Sample records for hypaque
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Effect of diatrizoate meglumine (Hypaque) on the perilymphatic oxygen tension.
Nagahara, K; Fisch, U
1982-12-01
The effect of diatrizoate meglumine (Hypaque) upon the perilymphatic oxygenation has been investigated using the polarographic method in cats submitted to various conditions such as normoxia, apnea, hypercapnia and chronically reduced vascularization. The minimal changes of the perilymphatic PO2 recorded after the injection of Hypaque permit to conclude that this drug has no practical effect upon the oxygenation of the perilymph.
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2005-02-01
were from Sigma (St. Louis, MO, USA). Cell Culture Human PBMC were isolated by Ficoll-Hypaque density gradient centri- fugation of heparinized blood...Cytotoxicity was measured by the uptake of trypan blue . T-cell proliferation was assayed with PBMC (105 cells/well) that were plated in triplicate with...concentration range used in these studies (1–30 nM), as confirmed by trypan blue dye exclusion test. However, at 100 nM triptolide, 20% of PBMC took up
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Avettand-Fènoël, Véronique; Chaix, Marie-Laure; Blanche, Stéphane; Burgard, Marianne; Floch, Corinne; Toure, Kadidia; Allemon, Marie-Christine; Warszawski, Josiane; Rouzioux, Christine
2009-02-01
HIV-1 diagnosis in babies born to seropositive mothers is one of the challenges of HIV epidemics in children. A simple, rapid protocol was developed for quantifying HIV-1 DNA in whole blood samples and was used in the ANRS French pediatric cohort in conditions of prevention of mother-to-child transmission. A quantitative HIV-1 DNA protocol (LTR real-time PCR) requiring small blood volumes was developed. First, analytical reproducibility was evaluated on 172 samples. Results obtained on blood cell pellets and Ficoll-Hypaque separated mononuclear cells were compared in 48 adult HIV-1 samples. Second, the protocol was applied to HIV-1 diagnosis in infants in parallel with plasma HIV-RNA quantitation. This prospective study was performed in children born between May 2005 and April 2007 included in the ANRS cohort. The assay showed good reproducibility. The 95% detection cut-off value was 6 copies/PCR, that is, 40 copies/10(6) leukocytes. HIV-DNA levels in whole blood were highly correlated with those obtained after Ficoll-Hypaque separation (r = 0.900, P < 0.0001). A total of 3,002 specimens from 1,135 infants were tested. The specificity of HIV-DNA and HIV-RNA assays was 100%. HIV-1 infection was diagnosed in nine infants before age 60 days. HIV-DNA levels were low, underlining the need for sensitive assays when highly active antiretroviral therapy (HAART) has been given. The performances of this HIV-DNA assay showed that it is adapted to early diagnosis in children. The results were equivalent to those of HIV-RNA assay. HIV-DNA may be used even in masked primary infection in newborns whose mothers have received HAART. (c) 2008 Wiley-Liss, Inc.
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In vivo traffic of indium-111-oxine labeled human lymphocytes collected by automated apheresis
DOE Office of Scientific and Technical Information (OSTI.GOV)
Read, E.J.; Keenan, A.M.; Carter, C.S.
1990-06-01
The in vivo traffic patterns of autologous lymphocytes were studied in five normal human volunteers using lymphocytes obtained by automated apheresis, separated on Ficoll-Hypaque gradients, and labeled ex vivo with {sup 111}In-oxine. Final lymphocyte infusions contained 1.8-3.1 X 10(9) cells and 270-390 microCi (9.99-14.43 MBq) {sup 111}In, or 11-17 microCi (0.41-0.63 MBq) per 10(8) lymphocytes. Gamma imaging showed transient lung uptake and significant retention of radioactivity in the liver and spleen. Progressive uptake of activity in normal, nonpalpable axillary and inguinal lymph nodes was seen from 24 to 96 hr. Accumulation of radioactivity also was demonstrated at the forearm skinmore » test site, as well as in its associated epitrochlear and axillary lymph nodes, in a subject who had been tested for delayed hypersensitivity with tetanus toxoid. Indium-111-oxine labeled human lymphocytes may provide a useful tool for future studies of normal and abnormal lymphocyte traffic.« less
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Shariati, Gholam Reza; Ahangari, Ghasem; Hossein-nezhad, Arash; Asadi, Seyed Mohammad; Pooyafard, Farzaneh; Ahmadkhaniha, Hamid Reza
2009-09-01
Serotonin receptors are involved in pathophysiology of schizophrenia and may mediate other neurotransmitter effects. We investigated serotonin receptors gene expression in peripheral blood mononuclear cells (PBMC) of naïve schizophrenic patients, before and after treatment. Also serotonin receptor gene expression was compared in two treatment groups including Haloperidol and Olanzapine. The PBMC was separated from whole blood by Ficoll-hypaque. The total cellular RNA was extracted and the cDNA was synthesized. This process was followed by real-time PCR using primer pairs specific for 5HT(3a) serotonin receptor mRNA and beta-actin as internal control. The results showed the presence of subtype of serotonin receptor in lymphocytes. Serotonin gene expression showed significant changes in Olanzapine treatment group which correlated with Clinical Global Impression (CGI) score improvement. In conclusion, the present study has shown that human PBMC express serotonin receptors 5HT(3a). Moreover, clinical symptom improvement of Olanzapin may be demonstrated by a change in serotonin receptor gene expression.
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Lymphocyte subpopulations of intestinal mucosa in inflammatory bowel disease.
