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

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...

Identification of mononuclear cells in human blood. II. Evaluation of morphological and immunological aspects of native and formaldehyde-fixed cell populations

NARCIS (Netherlands)

Schuit, H.R.E.; Hijmans, W.

1980-01-01

The presence of surface-associated immunoglobulins and Fc receptors on mononuclear cells from normal human blood waas investigated by the direct immunofluorescence technique combined with phase-contrast microscopy. Formaldehyde-fixed cells were compared to unfixed cells and to cells preincubated at

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

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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...

Beneficial effects of non-matched allogeneic cord blood mononuclear cells upon patients with idiopathic osteoporosis

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Li Jun

2012-05-01

Full Text Available Abstract Background Immunological arguments and historical examples have shown that treatment with cord blood for non-hematopoietic activities, such as growth factor production and stimulation of angiogenesis, may not require matching or immune suppression. Methods To study the benefit of blood mononuclear cell therapy, 8 patients with idiopathic osteoporosis were given intermittent treatments with non-matched allogeneic cord blood mononuclear cells for 3 months. Morning fasting samples were collected for measuring urine N telopeptide of type-1 collagen, serum bone-specific alkaline phosphatase, and insulin-like growth factor 1 during one-year study. Results Clinical response was striking. Serum insulin-like growth factor 1 significantly increased in all patients at 3 months compared with baseline values, from 264.1 ± 107.0 to 384.4 ± 63.1 ng/mL (P = 0.002, with a tendency to return to baseline values at 12 months (312.9 ± 75.5 ng/mL, P = 0.083. In contrast, differences in serum bone-specific alkaline phosphatase and urine N telopeptide of type-1 collagen were not significant at 3 (P = 0.765, P = 0.057 or 12 months (P = 0.889, P = 0.122. A beneficial effect on bone density was observed in all patients at the lumbar spine. The mean bone mineral density calculated during therapy (0.6811 ± 0.1442 g/cm2 tended higher than baseline values (0.6239 ± 0.1362 g/cm2, P  Conclusions The findings indicate that for these patients with idiopathic osteoporosis, treatment with cord blood mononuclear cells led to a significant increase in insulin-like growth factor 1 levels, which favors the increase in bone mineral density.

Systemic chemotherapy induces microsatellite instability in the peripheral blood mononuclear cells of breast cancer patients

International Nuclear Information System (INIS)

Fonseca, Fernando LA; Sant Ana, Aleksandra VL; Bendit, Israel; Arias, Vitor; Costa, Luciano J; Pinhal, Aparecida A; Giglio, Auro del

2005-01-01

Systemic chemotherapy is an important part of treatment for breast cancer. We conducted the present study to evaluate whether systemic chemotherapy could produce microsatellite instability (MSI) in the peripheral blood mononuclear cell fraction of breast cancer patients. We studied 119 sequential blood samples from 30 previously untreated breast cancer patients before, during and after chemotherapy. For comparison, we also evaluated 20 women who had no relevant medical history (control group). In 27 out of 30 patients we observed MSI in at least one sample, and six patients had loss of heterozygosity. We found a significant correlation between the number of MSI events per sample and chemotherapy with alkylating agents (P < 0.0001). We also observed an inverse correlation between the percentage of cells positive for hMSH2 and the number of MSI events per sample (P = 0.00019) and use of alkylating agents (P = 0.019). We conclude that systemic chemotherapy may induce MSI and loss of heterozygosity in peripheral blood mononuclear cells from breast cancer patients receiving alkylating agents, possibly mediated by a chemotherapy-induced decrease in the expression of hMSH2. These effects may be related to the generation of secondary leukaemia in some patients, and may also intensify the genetic instability of tumours and increase resistance to treatment

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

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

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.

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

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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

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

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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

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

International Nuclear Information System (INIS)

Rodrigues, L.P.; Iglesias, D.; Nicola, F.C.; Steffens, D.; Valentim, L.; Witczak, A.; Zanatta, G.; Achaval, M.; Pranke, P.; Netto, C.A.

2011-01-01

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

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.

Counter-flow elutriation of clinical peripheral blood mononuclear cell concentrates for the production of dendritic and T cell therapies

OpenAIRE

Stroncek, David F; Fellowes, Vicki; Pham, Chauha; Khuu, Hanh; Fowler, Daniel H; Wood, Lauren V; Sabatino, Marianna

2014-01-01

Introduction Peripheral blood mononuclear cells (PBMC) concentrates collected by apheresis are frequently used as starting material for cellular therapies, but the cell of interest must often be isolated prior to initiating manufacturing. Study design and methods The results of enriching 59 clinical PBMC concentrates for monocytes or lymphocytes from patients with solid tumors or multiple myeloma using a commercial closed system semi-automated counter-flow elutriation instrument (Elutra, Teru...

Deleterious effect of ultraviolet-B radiation on accessory function of human blood adherent mononuclear cells

International Nuclear Information System (INIS)

Rich, E.A.; Elmets, C.A.; Fujiwara, H.; Wallis, R.S.; Ellner, J.J.

1987-01-01

The effects of ultraviolet-B radiation (UV-B) on accessory function of human blood adherent mononuclear cells (ADH) for antigen and mitogen-induced responses, and production by ADH of the amplifying cytokine interleukin 1 (IL-1) were examined. Responder lymphocytes were rendered accessory cell dependent by treatment of nonadherent cells with OKIal + complement. UV-B depressed accessory function of ADH in a dose-dependent manner. UV-B decreased accessory function of ADH for tetanus toxoid-induced responses and phytohaemagglutinin-induced responses. UV-B also decreased accessory activity of peripheral blood mononuclear cells but not Epstein-Barr virus-transformed B cells for a PPD-reactive T cell line. Interleukin 1 (IL-1) activity of supernatants of ADH was assayed on C3H/HeJ mouse thymocytes. Pretreatment of ADH with UV-B decreased lipopolysaccharide-stimulated IL-1 activity. Lysates of UV-B irradiated, LPS-stimulated ADH had no discernible IL-1 activity. Addition of IL-1 partially restored accessory activity of UV-B irradiated ADH for lymphocyte responses to TT. Exposure of ADH to TT or PHA for 30 min before irradiation blocked the inhibitory effect of UV-B on accessory activity. Thus, low doses of UV-B are deleterious to accessory function and to production of IL-1 by ADH. Interference with production of cytokines and with initial interactions of accessory cells with antigen and mitogen may be critical to the effects of UV-B on immunoregulatory function of ADH. (author)

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

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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...

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.

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

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...

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

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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.

Effect of paricalcitol and GcMAF on angiogenesis and human peripheral blood mononuclear cell proliferation and signaling.

Science.gov (United States)

Pacini, Stefania; Morucci, Gabriele; Punzi, Tiziana; Gulisano, Massimo; Ruggiero, Marco; Amato, Marcello; Aterini, Stefano

2012-01-01

In addition to its role in calcium homeostasis and bone mineralization, vitamin D is involved in immune defence, cardiovascular function, inflammation and angiogenesis, and these pleiotropic effects are of interested in the treatment of chronic kidney disease. Here we investigated the effects of paricalcitol, a nonhypercalcemic vitamin D analogue, on human peripheral blood mononuclear cell proliferation and signaling, and on angiogenesis. These effects were compared with those of a known inhibitor of angiogenesis pertaining to the vitamin D axis, the vitamin D-binding protein-derived Gc-macrophage activating factor (GcMAF). Since the effects of vitamin D receptor agonists are associated with polymorphisms of the gene coding for the receptor, we measured the effects of both compounds on mononuclear cells harvested from subjects harboring different BsmI polymorphisms. Paricalcitol inhibited mononuclear cell viability with the bb genotype showing the highest effect. GcMAF, on the contrary, stimulated cell proliferation, with the bb genotype showing the highest stimulatory effect. Both compounds stimulated 3'-5'-cyclic adenosine monophosphate formation in mononuclear cells with the highest effect on the bb genotype. Paricalcitol and GcMAF inhibited the angiogenesis induced by proinflammatory prostaglandin E1. Polymorphisms of the vitamin D receptor gene, known to be associated with the highest responses to vitamin D receptor agonists, are also associated with the highest responses to GcMAF. These results highlight the role of the vitamin D axis in chronic kidney disease, an axis which includes vitamin D, its receptor and vitamin D-binding protein-derived GcMAF.

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

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Nan Yu

Full Text Available 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.

Differential gene expression profiles of peripheral blood mononuclear cells in childhood asthma.

Science.gov (United States)

Kong, Qian; Li, Wen-Jing; Huang, Hua-Rong; Zhong, Ying-Qiang; Fang, Jian-Pei

2015-05-01

Asthma is a common childhood disease with strong genetic components. This study compared whole-genome expression differences between asthmatic young children and healthy controls to identify gene signatures of childhood asthma. Total RNA extracted from peripheral blood mononuclear cells (PBMC) was subjected to microarray analysis. QRT-PCR was performed to verify the microarray results. Classification and functional characterization of differential genes were illustrated by hierarchical clustering and gene ontology analysis. Multiple logistic regression (MLR) analysis, receiver operating characteristic (ROC) curve analysis, and discriminate power were used to scan asthma-specific diagnostic markers. For fold-change>2 and p childhood asthma model for prediction and diagnosis.

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

Alterations in bone marrow and blood mononuclear cell polyamine and methylglyoxal bis(guanylhydrazone) levels: phase I evaluation of alpha-difluoromethylornithine and methylglyoxal bis(guanylhydrazone) treatment of human hematological malignancies.

Science.gov (United States)

Maddox, A M; Freireich, E J; Keating, M J; Haddox, M K

1988-03-01

Nine patients with hematological malignancies were treated with difluoromethylornithine and methylglyoxal bis(guanylhydrazone). The number of circulating blast cells decreased in all of the patients treated with DFMO and MGBG for longer than 1 wk. Morphological evidence of myeloid maturation was evident in four patients with leukemia and the circulating M Protein decreased in one patient with multiple myeloma. The polyamine content of the mononuclear cells in both the peripheral blood and bone marrow was transiently increased after the initial MGBG dose. During administration of DFMO decreases were achieved in the peripheral blood mononuclear cell putrescine levels in 7 patients, spermidine levels in 5 patients, and spermine levels in 4 patients. Alterations in bone marrow mononuclear cell polyamine levels were similar to those which occurred in the peripheral cells. An average of 9 days of DFMO treatment was required to lower mononuclear cell polyamine levels. Three of the 4 evaluable patients receiving multiple MGBG doses had an increased mononuclear cell content of MGBG after DFMO pretreatment. Enhancement of cellular MGBG levels was not directly correlated to the degree of cellular polyamine depletion.

Recovery from Bell Palsy after Transplantation of Peripheral Blood Mononuclear Cells and Platelet-Rich Plasma

OpenAIRE

Seffer, Istvan; Nemeth, Zoltan

2017-01-01

Summary: Peripheral blood mononuclear cells (PBMCs) are multipotent, and plasma contains growth factors involving tissue regeneration. We hypothesized that transplantation of PBMC-plasma will promote the recovery of paralyzed facial muscles in Bell palsy. This case report describes the effects of PBMC-plasma transplantations in a 27-year-old female patient with right side Bell palsy. On the affected side of the face, the treatment resulted in both morphological and functional recovery includi...

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.

Amplification of the spleen macrophage population in malaria: possible role of a factor chemotactic for blood mononuclear cells

International Nuclear Information System (INIS)

Wyler, D.J.; Gallin, J.I.

1976-01-01

The mechanism of amplification of the splenic macrophages' population was investigated using mice infected with malaria as a model of an obligate intravascular infection. It was observed that these macrophages derived from blood monocytes rather than by local proliferation in the spleen. A factor, chemotactic for blood mononuclear cells, was present in spleen cells shortly after infection and preceded detectable increases in spleen macrophage number by 48 hours. This factor, in concert with spleen derived macrophage migration inhibition factor, may be important in the amplification of splenic macrophage population in intravascular infections

Diabetic Ephrin-B2-Stimulated Peripheral Blood Mononuclear Cells Enhance Poststroke Recovery in Mice

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Rose Hilal

2018-01-01

Full Text Available Clinical trials of cell therapy in stroke favor autologous cell transplantation. To date, feasibility studies have used bone marrow-derived mononuclear cells, but harvesting bone marrow cells is invasive thus complicating bedside treatment. We investigated the therapeutic potential of peripheral blood-derived mononuclear cells (PB-MNC harvested from diabetic patients and stimulated by ephrin-B2 (PB-MNC+ (500,000 cells, injected intravenously 18–24 hours after induced cerebral ischemia in mice. Infarct volume, neurological deficit, neurogenesis, angiogenesis, and inflammation were investigated as were the potential mechanisms of PB-MNC+ cells in poststroke neurorepair. At D3, infarct volume was reduced by 60% and 49% compared to unstimulated PB-MNC and PBS-treated mice, respectively. Compared to PBS, injection of PB-MNC+ increased cell proliferation in the peri-infarct area and the subventricular zone, decreased microglia/macrophage cell density, and upregulated TGF-β expression. At D14, microvessel density was decreased and functional recovery was enhanced compared to PBS-treated mice, whereas plasma levels of BDNF, a major regulator of neuroplasticity, were increased in mice treated with PB-MNC+ compared to the other two groups. Cell transcriptional analysis showed that ephrin-B2 induced phenotype switching of PB-MNC by upregulating genes controlling cell proliferation, inflammation, and angiogenesis, as confirmed by adhesion and Matrigel assays. Conclusions. This feasibility study suggests that PB-MNC+ transplantation poststroke could be a promising approach but warrants further investigation. If confirmed, this rapid, noninvasive bedside cell therapy strategy could be applied to stroke patients at the acute phase.

Diabetic Ephrin-B2-Stimulated Peripheral Blood Mononuclear Cells Enhance Poststroke Recovery in Mice.

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Hilal, Rose; Poittevin, Marine; Pasteur-Rousseau, Adrien; Cogo, Adrien; Mangin, Gabrielle; Chevauché, Marie; Ziat, Yasmine; Vilar, José; Launay, Jean-Marie; Gautier, Jean-François; Broquères-You, Dong; Levy, Bernard I; Merkulova-Rainon, Tatyana; Kubis, Nathalie

2018-01-01

Clinical trials of cell therapy in stroke favor autologous cell transplantation. To date, feasibility studies have used bone marrow-derived mononuclear cells, but harvesting bone marrow cells is invasive thus complicating bedside treatment. We investigated the therapeutic potential of peripheral blood-derived mononuclear cells (PB-MNC) harvested from diabetic patients and stimulated by ephrin-B2 (PB-MNC+) (500,000 cells), injected intravenously 18-24 hours after induced cerebral ischemia in mice. Infarct volume, neurological deficit, neurogenesis, angiogenesis, and inflammation were investigated as were the potential mechanisms of PB-MNC+ cells in poststroke neurorepair. At D3, infarct volume was reduced by 60% and 49% compared to unstimulated PB-MNC and PBS-treated mice, respectively. Compared to PBS, injection of PB-MNC+ increased cell proliferation in the peri-infarct area and the subventricular zone, decreased microglia/macrophage cell density, and upregulated TGF- β expression. At D14, microvessel density was decreased and functional recovery was enhanced compared to PBS-treated mice, whereas plasma levels of BDNF, a major regulator of neuroplasticity, were increased in mice treated with PB-MNC+ compared to the other two groups. Cell transcriptional analysis showed that ephrin-B2 induced phenotype switching of PB-MNC by upregulating genes controlling cell proliferation, inflammation, and angiogenesis, as confirmed by adhesion and Matrigel assays. Conclusions . This feasibility study suggests that PB-MNC+ transplantation poststroke could be a promising approach but warrants further investigation. If confirmed, this rapid, noninvasive bedside cell therapy strategy could be applied to stroke patients at the acute phase.

Detection of tumor-associated cells in cryopreserved peripheral blood mononuclear cell samples for retrospective analysis.

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Zhu, Peixuan; Stanton, Melissa L; Castle, Erik P; Joseph, Richard W; Adams, Daniel L; Li, Shuhong; Amstutz, Platte; Tang, Cha-Mei; Ho, Thai H

2016-07-02

Cryopreserved peripheral blood mononuclear cells (PBMCs) are commonly collected in biobanks. However, little data exist regarding the preservation of tumor-associated cells in cryopreserved collections. The objective of this study was to determine the feasibility of using the CellSieve™ microfiltration assay for the isolation of circulating tumor cells (CTCs) and circulating cancer-associated macrophage-like cells (CAMLs) from cryopreserved PBMC samples. Blood samples spiked with breast (MCF-7), prostate (PC-3), and renal (786-O) cancer cell lines were used to establish analytical accuracy, efficiency, and reproducibility after cryopreservation. The spiked samples were processed through Ficoll separation, and cryopreservation was followed by thawing and microfiltration. MCF-7 cells were successfully retrieved with recovery efficiencies of 90.5 % without cryopreservation and 87.8 and 89.0 %, respectively, on day 7 and day 66 following cryopreservation. The corresponding recovery efficiencies of PC-3 cells were 83.3 % without cryopreservation and 85.3 and 84.7 %, respectively, after cryopreservation. Recovery efficiencies of 786-O cells were 92.7 % without cryopreservation, and 82.7 and 81.3 %, respectively, after cryopreservation. The recovered cells retained the morphologic characteristics and immunohistochemical markers that had been observed before freezing. The protocols were further validated by quantitation of CAMLs in blood samples from two patients with renal cell carcinoma (RCC). The recovery rates of CTCs and CAMLs from cryopreserved samples were not statistically significant different (P > 0.05) from matched fresh samples. To our knowledge, this is the first report that CAMLs could be cryopreserved and analyzed after thawing with microfiltration technology. The application of microfiltration technology to cryopreserved samples will enable much greater retrospective study of cancer patients in relation to long-term outcomes.

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

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Kazemi, E.; Mortazavi, S. M. J.; Ali-Ghanbari, A.; Sharifzadeh, S.; Ranjbaran, R.; Mostafavi-pour, Z.; Zal, F.; Haghani, M.

2015-01-01

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. PMID:26396966

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.

Rescue by peripheral blood mononuclear cells in dogs from bone marrow failure after total-body irradiation

International Nuclear Information System (INIS)

Zander, A.R.; Gray, K.N.; Hester, J.P.

1984-01-01

In order to determine the minimum dose of buffy coat cells necessary to achieve hematopoietic rescue following supralethal irradiation, mongrel dogs under general anesthesia were subjected to leukacytapheresis using three different techniques of cell separation. The buffy coats were frozen with dimethylsulfoxide and stored at -196 degrees C until transfused. Sixteen dogs were irradiated with 800 rads and were supported with antibiotics and transfusions of irradiated homologous blood. They were transfused with the frozen and thawed buffy coat cells, and, if they survived, they were followed for 100 days, sacrificed, and their tissues studied. The mean yield of mononuclear cells during leukocytapheresis ranged from 4.1 +/- 2.0 X 10(9) (mean +/- SD) to 6.0 +/- 4.0 X 10(9) for the three leukacytapheresis methods; one technique was not as satisfactory as the other two. Six of the 16 dogs fully recovered with evidence of marrow rescue; however, only one had a dose of mononuclear cells less than 11.1 X 10(9). These data indicate that seven to 17 leukacytapheresis procedures would be required to reconstitute a 70 kilogram patient. These preliminary findings suggest that, because the yields of transplantable cells with current technology are not adequate, the transplantation potential of buffy coat cells exposed to mobilizing agents should be evaluated

Expression and clinical significance of NF-毷B, CTGF and OPN in mononuclear cells in peripheral blood as well as renal tissues in patients with IgA nephropathy

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Cheng-Luo Hao

2016-06-01

Full Text Available Objective: To study the expression and clinical significance of NF-kB, CTGF and OPN in mononuclear cells in peripheral blood as well as renal tissues in patients with IgA nephropathy. Methods: A total of 25 nephropathy patients diagnosed with IgA nephropathy and 25 patients receiving nephrectomy due to trauma or tumor in our hospital were studied. Peripheral blood and kidney tissues were collected to test NF-kB, CTGF, OPN, T-bet, GATA-3, RORγT and Foxp3 expressions. Results: CTGF and OPN percentages in peripheral blood mononuclear cells and kidney tissues of nephropathy patients were higher than those of the control group. NF-kB, CTGF and OPN expressions were significantly higher in M1, E1, S1 group patients’ peripheral blood mononuclear cells and renal tissues than those in M0, E1 and S1 group. T-bet, GATA-3 and RORγT expressions in nephropathy patients’ peripheral blood were significantly higher than those in the control group, and were positively correlated with NF-kB, CTGF and OPN expressions. The expression of Foxp3 was significantly lower than that of control group, and was negatively correlated with NF-kB, CTGF and OPN expressions. Conclusions: The expression of NF-kB, CTGF and OPN in peripheral blood mononuclear cells and renal tissue in patients with IgA nephropathy is abnormally high and can evaluate the prognosis of the disease and the differentiation of CD4+T cells.

INFLUENCE OF ALPHA-1-ACID GLYCOPROTEIN UPON PRODUCTION OF CYTOKINES BY PERIPHERAL BLOOD MONONUCLEARS

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М. V. Osikov

2007-01-01

Full Text Available Abstract. Alpha-1-acid glycoprotein (orosomucoid is a multifunctional acute phase reactant belonging to the family of lipocalines from plasma alpha-2 globulin fraction. In present study, we investigated dosedependent effects of orosomucoid upon secretion of IL-1â, IL-2, IL-3, IL-4 by mononuclear cells from venous blood of healthy volunteers. Mononuclear cells were separated by means of gradient centrifugation, followed by incubation for 24 hours with 250, 500, or 1000 mcg of orosomucoid per ml RPMI-1640 medium (resp., low, medium and high dose. The levels of cytokine production were assayed by ELISA technique. Orosomucoid-induced secretion of IL-1â and IL-4 was increased, whereas IL-3 secretion was inhibited. IL-2 production was suppressed at low doses of orosomucoid, and stimulated at medium and high doses. The effect of alpha-1-acid glycoprotein upon production of IL-2, IL-3 and IL-4 was dose-dependent. Hence, these data indicate that orosomucoid is capable of modifying IL-1â, IL-2, IL-3, and IL-4 secretion by blood mononuclear cells.

Increased nuclear tri-iodothyronine binding and thyroid hormone-stimulated glucose consumption in mononuclear blood cells from patients with liver cirrhosis

DEFF Research Database (Denmark)

Kvetny, J; Matzen, L

1991-01-01

Nuclear tri-iodothyronine (T3) maximal binding capacity (MBC) and thyroxine- and T3-stimulated cellular oxygen consumption and glucose consumption were examined in mononuclear blood cells from six patients with liver cirrhosis (LC), in six patients with alcoholic hepatitis (AH), and in six healthy...

Freeze-drying of mononuclear cells derived from umbilical cord blood followed by colony formation.

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Dity Natan

Full Text Available BACKGROUND: We recently showed that freeze-dried cells stored for 3 years at room temperature can direct embryonic development following cloning. However, viability, as evaluated by membrane integrity of the cells after freeze-drying, was very low; and it was mainly the DNA integrity that was preserved. In the present study, we improved the cells' viability and functionality after freeze-drying. METHODOLOGY/PRINCIPAL FINDINGS: We optimized the conditions of directional freezing, i.e. interface velocity and cell concentration, and we added the antioxidant EGCG to the freezing solution. The study was performed on mononuclear cells (MNCs derived from human umbilical cord blood. After freeze-drying, we tested the viability, number of CD34(+-presenting cells and ability of the rehydrated hematopoietic stem cells to differentiate into different blood cells in culture. The viability of the MNCs after freeze-drying and rehydration with pure water was 88%-91%. The total number of CD34(+-presenting cells and the number of colonies did not change significantly when evaluated before freezing, after freeze-thawing, and after freeze-drying (5.4 x 10(4+/-4.7, 3.49 x 10(4+/-6 and 6.31 x 10(4+/-12.27 cells, respectively, and 31+/-25.15, 47+/-45.8 and 23.44+/-13.3 colonies, respectively. CONCLUSIONS: This is the first report of nucleated cells which have been dried and then rehydrated with double-distilled water remaining viable, and of hematopoietic stem cells retaining their ability to differentiate into different blood cells.

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...

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

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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.

Autoradiographic quantification of vasoactive intestinal peptide binding sites in sections from human blood mononuclear cell pellets

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Gutkind, J.S.; Kurihara, M.; Castren, E.; Saavedra, J.M.

1988-09-01

Quantitative autoradiographic methods were utilized to characterize specific, high-affinity vasoactive intestinal peptide binding sites (Kd = 310 +/- 60 pmol/L; Bmax = 93 +/- 11 fmol/mg protein) in frozen sections obtained from a mononuclear cell pellet derived from 20 ml of human blood. The method is at least one order of magnitude more sensitive than conventional membrane binding techniques, and it has the potential for wide applications in studies of neuropeptide, biogenic amine, and drug binding in clinical samples.

Autoradiographic quantification of vasoactive intestinal peptide binding sites in sections from human blood mononuclear cell pellets

International Nuclear Information System (INIS)

Gutkind, J.S.; Kurihara, M.; Castren, E.; Saavedra, J.M.

1988-01-01

Quantitative autoradiographic methods were utilized to characterize specific, high-affinity vasoactive intestinal peptide binding sites (Kd = 310 +/- 60 pmol/L; Bmax = 93 +/- 11 fmol/mg protein) in frozen sections obtained from a mononuclear cell pellet derived from 20 ml of human blood. The method is at least one order of magnitude more sensitive than conventional membrane binding techniques, and it has the potential for wide applications in studies of neuropeptide, biogenic amine, and drug binding in clinical samples

Release of an endogenous pyrogen in vitro from rabbit mononuclear cells.

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Atkins, E; Bodel, P; Francis, L

1967-08-01

The capacity of rabbit mononuclear cells to release an endogenous pyrogen (EP) in vitro has been studied. After incubation with tuberculin, preparations of predominantly monocytic cells, derived from the respiratory passages of the lungs of rabbits sensitized with BCG, were activated to release EP. Pyrogen production occurred more slowly with lung monocytes than with blood leukocytes of similarly sensitized rabbits and 9 to 10 hr incubation in a fully supportive medium was required to produce clear-cut results. As previously reported with blood leukocytes, mononuclear cells from the lungs of normal animals were also activated by tuberculin but to a lesser degree than were those from specifically sensitized rabbits. Under a variety of conditions, mononuclear cells from either spleen or lymph nodes of the same sensitized rabbits failed to release detectable amounts of pyrogen when incubated with tuberculin in vitro but were activated in a majority of instances when phagocytosis of heat-killed staphylococci was used as the stimulus. Release of pyrogen from lung monocytes appears to be an active process that is both temperature-dependent and requires protein synthesis. Neither serum antibody nor complement appears to play a role in this process. Evidence is presented that the granulocyte is the main source of pyrogen evolved by blood leukocytes incubated in vitro with OT or heat-killed staphylococci, whereas the lung macrophage and/or monocyte is responsible for most of the pyrogen released from the lung cell preparations. From these studies, it is concluded that mononuclear cells can be activated in vitro by several microbial stimuli and must be considered an additional cellular source of EP. The clinical implications of these findings for the pathogenesis of fever in granulomatous diseases where the monocyte is the predominant cell are discussed.

Porcine blood mononuclear cell cytokine responses to PAMP molecules: comparison of mRNA and protein production

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Sørensen, Nanna Skall; Skovgaard, Kerstin; Heegaard, Peter M. H.

2011-01-01

Pathogen-associated molecular patterns (PAMPs) are conserved molecules of microorganisms inducing innate immune cells to secrete distinct patterns of cytokines. In veterinary species, due to a lack of specific antibodies, cytokines are often monitored as expressed mRNA only. This study investigated...... 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...

Optimizing recovery of frozen human peripheral blood mononuclear cells for flow cytometry.

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Bo Langhoff Hønge

Full Text Available Live peripheral blood mononuclear cells (PBMCs can be frozen and thawed for later analyses by adding and removing a cryoprotectant, such as dimethyl sulfoxide (DMSO. Laboratories across the world use various procedures, but published evidence of optimal thawing procedures is scarce.PBMCs were separated from blood collected from healthy Danish blood donors, and stored at -80°C after adding of DMSO. The essential steps in the thawing procedure were modified and performance was evaluated by flow cytometry with respect to the percentage and total yield of viable PMBCs.The best-performing washing medium was Roswell Park Memorial Institute (RPMI 1640 at 37°C with 20% fetal bovine serum. When using 10 mL washing medium in a 15-mL Falcon tube, samples should be centrifuged for at least 10 minutes at 500 g. We failed to detect any differences between the tested methods of mixing PBMCs with washing medium. Likewise, neither the thawing duration nor centrifugation temperature (20°C and 37°C had any effect. PBMCs could be incubated (rested for up to eight hours in a 37°C 5% CO2 incubator without affecting cell counts, but incubating PBMCs for 16 hours significantly decreased viability and recovery. In general, high viability was not necessarily associated with high recovery.Changing the thawing procedure significantly impacted PBMC viability and live cell recovery. Evaluating both viability and live PBMC recovery are necessary to evaluate method performance. Investigation of differential loss of PBMC subtypes and phenotypic changes during thawing and incubation requires further evaluation.

Effect of blood component coatings of enosseal implants on proliferation and synthetic activity of human osteoblasts and cytokine production of peripheral blood mononuclear cells

Czech Academy of Sciences Publication Activity Database

Himlová, L.; Kubies, Dana; Hulejová, H.; Bartová, J.; Riedel, Tomáš; Štikarová, J.; Suttnar, J.; Pešáková, V.

2016-01-01

Roč. 2016, č. 2016 (2016), 8769347_1-8769347_15 ISSN 0962-9351 R&D Projects: GA MZd(CZ) NT13297; GA MŠk(CZ) LQ1604; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61389013 Keywords : peripheral blood mononuclear cells * cytokine * osteoblasts Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.232, year: 2016

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

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Tanja Sofrenovic

Full Text Available 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.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.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.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.

Tumor necrosis factor-alpha expression in peripheral blood mononuclear cells correlates with early childhood social interaction in autism spectrum disorder.

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Makinodan, Manabu; Iwata, Keiko; Ikawa, Daisuke; Yamashita, Yasunori; Yamamuro, Kazuhiko; Toritsuka, Michihiro; Kimoto, Sohei; Okumura, Kazuki; Yamauchi, Takahira; Yoshino, Hiroki; Tsujii, Masatsugu; Sugiyama, Toshiro; Tsuchiya, Kenji; Mori, Norio; Matsuzaki, Hideo; Kishimoto, Toshifumi

2017-03-01

Autism spectrum disorder is a neurodevelopmental disorder characterized by impaired social interaction, poor communication skills, and repetitive/restrictive behaviors. Elevated blood levels of pro-inflammatory cytokines have been reported in subjects with autism spectrum disorder. On the other hand, early childhood adverse experience also increases blood levels of these cytokines. Since social experience of children with autism spectrum disorder is generally unlike to typically developing children, we hypothesized that social interaction during childhood contribute to pro-inflammatory cytokine expression in subjects with autism spectrum disorder. We compared revised Autism Diagnostic Interview scores and expression levels of pro-inflammatory cytokines in peripheral blood mononuclear cells of subjects with autism spectrum disorder (n = 30). The score of domain A on the revised Autism Diagnostic Interview, indicating social interaction impairment in early childhood, was negatively correlated with tumor necrosis factor-α mRNA expression level in peripheral blood mononuclear cells but not interleukin-1β or -6. Consistently, tumor necrosis factor-α mRNA expression was markedly low in subjects with autism spectrum disorder compared to typically developing children who presumably experienced the regular levels of social interaction. These findings suggest that the low blood levels of tumor necrosis factor-α mRNA in subjects with autism spectrum disorder might be due to impaired social interaction in early childhood. Copyright © 2016 Elsevier Ltd. All rights reserved.

Conversion of adult human peripheral blood mononuclear cells into induced neural stem cell by using episomal vectors

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Xihe Tang

2016-03-01

Full Text Available Human neural stem cells (NSCs hold great promise for research and therapy in neural diseases. Many studies have shown direct induction of NSCs from human fibroblasts, which require an invasive skin biopsy and a prolonged period of expansion in cell culture prior to use. Peripheral blood (PB is routinely used in medical diagnoses, and represents a noninvasive and easily accessible source of cells. Here we show direct derivation of NSCs from adult human PB mononuclear cells (PB-MNCs by employing episomal vectors for transgene delivery. These induced NSCs (iNSCs can expand more than 60 passages, can exhibit NSC morphology, gene expression, differentiation potential, and self-renewing capability and can give rise to multiple functional neural subtypes and glial cells in vitro. Furthermore, the iNSCs carry a specific regional identity and have electrophysiological activity upon differentiation. Our findings provide an easily accessible approach for generating human iNSCs which will facilitate disease modeling, drug screening, and possibly regenerative medicine.

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

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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

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

Grass immunotherapy induces inhibition of allergen-specific human peripheral blood mononuclear cell proliferation.

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Baskar, S; Hamilton, R G; Norman, P S; Ansari, A A

1997-02-01

The peripheral blood mononuclear cells (PBMC) from humans allergic to grass pollens (GR+ subjects) show strong in vitro proliferative responses to purified allergens from Lolium perenne pollen Lol p 1, and to a lesser extent to Lol p 2 and Lol p 3. By contrast, PBMC from grass allergic patients undergoing immunotherapy (GR + IT subjects) exhibit a very poor Lol p-specific proliferative response, similar to that observed in nongrass allergic subjects (GR-subjects). Unlike GR-subjects, both GR+ and GR + IT subjects have high levels of antigen-specific serum IgG and IgE antibodies to Lol p 1, Lol p 2 and Lol p 3. While GR+ subjects exhibit a significant correlation between antigen-specific serum antibody and PBMC responses, GR + IT subjects do not show a correlation between the two responses. The possible mechanisms by which immunotherapy may modulate allergen-specific T cell proliferative response are discussed.

Thyroid hormone stimulated glucose uptake in human mononuclear blood cells from normal persons and from patients with non-insulin-dependent diabetes mellitus

DEFF Research Database (Denmark)

Kvetny, J; Matzen, L

1989-01-01

Thyroxine and T3 induced oxygen consumption and glucose uptake were studied in vitro in mononuclear blood cells isolated from patients with non-insulin-dependent diabetes mellitus (NIDDM) and from non-diabetic control persons. Cellular oxygen consumption and glucose uptake were promptly increased...

Generation of dendritic cells from human bone marrow mononuclear cells: advantages for clinical application in comparison to peripheral blood monocyte derived cells.

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Bai, L; Feuerer, M; Beckhove, P; Umansky, V; Schirrmacher, V

2002-02-01

Dendritic cells (DCs) currently used for vaccination in clinical studies to induce immunity against malignant cells are normally generated from peripheral blood-derived monocytes. Here we studied conditions for the generation of DCs from unseparated human bone marrow (BM) mononuclear cells and compared them functionally with DCs from blood. The two types of DCs, from bone marrow (BM-DC) and peripheral blood (BL-DC), were generated in parallel from the same normal healthy donors by culturing in serum-free X-VIVO 20 medium containing GM-CSF and IL-4, and then the phenotypes and functions were compared. BM-DC generation occurred in 14 days and involved proliferative expansion from CD34 stem cells and differentiation while BL-DC generation occurred in 7 days from CD14 monocytes and involved only differentiation. A 7- to 25-fold higher number of DCs could be obtained from BM than from blood. BM-DC had similar phenotypes as BL-DC. The capacity to stimulate MLR reactivity in allogeneic T lymphocytes was higher with BM-DC than that with BL-DC. Also, the capacity to stimulate autologous memory T cell responses to tetanus toxoid (TT) or tuberculin (PPD) was higher with BM-DC than with BL-DC. These results suggest that BM-DC as produced here may be a very economic and useful source of professional antigen-presenting cells for anti-tumor immunotherapeutic protocols.

Quantitative evaluation of interleukin-12 p40 gene expression in peripheral blood mononuclear cells.

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Conte, Enrico; Nigro, Luciano; Fagone, Evelina; Drago, Francesco; Cacopardo, Bruno

2008-01-01

The heterodimeric cytokine IL-12 (composed of a p35 and a p40 subunit) is produced primarily by monocytes, macrophages and B cells. In vitro and in vivo experiments have demonstrated the crucial role of IL-12 in initiating and establishing both innate immunity and T cell-mediated resistance to intracellular pathogens, including Leishmania donovani, Toxoplasma gondii, Listeria monocytogenes, and Mycobacterium tuberculosis. Assessment of cytokine expression has thus become crucial to understand host responses to infections. In this study, by using the reverse transcriptase-real time PCR we developed a highly specific and sensitive assay to quantitatively evaluate IL-12p40 mRNA transcription levels in peripheral blood mononuclear cells (PBMCs) stimulated with PHA vs. unstimulated cells. We also used the ELISA to evaluate bioactive IL-12 release in culture supernatants. We provide evidence that IL-12 p40 mRNA levels were significantly up-regulated in PHA-activated PBMCs. These results were correlated with data of IL-12 levels obtained by ELISA.

Association between age and repair of oxidatively damaged DNA in human peripheral blood mononuclear cells

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Løhr, Mille; Jensen, Annie; Eriksen, Louise

2015-01-01

damaged DNA in peripheral blood mononuclear cells (PBMCs). We isolated PBMCs from subjects aged 18-83 years, as part of a health survey of the Danish population that focussed on lifestyle factors. The level of DNA repair activity was measured as incisions on potassium bromate-damaged DNA by the comet...... assay. There was an inverse association between age and DNA repair activity with a 0.65% decline in activity per year from age 18 to 83 (95% confidence interval: 0.16-1.14% per year). Univariate regression analysis also indicated inverse associations between DNA repair activity and waist-hip ratio (P...

Proteomic responses of peripheral blood mononuclear cells in the European eel (Anguilla anguilla) after perfluorooctane sulfonate exposure

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Roland, Kathleen, E-mail: kathleen.roland@fundp.ac.be [Research Unit in Environmental and Evolutionary Biology (URBE), Narilis (Namur Research Institute for Lifesciences), University of Namur - FUNDP, Rue de Bruxelles 61, B-5000, Namur (Belgium); Kestemont, Patrick; Hénuset, Laurence; Pierrard, Marie-Aline [Research Unit in Environmental and Evolutionary Biology (URBE), Narilis (Namur Research Institute for Lifesciences), University of Namur - FUNDP, Rue de Bruxelles 61, B-5000, Namur (Belgium); Raes, Martine; Dieu, Marc [Research Unit in Cellular Biology (URBC) Narilis (Namur Research Institute for Lifesciences), University of Namur - FUNDP, Rue de Bruxelles 61, B-5000, Namur (Belgium); Silvestre, Frédéric [Research Unit in Environmental and Evolutionary Biology (URBE), Narilis (Namur Research Institute for Lifesciences), University of Namur - FUNDP, Rue de Bruxelles 61, B-5000, Namur (Belgium)

2013-03-15

Highlights: ► We have evaluating the toxicity of eel peripheral blood mononuclear cells exposed during 48 h to 10 μg and 1 mg perfluoroctane sulfonate/L. ► After in vitro contaminations, the post-nuclear fraction was isolated and a proteomic analysis using 2D-DIGE was performed. ► 48 different proteins were identified and classified into main functional classes which provide clues on the cellular pathways mainly affected by PFOS. -- Abstract: Since the 1980s, the stocks of European eel have been declining in most of their geographical distribution area. Many factors can be attributed to this decline such as pollution by xenobiotics like perfluorooctane sulfonate (PFOS). This study aimed at evaluating the in vitro toxicity of eel peripheral blood mononuclear cells (PBMC) exposed to PFOS. Exposure time and two concentrations were chosen to avoid cell mortality (48 h exposure at 10 μg PFOS/L and 1 mg PFOS/L). After in vitro contaminations, the post-nuclear fraction was isolated and a proteomic analysis using 2D-DIGE was performed to compare PBMC from the control group with cells exposed to the pollutant. On the 158 spots that were significantly affected by PFOS exposure, a total of 48 different proteins were identified using nano-LCESI-MS/MS and the Peptide and Protein Prophet of Scaffold software. These proteins can be categorized into diverse functional classes, related to cytoskeleton, protein folding, cell signaling, proteolytic pathway and carbohydrate and energy metabolism, which provide clues on the cellular pathways mainly affected by PFOS. Some of the identified proteins are rarely found in other ecotoxicological proteomic studies and could constitute potential biomarkers of exposure to PFOS in fish.

Transcriptome analysis describing new immunity and defense genes in peripheral blood mononuclear cells of rheumatoid arthritis patients.

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Vitor Hugo Teixeira

Full Text Available BACKGROUND: Large-scale gene expression profiling of peripheral blood mononuclear cells from Rheumatoid Arthritis (RA patients could provide a molecular description that reflects the contribution of diverse cellular responses associated with this disease. The aim of our study was to identify peripheral blood gene expression profiles for RA patients, using Illumina technology, to gain insights into RA molecular mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: The Illumina Human-6v2 Expression BeadChips were used for a complete genome-wide transcript profiling of peripheral blood mononuclear cells (PBMCs from 18 RA patients and 15 controls. Differential analysis per gene was performed with one-way analysis of variance (ANOVA and P values were adjusted to control the False Discovery Rate (FDR<5%. Genes differentially expressed at significant level between patients and controls were analyzed using Gene Ontology (GO in the PANTHER database to identify biological processes. A differentially expression of 339 Reference Sequence genes (238 down-regulated and 101 up-regulated between the two groups was observed. We identified a remarkably elevated expression of a spectrum of genes involved in Immunity and Defense in PBMCs of RA patients compared to controls. This result is confirmed by GO analysis, suggesting that these genes could be activated systemically in RA. No significant down-regulated ontology groups were found. Microarray data were validated by real time PCR in a set of nine genes showing a high degree of correlation. CONCLUSIONS/SIGNIFICANCE: Our study highlighted several new genes that could contribute in the identification of innovative clinical biomarkers for diagnostic procedures and therapeutic interventions.

Mycobacterial antigens stimulate rheumatoid mononuclear cells to cartilage proteoglycan depletion

NARCIS (Netherlands)

Wilbrink, B.; Bijlsma, J. W.; Huber-Bruning, O.; van Roy, J. L.; den Otter, W.; van Eden, W.

1990-01-01

In a coculture with porcine articular cartilage explants unstimulated blood mononuclear cells (BMC) from patients with rheumatoid arthritis (RA), but not from healthy controls, induced proteoglycan depletion of dead cartilage. Specific stimulation of the RA BMC with Mycobacterium tuberculosis (MT),

Winter to summer change in vitamin D status reduces systemic inflammation and bioenergetic activity of human peripheral blood mononuclear cells

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Emily K. Calton

2017-08-01

Full Text Available Background: Vitamin D status [25(OHD] has recently been reported to be associated with altered cellular bioenergetic profiles of peripheral blood mononuclear cells (PBMCs. No study has tracked the seasonal variation of 25(OHD and its putative influence on whole body energy metabolism, cellular bioenergetic profiles, inflammatory markers and clinical chemistry. Material and methods: Whole body energy metabolism and substrate utilisation were measured by indirect calorimetry. PBMCs obtained from the same subjects were isolated from whole blood, counted and freshly seeded. Bioenergetic analysis (mitochondrial stress test and glycolysis stress test was performed using the Seahorse XFe96 flux analyser. 25(OHD was assessed using the Architect immunoassay method. Results: 25(OHD increased by a median (IQR of 14.40 (20.13 nmol/L (p75 nmol/L. The absolute change in 25(OHD was not associated with altered bioenergetics. Conclusion: Seasonal improvements in 25(OHD was associated with reduced systemic inflammation, PBMC bioenergetic profiles and whole body energy metabolism. These observational changes in PBMC bioenergetics were most pronounced in those who had insufficient 25(OHD in winter. The data warrants confirmation through cause and effect study designs. Keywords: Peripheral blood mononuclear cells, Bioenergetics, Vitamin D, Season, Inflammation, Insulin sensitivity

Peripheral blood mononuclear cells gene expression reflects adaptive response and metabolic damage associated to the intake of diets with an unbalanced proportion of macronutrients

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Schothorst, van Evert; Diaz, Ruben; Keijer, Jaap; Paula, Oliver

2015-01-01

Gene expression studies in peripheral blood mononuclear cells (PBMC) can provide knowledge that would be difficult to obtain using other types of biological samples, as these cells can reflect overall response of the body to a specific stimulus, such as diet. Here, we aimed to study the impact of

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

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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.

Mitochondrial Alterations in Peripheral Mononuclear Blood Cells from Alzheimer’s Disease and Mild Cognitive Impairment Patients

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A. Delbarba

2016-01-01

Full Text Available It is well recognized that mitochondrial dysfunction contributes to neurodegeneration occurring in Alzheimer’s disease (AD. However, evidences of mitochondrial defects in AD peripheral cells are still inconclusive. Here, some mitochondrial-encoded and nuclear-encoded proteins, involved in maintaining the correct mitochondria machine, were investigated in terms of protein expression and enzymatic activity in peripheral blood mononuclear cells (PBMCs isolated from AD and Mild Cognitive Impairment (MCI patients and healthy subjects. In addition mitochondrial DNA copy number was measured by real time PCR. We found some differences and some similarities between AD and MCI patients when compared with healthy subjects. For example, cytochrome C and cytochrome B were decreased in AD, while MCI showed only a statistical reduction of cytochrome C. On the other hand, both AD and MCI blood cells exhibited highly nitrated MnSOD, index of a prooxidant environment inside the mitochondria. TFAM, a regulator of mitochondrial genome replication and transcription, was decreased in both AD and MCI patients’ blood cells. Moreover also the mitochondrial DNA amount was reduced in PBMCs from both patient groups. In conclusion these data confirmed peripheral mitochondria impairment in AD and demonstrated that TFAM and mtDNA amount reduction could be two features of early events occurring in AD pathogenesis.

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.

Recovery from Bell Palsy after Transplantation of Peripheral Blood Mononuclear Cells and Platelet-Rich Plasma.

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Seffer, Istvan; Nemeth, Zoltan

2017-06-01

Peripheral blood mononuclear cells (PBMCs) are multipotent, and plasma contains growth factors involving tissue regeneration. We hypothesized that transplantation of PBMC-plasma will promote the recovery of paralyzed facial muscles in Bell palsy. This case report describes the effects of PBMC-plasma transplantations in a 27-year-old female patient with right side Bell palsy. On the affected side of the face, the treatment resulted in both morphological and functional recovery including voluntary facial movements. These findings suggest that PBMC-plasma has the capacity of facial muscle regeneration and provides a promising treatment strategy for patients suffering from Bell palsy or other neuromuscular disorders.

Interleukin-4 and interferon-¿ production by Leishmania stimulated peripheral blood mononuclear cells from nonexposed individuals

DEFF Research Database (Denmark)

Kurtzhals, J A; Kemp, M; Poulsen, L K

1995-01-01

of antigen stimulation suggesting a response due to antigen recognition. Both IL-4 and IFN-gamma production was abrogated by depletion of CD2+ or CD4+ but not CD8+ cells. CD2+ or CD4+ but not CD8+ enriched cultures produced cytokines as unseparated PBMC. Thus, in non-exposed individuals circulating...... call for studies of the importance of cytokine production by cross-reactive T cells for the outcome of L. donovani infections in humans and show that the method for IL-4 detection is useful for this purpose.......Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) production by Leishmania reactive peripheral blood mononuclear cells (PBMC) from non-exposed individuals was investigated. IFN-gamma was measured in culture supernatants after antigen stimulation. For the measurement of IL-4, antigen stimulated...

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

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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...... to as 'centre') collected and cryopreserved PBMC samples from three donors, using a standardised cell isolation protocol. The samples were analysed in 13 different laboratories for DNA damage, which is measured by the comet assay. The study aim was to assess variation in DNA damage in PBMC samples that were......%) by standardisation of the primary comet assay endpoint with calibration curve samples. The level of DNA strand breaks in the samples from two of the centres (0.56-0.61 lesions/10(6) bp) was significantly higher compared with the other three centres (0.41-0.45 lesions/10(6) bp). In contrast, there was no difference...

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

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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.

Generation of integration-free induced pluripotent stem cells (GZHMUi001-A by reprogramming peripheral blood mononuclear cells from a 47, XXX syndrome patient

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Yuchang Chen

2017-08-01

Full Text Available 47, XXX syndrome is one of several sex-chromosomal aneuploidies, and it has an incidence of approximately 1/1000 in newborn females. Because of heterogeneity in X-inactivation, these patients may exhibit a variety of clinical symptoms. Here, we report the generation of an integration-free human induced pluripotent stem cell line (GZHMUi001-A by using Sendai virus to reprogram peripheral blood mononuclear cells from a 47, XXX syndrome patient with premature ovarian failure. This 47, XXX iPS cell line has characteristics of pluripotent stem cells and is a useful tool for the investigation of this X chromosome aneuploid disease.

Generation of integration-free induced pluripotent stem cells (GZHMUi001-A) by reprogramming peripheral blood mononuclear cells from a 47, XXX syndrome patient.

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Chen, Yuchang; Ou, Zhanhui; Song, Bing; Xian, Yexing; Ouyang, Shuming; Xie, Yuhuan; Xue, Yanting; Sun, Xiaofang

2017-08-01

47, XXX syndrome is one of several sex-chromosomal aneuploidies, and it has an incidence of approximately 1/1000 in newborn females. Because of heterogeneity in X-inactivation, these patients may exhibit a variety of clinical symptoms. Here, we report the generation of an integration-free human induced pluripotent stem cell line (GZHMUi001-A) by using Sendai virus to reprogram peripheral blood mononuclear cells from a 47, XXX syndrome patient with premature ovarian failure. This 47, XXX iPS cell line has characteristics of pluripotent stem cells and is a useful tool for the investigation of this X chromosome aneuploid disease. Copyright © 2017. Published by Elsevier B.V.

Expression sequence tag library derived from peripheral blood mononuclear cells of the chlorocebus sabaeus

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Tchitchek Nicolas

2012-06-01

Full Text Available Abstract Background African Green Monkeys (AGM are amongst the most frequently used nonhuman primate models in clinical and biomedical research, nevertheless only few genomic resources exist for this species. Such information would be essential for the development of dedicated new generation technologies in fundamental and pre-clinical research using this model, and would deliver new insights into primate evolution. Results We have exhaustively sequenced an Expression Sequence Tag (EST library made from a pool of Peripheral Blood Mononuclear Cells from sixteen Chlorocebus sabaeus monkeys. Twelve of them were infected with the Simian Immunodeficiency Virus. The mononuclear cells were or not stimulated in vitro with Concanavalin A, with lipopolysacharrides, or through mixed lymphocyte reaction in order to generate a representative and broad library of expressed sequences in immune cells. We report here 37,787 sequences, which were assembled into 14,410 contigs representing an estimated 12% of the C. sabaeus transcriptome. Using data from primate genome databases, 9,029 assembled sequences from C. sabaeus could be annotated. Sequences have been systematically aligned with ten cDNA references of primate species including Homo sapiens, Pan troglodytes, and Macaca mulatta to identify ortholog transcripts. For 506 transcripts, sequences were quasi-complete. In addition, 6,576 transcript fragments are potentially specific to the C. sabaeus or corresponding to not yet described primate genes. Conclusions The EST library we provide here will prove useful in gene annotation efforts for future sequencing of the African Green Monkey genomes. Furthermore, this library, which particularly well represents immunological and hematological gene expression, will be an important resource for the comparative analysis of gene expression in clinically relevant nonhuman primate and human research.

