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Sample records for lymphocyte proliferation assay

  1. A Compartmental Model for Computing Cell Numbers in CFSE-based Lymphocyte Proliferation Assays

    Science.gov (United States)

    2012-01-31

    proliferation, survival and differentiation using CFSE time-series data, Nature Protocols , 2 (2007), 2057–2067. [41] E.D. Hawkins, J.F. Markham, L.P...the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester, Nature Protocols , 2 (2007), 2049– 2056. [61] P. Revy, M. Sospedra

  2. Standardisation and quality assurance of lymphocyte proliferation assays for use in the assessment of immune function. European Concerted Action on Immunological and Virological Markers of HIV Disease Progression.

    Science.gov (United States)

    Froebel, K S; Pakker, N G; Aiuti, F; Bofill, M; Choremi-Papadopoulou, H; Economidou, J; Rabian, C; Roos, M T; Ryder, L P; Miedema, F; Raab, G M

    1999-07-30

    Lymphocyte proliferation is a widely used technique to assess immune competence. However, the technique is subject to a large degree of variation, some biological and some technical. In this study, the components of variation in whole blood proliferation assays were analysed over time, using both antibody and mitogenic stimulants. The levels of variation within individual samples, between individuals and between groups of individuals over time were examined. A method of transforming the data is proposed which reduces the coefficients of variation to an acceptable level, and which expresses individual results as a standardised count. This method overcomes the problem of different levels of absolute counts, it corrects for time sensitive errors and allows data from multiple laboratories to be pooled.

  3. Effect of praziquantel on human lymphocyte proliferation in vitro

    DEFF Research Database (Denmark)

    Odum, Niels; Theander, T G; Bygbjerg, I C

    1984-01-01

    The antischistosomal drugs tartar emetic and niridazole exert immunosuppression both in vitro and in vivo. In the present study the influence of praziquantel (Biltricide), a potent schistosomicidal drug, on human lymphocyte proliferation in vitro was investigated. Praziquantel 80 micrograms...... no suppressive effect on human lymphocyte proliferation in vitro....

  4. Lymphocytes in patients with psoriasis promote proliferation of keratinocytes

    Institute of Scientific and Technical Information of China (English)

    DENG An-mei; ZHONG Ren-qian; CHEN Sun-xiao; ZHOU Ye; KONG Xian-tao

    2002-01-01

    Objective: To analyze the effect of lymphocytes on proliferation of keratinocytes in patients with psoriasis. Methods: Lymphocytes in lesion and peripheral blood were isolated and amplified, then cultured together with normal keratinocytes. By MTT method, the living cells were quantified in the mixed culture.Results: Compared with normal controls, lymphocytes from lesion and peripheral blood of psoriasis both promote the proliferation of keratinocytes (P<0. 01 and P<0. 05 respectively). The concentrations of IL-2 and IFN-γ in the mixture of lesion lymphocytes and keratinocytes were significantly higher than that of controls.Tripterygium glycosides inhibited this promotion. Conclusion: Lymphocytes in patients with psoriasis (mainly Thl cell) play an important role in proliferation of keratinocytes. This psoriasis cell model is useful for studies on signal transduction in psoriasis.

  5. Effect of chloroquine on human lymphocyte proliferation

    DEFF Research Database (Denmark)

    Bygbjerg, Ib Christian; Flachs, H

    1986-01-01

    the response to pokeweed mitogen. The response to concanavalin A and to various antigens was suppressed, especially the response to large particulate antigens. Oral intake of 300 mg of chloroquine base/week did not affect the lymphocyte proliferative responses. 600 mg of base/week decreased the response...

  6. Effect of oral proguanil on human lymphocyte proliferation

    DEFF Research Database (Denmark)

    Bygbjerg, Ib Christian; Flachs, H

    1986-01-01

    In vitro studies have indicated that the antifolates pyrimethamine [4, 6] and cycloguanil (the active metabolite of proguanil) suppress the proliferation of stimulated human lymphocytes; proguanil has no effect [2]. During the early growth phase of the cells, 14C-thymidine (14C-TdR) incorporation...... on human lymphocytes, the present study was undertaken. Little information is available about the serum levels of proguanil and cycloguanil following ingestion of prophylactic doses [8]. Therefore, the serum concentrations of proguanil and cycloguanil were estimated, to allow comparison with previous...

  7. Flow cytometric analysis of T lymphocyte proliferation in vivo by EdU incorporation.

    Science.gov (United States)

    Sun, Xiaojing; Zhang, Chunpan; Jin, Hua; Sun, Guangyong; Tian, Yue; Shi, Wen; Zhang, Dong

    2016-12-01

    Monitoring T lymphocyte proliferation, especially in vivo, is essential for the evaluation of adaptive immune reactions. Flow cytometry-based proliferation assays have advantages in measuring cell division of different T lymphocyte subsets at the same time by multicolor labelling. In this study, we aimed to establish the use of 5-Ethynyl-2'-deoxyuridine (EdU) incorporation in vivo to monitor T lymphocyte proliferation by flow cytometry with an adoptive transfer model. We found that fixation followed by permeabilization preserved T cell surface antigens and had no obvious effects on the fluorescence intensity of APC, PE, PE-Cy7, FITC and PerCP-Cy5.5 when the concentration of the permeabilization reagents was optimized. However, the click reaction resulted in a significant decrease in the fluorescence intensity of PE and PE-Cy7, and surface staining after the click reaction improved the fluorescence intensity. Thus, an extra step of blocking with PBS with 3% FBS between the click reaction and cell surface staining is needed. Furthermore, the percentage of EdU-positive cells increased in a dose-dependent manner, and the saturated dose of EdU was 20mg/kg. Intraperitoneal and intravenous injection had no differences in lymphocyte proliferation detection with EdU in vivo. In addition, T cell proliferation measured by EdU incorporation was comparable to BrdU but was lower than CFSE labelling. In conclusion, we optimized the protocols for EdU administration in vivo and staining in vitro, providing a feasible method for the measurement of T lymphocyte proliferation with EdU incorporation by flow cytometry in vivo.

  8. Bioluminescent assay for human lymphocyte blast transformation.

    Science.gov (United States)

    Bulanova, E G; Budagyan, V M; Romanova, N A; Brovko LYu; Ugarova, N N

    1995-05-01

    One of the basic tests of in vitro evaluation of immune cell functional activity is a proliferative response of lymphocytes on the action of external stimuli such as mitogenic lectines, antigens, etc. We compared two methods used to assess the lymphocyte functional status. (1) [3H]thymidine incorporation and (2) bioluminescence for determination of intracellular ATP in blast cells. Comparison has been done for healthy donors and patients with proven low immunological status. The proposed bioluminescent method for evaluation of the proliferative response was shown to be sensitive enough for diagnostic purposes. This method allows one to process a large number of samples at the same time and correlates highly with the radionuclide test use hazardous radioactive materials.

  9. The immunosuppressive effect of gossypol in mice is mediated by inhibition of lymphocyte proliferation and by induction of cell apoptosis

    Institute of Scientific and Technical Information of China (English)

    Wen-bin XU; Li-hui XU; Hong-song LU; Dong-yun OUYANG; Huan-jing SHI; Jing-fang DI; Xian-hui HE

    2009-01-01

    Aim: To investigate the immunosuppressive effect of gossypol in mice both in vitro and in vivo.Methods: The in vitro effect of gossypol on the proliferation of lymphocytes isolated from lymph nodes of BALB/c mice was determined by CFSE staining and by an MTS assay. Lymphocyte activation and lymphoblastic transformation were evaluated with immunostaining. Cell apoptosis was detected by Annexin-V and Hoechst 33342 staining. The in vivo immunosuppressive effect of gossypol on the DTH reaction was evaluated using a mouse DTH model induced by 2,4-dinitro-1-fluorobenzene (DNFB). The thickness of the ears was measured, and the histological changes of the mouse auricles were observed after hematoxylin-eosin staining. The proliferation capacity of lymphocytes from DTH mice was also assayed.Results: In vitro, gossypol could significantly inhibit the proliferation of mouse lymphocytes stimulated with phorbol ester plus ionomycin in a dose-dependent manner. Although the expression of the early activation antigen CD69 was not affected, the lymphoblastic transformation of both T and B lymphocyte subsets was significantly suppressed by gossypol.Moreover, gossypol could induce apoptosis of lymphocytes, and the effect was time- and dose-dependent. In vivo, the DTH reaction in mice was markedly alleviated by gossypol injected intraperitoneally. Lymphocytes from drug-treated DTH mice had a reduced proliferation capacity as compared with lymphocytes from untreated DTH mice. Gossypol treatment also markedly reduced the number of infiltrated lymphocytes in the auricles of DTH mice. Conclusion: Gossypol exhibited immunosuppressive effects in mice, probably by inhibition of lymphocyte proliferation and by induction of cell apoptosis.

  10. Gymnemic Acid Stimulates In Vitro Splenic Lymphocyte Proliferation.

    Science.gov (United States)

    Singh, Vineet Kumar; Dwivedi, Padmanabh; Chaudhary, B R; Singh, Ramesh

    2016-02-01

    Gymnemic acid is a mixture of triterpenoid saponins of oleanane class, isolated from Gymnema sylvestre Wild R.Br (family: Asclepidaceae), an herbal plant used in traditional medicine to treat diabetes. Effect of gymnemic acid (0.1-20 µg/mL) on in vitro mitogen (concanavalin A and lipopolysaccharide)-induced splenic lymphocyte proliferation was studied using rat as model. Significant (p sylvestre is scientifically supplemented with its immunomodulatory properties.

  11. Effect of catecholamines and thermal exposure on lymphocyte proliferation, IL-1α & β in buffaloes

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    R.C. Upadhyay

    2010-02-01

    Full Text Available In order to study the effect of catecholamines (epinephrine/norepinephrine and thermal exposure on in vitro buffalo Lymphocyte Proliferation (LP apparently healthy 2- 2 1/2 years old Murrah buffalo heifers maintained as per the standard feeding and management practices were selected from Institute herd. Jugular blood was collected in the forenoon on the day of experiment and processed for Total Leucocyte Count (TLC and Differential Leucocyte Count (DLC. Lymphocyte proliferation assays were performed using whole blood and cells were incubated with epinephrine and norepinephrine (1, 1.5,2 ng/ml at 37oC with 5% CO2. Cells were counted after 72 hrs of incubation and Lymphocyte Proliferation Index (LPI was calculated. Thermal stress effect on the cultures was observed after exposure at 45oC for 4 hr after 72hrs of incubation. The cells were separated from media and media was used for analysis of IL-1α & 1β by ELISA kit. Lymphocyte proliferation Index decreased in responses to Epinephrine and Norepinephrine (P<0.01. Concentration of epinephrine and norepinephrine (1, 1.5,2 ng /ml had no distinguishable effect on LPI. IL-1α & IL-1β levels when compared with control in supernatant (exposed to 45°C were low (P<0.01 and P<0.05, respectively. There was a significant positive correlation between LPI and IL-1α (r=0.80; P<0.01 and between LPI and IL-1β (r=0.78; P<0.05. The study indicated that lymphocyte proliferation in vitro and IL-1α & β levels were affected by catecholamines and thermal exposure. Further the levels of catecholamines had significant (P<0.01 negative effect on LPI indicating that catecholamines levels modulate immunity through IL-1α and IL-1β in buffaloes.

  12. DNA strand breaks (comet assay) in blood lymphocytes from wild bottlenose dolphins.

    Science.gov (United States)

    Lee, Richard F; Bulski, Karrie; Adams, Jeffrey D; Peden-Adams, Margie; Bossart, Gregory D; King, Lydia; Fair, Patricia A

    2013-12-15

    The comet assay was carried out on blood lymphocytes from a large number of wild dolphins (71 from Indian River Lagoon, FL, USA; 51 from Charleston Harbor, SC, USA) and provides a baseline study of DNA strand breaks in wild dolphin populations. There were no significant differences in the comet assay (% DNA in tail) results between the different age and sex categories. Significant difference in DNA strand breaks were found between Charleston Harbor dolphins (median--17.4% DNA in tail) and Indian River Lagoon dolphins (median--14.0% DNA in tail). A strong correlation found between T-cell proliferation and DNA strand breaks in dolphin lymphocytes suggests that dolphins with a high numbers of DNA strand breaks have a decreased ability to respond to infection. Higher concentrations of genotoxic agents in Charleston Harbor compared with Indian River lagoon may have been one of the causes of higher DNA strand breaks in these dolphins.

  13. Effect of Piper chaba Hunter, Piper sarmentosum Roxb. and Piper interruptum Opiz. on natural killer cell activity and lymphocyte proliferation.

    Science.gov (United States)

    Panthong, Sumalee; Itharat, Arunporn

    2014-08-01

    Immune system is the most important system ofhuman body. Thaifolk doctors have used some medicinal plants as an adaptogenic drug or immunomodulatory agent. Piper chaba Hunter, Piper sarmentosum Roxb. and Piper interruptum Opiz. are used by folk doctors to activate immune response in cancer patients. To investigate the effect on natural killer cell activity and on lymphocyte proliferation activity of water extract of P chaba Hunter P. sarmentosum Roxb. and P interruptum Opiz. MATERIAL ANDMETHOD: Plant materials were extracted by decoction method. All extracts were testedfor an immunomodulatory effect using PBMCs from twelve healthy donors by chromium release assay. Lymphocyte proliferation was also determined by 3H-thymidine uptake assay. The degree of activation was expressed as the stimulation index. The water extract of P chaba Hunter significantly increased lymphocyte proliferation at concentrations ofl ng/ml, 10 ng/ml, 1 μg/ml, 5 μg/ml, 10 μg/ml and 100 μg/ml. P sarmentosum Roxb., and P interruptum Opiz. extracts at those concentrations significantly stimulated lymphocyteproliferation. P sarmentosum Roxb. extractsignificantly increased natural killer (NK) cell activity at a concentration of 100 μg/ml but P chaba Hunter and P interruptum Opiz. extracts did not significantly stimulate natural killer cell activity. P chaba Hunter, P interruptum Opiz. andP sarmentosum Roxb. have an immunomodulatory effect especially for P sarmentosum Roxb. extract which can activate both lymphocyte proliferation and NK cell activity.

  14. Mycoplasma contamination revisited: mesenchymal stromal cells harboring Mycoplasma hyorhinis potently inhibit lymphocyte proliferation in vitro.

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    Severin Zinöcker

    Full Text Available BACKGROUND: Mesenchymal stromal cells (MSC have important immunomodulatory effects that can be exploited in the clinical setting, e.g. in patients suffering from graft-versus-host disease after allogeneic stem cell transplantation. In an experimental animal model, cultures of rat T lymphocytes were stimulated in vitro either with the mitogen Concanavalin A or with irradiated allogeneic cells in mixed lymphocyte reactions, the latter to simulate allo-immunogenic activation of transplanted T cells in vivo. This study investigated the inhibitory effects of rat bone marrow-derived MSC subsequently found to be infected with a common mycoplasma species (Mycoplasma hyorhinis on T cell activation in vitro and experimental graft-versus-host disease in vivo. PRINCIPAL FINDINGS: We found that M. hyorhinis infection increased the anti-proliferative effect of MSC dramatically, as measured by both radiometric and fluorimetric methods. Inhibition could not be explained solely by the well-known ability of mycoplasmas to degrade tritiated thymidine, but likely was the result of rapid dissemination of M. hyorhinis in the lymphocyte culture. CONCLUSIONS: This study demonstrates the potent inhibitory effect exerted by M. hyorhinis in standard lymphocyte proliferation assays in vitro. MSC are efficient vectors of mycoplasma infection, emphasizing the importance of monitoring cell cultures for contamination.

  15. Effects of low dose oxymatrine on mouse lymphocyte proliferation stimulated by Con A

    Institute of Scientific and Technical Information of China (English)

    LI Luo-si; WU Bin; LI Jian-guo; XIE Hong-fu; ZHANG Yang-de; CONG Ling; SHI Jun

    2005-01-01

    Objective To investigate the effects of low dose Oxymatrine (OMT) on mouse lymphocyte proliferation stimulated by Con A making use of fluorescence dyestuff CFDA-SE. Methods CFDA-SE staining and flow cytometry were used to detect the fluorescence intensity of lymphocytes after stimulated by polyclonal stimulators Con A and OMT. Then, related software was used to analyze the effects of OMT on mouse lymphocyte proliferation.Results After cultured for 48 h, CFSE fluorescence could be detected by cytometer, filial generation peaks did not appear in control group, which indicated that lymphocytes did not proliferate. Three peaks were obviously detected in Con A group which indicated that Lymphocytes divided after 48 h stimulated by Con A compared with the halving of the fluorescence intensity of control group. In groups with Con A and OMT treated, Primary generation peaks are all lower while filial generation peaks are significantly higher than groups with Con A treated only. This indicated OMT obviously promote lymphocyte proliferation. After cultured for 72 h, the fluorescence intensity changes between all groups are consistent with those of cultured for 48 h. Analyzed with CELLQuest software, it is shown that OMT could promote lymphocyte proliferation in 16, 8, 4 and 2μg/mL respectively. Conclusions 1) CFDA-SEdyeing and flow cytometer were both reliable tools to analyze lymphocyte proliferation; 2) lower dosage of OMTcould promote the proliferation of lymphocyte as a immunopotentiator.

  16. Boron Induces Lymphocyte Proliferation and Modulates the Priming Effects of Lipopolysaccharide on Macrophages.

    Science.gov (United States)

    Routray, Indusmita; Ali, Shakir

    2016-01-01

    Chemical mediators of inflammation (CMI) are important in host defense against infection. The reduced capacity of host to induce the secretion of these mediators following infection is one of the factors in host susceptibility to infection. Boron, which has been suggested for its role in infection, is reported in this study to increase lymphocyte proliferation and the secretion of CMI by the lipopolysaccharide (LPS)-stimulated peritoneal macrophages in BALB/c mice. Boron was administered to mice orally as borax at different doses for 10 consecutive days, followed by the stimulation of animals with ovalbumin and isolation of splenocytes for proliferation assay. The lymphocyte subsets were determined by flow cytometry in spleen cell suspension. The mediators of inflammation, TNF-α, IL-6, IL-1β and nitric oxide (NO), were measured in culture supernatant of LPS-primed macrophages isolated from borax treated mice. TNF and ILs were measured by ELISA. NO was determined by Griess test. The expression of inducible nitric oxide synthase (iNOS) in macrophages was studied by confocal microscopy. Results showed a significant increase in T and B cell populations, as indicated by an increase in CD4 and CD19, but not CD8, cells. Boron further stimulated the secretion of TNF-α, IL-6, IL-1β, NO and the expression of iNOS by the LPS-primed macrophages. The effect was dose dependent and most significant at a dose level of 4.6 mg/kg b. wt. Taken together, the study concludes that boron at physiological concentration induces lymphocyte proliferation and increases the synthesis and secretion of pro-inflammatory mediators by the LPS-primed macrophages, more specifically the M1 macrophages, possibly acting through Toll-like receptor. The study implicates boron as a regulator of the immune and inflammatory reactions and macrophage polarization, thus playing an important role in augmenting host defense against infection, with possible role in cancer and other diseases.

  17. Boron Induces Lymphocyte Proliferation and Modulates the Priming Effects of Lipopolysaccharide on Macrophages.

    Directory of Open Access Journals (Sweden)

    Indusmita Routray

    Full Text Available Chemical mediators of inflammation (CMI are important in host defense against infection. The reduced capacity of host to induce the secretion of these mediators following infection is one of the factors in host susceptibility to infection. Boron, which has been suggested for its role in infection, is reported in this study to increase lymphocyte proliferation and the secretion of CMI by the lipopolysaccharide (LPS-stimulated peritoneal macrophages in BALB/c mice. Boron was administered to mice orally as borax at different doses for 10 consecutive days, followed by the stimulation of animals with ovalbumin and isolation of splenocytes for proliferation assay. The lymphocyte subsets were determined by flow cytometry in spleen cell suspension. The mediators of inflammation, TNF-α, IL-6, IL-1β and nitric oxide (NO, were measured in culture supernatant of LPS-primed macrophages isolated from borax treated mice. TNF and ILs were measured by ELISA. NO was determined by Griess test. The expression of inducible nitric oxide synthase (iNOS in macrophages was studied by confocal microscopy. Results showed a significant increase in T and B cell populations, as indicated by an increase in CD4 and CD19, but not CD8, cells. Boron further stimulated the secretion of TNF-α, IL-6, IL-1β, NO and the expression of iNOS by the LPS-primed macrophages. The effect was dose dependent and most significant at a dose level of 4.6 mg/kg b. wt. Taken together, the study concludes that boron at physiological concentration induces lymphocyte proliferation and increases the synthesis and secretion of pro-inflammatory mediators by the LPS-primed macrophages, more specifically the M1 macrophages, possibly acting through Toll-like receptor. The study implicates boron as a regulator of the immune and inflammatory reactions and macrophage polarization, thus playing an important role in augmenting host defense against infection, with possible role in cancer and other diseases.

  18. Nuclear anomalies, chromosomal aberrations and proliferation rates in cultured lymphocytes of head and neck cancer patients.

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    George, Alex; Dey, Rupraj; Bhuria, Vikas; Banerjee, Shouvik; Ethirajan, Sivakumar; Siluvaimuthu, Ashok; Saraswathy, Radha

    2014-01-01

    Head and neck cancers (HNC) are extremely complex disease types and it is likely that chromosomal instability is involved in the genetic mechanisms of its genesis. However, there is little information regarding the background levels of chromosome instability in these patients. In this pilot study, we examined spontaneous chromosome instability in short-term lymphocyte cultures (72 hours) from 72 study subjects - 36 newly diagnosed HNC squamous cell carcinoma patients and 36 healthy ethnic controls. We estimated chromosome instability (CIN) using chromosomal aberration (CA) analysis and nuclear level anomalies using the Cytokinesis Block Micronucleus Cytome Assay (CBMN Cyt Assay). The proliferation rates in cultures of peripheral blood lymphocytes (PBL) were assessed by calculating the Cytokinesis Block Proliferation Index (CBPI). Our results showed a significantly higher mean level of spontaneous chromosome type aberrations (CSAs), chromatid type aberration (CTAs) dicentric chromosomes (DIC) and chromosome aneuploidy (CANEUP) in patients (CSAs, 0.0294±0.0038; CTAs, 0.0925±0.0060; DICs, 0.0213±0.0028; and CANEUPs, 0.0308±0.0035) compared to controls (CSAs, 0.0005±0.0003; CTAs, 0.0058±0.0015; DICs, 0.0005±0.0003; and CANEUPs, 0.0052±0.0013) where pnuclear anomalies showed significantly higher mean level of micronuclei (MNi), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) among cases (MNi, 0.01867±0.00108; NPBs, 0.01561±0.00234; NBUDs, 0.00658±0.00068) compared with controls (MNi, 0.00027±0.00009; NPBs, 0.00002±0.00002; NBUDs, 0.00011±0.00007).The evaluation of CBPI supported genomic instability in the peripheral blood lymphocytes showing a significantly lower proliferation rate in HNC patients (1.525±0.005552) compared to healthy subjects (1.686±0.009520 ) (pproliferation in the cultured peripheral lymphocytes of solid tumors could be biomarkers to predict malignancy in early stages.

  19. 50 Hz sinusoidal magnetic fields do not affect human lymphocyte activation and proliferation in vitro

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    Capri, Miriam; Mesirca, Pietro; Remondini, Daniel; Carosella, Simona; Pasi, Sara; Castellani, Gastone; Franceschi, Claudio; Bersani, Ferdinando

    2004-12-01

    In the last 30 years, an increasing public concern about the possible harmful effects of electromagnetic fields generated by power lines and domestic appliances has pushed the scientific community to search for a correct and comprehensive answer to this problem. In this work the effects of exposure to 50 Hz sinusoidal magnetic fields, with a magnetic flux density of 0.05 mT and 2.5 mT (peak values), were studied on human peripheral blood mononuclear cells (PBMCs) collected from healthy young and elderly donors. Cell activation and proliferation were investigated by using flow cytometry techniques and 3H-TdR incorporation assays, respectively. The results obtained indicated that exposure to the fields altered neither DNA synthesis nor the capacity of lymphocytes to enter the activation phase and progress into the cell cycle. Thus, the conclusions are that two important functional phases of human lymphocytes, such as activation and proliferation, are not affected by exposures to 50 Hz magnetic fields similar to those found under power lines.

  20. Lymphocyte Redox Imbalance and Reduced Proliferation after a Single Session of High Intensity Interval Exercise.

    Science.gov (United States)

    Tossige-Gomes, Rosalina; Costa, Karine Beatriz; Ottone, Vinícius de Oliveira; Magalhães, Flávio de Castro; Amorim, Fabiano Trigueiro; Rocha-Vieira, Etel

    2016-01-01

    This study investigated whether an acute session of high-intensity interval training (HIIT) is sufficient to alter lymphocyte function and redox status. Sixteen young healthy men underwent a HIIT session on a cycloergometer, consisting of eight bouts of 1 min at 90-100% of peak power, with 75 seconds of active recovery at 30 W between bouts. Venous blood was collected before, immediately after, and 30 minutes after the HIIT session. In response to Staphylococcus aureus superantigen B (SEB) stimulation, lymphocyte proliferation decreased and the IL-2 concentration increased after the HIIT session. However, the HIIT session had no effect on lymphocyte proliferation or IL-2 response to phytohemagglutinin stimulation. The HIIT session also induced lymphocyte redox imbalance, characterized by an increase in the concentration of thiobarbituric acid reactive substances and a decrease in the activity of the antioxidant enzyme catalase. Lymphocyte viability was not affected by the HIIT session. The frequencies of CD25+ and CD69+ T helper and B lymphocytes in response to superantigen stimulation were lower after exercise, suggesting that superantigen-induced lymphocyte activation was reduced by HIIT. However, HIIT also led to a reduction in the frequency of CD4+ and CD19+ cells, so the frequencies of CD25+ and CD69+ cells within the CD4 and CD19 cell populations were not affected by HIIT. These data indicate that the reduced lymphocyte proliferation observed after HIIT is not due to reduced early lymphocyte activation by superantigen. Our findings show that an acute HIIT session promotes lymphocyte redox imbalance and reduces lymphocyte proliferation in response to superantigenic, but not to mitogenic stimulation. This observation cannot be explained by alteration of the early lymphocyte activation response to superantigen. The manner in which lymphocyte function modulation by an acute HIIT session can affect individual immunity and susceptibility to infection is important

  1. Stimulation of rat B-lymphocyte proliferation by corticotropin-releasing factor.

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    McGillis, J P; Park, A; Rubin-Fletter, P; Turck, C; Dallman, M F; Payan, D G

    1989-07-01

    The mitogenic effect of corticotropin-releasing factor (CRF) on rat lymphocytes was investigated. When rat splenocytes were cultured for 48 hr with CFR, a dose-dependent increase in incorporation of 3H-thymidine (3H-Tdr) was observed, with a maximal response at 10 nM CRF. Comparison of the proliferative effect of CRF on enriched populations of B lymphocytes, T lymphocytes, or macrophages revealed that only B lymphocytes responded following treatment with CRF. When lymphocytes derived from different lymphoid tissues were compared, CRF had a greater proliferative effect on lymphocytes derived from gut-associated lymphoid tissue (mesenteric lymph nodes and Peyer's patches) than on lymphocytes from spleen or inguinal lymph nodes; CRF had no effect on thymocytes. Synthetic fragments of CRF were used to determine which portions of the peptide are recognized by lymphocytes. The C-terminal fragments alpha-helical CRF9-41 and CRF21-41 were as potent as native CRF in stimulating B-lymphocyte proliferation, whereas CRF1-20 did not stimulate proliferation. The activity of these peptides suggests that CRF stimulates lymphocyte proliferation by cellular recognition of structural determinants in the C-terminal one-half of the peptide.

  2. Multicolor flow cytometry analysis of the proliferations of T-lymphocyte subsets in vitro by EdU incorporation.

    Science.gov (United States)

    Sun, Yanli; Sun, Yu; Lin, Guigao; Zhang, Rui; Zhang, Kuo; Xie, Jiehong; Wang, Lunan; Li, Jinming

    2012-10-01

    EdU (5-ethynyl-2'-deoxyuridine) incorporation has proved advantageous in the studies of cell kinetics, DNA synthesis, and cellular proliferation in vitro and in vivo compared to [(3) H]thymidine incorporation and BrdU (5-bromo-2'-deoxyuridine) incorporation. Here, we describe a method that combines EdU incorporation and immunostaining with flow cytometric analysis to detect the proliferations of T lymphocyte subsets in vitro and optimized the assay's conditions. We found that the number of EdU(+) cells were associated with EdU concentration, incubation time, and the volume of Click reaction solution, the best EdU concentration 10-50 μM, the optimal incubation time 8-12 h and the proper volume of Click volume 100 μl for labeling 1 × 10(6) lymphocytes. Fixation was better to be performed before permeabilization, not together with. Furthermore, the permeabilization detergent reagent, PBS with 0.05% saponin was better than Tris buffer saline (TBS) with 0.1% Triton X-100. In addition, sufficient wash with PBS with 0.05% saponin has no influence on the staining of EdU(+) cells. Also, the lymphocytes incorporating EdU could be stored at 4°C, -80°C, and in liquid nitrogen up to 21 days. The present study will aid in optimization of flow cytometry assay to detect the proliferations of T cell subsets by EdU incorporation and the labeling of cell surface antigens.

  3. DMPD: Induction of proliferation and cytokine production in human T lymphocytes bylipopolysaccharide (LPS). [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 11090938 Induction of proliferation and cytokine production in human T lymphocytes ... (.png) (.svg) (.html) (.csml) Show Induction of proliferation and cytokine production in human T lymphocyte...and cytokine production in human T lymphocytes bylipopolysaccharide (LPS). Authors Ulmer AJ, Flad H, Rietsch

  4. Effect of thapsigargin on cytoplasmic Ca2+ and proliferation of human lymphocytes in relation to AIDS

    DEFF Research Database (Denmark)

    Scharff, O; Foder, B; Thastrup, Ole

    1988-01-01

    response at 100 nM. The effect of thapsigargin on [Ca2+]i exceeded that of phytohaemagglutinin (PHA) which raised [Ca2+]i to maximum 300 nM. In combination with phorbol 12-myristate 13-acetate (PMA), thapsigargin stimulated the proliferation of normal lymphocytes to the same extent as did PHA, whereas...... of the lymphocytes, which was much higher than that caused by the PHA treatment, even in AIDS lymphocytes. Moreover, the thapsigargin/PMA treatment stimulated the expression of the IL-2 receptors on both normal and AIDS lymphocytes, similar to the effect of PHA. It is concluded that thapsigargin exerts its effects...

  5. Lymphocyte proliferation to mycobacterial antigens is detectable across a spectrum of HIV-associated tuberculosis

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    Bakari Muhammad

    2009-02-01

    Full Text Available Abstract Background Identifying novel TB diagnostics is a major public health priority. We explored the diagnostic characteristics of antimycobacterial lymphocyte proliferation assays (LPA in HIV-infected subjects with latent or active TB. Methods HIV-infected subjects with bacille Calmette Guérin (BCG scars and CD4 counts ≥ 200 cells/mm3 entering a TB booster vaccine trial in Tanzania had baseline in vivo and in vitro immune tests performed: tuberculin skin tests (TST, LPA and five day assays of interferon gamma (IFN-γ release. Assay antigens were early secreted antigenic target 6 (ESAT-6, antigen 85 (Ag85, and Mycobacterium tuberculosis whole cell lysate (WCL. Subjects were screened for active TB at enrollment by history, exam, sputum smear and culture. We compared antimycobacterial immune responses between subjects with and without latent or active TB at enrollment. Results Among 1885 subjects screened, 635 had latent TB and 13 had active TB. Subjects with latent TB were more likely than subjects without TB to have LPA responses to ESAT-6 (13.2% vs. 5.5%, P Conclusion Lymphoproliferative responses to mycobacteria are detectable during HIV-associated active TB, and are less sensitive but more specific than TST. Trial registration ClinicalTrials.gov Identifier NCT00052195.

  6. Epithelial cell proliferation and glandular atrophy in lymphocytic gastritis: Effect of H pylori treatment

    Institute of Scientific and Technical Information of China (English)

    Johanna M. Makinen; Seppo Niemela; Tuomo Kerola; Juhani Lehtola; Tuomo J. Karttunen

    2003-01-01

    AIM: Lymphocytic gastritis is commonly ass ociated with Helicobacter pylori infection. The presence of glandular atrophy and foveolar hyperplasia in lymphocytic gastritis suggests abnormalities in cell proliferation and differentiation,forming a potential link with the suspected association with gastric cancer. Our aim was to compare epithelial cell proliferation and morphology in H pylori associated lymphocytic gastritis and H pylori gastritis without features of lymphocytic gastritis,and to evaluate the effect of H pylori treatment.METHODS: We studied 14 lymphocytic gastritis patients with H pylori infection. For controls, we selected 14 matched dyspeptic patients participating in another treatment trial whose H pylori infection had successfully been eradicated.Both groups were treated with a triple therapy and followed up with biopsies for 6-18 months (patients) or 3 months (controls). Blinded evaluation for histopathological features was carried out. To determine the cell proliferation index,the sections were labeled with Ki-67 antibody.RESULTS: Before treatment, lymphocytic gastritis was characterized by foveolar hyperplasia (P=0.001) and glandular atrophy in the body (P=0.008), and increased proliferation in both the body (P=0.001) and antrum (P=0.002). Proliferation correlated with foveolar hyperplasia and inflammation activity. After eradication therapy, the number of intraepithelial lymphocytes decreased in the body (P=0.004)and antrum (P=0.065), remaining higher than in controls (P<0.001). Simultaneously, the proliferation index decreased in the body from 0.38 to 0.15 (P=0.043), and in the antrum from 0.34 to 0.20 (P=0.069), the antral index still being higher in lymphocytic gastritis than in controls (P=0.010).Foveolar hyperplasia and glandular atrophy in the body improved (P=0.021), reaching the non-LG level.CONCLUSION: In lymphocytic gastritis, excessive epithelial cell proliferation is predominantly present in the body, where it associates with

  7. Differential expression of alkaline phosphatase gene in proliferating primary lymphocytes and malignant lymphoid cell lines.

    Science.gov (United States)

    Latheef, S A A; Devanabanda, Mallaiah; Sankati, Swetha; Madduri, Ramanadham

    2016-02-01

    Alkaline Phosphatase (APase) activity has been shown to be enhanced specifically in mitogen stimulated B lymphocytes committed to proliferation, but not in T lymphocytes. APase gene expression was analyzed in proliferating murine and human primary lymphocytes and human malignant cell lines using reverse transcriptase and real time PCR. In mitogen stimulated murine splenic lymphocytes, enhancement of APase activity correlated well with an increase in APase gene expression. However, in mitogen stimulated murine T lymphocytes and human PBL despite a vigorous proliferative response, no increase in APase enzyme activity or gene expression was observed. A constitutive expression of APase activity concomitant with APase gene expression was observed inhuman myeloma cell line, U266 B1. However, neither enzyme activity nor gene expression of APase were observed in human T cell lymphoma, SUPT-1. The results suggest a differential expression of APase activity and its gene in proliferating primary lymphocytes of mice and humans. The specific expression of APase activity and its gene only in human myeloma cells, but not in proliferating primary B cells can be exploited as a sensitive disease marker.

  8. Effects of environmental stressors on lymphocyte proliferation in Florida manatees, Trichechus manatus latirostris.

    Science.gov (United States)

    Walsh, Cathy J; Luer, Carl A; Noyes, David R

    2005-02-10

    The health of many Florida manatees (Trichechus manatus latirostris) is adversely affected each year by exposure to cold weather or harmful algal blooms (red tide; Karenia brevis). Exposures can be sublethal, resulting in stressed animals that are rescued and taken to authorized facilities for rehabilitation, or lethal if exposures are prolonged or unusually severe. To investigate whether sublethal environmental exposures can impair immune function in manatees, rendering animals vulnerable to disease or death, mitogen-induced proliferation was assessed in lymphocytes from manatees exposed to cold temperatures (N=20) or red tide (N=19) in the wild, and compared to lymphocyte responses from healthy free-ranging manatees (N=32). All animals sampled for this study were adults. Lymphocytes were stimulated in vitro with either concanavalin A (ConA) or phytohemagglutinin (PHA) and proliferation was assessed after 96 h using incorporation of the thymidine analog, bromodeoxyuridine (BrdU), into newly synthesized DNA. Proliferation of lymphocytes from manatees rescued from exposure to red tide or cold-stress was approximately one-third that of lymphocytes from healthy free-ranging manatees. To examine the direct effects of red tide toxins on lymphocyte function, mitogen-induced proliferation was assessed following co-culture of lymphocytes with K. brevis toxin extracts. Stimulation indices decreased with increasing toxin concentration, with a significant decrease in proliferation occurring in the presence of 400 ng red tide toxins/ml. When lymphocytes from cold-stressed manatees were co-cultured with red tide toxin extracts, proliferative responses were reduced even further, suggesting multiple stressors may have synergistic effects on immune function in manatees.

  9. Studying the proliferation of human peripheral blood T lymphocytes in serum-free medium.

    Science.gov (United States)

    Tabakov, V U; Litvina, M M; Schepkina, J V; Jarilin, A A; Chestkov, V V

    2009-01-01

    We compared the cultivation of human peripheral blood lymphocytes in serum-free medium Hybris-2 and RPMI 1640 medium with 10% fetal bovine serum in the presence of phytohemagglutinin and interleukin-2. The optimal concentration of phytohemagglutinin significantly differed in serum-free and serum-containing media (0.5 and 5 microg/ml, [corrected] respectively). Both mitogens were more potent in stimulating the proliferation of lymphocytes in serum-free medium than in serum-containing medium. Strong proliferation of CD3(+) and CD4(+) T lymphocytes was observed in both media. The dynamics of other markers was similar in serum-free and serum-containing media. However, significant differences were revealed between individual donors. Our results indicate that the developed serum-free medium may be used in lymphocyte cultivation for scientific, diagnostic, and therapeutic purposes.

  10. Inhibition of human lymphocyte proliferation and cleavage of interleukin-2 by Pseudomonas aeruginosa proteases

    DEFF Research Database (Denmark)

    Theander, T G; Kharazmi, A; Pedersen, B K

    1988-01-01

    This study was undertaken to determine the effect of Pseudomonas aeruginosa alkaline protease (AP) and elastase (ELA) on human lymphocyte function. AP at 50 micrograms/ml and ELA at 12 micrograms/ml caused a 50% inhibition of phytohemagglutinin-induced proliferation. There was no difference......, the inhibition was partly reversed. ELA at 10 micrograms/ml cleaved IL-2, as judged by size chromatography of a reaction mixture containing 125I-labeled IL-2 and the proteases. The ELA-digested IL-2 exhibited a reduced binding capacity to IL-2 receptors on the lymphocytes. Furthermore, treatment...... of phytohemagglutinin-stimulated lymphocytes with AP and ELA resulted in inhibition of binding of intact IL-2 to IL-2 receptors on the stimulated lymphocytes. These results indicated that P. aeruginosa-derived enzymes are able to interfere with human lymphocyte function in vitro and that this effect might be due...

  11. C1-esterase inhibitor blocks T lymphocyte proliferation and cytotoxic T lymphocyte generation in vitro

    DEFF Research Database (Denmark)

    Nissen, Mogens Holst; Bregenholt, S; Nording, J A

    1998-01-01

    We have previously shown that activated C1s complement and activated T cells cleave beta2-microglobulin (beta2m) in vitro leading to the formation of desLys58 beta2m. This process can specifically be inhibited by C1-esterase inhibitor (C1-inh). Furthermore we showed that exogenously added desLys58...... beta2m in nanomolar amounts to a one-way allogenic mixed lymphocyte culture (MLC) increased the endogenous production of IL-2 and the generation of allo-specific cytotoxic T lymphocytes. C1-inh was purified from fresh human plasma and added to human or murine MLC and mitogen-stimulated lymphocyte...... of allospecific cytotoxic activity, and changed the endogenous production of IL-2, IL-4, IL-10, IL-12 and IFN-gamma. These data clearly demonstrate a regulatory function of C1-inh on T cell-mediated immune functions....

  12. Altered lymphocyte proliferation and innate immune function in scrapie 139A- and ME7-infected mice.

    Science.gov (United States)

    Cho, In Soo; Spinner, Daryl S; Kascsak, Richard J; Meeker, H Cliff; Kim, Bo Sook; Park, Seung Yong; Schuller-Levis, Georgia; Park, Eunkyue

    2013-06-01

    Lymphoid organs play an important role in prion disease development and progression. While the role of lymphoid organs and changes in immune-related genes have been extensively investigated in scrapie-infected animals, innate immunity has not. Previous studies examined lymphocyte function in scrapie-infected C3H/HeJ mice, which exhibit defects in lipopolysaccharide (LPS) response now known to result from a mutation in Toll-like receptor (TLR) 4. We examined immune function in scrapie-infected CD1 mice, which are LPS responders. Lymphocyte proliferation from CD1 mice infected with either 139A or ME7 scrapie was measured in response to concanavalin (Con) A or LPS at 1 and 3 months after infection. Following LPS exposure, mice infected 3 months with ME7, but not 139A, demonstrated significantly decreased lymphocyte proliferation compared to controls. After Con A exposure, lymphocyte proliferation in scrapie-infected mice did not differ from controls. Gender-specific comparison of lymphocyte proliferation showed significant decreases in mitogenic responses in females infected 3 months with either 139A or ME7, compared to controls. Males infected for 3 months with ME7, but not 139A, showed significantly decreased proliferation after lymphocyte exposure to LPS, but not Con A. Neither gender showed changes in lymphocyte proliferation after 1 month of scrapie infection. Innate immune activation of peritoneal macrophages was determined via production of nitric oxide (NO), IL-6, and TNF-α after exposure to TLR ligands. TNF-α and IL-6 production were reduced in macrophages from females infected with either scrapie strain for 3 months, while NO production after TLR agonist plus IFN-γ exposure was decreased in both females and males infected for 3 months with 139A, compared to ME7. These data demonstrated altered innate immunity, suggesting hormonal and/or other gender-specific regulation may contribute to gender differences in some immune functions. Our data demonstrate

  13. Lamprey buccal gland secretory protein-2 (BGSP-2 inhibits human T lymphocyte proliferation

    Directory of Open Access Journals (Sweden)

    Jing SUN, Shuiyan YU, Zhuang XUE, Cenjie LIU, Yu WU, Xin LIU, Qingwei LI

    2010-04-01

    Full Text Available Lamprey is a representative of the agnathans, the most ancient class of vertebrates. Parasitic lampreys secrete anticoagulant from their buccal glands and prevent blood coagulation of host fishes. We identified a buccal gland secretory protein-2 (BGSP-2 from a buccal gland cDNA library of Lampetra japonica. The full-length BGSP-2 gene was cloned and the recombinant BGSP-2 protein was generated. The role of BGSP-2 on lymphocyte proliferation was studied by examining its effects on human T lymphocytes. We found that lamprey BGSP-2 was able to effectively block the proliferation of T cells in vitro by inducing G1/S cell cycle arrest. Furthermore, it inhibited the proliferation of human T lymphocytes stimulated by phytohemagglutinin (PHA at a minimum concentration of 0.1μg/ml. Our data suggest that lamprey BGSP-2 is able to block the mitosis of human T lymphocytes at the G1/S point, and has the potential of anti-proliferative effect on PHA-activated T lymphocytes [Current Zoology 56 (2: 252–258, 2010].

  14. Effects of feeding lipids of different fatty acid compositions upon rat lymphocyte proliferation.

    Science.gov (United States)

    Calder, P C; Costa-Rosa, L F; Curi, R

    1995-01-01

    Weanling Wistar rats were fed for 8 weeks on a low fat (3% by weight) diet (LF) or on diets containing 15% by weight hydrogenated coconut oil (coconut oil), cocoa butter, cashew kernel oil (cashew oil), canola oil or soya bean oil (soya oil). Each of the high fat diets resulted in inhibition of spleen lymphocyte proliferation in response to concanavalin A (Con A), a T-cell mitogen. Feeding the soya oil diet caused more than 95% inhibition of proliferation, compared with feeding the LF diet. Amongst the high fat diets, feeding the cashew, canola or soya oil diets resulted in inhibition of proliferation compared with feeding the coconut oil diet while feeding the soya oil diet resulted in inhibition of proliferation compared with feeding the cocoa butter or canola oil diets. Proliferation of lymphocytes in response to Con A was also investigated using whole blood culture. Once again, feeding the soya oil diet resulted in a dramatic decrease in proliferation (80%) compared with feeding the LF diet. Feeding the soya oil diet also inhibited proliferation compared with feeding the coconut oil, cocoa butter or cashew oil diets. Feeding the canola oil diet resulted in inhibition of proliferation compared with feeding the LF or cocoa butter diets.

  15. Mesenchymal stem cells derived from human placenta suppress allogeneic umbilical cord blood lymphocyte proliferation

    Institute of Scientific and Technical Information of China (English)

    Chang Dong LI; Wei Yuan ZHANG; He Lian LI; Xiao Xia JIANG; Yi ZHANG; Pei Hsien TANG; Ning MAO

    2005-01-01

    Human placenta-derived mononuclear cells (MNC) were isolated by a Percoll density gradient and cultured in mesenchymal stem cell (MSC) maintenance medium.The homogenous layer of adherent cells exhibited a typical fibroblastlike morphology,a large expansive potential,and cell cycle characteristics including a subset of quiescent cells.In vitro differentiation assays showed the tripotential differentiation capacity of these cells toward adipogenic,osteogenic and chondrogenic lineages.Flow cytometry analyses and immunocytochemistry stain showed that placental MSC was a homogeneous cell population devoid of hematopoietic cells,which uniformly expressed CD29,CD44,CD73,CD 105,CD166,laminin,fibronectin and vimentin while being negative for expression of CD31,CD34,CD45 and α-smooth muscle actin.Most importantly,immuno-phenotypic analyses demonstrated that these cells expressed class I major histocompatibility complex (MHC-Ⅰ),but they did not express MHC-Ⅱ molecules.Additionally these cells could suppress umbilical cord blood (UCB) lymphocytes proliferation induced by cellular or nonspecific mitogenic stimuli.This strongly implies that they may have potential application in allograft transplantation.Since placenta and UCB are homogeneous,the MSC derived from human placenta can be transplanted combined with hematopoietic stem cells (HSC) from UCB to reduce the potential graft-versus-host disease (GVHD) in recipients.

  16. [Preparation of polyclonal antibody against sAPRIL and analysis of function in suppressing sAPRIL-mediated lymphocyte proliferation].

    Science.gov (United States)

    Du, Ben-Jun; Gao, Quan-Sheng; Lan, Zhi; Fan, Jun-Wen; Ding, Lu-Jing; Li, Min; Qi, Yuan-Yuan; Kong, Wei

    2011-08-01

    This study was aimed to prepare the polyclonal antibody against the soluble proliferation-inducing ligand (sAPRIL) antigen and to investigate its effects in suppressing sAPRIL mediated lymphocyte proliferation. Mutated recombinant sAPRIL protein, which lacks biological activity but maintains immunogenicity, was used as antigen to immunize humanized SCID mice. Sera were obtained at 6 weeks after immunization. Indirect ELISA and Western blot were used to detect the antibody titer and specificity. The inhibition of polyclonal antibodies on Raji and Jurkat cell proliferation stimulated by sAPRIL was assessed by the MTT assay. The results showed that the mutant of sAPRIL could induce the production of polyclonal antibodies against human sAPRIL. Western blot and indirect ELISA analyses indicated that the anti-serum had higher specificity with a titer of 1:640. Functional analysis revealed that these polyclonal antibodies significantly inhibited the proliferation of Raji and Jurkat cell stimulated by sAPRIL (p polyclonal antibody against human sAPRIL is successfully prepared, which can inhibit the proliferation of Raji and Jurkat cells stimulated by sAPRIL in vitro.

  17. A Novel Natural Product, KL-21, Inhibits Proliferation and Induces Apoptosis in Chronic Lymphocytic Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Aysun Adan Gökbulut

    2015-06-01

    Full Text Available INTRODUCTION: The aims of this study were to examine the cytotoxic and apoptotic effects of KL-21, a novel plant product (produced by Naturin Natural Products, İzmir, Turkey, on 232B4 chronic lymphocytic leukemia (CLL cells and to determine the cytotoxic effects on healthy BEAS-2B human bronchial epithelial cells. METHODS: The cytotoxic effect of KL-21 was determined by MTT cell proliferation assay. Changes in caspase-3 enzyme activity were measured using the caspase-3 colorimetric assay. Changes in mitochondrial membrane potential were determined using the JC-1 dye-based method. Annexin V-FITC/PI double staining was performed to measure the apoptotic cell population. Effects of KL-21 on cell cycle profiles of CLL cells were investigated by flow cytometry. RESULTS: We detected time- and concentration-dependent increases in the cytotoxic effect of KL-21 on 232B4 CLL cells. However, we also showed that, especially at higher concentrations, KL-21 was less cytotoxic towards BEAS-2B healthy cells than towards CLL cells. Annexin-V/PI double staining results showed that the apoptotic cell population increased in 232B4 cells. Increasing concentrations of KL-21 increased caspase-3 enzyme activity and induced loss of mitochondrial membrane potential. KL-21 administration resulted in small increases in the percentage of the cells in the G0/G1 phase while it decreased the S phase cell population up to 1 mg/mL. At the highest concentration, most of the cells accumulated in the G0/G1 phase. DISCUSSION AND CONCLUSION: KL-21 has a growth-inhibitory effect on 232B4 CLL cells. KL-21 causes apoptosis and cell cycle arrest at G0/G1.

  18. N-acetylcysteine reverses immunotoxic effects of methyl mercury and augments murine lymphocyte proliferation in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Omara, F.; Fournier, M.; Bernier, J. [Univ. du Quebec, Montreal, Quebec (Canada); Blakley, B.

    1995-12-31

    N-Acetylcysteine (NAC) is a thiol antioxidant used clinically to treat chronic inflammatory lung disorders and acetaminophen poisoning in humans. The authors evaluated in vitro the effect of NAC on mitogen-induced blastogenesis in C57BI/6 mouse splenocytes by {sup 3}H-thymidine uptake, and its ability to protect against the immunotoxic effects of methyl mercury on lymphocyte proliferation. Lymphocyte proliferation stimulated by optimal and suboptimal concentrations of concanavalin A (Con A), lipopolysaccharide (LPS), or a combination of calcium ionophore A23187 and phorbol-12-myristate-13-acetate (PMA) were markedly enhanced by NAC. NAC itself was a weak mitogen. The kinetics of the NAC effect on splenocyte proliferation were mitogen dependent. NAC enhanced Con A-induced splenocyte proliferation in a dose-dependent and linear manner but enhanced the LPS-induced response at 50--400 {micro}g/ml of NAC followed by a decline in response to control value at higher concentrations. In splenocytes stimulated with PMA plus A23187, NAC increased proliferation at 50--200 pg/ml followed by a constant response at 200--1,000 {micro}g/ml NAC. When splenocytes were stimulated with higher concentrations of Con A (10 {micro}g/ml) or LPS (150 {micro}g/ml) which markedly suppress splenocyte proliferation, NAC significantly enhanced the Con A-induced response and reversed the inhibitory effect of high concentrations of LPS. NAC also protected lymphocytes against mitogen activation-induced cell death. Methyl mercury at 5 {times} 10{sup {minus}7}--1 {times} 10{sup {minus}6} suppressed Con A- and LPS-induced splenocyte proliferation by over 80%. However, NAC completely reversed the immunotoxic effects of methyl mercury on the mitogen-induced splenocyte proliferation even when the cells were pre-incubated with methyl mercury for 6 or 24 hr before stimulation with the mitogens.

  19. Alkylglycerols modulate the proliferation and differentiation of non-specific agonist and specific antigen-stimulated splenic lymphocytes.

    Directory of Open Access Journals (Sweden)

    Linxi Qian

    Full Text Available Alkylglycerols (AKGs are ether-linked glycerols derived from shark liver oil and found in small amounts in human milk. Previous studies showed that oral AKGs administration significantly increased the immune response in mice. The aim of the present study was to investigate the in vitro immunomodulatory effect of AKGs on stimulating splenic lymphocyte responses. C57BL/6 mice were immunized with hepatitis B surface antigen (HBsAg. Splenic B cells were purified and stimulated with anti-BCR and anti-CD38. Meanwhile, splenic CD4+ T cells were purified and stimulated with anti-CD3 and anti-CD28. For antigen specific stimulation, the purified CD4+ T cells were cocultured with HBsAg -pulsed dendritic cells. The stimulated lymphocytes were treated with different concentrations of AKGs. The cell proliferation was assessed by [3H]-thymidine incorporation assay. The maturation of B cells was assessed by examining the germline (GL transcription of IgG (γ1 mRNA expression, and the surface expressions of CD80/CD86 markers were examined by flow cytometry analysis. Th1/Th2 polarity was assessed by T-BET (Th1/GATA-3 (Th2 flow cytometry assay and by characteristic cytokines ELISA assay (TNF-α and IFN-γ for Th1; IL-4 and IL-10 for Th2. It was found that AKGs significantly increased the BCR/CD38 -stimulated B cell proliferation. The T cell proliferation in response to CD3/CD28 or specific antigen stimulation was also increased by AKGs. The transcriptional level of IgG (γ1 and the expressions of CD80/CD86 molecules were markedly increased by AKGs in BCR/CD38 -stimulated B cells. Meanwhile, the results showed that AKGs increased the expression of T-BET transcriptional factor and the production of Th1 cytokines (TNF-α and IFN-γ upon CD3/CD28 stimulation; whereas, levels of Th2 cytokines (IL-4 and IL-10 were decreased by AKGs. Our study demonstrated that AKGs can modulate immune responses by boosting the proliferation and maturation of murine lymphocytes in vitro.

  20. Effect of pyrimethamine and sulphadoxine on human lymphocyte proliferation

    DEFF Research Database (Denmark)

    Bygbjerg, I C; Odum, Niels; Theander, T G

    1986-01-01

    The in vitro effect of pyrimethamine (PYR) on human blood mononuclear cells stimulated with phytohaemagglutinin (PHA), pokeweed mitogen (PWM) and purified protein derivative of tuberculin (PPD) was studied by 14C-thymidine incorporation, by cell counting and by total DNA estimation. PYR in concen......The in vitro effect of pyrimethamine (PYR) on human blood mononuclear cells stimulated with phytohaemagglutinin (PHA), pokeweed mitogen (PWM) and purified protein derivative of tuberculin (PPD) was studied by 14C-thymidine incorporation, by cell counting and by total DNA estimation. PYR......-stimulated cells. The suppression of PHA-stimulated cells was reversed after one week. The increased 14C-thymidine incorporation observed in stimulated cells exposed to PYR in vitro in the early phase of proliferation did not reflect immunopotentiation but rather blocked endogenous thymidine synthesis...

