anti-MIF interventions. In a mouse MI model we observed similar changes in circulating MIF as seen in patients, with reciprocal significant increases in plasma MIF and reduction of MIF content in the infarct myocardium at 3 h after MI. MIF content in the infarct myocardium was restored at 72 h post-MI and was associated with robust macrophage infiltration. Further, anti-MIF intervention significantly reduced inflammatory cell infiltration and expression of monocyte chemoattractant protein-1 at 24 h and incidence of cardiac rupture in mice post-MI. CONCLUSION: MI leads to a rapid release of MIF from the myocardium into circulation. Subsequently MIF facilitates PBMC production of pro-inflammatory mediators and myocardial inflammatory infiltration. Attenuation of these events, and post-MI cardiac rupture, by anti-MIF interventions suggests

  12. Perturbations in the apoptotic pathway and mitochondrial network dynamics in peripheral blood mononuclear cells from bipolar disorder patients.

    Science.gov (United States)

    Scaini, G; Fries, G R; Valvassori, S S; Zeni, C P; Zunta-Soares, G; Berk, M; Soares, J C; Quevedo, J

    2017-05-02

    Bipolar disorder (BD) is a severe psychiatric disorder characterized by phasic changes of mood and can be associated with progressive structural brain change and cognitive decline. The numbers and sizes of glia and neurons are reduced in several brain areas, suggesting the involvement of apoptosis in the pathophysiology of BD. Because the changes in mitochondrial dynamics are closely related with the early process of apoptosis and the specific processes of apoptosis and mitochondrial dynamics in BD have not been fully elucidated, we measured the apoptotic pathway and the expression of mitochondrial fission/fusion proteins from BD patients and healthy controls. We recruited 16 patients with BD type I and sixteen well-matched healthy controls and investigated protein levels of several pro-apoptotic and anti-apoptotic factors, as well as the expression of mitochondrial fission/fusion proteins in peripheral blood mononuclear cells (PBMCs). Our results showed that the levels of the anti-apoptotic proteins Bcl-xL, survivin and Bcl-xL/Bak dimer were significantly decreased, while active caspase-3 protein levels were significantly increased in PBMCs from BD patients. Moreover, we observed the downregulation of the mitochondrial fusion-related proteins Mfn2 and Opa1 and the upregulation of the fission protein Fis1 in PBMCs from BD patients, both in terms of gene expression and protein levels. We also showed a significantly decrease in the citrate synthase activity. Finally, we found a positive correlation between Mfn2 and Opa1 with mitochondrial content markers, as well as a negative correlation between mitochondrial fission/fusion proteins and apoptotic markers. Overall, data reported here are consistent with the working hypothesis that apoptosis may contribute to cellular dysfunction, brain volume loss and progressive cognitive in BD. Moreover, we show an important relationship between mitochondrial dynamics and the cell death pathway activation in BD patients, supporting

  13. Multiple correlation analyses revealed complex relationship between DNA methylation and mRNA expression in human peripheral blood mononuclear cells.

    Science.gov (United States)

    Xie, Fang-Fei; Deng, Fei-Yan; Wu, Long-Fei; Mo, Xing-Bo; Zhu, Hong; Wu, Jian; Guo, Yu-Fan; Zeng, Ke-Qin; Wang, Ming-Jun; Zhu, Xiao-Wei; Xia, Wei; Wang, Lan; He, Pei; Bing, Peng-Fei; Lu, Xin; Zhang, Yong-Hong; Lei, Shu-Feng

    2017-07-22

    DNA methylation is an important regulator on the mRNA expression. However, a genome-wide correlation pattern between DNA methylation and mRNA expression in human peripheral blood mononuclear cells (PBMCs) is largely unknown. The comprehensive relationship between mRNA and DNA methylation was explored by using four types of correlation analyses and a genome-wide methylation-mRNA expression quantitative trait locus (eQTL) analysis in PBMCs in 46 unrelated female subjects. An enrichment analysis was performed to detect biological function for the detected genes. Single pair correlation coefficient (r T1) between methylation level and mRNA is moderate (-0.63-0.62) in intensity, and the negative and positive correlations are nearly equal in quantity. Correlation analysis on each gene (T4) found 60.1% genes showed correlations between mRNA and gene-based methylation at P correlation (R T4 > 0.8). Methylation sites have regulation effects on mRNA expression in eQTL analysis, with more often observations in region of transcription start site (TSS). The genes under significant methylation regulation both in correlation analysis and eQTL analysis tend to cluster to the categories (e.g., transcription, translation, regulation of transcription) that are essential for maintaining the basic life activities of cells. Our findings indicated that DNA methylation has predictive regulation effect on mRNA with a very complex pattern in PBMCs. The results increased our understanding on correlation of methylation and mRNA and also provided useful clues for future epigenetic studies in exploring biological and disease-related regulatory mechanisms in PBMC.

  14. Detection of Hepatitis C virus RNA in peripheral blood mononuclear cells of patients with abnormal alanine transaminase in Ahvaz

    Directory of Open Access Journals (Sweden)

    M Makvandi

    2014-01-01

    Full Text Available Purpose: Hepatitis C virus (HCV is an important agent for chronic and acute hepatitis. Occult hepatitis C remains a major health problem worldwide. Patients with chronic occult HCV may progress to cirrhosis and hepatocellular carcinoma. The aim of this study was to determine prevalence of occult hepatitis C by IS-PCR-ISH (in situ PCR in situ hybridisation in the patients with abnormal ALT. Materials and Methods: The blood samples were taken from 53 patients including 17 females (32.1% and 36 (67.9% males who had abnormal alanine transaminase (ALT for more than 1 year. The mean ALT and aspartate transaminase (AST level were 41.02 ± 9.3 and 24.17 ± 7.3, respectively. The patients′ age were between 4 and 70-years old with mean age 38 ± 13. All the patients were negative for HCV antibody, HCV RNA and HBs Ag. The peripheral blood mononuclear cells (PBMC were separated with ficoll gradient from each blood sample, then the cells were fixed on slides by cold acetone and followed by IS-PCR-ISH for HCV RNA detection. Results: Seventeen (32% patients including 6 (11.3% females and 11 (20.7% males showed positive results for HCV RNA by in situ-PCR in situ hybridisation. Ten (18.8% positive cases were between 20 and 40-years old and 6 (11.3% positive patients were between 40 and 60 years old. Ten (19.6% patients who were positive for IS-PCR-ISH also had positive anti-HBc IgG and 7 (13.2% patients were negative for HBc-IgG. Conclusion: In the present study high rate of 32% occult hepatitis C were found among the patients with elevated ALT.

  15. Differential miRNA expressions in peripheral blood mononuclear cells for diagnosis of lung cancer.

    Science.gov (United States)

    Ma, Jie; Lin, Yanli; Zhan, Min; Mann, Dean L; Stass, Sanford A; Jiang, Feng

    2015-10-01

    Tremendous efforts have been made to develop cancer biomarkers by detecting circulating extracellular miRNAs directly released from tumors. Yet, none of the cell-free biomarkers has been accepted to be used for early detection of non-small cell lung cancer (NSCLC). Peripheral blood mononucleated cells (PBMCs) act as the first line of defense against malignancy in immune system, their dysfunction may occur as an early event in cancer immunogenicity or immune evasion. We proposed to investigate whether analysis of miRNA expressions of PBMCs has diagnostic value for NSCLC. We first used a microarray to analyze PBMCs of 16 stage I NSCLC patients and 16 cancer-free smokers, and identified seven PBMC miRNAs with a significantly altered expression level in NSCLC patients. In a training set of 84 NSCLC patients and 69 cancer-free smokers, a panel of two miRNAs (miRs-19b-3p and -29b-3p) were developed from the seven PBMC miRNAs, producing 72.62% sensitivity and 82.61% specificity in identifying NSCLC. Furthermore, the miRNAs could identify squamous cell lung carcinoma (SCC), a major type of NSCLC, with 80.00% sensitivity and 89.86% specificity. The expression levels of the miRNAs were independent of disease stage. In a testing set of 56 NSCLC patients and 46 controls, the performance of the biomarkers was reproducibly confirmed. The study presents the first in-depth analysis of PBMC miRNA profile of NSCLC patients. The assessment of PBMC miRNAs may provide a new diagnostic approach for the early detection of NSCLC.

  16. Intermittent Hypoxia Alters Gene Expression in Peripheral Blood Mononuclear Cells of Healthy Volunteers

    OpenAIRE

    Polotsky, Vsevolod Y.; Shannon Bevans-Fonti; Grigoryev, Dmitry N.; Punjabi, Naresh M.

    2015-01-01

    Obstructive sleep apnea is associated with high cardiovascular morbidity and mortality. Intermittent hypoxia of obstructive sleep apnea is implicated in the development and progression of insulin resistance and atherosclerosis, which have been attributed to systemic inflammation. Intermittent hypoxia leads to pro-inflammatory gene up-regulation in cell culture, but the effects of intermittent hypoxia on gene expression in humans have not been elucidated. A cross-over study was performed expos...

  17. Priming of mononuclear cells with a combination of growth factors enhances wound healing via high angiogenic and engraftment capabilities.

    Science.gov (United States)

    Jin, Enze; Kim, Jong-Min; Kim, Sung-Whan

    2013-12-01

    Recently, we demonstrated that a specific combination of growth factors enhances the survival, adhesion and angiogenic potential of mononuclear cells (MNCs). In this study, we sought to investigate the changes of the angiogenic potential of MNCs after short-time priming with a specific combination of growth factors. MNCs were isolated using density gradient centrifugation and incubated with a priming cocktail containing epidermal growth factor (EGF), insulin-like growth factor (IGF)-1, fibroblast growth factor (FGF)-2, FMS-like tyrosine kinase (Flt)-3L , Angiopoietin (Ang)-1, granulocyte chemotactic protein (GCP)-2 and thrombopoietin (TPO) (all 400 ng/ml) for 15, 30 and 60 min. Wounds in nonobese diabetic-severe combined immune deficiency (NOD-SCID) mice were created by skin excision followed by cell transplantation. We performed a qRT-PCR analysis on the growth factor-primed cells. The angiogenic factors vascular endothelial growth factor (VEGF)-A, FGF-2, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF) and interleukin (IL)-8 and the anti-apoptotic factors IGF-1 and transforming growth factor-β1 were significantly elevated in the MNCs primed for 30 min. (T30) compared with the non-primed MNCs (T0). The scratch wound assay revealed that T30- conditioned media (CM) significantly increased the rate of fibroblast-mediated wound closure compared with the rates from T0-CM and human umbilical vein endothelial cells (HUVEC)-CM at 20 hrs. In vivo wound healing results revealed that the T30-treated wounds demonstrated accelerated wound healing at days 7 and 14 compared with those treated with T0. The histological analyses demonstrated that the number of engrafted cells and transdifferentiated keratinocytes in the wounds were significantly higher in the T30-transplanted group than in the T0-transplanted group. In conclusion, this study suggests that short-term priming of MNCs with growth factors might be alternative therapeutic option for cell

  18. Bovine colostrum modulates immune activation cascades in human peripheral blood mononuclear cells in vitro

    DEFF Research Database (Denmark)

    Jenny, Marcel; Pedersen, Ninfa R; Hidayat, Budi J

    2010-01-01

    factors and has a long history of use in traditional medicine. In an approach to evaluate the effects of bovine colostrum (BC) on the T-cell/macrophage interplay, we investigated and compared the capacity of BC containing low and high amounts of lactose and lactoferrin to modulate tryptophan degradation...... of lactose present in BC seems to diminish the activity of BC in our test system, since BC with higher amounts of lactose attenuated the stimulatory as well as the suppressive activity of BC....