Evaluating the role of low-speed centrifugation towards transfecting human peripheral blood mononuclear cell culture.

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Majumdar, M; Ratho, R; Chawla, Y; Singh, M P

2014-01-01

The conventional method of transfection of suspension cells by chemical has proven to be very difficult. We present a new transfection protocol, wherein, low-speed centrifugation of cell culture plates immediately after adding the lipid: DNA complex significantly enhances the transfection efficiency. Peripheral blood mononuclear cells (PBMCs) were transfected with BLOCK-iT™ Fluorescent Oligo (scrambled siRNA) and lipofectamine complex using conventional and low-speed centrifugation modified transfection protocols. The efficiency of transfection was determined using flowcytometer and cell viability was checked using MTT assay. Incorporation of low-speed centrifugation significantly enhances the transfection efficiency of BLOCK-iT™ in the suspension culture of PBMCs as compared to conventional transfection method (99.8% vs 28.3%; P Centrifugation enhanced transfection (CET) technique is simple, time-saving and novel application without compromising the cell viability in the context of recently popular RNA interference in suspension cultures of PBMCs. This undemanding modification might be applicable to a wide variety of cell lines and solve crucial problem of researchers working with RNA interference in suspension cultures.

A new portable device for automatic controlled-gradient cryopreservation of blood mononuclear cells.

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Hviid, L; Albeck, G; Hansen, B; Theander, T G; Talbot, A

1993-01-04

Protection of the functional integrity of mononuclear cells stored in liquid N2 requires careful control of the freezing procedure. Consequently, optimal quality of cryopreserved cells is usually assured by freezing according to a specified time-temperature gradient generated by computer-controlled freezing devices. While such equipment offers large capacity and secures maximum survival and functional integrity of the lymphocytes upon thawing, it is quite costly and strictly stationary. We have previously developed and tested an alternative, manual device for controlled-gradient lymphocyte freezing, 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 and the neck in an ordinary liquid N2 refrigerator. The lymphocyte samples are placed in a small elevator, which is moved through the N2 gas phase by a stepper motor. Time and temperature are measured at regular intervals, and the position of the samples adjusted to ensure that the actual measurements closely match encoded ideal values. Results of assays of the functional integrity and phenotypic composition of human mononuclear cells frozen by the new system were comparable to those obtained when using cells frozen by a commercially available, stationary cell-freezing equipment, or fresh autologous cell samples tested in parallel. Furthermore, there was a good correlation between functional and phenotypic data obtained using frozen and autologous fresh samples of mononuclear cells. The equipment described is low weight and has low N2 consumption, and is thus suitable for the collection and cryopreservation of lymphocytes under field conditions. Furthermore, the technique provides an inexpensive alternative for researchers with a limited requirement for the simultaneous freezing of

Activation of p38 MAPK by feline infectious peritonitis virus regulates pro-inflammatory cytokine production in primary blood-derived feline mononuclear cells.

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Regan, Andrew D; Cohen, Rebecca D; Whittaker, Gary R

2009-02-05

Feline infectious peritonitis (FIP) is an invariably fatal disease of cats caused by systemic infection with a feline coronavirus (FCoV) termed feline infectious peritonitis virus (FIPV). The lethal pathology associated with FIP (granulomatous inflammation and T-cell lymphopenia) is thought to be mediated by aberrant modulation of the immune system due to infection of cells such as monocytes and macrophages. Overproduction of pro-inflammatory cytokines occurs in cats with FIP, and has been suggested to play a significant role in the disease process. However, the mechanism underlying this process remains unknown. Here we show that infection of primary blood-derived feline mononuclear cells by FIPV WSU 79-1146 and FIPV-DF2 leads to rapid activation of the p38 MAPK pathway and that this activation regulates production of the pro-inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta). FIPV-induced p38 MAPK activation and pro-inflammatory cytokine production was inhibited by the pyridinyl imidazole inhibitors SB 203580 and SC 409 in a dose-dependent manner. FIPV-induced p38 MAPK activation was observed in primary feline blood-derived mononuclear cells individually purified from multiple SPF cats, as was the inhibition of TNF-alpha production by pyridinyl imidazole inhibitors.

The Impact of Glyphosate, Its Metabolites and Impurities on Viability, ATP Level and Morphological changes in Human Peripheral Blood Mononuclear Cells

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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

In vitro cytokine production and phenotype expression by blood mononuclear cells from umbilical cords, children and adults

DEFF Research Database (Denmark)

Müller, K; Zak, M; Nielsen, S

1996-01-01

Age related differences in immunological reactions include variations in the in vitro functions of blood mononuclear cells (MNC). In an attempt to understand the mechanism behind these differences we examined age related differences in the phenotype profiles of MNC in parallel with the in vitro......, and unmeasurable levels in cord blood MNC. Flow cytometry analysis of the phenotypic distribution of MNC revealed age related differences in the expression of CD3, CD4, CD8, CD14, CD19, CD45RA, CD45R0, CD2, LFA-1, ICAM-1 and LFA-3. Correlation studies did not indicate that the observed differences in cytokine....... In conclusion, the study provides evidence of age related differences in the production of TNF alpha, IL-6 and IFNg among neonates, children and adults. These differences may to some extent be caused by differences in the expression of cell surface molecules involved in cellular interactions and signalling....

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.

Effects of chronic morphine and morphine withdrawal on gene expression in rat peripheral blood mononuclear cells.

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Desjardins, Stephane; Belkai, Emilie; Crete, Dominique; Cordonnier, Laurie; Scherrmann, Jean-Michel; Noble, Florence; Marie-Claire, Cynthia

2008-12-01

Chronic morphine treatment alters gene expression in brain structures. There are increasing evidences showing a correlation, in gene expression modulation, between blood cells and brain in psychological troubles. To test whether gene expression regulation in blood cells could be found in drug addiction, we investigated gene expression profiles in peripheral blood mononuclear (PBMC) cells of saline and morphine-treated rats. In rats chronically treated with morphine, the behavioral signs of spontaneous withdrawal were observed and a withdrawal score was determined. This score enabled to select the time points at which the animals displayed the mildest and strongest withdrawal signs (12 h and 36 h after the last injection). Oligonucleotide arrays were used to assess differential gene expression in the PBMCs and quantitative real-time RT-PCR to validate the modulation of several candidate genes 12 h and 36 h after the last injection. Among the 812 differentially expressed candidates, several genes (Adcy5, Htr2a) and pathways (Map kinases, G-proteins, integrins) have already been described as modulated in the brain of morphine-treated rats. Sixteen out of the twenty-four tested candidates were validated at 12 h, some of them showed a sustained modulation at 36 h while for most of them the modulation evolved as the withdrawal score increased. This study suggests similarities between the gene expression profile in PBMCs and brain of morphine-treated rats. Thus, the searching of correlations between the severity of the withdrawal and the PBMCs gene expression pattern by transcriptional analysis of blood cells could be promising for the study of the mechanisms of addiction.

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

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

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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. Copyright © 2014 Elsevier B.V. All rights reserved.

Nuclear NF-κB p65 in peripheral blood mononuclear cells correlates with urinary MCP-1, RANTES and the severity of type 2 diabetic nephropathy.

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Bin Yi

Full Text Available AIMS: To investigate if nuclear NF-κB p65 expression in ex vivo isolated peripheral blood mononuclear cells correlates with urinary MCP-1 or RANTES and the severity of type 2 diabetic nephropathy. METHODS: According to their urinary albumin-to-creatinine ratio (uACR, 107 patients with type 2 diabetes (eGFR >60 ml/min were divided into normal albuminuria group (DN0 group, 38 cases, microalbuminuria group (DN1 group, 38 cases, and macroalbuminuria group (DN2 group, 31 cases, compared with matched healthy normal control group (NC group, 30 cases. Nuclear NF-κB p65 protein expression levels in peripheral blood mononuclear cells were detected by western blotting. Real-time quantitative polymerase chain reaction was used to detect NF-κB p65 mRNA expression and ELISA assay was used to detect the levels of urinary MCP-1 and RANTES. RESULTS: Nuclear NF-κB p65 protein and NF-κB p65 mRNA expression levels in peripheral blood mononuclear cells, urinary MCP-1/Cr and RANTES/Cr were all significantly higher in all diabetes groups as compared with NC group. In particular, the increase of nuclear NF-κB p65 protein and NF-κB p65 mRNA expressions, urinary MCP-1/Cr and RANTES/Cr all correlated with the severity of type 2 diabetic nephropathy as indicated by the increase in uACR. Pearson correlation analysis indicated that both urinary MCP-1/Cr and RANTES/Cr were positively correlated with nuclear NF-κB p65 protein or NF-κB p65 mRNA levels. Stepwise multiple regression analysis showed that nuclear NF-κB p65 protein or NF-κB p65 mRNA was an independent variable for urinary MCP-1/Cr, and MCP-1/Cr and RANTES/Cr were two independent variables for uACR. CONCLUSION: Our research demonstrates that nuclear NF-κB p65 protein and mRNA expressions in ex vivo isolated peripheral blood mononuclear cells well correlate with urinary MCP-1/Cr, RANTES/Cr and the severity of type 2 diabetic nephropathy.

Umbilical Cord Blood-Derived Mononuclear Cells Exhibit Pericyte-Like Phenotype and Support Network Formation of Endothelial Progenitor Cells In Vitro.

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Peters, Erica B; Liu, Betty; Christoforou, Nicolas; West, Jennifer L; Truskey, George A

2015-10-01

Umbilical cord blood represents a promising cell source for pro-angiogenic therapies. The present study examined the potential of mononuclear cells (MNCs) from umbilical cord blood to support endothelial progenitor cell (EPC) microvessel formation. MNCs were isolated from the cord blood of 20 separate donors and selected for further characterization based upon their proliferation potential and morphological resemblance to human vascular pericytes (HVPs). MNCs were screened for their ability to support EPC network formation using an in vitro assay (Matrigel™) as well as a reductionist, coculture system consisting of no additional angiogenic cytokines beyond those present in serum. In less than 15% of the isolations, we identified a population of highly proliferative MNCs that phenotypically resembled HVPs as assessed by expression of PDGFR-β, NG2, α-SMA, and ephrin-B2. Within a Matrigel™ system, MNCs demonstrated pericyte-like function through colocalization to EPC networks and similar effects as HVPs upon total EPC tubule length (p = 0.95) and number of branch points (p = 0.93). In a reductionist coculture system, MNCs served as pro-angiogenic mural cells by supporting EPC network formation to a significantly greater extent than HVP cocultures, by day 14 of coculture, as evidenced through EPC total tubule length (p < 0.0001) and number of branch points (p < 0.0001). Our findings are significant as we demonstrate mural cell progenitors can be isolated from umbilical cord blood and develop culture conditions to support their use in microvascular tissue engineering applications.

Evaluating the role of low-speed centrifugation towards transfecting human peripheral blood mononuclear cell culture

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M Majumdar

2014-01-01

Full Text Available The conventional method of transfection of suspension cells by chemical has proven to be very difficult. We present a new transfection protocol, wherein, low-speed centrifugation of cell culture plates immediately after adding the lipid: DNA complex significantly enhances the transfection efficiency. Peripheral blood mononuclear cells (PBMCs were transfected with BLOCK-iT™ Fluorescent Oligo (scrambled siRNA and lipofectamine complex using conventional and low-speed centrifugation modified transfection protocols. The efficiency of transfection was determined using flowcytometer and cell viability was checked using MTT assay. Incorporation of low-speed centrifugation significantly enhances the transfection efficiency of BLOCK-iT™ in the suspension culture of PBMCs as compared to conventional transfection method (99.8% vs 28.3%; P < 0.0001, even at a low concentration of 40 picomoles without affecting the cell viability. Centrifugation enhanced transfection (CET technique is simple, time-saving and novel application without compromising the cell viability in the context of recently popular RNA interference in suspension cultures of PBMCs. This undemanding modification might be applicable to a wide variety of cell lines and solve crucial problem of researchers working with RNA interference in suspension cultures.

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

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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.

Studies on the regeneration of the CFU-C population in blood and bone marrow or lethally irradiated dogs after autologous transfusion of cryopreserved mononuclear blood cells

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Nothdurft, W.; Bruch, C.; Fliedner, T.M.; Rueber, E.

1977-01-01

In a group of 8 lethally irradiated (1200 R) dogs, that were transfused autologously with cryopreserved mononuclear cells (MNC) derived from the peripheral blood by leucapheresis the concentration of colony-forming units in agar (CFU-C) in bone marrow and peripheral blood was estimated at regular intervals after irradiation and transfusion of MNC. The numbers of MNC transfused per kg body weight ranged from 0.32 x 10 9 to 1.63 x 10 9 with an incidence of CFU-C between 0.02 x 10 5 and 1.38 x 10 5 . In 6 dogs the CFU-C levels in the bone marrow reached the normal preirradiation values between days 15 and 20. But in 2 dogs that had received the lowest CFU-C numbers the regeneration of the bone marrow CFU-C was markedly delayed. In general the time course of the bone marrow repopulation by CFU-C for single dogs was reflected by a corresponding regeneration pattern of the blood CFU-C. The time course of the curves for the blood CFU-C levels on the other hand was of the same kind as for the granulocyte values in the peripheral blood, that reached the normal levels mainly around day 30 and thereafter. Considerable fluctuations were seen in the blood CFU-C levels of single dogs before irradiation and after mononuclear leucocyte transfusion. Despite of such limitations the blood CFU-C content appeared to be a useful indicator of haematopoietic regeneration of the bone marrow. (author)

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

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

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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...

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

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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......, our results indicate that inter-investigator difference in scoring is a strong determinant of DNA damage levels measured by the comet assay....

Expression of Cyclin D1 protein and CCN DI with PNKP genes in peripheral blood mononuclear cells in clean-up worker of Chernobyl accident with different state of immune system

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Bazika, D.A.; Kubashko, A.V.; Yil'jenko, Yi.M.; Belyajev, O.A.; Pleskach, O.Ya.

2015-01-01

The investigate of Cyclin D1+cells levels changes, associated CCND1 and PNKP genes in peripheral blood mononuclear cells in cleanup workers of Chornobyl accident with different state of immune system in depends on the dose irradiation. Analyzed data of the nuclear controller of cell cycle- Cyclin D1 protein expression changes and related CCND1 and PNKP genes in peripheral blood mononuclear cells in cleanup workers Chornobyl accident with different status of immune system in remote period after exposure is represented. Reveled changes in expression of Cyclin D1+cells and regulation of related genes may point on possible radiation-associated firm molecular disturbances occurred during elimination of consequences of Chornobyl accident, that could be a potential basis for cell and humoral communicative links breach in immune system result ing in elevation of stochastic effects like oncopathology in cleanup workers of Chornobyl accident in remote peri od after exposure

Microculture assay for isolation of human immunodeficiency virus type 1 and for titration of infected peripheral blood mononuclear cells.

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Dimitrov, D H; Melnick, J L; Hollinger, F B

1990-04-01

To define the optimal conditions for human immunodeficiency virus (HIV) detection in microcultures, experiments were conducted with different ratios of patient and donor peripheral blood mononuclear cells (PBMCs). Donor/patient PBMC ratios ranged from 1:1 to 1:125. Optimal results were obtained when 1,500,000 donor cells were cocultured with equal or smaller quantities of patient PBMCs. Thus, virologic endpoints could be achieved by diluting patient cells. Smaller numbers of donor cells, with or without larger numbers of patients cells, resulted in lower rates of HIV isolation. Similarly, the direct stimulation of patient PBMCs with phytohemagglutinin without the addition of normal donor cells lowered the sensitivity of the assay significantly. We suggest that a microculture procedure using a fixed quantity of donor cells with different dilutions of patient cells may be useful for monitoring changing HIV levels during antiviral therapy.

Cord Blood Cells Responses to IL2, IL7 and IL15 Cytokines for mTOR Expression

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Anahita Mohammadian

2017-04-01

Full Text Available Purpose: Mammalian target of rapamycin (mTORis important in hematopoiesis and affect cell growth,differentiation and survival. Although previous studies were identified the effect of cytokines on the mononuclear cells development however the cytokines effect on mTOR in cord blood mononuclear cells was unclear. The aim of this study was to evaluate mTOR expression in cord blood mononuclear and cord blood stem cells (CD34+ cells in culture conditions for lymphoid cell development. Methods: Isolation of The mononuclear cells (MNCs from umbilical cord blood were done with use of Ficollpaque density gradient. We evaluated cultured cord blood mononuclear and CD34+ cells in presece of IL2, IL7 and IL15 at distinct time points during 21 days by using flow cytometry. In this study, we presented the role of IL2, IL7 and IL15 on the expression of mTOR in cord blood cells. Results: mTOR expression were increased in peresence of IL2, IL7 and IL15 in day 14 and afterword reduced. However in persence of IL2 and IL15 expression of mTOR significantly reduced. mTOR expression in CD34+ cells decreased significantly from day7 to day 21 in culture. Conclusion: cytokines play important role in mTOR expression during hematopoiesis and development of cord blood mononuclear cells.

Estradiol and Progesterone Strongly Inhibit the Innate Immune Response of Mononuclear Cells in Newborns ▿

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Giannoni, Eric; Guignard, Laurence; Knaup Reymond, Marlies; Perreau, Matthieu; Roth-Kleiner, Matthias; Calandra, Thierry; Roger, Thierry

2011-01-01

Newborns are particularly susceptible to bacterial infections due to qualitative and quantitative deficiencies of the neonatal innate immune system. However, the mechanisms underlying these deficiencies are poorly understood. Given that fetuses are exposed to high concentrations of estradiol and progesterone during gestation and at time of delivery, we analyzed the effects of these hormones on the response of neonatal innate immune cells to endotoxin, bacterial lipopeptide, and Escherichia coli and group B Streptococcus, the two most common causes of early-onset neonatal sepsis. Here we show that at concentrations present in umbilical cord blood, estradiol and progesterone are as powerful as hydrocortisone for inhibition of cytokine production by cord blood mononuclear cells (CBMCs) and newborn monocytes. Interestingly, CBMCs and newborn monocytes are more sensitive to the effects of estradiol and progesterone than adult peripheral blood mononuclear cells and monocytes. This increased sensitivity is associated with higher expression levels of estrogen and membrane progesterone receptors but is independent of a downregulation of Toll-like receptor 2 (TLR2), TLR4, and myeloid differentiation primary response gene 88 in newborn cells. Estradiol and progesterone mediate their anti-inflammatory activity through inhibition of the NF-κB pathway but not the mitogen-activated protein kinase pathway in CBMCs. Altogether, these results suggest that elevated umbilical cord blood concentrations of estradiol and progesterone acting on mononuclear cells expressing high levels of steroid receptors contribute to impair innate immune responses in newborns. Therefore, intrauterine exposure to estradiol and progesterone may participate in increasing susceptibility to infection during the neonatal period. PMID:21518785

Child Maltreatment Is Associated with a Reduction of the Oxytocin Receptor in Peripheral Blood Mononuclear Cells.

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Krause, Sabrina; Boeck, Christina; Gumpp, Anja M; Rottler, Edit; Schury, Katharina; Karabatsiakis, Alexander; Buchheim, Anna; Gündel, Harald; Kolassa, Iris-Tatjana; Waller, Christiane

2018-01-01

Background: Child maltreatment (CM) and attachment experiences are closely linked to alterations in the human oxytocin (OXT) system. However, human data about oxytocin receptor (OXTR) protein levels are lacking. Therefore, we investigated oxytocin receptor (OXTR) protein levels in circulating immune cells and related them to circulating levels of OXT in peripheral blood. We hypothesized reduced OXTR protein levels, associated with both, experiences of CM and an insecure attachment representation. Methods: OXTR protein expressions were analyzed by western blot analyses in peripheral blood mononuclear cells (PBMC) and plasma OXT levels were determined by radioimmunoassay (RIA) in 49 mothers. We used the Childhood Trauma Questionnaire (CTQ) to assess adverse childhood experiences. Attachment representations (secure vs. insecure) were classified using the Adult Attachment Projective Picture System (AAP) and levels of anxiety and depression were assessed with the German version of the Hospital Depression and Anxiety scale (HADS-D). Results: CM-affected women showed significantly lower OXTR protein expression with significantly negative correlations between the OXTR protein expression and the CTQ sum score, whereas plasma OXT levels showed no significant differences in association with CM. Lower OXTR protein expression in PBMC were particularly pronounced in the group of insecurely attached mothers compared to the securely attached group. Anxiety levels were significantly higher in CM-affected women. Conclusion: This study demonstrated a significant association between CM and an alteration of OXTR protein expression in human blood cells as a sign for chronic, long-lasting alterations in this attachment-related neurobiological system.

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 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.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.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.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.

Clinical study on insulin receptors of mononuclear cells in diabetes

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Dalimunthe, D [Hiroshima Univ. (Japan). School of Medicine

1980-12-01

/sup 125/I-insulin binding activity to mononuclear cells was studied in 75 noninsulin-dependent diabetic subjects and 31 normal subjects and the following results were obtained. 1. /sup 125/I-insulin binding is directly proportional to the mononuclear cell concentrations. There is a linear increase of specific /sup 125/I-insulin binding. 2. The binding of /sup 125/I-insulin to mononuclear cells is displaced by the increasing concentration of native insulin. 3. The /sup 125/I-insulin degradation in the incubation medium after incubation of mononuclear cells for 24 hours at 4/sup 0/C was almost 5% in this study. 4. The insulin binding activity in diabetic subjects was lower than that in normal subjects (P < 0.001) without any significant difference in affinity constant. 5. The relationship of binding activity to age of diabetics (r = 0.06, N.S), relative body weitht (r = 0.06, N.S) and duration of diabetes from onset was not significant. 6. In untreated noninsulin-dependent diabetics the insulin binding activity was inversely correlated to fasting blood glucose level (r = 0.78, P < 0.001) and slightly inversely correlated to serum insulin level (r = 0.47, P < 0.01). A slight inverse correlation was also observed in serum triglyceride level (r = 0.53, P < 0.01) and in total cholesterol level (r = 0.29, P < 0.05). 7. No significant difference between the binding activity was observed by grade of diabetic retinopathy. 8. After treatment with diet and/or sulfonylurea, the diabetics exhibited a significant increase in insulin binding activity (P < 0.005) but no significant difference in plasma insulin level, body weight and plasma lipid levels was observed.

EXPRESSION OF GENETIC LOCI IN THE PERIPHERAL BLOOD MONONUCLEAR FRACTION FROM PATIENTS WITH PROSTATE CANCER

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M. I. Kogan

2014-08-01

Full Text Available The early diagnosis and radical treatment of aggressive prostate cancers (PC is an effective way of improving survival and quality of life in patients. To develop mini-invasive tests is one of the ways of solving the problem. The cells of a peripheral blood mononuclear fraction in the expression patterns of their genetic loci reflect the presence or absence of cancers, including information on therapeutic effectiveness. RT-PRC was used to study the relative expression of 15 genetic loci in a chromosome and one locus of mitochondrial DNA in the cells of the peripheral blood mononuclear fraction in patients with PC or benign prostate hyperplasia and in healthy men. The genetic locus patterns whose change may be of informative value for differential diagnosis in patients with different stages of PC were revealed. The authors studied the relationship and showed the prognostic role and non-relationship of the altered transcriptional activity of loci in the TP53, GSTP1, and IL10 genes in PC to the changes in prostate-specific antigen the level with 90 % specificity and 93 % specificity.

Activation of peripheral blood mononuclear cells by dengue virus infection depotentiates balapiravir.

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Chen, Yen-Liang; Abdul Ghafar, Nahdiyah; Karuna, Ratna; Fu, Yilong; Lim, Siew Pheng; Schul, Wouter; Gu, Feng; Herve, Maxime; Yokohama, Fumiaki; Wang, Gang; Cerny, Daniela; Fink, Katja; Blasco, Francesca; Shi, Pei-Yong

2014-02-01

In a recent clinical trial, balapiravir, a prodrug of a cytidine analog (R1479), failed to achieve efficacy (reducing viremia after treatment) in dengue patients, although the plasma trough concentration of R1479 remained above the 50% effective concentration (EC(50)). Here, we report experimental evidence to explain the discrepancy between the in vitro and in vivo results and its implication for drug development. R1479 lost its potency by 125-fold when balapiravir was used to treat primary human peripheral blood mononuclear cells (PBMCs; one of the major cells targeted for viral replication) that were preinfected with dengue virus. The elevated EC(50) was greater than the plasma trough concentration of R1479 observed in dengue patients treated with balapiravir and could possibly explain the efficacy failure. Mechanistically, dengue virus infection triggered PBMCs to generate cytokines, which decreased their efficiency of conversion of R1479 to its triphosphate form (the active antiviral ingredient), resulting in decreased antiviral potency. In contrast to the cytidine-based compound R1479, the potency of an adenosine-based inhibitor of dengue virus (NITD008) was much less affected. Taken together, our results demonstrate that viral infection in patients before treatment could significantly affect the conversion of the prodrug to its active form; such an effect should be calculated when estimating the dose efficacious for humans.

The influence of caloric deprivation and food composition on TSH, thyroid hormones and nuclear binding of T3 in mononuclear blood cells in obese women

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Matzen, L E; Kvetny, J

1989-01-01

In vivo changes in thyroid-stimulating hormone (TSH), thyroxin (T4), triiodothyronine (T3) and nuclear binding of T3 (NBT3) in mononuclear blood cells were studied in obese women during seven days of caloric deprivation (maximum 1,100 kcal/d). In seven women given a high protein diet (80% protein...

Effect of curcumin on ethanol-induced stress on mononuclear cells.

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Rajakrishnan, V; Shiney, S J; Sudhakaran, P R; Menon, V P

2002-03-01

Blood cells in circulation are exposed to a wide variety of stress-causing agents, causing a number of changes including interactions with other cells and the extracellular matrix of the endothelial wall. In order to understand the role of curcumin, an antioxidant principle from Curcuma longa Linn., on blood mononuclear cells from rabbits given ethanol for 30 days and ethanol with curcumin, cells were isolated and an attachment assay was carried out. The monocytes from ethanol-treated rabbits showed a lesser attachment to collagen, the major component of the vessel wall subendothelium, and those from curcumin treated animals along with ethanol showed a higher affinity to collagen, causing an alteration in the attachment of monocyte to collagen due to ethanol-induced stress. Copyright 2002 John Wiley & Sons, Ltd.

Cytogenetic studies in dogs after total body irradiation and allogeneic transfusion with cryopreserved blood mononuclear cells: observations in long-term chimeras

International Nuclear Information System (INIS)

Carbonell, F.; Calvo, W.; Fliedner, T.M.; Kratt, E.; Gerhartz, H.; Koerbling, M.; Nothdurft, W.; Ross, W.M.

1984-01-01

Cytogenetic studies were performed on two dog groups after total body irradiation and allogeneic transfusion with cryopreserved blood mononuclear cells. The first group of dogs was transfused with unseparated leukocytes and suffered from graft-versus-host disease (GvHD). Cytogenetic studies demonstrated only cells of donor origin in all dogs of this group. The second group of animals was transfused with fraction 2 of a discontinuous albumin gradient. The dogs of this group did not develop GvHD, and the cytogenetic studies showed the presence of a mosaic of cells from donor and recipient origin in all of them. These results suggest that the GvHD may suppress autochthonous regeneration

The Generation of Human γδT Cell-Derived Induced Pluripotent Stem Cells from Whole Peripheral Blood Mononuclear Cell Culture.

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Watanabe, Daisuke; Koyanagi-Aoi, Michiyo; Taniguchi-Ikeda, Mariko; Yoshida, Yukiko; Azuma, Takeshi; Aoi, Takashi

2018-01-01

γδT cells constitute a small proportion of lymphocytes in peripheral blood. Unlike αβT cells, the anti-tumor activities are exerted through several different pathways in a MHC-unrestricted manner. Thus, immunotherapy using γδT cells is considered to be effective for various types of cancer. Occasionally, however, ex vivo expanded cells are not as effective as expected due to cell exhaustion. To overcome the issue of T-cell exhaustion, researchers have generated induced pluripotent stem cells (iPSCs) that harbor the same T-cell receptor (TCR) genes as their original T-cells, which provide nearly limitless sources for antigen-specific cytotoxic T lymphocytes (CTLs). However, these technologies have focused on αβT cells and require a population of antigen-specific CTLs, which are purified by cell sorting with HLA-peptide multimer, as the origin of iPS cells. In the present study, we aimed to develop an efficient and convenient system for generating iPSCs that harbor rearrangements of the TCRG and TCRD gene regions (γδT-iPSCs) without cell-sorting. We stimulated human whole peripheral blood mononuclear cell (PBMC) culture using Interleukin-2 and Zoledronate to activate γδT cells. Gene transfer into those cells with the Sendai virus vector resulted in γδT cell-dominant expression of exogenous genes. The introduction of reprogramming factors into the stimulated PBMC culture allowed us to establish iPSC lines. Around 70% of the established lines carried rearrangements at the TCRG and TCRD gene locus. The γδT-iPSCs could differentiate into hematopoietic progenitors. Our technology will pave the way for new avenues toward novel immunotherapy that can be applied for various types of cancer. Stem Cells Translational Medicine 2018;7:34-44. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

The production of collagenase by adherent mononuclear cells cultured from human peripheral blood.

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Louie, J S; Weiss, J; Ryhänen, L; Nies, K M; Rantala-Ryhänen, S; Uitto, J

1984-12-01

Mononuclear cells were isolated from human peripheral blood by Ficoll-Hypaque centrifugation, and the cells adherent to plastic substrata were cultured in serum-free media supplemented with lactalbumin hydrolysate. These cell cultures, which consisted predominantly of monocyte-macrophages as judged by nonspecific esterase staining, accumulated collagenase in the medium. This collagenase resembled other vertebrate collagenases in that it cleaved native triple-helical type I collagen at a locus 3/4-length away from the amino-terminal end of the molecule. The collagenase activity was inhibited by Na2EDTA, dithiothreitol, and fetal calf serum, while the addition of Ca++ or N-ethylmaleimide enhanced the enzyme activity. The accumulation of collagenase in the culture media was markedly enhanced by the incubation of cells with concanavalin A or phorbol myristic acetate. In the presence of cycloheximide, the levels of collagenase activity were markedly reduced, suggesting that active protein synthesis was required to express the enzyme activity. In additional experiments, monocytes were further purified by counterflow centrifugation-elutriation. The collagenase production was markedly increased in cultures enriched in monocyte-macrophages and devoid of polymorphonuclear leukocytes. The accumulation of collagenase in monocyte cultures incubated for 48 hours in the presence of concanavalin A or phorbol myristic acetate was of the same order of magnitude as in parallel cultures containing the same number of polymorphonuclear leukocytes purified by Ficoll-Hypaque centrifugation and Plasmagel sedimentation.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased activity of cell membrane-associated prothrombinase, fibrinogen-like protein 2, in peripheral blood mononuclear cells of B-cell lymphoma patients.

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Esther Rabizadeh

Full Text Available Fibrinogen-like protein 2, FGL-2, was reported to be overexpressed in various cancer tissues, where it acts as a transmembrane prothrombinase. This study aims to determine the prothrombinase activity of FGL-2 in peripheral blood mononuclear cells (PBMC of patients with B-cell lymphoma. FGL-2 activity was determined in patients with B-cell lymphoma (n = 53, and healthy controls (n = 145. FGL-2 activity in patients at diagnosis increased 3 ± 0.3 fold (p < 0.001. Sensitivity and specificity of the test was established at 73.6% and 80.7%, respectively, using a cutoff of 150% activity over control. Moreover, FGL-2 activity in 10 of 11 patients in remission decreased by 76%. In contrast, no significant difference was observed in expression levels of fgl-2 gene in patients and controls. Taken together, our study indicates that FGL-2 prothrombinase activity in PBMC of lymphoma patients is increased in active disease and normalizes during remission, thus being a potential marker for follow up of lymphoma patients.

Aislamiento de células mononucleares de sangre periférica para trasplante de células madre: Método simplificado Isolation of mononuclear cells of peripheral blood for stem cell transplant: Simplified method

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Lázaro Cortina Rosales

2008-12-01

mobilized peripheral blood. The blood was processed in closed collection systems, using hydroxyethyl starch as a potentiator of erythrocyte sedimentation. The patients were treated with granulocyte colony stimulating factor at total doses of 40µcg/kg of weight during 2 days. Self-donation was performed when the leukocyte count was higher than 20 x 109/L. To assess the efficacy of the method, the amounts of nucleated cells, of mononuclear cells and of CD 34+ cells in the cellular concentrate were analyzed. Cellular viability was determined and a microbiological study of the material obtained was conducted. It was proved that the method is efficient and safe, since adequate cellular levels with a high viability and absence of bacterial contamination are attained. On the other hand, it is simple and cheap, which allows its application in other health centres, particularly in those with less resources. This makes possible that more patients benefit from the stem cell treatment.

Time and dose dependent expression in the proteome of human peripheral blood mononuclear cells with γ-irradiation

International Nuclear Information System (INIS)

Nishad, S.; Ghosh, Anu

2014-01-01

The aim of present study is to investigate time and dose dependent differential protein expression pattern of human peripheral blood mononuclear cells (PBMCs) after acute gamma irradiation. For this purpose, PBMCs extracted from eight healthy individuals were irradiated using 60 Co gamma rays (0.3 Gy and 1 Gy) and compared with sham irradiated controls. Total proteins were extracted 1 and 4 hour post irradiation and analyzed using 2-D gel electrophoresis. A fold change of 1.5 in spot intensity was considered as 'biological significant'. Protein identification was performed by MALDI-TOF mass spectrometry. The MS/MS spectra were interrogated using Mascot 2.1 for searching against SWISS-PROT database. One-hour post irradiation, 18 proteins showed a significant difference between the sham (0 Gy) and 0.3 Gy irradiated group (6 proteins up-regulated and 12 proteins down-regulated) and 17 proteins between the sham (0 Gy) and 1 Gy irradiated group (9 proteins up-regulated and 8 down-regulated). Four hours after irradiation, 16 proteins were differentially expressed between the sham irradiated and 0.3 Gy treated group (5 proteins up-regulated and 11 proteins downregulated). Relatively high dose of 1 Gy showed modulation of 13 proteins (5 proteins upregulated and 8 proteins down regulated) after 4 hours. There were 15 proteins that were observed both at the early time point of 1-hour and the late time point of 4-hour. Important among these were, proteins involved in cytoskeletal organization like Actin, Plastin-2, Vinculin, PDZ and LIM domain protein, WD repeat containing protein and the chaperone proteins like HSP 90-alpha and Protein disulfide-isomerase A3. Proteins like thiol specific antioxidant peroxiredoxin-6 (indicating increased levels of ROS and oxidative stress) showed dose specific expression while proteins like Ras-related Rap-1b-like protein (involved in cell survival) were observed with both 0.3 Gy and 1 Gy. During the study, human peripheral blood

Child Maltreatment Is Associated with a Reduction of the Oxytocin Receptor in Peripheral Blood Mononuclear Cells

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Sabrina Krause

2018-02-01

Full Text Available Background: Child maltreatment (CM and attachment experiences are closely linked to alterations in the human oxytocin (OXT system. However, human data about oxytocin receptor (OXTR protein levels are lacking. Therefore, we investigated oxytocin receptor (OXTR protein levels in circulating immune cells and related them to circulating levels of OXT in peripheral blood. We hypothesized reduced OXTR protein levels, associated with both, experiences of CM and an insecure attachment representation.Methods: OXTR protein expressions were analyzed by western blot analyses in peripheral blood mononuclear cells (PBMC and plasma OXT levels were determined by radioimmunoassay (RIA in 49 mothers. We used the Childhood Trauma Questionnaire (CTQ to assess adverse childhood experiences. Attachment representations (secure vs. insecure were classified using the Adult Attachment Projective Picture System (AAP and levels of anxiety and depression were assessed with the German version of the Hospital Depression and Anxiety scale (HADS-D.Results: CM-affected women showed significantly lower OXTR protein expression with significantly negative correlations between the OXTR protein expression and the CTQ sum score, whereas plasma OXT levels showed no significant differences in association with CM. Lower OXTR protein expression in PBMC were particularly pronounced in the group of insecurely attached mothers compared to the securely attached group. Anxiety levels were significantly higher in CM-affected women.Conclusion: This study demonstrated a significant association between CM and an alteration of OXTR protein expression in human blood cells as a sign for chronic, long-lasting alterations in this attachment-related neurobiological system.

Blood Mononuclear Cell Mitochondrial Respiratory Chain Complex IV Activity is Decreased in Multiple Sclerosis Patients: Effects of β-Interferon Treatment

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Iain Hargreaves

2018-02-01

Full Text Available Objectives: Evidence of mitochondrial respiratory chain (MRC dysfunction and oxidative stress has been implicated in the pathophysiology of multiple sclerosis (MS. However, at present, there is no reliable low invasive surrogate available to evaluate mitochondrial function in these patients. In view of the particular sensitivity of MRC complex IV to oxidative stress, the aim of this study was to assess blood mononuclear cell (BMNC MRC complex IV activity in MS patients and compare these results to age matched controls and MS patients on β-interferon treatment. Methods: Spectrophotometric enzyme assay was employed to measure MRC complex IV activity in blood mononuclear cell obtained multiple sclerosis patients and aged matched controls. Results: MRC Complex IV activity was found to be significantly decreased (p < 0.05 in MS patients (2.1 ± 0.8 k/nmol × 10−3; mean ± SD] when compared to the controls (7.2 ± 2.3 k/nmol × 10−3. Complex IV activity in MS patients on β-interferon (4.9 ± 1.5 k/nmol × 10−3 was not found to be significantly different from that of the controls. Conclusions: This study has indicated evidence of peripheral MRC complex IV deficiency in MS patients and has highlighted the potential utility of BMNCs as a potential means to evaluate mitochondrial function in this disorder. Furthermore, the reported improvement of complex IV activity may provide novel insights into the mode(s of action of β-interferon.

Isolation and preservation of peripheral blood mononuclear cells for analysis of islet antigen-reactive T cell responses: position statement of the T-Cell Workshop Committee of the Immunology of Diabetes Society.

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Mallone, R; Mannering, S I; Brooks-Worrell, B M; Durinovic-Belló, I; Cilio, C M; Wong, F S; Schloot, N C

2011-01-01

Autoimmune T cell responses directed against insulin-producing β cells are central to the pathogenesis of type 1 diabetes (T1D). Detection of such responses is therefore critical to provide novel biomarkers for T1D 'immune staging' and to understand the mechanisms underlying the disease. While different T cell assays are being developed for these purposes, it is important to optimize and standardize methods for processing human blood samples for these assays. To this end, we review data relevant to critical parameters in peripheral blood mononuclear cell (PBMC) isolation, (cryo)preservation, distribution and usage for detecting antigen-specific T cell responses. Based on these data, we propose recommendations on processing blood samples for T cell assays and identify gaps in knowledge that need to be addressed. These recommendations may be relevant not only for the analysis of T cell responses in autoimmune disease, but also in cancer and infectious disease, particularly in the context of clinical trials. © 2010 The Authors. Clinical and Experimental Immunology © 2010 British Society for Immunology.

CFU-C populations in blood and bone marrow of dogs after lethal irradiation and allogeneic transfusion with cryopreserved blood mononuclear cells

International Nuclear Information System (INIS)

Nothdurft, W.; Fliedner, T.M.; Calvo, W.; Flad, H.-D.; Huget, R.; Koerbling, M.; Krumbacher-von Loringen, K; Ross, W.M.; Schnappauf, H.-P.; Steinbach, I.

1978-01-01

Colony forming units in agar (CFU-C) were assayed in both bone marrow and peripheral blood of dogs during haemopoietic recovery after lethal total-body irradiation (1200 R) and allogeneic transfusion of blood mononuclear cells (MNC) from histocompatible donors. MNC had been collected from the peripheral blood by continuous-flow centrifugation leucapheris and cryopreserved at -196 deg C until transfusion. Two groups of dogs were studied. Group 1 dogs (n = 12) were given between 0.39 and 2.76 x 10 9 MNC per kg body wt. Group 2 dogs (n = 14) were transfused with a similar number of MNC, ranging from 0.51 to 1.87 x 10 9 per kg body wt., but in addition underwent immuno-suppressive therapy with methotrexate. In group 1 dogs, there was a rather good correlation between the number of CFU-C in the regenerating bone marrow and the recovery of the peripheral blood granulocyte values. The regeneration of the CPU-C population in the bone marrow of methotrexate-treated dogs showed a somewhat more heterogeneous picture than in dogs of group 1 and in dogs that, in a previous study, were transfused with autologous MNC. The minimum time interval required for the reconstitution of peripheral blood CFU-C to normal levels was 2-4 weeks but usually took from 4-14 weeks. (author)

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

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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.

Screening and fractionation of plant extracts with antiproliferative activity on human peripheral blood mononuclear cells

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Souza-Fagundes Elaine M

2002-01-01

Full Text Available Three hundred and thirteen extracts from 136 Brazilian plant species belonging to 36 families were tested for their suppressive activity on phytohemaglutinin (PHA stimulated proliferation of human peripheral blood mononuclear cells (PBMC. The proliferation was evaluated by the amount of [³H]-thymidine incorporated by the cells. Twenty extracts inhibited or strongly reduced the proliferation in a dose-dependent manner at doses between 10 and 100 µg/ml. Three of these extracts appeared to be non-toxic to lymphocytes, according to the trypan blue permeability assay and visual inspection using optical microscopy. Bioassay-guided fractionation of Alomia myriadenia extract showed that myriadenolide, a labdane diterpene known to occur in this species, could account for the observed activity of the crude extract. Using a similar protocol, an active fraction of the extract from Gaylussacia brasiliensis was obtained. Analysis of the ¹H and13C NMR spectra of this fraction indicates the presence of an acetylated triterpene whose characterization is underway. The extract of Himatanthus obovatus is currently under investigation.

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation.

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Rim, Yeri Alice; Nam, Yoojun; Ju, Ji Hyeon

2016-12-21

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent state. Now, reprogramming is done with various types of adult somatic cells: keratinocytes, urine cells, fibroblasts, etc. Early experiments were usually done with dermal fibroblasts. However, this required an invasive surgical procedure to obtain fibroblasts from the patients. Therefore, suspension cells, such as blood and urine cells, were considered ideal for reprogramming because of the convenience of obtaining the primary cells. Here, we report an efficient protocol for iPSC generation from peripheral blood mononuclear cells (PBMCs). By plating the transduced PBMCs serially to a new, matrix-coated plate using centrifugation, this protocol can easily provide iPSC colonies. This method is also applicable to umbilical cord blood mononuclear cells (CBMCs). This study presents a simple and efficient protocol for the reprogramming of PBMCs and CBMCs.

Menstrual blood closely resembles the uterine immune micro-environment and is clearly distinct from peripheral blood

NARCIS (Netherlands)

Molen, R.G. van der; Schutten, J.H.; Cranenbroek, B. van; Meer, M. ter; Donckers, J.; Scholten, R.R.; Heijden, O.W.H. van der; Spaanderman, M.E.A.; Joosten, I.

2014-01-01

STUDY QUESTION: Is menstrual blood a suitable source of endometrial derived lymphocytes? SUMMARY ANSWER: Mononuclear cells isolated from menstrual samples (menstrual blood mononuclear cells (MMC)) are clearly distinct from peripheral blood mononuclear cells (PBMC) and show a strong resemblance with

A clinical study on insulin receptors of mononuclear cells in diabetes

International Nuclear Information System (INIS)

Dalimunthe, D.

1980-01-01

125 I-insulin binding activity to mononuclear cells was studied in 75 noninsulin-dependent diabetic subjects and 31 normal subjects and the following results were obtained. 1. 125 I-insulin binding is directly proportional to the mononuclear cell concentrations. There is a linear increase of specific 125 I-insulin binding. 2. The binding of 125 I-insulin to mononuclear cells is displaced by the increasing concentration of native insulin. 3. The 125 I-insulin degradation in the incubation medium after incubation of mononuclear cells for 24 hours at 4 0 C was almost 5% in this study. 4. The insulin binding activity in diabetic subjects was lower than that in normal subjects (P < 0.001) without any significant difference in affinity constant. 5. The relationship of binding activity to age of diabetics (r = 0.06, N.S), relative body weitht (r = 0.06, N.S) and duration of diabetes from onset was not significant. 6. In untreated noninsulin-dependent diabetics the insulin binding activity was inversely correlated to fasting blood glucose level (r = 0.78, P < 0.001) and slightly inversely correlated to serum insulin level (r = 0.47, P < 0.01). A slight inverse correlation was also observed in serum triglyceride level (r = 0.53, P < 0.01) and in total cholesterol level (r = 0.29, P < 0.05). 7. No significant difference between the binding activity was observed by grade of diabetic retinopathy. 8. After treatment with diet and/or sulfonylurea, the diabetics exhibited a significant increase in insulin binding activity (P < 0.005) but no significant difference in plasma insulin level, body weight and plasma lipid levels was observed. (author)

Centella asiatica modulates cancer cachexia associated inflammatory cytokines and cell death in leukaemic THP-1 cells and peripheral blood mononuclear cells (PBMC's).

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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 (C LE ) in leukaemic THP-1 cells and normal peripheral blood mononuclear cells (PBMC's). Cytotoxcity of C LE was determined at 24 and 72 h (h). Oxidant scavenging activity of C LE 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. C LE decreased PBMC viability between 33.25-74.55% (24 h: 0.2-0.8 mg/ml C LE and 72 h: 0.4-0.8 mg/ml C LE ) and THP-1 viability by 28.404% (72 h: 0.8 mg/ml C LE ) (p cachexia.

Immunocapture and microplate-based activity and quantity measurement of pyruvate dehydrogenase in human peripheral blood mononuclear cells.

Science.gov (United States)

Liu, Xiaowen; Pervez, Hira; Andersen, Lars W; Uber, Amy; Montissol, Sophia; Patel, Parth; Donnino, Michael W

2015-01-01

Pyruvate dehydrogenase (PDH) activity is altered in many human disorders. Current methods require tissue samples and yield inconsistent results. We describe a modified method for measuring PDH activity from isolated human peripheral blood mononuclear cells (PBMCs). RESULTS/METHODOLOGY: We found that PDH activity and quantity can be successfully measured in human PBMCs. Freeze-thaw cycles cannot efficiently disrupt the mitochondrial membrane. Processing time of up to 20 h does not affect PDH activity with proteinase inhibitor addition and a detergent concentration of 3.3% showed maximum yield. Sample protein concentration is correlated to PDH activity and quantity in human PBMCs from healthy subjects. Measuring PDH activity from PBMCs is a novel, easy and less invasive way to further understand the role of PDH in human disease.

Counter-flow elutriation of clinical peripheral blood mononuclear cell concentrates for the production of dendritic and T cell therapies.

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Stroncek, David F; Fellowes, Vicki; Pham, Chauha; Khuu, Hanh; Fowler, Daniel H; Wood, Lauren V; Sabatino, Marianna

2014-09-17

Peripheral blood mononuclear cells (PBMC) concentrates collected by apheresis are frequently used as starting material for cellular therapies, but the cell of interest must often be isolated prior to initiating manufacturing. The results of enriching 59 clinical PBMC concentrates for monocytes or lymphocytes from patients with solid tumors or multiple myeloma using a commercial closed system semi-automated counter-flow elutriation instrument (Elutra, Terumo BCT) were evaluated for quality and consistency. Elutriated monocytes (n = 35) were used to manufacture autologous dendritic cells and elutriated lymphocytes (n = 24) were used manufacture autologous T cell therapies. Elutriated monocytes with >10% neutrophils were subjected to density gradient sedimentation to reduce neutrophil contamination and elutriated lymphocytes to RBC lysis. Elutriation separated the PBMC concentrates into 5 fractions. Almost all of the lymphocytes, platelets and red cells were found in fractions 1 and 2; in contrast, most of the monocytes, 88.6 ± 43.0%, and neutrophils, 74.8 ± 64.3%, were in fraction 5. In addition, elutriation of 6 PBMCs resulted in relatively large quantities of monocytes in fractions 1 or 2. These 6 PBMCs contained greater quantities of monocytes than the other 53 PBMCs. Among fraction 5 isolates 38 of 59 contained >10% neutrophils. High neutrophil content of fraction 5 was associated with greater quantities of neutrophils in the PBMC concentrate. Following density gradient separation the neutrophil counts fell to 3.6 ± 3.4% (all products contained <10% neutrophils). Following red cell lysis of the elutriated lymphocyte fraction the lymphocyte recovery was 86.7 ± 24.0% and 34.3 ± 37.4% of red blood cells remained. Elutriation was consistent and effective for isolating monocytes and lymphocytes from PBMC concentrates for manufacturing clinical cell therapies, but further processing is often required.

A Modified Ficoll-Paque Gradient Method for Isolating Mononuclear Cells from the Peripheral and Umbilical Cord Blood of Humans for Biobanks and Clinical Laboratories.

Science.gov (United States)

Jia, Yanjuan; Xu, Hui; Li, Yonghong; Wei, Chaojun; Guo, Rui; Wang, Fang; Wu, Yu; Liu, Jing; Jia, Jing; Yan, Junwen; Qi, Xiaoming; Li, Yuanting; Gao, Xiaoling

2018-04-01

Although the Ficoll-Paque method is classically used to isolate peripheral blood mononuclear cells (PBMCs), modifications in this method are required for a more rapid and economic output for biobanks and clinical laboratories, particularly in developing countries. In this study, we addressed this issue by modifying the Ficoll-Paque method for the isolation of PBMCs or mononuclear cells from the peripheral and the umbilical cord blood of healthy and diseased (infected, anemic, and chronic obstructive pulmonary disease) adult individuals. In the modified method, we initiated the cell isolation process from the buffy coat layer, which appears in the interface between the plasma and sediments after centrifugation, instead of using the whole blood as described in the classic method. Although the PBMC yield by the modified method was about 12% less than in the classic method, the number of PBMCs isolated by the modified method was more than one million, which is enough for different research/diagnostic purposes, such as multi-omics detection. Assessment of cell viability and purity by hematology analyzer and trypan blue showed no significant difference between the viability and purity of the PBMCs isolated by these two methods in almost all groups, except samples from the infected and cord blood groups, where lower PBMC purity with higher granulocyte contamination were observed. In addition, at delayed processing time points, all parameters for the two methods were decreased in a time-dependent manner, especially at 8, 12, or 24 hours after the sample collection. In summary, the performance of PBMC isolation by the classic and modified methods mainly relies on the PBMC ratio in original samples. The modified method could be preferred for PBMC isolation because of its time and cost savings, especially for the biobanks and clinical laboratories in developing countries.