  1. Effects of Euphorbia milii latex on mitogen-induced lymphocyte proliferation

    Directory of Open Access Journals (Sweden)

    I.F. Delgado

    2014-03-01

    Full Text Available The crude latex of "Crown-of-Thorns" (Euphorbia milii var hislopii, syn E.splendens is a potent plant molluscicide. For this reason, toxicological studies have been performed to evaluate the health risks posed by its use in schistosomiasis control programs. The present study is part of a more comprehensive immunotoxicological evaluation of this molluscicide. Here, we investigated the effects of E. milii latex on the proliferation of human lymphocytes in vitro. Lyophilized latex of E. milii (0, 0.5, 5, 25 and 50 µg/ml was incubated with whole blood in the presence of proliferation stimulators, i.e. lectins (phytohemagglutinin, concanavalin A and pokeweed mitogen, as well as with human monoclonal antibody against CD3 and tetanus toxoid. Cell proliferation was measured by ³H-thymidine incorporation, and the effects of latex on mitogen-induced cell proliferation were compared to the effects of 10 ng/ml of 12-O-tetradecanoylphorbol-13-acetate (TPA. Results showed that mitogen-induced cell proliferation was markedly enhanced by E. milii latex. This synergistic effect of latex on mitogen-induced lymphocyte proliferation may be due to the presence of TPA-like phorbol esters and/or to mitogenic plant lectins.

  2. Piperine from black pepper inhibits activation-induced proliferation and effector function of T lymphocytes.

    Science.gov (United States)

    Doucette, Carolyn D; Rodgers, Gemma; Liwski, Robert S; Hoskin, David W

    2015-11-01

    Piperine is a major alkaloid component of black pepper (Piper nigrum Linn), which is a widely consumed spice. Here, we investigated the effect of piperine on mouse T lymphocyte activation. Piperine inhibited polyclonal and antigen-specific T lymphocyte proliferation without affecting cell viability. Piperine also suppressed T lymphocyte entry into the S and G2 /M phases of the cell cycle, and decreased expression of G1 -associated cyclin D3, CDK4, and CDK6. In addition, piperine inhibited CD25 expression, synthesis of interferon-γ, interleukin (IL)-2, IL-4, and IL-17A, and the generation of cytotoxic effector cells. The inhibitory effect of piperine on T lymphocytes was associated with hypophosphorylation of Akt, extracellular signal-regulated kinase, and inhibitor of κBα, but not ZAP-70. The ability of piperine to inhibit several key signaling pathways involved in T lymphocyte activation and the acquisition of effector function suggests that piperine might be useful in the management of T lymphocyte-mediated autoimmune and chronic inflammatory disorders.

  3. Strong additive effect of calcitriol and cyclosporine A on lymphocyte proliferation in vitro and rat liver allotransplantations in vivo

    Institute of Scientific and Technical Information of China (English)

    ZHANG Ai-bin; ZHENG Shu-sen

    2006-01-01

    Background Vitamin D3 and its metabolites have been found to exert immunosuppressive effects both in vivo and in vitro. We investigated the synergistic effect of calcitriol and cyclosporine A (CsA) on lymphocyte proliferation in vitro and graft rejection following rat liver allotransplantations in vivo.Methods Alloantigen driven, human peripheral mononuclear cells' proliferation and cytokine production capacity were tested in the presence or absence of various concentrations of calcitriol or CsA. In vivo, liver allografts were transplanted in a high responder strain combination (SD to Wistar) rats and combination of subtherapeutical dose of CsA and calcitriol was administered in recipients, whereas the control recipients received single or no immunosuppressant. Proliferation of splenocyte from recipient was tested with mixed lymphocyte reaction. Serum interleukin-2 (IL-2) and interferon gamma (IFN-γ) concentrations were measured with enzyme linked immunosorbent assay.Results Combined medication of 10-9 mol/L calcitriol and 100 ng/ml CsA inhibited human peripheral mononuclear cells' proliferation to alloantigen and the production of IL-2 and IFN-γ but promoted that of IL-4and IL-10. Similarly, combination of 250 ng· kg-1 · d-1 calcitriol and 1.0 mg · kg-1 · d-1 CsA showed an additive effect in liver transplant model. It restrained splenocyte proliferation to alloantigen from donor and significantly reduced serum concentration of IL-2 and IFN-γ in recipients. Consequently, allograft rejection in combined medication group was minor (median William's grade was 1.0 vs 3.0 in combined medication group and in the control group, P<0.05) and the recipients' survival was evidently prolonged [(93.7±5.8) days vs (12.6±1.4) days in combined medication group and in the control group, P<0.01].Conclusion A combination of calcitriol and CsA has an additive effect on limiting lymphocyte proliferation and prolonging liver graft survival. With its additional

  4. [Rapid dicentric assay of human blood lymphocytes after exposure to low doses of ionizing radiation].

    Science.gov (United States)

    Repin, M V; Repina, L A

    2011-01-01

    The probability of losses of different chromosome aberrations during the dicentric chromosome assay of metaphase cells with incomplete sets of chromosome centromeres was estimated using a mathematical model for low doses of ionizing radiation. A dicentric assay of human blood lymphocytes without determination of the total amount of chromosome centromeres in cells without chromosome aberrations (rapid dicentric assay) has been proposed. The rapid dicentric analysis allows to register chromosome aberrations in full compliance with the conventional classification. The experimental data have shown no statistically significant difference between the frequencies of dicentric chromosomes detected by rapid and classical dicentric chromosome assays of human lymphocytes exposed to 0.5 Gy of 60Co gamma-rays. The rate of the rapid dicentric assay was almost twice as high as that of the classical dicentric assay.

  5. Interaction between Cl- channels and CRAC-related Ca2+ signaling during T lymphocyte activation and proliferation

    Institute of Scientific and Technical Information of China (English)

    Guan-lei WANG; Yan QIAN; Qin-ying QIU; Xiu-jian LAN; Hua HE; Yong-yuan GUAN

    2006-01-01

    Aim:To test the hypothesis that Cl- channel blockers affect T cell proliferation through Ca2+-release-activated Ca2+ (CRAC) signaling and examine the effects of the combination of a CRAC channel blocker and a Cl- channel blocker on concanavalin A (ConA;5 mg/mL) -induced Ca2+ signaling,gene expression and cellular proliferation in human peripheral T lymphocytes.Methods:[3H]Thymidine incorporation,Fura-2 fluorescent probe,RNase protection assay,and reverse transcription.polymerase chain reaction were used.Results:The Cl- channel blocker 4,4'-diisothiocvanostilbene-2,2'-disulfonic acid (DIDS) inhibited ConA-induced Ca2+influx.interleukin-2 mRNA expression and T lymphocyte proliferation in a concentration.dependent manner,and also enhanced the inhibitory effects of 1-{beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl}-1H-imidazole (SK&F96365) on the above key events during T cell activation.A combination ofDIDS (1μmol/L) and SK&F96365 (1μmol/L) significantly diminished ConA-induced ClC-3 mRNA expression by 64%,whereas DIDS (1μmol/L) or SK&F96365 (1μmol/L) alone decreased ConA-induced ClC-3 mRNA expression by only 16% and 9%.respectively.Conclusion:These results suggest that there is an interaction between CRAC-mediated Ca2+ signaling and DIDS-sensitive C1-channels during ConA-induced T cell activation and proliferation.Moreover,the DIDS-sensitive Cl-channels may be related to the ClC-3 Cl- channels.

  6. Triptolide inhibits proliferation of Epstein–Barr virus-positive B lymphocytes by down-regulating expression of a viral protein LMP1

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Heng [Department of Pathogen Biology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China); Guo, Wei [Department of Pathology and Physiology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China); Long, Cong; Wang, Huan; Wang, Jingchao [Department of Pathogen Biology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China); Sun, Xiaoping, E-mail: xsun6@whu.edu.cn [Department of Pathogen Biology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China); State Key Laboratory of Virology, Wuhan University, Wuhan 430072 (China)

    2015-01-16

    Highlights: • Triptolide inhibits proliferation of EBV-positive lymphoma cells in vitro and in vivo. • Triptolide reduces expression of LMP1 by decreasing its transcription level. • Triptolide inhibits ED-L1 promoter activity. - Abstract: Epstein–Barr virus (EBV) infects various types of cells and mainly establishes latent infection in B lymphocytes. The viral latent membrane protein 1 (LMP1) plays important roles in transformation and proliferation of B lymphocytes infected with EBV. Triptolide is a compound of Tripterygium extracts, showing anti-inflammatory, immunosuppressive, and anti-cancer activities. In this study, it is determined whether triptolide inhibits proliferation of Epstein–Barr virus-positive B lymphocytes. The CCK-8 assays were performed to examine cell viabilities of EBV-positive B95-8 and P3HR-1 cells treated by triptolide. The mRNA and protein levels of LMP1 were examined by real time-PCR and Western blotting, respectively. The activities of two LMP1 promoters (ED-L1 and TR-L1) were determined by Dual luciferase reportor assay. The results showed that triptolide inhibited the cell viability of EBV-positive B lymphocytes, and the over-expression of LMP1 attenuated this inhibitory effect. Triptolide decreased the LMP1 expression and transcriptional levels in EBV-positive B cells. The activity of LMP1 promoter ED-L1 in type III latent infection was strongly suppressed by triptolide treatment. In addition, triptolide strongly reduced growth of B95-8 induced B lymphoma in BALB/c nude mice. These results suggest that triptolide decreases proliferation of EBV-induced B lymphocytes possibly by a mechanism related to down-regulation of the LMP1 expression.

  7. Suppression of lymphocyte proliferation by marijuana components is related to cell number and cell source

    Energy Technology Data Exchange (ETDEWEB)

    Klein, T.; Pross, S.; Newton, C.; Friedman, H.

    1986-03-05

    Conflicting reports have appeared concerning the effect of marijuana components on immune responsiveness. The authors have observed that the effect of cannabinoids on lymphocyte proliferation varied with both the concentration of the drug and the mitogen used. They now report that at a constant concentration of drug, the cannabinoid effect varied from no effect to suppression depending upon the number of cells in culture and the organ source of the cells. Dispersed cell suspensions of mouse lymph node, spleen, and thymus were prepared and cultured at varying cell numbers with either delta-9-tetrahydrocannabinol or 11-hydroxy-delta-9-tetrahydrocannabinol and various mitogens. Lymphocyte proliferation was analyzed by /sup 3/H-thymidine incorporation. T-lymphocyte mitogen responses in cultures containing high cell numbers were unaffected by the cannabinoids but as cell numbers were reduced a suppression of the response was observed. Furthermore, thymus cells were considerably more susceptible to cannabinoid suppression than cells from either lymph node or spleen. These results suggest that certain lymphocyte subpopulations are more sensitive to cannabinoid suppression and that in addition to drug concentration other variables such as cell number and cell source must be considered when analyzing cannabinoid effects.

  8. Lymphocyte Proliferation Response to S Antigen in Patients with Uveitis and Optic Neuritis

    Institute of Scientific and Technical Information of China (English)

    PeixianRen; XiuzhenYan

    1995-01-01

    Purpose:To evaluate the autoimmunity which may play a major role in the etiolo-gy of certain forms of uveitis and optic neuritis.Methods:lymphocyte proliferation response to retinal soluble antigen in vitro by gy of certain forms of uveitis and optic neuritis.Methods:Lymphocyte proliferation response toretinal soluble antigen in vitro by incoperation3H-thymidine withDNA was tested in 115patients with anterior u-veitis,posterior/pan-uveitis,optic neuritis,and 50volunteers with unrelated diseases such as congenital ptosis,strabismus,or completely healthy persons as control.Results:The positive rate of lymphocyte stimulation was34%(18/53)in anteri-or uveitis,41.5%(17/41)in posterior/pan-uveitis,and57.1%(12/21)in optic euritis,The results in the experimental groups were significantly different from those of the control group(x2=14.76,P<0.05,x2=19.14P<0.005,x2=26.38,P<0.005,respectively).Conclusion:The autoimmunity plays a role in the patogenesis in certain forms of uveitis and optic neuritis,Such immune responses may be secondary to the expo-sition or release of retinal antigens by various causes,leading to activation or augmentation of meager or low-affinity S antigen specific lymphocytes which may preexist in the circulation and starting the pathogenic autoimmune process.Eye Science 1995;11:120-123.

  9. Myxoma virus suppresses proliferation of activated T lymphocytes yet permits oncolytic virus transfer to cancer cells.

    Science.gov (United States)

    Villa, Nancy Y; Wasserfall, Clive H; Meacham, Amy M; Wise, Elizabeth; Chan, Winnie; Wingard, John R; McFadden, Grant; Cogle, Christopher R

    2015-06-11

    Allogeneic hematopoietic cell transplant (allo-HCT) can be curative for certain hematologic malignancies, but the risk of graft-versus-host disease (GVHD) is a major limitation for wider application. Ideally, strategies to improve allo-HCT would involve suppression of T lymphocytes that drive GVHD while sparing those that mediate graft-versus-malignancy (GVM). Recently, using a xenograft model, we serendipitously discovered that myxoma virus (MYXV) prevented GVHD while permitting GVM. In this study, we show that MYXV binds to resting, primary human T lymphocytes but will only proceed into active virus infection after the T cells receive activation signals. MYXV-infected T lymphocytes exhibited impaired proliferation after activation with reduced expression of interferon-γ, interleukin-2 (IL-2), and soluble IL-2Rα, but did not affect expression of IL-4 and IL-10. MYXV suppressed T-cell proliferation in 2 patterns (full vs partial) depending on the donor. In terms of GVM, we show that MYXV-infected activated human T lymphocytes effectively deliver live oncolytic virus to human multiple myeloma cells, thus augmenting GVM by transfer of active oncolytic virus to residual cancer cells. Given this dual capacity of reducing GVHD plus increasing the antineoplastic effectiveness of GVM, ex vivo virotherapy with MYXV may be a promising clinical adjunct to allo-HCT regimens.

  10. An automated flow cytometric micronucleus assay for human lymphocytes.

    Science.gov (United States)

    Schreiber, G A; Beisker, W; Braselmann, H; Bauchinger, M; Bögl, K W; Nüsse, M

    1992-12-01

    A new flow cytometric method is presented for scoring micronuclei (MN) in human lymphocytes after in vitro gamma-irradiation. Fifty to fifty-five hours after PHA-stimulation, the frequency of micronuclei per nucleus and the fraction of cells in the second cell cycle were measured using flow cytometry. All data were automatically analysed using our DAS-software package. Eight individual linear-quadratic dose response curves derived from five donors revealed inter- and intra-individual variabilities of all curve parameters. Since also an age dependence was found for spontaneous MN-frequencies and for the linear curve parameter, a combined linear-quadratic age-dose-effect model was used to fit the data. The 90% prediction intervals show that a reliable individual dose estimation for donors aged between 23 and 54 years cannot be achieved for exposures below 1 Gy.

  11. Post-thaw non-cultured and post-thaw cultured equine cord blood mesenchymal stromal cells equally suppress lymphocyte proliferation in vitro.

    Directory of Open Access Journals (Sweden)

    Lynn B Williams

    Full Text Available Multipotent mesenchymal stromal cells (MSC are receiving increased attention for their non-progenitor immunomodulatory potential. Cryopreservation is commonly used for long-term storage of MSC. Post-thaw MSC proliferation is associated with a lag-phase in vitro. How this lag-phase affect MSC immunomodulatory properties is unknown. We hypothesized that in vitro there is no difference in lymphocyte suppression potential between quick-thawed cryopreserved equine cord blood (CB MSC immediately included in mixed lymphocyte reaction (MLR and same MSC allowed post-thaw culture time prior to inclusion in MLR. Cryopreserved CB-MSC from five unrelated foals were compared using two-way MLR. For each of the five unrelated MSC cultures, paired MLR assays of MSC allowed five days of post-thaw culture and MSC included in MLR assay immediately post-thawing were evaluated. We report no difference in the suppression of lymphocyte proliferation by CB-MSC that had undergone post-thaw culture and MSC not cultured post-thaw (p<0.0001. Also, there was no inter-donor variability between the lymphocyte suppressive properties of MSC harvested from the five different donors (p = 0.13. These findings suggest that cryopreserved CB-MSC may have clinical utility immediately upon thawing. One implication hereof is the possibility of using cryopreserved CB-MSC at third party locations without the need for cell culture equipment or competencies.

  12. Flow Cytometric Analysis of Leishmania Reactive CD4+/CD8+ Lymphocyte Proliferation in Cutaneous Leishmaniasis

    Directory of Open Access Journals (Sweden)

    H Keshavarz

    2008-12-01

    Full Text Available Background: Determination of the division history of T cells in vitro is helpful in the study of effector mechanisms against infections. Technique described here uses the intracellular fluorescent label carboxyfluorescein diacetate succinimidyl ester (CFSE to monitor the proliferation. Methods: In a cross sectional study, blood samples were collected from 7 volunteers with history of cutaneous leishmania­sis (CL and one healthy control from endemic areas in Isfahan province who referred to the Center for Research and Training in Skin Diseases and Leprosy (CRTSDL, then CD4+/CD8+ lymphocytes and CD14+ monocytes were isolated from peri­pheral blood mononuclear cells (PBMC using mAbs and magnetic nanoparticles. CFSE labeled CD4+ or CD8+ lympho­cytes cultured with autologous monocytes in the presence of PHA, SLA, live Leishmania major or as control with­out sti­mulation. Cells were harvested after 7 days and were analyzed using flow cytometry. Results: Five consecutive divisions were monitored separately. Stimulation of CD4+ or CD8+ lymphocytes from CL sub­jects with SLA showed a significant difference in proliferation comparing with unstimulated cells (P< 0.05. The signifi­cant difference in the percentages of CD4+ cells stimulated with SLA was revealed at different divisions for each subject. In CD8+ lymphocyte, significant stronger stimulation of SLA was evident later in the proliferation process. The mean number of divisions in both CD4+/CD8+ lymphocytes stimulated with SLA was significantly greater than when stimulated with live L. major (P=0.007 / P=0.012, respectively Conclusion: The percentage of divided cells might be calculated separately in each division. The cells remained active following CFSE staining and there is possibility of functional analysis simultaneously.

  13. Effects of the protein kinase C stimulant bryostatin 1 on the proliferation and colony formation of irradiated human T-lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Sung, S.J.; Lin, P.-S.; Schmidt-Ullrich, R.; Hall, C.E.; Walters, J.A.; McCrady, C.; Grant, S. [Virginia Commonwealth Univ., Richmond, VA (United States)

    1994-12-01

    The protein kinase C stimulant bryostatin 1 (Bryo) was used in examining human peripheral blood T-lymphocyte radiosensitivities in proliferation assays. Bryo was similar to PMA in inducing T-cell proliferation by the CD3, CD28 and CD69 pathways. No difference in radiosensitivities was observed in T-cells stimulated by the three independent surface antigen-mediated activation pathways. CD3 was chosen as the second signal for comparing the potencies of the three different first signals Bryo, phorbol 12-myristate, 13-acetate (PMA), and interleukin 2 (IL-2) in stimulating T-cell proliferation and in maintaining this response after radiation. Though there were radioresponse differences among various individuals, the irradiated lymphocytes consistently showed significantly greater proliferation when treated with Bryo or PMA than with IL-2. These results support the important tole of protein kinase C in T-cell radiation responses, and suggest a potential role for Bryo in enhancing T-lymphocyte survival during radiation therapy. (author).

  14. A novel lectin from Artocarpus lingnanensis induces proliferation and Th1/Th2 cytokine secretion through CD45 signaling pathway in human T lymphocytes.

    Science.gov (United States)

    Cui, Bo; Li, Lu; Zeng, Qiyan; Lin, Faquan; Yin, Lijun; Liao, Liejun; Huang, Min; Wang, Jingping

    2017-04-01

    Lectins are carbohydrate-binding proteins and have been used for purification and characterization of glycoproteins. In this study, a novel 58.9-kDa tetrameric lectin from Artocarpus lingnanensis seeds was purified, characterized, and its mitogenic potential was evaluated. The hemagglutination inhibition assay indicated that Artocarpus lingnanensis lectin (ALL) showed specificity toward galactose. ALL was effectively purified in a single-step using affinity chromatography on a galactose-Sepharose column. ALL showed pH optima between 5.0 and 9.0, and optimal temperature between 20 and 40 °C. ALL triggered proliferation and activation of human T lymphocytes (e.g., CD4(+) T lymphocytes). Flow cytometry and laser scanning confocal microscopy revealed binding of ALL to T cells and colocalized with CD45. Affinity chromatography and Western blot suggested that CD45 isolated from human T cell membrane fraction may be the major receptor of ALL. CD45 blocking antibody attenuated the binding and proliferation of T cells induced by ALL. CD45-PTPase inhibitor dephostatin reduced ALL-induced T cells proliferation and expression of CD25 and pZAP-70. Furthermore, secretion of ALL-induced Th1/Th2 cytokines was blocked with dephostatin. Also, dephostatin inhibited phosphorylation of ALL-mediated activation of ERK and p38MAPK. This study demonstrates the involvement of CD45-mediated signaling in ALL-induced T lymphocyte proliferation and Th1/Th2 cytokine secretion through activation of p38 and ERK.

  15. Dose assessment intercomparisons within the RENEB network using G0-lymphocyte prematurely condensed chromosomes (PCC assay)

    OpenAIRE

    Terzoudi, Georgia I; Pantelias, Gabriel; Darroudi, Firouz; Barszczewska, Katarzyna; Buraczewska, Iwona; Depuydt, Julie; Georgieva, Dimka; Hadjidekova, Valeria; Hatzi, Vasiliki I.; Karachristou, Ioanna; Karakosta, Maria; Meschini, Roberta; M’kacher, Radhia; Montoro, Alegria; Palitti, Fabrizio

    2017-01-01

    Purpose: Dose assessment intercomparisons within the RENEB network were performed for triage biodosimetry analyzing G0-lymphocyte PCC for harmonization, standardization and optimization of the PCC assay. Materials and methods: Comparative analysis among different partners for dose assessment included shipment of PCC-slides and captured images to construct dose-response curves for up to 6 Gy c-rays. Accident simulation exercises were performed to assess the suitability of the PCC assay by d...

  16. Novel T lymphocyte proliferation assessment using whole mouse cryo-imaging

    Science.gov (United States)

    Wuttisarnwattana, Patiwet; Raza, Syed A.; Eid, Saada; Cooke, Kenneth R.; Wilson, David L.

    2014-03-01

    New imaging technologies enable one to assess T-cell proliferation, an important feature of the immunological response. However, none of the traditional imaging modalities allow one to examine quantiatively T-cell function with microscopic resolution and single cell sensitivity over an entire mouse. To address this need, we established T-cells proliferation assays using 3D microscopic cryo-imaging. Assays include: (1) biodistribution of T-cells, (2) secondary lymphoid organ (SLO) volume measurement, (3) carboxyfluorescein succinimidyl ester (CFSE) dilution per cell as cells divide. To demonstrate the application, a graft-versus-host-disease (GVHD) model was used. 3D visualization show that T-cells specifically homed to the SLOs (spleen and lymph nodes) as well as GVHD target organs (such as GI-tract, liver, skin and thymus).The spleen was chosen as representative of the SLOs. For spleen size analysis, volumes of red and white pulp were measured. Spleen volumes of the allogeneic mice (with GVHD) were significantly larger than those of the syngeneic mice (without GVHD) at 72 to 120 hours post-transplant. For CFSE dilution approach, we employed color-coded volume rendering and probability density function (PDF) of single cell intensity to assess T-cell proliferation in the spleen. As compared to syngeneic T-cells, the allogeneic T-cells quickly aggregated in the spleen as indicated by increasing of CFSE signal over the first 48 hours. Then they rapidly proliferated as evidenced by reduced CFSE intensity (at 48-96 hours). Results suggest that assays can be used to study GVHD treatments using T-cell proliferation and biodistibution as assays. In summary, this is the first time that we are able to track and visualize T-cells in whole mouse with single cell sensitivity. We believe that our technique can be an alternative choice to traditional in vitro immunological proliferation assays by providing assessment of proliferation in an in vivo model.

  17. IMMUNOMODULATION OF SYNTHESIZED POLYMERS CONTAINING PHOSPHORUS IN THE BACKBONE —EFFECT ON THE PROLIFERATION OF LYMPHOCYTES

    Institute of Scientific and Technical Information of China (English)

    ZhuoRenxi; WangJun; 等

    1997-01-01

    The immunomodulation of several Charged synthetic polymers containing phosphorus in the backbone was studied in vitro through examining their inhibition or promotion effect on the proliferatioin of both T and B lymphocytes,It is found that polymers based on long chain alkyl ester of tyrosine exhibit immunomodulative activity.Negatively charged polymers show stimulative activity on LPS-induced B lymphocytes proliferation.Positively charged polymers exhibit inhibitory activity on both Con A-induced T lymphocytes and LPS-induced B lymplhyocytes proliferation.

  18. Inhibitory effects of telmisartan on culture and proliferation of and Kv1.3 potassium channel expression in peripheral blood CD4+ T lymphocytes from Xinjiang Kazakh patients with hypertension

    Directory of Open Access Journals (Sweden)

    Sha-Sha Huang

    2016-10-01

    Full Text Available Introduction: Activation of T lymphocytes, for which potassium channels are essential, is involved in the development of hypertension. In this study, we explored the inhibitory effects of telmisartan on the culture and proliferation of and Kv1.3 potassium channel expression in peripheral blood CD4+ T lymphocytes derived from Xinjiang Kazakh patients with hypertension. Methods: CD4+ T-cell samples from hypertensive Kazakh patients and healthy Kazakh people were divided into healthy control, case control, telmisartan, and 4-aminopytidine groups. Changes in the expression levels of interleukin (IL-6 and IL-17 in the blood of the healthy control and case control subjects were detected by enzyme-linked immunosorbent assay. Peripheral blood CD4+ T lymphocytes were first activated and proliferated in vitro and then incubated for 0, 24, and 48 h under various treatment conditions. Thereafter, changes in CD4+ T-lymphocytic proliferation were determined using Cell Counting Kit-8 and microscope photography. Changes in messenger RNA (mRNA and protein expression of the Kv1.3 potassium channel in CD4+ T lymphocytes were detected using real-time quantitative polymerase chain reaction and Western blots, respectively. Results: The IL-6 and IL-17 expression levels were significantly higher in the blood of the hypertensive Kazakh patients than in the healthy Kazakh people. Telmisartan inhibited T-lymphocytic proliferation, as well as the mRNA and protein expression of the Kv1.3 potassium channel in CD4+ T lymphocytes, and the inhibitory effects were time-dependent, with the strongest inhibition observed after 48 h and significantly weaker inhibition observed after 24 h of treatment. Conclusions: Telmisartan may potentially regulate hypertensive inflammatory responses by inhibiting T-lymphocytic proliferation and Kv1.3 potassium channel expression in CD4+ T lymphocytes.

  19. Inhibitory effects of telmisartan on culture and proliferation of and Kv1.3 potassium channel expression in peripheral blood CD4+ T lymphocytes from Xinjiang Kazakh patients with hypertension.

    Science.gov (United States)

    Huang, Sha-Sha; Zhang, Qiu-Bing; Yuan, Qing-Yan; He, Si-Li; Zhang, Yuan-Ming

    2016-10-01

    Activation of T lymphocytes, for which potassium channels are essential, is involved in the development of hypertension. In this study, we explored the inhibitory effects of telmisartan on the culture and proliferation of and Kv1.3 potassium channel expression in peripheral blood CD4(+) T lymphocytes derived from Xinjiang Kazakh patients with hypertension. CD4(+) T-cell samples from hypertensive Kazakh patients and healthy Kazakh people were divided into healthy control, case control, telmisartan, and 4-aminopytidine groups. Changes in the expression levels of interleukin (IL)-6 and IL-17 in the blood of the healthy control and case control subjects were detected by enzyme-linked immunosorbent assay. Peripheral blood CD4(+) T lymphocytes were first activated and proliferated in vitro and then incubated for 0, 24, and 48 h under various treatment conditions. Thereafter, changes in CD4(+) T-lymphocytic proliferation were determined using Cell Counting Kit-8 and microscope photography. Changes in messenger RNA (mRNA) and protein expression of the Kv1.3 potassium channel in CD4(+) T lymphocytes were detected using real-time quantitative polymerase chain reaction and Western blots, respectively. The IL-6 and IL-17 expression levels were significantly higher in the blood of the hypertensive Kazakh patients than in the healthy Kazakh people. Telmisartan inhibited T-lymphocytic proliferation, as well as the mRNA and protein expression of the Kv1.3 potassium channel in CD4(+) T lymphocytes, and the inhibitory effects were time-dependent, with the strongest inhibition observed after 48 h and significantly weaker inhibition observed after 24 h of treatment. Telmisartan may potentially regulate hypertensive inflammatory responses by inhibiting T-lymphocytic proliferation and Kv1.3 potassium channel expression in CD4(+) T lymphocytes. © The Author(s) 2016.

  20. An Experimental Study on the Role of Nuclear Factor-κB in the Signal Conduction of Protein Kinase C Regulating the Proliferation and Apoptosis of T Lymphocytes in Asthma

    Institute of Scientific and Technical Information of China (English)

    熊维宁; 徐永健; 张珍祥; 王孝养

    2004-01-01

    To explore the role of nuclear factor-κB(NF-κB) in the signal pathway of protein kinase C (PKC) regulating the proliferation and apoptosis of T lymphocytes in asthma. T lymphocytes were isolated from the asthmatic model of guinea pigs and the asthmatic patients. Either the T cells stimulated with PMA alone or those stimulated with PMA together with pyrrolidine dithiocarbamate (PDTC) were incubated for 1 and 24 h. The proliferation of and the presence of NF-κB in the cells incubated for 1 h were observed by MTT and immunohistochemical staining, respectivelyAnd the cells incubated for 24 h were observed for the apoptosis by TUNEL. All the assays were paralleled with controls, and all the data were analyzedstatistically with the software SAS. The percentage of cells of nuclear positive staining of NF-scB and the proliferation of T lymphocytes from asthmatic guinea pigs and asthmatic patients stimulated with PMA were significantly higher than those of T lymphocytes from asthmatic guinea pigs and asthmatic patients stimulated without PMA respectively ( P < 0.01 ) and those of T lymphocytes from normal control guinea pigs and normal control persons stimulated with PMA respectively ( P < 0.01 ), and were significantly reduced by PDTC (P < 0.01 ). The apoptosis index of T lymphocytes from asthmatic guinea pigs and asthmatic patients stimulated with PMA were significantly lower than those of T lymphocytes from asthmatic guinea pigs and asthmatic patients stimulated without PMA respectively ( P < 0.01 ) and those of T lymphocytes from normal control guine apigs and normal control persons stimulated with PMA respectively ( P < 0.01 ), and were significantly induced by PDTC ( P< 0.01 ). There were good positive correlation between the percentage of cells of nuclear staining of NF-κB ofT lymphocytes and the proliferation of T lymphocytes ( r = 0.51-0.72, P < 0.001 ), and also good negative correlation between the percentage of cells of nuclear staining of NF-scB and the

  1. PROLIFERATION RESPONSES IN PREIMMUNIZED MICE LYMPHOCYTES BY BORDETELLA PERTUSSIS CELL WALL COMPONENTS

    Directory of Open Access Journals (Sweden)

    Ashraf Mohabbati Mobarez

    2003-03-01

    Full Text Available Bordetella pertussis infects the respiratory tract of the human host and causes whooping cough in children. The nature of immunity against Bordetella pertussis infection and disease is poorly understood. The aim of this study was to investigate cell mediated immunity in mice immunized with outer membrane component of cell wall, of B. Pertussis.A group of mice were immunized with outer membrane complex (OMC and killed whole cell (WCV of B. pertussis, with an interval of 2 weeks. During a period of 7 weeks following the immunization, lymphocytes were isolated from lymph nodes of immunized mice. The in vitro proliferative response of isolated lymphocyte to stimulation with 20 ^g of 30 and 69 kDa outer membrane protein (OMP were measured as parameters for cell mediated immunity (CMI. The data were expressed as mean count per minute (CPMxlO3 after subtraction of the CPM of unstimulated control cultures. Lymphoblastogenic response was observed in immunized mice with WCV and OMC. At 30 days of post immunization a significant increase in response to 30 and 69 kDa OMP was observed, a small decrease in the response was evident against P30 and P69 at 60 and 120 days of post immunization, but the response was still higher than what was observed in control mice.Current findings indicate strongly the potential of outer membrane protein component of B. pertussis in proliferating lymphocytes in the mice.

  2. β1-Adrenoceptor autoantibodies from DCM patients enhance the proliferation of T lymphocytes through the β1-AR/cAMP/PKA and p38 MAPK pathways.

    Directory of Open Access Journals (Sweden)

    Yunhui Du

    Full Text Available BACKGROUND: Autoantibodies against the second extracellular loop of the β(1-adrenergic receptor (β(1-AA not only contribute to increased susceptibility to heart failure, but also play a causative role in myocardial remodeling through their sympathomimetic-like effects that are induced upon binding to the β(1-adrenergic receptor. However, their role in the function of T lymphocytes has never been previously investigated. Our present study was designed to determine whether β(1-AA isolated from the sera of dilated cardiomyopathy (DCM patients caused the proliferation of T cells and the secretion of cytokines. METHODS: Blood samples were collected from 95 DCM patients as well as 95 healthy subjects, and β(1-AA was detected using ELISA. The CD3(+T lymphocytes were selected separately through flow cytometry and the effect of β(1-AA on T lymphocyte proliferation was examined by CCK-8 kits and CFSE assay. Western blotting was used to analyze the expressions of phospho-VASP and phospho-p38 MAPK. RESULTS: β(1-AA enhanced the proliferation of T lymphocytes. This effect could be blocked by the selective β(1-adrenergic receptor antagonist metoprolol, PKA inhibitor H89, and p38 MAPK inhibitor SB203580. Furthermore, the expression of the phosphorylated forms of phospho-VASP and phospho-p38 MAPK were markedly increased in the presence of β(1-AA. β(1-AA also inhibited the secretion of interferon-γ (IFN-γ while promoting an increase in interleukin-4 (IL-4 levels. CONCLUSIONS: These results demonstrate that β(1-AA isolated from DCM patients binds to β(1-AR on the surface of T cells, causing changes in T-cell proliferation and secretion through the β(1-AR/cAMP/PKA and p38 MAPK pathways.

  3. Sulfasalazine and mesalamine modulate beryllium-specific lymphocyte proliferation and inflammatory cytokine production.

    Science.gov (United States)

    Dobis, Dave R; Sawyer, Richard T; Gillespie, May M; Newman, Lee S; Maier, Lisa A; Day, Brian J

    2010-10-01

    Occupational exposure to beryllium (Be) results in Be sensitization (BeS) that can progress to pulmonary granulomatous inflammation associated with chronic Be disease (CBD). Be-specific lymphocytes are present in the blood of patients with BeS and in the blood and lungs of patients with CBD. Sulfasalazine and its active metabolite, mesalamine, are clinically used to ameliorate chronic inflammation associated with inflammatory bowel disease. We tested whether sulfasalazine or mesalamine could decrease Be-stimulated peripheral blood mononuclear cell (PBMC) proliferation in subjects with CBD and BeS and Be-induced cytokine production in CBD bronchoalveolar lavage (BAL) cells. CBD (n = 25), BeS (n = 12) and healthy normal control (n = 6) subjects were enrolled and ex vivo proliferation and cytokine production were assessed in the presence of Be and sulfasalazine or mesalamine. Be-stimulated PBMC proliferation was inhibited by treatment with either sulfasalazine or mesalamine. Be-stimulated CBD BAL cell IFN-γ and TNF-α cytokine production was decreased by treatment with sulfasalazine or mesalamine. Our data suggest that both sulfasalazine and mesalamine interfere with Be-stimulated PBMC proliferation in CBD and BeS and dampens Be-stimulated CBD BAL cell proinflammatory cytokine production. These studies demonstrate that sulfasalazine and mesalamine can disrupt inflammatory pathways critical to the pathogenesis of chronic granulomatous inflammation in CBD, and may serve as novel therapy for human granulomatous lung diseases.

  4. Effect of propolis on mitotic and cellular proliferation indices in human blood lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Montoro, A.; Almonacid, M.; Villaescusa, J. [Valencia Hospital Univ. la Fe, Servicio de Proteccion Radiologica (Spain); Barquinero, J. [Barcelona Univ. Autonom, Servicio de Dosimetria Biologica, Unidad de Antropologia, Dept. de Biologia Animal, Vegetal y Ecologia, barcelona (Spain); Barrios, L. [Barcelona Univ. Autonoma, Dept. de Biologia Celular y Fisiologia. Unidad de Biologia Celular (Spain); Verdu, G. [Valencia Univ. Politecnica, Dept. de Ingenieria Quimica y Nuclear (Spain); Perez, J. [Hospital la Fe, Seccion de Radiofisica, Servicio de Radioterapia, valencia (Spain)

    2006-07-01

    The study of the frequency of chromosomal aberrations per cell is the tool used in Biological dosimetry studies. Using dose-effect calibration curve obtained in our laboratory, we can evaluate the radioprotector effect of the EEP (ethanolic extract of propolis) in cultures in vitro. Propolis is the generic name for resinous substance collected by honeybees. The results showed a reduction in chromosomal aberrations's frequency of up to 50 %. The following study consisted of analyzing human peripheral blood lymphocytes exposed to 2 Gy {gamma} rays, in presence and absence of EEP, the change in the frequency of chromosome aberrations was analysed with biological dosimetry. The protection against the formation of dicentric and ring was dose-dependent, but there seemed to be a maximum protection, i.e. a further increase in the concentration of EEP does not show additional protection. This work studies the effect of the EEP of the cellular cycle using the mitotic and cellular proliferation index, as an alternative for the screening cytostatic activity. The results indicate that the lymphocytes which were cultures in presence of EEP exhibited a significant and dependent-concentration decrease in mitotic index and proliferation kinetics. The possible mechanisms involved in the radioprotective influence of EEP are discussed. (authors)

  5. Nuclear Phosphoproteomic Screen Uncovers ACLY as Mediator of IL-2-induced Proliferation of CD4+ T lymphocytes

    DEFF Research Database (Denmark)

    Osinalde, Nerea; Mitxelena, Jone; Sánchez-Quiles, Virginia;

    2016-01-01

    Anti-cancer immunotherapies commonly rely on the use of interleukin-2 (IL-2) to promote the expansion of T lymphocytes. IL-2- dependent proliferation is the culmination of a complex network of phosphorylation-driven signaling events that impact on gene transcription through mechanisms...... and inducing the expression of cell cycle regulating genes in response to IL-2. Thus, the metabolic enzyme ACLY emerges as a bridge between cytokine signaling and proliferation of T lymphocytes, and may be an attractive candidate target for the development of more efficient anti-cancer immunotherapies....

  6. ELISPOT Assay for Monitoring Cytotoxic T Lymphocytes (CTL Activity in Cancer Vaccine Clinical Trials

    Directory of Open Access Journals (Sweden)

    Thomas J. Sayers

    2012-05-01

    Full Text Available The profiling and monitoring of immune responses are key elements in the evaluation of the efficacy and development of new biotherapies, and a number of assays have been introduced for analyzing various immune parameters before, during, and after immunotherapy. The choice of immune assays for a given clinical trial depends on the known or suggested immunomodulating mechanisms associated with the tested therapeutic modality. Cell-mediated cytotoxicity represents a key mechanism in the immune response to various pathogens and tumors. Therefore, the selection of monitoring methods for the appropriate assessment of cell-mediated cytotoxicity is thought to be crucial. Assays that can detect both cytotoxic T lymphocytes (CTL frequency and function, such as the IFN-γ enzyme-linked immunospot assay (ELISPOT have gained increasing popularity for monitoring clinical trials and in basic research. Results from various clinical trials, including peptide and whole tumor cell vaccination and cytokine treatment, have shown the suitability of the IFN-γ ELISPOT assay for monitoring T cell responses. However, the Granzyme B ELISPOT assay and Perforin ELISPOT assay may represent a more direct analysis of cell-mediated cytotoxicity as compared to the IFN-γ ELISPOT, since Granzyme B and perforin are the key mediators of target cell death via the granule-mediated pathway. In this review we analyze our own data and the data reported by others with regard to the application of various modifications of ELISPOT assays for monitoring CTL activity in clinical vaccine trials.

  7. Human Recombinant B7-H3 Expressed in E.coli Enhances T Lymphocyte Proliferation and IL-10 Secretion in Vitro

    Institute of Scientific and Technical Information of China (English)

    Guang-Bo ZHANG; Yong-Jing CHEN; Qin SHI; Hong-Bing MA; Yan GE; Qin WANG; Zhi JIANG; Ying XU; Xue-Guang ZHANG

    2004-01-01

    To explore the biofunctions of human B7-H3 on activated T lymphocyte,the gene of human B7-H3 encoding the extracellular region (IgV-like and IgC-like domains) was obtained by RT-PCR from human lung cells and subcloned into the prokaryotic expression vector pGEX-5X-3 to express glutathione S-transferase (GST) fusion protein.A 49 kD fusion protein (named as GST/hB7-H3 hereafter) was induced by IPTG and purified by standard methods reported in prokaryotic system.In the presence of the first signal imitated by anti-CD3 monoclonal antibody,T lymphocyte proliferation was observed by incubating purified T cells with soluble GST/hB7-H3 fusion protein by MTT assay.The concentrations of IFN-γ and IL-10 in the supernatants of T cells were determined by ELISA.The results showed that the GST/hB7-H3 protein produced in bacteria had modest biological activities to proliferate the T lymphocyte and enhance IFN-γ as well as IL-10 secretion.

  8. Genotoxic evaluation of Halfenprox using the human peripheral lymphocyte micronucleus assay and the Ames test.

    Science.gov (United States)

    Akyıl, Dilek; Eren, Yasin; Konuk, Muhsin; Dere, Hatice; Serteser, Ahmet

    2017-04-01

    The genotoxicity and mutagenicity of Halfenprox, a synthetic pyrethroid insecticide and acaricide, was assessed using two standard genotoxicity assays of the Salmonella typhimurium mutagenicity assay (Ames test) and in vitro micronucleus (MN) assay in human peripheral lymphocytes. In the Ames test, Salmonella strains TA98 and TA100 were treated with or without S9 fraction. The doses of Halfenprox were 6.25, 12.5, 25, 50, and 100 μg/plate and test materials were dissolved in DMSO. The concentrations of Halfenprox did not show mutagenic activity on both strains with and without S9 fraction. The MN assay was used to investigate the genotoxic effects of Halfenprox in human peripheral lymphocytes treated with 250, 500, 750, and 1000 μg/ml concentrations of Halfenprox for 24 and 48 h, and at 1000 μg/ml the concentration was significantly increased and the MN formation was compared with the negative control for both treatment periods. In addition, a significant decrease of the nuclear devision index (NDI) values at the higher concentrations of Halfenprox and at both treatment periods was observed.

  9. The use of the alkaline comet assay with lymphocytes in human biomonitoring studies.

    Science.gov (United States)

    Faust, Floriane; Kassie, Fekadu; Knasmüller, Siegfried; Boedecker, Rolf Hasso; Mann, Marion; Mersch-Sundermann, Volker

    2004-05-01

    We reviewed the data of 45 alkaline comet assay studies with lymphocytes published during the last three years with the objective of monitoring human exposure to genotoxic agents as a result of occupation, drug treatment, diseases or environmental pollution. The strengths of the studies were that: (i) a lot of data could be obtained within a relatively short period of time in a cost-effective manner, (ii) lymphocytes could be easily collected in a non-invasive way and proved to be good surrogate cells in that they picked up effects caused by agents with different cancer target organs and (iii) a remarkable concordance between comet assay and cytogenetic assay data was proved. However, our analysis revealed some shortcomings of the studies such as: (i) the inclusion of low number of study participants and bias in the number and gender of subjects between control and exposed groups, (ii) lack of qualitative and quantitative exposure data, (iii) lack of consideration of differences in physical activity and diet between control and exposed groups, (iv) difficulty in comparison of the studies due to lack of uniformity in the comet assay procedures such as duration of alkali unwinding and electrophoresis, slide scoring method and the metrics used to assess the extent of DNA damage and (v) controversy in the sensitivity of comet assay since it picked up DNA damage caused by agents such as wood dust, pesticides and hormone preparations which were found to be weak genotoxins or non-genotoxins in other tests, but gave inconsistent results with known mutagens/carcinogens such as tobacco smoke. We feel that for the alkaline comet assay to be an important tool in human biomonitoring studies, serious consideration should be given to the flaws in the design and performance of the assay.

  10. Ambiguous nucleus regulates the proliferation and percentage of T lymphocytes in peripheral blood

    Institute of Scientific and Technical Information of China (English)

    Wei Wang; Wei Chen; Yingwu Mei; Bin Guo; Zhanqing Yang; Shoupeng Fu; Zhanpeng Yue; Juxiong Liu

    2011-01-01

    The aim of this study was to examine the immunomodulatory role of the unilateral ambiguous nucleus (Amb). We performed electrical stimulation of the unilateral Amb, electrical stimulation of the left parietal cortex and the lateral hypothalamus following unilateral Amb lesion, as well as microinjection of acetylcholine chloride and hemicholine-3 into the unilateral Amb, and electrical stimulation of the unilateral Amb after injection of atropine, mecamylamine, propranolol, and phentolamine. Results showed that the number and proliferation of peripheral blood T lymphocytes were increased after electrical stimulation of the unilateral Amb. The cholinergic neurons in the Amb released choline substances to alter cellular immunity, thus confirming that the Amb mediates the neuro-immunomodulatory process.

  11. Assessment of individual radiosensitivity in human lymphocytes using micronucleus and microgel electrophoresis Comet assays

    Energy Technology Data Exchange (ETDEWEB)

    Giorgio, M. di; Sardi, M.; Busto, M.; Vallerga, M.; Taja, M.; Mairal, I.

    2004-07-01

    Background and purpose: Individual radiosensitivity is an inherent characteristic, associated with an increased reaction to ionizing radiation on the human body. Individuals show marked differences in radiation sensitivity, which has consequences in the fields of both radiation protection and radiation therapy. It is suggested that DNA repair mechanisms are involved. Consequently, the characterization of DNA repair in lymphocytes through cytokinesis blocked micronucleus (MN) and alkaline single-cell microgel electrophoresis (comet) assays could be suitable approaches to evaluate individual radiosensitivity in vitro. The amins of this study were: 1) to assess the in vitro radisensitivity of peripheral blood lymphocytes from two with the observed clinical response and 2) to test the predictive potential of both techniques. Materials and methods: 38 cancer patients receiving radiation therapy were enrolled in this study. The tumor sites were: head and neck (n=25) and cervic (n=13). 19 pateints were evaluated prior, mid-way and on completion of treatment (prospective group) and 19 patients were evaluated about 2-480 month after radiotherapy (retrospective group). Cytogenetic data from the prospective group were analyzed using a mathematical model to evaluate the attenuation of the cytogenetic effect as a function of the time between a single exposure and blood sampling, estimating a cytogentic recovery factor k. In the retrospective group, blood samples were irradiated in vitro with 0 (control) or 2 Gy and evaluated using MN test. Cytogenetic data were analyzed comparing expected MN frequencies (calibration curve from health donors) with values observed after in vitro irradiation. One over-reactor ad patients that did not develop late effects were also evaluated through comet assay. DNA damage and repair capacity were quantified by the Olive tail moment. Lymphocytes of health individuals were used as reference sample. In the prospective evaluation, factor K correlated

  12. Assessment of DNA damage in underground coal miners using the cytokinesis-block micronucleus assay in peripheral blood lymphocytes.

    Science.gov (United States)

    Sinitsky, Maxim Yu; Minina, Varvara I; Gafarov, Nikolay I; Asanov, Maxim A; Larionov, Aleksey V; Ponasenko, Anastasia V; Volobaev, Valentin P; Druzhinin, Vladimir G

    2016-11-01

    Coal miners are exposed to coal dust, containing mineral particles, inorganic compounds and polycyclic aromatic hydrocarbons, and to ionizing radiation. These factors can induce oxidative stress and promote inflammation that leads to DNA damage. The aim of this investigation is to analyse the degree of DNA damage in miners working in underground coal mines in Kemerovo Region (Russian Federation) using the cytokinesis-block micronucleus assay (CBMN) in peripheral blood lymphocytes. The exposed group included 143 coal miners (mean age = 50.11±7.36 years; mean length of service in coal mining conditions = 23.26±9.66 years). As a control group, we have used venous blood extracted from 127 healthy non-exposed men. The mean age in this group was 47.67±8.45 years. We have discovered that coal miners are characterized by a significant increase in the frequency of binucleated lymphocytes with micronuclei (MN), nucleoplasmic bridges (NPBs) and protrusions (NBUDs) compared to non-exposed donors. In addition, we report, for the first time, a reduction of cell proliferation in a cohort of coal miners. These data are evidence of the genotoxic and cytostatic effects of occupational harmful factors of the coal mining industry. No correlation between the level of chromosome damage and age, smoking status or length of service in coal mining conditions were discovered. We suggest that the CBMN assay would be useful in biomonitoring studies to monitor hygiene and prevention strategies in occupational settings in coal mining countries. © The Author 2016. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  13. Effect of Protein Kinase C on Proliferation and Apoptosis of T Lymphocytes in Idiopathic Thrombocytopenic Purpura Children

    Institute of Scientific and Technical Information of China (English)

    Changlin Wu; Fang Liu; Xuemin Zhou; Zhengwei Cheng; Xiaomeng Yang; Hong Xiao; Qun Chen; Kangrong Cai

    2005-01-01

    It is well-documented that T lymphocyte proliferation and apoptosis are abnormal in idiopathic thrombocytopenic purpura (ITP) children. However, the underlying regulation mechanisms especially in terms of signal transduction remain unknown. In this paper, we reported the changes of protein kinase C (PKC) activity in peripheral blood T lymphocytes and the effect of PKC on T lymphocyte proliferation and apoptosis. We demonstrated that in ITP children, the activator (PMA) and inhibitor (H-7) of PKC affected on T lymphocyte proliferation and apoptosis dramatically, but they altered little in healthy children. PKC activity was significantly enhanced in ITP children together with an increased expression of FasL on CD3+ T, CD4+ T and CD8+T cells, resulting in a positive correlation between PKC activity and the expression of FasL on T cells. While the PKC activity and the platelet count were negatively correlated. Taken together, our findings suggest that the PKC activation may enhance T lymphocytes activity, suppress T cell apoptosis and be involve in thrombocytes damage as a mechanism related to immune pathogenesis of ITP.

  14. Setae from larvae of the northern processionary moth (Thaumetopoea pinivora, TP) stimulate proliferation of human blood lymphocytes in vitro.

    Science.gov (United States)

    Holm, Göran; Andersson, Margareta; Ekberg, Monica; Fagrell, Bengt; Sjöberg, Jan; Bottai, Matteo; Björkholm, Magnus

    2014-01-01

    Larvae of the Northern pine processionary moth (Thaumetopoea pinivora, TP) carry microscopic needles (setae), which by penetrating skin and mucous membranes, may cause inflammatory/immune derived symptoms in man. In the present study the stimulatory effects of setae on human blood lymphocytes in vitro was investigated. Blood mononuclear cells were separated from venous blood or buffy coat of ten healthy individuals, six previously exposed to setae and four with no known exposure. Lymphoproliferation was measured as uptake of 3H-thymidine. Setae were prepared from TP larvae. Setae and saline setae extracts stimulated proliferation of T-lymphocytes in the presence of monocytic cells. Stimulation was pronounced in cells from persons who had been exposed to setae, and weak in cells from non-exposed donors. Chitin also induced lymphocyte proliferation in most donors, but to a lesser extent and independently of donor's previous exposure to setae. In conclusion, setae contain molecules that in the presence of monocytes activate human T-lymphocytes to proliferation. The antigenic nature of stimulatory molecules was supported by the significantly stronger lymphocyte response in persons previously exposed to setae than in non-exposed donors. The nature of such molecules remains to be defined.

  15. Setae from larvae of the northern processionary moth (Thaumetopoea pinivora, TP stimulate proliferation of human blood lymphocytes in vitro.

    Directory of Open Access Journals (Sweden)

    Göran Holm

    Full Text Available Larvae of the Northern pine processionary moth (Thaumetopoea pinivora, TP carry microscopic needles (setae, which by penetrating skin and mucous membranes, may cause inflammatory/immune derived symptoms in man. In the present study the stimulatory effects of setae on human blood lymphocytes in vitro was investigated. Blood mononuclear cells were separated from venous blood or buffy coat of ten healthy individuals, six previously exposed to setae and four with no known exposure. Lymphoproliferation was measured as uptake of 3H-thymidine. Setae were prepared from TP larvae. Setae and saline setae extracts stimulated proliferation of T-lymphocytes in the presence of monocytic cells. Stimulation was pronounced in cells from persons who had been exposed to setae, and weak in cells from non-exposed donors. Chitin also induced lymphocyte proliferation in most donors, but to a lesser extent and independently of donor's previous exposure to setae. In conclusion, setae contain molecules that in the presence of monocytes activate human T-lymphocytes to proliferation. The antigenic nature of stimulatory molecules was supported by the significantly stronger lymphocyte response in persons previously exposed to setae than in non-exposed donors. The nature of such molecules remains to be defined.

  16. Regulatory substances produced by lymphocytes. V. Production of inhibitor of DNA synthesis (IDS) by proliferating T lymphocytes.

    Science.gov (United States)

    Namba, Y; Jegasothy, B V; Waksman, B H

    1977-04-01

    The conditions neccessary for production of inhibitor of DNA synthesis (IDS) by rat lymphocytes were investigated. In concanavalin A (Con A)-stimulated lymph node cell (LNC) cultures, IDS production was not detected in the culture supernatant during the first 24 hr, and it increased gradually after that to reach a maximum at 3 to 4 days. When the cells were pretreated with mitomycin C, IDS was not produced, suggesting that DNA synthesis of LNC or a LNC subpopulation is necessary for IDS production. In contrast, Con A-stimulated spleen cells priduced a high level of IDS within 24 hr, and its production fell off sharply thereafter. Con A-stimulated rat thymocytes also produced IDS reaching a maximum at 2 to 3 dyas. However, thymus cells from rats treated with hydrocortisone 48 hr previously did not produce IDS. This finding implies that cortisol-sensitive (cortical) thymocytes are capable of producing IDS and cortisol-resistant (medullary) thymocytes are not. IDS production by lymphoblasts was proportional to cell number and unaffected eith by cell density (1 to 10 x 106/ml) or by the concomitant presence of normal cells from spleen, lymph node, or thymus. Thus Con A-stimulated cells, after becoming blasts, appear to produce IDS automatically wihtout affecting or being affected by other cells. Both spleen and thymus cells from rats injected with a large dose of antigen (ovalbumin, 100 mg, i.p.) 24 hr in advance produced substantial amounts of IDS in culture within 24 hr in the absence of mitogen or additional antigen, but not the cells from rats injected with an immunizing dose (1 mg) of the same antigen. The cells producing IDS in the spleen were shown to be adherent to glass wool, and those in the thymus were partially so. IDS production by antigen-stimulated spleen cells was abrogated by injecting rats with bromodexyuridine (BUdR) at 0 and 12 hr after the ovalbumin. These findings suggest that a subpopulation ofadherent spleen cells (possibly resembling cortical

  17. Nuclear Phosphoproteomic Screen Uncovers ACLY as Mediator of IL-2-induced Proliferation of CD4+ T lymphocytes

    DEFF Research Database (Denmark)

    Osinalde, Nerea; Mitxelena, Jone; Sánchez-Quiles, Virginia

    2016-01-01

    Anti-cancer immunotherapies commonly rely on the use of interleukin-2 (IL-2) to promote the expansion of T lymphocytes. IL-2- dependent proliferation is the culmination of a complex network of phosphorylation-driven signaling events that impact on gene transcription through mechanisms that are no...