Eade, O E; Andre-Ukena, S S; Moulton, C; MacPherson, B; Beeken, W L
1980-01-01
Lymphocyte subpopulations in peripheral blood (PBL) and intestinal mucosa (IML) of 10 patients with inflammatory bowel disease (IBD) were compared with those of 11 non-IBD controls. PBL were separated on Ficoll/hypaque gradients, and IML were isolated by incubation in dithiothreitol, EDTA, and collagenase. These methods yielded cells of good viability and with intact HLA A and B-antigens. T-cells, identified by neuraminidase-treated sheep RBC rosettes and non-specific esterase staining, comprised approximately 91% of the IML from normal mucosa of all groups. B-cells, identified by erythrocyte-antibody-complement rosettes and surface immunoglobulins, were only 7% of these IML populations. Cell yields were two-fold or more greater from abnormal IBD mucosa, with T-cells ranging from 55 to 95% and B-cells from 2 to 36%. The percentage of Fc receptor bearing cells was low in all specimens. By these methods, T-lymphocytes predominated in intestinal mucosa of both IBD and non-IBD patients, but there is marked increase in the percentage of B-cells isolated from abnormal mucosa in IBD. Images Fig. 1 Fig. 2 Fig. 3 Fig. 11 PMID:6968706
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Immunological responsiveness of frozen-thawed human lymphocytes.
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
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Induction and identification of rabbit peripheral blood derived dendritic cells
NASA Astrophysics Data System (ADS)
Zhou, Jing; Yang, FuYuan; Chen, WenLi
2012-03-01
Purpose: To study a method of the induction of dendritic cells (DCs) from rabbit peripheral blood. Methods: Peripheral blood cells were removed from rabbit, filtered through nylon mesh. Peripheral blood mononuclear cells (PBMC) were isolated from the blood cells by Ficoll-Hypaque centrifugation (density of 1.077g/cm3).To obtain DCs, PBMC were cultured in RPMI1640 medium containing 10% fetal calf serum, 50U/mL penicillin and streptomycin, referred to subsequently as complete medium, at 37°C in 5% CO2 atmosphere for 4 hours. Nonadherent cells were aspirated, adherent cells were continued incubated in complete medium, supplemented with granulocyte/macrophage colony-stimulating factor (GM-CSF, 50ng/ml),and interleukin 4 (IL-4, 50ng/ml) for 9 days. Fluorescein labeled antibodies(anti-CD14, anti-HLA-DR, anti-CD86) were used to sign cells cultured for 3,6,9 days respectively, Then flow cytometry was performed. Results: Ratio of anti-HLA-DR and anti-CD86 labeled cells increased with induction time extension, in contrast with anti-CD14. Conclusion: Dendritic cells can be effectively induced by the method of this experiment, cell maturation status increased with induction time extension.
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Single-sample method for the estimation of glomerular filtration rate in children
DOE Office of Scientific and Technical Information (OSTI.GOV)
Tauxe, W.N.; Bagchi, A.; Tepe, P.G.
1987-03-01
A method for the determination of the glomerular filtration rate (GFR) in children which involves the use of a single-plasma sample (SPS) after the injection of a radioactive indicator such as radioiodine labeled diatrizoate (Hypaque) has been developed. This is analogous to previously published SPS techniques of effective renal plasma flow (ERPF) in adults and children and GFR SPS techniques in adults. As a reference standard, GFR has been calculated from compartment analysis of injected radiopharmaceuticals (Sapirstein Method). Theoretical volumes of distribution were calculated at various times after injection (Vt) by dividing the total injected counts (I) by the plasmamore » concentration (Ct) expressed in liters, determined by counting an aliquot of plasma in a well type scintillation counter. Errors of predicting GFR from the various Vt values were determined as the standard error of estimate (Sy.x) in ml/min. They were found to be relatively high early after injection and to fall to a nadir of 3.9 ml/min at 91 min. The Sy.x Vt relationship was examined in linear, quadratic, and exponential form, but the simpler linear relationship was found to yield the lowest error. Other data calculated from the compartment analysis of the reference plasma disappearance curves are presented, but at this time have apparently little clinical relevance.« less
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[Harringtonine induces apoptosis in NB4 cells through down-regulation of Mcl-1].
Wu, Chunxiao; Shen, Hongqiang; Xia, Dajing
2013-07-01
To investigate the growth inhibition effect, cytotoxicity and apoptotic induction of harringtonine (HT) in human acute promyelocytic leukemia (APL) NB4 cells,and the related mechanism. NB4 cells were treated with HT. Total cell numbers were counted by hemocytometer, and cell viabilities were determined by trypan blue exclusion. Apoptotic cells were determined by fluorescence microscopy and FACS after staining with AO and EB or PI, respectively. The cleavage of PARP and the activation of Bax and the expression of anti-apoptotic proteins were determined by Western Blot. siRNA was used to silence the expression of target genes. Primary cells were isolated following Ficoll-Hypaque density gradient centrifugation method. HT inhibited cell growth and induced apoptosis of NB4 cells in a dose- and time-dependent manner. Apoptosis induced by HT was correlated with the down-regulation of Mcl-1 and the cleavage of PARP, while HT did not affect the protein level of Bax and Bak or change the protein level of Bcl-2. The silence of Bcl-XL sensitized HT-induced apoptosis in NB4 cells.Apoptosis induced by HT in primarily cultured APL cells was also correlated with the down-regulation of Mcl-1. HT inhibits cell growth and induces apoptosis in NB4 cells and primarily cultured APL cells, which may be associated with down-regulation of Mcl-1.
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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
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Apoptosis of lymphocytes in the presence of Cr(V) complexes: role in Cr(VI)-induced toxicity.