Transcriptomics identifies differences between ultrapure non-dioxin-like polychlorinated biphenyls (PCBs) and dioxin-like PCB126 in cultured peripheral blood mononuclear cells

International Nuclear Information System (INIS)

Wens, B.; De Boever, P.; Maes, M.; Hollanders, K.; Schoeters, G.

2011-01-01

Polychlorinated biphenyls (PCBs) remain ubiquitously present in human lipids despite the ban on their production and use. Their presence can be chemically monitored in peripheral blood samples of the general population. We tested whether in vitro exposure to different PCB congeners induced different gene expression profiles in peripheral blood cells. We have isolated peripheral blood mononuclear cells (PBMC) from whole blood of 8 healthy individuals and exposed these cells in vitro to individual non-dioxin-like (NDL)-PCB congeners (PCB52, 138 or 180; 10 μM) or dioxin-like (DL)-PCB congener PCB126 (1 μM) during 18 h. Differential gene expression response was measured using Agilent whole-human genome microarrays. Two-way ANOVA analysis of the data showed that both gender and PCB exposure are important factors influencing gene expression responses in blood cells. Hierarchical cluster analysis of genes influenced by PCB exposure, revealed that DL-PCB126 induced a different gene expression response compared to the NDL-PCBs. Biological interpretation of the results revealed that exposure to PCB126 induced the AhR signaling pathway, whereas the induction of nuclear receptor pathways by the NDL-PCBs was limited in blood cells. Nevertheless, molecular responses of blood cells to individual PCB congeners revealed significantly expressed genes that play a role in biological functions and processes known to be affected by PCB exposure in vivo. Observed gene expression changes in this in vitro model were found to be related to hepatotoxicity, immune and inflammatory response and disturbance of lipid and cholesterol homeostasis.

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

Protection of the functional integrity of mononuclear cells stored in liquid N2 requires careful control of the freezing procedure. Consequently, optimal quality of cryopreserved cells is usually assured by freezing according to a specified time-temperature gradient generated by computer......-controlled freezing devices. While such equipment offers large capacity and secures maximum survival and functional integrity of the lymphocytes upon thawing, it is quite costly and strictly stationary. We have previously developed and tested an alternative, manual device for controlled-gradient lymphocyte freezing...

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

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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.

Detection of canine distemper virus nucleocapsid protein gene in canine peripheral blood mononuclear cells by RT-PCR.

Science.gov (United States)

Shin, Y; Mori, T; Okita, M; Gemma, T; Kai, C; Mikami, T

1995-06-01

For a rapid diagnosis of canine distemper virus (CDV) infection, the reverse transcription-PCR (RT-PCR) was carried out to detect CDV nucleoprotein (NP) gene from canine peripheral blood mononuclear cells (PBMCs). Two sets of primers were targeted to two regions of NP gene of CDV Onderstepoort strain. The NP gene fragments were well amplified by the RT-PCR in each of the RNA extracts from Vero cells infected with 6 laboratory strains of CDV including Onderstepoort strain, and from PBMCs of a dog experimentally infected with CDV. The amplified NP gene was detected in 17 of 32 samples from dogs which were clinically suspected for CDV infection at veterinary hospitals. No RT-PCR product was found in 52 samples from healthy dogs including 40 specific pathogen free beagles vaccinated with an attenuated live virus-vaccine for CDV and 12 stray dogs. The RT-PCR provides a fast, sensitive, and supplementary method for the diagnosis of CDV infection in dogs.

Automated processing of human bone marrow can result in a population of mononuclear cells capable of achieving engraftment following transplantation.

Science.gov (United States)

Areman, E M; Cullis, H; Spitzer, T; Sacher, R A

1991-10-01

A concentrate of mononuclear bone marrow cells is often desired for ex vivo treatment with pharmacologic agents, monoclonal antibodies, cytokines, and other agents prior to transplantation. A method has been developed for automated separation of mononuclear cells from large volumes of harvested bone marrow. A programmable instrument originally designed for clinical ex vivo cell separation and the plasma-pheresis of patients and blood donors was adapted to permit rapid preparation, in a closed sterile system, of a bone marrow product enriched with mononuclear cells. A mean (+/- SEM) of 53 +/- 30 percent of the original mononuclear cells was recovered in a volume of 125 +/- 42 mL containing 82 +/- 12 percent mononuclear cells. This technique removed 95 +/- 9 percent of the red cells in the original marrow. No density gradient materials or sedimenting agents were employed in this process. Of 36 marrows processed by this technique, 19 autologous (6 of which were purged with 4-hydroperoxycyclophosphamide) and 7 allogeneic marrows have been transplanted, with all evaluable patients achieving a neutrophil count of 0.5 x 10(9) per L in a mean (+/- SEM) of 21 +/- 6 days.

Lol p I-specific IgE and IgG synthesis by peripheral blood mononuclear cells from atopic subjects in SCID mice.

Science.gov (United States)

Gagnon, R; Boutin, Y; Hébert, J

1995-06-01

The development of an animal model representative of the in vivo situation of human atopic diseases is always of interest for a better understanding of IgE production and regulation. Along these lines, mice with severe combined immunodeficiency (SCID mice) engrafted with lymphocytes from atopic subjects might be a suitable model for such studies. This study aims to analyze the production of Lol p I-specific IgE and IgG antibodies in SCID mice after transplantation of human peripheral blood mononuclear cells from atopic patients sensitive to grass pollens and from nonatopic donors. Peripheral blood mononuclear cells were transplanted into SCID mice, which were then challenged with Lol p I, and antibody responses (IgG and IgE) were analyzed over a 6-week period. Total IgG antibody was measured in each mouse serum after transplantation. Also, most mice (regardless of whether donors were atopic) that were challenged with Lol p I produced specific IgG antibody. Total IgE antibody production was observed only in mice grafted with cells from atopic patients. Lol p I-specific IgE antibodies were also produced after immunization with Lol p I. Although IgG antibody/response tended to plateau, the IgE antibody response increased until it peaked and declined thereafter. Interferon-gamma was detected in sera from mice producing IgE antibody, which supports a possible role of interferon-gamma in the decrease of IgE response. This study suggests that the SCID mouse model could represent an interesting approach to studying specific, total IgG and IgE antibody production, and ultimately their regulation.

Immunotoxicity assessment of rice-derived recombinant human serum albumin using human peripheral blood mononuclear cells.

Directory of Open Access Journals (Sweden)

Kai Fu

Full Text Available Human serum albumin (HSA is extensively used in clinics to treat a variety of diseases, such as hypoproteinemia, hemorrhagic shock, serious burn injuries, cirrhotic ascites and fetal erythroblastosis. To address supply shortages and high safety risks from limited human donors, we recently developed recombinant technology to produce HSA from rice endosperm. To assess the risk potential of HSA derived from Oryza sativa (OsrHSA before a First-in-human (FIH trial, we compared OsrHSA and plasma-derived HSA (pHSA, evaluating the potential for an immune reaction and toxicity using human peripheral blood mononuclear cells (PBMCs. The results indicated that neither OsrHSA nor pHSA stimulated T cell proliferation at 1x and 5x dosages. We also found no significant differences in the profiles of the CD4(+ and CD8(+ T cell subsets between OsrHSA- and pHSA-treated cells. Furthermore, the results showed that there were no significant differences between OsrHSA and pHSA in the production of cytokines such as interferon-gamma (IFN-γ, tumor necrosis factor-alpha (TNF-α, interleukin (IL-10 and IL-4. Our results demonstrated that OsrHSA has equivalent immunotoxicity to pHSA when using the PBMC model. Moreover, this ex vivo system could provide an alternative approach to predict potential risks in novel biopharmaceutical development.

Effects of Secondary Metabolites of Permafrost Bacillus sp. on Cytokine Synthesis by Human Peripheral Blood Mononuclear Cells.

Science.gov (United States)

Kalenova, L F; Kolyvanova, S S; Bazhin, A S; Besedin, I M; Mel'nikov, V P

2017-06-01

We studied the effects of secondary metabolites of Bacillus sp. isolated from late Neogene permafrost on secretion of proinflammatory (TNF-α, IL-1β, IL-8, IL-2, and IFNγ) and antiinflammatory (IL-4 and IL-10) cytokines by human peripheral blood mononuclear cells. It was found that metabolites of Bacillus sp. produced more potent effect on cytokine secretion than mitogen phytohemagglutinin and metabolites of Bacillus cereus, medicinal strain IP5832. Activity of metabolites depended on the temperature of bacteria incubation. "Cold" metabolites of Bacillus sp. (isolated at -5°C) primarily induced Th1-mediated secretion of IFNγ, while "warm" metabolites (obtained at 37°C) induced Th2-mediated secretion of IL-4. The results suggest that Bacillus sp. metabolites are promising material for the development of immunomodulating drugs.

Immunocapture and microplate-based activity and quantity measurement of pyruvate dehydrogenase in human peripheral blood mononuclear cells

Science.gov (United States)

Liu, Xiaowen; Pervez, Hira; Andersen, Lars W; Uber, Amy; Montissol, Sophia; Patel, Parth; Donnino, Michael W

2015-01-01

Background Pyruvate dehydrogenase (PDH) activity is altered in many human disorders. Current methods require tissue samples and yield inconsistent results. We describe a modified method for measuring PDH activity from isolated human peripheral blood mononuclear cells (PBMCs). Results/Methodology We found that PDH activity and quantity can be successfully measured in human PBMCs. Freeze-thaw cycles cannot efficiently disrupt the mitochondrial membrane. Processing time of up to 20 h does not affect PDH activity with proteinase inhibitor addition and a detergent concentration of 3.3% showed maximum yield. Sample protein concentration is correlated to PDH activity and quantity in human PBMCs from healthy subjects. Conclusion Measuring PDH activity from PBMCs is a novel, easy and less invasive way to further understand the role of PDH in human disease. PMID:25826140

In vitro radiation protection of peripheral blood mononuclear cells by Tetragona Clavipes Propolis

Energy Technology Data Exchange (ETDEWEB)

Nogueira, Naira de Souza Gomes [Universidade Federal do Amazonas (ICET/UFAM), Manaus (Brazil). Instituto de Ciências Exatas e Tecnologia; Campos, Tarcísio Passos Ribeiro; Montaño Valencia, Carlos Julio; Falcão, Patrícia Lima [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte (Brazil). Departamento de Técnicas Nucleares

2017-07-01

Background: Research in breast cancer has shown notable relevance in recent years, bringing serious concern on the public health policies due to its high incidence and mortality, especially in Brazil. Limitations due to the toxic effects in the normal tissues and the depletion in the immune system have often been present in the breast cancer radiotherapy and chemotherapy. It is a promise a drug development of vegetal origin that induce immune system protection in patients submitted to radiotherapy and chemotherapy, if such compounds inhibit the depletion of the circulating cell number. Objectives: To evaluate the in vitro effect of the extract of Tetragona Clavipes Propolis (TCP) in the viability of the human Peripheral Blood Mononuclear Cells (PBMC). Methods: The non-irradiated control, the irradiated control and the radiated and exposed to TCP (n=3) (GIE) groups were established. The cells were irradiated with predetermined doses of 2 and 5Gy, supplemented with TCP-extracts in aqueous solution at concentrations of 0.5, 1, 5 and 10 %. Post-irradiated sample was collected and viability assay was carried out at the time of 24, 48 and 72h. Results: A significant decrease of PBMC viability was observed after 2 and 5Gy. However, GIE showed an increase of cell viability, especially in the 5 and 10% concentrations of the extract incubated in culture, even after 5Gy. Conclusions: The findings showed that the aqueous extract of TCP is an exogenous protective agent to irradiated PBMC. This study opens a relevant perspective on the role of Propolis use as an adjuvant agent in protecting the immune system of patients undergoing breast radiotherapy. (author)

In vitro radiation protection of peripheral blood mononuclear cells by Tetragona Clavipes Propolis

International Nuclear Information System (INIS)

Nogueira, Naira de Souza Gomes; Campos, Tarcísio Passos Ribeiro; Montaño Valencia, Carlos Julio; Falcão, Patrícia Lima

2017-01-01

Background: Research in breast cancer has shown notable relevance in recent years, bringing serious concern on the public health policies due to its high incidence and mortality, especially in Brazil. Limitations due to the toxic effects in the normal tissues and the depletion in the immune system have often been present in the breast cancer radiotherapy and chemotherapy. It is a promise a drug development of vegetal origin that induce immune system protection in patients submitted to radiotherapy and chemotherapy, if such compounds inhibit the depletion of the circulating cell number. Objectives: To evaluate the in vitro effect of the extract of Tetragona Clavipes Propolis (TCP) in the viability of the human Peripheral Blood Mononuclear Cells (PBMC). Methods: The non-irradiated control, the irradiated control and the radiated and exposed to TCP (n=3) (GIE) groups were established. The cells were irradiated with predetermined doses of 2 and 5Gy, supplemented with TCP-extracts in aqueous solution at concentrations of 0.5, 1, 5 and 10 %. Post-irradiated sample was collected and viability assay was carried out at the time of 24, 48 and 72h. Results: A significant decrease of PBMC viability was observed after 2 and 5Gy. However, GIE showed an increase of cell viability, especially in the 5 and 10% concentrations of the extract incubated in culture, even after 5Gy. Conclusions: The findings showed that the aqueous extract of TCP is an exogenous protective agent to irradiated PBMC. This study opens a relevant perspective on the role of Propolis use as an adjuvant agent in protecting the immune system of patients undergoing breast radiotherapy. (author)

Interaction of rotavirus with human peripheral blood mononuclear cells: plasmacytoid dendritic cells play a role in stimulating memory rotavirus specific T cells in vitro.

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Mesa, Martha C; Rodríguez, Luz-Stella; Franco, Manuel A; Angel, Juana

2007-09-15

We studied the interaction of RV with human peripheral blood mononuclear cells (PBMC) from adult volunteers. After exposure of PBMC to rhesus RV (RRV), T and B lymphocytes, NK cells, monocytes, and myeloid and plasmacytoid dendritic cells expressed RV non-structural proteins, at variable levels. Expression of these RV proteins was abolished if infection was done in the presence of anti-VP7 neutralizing antibodies or 10% autologous serum. Supernatants of RRV exposed PBMC contained TNF-alpha, IL-6, IFN-alpha, IFN-gamma, IL-2 and IL-10. Plasmacytoid DC were found to be the main source of IFN-alpha production, and in their absence the production of IFN-gamma and the frequency of RV specific T cells that secrete IFN-gamma diminished. Finally, we could not detect RV-antigen associated with the PBMC or expression of RV non-structural proteins in PBMC of acutely RV-infected children. Thus, although PBMC are susceptible to the initial steps of RV infection, most PBMC of children with RV-gastroenteritis are not infected.

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

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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.

Detection of melanoma cells suspended in mononuclear cells and blood plasma using photoacoustic generation

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Spradling, Emily M.; Viator, John A.

2009-02-01

Melanoma is the deadliest form of skin cancer. Although the initial malignant cells are removed, it is impossible to determine whether or not the cancer has metastasized until a secondary tumor forms that is large enough to detect with conventional imaging. Photoacoustic detection of circulating melanoma cells in the bloodstream has shown promise for early detection of metastasis that may aid in treatment of this aggressive cancer. When blood is irradiated with energy from an Nd:YAG laser at 532 nm, photoacoustic signals are created and melanoma cells can be differentiated from the surrounding cells based on waveforms produced by an oscilloscope. Before this can be used as a diagnostic technique, however, we needed to investigate several parameters. Specifically, the current technique involves the in vitro separation of blood through centrifugation to isolate and test only the white blood cell layer. Using this method, we have detected a single cultured melanoma cell among a suspension of white blood cells. However, the process could be made simpler if the plasma layer were used for detection instead of the white blood cell layer. This layer is easier to obtain after blood separation, the optical difference between plasma and melanoma cells is more pronounced in this layer than in the white blood cell layer, and the possibility that any stray red blood cells could distort the results is eliminated. Using the photoacoustic apparatus, we detected no melanoma cells within the plasma of whole blood samples spiked with cultured melanoma cells.

Clozapine inhibits Th1 cell differentiation and causes the suppression of IFN-γ production in peripheral blood mononuclear cells.

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Chen, Mao-Liang; Tsai, Tzung-Chieh; Wang, Lu-Kai; Lin, Yi-Yin; Tsai, Ya-Min; Lee, Ming-Cheng; Tsai, Fu-Ming

2012-08-01

Antipsychotic drugs (APDs) are widely used to alleviate a number of psychic disorders and may have immunomodulatory effects. However, the previous studies of cytokine and immune regulation in APDs are quite inconsistent. The aim of this study was to examine the in vitro effects of different ADPs on cytokine production by peripheral blood mononuclear cells (PBMCs). We examined the effects of risperidone, clozapine, and haloperidol on the production of phorbol myristate acetate and ionomycin-induced interferon-γ (IFN-γ)/interleukin (IL)-4 in PBMCs by using intracellular staining. Real-time quantitative PCR and Western blot were used to further examine the expression changes of some critical transcription factors related to T-cell differentiation in antipsychotic-treated PBMCs. Our results indicated that clozapine can suppress the stimulated production of IFN-γ by 30.62%, whereas haloperidol weakly enhances the expression of IFN-γ. Differences in IL-4 production or in the number of CD4+ T cells were not observed in cells treated with different APDs. Furthermore, clozapine and risperidone inhibited the T-bet mRNA and protein expression, which are critical to Th1 differentiation. Also, clozapine can enhance the expression of Signal Transducer and Activator of Transcription 6 and GATA3, which are critical for the differentiation of Th2 cells. The results suggested that clozapine and haloperidol may induce different immunomodulatory effects on the immune system.

Detection of tetanus toxoid-specific memory T cells in equine lymph nodes but not in peripheral blood.

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Frayne, J; Stokes, C R

1995-07-01

The use of tetanus toxoid as a recall antigen to investigate equine immune responses would be, in theory, a useful and cost-effective model in vitro. However, by using various regimens for culturing peripheral blood mononuclear cells from horses previously immunised with toxoid no proliferative response to the antigen was obtained in vitro, whereas lymph node mononuclear cells from the same animals proliferated significantly in response to it. The lack of response by the peripheral blood mononuclear cells was not due to the presence of a suppressive factor but to a lack of recognition of the antigen by the T cells of the peripheral blood.

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...... 18-93 years. DNA damage was analyzed as strand breaks by the comet assay and levels of formamidopyrimidine (FPG-) and human 8-oxoguanine DNA glycosylase 1 (hOGG1)-sensitive sites There was an association between age and levels of FPG-sensitive sites for women, but not for men. The same tendency......, cholesterol and glycosylated hemoglobin (HbA1c). In the group of men, there were significant positive associations between alcohol intake, HbA1c and FPG-sensitive sites in multivariate analysis. The levels of metabolic risk factors were positively associated with age, yet only few subjects fulfilled all...

Dynamic Changes in Fetal Microchimerism in Maternal Peripheral Blood Mononuclear Cells, CD4+ and CD8+ Cells in Normal Pregnancy

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Adams Waldorf, Kristina M.; Gammill, Hilary S.; Lucas, Joëlle; Aydelotte, Tessa M.; Leisenring, Wendy M.; Lambert, Nathalie C.; Nelson, J. Lee

2010-01-01

Objective Cell trafficking during pregnancy results in persistence of small populations of fetal cells in the mother, known as fetal microchimerism (FMc). Changes in cell-free fetal DNA during gestation have been well-described, however, less is known about dynamic changes in fetal immune cells in maternal blood. We investigated FMc in maternal peripheral blood mononuclear cells (PBMC) longitudinally across gestation. Study Design Thirty-five women with normal pregnancies were studied. FMc was identified in PBMC, CD4+ and CD8+ subsets employing quantitative PCR assays targeting fetal-specific genetic polymorphisms. FMc quantities were reported as fetal genome equivalents (gEq) per 1,000,000 gEq mother’s cells. Poisson regression modeled the rate of FMc detection. Main Outcome Measure FMc in PBMC Results The probability of detecting one fetal cell equivalent increased 6.2-fold each trimester [Incidence Rate Ratio (IRR) 95% CI: 1.73, 21.91; p=0.005]. Although FMC in PBMC was not detected for the majority of time points, 7 of 35 women had detectable FMc during pregnancy at one or more time points, with the majority of positive samples being from the third trimester. There was a suggestion of greater HLA-sharing in families where women had FMc in PBMC. FMc was detected in 9% of CD4+ (2/23) and 18% of CD8+ (3/25) subsets. Conclusions FMc in PBMC increased as gestation progressed and was found within CD4+ and CD8+ subsets in some women in the latter half of gestation. A number of factors could influence cellular FMc levels including subclinical fetal-maternal interface changes and events related to parturition. Whether FMc during pregnancy predicts persistent FMc and/or correlates with fetal-maternal HLA-relationships also merits further study. PMID:20569981

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

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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.

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

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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.

Optimizing the method for generation of integration-free induced pluripotent stem cells from human peripheral blood.

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Gu, Haihui; Huang, Xia; Xu, Jing; Song, Lili; Liu, Shuping; Zhang, Xiao-Bing; Yuan, Weiping; Li, Yanxin

2018-06-15

Generation of induced pluripotent stem cells (iPSCs) from human peripheral blood provides a convenient and low-invasive way to obtain patient-specific iPSCs. The episomal vector is one of the best approaches for reprogramming somatic cells to pluripotent status because of its simplicity and affordability. However, the efficiency of episomal vector reprogramming of adult peripheral blood cells is relatively low compared with cord blood and bone marrow cells. In the present study, integration-free human iPSCs derived from peripheral blood were established via episomal technology. We optimized mononuclear cell isolation and cultivation, episomal vector promoters, and a combination of transcriptional factors to improve reprogramming efficiency. Here, we improved the generation efficiency of integration-free iPSCs from human peripheral blood mononuclear cells by optimizing the method of isolating mononuclear cells from peripheral blood, by modifying the integration of culture medium, and by adjusting the duration of culture time and the combination of different episomal vectors. With this optimized protocol, a valuable asset for banking patient-specific iPSCs has been established.

Evaluation of two different protocols for peripheral blood stem cell collection with the Fresenius AS 104 blood cell separator.

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Menichella, G; Lai, M; Pierelli, L; Vittori, M; Serafini, R; Ciarli, M; Foddai, M L; Salerno, G; Sica, S; Scambia, G; Leone, G; Bizzi, B

1997-01-01

Reconstitution of hematopoiesis by means of peripheral blood stem cells is a valid alternative to autologous bone marrow transplantation. The aim of this investigation was to increase the efficiency of collection of circulating blood progenitor cells and to obtain a purer product for transplant. We carried out leukapheresis procedures with the Fresenius AS 104 blood cell separator, using two different protocols, the previously used PBSC-LYM and a new mononuclear cell collection program. Both programs were highly effective in collecting mononuclear cells (MNC) and CD34+ cells. Some differences were found, especially regarding MNC yield and efficiencies. There are remarkable differences in the efficiency of collection of CD34+ cells (62.38% with the new program as opposed to 31.69% with the older one). Linear regression analysis showed a negative correlation between blood volume processed and MNC efficiency only for the PBSC-LYM program. Differences were also observed in the degree of inverse correlation existing in both programs between patients' white blood cell precount and MNC collection efficiency. The inverse correlation was stronger for the PBSC-LYM program. Seven patients with solid tumors and hematologic malignancies received high dose chemotherapy and were subsequently transplanted with peripheral blood stem cells collected using the new protocol. All patients obtained a complete and stable engraftment with the reinfusion product collected with one or two leukapheresis procedures. High efficiencies and yields were observed in the new protocol for MNC and CD34+ cells. These were able to effect rapid and complete bone marrow recovery after myeloablative chemotherapy.

Fuel feeds function: Energy balance and bovine peripheral blood mononuclear cell activation.

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Schwarm, A; Viergutz, T; Kuhla, B; Hammon, H M; Schweigel-Röntgen, M

2013-01-01

A general phenomenon in peripartum mammals is the breakdown of (acquired) immunity. The incidence of parasite load, disease and inflammation often rise during the specific energetically demanding time of pregnancy and lactation. In this period, blood leukocytes display decreased DNA synthesis in response to mitogens in vitro. Leukocyte activation, the phase of the cell cycle preceding the DNA synthetic phase has hardly been investigated, but the few studies suggest that leukocyte activation may also be impaired by the limited energy/nutrient availability. Leukocyte activation is characterized by manifold processes, thus, we used the cellular oxygen consumption rate (OCR) as a measure of ATP turnover to support all these processes. We hypothesized that the activation of peripheral blood mononuclear cells (PBMC) - in terms of oxygen consumed over basal levels after in vitro stimulation - is altered by energy balance around parturition. We studied peripartum high-yielding dairy cows because they undergo substantial fluctuations in energy intake, energy output and body fat mass. We established a fluorescence-based test strategy allowing for long-term (≥24h) quantification of O(2)-consumption and studied the peripartum period from 5 weeks ante partum to 5 weeks postpartum. In addition, we determined cellular lactate production, DNA/RNA synthesis and cell size and zoo-technical parameters such as animal energy intake and milk yield were assessed, as well as selected plasma parameters, e.g. glucose concentration. The basal OCR of PBMC from pregnant, non-lactating cows (n=6, -5 weeks ante partum) was 1.19±0.15 nmol min(-1) (10(7)cells)(-1) and increased to maximum levels of 2.54±0.49 nmol min(-1) (10(7)cells)(-1) in phytohemagglutinin (PHA)-stimulated PBMC. The basal OCR did not change over the peripartum period. Whereas the activation indices, herein defined as the PHA-induced 24h-increase of OCR above baseline, amounted to 1.1±0.3, 4.2±0.3, 4.1±1.1, 2.1±0.3, and

Simulated night shift work induces circadian misalignment of the human peripheral blood mononuclear cell transcriptome.

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Kervezee, Laura; Cuesta, Marc; Cermakian, Nicolas; Boivin, Diane B

2018-05-22

Misalignment of the endogenous circadian timing system leads to disruption of physiological rhythms and may contribute to the development of the deleterious health effects associated with night shift work. However, the molecular underpinnings remain to be elucidated. Here, we investigated the effect of a 4-day simulated night shift work protocol on the circadian regulation of the human transcriptome. Repeated blood samples were collected over two 24-hour measurement periods from eight healthy subjects under highly controlled laboratory conditions before and 4 days after a 10-hour delay of their habitual sleep period. RNA was extracted from peripheral blood mononuclear cells to obtain transcriptomic data. Cosinor analysis revealed a marked reduction of significantly rhythmic transcripts in the night shift condition compared with baseline at group and individual levels. Subsequent analysis using a mixed-effects model selection approach indicated that this decrease is mainly due to dampened rhythms rather than to a complete loss of rhythmicity: 73% of transcripts rhythmically expressed at baseline remained rhythmic during the night shift condition with a similar phase relative to habitual bedtimes, but with lower amplitudes. Functional analysis revealed that key biological processes are affected by the night shift protocol, most notably the natural killer cell-mediated immune response and Jun/AP1 and STAT pathways. These results show that 4 days of simulated night shifts leads to a loss in temporal coordination between the human circadian transcriptome and the external environment and impacts biological processes related to the adverse health effects associated to night shift work.

Rhinovirus induction of fractalkine (CX3CL1 in airway and peripheral blood mononuclear cells in asthma.

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Nadine Upton

Full Text Available Rhinovirus infection is associated with the majority of asthma exacerbations. The role of fractalkine in anti-viral (type 1 and pathogenic (type 2 responses to rhinovirus infection in allergic asthma is unknown. To determine whether (1 fractalkine is produced in airway cells and in peripheral blood leucocytes, (2 rhinovirus infection increases production of fractalkine and (3 levels of fractalkine differ in asthmatic compared to non-asthmatic subjects. Fractalkine protein and mRNA levels were measured in bronchoalveolar lavage (BAL cells and peripheral blood mononuclear cells (PBMCs from non-asthmatic controls (n = 15 and mild allergic asthmatic (n = 15 subjects. Protein levels of fractalkine were also measured in macrophages polarised ex vivo to give M1 (type 1 and M2 (type 2 macrophages and in BAL fluid obtained from mild (n = 11 and moderate (n = 14 allergic asthmatic and non-asthmatic control (n = 10 subjects pre and post in vivo rhinovirus infection. BAL cells produced significantly greater levels of fractalkine than PBMCs. Rhinovirus infection increased production of fractalkine by BAL cells from non-asthmatic controls (P<0.01 and in M1-polarised macrophages (P<0.05, but not in BAL cells from mild asthmatics or in M2 polarised macrophages. Rhinovirus induced fractalkine in PBMCs from asthmatic (P<0.001 and healthy control subjects (P<0.05. Trends towards induction of fractalkine in moderate asthmatic subjects during in vivo rhinovirus infection failed to reach statistical significance. Fractalkine may be involved in both immunopathological and anti-viral immune responses to rhinovirus infection. Further investigation into how fractalkine is regulated across different cell types and into the effect of stimulation including rhinovirus infection is warranted to better understand the precise role of this unique dual adhesion factor and chemokine in immune cell recruitment.

Clinical symptoms in fibromyalgia are better associated to lipid peroxidation levels in blood mononuclear cells rather than in plasma.

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Cordero, Mario D; Alcocer-Gómez, Elísabet; Cano-García, Francisco J; De Miguel, Manuel; Carrión, Angel M; Navas, Plácido; Sánchez Alcázar, José A

2011-01-01

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. 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. We found increased LPO levels in BMCs and plasma from FM patients as compared to normal control (PBMI, 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). 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.

Increased apoptosis and peripheral blood mononuclear cell suppression of bone marrow mesenchymal stem cells in severe aplastic anemia.

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Chao, Yu-Hua; Lin, Chiao-Wen; Pan, Hui-Hsien; Yang, Shun-Fa; Weng, Te-Fu; Peng, Ching-Tien; Wu, Kang-Hsi

2018-06-05

Although immune-mediated pathogenesis is considered an important aspect of severe aplastic anemia (SAA), its underlying mechanisms remain unclear. Mesenchymal stem cells (MSCs) are essential to the formation of specialized microenvironments in the bone marrow (BM), and MSC insufficiency can trigger the development of SAA. To find MSC alterations in the SAA BM, we compared BM MSCs from five children with SAA and five controls. Peripheral blood mononuclear cells (PBMCs) were cocultured with MSCs to evaluate the supportive effects of MSCs on hematopoiesis. Cytometric bead array immunoassay was used to determine cytokine excretion by MSCs. The immune functions of MSCs and their conditioned medium (CM) were evaluated by PBMC proliferation assays. SAA MSCs were characterized by a high percentage of cells in the abnormal sub-G1 phase of the cell cycle, which suggests an increased rate of apoptosis in SAA MSCs. In comparison with control MSCs, PBMCs cocultured with SAA MSCs displayed significantly reduced PBMC proliferation (P = 0.009). Aberrant cytokine profiles were secreted by SAA MSCs, with increased concentrations of interleukin-6, interferon-γ, tumor necrosis factor-α, and interleukin-1β in the CM. PBMC proliferation assays demonstrated additional immunosuppressive effects of SAA MSCs (P = 0.016) and their CM (P = 0.013). Our data revealed increased apoptosis and PBMC suppression of SAA MSCs. The alterations of MSCs may contribute to the formation of functionally abnormal microenvironments in SAA BM. © 2018 Wiley Periodicals, Inc.

Killing effect of peripheral blood mononuclear cells irradiated by γ ray on human gastric cancer MKN-28 cell

International Nuclear Information System (INIS)

Wu Daocheng; Zhang Xianqing; Mu Shijie; Liu Zhongxiang; Xia Aijun; Huang Xiaofeng; An Qunxing

2007-01-01

Objective: To observe the killing effect of peripheral blood mononuclear cells (PBMCs) irradiated by γ ray on cultured human gastric cancer cell line MKN-28. Methods: The experiment were divided into MKN-28 tumor cell control group, PBMCs groups and MKN-28 cells with irradiated or non-irradiated PBMCs co-culture groups. Radidation dosage were from 0.5 to 3 Gy, acridine orange/ethidium bromide (AO/EB) staining were used to observe the kill effect of PBMCs on tumor cells in different period. Results: After culture for 144h, the dead cells of several dosage irradiated PBMCs are much more than those of non-irradiated PBMCs group. At 240 hours of culture, the alive PBMCs deareses in number in both irradiated and non-irradiared groups, but decreases in radiated groups are more obvious. After culture for 72 h in the co-cultured groups, the difference is not evident among all radiation dosage groups. After 96-240 h of co-culture, the killing effect of 0.5-2Gy irradiated PBMCs on tumor cells is very strong, especially in 1Gy group, but the killing effect of PBMCs irradiated by 2.5-3Gy on tumor cells were weaker than that of 0.5-2Gy irradiated groups. At 240 hours co-cultured groups irradiated by 2.5-3Gy, tumor cells still survive and proliferate. Conclusion: Gamma ray irradiation have killing effect to some PBMCs. The cytocidal effect of PBMCs irradiated by 0.5-2Gy on tumor cells were increased. Chemotaxis and cytocidal effect of tumor cells to postirradiated PBMCs were also found. The killing effect of PBMCs irradiated by 2.5 and 3 Gy on tumor cells were restrained. (authors)

Taurine Transporter Gene Expression in Mononuclear Blood Cells of Type 1 Diabetes Patients.

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Napoli, Zaleida; Seghieri, Giuseppe; Bianchi, Loria; Anichini, Roberto; De Bellis, Alessandra; Campesi, Ilaria; Carru, Ciriaco; Occhioni, Stefano; Zinellu, Angelo; Franconi, Flavia

2016-01-01

Taurine transporter gene expression (RNA-TauT) has a role in retinal cell function and is modulated in vitro and in vivo by hyperglycemia and/or oxidative stress. This study was aimed at testing whether RNA-TauT gene expression is modified in blood mononuclear peripheral cells (MPCs) of type 1 diabetic patients, is related to plasma markers of oxidative stress or endothelial dysfunction, or, finally, is related to presence of retinopathy. RNA-TauT was measured in MPCs by real-time PCR-analysis in 35 type 1 diabetic patients and in 33 age- and sex-matched controls, additionally measuring plasma and cell taurine and markers of oxidative stress and endothelial dysfunction. RNA-TauT, expressed as 2(-ΔΔCt), was significantly higher in MPCs of type 1 diabetic patients than in controls [median (interquartile range): 1.32(0.31) versus 1.00(0.15); P = 0.01]. In diabetic patients RNA-TauT was related to HbA1c (r = 0.42; P = 0.01) and inversely to plasma homocysteine (r = -0.39; P = 0.02) being additionally significantly higher in MPCs of patients without retinopathy [(n = 22); 1.36(0.34)] compared to those with retinopathy [(n = 13); 1.16(0.20)], independently from HbA1c or diabetes duration. RNA-TauT gene expression is significantly upregulated in MPCs of type 1 diabetes patients and is related to HbA1c levels and inversely to plasma homocysteine. Finally, in diabetes patients, RNA-TauT upregulation seems to be blunted in patients with retinopathy independently of their metabolic control or longer diabetes duration.

Enhancement in irradiated mononuclear cells in culture of mitogen-induced incorporation of [3H]thymidine by homologous conditioned medium

International Nuclear Information System (INIS)

Sandru, G.; Greiner, R.

1994-01-01

Incorporation of [ 3 H]thymidine in irradiated peripheral blood mononuclear cell cultures irradiated in vitro was stimulated significantly by either concanavalin A or phytohemagglutinin only in the presence of homologous conditioned medium. Production of this activity by mononuclear cells was enhanced by irradiation and/or pulsed exposure to puromycin but was abolished by actinomycin D. Addition of anti-interleukin 1 or anti-interleukin 2 monoclonal antibodies to the conditioned medium before assay did not influence the stimulatory action. A similar significant stimulation of mononuclear cell cultures irradiated with 6 Gy by concanavalin A was obtained when purified preparations of homologous conditioned medium were used in the assay. Purification was done by ultrafiltration and concentration, heparin agarose chromatography, ammonium sulfate precipitation, concanavalin A agarose chromatography, DEAE-ion exchange chromatography and HPLC gel filtration chromatography. With SDS-PAGE and silver staining, the active HPLC fraction gave one band of 50 kDa, suggesting that this protein is responsible for the co-stimulatory effect of homologous conditioned medium for both mitogen-induced irradiated and nonirradiated mononuclear cell cultures. 42 refs., 9 figs., 3 tabs

In vitro response of the human breast cancer cell line MDAMB-231 and human peripheral blood mononuclear cells exposed to 60Co at single fraction

International Nuclear Information System (INIS)

Andrade, Lidia Maria; Campos, Tarcisio Passos Ribeiro de; Leite, M.F.; Goes, A.M.

2005-01-01

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 60 Co irradiation at a single fraction of 10 Gy, 25 Gy and 50 Gy doses at 136,4 cGy.min -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 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)

Cytokine secretion from human peripheral blood mononuclear cells cultured in vitro with metal particles.

Science.gov (United States)

Cachinho, Sandra C P; Pu, Fanrong; Hunt, John A

2013-04-01

The failure of implanted medical devices can be associated with changes in the production of cytokines by cells of the immune system. Cytokines released by peripheral blood mononuclear cells upon contact with metal particles were quantified to understand their role in implantation intergration and their importance as messengers in the recruitment of T-lymphocytes at the implantation site. Opsonization was utilised to understand the influence of serum proteins on particle-induced cytokine production and release. Different metal compositions were used in the particulate format, Titanium (Ti), Titanium alloy (Ti6Al4V), and Stainless Steel 316L (SS), and were cultured in vitro with a mixed population of monocytes/macrophages and lymphocytes. The cells were also exposed to an exogenous stimulant mixture of phytohemagglutinin-P and interferon-gamma (IFN-γ) and opsonized particles with human serum. Interleukins, IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IFN-γ, and tumor necrosis factor-alpha (TNF-α) were investigated using enzyme-linked immunosorbent assay as they are an indicator of the inflammation evoked by particulate metals. It has been experimentally evidenced that metal particles induced higher amounts of IL-6 and IL-1 but very low amounts of TNF-α. T-lymphocyte activation was evaluated by the quantification of IL-2 and IFN-γ levels. The results showed that nonopsonized and opsonized metal particles did not induce the release of increased levels of IL-2 and IFN-γ. Copyright © 2013 Wiley Periodicals, Inc.

Smoking-related microRNAs and mRNAs in human peripheral blood mononuclear cells

International Nuclear Information System (INIS)

Su, Ming-Wei; Yu, Sung-Liang; Lin, Wen-Chang; Tsai, Ching-Hui; Chen, Po-Hua; Lee, Yungling Leo

2016-01-01

Teenager smoking is of great importance in public health. Functional roles of microRNAs have been documented in smoke-induced gene expression changes, but comprehensive mechanisms of microRNA-mRNA regulation and benefits remained poorly understood. We conducted the Teenager Smoking Reduction Trial (TSRT) to investigate the causal association between active smoking reduction and whole-genome microRNA and mRNA expression changes in human peripheral blood mononuclear cells (PBMC). A total of 12 teenagers with a substantial reduction in smoke quantity and a decrease in urine cotinine/creatinine ratio were enrolled in genomic analyses. In Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA), differentially expressed genes altered by smoke reduction were mainly associated with glucocorticoid receptor signaling pathway. The integrative analysis of microRNA and mRNA found eleven differentially expressed microRNAs negatively correlated with predicted target genes. CD83 molecule regulated by miR-4498 in human PBMC, was critical for the canonical pathway of communication between innate and adaptive immune cells. Our data demonstrated that microRNAs could regulate immune responses in human PBMC after habitual smokers quit smoking and support the potential translational value of microRNAs in regulating disease-relevant gene expression caused by tobacco smoke. - Highlights: • We conducted a smoke reduction trial program and investigated the causal relationship between smoke and gene regulation. • MicroRNA and mRNA expression changes were examined in human PBMC. • MicroRNAs are important in regulating disease-causal genes after tobacco smoke reduction.

Smoking-related microRNAs and mRNAs in human peripheral blood mononuclear cells

Energy Technology Data Exchange (ETDEWEB)

Su, Ming-Wei [Institute of Epidemiology and Preventive Medicine, College of Public Health, National Taiwan University, Taipei, Taiwan (China); Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan (China); Yu, Sung-Liang [Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan (China); Lin, Wen-Chang [Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan (China); Tsai, Ching-Hui [Institute of Epidemiology and Preventive Medicine, College of Public Health, National Taiwan University, Taipei, Taiwan (China); Chen, Po-Hua [School of Medicine, National Taiwan University, Taipei, Taiwan (China); Lee, Yungling Leo, E-mail: leolee@ntu.edu.tw [Institute of Epidemiology and Preventive Medicine, College of Public Health, National Taiwan University, Taipei, Taiwan (China); Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan (China)

2016-08-15

Teenager smoking is of great importance in public health. Functional roles of microRNAs have been documented in smoke-induced gene expression changes, but comprehensive mechanisms of microRNA-mRNA regulation and benefits remained poorly understood. We conducted the Teenager Smoking Reduction Trial (TSRT) to investigate the causal association between active smoking reduction and whole-genome microRNA and mRNA expression changes in human peripheral blood mononuclear cells (PBMC). A total of 12 teenagers with a substantial reduction in smoke quantity and a decrease in urine cotinine/creatinine ratio were enrolled in genomic analyses. In Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA), differentially expressed genes altered by smoke reduction were mainly associated with glucocorticoid receptor signaling pathway. The integrative analysis of microRNA and mRNA found eleven differentially expressed microRNAs negatively correlated with predicted target genes. CD83 molecule regulated by miR-4498 in human PBMC, was critical for the canonical pathway of communication between innate and adaptive immune cells. Our data demonstrated that microRNAs could regulate immune responses in human PBMC after habitual smokers quit smoking and support the potential translational value of microRNAs in regulating disease-relevant gene expression caused by tobacco smoke. - Highlights: • We conducted a smoke reduction trial program and investigated the causal relationship between smoke and gene regulation. • MicroRNA and mRNA expression changes were examined in human PBMC. • MicroRNAs are important in regulating disease-causal genes after tobacco smoke reduction.

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

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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.

Basic Surface Properties of Mononuclear Cells from Didelphis marsupialis

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Nacife Valéria Pereira

1998-01-01

Full Text Available The electrostatic surface charge and surface tension of mononuclear cells/monocytes obtained from young and adult marsupials (Didelphis marsupialis were investigated by using cationized ferritin and colloidal iron hydroxyde, whole cell electrophoresis, and measurements of contact angles. Anionic sites were found distributed throughout the entire investigated cell surfaces. The results revealed that the anionic character of the cells is given by electrostatic charges corresponding to -18.8 mV (cells from young animals and -29.3 mV (cells from adult animals. The surface electrostatic charge decreased from 10 to 65.2% after treatment of the cells with each one of trypsin, neuraminidase and phospholipase C. The hydrophobic nature of the mononuclear cell surfaces studied by using the contact angle method revealed that both young and adult cells possess cell surfaces of high hidrofilicity since the angles formed with drops of saline water were 42.5°and 40.8°, respectively. Treatment of the cells with trypsin or neuraminidase rendered their surfaces more hydrophobic, suggesting that sialic acid-containing glycoproteins are responsible for most of the hydrophilicity observed in the mononuclear cell surfaces from D. marsupialis.

Reciprocal patterns of allergen-induced GATA-3 expression in peripheral blood mononuclear cells from atopics vs. non-atopics.

Science.gov (United States)

Macaubas, C; Lee, P T; Smallacombe, T B; Holt, B J; Wee, C; Sly, P D; Holt, P G

2002-01-01

T helper (Th)2 cytokines are considered to play a central role in the induction and expression of allergic disease. However, the relative importance of individual cytokines is unclear, and overall disease pathogenesis appears to involve the coordinate activities of a range of Th2 cytokines acting in sequence or in parallel. The present study examines an alternative approach to the study of cytokine gene function in atopy, focusing instead upon T cell transcription factors (TFs) which play a role in the regulation of multiple cytokine genes. To investigate the allergen-induced expression of the TF GATA-3 and c-Maf in peripheral blood mononuclear cells (PBMCs) and in cytokine-driven Th polarization. PBMC from house dust mite (HDM)-atopic and non-atopics were stimulated in vitro with allergen or anti-CD3/IL-2. TF expression was analysed by semiquantitative RT-PCR and major findings were validated by real-time PCR. Cell separations were performed to analyse the contribution of CD45RO+ cells. CD4+ cord blood cells were Th1 or Th2 polarized in vitro by exogenous cytokines and TF expression analysed by Northern blot and real-time PCR. Results We demonstrate for the first time that during differentiation of CD4+ CD45RA+ naïve human T cells towards Th2 commitment, and during allergen-specific reactivation of peripheral CD4+ CD45RO+ Th2 memory cells in established atopics, expression of the Th2-associated TF GATA-3 is rapidly up-regulated, whereas T cells from non-atopics display equally rapid GATA-3 down-regulation under identical conditions of allergen stimulation. These findings identify Th2-associated TFs as key determinants of the atopic phenotype, suggesting their unique potential as therapeutic targets for disease control.

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

It has recently been shown that plasticizers are present in indoor air dust, which may lead to human exposure via the inhalation route. Moreover, studies have indicated that plasticizers may possess adjuvant effects increasing the health damaging potential of allergens. The aim of this study...... 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...... viability was determined by incubating THP-1 cells with a 10-fold dilution series of monophthalates for 24 h. At different points in time cytokine expression (IL-1beta, IL-6, IL-12alpha (p35)) in THP-1 cells incubated with non-toxic concentrations of monophthalate (2-20 microg/ml)+/-LPS (1 microg/ml) were...

Long-chain PUFA in Granulocytes, Mononuclear Cells, and RBC in Patients With Cystic Fibrosis: Relation to Liver Disease

DEFF Research Database (Denmark)

Jorgensen, Marianne H.; Ott, Peter; Michaelsen, Kim F.

2012-01-01

-related liver disease were matched with 20 CF patients without. Blood samples were analysed for liver biochemistry and haematology. Granulocytes, mononuclear cells, and RBC were separated by density gradient centrifugation, and fatty acid composition was measured by gas chromatography. Hepatic ultrasound...

Anti-inflammatory compound resveratrol suppresses homocysteine formation in stimulated human peripheral blood mononuclear cells in vitro.

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Schroecksnadel, Katharina; Winkler, Christiana; Wirleitner, Barbara; Schennach, Harald; Weiss, Günter; Fuchs, Dietmar

2005-01-01

Inflammation, immune activation and oxidative stress play a major role in the pathogenesis of cardiovascular disorders. In addition to markers of inflammation, moderate hyperhomocysteinemia is an independent risk factor for cardiovascular disease, and there is a link between the activation of immunocompetent cells and the enhanced formation of homocysteine in vitro. Likewise, anti-inflammatory drugs and nutrients rich in antioxidant vitamins are able to reduce cardiovascular risk and to slow down the atherogenic process. Resveratrol, a phenolic antioxidant synthesized in grapes and vegetables and present in wine, has also been supposed to be beneficial for the prevention of cardiovascular events. Apart from its strong antioxidant properties, resveratrol has also been demonstrated to act as an anti-inflammatory agent. In this study the influence of resveratrol on the production of homocysteine by stimulated human peripheral blood mononuclear cells (PBMCs) was investigated. Results were compared to earlier described effects of the anti-inflammatory compounds aspirin and salicylic acid and of the lipid-lowering drug atorvastatin. Stimulation of PBMCs with the mitogens concanavalin A and phytohemagglutinin induced significantly higher homocysteine accumulation in supernatants compared with unstimulated cells. Treatment with 10-100 muM resveratrol suppressed homocysteine formation in a dose-dependent manner. Resveratrol did not influence the release of homocysteine from resting PBMCs. The data suggest that resveratrol may prevent homocysteine accumulation in the blood by suppressing immune activation cascades and the proliferation of mitogen-driven T-cells. The effect of resveratrol to down-regulate the release of homo-cysteine was comparable to the decline of neopterin concentrations in the same experiments. The suppressive effect of resveratrol was very similar to results obtained earlier with aspirin, salicylic acid and atorvastatin; however, it appeared that doses

The Effect of Long-Term Exercise on the Production of Osteoclastogenic and Antiosteoclastogenic Cytokines by Peripheral Blood Mononuclear Cells and on Serum Markers of Bone Metabolism

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J. Kelly Smith

2016-01-01

Full Text Available Although it is recognized that the mechanical stresses associated with physical activity augment bone mineral density and improve bone quality, our understanding of how exercise modulates bone homeostasis at the molecular level is lacking. In a before and after trial involving 43 healthy adults, we measured the effect of six months of supervised exercise training on the spontaneous and phytohemagglutinin-induced production of osteoclastogenic cytokines (interleukin-1α, tumor necrosis factor-α, antiosteoclastogenic cytokines (transforming growth factor-β1 and interleukins 4 and 10, pleiotropic cytokines with variable effects on osteoclastogenesis (interferon-γ, interleukin-6, and T cell growth and differentiation factors (interleukins 2 and 12 by peripheral blood mononuclear cells. We also measured lymphocyte phenotypes and serum markers of bone formation (osteocalcin, bone resorption (C-terminal telopeptides of Type I collagen, and bone homeostasis (25 (OH vitamin D, estradiol, testosterone, parathyroid hormone, and insulin-like growth factor 1. A combination of aerobic, resistance, and flexibility exercises done on average of 2.5 hours a week attenuated the production of osteoclastogenic cytokines and enhanced the production of antiosteoclastogenic cytokines. These changes were accompanied by a 16% reduction in collagen degradation products and a 9.8% increase in osteocalcin levels. We conclude that long-term moderate intensity exercise exerts a favorable effect on bone resorption by changing the balance between blood mononuclear cells producing osteoclastogenic cytokines and those producing antiosteoclastogenic cytokines. This trial is registered with Clinical Trials.gov Identifier: NCT02765945.

Transforming growth factor beta-1 and interleukin-17 gene transcription in peripheral blood mononuclear cells and the human response to infection.

