pha stimulation
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Author(s):  
V.P. Laptev ◽  
◽  
M.I. Kiselevsky ◽  
A.E. Ovchinnikov ◽  
N.G. Panfyorova ◽  
...  

We expiremented with mice inoculated with intramuscularly embryocarcinoma and registered functional condition of T-lymphocytic link of cells of bone brain and spleen to the index of spontaneous and stimulated by mitogen (PHA) proliferation. Intact animals and the ones which we introduced with Chlorine e6 and keep in dark conditions have revealed the zero level of the indexes. But the animals being introduced with Chlorine e6 and activated with laser and light-emitting diode radiation have been notable for the index of mitogen (PHA) stimulation. It was established to 25% with the index of spontaneous proliferation remained on zero mark. Animals with embryocarcinoma after FDT with X-e6 with or without glycosamine show immunostimulating effect.


2020 ◽  
Vol 13 ◽  
pp. 117864692097823
Author(s):  
Rona Kartika ◽  
Heri Wibowo ◽  
Dyah Purnamasari ◽  
Saraswati Pradipta ◽  
Rahma A Larasati

Aim: To analyze indoleamine 2,3-dioxygenase (IDO) production in the cell culture supernatant of phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs) from type 2 DM (T2DM) patients and investigate IDO’s association to pro- and anti-inflammatory cytokines. Subjects and methods: PBMC samples were collected from 21 T2DM patients and 17 normoglycemic participants, then stimulated with PHA for 3 days. Cytokine and IDO concentrations were measured in the PBMC culture supernatants. In vitro production of TNF-α, IL-6, interferon-γ, and IL-10 were measured using multiplex immunoassay. IDO concentration was assessed using ELISA. To assess how PHA stimulation altered IDO production and to minimize the unstimulated baseline effect of T2DM, we subtracted the PHA-stimulated IDO concentration from the unstimulated one. IBM SPSS version 23 was used for statistical analysis. Results: The IDO concentrations in the PBMC culture supernatants were significantly higher in T2DM patients regardless of whether they were unstimulated ( P < .001) or PHA-stimulated ( P = .012). Reduced IDO production was observed in 52.8% of T2DM patients and was associated with older age and lower interferon-γ levels. Conversely, 42.8% of T2DM patients showed increased IDO concentrations, which were correlated with the IL-6/IL-10 ratio ( r = 0.683, P = .021) and interferon-γ/IL-10 ratio ( r = 0.517, P = .077). Conclusion: The interferon-γ level was reduced in the PBMC culture supernatant of T2DM patients with reduced IDO production. Reduced IDO production in T2DM patients following PHA stimulation was associated with older age and, notably, higher baseline IDO concentrations. Since IDO is primarily produced by dendritic cells, reduced IDO production after PHA stimulation may indicate dendritic cell dysfunction.


Mutagenesis ◽  
2019 ◽  
Vol 34 (3) ◽  
pp. 217-237 ◽  
Author(s):  
James Whitwell ◽  
Robert Smith ◽  
Teresa Chirom ◽  
Gary Watters ◽  
Victoria Hargreaves ◽  
...  

Abstract The in vitro micronucleus (IVMN) test was endorsed for regulatory genotoxicity testing with adoption of the Organisation for Economic Co-operation and Development (OECD) test guideline (TG) 487 in 2010. This included two equally acceptable options for extended treatment in the absence of metabolic activation: a treatment for 1.5–2.0 cell cycles with harvest at the end of treatment (Option A) or treatment for 1.5–2.0 cell cycles followed by recovery for 1.5–2.0 cell cycles prior to harvest (Option B). Although no preferences were discussed, TG 487 cautions that Option B may not be appropriate for stimulated lymphocytes where exponential growth may be declining at 96 h after phytohaemagglutinin (PHA) stimulation. Following revision of TG 487 in 2014 and 2016, emphasis has been placed on using Option A. Given the purpose of the IVMN assay is to determine both clastogenic and aneugenic potential, the authors believe the assay is compromised if an extended treatment with recovery is not included for sensitive detection of certain classes of chemical. In this study, average generation time (via bromodeoxyuridine incorporation) of human peripheral blood lymphocytes (HPBL) was measured up to 144 h after PHA stimulation. In addition, the HPBL micronucleus (MN) assay was performed using Option A and B treatment schedules. Cytotoxicity (replication index) and MN induction were determined following treatment with 14 chemicals. The data demonstrate that lymphocytes actively divide beyond 96 h after PHA stimulation. Furthermore, MN induction was only observed with some aneugenic chemicals and nucleoside analogues in HPBLs following extended treatment with a recovery period. For the majority of chemicals tested the magnitude of MN induction was generally greater and MN induction was observed across a wider concentration range following the Option B treatment schedule. In addition, steep concentration-related toxicity following treatment without recovery is more common, making selection of suitable concentrations (within regulatory toxicity limits) for MN analysis challenging.