  19. Cytokine profiles in peripheral blood mononuclear cells and lymph node cells from piglets infected in utero with porcine reproductive and respiratory syndrome virus

    DEFF Research Database (Denmark)

    Aasted, B.; Bach, P.; Nielsen, Jens;

    2002-01-01

    compared, the levels of cytokine expression at 4 weeks diverged from those at 2 and 6 weeks, in that there was a significant decrease in the numbers of lymphocytes producing gamma-IFN and TNF-alpha. This tendency was also observed among blood monocytes and lymph node macrophages. Possible reasons......The aim of the present study was to investigate at 2, 4, and 6 weeks after birth cytokine expression by peripheral blood mononuclear cells and bronchial lymph node cells from piglets infected in utero with porcine reproductive and respiratory syndrome virus (PRRSV). Technically, by flow cytometry...... we were able to measure gamma interferon (gamma-IFN), tumor necrosis factor alpha (TNF-alpha), interleukin-4 (IL-4), and IL-8 levels. In general, we found increases in the percentages of IL-4-, gamma-IFN-, and TNF-alpha-producing lymphocytes in the infected piglets compared to the percentages...

  20. Immunophenotypic comparison of heterogenous non-sorted versus sorted mononuclear cells from human umbilical cord blood: a novel cell enrichment approach.

    Science.gov (United States)

    Indumathi, S; Harikrishnan, R; Rajkumar, J S; Dhanasekaran, M

    2015-01-01

    Human umbilical cord blood (hUCB) has been the preferred source of stem cells for the treatment of haematological malignancies and genetic disorders. This is primarily due to its non-invasiveness, high accessibility with relative ease of isolation. Still failures do prevail due to its heterogeneity and lesser frequency of MSC identified in UCB. This study, thus, employs a cell enrichment technology to improve its therapeutic efficacy. This was achieved by immunophenotypic comparison of stem cells isolated from the heterogenous non-sorted mononuclear cells (MNCs), linage depleted (Lin+ and Lin-) fractions obtained from magnetic activated cell sorter (MACS) and sorted MNCs obtained by fluorescent activated cell sorter (FACS). The markers under consideration were CD29, CD44, CD34, CD45, CD133, CD90 and CD117. FACS sorted MNCs were rich in naive stem cell population, whereas non-sorted MNCs and lineage depleted fractions were found to be rich in progenitors. Thus, we suggest that a combination therapy of both sorted population might serve as an alternative valuable tool in treating haematologic/genetic disorders. However, further research on cell enrichment technology might give a clue for improved cell based therapy in regenerative medicine.

  1. Expression of Interleukin- 13 inPeripheral Blood Mononuclear Cells from Patients with Nephrotic Syndrome

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective Nephrotic syndrome (NS) is a kind of renal diseases characterized mainly by proteinuria and hypoalbuminemia. The disturbance of cellular immunity plays a major role in the pathogenesis of NS. The change of interleukin (IL)-13 produced by Th2 cells was investigated in 50 children with NS. Methods All the patients were selected during their nephrotic stage and remission stage. The expression of IL-13 protein and mRNA was determined by ELISA and reverse transcription-polymerase chain reaction ( RT-PCR ) respectively. Results ①The production of IL-13 inphytoheamagglutinin (PHA)- stimulated monouuclear cells and the expression of IL-13 mRNA were siguificantly increased in patients in nephrotic stage as compared with controls. ②The expression of IL13 protein and mRNA in patients in remission stage was similar to that in the normal controls. ③Th_ere was no correlation between IL-13 and laboratory parameters. Conclusion The results suggest that the disturbance of cellulr immunity plays an important role in the pathogenesis of NS and they also demonstrate a protective role for IL-13 in the children uith NS.

  2. Influence of Irradiated Peripheral Blood Mononuclear Cells on Both Ex Vivo Proliferation of Human Natural Killer Cells and Change in Cellular Property

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    María Delso-Vallejo

    2017-07-01

    Full Text Available Clinical studies with adoptive immunotherapy using allogeneic natural killer (NK cells showed feasibility, but also limitation regarding the transfused absolute cell numbers. First promising results with peripheral blood mononuclear cells (PBMCs as feeder cells to improve the final cell number need further optimization and investigation of the unknown controlling mechanism in the cross-talk to NK cells. We investigated the influence of irradiated autologous PBMCs to boost NK cell proliferation in the presence of OKT3 and IL-2. Our findings demonstrate a requirement for receptor–ligand interactions between feeders and NK cells to produce soluble factors that can sustain NK cell proliferation. Thus, both physical contact between feeder and NK cells, and soluble factors produced in consequence, are required to fully enhance NK cell ex vivo proliferation. This occurred with an indispensable role of the cross-talk between T cells, monocytes, and NK cells, while B cells had no further influence in supporting NK cell proliferation under these co-culture conditions. Moreover, gene expression analysis of highly proliferating and non-proliferating NK cells revealed important phenotypic changes on 5-day cultured NK cells. Actively proliferating NK cells have reduced Siglec-7 and -9 expression compared with non-proliferating and resting NK cells (day 0, independently of the presence of feeder cells. Interestingly, proliferating NK cells cultured with feeder cells contained increased frequencies of cells expressing RANKL, B7-H3, and HLA class II molecules, particularly HLA-DR, compared with resting NK cells or expanded with IL-2 only. A subset of HLA-DR expressing NK cells, co-expressing RANKL, and B7-H3 corresponded to the most proliferative population under the established co-culture conditions. Our results highlight the importance of the crosstalk between T cells, monocytes, and NK cells in autologous feeder cell-based ex vivo NK cell expansion

  3. A method for production and determination of histamine releasing activity from human peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Kampen, G T; Poulsen, L K; Reimert, C M

    1997-01-01

    Histamine releasing factors, i.e. cytokines capable of inducing histamine release from basophils or mast cells, have been suggested to be involved in the pathogenesis of, for example, allergic late-phase reactions. Here we describe a controlled method for production and determination of histamine......). The preparations of HRA induced dose- and Ca2+-dependent histamine release from leukocytes. Supernatants of parallel cultures of unstimulated MNC did not induce histamine release. The HRA was neither due to exogenous histamine releasing compounds (e.g. Con A) nor to residual histamine in the preparations of HRA....... The kinetics of HRA induced histamine release (half-maximal release after > 40 min) were slower and more protracted than those of anti-IgE induced histamine release. However, based on a comparison between HRA induced histamine release from leukocytes and purified (97%) basophils, this did not appear to be due...

  4. Electronic Interfacing with Living Cells

    Science.gov (United States)

    Fleming, James T.

    The direct interfacing of living cells with inorganic electronic materials, components or systems has led to the development of two broad categories of devices that can (1) transduce biochemical signals generated by biological components into electrical signals and (2) transduce electronically generated signals into biochemical signals. The first category of devices permits the monitoring of living cells, the second, enables control of cellular processes. This review will survey this exciting area with emphasis on the fundamental issues and obstacles faced by researchers. Devices and applications that use both prokaryotic (microbial) and eukaryotic (mammalian) cells will be covered. Individual devices described include microbial biofuel cells that produce electricity, bioelectrical reactors that enable electronic control of cellular metabolism, living cell biosensors for the detection of chemicals and devices that permit monitoring and control of mammalian physiology.

  5. Antioxidant compounds in the seaweed Gelidiella acerosa protects human Peripheral Blood Mononuclear Cells against TCDD induced toxicity.

    Science.gov (United States)

    Ilavarasi, K; Chermakani, P; Arif Nisha, S; Sheeja Malar, D; Pandima Devi, K

    2015-04-01

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental toxin formed as an unintentional by-product of incomplete combustion. Several therapeutic approaches have evolved to combat its toxicity since it elicits immunotoxicity, neurotoxicity, hepatotoxicity, carcinogenicity and lethality. Search for drugs from natural resources especially from seaweeds has become intense due to their enormous pharmacological potential. Hence, the present study aims at revealing the protective effect of methanolic extract of G. acerosa (MEGA) in Peripheral Blood Mononuclear Cells (PBMC) against TCDD induced toxicity, by assessing the antioxidant, anti-apoptotic and cytoprotective activities. The results of antioxidant assays suggests that MEGA reverted TCDD induced toxicity by causing an alteration in the levels of antioxidant enzymes (Catalase [CAT], Superoxide dismutase [SOD], Glutathione peroxidase [GPx], Glutathione-S-transferase [GST]) and Glutathione [GSH]. The results of lipid peroxidation assay and protein carbonyl content reveal that MEGA protects PBMC from TCDD induced macromolecular damage. MEGA was found to exhibit significant (p TCDD induced oxidative DNA damage. Levels of phase-I detoxification enzymes determined by EROD assay and semi-quantitative RT-PCR showed that TCDD up-regulates the expression of CYP1A1 and upon co-treatment with MEGA, the expression got slightly decreased suggesting its protective role. Preliminary phytochemical analysis demonstrates that the extract is rich in cardiac glycosides and terpenoids. LC-MS analysis revealed the presence of antioxidants including caffeic acid, phytol and mannoheptulose in MEGA, which could be attributed for the observed protective effect against TCDD induced toxicity.

  6. Systematic Review and Meta-Analysis of Bone Marrow-Derived Mononuclear Cells in Animal Models of Ischemic Stroke.

    Science.gov (United States)

    Vahidy, Farhaan S; Rahbar, Mohammad H; Zhu, Hongjian; Rowan, Paul J; Bambhroliya, Arvind B; Savitz, Sean I

    2016-06-01

    Bone marrow-derived mononuclear cells (BMMNCs) offer the promise of augmenting poststroke recovery. There is mounting evidence of safety and efficacy of BMMNCs from preclinical studies of ischemic stroke; however, their pooled effects have not been described. Using Preferred Reporting Items for Systematic Review and Meta-Analysis guidelines, we conducted a systematic review of preclinical literature for intravenous use of BMMNCs followed by meta-analyses of histological and behavioral outcomes. Studies were selected based on predefined criteria. Data were abstracted by 2 independent investigators. After quality assessment, the pooled effects were generated using mixed-effect models. Impact of possible biases on estimated effect size was evaluated. Standardized mean difference and 95% confidence interval for reduction in lesion volume was significantly beneficial for BMMNC treatment (standardized mean difference: -3.3; 95% confidence interval, -4.3 to -2.3). n=113 each for BMMNC and controls. BMMNC-treated animals (n=161) also had improved function measured by cylinder test (standardized mean difference: -2.4; 95% confidence interval, -3.1 to -1.6), as compared with controls (n=205). A trend for benefit was observed for adhesive removal test and neurological deficit score. Study quality score (median: 6; Q1-Q3: 5-7) was correlated with year of publication. There was funnel plot asymmetry; however, the pooled effects were robust to the correction of this bias and remained significant in favor of BMMNC treatment. BMMNCs demonstrate beneficial effects across histological and behavioral outcomes in animal ischemic stroke models. Although study quality has improved over time, considerable degree of heterogeneity calls for standardization in the conduct and reporting of experimentation. © 2016 American Heart Association, Inc.

  7. H2AX phosphorylation level in peripheral blood mononuclear cells as an event-free survival predictor for bladder cancer.