Vasant, C; Balamurugan, K; Rajaram, R; Ramasami, T
2001-08-03
Cr(VI) compounds have been declared as a potent occupational carcinogen by IARC (1990) through epidemiological studies among workers in chrome plating, stainless-steel, and pigment industries. Studies relating to the role of intermediate oxidation states such as Cr(V) and Cr(IV) in Cr(VI)-induced carcinogenicity are gaining importance. In this study, issues relating to toxicity elicited by Cr(V) have been addressed and comparisons made with those relating to Cr(VI) employing human peripheral blood lymphocytes. Lymphocytes have been isolated from heparinized blood by Ficoll-Hypaque density gradient centrifugation and exposed to Cr(V) complexes viz. sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V), Na[Cr(V)O(ehba)(2)], 1 and sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V), Na[Cr(V)O(hmba)(2)], 2 and Cr(VI). The phytohemagglutinin (PHA)-induced proliferation of lymphocytes has been found to be inhibited by the two complexes of Cr(V) and chromate Cr(VI) in a time- and concentration-dependent manner. Viability of cells decreases in the presence of Cr(V). Apoptosis appears to be the mode of cell death in the presence of both Cr(V) and Cr(VI). Pretreatment of cells with antioxidants before exposure to chromium(V) complexes reverse apoptosis partially. Possibility for the formation and implication of reactive oxygen species in Cr(V)-induced apoptosis of human lymphocyte cells has been indicated in this investigation. The intermediates of Cr(V) and radical species in the biotoxic pathways elicited by Cr(VI) seems feasible. Copyright 2001 Academic Press.
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Pickering, L K; Cleary, T G; Caprioli, R M
1983-04-01
To compare the effect of human colostrum (days 1 to 3 postpartum) and mature milk (days 170 +/- 24 postpartum) on the function of polymorphonuclear leukocytes (PMNL), Ficoll-Hypaque-separated PMNL from the blood of 60 healthy volunteers were incubated with whole colostrum, colostral lipid, and colostral aqueous phase from 30 mothers, or with mature whole milk and its separated components from 30 mothers, and tested for resting and zymosan-stimulated oxidative metabolism, functional activity, and the presence of Fc receptors. Stimulated oxygen consumption, quantitative nitroblue tetrazolium dye reduction, [1-(14)C]glucose utilization, and Fc receptors were significantly (P < 0.05 to P < 0.001) less in PMNL exposed to whole human colostrum or colostral lipid than in non-lipid-exposed cells or cells exposed to the aqueous phase of colostrum. In contrast, PMNL exposed to whole mature milk or to its lipid or aqueous phase caused no significant decrease in any of these parameters when compared to nonexposed cells. In assays of phagocytosis, colostral PMNL or blood PMNL exposed to colostral lipid had a significant (P < 0.001) decrease in their ability to ingest [methyl-(3)H]thymidine-labeled Staphylococcus aureus when compared to non-lipid-exposed PMNL. Blood PMNL exposed to lipid from mature milk had no decrease in ability to ingest S. aureus. Analysis of total lipid and total and individual fatty acid content revealed a uniform increase in all components in mature milk when compared to colostrum. Lipid or lipid-soluble material present in human colostrum but not mature milk causes inhibition of phagocytosis and respiratory burst-related activities of PMNL.
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Sundaram, S; Cendoroglo, M; Cooker, L A; Jaber, B L; Faict, D; Holmes, C J; Pereira, B J
1997-11-01
Low pH, high osmolality, increasing glucose concentration, and glucose degradation products (GDP) formed during heat sterilization of conventional peritoneal dialysis (PD) fluids have been shown to have a detrimental effect on cells involved in peritoneal host defense. The two-chambered PD fluid bag in which glucose at pH approximately 3 is separated from a bicarbonate (25 mmol/L)-lactate (15 mmol/L) buffer during heat sterilization permits PD fluids with lower GDP to be delivered to the patient at neutral pH. To establish the possible benefit of two-chambered bag PD fluids on peripheral blood mononuclear cell (PBMC) and polymorphonuclear (PMN) cell function, we compared conventional 1.5% Dianeal (1.5%D) with 1.5% two-chambered bag bicarbonate-lactate (1.5%D-B), and conventional 4.25% Dianeal (4.25%D) with 4.25% two-chambered bag bicarbonate-lactate (4.25%D-B). Furthermore, to study the effect of the sterilization process on PBMC and PMN function, we compared filter-sterilized 4.25%D (4.25%D-F) with 4.25%D and 4.25%D-B. PBMC were harvested by Ficoll-Hypaque separation, and 2.5 x 10(6) cells in RPMI were incubated with an equal volume of the test fluids for 4 hours, pelleted, and resuspended in RPMI containing 10 ng endotoxin for a further 20 hours. Tumor necrosis factor alpha (TNF-alpha) production by endotoxin-stimulated PBMC was not significantly different (P = 0.10) between 1.5%D-B and 1.5%D, but was significantly higher (P = 0.01) with 4.25%D-B compared with 4.25%D. PBMC exposed to filter-sterilized fluid (4.25%D-F) showed significantly higher endotoxin-stimulated TNF-alpha production compared with 4.25%D (P = 0.02), but was not significantly different from 4.25%D-B (P = 0.40). PMN were harvested by Ficoll-Hypaque separation and 10 x 10(6) cells incubated with test fluids for 30 minutes. After incubation, phagocytosis (phagocytosis index) was determined by the uptake of 14C-labeled Staphylococcus aureus, oxidative burst by reduction of ferricytochrome C to ferrocytochrome C on stimulation with PMA, and enzyme release by measurement of endotoxin-stimulated bactericidal/permeability increasing protein (BPI). Bicarbonate-lactate two-chambered fluids of similar osmolality and glucose concentration conferred a significant improvement in phagocytosis (P = 0.02 for 1.5%D-B and P < 0.001 for 4.25%D-B). Oxidative burst and BPI release were significantly higher in 4.25%D-B compared with 4.25%D (P < 0.001). Filter-sterilized 4.25%D-F conferred a significant improvement in phagocytosis and oxidative burst compared with 4.25%D (P < 0.001) or 4.25%D-B (P < 0.001). Furthermore, conventional 4.25%D was associated with significantly lower BPI release compared with 4.25%D-F (P = 0.01). GDP's acetaldehyde and 5-HMF were analyzed in 4.25%D-B, 4.25%D, and 4.25%D-F. Acetaldehyde was below the lower limit (0.79 ppm) of the standard curve in 4.25%D-B and 4.25%D-F fluids but was detected (3.76 to 5.12 ppm) in all of the 4.25%D fluids. Relative levels of 5-HMF in the 4.25%D-B (0.032 to 0.041 Abs @ 284 nm) and 4.25%D (0.031 to 0.036 Abs @ 284 nm) were similar. The lowest levels (0.001 Abs @ 284 nm) were observed in the filter-sterilized 4.25%D-F. The beneficial effects of two-chambered bicarbonate lactate-buffered PD fluids on PBMC and PMN function are probably related to reduction of GDP from heat sterilization of glucose in a separate chamber at a lower pH. This improvement in biocompatibility could have a beneficial affect on peritoneal defenses.