LENUS (Irish Health Repository)

White, Mary

2012-02-01

INTRODUCTION: The occurrence of severe sepsis may be associated with deficient pro-inflammatory cytokine production. Transforming growth factor beta-1 (TGFbeta-1) predominantly inhibits inflammation and may simultaneously promote IL-17 production. Interleukin-17 (IL-17) is a recently described pro-inflammatory cytokine, which may be important in auto-immunity and infection. We investigated the hypothesis that the onset of sepsis is related to differential TGFbeta-1 and IL-17 gene expression. METHODS: A prospective observational study in a mixed intensive care unit (ICU) and hospital wards in a university hospital. Patients (59) with severe sepsis; 15 patients with gram-negative bacteraemia but without critical illness and 10 healthy controls were assayed for TGFbeta-1, IL-17a, IL-17f, IL-6 and IL-1beta mRNA in peripheral blood mononuclear cells (PBMC) by quantitative real-time PCR and serum protein levels by ELISA. RESULTS: TGFbeta-1 mRNA levels are reduced in patients with bacteraemia and sepsis compared with controls (p=0.02). IL-6 mRNA levels were reduced in bacteraemic patients compared with septic patients and controls (p=0.008). IL-1beta mRNA levels were similar in all groups, IL-17a and IL-17f mRNA levels are not detectable in peripheral blood mononuclear cells. IL-6 protein levels were greater in patients with sepsis than bacteraemic and control patients (p<0.0001). Activated TGFbeta-1 and IL-17 protein levels were similar in all groups. IL-1beta protein was not detectable in the majority of patients. CONCLUSIONS: Down regulation of TGFbeta-1 gene transcription was related to the occurrence of infection but not the onset of sepsis. Interleukin-17 production in PBMC may not be significant in the human host response to infection.

Intravenous Lipid Infusion Induces Endoplasmic Reticulum Stress in Endothelial Cells and Blood Mononuclear Cells of Healthy Adults.

Science.gov (United States)

Tampakakis, Emmanouil; Tabit, Corey E; Holbrook, Monika; Linder, Erika A; Berk, Brittany D; Frame, Alissa A; Bretón-Romero, Rosa; Fetterman, Jessica L; Gokce, Noyan; Vita, Joseph A; Hamburg, Naomi M

2016-01-11

Endoplasmic reticulum (ER) stress and the subsequent unfolded protein response may initially be protective, but when prolonged, have been implicated in atherogenesis in diabetic conditions. Triglycerides and free fatty acids (FFAs) are elevated in patients with diabetes and may contribute to ER stress. We sought to evaluate the effect of acute FFA elevation on ER stress in endothelial and circulating white cells. Twenty-one healthy subjects were treated with intralipid (20%; 45 mL/h) plus heparin (12 U/kg/h) infusion for 5 hours. Along with increased triglyceride and FFA levels, intralipid/heparin infusion reduced the calf reactive hyperemic response without a change in conduit artery flow-mediated dilation consistent with microvascular dysfunction. To investigate the short-term effects of elevated triglycerides and FFA, we measured markers of ER stress in peripheral blood mononuclear cells (PBMCs) and vascular endothelial cells (VECs). In VECs, activating transcription factor 6 (ATF6) and phospho-inositol requiring kinase 1 (pIRE1) proteins were elevated after infusion (both P<0.05). In PBMCs, ATF6 and spliced X-box-binding protein 1 (XBP-1) gene expression increased by 2.0- and 2.5-fold, respectively (both P<0.05), whereas CHOP and GADD34 decreased by ≈67% and 74%, respectively (both P<0.01). ATF6 and pIRE1 protein levels also increased (both P<0.05), and confocal microscopy revealed the nuclear localization of ATF6 after infusion, suggesting activation. Along with microvascular dysfunction, intralipid infusion induced an early protective ER stress response evidenced by activation of ATF6 and IRE1 in both leukocytes and endothelial cells. Our results suggest a potential link between metabolic disturbances and ER stress that may be relevant to vascular disease. © 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

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.

The NAD+ precursor nicotinic acid improves genomic integrity in human peripheral blood mononuclear cells after X-irradiation.

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Weidele, Kathrin; Beneke, Sascha; Bürkle, Alexander

2017-04-01

NAD + is an essential cofactor for enzymes catalyzing redox-reactions as well as an electron carrier in energy metabolism. Aside from this, NAD + consuming enzymes like poly(ADP-ribose) polymerases and sirtuins are important regulators involved in chromatin-restructuring processes during repair and epigenetics/transcriptional adaption. In order to replenish cellular NAD + levels after cleavage, synthesis starts from precursors such as nicotinamide, nicotinamide riboside or nicotinic acid to match the need for this essential molecule. In the present study, we investigated the impact of supplementation with nicotinic acid on resting and proliferating human mononuclear blood cells with a focus on DNA damage and repair processes. We observed that nicotinic acid supplementation increased NAD + levels as well as DNA repair efficiency and enhanced genomic stability evaluated by micronucleus test after x-ray treatment. Interestingly, resting cells displayed lower basal levels of DNA breaks compared to proliferating cells, but break-induction rates were identical. Despite similar levels of p53 protein upregulation after irradiation, higher NAD + concentrations led to reduced acetylation of this protein, suggesting enhanced SIRT1 activity. Our data reveal that even in normal primary human cells cellular NAD + levels may be limiting under conditions of genotoxic stress and that boosting the NAD + system with nicotinic acid can improve genomic stability. Copyright © 2017 Elsevier B.V. All rights reserved.

Cell therapy with bone marrow mononuclear cells in elastase-induced pulmonary emphysema.

Science.gov (United States)

Longhini-Dos-Santos, Nathalia; Barbosa-de-Oliveira, Valter Abraão; Kozma, Rodrigo Heras; Faria, Carolina Arruda de; Stessuk, Talita; Frei, Fernando; Ribeiro-Paes, João Tadeu

2013-04-01

Emphysema is characterized by destruction of alveolar walls with loss of gas exchange surface and consequent progressive dyspnea. This study aimed to evaluate the efficiency of cell therapy with bone marrow mononuclear cells (BMMC) in an animal model of elastase-induced pulmonary emphysema. Emphysema was induced in C57Bl/J6 female mice by intranasal instillation of elastase. After 21 days, the mice received bone marrow mononuclear cells from EGFP male mice with C57Bl/J6 background. The groups were assessed by comparison and statistically significant differences (p pulmonary emphysema.

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

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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

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. Copyright © 2015 Elsevier Ltd. All rights reserved.

Culicoides antigen extract stimulates equine blood mononuclear (BMN) cell proliferation and the release of eosinophil adherence-inducing factor(s).

Science.gov (United States)

Mckelvie, J; Foster, A P; Hamblin, A S; Cunningham, F M

2001-04-01

Intradermal injection of a Culicoides antigen extract (CAgX) induces T lymphocyte and eosinophil accumulation in the skin of horses with sweet itch. Blood mononuclear (BMN) cells from normal ponies proliferate when stimulated by mitogen (phytohaemagglutinin, PHA) or antigen (tetanus toxoid, TT) and, as shown here, release soluble factor(s) that induce eosinophil adherence. CAgX also caused concentration dependent proliferation of BMN cells from sweet itch and normal ponies [stimulation index: 29 (13) and 17 (7) for BMN cells from sweet itch and normal ponies, respectively during the active phase of disease; 4 microg protein ml(-1)CAgX; 168 h]. A heat labile factor(s) which caused eosinophil adherence was also released [sweet itch ponies: 6.0 (1.6) per cent adherence versus 1.3 (0.4) per cent; normal ponies: 6.6 (0.5) per cent adherence versus 0.9 (0.1) per cent for supernatants from CAgX (4 microg protein ml(-1); 48 hours) stimulated versus unstimulated BMN cells, respectively]. These results suggest that soluble proteins released from T lymphocytes could affect eosinophil function in the lesional skin of sweet itch horses. Copyright 2001 Harcourt Publishers Ltd.

No oxidative stress or DNA damage in peripheral blood mononuclear cells after exposure to particles from urban street air in overweight elderly

DEFF Research Database (Denmark)

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

2015-01-01

and oxidation-induced DNA damage studied mainly in young normal-weight subjects. We performed a controlled cross-over, randomised, single-blinded, repeated-measure study where 60 healthy subjects (25 males and 35 females) with age 55-83 years and body mass index above 25 kg/m(2) were exposed for 5h to either...... particle-filtered or sham-filtered air from a busy street with number of concentrations and PM2.5 levels of 1800/cm(3) versus 23 000/cm(3) and 3 µg/m(3) versus 24 µg/m(3), respectively. Peripheral blood mononuclear cells (PBMCs) were collected and assayed for production of ROS with and without ex vivo...

Fresenius AS.TEC204 blood cell separator.

Science.gov (United States)

Sugai, Mikiya

2003-02-01

Fresenius AS.TEC204 is a third-generation blood cell separator that incorporates the continuous centrifugal separation method and automatic control of the cell separation process. Continuous centrifugation separates cell components according to their specific gravity, and different cell components are either harvested or eliminated as needed. The interface between the red blood cell and plasma is optically detected, and the Interface Control (IFC) cooperates with different pumps, monitors and detectors to harvest required components automatically. The system is composed of three major sections; the Front Panel Unit; the Pump Unit, and the Centrifuge Unit. This unit can be used for a wide variety of clinical applications including collection of platelets, peripheral blood stem cells, bone marrow stem cells, granulocytes, mononuclear cells, and exchange of plasma or red cells, and for plasma treatment.

Expression of cytokines in chicken peripheral mononuclear blood cells (PMBCs exposed to probiotic strains and Salmonella Enteritidis

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Eva Husáková

2015-01-01

Full Text Available The mRNA expression of interleukin (IL-1β, LITAF, iNOS, macrophage inflammatory protein (MIP1-ß, and K60 were examined in peripheral blood mononuclear cells (PMBCs. The PMBCs were isolated from the chicken blood and in vitro exposed to the probiotic strains E. faecium AL41, E. faecium H31, L. fermentum AD1, and infected with Salmonella enterica serovar Enteritidis (SE147. The PMBCs were evaluated for mRNA expression levels at 24 h and 48 h post infection (p.i. using the reverse transcriptase polymerase chain reaction (RT-PCR. The level of expression of IL-1ß and MIP1-ß was upregulated (P S. Enteritidis + E. faecium AL41 group 48 h p.i. compared to 24 h p.i. Similarly, expression of LITAF was upregulated (P S. Enteritidis (SE group 48 h p.i. In PMBCs treated with E. faecium H31 and S. Enteritidis expression of IL-1ß (P P P E. faecium AL41 demonstrated the highest immunostimulatory effect on expression of selected cytokines by chicken PMBCs after Salmonella infection. It is supposed that the differences in cytokine induction within SE groups are related to lymphocytes isolated from different animals.

Cell-based regenerative strategies for treatment of diabetic skin wounds, a comparative study between human umbilical cord blood-mononuclear cells and calves' blood haemodialysate.

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Hala O El-Mesallamy

Full Text Available BACKGROUND: Diabetes-related foot problems are bound to increase. However, medical therapies for wound care are limited; therefore, the need for development of new treatment modalities to improve wound healing in diabetic patients is essential and constitutes an emerging field of investigation. METHODS: Animals were randomly divided into 8 groups (I-VIII (32 rats/group, all were streptozotocin (STZ-induced diabetics except groups III and VIII were non-diabetic controls. The study comprised two experiments; the first included 3 groups. Group I injected with mononuclear cells (MNCs derived from human umbilical cord blood (HUCB, group II a diabetic control group (PBS i.v. The second experiment included 5 groups, groups IV, V, and VI received topical HUCB-haemodialysate (HD, calves' blood HD, and solcoseryl, respectively. Group VII was the diabetic control group (topical saline. Standard circular wounds were created on the back of rats. A sample of each type of HD was analyzed using the high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS system. Wound area measurement and photography were carried out every 4 days. Plasma glucose, catalase (CAT, malondialdehyde (MDA, nitric oxide (NO and platelets count were assessed. Wound samples were excised for hydroxyproline (HP and histopathological study. RESULTS: Treatment with HUCB MNCs or HUCB-HD resulted in wound contraction, increased CAT, NO, platelets count, body weights, and HP content, and decreased MDA and glucose. CONCLUSION: Systemic administration of HUCB MNCs and topical application of the newly prepared HUCB-HD or calves' blood HD significantly accelerated the rate of diabetic wound healing and would open the possibility of their future use in regenerative medicine.

Generation of human induced pluripotent stem cells (EURACi001-A, EURACi002-A, EURACi003-A from peripheral blood mononuclear cells of three patients carrying mutations in the CAV3 gene

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Viviana Meraviglia

2018-03-01

Full Text Available Caveolinopathies are a heterogeneous family of genetic pathologies arising from alterations of the caveolin-3 gene (CAV3, encoding for the isoform specifically constituting muscle caveolae. Here, by reprogramming peripheral blood mononuclear cells, we report the generation of induced pluripotent stem cells (iPSCs from three patients carrying the ΔYTT deletion, T78K and W101C missense mutations in caveolin-3. iPSCs displayed normal karyotypes and all the features of pluripotent stem cells in terms of morphology, specific marker expression and ability to differentiate in vitro into the three germ layers. These lines thus represent a human cellular model to study the molecular basis of caveolinopathies.Resource tableImage 1Unique stem cell lines identifierEURACi001-AEURACi002-AEURACi003-AAlternative names of stem cell linesB2CAV3 (EURACi001-AL1CAV3 (EURACi002-AN1CAV3 (EURACi003-AInstitutionInstitute for Biomedicine, Eurac ResearchContact information of distributorAlessandra Rossini (alessandra.rossini@eurac.eduType of cell linesiPSCsOriginHumanCell sourcePeripheral blood mononuclear cells (PBMCsMethod of reprogrammingElectroporation of episomal vectors (pCXLE hOCT3/4-shp53-F, pCXLE-hSK, and pCXLE-hULMultiline rationaleNon-isogenic cell lines obtained from patients with mutations in the same gene (CAV3Gene modificationNOType of modificationSpontaneous mutationsAssociated diseaseCaveolinopathiesGene/locusHeterozygous CAV3 c.Δ184–192 (EURACi001-AHeterozygous CAV3 c.303 TGG > TGC (EURACi002-AHeterozygous CAV3 c.233 ACG > AAG (EURACi003-AMethod of modificationN/AName of transgene or resistanceN/AInducible/constitutive systemN/ADate archived/stock dateJanuary 2016 (EURACi001-ASeptember 2016 (EURACi002-AMay 2016 (EURACi003-ACell line repository/bankN/AEthical approvalPeripheral blood was collected from patients after signing the informed consent provided by Cell Line and DNA Biobank from Patients Affected by Genetic Diseases, member of the

A study of the frequency of infection of peripheral blood mononuclear cells of chronic hepatitis B virus carriers using the polymerase chain reaction and hybridization analysis

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Yap, S F; Wong, P W; Goh, K L; Wong, N W [Malaya Univ., Kuala Lumpur (Malaysia). Depts. of Pathology and Dept. of Medicine

1994-05-01

Peripheral blood mononuclear cells (PBMCs) from 100 consecutive chronic carriers of the Hepatitis B virus (HBV) were analysed to determine the frequency of infection of the PBMCs. Cells were isolated using a ficoll gradient and DNA extracted by phenol following an overnight incubation with proteinase K and tween-20. Target nucleic acid were amplified using a set of primers spanning the S region of the viral genome between nucleotides 79 and 761. Following amplification, the samples were gel electrophoresed and the fragments visualized by ethidium bromide staining. The presence of a fragment of about 720 bp was taken as indicative of specific amplification of the HBV nucleic acid sequences. Specificity of amplification was confirmed by hybridization analysis using virus specific probes. Thirty-six out of 41 (87.8%) HBeAg seropositive cases and 15/54 (27.8%) anti-HBe positive cases had HBV DNA detectable by gel electrophoresis. Following hybridization all carriers were found to harbour the virus in their mononuclear cells. The sensitivity using ethidium bromide staining to visualize the amplified sequences was about 1 pg. With hybridization analysis, sensitivity was increased about 10{sup 5}-fold. (author). 12 refs, 1 fig., 1 tab.

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

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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.

Bovine Peripheral Blood Mononuclear Cells Are More Sensitive to Deoxynivalenol Than Those Derived from Poultry and Swine

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Barbara Novak

2018-04-01

Full Text Available Deoxynivalenol (DON is one of the most prevalent mycotoxins, contaminating cereals and cereal-derived products. Its derivative deepoxy-deoxynivalenol (DOM-1 is produced by certain bacteria, which either occur naturally or are supplemented in feed additive. DON-induced impairments in protein synthesis are particularly problematic for highly proliferating immune cells. This study provides the first comparison of the effects of DON and DOM-1 on the concanavalin A-induced proliferation of porcine, chicken, and bovine peripheral blood mononuclear cells (PBMCs. Therefore, isolated PBMCs were treated with DON (0.01–3.37 µM and DOM-1 (1.39–357 µM separately, and proliferation was measured using a bromodeoxyuridine (BrdU assay. Although pigs are considered highly sensitive to DON, the present study revealed a substantially higher sensitivity of bovine (IC50 = 0.314 µM PBMCs compared to chicken (IC50 = 0.691 µM and porcine (IC50 = 0.693 µM PBMCs. Analyses on the proliferation of bovine T-cell subsets showed that all major subsets, namely, CD4+, CD8β+, and γδ T cells, were affected to a similar extent. In contrast, DOM-1 did not affect bovine PBMCs, but reduced the proliferation of chicken and porcine PBMCs at the highest tested concentration (357 µM. Results confirm the necessity of feed additives containing DON-to-DOM-1-transforming bacteria and highlights species-specific differences in the DON sensitivity of immune cells.

Naturally induced secretions of the potato cyst nematode co-stimulate the proliferation of both tobacco leaf protoplasts and human peripheral blood mononuclear cells.

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Goverse, A; Rouppe van der Voort, J; Roppe van der Voort, C; Kavelaars, A; Smant, G; Schots, A; Bakker, J; Helder, J

1999-10-01

Naturally induced secretions from infective juveniles of the potato cyst nematode Globodera rostochiensis co-stimulate the proliferation of tobacco leaf protoplasts in the presence of the synthetic phytohormones alpha-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP). With the use of a protoplast-based bioassay, a low-molecular-weight peptide(s) (cyst nematode secretions also co-stimulated mitogenesis in human peripheral blood mononuclear cells (PBMC). The stimulation of plant cells isolated from nontarget tissue--these nematodes normally invade the roots of potato plants--suggests the activation of a general signal transduction mechanism(s) by an oligopeptide(s) secreted by the nematode. Whether a similar oligopeptide-induced mechanism underlies human PBMC activation remains to be investigated. Reactivation of the cell cycle is a crucial event in feeding cell formation by cyst nematodes. The secretion of a mitogenic low-molecular-weight peptide(s) by infective juveniles of the potato cyst nematode could contribute to the redifferentiation of plant cells into such a feeding cell.

Novel Cell Preservation Technique to Extend Bovine In Vitro White Blood Cell Viability.

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Emilie L Laurin

Full Text Available Although cell-mediated immunity based diagnostics can be integral assays for early detection of various diseases of dairy cows, processing of blood samples for these tests is time-sensitive, often within 24 hours of collection, to maintain white blood cell viability. Therefore, to improve utility and practicality of such assays, the objective of this study was to assess the use of a novel white blood cell preservation technology in whole bovine blood. Blood samples from ten healthy cows were each divided into an unpreserved control sample and a test sample preserved with commercially-available cell transport medium. Samples were maintained at room temperature and stimulated with the mitogens pokeweed and concanavalinA, as well as with interleukin-12 p40. Stimulation was completed on days 1, 5, and 8 post-sampling. Viability of white blood cells was assessed through interferon gamma production determined with a commercial enzyme linked immunosorbent assay. In addition, mononuclear cell viability was assessed with propidium iodide flow cytometry. Greater interferon gamma production was observed on days 5 and 8 post-collection in preserved samples, with both pokeweed and concanavalinA stimulating positive interferon gamma production on day 5 post-collection. A greater proportion of the amount of interferon gamma produced on day 1 continued to be produced on days 5 and 8 post-collection with concanavalinA stimulation (with or without interleukin 12 as compared to pokeweed stimulation. Additionally, viable mononuclear cells were still present at eight days post-collection, with a higher mean proportion detected at days 5 and 8 in all stimulated preserved samples. This practical and simple method to extend in vitro white blood cell viability could benefit the efficient utilization of cell-based blood tests in ruminants.

Combustible and non-combustible tobacco product preparations differentially regulate human peripheral blood mononuclear cell functions.

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Arimilli, Subhashini; Damratoski, Brad E; Prasad, G L

2013-09-01

Natural killer (NK) cells and T cells play essential roles in innate and adaptive immune responses in protecting against microbial infections and in tumor surveillance. Although evidence suggests that smoking causes immunosuppression, there is limited information whether the use of smokeless tobacco (ST) products affects immune responses. In this study, we assessed the effects of two preparations of cigarette smoke, ST extract and nicotine on T cell and NK cell responses using Toll-like receptor-ligand stimulated human peripheral blood mononuclear cells (PBMCs). The tobacco product preparations (TPPs) tested included whole smoke conditioned media (WS-CM), total particulate matter (TPM) and a ST product preparation in complete artificial saliva (ST/CAS). The PBMCs were stimulated with polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharide (LPS). A marked reduction of the expression of intracellular IFN-γ and TNF-α was evident in NK cells and T cells treated with WS-CM and TPM. Consistently, attenuation of ligand-induced secretion of cytokines (IL-1β, IL-10, IL-12 and TNF-α) from PBMCs treated with WS-CM and TPM were observed. While the treatment with TPPs did not alter the expression of the maturation marker CD69, WS-CM and TPM inhibited the cytolytic activity of human PBMCs. Suppression of perforin by WS-CM was also detected. Although interference from the vehicle confounded the interpretation of effects of ST/CAS, some effects were evident only at high concentrations. Nicotine treatment minimally impacted expression of cytokines and cytolytic activity. Data presented herein suggests that the function of NK cells and T cells is influenced by exposure to TPPs (based on equi-nicotine units) in the following order: WS-CM>TPM>ST/CAS. These findings are consistent with the hypothesis put forward by others that chronic smoking leads to immunosuppression, an effect that may contribute to increased microbial infections and cancer incidence among smokers

Secretome of Aggregated Embryonic Stem Cell-Derived Mesenchymal Stem Cell Modulates the Release of Inflammatory Factors in Lipopolysaccharide-Induced Peripheral Blood Mononuclear Cells

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Mohammadi Ghahhari, Nastaran; Maghsood, Faezeh; Jahandideh, Saeed; Lotfinia, Majid; Lak, Shirin; Johari, Behrooz; Azarnezhad, Asaad; Kadivar, Mehdi

2018-07-01

Bone marrow mesenchymal stem cells (BM-MSCs) have emerged as a potential therapy for various inflammatory diseases. Because of some limitations, several recent studies have suggested the use of embryonic stem cell-derived MSCs (ESC-MSCs) as an alternative for BM-MSCs. Some of the therapeutic effects of the ESC-MSCs are related to the secretion of a broad array of cytokines and growth factors, known as secretome. Harnessing this secretome for therapeutic applications requires the optimization of production of secretary molecules. It has been shown that aggregation of MSCs into 3D spheroids, as a preconditioning strategy, can enhance immunomodulatory potential of such cells. In this study, we investigated the effect of secretome derived from human ESC-MSCs (hESC-MSCs) spheroids on secretion of IL-1β, IL-10, and tumor necrosis factor α (TNF-α) from lipopolysaccharide (LPS)-induced peripheral blood mononuclear cells (PBMCs). In the present study, after immunophenotyping and considering mesodermal differentiation of hESC-MSCs, the cells were non-adherently grown to prepare 3D aggregates, and then conditioned medium or secretome was extracted from the cultures. Afterwards, the anti-inflammatory effects of the secretome were assessed in an in vitro model of inflammation. Results from this study showed that aggregate-prepared secretome from hESC-MSCs was able to significantly decrease the secretion of TNF-α (301.7 ± 5.906, p strategy to increase immunomodulatory characteristics of hESC-MSCs.

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)

Research on effects of ionizing radiation of human peripheral blood white cell adhesive molecules

International Nuclear Information System (INIS)

Li Haijun; Cheng Ying; Le Chen; Min Rui

2008-01-01

Objective: To investigate the links between expression and function of adhesive molecule on the surface of irradiated peripheral blood white cells. Methods: Heparinized human peripheral blood was exposed to γ rays with different dose. At the different post-radiation time adhesive molecule expression on cellular surface was determined by double fluorescence labeling antibodies which were against adhesive molecule and special mark of granulocyte or mononuclear cell respectively with flow cytometry, and cellular adhesive ability to different matrixes mediated by adhesive molecule was estimated by commercializing enzyme-linked immunosorbent assay kit and crystalviolet dying. Results: A decline pattern of CD11b on surface of mononuclear cells and CD29 on surface of granulocyte with irradiation dose increase was found. The changes of adhesive ability of mononuclear cells to substance of β1-integrin and collagen-I was well related with irradiation dose. Conclusion: Good relationship shown by the changes of adhesive molecule expression and adhesive ability mediated by the molecules on the surface of peripheral blood white cells with radiation dose was primary base of further research on indicting exposure dose by biomarker. (authors)

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

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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 cryotherapy attenuates in vitro T-cell and monocyte activation. This may explain the disparity in in vivo neopterin and total neopterin between cold water immersion and passive recovery following repetitive exposure during a high-intensity physical impact sport that remains independent of physical performance. © 2016 The Authors. Experimental Physiology © 2016 The Physiological Society.

The Impact of Ramadan Fasting on SIRT1 mRNA Expression in Peripheral Blood Mononuclear Cells

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Mostafa Haji Molahoseini

2016-11-01

Full Text Available Background:The aim of this study was to evaluate the effect of Ramadan fasting on SIRT1 mRNA expression in healthy men.Islamic Ramadan fasting is a holy religious ceremony that has many spiritual benefits. Additionally, it can be considered as the equivalent of calorie restriction that may affect physical health. The results of previous studies revealed that calorie restriction increases the lifespan in laboratory rodents via increasing the expression of a histone deacetylase named SIRT1. Additionally, SIRT1 is known for its anti-inflammatory properties. Materials and Methods: Overall, 43 men volunteered for participating in this one-group before and after (self-controlled study. Two mL blood samples were taken prior to fasting and at the end of the 30th day of fasting. Routine biochemical tests and SIRT1 mRNA expression analysis were performed. Results: Cholesterol and low-density lipoproteins increase, however, high-density lipoproteins level decreased after Ramadan fasting. The analysis of real-time PCR results revealed that SIRT1 mRNA expression in human peripheral blood mononuclear cells increased 4.63 fold in fasting state in comparison with non-fasting state. Conclusion: Ramadan fasting has a significant effect on SIRT1 gene expression. Considering the immunosuppressive and anti-inflammatory properties of SIRT1, further studies are needed to evaluate the effects of SIRT1 up-regulation on the autoimmune and inflammatory diseases during Ramadan fasting.

Human Peripheral Blood Mononuclear Cells Exhibit Heterogeneous CD52 Expression Levels and Show Differential Sensitivity to Alemtuzumab Mediated Cytolysis

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Rao, Sambasiva P.; Sancho, Jose; Campos-Rivera, Juanita; Boutin, Paula M.; Severy, Peter B.; Weeden, Timothy; Shankara, Srinivas; Roberts, Bruce L.; Kaplan, Johanne M.

2012-01-01

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. PMID:22761788

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

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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.

Generation of human induced pluripotent stem cells (EURACi001-A, EURACi002-A, EURACi003-A) from peripheral blood mononuclear cells of three patients carrying mutations in the CAV3 gene.

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Meraviglia, Viviana; Benzoni, Patrizia; Landi, Sara; Murano, Carmen; Langione, Marianna; Motta, Benedetta M; Baratto, Serena; Silipigni, Rosamaria; Di Segni, Marina; Pramstaller, Peter P; DiFrancesco, Dario; Gazzerro, Elisabetta; Barbuti, Andrea; Rossini, Alessandra

2018-03-01

Caveolinopathies are a heterogeneous family of genetic pathologies arising from alterations of the caveolin-3 gene (CAV3), encoding for the isoform specifically constituting muscle caveolae. Here, by reprogramming peripheral blood mononuclear cells, we report the generation of induced pluripotent stem cells (iPSCs) from three patients carrying the ΔYTT deletion, T78K and W101C missense mutations in caveolin-3. iPSCs displayed normal karyotypes and all the features of pluripotent stem cells in terms of morphology, specific marker expression and ability to differentiate in vitro into the three germ layers. These lines thus represent a human cellular model to study the molecular basis of caveolinopathies. Resource table. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

UVB therapy decreases the adhesive interaction between peripheral blood mononuclear cells and dermal microvascular endothelium, and regulates the differential expression of CD54, VCAM-1, and E-selectin in psoriatic plaques

International Nuclear Information System (INIS)

Cai, J.-P.; Harris, K.; Chin, Y.H.

1996-01-01

A dermal lymphocytic infiltrate is a characteristic feature of psoriasis, and may be involved in the pathogenesis of the disease. We have previously shown that specialized dermal microvascular endothelial cells (DMEC) in psoriatic lesions promote the selective adherence of the CD4 CD45Ro helper T-cell subset. In this study, we examined the adhesive interaction between peripheral blood mononuclear cells and psoriatic DMEC in patients treated with ultraviolet B light (UVB), and correlated the results with the expression and function of endothelial adhesion molecules on DMEC. (author)

Clinical isolates of GB virus type C vary in their ability to persist and replicate in peripheral blood mononuclear cell cultures

International Nuclear Information System (INIS)

George, Sarah L.; Xiang Jinhua; Stapleton, Jack T.

2003-01-01

GB virus C/hepatitis G virus (GBV-C) replication in vitro is inefficient and inconsistent. In this study, clinical isolates of GBV-C were evaluated using peripheral blood mononuclear cell (PBMC) based culture methods. Isolates varied consistently in their ability to persistently replicate, and yield increased in cells grown without PHA/IL-2 stimulation. The deduced polyprotein sequence of an isolate that replicated well was determined (GenBank AY196904) and compared to 20 full-length GBV-C sequences. Fourteen of the 16 unique amino acid polymorphisms identified were in the coding regions for nonstructural proteins associated with interferon resistance and RNA replication. These data indicate that clinical GBV-C isolates vary in their ability to persist in culture, do not require PHA/IL-2 stimulation, and that sequence variability in key regulatory regions may affect growth in PBMC cultures. Since GBV-C appears to inhibit HIV replication in a coinfection model, these studies should facilitate determination of the mechanism of this interaction

Effect of selective blockade of oxygen consumption, glucose transport, and Ca2+ influx on thyroxine action in human mononuclear cells

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Kvetny, J; Matzen, L E

1990-01-01

The effect of selective blockade of cellular glucose transporters, Ca2+ influx, and mitochondrial oxygen consumption on thyroxine (T4)-stimulated oxygen consumption and glucose uptake was examined in human mononuclear blood cells. Blockade of glucose transporters by cytochalasin B (1 x 10(-5) mol....../L) and of Ca2+ influx by alprenolol (1 x 10(-5) mol/L) and verapamil (4 x 10(-4) mol/L) inhibited T4-activated glucose uptaken and reduced T4-stimulated oxygen consumption by 20%. Uncoupling of mitochondrial oxygen consumption by azide (1 x 10(-3) mol/L) inhibited T4-stimulated oxygen consumption, but had...... no effect on glucose uptake. We conclude that T4-stimulated glucose uptake in human mononuclear blood cells is dependent on intact glucose transporters and Ca2+ influx, but not on mitochondrial oxygen consumption. However, oxygen consumption is, in part, dependent on intact glucose uptake....

Increased levels of soluble CD226 in sera accompanied by decreased membrane CD226 expression on peripheral blood mononuclear cells from cancer patients

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Xu Zhuwei

2009-06-01

Full Text Available Abstract Background As a cellular membrane triggering receptor, CD226 is involved in the NK cell- or CTL-mediated lysis of tumor cells of different origin, including freshly isolated tumor cells and tumor cell lines. Here, we evaluated soluble CD226 (sCD226 levels in sera, and membrane CD226 (mCD226 expression on peripheral blood mononuclear cells (PBMC from cancer patients as well as normal subjects, and demonstrated the possible function and origin of the altered sCD226, which may provide useful information for understanding the mechanisms of tumor escape and for immunodiagnosis and immunotherapy. Results Soluble CD226 levels in serum samples from cancer patients were significantly higher than those in healthy individuals (P P Conclusion These findings suggest that sCD226 might be shed from cell membranes by certain proteases, and, further, sCD226 may be used as a predictor for monitoring cancer, and more important, a possible immunotherapy target, which may be useful in clinical application.

Accurate measurement of peripheral blood mononuclear cell concentration using image cytometry to eliminate RBC-induced counting error.

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Chan, Leo Li-Ying; Laverty, Daniel J; Smith, Tim; Nejad, Parham; Hei, Hillary; Gandhi, Roopali; Kuksin, Dmitry; Qiu, Jean

2013-02-28

Peripheral blood mononuclear cells (PBMCs) have been widely researched in the fields of immunology, infectious disease, oncology, transplantation, hematological malignancy, and vaccine development. Specifically, in immunology research, PBMCs have been utilized to monitor concentration, viability, proliferation, and cytokine production from immune cells, which are critical for both clinical trials and biomedical research. The viability and concentration of isolated PBMCs are traditionally measured by manual counting with trypan blue (TB) using a hemacytometer. One of the common issues of PBMC isolation is red blood cell (RBC) contamination. The RBC contamination can be dependent on the donor sample and/or technical skill level of the operator. RBC contamination in a PBMC sample can introduce error to the measured concentration, which can pass down to future experimental assays performed on these cells. To resolve this issue, RBC lysing protocol can be used to eliminate potential error caused by RBC contamination. In the recent years, a rapid fluorescence-based image cytometry system has been utilized for bright-field and fluorescence imaging analysis of cellular characteristics (Nexcelom Bioscience LLC, Lawrence, MA). The Cellometer image cytometry system has demonstrated the capability of automated concentration and viability detection in disposable counting chambers of unpurified mouse splenocytes and PBMCs stained with acridine orange (AO) and propidium iodide (PI) under fluorescence detection. In this work, we demonstrate the ability of Cellometer image cytometry system to accurately measure PBMC concentration, despite RBC contamination, by comparison of five different total PBMC counting methods: (1) manual counting of trypan blue-stained PBMCs in hemacytometer, (2) manual counting of PBMCs in bright-field images, (3) manual counting of acetic acid lysing of RBCs with TB-stained PBMCs, (4) automated counting of acetic acid lysing of RBCs with PI-stained PBMCs

Isolation of mesenchymal stem cells from equine umbilical cord blood

DEFF Research Database (Denmark)

Koch, Thomas Gadegaard; Heerkens, Tammy; Thomsen, Preben Dybdahl

2007-01-01

. The hypothesis of this study was that equine MSCs could be isolated from fresh whole equine cord blood. Results: Cord blood was collected from 7 foals immediately after foaling. The mononuclear cell fraction was isolated by Ficoll density centrifugation and cultured in a DMEM low glucose based media at 38.5o......Background: There are no published studies on stem cells from equine cord blood although commercial storage of equine cord blood for future autologous stem cell transplantations is available. Mesenchymal stem cells (MSC) have been isolated from fresh umbilical cord blood of humans collected non......-invasively at the time of birth and from sheep cord blood collected invasively by a surgical intrauterine approach. Mesenchymal stem cells isolation percentage from frozen-thawed human cord blood is low and the future isolation percentage of MSCs from cryopreserved equine cord blood is therefore expectedly low...

Ochratoxin A inhibits the production of tissue factor and plasminogen activator inhibitor-2 by human blood mononuclear cells: Another potential mechanism of immune-suppression

International Nuclear Information System (INIS)

Rossiello, Maria R.; Rotunno, Crescenzia; Coluccia, Addolorata; Carratu, Maria R.; Di Santo, Angelomaria; Evangelista, Virgilio; Semeraro, Nicola; Colucci, Mario

2008-01-01

The mycotoxin ochratoxin A (OTA), an ubiquitous contaminant of food products endowed with a wide spectrum of toxicity, affects several functions of mononuclear leukocytes. Monocytes/macrophages play a major role in fibrin accumulation associated with immune-inflammatory processes through the production of tissue factor (TF) and plasminogen activator inhibitor 2 (PAI-2). We studied the effect of OTA on TF and PAI-2 production by human blood mononuclear cells (MNC). The cells were incubated for 3 or 18 h at 37 deg. C with non toxic OTA concentrations in the absence and in the presence of lipopolysaccharide (LPS) or other inflammatory agents. TF activity was measured by a one-stage clotting test. Antigen assays were performed by specific ELISAs in cell extracts or conditioned media and specific mRNAs were assessed by RT-PCR. OTA had no direct effect on TF and PAI-2 production by MNC. However, OTA caused a dose-dependent reduction in LPS-induced TF (activity, antigen and mRNA) and PAI-2 (antigen and mRNA) production with > 85% inhibition at 1 μg/ml. Similar results were obtained when monocyte-enriched preparations were used instead of MNC. TF production was also impaired by OTA (1 μg/ml) when MNC were stimulated with phorbol myristate acetate (98% inhibition), IL-1β (83%) or TNF-α (62%). The inhibition of TF and PAI-2 induction might represent a hitherto unrecognized mechanism whereby OTA exerts immunosuppressant activity

Lactobacillus GG has in vitro effects on enhanced interleukin-10 and interferon-gamma release of mononuclear cells but no in vivo effects in supplemented mothers and their neonates.

Science.gov (United States)

Kopp, M V; Goldstein, M; Dietschek, A; Sofke, J; Heinzmann, A; Urbanek, R

2008-04-01

The value of probiotics for primary prevention is controversial. Moreover, only little is known about the underlying immunological mechanisms of action. Therefore, we assessed the proliferative response and cytokine release in cultures of isolated mononuclear cells from pregnant women and their neonates supplemented with Lactobacillus GG (LGG) or placebo. In a double-blind, placebo-controlled prospective trial, pregnant women with at least one first-degree relative or a partner with an atopic disease were randomly assigned to receive either the probiotic LGG (ATCC 53103; 5 x 10(9) colony-forming units LGG twice daily) or placebo 4-6 weeks before expected delivery, followed by a post-natal period of 6 months. Cord blood mononuclear cells (CBMC) and peripheral blood mononuclear cells (PBMC) of the corresponding mother were isolated from cord blood and peripheral blood (n=68). The proliferative response of CBMC and PBMC was expressed as the stimulation index (SI), which was calculated according to the ratio between the mean counts per minute (c.p.m.) values measured in the wells with stimulated cells and the mean c.p.m. values measured in the wells with unstimulated cells. Additionally, the cytokines IFN-gamma, IL-10 and IL-13 in the cell culture supernatants were measured using the ELISA technique. No difference was observed between the LGG-supplemented group and the placebo group in terms of the proliferative capacity of maternal or neonatal cord blood cells in response to IL-2, beta-lactoglobulin or LGG. In vitro stimulation with LGG resulted in significantly enhanced release of IL-10 and IFN-gamma, compared with cytokine release in unstimulated controls. However, this phenomenon was observed in supernatants of maternal and neonatal MC in both groups, independent of prior supplementation with LGG. LGG has in vitro effects on enhanced IL-10 and IFN-gamma release of mononuclear cells. However, supplementation with LGG during pregnancy did not alter the proliferative

Percutaneous implantation of peripheral blood mononuclear cells mobilized with granulocyte colony stimulating factor in osteoarthritis of the knee. First case reported in Cuba

International Nuclear Information System (INIS)

Baganet Cobas, Aymara Maria; Hernandez Ramirez, Porfirio; Fernandez Delgado, Norma

2010-01-01

The degenerative joint disease, also known as osteoarthrosis affects to 10% of elderlies aged 60. It is mainly characterized by pain in the involved joint, crepitation, morning stiff and a progressive limitation of movement of that joint leading to a partial or total wear of articular cartilage. The treatment of the knee osteoarthrosis is a great challenge. The recent advances in use of regenerative medicine suggest that adult stem cells could represent a promisor alternative in the treatment of this entity. In a female patient aged 61 presenting with knee osteoarthrosis authors placed a percutaneous implant of autologous mononuclear cells mobilized to peripheral blood by granulocyte colony-stimulating factor achieving a fast clinical and radiological improvement. This result suggests that the procedure used is a feasible, simple, safe and less expensive method for treatment of articular degenerative lesions

Autologous bone marrow mononuclear cell delivery to dilated ...

African Journals Online (AJOL)

Autologous bone marrow mononuclear cell delivery to dilated cardiomyopathy patients: A clinical trial. PLN Kaparthi, G Namita, LK Chelluri, VSP Rao, PK Shah, A Vasantha, SK Ratnakar, K Ravindhranath ...

Sporothrix schenckii sensu stricto and Sporothrix brasiliensis Are Differentially Recognized by Human Peripheral Blood Mononuclear Cells

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José A. Martínez-Álvarez

2017-05-01

Full Text Available Sporothrix schenckii sensu stricto and S. brasiliensis are usually associated to sporotrichosis, a subcutaneous mycosis worldwide distributed. Comparative analyses between these two species indicate they contain genetic and physiological differences that are likely to impact the interaction with host cells. Here, we study the composition of the cell wall from conidia, yeast-like cells and germlings of both species and found they contained the same sugar composition. The carbohydrate proportion in the S. schenckii sensu stricto wall was similar across the three cell morphologies, with exception in the chitin content, which was significantly different in the three morphologies. The cell wall from germlings showed lower rhamnose content and higher glucose levels than other cell morphologies. In S. brasiliensis, the wall sugars were constant in the three morphologies, but glucose was lower in yeast-like cells. In S. schenckii sensu stricto cells most of chitin and β1,3-glucan were underneath wall components, but in S. brasiliensis germlings, chitin was exposed at the cell surface, and β1,3-glucan was found in the outer part of the conidia wall. We also compared the ability of these cells to stimulate cytokine production by human peripheral blood mononuclear cells. The three S. schenckii sensu stricto morphologies stimulated increased levels of pro-inflammatory cytokines, when compared to S. brasiliensis cells; while the latter, with exception of conidia, stimulated higher IL-10 levels. Dectin-1 was a key receptor for cytokine production during stimulation with the three morphologies of S. schenckii sensu stricto, but dispensable for cytokine production stimulated by S. brasiliensis germlings. TLR2 and TLR4 were also involved in the sensing of Sporothrix cells, with a major role for the former during cytokine stimulation. Mannose receptor had a minor contribution during cytokine stimulation by S. schenckii sensu stricto yeast-like cells and

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...... plus bound-T3 (TT3) were significantly higher in the obese (p less than 0.05), concentrations of T4 and binding proteins did not differ. The NBT3 was significantly lower in the obese women; the maximal binding capacity (MBC) was 34.5 +/- 11.6 fmol/mg DNA in the obese subjects and 50.0 +/- 11.6 fmol....../mg DNA in the non-obese subjects (p less than 0.02). The binding affinities did not differ. We have previously shown that increasing T3 concentrations within the physiological range down-regulates NBT3. Therefore, the reduced NBT3 in the obese women was probably secondary to the increased TT3...

Gastric cancer tissue-derived mesenchymal stem cells impact peripheral blood mononuclear cells via disruption of Treg/Th17 balance to promote gastric cancer progression.

Science.gov (United States)

Wang, Mei; Chen, Bin; Sun, Xiao-Xian; Zhao, Xiang-Dong; Zhao, Yuan-Yuan; Sun, Li; Xu, Chang-Gen; Shen, Bo; Su, Zhao-Liang; Xu, Wen-Rong; Zhu, Wei

2017-12-01

Gastric cancer tissue-derived mesenchymal stem cells (GC-MSCs) are important resident stromal cells in the tumor microenvironment (TME) and have been shown to play a key role in gastric cancer progression. Whether GC-MSCs exert a tumor-promoting function by affecting anti-tumor immunity is still unclear. In this study, we used GC-MSC conditioned medium (GC-MSC-CM) to pretreat peripheral blood mononuclear cells (PBMCs) from healthy donors. We found that GC-MSC-CM pretreatment markedly reversed the inhibitory effect of PBMCs on gastric cancer growth in vivo, but did not affect functions of PBMCs on gastric cancer cell proliferation, cell cycle and apoptosis in vitro. PBMCs pretreated with GC-MSC-CM significantly promoted gastric cancer migration and epithelial-mesenchymal transition in vitro and liver metastases in vivo. Flow cytometry analysis showed that GC-MSC-CM pretreatment increased the proportion of Treg cells and reduced that of Th17 cells in PBMCs. CFSE labeling and naïve CD4 + T cells differentiation analysis revealed that GC-MSC-CM disrupted the Treg/Th17 balance in PBMCs by suppressing Th17 cell proliferation and inducing differentiation of Treg cells. Overall, our collective results indicate that GC-MSCs impair the anti-tumor immune response of PBMCs through disruption of Treg/Th17 balance, thus providing new evidence that gastric cancer tissue-derived MSCs contribute to the immunosuppressive TME. Copyright © 2017 Elsevier Inc. All rights reserved.

Establishment of HTLV-I-infected cell lines from peripheral blood mononuclear cells of Brazilian patients Estabelecimento de linhagens celulares infectadas por HTLV-I a partir de células mononucleares periféricas de pacientes brasileiros

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Carolina V. Pannuti

2004-08-01

Full Text Available To investigate epidemiological and pathogenetic features of HTLV-I infection, a cohort of carriers has been followed at the USP Teaching Hospital since 1991. This study describes the establishment of cell lines from peripheral blood mononuclear cells (PBMC of infected subjects. Ex vivo PBMC were cultured with those from a seronegative donor and morphologic evidence of cell transformation was obtained after 90 days with detection of multinucleated cells exhibiting cerebriform nuclei. Integration of HTLV-I proviral DNA and expression of viral antigens was demonstrated in culture by PCR and immunofluorescence. Cell lines were maintained for 240 days, gradually weaned from exogenous IL-2. Immunophenotyping of cell lines on flow cytometry yielded evidence of cell activation. Establishment of HTLV-I-infected cell lines from ex vivo PBMC is feasible and may be useful for studies on lymphocyte phenotypic changes and on mechanisms of HTLV-induced cell proliferation. Moreover they may be used with diagnostic purposes in immunofluorescence tests.Para investigar a epidemiologia e patogênese da infecção por HTLV-I seguimos coorte de portadores dessa retrovirose no HC-FMUSP desde 1991. Este estudo descreve o estabelecimento de linhagens celulares a partir de células mononucleares periféricas (CMP de indivíduos infectados. As CMP foram cultivadas com as de doador soronegativo, verificando-se após 90 dias evidência morfológica de transformação celular com detecção de células multinucleadas com núcleos cerebriformes. Demonstrou-se integração do DNA proviral e expressão in vitro de antígenos virais pela PCR e imunofluorescência. As linhagens celulares transformadas foram mantidas por 240 dias, com retirada gradual de IL-2 exógena. A imunofenotipagem por citometria de fluxo revelou ativação celular. O estabelecimento de linhagens celulares infectadas por HTLV-I a partir de CMP ex-vivo é exeqüível e pode ser útil na investigação de

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.

Human eosinophils modulate peripheral blood mononuclear cell response to Schistosoma mansoni adult worm antigen in vitro.

Science.gov (United States)

Tweyongyere, R; Namanya, H; Naniima, P; Cose, S; Tukahebwa, E M; Elliott, A M; Dunne, D W; Wilson, S

2016-08-01

High numbers of eosinophils are observed in parasitic infections and allergic diseases, where they are proposed to be terminally differentiated effector cells that play beneficial role in host defence, or cause harmful inflammatory response. Eosinophils have been associated with killing of schistosomulae in vitro, but there is growing evidence that eosinophils can play additional immuno-regulatory role. Here, we report results of a study that examines peripheral blood mononuclear cell (PBMC) cytokine responses to Schistosoma mansoni adult worm antigen (SWA) when stimulated alone or enriched with autologous eosinophils. Production of the Th-2 type cytokines interleukin (IL)-4, IL-5 and IL-13 was lower (P = 0·017, 0·018 and eosinophil cultures than in PBMC-only cultures stimulated with SWA. Substantial levels of IL-13, IL-10, interferon gamma and tumour necrosis factor alpha were recorded in cultures of eosinophils, but none of these cytokines showed significant association with the observed eosinophil-induced drop in cytokine responses of PBMC. Transwell experiments suggested that the observed effect is due to soluble mediators that downmodulate production of Th-2 type cytokines. This study shows that eosinophils may down-modulate schistosome-specific Th-2 type cytokine responses in S. mansoni-infected individuals. The mechanism of this immune modulation remains to be elucidated. © 2016 The Authors. Parasite Immunology Published by John Wiley & Sons Ltd.

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

Time-Course Study of the Transcriptome of Peripheral Blood Mononuclear Cells (PBMCs from Sheep Infected with Fasciola hepatica.

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Cristian A Alvarez Rojas

Full Text Available 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.

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.

Specific Features of the Hypothalamic Leptin Signaling Response to Cold Exposure Are Reflected in Peripheral Blood Mononuclear Cells in Rats and Ferrets

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Bàrbara Reynés

2017-08-01

Full Text Available Objectives: Cold exposure induces hyperphagia to counteract fat loss related to lipid mobilization and thermogenic activation. The aim of this study was investigate on the molecular mechanisms involved in cold-induced compensatory hyperphagia.Methods: We analyzed the effect of cold exposure on gene expression of orexigenic and anorexigenic peptides, and of leptin signaling-related genes in the hypothalamus of rats at different ages (1, 2, 4, and 6 months, as well as in ferrets. We also evaluated the potential of peripheral blood mononuclear cells to reflect hypothalamic molecular responses.Results: As expected, cold exposure induced hypoleptinemia in rats, which could be responsible for the increased ratio of orexigenic/anorexigenic peptides gene expression in the hypothalamus, mainly due to decreased anorexigenic gene expression, especially in young animals. In ferrets, which resemble humans more closely, cold exposure induced greater changes in hypothalamic mRNA levels of orexigenic genes. Despite the key role of leptin in food intake control, the effect of cold exposure on the expression of key hypothalamic leptin signaling cascade genes is not clear. In our study, cold exposure seemed to affect leptin signaling in 4-month-old rats (increased Socs3 and Lepr expression, likely associated with the smaller-increase in food intake and decreased body weight observed at this particular age. Similarly, cold exposed ferrets showed greater hypothalamic Socs3 and Stat3 gene expression. Interestingly, peripheral blood mononuclear cells (PBMC mimicked the hypothalamic increase in Lepr and Socs3 observed in 4-month-old rats, and the increased Socs3 mRNA expression observed in ferrets in response to cold exposure.Conclusions: The most outstanding result of our study is that PBMC reflected the specific modulation of leptin signaling observed in both animal models, rats and ferrets, which points forwards PBMC as easily obtainable biological material to be

Cultures and co-cultures of human blood mononuclear cells and endothelial cells for the biocompatibility assessment of surface modified AISI 316L austenitic stainless steel

Energy Technology Data Exchange (ETDEWEB)

Stio, Maria; Martinesi, Maria; Treves, Cristina [Dipartimento di Scienze Biomediche, Sperimentali e Cliniche ‘Mario Serio’, Sezione di Scienze Biochimiche, Università di Firenze, viale Morgagni 50, 50134 Firenze (Italy); Borgioli, Francesca, E-mail: francesca.borgioli@unifi.it [Dipartimento di Ingegneria Industriale (DIEF), Università di Firenze, via S. Marta 3, 50139 Firenze (Italy)

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. - Highlights: • Nitriding improves corrosion resistance and biocompatibility of ground AISI 316L. • The metallic samples differently affect different human cell cultures. • PBMC and HUVEC are a suitable model to test in vitro biocompatibility. • Co-cultures show that HUVEC are affected by pre-incubation of PBMC with the samples. • Inflammation parameters must be taken into account for assessing biocompatibility.