    Science.gov (United States)

    Turinetto, Valentina; Pardini, Barbara; Allione, Alessandra; Fiorito, Giovanni; Viberti, Clara; Guarrera, Simonetta; Russo, Alessia; Anglesio, Silvia; Ruo Redda, Maria Grazia; Casetta, Giovanni; Cucchiarale, Giuseppina; Destefanis, Paolo; Oderda, Marco; Gontero, Paolo; Rolle, Luigi; Frea, Bruno; Vineis, Paolo; Sacerdote, Carlotta; Giachino, Claudia; Matullo, Giuseppe

    2016-11-01

    Bladder cancer (BC) has a typical aetiology characterized by a multistep carcinogenesis due to environmental exposures, genetic susceptibility, and their interaction. Several lines of evidence suggest that DNA repair plays a role in the development and progression of BC. In particular, the study of individual susceptibility to DNA double strand breaks (DSBs) may provide valuable information on BC risk, and help to identify those patients at high-risk of either recurrence or progression of the disease, possibly personalizing both surveillance and treatment. Among the different DSB markers, the most well characterized is phosphorylation of the histone H2AX (γ-H2AX). We assessed any potential role of γ-H2AX as a molecular biomarker in a case-control study (146 cases and 146 controls) to identify individuals with increased BC risk and at high-risk of disease recurrence or progression. We investigated γ-H2AX levels in peripheral blood mononuclear cells before and after their exposure to ionizing radiation (IR). We did not find any significant difference among cases and controls. However, we observed a significant association between γ-H2AX basal levels and risk of disease recurrence or progression. In particular, both BC patients as a whole and the subgroup of non-muscle invasive BC (NMIBC) with high basal H2AX phosphorylation levels had a decreased risk of recurrence or progression (for all BC HR 0.70, 95%CI 0.52-0.94, P = 0.02; for NMIBC HR 0.68, 95%CI 0.50-0.92, P = 0.01), suggesting a protective effect of basal DSB signaling. Our data suggest that γ-H2AX can be considered as a potential molecular biomarker to identify patients with a higher risk of BC recurrence. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  8. Molecular epidemiology of different hepatitis C genotypes in serum and peripheral blood mononuclear cells in jahrom city of iran.

    Science.gov (United States)

    Ashrafi Hafez, Asghar; Baharlou, Rasoul; Mousavi Nasab, Seyed Dawood; Ahmadi Vasmehjani, Abbas; Shayestehpour, Mohammad; Joharinia, Negar; Ahmadi, Nayeb Ali

    2014-05-01

    The Hepatitis C Virus (HCV) is considered essentially hepatotropic, yet the virus compartments have also been found in important extra hepatic sites. Detection of HCV RNA in extra hepatic reservoirs such as peripheral blood mononuclear cells (PBMCs) is important for determining disease progression and treatment effectiveness. The present study aimed to determine different HCV genotypes in patients' plasma and PBMC specimens, in Jahrom city of Iran. Blood samples of 137 patients with established HCV were collected at the Honari clinic. These patients were anti-HCV and plasma HCV RNA positive. After plasma RNA extraction and obtaining a pellet of approximately 3-5 × 10(6) PBMCs, Real-time PCR was performed, using specific-genotype primers. Finally, data analysis was done by the Statistical Package for Social Sciences (SPSS) software. Subtype 3 was the most common genotype in plasma (57.7%) and PBMCs (51.1%). Subtype 1a was detected in 36.5% and 30.7% of plasma samples and PBMCs, respectively whereas subtype 4 was not detected in any of the cases. There was a genotype difference between plasma and PBMCs of 12.4% of patients. In four patients no genotype was detected in their plasma but genotype 3 was detected in the PBMCs. It is suggested that determination of the target genotype by plasma subtyping for choosing the proper antiviral therapy is essential but may result in therapy failure. HCV genotyping in PBMC samples, along with plasma specimens, might be more beneficial. Therefore determining the HCV genotype in PBMCs, before beginning the therapy is useful due to the possibility of occult infection detection.

  9. Fruit and vegetable consumption and proinflammatory gene expression from peripheral blood mononuclear cells in young adults: a translational study

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    Puchau Blanca

    2010-05-01

    Full Text Available Abstract Background Fruits and vegetables are important sources of fiber and nutrients with a recognized antioxidant capacity, which could have beneficial effects on the proinflammatory status as well as some metabolic syndrome and cardiovascular disease features. The current study assessed the potential relationships of fruit and vegetable consumption with the plasma concentrations and mRNA expression values of some proinflammatory markers in young adults. Methods One-hundred and twenty healthy subjects (50 men/70 women; 20.8 ± 2.6 y; 22.3 ± 2.8 kg/m2 were enrolled. Experimental determinations included anthropometry, blood pressure and lifestyle features as well as blood biochemical and inflammatory measurements. The mRNA was isolated from peripheral blood mononuclear cells (PBMC and the gene expression concerning selected inflammatory markers was assessed by quantitative real-time PCR. Nutritional intakes were estimated by a validated semi-quantitative food-frequency questionnaire. Results The highest tertile of energy-adjusted fruit and vegetable consumption (>660 g/d was associated with lower plasma concentrations of C-reactive protein (CRP and homocysteine and with lower ICAM1, IL1R1, IL6, TNFα and NFκB1 gene expression in PBMC (P for trend ICAM1, TNFα and NFκB1 gene expression in PBMC showed a descending trend as increased fiber intake (>19.5 g/d from fruits and vegetables (P for trend 11.8 mmol/d of dietary total antioxidant capacity showed lower plasma CRP and mRNA values of ICAM1, IL1R1, IL6, TNFα and NFκB1 genes (P for trend Conclusion A higher fruit and vegetable consumption was independently associated not only with reduced CRP and homocysteine concentrations but also with a lower mRNA expression in PBMC of some relevant proinflammatory markers in healthy young adults.

  10. Fermented Papaya Preparation Restores Age-Related Reductions in Peripheral Blood Mononuclear Cell Cytolytic Activity in Tube-Fed Patients

    Science.gov (United States)

    Fujita, Yuhzo; Tsuno, Haruo; Nakayama, Jiro

    2017-01-01

    Tube-fed elderly patients are generally supplied with the same type of nutrition over long periods, resulting in an increased risk for micronutrient deficiencies. Dietary polyphenols promote immunity and have anti-inflammatory, anti-carcinogenic, and anti-oxidative properties. Carica papaya Linn. is rich in several polyphenols; however, these polyphenols are poorly absorbed from the digestive tract in their original polymerized form. Therefore, we determined the molecular components of a fermented Carica papaya Linn. preparation, as well as its effects on immunity and the composition of gut microbiota in tube-fed patients. Different doses of the fermented C. papaya L. preparation were administered to three groups of tube-fed patients for 30 days. Its effects on fecal microbiota composition and immunity were assessed by 16S rRNA gene sequencing and immune-marker analysis, respectively. The chemical composition of the fermented C. papaya L. preparation was analyzed by capillary electrophoresis- and liquid chromatography- time of flight mass spectrometry. The fermented C. papaya L. preparation restored peripheral blood mononuclear cell (PBMC) cytolytic activity; however, no other biomarkers of immunity were observed. Treatment with the preparation (9 g/day) significantly reduced the abundance of Firmicutes in the fecal microbiota. In particular, treatment reduced Clostridium scindens and Eggerthella lenta in most patients receiving 9 g/day. Chemical analysis identified low-molecular-weight phenolic acids as polyphenol metabolites; however, no polymerized, large-molecular-weight molecules were detected. Our study indicates that elderly patients who are tube-fed over the long-term have decreased PBMC cytolytic activity. In addition, low-molecular-weight polyphenol metabolites fermented from polymerized polyphenols restore PBMC cytolytic activity and modulate the composition of gut microbiota in tube-fed patients. PMID:28060858

  11. Effects of eccentric exercise on toll-like receptor 4 signaling pathway in peripheral blood mononuclear cells.

    Science.gov (United States)

    Fernandez-Gonzalo, Rodrigo; De Paz, José A; Rodriguez-Miguelez, Paula; Cuevas, María J; González-Gallego, Javier

    2012-06-01

    This study aimed to investigate the response of the toll-like receptor 4 (TLR4) signaling pathway to an acute bout of eccentric exercise, and to assess whether eccentric training attenuated the effects induced by acute eccentric exercise. Twenty men (22.4 ± 0.5 yr) were divided into a control group (CG, n = 8) and a training group (TG, n = 12). Both groups performed two acute eccentric bouts on a squat machine in a 9-wk interval. During this time, TG followed a 6-wk eccentric training program (3 session/wk; 3-5 sets of 10 repetitions with loads ranging between the 40 and 50% of maximal isometric voluntary contraction). CD14, TLR4, and TNF-α mRNA levels, and CD14, TLR4, myeloid differentiation factor 88, tumor necrosis factor receptor-associated factor 6, TIR-domain-containing adapter-inducing interferon-β, phospho-IκB kinases, phospho-IκB, phospho-ERK-1/2, and TNF-α protein concentration were measured in peripheral blood mononuclear cells, before, immediately, and 2 h after each eccentric bout. The first acute eccentric bout triggered a proinflammatory response mediated by an upregulation of all of the factors measured within the TLR4 signaling pathway. Following the training period and after the second acute bout, CG showed a similar proinflammatory response than that seen after the first bout. However, the eccentric training intervention decreased significantly the protein concentration of all factors analyzed in TG compared with results obtained after the first bout. These results suggest that the TLR4-signaling pathway plays a critical role in the proinflammatory response seen after acute eccentric exercise. This response was attenuated after an eccentric training program through myeloid differentiation factor 88-dependent and -independent pathways.

  12. [Anesthetic management of patients with mental retardation during autologous transplantation of peripheral blood mononuclear cells outside the operating room].

    Science.gov (United States)

    Li, Meng-meng; Zhang, Qing-hong; Liu, Ying-hui; Yue, Li; Liu, Zhi-hui; Hao, Jian-hua

    2011-06-01

    To observe the anesthetic effect and safety of differential airway management in patients with mental retardation (MR) during autologous peripheral blood mononuclear cell transplantation (APBMCT) outside the operating room. In this prospective study, 30 uncooperative patients with MR receiving total intravenous anesthesia (TIVA) with propofol for APBMCT were randomized into 3 groups with monitored anesthesia care (MAC group), inserted classic laryngeal mask airway under general anesthesia (LMA group), or endotracheal tube placement (ETT group). The blood pressure (BP), heart rate (HR), SpO(2) and pH, PaCO(2), and HCO(3)(-) were monitored at 5 min and 1 h after anesthesia, before completion of the operation and at 1 h after the operation. The total operative time, dosage of propofol, awake time and body movement during the procedure were recorded. Compared with LMA and ETT groups, the MAC groups showed a significantly increased total dosage of propofol (66.07±5.41, 35.83±5.80, and 34.61±3.68 g·kg(-1)·min(-1), respectively, Pawake time (16.82±7.60, 4.31±1.32, and 3.73±1.33 min, P0.05). At 1 h after anesthesia, the pH in MAC group decreased markedly compared with that in LMA and ETT groups (PMAC group and remained so till the end of the surgery (P<0.05). Endotracheal tube placement is safer than laryngeal mask airway placement and monitored anesthesia care in patients with MR during APBMCT, and allows rapid onset of sedation with minimal cardiovascular responses, body movement and recovery, therefore is more suitable in the setting outside the operating room.

  13. Dynamic changes of HBV DNA in serum and peripheral blood mononuclear cells of chronic hepatitis patients after lamivudine treatment

    Institute of Scientific and Technical Information of China (English)

    Chang-Zheng Ke; Yue Chen; Zuo-Jiong Gong; Zhong-Ji Meng; Li Liu; Ze-Jiu Ren; Zuo-Hua Zhou

    2006-01-01

    AIM: To study the dynamic changes of hepatits B virus (HBV) DNA in serum and peripheral blood mononuclear cells (PBMCs) of patients after lamivudine therapy.METHODS: A total of 72 patients with chronic HBV infection were included in this study. All patients were confirmed to have the following conditions: above 16 years of age, elevated serum alanine amonotransferase (ALT), positive hepatitis B e antigen (HBeAg), positive HBV DNA in serum and PBMCs, negative antibodies against HAV, HCV, HDV, HEV. Other possible causes of chronic liver damages, such as drugs, alcohol and autoimmune diseases were excluded. Seventy-two cases were randomly divided into lamivudine treatment group (n = 42) and control group (n = 30). HBV DNA was detected both in serum and in PBMCs by fluorescence quantitative polymerase chain reaction (PCR), during and after lamivudine treatment.RESULTS: In the treatment group, HBV DNA became negative both in serum and in PBMC, of 38 and 25 out of 42 cases respectively during the 48 Wk oflamivudine treatment, the negative rate was 90.5% and 59.5% respectively. In the control group, the negative rate was 23.3% and 16.7% respectively. It was statistically significant at 12, 24 and 48 wk as compared with the control group (P < 0.005). The average conversion period of HBV DNA was 6 wk (2-8 wk) in serum and 16 wk (8-24 wk) in PBMC.CONCLUSION: Lamivudine has remarkable inhibitory effects on HBV replication both in serum and in PBMCs.The inhibitory effect on HBV DNA in PBMCs is weaker than that in serum.