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High IL-35 pleural expression in patients with tuberculous pleural effusion.
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.
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Function of beta 2-adrenergic receptors in chronic localized myalgia.
Maekawa, Kenji; Kuboki, Takuo; Inoue, Eitoku; Inoue-Minakuchi, Mami; Suzuki, Koji; Yatani, Hirofumi; Clark, Glenn T
2003-01-01
To investigate alteration of beta 2-adrenergic receptor (beta 2 AR) function in chronic localized myalgia subjects by evaluating levels of the beta 2 AR second messenger, cyclic adenosine monophosphate (cAMP), in mononuclear cells after beta AR-agonist stimulation. Eleven chronic localized myalgia subjects and 21 matched healthy controls participated in this study. Peripheral blood (30 cc) was drawn from the subjects' anterocubital vein. Mononuclear cells were isolated from the total blood by using the Ficoll-Hypaque gradient technique. Basal and stimulated intracellular cAMP levels were determined by enzyme immunoassay using a commercially available kit. Aliquots of 5 x 10(6) cells were incubated with or without stimulation of the beta AR-agonist isoproterenol for 5 minutes. Five different concentrations of isoproterenol (10(-3) M to 10(-7) M) were utilized. cAMP levels in both groups were tested statistically by a 2-way repeated-measures ANOVA with 2 predictors, group difference and isoproterenol concentration difference. As with isoproterenol stimulation, the cAMP responses to forskolin, which activates adenylyl cyclase directly and produces cAMP, bypassing the cell surface receptors were also measured. The basal cAMP levels in both groups (myalgia: 0.33 +/- 0.02 pmol/5 x 10(6) cells; control: 0.43 +/- 0.10 pmol/5 x 10(6) cells) were almost identical, and isoproterenol-produced cAMP levels increased dose-dependently in both groups. No significant differences in the mean cAMP levels were observed between the groups (P = .909). Significant increases were observed according to the isoproterenol concentration increase (P < .0001). The cAMP responses to forskolin stimulation also showed no significant group difference (P = .971). These results suggest that beta 2 AR function is not different between localized myalgia subjects and healthy individuals.
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Muirhead, K A; Wallace, P K; Schmitt, T C; Frescatore, R L; Franco, J A; Horan, P K
1986-01-01
As the diagnostic utility of lymphocyte subset analysis has been recognized in the clinical research laboratory, a wide variety of reagents and cell preparation, staining and analysis methods have also been described. Methods that are perfectly suitable for analysis of smaller sample numbers in the biological or clinical research setting are not always appropriate and/or applicable in the setting of a high volume clinical reference laboratory. We describe here some of the specific considerations involved in choosing a method for flow cytometric analysis which minimizes sample preparation and data analysis time while maximizing sample stability, viability, and reproducibility. Monoclonal T- and B-cell reagents from three manufacturers were found to give equivalent results for a reference population of healthy individuals. This was true whether direct or indirect immunofluorescence staining was used and whether cells were prepared by Ficoll-Hypaque fractionation (FH) or by lysis of whole blood. When B cells were enumerated using a polyclonal anti-immunoglobulin reagent, less cytophilic immunoglobulin staining was present after lysis than after FH preparation. However, both preparation methods required additional incubation at 37 degrees C to obtain results concordant with monoclonal B-cell reagents. Standard reagents were chosen on the basis of maximum positive/negative separation and the availability of appropriate negative controls. The effects of collection medium and storage conditions on sample stability and reproducibility of subset analysis were also assessed. Specimens collected in heparin and stored at room temperature in buffered medium gave reproducible results for 3 days after specimen collection, using either FH or lysis as the preparation method. General strategies for instrument optimization, quality control, and biohazard containment are also discussed.
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Shimojima, Y; Matsuda, M; Ishii, W; Gono, T; Ikeda, S
2008-01-01
Clinical pictures of poly-myalgia rheumatica (PMR) and remitting seronegative symmetrical synovitis with pitting edema (RS3PE) are often indistinguishable from those of early rheumatoid arthritis (RA). To investigate whether there is a difference in immunological aspects among these 3 disorders, we performed a phenotypic analysis of peripheral blood lymphocytes. Eleven patients with early RA, 14 with PMR and 11 with RS3PE were enrolled in this study. After separation of mononuclear cells from peripheral blood using the Ficoll-Hypaque method, surface markers and intracellular cytokines of lymphocytes were analyzed by 2- or 3-color flow cytometry. Both PMR and RS3PE showed a significant decrease in CD8+CD25+ cells (p<0.05), and significant increases in CD4+IFN-gamma+IL-4- (p<0.05), CD8+IFN-gamma+IL-4- (p<0.05 and p<0.01, respectively) and CD4+TNF-alpha+ cells (p<0.05) compared with early RA. CD3+CD4+ cells were higher in PMR than in RS3PE (p<0.01), but there were no significant differences in any other phenotypes between these disorders. A decrease in activated cytotoxic/suppressor T cells and increases in circulating Th1 and Tc1 cells may be common characteristics of PMR and RS3PE in comparison with early RA. Both disorders are clearly different from early RA, and probably belong to the same disease entity with regard to phenotypes of peripheral blood lymphocytes.