Cultures and co-cultures of human blood mononuclear cells and endothelial cells for the biocompatibility assessment of surface modified AISI 316L austenitic stainless steel

International Nuclear Information System (INIS)

Stio, Maria; Martinesi, Maria; Treves, Cristina; Borgioli, Francesca

2016-01-01

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. - Highlights: • Nitriding improves corrosion resistance and biocompatibility of ground AISI 316L. • The metallic samples differently affect different human cell cultures. • PBMC and HUVEC are a suitable model to test in vitro biocompatibility. • Co-cultures show that HUVEC are affected by pre-incubation of PBMC with the samples. • Inflammation parameters must be taken into account for assessing biocompatibility.

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......, IL-6, IL-10, TGF-beta and interferon-gamma was pronouncedly lower as compared to the bacterial stimulation of leucocytes beneath the CACO-2 cells. In the latter experiments, IL-6, IL-8 and TNF-alpha were the cytokines being mostly induced by apical addition of E. coli. Quantitative mRNA expression...

Effects of vitamin K3 and K5 on proliferation, cytokine production, and regulatory T cell-frequency in human peripheral-blood mononuclear cells.

Science.gov (United States)

Hatanaka, Hiroshige; Ishizawa, Hitomi; Nakamura, Yurie; Tadokoro, Hiroko; Tanaka, Sachiko; Onda, Kenji; Sugiyama, Kentaro; Hirano, Toshihiko

2014-03-18

The effects of vitamin K (VK) derivatives VK3 and VK5 on human immune cells have not been extensively investigated. We examined the effects of VK3 and VK5 on proliferation, apoptosis, cytokine production, and CD4+CD25+Foxp3+ regulatory T (Treg) cell-frequency in human peripheral blood mononuclear cells (PBMCs) activated by T cell mitogen in vitro. Anti-proliferative effects of VK3 and VK5 on T-cell mitogen activated PBMCs were assessed by WST assay procedures. Apoptotic cells were determined as Annexin V positive/propidium iodide (PI) negative cells. Cytokine concentrations in the supernatant of the culture medium were measured with bead-array procedures followed by analysis with flow cytometry. The CD4+CD25+Foxp3+Treg cells in mitogen-activated PBMCs were stained with fluorescence-labeled specific antibodies followed by flow cytometry. VK3 and VK5 suppressed the mitogen-activated proliferation of PBMCs significantly at 10-100μM (p<0.05). The data also suggest that VK3 and VK5 promote apoptosis in the mitogen-activated T cells. VK3 and VK5 significantly inhibited the production of tumor necrosis factor (TNF) α, interleukin (IL)-4, -6, and -10 from the activated PBMCs at 10-100μM (p<0.05). In contrast, VK3 and VK5 significantly increased Treg cell-frequency in the activated PBMCs at concentrations more than 10μM (p<0.001). Our data suggest that VK3 and VK5 attenuate T cell mediated immunity by inhibiting the proliferative response and inducing apoptosis in activated T cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Detection of Hepatitis C virus RNA in peripheral blood mononuclear cells of patients with abnormal alanine transaminase in Ahvaz

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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.

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.

Alterations of serum concentrations of thyroid hormones and sex hormone-binding globulin, nuclear binding of tri-iodothyronine and thyroid hormone-stimulated cellular uptake of oxygen and glucose in mononuclear blood cells from patients with non-thyroidal illness

DEFF Research Database (Denmark)

Kvetny, J; Matzen, L

1990-01-01

Nuclear tri-iodothyronine (T3) binding and thyroid hormone-stimulated oxygen consumption and glucose uptake were examined in mononuclear blood cells from patients with non-thyroidal illness (NTI) in which serum T3 was significantly (P less than 0.05) depressed (0.62 +/- 0.12 (S.D.) nmol/l) compared...

Nucleoside Analog-treated Chronic Hepatitis B Patients showed Reduced Expression of PECAM-1 Gene in Peripheral Blood Mononuclear Cells in Bangladesh

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Tabassum, Shahina; Ullah Munshi, Saif; Hossain, Marufa; Imam, Akhter

2014-01-01

ABSTRACT Background and aim Assessment of therapeutic response is important for monitoring the prognosis and to take decision for cessation of nucleoside analogues therapy in chronic hepatitis B patients. In addition to serum alanine aminotransferase (ALT), hepatitis B virus (HBV) deoxyribonucleic acid (DNA) load and HBeAg status, identification of molecular markers associated with host immune response would be essential to assess therapeutic response. In this regard the current study was performed with the aim to detect expression of platelet endothelial cell adhesion molecule (PECAM)-I gene in peripheral blood monocytes (PBMCs) of treated chronic hepatitis B patients and also to correlate expression of this gene with serum HBV DNA load and serum ALT levels. Materials and methods The study analyzed 60 chronic hepatitis B (CHB) patients, including 30 untreated and 30 nucleoside analogs treated and 10 healthy controls. PECAM-1 gene expression/ transcripts were detected by conventional RT-PCR. Results The expression PECAM-1 mRNA in the PBMCs of CHB patients was significantly higher in untreated (3.17 ± 0.75) than the treated patients (1.64 ± 0.29) (p Tabassum S, Munshi SU, Hossain M, Imam A. Nucleoside Analog-treated Chronic Hepatitis B Patients showed Reduced Expression of PECAM-1 Gene in Peripheral Blood Mononuclear Cells in Bangladesh. Euroasian J Hepato-Gastroenterol 2014;4(2):87-91. PMID:29699354

Helicobacter pylori dupA is polymorphic, and its active form induces proinflammatory cytokine secretion by mononuclear cells.

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Hussein, Nawfal R; Argent, Richard H; Marx, Christian K; Patel, Sapna R; Robinson, Karen; Atherton, John C

2010-07-15

Infection with Helicobacter pylori possessing a newly described virulence factor--duodenal ulcer-promoting gene A (dupA)--has been associated with duodenal ulceration and increased gastric inflammation. The dupA locus of 34 strains was sequenced. A panel of dupA mutants was generated and cocultured with human gastric epithelial cells and peripheral blood mononuclear cells; proinflammatory cytokine release was measured. IL8 expression was measured in human gastric biopsy specimens and related to the dupA and cagA status of infecting strains. Most H. pylori strains had a dupA allele that was longer (1884 bp; dupA1) than previously described dupA alleles, although some had truncated versions (dupA2). Unlike the best-characterized H. pylori virulence determinant, the cag pathogenicity island (cag PaI), neither dupA type induced release of interleukin (IL)-8 from gastric epithelial cells. However, infections due to dupA-positive strains were associated with higher-level mucosal IL-8 messenger RNA expression in the human stomach than were infections due to dupA-negative strains. To explain this paradox, we found that dupA1 (but not dupA2 or the cag PaI) substantially increased H. pylori-induced IL-12p40 and IL-12p70 production from CD14(+) mononuclear cells. Other T helper 1-associated cytokines were also modestly induced. We suggest that virulent H. pylori strains cause inflammation by stimulating epithelial cells through cag-encoded proteins and mononuclear inflammatory cells through dupA1 products.

Allium sativum L. regulates in vitro IL-17 gene expression in human peripheral blood mononuclear cells.

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Moutia, Mouna; Seghrouchni, Fouad; Abouelazz, Omar; Elouaddari, Anass; Al Jahid, Abdellah; Elhou, Abdelhalim; Nadifi, Sellama; Jamal Eddine, Jamal; Habti, Norddine; Badou, Abdallah

2016-09-29

Allium sativum L. (A.S.) "garlic", one of the most interesting medicinal plants, has been suggested to contain compounds that could be beneficial in numerous pathological situations including cancer. In this work, we aimed to assess the immunomodulatory effect of A.S. preparation on human peripheral blood mononuclear cells from healthy individuals. Nontoxic doses of A.S. were identified using MTT assay. Effects on CD4+ or CD8+ T lymphocyte proliferation were studied using flow cytometry. The effect of A.S. on cytokine gene expression was studied using qRT-PCR. Finally, qualitative analysis of A.S. was performed by HPLC approach. Data were analyzed statistically by one-way ANOVA test. The nontoxic doses of A.S. preparation did not affect neither spontaneous nor TCR-mediated CD4+ or CD8+ T lymphocyte proliferation. Interestingly, A.S. exhibited a statistically significant regulation of IL-17 gene expression, a cytokine involved in several inflammatory and autoimmune diseases. In contrast, the expression of IL-4, an anti-inflammatory cytokine, was unaffected. Qualitative analysis of A.S. ethanol preparation indicated the presence of three polyphenol bioactive compounds, which are catechin, vanillic acid and ferulic acid. The specific inhibition of the pro-inflammatory cytokine, IL-17 without affecting cell proliferation in human PBMCs by the Allium sativum L. preparation suggests a potential valuable effect of the compounds present in this plant for the treatment of inflammatory diseases and cancer, where IL-17 is highly expressed. The individual contribution of these three compounds to this global effect will be assessed.

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.

Expression changes of serotonin receptor gene subtype 5HT3a in peripheral blood mononuclear cells from schizophrenic patients treated with haloperidol and Olanzapin.

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Shariati, Gholam Reza; Ahangari, Ghasem; Hossein-nezhad, Arash; Asadi, Seyed Mohammad; Pooyafard, Farzaneh; Ahmadkhaniha, Hamid Reza

2009-09-01

Serotonin receptors are involved in pathophysiology of schizophrenia and may mediate other neurotransmitter effects. We investigated serotonin receptors gene expression in peripheral blood mononuclear cells (PBMC) of naïve schizophrenic patients, before and after treatment. Also serotonin receptor gene expression was compared in two treatment groups including Haloperidol and Olanzapine. The PBMC was separated from whole blood by Ficoll-hypaque. The total cellular RNA was extracted and the cDNA was synthesized. This process was followed by real-time PCR using primer pairs specific for 5HT(3a) serotonin receptor mRNA and beta-actin as internal control. The results showed the presence of subtype of serotonin receptor in lymphocytes. Serotonin gene expression showed significant changes in Olanzapine treatment group which correlated with Clinical Global Impression (CGI) score improvement. In conclusion, the present study has shown that human PBMC express serotonin receptors 5HT(3a). Moreover, clinical symptom improvement of Olanzapin may be demonstrated by a change in serotonin receptor gene expression.

Impact of HBV replication in peripheral blood mononuclear cell on HBV intrauterine transmission.

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Shi, Xiaohong; Wang, Xuefei; Xu, Xixi; Feng, Yongliang; Li, Shuzhen; Feng, Shuying; Wang, Bo; Wang, Suping

2017-12-01

This study determined the effect of hepatitis B virus (HBV) replication in peripheral blood mononuclear cell (PBMC) from HBsAg-positive mothers on HBV intrauterine transmission. A total of 150 HBsAg-positive mothers and their neonates were recruited in this study. Within 24 h after birth, HBV serological markers, serum HBV DNA, PBMC HBV relaxed circular DNA (rcDNA), and covalently closed circular DNA (cccDNA) were measured in the HBsAg-positive mothers and their neonates before passive-active immune prophylaxis. The relationship between HBV replication in PBMC and HBV intrauterine transmission was examined through Chisquare test and logistic regression. The rate of HBV intrauterine transmission was 8.00% (12/150) in the 150 neonates born to HBsAg-positive mothers. The positivities of PBMC HBV rcDNA and cccDNA in the HBsAg-positive mothers were 36.67% (55/150) and 10% (15/150), respectively. Maternal PBMC HBV cccDNA was a risk factor of HBV intrauterine transmission (OR = 6.003, 95% CI: 1.249-28.855). Maternal serum HBeAg was a risk factor of PBMC HBV rcDNA (OR = 3.896, 95% CI: 1.929-7.876) and PBMC HBV cccDNA (OR = 3.74, 95% CI: 1.186-11.793) in the HBsAg-positive mothers. Administration of hepatitis B immune globulin was a protective factor of PBMC HBV cccDNA (OR = 0.312, 95%CI: 0.102-0.954) during pregnancy. The positivity of PBMC HBV rcDNA was related to that of cccDNA in the HBsAg-positive mothers (χ 2 = 5.087, P = 0.024). This study suggests that PBMC is a reservoir of HBV and an extrahepatic site for virus replication and plays a critical role in HBV intrauterine transmission.

Cholesterol Transporters ABCA1 and ABCG1 Gene Expression in Peripheral Blood Mononuclear Cells in Patients with Metabolic Syndrome

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Zahra Tavoosi

2015-01-01

Full Text Available ABCA1 and ABCG1 genes encode the cholesterol transporter proteins that play a key role in cholesterol and phospholipids homeostasis. This study was aimed at evaluating and comparing ABCA1 and ABCG1 genes expression in metabolic syndrome patients and healthy individuals. This case-control study was performed on 36 patients with metabolic syndrome and the same number of healthy individuals in Hamadan (west of Iran during 2013-2014. Total RNA was extracted from mononuclear cells and purified using RNeasy Mini Kit column. The expression of ABCA1 and ABCG1 genes was performed by qRT-PCR. Lipid profile and fasting blood glucose were measured using colorimetric procedures. ABCG1 expression in metabolic syndrome patients was significantly lower (about 75% compared to that of control group, while for ABCA1 expression, there was no significant difference between the two studied groups. Comparison of other parameters such as HDL-C, FBS, BMI, waist circumference, and systolic and diastolic blood pressure between metabolic syndrome patients and healthy individuals showed significant differences (P<0.05. Decrease in ABCG1 expression in metabolic syndrome patients compared to healthy individuals suggests that hyperglycemia, related metabolites, and hyperlipidemia over the transporter capacity resulted in decreased expression of ABCG1. Absence of a significant change in ABCA1 gene expression between two groups can indicate a different regulation mechanism for ABCA1 expression.

Identification of Lactobacillus plantarum genes modulating the cytokine response of human peripheral blood mononuclear cells

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Molenaar Douwe

2010-11-01

Full Text Available Abstract Background Modulation of the immune system is one of the most plausible mechanisms underlying the beneficial effects of probiotic bacteria on human health. Presently, the specific probiotic cell products responsible for immunomodulation are largely unknown. In this study, the genetic and phenotypic diversity of strains of the Lactobacillus plantarum species were investigated to identify genes of L. plantarum with the potential to influence the amounts of cytokines interleukin 10 (IL-10 and IL-12 and the ratio of IL-10/IL-12 produced by peripheral blood mononuclear cells (PBMCs. Results A total of 42 Lactobacillus plantarum strains isolated from diverse environmental and human sources were evaluated for their capacity to stimulate cytokine production in PBMCs. The L. plantarum strains induced the secretion of the anti-inflammatory cytokine IL-10 over an average 14-fold range and secretion of the pro-inflammatory cytokine IL-12 over an average 16-fold range. Comparisons of the strain-specific cytokine responses of PBMCs to comparative genome hybridization profiles obtained with L. plantarum WCFS1 DNA microarrays (also termed gene-trait matching resulted in the identification of 6 candidate genetic loci with immunomodulatory capacities. These loci included genes encoding an N-acetyl-glucosamine/galactosamine phosphotransferase system, the LamBDCA quorum sensing system, and components of the plantaricin (bacteriocin biosynthesis and transport pathway. Deletion of these genes in L. plantarum WCFS1 resulted in growth phase-dependent changes in the PBMC IL-10 and IL-12 cytokine profiles compared with wild-type cells. Conclusions The altered PBMC cytokine profiles obtained with the L. plantarum WCFS1 mutants were in good agreement with the predictions made by gene-trait matching for the 42 L. plantarum strains. This study therefore resulted in the identification of genes present in certain strains of L. plantarum which might be responsible for

Transplantation of human umbilical cord blood-derived mononuclear cells induces recovery of motor dysfunction in a rat model of Parkinson's disease

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Chen C

2016-04-01

Full Text Available Chao Chen,1,* Jing Duan,1,* Aifang Shen,2,* Wei Wang,1 Hao Song,1 Yanming Liu,1 Xianjie Lu,1 Xiaobing Wang,2 Zhiqing You,1 Zhongchao Han,3,4 Fabin Han1 1Center for Stem Cells and Regenerative Medicine, The Liaocheng People's Hospital, Affiliated Liaocheng Hospital, Taishan Medical University, Shandong, People's Republic of China; 2Department of Gynecology and Obstetrics, The Liaocheng People's Hospital, Affiliated Liaocheng Hospital, Taishan Medical University, Shandong, People's Republic of China; 3The State Key Laboratory of Experimental Hematology, Institute of Hematology and Hospital of Blood Diseases, Chinese Academy of Medical Sciences, Peking Union of Medical College, Tianjin, People's Republic of China; 4National Engineering Research Center of Cell Products, AmCellGene Co. Ltd., TEDA, Tianjin, People's Republic of China*These authors contributed equally to this workAbstract: Human umbilical cord blood-derived mononuclear cells (hUCB-MNCs were reported to have neurorestorative capacity for neurological disorders such as stroke and traumatic brain injury. This study was performed to explore if hUCB-MNC transplantation plays any therapeutic effects for Parkinson's disease (PD in a 6-OHDA-lesioned rat model of PD. hUCB-MNCs were isolated from umbilical cord blood and administered to the striatum of the 6-OHDA-lesioned rats. The apomorphine-induced locomotive turning-overs were measured to evaluate the improvement of motor dysfunctions of the rats after administration of hUCB-MNCs. We observed that transplanted hUCB-MNCs significantly improve the motor deficits of the PD rats and that grafted hUCB-MNCs integrated to the host brains and differentiated to neurons and dopamine neurons in vivo after 16 weeks of transplantation. Our study provided evidence that transplanted hUCB-MNCs play therapeutic effects in a rat PD model by differentiating to neurons and dopamine neurons. Keywords: hUCB-MNCs, Parkinson's disease, transplantation

Evaluation of Antigen-Specific IgM and IgG Production during an In Vitro Peripheral Blood Mononuclear Cell Culture Assay

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Yoshiko Matsuda

2017-07-01

Full Text Available The recent attention given to diseases associated with memory B-cell (mBC-produced antibodies (Abs suggests the need for a similar in vitro assay to evaluate the functions of mBCs. Here, we cultured peripheral blood mononuclear cells (PBMCs with the intent to collect mBC-derived Abs in vitro and maintain their cell–cell contact-dependent interactions with helper T-cells. PBMCs were cultured with interleukin (IL-21, CpG-oligodeoxynucleotides (ODN, phorbol myristate acetate (PMA, and phytohemagglutinin/leucoagglutinin (PHA-L in 24-well flat-bottom plates (5 × 105 cells/well. A culture supernatant analysis of PBMCs from healthy donors (n = 10 indicated that antigen-specific IgM Ab levels in a PBMC culture supernatant might be better able to demonstrate the antigen sensitization status in a smaller peripheral blood sample, compared to IgG because Epstein–Barr virus-specific IgM mBCs circulate peripherally at a significantly higher frequency once antiviral humoral immunity has stabilized. Thus, our in vitro assay demonstrated the potential significance of antigen-specific IgM Ab production in the culture supernatants. Furthermore, an analysis of cultured PBMCs from allograft kidney recipients (n = 16 sensitized with de novo donor-specific human leukocyte antigen (HLA-specific Abs (DSAs showed that IgM-type HLA-specific Abs were detected mainly from the culture supernatants from PBMCs of patients with stable graft function, whereas IgG isotype HLA Abs were detectable only from patients with biopsy-proven antibody-mediated rejection. In other words, these IgG isotype Abs also represented an activated humoral immune response in vivo. Additionally, IgM- and IgG-expressing mBCs from healthy donors (n = 5 were cultured with IL-21, CpG-ODN, and a supernatant produced by stimulating CD19+ B-cell-depleted PBMCs with PHA-L and PMA in 24-well flat-bottom plates (1 × 105 cells/well, and the resulting in vitro analysis provided some

Increased ICAM-1 Expression in Transformed Human Oral Epithelial Cells: Molecular Mechanism and Functional Role in Peripheral Blood Mononuclear Cell Adhesion and Lymphokine-Activated-Killer Cell Cytotoxicity

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Huang, George T.-J.; Zhang, Xinli; Park, No-Hee

2012-01-01

The intercellular adhesion molecule-1 (ICAM-1, CD54) serves as a counter-receptor for the β2-integrins, LFA-1 and Mac-1, which are expressed on leukocytes. Although expression of ICAM-1 on tumor cells has a role in tumor progression and development, information on ICAM-1 expression and its role in oral cancer has not been established. Normal human oral keratinocytes (NHOK), human papilloma virus (HPV)-immortalized human oral keratinocyte lines (HOK-16B, HOK-18A, and HOK-18C), and six human oral neoplastic cell lines (HOK-16B-BaP-T1, SCC-4, SCC-9, HEp-2, Tu-177 and 1483) were used to study ICAM-1 expression and its functional role in vitro. Our results demonstrated that NHOK express negligible levels of ICAM-1, whereas immortalized human oral keratinocytes and cancer cells express significantly higher levels of ICAM-1, except for HOK-16B-BaP-T1 and HEp-2. Altered mRNA half-lives did not fully account for the increased accumulation of ICAM-1 mRNA. Adhesion of peripheral blood mononuclear cells (PBMC) to epithelial cells correlated with cell surface ICAM-1 expression levels. This adhesion was inhibited by antibodies specific for either ICAM-1 or LFA-1/Mac-1, suggesting a role for these molecules in adhesion. In contrast, lymphokine-activated-killer (LAK) cell cytotoxic killing of epithelial cells did not correlate with ICAM-1 levels or with adhesion. Nonetheless, within each cell line, blocking of ICAM-1 or LFA-1/Mac-1 reduced LAK cells killing, suggesting that ICAM-1 is involved in mediating this killing. PMID:10938387

High concentration of branched-chain amino acids promotes oxidative stress, inflammation and migration of human peripheral blood mononuclear cells via mTORC1 activation.

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Zhenyukh, Olha; Civantos, Esther; Ruiz-Ortega, Marta; Sánchez, Maria Soledad; Vázquez, Clotilde; Peiró, Concepción; Egido, Jesús; Mas, Sebastián

2017-03-01

Leucine, isoleucine and valine are essential aminoacids termed branched-chain amino acids (BCAA) due to its aliphatic side-chain. In several pathological and physiological conditions increased BCAA plasma concentrations have been described. Elevated BCAA levels predict insulin resistance development. Moreover, BCAA levels higher than 2mmol/L are neurotoxic by inducing microglial activation in maple syrup urine disease. However, there are no studies about the direct effects of BCAA in circulating cells. We have explored whether BCAA could promote oxidative stress and pro-inflammatory status in peripheral blood mononuclear cells (PBMCs) obtained from healthy donors. In cultured PBMCs, 10mmol/L BCAA increased the production of reactive oxygen species (ROS) via both NADPH oxidase and the mitochondria, and activated Akt-mTOR signalling. By using several inhibitors and activators of these molecular pathways we have described that mTOR activation by BCAA is linked to ROS production and mitochondrial dysfunction. BCAA stimulated the activation of the redox-sensitive transcription factor NF-κB, which resulted in the release of pro-inflammatory molecules, such as interleukin-6, tumor necrosis factor-α, intracellular adhesion molecule-1 or CD40L, and the migration of PBMCs. In conclusion, elevated BCAA blood levels can promote the activation of circulating PBMCs, by a mechanism that involving ROS production and NF-κB pathway activation. These data suggest that high concentrations of BCAA could exert deleterious effects on circulating blood cells and therefore contribute to the pro-inflammatory and oxidative status observed in several pathophysiological conditions. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Determination of Zidovudine Triphosphate Intracellular Concentrations in Peripheral Blood Mononuclear Cells from Human Immunodeficiency Virus-Infected Individuals by Tandem Mass Spectrometry

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Font, Eva; Rosario, Osvaldo; Santana, Jorge; García, Hermes; Sommadossi, Jean-Pierre; Rodriguez, Jose F.

1999-01-01

Nucleoside reverse transcriptase inhibitors (NRTIs) used against the human immunodeficiency virus (HIV) need to be activated intracellularly to their triphosphate moiety to inhibit HIV replication. Intracellular concentrations of these NRTI triphosphates, especially zidovudine triphosphate (ZDV-TP), are relatively low (low numbers of femtomoles per 106 cells) in HIV-infected patient peripheral blood mononuclear cells. Recently, several methods have used either high-performance liquid chromatography (HPLC) or solid-phase extraction (SPE) coupled with radioimmunoassay to obtain in vivo measurements of ZDV-TP. The limit of detection (LOD) by these methods ranged from 20 to 200 fmol/106 cells. In this report, we describe the development of a method to determine intracellular ZDV-TP concentrations in HIV-infected patients using SPE and HPLC with tandem mass spectrometry for analysis. The LOD by this method is 4.0 fmol/106 cells with a linear concentration range of at least 4 orders of magnitude from 4.0 to 10,000 fmol/106 cells. In hispanic HIV-infected patients, ZDV-TP was detectable even when the sampling time after drug administration was 15 h. Intracellular ZDV-TP concentrations in these patients ranged from 41 to 193 fmol/106 cells. The low LOD obtained with this method will provide the opportunity for further in vivo pharmacokinetic studies of intracellular ZDV-TP in different HIV-infected populations. Furthermore, this methodology could be used to perform simultaneous detection of two or more NRTIs, such as ZDV-TP and lamivudine triphosphate. PMID:10582890

Role of Oxidative Stress in the Suppression of Immune Responses in Peripheral Blood Mononuclear Cells Exposed to Combustible Tobacco Product Preparation.

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Arimilli, Subhashini; Schmidt, Eckhardt; Damratoski, Brad E; Prasad, G L

2017-10-01

Cigarette smoking is a major risk factor for several human diseases. Chronic inflammation, resulting from increased oxidative stress, has been suggested as a mechanism that contributes to the increased susceptibility of smokers to cancer and microbial infections. We have previously shown that whole-smoke conditioned medium (WS-CM) and total particulate matter (TPM) prepared from Kentucky 3R4F reference cigarettes [collectively called as combustible tobacco product preparations (TPPs)] potently suppressed agonist-stimulated cytokine secretion and target cell killing in peripheral blood mononuclear cells (PBMCs). Here we have investigated the role of oxidative stress from TPPs, which alters inflammatory responses in vitro. Particularly, we investigated the mechanisms of WS-CM-induced suppression of select cytokine secretions in Toll-like receptor (TLR) agonist-stimulated cells and target cell killing by effector cells in PBMCs. Pretreatment with N-acetyl cysteine (NAC), a precursor of reduced glutathione and an established anti-oxidant, protected against DNA damage and cytotoxicity caused by exposure to WS-CM. Similarly, secretion of tumor necrosis factor (TNF), interleukin (IL)-6, and IL-8 in response to TLR-4 stimulation was restored by pretreatment with NAC. Target cell killing, a functional measure of cytolytic cells in PBMCs, is suppressed by WS-CM. Pretreatment with NAC restored the target cell killing in WS-CM treated PBMCs. This was accompanied by higher perforin levels in the effector cell populations. Collectively, these data suggest that reducing oxidative stress caused by cigarette smoke components restores select immune responses in this ex vivo model.

Mononuclear cell secretome protects from experimental autoimmune myocarditis.

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Hoetzenecker, Konrad; Zimmermann, Matthias; Hoetzenecker, Wolfram; Schweiger, Thomas; Kollmann, Dagmar; Mildner, Michael; Hegedus, Balazs; Mitterbauer, Andreas; Hacker, Stefan; Birner, Peter; Gabriel, Christian; Gyöngyösi, Mariann; Blyszczuk, Przemyslaw; Eriksson, Urs; Ankersmit, Hendrik Jan

2015-03-14

Supernatants of serum-free cultured mononuclear cells (MNC) contain a mix of immunomodulating factors (secretome), which have been shown to attenuate detrimental inflammatory responses following myocardial ischaemia. Inflammatory dilated cardiomyopathy (iDCM) is a common cause of heart failure in young patients. Experimental autoimmune myocarditis (EAM) is a CD4+ T cell-dependent model, which mirrors important pathogenic aspects of iDCM. The aim of this study was to determine the influence of MNC secretome on myocardial inflammation in the EAM model. BALB/c mice were immunized twice with an alpha myosin heavy chain peptide together with Complete Freund adjuvant. Supernatants from mouse mononuclear cells were collected, dialysed, and injected i.p. at Day 0, Day 7, or Day 14, respectively. Myocarditis severity, T cell responses, and autoantibody formation were assessed at Day 21. The impact of MNC secretome on CD4+ T cell function and viability was evaluated using in vitro proliferation and cell viability assays. A single high-dose application of MNC secretome, injected at Day 14 after the first immunization, effectively attenuated myocardial inflammation. Mechanistically, MNC secretome induced caspase-8-dependent apoptosis in autoreactive CD4+ T cells. MNC secretome abrogated myocardial inflammation in a CD4+ T cell-dependent animal model of autoimmune myocarditis. This anti-inflammatory effect of MNC secretome suggests a novel and simple potential treatment concept for inflammatory heart diseases. © The Author 2013. Published by Oxford University Press on behalf of the European Society of Cardiology.

CD14+CD16+ monocytes are the main target of Zika virus infection in peripheral blood mononuclear cells in a paediatric study in Nicaragua.

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Michlmayr, Daniela; Andrade, Paulina; Gonzalez, Karla; Balmaseda, Angel; Harris, Eva

2017-11-01

The recent Zika pandemic in the Americas is linked to congenital birth defects and Guillain-Barré syndrome. White blood cells (WBCs) play an important role in host immune responses early in arboviral infection. Infected WBCs can also function as 'Trojan horses' and carry viruses into immune-sheltered spaces, including the placenta, testes and brain. Therefore, defining which WBCs are permissive to Zika virus (ZIKV) is critical. Here, we analyse ZIKV infectivity of peripheral blood mononuclear cells (PBMCs) in vitro and from Nicaraguan Zika patients and show CD14 + CD16 + monocytes are the main target of infection, with ZIKV replication detected in some dendritic cells. The frequency of CD14 + monocytes was significantly decreased, while the CD14 + CD16 + monocyte population was significantly expanded during ZIKV infection compared to uninfected controls. Viral RNA was detected in PBMCs from all patients, but in serum from only a subset, suggesting PBMCs may be a reservoir for ZIKV. In Zika patients, the frequency of infected cells was lower but the percentage of infected CD14 + CD16 + monocytes was significantly higher compared to dengue cases. The gene expression profile in monocytes isolated from ZIKV- and dengue virus-infected patients was comparable, except for significant differences in interferon-γ, CXCL12, XCL1, interleukin-6 and interleukin-10 levels. Thus, our study provides a detailed picture of the innate immune profile of ZIKV infection and highlights the important role of monocytes, and CD14 + CD16 + monocytes in particular.

Characterization of lymphokine-activated killer cells from peripheral blood and lymph nodes of non-Hodgkin's lymphoma patients

International Nuclear Information System (INIS)

Nadkarni, J.J.; Jehaver, K.G.; De, A.K.; Soman, C.S.; Nadkarni, K.S.

1993-01-01

Peripheral blood lymphocytes (PBL) and lymph node lymphocytes (LNL) from non-Hodgkin's lymphoma patients were tested for lymphokine-activated killer cells (LAK) cells cytotoxicity using appropriate targets in a short-term 51 chromium-release assay. The results showed a significant depression in LNL-LAK activity suggesting the reduced capacity of LNL to generate LAK cells. LNL-LAK cells demonstrated significantly low percentages of cells expressing CD16, CD56 and CD25 as compared to PBL-LAK and healthy donors. The reduced capacity to generate LAK cells in lymph nodes could by due to the presence of low numbers of natural killer cells which are thought to be the main precursors of LAK cells. The IL-2 producing ability of lymph node mononuclear cells was found to by significantly higher than that of peripheral blood mononuclear cells from both healthy donors and and NHL patients. (author)

Recovery and functional activity of mononuclear bone marrow and peripheral blood cells after different cell isolation protocols used in clinical trials for cell therapy after acute myocardial infarction

NARCIS (Netherlands)

van Beem, Rachel T.; Hirsch, Alexander; Lommerse, Ingrid M.; Zwaginga, Jaap Jan; Noort, Willy A.; Biemond, Bart J.; Piek, Jan J.; van der Schoot, C. Ellen; Voermans, Carlijn

2008-01-01

AIMS: Clinical trials showed contradictory results in functional recovery after intracoronary infusion of autologous mononuclear (bone marrow) cells in patients with acute myocardial infarction. A recent study suggests that this might be related to the isolation protocol used. In The Netherlands, a

Multielemental fractionation in human peripheral blood mononuclear cells by size exclusion liquid chromatography coupled to UV and ICP-MS detection.

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Alvarado, Gladys; Murillo, Miguel

2010-10-01

An analytical methodology is presented in this work to determine metal-biomolecule complexes size distribution patterns of several elements, among different compounds present in human peripheral blood mononuclear cells (PBMC). A hyphenated technique based on size exclusion chromatography (SEC) coupled online to UV and inductively coupled plasma mass spectrometry (ICP-MS) detection is used. Two different SEC columns with separation ranges between 1,500-1,000,000 relative molecular mass (M(r)) (Nanofilm SEC-250) and 5,000 and 100,000 relative molecular mass (M(r)) (TSK-Gel G2000 SW) are used with 10 mmol/L tris-HCl at pH 7.3 as mobile phase. Retention behavior (retention time and peak-area ratios) remained unchanged for several successive separations. Metal-containing compounds are found to a wide range of M(r). Copper-zinc superoxide dismutase, copper and zinc metallothionein, and copper and zinc transferrin are identified in PBMC samples. A high M(r) (147,000) metal-binding protein containing copper and zinc and a high M(r) (107,000) manganese-binding protein were also found; however, these remained unknown.

A New Synthetic Compound, 2-OH, Enhances Interleukin-2 and Interferon-γ Gene Expression in Human Peripheral Blood Mononuclear Cells

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Woan-Fang Tzeng

2009-07-01

Full Text Available A new synthetic compound, 6-hydroxy-2-tosylisoquinolin-1(2H-one (2-OH, was selected for immunopharmacological activity tests. The effects of 2-OH on human peripheral blood mononuclear cell (PBMC proliferation were determined by tritiated thymidine uptake. Compared to phytohemagglutinin (PHA; 5 μg/mL stimulation, 2-OH significantly enhanced PBMC proliferation in a dose-dependent manner. The 50% enhancement activity (EC50 for 2-OH was 4.4±0.1 μM. In addition, effects of 2-OH on interleukin-2 (IL-2 and interferon-γ (IFN-γ production in PBMC were determined by enzyme immunoassay. Results demonstrated that 2-OH stimulated IL-2 and IFN-γ production in PBMC. Data from reverse transcription-polymerase chain reaction (RT-PCR and real-time PCR indicated that IL-2 and IFN-γ mRNA expression in PBMC could be induced by 2-OH. Therefore, 2-OH enhanced IL-2 and IFN-γ production in PBMC by modulation their gene expression. We suggest that 2-OH may be an immunomodulatory agent.

Inorganic zinc supplementation modulates heat shock and immune response in heat stressed peripheral blood mononuclear cells of periparturient dairy cows.

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Sheikh, Aasif Ahmad; Aggarwal, Anjali; B, Indu; Aarif, Ovais

2017-06-01

Thermal stress in India is one of the major constraints affecting dairy cattle productivity. Every attempt should be made to ameliorate the heat and calving related stress in high producing dairy cows for higher economic returns. In the current study, inorganic zinc was tried to alleviate the adverse effects of thermal stress in periparturient cows. Twelve cows, six each of Sahiwal and Karan Fries (KF) in their second parity with confirmed pregnancy were chosen for the experiment. The blood samples were collected periparturiently on three occasions viz. -21, 0 and +21 days relative to calving. The in vitro study was conducted after isolating peripheral blood mononuclear cells (PBMC) from whole blood. The cultured PBMC were subjected to three different levels of exposures viz. 37°C as control, 42°C to induce thermal stress and 42°C + zinc to ameliorate the adverse effects of high temperature. Heat shock lead to a significant (Pheat shock proteins (HSP). HSP was more on the day of calving as well. KF showed more HSP concentration than Sahiwal breed indicating the heat bearing capacity of later. Zinc treatment to thermally stressed PBMC caused a fall in the HSP concentration in both the breeds during periparturient period. Moreover, heat stress increased significantly (PHeat and calving related stress caused a fall in the IL-12 levels which increased significantly (Pcows. The study could help to alleviate the heat stress and potentiate immunity by providing mineral supplements in periparturient dairy cattle habituating tropics. Copyright © 2017 Elsevier Inc. All rights reserved.

The Influence of Hepatitis C Virus Therapy on the DNA Base Excision Repair System of Peripheral Blood Mononuclear Cells.

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Czarny, Piotr; Merecz-Sadowska, Anna; Majchrzak, Kinga; Jabłkowski, Maciej; Szemraj, Janusz; Śliwiński, Tomasz; Karwowski, Bolesław

2017-07-01

Hepatitis C virus (HCV) can infect extrahepatic tissues, including lymphocytes, creating reservoir of the virus. Moreover, HCV proteins can interact with DNA damage response proteins of infected cells. In this article we investigated the influence of the virus infection and a new ombitasvir/paritaprevir/ritonavir ± dasabuvir ± ribavirin (OBV/PTV/r ± DSV ± RBV) anti-HCV therapy on the PBMCs (peripheral blood mononuclear cells, mainly lymphocytes) DNA base excision repair (BER) system. BER protein activity was analyzed in the nuclear and mitochondrial extracts (NE and ME) of PBMC isolated from patients before and after therapy, and from subjects without HCV, using modeled double-strand DNA, with 2'-deoxyuridine substitution as the DNA damage. The NE and ME obtained from patients before therapy demonstrated lower efficacy of 2'-deoxyuridine removal and DNA repair polymerization than those of the control group or patients after therapy. Moreover, the extracts from the patients after therapy had similar activity to those from the control group. However, the efficacy of apurinic/apyrimidinic site excision in NE did not differ between the studied groups. We postulate that infection of lymphocytes by the HCV can lead to a decrease in the activity of BER enzymes. However, the use of novel therapy results in the improvement of glycosylase activity as well as the regeneration of endonuclease and other crucial repair enzymes.

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.

Lipid-load in peripheral blood mononuclear cells: Impact of food-consumption, dietary-macronutrients, extracellular lipid availability and demographic factors.

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Ameer, Fatima; Munir, Rimsha; Usman, Hina; Rashid, Rida; Shahjahan, Muhammad; Hasnain, Shahida; Zaidi, Nousheen

2017-04-01

Lipid-load in peripheral blood mononuclear cells (PBMCs) has recently gained attention of the researchers working on nutritional regulation of metabolic health. Previous works have indicated that the metabolic circuitries in the circulating PBMCs are influenced by dietary-intake and macronutrient composition of diet. In the present work, we analyzed the impact of diet and dietary macronutrients on PBMCs' lipid-load. The overall analyses revealed that dietary carbohydrates and fats combinatorially induce triglyceride accumulation in PBMCs. On the other hand, dietary fats were shown to induce significant decrease in PBMCs' cholesterol-load. The effects of various demographic factors -including age, gender and body-weight- on PBMCs' lipid-load were also examined. Body-weight and age were both shown to affect PBMC's lipid-load. Our study fails to provide any direct association between extracellular lipid availability and cholesterol-load in both, freshly isolated and cultured PBMCs. The presented work significantly contributes to the current understanding of the impact of food-consumption, dietary macronutrients, extracellular lipid availability and demographic factors on lipid-load in PBMCs. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Characterization of bone marrow and lymph node repopulating cells by transplanting mononuclear cells into radiated dogs

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Ross, W.M.; Koerbling, M.; Northdurft, W.; Calvo, W.; Fliedner, T.M.

1977-01-01

The present investigations deal with an attempt to identify and characterize the multipotential stem cell present in mononuclear cell (MNC) suspensions collected from the peripheral blood of dogs by leukocytopheresis; various morphologic and functional tests have been employed in an endeavor to accomplish this task. In an attempt to increase the yield of MNC and, in particular, of stem cells, the presence of which was assumed to be indicated by CFU-c (colony forming units in agar), dextran sulfate (DS) was administered i.v. (15 mg/kg) 30 min before the beginning of leukocytopheresis. DS has been found to be an effective CFU-c mobilizing agent, capable of increasing the number of CFU-c in peripheral blood by 7 to 10 times within 3 hr. During a 4 hr leukocytopheresis, about 6.5-14 x 10 9 MNC were collected. To eliminate erythrocytes, a Ficoll-Isopaque gradient was employed. A discontinuous albumin gradient was prepared with 6 fractions (17 to 27 percent albumin, 350 mOsm) in an attempt to obtain a cell suspension with an improved ratio of CFU-c to PHA-reactive lymphocytes. Lymphocytes accumulated predominantly in fractions 4 to 6. CFU-c were found primarily in fraction 2; one cell out of 13 MNC was a CFU-c and 82 percent of the CFU-c was found here. In contrast, the majority of PHA-responsive cells was found in fractions 3 and 4

Generation of blood-derived dendritic cells in dogs with oral malignant melanoma.

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Catchpole, B; Stell, A J; Dobson, J M

2002-01-01

Advances in treatment of human melanoma indicate that immunotherapy, particularly dendritic cell (DC) immunization, may prove useful. The aim of this study was to investigate whether blood-derived DCs could be generated from canine melanoma patients. Peripheral blood mononuclear cells were isolated from three such dogs and cultured with recombinant canine granulocyte-macrophage colony stimulating factor (GM-CSF), canine interleukin 4 and human Flt3-ligand for 7 days. The resulting cells demonstrated a typical dendritic morphology, and were enriched for cells expressing CD1a, CD11c and MHC II by flow cytometric analysis. Thus, canine blood-derived DCs can be generated in vitro and DC immunization should be feasible in dogs. Copyright Harcourt Publishers Ltd.

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

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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. Copyright © 2016 Elsevier B.V. All rights reserved.

Pegylated interferons Lambda-1a and alfa-2a display different gene induction and cytokine and chemokine release profiles in whole blood, human hepatocytes and peripheral blood mononuclear cells.

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Freeman, J; Baglino, S; Friborg, J; Kraft, Z; Gray, T; Hill, M; McPhee, F; Hillson, J; Lopez-Talavera, J C; Wind-Rotolo, M

2014-06-01

Pegylated interferon-lambda-1a (Lambda), a type III interferon (IFN) in clinical development for the treatment of chronic HCV infection, has shown comparable efficacy and an improved safety profile to a regimen based on pegylated IFN alfa-2a (alfa). To establish a mechanistic context for this improved profile, we investigated the ex vivo effects of Lambda and alfa on cytokine and chemokine release, and on expression of IFN-stimulated genes (ISGs) in primary human hepatocytes and peripheral blood mononuclear cells (PBMCs) from healthy subjects. Our findings were further compared with changes observed in blood analysed from HCV-infected patients treated with Lambda or alfa in clinical studies. mRNA transcript and protein expression of the IFN-λ-limiting receptor subunit was lower compared with IFN-α receptor subunits in all cell types. Upon stimulation, alfa and Lambda induced ISG expression in hepatocytes and PBMCs, although in PBMCs Lambda-induced ISG expression was modest. Furthermore, alfa and Lambda induced release of cytokines and chemokines from hepatocytes and PBMCs, although differences in their kinetics of induction were observed. In HCV-infected patients, alfa treatment induced ISG expression in whole blood after single and repeat dosing. Lambda treatment induced modest ISG expression after single dosing and showed no induction after repeat dosing. Alfa and Lambda treatment increased IP-10, iTAC, IL-6, MCP-1 and MIP-1β levels in serum, with alfa inducing higher levels of all mediators compared with Lambda. Overall, ex vivo and in vivo induction profiles reported in this analysis strongly correlate with clinical observations of fewer related adverse events for Lambda vs those typically associated with alfa. © 2014 John Wiley & Sons Ltd.

Decreased proinflammatory cytokine production by peripheral blood mononuclear cells from vitiligo patients following aspirin treatment

International Nuclear Information System (INIS)

Zailaie, Mohammad Z.

2005-01-01

Limited studies have shown that treatment of cells with aspirin modulates their cytokine production. Consequently, the aim of the present study is to investigate the pattern of important proinflammatory cytokines production by stimulated peripheral blood mononuclear cells (PBMC) from patients with active vitiligo following long-term treatment with low-dose oral aspirin. The study was conducted at the Vitiligo Unit, King Abdul-Aziz University Medical Center, Jeddah, Kingdom of Saudi Arabia between March and October 2003. Thirty-two patients (18 females and 14 males) with non-segmental vitiligo were divided into 2 equal groups, one group received a daily single dose of oral aspirin (300 mg) and the other group received placebo for a period of 12 weeks. The concentrations of interleukin (IL)-1beta, IL-6, IL-8 and tumor necrosis factor-alpha (TNF-alpha) were determined in the supernatant of isolated cultured PMBC after being stimulated with bacterial lipopolysaccharide (LPS), before the start of aspirin treatment and at end of treatment period. Cytokine levels were measured using the quantitative sandwich enzyme-linked immunosorbent assay (ELISA) technique, utilizing commercially available kits. The proinflammatory cytokine production by the PBMC of patients with active vitiligo was significantly increased compared to normal controls. Thus, the relative percentage increase in the production of IL-1beta, IL-6, IL-8 and TNF-alpha was: 39.4%, 110.5% (p<0.05), 91.5% (p<0.01), and 37% (p<0.05). At the end of treatment, proinflammatory cytokine production in the aspirin-treated group of active vitiligo patients was significantly decreased compared to the placebo group. Thus, the relative percentage decrease in the production of IL-1beta IL-6, IL-8 and TNF-alpha was: 42.5%, 45.2% (p<0.05), 30.8% (p<0.01), and 50.6% (p<0.05). The vitiligo activity was arrested in all aspirin-treated patients, while 2 patients demonstrated significant repigmentation.Chronic administration of

Advanced feeder-free generation of induced pluripotent stem cells directly from blood cells.

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Trokovic, Ras; Weltner, Jere; Nishimura, Ken; Ohtaka, Manami; Nakanishi, Mahito; Salomaa, Veikko; Jalanko, Anu; Otonkoski, Timo; Kyttälä, Aija

2014-12-01

Generation of validated human induced pluripotent stem cells (iPSCs) for biobanking is essential for exploring the full potential of iPSCs in disease modeling and drug discovery. Peripheral blood mononuclear cells (PBMCs) are attractive targets for reprogramming, because blood is collected by a routine clinical procedure and is a commonly stored material in biobanks. Generation of iPSCs from blood cells has previously been reported using integrative retroviruses, episomal Sendai viruses, and DNA plasmids. However, most of the published protocols require expansion and/or activation of a specific cell population from PBMCs. We have recently collected a PBMC cohort from the Finnish population containing more than 2,000 subjects. Here we report efficient generation of iPSCs directly from PBMCs in feeder-free conditions in approximately 2 weeks. The produced iPSC clones are pluripotent and transgene-free. Together, these properties make this novel method a powerful tool for large-scale reprogramming of PBMCs and for iPSC biobanking. ©AlphaMed Press.

Cytokine and transcription factor expression by Aspergillus fumigatus-stimulated peripheral blood mononuclear cells in dogs with sino-nasal aspergillosis.

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Vanherberghen, M; Bureau, F; Peters, I R; Day, M J; Lynch, A; Fievez, L; Billen, F; Clercx, C; Peeters, D

2013-08-15

The causal agent of sino-nasal aspergillosis is usually Aspergillus fumigatus, which is a saprophytic and ubiquitous fungus that causes a severe rhinosinusitis in apparent healthy dogs. Affected dogs do not have systemic immuno-suppression. It has been shown previously that dogs affected by this disease have local over-expression of interleukin (IL)-10 and Th1 cytokines in nasal mucosal tissue. The aim of the present study was to assess the response of peripheral blood mononuclear cells (PBMC) from affected and unaffected dogs to antigen-specific stimulation with heat-inactivated Aspergillus spp. conidia, by quantifying gene expression for specific Th1, Th2, Th17 and Treg cytokines and their related transcription factors. Quantification of IL-4 and IFN-γ protein in culture supernatant was performed by enzyme-linked immunosorbent assay (ELISA). PBMC from dogs with SNA produced adequate mRNA encoding IFN-γ and IFN-γ protein. The expression of IL-17A mRNA was significantly greater in PBMC of affected compared with unaffected dogs. The amount of IL-10 mRNA in PBMC from affected dogs decreased after antigen-specific challenge. These results suggest that the incapacity of affected dogs to clear these fungal infections is not related to a defect in Th1 immunity or to an overwhelming regulatory reaction, but rather to an uncontrolled pro-inflammatory reaction driven by Th17 cells. Copyright © 2013 Elsevier B.V. All rights reserved.

Omega-3 fatty acids, EPA and DHA induce apoptosis and enhance drug sensitivity in multiple myeloma cells but not in normal peripheral mononuclear cells.

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Abdi, J; Garssen, J; Faber, J; Redegeld, F A

2014-12-01

The n-3 polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to enhance the effect of chemotherapeutic drugs in clinical studies in cancer patients and to induce apoptotic tumor cell death in vitro. Until now, EPA and DHA have never been investigated in multiple myeloma (MM). Human myeloma cells (L363, OPM-1, OPM-2 and U266) and normal peripheral blood mononuclear cells were exposed to EPA and DHA, and effects on mitochondrial function and apoptosis, caspase-3 activation, gene expression and drug toxicity were measured. Exposure to EPA and DHA induced apoptosis and increased sensitivity to bortezomib in MM cells. Importantly, they did not affect viability of normal human peripheral mononuclear cells. Messenger RNA expression arrays showed that EPA and DHA modulated genes involved in multiple signaling pathways including nuclear factor (NF) κB, Notch, Hedgehog, oxidative stress and Wnt. EPA and DHA inhibited NFκB activity and induced apoptosis through mitochondrial perturbation and caspase-3 activation. Our study suggests that EPA and DHA induce selective cytotoxic effects in MM and increase sensitivity to bortezomib and calls for further exploration into a potential application of these n-3 polyunsaturated fatty acids in the therapy of MM. Copyright © 2014 Elsevier Inc. All rights reserved.