2017 ◽  
Vol 118 (11) ◽  
pp. 942-948
Author(s):  
Kristoffer J. Jensen ◽  
Mia J. Søndergaard ◽  
Andreas Andersen ◽  
Cesario Martins ◽  
Christian Erikstrup ◽  
...  

AbstractHigh-dose vitamin A supplementation (VAS) may affect mortality to infectious diseases in a sex-differential manner. Here, we analysed the long-term immunological effects of neonatal vitamin A supplementation (NVAS) in 247 children, who had been randomly allocated to 50 000 or 25 000 IU vitamin A (15mg and 7·5mg retinol equivalents, respectively) or placebo at birth. At 4–6 months of age, we assessed bacille Calmette–Guérin (BCG) scarification, and we analysed in vitro responses of TNF-α, IL-5, IL-10, IL-13 and IFN-γ in whole blood stimulations to phytohaemagglutinin (PHA), purified protein derivative (PPD), tetanus toxoid and lipopolysaccharide. There were no differences between the two doses of NVAS, and thus they were analysed combined as NVAS (any dose) v. placebo. All analyses were performed unstratified and by sex. NVAS increased the chance of having a scar after BCG vaccination in females (NVAS v. placebo: 96 v. 71 %, proportion ratio: 1·24; 95 % CI 1·09, 1·42), but not in males (Pfor interaction=0·012). NVAS was associated with significant sex-differential effects on the pro- to anti-inflammatory cytokine ratios (TNF-α:IL-10) to PPD, tetanus toxoid and medium alone, which were increased in females but decreased in males. In addition, IL-17 responses tended to be increased in NVAS v. placebo recipients in males but not in females, significantly so for the PHA stimulation. The study corroborates sex-differential effects of VAS on the immune system, emphasising the importance of analysing VAS effects by sex.


2016 ◽  
Vol 68 (3) ◽  
pp. 509-513 ◽  
Author(s):  
Chunhe Yang ◽  
Hongwu Du

The hierarchical clustering method has been used for exploration of gene expression and proteomic profiles; however, little research into its application in the examination of expression of multiplecytokine/chemokine responses to stimuli has been reported. Thus, little progress has been made on how phytohemagglutinin(PHA) affects cytokine expression profiling on a large scale in the human hematological system. To investigate the characteristic expression pattern under PHA stimulation, Luminex, a multiplex bead-based suspension array, was performed. The data set collected from human peripheral blood mononuclear cells (PBMC) was analyzed using the hierarchical clustering method. It was revealed that two specific chemokines (CCL3 andCCL4) underwent significantly greater quantitative changes during induction of expression than other tested cytokines/chemokines after PHA stimulation. This result indicates that hierarchical clustering is a useful tool for detecting fine patterns during exploration of biological data, and that it can play an important role in comparative studies.


Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2196-2196
Author(s):  
Enrico Morello ◽  
Vladia Monsurrò ◽  
Elisabetta Pagani ◽  
Irene Cavattoni ◽  
Silvia Coin ◽  
...  

