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

PubMed

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.

Osteoclastogenesis and Osteoclastic Resorption of Tricalcium Phosphate: Effect of Strontium and Magnesium Doping

PubMed Central

Roy, Mangal; Bose, Susmita

2012-01-01

Bone substitute materials are required to support the remodeling process, which consists of osteoclastic resorption and osteoblastic synthesis. Osteoclasts, the bone resorbing cells, generate from differentiation of hemopoietic mononuclear cells. In the present study we have evaluated the effects of 1.0 wt% strontium (Sr) and 1.0 wt% magnesium (Mg) doping in beta-tricalcium phosphate (β-TCP) on the differentiation of mononuclear cells into osteoclast-like cells and its resorptive activity. In vitro osteoclast-like cell formation, adhesion, and resorption were studied using osteoclast precursor RAW 264.7 cell, supplemented with receptor activator of nuclear factor κβ ligand (RANKL). Osteoclast-like cell formation was noticed on pure and Sr doped β-TCP samples at day 8 which was absent on Mg doped β-TCP samples indicating decrease in initial osteoclast differentiation due to Mg doping. After 21 days of culture, osteoclast-like cell formation was evident on all samples with osteoclastic markers such as actin ring, multiple nuclei, and presence of vitronectin receptor αvβ3 integrin. After osteoclast differentiation, all substrates showed osteoclast-like cell mediated degradation, however; significantly restricted for Mg doped β-TCP samples. Our present results indicated substrate chemistry controlled osteoclast differentiation and resorptive activity which can be used in designing TCP based resorbable bone substitutes with controlled degradation properties. PMID:22566212

Osteoclastogenesis and osteoclastic resorption of tricalcium phosphate: effect of strontium and magnesium doping.

PubMed

Roy, Mangal; Bose, Susmita

2012-09-01

Bone substitute materials are required to support the remodeling process, which consists of osteoclastic resorption and osteoblastic synthesis. Osteoclasts, the bone-resorbing cells, generate from differentiation of hemopoietic mononuclear cells. In the present study, we have evaluated the effects of 1.0 wt % strontium (Sr) and 1.0 wt % magnesium (Mg) doping in beta-tricalcium phosphate (β-TCP) on the differentiation of mononuclear cells into osteoclast-like cells and its resorptive activity. In vitro osteoclast-like cell formation, adhesion, and resorption were studied using osteoclast precursor RAW 264.7 cell, supplemented with receptor activator of nuclear factor κβ ligand (RANKL). Osteoclast-like cell formation was noticed on pure and Sr-doped β-TCP samples at day 8, which was absent on Mg-doped β-TCP samples indicating decrease in initial osteoclast differentiation due to Mg doping. After 21 days of culture, osteoclast-like cell formation was evident on all samples with osteoclastic markers such as actin ring, multiple nuclei, and presence of vitronectin receptor α(v)β(3) integrin. After osteoclast differentiation, all substrates showed osteoclast-like cell-mediated degradation, however, significantly restricted for Mg-doped β-TCP samples. Our present results indicated that substrate chemistry controlled osteoclast differentiation and resorptive activity, which can be used in designing TCP-based resorbable bone substitutes with controlled degradation properties. Copyright © 2012 Wiley Periodicals, Inc.

Gene expression profile in circulating mononuclear cells after exposure to ultrafine carbon particles

PubMed Central

Huang, Yuh-Chin T.; Schmitt, Michael; Yang, Zhonghui; Que, Loretta G.; Stewart, Judith C.; Frampton, Mark W.; Devlin, Robert B.

2013-01-01

Context Exposure to particulate matter (PM) is associated with systemic health effects, but the cellular and molecular mechanisms are unclear. Objective We hypothesized that, if circulating mononuclear cells play an important role in mediating systemic effects of PM, they would show gene expression changes following exposure. Materials and methods Peripheral blood samples were collected before (0 hour) and at 24 hours after exposure from healthy subjects who participated in previous controlled exposures to ultrafine carbon particles (UFP, 50 μg/m3) or filtered air (FA)(n = 3 each). RNA from mononuclear cell fraction (>85% lymphocytes) was extracted, amplified and hybridized to Affymetrix HU133 plus 2 microarrays. Results We identified 1713 genes (UFP 24 hours vs. FA 0 and 24 hours, p < 0.05, FDR 0.01). The top 10 upregulated genes (fold) were CDKN1C (1.86), ZNF12 (1.83), SRGAP2 (1.82), FYB (1.79), LSM14B (1.79), CD93 (1.76), NCSTN (1.70), DUSP6 (1.69), TACC1 (1.68) and H2AFY (1.68). Upregulation of CDKN1C and SRGAP2 was confirmed by RT-PCR using samples from additional 5 subjects exposed to FA and UFP. We entered 1020 genes with a ratio >1.1 or <−1.1 into the Ingenuity Pathway Analysis and identified many canonical pathways related to inflammation, tissue growth and host defense against environmental insults, including IGF-1 signaling, insulin receptor signaling and NRF2-mediated oxidative stress response pathway. Discussion and conclusions Two-hour exposures to UFP produced gene expression changes in circulating mononuclear cells. These gene changes provide biologically plausible links to PM-induced systemic health effects, especially those in the cardiovascular system and glucose metabolism. PMID:20507211

Optimizing autologous nonmobilized mononuclear cell collections for cellular therapy in pediatric patients with high-risk leukemia.

PubMed

Even-Or, Ehud; Di Mola, Maria; Ali, Muhammad; Courtney, Sarah; McDougall, Elizabeth; Alexander, Sarah; Schechter, Tal; Whitlock, James A; Licht, Christoph; Krueger, Joerg

2017-06-01

The manufacturing of cellular products for immunotherapy, such as chimeric antigen receptor T cells, requires successful collection of mononuclear cells. Collections from children with high-risk leukemia present a challenge, especially because the established COBE Spectra apheresis device is being replaced by the novel Spectra Optia device (Optia) in many institutions. Published experience for mononuclear cell collections in children with Optia is lacking. Our aim was to compare the two collection devices and describe modified settings on the Optia to optimize mononuclear cell collections. As a quality initiative, we retrospectively collected and compared data from mononuclear cell collections on both devices. Collected data included patient's clinical characteristics; collection parameters, including precollection lymphocyte/CD3 counts, total blood volumes processed, runtimes, and side effects (including complete blood count and electrolyte changes); and product characteristics, including volumes and cell counts. Collection efficiencies and collection ratios were calculated. Twenty-six mononuclear cell collections were performed on 20 pediatric patients: 11 with COBE and 15 with Optia. Adequate mononuclear cell products were successfully collected with a single procedure from all patients except one, with mean calculated mononuclear cell collection efficiency that was significantly higher from Optia collections compared with COBE collections (57.9 ± 4.6% vs 40.3 ± 6.2%, respectively; p = 0.04). CD3-positive yields were comparable on both machines (p = 0.34) with significantly smaller blood volumes processed on Optia. Collected products had larger volumes on Optia. No significant side effects attributed to the procedure were noted. Mononuclear cell apheresis using the Optia device in children is more efficient and is as safe as that with the COBE device. © 2017 AABB.

Mitochondrial dysfunction in blood cells from amyotrophic lateral sclerosis patients.

PubMed

Ehinger, Johannes K; Morota, Saori; Hansson, Magnus J; Paul, Gesine; Elmér, Eskil

2015-06-01

Mitochondrial dysfunction is implicated in amyotrophic lateral sclerosis, where the progressive degeneration of motor neurons results in muscle atrophy, paralysis and death. Abnormalities in both central nervous system and muscle mitochondria have previously been demonstrated in patient samples, indicating systemic disease. In this case-control study, venous blood samples were acquired from 24 amyotrophic lateral sclerosis patients and 21 age-matched controls. Platelets and peripheral blood mononuclear cells were isolated and mitochondrial oxygen consumption measured in intact and permeabilized cells with additions of mitochondrial substrates, inhibitors and titration of an uncoupler. Respiratory values were normalized to cell count and for two markers of cellular mitochondrial content, citrate synthase activity and mitochondrial DNA, respectively. Mitochondrial function was correlated with clinical staging of disease severity. Complex IV (cytochrome c-oxidase)-activity normalized to mitochondrial content was decreased in platelets from amyotrophic lateral sclerosis patients both when normalized to citrate synthase activity and mitochondrial DNA copy number. In mononuclear cells, complex IV-activity was decreased when normalized to citrate synthase activity. Mitochondrial content was increased in amyotrophic lateral sclerosis patient platelets. In mononuclear cells, complex I activity declined and mitochondrial content increased progressively with advancing disease stage. The findings are, however, based on small subsets of patients and need to be confirmed. We conclude that when normalized to mitochondria-specific content, complex IV-activity is reduced in blood cells from amyotrophic lateral sclerosis patients and that there is an apparent compensatory increase in cellular mitochondrial content. This supports systemic involvement in amyotrophic lateral sclerosis and suggests further study of mitochondrial function in blood cells as a future biomarker for the disease.

Increased oxidative stress and apoptosis in peripheral blood mononuclear cells of fructose-fed rats.

PubMed

Porto, Marcella L; Lírio, Layla M; Dias, Ananda T; Batista, Alan T; Campagnaro, Bianca P; Mill, José G; Meyrelles, Silvana S; Baldo, Marcelo P

2015-12-01

Measuring of oxidative stress in peripheral blood mononuclear cells is a suitable model of dietary induced systemic oxidative stress. Thus, we aimed to evaluate whether a chronic high fructose intake could induce oxidative damage in peripheral blood and bone marrow mononuclear cells of rats. Animals were randomly assigned to the following groups: Control group (standard rat chow and tap water n=8), and Fructose group (standard rat chow and a 10% fructose solution in the drinking water n=8). Reactive oxygen species and cytokines were measure using flow cytometry in peripheral blood and bone-marrow mononuclear cells. Apoptotic cell death and the advanced oxidation protein products (AOPP) were also determined. We observed a significant increase in ROS production in peripheral blood mononuclear cells of fructose group as compared to control rats. Apoptosis and the AOPP were higher in those animals underwent high fructose intake. Serum levels of IL-6 and IL-12 were also increased after 12 weeks of high fructose intake. We concluded that fructose intake leads to systemic oxidative stress and pro-inflammatory condition which affect peripheral blood mononuclear cells and bone-marrow mononuclear cells viability. Copyright © 2015 Elsevier B.V. All rights reserved.

Comparison of three methods of DNA extraction from peripheral blood mononuclear cells and lung fragments of equines.

PubMed

Santos, E M; Paula, J F R; Motta, P M C; Heinemann, M B; Leite, R C; Haddad, J P A; Del Puerto, H L; Reis, J K P

2010-08-17

We compared three different protocols for DNA extraction from horse peripheral blood mononuclear cells (PBMC) and lung fragments, determining average final DNA concentration, purity, percentage of PCR amplification using beta-actin, and cost. Thirty-four samples from PBMC, and 33 samples from lung fragments were submitted to DNA extraction by three different protocols. Protocol A consisted of a phenol-chloroform and isoamylic alcohol extraction, Protocol B used alkaline extraction with NaOH, and Protocol C used the DNAzol((R)) reagent kit. Protocol A was the best option for DNA extraction from lung fragments, producing high DNA concentrations, with high sensitivity in PCR amplification (100%), followed by Protocols C and B. On the other hand, for PBMC samples, Protocol B gave the highest sensitivity in PCR amplification (100%), followed by Protocols C and A. We conclude that Protocol A should be used for PCR diagnosis from lung fragment samples, while Protocol B should be used for PBMC.

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.

PubMed

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.

The metabolites in peripheral blood mononuclear cells showed greater differences between patients with impaired fasting glucose or type 2 diabetes and healthy controls than those in plasma.

PubMed

Kim, Minjoo; Kim, Minkyung; Han, Ji Yun; Lee, Sang-Hyun; Jee, Sun Ha; Lee, Jong Ho

2017-03-01

To determine differences between peripheral blood mononuclear cells and the plasma metabolites in patients with impaired fasting glucose or type 2 diabetes and healthy controls. In all, 65 nononobese patients (aged 30-70 years) with impaired fasting glucose or type 2 diabetes and 65 nonobese sex-matched healthy controls were included, and fasting peripheral blood mononuclear cell and plasma metabolomes were profiled. The diabetic or impaired fasting glucose patients showed higher circulating and peripheral blood mononuclear cell lipoprotein phospholipase A 2 activities, high-sensitivity C-reactive protein and tumour necrosis factor-α than controls. Compared with controls, impaired fasting glucose or diabetic subjects showed increases in 11 peripheral blood mononuclear cell metabolites: six amino acids (valine, leucine, methionine, phenylalanine, tyrosine and tryptophan), l-pyroglutamic acid, two fatty acid amides containing palmitic amide and oleamide and two lysophosphatidylcholines. In impaired fasting glucose or diabetic patients, peripheral blood mononuclear cell lipoprotein phospholipase A 2 positively associated with peripheral blood mononuclear cell lysophosphatidylcholines and circulating inflammatory markers, including tumour necrosis factor-α, high-sensitivity C-reactive protein and lipoprotein phospholipase A 2 activities. In plasma metabolites between patients and healthy controls, we observed significant increases in only three amino acids (proline, valine and leucine) and decreases in only five lysophosphatidylcholines. This study demonstrates significant differences in the peripheral blood mononuclear cell metabolome in patients with impaired fasting glucose or diabetes compared with healthy controls. These differences were greater than those observed in the plasma metabolome. These data suggest peripheral blood mononuclear cells as a useful tool to better understand the inflammatory pathophysiology of diabetes.

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

PubMed Central

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

Elucidation of proliferative capability of mononuclear tetraploid cells, emerging spontaneously from diploid cells, using image cytometry and fluorescence in situ hybridization.

PubMed

Ito, Hideaki; Oga, Atsunori; Furuya, Tomoko; Ikemoto, Kenzo; Amakawa, Genta; Chochi, Yasuyo; Kawauchi, Shigeto; Sasaki, Kohsuke

2013-06-01

Proliferation of tetraploid cells (TCs) emerging from diploid cells is considered to be a critical event toward tumourigenesis, or cancer progression. Recently, several studies have reported that binuclear TCs emerging from normal cells are capable of mitosis, however, it has not been confirmed directly whether mononuclear TCs emerging from normal cells could proliferate, even cancer cells. The aim of this study is to detect mononuclear TCs in vitro, spontaneously emerging from diploid cells and to elucidate their proliferative capability directly. For this purpose, we have developed a novel method. In this study, two completely disomic cell lines were used, TIG-7, a fibroblast cell line and CAL-51, a breast cancer cell line. Cells were cultured on microscope slides and their DNA content was determined using an image cytometer. On the same slides, chromosome numbers were scored using centromere fluorescence in situ hybridization (FISH). For evaluating proliferative capability of TCs, bromodeoxyuridine (BrdUrd) incorporation and colony-forming ability were examined. Using our method, spontaneous emergence of mononuclear TCs was detected in both TIG-7 and CAL-51. Colonies of TIG-7 TCs were not observed, but were observed of CAL-51 TCs. Our method enables detection of mononuclear TCs and elucidation of their proliferative capability, directly; this evidence reveals that mononuclear TIG-7 TCs do not proliferate but that mononuclear CAL-51 TCs are able to. © 2013 Blackwell Publishing Ltd.

Segregation of human peripheral blood lymphocytes according to their affinity for insolubilized histamine. Principal differences between males and females.

PubMed Central

Tartakovsky, B; Segal, S; Shani, A; Hellerstein, S; Weinstein, Y; Bentwich, Z

1979-01-01

An attempt was made to investigate the possible existence of differences in the composition of peripheral blood lymphocytes between males and females. Using affinity chromatography of human peripheral mononuclear cells on insolubilized histamine together with staining by fluoresceinated histamine-rabbit serum albumin (HRSA) we revealed that males possess a significantly higher proportion of mononuclear cells which bind to HRSA. These results are also reflected in sex-related differences in proliferative responses of the HRSA-non-adherent mononuclear cell population to T cell-dependent mitogens antigens and allogeneic mononuclear cells. PMID:160849

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

PubMed

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.

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.

PubMed

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.

Effect of dialyzer membranes on beta-2 microglobulin production in Thai hemodialysis patients.

PubMed

Domrongkitchaiporn, S; Chuncharunee, S; Archararit, N; Atamasirikul, K; Vanichakarn, S

1997-09-01

Responses to different types of dialyzer membranes in an Asian population may differ from those of a Caucasian population. Comparative studies on the effects of different dialyzer membranes on beta-2 microglobulin production are also limited. Therefore, we conducted this study to determine the effects of different dialyzer membranes on in vitro mononuclear cell production of beta-2 microglobulin in 9 Thai hemodialysis patients. Each patient was dialysed with 4 different types of dialyzer, including cuprophane (CUP), cellulose diacetate (CD), polysulphone (PS), and polyacrylonitrile membrane (PAN), each for a 1-month period in a randomized sequence. Mononuclear cell culture was done by taking an immediate post-dialysis blood sample at the end of the 1-month period. Beta-2 microglobulin production from cell culture was determined 24 hours later. Mononuclear cell culture and determination of beta-2 microglobulin production from the culture were also done in 10 normal controls and 10 predialysis ESRD patients. The beta-2 microglobulin productions (microgram/L) were shown as follows; Control CUP CD PS PAN [table: see text] (*p < 0.05 compared to cuprophane membrane). polysulphone and polyacrylonitrile membrane induced significantly less beta-2 microglobulin production compared to cuprophane and slightly less compared to cellulose diacetate membrane.

The effects of compound danshen dripping pills and human umbilical cord blood mononuclear cell transplant after acute myocardial infarction.

PubMed

Jun, Yi; Chunju, Yuan; Qi, Ai; Liuxia, Deng; Guolong, Yu

2014-04-01

The low frequency of survival of stem cells implanted in the myocardium after acute myocardial infarction may be caused by inflammation and oxidative stress in the myocardial microenvironment. We evaluated the effects of a traditional Chinese medicine, Compound Danshen Dripping Pills, on the cardiac microenvironment and cardiac function when used alone or in combination with human umbilical cord blood mononuclear cell transplant after acute myocardial infarction. After surgically induced acute myocardial infarction, rabbits were treated with Compound Danshen Dripping Pills alone or in combination with human umbilical cord blood mononuclear cell transplant. Evaluation included histology, measurement of left ventricular ejection fraction and fractional shortening, leukocyte count, count of green fluorescent protein positive cells, superoxide dismutase activity, and malondialdehyde content. Combination treatment with Compound Danshen Dripping Pills and human umbilical cord blood mononuclear cell transplant significantly increased the survival of implanted cells, inhibited cardiac cell apoptosis, decreased oxidative stress, decreased the inflammatory response, and improved cardiac function. Rabbits treated with either Compound Danshen Dripping Pills or human umbilical cord blood mononuclear cells alone had improvement in these effects compared with untreated control rabbits. Combination therapy with Compound Danshen Dripping Pills and human umbilical cord blood mononuclear cells may improve cardiac function and morphology after acute myocardial infarction.

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

PubMed

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.

Intra-Hepatic Depletion of Mucosal-Associated Invariant T Cells in Hepatitis C Virus-Induced Liver Inflammation.

PubMed

Bolte, Fabian J; O'Keefe, Ashley C; Webb, Lauren M; Serti, Elisavet; Rivera, Elenita; Liang, T Jake; Ghany, Marc; Rehermann, Barbara

2017-11-01

Chronic hepatitis affects phenotypes of innate and adaptive immune cells. Mucosal-associated invariant T (MAIT) cells are enriched in the liver as compared with the blood, respond to intra-hepatic cytokines, and (via the semi-invariant T-cell receptor) to bacteria translocated from the gut. Little is known about the role of MAIT cells in livers of patients with chronic hepatitis C virus (HCV) infection and their fate after antiviral therapy. We collected blood samples from 42 patients with chronic HCV infection who achieved a sustained virologic response after 12 weeks of treatment with sofosbuvir and velpatasvir. Mononuclear cells were isolated from blood before treatment, at weeks 4 and 12 during treatment, and 24 weeks after the end of treatment. Liver biopsies were collected from 37 of the patients prior to and at week 4 of treatment. Mononuclear cells from 56 blood donors and 10 livers that were not suitable for transplantation were used as controls. Liver samples were assessed histologically for inflammation and fibrosis. Mononuclear cells from liver and blood were studied by flow cytometry and analyzed for responses to cytokine and bacterial stimulation. The frequency of MAIT cells among T cells was significantly lower in blood and liver samples of patients with HCV infection than of controls (median, 1.31% vs 2.32% for blood samples, P = .0048; and median, 4.34% vs 13.40% for liver samples, P = .001). There was an inverse correlation between the frequency of MAIT cells in the liver and histologically determined levels of liver inflammation (r = -.5437, P = .0006) and fibrosis (r = -.5829, P = .0002). MAIT cells from the liver had higher levels of activation and cytotoxicity than MAIT cells from blood (P < .0001). Production of interferon gamma by MAIT cells was dependent on monocyte-derived interleukin 18, and was reduced in patients with HCV infection in response to T-cell receptor-mediated but not cytokine-mediated stimulation, as compared with controls. Anti-viral therapy rapidly decreased liver inflammation and MAIT cell activation and cytotoxicity, and increased the MAIT cell frequency among intra-hepatic but not blood T cells. The MAIT cell response to T-cell receptor-mediated stimulation did not change during the 12 weeks of antiviral therapy. In analyses of paired blood and liver samples from patients with chronic HCV infection before, during, and after antiviral therapy with sofosbuvir and velpatasvir, we found that intrahepatic MAIT cells are activated by monocyte-derived cytokines and depleted in HCV-induced liver inflammation. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

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

PubMed

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

2012-12-01

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-equine antibodies. These data indicate that a cellular immune response is not elicited following implantation with the eHAC block graft, which might serve as an alternative material for regenerative therapy.

Analysis of synovial fluid of the Capybara's stifle joints.

PubMed

Brombini, Giovanna C; Rahal, Sheila C; Bergamini, Bruno C S; Lopes, Raimundo S; Santos, Ivan F C; Schimming, Bruno C

2017-03-01

Although normal synovial fluid has been well characterized in domestic animals such as dogs, cats, horses, and cows, the available information on larger rodents is scarce. The purpose of the study was to analyze the physical, chemical, and cytologic characteristics of the synovial fluid in stifle joints of Capybaras. Five free-ranging adult female Capybaras (Hydrochoerus hydrochaeris), weighing from 37 to 56 kg were used. Synovial fluid was obtained by aspiration of 10 stifle joints. Samples were analyzed for physical, chemical, and cytologic properties. Spontaneous clotting was negative in 9 samples. Most synovial fluids had pH 8, and protein concentrations ranged from 1.6 to 3.6 g/dL. The mucin clot test was good in all 6 samples that were tested. Nucleated cell counts ranged from 140 to 508 cells/μL. Relative differential leukocyte counts demonstrated a predominance of mononuclear cells (97.6%), including 76.2% undifferentiated mononuclear cells, 18.1% macrophages, and 3.66% lymphocytes. Polymorphonuclear cells included 1.83% neutrophils and 0.2% eosinophils. The synovial stifle joint fluid of healthy free-ranging adult Capybaras is clear, colorless, viscous, and with chemical features and cytologic findings similar to those seen in domestic animals. © 2017 American Society for Veterinary Clinical Pathology.

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

PubMed

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

2016-01-01

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

Effect of repeated arthrocentesis on cytologic analysis of synovial fluid in dogs.

PubMed

Berg, R I M; Sykes, J E; Kass, P H; Vernau, W

2009-01-01

Serial arthrocentesis and synovial fluid examination can be used to monitor treatment efficacy in immune-mediated polyarthritis (IMPA), but whether this procedure induces inflammation that interferes with test result interpretation is unknown. The aim of this study was to determine the effect of repeated arthrocentesis on synovial fluid cytology in healthy dogs. Nine healthy client-owned dogs. Prospective study. Arthrocentesis was performed under sedation on 4 joints (both carpi, 1 tarsus, 1 stifle) on each dog every 3 weeks, a total of 4 times. Automated cell counts were done on stifle fluid, smears were made, and differential cell counts done on smears from all joints. Slides were evaluated microscopically for erythrocyte numbers, total nucleated cell count, differential cell count, and cell morphology. Data were analyzed by 2-way analysis of variance. A total of 144 synovial fluid samples were examined. Repeated arthrocentesis was not associated with increases in synovial fluid neutrophil numbers. Mild mononuclear inflammation was detected in 13 samples from 6 dogs. Serial arthrocentesis at 3-week intervals can rarely be associated with mild mononuclear joint inflammation, but does not appear to induce neutrophilic inflammation, at least in healthy dogs, and can be useful to monitor treatment response in canine IMPA.

HSP70 in human polymorphonuclear and mononuclear leukocytes: comparison of the protein content and transcriptional activity of HSPA genes.

PubMed

Boyko, Anna A; Azhikina, Tatyana L; Streltsova, Maria A; Sapozhnikov, Alexander M; Kovalenko, Elena I

2017-01-01

Cell-type specific variations are typical for the expression of different members of the HSP70 family. In circulating immune cells, HSP70 proteins interact with units of signaling pathways involved in the immune responses and may promote cell survival in sites of inflammation. In this work, we compared basal HSP70 expression and stress-induced HSP70 response in polymorphonuclear and mononuclear human leukocytes. The intracellular content of inducible and constitutive forms of HSP70 was analyzed in relation to the transcriptional activity of HSPA genes. Hyperthermia was used as the stress model for induction of HSP70 synthesis in the cells. Our results demonstrated that granulocytes (mainly neutrophils) and mononuclear cells differ significantly by both basal HSP70 expression and levels of HSP70 induction under hyperthermia. The differences were observed at the levels of HSPA gene transcription and intracellular HSP70 content. The expression of constitutive Hsс70 protein was much higher in mononuclear cells consisting of monocytes and lymphocytes than in granulocytes. At the same time, intact neutrophils showed increased expression of inducible Hsp70 protein compared to mononuclear cells. Heat treatment induced additional expression of HSPA genes in leukocytes. The most pronounced increase in the expression was observed in polymorphonuclear and mononuclear leukocytes for HSPA1A/B. However, in granulocytes, the induction of the transcription of the HSPA8 gene encoding the Hsc70 protein was significantly higher than in mononuclear cells. These variations in transcriptional activity of HSPA genes and intracellular HSP70 content in different populations of leukocytes may reflect specified requirements for the chaperone activity in the cells with a distinct functional role in the immune system.

[Bone marrow mononuclear cells from murine tibia after the space flight on biosatellite "Bion-M1"].

PubMed

Andreeva, E R; Goncharova, E A; Gornostaeva, A N; Grigor'eva, O V; Buravkova, L B

2014-01-01

Cellularity, viability and immunophenotype of mononuclear cells derived from the tibial marrow of C57bL/6 mice were measured after the 30-day "Bion-M1" space flight and subsequent 7-day recovery. Cell number in the flight group was significantly less than in the group of vivarium control. There was no difference in the parameter between the flight and control groups after the recovery. Viability of mononuclear cells was more than 95% in all examined groups. Flow cytometric analysis failed to show differences in bone marrow cell immunophenotype (CD45, CD34, CD90.1 (Thy1); however, the flight animals had more large-sized CD45+ mononuclears than the control groups of mice. These results indicate that spaceflight factors did not have significant damaging effects on the number or immunophenotype of murine bone marrow mononuclears. These observations are consistent with the previously made assumption of a moderate and reversible stress reaction of mammals to space flight.

No evidence of persisting measles virus in peripheral blood mononuclear cells from children with autism spectrum disorder.

PubMed

D'Souza, Yasmin; Fombonne, Eric; Ward, Brian J

2006-10-01

Despite epidemiologic evidence to the contrary, claims of an association between measles-mumps-rubella vaccination and the development of autism have persisted. Such claims are based primarily on the identification of measles virus nucleic acids in tissues and body fluids by polymerase chain reaction. We sought to determine whether measles virus nucleic acids persist in children with autism spectrum disorder compared with control children. Peripheral blood mononuclear cells were isolated from 54 children with autism spectrum disorder and 34 developmentally normal children, and up to 4 real-time polymerase chain reaction assays and 2 nested polymerase chain reaction assays were performed. These assays targeted the nucleoprotein, fusion, and hemagglutinin genes of measles virus using previously published primer pairs with detection by SYBR green I. Our own real-time assay targeted the fusion gene using novel primers and an internal fluorescent probe. Positive reactions were evaluated rigorously, and amplicons were sequenced. Finally, anti-measles antibody titers were measured by enzyme immunoassay. The real-time assays based on previously published primers gave rise to a large number of positive reactions in both autism spectrum disorder and control samples. Almost all of the positive reactions in these assays were eliminated by evaluation of melting curves and amplicon band size. The amplicons for the remaining positive reactions were cloned and sequenced. No sample from either autism spectrum disorder or control groups was found to contain nucleic acids from any measles virus gene. In the nested polymerase chain reaction and in-house assays, none of the samples yielded positive results. Furthermore, there was no difference in anti-measles antibody titers between the autism and control groups. There is no evidence of measles virus persistence in the peripheral blood mononuclear cells of children with autism spectrum disorder.

Tularemia: Current Diagnosis and Treatment Options

DTIC Science & Technology

2008-04-01

for growing F. tularensis, which include cysteine blood agar, Thayer–Martin agar and cysteine heart agar with 9% heated sheep red blood cells (CHAB...samples contain inhibitors to PCR reactions, such the heme component of red blood cells [36]. These inhibitors cause the limit of detection of the organism...signaling and cytokine secretion in mouse monocytic and human peripheral blood mononuclear cells . Microb. Pathog. 38, 239–247 (2005). 16 Hrstka R

Study of muscle cell dedifferentiation after skeletal muscle injury of mice with a Cre-Lox system.

PubMed

Mu, Xiaodong; Peng, Hairong; Pan, Haiying; Huard, Johnny; Li, Yong

2011-02-03

Dedifferentiation of muscle cells in the tissue of mammals has yet to be observed. One of the challenges facing the study of skeletal muscle cell dedifferentiation is the availability of a reliable model that can confidentially distinguish differentiated cell populations of myotubes and non-fused mononuclear cells, including stem cells that can coexist within the population of cells being studied. In the current study, we created a Cre/Lox-β-galactosidase system, which can specifically tag differentiated multinuclear myotubes and myotube-generated mononuclear cells based on the activation of the marker gene, β-galactosidase. By using this system in an adult mouse model, we found that β-galactosidase positive mononuclear cells were generated from β-galactosidase positive multinuclear myofibers upon muscle injury. We also demonstrated that these mononuclear cells can develop into a variety of different muscle cell lineages, i.e., myoblasts, satellite cells, and muscle derived stem cells. These novel findings demonstrated, for the first time, that cellular dedifferentiation of skeletal muscle cells actually occurs in mammalian skeletal muscle following traumatic injury in vivo.

Nanoparticles of barium induce apoptosis in human phagocytes

PubMed Central

Mores, Luana; França, Eduardo Luzia; Silva, Núbia Andrade; Suchara, Eliane Aparecida; Honorio-França, Adenilda Cristina

2015-01-01

Purpose Nutrients and immunological factors of breast milk are essential for newborn growth and the development of their immune system, but this secretion can contain organic and inorganic toxins such as barium. Colostrum contamination with barium is an important issue to investigate because this naturally occurring element is also associated with human activity and industrial pollution. The study evaluated the administration of barium nanoparticles to colostrum, assessing the viability and functional activity of colostral mononuclear phagocytes. Methods Colostrum was collected from 24 clinically healthy women (aged 18–35 years). Cell viability, superoxide release, intracellular Ca2+ release, and phagocyte apoptosis were analyzed in the samples. Results Treatment with barium lowered mononuclear phagocyte viability, increased superoxide release, and reduced intracellular calcium release. In addition, barium increased cell death by apoptosis. Conclusion These data suggest that nanoparticles of barium in colostrum are toxic to cells, showing the importance of avoiding exposure to this element. PMID:26451108

Nanoparticles of barium induce apoptosis in human phagocytes.

PubMed

Mores, Luana; França, Eduardo Luzia; Silva, Núbia Andrade; Suchara, Eliane Aparecida; Honorio-França, Adenilda Cristina

2015-01-01

Nutrients and immunological factors of breast milk are essential for newborn growth and the development of their immune system, but this secretion can contain organic and inorganic toxins such as barium. Colostrum contamination with barium is an important issue to investigate because this naturally occurring element is also associated with human activity and industrial pollution. The study evaluated the administration of barium nanoparticles to colostrum, assessing the viability and functional activity of colostral mononuclear phagocytes. Colostrum was collected from 24 clinically healthy women (aged 18-35 years). Cell viability, superoxide release, intracellular Ca(2+) release, and phagocyte apoptosis were analyzed in the samples. Treatment with barium lowered mononuclear phagocyte viability, increased superoxide release, and reduced intracellular calcium release. In addition, barium increased cell death by apoptosis. These data suggest that nanoparticles of barium in colostrum are toxic to cells, showing the importance of avoiding exposure to this element.

Basic surface properties of mononuclear cells from Didelphis marsupialis.

PubMed

Nacife, V P; de Meirelles, M de N; Silva Filho, F C

1998-01-01

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 degrees and 40.8 degrees, 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.

The Peyer’s Patch Mononuclear Phagocyte System at Steady State and during Infection

PubMed Central

Da Silva, Clément; Wagner, Camille; Bonnardel, Johnny; Gorvel, Jean-Pierre; Lelouard, Hugues

2017-01-01

The gut represents a potential entry site for a wide range of pathogens including protozoa, bacteria, viruses, or fungi. Consequently, it is protected by one of the largest and most diversified population of immune cells of the body. Its surveillance requires the constant sampling of its encounters by dedicated sentinels composed of follicles and their associated epithelium located in specialized area. In the small intestine, Peyer’s patches (PPs) are the most important of these mucosal immune response inductive sites. Through several mechanisms including transcytosis by specialized epithelial cells called M-cells, access to the gut lumen is facilitated in PPs. Although antigen sampling is critical to the initiation of the mucosal immune response, pathogens have evolved strategies to take advantage of this permissive gateway to enter the host and disseminate. It is, therefore, critical to decipher the mechanisms that underlie both host defense and pathogen subversive strategies in order to develop new mucosal-based therapeutic approaches. Whereas penetration of pathogens through M cells has been well described, their fate once they have reached the subepithelial dome (SED) remains less well understood. Nevertheless, it is clear that the mononuclear phagocyte system (MPS) plays a critical role in handling these pathogens. MPS members, including both dendritic cells and macrophages, are indeed strongly enriched in the SED, interact with M cells, and are necessary for antigen presentation to immune effector cells. This review focuses on recent advances, which have allowed distinguishing the different PP mononuclear phagocyte subsets. It gives an overview of their diversity, specificity, location, and functions. Interaction of PP phagocytes with the microbiota and the follicle-associated epithelium as well as PP infection studies are described in the light of these new criteria of PP phagocyte identification. Finally, known alterations affecting the different phagocyte subsets during PP stimulation or infection are discussed. PMID:29038658

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

NASA Technical Reports Server (NTRS)

Meehan, R. T.

1986-01-01

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

Formation of solid tumors by a single multinucleated cancer cel

PubMed Central

Weihua, Zhang; Lin, Qingtang; Ramoth, Asa J.; Fan, Dominic; Fidler, Isaiah J.

2011-01-01

BACKGROUND Large multinucleated cells (MNC) commonly exist in tumorigenic cancer cell lines widely used in research, but their contributions to tumorigenesis are unknown. METHODS In this study, we characterized MNCs in the murine fibrosarcoma cell line UV-2237 in vitro and in vivo at a single cell level. RESULTS We observed that MNCs originated from a rare subpopulation of mononuclear cells; MNCs were positive for a senescent marker, β-galacosidase (SA-β-Gal); MNCs were responsible for the majority of clonogenic activity when cultured in hard agar; MNCs were more resistant to chemotherapeutic agents than were mononuclear cells; MNCs could undergo asymmetric division (producing mononuclear cells) and self-renewal in vitro and in vivo; and, most importantly a single MNC produced orthotopic subcutaneous tumors (composed mainly of mononuclear cells) that gave rise to spontaneous lung metastases in nude mice. CONCLUSIONS MNCs can be growth-arrested under stress, are highly resistant to chemotherapy, and can generate clonal orthotopic metastatic tumors PMID:21365635

Autorosette formation of erythrocytes on peripheral blood mononuclear cells in dogs vaccinated with canine distemper live-virus vaccine.

PubMed

Chandler, J P; Yang, T J

1981-08-01

A time course study of the peripheral blood leukocytes of dogs vaccinated with canine distemper live virus (a paramyxovirus) vaccines showed that autorosette-forming leukocytes appeared from day 3 to day 10 after vaccination. The number of these cells peaked at day 7 when as many as 35% of mononuclear cells formed rosettes with autologous erythrocytes. In contrast, in nonvaccinated dogs, only 0.6 +/- 0.3% (standard error of the mean) of mononuclear cells formed rosettes throughout the 2-week period.

Autologous intravenous bone marrow mononuclear cell therapy for patients with subacute ischaemic stroke: A pilot study

PubMed Central

Prasad, Kameshwar; Mohanty, Sujata; Bhatia, Rohit; Srivastava, M.V.P.; Garg, Ajay; Srivastava, Achal; Goyal, Vinay; Tripathi, Manjari; Kumar, Amit; Bal, Chandrashekar; Vij, Aarti; Mishra, Nalini Kant

2012-01-01

Background & objectives: Bone marrow mononuclear cell therapy has emerged as one of the option for the treatment of Stroke. Several preclinical studies have shown that the treatment with mononuclear cell (MNCs) can reduce the infarct size and improve the functional outcome. We evaluated the feasibility, safety and clinical outcome of administering bone marrow mononuclear cell (MNCs) intravenously to patients with subacute ischaemic stroke. Methods: In a non-randomized phase-I clinical study, 11 consecutive, eligible and consenting patients, aged 30-70 yr with ischaemic stroke involving anterior circulation within 7 to 30 days of onset of stroke were included. Bone marrow was aspirated from iliac crest and the harvested mononuclear cells were infused into antecubital vein. Outcomes measured for safety included immediate reactions after cell infusion and evidence of tumour formation at one year in whole body PET scan. Patients were followed at week 1, 4-6, 24 and 52 to determine clinical progress using National Institute of Health Stroke Scale (NIHSS), Barthel Index (BI), modified Rankin Scale (mRS), MRI, EEG and PET. Feasibility outcomes included target-dose feasibility. Favourable clinical outcome was defined as mRS score of 2 or less or BI score of 75 to 100 at six months after stem cell therapy. Results: Between September 2006 and April 2007, 11 patients were infused with bone-marrow mononuclear cells (mean 80 million with CD-34+ mean 0.92 million). Protocol was target-dose feasible in 9 patients (82%). FDG-PET scan at 24 and 52 wk in nine patients did not reveal evidence of tumour formation. Seven patients had favourable clinical outcome. Interpretation & conclusions: Intravenous bone marrow mononuclear cell therapy appears feasible and safe in patients with subacute ischaemic stroke. Further, a randomized controlled trial to examine its efficacy is being conducted. PMID:22960888

In vitro expansion of Lin+ and Lin- mononuclear cells from human peripheral blood

NASA Astrophysics Data System (ADS)

Norhaiza, H. Siti; Rohaya, M. A. W.; Zarina, Z. A. Intan; Hisham, Z. A. Shahrul

2013-11-01

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-) 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+) mononuclear cells expressing lineage positive markers such as CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a to obtained Lin- cell population. The ability of Lin+ and Lin- 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+ and Lin- 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+ mononuclear cells were not able to survive either in normal proliferation medium or proliferation medium supplemented with cytokines. Similarly, Lin- stem cells were not able to survive in proliferation medium however, addition of cytokines into the proliferation medium support Lin- stem cells for at least 18 days. The Lin- stem cells started to response to the cytokines added as early as Day 2 of culture. It is concluded that Lin- stem cells can be expanded in vitro by culturing in proliferation medium supplemented with cytokines.

The Symptomatic Persian Gulf Veterans Protocol: An Analysis of Risk Factors with an Immunologic and Neuropsychiatric Assessment.

DTIC Science & Technology

1999-01-01

Evaluation of peripheral blood mononuclear cells, urine and throat cultures samples for the presence of mycoplasma or ureaplasma organisms by...c) by PCR with a universal mycoplasma primer. Mycoplasma or ureaplasma were detected frequently in urine and throat swabs, however no significant

THE CONTRIBUTION OF TYRO3 FAMILY RECEPTOR TYROSINE KINASES TO THE HETEROGENEITY OF APOPTOTIC CELL UPTAKE BY MONONUCLEAR PHAGOCYTES

PubMed Central

Curtis, Jeffrey L.; Todt, Jill C.; Hu, Bin; Osterholzer, John J.; Freeman, Christine M.

2014-01-01

Mononuclear phagocytes comprise a mobile, broadly dispersed and highly adaptable system that lies at the very epicenter of host defense against pathogens and the interplay of the innate and adaptive arms of immunity. Understanding the molecular mechanisms that control the response of mononuclear phagocytes to apoptotic cells and the anti-inflammatory consequences of that response is an important goal with implications for multiple areas of biomedical sciences. This review details current understanding of the heterogeneity of apoptotic cell uptake by different members of the mononuclear phagocyte family in humans and mice. It also recounts the unique role of the Tyro3 family of receptor tyrosine kinases, best characterized for Mertk, in the signal transduction leading both to apoptotic cell ingestion and the anti-inflammatory effects that result. PMID:19273223

Tumor necrosis factor-alpha (TNF-alpha) concentrations from whole blood cultures correlate with isolated peripheral blood mononuclear cell cultures

USDA-ARS?s Scientific Manuscript database

Many cellular immune assays are impractical because they require labor-intensive isolation of cells from their natural environment. The objectives of this study were to determine the relationship between cell culture supernatant TNF-alpha from isolated peripheral blood mononuclear cells (PBMC) and w...

Composition and functional capacity of blood mononuclear leukocyte populations from neonatal calves on standard and intensified milk replacer diets.

PubMed

Nonnecke, B J; Foote, M R; Smith, J M; Pesch, B A; Van Amburgh, M E

2003-11-01

Effects of increased dietary energy and protein on the composition and functional capacities of blood mononuclear leukocyte populations from milk replacer-fed calves were investigated. Holstein bull calves (average age: 4.2 d; n = 19) were assigned randomly to one of two treatment groups. Treatment 1 calves (n = 9) were fed a 20% crude protein, 20% fat milk replacer at a rate of 1.4% body weight of dry matter/d for 8 wk, whereas treatment 2 calves (n = 10) were fed a 30% crude protein, 20% fat milk replacer at a rate of 2.5% body weight of dry matter per day. Composition and functional capacities of mononuclear leukocyte populations from blood samples collected at 4, 18, 32, 46, and 60 d of age were characterized by flow cytometry and ex vivo cell function assays. From 11 to 60 d of age, the mean daily weight gain of treatment 2 calves (1.20 kg/d) was greater than daily weight gain of treatment 1 calves (0.55 kg/d). At 60 d of age, the mean body weight of treatment two calves was 53% (39 kg) greater than the mean body weight of treatment 1 calves. Total numbers of blood leukocytes and the composition of the mononuclear leukocyte population were unaffected by the plane of nutrition. Mitogen-induced DNA-synthesis and immunoglobulin M secretion also were unaffected by dietary treatment. Blood mononuclear leukocytes from calves on intensified diets, however, produced less interferon-gamma and more inducible nitric oxide, suggesting that increased dietary energy and protein affects specific aspects of leukocyte function associated with cell-mediated immunity. The impact of altered interferon-gamma and NO production on the calf s susceptibility to infectious disease are not known. Mononuclear leukocyte populations from all calves also demonstrated age-related changes in composition and functional capacity, likely reflecting natural exposure to infectious agents and maturation of the calfs immune system.

Mononuclear phagocyte subpopulations in the mouse kidney.

PubMed

George, James F; Lever, Jeremie M; Agarwal, Anupam

2017-04-01

Mononuclear phagocytes are the most common cells in the kidney associated with immunity and inflammation. Although the presence of these cells in the kidney has been known for decades, the study of mononuclear phagocytes in the context of kidney function and dysfunction is still at an early stage. The purpose of this review is to summarize the present knowledge regarding classification of these cells in the mouse kidney and to identify relevant questions that would further advance the field and potentially lead to new opportunities for treatment of acute kidney injury and other kidney diseases.

Effect of curcumin on accumulation in mononuclear cells and secretion in incubation medium of Аβ(40) and cytokines under local excess of Аβ(42)-homoaggregates.

PubMed

Sokolik, V V; Shulga, S M

2016-01-01

The aim of the work was to investigate accumulation of endogenous Aβ40 and cytokines (IL-1β, TNFα, IL-6, IL-10) in mononuclear cells and their secretion into incubation medium under Aβ42-aggregates’ toxicity and anti-inflammatory effects of curcumin. Mononuclear cells were isolated in Ficoll-Urografin density gradient from venous blood of healthy donors, resuspended and used for testing of homoaggregates of Aβ42 (15 nM), curcumin (54 pM) and their combinations on various timescales (0, 1, 2, 3, 6 and 24 hours). Endogenous Aβ40 and cytokines were detected in mononuclear cells and (separately) in incubation medium by ELISA. We demonstrated for the first time that homoaggregates of Aβ42 cause rapid accumulation of endogenous Aβ40 in mononuclear cells and accelerate its secretion into incubation medium. We found increased concentration of TNFα after 3 hours of incubation, and no changes in IL-1β concentration due to secretion of these pro-inflammatory factors into incubation medium. The concentrations of IL-6 in mononuclear cells were increased under effects of Aβ42 homoaggregates, and it was being secreted profoundly into incubation medium. Aβ42 did not affect IL-10 secretion, yet caused an increase in its intracellular concentration after 1 hour of incubation, which was subsequently suppressed. Curcumin prevented the increase in Aβ40 concentration in mononuclear cells and significantly decreased its secretion resulting from Aβ42 toxicity. Curcumin negated the activating effect of Aβ42 on pro-inflammatory cytokines, starting immediately for IL-1β and on 3-6 hours for TNFα, which resulted in decreased extracellular concentrations of these cytokines. The polyphenol also potentiated repleni­shing of intracellular IL-6 and IL-10 concentrations and their secretion into incubation medium.

Review: Occult hepatitis C virus infection: still remains a controversy.

PubMed

Vidimliski, Pavlina Dzekova; Nikolov, Igor; Geshkovska, Nadica Matevska; Dimovski, Aleksandar; Rostaing, Lionel; Sikole, Aleksandar

2014-09-01

Occult hepatitis C virus (HCV) infection is characterized by the presence of HCV RNA in the liver cells or peripheral blood mononuclear cells of the patients whose serum samples test negative for HCV RNA, with or without presence of HCV antibodies. The present study reviews the existing literature on the persistence of occult hepatitis C virus infection, with description of the clinical characteristics and methods for identification of occult hepatitis C. Occult hepatitis C virus infection was detected in patients with abnormal results of liver function tests of unknown origin, with HCV antibodies and HCV RNA negativity in serum, and also in patients with spontaneous or treatment-induced recovery from hepatitis C. The viral replication in the liver cells and/or peripheral blood mononuclear cells was present in all clinical presentations of occult hepatitis C. The peripheral blood mononuclear cells represent an extra-hepatic site of HCV replication. The reason why HCV RNA was not detectable in the serum of patients with occult hepatitis C, could be the low number of circulating viral particles not detectable by the diagnostic tests with low sensitivity. It is uncertain whether occult hepatitis C is a different clinical entity or just a form of chronic hepatitis C virus infection. Data accumulated over the last decade demonstrated that an effective approach to the diagnosis of HCV infection would be the implementation of more sensitive HCV RNA diagnostic assays, and also, examination of the presence of viral particles in the cells of the immune system. © 2014 Wiley Periodicals, Inc.

Lymphocyte apheresis for chimeric antigen receptor T-cell manufacturing in children and young adults with leukemia and neuroblastoma.

PubMed

Ceppi, Francesco; Rivers, Julie; Annesley, Colleen; Pinto, Navin; Park, Julie R; Lindgren, Catherine; Mgebroff, Stephanie; Linn, Naomi; Delaney, Meghan; Gardner, Rebecca A

2018-06-01

The first step in the production of chimeric antigen receptor T cells is the collection of autologous T cells using apheresis technology. The procedure is technically challenging, because patients often have low leukocyte counts and are heavily pretreated with multiple lines of chemotherapy, marrow transplantation, and/or radiotherapy. Here, we report our experience of collecting T lymphocytes for chimeric antigen receptor T-cell manufacturing in pediatric and young adult patients with leukemia, non-Hodgkin lymphoma, or neuroblastoma. Apheresis procedures were performed on a COBE Spectra machine using the mononuclear cell program, with a collection target of 1 × 10 9 total mononuclear cells per kilogram. Data were collected regarding preapheresis and postapheresis blood counts, apheresis parameters, products, and adverse events. Ninety-nine patients (ages 1.3-25.7 years) and 102 apheresis events were available for analysis. Patients underwent apheresis at a variety of absolute lymphocyte cell counts, with a median absolute lymphocyte count of 944 cells/μL (range, 142-6944 cells/μL). Twenty-two patients (21.6%) had absolute lymphocyte counts less than 500 cells/μL. The mononuclear cell target was obtained in 100% of all apheresis harvests, and chimeric antigen receptor T-cell production was possible from the majority of collections (94%). Mononuclear cell collection efficiency was 65.4%, and T-lymphocyte collection efficiency was 83.4%. Ten patients (9.8%) presented with minor adverse events during the 102 apheresis procedures, with one exception of a severe allergy. Mononuclear cell apheresis for chimeric antigen receptor T-cell therapy is well tolerated and safe, and it is possible to obtain an adequate quantity of CD3+ lymphocytes for chimeric antigen receptor T-cell manufacturing in heavily pretreated patients who have low lymphocyte counts. © 2018 AABB.

Decreased non-MHC-restricted (CD56+) killer cell cytotoxicity after spaceflight

NASA Technical Reports Server (NTRS)

Mehta, S. K.; Kaur, I.; Grimm, E. A.; Smid, C.; Feeback, D. L.; Pierson, D. L.

2001-01-01

Cytotoxic activity of non-major histocompatibility complex-restricted (CD56+) (NMHC) killer cells and cell surface marker expression of peripheral blood mononuclear cells were determined before and after spaceflight. Ten astronauts (9 men, 1 woman) from two space shuttle missions (9- and 10-day duration) participated in the study. Blood samples were collected 10 days before launch, within 3 h after landing, and 3 days after landing. All peripheral blood mononuclear cell preparations were cryopreserved and analyzed simultaneously in a 4-h cytotoxicity (51)Cr release assay using K562 target cells. NMHC killer cell lytic activity was normalized per 1,000 CD56+ cells. When all 10 subjects were considered as one study group, NMHC killer cell numbers did not change significantly during the three sampling periods, but at landing lytic activity had decreased by approximately 40% (P < 0.05) from preflight values. Nine of ten astronauts had decreased lytic activity immediately after flight. NMHC killer cell cytotoxicity of only three astronauts returned toward preflight values by 3 days after landing. Consistent with decreased NMHC killer cell cytotoxicity, urinary cortisol significantly increased after landing compared with preflight levels. Plasma cortisol and ACTH levels at landing were not significantly different from preflight values. No correlation of changes in NMHC killer cell function or hormone levels with factors such as age, gender, mission, or spaceflight experience was found. After landing, expression of the major lymphocyte surface markers (CD3, CD4, CD8, CD14, CD16, CD56), as determined by flow cytometric analysis, did not show any consistent changes from measurements made before flight.

Regulation of mononuclear phagocyte development by IRF8.

PubMed

Tamura, Tomohiko

2017-01-01

Mononuclear phagocytes, such as monocytes and dendritic cells (DCs), are essential for tissue homeostasis and immunity. In adults, these cells develop from hematopoietic stem cells via a common progenitor population. We have been investigating the mechanism underlying the development of mononuclear phagocytes from the viewpoint of gene expression control by transcription factors. Particularly, IRF8, the loss of which causes immunodeficiency and chronic myeloid leukemia-like neutrophilia in mice and humans, promotes the development of monocytes and DCs, while it limits neutrophil differentiation. IRF8 cooperates with the myeloid master transcription factor, PU.1, in mononuclear phagocyte progenitors. KLF4 and BATF3 serve as critical transcription factors downstream of IRF8 to induce the differentiation of monocytes and DCs, respectively. Conversely, IRF8 blocks the activity of the transcription factor C/EBPα to suppress the neutrophil differentiation program. Indeed, Irf8 -/- mononuclear phagocyte progenitors do not efficiently generate monocytes and DCs and, instead, aberrantly give rise to a large number of neutrophils. Our recent data have begun to uncover the vital role of IRF8 in the establishment of distal enhancers in mononuclear phagocyte progenitors. These results place IRF8 as a central regulator of the development of monocytes and DCs.

[Study on the attenuation of graft versus host disease by methoxy polyethylene glycol modification of donor lymphocytes].

PubMed

Zhang, Quan; Yuan, Yi; Li, Su-Bo; Dou, Na; Ma, Fu-Ling; Ji, Shou-Ping

2004-05-01

To find out why mPEG modification of donor's lymphocytes can attenuate the occurrence of graft versus host disease(GVHD), but not affect the hemopoietic reconstitution of stem/progenitor cells after transplanting the mPEG-modified mononuclear cells from human cord blood into the SCID mice. The followings were observed: (1) Changes of CD4(+) and CD8(+) T cells and the ratio of CD4(+)/CD8(+) T cells were examined by flow cytometry before and after mononuclear cells from human cord blood were modified with mPEG. (2) The difference in forming the CFU-GM in-vitro between the mPEG modified-stem/progenitor cell group and non-modified cell group was observed. (3) The time of appearance of GVHD and the survival of the SCID mice were observed after the pre- and post-modification mononuclear cells were transplanted. (4) The number of humanized CD45(+) cells in the mouse's bone marrow was detected about 7 weeks after transplantation. (1) mPEG nearly completely covered up the CD4 and CD8 antigens on T cells, while the number of CFU-GM did not show any obvious change between the modified and non-modified cell groups. (2) GVHD appeared later in the modified mononuclear cell group than in the non-modified group, and the survival rate was elevated in the modified group than in the non-modified group. (3) Humanized CD45 cells were found in mouse's bone marrow at the 47th day after transplantation of both mPEG-modified and non-modified mononuclear cells. After CD4 and CD8 antigens were covered up with mPEG, the graft's immune response against host was weakened, but the proliferation and differentiation of transplanted hemopoietic stem/progenitor cells were not affected.

Mechanisms of mononuclear phagocyte recruitment in Alzheimer's disease.

PubMed

Hickman, Suzanne E; El Khoury, Joseph

2010-04-01

Alzheimer's disease (AD) is associated with a significant neuroinflammatory component. Mononuclear phagocytes including monocytes and microglia are the principal cells involved, and they accumulate at perivascular sites of beta-amyloid (Abeta) deposition and in senile plaques. Recent evidence suggests that mononuclear phagocyte accumulation in the AD brain is dependent on chemokines. CCL2, a major monocyte chemokine, is upregulated in the AD brain. Interaction of CCL2 with its receptor CCR2 regulates mononuclear phagocyte accumulation in a mouse model of AD. CCR2 deficiency leads to lower mononuclear phagocyte accumulation and is associated with higher brain Abeta levels, specifically around blood vessels, suggesting that monocytes accumulate at sites of Abeta deposition in an initial attempt to clear these deposits and stop or delay their neurotoxic effects. Indeed, enhancing mononuclear phagocyte accumulation delays progression of AD. Here we review the mechanisms of mononuclear phagocyte accumulation in AD and discuss the potential roles of additional chemokines and their receptors in this process. We also propose a multi-step model for recruitment of mononuclear phagocytes into the brain. The first step involves egress of monocyte/microglial precursors from the bone marrow into the blood. The second step is crossing the blood-brain barrier to the perivascular areas and into the brain parenchyma. The final step includes movement of monocytes/microglia from areas of the brain that lack any amyloid deposition to senile plaques. Understanding the mechanism of recruitment of mononuclear phagocytes to the AD brain is necessary to further understand the role of these cells in the pathogenesis of AD and to identify any potential therapeutic use of these cells for the treatment of this disease.

The mRNA expression levels of uncoupling proteins 1 and 2 in mononuclear cells from patients with metabolic disorders: obesity and type 2 diabetes mellitus.

PubMed

Margaryan, Sona; Witkowicz, Agata; Partyka, Anna; Yepiskoposyan, Levon; Manukyan, Gayane; Karabon, Lidia

2017-10-19

Type 2 diabetes mellitus (T2DM) and obesity are metabolic disorders whose major hallmark is insulin resistance. Impaired mitochondrial activity, such as reduced ratio of energy production to respiration, has been implicated in the development of insulin resistance. Uncoupling proteins (UCPs) are proton carriers, expressed in the mitochondrial inner membrane, that uncouple oxygen consumption by the respiratory chain from ATP synthesis. The aim of the study was to determine transcriptional levels of UCP1 and UCP2 in peripheral blood mononuclear cells (PBMCs) from patients with metabolic disorders: T2DM, obesity and from healthy individuals. The mRNA levels of UCP1, UCP2 were determined by Real-Time PCR method using Applied Biosystems assays. The UCP1 mRNA expression level was not detectable in the majority of studied samples, while very low expression was found in PBMCs from 3 obese persons. UCP2 mRNA expression level was detectable in all samples. The median mRNA expression of UCP2 was lower in all patients with metabolic disorders as compared to the controls (0.20+0.14 vs. 0.010+0.009, p=0.05). When compared separately, the differences of medians UCP2 mRNA expression level between the obese individuals and the controls as well as between the T2DM patients and the controls did not reach statistical significance. Decreased UCP2 gene expression in mononuclear cells from obese and diabetic patients might contribute to the immunological abnormalities in these metabolic disorders and suggests its role as a candidate gene in future studies of obesity and diabetes.

Mononuclear phagocyte subpopulations in the mouse kidney

PubMed Central

George, James F.; Lever, Jeremie M.

2017-01-01

Mononuclear phagocytes are the most common cells in the kidney associated with immunity and inflammation. Although the presence of these cells in the kidney has been known for decades, the study of mononuclear phagocytes in the context of kidney function and dysfunction is still at an early stage. The purpose of this review is to summarize the present knowledge regarding classification of these cells in the mouse kidney and to identify relevant questions that would further advance the field and potentially lead to new opportunities for treatment of acute kidney injury and other kidney diseases. PMID:28100500

Preliminary investigations on the effects of a Strongylus vulgaris larval extract, mononuclear factors and platelet factors on equine smooth muscle cells in vitro.

PubMed

Morgan, S J; Storts, R W; Stromberg, P C; Sowa, B A; Lay, J C

1989-01-01

Factors involved in the proliferation of equine vascular smooth muscle cells were studied in vitro. The most prominent proliferative responses in cultured vascular smooth muscle cells were induced by Strongylus vulgaris larval antigen extract (LAE) and platelet-derived factors. Less significant proliferative responses were obtained with conditioned media from S. vulgaris LAE stimulated and from unstimulated equine mononuclear leukocytes. Additionally, vascular smooth muscle cells exposed to S. vulgaris LAE developed numerous perinuclear vacuoles and were more spindle-shaped than control or smooth muscle cells exposed to other factors. Equine mononuclear leukocytes exposed to LAE developed prominent morphological changes, including enlargement, clumping and increased numbers of mitotic figures.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Norhaiza, H. Siti; Zarina, Z. A. Intan; Hisham, Z. A. Shahrul

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)more » 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, addition of cytokines into the proliferation medium support Lin{sup −} stem cells for at least 18 days. The Lin{sup −} stem cells started to response to the cytokines added as early as Day 2 of culture. It is concluded that Lin{sup −} stem cells can be expanded in vitro by culturing in proliferation medium supplemented with cytokines.« less

Investigating Evolutionary Conservation of Dendritic Cell Subset Identity and Functions

PubMed Central

Vu Manh, Thien-Phong; Bertho, Nicolas; Hosmalin, Anne; Schwartz-Cornil, Isabelle; Dalod, Marc

2015-01-01

Dendritic cells (DCs) were initially defined as mononuclear phagocytes with a dendritic morphology and an exquisite efficiency for naïve T-cell activation. DC encompass several subsets initially identified by their expression of specific cell surface molecules and later shown to excel in distinct functions and to develop under the instruction of different transcription factors or cytokines. Very few cell surface molecules are expressed in a specific manner on any immune cell type. Hence, to identify cell types, the sole use of a small number of cell surface markers in classical flow cytometry can be deceiving. Moreover, the markers currently used to define mononuclear phagocyte subsets vary depending on the tissue and animal species studied and even between laboratories. This has led to confusion in the definition of DC subset identity and in their attribution of specific functions. There is a strong need to identify a rigorous and consensus way to define mononuclear phagocyte subsets, with precise guidelines potentially applicable throughout tissues and species. We will discuss the advantages, drawbacks, and complementarities of different methodologies: cell surface phenotyping, ontogeny, functional characterization, and molecular profiling. We will advocate that gene expression profiling is a very rigorous, largely unbiased and accessible method to define the identity of mononuclear phagocyte subsets, which strengthens and refines surface phenotyping. It is uniquely powerful to yield new, experimentally testable, hypotheses on the ontogeny or functions of mononuclear phagocyte subsets, their molecular regulation, and their evolutionary conservation. We propose defining cell populations based on a combination of cell surface phenotyping, expression analysis of hallmark genes, and robust functional assays, in order to reach a consensus and integrate faster the huge but scattered knowledge accumulated by different laboratories on different cell types, organs, and species. PMID:26082777

Isolation and purification of rabbit mesenchymal stem cells using an optimized protocol.

PubMed

Lin, Chunbo; Shen, Maorong; Chen, Weiping; Li, Xiaofeng; Luo, Daoming; Cai, Jinhong; Yang, Yuan

2015-11-01

Mesenchymal stem cells were first isolated and grown in vitro by Friedenstein over 40 yr ago; however, their isolation remains challenging as they lack unique markers for identification and are present in very small quantities in mesenchymal tissues and bone marrow. Using whole marrow samples, common methods for mesenchymal stem cell isolation are the adhesion method and density gradient fractionation. The whole marrow sample adhesion method still results in the nonspecific isolation of mononuclear cells, and activation and/or potential loss of target cells. Density gradient fractionation methods are complicated, and may result in contamination with toxic substances that affect cell viability. In the present study, we developed an optimized protocol for the isolation and purification of mesenchymal stem cells based on the principles of hypotonic lysis and natural sedimentation.

Phenotyping of peripheral blood mononuclear cells during acute dengue illness demonstrates infection and increased activation of monocytes in severe cases compared to classic dengue fever

PubMed Central

Durbin, Anna P.; Vargas, Maria José; Wanionek, Kimberli; Hammond, Samantha N.; Gordon, Aubree; Rocha, Crisanta; Balmaseda, Angel; Harris, Eva

2008-01-01

In vitro studies have attempted to identify dengue virus (DEN) target cells in peripheral blood; however, extensive phenotyping of peripheral blood mononuclear cells (PBMCs) from dengue patients has not been reported. PBMCs collected from hospitalized children suspected of acute dengue were analyzed for DEN prM, CD32, CD86, CD14, CD11c, CD16, CD209, CCR7, CD4, and CD8 by flow cytometry to detect DEN antigen in PBMCs and to phenotype DEN-positive cells. DEN prM was detected primarily in activated monocytes (CD14+, CD32+, CD86+, CD11c+). A subset of samples analyzed for DEN nonstructural protein 3 (NS3) confirmed that approximately half of DEN antigen-positive cells contained replicating virus. A higher percentage of PBMCs from DHF patients expressed prM, CD86, CD32, and CD11c than did those from DF patients. Increased activation of monocytes and greater numbers of DEN-infected cells were associated with more severe dengue, implicating a role for monocyte activation in dengue immunopathogenesis. PMID:18452966

Altered Mitochondrial Membrane Potential, Mass, and Morphology in the Mononuclear Cells of Humans with Type 2 Diabetes

PubMed Central

Widlansky, Michael E.; Wang, Jingli; Shenouda, Sherene M.; Hagen, Tory M.; Smith, Anthony R.; Kizhakekuttu, Tinoy J.; Kluge, Matthew A.; Weihrauch, Dorothee; Gutterman, David D.; Vita, Joseph A.

2010-01-01

Mitochondrial membrane hyperpolarization and morphological changes are important in inflammatory cell activation. Despite the pathophysiological relevance, no valid and reproducible method for measuring mitochondrial homeostasis in human inflammatory cells is currently available. This study's purpose was to define and validate reproducible methods for measuring relevant mitochondrial perturbations and to determine whether these methods could discern mitochondrial perturbations in type 2 diabetes mellitus (T2DM), a condition associated with altered mitochondrial homeostasis. We employed 5,5',6,6'-tetrachloro-1,1'3,3'-tetraethylbenzamidazol-carboncyanine (JC-1) to estimate mitochondrial membrane potential (ψm) and acridine orange 10-nonyl bromide (NAO) to assess mitochondrial mass in human mononuclear cells isolated from blood. Both assays were reproducible. We validated our findings by electron microscopy and pharmacological manipulation of ψm. We measured JC-1 and NAO fluorescence in the mononuclear cells of 27 T2DM patients and 32 controls. Mitochondria were more polarized (P=0.02) and mitochondrial mass was lower in T2DM (P=0.008). Electron microscopy demonstrated diabetic mitochondria were smaller, more spherical, and occupied less cellular area in T2DM. Mitochondrial superoxide production was higher in T2DM (P=0.01). Valid and reproducible measurements of mitochondrial homeostasis can be made in human mononuclear cells using these fluorophores. Further, potential clinically relevant perturbations in mitochondrial homeostasis in T2DM human mononuclear cells can be detected. PMID:20621033

Immunodeficiency with thymoma: failure to induce Ig production in immunodeficient lymphocytes cocultured with normal T cells. [X radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Litwin, S.D.

Blood mononuclear cells of two individuals having immunodeficiency with thymoma (ID-THY) were cocultured with normal mononuclear cells or treated mononuclear cell fractions in an attempt to correct an imbalance of regulatory cells postulated to be responsible for the failure of pokeweed mitogen-induced Ig synthesis in vitro. Treatment included abrogation of suppressor cell activity by irradiation or incubation with prednisolone in vitro. T cell help was provided by cocultivating lymphocytes of related and unrelated persons, and in some cases autologous treated cells. Ig secretion failed to be induced by any experimental maneuver suggesting that the primary problem in the above ID-THYmore » cells was related to defective or deficient B cells rather than an imbalance of T regulatory cells. Prednisolone treatment in vitro decreased suppressor cell activity in allogeneic cocultures of two ID-THY persons (S1 and S2) but not of an individual (S3) with variable immunodeficiency suggesting heterogeneity of suppressor cells.« less

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

PubMed Central

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

2017-01-01

Imatinib is actively transported by organic cation transporter-1 (OCT-1) influx transporter, and low OCT-1 activity in diagnostic chronic myeloid leukemia blood mononuclear cells is significantly associated with poor molecular response to imatinib. Herein we report that, in diagnostic chronic myeloid leukemia mononuclear cells and BCR-ABL1+ cell lines, peroxisome proliferator-activated receptor γ agonists (GW1929, rosiglitazone, pioglitazone) significantly decrease OCT-1 activity; conversely, peroxisome proliferator-activated receptor γ antagonists (GW9662, T0070907) increase OCT-1 activity. Importantly, these effects can lead to corresponding changes in sensitivity to BCR-ABL kinase inhibition. Results were confirmed in peroxisome proliferator-activated receptor γ-transduced K562 cells. Furthermore, we identified a strong negative correlation between OCT-1 activity and peroxisome proliferator-activated receptor γ transcriptional activity in diagnostic chronic myeloid leukemia patients (n=84; P<0.0001), suggesting that peroxisome proliferator-activated receptor γ activation has a negative impact on the intracellular uptake of imatinib and consequent BCR-ABL kinase inhibition. The inter-patient variability of peroxisome proliferator-activated receptor γ activation likely accounts for the heterogeneity observed in patient OCT-1 activity at diagnosis. Recently, the peroxisome proliferator-activated receptor γ agonist pioglitazone was reported to act synergistically with imatinib, targeting the residual chronic myeloid leukemia stem cell pool. Our findings suggest that peroxisome proliferator-activated receptor γ ligands have differential effects on circulating mononuclear cells compared to stem cells. Since the effect of peroxisome proliferator-activated receptor γ activation on imatinib uptake in mononuclear cells may counteract the clinical benefit of this activation in stem cells, caution should be applied when combining these therapies, especially in patients with high peroxisome proliferator-activated receptor γ transcriptional activity. PMID:28154092

Theophylline therapy inhibits neutrophil and mononuclear cell chemotaxis from chronic asthmatic children.

PubMed Central

Condino-Neto, A; Vilela, M M; Cambiucci, E C; Ribeiro, J D; Guglielmi, A A; Magna, L A; De Nucci, G

1991-01-01

1. Theophylline is commonly used to relieve symptoms of chronic asthma. Since neutrophil and mononuclear cell activation are associated with late phase asthmatic reactions, effects of theophylline on these cells may be of importance. 2. In the present investigation we compared neutrophil and mononuclear cell chemotaxis from chronic asthmatic children during and after theophylline therapy. 3. Thirty patients were recruited for the study. Each patient received theophylline orally for 10 days. The theophylline dose was 20 mg kg-1 day-1 given in four divided doses. On the tenth day, blood was collected into heparinized (100 u ml-1) and siliconized tubes 2 h after the last theophylline dose for chemotactic assays, cAMP and theophylline plasma determinations. When clinical conditions allowed, theophylline was discontinued for 7 days and the chemotactic assays, cAMP and theophylline plasma concentrations repeated. Serum complement and IgE levels were also determined. 4. Theophylline therapy clearly inhibited both spontaneous and stimulated neutrophil and mononuclear cell chemotaxis. Twenty-seven patients had therapeutic plasma concentrations of theophylline (5-20 micrograms ml-1). Discontinuation of theophylline therapy caused a significant decrease in plasma cAMP levels (44 and 31 pmol ml-1 respectively during and after treatment, n = 30, P less than 0.001). 5. The inhibition of neutrophil and mononuclear cell migration by theophylline therapy in chronic asthmatic children may be beneficial for the control of the inflammatory response observed in these patients. PMID:1659436

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

PubMed

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.

Localization of HTLV-I tax proviral DNA in mononuclear cells.

PubMed

Zucker-Franklin, Dorothea; Pancake, Bette A; Najfeld, Vesna

2003-01-01

The tax sequence of HTLV-I is demonstrable in the skin and blood mononuclear cells of patients with mycosis fungoides, as well as in the mononuclear leukocytes of some healthy blood donors, but was not demonstrable when PCR/Southern analyses were carried out on preparations of high-molecular-weight genomic DNA. Therefore, it was postulated that tax DNA may not be integrated. To investigate this possibility fluorescence in situ hybridization was carried out on cells arrested in metaphase, using a probe containing the HTLV-I tax proviral DNA full-length open reading frame coding sequence. While metaphases prepared from C91PL cells, a cell line infected with HTLV-I, showed an abundance of chromosome-associated as well as extra-chromosomal signals, metaphases prepared with blood mononuclear cells from healthy tax sequence positive donors did not reveal any tax DNA associated with chromosomes. Such signals were readily detected extra-chromosomally. Although it has been demonstrated that transactivation of genes by gene products encoded by extra-chromosomal DNA may have nosocomial implications, whether transactivation by p40 tax generated from extra-chromosomal tax sequences is responsible for the development of neoplasia remains to be investigated.

Tissues Use Resident Dendritic Cells and Macrophages to Maintain Homeostasis and to Regain Homeostasis upon Tissue Injury: The Immunoregulatory Role of Changing Tissue Environments

PubMed Central

Lech, Maciej; Gröbmayr, Regina; Weidenbusch, Marc; Anders, Hans-Joachim

2012-01-01

Most tissues harbor resident mononuclear phagocytes, that is, dendritic cells and macrophages. A classification that sufficiently covers their phenotypic heterogeneity and plasticity during homeostasis and disease does not yet exist because cell culture-based phenotypes often do not match those found in vivo. The plasticity of mononuclear phagocytes becomes obvious during dynamic or complex disease processes. Different data interpretation also originates from different conceptual perspectives. An immune-centric view assumes that a particular priming of phagocytes then causes a particular type of pathology in target tissues, conceptually similar to antigen-specific T-cell priming. A tissue-centric view assumes that changing tissue microenvironments shape the phenotypes of their resident and infiltrating mononuclear phagocytes to fulfill the tissue's need to maintain or regain homeostasis. Here we discuss the latter concept, for example, why different organs host different types of mononuclear phagocytes during homeostasis. We further discuss how injuries alter tissue environments and how this primes mononuclear phagocytes to enforce this particular environment, for example, to support host defense and pathogen clearance, to support the resolution of inflammation, to support epithelial and mesenchymal healing, and to support the resolution of fibrosis to the smallest possible scar. Thus, organ- and disease phase-specific microenvironments determine macrophage and dendritic cell heterogeneity in a temporal and spatial manner, which assures their support to maintain and regain homeostasis in whatever condition. Mononuclear phagocytes contributions to tissue pathologies relate to their central roles in orchestrating all stages of host defense and wound healing, which often become maladaptive processes, especially in sterile and/or diffuse tissue injuries. PMID:23251037

Collection and use of circulating hematopoietic progenitor cells.

PubMed

Lee, J H; Klein, H G

1995-02-01

Although lymphocytes and monocytes are becoming increasingly important in transfusion therapy, peripheral stem cells have been responsible for the recent explosive interest in harvesting mononuclear cells from the peripheral circulation. Despite their low concentration in peripheral blood and the consequent difficulty in cell collection, circulating hematopoietic progenitor cells are collected and used almost routinely. These mononuclear cells, possessing the capacity for hematopoietic reconstitution and the potential for definitive therapy of a variety of disorders, have been the focus of recent intense interest in transfusion medicine.

Analysis of DNA Double-Strand Breaks and Cytotoxicity after 7 Tesla Magnetic Resonance Imaging of Isolated Human Lymphocytes

PubMed Central

Guttek, Karina; Hartig, Roland; Godenschweger, Frank; Roggenbuck, Dirk; Ricke, Jens; Reinhold, Dirk; Speck, Oliver

2015-01-01

The global use of magnetic resonance imaging (MRI) is constantly growing and the field strengths increasing. Yet, only little data about harmful biological effects caused by MRI exposure are available and published research analyzing the impact of MRI on DNA integrity reported controversial results. This in vitro study aimed to investigate the genotoxic and cytotoxic potential of 7 T ultra-high-field MRI on isolated human peripheral blood mononuclear cells. Hence, unstimulated mononuclear blood cells were exposed to 7 T static magnetic field alone or in combination with maximum permissible imaging gradients and radiofrequency pulses as well as to ionizing radiation during computed tomography and γ-ray exposure. DNA double-strand breaks were quantified by flow cytometry and automated microscopy analysis of immunofluorescence stained γH2AX. Cytotoxicity was studied by CellTiter-Blue viability assay and [3H]-thymidine proliferation assay. Exposure of unstimulated mononuclear blood cells to 7 T static magnetic field alone or combined with varying gradient magnetic fields and pulsed radiofrequency fields did not induce DNA double-strand breaks, whereas irradiation with X- and γ-rays led to a dose-dependent induction of γH2AX foci. The viability assay revealed a time- and dose-dependent decrease in metabolic activity only among samples exposed to γ-radiation. Further, there was no evidence for altered proliferation response after cells were exposed to 7 T MRI or low doses of ionizing radiation (≤ 0.2 Gy). These findings confirm the acceptance of MRI as a safe non-invasive diagnostic imaging tool, but whether MRI can induce other types of DNA lesions or DNA double-strand breaks during altered conditions still needs to be investigated. PMID:26176601

Effects of periodontal therapy on serum C-reactive protein, sE-selectin, and tumor necrosis factor-alpha secretion by peripheral blood-derived macrophages in diabetes. A pilot study.

PubMed

Lalla, E; Kaplan, S; Yang, J; Roth, G A; Papapanou, P N; Greenberg, S

2007-06-01

Diabetes is associated with an increased risk for vascular disease and periodontitis. The aim of this study was to assess the effects of periodontal treatment in diabetes with respect to alterations in the pro-inflammatory potential of peripheral blood mononuclear cells. Ten patients with diabetes and moderate to severe periodontitis received full-mouth subgingival debridement. Blood samples for serum/plasma and mononuclear cell isolation were collected prior to and 4 wk after therapy. Mononuclear cells were analyzed by flow cytometry and stimulated with lipopolysaccharide or ionomycin/phorbol ester to determine the pro-inflammatory capacity of macrophages and lymphocytes, respectively. Following periodontal treatment, all patients demonstrated a significant improvement in clinical periodontal status (p < 0.05), despite only modest reduction in subgingival bacterial load or homologous serum immunoglobulin G titers. CD14(+) blood monocytes decreased by 47% (p < 0.05), and the percentage of macrophages spontaneously releasing tumor necrosis factor-alpha decreased by 78% (p < 0.05). There were no significant changes in the capacity of lymphocytes to secrete interferon-gamma. Among a number of serum inflammatory markers tested, high-sensitivity-C-reactive protein significantly decreased by 37% (p < 0.01) and soluble E-selectin decreased by 16.6% (p < 0.05). These data suggest a reduced tendency for monocyte/macrophage-driven inflammation with periodontal therapy and a potential impact on atherosclerosis-related complications in diabetic individuals.

Arterial and venous endothelia display differential functional fractalkine (CX3CL1) expression by angiotensin-II.

PubMed

Rius, Cristina; Piqueras, Laura; González-Navarro, Herminia; Albertos, Fernando; Company, Chantal; López-Ginés, Concha; Ludwig, Andreas; Blanes, Jose-Ignacio; Morcillo, Esteban J; Sanz, Maria-Jesus

2013-01-01

Angiotensin-II (Ang-II) promotes the interaction of mononuclear cells with arterioles and neutrophils with postcapillary venules. To investigate the mechanisms underlying this dissimilar response, the involvement of fractalkine (CX(3)CL1) was explored. Enhanced CX(3)CL1 expression was detected in both cremasteric arterioles and postcapillary venules 24 hours after Ang-II intrascrotal injection. Arteriolar leukocyte adhesion was the unique parameter significantly reduced (83%) in animals lacking CX(3)CL1 receptor (CX(3)CR1). Human umbilical arterial and venous endothelial cell stimulation with 1 μmol/L Ang-II increased CX(3)CL1 expression, yet neutralization of CX(3)CL1 activity only significantly inhibited Ang-II-induced mononuclear cell-human umbilical arterial endothelial cell interactions (73%) but not with human umbilical venous endothelial cells. The use of small interfering RNA revealed the involvement of tumor necrosis factor-α in Ang-II-induced CX(3)CL1 upregulation and mononuclear cell arrest. Nox5 knockdown with small interfering RNA or pharmacological inhibition of extracellular signal-regulated kinases1/2, p38 mitogen-activated protein kinase, and nuclear factor-κB also abolished these responses. Finally, when human umbilical arterial endothelial cells were costimulated with Ang-II, tumor necrosis factor-α, and interferon-γ, CX(3)CL1 expression and mononuclear cell adhesiveness were more pronounced than when each stimulus was provided alone. These results suggest that Ang-II induces functional CX(3)CL1 expression in arterial but not in venous endothelia. Thus, targeting endothelial CX(3)CL1-mononuclear leukocyte CX(3)CR1 interactions may constitute a new therapeutic strategy in the treatment of Ang-II-associated cardiovascular diseases.