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Streck, R J; Hurley, E L; Epstein, D A; Pauly, J L
1992-01-01
We report a simple and efficient culture procedure for the generation of tumour-cytolytic human monocyte-derived macrophages (MAC). In this method, normal human peripheral blood mononuclear cells, isolated using a conventional Ficoll-Hypaque density gradient procedure, are cultured as a heterogenous leukocyte population in Teflon or other hydrophobic cultureware, in a commercially available serum-free culture medium (M-SFM) that has been formulated specifically for the cultivation and ex vivo stimulation of human monocytes and MAC, and in the absence of exogenous mitogens, antigens, cytokines or other stimulants. This procedure features a negative-selection technique that takes advantage of the differential survival of blood leukocytes. Using the prescribed in vitro conditions, lymphocytes survived relatively poorly, whereas monocytes differentiated in the absence of exogenous stimulants into mature tumour-cytolytic MAC. The MAC were present as non-adherent, single cells that expressed good viability (greater than 95%) for a prolonged period (greater than 60 days). When compared to conventional procedures for generating MAC, the prescribed technique is thought to offer several important advantages in that it: (a) eliminates the tedious and cumbersome monocyte isolation procedures, thus providing a significant savings not only in time and money but also in eliminating repetitive cell manipulations that have often been associated with damage to monocyte morphology and/or function; (b) reduces the loss of monocyte subsets that are not recovered during specific isolation procedures; (c) facilitates harvesting a single cell, non-adherent suspension of immunocompetent MAC suitable for various examinations including analyses defining MAC morphology, cytochemistry, phenotype and function; and (d) eliminates variability and artifacts associated with different sera that are utilised frequently as medium supplements. The utility of the prescribed method is illustrated by the results of ongoing studies in which scanning electron microscopy and confocal laser scanning microscopy are being used to define MAC function in different immunological reactions, and examples of these observations are presented herein.
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Bone marrow uptake of 99mTc-MIBI in patients with multiple myeloma.
Fonti, R; Del Vecchio, S; Zannetti, A; De Renzo, A; Di Gennaro, F; Catalano, L; Califano, C; Pace, L; Rotoli, B; Salvatore, M
2001-02-01
In a previous study, we showed the ability of technetium-99m methoxyisobutylisonitrile (99mTc-MIBI) scan to identify active disease in patients with multiple myeloma (Eur J Nucl Med 1998; 25: 714-720). In particular, a semiquantitative score of the extension and intensity of bone marrow uptake was derived and correlated with both the clinical status of the disease and plasma cell bone marrow infiltration. In order to estimate quantitatively 99mTc-MIBI bone marrow uptake and to verify the intracellular localization of the tracer, bone marrow samples obtained from 24 multiple myeloma patients, three patients with monoclonal gammopathy of undetermined significance (MGUS) and two healthy donors were studied for in vitro uptake. After centrifugation over Ficoll-Hypaque gradient, cell suspensions were incubated with 99mTc-MIBI and the uptake was expressed as the percentage of radioactivity specifically retained within the cells. The cellular localization of the tracer was assessed by micro-autoradiography. Twenty-two out of 27 patients underwent 99mTc-MIBI scan within a week of bone marrow sampling. Whole-body images were obtained 10 min after intravenous injection of 555 MBq of the tracer; the extension and intensity of 99mTc-MIBI uptake were graded using the semiquantitative score. A statistically significant correlation was found between in vitro uptake of 99mTc-MIBI and both plasma cell infiltration (Pearson's coefficient of correlation r=0.69, P<0.0001) and in vivo score (Spearman rank correlation coefficient r=0.60, P<0.01). No specific tracer uptake was found in bone marrow samples obtained from the two healthy donors. Micro-autoradiography showed localization of 99mTc-MIBI inside the plasma cells infiltrating the bone marrow. Therefore, our findings show that the degree of tracer uptake both in vitro and in vivo is related to the percentage of infiltrating plasma cells which accumulate the tracer in their inner compartments.
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Sheikhi, A; Shakerian, M; Giti, H; Baghaeifar, M; Jafarzadeh, A; Ghaed, V; Heibor, M R; Baharifar, N; Dadafarin, Z; Bashirpour, G
2016-06-01
There are some evidences for the immunomodulation disorders in the response to intestinal microbiota in inflammatory bowel disease. Yogurt is a fermented milk product made with a starter culture consisting of different probiotics which could be colonized in intestine. However, the role of probiotics in the aetiopathogenesis of ulcerative colitis (UC) has not been clarified. To determine how the immune system responds to these bacteria this study was planned. Bifidobacterium lactis BB-12 (B. lactis) and Lactobacillus acidophilus LA-5 (L. acidophilus) were cultivated on MRS broth. PBMCs of 36 UC patients were separated by Ficoll-Hypaque centrifugation and co-cultured with different concentrations of UV killed bacteria in RPMI-1 640 plus 10% FCS for 48/72 h. IL-10, TGF-β, IFN-γ and TNF-α were measured in supernatant of PBMCs by ELISA. Both bacteria significantly augmented IL-10, TGF-β, IFN-γ and TNF-α compared to control (p<0.001). The secretion levels of IL-10 and TGF-β by B. lactis- compared to L. acidophilus-stimulated PBMCs were significantly higher (p<0.05, p<0.01 respectively). The secretion levels of TNF-α and IFN-γ by PBMCs after 72 h were significantly lower compared to 48 h stimulation by B. lactis (p<0.001, p<0.035 respectively). These data show that both probiotics may trigger the pro- and anti-inflammatory immune response of UC patients. It seems that IL-10/TGF-β uprising by B. lactis could be the reason of TNF-α/IFN-γ reduction. Therefore albeit B. lactis still stimulates the effector Th cells but because of more stimulatory effect on Tregs, it could be a good potential therapeutic candidate for further investigation. © Georg Thieme Verlag KG Stuttgart · New York.