  18. Antigen-specific activation and proliferation of CD4+ and CD8+ T lymphocytes from brucellosis patients.

    Science.gov (United States)

    Moreno-Lafont, Martha Cecilia; López-Santiago, Rubén; Zumarán-Cuéllar, Elena; Paredes-Cervantes, Vladimir; López-Merino, Ahidé; Estrada-Aguilera, Ariel; Santos-Argumedo, Leopoldo

    2002-01-01

    Salt-extractable antigen from Brucella melitensis 16M (RCM-BM) was used to evaluate the immune response from acute and chronic patients suffering from Brucella infections (in Mexico); their responses were compared with those of healthy controls. As a readout we used upregulation of CD69 (a well-established early activation marker for lymphocytes), lymphocyte proliferation by 3[H]thymidine or 5-bromo-2-deoxyuridine (BrdU) incorporation measured by liquid scintillation or flow cytometry, respectively, and production of gamma interferon (IFN gamma). We compared the antigen-specific response with the response induced by phytohaemagglutinin (PHA) as a positive control. There was no difference between acute patients and the healthy controls in the percentages of CD3+, CD4+ or CD8+ lymphocytes. However, we found that chronic patients had a significant (P brucellosis patients and in CD8+ T lymphocytes in chronic patients, indicating that both populations became activated by this antigen preparation. Moreover, lymphocyte proliferation from both acute and chronic patients in response to RCM-BM was highly significant (P < 0.001) when compared with healthy controls. However, there were no apparent differences between acute and chronic patients. Although the incorporation of BrdU showed similar results it provided additional information, since we demonstrated that both CD4+ and CD8+ T lymphocytes from acute and chronic patients proliferated equally well in response to RCM-BM. Similar results were observed with intracellular IFN gamma determination. As a whole, our data suggest an important role for both CD4+ and CD8+ T lymphocytes in Brucella infection in humans. As has been reported in mice, it is feasible that activated CD8+ T cells participate in protection against Brucella in humans through cytotoxicity or/and by the production of factors such as interferon and granulysin. The role of these cells should be carefully analysed to understand better their participation in human

  19. Effect of cholinomimetics and adrenomimetics on proliferation of mouse B lymphocytes during primary immune response to protein antigen

    Energy Technology Data Exchange (ETDEWEB)

    Ado, A.D.; Dontsov, V.I.; Gol' dshtein, M.M.

    1985-12-01

    The aim of this investigation was to study the effect of neurotransmitters on proliferation of B lymphocytes induced by specific antigen. Experiments were carried out on female mice. To estimate proliferative activity, lymphocytes enriched with B cells were incubated in medium 199 for 2 h at 37 degrees C in a dose of 2.10/sup 6/-5.10/sup 6/ cells with 2 microCi of /sup 3/H-(methyl)-thymidine. The effect of acetylcholine on incorporation of /sup 3/H-thymidine into B lymphocytes of mice immunized with different doses of antigen during culture is shown. Discordance of effects of adrenalin and acetylcholine on incorporation of /sup 3/H-thymidine into B lymphocytes of mice immunized with different doses of ovalbumin is also shown.

  20. Recombinant human B7-H4 expressed in Escherichia coli inhibits T lymphocyte proliferation and IL-2 secretion in vitro

    Institute of Scientific and Technical Information of China (English)

    Yi-xiang MAO; Xue-guang ZHANG; Yong-Jing CHEN; Van GE; Hong-bing MA; Jian-feng YU; Hong-ya WU; Yu-min HU; Qin WANG; Qin SHI

    2006-01-01

    Aim: To explore the biofunctions of human B7-H4 generated from prokaryotic system. Methods: The gene of human B7-H4 extracellular region (IgⅤ-like and IgC-like domains) was obtained by PCR from human cDNA FLJ22418 and then inserted into the prokaryotic expression vector pGEX-5X-3 expressing glutathione. r-transferase (GST) fusion protein. After being identified by restriction enzyme digestion and sequencing, the recombinant vector was transferred into host strain E coli BL21-RIL(DE3). A 47 kDa fusion protein (GST/hB7-H4) was induced by isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified by standard methods reported in the prokaryotic system. The inhibitory effect of GST/hB7-H4 on proliferation of T cells was observed in vitro by CD3mAb activated T-cell cultur-ing system and [3H]-thymidine incorporation assay. The concentrations of interleukin-2 and iterferon-g in the supernatants of T cells were determined by ELISA. Results: We successfully constructed the method for high-level expression and purification of the hB7-H4 extracellular domain as GST fusion protein from E coli. The GST/hB7-H4 fusion protein produced in bacteria had obvious biological activity to inhibit T-lymphocyte proliferation and IL-2 secretion. Conclusion: The prokaryote expression system could be used to generate hB7-H4 protein with natural spatial conformations and biological functions, which provided an efficient and economical way for the preparation of this target protein.

  1. New and cost effective cell-based assay for Dialyzed Leukocyte Extract (DLE)-induced Jurkat cells proliferation under azathioprine treatment.

    Science.gov (United States)

    Cardoso, F M; Tomkova, M; Petrovajova, D; Bubanova, M; Ragac, O; Hornakova, T

    2017-01-31

    The human Dialyzed Leukocyte Extract (DLE) is a heterogeneous mix of oligopeptides of assays exist: E-rosette test, induction of delayed type hypersensitivity in mice, leukocyte migration and IFN-γ secretion. The animal-origin materials and in vivo assays convey a considerable logistic, ethic and economic burden, meanwhile the available in vitro assays have been reported with limited reproducibility and sometimes contradictory results. Here we are reporting a new DLE biological activity cell-based assay. The A20 and Jurkat cell lines were treated with (+Aza) or without (-Aza) azathioprine, DLE (+DLE) or both (+Aza/+DLE). After 72h, the cell proliferation was analyzed by the MTT or BrdU incorporation assays. In +Aza/+DLE treated cells, we observed a significant higher proliferation, when compared with +Aza/-DLE. In the absence of Aza, cells did not present any proliferation difference between -DLE or +DLE treatments. Both assays, MTT and BrdU showed similar results, being the MTT test more cost effective and we select it for validation as DLE biological assay using Jurkat cells only. We tested three different lyophilized DLE batches and we found consistent results with acceptable assay reproducibility and linearity. The DLE capacity for rescuing Jurkat cell proliferation during +Aza treatment was consistent using different liquid and lyophilized DLE batches, presenting also consistent chromatographic profiles. Finally, DLE treatment in Jurkat cells did not result into significant IL-2 of IFN-γ secretion, and known lymphocyte proliferative drugs failed to rescue Jurkat cells viability in presence of +Aza, as +DLE treatment did in our MTT assay. In conclusion, our new cell-based MTT assay has excellent DLE biological activity consistency, robustness and is cost effective, presenting important advantages over previous DLE activity in vitro and in vivo assays.

  2. Inhibition of murine splenic T lymphocyte proliferation by 2-deoxy-D-glucose-induced metabolic stress

    Science.gov (United States)

    Miller, E. S.; Klinger, J. C.; Akin, C.; Koebel, D. A.; Sonnenfeld, G.

    1994-01-01

    Female Swiss-Webster mice were injected with the glucose analogue 2-deoxy-D-glucose (2-DG), which when administered to rodents induces acute periods of metabolic stress. A single or multiple injections of 2-DG invoked a stress response, as evidenced by increases in serum corticosterone levels. The influence of this metabolic stressor on the blastogenic potential of splenic T lymphocytes was then examined. It was found that one, two, or three injections of 2-DG resulted in depressed T cell proliferative responses, with an attenuation of the effect occurring by the fifth injection. The 2-DG-induced inhibition of T cell proliferation was not attributable to 2-DG-induced cytolysis, as in vitro incubation of naive T cells with varying concentrations of 2-DG did not result in a reduction in cell number or viability, and flow cytometric analysis demonstrated that percentages of CD3, CD4, and CD8 splenic T cells were not altered as a result of 2-DG-induced stress. Incubating naive T cells in varying concentrations of 2-DG resulted in a dose-dependent inhibition of T cell blastogenic potential. Following in vivo exposure to 2-DG, T cell proliferation did not return to normal levels until 3 days after the cessation of 2-DG injections. Administering the beta-adrenergic receptor antagonist propranolol did not reverse the inhibited lymphoproliferation in 2-DG-treated mice. The inhibition in T cell proliferation was not observed, however, in mice that had been adrenalectomized or hypophysectomized and injected with 2-DG.(ABSTRACT TRUNCATED AT 250 WORDS).

  3. Effect of melatonin on monochromatic light-induced T-lymphocyte proliferation in the thymus of chickens.

    Science.gov (United States)

    Chen, Fuju; Reheman, Aikebaier; Cao, Jing; Wang, Zixu; Dong, Yulan; Zhang, Yuxian; Chen, Yaoxing

    2016-08-01

    A total of 360 post-hatching day 0 (P0) Arbor Acre male broilers, including intact, sham operation and pinealectomy groups, were exposed to white light (WL), red light (RL), green light (GL) and blue light (BL) from a light-emitting diode (LED) system until for P14. We studied the effects of melatonin and its receptors on monochromatic light-induced T-lymphocyte proliferation in the thymus of broilers. The density of proliferating cell nuclear antigen (PCNA) cells and the proliferation of T-lymphocytes in response to Concanavalin A (ConA) in GL significantly increased both in vivo and in vitro (from 9.57% to 32.03% and from 34.30% to 50.53%, respectively) compared with other lights (plights (p<0.005). However, exogenous melatonin (10(-9)M) significantly increased the proliferative activity of T-lymphocyte by 9.64% (p=0.002). In addition, GL significantly increased mRNA expression levels of Mel1a, Mel1b and Mel1c receptors from 21.09% to 32.57%, and protein expression levels from 24.43% to 42.92% compared with RL (p<0.05). However, these effects were blocked after pinealectomy. Furthermore, 4P-PDOT (a selective Mel1b antagonist) and prazosin (a selective Mel1c antagonist) attenuated GL-induced T-lymphocyte proliferation in response to ConA (p=0.000). Luzindole (a nonselective Mel1a/Mel1b antagonist), however, did not induce these effects (p=0.334). These results suggest that melatonin may mediate GL-induced T-lymphocyte proliferation via the Mel1b and Mel1c receptors but not via the Mel1a receptor.

  4. Proliferation assays for estrogenicity testing with high predictive value for the in vivo uterotrophic effect

    NARCIS (Netherlands)

    Wang, S.; Aarts, J.M.M.J.G.; Evers, N.M.; Peijnenburg, A.A.C.M.; Rietjens, I.; Bovee, T.F.H.

    2012-01-01

    Proliferation assays based on human cell lines are the most used in vitro tests to determine estrogenic properties of compounds. Our objective was to characterise to what extent these in vitro tests provide alternatives for the in vivo Allen and Doisy test, a uterotrophic assay in immature or

  5. Lymphocyte DNA damage in Turkish asphalt workers detected by the comet assay.

    Science.gov (United States)

    Bacaksiz, Aysegul; Kayaalti, Zeliha; Soylemez, Esma; Tutkun, Engin; Soylemezoglu, Tulin

    2014-01-01

    Asphalt has a highly complex structure and it contains several organic compounds including polycyclic aromatic hydrocarbons and heterocyclic compounds. In this study, comet assay was used to detect the DNA damage in blood lymphocytes of 30 workers exposed to asphalt fumes and 30 nonexposed controls. This is the first report on Turkish asphalt workers' investigated DNA damage using the alkaline single cell gel electrophoresis (SCGE). The DNA damage was evaluated by the percentage of DNA in the comet tail (% tail DNA) for each cell. According to our results, workers exposed to asphalt fumes had higher DNA damage than the control group (p asphalt fumes caused a significant increase in DNA damage and the comet assay is a suitable method for determining DNA damage in asphalt workers.

  6. Evaluation of the DNA damaging effects of amitraz on human lymphocytes in the Comet assay.

    Science.gov (United States)

    Radakovic, Milena; Stevanovic, Jevrosima; Djelic, Ninoslav; Lakic, Nada; Knezevic-Vukcevic, Jelena; Vukovic-Gacic, Branka; Stanimirovic, Zoran

    2013-03-01

    Amitraz is formamidine pesticide widely used as insecticide and acaricide. In veterinary medicine, amitraz has important uses against ticks, mites and lice on animals. Also, amitraz is used in apiculture to control Varroa destructor. It this study, the alkaline Comet assay was used to evaluate DNA damaging effects of amitraz in human lymphocytes. Isolated human lymphocytes were incubated with varying concentrations of amitraz (0.035, 0.35, 3.5, 35 and 350 mu g/mL). The Comet assay demonstrated that all concentrations of amitraz caused statistically significant increase in the level of DNA damage, thus indicating that amitraz possesses genotoxic potential. The concentration of amitraz that produced the highest DNA damage (3.5 mu g/mL) was chosen for further analysis with the antioxidant catalase. The obtained results showed that co-treatment with antioxidant catalase (100 IU/mL or 500 IU/mL) significantly reduced the level of DNA damage, indicating the possible involvement of reactive oxygen species in DNA damaging effects of amitraz. Flow cytometric analysis revealed increase of the apoptotic index following treatment with amitraz. However, co-treatment with catalase reduced the apoptotic index, while treatment with catalase alone reduced the percentage of apoptotoc cells even in comparison with the negative control. Therefore, catalase had protective effects against ROS-mediated DNA damage and apoptosis.

  7. Genotoxicity evaluation of drinking water sources in human peripheral blood lymphocytes using the comet assay

    Institute of Scientific and Technical Information of China (English)

    WU Yulin; CHEN Haigang; LI Zhaoli; SUN Liwei; QU Mengmeng; LI Mei; KONG Zhiming

    2008-01-01

    The potential harm of organic pollutants in drinking water to human health is widely focused on in the world; more and more pollutants with genotoxic substances are released into the aquatic environment. Water source samples were collected from 7 different localities of Nanjing City. The potential genotoxicity of organic extracts from drinking water sources were investigated by means of the comet assay in human peripheral lymphocytes. The results showed that all the organic extracts from all the water source samples could induce DNA damages of human peripheral blood lymphocytes at different levels. A significant difference (P < 0.01) was observed when compared with the solvent control. The DNA damage increased with the increase of the dosage of the original water source. Significant differences of DNA damage were observed in different drinking water sources, as shown by the multiple comparisons analysis at the dosage of 100×; the degree of DNA damage treated by Hushu waterworks (at town level) was the most serious, the arbitrary units (AU) was 141.62±6.96, however, that of Shangyuanmen waterworks (at city level) was only 109.64±2.97. The analysis also revealed that the genotoxicity of town's water sources was higher than that of the city. The results demonstrated that the comet assay can be successfully applied to the genotoxicity monitoring programs of drinking water sources.

  8. Evaluation of the DNA damaging effects of amitraz on human lymphocytes in the Comet assay

    Indian Academy of Sciences (India)

    Milena Radakovic; Jevrosima Stevanovic; Ninoslav Djelic; Nada Lakic; Jelena Knezevic-Vukcevic; Branka Vukovic-Gacic; Zoran Stanimirovic

    2013-03-01

    Amitraz is formamidine pesticide widely used as insecticide and acaricide. In veterinary medicine, amitraz has important uses against ticks, mites and lice on animals. Also, amitraz is used in apiculture to control Varroa destructor. It this study, the alkaline Comet assay was used to evaluate DNA damaging effects of amitraz in human lymphocytes. Isolated human lymphocytes were incubated with varying concentrations of amitraz (0.035, 0.35, 3.5, 35 and 350 g/mL). The Comet assay demonstrated that all concentrations of amitraz caused statistically significant increase in the level of DNA damage, thus indicating that amitraz possesses genotoxic potential. The concentration of amitraz that produced the highest DNA damage (3.5 g/mL) was chosen for further analysis with the antioxidant catalase. The obtained results showed that co-treatment with antioxidant catalase (100 IU/mL or 500 IU/mL) significantly reduced the level of DNA damage, indicating the possible involvement of reactive oxygen species in DNA damaging effects of amitraz. Flow cytometric analysis revealed increase of the apoptotic index following treatment with amitraz. However, co-treatment with catalase reduced the apoptotic index, while treatment with catalase alone reduced the percentage of apoptotoc cells even in comparison with the negative control. Therefore, catalase had protective effects against ROS-mediated DNA damage and apoptosis.

  9. Recombinant human erythropoietin treatment of chronic renal failure patients normalizes altered phenotype and proliferation of CD4-positive T lymphocytes.

    Science.gov (United States)

    Lisowska, Katarzyna A; Debska-Slizien, Alicja; Radzka, Monika; Witkowski, Jacek M; Rutkowski, Boleslaw; Bryl, Ewa

    2010-03-01

    Patients with chronic renal failure (CRF) receive recombinant human erythropoietin (rhEPO) for the correction of anemia. However, rhEPO also has an immunomodulatory effect. Detailed changes of phenotype and function of CD4(+) T lymphocytes in CRF patients receiving rhEPO have not been reported yet; their study may bring insight into understanding of this immunomodulatory action of rhEPO. Two groups of CRF patients were included into the study: those treated; and those not receiving rhEPO. The expression of activation markers on CD4(+) lymphocytes was measured with flow cytometry, both ex vivo and in vitro. The kinetics of CD4(+) T lymphocytes proliferation was calculated using a dividing cells tracing method and numerical approach. Significantly higher percentages of CD4(+)CD95(+), CD4(+)HLA-DR(+) cells, and lower percentages of CD4(+)CD69(+) and CD4(+)CD28(+) cells were observed in both rhEPO-treated and untreated patients when compared with healthy controls. Changes in the proportions of CD4(+)CD28(+) and CD4(+)HLA-DR(+) subpopulations were dependent on the type of rhEPO, being more pronounced for rhEPObeta. CD4(+) lymphocytes from untreated patients exhibited decreased expression of CD28 and CD69 after stimulation in vitro, whereas the expression of these antigens on lymphocytes of rhEPO-treated patients was similar to that observed in healthy controls. Fewer CD4(+)CD28(+) T lymphocytes of untreated patients proliferated in vitro; these cells had longer G0-->G1 time, which negatively correlated with surface expression of CD28. Our study confirms that rhEPO treatment normalizes activation parameters of CD4(+) T lymphocytes and their proliferative capacity, which could explain earlier described immunomodulatory effects of rhEPO in patients suffering from CRF.

  10. Human immune compartment comparisons: Optimization of proliferative assays for blood and gut T lymphocytes.

    Science.gov (United States)

    Dock, Jeffrey; Hultin, Lance; Hultin, Patricia; Elliot, Julie; Yang, Otto O; Anton, Peter A; Jamieson, Beth D; Effros, Rita B

    2017-03-21

    The accumulation of peripheral blood late-differentiated memory CD8 T cells with features of replicative (cellular) senescence, including inability to proliferate in vitro, has been extensively studied. Importantly, the abundance of these cells is directly correlated with increased morbidity and mortality in older persons. Of note, peripheral blood contains only 2% of the total body lymphocyte population. By contrast, the gut-associated lymphoid tissue (GALT) is the most extensive lymphoid organ, housing up to 60% of total body lymphocytes, but has never been assessed with respect to senescence profiles. We report here the development of a method for measuring and comparing proliferative capacity of peripheral blood and gut colorectal mucosa-derived CD8 T cells. The protocol involves a 5-day culture of mononuclear leukocyte populations, from blood and gut colorectal mucosa respectively, labeled with 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) and 5-bromo-2'-deoxyuridine (BrdU) and stimulated with anti-CD2/3/28-linked microbeads. Variables tested and optimized as part of the protocol development include: mode of T cell stimulation, CFSE concentration, inclusion of a second proliferation marker, BrdU, culture duration, initial culture concentration, and inclusion of autologous irradiated feeder cells. Moving forward, this protocol demonstrates a significant advance in the ability of researchers to study compartment-specific differences of in vitro proliferative dynamics of CD8 T cells, as an indicator of replicative senescence and immunological aging. The study's two main novel contributions are (1) Optimization and adaptation of standard proliferative dynamics blood T cell protocols for T cells within the mucosal immune system. (2) Introduction of the novel technique of combining CFSE and BrdU staining to do so.

  11. Suppression of lymphocyte proliferation by parainfluenza virus type 3-infected bovine alveolar macrophages.

    Science.gov (United States)

    Basaraba, R J; Brown, P R; Laegreid, W W; Silflow, R M; Evermann, J F; Leid, R W

    1993-06-01

    Lymphocytes stimulated with concanavalin A (Con A) or antigen in the presence of bovine parainfluenza virus type 3 (PIV-3) infected bovine alveolar macrophages (BAM) or monocytes, had depressed [3H]thymidine incorporation. This failure of lymphocytes to incorporate radiolabel required live virus, was time dependent and was most pronounced when BAM were infected for 48 hr prior to the addition of lymphocytes. The rate of infection of alveolar macrophages and the release of infectious virus into culture supernatants paralleled suppression of lymphocyte mitogenesis by PIV-3. However, the peak titre of exogenous, live or inactivated virus was not suppressive when added to lymphocyte macrophage cultures just prior to Con A stimulation. Neither the loss of viable alveolar macrophages nor a shift in antigen or mitogen dose response in virally infected cultures could account for the deficit in [3H]thymidine incorporation by lymphocytes. Despite the presence of lymphocyte-associated virus antigen detected by direct immunofluorescence, no increase in PIV-3 titre above baseline was seen from infected lymphocytes, irrespective of mitogen stimulation. Likewise, lymphocytes did not contribute to the extracellular virus pool in lymphocyte-macrophage cultures as the increases in viral titre above basal levels in supernatants were equal to levels released by macrophages alone. The expression of viral antigen on lymphocytes stimulated in the presence of PIV-3-infected BAM suggests a non-productive or abortive infection of lymphocytes mediated through contact with infected macrophages.

  12. The 15N and 46R Residues of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein Enhance Regulatory T Lymphocytes Proliferation.

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    Baochao Fan

    Full Text Available Porcine reproductive and respiratory syndrome virus (PRRSV negatively modulates host immune responses, resulting in persistent infection and immunosuppression. PRRSV infection increases the number of PRRSV-specific regulatory T lymphocytes (Tregs in infected pigs. However, the target antigens for Tregs proliferation in PRRSV infection have not been fully understood. In this study, we demonstrated that the highly pathogenic PRRSV (HP-PRRSV induced more CD4+CD25+Foxp3+ Tregs than classical PRRSV (C-PRRSV strain. Of the recombinant GP5, M and N proteins of HP-PRRSV expressed in baculovirus expression systems, only N protein induced Tregs proliferation. The Tregs assays showed that three amino-acid regions, 15-21, 42-48 and 88-94, in N protein played an important role in induction of Tregs proliferation with synthetic peptides covering the whole length of N protein. By using reverse genetic methods, it was firstly found that the 15N and 46R residues in PRRSV N protein were critical for induction of Tregs proliferation. The phenotype of induced Tregs closely resembled that of transforming-growth-factor-β-secreting T helper 3 Tregs in swine. These data should be useful for understanding the mechanism of immunity to PRRSV and development of infection control strategies in the future.

  13. The 15N and 46R Residues of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein Enhance Regulatory T Lymphocytes Proliferation.

    Science.gov (United States)

    Fan, Baochao; Liu, Xing; Bai, Juan; Li, Yufeng; Zhang, Qiaoya; Jiang, Ping

    2015-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) negatively modulates host immune responses, resulting in persistent infection and immunosuppression. PRRSV infection increases the number of PRRSV-specific regulatory T lymphocytes (Tregs) in infected pigs. However, the target antigens for Tregs proliferation in PRRSV infection have not been fully understood. In this study, we demonstrated that the highly pathogenic PRRSV (HP-PRRSV) induced more CD4+CD25+Foxp3+ Tregs than classical PRRSV (C-PRRSV) strain. Of the recombinant GP5, M and N proteins of HP-PRRSV expressed in baculovirus expression systems, only N protein induced Tregs proliferation. The Tregs assays showed that three amino-acid regions, 15-21, 42-48 and 88-94, in N protein played an important role in induction of Tregs proliferation with synthetic peptides covering the whole length of N protein. By using reverse genetic methods, it was firstly found that the 15N and 46R residues in PRRSV N protein were critical for induction of Tregs proliferation. The phenotype of induced Tregs closely resembled that of transforming-growth-factor-β-secreting T helper 3 Tregs in swine. These data should be useful for understanding the mechanism of immunity to PRRSV and development of infection control strategies in the future.

  14. Genotoxic and antigenotoxic effects of Fucus vesiculosus extract on cultured human lymphocytes using the chromosome aberration and Comet assays

    Directory of Open Access Journals (Sweden)

    Cleide Leite-Silva

    2007-01-01

    Full Text Available The brown seaweed Fucus vesiculosus (Fucales, Fucaceae was screened for its protective activity using doxorubicin-induced DNA damage in human lymphocytes. In this study, we assessed the genotoxic and antigenotoxic potential of three different concentrations (0.25, 0.5 and 1.0 mg mL-1 of F. vesiculosus aqueous extract using the chromosome aberration and Comet assays. Treatment of human lymphocyte cultures with 0.25, 0.5 and 1.0 mg mL-1 F. vesiculosus aqueous extract had no effect on the chromosome aberration frequency or on the extent of DNA damage detected by the Comet assay. The antigenotoxic effects of the extract were tested in human lymphocyte cultures treated with 15 µg mL-1 of doxorubicin, either alone or combined with the different concentrations of the extract, which was added to the cultures before, simultaneously with or after the doxorubicin. Only when lymphocytes were pre-treated with extract there was a reduction in doxorubicin-induced chromosome aberrations and DNA damage as detected by the Comet assay. These results demonstrate that F. vesiculosus aqueous extract is not genotoxic in cultured human lymphocytes and indicate that when added to lymphocyte cultures before doxorubicin it has antigenotoxic activity against doxorubicin-induced DNA damage.

  15. Mitogen-induced lymphocyte proliferation in loggerhead sea turtles: comparison of methods and effects of gender, plasma testosterone concentration, and body condition on immunity.

    Science.gov (United States)

    Keller, Jennifer M; McClellan-Green, Patricia D; Lee, A Michelle; Arendt, Mike D; Maier, Philip P; Segars, Al L; Whitaker, J David; Keil, Deborah E; Peden-Adams, Margie M

    2005-02-10

    A fully functioning immune system is vital to the survival of threatened and endangered sea turtles. Immunological protection against diseases in any organism can be reduced by a number of natural and anthropogenic factors, such as seasonal changes, malnutrition, disease states, and contaminant exposure. These factors are even more critical when they occur in endangered species or populations. To identify alterations in the immunological health of loggerhead sea turtles (Caretta caretta), the mitogen-induced lymphocyte proliferation (LP) assay was developed using peripheral blood leukocytes (PBLs). Collection and culture conditions were optimized for this assay using non-lethal blood samples collected from free-ranging turtles along the southeastern US coast. During the collection, two anticoagulants (sodium heparin and lithium heparin) were compared to determine effects of different ions on assay results. Optimal culture conditions were established for loggerhead PBLs while two different methods of measuring LP were compared: (1) the traditional radioactive (3)H-thymidine assay and (2) a non-radioactive, colorimetric method utilizing 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium (MTT). The results indicate that the (3)H-thymidine and the non-radioactive MTT methods did not correlate with each other and that the use of heparin type did not influence the results of the LP assay. Lastly, using these optimized methods, we investigated the effect of gender, plasma testosterone concentration, and body condition on LP in loggerhead turtles and found that none of the parameters largely influenced LP.

  16. The influence of galvanic currents and voltage on the proliferation activity of lymphocytes and expression of cell surface molecules.

    Science.gov (United States)

    Podzimek, S; Hána, K; Miksovský, M; Pousek, L; Matucha, P; Meloun, M; Procházková, J

    2008-01-01

    Release of metal ions from dental metal fillings supported by galvanism can cause local or general pathological problems in sensitive and genetically susceptible individuals. We aimed to investigate in vitro lymphocyte responses and expression of surface molecules influenced by galvanic currents and voltage. Human peripheral blood lymphocytes were influenced by galvanic currents and voltages and lymphocyte proliferation was measured. Control samples were not exposed to the influence of galvanism. We also studied the expression of surface molecules by the FACS analysis. A 15-h and shorter influence of almost all tested currents and voltages caused a significant decrease in lymphocyte proliferation and the 15-h influence of 20 microA currents significantly increased expression of surface molecules CD 19, 11a/18, 19/69 and 19/95. An influence of 10 and 3 microA currents led to a significant decrease in the expression of surface molecules CD 3, 11a/18, 3/69 and 3/95 and to a significant increase in CD 19 expression. An 80 mV voltage influence led to a significant decrease in the expression of surface molecules CD 3, 11a/18, 3/69, 3/95, 19/69 and 19/95, and 200 and 300 mV voltages significantly decreased the expression of surface molecules CD 3, 19, 11a/18, 3/95 and 19/95 and significantly increased CD 19/69 expression. A long-lasting influence of galvanism can, in sensitive and genetically susceptible individuals, influence lymphocyte proliferation and surface molecule expression. The threshold for pathological values of 5 microA for galvanic currents and 100 mV for galvanic voltage was confirmed.

  17. Dietary palmitic acid influences LDL-mediated lymphocyte proliferation differently to other mono- and polyunsaturated fatty acids in rats.

    Science.gov (United States)

    Tinahones, F J; Gómez-Zumaquero, J M; Monzón, A; Rojo-Martínez, G; Pareja, A; Morcillo, S; Cardona, F; Olveira, G; Soriguer, F

    2004-10-01

    Recent studies suggest that the biological effects of saturated fatty acids depend on the length of their chain. We compared the effect of diets containing different fatty acids on plasma lipids and lymphocyte proliferation in the presence of lovastatin and with increasing amounts of LDL. Lymphocytes from rats fed with a diet rich in palmitic acid had a greater lymphocyte proliferation capacity than those from rats fed with diets rich in oleic acid, linoleic acid, or fish oil. This effect was maintained when small amounts of polyunsaturatwed fatty acids (PUFA; sunflower oil) were added to the palmitic acid diet. LDL receptor activity, measured by the capacity of lovastatin to revert the inhibition of lymphocyte proliferation with increasing amounts of LDL in the medium, was greater in the rats fed with palmitic acid, and was similar to the other groups when small amounts of PUFA were added. All the groups had similar levels of plasma cholesterol, but the LDL levels were significantly lower in the group fed with palmitic acid plus PUFA. The highest HDL-cholesterol (HDLc) levels were found in the palmitic acid group and the lowest LDL-cholesterol (LDLc)/HDLc ratio in the palmitic acid plus PUFA group. These results suggest that diets rich in palmitic acid do not raise total cholesterol, but reduce LDLc or keep it normal, and raise HDLc levels. This effect may be partly due to an increase in LDL receptor activity. The inclusion of small amounts of PUFA in the diet rich in palmitic acid substantially modified the LDL receptor response in the lymphocytes, suggesting that the proportion of different families of dietary fatty acids may be more important than the individual amount of each in absolute terms to explain their effects on plasma lipids and lipoproteins.

  18. Nuclear Phosphoproteomic Screen Uncovers ACLY as Mediator of IL-2-induced Proliferation of CD4+ T lymphocytes.

    Science.gov (United States)

    Osinalde, Nerea; Mitxelena, Jone; Sánchez-Quiles, Virginia; Akimov, Vyacheslav; Aloria, Kerman; Arizmendi, Jesus M; Zubiaga, Ana M; Blagoev, Blagoy; Kratchmarova, Irina

    2016-06-01

    Anti-cancer immunotherapies commonly rely on the use of interleukin-2 (IL-2) to promote the expansion of T lymphocytes. IL-2- dependent proliferation is the culmination of a complex network of phosphorylation-driven signaling events that impact on gene transcription through mechanisms that are not clearly understood. To study the role of IL-2 in the regulation of nuclear protein function we have performed an unbiased mass spectrometry-based study of the nuclear phosphoproteome of resting and IL-2-treated CD4(+) T lymphocytes. We detected 8521distinct phosphosites including many that are not yet reported in curated phosphorylation databases. Although most phosphorylation sites remained unaffected upon IL-2 treatment, 391 sites corresponding to 288 gene products showed robust IL-2-dependent regulation. Importantly, we show that ATP-citrate lyase (ACLY) is a key phosphoprotein effector of IL-2-mediated T-cell responses. ACLY becomes phosphorylated on serine 455 in T lymphocytes upon IL-2-driven activation of AKT, and depletion or inactivation of ACLY compromises IL-2-promoted T-cell growth. Mechanistically, we demonstrate that ACLY is required for enhancing histone acetylation levels and inducing the expression of cell cycle regulating genes in response to IL-2. Thus, the metabolic enzyme ACLY emerges as a bridge between cytokine signaling and proliferation of T lymphocytes, and may be an attractive candidate target for the development of more efficient anti-cancer immunotherapies.

  19. MAJOR AND LYMPHOCYTE POPULATIONS OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES AND THEIR REFERENCE VALUES, AS ASSAYED BY MULTI-COLOUR CYTOMETRY

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    S. V. Khaidukov

    2009-01-01

    Full Text Available Abstract. Determination of lymphocyte subpopulations and their phenotypes is an important diagnostic feature, in order to elucidate some disturbances connected with immune system functioning. However, insufficient data are obtained when analyzing only major populations of peripheral lymphocytes. In order to perform clinical diagnostics, the data about minor lymphocytic populations and activated cellular pools seem to be more pertinent.Studies of peripheral blood cell subpopulations of healthy donors performed in different Russian regions allowed to assess quantitative distribution intervals for both major and minor immune cell subpopulations in humans. The results obtained, as compared with data from literature, provide an evidence for similar reference intervals for main immune cell subpopulations in healthy donors, independent on their habitation area.Present work has resulted into development of algorithms for cytometric studies and generation of certain panels of monoclonal antibodies enabling evaluation of all main lymphocyte subpopulations, as well as their minor subsets participating in emerging immune response. The distribution intervals have been estimated for such minor subpopulations, as B1- and B2-lymphocytes, memory B-cells, γδ- and αβT-cells, regulatory and naїve T-cells, cytotoxic and secretory NK-cell polupations.The results of present study, while been performed with peripheral blood of healthy donors, may provide a basis of reference values when studying subpopulation profile of immune cells.

  20. Response of Lymphocytes to Radiation in Untreated Breast Cancer Patients as Detected with Three Different Genetic Assays

    Institute of Scientific and Technical Information of China (English)

    JIAN-LIN LOU; ZHI-JIAN CHEN; JIANG WEI; JI-LIANG HE; LI-FEN JIN; SHI-JIE CHEN; WEI ZHENG; SHI-JIE XU

    2008-01-01

    To detect the response of lymphocytes to radiation in untreated breast cancer patients with three different genetic assays.Methods Blood samples were collected from 25 untreated patients and 25 controls.Each blood sample was divided into two parts:one was irradiated by 3-Gy X-ray (irradiated sample),the other was not irradiated (non-irradiated sample).The radiosensitivity of lymphocytes was assessed by comet assay,cytokinesis-block micronucleus (CBMN) assay and 6-TG-resistant cells scored (TG) assay.Results The baseline values of micronucleated cell frequency (MCF) and micronucleus frequency (MNF) in the patients were significantly higher than those in the controls (P<0.01),and 3-Gy X-ray induced genetic damage to lymphocytes in the patients increased significantly as compared with that in the controls as detected with the three genetic assays (P<0.01).The proportion of radiosensitive cases in the patient group was 48% for the mean tail length (MTL),40% for the mean tail moment (MTM),40% for MCE 44% for MNE and 48% for mutation frequencies of the hprt gene (Mfs-hprt),respectively,whereas the proportion of radiosensitive cases in the control group was only 8% for all the parameters.Conclusion The difference in the lymphocyte radiosensitivity between the breast cancer patients and the controls is significant.Moreover,there are wide individual variations in lymphocyte radiosensitivity of patients with breast cancer.In some cases,the radiosensitivity of the same patient may be different as detected with the different assays.It is suggested that multiple assays should be used to assess the radiosensitivity of patients with breast cancer before therapy.

  1. Cytotoxicity and Inhibition of Lymphocyte Proliferation of Fasciculatin, a Linear Furanosesterterpene Isolated from Ircinia variabilis Collected from the Atlantic Coast of Morocco

    OpenAIRE

    Werner Herz; Maria São José Nascimento; Nair Nazareth; Anake Kijjoa; Pinho, Paulo M.; Aziz Fassouane; Saida Rifai

    2005-01-01

    Abstract: Fasciculatin, a furanosesterterpene isolated from the marine sponge Ircinia variabilis from the Atlantic Coast of Morocco, has been evaluated for its influence on a mitogen-induced proliferation of human lymphocytes and growth of human tumor cell lines.

  2. Colchicum autumnale agglutinin activates all murine T-lymphocytes but does not induce the proliferation of all activated cells.

    Science.gov (United States)

    Bemer, V; Van Damme, E J; Peumans, W J; Perret, R; Truffa-Bachi, P

    1996-08-25

    Plant lectins with mitogenic properties for T-lymphocytes have been particularly useful for the study of T-cell activation and effector functions. In the search for mitogenic lectins possessing activation features different from the ones associated with the already known mitogens, we found that an agglutinin isolated from Colchicum autumnale tubers, Colchicum autumnale agglutinin (CAA), possesses interesting properties. First, contrasting with the classical mitogens, CAA induces the proliferation of a fraction of the CD4+ and CD8+ mouse T-lymphocytes. Second, the CAA-induced proliferation requires MHC class II and CD4 molecules. Third, although only a fraction of T-cells enters into the cell cycle, all T-lymphocytes are activated and express high levels of the activation markers CD69 and CD44. Finally, CAA-stimulation is characterized by a particular pattern of the cytokine gene expression, reflected by the transcription of the IL2, IL5, and IFN-gamma genes, while the IL4 and IL10 genes remained silent. Taken together these data demonstrate that CAA activation does not conform to the pathway of T-cell triggering observed with classical mitogenes and represents a new tool for the analysis of T-cell activation.

  3. In Vitro genotoxic and antigenotoxic studies of Thai Noni fruit juice by chromosomal aberration and sister chromatid exchange assays in human lymphocytes

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    Treetip Ratanavalachai

    2008-09-01

    Full Text Available The genotoxic and antigenotoxic effects of Noni fruit juice produced in Thailand have been studied in human lymphocytes for chromosome aberration assay and sister chromatid exchange (SCE assay in vitro. Treatment of Noni fruit juice(3.1-50 mg/ml alone for 3 h did not significantly induce chromosomal aberration or SCE (p<0.05. Noni fruit juice at 6.2 mg/ml is the optimum dose for cell survival and cell replication as demonstrated by the highest value of mitotic index and proliferation index (P.I.. Interestingly, pretreatment of Noni fruit juice at the same concentration of 6.2 mg/ml for 2 hfollowed by mitomycin C treatment at 3 μg/ml for 2 h significantly reduced SCE level induced by mitomycin C (p<0.05. However, these treatments did not show significant decrease in chromatid-type aberrations. Our data indicate that Thai Noni fruit juice is not genotoxic against human lymphocytes in vitro. In addition, pretreatment of Noni fruit juice at 6.2 mg/ml demonstrated no anticlastogenic effect while had some antigenotoxic effects as demonstrated by significant decrease in the SCE level induced by mitomycin C (p<0.05. Therefore, the optimum dose of Noni fruit juice used as a traditional medicine is required and needs to be studied further for the benefit of human health.

  4. A simple high-content cell cycle assay reveals frequent discrepancies between cell number and ATP and MTS proliferation assays.

    Directory of Open Access Journals (Sweden)

    Grace Ka Yan Chan

    Full Text Available In order to efficiently characterize both antiproliferative potency and mechanism of action of small molecules targeting the cell cycle, we developed a high-throughput image-based assay to determine cell number and cell cycle phase distribution. Using this we profiled the effects of experimental and approved anti-cancer agents with a range mechanisms of action on a set of cell lines, comparing direct cell counting versus two metabolism-based cell viability/proliferation assay formats, ATP-dependent bioluminescence, MTS (3-(4,5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl-2H-tetrazolium reduction, and a whole-well DNA-binding dye fluorescence assay. We show that, depending on compound mechanisms of action, the metabolism-based proxy assays are frequently prone to 1 significant underestimation of compound potency and efficacy, and 2 non-monotonic dose-response curves due to concentration-dependent phenotypic 'switching'. In particular, potency and efficacy of DNA synthesis-targeting agents such as gemcitabine and etoposide could be profoundly underestimated by ATP and MTS-reduction assays. In the same image-based assay we showed that drug-induced increases in ATP content were associated with increased cell size and proportionate increases in mitochondrial content and respiratory flux concomitant with cell cycle arrest. Therefore, differences in compound mechanism of action and cell line-specific responses can yield significantly misleading results when using ATP or tetrazolium-reduction assays as a proxy for cell number when screening compounds for antiproliferative activity or profiling panels of cell lines for drug sensitivity.

  5. Effects of dietary Fusarium mycotoxins on intestinal lymphocyte subset populations, cell proliferation and histological changes in avian lymphoid organs.

    Science.gov (United States)

    Girish, C K; Smith, T K; Boermans, H J; Anil Kumar, P; Girgis, G N

    2010-10-01

    An experiment was conducted to investigate the effects of dietary Fusarium mycotoxins on gut immunity, cell proliferation, and histology of avian lymphoid organs. The efficacy of a polymeric glucomannan mycotoxin adsorbent (GMA) was also determined. Seventy-two one-day-old male turkey poults were fed corn, wheat, and soybean meal-based diets for 21 days. Diets included control grains, contaminated grains and contaminated grains +0.2% GMA. The major contaminant was deoxynivalenol (3.9 μg/g) with lesser amounts of zearalenone (0.67-0.75 μg/g), 15-acetyl-deoxynivalenol (0.34 μg/g) and HT-2 toxin (0.078-0.085 μg/g). T- and B-lymphocyte populations and crypt cellular proliferation in duodenum, jejunum, ileum and cecal tonsil were measured immunohistochemically on day 14 and 21. Histological changes were recorded after 14 and 21 days of feeding. Feeding contaminated grains significantly increased the percentage of B-lymphocytes in ileum on day 14, and reduced (Pcontaminated diets also caused a reduction (Pcontaminated with Fusarium mycotoxins results in adverse effects on gut immunity and mucosal cell proliferation.

  6. Utility of lyophilized PMA and ionomycin to stimulate lymphocytes in whole blood for immunological assays.

    Science.gov (United States)

    Belouski, Shelley Sims; Wilkinson, Julie; Thomas, John; Kelley, Keith; Wang, Shen-Wu; Suggs, Sid; Ferbas, John

    2010-01-01

    The need to implement robust biomarkers in clinical trials has never been greater, and such efforts can be easily compromised by reagent instability or simple human error during assay set-up. Many biotechnology and pharmaceutical companies are introducing efforts to conduct biomarker studies under more rigorous settings, and the use of plates or tubes pre-loaded with stimulation or staining reagents could be of value for studies that involve flow cytometry. Five reagents lyophilized from ethanol or CHAPS buffer stock solution of phorbol 12-myristate 13-acetate (PMA) and ionomycin were benchmarked against standard DMSO liquid formulation for their stimulation equivalency. The median fluorescence intensity of phosphorylated ribosomal protein S6 in lymphocytes was assessed on a BD FACSCalibur. We demonstrate here that tubes pre-loaded with lyophilized versions of the liquid reagents can provide equivalent stimulation in healthy volunteer specimens. The value of this approach is that it safeguards against omission or erroneous addition of bulk liquid formulations of PMA and ionomycin to the reaction vessel (i.e., plate or tube) and also lends itself to extended stability/shelf-life of these reagents. On the basis of this initial success, we plan to expand our evaluation of lyophilized reagents so that they can be incorporated into our clinical biomarker campaigns as appropriate.

  7. Physiological crosstalk between the AC/PKA and PLC/PKC pathways modulates melatonin-mediated, monochromatic-light-induced proliferation of T-lymphocytes in chickens.

    Science.gov (United States)

    Guo, Qingyun; Wang, Zixu; Dong, Yulan; Cao, Jing; Chen, Yaoxing

    2017-06-28

    Previous study has demonstrated that melatonin plays a critical role in monochromatic-light-induced lymphocyte proliferation in response to T cell mitogen concanavalin A (ConA). However, its intracellular mechanism is still unclear. In this study, we investigate the intracellular signal pathways of melatonin receptor-mediated T-lymphocyte proliferation in the spleens of chicks exposed to different light wavelengths. Results showed that green light enhanced T-lymphocyte proliferation by 2.46-6.83% and increased splenic mRNA and protein expressions of melatonin receptor subtypes (Mel1a, Mel1b and Mel1c) by 16.05-40.43% compared with the white, red and blue light groups. However, pinealectomy resulted in a decrease in T-lymphocyte proliferation and melatonin receptor expression with no statistically significant differences between the different light groups. In vitro experiments showed that the Mel1b selective antagonist 4P-PDOT, the Mel1c selective antagonist prazosin and the mitogen-activated protein kinase kinase-1 (MEK-1) inhibitor PD98059 suppressed both melatonin-induced lymphocyte proliferation in response to ConA and melatonin- and ConA-stimulated extracellular signal-regulated kinase 1/2 (ERK1/2) activity but that the Mel1a/Mel1b non-selective antagonist luzindole did not. In addition, pretreatment with forskolin (FSK, the adenylyl cyclase activator), H89 (the PKA inhibitor), U73122 (the PLC inhibitor) or Go6983 (the broad spectrum PKC inhibitor) markedly attenuated melatonin- and ConA-stimulated T-lymphocyte proliferation and ERK1/2 activity. These results demonstrate that melatonin mediates green-light-induced T-lymphocyte proliferation via the Mel1b and Mel1c receptors by triggering crosstalk between the cAMP/PKA and PLC/PKC signal pathways followed by ERK1/2 activation.

  8. Autoantigenic targets of B-cell receptors derived from chronic lymphocytic leukemias bind to and induce proliferation of leukemic cells.

    Science.gov (United States)

    Zwick, Carsten; Fadle, Natalie; Regitz, Evi; Kemele, Maria; Stilgenbauer, Stephan; Bühler, Andreas; Pfreundschuh, Michael; Preuss, Klaus-Dieter

    2013-06-06

    Antigenic targets of the B-cell receptor (BCR) derived from malignant cells in chronic lymphocytic leukemia (CLL) might play a role in the pathogenesis of this neoplasm. We screened human tissue-derived protein macroarrays with antigen-binding fragments derived from 47 consecutive cases of CLL. An autoantigenic target was identified for 12/47 (25.5%) of the cases, with 3 autoantigens being the target of the BCRs from 2 patients each. Recombinantly expressed autoantigens bound specifically to the CLL cells from which the BCR used for the identification of the respective autoantigen was derived. Moreover, binding of the autoantigen to the respective leukemic cells induced a specific activation and proliferation of these cells. In conclusion, autoantigens are frequent targets of CLL-BCRs. Their specific binding to and induction of proliferation in the respective leukemic cells provide the most convincing evidence to date for the long-time hypothesized role of autoantigens in the pathogenesis of CLL.

  9. DNA repair capacity of cultured human lymphocytes exposed to mutagens measured by the comet assay and array expression analysis.

    Science.gov (United States)

    Bausinger, Julia; Speit, Günter

    2015-11-01

    Repair of mutagen-induced DNA lesions during transportation, storage and cultivation of lymphocytes may have a significant impact on results obtained in human biomonitoring after occupational and environmental exposure of human populations to genotoxic chemicals. Using the comet assay in combination with the repair inhibitor aphidicolin and array gene expression analysis of 92 DNA repair genes, we investigated the repair of DNA lesions induced by methyl methanesulfonate (MMS) and benzo[a]pyrenediolepoxide (BPDE) in phytohaemagglutinin (PHA)-stimulated cultured human lymphocytes in the time segment before replication. The comet assay indicated fast repair of MMS-induced damage during the first hours of cultivation. In contrast, removal of BPDE-induced lesions was slower and significant amounts of damage seem to persist until S-phase. Gene expression analysis revealed that PHA stimulation had a clear effect on gene regulation in lymphocytes already during the first 18h of cultivation. Under the conditions of this study, genotoxic concentrations of MMS did not induce significant changes in gene expression. In contrast, exposure to BPDE led to altered expression of several genes in a time- and concentration-related manner. Of the significantly up-regulated genes, only two genes (XPA and XPC) were directly related to nucleotide excision repair. Our results suggest that PHA stimulation of human lymphocytes influences the expression of DNA repair genes in human lymphocytes. The effect of induced DNA damage on gene expression is comparatively low and depends on the mutagens used. PHA-stimulated lymphocytes repair induced DNA damage before they start to replicate but the repair activity during the first 18h of cultivation is not affected by changes in the expression of DNA repair genes during this period of time.

  10. Effects of several salt marsh plants on mouse spleen and thymus cell proliferation using mtt assay

    Science.gov (United States)

    Seo, Youngwan; Lee, Hee-Jung; Kim, You Ah; Youn, Hyun Joo; Lee, Burm-Jong

    2005-12-01

    In the present study, we have tested the effects of 21 salt marsh plants on cell proliferation of mouse immune cells (spleen and thymus) using MTT assay in culture. The methanolic extracts of six salt marsh plants ( Rosa rugosa, Ixeris tamagawaensis, Artemisia capillaris, Tetragonia tetragonoides, Erigeron annus, and Glehnia littoralis) showed very powerful suppressive effects of mouse immune cell death and significant activities of cell proliferation in vitro. Especially, the methanolic extract of Rosa rugosa was found to have fifteen times compared to the control treatment, demonstrating that Rosa rugosa may have a potent stimulation effect on immune cell proliferation. These results suggest that several salt marsh plants including Rosa rugosa could be useful for further study as an immunomodulating agent.

  11. Activation and proliferation signals in primary human T lymphocytes inhibited by ergosterol peroxide isolated from Cordyceps cicadae

    Science.gov (United States)

    Kuo, Y C; Weng, S C; Chou, C J; Chang, T T; Tsai, W J

    2003-01-01

    Effects of ergosterol peroxide (C28H44O3; Cpd 6A) from Cordyceps cicadae on phytohemagglutinin (PHA)-stimulated cell proliferation were studied in primary human T cells. The results showed that Cpd 6A suppressed T-cell proliferation for about 24 h after stimulation with PHA. Cell cycle analysis indicated that Cpd 6A arrested the cell cycle progression of activated T cells from the G1 transition to the S phase. To localize the point in the cell cycle where arrest occurred, a set of key regulatory events leading to the G1/S boundary, including the expression of cyclins D2, E, A1, and B1, interleukin (IL)-2, IL-4, interferon-γ (IFN-γ), and activating protein-1 (AP-1), was examined. Cpd 6A suppressed, in activated T lymphocytes, the production and mRNA expression of cyclin E, IL-2, IL-4, IL-10, and IFN-γ in a dose-dependent manner. Expression of AP-1 proteins, consisting of c-Fos and c-Jun, in activated T lymphocytes was decreased by Cpd 6A. The kinetic study indicated that the inhibitory effects of Cpd 6A on IL-2 mRNA expressed in T cells might be related to blocking c-Fos protein synthesis. T-cell proliferation after Cpd 6A treatment was partially restored by addition of IL-2, IL-4, and IFN-γ. These suppressant effects of Cpd 6A on T-cell proliferation, activated by PHA, appeared to be mediated, at least in part, through the inhibition of early gene transcripts, especially those of cyclin E, IFN-γ, IL-2, and IL-4, and by arresting cell cycle progression in the cells. PMID:14504132

  12. Determination of genotoxic effects of methidathion alkaline hydrolysis in human lymphocytes using the micronucleus assay and square-wave voltammetry.

    Science.gov (United States)

    Stivaktakis, Polychronis D; Giannakopoulos, Evangelos; Vlastos, Dimitris; Matthopoulos, Demetrios P

    2017-02-01

    The interaction of pesticides with environmental factors, such as pH, may result in alterations of their physicochemical properties and should be taken into consideration in regard to their classification. This study investigates the genotoxicity of methidathion and its alkaline hydrolysis by-products in cultured human lymphocytes, using the square-wave voltammetry (square wave-adsorptive cathodic stripping voltammetry (SW-AdCSV) technique) and the cytokinesis block micronucleus assay (CBMN assay). According to the SW-AdCSV data the alkaline hydrolysis of methidathion results in two new molecules, one non-electro-active and a second electro-active which is more genotoxic than methidathion itself in cultured human lymphocytes, inducing higher micronuclei frequencies. The present study confirms the SW-AdCSV technique as a voltammetric method which can successfully simulates the electrodynamics of the cellular membrane.

  13. Induction and repair of DNA damage measured by the comet assay in human T lymphocytes separated by immunomagnetic cell sorting.

    Science.gov (United States)

    Bausinger, Julia; Speit, Günter

    2014-11-01

    The comet assay is widely used in human biomonitoring to measure DNA damage in whole blood or isolated peripheral blood mononuclear cells (PBMC) as a marker of exposure to genotoxic agents. Cytogenetic assays with phytohemagglutinin (PHA)-stimulated cultured T lymphocytes are also frequently performed in human biomonitoring. Cytogenetic effects (micronuclei, chromosome aberrations, sister chromatid exchanges) may be induced in vivo but also occur ex vivo during the cultivation of lymphocytes as a consequence of DNA damage present in lymphocytes at the time of sampling. To better understand whether DNA damage measured by the comet assay in PBMC is representative for DNA damage in T cells, we comparatively investigated DNA damage and its repair in PBMC and T cells obtained by immunomagnetic cell sorting. PBMC cultures and T cell cultures were exposed to mutagens with different modes of genotoxic action and DNA damage was measured by the comet assay after the end of a 2h exposure and after 18h post-incubation. The mutagens tested were methyl methanesulfonate (MMS), (±)-anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE), 4-nitroquinoline-1-oxide (4NQO), styrene oxide and potassium bromate. MMS and potassium bromate were also tested by the modified comet assay with formamido pyrimidine glycosylase (FPG) protein. The results indicate that the mutagens tested induce DNA damage in PBMC and T cells in the same range of concentrations and removal of induced DNA lesions occurs to a comparable extent. Based on these results, we conclude that the comet assay with PBMC is suited to predict DNA damage and its removal in T cells.