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

PubMed

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.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Madaric, Juraj, E-mail: jurmad@hotmail.com; Klepanec, Andrej; Mistrik, Martin

Induction of therapeutic angiogenesis by autologous bone marrow mononuclear cell transplantation has been identified as a potential new option in patients with advanced lower-limb ischemia. There is little evidence of the benefit of intra-arterial cell application in upper-limb critical ischemia. We describe a patient with upper-extremity critical limb ischemia with digital gangrene resulting from hypothenar hammer syndrome successfully treated by intra-arterial autologous bone marrow mononuclear cell transplantation.

Histological and Immunological Description of the Leishmanin Skin Test in Ibizan Hounds.

PubMed

Ordeix, L; Silva, J E Dos S; Llull, J; Quirola, P; Montserrat-Sangrà, S; Martínez-Orellana, P; Solano-Gallego, L

2018-01-01

The leishmanin skin test (LST), a delayed-type hypersensitivity (DTH) reaction to Leishmania infantum, can specifically identify dogs that have made a cell-mediated immune response to L. infantum infection. The Ibizan hound appears to be more resistant to L. infantum infection than other breeds of dog. The aim of this study was to describe the histological and immunohistochemical changes induced by the LST in Ibizan hounds living in an area highly endemic for leishmaniosis. The majority of dogs were apparently healthy, lacked serum antibody to L. infantum and blood parasitaemia, but had marked specific interferon gamma production after in-vitro blood stimulation with L. infantum. Leishmanin (3 × 10 8 killed promastigotes of L. infantum/ml) was injected intradermally and biopsy samples were obtained from a positive reaction at 72 h from nine Ibizan hounds. A moderate to intense, perivascular to interstitial dermatitis and panniculitis characterized the inflammatory response at the injection site. In addition, three samples had diffuse inflammation in the deep dermis and panniculus. Oedema and necrosis were present in the deep dermis and panniculus. Congestion and haemorrhage were observed in five biopsies. T lymphocytes (CD3 + ) and large mononuclear cells (lysozyme - ) were the most prevalent cells. CD3 + cells were significantly more numerous than CD20 + B cells and lysozyme + cells. B cells were sparsely distributed, especially in the deep dermis and panniculus. Rare neutrophils and macrophages (lysozyme + ) were observed with few eosinophils. Toll-like receptor (TLR)-2 protein was expressed in large mononuclear cells mainly located in the superficial dermis. Leishmania immunohistochemistry was negative and quantitative polymerase chain reaction was positive in all cases. The intradermal injection of killed L. infantum promastigotes in Ibizan hounds causes similar histological and immunohistochemical findings to those described for human subjects and are indicative of a DTH response. Moreover, TLR2 protein is expressed in inflammatory cells similar to findings in clinically affected skin biopsy samples. Copyright © 2017 Elsevier Ltd. All rights reserved.

Lipopolysaccharide-induced early response genes in bovine peripheral blood mononuclear cells implicate GLG1/E-selectin as a key ligand–receptor interaction

USDA-ARS?s Scientific Manuscript database

This study uses a systems biology approach, integrating global gene expression information and knowledge of the regulatory events in cells to identify transcription networks controlling peripheral blood mononuclear cells’ (PBMCs) immune response to lipopolysaccharide (LPS) and to identify the molecu...

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

USDA-ARS?s Scientific Manuscript database

Objective: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have beneficial effects on inflammation and cardiovascular disease (CVD). Our aim was to assess the effect of a six-week supplementation with either olive oil, EPA, or DHA on gene expression in peripheral blood mononuclear cells (...

Induction of expression of iNOS by N-nitrosodimethylamine (NDMA) in human leukocytes.

PubMed

Ratajczak-Wrona, Wioletta; Jablonska, Ewa; Jablonski, Jakub; Marcinczyk, Magdalena

2009-01-01

The aim of this study was to assess the influence of N-nitrosodimethylamine (NDMA) on expression of inducible nitric oxide synthase (iNOS), as well as production of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) by human neutrophils (PMN) and peripheral blood mononuclear cells (PBMC), and the participation of the p38 MAPK kinase in this process. Furthermore, the ability of neutrophils to release superoxide anion was determined. The influence of N-nitrosodimethylamine on iNOS expression was determined in isolated PMN and PBMC cells from peripheral blood of healthy individuals. The mononuclear cells showed higher sensitivity to NDMA. Moreover, cytotoxic effect of NDMA can be influenced in some way by the impact of this xenobiotic on nitric oxide and superoxide anion release from human leukocytes. Furthermore, increased generation of these radicals by human leukocytes suggest that neutrophils and mononuclear cells that are exposed to NDMA activity can play a key role in endogenous NDMA generation. However the relationship between iNOS expression and phospho-p38 MAPK in neutrophils and mononuclear cells shows that p38 MAPK pathway participates in induction of iNOS expression in the presence of NDMA.

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

PubMed

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.

Generation of human-to-pig chimerism to induce tolerance through transcutaneous in utero injection of cord blood-derived mononuclear cells or human bone marrow mesenchymals cells in a preclinical program of liver xenotransplantation: preliminary results.

PubMed

Abellaneda, J M; Ramis, G; Martínez-Alarcón, L; Majado, M J; Quereda, J J; Herrero-Medrano, J M; Mendonça, L; García-Nicolás, O; Reus, M; Insausti, C; Ríos, A; López-Navas, A; González, M R; Pallarés, F J; Munoz, A; Ramírez, P; Parrilla, P

2012-01-01

Using a percutaneous ecoguided injection system to obtain chimeric piglets through a less invasive and traumatic technique than previously reported. The two types of human cells included umbilical cord blood mononuclear elements and mesenchymal stem cells cultured from bone marrow. Four sows at gestational day 50 were anesthetized. A needle was inserted through the skin and uterine wall to reach the peritoneal cavity of the fetuses under continuous ultrasound guidance. Fourteen piglets were injected with various cell concentrations. All sows carried pregnancies to term yielding 69 piglets, among which 67 were alive and two mummified. Two piglets died during the first 48 hours of life. Chimerism was detected using flow cytometry and by quantitative polymerase chain reaction (q-PCR) to detect Alu gene in blood or tissues samples. The analysis detected blood chimerism in 13 piglets (21%) by flow cytometry and the presence of the human Alu gene in 33 (51%) by q-PCR. The results suggest cell trafficking between littermates after in utero injection. Transcutaneous echo-guided injection succeeded to produce chimeric piglets without disadvantages to the sow or the fetuses and avoiding abortions or fetal death. Copyright © 2012 Elsevier Inc. All rights reserved.

Carbendazim-induced haematological, biochemical and histopathological changes to the liver and kidney of male rats.

PubMed

Selmanoglu, G; Barlas, N; Songür, S; Koçkaya, E A

2001-12-01

Carbendazim is a systemic broad-spectrum fungicide controlling a wide range of pathogens. It is also used as a preservative in paint, textile, papermaking and leather industry, as well as a preservative of fruits. In the present study, carbendazim was administered at 0, 150, 300 and 600 mg/kg per day doses orally to male rats (Rattus rattus) for 15 weeks. At the end of the experiment, blood samples, liver and kidney tissues of each animal were taken. Serum enzyme activities, and haematological and biochemical parameters were analysed. In toxicological tests, 600 mg/kg per day doses of carbendazim caused an increase of albumin, glucose, creatinine and cholesterol levels. Also, at the same doses, white blood cell and lymphocyte counts decreased. However, mean cell hemoglobin and mean cell hemoglobin concentrations increased. Histopathological examinations revealed congestion, an enlargement of the sinusoids, an increase in the number of Kupffer cells, mononuclear cell infiltration and hydropic degeneration in the liver. At the highest doses, congestion, mononuclear cell infiltration, tubular degeneration and fibrosis were observed in the kidney tissue. These results indicate that 300 and 600 mg/kg per day carbendazim affected the liver and kidney tissue and caused some changes on haematological and biochemical parameters of rats.

Viability and neural differentiation of mesenchymal stem cells derived from the umbilical cord following perinatal asphyxia.

PubMed

Aly, H; Mohsen, L; Badrawi, N; Gabr, H; Ali, Z; Akmal, D

2012-09-01

Hypoxia-ischemia is the leading cause of neurological handicaps in newborns worldwide. Mesenchymal stem cells (MSCs) collected from fresh cord blood of asphyxiated newborns have the potential to regenerate damaged neural tissues. The aim of this study was to examine the capacity for MSCs to differentiate into neural tissue that could subsequently be used for autologous transplantation. We collected cord blood samples from full-term newborns with perinatal hypoxemia (n=27), healthy newborns (n=14) and non-hypoxic premature neonates (n=14). Mononuclear cells were separated, counted, and then analyzed by flow cytometry to assess various stem cell populations. MSCs were isolated by plastic adherence and characterized by morphology. Cells underwent immunophenotyping and trilineage differentiation potential. They were then cultured in conditions favoring neural differentiation. Neural lineage commitment was detected using immunohistochemical staining for glial fibrillary acidic protein, tubulin III and oligodendrocyte marker O4 antibodies. Mononuclear cell count and viability did not differ among the three groups of infants. Neural differentiation was best demonstrated in the cells derived from hypoxia-ischemia term neonates, of which 69% had complete and 31% had partial neural differentiation. Cells derived from preterm neonates had the least amount of neural differentiation, whereas partial differentiation was observed in only 12%. These findings support the potential utilization of umbilical cord stem cells as a source for autologous transplant in asphyxiated neonates.

Phenotypic and functional consequences of different isolation protocols on skin mononuclear phagocytes.

PubMed

Botting, Rachel A; Bertram, Kirstie M; Baharlou, Heeva; Sandgren, Kerrie J; Fletcher, James; Rhodes, Jake W; Rana, Hafsa; Plasto, Toby M; Wang, Xin Maggie; Lim, Jake J K; Barnouti, Laith; Kohout, Mark P; Papadopoulos, Tim; Merten, Steve; Olbourne, Norman; Cunningham, Anthony L; Haniffa, Muzlifah; Harman, Andrew N

2017-06-01

Mononuclear phagocytes are present in skin and mucosa and represent one of the first lines of defense against invading pathogens, which they detect via an array of pathogen-binding receptors expressed on their surface. However, their extraction from tissue is difficult, and the isolation technique used has functional consequences on the cells obtained. Here, we compare mononuclear phagocytes isolated from human skin using either enzymatic digestion or spontaneous migration. Cells isolated via enzymatic digestion are in an immature state, and all subsets are easily defined. However, cells isolated by spontaneous migration are in a mature state, and CD141 cross-presenting DCs (cDC1) are more difficult to define. Different pathogen-binding receptors are susceptible to cleavage by blends of collagenase, demonstrating that great care must be taken in choosing the correct enzyme blend to digest tissue if carrying out pathogen-interaction assays. Finally, we have optimized mononuclear phagocyte culture conditions to enhance their survival after liberation from the tissue. © The Author(s).

Phenotypic and functional consequences of different isolation protocols on skin mononuclear phagocytes

PubMed Central

Botting, Rachel A.; Bertram, Kirstie M.; Baharlou, Heeva; Sandgren, Kerrie J.; Fletcher, James; Rhodes, Jake W.; Rana, Hafsa; Plasto, Toby M.; Wang, Xin Maggie; Lim, Jake J. K.; Barnouti, Laith; Kohout, Mark P.; Papadopoulos, Tim; Merten, Steve; Olbourne, Norman; Cunningham, Anthony L.; Haniffa, Muzlifah; Harman, Andrew N.

2017-01-01

Mononuclear phagocytes are present in skin and mucosa and represent one of the first lines of defense against invading pathogens, which they detect via an array of pathogen-binding receptors expressed on their surface. However, their extraction from tissue is difficult, and the isolation technique used has functional consequences on the cells obtained. Here, we compare mononuclear phagocytes isolated from human skin using either enzymatic digestion or spontaneous migration. Cells isolated via enzymatic digestion are in an immature state, and all subsets are easily defined. However, cells isolated by spontaneous migration are in a mature state, and CD141 cross-presenting DCs (cDC1) are more difficult to define. Different pathogen-binding receptors are susceptible to cleavage by blends of collagenase, demonstrating that great care must be taken in choosing the correct enzyme blend to digest tissue if carrying out pathogen-interaction assays. Finally, we have optimized mononuclear phagocyte culture conditions to enhance their survival after liberation from the tissue. PMID:28270408

Expression of EMAP-II in the rat dental follicle and its potential role in tooth eruption

PubMed Central

Liu, Dawen; Wise, Gary E.

2008-01-01

Endothelial monocyte-activating polypeptide II (EMAP-II) is an inflammatory cytokine with chemotactic activity. Because the dental follicle (DF) recruits mononuclear cells (osteoclast precursors) to promote the osteoclastogenesis needed for tooth eruption, it was the aim of this study to determine if EMAP-II may contribute to this recruitment. Using a DNA microarray, EMAP-II was found to be highly expressed in vivo in the DFs of day 1 to day 11 postnatal rats, with its expression elevated at days 1 and 3. Using a siRNA to knock down EMAP-II expression also resulted in a reduction in expression of CSF-1 and MCP-1 in the DF cells. Addition of EMAP-II protein to the DF cells partially restored the expression of CSF-1 and MCP-1. In chemotaxis assays using either conditioned medium of the DF cells with anti-EMAP-II antibody added or conditioned medium of DF cells with EMAP-II knocked down by siRNA, migration indexes of bone marrow mononuclear cells were significantly reduced. These results suggest that EMAP-II is another chemotactic molecule in the dental follicle involved in recruitment of mononuclear cells, and that EMAP-II may exert its chemotactic function directly by recruiting mononuclear cells and indirectly by enhancing the expression of other chemotactic molecules (CSF-1 and MCP-1). PMID:18705801

The toxic effects of silver nanoparticles on blood mononuclear cells.

PubMed

Barkhordari, A; Barzegar, S; Hekmatimoghaddam, H; Jebali, A; Rahimi Moghadam, S; Khanjani, N

2014-07-01

Nanoparticles have become one of the leading technologies over the past two years. The extensive use of nanoparticles has raised great concern about their occupational fate and biological effects. With an increase in the production and use of nanomaterial, it is more likely to get exposed to them occupationally and environmentally. To assess the toxicity of silver nanoparticles on human mononuclear cells. In this in vitro experimental study, suspensions of blood mononuclear cells from 10 young healthy men were incubated with 10-nm silver nanoparticles in different concentrations (range: 1-500 μg/mL) for 6 and 24 hours by MTT assay. Positive and negative controls were used for comparison. After 6 hours of exposure, 10.9% to 48.4% of the cells died. After 24 hours of exposure, the rate ranged from 56.8% to 86.3%. Regardless of the exposure time, the maximum cytotoxicity was observed at the concentration of 500 μg/mL of silver nanoparticles. By increasing the exposure time to 24 hours, the cytotoxicity of nanoparticles substantially increased at all concentrations. Cell death was significantly higher when compared to the controls (p<0.01). Silver nanoparticles possess both time- and dose-dependent cytotoxicity and can thus be considered as very toxic for mononuclear cells.

Klotho gene expression decreases in peripheral blood mononuclear cells (PBMCs) of patients with relapsing-remitting multiple sclerosis.

PubMed

Karami, Masoumeh; Mehrabi, Farzad; Allameh, Abdolamir; Pahlevan Kakhki, Majid; Amiri, Mehdi; Emami Aleagha, Mohammad Sajad

2017-10-15

we recently showed that a hypothesized anti-aging and anti-inflammatory protein, namely Klotho, may contribute to the etiology and/or pathogenesis of multiple sclerosis (MS). In addition, Klotho function and its gene expression are dependent on inflammatory pathways. Accordingly, the aim of this study was to investigate the Klotho gene expression within peripheral blood mononuclear cells (PBMCs) of patients with MS. Altogether, 30 patients with relapsing-remitting MS (RRMS) along with 30 age and sex-matched healthy individuals were enrolled in this study. Blood samples were obtained from all participants and then PBMCs were isolated. The quantitative Real-Time PCR was carried out for Klotho mRNA derived from PBMCs. The results showed that klotho gene expression in the PBMCs of patients with RRMS is nearly 2.5-fold less than healthy individuals (P=0.0006). This is the first study demonstrating a possible role of Klotho in the PBMCs of MS patients. Copyright © 2017 Elsevier B.V. All rights reserved.

Expression levels of seven candidate genes in human peripheral blood mononuclear cells and their association with preeclampsia

PubMed Central

Garza-Veloz, I.; Carrillo-Sanchez, K.; Martinez-Gaytan, V.; Cortes-Flores, R.; Ochoa-Torres, M. A.; Guerrero, G. G.; Rodriguez-Sanchez, I. P.; Cancela-Murrieta, C. O.; Zamudio-Osuna, M.; Badillo-Almaraz, J. I.; Castruita-De la Rosa, C.

2014-01-01

Objective To evaluate the peripheral blood mononuclear cell (PBMC) expression levels of hemeoxygenase 1 (HMOX-1), superoxide dismutase 1 (SOD-1), vascular endothelial growth factor A (VEGF-A), transforming growth factor beta 1 (TGF-β1), interleukin (IL)-6, IL-15 and AdipoQ genes to study their association with preeclampsia (PE). Methods A total of 177 pregnant women were recruited: 108 cases and 69 controls. Quantification of gene expression was measured by quantitative real-time polymerase chain reaction (PCR) using TaqMan probes. Results Underexpression of VEGF-A and TGF-β1 was a constant in most of the cases (80.91% and 76.36%, respectively) and their expression was associated with onset and/or severity of disease (p values < 0.05). IL-6, IL-15 and AdipoQ, showed low or no expression in PBMC samples evaluated. Conclusion PBMC underexpression of VEGF-A and TGF-β1 is a hallmark of PE in the study population. PMID:24295154

Immune response in asymptomatic smokers.

PubMed

Zeidel, A; Beilin, B; Yardeni, I; Mayburd, E; Smirnov, G; Bessler, H

2002-09-01

It has been demonstrated that cigarette smoking affects the immune system. Impairment of alveolar mononuclear cell function, described previously, may contribute to the higher rate of postoperative respiratory infections. However, increased susceptibility of smokers to infections of other origin (e.g. wound-related) implies that tobacco effect is not restricted to the respiratory immune competent cells. The present study was designed to investigate the systemic effect of tobacco smoking as it exerted on blood-derived immune cells. We measured systemic cytotoxic activity of natural killer cells, production of pro- and anti-inflammatory cytokines by blood mononuclear cells and their proliferation in response to mitogens. To minimize the immunosuppressive effect of other smoke-related factors, the smokers with chronic obstructive pulmonary disease (COPD) were excluded from this study. Peripheral blood mononuclear cells (PBMC) from 24 chronic asymptomatic smokers, and 28 controls, age and gender matched, were isolated and incubated in vitro with lipopolysaccharide (LPS) or phytohemagglutinin (PHA) to induce secretion of IL-1beta, IL-1ra, IL-6, IL-10, TNFalpha and IL-2, respectively, from mononuclear cells. The level of the cytokines in the supernatants was measured using ELISA kits. The proliferative response to the mitogens PHA and concanavalin A (ConA) was evaluated by 3H-thymidine incorporation and NK cell cytotoxicity by 51Cr release assay. Mononuclear cells from smokers showed increased production of the pro-inflammatory cytokines IL-1beta, IL-6 and TNFalpha and enhanced proliferative response to mitogens as compared to non-smoking population. The secretion of IL-2 and the anti-inflammatory cytokines IL-1ra and IL-10 was similar in both groups. NK cell cytotoxic activity was suppressed in the smokers. Cigarette smokers without chronic obstructive pulmonary disease (COPD) exhibit impaired NK cytotoxic activity in peripheral blood and unbalanced systemic production of pro- and anti-inflammatory cytokines. These changes may serve as predisposing factors for respiratory and systemic infections in the postoperative period and should alert an anesthetist during perioperative management.

M-CSF Mediates Host Defense during Bacterial Pneumonia by Promoting the Survival of Lung and Liver Mononuclear Phagocytes.

PubMed

Bettina, Alexandra; Zhang, Zhimin; Michels, Kathryn; Cagnina, R Elaine; Vincent, Isaah S; Burdick, Marie D; Kadl, Alexandra; Mehrad, Borna

2016-06-15

Gram-negative bacterial pneumonia is a common and dangerous infection with diminishing treatment options due to increasing antibiotic resistance among causal pathogens. The mononuclear phagocyte system is a heterogeneous group of leukocytes composed of tissue-resident macrophages, dendritic cells, and monocyte-derived cells that are critical in defense against pneumonia, but mechanisms that regulate their maintenance and function during infection are poorly defined. M-CSF has myriad effects on mononuclear phagocytes but its role in pneumonia is unknown. We therefore tested the hypothesis that M-CSF is required for mononuclear phagocyte-mediated host defenses during bacterial pneumonia in a murine model of infection. Genetic deletion or immunoneutralization of M-CSF resulted in reduced survival, increased bacterial burden, and greater lung injury. M-CSF was necessary for the expansion of lung mononuclear phagocytes during infection but did not affect the number of bone marrow or blood monocytes, proliferation of precursors, or recruitment of leukocytes to the lungs. In contrast, M-CSF was essential to survival and antimicrobial functions of both lung and liver mononuclear phagocytes during pneumonia, and its absence resulted in bacterial dissemination to the liver and hepatic necrosis. We conclude that M-CSF is critical to host defenses against bacterial pneumonia by mediating survival and antimicrobial functions of mononuclear phagocytes in the lungs and liver. Copyright © 2016 by The American Association of Immunologists, Inc.

M-CSF mediates host defense during bacterial pneumonia by promoting the survival of lung and liver mononuclear phagocytes

PubMed Central

Bettina, Alexandra; Zhang, Zhimin; Michels, Kathryn; Cagnina, R. Elaine; Vincent, Isaah S.; Burdick, Marie D.; Kadl, Alexandra; Mehrad, Borna

2016-01-01

Gram-negative bacterial pneumonia is a common and dangerous infection with diminishing treatment options due to increasing antibiotic resistance among causal pathogens. The mononuclear phagocyte system is a heterogeneous group of leukocytes composed of tissue-resident macrophages, dendritic cells and monocyte-derived cells that are critical in defense against pneumonia, but mechanisms that regulate their maintenance and function during infection are poorly defined. Macrophage-colony stimulating factor (M-CSF) has myriad effects on mononuclear phagocytes but its role in pneumonia is unknown. We therefore tested the hypothesis that M-CSF is required for mononuclear phagocyte-mediated host defenses during bacterial pneumonia in a murine model of infection. Genetic deletion or immunoneutralization of M-CSF resulted in reduced survival, increased bacterial burden and greater lung injury. M-CSF was necessary for the expansion of lung mononuclear phagocytes during infection but did not affect the number of bone marrow or blood monocytes, the proliferation of precursors or the recruitment of leukocytes to the lungs. In contrast, M-CSF was essential to survival and anti-microbial functions of both lung and liver mononuclear phagocytes during pneumonia and its absence resulted in bacterial dissemination to the liver and hepatic necrosis. We conclude that M-CSF is critical to host defenses against bacterial pneumonia by mediating survival and anti-microbial functions of mononuclear phagocytes in the lungs and liver. PMID:27183631

Multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators

PubMed Central

Bodenmiller, Bernd; Zunder, Eli R.; Finck, Rachel; Chen, Tiffany J.; Savig, Erica S.; Bruggner, Robert V.; Simonds, Erin F.; Bendall, Sean C.; Sachs, Karen; Krutzik, Peter O.; Nolan, Garry P.

2013-01-01

The ability to comprehensively explore the impact of bio-active molecules on human samples at the single-cell level can provide great insight for biomedical research. Mass cytometry enables quantitative single-cell analysis with deep dimensionality, but currently lacks high-throughput capability. Here we report a method termed mass-tag cellular barcoding (MCB) that increases mass cytometry throughput by sample multiplexing. 96-well format MCB was used to characterize human peripheral blood mononuclear cell (PBMC) signaling dynamics, cell-to-cell communication, the signaling variability between 8 donors, and to define the impact of 27 inhibitors on this system. For each compound, 14 phosphorylation sites were measured in 14 PBMC types, resulting in 18,816 quantified phosphorylation levels from each multiplexed sample. This high-dimensional systems-level inquiry allowed analysis across cell-type and signaling space, reclassified inhibitors, and revealed off-target effects. MCB enables high-content, high-throughput screening, with potential applications for drug discovery, pre-clinical testing, and mechanistic investigation of human disease. PMID:22902532

Walter Reed Army Institute of Research Annual Progress Report, Fiscal Year 1981.

DTIC Science & Technology

1981-10-01

induce mononuclear cell-mediated antibacterial activity against X-16 shigella has been further in- vestigated. Monocytes, but not K or T lymphocytes are...M.J. Bertovich, and S.B. Formal. Antibody-dependent cell-mediated antibacterial activity : K lymphocytes, monocytes and granulo- cytes are effective...Wilson, S.R., Formal, S.B., Keren, D.F. and Lowell, G.H. Secretory IgA- dependent mononuclear cell-mediated antibacterial activity . Fed. Proc. 4787

Intestinal Mononuclear Phagocytes in Health and Disease.

PubMed

Sanders, Theodore J; Yrlid, Ulf; Maloy, Kevin J

2017-01-01

The intestine is the tissue of the body with the highest constitutive exposure to foreign antigen and is also a common entry portal for many local and systemic pathogens. Therefore, the local immune system has the unenviable task of balancing efficient responses to dangerous pathogens with tolerance toward beneficial microbiota and food antigens. As in most tissues, the decision between tolerance and immunity is critically governed by the activity of local myeloid cells. However, the unique challenges posed by the intestinal environment have necessitated the development of several specialized mononuclear phagocyte populations with distinct phenotypic and functional characteristics that have vital roles in maintaining barrier function and immune homeostasis in the intestine. Intestinal mononuclear phagocyte populations, comprising dendritic cells and macrophages, are crucial for raising appropriate active immune responses against ingested pathogens. Recent technical advances, including microsurgical approaches allowing collection of cells migrating in intestinal lymph, intravital microscopy, and novel gene-targeting approaches, have led to clearer distinctions between mononuclear phagocyte populations in intestinal tissue. In this review, we present an overview of the various subpopulations of intestinal mononuclear phagocytes and discuss their phenotypic and functional characteristics. We also outline their roles in host protection from infection and their regulatory functions in maintaining immune tolerance toward beneficial intestinal antigens.

High IL-35 pleural expression in patients with tuberculous pleural effusion.

PubMed

Dong, Xuan; Yang, Jiong

2015-05-03

IL-35 is a novel anti-inflammatory and immunosuppressive cytokine primarily produced by Treg cells, and is involved in inflammatory diseases and autoimmune diseases. However, its roles in tuberculous pleural effusion (TPE) remain unknown. We aimed to investigate the potential involvement of IL-35 in TPE. Thirty TPE patients and 20 lung cancer patients with malignant pleural effusion (MPE) were recruited. Samples of pleural effusion (100 mL) were collected after traditional pleurocentesis. Blood was sampled from TPE patients. Mononuclear cells were isolated by Ficoll-Hypaque gradient. Proportions of Th1, Th17, and IL-35-producing cells were analyzed by flow cytometry. IL-35 was assessed by real-time RT-PCR, ELISA, and immunofluorescence. An ELISPOT assay was used to assess the effect of IL-35 on pleural effusion mononuclear cells (PEMCs). Proportions of IL-35-producing cells were higher in TPE compared with MPE (49.4±6.0 vs. 15.8±5.4%, P<0.001) and blood from TPE patients (49.4±6.0% vs. 16.6±3.1, P<0.001). IL-35, IL-17 and IFN-γ were elevated in TPE compared with MPE (all P<0.01). ELISPOT assay showed that IL-35 reduced the proportion of IFN-γ-producing CD4+ T cells in TPE. IL-35 mRNA expression was higher in TPE compared with MPE (P<0.001). Immunofluorescence showed that IL-35-positive cells were present in pleural tissues from TPE patients. Results suggest that there is an imbalance in IL-35 metabolism in TPE. However, further studies are required to assess the exact relationship with the immune system response to tuberculosis. IL-35 might play a role in TPE and might be targeted as a treatment for TPE.

Impact of dialyzer membrane on apoptosis and function of polymorphonuclear cells and cytokine synthesis by peripheral blood mononuclear cells in hemodialysis patients.

PubMed

Andreoli, Maria C C; Dalboni, Maria A; Watanabe, Renato; Manfredi, Silvia R; Canziani, Maria E F; Kallás, Esper G; Sesso, Ricardo C; Draibe, Sergio A; Balakrishnan, Vaidyanathapuram S; Jaber, Bertrand L; Liangos, Orfeas; Cendoroglo, Miguel

2007-12-01

In an in vivo crossover trial, we compared a cellulosic with a synthetic dialyzer with respect to polymorphonuclear cells (PMN) function and apoptosis, cytokine serum levels and synthesis by peripheral blood mononuclear cells (PBMC), and complement activation. Twenty hemodialysis (HD) patients were assigned in alternate order to HD with cellulose acetate (CA) or polysulfone (PS) dialyzer. After 2 weeks, patients were crossed over to the second dialyzer and treated for another 2 weeks. Apoptosis was assessed by flow cytometry in freshly isolated PMN. Phagocytosis and production of peroxide by PMN were studied by flow cytometry in whole blood. PBMC were isolated from blood samples and incubated for 24 h with or without lipopolysaccharide (LPS). There was no impact of dialyzer biocompatibility on PMN apoptosis and function, cytokine synthesis by PBMC or on their serum levels, serum levels of C3a, and terminal complement complex (TCC). Nevertheless, after HD, serum levels of complement correlated negatively with PMN phagocytosis and peroxide production, and positively with PMN apoptosis and cytokine production by PBMC. Although the results did not show a dialyzer advantage on the immunologic parameters, complement activation may have modulated cell function and apoptosis after HD.

Effects of dietary glutamine on inflammatory mediator gene expressions in rats with streptozotocin-induced diabetes.

PubMed

Tsai, Pei-Hsuan; Yeh, Chui-Li; Liu, Jun-Jen; Chiu, Wan-Chun; Yeh, Sung-Ling

2012-03-01

This study investigated the effects of glutamine (Gln) supplementation on gene expressions of inflammatory mediators and cytokines associated with T-helper cell type 17 (Th17) regulation in diabetic rats. There were one normal control group and two diabetic groups in this study. Rats in the normal control group were fed a regular chow diet. One diabetic group (DM) was fed a common semipurified diet, and the other diabetic group received a diet in which part of the casein was replaced by Gln (DM-Gln), which provided 25% of the total amino acid nitrogen for 8 wk. Diabetes was induced by an intraperitoneal injection of nicotinamide followed by streptozotocin. Rats with blood glucose levels exceeding 200 mg/dL were considered diabetic. Blood samples and blood mononuclear cells of the animals were collected at the end of the study for further analysis. Gene expressions of transforming growth factor-β1 and interleukin-17A did not differ in blood mononuclear cells among the three groups. Expressions of interleukin-6, interleukin-23, monocyte chemotactic protein-1, and the receptor of the advanced glycated endproducts gene were higher in blood mononuclear cells and the ratio of reduced to oxidized glutathione was lower in erythrocytes in the DM group than in the normal control group. Messenger RNA expressions of these genes were lower, whereas the ratio of reduced to oxidized glutathione was higher in the DM-Gln group than in the DM group. Supplemental dietary Gln increased the antioxidant potential and downregulated the expressions of inflammatory mediators. However, Th17 might not be an important involved pathway and the regulatory effect of Gln on Th17 immune response was not obvious in this animal model. Copyright © 2012 Elsevier Inc. All rights reserved.

In vitro effects of CpG oligodeoxynucleotides delivered by gelatin nanoparticles on canine peripheral blood mononuclear cells of atopic and healthy dogs - a pilot study.

PubMed

Prélaud, Ana Rostaher; Fuchs, Sebastian; Weber, Karin; Winter, Gerhard; Coester, Conrad; Mueller, Ralf S

2013-10-01

Cytosine-phosphate-guanine (CpG) oligodeoxynucleotides offer a novel promising immunotherapeutic approach for atopic dermatitis (AD) both in humans and animals. Gelatin nanoparticles (GNP) enhance and prolong CpG-associated immunomodulatory effects and minimize adverse effects both in vitro and in vivo. Information about the effects of this combination in dogs is lacking. The aim of this study was to evaluate immunological effects of CpG coupled to GNP on canine peripheral blood mononuclear cells (PBMCs) in vitro. Eight dogs with AD, diagnosed by standard criteria and with a concurrent immediate hypersensitivity to house dust mites were included. Control samples were taken from eight healthy, age-matched control dogs without history or evidence of cutaneous or systemic illness. Peripheral blood mononuclear cells of healthy and allergic dogs were incubated with CpG-GNP and the uptake of CpG-GNP was demonstrated using confocal laser scanning microscopy. Cell culture supernatant concentrations of interferon gamma (IFN-γ), interleukin (IL)-4, IL-6 and IL-10 were measured by Canine Cytokine Milliplex. No significant changes in IFN-γ and IL-4 were found when comparing PBMCs incubated with CpG and CpG-GNP with the negative controls in atopic and healthy dogs. Interleukin-6 was not detected in any of the groups. However, a statistically significant increase in IL-10 concentration was found after 24 h stimulation with CpG-GNP compared with CpG alone both in atopic and healthy dogs. As IL-10 is considered an immunosuppressive cytokine playing a key role in peripheral tolerance; the reported CpG-GNP formulation could be a new approach in allergy treatment. © 2013 ESVD and ACVD.

UV-inactivated HSV-1 potently activates NK cell killing of leukemic cells

PubMed Central

Samudio, Ismael; Rezvani, Katayoun; Shaim, Hila; Hofs, Elyse; Ngom, Mor; Bu, Luke; Liu, Guoyu; Lee, Jason T. C.; Imren, Suzan; Lam, Vivian; Poon, Grace F. T.; Ghaedi, Maryam; Takei, Fumio; Humphries, Keith; Jia, William

2016-01-01

Herein we demonstrate that oncolytic herpes simplex virus-1 (HSV-1) potently activates human peripheral blood mononuclear cells (PBMCs) to lyse leukemic cell lines and primary acute myeloid leukemia samples, but not healthy allogeneic lymphocytes. Intriguingly, we found that UV light–inactivated HSV-1 (UV-HSV-1) is equally effective in promoting PBMC cytolysis of leukemic cells and is 1000- to 10 000-fold more potent at stimulating innate antileukemic responses than UV-inactivated cytomegalovirus, vesicular stomatitis virus, reovirus, or adenovirus. Mechanistically, UV-HSV-1 stimulates PBMC cytolysis of leukemic cells, partly via Toll-like receptor-2/protein kinase C/nuclear factor-κB signaling, and potently stimulates expression of CD69, degranulation, migration, and cytokine production in natural killer (NK) cells, suggesting that surface components of UV-HSV-1 directly activate NK cells. Importantly, UV-HSV-1 synergizes with interleukin-15 (IL-15) and IL-2 in inducing activation and cytolytic activity of NK cells. Additionally, UV-HSV-1 stimulates glycolysis and fatty acid oxidation–dependent oxygen consumption in NK cells, but only glycolysis is required for their enhanced antileukemic activity. Last, we demonstrate that T cell–depleted human PBMCs exposed to UV-HSV-1 provide a survival benefit in a murine xenograft model of human acute myeloid leukemia (AML). Taken together, our results support the preclinical development of UV-HSV-1 as an adjuvant, alone or in combination with IL-15, for allogeneic donor mononuclear cell infusions to treat AML. PMID:26941401

Defining Mononuclear Phagocyte Subset Homology Across Several Distant Warm-Blooded Vertebrates Through Comparative Transcriptomics

PubMed Central

Vu Manh, Thien-Phong; Elhmouzi-Younes, Jamila; Urien, Céline; Ruscanu, Suzana; Jouneau, Luc; Bourge, Mickaël; Moroldo, Marco; Foucras, Gilles; Salmon, Henri; Marty, Hélène; Quéré, Pascale; Bertho, Nicolas; Boudinot, Pierre; Dalod, Marc; Schwartz-Cornil, Isabelle

2015-01-01

Mononuclear phagocytes are organized in a complex system of ontogenetically and functionally distinct subsets, that has been best described in mouse and to some extent in human. Identification of homologous mononuclear phagocyte subsets in other vertebrate species of biomedical, economic, and environmental interest is needed to improve our knowledge in physiologic and physio-pathologic processes, and to design intervention strategies against a variety of diseases, including zoonotic infections. We developed a streamlined approach combining refined cell sorting and integrated comparative transcriptomics analyses which revealed conservation of the mononuclear phagocyte organization across human, mouse, sheep, pigs and, in some respect, chicken. This strategy should help democratizing the use of omics analyses for the identification and study of cell types across tissues and species. Moreover, we identified conserved gene signatures that enable robust identification and universal definition of these cell types. We identified new evolutionarily conserved gene candidates and gene interaction networks for the molecular regulation of the development or functions of these cell types, as well as conserved surface candidates for refined subset phenotyping throughout species. A phylogenetic analysis revealed that orthologous genes of the conserved signatures exist in teleost fishes and apparently not in Lamprey. PMID:26150816

Mononuclear phagocytes as a target, not a barrier, for drug delivery.

PubMed

Yong, Seok-Beom; Song, Yoonsung; Kim, Hyung Jin; Ain, Qurrat Ul; Kim, Yong-Hee

2017-08-10

Mononuclear phagocytes have been generally recognized as a barrier to drug delivery. Recently, a new understanding of mononuclear phagocytes (MPS) ontogeny has surfaced and their functions in disease have been unveiled, demonstrating the need for re-evaluation of perspectives on mononuclear phagocytes in drug delivery. In this review, we described mononuclear phagocyte biology and focus on their accumulation mechanisms in disease sites with explanations of monocyte heterogeneity. In the 'MPS as a barrier' section, we summarized recent studies on mechanisms to avoid phagocytosis based on two different biological principles: protein adsorption and self-recognition. In the 'MPS as a target' section, more detailed descriptions were given on mononuclear phagocyte-targeted drug delivery systems and their applications to various diseases. Collectively, we emphasize in this review that mononuclear phagocytes are potent targets for future drug delivery systems. Mononuclear phagocyte-targeted delivery systems should be created with an understanding of mononuclear phagocyte ontogeny and pathology. Each specific subset of phagocytes should be targeted differently by location and function for improved disease-drug delivery while avoiding RES clearance such as Kupffer cells and splenic macrophages. Copyright © 2017 Elsevier B.V. All rights reserved.

In vitro cell-mediated immunity after thermal injury is not impaired. Density gradient purification of mononuclear cells is associated with spurious (artifactual) immunosuppression.

PubMed Central

Xu, D Z; Deitch, E A; Sittig, K; Qi, L; McDonald, J C

1988-01-01

Mononuclear cells isolated by density gradient centrifugation from the peripheral blood of burn patients, but not healthy volunteers, are contaminated with large numbers of nonmononuclear cells. These contaminating leukocytes could cause artifactual alterations in standard in vitro tests of lymphocyte function. Thus, we compared the in vitro blastogenic response of density gradient purified leukocytes and T-cell purified lymphocytes from 13 burn patients to mitogenic (PHA) and antigenic stimuli. The mitogenic and antigenic response of the patients' density gradient purified leukocytes were impaired compared to healthy volunteers (p less than 0.01). However, when the contaminating nonlymphocytes were removed, the patients' cells responded normally to both stimuli. Thus, density gradient purified mononuclear cells from burn patients are contaminated by leukocytes that are not phenotypically or functionally lymphocytes. Since the lymphocytes from burn patients respond normally to PHA and alloantigens after the contaminating nonlymphocyte cell population has been removed, it appears that in vitro assays of lymphocyte function using density gradient purified leukocytes may give spurious results. PMID:2973771

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

PubMed

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.

Barcoding of live human peripheral blood mononuclear cells for multiplexed mass cytometry.

PubMed

Mei, Henrik E; Leipold, Michael D; Schulz, Axel Ronald; Chester, Cariad; Maecker, Holden T

2015-02-15

Mass cytometry is developing as a means of multiparametric single-cell analysis. In this study, we present an approach to barcoding separate live human PBMC samples for combined preparation and acquisition on a cytometry by time of flight instrument. Using six different anti-CD45 Ab conjugates labeled with Pd104, Pd106, Pd108, Pd110, In113, and In115, respectively, we barcoded up to 20 samples with unique combinations of exactly three different CD45 Ab tags. Cell events carrying more than or less than three different tags were excluded from analyses during Boolean data deconvolution, allowing for precise sample assignment and the electronic removal of cell aggregates. Data from barcoded samples matched data from corresponding individually stained and acquired samples, at cell event recoveries similar to individual sample analyses. The approach greatly reduced technical noise and minimizes unwanted cell doublet events in mass cytometry data, and it reduces wet work and Ab consumption. It also eliminates sample-to-sample carryover and the requirement of instrument cleaning between samples, thereby effectively reducing overall instrument runtime. Hence, CD45 barcoding facilitates accuracy of mass cytometric immunophenotyping studies, thus supporting biomarker discovery efforts, and it should be applicable to fluorescence flow cytometry as well. Copyright © 2015 by The American Association of Immunologists, Inc.

Mononuclear cells in the corneal response to endotoxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Howes, E.L.; Cruse, V.K.; Kwok, M.T.

A severe keratitis can be produced after the direct injection of bacterial endotoxin, or lipopolysaccharide (LPS), in rabbits. Corneal inflammation can progress to scarring and vascularization within a 2 to 3 week period. Pretreatment with systemic adrenal corticosteroids (triamcinolone) prevents this response. Limbal cellular and vascular events were studied during the first 20 hr after injection of LPS in treated and nontreated rabbits. Perivascular limbal inflammatory cells were counted and limbal vascular permeability was assessed by extravasation of 131I-albumin and 125I-fibrinogen in the cornea. Corticosteroids decreased but did not prevent the early protein extravasation and profoundly altered the inflammatory cellmore » population around blood vessels at the limbus. Mononuclear cells, particularly mononuclear phagocytes, were sharply reduced. It is proposed that these cell types play an important role in the perpetuation and amplification of the inflammatory response in this reaction.« less

Micro-buffy coats of whole blood: a method for the electron microscopic study of mononuclear cells.

PubMed

Nunes, J F; Soares, J O; Alves de Matos, A P

1979-09-01

A method for the electron microscopic study of human peripheral lymphocytes by which very small buffy coats are obtained through centrifugation of heparinized whole blood in glass or plastic microhematocrit tubes is presented. This method is time saving and efficient, yielding well preserved material and a comparatively large number of mononuclear cells (mainly lymphocytes) in each thin section.

Paracrine Factors from Irradiated Peripheral Blood Mononuclear Cells Improve Skin Regeneration and Angiogenesis in a Porcine Burn Model

PubMed Central

Hacker, Stefan; Mittermayr, Rainer; Nickl, Stefanie; Haider, Thomas; Lebherz-Eichinger, Diana; Beer, Lucian; Mitterbauer, Andreas; Leiss, Harald; Zimmermann, Matthias; Schweiger, Thomas; Keibl, Claudia; Hofbauer, Helmut; Gabriel, Christian; Pavone-Gyöngyösi, Mariann; Redl, Heinz; Tschachler, Erwin; Mildner, Michael; Ankersmit, Hendrik Jan

2016-01-01

Burn wounds pose a serious threat to patients and often require surgical treatment. Skin grafting aims to achieve wound closure but requires a well-vascularized wound bed. The secretome of peripheral blood mononuclear cells (PBMCs) has been shown to improve wound healing and angiogenesis. We hypothesized that topical application of the PBMC secretome would improve the quality of regenerating skin, increase angiogenesis, and reduce scar formation after burn injury and skin grafting in a porcine model. Full-thickness burn injuries were created on the back of female pigs. Necrotic areas were excised and the wounds were covered with split-thickness mesh skin grafts. Wounds were treated repeatedly with either the secretome of cultured PBMCs (SecPBMC), apoptotic PBMCs (Apo-SecPBMC), or controls. The wounds treated with Apo-SecPBMC had an increased epidermal thickness, higher number of rete ridges, and more advanced epidermal differentiation than controls. The samples treated with Apo-SecPBMC had a two-fold increase in CD31+ cells, indicating more angiogenesis. These data suggest that the repeated application of Apo-SecPBMC significantly improves epidermal thickness, angiogenesis, and skin quality in a porcine model of burn injury and skin grafting. PMID:27125302

Role of bone marrow transplantation for correcting hemophilia A in mice

PubMed Central

Follenzi, Antonia; Raut, Sanj; Merlin, Simone; Sarkar, Rita

2012-01-01

To better understand cellular basis of hemophilia, cell types capable of producing FVIII need to be identified. We determined whether bone marrow (BM)–derived cells would produce cells capable of synthesizing and releasing FVIII by transplanting healthy mouse BM into hemophilia A mice. To track donor-derived cells, we used genetic reporters. Use of multiple coagulation assays demonstrated whether FVIII produced by discrete cell populations would correct hemophilia A. We found that animals receiving healthy BM cells survived bleeding challenge with correction of hemophilia, although donor BM-derived hepatocytes or endothelial cells were extremely rare, and these cells did not account for therapeutic benefits. By contrast, donor BM-derived mononuclear and mesenchymal stromal cells were more abundant and expressed FVIII mRNA as well as FVIII protein. Moreover, injection of healthy mouse Kupffer cells (liver macrophage/mononuclear cells), which predominantly originate from BM, or of healthy BM-derived mesenchymal stromal cells, protected hemophilia A mice from bleeding challenge with appearance of FVIII in blood. Therefore, BM transplantation corrected hemophilia A through donor-derived mononuclear cells and mesenchymal stromal cells. These insights into FVIII synthesis and production in alternative cell types will advance studies of pathophysiological mechanisms and therapeutic development in hemophilia A. PMID:22368271

A method for isolation of rat lymphocyte-rich mononuclear cells from lung tissue useful for determination of nucleoside triphosphate diphosphohydrolase activity.

PubMed

Jaques, Jeandre Augusto Dos S; Peres Rezer, João Felipe; Ruchel, Jader Betsch; Gutierres, Jessié; Bairros, André Valle; Gomes Farias, Iria Luiza; Almeida da Luz, Sonia Cristina; Mello Bertoncheli, Claudia de; Chitolina Schetinger, Maria Rosa; Morsch, Vera Maria; Leal, Daniela Bitencourt Rosa

2011-03-01

Methods for the isolation of peripheral blood mononuclear cells (PBMCs) and human lung mononuclear cells (LMCs) have been proposed previously. This study describes a method that allows the separation of lymphocyte-rich LMCs from rats. Trypan blue was applied to determine cell viability. White blood cell and differential cell counts were also performed. Relationships between nucleoside triphosphate diphosphohydrolase (NTPDase, EC 3.6.1.5) activities expressed in milligrams of protein, millions of cells, and millions of viable cells were examined as linear correlations. The lung tissue yielded 82.46% lymphocytes, 8.6% macrophages, 2.20% monocytes, and 1.27% polymorphonuclear cells (PMNs). In LMCs, a very strong correlation was observed as follows: between NTPDase activity, as determined using ATP or ADP as a substrate, expressed in milligrams of protein and that expressed in millions of cells (r ≥ 0.91), between that expressed in milligrams of protein and that expressed in millions of viable cells (r ≥ 0.91), and between that expressed in millions of cells and that expressed in millions of viable cells (r ≥ 0.98). Based on our results, we affirm that NTPDase activity could be expressed in millions of viable cells, millions of cells, or milligrams of protein. 2010 Elsevier Inc. All rights reserved.

NMDA Receptors Regulate Genes Responsible for Major Immune Functions of Mononuclears in Human Peripheral Blood.

PubMed

Kuzmina, U Sh; Zainullina, L F; Sadovnikov, S V; Vakhitov, V A; Vakhitova, Yu V

2018-06-19

To determine the role of NMDA receptors in the functional regulation of immunocompetent cells, comparative assay was carried out for genes expressed in the mononuclears in peripheral blood of healthy persons under normal conditions and after blockade of these receptors. The genes, whose expression changed in response to blockade of NMDA receptors in mononuclears, encode the products involved in regulation of the major functions of immune cells, such as proliferation (IL4, VCAM1, and CDKN2A), apoptosis (BAX, MYC, CDKN2A, HSPB1, and CADD45A), activation (IL4R, IL4, VCAM1, and CDKN2A), and differentiation (IL4, VCAM1, and BAX).

Detection and Characterization of Circulating Tumour Cells from Frozen Peripheral Blood Mononuclear Cells

PubMed Central

Lu, David; Graf, Ryon P.; Harvey, Melissa; Madan, Ravi A.; Heery, Christopher; Marte, Jennifer; Beasley, Sharon; Tsang, Kwong Y.; Krupa, Rachel; Louw, Jessica; Wahl, Justin; Bales, Natalee; Landers, Mark; Marrinucci, Dena; Schlom, Jeffrey; Gulley, James L.; Dittamore, Ryan

2015-01-01

Retrospective analysis of patient tumour samples is a cornerstone of clinical research. CTC biomarker characterization offers a non-invasive method to analyse patient samples. However, current CTC technologies require prospective blood collection, thereby reducing the ability to utilize archived clinical cohorts with long-term outcome data. We sought to investigate CTC recovery from frozen, archived patient PBMC pellets. Matched samples from both mCRPC patients and mock samples, which were prepared by spiking healthy donor blood with cultured prostate cancer cell line cells, were processed “fresh” via Epic CTC Platform or from “frozen” PBMC pellets. Samples were analysed for CTC enumeration and biomarker characterization via immunofluorescent (IF) biomarkers, fluorescence in-situ hybridization (FISH) and CTC morphology. In the frozen patient PMBC samples, the median CTC recovery was 18%, compared to the freshly processed blood. However, abundance and localization of cytokeratin (CK) and androgen receptor (AR) protein, as measured by IF, were largely concordant between the fresh and frozen CTCs. Furthermore, a FISH analysis of PTEN loss showed high concordance in fresh vs. frozen. The observed data indicate that CTC biomarker characterization from frozen archival samples is feasible and representative of prospectively collected samples. PMID:28936240

Temperature fluctuations during deep temperature cryopreservation reduce PBMC recovery, viability and T-cell function.

PubMed

Germann, Anja; Oh, Young-Joo; Schmidt, Tomm; Schön, Uwe; Zimmermann, Heiko; von Briesen, Hagen

2013-10-01

The ability to analyze cryopreserved peripheral blood mononuclear cell (PBMC) from biobanks for antigen-specific immunity is necessary to evaluate response to immune-based therapies. To ensure comparable assay results, collaborative research in multicenter trials needs reliable and reproducible cryopreservation that maintains cell viability and functionality. A standardized cryopreservation procedure is comprised of not only sample collection, preparation and freezing but also low temperature storage in liquid nitrogen without any temperature fluctuations, to avoid cell damage. Therefore, we have developed a storage approach to minimize suboptimal storage conditions in order to maximize cell viability, recovery and T-cell functionality. We compared the influence of repeated temperature fluctuations on cell health from sample storage, sample sorting and removal in comparison to sample storage without temperature rises. We found that cyclical temperature shifts during low temperature storage reduce cell viability, recovery and immune response against specific-antigens. We showed that samples handled under a protective hood system, to avoid or minimize such repeated temperature rises, have comparable cell viability and cell recovery rates to samples stored without any temperature fluctuations. Also T-cell functionality could be considerably increased with the use of the protective hood system compared to sample handling without such a protection system. This data suggests that the impact of temperature fluctuation on cell integrity should be carefully considered in future clinical vaccine trials and consideration should be given to optimal sample storage conditions. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Increased telomere length and proliferative potential in peripheral blood mononuclear cells of adults of different ages stimulated with concanavalin A

PubMed Central

2013-01-01

Background Recently, a direct correlation with telomere length, proliferative potential and telomerase activity has been found in the process of aging in peripheral blood cells. The objective of the study was to evaluate telomere length and proliferative potential in peripheral blood mononuclear cells (PBMCs) after stimulation with Concanavalin A (ConA) of young adults compared with older adults. Methods Blood samples were obtained from 20 healthy young males (20–25 years old) (group Y) and 20 males (60–65 years old) (group O). We compared PBMC proliferation before and after stimulation with ConA. DNA was isolated from cells separated before and after culture with ConA for telomeric measurement by real-time polymerase chain reaction. Results In vitro stimulation of PBMCs from young subjects induced an increase of telomere length as well as a higher replicative capacity of cell proliferation. Samples from older adults showed higher loss of telomeric DNA (p = 0.03) and higher levels of senescent (≤6.2 kb) telomeric DNA (p = 0.02) and displayed a marked decrease of proliferation capacity. Viability cell counts and CFSE tracking in 72-h-old cell cultures indicated that group O PBMCs (CD8+ and CD4+ T cells) underwent fewer mitotic cycles and had shorter telomeres than group Y (p = 0.04). Conclusions Our findings confirm that telomere length in older-age adults is shorter than in younger subjects. After stimulation with ConA, cells are not restored to the previous telomere length and undergo replicative senescence. This is in sharp contrast to the response observed in young adults after ConA stimulation where cells increase in telomere length and replicative capacity. The mechanisms involved in this phenomenon are not yet clear and merit further investigation. PMID:24063536

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

PubMed

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

2013-12-01

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

Failure of rabbit neutrophils to secrete endogenous pyrogen when stimulated with staphylococci.

PubMed

Hanson, D F; Murphy, P A; Windle, B E

1980-06-01

Cells obtained from acute peritoneal exudates in rabbits were separated into neutrophil and mononuclear populations by centrifugation on colloidal silica gradients. When these populations were separately incubated in tissue culture medium in the presence of opsonized Staphylococcus epidermidis, endogenous pyrogen was secreted only by the adherent cells of the mononuclear population. Pyrogen production by neutrophils could not have amounted to as much as 1% of the pyrogen produced by macrophages. When mononuclear cells were added back to purified neutrophils, no pyrogen was produced that could not be accounted for by the number of macrophages added. Rabbit blood cells were similarly fractionated on colloidal silica gradients. Again, endogenous pyrogen was made only by the adherent mononuclear population. The neutrophils isolated on these gradients appeared to be morphologically normal and were 85% viable as judged by dye exclusion. They showed normal random motility. Both blood and exudate neutrophils responded chemotactically to N-formyl Met-Leu-Phe, and blood neutrophils responded chemotactically to zymosan-activated serum. Both kinds of neutrophils phagocytosed zymosan particles and both killed opsonized S. epidermidis in a roller tube system. Both blood and exudate neutrophils showed normal superoxide production when stimulated with opsonized zymosan particles. This evidence suggests that macrophages are the only source of endogenous pyrogens, and that pyrogens secreted by cell populations that are rich in neutrophils are to be attributed to the monocytes or macrophages that they contain.

Failure of rabbit neutrophils to secrete endogenous pyrogen when stimulated with staphylococci

PubMed Central

1980-01-01

Cells obtained from acute peritoneal exudates in rabbits were separated into neutrophil and mononuclear populations by centrifugation on colloidal silica gradients. When these populations were separately incubated in tissue culture medium in the presence of opsonized Staphylococcus epidermidis, endogenous pyrogen was secreted only by the adherent cells of the mononuclear population. Pyrogen production by neutrophils could not have amounted to as much as 1% of the pyrogen produced by macrophages. When mononuclear cells were added back to purified neutrophils, no pyrogen was produced that could not be accounted for by the number of macrophages added. Rabbit blood cells were similarly fractionated on colloidal silica gradients. Again, endogenous pyrogen was made only by the adherent mononuclear population. The neutrophils isolated on these gradients appeared to be morphologically normal and were 85% viable as judged by dye exclusion. They showed normal random motility. Both blood and exudate neutrophils responded chemotactically to N-formyl Met-Leu-Phe, and blood neutrophils responded chemotactically to zymosan-activated serum. Both kinds of neutrophils phagocytosed zymosan particles and both killed opsonized S. epidermidis in a roller tube system. Both blood and exudate neutrophils showed normal superoxide production when stimulated with opsonized zymosan particles. This evidence suggests that macrophages are the only source of endogenous pyrogens, and that pyrogens secreted by cell populations that are rich in neutrophils are to be attributed to the monocytes or macrophages that they contain. PMID:6247413

Clinical disease upregulates expression of CD40 and CD40 ligand on peripheral blood mononuclear cells from cattle naturally infected with Mycobacterium avium subsp. paratuberculosis

USDA-ARS?s Scientific Manuscript database

CD40 and CD40L interactions have costimulatory effects that are part of a complex series of events in host cellular and humoral immune responses and inflammation. The purpose of this study was to examine the changes in expression of CD40 and CD40L on peripheral blood mononuclear cells (PBMCs) isolat...

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

PubMed Central

Cesar, Beatriz; Abud, Ana Paula R.; de Oliveira, Carolina C.; Cardoso, Francolino; Bernardi, Raffaello Popa Di; Guimarães, Fernando S. F.; Gabardo, Juarez; de Freitas Buchi, Dorly

2011-01-01

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. PMID:19736221

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

PubMed

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 < 0.05) were observed among the groups treated with BMMC and evaluated after 7, 14 and 21 days. Analysis of the mean linear intercept (Lm) values for the different groups allowed to observe that the group treated with BMMC and evaluated after 21 days showed the most significant result. The group that received no treatment showed a statistically significant difference when compared to other groups, except the group treated and evaluated after 21 days, evidencing the efficacy of cell therapy with BMMC in pulmonary emphysema.

Global changes in DNA methylation in Alzheimer's disease peripheral blood mononuclear cells.

PubMed

Di Francesco, Andrea; Arosio, Beatrice; Falconi, Anastasia; Micioni Di Bonaventura, Maria Vittoria; Karimi, Mohsen; Mari, Daniela; Casati, Martina; Maccarrone, Mauro; D'Addario, Claudio

2015-03-01

Changes in epigenetic marks may help explain the late onset of Alzheimer's disease (AD). In this study we measured genome-wide DNA methylation by luminometric methylation assay, a quantitative measurement of genome-wide DNA methylation, on DNA isolated from peripheral blood mononuclear cells of 37 subjects with late-onset AD (LOAD) and 44 healthy controls (CT). We found an increase in global DNA methylation in LOAD subjects compared to CT (p=0.0122), associated with worse cognitive performances (p=0.0002). DNA hypermethylation in LOAD group was paralleled by higher DNA methyltransferase 1 (DNMT1) gene expression and protein levels. When data were stratified on the basis of the APOE polymorphisms, higher DNA methylation levels were associated with the presence of APOE ε4 allele (p=0.0043) in the global population. Among the APOE ε3 carriers, a significant increase of DNA methylation was still observed in LOAD patients compared to healthy controls (p=0.05). Our data suggest global DNA methylation in peripheral samples as a useful marker for screening individuals at risk of developing AD. Copyright © 2014 Elsevier Inc. All rights reserved.

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.

PubMed

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 capacity or cytokine pattern in their recipients.

The Cytocidal Activity of OK‐432‐activated Mononuclear Cells against Human Glioma Cells is Partly Mediated through the Fas Ligand/Fas System

PubMed Central

Toda, Keisuke; Shiraishi, Tetsuya; Hirotsu, Tatsumi; Fukuyama, Kouzou; Mineta, Toshihiro; Kawaguchi, Shojiro; Tabuchi, Kazuo

1996-01-01

We have been applying an adoptive immunotherapy protocol to patients with malignant brain tumors using OK‐432‐activated peripheral blood mononuclear cells (OK‐MCs). In order to elucidate the mechanism of OK‐MCs' cytotoxicity, we examined the expression of Fas ligand mRNA in OK‐MCs and the cytocidal activity of these cells against a human glioma cell line, T98G which expresses a high level of Fas. The expression of Fas ligand mRNA was low in non‐treated peripheral blood mononuclear cells and was elevated by treatment with OK‐432, irrespective of the dose employed. Apoptosis of T98G cells induced by OK‐MCs was unequivocally inhibited by the pretreatment of T98G cells with ZB4 monoclonal antibody, which binds to Fas and blocks the binding of Fas ligand to Fas. These data indicate that the cytotoxic activity of OK‐MCs via apoptosis seems to be at least partly mediated by the Fas ligand/Fas system. Adoptive immunotherapy using the Fas ligand/Fas system could be a new treatment modality for human malignant brain tumors. PMID:8878461

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

PubMed

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.

The Effects of Omega-3 Fatty Acids Supplementation on Gene Expression Involved in the Insulin and Lipid Signaling Pathway in Patients with Polycystic Ovary Syndrome.

PubMed

Nasri, Khadijeh; Hantoushzadeh, Sedigheh; Aghadavod, Esmat; Taghizadeh, Mohsen; Asemi, Zatollah

2017-06-01

Limited data are available evaluating the effects of omega-3 fatty acids supplementation on gene expression involved in the insulin and lipid-signaling pathway in women with polycystic ovary syndrome (PCOS). This study was conducted to evaluate the effects of omega-3 fatty acids supplementation on gene expression involved in the insulin and lipid signaling pathway in women with PCOS. This randomized double blind, placebo-controlled trial was done among 60 women aged 18-40 years old and diagnosed with PCOS according to the Rotterdam criteria. Participants were randomly assigned into 2 groups to receive either 1 000 mg omega-3 fatty acids from flaxseed oil containing 400 mg α-linolenic acid (n=30) or placebo (n=30) twice a day for 12 weeks. Gene expressions involved in the insulin and lipid-signaling pathway were quantified in blood samples of PCOS women with RT-PCR method. Quantitative results of RT-PCR demonstrated that compared with the placebo, omega-3 fatty acids supplementation upregulated peroxisome proliferator-activated receptor gamma (PPAR-γ) mRNA (p=0.005) in peripheral blood mononuclear cells of women with PCOS. In addition, compared to the placebo, omega-3 fatty acids supplementation downregulated expressed levels of oxidized low-density lipoprotein receptor (LDLR) mRNA (p=0.002) in peripheral blood mononuclear cells of women with PCOS. We did not observe any significant effect of omega-3 fatty acids supplementation on expressed levels of glucose transporter 1 (GLUT-1) and lipoprotein(a) [Lp(a)] genes in peripheral blood mononuclear cells. Overall, omega-3 fatty acids supplementation for 12 weeks in PCOS women significantly improved gene expression of PPAR-γ and LDLR. © Georg Thieme Verlag KG Stuttgart · New York.

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

PubMed

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.

Probiotic metabolites from Bacillus coagulans GanedenBC30TM support maturation of antigen-presenting cells in vitro

PubMed Central

Benson, Kathleen F; Redman, Kimberlee A; Carter, Steve G; Keller, David; Farmer, Sean; Endres, John R; Jensen, Gitte S

2012-01-01

AIM: To study the effects of probiotic metabolites on maturation stage of antigen-presenting immune cells. METHODS: Ganeden Bacillus coagulans 30 (GBC30) bacterial cultures in log phase were used to isolate the secreted metabolite (MET) fraction. A second fraction was made to generate a crude cell-wall-enriched fraction, by centrifugation and lysis, followed by washing. A preparation of MET was subjected to size exclusion centrifugation, generating three fractions: < 3 kDa, 3-30 kDa, and 30-200 kDa and activities were tested in comparison to crude MET and cell wall in primary cultures of human peripheral blood mononuclear cell (PBMC) as a source of antigen-presenting mononuclear phagocytes. The maturation status of mononuclear phagocytes was evaluated by staining with monoclonal antibodies towards CD14, CD16, CD80 and CD86 and analyzed by flow cytometry. RESULTS: Treatment of PBMC with MET supported maturation of mononuclear phagocytes toward both macrophage and dendritic cell phenotypes. The biological activity unique to the metabolites included a reduction of CD14+ CD16+ pro-inflammatory cells, and this property was associated with the high molecular weight metabolite fraction. Changes were also seen for the dendritic cell maturation markers CD80 and CD86. On CD14dim cells, an increase in both CD80 and CD86 expression was seen, in contrast to a selective increase in CD86 expression on CD14bright cells. The co-expression of CD80 and CD86 indicates effective antigen presentation to T cells and support of T helper cell differentiation. The selective expression of CD86 in the absence of CD80 points to a role in generating T regulatory cells. CONCLUSION: The data show that a primary mechanism of action of GBC30 metabolites involves support of more mature phenotypes of antigen-presenting cells, important for immunological decision-making. PMID:22563167

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

PubMed

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 < 0.0001). This highly significant correlation was maintained for samples with the NAT1*4 (p = 0.002) and NAT1*14B haplotypes (p = 0.0106). These results provide the first documentation that NAT1-catalyzed 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.

Extracorporeal photopheresis reduces the number of mononuclear cells that produce pro-inflammatory cytokines, when tested ex-vivo.

PubMed

Bladon, John; Taylor, Peter

2002-01-01

Extracorporeal photopheresis (ECP) has been shown to be clinically effective in the treatment of many T cell-mediated conditions. ECP's mechanism of action includes the induction of apoptosis and the release of pro-inflammatory cytokines. Recently, we have observed early lymphoid apoptosis, detectable immediately post ECP. We were interested to determine what influence ECP has on pro-inflammatory cytokine secretion at this early pre-infusion stage. Samples from 6 cutaneous T cell lymphoma (CTCL) and 5 graft versus host disease (GvHD) patients were taken pre ECP and immediately post ECP, prior to re-infusion. Following separation, the PBMCs were added to a cell culture medium and stimulated with PMA, Ionomycin, and Brefeldin A for 6 hours. Using flow cytometry, intracellular cytokine expression of IFNgamma and TNFalpha was determined in the T cell population. The monocytes were evaluated for IL6, IFNgamma, IL12, and TNFalpha. For both patient groups, the number of IFNgamma-expressing T cells fell significantly at re-infusion, whilst both T cell- and monocyte-expressing TNFalpha levels were reduced at re-infusion. All other cytokines tested showed no significant change post ECP. For GvHD, pro-inflammatory cytokines have a pathological role. Their down-regulation may have a direct clinical benefit. However, the reduction in the number of IFNgamma- and TNFalpha-expressing mononuclear cells means, at this early stage, it is unlikely that these cytokines assist in the removal of the malignant Th2 cells present in CTCL. Copyright 2002 Wiley-Liss, Inc.

Characterization of lymphocyte populations in nonspecific interstitial pneumonia*

PubMed Central

Keogh, Karina A; Limper, Andrew H

2005-01-01

Study objectives Nonspecific interstitial pneumonia (NSIP) has been identified as a distinct entity with a more favorable prognosis and better response to immunosuppressive therapies than usual interstitial pneumonia (UIP). However the inflammatory profile of NSIP has not been characterized. Design Using immunohistochemistry techniques on open lung biopsy specimens, the infiltrate in NSIP was characterized in terms of T and B cells, and macrophages, and the T cell population further identified as either CD4 (helper) or CD8 (suppressor-cytotoxic) T cells. The extent of Th1 and Th2 cytokine producing cells was determined and compared to specimens from patients with UIP. Results In ten NSIP tissue samples 41.4 ± 4% of mononuclear cells expressed CD3, 24.7 ± 1.8% CD4, 19.1 ± 2% CD8, 27.4 ± 3.9% CD20, and 14.3 ± 1.6% had CD68 expression. Mononuclear cells expressed INFγ 21.9 ± 1.9% of the time and IL-4 in 3.0 ± 1%. In contrast, biopsies from eight patients with UIP demonstrated substantially less cellular staining for either cytokine (INFγ; 4.6 ± 1.7% and IL-4; 0.6 ± 0.3%). Significant populations of CD20 positive B-cells were also identified. Conclusion The lymphocytic infiltrate in NSIP is characterized by an elevated CD4/CD8 T-cell ratio, and is predominantly of Th1 type, with additional populations rich in B-cells. Such features are consistent with the favorable clinical course observed in patients with NSIP compared to UIP. PMID:16287509

Capsular synovial-like hyperplasia around mammary implants similar to detritic synovitis. A morphologic and immunohistochemical study of 15 cases.

PubMed

Hameed, M R; Erlandson, R; Rosen, P P

1995-04-01

Formation of a fibrous envelope around the implant, a so-called capsule with resultant contracture of the prosthesis, is an occasional complication of augmentation mammoplasty. The capsulectomy specimen contains mature scar tissue with mononuclear cells, histiocytes, and foreign body giant cells. We studied 15 capsulectomy specimens. Seven showed a striking form of papillary villous synovial-like hyperplasia similar to detritic synovitis, a form of proliferative synovitis caused by orthopedic prosthetic devices. There was an accompanying infiltration of the subcapsular surface by mononuclear cells, giant cells, and chronic inflammatory cells. This reaction was independent of the type of prosthetic device. In one case, foreign material consistent with polyurethane was demonstrated by histology and electron microscopy. Among eight cases without capsular synovial-like hyperplasia (CSH), two showed dense fibrous tissue with foamy macrophages, and the rest showed fat necrosis, foreign body giant cell reaction, and occasional evidence of foreign material, including silicone granulomas. We stained four of the CSH, two with silicone granulomas, and one sample with dense fibrous tissue with peanut agglutinin and antibodies against vimentin and S-100 protein. Selected cases were also stained for concanavalin A and cytokeratin. CSH stained for concanavalin A, peanut agglutinin, and vimentin but was negative for cytokeratin. Our cases showed a striking similarity in the staining pattern of CSH, detritic synovitis, and normal synovium. We conclude that CSH of the mammary prosthetic capsule is pathophysiologically similar to proliferative synovitis.

Reciprocal modulation of helper Th1 and Th17 cells by the β2-adrenergic receptor agonist drug terbutaline.

PubMed

Carvajal Gonczi, Catalina M; Tabatabaei Shafiei, Mahdieh; East, Ashley; Martire, Erika; Maurice-Ventouris, Meagane H I; Darlington, Peter J

2017-09-01

Catecholamine hormones are powerful regulators of the immune system produced by the sympathetic nervous system (SNS). They regulate the adaptive immune system by altering T-cell differentiation into T helper (Th) 1 and Th2 cell subsets, but the effect on Th17 cells is not known. Th17 cells, defined, in part, by chemokine receptor CCR6 and cytokine interleukin (IL)-17A, are crucial for mediating certain pathogen-specific responses and are linked with several autoimmune diseases. We demonstrated that a proportion of human Th17 cells express beta 2-adrenergic receptor (β2AR), a G protein-coupled receptor that responds to catecholamines. Activation of peripheral blood mononuclear cells, which were obtained from venous blood drawn from healthy volunteers, with anti-cluster of differentiation 3 (CD3) and anti-CD28 and with a β2-agonist drug, terbutaline (TERB), augmented IL-17A levels (P < 0.01) in the majority of samples. TERB reduced interferon gamma (IFNγ) indicating that IL-17A and IFNγ are reciprocally regulated. Similar reciprocal regulation was observed with dbcAMP. Proliferation of Th cells was monitored by carboxyfluorescein diacetate N-succinimidyl ester labeling and flow cytometry with antibody staining for CD3 and CD4. TERB increased proliferation by a small but significant margin (P < 0.001). Next, Th17 cells (CD4 + CXCR3 - CCR6 + ) were purified using an immunomagnetic positive selection kit, which removes all other mononuclear cells. TERB increased IL-17A from purified Th17 cells, which argues that TERB acts directly on Th17 cells. Thus, hormone signals from the SNS maintain a balance of Th cells subtypes through the β2AR. © 2017 Federation of European Biochemical Societies.

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

PubMed Central

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

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

PubMed

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.

Quantitative analysis of immune cell subset infiltration of supraspinatus muscle after severe rotator cuff injury.

PubMed

Krieger, J R; Tellier, L E; Ollukaren, M T; Temenoff, J S; Botchwey, E A

2017-06-01

Rotator cuff tears cause muscle degeneration that is characterized by myofiber atrophy, fatty infiltration, and fibrosis and is minimally responsive to current treatment options. The underlying pathogenesis of rotator cuff muscle degeneration remains to be elucidated, and increasing evidence implicates immune cell infiltration as a significant factor. Because immune cells are comprised of highly heterogeneous subpopulations that exert divergent effects on injured tissue, understanding trafficking and accumulation of immune subpopulations may hold the key to more effective therapies. The present study quantifies subpopulations of immune cells infiltrating the murine supraspinatus muscle after severe rotator cuff injury that includes tenotomy and denervation. Rotator cuff injury stimulates dramatic infiltration of mononuclear phagocytes, enriches mononuclear phagocytes in non-classical subpopulations, and enriches T lymphocytes in T H and T reg subpopulations. The combination of tenotomy plus denervation significantly increases mononuclear phagocyte infiltration, enriches macrophages in the non-classical subpopulation, and decreases T lymphocyte enrichment in T H cells compared to tenotomy alone. Depletion of circulating monocytes via liposomal clodronate accelerates supraspinatus atrophy after tenotomy and denervation. The study may aid rational design of immunologically smart therapies that harness immune cells to enhance outcomes after rotator cuff tears.

The CardiAMP Heart Failure trial: A randomized controlled pivotal trial of high-dose autologous bone marrow mononuclear cells using the CardiAMP cell therapy system in patients with post-myocardial infarction heart failure: Trial rationale and study design.

PubMed

Raval, Amish N; Cook, Thomas D; Duckers, Henricus J; Johnston, Peter V; Traverse, Jay H; Abraham, William T; Altman, Peter A; Pepine, Carl J

2018-07-01

Heart failure following myocardial infarction is a common, disabling, and deadly condition. Direct injection of autologous bone marrow mononuclear cells into the myocardium may result in improved functional recovery, relieve symptoms, and improve other cardiovascular outcomes. CardiAMP-HF is a randomized, double-blind, sham-controlled, pivotal trial designed to investigate the safety and efficacy of autologous bone marrow mononuclear cells treatment for patients with medically refractory and symptomatic ischemic cardiomyopathy. The primary end point is change in 6-minute walk distance adjusted for major adverse cardiovascular events at 12 months following treatment. Particularly novel aspects of this trial include a cell potency assay to screen subjects who have bone marrow cell characteristics that suggest a favorable response to treatment, a point-of-care treatment method, a high target dose of 200 million cells, and an efficient transcatheter intramyocardial delivery method that is associated with high cell retention. This novel approach may lead to a new treatment for those with ischemic heart disease suffering from medically refractory heart failure. Copyright © 2018 Elsevier Inc. All rights reserved.

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

PubMed

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

2010-01-01

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

Immune suppressive effects of Helicobacter pylori on human peripheral blood mononuclear cells.

PubMed

Knipp, U; Birkholz, S; Kaup, W; Opferkuch, W

1993-05-01

Helicobacter pylori, the causative agent of type-B gastritis and duodenal ulcer in man is described as a bacterium able to stimulate the human immune system. This study demonstrates that H. pylori besides this property possesses an immune suppressive activity. The in vitro proliferation of human peripheral blood mononuclear cells to purified protein derivative of tuberculin (PPD), phytohemagglutinin, and concanavalin A was reduced in a dose-dependent manner by bacteria which had been inactivated by incubation at 56 degrees C as well as by a soluble cytoplasmic fraction of H. pylori. The immune suppressive effect on the mitogen-induced proliferation could be increased by preincubation of the mononuclear cells with H. pylori. The observed effect does not seem to be a specific phenomenon depending on prior exposure of the blood donors to H. pylori, since suppression occurred with mononuclear cells of H. pylori-infected patients as well as of antibody-negative healthy control individuals. The suppressive activity was non-dialyzable, heat-labile (100 degrees C, 30 min) and sensitive to trypsin. Furthermore, the treatment at 100 degrees C caused an increase in the capability of H. pylori to induce lymphoproliferation. This fact indicates that the suppressive factor is also effective on H. pylori antigens. While exogenous interleukin-2, could to a certain extent, restore the responsiveness of the lymphocytes after PPD-stimulation in the presence of H. pylori, the addition of interleukin-1 had no effect on the suppressed lymphoproliferation. Cell-separation and cell-mixing experiments indicated that an influence on monocytes rather than on T cells is the major cause of the observed suppressive effect. Although the immunological mechanisms involved in H. pylori-associated gastritis are not clearly defined, it is reasonable to presume that suppression of host defense mechanisms may contribute to the pathogenesis of this disease.

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

PubMed Central

Abdul Karim, Norwahidah; Abdul Hamid, Noor Aini; Harun, Roslan; Wan Ngah, Wan Zurinah

2013-01-01

Mechanisms determining both functional rate of decline and the time of onset in aging remain elusive. Studies of the aging process especially those involving the comparison of long-lived individuals and young controls are fairly limited. Therefore, this research aims to determine the differential gene expression profile in related individuals from villages in Pahang, Malaysia. Genome-wide microarray analysis of 18 samples of peripheral blood mononuclear cells (PBMCs) from two groups: octo/nonagenarians (80–99 years old) and their offspring (50.2  ±  4.0 years old) revealed that 477 transcripts were age-induced and 335 transcripts were age-repressed with fold changes ≥1.2 in octo/nonagenarians compared to offspring. Interestingly, changes in gene expression were associated with increased capacity for apoptosis (BAK1), cell cycle regulation (CDKN1B), metabolic process (LRPAP1), insulin action (IGF2R), and increased immune and inflammatory response (IL27RA), whereas response to stress (HSPA8), damage stimulus (XRCC6), and chromatin remodelling (TINF2) pathways were downregulated in octo/nonagenarians. These results suggested that systemic telomere maintenance, metabolism, cell signalling, and redox regulation may be important for individuals to maintain their healthy state with advancing age and that these processes play an important role in the determination of the healthy life-span. PMID:24381713

Studies on Changes of β-Adrenergic Receptors in Polymorphonuclear Cell and Mononuclear Cell with the Changes of Thyroid Function

PubMed Central

Lee, Jong Do; You, Myung Hee; Kim, Young Seol; Kim, Jin Woo; Kim, Kwang Won; Kim, Sun Woo; Choi, Young Kil

1986-01-01

Although it has been well established that thyroid hormones increase β-adrenergic receptors of various tissues in the animal studies, there are controversies about the β-adrenergic receptor changes of human mononuclear cells and polymorphonuclear cells. The present study was performed to analyze the change of β-adrenergic receptor of those cells according to the thyroid functional status and to evaluate their usefulness in assessment of sympathetic hyperactivity. We measured [3H]-dihydroalprenolol binding to circulating mononuclear and polymorphonuclear cells from 18 patients with hyperthyrodism, 7 with hypothyroidism, 8 with euthyroid goiter and 21 normal controls. Only with polymorphonuclear cells the receptor concentration was significantly higher (P<0.01) in hyperthyroidism (46.07±4.78 fmol/mg protein) than in the normal control (28.42±2.06 fmol/mg protein) and the affinity constants of both cells were comparable to normal control values. And serum concentrations of T3 were not correlated well with the changes of receptor concentrations in hyperthyroidism. The patients with hypothyroidism and euthyroid goiter showed no significant difference in the receptor concentration and the affinity constants with both cell binding assays. These results indicate that thyroid hormones increase the receptor concentration in polymorphonuclear cells which might be responsible for the symptoms of sympathetic hyperactivity and the polymorphornuclear cells are useful for β-adrenergic receptor assay. PMID:15759381

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

PubMed

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.