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Zhu, Yi; Liang, Liang; Qian, Dan; Yu, Hongsong; Yang, Peizeng; Lei, Bo
2013-01-01
Purpose To investigate Toll-like receptor (TLR) expression and reactivity in patients with the wet form age-related macular degeneration (AMD). Methods Blood samples were collected from 25 patients with wet AMD and 25 age-matched healthy controls. Peripheral blood mononuclear cells (PBMCs) were isolated with Ficoll-Hypaque density gradient centrifugation. Expression of TLR1 to TLR10 mRNAs in PBMCs from 15 patients with wet AMD and 15 controls was assessed with real-time PCR. TLR2 and TLR3 protein levels in PBMCs from six patients with wet AMD and six controls were measured with flow cytometry. After PBMCs were stimulated with peptidoglycan (PGN) and poly(I:C), the specific ligands of TLR2 and TLR3, cytokines interleukin-6 (IL-6), IL-8, VEGF, and monocyte chemoattractant protein-1 (MCP-1) production in 11 patients with wet AMD and 11 controls were assessed. Results TLR2 and TLR3 mRNA and protein expression in the PBMCs of the patients with wet AMD was significantly higher than that in the controls. However, the difference in TLR1 and TLR4–10 mRNA expression between the two groups was not significant. The PBMCs of the patients with wet AMD produced more IL-6 and IL-8 proteins than the controls in response to PGN, a ligand for TLR2, and more IL-6 protein than the controls in response to poly(I:C), the ligand for TLR3. However, there was no significant difference in vascular endothelial growth factor and monocyte chemoattractant protein-1 production between the wet AMD group and the control group when the PBMCs were stimulated with PGN or poly(I:C). Conclusions Our data suggested that upregulation of TLR2 and TLR3 may be associated with the pathogenesis of wet AMD. PMID:23946637
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Kim, Yong-Man; Jung, Min-Hyung; Song, Ha-Young; Yang, Hyun Ok; Lee, Sung-Tae; Kim, Jong-Hyeok; Kim, Young-Tak; Nam, Joo-Hyun; Mok, Jung-Eun
2005-02-01
This study was designed to establish a more effective and safe culture system for adoptive immunotherapy by investigating the use of homologous cord blood plasma (HCBP) instead of fetal bovine serum (FBS), which has various limitations including ethical problems for the ex vivo expansion of human umbilical T lymphocytes. Fresh human umbilical mononuclear cell fractions were isolated by Ficoll-Hypaque density centrifugation. Nonadherent mononuclear cell fractions were cultured with anti-CD3 antibody (5 microg/ml), IL-2 (175 U/ml), and either 10% FBS or 10% HCBP. On day 8, the cellular proliferation rate and cell surface markers were assessed. There was no significant difference in proliferation when human umbilical cord blood T lymphocytes were grown in medium supplemented with FBS or HCBP (p > 0.05). In medium containing FBS, the proportion of CD3(+)CD4(+) (markers for helper T cell), CD3(+)CD8(+) (cytotoxic T cell), CD3(+)CD25(+) (activated T cell), CD3(+)CD38(+) (immature T cell), and CD3(+)CD45RO(+) (memory T cell) cells was significantly increased (p < 0.05), whereas proportion of CD3(+)CD45RA(+) (naive T cell) and CD16(+)CD56(+) (NK cell) cells was significantly decreased (p < 0.05). In HCBP supplemented medium, the proportion of CD3(+)CD8(+), CD3(+)CD25(+), CD3(+)CD45RA(+), and CD3(+)CD45RO(+) cells was significantly increased (p < 0.05). The proportion of CD3(+)CD4(+), CD3(+)CD45RO(+) and CD3(+)CD38(+) cells was significantly higher, but proportion of CD3(+)CD45RA(+) and CD3(+)CD8(+) cells was significantly lower in FBS compared with HCBP supplemented medium (p < 0.05). Our results support the feasibility of ex vivo expansion of human umbilical cord blood T lymphocytes in medium supplemented with HCBP for future adoptive cellular immunotherapy.
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Zhou, Naibao; Fu, Zhijian; Li, Hao; Wang, Kaiguo
2017-04-01
Ketamine has been used as an analgesic adjuvant with morphine in the treatment of refractory cancer pain recently. But both morphine and ketamine have been reported to produce a number of immunomodulatory effects. The current study was performed to assess whether the concentration of ketamine, as adjuvant analgesics for patient with refractory cancer pain, was related to its effect on T cells interleukin-2 (IL-2)/interferon-γ (IFN-γ) expression in vitro. Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood of patients with refractory cancer pain over a Ficoll-Hypaque density gradient. T cells were isolated from by positive selection using anti-CD3 beads. T cells were then treated with vehicle (C group), morphine (200 ng/mL, M group), morphine (200 ng/mL), and different dose of ketamine (100, 200, 1000 ng/mL; MK1, MK5, MK10 group) for 24 hours before stimulation with anti-CD3 and anti-CD28. Then supernatant IL-2 and IFN-γ protein analysis, quantitative reverse transcription polymerase chain reaction (RT-PCR) for IL-2 and IFN-γ were done. There were no significant difference of supernatant IL-2 and IFN-γ among C group, M group, and MK1 group, but the mRNA of M group and MK1 group were decreased compared with C group (P < .05). Compared with C group, both of the supernatant protein and the mRNA of MK5 group and MK10 group were all significantly decreased (P < .01). Compared with M group, both of the supernatant protein and the mRNA of MK5 group and MK10 group were all decreased (P < .05), while supernatant IL-2 and the mRNA of MK10 group were significantly decreased (P < .01). In conclusion, we confirmed that just as morphine, ketamine dose-dependently suppressed IL-2 and IFN-γ of activated T lymphocyte of patients with refractory cancer pain in vitro, but the inhibitory action of low dose ketamine could be neglected.