  14. Lymphocyte Redox Imbalance and Reduced Proliferation after a Single Session of High Intensity Interval Exercise

    OpenAIRE

    Tossige-Gomes, Rosalina; Costa, Karine Beatriz; Ottone, Vinícius de Oliveira; Magalhães, Flávio de Castro; Amorim, Fabiano Trigueiro; Rocha-Vieira, Etel

    2016-01-01

    This study investigated whether an acute session of high-intensity interval training (HIIT) is sufficient to alter lymphocyte function and redox status. Sixteen young healthy men underwent a HIIT session on a cycloergometer, consisting of eight bouts of 1 min at 90–100% of peak power, with 75 seconds of active recovery at 30 W between bouts. Venous blood was collected before, immediately after, and 30 minutes after the HIIT session. In response to Staphylococcus aureus superantigen B (SEB) st...

  15. Leukemia cell proliferation and death in chronic lymphocytic leukemia patients on therapy with the BTK inhibitor ibrutinib.

    Science.gov (United States)

    Burger, Jan A; Li, Kelvin W; Keating, Michael J; Sivina, Mariela; Amer, Ahmed M; Garg, Naveen; Ferrajoli, Alessandra; Huang, Xuelin; Kantarjian, Hagop; Wierda, William G; O'Brien, Susan; Hellerstein, Marc K; Turner, Scott M; Emson, Claire L; Chen, Shih-Shih; Yan, Xiao-Jie; Wodarz, Dominik; Chiorazzi, Nicholas

    2017-01-26

    BACKGROUND. Ibrutinib is an effective targeted therapy for patients with chronic lymphocytic leukemia (CLL) that inhibits Bruton's tyrosine kinase (BTK), a kinase involved in B cell receptor signaling. METHODS. We used stable isotopic labeling with deuterated water ((2)H2O) to measure directly the effects of ibrutinib on leukemia cell proliferation and death in 30 patients with CLL. RESULTS. The measured average CLL cell proliferation ("birth") rate before ibrutinib therapy was 0.39% of the clone per day (range 0.17%-1.04%); this decreased to 0.05% per day (range 0%-0.36%) with treatment. Death rates of blood CLL cells increased from 0.18% per day (average, range 0%-0.7%) prior to treatment to 1.5% per day (range 0%-3.0%) during ibrutinib therapy, and they were even higher in tissue compartments. CONCLUSIONS. This study provides the first direct in vivo measurements to our knowledge of ibrutinib's antileukemia actions, demonstrating profound and immediate inhibition of CLL cell proliferation and promotion of high rates of CLL cell death. TRIAL REGISTRATION. This trial was registered at clinicaltrials.gov (NCT01752426). FUNDING. This study was supported by a Cancer Center Support Grant (National Cancer Institute grant P30 CA016672), an NIH grant (CA081554) from the National Cancer Institute, MD Anderson's Moon Shots Program in CLL, and Pharmacyclics, an AbbVie company.

  16. CD8+T Lymphocyte Expansion, Proliferation and Activation in Dengue Fever

    OpenAIRE

    Andréia Manso de Matos; Karina Inacio Carvalho; Daniela Santoro Rosa; Lucy Santos Villas-Boas; Wanessa Cardoso da Silva; Célia Luiza de Lima Rodrigues; Olímpia Massae Nakasone Peel Furtado Oliveira; José Eduardo Levi; Evaldo Stanislau Affonso de Araújo; Claudio Sergio Pannuti; Expedito José Albuquerque Luna; Esper George Kallas

    2015-01-01

    Dengue fever induces a robust immune response, including massive T cell activation. The level of T cell activation may, however, be associated with more severe disease. In this study, we explored the level of CD8+ T lymphocyte activation in the first six days after onset of symptoms during a DENV2 outbreak in early 2010 on the coast of São Paulo State, Brazil. Using flow cytometry we detected a progressive increase in the percentage of CD8+ T cells in 74 dengue fever cases. Peripheral blood m...

  17. Damage of Splenic T Lymphocyte Proliferation and Differentiation and Its Normalization by Electroacupuncture in Morphine-Dependent Mice Mode

    Directory of Open Access Journals (Sweden)

    Hong-Yu Li

    2011-01-01

    Full Text Available In a previous paper we reported that electroacupuncture (EA could suppress opioid withdrawal syndrome and increase the appetite, sleep, and body weight in heroin addicts or morphine dependent animals. Considering that opioids were known to inhibit immune function, the present study was designed to observe whether EA could modulate the immune status of morphine dependent and withdrawal mice. We found that chronic morphine-induced decrease of splenic T lymphocyte proliferation and IL-2 production can be significantly raised by 2 Hz EA, and the fluctuation of CD4+/CD8+ ratio was also run to the baseline level by the EA. These findings indicated that chronic morphine exposure-induced immune dysfunction in mice could be normalized by 2 Hz EA.

  18. Proliferation capacity of T-lymphocytes is affected transiently after a long-term weight gain in Beagle dogs.

    Science.gov (United States)

    Van de Velde, H; Janssens, G P J; Rochus, K; Duchateau, L; Scharek-Tedin, L; Zentek, J; Nguyen, P; Cox, E; Buyse, J; Biourge, V; Hesta, M

    2013-04-15

    Across species obesity is associated with several disorders but in companion animals little information is available on the impact of chronic obesity on immune competence. The aim of the present study was to investigate whether weight gain and stable obese bodyweight affects the immune cell response. Obesity was induced in eight adult healthy beagle dogs (weight gain group; WGG) by a weight gain period (WGP) of 47 weeks, which was immediately followed by a period (stable period: SP) of stable obesity of 26 weeks. Eight adult healthy beagle dogs were included as a control group (CG) and remained at their ideal bodyweight throughout the entire study. Body composition was measured at five intervening time-points. Concentration of serum leptin and inflammatory cytokines, functionality of lymphocytes and phagocytic activity of neutrophils and monocytes were evaluated at ten intervening time-points. Serum leptin concentration was rising during the WGP in the WGG but went to lower concentrations during the SP. At the end of long-term weight gain, a decreased mitogen-induced proliferation of T-lymphocytes was noted but this alteration seemed to be transient after stabilization of bodyweight. This finding may imply an altered immune response for dogs with different energy balances. However, no systemic low grade inflammation or alteration in other immune cell functions was observed. Consequently it is suggested that the change in energy balance during the onset of obesity (becoming obese versus being obese), evokes an additional obesity-related disorder in dogs, i.e. impaired T-lymphocyte immune function.

  19. Nucleobase-Based Barbiturates: Their Protective Effect against DNA Damage Induced by Bleomycin-Iron, Antioxidant, and Lymphocyte Transformation Assay

    Directory of Open Access Journals (Sweden)

    Bhaveshkumar D. Dhorajiya

    2014-01-01

    Full Text Available A number of nucleobase-based barbiturates have been synthesized by combination of nucleic acid bases and heterocyclic amines and barbituric acid derivatives through green and efficient multicomponent route and one pot reaction. This approach was accomplished efficiently using aqueous medium to give the corresponding products in high yield. The newly synthesized compounds were characterized by spectral analysis (FT-IR, 1H NMR, 13C NMR, HMBC, and UV spectroscopy and elemental analysis. Representative of all synthesized compounds was tested and evaluated for antioxidant, bleomycin-dependent DNA damage, and Lymphocyte Transformation studies. Compounds TBC > TBA > TBG showed highest lymphocyte transformation assay, TBC > TBA > BG showed inhibitory antioxidant activity using ABTS methods, and TBC > BPA > BAMT > TBA > 1, 3-TBA manifested the best protective effect against DNA damage induced by bleomycin.

  20. Thymus derived inhibitor of lymphocyte proliferation : III—Partial purification and characteristic biological activity

    NARCIS (Netherlands)

    Rijke, E.O.; Ballieux, R.E.

    1980-01-01

    A crude factor isolated from bovine thymus was partially purified using ion exchange chromatography. The resulting fraction was tested for inhibitory properties towards the proliferation of various human and murine lymphoid cells in culture. It was found that this partially purified thymic factor (T

  1. CD8+ T lymphocyte expansion, proliferation and activation in dengue fever.

    Science.gov (United States)

    de Matos, Andréia Manso; Carvalho, Karina Inacio; Rosa, Daniela Santoro; Villas-Boas, Lucy Santos; da Silva, Wanessa Cardoso; Rodrigues, Célia Luiza de Lima; Oliveira, Olímpia Massae Nakasone Peel Furtado; Levi, José Eduardo; Araújo, Evaldo Stanislau Affonso; Pannuti, Claudio Sergio; Luna, Expedito José Albuquerque; Kallas, Esper George

    2015-02-01

    Dengue fever induces a robust immune response, including massive T cell activation. The level of T cell activation may, however, be associated with more severe disease. In this study, we explored the level of CD8+ T lymphocyte activation in the first six days after onset of symptoms during a DENV2 outbreak in early 2010 on the coast of São Paulo State, Brazil. Using flow cytometry we detected a progressive increase in the percentage of CD8+ T cells in 74 dengue fever cases. Peripheral blood mononuclear cells from 30 cases were thawed and evaluated using expanded phenotyping. The expansion of the CD8+ T cells was coupled with increased Ki67 expression. Cell activation was observed later in the course of disease, as determined by the expression of the activation markers CD38 and HLA-DR. This increased CD8+ T lymphocyte activation was observed in all memory subsets, but was more pronounced in the effector memory subset, as defined by higher CD38 expression. Our results show that most CD8+ T cell subsets are expanded during DENV2 infection and that the effector memory subset is the predominantly affected sub population.

  2. CD8+ T lymphocyte expansion, proliferation and activation in dengue fever.

    Directory of Open Access Journals (Sweden)

    Andréia Manso de Matos

    2015-02-01

    Full Text Available Dengue fever induces a robust immune response, including massive T cell activation. The level of T cell activation may, however, be associated with more severe disease. In this study, we explored the level of CD8+ T lymphocyte activation in the first six days after onset of symptoms during a DENV2 outbreak in early 2010 on the coast of São Paulo State, Brazil. Using flow cytometry we detected a progressive increase in the percentage of CD8+ T cells in 74 dengue fever cases. Peripheral blood mononuclear cells from 30 cases were thawed and evaluated using expanded phenotyping. The expansion of the CD8+ T cells was coupled with increased Ki67 expression. Cell activation was observed later in the course of disease, as determined by the expression of the activation markers CD38 and HLA-DR. This increased CD8+ T lymphocyte activation was observed in all memory subsets, but was more pronounced in the effector memory subset, as defined by higher CD38 expression. Our results show that most CD8+ T cell subsets are expanded during DENV2 infection and that the effector memory subset is the predominantly affected sub population.

  3. CD8+ T Lymphocyte Expansion, Proliferation and Activation in Dengue Fever

    Science.gov (United States)

    de Matos, Andréia Manso; Carvalho, Karina Inacio; Rosa, Daniela Santoro; Villas-Boas, Lucy Santos; da Silva, Wanessa Cardoso; Rodrigues, Célia Luiza de Lima; Oliveira, Olímpia Massae Nakasone Peel Furtado; Levi, José Eduardo; Araújo, Evaldo Stanislau Affonso; Pannuti, Claudio Sergio; Luna, Expedito José Albuquerque; Kallas, Esper George

    2015-01-01

    Dengue fever induces a robust immune response, including massive T cell activation. The level of T cell activation may, however, be associated with more severe disease. In this study, we explored the level of CD8+ T lymphocyte activation in the first six days after onset of symptoms during a DENV2 outbreak in early 2010 on the coast of São Paulo State, Brazil. Using flow cytometry we detected a progressive increase in the percentage of CD8+ T cells in 74 dengue fever cases. Peripheral blood mononuclear cells from 30 cases were thawed and evaluated using expanded phenotyping. The expansion of the CD8+ T cells was coupled with increased Ki67 expression. Cell activation was observed later in the course of disease, as determined by the expression of the activation markers CD38 and HLA-DR. This increased CD8+ T lymphocyte activation was observed in all memory subsets, but was more pronounced in the effector memory subset, as defined by higher CD38 expression. Our results show that most CD8+ T cell subsets are expanded during DENV2 infection and that the effector memory subset is the predominantly affected sub population. PMID:25675375

  4. Dissecting the T Cell Response: Proliferation Assays vs. Cytokine Signatures by ELISPOT

    Directory of Open Access Journals (Sweden)

    Magdalena Tary-Lehmann

    2012-05-01

    Full Text Available Chronic allograft rejection is in part mediated by host T cells that recognize allogeneic antigens on transplanted tissue. One factor that determines the outcome of a T cell response is clonal size, while another is the effector quality. Studies of alloimmune predictors of transplant graft survival have most commonly focused on only one measure of the alloimmune response. Because differing qualities and frequencies of the allospecific T cell response may provide distinctly different information we analyzed the relationship between frequency of soluble antigen and allo-antigen specific memory IFN-g secreting CD4 and CD8 T cells, their ability to secrete IL-2, and their proliferative capacity, while accounting for cognate and bystander proliferation. The results show proliferative responses primarily reflect on IL-2 production by antigen-specific T cells, and that proliferating cells in such assays entail a considerable fraction of bystander cells. On the other hand, proliferation (and IL-2 production did not reflect on the frequency of IFN-γ producing memory cells, a finding particularly accentuated in the CD8 T cell compartment. These data provide rationale for considering both frequency and effector function of pre-transplant T cell reactivity when analyzing immune predictors of graft rejection.

  5. Cytotoxicity and Inhibition of Lymphocyte Proliferation of Fasciculatin, a Linear Furanosesterterpene Isolated from Ircinia variabilis Collected from the Atlantic Coast of Morocco

    Directory of Open Access Journals (Sweden)

    Werner Herz

    2005-03-01

    Full Text Available Abstract: Fasciculatin, a furanosesterterpene isolated from the marine sponge Ircinia variabilis from the Atlantic Coast of Morocco, has been evaluated for its influence on a mitogen-induced proliferation of human lymphocytes and growth of human tumor cell lines.

  6. A three-dimensional organotypic assay to measure target cell killing by cytotoxic T lymphocytes.

    NARCIS (Netherlands)

    Weigelin, B.; Friedl, P.H.A.

    2010-01-01

    Cytotoxic T lymphocytes (CTL) mediate antigen- and cell-cell contact dependent killing of target cells, such as cancer cells and virus-infected cells. In vivo, this process requires the active migration of CTL towards and away from target cells. We here describe an organotypic 3D collagen matrix

  7. In vitro pituitary and thyroid cell proliferation assays and their relevance as alternatives to animal testing.

    Science.gov (United States)

    Jomaa, Barae; Aarts, Jac M M J G; de Haan, Laura H J; Peijnenburg, Ad A C M; Bovee, Toine F H; Murk, Albertinka J; Rietjens, Ivonne M C M

    2013-01-01

    This study investigates the in vitro effect of eleven thyroid-active compounds known to affect pituitary and/or thyroid weights in vivo, using the proliferation of GH3 rat pituitary cells in the so-called "T-screen," and of FRTL-5 rat thyroid cells in a newly developed test denoted "TSH-screen" to gain insight into the relative value of these in vitro proliferation tests for an integrated testing strategy (ITS) for thyroid activity. Pituitary cell proliferation in the T-screen was stimulated by three out of eleven tested compounds, namely thyrotropin releasing hormone (TRH), triiodothyronine (T3) and thyroxine (T4). Of these three compounds, only T4 causes an increase in relative pituitary weight, and thus T4 was the only compound for which the effect in the in vitro assay correlated with a reported in vivo effect. As to the newly developed TSH-screen, two compounds had an effect, namely, thyroid-stimulating hormone (TSH) induced and T4 antagonized FRTL-5 cell proliferation. These effects correlated with in vivo changes induced by these compounds on thyroid weight. Altogether, the results indicate that most of the selected compounds affect pituitary and thyroid weights by modes of action different from a direct thyroid hormone receptor (THR) or TSH receptor (TSHR)-mediated effect, and point to the need for additional in vitro tests for an ITS. Additional analysis of the T-screen revealed a positive correlation between the THR-mediated effects of the tested compounds in vitro and their effects on relative heart weight in vivo, suggesting that the T-screen may directly predict this THR-mediated in vivo adverse effect.

  8. Peripheral T-lymphocytes express WNT7A and its restoration in leukemia-derived lymphoblasts inhibits cell proliferation

    Directory of Open Access Journals (Sweden)

    Ochoa-Hernández Alejandra B

    2012-02-01

    Full Text Available Abstract Background WNT7a, a member of the Wnt ligand family implicated in several developmental processes, has also been reported to be dysregulated in some types of tumors; however, its function and implication in oncogenesis is poorly understood. Moreover, the expression of this gene and the role that it plays in the biology of blood cells remains unclear. In addition to determining the expression of the WNT7A gene in blood cells, in leukemia-derived cell lines, and in samples of patients with leukemia, the aim of this study was to seek the effect of this gene in proliferation. Methods We analyzed peripheral blood mononuclear cells, sorted CD3 and CD19 cells, four leukemia-derived cell lines, and blood samples from 14 patients with Acute lymphoblastic leukemia (ALL, and 19 clinically healthy subjects. Reverse transcription followed by quantitative Real-time Polymerase chain reaction (qRT-PCR analysis were performed to determine relative WNT7A expression. Restoration of WNT7a was done employing a lentiviral system and by using a recombinant human protein. Cell proliferation was measured by addition of WST-1 to cell cultures. Results WNT7a is mainly produced by CD3 T-lymphocytes, its expression decreases upon activation, and it is severely reduced in leukemia-derived cell lines, as well as in the blood samples of patients with ALL when compared with healthy controls (p ≤0.001. By restoring WNT7A expression in leukemia-derived cells, we were able to demonstrate that WNT7a inhibits cell growth. A similar effect was observed when a recombinant human WNT7a protein was used. Interestingly, restoration of WNT7A expression in Jurkat cells did not activate the canonical Wnt/β-catenin pathway. Conclusions To our knowledge, this is the first report evidencing quantitatively decreased WNT7A levels in leukemia-derived cells and that WNT7A restoration in T-lymphocytes inhibits cell proliferation. In addition, our results also support the possible

  9. Effects of Neuromedin S on the Proliferation of Splenic Lymphocytes and the Cytokine Secretion by Pulmonary Alveolar Macrophages in Pigs in vitro.

    Science.gov (United States)

    Lin, R; Wang, Q; Qi, B; Huang, Y; Yang, G

    2016-09-01

    Neuromedin S (NMS), a 36-amino acid neuropeptide, has been found to be involved in the regulation of the endocrine activity. It has been also detected in immune tissues in mammals, what suggests that NMS may play an important role in the regulation of immune response. The aim of this study was to demonstrate the presence of NMS receptor 1 (NMU1R) and effect of NMS in pig splenic lymphocytes (SPLs) and pulmonary alveolar macrophages (PAMs). The presence of NMU1R in pig SPLs and PAMs was respectively confirmed by reverse transcription-polymerase chain reaction (RT-PCR), western blot analysis and immunocytochemical methods. Furthermore, SPL proliferation was analyzed using the 3-(4,5)-dimethyl-thiahiazo-(-2-yl)-3,5-di-phenytetrazoliumromide (MTT) method. Additionally, the secretion of interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) in PAMs was all measured by enzyme-linked immunosorbent assay (ELISA) kits. In the present study, the results of RT-PCR and western blot analysis revealed that NMU1R mRNA and protein were both expressed in pig SPLs and PAMs, and the immunocytochemical investigations further revealed that the positive signal of NMU1R immunoreactivity was observed in plasma membranes of both SPLs and PAMs. In the in vitro study, we found that at concentrations of 0.001-1000 nM NMS alone or combined with lipopolysaccharide or phytohemagglutinin significantly increased SPL proliferation. Application of ELISA method showed that NMS could induce the secretion of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α in PAMs. These results suggest that NMS can act as a potently positive pro-inflammatory factor and immunomodulatory agent that affects the immune response of immune cells by combining with its receptor NMU1R.

  10. Improving the reproducibility of the MCF-7 cell proliferation assay for the detection of xenoestrogens.

    Science.gov (United States)

    Payne, J; Jones, C; Lakhani, S; Kortenkamp, A

    2000-03-29

    The MCF-7 cell proliferation assay is potentially a simple and highly reproducible tool for the identification of estrogenic compounds. However, its widespread use has been complicated by the lack of a standardised protocol, resulting in considerable inter-laboratory variability. We have explored the sources of variability both in relation to cell lines and test regimens and report on optimised procedures for the identification of estrogenic agents. Two supposedly identical MCF-7 parent cell lines (designated UCL and SOP), and the BUS subline were cultured according to an existing protocol, and responses to 17-estradiol (E2) assessed. Despite yielding almost identical EC50 values, the proliferative response varied widely between cell lines from 0.98-fold over controls (UCL) to 8.9-fold (BUS) indicating major differences between them. The underlying causes may be genetic, and to assess this we used comparative genomic hybridisation (CGH), a technique which allows the detection of DNA sequence copy number changes on a genome-wide scale. Although numerous similarities existed between the different cell lines, the least oestrogen-responsive line (MCF-7/UCL) exhibited the greatest number of cytogenetic changes, many of which were not seen in MCF-7/SOP cells. We suggest that care must be taken, therefore, when choosing a cell line for MCF-7 cell-based experiments. Selecting the MCF-7/SOP line for further work, we carried out a thorough and systematic optimisation of the MCF-7 cell proliferation assay, finding that a 72-h period in oestrogen-free medium before treatment strongly influenced the cells response to E2. With 1 nM E2, proliferation increased from 1.5-fold to 6.5-fold relative to vehicle-treated controls, a response similar to that seen with MCF-7/BUS cells in the E-SCREEN protocol devised by Soto et al. With parent MCF-7 cells, other laboratories have reported only 4.5-fold increases as maximal. Here we present evidence that the choice of cell line and culture

  11. Cytokine production and lymphocyte proliferation in patients with Nocardia brasiliensis actinomycetoma.

    Science.gov (United States)

    Méndez-Tovar, Luis J; Mondragón-González, Rafael; Vega-López, Francisco; Dockrell, Hazel M; Hay, Roderick; López-Martínez, Rubén; Manzano-Gayosso, Patricia; Hernández-Hernández, Francisca; Padilla-Desgarennes, Carmen; Bonifaz, Alexandro

    2004-11-01

    IFN-gamma, TNF-alpha, IL-4, IL-10 and IL-12 concentrations in the supernatant of peripheral blood mononuclear cell (PBMC) cultures and the in vitro proliferation of PBMC were studied in 25 patients with actinomycetoma caused by Nocardia brasiliensis and in 10 healthy controls from endemic zones. Cell cultures were stimulated by a N. brasiliensis crude cytoplasmic antigen (NB) and five semi-purified protein fractions (NB2, NB4, NB6, NB8, and NB10) separated by isoelectric. Phytohemagglutinin (PHA) and purified protein derivative (PPD) of Mycobacterium tuberculosis were used as control antigens. Skin tests were performed by injecting 0.1 ml of candidin and PPD intradermally (ID). Patients showed a poor response to tuberculin, while their response to candidin was more than two fold greater than that observed in the controls. Cell proliferation showed no statistically significant differences in either group. IFN-gamma production was higher in the healthy controls than in the patients, whereas TNF-alpha secretion was slightly higher in the patients' cultures. IL-4 was detected in the patients' cultures but not in the controls. IL-10 and IL-12 were present at low concentrations in both groups. These results suggest that patients with actinomycetoma show normal antigen recognition, but with low IFN-gamma production, and higher concentrations of IL-4, IL-10 and TNF-alpha in the patients' PBMC cultures, indicating that they probably have a Th2 type of immune response.

  12. Human hepatitis B viral e antigen and its precursor P20 inhibit T lymphocyte proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Purvina, Maija; Hoste, Astrid; Rossignol, Jean-Michel [Universite de Versailles-Saint-Quentin-en-Yvelines, Laboratoire de Genetique et Biologie Cellulaire, EA 4589, 45 avenue des Etats-Unis, 78035 Versailles (France); Lagaudriere-Gesbert, Cecile, E-mail: cecile.lagaudriere-gesbert@u-psud.fr [Universite de Versailles-Saint-Quentin-en-Yvelines, Laboratoire de Genetique et Biologie Cellulaire, EA 4589, 45 avenue des Etats-Unis, 78035 Versailles (France)

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer P20, precursor of the HBeAg, interacts with the cellular protein gC1qR. Black-Right-Pointing-Pointer HBeAg and P20 bind to T cell surface and inhibit mitogen-induced T cell division. Black-Right-Pointing-Pointer HBeAg and P20 inhibition of T cell proliferation is gC1qR and IL-1RAcP-independent. -- Abstract: The hepatitis B virus (HBV) Precore protein is processed through the secretory pathway directly as HBeAg or with the generation of an intermediate (P20). Precore gene has been shown to be implicated in viral persistence, but the functions of HBeAg and its precursors have not been fully elucidated. We show that the secreted proteins HBeAg and P20 interact with T cell surface and alter Kit-225 and primary T cells proliferation, a process which may facilitate the establishment of HBV persistence. Our data indicate that the N-terminal end of Precore is important for these inhibitory effects and exclude that they are dependent on the association of HBeAg and P20 with two characterized cell surface ligands, the Interleukin-1 Receptor Accessory Protein and gC1qR (present study).

  13. In vitro assessment of genotoxic effects of electric arc furnace dust on human lymphocytes using the alkaline comet assay.

    Science.gov (United States)

    Garaj-Vrhovac, Vera; Orescanin, Visnja; Ruk, Damir; Gajski, Goran

    2009-02-15

    In vitro genotoxic effects of leachates of electric arc furnace dust (EAFD) on human peripheral lymphocytes, assessed prior and following the treatment with a strong alkaline solution were investigated using the alkaline comet assay. Prior and following the treatment, lymphocytes were incubated with leachate of EAFD for 6 and 24 hours at 37 degrees C. Negative controls were also included. Mean values of the tail lengths established in the samples treated with the leachate stemming from the original dust for 6 and 24 hours, were 15.70 microm and 16.78 microm, respectively, as compared to 12.33 microm found in the control sample. Slight, but significant increase in the tail length was also found with the dust treated with a strong alkaline solution (13.37 microm and 13.60 microm). In case of high heavy metal concentrations (the extract of the original furnace dust), the incubation period was revealed to be of significance as well. The obtained results lead to the conclusion that alkaline comet assay could be used as a rapid, sensitive and low-cost tool when assessing genotoxicity of various waste materials, such as leachates of the electric arc furnace dust.

  14. Anchoring of c-myc on nuclear matrix proteins in process of mouse thymic T lymphocyte proliferation induced by ConA

    Institute of Scientific and Technical Information of China (English)

    曾丛梅; 蔡树涛; 周凤兰; 张锦珠; 王平

    1996-01-01

    Isolation and characteriation of functional nudear matrix proteins involved in DNA anchoring and gene expression is one of the major subjects of current nudear matrix research. Southwestern blotting (DNA-protein hybridization) was applied to studying the anchoring of c-myc on the nudear matrix proteins in mouse thymic T lymphocytes. The results showed that c-myc bound to the lamin, p34 and p36 nudear matrix proteins specifically. In the process of mouse thymic PNA T lymphocytes proliferation induced by ConA, the anchoring of c-myc on p34 and p36 nudear matrix proteins changed dynamically.

  15. Measuring melanoma-specific cytotoxic T lymphocytes elicited by dendritic cell vaccines with a tumor inhibition assay in vitro.

    Science.gov (United States)

    Paczesny, Sophie; Shi, Honhgzhen; Saito, Hiroaki; Mannoni, Patrice; Fay, Joseph; Banchereau, Jacques; Palucka, A Karolina

    2005-01-01

    Improving cancer vaccines depends on assays measuring elicited tumor-specific T-cell immunity. Cytotoxic effector cells are essential for tumor clearance and are commonly evaluated using 51Cr release from labeled target cells after a short (4 hours) incubation with T cells. The authors used a tumor inhibition assay (TIA) that assesses the capacity of cytotoxic T lymphocytes (CTLs) to control the survival/growth of EGFP-labeled tumor cell lines. TIA was validated using CD8+ T cells primed in vitro against melanoma and breast cancer cells. TIA was then used to assess the CTL function of cultured CD8+ T cells isolated from patients with metastatic melanoma who underwent vaccination with peptide-pulsed CD34+ HPCs-derived DCs. After the DC vaccination, T cells from six of eight patients yielded CTLs that could inhibit the survival/growth of melanoma cells. The results of TIA correlated with killing of tumor cells in a standard 4-hour 51Cr release assay, yet TIA allowed detection of CTL activities that appeared marginal in the 51Cr release assay. Thus, TIA might prove valuable for measuring spontaneous and induced antigen-specific cytotoxic T cells.

  16. Study of the genotoxic and cytotoxic effects of the α-, β-, and γ- Hexachlorocyclohexane isomers in human lymphocyte cells using the cytokinesis-block micronucleus assay.

    Science.gov (United States)

    Ennaceur, Soukaina

    2017-01-01

    The genotoxic potential of hexachlorocyclohexane (HCH) isomers (α-, β-, and γ-) which are organochlorine pesticides was tested in peripheral blood lymphocyte cultures from two donors by using the cytokinesis-block micronucleus assay. Micronucleus (MN) frequency, binucleated cells with micronucleus (BNMN), and cytokinesis-blocked proliferation index (CBPI) were determined as genotoxic and cytotoxic endpoints. At the concentration ranges tested (12.5-100 μg.L (-1)), all HCH isomers induced dose-dependent cytotoxic effects, γ-HCH being the most toxic. This isomer was also able to induce significant increase in MN frequency and BNMN cells indicating a genotoxic potential at 50 and 100 μg.L (-1). The genotoxic test of β-HCH showed a positive induction of MN and BNMN cells at the highest concentration of 100 μg.L (-1) and a significant cytotoxicity at 50 μg.L (-1). Under the experimental condition used, α-HCH was unable to induce any significant increase in MN frequency confirming that α-HCH is a non-genotoxic agent.

  17. Electrochemical sensors, MTT and immunofluorescence assays for monitoring the proliferation effects of cissus populnea extracts on Sertoli cells

    Directory of Open Access Journals (Sweden)

    McGee Dennis

    2011-05-01

    Full Text Available Abstract Background We describe the development of an electrochemical sensor array for monitoring the proliferation effects of cissus populnea plant extracts on TM4 Sertoli cells. Methods The proliferation activities of the extracts on Sertoli cells were studied using a high-throughput electrochemical sensor array (DOX-96 and the analytical sensor characteristics were compared with conventional colorimetric MTT (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay and fluorescence spectroscopy. Results This work shows that there is a definite positive trend in the proliferation effect of the extract of Cissus populnea on the TM4 Sertoli cells. All of the three techniques confirmed that the most effective concentration for the proliferation is 10 ppm. At this concentration, the proliferation effect was established around 120% for both DOX-96 and MTT techniques, whereas fluorescence assays showed a higher level (120-150%. DOX-96 showed a lower limit of detection (1.25 × 10(4 cells/ml; whereas the LOD recorded for both MTT and fluorescence techniques was 2.5 × 10(4 cells/ml. Visual examination of the cells by means of confocal fluorescence microscopy confirmed the proliferation of Sertoli cells as was determined using the MTT assay. This investigation provides a confident interpretation of the results and proved that the most effective concentration for the proliferation using Cissus populnea plant extract is 10 ppm. Conclusions Overall, the DOX results compared well with the conventional methods of checking proliferation of cells. The fascinating feature of the sensor array is the ability to provide continuous proliferation experiments with no additional reagents including 96 simultaneous electrochemical experiments. The use of the DOX-96 could reduce a typical bioassay time by 20-fold. Thus the DOX-96 can be used as both a research tool and for practical cell culture monitoring.

  18. New Molecular Assay for the Proliferation Signature in Mantle Cell Lymphoma Applicable to Formalin-Fixed Paraffin-Embedded Biopsies.

    Science.gov (United States)

    Scott, David W; Abrisqueta, Pau; Wright, George W; Slack, Graham W; Mottok, Anja; Villa, Diego; Jares, Pedro; Rauert-Wunderlich, Hilka; Royo, Cristina; Clot, Guillem; Pinyol, Magda; Boyle, Merrill; Chan, Fong Chun; Braziel, Rita M; Chan, Wing C; Weisenburger, Dennis D; Cook, James R; Greiner, Timothy C; Fu, Kai; Ott, German; Delabie, Jan; Smeland, Erlend B; Holte, Harald; Jaffe, Elaine S; Steidl, Christian; Connors, Joseph M; Gascoyne, Randy D; Rosenwald, Andreas; Staudt, Louis M; Campo, Elias; Rimsza, Lisa M

    2017-05-20

    Purpose Mantle cell lymphoma is an aggressive B-cell neoplasm that displays heterogeneous outcomes after treatment. In 2003, the Lymphoma/Leukemia Molecular Profiling Project described a powerful biomarker-the proliferation signature-using gene expression in fresh frozen material. Herein, we describe the training and validation of a new assay that measures the proliferation signature in RNA derived from routinely available formalin-fixed paraffin-embedded (FFPE) biopsies. Methods Forty-seven FFPE biopsies were used to train an assay on the NanoString platform, using microarray gene expression data of matched fresh frozen biopsies as a gold standard. The locked assay was applied to pretreatment FFPE lymph node biopsies from an independent cohort of 110 patients uniformly treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone. Seventeen biopsies were tested across three laboratories to assess assay reproducibility. Results The MCL35 assay, which contained a 17-gene proliferation signature, yielded gene expression of sufficient quality to assign an assay score and risk group in 108 (98%) of 110 archival FFPE biopsies. The MCL35 assay assigned patients to high-risk (26%), standard-risk (29%), and low-risk (45%) groups, with different lengths of overall survival (OS): a median of 1.1, 2.6, and 8.6 years, respectively (log-rank for trend, P < .001). In multivariable analysis, these risk groups and the Mantle Cell Lymphoma International Prognostic Index were independently associated with OS ( P < .001 for both variables). Concordance of risk assignment across the three independent laboratories was 100%. Conclusion The newly developed and validated MCL35 assay for FFPE biopsies uses the proliferation signature to define groups of patients with significantly different OS independent of the Mantle Cell Lymphoma International Prognostic Index. Importantly, the analytic and clinical validity of this assay defines it as a reliable biomarker to

  19. Long-lasting inhibitory effects of fetal liver mesenchymal stem cells on T-lymphocyte proliferation.

    Directory of Open Access Journals (Sweden)

    Massimo Giuliani

    Full Text Available Human bone marrow mesenchymal stem cells (BM-MSC are multipotent progenitor cells that have transient immunomodulatory properties on Natural Killer (NK cells, Dendritic Cells (DC, and T cells. This study compared the use of MSC isolated from bone marrow and fetal liver (FL-MSC to determine which displayed the most efficient immunosuppressive effects on T cell activation. Although both types of MSC exhibit similar phenotype profile, FL-MSC displays a much more extended in vitro life-span and immunomodulatory properties. When co-cultured with CD3/CD28-stimulated T cells, both BM-MSC and FL-MSC affected T cell proliferation by inhibiting their entry into the cell cycle, by inducing the down-regulation of phospho-retinoblastoma (pRb, cyclins A and D1, as well as up-regulating p27(kip1 expression. The T cell inhibition by MSC was not due to the soluble HLA-G5 isoform, but to the surface expression of HLA-G1, as shown by the need of cell-cell contact and by the use of neutralizing anti-HLA-G antibodies. To note, in a HLA-G-mediated fashion, MSC facilitated the expansion of a CD4(low/CD8(low T subset that had decreased secretion of IFN-γ, and an induced secretion of the immunomodulatory cytokine IL-10. Because of their longer lasting in vitro immunosuppressive properties, mainly mediated by HLA-G, and their more efficient induction of IL-10 production and T cell apoptosis, fetal liver MSC could be considered a new tool for MSC therapy to prevent allograft rejection.

  20. Lymphocyte transformation assay for C neoformans antigen is not reliable for detecting cellular impairment in patients with Neurocryptococcosis

    Directory of Open Access Journals (Sweden)

    Rocha Katya C

    2012-10-01

    Full Text Available Abstract Background Cryptococcus neoformans causes meningitis and disseminated infection in healthy individuals, but more commonly in hosts with defective immune responses. Cell-mediated immunity is an important component of the immune response to a great variety of infections, including yeast infections. We aimed to evaluate a specific lymphocyte transformation assay to Cryptococcus neoformans in order to identify immunodeficiency associated to neurocryptococcosis (NCC as primary cause of the mycosis. Methods Healthy volunteers, poultry growers, and HIV-seronegative patients with neurocryptococcosis were tested for cellular immune response. Cryptococcal meningitis was diagnosed by India ink staining of cerebrospinal fluid and cryptococcal antigen test (Immunomycol-Inc, SP, Brazil. Isolated peripheral blood mononuclear cells were stimulated with C. neoformans antigen, C. albicans antigen, and pokeweed mitogen. The amount of 3H-thymidine incorporated was assessed, and the results were expressed as stimulation index (SI and log SI, sensitivity, specificity, and cut-off value (receiver operating characteristics curve. We applied unpaired Student t tests to compare data and considered significant differences for p Results The lymphotoxin alpha showed a low capacity with all the stimuli for classifying patients as responders and non-responders. Lymphotoxin alpha stimulated by heated-killed antigen from patients with neurocryptococcosis was not affected by TCD4+ cell count, and the intensity of response did not correlate with the clinical evolution of neurocryptococcosis. Conclusion Response to lymphocyte transformation assay should be analyzed based on a normal range and using more than one stimulator. The use of a cut-off value to classify patients with neurocryptococcosis is inadequate. Statistical analysis should be based on the log transformation of SI. A more purified antigen for evaluating specific response to C. neoformans is needed.

  1. Evaluation of estrogenic potential of flavonoids using a recombinant yeast strain and MCF7/BUS cell proliferation assay.

    Science.gov (United States)

    Resende, Flávia A; de Oliveira, Ana Paula S; de Camargo, Mariana S; Vilegas, Wagner; Varanda, Eliana A

    2013-01-01

    Phytoestrogens are of interest because of their reported beneficial effects on many human maladies including cancer, neurodegeneration, cardiovascular disease and diabetes. Furthermore, there is a search for compounds with estrogenic activity that can replace estrogen in hormone replacement therapy during menopause, without the undesirable effects of estrogen, such as the elevation of breast cancer occurrence. Thus, the principal objective of this study was to assess the estrogenic activity of flavonoids with different hydroxylation patterns: quercetin, kaempferol, luteolin, fisetin, chrysin, galangin, flavone, 3-hydroxyflavone, 5-hydroxyflavone and 7-hydroxyflavone via two different in vitro assays, the recombinant yeast assay (RYA) and the MCF-7 proliferation assay (E-screen), since the most potent phytoestrogens are members of the flavonoid family. In these assays, kaempferol was the only compound that showed ERα-dependent transcriptional activation activity by RYA, showing 6.74±1.7 nM EEQ, besides acting as a full agonist for the stimulation of proliferation of MCF-7/BUS cells. The other compounds did not show detectable levels of interaction with ER under the conditions used in the RYA. However, in the E-screen assay, compounds such as galangin, luteolin and fisetin also stimulated the proliferation of MCF-7/BUS cells, acting as partial agonists. In the evaluation of antiestrogenicity, the compounds quercetin, chrysin and 3-hydroxyflavone significantly inhibited the cell proliferation induced by 17-β-estradiol in the E-screen assay, indicating that these compounds may act as estrogen receptor antagonists. Overall, it became clear in the assay results that the estrogenic activity of flavonoids was affected by small structural differences such as the number of hydroxyl groups, especially those on the B ring of the flavonoid.

  2. Evaluation of estrogenic potential of flavonoids using a recombinant yeast strain and MCF7/BUS cell proliferation assay.

    Directory of Open Access Journals (Sweden)

    Flávia A Resende

    Full Text Available Phytoestrogens are of interest because of their reported beneficial effects on many human maladies including cancer, neurodegeneration, cardiovascular disease and diabetes. Furthermore, there is a search for compounds with estrogenic activity that can replace estrogen in hormone replacement therapy during menopause, without the undesirable effects of estrogen, such as the elevation of breast cancer occurrence. Thus, the principal objective of this study was to assess the estrogenic activity of flavonoids with different hydroxylation patterns: quercetin, kaempferol, luteolin, fisetin, chrysin, galangin, flavone, 3-hydroxyflavone, 5-hydroxyflavone and 7-hydroxyflavone via two different in vitro assays, the recombinant yeast assay (RYA and the MCF-7 proliferation assay (E-screen, since the most potent phytoestrogens are members of the flavonoid family. In these assays, kaempferol was the only compound that showed ERα-dependent transcriptional activation activity by RYA, showing 6.74±1.7 nM EEQ, besides acting as a full agonist for the stimulation of proliferation of MCF-7/BUS cells. The other compounds did not show detectable levels of interaction with ER under the conditions used in the RYA. However, in the E-screen assay, compounds such as galangin, luteolin and fisetin also stimulated the proliferation of MCF-7/BUS cells, acting as partial agonists. In the evaluation of antiestrogenicity, the compounds quercetin, chrysin and 3-hydroxyflavone significantly inhibited the cell proliferation induced by 17-β-estradiol in the E-screen assay, indicating that these compounds may act as estrogen receptor antagonists. Overall, it became clear in the assay results that the estrogenic activity of flavonoids was affected by small structural differences such as the number of hydroxyl groups, especially those on the B ring of the flavonoid.

  3. Results of The Analysis of The Blood Beryllium Lymphocyte Proliferation Test Data From The Oak Ridge Y-12 Study

    Energy Technology Data Exchange (ETDEWEB)

    Frome, EL

    2001-12-18

    The potential hazards from exposure to beryllium or beryllium compounds in the workplace were first reported in the 1930s. The tritiated thymidine beryllium lymphocyte proliferation test (BeLPT) is an in vitro blood test that is widely used to screen beryllium exposed workers in the nuclear industry for sensitivity to beryllium. Newman [18] has discussed the clinical significance of the BeLPT and described a standard protocol that was developed in the late 1980s. Cell proliferation is measured by the incorporation of tritiated thymidine into dividing cells on two culture dates and using three concentrations of beryllium sulfate. Results are expressed as a ''stimulation index'' (SI) which is the ratio of the amount of tritiated thymidine (measured by beta counts) in the stimulated cells divided by the counts for the unstimulated cells on the same culture day. Several statistical methods for use in the routine analysis of the BeLPT were considered in the early 1990's by Frome et al. [7]. The least absolute values (LAV) method was recommended for routine analysis of the BeLPT. The purposes of this report are to further evaluate the LAV method using new data, and to describe a new method for identification of an abnormal or borderline test. This new statistical biological positive (SBP) method reflects the clinical judgment that (1) at least two SIs show a ''positive'' response to beryllium, and (2), that the maximum of the six SIs must exceed a cut point that is determined from a reference data set of normal individuals whose blood has been tested by the same method in the same serum. The new data is from the Y-12 facility in Oak Ridge and consist of 1080 worker and 33 nonexposed control BeLPTs (all tested in the same serum). Graphical results are presented to explain the statistical method, and the new SBP method is applied to the Y-12 group. The true positive rate and specificity of the new method were estimated to be 86

  4. Results of The Analysis of The Blood Beryllium Lymphocyte Proliferation Test Data From The Oak Ridge Y-12 Study

    Energy Technology Data Exchange (ETDEWEB)

    Frome, EL

    2001-12-18

    The potential hazards from exposure to beryllium or beryllium compounds in the workplace were first reported in the 1930s. The tritiated thymidine beryllium lymphocyte proliferation test (BeLPT) is an in vitro blood test that is widely used to screen beryllium exposed workers in the nuclear industry for sensitivity to beryllium. Newman [18] has discussed the clinical significance of the BeLPT and described a standard protocol that was developed in the late 1980s. Cell proliferation is measured by the incorporation of tritiated thymidine into dividing cells on two culture dates and using three concentrations of beryllium sulfate. Results are expressed as a ''stimulation index'' (SI) which is the ratio of the amount of tritiated thymidine (measured by beta counts) in the stimulated cells divided by the counts for the unstimulated cells on the same culture day. Several statistical methods for use in the routine analysis of the BeLPT were considered in the early 1990's by Frome et al. [7]. The least absolute values (LAV) method was recommended for routine analysis of the BeLPT. The purposes of this report are to further evaluate the LAV method using new data, and to describe a new method for identification of an abnormal or borderline test. This new statistical biological positive (SBP) method reflects the clinical judgment that (1) at least two SIs show a ''positive'' response to beryllium, and (2), that the maximum of the six SIs must exceed a cut point that is determined from a reference data set of normal individuals whose blood has been tested by the same method in the same serum. The new data is from the Y-12 facility in Oak Ridge and consist of 1080 worker and 33 nonexposed control BeLPTs (all tested in the same serum). Graphical results are presented to explain the statistical method, and the new SBP method is applied to the Y-12 group. The true positive rate and specificity of the new method were estimated to be 86

  5. Protein kinase Cε regulates proliferation and cell sensitivity to TGF-1β of CD4+ T lymphocytes: implications for Hashimoto thyroiditis.

    Science.gov (United States)

    Mirandola, Prisco; Gobbi, Giuliana; Masselli, Elena; Micheloni, Cristina; Di Marcantonio, Daniela; Queirolo, Valeria; Chiodera, Paolo; Meschi, Tiziana; Vitale, Marco

    2011-11-01

    We have studied the functional role of protein kinase Cε (PKCε) in the control of human CD4(+) T cell proliferation and in their response to TGF-1β. We demonstrate that PKCε sustains CD4(+) T cell proliferation triggered in vitro by CD3 stimulation. Transient knockdown of PKCε expression decreases IL-2R chain transcription, and consequently cell surface expression levels of CD25. PKCε silencing in CD4 T cells potentiates the inhibitory effects of TGF-1β, whereas in contrast, the forced expression of PKCε virtually abrogates the inhibitory effects of TGF-1β. Being that PKCε is therefore implicated in the response of CD4 T cells to both CD3-mediated proliferative stimuli and TGF-1β antiproliferative signals, we studied it in Hashimoto thyroiditis (HT), a pathology characterized by abnormal lymphocyte proliferation and activation. When we analyzed CD4 T cells from HT patients, we found a significant increase of PKCε expression, accounting for their enhanced survival, proliferation, and decreased sensitivity to TGF-1β. The increased expression of PKCε in CD4(+) T cells of HT patients, which is described for the first time, to our knowledge, in this article, viewed in the perspective of the physiological role of PKCε in normal Th lymphocytes, adds knowledge to the molecular pathophysiology of HT and creates potentially new pharmacological targets for the therapy of this disease.

  6. Monocyte-derived dendritic cells enhance cell proliferation and porcine circovirus type 2 replication in concanavalin A-stimulated swine peripheral blood lymphocytes in vitro.

    Science.gov (United States)

    Lin, Chun-Ming; Jeng, Chian-Ren; Hsiao, Shih-Hsuan; Lee, Yao; Tsai, Yi-Chieh; Chia, Mi-Yuan; Pang, Victor Fei

    2012-01-15

    Dendritic cells (DCs) are professional antigen presenting cells cooperating with other immune cells for the activation of innate and adaptive immune responses. The objective of the present study was to investigate the replication activity of porcine circovirus type 2 (PCV2) in DCs and/or lymphocytes during their cross talk and its possible mechanism. Two models were set, herein. Swine blood monocyte (Mo)-derived DCs (MoDCs) or peripheral blood lymphocytes (PBLs) were inoculated with PCV2 prior to their co-cultivation. Bacterial lipopolysaccharide (LPS) and concanavalin A (Con A) were used to stimulate MoDCs and PBLs, respectively. During 6 days of cultivation, a high PCV2 antigen-containing rate without detectable intranuclear signals and a slight but significant increase in the copy number of PCV2 genome were detected in PCV2-inoculated MoDCs. The presence of LPS alone or PCV2-free PBLs, however, had no effect on the location of PCV2 antigens or copy number of PCV2 genome in PCV2-inoculated MoDCs. On the contrary, active PCV2 replication occurred in Con A-stimulated PCV2-inoculated PBLs. When compared with blood Mos, MoDCs induced significantly higher cell proliferation and intensified PCV2 replication in Con A-stimulated PCV2-inoculated PBLs, for which direct contact between MoDCs and lymphocytes was required. Among the cytokines secreted by Con A-activated PBLs, interleukin (IL)-2, but not IL-4 or interferon-γ, could induce cell proliferation and PCV2 replication in PCV2-inoculated PBLs. The findings suggest that although MoDCs support only limited PCV2 replication in themselves, their accessory cell function is required for cell proliferation and PCV2 replication in PCV2-infected lymphocytes.

  7. MTS dye based colorimetric CTLL-2 cell proliferation assay for product release and stability monitoring of interleukin-15: assay qualification, standardization and statistical analysis.

    Science.gov (United States)

    Soman, Gopalan; Yang, Xiaoyi; Jiang, Hengguang; Giardina, Steve; Vyas, Vinay; Mitra, George; Yovandich, Jason; Creekmore, Stephen P; Waldmann, Thomas A; Quiñones, Octavio; Alvord, W Gregory

    2009-08-31

    A colorimetric cell proliferation assay using soluble tetrazolium salt [(CellTiter 96(R) Aqueous One Solution) cell proliferation reagent, containing the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) and an electron coupling reagent phenazine ethosulfate], was optimized and qualified for quantitative determination of IL-15 dependent CTLL-2 cell proliferation activity. An in-house recombinant Human (rHu)IL-15 reference lot was standardized (IU/mg) against an international reference standard. Specificity of the assay for IL-15 was documented by illustrating the ability of neutralizing anti-IL-15 antibodies to block the product specific CTLL-2 cell proliferation and the lack of blocking effect with anti-IL-2 antibodies. Under the defined assay conditions, the linear dose-response concentration range was between 0.04 and 0.17ng/ml of the rHuIL-15 produced in-house and 0.5-3.0IU/ml for the international standard. Statistical analysis of the data was performed with the use of scripts written in the R Statistical Language and Environment utilizing a four-parameter logistic regression fit analysis procedure. The overall variation in the ED(50) values for the in-house reference standard from 55 independent estimates performed over the period of 1year was 12.3% of the average. Excellent intra-plate and within-day/inter-plate consistency was observed for all four parameter estimates in the model. Different preparations of rHuIL-15 showed excellent intra-plate consistency in the parameter estimates corresponding to the lower and upper asymptotes as well as to the 'slope' factor at the mid-point. The ED(50) values showed statistically significant differences for different lots and for control versus stressed samples. Three R-scripts improve data analysis capabilities allowing one to describe assay variations, to draw inferences between data sets from formal statistical tests, and to set up improved assay acceptance

  8. Understanding colonization and proliferation potential of endophytes and pathogen in planta via plating, polymerase chain reaction, and ergosterol assay

    Directory of Open Access Journals (Sweden)

    Yiing Yng Chow

    2017-01-01

    Full Text Available This study aimed to establish the colonization behavior and proliferation potential of three endophytes and one pathogen Ganoderma boninense (Gb introduced into oil palm ramets (host model. The endophytes selected were Diaporthe phaseolorum (WAA02, Trichoderma asperellum (T2, and Penicillium citrinum (BTF08. Ramets were first inoculated with 100 mL of fungal cells (106 cfu mL−1 via soil drenching. For the next 7 days, ramets were sampled and subjected to three different assays to detect and identify fungal colonization, and establish their proliferation potential in planta. Plate assay revealed the presence of endophytes in root, stem and leaf tissues within 7 days after inoculation. Polymerase Chain Reaction (PCR detected and identified the isolates from the plant tissues. The ergosterol assay (via high-performance liquid chromatography, HPLC confirmed the presence of endophytes and Gb in planta. The increase in ergosterol levels throughout 49 days was however insignificant, suggesting that proliferation may be absent or may occur very slowly in planta. This study strongly suggests that the selected endophytes could colonize the host upon inoculation, but proliferation occurs at a slower rate, which may subsequently influence the biocontrol expression of endophytes against the pathogen.

  9. Investigation on the mechanisms of genotoxicity of butadiene, styrene and their combination in human lymphocytes using the Comet assay.

    Science.gov (United States)

    Cemeli, Eduardo; Mirkova, Ekaterina; Chiuchiarelli, Giorgia; Alexandrova, Elena; Anderson, Diana

    2009-05-12

    The toxicity of butadiene and styrene is exerted by their metabolites. Such metabolites have been extensively scrutinized at the in vitro level demonstrating evident genotoxic properties. In monitoring, a diverse range of outcomes has been produced. Additionally, epidemiological studies in rubber workers face difficulties of data interpretation due to the changeability and multiple exposures of the workers as well as to confounding factors inherent to the cohorts. Nevertheless, toxicity has been associated with a significant trend of increasing the risk of leukaemia in employees at the styrene-butadiene rubber industry. Thus, further effort must be made to distinguish the exposures to each chemical over time and to characterize their interrelationships. The present investigation focuses on the effects and mechanisms of damage of the mixture styrene-butadiene by examining its metabolites: styrene oxide (SO), butadiene monoepoxide (BME) and butadiene diepoxide (BDE) respectively. The in vitro Comet assay on frozen lymphocytes has been employed to ascertain the DNA damage patterns for the styrene-butadiene metabolites combined and on their own. Different patterns were observed for the mixture and each of its components. This study has also led to determining the mechanism of damage of the mixture and the compounds. With regard to the presence of reactive oxygen species (ROS), co-treatment with catalase does not modulate the genotoxicity of the mixture but it does modulate its components. The outcomes also indicate that the mixture induces cross-links and this is due to the influence of BDE in the mixture, being more evident as the concentration of BDE increases. An investigation on the sensitivity of lymphocytes from occupationally un/exposed subjects to in vitro exposure of the mixture and its components revealed that occupationally exposed subjects had a substantially higher background of DNA damage and a lower sensitivity to the metabolites of styrene, 1

  10. Immature dendritic cells generated from cryopreserved human monocytes show impaired ability to respond to LPS and to induce allogeneic lymphocyte proliferation.