Possible pseudogout in two dogs.

PubMed

Forsyth, S F; Thompson, K G; Donald, J J

2007-03-01

Pseudogout, the acute form of calcium pyrophosphate deposition disease, is a common condition in elderly human beings and is characterised by the sudden onset of intense joint pain and synovitis. It is rarely identified in animals but was diagnosed in two dogs that presented with acute lameness and pyrexia. Cytology of the synovial fluid showed a mildly elevated cell count with both non-degenerate neutrophils and mononuclear cells present. Many of the mononuclear cells and occasional neutrophils contained square or rhomboid-shaped crystals that were variable in shape and size and weakly birefringent on examination under polarised light. Clinical signs resolved following treatment with prednisolone.

Plasmodium and mononuclear phagocytes.

PubMed

Mac-Daniel, Laura; Ménard, Robert

2015-01-01

Plasmodium, the causative agent of malaria, initially multiplies inside liver cells and then in successive cycles inside erythrocytes, causing the symptoms of the disease. In this review, we discuss interactions between the extracellular and intracellular forms of the Plasmodium parasite and innate immune cells in the mammalian host, with a special emphasis on mononuclear phagocytes. We overview here what is known about the innate immune cells that interact with parasites, mechanisms used by the parasite to evade them, and the protective or detrimental contribution of these interactions on parasite progression through its life cycle and pathology in the host. Copyright © 2014 Elsevier Ltd. All rights reserved.

Collection, Storage, and Preparation of Human Blood Cells

PubMed Central

Dagur, Pradeep K.; McCoy, J. Philip

2015-01-01

Human peripheral blood is often studied by flow cytometry in both the research and clinical laboratories. The methods for collection, storage, and preparation of peripheral blood will vary depending on the cell lineage to be examined as well as the type of assay to be performed. This unit presents protocols for collection of blood, separation of leukocytes from whole blood by lysis of erythrocytes, isolating mononuclear cells by density gradient separation, and assorted non-flow sorting methods, such as magnetic bead separations, for enriching specific cell populations, including monocytes, T lymphocytes, B lymphocytes, neutrophils,, , and platelets prior to flow cytometric analysis. A protocol is also offered for cryopreservation of cells since clinical research often involves retrospective flow cytometric analysis of samples stored over a period of months or years. PMID:26132177

Mononuclear-macrophages but not neutrophils act as major infiltrating anti-leptospiral phagocytes during leptospirosis.

PubMed

Chen, Xu; Li, Shi-Jun; Ojcius, David M; Sun, Ai-Hua; Hu, Wei-Lin; Lin, Xu'ai; Yan, Jie

2017-01-01

To identify the major infiltrating phagocytes during leptospirosis and examine the killing mechanism used by the host to eliminate Leptospira interrogans. Major infiltrating phagocytes in Leptospira-infected C3H/HeJ mice were detected by immunohistochemistry. Chemokines and vascular endothelial cell adhesion molecules (VECAMs) of Leptospira-infected mice and leptospirosis patients were detected by microarray and immunohistochemistry. Leptospira-phagocytosing and -killing abilities of human or mouse macrophages and neutrophils, and the roles of intracellular ROS, NO and [Ca2+]i in Leptospira-killing process were evaluated by confocal microscopy and spectrofluorimetry. Peripheral blood mononuclear-macrophages rather than neutrophils were the main infiltrating phagocytes in the lungs, liver and kidneys of infected mice. Levels of macrophage- but not neutrophil-specific chemokines and VECAMs were significantly increased in the samples from infected mice and patients. All macrophages tested had a higher ability than neutrophils to phagocytose and kill leptospires. Higher ROS and NO levels and [Ca2+]i in the macrophages were involved in killing leptospires. Human macrophages displayed more phagolysosome formation and a stronger leptospire-killing ability to than mouse macrophages. Mononuclear-macrophages but not neutrophils represent the main infiltrating and anti-leptospiral phagocytes during leptospirosis. A lower level of phagosome-lysosome fusion may be responsible for the lower Leptospira-killing ability of human macrophages.

Increased HIV-1 transcriptional activity and infectious burden in peripheral blood and gut-associated CD4+ T cells expressing CD30

PubMed Central

Carvidi, Alexander B.; Smith, Louis C. B.; Khan, Shahzada; Trapecar, Martin; Stoddart, Cheryl A.; Kuritzkes, Daniel R.

2018-01-01

HIV-1-infected cells persist indefinitely despite the use of combination antiretroviral therapy (ART), and novel therapeutic strategies to target and purge residual infected cells in individuals on ART are urgently needed. Here, we demonstrate that CD4+ T cell-associated HIV-1 RNA is often highly enriched in cells expressing CD30, and that cells expressing this marker considerably contribute to the total pool of transcriptionally active CD4+ lymphocytes in individuals on suppressive ART. Using in situ RNA hybridization studies, we show co-localization of CD30 with HIV-1 transcriptional activity in gut-associated lymphoid tissues. We also demonstrate that ex vivo treatment with brentuximab vedotin, an antibody-drug conjugate (ADC) that targets CD30, significantly reduces the total amount of HIV-1 DNA in peripheral blood mononuclear cells obtained from infected, ART-suppressed individuals. Finally, we observed that an HIV-1-infected individual, who received repeated brentuximab vedotin infusions for lymphoma, had no detectable virus in peripheral blood mononuclear cells. Overall, CD30 may be a marker of residual, transcriptionally active HIV-1 infected cells in the setting of suppressive ART. Given that CD30 is only expressed on a small number of total mononuclear cells, it is a potential therapeutic target of persistent HIV-1 infection. PMID:29470552

Label-free single-cell separation and imaging of cancer cells using an integrated microfluidic system

PubMed Central

Antfolk, Maria; Kim, Soo Hyeon; Koizumi, Saori; Fujii, Teruo; Laurell, Thomas

2017-01-01

The incidence of cancer is increasing worldwide and metastatic disease, through the spread of circulating tumor cells (CTCs), is responsible for the majority of the cancer deaths. Accurate monitoring of CTC levels in blood provides clinical information supporting therapeutic decision making, and improved methods for CTC enumeration are asked for. Microfluidics has been extensively used for this purpose but most methods require several post-separation processing steps including concentration of the sample before analysis. This induces a high risk of sample loss of the collected rare cells. Here, an integrated system is presented that efficiently eliminates this risk by integrating label-free separation with single cell arraying of the target cell population, enabling direct on-chip tumor cell identification and enumeration. Prostate cancer cells (DU145) spiked into a sample with whole blood concentration of the peripheral blood mononuclear cell (PBMC) fraction were efficiently separated and trapped at a recovery of 76.2 ± 5.9% of the cancer cells and a minute contamination of 0.12 ± 0.04% PBMCs while simultaneously enabling a 20x volumetric concentration. This constitutes a first step towards a fully integrated system for rapid label-free separation and on-chip phenotypic characterization of circulating tumor cells from peripheral venous blood in clinical practice. PMID:28425472

Label-free single-cell separation and imaging of cancer cells using an integrated microfluidic system.

PubMed

Antfolk, Maria; Kim, Soo Hyeon; Koizumi, Saori; Fujii, Teruo; Laurell, Thomas

2017-04-20

The incidence of cancer is increasing worldwide and metastatic disease, through the spread of circulating tumor cells (CTCs), is responsible for the majority of the cancer deaths. Accurate monitoring of CTC levels in blood provides clinical information supporting therapeutic decision making, and improved methods for CTC enumeration are asked for. Microfluidics has been extensively used for this purpose but most methods require several post-separation processing steps including concentration of the sample before analysis. This induces a high risk of sample loss of the collected rare cells. Here, an integrated system is presented that efficiently eliminates this risk by integrating label-free separation with single cell arraying of the target cell population, enabling direct on-chip tumor cell identification and enumeration. Prostate cancer cells (DU145) spiked into a sample with whole blood concentration of the peripheral blood mononuclear cell (PBMC) fraction were efficiently separated and trapped at a recovery of 76.2 ± 5.9% of the cancer cells and a minute contamination of 0.12 ± 0.04% PBMCs while simultaneously enabling a 20x volumetric concentration. This constitutes a first step towards a fully integrated system for rapid label-free separation and on-chip phenotypic characterization of circulating tumor cells from peripheral venous blood in clinical practice.

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

PubMed Central

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

Particulate matter induces prothrombotic microparticle shedding by human mononuclear and endothelial cells.

PubMed

Neri, Tommaso; Pergoli, Laura; Petrini, Silvia; Gravendonk, Lotte; Balia, Cristina; Scalise, Valentina; Amoruso, Angela; Pedrinelli, Roberto; Paggiaro, Pierluigi; Bollati, Valentina; Celi, Alessandro

2016-04-01

Particulate airborne pollution is associated with increased cardiopulmonary morbidity. Microparticles are extracellular vesicles shed by cells upon activation or apoptosis involved in physiological processes such as coagulation and inflammation, including airway inflammation. We investigated the hypothesis that particulate matter causes the shedding of microparticles by human mononuclear and endothelial cells. Cells, isolated from the blood and the umbilical cords of normal donors, were cultured in the presence of particulate from a standard reference. Microparticles were assessed in the supernatant as phosphatidylserine concentration. Microparticle-associated tissue factor was assessed by an one-stage clotting assay. Nanosight technology was used to evaluate microparticle size distribution. Particulate matter induces a dose- and time- dependent, rapid (1h) increase in microparticle generation in both cells. These microparticles express functional tissue factor. Particulate matter increases intracellular calcium concentration and phospholipase C inhibition reduces microparticle generation. Nanosight analysis confirmed that upon exposure to particulate matter both cells express particles with a size range consistent with the definition of microparticles (50-1000 nm). Exposure of mononuclear and endothelial cells to particulate matter upregulates the generation of microparticles at least partially mediated by calcium mobilization. This observation might provide a further link between airborne pollution and cardiopulmonary morbidity. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PubMed

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

2016-10-01

Subgroup J avian leukosis virus (ALV-J) is an oncogenic retrovirus known to induce tumor formation and immunosuppression in infected chickens. One of the organs susceptible to ALV-J is the bone marrow, from which specialized antigen-presenting cells named dendritic cells (BM-DCs) are derived. Notably, these cells possess the unique ability to induce primary immune responses. In the present study, a method of cultivating and purifying DCs from chicken bone marrow in vitro was established to investigate the effects of ALV-J infection on BM-DC differentiation or generation. The results indicated that ALV-J not only infects the chicken bone marrow mononuclear cells but also appears to inhibit the differentiation and maturation of BM-DCs and to trigger apoptosis. Moreover, substantial reductions in the mRNA expression of TLR1, TLR2, TLR3, MHCI, and MHCII and in cytokine production were detected in the surviving BM-DCs following ALV-J infection. These findings indicate that ALV-J infection disrupts the process of bone marrow mononuclear cell differentiation into BM-DCs likely via altered antigen presentation, resulting in a downstream immune response in affected chickens. Copyright © 2016 Elsevier B.V. All rights reserved.

Pig but not Human Interferon-γ Initiates Human Cell-Mediated Rejection of Pig Tissue in vivo

NASA Astrophysics Data System (ADS)

Sultan, Parvez; Murray, Allan G.; McNiff, Jennifer M.; Lorber, Marc I.; Askenase, Philip W.; Bothwell, Alfred L. M.; Pober, Jordan S.

1997-08-01

Split-thickness pig skin was transplanted on severe combined immunodeficient mice so that pig dermal microvessels spontaneously inosculated with mouse microvessels and functioned to perfuse the grafts. Pig endothelial cells in the healed grafts constitutively expressed class I and class II major histocompatibility complex molecules. Major histocompatibility complex molecule expression could be further increased by intradermal injection of pig interferon-γ (IFN-γ ) but not human IFN-γ or tumor necrosis factor. Grafts injected with pig IFN-γ also developed a sparse infiltrate of mouse neutrophils and eosinophils without evidence of injury. Introduction of human peripheral blood mononuclear cells into the animals by intraperitoneal inoculation resulted in sparse perivascular mononuclear cell infiltrates in the grafts confined to the pig dermis. Injection of pig skin grafts on mice that received human peripheral blood mononuclear cells with pig IFN-γ (but not human IFN-γ or heat-inactivated pig IFN-γ ) induced human CD4+ and CD8+ T cells and macrophages to more extensively infiltrate the pig skin grafts and injure pig dermal microvessels. These findings suggest that human T cell-mediated rejection of xenotransplanted pig organs may be prevented if cellular sources of pig interferon (e.g., passenger lymphocytes) are eliminated from the graft.

Identification of HIV-1 genitourinary tract compartmentalization by analyzing the env gene sequences in urine.

PubMed

Blasi, Maria; Carpenter, J Harris; Balakumaran, Bala; Cara, Andrea; Gao, Feng; Klotman, Mary E

2015-08-24

HIV-1 persists indefinitely in memory CD4 T cells and other long-lived cellular reservoirs despite antiretroviral therapy. Our group had previously demonstrated that HIV-1 can establish a productive infection in renal epithelial cells and that the kidney represents a separate compartment for HIV-1 replication. Here, to better understand the viruses in this unique site, we genetically characterized and compared the viruses in blood and urine specimens from 24 HIV-1 infected patients with detectable viremia. Blood and urine samples were obtained from 35 HIV-1 positive patients. Single-genome amplification was performed on HIV-1 env RNA and DNA isolated from urine supernatants and urine-derived cell pellets, respectively, as well as from plasma and peripheral blood mononuclear cell from the same individuals. Neighbor-joining trees were constructed under the Kimura 2-parameter model. We amplified and sequenced the full-length HIV-1 envelope (env) gene from 12 of the 24 individuals, indicating that 50% of the viremic HIV-1-positive patients had viral RNA in their urine. Phylogenetic analysis of the env sequences from four individuals with more than 15 urine-derived env sequences showed that the majority of the sequences from urine formed distinct cluster(s) independent of those peripheral blood mononuclear cell and plasma-derived sequences, consistent with viral compartmentalization in the urine. Our results suggest the presence of a distinct HIV compartment in the genitourinary tract.

Bone marrow mononuclears from murine tibia after spaceflight on biosatellite

NASA Astrophysics Data System (ADS)

Andreeva, Elena; Roe, Maria; Buravkova, Ludmila; Andrianova, Irina; Goncharova, Elena; Gornostaeva, Alexandra

Elucidation of the space flight effects on the adult stem and progenitor cells is an important goal in space biology and medicine. A unique opportunity for this is provided by project "BION -M1". The purpose of this study was to evaluate the effects of a 30-day flight on biosatellite "BION - M1" and the subsequent 7-day recovery on the quantity, viability, immunophenotype of mononuclears from murine tibia bone marrow. Also the in vitro characterization of functional capacity of multipotent mesenchymal stromal cells (MSCs) was scheduled. Under the project, the S57black/6 mice were divided into groups: spaceflight/vivarium control, recovery after spaceflight/ vivarium control to recovery. Bone marrow mononuclears were isolated from the tibia and immunophenotyped using antibodies against CD45, CD34, CD90 on a flow cytometer Epics XL (Beckman Coulter). A part of the each pool was frozen for subsequent estimation of hematopoietic colony-forming units (CFU), the rest was used for the evaluation of fibroblast CFU (CFUf) number, MSC proliferative activity and osteogenic potency. The cell number in the flight group was significantly lower than in the vivarium control group. There were no differences in this parameter between flight and control groups after 7 days of recovery. The mononuclears viability was more than 95 percent in all examined groups. Flow cytometric analysis showed no differences in the bone marrow cell immunophenotype (CD45, CD34, CD90.1 (Thy1)), but the flight animals had more large-sized CD45+mononuclears, than the control groups of mice. There was no difference in the CFUf number between groups. After 7 days in vitro the MSC number in flight group was twice higher than in vivarium group, after 10 days - 4 times higher. These data may indicate a higher proliferative activity of MSCs after spaceflight. MSCs showed the same and high alkaline phosphatase activity, both in flight and in the control groups, suggesting no effect of spaceflight factors on early osteogenic potency of stromal cells. These results indicate that spaceflight factors had no significant damaging effects on the murine bone marrow mononuclears. These observations are consistent with previously made assumption of moderate and reversible stress reaction of mammals on spaceflight conditions. This work was supported by Program of Basic Research of IMBP RAS

The multifaceted role of the renal mononuclear phagocyte system.

PubMed

Viehmann, Susanne F; Böhner, Alexander M C; Kurts, Christian; Brähler, Sebastian

2018-04-22

The kidney contains a large and complex network of mononuclear phagocytes, which includes dendritic cells (DCs) and macrophages (MØs). The distinction between these cell types is traditionally based on the expression of molecular markers and morphology. However, several classification systems are used in parallel to identify DCs and MØs, leading to considerable uncertainty about their identity and functional roles. The discovery that a substantial proportion of macrophages in tissues like the kidney are embryonically derived further complicates the situation. Recent studies have used newly identified transcription factors such as ZBTB46 and lineage tracing techniques for classifying mononuclear phagocytes. These approaches have shed new light on the functional specialization of these cells in health and disease, uncovered an influence of the renal microenvironment and revealed considerable cellular plasticity, especially in inflammatory situations. In this review, the current knowledge about the developmental origins and versatile functional roles of DCs and MØs in kidney homeostasis and disease is discussed. Copyright © 2018 Elsevier Inc. All rights reserved.

Toxocara canis adult worm antigen induces proliferative response of healthy human peripheral blood mononuclear cells.

PubMed

Inuo, G; Akao, N; Kohsaka, H; Saito, I; Miyasaka, N; Fujita, K

1995-02-01

The proliferative response of human peripheral blood mononuclear cells (PBMC) from healthy donors to Toxocara canis adult worm antigens (TcA) was examined. PBMC from all donors examined (n = 7) strongly responded to TcA in a dose-dependent fashion after six days of culture, irrespective of their serological reactivity. In contrast, cord blood mononuclear cells did not react to TcA. The proliferation of PBMC in response to TcA was completely inhibited by anti-HLA-DR antibody. Purified CD4+ T cells reconstituted with autologous irradiated antigen presenting cells (APC) vigorously proliferated in response to TcA, but this was abrogated by pretreatment of APC with paraformaldehyde. Significant IL-2, IL-3, IL-4, IL-5 and IFN-gamma mRNA expression was detected in PBMC stimulated with TcA, with expression peaking at 72 h after stimulation. IL-1 beta, IL-6, IL-10 and GM-CSF mRNA expression was also upregulated, peaking at 24 h after stimulation. Taken together, these results suggest that adult T. canis-derived antigens have the ability to activate human PBMC as conventional antigens, possibly due to their cross-reactivity, which may be involved in the host defence against helminth infection.

Collection and processing of whole blood for transformation of peripheral blood mononuclear cells and extraction of DNA: the Type 1 Diabetes Genetics Consortium

PubMed Central

Rosinger, Silke; Nutland, Sarah; Mickelson, Eric; Varney, Michael D; Boehm, Bernard O; Olsem, Gary J; Hansen, John A; Nicholson, Ian; Hilner, Joan E; Perdue, Letitia H; Pierce, June J; Akolkar, Beena; Nierras, Concepcion; Steffes, Michael W

2010-01-01

Background and Purpose To yield large amounts of DNA for many genotype analyses and to provide a renewable source of DNA, the Type 1 Diabetes Genetics Consortium (T1DGC) harvested DNA and peripheral blood mononuclear cells (PBMCs) from individuals with type 1 diabetes and their family members in several regions of the world. Methods DNA repositories were established in Asia-Pacific, Europe, North America, and the United Kingdom. To address region-specific needs, different methods and sample processing techniques were used among the laboratories to extract and to quantify DNA and to establish Epstein-Barr virus transformed cell lines. Results More than 98% of the samples of PBMCs were successfully transformed. Approximately 20–25 µg of DNA were extracted per mL of whole blood. Extraction of DNA from the cell pack ranged from 92 to 165 µg per cell pack. In addition, the extracted DNA from whole blood or transformed cells was successfully utilized in each regional human leukocyte antigen genotyping laboratory and by several additional laboratories performing consortium-wide genotyping projects. Limitations Although the isolation of PBMCs was consistent among sites, the measurement of DNA was difficult to harmonize. Conclusions DNA repositories can be established in different regions of the world and produce similar amounts of high-quality DNA for a variety of high-throughput genotyping techniques. Furthermore, even with the distances and time necessary for transportation, highly efficient transformation of PBMCs is possible. For future studies/trials involving several laboratories in different locations, the T1DGC experience includes examples of protocols that may be applicable. In summary, T1DGC has developed protocols that would be of interest to any scientific organization attempting to overcome the logistical problems associated with studies/trials spanning multiple research facilities, located in different regions of the world. PMID:20595244

Mononuclear cell secretome protects from experimental autoimmune myocarditis

PubMed Central

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

2015-01-01

Aims 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. Methods and results 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. Conclusion 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. PMID:23321350

Relation of RhoA in Peripheral Blood Mononuclear Cells With Severity of Aneurysmal Subarachnoid Hemorrhage and Vasospasm.

PubMed

González-Montelongo, María Del Carmen; Egea-Guerrero, Juan José; Murillo-Cabezas, Francisco; González-Montelongo, Rafaela; Ruiz de Azúa-López, Zaida; Rodríguez-Rodríguez, Ana; Vilches-Arenas, Angel; Castellano, Antonio; Ureña, Juan

2018-06-01

Rho-kinase, an effector of RhoA, is associated with various cardiovascular diseases in circulating blood cells. However, the role of RhoA/Rho-kinase in peripheral blood mononuclear cells from patients with spontaneous aneurysmal subarachnoid hemorrhage (aSAH) has not yet been studied in relation to the severity of this disease. Therefore, we analyzed the expression and activity of RhoA as a possible biomarker in aSAH. Twenty-four patients with aSAH and 15 healthy subjects were examined. Peripheral blood mononuclear cells were collected, and RhoA activity and expression were determined by RhoA activation assay kit (G-LISA) and enzyme-linked immunosorbent assay tests, respectively. The severity of aSAH was determined from the World Federation of Neurological Surgeon scale, and vasospasm was evaluated using clinical symptoms, arteriography, and sonography. RhoA expression was significantly increased in peripheral blood mononuclear cells from patients on days 0, 2, and 4 after aSAH versus healthy subjects ( P =0.036, 0.010, and 0.018, respectively, by U Mann-Whitney analysis). There was a significant correlation between RhoA expression and injury severity on days 2 and 4 (Spearman test, day 2: r =0.682, n=14, P =0.007; day 4: r =0.721, n=14, P =0.004). No significant correlation was observed on day 0 (day 0: r =0.131, n=6, P =0.805). Active RhoA was not significantly different in patients and healthy subjects on days 0, 2, and 4 ( P =0.243, 0.222, and 0.600, respectively) nor did it increase significantly on days 0 and 2 in patients with vasospasm versus patients without vasospasm ( P =0.064 and 0.519, respectively). In contrast, active RhoA was significantly higher on day 4 in patients who developed vasospasm versus patients without vasospasm ( P =0.028). Our preliminary results indicate that RhoA expression and activity in peripheral blood mononuclear cells might be related with aSAH severity and cerebral vasospasm. RhoA is a potential biomarker of the risks associated with aSAH. © 2018 American Heart Association, Inc.

Mononuclear Phagocyte-Mediated Antifungal Immunity: The Role of Chemotactic Receptors and Ligands

PubMed Central

Swamydas, Muthulekha; Break, Timothy J.; Lionakis, Michail S.

2015-01-01

Over the past two decades, fungal infections have emerged as significant causes of morbidity and mortality in patients with hematological malignancies, hematopoietic stem cell or solid organ transplantation and acquired immunodeficiency syndrome. Besides neutrophils and CD4+ T lymphocytes, which have long been known to play an indispensable role in promoting protective antifungal immunity, mononuclear phagocytes are now being increasingly recognized as critical mediators of host defense against fungi. Thus, a recent surge of research studies has focused on understanding the mechanisms by which resident and recruited monocytes, macrophages and dendritic cells accumulate and become activated at the sites of fungal infection. Herein, we critically review how a variety of G-protein coupled chemoattractant receptors and their ligands mediate mononuclear phagocyte recruitment and effector function during infection by the most common human fungal pathogens. PMID:25715741

Perspectives on Systems Modeling of Human Peripheral Blood Mononuclear Cells

PubMed Central

Sen, Partho; Kemppainen, Esko; Orešič, Matej

2018-01-01

Human peripheral blood mononuclear cells (PBMCs) are the key drivers of the immune responses. These cells undergo activation, proliferation and differentiation into various subsets. During these processes they initiate metabolic reprogramming, which is coordinated by specific gene and protein activities. PBMCs as a model system have been widely used to study metabolic and autoimmune diseases. Herein we review various omics and systems-based approaches such as transcriptomics, epigenomics, proteomics, and metabolomics as applied to PBMCs, particularly T helper subsets, that unveiled disease markers and the underlying mechanisms. We also discuss and emphasize several aspects of T cell metabolic modeling in healthy and disease states using genome-scale metabolic models. PMID:29376056

Short Communication: Lack of Support for Socially Connected HIV-1 Transmission Among Young Adult Black Men Who Have Sex with Men.

PubMed

Fujimoto, Kayo; Coghill, Lyndon M; Weier, Christopher A; Hwang, Lu-Yu; Kim, Ju Yeong; Schneider, John A; Metzker, Michael L; Brown, Jeremy M

2017-09-01

We explore the phylogenetic relationships among HIV sequences sampled from young adult black men who have sex with men (YAB-MSM), who are connected through peer referral/social ties and who attend common venues. Using 196 viral sequences sampled from the peripheral blood mononuclear cells of 10 individuals, our preliminary phylogenetic results indicate that these socially connected YAB-MSM are infected with distantly related viruses and provide no evidence for viral transmission between network members. Our results suggest that HIV-prevention strategies that target young adult MSM should extend beyond their network members and local community.

In-vitro generation of interleukin-10 secreting B-regulatory cells from donor adipose tissue derived mesenchymal stem cells and recipient peripheral blood mononuclear cells for potential cell therapy.

PubMed

Gupte, Kunal S; Vanikar, Aruna V; Trivedi, Hargovind L; Patel, Chetan N; Patel, Jignesh V

2017-02-01

Interleukin-10 secreting B-cells are a major subset of B-regulatory cells (B-regs), commonly recognized as CD19 + /38 hi /24 hi /IL10 + . They carry out immunomodulation by release of specific cytokines and/or cell-to-cell contact. We have generated B-regs in-vitro from donor adipose tissue derived mesenchymal stem cells (AD-MSC) and renal allograft recipient (RAR) peripheral blood mononuclear cells (PBMC) for potential cell therapy. Mononuclear cells separated by density gradient centrifugation from 50 ml anti-coagulated blood of 15-RAR and respective donors were analysed for baseline B-regs using appropriate antibodies. Equal amount (20 × 10 6  cells/ml) of stimulator (irradiated at 7.45 Gy/min for 10 min) and responder (non-irradiated) cells were co-cultured with in-vitro generated AD-MSC (1 × 10 6  cells/ml) in proliferation medium containing lipopolysaccharide from E. coli K12 strain at 37 °C with 5% CO 2 . Cells were harvested on day-7 and analyzed for viability, sterility, quantity, morphology and phenotyping. In-vitro generated B-reg levels were compared with baseline B-regs. In-vitro generated B-reg count increased to 16.75% from baseline count of 3.35%. B-regs can be successfully generated in-vitro from donor AD-MSC and RAR PBMC for potential cell therapy. Copyright © 2017 Chang Gung University. Published by Elsevier B.V. All rights reserved.

Langerhans Cell Histiocytosis—Health Professional Version

Cancer.gov

Histiocytic diseases, like Langerhans cell histiocytosis (LCH) and myeloid-derived dendritic cell disorder, in children and adults are caused by an abnormal accumulation of cells of the mononuclear phagocytic system. Find evidence-based information on Langerhans cell histiocytosis treatment.

Effect of cryopreservation of peripheral blood mononuclear cells (PBMCs) on the variability of an antigen-specific memory B cell ELISpot.

PubMed

Trück, Johannes; Mitchell, Ruth; Thompson, Amber J; Morales-Aza, Begonia; Clutterbuck, Elizabeth A; Kelly, Dominic F; Finn, Adam; Pollard, Andrew J

2014-01-01

The ELISpot assay is used in vaccine studies for the quantification of antigen-specific memory B cells (B(MEM)), and can be performed using cryopreserved samples. The effects of cryopreservation on B(MEM) detection and the consistency of cultured ELISpot assays when performed by different operators or laboratories are unknown. In this study, blood was taken from healthy volunteers, and a cultured ELISpot assay was used to count B(MEM) specific for 2 routine vaccine antigens (diphtheria and tetanus toxoid). Results were assessed for intra- and inter-operator variation, and the effects of cryopreservation. Cryopreserved samples were shipped to a second laboratory in order to assess inter-laboratory variation. B(MEM) frequencies were very strongly correlated when comparing fresh and frozen samples processed by the same operator, and were also very strongly correlated when comparing 2 operators in the same laboratory. Results were slightly less consistent when samples were processed in different laboratories but correlation between the 2 measurements was still very strong. Although cell viability was reduced in some cryopreserved samples due to higher temperatures during transportation, B(MEM) could still be quantified. These results demonstrate the reproducibility of the ELISpot assay across operators and laboratories, and support the use of cryopreserved samples in future B(MEM) studies.

Influence of parturition and diets enriched in n-3 or n-6 polyunsaturated fatty acids on immune response of dairy cows during the transition period.

PubMed

Lessard, M; Gagnon, N; Godson, D L; Petit, H V

2004-07-01

The objectives of this study were to evaluate the functional properties of immunocompetent cells in dairy cows fed diets enriched in n-3 or n-6 polyunsaturated fatty acids during the transition period. Six weeks before calving, 21 primiparous and 27 multiparous pregnant Holstein dairy cows were randomly allotted to 1 of 3 dietary fat treatments: calcium salts of palm oil (Megalac), micronized soybeans, or whole flaxseed, which are, respectively, rich in saturated, n-6, or n-3 fatty acids. On wk 6 and 3 before parturition, cows received a subcutaneous injection of ovalbumin to measure the antibody response in colostrum and serum. Colostrum samples were collected at the first milking after calving, and blood samples were taken 6, 3, and 1 wk before the expected calving date and 1, 3, and 6 wk after calving. Blood mononuclear cells were cultured to evaluate the proliferative response to concanavalin A and the in vitro productions of interferon-gamma, tumor necrosis factor-alpha, nitric oxide, and prostaglandin E2. The serum antibody response to ovalbumin was unaffected by dietary fatty acids, but the response was lower in primiparous cows than in multiparous cows. A significant diet x parity interaction indicated that colostral antibody level against ovalbumin was significantly higher in multiparous cows fed soybeans than in those fed flaxseed or Megalac; there was no difference among treatments for primiparous cows. The lymphocyte response to concanavalin A was lower in cows fed soybeans than in those receiving flaxseed or Megalac when the cells were incubated with autologous serum. The proliferative response of mononuclear cells incubated with autologous serum was suppressed in the 1st wk after calving in both primiparous and multiparous cows, and multiparous cows showed a higher response than primiparous cows throughout the experiment. There was a significant interaction between parity and diet as a result of a greater production of interferon-gamma by mononuclear cells incubated with autologous serum in multiparous cows than in primiparous cows fed flaxseed; there was no difference among cows fed the other diets. Interferon-gamma production was reduced around calving while the inverse was observed for productions of nitric oxide and tumor necrosis factor-alpha. Productions of nitric oxide, prostaglandin E2, and tumor necrosis factor-gamma were greater in primiparous cows than in multiparous cows. In conclusion, functional properties of lymphocytes and monocyte/macrophage lineage of dairy cows during the transition period are modulated by parturition and the composition of polyunsaturated fatty acids in the diet.

Quantitative profiling of 4'-geranyloxyferulic acid and its conjugate with l-nitroarginine methyl ester in mononuclear cells by high-performance liquid chromatography with fluorescence detection.

PubMed

Taddeo, Vito Alessandro; Genovese, Salvatore; Carlucci, Giuseppe; Ferrone, Vincenzo; Patruno, Antonia; Ferrone, Alessio; de Medina, Philippe; Fiorito, Serena; Epifano, Francesco

2017-01-30

Oxyprenylated natural products were shown to exert in vitro and in vivo remarkable anti-cancer and anti-inflammatory effects. This paper describes a rapid, selective, and sensitive HPLC method with fluorescence detection for determination of 4'-geranyloxyferulic acid (GOFA) and its conjugate with l-nitroarginine methyl ester (GOFA-L-NAME) in mononuclear cells. Analytes were extracted from cells using methanol and eluted on a GraceSmart RP 18 analytical column (250×4.6mm i.d., 5μm particle size) kept at 25°C. A mixture of formic acid 1% in water (A) and methanol (B) were used as mobile phase, at a flow-rate of 1.2mL/min in gradient elution. A fluorescence detector (excitation/emission wavelength of 319/398nm for GOFA and GOFA-L-NAME), was used for the two analytes. Calibration curves of GOFA and GOFA-L-NAME were linear over the concentration range of 1.0-50μg/mL, with correlation coefficients (r 2 )≥0.9995. Intra- and inter-assay precision do not exceed 6.8%. The accuracy was from 94% to 105% for quality control samples (2.0, 25.0 and 40μg/mL). The mean (RSD%) extraction recoveries (n=5) for GOFA and GOFA-L-NAME from spiked cells at 2.0, 25.0 and 40.0μg/mL were 92.4±1.5%, 94.7±0.9% and 93.8±1.1%, for GOFA and 95.3±1.2%, 94.8±1.0% and 93.9±1.3%, for GOFA-L-NAME. The limits of detection and quantification were 0.3μg/mL and 1.0μg/mL for GOFA and GOFA-L-NAME. This method was successfully applied to measure GOFA and GOFA-L-NAME concentrations in a mononuclear cells. Copyright © 2016 Elsevier B.V. All rights reserved.

New Insights into the Immunobiology of Mononuclear Phagocytic Cells and Their Relevance to the Pathogenesis of Cardiovascular Diseases

PubMed Central

Sanmarco, Liliana Maria; Eberhardt, Natalia; Ponce, Nicolás Eric; Cano, Roxana Carolina; Bonacci, Gustavo; Aoki, Maria Pilar

2018-01-01

Macrophages are the primary immune cells that reside within the myocardium, suggesting that these mononuclear phagocytes are essential in the orchestration of cardiac immunity and homeostasis. Independent of the nature of the injury, the heart triggers leukocyte activation and recruitment. However, inflammation is harmful to this vital terminally differentiated organ with extremely poor regenerative capacity. As such, cardiac tissue has evolved particular strategies to increase the stress tolerance and minimize the impact of inflammation. In this sense, growing evidences show that mononuclear phagocytic cells are particularly dynamic during cardiac inflammation or infection and would actively participate in tissue repair and functional recovery. They respond to soluble mediators such as metabolites or cytokines, which play central roles in the timing of the intrinsic cardiac stress response. During myocardial infarction two distinct phases of monocyte influx have been identified. Upon infarction, the heart modulates its chemokine expression profile that sequentially and actively recruits inflammatory monocytes, first, and healing monocytes, later. In the same way, a sudden switch from inflammatory macrophages (with microbicidal effectors) toward anti-inflammatory macrophages occurs within the myocardium very shortly after infection with Trypanosoma cruzi, the causal agent of Chagas cardiomyopathy. While in sterile injury, healing response is necessary to stop tissue damage; during an intracellular infection, the anti-inflammatory milieu in infected hearts would promote microbial persistence. The balance of mononuclear phagocytic cells seems to be also dynamic in atherosclerosis influencing plaque initiation and fate. This review summarizes the participation of mononuclear phagocyte system in cardiovascular diseases, keeping in mind that the immune system evolved to promote the reestablishment of tissue homeostasis following infection/injury, and that the effects of different mediators could modulate the magnitude and quality of the immune response. The knowledge of the effects triggered by diverse mediators would serve to identify new therapeutic targets in different cardiovascular pathologies. PMID:29375564

New Insights into the Immunobiology of Mononuclear Phagocytic Cells and Their Relevance to the Pathogenesis of Cardiovascular Diseases.

PubMed

Sanmarco, Liliana Maria; Eberhardt, Natalia; Ponce, Nicolás Eric; Cano, Roxana Carolina; Bonacci, Gustavo; Aoki, Maria Pilar

2017-01-01

Macrophages are the primary immune cells that reside within the myocardium, suggesting that these mononuclear phagocytes are essential in the orchestration of cardiac immunity and homeostasis. Independent of the nature of the injury, the heart triggers leukocyte activation and recruitment. However, inflammation is harmful to this vital terminally differentiated organ with extremely poor regenerative capacity. As such, cardiac tissue has evolved particular strategies to increase the stress tolerance and minimize the impact of inflammation. In this sense, growing evidences show that mononuclear phagocytic cells are particularly dynamic during cardiac inflammation or infection and would actively participate in tissue repair and functional recovery. They respond to soluble mediators such as metabolites or cytokines, which play central roles in the timing of the intrinsic cardiac stress response. During myocardial infarction two distinct phases of monocyte influx have been identified. Upon infarction, the heart modulates its chemokine expression profile that sequentially and actively recruits inflammatory monocytes, first, and healing monocytes, later. In the same way, a sudden switch from inflammatory macrophages (with microbicidal effectors) toward anti-inflammatory macrophages occurs within the myocardium very shortly after infection with Trypanosoma cruzi , the causal agent of Chagas cardiomyopathy. While in sterile injury, healing response is necessary to stop tissue damage; during an intracellular infection, the anti-inflammatory milieu in infected hearts would promote microbial persistence. The balance of mononuclear phagocytic cells seems to be also dynamic in atherosclerosis influencing plaque initiation and fate. This review summarizes the participation of mononuclear phagocyte system in cardiovascular diseases, keeping in mind that the immune system evolved to promote the reestablishment of tissue homeostasis following infection/injury, and that the effects of different mediators could modulate the magnitude and quality of the immune response. The knowledge of the effects triggered by diverse mediators would serve to identify new therapeutic targets in different cardiovascular pathologies.

B-Cell Maturation Antigen, A Proliferation-Inducing Ligand, and B-Cell Activating Factor Are Candidate Mediators of Spinal Cord Injury-Induced Autoimmunity

PubMed Central

Saltzman, Jonah W.; Battaglino, Ricardo A.; Salles, Loise; Jha, Prateek; Sudhakar, Supreetha; Garshick, Eric; Stott, Helen L.; Zafonte, Ross

2013-01-01

Abstract Autoimmunity is thought to contribute to poor neurological outcomes after spinal cord injury (SCI). There are few mechanism-based therapies, however, designed to reduce tissue damage and neurotoxicity after SCI because the molecular and cellular bases for SCI-induced autoimmunity are not completely understood. Recent groundbreaking studies in rodents indicate that B cells are responsible for SCI-induced autoimmunity. This novel paradigm, if confirmed in humans, could aid in the design of neuroprotective immunotherapies. The aim of this study was to investigate the molecular signaling pathways and mechanisms by which autoimmunity is induced after SCI, with the goal of identifying potential targets in therapies designed to reduce tissue damage and inflammation in the chronic phase of SCI. To that end, we performed an exploratory microarray analysis of peripheral blood mononuclear cells to identify differentially expressed genes in chronic SCI. We identified a gene network associated with lymphoid tissue structure and development that was composed of 29 distinct molecules and five protein complexes, including two cytokines, a proliferation-inducing ligand (APRIL) and B-cell–activating factor (BAFF), and one receptor, B-cell maturation antigen (BMCA) involved in B cell development, proliferation, activation, and survival. Real-time polymerase chain reaction analysis from ribonucleic acid samples confirmed upregulation of these three genes in SCI. To our knowledge, this is the first report that peripheral blood mononuclear cells produce increased levels of BAFF and APRIL in chronic SCI. This finding provides evidence of systemic regulation of SCI-autoimmunity via APRIL and BAFF mediated activation of B cells through BMCA and points toward these molecules as potential targets of therapies designed to reduce neuroinflammation after SCI. PMID:23088438

[Depiction of the structure of the vitreous body in horses without ocular diseases and in horses with equine recurrent uveitis (ERU) using transmission electron microscopy].

PubMed

Niedermaier, G; Wollanke, B; Hoffmann, R; Matiasek, K; Gerhards, H

2006-06-01

Neither the ultrastructure of the vitreous body from horses without ocular diseases, nor the pathomorphological changes in the vitreous body associated with equine recurrent uveitis (ERU) have been described. However, the vitreous body plays an important role in the pathogenesis of ERU. Ten vitreous body samples obtained from 5 horses without ocular disease, and 38 vitreous body samples from horses with ERU (collected during vitrectomy) were examined by transmission electron microscopy. The vitreous body samples of horses without ocular diseases were characterized by a loose network of unbranched fibrils 10-12 nm in width. In the vitreous body samples of horses with ERU numerous dense bundles of fibrils, mononuclear inflammatory cells and necrotic cells represent the destruction of the vitreous fibrillar network. In this study, equine vitreous body ultrastructure was described for the first time. Thus, demonstrating ultramorphologically, the clinically apparent changes of the vitreous body associated with ERU.

Novel methods of treating ovarian infertility in older and POF women, testicular infertility, and other human functional diseases.

PubMed

Bukovsky, Antonin

2015-02-25

In vitro maturation (IVM) and in vitro fertilization (IVF) technologies are facing with growing demands of older women to conceive. Although ovarian stem cells (OSCs) of older women are capable of producing in vitro fresh oocyte-like cells (OLCs), such cells cannot respond to IVM and IVF due to the lack of granulosa cells required for their maturation. Follicular renewal is also dependent on support of circulating blood mononuclear cells. They induce intermediary stages of meiosis (metaphase I chromosomal duplication and crossover, anaphase, telophase, and cytokinesis) in newly emerging ovarian germ cells, as for the first time demonstrated here, induce formation of granulosa cells, and stimulate follicular growth and development. A pretreatment of OSC culture with mononuclear cells collected from blood of a young healthy fertile woman may cause differentiation of bipotential OSCs into both developing germ and granulosa cells. A small blood volume replacement may enable treatment of ovarian infertility in vivo. The transferred mononuclear cells may temporarily rejuvenate virtually all tissues, including improvement of the function of endocrine tissues. Formation of new follicles and their development may be sufficient for IVM and IVF. The novel proposed in vitro approaches may be used as a second possibility. Infertility of human males affects almost a half of the infertility cases worldwide. Small blood volume replacement from young healthy fertile men may also be easy approach for the improvement of sperm quality in older or other affected men. In addition, body rejuvenation by small blood volume replacement from young healthy individuals of the same sex could represent a decline of in vitro methodology in favor of in vivo treatment for human functional diseases. Here we propose for the first time that blood mononuclear cells are essential for rejuvenation of those tissues, where immune system components participate in an appropriate division and differentiation of tissue stem cells. If needed, small blood volume replacement from distinct young healthy individuals could be utilized in six month intervals for repair of young altered or aged reproductive and other tissue functions. Systemic and local use of honey bee propolis tincture is an alternative option for functional rejuvenation of some tissues.

Membrane Transfer from Mononuclear Cells to Polymorphonuclear Neutrophils Transduces Cell Survival and Activation Signals in the Recipient Cells via Anti-Extrinsic Apoptotic and MAP Kinase Signaling Pathways.

PubMed

Li, Ko-Jen; Wu, Cheng-Han; Shen, Chieh-Yu; Kuo, Yu-Min; Yu, Chia-Li; Hsieh, Song-Chou

2016-01-01

The biological significance of membrane transfer (trogocytosis) between polymorphonuclear neutrophils (PMNs) and mononuclear cells (MNCs) remains unclear. We investigated the biological/immunological effects and molecular basis of trogocytosis among various immune cells in healthy individuals and patients with active systemic lupus erythematosus (SLE). By flow cytometry, we determined that molecules in the immunological synapse, including HLA class-I and-II, CD11b and LFA-1, along with CXCR1, are exchanged among autologous PMNs, CD4+ T cells, and U937 cells (monocytes) after cell-cell contact. Small interfering RNA knockdown of the integrin adhesion molecule CD11a in U937 unexpectedly enhanced the level of total membrane transfer from U937 to PMN cells. Functionally, phagocytosis and IL-8 production by PMNs were enhanced after co-culture with T cells. Total membrane transfer from CD4+ T to PMNs delayed PMN apoptosis by suppressing the extrinsic apoptotic molecules, BAX, MYC and caspase 8. This enhancement of activities of PMNs by T cells was found to be mediated via p38- and P44/42-Akt-MAP kinase pathways and inhibited by the actin-polymerization inhibitor, latrunculin B, the clathrin inhibitor, Pitstop-2, and human immunoglobulin G, but not by the caveolin inhibitor, methyl-β-cyclodextrin. In addition, membrane transfer from PMNs enhanced IL-2 production by recipient anti-CD3/anti-CD28 activated MNCs, and this was suppressed by inhibitors of mitogen-activated protein kinase (PD98059) and protein kinase C (Rottlerin). Of clinical significance, decreased total membrane transfer from PMNs to MNCs in patients with active SLE suppressed mononuclear IL-2 production. In conclusion, membrane transfer from MNCs to PMNs, mainly at the immunological synapse, transduces survival and activation signals to enhance PMN functions and is dependent on actin polymerization, clathrin activation, and Fcγ receptors, while membrane transfer from PMNs to MNCs depends on MAP kinase and PKC signaling. Defective membrane transfer from PMNs to MNCs in patients with active systemic lupus erythematous suppressed activated mononuclear IL-2 production.

Coregulator profiling of the glucocorticoid receptor in lymphoid malignancies

PubMed Central

Clarisse, Dorien; Thommis, Jonathan; Van Wesemael, Karlien; Houtman, René; Ratman, Dariusz; Tavernier, Jan; Offner, Fritz; Beck, Ilse; De Bosscher, Karolien

2017-01-01

Coregulators cooperate with nuclear receptors, such as the glucocorticoid receptor (GR), to enhance or repress transcription. These regulatory proteins are implicated in cancer, yet, their role in lymphoid malignancies, including multiple myeloma (MM) and acute lymphoblastic leukemia (ALL), is largely unknown. Here, we report the use and extension of the microarray assay for real-time nuclear receptor coregulator interactions (MARCoNI) technology to detect coregulator associations with endogenous GR in cell lysates. We use MARCoNI to determine the GR coregulator profile of glucocorticoid-sensitive (MM and ALL) and glucocorticoid-resistant (ALL) cells, and identify common and unique coregulators for different cell line comparisons. Overall, we identify SRC-1/2/3, PGC-1α, RIP140 and DAX-1 as the strongest interacting coregulators of GR in MM and ALL cells and show that the interaction strength does not correlate with GR protein levels. Lastly, as a step towards patient samples, we determine the GR coregulator profile of peripheral blood mononuclear cells. We profile the interactions between GR and coregulators in MM and ALL cells and suggest to further explore the GR coregulator profile in hematological patient samples. PMID:29312638

Coregulator profiling of the glucocorticoid receptor in lymphoid malignancies.

PubMed

Clarisse, Dorien; Thommis, Jonathan; Van Wesemael, Karlien; Houtman, René; Ratman, Dariusz; Tavernier, Jan; Offner, Fritz; Beck, Ilse; De Bosscher, Karolien

2017-12-12

Coregulators cooperate with nuclear receptors, such as the glucocorticoid receptor (GR), to enhance or repress transcription. These regulatory proteins are implicated in cancer, yet, their role in lymphoid malignancies, including multiple myeloma (MM) and acute lymphoblastic leukemia (ALL), is largely unknown. Here, we report the use and extension of the microarray assay for real-time nuclear receptor coregulator interactions (MARCoNI) technology to detect coregulator associations with endogenous GR in cell lysates. We use MARCoNI to determine the GR coregulator profile of glucocorticoid-sensitive (MM and ALL) and glucocorticoid-resistant (ALL) cells, and identify common and unique coregulators for different cell line comparisons. Overall, we identify SRC-1/2/3, PGC-1α, RIP140 and DAX-1 as the strongest interacting coregulators of GR in MM and ALL cells and show that the interaction strength does not correlate with GR protein levels. Lastly, as a step towards patient samples, we determine the GR coregulator profile of peripheral blood mononuclear cells. We profile the interactions between GR and coregulators in MM and ALL cells and suggest to further explore the GR coregulator profile in hematological patient samples.

Laser capture microdissection: should an ultraviolet or infrared laser be used?

PubMed

Vandewoestyne, Mado; Goossens, Karen; Burvenich, Christian; Van Soom, Ann; Peelman, Luc; Deforce, Dieter

2013-08-15

Laser capture microdissection (LCM) is a well-established cell separation technique. It combines microscopy with laser beam technology and allows targeting of specific cells or tissue regions that need to be separated from others. Consequently, this biological material can be used for genome or transcriptome analyses. Appropriate methods of sample preparation, however, are crucial for the success of downstream molecular analysis. The aim of this study was to objectively compare the two main LCM systems, one based on an ultraviolet (UV) laser and the other based on an infrared (IR) laser, on different criteria ranging from user-friendliness to sample quality. The comparison was performed on two types of samples: peripheral blood mononuclear cells and blastocysts. The UV laser LCM system had several advantages over the IR laser LCM system. Not only does the UV system allow faster and more precise sample collection, but also the obtained samples-even single cell samples-can be used for DNA extraction and downstream polymerase chain reaction (PCR) applications. RNA-based applications are more challenging for both LCM systems. Although sufficient RNA can be extracted from as few as 10 cells for reverse transcription quantitative PCR (RT-qPCR) analysis, the low RNA quality should be taken into account when designing the RT-qPCR assays. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Morphologic and cytochemical characteristics of blood cells from Hawaiian green turtles

USGS Publications Warehouse

Work, Thierry M.; Raskin, R.E.; Balazs, George H.; Whittaker, S.D.

1998-01-01

Objective - To identify and characterize blood cells from free-ranging Hawaiian green turtles, Chelonia mydas. Sample Population - 26 green turtles from Puako on the island of Hawaii and Kaneohe Bay on the island of Oahu. Procedure - Blood was examined, using light and electron microscopy and cytochemical stains that included benzidine peroxidase, chloroacetate esterase, alpha naphthyl butyrate esterase, acid phosphatase, Sudan black B, periodic acid-Schiff, and toluidine blue. Results - 6 types of WBC were identified: lymphocytes, monocytes, thrombocytes, heterophils, basophils, and eosinophils (small and large). Morphologic characteristics of mononuclear cells and most granulocytes were similar to those of cells from other reptiles except that green turtles have both large and small eosinophils. Conclusions - Our classification of green turtle blood cells clarifies imporoper nomenclature reported previously and provides a reference for future hematologic studies in this species.

Donor Cell Composition and Reactivity Predict Risk of Acute Graft-versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation.

PubMed

Sairafi, Darius; Stikvoort, Arwen; Gertow, Jens; Mattsson, Jonas; Uhlin, Michael

2016-01-01

Background . Graft-versus-host disease (GVHD) is a serious complication after allogeneic hematopoietic stem cell transplantation (HSCT). We designed a functional assay for assessment of individual risk for acute GVHD. Study Design and Methods . Blood samples were collected from patients and donors before HSCT. Two groups of seven patients each were selected, one in which individuals developed acute GVHD grades II-IV and one in which none showed any clinical signs of GVHD. Peripheral blood mononuclear cells (PBMCs) isolated from donors were incubated in mixed lymphocyte cultures (MLCs) with recipient PBMCs. The cells were characterized by flow cytometry before and after MLC. Results . Samples from donors in the GVHD group contained significantly lower frequencies of naïve γδ T-cells and T-cells expressing NK-cell markers CD56 and CD94. Donor samples in this group also exhibited lower frequencies of naïve CD95 + T-cells compared to controls. After MLC, there were dissimilarities in the CD4/CD8 T-cell ratio and frequency of CD69 + T-cells between the two patient groups, with the non-GVHD group showing higher frequencies of CD8 + and CD69 + T-cells. Conclusion . We conclude that a thorough flow cytometric analysis of donor cells for phenotype and allogeneic reactivity may be of value when assessing pretransplant risk for severe acute GVHD.

Mononuclear nonheme iron(III) complexes that show superoxide dismutase-like activity and antioxidant effects against menadione-mediated oxidative stress.

PubMed

Hitomi, Yutaka; Iwamoto, Yuji; Kashida, Akihiro; Kodera, Masahito

2015-05-21

This communication describes the superoxide dismutase (SOD)-like activity of mononuclear iron(III) complexes with pentadentate monocarboxylamido ligands. The SOD activity can be controlled by the electronic nature of the substituent group on the ligand. The nitro-substituted complex showed clear cytoprotective activity against menadione-mediated oxidative stress in cultured cells.

Preclinical development of a bridging therapy for radiation casualties: appropriate for high risk personnel.

PubMed

Singh, Vijay K; Wise, Stephen Y; Fatanmi, Oluseyi O; Beattie, Lindsay A; Seed, Thomas M

2014-06-01

The authors demonstrate the efficacy of a bridging therapy in a preclinical animal model that allows the lymphohematopoietic system of severely immunocompromised individuals exposed to acute, high-dose ionizing irradiation to recover and to survive. CD2F1 mice were irradiated acutely with high doses causing severe, potentially fatal hematopoietic or gastrointestinal injuries and then transfused intravenously with progenitor-enriched, whole blood, or peripheral blood mononuclear cells from mice injected with tocopherol succinate- and AMD3100- (a chemokine receptor anatogonist used to improve the yield of mobilized progenitors). Survival of these mice over a 30-d period was used as the primary measured endpoint of therapeutic effectiveness. The authors demonstrate that tocopherol succinate and AMD3100 mobilize progenitors into peripheral circulation and that the infusion of mobilized progenitor enriched blood or mononuclear cells acts as a bridging therapy for lymphohematopoietic system recovery in mice exposed to whole-body ionizing irradiation. The results demonstrate that infusion of whole blood or blood mononuclear cells from tocopherol succinate (TS)- and AMD3100-injected mice improved the survival of mice receiving high radiation doses significantly. The efficacy of TS-injected donor mice blood or mononuclear cells was comparable to that of blood or cells obtained from mice injected with granulocyte colony-stimulating factor. Donor origin-mobilized progenitors were found to localize in various tissues. The authors suggest that tocopherol succinate is an optimal agent for mobilizing progenitors with significant therapeutic potential. The extent of progenitor mobilization that tocopherol succinate elicits in experimental mice is comparable quantitatively to clinically used drugs such as granulocyte-colony stimulating factor and AMD3100. Therefore, it is proposed that tocopherol succinate be considered for further translational development and ultimately for use in humans.

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

PubMed

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.

Vitamin B6 Modifies the Immune Cross-Talk between Mononuclear and Colon Carcinoma Cells.

PubMed

Bessler, H; Djaldetti, M

2016-01-01

The role of vitamin B6 as a key component in a number of biological events has been well established. Based on the relationship between chronic inflammation and carcinogenesis on the one hand, and the interaction between immune and cancer cells expressed by modulated cytokine production on the other hand, the aim of the present work was to examine the possibility that vitamin B6 affects cancer development by an interference in the cross-talk between human peripheral blood mononuclear cells (PBMC) and those from two colon carcinoma cell lines. Both non-stimulated PBMC and mononuclear cells induced for cytokine production by HT-29 and RKO cells from human colon carcinoma lines were incubated without and with 4, 20 and 100 μg/ml of pyridoxal hydrochloride (vitamin B6) and secretion of TNF-α, IL-1β, IL-6, IFN-γ, IL-10, and IL-1ra was examined. Vit B6 caused a dose-dependent decrease in production of all cytokines examined, except for that of IL-1ra. The results indicate that vitamin B6 exerts an immunomodulatory effect on human PBMC. The finding that production of inflammatory cytokines is more pronounced when PBMC are in contact with malignant cells and markedly inhibited by the vitamin suggests an additional way by which vitamin B6 may exert its carcinopreventive effect.

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

PubMed

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.

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

PubMed

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.

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

PubMed Central

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

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. PMID:29736174

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

PubMed

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

2015-01-01

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

Mucorales spores induce a proinflammatory cytokine response in human mononuclear phagocytes and harbor no rodlet hydrophobins.

PubMed

Wurster, Sebastian; Thielen, Vanessa; Weis, Philipp; Walther, Paul; Elias, Johannes; Waaga-Gasser, Ana Maria; Dragan, Mariola; Dandekar, Thomas; Einsele, Hermann; Löffler, Jürgen; Ullmann, Andrew J

2017-11-17

Mucormycoses are life-threatening infections in immunocompromised patients. This study characterizes the response of human mononuclear cells to different Mucorales and Ascomycota. PBMC, monocytes, and monocyte derived dendritic cells (moDCs) from healthy donors were stimulated with resting and germinated stages of Mucorales and Ascomycota. Cytokine response and expression of activation markers were studied. Both inactivated germ tubes and resting spores of Rhizopus arrhizus and other human pathogenic Mucorales species significantly stimulated mRNA synthesis and secretion of proinflammatory cytokines. Moreover, R. arrhizus spores induced the upregulation of co-stimulatory molecules on moDCs and a specific T-helper cell response. Removal of rodlet hydrophobins by hydrofluoric acid treatment of A. fumigatus conidia resulted in enhanced immunogenicity, whereas the cytokine response of PBMCs to dormant R. arrhizus spores was not influenced by hydrofluoric acid. Scanning electron micrographs of Mucorales spores did not exhibit any morphological correlates of rodlet hydrophobins. Taken together, this study revealed striking differences in the response of human mononuclear cells to resting stages of Ascomycota and Mucorales, which may be explained by absence of an immunoprotective hydrophobin layer in Mucorales spores.

Efficient and safe gene delivery to human corneal endothelium using magnetic nanoparticles.

PubMed

Czugala, Marta; Mykhaylyk, Olga; Böhler, Philip; Onderka, Jasmine; Stork, Björn; Wesselborg, Sebastian; Kruse, Friedrich E; Plank, Christian; Singer, Bernhard B; Fuchsluger, Thomas A

2016-07-01

To develop a safe and efficient method for targeted, anti-apoptotic gene therapy of corneal endothelial cells (CECs). Magnetofection (MF), a combination of lipofection with magnetic nanoparticles (MNPs; PEI-Mag2, SO-Mag5, PalD1-Mag1), was tested in human CECs and in explanted human corneas. Effects on cell viability and function were investigated. Immunocompatibility was assessed in human peripheral blood mononuclear cells. Silica iron-oxide MNPs (SO-Mag5) combined with X-tremeGENE-HP achieved high transfection efficiency in human CECs and explanted human corneas, without altering cell viability or function. Magnetofection caused no immunomodulatory effects in human peripheral blood mononuclear cells. Magnetofection with anti-apoptotic P35 gene effectively blocked apoptosis in CECs. Magnetofection is a promising tool for gene therapy of corneal endothelial cells with potential for targeted on-site delivery.

Mononuclear cell secretome protects from experimental autoimmune myocarditis.

PubMed

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.

A cluster pattern algorithm for the analysis of multiparametric cell assays.

PubMed

Kaufman, Menachem; Bloch, David; Zurgil, Naomi; Shafran, Yana; Deutsch, Mordechai

2005-09-01

The issue of multiparametric analysis of complex single cell assays of both static and flow cytometry (SC and FC, respectively) has become common in recent years. In such assays, the analysis of changes, applying common statistical parameters and tests, often fails to detect significant differences between the investigated samples. The cluster pattern similarity (CPS) measure between two sets of gated clusters is based on computing the difference between their density distribution functions' set points. The CPS was applied for the discrimination between two observations in a four-dimensional parameter space. The similarity coefficient (r) ranges between 0 (perfect similarity) to 1 (dissimilar). Three CPS validation tests were carried out: on the same stock samples of fluorescent beads, yielding very low r's (0, 0.066); and on two cell models: mitogenic stimulation of peripheral blood mononuclear cells (PBMC), and apoptosis induction in Jurkat T cell line by H2O2. In both latter cases, r indicated similarity (r < 0.23) within the same group, and dissimilarity (r > 0.48) otherwise. This classification and algorithm approach offers a measure of similarity between samples. It relies on the multidimensional pattern of the sample parameters. The algorithm compensates for environmental drifts in this apparatus and assay; it also may be applied to more than four dimensions.

Comparative studies of mononuclear phagocyte function in patients with Crohn's disease and colon neoplasms.

PubMed Central

Beeken, W L; St Andre-Ukena, S; Gundel, R M

1983-01-01

Phagocytosis and cellular cytotoxicity by mononuclear phagocytes of blood and intestinal mucosa were studied in patients with Crohn's disease and large bowel neoplasms. Antibody coated sheep erythrocytes were used for phagocytic assays and cellular cytotoxicity in vitro was measured by 24 hour isotope release from 75Selenium methionine-labelled RPMI 4788 human cancer cell cultures in the presence of mononuclear phagocyte-enriched effector populations. The mean percent of mononuclear phagocytes in Ficoll-Hypaque purified mononuclear cell suspensions of blood of healthy controls was 25.9 compared with 44.6 in patients with Crohn's disease, 45.6 in patients with colon neoplasms and 11.6 in intestinal mucosa. Phagocytic indices were similar in all groups, but the phagocytic capacity of mucosal macrophages was twice that of blood monocytes. Mean cytotoxicity of monocytes of patients with Crohn's disease was 12.8% compared with 22.9% for monocytes from normal controls, and 29.4% for patients with colon tumours. Mean cytotoxicity by mucosal macrophages was 18.0% compared with 13.2% by mucosal lymphocyte populations. Exposure of monocytes of Crohn's disease patients to bacterial lipopolysaccharide modestly increased cytotoxicity, but exposure did not alter phagocytosis by monocytes of patients or controls. The results indicate that monocytes of patients with Crohn's disease exhibit subnormal in vitro cytotoxicity. Mucosal macrophages from patients with various diseases show enhanced phagocytosis compared with blood monocytes, and they can mediate cellular cytotoxicity in vitro. PMID:6629113

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

PubMed

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.

Novel flowcytometry-based approach of malignant cell detection in body fluids using an automated hematology analyzer

PubMed Central

Tabe, Yoko; Takemura, Hiroyuki; Kimura, Konobu; Takahashi, Toshihiro; Yang, Haeun; Tsuchiya, Koji; Konishi, Aya; Uchihashi, Kinya; Horii, Takashi; Ohsaka, Akimichi

2018-01-01

Morphological microscopic examinations of nucleated cells in body fluid (BF) samples are performed to screen malignancy. However, the morphological differentiation is time-consuming and labor-intensive. This study aimed to develop a new flowcytometry-based gating analysis mode “XN-BF gating algorithm” to detect malignant cells using an automated hematology analyzer, Sysmex XN-1000. XN-BF mode was equipped with WDF white blood cell (WBC) differential channel. We added two algorithms to the WDF channel: Rule 1 detects larger and clumped cell signals compared to the leukocytes, targeting the clustered malignant cells; Rule 2 detects middle sized mononuclear cells containing less granules than neutrophils with similar fluorescence signal to monocytes, targeting hematological malignant cells and solid tumor cells. BF samples that meet, at least, one rule were detected as malignant. To evaluate this novel gating algorithm, 92 various BF samples were collected. Manual microscopic differentiation with the May-Grunwald Giemsa stain and WBC count with hemocytometer were also performed. The performance of these three methods were evaluated by comparing with the cytological diagnosis. The XN-BF gating algorithm achieved sensitivity of 63.0% and specificity of 87.8% with 68.0% for positive predictive value and 85.1% for negative predictive value in detecting malignant-cell positive samples. Manual microscopic WBC differentiation and WBC count demonstrated 70.4% and 66.7% of sensitivities, and 96.9% and 92.3% of specificities, respectively. The XN-BF gating algorithm can be a feasible tool in hematology laboratories for prompt screening of malignant cells in various BF samples. PMID:29425230

Novel flowcytometry-based approach of malignant cell detection in body fluids using an automated hematology analyzer.

PubMed

Ai, Tomohiko; Tabe, Yoko; Takemura, Hiroyuki; Kimura, Konobu; Takahashi, Toshihiro; Yang, Haeun; Tsuchiya, Koji; Konishi, Aya; Uchihashi, Kinya; Horii, Takashi; Ohsaka, Akimichi

2018-01-01

Morphological microscopic examinations of nucleated cells in body fluid (BF) samples are performed to screen malignancy. However, the morphological differentiation is time-consuming and labor-intensive. This study aimed to develop a new flowcytometry-based gating analysis mode "XN-BF gating algorithm" to detect malignant cells using an automated hematology analyzer, Sysmex XN-1000. XN-BF mode was equipped with WDF white blood cell (WBC) differential channel. We added two algorithms to the WDF channel: Rule 1 detects larger and clumped cell signals compared to the leukocytes, targeting the clustered malignant cells; Rule 2 detects middle sized mononuclear cells containing less granules than neutrophils with similar fluorescence signal to monocytes, targeting hematological malignant cells and solid tumor cells. BF samples that meet, at least, one rule were detected as malignant. To evaluate this novel gating algorithm, 92 various BF samples were collected. Manual microscopic differentiation with the May-Grunwald Giemsa stain and WBC count with hemocytometer were also performed. The performance of these three methods were evaluated by comparing with the cytological diagnosis. The XN-BF gating algorithm achieved sensitivity of 63.0% and specificity of 87.8% with 68.0% for positive predictive value and 85.1% for negative predictive value in detecting malignant-cell positive samples. Manual microscopic WBC differentiation and WBC count demonstrated 70.4% and 66.7% of sensitivities, and 96.9% and 92.3% of specificities, respectively. The XN-BF gating algorithm can be a feasible tool in hematology laboratories for prompt screening of malignant cells in various BF samples.

Thalidomide prolongs survival after experimental musculoskeletal injury, through an effect on mononuclear apoptosis.

PubMed

Panousis, Konstantinos; Nikolaou, Vassilios S; Tsaganos, Thomas; Lallos, Stergios; Giamarellos-Bourboulis, Evangelos J; Efstathopoulos, Nicolas

2014-05-01

This study was conducted to investigate the effects of intravenous thalidomide administration in an experimental model of musculoskeletal trauma. We hypothesized that because thalidomide inhibits secretion of tumor necrosis factor alpha (TNF-α), survival of animals that received thalidomide would be significantly prolonged. After an open fracture of the right femur, 24 rabbits were randomly assigned to control and thalidomide groups. Intravenous therapy with thalidomide was started 30 min after fracture. Hemodynamic monitoring of all animals was performed for 4 h. Survival was recorded and bacterial growth in blood and organs was measured after animal death or sacrifice. Blood was sampled for TNF-α measurement and for isolation of peripheral blood mononuclear cells (PBMCs). Apoptosis of PBMCs was measured by flow cytometry. Survival was significantly prolonged in the thalidomide group. Apoptosis of PBMCs was increased in the control group compared with the thalidomide group at 24 h. There were no differences in vital signs, blood and tissue cultures, and serum TNF-α concentration between the two groups. Intravenous thalidomide prolonged survival in an experimental model of severe musculoskeletal injury in rabbits. Its mechanism of action did not involve TNF-α suppression but prevention of mononuclear apoptosis. In view of these promising results, further research is needed to clarify the immunomodulatory mechanism of action of thalidomide and its potential use for the management of severe trauma. Copyright © 2014 Elsevier Inc. All rights reserved.

Myelogenous leukemia in a bearded dragon (Acanthodraco vitticeps).

PubMed

Tocidlowski, M E; McNamara, P L; Wojcieszyn, J W

2001-03-01

A 3-yr-old bearded dragon (Acanthodraco vitticeps) presented with lethargy, a swollen right elbow joint, inability to move its rear limbs normally, and marked leukocytosis. The majority of leukocytes were an abnormal mononuclear lymphoid-type cell with a high nuclear to cytoplasmic ratio, a slightly blue cytoplasm, nuclei with coarsely granular chromatin, and some nuclear clefts. Acute leukemia of lymphoid or myeloid origin was tentatively diagnosed. The abnormal mononuclear leukocyte cell population stained positively for the myeloid cytochemical stains: peroxidase, chloroacetate esterase, and L1-calprotectin. The abnormal cell population of the peripheral blood did not stain with the lymphoid cytochemical stains: alpha-naphthyl butyrate esterase, CD3, and CD79a.

Time-resolved fluorescence monitoring of cholesterol in peripheral blood mononuclear cells

NASA Astrophysics Data System (ADS)

Martinakova, Z.; Horilova, J.; Lajdova, I.; Marcek Chorvatova, A.

2014-12-01

Precise evaluation of intracellular cholesterol distribution is crucial for improving diagnostics of diseased states associated with cholesterol alteration. Time-resolved fluorescence techniques are tested for non-invasive investigation of cholesterol in living cells. Fluorescent probe NBD attached to cholesterol was employed to evaluate cholesterol distribution in peripheral blood mononuclear cells (PBMC) isolated from the human blood. Fluorescence Lifetime Imaging Microscopy (FLIM) was successfully applied to simultaneously monitor the spatial distribution and the timeresolved characteristics of the NBD-cholesterol fluorescence in PBMC. Gathered data are the first step in the development of a new perspective non-invasive diagnostic method for evaluation of cholesterol modifications in diseases associated with disorders of lipid metabolism.

Extracellular calcium (Ca2+o)-sensing receptor in a mouse monocyte-macrophage cell line (J774): potential mediator of the actions of Ca2+o on the function of J774 cells

NASA Technical Reports Server (NTRS)

Yamaguchi, T.; Kifor, O.; Chattopadhyay, N.; Bai, M.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

1998-01-01

The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Macrophage-like mononuclear cells appear at sites of osteoclastic bone resorption during bone remodeling and may play a role in the "reversal" phase following osteoclastic resorption and preceding bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for bone marrow mononuclear cells in the vicinity, leading us to investigate whether such mononuclear cells express the CaR. In this study, we used the mouse J774 cell line, which exhibits a pure monocyte-macrophage phenotype. Both immunocytochemistry and Western blot analysis, using polyclonal antisera specific for the CaR, detected CaR protein in J774 cells. The use of reverse transcriptase-polymerase chain reaction with CaR-specific primers, including a set of intron-spanning primers, followed by nucleotide sequencing of the amplified products, also identified CaR transcripts in J774 cells. Exposure of J774 cells to high Ca2+o (2.8 mM or more) or the polycationic CaR agonist, neomycin (100 microM), stimulated both chemotaxis and DNA synthesis in J774 cells. Therefore, taken together, our data strongly suggest that the monocyte-macrophage cell line, J774, possesses both CaR protein and mRNA very similar, if not identical, to those in parathyroid and kidney.

Oral warfarin affects peripheral blood leukocyte IL-6 and TNFα production in rats.

PubMed

Popov, Aleksandra; Belij, Sandra; Subota, Vesna; Zolotarevski, Lidija; Mirkov, Ivana; Kataranovski, Dragan; Kataranovski, Milena

2013-01-01

Warfarin is a Vitamin K (VK) antagonist that affects Vitamin K-dependent (VKD) processes, including blood coagulation, as well as processes unrelated to hemostasis such as bone growth, calcification, and growth of some cell types. In addition, warfarin exerts influence on some non-VKD-related activities, including anti-tumor and immunomodulating activity. With respect to the latter, both immune stimulating and suppressive effects have been noted in different experimental systems. To explore the in vivo immunomodulatory potential of warfarin on one type of activity (i.e., cytokine production) in two different immune cell populations (i.e., mononuclear or polymorphonuclear cells), effects of subchronic oral warfarin intake in rats on pro-inflammatory cytokine (i.e., TNFα, IL-6) production by peripheral blood mononuclear and polymorphonuclear cells (granulocytes) was examined. Differential effects of warfarin intake on TNFα and IL-6 were noted, depending on the type of peripheral blood leukocytes and on the cytokine examined. Specifically, a lack of effect on TNFα and a priming of IL-6 production by mononuclear cells along with a decrease in TNFα and a lack of effect on IL-6 in polymorphonuclear cells were seen in warfarin-exposed hosts. The cell- and cytokine-dependent effects from subchronic oral warfarin intake on peripheral blood leukocytes demonstrated in this study could, possibly, differentially affect reactions mediated by these cells. Ultimately, the observed effects in rats might have implications for those humans who are on long-term/prolonged warfarin therapy.

HLA-targeted flow cytometric sorting of blood cells allows separation of pure and viable microchimeric cell populations.

PubMed

Drabbels, Jos J M; van de Keur, Carin; Kemps, Berit M; Mulder, Arend; Scherjon, Sicco A; Claas, Frans H J; Eikmans, Michael

2011-11-10

Microchimerism is defined by the presence of low levels of nonhost cells in a person. We developed a reliable method for separating viable microchimeric cells from the host environment. For flow cytometric cell sorting, HLA antigens were targeted with human monoclonal HLA antibodies (mAbs). Optimal separation of microchimeric cells (present at a proportion as low as 0.01% in artificial mixtures) was obtained with 2 different HLA mAbs, one targeting the chimeric cells and the other the background cells. To verify purity of separated cell populations, flow-sorted fractions of 1000 cells were processed for DNA analysis by HLA-allele-specific and Y-chromosome-directed real-time quantitative PCR assays. After sorting, PCR signals of chimeric DNA markers in the positive fractions were significantly enhanced compared with those in the presort samples, and they were similar to those in 100% chimeric control samples. Next, we demonstrate applicability of HLA-targeted FACS sorting after pregnancy by separating chimeric maternal cells from child umbilical cord mononuclear cells. Targeting allelic differences with anti-HLA mAbs with FACS sorting allows maximal enrichment of viable microchimeric cells from a background cell population. The current methodology enables reliable microchimeric cell detection and separation in clinical specimens.

No Evidence for XMRV Nucleic Acids, Infectious Virus or Anti-XMRV Antibodies in Canadian Patients with Chronic Fatigue Syndrome

PubMed Central

Steffen, Imke; Tyrrell, D. Lorne; Stein, Eleanor; Montalvo, Leilani; Lee, Tzong-Hae; Zhou, Yanchen; Lu, Kai; Switzer, William M.; Tang, Shaohua; Jia, Hongwei; Hockman, Darren; Santer, Deanna M.; Logan, Michael; Landi, Amir; Law, John; Houghton, Michael; Simmons, Graham

2011-01-01

The gammaretroviruses xenotropic murine leukemia virus (MLV)-related virus (XMRV) and MLV have been reported to be more prevalent in plasma and peripheral blood mononuclear cells of chronic fatigue syndrome (CFS) patients than in healthy controls. Here, we report the complex analysis of whole blood and plasma samples from 58 CFS patients and 57 controls from Canada for the presence of XMRV/MLV nucleic acids, infectious virus, and XMRV/MLV-specific antibodies. Multiple techniques were employed, including nested and qRT-PCR, cell culture, and immunoblotting. We found no evidence of XMRV or MLV in humans and conclude that CFS is not associated with these gammaretroviruses. PMID:22114717

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

DTIC Science & Technology

2013-09-01

Bongarzone ER. Expression of sonic hedgehog targeted genes in peripheral blood mononuclear cells of patients with multiple sclerosis. Society for...Print Program#/Poster#: 322.13 Presentation Title: Expression of sonic  hedgehog  targeted genes in peripheral blood mononuclear cells of patients with...analyses. Gene array hybridization showed up­ regulation of various components of the Sonic  hedgehog  (Shh) pathway including, Olig1 and Olig2. Taken

International Symposium on Genetic Control of Host Resistance to Infection and Malignancy (2nd) held in Montreal, Canada, on May 12-16, 1985

DTIC Science & Technology

1985-11-01

BR, BIO.SM, BIO.M, BIO.D2, Blu.S and BIO.PL animals and is typified by fibrous scars which consist of inflammatory cells within linear bands c fibrous...connective tissue rather than discrete foci of necrosis. These linear bands of connective tissue contain the same degree of mononuclear cell...per granulome were significantly higher in the liver aid in the lungs of the B1O.D2n mice. Both, mononuclear and neutrophil.s are present in the

Different Patterns of Cytokines and Chemokines Combined with IFN-γ Production Reflect Mycobacterium tuberculosis Infection and Disease

PubMed Central

Hu, Shizong; Jin, Dongdong; Chen, Xinchun; Jin, Qi; Liu, Haiying

2012-01-01

Background IFN-γ is presently the only soluble immunological marker used to help diagnose latent Mycobacterium tuberculosis (M.tb) infection. However, IFN-γ is not available to distinguish latent from active TB infection. Moreover, extrapulmonary tuberculosis, such as tuberculous pleurisy, cannot be properly diagnosed by IFN-γ release assay. As a result, other disease- or infection-related immunological biomarkers that would be more effective need to be screened and identified. Methodology A panel of 41 soluble immunological molecules (17 cytokines and 24 chemokines) was tested using Luminex liquid array-based multiplexed immunoassays. Samples, including plasma and pleural effusions, from healthy donors (HD, n = 12) or patients with latent tuberculosis infection (LTBI, n = 20), pulmonary tuberculosis (TB, n = 12), tuberculous pleurisy (TP, n = 15) or lung cancer (LC, n = 15) were collected and screened for soluble markers. Peripheral blood mononuclear cells (PBMCs) and pleural fluid mononuclear cells (PFMCs) were also isolated to investigate antigen-specific immune factors. Principal Findings For the 41 examined factors, our results indicated that three patterns were closely associated with infection and disease. (1) Significantly elevated plasma levels of IL-2, IP-10, CXCL11 and CXCL12 were present in both patients with tuberculosis and in a sub-group participant with latent tuberculosis infection who showed a higher level of IFN-γ producing cells by ELISPOT assay compared with other latently infected individuals. (2) IL-6 and IL-9 were only significantly increased in plasma from active TB patients, and the two factors were consistently highly secreted after M.tb antigen stimulation. (3) When patients developed tuberculous pleurisy, CCL1, CCL21 and IL-6 were specifically increased in the pleural effusions. In particular, these three factors were consistently highly secreted by pleural fluid mononuclear cells following M.tb-specific antigen stimulation. In conclusion, our data imply that the specific secretion of soluble immunological factors, in addition to IFN-γ, may be used to evaluate M.tb infection and tuberculosis disease. PMID:23028695

A benchmark for evaluation of algorithms for identification of cellular correlates of clinical outcomes.