Correlation analyses revealed global microRNA-mRNA expression associations in human peripheral blood mononuclear cells.

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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

2018-02-01

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.

Downregulation of TIM-3 mRNA expression in peripheral blood mononuclear cells from patients with systemic lupus erythematosus

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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.

Downregulation of TIM-3 mRNA expression in peripheral blood mononuclear cells from patients with systemic lupus erythematosus

International Nuclear Information System (INIS)

Cai, X.Z.; Huang, W.Y.; Qiao, Y.; Chen, Y.; Du, S.Y.; Chen, D.; Yu, S.; Liu, N.; Dou, L.Y.; Jiang, Y.

2014-01-01

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 s =0.283, P=0.049) and serum albumin (r s =0.297, P=0.047). TIM-3 mRNA expression was weakly correlated with the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI; r 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

Relation of Mitochondrial Oxygen Consumption in Peripheral Blood Mononuclear Cells to Vascular Function in Type 2 Diabetes Mellitus

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Hartman, Mor-Li; Shirihai, Orian S.; Holbrook, Monika; Xu, Guoquan; Kocherla, Marsha; Shah, Akash; Fetterman, Jessica L.; Kluge, Matthew A.; Frame, Alissa A.; Hamburg, Naomi M.; Vita, Joseph A.

2014-01-01

Recent studies have shown mitochondrial dysfunction and increased production of reactive oxygen species in peripheral blood mononuclear cells (PBMC’s) and endothelial cells from patients with diabetes mellitus. Mitochondria oxygen consumption is coupled to ATP production and also occurs in an uncoupled fashion during formation of reactive oxygen species by components of the electron transport chain and other enzymatic sites. We therefore hypothesized that diabetes would be associated with higher total and uncoupled oxygen consumption in PBMC’s that would correlate with endothelial dysfunction. We developed a method to measure oxygen consumption in freshly isolated PBMC’s and applied it to 26 patients with type 2 diabetes mellitus and 28 non-diabetic controls. Basal (192±47 vs. 161±44 pMoles/min, P=0.01), uncoupled (64±16 vs. 53±16 pMoles/min, P=0.007), and maximal (795±87 vs. 715±128 pMoles/min, P=0.01) oxygen consumption rates were higher in diabetic patients compared to controls. There were no significant correlations between oxygen consumption rates and endothelium-dependent flow-mediated dilation measured by vascular ultrasound. Non-endothelium-dependent nitroglycerin-mediated dilation was lower in diabetics (10.1±6.6 vs. 15.8±4.8%, P=0.03) and correlated with maximal oxygen consumption (R= −0.64, P=0.001). In summary, we found that diabetes mellitus is associated with a pattern of mitochondrial oxygen consumption consistent with higher production of reactive oxygen species. The correlation between oxygen consumption and nitroglycerin-mediated dilation may suggest a link between mitochondrial dysfunction and vascular smooth muscle cell dysfunction that merits further study. Finally, the described method may have utility for assessment of mitochondrial function in larger scale observational and interventional studies in humans. PMID:24558030

Induction and identification of rabbit peripheral blood derived dendritic cells

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Zhou, Jing; Yang, FuYuan; Chen, WenLi

2012-03-01

Purpose: To study a method of the induction of dendritic cells (DCs) from rabbit peripheral blood. Methods: Peripheral blood cells were removed from rabbit, filtered through nylon mesh. Peripheral blood mononuclear cells (PBMC) were isolated from the blood cells by Ficoll-Hypaque centrifugation (density of 1.077g/cm3).To obtain DCs, PBMC were cultured in RPMI1640 medium containing 10% fetal calf serum, 50U/mL penicillin and streptomycin, referred to subsequently as complete medium, at 37°C in 5% CO2 atmosphere for 4 hours. Nonadherent cells were aspirated, adherent cells were continued incubated in complete medium, supplemented with granulocyte/macrophage colony-stimulating factor (GM-CSF, 50ng/ml),and interleukin 4 (IL-4, 50ng/ml) for 9 days. Fluorescein labeled antibodies(anti-CD14, anti-HLA-DR, anti-CD86) were used to sign cells cultured for 3,6,9 days respectively, Then flow cytometry was performed. Results: Ratio of anti-HLA-DR and anti-CD86 labeled cells increased with induction time extension, in contrast with anti-CD14. Conclusion: Dendritic cells can be effectively induced by the method of this experiment, cell maturation status increased with induction time extension.

Data regarding association between serum osteoprotegerin level, numerous of circulating endothelial-derived and mononuclear-derived progenitor cells in patients with metabolic syndrome

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Alexander E. Berezin

2016-09-01

Full Text Available Metabolic syndrome (MetS is defined as cluster of multiple metabolic and cardiovascular (CV abnormalities included abdominal obesity, high-normal blood pressure, dyslipidaemia, and impaired fasting glucose tolerance that exhibits has a growing prevalence worldwide. We investigated whether an elevated level of osteoprotegerin (OPG predicts imbalance between different phenotypes of circulating endothelial (EPCs and mononuclear (MPCs progenitor cells in MetS patients. We have analyzed data regarding dysmetabolic disorder subjects without known CV disease, as well as with known type two diabetes mellitus. All patients have given their informed written consent for participation in the study. This article contains data on the independent predictors of depletion in numerous of circulating EPCs and MPCs in MetS patients. The data are supplemental to our original research article describing detailed associations of elevated OPG level in MetS patients with numerous of EPCs and MPCs beyond traditional CV risk factors. Keywords: Metabolic syndrome, Osteoprotegerin, Circulating endothelial derived progenitor cells, Mononuclear-derived progenitor cells

Two immunosuppressive compounds from the mushroom Rubinoboletus ballouii using human peripheral blood mononuclear cells by bioactivity-guided fractionation.

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Li, Long-Fei; Chan, Ben Chung-Lap; Yue, Grace Gar-Lee; Lau, Clara Bik-San; Han, Quan-Bin; Leung, Ping-Chung; Liu, Ji-Kai; Fung, Kwok-Pui

2013-10-15

Rubinoboletus ballouii is an edible mushroom wildly grown in Yunnan province, China. Up till now, little was known about the chemical and biological properties of this mushroom. The aim of this study was to investigate the immunomodulatory effects of the ethanolic extract of Rubinoboletus ballouii and its fractions on human peripheral blood mononuclear cells (PBMCs) using bioactivity-guided fractionation. The crude extract of the fruiting bodies of RB was fractionated by high-speed counter current chromatography (HSCCC). Twelve fractions were obtained and the third fraction (Fraction C) exerted the most potent anti-inflammatory activities in mitogen-activated PBMCs. Further fractionation of fraction C led to the isolation of two single compounds which were elucidated as 1-ribofuranosyl-s-triazin-2(1H)-one and pistillarin, respectively. The results showed that both 1-ribofuranosyl-s-triazin-2(1H)-one and pistillarin exhibited significant immunosuppressive effects on phytohemagglutinin (PHA)-stimulated human PBMCs by inhibiting [methyl-(3)H]-thymidine uptake and inflammatory cytokines productions such as tumor necrosis factor (TNF)-α, interleukin (IL)-10, interferon (IFN)-γ and IL-1β. Besides, 1-ribofuranosyl-s-triazin-2(1H)-one was firstly found in natural resources, and pistillarin was also isolated from the family Boletaceae for the first time. They exhibited great potential in developing as anti-inflammatory reagents. Copyright © 2013 Elsevier GmbH. All rights reserved.

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.

Clinical Symptoms in Fibromyalgia Are Better Associated to Lipid Peroxidation Levels in Blood Mononuclear Cells Rather than in Plasma

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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

Clinical symptoms in fibromyalgia are better associated to lipid peroxidation levels in blood mononuclear cells rather than in plasma.

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Mario D Cordero

Full Text Available 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.

Allogeneic human dermal fibroblasts are viable in peripheral blood mononuclear co-culture

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Restu Syamsul Hadi

2014-08-01

Full Text Available Background Transplanted allogeneic dermal fibroblasts retain stem cell subpopulations, and are easily isolated, expanded and stored using standard techniques. Their potential for regenerative therapy of chronic wounds should be evaluated. The aim of this study was to determine allogeneic fibroblast viability in the presence of peripheral blood mononuclear cells (PBMC. Methods In this experimental study, fibroblasts were isolated from foreskin explants, expanded in the presence of serum, and stored using slow-freezing. We used one intervention group of allogeneic fibroblasts co-cultured with PBMC and 2 control groups of separate fibroblast and PBMC cultures.Fibroblasts were characterized by their collagen secretion and octamer-binding transcription factor 4 (OCT4 expression. Viability was evaluated using water soluble tetrazolium-1 (WST-1 proliferation assay. Absorbances were measured at 450 nm. Data analysis was performed by student’s paired t-test. Results Dermal fibroblasts were shown to secrete collagen, express OCT4, be recoverable after cryopreservation, and become attached to the culture dish in a co-culture with PBMC. Co-cultured and control fibroblasts had no significantly different cell viabilities (p>0.05. Calculated viable cell numbers increased 1.8 and 5.1-fold, respectively, at days 2 and 4 in vitro. Both groups showed comparable doubling times at days 2 and 4 in vitro. PBMC did not interfere with allogeneic fibroblast viability and proliferative capacity Conclusions Allogeneic fibroblasts remain viable and proliferate in the presence of host PBMC. Future research should evaluate allogeneic human dermal fibroblast competency in clinical settings. Dermal fibroblasts are a potential source for cell therapy in chronic wound management.

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

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

2012-01-01

Highlights: ► Butachlor exhibited strong binding affinity with DNA and produced 8-oxodG adducts. ► Butachlor induced DNA strand breaks and micronuclei formation in PBMN cells. ► Butachlor induced ROS and dissipation of mitochondrial membrane potential in cells. ► Butachlor resulted in cell cycle arrest and eventually caused cellular necrosis. -- Abstract: 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 24 h resulted in significantly increased number of binucleated micronucleated (BNMN) cells with a dose dependent reduction in the nuclear division index (NDI). The flow

Characterization of coal fly ash nanoparticles and induced oxidative DNA damage in human peripheral blood mononuclear cells

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Dwivedi, Sourabh; Saquib, Quaiser; Al-Khedhairy, Abdulaziz A.; Ali, Al-Yousef Sulaiman; Musarrat, Javed

2012-01-01

The nano-sized particles present in coal fly ash (CFA) were characterized through the X-ray diffraction (XRD), transmission and scanning electron microscopy (TEM, SEM), atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FTIR) analyses. The XRD data revealed the average crystallite size of the CFA nanoparticles (CFA-NPs) as 14 nm. TEM and SEM imaging demonstrated predominantly spherical and some polymorphic structures in the size range of 11 to 25 nm. The amount of heavy metal associated with CFA particles (μg/g) were determined as Fe (34160.0 ± 1.38), Ni (150.8 ± 0.78), Cu (99.3 ± 0.56) and Cr (64.0 ± 0.86). However, the bioavailability of heavy metals in terms of percent release was in the order as Cr > Ni > Cu > Fe in CFA-dimethyl sulfoxide (DMSO) extract. The comet and cytokinesis blocked micronucleus (CBMN) assays revealed substantial genomic DNA damage in peripheral blood mononuclear (PBMN) cells treated with CFA-NPs in Aq and DMSO extracts. About 1.8 and 3.6 strand breaks per unit of DNA were estimated through alkaline unwinding assay at 1:100 DNA nucleotide/CFA ppm ratios with the Aq and DMSO extracts, respectively. The DNA and mitochondrial damage was invariably greater with CFA-DMSO extract vis-à-vis -Aq extract. Generation of superoxide anions (O 2 • − ) and intracellular reactive oxygen species (ROS) through metal redox-cycling, alteration in mitochondrial potential and 8-oxodG production elucidated CFA-NPs induced oxidative stress as a plausible mechanism for CFA-induced genotoxicity. -- Highlights: ► CFA consists of spherical crystalline nanoparticles in size range of 11–25 nm. ► Alkaline unwinding assay revealed single-strandedness in CFA treated ctDNA. ► CFA nanoparticles exhibited the ability to induce ROS and oxidative DNA damage. ► Comet and CBMN assays revealed DNA and chromosomal breakage in PBMN cells. ► CFA-NPs resulted in mitochondrial membrane damage in PBMN cells.

Characterization of coal fly ash nanoparticles and induced oxidative DNA damage in human peripheral blood mononuclear cells

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Dwivedi, Sourabh; Saquib, Quaiser; Al-Khedhairy, Abdulaziz A. [Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451 (Saudi Arabia); Ali, Al-Yousef Sulaiman [Department of Medical Laboratory Sciences, College of Applied Medical Science, University of Dammam, P.O. Box 1683, Hafr Al Batin-31991 (Saudi Arabia); Musarrat, Javed, E-mail: musarratj1@yahoo.com [Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451 (Saudi Arabia); Department of Agricultural Microbiology, Faculty of Agricultural Sciences, AMU, Aligarh202002 (India)

2012-10-15

The nano-sized particles present in coal fly ash (CFA) were characterized through the X-ray diffraction (XRD), transmission and scanning electron microscopy (TEM, SEM), atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FTIR) analyses. The XRD data revealed the average crystallite size of the CFA nanoparticles (CFA-NPs) as 14 nm. TEM and SEM imaging demonstrated predominantly spherical and some polymorphic structures in the size range of 11 to 25 nm. The amount of heavy metal associated with CFA particles ({mu}g/g) were determined as Fe (34160.0 {+-} 1.38), Ni (150.8 {+-} 0.78), Cu (99.3 {+-} 0.56) and Cr (64.0 {+-} 0.86). However, the bioavailability of heavy metals in terms of percent release was in the order as Cr > Ni > Cu > Fe in CFA-dimethyl sulfoxide (DMSO) extract. The comet and cytokinesis blocked micronucleus (CBMN) assays revealed substantial genomic DNA damage in peripheral blood mononuclear (PBMN) cells treated with CFA-NPs in Aq and DMSO extracts. About 1.8 and 3.6 strand breaks per unit of DNA were estimated through alkaline unwinding assay at 1:100 DNA nucleotide/CFA ppm ratios with the Aq and DMSO extracts, respectively. The DNA and mitochondrial damage was invariably greater with CFA-DMSO extract vis-a-vis -Aq extract. Generation of superoxide anions (O{sub 2} Bullet {sup -}) and intracellular reactive oxygen species (ROS) through metal redox-cycling, alteration in mitochondrial potential and 8-oxodG production elucidated CFA-NPs induced oxidative stress as a plausible mechanism for CFA-induced genotoxicity. -- Highlights: Black-Right-Pointing-Pointer CFA consists of spherical crystalline nanoparticles in size range of 11-25 nm. Black-Right-Pointing-Pointer Alkaline unwinding assay revealed single-strandedness in CFA treated ctDNA. Black-Right-Pointing-Pointer CFA nanoparticles exhibited the ability to induce ROS and oxidative DNA damage. Black-Right-Pointing-Pointer Comet and CBMN assays revealed DNA and chromosomal

Short communication: Cytokine profiles from blood mononuclear cells of dairy cows classified with divergent immune response phenotypes.

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Martin, C E; Paibomesai, M A; Emam, S M; Gallienne, J; Hine, B C; Thompson-Crispi, K A; Mallard, B A

2016-03-01

Genetic selection for enhanced immune response has been shown to decrease disease occurrence in dairy cattle. Cows can be classified as high (H), average, or low responders based on antibody-mediated immune response (AMIR), predominated by type-2 cytokine production, and cell-mediated immune response (CMIR) through estimated breeding values for these traits. The purpose of this study was to identify in vitro tests that correlate with in vivo immune response phenotyping in dairy cattle. Blood mononuclear cells (BMC) isolated from cows classified as H-AMIR and H-CMIR through estimated breeding values for immune response traits were stimulated with concanavalin A (ConA; Sigma Aldrich, St. Louis, MO) and gene expression, cytokine production, and cell proliferation was determined at multiple time points. A repeated measures model, which included the effects of immune response group, parity, and stage of lactation, was used to compare differences between immune response phenotype groups. The H-AMIR cows produced more IL-4 protein than H-CMIR cows at 48 h; however, no difference in gene expression of type-2 transcription factor GATA3 or IL4 was noted. The BMC from H-CMIR cows had increased production of IFN-γ protein at 48, 72, and 96 h compared with H-AMIR animals. Further, H-CMIR cows had increased expression of the IFNG gene at 16, 24, and 48 h post-treatment with ConA, although expression of the type-1 transcription factor gene TBX21 did not differ between immune response groups. Although proliferation of BMC increased from 24 to 72 h after ConA stimulation, no differences were found between the immune response groups. Overall, stimulation of H-AMIR and H-CMIR bovine BMC with ConA resulted in distinct cytokine production profiles according to genetically defined groups. These distinct cytokine profiles could be used to define disease resistance phenotypes in dairy cows according to stimulation in vitro; however, other immune response phenotypes should be assessed

Vaccine-Mediated Mechanisms Controlling Replication of Francisella tularensis in Human Peripheral Blood Mononuclear Cells Using a Co-culture System

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Kjell Eneslätt

2018-02-01

Full Text Available Cell-mediated immunity (CMI is normally required for efficient protection against intracellular infections, however, identification of correlates is challenging and they are generally lacking. Francisella tularensis is a highly virulent, facultative intracellular bacterium and CMI is critically required for protection against the pathogen, but how this is effectuated in humans is poorly understood. To understand the protective mechanisms, we established an in vitro co-culture assay to identify how control of infection of F. tularensis is accomplished by human cells and hypothesized that the model will mimic in vivo immune mechanisms. Non-adherent peripheral blood mononuclear cells (PBMCs were expanded with antigen and added to cultures with adherent PBMC infected with the human vaccine strain, LVS, or the highly virulent SCHU S4 strain. Intracellular numbers of F. tularensis was followed for 72 h and secreted and intracellular cytokines were analyzed. Addition of PBMC expanded from naïve individuals, i.e., those with no record of immunization to F. tularensis, generally resulted in little or no control of intracellular bacterial growth, whereas addition of PBMC from a majority of F. tularensis-immune individuals executed static and sometimes cidal effects on intracellular bacteria. Regardless of infecting strain, statistical differences between the two groups were significant, P < 0.05. Secretion of 11 cytokines was analyzed after 72 h of infection and significant differences with regard to secretion of IFN-γ, TNF, and MIP-1β was observed between immune and naïve individuals for LVS-infected cultures. Also, in LVS-infected cultures, CD4 T cells from vaccinees, but not CD8 T cells, showed significantly higher expression of IFN-γ, MIP-1β, TNF, and CD107a than cells from naïve individuals. The co-culture system appears to identify correlates of immunity that are relevant for the understanding of mechanisms of the protective host immunity to

Diagnostic values for the viral load in peripheral blood mononuclear cells of patients with chronic active Epstein-Barr virus disease.

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Ito, Yoshinori; Suzuki, Michio; Kawada, Jun-ichi; Kimura, Hiroshi

2016-04-01

Chronic active Epstein-Barr virus disease (CAEBV) is a distinct EBV-associated lymphoproliferative disease with a poor prognosis. Although the viral load in blood samples has been widely used for diagnosing CAEBV, well-defined viral load thresholds to guide clinicians are currently lacking. The aim of the present study was to determine standardized diagnostic values for EBV load in blood samples of CAEBV patients using the World Health Organization international standard for reporting. Levels of EBV DNA in 103 peripheral blood mononuclear cells (PBMCs) and 95 plasma/serum samples from 107 cases with CAEBV were quantified and expressed in international units. Receiver operating characteristic curves were analyzed to assess the most appropriate cut-off values for levels of EBV DNA to distinguish CAEBV from EBV-associated infectious mononucleosis (IM) and controls with past EBV infection. Levels of EBV DNA in PBMCs were significantly higher in the CAEBV group (median, 10(4.2) IU/μgDNA) compared to the IM (median, 10(2.1) IU/μgDNA) and control groups. An inconsistent qualitative result was seen in 13 of 86 CAEBV patients; in these, EBV-DNA was positive in PBMCs, but negative in plasma. Diagnostic cut-off values for viral load in PBMCs from CAEBV patients, as compared to those of healthy controls and IM patients, were 10(2.0) IU/μgDNA and 10(3.2) IU/μgDNA, respectively. For diagnostic purposes, the viral load of PBMCs was better than of plasma/serum. A diagnostic cut-off EBV load for CAEBV may be useful for the management of CAEBV patients. Copyright © 2015 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

M cells and granular mononuclear cells in Peyer's patch domes of mice depleted of their lymphocytes by total lymphoid irradiation

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Ermak, T.H.; Steger, H.J.; Strober, S.; Owen, R.L.

1989-01-01

The cytoarchitecture of Peyer's patches that were depleted of their lymphocytes by total lymphoid irradiation (TLI) was examined with particular attention to the effects on M cells in the follicle epithelium and on mononuclear cells in follicle domes underlying the epithelium. Five-month-old, specific pathogen-free Balb/c mice were irradiated with 200-250 rad/day, five times a week to a total dose of 3400-4250, and their Peyer's patches were either fixed for electron microscopy or frozen for immunohistochemistry 1-4 days after completion of irradiation. Control mice were examined at the same time intervals. Follicle domes of TLI mice had approximately one fourth the epithelial surface area of domes of control mice. Within the epithelium, lymphoid cells were virtually depleted after TLI, and yet the epithelium contained M cells. In control mice, most M cells were accompanied by lymphoid cells in invaginations of the apical-lateral cell membrane. In TLI mice, most M cells did not have such apical-lateral invaginations and were columnar shaped. Other than lacking lymphocytes, these cells appeared to be mature M cells. Some M cells did have lymphoid cells or granular mononuclear cells below their basal membranes, adjacent to the basal lamina. Below the epithelium, the proportion of granular mononuclear cells was greatly increased following TLI. The retention of M cells and the increase in proportion of granular mononuclear cells in follicle domes are consistent with selective depletion of lymphocytes following TLI. Persistence of M cells without lymphocytic invaginations after TLI suggests that M cells can differentiate in the absence of, or at least in the presence of very few, lymphocytes, and that invagination by lymphocytes is not necessary to maintain mature M cell morphology

Synergistic effect of DDT and its metabolites in lipopolysaccharide-mediated TNF-α production is inhibited by progesterone in peripheral blood mononuclear cells.

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Dominguez-Lopez, Pablo; Diaz-Cueto, Laura; Aguilar-Rojas, Arturo; Arechavaleta-Velasco, Fabian

2017-07-01

Increased TNF-α levels have been associated with adverse pregnancy outcomes. Lipopolysaccharide (LPS), 1,1,1-trichloro-2,2-bis-(chlorophenyl)ethane (DDT), 1,1-bis-(chlorophenyl)-2,2-dichloroethene (DDE), and 1,1-dichloro-2,2-bis(chlorophenyl)ethane (DDD) induce TNF-α release in peripheral blood mononuclear cells (PBMC). Conversely, progesterone (P4) inhibits TNF-α secretion. Pregnant women in malaria endemic areas may be co-exposure to these compounds. Thus, this study was to investigate the synergistic effect of LPS and these pesticides in PBMC and to assess P4 influence on this synergy. Cultured PBMC were exposed to each pesticide in the presence of LPS, P4, or their combination. TNF-α was measured by ELISA. All pesticides enhanced TNF-α synthesis in PBMC. Co-exposure with LPS synergizes TNF-α production, which is blocked by progesterone. These results indicate that these organochlorines act synergistically with LPS to induce TNF-α secretion in PBMC. This effect is blocked by P4. © 2017 Wiley Periodicals, Inc.

Differences between the genomes of lymphoblastoid cell lines and blood-derived samples

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Joesch-Cohen LM

2017-02-01

Full Text Available Lena M Joesch-Cohen, Gustavo Glusman Institute for Systems Biology, Seattle, WA, USA Abstract: Lymphoblastoid cell lines (LCLs represent a convenient research tool for expanding the amount of biologic material available from an individual. LCLs are commonly used as reference materials, most notably from the Genome in a Bottle Consortium. However, the question remains how faithfully LCL-derived genome assemblies represent the germline genome of the donor individual as compared to the genome assemblies derived from peripheral blood mononuclear cells. We present an in-depth comparison of a large collection of LCL- and peripheral blood mononuclear cell-derived genomes in terms of distributions of coverage and copy number alterations. We found significant differences in the depth of coverage and copy number calls, which may be driven by differential replication timing. Importantly, these copy number changes preferentially affect regions closer to genes and with higher GC content. This suggests that genomic studies based on LCLs may display locus-specific biases, and that conclusions based on analysis of depth of coverage and copy number variation may require further scrutiny. Keywords: genomics, whole-genome sequencing, viral transformation, copy number changes, bioinformatics

Detection of micro RNA hsa-let-7e in peripheral blood mononuclear cells infected with dengue virus serotype-2: preliminary study

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Masyeni, S.; Hadi, U.; Kuntaman; Yohan, B.; Margyaningsih, N. I.; Sasmono, R. T.

2018-03-01

Pathogenesis of dengue infection is still obscure. Recently, the role of microRNA has been associated with the cytokine storm which leads to plasma leakage in endothelial cells. The objective of our study was to determine whether particular microRNA is overexpressed in PBMCs infected with DENV and to assess its correlation to the expression of suppressor of cytokine signaling 3 (SOCS3) proteins to increase the production of pro-inflammatory cytokines. We report the result of a preliminary study on the expression of microRNA hsa-let-7e. The peripheral blood mononuclear cells (PBMCs) from the healthy volunteer were infected with the clinical isolate of DENV-2. RNA was extracted with miRCURYLNATMExiqon. Quantitative Real-Time PCR was used to measure the relative expression of hsa-let-7e micro RNA and the mRNA of SOCS3 proteins. MicroRNA hsa-let-7e expression was increased in PBMCs upon DENV-2 infection. The relative expression of hsa-let-7e is detected at 1.46 folds relative to uninfected PBMCs in 4 hours post-infection and decreased in 19 hours post infection. In contrast, the expression of mRNA of SOCS3 was inversely expressed with hsa-let-7 expression. MicroRNA was overexpressed in PBMCs upon infection with DENV-2. This microRNA may bind the SOCS3 and contribute to the pathogenesis of dengue infection.

Multiple correlation analyses revealed complex relationship between DNA methylation and mRNA expression in human peripheral blood mononuclear cells.

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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

2018-01-01

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.

Menstrual blood closely resembles the uterine immune micro-environment and is clearly distinct from peripheral blood.

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van der Molen, R G; Schutten, J H F; van Cranenbroek, B; ter Meer, M; Donckers, J; Scholten, R R; van der Heijden, O W H; Spaanderman, M E A; Joosten, I

2014-02-01

Is menstrual blood a suitable source of endometrial derived lymphocytes? Mononuclear cells isolated from menstrual samples (menstrual blood mononuclear cells (MMC)) are clearly distinct from peripheral blood mononuclear cells (PBMC) and show a strong resemblance with biopsy-derived endometrial mononuclear cells. A critical event in the onset of pregnancy is the implantation of the embryo in the uterine wall. The immune cell composition in the endometrium at the time of implantation is considered pivotal for success. Despite advancing knowledge on the composition of the immune cell population in the uterus, the role of endometrial immune cells in reproductive disorders is still not fully resolved, mainly due to the fact that this type of research requires invasive techniques. Here, we collected menstrual fluid and validated this unique non-invasive technique to obtain and study the endometrium-derived immune cells which would be present around the time of implantation. Five healthy non-pregnant females with regular menstruation cycles and not using oral contraceptives collected their menstrual blood using a menstrual cup in five consecutive cycles. Sampling took place over the first 3 days of menses, with 12 h intervals. Peripheral blood samples, taken before and after each menstruation, were obtained for comparative analysis. MMC and PBMC samples were characterized for the different lymphocyte subsets by flow cytometry, with emphasis on NK cells and T cells. Next, the functional capacity of the MMC-derived NK cells was determined by measuring intracellular production of IFN-γ, granzyme B and perforin after culture in the presence of IL-2 and IL-15. In support of their endometrial origin, MMC samples contained the typical composition of mononuclear cells expected of endometrial tissue, were phenotypically similar to the reported phenotype for biopsy-derived endometrial cells, and were distinct from PBMC. Increased percentages of NK cells and decreased percentages

Relationship between plasma cholesterol levels and cholesterol esterification in isolated human mononuclear cells

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Dallongeville, J.; Davignon, J.; Lussier-Cacan, S.

1990-01-01

The authors studied the relationship between plasma lipoprotein concentrations and cholesterol esterification in freshly isolated human mononuclear cells from 27 normolipidemic and 32 hyperlipidemic individuals. Cells were either incubated for 5 hours with radiolabeled oleate immediately after isolation or were preincubated for 18 hours in the presence of exogenous cholesterol, and then incubated with [ 14 C]sodium-oleate-albumin complex. In the absence of exogenous cholesterol, control and hypercholesterolemic subjects had similarly low values of intracellular cholesterol esterification. In the presence of exogenous cholesterol, both hypertriglyceridemic and hypercholesterolemic subjects had higher cholesterol esterification than controls. There was a significant correlation between the rate of cholesterol esterification and plasma total cholesterol. These results suggest that plasma cholesterol levels may regulate mononuclear cell intra-cellular cholesterol esterification in humans

A module of human peripheral blood mononuclear cell transcriptional network containing primitive and differentiation markers is related to specific cardiovascular health variables.

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Leni Moldovan

Full Text Available Peripheral blood mononuclear cells (PBMCs, including rare circulating stem and progenitor cells (CSPCs, have important yet poorly understood roles in the maintenance and repair of blood vessels and perfused organs. Our hypothesis was that the identities and functions of CSPCs in cardiovascular health could be ascertained by analyzing the patterns of their co-expressed markers in unselected PBMC samples. Because gene microarrays had failed to detect many stem cell-associated genes, we performed quantitative real-time PCR to measure the expression of 45 primitive and tissue differentiation markers in PBMCs from healthy and hypertensive human subjects. We compared these expression levels to the subjects' demographic and cardiovascular risk factors, including vascular stiffness. The tested marker genes were expressed in all of samples and organized in hierarchical transcriptional network modules, constructed by a bottom-up approach. An index of gene expression in one of these modules (metagene, defined as the average standardized relative copy numbers of 15 pluripotency and cardiovascular differentiation markers, was negatively correlated (all p<0.03 with age (R2 = -0.23, vascular stiffness (R2 = -0.24, and central aortic pressure (R2 = -0.19 and positively correlated with body mass index (R2 = 0.72, in women. The co-expression of three neovascular markers was validated at the single-cell level using mRNA in situ hybridization and immunocytochemistry. The overall gene expression in this cardiovascular module was reduced by 72±22% in the patients compared with controls. However, the compactness of both modules was increased in the patients' samples, which was reflected in reduced dispersion of their nodes' degrees of connectivity, suggesting a more primitive character of the patients' CSPCs. In conclusion, our results show that the relationship between CSPCs and vascular function is encoded in modules of the PBMCs transcriptional

Increased oxidative DNA damage in mononuclear leukocytes in vitiligo

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Giovannelli, Lisa [Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy)]. E-mail: lisag@pharm.unifi.it; Bellandi, Serena [Department of Dermatological Sciences, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy); Pitozzi, Vanessa [Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy); Fabbri, Paolo [Department of Dermatological Sciences, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy); Dolara, Piero [Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy); Moretti, Silvia [Department of Dermatological Sciences, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy)

2004-11-22

Vitiligo is an acquired pigmentary disorder of the skin of unknown aetiology. The autocytotoxic hypothesis suggests that melanocyte impairment could be related to increased oxidative stress. Evidences have been reported that in vitiligo oxidative stress might also be present systemically. We used the comet assay (single cell alkaline gel electrophoresis) to evaluate DNA strand breaks and DNA base oxidation, measured as formamidopyrimidine DNA glycosylase (FPG)-sensitive sites, in peripheral blood cells from patients with active vitiligo and healthy controls. The basal level of oxidative DNA damage in mononuclear leukocytes was increased in vitiligo compared to normal subjects, whereas DNA strand breaks (SBs) were not changed. This alteration was not accompanied by a different capability to respond to in vitro oxidative challenge. No differences in the basal levels of DNA damage in polymorphonuclear leukocytes were found between patients and healthy subjects. Thus, this study supports the hypothesis that in vitiligo a systemic oxidative stress exists, and demonstrates for the first time the presence of oxidative alterations at the nuclear level. The increase in oxidative DNA damage shown in the mononuclear component of peripheral blood leukocytes from vitiligo patients was not particularly severe. However, these findings support an adjuvant role of antioxidant treatment in vitiligo.

Increased oxidative DNA damage in mononuclear leukocytes in vitiligo

International Nuclear Information System (INIS)

Giovannelli, Lisa; Bellandi, Serena; Pitozzi, Vanessa; Fabbri, Paolo; Dolara, Piero; Moretti, Silvia

2004-01-01

Vitiligo is an acquired pigmentary disorder of the skin of unknown aetiology. The autocytotoxic hypothesis suggests that melanocyte impairment could be related to increased oxidative stress. Evidences have been reported that in vitiligo oxidative stress might also be present systemically. We used the comet assay (single cell alkaline gel electrophoresis) to evaluate DNA strand breaks and DNA base oxidation, measured as formamidopyrimidine DNA glycosylase (FPG)-sensitive sites, in peripheral blood cells from patients with active vitiligo and healthy controls. The basal level of oxidative DNA damage in mononuclear leukocytes was increased in vitiligo compared to normal subjects, whereas DNA strand breaks (SBs) were not changed. This alteration was not accompanied by a different capability to respond to in vitro oxidative challenge. No differences in the basal levels of DNA damage in polymorphonuclear leukocytes were found between patients and healthy subjects. Thus, this study supports the hypothesis that in vitiligo a systemic oxidative stress exists, and demonstrates for the first time the presence of oxidative alterations at the nuclear level. The increase in oxidative DNA damage shown in the mononuclear component of peripheral blood leukocytes from vitiligo patients was not particularly severe. However, these findings support an adjuvant role of antioxidant treatment in vitiligo

Tax posttranslational modifications and interaction with calreticulin in MT-2 cells and human peripheral blood mononuclear cells of human T cell lymphotropic virus type-I-associated myelopathy/tropical spastic paraparesis patients.

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Medina, Fernando; Quintremil, Sebastian; Alberti, Carolina; Barriga, Andres; Cartier, Luis; Puente, Javier; Ramírez, Eugenio; Ferreira, Arturo; Tanaka, Yuetsu; Valenzuela, Maria Antonieta

2014-04-01

The human retrovirus human T cell lymphotropic virus type-I (HTLV-1) is the etiologic agent of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Axonal degeneration in HAM/TSP patients occurs without neuron infection, with the secreted viral Tax protein proposed to be involved. We previously found that Tax secreted into the culture medium of MT-2 cells (HTLV-1-infected cell line) produced neurite retraction in neuroblastoma cells differentiated to neuronal type. To assess the relevance of Tax posttranslational modifications on this effect, we addressed the question of whether Tax secreted by MT-2 cells and peripheral blood mononuclear cells (PBMCs) of HTLV-1-infected subjects is modified. The interaction of Tax with calreticulin (CRT) that modulates intracellular Tax localization and secretion has been described. We studied Tax localization and modifications in MT-2 cells and its interaction with CRT. Intracellular Tax in MT-2 cells was assessed by flow cytometry, corresponding mainly to a 71-kDa protein followed by western blot. This protein reported as a chimera with gp21 viral protein-confirmed by mass spectrometry-showed no ubiquitination or SUMOylation. The Tax-CRT interaction was determined by confocal microscopy and coimmunoprecipitation. Extracellular Tax from HAM/TSP PBMCs is ubiquitinated according to western blot, and its interaction with CRT was shown by coimmunoprecipitation. A positive correlation between Tax and CRT secretion was observed in HAM/TSP PBMCs and asymptomatic carriers. For both proteins inhibitors and activators of secretion showed secretion through the endoplasmic reticulum-Golgi complex. Tax, present in PBMC culture medium, produced neurite retraction in differentiated neuroblastoma cells. These results suggest that Tax, whether ubiquitinated or not, is active for neurite retraction.

The comparison of knee osteoarthritis treatment with single-dose bone marrow-derived mononuclear cells vs. hyaluronic acid injections.

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Goncars, Valdis; Jakobsons, Eriks; Blums, Kristaps; Briede, Ieva; Patetko, Liene; Erglis, Kristaps; Erglis, Martins; Kalnberzs, Konstantins; Muiznieks, Indrikis; Erglis, Andrejs

2017-01-01

The aim of this study was to compare treatment methods of the knee joint degenerative osteoarthritis, using autologous bone marrow-derived mononuclear cells and hyaluronic acid injections and observe prevalence of adverse effects in both groups. A prospective randomized controlled clinical trial was carried out. The analysis of pain and changes in osteoarthritis symptoms after a single intra-articular bone marrow-derived mononuclear cell injection into the knee joint in the Kellgren-Lawrence stage II-III osteoarthritis during the 12-month period were performed. The results were compared with the control group treated routinely by hyaluronic acid injections therapy. A therapy group of patients (n=28) received single bone marrow-derived mononuclear cell intra-articular injections. A control group of patients (n=28) was treated with a total of three sodium hyaluronate intra-articular injections each one performed a week apart. The clinical results were obtained using the Knee Osteoarthritis Outcome Score (KOOS) and the Knee Society Score (KSS) before and 3, 6, and 12 months after injection. A statistically significant improvement was observed in the mononuclear cell group over the starting point in all scores. At the endpoint at month 12, the KOOS score improved significantly (Phyaluronic acid versus the bone marrow-derived mononuclear cells group at time points 6 and 12 months demonstrated a statistically significant (Phyaluronic acid group. In both groups serious adverse effects were not observed. The intra-articular injection of bone marrow-derived mononuclear cells is a safe manipulation with no side effects during the 12-month period. This treatment provides statistically significant clinical improvement between the starting point and 1, 3, 6, and 12 months after. When compared to hyaluronic acid treatment, better pain relief in the long-term period of mononuclear cell group was observed. Copyright © 2017 The Lithuanian University of Health Sciences. Production

Human umbilical blood mononuclear cell-derived mesenchymal stem cells serve as interleukin-21 gene delivery vehicles for epithelial ovarian cancer therapy in nude mice.

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Hu, Weihua; Wang, Jing; He, Xiangfeng; Zhang, Hongyi; Yu, Fangliu; Jiang, Longwei; Chen, Dengyu; Chen, Junsong; Dou, Jun

2011-01-01

Ovarian cancer causes more deaths than any other cancer of the female reproductive system, and its overall cure rate remains low. The present study investigated human umbilical blood mononuclear cell (UBMC)-derived mesenchymal stem cells (UBMC-MSCs) as interleukin-21 (IL-21) gene delivery vehicles for ovarian cancer therapy in nude mice. MSCs were isolated from UBMCs and the expanded cells were phenotyped by flow cytometry. Cultured UBMCs were differentiated into osteocytes and adipocytes using appropriate media and then the UBMC-MSCs were transfected with recombinant pIRES2-IL-21-enhancement green fluorescent protein. UBMC-MSCs expressing IL-21 were named as UBMC-MSC-IL-21. Mice with A2780 ovarian cancer were treated with UBMC-MSC-IL-21 intravenously, and the therapeutic efficacy was evaluated by the tumor volume and mouse survival. To address the mechanism of UBMC-MSC-IL-21 against ovarian cancer, the expression of IL-21, natural killer glucoprotein 2 domain and major histocompatibility complex class I chain-related molecules A/B were detected in UBMC-MSC-IL-21 and in the tumor sites. Interferon-γ-secreting splenocyte numbers and natural killer cytotoxicity were significantly increased in the UBMC-MSC-IL-21-treated mice as compared with the UBMC-MSCs or the UBMC-MSC-mock plasmid-treated mice. Most notably, tumor growth was delayed and survival was prolonged in ovarian-cancer-bearing mice treated with UBMC-MSC-IL-21. Our data provide important evidence that UBMC-MSCs can serve as vehicles for IL-21 gene delivery and inhibit the established tumor. Copyright © 2011 International Union of Biochemistry and Molecular Biology, Inc.

INDUCTION OF CYTOKINE PRODUCTION IN CHEETAH (ACINONYX JUBATUS) PERIPHERAL BLOOD MONONUCLEAR CELLS AND VALIDATION OF FELINE-SPECIFIC CYTOKINE ASSAYS FOR ANALYSIS OF CHEETAH SERUM.

Science.gov (United States)

Franklin, Ashley D; Crosier, Adrienne E; Vansandt, Lindsey M; Mattson, Elliot; Xiao, Zhengguo

2015-06-01

Peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood of cheetahs (Acinonyx jubatus ; n=3) and stimulated with lipopolysaccharides (LPS) to induce the production of proinflammatory cytokines TNF-α, IL-1β, and IL-6 for establishment of cross-reactivity between these cheetah cytokines and feline-specific cytokine antibodies provided in commercially available Feline DuoSet® ELISA kits (R&D Systems, Inc., Minneapolis, Minnesota 55413, USA). This study found that feline-specific cytokine antibodies bind specifically to cheetah proinflammatory cytokines TNF-α, IL-1β, and IL-6 from cell culture supernatants. The assays also revealed that cheetah PBMCs produce a measurable, cell concentration-dependent increase in proinflammatory cytokine production after LPS stimulation. To enable the use of these kits, which are designed for cell culture supernatants for analyzing cytokine concentrations in cheetah serum, percent recovery and parallelism of feline cytokine standards in cheetah serum were also evaluated. Cytokine concentrations in cheetah serum were approximated based on the use of domestic cat standards in the absence of cheetah standard material. In all cases (for cytokines TNF-α, IL-1β, and IL-6), percent recovery increased as the serum sample dilution increased, though percent recovery varied between cytokines at a given dilution factor. A 1:2 dilution of serum resulted in approximately 45, 82, and 7% recovery of TNF-α, IL-1β, and IL-6 standards, respectively. Adequate parallelism was observed across a large range of cytokine concentrations for TNF-α and IL-1β; however, a significant departure from parallelism was observed between the IL-6 standard and the serum samples (P=0.004). Therefore, based on our results, the Feline DuoSet ELISA (R&D Systems, Inc.) kits are valid assays for the measurement of TNF-α and IL-1β in cheetah serum but should not be used for accurate measurement of IL-6.

Proteomic Analysis of Peripheral Blood Mononuclear Cells after a High-Fat, High-Carbohydrate Meal with Orange Juice.

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Chaves, Daniela F S; Carvalho, Paulo C; Brasili, Elisa; Rogero, Marcelo M; Hassimotto, Neuza A; Diedrich, Jolene K; Moresco, James J; Yates, John R; Lajolo, Franco M

2017-11-03

Oxidative stress and inflammation play a role in the physiopathology of insulin resistance, diabetes and cardiovascular disease. A single high-fat, high-carbohydrate (HFHC) meal induces an increase in inflammatory and oxidative stress markers in peripheral blood mononuclear cells (PBMC). Previous studies have shown that orange juice is able to prevent this response by inhibiting toll like receptors (TLR) expression and endotoxemia. Our goal was to study the proteome response in PBMC after the consumption of a HFHC meal consumed with water, orange juice or an isocaloric beverage (water with glucose). Twelve healthy individuals completed the protocol in a crossover design, and blood samples were obtained before and 1, 3, and 5 h after consumption. Proteomic profile, glucose, insulin, lipid and cytokines levels were investigated. The glycemic and insulinemic response was higher when the meal was consumed with glucose, while there was no difference in the response between water and orange juice. Proteome analysis in PBMC was carried out using TMT ten-plex. A total of 3813 proteins, originating from 15 662 peptides were identified. Three proteins showed significantly altered expression in the three treatments: apolipoprotein A-II, ceruloplasmin and hemopexin. When the HFHC meal was consumed with water there was an increase in some inflammatory pathways such as the Fc-gamma receptor dependent phagocytosis and the complement cascade, but the immune system as a whole was not significantly altered. However, when the meal was consumed with glucose, the immune system was up regulated. Among the pathways induced after 3 h were those of the adaptive immune system and cytokine signaling. Five hours after the meal, pathways of the complement cascade and classical antibody mediated complement activation were up regulated. When the meal was consumed with orange juice there was an up regulation of proteins involved in signal transduction, DNA replication and cell cycle. The

Effect of soluble factors derived from oral cancer cells on the production of interferon-γ from peripheral blood mononuclear cells following stimulation with OK-432.

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Ohe, Go; Sasai, Akiko; Uchida, Daisuke; Tamatani, Tetsuya; Nagai, Hirokazu; Miyamoto, Youji

2013-08-01

The streptococcal antitumor agent OK-432 is commonly used as an immunopotentiator for immunotherapy in various types of malignant tumors including oral cancer. It has been demonstrated that OK-432 elicits an antitumor effect by stimulating immunocompetent cells, thereby inducing multiple cytokines including interferon (IFN)-γ, interleukin (IL)-2 and IL-12. Serum concentrations of IFN-γ in patients with oral cancer were examined 24 h after administration of OK-432. Serum concentrations of IFN-γ in patients with advanced cancer were significantly lower than those in patients with early cancer. These results suggested that some soluble factors produced by cancer cells may inhibit IFN-γ production with OK-432. Thus, in the present study, an in vitro simulation model was established for the immune status of patients with oral cancer by adding conditioned medium (CM) derived from oral cancer cell lines into a culture of peripheral blood mononuclear cells (PBMCs) derived from a healthy volunteer. We investigated whether soluble factors derived from oral cancer cells affected IFN-γ production from PBMCs following stimulation with OK-432. PBMCs stimulated with OK-432 produced a large amount of IFN-γ; however, both IFN-γ production and cytotoxic activity from PBMCs induced by OK-432 were inhibited by the addition of CM in a dose-dependent manner. In order to examine these inhibitory effects against IFN-γ production, the contribution of inhibitory cytokines such as IL-4, IL-6, IL-10, transforming growth factor-β and vascular endothelial growth factor was investigated. However, neutralization of these inhibitory cytokines did not recover IFN-γ production inhibited by CM. These results indicated that unknown molecules may inhibit IFN-γ production from PBMCs following stimulation with OK-432.

Up-regulation of Slc39A2(Zip2) mRNA in peripheral blood mononuclear cells from patients with pulmonary tuberculosis.

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Tao, Yan-ting; Huang, Qing; Jiang, Ya-li; Wang, Xiao-lei; Sun, Ping; Tian, Yuanyuan; Wu, Hai-liang; Zhang, Min; Meng, Si-bo; Wang, Yu-shu; Sun, Qing; Zhang, Lian-ying

2013-08-01

Zinc is the most common trace mineral after iron in the human body. In organisms, zinc transporters help zinc influx and efflux from cells. A previous study has reported that Zip2 was up-regulated over 27-fold in human monocytic THP-1 cells, when intracellular zinc was depleted by TPEN. Our study found Zip2 was over-expressed in leukocytes of asthmatic infants, especially those in which the serum zinc level was lower than those in healthy infants. Pulmonary tuberculosis (PTB) patients have significantly low serum zinc levels. Here we investigated whether Zip2 level was changed in the patients with PTB. Zip2 mRNA and protein levels in peripheral blood mononuclear cells (PBMC) from PTB (n1=23) and healthy controls (n2=42) were detected by quantitative real-time PCR and western blot, respectively. mRNA expression levels of another four zinc transporters, Zip1, Zip6, Zip8 and ZnT1, were detected by quantitative real-time PCR. Zip2 mRNA level was significantly up-regulated in PTB patients (P=0.001), and Zip8 mRNA level was significantly down-regulated compared with control individuals (Plevels of Zip1, Zip6 and ZnT1 in either group (P>0.05). Zip2 protein expression levels increased in PTB patients compared with control individuals. Our study found that knockdown of ZIP2 with siRNA caused a decrease in Zip2 levels in PBMC of PTB patients, while reducing the expression of INF-γ (Pinitial infection control of the human body, by promoting and maintaining the immune response of adaptive T cells.

Effect of low-dose gamma radiation on HIV replication in human peripheral blood mononuclear cells

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Xu, Y. [British Columbia Centre for Excellence in HIV/AIDS, British Columbia (Canada); Conway, B. [British Columbia Centre for Excellence in HIV/AIDS, British Columbia (Canada)]|[British Columbia Centre for Excellence in HIV/AIDS, British Columbia (Canada). Dept. of Medicine; Montaner, J.S.G. [British Columbia Centre for Excellence in HIV/AIDS, British Columbia (Canada)]|[British Columbia Centre for Excellence in HIV/AIDS, British Columbia (Canada). Dept. of Medicine]|[Canadian HIV Trials Network, Vancouver (Canada); O`Shaughnessy, M.V. [British Columbia Centre for Excellence in HIV/AIDS, British Columbia (Canada)]|[British Columbia Centre for Excellence in HIV/AIDS, British Columbia (Canada). Faculty of Medicine]|[Canadian HIV Trials Network, Vancouver (Canada); Greenstock, C.L. [AECL Research, Chalk River, Ontario (Canada). Radiation Biology and Health Physics Branch

1996-08-01

Recent studies have demonstrated that UV light and x-irradiation enhance human immunodeficiency virus (HIV) gene expression. There are few published data on related effects of {gamma}-radiation. This may be of clinical relevance, as radiotherapy has been used extensively for the treatment of acquired immunodeficiency syndrome associated conditions. We have studied the effects of {gamma}-radiation on HIV replication in mono-nuclear cells (MC). These cells were obtained from five seronegative healthy donors, exposed to 0-200 cGy {gamma}-radiation, stimulated with phytohemagglutinin-P (PHA-P) for 24 h, infected with a laboratory strain of HIV (HTLV-IIIB, multiplicity of infection = 0.001), then carried in culture for 14 days. Overall, when considering p24 antigen levels on days 7 and 11 in cultures established from cells exposed to 50 cGy, the maximal levels were significantly higher than those measured in the parallel control cultures taken as a whole (P < 0.05), with viral replication enhanced as much as 1000-fold in one case. No significant cytotoxicity was observed following exposure to doses up to 50 cGy. The mechanism of the observed effect remains unknown but may relate to direct gene activation and/or free radical generation, leading to such activation. To date, there is no evidence that viral stimulation occurs following therapeutic radiation in a clinical setting. (author).

Effect of low-dose gamma radiation on HIV replication in human peripheral blood mononuclear cells

International Nuclear Information System (INIS)

Xu, Y.; Conway, B.; O'Shaughnessy, M.V.; Greenstock, C.L.