    Directory of Open Access Journals (Sweden)

    Guilherme Ferreira Silveira

    Full Text Available Dendritic cells play a key role in the immune system, in the sensing of foreign antigens and triggering of an adaptive immune response. Cryopreservation of human monocytes was investigated to understand its effect on differentiation into immature monocyte-derived dendritic cells (imdDCs, the response to inflammatory stimuli and the ability to induce allogeneic lymphocyte proliferation. Cryopreserved (crp-monocytes were able to differentiate into imdDCs, albeit to a lesser extent than freshly (frh-obtained monocytes. Furthermore, crp-imdDCs had lower rates of maturation and cytokine/chemokine secretion in response to LPS than frh-imdDCs. Lower expression of Toll-like receptor 4 (at 24 and 48 h and higher susceptibility to apoptosis in crp-imdDCs than in fresh cells would account for the impaired maturation and cytokine/chemokine secretion observed. A mixed leukocyte reaction showed that lymphocyte proliferation was lower with crp-imdDCs than with frh-imdDCs. These findings suggested that the source of monocytes used to generate human imdDCs could influence the accuracy of results observed in studies of the immune response to pathogens, lymphocyte activation, vaccination and antigen sensing. It is not always possible to work with freshly isolated monocytes but the possible effects of freezing/thawing on the biology and responsiveness of imdDCs should be taken into account.

  11. Human T cell priming assay: depletion of peripheral blood lymphocytes in CD25(+) cells improves the in vitro detection of weak allergen-specific T cells.

    Science.gov (United States)

    Vocanson, Marc; Achachi, Amine; Mutez, Virginie; Cluzel-Tailhardat, Magalie; Varlet, Béatrice Le; Rozières, Aurore; Fournier, Philippe; Nicolas, Jean-François

    2014-01-01

    To develop an in vitro assay that recapitulates the key event of allergic contact dermatitis (ACD), that is the priming of effector T cells by hapten-presenting dendritic cells, and then allows for the sensitive detection of chemical allergens represents a major challenge. Classical human T cell priming assays (hTCPA) that have been developed in the past, using hapten-loaded monocyte-derived dendritic cells (MDDCs) as antigen-presenting cells and peripheral blood lymphocytes (PBLs) as responding cells, were not efficient to prime T cells to common allergens with moderate/weak sensitizing properties. Recent progress in the understanding of the effector and regulatory mechanisms of ACD have shown that T cell priming requires efficient uptake of allergens by immunogenic DCs and that it is controlled by several subsets of regulatory cells including CD25(+) Tregs. We therefore analyzed various parameters involved in allergen-specific T cell activation in vitro and showed that priming of allergen-specific T cells is hampered by several subsets of immune cells comprising CD1a(neg) DCs, CD25(+) T cells, and CD56(+) regulatory cells.CD4(+)CD25(+)FoxP3(+) Tregs prevented the in vitro T cell priming to moderate/weak allergens, and depletion of human PBLs in CD25(+) cells significantly increased specific T cell proliferation and IFN-γ secretion. CD56(+) cells exerted an additional control of T cell priming since co-depletion of both CD56(+) and CD25(+) cells improved the magnitude of chemical-specific T cell activation. Finally, CD1a(low) MDDCs were able to inhibit T cell activation obtained by allergen-pulsed CD1a(high) MDDC. Moreover, we showed that uptake by DC of allergen-encapsulated nanoparticles significantly increased their activation status and their ability to prompt specific T cell activation. Hence, by combining the different strategies, i.e., depletion of CD25(+) and CD56(+) cells, use of CD1a(high) MDDC, and nanoparticle encapsulation of allergens, it was

  12. Analysis of IL-2-like factor in lymphocyte culture supernatant of olive flounder, Paralichthys oliveaceus

    Institute of Scientific and Technical Information of China (English)

    WU Riqin; ZHANG Peijun; LI Jun; XU Yongli

    2005-01-01

    To study immune mechanism of fish lymphocyte we performed a proliferation assay and ELISA using monoclonal antibody against human IL-2. The result showed that an interleukin-2 (IL-2)-like factor was detected in the supernatant of plant haemoglutinin (PHA)-stimulated lymphocyte culture from peripheral blood,spleen and head kidney of olive flounder, Paralichthys olivaceus. The quantities of IL-2-1ike factor in the supematant from different lymphoid tissues were quite different. The IL-2 like factor in the supernatant from cultured head kidney lymphocytes was much higher than those of peripheral blood lymphocytes and spleen lymphocytes (P<0.01). The IL-2 activity was found in either mouse thymocyte proliferation assay or flounder head kidney lymphocyte proliferation assay and shown to have obvious enhancing effect on proliferation of the above two types of cell. The recombinant human IL-2 (rhIL-2) was able to stimulate flounder thymocyte proliferation and used to detect the IL-2 receptor (IL-2R) on the surface of flounder lymphocyte. The cross-reaction between the lymphocytes of flounder peripheral blood and CD25(IL-2R) was detected with flow cytometry and shown that the percentage of CD25-positive cell in peripheral blood was 7.74± 0.67%.

  13. Sublethal red tide toxin exposure in free-ranging manatees (Trichechus manatus) affects the immune system through reduced lymphocyte proliferation responses, inflammation, and oxidative stress

    Energy Technology Data Exchange (ETDEWEB)

    Walsh, Catherine J., E-mail: cjwalsh@mote.org [Marine Immunology Program, Mote Marine Laboratory, 1600 Ken Thompson Parkway, Sarasota, FL 34236 (United States); Butawan, Matthew, E-mail: mattbutawan@outlook.com [Marine Immunology Program, Mote Marine Laboratory, 1600 Ken Thompson Parkway, Sarasota, FL 34236 (United States); Yordy, Jennifer, E-mail: jennifer.e.balmer@gmail.com [Marine Immunology Program, Mote Marine Laboratory, 1600 Ken Thompson Parkway, Sarasota, FL 34236 (United States); Ball, Ray, E-mail: Ray.Ball@lowryparkzoo.com [Lowry Park Zoo, 1101 W Sligh Ave, Tampa, FL 33604 (United States); Flewelling, Leanne, E-mail: Leanne.Flewelling@MyFWC.com [Fish and Wildlife Research Institute, Florida Fish and Wildlife Conservation Commission, 100 8th Ave SE, St. Petersburg, FL 33701 (United States); Wit, Martine de, E-mail: Martine.deWit@MyFWC.com [Fish and Wildlife Research Institute, Florida Fish and Wildlife Conservation Commission, 100 8th Ave SE, St. Petersburg, FL 33701 (United States); Bonde, Robert K., E-mail: rbonde@usgs.gov [U.S. Geological Survey, Sirenia Project, 7920 NE 71st Street, Gainesville, FL 32653 (United States)

    2015-04-15

    Highlights: • Sublethal brevetoxin exposure affects manatee immune function. • Plasma brevetoxin levels correlate with oxidative stress in rescued manatees. • Brevetoxin exposure affects lymphocyte proliferation in rescued manatees. • Plasma brevetoxin concentrations ranged from 0 to 19 ng PbTx-3 eq/mL. - Abstract: The health of many Florida manatees (Trichechus manatus latirostris) is adversely affected by exposure to blooms of the toxic dinoflagellate, Karenia brevis. K. brevis blooms are common in manatee habitats of Florida’s southwestern coast and produce a group of cyclic polyether toxins collectively referred to as red tide toxins, or brevetoxins. Although a large number of manatees exposed to significant levels of red tide toxins die, several manatees are rescued from sublethal exposure and are successfully treated and returned to the wild. Sublethal brevetoxin exposure may potentially impact the manatee immune system. Lymphocyte proliferative responses and a suite of immune function parameters in the plasma were used to evaluate effects of brevetoxin exposure on health of manatees rescued from natural exposure to red tide toxins in their habitat. Blood samples were collected from rescued manatees at Lowry Park Zoo in Tampa, FL and from healthy, unexposed manatees in Crystal River, FL. Peripheral blood leukocytes (PBL) isolated from whole blood were stimulated with T-cell mitogens, ConA and PHA. A suite of plasma parameters, including plasma protein electrophoresis profiles, lysozyme activity, superoxide dismutase (SOD) activity, and reactive oxygen/nitrogen (ROS/RNS) species, was also used to assess manatee health. Significant decreases (p < 0.05) in lymphocyte proliferation were observed in ConA and PHA stimulated lymphocytes from rescued animals compared to non-exposed animals. Significant correlations were observed between oxidative stress markers (SOD, ROS/RNS) and plasma brevetoxin concentrations. Sublethal exposure to brevetoxins in the

  14. A panel of real-time PCR assays for specific detection of three phytoplasmas from the apple proliferation group.

    Science.gov (United States)

    Nikolić, Petra; Mehle, Natasa; Gruden, Kristina; Ravnikar, Maja; Dermastia, Marina

    2010-10-01

    We report here on the development of combination of assays for fast, reliable, specific and sensitive detection and discrimination of 'Candidatus Phytoplasma mali', 'Ca. P. prunorum' and 'Ca. P. pyri' from the 16Sr-X (apple proliferation - AP) group. These phytoplasmas are causal agents of diseases of fruit trees within the family Rosaceae, namely apple proliferation (AP), European stone fruit yellows (ESFY) and pear decline (PD). The designed panel of assays uses TaqMan minor groove binder probes (MGB). It comprises the same set of primers and specific probes for species-specific amplification within the 16S-23S rRNA intergenic spacer region, a set of primers and probes for amplification of the 16S ribosomal DNA region for the universal phytoplasma detection, and an additional set of primers and probe for 18S rRNA as an endogenous quality control of DNA extraction. The performance characteristics of the panel were evaluated. The advantages of new assays were shown in a comparative study with the conventional PCR, which proved their higher sensitivity combined with three-fold shorter time of testing process; and in comparison with two reported multiplex real-time PCR assays for detection of 'Ca. P. mali' or 'Ca. P. pyri'. New panel of assays were tested on the DNA samples of 'Ca. P. mali', 'Ca. P. prunorum', 'Ca. P. pyri', other phytoplasmas and other bacteria isolated from plant material. Additionally, 198 symptomatic and asymptomatic fruit tree field samples collecting during several growing seasons were tested with new assays as well. The results of this study indicate that the combination of three specific assays may be applied in routine phytoplasma surveys and in the certification programs.

  15. Immunopotentiality of Ayurvedic polyherbal formulations “Saribadi” and “Anantamul Salsa” with augmentation of IgM production and lymphocytes proliferation:A preliminary study

    Institute of Scientific and Technical Information of China (English)

    Md Moklesur Rahman Sarker; Long Chiau Ming; Md Zaidul Islam Sarker; M Shahabuddin Kabir Choudhuri

    2016-01-01

    Objective: To assess the immunopotentiality of Ayurvedic polyherbal preparations,“Saribadi”and“Anantamul Salsa”. Methods: Freshly prepared BALB/c mice splenocytes were cultured with“Saribadi”or“Anantamul Salsa” treatment [doses of 0.25%, 0.50%, 0.75%, 1.00%, 1.50%, 2.00%, 3.00%and 4.00%(v/v)] at 37 ? C for 5 days. The immunoglobulin M (IgM) production and lymphocytes proliferation were determined by ELISA and MTT methods, respectively. Endotoxin contamination was assessed by treating the preparations with polymyxin B. Results: The doses of“Saribadi”[0.25%, 0.50%, 0.75%and 1.00%(v/v)] significantly increased IgM productions (0.966, 0.728, 0.695 and 0.615 mg/mL vs. control 0.265 mg/mL) and lymphocytes proliferation [absorbance 0.311, 0.394, 0.372 and 0.334 optical density (OD) vs. control 0.162 OD]. Similarly, the doses of“Anantamul Salsa”[0.50%, 0.75%, 1.00%and 1.50%(v/v)] promoted IgM productions (0.933, 0.919, 0.917 and 0.892 mg/mL vs. control 0.502 mg/mL) and the doses of “Anantamul Salsa” [0.50%, 0.75%, 1.00%, 1.50%, 2.00%, and 3.00%(v/v)] stimulated lymphocytes proliferation (absorbance 0.395, 0.326, 0.440, 0.398, 0.452 and 0.355 OD vs. control 0.199 OD). The activity of“Saribadi”and“Anantamul Salsa”was not retarded by the treatment of preparations with polymyxin B. Conclusions: Immunomodulatory activity of “Saribadi” and “Anantamul Salsa” was unveiled for the first time.“Saribadi”and“Anantamul Salsa”possess immunostimulating potential acting through the induction of lymphocyte proliferation and IgM production. These preparations may be useful in strengthening immune responses. However, further cellular and in vivo studies are required.

  16. Immunopotentiality of Ayurvedic polyherbal formulations “Saribadi” and “Anantamul Salsa” with augmentation of IgM production and lymphocytes proliferation:A preliminary study

    Institute of Scientific and Technical Information of China (English)

    Md.Moklesur Rahman Sarker; Long Chiau Ming; Md.Zaidul Islam Sarker; M Shahabuddin Kabir Choudhuri

    2016-01-01

    Objective:To assess the immunopotentiality of Ayurvedic polyherbal preparations,"Saribadi" and "Anantamul Salsa".Methods: Freshly prepared BALB/c mice splenocytes were cultured with "Saribadi" or"Anantamul Salsa" treatment [doses of 0.25%, 0.50%, 0.75%, 1.00%, 1.50%, 2.00%,3.00% and 4.00%(v/v)] at 37C for 5 days. The immunoglobulin M(IgM) production and lymphocytes proliferation were determined by ELISA and MTT methods, respectively.Endotoxin contamination was assessed by treating the preparations with polymyxin B.Results: The doses of "Saribadi" [0.25%, 0.50%, 0.75% and 1.00%(v/v)] significantly increased IgM productions(0.966, 0.728, 0.695 and 0.615 mg/m L vs. control 0.265 mg/m L)and lymphocytes proliferation [absorbance 0.311, 0.394, 0.372 and 0.334 optical density(OD) vs. control 0.162 OD]. Similarly, the doses of "Anantamul Salsa" [0.50%, 0.75%,1.00% and 1.50%(v/v)] promoted IgM productions(0.933, 0.919, 0.917 and 0.892 mg/m L vs. control 0.502 mg/m L) and the doses of "Anantamul Salsa" [0.50%, 0.75%, 1.00%,1.50%, 2.00%, and 3.00%(v/v)] stimulated lymphocytes proliferation(absorbance 0.395,0.326, 0.440, 0.398, 0.452 and 0.355 OD vs. control 0.199 OD). The activity of "Saribadi"and "Anantamul Salsa" was not retarded by the treatment of preparations with polymyxin B.Conclusions: Immunomodulatory activity of "Saribadi" and "Anantamul Salsa" was unveiled for the first time. "Saribadi" and "Anantamul Salsa" possess immunostimulating potential acting through the induction of lymphocyte proliferation and IgM production.These preparations may be useful in strengthening immune responses. However, further cellular and in vivo studies are required.

  17. Effects of PCBs and PBDEs on thyroid hormone, lymphocyte proliferation, hematology and kidney injury markers in residents of an e-waste dismantling area in Zhejiang, China.

    Science.gov (United States)

    Xu, Peiwei; Lou, Xiaoming; Ding, Gangqiang; Shen, Haitao; Wu, Lizhi; Chen, Zhijian; Han, Jianlong; Wang, Xiaofeng

    2015-12-01

    Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are two typical categories of contaminants released from e-waste dismantling environments. In China, the body burdens of PCBs and PBDEs are associated with abnormal thyroid hormones in populations from e-waste dismantling sites, but the results are limited and contradictory. In this study, we measured the serum levels of PCBs and PBDEs and the thyroid hormone free triiodothyronine (FT3), free thyroxine (FT4) and thyroid-stimulating hormone (TSH) in 40 residents in an e-waste dismantling area and in 15 residents in a control area. Additionally, we also measured some lymphocyte proliferation indexes, hematologic parameters and kidney injury markers, including white blood cells, neutrophils, monocytes, lymphocytes, hemoglobin, platelets, serum creatinine and beta 2-microglobulin (β2-MG). The results indicated that the mean level of ΣPCBs in the exposure group was significantly higher than that in the control group (964.39 and 67.98 ng g(-1), p0.05). We determined that serum levels of FT3, FT4, monocytes and lymphocytes were significantly lower, whereas the levels of neutrophils, hemoglobin, platelets and serum creatinine were significantly higher in the exposed group (pe-waste dismantling environment may increase the body burdens of PCBs and the specific PBDEs congeners in native residents and that the contaminants released from e-waste may contribute to abnormal changes in body levels of thyroid hormone, hematology and kidney injury markers.

  18. Autophagy regulates T lymphocyte proliferation through selective degradation of the cell-cycle inhibitor CDKN1B/p27Kip1.

    Science.gov (United States)

    Jia, Wei; He, Ming-Xiao; McLeod, Ian X; Guo, Jian; Ji, Dong; He, You-Wen

    2015-01-01

    The highly conserved cellular degradation pathway, macroautophagy, regulates the homeostasis of organelles and promotes the survival of T lymphocytes. Previous results indicate that Atg3-, Atg5-, or Pik3c3/Vps34-deficient T cells cannot proliferate efficiently. Here we demonstrate that the proliferation of Atg7-deficient T cells is defective. By using an adoptive transfer and Listeria monocytogenes (LM) mouse infection model, we found that the primary immune response against LM is intrinsically impaired in autophagy-deficient CD8(+) T cells because the cell population cannot expand after infection. Autophagy-deficient T cells fail to enter into S-phase after TCR stimulation. The major negative regulator of the cell cycle in T lymphocytes, CDKN1B, is accumulated in autophagy-deficient naïve T cells and CDKN1B cannot be degraded after TCR stimulation. Furthermore, our results indicate that genetic deletion of one allele of CDKN1B in autophagy-deficient T cells restores proliferative capability and the cells can enter into S-phase after TCR stimulation. Finally, we found that natural CDKN1B forms polymers and is physiologically associated with the autophagy receptor protein SQSTM1/p62 (sequestosome 1). Collectively, autophagy is required for maintaining the expression level of CDKN1B in naïve T cells and selectively degrades CDKN1B after TCR stimulation.

  19. Long-term cadmium exposure leads to the enhancement of lymphocyte proliferation via down-regulating p16 by DNA hypermethylation.

    Science.gov (United States)

    Yuan, Dexiao; Ye, Shuang; Pan, Yan; Bao, Yizhong; Chen, Honghong; Shao, Chunlin

    2013-10-09

    Cadmium (Cd) is a well-established carcinogen, however, the underlying mechanism, especially the role of epigenetics in it, is still poorly understood. Our previous work has disclosed that when rats were exposed to 0.5mg CdCl2 (kgd) for 8 and 12 weeks, the growth of peripheral white blood cells (WBC) was obviously stimulated but no over-proliferation of granulocyte-monocyte (GM) progenitor cells was observed in the bone marrow, suggesting that the over-proliferation of lymphocyte was promoted by Cd exposure. Is DNA-methylation involved in this Cd-stimulated cell proliferation? The present study found that when human B lymphoblast HMy2.CIR cells were exposed to Cd with a dose lower than 0.1μM for 3 months, both cell proliferation and mRNA expressions of DNA methyltransferases of DNMT1 and DNMT3b were increased, while the mRNA of tumor suppressor gene p16 was remarkably decreased. Furthermore, the level of genomic DNA methylation was increased and the CpG island in p16 promoter was hypermethylated in the Cd-exposed cells. A DNA demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC), diminished Cd-stimulated cell proliferation associated with p16 overexpression. Our results suggested that the chronic exposure of low dose Cd could induce hypermethylation of p16 promoter and hence suppress p16 expression and then promote cell proliferation, which might contribute to Cd-induced carcinogenesis. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Effect of Benzene on Proliferation and Apoptosis of Splenic Lymphocytes in Mother Generation and Offspring Mice%苯对母鼠和子鼠脾淋巴细胞的增殖与凋亡影响

    Institute of Scientific and Technical Information of China (English)

    旷亦乐; 李纯颖; 杨双波; 李紫; 吴成秋

    2011-01-01

    Objective To explore the effect of benzene on proliferation and apoptceis of splenic lymphocytes in mother generation and offspring mice. Methods Forty pregnant mice were divided averagely into 4 groups at random. From the 7th day after pregnancy, each of group was exposed to benzene vapour until to parturition (0.0, 5.0, 10.0 and 15.0 mg/m3,respectively), 2 hours par day. At the 1st and 7th days after parturition, 5 mother generation mice end 5 offspring mice of each group were killed to detect the proliferation, cell cycle and apoptosis of splenic lymphocytes in mother generation and offspring mice by MTT assay and flow cytometric analysis. Results During the 1st day and 7th day after parturition, the proliferation of splenic lymphocytes of mother generation and offspring mice in the middle - and high - concentration of benzene groups was inhibited significantly in a concentration-dependent manner (P<O. 05). The cell cycle of splenic lymphocytes of mother generation and offspring mice was blocked in G0/G1 phase in the middle - and high - concentration of benzene groups. The quantity of splenic lymphocytes apoptosis was increased significantly in a concentration- dependent manner in each benzene group (P < 0.05). Conclusion Benzene exposure during pregnancy can damage the immunological function of mother generation mice and offspring mice.%目的 探讨妊娠期接触苯对母鼠及其子鼠免疫功能的影响.方法 40只孕鼠被随机等分为空气对照组和5.0、10.0、15.0 mg/m3三个不同浓度的苯染毒组,各组从孕7 d开始,连续染毒至分娩,每天染毒2 h.分别在分娩后的1 d及7 d,每组取5只母鼠和5只子鼠处死,取脾制备脾淋巴细胞;检测母鼠和子鼠脾淋巴细胞增殖力、细胞周期及细胞凋亡.结果 在分娩后1 d及7 d,中、高浓度苯染毒组母鼠及子鼠的脾淋巴细胞增殖力均低于对照组(P<0.05),并有明显的剂量-效应关系(P<0.05);子鼠与母鼠的淋巴细胞增殖抑制

  1. Determination of the estrogenic activity of wild phytoestrogen-rich Pueraria mirifica by MCF-7 proliferation assay.

    Science.gov (United States)

    Cherdshewasart, Wichai; Traisup, Virasinee; Picha, Porntipa

    2008-02-01

    The aim of this study was to evaluate the estrogenic activity of tuberous samples of wild, phytoestrogen-rich Pueraria mirifica collected from 28 out of 76 provinces of Thailand by MCF-7 proliferation assay. The plant extracts were administered to MCF-7, ER alpha positive human mammary adenocarcinoma cell cultures, for 3 days at dosages of 0.1, 1, 10, 100 and 1,000 microg/ml and were compared with 17 beta-estradiol at concentrations of 10(-12)-10(-6) M. The mean P. mirifica population at 1 mug/ml exhibited significant proliferation. Two plant samples exhibited levels of proliferation in MCF-7 that were similar to 17beta-estradiol. The mean P. mirifica populations at 100 and 1,000 microg/ml exhibited significant cytotoxicity in MCF-7. Analysis of the estrogenic activity of puerarin, representative of major isoflavonoids in P. mirifica tubers, revealed proliferation in MCF-7 only at the highest dose (10(-6) M) that was 10(2)-10(5) times less active than 17 beta-estradiol. Puerarin and 17 beta-estradiol at concentration of 10(-12)-10(-6) M exhibited no cytotoxicity in MCF-7.

  2. Effects of PCBs and PBDEs on thyroid hormone, lymphocyte proliferation, hematology and kidney injury markers in residents of an e-waste dismantling area in Zhejiang, China

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Peiwei, E-mail: pwxu@cdc.zj.cn; Lou, Xiaoming; Ding, Gangqiang, E-mail: gqding@cdc.zj.cn; Shen, Haitao; Wu, Lizhi; Chen, Zhijian; Han, Jianlong; Wang, Xiaofeng, E-mail: zjcdcwxf@gmail.com

    2015-12-01

    Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are two typical categories of contaminants released from e-waste dismantling environments. In China, the body burdens of PCBs and PBDEs are associated with abnormal thyroid hormones in populations from e-waste dismantling sites, but the results are limited and contradictory. In this study, we measured the serum levels of PCBs and PBDEs and the thyroid hormone free triiodothyronine (FT3), free thyroxine (FT4) and thyroid-stimulating hormone (TSH) in 40 residents in an e-waste dismantling area and in 15 residents in a control area. Additionally, we also measured some lymphocyte proliferation indexes, hematologic parameters and kidney injury markers, including white blood cells, neutrophils, monocytes, lymphocytes, hemoglobin, platelets, serum creatinine and beta 2-microglobulin (β{sub 2}-MG). The results indicated that the mean level of ΣPCBs in the exposure group was significantly higher than that in the control group (964.39 and 67.98 ng g{sup −1}, p < 0.0001), but the mean level of ΣPBDEs in the exposure group was not significantly higher than that in the controls (139.32 vs. 75.74 ng g{sup −1}, p > 0.05). We determined that serum levels of FT3, FT4, monocytes and lymphocytes were significantly lower, whereas the levels of neutrophils, hemoglobin, platelets and serum creatinine were significantly higher in the exposed group (p < 0.05). The mean level of ΣPCBs was negatively correlated with levels of FT3, FT4, monocytes and lymphocytes (p < 0.05) and positively correlated with levels of neutrophils, hemoglobin, serum creatinine and β{sub 2}-MG (p < 0.05). Additionally, the mean level of ΣPBDEs was positively correlated with levels of white blood cells, hemoglobin and platelets (p < 0.05). Our data suggest that exposure to an e-waste dismantling environment may increase the body burdens of PCBs and the specific PBDEs congeners in native residents and that the contaminants released

  3. LDH, proliferation curves and cell cycle analysis are the most suitable assays to identify and characterize new phytotherapeutic compounds.

    Science.gov (United States)

    Specian, Ana Flávia L; Serpeloni, Juliana M; Tuttis, Katiuska; Ribeiro, Diego L; Cilião, Heloísa L; Varanda, Eliana A; Sannomiya, Miriam; Martinez-Lopez, Wilner; Vilegas, Wagner; Cólus, Ilce M S

    2016-12-01

    Brazilian flora biodiversity has been widely investigated to identify effective and safe phytotherapeutic compounds. Among the investigated plant species, the Byrsonima genus exhibits promising biological activities. This study aimed at evaluating the cytotoxicity of B. correifolia, B. verbascifolia, B. fagifolia and B. intermedia extracts using different assays in two cell lines (primary gastric and HepG2 cells). The different extract concentrations effects on cell viability were assayed using the MTT, aquabluer, neutral red and LDH assays. Non-cytotoxic concentrations were selected to generate cell proliferation curves and to assess cell cycle kinetics by flow cytometry. Byrsonima extracts differentially affected cell viability depending on the metabolic cellular state and the biological parameter evaluated. B. fagifolia and B. intermedia extracts exhibited lower cytotoxic effects than B. correifolia and B. verbascifolia in all assays. The results obtained with LDH and flow cytometry assays were more reliable, suggesting that they can be useful in the screening for herbal medicine and to further characterize these extracts as phytotherapeutic compounds.

  4. Application of lymphocyte parameters in screening lymphocyte proliferation and reactive hyperplasia disease%淋巴细胞参数在筛选淋巴细胞增殖及反应性增生疾病中的应用

    Institute of Scientific and Technical Information of China (English)

    徐卫益; 胡海莹; 汤冬琴

    2014-01-01

    Objective To investigate the application of lymphocyte parameters in screening the lymphocyte proliferation and re-active hyperplasia disease.Methods 128 cases as the healthy control,100 cases of lymphoma,35 cases of multiple myeloma(MM), 34 cases of lymphocytes reactive hyperplasia(RL)and 5 cases of chronic lymphocytic leukemia(CLL)were selected and performed the peripheral blood cells analysis by the Sysmex XE-2100 hematology analyzer.The relevant lymphocyte parameter values were re-corded and the comparative analysis in the difference between the disease groups and the control group was performed.The role of each lymphocyte parameter in screening the lymphocyte proliferation and reactive hyperplasia diseases was evaluated by the receiver operating characteristic curve(ROC curve).Results The high fluorescence lymphocytes(HFL),in the control group was 0.008 ± 0.008,in the lymphoma group was 0.016±0.058,in the MM group was 0.019 ±0.063,in the RL group was 0.040 ±0.070,and CLL group was 0.388±0.158.Compared with control group,the difference of RL and CLL group was statistically significant(P morphological changes of lymphocytes sensitively and contribute to screening the patients with lymphocyte proliferation and reactive hyperplasia disease.%目的:探讨淋巴细胞参数在淋巴细胞增殖及反应性增生疾病筛查中的应用。方法选取健康对照组128例、淋巴瘤患者100例、多发性骨髓瘤(MM)患者35例、淋巴细胞反应性增生(RL)患者34例、慢性淋巴细胞白血病(CLL)患者5例。分别用 Sysmex XE-2100血液分析仪进行外周血血细胞分析,记录相应淋巴细胞参数值,并对其进行疾病组与对照组差异比较分析;以受试者操作特性曲线(ROC 曲线)评价各淋巴细胞参数在筛选淋巴细胞增殖及反应性增生疾病患者中的作用。结果高荧光淋巴细胞(HFL):对照组0.008±0.008,淋巴瘤组0.016±0.058,MM 组0.019±0.063,RL 组0.040±0

  5. Combined assay of surface immunoglobulin intensity and mouse rosettes. A practical parameter in the differential diagnosis of small lymphocytic and follicular center cell lymphomas.

    Science.gov (United States)

    Batata, A; Shen, B

    1993-03-01

    Cell suspensions from the lymph nodes of small lymphocytic lymphoma (n = 94) and nodular and diffuse follicular center cell lymphomas (n = 330) were analyzed to evaluate the diagnostic significance of the surface immunoglobulin (SIg) intensity and mouse rosette assay (MR). In small lymphocytic lymphoma, SIg was monoclonal in 65 cases (69.15%), with weak fluorescence in 59 (90.77%). It was not detected in 29 cases (30.85%). The MR findings were positive in 68 cases (72.34%) and negative in 26 (27.66%). The combined results of these two assays showed the following: weak SIg/MR+, 35 (37.23%); weak SIg/MR-, 24 (25.53%); strong SIg/MR+, 6 (6.38%); strong SIg/MR-, 0; undetected SIg/MR+, 27 (28.72%); and undetected SIg/MR-, 2 (2.13%). By performing the assays for these two markers and accepting weak SIg/MR+, weak SIg/MR-, strong SIg/MR+, or undetected SIg/MR+ as sufficient for diagnosis, 92 cases (97.87%) were diagnosed. In diffuse follicular center cell lymphomas, SIg was monoclonal in 287 cases (86.97%), with strong fluorescence in 258 (89.9%) and weak fluorescence in 29 (10.1%). It was not detected in 43 cases (13.03%). The MR results were positive in 34 cases (10.3%) and negative in 296 (89.7%). The combined findings of these two assays showed that strong SIg/MR- was present in 244 cases (73.94%). The diagnostic value of the combined assay in the differential diagnosis between small lymphocytic lymphoma and diffuse follicular center cell lymphomas was proved using five statistical parameters.

  6. Quantitative miR analysis in chronic lymphocytic leukaemia/small lymphocytic lymphoma - proliferation centres are characterized by high miR-92a and miR-155 and low miR-150 expression.

    Science.gov (United States)

    Szurián, Kinga; Csala, Irén; Piurkó, Violetta; Deák, Linda; Matolcsy, András; Reiniger, Lilla

    2017-07-01

    Proliferation centres (PCs) are histological hallmarks of lymph nodes in chronic lymphocytic leukaemia/small lymphocytic lymphoma (CLL/SLL). Chromosomal abnormalities have already been described to accumulate preferably in the PCs as opposed to the intervening small cell areas. To further characterize the pathogenic role of PCs, the expression levels of 17 selected miRs known to be involved in the development of CLL/SLL were compared in the PCs and the intervening small cell areas in lymph nodes of 15 patients with CLL/SLL. The miR expression levels were also compared to the cytogenetic alterations defined by FISH analysis. Our results show that two known oncomiRs, miR-155 and -92a were upregulated and the tumour suppressor miR-150 was downregulated in the PCs. Low expression of miR-150 was also associated with loss of 11q. In summary we found significantly higher expression of oncomiRs and lower expression of a tumour suppressor miR in PCs of CLL/SLL lymph nodes, which support the hypothesis that the PCs may drive the disease and play a role in progression. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Detection of weak estrogenic flavonoids using a recombinant yeast strain and a modified MCF7 cell proliferation assay

    DEFF Research Database (Denmark)

    Breinholt, Vibeke; Larsen, John Christian

    1998-01-01

    stimulate the transcriptional activity of the human estrogen receptor in the yeast assay increasing transcriptional activity 5-13-fold above background level, corresponding to EC50 values between 0.1 and 25 mu M, Five compounds increased the transcriptional activity 2-5-fold over the control, with EC50......-estradiol, when compared on the basis of EC50 values. The estrogenic activity of the dietary flavonoids was further investigated in estrogen-dependent human MCF7 breast cancer cells. In this system several of the flavonoids were likewise capable of mimicking natural estrogens and thereby induce cell proliferation....... Similar structural requirements for estrogenic activity were found for the two assays. The present results provide evidence that several of the flavo-estrogens possess estrogenic properties comparable in activity to the well-established isoflavonoid estrogens. The use of Alamar Blue, a vital dye which...

  8. Murine Butyrophilin-like (Btnl 1 and Btnl6 form heteromeric complexes in small intestinal epithelial cells and promote proliferation of local T lymphocytes

    Directory of Open Access Journals (Sweden)

    Cristina eLebrero-Fernández

    2016-01-01

    Full Text Available To date, few molecular conduits mediating the cross-talk between intestinal epithelial cells and intraepithelial lymphocytes (IELs have been described. We recently showed that Butyrophilin-like (Btnl 1 can attenuate the epithelial response to activated IELs, resulting in reduced production of pro-inflammatory mediators such as IL-6 and CXCL1. We here report that like Btnl1, murine Btnl6 expression is primarily confined to the intestinal epithelium. Although Btnl1 can exist in a cell surface-expressed homomeric form, we found that it additionally forms heteromeric complexes with Btnl6, and that the engagement of Btnl1 is a prerequisite for surface expression of Btnl6 on intestinal epithelial cells. In an IEL-epithelial cell co-culture system, enforced epithelial cell expression of Btnl1 significantly enhanced the proliferation of IELs in the absence of exogenous activation. The effect on proliferation was dependent on the presence of IL-2 or IL-15 and restricted to IELs upregulating CD25. In the gamma delta (gd T-cell subset, the Btnl1-Btnl6 complex, but not Btnl1, specifically elevated the proliferation of IELs bearing the Vg7Vd4 receptor. Thus, our results show that murine epithelial cell-specific Btnl proteins can form intrafamily heterocomplexes, and suggest that the interaction between Btnl proteins and IELs regulates the expansion of IELs in the intestinal mucosa.

  9. CD226 (DNAM-1) is involved in lymphocyte function-associated antigen 1 costimulatory signal for naive T cell differentiation and proliferation.

    Science.gov (United States)

    Shibuya, Kazuko; Shirakawa, Jun; Kameyama, Tomie; Honda, Shin-Ichiro; Tahara-Hanaoka, Satoko; Miyamoto, Akitomo; Onodera, Masafumi; Sumida, Takayuki; Nakauchi, Hiromitsu; Miyoshi, Hiroyuki; Shibuya, Akira

    2003-12-15

    Upon antigen recognition by the T cell receptor, lymphocyte function-associated antigen 1 (LFA-1) physically associates with the leukocyte adhesion molecule CD226 (DNAM-1) and the protein tyrosine kinase Fyn. We show that lentiviral vector-mediated mutant (Y-F322) CD226 transferred into naive CD4+ helper T cells (Ths) inhibited interleukin (IL)-12-independent Th1 development initiated by CD3 and LFA-1 ligations. Moreover, proliferation induced by LFA-1 costimulatory signal was suppressed in mutant (Y-F322) CD226-transduced naive CD4+ and CD8+ T cells in the absence of IL-2. These results suggest that CD226 is involved in LFA-1-mediated costimulatory signals for triggering naive T cell differentiation and proliferation. We also demonstrate that although LFA-1, CD226, and Fyn are polarized at the immunological synapse upon stimulation with anti-CD3 in CD4+ and CD8+ T cells, lipid rafts are polarized in CD4+, but not CD8+, T cells. Moreover, proliferation initiated by LFA-1 costimulatory signal is suppressed by lipid raft disruption in CD4+, but not CD8+, T cells, suggesting that the LFA-1 costimulatory signal is independent of lipid rafts in CD8+ T cells.

  10. Application of Comet assay to assess the effects of white bean meal on DNA of human lymphocytes

    Directory of Open Access Journals (Sweden)

    Luciana Lopes Silva Pereira

    2012-03-01

    Full Text Available This study was conducted to evaluate the potential induction of genotoxic effects of white bean flour using the Comet assay. The test was conducted with human lymphocytes present in whole blood immediately after collection, by incubation with white bean flour in three concentrations (3.92, 9.52 and 18.18 mg/mL at 37 ºC for 4 h followed by preparation of slides. Samples were considered positive (above 20% damage when the damage observed to cellular DNA was higher than the negative control. No genotoxic potential was found at the doses tested. However, it would be premature to suggest absence of risk to human health of DNA damage since the exposure of cells to the extract was restricted to four hours rather than a whole cell cycle. Additionally, further information on toxicology should be obtained in future studies.Este estudo foi realizado para avaliar o potencial de indução de efeitos genotóxicos da farinha de feijão branco utilizando o teste do Cometa. O ensaio foi realizado com linfócitos humanos presentes no sangue imediatamente após a coleta, por incubação com farinha de feijão branco em três concentrações (3,92, 9,52 e 18,18 mg/mL a 37 ºC por 4 h, seguida de preparação das lâminas. As amostras foram consideradas positivas (acima de 20% de dano, quando os danos observados no DNA celular foram maiores do que o controle negativo. Verificou-se que as doses testadas não mostraram potencial genotóxico. No entanto, seria prematuro fazer recomendações sobre o padrão de riscos para a saúde humana resultantes de danos ao DNA já que exposição das células ao extrato foi restrito ao período de quatro horas e não durante um ciclo celular completo. Além disso, outras informações sobre a toxicologia devem ser obtidas no futuro.

  11. A Division-Dependent Compartmental Model for Computing Cell Numbers in CFSE-based Lymphocyte Proliferation Assays

    Science.gov (United States)

    2012-02-12

    based upon the premises of information theory, is found in the Akaike Information Criterion (AIC). Briefly, for models with independent, homoscedastic...we can compute the Akaike weights wr = exp ( −∆r 2 ) ∑ r exp ( −∆r 2 ) . (26) It can be shown (either by likelihood ratio tests or in a Bayesian ...No. 0704-0188 Public reporting burden for the collection of information is estimated to average 1 hour per response, including the time for reviewing

  12. Dose-dependent in vivo effect of Rhodiola and Echinacea on the mitogen-induced lymphocyte proliferation in mice.

    Science.gov (United States)

    Skopińska-Rózewska, E; Sokolnicka, I; Siwicki, A K; Stankiewicz, W; Dabrowski, M P; Buchwald, W; Krajewska-Patan, A; Mielcarek, S; Mścisz, A; Furmanowa, M

    2011-01-01

    Echinacea purpurea (EP) and Echinacea angustifolia (EA) are ones of the most important world's herbs with immunotropic activity. They were traditional medicinal plants used by North American Indians for the treatment of various illnesses. Now they are cultivated in many countries and are used mainly to treat respiratory tract infections. Rhodiola rosea (RR) and Rhodiola quadrifida (RQ) are medicinal plants originated from Asia and used traditionally as adaptogens, antidepressants, and anti-inflammatory remedies. We previously reported, that extracts of underground parts of RR and RQ exhibited immunotropic activity. We have demonstrated in pigs that in vitro RR or RQ supplementation of blood lymphocyte cultures stimulated T cell proliferative response to Con A in lower, and inhibited it in higher Rhodiola extract concentrations. The aim of this work was to evaluate the in vivo effect of these herbal remedies on the in vitro proliferative response of mouse splenic lymphocytes to another T-cell mitogen- Phaseolus vulgaris haemagglutinin (PHA). We have found significant stimulation of proliferative response, in comparison to the controls, in mice fed lower doses of tested remedies, and inhibition, no effect or lower stimulation, in mice fed higher doses of these drugs.

  13. Costimulation of resting B lymphocytes alters the IL-4-activated IRS2 signaling pathway in a STAT6 independent manner: implications for cell survival and proliferation

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    IL-4 is an important B cell survival and growth factor.IL-4 induced the tyrosine phosphorylation of IRS2 in resting B lymphocytes and in LPS- or CD40L-activated blasts.Phosphorylated IRS2 coprecipitated with the p85 subunit of PI 3' kinase in both resting and activated cells.By contrast,association of phosphorylated IRS2 with GRB2 was not detected in resting B cells after IL-4 treatment although both proteins were expressed.However,IL-4 induced association of IRS2 with GRB2 in B cell blasts.The pattern of IL-4-induced recruitment of p85 and GRB2 to IRS2 observed in B cells derived from STAT6 null mice was identical to that observed for normal mice.While IL-4 alone does not induce activation of MEK,a MEK1 inhibitor suppressed the IL-4-induced proliferative response of LPS-activated B cell blasts.These results demonstrate that costimulation of splenic B cells alters IL-4-induced signal transduction independent of STAT6 leading to proliferation.Furthermore,proliferation induced by IL-4 in LPS-activated blasts is dependent upon the MAP kinase pathway.

  14. Iron oxide nanoparticles show no toxicity in the comet assay in lymphocytes: A promising vehicle as a nitric oxide releasing nanocarrier in biomedical applications

    Science.gov (United States)

    de Lima, R.; Oliveira, J. L.; Murakami, P. S. K.; Molina, M. A. M.; Itri, R.; Haddad, P.; Seabra, A. B.

    2013-04-01

    This work reports the synthesis and toxicological evaluation of surface modified magnetic iron oxide nanoparticles as vehicles to carry and deliver nitric oxide (NO). The surface of the magnetic nanoparticles (MNPs) was coated with two thiol-containing hydrophilic ligands: mercaptosuccinic acid (MSA) or dimercaptosuccinic acid (DMSA), leading to thiolated MNPs. Free thiols groups on the surface of MSA- or DMSA-MNPs were nitrosated leading to NO-releasing MNPs. The genotoxicity of thiolated-coated MNPs was evaluated towards human lymphocyte cells by the comet assay. No genotoxicity was observed due to exposure of human lymphocytes to MSA- or DMSA-MNPs, indicating that these nanovectors can be used as inert vehicles in drug delivery, in biomedical applications. On the other hand, NO-releasing MPNs showed genotoxicity and apoptotic activities towards human lymphocyte cell cultures. These results indicate that NO-releasing MNPs may result in important biomedical applications, such as the treatment of tumors, in which MNPs can be guided to the target site through the application of an external magnetic field, and release NO directly to the desired site of action.

  15. Proliferation assays (BrdU and EdU) on skeletal tissue sections.

    Science.gov (United States)

    Mead, Timothy J; Lefebvre, Véronique

    2014-01-01

    Assessing cell proliferation in situ is an important phenotyping component of skeletal tissues from development to adult stages and disease. Various methods exist including immunostaining for proteins and protein modifications associated with specific steps of the cell cycle, but the gold standard is to quantify the percentage of DNA-synthesizing cells. The thymidine analog 5-bromo-2'-deoxyuridine (BrdU) has been widely used in the last decades for this purpose, with the inconvenience that its detection is lengthy and requires harsh treatment of tissue sections to give access of anti-BrdU antibody to nucleosides in genomic DNA. In 2008, Salic and Mitchison developed a new method and proved it to be quicker, simpler, and highly sensitive in non-skeletal tissues. This method relies on incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into de novo DNA. This other thymidine analog is readily detected by click chemistry, i.e., covalent cross-linking of its ethynyl group with a fluorescent azide, a molecule small enough to diffuse freely through native tissues and DNA. Here, we describe and compare the BrdU and EdU approaches in skeletal tissues and conclude that in these tissues too EdU provides an easy and very sensitive alternative to BrdU.

  16. Telomerase activity is spontaneously increased in lymphocytes from patients with atopic dermatitis and correlates with cellular proliferation

    DEFF Research Database (Denmark)

    Wu, Kehuai; Volke, Anne Rehné; Lund, Marianne

    1999-01-01

    , and staphylococcal enterotoxin A (SEA) (0.1 microg/ml). Telomerase activity was measured by the telomeric repeat amplification protocol-based telomerase polymerase chain reaction enzyme-linked immunosorbent assay at 0 and 72 h of incubation. In addition, DNA synthesis of the cells was assayed using 3H......-thymidine incorporation. We found that telomerase activity in non-stimulated PBMC from patients with AD was significantly up-regulated without any stimulation during the 72 h of in vitro incubation. The most potent stimulator of telomerase activity was SEA, followed by anti-CD3 plus IL-2, anti-CD3 alone, and PPD. IL-2...

  17. Use of an aqueous soluble tetrazolium/formazan assay to measure viability and proliferation of lymphokine-dependent cell lines.

    Science.gov (United States)

    Buttke, T M; McCubrey, J A; Owen, T C

    1993-01-04

    A new tetrazolium compound, MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt), has recently been described which in the presence of phenazine methosulfate (PMS) is reduced by living cells to yield a formazan product that can be assayed colorimetrically. An important advantage of MTS/PMS over other tetrazolium dyes (e.g., MTT) is the aqueous solubility of the reduced formazan product which eliminates the need for detergent solubilization or organic solvent extraction steps. Its advantages over XTT/PMS, another tetrazolium which yields a water-soluble formazan product, include the absorbance range of color produced (515-580 nm as opposed to 450 nm), the rapidity of color development, and the storage stability of the MTS/PMS reagent solution. In the present study, MTS/PMS was used to assay viability and proliferation of the IL-2-dependent HT-2 and CTLL-2 cell lines and the IL-3-dependent FDC-P1 and FL5.12 cell lines. With each cell line, the amount of formazan product was time-dependent and proportional to the number of viable cells. Furthermore, with both HT-2 and CTLL-2 cells it was found that cultures could be simultaneously labeled with MTS/PMS and [3H]thymidine, with relatively little effect of the dye on uptake of the latter. This feature was further capitalized upon in studies with FDC-P1 cells, in which the co-addition of MTS/PMS and [3H]thymidine was used to distinguish between cell viability and proliferation.

  18. Proliferation assay of mouse embryonic stem (ES) cells exposed to atmospheric-pressure plasmas at room temperature

    Science.gov (United States)

    Miura, Taichi; Ando, Ayumi; Hirano, Kazumi; Ogura, Chika; Kanazawa, Tatsuya; Ikeguchi, Masamichi; Seki, Atsushi; Nishihara, Shoko; Hamaguchi, Satoshi

    2014-11-01

    Proliferation assays of mouse embryonic stem (ES) cells have been performed with cell culture media exposed to atmospheric-pressure plasmas (APPs), which generate reactive species in the media at room temperature. It is found that serum in cell culture media functions as a scavenger of highly reactive species and tends to protect cells in the media against cellular damage. On the other hand, if serum is not present in a cell culture medium when it is exposed to APP, the medium becomes cytotoxic and cannot be detoxified by serum added afterwards. Plasma-induced cytotoxic media hinder proliferation of mouse ES cells and may even cause cell death. It is also shown by nuclear magnetic resonance spectroscopy that organic compounds in cell culture media are in general not significantly modified by plasma exposure. These results indicate that if there is no serum in media when they are exposed to APPs, highly reactive species (such as OH radicals) generated in the media by the APP exposure are immediately converted to less reactive species (such as H2O2), which can no longer readily react with serum that is added to the medium after plasma exposure. This study has clearly shown that it is these less reactive species, rather than highly reactive species, that make the medium cytotoxic to mouse ES cells.

  19. The improvement effects of edible bird’s nest on proliferation and activation of B lymphocyte and its antagonistic effects on immunosuppression induced by cyclophosphamide

    Directory of Open Access Journals (Sweden)

    Zhao R

    2016-01-01

    Full Text Available Ran Zhao,1,* Geng Li,1,* Xiu-juan Kong,1 Xiu-yan Huang,2 Wei Li,1 Yao-ying Zeng,2 Xiao-ping Lai31Traditional Chinese Medicinal College, Guangzhou University of Chinese Medicine, 2Life Science College, Jinan University, Guangzhou, 3Dongguan Mathematical Engineering Academy of Chinese Medicine, Guangzhou University of Chinese Medicine, Dongguan, People’s Republic of China*These authors contributed equally to this workAbstract: Edible bird’s nest (EBN is regarded as an immune-enhancing food in the People’s Republic of China. The aim of this study is to demonstrate the efficiency of EBN in improving the immunity of mouse both in vivo and in vitro. We observed the effects of EBN on spleen lymphocytes proliferation and activation, as well as immunoglobulin isotypes as indicators. In addition, we evaluated the content of total sIgA in the intestinal juice to assess mucosal immunity. The results showed that EBN could promote the proliferation and activation of B-cells and increase IgE, IgA, IgM, and IgG3 levels. We also found that EBN extract can promote the secretion of sIgA in the small intestine. Using cyclophosphamide (CY, we established an immunosuppressed mouse model in which we identified a reversal influence on the ratio of CD3+/CD19+ cells, which indicates that EBN also protects B-cells from the damage induced by CY. We also applied polymyxin B to exclude the interference of lipopolysaccharide throughout the experiment. In conclusion, we found that EBN can reduce the intestinal immune injury induced by CY by accelerating the proliferation and activation of B-cells and enhancing antibody secretion of B-cells.Keywords: chemotherapy, immunological enhancement, intestinal mucosal immune, EBN

  20. Proliferation induced by Plasmodium falciparum antigen and interleukin-2 production by lymphocytes isolated from malaria-immune individuals

    DEFF Research Database (Denmark)

    Theander, T G; Bygbjerg, I C; Jepsen, S

    1986-01-01

    -immune individuals, the proliferative response and the interleukin-2 (IL-2) production of SPAg-activated mononuclear cells (MNCs) from individuals unexposed, sensitized, and immune to malaria were measured. It was found that MNC isolated from malaria-immune individuals proliferated in response to SPAg...... and that this activation resulted in measurable IL-2 production in 5 of 10 MNC cultures. MNC isolates from most unexposed individuals did not respond to SPAg. To establish which cells responded to SPAg, different subpopulations of MNCs were tested. Only T helper cells were found to respond, and they responded only when...

  1. Fluorescent ester dye-based assays for the in vitro measurement of Neospora caninum proliferation.

    Science.gov (United States)

    Mota, Caroline M; Ferreira, Marcela D; Costa, Lourenço F; Barros, Patrício S C; Silva, Murilo V; Santiago, Fernanda M; Mineo, José R; Mineo, Tiago W P

    2014-09-15

    Techniques for the measurement of parasite loads in different experimental models have evolved throughout the years. The quantification of stained slides using regular cytological stains is currently the most common technique. However, this modality of evaluation is labor-intensive, and the interpretation of the results is subjective because the successes of the assays mainly rely on the abilities of the professionals involved. Moreover, the novel genetic manipulation techniques that are commonly applied for closely related Toxoplasma gondii have not yet been developed for Neospora caninum. Thus, we aimed to develop a simple protocol for parasite quantification using pre-stained N. caninum tachyzoites and fluorescent probes based on ester compounds (i.e., CFSE and DDAO). For this purpose, we employed a quantification procedure based on flow cytometry analysis. Pre-stained parasites were also examined with a fluorescent microscope, which revealed that both dyes were detectable. Direct comparison of the numbers of CFSE+ and DDAO+ cells to the values obtained with classical cytology techniques yielded statistically comparable results that also accorded with genomic DNA amplification results. Although the fluorescence emitted by DDAO was more intense and provided better discrimination between the populations of parasitized cells, CFSE+ tachyzoites were detected for several days. In conclusion, this study describes a simple, fast, low-cost and reproducible protocol for N. caninum quantification that is based on parasite pre-staining with fluorescent ester-based probes.

  2. 氧化苦参碱对小鼠淋巴结T细胞增殖的双向作用%Two-ways effects of Oxymatrine on T lymphocyte proliferation in mouse lymph node stimulated by Con A

    Institute of Scientific and Technical Information of China (English)

    伍斌; 谢红付; 李罗丝; 张江林; 李建国

    2006-01-01

    [Objective] To investigate the effects of Oxymatrine (OMT) on mouse lymphocyte proliferation stimulated by Con A, explore the mechanism of the effects of OMT on the immune system and provide theoretical and experimental evidence for the clinical application of OMT in treating immune-related diseases. [Methods] CFDA-SE staining and flow cytometry were used to detect the fluorescence intensity of lymphocytes after stimulated by polyclonal stimulators Con A and OMT. Related softwares were used to analyze the effects of OMT on mouse lymphocyte proliferation. [Results] OMT has the function to restrain the proliferation of lymphocyte of mouse depended on its concentration with 500, 125 and 31 μg/mL in substrate, but 16, 8, 4, 2 μg/mL concentration, it improves the proliferation of T lymphocytes of mouse's lymph node, the dependence on its concentration is not significant. [Conclusions ] 1. Both CFDA-SE dyeing and flow cytometer were reliable tools to analyze lymphocyte proliferation. 2. OMT has the two-ways effects on T lymphocyte proliferation in mouse lymph node stimulated by Con A.%目的检测氧化苦参碱(Oxymatrine,OMT)对刀豆蛋白A(Con A)刺激的小鼠淋巴结T细胞增殖的影响,探讨OMT对免疫系统的作用机制,为临床用OMT治疗免疫相关性疾病提供理论和实验依据.方法利用CFDA-SE染色,流式细胞术检测淋巴细胞在多克隆刺激剂Con A和OMT的共同作同下荧光强度的变化,并应用CELLQuest软件分析OMT对小鼠淋巴结T细胞增殖的影响程度.结果 500、125和31μg/mLOMT对小鼠淋巴结T细胞增殖呈剂量依赖性抑制,而16、8、4、2μg/mL OMT对小鼠淋巴结T细胞增殖起促进作用,但其剂量依赖关系不明显.结论 CFDA-SE染色和流式细胞术是分析淋巴细胞增殖的有力工具:OMT对小鼠淋巴结T细胞的增殖呈双向作用.

  3. A simple method to measure cell viability in proliferation and cytotoxicity assays

    Directory of Open Access Journals (Sweden)

    Ricardo Carneiro Borra

    2009-09-01

    Full Text Available Resazurin dye has been broadly used as indicator of cell viability in several types of assays for evaluation of the biocompatibility of medical and dental materials. Mitochondrial enzymes, as carriers of diaphorase activities, are probably responsible for the transference of electrons from NADPH + H+ to resazurin, which is reduced to resorufin. The level of reduction can be quantified by spectrophotometers since resazurin exhibits an absorption peak at 600 ηm and resorufin at 570 ηm wavelengths. However, the requirement of a spectrophotometer and specific filters for the quantification could be a barrier to many laboratories. Digital cameras containing red, green and blue filters, which allow the capture of red (600 to 700 ηm and green (500 to 600 ηm light wavelengths in ranges bordering on resazurin and resorufin absorption bands, could be used as an alternative method for the assessment of resazurin and resorufin concentrations. Thus, our aim was to develop a simple, cheap and precise method based on a digital CCD camera to measure the reduction of resazurin. We compared the capability of the CCD-based method to distinguish different concentrations of L929 and normal Human buccal fibroblast cell lines with that of a conventional microplate reader. The correlation was analyzed through the Pearson coefficient. The results showed a strong association between the measurements of the method developed here and those made with the microplate reader (r² = 0.996; p < 0.01 and with the cellular concentrations (r² = 0.965; p < 0.01. We concluded that the developed Colorimetric Quantification System based on CCD Images allowed rapid assessment of the cultured cell concentrations with simple equipment at a reduced cost.