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Zhou, Naibao; Fu, Zhijian; Li, Hao; Wang, Kaiguo
2017-01-01
Abstract Background: Ketamine has been used as an analgesic adjuvant with morphine in the treatment of refractory cancer pain recently. But both morphine and ketamine have been reported to produce a number of immunomodulatory effects. The current study was performed to assess whether the concentration of ketamine, as adjuvant analgesics for patient with refractory cancer pain, was related to its effect on T cells interleukin-2 (IL-2)/interferon-γ (IFN-γ) expression in vitro. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood of patients with refractory cancer pain over a Ficoll-Hypaque density gradient. T cells were isolated from by positive selection using anti-CD3 beads. T cells were then treated with vehicle (C group), morphine (200 ng/mL, M group), morphine (200 ng/mL), and different dose of ketamine (100, 200, 1000 ng/mL; MK1, MK5, MK10 group) for 24 hours before stimulation with anti-CD3 and anti-CD28. Then supernatant IL-2 and IFN-γ protein analysis, quantitative reverse transcription polymerase chain reaction (RT-PCR) for IL-2 and IFN-γ were done. Results: There were no significant difference of supernatant IL-2 and IFN-γ among C group, M group, and MK1 group, but the mRNA of M group and MK1 group were decreased compared with C group (P < .05). Compared with C group, both of the supernatant protein and the mRNA of MK5 group and MK10 group were all significantly decreased (P < .01). Compared with M group, both of the supernatant protein and the mRNA of MK5 group and MK10 group were all decreased (P < .05), while supernatant IL-2 and the mRNA of MK10 group were significantly decreased (P < .01). Conclusion: In conclusion, we confirmed that just as morphine, ketamine dose-dependently suppressed IL-2 and IFN-γ of activated T lymphocyte of patients with refractory cancer pain in vitro, but the inhibitory action of low dose ketamine could be neglected. PMID:28422864
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Bisping, G; Lügering, N; Lütke-Brintrup, S; Pauels, H -G; Schürmann, G; Domschke, W; Kucharzik, T
2001-01-01
Intestinal epithelial cells seem to play a key role during IBD. The network of cellular interactions between epithelial cells and lamina propria mononuclear cells is still incompletely understood. In the following co-culture model we investigated the influence of intestinal epithelial cells on cytokine expression of T cytotoxic and T helper cells from patients with IBD and healthy controls. Peripheral blood mononuclear cells (PBMC) were purified by a Ficoll–Hypaque gradient followed by co-incubation with epithelial cells in multiwell cell culture insert plates in direct contact as well as separated by transwell filters. We used Caco-2 cells as well as freshly isolated colonic epithelia obtained from surgical specimens. Three-colour immunofluorescence flow cytometry was performed after collection, stimulation and staining of PBMC with anti-CD4, anti-CD8, anti-IFN-γ and anti-IL-4. Patients with IBD (Crohn's disease (CD), n = 12; ulcerative colitis (UC), n = 16) and healthy controls (n = 10) were included in the study. After 24 h of co-incubation with Caco-2 cells we found a significant increase of IFN-γ-producing CD8+ lymphocytes in patients with IBD. In contrast, healthy controls did not respond to the epithelial stimulus. No significant differences could be found between CD and UC or active and inactive disease. A significant increase of IFN-γ+/CD8+ lymphocytes in patients with UC was also seen after direct co-incubation with primary cultures of colonic crypt cells. The observed epithelial–lymphocyte interaction seems to be MHC I-restricted. No significant epithelial cell-mediated effects on cytokine expression were detected in the PBMC CD4+ subsets. Patients with IBD—even in an inactive state of disease—exert an increased capacity for IFN-γ induction in CD8+ lymphocytes mediated by intestinal epithelial cells. This mechanism may be important during chronic intestinal inflammation, as in the case of altered mucosal barrier function epithelial cells may become targets for IFN-γ-producing CD8+ lymphocytes. PMID:11167992
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The Action of Red Cell Calcium Ions on Human Erythrophagocytosis in Vitro
Romero, Pedro J.; Hernández-Chinea, Concepción
2017-01-01
In the present work we have studied in vitro the effect of increasing red cell Ca2+ ions on human erythrophagocytosis by peripheral monocyte-derived autologous macrophages. In addition, the relative contribution to phagocytosis of phosphatidylserine exposure, autologous IgG binding, complement deposition and Gárdos channel activity was also investigated. Monocytes were obtained after ficoll-hypaque fractionation and induced to transform by adherence to glass coverslips, for 24 h at 37°C in a RPMI medium, containing 10% fetal calf serum. Red blood cells (RBC) were loaded with Ca2+ using 10 μM A23187 and 1 mM Ca-EGTA buffers, in the absence of Mg2+. Ca2+-loaded cells were transferred to above coverslips and incubated for 2 h at 37°C under various experimental conditions, after which phagocytosis was assessed by light microscopy. Confirming earlier findings, phagocytosis depended on internal Ca2+. Accordingly; it was linearly raised from about 2–15% by increasing the free Ca2+ content of the loading solution from 0.5 to 20 μM, respectively. Such a linear increase was virtually doubled by the presence of 40% autologous serum. At 7 μM Ca2+, the phagocytosis degree attained with serum was practically equal to that obtained with either 2 mg/ml affinity-purified IgG or 40% IgG-depleted serum. However, phagocytosis was reduced to levels found with Ca2+ alone when IgG-depleted serum was inactivated by heat, implying an involvement of complement. On the other hand, phagocytosis in the absence of serum was markedly reduced by preincubating macrophages with phosphatidylserine-containing liposomes. In contrast, a similar incubation in the presence of serum affected it partially whereas employing liposomes made only of phosphatidylcholine essentially had no effect. Significantly, the Gárdos channel inhibitors clotrimazole (2 μM) and TRAM-34 (100 nM) fully blocked serum-dependent phagocytosis. These findings show that a raised internal Ca2+ promotes erythrophagocytosis by independently triggering phosphatidylserine externalization, complement deposition and IgG binding. Serum appeared to stimulate phagocytosis in a way dependent on Gárdos activity. It seems likely that Ca2+ promoted IgG-binding to erythrocytes via Gárdos channel activation. This can be an important signal for clearance of senescent human erythrocytes under physiological conditions. PMID:29255426