  14. Quantification of miltefosine in peripheral blood mononuclear cells by high-performance liquid chromatography-tandem mass spectrometry

    Science.gov (United States)

    Kip, A.E.; Rosing, H.; Hillebrand, M.J.X.; Castro, M.M.; Gomez, M.A.; Schellens, J.H.M.; Beijnen, J.H.; Dorlo, T.P.C.

    2015-01-01

    Phagocytes, the physiological compartment in which Leishmania parasites reside, are the main site of action of the drug miltefosine, but the intracellular pharmacokinetics of miltefosine remain unexplored. We developed a bioanalytical method to quantify miltefosine in human peripheral blood mononuclear cells (PBMCs), expanding from an existing high performance liquid chromatography-tandem mass spectrometry method for the quantification of miltefosine in plasma. The method introduced deuterated miltefosine as an internal standard. Miltefosine was extracted from PBMC pellets by addition of 62.5% methanol. Supernatant was collected, evaporated and reconstituted in plasma. Chromatographic separation was performed on a reversed phase C18 column and detection with a triple-quadrupole mass spectrometer. Miltefosine was quantified using plasma calibration standards ranging from 4 to 1000 ng/mL. This method was validated with respect to its PBMC matrix effect, selectivity, recovery and stability. No matrix effect could be observed from the PBMC content (ranging from 0.17 to 26.3 × 106 PBMCs) reconstituted in plasma, as quality control samples were within 3.0% of the nominal concentration (precision less than 7.7%). At the lower limit of quantitation of 4 ng/mL plasma, corresponding to 0.12 ng/106 PBMCs in a typical clinical sample, measured concentrations were within 8.6% of the nominal value. Recovery showed to be reproducible as adding additional pre-treatment steps did not increase the recovery with more than 9%. This method was successfully applied to measure intracellular miltefosine concentrations in PBMC samples from six cutaneous leishmaniasis patients up to one month post-treatment. PMID:26160472

  15. Differential expression of microRNA in peripheral blood mononuclear cells as specific biomarker for major depressive disorder patients.

    Science.gov (United States)

    Fan, Hui-min; Sun, Xin-yang; Guo, Wei; Zhong, Ai-fang; Niu, Wei; Zhao, Lin; Dai, Yun-hua; Guo, Zhong-min; Zhang, Li-yi; Lu, Jim

    2014-12-01

    Currently, diagnosis and treatment of major depressive disorder (MDD) are based on the patients' description of symptoms, mental status examinations, and clinical behavioral observations, which increases the chance of misdiagnosis. There is a serious need to find a practical biomarker for the proper diagnosis of MDD. This study aimed to explore the possibility of microRNA (miRNA) in peripheral blood mononuclear cells (PBMCs) as specific blood-based biomarker for MDD patients. By using an Affymetrix array that covers 723 human miRNAs, we identified 26 miRNAs with significant changes in expression in PBMCs of MDD patients. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis in a larger cohort of 81 MDD patients and 46 healthy controls confirmed that the expression levels of 5 miRNAs (miRNA-26b, miRNA-1972, miRNA-4485, miRNA-4498, and miRNA-4743) were up-regulated. By receiver operating characteristic (ROC) curve analysis, the combining area under the ROC curve (AUC) of these five miRNAs was 0.636 [95% confidence interval (CI): 0.58-0.90]. MiRNA target gene prediction and functional annotation analysis showed that there was a significant enrichment in several pathways associated with nervous system and brain functions, supporting the hypothesis that differentially-regulated miRNAs may be involved in mechanism underlying development of MDD. We conclude that altered expression of miRNAs in PMBCs might be involved in multiple stages of MDD pathogenesis, and thus might be able to serve as specific biomarker for diagnosis of MDD.

  16. Synthesis of Th17 cytokines in the culture of peripheral blood mononuclear cells stimulated with Borrelia burgdorferi sensu lato

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    Sambor Grygorczuk

    2016-06-01

    Full Text Available [b]Introduction and objective. [/b]Th17 lymphocytes and their cytokines, interleukin 17A (IL-17A, IL-17F and IL-22, participate in the response to extracellular bacteria and in the autoimmunity and may be engaged in the pathogenesis of Lyme borreliosis. Concentrations were measured of IL-17A, IL-17F and IL-22 in the supernatant of the peripheral blood mononuclear cells (PBMC culture stimulated with [i]Borrelia burgdorferi sensu lato[/i] ([i]B. burgdorferi[/i]. [b]Materials and method.[/b] The study group consisted of 13 patients with early disseminated and late Lyme borreliosis and a control group of 7 healthy persons. PBMC cultures were stimulated for 48 hours with [i]B. burgdorferi [/i]spirochetes of three pathogenic species: [i]B. burgdorferi[/i] sensu stricto, B. afzelii or B. garinii, in the multiplicity of infection 10:1. Concentrations of Th17 cytokines IL-17A, IL-17F and IL-22, as well as Th2/immunoregulatory cytokine IL-10 were measured with ELISA assays. [b]Results. [/b]Expression of IL-17A, IL-17F and IL-22 increased under stimulation, simultaneously with the increased IL-10 expression. Concentration of IL-17F tended to be lower in early neuroborreliosis than in late Lyme borreliosis and than in controls. [i]B. afzelii[/i] elicited higher expression of IL-17A than the other two species. [b]Conclusions.[/b] IL-17A, IL-17F and IL-22 are synthesized simultaneously by PBMC stimulated with [i]B. burgdorferi[/i]. There is no antagonism between Th17 response and IL-10 expression. The role of Th17 cytokines seems to differ depending on the clinical stage of Lyme borreliosis and on the [i]B. burgdorferi[/i] species.

  17. CRP and TNF-α  Induce PAPP-A Expression in Human Peripheral Blood Mononuclear Cells

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    Weiping Li

    2012-01-01

    Full Text Available Objective. The effects of C-reactive protein (CRP and tumor necrosis factor-α (TNF-α on pregnancy-associated plasma protein-A (PAPP-A expression in human peripheral blood mononuclear cells (PBMCs require further investigation. Methods. The PAPP-A levels in culture supernatants, PAPP-A mRNA expression, and cellular PAPP-A expression were measured in human PBMCs isolated from fresh blood donations provided by 6 healthy volunteers (4 donations per volunteer. Analyses were conducted by ultrasensitive ELISA, western blotting, and RT-PCR following stimulation with CRP or TNF-α cytokines. Results. PAPP-A mRNA and protein levels after CRP stimulation peaked at 24 hours, whereas peak PAPP-A mRNA and protein levels were achieved after TNF-α stimulation at only 2 and 8 hours, respectively. These findings indicate the dose-dependent effect of CRP and TNF-α stimulation. Actinomycin D treatment completely prevented CRP and TNF-α induction of PAPP-A mRNA and protein expression. Additionally, nuclear factor- (NF- κB inhibitor (BAY11-7082 potently inhibited both CRP and TNF-α stimulated PAPP-A mRNA and protein expression. Conclusions. Human PBMCs are capable of expressing PAPP-A in vitro, expression that may be regulated by CRP and TNF-α through the NF-κB pathway. This mechanism may play a significant role in the observed increase of serum PAPP-A levels in acute coronary syndrome (ACS.

  18. Treatment of osteonecrosis of the femoral head by percutaneous decompression and autologous bone marrow mononuclear cell infusion

    Institute of Scientific and Technical Information of China (English)

    YAN Zuo-qin; CHEN Yun-su; LI Wen-jun; YANG Yi; HUO Jian-zhong; CHEN Zheng-rong; SHI Jian-hui; GE Jun-bo

    2006-01-01

    Objective:To evaluate the clinical efficacy and safety of the treatment of osteonecrosis of the femoral head by percutaneous decompression and autologous bone marrow mononuclear cell (BMCs) infusion.Methods: 44 hips in 28 patients with avascular necrosis at early stage were treated by percutaneous multiple holes decompression followed by autologous BMCs infusion. Autologous BMCs were concentrated from bone marrow that was taken from the posterior iliac crest of the patient. Patients were followed up at least 2 years. The results were determined by the changes in the Harris hip score and the progression in the radiograghic stages.Results: No complications were observed after the operation. Before operation, there were stage Ⅰ of femoral head necrosis in 8 hips, stage Ⅱ in 15 hips, stage Ⅲ in 14hips, stage Ⅳ in 7 hips, and the postoperative stages at the most recent follow-up were stage O in 1 hip, stage Ⅰ in 6hips, stage Ⅱ in 13 hips, stage Ⅲ in 13 hips, stage Ⅳ in 7hips, stage Ⅴ in 4 hips. The mean preoperative Harris hip score was 58 (46-89), and improved to 86 (70-94)postoperatively. All the femoral head collapsed preoperatively showed that the necrotic size was at least more than 30 %.Conclusions: Percutaneous multiple holes decompression combined with autologous BMCs is a new way to treat avascular necrosis of the femoral head. The earlier the stage, the better the result. A randomized prospective study needed to compare with routine core decompression in the future.

  19. Correlation analyses revealed global microRNA-mRNA expression associations in human peripheral blood mononuclear cells.

    Science.gov (United States)

    Wang, Lan; Zhu, Jiang; Deng, Fei-Yan; Wu, Long-Fei; Mo, Xing-Bo; Zhu, Xiao-Wei; Xia, Wei; Xie, Fang-Fei; He, Pei; Bing, Peng-Fei; Qiu, Ying-Hua; Lin, Xiang; Lu, Xin; Zhang, Lei; Yi, Neng-Jun; Zhang, Yong-Hong; Lei, Shu-Feng

    2017-09-06

    MicroRNAs (miRNAs) can regulate gene expression through binding to complementary sites in the 3'-untranslated regions of target mRNAs, which will lead to existence of correlation in expression between miRNA and mRNA. However, the miRNA-mRNA correlation patterns are complex and remain largely unclear yet. To establish the global correlation patterns in human peripheral blood mononuclear cells (PBMCs), multiple miRNA-mRNA correlation analyses and expression quantitative trait locus (eQTL) analysis were conducted in this study. We predicted and achieved 861 miRNA-mRNA pairs (65 miRNAs, 412 mRNAs) using multiple bioinformatics programs, and found global negative miRNA-mRNA correlations in PBMC from all 46 study subjects. Among the 861 pairs of correlations, 19.5% were significant (P correlation network was complex and highlighted key miRNAs/genes in PBMC. Some miRNAs, such as hsa-miR-29a, hsa-miR-148a, regulate a cluster of target genes. Some genes, e.g., TNRC6A, are regulated by multiple miRNAs. The identified genes tend to be enriched in molecular functions of DNA and RNA binding, and biological processes such as protein transport, regulation of translation and chromatin modification. The results provided a global view of the miRNA-mRNA expression correlation profile in human PBMCs, which would facilitate in-depth investigation of biological functions of key miRNAs/mRNAs and better understanding of the pathogenesis underlying PBMC-related diseases.