PubMed

Aghaeepour, Nima; Chattopadhyay, Pratip; Chikina, Maria; Dhaene, Tom; Van Gassen, Sofie; Kursa, Miron; Lambrecht, Bart N; Malek, Mehrnoush; McLachlan, G J; Qian, Yu; Qiu, Peng; Saeys, Yvan; Stanton, Rick; Tong, Dong; Vens, Celine; Walkowiak, Sławomir; Wang, Kui; Finak, Greg; Gottardo, Raphael; Mosmann, Tim; Nolan, Garry P; Scheuermann, Richard H; Brinkman, Ryan R

2016-01-01

The Flow Cytometry: Critical Assessment of Population Identification Methods (FlowCAP) challenges were established to compare the performance of computational methods for identifying cell populations in multidimensional flow cytometry data. Here we report the results of FlowCAP-IV where algorithms from seven different research groups predicted the time to progression to AIDS among a cohort of 384 HIV+ subjects, using antigen-stimulated peripheral blood mononuclear cell (PBMC) samples analyzed with a 14-color staining panel. Two approaches (FlowReMi.1 and flowDensity-flowType-RchyOptimyx) provided statistically significant predictive value in the blinded test set. Manual validation of submitted results indicated that unbiased analysis of single cell phenotypes could reveal unexpected cell types that correlated with outcomes of interest in high dimensional flow cytometry datasets. © 2015 International Society for Advancement of Cytometry.

Natural Hemozoin Stimulates Syncytiotrophoblast to Secrete Chemokines and Recruit Peripheral Blood Mononuclear Cells

PubMed Central

Lucchi, Naomi W.; Sarr, Demba; Owino, Simon O.; Mwalimu, Stephen M.; Peterson, David S.; Moore, Julie M.

2011-01-01

Background Placental malaria is associated with local accumulation of parasitized erythrocytes, deposition of the parasite hemoglobin metabolite, hemozoin, and accumulation of mononuclear cells in the intervillous space. Fetal syncytiotrophoblast cells in contact with maternal blood are known to respond immunologically to cytoadherent Plasmodium falciparum-infected erythrocytes, but their responsiveness to hemozoin, a potent pro-inflammatory stimulator of monocytes, macrophages and dendritic cells, is not known. Methods The biochemical and immunological changes induced in primary syncytiotrophoblast by natural hemozoin was assessed. Changes in syncytiotrophoblast mitogen-activated protein kinase activation was assessed by immunoblotting and secreted cytokine and chemokine proteins were assayed by ELISA. Chemotaxis of peripheral blood mononuclear cells was assessed using a two-chamber assay system and flow cytometry was used to assess the activation of primary monocytes by hemozoin-stimulated syncytiotrophoblast conditioned medium. Results Hemozoin stimulation induced ERK1/2 phosphorylation. Treated cells secreted CXCL8, CCL3, CCL4, and tumor necrosis factor and released soluble intercellular adhesion molecule-1. Furthermore, the dependence of the hemozoin responses on ERK1/2 stimulation was confirmed by inhibition of chemokine release in syncytiotrophoblast treated with an ERK pathway inhibitor. Hemozoin-stimulated cells elicited the specific migration of PBMCs, and conditioned medium from the cells induced the upregulation of intercellular adhesion molecule-1 on primary monocytes. Conclusions These findings confirm an immunostimulatory role for hemozoin and expand the cell types known to be responsive to hemozoin to include fetal syncytiotrophoblast. The results provide further evidence that syncytiotrophoblast cells can influence the local maternal immune response to placental malaria. PMID:21632106

Human umbilical cord blood mesenchymal stem cells reduce colitis in mice by activating NOD2 signaling to COX2.

PubMed

Kim, Hyung-Sik; Shin, Tae-Hoon; Lee, Byung-Chul; Yu, Kyung-Rok; Seo, Yoojin; Lee, Seunghee; Seo, Min-Soo; Hong, In-Sun; Choi, Soon Won; Seo, Kwang-Won; Núñez, Gabriel; Park, Jong-Hwan; Kang, Kyung-Sun

2013-12-01

Decreased levels or function of nucleotide-binding oligomerization domain 2 (NOD2) are associated with Crohn's disease. NOD2 regulates intestinal inflammation, and also is expressed by human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs), to regulate their differentiation. We investigated whether NOD2 is required for the anti-inflammatory activities of MSCs in mice with colitis. Colitis was induced in mice by administration of dextran sulfate sodium or trinitrobenzene sulfonic acid. Mice then were given intraperitoneal injections of NOD2-activated hUCB-MSCs; colon tissues and mesenteric lymph nodes were collected for histologic analyses. A bromodeoxyuridine assay was used to determine the ability of hUCB-MSCs to inhibit proliferation of human mononuclear cells in culture. Administration of hUCB-MSCs reduced the severity of colitis in mice. The anti-inflammatory effects of hUCB-MSCs were greatly increased by activation of NOD2 by its ligand, muramyl dipeptide (MDP). Administration of NOD2-activated hUCB-MSCs increased anti-inflammatory responses in colons of mice, such as production of interleukin (IL)-10 and infiltration by T regulatory cells, and reduced production of inflammatory cytokines. Proliferation of mononuclear cells was inhibited significantly by co-culture with hUCB-MSCs that had been stimulated with MDP. MDP induced prolonged production of prostaglandin (PG)E2 in hUCB-MSCs via the NOD2-RIP2 pathway, which suppressed proliferation of mononuclear cells derived from hUCB. PGE2 produced by hUCB-MSCs in response to MDP increased production of IL-10 and T regulatory cells. In mice, production of PGE2 by MSCs and subsequent production of IL-10 were required to reduce the severity of colitis. Activation of NOD2 is required for the ability of hUCB-MSCs to reduce the severity of colitis in mice. NOD2 signaling increases the ability of these cells to suppress mononuclear cell proliferation by inducing production of PGE2. Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.

T- and B-cell subpopulations in infectious mononucleosis

PubMed Central

Papamichail, M.; Sheldon, P. J.; Holborow, E. J.

1974-01-01

Mononuclear cells separated from the blood in fourteen cases of infectious mononucleosis at various intervals from the onset were tested for the presence of surface immunoglobulin and for ability to form spontaneous rosettes with washed sheep red blood cells. The mononucleosis during the acute phase of the illness consisted largely of a T lymphocytosis. The absolute count of T lymphocytes returned to the normal range approximately 2 months after the onset of the illness. B cells (bearing surface immunoglobulin) were only slightly increased in the acute phase. In four cases appreciable numbers of fluorescent rosetting cells were also present, and investigation suggested that these were T cells coated with anti-T-cell autoantibody. During the first 2 weeks of the illness responsiveness to phytohaemagglutinin was severely depressed, but thereafter returned towards normal. It is thought likely that in infectious mononucleosis the vast majority of atypical mononuclear cells are T cells proliferating in response to E-B virus-infected B cells, and cytotoxic towards them. ImagesFig. 3 PMID:4549622

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

PubMed

Hopper, Niina; Wardale, John; Brooks, Roger; Power, Jonathan; Rushton, Neil; Henson, Frances

2015-01-01

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

Human mononuclear cell function after 4 C storage during 1-G and microgravity conditions of spaceflight

NASA Technical Reports Server (NTRS)

Meehan, Richard; Taylor, Gerald; Lionetti, Fabian; Neale, Laurie; Curren, Tim

1989-01-01

To investigate the possibility of restoring immune competence of crewmembers during a prolonged spaceflight by infusions of autologous blood components, the effect of storage at 4 C aboard Space Shuttle Columbia (Mission 61-c) on the activity of human peripheral blood mononuclear cells (PBMNCs), stored as leukocyte concentrates in autologous plasa, was investigated. The results of preflight storage at 4 C demonstrated a progressive daily loss in mitogen-stimulated protein synthesis, and thymidine uptake, as well as a progressive reduction in the percentage of PBMNCs expressing cell-surface phenotype markers. The ability of PBMNCs stored at 4 C for 8 d in Columbia's middeck, to become activated and proliferate in vitro was similar to that of cells that remained for 7 d on ground.

CORRELATION OF A BIPHASIC METABOLIC RESPONSE WITH A BIPHASIC RESPONSE IN RESISTANCE TO TUBERCULOSIS IN RABBITS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Allison, M.J.; Zappasodi, P.; Lurie, M.B.

Peritoneal exudate mononuclear cells obtained from BCG-vaccinated rabbits showed higher utilization of succinate, glycerophosphate, beta - hydroxybutyrate, and glycerol than cells from control animals. No differences in utilization of the following substrates were noted: lactate, glucose-6-phosphate, malate, isocitrate, alpha -ketoglutarate, and glutamic acid. A second, later stage of elevated metabolic activity was associated with heightened resistance to infection. When rabbits which had been irradiated with 400 r 2 years previously were vaccinated with BCG, they failed to respond as shown by their lack of resistance to infection and failure of their mononuclear cells to show the biphasic metabolic stimulation. Themore » results demonstrate the close relation between the metabolic capabilities of reticuloendothelial cells and their resistance to tuberculosis. (H.H.D.)« less

HIV-1 isolation from infected peripheral blood mononuclear cells.

PubMed

Dispinseri, Stefania; Saba, Elisa; Vicenzi, Elisa; Kootstra, Neeltje A; Schuitemaker, Hanneke; Scarlatti, Gabriella

2014-01-01

Human immunodeficiency virus 1 (HIV-1) isolation from peripheral blood mononuclear cells (PBMCs) allows retrieval of replication-competent viral variants. In order to impose the smallest possible selective pressure on the viral isolates, isolation must be carried out in primary cultures of cells and not in tumor derived cell lines. The procedure involves culture of PBMCs from an infected patient with phytohemagglutinin (PHA)-stimulated PBMC from seronegative donors, which provide susceptible target cells for HIV replication. HIV can be isolated from the bulk population of PBMCs or after cloning of the cells to obtain viral biological clones. Viral production is determined with p24 antigen (Ag) detection assays or with reverse transcriptase (RT) activity assay. Once isolated, HIV-1 can be propagated by infecting PHA-stimulated PBMCs from healthy donors. Aliquots from culture with a high production of virus are stored for later use.

Circulating and Tissue-Resident CD4+ T Cells With Reactivity to Intestinal Microbiota Are Abundant in Healthy Individuals and Function Is Altered During Inflammation.

PubMed

Hegazy, Ahmed N; West, Nathaniel R; Stubbington, Michael J T; Wendt, Emily; Suijker, Kim I M; Datsi, Angeliki; This, Sebastien; Danne, Camille; Campion, Suzanne; Duncan, Sylvia H; Owens, Benjamin M J; Uhlig, Holm H; McMichael, Andrew; Bergthaler, Andreas; Teichmann, Sarah A; Keshav, Satish; Powrie, Fiona

2017-11-01

Interactions between commensal microbes and the immune system are tightly regulated and maintain intestinal homeostasis, but little is known about these interactions in humans. We investigated responses of human CD4 + T cells to the intestinal microbiota. We measured the abundance of T cells in circulation and intestinal tissues that respond to intestinal microbes and determined their clonal diversity. We also assessed their functional phenotypes and effects on intestinal resident cell populations, and studied alterations in microbe-reactive T cells in patients with chronic intestinal inflammation. We collected samples of peripheral blood mononuclear cells and intestinal tissues from healthy individuals (controls, n = 13-30) and patients with inflammatory bowel diseases (n = 119; 59 with ulcerative colitis and 60 with Crohn's disease). We used 2 independent assays (CD154 detection and carboxy-fluorescein succinimidyl ester dilution assays) and 9 intestinal bacterial species (Escherichia coli, Lactobacillus acidophilus, Bifidobacterium animalis subsp lactis, Faecalibacterium prausnitzii, Bacteroides vulgatus, Roseburia intestinalis, Ruminococcus obeum, Salmonella typhimurium, and Clostridium difficile) to quantify, expand, and characterize microbe-reactive CD4 + T cells. We sequenced T-cell receptor Vβ genes in expanded microbe-reactive T-cell lines to determine their clonal diversity. We examined the effects of microbe-reactive CD4 + T cells on intestinal stromal and epithelial cell lines. Cytokines, chemokines, and gene expression patterns were measured by flow cytometry and quantitative polymerase chain reaction. Circulating and gut-resident CD4 + T cells from controls responded to bacteria at frequencies of 40-4000 per million for each bacterial species tested. Microbiota-reactive CD4 + T cells were mainly of a memory phenotype, present in peripheral blood mononuclear cells and intestinal tissue, and had a diverse T-cell receptor Vβ repertoire. These cells were functionally heterogeneous, produced barrier-protective cytokines, and stimulated intestinal stromal and epithelial cells via interleukin 17A, interferon gamma, and tumor necrosis factor. In patients with inflammatory bowel diseases, microbiota-reactive CD4 + T cells were reduced in the blood compared with intestine; T-cell responses that we detected had an increased frequency of interleukin 17A production compared with responses of T cells from blood or intestinal tissues of controls. In an analysis of peripheral blood mononuclear cells and intestinal tissues from patients with inflammatory bowel diseases vs controls, we found that reactivity to intestinal bacteria is a normal property of the human CD4 + T-cell repertoire, and does not necessarily indicate disrupted interactions between immune cells and the commensal microbiota. T-cell responses to commensals might support intestinal homeostasis, by producing barrier-protective cytokines and providing a large pool of T cells that react to pathogens. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Resveratrol modulates apoptosis and oxidation in human blood mononuclear cells.

PubMed

Losa, G A

2003-09-01

We examined the effect of resveratrol (RS), a nonflavonoid polyphenolic phytoalexin found in grapes and red wine, and RS coincubated with the oxidant 2-deoxy-D-ribose (dR), on apoptosis and on the oxidative metabolic status of normal human peripheral blood mononuclear cells (PBMNCs) isolated ex vivo from healthy donors. Apoptosis was measured by changes of membrane permeability to propidium iodide (PI), plasma membrane exposure of phosphatidylserine (PS) and intracellular caspase activity. Oxidative status was assessed by recording the intracellular glutathione concentration (GSH), the activities of the enzymes y-glutamyltransferase (y-GT) and glutathione-S-transferase (GST), and intracellular lipid peroxidation (MDA). Neither apoptotic nor oxidative parameters were affected by culturing PBMNCs in medium containing RS up to 20 micro M for 5 days, while the frequency of cells with intermediate permeability to PI (17% +/- 5) increased at 50 micro M of RS. Thus resveratrol was slightly toxic, but there was little apoptosis in these cells. Peripheral blood mononuclear cells were also grown first in medium plus RS for 24 h and then for 96 h in medium containing RS plus 10 mM of dR, an oxidant sugar that is apoptogenic for human lymphocytes. The apoptotic changes triggered by dR were counteracted by the phytoalexin in a dose-dependent manner, but RS activity was absent at the lowest concentration (5 micro M) and significantly reduced at the highest concentration used (50 micro M). In PBMNCs coincubated with 20 micro M of RS and 10 mM of dR the antioxidant effect of RS manifested with a significant reduction of caspases-3, -8, y-GT, GST activities and MDA content. Peripheral blood mononuclear cells acquire antioxidant capacity when treated with RS. Grape resveratrol may make a useful dietary supplement for minimizing oxidative injury in immune-perturbed states and human chronic degenerative diseases.

Anti-fibrotic potential of human umbilical cord mononuclear cells and mouse bone marrow cells in CCl4- induced liver fibrosis in mice.

PubMed

Elmahdy, Nageh Ahmed; Sokar, Samia Salem; Salem, Mohamed Labib; Sarhan, Naglaa Ibrahim; Abou-Elela, Sherin Hamed

2017-05-01

Liver fibrosis is the consequence of hepatocyte injury that leads to the activation of hepatic stellate cells (HSC). The treatment of choice is Liver transplantation; however, it has many problems such as surgery-related complications, immunological rejection and high costs associated with the procedure. Stem cell-based therapy would be a potential alternative, so the aim of this study is to investigate the therapeutic potential of human umbilical cord mononuclear cells (MNC) and mouse bone marrow cells (BMC) against carbon tetrachloride (CCl 4 ) induced liver fibrosis in mice and compare it with that of silymarin. In the present study, male albino mice (N=60) were divided into six groups (10 mice each), the first group served as the normal control group while the remaining five groups were rendered fibrotic by intraperitoneal injections of CCl 4 and being left for 6 weeks to develop hepatic fibrosis. Thereafter, the mice were divided into CCl 4 group, CCl 4 group receiving MNC or BMC or silymarin or MNC and silymarin combination. After the specified treatment period, animals were then euthanized, blood and tissue samples were collected for measurement of alanine aminotransferase(ALT), aspartate aminotransferase(AST), malondialdehyde(MDA), reduced glutathione(GSH), collagen, Laminin, transforming growth factor β1(TGFβ1), tumor necrosis factor alpha(TNFα). MNC, BMC, and the combination therapy showed a significant decrease in ALT, AST, MDA, collagen, Laminin, TGFβ1, and TNFα and a significant increase in GSH. The data displayed a similar regression of fibrosis with the histological and immunohistological parameters. In conclusion, MNC, BMC and the combination therapy showed a potential therapeutic effect against liver fibrosis via reducing oxidative stress, inflammatory mediators, and fibrogenic markers. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Characterization of bone marrow mononuclear cells on biomaterials for bone tissue engineering in vitro.

PubMed

Henrich, Dirk; Verboket, René; Schaible, Alexander; Kontradowitz, Kerstin; Oppermann, Elsie; Brune, Jan C; Nau, Christoph; Meier, Simon; Bonig, Halvard; Marzi, Ingo; Seebach, Caroline

2015-01-01

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

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

PubMed Central

Verboket, René; Kontradowitz, Kerstin; Oppermann, Elsie; Brune, Jan C.; Nau, Christoph; Meier, Simon; Bonig, Halvard; Marzi, Ingo; Seebach, Caroline

2015-01-01

Bone marrow mononuclear cells (BMCs) are suitable for bone tissue engineering. Comparative data regarding the needs of BMC for the adhesion on biomaterials and biocompatibility to various biomaterials are lacking to a large extent. Therefore, we evaluated whether a surface coating would enhance BMC adhesion and analyze the biocompatibility of three different kinds of biomaterials. BMCs were purified from human bone marrow aspirate samples. Beta tricalcium phosphate (β-TCP, without coating or coated with fibronectin or human plasma), demineralized bone matrix (DBM), and bovine cancellous bone (BS) were assessed. Seeding efficacy on β-TCP was 95% regardless of the surface coating. BMC demonstrated a significantly increased initial adhesion on DBM and β-TCP compared to BS. On day 14, metabolic activity was significantly increased in BMC seeded on DBM in comparison to BMC seeded on BS. Likewise increased VEGF-synthesis was observed on day 2 in BMC seeded on DBM when compared to BMC seeded on BS. The seeding efficacy of BMC on uncoated biomaterials is generally high although there are differences between these biomaterials. Beta-TCP and DBM were similar and both superior to BS, suggesting either as suitable materials for spatial restriction of BMC used for regenerative medicine purposes in vivo. PMID:25802865

CD8+ T Cells Provide an Immunologic Signature of Tuberculosis in Young Children

PubMed Central

Nyendak, Melissa; Kiguli, Sarah; Zalwango, Sarah; Mori, Tomi; Mayanja-Kizza, Harriet; Balyejusa, Stephen; Null, Megan; Baseke, Joy; Mulindwa, Deo; Byrd, Laura; Swarbrick, Gwendolyn; Scott, Christine; Johnson, Denise F.; Malone, LaShaunda; Mudido-Musoke, Philipa; Boom, W. Henry; Lewinsohn, David M.; Lewinsohn, Deborah A.

2012-01-01

Rationale: The immunologic events surrounding primary Mycobacterium tuberculosis infection and development of tuberculosis remain controversial. Young children who develop tuberculosis do so quickly after first exposure, thus permitting study of immune response to primary infection and disease. We hypothesized that M. tuberculosis–specific CD8+ T cells are generated in response to high bacillary loads occurring during tuberculosis. Objectives: To determine if M. tuberculosis–specific T cells are generated among healthy children exposed to M. tuberculosis and children with tuberculosis. Methods: Enzyme-linked immunosorbent spot assays were used to measure IFN-γ production in response to M. tuberculosis–specific proteins ESAT-6/CFP-10 by peripheral blood mononuclear cells and CD8+ T cells isolated from Ugandan children hospitalized with tuberculosis (n = 96) or healthy tuberculosis contacts (n = 62). Measurements and Main Results: The proportion of positive CD8+ T-cell assays and magnitude of CD8+ T-cell responses were significantly greater among young (<5 yr) tuberculosis cases compared with young contacts (P = 0.02, Fisher exact test, P = 0.01, Wilcoxon rank-sum, respectively). M. tuberculosis–specific T-cell responses measured in peripheral blood mononuclear cells were equivalent between groups. Conclusions: Among young children, M. tuberculosis–specific CD8+ T cells develop in response to high bacillary loads, as occurs during tuberculosis, and are unlikely to be found after M. tuberculosis exposure. T-cell responses measured in peripheral blood mononuclear cells are generated after M. tuberculosis exposure alone, and thus cannot distinguish exposure from disease. In young children, IFN-γ–producing M. tuberculosis–specific CD8+ T cells provide an immunologic signature of primary M. tuberculosis infection resulting in disease. PMID:22071329

Renal dendritic cells sample blood-borne antigen and guide T-cell migration to the kidney by means of intravascular processes.

PubMed

Yatim, Karim M; Gosto, Minja; Humar, Rishab; Williams, Amanda L; Oberbarnscheidt, Martin H

2016-10-01

Bony fish are among the first vertebrates to possess an innate and adaptive immune system. In these species, the kidney has a dual function: filtering solutes similar to mammals and acting as a lymphoid organ responsible for hematopoiesis and antigen processing. Recent studies have shown that the mammalian kidney has an extensive network of mononuclear phagocytes, whose function is not fully understood. Here, we employed two-photon intravital microscopy of fluorescent reporter mice to demonstrate that renal dendritic cells encase the microvasculature in the cortex, extend dendrites into the peritubular capillaries, and sample the blood for antigen. We utilized a mouse model of systemic bacterial infection as well as immune complexes to demonstrate antigen uptake by renal dendritic cells. As a consequence, renal dendritic cells mediated T-cell migration into the kidney in an antigen-dependent manner in the setting of bacterial infection. Thus, renal dendritic cells may be uniquely positioned to play an important role not only in surveillance of systemic infection but also in local infection and autoimmunity. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

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

PubMed Central

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

Identification of Highly Methylated Genes across Various Types of B-Cell Non-Hodgkin Lymphoma

PubMed Central

Bethge, Nicole; Honne, Hilde; Hilden, Vera; Trøen, Gunhild; Eknæs, Mette; Liestøl, Knut; Holte, Harald; Delabie, Jan; Smeland, Erlend B.; Lind, Guro E.

2013-01-01

Epigenetic alterations of gene expression are important in the development of cancer. In this study, we identified genes which are epigenetically altered in major lymphoma types. We used DNA microarray technology to assess changes in gene expression after treatment of 11 lymphoma cell lines with epigenetic drugs. We identified 233 genes with upregulated expression in treated cell lines and with downregulated expression in B-cell lymphoma patient samples (n = 480) when compared to normal B cells (n = 5). The top 30 genes were further analyzed by methylation specific PCR (MSP) in 18 lymphoma cell lines. Seven of the genes were methylated in more than 70% of the cell lines and were further subjected to quantitative MSP in 37 B-cell lymphoma patient samples (diffuse large B-cell lymphoma (activated B-cell like and germinal center B-cell like subtypes), follicular lymphoma and Burkitt`s lymphoma) and normal B lymphocytes from 10 healthy donors. The promoters of DSP, FZD8, KCNH2, and PPP1R14A were methylated in 28%, 67%, 22%, and 78% of the 36 tumor samples, respectively, but not in control samples. Validation using a second series of healthy donor controls (n = 42; normal B cells, peripheral blood mononuclear cells, bone marrow, tonsils and follicular hyperplasia) and fresh-frozen lymphoma biopsies (n = 25), confirmed the results. The DNA methylation biomarker panel consisting of DSP, FZD8, KCNH2, and PPP1R14A was positive in 89% (54/61) of all lymphomas. Receiver operating characteristic analysis to determine the discriminative power between lymphoma and healthy control samples showed a c-statistic of 0.96, indicating a possible role for the biomarker panel in monitoring of lymphoma patients. PMID:24260260

Assessment of Normal Variability in Peripheral Blood Gene Expression

DOE PAGES

Campbell, Catherine; Vernon, Suzanne D.; Karem, Kevin L.; ...

2002-01-01

Peripheral blood is representative of many systemic processes and is an ideal sample for expression profiling of diseases that have no known or accessible lesion. Peripheral blood is a complex mixture of cell types and some differences in peripheral blood gene expression may reflect the timing of sample collection rather than an underlying disease process. For this reason, it is important to assess study design factors that may cause variability in gene expression not related to what is being analyzed. Variation in the gene expression of circulating peripheral blood mononuclear cells (PBMCs) from three healthy volunteers sampled three times onemore » day each week for one month was examined for 1,176 genes printed on filter arrays. Less than 1% of the genes showed any variation in expression that was related to the time of collection, and none of the changes were noted in more than one individual. These results suggest that observed variation was due to experimental variability.« less

Patient-specific 3D microfluidic tissue model for multiple myeloma.

PubMed

Zhang, Wenting; Lee, Woo Y; Siegel, David S; Tolias, Peter; Zilberberg, Jenny

2014-08-01

In vitro culturing of primary multiple myeloma cells (MMC) has been a major challenge as this plasma cell malignancy depends on the bone marrow environment for its survival. Using a microfluidic platform to emulate the dynamic physiology of the bone marrow microenvironment, we report here a new approach for culturing difficult to preserve primary human MMC. The system uses a three-dimensional ossified tissue to mimic the tumor niche and recapitulate interactions between bone marrow cells and osteoblasts (OSB). To this end, the human fetal OSB cell line hFOB 1.19 was cultured in an eight-chamber microfluidic culture device to facilitate the seeding of mononuclear cells from bone marrow aspirates from three multiple myeloma patients. Optical microscopy, used for real-time monitoring of mononuclear cell interactions with the ossified tissue, confirmed that these are drawn toward the OSB layer. After 3 weeks, cocultures were characterized by flow cytometry to evaluate the amount of expansion of primary MMC (with CD138(+) and CD38(+)CD56(+) phenotypes) in this system. For each of the three patients analyzed, bone marrow mononuclear cells underwent, on an average, 2 to 5 expansions; CD38(+)CD56(+) cells underwent 1 to 3 expansions and CD138(+) cells underwent 2.5 to 4.6 expansions. This approach is expected to provide a new avenue that can facilitate: (1) testing of personalized therapeutics for multiple myeloma patients; (2) evaluation of new drugs without the need for costly animal models; and (3) studying the biology of multiple myeloma, and in particular, the mechanisms responsible for drug resistance and relapse.

Contrasting Responses of Kupffer Cells and Inflammatory Mononuclear Phagocytes to Biliary Obstruction in a Mouse Model of Cholestatic Liver Injury

PubMed Central

Duwaerts, Caroline C.; Gehring, Stephan; Cheng, Chao-Wen; van Rooijen, Nico; Gregory, Stephen H.

2012-01-01

Background Biliary obstruction and cholestasis are serious complications of many liver diseases. While resident hepatic macrophages (Kupffer cells) are frequently implicated in disease progression, most studies fail to differentiate the contribution of Kupffer cells and inflammatory mononuclear phagocytes (iMNPs) that infiltrate the liver subsequent to obstruction. Aim This study was undertaken to examine the roles and potential interactions of these two disparate mononuclear phagocyte populations in hepatic injury attending cholestasis. Methods Female, C57Bl/6 mice were injected with magnetic beads on day three prior to sham operation or bile duct ligation (BDL) in order to facilitate subsequent Kupffer cell isolation. Three days post surgery, animals were euthanized, and bead-containing Kupffer cells and iMNPs were separated, purified, and analyzed. To examine the ability of Kupffer cells to modulate iMNP activity, iMNPs were isolated from the livers of intact and Kupffer cell-depleted mice on day 3 post-surgery and compared. Results Purified Kupffer cells and iMNP populations obtained from BDL mice exhibited heterogeneous morphologies rendering them visually indistinguishable. iMNPs, however, were characterized by the increased expression of Ly-6C and CD11b and the elevated production of chemokines/cytokines characteristic of inflammatory cells. In the absence of Kupffer cells, iMNPs immigrating to the liver following BDL exhibited significant decreases in CD11b and Ly-6C expression, and in pro-inflammatory chemokine/cytokine production. Conclusions Kupffer cells and iMNPs exhibit disparate biological responses to biliary obstruction and cholestasis. Kupffer cells play a key role in regulating iMNP influx and activity. PMID:23240869

A Hydrogel-Endothelial Cell implant Mimics Infantile Hemangioma: Modulation by Survivin and the Hippo pathway*

PubMed Central

Tsuneki, Masayuki; Hardee, Steven; Michaud, Michael; Morotti, Raffaella; Lavik, Erin; Madri, Joseph A.

2015-01-01

Microvascular endothelial cells cultured in three-dimensional hydrogel scaffolds form a network of microvessel structures when implanted subcutaneously in mice, inosculate with host vessels and over time remodel into large ectatic vascular structures resembling hemangiomas. When compared to infantile hemaniomas similarities were noted including a temporal progression from a morphological appearance of a proliferative phase to the appearance of an involuted phase mimicking the proliferative and involutional phases of infantile hemangioma. Consistent with the progression of a proliferative phase to an involuted phase, both the murine implants and human biopsy tissue exhibit reduced expression of Ajuba, YAP and Survivin labeling as they progressed over time. Significant numbers of CD45+, CD11b+, Mac3+ mononuclear cells were found at the 2 week time point in our implant model which correlated with the presence of CD45+, CD68+ mononuclear cells observed in biopsies of human proliferative phase hemangiomas. At the 4 week time point in our implant model only small numbers of CD45+ cells were detected, which again correlated with our findings of significantly diminished CD45+, CD68+ mononuclear cells in human involutional phase hemangiomas. The demonstration of mononuclear cell infiltration transiently in the proliferative phase of these lesions suggests that the vascular proliferation and/or regression may be driven in part by an immune response. Gross and microscopic morphological appearances of human proliferative and involutional hemangiomas and our implant model correlate well with each other as do the expression levels of Hippo pathway components (Ajuba and YAP) and Survivin and correlate with proliferation in these entities. Inhibitors of Survivin and Ajuba (which we have demonstrated to inhibit proliferation and increase apoptosis in murine hemangioma cell tissue culture) may have potential as other beneficial treatments for proliferating infantile hemangiomas. This implant model may have potential as a modest through-put screen for testing and development of therapeutics targeted at the proliferative phase of infantile hemangiomas, reducing the subsequent post-involutional scarring sometimes associated with these lesions. PMID:25961170

Expression of IFN-Inducible Genes with Antiviral Function OAS1 and MX1 in Health and under Conditions of Recurrent Herpes Simplex Infection.

PubMed

Karaulov, A V; Shulzhenko, A E; Karsonova, A V

2017-07-01

We studied the expression of IFN-inducible genes OAS1 and Mx1 in lysates of peripheral blood mononuclear cells from patients suffering from recurrent Herpes simplex infections in comparison with healthy people. To induce the expression of the studied genes, blood mononuclears were incubated with recombinant IFN-α2b in concentrations of 1, 10, and 100 U/ml for 3 h and then the content of the studied transcripts was evaluated. Relative expression of OAS1 and Mx1 in patients with recurrent forms of Herpes simplex both during the acute stage and clinical remission did not differ significantly from that in healthy people after stimulation with IFN-α2b in a concentration of 1 U/ml and in higher concentrations (10 and 100 U/ml). It was concluded that intracellular signal transduction in IFN-α-activated cells in vitro was not disturbed in patients with recurrent forms of Herpes simplex infection. Thus, the reported phenomenon of IFN-signalling distortion by Herpes simplex virus proteins observed in experiments on model cell lines infected with Herpes simplex virus was not confirmed in our experiments on peripheral blood mononuclear cells from patients with Herpes simplex infection.

[Analysis of characteristics of mononuclear cells remaining in the leukoreduction system chamber of Trima Accel and their differentiation into dendritic cells].

PubMed

Lee, Yangsoon; Kim, Sinyoung; Lee, Seung-Tae; Kim, Han-Soo; Baek, Eun-Jung; Kim, Hyung Jin; Lee, MeeKyung; Kim, Hyun Ok

2009-08-01

We investigated the characteristics of the mononuclear cells remaining in the leukoreduction system (LRS) chambers of Trima Accel in comparison with those of standard buffy coat cells, and evaluated their potential for differentiation into dendritic cells. Twenty-six LRS chambers of Trima Accel were collected after platelet pheresis from healthy adults. Flow cytometric analysis for T, B, NK, and CD14+ cells was performed and the number of CD34+ cells was counted. Differentiation and maturation into dendritic cells were induced using CD14+ cells seperated via Magnetic cell sorting (MACS) Seperation (Miltenyi Biotec Inc., USA). Total white blood cell (WBC) count in LRS chambers was 10.8 x 10(8) (range 7.7-18.0 x 10(8)). The median values (range) of proportions of each cells were CD4+ T cell 29.6% (18.7-37.6), CD8+ T cell 27.7% (19.2-40.0), B cell 5.5% (2.2-12.1), NK cell 15.7% (13.7-19.9), and CD14+ cells 12.4% (8.6-32.3) respectively. Although total WBC count was significantly higher in the buffy coat (whole blood of 400 mL) than the LRS chambers, the numbers of lymphocytes and monocytes were not statistically different. The numbers of B cells and CD4+ cells were significantly higher in the buffy coat than the LRS chambers (P<0.05). The median value (range) of CD34+ cells obtained from the LRS chambers was 0.9 x 10(6) (0.2-2.6 x 10(6)). After 7 days of cytokine-supplemented culture, the CD14+ cells were successfully differentiated into dendritic cells. The mononuclear cells in LRS chambers of Trima Accel are an excellent alternative source of viable and functional human blood cells, which can be used for research purposes.

Immune response CC chemokines CCL2 and CCL5 are associated with pulmonary sarcoidosis

PubMed Central

2011-01-01

Background Pulmonary sarcoidosis involves an intense leukocyte infiltration of the lung with the formation of non-necrotizing granulomas. CC chemokines (chemokine (C-C motif) ligand 2 (CCL2)-CCL5) are chemoattractants of mononuclear cells and act through seven transmembrane G-coupled receptors. Previous studies have demonstrated conflicting results with regard to the associations of these chemokines with sarcoidosis. In an effort to clarify previous discrepancies, we performed the largest observational study to date of CC chemokines in bronchoalveolar lavage fluid (BALF) from patients with pulmonary sarcoidosis. Results BALF chemokine levels from 72 patients affected by pulmonary sarcoidosis were analyzed by enzyme-linked immunosorbent assay (ELISA) and compared to 8 healthy volunteers. BALF CCL3 and CCL4 levels from pulmonary sarcoidosis patients were not increased compared to controls. However, CCL2 and CCL5 levels were elevated, and subgroup analysis showed higher levels of both chemokines in all stages of pulmonary sarcoidosis. CCL2, CCL5, CC chemokine receptor type 1 (CCR1), CCR2 and CCR3 were expressed from mononuclear cells forming the lung granulomas, while CCR5 was only found on mast cells. Conclusions These data suggest that CCL2 and CCL5 are important mediators in recruiting CCR1, CCR2, and CCR3 expressing mononuclear cells as well as CCR5-expressing mast cells during all stages of pulmonary sarcoidosis. PMID:21463523

Immune response CC chemokines CCL2 and CCL5 are associated with pulmonary sarcoidosis.

PubMed

Palchevskiy, Vyacheslav; Hashemi, Nastran; Weigt, Stephen S; Xue, Ying Ying; Derhovanessian, Ariss; Keane, Michael P; Strieter, Robert M; Fishbein, Michael C; Deng, Jane C; Lynch, Joseph P; Elashoff, Robert; Belperio, John A

2011-04-04

Pulmonary sarcoidosis involves an intense leukocyte infiltration of the lung with the formation of non-necrotizing granulomas. CC chemokines (chemokine (C-C motif) ligand 2 (CCL2)-CCL5) are chemoattractants of mononuclear cells and act through seven transmembrane G-coupled receptors. Previous studies have demonstrated conflicting results with regard to the associations of these chemokines with sarcoidosis. In an effort to clarify previous discrepancies, we performed the largest observational study to date of CC chemokines in bronchoalveolar lavage fluid (BALF) from patients with pulmonary sarcoidosis. BALF chemokine levels from 72 patients affected by pulmonary sarcoidosis were analyzed by enzyme-linked immunosorbent assay (ELISA) and compared to 8 healthy volunteers. BALF CCL3 and CCL4 levels from pulmonary sarcoidosis patients were not increased compared to controls. However, CCL2 and CCL5 levels were elevated, and subgroup analysis showed higher levels of both chemokines in all stages of pulmonary sarcoidosis. CCL2, CCL5, CC chemokine receptor type 1 (CCR1), CCR2 and CCR3 were expressed from mononuclear cells forming the lung granulomas, while CCR5 was only found on mast cells. These data suggest that CCL2 and CCL5 are important mediators in recruiting CCR1, CCR2, and CCR3 expressing mononuclear cells as well as CCR5-expressing mast cells during all stages of pulmonary sarcoidosis.

Concurrent detection of secreted products from human lymphocytes by microengraving: cytokines and antigen-reactive antibodies

PubMed Central

Bradshaw, Elizabeth M.; Kent, Sally C.; Tripuraneni, Vinay; Orban, Tihamer; Ploegh, Hidde L.; Hafler, David A.; Love, J. Christopher

2008-01-01

Cell surface determinants, cytokines and antibodies secreted by hematopoietic cells are used to classify their lineage and function. Currently available techniques are unable to elucidate multiple secreted proteins while also assigning phenotypic surface-displayed markers to the individual living cells. Here, a soft lithographic method, microengraving, was adapted for the multiplexed interrogation of populations of individual human peripheral blood mononuclear cells for secreted cytokines (IFN-γ and IL-6), antigen-specific antibodies, and lineage-specific surface-expressed markers. Application of the method to a clinical sample from a recent onset Type 1 diabetic subject with a positive titer of anti-insulin antibodies showed that ~0.58% of circulating CD19+ B cells secreted proinsulin-reactive antibodies of the IgG isotype and 2–3% of circulating cells secreted IL-6. These data demonstrate the utility of microengraving for interrogating multiple phenotypes of single human cells concurrently and for detecting rare populations of cells by their secreted products. PMID:18675591

Dehydroleucodine, a Sesquiterpene Lactone from Gynoxys verrucosa, Demonstrates Cytotoxic Activity against Human Leukemia Cells.

PubMed

Ordóñez, Paola E; Sharma, Krishan K; Bystrom, Laura M; Alas, Maria A; Enriquez, Raul G; Malagón, Omar; Jones, Darin E; Guzman, Monica L; Compadre, Cesar M

2016-04-22

The sesquiterpene lactones dehydroleucodine (1) and leucodine (2) were isolated from Gynoxys verrucosa, a species used in traditional medicine in southern Ecuador. The activity of these compounds was determined against eight acute myeloid leukemia (AML) cell lines and compared with their activity against normal peripheral blood mononuclear cells. Compound 1 showed cytotoxic activity against the tested cell lines, with LD50 values between 5.0 and 18.9 μM. Compound 2 was inactive against all of the tested cell lines, demonstrating that the exocyclic methylene in the lactone ring is required for cytotoxic activity. Importantly, compound 1 induced less toxicity to normal blood cells than to AML cell lines and was active against human AML cell samples from five patients, with an average LD50 of 9.4 μM. Mechanistic assays suggest that compound 1 has a similar mechanism of action to parthenolide (3). Although these compounds have significant structural differences, their lipophilic surface signatures show striking similarities.

Optimizing cord blood sample cryopreservation.

PubMed

Harris, David T

2012-03-01

Cord blood (CB) banking is becoming more and more commonplace throughout the medical community, both in the USA and elsewhere. It is now generally recognized that storage of CB samples in multiple aliquots is the preferred approach to banking because it allows the greatest number of uses of the sample. However, it is unclear which are the best methodologies for cryopreservation and storage of the sample aliquots. In the current study we analyzed variables that could affect these processes. CB were processed into mononuclear cells (MNC) and frozen in commercially available human serum albumin (HSA) or autologous CB plasma using cryovials of various sizes and cryobags. The bacteriophage phiX174 was used as a model virus to test for cross-contamination. We observed that cryopreservation of CB in HSA, undiluted autologous human plasma and 50% diluted plasma was equivalent in terms of cell recovery and cell viability. We also found that cryopreservation of CB samples in either cryovials or cryobags displayed equivalent thermal characteristics. Finally, we demonstrated that overwrapping the CB storage container in an impermeable plastic sheathing was sufficient to prevent cross-sample viral contamination during prolonged storage in the liquid phase of liquid nitrogen dewar storage. CB may be cryopreserved in either vials or bags without concern for temperature stability. Sample overwrapping is sufficient to prevent microbiologic contamination of the samples while in liquid-phase liquid nitrogen storage.

In Vivo Hypermutation of Xenotropic Murine Leukemia Virus-Related Virus DNA in Peripheral Blood Mononuclear Cells of Rhesus Macaque by APOBEC3 Proteins

PubMed Central

Zhang, Ao; Bogerd, Hal; Villinger, Francois; Gupta, Jaydip Das; Dong, Beihua; Klein, Eric A.; Hackett, John; Schochetman, Gerald; Cullen, Bryan R.; Silverman, Robert H.

2011-01-01

The gammaretrovirus, xenotropic murine leukemia virus-related virus (XMRV), replicates to high titers in some human cell lines and is able to infect non-human primates. To determine whether APOBEC3 (A3) proteins restrict XMRV infections in a non-human primate model, we sequenced proviral DNA from peripheral blood mononuclear cells of XMRV-infected rhesus macaques. Hypermutation characteristic of A3DE, A3F and A3G activities was observed in the XMRV proviral sequences in vivo. Furthermore, expression of rhesus A3DE, A3F, or A3G in human cells inhibited XMRV infection and caused hypermutation of XMRV DNA. These studies show that some rhesus A3 isoforms are highly effective against XMRV in the blood of a non-human primate model of infection and in cultured human cells. PMID:21982221

Involvement of blood mononuclear cells in the infertility, age-associated diseases and cancer treatment

PubMed Central

Bukovsky, Antonin

2016-01-01

Blood mononuclear cells consist of T cells and monocyte derived cells. Beside immunity, the blood mononuclear cells belong to the complex tissue control system (TCS), where they exhibit morphostatic function by stimulating proliferation of tissue stem cells followed by cellular differentiation, that is stopped after attaining the proper functional stage, which differs among various tissue types. Therefore, the term immune and morphostatic system (IMS) should be implied. The TCS-mediated morphostasis also consists of vascular pericytes controlled by autonomic innervation, which is regulating the quantity of distinct tissues in vivo. Lack of proper differentiation of tissue cells by TCS causes either tissue underdevelopment, e.g., muscular dystrophy, or degenerative functional failures, e.g., type 1 diabetes and age-associated diseases. With the gradual IMS regression after 35 years of age the gonadal infertility develops, followed by a growing incidence of age-associated diseases and cancers. Without restoring an altered TCS function in a degenerative disease, the implantation of tissue-specific stem cells alone by regenerative medicine can not be successful. Transfused young blood could temporarily restore fertility to enable parenthood. The young blood could also temporarily alleviate aging diseases, and this can be extended by substances inducing IMS regeneration, like the honey bee propolis. The local and/or systemic use of honey bee propolis stopped hair and teeth loss, regressed varicose veins, improved altered hearing, and lowered high blood pressure and sugar levels. Complete regression of stage IV ovarian cancer with liver metastases after a simple elaborated immunotherapy is also reported. PMID:28074124

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

PubMed Central

Alestalo, Kirsi; Miettinen, Johanna A.; Vuolteenaho, Olli; Huikuri, Heikki; Lehenkari, Petri

2015-01-01

Background Acute myocardial infarction (AMI) launches an inflammatory response and a repair process to compensate cardiac function. During this process, the balance between proinflammatory and anti-inflammatory cytokines is important for optimal cardiac repair. Stem cell transplantation after AMI improves tissue repair and increases the ventricular ejection fraction. Here, we studied in detail the acute effect of bone marrow mononuclear cell (BMMNC) transplantation on proinflammatory and anti-inflammatory cytokines in patients with ST segment elevation myocardial infarction (STEMI). Methods Patients with STEMI treated with thrombolysis followed by percutaneous coronary intervention (PCI) were randomly assigned to receive either BMMNC or saline as an intracoronary injection. Cardiac function was evaluated by left ventricle angiogram during the PCI and again after 6 months. The concentrations of 27 cytokines were measured from plasma samples up to 4 days after the PCI and the intracoronary injection. Results Twenty-six patients (control group, n = 12; BMMNC group, n = 14) from the previously reported FINCELL study (n = 80) were included to this study. At day 2, the change in the proinflammatory cytokines correlated with the change in the anti-inflammatory cytokines in both groups (Kendall’s tau, control 0.6; BMMNC 0.7). At day 4, the correlation had completely disappeared in the control group but was preserved in the BMMNC group (Kendall’s tau, control 0.3; BMMNC 0.7). Conclusions BMMNC transplantation is associated with preserved balance between pro- and anti-inflammatory cytokines after STEMI in PCI-treated patients. This may partly explain the favorable effect of stem cell transplantation after AMI. PMID:26690350

Male rats develop more severe experimental autoimmune encephalomyelitis than female rats: sexual dimorphism and diergism at the spinal cord level.

PubMed

Nacka-Aleksić, Mirjana; Djikić, Jasmina; Pilipović, Ivan; Stojić-Vukanić, Zorica; Kosec, Duško; Bufan, Biljana; Arsenović-Ranin, Nevena; Dimitrijević, Mirjana; Leposavić, Gordana

2015-10-01

Compared with females, male Dark Agouti (DA) rats immunized for experimental autoimmune encephalomyelitis (EAE) with rat spinal cord homogenate in complete Freund's adjuvant (CFA) exhibited lower incidence of the disease, but the maximal neurological deficit was greater in the animals that developed the disease. Consistently, at the peak of the disease greater number of reactivated CD4+CD134+CD45RC- T lymphocytes was retrieved from male rat spinal cord. Their microglia/macrophages were more activated and produced greater amount of prototypic proinflammatory cytokines in vitro. Additionally, oppositely to the expression of mRNAs for IL-12/p35, IL-10 and IL-27/p28, the expression of mRNA for IL-23/p19 was upregulated in male rat spinal cord mononuclear cells. Consequently, the IL-17+:IFN-γ+ cell ratio within T lymphocytes from their spinal cord was skewed towards IL-17+ cells. Within this subpopulation, the IL-17+IFN-γ+:IL-17+IL-10+ cell ratio was shifted towards IL-17+IFN-γ+ cells, which have prominent tissue damaging capacity. This was associated with an upregulated expression of mRNAs for IL-1β and IL-6, but downregulated TGF-β mRNA expression in male rat spinal cord mononuclear cells. The enhanced GM-CSF mRNA expression in these cells supported the greater pathogenicity of IL-17+ T lymphocytes infiltrating male spinal cord. In the inductive phase of the disease, contrary to the draining lymph node, in the spinal cord the frequency of CD134+ cells among CD4+ T lymphocytes and the frequency of IL-17+ cells among T lymphocytes were greater in male than in female rats. This most likely reflected an enhanced transmigration of mononuclear cells into the spinal cord (judging by the lesser spinal cord CXCL12 mRNA expression), the greater frequency of activated microglia/macrophages and the increased expression of mRNAs for Th17 polarizing cytokines in male rat spinal cord mononuclear cells. Collectively, the results showed cellular and molecular mechanisms underlying the target organ specific sexual dimorphism in the T lymphocyte-dependent immune/inflammatory response, and suggested a substantial role for the target organ in shaping the sexually dimorphic clinical outcome of EAE. Copyright © 2015 Elsevier Inc. All rights reserved.

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

PubMed

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.

Regulation of interleukin-1beta and interleukin-8 production by agonists of mu and delta opiate receptors in vitro.

PubMed

Gein, S V; Gorshkova, K G; Tendryakova, S P

2009-07-01

The studies reported here showed that beta-endorphin at concentrations of 10(-7)-10(-11) M increased interleukin-1beta (IL-1beta) production in unfractionated leukocyte suspensions both in the presence of 0.1 microg/ml lipopolysaccharide (LPS) and in cultures not stimulated with LPS. Interleukin-8 (IL-8) production by leukocytes was inhibited by beta-endorphin at concentrations of 10(-7) and 10(-11) M in the presence of LPS. The stimulatory effect of beta-endorphin on IL-1beta production was not blocked by naloxone or naltrindole. Suppression of IL-8 production was blocked by naloxone and naltrindole. In the mononuclear cell and neutrophil fractions, beta-endorphin and the delta agonist DADLE increased IL-1beta synthesis in both the spontaneous and stimulated versions of the test, while beta-endorphin and the delta agonist DADLE inhibited IL-8 production in the mononuclear cell and neutrophil fractions only in LPS-stimulated cultures. The mu agonist DAGO had no effect on IL-1beta production by mononuclear cells or neutrophils, though it suppressed LPS-induced secretion of IL-8 by neutrophils.

Mononuclear cell membranes: stabilization by reproterol and cromoglycate, destabilization by fenoterol and salbutamol.

PubMed

Zimmer, Guido; Bernhörster, Markus; Pilz, Patrizius; Schuchmann-Fix, Jutta; Hüggelmeier, Rolf; Blüm, Nicole; Libertus, Herman

2006-01-01

Electron paramagnetic resonance (EPR) spectroscopy with spin labels 5- and 16-doxyl-stearic acid (DSA) was used to differentiate between actions of beta-agonists on human mononuclear cell membrane. Reproterol (CAS 13055-82-8), salbutamol (CAS 51022-70-9) and fenoterol (CAS 1944-12-3) compared to cromoglycate (CAS 15826-37-6) were used at concentrations of 10-100 nmol/l per 10(7) cells. With reproterol, order and polarity was not much changed, whereas salbutamol and fenoterol significantly destabilized the membrane to similar extent. Cromoglycate acted in a stabilizing fashion. With trypan blue exclusion, reproterol and cromoglycate showed stable values, whereas salbutamol and fenoterol augmented permeability. Thus, by conventional lipid spin labeling the discrimination between salbutamol and fenoterol could not be carried out. In contrast, previous lipid peroxidation studies in a model system had revealed a decrease by reproterol, no change by salbutamol and an increase by fenoterol. Also, using fenoterol, protein spin label 4-maleimido-TEMPO (2, 2, 6, 6-tetramethyl-1-piperidinyloxy) showed an increase of membrane rigidity of mononuclear cells. Moreover, mast cells of different origin were previously found tween beta-agonists. Reproterol in all tests behaved in a therapeutically profitable way. In conclusion, in addition to lipid spin labeling other methods and materials should be considered, to finally arrive at a more realistic differentiation between, for instance, salbutamol and fenoterol. The term "membrane (de) stabilization" should not generally be used without careful consideration of the type of cell/membrane in question.

CCS and SOD1 mRNA are reduced after copper supplementation in peripheral mononuclear cells of individuals with high serum ceruloplasmin concentration.

PubMed

Suazo, Miriam; Olivares, Felipe; Mendez, Marco A; Pulgar, Rodrigo; Prohaska, Joseph R; Arredondo, Miguel; Pizarro, Fernando; Olivares, Manuel; Araya, Magdalena; González, Mauricio

2008-04-01

The limits of copper homeostatic regulation in humans are not known, making it difficult to define the milder effects of early copper excess. Furthermore, a robust assay to facilitate the detection of early stages of copper excess is needed. To address these issues, we assessed changes in relative mRNA abundance of methallothionein 2A (MT2A), prion (PrP), amyloid precursor-like protein 2 (APLP2), Cu/Zn superoxide dismutase (SOD1) and its copper chaperone (CCS) in peripheral mononuclear cells (PMNCs) from healthy adults representing the 5% highest and lowest extremes in the distribution curve of serum ceruloplasmin (Cp) concentrations of 800 individuals. The intracellular Cu content was also determined. PMNCs were isolated from individuals before and after exposure to a single daily dose of 10 mg Cu (as CuSO(4)) for 2 months. Results showed that although there were fluctuations in serum Cp values of the samples assessed before copper exposure, no significant differences were observed in cell copper content or in the relative abundance of MT2A, PrP and APLP2 transcripts in PMNCs. Also, these values were not modified after copper supplementation. However, CCS and SOD1 mRNA levels were reduced in PMNCs after copper supplementation in the individuals with the high Cp values, suggesting that they should be further explored as biomarkers of moderate copper overload in humans.

The Center for HIV/AIDS Vaccine Immunology (CHAVI) Multi-site Quality Assurance Program for Cryopreserved Human Peripheral Blood Mononuclear Cells

PubMed Central

Sarzotti-Kelsoe, Marcella; Needham, Leila K.; Rountree, Wes; Bainbridge, John; Gray, Clive M.; Fiscus, Susan A.; Ferrari, Guido; Stevens, Wendy S.; Stager, Susan L.; Binz, Whitney; Louzao, Raul; Long, Kristy O.; Mokgotho, Pauline; Moodley, Niranjini; Mackay, Melanie; Kerkau, Melissa; McMillion, Takesha; Kirchherr, Jennifer; Soderberg, Kelly A.; Haynes, Barton F.; Denny, Thomas N.

2014-01-01

The Center for HIV/AIDS Vaccine Immunology (CHAVI) consortium was established to determine the host and virus factors associated with HIV transmission, infection and containment of virus replication, with the goal of advancing the development of an HIV protective vaccine. Studies to meet this goal required the use of cryopreserved Peripheral Blood Mononuclear Cell (PBMC) specimens, and therefore it was imperative that a quality assurance (QA) oversight program be developed to monitor PBMC samples obtained from study participants at multiple international sites. Nine site-affiliated laboratories in Africa and the USA collected and processed PBMCs, and cryopreserved PBMC were shipped to CHAVI repositories in Africa and the USA for long-term storage. A three-stage program was designed, based on Good Clinical Laboratory Practices (GCLP), to monitor PBMC integrity at each step of this process. The first stage evaluated the integrity of fresh PBMCs for initial viability, overall yield, and processing time at the site-affiliated laboratories (Stage 1); for the second stage, the repositories determined post-thaw viability and cell recovery of cryopreserved PBMC, received from the site-affiliated laboratories (Stage 2); the third stage assessed the long-term specimen storage at each repository (Stage 3). Overall, the CHAVI PBMC QA oversight program results highlight the relative importance of each of these stages to the ultimate goal of preserving specimen integrity from peripheral blood collection to long-term repository storage. PMID:24910414

Specific destruction of islet transplants in NOD<-->C57BL/6 and NOD<-->C3H/Tif embryo aggregation chimeras irrespective of allelic differences in beta-cell antigens.

PubMed

Leijon, K; Hillörn, V; Bergqvist, I; Holmberg, D

1995-06-01

We have tested the hypothesis that allelic differences in the antigens expressed by the beta-cells of the islets of Langerhans influence the development of insulitis in the non-obese diabetic (NOD) mouse. Islets of Langerhans from NOD, C57BL/6 and C3H/Tif mice were transplanted under the kidney capsule of NOD<-->C57BL/6 and NOD<-->C3H/Tif embryo aggregation (EA) chimeras and the infiltration was scored 5-7 weeks later. Mononuclear cell infiltration of pancreatic islets was observed in 60% of the NOD<-->C57BL/6 and in 55% of the NOD<-->C3H/Tif EA chimeras. All transplanted EA chimeras that developed insulitis also displayed mononuclear cell infiltrates in the transplants, irrespective of the origin of the transplanted islets. In contrast, no infiltration of transplants was detected in EA chimeras scoring negative for insulitis. These results demonstrate that the specific destruction of islet transplants does not require the expression of NOD specific antigens by the islets. Moreover, the beta-cell destruction appears not to be restricted to NOD-MHC. The correlation between insulitis and transplant beta-cell destruction suggests the possibility that the development of insulitis is a prerequisite for transplant specific destruction. MHC restricted destruction may, therefore, precede the beta-cell destruction of transplanted islets. The chimerism among the mononuclear cells infiltrating the islet transplants was found to correlate with the overall haematopoetic chimerism in each of the individual EA chimeras. This observation suggests that NOD bone marrow, as well as non-NOD bone marrow, generates cells contributing to the beta-cell destruction process.

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

PubMed Central

Wang, Yaqin; Guo, Yue; Zhou, Danni; Xu, Mei; Ding, Jinli; Yang, Jing

2015-01-01

Successful embryo implantation and placentation depend on appropriate trophoblast invasion into the maternal endometrial stroma. Human chorionic gonadotropin (hCG) is one of the earliest embryo-derived secreted signals in the peripheral blood mononuclear cells (PBMC) that abundantly expresses hCG receptors. The aims of this study were to estimate the effect of human embryo–secreted hCG on PBMC function and investigate the role and underlying mechanisms of activated PBMC in trophoblast invasion. Blood samples were collected from women undergoing benign gynecological surgery during the mid-secretory phase. PBMC were isolated and stimulated with or without hCG for 0 or 24 h. Interleukin-1β (IL-1β) and leukemia inhibitory factor (LIF) expressions in PBMC were detected by enzyme-linked immunosorbent assay and real-time polymerase chain reaction (PCR). The JAR cell line served as a model for trophoblast cells and was divided into four groups: control, hCG only, PBMC only, and PBMC with hCG. JAR cell invasive and proliferative abilities were detected by trans-well and CCK8 assays and matrix metalloproteinase (MMP)-2 (MMP-2), MMP-9, vascular endothelial growth factor (VEGF), tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 expressions in JAR cells were detected by western blotting and real-time PCR analysis. We found that hCG can remarkably promote IL-1β and LIF promotion in PBMC after 24-h culture. PBMC activated by hCG significantly increased the number of invasive JAR cells in an invasion assay without affecting proliferation, and hCG-activated PBMC significantly increased MMP-2, MMP-9, and VEGF and decreased TIMP-1 and TIMP-2 expressions in JAR cells in a dose-dependent manner. This study demonstrated that hCG stimulates cytokine secretion in human PBMC and could stimulate trophoblast invasion. PMID:26087261

Chemotherapy treatment is associated with altered PD-L1 expression in lung cancer patients.

PubMed

Rojkó, Lívia; Reiniger, Lilla; Téglási, Vanda; Fábián, Katalin; Pipek, Orsolya; Vágvölgyi, Attila; Agócs, László; Fillinger, János; Kajdácsi, Zita; Tímár, József; Döme, Balázs; Szállási, Zoltán; Moldvay, Judit

2018-04-19

While the predictive value of programmed cell death ligand-1 (PD-L1) protein expression for immune checkpoint inhibitor therapy of lung cancer has been extensively studied, the impact of standard platinum-based chemotherapy on PD-L1 or programmed cell death-1 (PD-1) expression is unknown. The aim of this study was to determine the changes in PD-L1 expression of tumor cells (TC) and immune cells (IC), in PD-1 expression of IC, and in the amount of stromal mononuclear cell infiltration after platinum-based chemotherapy in patients with lung cancer. We determined the amount of stromal mononuclear cells and PD-L1/PD-1 expressions by immunohistochemistry in bronchoscopic biopsy samples including 20 adenocarcinomas (ADC), 15 squamous cell carcinomas (SCC), 2 other types of non-small cell lung cancer, and 4 small cell lung cancers together with their corresponding surgical resection tissues after platinum-based chemotherapy. PD-L1 expression of TC decreased in ten patients (24.4%) and increased in three patients (7.32%) after neoadjuvant chemotherapy (p = 0.051). The decrease in PD-L1 expression, however, was significant only in patients who received cisplatin-gemcitabine combination (p = 0.020), while in the carboplatin-paclitaxel group, no similar tendency could be observed (p = 0.432). There was no difference between ADC and SCC groups. Neither PD-1 expression nor the amount of stromal IC infiltration showed significant changes after chemotherapy. This is the first study, in which both PD-L1 and PD-1 expression were analyzed together with the amount of stromal IC infiltration in different histological subtypes of lung cancer before and after platinum-based chemotherapy. Our results confirm that chemotherapy decreases PD-L1 expression of TC in a subset of patients, therefore, rebiopsy and re-evaluation of PD-L1 expression may be necessary for the indication of immune checkpoint inhibitor therapy.

Transient detection of Chlamydial-specific Th1 memory cells in the peripheral circulation of women with history of Chlamydia trachomatis genital tract infection.

PubMed

Vicetti Miguel, Rodolfo D; Reighard, Seth D; Chavez, Jean M; Rabe, Lorna K; Maryak, Samantha A; Wiesenfeld, Harold C; Cherpes, Thomas L

2012-12-01

Development of safe and effective Chlamydia trachomatis vaccines requires better understanding of the host immune responses elicited by natural infection. Peripheral blood mononuclear cells isolated from women with or without history of genital tract chlamydial infection were stimulated with inactivated C. trachomatis elementary bodies (EB) in ELISPOT assays that enumerated frequencies of cells producing interferon (IFN)-γ or interleukin (IL)-17. IFN-γ-positive cells were highest among women sampled 30-60 days after diagnosis of C. trachomatis infection and treatment initiation, while the numbers of IFN-γ-positive cells were equally low among uninfected women and women sampled <30 or >60 days after diagnosis of infection. Conversely, IL-17-positive cell numbers were uniformly low among all participants. Dramatically reduced numbers of Chlamydia-specific Th1 memory cells in the peripheral circulation of study participants sampled more than 2 months after diagnosis, and initiation of treatment provides new insight into the results from C. trachomatis vaccine trials, in which immunization with EB provided only short-lived protection. Our results also suggest that an effective vaccine against this weakly antigenic intracellular pathogen will need to generate immunological memory more durable than that elicited by natural infection. © 2012 John Wiley & Sons A/S.

The UK ME/CFS Biobank for biomedical research on Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and Multiple Sclerosis

PubMed Central

Lacerda, Eliana M.; Bowman, Erinna W.; Cliff, Jacqueline M.; Kingdon, Caroline C.; King, Elizabeth C.; Lee, Ji-Sook; Clark, Taane G.; Dockrell, Hazel M.; Riley, Eleanor M.; Curran, Hayley; Nacul, Luis

2017-01-01

The UK ME/CFS Biobank was launched in August 2011 following extensive consultation with professionals and patient representatives. The bioresource aims to enhance research on myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), related to pathophysiology, biomarkers and therapeutic approaches. The cohort includes 18–60 year olds, encompassing 284 clinically-confirmed ME/CFS cases, 60 neurologist-diagnosed multiple sclerosis (MS) cases, and 135 healthy individuals. The Biobank contains blood samples, aliquoted into serum, plasma, peripheral blood mononuclear cells (PBMC), red blood cells/granulocyte pellet, whole blood, and RNA (totalling 29,863 aliquots). Extensive dataset (700 clinical and socio-demographic variables/participant) enables comprehensive phenotyping. Potential reuse is conditional to ethical approval. PMID:28649428

The UK ME/CFS Biobank for biomedical research on Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and Multiple Sclerosis.

PubMed

Lacerda, Eliana M; Bowman, Erinna W; Cliff, Jacqueline M; Kingdon, Caroline C; King, Elizabeth C; Lee, Ji-Sook; Clark, Taane G; Dockrell, Hazel M; Riley, Eleanor M; Curran, Hayley; Nacul, Luis

2017-01-01

The UK ME/CFS Biobank was launched in August 2011 following extensive consultation with professionals and patient representatives. The bioresource aims to enhance research on myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), related to pathophysiology, biomarkers and therapeutic approaches. The cohort includes 18-60 year olds, encompassing 284 clinically-confirmed ME/CFS cases, 60 neurologist-diagnosed multiple sclerosis (MS) cases, and 135 healthy individuals. The Biobank contains blood samples, aliquoted into serum, plasma, peripheral blood mononuclear cells (PBMC), red blood cells/granulocyte pellet, whole blood, and RNA (totalling 29,863 aliquots). Extensive dataset (700 clinical and socio-demographic variables/participant) enables comprehensive phenotyping. Potential reuse is conditional to ethical approval.

RNA-Seq analysis identifies aberrant RNA splicing of TRIP12 in acute myeloid leukemia patients at remission.

PubMed

Gao, Panke; Jin, Zhen; Cheng, Yingying; Cao, Xiangshan

2014-10-01

Aberrant splicing events play important roles in the pathogenesis of acute myeloid leukemia (AML). To investigate the aberrant splicing events in AML during treatment, we carried out RNA sequencing in peripheral mononuclear cell samples from a patient with complete remission. In addition to the sequencing samples, selected splicing events were confirmed and validated with real-time quantitative RT-PCR in another seven pairs of samples. A total of 4.05 and 3.39 GB clean data of the AML and remission sample were generated, respectively, and 2,223 differentially expressed genes (DEGs) were identified. Integrated with gene expression profiling on T cells from AML patients compared with healthy donors, 82 DEGs were also differentially expressed in AML CD4 T cells and CD8 T cells. Twenty-three alternative splicing events were considered to be confidential, and they were involved in many biological processes, such as RNA processing, cellular macromolecule catabolic process, and DNA binding process. An exon3-skipping event in TRIP12 was detected in patients at remission and further validated in another three independent samples. TRIP12 is an ubiquitin ligase of ARF, which suppresses aberrant cell growth by activating p53 responses. The exon3-skipping isoform of TRIP12 increased significantly after treatment. Our results may provide new understanding of AML, and the confirmed alternative splicing event of TRIP12 may be used as potential target for future investigations.

Diversity of Human and Macaque Airway Immune Cells at Baseline and during Tuberculosis Infection

PubMed Central

Myers, Amy J.; Jarvela, Jessica; Flynn, JoAnne; Rutledge, Tara; Bonfield, Tracey

2016-01-01

Immune cells of the distal airways serve as “first responders” of host immunity to the airborne pathogen Mycobacterium tuberculosis (Mtb). Mtb infection of cynomolgus macaques recapitulates the range of human outcomes from clinically silent latent tuberculosis infection (LTBI) to active tuberculosis of various degrees of severity. To further advance the application of this model to human studies, we compared profiles of bronchoalveolar lavage (BAL) cells of humans and cynomolgus macaques before and after Mtb infection. A simple gating strategy effectively defined BAL T-cell and phagocyte populations in both species. BAL from Mtb-naive humans and macaques showed similar differential cell counts. BAL T cells of macaques were composed of fewer CD4+cells but more CD8+ and CD4+CD8+ double-positive cells than were BAL T cells of humans. The most common mononuclear phagocyte population in BAL of both species displayed coexpression of HLA-DR, CD206, CD11b, and CD11c; however, multiple phagocyte subsets displaying only some of these markers were observed as well. Macaques with LTBI displayed a marked BAL lymphocytosis that was not observed in humans with LTBI. In macaques, the prevalence of specific mononuclear phagocyte subsets in baseline BAL correlated with ultimate outcomes of Mtb infection (i.e., LTBI versus active disease). Overall, these findings demonstrate the comparability of studies of pulmonary immunity to Mtb in humans and macaques. They also indicate a previously undescribed complexity of airway mononuclear phagocyte populations that suggests further lines of investigation relevant to understanding the mechanisms of both protection from and susceptibility to the development of active tuberculosis within the lung. PMID:27509488

Evaluation of the new continuous mononuclear cell collection protocol versus an older version on two different apheresis machines.

PubMed

Spoerl, Silvia; Wäscher, Dagmar; Nagel, Stefanie; Peschel, Christian; Verbeek, Mareike; Götze, Katharina; Krackhardt, Angela M

2018-05-06

Cell separators are routinely used to collect CD34 + blood stem cells in the context of customized stem cell transplantation procedures. The Spectra Optia (Terumo BCT) is a novel development of the precursor instrument, the Cobe Spectra (Terumo BCT). In this report, 146 autologous and 42 allogeneic donors undergoing apheresis on the Cobe Spectra using the mononuclear cell (MNC) program 4.7 or on the Spectra Optia using the new continuous mononuclear cell (cMNC) program 11.2 are compared. Viability of cells and collection efficacy within the apheresis products was comparable for autologous and allogeneic products collected with the MNC or cMNC method. However, we found a reduced duration of the apheresis procedure and lower hematocrit within the apheresis products when using the cMNC in autologous and allogeneic donors. Moreover, allogeneic donors collected substantially more CD34 + cells per kilogram of body weight when using the cMNC method. Differences in platelets before and after apheresis were substantially smaller in this cohort when compared to the cohort collected with the MNC method. Neutrophil and platelet engraftment after autologous or allogeneic transplantation with a product collected with the MNC procedure was comparable to a transplantation with a product processed according to the cMNC method. Comparison of the MNC (Cobe Spectra) and the cMNC (Spectra Optia) methods demonstrated an equal performance and outcome. However, advantages were present using the cMNC method with respect to apheresis duration and hematocrit within the apheresis product (autologous/allogeneic donors) and numbers of CD34 + cells collected, especially in allogeneic donors. © 2018 AABB.

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

NASA Astrophysics Data System (ADS)

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 < 0.001) and five related genes were found to be significantly down-regulated. These genes play a significant role in promoting the proliferation of T-lymphocytes, the regulation of membrane trafficking, promote early response, mediating C-myc related proliferation, promote antiapoptotic activity and protects mitochondria from the accumulation of oxidatively damaged membrane proteins. Overall, our analysis indicates that KMEG promotes T- cell proliferation and has an anti-inflammatory effect, thereby increasing immunity and possible protection from chronic inflammation setting which is optimally required during long term space flights.

Resting and Activated Natural Tregs Decrease in the Peripheral Blood of Patients with Atherosclerosis.

PubMed

Yazdani, Mohammadreza; Khosropanah, Shahdad; Hosseini, Ahmad; Doroudchi, Mehrnoosh

2016-12-01

Atherosclerosis is a chronic inflammatory disease affecting large and medium arteries. CD4+ T cells are known to play a role in the progression of the disease. CD4+CD25+Foxp3+ natural Treg (nTreg) cells seem to have a protective role in the disease and their reduction in acute coronary syndrome is recently shown. To investigate the frequency of nTreg subsets in the peripheral blood of patients with atherosclerosis. Confirmation of atherosclerosis was done by angiography and 15 ml heparinized blood was obtained from each of the 13 non-diabetic patients and 13 non-diabetic, non-smoker individuals with normal/insignificant coronary artery disease confirmed by angiography. Lipid profiles of the patients and controls were measured at the time of sampling. Mononuclear cells were used for both RNA extraction and immunophenotyping by real-time PCR and flowcytometry techniques, respectively. In natural Treg subsets, the frequency of CD4+CD45RO-CD25+Foxp3lo T-cells (resting nTregs) was greater in controls than patients (p=0.02). The frequency of CD4+CD45RO+CD25hiFoxp3hi T-cells (activated nTregs) was significantly higher in controls compared with patients (p=0.02). However, the frequency of CD4+CD25+CD45RO+Foxp3- T-cells (effector/memory T-cell) increased in patients compared with controls (p=0.01). Both the MFI and gene expression of Foxp3 were higher in control group than in patients (p=0.015 and p=0.017, respectively). Moreover, the TGF-β gene expression showed a decrease in the peripheral blood mononuclear cells of patients compared with controls (p=0.03). Decrease in both subsets of resting and activated nTregs along with a decrease in the expression of Foxp3 and TGF-β genes in patients with atherosclerosis suggests phenotypic changes in these subsets, which may as well be correlated with a more inflammatory profile in their lymphocytes.

Antarctic isolation: immune and viral studies

NASA Technical Reports Server (NTRS)

Tingate, T. R.; Lugg, D. J.; Muller, H. K.; Stowe, R. P.; Pierson, D. L.

1997-01-01

Stressful environmental conditions are a major determinant of immune reactivity. This effect is pronounced in Australian National Antarctic Research Expedition populations exposed to prolonged periods of isolation in the Antarctic. Alterations of T cell function, including depression of cutaneous delayed-type hypersensitivity responses and a peak 48.9% reduction of T cell proliferation to the mitogen phytohaemagglutinin, were documented during a 9-month period of isolation. T cell dysfunction was mediated by changes within the peripheral blood mononuclear cell compartment, including a paradoxical atypical monocytosis associated with altered production of inflammatory cytokines. There was a striking reduction in the production by peripheral blood mononuclear cells of the predominant pro-inflammatory monokine TNF-alpha and changes were also detected in the production of IL-1, IL-2, IL-6, IL-1ra and IL-10. Prolonged Antarctic isolation is also associated with altered latent herpesvirus homeostasis, including increased herpesvirus shedding and expansion of the polyclonal latent Epstein-Barr virus-infected B cell population. These findings have important long-term health implications.

Systemic cell-mediated reactions in vivo. Effect of the interaction of circulating antigen with sensitized lymphocytes on glomeruli and pulmonary alveoli.

PubMed Central

Bhan, A. K.; Schneeberger, E. E.; Collins, A. B.; McCluskey, R. T.

1984-01-01

The effects of systemic cell-mediated hypersensitivity reactions on glomeruli and lungs were investigated in rats. The animals were given an intravenous injection of antigen 7 days after sensitization or were given an intravenous injection of lymph node cells from sensitized syngeneic donors 1 day after antigen injection. Control animals were given an irrelevant antigen or saline. All animals received three injections of 3H-thymidine during the course of the experiments. The animals were sacrificed 2 or 3 days after antigen injection. Autoradiographs of renal and pulmonary tissue showed significantly more labeled mononuclear cells in glomeruli and pulmonary alveolar walls in the experimental groups than in the control groups. Immunofluorescence studies did not reveal antigen, rat IgG, or C3 in glomeruli. The results indicate that systemic cell-mediated reactions can lead to an accumulation of mononuclear cells in glomeruli and lungs, an effect that may contribute to tissue injury. Images Figure 1 Figure 2 Figure 3 PMID:6611090

[The role of poloxamer 188 for cord blood mononuclear cells into megakaryocytes cultivation and induction in three-dimensional WAVE Bioreactor].

PubMed

Chen, L; Yue, W; Xie, X Y; Zhang, X Y; Lyu, Y; Liu, D Q; Xi, J F; Qu, M Y; Fan, Z; Fang, F; Pei, X T

2018-01-14

Objective: To observe the effect of poloxamer 188 (P188) on megakaryocyte cultivation and induction from cord blood mononuclear cells in order to obtain more megakaryocyte progenitor cells (MPC). Methods: The cord blood mononuclear cells were isolated and inoculated in cell culture bag or cell culture flask respectively. The WIGGENS shaker and cell culture bags were used to mimick WAVE Bioreactor for three-dimensional (3D) cell culture, and the P188 was added to induction medium, The cells were detected for morphology, surface marker, viability, and number on day 14. Results: In the two-dimensional (2D) culture, CD41(+), CD41(+)/CD61(+), CD61(+) megakaryocytic numbers increased significantly after adding P188 (all P <0.01). And in the 3D culture of adding P188, the cell volume became larger and the nuclear shape was irregular, the cytoplasm appeared magenta granules, and the megakaryocyte cells became more mature. By 3D culture, the expression of CD41/CD61 was (36.30±1.27)% vs (23.95±1.34)%, hence the differentiation for MPC was significantly higher than that in the 2D group ( P <0.01). Furthermore, adding P188 in 3D culture resulted in highest differentiation efficiency for MPC [(59.45±1.20)%]. There were no significantly differences in terms of cell viability and cell number among 3D culture containing P188, 2D and 3D culture groups (all P >0.05). Conclusion: 3D culture was beneficial for the differentiation of MPC, but the cell viability was lower than 2D group; However, the satisfied cell growth and better induction efficiency were obtained by adding of P188, which might provide a new method of megakaryocytes production for clinical application.

MHC class I, MHC class II and intercellular adhesion molecule-1 (ICAM-1) expression in inflammatory myopathies.

PubMed

Bartoccioni, E; Gallucci, S; Scuderi, F; Ricci, E; Servidei, S; Broccolini, A; Tonali, P

1994-01-01

We investigated the relationship between the MHC-I, MHC-II and intercellular adhesion molecule-1 (ICAM-1) expression on myofibres and the presence of inflammatory cells in muscle specimens of 18 patients with inflammatory myopathies (nine polymyositis, seven dermatomyositis, two inclusion body myositis). We observed MHC-I expression in muscle fibres, infiltrating mononuclear cells and endothelial cells in every specimen. In seven patients, some muscle fibres were MHC-II-positive for the DR antigen, while the DP and DQ antigens were absent. ICAM-1 expression, detected in seven patients, was found in clusters of myofibres, associated with a marked MHC-I positivity and a widespread mononuclear infiltration. Most of the ICAM-1-positive fibres were regenerating fibres. Furthermore, some fibres expressed both ICAM-1 and DR antigens near infiltrating cells. This finding could support the hypothesis that myofibres may themselves be the site of autosensitization.

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

PubMed

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, and (5) AO/PI dual staining method. The results show comparable total PBMC counting among all five methods, which validate the AO/PI staining method for PBMC measurement in the image cytometry method. Copyright © 2012 Elsevier B.V. All rights reserved.

Local injection of autologous bone marrow cells to regenerate muscle in patients with traumatic brachial plexus injury: a pilot study.

PubMed

Hogendoorn, S; Duijnisveld, B J; van Duinen, S G; Stoel, B C; van Dijk, J G; Fibbe, W E; Nelissen, R G H H

2014-01-01

Traumatic brachial plexus injury causes severe functional impairment of the arm. Elbow flexion is often affected. Nerve surgery or tendon transfers provide the only means to obtain improved elbow flexion. Unfortunately, the functionality of the arm often remains insufficient. Stem cell therapy could potentially improve muscle strength and avoid muscle-tendon transfer. This pilot study assesses the safety and regenerative potential of autologous bone marrow-derived mononuclear cell injection in partially denervated biceps. Nine brachial plexus patients with insufficient elbow flexion (i.e., partial denervation) received intramuscular escalating doses of autologous bone marrow-derived mononuclear cells, combined with tendon transfers. Effect parameters included biceps biopsies, motor unit analysis on needle electromyography and computerised muscle tomography, before and after cell therapy. No adverse effects in vital signs, bone marrow aspiration sites, injection sites, or surgical wound were seen. After cell therapy there was a 52% decrease in muscle fibrosis (p = 0.01), an 80% increase in myofibre diameter (p = 0.007), a 50% increase in satellite cells (p = 0.045) and an 83% increase in capillary-to-myofibre ratio (p < 0.001) was shown. CT analysis demonstrated a 48% decrease in mean muscle density (p = 0.009). Motor unit analysis showed a mean increase of 36% in motor unit amplitude (p = 0.045), 22% increase in duration (p = 0.005) and 29% increase in number of phases (p = 0.002). Mononuclear cell injection in partly denervated muscle of brachial plexus patients is safe. The results suggest enhanced muscle reinnervation and regeneration. Cite this article: Bone Joint Res 2014;3:38-47.

Interferon-induced 2'-5' adenylate synthetase in vivo and interferon production in vitro by lymphocytes from systemic lupus erythematosus patients with and without circulating interferon

DOE Office of Scientific and Technical Information (OSTI.GOV)

Preble, O.T.; Rothko, K.; Klippel, J.H.

1983-06-01

The interferon (IFN)-induced enzyme 2-5A synthetase was elevated in mononuclear cells from both serum IFN-positive and -negative systemic lupus erythematosus (SLE) patients. This suggests that a much higher percentage of patients than previously thought produce endogenous IFN. These results may partly explain findings that mononuclear cells from SLE patients are deficient in IFN production in vitro in response to certain IFN inducers. Although normal lymphocytes can produce an acid-labile alpha IFN after stimulation with C. parvum in vitro, the reason for endogenous production of this unusual alpha IFN by SLE patients remains unknown.