Abstract Background. GVHD is associated with a high morbidity and mortality in alloSCT patients. An early diagnosis of GVHD could reduce this adverse impact on the outcome of alloSCT. The effect of Th1 cytokine IFN-γ is crucial in the pathogenesis of GVHD and, as expected, higher protein levels are reported in the serum of patients with active chronic GVHD. The monitoring of IFN-γ basal levels as well as IFN-γ induced by mitogen stimulation in the blood samples of patients after alloSCT could help the management and the prediction of GVHD. A recent ELISA based test (QuantiFERON®-CMV) could measure specific (anti-CMV) and aspecific production of IFN-γ in whole blood. The aim of this study is to assess the reliability of the positive control of the QuantiFERON®-CMV kit as new marker for GVHD early diagnosis. Methods. The study was performed in 2 phases. In the first phase of the study, 92 whole blood specimens were collected and analyzed from 29 patients undergoing alloSCT. In the second phase 10 patients were observed prospectively with collection of blood samples every 2–3 weeks since engraftment until 4–6 months after SCT in order to study the PHA stimulated IFN-γ production in relationship with the onset of chronic GVHD. QuantiFERON®-CMV is an in vitro diagnostic test that use an antigenic human cytomegalovirus proteins (CMV) peptide cocktail to stimulate cells from whole blood. The mitogen-stimulated (PHA) plasma sample is used as an IFN-γ positive control for each specimen tested. Detection of interferon-γ (IFN-γ) by ELISA is used to identify responses. In order to assess the association between IFN-γ response due to PHA stimulation and GVHD, the positivity of the test was determined according to 2 different cut-off: #1) 0,5 IU/mL as defined by manufacturer, #2) 9 IU/mL as experimentally defined by the median of the observations in our data set. GVHD extension was defined by Seattle criteria and/or the number of involved sites, Chi-square test was used to assess the statistical correlation between IFN-γ production and clinical outcomes. Results. In the first phase, among 92 samples 70 were positive for the PHA stimulated IFN-γ production according to the cut-off #1; 61% (43/70) were associated with GVHD whereas 27% (6/22) with lower PHA stimulated IFN-γ production were associated with GVHD: this difference was proved to be significant (p=0.005). According to the cut-off #2 46 samples out of 92 were positive for the PHA stimulated IFN-γ production; 71% (33/46) were associated with GVHD whereas 34% (16/46) with lower PHA stimulated IFN-γ production were associated with GVHD: this difference was proved to be significant (p=0.000). In the second prospective phase of the study, 7/10 patients became positive for the PHA stimulated IFN-γ production after alloSCT: 6 developed subsequently chronic GVHD. The median time to the onset of GVHD was 100 days from the first sample proved positive above the cut-off #1 and 33 days from the first sample proved positive above the cutoff#2. Four patients received steroid treatment for extensive chronic GVHD and their PHA stimulated IFN-γ production dropped after treatment (figure 1, continuous line). Conclusions: The PHA stimulated IFN-γ production is associated with GVHD. The monitoring of PHA stimulated IFN-γ after alloSCT seems to predict the onset of GVHD and could help in the modulation of immunosuppressive treatment. However, larger prospective studies are needed. Figure Figure


2008 ◽  
Vol 55 (2) ◽  
pp. 381-390 ◽  
Author(s):  
Zdenka Vilasová ◽  
Martina Rezácová ◽  
Jirina Vávrová ◽  
Ales Tichý ◽  
Doris Vokurková ◽  
...  

The main aim of this study was to compare the reaction of quiescent and proliferating, i.e. phytohemagglutinin (PHA)-stimulated, human peripheral blood mononuclear cells (PBMCs) to gamma-radiation, and analyse changes of proteins related to repair of DNA damage and apoptosis, such as gammaH2A.X, p53, p53 phosphorylation at serines-15 and -392, and p21 and their dose dependence. Freshly isolated PBMCs in peripheral blood are predominantly quiescent, in G(0) phase, and with very low amounts of proteins p53 and p21. Using confocal microscopy we detected dose dependent (0.5-5 Gy) induction of foci containing gammaH2A.X (1 h after gamma-ray exposure), which are formed around radiation-induced double strand breaks of DNA. Apoptosis was detected from 24 h after irradiation by the dose of 4 Gy onwards by Annexin V binding and lamin B cleavage. Seventy two hours after irradiation 70% of CD3(+) lymphocytes were A(+). Neither increase in p53 nor its phosphorylation on serine-392 after irradiation was detected in these cells. However, massive increase in p21 (cyclin-dependent kinase inhibitor 1A) was detected after irradiation, which can be responsible for late occurrence of apoptosis in these quiescent cells. PHA-stimulation itself (72 h) caused an increase in early apoptosis (A(+)PI(-)) in comparison to non-stimulated PBMCs (38% A(+) resp. 13.4%). After PHA-stimulation also the amount of gammaH2A.X, p53, and p21 increased, but no phosphorylation of p53 on serine-392 or -15 was detected. Reaction to gamma-radiation was different in PHA-stimulated lymphocytes: the p53 pathway was activated and p53 was phosphorylated on serines-15 and -392 4 h after irradiation by the dose of 4 Gy. Phosphorylation of p53 at serine-15 increased in a dose-dependent manner in the studied dose range 0.2-7.5 Gy. Also the amount of p21 increased after irradiation. Seventy two hours after irradiation of PHA-stimulated CD3(+) T lymphocytes by the dose of 4 Gy 65% of cells were A(+).