  4. Genotoxicity assessment of antidiabetic formulation (ADPHF6 in human lymphocytes by single cell gel electrophoresis (comet assay - an in vitro study

    Directory of Open Access Journals (Sweden)

    Devanand Shanmugasundaram

    2015-06-01

    Full Text Available Levels of Reactive Oxygen Species (ROS molecules during aerobic metabolism are often regulated by unique endogenous antioxidant system. During hyperglycaemic condition, accumulation of excess fatty acids & glucose in adipose tissue (Wright Jr E., 2006 results in increased levels of ROS. When ROS molecules overwhelms the cells antioxidant defence system, it ends up in cellular oxidative stress; which in turn is reported to cause oxidative DNA damage & intervene damage to macromolecules & cellular membranes (Ahmad et al., 2013. Our novel anti-hyperglycaemic polyherbal formulation (ADPHF6 had already illustrated significant inhibitory activity against α-amylase & α-glucosidase enzymes and also scavenging free radicals (in vitro models. The present study demonstrates the protective effect of formulation against H2O2 induced DNA damage in human lymphocytes by Single Cell Gel Electrophoresis (SCGE assay. Experimental procedures were approved by Institutional Human Ethics Committee of Frontier Lifeline Hospital, Chennai, India (FLL/IEC/02/2014. Peripheral human lymphocytes were isolated (Duthie et.al, 2002 and subjected for Cell viability by Trypan blue exclusion method. The alkaline SCGE assay was carried out to determine the level of DNA damage in ADPHF6 treated cells with minor modifications from Singh et al., 1988. Frosted microscopic slides were pre-coated with 1% NMA followed by 1% LMA and incubated for 15 min at 15-20o C. 100 μL of freshly prepared cell suspension (2 x 104 cells was mixed with 0.5% LMA & casted on microscopic slide. The cells were immersed in lysing solution for 2 hours at 4O C and washed in TBE buffer for 5 min at RT. All the slides were treated with fresh alkaline solution for 20 minutes for expression of alkali-labile damage. Electrophoresis was performed at 24 V for 20 min at RT. Slides were washed in neutralizing buffer for 5 min at RT. All the groups were stained with Acridine Orange (20µg/ml & Propidium Iodide (20µg

  5. Effects of ginkgolide B on activation, proliferation and apoptosis of murine T lymphocytes in vitro%银杏内酯B对小鼠T淋巴细胞体外活化、增殖与凋亡的影响

    Institute of Scientific and Technical Information of China (English)

    王晓东; 曾耀英; 宋兵

    2011-01-01

    To explore the potential immunomodulatory effects and related mechanisms of ginkgolide B (GB), a known potent antagonist of platelet-activating factor receptor, we investigated the behaviors of T lymphocytes, including activation, proliferation and apoptosis. T lymphocytes were prepared from murine lymph nodes, and then treated with different concentrations of GB before cultivation. Activation and proliferation of T cells were induced by polyclonal activator concanavalin (Con A). Fluorescence conjugated monoclonal antibodies and flow cytometry were used to detect the expression of CD3/CD69 and CD3/CD25; CFDA-SE labeling and flow cytometry were used to trace the proliferation of T lymphocytes and an MTT assay was taken to re-prove. Apoptosis of T cells were induced by dexamethasone (Dex), while [DiOC6(3)/PI] staining and flow cytometry were used to distinguish apoptotic, dead and living cell populations. In a dose-dependent manner, GB (5, 10, and 20 (xmol/L) significantly suppressed the expression of CD69 at 6th h and CD25 at 24th h, as well as inhibited cell proliferation at 48th h after Con A stimulation. To some extent, GB also exhibited protective effect on T lymphocytes from Dex-induced apoptosis. According to these interesting effects of GB on T lymphocytes behaving and functioning, it's quite reasonable to do further studies of GB as a nature occurring immunomodulator candidate.%目的 银杏内酯B (6B)是已知的天然而强效的血小板激活因子(PAF)受体(PAFR)拈抗剂,本文研究GB对小鼠T淋巴细胞活化、增殖及凋亡3大体外行为的影响,初步探讨其潜在的免疫调节作用与机制,从而为临床应用提供可靠的实验依据.方法 分离小鼠淋巴细胞,培养前以不同浓度的GB预孵;以刀豆蛋白A(Con A)诱导细胞的活化与增殖,以荧光抗体双色标记(anti-CD3 mAb-PE/anti-CD69 mAb-FITC、anti-CD3 mAb-PE/anti-CD25 mAb-FITC)结合流式细胞术分别检测T淋巴细胞早、中

  6. Comparison of differences between dicentric assay and translocation analysis for biodosimetry in cultured peripheral blood lymphocytes of Korean individuals

    Energy Technology Data Exchange (ETDEWEB)

    Park, Hyun Jin; Park, Mi Young; Seo, Min Ji; Kwon, Hee Kyung; Lee, Su Jae; Lee, Yun Sil; Ji, Young Hoon; Choi, Soo Yong; Cho, Chul Koo; Kim, Tae Hwan; Kang, Chang Mo [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2004-07-01

    Chromosome aberrations are considered to be important indicators of induced DNA damage and genomic instability. For this reason, they constitute the main parameter used to monitor individuals exposed to radiation. Biological dosimetry using the analysis of dicentrics in human lymphocytes is well established, especially in case of acute exposure, when the blood samples are taken within a few weeks. However, dicentric analysis is not an adequate parameter in case of chronic exposure, because these aberrations are unstable with time, and have a limited use for dose assessment of past exposures. In contrast to dicentrics, however, translocations are considered stable in cell division and so the yield should not fall with time. In the present study, using FISH-chromosome painting analysis with the dose-response curve for chromosome aberrations, we monitored the stable and unstable chromosome aberrations of 2 Korean's periperal blood lymphocytes irradiated in vitro with {gamma}-rays from {sup 137}Cs (doses between 0.0 and 2.0 Gy). By using the dose-response curve for chromosome aberration, our aim was to estimate the absorbed doses, and then establish comparison with the results obtained by conventional dicentric analysis, thus taking the opportunity to test the validity of chromosome aberration analysis by FISH painting method for retrospective biodosimetry in Korean individual.

  7. Assessment of the DNA Damage in Human Sperm and Lymphocytes Exposed to the Carcinogen Food Contaminant Furan with Comet Assay

    Directory of Open Access Journals (Sweden)

    Dilek Pandir

    2015-10-01

    Full Text Available ABSTRACTThe aim of this work was to assess the damage of DNA in human blood cell and spermin vitro under the influence of furan. These cells were administered 0-600 μM of furan at 37 and 32oC for 30 and 60 min, respectively. A significant increase in tail DNA%, tail length and moment indicating DNA damage was observed at increasing doses when compared to the controls. The treatment with 300 and 600 μM of furan showed a maximum increase of 86.74 ± 2.43 and 93.29 ± 8.68 compared to the control tail DNA% in lymphocytes. However, only 600 μM of furan showed a maximum increase of 94.71 ± 6.24 compared to the control tail DNA% in sperm. The results suggested that furan caused DNA damage in lymphocytes at increasing doses, but appeared to have not the same effect on human sperm at the low doses. Genotoxic activity had an impact on the risk assessment of furan formed on the food for human cells. Therefore, it would be important to further investigate these properties of furan as the food mutagen.

  8. Estudo da proliferação linfocitária em pacientes sensibilizados ao níquel Study on lymphocyte proliferation in nickel sensitive patients

    Directory of Open Access Journals (Sweden)

    Ana Paula Galli Sanchez

    2005-04-01

    Full Text Available FUNDAMENTO: O diagnóstico da alergia ao níquel é estabelecido com a realização do teste de contato. OBJETIVO: Desenvolver um método diagnóstico mais sensível e específico. CASUÍSTICAS E MÉTODOS: Dezenove pacientes com teste de contato positivo para o níquel e 25 controles foram submetidos ao teste da proliferação linfocitária. As células mononucleadas foram isoladas do sangue venoso periférico e cultivadas em triplicatas, em placas de cultura (2x10(5 células/orifício com: meio de cultura apenas; sulfato de níquel (156,25; 78,13; 19,53; 9,77 e 2,44µM e concentrações ideais do antígeno Candida albicans e dos mitógenos pokeweed, fito-hemaglutinina A e anticorpo anti-CD3 (OKT3. Timidina tritiada foi adicionada às placas, a radioatividade incorporada pelas células medida e os resultados expressos pelo índice de estimulação (IE. RESULTADOS: A resposta proliferativa dos linfócitos dos casos foi superior à dos controles em todas as concentrações de níquel testadas. Considerando teste positivo para níquel quando IE > 3, nenhum dos controles e 16 (84,21% dos casos apresentaram teste positivo em pelo menos uma das cinco concentrações usadas. As respostas à Candida albicans e aos mitógenos foram semelhantes nos casos e controles, demonstrando a integridade da imunidade celular em ambos os grupos. CONCLUSÃO: O teste da proliferação linfocitária mostra-se útil no diagnóstico da alergia ao níquel.BACKGROUND: The diagnosis of nickel sensitivity is made by epicutaneous patch testing. OBJECTIVE: To develop a more sensitive and specific test. PATIENTS AND METHODS: Nineteen patients with positive patch test reactions to nickel and 25 controls were submitted to lymphocyte proliferation test. Mononuclear cells were isolated from peripheral venous blood and cultivated in triplicate in culture plates (2x10(5 cells/well with: culture medium only, nickel sulfate (156.25; 78.13; 19.53; 9.77 and 2.44µM and optimal

  9. Detection of weak estrogenic flavonoids using a recombinant yeast strain and a modified MCF7 cell proliferation assay.

    Science.gov (United States)

    Breinholt, V; Larsen, J C

    1998-06-01

    A newly developed recombinant yeast strain, in which the human estrogen receptor has been stably integrated into the genome of the yeast, was used to gain information on the estrogenic activity of a large series of dietary flavonoids. Among 23 flavonoids investigated, 8 were found to markedly stimulate the transcriptional activity of the human estrogen receptor in the yeast assay increasing transcriptional activity 5-13-fold above background level, corresponding to EC50 values between 0.1 and 25 microM. Five compounds increased the transcriptional activity 2-5-fold over the control, with EC50 values ranging from 84 to 102 microM, whereas the remaining flavonoids were devoid of activity. The most potent flavonoid estrogens tested were naringenin, apigenin, kaempferol, phloretin, and the four isoflavonoids equol, genistein, daidzein, and biochanin A. With the exception of biochanin A, the main feature required to confer estrogenicity was the presence of a single hydroxyl group in the 4'-position of the B-ring of the flavan nucleus, corresponding to the 4-position on phloretin. The estrogenic potency of the flavonoids was found to be 4 000-4 000 000 times lower than that observed for 17beta-estradiol, when compared on the basis of EC50 values. The estrogenic activity of the dietary flavonoids was further investigated in estrogen-dependent human MCF7 breast cancer cells. In this system several of the flavonoids were likewise capable of mimicking natural estrogens and thereby induce cell proliferation. Similar structural requirements for estrogenic activity were found for the two assays. The present results provide evidence that several of the flavo-estrogens possess estrogenic properties comparable in activity to the well-established isoflavonoid estrogens. The use of Alamar Blue, a vital dye which is metabolically reduced by cellular enzymes to a fluorescent product, was found to greatly simplify the MCF7 cell-based estrogen screen, making this mammalian assay

  10. The sensitivity of the in vitro cytokinesis-blocked micronucleus assay in lymphocytes for different and combined radiation qualities

    Energy Technology Data Exchange (ETDEWEB)

    Wuttke, K.; Mueller, W.U.; Streffer, C. [Universitaetsklinikum Essen (Germany). Inst. fuer Medizinische Strahlenbiologie

    1998-05-01

    Purpose: The dose-response relationship and the relative biological effectiveness (RBE) for the induction of micronuclei in lymphocytes was analyzed after irradiation in vitro with a 6-MeV neutron beam that was followed by 240-kV X-rays. The dose range of the combined exposure comprised 1 to 3 Gy. For reference, the dose-effect relationships found after X-ray (0.5 to 5 Gy)- and neutron (0.5 to 4 Gy) exposure applied separately are presented. The possibility of an interaction between the 2 radiation qualities is investigated by the method of isobole calculation termed `envelope of additivity`. Methods: Micronuclei were analyzed in PHA-stimulated, cytokinesis-blocked human lymphocytes. Results: The dose-response relationships for the micronucleus frequencies induced by the neutron irradiation, as well as by the mixed exposure, were linear. A saturation effect was indicated after neutron doses higher than 3 Gy. After low LET exposure the dose-response curves were describable by a linear-quadratic model. For neutron-induced micronucleus frequencies, RBE-values of 2 to 3 and for the combined exposure RBE values of 1.5 to 2 were calculated for a range of effect of 0.5 to 1.5 micronuclei/binucleated lymphocyte. No indication was found for an interaction between the damage induced by X-rays and that produced by neutrons under our experimental conditions. (orig./MG) [Deutsch] Fragestellung: Die Mikronukeusinduktion in Lymphozyten wurde nach In-vitro-Bestrahlung mit 6-MeV-Neutronen (0,5 bis 4 Gy), 240-kV-Roentgenstrahlung (0,5 bis 5 Gy) bzw. einer Kombination dieser Strahlenqualitaeten (1 bis 3 Gy Gesamtdosis) untersucht. Anhand der Dosis-Wirkungs-Beziehungen fuer die einzelne und kombinierte Anwendung beider Strahlenarten wurde die relative biologische Wirksamkeit (RBW) fuer Neutronen bzw. fuer die Kombination von Neutronen und Roentgenstrahlen ermittelt. Mit Hilfe einer Isobolenkalkulation (`envelope of additivity`) wurde die Moeglichkeit einer Interaktion zwischen den

  11. In Vitro Endothelial Cell Proliferation Assay Reveals Distinct Levels of Proangiogenic Cytokines Characterizing Sera of Healthy Subjects and of Patients with Heart Failure

    Directory of Open Access Journals (Sweden)

    Rebecca Voltan

    2014-01-01

    Full Text Available Although myocardial angiogenesis is thought to play an important role in heart failure (HF, the involvement of circulating proinflammatory and proangiogenic cytokines in the pathogenesis and/or prognosis of HF has not been deeply investigated. By using a highly standardized proliferation assay with human endothelial cells, we first demonstrated that sera from older (mean age 52±7.6 years; n=46 healthy donors promoted endothelial cell proliferation to a significantly higher extent compared to sera obtained from younger healthy donors (mean age 29±8.6 years; n=20. The promotion of endothelial cell proliferation was accompanied by high serum levels of several proangiogenic cytokines. When we assessed endothelial cell proliferation in response to HF patients’ sera, we observed that a subset of sera (n=11 promoted cell proliferation to a significantly lesser extent compared to the majority of sera (n=18. Also, in this case, the difference between the patient groups in the ability to induce endothelial cell proliferation correlated to significant (P<0.05 differences in serum proangiogenic cytokine levels. Unexpectedly, HF patients associated to the highest endothelial proliferation index showed the worst prognosis as evaluated in terms of subsequent cardiovascular events in the follow-up, suggesting that high levels of circulating proangiogenic cytokines might be related to a worse prognosis.

  12. Quantitation of minimal disease levels in chronic lymphocytic leukemia using a sensitive flow cytometric assay improves the prediction of outcome and can be used to optimize therapy.

    Science.gov (United States)

    Rawstron, A C; Kennedy, B; Evans, P A; Davies, F E; Richards, S J; Haynes, A P; Russell, N H; Hale, G; Morgan, G J; Jack, A S; Hillmen, P

    2001-07-01

    Previous studies have suggested that the level of residual disease at the end of therapy predicts outcome in chronic lymphocytic leukemia (CLL). However, available methods for detecting CLL cells are either insensitive or not routinely applicable. A flow cytometric assay was developed that can differentiate CLL cells from normal B cells on the basis of their CD19/CD5/CD20/CD79b expression. The assay is rapid and can detect one CLL cell in 10(4) to 10(5) leukocytes in all patients. We have compared this assay to conventional assessment in 104 patients treated with CAMPATH-1H and/or autologous transplant. During CAMPATH-1H therapy, circulating CLL cells were rapidly depleted in responding patients, but remained detectable in nonresponders. Patients with more than 0.01 x 10(9)/L circulating CLL cells always had significant (> 5%) marrow disease, and blood monitoring could be used to time marrow assessments. In 25 out of 104 patients achieving complete remission by National Cancer Institute (NCI) criteria, the detection of residual bone marrow disease at more than 0.05% of leukocytes in 6 out of 25 patients predicted significantly poorer event-free (P =.0001) and overall survival (P =.007). CLL cells are detectable at a median of 15.8 months (range, 5.5-41.8) posttreatment in 9 out of 18 evaluable patients with less than 0.05% CLL cells at end of treatment. All patients with detectable disease have progressively increasing disease levels on follow-up. The use of sensitive techniques, such as the flow assay described here, allow accurate quantitation of disease levels and provide an accurate method for guiding therapy and predicting outcome. These results suggest that the eradication of detectable disease may lead to improved survival and should be tested in future studies.

  13. Computer-based design of an HLA-haplotype and HIV-clade independent cytotoxic T-lymphocyte assay for monitoring HIV-specific immunity.

    Science.gov (United States)

    Amicosante, Massimo; Gioia, Cristiana; Montesano, Carla; Casetti, Rita; Topino, Simone; D'Offizi, Gianpiero; Cappelli, Giulia; Ippolito, Giuseppe; Colizzi, Vittorio; Poccia, Fabrizio; Pucillo, Leopoldo P.

    2002-01-01

    BACKGROUND: Human immunodeficiency virus (HIV)- specific CD8-positive cytotoxic T-lymphocytes (CTL) play a key role in controlling HIV infection. Monitoring CTL response could be clinically relevant during structured therapy interruption (STI), HIV exposure, and vaccine trials. However, HLA patients' restriction and HIV variability limited the development of a CTL assay with broad specificity. MATERIALS AND METHODS: We designed an HLA-class I/HIV-1 clade independent assay for assessing HIV- specific CTL by using a computer-assisted selection ofthe CTL epitopes. Twenty-eight 15-mers were selected by peptide-binding motifs analysis using different databases (HIV-Immunology Database, SYFPEITHI, BIMAS). Altogether they putatively bind to more than 90% of HLA haplotypes in different populations, with an overall HIV-1 variability below 9%. The peptide pool was used as an antigen in an intracellular cytokine staining (ICS) assay for quantifying HIV-specific CTL response. RESULTS: The test can be performed using both fresh and cryopreserved peripheral blood mononuclear cells (PBMC), whereas GAG protein as antigen works only on fresh PBMC. A significantly higher CTL response with respect to HIV-negative controls was detected in all HIV-1 infected subjects of two groups of patients with different ethnicities (Caucasians and Africans) and coming from areas with different HIV-1 clade prevalences (clade B and A/G, respectively). In Caucasian patients, after month of STI, the number of HIV-1 specific CTL (2,896 +/- 2,780 IFN-gamma specific CD8 cells/ml) was significantly higher than that found at enrolment (2,125 +/- 4,426 IFN-gamma specific CD8 cells/ml, p< 0.05). CONCLUSIONS: These data indicate that this CTL assay is broadly specific and could represent a useful clinical tool for HIV immunodiagnostic independent of HLA-haplotype and HIV-clade variabilities. PMID:12606814

  14. 21. New developments in the lymphocyte micronucleus assay-the effect of diet, genotype and the importance of nucleoplasmic bridges

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    @@The cytokinesis-block micronucleus(CBMN)assay is an established method for studying chromosome breakage and loss in human cells both in vitro and in vivo. However, its use in the study of genomic stability could be improved by a better understanding of the impact of (a) micronutrients (b) genetic background and (c) of other associated important nuclear events indicative of chromosome rearrangement (ie nucleoplasmic

  15. INVESTIGATION OF DNA REPAIR BY SISTER CHROMATID EXCHANGE (SCE) ANALYSIS AND THE ALKALINE SINGLE CELL GEL ASSAY (SCG) IN MAMMALIAN GO-LYMPHOCYTES AFTER IN VITRO EXPOSURE TO ETHYLENE OXIDE (EO)

    Science.gov (United States)

    Investigation ofDNA Repair by Sister Chromatid Exchange (SCE) Analysis and the Alkaline Single Cell Gel Assay (SCG) in Mammalian Go-Lymphocytes after In Vitro Exposure to Ethylene Oxide (EO). EO is a large volume chemical used primarily as an intermediate in manufacturing...

  16. Cannabinoids inhibit T-cells via cannabinoid receptor 2 in an in vitro assay for graft rejection, the mixed lymphocyte reaction.

    Science.gov (United States)

    Robinson, Rebecca Hartzell; Meissler, Joseph J; Breslow-Deckman, Jessica M; Gaughan, John; Adler, Martin W; Eisenstein, Toby K

    2013-12-01

    Cannabinoids are known to have anti-inflammatory and immunomodulatory properties. Cannabinoid receptor 2 (CB2) is expressed mainly on leukocytes and is the receptor implicated in mediating many of the effects of cannabinoids on immune processes. This study tested the capacity of Δ(9)-tetrahydrocannabinol (Δ(9)-THC) and of two CB2-selective agonists to inhibit the murine Mixed Lymphocyte Reaction (MLR), an in vitro correlate of graft rejection following skin and organ transplantation. Both CB2-selective agonists and Δ(9)-THC significantly suppressed the MLR in a dose dependent fashion. The inhibition was via CB2, as suppression could be blocked by pretreatment with a CB2-selective antagonist, but not by a CB1 antagonist, and none of the compounds suppressed the MLR when splenocytes from CB2 deficient mice were used. The CB2 agonists were shown to act directly on T-cells, as exposure of CD3(+) cells to these compounds completely inhibited their action in a reconstituted MLR. Further, the CB2-selective agonists completely inhibited proliferation of purified T-cells activated by anti-CD3 and anti-CD28 antibodies. T-cell function was decreased by the CB2 agonists, as an ELISA of MLR culture supernatants revealed IL-2 release was significantly decreased in the cannabinoid treated cells. Together, these data support the potential of this class of compounds as useful therapies to prolong graft survival in transplant patients.

  17. Plasmid DNA as a special stimular to stimulate lymphocyte proliferation%DNA作为一种特异性刺激剂刺激淋巴细胞增殖的研究

    Institute of Scientific and Technical Information of China (English)

    吴琼; 孙英军; 张艳; 郑海学

    2011-01-01

    目的 为了探讨质粒DNA体外刺激淋巴细胞的增殖状况,建立了一种方便可靠的评价豚鼠细胞免疫水平的试验方法.方法 用羧基荧光素乙酰乙酸琥珀酰亚胺酯(cFsEl染色豚鼠全血,经植物血凝素(PHA)和质粒DNA刺激培养,利用流式细胞术分析细胞的增殖状况.结果 豚鼠全血经PHA和DNA刺激,淋巴细胞增殖能力不同;未免疫组经DNA和PHA刺激后增殖的差异显著,免疫组差异不显著.DNA质粒在体内外均可作为刺激源刺激淋巴细胞增殖.结论 建立了一种基于活细胞染料CFSE染色的豚鼠全血淋巴细胞增殖试验方法,可方便、快速、有效地评价细胞免疫水平.%To investigate the level of lymphocyte proliferation stimulated by plasmid DNA in vitro, and to establish a convenient and reliable method to assess the level of cellular immunity in guinea pig, the whole blood of guinea pig was stained by Carboxyfluorescein diacetate succinimidyl ester (CFSE), then stimulated by phytohemagglutinin(PHA) and plasmid DNA, and cultivated for 3 days.The cell proliferation was detected by flow cytometry.We observed that PHA and DNA stimulation could promote lymphocyte proliferation: the proliferation in non-immune group was significantly enhanced, while the inactivated vaccine immune group did not significantly changed, which indicate that plasmid DNA can be used as animmunogen to stimulate lymphocyte proliferation.Through this study, a live cell-based dye CFSE staining of guinea pig whole blood lymphocyte proliferation test method was established to evaluate the cellular immunity conveniently and effectively.

  18. Stimulation of AIDS lymphocytes with calcium ionophore (A23187) and phorbol ester (PMA): studies of cytoplasmic free Ca, IL-2 receptor expression, IL-2 production, and proliferation

    DEFF Research Database (Denmark)

    Hofmann, B; Moller, J; Langhoff, E

    1989-01-01

    nine patients with AIDS with the response of lymphocytes from nine control subjects showed that the response of AIDS lymphocytes was severely decreased when stimulated with PHA and no further response could be achieved by stimulation with A23187/PMA. On the other hand, no significant difference between...... the PHA-induced rise of cytoplasmic free calcium concentration ([Ca2+]1) in normal and AIDS lymphocytes was observed. The percentage of cells expressing IL-2 receptors (CD25) was also normal both after addition of PHA and after addition of A23187/PMA and the expression was normal on both CD4 and CD8 cells....... The production of IL-2 in normal lymphocytes stimulated with A23187/PMA was 33 times higher than that after stimulation with PHA. In AIDS lymphocytes the production of IL-2 induced by all activators was severely decreased compared to control subjects, although the production of IL-2 after stimulation with A23187...

  19. 两种细胞增殖分析方法的比较性研究%Contrastive study of two cell proliferation assays

    Institute of Scientific and Technical Information of China (English)

    韩建群; 修瑞娟

    2012-01-01

    Objective To compare the detection sensitivity between MTT and EdU incorporation for cell proliferation assay. Methods Prostate cancer cell line ( PC-3 ) was exposed to different concentrations of cisplatin for 24 hours. Cells viability and proliferation were respectively detected by MTT and EdU incorporation assay. Result PC-3 cell viability and proliferation were significantly decreased in 5 μg/mL condition by MTT detection (P <0. 05) , however, the proliferation positive cell ratio and the density of fluorescence were significantly decreased in 2 μg/mL condition by EdU incorporation assay ( P < 0.05). Conclusion Simple, fast and economic are the advantages of MTT method, but it only reflects the sta-tus of viable cells, independent with proliferation population; sensitivity, objectivity and specificity are the advantages of EdU incorporation assay, which is related with S phase cell cycle, directly reflects the cells proliferation.%目的 比较MTT法和EdU标记法两种常用测定细胞增殖的方法的优劣.方法 以化疗药物顺铂浓度梯度处理前列腺癌细胞株PC-3,分别采用MTT法和EdU标记技术进行细胞增殖变化测定.结果 MTT组顺铂浓度增至5μg/mL时细胞活力和增殖明显受抑(P<0.05);EdU掺入组顺铂浓度增至2μg/mL时增殖细胞阳性率和胞内荧光强度均显著下降(P<0.05).结论 MTT法简单、快速、经济,结果与活细胞数有关,间接反映细胞的增殖状态;EdU标记技术灵敏、客观,特异性标记分裂期细胞,直接反映细胞的增殖.

  20. Sulfonylureas and glinides exhibit peroxisome proliferator-activated receptor gamma activity: A combined virtual screening and biological assay approach

    NARCIS (Netherlands)

    Scarsi, M.; Podvinec, M.; Roth, A.; Hug, H.; Kersten, A.H.; Albrecht, H.; Schwede, T.; Meyer, U.A.; Rucker, C.

    2007-01-01

    Most drugs currently employed in the treatment of type 2 diabetes either target the sulfonylurea receptor stimulating insulin release (sulfonylureas, glinides), or target the peroxisome proliferator-activated receptor (PPAR) improving insulin resistance (thiazolidinediones). Our work shows that sulf

  1. G-banding chromosomal aberration assay of lymphocyte after radiation%辐射后淋巴细胞的G显带染色体畸变分析

    Institute of Scientific and Technical Information of China (English)

    赵明刚; 施常备; 赵华; 袁勇; 姚俊涛; 陆建荣; 袁彬; 陈葳; 王翔

    2011-01-01

    目的:通过染色体G显带技术研究辐射后染色体的结构畸变情况.方法:4MV的X线辐射离体入血,淋巴细胞培养后,应用染色体G显带技术,分析辐射后染色体的结构畸变情况,包括双着丝粒、缺失、易位、环状染色体、染色体片段,等.结果:辐射后观察分散良好、形态清晰的细胞核型100个,染色体畸变总数为428,其中双着丝粒、缺失、易位、环状染色体、染色体片段的发生数分别为 86、41、111、9、181.分析各号染色体辐射后畸变情况,未见明显差异.结论:染色体G带分析可以提供较丰富的染色体结构畸变信息.%Objective : To validate the value of G - banding chromosomal aberration of lymphocyte after radiation. Methods : The blood was irradiated by 4 MV X - ray and was cultured 3 days. The G - banding chromosomal aherration was assayed including dicentrics, deletions, translocations, rings, and fragments chromosomal aberration. Results:There were 100 good karyotypes observed. The total of chromosomal aberration was 428. The dicentric, deletion, translocation, ring, and fragment chromosomal aberration were 86, 41 , 111 , 9, and 181 respectively. There was no significant difference in chromosomal aherration after radiation in every NO. chromosome. Conclusion : The G - banding chromosomal aberration assay should be paid more attention in radiation biology.

  2. Inhibitory effect of arctigenin on lymphocyte activation stimulated with PMA/ionomycin.

    Science.gov (United States)

    Sun, Cheng-Hong; Lai, Xin-Qiang; Zhang, Li; Yao, Jing-Chun; Guan, Yong-Xia; Pan, Li-Hong; Yan, Ying

    2014-04-01

    This study investigated the effect of arctigenin (Arc) on the cell activation, cytokines expression, proliferation, and cell-cycle distribution of mouse T lymphocytes. Mouse lymphocytes were prepared from lymph node and treated with Phorbol-12-myristate-13-acetate (PMA)/Ionimycin (Ion) and/or Arc. CD69, CD25, cytokines, proliferation and cell cycle were assayed by flow cytometry. The results showed that, at concentrations of less than 1.00 micromol x L(-1), Arc expressed non-obvious cell damage to cultured lymphocytes, however, it could significantly down-regulate the expression of CD69 and CD25, as well as TNF-alpha, IFN-gamma, IL-2, IL-4, IL-6 and IL-10 on PMA/Ion stimulated lymphocytes. At the same time, Arc could also inhibit the proliferation of PMA/Ion-activated lymphocytes and exhibited lymphocyte G 0/G1 phase cycle arrest. These results suggest that Arc possesses significant anti-inflammatory effects that may be mediated through the regulation of cell activation, cytokines expression and cell proliferation.

  3. CD80 and CD86 Costimulatory Molecules Differentially Regulate OT-II CD4+ T Lymphocyte Proliferation and Cytokine Response in Cocultures with Antigen-Presenting Cells Derived from Pregnant and Pseudopregnant Mice

    Science.gov (United States)

    Maj, Tomasz; Slawek, Anna

    2014-01-01

    Immune phenomena during the preimplantation period of pregnancy are poorly understood. The aim of our study was to assess the capacity for antigen presentation of splenic antigen-presenting cells (APCs) derived from pregnant and pseudopregnant mice in in vitro conditions. Therefore, sorted CD11c+ dendritic cells and macrophages F4/80+ and CD11b+ presenting ovalbumin (OVA) were cocultured with CD4+ T cells derived from OT-II mice's (C57BL6/J-Tg(TcraTcrb)1100Mjb/J) spleen. After 132 hours of cell culture, proliferation of lymphocytes (ELISA-BrdU), activation of these cells (flow cytometry), cytokine profile (ELISA), and influence of costimulatory molecules blocking on these parameters were measured. We did not detect any differences in regulation of Th1/Th2 cytokine balance. CD86 seems to be the main costimulatory molecule involved in the proliferation response but CD80 is the main costimulatory molecule influencing cytokine secretion in pregnant mice. In conclusion, this study showed that CD80 and CD86 costimulatory molecules regulate OT-II CD4+ T lymphocyte proliferation and cytokine response in cocultures with antigen-presenting cells derived from pregnant and pseudopregnant mice. The implications of these changes still remain unclear. PMID:24771983

  4. CD80 and CD86 Costimulatory Molecules Differentially Regulate OT-II CD4+ T Lymphocyte Proliferation and Cytokine Response in Cocultures with Antigen-Presenting Cells Derived from Pregnant and Pseudopregnant Mice

    Directory of Open Access Journals (Sweden)

    Tomasz Maj

    2014-01-01

    Full Text Available Immune phenomena during the preimplantation period of pregnancy are poorly understood. The aim of our study was to assess the capacity for antigen presentation of splenic antigen-presenting cells (APCs derived from pregnant and pseudopregnant mice in in vitro conditions. Therefore, sorted CD11c+ dendritic cells and macrophages F4/80+ and CD11b+ presenting ovalbumin (OVA were cocultured with CD4+ T cells derived from OT-II mice’s (C57BL6/J-Tg(TcraTcrb1100Mjb/J spleen. After 132 hours of cell culture, proliferation of lymphocytes (ELISA-BrdU, activation of these cells (flow cytometry, cytokine profile (ELISA, and influence of costimulatory molecules blocking on these parameters were measured. We did not detect any differences in regulation of Th1/Th2 cytokine balance. CD86 seems to be the main costimulatory molecule involved in the proliferation response but CD80 is the main costimulatory molecule influencing cytokine secretion in pregnant mice. In conclusion, this study showed that CD80 and CD86 costimulatory molecules regulate OT-II CD4+ T lymphocyte proliferation and cytokine response in cocultures with antigen-presenting cells derived from pregnant and pseudopregnant mice. The implications of these changes still remain unclear.

  5. 乳源β Casein 125肽的基本特征及促B淋巴细胞增殖作用%Basic characteristics of a human milk-derived peptide β-Casein-125 and its effect on B lymphocyte proliferation

    Institute of Scientific and Technical Information of China (English)

    刘贵友; 万俊; 肖文

    2016-01-01

    The biologic characteristics of β⁃Casein⁃125 peptide were analyzed by Uniprot, SABLE, ProtParam tool and other online databases. The content ofβ⁃Casein⁃125 in colostrum, transition milk and mature milk was mesured by mass spectrometry technology. Furthermore, the effect of β⁃Casein⁃125 peptide in B lymphocyte proliferation was evaluated by MTT assay. The online databases showed that stability coefficient, aliphatic index and grand average of hydropathicity of β⁃Casein⁃125 were 111�43, 111�43 and 0�471, respectively. It indicated that β⁃Casein⁃125 is a hydrophobic stable peptide. The content of β⁃Casein⁃125 was significantly decreased with lactation time, detected by mass spectrometry technology. Moreover, β⁃Casein⁃125 could across the cell membrane of mouse B lymphocytes, and promote their proliferation. β⁃Casein⁃125, a hydrophobic peptide with high stability, could promote lymphocyte proliferation. It was indicated thatβ⁃Casein⁃125 played a critial role in promoting the neonatal immune system maturation.%利用Uniprot、SABLE、ProtParam tool等在线数据库分析β Casein 125肽生物学特征;利用质谱定量技术检测β Casein 125肽在初乳、过渡乳和成熟乳中的含量变化;利用细胞增殖实验方法揭示β Casein 125肽在促进B淋巴细胞增殖中的作用。结果表明:β Casein 125肽的稳定性系数为111�43,脂肪指数和亲水性分别为111�43和0�471,属于疏水性稳定型多肽。质谱定量分析发现:β Casein 125肽含量随泌乳时间变化含量显著降低。β Casein 125肽不仅可以顺利进入小鼠B淋巴细胞,并且可显著促进细胞增殖。β Casein 125肽具有高稳定性和疏水性的特征,可以促进淋巴细胞增殖,提示该肽可能在新生儿免疫系统建立中发挥重要作用。

  6. ACTIVATION ASSAY FOR PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR- ALPHA (PPARÁ) BY PERFLUOROALKYL ACIDS (PFAAS) IN COS-1 CELLS

    Science.gov (United States)

    PFAAs have been found to elicit various physiological effects including peroxisome proliferation, indicating the mechanism of action for these chemicals could involve PPAR. This study investigates the ability of PFAAs to bind and activate mouse and human PPARα in COS-1 cell...

  7. B cell helper assays.

    Science.gov (United States)

    Abrignani, Sergio; Tonti, Elena; Casorati, Giulia; Dellabona, Paolo

    2009-01-01

    Activation, proliferation and differentiation of naïve B lymphocytes into memory B cells and plasma cells requires engagement of the B cell receptor (BCR) coupled to T-cell help (1, 2). T cells deliver help in cognate fashion when they are activated upon recognition of specific MHC-peptide complexes presented by B cells. T cells can also deliver help in a non-cognate or bystander fashion, when they do not find specific MHC-peptide complexes on B cells and are activated by alternative mechanisms. T-cell dependent activation of B cells can be studied in vitro by experimental models called "B cell helper assays" that are based on the co-culture of B cells with activated T cells. These assays allow to decipher the molecular bases for productive T-dependent B cell responses. We show here examples of B cell helper assays in vitro, which can be reproduced with any subset of T lymphocytes that displays the appropriate helper signals.

  8. Identification and ranking of poly (ADP-ribose) polymerase inhibitors as protectors against sulfur mustard induced decrease in cellular energy and viability in in vitro assays with human lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Meier, H.L.; Kelly, S.A.

    1993-05-13

    Lymphocyte were utilized as a model for investigating HD effects on resting cells. Lymphocytes exposed to HD demonstrated a concentration dependent decrease in ATP, NAD, and viability. The decrease began in 15 minutes for ATP, 2 hours for NAD, and 6 hours for viability. All three of these HD initiated biochemical changes can be blocked by poly (ADP-ribose) polymerase inhibitors (PADPRPI). To completely inhibit HD initiated ATP, NAD, and viability decreases the PADPRPI had to be present at time 0, 1, and 4 hours respectfully. The amount of protection conferred by the PADPRPI in the viability assay decreased in a linear manner with the delay of the addition and the concentration of the inhibitor from 6-12 hours post HD exposure. There was a good correlation between IC50 to inhibit poly (ADP-ribose) polymerase and EC50 prevention of HD initiated cell death (r=O.94). Thus, three in vitro assays which can measure biochemical and pathologic changes induced by HD in G sub 0 lymphocytes have been developed. These assays have been employed to study the ability of candidate antidotes to prevent HD initiated changes. Benzamidine analogs, including the F.D.A. approved vitamin niacinamide, have been shown to be effective at inhibiting all of these changes.

  9. Suppressor cell hyperactivity relative to allogeneic lymphocyte proliferation as a manifestation of defective T-T-cell interactions in systemic lupus erythematosus

    Energy Technology Data Exchange (ETDEWEB)

    Stenina, M.A.; Potapova, A.A.; Biryukov, A.V.; Skripnik, A.Yu.; Cheredeev, A.N.

    1987-01-01

    The authors study the state of immunoregulatory process in patients with systemic lupus erythematosus at the T-T-cell interaction level and seek to test the possibility of the pharmacological modulation of this process. The proliferative activity of mononuclear lymphocytes, extracted from the blood of ten lupus patients, was assessed by measuring the incorporation of tritiated thymidine into cultures stimulated by phytohemagglutinin, concanavalin, and theophylline. The comparative effects of each of these agents on the immunoregulatory and proliferative activity of the lymphocytes are reported.

  10. An intrinsic GABAergic system in human lymphocytes.

    Science.gov (United States)

    Dionisio, Leonardo; José De Rosa, María; Bouzat, Cecilia; Esandi, María Del Carmen

    2011-01-01

    γ-amino butyric acid (GABA) is an ubiquitous neurotransmitter in the central nervous system and it is also present in non-neuronal cells. In this study we investigated the presence of neuronal components of the GABAergic system in lymphocytes and its functional significance. By using RT-PCR we detected mRNA expression of different components of the GABAergic system in resting and mitogen-activated lymphocytes: i) GAD67, an isoform of the enzyme that synthetizes GABA; ii) VIAAT, the vesicular protein involved in GABA storage; iii) GABA transporters (GAT-1 and GAT-2); iv) GABA-T, the enzyme that catabolizes GABA; and v) subunits that conform ionotropic GABA receptors. The presence of VIAAT protein in resting and activated cells was confirmed by immunocytochemistry. The functionality of GABA transporters was evaluated by measuring the uptake of radioactive GABA. The results show that [(3)H]GABA uptake is 5-fold higher in activated than in resting lymphocytes. To determine if GABA subunits assemble into functional channels, we performed whole-cell recordings in activated lymphocytes. GABA and muscimol, a specific agonist of ionotropic GABA receptors, elicit macroscopic currents in about 10-15% of the cells. Finally, by using [(3)H]thymidine incorporation assays, we determined that the presence of agonists of GABA receptor during activation inhibits lymphocyte proliferation. Our results reveal that lymphocytes have a functional GABAergic system, similar to the neuronal one, which may operate as a modulator of T-cell activation. Pharmacological modulation of this system may provide new approaches for regulation of T-cell response. Copyright © 2010 Elsevier Ltd. All rights reserved.

  11. Evaluation of different toxicity assays applied to proliferating cells and to stratified epithelium in relation to permeability enhancement with glycocholate

    DEFF Research Database (Denmark)

    Eirheim, Heidi Ugelstad; Bundgaard, Christoffer; Nielsen, Hanne Mørck

    2004-01-01

    exposed to different GC concentrations for 4 h. The MTS/PMS assay and neutral red (NR) retention were performed along with quantitation of ATP, lactate dehydrogenase (LDH) and extracellular protein. The toxicity was calculated as the IC50 value relative to the control. Increase in 3H-mannitol permeability...

  12. Effect of borax on immune cell proliferation and sister chromatid exchange in human chromosomes

    Directory of Open Access Journals (Sweden)

    Pongsavee Malinee

    2009-10-01

    Full Text Available Abstract Background Borax is used as a food additive. It becomes toxic when accumulated in the body. It causes vomiting, fatigue and renal failure. Methods The heparinized blood samples from 40 healthy men were studied for the impact of borax toxicity on immune cell proliferation (lymphocyte proliferation and sister chromatid exchange in human chromosomes. The MTT assay and Sister Chromatid Exchange (SCE technic were used in this experiment with the borax concentrations of 0.1, 0.15, 0.2, 0.3 and 0.6 mg/ml. Results It showed that the immune cell proliferation (lymphocyte proliferation was decreased when the concentrations of borax increased. The borax concentration of 0.6 mg/ml had the most effectiveness to the lymphocyte proliferation and had the highest cytotoxicity index (CI. The borax concentrations of 0.15, 0.2, 0.3 and 0.6 mg/ml significantly induced sister chromatid exchange in human chromosomes (P Conclusion Borax had effects on immune cell proliferation (lymphocyte proliferation and induced sister chromatid exchange in human chromosomes. Toxicity of borax may lead to cellular toxicity and genetic defect in human.

  13. Evaluation of radio-induced DNA damage and their repair in human lymphocytes by comet assay or single cell gel electrophoresis; Avaliacao do dano radioinduzido no DNA e reparo em linfocitos humanos pelo metodo do cometa (single cell gel electrophoresis)

    Energy Technology Data Exchange (ETDEWEB)

    Nascimento, Patricia A. do; Suzuki, Miriam F.; Okazaki, Kayo [Instituto de Pesquisas Energeticas e Nucleares (IPEN), Sao Paulo, SP (Brazil)

    1997-12-01

    The comet assay, also called single cell gel electrophoresis technique, permits to evaluate quantitatively DNA breakage induced by chemical and physical agents at the level of the single cell. The present paper refers to the construction of dose-response curves to DNA damage and repair studies in human peripheral lymphocytes, utilizing the comet assay for the radiosensitivity analysis. So, the blood samples were obtained from healthy donors (40-50 year old), irradiated in a {sup 60} Co source (GAMMACEL 220) with doses of 0.17, 0.25, 0.57, 1.10, 2.12 and 4.22 Gy (0.59 Gy/min.) and processed 1 and 24 hours after the exposition. Results obtained showed a increase in the total lenght of comet (DNA migration) as a function of radiation dose in samples processed 1 and 24 hours after the treatment. The DNA lesion in irradiated lymphocytes with 4.22 Gy (means value of 101.4 {mu}m) were 3.4 times higher than in the untreated lymphocytes (mean value of 30 {mu}m) instead of 24 hours after the irradiation were 1.5 times higher (mean value of 46.3 {mu}m). This reduction on DNA repair occurred in these cells. It was also possible visualized the presence of subpopulations of the cells with different sensitivity and repair capacity to ionizing radiation in these donors. (author). 8 refs., 3 figs.

  14. Mutagenicity of the peroxisome proliferators clofibrate, Wyeth 14,643 and di-2-ethylhexyl phthalate in the lacZ plasmid-based transgenic mouse mutation assay

    Directory of Open Access Journals (Sweden)

    Boerrigter Michaël

    2004-01-01

    Full Text Available Abstract Background Peroxisome proliferators are considered rodent carcinogens that are putative human non-carcinogens based on the presumed absence of direct genetic toxicity in rodent and human cells and the resistance of human cells to the induction of peroxisomes by peroxisome proliferators. The highly sensitive lacZ plasmid-based transgenic mouse mutation assay was employed to investigate the mutagenicity of several peroxisome proliferators based on several lines of evidence suggesting that these agents may in fact exert a genotoxic effect. Methods Male and female lacZ-plasmid based transgenic mice were treated at 4 months of age with 6 doses of 2,333 mg di-2-ethylhexyl phthalate (DHEP, 200 mg Wyeth-14,643, or 90 mg clofibrate per kg of bodyweight, respectively, over a two-week period. Control animals were treated with the respective vehicles only (35% propyl glycol for DEHP and Wyeth-14,643 treatment controls and sterile water for clofibrate treatment controls. The mutant frequency in liver, kidney and spleen DNA was determined as the proportion of retrieved mutant and wild-type lacZ plasmids expressed in Escherichia Coli C host cells employing a positive selection system for mutant plasmids. Results Exposure to DEHP or Wyeth-14,643 significantly increased the mutant frequency in liver, but not in kidney or spleen, of both female and male mice. Treatment with clofibrate did not lead to an increased mutant frequency in any of the organs studied. Conclusion The results indicate that some peroxisome proliferators display an organ-specific mutagenicity in lacZ plasmid-based transgenic mice consistent with historical observations of organ- and compound-specific carcinogenicity.

  15. 甜菜碱促进小鼠脾淋巴细胞增殖作用的钙通道机制研究%Calcium channel mechanism by which betaine promotes proliferation of lymphocytes in mice

    Institute of Scientific and Technical Information of China (English)

    季宇彬; 高世勇; 冯小燕; 何立巍

    2009-01-01

    Objective: To study how the way in which betaine promotes the proliferation of mouse spleen lymphocytes is related to calcium channels. Method:BALB/c mice were used for this experiment. Mouse spleen lymphocytes were obtained through in vitro cultivation after they had been separated, and were divided into a negative control group, a ConA group, and 0. 04, 0. 4, 4, and 20 mmol·L~(-1) betaine groups. MTT was used to observe the effect of betaine on the proliferation of mouse spleen lymphocytes; flow cytometry was used to measure the changes in the cell cycle of mouse spleen lymphocytes; and laser confocal scanning microscopy was used to observe the changes in the intracellular [ Ca~(2+) ]; of mouse spleen lymphocytes after betaine or different calcium channel blockers were applied. Result; Betaine was found to promote the proliferation of mouse spleen lymphocytes 12 h after it had been applied in vitro in concentrations of 4 and 20 mmol·L~(-1). It was also found to promote the proliferation of mouse spleen lymphocytes 24 h and 48 h after it had been applied in vitro in concentrations of 0. 04, 0. 4, 4, and 20 mmol·L~(-1), with the effect being most marked for the 4 mmol·L~(-1) group 24 h after its application. It was found to facilitate the entry of mouse spleen lymphocytes from the G_0/G_1 to the S phase 4, 6, 18, and 24 h after it had been applied to mouse spleen lymphocytes in a concentration of 4 mmol·L~(-1) , with the effect being most marked at 18 h after its application. Intracellular [ Ca~(2+) ]; in mouse spleen lymphocytes increased significantly (P < 0.01 ) 6,12, 18 h after 4 mmol·L~(-1) betaine had acted on the lymphocytes, with the effect being most marked at 6 h. The calcium channel blockers nifidipine, dihiazem, mibefradil, and genistein had no effect on the increase of the intracellular [ Ca~(2+) ] ; in mouse spleen lymphocytes due to the application of betaine, while verapamil, mycifradin, heparin, and procaine could block such increase

  16. Rapid assessment of repair of ultraviolet DNA damage with a modified host-cell reactivation assay using a luciferase reporter gene and correlation with polymorphisms of DNA repair genes in normal human lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Qiao Yawei; Spitz, Margaret R.; Guo Zhaozheng; Hadeyati, Mohammad; Grossman, Lawrence; Kraemer, Kenneth H.; Wei Qingyi

    2002-11-30

    As DNA repair plays an important role in genetic susceptibility to cancer, assessment of the DNA repair phenotype is critical for molecular epidemiological studies of cancer. In this report, we compared use of the luciferase (luc) reporter gene in a host-cell reactivation (HCR) (LUC) assay of repair of ultraviolet (UV) damage to DNA to use of the chloramphenicol (cat) gene-based HCR (CAT) assay we used previously for case-control studies. We performed both the assays on cryopreserved lymphocytes from 102 healthy non-Hispanic white subjects. There was a close correlation between DNA repair capacity (DRC) as measured by the LUC and CAT assays. Although these two assays had similar variation, the LUC assay was faster and more sensitive. We also analyzed the relationship between DRC and the subjects' previously determined genotypes for four polymorphisms of two nucleotide-excision repair (NER) genes (in intron 9 of xeroderma pigmentosum (XP) C and exons 6, 10 and 23 of XPD) and one polymorphism of a base-excision repair gene in exon 10 of X-ray complementing group 1 (XRCC1). The DRC was significantly lower in subjects homozygous for one or more polymorphisms of the two NER genes than in subjects with other genotypes (P=0.010). In contrast, the polymorphic XRCC1 allele had no significant effect on DRC. These results suggest that the post-UV LUC assay measures NER phenotype and that polymorphisms of XPC and XPD genes modulate DRC. For population studies of the DNA repair phenotype, many samples need to be evaluated, and so the LUC assay has several advantages over the CAT assay: the LUC assay was more sensitive, had less variation, was not radioactive, was easier to perform, and required fewer cryopreserved cells. These features make the LUC-based HCR assay suitable for molecular epidemiological studies.

  17. 黑素细胞对同种异体淋巴细胞转化增殖的影响%The effects of Melanocyte to the Transormation and Proliferation of Allogeneic Lymphocyte

    Institute of Scientific and Technical Information of China (English)

    项蕾红; 郑志忠; 陈伟华; 祝绿川; 廖康煌

    2001-01-01

    目的观察体外培养人黑素细胞与同种异体的淋巴细胞混合后,黑素细胞对淋巴细胞的促转化、增殖作用。方法用3H-TdR掺入同位素液态闪烁计数法测定淋巴细胞的转化增殖率。实验结果应用t检验进行统计学分析。结果黑素细胞对同种异体淋巴细胞的转化增殖率与刀豆蛋白(conA)刺激淋巴细胞转化、增殖的阳性对照比较,结果显示黑素细胞的促淋巴细胞转化、增殖的特异性抗原作用较弱。进一步比较黑素细胞对不同病期的白癜风患者的淋巴细胞的影响,发现黑素细胞对活动期白癜风患者淋巴细胞的刺激作用相对较强,而稳定期患者和正常人对照组结果无显著性差异。结论正常人黑素细胞的特异性抗原作用较弱,为同种异体黑素细胞移植治疗白癜风创造了有利的条件。%Objective To observe the cultured melanocyte,as specificantigen,how to affect the transormation and proliferation of allogeneic lymphocytes in the mixed melanocyte lymphocyte reaction.Methods 3H-thymicine-added uptake was evaluated by means of liquid scintillation counting and expressed as cpm.The Results of lymphocyte proliferation were expressed by the stimulation indexes.The indexes of each group was analyzed statistically by t-test.Results The stimulation indexes of active vitiligo was significantly different compared with that of stable vitiligo and normal controls.The stimulation indexes of the group which melanocyte was used as specific antigen,was also significantly different compared with that of positive controls.Conclusion The melanocyte worked as specific antigen in mixed melanocyte lymphocyte reaction and its effect was weak.In the melanocyte allograft,the patients with stable vitiligo were the first choice.

  18. Oral supplementation of diabetic mice with propolis restores the proliferation capacity and chemotaxis of B and T lymphocytes towards CCL21 and CXCL12 by modulating the lipid profile, the pro-inflammatory cytokine levels and oxidative stress.