  20. Influence of Transplantation of Allogenic Bone Marrow Mononuclear Cells on the Left Ventricular Remodeling of Rat after Acute Myocardial Infarction

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    To probe into the influence of transplantation of allogenic bone marrow mononuclear cells (BM-MNCs) on the left ventricular remodeling of rat after acute myocardial infarction (AMD,60 male Wistar rats were evenly divided into three groups at random: control group 1, control group 2and transplantation group. In control group 1, chest was opened without ligation of coronary artery;in control group 2 and transplantation group, the left anterior descending branch of coronary artery was ligated to establish AMI model. Prepared culture medium and allogenic BM-MNCs suspension were respectively implanted the surrounding area of infracted cardiac muscle via epicardium of control group 2 and transplantation group. Four weeks after the operation, the osteopontin gene (OPN mRNA, P<0.01), type Ⅰ collagen (P<0.01) and angiotensin Ⅱ (AngⅡ, P<0.01) content in the left ventricular non-infracted myocardium, and the Ang Ⅱ density in blood plasma (P<0.05) of transplantation group and control group 2 were all significantly higher than that of control group 1. In the transplantation group, the myocardial OPN mRNA, type Ⅰ collagen and Ang Ⅱ content of non-infracted zone in left ventricle, and the Ang Ⅱ concentration in blood plasma were all significantly lower than those of control group 2 (P<0.05 for all). It is concluded that allogenic BM-MNCs transplantation may ease left ventricular remodeling after AMI by inhibiting the synthesis of type Ⅰ collagen in the cardiac muscle and down-regulating the expression of Ang Ⅱ and OPN gene.

  1. Feasibility and safety of autologous bone marrow mononuclear cell transplantation in patients with advanced chronic liver disease

    Institute of Scientific and Technical Information of China (English)

    Andre Castro Lyra; Bernd Genser; Ricardo Ribeiro dos Santos; Luiz Guilherme Costa Lyra; Milena Botelho Pereira Soares; Luiz Flavio Maia da Silva; Marcos Fraga Fortes; André Goyanna Pinheiro Silva; Augusto César de Andrade Mota; Sheilla A Oliveira; Eduardo Lorens Braga; Wilson Andrade de Carvalho

    2007-01-01

    AIM:To evaluate the safety and feasibility of bone marrow cell(BMC)transplantation in patients with chronic liver disease on the waiting list for liver transplantation.METHODS:Ten patients(eight males)with chronic liver disease were enrolled to receive infusion of autologous bone marrow-derived cells.Seven patients were classified as Child-Pugh B and three as Child-Pugh C.Baseline assessment included complete clinical and laboratory evaluation and abdominal MRI.Approximately 50 mL of bone marrow aspirate was prepared by centrifugation in a ficoll-hypaque gradient.At least of 100 millions of mononuclear-enriched BMCs were infused into the hepatic artery using the routine technique for arterial chemoembolization for liver tumors.Patients were followed up for adverse events up to 4 mo.RESULTS:The median age of the patients was 52 years(range 24-70 years).All patients were discharged 48 h after BMC infusion.Two patients complained of mild pain at the bone marrow needle puncture site.No other complications or specific side effects related to the procedure were observed.Bilirubin levels were lower at 1(2.19 ± 0.9)and 4 mo(2.10 ± 1.0)after cell transplantation that baseline levels(2.78 ± 1.2).Albumin levels 4 mo after BMC infusion(3.73 ± 0.5)were higher than baseline levels(3.47 ± 0.5).International normalized ratio(INR)decreased from 1.48(SD = 0.23)to 1.43(SD = 0.23)one month after cell transplantation.CONCLUSION:BMC infusion into hepatic artery of patients with advanced chronic liver disease is safe and feasible.In addition,a decrease in mean serum bilirubin and INR levels and an increase in albumin levels are observed.Our data warrant further studies in order to evaluate the effect of BMC transplantation in patients with advanced chronic liver disease.

  2. Modulation of Human Peripheral Blood Mononuclear Cell Signaling by Medicinal Cannabinoids

    Science.gov (United States)

    Utomo, Wesley K.; de Vries, Marjan; Braat, Henri; Bruno, Marco J.; Parikh, Kaushal; Comalada, Mònica; Peppelenbosch, Maikel P.; van Goor, Harry; Fuhler, Gwenny M.

    2017-01-01

    Medical marijuana is increasingly prescribed as an analgesic for a growing number of indications, amongst which terminal cancer and multiple sclerosis. However, the mechanistic aspects and properties of cannabis remain remarkably poorly characterized. In this study we aimed to investigate the immune-cell modulatory properties of medical cannabis. Healthy volunteers were asked to ingest medical cannabis, and kinome profiling was used to generate comprehensive descriptions of the cannabis challenge on inflammatory signal transduction in the peripheral blood of these volunteers. Results were related to both short term and long term effects in patients experimentally treated with a medical marijuana preparation for suffering from abdominal pain as a result of chronic pancreatitis or other causes. The results reveal an immunosuppressive effect of cannabinoid preparations via deactivation of signaling through the pro-inflammatory p38 MAP kinase and mTOR pathways and a concomitant deactivation of the pro-mitogenic ERK pathway. However, long term cannabis exposure in two patients resulted in reversal of this effect. While these data provide a powerful mechanistic rationale for the clinical use of medical marijuana in inflammatory and oncological disease, caution may be advised with sustained use of such preparations. PMID:28174520

  3. Lactic acid bacteria inhibit TH2 cytokine production by mononuclear cells from allergic patients.

    Science.gov (United States)

    Pochard, Pierre; Gosset, Philippe; Grangette, Corinne; Andre, Claude; Tonnel, André-Bernard; Pestel, Joël; Mercenier, Annick

    2002-10-01

    Among factors potentially involved in the increased prevalence of allergic diseases, modification of the intestinal bacteria flora or lack of bacterial stimulation during childhood has been proposed. Lactic acid bacteria (LAB) present in fermented foods or belonging to the natural intestinal microflora were shown to exert beneficial effects on human health. Recent reports have indicated their capacity to reduce allergic symptoms. The purpose of this investigation was to determine the effect of LAB on the production of type 2 cytokines, which characterize allergic diseases. PBMCs from patients allergic to house dust mite versus those from healthy donors were stimulated for 48 hours with the related Dermatophagoides pteronyssinus allergen or with a staphylococcal superantigen. The effect of LAB preincubation was assessed by measuring the type 2 cytokine production by means of specific ELISA. The tested gram-positive LAB were shown to inhibit the secretion of T(H)2 cytokines (IL-4 and IL-5). This effect was dose dependent and was observed irrespective of the LAB strain used. No significant inhibition was induced by the control, gram-negative Escherichia coli TG1. Interestingly, LAB reduced the T(H)2 cytokine production from allergic PBMCs specifically restimulated with the related allergen. The inhibition mechanism was shown to be dependent on antigen-presenting cells (ie, monocytes) and on the involvement of IL-12 and IFN-gamma. The tested LAB strains were demonstrated to exhibit an anti-T(H)2 activity, and thus different strains of this family might be useful in the prevention of allergic diseases.

  4. [Significance of detecting the EBV-DNA level in peripheral blood mononuclear cells and the EBV-infected cell type in patients with chronic active EBV infection].

    Science.gov (United States)

    Xing, Yan; Song, Hong-mei; Wu, Xiao-yan; Wang, Wei; Wei, Min

    2011-07-01

    To study the difference in the EBV-DNA level in peripheral blood mononuclear cells (PBMC) and the type of Epstein-Barr virus (EBV)-infected cells in pediatric patients with chronic active EBV (CAEBV) infection, acute EBV infection (AEBV) and healthy children, and to analyze the relationship between the above difference and the clinical manifestation of CAEBV. Real-time fluorescent quantitative polymerase chain reaction (PCR) was used to detect the EBV-DNA levels in peripheral blood mononuclear cells (PBMC) in 12 normal children, 10 pediatric patients with CAEBV infection and 13 pediatric patients with AEBV infection in our hospital between March 2004 and April 2008. Immunomagnetic bead cell fractionation and fluorescent in situ hybridization (FISH) by EBV encoding RNA-1 ( EBER-1) probe were used in the healthy children, EBV-DNA positive CAEBV patients and AEBV patients to detect the type of EBV-infected cells. The average EBV-DNA level in CAEBV patients' PBMC was (6.8 x 10(7) +/- 1.1 x 10(8)) copies/ml, while the average EBV-DNA level of AEBV patients' PBMC was (1.3 x 10(6) +/- 1.6 x 10(6)) copies/ml. The average EBV-DNA level of CAEBV infected patients' PBMC was significantly higher than that of AEBV infected patients' PBMC (Pblood cells. In 1 CAEBV patient the infection was mainly found in NK cells, who presented with hypersensitivity to mosquito biting and high IgE level (2500 U/ml). But EBV in seven AEBV patients infection was found only in B cells who presented with only IM for one time and no EBV-infected PBMC were found in the remaining 6 healthy children. There are much more EBV replications and different EBV-infected cell types in CAEBV patients. Detection of EBV-DNA level by real-time fluorescent quantitative PCR and the detection of the type of EBV-infected cells may help in diagnosis, treatment and development evaluation of children with CAEBV infection.

  5. 4-1BB gene expression in peripheral blood mononuclear cells from orthotopic liver transplant recipients with graft acceptance

    Institute of Scientific and Technical Information of China (English)

    万云乐; 郑树森; 贾长库; 杨家印; 金晓凌; 赵志成

    2003-01-01

    Objective To investigate the gene expression of 4-1BB in peripheral blood mononuclear cells (PBMCs) and the possible significance of the 4-1BB pathway after clinical orthotopic liver transplantation (OLT). Methods 4-1BB mRNA levels in PBMCs from 22 OLT patients were analyzed by RT-PCR. 4-1BB protein expressed on the surface of CD4+ and CD8+ T cells were detected by flow cytometry, and visualized with direct immunofluorescence and confocal fluorescence microscopy. Patients with primary liver cancer (PLC) and healthy volunteers served as controls. Six cases of recently performed liver transplantation were also observed in this study.Results 4-1BB mRNA was detected in PBMCs from both liver transplant patients with long-term graft acceptance (22 cases) and from transplant patients on day 1 to day 3 post-transplantation (6 cases), but was not found in PBMCs from transplant patients on day 7 to day 30 post-transplantation (6 cases). 4-1BB mRNA was also not found in samples from 8 of the healthy controls and 7 of the PLC patients, though very low expression was detected in the other 4 healthy volunteers and 6 PLC patients. Simultaneously, 4-1BB protein was expressed at nearly undetectable levels on CD4+ and CD8+ T cells from healthy controls, PLC patients, as well as OLT patients within the first month post-transplantation (6 cases). However, 4-1BB expression was found on the surface of CD4+ and CD8+ T cells from liver transplant patients with long-term graft acceptance. Direct immunofluorescent staining and confocal fluorescence microscopy clearly revealed evidence of 4-1BB protein on cell membranes of CD4+ and CD8+ T cells from liver transplant patients with long-term graft acceptance. Simultaneously, a significantly higher percentage of CD3+ CD25+ T cells were found in liver transplant patients with long-term graft acceptance group as compared with the healthy control group (P<0.05). The expression of 4-1BB protein on T cells did not correlate with the survival

  6. Induction of RET dependent and independent pro-inflammatory programs in human peripheral blood mononuclear cells from Hirschsprung patients.