Blood ◽  
2006 ◽  
Vol 107 (9) ◽  
pp. 3639-3646 ◽  
Author(s):  
Zhi-Zhang Yang ◽  
Anne J. Novak ◽  
Mary J. Stenson ◽  
Thomas E. Witzig ◽  
Stephen M. Ansell

Most non-Hodgkin lymphomas (NHLs) are of B-cell origin, but the tumor tissue can be variably infiltrated with T cells. In the present study, we have identified a subset of CD4+CD25+ T cells with high levels of CTLA-4 and Foxp3 (intratumoral Treg cells) that are overrepresented in biopsy specimens of B-cell NHL (median of 17% in lymphoma biopsies, 12% in inflammatory tonsil, and 6% in tumor-free lymph nodes; P = .001). We found that these CD4+CD25+ T cells suppressed the proliferation and cytokine (IFN-γ and IL-4) production of infiltrating CD4+CD25- T cells in response to PHA stimulation. PD-1 was found to be constitutively and exclusively expressed on a subset of infiltrating CD4+CD25- T cells, and B7-H1 could be induced on intratumoral CD4+CD25+ T cells in B-cell NHL. Anti-B7-H1 antibody or PD-1 fusion protein partly restored the proliferation of infiltrating CD4+CD25- T cells when cocultured with intratumoral Treg cells. Finally, we found that CCL22 secreted by lymphoma B cells is involved in the chemotaxis and migration of intratumoral Treg cells that express CCR4, but not CCR8. Taken together, our results suggest that Treg cells are highly represented in the area of B-cell NHL and that malignant B cells are involved in the recruitment of these cells into the area of lymphoma.


Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2398-2398
Author(s):  
Alexander Roeth ◽  
Lisa Schneider ◽  
Gabriela Baerlocher ◽  
Ulrich Duehrsen

Abstract Telomerase maintains telomere length by adding TTAGGG repeats to the 3′-ends of chromosomes. In human T-cells telomerase is transiently expressed upon activation and stimulation and, as shown previously, telomerase levels are able to control their lifespan. To understand the effects of culture parameters on telomerase activity and cell lifespan is of critical importance to improve T-cell expansion. Here, we investigated the influence of culture conditions and of stimulation on lifespan, clonogenicity (number of positive wells), cell cycle and telomerase activity in T-cells in vitro. Isolated peripheral blood mononuclear cells (PBMCs) from healthy individuals were cultured in RPMI 1640 medium containing IL-2, different sera (10%) (fetal calf serum (FCS), AB serum and autologous serum) and stimulated with phytohaemagglutinin (PHA) and irradiated allogeneic mononuclear feeder cells or anti-CD3/CD28 beads. T-cells were counted and passaged weekly until senescence. Expanded T-cells were analyzed for cell cycle by DNA staining with propidium iodide (PI) at various time points after stimulation and telomerase activity was measured using the telomerase PCR ELISA. Clonogenicity was determined by limiting dilution using different culture conditions. The proliferative lifespan of T-cells expanded with PHA/feeder cells and autologous serum from two different donors was significantly increased compared to T-cells stimulated with PHA/feeder cells and AB serum (44.7 vs. 14.2 PDs; 42.4 vs. 27.4 PDs). In contrast, no or only little difference was found for T-cells expanded with anti-CD3/CD28 beads and autologous or AB serum (8.7 vs. 8.1 PDs; 15.5 vs. 9.6 PDs). Most likely the latter findings reflect the loss of CD28 expression on T-cells. Furthermore, the use of autologous serum also increased the clonogenicity to about 4-fold compared to the use of AB serum or FCS without any signs for differences in the fractions of cycling cells. Interestingly, T-cells cultured with autologous serum exhibited a higher telomerase activity at day 3 after stimulation and a reduced decline of telomerase activity at day 6 and 9 compared to cultures with AB serum. Our results demonstrate that the use of autologous serum combined with PHA stimulation and feeder cells remarkably extends the proliferative lifespan and clonogenicity of human T-cells in vitro. In addition, T-cells cultured in medium containing autologous serum exhibit higher and prolonged telomerase activity compared to cultures with AB serum. Further studies are ongoing to elucidate the underlying factors involved in those observations. Nevertheless, we suggest that autologous serum together with PHA stimulation and feeder cells is superior to expand human T-cells especially for clinical applications where large numbers of specific T-cells are required for adoptive transfer strategies in cancer patients with for example severe viral infections.


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