[Production of selected cytokines by monocytes (IL-1 beta, IL-6) and lymphocytes (IL-2, IL-4) in peripheral blood of patients with nonallergic bronchial asthma treated with Broncho-Vaxom].

PubMed

Moniuszko, T; Rutkowski, R; Chyrek-Borowska, S

1995-01-01

In 16 patients with nonallergic bronchial asthma treated with Broncho-vaxom and 10 healthy persons the mononuclear peripheral blood cells ability for IL-1 beta, IL-2, IL-4 and IL-6 production were studied. Nonallergic asthmatics were characterised by increased levels of IL-1 beta and IL-6 produced by monocytes. After Broncho-vaxom therapy a decreased for IL-1 beta and IL-6, and an increased production of IL-2 were observed. These findings indicate that orally administered Broncho-vaxom affects on biological activity of mononuclear peripheral blood cells.

Therapeutic effects of nandrolone decanoate, tibolone, lynestrenol and ethylestrenol on Sjögren's syndrome-like disorder in NZB/W mice.

PubMed Central

Verheul, H A; Schot, L P; Schuurs, H W

1986-01-01

Growth of mononuclear cell infiltration in submandibular glands is significantly inhibited by Org OD14 (tibolone), lynestrenol and ethylestrenol given orally to New Zealand Black/White (NZB/W) mice from 26 weeks of age onwards. In addition, the extent of already established mononuclear cell infiltrations is significantly inhibited and reduced by nandrolone decanoate injected from 43 weeks of age onwards. Tibolone and nandrolone decanoate are the most potent of the four drugs. The therapeutic effect of these four steroids on the Sjögren's syndrome-like disorder in NZB/W mice is not related to their endocrine activities. Images Fig. 1 PMID:3742875

IL-1β Production by Intermediate Monocytes Is Associated with Immunopathology in Cutaneous Leishmaniasis.

PubMed

Santos, Daniela; Campos, Taís M; Saldanha, Maíra; Oliveira, Sergio C; Nascimento, Mauricio; Zamboni, Dario S; Machado, Paulo R; Arruda, Sérgio; Scott, Phillip; Carvalho, Edgar M; Carvalho, Lucas P

2018-05-01

Cutaneous leishmaniasis due to Leishmania braziliensis infection is an inflammatory disease in which skin ulcer development is associated with mononuclear cell infiltrate and high levels of inflammatory cytokine production. Recently, NLRP3 inflammasome activation and IL-1β production have been associated with increased pathology in murine cutaneous leishmaniasis. We hypothesized that cutaneous leishmaniasis patients have increased expression of NLRP3, leading to high levels of IL-1β production. In this article we show high production of IL-1β in biopsy samples and Leishmania antigen-stimulated peripheral blood mononuclear cells from patients infected with L. braziliensis and reduced IL-1β levels after cure. IL-1β production positively correlated with the area of necrosis in lesions and duration of the lesions. The main source of IL-1β was intermediate monocytes (CD14 ++ CD16 + ). Furthermore, our murine experiments show that IL-1β production in response to L. braziliensis was dependent on NLRP3, caspase-1, and caspase-recruiting domain (ASC). Additionally, we observed an increased expression of the NLRP3 gene in macrophages and the NLRP3 protein in intermediate monocytes from cutaneous leishmaniasis patients. These results identify an important role for human intermediate monocytes in the production of IL-1β, which contributes to the immunopathology observed in cutaneous leishmaniasis patients. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Association Between Schizophrenia and DNA Demethylase Activity in Human Peripheral Blood Mononuclear Cells.

PubMed

Zhang, Lili; Pang, Bo; Zhang, Wenbin; Bai, Wei; Yu, Weiying; Li, Yuanyuan; Hua, Wanqing; Li, Wenjun; Kou, Changgui

2018-06-01

DNA demethylase is a crucial enzyme in the epigenetic modification and regulation mechanisms of gene transcription. Based on previous assertions that the pathophysiology of schizophrenia is associated with epigenetics, we aimed to explore whether DNA demethylase activity might be related to schizophrenia in northeast China. We recruited 25 patients with first-episode schizophrenia and 29 normal controls from a northeast Chinese Han population. The diagnostic criteria of schizophrenia were determined according to diseases and related health problems, the tenth revision (ICD-10), and criteria of mental disorders, the third revised edition (CCMD3). DNA demethylase activity in human peripheral blood mononuclear cells (PBMCs) was measured using a DNA demethylase activity colorimetric assay ultra kit. Using Student's t-test, activation of DNA demethylase and its activity were higher in schizophrenia patients compared to healthy individuals (p < 0.001). Furthermore, the level of DNA demethylase activity in male and female subjects with schizophrenia significantly increased (all p < 0.05). Our data showed that DNA demethylase might play a role in the pathophysiology of schizophrenia, and individuals with higher DNA demethylase activity were susceptible to schizophrenia in a northeast Chinese Han population. To the best of our knowledge, this is the first time directly measured human blood samples to examine the association between first-episode schizophrenia patients and DNA demethylase activity, which will provide new insight to explore the effect on the mechanism of schizophrenia.

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

PubMed

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.

Multiplexed Affinity-Based Separation of Proteins and Cells Using Inertial Microfluidics.

PubMed

Sarkar, Aniruddh; Hou, Han Wei; Mahan, Alison E; Han, Jongyoon; Alter, Galit

2016-03-30

Isolation of low abundance proteins or rare cells from complex mixtures, such as blood, is required for many diagnostic, therapeutic and research applications. Current affinity-based protein or cell separation methods use binary 'bind-elute' separations and are inefficient when applied to the isolation of multiple low-abundance proteins or cell types. We present a method for rapid and multiplexed, yet inexpensive, affinity-based isolation of both proteins and cells, using a size-coded mixture of multiple affinity-capture microbeads and an inertial microfluidic particle sorter device. In a single binding step, different targets-cells or proteins-bind to beads of different sizes, which are then sorted by flowing them through a spiral microfluidic channel. This technique performs continuous-flow, high throughput affinity-separation of milligram-scale protein samples or millions of cells in minutes after binding. We demonstrate the simultaneous isolation of multiple antibodies from serum and multiple cell types from peripheral blood mononuclear cells or whole blood. We use the technique to isolate low abundance antibodies specific to different HIV antigens and rare HIV-specific cells from blood obtained from HIV+ patients.

Reovirus-Mediated Cytotoxicity and Enhancement of Innate Immune Responses Against Acute Myeloid Leukemia

PubMed Central

Hall, Kathryn; Scott, Karen J.; Rose, Ailsa; Desborough, Michael; Harrington, Kevin; Pandha, Hardev; Parrish, Christopher; Vile, Richard; Coffey, Matt; Bowen, David; Errington-Mais, Fiona

2012-01-01

Abstract Reovirus is a naturally occurring oncolytic virus that has shown preclinical efficacy in the treatment of a wide range of tumor types and has now reached phase III testing in clinical trials. The anti-cancer activity of reovirus has been attributed to both its direct oncolytic activity and the enhancement of anti-tumor immune responses. In this study, we have investigated the direct effect of reovirus on acute myeloid leukemia (AML) cells and its potential to enhance innate immune responses against AML, including the testing of primary samples from patients. Reovirus was found to replicate in and kill AML cell lines, and to reduce cell viability in primary AML samples. The pro-inflammatory cytokine interferon alpha (IFNα) and the chemokine (C-C motif) ligand 5 (known as RANTES [regulated upon activation, normal T-cell expressed, and secreted]) were also secreted from AML cells in response to virus treatment. In addition, reovirus-mediated activation of natural killer (NK) cells, within the context of peripheral blood mononuclear cells, stimulated their anti-leukemia response, with increased NK degranulation and IFNγ production and enhanced killing of AML targets. These data suggest that reovirus has the potential as both a direct cytotoxic and an immunotherapeutic agent for the treatment of AML. PMID:23515241

The Complexity of Fungal β-Glucan in Health and Disease: Effects on the Mononuclear Phagocyte System

PubMed Central

Camilli, Giorgio; Tabouret, Guillaume; Quintin, Jessica

2018-01-01

β-glucan, the most abundant fungal cell wall polysaccharide, has gained much attention from the scientific community in the last few decades for its fascinating but not yet fully understood immunobiology. Study of this molecule has been motivated by its importance as a pathogen-associated molecular pattern upon fungal infection as well as by its promising clinical utility as biological response modifier for the treatment of cancer and infectious diseases. Its immune effect is attributed to the ability to bind to different receptors expressed on the cell surface of phagocytic and cytotoxic innate immune cells, including monocytes, macrophages, neutrophils, and natural killer cells. The characteristics of the immune responses generated depend on the cell types and receptors involved. Size and biochemical composition of β-glucans isolated from different sources affect their immunomodulatory properties. The variety of studies using crude extracts of fungal cell wall rather than purified β-glucans renders data difficult to interpret. A better understanding of the mechanisms of purified fungal β-glucan recognition, downstream signaling pathways, and subsequent immune regulation activated, is, therefore, essential not only to develop new antifungal therapy but also to evaluate β-glucan as a putative anti-infective and antitumor mediator. Here, we briefly review the complexity of interactions between fungal β-glucans and mononuclear phagocytes during fungal infections. Furthermore, we discuss and present available studies suggesting how different fungal β-glucans exhibit antitumor and antimicrobial activities by modulating the biologic responses of mononuclear phagocytes, which make them potential candidates as therapeutic agents. PMID:29755450

The Complexity of Fungal β-Glucan in Health and Disease: Effects on the Mononuclear Phagocyte System.

PubMed

Camilli, Giorgio; Tabouret, Guillaume; Quintin, Jessica

2018-01-01

β-glucan, the most abundant fungal cell wall polysaccharide, has gained much attention from the scientific community in the last few decades for its fascinating but not yet fully understood immunobiology. Study of this molecule has been motivated by its importance as a pathogen-associated molecular pattern upon fungal infection as well as by its promising clinical utility as biological response modifier for the treatment of cancer and infectious diseases. Its immune effect is attributed to the ability to bind to different receptors expressed on the cell surface of phagocytic and cytotoxic innate immune cells, including monocytes, macrophages, neutrophils, and natural killer cells. The characteristics of the immune responses generated depend on the cell types and receptors involved. Size and biochemical composition of β-glucans isolated from different sources affect their immunomodulatory properties. The variety of studies using crude extracts of fungal cell wall rather than purified β-glucans renders data difficult to interpret. A better understanding of the mechanisms of purified fungal β-glucan recognition, downstream signaling pathways, and subsequent immune regulation activated, is, therefore, essential not only to develop new antifungal therapy but also to evaluate β-glucan as a putative anti-infective and antitumor mediator. Here, we briefly review the complexity of interactions between fungal β-glucans and mononuclear phagocytes during fungal infections. Furthermore, we discuss and present available studies suggesting how different fungal β-glucans exhibit antitumor and antimicrobial activities by modulating the biologic responses of mononuclear phagocytes, which make them potential candidates as therapeutic agents.

Disorders of B cells and helper T cells in the pathogenesis of the immunoglobulin deficiency of patients with ataxia telangiectasia.

PubMed Central

Waldmann, T A; Broder, S; Goldman, C K; Frost, K; Korsmeyer, S J; Medici, M A

1983-01-01

The pathogenesis of the immunoglobulin deficiency of 20 patients with ataxia telangiectasia was studied using an in vitro immunoglobulin biosynthesis system. 10 patients had no detectable IgA in their serum as assessed by radial diffusion in agar and 3 had a reduced serum IgA concentration. The peripheral blood mononuclear cells of 17 of the patients and 17 normal controls were cultured with pokeweed mitogen for 12 d and the immunoglobulin in the supernatants measured. The immunoglobulin synthesis was below the lower limit of the normal 95% confidence interval for IgM in 5 patients, for IgG in 8, and for IgA in 14. The mononuclear cells from 9 of the 10 patients with a serum IgA concentration less than 0.1 mg/ml failed to synthesize IgA in vitro. None of the patients manifested excessive suppressor cell activity. All patients had reduced but measurable helper T cell activity for immunoglobulin synthesis by co-cultured normal pokeweed mitogen-stimulated B cells (geometric mean 22% of normal). Furthermore, the addition of normal irradiated T cells to patient peripheral blood mononuclear cells led to an augmentation of IgM synthesis in 15 of 17 and to increased IgG synthesis in 9 of the 17 patients studied, including 9 of the 12 patients who had synthesized IgG before the addition of the irradiated T cells. In addition, IgA synthesis was increased in all eight patients examined that had serum IgA concentrations greater than 0.1 mg/ml. These studies suggest that a helper T cell defect contributes to the diminished immunoglobulin synthesis. However, a helper T cell defect does not appear to be the sole cause since there was no IgA synthesis by the peripheral blood mononuclear cells of 9 of the 10 patients with a profoundly reduced serum IgA even when co-cultured with normal T cells. Furthermore, the cells of the nine patients with profoundly reduced IgA levels examined also failed to produce IgA when stimulated with the relatively helper T cell-independent polyclonal activators, Nocardia water soluble mitogen or Epstein-Barr virus. Taken together these data support the view that the reduced immunoglobulin synthesis of these patients is due to defects of both B cells and helper T cells. Such a broad defect in lymphocyte maturation taken in conjunction with our demonstration of persistent alpha fetoprotein production by ataxia telangiectasia patients provides support for the proposal that these patients exhibit a generalized defect in tissue differentiation. PMID:6822665

Telomete length in peripheral blood mononuclear cells is associated with folate status in men

USDA-ARS?s Scientific Manuscript database

Human chromosomes are capped by tandem repeats of DNA and associated proteins termed telomeres. The length of the telomeres is reduced with increasing cell divisions except when the enzyme telomerase is active as seen in stem cells and germ cells. Telomere dysfunction has been associated with deve...

Indoleamine 2,3-dioxygenase is differentially expressed by different white blood cell populations of rhesus macaques (Macaca mulatta).

PubMed

Lei, N; Wang, Y; Zhang, W-J; Duan, J-Z; Yang, G-B

2013-08-01

Indoleamine 2,3-dioxygenase (IDO) is involved in immune processes such as transplant and fetal rejection, autoimmunity, cancer, and infection; however, its expression in rhesus macaques has not been fully addressed. Indoleamine 2,3-dioxygenase mRNA and protein in the white blood cells (WBCs) of Chinese rhesus macaques were examined by RT-PCR, western blotting, real-time RT-PCR, and flow cytometry. Both IDO protein and mRNA could be readily detected in WBCs or peripheral blood mononuclear cells (PBMCs) of normal rhesus macaques. IDO+ cell frequency was the highest among CD14(+) mononuclear cells, followed by CD56(+) cells and DCs. No difference in the frequency of IDO+ cells between CD4(+) and CD8(+) T cells; however, Th17 cells have higher frequency of IDO+ cells than Th1 cells, with Th2 cells the lowest. Toll-like receptor (TLR) stimulation significantly increased IDO protein level in CD14(+) , CD56(+) , CD1c(+) , CD11c(+) , and CD123(+) myeloid cells. Rhesus macaques express IDO differentially in their leukocyte subsets and are suitable for IDO-related pathophysiological studies. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Decreased collagen types I and IV, laminin, CK-19 and α-SMA expression after bone marrow cell transplantation in rats with liver fibrosis.

PubMed

Carvalho, S N; Lira, D C; Oliveira, G P; Thole, A A; Stumbo, A C; Caetano, C E; Marques, R G; Carvalho, L

2010-11-01

Bone marrow cells have frequently been tested in animal models of liver fibrosis to assess their role in hepatic regeneration. The mononuclear fraction of bone marrow cells is of particular interest, as many studies show that these cells may be beneficial to treat hepatic fibrosis. In this study, we used the bile duct ligation model to induce hepatic fibrosis in an irreversible manner, and rats were treated with bone marrow mononuclear (BMMN) cells after fibrosis was established. Analysis of collagen types I and IV, laminin and α-SMA showed a decreased expression of these proteins in fibrotic livers after 7 days of BMMN cell injection. Moreover, cytokeratin-19 analysis showed a reduction in bile ducts in the BMMN cell-treated group. These results were accompanied by ameliorated levels of hepatic enzymes GPT (Glutamic-pyruvic transaminase), GOT (glutamic-oxaloacetic transaminase) and alkaline phosphatase (AP). Therefore, we showed that BMMN cells decrease hepatic fibrosis by significantly reducing myofibroblast numbers and through reduction of the collagen and laminin-rich extracellular matrix of fibrotic septa and hepatic sinusoids.

Intracellular levels of hepatitis B virus DNA and pregenomic RNA in peripheral blood mononuclear cells of chronically infected patients.

PubMed

Lu, L; Zhang, H-Y; Yueng, Y-H; Cheung, K-F; Luk, J M; Wang, F-S; Lau, G K K

2009-02-01

It remains uncertain whether hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) and pregenomic RNA (pgRNA) can be detected in the serum or peripheral blood mononuclear cells (PBMC) of patients with chronic hepatitis B (CHB) infection. We examined HBV cccDNA and pgRNA in the serum and PBMC, and investigated the effect of lamivudine therapy on the viral loads in the PBMC of CHB patients. Paired serum and PBMC samples from 50 treatment-naïve CHB patients [25 hepatitis B e antigen (HBeAg) positive and 25 HBeAg negative] were quantified for total HBV DNA, cccDNA and pgRNA by real time polymerase chain reaction. HBV cccDNA and pgRNA were below the lower detection limit in all serum samples, and in 84% of PBMC. HBV DNA (r = 0.889, P < 0.001) and pgRNA (r = 0.696, P < 0.001) in PBMC correlated with the HBV DNA in serum. In the longitudinal study, 30 patients treated with lamivudine therapy for a median duration of 34 weeks (range 12-48 weeks) were examined. The median HBV DNA reduction in PBMC before and after treatment was 1.318 (range -0.471 to 3.846) log units, which was significantly lower than serum HBV DNA reduction [3.371 (range -0.883 to 9.454) log units, P < 0.05]. HBV cccDNA and pgRNA were undetectable in the serum of CHB patients. HBV viral loads in PBMC correlated with serum HBV DNA. Lamivudine therapy had less effect on the HBV viral loads in PBMC compared with the serum viral loads.

Delayed Dermal Hypersensitivity in Mice to Spherule and Mycelial Extracts of Coccidioides immitis

PubMed Central

Kong, Yi-Chi M.; Savage, D. C.; Kong, Leighton N. L.

1966-01-01

Kong, Yi-chi M. (University of California, Berkeley), D. C. Savage, and Leighton N. L. Kong. Delayed dermal hypersensitivity in mice to spherule and mycelial extracts of Coccidioides immitis. J. Bacteriol. 91:876–883. 1966.—A delayed hypersensitivity reaction to spherule and mycelial extracts of Coccidioides immitis was elicited in the footpads of mice vaccinated with killed spherules. Emulsification of the spherules with Freund's adjuvants was unnecessary, but a high concentration of antigen was required to elicit the reaction. Injection of the extracts produced, initially, a swelling which subsided within 4 hr, and then induration, which began at 6 to 8 hr and reached a maximum at 24 hr. The time course of the reaction corresponded to that of the tuberculin reaction in BCG-vaccinated mice. The histological response to coccidioidal extracts was characterized by the early infiltration of both polymorphonuclear and mononuclear cells, and the subsequent predominance of mononuclear cells at 24 to 48 hr. By 72 hr, the mononuclear cells comprised >90% of the cellular infiltrate. Animals infected intranasally with arthrospores (1 to 5 ld50) reacted negatively before and during the crisis period; thereafter (by 28 to 31 days after infection), up to 50% of the survivors showed a delayed reaction. Images PMID:5894227

Improvement in clinical signs and cellular immunity of dogs with visceral leishmaniasis using the immunomodulator P-MAPA.

PubMed

Santiago, Maria Emília B; Neto, Luiz Silveira; Alexandre, Eduardo Costa; Munari, Danísio Prado; Andrade, Mariana Macedo C; Somenzari, Marcos Arruda; Ciarlini, Paulo César; de Lima, V M F

2013-09-01

This study investigated the immunotherapeutic potential of the protein aggregate magnesium-ammonium phospholinoleate-palmitoleate anhydride immuno-modulator (P-MAPA) on canine visceral leishmaniasis. Twenty mongrel dogs presenting clinical symptoms compatible with leishmaniasis and diagnosis confirmed by the detection of anti-leishmania antibodies were studied. Ten dogs received 15 doses of the immunomodulator (2.0 mg/kg) intramuscularly, and 10 received saline as a placebo. Skin and peripheral blood samples were collected following administration of the immunomodulator. The groups were followed to observe for clinical signals of remission; parasite load in the skin biopsies using real-time PCR, the cytokines IL-2, IL-10 and IFN-γ in the supernatant of peripheral blood mononuclear cells stimulated in vitro with either total promastigote antigen or phytohemagglutinin measured by capture ELISA, and changes in CD4⁺ and CD8⁺ T cell subpopulations evaluated by flow cytometry. Comparison between the groups showed that treatment with the immunomodulator promoted improvement in clinical signs and a significant reduction in parasite load in the skin. In peripheral blood mononuclear cell cultures, supernatants showed a decrease in IL-10 levels and an increase in IL-2 and IFN-γ. An increase in CD8⁺ T cells was observed in peripheral blood. In addition, the in vitro leishmanicidal action of P-MAPA was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and no leishmanicidal activity was detected. These findings suggest that P-MAPA has potential as an immunotherapeutic drug in canine visceral leishmaniasis, since it assists in reestablishing partial immunocompetence of infected dogs. Copyright © 2013 Elsevier B.V. All rights reserved.

Elevated Immune Response Among Children 4 Years of Age With Pronounced Local Adverse Events After the Fifth Diphtheria, Tetanus, Acellular Pertussis Vaccination.

PubMed

van der Lee, Saskia; Kemmeren, Jeanet M; de Rond, Lia G H; Öztürk, Kemal; Westerhof, Anneke; de Melker, Hester E; Sanders, Elisabeth A M; Berbers, Guy A M; van der Maas, Nicoline A T; Rümke, Hans C; Buisman, Anne-Marie

2017-09-01

In the Netherlands, acellular pertussis vaccines replaced the more reactogenic whole-cell pertussis vaccines. This replacement in the primary immunization schedule of infants coincided with a significant increase in pronounced local adverse events (AEs) in 4 years old children shortly after the administration of a fifth diphtheria, tetanus, acellular pertussis and inactivated polio (DTaP-IPV) vaccine. The objective of this study was to investigate possible differences in vaccine antigen-specific immune responses between children with and without a pronounced local AE after the fifth DTaP-IPV vaccination. Blood was sampled in 2 groups of 4-year-olds: a case group reporting pronounced local swelling and/or erythema up to extensive limb swelling at the injection site (n = 30) and a control group (n = 30). Peripheral blood mononuclear cells were stimulated with individual vaccine antigens. Plasma antigen-specific IgG, IgG subclass and total IgE concentrations and T-cell cytokine [interferon-gamma, interleukin (IL)-13, IL-17 and IL-10] production by stimulated peripheral blood mononuclear cells were determined by multiplex bead-based fluorescent multiplex immunoassays. In children with AEs, significantly higher total IgE and vaccine antigen-specific IgG and IgG4 responses as well as levels of the T-helper 2 (Th2) cytokine IL-13 were found after pertussis, tetanus and diphtheria stimulation compared with controls. Children with pronounced local reactions show higher humoral and cellular immune responses. Acellular vaccines are known to skew toward more Th2 responses. The pronounced local AEs may be associated with more Th2 skewing after the fifth DTaP-IPV vaccination, but other biologic factors may also impact the occurrence of these pronounced local reactions.

Immune status of free-ranging green turtles with fibropapillomatosis from Hawaii

USGS Publications Warehouse

Work, Thierry M.; Rameyer, Robert; Balazs, George H.; Cray, Carolyn; Chang, Sandra P.

2001-01-01

Cell-mediated and humoral immune status of free-ranging green turtles (Chelonia mydas) in Hawaii (USA) with and without fibropapillornatosis (FP) were assessed. Tumored and non-tumored turtles from Kaneohe Bay (KB) on the island of Oahu and from FP-free areas on the west (Kona/Kohala) coast of the island of Hawaii were sampled from April 1998 through February 1999. Turtles on Oahu were grouped (0-3) for severity of tumors with 0 for absence of tumors, 1 for light, 2 for moderate, and 3 for most severe. Turtles were weighed, straight carapace length measured and the regression slope of weight to straight carapace length compared between groups (KB0, KB1, KB2, KB3, Kona). Blood was assayed for differential white blood cell count, hematocrit, in vitro peripheral blood mononuclear cell (PBMC) proliferation in the presence of concanavalin A (ConA) and phytohaemagglutinin (PHA), and protein electrophoresis. On Oahu, heterophil/lymphocyte ratio increased while eosinophil/monocyte ratio decreased with increasing tumors score. Peripheral blood mononuclear cell proliferation indices for ConA and PHA were significantly lower for turtles with tumor scores 2 and 3. Tumor score 3 turtles (KB3) had significantly lower hematocrit, total protein, alpha 1, alpha 2, and gamma globulins than the other four groups. No significant differences in immune status were seen between non-tumored (or KB1) turtles from Oahu and Hawaii. There was no significant difference between groups in regression slopes of body condition to carapace length. We conclude that turtles with severe FP are imunosuppressed. Furthermore, the lack of significant difference in immune status between non-tumored (and KB1) turtles from Oahu and Kona/Kohala indicates that immunosuppression may not be a prerequisite for development of FP.

Validation of biological activity testing procedure of recombinant human interleukin-7.

PubMed

Lutsenko, T N; Kovalenko, M V; Galkin, O Yu

2017-01-01

Validation procedure for method of monitoring the biological activity of reсombinant human interleukin-7 has been developed and conducted according to the requirements of national and international recommendations. This method is based on the ability of recombinant human interleukin-7 to induce proliferation of T lymphocytes. It has been shown that to control the biological activity of recombinant human interleukin-7 peripheral blood mononuclear cells (PBMCs) derived from blood or cell lines can be used. Validation charac­teristics that should be determined depend on the method, type of product or object test/measurement and biological test systems used in research. The validation procedure for the method of control of biological activity of recombinant human interleukin-7 in peripheral blood mononuclear cells showed satisfactory results on all parameters tested such as specificity, accuracy, precision and linearity.

Molecular changes associated with heat-shock treatment in avian mononuclear and lymphoid lineage cells.

PubMed

Miller, L; Qureshi, M A

1992-03-01

The induction of heat-shock protein (HSP) synthesis in avian cells of the mononuclear phagocytic system (MPS) and lymphoid system (LS) lineage was investigated by exposure to in vitro heat-shock conditions. In addition, the kinetics of HSP90 mRNA expression was examined in chicken peritoneal macrophages (PM) as well as heat-shock-induced HSP synthesis in PM from chickens, turkeys, quail, and ducks. Each MPS and LS cell type expressed three major (23, 70, and 90 kDa) HSP following a 1-h heat shock at 45 C. However, a unique heat-induced 32-kDa protein (P32) was expressed only by cells of MPS lineage. The expression of HSP90 mRNA in chicken PM was temperature- and time-dependent. These findings imply that avian PM undergo molecular changes in response to elevated environmental temperatures and that the pattern of HSP expression appears to be distinct for cells of the MPS and LS lineages in chickens.

Effector and memory T cell subsets in the response to bovine tuberculosis

USDA-ARS?s Scientific Manuscript database

Long-term (i.e., 14 days) cultured IFN-gamma ELISPOT assays of peripheral blood mononuclear cells (PBMC) are used to access T cell central memory (Tcm) responses in both cattle and humans. With bovine tuberculosis, vaccine-elicited long-term IFN-gamma ELISPOT response correlates with protection; how...

Comparison of neointimal hyperplasia with drug-eluting stents versus bare metal stents in patients undergoing intracoronary bone-marrow mononuclear cell transplantation following acute myocardial infarction.

PubMed

Villa, Adolfo; Arnold, Roman; Sánchez, Pedro L; Gimeno, Federico; Ramos, Benigno; Cantero, Teresa; Fernández, Maria Eugenia; Sanz, Ricardo; Gutiérrez, Oliver; Mota, Pedro; García-Frade, Javier; San Román, José Alberto; Fernández-Avilés, Francisco

2009-06-15

The aims of this study were to assess the safety of drug-eluting stent (DES) use and to compare the incidence of in-stent restenosis (ISR) and neointimal hyperplasia formation according to the type of stent implanted (DES vs bare-metal stents [BMS]) in patients who underwent intracoronary bone marrow mononuclear cell transplantation after acute ST elevation myocardial infarction. Fifty-nine patients with successfully revascularized ST elevation myocardial infarction (37 using BMS and 22 using DES) underwent paired angiographic examinations at baseline and 6 to 9 months after the intracoronary injection of 91 million +/- 56 million autologous bone marrow mononuclear cells. A subgroup of 30 patients also underwent serial intravascular ultrasound examinations. Off-line angiographic assessment showed 4 cases of binary ISR, primarily in BMS (3 cases), and no major adverse cardiac events were associated with stent type (mean follow-up period 41 +/- 10 months). At follow-up, angiographic late luminal loss was significantly lower in patients with DES than in those patients with BMS (0.35 +/- 0.66 vs 0.71 +/- 0.38 mm, p = 0.011). Multivariate analysis identified the use of DES (beta = -0.32, 95% confidence interval [CI] -0.57 to -0.26, p = 0.03) and a smaller baseline reference vessel diameter (beta = 0.29, 95% CI 0.04 to 0.54, p = 0.02) as independent predictors of lower late loss. Moreover, intravascular ultrasound showed a significant reduction of in-stent neointimal hyperplasia formation related to DES use compared with BMS use (Delta neointimal hyperplasia volume 5.4 mm(3) [95% CI 2.7 to 28.1] vs 35.9 mm(3) [95% CI 22.0 to 43.6], p = 0.035). In conclusion, these findings suggest that the use of DES is safe and may prevent ISR and neointimal hyperplasia formation in patients who undergo intracoronary bone marrow mononuclear cell transplantation after a successfully revascularized ST elevation myocardial infarction.

Evaluation of drug-induced hematotoxicity using novel in vitro monkey CFU-GM and BFU-E colony assays.

PubMed

Goto, Koichi; Goto, Mayumi; Ando-Imaoka, Masako; Kai, Kiyonori; Mori, Kazuhiko

2017-01-01

In order to evaluate drug-induced hematotoxicity in monkey cells in vitro, colony-forming unit-granulocyte, macrophage (CFU-GM), and burst-forming unit-erythroid (BFU-E) colony assays were established using mononuclear cells in the bone marrow collected from male cynomolgus monkeys. Furthermore, the effects of doxorubicin, chloramphenicol, and linezolid on CFU-GM and BFU-E colony formation were investigated using established monkey CFU-GM and BFU-E colony assays in comparison with those on human CFU-GM and BFU-E colonies acquired from human umbilical cord blood cells. Bone marrow mononuclear cells were collected from the ischial or iliac bone of male cynomolgus monkeys. The cells were subsequently processed by density gradient separation at 1.067, 1.070, or 1.077 g/mL for CFU-GM or 1.077 g/mL for BFU-E, and then cultured in methylcellulose medium for 9 or 13 days, respectively. A sufficient number of CFU-GM colonies were formed from mononuclear cells processed at a density of 1.070 g/mL. Moreover, the number of BFU-E colonies from the cells processed at a density of 1.077 g/mL was sufficient for the colony assay. The number of CFU-GM or BFU-E colonies decreased after treatment with the drugs of interest in a concentration-dependent manner. Compared with human CFU-GM, monkey CFU-GM were more sensitive to chloramphenicol and resistant to doxorubicin, whereas monkey BFU-E were more sensitive to all compounds in comparison to the sensitivity of human BFU-E. In conclusion, monkey CFU-GM and BFU-E colony assays were established and considered useful tools to evaluate the differences in drug-induced hematotoxicity between species.

MicroRNAs hsa-miR-99b, hsa-miR-330, hsa-miR-126 and hsa-miR-30c: Potential Diagnostic Biomarkers in Natural Killer (NK) Cells of Patients with Chronic Fatigue Syndrome (CFS)/ Myalgic Encephalomyelitis (ME).

PubMed

Petty, Robert D; McCarthy, Neil E; Le Dieu, Rifca; Kerr, Jonathan R

2016-01-01

Chronic Fatigue Syndrome (CFS/ME) is a complex multisystem disease of unknown aetiology which causes debilitating symptoms in up to 1% of the global population. Although a large cohort of genes have been shown to exhibit altered expression in CFS/ME patients, it is currently unknown whether microRNA (miRNA) molecules which regulate gene translation contribute to disease pathogenesis. We hypothesized that changes in microRNA expression in patient leukocytes contribute to CFS/ME pathology, and may therefore represent useful diagnostic biomarkers that can be detected in the peripheral blood of CFS/ME patients. miRNA expression in peripheral blood mononuclear cells (PBMC) from CFS/ME patients and healthy controls was analysed using the Ambion Bioarray V1. miRNA demonstrating differential expression were validated by qRT-PCR and then replicated in fractionated blood leukocyte subsets from an independent patient cohort. The CFS/ME associated miRNA identified by these experiments were then transfected into primary NK cells and gene expression analyses conducted to identify their gene targets. Microarray analysis identified differential expression of 34 miRNA, all of which were up-regulated. Four of the 34 miRNA had confirmed expression changes by qRT-PCR. Fractionating PBMC samples by cell type from an independent patient cohort identified changes in miRNA expression in NK-cells, B-cells and monocytes with the most significant abnormalities occurring in NK cells. Transfecting primary NK cells with hsa-miR-99b or hsa-miR-330-3p, resulted in gene expression changes consistent with NK cell activation but diminished cytotoxicity, suggesting that defective NK cell function contributes to CFS/ME pathology. This study demonstrates altered microRNA expression in the peripheral blood mononuclear cells of CFS/ME patients, which are potential diagnostic biomarkers. The greatest degree of miRNA deregulation was identified in NK cells with targets consistent with cellular activation and altered effector function.

MicroRNAs hsa-miR-99b, hsa-miR-330, hsa-miR-126 and hsa-miR-30c: Potential Diagnostic Biomarkers in Natural Killer (NK) Cells of Patients with Chronic Fatigue Syndrome (CFS)/ Myalgic Encephalomyelitis (ME)

PubMed Central

Petty, Robert D.; McCarthy, Neil E.; Le Dieu, Rifca; Kerr, Jonathan R.

2016-01-01

Background Chronic Fatigue Syndrome (CFS/ME) is a complex multisystem disease of unknown aetiology which causes debilitating symptoms in up to 1% of the global population. Although a large cohort of genes have been shown to exhibit altered expression in CFS/ME patients, it is currently unknown whether microRNA (miRNA) molecules which regulate gene translation contribute to disease pathogenesis. We hypothesized that changes in microRNA expression in patient leukocytes contribute to CFS/ME pathology, and may therefore represent useful diagnostic biomarkers that can be detected in the peripheral blood of CFS/ME patients. Methods miRNA expression in peripheral blood mononuclear cells (PBMC) from CFS/ME patients and healthy controls was analysed using the Ambion Bioarray V1. miRNA demonstrating differential expression were validated by qRT-PCR and then replicated in fractionated blood leukocyte subsets from an independent patient cohort. The CFS/ME associated miRNA identified by these experiments were then transfected into primary NK cells and gene expression analyses conducted to identify their gene targets. Results Microarray analysis identified differential expression of 34 miRNA, all of which were up-regulated. Four of the 34 miRNA had confirmed expression changes by qRT-PCR. Fractionating PBMC samples by cell type from an independent patient cohort identified changes in miRNA expression in NK-cells, B-cells and monocytes with the most significant abnormalities occurring in NK cells. Transfecting primary NK cells with hsa-miR-99b or hsa-miR-330-3p, resulted in gene expression changes consistent with NK cell activation but diminished cytotoxicity, suggesting that defective NK cell function contributes to CFS/ME pathology. Conclusion This study demonstrates altered microRNA expression in the peripheral blood mononuclear cells of CFS/ME patients, which are potential diagnostic biomarkers. The greatest degree of miRNA deregulation was identified in NK cells with targets consistent with cellular activation and altered effector function. PMID:26967895

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

PubMed

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.

[Establishment and Management of Multiple Myeloma Specimen Bank Applied for Molecular Biological Researches].

PubMed

Li, Han-Qing; Mei, Jian-Gang; Cao, Hong-Qin; Shao, Liang-Jing; Zhai, Yong-Ping

2017-12-01

To establish a multiple myeloma specimen bank applied for molecular biological researches and to explore the methods of specimen collection, transportation, storage, quality control and the management of specimen bank. Bone marrow and blood samples were collected from multiple myeloma patients, plasma cell sorting were operated after the separation of mononuclear cells from bone marrow specimens. The plasma cells were divided into 2 parts, one was added with proper amount of TRIzol and then kept in -80 °C refrigerator for subsequent RNA extraction, the other was added with proper amount of calf serum cell frozen liquid and then kept in -80 °C refrigerator for subsequent cryopreservation of DNA extraction after numbered respectively. Serum and plasma were separated from peripheral blood, specimens of serum and plasma were then stored at -80 °C refrigerator after registration. Meantime, the myeloma specimen information management system was established, managed and maintained by specially-assigned persons and continuous modification and improvement in the process of use as to facilitate the rapid collection, management, query of the effective samples and clinical data. A total of 244 portions plasma cells, 564 portions of serum, and 1005 portions of plasma were collected, clinical characters were documented. A multiple myeloma specimen bank have been established initially, which can provide quality samples and related clinical information for molecular biological research on multiple myeloma.

Evaluation of IL-17B and IL-17F mRNA expression in peripheral blood mononuclear cells and association with clinical outcome of IBD patients.

PubMed

Safari, Mohammad Taghi; Chaleshi, Vahid; Tarban, Peyman; Nourian, Mahyar; Balaii, Hedieh; Shahrokh, Shabnam; Asadzadeh Aghdaei, Hamid

2017-01-01

In this study, we determined the gene expression analysis of IL-17 gene family for early detection of subclinical inflammation among IBD patients. Cytokines have a vital role in the pathogenesis of inflammatory bowel disease (IBD). Interleukin-17 is the signature cytokine of the recently identified T helper 17 (Th17) cell subset. IL-17F is mainly involved in mucosal host defense mechanisms whereas the functions of IL-17B remain largely elusive. In this cross-sectional study, IBD patients divided into two active and inactive groups. Peripheral blood mononuclear cells (PBMCs) from 38 IBD patients which 20 inactive samples and 18 active individuals were collected. Changes of IL-17 F and IL-17B mRNA expression level evaluated by quantitative-real time-PCR. mRNA expression level of IL-17B and IL-17F in CD, UC, active and inactive groups have been assessed and there were no significant differences (P>0.05). Patients were classified into five different categories as follows: i) 5ASA; ii) 5ASA + Pred; iii) 5ASA + AZA; iv) 5ASA + Pred + AZA; v) 5ASA + Pred + AZA + IFX according to medication usage, expression of IL-17F and IL-17B had no differences (p>0.05). Evaluation of IL-17B and IL-17F mRNA expression level illustrate no difference among active and inactive patients. Therefore, IL-17B and IL-17F are not biomarkers in an Iranian IBD patients.

Serum amyloid A induction of cytokines in monocytes/macrophages and lymphocytes.

PubMed

Song, Changjie; Hsu, Kenneth; Yamen, Eric; Yan, Weixing; Fock, Jianyi; Witting, Paul K; Geczy, Carolyn L; Freedman, S Ben

2009-12-01

Serum amyloid A (SAA) is a biomarker of inflammation. Elevated blood levels in cardiovascular disease and local deposition in atheroma implies a role of SAA as a mediator rather than just a marker of inflammation. This study explored SAA-induced cytokine production and secretion by mononuclear cells. RT-PCR showed that SAA time-dependently induced cytokine mRNAs in peripheral blood mononuclear cells (PBMC) and THP-1 monocytoid cells, and dramatically increased IL-1beta, MCP-1, IL-6, IL-8, IL-10, GM-CSF, TNF, and MIP-1alpha secretion by PBMC to levels 28 to 25,000 fold above baseline, as measured with Bio-Plex kits; monocytes were the principle source. SAA induction of cytokines in monocyte-derived macrophages (MDM) was significantly higher than from monocytes from the same donors. SAA time-dependently induced transient and significant upregulation of NF-kappaB1 mRNA; inhibitor studies indicate that activation of NF-kappaB through the ERK1/2, p38 and JNK MAPKs and the PI3K pathway was involved. PBMC from 10 patients with coronary artery disease (CAD) spontaneously secreted higher levels of IL-6 and MIP-1alpha after 24h incubation than PBMC from normal controls, whereas SAA-induced levels of all cytokines were similar to controls. Aortic and coronary sinus sampling in 23 CAD patients indicated significant SAA release into the coronary circulation, not evident in 11 controls. SAA can increase monocyte and macrophage cytokine production, possibly at sites of atherosclerosis, thereby contributing to the pro-inflammatory state in coronary artery disease.

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

PubMed

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.

Evaluation of IL-17B and IL-17F mRNA expression in peripheral blood mononuclear cells and association with clinical outcome of IBD patients

PubMed Central

Safari, Mohammad Taghi; Chaleshi, Vahid; Tarban, Peyman; Nourian, Mahyar; Balaii, Hedieh; Shahrokh, Shabnam; Asadzadeh Aghdaei, Hamid

2017-01-01

Aim: In this study, we determined the gene expression analysis of IL-17 gene family for early detection of subclinical inflammation among IBD patients. Background: Cytokines have a vital role in the pathogenesis of inflammatory bowel disease (IBD). Interleukin-17 is the signature cytokine of the recently identified T helper 17 (Th17) cell subset. IL-17F is mainly involved in mucosal host defense mechanisms whereas the functions of IL-17B remain largely elusive. Methods: In this cross-sectional study, IBD patients divided into two active and inactive groups. Peripheral blood mononuclear cells (PBMCs) from 38 IBD patients which 20 inactive samples and 18 active individuals were collected. Changes of IL-17 F and IL-17B mRNA expression level evaluated by quantitative-real time-PCR. Results: mRNA expression level of IL-17B and IL-17F in CD, UC, active and inactive groups have been assessed and there were no significant differences (P>0.05). Patients were classified into five different categories as follows: i) 5ASA; ii) 5ASA + Pred; iii) 5ASA + AZA; iv) 5ASA + Pred + AZA; v) 5ASA + Pred + AZA + IFX according to medication usage, expression of IL-17F and IL-17B had no differences (p>0.05). Conclusion: Evaluation of IL-17B and IL-17F mRNA expression level illustrate no difference among active and inactive patients. Therefore, IL-17B and IL-17F are not biomarkers in an Iranian IBD patients. PMID:29511476

Cytotoxic and genotoxic effects of defence secretion of Ulomoides dermestoides on A549 cells.

PubMed

Crespo, Rosana; Villaverde, M Luciana; Girotti, Juan R; Güerci, Alba; Juárez, M Patricia; de Bravo, Margarita G

2011-06-14

Ulomoides dermestoides (Fairmaire, 1893) is a cosmopolitan tenebrionid beetle reared by Argentine people who consume them alive as an alternative medicine in the treatment of different illnesses such as asthma, Parkinson's, diabetes, arthritis, HIV and specially cancer. To evaluate the cytotoxicity and DNA damage of the major volatile components released by Ulomoides dermestoides on human lung carcinoma epithelial cell line A549. The defence compounds of Ulomoides dermestoides were extracted with dichloromethane and analyzed and quantified by capillary gas chromatography. The toxicity effects of the beetle's extract against A549 cell line were evaluated. Cytotoxicity was evaluated by MTT test and Trypan blue assay and genotoxicity was evaluated by the comet assay. The synthetic compounds, individually or combined, were also tested in A549 cells and normal mononuclear human cells. The defence compounds of Ulomoides dermestoides extracted with dichloromethane (methyl-1,4-benzoquinones, ethyl-1,4-benzoquinones and 1-pentadecene as major components) showed cytotoxic activity on A549 cells demonstrated by MTT test and Trypan blue assay, with IC(50) values of 0.26equivalent/ml and 0.34equivalent/ml, respectively (1equivalent=amount of components extracted per beetle). The inhibition of A549 cell proliferation with the synthetic blend (1,4-benzoquinone and 1-pentadecene) or 1,4-benzoquinone alone was similar to that obtained with the insect extract. 1-Pentadecene showed no inhibitory effect. Low doses of insect extract or synthetic blend (0.15equivalent/ml) inhibited mononuclear cell proliferation by 72.2±2.7% and induced significant DNA damage both in tumor and mononuclear cells. Results of this study demonstrated that defence compounds of Ulomoides dermestoides reduced cell viability and induced DNA damage. We also concluded that the insect benzoquinones are primarily responsible for inducing cytotoxicity and genotoxicity in culture cells. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

An affordable method to obtain cultured endothelial cells from peripheral blood

PubMed Central

Bueno-Betí, Carlos; Novella, Susana; Lázaro-Franco, Macarena; Pérez-Cremades, Daniel; Heras, Magda; Sanchís, Juan; Hermenegildo, Carlos

2013-01-01

The culture of endothelial progenitor cells (EPC) provides an excellent tool to research on EPC biology and vascular regeneration and vasculogenesis. The use of different protocols to obtain EPC cultures makes it difficult to obtain comparable results in different groups. This work offers a systematic comparison of the main variables of most commonly used protocols for EPC isolation, culture and functional evaluation. Peripheral blood samples from healthy individuals were recovered and mononuclear cells were cultured. Different recovery and culture conditions were tested: blood volume, blood anticoagulant, coating matrix and percentage of foetal bovine serum (FBS) in culture media. The success of culture procedure, first colonies of endothelial cells appearance time, correlation with number of circulating EPC (cEPC) and functional comparison with human umbilical vein endothelial cells (HUVEC) were studied. The use of heparin, a minimum blood volume of 30 ml, fibronectin as a coating matrix and endothelial growing media-2 supplemented with 20% FBS increased the success of obtaining EPC cultures up to 80% of the processed samples while reducing EPC colony appearance mean time to a minimum of 13 days. Blood samples exhibiting higher cEPC numbers resulted in reduced EPC colony appearance mean time. Cells isolated by using this combination were endothelial cell-like EPCs morphological and phenotypically. Functionally, cultured EPC showed decreased growing and vasculogenic capacity when compared to HUVEC. Thus, above-mentioned conditions allow the isolation and culture of EPC with smaller blood volumes and shorter times than currently used protocols. PMID:24118735

Intraindividual dynamics of transcriptome and genome-wide stability of DNA methylation

PubMed Central

Furukawa, Ryohei; Hachiya, Tsuyoshi; Ohmomo, Hideki; Shiwa, Yuh; Ono, Kanako; Suzuki, Sadafumi; Satoh, Mamoru; Hitomi, Jiro; Sobue, Kenji; Shimizu, Atsushi

2016-01-01

Cytosine methylation at CpG dinucleotides is an epigenetic mechanism that affects the gene expression profiles responsible for the functional differences in various cells and tissues. Although gene expression patterns are dynamically altered in response to various stimuli, the intraindividual dynamics of DNA methylation in human cells are yet to be fully understood. Here, we investigated the extent to which DNA methylation contributes to the dynamics of gene expression by collecting 24 blood samples from two individuals over a period of 3 months. Transcriptome and methylome association analyses revealed that only ~2% of dynamic changes in gene expression could be explained by the intraindividual variation of DNA methylation levels in peripheral blood mononuclear cells and purified monocytes. These results showed that DNA methylation levels remain stable for at least several months, suggesting that disease-associated DNA methylation markers are useful for estimating the risk of disease manifestation. PMID:27192970

Type-I interferon receptor expression: its circadian rhythm and downregulation after interferon-alpha administration in peripheral blood cells from renal cancer patients.

PubMed

Shiba, Masahiro; Nonomura, Norio; Nakai, Yasutomo; Nakayama, Masashi; Takayama, Hitoshi; Inoue, Hitoshi; Tsujimura, Akira; Nishimura, Kazuo; Okuyama, Akihiko

2009-04-01

To investigate the regulation of interferon-alpha (IFN-alpha) receptor expression in metastatic renal cell carcinoma (RCC) after IFN-alpha administration. Blood sampling was carried out in eight patients with metastatic RCC and six healthy volunteers. Flow-cytometric analysis using a monoclonal antibody against the active subunit of the type-I IFN-alpha receptor (IFNAR2) was carried out to examine the circadian rhythm of IFNAR2 expression in peripheral blood mononuclear cells (PBMC) as well as its downregulation after IFN-alpha administration. According to its circadian rhythm IFNAR2 in PBMC had a peak expression at night. Once IFN-alpha is administered, IFNAR2 levels in PBMC showed downregulation within 48 h and recovered within another 48 h. Our findings might support the establishment of an optimal schedule for IFN-alpha administration.

Tumor Necrosis Factor-producing T-regulatory Cells Are Associated With Severe Liver Injury in Patients With Acute Hepatitis A.

PubMed

Choi, Yoon Seok; Jung, Min Kyung; Lee, Jeewon; Choi, Seong Jin; Choi, Sung Hoon; Lee, Hyun Woong; Lee, Jong-Joo; Kim, Hyung Joon; Ahn, Sang Hoon; Lee, Dong Hyeon; Kim, Won; Park, Su-Hyung; Huh, Jun R; Kim, Hyoung-Pyo; Park, Jun Yong; Shin, Eui-Cheol

2018-03-01

CD4 + CD25 + Foxp3 + T-regulatory (Treg) cells control immune responses and maintain immune homeostasis. However, under inflammatory conditions, Treg cells produce cytokines that promote inflammation. We investigated production of tumor necrosis factor (TNF) by Treg cells in patients with acute hepatitis A (AHA), and examined the characteristics of these cells and association with clinical factors. We analyzed blood samples collected from 63 patients with AHA at the time of hospitalization (and some at later time points) and 19 healthy donors in South Korea. Liver tissues were collected from patients with fulminant AHA during liver transplantation. Peripheral blood mononuclear cells were isolated from whole blood and lymphocytes were isolated from liver tissues and analyzed by flow cytometry. Cytokine production from Treg cells (CD4 + CD25 + Foxp3 + ) was measured by immunofluorescence levels following stimulation with anti-CD3 and anti-CD28. Epigenetic stability of Treg cells was determined based on DNA methylation patterns. Phenotypes of Treg cells were analyzed by flow cytometry and an RORγt inhibitor, ML-209, was used to inhibit TNF production. Treg cell suppression assay was performed by co-culture of Treg-depleted peripheral blood mononuclear cells s and isolated Treg cells. A higher proportion of CD4 + CD25 + Foxp3 + Treg cells from patients with AHA compared with controls produced TNF upon stimulation with anti-CD3 and anti-CD28 (11.2% vs 2.8%). DNA methylation analysis confirmed the identity of the Treg cells. TNF-producing Treg cells had features of T-helper 17 cells, including up-regulation of RORγt, which was required for TNF production. The Treg cells had reduced suppressive functions compared with Treg cells from controls. The frequency of TNF-producing Treg cells in AHA patients' blood correlated with their serum level of alanine aminotransferase. Treg cells from patients with AHA have altered functions compared with Treg cells from healthy individuals. Treg cells from patients with AHA produce higher levels of TNF, gain features of T-helper 17 cells, and have reduced suppressive activity. The presence of these cells is associated with severe liver injury in patients with AHA. Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.

Amount of HIV DNA in Peripheral Blood Mononuclear Cells is Proportional to the Severity of HIV-1-Associated Neurocognitive Disorders

PubMed Central

Shiramizu, Bruce; Williams, Andrew E.; Shikuma, Cecilia; Valcour, Victor

2009-01-01

Human immunodeficiency virus (HIV) DNA in peripheral blood mononuclear cells was previously associated with neuropsychological function. By including individuals encompassing the full range of HIV-1-associated neurocognitive disorders, this study reports results from subjects with normal cognition, minor cognitive motor disorder, and HIV-1-associated dementia. Individuals with normal cognition had relatively low HIV DNA levels compared to those with minor cognitive motor disorder and HIV-1-associated dementia. Neuropsychological deficits were significantly associated with entry HIV DNA in all domains. These findings demonstrate for the first time that the severity of HIV-1-associated neurocognitive disorders is proportional to the amount of circulating HIV DNA. PMID:19359454

The connective tissue component of the caprine arthritis-encephalitis syndrome.

PubMed Central

Crawford, T. B.; Adams, D. S.; Sande, R. D.; Gorham, J. R.; Henson, J. B.

1980-01-01

The gross and microscopic connective tissue lesions in 12 goats with caprine arthritis-encephalitis (CAE) are described, including those from which a virus (CAEV) was isolated. Lesions were most often associated with synovial-lined structures including joints, tendon sheaths, and bursae, and were typified by synovial cell proliferations, subsynovial mononuclear cell infiltration, the presence of fibrin, fibrinous concretions, necrosis, and mineralization. Extrasynovial lesions were located in kidneys, vessels, and brain. The inflammatory infiltrates in these organs were predominantly mononuclear. Amyloid was also found in liver, spleen, and kidney. Microbiologic techniques failed to demonstrate any bacteria, mycoplasma, or chlamydia in the lesions. Images Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 1 Figure 2 Figure 3 Figure 4 PMID:7406019

Early application of Met-RANTES ameliorates chronic allograft nephropathy.

PubMed

Song, Erwei; Zou, Hequn; Yao, Yousheng; Proudfoot, Amanda; Antus, Balazs; Liu, Shanying; Jens, Lutz; Heemann, Uwe

2002-02-01

Initial insults to kidney allografts, characterized by infiltration of mononuclear inflammatory cells, contribute to chronic allograft nephropathy. Chemokines such as RANTES (regulated upon activation, normal T cell expressed) are thought to be responsible for the recruitment and activation of infiltrating cells. The present study investigated whether early application of Met-RANTES, a chemokine receptor antagonist that blocks the effects of RANTES, can protect renal allografts from long-term deterioration. Fisher (F344) rat kidneys were orthotopically transplanted into Lewis recipients and treated with cyclosporine A (1.5 mg/kg/day) for the first 10 days following transplantation, together with either Met-RANTES at 40 microg/day, 200 microg/day or vehicle for the first 7 days. Animals were harvested at 2 and 28 weeks after transplantation for histologic, immunohistologic and molecular analysis. Met-RANTES treatment reduced the infiltration of lymphocytes and macrophages in allografts at 2 weeks after transplantation, accompanied by decreased mRNA expression of interleukin (IL)-2, IL-1beta, tumor necrosis factor-alpha (TNF-alpha) and RANTES. At post-transplantation week 28, Met-RANTES treatment at high and low doses reduced urinary protein excretion and significantly ameliorated glomerulosclerosis, interstitial fibrosis, tubular atrophy, intimal proliferation of graft arteries and mononuclear cell infiltration. However, creatinine clearance was not influenced by Met-RANTES. Furthermore, Met-RANTES suppressed the mRNA expression of transforming growth factor-beta (TGF-beta) and platelet-derived growth factor-B (PDGF-B). Blockade of chemokine receptors by Met-RANTES diminishes early infiltration and activation of mononuclear cells in the grafts, and thus reduces the pace of chronic allograft nephropathy.

Thalidomide suppressed IL-1beta while enhancing TNF-alpha and IL-10, when cells in whole blood were stimulated with lipopolysaccharide.

PubMed

Shannon, Edward; Noveck, Robert; Sandoval, Felipe; Kamath, Burde

2008-01-01

Thalidomide is used to treat erythema nodosum leprosum (ENL). The events that precipitate this inflammatory reaction, which may occur in multibacillary leprosy patients, and the mechanism by which thalidomide arrest ENL, are not known. Thalidomide's ability to inhibit tumor necrosis factor alpha (TNF-alpha) in vitro has been proposed as a partial explanation of its effective treatment of ENL. In in vitro assays, thalidomide can enhance or suppress TNF-alpha. This is dependent on the stimulant used to evoke TNF-alpha; the procedure used to isolate the mononuclear cells from blood, and the predominant mononuclear cell type in the culture. To avoid artifacts that may occur during isolation of mononuclear cells from blood, we stimulated normal human blood with LPS and evaluated the effect of thalidomide and dexamethasone on TNF-alpha, and other inflammatory cytokines and biomarkers. Thalidomide suppressed interleukin 1 beta (IL-1beta) (p = 0.007), and it enhanced TNF-alpha (p = 0.007) and interleukin 10 (IL-10) (p = 0.031). Dexamethasone enhanced IL-10 (p = 0.013) and suppressed IL-1beta, TNF-alpha, interleukin 6 (IL-6), and interleukin 8 (IL-8) (p = 0.013). The two drugs did not suppress: C-reactive protein (CRP), Ig-superfamily cell-adhesion molecule 1 (ICAM 1), tumor necrosis factor receptor 1 (TNFR1), tumor necrosis factor receptor 2 (TNFR2), or amyloid A. In vitro and in vivo evidence is accumulating that TNF-alpha is not the primary cytokine targeted by thalidomide in ENL and other inflammatory conditions.

[Effect of cryopreservation on umbilical blood cells and its mechanism].

PubMed

Li, Xin; Chen, Fangping; Jiang, Tiebin; Wang, Erhua; Liu, Jing

2013-07-01

To evaluate the effect of cryopreservation on clonogenic ability and apoptosis rate of mono-nuclear cells and CD34+ cells in umbilical blood (UB), and to choose the index to present the freezing injury and optimize the cryopreservation of UB. The mono-nuclear cells (MNC) and CD34+ cells were separated from UB and frozen.After 30 days, they were thawed in warm water. Clonogenic capacity and clonogenic recovery before and after the cryopreservation was compared. We also used Annexin V-FITC-PI to investigate the apoptosis rate of the cells before and after the cryopreservation of these 2 types of cells. The number of colony forming unit-granulocyte/monocyte (CFU-GMs) was not changed after freezing and thawing in both MNCs and CD34+ cells, while the number of colony forming unit-granulocyte, erythrocyte, monocyte and megakaryocyte (CFU-GEMM) was obviously reduced after freezing in CD34+ cells. The 2 types of cryopreserved cells had certain degree of apoptosis before the cryopreservation. MNC-type cryopreservation increased the cells apoptosis a little, while CD34+-type cryopreservation increased more. The cells have certain degree of apoptosis before the cryopreservation. The freezing and thawing procedure does affect the early stage progenitor cells-CFU-GEMM in the CD34+- type cryopreserved cells in UB. The damage may be induced by the cell apoptosis.

Bovine central memory T cells are highly proliferative in response to bovine tuberculosis infection

USDA-ARS?s Scientific Manuscript database

Long-term (i.e., 14 days) cultured IFN-gamma responses of peripheral blood mononuclear cells are used as a correlate of T cell central memory (Tcm) responses in both humans and cattle. With bovine tuberculosis, vaccine-elicited long-term IFN-gamma ELISPOT assays are a correlate of protection. Recent...

Phenotype and function of CD209+ bovine blood dendritic cells, monocyte-derived-dendritic cells and monocyte-derived macrophages

USDA-ARS?s Scientific Manuscript database

Phylogenic comparisons of the mononuclear phagocyte system (MPS) of humans and mice demonstrate phenotypic divergence of dendritic cell (DC) subsets that play similar roles in innate and adaptive immunity. Although differing in phenotype, DC can be classified into four groups according to ontogeny a...

Excess apoptosis of mononuclear cells contributes to the depressed cytomegalovirus-specific immunity in HIV-infected patients on HAART

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weinberg, Adriana; Jesser, Renee D.; Edelstein, Charles L.

2004-12-05

HIV-infected patients on highly active antiretroviral therapy (HAART) have persistently decreased cytomegalovirus (CMV)-specific proliferative responses [lymphocyte proliferation assay (LPA)] in spite of increases in CD4+ T cell counts. Here we demonstrate an association between apoptosis of unstimulated peripheral blood mononuclear cells (uPBMC) and decreased CMV-LPA. HAART recipients had more apoptosis of uPBMC than controls when measured by caspases 3, 8, and 9 activities and by annexin V binding. Patients with undetectable HIV replication maintained significantly higher apoptosis of CD4+ and CD14+ cells compared to controls. CMV-LPA decreased with higher apoptosis of uPBMC in patients only. This association was independent ofmore » CD4+ cell counts or HIV replication. Furthermore, rescuing PBMC from apoptosis with crmA, but not with TRAIL- or Fas-pathway blocking agents or with other caspase inhibitors, increased CMV-LPA in HAART recipients. This effect was not observed in uninfected controls, further indicating that the down regulatory effect of apoptosis on cell-mediated immunity (CMI) was specifically associated with the HIV-infected status.« less

Idiotypic Cascades in Cancer Patients Treated with Monoclonal Antibody CO17-1A

NASA Astrophysics Data System (ADS)

Wettendorff, Martine; Iliopoulos, Dimitrios; Tempero, Margaret; Kay, David; Defreitas, Elaine; Koprowski, Hilary; Herlyn, Dorothee

1989-05-01

We have previously shown that gastrointestinal cancer patients treated with monoclonal antibody CO17-1A (Ab1) developed anti-idiotypic antibodies (Ab2) to the Ab1. We now demonstrate that patients produce anti-anti-idiotypic antibodies (Ab3) to their autologous Ab2. Ab3 were demonstrated in culture supernatants of peripheral blood mononuclear cells from five Ab1-treated patients after stimulation of the cells with heterologous Ab2 that functionally mimicked the tumor antigen (Ag) defined by Ab1 and immunologically cross reacted with the patients' Ab2. Ab3 shared idiotopes with Ab1 and were Ab1-like in their binding specificities to tumor cells, Ag, and Ab2. Such antibodies were also elicited by stimulating cells with Ag. However, they were not produced by stimulating posttreatment mononuclear cells with control proteins or by stimulating pretreatment cells with either Ag or Ab2. Our results demonstrate idiotypic cascades in cancer patients treated with monoclonal antibody. Ag-specific Ab3 responses may underlie delayed clinical responses often observed in cancer patients treated with monoclonal antibodies of various specificities.

Macrophage phenotype is associated with disease severity in preterm infants with chronic lung disease.

PubMed

Prince, Lynne R; Maxwell, Nicola C; Gill, Sharonjit K; Dockrell, David H; Sabroe, Ian; McGreal, Eamon P; Kotecha, Sailesh; Whyte, Moira K

2014-01-01

The etiology of persistent lung inflammation in preterm infants with chronic lung disease of prematurity (CLD) is poorly characterized, hampering efforts to stratify prognosis and treatment. Airway macrophages are important innate immune cells with roles in both the induction and resolution of tissue inflammation. To investigate airway innate immune cellular phenotypes in preterm infants with respiratory distress syndrome (RDS) or CLD. Bronchoalveolar lavage (BAL) fluid was obtained from term and preterm infants requiring mechanical ventilation. BAL cells were phenotyped by flow cytometry. Preterm birth was associated with an increase in the proportion of non-classical CD14(+)/CD16(+) monocytes on the day of delivery (58.9 ± 5.8% of total mononuclear cells in preterm vs 33.0 ± 6.1% in term infants, p = 0.02). Infants with RDS were born with significantly more CD36(+) macrophages compared with the CLD group (70.3 ± 5.3% in RDS vs 37.6 ± 8.9% in control, p = 0.02). At day 3, infants born at a low gestational age are more likely to have greater numbers of CD14(+) mononuclear phagocytes in the airway (p = 0.03), but fewer of these cells are functionally polarized as assessed by HLA-DR (p = 0.05) or CD36 (p = 0.05) positivity, suggesting increased recruitment of monocytes or a failure to mature these cells in the lung. These findings suggest that macrophage polarization may be affected by gestational maturity, that more immature macrophage phenotypes may be associated with the progression of RDS to CLD and that phenotyping mononuclear cells in BAL could predict disease outcome.

[In vitro effect of lupeol and casearin G on peripheral blood mononuclear and tumor cells].

PubMed

Dupuy L, Omar A; Bonilla V, José A; Murillo, Renato; Taylor, Peter; Abad, María Jesús; González, Lorena; Juliao A, Johanna

2013-09-01

The rainforest is an important source of natural compounds with therapeutic properties. Although there are many anti-inflammatory and antineoplastic drugs available to the clinician, there is an ongoing need for new therapeutic drugs with fewer serious adverse effects. To evaluate the in vitro cytotoxic effects of lupeol and casearin G on tumor cells, on phagocytic activity and nitric oxide (NO) production by blood mononuclear cells. The cytotoxic effect of these compounds on cell lines MCF-7 (human breast adenocarcinoma) and PC-3 (human prostate cancer) was measured by a colorimetric assay (MTS/PMS) and the sulphorhodamine B assay. Peripheral blood mononuclear cells were obtained from eight healthy volunteers. The effect of these compounds on nitric oxide (NO) production was measured using the Griess reaction. Their effect on phagocytic activity of PBMC was also evaluated. Lupeol (≥ 2 mM) resulted in a reduction of both the phagocytic index and the percentage of phagocytic monocytes and macrophages. Treatment of monocytes/macrophages with lupeol (72 µM) and casearin G (4 µM) reduced the production of NO. Neither lupeol (< 969 µM) nor casearin G (< 55 µM) had cytotoxic effects on PBMC. Casearin G showed both cytotoxic (IC50, LC50) and cytostatic (GI50) effects against tumor cells, PC-3 (IC50 = 12.5 µM; GI50 = 13.3 µM; LC50 = 51.9 µM) and MCF-7 (IC50 = 112.8 µM; GI50 = 11.8 µM; LC50 = 49.4 µM), as well as a hemolytic effect (≥ 182 µM). These observations indicate that lupeol and casearin G might be useful compounds in the preparation of anti-inflammatory drugs, whereas casearin G might be useful in the elaboration of antitumor drugs.

Cytokine regulation on the synthesis of nitric oxide in vivo by chronically infected human polymorphonuclear leucocytes.

PubMed Central

Takeichi, O; Saito, I; Okamoto, Y; Tsurumachi, T; Saito, T

1998-01-01

To determine if nitric oxide (NO) is produced by chronically infected human polymorphonuclear leucocytes (PMNs) in vivo, inflamed exudates (periapical exudates: PE) collected from periapical periodontitis patients were examined. Cell-free supernatants and cells were separated by centrifugation. Significant levels of nitrite concentrations were observed in the supernatants. The production of inducible NO synthase (iNOS) in highly purified PMNs derived from PEs was then immunocytochemically determined using rabbit anti-human iNOS antiserum. In vitro, human peripheral blood PMNs (PB-PMNs) isolated from patients were cultured with a combination of Esherichia coli-lipopolysaccharide (LPS), recombinant human interferon-gamma (rhIFN-gamma) and/or interleukin-1 beta (rhIL-1 beta). The stimulated PB-PMNs showed steady-state levels of nitrite. The stimulation of LPS, rhIFN-gamma and rhIL-1 beta showed more NO induction than that of LPS with either IFN-gamma or IL-1 beta, suggesting the synergistic effects of cytokines. Cryostat sections of surgically removed periapical tissues were also immunohistochemically examined for iNOS, IFN-gamma and IL-1 beta. Two-colour immunohistochemistry revealed the interaction of iNOS-producing PMNs and IFN-gamma- or IL-1 beta-producing mononuclear cells. On the basis of these data, we concluded that with the stimulation of inflammatory cytokines derived from mononuclear cells, PMNs can spontaneously produce NO at the site of chronic infection. The present studies are consistent with a hypothesis suggesting that PMNs could be regulated and delicately balanced to produce NO by mononuclear cell-derived cytokines in vivo. NO-producing cells may play a pivotal role in chronic inflammation. Images Figure 2 Figure 4 Figure 5 Figure 6 PMID:9616379

T-helper cell type 17/regulatory T-cell immunoregulatory balance in human radicular cysts and periapical granulomas.

PubMed

Marçal, Juliana R B; Samuel, Renata O; Fernandes, Danielle; de Araujo, Marcelo S; Napimoga, Marcelo H; Pereira, Sanivia A L; Clemente-Napimoga, Juliana T; Alves, Polyanna M; Mattar, Rinaldo; Rodrigues, Virmondes; Rodrigues, Denise B R

2010-06-01

Cysts and granulomas are chronic periapical lesions mediated by a set of inflammatory mediators that develop to contain a periapical infection. This study analyzed the nature of the inflammatory infiltrate, presence of mast cells, and in situ expression of cytokines (interleukin [IL]-17 and transforming growth factor [TGF]-beta), chemokines (macrophage inflammatory protein [MIP]-1beta and monocyte chemotactic protein [MCP]-1), and nuclear transcription factor (FoxP3) in human periapical granulomas and cysts compared with a control group. Fifty-five lesions (25 periapical cysts, 25 periapical granulomas, and 5 controls) were analyzed. The type of inflammatory infiltrate was evaluated by hematoxylin-eosin staining, and the presence of mast cells was analyzed by toluidine blue staining. Indirect immunohistochemistry was used to evaluate the expression of cytokines, chemokines, and FoxP3. The inflammatory infiltrate mainly consisted of mononuclear cells. In cysts, mononuclear infiltrates were significantly more frequent than mixed (polymorphonuclear/mononuclear) infiltrates (P = .04). Mixed inflammatory infiltrates were significantly more frequent in patients with sinus tract (P = .0001). The number of mast cells was significantly higher in granulomas than in cystic lesions (P = .02). A significant difference in the expression of IL-17 (P = .001) and TGF-beta (P = .003) was observed between cysts and granulomas and the control group. Significantly higher IL-17 levels were also observed in cases of patients with sinus tract (P = .03). We observed that chronic periapical lesions might experience a reagudization process that is correlated with an increased leukocyte infiltration, with the predominance of neutrophils attracted by a chemokine milieu, as well as the increased presence of IL-17. Copyright 2010 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Trypanosoma congolense: tissue distribution of long-term T- and B-cell responses in cattle.

PubMed

Lutje, V; Taylor, K A; Boulangé, A; Authié, E

1995-11-01

Memory T- and B-cell responses to trypanosome antigens were measured in peripheral blood mononuclear cells, spleen and lymph node cells obtained from four trypanotolerant N'Dama cattle which had been exposed to six experimental infections with Trypanosoma congolense. These cattle were treated with trypanocidal drugs following each infection and had remained aparasitemic for 3 years prior to this study. The antigens used were whole trypanosome lysate, variable surface glycoprotein, a 33-kDa cysteine protease (congopain) and a 70-kDa heat-shock protein. As parameters of T-cell-mediated immunity, we measured T-cell proliferation and IFN-gamma production. Lymph node cells, spleen cells and peripheral blood mononuclear cells all proliferated to a mitogenic stimulus (concanavalin A) but only lymph node cells responded to trypanosome antigens. Similarly, IFN-gamma was produced by both lymph node and spleen cells stimulated with concanavalin A but only by lymph node cells stimulated with variable surface glycoprotein and whole trypanosome lysate. T. congolense-specific antibodies were detected in sera and in supernatants of cultured lymph node and spleen cells after in vitro stimulation with lipopolysaccharide and recombinant bovine interleukin-2. In conclusion, we have demonstrated that memory T- and B-cell responses are detectable in various lymphoid organs in cattle 3 years following infection and treatment with T. congolense.

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

PubMed Central

Komuta, Yukari; Ishii, Toshiyuki; Kaneda, Makoto; Ueda, Yasuji; Miyamoto, Kiyoko; Toyoda, Masashi; Umezawa, Akihiro

2016-01-01

ABSTRACT 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. PMID:27170256

Cryopreservation for delayed circulating tumor cell isolation is a valid strategy for prognostic association of circulating tumor cells in gastroesophageal cancer.

PubMed

Brungs, Daniel; Lynch, David; Luk, Alison Ws; Minaei, Elahe; Ranson, Marie; Aghmesheh, Morteza; Vine, Kara L; Carolan, Martin; Jaber, Mouhannad; de Souza, Paul; Becker, Therese M

2018-02-21

To demonstrate the feasibility of cryopreservation of peripheral blood mononuclear cells (PBMCs) for prognostic circulating tumor cell (CTC) detection in gastroesophageal cancer. Using 7.5 mL blood samples collected in EDTA tubes from patients with gastroesopheagal adenocarcinoma, CTCs were isolated by epithelial cell adhesion molecule based immunomagnetic capture using the IsoFlux platform. Paired specimens taken during the same blood draw ( n = 15) were used to compare number of CTCs isolated from fresh and cryopreserved PBMCs. Blood samples were processed within 24 h to recover the PBMC fraction, with PBMCs used for fresh analysis immediately processed for CTC isolation. Cryopreservation of PBMCs lasted from 2 wk to 25.2 mo (median 14.6 mo). CTCs isolated from pre-treatment cryopreserved PBMCs ( n = 43) were examined for associations with clinicopathological variables and survival outcomes. While there was a significant trend to a decrease in CTC numbers associated with cryopreserved specimens (mean number of CTCs 34.4 vs 51.5, P = 0.04), this was predominately in samples with a total CTC count of > 50, with low CTC count samples less affected ( P = 0.06). There was no significant association between the duration of cryopreservation and number of CTCs. In cryopreserved PBMCs from patient samples prior to treatment, a high CTC count (> 17) was associated with poorer overall survival (OS) ( n = 43, HR = 4.4, 95%CI: 1.7-11.7, P = 0.0013). In multivariate analysis, after controlling for sex, age, stage, ECOG performance status, and primary tumor location, a high CTC count remained significantly associated with a poorer OS (HR = 3.7, 95%CI: 1.2-12.4, P = 0.03). PBMC cryopreservation for delayed CTC isolation is a valid strategy to assist with sample collection, transporting and processing.

Change in the Content of Immunoproteasomes and Macrophages in Rat Liver At the Induction of Donor-Specific Tolerance.

PubMed

Karpova, Ya D; Ustichenko, V D; Alabedal'karim, N M; Stepanova, A A; Lyupina, Yu V; Boguslavski, K I; Bozhok, G A; Sharova, N P

2017-01-01

Induction of donor specific tolerance (DST) by the introduction of donor cells into a recipient's portal vein is one of the approaches used to solve the problem of transplant engraftment. However, the mechanism of DST development remains unclear to this moment. In the present work, we first studied the change in the content of immunoproteasomes and macrophages of the liver at early stages of the development of allospecific portal tolerance in rats by Western blotting and flow cytofluorimetry. On the basis of the data obtained, we can conclude that the induction of DST is an active process characterized by two phases during which the level of the proteasome immune subunits LMP2 and LMP7 in liver mononuclear cells, including Kupffer cells, and the number of Kupffer cells change. The first phase lasts up to 5 days after the beginning of DST induction; the second phase - from 5 to 14 days. In both phases, the level of the subunits LMP2 and LMP7 in the total pool of mononuclear cells and Kupffer cells increases, with maximum values on days 1 and 7. In addition, the total number of Kupffer cells increases in both phases with a shift in several days. The most noticeable changes take place in the second phase. The third day is characterized by a lower content of mononuclear cells expressing immunoproteasomes compared to the control value in native animals. Presumably, at this time point a "window of opportunity" appears for subsequent filling of an empty niche with cells of different subpopulations and, depending on this fact, the development of tolerance or rejection. The results obtained raise the new tasks of finding ways to influence the cellular composition in the liver and the expression of immunoproteasomes on the third day after the beginning of DST induction to block the development of rejection.

Resveratrol Alters Proliferative Responses and Apoptosis in Human Activated B Lymphocytes In Vitro

USDA-ARS?s Scientific Manuscript database

We hypothesized that resveratrol, a polyphenol found in grapes, peanuts, and berries would modulate B lymphocyte proliferation, immunoglobulin synthesis, and apoptosis after activation with T-cell dependent pokeweed mitogen. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of ...

HIV-1 DNA levels in peripheral blood mononuclear cells and cannabis use are associated with intermittent HIV shedding in semen of men who have sex with men on successful antiretroviral regimens.

PubMed

Ghosn, Jade; Leruez-Ville, Marianne; Blanche, Jérôme; Delobelle, Aurore; Beaudoux, Céline; Mascard, Laurence; Lecuyer, Hervé; Canestri, Ana; Landman, Roland; Zucman, David; Ponscarme, Diane; Rami, Agathe; Viard, Jean-Paul; Spire, Bruno; Rouzioux, Christine; Costagliola, Dominique; Suzan-Monti, Marie

2014-06-01

Few data exist on the efficacy of combined antiretroviral therapy (cART) in semen of human immunodeficiency virus type 1 (HIV-1) infected men who have sex with men (MSM) with sustained control of HIV replication in blood. HIV-1 infected MSM on successful cART for >6 months were enrolled. HIV-RNA was quantified in seminal plasma (spVL) and in blood plasma (bpVL) from 2 paired samples collected 4 weeks apart. Relationship between spVL and bpVL (measured by an ultrasensitive assay, LOQ 10 copies/mL), total peripheral blood mononuclear cells (PBMC)-associated HIV-DNA, sexually transmitted infections (STIs), and self-reported socio-behavioral characteristics was assessed using GEE logistic regression. In total, 157 patients were included. Median time with bpVL <50 copies/mL was 3.3 years. spVL was detectable in 23/304 samples (prevalence 7.6%). Median spVL was 145 cp/mL (100-1475). spVL was detectable on the first, on the second, and on both samples in 5, 14, and 2 men, respectively. In sum, 33 individuals (21%) had STIs (asymptomatic in 24/33). Residual bpVL was undetectable by ultrasensitive assay in 225/300 samples (75%). After multivariable adjustments, PBMC-associated HIV-DNA (OR 2.6[1.2; 6.0], for HIV-DNA > 2.5 log10 cp/10(6) PBMC, P = .02), and cannabis use during sexual intercourse (OR 2.8[1.2; 6.7], P = .02) were the only factors associated significantly with spVL. We show that HIV-RNA can be detected intermittently in semen of HIV-1 infected MSM despite successful cART. The size of blood HIV-1 reservoir predicted spVL detection. Our results indicated also that the possible effect of cannabis should be taken into account when developing prevention interventions targeted toward HIV-infected MSM on successful cART. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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

PubMed

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

2015-08-15

This paper compares results of peripheral blood mononuclear cell (PBMC) incubation with fexofenadine (FXF) and osthole. FXF is a third-generation antihistamine drug and osthole is assumed a natural antihistamine alternative. To our best knowledge, this is the first comparative study on FXF, osthole and histamine cytokine secretion and cytotoxicity in PBMC in vitro cultures using cell proliferation ELISA BrdU. The cultures were treated 12, 42, 48 and 72h with FXF and osthole at 150, 300 and 450ng/ml concentrations and histamine at 50, 100 and 200ng/ml. Our study results confirm that FXF, osthole and histamine exert no cytotoxic effect on PBMCs and that IL-6, IL-10 and TNF-α cytokine secretion following osthole cell stimulation was similar to that by FXF stimulation.This confirms our hypothesis that osthole is a natural histamine antagonist, and can therefore be beneficially applied in antihistamine treatment. Copyright © 2015 Elsevier B.V. All rights reserved.