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    Marta Rusmini

    Full Text Available Hirschsprung disease (HSCR is a rare congenital anomaly characterized by the absence of enteric ganglia in the distal intestinal tract. While classified as a multigenic disorder, the altered function of the RET tyrosine kinase receptor is responsible for the majority of the pathogenesis of HSCR. Recent evidence demonstrate a strong association between RET and the homeostasis of immune system. Here, we utilize a unique cohort of fifty HSCR patients to fully characterize the expression of RET receptor on both innate (monocytes and Natural Killer lymphocytes and adaptive (B and T lymphocytes human peripheral blood mononuclear cells (PBMCs and to explore the role of RET signaling in the immune system. We show that the increased expression of RET receptor on immune cell subsets from HSCR individuals correlates with the presence of loss-of-function RET mutations. Moreover, we demonstrate that the engagement of RET on PBMCs induces the modulation of several inflammatory genes. In particular, RET stimulation with glial-cell line derived neurotrophic factor family (GDNF and glycosyl-phosphatidylinositol membrane anchored co-receptor α1 (GFRα1 trigger the up-modulation of genes encoding either for chemokines (CCL20, CCL2, CCL3, CCL4, CCL7, CXCL1 and cytokines (IL-1β, IL-6 and IL-8 and the down-regulation of chemokine/cytokine receptors (CCR2 and IL8-Rα. Although at different levels, the modulation of these "RET-dependent genes" occurs in both healthy donors and HSCR patients. We also describe another set of genes that, independently from RET stimulation, are differently regulated in healthy donors versus HSCR patients. Among these "RET-independent genes", there are CSF-1R, IL1-R1, IL1-R2 and TGFβ-1, whose levels of transcripts were lower in HSCR patients compared to healthy donors, thus suggesting aberrancies of inflammatory responses at mucosal level. Overall our results demonstrate that immune system actively participates in the physiopathology of

  7. In vivo infiltration of mononuclear cells in squamous cell carcinoma of the head and neck correlates with the ability to expand tumour-infiltrating T cells in vitro and with the expression of MHC class I antigens on tumour cells

    DEFF Research Database (Denmark)

    Hald, J; Rasmussen, N; Claesson, Mogens Helweg

    1994-01-01

    -peptide-specific T cells in the patients. Eight out of ten expanded tumour-infiltrating lymphocyte (TIL) cultures showed T-cell-mediated cytotoxicity. "Promiscuous" cytotoxic T cell activity against the natural-killer-cell-sensitive K562 target cell line was observed in three out of ten TIL expansion......A series of 18 head and neck squamous cell carcinoma biopsies, 6 primary and 12 recurrent, were investigated for tumour-infiltrating mononuclear cells with monoclonal or polyclonal antibodies. Our results suggest that the number of T cells at the tumour edge in vivo correlates well...... with their ability to expand in vitro in the presence of high-dose interleukin-2 (2000 U/ml). High MHC class I antigen expression on tumour cells was found to be positively correlated with p53 overexpression, suggesting that p53-derived peptides, wild-type or mutated ones, presented by MHC class I antigens...

  8. RESPONSE OF MONONUCLEAR CELLS TO CANCER-ASSOCIATED ANTIGENS, HUMORAL FACTORS OF IMMUNITY, AND PATHOHISTOLOGICAL FINDINGS IN WOMEN WITH GENITAL MALIGNANCIES CERVICAL EPITHELIAL DYSPLASIA

    Directory of Open Access Journals (Sweden)

    A. I. Autenshlus

    2007-01-01

    Full Text Available Abstract. In vitro response of blood mononuclear cells to cancer-associated antigens was studied in women with genital pathology, and these results were compared with serum levels of pro- and anti-inflammatory factors of immunity, like as with histological features of genital cancer and cervical epithelial dysplasia. In vitro cellular response was regarded as positive, if relative amounts of CD8-positive lymphocytes increased by > 15% following incubation of blood mononuclears with cancer-associated antigens. Positive reaction and elevated serum levels of anti-TNFα and anti-IFNγ antibodies were associated with lesser malignancy of tumor, as proven by histological findings in the women with genital cancer. A positive cellular reaction was associated with increased serum levels of IFNγ and anti-TNFα in women with grade II–III cervical epithelial dysplasia. It is concluded about potential applicability of testing mononuclears with fetal proteins, to determine a grade of malignancy for the female genital tumors, as well as a degree of regenerative disturbances of cervical epithelium.

  9. Cultures and co-cultures of human blood mononuclear cells and endothelial cells for the biocompatibility assessment of surface modified AISI 316L austenitic stainless steel.

    Science.gov (United States)

    Stio, Maria; Martinesi, Maria; Treves, Cristina; Borgioli, Francesca

    2016-12-01

    Samples of AISI 316L austenitic stainless steel were subjected either to grinding and polishing procedure, or to grinding and then low temperature glow-discharge nitriding treatment, or to grinding, nitriding and subsequently coating with collagen-I. Nitrided samples, even if only ground, show a higher corrosion resistance in PBS solution, in comparison with ground and polished AISI 316L. Biocompatibility was evaluated in vitro by incubating the samples with either peripheral blood mononuclear cells (PBMC) or human umbilical vein endothelial cells (HUVEC), tested separately or in co-culture. HUVEC-PBMC co-culture and co-incubation of HUVEC with PBMC culture medium, after the previous incubation of PBMC with metallic samples, allowed to determine whether the incubation of PBMC with the different samples might affect HUVEC behaviour. Many biological parameters were considered: cell proliferation, release of cytokines, matrix metalloproteinases (MMPs) and sICAM-1, gelatinolytic activity of MMPs, and ICAM-1 protein expression. Nitriding treatment, with or without collagen coating of the samples, is able to ameliorate some of the biological parameters taken into account. The obtained results point out that biocompatibility may be successfully tested in vitro, using cultures of normal human cells, as blood and endothelial cells, but more than one cell line should be used, separately or in co-culture, and different parameters should be determined, in particular those correlated with inflammatory phenomena.

  10. Efficient generation of transgene-free induced pluripotent stem cells from normal and neoplastic bone marrow and cord blood mononuclear cells.

    Science.gov (United States)

    Hu, Kejin; Yu, Junying; Suknuntha, Kran; Tian, Shulan; Montgomery, Karen; Choi, Kyung-Dal; Stewart, Ron; Thomson, James A; Slukvin, Igor I

    2011-04-07

    Reprogramming blood cells to induced pluripotent stem cells (iPSCs) provides a novel tool for modeling blood diseases in vitro. However, the well-known limitations of current reprogramming technologies include low efficiency, slow kinetics, and transgene integration and residual expression. In the present study, we have demonstrated that iPSCs free of transgene and vector sequences could be generated from human BM and CB mononuclear cells using non-integrating episomal vectors. The reprogramming described here is up to 100 times more efficient, occurs 1-3 weeks faster compared with the reprogramming of fibroblasts, and does not require isolation of progenitors or multiple rounds of transfection. Blood-derived iPSC lines lacked rearrangements of IGH and TCR, indicating that their origin is non-B- or non-T-lymphoid cells. When cocultured on OP9, blood-derived iPSCs could be differentiated back to the blood cells, albeit with lower efficiency compared to fibroblast-derived iPSCs. We also generated transgene-free iPSCs from the BM of a patient with chronic myeloid leukemia (CML). CML iPSCs showed a unique complex chromosomal translocation identified in marrow sample while displaying typical embryonic stem cell phenotype and pluripotent differentiation potential. This approach provides an opportunity to explore banked normal and diseased CB and BM samples without the limitations associated with virus-based methods.

  11. Liquid Cell Transmission Electron Microscopy

    Science.gov (United States)

    Liao, Hong-Gang; Zheng, Haimei

    2016-05-01

    Liquid cell transmission electron microscopy (TEM) has attracted significant interest in recent years. With nanofabricated liquid cells, it has been possible to image through liquids using TEM with subnanometer resolution, and many previously unseen materials dynamics have been revealed. Liquid cell TEM has been applied to many areas of research, ranging from chemistry to physics, materials science, and biology. So far, topics of study include nanoparticle growth and assembly, electrochemical deposition and lithiation for batteries, tracking and manipulation of nanoparticles, catalysis, and imaging of biological materials. In this article, we first review the development of liquid cell TEM and then highlight progress in various areas of research. In the study of nanoparticle growth, the electron beam can serve both as the illumination source for imaging and as the input energy for reactions. However, many other research topics require the control of electron beam effects to minimize electron beam damage. We discuss efforts to understand electron beam-liquid matter interactions. Finally, we provide a perspective on future challenges and opportunities in liquid cell TEM.

  12. West Nile Virus Challenge Alters the Transcription Profiles of Innate Immune genes in Rabbit Peripheral Blood Mononuclear Cells

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    Muhammad Jasim eUddin

    2015-12-01

    Full Text Available The peripheral innate immune response to West Nile virus (WNV is crucial for control of virus spread to the central nervous system. Therefore, transcriptomes encoding the innate immune response proteins against WNV were investigated in peripheral blood mononuclear cells (PBMCs of New Zealand White rabbits, a recently established novel rabbit model for WNV pathogenesis studies. PBMCs were challenged with an Australian WNV strain, WNVNSW2011, in vitro and mRNA expression of selected immune response genes were quantified at 2h, 6h, 12h and 24h post infection (pi using qRT-PCR. Compared to mock-inoculated PBMCs, WNV-stimulated PBMCs expressed high levels of interferon (IFN alpha (IFNA, gamma (IFNG, IL6, IL12, IL22, CXCL10 and pentraxin 3 (PTX3 mRNA. Likewise, TLR1, 2, 3, 4, 6 and 10 mRNA became up-regulated with highest expression seen for TLR3, 4 and 6. TLRs-signalling downstream genes (MyD88, STAT1, TRAF3, IRF7 and IRF9 subsequently became up-regulated. The high expression of IFNs, TLR3, TLR4, TRAF3, STAT1, IRF7 and IRF9 are in accordance with antiviral activities, while expression of TNFA, HO1, iNOS, caspase 3 and 9 transcripts suggests the involvement of oxidative stress and apoptosis in WNV stimulated rabbit PBMCs, respectively. The level of WNVNSW2011 RNA increased at 24h pi in PBMCs challenged with virus in vitro compared to input virus. The expression dynamics of selected genes were validated in PBMCs from rabbits experimentally infected with WNV in vivo. Higher expression of IFNA, IFN beta (IFNB, IFNG, TNFA, IL6, IL22, PTX3, TLR3 & TLR4, IRF7, IRF9, STST1, TRAF3, caspase 3 and caspase 9 were seen in PBMCs from WNV infected rabbits on day 3 post intradermal virus inoculation compared to PBMCs from uninfected control rabbits. This study highlights the array of cytokines and TLRs involved in the host innate immune response to WNV in the rabbit leukocytes, and suggests that these cells may be a useful in vitro model for WNV infection study.