1996-01-01

Recent studies have demonstrated that UV light and x-irradiation enhance human immunodeficiency virus (HIV) gene expression. There are few published data on related effects of γ-radiation. This may be of clinical relevance, as radiotherapy has been used extensively for the treatment of acquired immunodeficiency syndrome associated conditions. We have studied the effects of γ-radiation on HIV replication in mono-nuclear cells (MC). These cells were obtained from five seronegative healthy donors, exposed to 0-200 cGy γ-radiation, stimulated with phytohemagglutinin-P (PHA-P) for 24 h, infected with a laboratory strain of HIV (HTLV-IIIB, multiplicity of infection = 0.001), then carried in culture for 14 days. Overall, when considering p24 antigen levels on days 7 and 11 in cultures established from cells exposed to 50 cGy, the maximal levels were significantly higher than those measured in the parallel control cultures taken as a whole (P < 0.05), with viral replication enhanced as much as 1000-fold in one case. No significant cytotoxicity was observed following exposure to doses up to 50 cGy. The mechanism of the observed effect remains unknown but may relate to direct gene activation and/or free radical generation, leading to such activation. To date, there is no evidence that viral stimulation occurs following therapeutic radiation in a clinical setting. (author)

Rapid Column-Free Enrichment of Mononuclear Cells from Solid Tissues

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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

Transcriptome analysis of the human T lymphocyte cell line Jurkat and human peripheral blood mononuclear cells exposed to deoxynivalenol (DON): New mechanistic insights

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Katika, Madhumohan R. [RIKILT-Institute of Food Safety, Wageningen University and Research Centre, Wageningen (Netherlands); Department of Health Risk Analysis and Toxicology, Maastricht University (Netherlands); Netherlands Toxicogenomics Centre (Netherlands); Hendriksen, Peter J.M. [RIKILT-Institute of Food Safety, Wageningen University and Research Centre, Wageningen (Netherlands); Netherlands Toxicogenomics Centre (Netherlands); Shao, Jia [RIKILT-Institute of Food Safety, Wageningen University and Research Centre, Wageningen (Netherlands); Department of Health Risk Analysis and Toxicology, Maastricht University (Netherlands); Netherlands Toxicogenomics Centre (Netherlands); Loveren, Henk van [Department of Health Risk Analysis and Toxicology, Maastricht University (Netherlands); National Institute for Public Health and the Environment (RIVM), Bilthoven (Netherlands); Netherlands Toxicogenomics Centre (Netherlands); Peijnenburg, Ad, E-mail: ad.peijnenburg@wur.nl [RIKILT-Institute of Food Safety, Wageningen University and Research Centre, Wageningen (Netherlands); Netherlands Toxicogenomics Centre (Netherlands)

2012-10-01

Deoxynivalenol (DON) or vomitoxin is a commonly encountered type-B trichothecene mycotoxin, produced by Fusarium species predominantly found in cereals and grains. DON is known to exert toxic effects on the gastrointestinal, reproductive and neuroendocrine systems, and particularly on the immune system. Depending on dose and exposure time, it can either stimulate or suppress immune function. The main objective of this study was to obtain a deeper insight into DON-induced effects on lymphoid cells. For this, we exposed the human T-lymphocyte cell line Jurkat and human peripheral blood mononuclear cells (PBMCs) to various concentrations of DON for various times and examined gene expression changes by DNA microarray analysis. Jurkat cells were exposed to 0.25 and 0.5 μM DON for 3, 6 and 24 h. Biological interpretation of the microarray data indicated that DON affects various processes in these cells: It upregulates genes involved in ribosome structure and function, RNA/protein synthesis and processing, endoplasmic reticulum (ER) stress, calcium-mediated signaling, mitochondrial function, oxidative stress, the NFAT and NF-κB/TNF-α pathways, T cell activation and apoptosis. The effects of DON on the expression of genes involved in ER stress, NFAT activation and apoptosis were confirmed by qRT-PCR. Other biochemical experiments confirmed that DON activates calcium-dependent proteins such as calcineurin and M-calpain that are known to be involved in T cell activation and apoptosis. Induction of T cell activation was also confirmed by demonstrating that DON activates NFATC1 and induces its translocation from the cytoplasm to the nucleus. For the gene expression profiling of PBMCs, cells were exposed to 2 and 4 μM DON for 6 and 24 h. Comparison of the Jurkat microarray data with those obtained with PBMCs showed that most of the processes affected by DON in the Jurkat cell line were also affected in the PBMCs. -- Highlights: ► The human T cell line Jurkat and human

Density-Gradient Mediated Band Extraction of Leukocytes from Whole Blood Using Centrifugo-Pneumatic Siphon Valving on Centrifugal Microfluidic Discs

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Kearney, Sinéad M.; Kilcawley, Niamh A.; Early, Philip L.; Glynn, Macdara T.; Ducrée, Jens

2016-01-01

Here we present retrieval of Peripheral Blood Mononuclear Cells by density-gradient medium based centrifugation for subsequent analysis of the leukocytes on an integrated microfluidic “Lab-on-a-Disc” cartridge. Isolation of white blood cells constitutes a critical sample preparation step for many bioassays. Centrifugo-pneumatic siphon valves are particularly suited for blood processing as they function without need of surface treatment and are ‘low-pass’, i.e., holding at high centrifugation speeds and opening upon reduction of the spin rate. Both ‘hydrostatically’ and ‘hydrodynamically’ triggered centrifugo-pneumatic siphon valving schemes are presented. Firstly, the geometry of the pneumatic chamber of hydrostatically primed centrifugo-pneumatic siphon valves is optimised to enable smooth and uniform layering of blood on top of the density-gradient medium; this feature proves to be key for efficient Peripheral Blood Mononuclear Cell extraction. A theoretical analysis of hydrostatically primed valves is also presented which determines the optimum priming pressure for the individual valves. Next, ‘dual siphon’ configurations for both hydrostatically and hydrodynamically primed centrifugo-pneumatic siphon valves are introduced; here plasma and Peripheral Blood Mononuclear Cells are extracted through a distinct siphon valve. This work represents a first step towards enabling on disc multi-parameter analysis. Finally, the efficiency of Peripheral Blood Mononuclear Cells extraction in these structures is characterised using a simplified design. A microfluidic mechanism, which we termed phase switching, is identified which affects the efficiency of Peripheral Blood Mononuclear Cell extraction. PMID:27167376

Impact of C-rel inhibition of cord blood-derived B-, T-, and NK cells.

Science.gov (United States)

Fallahi, Shirin; Mohammadi, Seyede Momeneh; Tayefi Nasrabadi, Hamid; Alihemmati, Alireza; Samadi, Naser; Gholami, Sanaz; Shanehbandi, Dariush; Nozad Charoudeh, Hojjatollah

2017-12-01

The c-Rel transcription factor is a unique member of the nuclear factor (NF)-κB family that has a role in curtailing the proliferation, differentiation, cytokine production, and overall activity of B- and T-cells. In addition, c-Rel is a key regulator of apoptosis in that it influences the expression of anti-apoptotic genes such as Bcl-2 and Bcl-xL; conversely, inhibition of c-Rel increases cell apoptosis. To better understand the relationship between c-Rel expression and effects on B- and T-cell expansion, the current study evaluated c-Rel expression in cord blood mononuclear cells. This particular source was selected as cord blood is an important source of cells used for transplantation and immunotherapy, primarily in treating leukemias. As stem cell factor (SCF) and FLT3 are important agents for hematopoietic stem cell expansion, and cytokines like interleukin (IL)-2, -7, and -15 are essential for T- and B- (and also NK) cell development and proliferation, the current study evaluated c-Rel expression in cord blood mononuclear cells and CD34 +  cells, as well as effects on B-, T-, and NK cells associated with alterations in c-Rel expression, using flow cytometry and PCR. The results showed c-Rel expression increased among cells cultured in the presence of SCF and FLT3 but was reduced when IL-2, IL-7, and IL-15 were used all together. Further, inhibition of c-Rel expression by siRNA reduced cord blood-derived B-, T-, and NK cell differentiation and expansion. These results indicated that with cells isolated from cord blood, c-Rel has an important role in B-, T-, and NK cell differentiation and, further, that agents (select cytokines/growth factors) that could impact on its expression might not only affect immune cell profiles in a host but could potentially also limit apoptotic activities in (non-)immune cells in that host. In the context of cancer (immuno)therapy, in particular, when cord blood is used an important source in stem cell transplantation in

Epigenome-wide DNA methylation analysis in siblings and monozygotic twins discordant for sporadic Parkinson's disease revealed different epigenetic patterns in peripheral blood mononuclear cells.

Science.gov (United States)

Kaut, Oliver; Schmitt, Ina; Tost, Jörg; Busato, Florence; Liu, Yi; Hofmann, Per; Witt, Stephanie H; Rietschel, Marcella; Fröhlich, Holger; Wüllner, Ullrich

2017-01-01

Numerous studies have elucidated the genetics of Parkinson's disease; however, the aetiology of the majority of sporadic cases has not yet been resolved. We hypothesized that epigenetic variations could be associated with PD and evaluated the DNA methylation pattern in PD patients compared to brothers or twins without PD. The methylation of DNA from peripheral blood mononuclear cells of 62 discordant siblings including 24 monozygotic twins was characterized with Illumina DNA Methylation 450K bead arrays and subsequently validated in two independent cohorts: 221 PD vs. 227 healthy individuals (cohort 1) applying Illumina's VeraCode and 472 PD patients vs. 487 controls (cohort 2) using pyrosequencing. We choose a delta beta of >15 % and selected 62 differentially methylated CpGs in 51 genes from the discordant siblings. Among them, three displayed multiple CpGs per gene: microRNA 886 (MIR886, 10 CpGs), phosphodiesterase 4D (PDE4D, 2 CpGs) and tripartite motif-containing 34 (TRIM34, 2 CpGs). PDE4D was confirmed in both cohorts (p value 2.44e-05). In addition, for biomarker construction, we used the penalized logistic regression model, resulting in a signature of eight CpGs with an AUC of 0.77. Our findings suggest that a distinct level of PD susceptibility stems from individual, epigenetic modifications of specific genes. We identified a signature of CpGs in blood cells that could separate control from disease with a reasonable discriminatory power, holding promise for future epigenetically based biomarker development.

Analysis of the kinetic of expression of tristetraprolin and HuR by rheumatoid arthritis patients pheripheral blood mononuclear cells stimulated with lipopolysaccharide

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G. Ferraccioli

2011-09-01

Full Text Available Objective. Given the role of TNF-α in Rheumatoid Arthritis (RA we decided to define the characteristics of the TNF- α synthesis by peripheral blood mononuclear cells (PBMNCs obtained from active-aggressive RA patients giving a particular attention to the modulation of the expression of two fundamental proteins in TNF-α mRNA stability regulation, Tristetraprolin (TTP and HuR. Methods. 11 RA patients with active disease were enrolled in the study before their entry in 2 double blind protocols: Infliximab versus MTX and Etanercept versus MTX. 9 healthy blood donors were taken as controls. PBMNCs obtained by Ficoll centrifugation and plastic adherence were stimulated with lipopolysaccharide (LPS and TNF-α was measured in the supernatant during 8 hours by ELISA. At each time point the cells were harvested and analysed for TNF- α, TTP and HuR mRNA expression by semi-quantitative PCR. Results. MNCs TNF-α secretion after LPS stimulation did not differ significantly between RA and control subjects, even if a tendency towards a more prompt response was observed in the patients. More importantly only the DMARDs responsive patients (DAS <3.7 at the 6th month, with a minimal reduction of 1.2 points disclosed precociously (at the first month a significant change in the profile of TNF-α secretion and maintained it until the 6th month. The “normalization” of the synthetic behaviour was accompanied by the resetting in the regulation of the expression of the TTP, that appeared significantly different in the patients before and after therapy. Conclusions. Independently from the type of therapy, responsive patients demonstrate a rapid change in the cellular biology at the systemic level that might drive the resolution of the phlogistic process at the synovial level.

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

Directory of Open Access Journals (Sweden)

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

[Characteristic and function of peripheral blood mononuclear cells-induced macrophages in patients with myelodysplastic syndrome].

Science.gov (United States)

Han, Y; Wang, H Q; Fu, R; Qu, W; Ruan, E B; Wang, X M; Wang, G J; Wu, Y H; Liu, H; Song, J; Guan, J; Xing, L M; Li, L J; Jiang, H J; Liu, H; Wang, Y H; Liu, C Y; Zhang, W; Shao, Z H

2017-08-14

Objective: To explore characteristic and function of peripheral blood mononuclear cells (PBMNC) -induced macrophages in patients with myelodysplastic syndrome (MDS) to couple with its progression. Methods: A total of 24 MDS patients (11 low-risk patients and 13 high-risk group patients) referred to Department of Hematology of Tianjin Medical University General Hospital and normal controls were enrolled from September 2014 to December 2015. PBMNC was stimulated with GM-CSF to transform to macrophages. The morphology of macrophages was observed by microscope. The quantity of macrophages, CD206 and SIRPα on surface of macrophages were detected by flow cytometry. The phagocytic function of macrophages was analyzed by fluorescence microscopy and flow cytometry. Results: The morphology of macrophages from MDS patients was abnormal. The percentage of transformed macrophages was (5.17±3.47) % in patients with MDS, which was lower than that in controls significantly[ (66.18±13.43) %, t =3.529, P =0.001]. The expression of CD206 on macrophages from MDS patients was significantly lower than that of controls[ (9.73±2.59) % vs (51.15±10.82) %, t =4.551, P patients was significantly lower than that of controls [ (0.51±0.09) % vs (0.77±0.06) %, t =2.102, P =0.043]. The phagocytic index and the percentage of phagocytic of macrophages from MDS patients were significantly lower than those of macrophages from normal controls[0.45±0.08 vs 0.92±0.07, t =-6.253, P =0.008; (23.69±3.22) % vs (42.75±2.13) %, t =-6.982, P =0.006 respectively]by flow cytometry. The phagocytic index of MDS patients was significantly lower than that of controls (0.24±0.04 vs 0.48±0.96, t =3.464, P =0.001) by fluorescence microscopy. Conclusion: The quantity, recognization receptors and phagocytosis of PBMNC-induced macrophages decreased in MDS patients.

Changes in some pro-and anti-inflammatory cytokines produced by bovine peripheral blood mononuclear cells following foot and mouth disease vaccination

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N. Delirezh

2016-09-01

Full Text Available Interleukin (IL-17 is exclusively produced by CD4 helper T-cells upon activation. It most often acts as a pro-inflammatory cytokine, which stimulates the release of pro-inflammatory cytokines IL-6, IL-8, TNF-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF. In this study, we studied the in-vitro IL-17 response to specific antigens and a variety of mitogens and compared the IL-17 response to IL-2, IL-4, IL-5, IL-6, IL-10, and IFN-γ responses. We used a foot and mouth disease (FMD vaccine as specific antigens and mitogens (phytohemagglutinin [PHA], pokeweed mitogen [PWM], and concanavalin A [Con A] to stimulate peripheral blood mononuclear cells (PBMCs of vaccinated calves. Cell culture supernatant was harvested and analyzed for cytokines, using commercially available bovine ELISA kits. The mitogens induced a significant increase in IL-17 production. IL-17 was produced at high levels in response to the T cell-stimulated mitogens, PHA, and Con A, and at low levels in response to PWM mitogens. In contrast, level of the produced IL-17 cytokines in response to the FMDV antigens was lower as compared to those produced by mitogens. The FMDV antigens and mitogens significantly increased IL-17 production. There was not a correlation between IL-17 production and type-1 cytokine, IFN-γ, and IL-2, while there was a correlation between type-2 cytokine, IL-4, and IL-5 at either cytokine level produced by PBMCs stimulated by FMDV antigens. Moreover, there was an interaction between IL-17 and IL-6, that is, as IL-6 cytokine level elevated or diminished, IL-17 cytokine level increased or decreased, as well.

In vitro formation of osteoclasts from long-term cultures of bone marrow mononuclear phagocytes

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Burger, E.H.; Van der Meer, J.W.; van de Gevel, J.S.; Gribnau, J.C.; Thesingh, G.W.; van Furth, R.

1982-01-01

The origin of osteoclasts was studied in an in vitro model using organ cultures of periosteum-free embryonic mouse long-bone primordia, which were co-cultured with various cell populations. The bone rudiments were freed of their periosteum-perichondrium by collagenase treatment in a stage before cartilage erosion and osteoclast formation, and co-cultured for 7 d with either embryonic liver or mononuclear phagocytes from various sources. Light and electron microscopic examination of the cultures showed that mineralized matrix-resorbing osteoclasts developed only in bones co-cultured with embryonic liver or with cultured bone marrow mononuclear phagocytes but not when co-cultured with blood monocytes or resident or exudate peritoneal macrophages. Osteoclasts developed from the weakly adherent, but not from the strongly adherent cells of bone marrow cultures, whereas 1,000 rad irradiation destroyed the capacity of such cultures to form osteoclasts. In bone cultures to which no other cells were added, osteoclasts were virtually absent. Bone-resorbing activity of in vitro formed osteoclasts was demonstrated by 45 Ca release studies. These studies demonstrate that osteoclasts develop from cells present in cultures of proliferating mononuclear phagocytes and that, at least in our system, monocytes and macrophages are unable to form osteoclasts. The most likely candidates for osteoclast precursor cells seem to be monoblasts and promonocytes

The comparison of knee osteoarthritis treatment with single-dose bone marrow-derived mononuclear cells vs. hyaluronic acid injections

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Valdis Goncars

2017-01-01

Conclusions: The intra-articular injection of bone marrow-derived mononuclear cells is a safe manipulation with no side effects during the 12-month period. This treatment provides statistically significant clinical improvement between the starting point and 1, 3, 6, and 12 months after. When compared to hyaluronic acid treatment, better pain relief in the long-term period of mononuclear cell group was observed.

Long noncoding RNA expression in peripheral blood mononuclear cells and suicide risk in Chinese patients with major depressive disorder.

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Cui, Xuelian; Niu, Wei; Kong, Lingming; He, Mingjun; Jiang, Kunhong; Chen, Shengdong; Zhong, Aifang; Li, Wanshuai; Lu, Jim; Zhang, Liyi

2017-06-01

WHO stated that nearly one million people commit suicide every year worldly, and 40% of the suicide completer suffered from depression. The primary aim of this study was to explore the association between long noncoding RNAs (lncRNAs) expression in peripheral blood mononuclear cells (PBMCs) and suicide risk of patients with major depressive disorder (MDD). Using Human LncRNA 3.0 microarray profiling which includes 30,586 human lncRNAs and RT-PCR, six down-regulated lncRNAs were identified differentially expressed in MDD patients. According to suicidal ideation and suicidal attempt, the suicide risk of MDD patients was classified into suicidal ideation versus no suicidal ideation groups, and past attempt versus no past attempt groups, respectively. The expression of six lncRNAs in MDD patients and controls were examined by RT-PCR. The expression of six lncRNAs had significant differences between no suicidal ideation, suicidal ideation, and controls; corresponding lncRNAs associated with suicidal attempt had remarkable differences between no past attempt, past attempt, and controls. Additionally, only the expression of lncRNAs in suicidal ideation group and past attempt group markedly declined compared with controls. This study indicated that the expression of six down-regulated lncRNAs had a negative association with suicide risk in MDD patients, and the expression of lncRNAs in PBMCs could have the potential to help clinician judge the suicide risk of MDD patients to provide timely treatment and prevent suicide.

Mycoplasma hyopneumoniae-derived lipid-associated membrane proteins induce inflammation and apoptosis in porcine peripheral blood mononuclear cells in vitro.

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Bai, Fangfang; Ni, Bo; Liu, Maojun; Feng, Zhixin; Xiong, Qiyan; Shao, Guoqing

2015-01-30

Mycoplasma hyopneumoniae is the causative agent of swine enzootic pneumonia (EP), a disease that causes considerable economic losss in swine industry. Lipid-associated membrane proteins (LAMPs) of mycoplasma play important roles in causing mycoplasma diseases. The present study explores the pathogenic mechanisms of M. hyopneumoniae LAMPs by elucidating their role in modulating the inflammation, apoptosis, and relevant signaling pathways of peripheral blood mononuclear cells (PBMCs) of pig. LAMP treatment inhibited the growth of PBMCs. Up-regulation of cytokines, such as IL-6 and IL-1β, as well as increased production of nitric oxide (NO) and superoxide anion were all detected in the supernatant of LAMPs-treated PBMCs. Furthermore, flow cytometric analysis using dual staining with annexin-V-FITC and propidium iodide (PI) showed that LAMPs of M. hyopneumoniae induced a time-dependent apoptosis in lymphocyts and monocytes from PBMCs, which was blocked by NOS inhibitor or antioxidant. In addition, LAMPs induced the phosphorylation of p38, the ratio of pro-apoptotic Bax protein to anti-apoptotic Bcl-2, activation of caspase-3 and caspase-8, and poly ADP-ribose polymerase (PARP) cleavage in PBMCs. These findings demonstrated that M. hyopneumoniae LAMPs induced the production of proinflammatory cytokines, NO and reactive oxygen species (ROS), and apoptosis of PBMCs in vitro through p38 MAPK and Bax/Bcl-2 signaling pathways, as well as caspase activation. Copyright © 2014 Elsevier B.V. All rights reserved.

Suppression of leukocyte inhibitory factor (LIF) production and [3H]thymidine incorporation by concanavalin A-activated mononuclear cells

International Nuclear Information System (INIS)

Lomnitzer, R.; Rabson, A.R.

1979-01-01

The capacity of human mononuclear (MN) cells pretreated with concanavalin A (Con A) to suppress the activity of fresh phytohemagglutinin (PHA)-pulsed mononuclear cells was assessed. Con A-pretreated MN cells suppressed leukocyte inhibitory factor (LIF) activity in supernatants of PHA-pulsed cell cultures and [ 3 H]thymidine incorporation by these cells. Suppression was obtained in both allogeneic and autologous systems with mitomycin-treated, irradiated, or untreated Con A-induced cells. Lymphocytes from two patients that, following treatment with Con A, did not suppress mitogen-induced proliferative response of normal cells also did not suppress LIF production

Arylamine N-acetyltransferase 1 in situ N-acetylation on CD3+ peripheral blood mononuclear cells correlate with NATb mRNA and NAT1 haplotype.

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Salazar-González, Raúl A; Turiján-Espinoza, Eneida; Hein, David W; Niño-Moreno, Perla C; Romano-Moreno, Silvia; Milán-Segovia, Rosa C; Portales-Pérez, Diana P

2018-02-01

Human arylamine N-acetyltransferase 1 (NAT1) is responsible for the activation and elimination of xenobiotic compounds and carcinogens. Genetic polymorphisms in NAT1 modify both drug efficacy and toxicity. Previous studies have suggested a role for NAT1 in the development of several diseases. The aim of the present study was to evaluate NAT1 protein expression and in situ N-acetylation capacity in peripheral blood mononuclear cells (PBMC), as well as their possible associations with the expression of NAT1 transcript and NAT1 genotype. We report NAT1 protein, mRNA levels, and N-acetylation in situ activity for PBMC obtained from healthy donors. NAT1-specific protein expression was higher in CD3+ cells than other major immune cell subtypes (CD19 or CD56 cells). N-acetylation of pABA varied markedly among the PBMC of participants, but correlated very significantly with levels of NAT1 transcripts. NAT1*4 subjects showed significantly (p = 0.017) higher apparent pABA V max of 71.3 ± 3.7 versus the NAT1*14B subjects apparent V max of 58.5 ± 2.5 nmoles Ac-pABA/24 h/million cells. Levels of pABA N-acetylation activity at each concentration of substrate evaluated also significantly correlated with NAT1 mRNA levels for all samples (p N-acetylation in PBMC is higher in T cell than in other immune cell subtypes and that individual variation in N-acetylation capacity is dependent upon NAT1 mRNA and NAT1 haplotype.

Soluble suppressor supernatants elaborated by concanavalin A-activated human mononuclear cells. Characterization of a soluble suppressor of B cell immunoglobulin production

International Nuclear Information System (INIS)

Fleisher, T.A.; Greene, W.C.; Blaese, R.M.; Waldmann, T.A.

1981-01-01

Human peripheral blood mononuclear cells (PBMC) activated with the mitogenic lectin concanavalin A (Con A) elaborate a soluble immune suppressor supernatant (SISS) that contains at least 2 distinct suppressor factors. One of these, SISS-B, inhibits polyclonal B cell immunoglobulin production, whereas the other, SISS-T, suppresses T cell proliferation to both mitogens and antigens. The latter mediator is discussed in the companion paper. Characteristics of the human soluble suppressor of B cell immunoglobulin production (SISS-B) include: 1) inhibition by a noncytotoxic mechanism, 2) loss of activity in the presence of the monosaccharide L-rhamnose, 3) appearance within 8 to 16 hr after the addition of Con A, 4) elaboration by cells irradiated with 500 or 2000 rads, 5) production by highly purified T cells, 6) stability at pH 2.5 but instability at 56/sup o/C, and 7) m.w. of 60 to 80,000. These data indicate that after Con A activation, selected T cells not only become potent suppressor cells, but also generate a soluble saccharide-specific factor(s) that inhibits polyclonal immunoglobulin production by human B cells

Expression of human immunodeficiency virus (HIV) in naturally infected peripheral blood mononuclear cells: comparison of a standard co-culture technique with a newly developed microculture method.

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Eberlein, B; Baur, A; Neundorfer, M; Jahn, G

1991-05-01

Peripheral blood mononuclear cells (PBMCs) from 29 patients infected with human immunodeficiency virus (HIV) were cultured by two different methods. One was the standard co-culture technique, the other a newly developed microculture method. In this assay 10(6) PBMCs were cultivated in 250 microliters medium, no activating agents or allogeneic cells were present. P24 antigen production measured by this method was found in 7 out of 11 PBMC cultures of patients in the Walter Reed (WR) stage 1 or 2, whereas only 4 samples were positive by the co-culture procedure. Cultures from patients in the later stages of the disease (WR 5/6) showed a higher p24 production by the co-culture method than by the microculture assay. It is assumed that rapidly growing HIV strains can be better assessed by the co-culture method which may select for these strains. P24 expression can be more easily obtained by the microculture technique even in cases where slowly replicating strains may be present. In conclusion, results from the microculture procedure described may be a useful supplementation to findings observed by the co-culture method.

Clues to pathogenesis of spondyloarthropathy derived from synovial fluid mononuclear cell gene expression profiles

NARCIS (Netherlands)

Gu, Jieruo; Rihl, Markus; Märker-Hermann, Elisabeth; Baeten, Dominique; Kuipers, Jens G.; Song, Yeong Wook; Maksymowych, Walter P.; Burgos-Vargas, Ruben; Veys, Eric M.; de Keyser, Filip; Deister, Helmuth; Xiong, Momiao; Huang, Feng; Tsai, Wen Chan; Yu, David Tak Yan

2002-01-01

OBJECTIVE: To use gene expression profiles of spondyloarthropathy (SpA) synovial fluid mononuclear cells (SFMC) to determine if there are transcripts that support the unfolded protein response (UPR) hypothesis, and to identify which cytokines/chemokines are being expressed and which cell fractions

The repopulation of lymph nodes of dogs after 1200 R whole-body x-irradiation and intravenous administration of mononuclear blood leukocytes.

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Nelson, B; Calvo, W; Fliedner, T M; Herbst, E; Bruch, C; Schnappauf, H P; Flad, H D

1976-08-01

Fresh and cryopreserved autologous or allogeneic mononuclear blood cells (MBCs) intravenously injected in 1200 R total-body x-irradiated dogs repopulated lymph nodes within 10 days after tranfusion. Several parameters of the lymphopoietic regeneration were correlated with the number of cells transfused and with the number of colony-forming units contained in the cell suspension when they were cultured in agar (CFUc). Values within the normal or close to normal range were reached in the mesenteric nodes of most of the animals transfused with 10 X 10(9) MBC or more. These values were obtained when 5 X 10(5) CFUc or more were transfused. Axillary nodes showed lower values than mesenteric nodes. They were mostly under the normal range but well over those of the irradiated controls. Frozen and thawed MBCs seem to be as effective as fresh cells for lymphopoietic restoration. The mesenteric nodes of dogs transfused with allogeneic MBCs showed higher cellularity and larger cortical-paracortical areas than those of dogs tranfused with approximately the same number of autologous cells. The repopulation of lymph nodes parallels that of the marrow.

Distribution of Curcumin and THC in Peripheral Blood Mononuclear Cells Isolated from Healthy Individuals and Patients with Chronic Lymphocytic Leukemia.

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Bolger, Gordon T; Licollari, Albert; Tan, Aimin; Greil, Richard; Pleyer, Lisa; Vcelar, Brigitta; Majeed, Muhammad; Sordillo, Peter

2018-01-01

Background/Aim: Curcumin is being widely investigated for its anticancer properties and studies in the literature suggest that curcumin distributes to a higher degree in tumor versus non-tumor cells. In the current study, we report on investigation of the distribution of curcumin and metabolism to THC in PBMC from healthy individuals and chronic lymphocytic leukemia (CLL) patients following exposure to Lipocurc™ (liposomal curcumin). Materials and Methods: The time and temperature-dependent distribution of liposomal curcumin and metabolism to tetrahydrocurcumin (THC) were measured in vitro in human peripheral blood mononuclear cells (PBMC) obtained from healthy individuals, PBMC HI (cryopreserved and freshly isolated PBMC) and CLL patients (cryopreserved PBMC) with lymphocyte counts ranging from 17-58×10 6 cells/ml (PBMC CLL,Grp 1 ) and >150×10 6 cells/ml (PBMC CLL,Grp 2 ). PBMC were incubated in plasma protein supplemented media with Lipocurc™ for 2-16 min at 37°C and 4°C and the cell and medium levels of curcumin determined by LC-MS/MS. Results: PBMC from CLL patients displayed a 2.2-2.6-fold higher distribution of curcumin compared to PBMC HI Curcumin distribution into PBMCCLL, Grp 1/Grp 2 ranged from 384.75 - 574.50 ng/g w.w. of cell pellet and was greater compared to PBMC HI that ranged from 122.27-220.59 ng/g w.w. of cell pellet following incubation for up to 15-16 min at 37°C. The distribution of curcumin into PBMC CLL,Grp 2 was time-dependent in comparison to PBMC HI which did not display a time-dependence and there was no temperature-dependence for curcumin distribution in either cell type. Curcumin was metabolized to THC in PBMC. The metabolism of curcumin to THC was not markedly different between PBMC HI (range=23.94-42.04 ng/g w.w. cell pellet) and PBMC CLL,Grp 1/Grp 2 (range=23.08-48.22 ng/g. w.w. cell pellet). However, a significantly greater time and temperature-dependence was noted for THC in PBMC CLL,Grp 2 compared to PBMC HI Conclusion

Impairment of T-regulatory cells in cord blood of atopic mothers.

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Schaub, Bianca; Liu, Jing; Höppler, Sabine; Haug, Severine; Sattler, Christine; Lluis, Anna; Illi, Sabina; von Mutius, Erika

2008-06-01

Maternal atopy is a strong predictor for the development of childhood allergic diseases. The underlying mechanisms are ill defined, yet regulatory T (Treg) and T(H)17 cells may play a key role potentially shaping the early immune system toward a proallergic or antiallergic immune regulation. We examined T(H)1/T(H)2, Treg, and T(H)17 cell responses to innate (lipid A/peptidoglycan) and mitogen/adaptive (phytohemagglutinin/Dermatophagoides pteronyssinus 1) immune stimulation in cord blood from offspring of atopic/nonatopic mothers. Cord blood mononuclear cells from 161 healthy neonates (59% nonatopic, 41% atopic mothers) were investigated regarding Treg and T(H)17 cells (mRNA/surface markers), suppressive function, and proliferation/cytokine secretion. Cord blood from offspring of atopic mothers showed fewer innate-induced Treg cells (CD4(+)CD25(+)high), lower mRNA expression of associated markers (glucocorticoid-induced tumor necrosis factor receptor-related protein/lymphocyte activation gene 3; P cell function was impaired in mitogen-induced suppression of T effector cells in cord blood of offspring from atopic mothers (P = .03). Furthermore, IL-10 and IFN-gamma secretion were decreased in innate-stimulated cord blood of offspring from atopic mothers (P = .04/.05). Innate-induced IL-17 was independent of maternal atopy and highly correlated with IL-13 secretion. In offspring of atopic mothers, Treg cell numbers, expression, and function were impaired at birth. T(H)17 cells were correlated with T(H)2 cells, independently of maternal atopy.

Shorter telomeres in peripheral blood mononuclear cells from older persons with sarcopenia: results from an exploratory study

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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.

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.

Phenotypic characterization of mononuclear inflammatory cells following equine hydroxyapatite/collagen block grafting in rats

International Nuclear Information System (INIS)

Alsuwaiyan, Asim; Wang, Bing-Yan; Cohen, Robert E

2012-01-01

To measure the inflammatory changes associated with the implantation of an equine hydroxyapatite and collagen-containing block graft (eHAC block) in a rodent model system, an eHAC block graft was implanted subcutaneously in rats. Control groups included saline, turpentine oil, and human mineralized particulate allograft (hMPA). Animals were sacrificed and tissue samples obtained after three days, as well as after 1, 2, 4 and 8 weeks. A panel of immunologic probes was used to identify circulatory monocytic cells (ED1), resident mononuclear phagocytes (ED2), mononuclear phagocytes of lymphoid origin (ED3), expression of Ia antigen (OX6), T-cells (OX19), and B-cells (OX33). Immunocytochemical localization was performed and mononuclear cells localized with each immunologic probe counted. Rat sera obtained after eight weeks were used for nitrocellulose dot-blotting to assess circulating anti-equine immunoglobulins. Statistical analysis was performed using two-way analysis of variance, in conjunction with the Bonferroni correction to account for multiple comparisons. A transient increase in monocytes at 3 days and 1 week was observed in all groups, but was significantly higher in the turpentine control (P < 0.0001). A significant increase in the numbers of mononuclear cells detected with clones ED2 and ED3 was observed in specimens from the turpentine group, in contrast to the other groups in the 3 day to 4 week interval (P < 0.0001), as well as within all time periods (P < 0.0001). A statistically significant difference in numbers of ED3-positive cells was observed in the hMPA group compared to the saline and the eHAC block groups after one week (P < 0.0001). Significantly more OX6-positive cells were observed in the turpentine group, compared to other groups (3 days to 1 week; P < 0.0001). T-lymphocytes were essentially absent except for rats given turpentine (after 1 week). No B-lymphocyte response was found and none of the rats developed systemic anti

Decreased A20 mRNA and protein expression in peripheral blood mononuclear cells in patients with type 2 diabetes and latent autoimmune diabetes in adults.

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Cheng, Liqing; Zhang, Dongmei; Jiang, Youzhao; Deng, Wuquan; Wu, Qi'nan; Jiang, Xiaoyan; Chen, Bing

2014-12-01

A20 is a negative regulator of nuclear factor kappa B activation and the central gatekeeper in inflammation and immunity. While its role in type 1 diabetes has been widely studied, its expression level in immune cells from type 2 diabetes (T2D) and latent autoimmune diabetes in adult (LADA) patients remains unclear. This study aimed to clarify whether the expression of A20 is altered in patients with T2D or LADA. Quantitative real-time polymerase chain reaction and western blotting were utilized to determine the expression of A20 mRNA and protein respectively in peripheral blood mononuclear cells (PBMCs) from patients with T2D (n=36) or LADA (n=17) and sex- and age-matched healthy controls (n=34). The mRNA and protein expression of A20 in PBMCs from T2D and LADA patients was significantly decreased compared with healthy controls (P1 year since diagnosis) (P<0.05). Our results suggest that decreased expression of A20 in PBMCs may be involved in the pathogenesis of diabetes, and targeting A20 may offer a potential therapeutic tool in the treatment of diabetes. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Interleukin-6 as an endogenous pyrogen: induction of prostaglandin E2 in brain but not in peripheral blood mononuclear cells.

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Dinarello, C A; Cannon, J G; Mancilla, J; Bishai, I; Lees, J; Coceani, F

1991-10-25

Fever induced by endogenous as well as exogenous pyrogens is often prevented by cyclooxygenase inhibitors; endogenous pyrogens stimulate prostaglandin E2 (PGE2) in or near the thermoregulatory centers of the brain. The cytokines, interleukin-1 (IL-1) and tumor necrosis factor (TNF), are two pyrogens which stimulate brain PGE2 formation during fever and also increase PGE2 synthesis in human mononuclear cells in vitro. In the present study, we examined whether interleukin-6 (IL-6) stimulates PGE2 formation in a manner similar to IL-1 and TNF. Both glycosylated and non-glycosylated forms of recombinant human IL-6 were tested. Following intravenous injection into rabbits, the glycosylated IL-6 was more pyrogenic than the non-glycosylated form and there was no evidence of synergy in the production of fever when IL-6 and IL-1 were given simultaneously. IL-6 fever was blocked by prior administration of the cyclooxygenase inhibitor ibuprofen. IL-6 was also pyrogenic in the cat by either the systemic or the intraventricular route. However, in both species, IL-6 was less effective than IL-1 beta. When given intraventricularly to cats, IL-6 produced an increase in PGE2 levels of the cerebrospinal fluid in parallel with the rise in body temperature. In the latter respect, IL-6 imitated IL-1 beta; however, IL-6 from 0.15-15 micrograms/ml did not increase mononuclear cell PGE2 production in vitro whereas IL-1 beta induced 20-30-fold increases in PGE2 at 100 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Diagnostic values of microRNA-31 in peripheral blood mononuclear cells for pediatric pulmonary tuberculosis in Chinese patients.

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Wang, J X; Xu, J; Han, Y F; Zhu, Y B; Zhang, W J

2015-12-17

We investigated the diagnostic values of microRNA-31 in peripheral blood mononuclear cells (PBMCs) for pediatric pulmonary tuberculosis in Chinese patients. Sixty-five children with TB were selected for this study, which was conducted at the Department of Infectious Diseases People's Hospital of Laiwu City between December 2013 and December 2014. Sixty healthy children, selected in parallel, served as the control group. Real-time PCR was used to detect miR-31 expression in PBMCs. Serum levels of IL-6, TNF-α, NF-κB, and IFN-γ was detected by ELISA. ROC curve was employed to evaluate the diagnostic value of miR-31 in pediatric TB. Results show that expression of miRNA-31 in pediatric TB patients was significantly lower than that in normal children (0.48 ± 0.15 vs 1.23 ± 0.36, P < 0.05). By contrast, serum levels of the innate immune response cytokines, IL-6, TNF-α, NF-κB, and IFN-γ, were significantly higher in pediatric TB patients compared with normal children (P < 0.05). Furthermore, miRNA-31 expression was negatively correlated with serum levels of IL-6 (t = 69.91, P < 0.001), TNF-α (t = 10.96, P < 0.001), NF-κB (t = 39.94, P < 0.001), and IFN -γ (t = 37.94, P < 0.001). The cut-off threshold of miR-31 for pediatric TB diagnosis is 0.835 with a sensitivity of 98.5% and a specificity of 86.7%. Therefore, miR-31 has the potential to be a diagnostic marker in pediatric TB patients.

Repopulation of lymph nodes of dogs after 1200 R whole-body x-irradiation and intravenous administration of mononuclear blood leukocytes

International Nuclear Information System (INIS)

Nelson, B.; Calvo, W.; Fliedner, T.M.; Herbst, E.; Bruch, C.; Schnappauf, H.P.; Flad, H.D.

1976-01-01

Fresh and cryopreserved autologous or allogeneic mononuclear blood cells (MBCs) intravenously injected in 1200 R total-body x-irradiated dogs repopulated lymph nodes within 10 days after transfusion. Several parameters of the lymphopoietic regeneration were correlated with the number of cells transfused and with the number of colony-forming units contained in the cell suspension when they were cultured in agar (CFU/sub c/). Values within the normal or close to normal range were reached in the mesenteric nodes of most of the animals transfused with 10 x 10 9 MBC or more. These values were obtained when 5 x 10 5 CFU/sub c/ or more were transfused. Axillary nodes showed lower values than mesenteric nodes. They were mostly under the normal range but well over those of the irradiated controls. Frozen and thawed MBCs seem to be as effective as fresh cells for lymphopoietic restoration. The mesenteric nodes of dogs transfused with allogeneic MBCs showed higher cellularity and larger cortical-paracortical areas than those of dogs transfused with approximately the same number of autologous cells. The repopulation of lymph nodes parallels that of the marrow

Global levels of histone modifications in peripheral blood mononuclear cells of subjects with exposure to nickel.

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Arita, Adriana; Niu, Jingping; Qu, Qingshan; Zhao, Najuan; Ruan, Ye; Nadas, Arthur; Chervona, Yana; Wu, Fen; Sun, Hong; Hayes, Richard B; Costa, Max

2012-02-01

Occupational exposure to nickel (Ni) is associated with an increased risk for lung and nasal cancers. Ni compounds exhibit weak mutagenic activity, cause gene amplification, and disrupt cellular epigenetic homeostasis. However, the Ni-induced changes in global histone modification levels have only been tested in vitro. This study was conducted in a Chinese population to determine whether occupational exposure to Ni is associated with alterations of global histone modification levels and to evaluate the inter- and intraindividual variance of global histone modification levels. Forty-five subjects with occupational exposure to Ni and 75 referents were recruited. Urinary Ni and global H3K4 trimethylation, H3K9 acetylation, and H3K9 dimethylation levels were measured in peripheral blood mononuclear cells (PBMCs) of subjects. H3K4me3 was elevated in Ni-exposed subjects (0.25% ± 0.11%) compared with referents (0.15% ± 0.04%; p = 0.0004), and H3K9me2 was decreased (Ni-exposed subjects, 0.11% ± 0.05%; referents, 0.15% ± 0.04%; p = 0.003). H3K4me3 was positively (r = 0.4, p = 0.0008) and H3K9ac was negatively (r = 0.1, p = 0.01) associated with urinary Ni. Interindividual variances of H3K4me3, H3K9ac, and H3K9me2 were larger compared with intraindividual variance in both exposure test groups, resulting in reliability coefficients (an estimate of consistency of a set of measurements) of 0.60, 0.67, and 0.79 for H3K4me3, H3K9ac, and H3K9me2, respectively, for Ni-exposed subjects and of 0.75, 0.74, and 0.97, respectively, for referent subjects. The results of this study indicate that occupational exposure to Ni is associated with alterations of global histone modification levels and that measurements of global levels of histone modifications are relatively stable over time in human PBMCs.

Relationship of glucocorticoid receptor expression in peripheral blood mononuclear cells and the cochlea of guinea pigs and effects of dexamethasone administration.

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Ling Lu

Full Text Available BACKGROUND: Glucocorticoids (GCs are widely used to treat sudden sensorineural hearing loss (SSNHL and significantly improve hearing. However, GC insensitivity has been observed in some patients of SSNHL. OBJECTIVE: To study the correlation between GR expression in peripheral blood mononuclear cells (PBMCs and in the cochlea of guinea pigs at mRNA and protein levels. METHODS: One group of guinea pigs received dexamethasone (10 mg/kg/day intraperitoneally for 7 consecutive days (dexamethasone group, and another group of guinea pigs received normal saline (control group. Real time PCR and Western blotting were used to detect the expression of GR mRNA and GR protein in PBMCs and the cochleae. RESULTS: The GR mRNA and GR protein were detected in both PBMCs and the cochlear tissue of guinea pigs. GR mRNA and GR protein levels in PBMCs were positively correlated with those in the cochlea. The expression of GR mRNA and GR protein was significantly increased in the dexamethasone group compared to the control group. CONCLUSIONS: Levels of GR mRNA and GR protein in the PBMCs were positively correlated with those in the cochlea of guinea pigs. Systemic dexamethasone treatment can significantly up-regulate GR expression in PBMCs and in the cochlea. Measurement of the GR level in PBMCs could be used as an indicator of GR level in the cochlea.

Elevated interleukin-1β in peripheral blood mononuclear cells contributes to the pathogenesis of autoimmune thyroid diseases, especially of Hashimoto thyroiditis.

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Sun, Li; Zhang, Xiaoxu; Dai, Fang; Shen, Jijia; Ren, Cuiping; Zuo, Chunlin; Zhang, Qiu

2016-08-01

To explore the relationship between IL-1β expression and two common autoimmune thyroid diseases: Hashimoto thyroiditis (HT) and Graves' disease (GD). qRT-PCR, Quantiglo ELISA, and flow cytometry were used to evaluate the expression levels of IL-1β in serum, peripheral blood mononuclear cells (PBMCs), and thyroid tissue samples from patients with HT or GD. Local infiltration of monocytes was assessed by immunohistochemical study of patients' thyroid tissue samples. Although no significant differences in IL-1β levels were found between samples of serum from patients with HT or GD and normal controls, we found that IL-1β mRNA and protein levels in PBMCs of HT patients were significantly higher than those of patients with GD, which were in turn higher than the level in normal controls. In addition, IL-1β mRNA was also increased in thyroid gland tissue from patients with HT compared to those with GD, and this was accompanied by increased local infiltration of monocytes into thyroid tissues. Correlation analysis of the clinical samples validated the association of high IL-1β levels with the pathogenesis of HT. Our study suggests that IL-1β may be an active etiologic factor in the pathogenesis of HT and thus present a new target for novel diagnostics and treatment.

Divergence of canonical danger signals: The genome-level expression patterns of human mononuclear cells subjected to heat shock or lipopolysaccharide

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Sakthivel Bhuvaneswari

2008-05-01

Full Text Available Abstract Background Peripheral blood mononuclear cells (PBMC serve a sentinel role allowing the host to efficiently sense and adapt to the presence of danger signals. Herein we have directly compared the genome-level expression patterns (microarray of a human PBMC model (THP-1 cells subjected to one of two canonical danger signals, heat shock or lipopolysaccharide (LPS. Results and Discussion Based on sequential expression and statistical filters, and in comparison to control cells, we found that 3,988 genes were differentially regulated in THP-1 cells subjected to LPS stress, and 2,921 genes were differentially regulated in THP-1 cells subjected to heat shock stress. Venn analyses demonstrated that the majority of differentially regulated genes (≥ 70% were uniquely expressed in response to one of the two danger signals. Functional analyses demonstrated that the two danger signals induced expression or repression of genes corresponding to unique pathways, molecular functions, biological processes, and gene networks. In contrast, there were 184 genes that were commonly upregulated by both stress signals, and 430 genes that were commonly downregulated by both stress signals. Interestingly, the 184 commonly upregulated genes corresponded to a gene network broadly related to inflammation, and more specifically to chemokine signaling. Conclusion These data demonstrate that the mononuclear cell responses to the canonical stress signals, heat shock and LPS, are highly divergent. However, there is a heretofore unrecognized common pattern of gene network expression corresponding to chemokine-related biology. The data also serve as a reference database for investigators in the field of stress signaling.

Cytotoxicity of Betel leaf (Piper betel L. against primary culture of chicken embryo fibroblast and its effects on the production of proinflammatory cytokines by human peripheral blood mononuclear cells

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Suprapto Ma’at

2012-06-01

Full Text Available Background: Betel leaf (Piper betel L. has been used in modern and traditional medicine as antiseptic, antibacterial, and also prevention of plaque accumulation, but it still can stimulate cancer in lime-piper betel quid. Betel leaf also has anti-inflammatory properties. Purpose: The purpose of this study was examine the cytotoxicity of Betel leaf extract (BLE against primary culture of chicken embryo fibroblast and its effects on the production of proinflammatory cytokines by peripheral blood mononuclear cells (PBMC stimulated with LPS. Methods: MTT assay was used to investigate the survival rate of the culture with the survival rate result of the given culture extract 4%, 2% and 1% about 82%, 83.4% and 85%. There was no significant difference between treatment with various concentrations of the extract and the control (p>0.05. To evaluate the effect of Betel leaf extracts on the production of cytokines, proinflammatory was conducted by incubating the extracts of betel leaf with peripheral blood mononuclear cells stimulated with lipopolysaccharide. Peripheral blood mononuclear cells were obtained from healthy volunteers isolated by density centrifugation method using Ficoll-Hypaque. Once coupled with various concentrations of betel leaf extract and lipopolysaccharide, and then incubated for 24 hours, the culture supernatant was used to determine the level of IFN-γ and TNF-α by ELISA method. Results: It is known that the survival rates of BLE 4%, 2% and 1% were 82%, 83.4% and 85%. There was no significant of difference between several concentrations of BLE and those in the control group (p>0.05. The production of IFN-γ and TNF-α stimulated with LPS was no significant difference between BLE 4%, 2% and 1% and that in the control group (p>0.05. Conclusion: It can be concluded that BLE is not toxic against primary culture of chicken embryo fibroblast, and the production of IFN-γ and TNF-α by PBMC was not affected by BLE.Latar belakang: Daun

Enrichment of circulating tumor cells from a large blood volume using leukapheresis and elutriation: proof of concept.

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Eifler, Robert L; Lind, Judith; Falkenhagen, Dieter; Weber, Viktoria; Fischer, Michael B; Zeillinger, Robert

2011-03-01

The aim of this study was to determine the applicability of a sequential process using leukapheresis, elutriation, and fluorescence-activated cell sorting (FACS) to enrich and isolate circulating tumor cells from a large blood volume to allow further molecular analysis. Mononuclear cells were collected from 10 L of blood by leukapheresis, to which carboxyfluorescein succinimidyl ester prelabeled CaOV-3 tumor cells were spiked at a ratio of 26 to 10⁶ leukocytes. Elutriation separated the spiked leukapheresates primarily by cell size into distinct fractions, and leukocytes and tumor cells, characterized as carboxyfluorescein succinimidyl ester positive, EpCAM positive and CD45 negative events, were quantified by flow cytometry. Tumor cells were isolated from the last fraction using FACS or anti-EpCAM coupled immunomagnetic beads, and their recovery and purity determined by fluorescent microscopy and real-time PCR. Leukapheresis collected 13.5 x 10⁹ mononuclear cells with 87% efficiency. In total, 53 to 78% of spiked tumor cells were pre-enriched in the last elutriation fraction among 1.6 x 10⁹ monocytes. Flow cytometry predicted a circulating tumor cell purity of ~90% giving an enrichment of 100,000-fold following leukapheresis, elutriation, and FACS, where CaOV-3 cells were identified as EpCAM positive and CD45 negative events. FACS confirmed this purity. Alternatively, immunomagnetic bead adsorption recovered 10% of tumor cells with a median purity of 3.5%. This proof of concept study demonstrated that elutriation and FACS following leukapheresis are able to enrich and isolate tumor cells from a large blood volume for molecular characterization. Copyright © 2010 International Clinical Cytometry Society.