Identification of CD3+ T lymphocytes in the green turtle Chelonia mydas

USGS Publications Warehouse

Munoz, F.A.; Estrada-Parra, S.; Romero-Rojas, A.; Work, Thierry M.; Gonzalez-Ballesteros, E.; Estrada-Garcia, I.

2009-01-01

To understand the role of the immune system with respect to disease in reptiles, there is the need to develop tools to assess the host's immune response. An important tool is the development of molecular markers to identify immune cells, and these are limited for reptiles. We developed a technique for the cryopreservation of peripheral blood mononuclear cells and showed that a commercially available anti-CD3 epsilon chain antibody detects a subpopulation of CD3 positive peripheral blood lymphocytes in the marine turtle Chelonia mydas. In the thymus and in skin inoculated with phytohemagglutinin, the same antibody showed the classical staining pattern observed in mammals and birds. For Western blot, the anti-CD3 antibodies identified a 17.6 kDa band in membrane proteins of peripheral blood mononuclear cell compatible in weight to previously described CD3 molecules. This is the first demostration of CD3+ cells in reptiles using specific antibodies.

MHC class I, MHC class II and intercellular adhesion molecule-1 (ICAM-1) expression in inflammatory myopathies.

PubMed Central

Bartoccioni, E; Gallucci, S; Scuderi, F; Ricci, E; Servidei, S; Broccolini, A; Tonali, P

1994-01-01

We investigated the relationship between the MHC-I, MHC-II and intercellular adhesion molecule-1 (ICAM-1) expression on myofibres and the presence of inflammatory cells in muscle specimens of 18 patients with inflammatory myopathies (nine polymyositis, seven dermatomyositis, two inclusion body myositis). We observed MHC-I expression in muscle fibres, infiltrating mononuclear cells and endothelial cells in every specimen. In seven patients, some muscle fibres were MHC-II-positive for the DR antigen, while the DP and DQ antigens were absent. ICAM-1 expression, detected in seven patients, was found in clusters of myofibres, associated with a marked MHC-I positivity and a widespread mononuclear infiltration. Most of the ICAM-1-positive fibres were regenerating fibres. Furthermore, some fibres expressed both ICAM-1 and DR antigens near infiltrating cells. This finding could support the hypothesis that myofibres may themselves be the site of autosensitization. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7507012

Stress-induced alterations in interferon production and class II histocompatibility antigen expression

NASA Technical Reports Server (NTRS)

Sonnenfeld, G.; Cunnick, J. E.; Armfield, A. V.; Wood, P. G.; Rabin, B. S.

1992-01-01

Mild electric foot-shock has been shown to be a stressor that can alter immune responses. Male Lewis rats were exposed to one session of 16 5.0-s 1.6-mA foot-shocks. Production of interferon-gamma by splenocytes in response to concanavalin-A was decreased in spleens from the shocked rats compared to control spleens. Spleen cells from rats treated with nadolol, a peripherally acting beta-adrenergic receptor antagonist, and then shocked, showed dose-dependent attenuation of the suppression of interferon-gamma production. This suggests that catecholamines mediate shock-induced suppression of interferon-gamma production. The percentage of splenic mononuclear cells expressing class II histocompatibility (Ia) antigens on their surfaces from spleens of shocked rats was determined by flow cytometry. Significantly decreased class II positive mononuclear cells were present in the spleens of shocked rats in comparison to the spleens of control rats. This may reflect an alteration of cell trafficking or decreased production of class II antigens.

Bruton's Tyrosine Kinase: An Emerging Key Player in Innate Immunity.

PubMed

Weber, Alexander N R; Bittner, Zsofia; Liu, Xiao; Dang, Truong-Minh; Radsak, Markus Philipp; Brunner, Cornelia

2017-01-01

Bruton's tyrosine kinase (BTK) was initially discovered as a critical mediator of B cell receptor signaling in the development and functioning of adaptive immunity. Growing evidence also suggests multiple roles for BTK in mononuclear cells of the innate immune system, especially in dendritic cells and macrophages. For example, BTK has been shown to function in Toll-like receptor-mediated recognition of infectious agents, cellular maturation and recruitment processes, and Fc receptor signaling. Most recently, BTK was additionally identified as a direct regulator of a key innate inflammatory machinery, the NLRP3 inflammasome. BTK has thus attracted interest not only for gaining a more thorough basic understanding of the human innate immune system but also as a target to therapeutically modulate innate immunity. We here review the latest developments on the role of BTK in mononuclear innate immune cells in mouse versus man, with specific emphasis on the sensing of infectious agents and the induction of inflammation. Therapeutic implications for modulating innate immunity and critical open questions are also discussed.

A novel method for evaluating antibody-dependent cell-mediated cytotoxicity by flowcytometry using cryopreserved human peripheral blood mononuclear cells

PubMed Central

Yamashita, Makiko; Kitano, Shigehisa; Aikawa, Hiroaki; Kuchiba, Aya; Hayashi, Mitsuhiro; Yamamoto, Noboru; Tamura, Kenji; Hamada, Akinobu

2016-01-01

Analyzing the cytotoxic functions of effector cells, such as NK cells against target cancer cells, is thought to be necessary for predicting the clinical efficacy of antibody-dependent cellular cytotoxicity (ADCC) -dependent antibody therapy. The 51Cr release assay has long been the most widely used method for quantification of ADCC activity. However, the reproducibilities of these release assays are not adequate, and they do not allow evaluation of the lysis susceptibilities of distinct cell types within the target cell population. In this study, we established a novel method for evaluating cytotoxicity, which involves the detection and quantification of dead target cells using flowcytometry. CFSE (carboxyfluorescein succinimidyl ester) was used as a dye to specifically stain and thereby label the target cell population, allowing living and dead cells, as well as both target and effector cells, to be quantitatively distinguished. Furthermore, with our new approach, ADCC activity was more reproducibly, sensitively, and specifically detectable, not only in freshly isolated but also in frozen human peripheral blood mononuclear cells (PBMCs), than with the calcein-AM release assay. This assay, validated herein, is expected to become a standard assay for evaluating ADCC activity which will ultimately contribute the clinical development of ADCC dependent-antibody therapies. PMID:26813960

A novel method for evaluating antibody-dependent cell-mediated cytotoxicity by flowcytometry using cryopreserved human peripheral blood mononuclear cells.

PubMed

Yamashita, Makiko; Kitano, Shigehisa; Aikawa, Hiroaki; Kuchiba, Aya; Hayashi, Mitsuhiro; Yamamoto, Noboru; Tamura, Kenji; Hamada, Akinobu

2016-01-27

Analyzing the cytotoxic functions of effector cells, such as NK cells against target cancer cells, is thought to be necessary for predicting the clinical efficacy of antibody-dependent cellular cytotoxicity (ADCC) -dependent antibody therapy. The (51)Cr release assay has long been the most widely used method for quantification of ADCC activity. However, the reproducibilities of these release assays are not adequate, and they do not allow evaluation of the lysis susceptibilities of distinct cell types within the target cell population. In this study, we established a novel method for evaluating cytotoxicity, which involves the detection and quantification of dead target cells using flowcytometry. CFSE (carboxyfluorescein succinimidyl ester) was used as a dye to specifically stain and thereby label the target cell population, allowing living and dead cells, as well as both target and effector cells, to be quantitatively distinguished. Furthermore, with our new approach, ADCC activity was more reproducibly, sensitively, and specifically detectable, not only in freshly isolated but also in frozen human peripheral blood mononuclear cells (PBMCs), than with the calcein-AM release assay. This assay, validated herein, is expected to become a standard assay for evaluating ADCC activity which will ultimately contribute the clinical development of ADCC dependent-antibody therapies.

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

PubMed

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.

Morphological and functional determinants of fluoxetine (Prozac)-induced pulmonary disease in an experimental model.

PubMed

Capelozzi, Marco A; Leick-Maldonado, Edna A; Parra, Edwin R; Martins, Mílton A; Tibério, Iolanda F L C; Capelozzi, Vera L

2007-05-14

Fluoxetine treatment effects were determined by evaluating respiratory mechanics (elastance/resistance) and exhaled nitric oxide, as well as mononuclear and polymorphonuclear cell recruitment into the lungs, in an experimental guinea pig model. Guinea pigs were divided into four groups: Fl (fluoxetine only, n=7); Fl+Sw (fluoxetine and forced swimming, n=7); Ns+Sw (normal saline and forced swimming, n=8); and Ns (normal saline only, n=8). Treated animals received oral fluoxetine (10 mg/(kg day)) for 30 consecutive days. On day 31, all animals were anesthetized and mechanically ventilated so that respiratory system elastance and resistance, as well exhaled nitric oxide, could be determined. The lungs were then excised en bloc for histological and immunohistochemical evaluation. Forced swimming induced bronchodilation in untreated animals and bronchoconstriction in fluoxetine-treated animals. Fluoxetine treatment was also associated with mononuclear infiltration (predominantly into alveolar walls) and neutrophil recruitment. In addition, levels of exhaled nitric oxide, an inflammatory marker, were higher in fluoxetine-treated animals. Swimming-induced stress also amplified mononuclear cell recruitment to the lungs. These results show that, in this experimental model, fluoxetine treatment reproduces the pathology of chronic interstitial pneumonia in humans.

Digital-Direct-RT-PCR: a sensitive and specific method for quantification of CTC in patients with cervical carcinoma

PubMed Central

Pfitzner, Claudia; Schröder, Isabel; Scheungraber, Cornelia; Dogan, Askin; Runnebaum, Ingo Bernhard; Dürst, Matthias; Häfner, Norman

2014-01-01

The detection of circulating tumour cells (CTC) in cancer patients may be useful for therapy monitoring and prediction of relapse. A sensitive assay based on HPV-oncogene transcripts which are highly specific for cervical cancer cells was established. The Digital-Direct-RT-PCR (DD-RT-PCR) combines Ficoll-separation, ThinPrep-fixation and one-step RT-PCR in a low-throughput digital-PCR format enabling the direct analysis and detection of individual CTC without RNA isolation. Experimental samples demonstrated a sensitivity of one HPV-positive cell in 500,000 HPV-negative cells. Spike-in experiments with down to 5 HPV-positive cells per millilitre EDTA-blood resulted in concordant positive results by PCR and immunocytochemistry. Blood samples from 3 of 10 CxCa patients each contained a single HPV-oncogene transcript expressing CTC among 5 to 15*105 MNBC. Only 1 of 7 patients with local but 2 of 3 women with systemic disease had CTC. This highly sensitive DD-RT-PCR for the detection of CTC may also be applied to other tumour entities which express tumour-specific transcripts. Abbreviations: CTC – circulating tumour cells, CxCa – cervical cancer, DD-RT-PCR – Digital-Direct Reverse Transcriptase PCR, HPV – Human Papilloma Virus, MNBC – mononuclear blood cells, ICC – immunocytochemistry. PMID:24496006

Antimicrobial-induced endotoxin and cytokine activity in an in vitro model of septicemia in foals.

PubMed

Bentley, Adrienne P; Barton, Michelle H; Lee, Margie D; Norton, Natalie A; Moore, James N

2002-05-01

To determine which antimicrobials that are used to treat neonatal foals with septicemia attributable to Escherichia coli will minimize endotoxin release from bacteria and subsequent activity of inflammatory mediators while maintaining bactericidal efficacy. Blood samples from 10 healthy foals. Escherichia coli isolates A and B were isolated from 2 septicemic foals, and minimal inhibitory concentrations (MIC) were determined for 9 antimicrobials. Five of these antimicrobials were tested in vitro at 2 and 20 times their respective MIC. Whole blood or mononuclear cells grown in tissue-culture media were incubated with 105 colony-forming units of E. coli and each antimicrobial or saline (0.9% NaCl) solution. After 6 hours, number of viable bacteria remaining was determined, and supernatant was tested for endotoxin and tumor necrosis activity. Testing in whole blood was compromised by bactericidal effects of the blood itself. In mononuclear cell suspensions, each antimicrobial significantly reduced the number of viable bacteria to low or undetectable amounts. Antimicrobials did not differ significantly in efficacy of bacterial killing. Amikacin used alone or in combination with ampicillin resulted in significantly less endotoxin activity than did ampicillin, imipenem, or ceftiofur alone. There was a correlation between TNF-alpha and endotoxin activity. Aminoglycosides appear less likely to induce endotoxemia and TNF-alpha synthesis during bactericidal treatment of E. coli septicemia, compared with beta-lactam antimicrobials. Use of ampicillin, imipenem, or ceftiofur in the treatment of septicemic neonatal foals should be accompanied by appropriate treatment for endotoxemia.

Patient-specific cardiovascular progenitor cells derived from integration-free induced pluripotent stem cells for vascular tissue regeneration.

PubMed

Hu, Jiang; Wang, Yongyu; Jiao, Jiao; Liu, Zhongning; Zhao, Chao; Zhou, Zhou; Zhang, Zhanpeng; Forde, Kaitlynn; Wang, Lunchang; Wang, Jiangang; Baylink, David J; Zhang, Xiao-Bing; Gao, Shaorong; Yang, Bo; Chen, Y Eugene; Ma, Peter X

2015-12-01

Tissue-engineered blood vessels (TEBVs) are promising in regenerating a live vascular replacement. However, the vascular cell source is limited, and it is crucial to develop a scaffold that accommodates new type of vascular progenitor cells and facilitates in vivo lineage specification of the cells into functional vascular smooth muscle cells (VSMCs) to regenerate vascular tissue. In the present study, integration-free human induced pluripotent stem cells (hiPSCs) were established from patient peripheral blood mononuclear cells through episomal vector nucleofection of reprogramming factors. The established hiPSCs were then induced into mesoderm-originated cardiovascular progenitor cells (CVPCs) with a highly efficient directed lineage specification method. The derived CVPCs were demonstrated to be able to differentiate into functional VSMCs. Subcutaneous implantation of CVPCs seeded on macroporous nanofibrous poly(l-lactide) scaffolds led to in vivo VSMC lineage specification and matrix deposition inside the scaffolds. In summary, we established integration-free patient-specific hiPSCs from peripheral blood mononuclear cells, derived CVPCs through directed lineage specification, and developed an advanced scaffold for these progenitor cells to further differentiate in vivo into VSMCs and regenerate vascular tissue in a subcutaneous implantation model. This study has established an efficient patient-specific approach towards in vivo regeneration of vascular tissue. Copyright © 2015 Elsevier Ltd. All rights reserved.

Umbilical cord CD71+ erythroid cells are reduced in neonates born to women in spontaneous preterm labor.

PubMed

Gomez-Lopez, Nardhy; Romero, Roberto; Xu, Yi; Miller, Derek; Unkel, Ronald; C MacKenzie, Tippi; Frascoli, Michela; Hassan, Sonia S

2016-10-01

Preterm neonates are highly susceptible to infection. Neonatal host defense against infection seems to be maintained by the temporal presence of immunosuppressive CD71+ erythroid cells. The aim of this study was to investigate whether umbilical cord CD71+ erythroid cells are reduced in neonates born to women who undergo spontaneous preterm labor/birth. Umbilical cord blood samples (n=155) were collected from neonates born to women who delivered preterm with (n=39) and without (n=12) spontaneous labor or at term with (n=82) and without (n=22) spontaneous labor. Time-matched maternal peripheral blood samples were also included (n=111). Mononuclear cells were isolated from these samples, and CD71+ erythroid cells were identified and quantified as CD3-CD235a+CD71+ cells by flow cytometry. (i) The proportion of CD71+ erythroid cells was 50-fold higher in cord blood than in maternal blood; (ii) a reduced number and frequency of umbilical cord CD71+ erythroid cells were found in neonates born to women who underwent spontaneous preterm labor compared to those born to women who delivered preterm without labor; (iii) umbilical cord CD71+ erythroid cells were fewer in neonates born to term pregnancies, regardless of the process of labor, than in those born to women who delivered preterm without labor; and (iv) no differences were seen in umbilical cord CD71+ erythroid cells between neonates born to women who underwent spontaneous preterm labor and those born to women who delivered at term with labor. Umbilical cord CD71+ erythroid cells are reduced in neonates born to women who had undergone spontaneous preterm labor. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Placental expression of asialoglycoprotein receptor associated with Hepatitis B virus transmission from mother to child.

PubMed

Vyas, Ashish Kumar; Ramakrishna, Usha; Sen, Bijoya; Islam, Mojahidul; Ramakrishna, Gayatri; Patra, Sharda; Rastogi, Archana; Sarin, Shiv Kumar; Trehanpati, Nirupma

2018-04-30

Asialoglycoprotein receptor expression on hepatocytes has been associated with endocytosis, binding and uptake of hepatitis B virus. The role of asialoglycoprotein receptor in hepatitis B virus vertical transmission and its expression on placenta has not yet been studied. Thirty-four HBsAg+ve and 13 healthy pregnant mothers along with their newborns were enrolled. The former were categorized into transmitting and non-transmitting mothers based on their newborns being hepatitis B surface antigen and hepatitis B virus DNA positive. Expression of asialoglycoprotein receptor and hepatitis B surface antigen in placenta and isoform of asialoglycoprotein receptor on dendritic cell in peripheral and cord blood dendritic cells were analysed using flowcytometry, immune histochemistry, immune florescence and qRT-PCR. Twelve HBsAg+ve mothers transmitted hepatitis B virus to their newborns whereas the rest (n = 22) did not. Hepatitis B virus-transmitting mothers showed increased expression of asialoglycoprotein receptor in trophoblasts of placenta. Immunofluorescence microscopy revealed colocalization of hepatitis B surface antigen and asialoglycoprotein receptor in placenta as well as in DCs of transmitting mothers. There was no significant difference in the expression of asialoglycoprotein receptor on peripheral blood mononuclear cells or chord blood mononuclear cells between the 2 groups. However, hepatitis B virus-transmitting mothers and their HBsAg+ve newborns showed increased mRNA levels of isoform of asialoglycoprotein receptor on dendritic cell in peripheral blood mononuclear cells. Hepatitis B virus-transmitting mothers and their HBsAg+ve newborns showed an increased expression of isoform of asialoglycoprotein receptor on dendritic cell on circulating dendritic cells compared to hepatitis B virus non-transmitting mothers and their negative newborns. This study revealed that increased expression of asialoglycoprotein receptor in placenta and colocalization with hepatitis B surface antigen strongly indicates its role in intrauterine transmission of hepatitis B virus. Asialoglycoprotein receptor-blocking strategy can be used for therapeutic intervention of vertical transmission. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Comparative study of the effect of chloro-, dichloro-, bromo-, and dibromoacetic acid on necrotic, apoptotic and morphological changes in human peripheral blood mononuclear cells (in vitro study).

PubMed

Michałowicz, Jaromir; Wróblewski, Wojciech; Mokra, Katarzyna; Maćczak, Aneta; Kwiatkowska, Marta

2015-10-01

In this study, the effect of monochloroacetic acid (MCAA), dichloroacetic acid (DCAA), monobromoacetic acid (MBAA) and dibromoacetic acid (DBAA) on human peripheral blood mononuclear cells (PBMCs) was assessed. HAAs studied induced at millimolar concentrations necrotic alterations in PBMCs with the strongest effect noted for MBAA and DBAA. Chloro- and bromoacetic acids also provoked changes in PBMCs morphology because they caused a strong decrease in cell size (particularly DCAA and DBAA) and increase in cell granulation (mainly MBAA and DBAA). All HAAs studied, and DCAA and DBAA in particular (at lower concentrations than those, which caused necrosis) induced apoptotic changes, which was confirmed by analysis of alterations in cell membrane permeability and caspase 8, 9 and 3 activation. Moreover, HAAs examined (mainly dihalogenated acids) strongly increased transmembrane mitochondrial potential and enhanced ROS (mainly hydroxyl radical) formation, which was possibly associated with apoptotic changes provoked by those substances. The results showed that DBAA exhibited the strongest effects on PBMCs. Copyright © 2015 Elsevier Ltd. All rights reserved.

In vitro immunomodulatory effects of cuphiin D1 on human mononuclear cells.

PubMed

Wang, Ching-Chiung; Chen, Lih-Geeng; Yang, Ling-Ling

2002-01-01

Cuphiin D1 (CD1), a macrocyclic hydrolyzable tannin isolated from Cuphea hyssopifolia, has been shown to exert antitumor activity both in vitro and in vivo. Moreover, the antitumor effects of CD1 are not only related to its cytotoxicity to carcinoma cell lines, but also depend on host-mediated mechanisms. In the present study, CD1 was investigated for its effects on the proliferation and cytokine secretion of human peripheral blood mononuclear cells (PBMCs). At concentrations of from 6.25 to 50 micrograms/ml, it enhanced the 3H-thymidine incorporation of concanavalin A (Con A)-stimulated PBMCs in a dose-dependent manner. Excretion of IL-1 beta, IL-2 and TNF-alpha by CD1-stimulated PBMCs was markedly increased in a dose-dependent manner. The results show that CD1 could stimulate PBMCs release of IL-1 beta, IL-2 and TNF-alpha and then activate T cells. Therefore, CD1-activated T cells via IL-1 beta in vitro might account for the host-mediated CD1 mechanism of action.

Partial restoration of impaired interleukin-2 production and Tac antigen (putative interleukin-2 receptor) expression in patients with acquired immune deficiency syndrome by isoprinosine treatment in vitro.

PubMed Central

Tsang, K Y; Fudenberg, H H; Galbraith, G M; Donnelly, R P; Bishop, L R; Koopmann, W R

1985-01-01

The in vitro effects of isoprinosine (ISO) on interleukin-2 (IL-2) production, the expression of Tac antigen (IL-2 receptor) on lymphocytes, and the ability of Leu 3(+) cells to absorb interleukin-1 (IL-1) were investigated in 10 patients with acquired immune deficiency syndrome (AIDS). In 9 of the 10 patients, production of IL-2 from mononuclear cells and Leu 3(+) cells was depressed; expression of Tac antigen on mononuclear cells and Leu 2(+) cells was found to be depressed in 9 of 10 patients. The ability of the Leu 3(+) lymphocytes to absorb IL-1 was depressed in all (four of four) patients studied. After ISO treatment, IL-2 production, Tac antigen expression and IL-1 absorption were restored to normal or near normal levels in most of the patients. These results suggest that ISO has an immunostimulating capacity in AIDS patients and that the potential of ISO in immune response restoration in AIDS patients deserves critical consideration. PMID:2581997

Peripheral Blood Mononuclear Cells Enhance the Anabolic Effects of Platelet-Rich Plasma on Anterior Cruciate Ligament Fibroblasts

PubMed Central

Yoshida, Ryu; Murray, Martha M.

2012-01-01

Use of platelet-rich plasma (PRP) has shown promise in various orthopaedic applications, including treatment of anterior cruciate ligament (ACL) injuries. However, various components of blood, including peripheral blood mononuclear cells (PBMCs), are removed in the process of making PRP. It is yet unknown whether these PBMCs have a positive or negative effect on fibroblast behavior. To begin to define the effect of PBMCs on ACL fibroblasts, ACL fibroblasts were cultured on three-dimensional collagen scaffolds for 14 days with and without PBMCs. ACL fibroblasts exposed to PBMCs showed increased type I and type III procollagen gene expression, collagen protein expression, and cell proliferation when the cells were cultured in the presence of platelets and plasma. However, addition of PBMCs to cells cultured without the presence of platelets had no effect. The increase in collagen gene and protein expression was accompanied by an increase in IL-6 expression by the PBMCs with exposure to the platelets. Our results suggest that the interaction between platelets and PBMCs leads to an IL-6 mediated increase in collagen expression by ACL fibroblasts. PMID:22767425

Lymphocyte responses to phytohaemagglutinin: age-related effects.

PubMed Central

Fernandez, L A; MacSween, J M; Langley, G R

1976-01-01

Cell-mediated immunity is depressed in elderly individuals compared to young individuals, and lymphocytes from elderly individuals have been reported to have impaired lymphocyte responsiveness to stimulation by PHA after 4 days of culture. We have confirmed this observation. However, after 8 days of culture, the lymphocyte responses were greater in elderly normal individuals than in young normal individuals. Responses of lymphocytes from young individuals decreased with time from 4 to 8 days in culture, while there were increased responses with time when lymphocytes from elderly individuals were studied. When adherent cells from lymphocytes of young individuals were removed by passage through protein-coated Degalan-bead columns, the lymphocyte responses to PHA were significantly increased at 8 days. Passage of lymphocytes from elderly individuals through coated Degalan bead columns did not alter the lymphocyte responses. Removal of macrophages from the mononuclear cells obtained from young individuals did not result in increased lymphocyte responses to PHA after 8 days in culture. Removal of adherent cells appeared to have the same effect regardless of the efficiency of removing B cells. The adherent cells removed by the protein coated columns, therefore, appear to be nonphagocytic mononuclear cells which are not B lymphocytes. PMID:1086285

Prevalence of asinine herpesvirus type 5 (AsHV-5) infection in clinically normal Lipizzaner horses.

PubMed

Rushton, James Oliver; Kolodziejek, Jolanta; Nell, Barbara; Nowotny, Norbert

2014-04-01

The aim of this study was to assess the extent of asinine herpesvirus (AsHV) type 5 infection in 'closed' populations of clinically normal Lipizzaner horses. Peripheral blood mononuclear cells plus nasal and conjunctival swabs were obtained on four occasions over an 18 month period from 266 animals as part of a health surveillance programme. Sequence analysis of samples that were positive by nested consensus herpesvirus PCR but negative using quantified equid herpesvirus (EHV) type 2 and 5 PCR, revealed a total of 51 samples from 39 horses positive for AsHV-5. No statistically significant association between animal age, gender or geographical location and infection status was identified. The findings suggest sub-clinical AsHV-5 infection may be encountered more frequently than previously reported. Copyright © 2013 Elsevier Ltd. All rights reserved.

Blood mononuclear cell gene expression profiles characterize the oxidant, hemolytic, and inflammatory stress of sickle cell disease

PubMed Central

Jison, Maria L.; Munson, Peter J.; Barb, Jennifer J.; Suffredini, Anthony F.; Talwar, Shefali; Logun, Carolea; Raghavachari, Nalini; Beigel, John H.; Shelhamer, James H.; Danner, Robert L.; Gladwin, Mark T.

2016-01-01

In sickle cell disease, deoxygenation of intra-erythrocytic hemoglobin S leads to hemoglobin polymerization, erythrocyte rigidity, hemolysis, and microvascular occlusion. Ischemia-reperfusion injury, plasma hemoglobin-mediated nitric oxide consumption, and free radical generation activate systemic inflammatory responses. To characterize the role of circulating leukocytes in sickle cell pathogenesis we performed global transcriptional analysis of blood mononuclear cells from 27 patients in steady-state sickle cell disease (10 patients treated and 17 patients untreated with hydroxyurea) compared with 13 control subjects. We used gender-specific gene expression to validate human microarray experiments. Patients with sickle cell disease demonstrated differential gene expression of 112 genes involved in heme metabolism, cell-cycle regulation, antioxidant and stress responses, inflammation, and angiogenesis. Inducible heme oxygenase-1 and downstream proteins biliverdin reductase and p21, a cyclin-dependent kinase, were up-regulated, potentially contributing to phenotypic heterogeneity and absence of atherosclerosis in patients with sickle cell disease despite endothelial dysfunction and vascular inflammation. Hydroxyurea therapy did not significantly affect leukocyte gene expression, suggesting that such therapy has limited direct anti-inflammatory activity beyond leukoreduction. Global transcriptional analysis of circulating leukocytes highlights the intense oxidant and inflammatory nature of steady-state sickle cell disease and provides insight into the broad compensatory responses to vascular injury. PMID:15031206

Generation of avian cells resembling osteoclasts from mononuclear phagocytes

NASA Technical Reports Server (NTRS)

Alvarez, J. I.; Teitelbaum, S. L.; Blair, H. C.; Greenfield, E. M.; Athanasou, N. A.; Ross, F. P.

1991-01-01

Several lines of indirect evidence suggest that a monocyte family precursor gives rise to the osteoclast, although this hypothesis is controversial. Starting with a uniform population of nonspecific esterase positive, tartrate-sensitive, acid phosphatase-producing, mannose receptor-bearing mononuclear cells, prepared from dispersed marrow of calcium-deprived laying hens by cell density separation and selective cellular adherence, we generated multinucleated cells in vitro. When cultured with devitalized bone, these cells show, by electron microscopy, the characteristic osteoclast morphology in that they are mitochondria-rich, multinucleated, and, most importantly, develop characteristic ruffled membranes at the matrix attachment site. Moreover, as documented by scanning electron microscopy, these cells pit bone slices in a manner identical to freshly isolated osteoclasts. In addition, isoenzymes of acid phosphatase from generated osteoclasts, separated by 7.5% polyacrylamide gel electrophoresis at pH 4, are identical to those of mature osteoclasts in migration pattern and tartrate resistance, although the precursor cells from which the osteoclasts are generated produce an entirely different isoenzyme, which is tartrate-sensitive and migrates less rapidly at pH 4. The fused cells also exhibit a cAMP response to prostaglandin E2. Therefore, osteoclast-like cells can be derived by in vitro culture of a marrow-derived monocyte cell population.

Reactivity of dinuclear copper(II) complexes towards melanoma cells: Correlation with its stability, tyrosinase mimicking and nuclease activity.

PubMed

Nunes, Cléia Justino; Borges, Beatriz Essenfelder; Nakao, Lia Sumie; Peyroux, Eugénie; Hardré, Renaud; Faure, Bruno; Réglier, Marius; Giorgi, Michel; Prieto, Marcela Bach; Oliveira, Carla Columbano; Da Costa Ferreira, Ana M

2015-08-01

In this work, the influence of two new dinuclear copper(II) complexes in the viability of melanoma cells (B16F10 and TM1MNG3) was investigated, with the aim of verifying possible correlations between their cytotoxicity and their structure. One of the complexes had a polydentate dinucleating amine-imine ligand (complex 2), and the other a tridentate imine and a diamine-bridging ligand (complex 4). The analogous mononuclear copper(II) species (complexes 1 and 3, respectively) were also prepared for comparative studies. Crystal structure determination of complex 2 indicated a square-based pyramidal geometry around each copper, coordinated to three N atoms from the ligand and the remaining sites being occupied by either solvent molecules or counter-ions. Complex 4 has a tetragonal geometry. Interactions of these complexes with human albumin protein (HSA) allowed an estimation of their relative stabilities. Complementary studies of their reactivity towards DNA indicated that all of them are able of causing significant oxidative damage, with single and double strand cleavages, in the presence of hydrogen peroxide. However, nuclease activity of the dinuclear species was very similar and much higher than that of the corresponding mononuclear compounds. Although complex 2, with a more flexible structure, exhibits a much higher tyrosinase activity than complex 4, having a more rigid environment around the metal ion, both complexes showed comparable cytotoxicity towards melanoma cells. Corresponding mononuclear complexes showed to be remarkably less reactive as tyrosinase mimics as well as cytotoxic agents. Moreover, the dinuclear complexes showed higher cytotoxicity towards more melanogenic cells. The obtained results indicated that the structure of these species is decisive for its activity towards the malignant tumor cells tested. Copyright © 2015 Elsevier Inc. All rights reserved.

Strict lymphotropism of Epstein-Barr virus during acute infectious mononucleosis in nonimmunocompromised individuals.

PubMed

Karajannis, M A; Hummel, M; Anagnostopoulos, I; Stein, H

1997-04-15

Previous investigations of exfoliated oropharyngeal cells from individuals suffering from infectious mononucleosis (IM) suggested that the oropharyngeal epithelia are the primary target and also the site of life-long persistence of the Epstein-Barr virus (EBV). This concept was widely accepted. However, the investigation of histological sections with more sensitive EBV detection techniques has drawn this concept into doubt since EBV proved to be constantly absent in normal epithelial cells. To elucidate the discrepancy, throat washings and peripheral mononuclear blood cells from 16 patients suffering from IM were investigated for EBV-DNA and EBV gene products employing highly sensitive in situ hybridization, immunocytochemistry, and polymerase chain reaction. Although all patients exhibited latently infected B lymphocytes in peripheral blood, samples of exfoliated oropharyngeal cells were constantly EBV-negative with the exception of three cases. In these cases, the patients additionally suffered from purulent ulcerating tonsillitis, EBV-infected B cells, but no EBV-infected epithelial cells were detectable. These findings support the view that recirculating lymphocytes of B-cell origin, but not epithelial cells are the initial target of EBV during primary infection and that B cells also represent the site of life-long viral persistence.

Effects of strenuous exercise on Th1/Th2 gene expression from human peripheral blood mononuclear cells of marathon participants.

PubMed

Xiang, Lianbin; Rehm, Kristina E; Marshall, Gailen D

2014-08-01

Physical stressors, such as strenuous exercise, can have numerous effects on the human body including the immune system. The aim of this study was to evaluate the gene expression profile of Th1/Th2 cytokines and related transcription factor genes in order to investigate possible immune imbalances before and after a marathon. Blood samples were collected from 16 normal volunteers 24-48 h before and one week after completing a marathon race. Gene expression of Th1 and Th2 related cytokines from human peripheral blood mononuclear cells (PBMC) was analyzed using Human Th1-Th2-Th3 RT(2) Profiler PCR Array and qRT-PCR that measured the transcript levels of 84 genes related to T cell activation. We found that PBMC express a characteristic Th2-like gene profile one week post-marathon compared to pre-marathon. The majority of genes up-regulated one week post-marathon such as IL-4, GATA3, and CCR4 were Th2 associated. For Th1-related genes, CXCR3 and IRF1 were up-regulated one week post-marathon. There was a trend of down-regulation of two Th1 related genes, T-bet and STAT1. Th3-related gene expression patterns did not change in the study. The ratios of both IFN-γ/IL-4 and T-bet/GATA3 gene expressions were significantly lower one week after marathon. These findings suggest that a Th1/Th2 immune imbalance persisted at least 1 week after completion of a marathon which offers a mechanistic rationale for the increased risk of upper respiratory tract infections often reported after strenuous exercise. Copyright © 2014 Elsevier Ltd. All rights reserved.

Changes in Transcriptional Output of Human Peripheral Blood Mononuclear Cells Following Resistance Exercise

DTIC Science & Technology

2011-01-01

immunodeficiency virus (HIV), or had been diagnosed with acquired immune deficiency syndrome (AIDS). Participants were also excluded if they were... Laron Z (1995) Mathematical formulae for the prediction of the residual beta cell function during the first two years of disease in children and

"Characterization of the immune reagent chicken IL-16"

USDA-ARS?s Scientific Manuscript database

Interleukin-16 has been characterized as a pro-inflammatory cytokine that mediates an immune response in human and mouse monocytes and peripheral blood mononuclear cells (PBMC), and it plays a role in proliferating B-cells and myelomas. The function of chicken IL-16 ortholog (ch-IL-16) is far less u...

Differential effects of LPS, IFN-gamma and TNF alpha on the secretion of lysozyme by individual human mononuclear phagocytes: relationship to cell maturity.

PubMed Central

Lewis, C E; McCarthy, S P; Lorenzen, J; McGee, J O

1990-01-01

Human mononuclear phagocytes can be activated to perform a variety of complex functions by exposure to the immunomodulators, lipopolysaccharide (LPS), interferon-gamma (IFN-gamma) and tumour necrosis factor alpha (TNF alpha). Although such activation often involves the release of various cytokines by monocytes and macrophages, little is known of the effects of such signals on their secretion of lysozyme (LZM). In this study, a reverse haemolytic plaque assay for LZM secretion is coupled with immunocytochemistry for the pan macrophage (CD68) marker, EBM/11. This enabled the direct effects of LPS, IFN-gamma and TNF alpha on the secretion of LZM by individual, immunoidentified human mononuclear phagocytes to be investigated. The overall secretion of this peptide by populations of freshly isolated or 3-day cultured monocytes was augmented by exposure for 6 hr to bacterial LPS, recombinant human IFN-gamma or recombinant human TNF alpha. Extension of the culture period for monocytes from 3 to 7 days prior to use in the assay resulted in higher levels of LZM secretion, which could be further increased by TNF alpha but not by LPS or IFN-gamma. Individual peritoneal macrophages activated by inflammation in vivo were uniform in their augmented LZM responses to TNF alpha, but a small subpopulation of human peritoneal macrophages, which may represent younger 'inflammatory' exudate macrophages, was seen to be preferentially responsive to the LZM-stimulating effects of LPS and IFN-gamma. These studies suggest that (i) secretion of LZM by human mononuclear phagocytes can be regulated by LPS and IFN-gamma, although the effects of these agents may be dependent upon the state of maturation and/or differentiation of the cells, and (ii) TNF alpha is a potent stimulant of LZM secretion by monocytes and macrophages irrespective of cell maturity. Images Figure 1 Figure 1 PMID:2107146

αMβ2-integrin-intercellular adhesion molecule-1 interactions drive the flow-dependent trafficking of Guillain-Barré syndrome patient derived mononuclear leukocytes at the blood-nerve barrier in vitro

PubMed Central

Yosef, Nejla; Ubogu, Eroboghene E.

2012-01-01

The mechanisms of hematogenous leukocyte trafficking at the human blood-nerve barrier (BNB) are largely unknown. Intercellular adhesion molecule-1 (ICAM-1) has been implicated in the pathogenesis of Guillain-Barré syndrome (GBS). We developed a cytokine-activated human in vitro BNB model using primary endoneurial endothelial cells. Endothelial treatment with 10 U/mL tissue necrosis factor-α and 20 U/mL interferon-γ resulted in de novo expression of proinflammatory chemokines CCL2, CXCL9, CXCL11 and CCL20, with increased expression of CXCL2-3, CXCL8 and CXCL10 relative to basal levels. Cytokine treatment induced/ enhanced ICAM-1, E- and P-selectin, vascular cell adhesion molecule-1 and the alternatively spliced pro-adhesive fibronectin variant, fibronectin connecting segment-1 expression in a time-dependent manner, without alterations in junctional adhesion molecule-A expression. Lymphocytes and monocytes from untreated GBS patients express ICAM-1 counterligands, αM- and αL-integrin, with differential regulation of αM-integrin expression compared to healthy controls. Under flow conditions that mimic capillary hemodynamics in vivo, there was a >3-fold increase in total GBS patient and healthy control mononuclear leukocyte adhesion/ migration at the BNB following cytokine treatment relative to the untreated state. Function neutralizing monoclonal antibodies against human αM-integrin (CD11b) and ICAM-1 reduced untreated GBS patient mononuclear leukocyte trafficking at the BNB by 59% and 64.2% respectively. Monoclonal antibodies against αL-integrin (CD11a) and human intravenous immunoglobulin reduced total leukocyte adhesion/migration by 22.8% and 17.6% respectively. This study demonstrates differential regulation of αM-integrin on circulating mononuclear cells in GBS, as well as an important role for αM-integrin-ICAM-1 interactions in pathogenic GBS patient leukocyte trafficking at the human BNB in vitro. PMID:22552879

Reduction of transforming growth factor-β1 expression in leukemia and its possible role in leukemia development.

PubMed

Wu, Yong; Chen, Ping; Huang, Hui-Fang; Huang, Mei-Juan; Chen, Yuan-Zhong

2012-01-01

The expression of transforming growth factor-β1 (TGF-β1) in leukemic cells and sera from patients with leukemia and its possible role in leukemia development were studied. TGF-β1 levels in culture supernatants from leukemic cells were significantly lower than those from normal bone marrow mononuclear cells. Serum TGF-β1 levels in leukemic patients were significantly lower compared with healthy controls, but returned to normal in patients achieving complete remission, and decreased when patients relapsed. TGF-β1 mRNA expression levels were significantly higher in normal bone marrow mononuclear cells but lower in leukemic cells compared with normal CD34 + cells. After transfection of the TGF-β1 gene to HL-60 cells, cell apoptosis was detected. Moreover, by flow cytometry analysis, cells arrested in G1 phase were 62% for TGF-β1 transfected cells and 44% for controls. Transfection of exogenous TGF-β1 gene inhibited HL60 cells xenograft growth in nude mice, and prolonged survival of tumor-bearing mice compared with the controls. Decreased endogenous TGF-β1 expression in leukemia cells may be involved in leukemia development, Transfection of exogenous TGF-B1 gene to HL60 can inhibit the proliferation of the cells and induce cell apoptosis by down regulating bcl-2, hTERT (human telomerase reverse transcriptase) and c-myc expression.

Proliferative responses to canine thyroglobulin of peripheral blood mononuclear cells from hypothyroid dogs.

PubMed

Tani, Hiroyuki; Nabetani, Tomoyo; Sasai, Kazumi; Baba, Eiichiroh

2005-04-01

The immune responses of hypothyroid dogs to canine thyroglobulin (cTg) were evaluated for the proliferative ability of peripheral blood mononuclear cells (PBMC). PBMC from three hypothyroid dogs with high titers of thyroglobulin autoantibody (TgAA) and 3 clinically normal dogs were cultured with 5, 10, or 20 microg/ml of cTg for 72 hr. The proliferative responses of the cells were determined by the level of incorporated BrdU. The numbers of cells expressing Thy-1, CD4, CD8 and IgG in the PBMC were counted by the immunofluorescence method. Proliferative responses to cTg were observed in the cells from hypothyroid dogs. The number of cells expressing IgG and CD8 in the hypothyroid dogs tended to be high compared with the clinically normal dogs. The CD4+ cells in cultures from hypothyroid dogs increased depending upon the amount of cTg. There was a significant (P<0.05) positive correlation between the number of CD4+ cells and the concentration of cTg in the cultures from hypothyroid dogs. These findings suggest a possible relationship between canine hypothyroidism and cellular immunity. Loss of self tolerance to thyroid antigens in CD4+ T cells may play an important role in the development of canine hypothyroidism.

Solution-Phase Crosstalk and Regulatory Interactions Between Multipotent Adult Progenitor Cells and Peripheral Blood Mononuclear Cells

PubMed Central

van’t Hof, Wouter; Reddy, Ashok P.; Wilmarth, Phillip A.; David, Larry L.; Raber, Amy; Bogaerts, Annelies; Timmerman, Lien; Pinxteren, Jef; Roobrouck, Valerie D.; Deans, Robert J.; Maziarz, Richard T.

2015-01-01

Multipotent adult progenitor cells (MAPCs) are adult adherent stromal stem cells currently being assessed in clinical trials for acute graft versus host disease with demonstrated immunomodulatory capabilities and the potential to ameliorate detrimental autoimmune and inflammation-related processes. Anti-CD3/anti-CD28 (3/28) activation of T cells within the peripheral blood mononuclear cell (PBMC) compartment was performed in the presence or absence of MAPCs. Liquid chromatography-coupled tandem mass spectrometry was used to characterize the differential secretion of proteins, and transcriptional profiling was used to monitor mRNA expression changes in both cell populations. Overall, 239 secreted and/or ectodomain-shed proteins were detected in the secretomes of PBMCs and MAPCs. In addition, 3/28 activation of PBMCs induced differential expression of 2,925 genes, and 22% of these transcripts were differentially expressed on exposure to MAPCs in Transwell. MAPCs exposed to 3/28-activated PBMCs showed differential expression of 1,247 MAPC genes. Crosstalk was demonstrated by reciprocal transcriptional regulation. Secretome proteins and transcriptional signatures were used to predict molecular activities by which MAPCs could dampen local and systemic inflammatory responses. These data support the hypothesis that MAPCs block PBMC proliferation via cell cycle arrest coupled to metabolic stress in the form of tryptophan depletion, resulting in GCN2 kinase activation, downstream signaling, and inhibition of cyclin D1 translation. These data also provide a plausible explanation for the immune privilege reported with administration of donor MAPCs. Although most components of the major histocompatibility complex class II antigen presentation pathway were markedly transcriptionally upregulated, cell surface expression of human leukocyte antigen-DR is minimal on MAPCs exposed to 3/28-activated PBMCs. Significance This study documents experiments quantifying solution-phase crosstalk between multipotent adult progenitor cells (MAPCs) and peripheral blood mononuclear cells. The secretome and transcriptional changes quantified suggest mechanisms by which MAPCs are hypothesized to provide both local and systemic immunoregulation of inflammation. The potential impact of these studies includes development of a robust experimental framework to be used for preclinical evaluation of the specific mechanisms by which beneficial effects are obtained after treatment of patients with MAPCs. PMID:26494783

Probiotic Lactobacillus-induced improvement in murine chronic inflammatory bowel disease is associated with the down-regulation of pro-inflammatory cytokines in lamina propria mononuclear cells

PubMed Central

Matsumoto, S; Hara, T; Hori, T; Mitsuyama, K; Nagaoka, M; Tomiyasu, N; Suzuki, A; Sata, M

2005-01-01

IL-6/STAT-3 signals play key roles in inflammatory bowel disease (IBD). It is known that Lactobacillus casei strain Shirota (LcS) improves inflammatory disorders. This study aimed to elucidate the effect of LcS on murine chronic IBD and to clarify the mechanism. We focused the inhibitory effect of LcS on the production of IL-6 in lipopolysaccharide (LPS)-stimulated large intestinal lamina propria mononuclear cells (LI-LPMC) isolated from mice with chronic colitis and in RAW264·7 cells in vitro. We also determined in vivo the effect of LcS on murine chronic IBD models induced with dextran sodium sulphate and SAMP1/Yit mice. Finally, we examined the cellular determinants of LcS for the down-regulation of IL-6 secretion by LI-LPMC, RAW264·7 cells and peripheral blood mononuclear cells (PBMC) derived from patients with ulcerative colitis (UC). LcS, but not other strains of Lactobacillus, inhibited the production of IL-6 in LPS-stimulated LI-LPMC and RAW264·7 cells, down-regulating the nuclear translocation of NF-κB. The LcS-diet-improved murine chronic colitis is associated with the reduction of IL-6 synthesis by LI-LPMC. LcS also improved chronic ileitis in SAMP1/Yit mice. The release of IL-6 in vitro in LPS-stimulated LI-LPMC, RAW 264·7 cells and UC-PBMC was inhibited by a polysaccharide-peptidoglycan complex (PSPG) derived from LcS. This probiotic-induced improvement in murine chronic inflammatory bowel disease is associated with the down-regulation of pro-inflammatory cytokines such as IL-6 and IFN-γ production in LPMC. Therefore, LcS may be a useful probiotic for the treatment of human inflammatory bowel disease. PMID:15932502

Remodeling of the mononuclear phagocyte network underlies chronic inflammation and disease progression in heart failure: critical importance of the cardiosplenic axis.

PubMed

Ismahil, Mohamed Ameen; Hamid, Tariq; Bansal, Shyam S; Patel, Bindiya; Kingery, Justin R; Prabhu, Sumanth D

2014-01-17

The role of mononuclear phagocytes in chronic heart failure (HF) is unknown. Our aim was to delineate monocyte, macrophage, and dendritic cell trafficking in HF and define the contribution of the spleen to cardiac remodeling. We evaluated C57Bl/6 mice with chronic HF 8 weeks after coronary ligation. As compared with sham-operated controls, HF mice exhibited: (1) increased proinflammatory CD11b+ F4/80+ CD206- macrophages and CD11b+ F4/80+ Gr-1(hi) monocytes in the heart and peripheral blood, respectively, and reduced CD11b+ F4/80+ Gr-1(hi) monocytes in the spleen; (2) significantly increased CD11c+ B220- classical dendritic cells and CD11c+ low)B220+ plasmacytoid dendritic cells in both the heart and spleen, and increased classic dendritic cells and plasmacytoid dendritic cells in peripheral blood and bone marrow, respectively; (3) increased CD4+ helper and CD8+ cytotoxic T-cells in the spleen; and (4) profound splenic remodeling with abundant white pulp follicles, markedly increased size of the marginal zone and germinal centers, and increased expression of alarmins. Splenectomy in mice with established HF reversed pathological cardiac remodeling and inflammation. Splenocytes adoptively transferred from mice with HF, but not from sham-operated mice, homed to the heart and induced long-term left ventricular dilatation, dysfunction, and fibrosis in naive recipients. Recipient mice also exhibited monocyte activation and splenic remodeling similar to HF mice. Activation of mononuclear phagocytes is central to the progression of cardiac remodeling in HF, and heightened antigen processing in the spleen plays a critical role in this process. Splenocytes (presumably splenic monocytes and dendritic cells) promote immune-mediated injurious responses in the failing heart and retain this memory on adoptive transfer.

The Mismetallation of Enzymes during Oxidative Stress*

PubMed Central

Imlay, James A.

2014-01-01

Mononuclear iron enzymes can tightly bind non-activating metals. How do cells avoid mismetallation? The model bacterium Escherichia coli may control its metal pools so that thermodynamics favor the correct metallation of each enzyme. This system is disrupted, however, by superoxide and hydrogen peroxide. These species oxidize ferrous iron and thereby displace it from many iron-dependent mononuclear enzymes. Ultimately, zinc binds in its place, confers little activity, and imposes metabolic bottlenecks. Data suggest that E. coli compensates by using thiols to extract the zinc and by importing manganese to replace the catalytic iron atom. Manganese resists oxidants and provides substantial activity. PMID:25160623

[Genomic disorders in the mononuclear blood cells of those who worked in the cleanup of the accident at the Chernobyl Atomic Electric Power Station].

PubMed

Butenko, Z A; Smirnova, I A; Zak, K P; Mikhaĭlovskaia, E V; Ianok, E A; Kishinskaia, E G

1998-01-01

The results of molecular investigations of blood mononuclears from 120 clean-up workers after 7-9 years of Chernobyl accident with the total exposure radiation doses ranging from 5 to 76 cGr are presented. Structural polymorphism of the leukemia associated bcr and ribosomal RNA (rRNA) genes were studied using Southern blot hybridization. Allelic polymorphism of bcr gene with characteristic for leukemia allele distribution was detected in 16.6%. Rearrangements of rRNA genes were observed in 13% of Chernobyl accident clean-up workers.

The RORγt-CCR6-CCL20 axis augments Th17 cells invasion into the synovia of rheumatoid arthritis patients.

PubMed

Kaneko, Shunta; Kondo, Yuya; Yokosawa, Masahiro; Furuyama, Kotona; Segawa, Seiji; Tsuboi, Hiroto; Kanamori, Akihiro; Matsumoto, Isao; Yamazaki, Masashi; Sumida, Takayuki

2018-01-22

To clarify the pathogenic role of transcription factor expression of CD4 + T helper (Th) cell subsets in the development of rheumatoid arthritis (RA). We collected CD4 + T cells from peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) by magnetic cell sorting. The proportion of Th cell subsets were classified from cell surface markers (CD45RA, CXCR5, CXCR3, CCR6) and the expression of their transcription factors (T-bet, GATA3, RORγt) were analyzed by flow cytometry before and at 24 weeks after anti-rheumatic treatment. Chemotaxis assays quantified migratory ability. The expression of CCR6 and RORγt in Th17 cells from PBMC of RA patients was significantly higher than in healthy control volunteers and osteoarthritis patients. The proportion of Th17 cells in SFMCs of RA patients was significantly higher than that in PBMCs. Chemotaxis assays revealed that the migration index of Th17 cells towards CCL20 was remarkably enhanced in RA patients. The expression of CCR6 and RORγt in Th17 cells at 24 weeks post-therapeutic intervention was significantly decreased compared to before treatment. The high expression of RORγt might facilitate the migration of Th17 cells to inflamed joints via the enhanced expression of CCR6 and contribute to the pathology of RA.

Human mononuclear cell function after 4 degrees C storage during 1-G and microgravity conditions of spaceflight.

PubMed

Meehan, R; Taylor, G; Lionetti, F; Neale, L; Curren, T

1989-07-01

Future space missions of long duration may require that autologous leukocytes be stored in flight for infusion to restore normal immune competence in crewmembers. Peripheral blood mononuclear cells (PBMNCs), as leukocyte concentrates in autologous plasma and 2% dextrose, were stored in the microgravity conditions provided by the U.S. Space Shuttle Columbia (Mission 61-C). Activity of PBMNC after space flight was compared with that from a series of preflight ground control experiments, which demonstrated in culture a progressive daily loss in mitogen-stimulated protein synthesis at 24 h and thymidine uptake at 72 h after storage for 7 d at 4 degrees C. Post-storage viabilities were at least 90% as determined by trypan dye exclusion. A progressive reduction in the percentage of PBMNC expressing cell-surface phenotype markers, which was similar for monocytes, B cells, and T-cell subsets, also occurred after storage. The ability of PBMNC, stored for 8 d in Columbia's middeck, to become activated and proliferate in vitro was similar to that of cells that remained in identical flight lockers on the ground as 1-G controls, thus indicating that PBMNCs were not adversely affected by storage under microgravity conditions.

Human mononuclear cell function after 4 degrees C storage during 1-G and microgravity conditions of spaceflight

NASA Technical Reports Server (NTRS)

Meehan, R.; Taylor, G.; Lionetti, F.; Neale, L.; Curren, T.

1989-01-01

Future space missions of long duration may require that autologous leukocytes be stored in flight for infusion to restore normal immune competence in crewmembers. Peripheral blood mononuclear cells (PBMNCs), as leukocyte concentrates in autologous plasma and 2% dextrose, were stored in the microgravity conditions provided by the U.S. Space Shuttle Columbia (Mission 61-C). Activity of PBMNC after space flight was compared with that from a series of preflight ground control experiments, which demonstrated in culture a progressive daily loss in mitogen-stimulated protein synthesis at 24 h and thymidine uptake at 72 h after storage for 7 d at 4 degrees C. Post-storage viabilities were at least 90% as determined by trypan dye exclusion. A progressive reduction in the percentage of PBMNC expressing cell-surface phenotype markers, which was similar for monocytes, B cells, and T-cell subsets, also occurred after storage. The ability of PBMNC, stored for 8 d in Columbia's middeck, to become activated and proliferate in vitro was similar to that of cells that remained in identical flight lockers on the ground as 1-G controls, thus indicating that PBMNCs were not adversely affected by storage under microgravity conditions.

Decreased NK-Cell Cytotoxicity after Short Flights on the Space Shuttle

NASA Technical Reports Server (NTRS)

Mehta, Satish K.; Grimm, Elizabeth A.; Smid, Christine; Kaur, Indreshpal; Feeback, Daniel L.; Pierson, Duane L.

2000-01-01

Cytotoxic activity of natural killer (NK) cells and cell surface marker expression of peripheral blood mononuclear cells (PBMCs) isolated from 11 U.S. astronauts on two different missions were determined before and after 9 or 10 days of spaceflight aboard the space shuttle. Blood samples were collected 10 and 3 days before launch, within 3 hours after landing, and 3 days after landing. All PBMC preparations were cryopreserved and analyzed simultaneously in a 4-hour cytotoxicity "Cr-release assay using NK-sensitive K-562 target cells. Compared to preflight values, NK-cell cytotoxicity (corrected for lymphopenia observed on landing day) was significantly decreased at landing (P < 0.0125). It then apparently began to recover and approached preflight values by 3 days after landing. Consistent with decreased NK-cell cytotoxicity, significant increases from preflight values were found in plasma adrenocorticotropic hormone at landing. Plasma and urinary cortisol levels did not change significantly from preflight values. Expression of major lymphocyte surface markers (CD3, CD4, CD8, CD14, CD16, CD56), determined by flow cytometric analysis, revealed no consistent phenotypic changes in relative percent of NK or other lymphoid cells after 10 days of spaceflight.

Photoacoustic detection of hemozoin in human mononuclear cells as an early indicator of malaria infection

NASA Astrophysics Data System (ADS)

Custer, Jonathan R.; Kariuki, Michael; Beerntsen, Brenda T.; Viator, John A.

2010-02-01

Malaria is a blood borne infection affecting hundreds of millions of people worldwide2. The parasites reproduce within the blood cells, eventually causing their death and lysis. This process releases the parasites into the blood, continuing the cycle of infection. Usually, malaria is diagnosed only after a patient presents symptoms, including high fever, nausea, and, in advanced cases, coma and death. While invading the bloodstream of a host, malaria parasites convert hemoglobin into an insoluble crystal, known as hemozoin. These crystals, approximately several hundred nanometers in size, are contained within red blood cells and white blood cells that ingest free hemozoin in the blood. Thus, infected red blood cells and white blood cells contain a unique optical absorber that can be detected in blood samples using static photoacoustic detection methods. We separated the white blood cells from malaria infected blood and tested it in a photoacoustic set up using a tunable laser system consisting of an optical parametric oscillator pumped by an Nd:YAG laser with pulse duration of 5 ns. Our threshold of detection was 10 infected white blood cells per microliter, which is more sensitive than current diagnosis methods using microscopic analysis of blood.

Flow Cytometric Human Leukocyte Antigen-B27 Typing with Stored Samples for Batch Testing

PubMed Central

Seo, Bo Young

2013-01-01

Background Flow cytometry (FC) HLA-B27 typing is still used extensively for the diagnosis of spondyloarthropathies. If patient blood samples are stored for a prolonged duration, this testing can be performed in a batch manner, and in-house cellular controls could easily be procured. In this study, we investigated various methods of storing patient blood samples. Methods We compared four storage methods: three methods of analyzing lymphocytes (whole blood stored at room temperature, frozen mononuclear cells, and frozen white blood cells [WBCs] after lysing red blood cells [RBCs]), and one method using frozen platelets (FPLT). We used three ratios associated with mean fluorescence intensities (MFI) for HLAB27 assignment: the B27 MFI ratio (sample/control) for HLA-B27 fluorescein-5-isothiocyanate (FITC); the B7 MFI ratio for HLA-B7 phycoerythrin (PE); and the ratio of these two ratios, B7/B27 ratio. Results Comparing the B27 MFI ratios of each storage method for the HLA-B27+ samples and the B7/B27 ratios for the HLA-B7+ samples revealed that FPLT was the best of the four methods. FPLT had a sensitivity of 100% and a specificity of 99.3% for HLA-B27 assignment in DNA-typed samples (N=164) when the two criteria, namely, B27 MFI ratio >4.0 and B7/B27 ratio <1.5, were used. Conclusions The FPLT method was found to offer a simple, economical, and accurate method of FC HLA-B27 typing by using stored patient samples. If stored samples are used, this method has the potential to replace the standard FC typing method when used in combination with a complementary DNA-based method. PMID:23667843

A positive immunoselection method to isolate villous cytotrophoblast cells from first trimester and term placenta to high purity.

PubMed

Pötgens, A J G; Kataoka, H; Ferstl, S; Frank, H-G; Kaufmann, P

2003-04-01

We developed a method for isolating highly pure villous cytotrophoblast cells from first trimester and term placenta that excludes extravillous trophoblast and syncytiotrophoblast fragments. The method is based on positive immunoselection using an antibody (mAb C76/18) reacting with hepatocyte growth factor activator inhibitor 1, HAI-1, a membrane antigen on villous cytotrophoblast. As a comparison, we also immunopurified cells using an antibody against CD105, present on syncytiotrophoblast and some extravillous trophoblast cells. The isolates were characterized by flow cytometry. HAI-1-positive cells from first trimester and term placentae were highly pure (>98 per cent cytokeratin 7-positive) mononuclear trophoblast cells. These isolations were contaminated with only very small percentages of vimentin and CD45-positive cells. HAI-1-positive trophoblast cells lacked CD105 and also HLA class I, a marker for extravillous trophoblast. In culture HAI-1-positive cells adhered, displayed an epithelial morphology, and survived for more than three days. In contrast, CD105-positive cell fractions from first trimester placenta were a heterogeneous mixture of mononuclear and multinuclear elements consisting of syncytiotrophoblast fragments, extravillous trophoblast cells, as well as around 5 per cent non-trophoblastic contaminants. In conclusion, the positive immunoselection method using antibody C76/18 yielded highly pure villous cytotrophoblast cells devoid of elements derived from syncytiotrophoblast or extravillous trophoblast.

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

PubMed

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.

Cytotoxic and genotoxic potential of geraniol in peripheral blood mononuclear cells and human hepatoma cell line (HepG2).

PubMed

Queiroz, T B; Santos, G F; Ventura, S C; Hiruma-Lima, C A; Gaivão, I O M; Maistro, E L

2017-09-27

Geraniol is an acyclic monoterpene alcohol present in the essential oil of many aromatic plants and is one of the most frequently used molecules by the flavor and fragrance industries. The literature also reports its therapeutic potential, highlighting itself especially as a likely molecule for the development of drugs against cancer. In view of these considerations, this study was designed to evaluate the cytotoxic and genotoxic potential of geraniol, in an in vitro protocol, using two types of human cells: one without the ability to metabolize (peripheral blood mononuclear cells - PBMC), and the other with this capability (human hepatoma cell line - HepG2) through the comet assay and the micronucleus test. Four concentrations (10, 25, 50, and 100 µg/mL) were selected for the genotoxic assessment for PBMC and three (1.25, 2.5, and 5 µg/mL) for HepG2 cells based on cytotoxicity tests (MTT assay). Results showed that geraniol did not present genotoxic or clastogenic/aneugenic effects on both cell types under the conditions studied. However, caution is advised in the use of this substance by humans, since a significant reduction in viability of HepG2 and a marked decrease in cell viability on normal PBMC were verified.

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

PubMed

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.

Histopathological examination of nerve samples from pure neural leprosy patients: obtaining maximum information to improve diagnostic efficiency.

PubMed

Antunes, Sérgio Luiz Gomes; Chimelli, Leila; Jardim, Márcia Rodrigues; Vital, Robson Teixeira; Nery, José Augusto da Costa; Corte-Real, Suzana; Hacker, Mariana Andréa Vilas Boas; Sarno, Euzenir Nunes

2012-03-01

Nerve biopsy examination is an important auxiliary procedure for diagnosing pure neural leprosy (PNL). When acid-fast bacilli (AFB) are not detected in the nerve sample, the value of other nonspecific histological alterations should be considered along with pertinent clinical, electroneuromyographical and laboratory data (the detection of Mycobacterium leprae DNA with polymerase chain reaction and the detection of serum anti-phenolic glycolipid 1 antibodies) to support a possible or probable PNL diagnosis. Three hundred forty nerve samples [144 from PNL patients and 196 from patients with non-leprosy peripheral neuropathies (NLN)] were examined. Both AFB-negative and AFB-positive PNL samples had more frequent histopathological alterations (epithelioid granulomas, mononuclear infiltrates, fibrosis, perineurial and subperineurial oedema and decreased numbers of myelinated fibres) than the NLN group. Multivariate analysis revealed that independently, mononuclear infiltrate and perineurial fibrosis were more common in the PNL group and were able to correctly classify AFB-negative PNL samples. These results indicate that even in the absence of AFB, these histopathological nerve alterations may justify a PNL diagnosis when observed in conjunction with pertinent clinical, epidemiological and laboratory data.

Inhibition of pro-inflammatory mediators: role of Bacopa monniera (L.) Wettst.

PubMed

Viji, Vijayan; Helen, Antony

2011-10-01

Bacopa monniera (L.) Wettst is a renowned plant in the Ayurvedic system of medicine. The present study seeks to identify the anti-inflammatory activity of two fractions from the methanolic extract of Bacopa, viz. the triterpenoid and bacoside-enriched fractions. The ability of these two fractions to inhibit the production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 was tested using lipopolysaccharide (LPS)-activated peripheral blood mononuclear cells and peritoneal exudate cells in vitro. We found that triterpenoid and bacoside-enriched fractions significantly inhibited LPS-activated TNF-α, IL-6 and nitrite production in mononuclear cells. Significant antioxidant activity was exhibited by the bacoside enriched fraction compared to the triterpenoid fraction. Carrageenan-induced hind paw oedema assay revealed that triterpenoid and bacoside-enriched fractions exerted anti-oedematogenic effect, while in the arthritis model only the triterpenoid fraction exerted an anti-arthritic potential. The present study provides an insight into the ability of Bacopa monniera to inhibit inflammation through modulation of pro-inflammatory mediator release.

Activation of cathepsins B and L in mouse lymphosarcoma tissue under the effect of cyclophosphamide is associated with apoptosis induction and infiltration by mononuclear phagocytes.

PubMed

Zhanaeva, S Ya; Mel'nikova, E V; Trufakin, V A; Korolenko, T A

2013-11-01

We analyzed activities of lysosomal cystein cathepsins B and L in mouse LS lymphosarcoma and its drug-resistant RLS 40 strain and their correlations with the dynamics of the percentage of cells with fragmented DNA and CD14 (+) phagocytes over 3 days after cyclophosphamide injection. LS regression and inhibition of RLS 40 growth after cyclophosphamide injection were paralleled by an increase in cathepsins B and L activities in tumor tissues. The antitumor effect of cyclophosphamide associated with apoptosis intensity and protease activities were significantly higher in LS. Positive correlations between activities of cathepsins B and L and the LS tissue content of cells with fragmented DNA and CD14 (+) phagocytes and negative correlations thereof with tumor weight were detected. It seems that the increase in cathepsins B and L activities in LS tissues was caused by cyclophosphamide induction of apoptosis and depended on the level of tumor cell infiltration with mononuclear phagocytes.

Distinct Gene Expression Profiles in Peripheral Blood Mononuclear Cells from Patients Infected with Vaccinia Virus, Yellow Fever 17D Virus, or Upper Respiratory Infections Running Title: PBMC Expression Response to Viral Agents

PubMed Central

Scherer, Christina A.; Magness, Charles L.; Steiger, Kathryn V.; Poitinger, Nicholas D.; Caputo, Christine M.; Miner, Douglas G.; Winokur, Patricia L.; Klinzman, Donna; McKee, Janice; Pilar, Christine; Ward, Patricia A.; Gillham, Martha H.; Haulman, N. Jean; Stapleton, Jack T.; Iadonato, Shawn P.

2007-01-01

Gene expression in human peripheral blood mononuclear cells was systematically evaluated following smallpox and yellow fever vaccination, and naturally occurring upper respiratory infection (URI). All three infections were characterized by the induction of many interferon stimulated genes, as well as enhanced expression of genes involved in proteolysis and antigen presentation. Vaccinia infection was also characterized by a distinct expression signature composed of up-regulation of monocyte response genes, with repression of genes expressed by B and T-cells. In contrast, the yellow fever host response was characterized by a suppression of ribosomal and translation factors, distinguishing this infection from vaccinia and URI. No significant URI-specific signature was observed, perhaps reflecting greater heterogeneity in the study population and etiological agents. Taken together, these data suggest that specific host gene expression signatures may be identified that distinguish one or a small number of virus agents. PMID:17651872

CXCL16/CXCR6-mediated adhesion of human peripheral blood mononuclear cells to inflamed endothelium.

PubMed

Linke, Bona; Meyer Dos Santos, Sascha; Picard-Willems, Bettina; Keese, Michael; Harder, Sebastian; Geisslinger, Gerd; Scholich, Klaus

2017-06-21

The endothelial chemokine CXC motif ligand 16 (CXCL16) is involved in the recruitment and firm adhesion of CXCR6 + cells to the atherosclerosis-prone aortic vessel wall. Recently we showed that CXCR6 + platelets from flowing blood attach to CXCL16 expressed by activated endothelium on the luminal side of the blood vessel. With this study we supplement these findings with the observation that platelets bound to the inflamed endothelium are presenting CXCR6 to CXCL16-positive peripheral blood mononuclear cells (PBMCs) and, thus, are mediating an increased adhesion of PBMCs to the arterial wall. Furthermore we identified endothelial CXCL16 as an important adhesion molecule promoting the firm adhesion of CXCR6-positive PBMCs to inflamed endothelium. Our results demonstrate that endothelial CXCL16 as well as platelet CXCR6 are acting as potent PBMC-adhesion ligands, inducing PBMC-adhesion to the atherosclerosis-prone vessel wall and thus promoting the progression of atherosclerosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Failure to detect genomic material of HTLV-I or HTLV-II in mononuclear cells of Italian patients with multiple sclerosis and chronic progressive myelopathy.

PubMed

Merelli, E; Sola, P; Marasca, R; Salati, R; Torelli, G

1993-01-01

To contribute to the undecided question if a retrovirus of the human T-cell lymphotropic virus (HTLV) family may be involved in the development of multiple sclerosis (MS), we investigated by the polymerase chain reaction (PCR) the presence of HTLV-I and HTLV-II sequences in the peripheral blood mononuclear cell DNAs from 30 patients affected by MS and 15 by chronic progressive myelopathy. Moreover a control group of 14 blood donors was examined. All these patients were devoid of anti-HTLV-I antibody in the serum and cerebrospinal fluid at ELISA. For the PCR, primers and probes specific for the tax region common to HTLV-I and HTLV-II, for the pol region of HTLV-I, and for the pol region of HTLV-II were used. In spite of the high sensitivity of the technique used, the three groups of subjects were negative for HTLV-I and HTLV-II genomic sequences.

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

PubMed

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.

Distinct gene expression profiles in peripheral blood mononuclear cells from patients infected with vaccinia virus, yellow fever 17D virus, or upper respiratory infections.

PubMed

Scherer, Christina A; Magness, Charles L; Steiger, Kathryn V; Poitinger, Nicholas D; Caputo, Christine M; Miner, Douglas G; Winokur, Patricia L; Klinzman, Donna; McKee, Janice; Pilar, Christine; Ward, Patricia A; Gillham, Martha H; Haulman, N Jean; Stapleton, Jack T; Iadonato, Shawn P

2007-08-29

Gene expression in human peripheral blood mononuclear cells was systematically evaluated following smallpox and yellow fever vaccination, and naturally occurring upper respiratory infection (URI). All three infections were characterized by the induction of many interferon stimulated genes, as well as enhanced expression of genes involved in proteolysis and antigen presentation. Vaccinia infection was also characterized by a distinct expression signature composed of up-regulation of monocyte response genes, with repression of genes expressed by B and T-cells. In contrast, the yellow fever host response was characterized by a suppression of ribosomal and translation factors, distinguishing this infection from vaccinia and URI. No significant URI-specific signature was observed, perhaps reflecting greater heterogeneity in the study population and etiological agents. Taken together, these data suggest that specific host gene expression signatures may be identified that distinguish one or a small number of virus agents.

The Expression of Caspases Is Enhanced in Peripheral Blood Mononuclear Cells of Autism Spectrum Disorder Patients

ERIC Educational Resources Information Center

Siniscalco, Dario; Sapone, Anna; Giordano, Catia; Cirillo, Alessandra; de Novellis, Vito; de Magistris, Laura; Rossi, Francesco; Fasano, Alessio; Maione, Sabatino; Antonucci, Nicola

2012-01-01

Autism and autism spectrum disorders (ASDs) are heterogeneous complex neuro-developmental disorders characterized by dysfunctions in social interaction and communication skills. Their pathogenesis has been linked to interactions between genes and environmental factors. Consistent with the evidence of certain similarities between immune cells and…

Low cytotoxicity of anisotropic gold nanoparticles coated with lysine on peripheral blood mononuclear cells "in vitro".

PubMed

Avila-Alejo, Jorge O; González-Palomo, Ana K; Plascencia-Villa, Germán; José-Yacamán, Miguel; Navarro-Contreras, Hugo R; Pérez-Maldonado, Iván N

2017-12-01

The aim of this study was to evaluate the cytotoxic effects of anisotropic (non spherical morphologies) gold nanoparticles coated with the amino acid Lysine (Lys) on peripheral blood mononuclear cells (PBMC) "in vitro". Gold (Au) nanoparticles tested in this study were synthesized by a seed-mediated growth using Lys as a structure and shape directing agent. Cytotoxic effects were evaluated by cell viability (resazurin assay), reactive oxygen species (ROS) induction (2',7'-dichlorofluorescein diacetate assay), DNA damage (comet assay) and apoptosis/necrosis (AnnexinV/propidium iodide assay) after PBMC were exposed to increasing concentrations (10, 25, 50, 100, and 250μM) of AuNPs coated with Lys (AuNPs-Lys) at different exposure times (3, 6, 12, and 24h). The results demonstrated that AuNPs-Lys exhibited low cytotoxicity towards PBMC, (high cell viability), with low levels of ROS, DNA damage and apoptosis/necrosis detected after treatment. These data suggest that AuNPs-Lys, might be viable for biomedical application subject to further investigations. Copyright © 2017 Elsevier B.V. All rights reserved.

Cell therapy of pseudarthrosis.

PubMed

Bastos Filho, Ricardo; Lermontov, Simone; Borojevic, Radovan; Schott, Paulo Cezar; Gameiro, Vinicius Schott; Granjeiro, José Mauro

2012-01-01

To assess the safety and efficiency of cell therapy for pseudarthrosis. Implant of the bone marrow aspirate was compared to mononuclear cells purified extemporaneously using the Sepax(®) equipment. Six patients with nonunion of the tibia or femur were treated. Four received a percutaneous infusion of autologous bone marrow aspirated from the iliac crest, and two received autologous bone marrow mononuclear cells separated from the aspirate with the Sepax(®). The primary fixation method was unchanged, and the nonunion focus was not exposed. Physical examination and radiographies were performed 2, 4 and 6 months after the treatment by the same physician. After consolidation of the fracture the satisfaction of the patients was estimated using the adapted QALY scale. No complications occurred as a result of the referred procedures. Bone consolidation was obtained in all cases within 3 to 24 weeks. The degree of patient satisfaction before and after bone consolidation was assessed, with the average value increasing from two to nine (p=0.0156). We conclude that the proposed method is effective and safe for the treatment of nonunion of long bones regardless of the stabilization method used. Level of Evidence II, Prospective Comparative Study.

An investigation of the effects of MitoQ on human peripheral mononuclear cells.

PubMed

Marthandan, Shiva; Murphy, Michael P; Billett, Ellen; Barnett, Yvonne

2011-03-01

MitoQ is a ubiquinone derivative targeted to mitochondria which is known to have both antioxidant and anti-apoptotic properties within mammalian cells. Previous research has suggested that the age-related increase in oxidative DNA damage in T lymphocytes might contribute to their functional decline with age. This paper describes the impact of mitoQ on unchallenged or oxidatively challenged ex vivo human peripheral blood mononuclear cells from healthy 25-30 or 55-60 year old volunteers. When cells were challenged with hydrogen peroxide (H(2)O(2)), following mitoQ treatment (0.1-1.0 μM), the ratio of reduced to oxidized forms of glutathione increased, the levels of oxidative DNA damage decreased and there was an increase in the mitochondrial membrane potential. Low levels of mitoQ (0.1 or 0.25 μM) had no impact on endogenous DNA damage, whilst higher levels (0.5 and 1.0 μM) of mitoQ significantly reduced endogenous levels of DNA damage. The results of this investigation suggest that mitoQ may have anti-immunosenescent potential.

Evidence of Mycobacterium tuberculosis complex bacteraemia in intradermal skin test positive cattle detected using phage-RPA.

PubMed

Swift, Benjamin M C; Convery, Thomas W; Rees, Catherine E D

2016-10-02

Bovine tuberculosis is a zoonotic infectious disease caused by Mycobacterium bovis that affects cattle and can cause tuberculosis in a range of wildlife animals. A bacteriophage-based method combined with PCR (phage-PCR) has been recently used to detect and identify viable pathogenic mycobacteria in the peripheral blood mononuclear cells (PBMCs) of animals suffering from paratuberculosis. To adapt this method for the detection of M. bovis in blood, a new isothermal DNA amplification protocol using Recombinase Polymerase Amplification (RPA) was developed and was found to be able to detect M. bovis BCG within 48 h, with a limit of detection of approximately 10 cells per ml of blood for artificially inoculated blood samples. When blood samples (2 ml) from a Single Comparative Cervical Intradermal Tuberculin (SCCIT)- negative beef herd were tested, Mycobacterium tuberculosis complex (MTC) cells were not detected from any (45) of the blood samples. However when blood samples from SCCIT-positive animals were tested, viable MTC bacteria were detected in 66 % (27/41) of samples. Of these 41 animals sampled, 32 % (13) had visible lesions. In the visible lesion (VL) group, 85 % (11/13) had detectable levels of MTC whereas only 57 % (16/28) of animals which had no visible lesions (NVL) were found to have detectable mycobacteraemia. These results indicated that this simple, rapid method can be applied for the study of M. bovis infections. The frequency with which viable mycobacteria were detected in the peripheral blood of SCCIT-positive animals changes the paradigm of this disease.

Evidence of Epstein-Barr Virus Association with Gastric Cancer and Non-Atrophic Gastritis

PubMed Central

Martínez-López, Juan L.E.; Torres, Javier; Camorlinga-Ponce, Margarita; Mantilla, Alejandra; Leal, Yelda A.; Fuentes-Pananá, Ezequiel M.

2014-01-01

Different lines of evidence support an association between Epstein-Barr virus (EBV) and gastric cancer (GC). The main understood risk factor to develop GC is infection by Helicobacter pylori (H. pylori), which triggers a local inflammatory response critical for progression from gastritis to GC. The role of EBV in early inflammatory gastric lesions has been poorly studied. A recent study proposed a cutoff value of 2000 EBV particles to identify patients with increased chances of infection of the gastric epithelium, which may favor the inflammatory process. To better understand the role of EBV in cancer progression, we analyzed 75 samples of GC, 147 control samples of non-tumor gastric tissue derived from GC patients and 75 biopsies from patients with non-atrophic gastritis (NAG). A first-round PCR was used for EBV detection in tumor and non-tumor controls and a more sensitive nested PCR for gastritis samples; both PCRs had lower detection limits above the proposed cutoff value. With this strategy 10.67% of GC, 1.3% of non-tumor controls and 8% of gastritis samples were found positive. An EBER1 in situ hybridization showed EBV infection of epithelial cells in GC and in a third of NAG samples, while in the other NAGs infection was restricted to the mononuclear cell infiltrate. EBV-positive GCs were enriched in lace and cribriform patterns, while these rare patterns were not observed in EBV negative samples. Our results support a role for EBV in GC and early precursor lesions, either as directly oncogenic infecting epithelial cells or indirectly as an inflammatory trigger. PMID:24448220

Mononuclear cells, mast cells and mucous cells as part of the delayed hypersensitivity response to aerosolized antigen in mice.