  13. No evidence of myocardial restoration following transplantation of mononuclear bone marrow cells in coronary bypass grafting surgery patients based upon cardiac SPECT and 18F-PET

    Directory of Open Access Journals (Sweden)

    Ünal Nermin

    2006-07-01

    Full Text Available Abstract Background We tested the hypothesis, that intramyocardial injection of mononuclear bone marrow cells combined with coronary artery bypass grafting (CABG surgery improves tissue viability or function in infarct regions with non-viable myocardium as assessed by nuclear imaging techniques. Methods Thus far, 7 patients (60 ± 10 [SD] years undergoing elective CABG surgery after a myocardial infarction were included in this study. Prior to sternotomy, bone marrow was harvested by sternal puncture. Mononuclear bone marrow cells were isolated by gradient centrifugation and resuspended in 2 ml volume of Hank's buffered salt solution. At the end of CABG surgery 10 injections of 0.2 ml each were applied to the core area and borderzones of the infarct. Global and regional perfusion and viability were evaluated by ECG-gated 99mTc-tetrofosmin myocardial single-photon emission computed tomograph (SPECT imaging and 18F-fluorodeoxyglucose positron emission tomography (FDG-PET in all study patients Results Non-viable segments indicating transmural defects were identified in 5 patients. Two patients were found to have non-transmural defects before surgery. Concomitant surgical revascularisation and bone marrow cell injection was performed in all patients without major complications. The median total injected mononuclear cell number was 7.0 × 107 (range: 0.8–20.4. At 3 months 99mTc-tetrofosmin SPECT and 18F-FDG-PET scanning showed in 5 patients (transmural defect n = 4; non-transmural defect n = 1 no change in myocardial viability and in two patients (transmural defect n = 1, non-transmural defect n = 1 enhanced myocardial viability by 75%. Overall, global and regional LV ejection fraction was not significantly increased after surgery compared with the preoperative value. Conclusion In CABG surgery patients with non-viable segments the concurrent use of intramyocardial cell transfer did not show any clear improvement in tissue viability or function by

  14. The Impact of Glyphosate, Its Metabolites and Impurities on Viability, ATP Level and Morphological changes in Human Peripheral Blood Mononuclear Cells

    Science.gov (United States)

    Kwiatkowska, Marta; Jarosiewicz, Paweł; Michałowicz, Jaromir; Koter-Michalak, Maria; Huras, Bogumiła; Bukowska, Bożena

    2016-01-01

    The toxicity of herbicides to animals and human is an issue of worldwide concern. The present study has been undertaken to assess toxic effect of widely used pesticide—glyphosate, its metabolites: aminomethylphosphonic acid (AMPA) and methylphosphonic acid and its impurities: N-(phosphonomethyl)iminodiacetic acid (PMIDA), N-methylglyphosate, hydroxymethylphosphonic acid and bis-(phosphonomethyl)amine on human peripheral blood mononuclear cells (PBMCs). We have evaluated the effect of those compounds on viability, ATP level, size (FSC-A parameter) and granulation (SSC-A parameter) of the cells studied. Human peripheral blood mononuclear cells were exposed to different concentrations of glyphosate, its metabolites and impurities (0.01–10 mM) for 4 and 24 h. It was found that investigated compounds caused statistically significant decrease in viability and ATP level of PBMCs. The strongest changes in cell viability and ATP level were observed after 24 h incubation of PBMCs with bis-(phosphonomethyl)amine, and particularly PMIDA. Moreover, all studied compounds changed cell granularity, while PMIDA and bis-(phosphonomethyl)amine altered PBMCs size. It may be concluded that bis-(phosphonomethyl)amine, and PMIDA caused a slightly stronger damage to PBMCs than did glyphosate. Changes in the parameters studied in PBMCs were observed only at high concentrations of the compounds examined, which clearly shows that they may occur in this cell type only as a result of acute poisoning of human organism with these substances. PMID:27280764

  15. Rationale and Design for the Intramyocardial Injection of Autologous Bone Marrow Mononuclear Cells for Patients with Chronic Ischemic Heart Disease and Left Ventricular Dysfunction Trial (FOCUS)

    Science.gov (United States)

    Willerson, James T.; Perin, Emerson C.; Ellis, Stephen G.; Pepine, Carl J.; Henry, Timothy D.; Zhao, David X.M.; Lai, Dejian; Penn, Marc S.; Byrne, Barry J.; Silva, G; Gee, Adrian; Traverse, Jay H.; Hatzopoulos, Antonis K.; Forder, John R.; Martin, Daniel; Kronenberg, Marvin; Taylor, Doris A.; Cogle, Christopher R.; Baraniuk, Sarah; Westbrook, Lynette; Sayre, Shelly L.; Vojvodic, Rachel W.; Gordon, David J.; Skarlatos, Sonia I.; Moyé, Lemuel A.; Simari, Robert D.

    2010-01-01

    Background The increasing worldwide prevalence of coronary artery disease (CAD) continues to challenge the medical community. Management options include medical and revascularization therapy. Despite advances in these methods, CAD is a leading cause of recurrent ischemia and heart failure, posing significant morbidity and mortality risks along with increasing health costs in a large patient population worldwide. Trial Design The Cardiovascular Cell Therapy Research Network (CCTRN) was established by the National Institutes of Health to investigate the role of cell therapy in the treatment of chronic cardiovascular disease. FOCUS is a CCTRN-designed randomized Phase II, placebo-controlled clinical trial that will assess the effect of autologous bone marrow mononuclear cells delivered transendocardially to patients with left ventricular (LV) dysfunction and symptomatic heart failure or angina. All patients need to have limiting ischemia by reversible ischemia on SPECT assessment. Results After thoughtful consideration of both statistical and clinical principles, we will recruit 87 patients (58 cell treated and 29 placebo) to receive either bone marrow–derived stem cells or placebo. Myocardial perfusion, LV contractile performance, and maximal oxygen consumption are the primary outcome measures. Conclusions The designed clinical trial will provide a sound assessment of the effect of autologous bone marrow mononuclear cells in improving blood flow and contractile function of the heart. The target population is patients with CAD and LV dysfunction with limiting angina or symptomatic heat failure. Patient safety is a central concern of the CCTRN, and patients will be followed for at least 5 years. PMID:20691824

  16. In vitro response of the human breast cancer cell line MDAMB-231 and human peripheral blood mononuclear cells exposed to {sup 60}Co at single fraction

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    Andrade, Lidia Maria; Campos, Tarcisio Passos Ribeiro de [Universidade Federal de Minas Gerais, Belo Horizonte, MG (Brazil). Dept. de Engenharia Nuclear]. E-mail: lidia.andrade@unifenas.br; Leite, M.F. [Universidade Federal de Minas Gerais, Belo Horizonte, MG (Brazil). Dept. de Fisiologia e Biofisica; Goes, A.M. [Universidade Federal de Minas Gerais, Belo Horizonte, MG (Brazil). Dept. de Bioquimica e Imunologia

    2005-10-15

    Radiotherapy using gamma rays is a common modality of breast cancer treatment. The aim of this research is to investigate the biological response of the human breast cancer cell line MDAMB-231 and human peripheral blood mononuclear cells (PBMC) exposed in vitro to {sup 60} Co irradiation at a single fraction of 10 Gy, 25 Gy and 50 Gy doses at 136,4 cGy.min{sup -1} rate. Cells were irradiated at room temperature by the Theratron 80 radiotherapy system. Biological response was evaluated through cellular viability using MTT assay and nucleus damages visualized by Propidium Iodide assay and electrophoresis agarose gel after gamma irradiation. Nucleus damages induced by {sup 60} Co irradiation were compared to damage caused by cell exposure to 10% methanol. The 50 Gy dose of irradiation did not stimulate nucleus damages at the same level as that affected by 10% methanol induction in the MDAMB-231. Further studies are necessary to understand these mechanisms in the MDAMB-231 human breast carcinoma cell line.(author)

  17. In vitro replication activity of bovine viral diarrhea virus in an epithelial cell line and in bovine peripheral blood mononuclear cells.

    Science.gov (United States)

    Turin, Lauretta; Lucchini, Barbara; Bronzo, Valerio; Luzzago, Camilla

    2012-11-01

    The present study focused on the in vitro infection of Madin-Darby bovine kidney (MDBK) cells and bovine peripheral blood mononuclear cells (PBMCs) from naÏve animals with non-cytopathic (ncp, BVDV-1b NY-1) and cytopathic (cp, BVDV-1a NADL) strains. Infections with 0.1 and 1 multiplicity of infections (MOI) and incubation times of 18 and 36 hr were compared. Twelve BVDV naÏve heifers were enrolled to collect PBMCs. The viral loads in MDBK cells and in PBMCs after in vitro infections were measured by real-time polymerase chain reaction (PCR) assays. The highest viral loads were measured at 1 MOI and 36 hr post infection in both cell systems and the lowest at 0.1 MOI and 18 hr with the exception of the cp strain NADL in PBMCs, for which the highest viral load was observed at 0.1 MOI and 36 hr. Viral load mean values were higher for the cp strain than the ncp strain irrespective of the extent of the infection period and MOI. The models of infection studied uncovered different replication activities respectively according to the biotype of virus, the cell substrate and the duration of infection. Replication tends to be higher in PBMCs, particularly at low MOIs and for the ncp strain.

  18. Stable Delivery of CCR5-Directed shRNA into Human Primary Peripheral Blood Mononuclear Cells and Hematopoietic Stem/Progenitor Cells via a Lentiviral Vector

    Science.gov (United States)

    Shimizu, Saki; Yadav, Swati Seth; An, Dong Sung

    2016-01-01

    RNAi is a powerful tool to achieve suppression of a specific gene expression and therefore it has tremendous potential for gene therapy applications. A number of vector systems have been developed to express short-hairpin RNAs (shRNAs) to produce siRNAs within mammalian T-cells, primary hematopoietic stem/progenitor cells (HSPC), human peripheral blood mononuclear cells, and in animal model systems. Among these, vectors based on lentivirus backbones have significantly transformed our ability to transfer shRNAs into nondividing cells, such as HSPC, resulting in high transduction efficiencies. However, delivery and long-term expression of shRNAs should be carefully optimized for efficient knock down of target gene without causing cytotoxicity in mammalian cells. Here, we describe our protocols for the development of shRNA against a major HIV co-receptor/chemokine receptor CCR5 and the use of lentiviral vectors for stable shRNA delivery and expression in primary human PBMC and HSPC. PMID:26472455

  19. Induction of an antitumor response using dendritic cells transfected with DNA constructs encoding the HLA-A*02:01-restricted epitopes of tumor-associated antigens in culture of mononuclear cells of breast cancer patients.

    Science.gov (United States)

    Sennikov, Sergey Vital'evich; Shevchenko, Julia Alexandrovna; Kurilin, Vasilii Vasil'evich; Khantakova, Julia Nikolaevna; Lopatnikova, Julia Anatol'evna; Gavrilova, Elena Vasil'evna; Maksyutov, Rinat Amirovich; Bakulina, Anastasiya Yur'evna; Sidorov, Sergey Vasil'evich; Khristin, Alexander Alexandrovich; Maksyutov, Amir Zakievich

    2016-02-01

    Advances in oncoimmunology related to the definition of the basic mechanisms of the formation of antitumor immune response, as well as the opening of tumor-associated antigens recognized by immune cells, allowed to start developing ways to influence the effector cells of the immune system to generate effective antitumor cytotoxic response. We investigated the possibility to stimulate an antitumor response in a culture of mononuclear cells of breast cancer patients by dendritic cells transfected with HLA-A*02:01-restricted DNA constructs. We isolated dendritic cells from peripheral blood monocytes and delivered our constructs to these cells by magnetic transfection. Additionally, a series of experiments with loading of dendritic cells with autologous tumor cell lysate antigens was conducted. We have shown that dendritic cells transfected with the HLA-A*02:01-restricted DNA constructs are effective in inducing an antitumor response in a culture of mononuclear cells of breast cancer patients. Dendritic cells transfected with DNA constructor dendritic cells loaded with lysate antigens revealed a comparable stimulated cytotoxic response of mononuclear cells to these two ways of antigen delivery. We conclude that using DNA constructs in conjunction with patient stratification by HLA type allows the application of transfected DCs as an effective method to stimulate antitumor immunity in vitro.