    Science.gov (United States)

    Al Ghamdi, Ahmad A; Badr, Gamal; Hozzein, Wael N; Allam, Ahmed; Al-Waili, Noori S; Al-Wadaan, Mohammed A; Garraud, Olivier

    2015-09-15

    Type 1 diabetes mellitus (T1D) is a chronic autoimmune disease caused by the selective destruction of pancreatic β cells, followed by hyperglycemia, oxidative stress and the subsequent extensive impairment of immune cell functions, a phenomenon responsible for the development of chronic diabetic complications. Propolis, a natural bee product that is extensively used in foods and beverages, significantly benefits human health. Specifically, propolis exerts antioxidant, anti-inflammatory and analgesic effects that may improve diabetic complications. To further elucidate the potential benefits of propolis, the present study investigated the effect of dietary supplementation with propolis on the plasma cytokine profiles, free radical levels, lipid profile and lymphocyte proliferation and chemotaxis in a streptozotocin (STZ)-induced type I diabetic mouse model. Thirty male mice were equally distributed into 3 experimental groups: group 1, non-diabetic control mice; group 2, diabetic mice; and group 3, diabetic mice supplemented daily with an ethanol-soluble derivative of propolis (100 mg/kg body weight) for 1 month. First, the induction of diabetes in mice was associated with hyperglycemia and significant decreases in the insulin level and the lymphocyte count. In this context, diabetic mice exhibited severe diabetic complications, as demonstrated by a significant decrease in the levels of IL-2, IL-4 and IL-7, prolonged elevation of the levels of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) and reactive oxygen species (ROS) and altered lipid profiles compared with control non-diabetic mice. Moreover, antigen stimulation of B and T lymphocytes markedly reduced the proliferative capacity and chemotaxis of these cells towards CCL21 and CXCL12 in diabetic mice compared with control mice. Interestingly, compared with diabetes induction alone, treatment of diabetic mice with propolis significantly restored the plasma cytokine and ROS levels and the lipid profile to

  19. Automated Scoring and Analysis of Micronucleated Human Lymphocytes.

    Science.gov (United States)

    Callisen, Hannes Heinrich

    Physical and chemical mutagens and carcinogens in our environment produce chromosome abberations in the circulating peripheral blood lymphocytes. The abberations, in turn, give rise to micronuclei when the lymphocytes proliferate in culture. In order to improve the micronucleus assay as a method for screening human populations for chromosome damage, I have (1) developed a high-resolution optical low-light-level micrometry expert system (HOLMES) to digitize and process microscope images of micronuclei in human peripheral blood lymphocytes, (2) defined a protocol of image processing techniques to objectively and uniquely identify and score micronuclei, and (3) analysed digital images of lymphocytes in order to study methods for (a) verifying the identification of suspect micronuclei, (b) classifying proliferating and non-proliferating lymphocytes, and (c) understanding the mechanisms of micronuclei formation and micronuclei fate during cell division. For the purpose of scoring micronuclei, HOLMES promises to (a) improve counting statistics since a greater number of cells can be scored without operator/microscopist fatigue, (b) provide for a more objective and consistent criterion for the identification of micronuclei than the human observer, and (c) yield quantitative information on nuclear and micronuclear characteristics useful in better understanding the micronucleus life cycle. My results on computer aided identification of micronuclei on microscope slides are gratifying. They demonstrate that automation of the micronucleus assay is feasible. Manual verification of HOLMES' results show correct extraction of micronuclei from the scene for 70% of the digitized images and correct identification of the micronuclei for 90% of the extracted objects. Moreover, quantitative analysis on digitized images of lymphocytes using HOLMES has revealed several exciting results: (a) micronuclear DNA content may be estimated from simple area measurements, (b) micronuclei seem to

  20. Quantitative Validation of the Presto Blue Metabolic Assay for Online Monitoring of Cell Proliferation in a 3D Perfusion Bioreactor System.

    Science.gov (United States)

    Sonnaert, Maarten; Papantoniou, Ioannis; Luyten, Frank P; Schrooten, Jan Ir

    2015-06-01

    As the fields of tissue engineering and regenerative medicine mature toward clinical applications, the need for online monitoring both for quantitative and qualitative use becomes essential. Resazurin-based metabolic assays are frequently applied for determining cytotoxicity and have shown great potential for monitoring 3D bioreactor-facilitated cell culture. However, no quantitative correlation between the metabolic conversion rate of resazurin and cell number has been defined yet. In this work, we determined conversion rates of Presto Blue, a resazurin-based metabolic assay, for human periosteal cells during 2D and 3D static and 3D perfusion cultures. Our results showed that for the evaluated culture systems there is a quantitative correlation between the Presto Blue conversion rate and the cell number during the expansion phase with no influence of the perfusion-related parameters, that is, flow rate and shear stress. The correlation between the cell number and Presto Blue conversion subsequently enabled the definition of operating windows for optimal signal readouts. In conclusion, our data showed that the conversion of the resazurin-based Presto Blue metabolic assay can be used as a quantitative readout for online monitoring of cell proliferation in a 3D perfusion bioreactor system, although a system-specific validation is required.

  1. Synthesis and anticancer activity of new flavonoid analogs and inconsistencies in assays related to proliferation and viability measurements.

    Science.gov (United States)

    Forbes, Alaina M; Lin, Huimin; Meadows, Gary G; Meier, G Patrick

    2014-08-01

    Flavonoids have been studied intensely for their ability to act as anti-carcinogenic, anti-inflammatory, anti-viral and anti-aging agents and are often marketed as supplements related to their anti-inflammatory activity. Previous studies have primarily focused on the effects of polar natural flavonoids. We examined the activity of novel hydrophobic and lipophilic flavonols against human DU-145 and PC-3 prostate cancer cell lines. All flavonol analogs were more active than the naturally occurring flavonols quercetin, kaempferol, kaempferide and galangin. The most potent analogs were 6.5-fold more active against DU-145 and PC-3 cells than quercetin and fell within the biologically relevant concentration range (low micromolar). We also evaluated the potential toxic effects of flavonol analogs on normal cells, an assessment that has frequently been ignored when studying the anticancer effects of flavonoids. During these analyses, we discovered that various metabolic and DNA staining assays were unreliable methods for assessing cell viability of flavonoids. Flavonoids reduce colorimetric dyes such as MTT and Alamar Blue in the absence of cells. We showed that flavonol-treated prostate cancer cells were stained less intensely with crystal violet than untreated cells at non-toxic concentrations. The trypan blue exclusion assay was selected as a reliable alternative for measuring cell viability.

  2. In vitro genotoxicity and cytotoxicity of five new chemical compounds of plant origin by means of the human lymphocyte micronucleus assay.

    Science.gov (United States)

    Scarpato, R; Pistelli, L; Bertoli, A; Nieri, E; Migliore, L

    1998-04-01

    The micronucleus test in human peripheral lymphocytes is widely used in toxicology for the assessment of the genotoxic profile of chemical compounds of environmental concern. The aim of this study was to evaluate the genotoxic, cytotoxic and antimitotic activity of five new compounds isolated from Prunus africana Hook or from Bupleurum fruticosum L. The experiments were conducted only in vitro. Results showed that none of the plant extracts, tested over a wide range of concentrations, increased the frequency of micronuclei. Only compounds 2 and 5 were found to be toxic for phytohaemagglutinin-stimulated lymphocytes at the maximum dose used. reserved.

  3. Initiation of lymphocyte DNA synthesis.

    Science.gov (United States)

    Coffman, F D; Fresa, K L; Cohen, S

    1991-01-01

    The initiation of DNA replication in T lymphocytes appears to be regulated by two distinct activities: one associated with proliferation which mediates initiation, and another associated with quiescence which blocks initiation. Activated lymphocytes and proliferating lymphoid cell lines produce an activity, termed ADR, which can initiate DNA replication in isolated, quiescent nuclei. ADR is heat-labile, has protease activity or interacts closely with a protease, and is distinct from the DNA polymerases. ADR activity is absent in quiescent lymphocytes and appears in mitogen-stimulated lymphocytes after IL-2 binding. The generation of active ADR appears to be mediated by phosphorylation of a precursor which is present in resting cells. Nuclei from mitogen-unresponsive lymphocytes fail to initiate DNA replication in response to ADR, of potential importance in the age-related decline of immunity. Quiescent lymphocytes lack ADR and synthesize an ADR-inhibitory activity. The ADR inhibitor is a heat-stable protein which suppresses the initiation of DNA synthesis, but is ineffective at suppressing elongation once DNA strand replication has begun. Nuclei from several neoplastic cell lines fail to respond to the ADR inhibitor, which may play a role in the continuous proliferation of these cells. At least one of these neoplastic cell lines produces both ADR and an inhibitory factor. These findings suggest that the regulation of proliferation is dependent on the balance between activating and inhibitory pathways.

  4. Evaluation of the antigenotoxic effects of the royal sun mushroom, agaricus brasiliensis (Higher basidiomycetes) in human lymphocytes treated with thymol in the comet assay

    NARCIS (Netherlands)

    Radaković, Milena; Stevanović, Jevrosima; Soković, Marina; Radović, Dejan; Griensven, Van Leo J.L.D.; Stanimirović, Zoran

    2015-01-01

    The aim of this investigation was to evaluate the possible protective activity of Agaricus brasiliensis (=A. blazei sensu Murrill) ethanol extract against thymol-induced DNA damage in human lymphocytes. Before we studied the possible interaction of thymol and A. brasiliensis extract, each compone

  5. Phosphoinositide-dependent kinase 1 controls migration and malignant transformation but not cell growth and proliferation in PTEN-null lymphocytes

    NARCIS (Netherlands)

    Finlay, D.K.; Sinclair, L.V.; Feijoo, C.; Waugh, C.M.; Hagenbeek, T.J.; Spits, H.; Cantrell, D.A.

    2009-01-01

    In normal T cell progenitors, phosphoinositide-dependent kinase l (PDK1)-mediated phosphorylation and activation of protein kinase B (PKB) is essential for the phosphorylation and inactivation of Foxo family transcription factors, and also controls T cell growth and proliferation. The current study

  6. Effects of Taohong Siwu Decoction Ⅱ in the Chick Chorioallantoic Membrane (CAM) Assay and on B16 Melanoma in Mice and Endothelial Cells ECV304 Proliferation

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Objective: To investigate the anti-angiogenesis action of Taohong Siwu Decoction Ⅱ (THSWD Ⅱ). Methods:The chick chorioallantoic membrane (CAM) assay was adopted to study the anti-angiogenesis action of THSWD Ⅱ; the MTT test was used to investigate its effect on proliferation of the human umbilical vein endothelial cells ECV304; and the immunohistochemical method was used to observe the effect of THSWD Ⅱ on the expression of kinase insert domain containing receptor/fetal liver kinase 1 (KDR/Flk-1) and the microvessel density (MVD) of B 16 melanoma in mice. Results: After treatment with THSWD Ⅱ, the blood vessel index of CAM and the absorbency of ECV304 in the THSWD Ⅱ lmg/ml group and the 2mg/ml group decreased significantly (P<0.01); the weight, the expression of KDR/Flk-1 and the MVD of B16 melanoma in mice reduced significantly in the THSWD Ⅱ 5g/kg group, the 10g/kg group and the TSHSWD10g/kg plus cyclophosphamide group (P<0.01). Conclusion: THSWD Ⅱ has the actions of anti-angiogenesis, and inhibiting the proliferation of ECV304 cells and the growth of B16 melanoma. The clinical anti-tumour mechanism is considered to be related possibly to its anti-angiogenesis action by inhibiting the expression of KDR/FIK- 1.

  7. Ruta 6 selectively induces cell death in brain cancer cells but proliferation in normal peripheral blood lymphocytes: A novel treatment for human brain cancer.

    Science.gov (United States)

    Pathak, Sen; Multani, Asha S; Banerji, Pratip; Banerji, Prasanta

    2003-10-01

    Although conventional chemotherapies are used to treat patients with malignancies, damage to normal cells is problematic. Blood-forming bone marrow cells are the most adversely affected. It is therefore necessary to find alternative agents that can kill cancer cells but have minimal effects on normal cells. We investigated the brain cancer cell-killing activity of a homeopathic medicine, Ruta, isolated from a plant, Ruta graveolens. We treated human brain cancer and HL-60 leukemia cells, normal B-lymphoid cells, and murine melanoma cells in vitro with different concentrations of Ruta in combination with Ca3(PO4)2. Fifteen patients diagnosed with intracranial tumors were treated with Ruta 6 and Ca3(PO4)2. Of these 15 patients, 6 of the 7 glioma patients showed complete regression of tumors. Normal human blood lymphocytes, B-lymphoid cells, and brain cancer cells treated with Ruta in vitro were examined for telomere dynamics, mitotic catastrophe, and apoptosis to understand the possible mechanism of cell-killing, using conventional and molecular cytogenetic techniques. Both in vivo and in vitro results showed induction of survival-signaling pathways in normal lymphocytes and induction of death-signaling pathways in brain cancer cells. Cancer cell death was initiated by telomere erosion and completed through mitotic catastrophe events. We propose that Ruta in combination with Ca3(PO4)2 could be used for effective treatment of brain cancers, particularly glioma.

  8. Occupational exposure to cytostatic/antineoplastic drugs and cytogenetic damage measured using the lymphocyte cytokinesis-block micronucleus assay: A systematic review of the literature and meta-analysis.

    Science.gov (United States)

    Villarini, M; Gianfredi, V; Levorato, S; Vannini, S; Salvatori, T; Moretti, M

    Many studies have reported the occurrence of work-environment contamination by antineoplastic drugs (ANPD), with significant incorporation of trace amounts of these hazardous drugs in hospital personnel. Given the ability of most ANPD to actively bind DNA, thus inducing genotoxic effects, it is of pivotal importance to assess the degree of genotoxic damage (i.e., residual genotoxic risk) in occupationally exposed subjects. The lymphocyte cytokinesis-block micronucleus (L-CBMN) assay is largely used for biological effect monitoring in subjects occupationally exposed to ANPD. In this study, we identified and analyzed the studies published reporting the use of the L-CBMN assay as biomarker of genotoxic risk in health care workers exposed to ANPD with the aim of performing meta-analysis and providing a meta-estimate of the genotoxic effect of exposure. We retrieved 24 studies, published from 1988 to 2015, measuring MN in peripheral blood lymphocytes in health care workers occupationally exposed to ANPD. In 15 out of the 24 studies (62.5%), increased MN frequencies were recognized in exposed subjects as compared to controls. The meta-analysis of MN frequency of the combined studies confirmed an association between occupational exposure to ANPD and cytogenetic effects with an overall meta-estimate of 1.67 [95% CI: 1.41-1.98]. In 16 out of the 24 studies (66.6%) at least one other genotoxicity biomarker, besides L-CBMN assay, was employed for biological effect monitoring. In several studies the effect of exposure to ANPD was evaluated also in terms of MN in exfoliated buccal cells. Other studies focused on genotoxicity endpoints, such as sister chromatid exchanges (3 studies), chromosome aberrations (6 studies), or primary DNA damage investigated by comet assay (7 studies). Overall, there was good agreement between other genotoxicity tests employed and L-CBMN assay outcomes. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. 白藜芦醇对小鼠CD4+T细胞增殖、凋亡和亚群作用研究%Effects of resveratrol on the proliferation, apotosis and subsets distribution of murine CD4+T lymphocytes

    Institute of Scientific and Technical Information of China (English)

    王卓龙; 曾小峰; 李梦涛

    2013-01-01

    The purpose of the study is to investigate the effect of resveratrol (Res) on the proliferation,apotosis and subsets distribution of murine CD4+ T lymphocytes.Splenic mononuclear cells (SMC) from specific pathogen free (SPF) Balb/c mice (female,age 9-10 weeks) were cultured with Res (0,20,40,or 80 mmol/L) for 30 min in vitro,then cocultured with Ionomycin and PMA for 1 h for activation,followed by cultivation with protein transporting inhibitor Golgiplug for 4-6 hours.IL-4 and IFN-γ levels in the cultured cells were detected using intracellular cytokine staining with flow cytometry.On the other way,freshly isolated CD4+T lymphocytes using magnetic cell sorting were cultured with Res (0,10,20,or 40 mmol/L) for 36 hours.Then the cells were stained with PI and Annexin V and assayed using flow cytometry.For detecting the proliferation,CD4+T lymphocytes were stained withCFSE and cocultured with anti-CD3,anti-CD28 and IL-2 and Res (0,10,20,or 40 mmol/L) for 4 days in vitro.The results showed that Res (≥20 mmol/L) decreased IFN-γ levels and proliferation of CD4+T lymphocytes significantly in a dose-dependent manner,while Res (≥ 10 mmol/L) induced CD4+T lymphocytes apoptosis significantly in a dose-dependent manner.Thus we concluded that Res can inhibit proliferation and Th1 subset differentiation of murine CD4+ T lymphocytes and induce CD4+ T lymphocyte apoptosis in a dose-dependent manner.%目的 研究白藜芦醇对小鼠CD4+T细胞增殖、凋亡和亚群的影响.方法 Balb/c鼠(SPF,雌性,9~10周龄)脾脏单个核细胞(SMC)与不同浓度(0、20、40、80 mmol/L)白藜芦醇共培养30 min,加离子霉素和PMA共培养1h后,再加蛋白质转运阻断剂Golgiplug继续共培养4~6 h,收集细胞采用胞内细胞因子染色法结合流式细胞术检测IL-4和IFN-γ情况.免疫磁珠法从SMC分选CD4+T细胞.CD4+T细胞与不同浓度(0、10、20、40 mmol/L)白藜芦醇、抗CD3/抗CD28抗体和IL-2共培养,4d后收集细胞采用流式细

  10. An exploratory study into the effect of exhausting bicycle exercise on endocrine and immune responses in post-menopausal women: relationships between vigour and plasma cortisol concentrations and lymphocyte proliferation following exercise.

    Science.gov (United States)

    van der Pompe, G; Bernards, N; Kavelaars, A; Heijnen, C

    2001-08-01

    It is well-established that bicycle exercise alters the endocrine and immune responses in men, but little information is available for women, especially middle-aged, post-menopausal women. The purpose of our study was to document the endocrine and immune reactivity to exhausting bicycle exercise in post-menopausal women, and to explore whether complaints of fatigue or low vigour are related to these exercise-induced responses. Thirteen healthy post-menopausal women participated in this study. We used a graded exercise protocol to study the kinetics of activation of the endocrine and immune system. We chose to examine hormones related to the hypothalamus-pituitary-adrenal (HPA) system such as adrenocorticotropin hormone (ACTH) and cortisol and hormones related to the pituitary such as prolactin (PRL) and growth hormone (GH). With regard to the immune system, we examined the natural killer (NK) cell activity and pokeweed (PWM)-induced lymphocyte proliferation in addition to changes in peripheral blood cell counts. Our results demonstrate that acute physical stress results in a strong release of ACTH, cortisol, GH and PRL. The bicycle test significantly increased the number of CD3+, CD4+, CD16/56+ (NK cells) and CD8+ cells in our group of post-menopausal women. Interestingly, NK activity did not increase significantly despite an increase in NK cell numbers. PWM-induced lymphocyte proliferation did not change either. In addition, our data support the hypothesis that low vigour in post-menopausal women interferes with the endocrine and immune responses to exhausting exercise. In women with complaints of low vigour we found lower cortisol responses and higher increments in the proliferative capacity of lymphocytes as compared to those with high vigour scores. NK activity was unrelated to exhaustive mood states. These data indicate that endocrine as well as immune system activity changes in response to exhausting exercise in middle-aged, post-menopausal women. In addition

  11. CTLA4Fcε, a novel soluble fusion protein that binds B7 molecules and the IgE receptors, and reduces human in vitro soluble CD23 production and lymphocyte proliferation.

    Science.gov (United States)

    Perez-Witzke, Daniel; Miranda-García, María Auxiliadora; Suárez, Nuris; Becerra, Raquel; Duque, Kharelys; Porras, Verónica; Fuenmayor, Jaheli; Montano, Ramon Fernando

    2016-05-01

    Immunoglobulin E-mediated allergy and certain autoimmune diseases are characterized by the presence of a T helper type 2 (Th2) immune response and allergen-specific or self-reactive IgE. Soluble CD23 (sCD23) is a B-cell factor that fosters IgE class-switching and synthesis, suggesting that sCD23 may be a therapeutic target for these pathologies. We produced a recombinant protein, CTLA4Fcε, by fusing the ectodomain of the immunoregulatory molecule cytotoxic T-lymphocyte antigen 4 (CTLA-4) with a fragment of the IgE H-chain constant region. In SDS-PAGE/inmunoblot analyses, CTLA4Fcε appeared as a 70,000 MW polypeptide that forms homodimers. Flow cytometry showed that CTLA4Fcε binds to IgE receptors FcεRI and FcεRII/CD23, as well as to CTLA-4 counter-receptors CD80 and CD86. Binding of CTLA4Fcε to FcεRII/CD23 appeared stronger than that of IgE. Since the cells used to study CD23 binding express CD80 and CD86, simultaneous binding of CTLA4Fcε to CD23 and CD80/CD86 seems to occur and would explain this difference. As measured by a human CD23-specific ELISA, CTLA4Fcε - but not IgE - induced a concentration-dependent reduction of sCD23 in culture supernatants of RPMI-8866 cells. Our results suggest that the simultaneous binding of CTLA4Fcɛ to CD23-CD80/CD86 may cause the formation of multi-molecular complexes that are either internalized or pose a steric hindrance to enzymatic proteolysis, so blocking sCD23 generation. CTLA4Fcε caused a concentration-dependent reduction of lymphocyte proliferation in human peripheral blood mononuclear cell samples stimulated in vitro with concanavalin A. The ability to bind IgE receptors on effector cells, to regulate the production of sCD23 and to inhibit lymphocyte proliferation suggests that CTLA4Fcɛ has immunomodulatory properties on human Th2 responses.

  12. A chimera embryo assay reveals a decrease in embryonic cellular proliferation induced by sperm from X-irradiated male mice

    Energy Technology Data Exchange (ETDEWEB)

    Obasaju, M.F.; Wiley, L.M.; Oudiz, D.J.; Raabe, O.; Overstreet, J.W.

    1989-05-01

    Male mice were divided into three experimental groups and a control group. Mice in the experimental groups received one of three doses of acute X irradiation (1.73, 0.29, and 0.05 Gy) and together with the control unirradiated mice were then mated weekly to unirradiated female mice for a 9-week experimental period. Embryos were recovered from the weekly matings at the four-cell stage and examined by the chimera assay for proliferative disadvantage. Aggregation chimeras were constructed of embryos from female mice mated to irradiated males (experimental embryos) and embryos from females mated to unexposed males (control embryos) and contained either one experimental embryo and one control embryo (heterologous chimera) or two control embryos (control chimera). The control embryo in heterologous chimeras and either embryo in control chimeras were prelabeled with the vital dye fluorescein isothiocyanate (FITC), and the chimeras were cultured for 40 h and viewed under phase-contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution from the FITC-labeled embryo. Experimental and control embryos that were cultured singly were also examined for embryo cell number at the end of the 40-h culture period. In control chimeras, the mean ratio of the unlabeled cells:total chimera cell number (henceforth referred to as ''mean ratio'') was 0.50 with little or no weekly variation over the 9-week experimental period. During Weeks 4-7, the mean ratios of heterologous chimeras differed significantly from the mean ratio of control chimeras with the greatest differences occurring during Week 7 (0.41 for chimeras of 0.05 Gy dose group, 0.40 for chimeras of the 0.29 Gy dose group, and 0.17 for chimeras of the 1.73 Gy dose group).

  13. 牙髓干细胞MHC分子表达与体外混合淋巴细胞的增殖%MHC molecule expression in dental pulp stem cells and mixed lymphocyte proliferation in vitro

    Institute of Scientific and Technical Information of China (English)

    聂姗姗; 刘佳; 张瑞涵; 王璇; 李伯琦; 孙大磊; 热甫卡提; 刘奕杉

    2014-01-01

    背景:若牙髓干细胞诱导分化后仍然具有与未分化时相似的免疫调节能力,则有可能为组织工程提供同种异体种子细胞来源。  目的:观察牙髓干细胞表面免疫分子的表达以及体外调节淋巴细胞反应的功能。  方法:从C57BL/6小鼠牙髓组织中分离获取牙髓干细胞,体外培养至第2代,流式细胞仪检测免疫分子MHC-Ⅰ、MHC-Ⅱ的表达。以1×105/孔牙髓干细胞刺激异体淋巴细胞,观察细胞增殖情况。以1×105/孔数量的牙髓干细胞或经γ-干扰素作用后的牙髓干细胞加入混合双向淋巴细胞反应体系中,观察淋巴细胞增殖情况。  结果与结论:牙髓干细胞表达MHC-Ⅰ类分子,但未检测到MHC-Ⅱ类分子阳性表达。γ-干扰素刺激48 h后, MHC-Ⅰ表达未见明显增高,MHC-Ⅱ类分子表达明显增高。异体或经γ-干扰素作用的牙髓干细胞均未能刺激淋巴细胞体外增殖。说明牙髓干细胞可在体外调节淋巴细胞增殖反应,有可能成为组织工程或细胞治疗中同种异体细胞来源。%BACKGROUND:If differentiated dental pulp stem cels have immune adjustment ability similar to undifferentiated ones, they could become a new source of alogeneic seed cels in tissue engineering. OBJECTIVE:To study the immunological properties of dental pulp stem cels and their immunomodulatory effects on lymphocytes in vitro. METHODS:Dental pulp stem cels were isolated from mouse dental pulp tissue. Cels at passage 2 were detected using flow cytometry for the expression of MHC-I, MHC-II. Inguinal lymphcels were cultured with alogeneic dental pulp stem cels at a density of 1×105 cels per wel. Dental pulp stem cels at 1×105 cels per wel or interferon-γ-treated dental pulp stem cels were added into the mixed lymphocyte reaction system to observe lymphocyte proliferation. RESULTS AND CONCLUSION: Flow cytometry showed that undifferentiated dental pulp stem cels

  14. Preparation of Mycobacterium polysaccharides and their effect on lymphocyte proliferation%分枝杆菌多糖的制备及对小鼠脾淋巴细胞增殖的影响

    Institute of Scientific and Technical Information of China (English)

    周莉婷; 赵晶晶; 吴彦卓; 杨仲璠; 姚文兵; 徐明波

    2011-01-01

    目的:制备分枝杆菌多糖并考察其对小鼠淋巴细胞增殖的影响.方法:用苏通培养基培养分枝杆菌,菌体脱脂后,用5种方法提取,比较多糖得率,并用正交试验优化超声提取工艺,再用稀碱提取残渣,粗多糖经Sevag法除蛋白、DEAE-Sepharose Fast Flow层析柱纯化后得到分枝杆菌多糖;用MTT法检测分枝杆菌多糖对小鼠脾淋巴细胞增殖的影响.结果:超声后热水提取多糖得率最高,超声提取的优化工艺是提取时间40 min,固液比为1∶150,超声功率600 W;单因素试验优化的Sevag法除蛋白条件为体积比1∶5,萃取时间为20 min,萃取6次;提取得到的4种多糖组分在6.25~50 mg·L-1范围内均能显著促进脾淋巴细胞的增殖.结论:用本实验工艺成功制备了分枝杆菌多糖,制备物具有刺激小鼠脾淋巴细胞增殖的作用.%Objective: To prepare Mycobacterium polysaccharides (MPS) and determine their effect on lymphocyte proliferation in mice. Methods; Mycobacterium was cultivated in Sauton's medium, and 5 methods were used to extract polysaccharides, and the extraction rates were compared. The ultrasonic extraction was optimized by an orthogonal design. Hot-water extraction residue was extracted in alkaline solution. Crude polysaccharides were purified by deproteinization and DEAE-sepharose Fast Flow chromatography. The effect of polysaccharides on mouse splenic lymphocytes was analyzed by MTT method. Results; The highest polysaccharide yield was obtained by ultrasonic and followed hot-water extraction; the best ultrasonic extraction condition defined by the orthogonal design was extracting 40 min with 150 times solvent and 600 W power. The best deproteiniation condition was extracting 6 times with 1/5 Sevag reagent, 20 min for every time. All the obtained 4 kinds of mycobacterium polysaccharides apparently stimulated the proliferation of mouse splenic lymphocytes at concentrations of 6. 25 ~ 50 mg·L-1. Conclusion

  15. AFRRI’s Gamma-Ray, X-Ray, and Fission-Neutron Calibration Curves for the Lymphocyte Dicentric Assay: Application of a Metaphase Finder System

    Science.gov (United States)

    2002-05-01

    mature automated metaphase-finding dicentric chromosome is one such aberration, component coupled with lymphocyte- dicentric and it is recognized as a...biomarker of exposures scoring at peripheral, or satellite, scoring sta- to ionizing radiation. Measuring the frequency tions. of dicentric chromosomes ...published reports show that differ- mechanisms of chromosome -aberration forma- ences exist in the measured yield of dicentrics tion (Bauchinger 1983). per Gy

  16. In vitro mutagenicity and genotoxicity study of a number of short-chain chlorinated hydrocarbons using the micronucleus test and the alkaline single cell gel electrophoresis technique (Comet assay) in human lymphocytes: a structure-activity relationship (QSAR) analysis of the genotoxic and cytotoxic potential.

    Science.gov (United States)

    Tafazoli, M; Baeten, A; Geerlings, P; Kirsch-Volders, M

    1998-03-01

    Using the micronucleus (MN) test and the alkaline single cell gel electrophoresis (Comet) assay, potential mutagenicity (MN formation), genotoxicity (DNA breakage capacity) and cytotoxicity (cell proliferation reduction) of five chlorinated hydrocarbons (carbon tetrachloride, hexachloroethane, 1,2-dichloroethane, 1-chlorohexane and 2,3-dichlorobutane) have been evaluated in isolated human lymphocytes. With the MN test a low but statistically significant mutagenic activity was detected for all tested substances (except 2,3-dichlorobutane) with one out of the two donors and in the presence or absence of an exogenous metabolic activation system (S9 mix). However, at the concentration ranges tested none of the positive compounds induced a clear dose-dependent mutagenic effect. The Comet assay detected a strong DNA damaging effect for 1-chlorohexane, 2,3-dichlorobutane and 1,2-dichloroethane, but not for carbon tetrachloride and hexachloroethane. The influence of metabolism on the genotoxic activity of the chemicals was more clear in the Comet assay than in the MN test. The experimental genotoxicity and cytotoxicity data obtained in this study, together with data on five more related chemicals previously investigated, and their physico-chemical descriptors or electronic parameters have been used for QSAR analysis. The QSAR analysis high-lighted that the toxicity of the tested compounds was influenced by different parameters, like lipophilicity (logP), electron donor ability (charge) and longest carbon-chlorine (LBC-Cl) bond length. In addition, steric parameters, like molar refractivity (MR) and LBC-Cl, and electronic parameters, like ELUMO (energy of the lowest unoccupied molecular orbital, indicating electrophilicity), were predominant factors discriminating genotoxins from non-genotoxins in the presence but not in the absence of S9 mix. Although a limited number of compounds have been examined and cytotoxicity and genotoxicity were identified in two different

  17. Natural killer cell activity, lymphocyte proliferation, and cytokine profile in tumor-bearing mice treated with MAPA, a magnesium aggregated polymer from Aspergillus oryzae.

    Science.gov (United States)

    Justo, G Z; Durán, N; Queiroz, M L S

    2003-08-01

    The present study examined the effects of MAPA, an antitumor aggregated polymer of protein magnesium ammonium phospholinoleate-palmitoleate anhydride, isolated from Aspergillus oryzae, on concanavalin A (Con A)-induced spleen cell proliferation, cytokine production and on natural killer (NK) cell activity in Ehrlich ascites tumor-bearing mice. The Ehrlich ascites tumor (EAT) growth led to diminished mitogen-induced expansion of spleen cell populations and total NK activity. This was accompanied by striking spleen enlargement, with a marked increase in total cell counts. Moreover, a substantial enhancement in IL-10 levels, paralleled by a significant decrease in IL-2 was observed, while production of IL-4 and interferon-gamma (IFN-gamma) was not altered. Treatment of mice with 5 mg/kg MAPA for 7 days promoted spleen cell proliferation, IL-2 production and NK cell activity regardless of tumor outgrowth. In addition, MAPA treatment markedly enhanced IFN-gamma levels and reduced IL-10 production relative to EAT mice. A 35% reduction in splenomegaly with normal number of nucleated cells was also found. Altogether, our results suggest that MAPA directly and/or indirectly modulates immune cell activity, and probably disengages tumor-induced suppression of these responses. Clearly, MAPA has an impact and may delay tumor outgrowth through immunotherapeutic mechanisms.

  18. Sublethal red tide toxin exposure in free-ranging manatees (Trichechus manatus) affects the immune system through reduced lymphocyte proliferation responses, inflammation, and oxidative stress.

    Science.gov (United States)

    Walsh, Catherine J; Butawan, Matthew; Yordy, Jennifer; Ball, Ray; Flewelling, Leanne; de Wit, Martine; Bonde, Robert K

    2015-04-01

    The health of many Florida manatees (Trichechus manatus latirostris) is adversely affected by exposure to blooms of the toxic dinoflagellate, Karenia brevis. K. brevis blooms are common in manatee habitats of Florida's southwestern coast and produce a group of cyclic polyether toxins collectively referred to as red tide toxins, or brevetoxins. Although a large number of manatees exposed to significant levels of red tide toxins die, several manatees are rescued from sublethal exposure and are successfully treated and returned to the wild. Sublethal brevetoxin exposure may potentially impact the manatee immune system. Lymphocyte proliferative responses and a suite of immune function parameters in the plasma were used to evaluate effects of brevetoxin exposure on health of manatees rescued from natural exposure to red tide toxins in their habitat. Blood samples were collected from rescued manatees at Lowry Park Zoo in Tampa, FL and from healthy, unexposed manatees in Crystal River, FL. Peripheral blood leukocytes (PBL) isolated from whole blood were stimulated with T-cell mitogens, ConA and PHA. A suite of plasma parameters, including plasma protein electrophoresis profiles, lysozyme activity, superoxide dismutase (SOD) activity, and reactive oxygen/nitrogen (ROS/RNS) species, was also used to assess manatee health. Significant decreases (pimmune function components, and thus, overall health, in the Florida manatee.

  19. Determination of CD4+ and CD8+ lymphocytes with the cytosphere assay: a comparative study with flow cytometry and the immunoalkaline phosphatase method

    DEFF Research Database (Denmark)

    Gernow, A; Lisse, I M; Böttiger, B;

    1995-01-01

    number of CD4+ lymphocytes determined by CA showed a good correlation with results obtained by FC (correlation coefficients were 0.92 and 0.74 in Denmark and The Ivory Coast, respectively) and IA (correlation coefficients were 0.94 and 0.66 in Denmark and The Ivory Coast, respectively). However, for HIV......-seronegative samples the corresponding correlation coefficients were low. CD4% determinations deviated more from FC counts at higher CD4 counts than at lower levels for both seronegative and seropositive individuals. In conclusion, the CA performed best for samples from HIV-infected individuals. Before a more general...

  20. Effects of Trace Element Compound Injection on Lymphocyte Proliferation of Plateau Tibetan Sheep%复方微量元素注射液对高原型藏羊淋巴细胞增殖的影响

    Institute of Scientific and Technical Information of China (English)

    余劲; 曾朝蓉; 蓝岚; 蒋忠荣; 胡延春

    2014-01-01

    为了探讨微量元素对高原型藏羊细胞免疫功能的影响,并筛选出增强藏羊细胞免疫的复方微量元素制剂的最佳剂量,笔者开展了本试验。将体重为13 kg±1 kg的高原型藏羊随机分成I组(对照组)、II组(低剂量组)、III组(中剂量组)、IV组(高剂量组),分别按0、0.1、0.2、0.4 mL/kg肌肉注射复方微量元素制剂,并用MTT法测定注射后0、15、30、45、60 d藏羊血清中的淋巴细胞增殖率。结果显示:与对照组相比,各试验组的淋巴细胞增殖率均显著或者极显著升高(P<0.05或P<0.01),说明复方微量元素制剂可显著提高高原型藏羊的细胞免疫功能,其中中剂量组(0.2 mL/kg)的效果最好。%The purpose of this experiment was to study the effects of trace element compound injection on cellular immune function and to select the optimal dose. 24 Plateau type of Tibetan sheep (weight of 13 kg ±1 kg) were randomly divided into four groups:I group (control group), II group(low dose group), III group (middle dose group), IV group (high dose group), each group was given intramuscular injection of trace element compound injection with 0, 0.1, 0.2, 0.4 mL/kg body weight respectively. Blood samples were drawn at the day before injecting (recorded as 0 d), after 15 d,30 d,45 d,60 d,and lymphocyte proliferation rates were measured. The results showed that lymphocyte proliferation rates of the low-dose group and high dose group were higher than that of the control group(P<0.05). Lymphocyte proliferation rate of middle-dose group was extremely sig-nificant higher during all the experiment groups (P<0.01). The conclusion was that trace element compound injection could improve cellular immune function of the Plateau Tibetan sheep significant-ly,and the middle dose group (0.2 mL/kg) was the best.

  1. 嵌合锚定T淋巴细胞对肝癌细胞增殖的阻滞效应%Repression Effects of Anchor Chemric T Lymphocytes on Proliferation of Tumor Associated Glycoprotein 72 Positive Hepatocarcinoma Cells

    Institute of Scientific and Technical Information of China (English)

    徐宏勇; 徐立; 李开宗

    2011-01-01

    Objective To investigate the method for generating anchor chemric T lymphocytes that can target tumor associated glycoprotein-72 (TAG72) antigen and analyze their repressive effects on proliferation of TAG72 positive hepatocarcinoma cells. Methods Firstly, peripheral blood mononuclear cells (PBMCs) from healthy volunteers were isolated. And then, CD8+ T cells were isolated from PBMCs via magnetic activated cell sorting (MACS). These lymphocytes were transfected with recombinant vector, anti-TAG72-scFv-CD28-pcDNA3, through Lipofectamine2000 to gernerate anchor chimeric TAG72-specific CD8+ T cells. SMMC7721 (TAG72 positive) hepatocarcinoma cells were co-cultured with chimeric T lymphocytes and their cell cycles were analyzed by flow cytometry (FCM). Results Anchor chmeric T lymphcytes targetting TAG72 recognized TAG72 positive SMM7721 cells and repressive effects on their proliferation were observed by flow cytometry. Conclusion Anchor chmeric T lymphcytes targetting TAG72 on tumor surface can specifically recognize TAG72 positive hepatocarcinoma cells and may exert repressive effect on their proliferation.%目的 探讨肿瘤相关糖蛋白72(TAG72)靶向性嵌合锚定T淋巴细胞的制备方法,并检测它对TAG72阳性肝癌细胞增殖的阻滞效应.方法 分离外周血单核细胞(PBMC),然后用免疫磁珠法分离得到CD8+T淋巴细胞.将重组真核表达载体anti-TAG72-scFv-CD28-pcDNA3.0采用脂质体介导的细胞转染和细胞培养,以制备TAG72靶向性的嵌合锚定T淋巴细胞;将嵌合锚定T淋巴细胞与TAG72阳性肝癌细胞SMMC7721共培养,通过流式细胞仪检测肝癌细胞的周期变化,分析嵌合锚定T淋巴细胞对肝癌细胞增殖的抑制效应.结果 TAG72靶向性嵌合锚定T淋巴细胞可识别肝癌细胞SMMC7721;用流式细胞仪检测发现,嵌合锚定T淋巴细胞可引起肝癌细胞SMMC7721的增殖阻滞.结论 TAG72靶向性嵌合锚定T淋巴细胞可特异性识别TAG72阳性肝癌细胞SMMC7721并引起其增殖阻滞.

  2. The Inhibitory effects of Hydrocortisone on Proliferation of Mouse T Lymphocytes%氢化可的松对小鼠T淋巴细胞增殖的抑制作用

    Institute of Scientific and Technical Information of China (English)

    彭新青; 周大杰; 乔玉莉; 韦日明

    2011-01-01

    [Objective] The aim was to investigate effects of Hydrocortisone on proliferation of mouse T lymphocytes. [Method] T lymphocytes separated aseptically from mouse spleen were stimulated in RPMI1640 complete medium plus Con A and transformed. The CD25 and CD69 on the surface of activated T cells as well as their co - expression rate were detected by Flow Cytometry ( FCM). The transformed T cells were divided into two groups; the control group cultured without Hydrocortisone and the experimental groups cultured with different concentration Hydrocortisone. The T cells were counted at different culture times and the growth curves were made. [Result] The CD25 and CD69 on T cells activated by Con A in three days and their co - expression rate reached the highest level, being 73.5% 、58% as well as 32% respectively. The growth curves showed that both groups of T cells proliferation reached to exponential phase of growth cultured at the 2nd day with Hydrocortisone; the activated mouse T cells were inhibited by all of 10-5mol/L、10~6mol/L、10-7mol/L Hydrocortisone without significant difference(P>0.05)among the three concentration groups, and with significant difference compared to the control group(P<0.05). [Conclusion] Hydrocortisone could inhibit proliferation of mouse T lymphocytes at a certain range of concentration, providing an experimental basis for animal model of cell transplantation in inducing immune tolerance.%[目的]探讨氢化可的松对小鼠T淋巴细胞增殖功能的影响.[方法]用淋巴细胞分离液无菌制备小鼠脾淋巴细胞,于RPMI1640的完全培养基中加入ConA后进行转化.用流式细胞仪检测T细胞活化分子CD25和CD69及其共表达率.将转化的淋巴细胞分成不加氢化可的松的对照组和加入不同浓度的氢化可的松组,在各时间点取样计数,绘制其生长曲线.[结果]经ConA刺激3d的T细胞上CD25和CD69分子及其共表达率最高,分别达73.5%、58%和32%.生长曲线显示,

  3. Vehicle and positive control values from the in vivo rodent comet assay and biomonitoring studies using human lymphocytes: historical database and influence of technical aspects.

    Science.gov (United States)

    Pant, Kamala; Springer, S; Bruce, S; Lawlor, T; Hewitt, N; Aardema, M J

    2014-10-01

    There is increased interest in the in vivo comet assay in rodents as a follow-up approach for determining the biological relevance of chemicals that are genotoxic in in vitro assays. This is partly because, unlike other assays, DNA damage can be assessed in this assay in virtually any tissue. Since background levels of DNA damage can vary with the species, tissue, and cell processing method, a robust historical control database covering multiple tissues is essential. We describe extensive vehicle and positive control data for multiple tissues from rats and mice. In addition, we report historical data from control and genotoxin-treated human blood. Technical issues impacting comet results are described, including the method of cell preparation and freezing. Cell preparation by scraping (stomach and other GI tract organs) resulted in higher % tail DNA than mincing (liver, spleen, kidney etc) or direct collection (blood or bone marrow). Treatment with the positive control genotoxicant, ethyl methanesulfonate (EMS) in rats and methyl methanesulfonate in mice, resulted in statistically significant increases in % tail DNA. Background DNA damage was not markedly increased when cell suspensions were stored frozen prior to preparing slides, and the outcome of the assay was unchanged (EMS was always positive). In conclusion, historical data from our laboratory for the in vivo comet assay for multiple tissues from rats and mice, as well as human blood show very good reproducibility. These data and recommendations provided are aimed at contributing to the design and proper interpretation of results from comet assays.

  4. Novel Confocal Microscopic and Flow Cytometric Based Assays to Visualize and Detect the (Beta)2-Adrenergic Receptor in Human Lymphocyte and Mononuclear Cell Populations

    Science.gov (United States)

    Salicru, A. N.; Crucian, B. E.; Nelman, M. A.; Sams, C. F.; Actor, J. K.; Marshall, G. D.

    2006-01-01

    The data show that immunophenotyping of leukocyte populations with (beta)2AR is possible with the commercially available Ab, although the FC assay is limited to the IST as a result of the Ab binding site to the intracellular C-terminus of the 2AR. The FC assay has applications for measuring alterations in total (beta)2AR in human leukocyte populations as changes in fluorescence. In addition, CM confirms that both surface and intracellular compartments stain positively for the (beta)2AR and can be used for qualitative assays that screen for changes in receptor compartmentalization and localization.

  5. 荧光染料CFSE在实验性葡萄膜炎T细胞增殖研究中的应用%Tracking T Lymphocytes Proliferation with Vital Dye CFSE in Experimental Autoimmune Uveitis

    Institute of Scientific and Technical Information of China (English)

    雷松; 毛咏秋

    2009-01-01

    Objective To track T lymphocytes proliferation with vital dye CFSE in experimental autoimmune uveitis (EAU). Methods B10RIII mice were immunized with synthetic peptide of interphotoreceptor retinal-binding protein (IRBP161-180) to develop EAU. The proliferation of IRBP-specific T cell subsets was detected by CFSE staining, fluorescent antibody labeling, and flow cytometry. Results The IRBP-specific T cells divided after 4 days of stimulation with IRBP161-180, halving the fluorescence intensity. The proliferations of CD4~+ and CD8~+ T cells were asynchronous, with CD8~+ T cells dividing more vigorously and having more drop in percentage of parent cells(P<0. 01). Conclusion CFSE-Labeling can detect the proliferation of autoreactive T cells and their subsets in EAU effectively.%目的 探讨荧光染料CFSE在实验性葡萄膜炎(EAU)抗原特异T细胞增殖研究中的应用价值. 方法 以人类光感受器间维生素A类结合蛋白(IRBP)的多肽片段IRBP161-180为抗原,免疫B10RⅢ小鼠使其产生EAU.用CFSE荧光染料标记细胞,结合荧光单抗和流式细胞术,检测T细胞及其亚群在特异抗原刺激下,不同时间点细胞增殖的情况.结果 IRBP161-180刺激4 d后出现T淋巴细胞增殖分裂,表现为CFSE荧光强度的系列减半.使用荧光单抗双标记发现CD4~+ T和CD8~+ T细胞增殖反应不同步,CD8~+ T细胞较CD4~+T细胞增殖分裂更活跃,亲代细胞百分率下降更明显(P<0.01).结论 CFSE荧光标记技术是分析EAU抗原特异性T细胞及亚群增殖分裂的有力工具.

  6. Genotoxic effects of bistratene A on human lymphocytes.

    Science.gov (United States)

    Rojas, E; Valverde, M; Vega, L; Salvador, A; Ramirez, P; Herrera, L A; Watters, D; Lavin, M F; Ostrosky-Wegman, P

    1996-03-01

    Bistratene A, a toxin isolated from the colonial ascidian Lissoclinum bistratum causes a decrease in mitotic index and retardation of lymphocyte proliferation kinetics when it is added at 48 h to 72-h human lymphocyte cultures. In the same cultures, the incidence of sister chromatid exchanges was not altered by this compound. We also observed an increase in the number of polyploid cells in the cultures, and alterations of the beta-tubulin organization by immunocytochemistry with an antibody against beta-tubulin. Bistratene A induces DNA damage in a dose-dependent fashion in leukocytes, as measured by the alkaline single cell gel electrophoresis assay. These results show that bistratene A interferes with microtubule assembly, is cytotoxic and cytostatic, and that it causes DNA damage.

  7. Onset of the lymphocytic infiltration and hyperplasia preceding the proliferation in F1 mouse lungs from the N-ethyl-N-nitrosourea exposed mothers: Prevention during the lactation period by inositol hexaphosphate

    Directory of Open Access Journals (Sweden)

    Satya Sahay

    2015-01-01

    Full Text Available Maternal exposure to a carcinogen is associated with increased risk of different cancers in the offspring. The foetus is highly sensitive to carcinogens and this contributes to the foetal basis of the onset of disease. The better understanding of the molecular mechanisms involved in the early stage of lung tumourigenesis in the offspring is needed for the newer preventive strategies. We evaluated the effects of N-ethyl-N-nitrosourea (ENU given on the 17th day of gestation and antitumour agent inositol hexaphosphate (IP6 to the mothers at the early stage of lung tumourigenesis in F1 mice. There was no treatment related effects on the litter size or body weight of the F1 mice at the PND12 or 24. Analysis of PCNA, NF-κB (p50, IL-6, COX-2, pSTAT3, STAT3, caspase-3, caspase-9, PARP, Akt signalling and downstream cyclin D1 along with miR-155, suggested the modulation of proliferation, inflammation and apoptosis at PND12 and 24. IP6 administration to the predisposed mothers prevented the proliferation, inflammation and enhanced apoptosis in F1 lung as showed by a reduction in PCNA, NF-κB (p50, IL-6, COX-2, pSTAT3, STAT3, miR-155 and increase in caspases, cleavage of poly (ADP-ribose polymerase. IP6 administration also inhibited the activation of Akt and cyclin D1. Our study shows that tumourigenic changes take place in the lungs of the F1 generation from the carcinogen predisposed mothers even before the onset of tumours and the simultaneous intake of chemopreventive agent during the gestation or lactation period could prevent the lymphocytic infiltration and hyperplasia preceding the tumourigenesis.

  8. Correlation between the pharmacological efficacy of cyclosporine and tacrolimus as evaluated by the lymphocyte immunosuppressant sensitivity test (LIST) and the MTT assay procedure in patients before and after renal transplantation.

    Science.gov (United States)

    Sugiyama, K; Isogai, K; Toyama, A; Satoh, H; Saito, K; Nakagawa, Y; Tasaki, M; Takahashi, K; Hirano, T

    2011-02-01

    Cyclosporine and tacrolimus are calcineurin inhibitors that are used to prevent acute rejection in renal transplant recipients. The lymphocyte immunosuppressant sensitivity test (LIST) can predict the pharmacological efficacy of these immunosuppressive agents for renal transplant recipients. There is a correlation between cyclosporine and tacrolimus pharmacological efficacy as evaluated by LIST by the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay procedure prior to renal transplantation. However, the LIST can also evaluate patients before and after the transplantation. The present study examined the relationship between cyclosporine and tacrolimus pharmacological efficacy by LIST using the MTT assay in 16 renal transplant recipients at 1, 3 and 12 months after transplantation, as well as before the operation. The relationship of cyclosporine and tacrolimus pharmacological efficacy gave a significant Kendall and Spearman's coefficient correlation in these transplant recipients by the LIST using the MTT assay procedure immediately prior to renal transplantation (rk = 0.711, rs = 0.877, p cyclosporine and tacrolimus IC50 values were also observed with a significant Kendall and Spearman's coefficient correlation at 1 and 12 months after transplantation (rk1month = 0.65, rs1month = 0.829, p transplantation (rk3month = 0.117, rs3month = 0.1, p > 0.05). Both cyclosporine and tacrolimus exhibit pharmacological efficacy by the inhibition of calcineurin. However, the correlation between cyclosporine and tacrolimus pharmacological efficacies may be altered, due to immunosuppressive therapy or clinical events at 3 months after renal transplantation.

  9. Subclass of individual IgA-secreting human lymphocytes. Investigation of in vivo pneumococcal polysaccharide-induced and in vitro mitogen-induced blood B cells by monolayer plaque-forming cell assays

    DEFF Research Database (Denmark)

    Heilmann, C; Barington, T; Sigsgaard, T

    1988-01-01

    The subclass of individual human IgA B cells was investigated by means of monolayer plaque-forming cell assays permitting analysis of all IgA-secreting cells as well as of cells secreting IgA anti-pneumococcal polysaccharide antibody. Center cells were examined by indirect immunofluorescence...... that these Ag have an unusually high ability to activate IgA2 B cells, or that the B cells stimulated originate from lymphatic tissues with a high frequency of IgA2 committed cells....... staining with mouse mAb against either of the two IgA subclasses as primary antibodies and FITC-conjugated rabbit anti-mouse Ig as the second antibody. Blood lymphocytes spontaneously secreting IgA (mean 399/10(6) mononuclear cells) produced mainly IgA1 (73%). A similar distribution of subclasses...

  10. 不同外邪对正常及免疫功能低下小鼠脾淋巴细胞增殖能力的影响%Effect of attacks factors on the immunesplenic Lymphocytes proliferation capacity of normal and low immune function mice

    Institute of Scientific and Technical Information of China (English)

    马彦平; 郭彩云; 李俊莲; 李艳彦; 杨琬芳; 李孝波; 王平; 陶功定

    2012-01-01

    Objective: To detect the proliferation capacity of splenic lymphocytes of mice in the cold, cold dampness and hot and humid environment, and discusses the effect of different factors of attacks on the immune functions of mice. Methods: We choose knife bean protein (ConA) induced spleen lymphocyte proliferation, use the MTT method to determine the spleen lymphatic proliferation capacity in mice. Results: ①The mice splenic lymphocytes proliferation capacity of all the attack factors processing groups were significantly lower than the normal group (P<0.01).②All the attack factors processing low immune functions groups compared with low immune function group, the splenic lymphocytes proliferation capacity of wind-cold, cold-dampness low immune function groups reduced significantly (P<0.01); the splenic lymphocytes proliferation capacity of dampness-heat low immune function groups decreasd, but this was not statistically significant. ③The wind-cold low immune function group compared with the wind-cold group, the cold-dampness low immune function group compared with the cold-dampness group, the dampness-heat low immune function group compared with the dampness-heat group, all the mice splenic lymphocytes proliferation capacity significantly reduced (P<0.01). Conclusion: Compared with the normal group, the mice splenic lymphocytes proliferation capacity of all the attack factor processing groups reduced, the mice splenic lymphocytes proliferation capacity of low immune modle groups especially signigicant. The study shows that the three attacks factors of wind-cold, cold-dampness, dampness-heat can make the immune function of mice reduce and cause the happen of the disease easily.%目的:通过检测在风寒、寒湿和湿热环境下小鼠脾淋巴细胞的增殖能力,探讨不同外邪因素对小鼠免疫功能的影响.方法:选择刀豆蛋白(ConA)诱导脾淋巴细胞增殖,采用MTT法测定小鼠脾淋巴细胞增殖能力.结果:①各外邪因素处理

  11. 枸杞多糖对雏鸡淋巴细胞体外增殖及分泌IL-2的影响%Effects of Lycium barbarum Polysaccharide on Cell Proliferation of Chicken Lymphocytes and IL-2 Excretion in vitro

    Institute of Scientific and Technical Information of China (English)

    徐占云; 秦睿玲; 褚耀诚; 李国辉; 李春红; 王丽

    2013-01-01

    This experiment was conducted to investigate the effects of Lycium barbarum polysaccharide (LBP) on cell proliferation of chicken Lymphocytes in vitro. Different concentrations of LBP were added into cultured with plants hemagg-lutinin (PHA) stimulated chicken thymus, spleen and peripheral blood lymphocyte, and cultured with bacteria lipopolysaccharide (LPS) stimulated chicken bursa of Fabricius, peripheral blood lymphocyte, spleen, MTT method and ELISA method were used to determine the lymphocyte proliferation and IL-2 excretion respectively. The results showed that: (1) LBP could stimulate T, B lymphocyte proliferation and IL-2 excretion in certain concentration range and with the increase of the concentration of polysaccharide increase; (2) The OD value of T, B lymphocyte with the source of the same organ were significantly growth trend with chickens dayage growth in same concentration of LBP. (3) The OD value of thymus T lymphocyte and bursa of Fabricius B lymphocyte were higher than in peripheral blood and the spleen T, B lymphocytes with the same concentration LBP and the same day age of chicken. These results indicated that LBP could stimulate T, B lymphocyte proliferation and IL-2 excretion, and the promoting function of lymphocyte proliferation have certain concentration-response, time relationship and organizational correlation.%为探讨枸杞多糖(LBP)对雏鸡T、B淋巴细胞体外增殖的影响,将不同浓度的LBP分别加入到用植物血凝素(PHA)刺激的体外培养的雏鸡胸腺、外周血、脾脏淋巴细胞中,以及用细菌脂多糖(LPS)刺激的体外培养的雏鸡法氏囊、外周血、脾脏淋巴细胞中,分别用MTT比色法和ELISA法测定淋巴细胞增殖的变化以及IL-2的分泌量.结果显示:(1)LBP在一定浓度范围内时,均能刺激T、B淋巴细胞的增殖和促进IL-2的分泌,且随着多糖浓度的增加而增加;(2)LBP对同一器官来源的T、B淋巴细胞的OD值随着雏鸡日龄的

  12. Protective effect of Crataegus monogyna Jacq. ethanolic extracts in oxidant-induced DNA damage evaluated through comet assay with human peripheral lymphocytes

    Directory of Open Access Journals (Sweden)

    Joao C. M. Barreira

    2015-06-01

    The results showed significant differences within the assessed botanical parts and also among the assayed concentrations. The performed investigation might be considered as representing a step further in the evaluation of the in vivo bioactive potential of this highly promising species. Furthermore, it has established some practical bases for the evaluation of additional natural matrices with high scoring in bioactivity screening studies.