PubMed Central

Enander, I; Ahlstedt, S; Nygren, H

1984-01-01

Mice (BALB/c) exposed to picryl chloride (PiCl) on their shaved abdomen and untreated animals were after 7 days exposed to daily aerosolized trinitrophenylated dog serum albumin (TNP-DSA). Mice exposed to PiCl tended to respond earlier and more strongly with both delayed hypersensitivity (DH) and IgG antibodies in serum and bronchial washings than did mice exposed to aerosol only. Picryl chloride sensitization resulted in spontaneously proliferating axillary lymph node cells, which could not be further stimulated with antigen or mitogen. Histological examination of lung tissue of aerosol-sensitized animals revealed an increase in mononuclear cells and mast cells around bronchioli and mucous cells, particularly in those animals exposed for prolonged periods and sensitized with PiCl prior to aerosol. Sensitization of mice with aerosolized TNP-DSA administrated in two 2- and 1-week periods with a 4-week interval responded with DH and IgG antibody in a dose dependent fashion irrespective of presensitization with PiCl. In bronchial washings IgG antibodies were found particularly after two 2-week periods of exposure. The cells taken from the axillary or brachial lymph nodes showed spontaneous proliferation. Culture of the cells to achieve mast cell maturation resulted in no or very low numbers of mast cells in the lymph nodes. PMID:6706375

Mononuclear Cells from Dedifferentiation of Mouse Myotubes display Remarkable Regenerative Capability

PubMed Central

Yang, Zhong; Liu, Qiang; Mannix, Robert J.; Xu, Xiaoyin; Li, Hongli; Ma, Zhiyuan; Ingber, Donald E.; Allen, Paul D.; Wang, Yaming

2015-01-01

Certain lower organisms achieve organ regeneration by reverting differentiated cells into tissue-specific progenitors that re-enter embryonic programs. During muscle regeneration in the urodele amphibian, post-mitotic multinucleated skeletal myofibers transform into mononucleated proliferating cells upon injury, and a transcription factor-msx1 plays a role in their reprograming. Whether this powerful regeneration strategy can be leveraged in mammals remains unknown, as it has not been demonstrated that the dedifferentiated progenitor cells arising from muscle cells overexpressing Msx1 are lineage-specific and possess the same potent regenerative capability as their amphibian counterparts. Here we show that ectopic expression of Msx1 reprograms post-mitotic, multinucleated, primary mouse myotubes to become proliferating mononuclear cells. These dedifferentiated cells reactivate genes expressed by embryonic muscle progenitor cells and generate only muscle tissue in vivo both in an ectopic location and inside existing muscle. More importantly, distinct from adult muscle satellite cells, these cells appear both to fuse with existing fibers and to regenerate myofibers in a robust and time-dependent manner. Upon transplantation into a degenerating muscle, these dedifferentiated cells generated a large number of myofibers that increased over time and replenished almost half of the cross-sectional area of the muscle in only 12 weeks. Our study demonstrates that mammals can harness a muscle regeneration strategy used by lower organisms when the same molecular pathway is activated. PMID:24916688

Automated processing of human bone marrow grafts for transplantation.

PubMed

Zingsem, J; Zeiler, T; Zimmermanm, R; Weisbach, V; Mitschulat, H; Schmid, H; Beyer, J; Siegert, W; Eckstein, R

1993-01-01

Prior to purging or cryopreservation, we concentrated 21 bone marrow (BM) harvests using a modification of the 'grancollect-protocol' of the Fresenius AS 104 cell separator with the P1-Y set. Within 40-70 min, the initial marrow volume of 1,265 ml (+/- 537 ml) was processed two to three times. A mean of 47% (+/- 21%) of the initial mononuclear cells was recovered in a mean volume of 128 ml (+36 ml). The recovery of clonogenic cells, measured by CFU-GM assays, was 68% (+/- 47%). Red blood cells in the BM concentrates were reduced to 7% (+/- 4%) of the initial number. The procedure was efficient and yielded a BM cell fraction suitable for purging, cryopreservation and transplantation. At this time, 10 of the 21 patients whose BM was processed using this technique have been transplanted. Seven of these 10 patients have been grafted using the BM alone. Three of the 10 patients showed reduced cell viability and colony growth in the thawed BM samples, and therefore obtained BM and peripheral blood-derived stem cells. All transplanted patients showed an evaluable engraftment, achieving 1,000 granulocytes per microliter of peripheral blood in a mean of 18 days.

Pretranslational regulation of the synthesis of the third component of complement in human mononuclear phagocytes by the lipid A portion of lipopolysaccharide.

PubMed Central

Strunk, R C; Whitehead, A S; Cole, F S

1985-01-01

The third component of complement (C3) is a plasma glycoprotein with a variety of biologic functions in the initiation and maintenance of host response to infectious agents. While the hepatocyte is the primary source of plasma C3, mononuclear phagocytes contribute to the regulation of tissue availability of C3. Lipopolysaccharide (LPS), a constituent of cell walls of gram-negative bacteria, consists of a polysaccharide moiety (core polysaccharide and O antigen) covalently linked to a lipid portion (lipid A). Using metabolic labeling with [35S]methionine, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis, we examined the effects of LPS on synthesis of C3 by human mononuclear phagocytes as well as synthesis of the second component of complement (C2), factor B, lysozyme, and total protein. LPS increased C3 synthesis 5-30-fold without affecting the kinetics of secretion of C3 or the synthesis of C2, lysozyme, or total protein. Factor B synthesis was consistently increased by LPS. Experiments with lipid A-inactivated LPS (alkaline treated), LPS from a polysaccharide mutant strain, and lipid X (a lipid A precursor) indicated that the lipid A portion is the structural element required for this effect. Northern blot analysis demonstrated at least a fivefold increase in C3 mRNA in LPS-treated monolayers, which suggests that the regulation of the increase in C3 synthesis is pretranslational. C2 mRNA and factor B mRNA were increased approximately twofold. The availability of specific gene products in human mononuclear phagocytes that respond to LPS should permit understanding of the molecular regulation of more complex functions of these cells elicited by LPS in which multiple gene products are coordinately expressed. Images PMID:3900137

Glutathione and growth inhibition of Mycobacterium tuberculosis in healthy and HIV infected subjects

PubMed Central

Venketaraman, Vishwanath; Rodgers, Tatanisha; Linares, Rafael; Reilly, Nancy; Swaminathan, Shobha; Hom, David; Millman, Ariel C; Wallis, Robert; Connell, Nancy D

2006-01-01

Intracellular levels of glutathione are depleted in patients with acquired immunodeficiency syndrome in whom the risk of tuberculosis, particularly disseminated disease is many times that of healthy individuals. In this study, we examined the role of glutathione in immunity against tuberculosis infection in samples derived from healthy and human immunodeficiency virus infected subjects. Our studies confirm that glutathione levels are reduced in peripheral blood mononuclear cells and in red blood cells isolated from human immunodeficiency virus-infected subjects (CD4>400/cumm). Furthermore, treatment of blood cultures from human immunodeficiency virus infected subjects with N-acetyl cysteine, a glutathione precursor, caused improved control of intracellular M. tuberculosis infection. N-acetyl cysteine treatment decreased the levels of IL-1, TNF-α, and IL-6, and increased the levels of IFN-γ in blood cultures derived from human immunodeficiency virus-infected subjects, promoting the host immune responses to contain M. tuberculosis infection successfully. PMID:16504020

Lactobacillus acidophilus modulates inflammatory activity by regulating the TLR4 and NF-κB expression in porcine peripheral blood mononuclear cells after lipopolysaccharide challenge.

PubMed

Lee, Sang In; Kim, Hyun Soo; Koo, Jin Mo; Kim, In Ho

2016-02-28

A total of forty weaned pigs ((Landrace × Yorkshire) × Duroc) were used to evaluate the effects of Lactobacillus acidophilus on inflammatory activity after lipopolysaccharide (LPS) challenge. Experimental treatments were as follows: (T1) control diet+saline challenge; (T2) control diet with 0·1% L. acidophilus+saline challenge; (T3) control diet+LPS challenge; and (T4) control diet with 0·1% L. acidophilus+LPS challenge. On d-14, piglets were challenged with saline (T1 and T2) or LPS (T3 and T4). Blood samples were obtained at 0, 2, 4, 6 and 12 h after being challenged and analysed for immune cell cytokine production and gene expression pattern. The L. acidophilus treatment increased the average daily weight gain (ADWG) and average daily feed intake (ADFI) compared with the control diet. With the control diet, the LPS challenge (T3) increased the number of immune cells and expression of TNF-α and IL-6 compared with the saline challenge (T1). Whereas with the saline challenge L. acidophilus treatment (T2) increased the number of leucocytes and CD4 compared with the control diet (T1), with the LPS challenge L. acidophilus treatment (T4) decreased the number of leucocytes, lymphocytes, CD4+ and CD8+ and expression of TNF-α and IL-6 compared with the control diet (T3). L. acidophilus treatment decreased the expression of TRL4 and NF-κB in peripheral blood mononuclear cells (PBMC) after LPS challenge, which leads to inhibition of TNF-α, IFN-γ, IL-6, IL-8 and IL1B1 and to induction of IL-4 and IL-10. We suggested that L. acidophilus improved ADWG and ADFI and protected against LPS-induced inflammatory responses by regulating TLR4 and NF-κB expression in porcine PBMC.

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

PubMed

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 < 0.001) reduced total neopterin production from the mixed cell culture, whereas cold water immersion significantly (P < 0.05) attenuated urinary neopterin and total neopterin during the training camp without having any effect on physical performance parameters. Soreness and fatigue showed little variation between the groups, whereas training session performance was significantly (P < 0.05) elevated in the cold water immersion group. The data suggest that acute and repetitive cryotherapy attenuates in vitro T-cell and monocyte activation. This 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.

Imaging of blood antigen distribution on blood cells by thermal lens microscopy

NASA Astrophysics Data System (ADS)

Kimura, Hiroko; Sekiguchi, Kazuya; Nagao, Fumiko; Mukaida, Masahiro; Kitamori, Takehiko; Sawada, Tsuguo

2000-05-01

Blood group antigens on a cell were measured by a new microscopic method, i.e. thermal lens microscopy which involves spectrometry using a laser-induced thermal-lens effect. The blood group antigen was immunologically stained using antibody labeled with colloidal gold. Human leukocyte antigens (HLA) on lymphocytes and mononuclear leukocytes were observed by the thermal lens microscope, and Lewis blood group antigens on erythrocytes and polymorphonuclear leukocytes were also observed. The antigen distribution on each cell-surface was imaged using this technique. In spite of convex surface of living cells, colloidal gold was correctly quantified by adjusting the deviation of the focal point of the probe laser by the phase of the signal. In the measurement of leukocyte antigens, antigens of HLA-A, -B, -C loci on the lymphocytes were identified and quantitated by using a single cell. The image of HLA-A, -B, -C antigen distribution on a mononuclear leukocyte was obtained. In the measurement of erythrocyte antigens, a small quantity of Lewis antigens was detected on the cord erythrocytes. Localized small quantities of membrane antigens are better quantitated without extraction or cytolysis. Our thermal lens microscope is a powerful and highly sensitive analytical tool for detecting and quantitating localized antigens in single cells and/or cell-surface-associated molecules.

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

PubMed

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) (< 3 kDa) was shown to be responsible for the observed effect. This mitogenic oligopeptide(s) is functionally dissimilar to auxin and cytokinin and, in addition, it does not change the sensitivity of the protoplasts toward these phytohormones. In combination with the mitogen phytohemagglutinin (PHA), 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.

Effect of cryopreservation on delineation of immune cell subpopulations in tumor specimens as determinated by multiparametric single cell mass cytometry analysis.

PubMed

Kadić, Elma; Moniz, Raymond J; Huo, Ying; Chi, An; Kariv, Ilona

2017-02-02

Comprehensive understanding of cellular immune subsets involved in regulation of tumor progression is central to the development of cancer immunotherapies. Single cell immunophenotyping has historically been accomplished by flow cytometry (FC) analysis, enabling the analysis of up to 18 markers. Recent advancements in mass cytometry (MC) have facilitated detection of over 50 markers, utilizing high resolving power of mass spectrometry (MS). This study examined an analytical and operational feasibility of MC for an in-depth immunophenotyping analysis of the tumor microenvironment, using the commercial CyTOF™ instrument, and further interrogated challenges in managing the integrity of tumor specimens. Initial longitudinal studies with frozen peripheral blood mononuclear cells (PBMCs) showed minimal MC inter-assay variability over nine independent runs. In addition, detection of common leukocyte lineage markers using MC and FC detection confirmed that these methodologies are comparable in cell subset identification. An advanced multiparametric MC analysis of 39 total markers enabled a comprehensive evaluation of cell surface marker expression in fresh and cryopreserved tumor samples. This comparative analysis revealed significant reduction of expression levels of multiple markers upon cryopreservation. Most notably myeloid derived suppressor cells (MDSC), defined by co-expression of CD66b + and CD15 + , HLA-DR dim and CD14 - phenotype, were undetectable in frozen samples. These results suggest that optimization and evaluation of cryopreservation protocols is necessary for accurate biomarker discovery in frozen tumor specimens.

Role of T Cell TGF-β Signaling in Intestinal Cytokine Responses and Helminthic Immune Modulation

PubMed Central

Ince, M. Nedim; Elliott, David E.; Setiawan, Tommy; Metwali, Ahmed; Blum, Arthur; Chen, Hung-lin; Urban, Joseph F.; Flavell, Richard A.; Weinstock, Joel V.

2010-01-01

Colonization with helminthic parasites induces mucosal regulatory cytokines, like IL-10 or TGF-β that are important in suppressing colitis. Helminths induce mucosal T cell IL-10 secretion and regulate lamina propria mononuclear cell Th1 cytokine generation in an IL-10 dependent manner in wild-type mice. Helminths also stimulate mucosal TGF-β release. As TGF-β exerts major regulatory effects on T lymphocytes, we investigated the role of T lymphocyte TGF-β signaling in helminthic modulation of intestinal immunity. T cell TGF-β signaling is interrupted in TGF-βRII DN mice by T cell-specific over-expression of a dominant negative TGF-β receptor II. We studied lamina propria mononuclear cell responses in wild-type and TGF-βRII DN mice that were uninfected or colonized with the nematode, Heligmosomoides polygyrus. Our results indicate an essential role of T cell TGF-β signaling in limiting mucosal Th1 and Th2 responses. Furthermore, we demonstrate that helminthic induction of intestinal T cell IL-10 secretion requires intact T cell TGF-β signaling pathway. Helminths fail to curtail robust, dysregulated intestinal Th1 cytokine production and chronic colitis in TGF-βRII DN mice. Thus, T cell TGF-β signaling is essential for helminthic stimulation of mucosal IL-10 production, helminthic modulation of intestinal interferon-γ generation and H. polygyrus-mediated suppression of chronic colitis. PMID:19544487

Human Peripheral Blood Mononuclear Cells Cultured in Normal and Hyperglycemic Media in Simulated Microgravity Using NASA Bioreactors

NASA Technical Reports Server (NTRS)

Lawless, DeSales

2003-01-01

We sought answers to several questions this summer at NASA Johnson Space Center. Initial studies involved the in vitro culture of human peripheral blood mononuclear in cells in different conditioned culture media. Several human cancer clones were similarly studied to determine responses to aberrant glycosylation by the argon laser. The cells were grown at unit gravity in flasks and in simulated microgravity using NASA bioreactors. The cells in each instance were analyzed by flow cytometry. Cell cycle analysis was acquired by staining nuclear DNA with propidium iodide. Responses to the laser stimulation was measured by observing autofluorescence emitted in the green and red spectra after stimulation. Extent of glycosylation correlated with the intensity of the laser stimulated auto-fluorescence. Our particular study was to detect and monitor aberrant glycosylation and its role in etiopathogenesis. Comparisons were made between cells known to be neoplastic and normal cell controls using the same Laser Induced Autofluorescence technique. Studies were begun after extensive literature searches on using the antigen presenting potential of dendritic cells to induce proliferation of antigen specific cytotoxic T-cells. The Sendai virus served as the antigen. Our goal is to generate sufficient numbers of such cells in the simulated microgravity environment for use in autologous transplants of virally infected individuals including those positive for hepatitis and HIV.

Effects of nonsteroidal anti-inflammatory meloxicam on stomach, kidney, and liver of rats.

PubMed

Burukoglu, Dilek; Baycu, Cengiz; Taplamacioglu, Fulya; Sahin, Erhan; Bektur, Ezgi

2016-06-01

Nonsteroidal anti-inflammatory (NSAI) drugs are the most commonly used group of drugs today. Increase in the use of standard NSAI for treating pain and inflammation was restricted by the fact that these drugs were proven to possibly cause gastrointestinal and renal toxicity. Meloxicam is a NSAI that has anti-inflammatory, analgesic, and antipyretic effects. This study aims to investigate the effects of meloxicam on stomach, kidney, and liver of rats under light microscopy level. Based on the light microscopic observations, mononuclear cell infiltration and pseudolobular formation was established in liver samples of animals in the experimental group. Metaplasia in surface and glandular epithelia and atrophy were observed in stomach samples. Glomerular stasis-related hypertrophy and focal interstitial nephritis were found in kidneys. It was concluded in this study that meloxicam might cause hepatotoxicity, nephrotoxicity, and gastric metaplasia in rats at a used dose and duration. © The Author(s) 2014.

Flow cytometric discrimination of seven lineage markers by using two fluorochromes

PubMed Central

Boin, Francesco; Giardino Torchia, Maria Letizia; Borrello, Ivan; Noonan, Kimberly A.; Neil, Matthew; Soloski, Mark J.

2017-01-01

Flow cytometry is the primary immunological technique used to analyze multiple parameters on complex cell populations. We present a staining method that identifies major human mononuclear lymphoid and myeloid populations (CD4+ and CD8+ T cells, γδ T cells, B cells, NK cells and monocytes), using only two fluorochromes and a minimal number of cells. Our approach increases the number of markers recordable on most flow cytometers allowing for a deeper and more comprehensive immunophenotyping. PMID:29190813

Killing multiple myeloma cells with the small molecule 3-bromopyruvate: implications for therapy.

PubMed

Majkowska-Skrobek, Grażyna; Augustyniak, Daria; Lis, Paweł; Bartkowiak, Anna; Gonchar, Mykhailo; Ko, Young H; Pedersen, Peter L; Goffeau, Andre; Ułaszewski, Stanisław

2014-07-01

The small molecule 3-bromopyruvate (3-BP), which has emerged recently as the first member of a new class of potent anticancer agents, was tested for its capacity to kill multiple myeloma (MM) cancer cells. Human MM cells (RPMI 8226) begin to lose viability significantly within 8 h of incubation in the presence of 3-BP. The Km (0.3 mmol/l) for intracellular accumulation of 3-BP in MM cells is 24 times lower than that in control cells (7.2 mmol/l). Therefore, the uptake of 3-BP by MM cells is significantly higher than that by peripheral blood mononuclear cells. Further, the IC50 values for human MM cells and control peripheral blood mononuclear cells are 24 and 58 µmol/l, respectively. Therefore, specificity and selectivity of 3-BP toward MM cancer cells are evident on the basis of the above. In MM cells the transcription levels of the gene encoding the monocarboxylate transporter MCT1 is significantly amplified compared with control cells. The level of intracellular ATP in MM cells decreases by over 90% within 1 h after addition of 100 µmol/l 3-BP. The cytotoxicity of 3-BP, exemplified by a marked decrease in viability of MM cells, is potentiated by the inhibitor of glutathione synthesis buthionine sulfoximine. In addition, the lack of mutagenicity and its superior capacity relative to Glivec to kill MM cancer cells are presented in this study.

Changes in metabolite, energy metabolism related enzyme activities and peripheral blood mononuclear cell (PBMC) populations in beef heifers with two differing liveweight change profiles in New Zealand.

PubMed

Mori, A; Kenyon, P R; Mori, N; Yamamoto, I; Tanaka, Y; Suzuki, N; Tazaki, H; Ozawa, T; Hayashi, T; Hickson, R E; Morris, S T; Blair, H; Arai, T

2008-02-01

Metabolite and immunoreactive insulin (IRI) concentrations, energy metabolism related enzymes activities and peripheral blood mononuclear cell (PBMC) populations were measured in blood of pregnant Angus heifers with differing liveweight change profiles (gaining or losing), in New Zealand to investigate the meanings of those parameters in the restricted feeding beef heifers. Beef heifers losing liveweight (-412 g/day) showed significantly lower concentrations of plasma IRI, and higher concentrations of plasma free fatty acid (FFA) than heifers gaining liveweight (483 g/day). The cytosolic and mitochondrial malate dehydrogenase (MDH) activities and MDH/lactate dehydrogenase (M/L) ratio in leukocytes of the liveweight losing heifers were significantly higher than those the liveweight gaining heifers. Percentages of cluster of differentiation (CD) 3 positive cells and natural killer (NK) cells in PBMC decreased significantly in the liveweight losing heifers compared to those in the liveweight gaining heifers. Plasma IRI and FFA concentrations, leukocyte cytosolic and mitochondrial MDH activities and CD3 positive and NK cell populations may be useful markers to evaluate metabolic conditions and immunity in the restricted feeding beef heifers.

Olive Oil Phenolics Prevent Oxysterol‐Induced Proinflammatory Cytokine Secretion and Reactive Oxygen Species Production in Human Peripheral Blood Mononuclear Cells, Through Modulation of p38 and JNK Pathways

PubMed Central

Deiana, Monica; Spencer, Jeremy P. E.; Corona, Giulia

2017-01-01

Scope The aim of the present study was to investigate the ability of extra virgin olive oil (EVOO) polyphenols to counteract the proinflammatory effects induced by dietary and endogenous oxysterols in ex vivo immune cells. Methods and results Peripheral blood mononuclear cells (PBMCs), separated from the whole blood of healthy donors, were utilized and were stimulated with an oxysterols mixture, in the presence of physiologically relevant concentrations of the EVOO polyphenols, hydroxytyrosol, tyrosol, and homovanillic alcohol. Oxysterols significantly increased the production of proinflammatory cytokines, interleukin‐1β, regulated on activation, normal T‐cell expressed and secreted and macrophage migration inhibitory factor in ex vivo cultured PBMCs. Increased levels of reactive oxygen species (ROS) were also detected along with increased phosphorylation of the p38 and JNK. All phenolic compounds significantly reduced cytokine secretion induced by the oxysterols and inhibited ROS production and mitogen activated protein kinase phosphorylation. Conclusions These results suggest that extra virgin olive oil polyphenols modulate the immune response induced by dietary and endogenous cholesterol oxidation products in human immune cells and may hold benefit in controlling chronic immune and/or inflammatory processes. PMID:28815947

Modulation of the proteome of peripheral blood mononuclear cells from HIV-1 infected patients by drugs of abuse

PubMed Central

Reynolds, Jessica L.; Mahajan, Supriya D.; Aalinkeel, Ravikunar; Nair, Bindukumar; Sykes, Donald E.; Agosto-Mujica, Arnadri; Hsiao, Chiu Bin; Schwartz, Stanley A.

2010-01-01

We used proteomic analyses to assess how drug abuse modulates immunologic responses to infections with the human immunodeficiency virus type 1 (HIV-1). Two dimensional (2D) difference gel electrophoresis was utilized to determine changes in the proteome of peripheral blood mononuclear cells (PBMC) isolated from HIV-1 positive donors that occurred after treatment with cocaine or methamphetamine. Both drugs differentially regulated the expression of several functional classes of proteins. We further isolated specific subpopulations of PBMC to determine which subpopulations were selectively affected by treatment with drugs of abuse. Monocytes, B cells and T cells were positively or negatively selected from PBMC isolated from HIV-1 positive donors. Our results demonstrate that cocaine and methamphetamine modulate gene expression primarily in monocytes and T cells, the primary targets of HIV-1 infection. Proteomic data were validated with quantitative, real-time PCR. These studies elucidate the molecular mechanisms underlying the effects of drugs of abuse on HIV-1 infections. Several functionally relevant classes of proteins were identified as potential mediators of HIV-1 pathogenesis and disease progression associated with drug abuse. PMID:19543960

Fabrication of Cardiac Patch with Decellularized Porcine Myocardial Scaffold and Bone Marrow Mononuclear Cells

PubMed Central

Wang, Bo; Borazjani, Ali; Tahai, Mina; de Jongh Curry, Amy L.; Simionescu, Dan T.; Guan, Jianjun; To, Filip; Elder, Steve H.; Liao, Jun

2010-01-01

Tissue engineered cardiac grafts are a promising therapeutic mode for ventricular wall reconstruction. Recently, it has been found that acellular tissue scaffolds provide natural ultrastructural, mechanical, and compositional cues for recellularization and tissue remodeling. We thus assess the potential of decellularized porcine myocardium as a scaffold for thick cardiac patch tissue engineering. Myocardial sections with 2 mm thickness were decellularized using 0.1% sodium dodecyl sulfate (SDS), and then reseeded with differentiated bone marrow mononuclear cells. We found that thorough decellularization could be achieved after 2.5 weeks treatment. Reseeded cells were found to infiltrate and proliferate in the tissue constructs. Immunohistological staining studies showed that the reseeded cells maintained cardiomyocyte-like phenotype and possible endothelialization was found in locations close to vasculature channels, indicating angiogenesis potential. Both biaxial and uniaxial mechanical testing showed a stiffer mechanical response of the acellular myocardial scaffolds; however, tissue extensibility and tensile modulus were found to recover in the constructs along with the culture time, as expected from increased cellular content. The cardiac patch that we envision for clinical application will benefit from the natural architecture of myocardial extracellular matrix, which has the potential to promote stem cell differentiation, cardiac regeneration, and angiogenesis. PMID:20694977

Silicone nanoparticles do not induce immune responses by naïve human peripheral blood mononuclear cells: implications in breast implants.

PubMed

Nair, Narayanan; Pilakka-Kanthikeel, Sudheesh; Saiyed, Zainulabedin; Yndart, Adriana; Nair, Madhavan

2012-07-01

Several studies have reported adverse immunological effects of silicone due to their ability to induce proinflammatory molecules, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). In recent years, use of nanoparticles has been under fast development for therapeutic drug targeting, diagnostic imaging, and immune response in various fields of nanomedicine. The authors hypothesize that immune responses induced by in vivo use of silicone materials can be reduced or eliminated by the use of nanosilicone. Peripheral blood mononuclear cells obtained from naïve normal subjects were cultured with different concentrations of silicone nanoparticles and microparticles for 24 hours. The culture supernatants were quantitated for TNF-α, IL-6, and interferon-γ (IFN-γ) secretion by enzyme-linked immunosorbent assay. The pellets were used for specific IL-6, TNF-α, and IFN-γ gene expression by real-time polymerase chain reaction, respectively. Cytotoxicity was evaluated by XTT viability assay. Results were compared between silicone nanoparticles and microparticles and untreated controls. Silicone nanoparticles up to 100 μg/ml did not induce any detectable levels of specific TNF-α, IFN-γ, and IL-6 gene expression and protein production and the results were comparable to those for untreated controls. Silicone microparticles at 100 μg/ml, however, significantly induced the production and gene expression of TNF-α, IL-6, and IFN-γ by peripheral blood mononuclear cells. XTT viability assay showed that silicone nanoparticles or microparticles, even at the highest concentration used, were not cytotoxic to cells. The results suggest that silicone nanoparticles can be engineered to avoid immune recognition and subsequent silicone microparticle-related adverse effects and thus may be of therapeutic significance in the cosmetic industry, plastic surgery, and aesthetic medicine.

Successful treatment by double-filtration plasmapheresis of a patient with bullous pemphigoid: effects in vivo on transcripts of several genes for chemokines and cytokines in peripheral blood mononuclear cells.

PubMed

Hatano, Y; Katagiri, K; Arakawa, S; Umeki, T; Takayasu, S; Fujiwara, S

2003-03-01

The involvement of various cytokines and chemokines has been reported in the pathogenesis of bullous pemphigoid (BP). Double-filtration plasmapheresis (DFPP) is an effective treatment for BP but the mechanism of action remains unclear. Using semiquantitative reverse transcription-polymerase chain reaction, we examined levels of transcripts for various cytokines and chemokines in freshly isolated peripheral blood mononuclear cells in a patient with BP before and after DFPP treatment. DFPP was performed four times. Relative levels of transcripts for interleukin (IL)-8, macrophage inflammatory protein (MIP)-1alpha and IL-5, and the ratio of relative levels of transcripts for IL-4 and interferon (IFN)-gamma, were higher, before treatment, than in healthy controls, and decreased when the extent of the lesions was reduced. Relative levels of transcripts for tumour necrosis factor (TNF)-alpha and IL-4 also decreased with regression of lesions, although they were similar to or lower than the corresponding levels in healthy individuals. When eruptions recurred, relative levels of transcripts for IL-8, MIP-1alpha, RANTES (regulated upon activation normal T cell expressed and secreted), IL-2, IFN-gamma and TNF-alpha were very much higher than those prior to the recurrence, while relative levels of mRNAs for IL-4 and IL-5 did not increase. Relative levels of transcripts for IL-8, MIP-1alpha, TNF-alpha and IL-2 were lower at the end of each individual DFPP and after the four treatments than at the beginning of treatment. Our observations suggest that cytokines and chemokines produced in mononuclear cells play important roles in the pathogenesis of BP and that regulation of their expression might be involved in the therapeutic effects of DFPP in BP.

B7-H1 expression is associated with expansion of regulatory T cells in colorectal carcinoma

PubMed Central

Hua, Dong; Sun, Jing; Mao, Yong; Chen, Lu-Jun; Wu, Yu-Yu; Zhang, Xue-Guang

2012-01-01

AIM: To investigate the expression of B7-H1 in human colorectal carcinoma (CRC) to define its regulating effects on T cells in tumor microenvironment. METHODS: One hundred and two paraffin blocks and 33 fresh samples of CRC tissues were subject to this study. Immunohistochemistry was performed for B7-H1 and CD3 staining in CRC tissues. Ficoll-Hypaque density gradient centrifugation was used to isolate peripheral blood mononuclear cells of fresh CRC tissues; flow cytometry and immunofluorescence staining were used for detection of regulatory T cells. Data was analyzed with statistical software. RESULTS: Costimulatory molecule B7-H1 was found strongly expressed in CRC tissues, localized in tumor cell membrane and cytoplasm, while weak or none expression of B7-H1 was detected in pared normal colorectal tissues. Meanwhile, CD3 positive T cells were found congregated in CRC tumor nest and stroma. Statistic analysis showed that B7-H1 expression level was negatively correlated to the total T cell density in tumor nest (P < 0.0001) and tumor stroma (P = 0.0200) of 102 cases of CRC tissues. Among the total T cells, a variable amount of regulatory T cells with a clear Foxp3+ (forkhead box P3) staining could be detected in CRC tissues and patients’ blood. Interestingly, in the 33 samples (15 cases of B7-H1high CRC tissues and 18 cases of B7-H1low CRC tissues) of freshly isolated mononuclear cells from CRC tissues, the percentages of CD4+Foxp3+ and CD8+Foxp3+ regulatory T cells were found remarkably higher in B7-H1high CRC tissues than in B7-H1low CRC tissues (P = 0.0024, P = 0.0182), indicating that B7-H1 expression was involved in proliferation of regulatory T cell. No significant difference was found in CRC peripheral blood (P = 0.0863, P = 0.0678). PD-1 is the specific ligand for B7-H1 pathway transferring inhibitory signal to T cell, which is expressed by activated T cell. Our further analysis of PD-1 expression on T cells in CRC tissues showed that conventional T cells (CD4+Foxp3-/CD8+Foxp3-), which was thought to contribute to the anti-tumor immune response, highly expressed PD-1; while regulatory T cells (CD4+Foxp3+/CD8+Foxp3-) almost failed to express PD-1. The average percentage of PD-1 expression on regulatory T cells was significantly higher than the percentage of PD-1 on conventional T cells (CD4+Foxp3- T cell, P < 0.0001; CD8+Foxp3- T cell, P < 0.0001). The diverse expression of PD-1 might lead to different fate of T cell subsets in B7-H1 over-expression CRC tumor microenvironment. CONCLUSION: B7-H1 expression in tumor cells can inhibit the conventional T cell proliferation in tumor microenvironment through the PD-1 expression on conventional T cells. PMID:22408358

Comprehensive analysis of mouse retinal mononuclear phagocytes.

PubMed

Lückoff, Anika; Scholz, Rebecca; Sennlaub, Florian; Xu, Heping; Langmann, Thomas

2017-06-01

The innate immune system is activated in a number of degenerative and inflammatory retinal disorders such as age-related macular degeneration (AMD). Retinal microglia, choroidal macrophages, and recruited monocytes, collectively termed 'retinal mononuclear phagocytes', are critical determinants of ocular disease outcome. Many publications have described the presence of these cells in mouse models for retinal disease; however, only limited aspects of their behavior have been uncovered, and these have only been uncovered using a single detection method. The workflow presented here describes a comprehensive analysis strategy that allows characterization of retinal mononuclear phagocytes in vivo and in situ. We present standardized working steps for scanning laser ophthalmoscopy of microglia from MacGreen reporter mice (mice expressing the macrophage colony-stimulating factor receptor GFP transgene throughout the mononuclear phagocyte system), quantitative analysis of Iba1-stained retinal sections and flat mounts, CD11b-based retinal flow cytometry, and qRT-PCR analysis of key microglia markers. The protocol can be completed within 3 d, and we present data from retinas treated with laser-induced choroidal neovascularization (CNV), bright white-light exposure, and Fam161a-associated inherited retinal degeneration. The assays can be applied to any of the existing mouse models for retinal disorders and may be valuable for documenting immune responses in studies for immunomodulatory therapies.

Vascular Morphogenesis in the Context of Inflammation: Self-Organization in a Fibrin-Based 3D Culture System.

PubMed

Rüger, Beate M; Buchacher, Tanja; Giurea, Alexander; Kubista, Bernd; Fischer, Michael B; Breuss, Johannes M

2018-01-01

Introduction: New vessel formation requires a continuous and tightly regulated interplay between endothelial cells with cells of the perivascular microenvironment supported by mechanic-physical and chemical cues from the extracellular matrix. Aim: Here we investigated the potential of small fragments of synovial tissue to form de novo vascular structures in the context of inflammation within three dimensional (3D) fibrin-based matrices in vitro , and assessed the contribution of mesenchymal stromal cell (MSC)-immune cell cross-talk to neovascularization considering paracrine signals in a fibrin-based co-culture model. Material and Methods: Synovial tissue fragments from patients with rheumatoid arthritis (RA) and inflammatory osteoarthritis (OA) were cultivated within 3D fibrin matrices for up to 4 weeks. Cellular and structural re-arrangement of the initially acellular matrix were documented by phase contrast microscopy and characterized by confocal laser-scanning microscopy of topographically intact 3D cultures and by immunohistochemistry. MSC-peripheral blood mononuclear cell (PBMC) co-cultures in the 3D fibrin system specifically addressed the influence of perivascular cell interactions to neo-vessel formation in a pro-inflammatory microenvironment. Cytokine levels in the supernatants of cultured explant tissues and co-cultures were evaluated by the Bio-Plex cytokine assay and ELISA. Results: Vascular outgrowth from the embedded tissue into the fibrin matrix was preceded by leukocyte egress from the tissue fragments. Neo-vessels originating from both the embedded sample and from clusters locally formed by emigrated mononuclear cells were consistently associated with CD45 + leukocytes. MSC and PBMC in co-culture formed vasculogenic clusters. Clusters and cells with endothelial phenotype emerging from them, were surrounded by a collagen IV scaffold. No vascular structures were observed in control 3D monocultures of PBMC or MSC. Paracrine signals released by cultured OA tissue fragments corresponded with elevated levels of granulocyte-colony stimulating factor, vascular endothelial growth factor and interleukin-6 secreted by MSC-PBMC co-cultures. Conclusion: Our results show that synovial tissue fragments with immune cell infiltrates have the potential to form new vessels in initially avascular 3D fibrin-based matrices. Cross-talk and cluster formation of MSC with immune cells within the 3D fibrin environment through self-organization and secretion of pro-angiogenic paracrine factors can support neo-vessel growth.

Smoking habit and gastritis histology.

PubMed

Namiot, A; Kemona, A; Namiot, Z

2007-01-01

Long-term cigarette smoking may increase the risk of digestive tract pathologies, however, what is the influence smoking habit on gastric mucosa histology is still poorly elicited. The aim of the study was to compare histological evaluation of gastritis in smoker and non-smoker groups. A total of 236 patients of various H. pylori status (109 infected, 127 non-infected), clinical diagnosis (107 duodenal ulcer disease, 129 dyspepsia), and smoking habit (92 smokers, 144 non-smokers) were included. Subjects were classified as smokers if they smoked 5 or more cigarettes per day for at least 3 years. A histological examination of endoscopically obtained samples was performed by two experienced pathomorphologists blinded to the diagnoses and smoking habit. Microscopic slices of the gastric mucosa were stained with hematoxylin-eosin and Giemsa. Apart from histological diagnosis, H. pylori status was additionally confirmed by an urease test (CLO-test) at least in one of two gastric locations (antrum or corpus). In the H. pylori infected population, H. pylori density, neutrophils, and mononuclear cells infiltration in the gastric corpus mucosa were lower in smokers than non-smokers, while in the antrum the differences were not significant. In the non-infected population, no significant differences in neutrophils and mononuclear cells infiltration between smokers and non-smokers were found. Since the significant differences in studied parameters of chronic gastritis between smokers and non-smokers were found in the corpus mucosa of H. pylori infected subjects, smoking should be taken into account when a histological evaluation of the gastric mucosa in the H. pylori infected population is performed.

Mitochondrial Oxidative Phosphorylation Protein Levels in Peripheral Blood Mononuclear Cells Correlate with Levels in Subcutaneous Adipose Tissue within Samples Differing by HIV and Lipoatrophy Status

PubMed Central

Gerschenson, Mariana; Chow, Dominic; Libutti, Daniel E.; Willis, John H.; Murray, James; Capaldi, Roderick A.; Marusich, Michael

2008-01-01

Abstract Depletion of mitochondrial DNA (mtDNA) and mtDNA-encoded respiratory chain proteins in subcutaneous (SC) fat from patients with HIV lipoatrophy have clearly demonstrated the role of mitochondrial dysfunction in this syndrome. Research in HIV lipoatrophy, however, has been severely hampered by the lack of a suitable surrogate marker in blood or other easily obtained clinical specimens as fat biopsies are invasive and mtDNA levels in peripheral blood mononuclear cells (PBMC) do not consistently correlate with the disease process. We used a simple, rapid, quantitative 2-site dipstick immunoassay to measure OXPHOS enzymes Complex I (CI) and Complex IV (CIV), and rtPCR to measure mtDNA in 26 matched SC fat and PBMC specimens previously banked from individuals on potent antiretroviral (ARV) therapy with HIV lipoatrophy, on similar ARV therapy without lipoatrophy, and in HIV seronegative controls. Significant correlations were found between the respective PBMC and fat levels for both CI (r = 0.442, p = 0.024) and for CIV (r = 0.507, p = 0.008). Both CI and CIV protein levels were also significantly reduced in both PBMCs and fat in lipoatrophic subjects compared to HIV seronegative controls (p ≤ 0.05), while a comparative reduction in mtDNA levels in lipoatrophic subjects was observed only in fat. We conclude that CI and CIV levels in PBMCs correlate to their respective levels in fat and may have utility as surrogate markers of mitochondrial dysfunction in lipoatrophy. PMID:18844460

Effects of Caffeine Supplementation on Plasma and Blood Mononuclear Cell Interleukin-10 Levels After Exercise.

PubMed

Tauler, Pedro; Martinez, Sonia; Martinez, Pau; Lozano, Leticia; Moreno, Carlos; Aguiló, Antoni

2016-02-01

This study compared the response of interleukin (IL)-10, and also of IL-6 and IL-12 p40, to exercise and caffeine supplementation between plasma and blood mononuclear cells (BMNCs). Participants in the study (n = 28) were randomly allocated in a double-blind fashion to either caffeine (n = 14) or placebo (n = 14) treatments. One hour before completing a 15-km run competition, athletes took 6 mg/kg body mass of caffeine or a placebo. Plasma and BMNCs were purified from blood samples taken before and after competition. Concentrations of interleukins (IL-10, IL-6, and IL-12 p40), cyclic adenosine monophosphate (cAMP), caffeine, adrenaline, and cortisol were measured in plasma. IL-10, IL-6, and IL-12 p40 and cAMP levels were also determined in BMNCs. Exercise induced significant increases in IL-6 and IL-10 plasma levels, with higher increases in the caffeine-supplemented group. After 2-hr recovery, these levels returned to almost preexercise values. However, no effect of caffeine on BMNC cytokines was observed. IL-10, IL-6, and IL-12 p40 levels in BMNCs increased mainly at 2 hr postexercise. cAMP levels increased postexercise in plasma and after recovery in BMNCs, but no effects of caffeine were observed. In conclusion, caffeine did not modify cytokine levels in BMNCs in response to exercise. However, higher increases of IL-10 were observed in plasma after exercise in the supplemented participants, which could suppose an enhancement of the anti-inflammatory properties of exercise.

High production of pro-inflammatory cytokines by maternal blood mononuclear cells is associated with reduced maternal malaria but increased cord blood infection.

PubMed

Dobaño, Carlota; Berthoud, Tamara; Manaca, Maria Nelia; Nhabomba, Augusto; Guinovart, Caterina; Aguilar, Ruth; Barbosa, Arnoldo; Groves, Penny; Rodríguez, Mauricio H; Jimenez, Alfons; Quimice, Lazaro M; Aponte, John J; Ordi, Jaume; Doolan, Denise L; Mayor, Alfredo; Alonso, Pedro L

2018-05-10

Increased susceptibility to malaria during pregnancy is not completely understood. Cellular immune responses mediate both pathology and immunity but the effector responses involved in these processes have not been fully characterized. Maternal and fetal cytokine and chemokine responses to malaria at delivery, and their association with pregnancy and childhood outcomes, were investigated in 174 samples from a mother and child cohort from Mozambique. Peripheral and cord mononuclear cells were stimulated with Plasmodium falciparum lysate and secretion of IL-12p70, IFN-γ, IL-2, IL-10, IL-8, IL-6, IL-4, IL-5, IL-1β, TNF, TNF-β was quantified in culture supernatants by multiplex flow cytometry while cellular mRNA expression of IFN-γ, TNF, IL-2, IL-4, IL-6, IL-10 and IL-13 was measured by quantitative PCR. Higher concentrations of IL-6 and IL-1β were associated with a reduced risk of P. falciparum infection in pregnant women (p < 0.049). Pro-inflammatory cytokines IL-6, IL-1β and TNF strongly correlated among themselves (ρ > 0.5, p < 0.001). Higher production of IL-1β was significantly associated with congenital malaria (p < 0.046) and excessive TNF was associated with peripheral infection and placental lesions (p < 0.044). Complex network of immuno-pathological cytokine mechanisms in the placental and utero environments showed a potential trade-off between positive and negative effects on mother and newborn susceptibility to infection.

Role of Mononuclear Phagocytes in the Pathogenesis of Human Immunodeficiency Virus Infection

DTIC Science & Technology

1990-01-01

interpretive problems. First, patient selection has been limited to individuals with late-stage, symptomatic HIV infection. The average time from...months. Such monocytes infected with HIV showed a frequency of infected cells in culture of 5-20% by immunofluorescence with mono- clonal anti -pl7 capsid...cells equally; viruses isolated in PHA/IL-2-treated lymphoblasts replicate only in T cells. Target cell selection of diverse HIV variants may underlie

Establishment and characterization of Macaca fascicularis lymphoblastoid cell lines.

PubMed

Manning, C H; Heise, E R

1992-01-01

A panel of cynomolgus macaque lymphoblastoid cell lines (LCL) was established by transforming peripheral blood mononuclear cells (PBMC) with Herpesvirus papio (HVP), and selected lines were examined by flow cytometry. Results indicate that HVP-transformed macaque LCL are phenotypically heterogeneous and resemble human Epstein-Barr virus (EBV)-transformed LCL in the abundant expression of major histocompatibility complex (MHC) class I and class II molecules. At least some lines are of B cell origin.

Single-cell analysis of HIV-1 transcriptional activity reveals expression of proviruses in expanded clones during ART.

PubMed

Wiegand, Ann; Spindler, Jonathan; Hong, Feiyu F; Shao, Wei; Cyktor, Joshua C; Cillo, Anthony R; Halvas, Elias K; Coffin, John M; Mellors, John W; Kearney, Mary F

2017-05-02

Little is known about the fraction of human immunodeficiency virus type 1 (HIV-1) proviruses that express unspliced viral RNA in vivo or about the levels of HIV RNA expression within single infected cells. We developed a sensitive cell-associated HIV RNA and DNA single-genome sequencing (CARD-SGS) method to investigate fractional proviral expression of HIV RNA (1.3-kb fragment of p6, protease, and reverse transcriptase) and the levels of HIV RNA in single HIV-infected cells from blood samples obtained from individuals with viremia or individuals on long-term suppressive antiretroviral therapy (ART). Spiking experiments show that the CARD-SGS method can detect a single cell expressing HIV RNA. Applying CARD-SGS to blood mononuclear cells in six samples from four HIV-infected donors (one with viremia and not on ART and three with viremia suppressed on ART) revealed that an average of 7% of proviruses (range: 2-18%) expressed HIV RNA. Levels of expression varied from one to 62 HIV RNA molecules per cell (median of 1). CARD-SGS also revealed the frequent expression of identical HIV RNA sequences across multiple single cells and across multiple time points in donors on suppressive ART consistent with constitutive expression of HIV RNA in infected cell clones. Defective proviruses were found to express HIV RNA at levels similar to those proviruses that had no obvious defects. CARD-SGS is a useful tool to characterize fractional proviral expression in single infected cells that persist despite ART and to assess the impact of experimental interventions on proviral populations and their expression.

Age-Associated Decline in Dendritic Cell Function and the Impact of Mediterranean Diet Intervention in Elderly Subjects.

PubMed

Clements, Sarah J; Maijo, Monica; Ivory, Kamal; Nicoletti, Claudio; Carding, Simon R

2017-01-01

Aging is accompanied by increased susceptibility to infection and age-associated chronic diseases. It is also associated with reduced vaccine responses, which is often attributed to immunosenescence and the functional decline of the immune system. Immunosenescence is characterized by a chronic, low-grade, inflammatory state termed inflammaging. Habitants of Mediterranean (MED) regions maintain good health into old age; often attributed to MED diets. Adoption of a MED-diet by elderly subjects, in Norfolk (UK), may improve immune responses of these individuals and in particular, dendritic cell (DC) function. A total of 120 elderly subjects (65-79 years old) recruited onto the Nu-AGE study, a multicenter European dietary study specifically addressing the needs of the elderly, across five countries, and were randomized to the control or MED-diet groups, for one year. Blood samples were taken pre- and post-intervention for DC analysis and were compared with each other, and to samples obtained from 45 young (18-40 years old) subjects. MED-diet compliance was assessed using high performance liquid chromatography-with tandem mass spectrometry analysis of urine samples. Immune cell and DC subset numbers and concentrations of secreted proteins were determined by flow cytometric analysis. As expected, reduced myeloid DC numbers were observed in blood samples from elderly subjects compared with young. The elevated secretion of the adipokine, resistin, after ex vivo stimulation of peripheral blood mononuclear cells from elderly subjects, was significantly reduced after MED-diet intervention. This study provides further evidence of numerical and functional effects of aging on DCs. The MED-diet showed potential to impact on the aging immune cells investigated and could provide an economical approach to address problems associated with our aging population.

Is MMTV associated with human breast cancer? Maybe, but probably not.

PubMed

Perzova, Raisa; Abbott, Lynn; Benz, Patricia; Landas, Steve; Khan, Seema; Glaser, Jordan; Cunningham, Coleen K; Poiesz, Bernard

2017-10-13

Conflicting results regarding the association of MMTV with human breast cancer have been reported. Published sequence data have indicated unique MMTV strains in some human samples. However, concerns regarding contamination as a cause of false positive results have persisted. We performed PCR assays for MMTV on human breast cancer cell lines and fresh frozen and formalin fixed normal and malignant human breast epithelial samples. Assays were also performed on peripheral blood mononuclear cells from volunteer blood donors and subjects at risk for human retroviral infections. In addition, assays were performed on DNA samples from wild and laboratory mice. Sequencing of MMTV positive samples from both humans and mice were performed and phylogenetically compared. Using PCR under rigorous conditions to prevent and detect "carryover" contamination, we did detect MMTV DNA in human samples, including breast cancer. However, the results were not consistent and seemed to be an artifact. Further, experiments indicated that the probable source of false positives was murine DNA, containing endogenous MMTV, present in our building. However, comparison of published and, herein, newly described MMTV sequences with published data, indicates that there are some very unique human MMTV sequences in the literature. While we could not confirm the true presence of MMTV in our human breast cancer subjects, the data indicate that further, perhaps more traditional, retroviral studies are warranted to ascertain whether MMTV might rarely be the cause of human breast cancer.

Hemorrhagic cystitis in children undergoing bone marrow transplantation: a putative role for simian virus 40.

PubMed

Comar, Manola; D'Agaro, Pierlanfranco; Andolina, Marino; Maximova, Natasha; Martini, Fernanda; Tognon, Mauro; Campello, Cesare

2004-08-27

Late-onset hemorrhagic cystitis (HC) is a well-known severe complication of bone marrow transplantation (BMT), both in adults and in children. Protracted postengraftment HC is associated with graft-versus-host disease and viral infections, mainly caused by BK virus (BKV) or adenovirus (AV). This study investigated whether simian virus 40 (SV40) DNA sequences can be detected in specimens from pediatric patients affected by severe postengraftment HC. The clinical diagnosis of HC was made in 7 of 28 BMT children. DNA from peripheral blood mononuclear cells (PBMC) and urine sediment cells and supernatants was analyzed by polymerase chain reaction (PCR) for human cytomegalovirus (HCMV), AV, BKV, JC virus (JCV), and SV40. DNA filter hybridization and sequencing was carried out in SV40-positive samples. SV40 footprints were detected in two of seven cases of HC. Specific SV40 DNA sequences were detected by PCR and by filter hybridization both in urine and in PBMC samples at the HC onset and during the follow-up. The DNA sequencing proved that the amplicons belonged to the SV40 wild-type. Urine samples of the two HC cases tested negative by cell cultures, PCR, or both for HCMV, BKV, JCV, and AV. The detection of SV40 DNA sequences suggest that this simian polyomavirus could be involved, at least in some cases, in the HC occurring in children after BMT.

Peripheral blood mononuclear cells analysis in microfluidic flow by coherent imaging tools

NASA Astrophysics Data System (ADS)

Dannhauser, David; Rossi, Domenico; Memmolo, Pasquale; Causa, Filippo; Finizio, Andrea; Ferraro, Pietro; Netti, Paolo A.

2017-06-01

Cell of human blood stream are divided into two groups: Red Blood Cells (RBC) and White Blood Cells (WBC). RBC have a peculiar biconcave disk shape and they are responsible for the delivering of O2 and CO2 through the body. WBC are a more widespread class of cell ensuring immunity against pathogens. They can be divided in two main classes: granulocyte cells and A-granulocyte cells. Neutrophils, basophils and eosinophils belong to the granulocyte cell class, while lymphocytes and monocytes belong to A-granulocyte. Both in RBC and WBC, the intrinsic physical properties of a cell are indicators of cell condition and, furthermore, of the overall human body state. Thus, the accurate comprehension of the physiological structure of WBCs is fundamental to recognize diseases. Here we show the possibility to simple and straightforwardly characterize the physical properties of individual RBC and mononuclear WBC in a microfluidic context, using a wide angle light scattering apparatus and a corresponding theoretical simulation of Optical Signature (OS). A non-Newtonian polymer alignment solution for cell is used to ensure an individual cell alignment in the microfluidic flow, thus permitting a precise investigation. Additionally, Quantitative Phase Imaging (QPI) holographic measurements are performed to estimate cell morphometric features, such as their refractive index. We analyzed more than 200 WBCs and 100 RBCs of three different probands. Results showed distinct cell populations according to their measured dimensions and shape, which can be associated to the presence of RBC, lymphocytes and monocytes.

CELLULAR REACTIONS TO REINJECTION OF ANTIGEN

DOE Office of Scientific and Technical Information (OSTI.GOV)

Speirs, R.S.; Speirs, E.E.

1963-01-01

Studies in mice showed that an injection of tritiated antigen resulted in an incorporation of radioactivity in neutrophils, eosinophils, and macrophages at different times during the inflammatory cycle. Necrosis of labeled cells was frequently observed, and the incorporated radioactive material was passed from cell to cell by phagocytosis. As the inflammation subsided, there was a marked decrease in the number of labeled cells in the exudate, and a concomitant appearance and persistence of labeled cells in lymph nodes, spleen, and bone marrow. The fate of these labeled cells was followed after re-exposure to antigen, using autoradiographic procedures. An intraperitoneal injectionmore » of tetanus or diphtheria toxoid at 10, 30, or 60 days after sensitization produced an increase in the total number of mononuclear cells and an increase in the mononuclear cells containing radioactive material. The labeled cells were found in all animals autopsied within 70 days of sensitization and in several animals autopsied approximately 270 days after sensitization. The labeled cells were macrophages or large lymphoid cells. Approximately 12% of these cells were multinucleated, usually binucleated. An attempt was made to develop a concept of antibody formation on a molecular level. It is postulated that lymphocytes, macrophages, and plasma cells act as carriers for the necessary template RNA and associated microsomes, whereas the eosinophiles, and possibly the neutrophiles, supply a means of transporting antigen and specific enzymatic material to the reacting cells, thereby initiating changes leading to hypersensitivity and antibody formation. (C.H.)« less

Antibody-Mediated and Cellular Immune Responses Induced in Naive Volunteers by Vaccination with Long Synthetic Peptides Derived from the Plasmodium vivax Circumsporozoite Protein

PubMed Central

Arévalo-Herrera, Myriam; Soto, Liliana; Perlaza, Blanca Liliana; Céspedes, Nora; Vera, Omaira; Lenis, Ana Milena; Bonelo, Anilza; Corradin, Giampietro; Herrera, Sócrates

2011-01-01

Plasmodium vivax circumsporozoite (CS) protein is a leading malaria vaccine candidate. We describe the characterization of specific immune responses induced in 21 malaria-naive volunteers vaccinated with long synthetic peptides derived from the CS protein formulated in Montanide ISA 720. Both antibody- and cell-mediated immune responses were analyzed. Antibodies were predominantly of IgG1 and IgG3 isotypes, recognized parasite proteins on the immunofluorescent antibody test, and partially blocked sporozoite invasion of hepatoma cell lines in vitro. Peripheral blood mononuclear cells from most volunteers (94%) showed IFN-γ production in vitro upon stimulation with both long signal peptide and short peptides containing CD8+ T-cell epitopes. The relatively limited sample size did not allow conclusions about HLA associations with the immune responses observed. In summary, the inherent safety and tolerability together with strong antibody responses, invasion blocking activity, and the IFN-γ production induced by these vaccine candidates warrants further testing in a phase II clinical trial. PMID:21292876

AAV capsid CD8+ T-cell epitopes are highly conserved across AAV serotypes

PubMed Central

Hui, Daniel J; Edmonson, Shyrie C; Podsakoff, Gregory M; Pien, Gary C; Ivanciu, Lacramioara; Camire, Rodney M; Ertl, Hildegund; Mingozzi, Federico; High, Katherine A; Basner-Tschakarjan, Etiena

2015-01-01

Adeno-associated virus (AAV) has become one of the most promising vectors in gene transfer in the last 10 years with successful translation to clinical trials in humans and even market approval for a first gene therapy product in Europe. Administration to humans, however, revealed that adaptive immune responses against the vector capsid can present an obstacle to sustained transgene expression due to the activation and expansion of capsid-specific T cells. The limited number of peripheral blood mononuclear cells (PBMCs) obtained from samples within clinical trials allows for little more than monitoring of T-cell responses. We were able to identify immunodominant major histocompatibility complex (MHC) class I epitopes for common human leukocyte antigen (HLA) types by using spleens isolated from subjects undergoing splenectomy for non-malignant indications as a source of large numbers of lymphocytes and restimulating them with single AAV capsid peptides in vitro. Further experiments confirmed that these epitopes are naturally processed and functionally relevant. The design of more effective and less immunogenic AAV vectors, and precise immune monitoring of vector-infused subjects, are facilitated by these findings. PMID:26445723

AAV capsid CD8+ T-cell epitopes are highly conserved across AAV serotypes.

PubMed

Hui, Daniel J; Edmonson, Shyrie C; Podsakoff, Gregory M; Pien, Gary C; Ivanciu, Lacramioara; Camire, Rodney M; Ertl, Hildegund; Mingozzi, Federico; High, Katherine A; Basner-Tschakarjan, Etiena

2015-01-01

Adeno-associated virus (AAV) has become one of the most promising vectors in gene transfer in the last 10 years with successful translation to clinical trials in humans and even market approval for a first gene therapy product in Europe. Administration to humans, however, revealed that adaptive immune responses against the vector capsid can present an obstacle to sustained transgene expression due to the activation and expansion of capsid-specific T cells. The limited number of peripheral blood mononuclear cells (PBMCs) obtained from samples within clinical trials allows for little more than monitoring of T-cell responses. We were able to identify immunodominant major histocompatibility complex (MHC) class I epitopes for common human leukocyte antigen (HLA) types by using spleens isolated from subjects undergoing splenectomy for non-malignant indications as a source of large numbers of lymphocytes and restimulating them with single AAV capsid peptides in vitro. Further experiments confirmed that these epitopes are naturally processed and functionally relevant. The design of more effective and less immunogenic AAV vectors, and precise immune monitoring of vector-infused subjects, are facilitated by these findings.

Evaluation of erythroblast macrophage protein related to erythroblastic islands in patients with hematopoietic stem cell transplantation

PubMed Central

2013-01-01

Background Hematopoietic evaluation of the patients after Hematopoietic stem cell transplantation (HSCT) is very important. Erythroblast macrophage protein (Emp) is a key protein with function in normal differentiation of erythroid cells and macrophages. Emp expression correlates with erythroblastic island formation, a process widely believed to be associated with hematopoiesis in bone marrow. We aimed to investigate the hematopoietic function of bone marrow from 46 HSCT patients and 16 inpatients with severe anemia applied to the treatment of EPO by measuring Emp expression level. Methods Emp mRNA and protein expression levels in mononuclear cells of bone marrow and peripheral blood samples were detected by RT-PCR and Western blotting method respectively. Results While hematopoiesis occurs in bone marrow, Emp expression level was elevated and more erythroblastic islands were found , and Emp is upregulated in bone marrow in response to erythropoietin (EPO) treatment. Conclusions Emp expression correlates with erythroblastic island formation and has an important function for bone marrow hematopoiesis. Emp could be a potential biomarker for hematopoietic evaluation of HSCT patients. PMID:23566571

Comparative Phytochemical Analysis of Essential Oils from Different Biological Parts of Artemisia herba alba and Their Cytotoxic Effect on Cancer Cells.

PubMed

Tilaoui, Mounir; Ait Mouse, Hassan; Jaafari, Abdeslam; Zyad, Abdelmajid

2015-01-01

Carrying out the chemical composition and antiproliferative effects against cancer cells from different biological parts of Artemisia herba alba. Essential oils were studied by gas chromatography coupled to mass spectrometry (GC-MS) and their antitumoral activity was tested against P815 mastocytoma and BSR kidney carcinoma cell lines; also, in order to evaluate the effect on normal human cells, oils were tested against peripheral blood mononuclear cells PBMCs. Essential oils from leaves and aerial parts (mixture of capitulum and leaves) were mainly composed by oxygenated sesquiterpenes 39.89% and 46.15% respectively; capitulum oil contained essentially monoterpenes (22.86%) and monocyclic monoterpenes (21.48%); esters constituted the major fraction (62.8%) of stem oil. Essential oils of different biological parts studied demonstrated a differential antiproliferative activity against P815 and BSR cancer cells; P815 cells are the most sensitive to the cytotoxic effect. Leaves and capitulum essential oils are more active than aerial parts. Interestingly, no cytotoxic effect of these essential oils was observed on peripheral blood mononuclear cells. Our results showed that the chemical composition variability of essential oils depends on the nature of botanical parts of Artemisia herba alba. Furthermore, we have demonstrated that the differential cytotoxic effect depends not only on the essential oils concentration, but also on the target cells and the botanical parts of essential oils used.

Dynamic acquisition of HTLV-1 tax protein by mononuclear phagocytes: Role in neurologic disease.

PubMed

Matsuura, Eiji; Enose-Akahata, Yoshimi; Yao, Karen; Oh, Unsong; Tanaka, Yuetsu; Takashima, Hiroshi; Jacobson, Steven

2017-03-15

Pathology of HTLV-1 associated myelopathy/Tropical spastic paraparesis (HAM/TSP) is believed to be the result of "bystander damage" involving effector CD8 (+) T lymphocytes (CTLs) killing of virus infected cells. But the specific cellular events leading up to tissue injury are still unclear. Here, we developed the Microscopy Imaging of Cytotoxic T lymphocyte assay with Fluorescence emission (MI-CaFé), an optimized visualization analysis to explore the interactions between CTLs and virus infected or viral antigen presenting target cells. Various cell-to-cell formations can be observed and our results demonstrate elevated frequencies of CTL-target cell conjugates in HAM/TSP patient PBMCs compared to control PBMCs. Furthermore, HTLV-1 Tax protein expression can be localized at the cell-cell junctions and also tracked moving from an infected cell to a CD14 (+) mononuclear phagocyte (MP). Activation of CD14 (+) MPs in HAM/TSP patient PBMCs and antigenic presentation of HTLV-1 Tax by MPs can be inferred by their spontaneous cytotoxicity after 18h of in vitro culture. Given that CD4 (+) T lymphocytes are the primary reservoirs of HTLV-1 and MPs are scavenger cells responsible for pathogen clearance, spontaneous cytotoxicity against MPs in HAM/TSP PBMCs suggests a mechanism of chronic inflammation, secondary to low level of persistent virus infection within the central nervous system. Published by Elsevier B.V.

Immunological responsiveness of frozen-thawed human lymphocytes.

PubMed Central

Strong, D M; Woody, J N; Factor, M A; Ahmed, A; Sell, K W

1975-01-01

Mononuclear cells (10--20 X 10(6)) obtained from human peripheral blood by a standard Ficoll-Hypaque technique were suspended in RPMI 1640 media at 4 degrees C containing 10% foetal calf serum and 7-5% dimethyl sulphoxide (DMSO). Two-millilitre aliquots were cooled at -1 degree C/min in a Cryoson BV-4 programmed freezing system to -30 degrees C, then -5 degrees C/min to -80 degrees C and stored in liquid nitrogen vapor. On the day of testing, cell suspensions were thawed rapidly in a 37 degree C water bath. DMSO was diluted slowly out of the sample and cells resuspended in fresh RPMI 1640. It was found that frozen stored human lymphocytes (FSHL) demonstrated all the characteristics of fresh unfrozen cells. These included their ability to form spontaneous rosettes with sheep erythrocytes ('E' rosettes) and sheep erythrocyte--antibody--complement rosettes ('EAC' rosettes). The presence of surface immunoglobulins and Fc receptors were shown by membrane immunofluorescence to be comparable. In addition, the results show that FSHL respond to mitogens, specific antigens; act as both stimulators and responders in the mixed lymphocyte culture reaction; and exhibit cell-mediated lymphocytotoxicity following in vitro sensitization, or against antibody-coated target cells. PMID:128429

Bioactivity of xerogels as modulators of osteoclastogenesis mediated by connexin 43.

PubMed

Glenske, Kristina; Wagner, Alena-Svenja; Hanke, Thomas; Cavalcanti-Adam, Elisabetta A; Heinemann, Sascha; Heinemann, Christiane; Kruppke, Benjamin; Arnhold, Stefan; Moritz, Andreas; Schwab, Elisabeth H; Worch, Hartmut; Wenisch, Sabine

2014-02-01

In order to investigate the effects of different degrees of bioactivity of xerogels on connexin 43 (cx43) signaling of osteoclasts a cell culture approach was developed. Cells isolated from peripheral blood mononuclear cells were cultured in combination with the xerogels and were harvested for further investigations on day 1, day 5, and day 10. By means of quantitative PCR increased cx43 mRNA levels and coincident decreasing mRNA levels of the calcium sensing receptor, TRAP, and Cathepsin K were detected with increasing bioactivity of the xerogel samples. Additionally, osteoclasts cultured on tissue culture plates were used to perform principle investigations on cell differentiation by means of transmission electron microscopy, life cell imaging, and immunofluorescence, and the results demonstrated that cx43-signaling could be attributed to migration and fusion of osteoclast precursors. Therefore, the positive correlation of cx43 expression with high xerogel bioactivity was caused by proceeding differentiation of the osteoclasts. Finally, the presently observed pattern of cx43 signaling refers to strong effects regarding bioactivity on cx43-associated cell differentiation of osteoclasts influenced by extracellular calcium ions. Copyright © 2013 Elsevier Ltd. All rights reserved.

Inflammatory cytokine production in chronic active Epstein-Barr virus infection.

PubMed

Onozawa, Erika; Shibayama, Haruna; Imadome, Ken-Ichi; Tsuzura, Akiho; Koyama, Takatoshi; Miura, Osamu; Arai, Ayako

2017-01-01

In order to clarify the mechanisms underlying the development of inflammation in chronic active Epstein-Barr virus infection (CABEV), we examined cytokine production using patient samples. Eleven patients were analyzed. The serum concentrations of IFN-γ, TNF-α, and IL-6 were significantly higher in patients than in healthy donors. The mRNAs of these cytokines in peripheral blood mononuclear cells were elevated in patients as compared with healthy donors. The mRNA of IFN-γ was significantly higher in patients than in healthy donors. We examined which fraction produced the cytokines in the CD4-, CD8-, and CD56-positive fractions of PBMCs. The mRNAs of IFN-γ, TNF-α, and IL-6 were highly expressed in EBV-infected cells, whereas expression was also observed in non-infected cells. We performed in vitro infection of EBV on a T-cell line, MOLT4. EBV infection enhanced the mRNA expressions of IFN-γ and TNF-α. These results suggest that the inflammatory cytokines in CAEBV are produced not only by EBV-infected but also non-infected cells. EBV itself may have roles in the cytokine production observed in infected cells.

Biologic consequences of Stat1-independent IFN signaling

PubMed Central

Gil, M. Pilar; Bohn, Erwin; O'Guin, Andrew K.; Ramana, Chilakamarti V.; Levine, Beth; Stark, George R.; Virgin, Herbert W.; Schreiber, Robert D.

2001-01-01

Although Stat1 is required for many IFN-dependent responses, recent work has shown that IFNγ functions independently of Stat1 to affect the growth of tumor cells or immortalized fibroblasts. We now demonstrate that both IFNγ and IFNα/β regulate proliferative responses in cells of the mononuclear phagocyte lineage derived from Stat1-null mice. Using both representational difference analysis and gene arrays, we show that IFNγ exerts its Stat1-independent actions on mononuclear phagocytes by regulating the expression of many genes. This result was confirmed by monitoring changes in expression and function of the corresponding gene products. Regulation of the expression of these genes requires the IFNγ receptor and Jak1. The physiologic relevance of IFN-dependent, Stat1-independent signaling was demonstrated by monitoring antiviral responses in Stat1-null mice. Thus, the IFN receptors engage alternative Stat1-independent signaling pathways that have important physiological consequences. PMID:11390995

Failure to Detect Borna Disease Virus Antibody and RNA from Peripheral Blood Mononuclear Cells of Psychiatric Patients

PubMed Central

Na, Kyoung-Sae; Tae, Seong-Ho; Song, Jin-won

2009-01-01

Objective Borna disease virus (BDV) is a highly neurotropic agent causing various neuropsychiatric symptoms in animals. Over the past two decades, it has been suggested that BDV might be associated with human psychiatric diseases. We aimed to investigate whether BDV is associated with psychiatric patients in Korea. Methods We recruited 60 normal controls and 198 psychiatric patients (98 patients with depressive disorder, 60 with schizophrenia, and 40 with bipolar disorder). We used an indirect immunofluorescence antibody (IFA) test for the BDV antibody and a real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assay for p24 and p40 RNA from peripheral blood mononuclear cells (PBMCs). Results Neither the BDV antibody nor p24, p40 RNA was detected in controls and patients groups. Conclusion Our results suggest that BDV might not be associated with psychiatric patients in Korea. PMID:20140130

Stimulation of proinflammatory cytokines by volatile sulfur compounds in endodontically treated teeth.

PubMed

Lechner, Johann; von Baehr, Volker

2015-01-01

Persistent microorganisms in endodontically treated teeth produce volatile sulfur compounds (VSC) such as methyl mercaptan, hydrogen sulfide, and thioether. In this retrospective study, we evaluated the ex vivo immune response of peripheral blood mononuclear cells to sulfur compounds in 354 patients with systemic diseases. These systemic findings are correlated with semiquantitative values of a VSC indicator applied directly on endodontically treated teeth. Data elucidate the role of VSC in patients with immunologic diseases and the role of a semiquantitative chairside test, like the VSC indicator presented here, in correlation to IFNg and IL-10 sensitization in peripheral blood mononuclear cells. The association between ex vivo-stimulated cytokines and endodontically derived sulfur components is supported by the fact that the number of interferon gamma- and/or interleukin-10-positive sensitized patients declined significantly 3-8 months after extraction of the corresponding teeth.

Stimulation of proinflammatory cytokines by volatile sulfur compounds in endodontically treated teeth

PubMed Central

Lechner, Johann; von Baehr, Volker

2015-01-01

Persistent microorganisms in endodontically treated teeth produce volatile sulfur compounds (VSC) such as methyl mercaptan, hydrogen sulfide, and thioether. In this retrospective study, we evaluated the ex vivo immune response of peripheral blood mononuclear cells to sulfur compounds in 354 patients with systemic diseases. These systemic findings are correlated with semiquantitative values of a VSC indicator applied directly on endodontically treated teeth. Data elucidate the role of VSC in patients with immunologic diseases and the role of a semiquantitative chairside test, like the VSC indicator presented here, in correlation to IFNg and IL-10 sensitization in peripheral blood mononuclear cells. The association between ex vivo-stimulated cytokines and endodontically derived sulfur components is supported by the fact that the number of interferon gamma- and/or interleukin-10-positive sensitized patients declined significantly 3–8 months after extraction of the corresponding teeth. PMID:25792853

Differential immune response of mallard duck peripheral blood mononuclear cells to two highly pathogenic avian influenza H5N1 viruses with distinct pathogenicity in mallard ducks.

PubMed

Cui, Zhu; Hu, Jiao; He, Liang; Li, Qunhui; Gu, Min; Wang, Xiaoquan; Hu, Shunlin; Liu, Huimou; Liu, Wenbo; Liu, Xiaowen; Liu, Xiufan

2014-02-01

CK10 and GS10 are two H5N1 highly pathogenic influenza viruses of similar genetic background but differ in their pathogenicity in mallard ducks. CK10 is highly pathogenic whereas GS10 is low pathogenic. In this study, strong inflammatory response in terms of the expression level of several cytokines was observed in mallard duck peripheral blood mononuclear cells (PBMC) infected with CK10 while mild response was triggered in those by GS10 infection. Two remarkable and intense peaks of immune response were induced by CK10 infection within 24 hours (at 8 and 24 hours post infection, respectively) without reducing the virus replication. Our observations indicated that sustained and intense innate immune responses may be central to the high pathogenicity caused by CK10 in ducks.

Gamma delta T cells are early responders to Mycobacterium avium subsp. paratuberculosis in colostrum-replete Holstein calves

USDA-ARS?s Scientific Manuscript database

Peripheral blood mononuclear and mesenteric lymph node cells (PBMC and MNL, respectively) were obtained from 30 calves that were assigned randomly at birth to one of six treatment groups: 1) colostrum deprived (CD), no vitamins; 2) colostrum replacer (CR), no vitamins; 3) CR, vitamin A; 4) CR, vitam...

Curcumin and its synthetic analogue dimethoxycurcumin differentially modulates antioxidant status of normal human peripheral blood mononuclear cells.

PubMed

Simon, Emmanuel; Aswini, P; Sameer Kumar, V B; Mankadath, Gokuldas

2018-05-01

Curcumin is a polyphenol derived from the herb Curcuma longa, which has been extensively studied in terms of its antitumour, antioxidant, and chemopreventive activity as well as various other effects. In the present work we compared curcumin with its synthetic analogue dimethoxycurcumin (dimc) in terms of its antioxidant enzyme-modulating effects in human peripheral blood mononuclear cells (PBMC). We found that these compounds modulate antioxidant enzymes differentially. Both curcumin and dimethoxycurcumin effected a decrease in lipid peroxidation status in PBMC, however, curcumin had better activity in this regard. An increase in the activity of catalase was seen in the case of curcumin-treated PBMC, whereas dimc increased catalase activity significantly to almost twofold level. Real time-polymerase chain reaction (RT-PCR) analysis revealed significant up-regulation of catalase at mRNA level post treatment with curcumin as well as dimc, however, dimc had better activity in this regard. Glutathione reductase (GR) activity and reduced glutathione levels increased in the case of peripheral blood mononuclear cells (PBMC) treated with curcumin, however, the trend was reversed with dimethoxycurcumin where, both glutathione reductase activity and reduced glutathione levels were significantly reduced. RT-PCR analysis of glutathione reductase mRNA levels showed decrease in mRNA levels post treatment with dimethoxycurcumin (dimc) further corroborating GR enzyme assay results, however, we could not obtain significant result post curcumin treatment. NFkB reporter assay and western blot analysis of nuclear as well as cytosolic fractions of NFkB revealed that curcumin inhibits NFkB activation whereas inhibition was much less with dimc. It has been reported that curcumin and dimc exerts differential cytotoxicity in normal and tumour cells and the reason for this had been attributed to the differential uptake of these compounds by normal cells and tumour cells. Based on our results we propose that differential modulation of antioxidant enzymes via NFkB pathway could be the reason behind differential cytotoxicity of dimc as well as curcumin in normal cells and tumour cells in addition to differential uptake of these compounds as reported previously.

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

PubMed

Matsuda, Yoshiko; Imamura, Ryoichi; Takahara, Shiro

2017-01-01

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 × 10 5  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 × 10 5  cells/well), and the resulting in vitro analysis provided some information regarding the biological processes of IgG and IgM mBCs in peripheral blood. Taken together, our findings suggest that antigen-specific Ab subtype analyses of supernatants from cultured PBMCs might more effectively and accurately reflect a patient's Ab-associated pathological condition vs. than serum IgG and IgM levels.

Cryopreservation for delayed circulating tumor cell isolation is a valid strategy for prognostic association of circulating tumor cells in gastroesophageal cancer

PubMed Central

Brungs, Daniel; Lynch, David; Luk, Alison WS; Minaei, Elahe; Ranson, Marie; Aghmesheh, Morteza; Vine, Kara L; Carolan, Martin; Jaber, Mouhannad; de Souza, Paul; Becker, Therese M

2018-01-01

AIM To demonstrate the feasibility of cryopreservation of peripheral blood mononuclear cells (PBMCs) for prognostic circulating tumor cell (CTC) detection in gastroesophageal cancer. METHODS Using 7.5 mL blood samples collected in EDTA tubes from patients with gastroesopheagal adenocarcinoma, CTCs were isolated by epithelial cell adhesion molecule based immunomagnetic capture using the IsoFlux platform. Paired specimens taken during the same blood draw (n = 15) were used to compare number of CTCs isolated from fresh and cryopreserved PBMCs. Blood samples were processed within 24 h to recover the PBMC fraction, with PBMCs used for fresh analysis immediately processed for CTC isolation. Cryopreservation of PBMCs lasted from 2 wk to 25.2 mo (median 14.6 mo). CTCs isolated from pre-treatment cryopreserved PBMCs (n = 43) were examined for associations with clinicopathological variables and survival outcomes. RESULTS While there was a significant trend to a decrease in CTC numbers associated with cryopreserved specimens (mean number of CTCs 34.4 vs 51.5, P = 0.04), this was predominately in samples with a total CTC count of > 50, with low CTC count samples less affected (P = 0.06). There was no significant association between the duration of cryopreservation and number of CTCs. In cryopreserved PBMCs from patient samples prior to treatment, a high CTC count (> 17) was associated with poorer overall survival (OS) (n = 43, HR = 4.4, 95%CI: 1.7-11.7, P = 0.0013). In multivariate analysis, after controlling for sex, age, stage, ECOG performance status, and primary tumor location, a high CTC count remained significantly associated with a poorer OS (HR = 3.7, 95%CI: 1.2-12.4, P = 0.03). CONCLUSION PBMC cryopreservation for delayed CTC isolation is a valid strategy to assist with sample collection, transporting and processing. PMID:29467551

Cacao extracts suppress tryptophan degradation of mitogen-stimulated peripheral blood mononuclear cells.

PubMed

Jenny, M; Santer, E; Klein, A; Ledochowski, M; Schennach, H; Ueberall, F; Fuchs, D

2009-03-18

The fruits of Theobroma cacao L. (Sterculiaceae) have been used as food and a remedy for more than 4000 years. Today, about 100 therapeutic applications of cacao are described involving the gastrointestinal, nervous, cardiovascular and immune systems. Pro-inflammatory cytokine interferon-gamma and related biochemical pathways like tryptophan degradation by indoleamine 2,3-dioxygenase and neopterin formation are closely associated with the pathogenesis of such disorders. To determine the anti-inflammatory effect of cacao extracts on interferon-gamma and biochemical consequences in immunocompetent cells. Effects of aqueous or ethanolic extracts of cacao were examined on mitogen-induced human peripheral blood mononuclear cells (PBMC) of healthy donors and on lipopolysaccharide-stimulated myelomonocytic THP-1 cells. Antioxidant activity of extracts was determined by oxygen radical absorption capacity (ORAC) assay. In mitogen-stimulated PBMC, enhanced degradation of tryptophan, formation of neopterin and interferon-gamma were almost completely suppressed by the cacao extracts at doses of > or = 5 microg/mL. Cacao extracts had no effect on tryptophan degradation in lipopolysaccharide-stimulated THP-1 cells. There is a significant suppressive effect of cacao extracts on pro-inflammatory pathways in activated T-cells. Particularly the influence on indoleamine 2,3-dioxygenase could relate to some of the beneficial health effects ascribed to cacao.

Differential regulation by Seogak Jihwang-Tang on cytokines production in peripheral blood mononuclear cells from the cerebral infarction patients presenting with altered consciousness.

PubMed

Jeong, Hyun-Ja; Chung, Hwan-Suck; Kim, Yo-Han; Moon, Byung-Soon; Sung, Kang-Keyng; Bai, Sun-Joon; Cho, Kwang-Ho; Kim, Yun-Kyung; Hong, Seung-Heon; Shin, Taekyun; Kim, Hyung-Min

2004-10-01

Seogak Jihwang-Tang (SJT) has been widely used to treat patients suffering from cerebral infarction. However, very little scientific investigation has been carried out. We investigated the effect of SJT on the production of various cytokines using peripheral blood mononuclear cells from the cerebral infarction patients presenting with altered consciousness. The cytokines production was determined by enzyme-linked immunosorbent assay. The amount of IL-4, IL-10 and TGF-beta1 in culture supernatant significantly increased in the SJT, lipopolysaccharide (LPS) or PHA-treated cells compared to unstimulated cells (P < 0.05). We also showed that increased IL-4 and IL-10 levels by LPS or phytohaemagglutinin (PHA) were significantly inhibited by SJT in a dose-dependent manner. Maximal inhibition rate of IL-4 and IL-10 production by SJT was 45.6 +/- 3.3% and 61 +/- 4.7% for LPS-stimulated cells and 27.3 +/- 1.2% and 83.6 +/- 2% for PHA-stimulated cells, respectively (P < 0.05). On the other hand, SJT significantly increased the LPS or PHA-induced TGF-beta1 production (P < 0.05). These data suggest that SJT has a regulatory effect on the cytokines production, which might explain its beneficial effect in the treatment of cerebral infarction.