  20. Transplante autólogo de células mononucleares da medula óssea em úlcera de córnea experimental em cães Bone marrow mononuclear cells autotransplant in experimental corneal ulcer in dogs

    Directory of Open Access Journals (Sweden)

    Guilherme Kanciukaitis Tognoli

    2009-02-01

    Full Text Available As células mononucleares (CM da medula óssea (MO despertam grande interesse nas pesquisas sobre regeneração tecidual. O limbo é a fonte de células-tronco (CT para repor ceratócitos lesados e uma disfunção destas é denominada deficiência límbica. Essa condição é desenvolvida por diversas afecções, sendo que a queimadura por base é a mais comum. A fim de confirmar a presença das CM da MO transplantadas, a ocorrência de quimiotaxia destas e comparar histopatologicamente os grupos tratado e controle, utilizou-se um modelo experimental de úlcera de córnea associado ao autotransplante de CM. Para tanto, 16 cães machos ou fêmeas, sem raça definida, foram submetidos à úlcera experimental de córnea com papel filtro embebido em hidróxido de sódio (NaOH. Após as lesões, os animais foram submetidos a transplante subconjuntival de CM da MO, previamente marcadas com nanocristais. A avaliação pós-operatória foi realizada por imunofluorescência no sexto dia após o transplante e por histopatologia passados 15 dias do procedimento, quando foi possível notar que as CM fixaram-se na região lesionada, não sofreram quimiotaxia e, apesar de diminuírem a inflamação, não auxiliaram o processo de cicatrização corneana a curto prazo. Assim, sugerem-se estudos adicionais no transplante de CM da MO na cicatrização da córnea.Bone marrow (BM mononuclear cells (MC are a great subject in tissual regeneration. The main stem cell source to the eye is the limbus. Theses cells replace injured corneal cells, however, if the limbal stem cells are not functional, a limbal deficiency with concomitant conjunctivalization takes place. This pathological condition can be caused for several reasons, in which alkali burns are the most common. To conduct a research about transplanted BM MC presence, the cells homing and to histopathologically compare the treated and sham group, an experimental corneal ulcer model associated with MC

  1. Non-major histocompatibility complex-restricted cytotoxic activity of blood mononuclear cells stimulated with secreted mycobacterial proteins and other mycobacterial antigens

    DEFF Research Database (Denmark)

    Ravn, P; Pedersen, B K

    1994-01-01

    Several observations indicate that non-major histocompatibility complex (MHC)-restricted cytotoxicity, mediated for example by natural killer cells and lymphokine-activated killer cells, may serve as an important antimicrobial defense mechanism. The purpose of the present study was to investigate...... the influences of different mycobacterial antigens on non-MHC-restricted cytotoxicity and further to investigate the ways by which various lymphocyte subpopulations contribute to the development of this cytotoxicity. Non-MHC-restricted cytotoxicity was induced following stimulation of mononuclear cells......+ cells proliferated and expressed interleukin-2 receptors following stimulation with mycobacterial antigens. Depletion studies after antigen stimulation showed that the cytotoxic effector cells were CD16+ CD56+ and CD4-; the CD4+ cells alone did not mediate non-MHC-restricted cytotoxicity. To evaluate...

  2. Hypomethylation of long interspersed nucleotide element-1 in peripheral mononuclear cells of juvenile systemic lupus erythematosus patients in China.

    Science.gov (United States)

    Huang, Xiaolan; Su, Gaixiu; Wang, Zhen; Shangguan, Shaofang; Cui, Xiaodai; Zhu, Jia; Kang, Min; Li, Shengnan; Zhang, Ting; Wu, Fengqi; Wang, Li

    2014-03-01

    Methylation abnormalities in T lymphocytes have been reported to correlate with systemic lupus erythematosus (SLE). Previous studies identified hypomethylation in the promoter of several genes linked to SLE. Long interspersed nucleotide element-1 (LINE-1) constitutes 17-25% of the human genome, and LINE-1 hypomethylation has been reported in SLE. Limited information is available regarding LINE-1 methylation in juvenile SLE (JSLE). Methylation levels of LINE-1 in peripheral blood mononuclear cells (PBMCs) from 59 JSLE and 47 control samples were examined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Total homocysteine (tHcy) concentrations in plasma were measured by immunoassay. Significant hypomethylation of LINE-1 was observed in PBMCs from JSLE patients (60.93% in cases compared with 62.88% in controls, P = 0.001). Significant LINE-1 hypomethylation was observed in active SLE compared to controls (60.66% vs. 62.88%, P = 0.001). According to other clinical parameters, a significant correlation was found between LINE-1 methylation levels and the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2000) of the cases (r = -0.285, P = 0.032). The risk of JSLE increased with decreasing levels of LINE-1 methylation, with an odds ratio of 14.5 (95% CI: 2.8-75.6, P = 0.002). Cases had significantly higher plasma concentrations of tHcy than controls (15.11 vs. 11.02 μmol/L, P = 0.028); the correlation between LINE-1 methylation levels and tHcy was significant (r = -0.4, P = 0.013). Correlations between methylation levels of LINE-1 and complement component 3 were significant (r = 0.317, P = 0.044; r = 0.387, P = 0.031, in total JSLE and active JSLE, respectively). Hypomethylation of LINE-1 is associated with risk of JSLE, and LINE-1 methylation levels were related to disease activity and clinical manifestations. The correlation between tHcy levels and LINE-1 methylation was significant. © 2013 Asia Pacific League of

  3. Telomere Length in Peripheral Blood Mononuclear Cells of Patients on Chronic Hemodialysis Is Related With Telomerase Activity and Treatment Duration.

    Science.gov (United States)

    Stefanidis, Ioannis; Voliotis, Georgios; Papanikolaou, Vassilios; Chronopoulou, Ioanna; Eleftheriadis, Theodoros; Kowald, Axel; Zintzaras, Elias; Tsezou, Aspasia

    2015-09-01

    Telomere shortening to a critical limit is associated with replicative senescence. This process is prevented by the enzyme telomerase. Oxidative stress and chronic inflammation are factors accelerating telomere loss. Chronic hemodialysis, typically accompanied by oxidative stress and inflammation, may be also associated with replicative senescence. To test this hypothesis, we determined telomere length and telomerase activity in peripheral blood mononuclear cells (PBMCs) in a cross-sectional study. Hemodialysis patients at the University Hospital Larissa and healthy controls were studied. Telomere length was determined by the TeloTAGGG Telomere Length Assay and telomerase activity by Telomerase PCR-ELISA (Roche Diagnostics GmbH, Mannheim, Germany). We enrolled 43 hemodialysis patients (17 females; age 65.0 ± 12.7 years) and 23 controls (six females; age 62.1 ± 15.7 years). Between the two groups, there was no difference in telomere length (6.95 ± 3.25 vs. 7.31 ± 1.96 kb; P = 0.244) or in telomerase activity (1.82 ± 2.91 vs. 2.71 ± 3.0; P = 0.085). Telomere length correlated inversely with vintage of hemodialysis (r = -0.332, P = 0.030). In hemodialysis patients, positive telomerase activity correlated with telomere length (r = 0.443, P = 0.030). Only age, and neither telomere length nor telomerase activity, was an independent survival predictor (hazard ratio 1.116, 95% confidence interval 1.009-1.234, P = 0.033). In this study, telomere length and telomerase activity in PBMCs are not altered in hemodialysis patients compared with healthy controls. Long duration of hemodialysis treatment is associated with telomere shortening and positive telomerase activity with an increased telomere length in PBMCs of hemodialysis patients. The underlying mechanism and clinical implications of our findings require further investigation.

  4. Susceptibility factors and DNA adducts in peripheral blood mononuclear cells of aluminium smelter workers exposed to polycyclic aromatic hydrocarbons

    Energy Technology Data Exchange (ETDEWEB)

    Tuominen, Rainer; Warholm, Margareta; Rannug, Agneta [Institute of Environmental Medicine, Karolinska Institutet, Stockholm (Sweden); National Institute for Working Life, Stockholm (Sweden); Baranczewski, Pawel; Moeller, Lennart [Center for Nutrition and Toxicology, Karolinska Institutet, Huddinge (Sweden); Hagmar, Lars [Department of Occupational and Environmental Medicine, Lund University Hospital, (Sweden)

    2002-04-01

    Formation of DNA adducts as a result of exposure to polycyclic aromatic hydrocarbons (PAH) was studied in 98 potroom workers from an aluminium smelting plant and in 55 blue-collar workers without occupational PAH exposure. DNA from peripheral blood mononuclear cells (PBMC) was used for quantitation of individual PAH-DNA adducts by {sup 32}P-postlabelling/high performance liquid chromatography (HPLC) analysis. Four individual DNA adducts (denoted A, B, C and D) were quantified in 141 of a total of 153 subjects. Genetic polymorphisms for cytochrome P-4501A1 (CYP1A1), microsomal epoxide hydrolase, N-acetyltransferase 2, glutathione transferases M1, P1 and T1 (GSTM1, GSTP1 and GSTT1, respectively) and NAD(P)H: quinone oxidoreductase 1 (NQO1) were analysed. For 52 subjects, analysis of mRNA inducibility of CYP1A1 was performed. No statistically significant differences in the levels of total or individual DNA adducts A, C and D were found between potroom workers and control subjects. All potroom workers and the subgroup of potroom workers who reported to never/sometimes use personal respiratory protection (n=72) were found to have a significantly higher likelihood of having high levels of adduct B than control subjects [odds ratio (OR) =3.4 with 95% confidence interval (CI) of 1.3-9.2, and OR=4.2 with 95% CI 1.6-11.5, respectively]. In the subgroup, levels of adducts A and B were found to be significantly higher among workers with employment time of less than 6 months (n=5). Also, the levels of the individual DNA adducts were to some extent modified by genetic polymorphisms in CYP1A1, GSTM1, GSTP1 and NQO1 and by CYP1A1 inducibility. In conclusion, levels of adduct B, identified by {sup 32}P-postlabelling/HPLC methodology as an indicator of PAH exposure in aluminium production, were modified by the use of respiratory protection, length of employment and genetic polymorphisms. (orig.)

  5. Shorter telomeres in peripheral blood mononuclear cells from older persons with sarcopenia: results from an exploratory study

    Directory of Open Access Journals (Sweden)

    Emanuele eMarzetti

    2014-08-01

    Full Text Available Background. Telomere shortening in peripheral blood mononuclear cells (PBMCs has been associated with biological age and several chronic degenerative diseases. However, the relationship between telomere length and sarcopenia, a hallmark of the aging process, is unknown. The aim of the present study was therefore to determine whether PBMC telomeres obtained from sarcopenic older persons were shorter relative to non-sarcopenic peers. We further explored if PBMC telomere length was associated with frailty, a major clinical correlate of sarcopenia.Methods. Analyses were conducted in 142 persons aged >/= 65 years referred to a geriatric outpatient clinic (University Hospital. The presence of sarcopenia was established according to the European Working Group on Sarcopenia in Older People criteria, with bioelectrical impedance analysis used for muscle mass estimation. The frailty status was determined by both the Fried’s criteria (physical frailty, PF and a modified Rockwood’s frailty index (FI. Telomere length was measured in PBMCs by quantitative real-time polymerase chain reaction according to the Telomere/Single copy gene ratio (T/S method.Results. Among 142 outpatients (mean age 75.0 ± 6.5 years, 59.2% women, sarcopenia was diagnosed in 23 individuals (19.3%. The PF phenotype was detected in 74 participants (52.1%. The average FI score was 0.46 ± 0.17. PBMC telomeres were shorter in sarcopenic subjects (T/S = 0.21; 95% CI: 0.18 – 0.24 relative to non-sarcopenic individuals (T/S = 0.26; 95%: CI: 0.24 – 0.28; p = 0.01, independent of age, gender, smoking habit, or comorbidity. No significant associations were determined between telomere length and either PF or FI.Conclusion. PBMC telomere length, expressed as T/S values, is shorter in older outpatients with sarcopenia. The cross-sectional assessment of PBMC telomere length is not sufficient at capturing the complex, multidimensional syndrome of frailty.

  6. Dopamine Agonists Exert Nurr1-inducing Effect in Peripheral Blood Mononuclear Cells of Patients with Parkinson's Disease

    Institute of Scientific and Technical Information of China (English)

    Li-Min Zhang; Cong-Cong Sun; Ming-Shu Mo; Luan Cen; Lei Wei; Fei-Fei Luo; Yi Li

    2015-01-01

    Background:Nurr1 plays an essential role in the development,survival,and function maintenance ofmidbrain dopaminergic (DA) neurons,and it is a potential target for Parkinson's disease (PD).Nurr1 mRNA can be detected in peripheral blood mononuclear cells (PBMCs),but whether there is any association of altered Nurr1 expression in PBMC with the disease and DA drug treatments remains elusive.This study aimed to measure the Nurrl mRNA level in PBMC and evaluate the effect of Nurr1 expression by DA agents in vivo and in vitro.Methods:The mRNA levels of Nurrl in PBMC of four subgroups of 362 PD patients and 193 healthy controls (H