Expression of TNF, IL-17A, IL-4 and IL-10 cytokines in irradiated peripheral blood mononuclear cells 'In vitro'

International Nuclear Information System (INIS)

Amaral, Ademir de Jesus; Leite, Lidía Lúcia Bezerra; Nascimento, Ayala Gomes do; Diniz, Ewerton Clementino; Silva, Gicielne Freitas da; Fernandes, Thiago de Salazar e; Silva, Edvane Borges da; Cavalcanti, Mariana Brayner; Veras, Robson Cavalcante; Medeiros, Isac Almeida de

2017-01-01

The aim of the present study was to determine and to compare the profile of cytokines produced by non-irradiated and irradiated peripheral blood mononuclear cells (PBMCs) and the possible application of this analysis as a biomarker of individual radiosensitivity. For this, peripheral blood (PB) samples were collected from seven healthy volunteers, and each sample divided in two aliquots: one aliquot was irradiated with a dose of 2 Gy (from a 6MV Linear Accelerator) and while the other one was kept non irradiated. All PBMCs were cultured in RPMI 1640 supplemented with 10% Bovine Fetal Serum for 48 hours at 37°C and 5% CO2. The cytokines TNF, IL-17A, IL-4 and IL-10 were measured by flow cytometry. Wilcoxon test was performed with the level of significance of 95%. In the irradiated samples it was observed a slight increase of the median of the level of cytokines TNF, IL-4 and IL-10 (from 1040.9 to 1196.1 pg/mL, from 127.3 to 138 pg/mL, and from 99.9 to 120.8 pg/mL, respectively) and a slight decrease in median of cytokines IL- 17A (from 841.1 to 799.4 pg/mL). In addition to this evidence, there was a high inter-individual variability of cytokine concentrations in response to irradiation. It was observed that some individuals are more responsive to the expression of some inflammatory proteins after exposure to X-rays. Although further studies are necessary, the hypothesis that raises is that these biomarkers could be predictor of future individual responses to ionizing radiation exposure. (author)

Bystander apoptosis in human cells mediated by irradiated blood plasma

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Vinnikov, Volodymyr, E-mail: vlad.vinnikov@mail.ru [Grigoriev Institute for Medical Radiology of the National Academy of Medical Science of Ukraine (Ukraine); Lloyd, David; Finnon, Paul [Centre for Radiation, Chemical and Environmental Hazards of the Health Protection Agency of the United Kingdom (United Kingdom)

2012-03-01

Following exposure to high doses of ionizing radiation, due to an accident or during radiotherapy, bystander signalling poses a potential hazard to unirradiated cells and tissues. This process can be mediated by factors circulating in blood plasma. Thus, we assessed the ability of plasma taken from in vitro irradiated human blood to produce a direct cytotoxic effect, by inducing apoptosis in primary human peripheral blood mononuclear cells (PBM), which mainly comprised G{sub 0}-stage lymphocytes. Plasma was collected from healthy donors' blood irradiated in vitro to 0-40 Gy acute {gamma}-rays. Reporter PBM were separated from unirradiated blood with Histopaque and held in medium with the test plasma for 24 h at 37 Degree-Sign C. Additionally, plasma from in vitro irradiated and unirradiated blood was tested against PBM collected from blood given 4 Gy. Apoptosis in reporter PBM was measured by the Annexin V test using flow cytometry. Plasma collected from unirradiated and irradiated blood did not produce any apoptotic response above the control level in unirradiated reporter PBM. Surprisingly, plasma from irradiated blood caused a dose-dependent reduction of apoptosis in irradiated reporter PBM. The yields of radiation-induced cell death in irradiated reporter PBM (after subtracting the respective values in unirradiated reporter PBM) were 22.2 {+-} 1.8% in plasma-free cultures, 21.6 {+-} 1.1% in cultures treated with plasma from unirradiated blood, 20.2 {+-} 1.4% in cultures with plasma from blood given 2-4 Gy and 16.7 {+-} 3.2% in cultures with plasma from blood given 6-10 Gy. These results suggested that irradiated blood plasma did not cause a radiation-induced bystander cell-killing effect. Instead, a reduction of apoptosis in irradiated reporter cells cultured with irradiated blood plasma has implications concerning oncogenic risk from mutated cells surviving after high dose in vivo irradiation (e.g. radiotherapy) and requires further study.

Bystander apoptosis in human cells mediated by irradiated blood plasma

International Nuclear Information System (INIS)

Vinnikov, Volodymyr; Lloyd, David; Finnon, Paul

2012-01-01

Following exposure to high doses of ionizing radiation, due to an accident or during radiotherapy, bystander signalling poses a potential hazard to unirradiated cells and tissues. This process can be mediated by factors circulating in blood plasma. Thus, we assessed the ability of plasma taken from in vitro irradiated human blood to produce a direct cytotoxic effect, by inducing apoptosis in primary human peripheral blood mononuclear cells (PBM), which mainly comprised G 0 -stage lymphocytes. Plasma was collected from healthy donors’ blood irradiated in vitro to 0–40 Gy acute γ-rays. Reporter PBM were separated from unirradiated blood with Histopaque and held in medium with the test plasma for 24 h at 37 °C. Additionally, plasma from in vitro irradiated and unirradiated blood was tested against PBM collected from blood given 4 Gy. Apoptosis in reporter PBM was measured by the Annexin V test using flow cytometry. Plasma collected from unirradiated and irradiated blood did not produce any apoptotic response above the control level in unirradiated reporter PBM. Surprisingly, plasma from irradiated blood caused a dose-dependent reduction of apoptosis in irradiated reporter PBM. The yields of radiation-induced cell death in irradiated reporter PBM (after subtracting the respective values in unirradiated reporter PBM) were 22.2 ± 1.8% in plasma-free cultures, 21.6 ± 1.1% in cultures treated with plasma from unirradiated blood, 20.2 ± 1.4% in cultures with plasma from blood given 2–4 Gy and 16.7 ± 3.2% in cultures with plasma from blood given 6–10 Gy. These results suggested that irradiated blood plasma did not cause a radiation-induced bystander cell-killing effect. Instead, a reduction of apoptosis in irradiated reporter cells cultured with irradiated blood plasma has implications concerning oncogenic risk from mutated cells surviving after high dose in vivo irradiation (e.g. radiotherapy) and requires further study.

Phagocytosis-induced 51Cr release from activated macrophages and blood mononuclears. Effect of colchicine and antioxidants

International Nuclear Information System (INIS)

McGee, M.P.; Hale, A.H.

1981-01-01

The chromium-release test was adapted to the measurement of the cellular injury induced when activated macrophages phagocytose particulates. Macrophages obtained from rabbit lungs undergoing BCG-induced chronic inflammation released more chromium when incubated in the presence of phagocytosable particles than when incubated under resting conditions. Blood mononuclear cells, 40-60% monocytes, procured from the same BCG-injected animals, were less susceptible to phagocytosis-induced injury than the macrophages obtained from the lungs. The amount of chromium released by the activated macrophages was proportional to the number of particles present during incubation. In the presence of catalase, the amounts of chromium released by phagocytosing and resting macrophages were similar; in the presence of superoxide dismutase and cytochrome c, the amount of chromium released by phagocytosing macrophages was 13-35% less than the amount of chromium released by macrophages incubated without the antioxidants. In addition, colchicine, an inhibitor of degranulation also exerted partial inhibition of the chromium release. These results suggest that oxygen radicals and lysosomal contents contribute to the cellular injury that results from phagocytosis

Induction of antigen-specific antibody response in human pheripheral blood lymphocytes in vitro by a dog kidney cell vaccine against rabies virus (DKCV).

NARCIS (Netherlands)

F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert); H.G. Loggen; R.H.J. Bakker (Roland); J.A.A.M. van Asten (Jack); J.G. Kreeftenberg; P. van der Marel; G. van Steenis (Bert)

1983-01-01

textabstractIn the present report an in vitro method for obtaining a secondary human antibody response to a dog kidney cell vaccine against rabies virus (DKCV) is described. Cultures of peripheral blood mononuclear cells from normal rabies-immune and nonimmune donors were stimulated in vitro by

Isolation and characterization of equine peripheral blood-derived multipotent mesenchymal stromal cells

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Armando de M. Carvalho

2013-09-01

Full Text Available The objective of the study was to isolate, cultivate and characterize equine peripheral blood-derived multipotent mesenchymal stromal cells (PbMSCs. Peripheral blood was collected, followed by the isolation of mononuclear cells using density gradient reagents, and the cultivation of adherent cells. Monoclonal mouse anti-horse CD13, mouse anti-horse CD44, and mouse anti-rat CD90 antibodies were used for the immunophenotypic characterization of the surface of the PbMSCs. These cells were also cultured in specific media for adipogenic and chondrogenic differentiation. There was no expression of the CD13 marker, but CD44 and CD90 were expressed in all of the passages tested. After 14 days of cell differentiation into adipocytes, lipid droplets were observed upon Oil Red O (ORO staining. Twenty-one days after chondrogenic differentiation, the cells were stained with Alcian Blue. Although the technique for the isolation of these cells requires improvement, the present study demonstrates the partial characterization of PbMSCs, classifying them as a promising type of progenitor cells for use in equine cell therapy.

Smoking cessation reverses DNA double-strand breaks in human mononuclear cells.

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Mari Ishida

Full Text Available OBJECTIVE: Cigarette smoking is a major risk factor for atherosclerotic cardiovascular disease, which is responsible for a significant proportion of smoking-related deaths. However, the precise mechanism whereby smoking induces this pathology has not been fully delineated. Based on observation of DNA double-strand breaks (DSBs, the most harmful type of DNA damage, in atherosclerotic lesions, we hypothesized that there is a direct association between smoking and DSBs. The goal of this study was to investigate whether smoking induces DSBs and smoking cessation reverses DSBs in vivo through examination of peripheral mononuclear cells (MNCs. APPROACH AND RESULTS: Immunoreactivity of oxidative modification of DNA and DSBs were increased in human atherosclerotic lesions but not in the adjacent normal area. DSBs in human MNCs isolated from the blood of volunteers can be detected as cytologically visible "foci" using an antibody against the phosphorylated form of the histone H2AX (γ-H2AX. Young healthy active smokers (n = 15 showed increased γ-H2AX foci number when compared with non-smokers (n = 12 (foci number/cell: median, 0.37/cell; interquartile range [IQR], 0.31-0.58 vs. 4.36/cell; IQR, 3.09-7.39, p<0.0001. Smoking cessation for 1 month reduced the γ-H2AX foci number (median, 4.44/cell; IQR, 4.36-5.24 to 0.28/cell; IQR, 0.12-0.53, p<0.05. A positive correlation was noted between γ-H2AX foci number and exhaled carbon monoxide levels (r = 0.75, p<0.01. CONCLUSIONS: Smoking induces DSBs in human MNCs in vivo, and importantly, smoking cessation for 1 month resulted in a decrease in DSBs to a level comparable to that seen in non-smokers. These data reinforce the notion that the cigarette smoking induces DSBs and highlight the importance of smoking cessation.

Neovascularization Potential of Blood Outgrowth Endothelial Cells From Patients With Stable Ischemic Heart Failure Is Preserved

OpenAIRE

Dauwe, Dieter; Pelacho, Beatriz; Wibowo, Arief; Walravens, Ann-Sophie; Verdonck, Kristoff; Gillijns, Hilde; Caluwe, Ellen; Pokreisz, Peter; van Gastel, Nick; Carmeliet, Geert; Depypere, Maarten; Maes, Frederik; Vanden Driessche, Nina; Droogne, Walter; Van Cleemput, Johan

2016-01-01

Background Blood outgrowth endothelial cells (BOECs) mediate therapeutic neovascularization in experimental models, but outgrowth characteristics and functionality of BOECs from patients with ischemic cardiomyopathy (ICMP) are unknown. We compared outgrowth efficiency and in?vitro and in?vivo functionality of BOECs derived from ICMP with BOECs from age?matched (ACON) and healthy young (CON) controls. Methods and Results We isolated 3.6?0.6 BOEC colonies/100?106 mononuclear cells (MNCs) from 6...

Effect of Infla-Kine supplementation on the gene expression of inflammatory markers in peripheral mononuclear cells and on C-reactive protein in blood.

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Mikirova, Nina A; Kesari, Santosh; Ichim, Thomas E; Riordan, Neil H

2017-10-20

Chronic inflammation is a predisposing factor to numerous degenerative diseases including cancer, heart failure and Alzheimer's disease. Infla-Kine is a natural supplement comprised of a proprietary blend of Lactobacillus fermentum extract, burdock seed (arctigenin), zinc, alpha lipoic acid, papaya enzyme and an enhanced absorption bio-curcumin complex (BCM-95 ® ). Infla-Kine was administered twice daily to 24 health volunteers for 4 weeks. Quantitative RT-PCR was used to assess mRNA transcripts of IL-1b, IL8, IL-6, NF-κB, and TNF-α from peripheral blood mononuclear cells (PBMC). C reactive protein (CRP) was measured from serum. Additionally, quality of life questionnaires were employed to assess general feeling of well-being. Assessments were made before treatment and at conclusion of treatment (4 weeks). As compared to pre-treatment, after 4 weeks, a statistically significant reduction of IL8, IL-6, NF-κB, and TNF-α transcripts was observed in PBMC. Furthermore, reduction of IL-1b transcript and serum CRP was observed but did not reach statistical significance. Quality of life improvements were most prevalent in muscle and joint pains. Overall, our data demonstrate that twice daily administration of Infla-Kine for 4 weeks reduces inflammatory markers and quality of life in healthy volunteers.

Identification of a B cell-dependent subpopulation of multiple sclerosis by measurements of brain-reactive B cells in the blood.

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Kuerten, Stefanie; Pommerschein, Giovanna; Barth, Stefanie K; Hohmann, Christopher; Milles, Bianca; Sammer, Fabian W; Duffy, Cathrina E; Wunsch, Marie; Rovituso, Damiano M; Schroeter, Michael; Addicks, Klaus; Kaiser, Claudia C; Lehmann, Paul V

2014-01-01

B cells are increasingly coming into play in the pathogenesis of multiple sclerosis (MS). Here, we screened peripheral blood mononuclear cells (PBMC) from patients with clinically isolated syndrome (CIS), MS, other non-inflammatory neurological, inflammatory neurological or autoimmune diseases, and healthy donors for their B cell reactivity to CNS antigen using the enzyme-linked immunospot technique (ELISPOT) after 96 h of polyclonal stimulation. Our data show that nine of 15 patients with CIS (60.0%) and 53 of 67 patients with definite MS (79.1%) displayed CNS-reactive B cells, compared to none of the control donors. The presence of CNS-reactive B cells in the blood of the majority of patients with MS or at risk to develop MS along with their absence in control subjects suggests that they might be indicative of a B cell-dependent subpopulation of the disease. Copyright © 2014. Published by Elsevier Inc.

Polycyclic aromatic hydrocarbons reciprocally regulate IL-22 and IL-17 cytokines in peripheral blood mononuclear cells from both healthy and asthmatic subjects.

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Coline Plé

Full Text Available Pollution, including polycyclic aromatic hydrocarbons (PAH, may contribute to increased prevalence of asthma. PAH can bind to the Aryl hydrocarbon Receptor (AhR, a transcription factor involved in Th17/Th22 type polarization. These cells produce IL17A and IL-22, which allow neutrophil recruitment, airway smooth muscle proliferation and tissue repair and remodeling. Increased IL-17 and IL-22 productions have been associated with asthma. We hypothesized that PAH might affect, through their effects on AhR, IL-17 and IL-22 production in allergic asthmatics. Activated peripheral blood mononuclear cells (PBMCs from 16 nonallergic nonasthmatic (NA and 16 intermittent allergic asthmatic (AA subjects were incubated with PAH, and IL-17 and IL-22 productions were assessed. At baseline, activated PBMCs from AA exhibited an increased IL-17/IL-22 profile compared with NA subjects. Diesel exhaust particle (DEP-PAH and Benzo[a]Pyrene (B[a]P stimulation further increased IL-22 but decreased IL-17A production in both groups. The PAH-induced IL-22 levels in asthmatic patients were significantly higher than in healthy subjects. Among PBMCs, PAH-induced IL-22 expression originated principally from single IL-22- but not from IL-17- expressing CD4 T cells. The Th17 transcription factors RORA and RORC were down regulated, whereas AhR target gene CYP1A1 was upregulated. IL-22 induction by DEP-PAH was mainly dependent upon AhR whereas IL-22 induction by B[a]P was dependent upon activation of PI3K and JNK. Altogether, these data suggest that DEP-PAH and B[a]P may contribute to increased IL22 production in both healthy and asthmatic subjects through mechanisms involving both AhR -dependent and -independent pathways.

Effects of a healthy Nordic diet on gene expression changes in peripheral blood mononuclear cells in response to an oral glucose tolerance test in subjects with metabolic syndrome

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Leder, Lena; Kolehmainen, Marjukka; Narverud, Ingunn

2016-01-01

BACKGROUND: Diet has a great impact on the risk of developing features of metabolic syndrome (MetS), type 2 diabetes mellitus (T2DM), and cardiovascular diseases (CVD). We evaluated whether a long-term healthy Nordic diet (ND) can modify the expression of inflammation and lipid metabolism......-related genes in peripheral blood mononuclear cells (PBMCs) during a 2-h oral glucose tolerance test (OGTT) in individuals with MetS. METHODS: A Nordic multicenter randomized dietary study included subjects (n = 213) with MetS, randomized to a ND group or a control diet (CD) group applying an isocaloric study......, and the mRNA gene expression analysis was measured by quantitative real-time polymerase chain reaction (qPCR). We analyzed the mRNA expression changes of 44 genes before and after a 2hOGTT at the beginning and the end of the intervention. RESULTS: The healthy ND significantly down-regulated the expression...

Granulocyte colony-stimulating factor decreases the Th1/Th2 ratio in peripheral blood mononuclear cells from patients with chronic immune thrombocytopenic purpura in vitro.

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Ge, Fei; Zhang, Zhuo; Hou, Jinxiao; Cao, Fenglin; Zhang, Yingmei; Wang, Ping; Wei, Hong; Zhou, Jin

2016-12-01

Chronic immune thrombocytopenia purpura (ITP) is an autoimmune disease that exhibits an abnormally high Th1/Th2 ratio. Granulocyte colony-stimulating factor (G-CSF) has been shown to decrease the Th1/Th2 ratio in healthy donors. In this study, we investigated the effects of G-CSF treatment on the Th1/Th2 cells and the underlying mechanisms in patients with ITP in vitro. Peripheral blood mononuclear cells (PBMCs) isolated from patients with ITP and healthy controls were treated with G-CSF. Expression levels of interferon (IFN)-γ, interleukin (IL)-2, IL-4, and IL-13 in supernatants were measured by enzyme-linked immunosorbent assays. The expression of IFN-γ, IL-4, and G-CSF receptor (G-CSFR) on Th1 and Th2 cells were examined by flow cytometry and confocal microscopy. The mRNA expression of IFN-γ, IL-2, IL-4, IL-13, and T-box expressed in T cells (T-bet) and GATA-binding protein 3 (GATA-3) in PBMCs was evaluated by reverse transcription polymerase chain reaction. The results showed that G-CSF could significantly reduce the Th1/Th2 ratio in PBMCs from patients with ITP in vitro. As the concentration of G-CSF increased, Th1/Th2 ([IFN-γ+IL-2]/[IL-4+IL-13]) cytokine ratios and T-bet/GATA-3 mRNA ratios decreased in a concentration-dependent manner. Th1 cells and Th2 cells both expressed G-CSFR. These results suggest that G-CSF could decrease the Th1/Th2 ratio in the context of ITP, and elucidate the direct and indirect immunomodulatory mechanisms underlying G-CSF functions in Th1/Th2 cells, thus supporting the therapeutic potential of G-CSF in the treatment of patients with ITP. Copyright © 2016 Elsevier Ltd. All rights reserved.

Winter to summer change in vitamin D status reduces systemic inflammation and bioenergetic activity of human peripheral blood mononuclear cells.

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Calton, Emily K; Keane, Kevin N; Raizel, Raquel; Rowlands, Jordan; Soares, Mario J; Newsholme, Philip

2017-08-01

Vitamin D status [25(OH)D] has recently been reported to be associated with altered cellular bioenergetic profiles of peripheral blood mononuclear cells (PBMCs). No study has tracked the seasonal variation of 25(OH)D and its putative influence on whole body energy metabolism, cellular bioenergetic profiles, inflammatory markers and clinical chemistry. Whole body energy metabolism and substrate utilisation were measured by indirect calorimetry. PBMCs obtained from the same subjects were isolated from whole blood, counted and freshly seeded. Bioenergetic analysis (mitochondrial stress test and glycolysis stress test) was performed using the Seahorse XF e 96 flux analyser. 25(OH)D was assessed using the Architect immunoassay method. 25(OH)D increased by a median (IQR) of 14.40 (20.13)nmol/L (pwinter to summer and was accompanied by significant improvements in indices of insulin sensitivity, McAuley's index (p=0.019) and quantitative insulin sensitivity check index (p=0.028). PBMC mitochondrial parameters basal respiration, non-mitochondrial respiration, ATP production, proton leak, and maximal respiration decreased in summer compared to winter. Similarly, PBMC glycolytic parameters glycolytic activity, glucose response, and glycolytic capacity were all reduced in summer compared to winter. There was also a trend for absolute resting metabolic rate (RMR) to decrease (p=0.066). Markers of systemic inflammation MCP-1, IL-6, IL-8, IL-10, and IL-12p70 decreased significantly in summer compared to winter. Participants who entered winter with a low 25(OH)D (winter 25(OH)D concentrations of 50-75nmol/L or >75nmol/L. The absolute change in 25(OH)D was not associated with altered bioenergetics. Seasonal improvements in 25(OH)D was associated with reduced systemic inflammation, PBMC bioenergetic profiles and whole body energy metabolism. These observational changes in PBMC bioenergetics were most pronounced in those who had insufficient 25(OH)D in winter. The data warrants

Comparison of real-time reverse transcriptase polymerase chain reaction of peripheral blood mononuclear cells, serum and cell-free body cavity effusion for the diagnosis of feline infectious peritonitis.

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Doenges, Stephanie J; Weber, Karin; Dorsch, Roswitha; Fux, Robert; Hartmann, Katrin

2017-04-01

Objectives Diagnosis of feline infectious peritonitis (FIP) remains challenging, especially in cats without effusions. The objective of this study was to evaluate the sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction (RT-PCR) detecting feline coronavirus (FCoV) RNA in peripheral blood mononuclear cells (PBMCs) and serum in comparison with the same real-time RT-PCR in cell-free body cavity effusion. Methods This prospective case-control study included 92 cats. Forty-three cats had a definitive diagnosis of FIP, established either by histopathological examination (n = 28) or by positive immunofluorescence staining of FCoV antigen in macrophages of effusions (n = 11), or by both methods (n = 4). Forty-nine control cats had other diseases but similar clinical signs. Real-time RT-PCR was performed on PBMCs of 37 cats (21 cats with FIP, 16 controls), on serum of 51 cats (26 cats with FIP, 25 controls) and on cell-free body cavity effusion of 69 cats (36 cats with FIP, 33 controls). Sensitivity, specificity, positive and negative predictive value, including 95% confidence intervals (CI), were calculated. Results Real-time RT-PCR of PBMCs, serum and cell-free body cavity effusion showed a specificity of 100% (95% CI 79.4-100% in PBMCs, 86.3-100% in serum, 89.4-100% in cell-free body cavity effusion) and a sensitivity of 28.6% (95% CI 11.3-52.2%) in PBMCs, 15.4% (95% CI 4.4-34.9%) in serum and 88.9% (95% CI 73.9-96.9%) in cell-free body cavity effusion to diagnose FIP. Conclusions and relevance Although it is known that RT-PCR can often provide false-positive results in healthy cats, this real-time RT-PCR was shown to be a specific tool for the diagnosis of FIP when applied in a clinical setting. Sensitivity in cell-free body cavity effusion was high but low in PBMCs and serum. PBMC samples showed a higher sensitivity than serum samples, and are therefore a better choice if no effusion is present.

Mobilization of Neural Precursors in the Circulating Blood of Patients with Multiple Sclerosis

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2013-09-01

Bongarzone ER. Expression of sonic hedgehog targeted genes in peripheral blood mononuclear cells of patients with multiple sclerosis. Society for...recovery in a rat spinal cord injury model. Spine (Phila Pa 1976) 34:249-254. Schwartz PH, Bryant PJ, Fuja TJ, Su H, O’Dowd DK, Klassen H (2003...Print Program#/Poster#: 322.13 Presentation Title: Expression of sonic  hedgehog  targeted genes in peripheral blood mononuclear cells of patients with

Consumption of selenium-enriched broccoli increases cytokine production in human peripheral blood mononuclear cells stimulated ex vivo, a preliminary human intervention study.

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Bentley-Hewitt, Kerry L; Chen, Ronan K-Y; Lill, Ross E; Hedderley, Duncan I; Herath, Thanuja D; Matich, Adam J; McKenzie, Marian J

2014-12-01

Selenium (Se) is a micronutrient essential for human health, including immune function. Previous research indicates that Se supplementation may cause a shift from T helper (Th)1- to Th2-type immune responses. We aim to test the potential health promoting effects of Se-enriched broccoli. In a human trial, 18 participants consumed control broccoli daily for 3 days. After a 3-day wash-out period, the participants were provided with Se-enriched broccoli containing 200 μg of Se per serving for 3 days. Plasma and peripheral blood mononuclear cell (PBMC) samples were collected at the start and end of each broccoli feeding period for analysis of total Se and measurement of cytokine production from PBMC stimulated with antigens ex vivo. Plasma Se content remained consistent throughout the control broccoli feeding period and the baseline of the Se-enriched broccoli period (1.22 μmol/L) and then significantly increased following 3 days of Se-enriched broccoli feeding. Interleukin (IL-2, IL-4, IL-5, IL-13, and IL-22) production from PBMC significantly increased after 3 days of Se-enriched broccoli feeding compared with baseline. This study indicates that consumption of Se-enriched broccoli may increase immune responses toward a range of immune challenges. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Heterogeneity in white blood cells has potential to confound DNA methylation measurements.

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Bjorn T Adalsteinsson

Full Text Available Epigenetic studies are commonly conducted on DNA from tissue samples. However, tissues are ensembles of cells that may each have their own epigenetic profile, and therefore inter-individual cellular heterogeneity may compromise these studies. Here, we explore the potential for such confounding on DNA methylation measurement outcomes when using DNA from whole blood. DNA methylation was measured using pyrosequencing-based methodology in whole blood (n = 50-179 and in two white blood cell fractions (n = 20, isolated using density gradient centrifugation, in four CGIs (CpG Islands located in genes HHEX (10 CpG sites assayed, KCNJ11 (8 CpGs, KCNQ1 (4 CpGs and PM20D1 (7 CpGs. Cellular heterogeneity (variation in proportional white blood cell counts of neutrophils, lymphocytes, monocytes, eosinophils and basophils, counted by an automated cell counter explained up to 40% (p<0.0001 of the inter-individual variation in whole blood DNA methylation levels in the HHEX CGI, but not a significant proportion of the variation in the other three CGIs tested. DNA methylation levels in the two cell fractions, polymorphonuclear and mononuclear cells, differed significantly in the HHEX CGI; specifically the average absolute difference ranged between 3.4-15.7 percentage points per CpG site. In the other three CGIs tested, methylation levels in the two fractions did not differ significantly, and/or the difference was more moderate. In the examined CGIs, methylation levels were highly correlated between cell fractions. In summary, our analysis detects region-specific differential DNA methylation between white blood cell subtypes, which can confound the outcome of whole blood DNA methylation measurements. Finally, by demonstrating the high correlation between methylation levels in cell fractions, our results suggest a possibility to use a proportional number of a single white blood cell type to correct for this confounding effect in analyses.

Microarray analysis of gene expression in peripheral blood mononuclear cells from dioxin-exposed human subjects

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McHale, Cliona M.; Zhang, Luoping; Hubbard, Alan E.; Zhao, Xin; Baccarelli, Andrea; Pesatori, Angela C.; Smith, Martyn T.; Landi, Maria Teresa

2007-01-01

Tetrachlorodibenzo-p-dioxin (TCDD) is classified as a human carcinogen and exerts toxic effects on the skin (chloracne). Effects on reproductive, immunological, and endocrine systems have also been observed in animal models. TCDD acts through the aryl hydrocarbon receptor (AhR) pathway influencing largely unknown gene networks. An industrial accident in Seveso, Italy in 1976 exposed thousands of people to substantial quantities of TCDD. Twenty years after the exposure, this study examines global gene expression in the mononuclear cells of 26 Seveso female never smokers, with similar age, alcohol consumption, use of medications, and background plasma levels of 22 dioxin congeners unrelated to the Seveso accident. Plasma dioxin levels were still elevated in the exposed subjects. We performed analyses in two different comparison groups. The first included high-exposed study subjects compared with individuals with background TCDD levels (average plasma levels 99.4 and 6.7 ppt, respectively); the second compared subjects who developed chloracne after the accident, and those who did not develop this disease. Overall, we observed a modest alteration of gene expression based on dioxin levels or on chloracne status. In the comparison between high levels and background levels of TCDD, four histone genes were up-regulated and modified expression of HIST1H3H was confirmed by real-time PCR. In the comparison between chloracne case-control subjects, five hemoglobin genes were up-regulated. Pathway analysis revealed two major networks for each comparison, involving cell proliferation, apoptosis, immunological and hematological disease, and other pathways. Further examination of the role of these genes in dioxin induced-toxicity is warranted

ChIP-seq analysis of histone H3K9 trimethylation in peripheral blood mononuclear cells of membranous nephropathy patients

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Sui, W.G. [Guangxi Key Laboratory of Metabolic Diseases Research, Nephrology Department, 181st Hospital, Guilin, Guangxi (China); He, H.Y. [The Life Science College, Guangxi Normal University, Guilin, Guangxi (China); Yan, Q.; Chen, J.J. [Guangxi Key Laboratory of Metabolic Diseases Research, Nephrology Department, 181st Hospital, Guilin, Guangxi (China); Zhang, R.H. [The Life Science College, Guangxi Normal University, Guilin, Guangxi (China); Dai, Y. [Clinical Medical Research Center, The Second Clinical Medical College, Shenzhen People’s Hospital, Jinan University, Shenzhen, Guangdong (China)

2013-12-12

Membranous nephropathy (MN), characterized by the presence of diffuse thickening of the glomerular basement membrane and subepithelial in situ immune complex disposition, is the most common cause of idiopathic nephrotic syndrome in adults, with an incidence of 5-10 per million per year. A number of studies have confirmed the relevance of several experimental insights to the pathogenesis of human MN, but the specific biomarkers of MN have not been fully elucidated. As a result, our knowledge of the alterations in histone methylation in MN is unclear. We used chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) to analyze the variations in a methylated histone (H3K9me3) in peripheral blood mononuclear cells from 10 MN patients and 10 healthy subjects. There were 108 genes with significantly different expression in the MN patients compared with the normal controls. In MN patients, significantly increased activity was seen in 75 H3K9me3 genes, and decreased activity was seen in 33, compared with healthy subjects. Five positive genes, DiGeorge syndrome critical region gene 6 (DGCR6), sorting nexin 16 (SNX16), contactin 4 (CNTN4), baculoviral IAP repeat containing 3 (BIRC3), and baculoviral IAP repeat containing 2 (BIRC2), were selected and quantified. There were alterations of H3K9me3 in MN patients. These may be candidates to help explain pathogenesis in MN patients. Such novel findings show that H3K9me3 may be a potential biomarker or promising target for epigenetic-based MN therapies.

Human mesenchymal stem cells promote CD34+ hematopoietic stem cell proliferation with preserved red blood cell differentiation capacity.

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Lau, Show Xuan; Leong, Yin Yee; Ng, Wai Hoe; Ng, Albert Wee Po; Ismail, Ida Shazrina; Yusoff, Narazah Mohd; Ramasamy, Rajesh; Tan, Jun Jie

2017-06-01

Studies showed that co-transplantation of mesenchymal stem cells (MSCs) and cord blood-derived CD34 + hematopoietic stem cells (HSCs) offered greater therapeutic effects but little is known regarding the effects of human Wharton's jelly derived MSCs on HSC expansion and red blood cell (RBC) generation in vitro. This study aimed to investigate the effects of MSCs on HSC expansion and differentiation. HSCs were co-cultured with MSCs or with 10% MSCs-derived conditioned medium, with HSCs cultured under standard medium served as a control. Cell expansion rates, number of mononuclear cell post-expansion and number of enucleated cells post-differentiation were evaluated. HSCs showed superior proliferation in the presence of MSC with mean expansion rate of 3.5 × 10 8  ± 1.8 × 10 7 after day 7 compared to the conditioned medium and the control group (8.9 × 10 7  ± 1.1 × 10 8 and 7.0 × 10 7  ± 3.3 × 10 6 respectively, P cell was greater compared to earlier passages, indicating successful RBC differentiation. Cord blood-derived CD34 + HSCs can be greatly expanded by co-culturing with MSCs without affecting the RBC differentiation capability, suggesting the importance of direct MSC-HSCs contact in HSC expansion and RBC differentiation. © 2017 International Federation for Cell Biology.

Phosphorylation of Histone H2A.X in Peripheral Blood Mononuclear Cells May Be a Useful Marker for Monitoring Cardiometabolic Risk in Nondiabetic Individuals

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So Ra Yoon

2017-01-01

Full Text Available Phosphorylation of H2A.X (serine 139 in the histone H2A family located in the downstream of the DNA damage kinase signaling cascade is an important indicator of DNA damage. Recently, phosphorylation of H2A.X was proposed as a sensitive biomarker of aging. This study investigated if phosphorylation of H2A.X in peripheral blood mononuclear cells (PBMCs is associated with cardiometabolic risk in nondiabetic individuals. Basic parameters and oxidative stress/inflammatory markers were measured in nondiabetic healthy Koreans (n=119. Phosphorylation of H2A.X was measured randomly among the study subjects using a flow cytometer. According to the number of metabolic syndrome risk factor (MetS-RF, the study subjects were subdivided into “super healthy” (MetS−RF=0, n=71 and “MetS-risk” (MetS−RF≥1, n=48 groups. Phosphorylation of H2A.X in PBMCs (percentages and mean fluorescence intensity was significantly higher in the MetS-risk group than in the super healthy group after adjusting for age, sex, cigarette smoking, and alcohol consumption. Phosphorylated H2A.X was positively correlated with the number of MetS-RF as well as waist circumference, blood pressures, triglyceride, HbA1C, oxidized LDL, high sensitivity C-reactive protein, tumor necrosis factor-alpha, and alanine aminotransferase after the adjustment. The present study suggested that phosphorylated H2A.X in circulating PBMCs measured by flow cytometer may be a useful marker for monitoring cardiometabolic risk in nondiabetic individuals.

Distinct kinetics of memory B-cell and plasma-cell responses in peripheral blood following a blood-stage Plasmodium chabaudi infection in mice.

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Eunice W Nduati

2010-11-01

Full Text Available B cell and plasma cell responses take place in lymphoid organs, but because of the inaccessibility of these organs, analyses of human responses are largely performed using peripheral blood mononuclear cells (PBMC. To determine whether PBMC are a useful source of memory B cells and plasma cells in malaria, and whether they reflect Plasmodium-specific B cell responses in spleen or bone marrow, we have investigated these components of the humoral response in PBMC using a model of Plasmodium chabaudi blood-stage infections in C57BL/6 mice. We detected memory B cells, defined as isotype-switched IgD(- IgM(- CD19(+ B cells, and low numbers of Plasmodium chabaudi Merozoite Surface Protein-1 (MSP1-specific memory B cells, in PBMC at all time points sampled for up to 90 days following primary or secondary infection. By contrast, we only detected CD138(+ plasma cells and MSP1-specific antibody-secreting cells within a narrow time frame following primary (days 10 to 25 or secondary (day 10 infection. CD138(+ plasma cells in PBMC at these times expressed CD19, B220 and MHC class II, suggesting that they were not dislodged bone-marrow long-lived plasma cells, but newly differentiated migratory plasmablasts migrating to the bone marrow; thus reflective of an ongoing or developing immune response. Our data indicates that PBMC can be a useful source for malaria-specific memory B cells and plasma cells, but extrapolation of the results to human malaria infections suggests that timing of sampling, particularly for plasma cells, may be critical. Studies should therefore include multiple sampling points, and at times of infection/immunisation when the B-cell phenotypes of interest are likely to be found in peripheral blood.

Immune Modulation in Normal Human Peripheral Blood Mononuclear Cells (PBMCs) (Lymphocytes) in Response to Benzofuran-2-Carboxylic Acid Derivative KMEG during Spaceflight

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Okoro, Elvis; Mann, Vivek; Ellis, Ivory; Mansoor, Elvedina; Olamigoke, Loretta; Marriott, Karla Sue; Denkins, Pamela; Williams, Willie; Sundaresan, Alamelu

2017-08-01

Microgravity and radiation exposure during space flight have been widely reported to induce the suppression of normal immune system function, and increase the risk of cancer development in humans. These findings pose a serious risk to manned space missions. Interestingly, recent studies have shown that benzofuran-2-carboxylic acid derivatives can inhibit the progression of some of these devastating effects on earth and in modeled microgravity. However, these studies had not assessed the impacts of benzofuran-2- carboxylic acid and its derivatives on global gene expression under spaceflight conditions. In this study, the ability of a specific benzofuran-2-carboxylic acid derivative (KMEG) to confer protection from radiation and restore normal immune function was investigated following exposure to space flight conditions on the ISS. Normal human peripheral blood mononuclear cells (lymphocytes) treated with 10 µ g/ml of KMEG together with untreated control samples were flown on Nanoracks hardware on Spacex-3 flight. The Samples were returned one month later and gene expression was analyzed. A 1g-ground control experiment was performed in parallel at the Kennedy spaceflight center. The first overall subtractive unrestricted analysis revealed 78 genes, which were differentially expressed in space flight KMEG, untreated lymphocytes as compared to the corresponding ground controls. However, in KMEG-treated space flight lymphocytes, there was an increased expression of a group of genes that mediate increased transcription, translation and innate immune system mediating functions of lymphocytes as compared to KMEG-untreated samples. Analysis of genes related to T cell proliferation in spaceflight KMEG-treated lymphocytes compared to 1g-ground KMEG- treated lymphocytes revealed six T cell proliferation and signaling genes to be significantly upregulated (p trafficking, promote early response, mediating C-myc related proliferation, promote antiapoptotic activity and protects

Marked reduction of AKT1 expression and deregulation of AKT1-associated pathways in peripheral blood mononuclear cells of schizophrenia patients.

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Nico J M van Beveren

Full Text Available BACKGROUND: Recent studies have suggested that deregulated AKT1 signaling is associated with schizophrenia. We hypothesized that if this is indeed the case, we should observe both decreased AKT1 expression as well as deregulation of AKT1 regulated pathways in Peripheral Blood Mononuclear Cells (PBMCs of schizophrenia patients. OBJECTIVES: To examine PBMC expression levels of AKT1 in schizophrenia patients versus controls, and to examine whether functional biological processes in which AKT1 plays an important role are deregulated in schizophrenia patients. METHODS/RESULTS: A case-control study, investigating whole-genome PBMC gene expression in male, recent onset (<5 years schizophrenia patients (N = 41 as compared to controls (N = 29. Genes, differentially expressed between patients and controls were identified using ANOVA with Benjamini-Hochberg correction (false discovery rate (FDR = 0.05. Functional aspects of the deregulated set of genes were investigated with the Ingenuity Pathway Analysis (IPA Software Tool. We found significantly decreased PBMC expression of AKT1 (p<0.001, t = -4.25 in the patients. AKT1 expression was decreased in antipsychotic-free or -naive patients (N = 11, in florid psychotic (N = 20 and in remitted (N = 21 patients. A total of 1224 genes were differentially expressed between patients and controls (FDR = 0.05. Functional analysis of the entire deregulated gene set indicated deregulated canonical pathways involved in a large number of cellular processes: immune system, cell adhesion and neuronal guidance, neurotrophins and (neural growth factors, oxidative stress and glucose metabolism, and apoptosis and cell-cycle regulation. Many of these processes are associated with AKT1. CONCLUSIONS: We show significantly decreased PBMC gene expression of AKT1 in male, recent-onset schizophrenia patients. Our observations suggest that decreased PBMC AKT1 expression is a stable trait in recent onset

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

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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.

DNA damage response and role of shelterin complex in human peripheral blood mononuclear cells exposed to gamma radiation

International Nuclear Information System (INIS)

Saini, Divyalakshmi; Das, Birajalaxmi

2013-01-01

Telomeres are the DNA protein structures that cap the ends of linear DNA. It consists of short repetitive DNA sequences (TTAGGG)n and specialized telomere binding proteins. There are six telomeric proteins (TRF1, TRF2, TIN2, TERF2, PTOP and POT1) called as shelterin complex/telosome which maintains telomere integrity. The function of this 'telosome' is to protect the natural ends of the chromosomes from being recognized as artificial DNA breaks, thereby preventing chromosome end-to-end fusions. DNA Damage Response (DDR) induced by radiation and its interaction with telomeric protein complex is poorly understood in human PBMCs at G 0 stage. Alterations in either telomeric DNA or telomere binding proteins can impair the function of the telosome, which may lead to senescence or apoptosis. Ionizing radiation which induces a plethora of DNA lesions in human cell may also alter the expression of telomere associated proteins. In the present study, we have made an attempt to study the DNA damage response of telomere proteins in human peripheral blood mononuclear cells exposed to gamma radiation. Venous blood samples were collected from eight random healthy volunteers and PBMCs were separated. Dose response as well as time point kinetics study was carried out at transcription as well as protein level. PBMCs were irradiated at various doses between 10 cGy to 2.0 Gy at a dose rate of 1.0 Gy/min. Total RNA was isolated for gene expression analysis at 0 hour and 4 hours respectively. cDNA was prepared and transcriptional pattern as studied using real time q-PCR where Taqman probes were used. Time point kinetics of transcriptional pattern of TRF1, TRF2, TIN2, TERF2, PTOP and POT1 was carried out at 0 min, 15 min, 30 min, 60 min, and 120 min for two different doses (1.0 Gy and 2.0 Gy). Dose response and time point kinetics of TRF2 was studied at similar doses using confocal microscopy. Our results revealed that at 2.0 Gy there was a two fold increase at the level of transcription

Immunomodulatory Effects of Lactobacillus salivarius LS01 and Bifidobacterium breve BR03, Alone and in Combination, on Peripheral Blood Mononuclear Cells of Allergic Asthmatics.

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Drago, Lorenzo; De Vecchi, Elena; Gabrieli, Arianna; De Grandi, Roberta; Toscano, Marco

2015-07-01

The aim of this study was to evaluate probiotic characteristics of Lactobacillus salivarius LS01 and Bifidobacterium breve BR03 alone and in combination and their immunomodulatory activity in asthmatic subjects. Subjects affected by allergic asthma were recruited. Initially, LS01 and BR03 were analyzed for their growth compatibility by a broth compatibility assay. To study the antimicrobial activity of probiotic strains, an agar diffusion assay was performed. Finally, cytokine production by peripheral blood mononuclear cells (PBMCs) stimulated with LS01 and BR03 was determined by means of specific quantitative enzyme-linked immunosorbent assay (ELISA). The growth of some clinical pathogens were slightly inhibited by LS01 and LS01-BR03 co-culture supernatant not neutralized to pH 6.5, while only the growth of E. coli and S. aureus was inhibited by the supernatant of LS01 and LS01-BR03 neutralized to pH 6.5. Furthermore, LS01 and BR03 combination was able to decrease the secretion of proinflammatory cytokines by PBMCs, leading to an intense increase in IL-10 production. L. salivarius LS01 and B. breve BR03 showed promising probiotic properties and beneficial immunomodulatory activity that are increased when the 2 strains are used in combination in the same formulation.

Nanoparticle encapsulation in red blood cells enables blood-pool magnetic particle imaging hours after injection

International Nuclear Information System (INIS)

Rahmer, J; Gleich, B; Borgert, J; Antonelli, A; Sfara, C; Magnani, M; Tiemann, B; Weizenecker, J

2013-01-01

Magnetic particle imaging (MPI) is a new medical imaging approach that is based on the nonlinear magnetization response of super-paramagnetic iron oxide nanoparticles (SPIOs) injected into the blood stream. To date, real-time MPI of the bolus passage of an approved MRI SPIO contrast agent injected into the tail vein of living mice has been demonstrated. However, nanoparticles are rapidly removed from the blood stream by the mononuclear phagocyte system. Therefore, imaging applications for long-term monitoring require the repeated administration of bolus injections, which complicates quantitative comparisons due to the temporal variations in concentration. Encapsulation of SPIOs into red blood cells (RBCs) has been suggested to increase the blood circulation time of nanoparticles. This work presents first evidence that SPIO-loaded RBCs can be imaged in the blood pool of mice several hours after injection using MPI. This finding is supported by magnetic particle spectroscopy performed to quantify the iron concentration in blood samples extracted from the mice 3 and 24 h after injection of SPIO-loaded RBCs. Based on these results, new MPI applications can be envisioned, such as permanent 3D real-time visualization of the vessel tree during interventional procedures, bleeding monitoring after stroke, or long-term monitoring and treatment control of cardiovascular diseases. (paper)

Brief Report: Blockade of TANK-Binding Kinase 1/IKKɛ Inhibits Mutant Stimulator of Interferon Genes (STING)-Mediated Inflammatory Responses in Human Peripheral Blood Mononuclear Cells.

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Frémond, Marie-Louise; Uggenti, Carolina; Van Eyck, Lien; Melki, Isabelle; Bondet, Vincent; Kitabayashi, Naoki; Hertel, Christina; Hayday, Adrian; Neven, Bénédicte; Rose, Yoann; Duffy, Darragh; Crow, Yanick J; Rodero, Mathieu P

2017-07-01

Gain-of-function mutations in TMEM173, encoding the stimulator of interferon (IFN) genes (STING) protein, underlie a novel type I interferonopathy that is minimally responsive to conventional immunosuppressive therapies and associated with high frequency of childhood morbidity and mortality. STING gain-of-function causes constitutive oversecretion of IFN. This study was undertaken to determine the effects of a TANK-binding kinase 1 (TBK-1)/IKKɛ inhibitor (BX795) on secretion and signaling of IFN in primary peripheral blood mononuclear cells (PBMCs) from patients with mutations in STING. PBMCs from 4 patients with STING-associated disease were treated with BX795. The effect of BX795 on IFN pathways was assessed by Western blotting and an IFNβ reporter assay, as well as by quantification of IFNα in cell lysates, staining for STAT-1 phosphorylation, and measurement of IFN-stimulated gene (ISG) messenger RNA (mRNA) expression. Treatment of PBMCs with BX795 inhibited the phosphorylation of IFN regulatory factor 3 and IFNβ promoter activity induced in HEK 293T cells by cyclic GMP-AMP or by genetic activation of STING. In vitro exposure to BX795 inhibited IFNα production in PBMCs of patients with STING-associated disease without affecting cell survival. In addition, BX795 decreased STAT-1 phosphorylation and ISG mRNA expression independent of IFNα blockade. These findings demonstrate the effect of BX795 on reducing type I IFN production and IFN signaling in cells from patients with gain-of-function mutations in STING. A combined inhibition of TBK-1 and IKKɛ therefore holds potential for the treatment of patients carrying STING mutations, and may also be relevant in other type I interferonopathies. © 2017, American College of Rheumatology.

Low-dose radiation (LDR) induces hematopoietic hormesis: LDR-induced mobilization of hematopoietic progenitor cells into peripheral blood circulation.

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Li, Wei; Wang, Guanjun; Cui, Jiuwei; Xue, Lu; Cai, Lu

2004-11-01

The aim of this study was to investigate the stimulating effect of low-dose radiation (LDR) on bone marrow hematopoietic progenitor cell (HPC) proliferation and peripheral blood mobilization. Mice were exposed to 25- to 100-mGy x-rays. Bone marrow and peripheral blood HPCs (BFU-E, CFU-GM, and c-kit+ cells) were measured, and GM-CSF, G-CSF, and IL-3 protein and mRNA expression were detected using ELISA, slot blot hybridization, and Northern blot methods. To functionally evaluate LDR-stimulated and -mobilized HPCs, repopulation of peripheral blood cells in lethally irradiated recipients after transplantation of LDR-treated donor HPCs was examined by WBC counts, animal survival, and colony-forming units in the recipient spleens (CFUs-S). 75-mGy x-rays induced a maximal stimulation for bone marrow HPC proliferation (CFU-GM and BFU-E formation) 48 hours postirradiation, along with a significant increase in HPC mobilization into peripheral blood 48 to 72 hours postradiation, as shown by increases in CFU-GM formation and proportion of c-kit+ cells in the peripheral mononuclear cells. 75-mGy x-rays also maximally induced increases in G-CSF and GM-CSF mRNA expression in splenocytes and levels of serum GM-CSF. To define the critical role of these hematopoietic-stimulating factors in HPC peripheral mobilization, direct administration of G-CSF at a dose of 300 microg/kg/day or 150 microg/kg/day was applied and found to significantly stimulate GM-CFU formation and increase c-kit+ cells in the peripheral mononuclear cells. More importantly, 75-mGy x-rays plus 150 microg/kg/day G-CSF (LDR/150-G-CSF) produced a similar effect to that of 300 microg/kg/day G-CSF alone. Furthermore, the capability of LDR-mobilized donor HPCs to repopulate blood cells was confirmed in lethally irradiated recipient mice by counting peripheral WBC and CFUs-S. These results suggest that LDR induces hematopoietic hormesis, as demonstrated by HPC proliferation and peripheral mobilization, providing a

Characterization of LDL-receptors of freshly isolated mononuclear cells of healthy subjects and of FH-patients

International Nuclear Information System (INIS)

Banyai, M.

1991-05-01

The central role of the LDL (=low density lipoproteins) receptor in artherosclerosis was first appreciated when it was shown that its absence was responsible for FH (familial hypercholesterolemia). To determine the high affinity cell surface binding activity in circulating human mononuclear cells (MNCs), these cells were incubated with low concentrations (1-50 μg protein/ml) of 123 I-LDL or 111 In-LDL either in the presence or absence of an excess of unlabeled LDL at 4 deg C for 45 minutes. MNCs of healthy subjects and of heterozygous FH-patients were found to possess high affinity LDL receptors immediately after they were isolated from the blood stream. The results indicate that the FH-patients enclosed in this study possess a reduced number of the same high affinity binding sites as healthy subjects confirming the diagnosis of heterozygous FH. In this study 123 I-LDL binding and 111 In-LDL binding to MNCs has been shown to saturable, reversible and displaceable and time-dependent. 123 I-LDL and 111 In-LDL as well can be recommended for the in-vitro determination of LDL-receptor binding activity as both binding processes show approximately the same characteristics. (author)

The Generation of Human Induced Pluripotent Stem Cells from Blood Cells: An Efficient Protocol Using Serial Plating of Reprogrammed Cells by Centrifugation

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