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Babych, H; Antonyak, H; Sklyarov, A Y
2000-06-01
To investigate the participation of thyroxine in the regulation of energy metabolism in neutrophilic polymorphonuclear leukocytes and their bone marrow precursors. The influence of L-thyroxine (T4; 4 mg/kg every 12 hr from day 2 to 10 of age) was estimated on the activity of hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G-6-PDH), NADP-dependent isocitrate dehydrogenase (ICDH) and cytochrome C-oxidase in bone marrow myeloid cells and circulating neutrophils of 3, 5 and 10 day (d) old piglets. Serum T4 and 3,5, 3'-triiodothyronine (T3) concentrations were estimated at every stage of experiment by radioimmunoassay. Bone marrow cells of myeloid lineage and blood neutrophilic polymorphonuclear leukocytes were separated by differential centrifugation of haematopoietic cell suspension using Ficoll-Hypaque gradients. The hyperthyroid status resulted in significant increase in PFK and LDH activity in myelokaryocytes of 3 and 3-5 d piglets, while the activity of HK and PK in the cells of 3-10 d animals remained unchanged. Moreover, ICDH activity in myelokaryocytes increased on day 10 and that of cytochrome C oxidase in bone marrow cells at all intervals. Marked increase in HK and LDH activity on day 3-5 was found also in blood polymorphonuclear granulocytes, while PFK and PK activity was increased during the whole period. At the same time even the increase in ICDH and cytochrome C-oxidase activity was observed, respectively, in 3 and 5-10 d old piglet neutrophils. Besides that, T4 inhibited G-6-PDH activity in myeloid cells on day 3 to 10 and did not influence the enzyme activity in circulating leukocytes. The administration of T4 resulted in preferential stimulation of oxidative stages of carbohydrate catabolism in myelocaryocytes, while the activity of glycolytic enzymes in these cells was less affected. On the contrary, the enzymes of glycolysis in blood neutrophils showed higher sensitivity to T4 action as compared to catalysts of oxidative reactions. The intensity of pentose phosphate pathway seems to be inhibited in bone marrow myelocaryocytes of T4 treated animals, while that in blood leukocytes remained unaffected.
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The Action of Red Cell Calcium Ions on Human Erythrophagocytosis in Vitro.
Romero, Pedro J; Hernández-Chinea, Concepción
2017-01-01
In the present work we have studied in vitro the effect of increasing red cell Ca 2+ ions on human erythrophagocytosis by peripheral monocyte-derived autologous macrophages. In addition, the relative contribution to phagocytosis of phosphatidylserine exposure, autologous IgG binding, complement deposition and Gárdos channel activity was also investigated. Monocytes were obtained after ficoll-hypaque fractionation and induced to transform by adherence to glass coverslips, for 24 h at 37°C in a RPMI medium, containing 10% fetal calf serum. Red blood cells (RBC) were loaded with Ca 2+ using 10 μM A23187 and 1 mM Ca-EGTA buffers, in the absence of Mg 2+ . Ca 2+ -loaded cells were transferred to above coverslips and incubated for 2 h at 37°C under various experimental conditions, after which phagocytosis was assessed by light microscopy. Confirming earlier findings, phagocytosis depended on internal Ca 2+ . Accordingly; it was linearly raised from about 2-15% by increasing the free Ca 2+ content of the loading solution from 0.5 to 20 μM, respectively. Such a linear increase was virtually doubled by the presence of 40% autologous serum. At 7 μM Ca 2+ , the phagocytosis degree attained with serum was practically equal to that obtained with either 2 mg/ml affinity-purified IgG or 40% IgG-depleted serum. However, phagocytosis was reduced to levels found with Ca 2+ alone when IgG-depleted serum was inactivated by heat, implying an involvement of complement. On the other hand, phagocytosis in the absence of serum was markedly reduced by preincubating macrophages with phosphatidylserine-containing liposomes. In contrast, a similar incubation in the presence of serum affected it partially whereas employing liposomes made only of phosphatidylcholine essentially had no effect. Significantly, the Gárdos channel inhibitors clotrimazole (2 μM) and TRAM-34 (100 nM) fully blocked serum-dependent phagocytosis. These findings show that a raised internal Ca 2+ promotes erythrophagocytosis by independently triggering phosphatidylserine externalization, complement deposition and IgG binding. Serum appeared to stimulate phagocytosis in a way dependent on Gárdos activity. It seems likely that Ca 2+ promoted IgG-binding to erythrocytes via Gárdos channel activation. This can be an important signal for clearance of senescent human erythrocytes under physiological conditions.
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B7-H1 expression is associated with expansion of regulatory T cells in colorectal carcinoma
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
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B7-H1 expression is associated with expansion of regulatory T cells in colorectal carcinoma.
Hua, Dong; Sun, Jing; Mao, Yong; Chen, Lu-Jun; Wu, Yu-Yu; Zhang, Xue-Guang
2012-03-07
To investigate the expression of B7-H1 in human colorectal carcinoma (CRC) to define its regulating effects on T cells in tumor microenvironment. 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. 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-H1(high) CRC tissues and 18 cases of B7-H1(low) 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-H1(high) CRC tissues than in B7-H1(low) 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. 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.