Controlled-rate freezer cryopreservation of highly concentrated peripheral blood mononuclear cells results in higher cell yields and superior autologous T-cell stimulation for dendritic cell-based immunotherapy.

PubMed

Buhl, Timo; Legler, Tobias J; Rosenberger, Albert; Schardt, Anke; Schön, Michael P; Haenssle, Holger A

2012-11-01

Availability of large quantities of functionally effective dendritic cells (DC) represents one of the major challenges for immunotherapeutic trials against infectious or malignant diseases. Low numbers or insufficient T-cell activation of DC may result in premature termination of treatment and unsatisfying immune responses in clinical trials. Based on the notion that cryopreservation of monocytes is superior to cryopreservation of immature or mature DC in terms of resulting DC quantity and immuno-stimulatory capacity, we aimed to establish an optimized protocol for the cryopreservation of highly concentrated peripheral blood mononuclear cells (PBMC) for DC-based immunotherapy. Cryopreserved cell preparations were analyzed regarding quantitative recovery, viability, phenotype, and functional properties. In contrast to standard isopropyl alcohol (IPA) freezing, PBMC cryopreservation in an automated controlled-rate freezer (CRF) with subsequent thawing and differentiation resulted in significantly higher cell yields of immature and mature DC. Immature DC yields and total protein content after using CRF were comparable with results obtained with freshly prepared PBMC and exceeded results of standard IPA freezing by approximately 50 %. While differentiation markers, allogeneic T-cell stimulation, viability, and cytokine profiles were similar to DC from standard freezing procedures, DC generated from CRF-cryopreserved PBMC induced a significantly higher antigen-specific IFN-γ release from autologous effector T cells. In summary, automated controlled-rate freezing of highly concentrated PBMC represents an improved method for increasing DC yields and autologous T-cell stimulation.

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

PubMed

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.

Does cellular aging relate to patterns of allostasis? An examination of basal and stress reactive HPA axis activity and telomere length

PubMed Central

Tomiyama, A. Janet; O’Donovan, Aoife; Lin, Jue; Puterman, Eli; Lazaro, Alanie; Chan, Jessica; Dhabhar, Firdaus S.; Wolkowitz, Owen; Kirschbaum, Clemens; Blackburn, Elizabeth; Epel, Elissa

2012-01-01

Long-term exposure to stress and its physiological mediators, in particular cortisol, may lead to impaired telomere maintenance. In this study, we examine if greater cortisol responses to an acute stressor and/or dysregulated patterns of daily cortisol secretion are associated with shorter telomere length. Twenty-three post-menopausal women comprising caregivers for dementia partners (n=14) and age- and BMI-matched non-caregivers provided home sampling of cortisol–saliva samples at waking, 30 min after waking, and bedtime, and a 12-hour overnight urine collection. They were also exposed to an acute laboratory stressor throughout which they provided saliva samples. Peripheral blood mononuclear cells were isolated from a fasting blood sample and assayed for telomere length. As hypothesized, greater cortisol responses to the acute stressor were associated with shorter telomeres, as were higher overnight urinary free cortisol levels and flatter daytime cortisol slopes. While robust physiological responses to acute stress serve important functions, the long-term consequences of frequent high stress reactivity may include accelerated telomere shortening. PMID:22138440

Immunocyte Response to Experimental Mumps Virus Infection in Rhesus Monkeys

PubMed Central

Genco, R. J.; Flanagan, T. D.; Emmings, F. G.

1973-01-01

Nineteen rhesus monkeys were inoculated with mumps virus by retrograde ductal instillation into the parotid gland. Evidence of infection was obtained in all instances. Virus was isolated from buccal swabbings and parotid biopsies for 1 week after inoculation. A vigorous serum-neutralizing antibody response occurred within 3 weeks, and there was marked monocytic infiltration of the parotid stroma. The monocytic infiltrate comprised as much as 60% of the total gland volume 1 week after infection. The predominant inflammatory cells were non-immunoglobulin-containing mononuclear cells resembling lymphocytes. Plasma cells containing immunoglobulin G (IgG), IgA, IgM, and IgE increased in numbers in the gland after infection, the greatest increase occurring in IgG-containing cells. Neutralizing antibodies and interferon were found in extracts prepared from the infected glands. Neutralizing activity was highest in samples taken 3 weeks after infection but was detectable in samples taken as soon as 36 to 48 h after infection. Interferon activity was detected in significant amounts 36 to 48 h after infection. Challenge of previously infected animals resulted in an increase in the monocytic infiltrate as well as an increase in numbers of immunoglobulin-containing plasma cells. However, reinfection did not occur as evidenced by the inability to culture shed virus after challenge. This model should be useful for in vivo study of biochemical mediators which evoke inflammatory cell infiltration and which may be significant both in protection and in tissue damage. PMID:4202659

The effects of cryopreservation on the expression of canine regulatory T-cell markers.

PubMed

Tarpataki, Noemi; Wawrzyniak, Marcin; Akdis, Cezmi A; Rückert, Beate; Meli, Marina L; Fischer, Nina M; Favrot, Claude; Rostaher, Ana

2017-08-01

Regulatory T (Treg) cells have been described as key regulators in various immunological processes and are of growing interest in veterinary allergy. Cryopreservation of immune cells is performed routinely in human basic science research and in clinical studies. As such, it allows batch testing of collected samples at a single time point, resulting in a significant reduction in sample variability. Data which describe the effects of cryopreservation on Treg cell frequency and functionality in the canine species are important to inform future research. The purpose of this study was to establish a robust freeze/thaw procedure and flow cytometric staining protocol for canine Treg cells, and to compare the frequencies of different canine Treg cell phenotypes before and after cryopreservation. Nine privately owned dogs. Peripheral blood mononuclear cells were isolated and Treg cells stained and analysed by flow cytometry, before and after three months of cryopreservation. The recovery percentages and the corresponding correlations (fresh versus cryopreserved) for CD4 + CD25 + , CD4 + FOXP3 + and CD4 + CD25 + FOXP3 + cell populations were calculated. A high recovery rate of 97.2 (r = 0.94, P < 0.0001), 93.9 (r = 0.77, P < 0.01) and 101.7% (r = 0.99, P < 0.0001) for CD4 + CD25 + , CD4 + FOXP3 + and CD4 + CD25 + FOXP3 + cell populations, respectively, was observed. This study demonstrates an optimized protocol for freezing, thawing and quantifying canine Treg cells. These results indicate that cryopreservation does not substantially affect the expression of surface and intracellular markers of canine Treg cells; however, additional studies will be necessary to assess whether functionality of the cells is also maintained. © 2017 ESVD and ACVD.

Evaluation of gastric histology in children and adolescents with Helicobacter pylori gastritis using the Update Sydney System.

PubMed

Langner, Marini; Machado, Rodrigo Strehl; Patrício, Francy R S; Kawakami, Elisabete

2009-01-01

Although Helicobacter pylori infection is prevalent in our country, there are few studies evaluating the associated histological abnormalities in children. To evaluate the histological features of the gastric mucosa in children and adolescents with Helicobacter pylori gastritis. One hundred and thirty two gastric biopsies from 22 symptomatic patients infected with H. pylori (14F/8M, median age 10 y 5 mo, age range 2 y 11 mo to 16 y 9 mo) were evaluated. Evaluated gastric regions included: antrum (lesser and greater curvature), corpus (lesser and greater curvature), incisura angularis and fundus. Histological examination was performed according to the Updated Sydney System, and regional scores for polymorphonuclear and mononuclear cell infiltrate as well as bacterial density were generated. Fifteen (68.2%) patients presented H. pylori-chronic active gastritis, six (27.3%) presented antrum-predominant H. pylori-chronic active gastritis, and one (4.5%) presented corpus-predominant H. pylori-chronic active gastritis. Polymorphonuclear cell infiltrate and mononuclear cell infiltrate were observed in 93.9% and 98.5% of the biopsy specimens, respectively. Higher histological scores for polymorphonuclear infiltrate, mononuclear infiltrate, and bacterial density were observed in the gastric antrum. Intestinal metaplasia and gastric atrophy were not identified in any patient. Lymphoid aggregates and lymphoid follicles were observed in the gastric antrum of three (13.6%) and seven (31.8%) patients, respectively, but they were not related to antral nodularity. Chronic active gastritis was observed in all patients with H. pylori infection. However, antral or corporeal predominance was not observed in most patients.

Alterations in the function of circulating mononuclear cells derived from patients with Crohn’s disease treated with mastic

PubMed Central

Kaliora, Andriana C; Stathopoulou, Maria G; Triantafillidis, John K; Dedoussis, George VZ; Andrikopoulos, Nikolaos K

2007-01-01

AIM: To assess the effects of mastic administration on cytokine production of circulating mononuclear cells of patients with active Crohn's disease (CD). METHODS: The study was conducted in patients with established mildly to moderately active CD, attending the outpatient clinics of the hospital, and in healthy controls. Recruited to a 4 wk treatment with mastic caps (6 caps/d,0.37 g/cap) were 10 patients and 8 controls, all of who successfully completed the protocol. Interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1), macrophage migration inhibitory factor (MIF) and intracellular antioxidant glutathione (GSH) were evaluated in peripheral blood mononuclear cells (PBMC) before and after treatment. RESULTS: Treating CD patients with mastic resulted in the reduction of TNF-α secretion (2.1 ± 0.9 ng/mL vs 0.5 ± 0.4 ng/mL, P = 0.028). MIF release was significantly increased (1.2 ± 0.4 ng/mL vs 2.5 ± 0.7 ng/mL, P = 0.026) meaning that random migration and chemotaxis of monocytes/macrophages was inhibited. No significant changes were observed in IL-6, MCP-1 and GSH concentrations. CONCLUSION: This study shows that mastic acts as an immunomodulator on PBMC, acting as a TNF-α inhibitor and a MIF stimulator. Although further double-blind, placebo-controlled studies in a large number of patients is required to clarify the role of this natural product, this finding provides strong evidence that mastic might be an important regulator of immunity in CD. PMID:18023095

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

PubMed

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)

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

PubMed Central

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

Interdisciplinary Studies on the Combat Readiness and Health Issues Faced by Military Personnel

DTIC Science & Technology

2008-09-01

University of Texas T and operational at the University of Texas at Dallas Center for BrainHealth located at 2200 W. Mockingbird Lane, Dallas, Texas...cells), and the targeted cells have been efficiently killed with NIR. This work is now published (Chakravarty et al., 2008) (Appendix B...mononuclear cells bound only to the CNTs coupled to the anti-CD25 mAb. Most importantly, only the specifically targeted cells were killed after exposure to

The Activity of Immunoglobulin Y Anti-Mycobacterium tuberculosis on Proliferation and Cytokine Expression of Rat Peripheral Blood Mononuclear Cells

PubMed Central

Sudjarwo, Sri Agus; Eraiko, Koerniasari; Sudjarwo, Giftania Wardani; Koerniasari

2017-01-01

Objective: It has long been known that chickens, like mammals, are capable of producing antigen-specific immunoglobulin Y (IgY), which functions similar to IgG. The present study was performed to investigate the activity of IgY anti-Mycobacterium tuberculosis on proliferation, interleukin (IL)-2, and interferon (IFN)-γ expression of rat peripheral blood mononuclear cells (PBMCs). Materials and Methods: The activity of IgY anti-M. tuberculosis in different doses (25, 50, and 100 μg/ml) on rat PBMCs proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. The production of IL-2 and IFN-γ in the PBMC supernatant was determined using enzyme-linked immunosorbent assay. Investigation was performed on mRNA expression of IL-2 and IFN-γ by reverse transcription-polymerase chain reaction (RT-PCR). Results: IgY anti-M. tuberculosis significantly increased the proliferation of rat PBMC. Furthermore, IgY anti-M. tuberculosis dose dependently increased IL-2 and IFN-γ production in PBMC, suggesting that pharmacological activities of IgY anti-M. tuberculosis in PBMC may be mediated by regulating the production of cytokines. In the RT-PCR, expression of cytokines such as IL-2 and IFN-γ in PBMC cultures was increased by IgY anti-M. tuberculosis. Conclusions: We concluded that increasing IL-2 and IFN-γ productions in PBMC was related to IgY anti-M. tuberculosis, stimulating the mRNA transcription (gene expression) of these cytokines which can induce proliferation of PBMC. SUMMARY Lohman laying hens immunized intramuscularly with antigens of M. tuberculosis can produce specific IgY anti-Mycobacterium tuberculosis complexIgY anti-M. tuberculosis significantly increased the proliferation of rat peripheral blood mononuclear cell (PBMC)IgY anti-M. tuberculosis dose dependently increased interleukin 2 (IL-2) and interferon (IFN)-γ production in PBMCIn the reverse transcription-polymerase chain reaction, expression of cytokines such as IL-2 and IFN-γ in PBMC cultures was increased by IgY anti-M. tuberculosisThe increasing IL-2 and IFN-γ productions in PBMC were related to stimulation on mRNA transcription which can induce proliferation of PBMC. Abbreviations Used: IgY anti-M. tuberculosis: Immunoglobulin Y anti-Mycobacterium tuberculosis; IL-2: Interleukin-2; IFN-γ: Interferon-γ; PBMCs: Peripheral blood mononuclear cells. PMID:29333035

The Activity of Immunoglobulin Y Anti-Mycobacterium tuberculosis on Proliferation and Cytokine Expression of Rat Peripheral Blood Mononuclear Cells.

PubMed

Sudjarwo, Sri Agus; Eraiko, Koerniasari; Sudjarwo, Giftania Wardani; Koerniasari

2017-12-01

It has long been known that chickens, like mammals, are capable of producing antigen-specific immunoglobulin Y (IgY), which functions similar to IgG. The present study was performed to investigate the activity of IgY anti- Mycobacterium tuberculosis on proliferation, interleukin (IL)-2, and interferon (IFN)-γ expression of rat peripheral blood mononuclear cells (PBMCs). The activity of IgY anti- M. tuberculosis in different doses (25, 50, and 100 μg/ml) on rat PBMCs proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. The production of IL-2 and IFN-γ in the PBMC supernatant was determined using enzyme-linked immunosorbent assay. Investigation was performed on mRNA expression of IL-2 and IFN-γ by reverse transcription-polymerase chain reaction (RT-PCR). IgY anti- M. tuberculosis significantly increased the proliferation of rat PBMC. Furthermore, IgY anti-M. tuberculosis dose dependently increased IL-2 and IFN-γ production in PBMC, suggesting that pharmacological activities of IgY anti- M. tuberculosis in PBMC may be mediated by regulating the production of cytokines. In the RT-PCR, expression of cytokines such as IL-2 and IFN-γ in PBMC cultures was increased by IgY anti- M. tuberculosis . We concluded that increasing IL-2 and IFN-γ productions in PBMC was related to IgY anti- M. tuberculosis , stimulating the mRNA transcription (gene expression) of these cytokines which can induce proliferation of PBMC. Lohman laying hens immunized intramuscularly with antigens of M. tuberculosis can produce specific IgY anti- Mycobacterium tuberculosis complexIgY anti- M. tuberculosis significantly increased the proliferation of rat peripheral blood mononuclear cell (PBMC)IgY anti- M. tuberculosis dose dependently increased interleukin 2 (IL-2) and interferon (IFN)-γ production in PBMCIn the reverse transcription-polymerase chain reaction, expression of cytokines such as IL-2 and IFN-γ in PBMC cultures was increased by IgY anti-M. tuberculosis The increasing IL-2 and IFN-γ productions in PBMC were related to stimulation on mRNA transcription which can induce proliferation of PBMC. Abbreviations Used: IgY anti- M . tuberculosis: Immunoglobulin Y anti- Mycobacterium tuberculosis ; IL-2: Interleukin-2; IFN-γ: Interferon-γ; PBMCs: Peripheral blood mononuclear cells.

The effects of acute oral glutamine supplementation on exercise-induced gastrointestinal permeability and heat shock protein expression in peripheral blood mononuclear cells.

PubMed

Zuhl, Micah; Dokladny, Karol; Mermier, Christine; Schneider, Suzanne; Salgado, Roy; Moseley, Pope

2015-01-01

Chronic glutamine supplementation reduces exercise-induced intestinal permeability and inhibits the NF-κB pro-inflammatory pathway in human peripheral blood mononuclear cells. These effects were correlated with activation of HSP70. The purpose of this paper is to test if an acute dose of oral glutamine prior to exercise reduces intestinal permeability along with activation of the heat shock response leading to inhibition of pro-inflammatory markers. Physically active subjects (N = 7) completed baseline and exercise intestinal permeability tests, determined by the percent ratio of urinary lactulose (5 g) to rhamnose (2 g). Exercise included two 60-min treadmill runs at 70 % of VO2max at 30 °C after ingestion of glutamine (Gln) or placebo (Pla). Plasma levels of endotoxin and TNF-α, along with peripheral blood mononuclear cell (PBMC) protein expression of HSP70 and IκBα, were measured pre- and post-exercise and 2 and 4 h post-exercise. Permeability increased in the Pla trial compared to that at rest (0.06 ± 0.01 vs. 0.02 ± 0.018) and did not increase in the Gln trial. Plasma endotoxin was lower at the 4-h time point in the Gln vs. 4 h in the Pla (6.715 ± 0.046 pg/ml vs. 7.952 ± 1.11 pg/ml). TNF-α was lower 4 h post-exercise in the Gln vs. Pla (1.64 ± 0.09 pg/ml vs. 1.87 ± 0.12 pg/ml). PBMC expression of IkBα was higher 4 h post-exercise in the Gln vs. 4 h in the Pla (1.29 ± 0.43 vs. 0.8892 ± 0.040). HSP70 was higher pre-exercise and 2 h post-exercise in the Gln vs. Pla (1.35 ± 0.21 vs. 1.000 ± 0.000 and 1.65 ± 0.21 vs. 1.27 ± 0.40). Acute oral glutamine supplementation prevents an exercise-induced rise in intestinal permeability and suppresses NF-κB activation in peripheral blood mononuclear cells.

19F-perfluorocarbon-labeled human peripheral blood mononuclear cells can be detected in vivo using clinical MRI parameters in a therapeutic cell setting.

PubMed

Fink, Corby; Gaudet, Jeffrey M; Fox, Matthew S; Bhatt, Shashank; Viswanathan, Sowmya; Smith, Michael; Chin, Joseph; Foster, Paula J; Dekaban, Gregory A

2018-01-12

A 19 Fluorine ( 19 F) perfluorocarbon cell labeling agent, when employed with an appropriate cellular MRI protocol, allows for in vivo cell tracking. 19 F cellular MRI can be used to non-invasively assess the location and persistence of cell-based cancer vaccines and other cell-based therapies. This study was designed to determine the feasibility of labeling and tracking peripheral blood mononuclear cells (PBMC), a heterogeneous cell population. Under GMP-compliant conditions human PBMC were labeled with a 19 F-based MRI cell-labeling agent in a manner safe for autologous re-injection. Greater than 99% of PBMC labeled with the 19 F cell-labeling agent without affecting functionality or affecting viability. The 19 F-labeled PBMC were detected in vivo in a mouse model at the injection site and in a draining lymph node. A clinical cellular MR protocol was optimized for the detection of PBMC injected both at the surface of a porcine shank and at a depth of 1.2 cm, equivalent to depth of a human lymph node, using a dual 1 H/ 19 F dual switchable surface radio frequency coil. This study demonstrates it is feasible to label and track 19 F-labeled PBMC using clinical MRI protocols. Thus, 19 F cellular MRI represents a non-invasive imaging technique suitable to assess the effectiveness of cell-based cancer vaccines.

Mechanisms of stimulation of interleukin-1 beta and tumor necrosis factor-alpha by Mycobacterium tuberculosis components.

PubMed Central

Zhang, Y; Doerfler, M; Lee, T C; Guillemin, B; Rom, W N

1993-01-01

The granulomatous immune response in tuberculosis is characterized by delayed hypersensitivity and is mediated by various cytokines released by the stimulated mononuclear phagocytes, including tumor necrosis factor-alpha (TNF alpha) and IL-1 beta. We have demonstrated that Mycobacterium tuberculosis cell wall component lipoarabinomannan (LAM), mycobacterial heat shock protein-65 kD, and M. tuberculosis culture filtrate, devoid of LPS as assessed by the Amebocyte Lysate assay, stimulate the production of TNF alpha and IL-1 beta proteins and mRNA from mononuclear phagocytes (THP-1 cells). The effect of LAM on the release of these cytokines was specific, as only LAM stimulation was inhibited by anti-LAM monoclonal antibody. Interestingly, we found that LAM and Gram-negative bacterial cell wall-associated endotoxin LPS may share a similar mechanism in their stimulatory action as demonstrated by inhibition of TNF alpha and IL-1 beta release by monoclonal antibodies to CD14. Anti-CD14 monoclonal antibody MY4 inhibited both TNF alpha and IL-1 beta release with LAM and LPS but no effect was observed with other mycobacterial proteins. An isotype antibody control did not inhibit release of cytokines under the same experimental conditions. M. tuberculosis and its components upregulated IL-1 beta and TNF alpha mRNAs in THP-1 cells. Nuclear run-on assay for IL-1 beta demonstrated that LAM increased the transcription rate. The induction of IL-1 beta was regulated at the transcriptional level, in which these stimuli acted through cis-acting element(s) on the 5' flanking region of the IL-1 beta genomic DNA. M. tuberculosis cell wall component LAM acts similarly to LPS in activating mononuclear phagocyte cytokine TNF alpha and IL-1 beta release through CD14 and synthesis at the transcriptional level; both cytokines are key participants in the host immune response to tuberculosis. Images PMID:7683696

Evaluation of the COBAS AMPLICOR CMV MONITOR test for detection of viral DNA in specimens taken from patients after liver transplantation.

PubMed

Sia, I G; Wilson, J A; Espy, M J; Paya, C V; Smith, T F

2000-02-01

Detection of cytomegalovirus (CMV) DNA in blood by PCR is a sensitive method for the detection of infection in patients posttransplantation. The test, however, has low specificity for the identification of overt CMV disease. Quantitative CMV PCR has been shown to overcome this shortcoming. The COBAS AMPLICOR CMV MONITOR test was evaluated by using consecutive serum and peripheral blood mononuclear cell (PBMN) samples from liver transplant patients. Twenty-five patients had CMV viremia (by shell vial cell culture assay) and/or tissue-invasive disease (by biopsy); 20 had no active infection. A total of 262 serum and 62 PBMN specimens were tested. Of 159 serum specimens from patients with overt CMV infection, the COBAS assay detected CMV DNA in 21 patients (sensitivity, 84%). Only 1 of 103 samples from patients with no evidence of active infection had detectable CMV DNA (341 copies/ml). By comparison of 62 matching serum and PBMN samples by the same assay, 12 PBMN samples were exclusively positive, whereas only 2 serum samples were exclusively positive (P < 0.05). At the time of clinical CMV infection, viral copy numbers were higher in PBMNs than serum from four of five patients. The COBAS AMPLICOR CMV MONITOR test is a sensitive and specific test for the quantitative detection of CMV DNA in blood. Clinical applications of the assay will require further validation with samples from a larger population of transplant patients.

Evaluation of the COBAS AMPLICOR CMV MONITOR Test for Detection of Viral DNA in Specimens Taken from Patients after Liver Transplantation

PubMed Central

Sia, Irene G.; Wilson, Jennie A.; Espy, Mark J.; Paya, Carlos V.; Smith, Thomas F.

2000-01-01

Detection of cytomegalovirus (CMV) DNA in blood by PCR is a sensitive method for the detection of infection in patients posttransplantation. The test, however, has low specificity for the identification of overt CMV disease. Quantitative CMV PCR has been shown to overcome this shortcoming. The COBAS AMPLICOR CMV MONITOR test was evaluated by using consecutive serum and peripheral blood mononuclear cell (PBMN) samples from liver transplant patients. Twenty-five patients had CMV viremia (by shell vial cell culture assay) and/or tissue-invasive disease (by biopsy); 20 had no active infection. A total of 262 serum and 62 PBMN specimens were tested. Of 159 serum specimens from patients with overt CMV infection, the COBAS assay detected CMV DNA in 21 patients (sensitivity, 84%). Only 1 of 103 samples from patients with no evidence of active infection had detectable CMV DNA (341 copies/ml). By comparison of 62 matching serum and PBMN samples by the same assay, 12 PBMN samples were exclusively positive, whereas only 2 serum samples were exclusively positive (P < 0.05). At the time of clinical CMV infection, viral copy numbers were higher in PBMNs than serum from four of five patients. The COBAS AMPLICOR CMV MONITOR test is a sensitive and specific test for the quantitative detection of CMV DNA in blood. Clinical applications of the assay will require further validation with samples from a larger population of transplant patients. PMID:10655353

Measurement of Intracellular Ribavirin Mono-, Di- and Triphosphate Using Solid Phase Extraction and LC-MS/MS Quantification

PubMed Central

Jimmerson, Leah C.; Ray, Michelle L.; Bushman, Lane R.; Anderson, Peter L.; Klein, Brandon; Rower, Joseph E.; Zheng, Jia-Hua; Kiser, Jennifer J.

2014-01-01

Ribavirin (RBV) is a nucleoside analog used to treat a variety of DNA and RNA viruses. RBV undergoes intracellular phosphorylation to a mono- (MP), di- (DP), and triphosphate (TP). The phosphorylated forms have been associated with the mechanisms of antiviral effect observed in vitro, but the intracellular pharmacology of the drug has not been well characterized in vivo. A highly sensitive LC-MS/MS method was developed and validated for the determination of intracellular RBV MP, DP, and TP in multiple cell matrix types. For this method, the individual MP, DP, and TP fractions were isolated from lysed intracellular matrix using strong anion exchange solid phase extraction, dephosphorylated to parent RBV, desalted and concentrated and quantified using LC-MS/MS. The method utilized a stable labeled internal standard (RBV-13C5) which facilitated accuracy (% deviation within ±15%) and precision (coefficient of variation of ≤15%). The quantifiable linear range for the assay was 0.50 to 200 pmol/sample. The method was applied to the measurement of RBV MP, DP, and TP in human peripheral blood mononuclear cells (PBMC), red blood cells (RBC), and dried blood spot (DBS) samples obtained from patients taking RBV for the treatment of chronic Hepatitis C virus infection. PMID:25555148

In situ immune response and mechanisms of cell damage in central nervous system of fatal cases microcephaly by Zika virus.

PubMed

Azevedo, Raimunda S S; de Sousa, Jorge R; Araujo, Marialva T F; Martins Filho, Arnaldo J; de Alcantara, Bianca N; Araujo, Fernanda M C; Queiroz, Maria G L; Cruz, Ana C R; Vasconcelos, Beatriz H Baldez; Chiang, Jannifer O; Martins, Lívia C; Casseb, Livia M N; da Silva, Eliana V; Carvalho, Valéria L; Vasconcelos, Barbara C Baldez; Rodrigues, Sueli G; Oliveira, Consuelo S; Quaresma, Juarez A S; Vasconcelos, Pedro F C

2018-01-08

Zika virus (ZIKV) has recently caused a pandemic disease, and many cases of ZIKV infection in pregnant women resulted in abortion, stillbirth, deaths and congenital defects including microcephaly, which now has been proposed as ZIKV congenital syndrome. This study aimed to investigate the in situ immune response profile and mechanisms of neuronal cell damage in fatal Zika microcephaly cases. Brain tissue samples were collected from 15 cases, including 10 microcephalic ZIKV-positive neonates with fatal outcome and five neonatal control flavivirus-negative neonates that died due to other causes, but with preserved central nervous system (CNS) architecture. In microcephaly cases, the histopathological features of the tissue samples were characterized in three CNS areas (meninges, perivascular space, and parenchyma). The changes found were mainly calcification, necrosis, neuronophagy, gliosis, microglial nodules, and inflammatory infiltration of mononuclear cells. The in situ immune response against ZIKV in the CNS of newborns is complex. Despite the predominant expression of Th2 cytokines, other cytokines such as Th1, Th17, Treg, Th9, and Th22 are involved to a lesser extent, but are still likely to participate in the immunopathogenic mechanisms of neural disease in fatal cases of microcephaly caused by ZIKV.

Improvement of IFNγ ELISPOT Performance Following Overnight Resting of Frozen PBMC Samples Confirmed Through Rigorous Statistical Analysis

PubMed Central

Santos, Radleigh; Buying, Alcinette; Sabri, Nazila; Yu, John; Gringeri, Anthony; Bender, James; Janetzki, Sylvia; Pinilla, Clemencia; Judkowski, Valeria A.

2014-01-01

Immune monitoring of functional responses is a fundamental parameter to establish correlates of protection in clinical trials evaluating vaccines and therapies to boost antigen-specific responses. The IFNγ ELISPOT assay is a well-standardized and validated method for the determination of functional IFNγ-producing T-cells in peripheral blood mononuclear cells (PBMC); however, its performance greatly depends on the quality and integrity of the cryopreserved PBMC. Here, we investigate the effect of overnight (ON) resting of the PBMC on the detection of CD8-restricted peptide-specific responses by IFNγ ELISPOT. The study used PBMC from healthy donors to evaluate the CD8 T-cell response to five pooled or individual HLA-A2 viral peptides. The results were analyzed using a modification of the existing distribution free resampling (DFR) recommended for the analysis of ELISPOT data to ensure the most rigorous possible standard of significance. The results of the study demonstrate that ON resting of PBMC samples prior to IFNγ ELISPOT increases both the magnitude and the statistical significance of the responses. In addition, a comparison of the results with a 13-day preculture of PBMC with the peptides before testing demonstrates that ON resting is sufficient for the efficient evaluation of immune functioning. PMID:25546016

A potential individual cell malignancy indicator: focal length

NASA Astrophysics Data System (ADS)

Wang, Weina; Lear, Kevin L.

2011-03-01

The label-free technique of optofluidic intracavity spectroscopy (OFIS) utilizes the optical transmission spectrum of a cell in a microfluidic Fabry-Pérot (F-P) cavity to distinguish cells from cancerous cell lines and baseline normal blood cells. The classification between canine hemangiosarcoma (HSA) cancer cells and monocytes in canine normal peripheral blood mononuclear cells (PBMCs) had been demonstrated with 95% sensitivity and 98% specificity. Now with a new optical model that treats the cell settled at the bottom of the cavity as a thin lens, the focal length of cells was extracted and used as an individual cell malignancy indicator.

Failure of nitric oxide production by macrophages and decrease in CD4+ T cells in oral paracoccidioidomycosis: possible mechanisms that permit local fungal multiplication.

PubMed

Batista, Aline Carvalho; Soares, Cleverson Teixeira; Lara, Vanessa Soares

2005-01-01

Paracoccidioidomycosis is a chronic granulomatous disease that induces a specific inflammatory and immune response. The participation of nitric oxide (NO), a product of the inducible nitric oxide synthase enzyme (iNOS), as an important fungicidal molecule against Paracoccidioides brasiliensis has been demonstrated. In order to further characterize the Oral Paracoccidioidomycosis (OP), we undertook an immunohistochemical study of iNOS+, CD45RO+, CD3+, CD8+, CD20+, CD68+ cells and mast cells. The samples were distributed in groups according to the number of viable fungi per mm2. Our results showed weak immunolabeling for iNOS in the multinucleated giant cells (MNGC) and in most of the mononuclear (MN) cells, and the proportion of iNOS+ MN/MNGC cells in the OP were comparable to Control (clinically healthy oral tissues). Additionally, our analysis revealed a similarity in the number of CD4+ cells between the Control and the OP groups with higher numbers of fungi. These findings suggest that a low expression of iNOS and a decrease in the CD4+ T cells in OP may represent possible mechanisms that permit the local fungal multiplication and maintenance of active oral lesions.

Evolving Identification of Blood Cells Associated with Clinically Isolated Syndrome: Importance of Time since Clinical Presentation and Diagnostic MRI

PubMed Central

Trend, Stephanie; Jones, Anderson P.; Geldenhuys, Sian; Byrne, Scott N.; Fabis-Pedrini, Marzena J.; Nolan, David; Booth, David R.; Carroll, William M.; Lucas, Robyn M.; Kermode, Allan G.; Hart, Prue H.

2017-01-01

It is not clear how the profile of immune cells in peripheral blood differs between patients with clinically isolated syndrome (CIS) and healthy controls (HC). This study aimed to identify a CIS peripheral blood signature that may provide clues for potential immunomodulatory approaches early in disease. Peripheral blood mononuclear cells (PBMCs) were collected from 18 people with CIS, 19 HC and 13 individuals with other demyelinating conditions (ODC) including multiple sclerosis (MS). Individuals with CIS separated into two groups, namely those with early (≤14 days post-diagnostic magnetic resonance imaging (MRI); n = 6) and late (≥27 days; n = 12) blood sampling. Transitional B cells were increased in the blood of CIS patients independently of when blood was taken. However, there were two time-dependent effects found in the late CIS group relative to HC, including decreased CD56bright NK cells, which correlated significantly with time since MRI, and increased CD141+ myeloid dendritic cell (mDC2) frequencies. Higher CD1c+ B cells and lower non-classical monocyte frequencies were characteristic of more recent demyelinating disease activity (ODC and early CIS). Analysing cell populations by time since symptoms (subjective) and diagnostic MRI (objective) may contribute to understanding CIS. PMID:28617321

Association of Immunological Cell Profiles with Specific Clinical Phenotypes of Scleroderma Disease

PubMed Central

Calzada, David; Mayayo, Teodoro; González-Rodríguez, María Luisa; Rabasco, Antonio María; Lahoz, Carlos

2014-01-01

This study aimed to search the correlation among immunological profiles and clinical phenotypes of scleroderma in well-characterized groups of scleroderma patients, comparing forty-nine scleroderma patients stratified according to specific clinical phenotypes with forty-nine healthy controls. Five immunological cell subpopulations (B, CD4+ and CD8+ T-cells, NK, and monocytes) and their respective stages of apoptosis and activation were analyzed by flow cytometry, in samples of peripheral blood mononuclear cells (PBMCs). Analyses of results were stratified according to disease stage, time since the diagnosis, and visceral damage (pulmonary fibrosis, pulmonary hypertension, and cardiac affliction) and by time of treatment with corticosteroids. An increase in the percentages of monocytes and a decrease in the B cells were mainly related to the disease progression. A general apoptosis decrease was found in all phenotypes studied, except in localized scleroderma. An increase of B and NK cells activation was found in patients diagnosed more than 10 years ago. Specific cell populations like monocytes, NK, and B cells were associated with the type of affected organ. This study shows how, in a heterogeneous disease, proper patient's stratification according to clinical phenotypes allows finding specific cellular profiles. Our data may lead to improvements in the knowledge of prognosis factors and to aid in the analysis of future specific therapies. PMID:24818126

Evolving Identification of Blood Cells Associated with Clinically Isolated Syndrome: Importance of Time since Clinical Presentation and Diagnostic MRI.

PubMed

Trend, Stephanie; Jones, Anderson P; Geldenhuys, Sian; Byrne, Scott N; Fabis-Pedrini, Marzena J; Nolan, David; Booth, David R; Carroll, William M; Lucas, Robyn M; Kermode, Allan G; Hart, Prue H

2017-06-15

It is not clear how the profile of immune cells in peripheral blood differs between patients with clinically isolated syndrome (CIS) and healthy controls (HC). This study aimed to identify a CIS peripheral blood signature that may provide clues for potential immunomodulatory approaches early in disease. Peripheral blood mononuclear cells (PBMCs) were collected from 18 people with CIS, 19 HC and 13 individuals with other demyelinating conditions (ODC) including multiple sclerosis (MS). Individuals with CIS separated into two groups, namely those with early (≤14 days post-diagnostic magnetic resonance imaging (MRI); n = 6) and late (≥27 days; n = 12) blood sampling. Transitional B cells were increased in the blood of CIS patients independently of when blood was taken. However, there were two time-dependent effects found in the late CIS group relative to HC, including decreased CD56bright NK cells, which correlated significantly with time since MRI, and increased CD141+ myeloid dendritic cell (mDC2) frequencies. Higher CD1c+ B cells and lower non-classical monocyte frequencies were characteristic of more recent demyelinating disease activity (ODC and early CIS). Analysing cell populations by time since symptoms (subjective) and diagnostic MRI (objective) may contribute to understanding CIS.

Determinants of Risk Infection During Therapy with Anti TNF-Alpha Blocking Agents in Rheumatoid Arthritis

PubMed Central

Benucci, M; Saviola, G; Baiardi, P; Manfredi, M; Sarzi Puttini, P; Atzeni, Fabiola

2012-01-01

The use of TNF-alpha antagonists (infliximab, etanercept, adalimumab) has changed the course of many rheumatic diseases including rheumatoid arthritis (RA). Since their approval, some questions regarding their safety including infections have been observed. The aim of the study was to evaluate the changes in cytokines levels and cells subsets in patients with RA during anti TNF blocking agents treatment and the possible effect on infections’ development. We evaluated in 89 RA patients [39 treated with etanercept (ETN), 29 with adalimumab (ADA) and 21 with infliximab (IFN)] at baseline and after 6 months the following parameters: procalcitonin, ESR, CRP, cytokines as TNF, IL-6, IL-10, IL-8 and the TNF/IL-10 ratio, and peripheral mononuclear cells as CD3+, CD3+/CD4+, CD3+/CD8+, CD19+, CD3- /CD16+/56+, CD14+HLADR+, CD20+, CD19+/CD38+. Peripheral mononuclear cells were detected by flow cytometric system Cytomics FC500 and cytokines circulating levels by a quantitative sandwich enzyme immunoassay technique (Human IL-8 Instant ELISAe Bioscience, Human IL-6 Instant ELISA e Bioscience, Human IL-10 Instant ELISAe Bioscience and Human TNF-a Quantikine immunoassay RD system). A lower reduction of CD14+HLADR+ in ADA group 54.6±10.4% vs ETA 48.4±15.7% vs INF 40.7±16.5%, p<0.039 was found. No differences in all three groups on peripheral mononuclear cells CD3+, CD3+/CD4+, CD3+/CD8+, CD19+, CD 20+, CD19+/CD38+, CD3-/CD16+/56+, and cytokine circulating levels were found. The number of infections at 6 months was: 10.3% in ADA group, 12.8% in ETN group and 19.04% in IFN group. A correlation was found between the reduction in CD14+HLADR+ cells and IFN treatment. Our data showed that the level of CD14+HLADR+ cells was reduced during therapy with IFN. ADA and ETN don’t reduce lymphocyte populations and their subsets such as CD14+HLADR+ cells that play an important role host defence. PMID:22655000

Dynamic of CSF and serum biomarkers in HIV-1 subtype C encephalitis with CNS genetic compartmentalization-case study.

PubMed

de Almeida, Sergio M; Rotta, Indianara; Ribeiro, Clea E; Oliveira, Michelli F; Chaillon, Antoine; de Pereira, Ana Paula; Cunha, Ana Paula; Zonta, Marise; Bents, Joao França; Raboni, Sonia M; Smith, Davey; Letendre, Scott; Ellis, Ronald J

2017-06-01

Despite the effective suppression of viremia with antiretroviral therapy, HIV can still replicate in the central nervous system (CNS). This was a longitudinal study of the cerebrospinal fluid (CSF) and serum dynamics of several biomarkers related to inflammation, the blood-brain barrier, neuronal injury, and IgG intrathecal synthesis in serial samples of CSF and serum from a patient infected with HIV-1 subtype C with CNS compartmentalization.The phylogenetic analyses of plasma and CSF samples in an acute phase using next-generation sequencing and F-statistics analysis of C2-V3 haplotypes revealed distinct compartmentalized CSF viruses in paired CSF and peripheral blood mononuclear cell samples. The CSF biomarker analysis in this patient showed that symptomatic CSF escape is accompanied by CNS inflammation, high levels of cell and humoral immune biomarkers, CNS barrier dysfunction, and an increase in neuronal injury biomarkers with demyelization. Independent and isolated HIV replication can occur in the CNS, even in HIV-1 subtype C, leading to compartmentalization and development of quasispecies distinct from the peripheral plasma. These immunological aspects of the HIV CNS escape have not been described previously. To our knowledge, this is the first report of CNS HIV escape and compartmentalization in HIV-1 subtype C.

The transcriptome of the human mast cell leukemia cells HMC-1.2: an approach to identify specific changes in the gene expression profile in KitD816V systemic mastocytosis.

PubMed

Haenisch, B; Herms, S; Molderings, G J

2013-05-01

To circumvent the costly isolation procedure associated with tissue mast cells, human mast cell lines such as HMC-1 are employed in mastocytosis research, but their relation to mutated mast cells in systemic mastocytosis has not been investigated systematically. In the present study, we determined the transcriptome of HMC-1.2 cells and compared the expression data with those reported in the literature for normal human resting lung and tonsillar mast cells as well as leukocytes from peripheral blood and mononuclear cells from bone marrow aspirates of patients with D816 V-positive systemic mastocytosis. Our results suggest that HMC-1.2 cells are an appropriate model for the investigation of this variant of systemic mast cell activation disease. The data confirm previous suggestions that the pathologically increased activity of mast cells in patients with D816 V-positive systemic mastocytosis can be deduced from the detection of mutation-related changes in the gene expression profile in leukocytes from peripheral blood and in mononuclear cells from bone marrow aspirates. Thus, mutation-related changes of the expression profile can serve as surrogates (besides clustering of mast cells, expression of CD25, and increased release of tryptase) for the presence of the mutation D816 V in tyrosine kinase Kit in patients with systemic mastocytosis according to the WHO criteria. Whether this also holds true for systemic mast cell activation disease caused by other mutations in Kit or other mast cell activity-related genes is a subject for future studies.

Case for diagnosis. Actinic prurigo.

PubMed

Daldon, Patricia Erica Christofoletti; Pascini, Mirella; Correa, Mariane

2010-01-01

A 13-year-old black boy had pruritic papular and nodular lesions on his forearms associated to edema of the lower lip, photophobia, conjunctivitis and pterygium. Skin biopsy of the lower lip revealed acanthosis, spongiosis with dermal perivascular mononuclear cell infiltration composed by lymphocytes, plasma cells and eosinophils consistent with actinic prurigo. Lesions improved considerably with the use of thalidomide 100mg/ day.

Evaluation of Stability and In Vitro Security of Nanoemulsions Containing Eucalyptus globulus Oil

PubMed Central

Quatrin, Priscilla Maciel; Sagrillo, Michele Rorato; Nascimento, Kátia

2017-01-01

Essential oil of Eucalyptus globulus presents several pharmacological properties. However, their therapeutic efficacy may be affected by limitations due to several conditions, rendering it difficult to obtain stable and effective pharmaceutical formulations. The use of nanotechnology is an alternative to improve their characteristics aiming to ensure their stability and effectiveness. Furthermore, studies about the possible toxic effects of nanostructures are necessary to evaluate safety when the formulation comes into contact with human cells. Hence, in this paper, we evaluate for the first time the stability and in vitro cytogenotoxicity of nanoemulsions containing Eucalyptus globulus in peripheral blood mononuclear cells. As a result, the stability study found that the best condition for storage up to 90 days was refrigeration (4°C); it was the condition that best preserved the nanometric features. The content of the major compounds of oil was maintained after nanoencapsulation and preserved over time. In tests to evaluate the safety of this formulation, we can conclude that, at a low concentration (approximately 0.1%), Eucalyptus globulus nanoemulsion did not cause toxicity in peripheral blood mononuclear cells and also showed a protective effect in cells against possible damage when compared to oil in free form. PMID:28691021

Altered development of the brain after focal herpesvirus infection of the central nervous system.

PubMed

Koontz, Thad; Bralic, Marina; Tomac, Jelena; Pernjak-Pugel, Ester; Bantug, Glen; Jonjic, Stipan; Britt, William J

2008-02-18

Human cytomegalovirus infection of the developing central nervous system (CNS) is a major cause of neurological damage in newborn infants and children. To investigate the pathogenesis of this human infection, we developed a mouse model of infection in the developing CNS. Intraperitoneal inoculation of newborn animals with murine cytomegalovirus resulted in virus replication in the liver followed by virus spread to the brain. Virus infection of the CNS was associated with the induction of inflammatory responses, including the induction of a large number of interferon-stimulated genes and histological evidence of focal encephalitis with recruitment of mononuclear cells to foci containing virus-infected cells. The morphogenesis of the cerebellum was delayed in infected animals. The defects in cerebellar development in infected animals were generalized and, although correlated temporally with virus replication and CNS inflammation, spatially unrelated to foci of virus-infected cells. Specific defects included decreased granular neuron proliferation and migration, expression of differentiation markers, and activation of neurotrophin receptors. These findings suggested that in the developing CNS, focal virus infection and induction of inflammatory responses in resident and infiltrating mononuclear cells resulted in delayed cerebellar morphogenesis.

Altered development of the brain after focal herpesvirus infection of the central nervous system

PubMed Central

Koontz, Thad; Bralic, Marina; Tomac, Jelena; Pernjak-Pugel, Ester; Bantug, Glen; Jonjic, Stipan; Britt, William J.

2008-01-01

Human cytomegalovirus infection of the developing central nervous system (CNS) is a major cause of neurological damage in newborn infants and children. To investigate the pathogenesis of this human infection, we developed a mouse model of infection in the developing CNS. Intraperitoneal inoculation of newborn animals with murine cytomegalovirus resulted in virus replication in the liver followed by virus spread to the brain. Virus infection of the CNS was associated with the induction of inflammatory responses, including the induction of a large number of interferon-stimulated genes and histological evidence of focal encephalitis with recruitment of mononuclear cells to foci containing virus-infected cells. The morphogenesis of the cerebellum was delayed in infected animals. The defects in cerebellar development in infected animals were generalized and, although correlated temporally with virus replication and CNS inflammation, spatially unrelated to foci of virus-infected cells. Specific defects included decreased granular neuron proliferation and migration, expression of differentiation markers, and activation of neurotrophin receptors. These findings suggested that in the developing CNS, focal virus infection and induction of inflammatory responses in resident and infiltrating mononuclear cells resulted in delayed cerebellar morphogenesis. PMID:18268036

Engraftment of tonsillar mononuclear cells in human skin/SCID mouse chimera--validation of a novel xenogeneic transplantation model for autoimmune diseases.

PubMed

Yamanaka, N; Yamamoto, Y; Kuki, K

2001-01-01

Pustulosis palmaris et plantaris (PPP) has been considered as one of the typical tonsillar focal infections, based on the marked clinical improvement of the skin lesions after tonsillectomy. Despite the accumulation of data showing the clinical efficacy of tonsillectomy for this skin lesion, fundamental etiological and pathophysiological issues have yet to be addressed. One primary obstacle hindering investigators has been the lack of an appropriate animal model for this human skin disorder. In the early stage of PPP, it has been reported that lymphocytes, predominantly CD4+ T lymphocytes, infiltrate the palmar and plantar skins. However, the origin and mechanism of infiltration by these lymphocytes is not clear and there are very few reports on whether tonsillar mononuclear cells react directly with the skin. We have been intrigued by the ability to engraft human cells onto severe combined immunodeficiency (SCID) mice, together with the opportunity for long-term graft survival and ability to adoptively transfer various human immunocompetent cells. In this review, we addressed the existing deficiencies in our understanding of the relationship between tonsils and PPP by using emerging transplantation technology involving SCID mice.

Idiopathic hirsutism: local and peripheral expression of aromatase (CYP19A) and 5α-reductase genes (SRD5A1 and SRD5A2).

PubMed

Caglayan, A Okay; Dundar, Munis; Tanriverdi, Fatih; Baysal, Nuran A; Unluhizarci, Kursad; Ozkul, Yusuf; Borlu, Murat; Batukan, Cem; Kelestimur, Fahrettin

2011-08-01

To evaluate idiopathic hirsutism etiology via molecular studies testing peripheral and local aromatase and 5α-reductase expression. Assessment of the expression of messenger RNA (mRNA) for type 1 and 2,5α-reductase isoenzyme gene (SDR5A1, SDR5A2) and aromatase (CYP19A) in dermal papillae cells and peripheral blood mononuclear cells. University hospital. 28 untreated idiopathic hirsute patients and 20 healthy women (controls). Human skin biopsies and peripheral venous blood. SDR5A1, SDR5A2, CYP19A gene expression in skin biopsies and peripheral blood. A statistically significant reduction of SRD5A1, SRD5A2, and CYP19A gene expression was found in the dermal papillae cells and peripheral blood mononuclear cell between the study and control group. Further study, including protein expression and enzyme activity assays, are warranted to characterize the paradoxically low gene expression levels of local 5α-reductase and aromatase in women with idiopathic hirsutism. Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Cell therapy of pseudarthrosis

PubMed Central

Bastos Filho, Ricardo; Lermontov, Simone; Borojevic, Radovan; Schott, Paulo Cezar; Gameiro, Vinicius Schott; Granjeiro, José Mauro

2012-01-01

Objective To assess the safety and efficiency of cell therapy for pseudarthrosis. Implant of the bone marrow aspirate was compared to mononuclear cells purified extemporaneously using the Sepax® equipment. Methods Six patients with nonunion of the tibia or femur were treated. Four received a percutaneous infusion of autologous bone marrow aspirated from the iliac crest, and two received autologous bone marrow mononuclear cells separated from the aspirate with the Sepax®. The primary fixation method was unchanged, and the nonunion focus was not exposed. Physical examination and radiographies were performed 2, 4 and 6 months after the treatment by the same physician. After consolidation of the fracture the satisfaction of the patients was estimated using the adapted QALY scale. Results No complications occurred as a result of the referred procedures. Bone consolidation was obtained in all cases within 3 to 24 weeks. The degree of patient satisfaction before and after bone consolidation was assessed, with the average value increasing from two to nine (p=0.0156). Conclusion We conclude that the proposed method is effective and safe for the treatment of nonunion of long bones regardless of the stabilization method used. Level of Evidence II, Prospective Comparative Study PMID:24453616

Ixodes scapularis saliva mitigates inflammatory cytokine secretion during Anaplasma phagocytophilum stimulation of immune cells

PubMed Central

2012-01-01

Background Ixodes scapularis saliva enables the transmission of infectious agents to the mammalian host due to its immunomodulatory, anesthetic and anti-coagulant properties. However, how I. scapularis saliva influences host cytokine secretion in the presence of the obligate intracellular rickettsial pathogen Anaplasma phagocytophilum remains elusive. Methods Bone marrow derived macrophages (BMDMs) were stimulated with pathogen associated molecular patterns (PAMPs) and A. phagocytophilum. Cytokine secretion was measured in the presence and absence of I. scapularis saliva. Human peripheral blood mononuclear cells (PBMCs) were also stimulated with Tumor Necrosis Factor (TNF)-α in the presence and absence of I. scapularis saliva and interleukin (IL)-8 was measured. Results I. scapularis saliva inhibits inflammatory cytokine secretion by macrophages during stimulation of Toll-like (TLR) and Nod-like receptor (NLR) signaling pathways. The effect of I. scapularis saliva on immune cells is not restricted to murine macrophages because decreasing levels of interleukin (IL)-8 were observed after TNF-α stimulation of human peripheral blood mononuclear cells. I. scapularis saliva also mitigates pro-inflammatory cytokine response by murine macrophages during challenge with A. phagocytophilum. Conclusions These findings suggest that I. scapularis may inhibit inflammatory cytokine secretion during rickettsial transmission at the vector-host interface. PMID:23050849

Evidence for a terminal differentiation process in the rat liver.

PubMed

Sigal, S H; Gupta, S; Gebhard, D F; Holst, P; Neufeld, D; Reid, L M

1995-07-01

In rapidly renewing epithelia, such as skin and gut, as well as hemopoietic cells and stromal fibroblasts, the process of progenitor cell maturation, terminal differentiation and senescence from cells of a fetal phenotype is strikingly similar. To examine hepatocellular maturation, we studied embryonic, suckling and young adult rat liver cells with multiparametric fluorescence activated cell sorting (FACS), after exclusion of hemopoietic, endothelial, Kupffer, and nonviable cells. With maturation, cell granularity and autofluorescence exponentially increased from fetal liver to suckling and adult liver as the proportion of S phase cells progressively declined from 33.8% +/- 1.3% to 4.9% +/- 2.8% and 1.1% +/- 0.6% (P < 0.05), respectively. In liver from fetal and suckling rats, all hepatocytes were mononuclear and contained diploid DNA whereas 21.2% +/- 5.9% hepatocytes in adult liver were binucleated. Analysis of nuclear DNA content in adult hepatocytes demonstrated that 53.3% +/- 3.9% of the nuclei were diploid, 43.6% +/- 3.5% tetraploid and 0.5 +/- 0.6% octaploid. However, in the adult liver, small, mononuclear cells were also present with granularity and autofluorescence comparable to fetal hepatoblasts, as well as glucose-6-phosphatase activity, diploid DNA in 89.0% +/- 2.1% of the nuclei, and with increased granularity in culture. Since general features of terminal cellularity differentiation and senescence include cessation of mitotic activity, polyploidy and accumulation of autofluorescent secondary lysosomes, our data suggest that liver cells too undergo a process of terminal differentiation.

Susceptibility Testing by Polymerase Chain Reaction DNA Quantitation: A Method to Measure Drug Resistance of Human Immunodeficiency Virus Type 1 Isolates

NASA Astrophysics Data System (ADS)

Eron, Joseph J.; Gorczyca, Paul; Kaplan, Joan C.; D'Aquila, Richard T.

1992-04-01

Polymerase chain reaction (PCR) DNA quantitation (PDQ) susceptibility testing rapidly and directly measures nucleoside sensitivity of human immunodeficiency virus type 1 (HIV-1) isolates. PCR is used to quantitate the amount of HIV-1 DNA synthesized after in vitro infection of peripheral blood mononuclear cells. The relative amounts of HIV-1 DNA in cell lysates from cultures maintained at different drug concentrations reflect drug inhibition of virus replication. The results of PDQ susceptibility testing of 2- or 3-day cultures are supported by assays measuring HIV-1 p24 antigen production in supernatants of 7- or 10-day cultures. DNA sequence analyses to identify mutations in the reverse transcriptase gene that cause resistance to 3'-azido-3'-deoxythymidine also support the PDQ results. With the PDQ method, both infectivity titration and susceptibility testing can be performed on supernatants from primary cultures of peripheral blood mononuclear cells. PDQ susceptibility testing should facilitate epidemiologic studies of the clinical significance of drug-resistant HIV-1 isolates.

Gc protein-derived macrophage-activating factor (GcMAF) stimulates cAMP formation in human mononuclear cells and inhibits angiogenesis in chick embryo chorionallantoic membrane assay.

PubMed

Pacini, Stefania; Morucci, Gabriele; Punzi, Tiziana; Gulisano, Massimo; Ruggiero, Marco

2011-04-01

The effects of Gc protein-derived macrophage-activating factor (GcMAF) have been studied in cancer and other conditions where angiogenesis is deregulated. In this study, we demonstrate for the first time that the mitogenic response of human peripheral blood mononuclear cells (PBMCs) to GcMAF was associated with 3'-5'-cyclic adenosine monophosphate (cAMP) formation. The effect was dose dependent, and maximal stimulation was achieved using 0.1 ng/ml. Heparin inhibited the stimulatory effect of GcMAF on PBMCs. In addition, we demonstrate that GcMAF (1 ng/ml) inhibited prostaglandin E(1)- and human breast cancer cell-stimulated angiogenesis in chick embryo chorionallantoic membrane (CAM) assay. Finally, we tested different GcMAF preparations on CAM, and the assay proved to be a reliable, reproducible and inexpensive method to determine the relative potencies of different preparations and their stability; we observed that storage at room temperature for 15 days decreased GcMAF potency by about 50%. These data could prove useful for upcoming clinical trials on GcMAF.

Tryptamine and dimethyltryptamine inhibit indoleamine 2,3 dioxygenase and increase the tumor-reactive effect of peripheral blood mononuclear cells.

PubMed

Tourino, Melissa Cavalheiro; de Oliveira, Edson Mendes; Bellé, Luziane Potrich; Knebel, Franciele Hinterholz; Albuquerque, Renata Chaves; Dörr, Felipe Augusto; Okada, Sabrina Sayori; Migliorini, Silene; Soares, Irene Silva; Campa, Ana

2013-07-01

Indoleamine 2,3-dioxygenase (IDO) is an interferon-γ (IFN-γ)-induced tryptophan-degrading enzyme, producing kynurenine (KYN) that participates in the mechanism of tumor immune tolerance. Thus, IDO inhibition has been considered a strategy for anticancer therapy. The aim of this study was to identify whether the metabolites originated from the competitive routes of tryptophan metabolism, such as the serotonergic or N, N-dimethyltryptamine (DMT) pathways, have inhibitory effects on recombinant human IDO (rhIDO) activity. Serotonin and melatonin had no effect; on the other hand, tryptamine (TRY) and DMT modulated the activity of rhIDO as classical non-competitive inhibitors, with Ki values of 156 and 506 μM, respectively. This inhibitory effect was also observed on constitutively expressed or IFN-γ-induced IDO in the A172 human glioma cell line. TRY and DMT increased the cytotoxic activity of peripheral blood mononuclear cells (PBMCs) in co-culture assays. We conclude that the IDO inhibition by TRY and DMT contributed to a more effective tumor-reactive response by the PBMCs. Copyright © 2013 John Wiley & Sons, Ltd.

Role of helminths in regulating mucosal inflammation.

PubMed

Weinstock, Joel V; Summers, Robert W; Elliott, David E

2005-09-01

The rapid rise in prevalence of ulcerative colitis (UC) and Crohn's disease (CD) in highly developed countries suggests that environmental change engenders risk for inflammatory bowel disease (IBD). Eradication of parasitic worms (helminths) through increased hygiene may be one such change that has led to increased prevalence of these diseases. Helminths alter host mucosal and systemic immunity, inhibiting dysregulated inflammatory responses. Animals exposed to helminths are protected from experimental colitis, encephalitis, and diabetes. Patients with CD or UC improve when exposed to whipworm. Lamina propria (LP) mononuclear cells from helminth-colonized mice make less interleukin (IL)-12 p40 and IFN-gamma, but more IL-4, IL-13, IL-10, TGF-beta, and PGE(2) compared to LP mononuclear cells from naive mice. Systemic immune responses show similar skewing toward Th2 and regulatory cytokine production in worm-colonized animal models and humans. Recent reports suggest that helminths induce regulatory T cell activity. These effects by once ubiquitous organisms may have protected individuals from many of the emerging immune-mediated illnesses like IBD, multiple sclerosis, type I diabetes, and asthma.

Decreased TIM-3 mRNA expression in peripheral blood mononuclear cells from nephropathy patients.

PubMed

Cai, X Z; Liu, N; Qiao, Y; Du, S Y; Chen, Y; Chen, D; Yu, S; Jiang, Y

2015-06-12

Increasing evidence shows that TIM-1 and TIM-3 in-fluence chronic autoimmune diseases, and their expression levels in immune cells from nephritic patients are still unknown. Real-time transcription-polymerase chain reaction analysis was used to deter-mine expression levels of TIM-1 and TIM-3 mRNA in peripheral blood mononuclear cells (PBMCs) from 36 patients with minimal change glo-merulopathy (MCG), 65 patients with lupus nephritis (LN), 78 patients with IgA nephropathy (IgAN), 55 patients with membranous nephropa-thy (MN), 22 patients with crescentic glomerulonephritis (CGN), 26 patients with anaphylactoid purpura nephritis (APN), and 63 healthy controls. TIM-3 mRNA expression significantly decreased in PBMCs from nephritic patients (LN, P < 0.0001; MCG, P < 0.0001; MN, P = 0.0031; CGN, P = 0.0464; IgAN, P = 0.0002; APN, P = 0.0392) com-pared with healthy controls. In contrast, there was no significant differ-ence in TIM-1 mRNA expression between the patients and the healthy controls. Our results suggest that insufficient expression of TIM-3 mRNA may be involved in the pathogenesis of nephropathy.

Detection of Sirtuin-1 protein expression in peripheral blood leukocytes in dogs.

PubMed

Yoshimura, Kuniko; Matsuu, Aya; Sasaki, Kai; Momoi, Yasuyuki

2018-05-11

Sirtuin-1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD + )-dependent histone deacetylase with a large number of protein substrates. It has attracted a lot of attention in association with extending lifespan. The objective of this study was to enable the evaluation of SIRT1 expression in peripheral blood mononuclear cells (PBMCs) from dogs by flow cytometry. Three transcript variants were amplified from PBMCs by reverse transcription PCR and the nucleotide sequences were analyzed. On the basis deduced amino acid sequence, a monoclonal antibody against human SIRT1, 1F3, was selected to detect canine SIRT1. Canine SIRT1 in peripheral blood mononuclear cells was successfully detected by western blotting using this antibody. Intracellular canine SIRT1 was also detected in permeabilized 293T cells transfected with a canine SIRT1 expression plasmid by flow cytometry using this antibody. SIRT1 was detected in all leukocyte subsets including lymphocytes, granulocytes and monocytes. The expression level was markedly different among individual dogs. These results indicated that the method applied in this study is useful for evaluating canine SIRT1 levels in PBMCs from dogs.

Walter Reed Army Institute of Research Annual Progress Report, Fiscal Year 1980

DTIC Science & Technology

1980-10-01

for Military Medicine. 31. Smith, L.F. and G.H. Lowell. 1980. Antibody-dependent cell-mediated antibacterial activity of human mononuclear cells. II...dependent monocyte-mediated antibacterial activity . J. Exp. Med. 152:452-457. 33. Lowell, G.H., L.F. Smith, J.M. Griffiss, B.L. Brandt and R.P...dependent cell-mediated antibacterial activity . K lymphocytes, monocytes and granulocytes are effective against shigella. J. Im- munol. (In press). 35

Intestinal immune response to human Cryptosporidium sp. infection

DTIC Science & Technology

2008-01-01

proliferation of human peripheral blood mononuclear cells in vitro . J. Infect. Dis. 172:211–216. 29. Gomez Morales, M. A., G. La Rosa, A. Ludovisi, A. M...at different developmental stages modulates host cell apop- tosis in vitro . Infect. Immun. 72:6061–6067. 68. Moore, K. W., R. de Waal Malefyt, R. L...Department of Medicine, Division of Gastroenterology, University of California at San Diego, La Jolla, California2; Cell Biology Laboratory, United

Demonstration that CobG, the monooxygenase associated with the ring contraction process of the aerobic cobalamin (vitamin B12) biosynthetic pathway, contains an Fe-S center and a mononuclear non-heme iron center.

PubMed

Schroeder, Susanne; Lawrence, Andrew D; Biedendieck, Rebekka; Rose, Ruth-Sarah; Deery, Evelyne; Graham, Ross M; McLean, Kirsty J; Munro, Andrew W; Rigby, Stephen E J; Warren, Martin J

2009-02-20

The ring contraction process that occurs during cobalamin (vitamin B(12)) biosynthesis is mediated via the action of two enzymes, CobG and CobJ. The first of these generates a tertiary alcohol at the C-20 position of precorrin-3A by functioning as a monooxygenase, a reaction that also forms a gamma lactone with the acetic acid side chain on ring A. The product, precorrin-3B, is then acted upon by CobJ, which methylates at the C-17 position and promotes ring contraction of the macrocycle by catalyzing a masked pinacol rearrangement. Here, we report the characterization of CobG enzymes from Pseudomonas denitrificans and Brucella melitensis. We show that both contain a [4Fe-4S] center as well as a mononuclear non-heme iron. Although both enzymes are active in vivo, the P. denitrificans enzyme was found to be inactive in vitro. Further analysis of this enzyme revealed that the mononuclear non-heme iron was not reducible, and it was concluded that it is rapidly inactivated once it is released from the bacterial cell. In contrast, the B. melitensis enzyme was found to be fully active in vitro and the mononuclear non-heme iron was reducible by dithionite. The reduced mononuclear non-heme was able to react with the oxygen analogue NO, but only in the presence of the substrate precorrin-3A. The cysteine residues responsible for binding the Fe-S center were identified by site-directed mutagenesis. A mechanism for CobG is presented.

A human monoclonal antibody drug and target discovery platform for B-cell chronic lymphocytic leukemia based on allogeneic hematopoietic stem cell transplantation and phage display.

PubMed

Baskar, Sivasubramanian; Suschak, Jessica M; Samija, Ivan; Srinivasan, Ramaprasad; Childs, Richard W; Pavletic, Steven Z; Bishop, Michael R; Rader, Christoph

2009-11-12

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is the only potentially curative treatment available for patients with B-cell chronic lymphocytic leukemia (B-CLL). Here, we show that post-alloHSCT antibody repertoires can be mined for the discovery of fully human monoclonal antibodies to B-CLL cell-surface antigens. Sera collected from B-CLL patients at defined times after alloHSCT showed selective binding to primary B-CLL cells. Pre-alloHSCT sera, donor sera, and control sera were negative. To identify post-alloHSCT serum antibodies and subsequently B-CLL cell-surface antigens they recognize, we generated a human antibody-binding fragment (Fab) library from post-alloHSCT peripheral blood mononuclear cells and selected it on primary B-CLL cells by phage display. A panel of Fab with B-CLL cell-surface reactivity was strongly enriched. Selection was dominated by highly homologous Fab predicted to bind the same antigen. One Fab was converted to immunoglobulin G1 and analyzed for reactivity with peripheral blood mononuclear cells from B-CLL patients and healthy volunteers. Cell-surface antigen expression was restricted to primary B cells and up-regulated in primary B-CLL cells. Mining post-alloHSCT antibody repertoires offers a novel route to discover fully human monoclonal antibodies and identify antigens of potential therapeutic relevance to B-CLL and possibly other cancers. Trials described herein were registered at www.clinicaltrials.gov as nos. NCT00055744 and NCT00003838.

High temperature affects the phagocytic activity of human peripheral blood mononuclear cells.

PubMed

Djaldetti, Meir; Bessler, Hanna

2015-10-01

The ability for engulfment of pathogens and inert particles is the key hallmark of the phagocytic cells. Phagocytes play a significant role in the modulation of local or extended inflammation. Since fever activates a number of factors linked with the immune response it was the goal of this study to examine the in vitro effect of hyperthermia on the phagocytic capacity, the number of phagocytic cells and the viability of human peripheral blood mononuclear cells (PBMC) at 37 and 40°C. PBMC were incubated with 0.8 μm polysterene latex beads, for 2 hours at 37 and 40°C. The number of phagocytic cells, and that of latex particles internalized by each individual cell was counted with a light microscope. In addition, the percentage of viable cells and the number of active metabolic cells was evaluated. A temperature of 40°C significantly increased the number of phagocytic cells and the phagocytic index by 41 and 37% respectively, as compared to cells incubated at 37°C. While the number of vital cells (trypan blue test) did not differ statistically at both temperatures, the number of active metabolic cells (XTT test) after 2 h of incubation at 40°C was 17% higher as compared with that at 37°C. However, the number of active metabolic cells after 24 h of incubation at 40°C was 51% lower compared with cells incubated at 37°C. The increased phagocytic capacity of human peripheral blood monocytes at high temperature further enlightens the immunomodulatory effect of fever in the immune responses during inflammation.

Reactivity of airway phagocytes during the development of acute pneumonia under conditions of stimulation of mononuclear phagocyte system with zymosan.

PubMed

Makarova, O P

2008-12-01

Pneumonia was induced in (CBA x C57Bl)F1 mice under conditions of stimulation of the mononuclear phagocyte system with zymosan. The number of neutrophils in airways increased after 3 days; by day 14, the number of cells in the bronchoalveolar lavage fluid further increased due to migration of macrophages. After zymosan prestimulation, the number and functional activity of neutrophils during the early period of inflammation (3 days) did not change, but the increase in phagocytic activity of macrophages was inhibited by 20%. By day 14, the effect of prestimulation manifested in 4.5-fold decreased capacity of neutrophils and macrophages to reduce NBT.

Advances in Bone Marrow Stem Cell Therapy for Retinal Dysfunction

PubMed Central

Park, Susanna S.; Moisseiev, Elad; Bauer, Gerhard; Anderson, Johnathon D.; Grant, Maria B.; Zam, Azhar; Zawadzki, Robert J.; Werner, John S.; Nolta, Jan A.

2016-01-01

The most common cause of untreatable vision loss is dysfunction of the retina. Conditions, such as age-related macular degeneration, diabetic retinopathy and glaucoma remain leading causes of untreatable blindness worldwide. Various stem cell approaches are being explored for treatment of retinal regeneration. The rationale for using bone marrow stem cells to treat retinal dysfunction is based on preclinical evidence showing that bone marrow stem cells can rescue degenerating and ischemic retina. These stem cells have primarily paracrine trophic effects although some cells can directly incorporate into damaged tissue. Since the paracrine trophic effects can have regenerative effects on multiple cells in the retina, the use of this cell therapy is not limited to a particular retinal condition. Autologous bone marrow-derived stem cells are being explored in early clinical trials as therapy for various retinal conditions. These bone marrow stem cells include mesenchymal stem cells, mononuclear cells and CD34+ cells. Autologous therapy requires no systemic immunosuppression or donor matching. Intravitreal delivery of CD34+ cells and mononuclear cells appears to be tolerated and is being explored since some of these cells can home into the damaged retina after intravitreal administration. The safety of intravitreal delivery of mesenchymal stem cells has not been well established. This review provides an update of the current evidence in support of the use of bone marrow stem cells as treatment for retinal dysfunction. The potential limitations and complications of using certain forms of bone marrow stem cells as therapy are discussed. Future directions of research include methods to optimize the therapeutic potential of these stem cells, non-cellular alternatives using extracellular vesicles, and in vivo high-resolution retinal imaging to detect cellular changes in the retina following cell therapy. PMID:27784628

Mesenchymal stem cell and derived exosome as small RNA carrier and Immunomodulator to improve islet transplantation.

PubMed

Wen, Di; Peng, Yang; Liu, Di; Weizmann, Yossi; Mahato, Ram I

2016-09-28

Human bone marrow mesenchymal stem cells (hBMSCs) and their exosomes can suppress immune reaction and deliver small RNAs. Thus, they may improve islet transplantation by delivering small RNAs for promoting islet function and inhibiting immune rejection. Here, we proposed an hBMSC and its exosome-based therapy to overcome immune rejection and poor islet function, both of which hinder the success of islet transplantation. We found overexpressed siFas and anti-miR-375 in plasmid encoding shFas and anti-miR-375 transfected hBMSC-derived exosomes, which silenced Fas and miR-375 of human islets and improved their viability and function against inflammatory cytokines. This plasmid transfected hBMSCs downregulated Fas and miR-375 of human islets in a humanized NOD scid gamma (NSG) mouse model, whose immune reaction was inhibited by injecting hBMSC and peripheral blood mononuclear cell (PBMC) co-cultured exosomes. These exosomes suppressed immune reaction by inhibiting PBMC proliferation and enhancing regulatory T cell (Treg) function. Collectively, our studies elucidated the mechanisms of RNA delivery from hBMSCs to human islets and the immunosuppressive effect of hBMSC and peripheral blood mononuclear cell co-cultured exosomes for improving islet transplantation. Copyright © 2016 Elsevier B.V. All rights reserved.

[Neuropathology of Natalizumab-Associated Progressive Multifocal Leukoencephalopathy].

PubMed

Kanda, Takashi

2015-07-01

The pathological findings of natalizumab-associated progressive multifocal leukoencephalopathy (PML) are reviewed. In addition to the classical pathology of PML including the presence of enlarged abnormal astrocytes, intranuclear inclusions mainly found within large swollen oligodendrocytes and abundant myelin-laden macrophages/microglia, massive perivascular and parenchymal mononuclear cell infiltration was observed. The latter pathologic picture is that of immune reconstitution inflammatory syndrome (IRIS), and most of these infiltrating cells are CD8-positive T cells. Because IRIS inevitably occurs after the cessation of natalizumab therapy due to the development of PML and subsequent plasma exchange, most of the published pathologic pictures of natalizumab-associated PML patients were a mixture of PML and PML-IRIS. PML-IRIS is also characterized by fewer oligodendroglial viral inclusions and fewer cells that are immunoreactive against anti-JCV antibodies. These findings suggest the effective removal of JCV after the return of normal immune-surveillance in the central nervous system, but clinicians should be aware that JCV elimination is not complete under the IRIS condition, and the immunosuppressive therapy against IRIS should be carefully performed. Mononuclear cell infiltration in natalizumab-associated PML-IRIS patients was much more prominent than that in HIV-associated PML-IRIS patients, reflecting the retained, even enhanced, systemic immunities in patients treated by natalizumab.

Gene expression analysis of whole blood, peripheral blood mononuclear cells, and lymphoblastoid cell lines from the Framingham Heart Study

PubMed Central

Joehanes, Roby; Johnson, Andrew D.; Barb, Jennifer J.; Raghavachari, Nalini; Liu, Poching; Woodhouse, Kimberly A.; O'Donnell, Christopher J.; Munson, Peter J.

2012-01-01

Despite a growing number of reports of gene expression analysis from blood-derived RNA sources, there have been few systematic comparisons of various RNA sources in transcriptomic analysis or for biomarker discovery in the context of cardiovascular disease (CVD). As a pilot study of the Systems Approach to Biomarker Research (SABRe) in CVD Initiative, this investigation used Affymetrix Exon arrays to characterize gene expression of three blood-derived RNA sources: lymphoblastoid cell lines (LCL), whole blood using PAXgene tubes (PAX), and peripheral blood mononuclear cells (PBMC). Their performance was compared in relation to identifying transcript associations with sex and CVD risk factors, such as age, high-density lipoprotein, and smoking status, and the differential blood cell count. We also identified a set of exons that vary substantially between participants, but consistently in each RNA source. Such exons are thus stable phenotypes of the participant and may potentially become useful fingerprinting biomarkers. In agreement with previous studies, we found that each of the RNA sources is distinct. Unlike PAX and PBMC, LCL gene expression showed little association with the differential blood count. LCL, however, was able to detect two genes related to smoking status. PAX and PBMC identified Y-chromosome probe sets similarly and slightly better than LCL. PMID:22045913

Ocular pathology in melanomatous Sinclair miniature swine.

PubMed Central

Feeney-Burns, L.; Burns, R. P.; Gao, C. L.

1988-01-01

Eyes of cutaneous melanoma-bearing miniature Sinclair swine were examined by light and electron microscopy during growth and maturation of animals. The histology of ocular depigmentation, which occurs during melanoma arrest and skin depigmentation, was studied in eyes of animals that had successful and unsuccessful tumor regression. In 12 animals one eye was removed at an early stage and the second eye at a later stage of disease or maturation. The histologic and clinical course of the ocular changes was correlated with changes in the cutaneous tumors. Animals showing rapid cutaneous tumor regression developed acute uveitis that was characterized by an influx of mononuclear cells into the stroma of the ciliary body; later this spread to the iris and choroid. In late stages the cornea sometimes developed a band of calcium precipitates beneath the basal epithelial cells (band keratopathy). Uveal melanocytes developed watery cytoplasm typical of cells with ruptured plasma membranes. Released melanin granules were taken up initially into the lysosomal compartment of mononuclear cells having the various morphologic features of lymphocytes and monocytes; later, melanin also appeared in fibrocyte lysosomes. The relationship of these processes to various cell-mediated immunity phenomena is being studied. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:3354645

Molecular cloning of an inducible serine esterase gene from human cytotoxic lymphocytes.

PubMed Central

Trapani, J A; Klein, J L; White, P C; Dupont, B

1988-01-01

A cDNA clone encoding a human serine esterase gene was isolated from a library constructed from poly(A)+ RNA of allogeneically stimulated, interleukin 2-expanded peripheral blood mononuclear cells. The clone, designated HSE26.1, represents a full-length copy of a 0.9-kilobase mRNA present in human cytotoxic cells but absent from a wide variety of noncytotoxic cell lines. Clone HSE26.1 contains an 892-base-pair sequence, including a single 741-base-pair open reading frame encoding a putative 247-residue polypeptide. The first 20 amino acids of the polypeptide form a leader sequence. The mature protein is predicted to have an unglycosylated Mr of approximately equal to 26,000 and contains a single potential site for N-linked glycosylation. The nucleotide and predicted amino acid sequences of clone HSE26.1 are homologous with all murine and human serine esterases cloned thus far but are most similar to mouse granzyme B (70% nucleotide and 68% amino acid identity). HSE26.1 protein is expressed weakly in unstimulated peripheral blood mononuclear cells but is strongly induced within 6-hr incubation in medium containing phytohemagglutinin. The data suggest that the protein encoded by HSE26.1 plays a role in cell-mediated cytotoxicity. Images PMID:3261871

A randomized phase II trial comparing two different sequence combinations of autologous vaccine and human recombinant interferon gamma and human recombinant interferon alpha2B therapy in patients with metastatic renal cell carcinoma: clinical outcome and analysis of immunological parameters.

PubMed

Schwaab, T; Heaney, J A; Schned, A R; Harris, R D; Cole, B F; Noelle, R J; Phillips, D M; Stempkowski, L; Ernstoff, M S

2000-04-01

The clinical observation of spontaneous regression in patients with renal cell carcinoma (RCC) and the response to various immunotherapeutic therapies strongly suggest a role for the host immune system in this disease. Prior studies showed that sequential administration of interferon (IFN) gamma and IFN alpha to RCC patients was safe. Clinical responses as well as immune changes in the peripheral blood mononuclear cell compartment were observed. Autologous tumor cell vaccines (AV) have also demonstrated activity in renal cell carcinoma. We hypothesize that the addition of AV to sequential IFN gamma and a therapy might improve the tumor-specific immune response by providing an appropriate source of antigen in the appropriate cytokine environment. To our knowledge, this is the first trial using AV combined with IFN alpha and IFN gamma. The purpose of this study was to evaluate the feasibility of manufacturing and administering (AV) from resected tumor samples, and administration of AV with combination IFN gamma and IFN alpha therapy. Finally, the impact on immunological parameters of these treatment options was assessed. Patients with metastatic RCC were randomly assigned to receive AV plus bCG along with a sequential administration of IFN gamma and a either together or after initiation of vaccine. Toxicity and clinical responses were evaluated. Modulations of the immune system were investigated by analyzing phenotype, cytokine mRNA expression, T cell proliferation and cytotoxicity in the peripheral blood mononuclear cell compartment. Fourteen patients with metastatic renal cell carcinoma were enrolled in this study; 9 were available for response evaluation. In a 70 day period, 3 (33%) showed mixed responses, 5 (56%) stable disease and 1 (11%) progression of disease. Toxicities were consistent with previous clinical reports. In the flow-cytometry phenotype analysis, stimulation of distinct subsets of circulating T-lymphocytes and a decrease of CD8+ T cell subsets was demonstrated. T-cell proliferation to allogeneic tumor cell stimulation improved following treatment. IL-4 and IL-5 mRNA levels were reduced in all patients after treatment. Patients who responded to treatment did not produce any IL-4 mRNA at all, before or after treatment. AV with IFNgamma and IFNalpha therapy might induce a MHC class-mediated cytotoxic T lymphocyte (CTL) response. We suggest that adequate therapy might direct T cell response toward a Th1 type response. We hypothesize a state of improved immune readiness in patients who might eventually respond to immunotherapy.