  13. 13. APPLICATION OF THE CYTOKINESIS BLOCK MICRONUCLEUS ASSAY FOR BIOMONITORING PURPOSES: INCLUSION OF MICRONUCLEI IN NON-DIVIDED MONONUCLEAR LYMPHOCYTES AND NECROSIS/APOPTOSIS

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    @@Human biomonitoring of early genetic effects requires accurate, sensitive and, if possible, easy and not too time-consuming methodologies to assess mutations. One of the most promising methodologies today is the cytokinesis block micronucleus assay, which detects both chromosome breakage and chromosome loss in once-divided

  14. 糖酵解抑制剂3-溴丙酮酸对异基因小鼠混合培养脾细胞增殖的影响及其机制初步研究%Effect of glycolytic inhibitor 3-BrPA on the proliferation and apoptosis of mouse splenic lymphocytes in mixed lymphocytes culture

    Institute of Scientific and Technical Information of China (English)

    周睿卿; 王静; 邱大发; 牛晓敏; 郭子文; 何慧清; 许晓军

    2014-01-01

    Objective To study the effect of glycolytic inhibitor 3-Bromopyruvate (3-BrPA) on the proliferation and apoptosis of mouse spleen lymphocytes and explore its mechanism.Methods An one-way mixed lymphocyte culture (MLC) system was established,including BALB/c mouse spleen cells (H-2d) as stimulator and C57BL/6 mouse spleen cells (H-2b) as responder.With treatment of 3-BrPA at different concentrations (0-200 μmol/L),lymphocyte proliferation capacity was detected by the CCK-8 method,the expression of CD3,CD4,and CD8 by flow cytometry,and the concentrations of cytokine interleukin (IL)-4 and interferon (IFN)-γ in the supernatant by ELISA.Results At a middle or high dose (over 20 μmol/L),3-BrPA displayed a dose-dependent inhibitory effect on lymphocyte proliferation in the MLC system.The 50% inhibitory concentration (IC50) were 48.6,41.2,and 41.9 μmol/L after 24,36,and 48 h culture,respectively.With treatment of 50 μmol/L 3-BrPA,the IFN-γ level [(164.25±20.14) ng/L] was significantly lower,compared with control [(277.61 ± 18.46) ng/L].The IL-4 level [(31.06 ± 6.06)ng/L] was significantly higher,compared with control [(28.64 ± 3.97) ng/L].Consequently,the IFN-γ/IL-4 ratio decreased significantly.Conclusions These results indicate that 3-BrPA had a significant inhibitory effect on the proliferation of mouse spleen lymphocytes cultured in MLC system,accompanied with the Th2-biased secretion of cytokines.%目的 研究糖酵解抑制剂3-溴丙酮酸(3-BrPA)对单向混合培养的异基因小鼠脾细胞增殖的影响及其机制.方法 以BALB/c(H-2d)小鼠脾细胞为刺激细胞,以C57BL/6(H-2b)小鼠脾细胞为反应细胞,建立单向混合淋巴细胞培养(MLC)体系,给予不同浓度3-BrPA(0~200 μmol/L)处理后,采用CCK-8法检测淋巴细胞增殖活性,流式细胞术检测MLC体系中反应细胞CD3、CD4、CD8等表面标志表达,ELISA法检测MLC体系上清中IL-4、IFN-γ等细胞因子水平.结果 CCK-8检测结果显示,高浓度(20

  15. The injury progression of T lymphocytes in a mouse model with subcutaneous injection of a high dose of sulfur mustard

    Institute of Scientific and Technical Information of China (English)

    Yi-Zhou Mei; Xiao-Rui Zhang; Ning Jiang; Jun-Ping Cheng; Feng Liu; Pan Zheng; Wen-Xia Zhou; Yong-Xiang Zhang

    2014-01-01

    Background: In clinical studies, the findings on sulfur mustard (SM) toxicity for CD3+CD4+ and CD3+CD8+ T lymphocyte subsets are contradictory. In animal experiments, the effect of SM on the T cell number and proliferation is incompatible and is even the opposite of the results in human studies. In this study, we observed the dynamic changes of T lymphocytes in the first week in a high-dose SM-induced model. Methods: Mice were exposed to SM by subcutaneous injection (20 mg/kg) and were sacrificed 4 h, 24 h, 72 h and 168 h later. Spleen T lymphocyte proliferation was evaluated by3H-TdR. Flow cytometric analysis was used to observe the percentage of CD3+CD4+ and CD3+CD8+ T lymphocyte subsets. The IL-1β, IL-6, IL-10 and TNF-α levels in plasma were assayed using the Luminex method. DNA damage in bone marrow cells was observed with the single cell gel electrophoresis technique (SCGE). Results: SM continuously inhibited the proliferation of lymphocytes for 7 days, and there was a significant rebound of Con A-induced T lymphocyte proliferation only at 24 h. The percentage of CD3+CD4+ and CD3+CD8+ lymphocytes was upregulated, which was accompanied by increased IL-1β and TNF-α and decreased IL-10. The IL-6 level was gradually decreased in the PG group at 4 h. The peak of lymphocytic apoptosis and DNA damage occurred at 24 h and 72 h, respectively. Conclusion: Our results show that SM significantly inhibited T lymphocyte proliferation as well as induced CD3+CD4+ and CD3+CD8+ upregulation. SM intoxication also significantly increased the levels of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) and inhibited the level of anti-inflammatory cytokine IL-10. Our results may partly be due to the significant SM induced significant apoptosis and necrosis of lymphocytes as well as DNA damage of bone marrow cells. The results provided a favorable evaluation of SM immune toxicity in an animal model.

  16. Astaxanthin, a Carotenoid, Stimulates Immune Responses by Enhancing IFN-γ and IL-2 Secretion in Primary Cultured Lymphocytes in Vitro and ex Vivo.

    Science.gov (United States)

    Lin, Kuan-Hung; Lin, Kao-Chang; Lu, Wan-Jung; Thomas, Philip-Aloysius; Jayakumar, Thanasekaran; Sheu, Joen-Rong

    2015-12-29

    Astaxanthin, a potent antioxidant carotenoid, plays a major role in modulating the immune response. In this study, we examined the immunomodulatory effects of astaxanthin on cytokine production in primary cultured lymphocytes both in vitro and ex vivo. Direct administration of astaxanthin (70-300 nM) did not produce cytotoxicity in lipopolysaccharide (LPS, 100 µg/ mL)- or concanavalin A (Con A, 10 µg/ mL)-activated lymphocytes, whereas astaxanthin alone at 300 nM induced proliferation of splenic lymphocytes (p astaxanthin, alone or with Con A, had no apparent effect on interferon (INF-γ) and interleukin (IL-2) production in primary cultured lymphocytes, it enhanced LPS-induced INF-γ production. In an ex vivo experiment, oral administration of astaxanthin (0.28, 1.4 and 7 mg/kg/day) for 14 days did not cause alterations in the body or spleen weights of mice and also was not toxic to lymphocyte cells derived from the mice. Moreover, treatment with astaxanthin significantly increased LPS-induced lymphocyte proliferation ex vivo but not Con A-stimulated lymphocyte proliferation ex vivo. Enzyme linked immunosorbent assay (ELISA) analysis revealed that administration of astaxanthin significantly enhanced INF-γ production in response to both LPS and Con A stimulation, whereas IL-2 production increased only in response to Con A stimulation. Also, astaxanthin treatment alone significantly increased IL-2 production in lymphocytes derived from mice, but did not significantly change production of INF-γ. These findings suggest that astaxanthin modulates lymphocytic immune responses in vitro, and that it partly exerts its ex vivo immunomodulatory effects by increasing INF-γ and IL-2 production without inducing cytotoxicity.

  17. Culture and identification of mouse bone marrow derived DCs that induce T lymphocyte proliferation%小鼠骨髓来源树突状细胞培养鉴定和诱导T淋巴细胞增殖

    Institute of Scientific and Technical Information of China (English)

    吴昆; 拜合提亚·阿扎提; 王文光; 安尼瓦尔·牙生; 王玉杰

    2011-01-01

    Objective:To establish a method of inducing and culturing dendritic cells from mouse bone marrow in vitro,and observe their biological feature and function of stimulating T lymphocytes proliferate in different stages. Methods: Mononuclear cells isolated from mouse bone marrow were induced into dendritic cells by being cultured with GM-CSF and IL-4,and then examined from aspects of morphology,phenotype and function. Results:Modality of mononuclear cells diversified after being cultured in CM-CSF and IL-4 for 3 days,and growthing clustered-liked. When culturing for S days,typical morphology with dendritic processes can be observed. A large number of morphologically typical dendritic cells were observed after culturing for 8 days,under scan electron microscope. DCs displayed typical morphology with dendritic processes and high expressed specific marker of bone marrow derived DC-CD11c and costimulatory molecules of CD40,CD80,CD86 and MHC-Ⅱ. Mature BMDC could stimulate syngenic and allogenic mixed lymphocyte proliferation. Conclusion: A large number of dendritic cells can be generated by culturing the mononuclear cells derived from mouse bone marrow in vitro, and thus will lay a foundation for future research in producing the anti-tumor vaccine.%目的:建立一种体外诱导培养小鼠树突状细胞(DCs)的方法,并观察其不同生长阶段生物学形态及刺激淋巴细胞增殖的能力。方法:联合应用粒细胞-巨噬细胞集落刺激因子(GM-CSF)、白细胞介素-4(IL-4)诱导培养小鼠骨髓单个核细胞向DCs分化,并从形态学、表型及功能方面对其加以检测。结果:用GM-CSF和IL-4诱导培养小鼠骨髓来源的单核细胞,3 d后可见细胞形态改变,并呈集簇生长,培养至5 d可见典型的树突状突起,体外诱导培养8 d后获得大量成熟的DCs,扫描电镜可见典型的树突状细胞形态,高表达骨髓来源DCs特异性标记CD11c和共刺激分子CD40、CD80、CD86和MHC-Ⅱ

  18. Serotonin transporter in lymphocytes of rats exposed to physical restraint stress.

    Science.gov (United States)

    Medina-Martel, Matilde; Urbina, Mary; Fazzino, Fili; Lima, Lucimey

    2013-01-01

    Glucocorticoids and stress cause transcriptional and functional changes on the serotonin transporter (SERT) in the central nervous system. Stress can produce specific modifications of SERT in lymphocytes, which could be associated with alterations in immune response. The aim of this study was to evaluate the effect of a physical restraint stress protocol on (1) rat lymphocyte proliferation in the presence of the selective serotonin reuptake inhibitor fluoxetine and (2) SERT kinetic parameters, i.e. binding capacity (Bmax), affinity (Kd) and Hill coefficient (nH). Male adult Sprague-Dawley rats were placed in Plexiglass boxes (5 h daily for 5 days), and blood was obtained by cardiac puncture on day 6. Serum corticosterone was quantitated by an immunoenzymatic assay. Lymphocytes were isolated by density gradients and adhesion to plastic, of which there was sufficient material for further experiments, then cultured with or without the mitogen concanavalin A (Con A, 2 μg/ml) and fluoxetine (1-50 μM). Cell proliferation was measured with tetrazolium salts, and [(3)H]paroxetine was used as a SERT-specific ligand for binding assays. Restraint produced a significant increase in serum corticosterone of stressed rats. The proliferative response to Con A was similar in the controls and stressed animals. Fluoxetine reduced cell proliferation with and without Con A. Restraint diminished the inhibitory effect of fluoxetine on proliferation. Restraint also increased Bmax and Kd, but decreased nH. Treatment of rats with actinomycin D, a transcription inhibitor, reduced Bmax in stressed animals. Restraint stress modulated the effect of fluoxetine on cell proliferation, probably through the modification of the presence and the function of SERT. Copyright © 2013 S. Karger AG, Basel.

  19. ALS patients’ regulatory T lymphocytes are dysfunctional, and correlate with disease progression rate and severity

    Science.gov (United States)

    Beers, David R.; Zhao, Weihua; Wang, Jinghong; Zhang, Xiujun; Wen, Shixiang; Neal, Dan; Thonhoff, Jason R.; Alsuliman, Abdullah S.; Shpall, Elizabeth J.; Rezvani, Katy

    2017-01-01

    Neuroinflammation is a pathological hallmark of ALS in both transgenic rodent models and patients, and is characterized by proinflammatory T lymphocytes and activated macrophages/microglia. In ALS mouse models, decreased regulatory T lymphocytes (Tregs) exacerbate the neuroinflammatory process, leading to accelerated motoneuron death and shortened survival; passive transfer of Tregs suppresses the neuroinflammation and prolongs survival. Treg numbers and FOXP3 expression are also decreased in rapidly progressing ALS patients. A key question is whether the marked neuroinflammation in ALS can be attributed to the impaired suppressive function of ALS Tregs in addition to their decreased numbers. To address this question, T lymphocyte proliferation assays were performed. Compared with control Tregs, ALS Tregs were less effective in suppressing responder T lymphocyte proliferation. Although both slowly and rapidly progressing ALS patients had dysfunctional Tregs, the greater the clinically assessed disease burden or the more rapidly progressing the patient, the greater the Treg dysfunction. Epigenetically, the percentage methylation of the Treg-specific demethylated region was greater in ALS Tregs. After in vitro expansion, ALS Tregs regained suppressive abilities to the levels of control Tregs, suggesting that autologous passive transfer of expanded Tregs might offer a novel cellular therapy to slow disease progression. PMID:28289705

  20. Development of an Immunoperoxidase Monolayer Assay for the Detection of Antibodies against Peste des Petits Ruminants Virus Based on BHK-21 Cell Line Stably Expressing the Goat Signaling Lymphocyte Activation Molecule

    Science.gov (United States)

    Chen, Weiye; Li, Cuicui; Xie, Meimei; Bu, Zhigao

    2016-01-01

    From 2013 to 2015, peste des petits ruminants (PPR) broke out in more than half of the provinces of China; thus, the application and development of diagnostic methods are very important for the control of PPR. Here, an immunoperoxidase monolayer assay (IPMA) was developed to detect antibodies against PPR. However, during IPMA development, we found that Vero cells were not the appropriate choice because staining results were not easily observed. Therefore, we first established a baby hamster kidney-goat signaling lymphocyte activation molecule (BHK-SLAM) cell line that could stably express goat SLAM for at least 20 generations. Compared with Vero cells, the PPR-mediated cytopathic effect occurred earlier in BHK-SLAM cells, and large syncytia appeared after virus infection. Based on this cell line and recombinant PPR virus expressing the green fluorescent protein (GFP) (rPPRV-GFP), an IPMA for PPR diagnosis was developed. One hundred and ninety-eight PPR serum samples from goats or sheep were tested by the IPMA and virus neutralization test (VNT). Compared with the VNT, the sensitivity and specificity of the IPMA were 91% and 100%, respectively, and the coincidence rate of the two methods was 95.5%. The IPMA assay could be completed in 4 h, compared with more than 6 d for the VNT using rPPRV-GFP, and it is easily performed, as the staining results can be observed under a microscope. Additionally, unlike the VNT, the IPMA does not require antigen purification, which will reduce its cost. In conclusion, the established IPMA will be an alternative method that replaces the VNT for detecting antibodies against PPRV in the field. PMID:27768770

  1. 19. The HUman Micro Nucleus project. International Date Base Comparison for results with the cytokinesis-block micronucleus assay in human lymphocytes. Ⅰ. Effect of laboratory protocol, scoring criteria, and host factors on the frequency of micronuclei

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The first results of an analysis of pooled data from laboratories using the cytokinesis-block micronucleus assay in human lymphocytes and participating in the HUMN (HUman MicroNucleus project) international collaborative study are presented. The effects of laboratory protocol, scoring criteria, and host factors on baseline micronucleus(MN) frequency are evaluated, and a reference range of “normal” values against which future studies may be compared is provided. Primary data from historical records were submitted by 25 laboratories distributed in 16 countries. This resulted in a database of nearly 7000 subjects. Potentially significant differences were present in the methods used by participating laboratories, such as in the type of culture medium, the concentration of Cytochalasin-B, the percentage of fetal calf serum, and in the culture method. Differences in criteria for scoring MN were also evident. The overall median MN frequency in non-exposed(i.e., normal) subjects was 6.5‰ and the interquartile range was between 3‰ and 12‰. An increase in MN frequency with age was evident in all but two laboratories. The effect of gender, although not so evident in all databases, was also present, with females having a 19% higher level of MN (95% C.I.:14-24%). Statistical analyses were performed using random-effects models for correlated data. Our best model, which included exposure to genotoxic factors, host factors, methods, and scoring criteria, explained 75% of the total variance, with the largest contribution attributable to laboratory methods.

  2. Construction of HA-1-DC Nucleic-acid Vaccine and Induction of Specific Cytotoxic T Lymphocytes

    Institute of Scientific and Technical Information of China (English)

    WANG Yaya; ZHANG Donghua; HU Jinmei; LIU Wenli; ZHOU hongsheng; ZHANG Lu; LIU Dan; HUANG Zhenqian; TAN Huo

    2007-01-01

    An HA-1-DC nucleic-acid vaccine was constructed to induce anti-leukemia effect after hematopoietic stem cell transplantation (HSCT). DCs were generated from HSCT donors in vitro, and its immunologic activity was assayed by using flow cytometry and mixed lymphocytes reaction.HA-1 gene was electroporated into the cultured DCs to construct a DC nucleic-acid vaccine. After transfection for 48 h, the expression of HA-1 protein could be detected by using Western blot. The DCs were cultured with syngenic lymphocytes to induce specific cytotoxic T lymphocytes (CTLs).The cytoxicity of the CTLs was detected by LDH assay. The results showed that The DCs derived from peripheral blood monocytes (PBMCs) expressed the phenotype of DCs, and were effective in stimulating proliferation of the allogenic lymphocytes. After electroporating for 48-h, HA-1 protein was detected by using Western blot. The cytotoxity of inducing CTLs was higher than the control group. It was suggested that minor histocompatibility antigen HA-1 could be considered as a target of immunotherapy against leukemia after HSCT.

  3. 59. Protectivc effect of melatonin on genetic damage by chemical mutagen and the influence on cell prolife-ration kenetics

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    /L melatonin. ④ The proliferation cycle of lymphocyte cultured in the presence of 1.00 mmol/L melatonin exhibited an increase in the percent of cells in their first division, and concomitant significant decrease in their third or later division. which were increased in their second division in cells treated with melatonin of three concentration respectively. The ratio between the first and third or later division, between the second and third or later division of lymphocytes exposed to 1.00 mmol/L melatonin were higher than negative control. ⑤ The micronucleus rates of binuclear lymphocytes treated with 0.01,0.10,1.00 mmol/L melatonin plus MMC were lower than positive control, of which the inhibitory rates of micronuclei were 14.37%, 22.17% and 31.34%, respectively. ⑥ The micronucleus rates of mouse bone marrow polycychromatic erythrocytes exposed to 0.1,1.0,10.0 mg/kg*bw melatonin plus CP were lower markedly than positive control, in which it exhibited a significant and concentration-depended decrease, of which the inhibitory rates of micronuclei were 28.89%, 47.73% and 69.96%, respectively. Conclusion: In vitro assay. It seems that melatonin has not only no genotoxic, but also anti-mutagenic effect on lymphocyte. It can inhibit the increase of frequency of SCE in lymphocyte and micronucleus rate of binuclear lymphocyte induced by MMC, and micronucleus rate of mouse bone marrow induced by CP at some concentiations. It can inhibit the lymphocyte proliferation of mitogen stimulated and has some influence on cell proliferation kenetics at higher concentrations (1.00 mmol/L).

  4. Assessment of genotoxicity of Lannate-90® and its plant and animal metabolites in human lymphocyte cultures.

    Science.gov (United States)

    Valencia-Quintana, Rafael; Gómez-Arroyo, Sandra; Sánchez-Alarcón, Juana; Milić, Mirta; Olivares, José Luis Gómez; Waliszewski, Stefan M; Cortés-Eslava, Josefina; Villalobos-Pietrini, Rafael; Calderón-Segura, María Elena

    2016-06-01

    This study evaluated direct and metabolic genotoxic effects caused by Lannate-90®, a methomyl-based formulation (90 % active ingredient), in human lymphocyte cultures using sister chromatid exchange assay (SCE). Two processes were used for the plant promutagens evaluation: in vivo activation, applying the insecticide systemically in plants for 4 h and subsequently adding plant metabolites containing extracts to lymphocyte cultures; and in vitro activation, where the insecticide was incubated with Vicia faba S10 mix plus human lymphocyte culture. Direct treatment with the insecticide significantly increased SCE frequency in human lymphocytes (250-750 mgL-1), with cellular death observed at 1000 mgL-1 concentration. Using the extracts of Vicia faba treated with Lannate-90® to treat human lymphocytes, a dose-response relationship was observed. In lymphocyte cultures treated directly with the insecticide for 2 h, a negative response was obtained. When S10 mix was added, SCE frequency did not change significantly. Meanwhile, a mixture of S9 mammalian metabolic mix and Lannate-90® increased the SCE frequency, with an observed concentration-dependent response. Although Lannate-90® induced cellular death at the highest concentrations, it did not cause a delay in cell proliferation in any of the treatments, confirming its genotoxic action. This study is one of the first to evaluate and compare the direct effect of Lannate-90® in two bioassays, animal and vegetal, and the effect of plant and animal metabolism on its genotoxic potential.

  5. Synthesis and biological evaluation of salicylic acid conjugated isoxazoline analogues on immune cell proliferation and angiogenesis.

    Science.gov (United States)

    Puttaswamy, Naveen; Pavan Kumar, G S; Al-Ghorbani, Mohammed; Vigneshwaran, V; Prabhakar, B T; Khanum, Shaukath Ara

    2016-05-23

    Mitogenicity is the ability of the natural or synthetic compounds to induce cell division or proliferation. A series of salicylic acid derivatives containing isoxazoline moiety (8a-j) were synthesized and their immunopharmacological activities targeting lymphocyte proliferation and angiogenesis were evaluated. The compounds 8a-j mitogenicity were investigated on immunological cells that include human peripheral blood lymphocytes and murine splenocytes in-vitro. The results implicate that among the series of 8a-j, compound 8e showed a potent proliferative response on both human and murine lymphocytes. The proliferative index of the compound 8e was comparable to the reference mitogen Con A and mitogenecity is due to increased secretion IL-2. In -vivo CAM and rat corneal angiogenesis assays were performed to assess the compound's effect on endothelial cell migration and proliferation which inferred that 8e also induces the proliferation of endothelial cells. The study reports the synthetic immunostimulatory and pro-angiogenic activity of novel mitogen 8e which could be translated into new drug in future.

  6. Immunotoxicity of bisphenol A to Carassius auratus lymphocytes and macrophages following in vitro exposure

    Institute of Scientific and Technical Information of China (English)

    YIN Da-qiang; HU Shuang-qing; GU Ying; WEI Li; LIU Shu-shen; ZHANG Ai-qian

    2007-01-01

    Bisphenol A (BPA) is the monomer component of polycarbonate plastics and classified as an endocrine disrupting chemical (EDC).The reproductive toxicity of BPA has been extensively studied in mammals; however, relatively little information is available on the immunotoxic responses of fish to BPA. In this study, we investigated the effects of BPA on the immune functions of lymphocytes and macrophages in Carassius auratus. The effects of BPA were compared with those of two natural steroid hormones, estradiol and hydrocortisone. Proliferation of the two types of cells in response to PHA was measured using colorimetric MTT assay. Macrophage respiratory burst stimulated by Con A was measured using chemiluminescence assay. Results showed that BPA (0.054-5.4 mg/L),estradiol (0.0002-2.0 mg/L) and hydrocortisone (5-50 mg/L) significantly induced Carassius auratus lymphocyte proliferation while higher doses of hydrocortisone (500-5000 mg/L) appeared to be inhibitory. BPA (0.005-50 mg/L), estradiol (0.005-800 mg/L) and hydrocortisone (0.005-500 mg/L) markedly enhanced macrophage proliferation, whereas higher doses of BPA (500-1000 mg/L)appeared to inhibit cell proliferation. Furthermore, higher dosage of BPA (50 mg/L) and hydrocortisone (50 and 500 mg/L) suppressed the macrophages respiratory burst while estradiol is stimulative all the doses tested (0.05-500 mg/L). In conclusion, BPA could have immunotoxicity to Carassius auratus and functional changes of lymphocyte and macrophage in Carassius auratus may be different between low and high dosages.

  7. Study of response of thymic and submaxillary lymph node lymphocytes to administration of lead by different routes.

    Science.gov (United States)

    Teijón, César; Blanco, María Dolores; Romero, Carlos Santiago; Beneit, Juan Vicente; Villarino, Antonio Luis; Guerrero, Sandra; Olmo, Rosa

    2010-06-01

    A number of studies have reported that heavy metals are not only toxic for the organism but they may modulate immune responses. In the current study, the effect of 4-week administration of 200 ppm of PbAc(2), using different routes of administration (orally and intraperitoneal injection), on lymphatic organs was evaluated. In the thymus, the number of lymphocyte cells and the cellularity diminished significantly for both routes of treatment. Regarding the submaxillary lymph nodes, no significant variations took place. Cell-mediated immune response is commonly evaluated by cell proliferation assays. Mitogens are known to induce a vigorous proliferative response in lymphoid cells from mammals. An increase in the proliferation of T lymphocytes stimulated by concanavalin A and the proliferation of B lymphocytes stimulated with lipopolysaccharides was found in thymus for both routes of administration, whereas in the lymph nodes, there was a decrease in proliferation of T lymphocytes. Furthermore, lead administration by intraperitoneal route caused an effect on B and T lymphocyte subpopulations. Thus, there was an increase in B+ cells and a decrease in T+ cells. Regarding CD4+ and CD8+ T cells, there were only variations, concretely a drop in both subpopulations, in lymph nodes when lead was administered intraperitoneally. It is important to emphasize that an increase in apoptosis was found in this tissue. At the histological level, evident alterations were described in thymus both for the oral and for the intraperitoneal route. Therefore, it is possible to show that lead administered by both routes generated effects on an immunological level.

  8. Platelet-Activating Factor Antagonists Decrease Follicular Dendritic-Cell Stimulation of Human B Lymphocytes

    Directory of Open Access Journals (Sweden)

    Halickman Isaac

    2005-06-01

    Full Text Available Abstract Both B-lymphoblastoid cell lines and tonsillar B lymphocytes express receptors for platelet-activating factor (PAF. In lymph node germinal centres, B lymphocytes interact with follicular dendritic cells (FDCs, which present antigen-containing immune complexes to B lymphocytes. FDCs have phenotypic features that are similar to those of stromal cells and monocytes and may therefore be a source of lipid mediators. In this study, we evaluated the effects of the PAF antagonist WEB 2170 on the activation of tonsillar B lymphocytes by FDCs. FDCs were isolated from tonsils by Bovine Serum Albumin (BSA gradient centrifugation. After being cultured for 6 to 10 days, they were incubated with freshly isolated B cells in the presence or absence of the specific PAF receptor antagonist WEB 2170. B-lymphocyte proliferation was assessed by [3H]-thymidine incorporation, and immunoglobulin (Ig G and IgM secretion was assessed by enzyme-linked immunosorbent assay (ELISA. WEB 2170 (10-6 to 10-8 M inhibited [3H]-thymidine incorporation by up to 35% ± 3%. Moreover, the secretion of IgG and IgM was inhibited by up to 50% by WEB 2170 concentrations ranging from 10-6 to 10-8 M. There was no evidence of toxicity by trypan blue staining, and the addition of WEB 2170 to B cells in the absence of FDCs did not inhibit the spontaneous production of IgG or IgM. The effect of the PAF antagonist is primarily on B lymphocytes, as reverse transcription polymerase chain reaction detected little PAF receptor messenger ribonucleic acid (mRNA from FDCs. These data suggest that endogenous production of PAF may be important in the interaction of B lymphocytes with FDCs.

  9. 模拟微重力环境下虫草多糖促进淋巴细胞增殖和CD分子表达的实验研究%Cordyceps Sinensis Enhances Lymphocyte Proliferation and CD Markers Expression in Simulated Microgravity Environment

    Institute of Scientific and Technical Information of China (English)

    郝彤; 王妍; 李俊杰; 杜芝燕; 段翠密; 王滟濛; 王常勇; 宋锦苹; 王林杰; 李莹辉

    2012-01-01

    This study was aimed to explore the effect of cordyceps sinensis enhancing lymphocyte proliferation and surface CD marker expression in simulated microgravity environment. The splenic lymphocytes were separated from mice and cultured in the rotary cell culture system simulated microgravity environment. The cells were treated with different concentration of cordyceps sinensis solution (0, 6.25, 12.5, 25 and 50 μg/ml) for 24, 48 and 72 h respectively, then the cells were harvested, and analyzed for cell proliferation and the expression of cell surface markers(CD4 and CD8). The results showed that under simulated microgravity environment, the lymphocyte proliferation was inhibited. When the concentration of cordyceps sinensis was 25 or 50 μg/ml, the lymphocyte proliferation, CD4 and CD8 expressions all increased, but 50 μg/ml cordyceps sinensis could inhibit the proliferation ability with the time prolonging. It is concluded that the suitable concentration of cordyceps sinensis displayed the ability to enhance the lymphocyte proliferation and CD marker expression in simulated microgravity environment. These results may be valuable for screening drugs which can be potentially against immunosuppression under simulated microgravity.%本研究旨在探索旋转式细胞培养系统(RCCS)模拟微重力环境下虫草多糖促进淋巴细胞增殖和表面CD分子表达的作用.体外分离培养小鼠脾淋巴细胞,在RCCS模拟微重力环境中分别添加6.25、12.5、25和50 μg/ml的虫草多糖溶液培养,在24、48和72 h检测虫草多糖对淋巴细胞增殖及表面CD分子表达的作用.结果表明,模拟微重力环境抑制了淋巴细胞的增殖能力,25和50 μg/ml虫草多糖对淋巴细胞的增殖和CD4、CD8的表达均具有促进作用,但50μg/ml虫草多糖促进淋巴细胞增殖的作用随对间延长变为抑制作用.结论:适宜浓度范围内的虫草多糖具有对抗模拟微重力环境下淋巴细胞增殖能力下降和表

  10. Effects of two plant growth regulators, indole-3-acetic acid and β-naphthoxyacetic acid, on genotoxicity in Drosophila SMART assay and on proliferation and viability of HEK293 cells from the perspective of carcinogenesis.

    Science.gov (United States)

    Karadeniz, Asuman; Kaya, Bülent; Savaş, Burhan; Topcuoğlu, Ş Fatih

    2011-10-01

    In this study, the mutagenic and recombinogenic effects of indole-3-acetic acid (IAA), a plant growth regulator naturally synthesized in plants but produced synthetically, and β-naphthoxyacetic acid (BNOA), a synthetic plant growth regulator widely used in agricultural regions, were investigated using the somatic mutation and recombination test (SMART) in Drosophila wings. The effect of the same plant growth regulators against the proliferation and viability of a human immortalized embryonic kidney HEK293 cells which is at the early stage of carcinogenesis were also examined with MTT and trypan-blue exclusion assays. For the SMART assay, two different crosses were used: a standard and a high-bioactivation (HB) cross, involving the flare-3 and the multiple wing hairs markers. The HB cross involved flies characterized by an increased cytochrome P-450-dependent bioactivation capacity, which permits the more efficient biotransformation of promutagens and procarcinogens. In both crosses, the wings of the two types of progeny, inversion-free marker heterozygotes and balancer heterozygotes, were analyzed. The results show that IAA and BNOA are not mutagenic or recombinogenic in the wing cells of Drosophila. Furthermore, neither plant growth regulator affected the proliferation rate of HEK293 cells; however, both of them induced cell death at high concentrations.

  11. Effects of housefly maggots extracts on the proliferation and the CD4+/CD8+ ratio of mouser splenic lymphocytes in vitro%罗仙子提取物对小鼠脾淋巴细胞的体外增殖及CD4+/CD8+比例的影响

    Institute of Scientific and Technical Information of China (English)

    许银叶; 褚夫江; 吴玉萍; 金小宝; 朱家勇

    2013-01-01

    目的 研究罗仙子提取物(housefly maggots extracts,HME)对正常小鼠脾淋巴细胞的体外增殖,以及对小鼠淋巴细胞亚群CD4+/CD8+比例的影响.方法 不同浓度的罗仙子提取物对刀豆蛋白A(concanavalin A,ConA)体外处理的小鼠脾T淋巴细胞作用,初步确定其发挥作用的最佳浓度.采用MTT法检测罗仙子提取物在不同的时间点对ConA诱导的小鼠脾淋巴细胞的增殖能力,运用流式细胞仪检测细胞周期的分布以及CD4+/CD8+比例.结果 罗仙子提取物浓度为40 μg/mL,作用48 h后,能明显促进ConA 诱导的小鼠脾淋巴细胞增殖,并且能降低G0/G1期细胞的百分比以及升高S期和G2/M期细胞的百分比,并以48 h效果最明显.能使CD4+、CD4+/CD8+比例降低.结论 罗仙子可能通过免疫调节作用影响动脉粥样硬化.%Objective To study the effects of housefly maggots extracts ( HME ) on the proliferation and the CD4+/CD8+ ration of mouse splenic lymphocytes in vitro. Methods Mouse splenic lymphocytes stimulated by con-canavalin A ( ConA ) were treated with different concentrations of HME. The effects of splenocyte proliferation were assessed by MTT in order to select the most effective concentration of HME. Using the most effective concentration, splenic lymphocytes were treated with HME at different time points. Splenocyte cell cycle distribution and the CIM + /CD8 + ratio were observed by flow cytometry. Results HME of 40μg/mL notabty promoted the proliferation of splenic lymphocyte after cultivation for 48 hours, reduced the G0/G1 phase cells and raised the S and G2/M phase cells. HME could also reduced the CD4+ , CD8 + and CD4 + /CD8+ ratio. Conclusion HME regulates immue functions in atherosclerosis.

  12. 氧化苦参碱抑制二硝基氟苯所致小鼠接触性皮炎及淋巴细胞增殖%Restraint-effect of Oxymatrine on mouse's allergic contact dermatitis stimulated by DNFB and lymphocyte proliferation stimulated by Con A

    Institute of Scientific and Technical Information of China (English)

    陈明亮; 伍斌; 谢红付; 张江林; 杜乾君; 李建国; 李罗丝

    2006-01-01

    [Objective] To explore the restraint-effect of oxymatrine (OMT)on allergic contact dermatitis (ACD)and lymphocyte proliferation. [Methods] 1.To build up an ACD mouse model stimulated by DNFB and then to perform intraperitoneal injection with different dosages of OMT, PBS and hydrocortisone(HCT), To observe the curative effect by examining the swelling degree of mice auricles. 2. Dyed by Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) and then flow cytometer was used to examine the fluorescence intensity changes of lymphocyte co-influenced by polyclonal stimulator Concanavalin A (Con A) and OMT. Whereafter, analyze the efficacy of OMT on mouse lymphocyte proliferation by related software. [Results] 1. Comparing with PBS group, OMT possesses much stronger restraint-effect on ACD that caused by DNFB and depends on its injection dosage. Its restraint-effect is equivalent to the HCT of the same dosage, but brings fewer side effects. 2. In vitro expenment, it proves that OMT has the effects to restrain the proliferation of mice's lymphocyte depending on its concentration in substrate as it behaves differently in group 500, 125 and 31 μg/ml. [Conclusion] OMT possesses the obvious effect of restraining the ACD stimulated by DNFB; OMT is a kind of immunosuppressor.%目的 探讨氧化苦参碱(OMT)对二硝基氟苯(DNFB)所致小鼠变应性接触性皮炎(ACD)的抑制作用及小鼠淋巴细胞增殖的影响.方法 建立DNFB所致小鼠ACD模型,以不同剂量的OMT、PBS、氢化可的松(HCT)进行腹腔注射,检测小鼠耳廓肿胀度变化;利用羧基荧光素乙酰乙酸(CFDA-SE)染色,流式细胞术检测OMT对小鼠淋巴细胞增殖的影响.结果 OMT对DNFB所致小鼠ACD呈剂量依赖性抑制作用,且与同等剂量HCT作用效果相似,但副作用明显减小;体外实验证明,在500、125和31 μg/mL组OMT对小鼠淋巴细胞增殖呈剂量依赖性抑制.结论 OMT对DNFB所致小鼠ACD有显著的抑制作用,而且抑制小鼠淋

  13. Effects of Spinach Powder Fat-Soluble Extract on Proliferation of Human Gastric Adenocarcinoma Cells

    Institute of Scientific and Technical Information of China (English)

    HE TAo; HUANG CHENG-YU; CHEN HAl; HOU YUN-HUA

    1999-01-01

    Four kinds of assays were used to study the effect of a fat-soluble extract of spinach powder(SPFE) on the proliferation of human gastric adenocarcinoma cell line (SGC-7901) in vitro.These studies included: ( i ) cell growth assay, ( ii ) colony forming assay, ( iii ) MTT colorimetric assay, and ( iv ) 3H-TdR incorporation assay. The concentrations of SPFE expressed as the level of β-carotene in the medium were 2 × 10-s, 2 × 10-7 and 2 × 10-6 mol/L β-carotene in assays ( i ) ~ ( iii ), but 4 × 10-8, 4 × 10-7 and 4 × 10-6 mol/L β-carotene in assay ( iV ) respectively. The results indicated that SPFE inhibited the proliferation and colony forming ability of SGC-7901 cells. And in MTT assay, SPFE inhibited the viability of SGC-7901 cells, but no inhibitory effect of SPFE was observed on the viability of lymphocytes in peripheral blood of healthy people. Finally, in the 3H-TdR incorporation test, both SPFE and β-carotene showed significant inhibitory effects on DNA synthesis in SGC-7901 cells, but SPFE was more effective than 3-carotene.

  14. In vitro genotoxicity of fipronil sister chromatid exchange, cytokinesis block micronucleus test, and comet assay.

    Science.gov (United States)

    Çelik, Ayla; Ekinci, Seda Yaprak; Güler, Gizem; Yildirim, Seda

    2014-03-01

    Fipronil (FP) is a phenylpyrazole pesticide developed by the transnational company Rhône-Poulenc Agro in 1987. Data on the genotoxicity and toxicity of FP are rather inadequate. In this study, we aimed to evaluate the potential genotoxic activity of FP using the single-cell microgel electrophoresis or comet assay, sister chromatid exchanges (SCEs), and micronuclei (MN) in human peripheral blood lymphocytes. In addition, the cytokinesis block proliferation index (CBPI) and proliferation index (PRI) were measured for cytotoxicity. In this study, three different doses of FP were used (0.7, 0.3, 0.1 μg/mL). Mitomycin C (2 μg/mL) and hydrogen peroxide were used as positive controls for SCE MN test systems, and comet assay, respectively. FP induced a statistically significant increase in the MN and SCE frequency and DNA damage in a dose-dependent manner in human peripheral blood lymphocytes (pcomet assay, we showed that all the doses of the FP induced DNA damage in human peripheral blood lymphocytes in vitro (p<0.05).

  15. Developmental changes in cell proliferation and apoptosis in the normal duck thymus.

    Science.gov (United States)

    Fang, J; Cui, H; Peng, X; Chen, Z; He, M; Tang, L

    2011-12-01

    Cell proliferation and apoptosis in the normal duck thymus during embryonic and post-embryonic development were studied. The flow cytometry assay shows that the level of G(0)/G(1) thymic cell population and the proportion of apoptotic cells increased with age, while the levels of S phase, G(2) + M phase and the proliferating index decreased with age. Proliferation cell nuclear antigen (PCNA) was mainly detected in the nuclei of lymphocytes. The number of PCNA-positive cells in the cortex and medulla significantly decreased with age. Transferase-mediated dUTP nick-end labelling (TUNEL) reaction stained apoptotic bodies in the cytoplasm of macrophages and free apoptotic bodies or nuclei with condensed chromatin in lymphocytes. The number of TUNEL-positive cells in the cortex and medulla markedly increased with age. The amount of proliferation and apoptotic cells in the thymic cortex was higher than that in the medulla. The balance between proliferation and apoptosis in the duck thymus may account for the process of thymic development and involution.

  16. Activation of Signal Transducer and Activator of Transcription 5 (STAT5) in Splenocyte Proliferation of Asthma Mice Induced by Ovalbumin

    Institute of Scientific and Technical Information of China (English)

    Guoping Li; Zhigang Liu; Peixing Ran; Jing Qiu; Nanshan Zhong

    2004-01-01

    To investigate the role of signal transducer and transcriptional activator 5 (STAT5) activated in ovalbumin (OVA)-induced splenocyte proliferation of asthma mice, an asthma mouse model was set up by intraperitoneal injection and aspiration of OVA with nebulizer. The proliferation of splenocytes isolated from the asthma mice was detected by [3H] thymidine incorporation. The phosphorytation of STAT5 was examined by Western blotting and STAT5-DNA binding was measured by electrophoretic mobility shift assay (EMSA). OVA could pronouncedly induce the splenocyte proliferation of asthma mice in a dose-dependent manner compared with control groups. Phosphorylation of STAT5 and STAT5-DNA binding were observed in splenocytes from asthma mice induced by OVA at 1 h and 3 h. These results indicated that STAT5 signal pathway played an important role in lymphocyte proliferation of asthma mice induced by OVA. Cellular & Molecular Immunology.2004;1(6):471-474.

  17. Comparison of the effects of polysaccharides from wood-cultured and bag-cultured Ganoderma lucidum on murine spleen lymphocyte proliferation in vitro%段木栽培及袋栽灵芝多糖对体外培养小鼠脾淋巴细胞增殖活性的比较

    Institute of Scientific and Technical Information of China (English)

    曹立珍; 林志彬

    2003-01-01

    目的比较段木栽培灵芝多糖(wood-cultured Ganoderma lucidum polysaccharides, Gl-PS-WC) 及袋栽灵芝多糖(bag-cultured Ganoderma lucidum polysaccharides, Gl-PS-BC)对体外培养小鼠脾淋巴细胞增殖活性的影响,探讨袋栽灵芝多糖替代段木栽培灵芝多糖的可能性. 方法检测两种灵芝多糖对混合淋巴细胞培养(MLC)反应的影响;观察对刀豆蛋白A (Con A)、细菌脂多糖(LPS)诱导淋巴细胞增殖的影响以及对环孢素A (CsA)、丝裂霉素C(Mit C)、足叶乙苷(VP-16) 等抑制MLC反应的影响.结果当质量浓度为0.2~12.8 mg*L-1时,两种灵芝多糖均可促进MLC反应,增强Con A或LPS诱导的淋巴细胞增殖,并拮抗CsA, Mit C或VP-16对MLC反应的抑制作用.未发现两种多糖之间有显著性差异.结论 Gl-PS-WC及Gl-PS-BC对体外培养脾淋巴细胞的增殖活性有类似作用.%Aim To compare the influences of wood-cultured Ganoderma lucidum polysaccharides (Gl-PS-WC) and bag-cultured Ganoderma lucidum polysaccharides (Gl-PS-BC) on the proliferation activities of murine spleen lymphocytes in vitro, and investigate whether Gl-PS-BC can be substituted for Gl-PS-WC. Methods Mixed lymphocyte culture (MLC) reaction, lymphocyte proliferation induced by concanavalin A (Con A, 1 mg*L-1) or lipopolysaccharide (LPS, 5 mg*L-1), MLC reactions inhibited by immunosuppressive drugs, cyclosporine A (CsA, 0.1 mg*L-1), mitomycin (Mit C, 0.1 mg*L-1), or antitumor drug, etoposide (VP-16, 0.1 mg*L-1), were detected in the presence or absence of Gl-PS-WC and Gl-PS-BC in the concentration range of 0.2-12.8 mg*L-1. Results Two kinds of polysaccharides were shown to promote MLC in the range of 0.2-12.8 mg*L-1, increase lymphocyte proliferation induced by Con A or LPS and antagonize the inhibitory effects of CsA, Mit C or VP-16 on MLC. No significant difference was observed between these two kinds of polysaccharides in selected concentrations. Conclusion Gl-PS-WC and Gl-PS-BC showed similar effects

  18. Study on psoriatic peripheral blood T-lymphocytes:effects of CD147-targeting small interfering RNA on the expression of CD147 and on cell proliferation and activation%CD147 siRNA转染对银屑病患者外周血T淋巴细胞CD147的表达及细胞增殖、活化的影响

    Institute of Scientific and Technical Information of China (English)

    常静; 陈翔; 粟娟; 匡叶红; 鲁慧; 彭胜

    2008-01-01

    Objective To investigate the effects of small interfering RNA(siRNA)on the expression of CD147 on peripheral blood T-lymphocytes from patients with psoriasis vulgaris,and its effect on the proliferation and activation of these cells.Methods Peripheral blood monouclear cells(PBMC)were obtained from 10 patients with psoriasis vulgaris,and T lymphocytes were isolated.CD147 siRNA was chemically synthesized,then electroporated into the peripheral T-lymphocytes.Untransfected cells,blank-transfected cells and unspecifically transfected cells served as the control.After 24-,48-,72-and 96-hour incubation,RT-PCR was used to detect the mRNA expression of CD147 in these cells.MTT assay and flow cytometry were utilized to assess the proliferation of these cellas,and the expression Of CD25 at 24,48,and 72 hours after the transfection.Results Compared with untransfected cells,the mRNA expression of CD147 declined significantly in CD147 siRNA-transfected cells at 24 hours(P<0.05),reached to the minimum at 48 hours (P<0.01):there was no significant difierence in the expression of CD147 between the two groups of cells at 96 hours after the transfection(P>0.05).There was a decrease of cell proliferation level by(44.5±3.13)%,(50.7±3.5)%and(53.98±4.15)%in CD147 siRNA-transfected cells 24,48 and 72 hours following the transfection,respectively;the corresponding decrease in blank-transfected cells was (37.28±3.56)%,(33.73±3.29)%,and(28.80±1.49)%,respectively,and that in unspecifically transfected cells,(31.29±2.46)%,(36.1±2.62)%and(32.08±2.78)%,respectively.A significant decrease was observed in the proliferation of CD147 siRNA-transfected cells compared with that of blank-transfected cells and unspecifically transfected cells at these three time points(P<0.05,0.01,0.01 respectively).The expression rate of CD25 at 24,48 and 72 hours was(47.23±3.65)%,(31.50±4.22)%and(23.05±4.15)%,respectively,on CD147 siRNA-transfected cells,and,(80.2±4.8)%,(81.6±3.35)%and(83.5±4

  19. 人FasL基因转染成熟树突状细胞对T淋巴细胞增殖和凋亡的影响%Dendritic Cells Genetically Engineered to Express Fas Ligand Regulate T Lymphocyte Proliferation and Apoptosis

    Institute of Scientific and Technical Information of China (English)

    周浩; 王芳; 王毅; 廖端芳; 陈专华; 邢增术; 王刚; 罗志刚; 秦国庆; 李建军; 钱坤; 蒋鸿涛

    2011-01-01

    To investigate the effects of dendritic cells on T lymphocyte proliferation and apoptosis, providing an in vitro model of clinical transplant immunological tolerance. After mature dendritic cells (mDCs) from peripheral blood of healthy adults was successfully transfected with human FasL gene, mDCs were analyzed for the expression of cell surface molecules, antigen presenting function and their apoptosis. Effects of nDCs on T lymphocyte proliferation and apoptosis were further detected based on co-culture of mDCs and T lymphocytes.The results show that, FasL did not significantly change the expression level of mlC's surface molecules CD40,CD80, CD86 and HLA-DR; FasL did not induce apoptosis of mDC. No effects on the antigen presenting function of mDC were observed as well. The miC transfected with FasL decreased stimulation index and increased apoptosis of allogeneic T-lymphocyte significantly. So that, human mDCs transfected with FasL may regulate T lymphocyte proliferation and apoptosis without alteration of cell surface molecules and antigen presentation characteristics on human mDC.%探讨转染人FasL基因的成熟树突状细胞(DC)对异体T淋巴细胞增殖和凋亡的影响,为实现临床器官移植免疫耐受提供初步实验依据.从健康成年人外周静脉血中获得成熟树突状细胞.将人FasL基因成功转染成熟树突状细胞,检测其表面分子的表达和自身凋亡情况,并对其抗原递呈功能进行分析.从异体健康成人外周血中获取T淋巴细胞,将转染成功的树突状细胞与T淋巴细胞混合培养,检测其对T淋巴细胞增殖和凋亡的影响.结果表明:人FasL基因转染没有明显影响成熟树突状细胞表面分子CD40、CD80、CD86和HLA-DR的表达;没有诱导树突状细胞自身发生凋亡;没有影响DC的抗原递呈功能.转染FasL基因后的树突状细胞使异体T淋巴细胞刺激指数明显下降,凋亡增加.因此认为,人FasL,基因转染对成熟树突状细胞的

  20. Piperine blocks interleukin-2-driven cell cycle progression in CTLL-2 T lymphocytes by inhibiting multiple signal transduction pathways.

    Science.gov (United States)

    Doucette, Carolyn D; Greenshields, Anna L; Liwski, Robert S; Hoskin, David W

    2015-04-02

    Piperine, a pungent alkaloid found in the fruits of black pepper plants, has diverse physiological effects, including the ability to inhibit immune cell-mediated inflammation. Since the cytokine interleukin-2 (IL-2) is essential for the clonal expansion and differentiation of T lymphocytes, we investigated the effect of piperine on IL-2 signaling in IL-2-dependent mouse CTLL-2 T lymphocytes. Tritiated-thymidine incorporation assays and flow cytometric analysis of Oregon Green 488-stained cells showed that piperine inhibited IL-2-driven T lymphocyte proliferation; however, piperine did not cause T lymphocytes to die or decrease their expression of the high affinity IL-2 receptor, as determined by flow cytometry. Western blot analysis showed that piperine blocked the IL-2-induced phosphorylation of signal transducer and activator of transcription (STAT) 3 and STAT5 without affecting the upstream phosphorylation of Janus kinase (JAK) 1 and JAK3. In addition, piperine inhibited the IL-2-induced phosphorylation of extracellular signal-regulated kinase 1/2 and Akt, which are signaling molecules that regulate cell cycle progression. Piperine also suppressed the expression of cyclin-dependent kinase (Cdk) 1, Cdk4, Cdk6, cyclin B, cyclin D2, and Cdc25c protein phosphatase by IL-2-stimulated T lymphocytes, indicating G0/G1 and G2/M cell cycle arrest. Piperine-mediated inhibition of IL-2 signaling and cell cycle progression in CTLL-2 T lymphocytes suggests that piperine should be further investigated in animal models as a possible natural source treatment for T lymphocyte-mediated transplant rejection and autoimmune disease.

  1. Th1 type lymphocyte reactivity to metals in patients with total hip arthroplasty

    Directory of Open Access Journals (Sweden)

    Finnegan Alison

    2008-02-01

    Full Text Available Abstract Background All prostheses with metallic components release metal debris that can potentially activate the immune system. However, implant-related metal hyper-reactivity has not been well characterized. In this study, we hypothesized that adaptive immunity reaction(s, particularly T-helper type 1 (Th1 responses, will be dominant in any metal-reactivity responses of patients with total joint replacements (TJAs. We tested this hypothesis by evaluating lymphocyte reactivity to metal "ions" in subjects with and without total hip replacements, using proliferation assays and cytokine analysis. Methods Lymphocytes from young healthy individuals without an implant or a history of metal allergy (Group 1: n = 8 were used to assess lymphocyte responses to metal challenge agents. In addition, individuals (Group 2: n = 15 with well functioning total hip arthroplasties (average Harris Hip Score = 91, average time in-situ 158 months were studied. Age matched controls with no implants were also used for comparison (Group 3, n = 8, 4 male, 4 female average age 70, range 49–80. Group 1 subjects' lymphocyte proliferation response to Aluminum+3, Cobalt+2, Chromium+3, Copper+2, Iron+3, Molybdenum+5, Manganeese+2, Nickel+2, Vanadium+3 and Sodium+2 chloride solutions at a variety of concentrations (0.0, 0.05, 0.1, 0.5, 1.0 and 10.0 mM was studied to establish toxicity thresholds. Mononuclear cells from Group 2 and 3 subjects were challenged with 0.1 mM CrCl3, 0.1 mM NiCl2, 0.1 mM CoCl2 and approx. 0.001 mM titanium and the reactions measured with proliferation assays and cytokine analysis to determine T-cell subtype prominence. Results Primary lymphocytes from patients with well functioning total hip replacements demonstrated a higher incidence and greater magnitude of reactivity to chromium than young healthy controls (p 2 fold stimulation index response, p 10 mM. The differential secretion of signature T-cell subsets' cytokines (Th1 and Th2 lymphocytes

  2. Enhancement of human natural cytotoxicity by Plasmodium falciparum antigen activated lymphocytes

    DEFF Research Database (Denmark)

    Theander, T G; Pedersen, B K; Bygbjerg, I C

    1987-01-01

    Mononuclear cells (MNC) isolated from malaria immune donors and from donors never exposed to malaria were stimulated in vitro with soluble purified Plasmodium falciparum antigens (SPag) or PPD. After 7 days of culture the proliferative response and the cytotoxic activity against the natural killer...... cell (NK cell) sensitive cell line, K562, were measured. It was found that SPag stimulation enhanced cytotoxic activity of MNC from donors whose lymphocytes exhibited a strong proliferative response to the antigen. MNC with low proliferative responsiveness showed increased cytotoxic activity if the MNC...... were preincubated with interleukin 2 (IL-2) for one hour before the start of the cytotoxic assay. SPag activation did not enhance the cytotoxic activity of MNC which did not respond to the antigen in the proliferation assay, and preincubation of these cells with IL-2 did not increase the activity. PPD...

  3. Anti-inflammatory effects of novel barbituric acid derivatives in T lymphocytes.

    Science.gov (United States)

    Xu, Chenjia; Wyman, Arlene R; Alaamery, Manal A; Argueta, Shannon A; Ivey, F Douglas; Meyers, John A; Lerner, Adam; Burdo, Tricia H; Connolly, Timothy; Hoffman, Charles S; Chiles, Thomas C

    2016-09-01

    We have used a high throughput small molecule screen, using a fission yeast-based assay, to identify novel phosphodiesterase 7 (PDE7) inhibitors. One of the most effective hit compounds was BC12, a barbituric acid-based molecule that exhibits unusually potent immunosuppressive and immunomodulatory actions on T lymphocyte function, including inhibition of T cell proliferation and IL-2 cytokine production. BC12 treatment confers a >95% inhibition of IL-2 secretion in phytohaemagglutinin (PHA) plus phorbol-12-myristate-13-acetate (PMA) stimulated Jurkat T cells. The effect of BC12 on IL-2 secretion is not due to decreased cell viability; rather, BC12 blocks up-regulation of IL-2 transcription in activated T cells. BC12 also inhibits IL-2 secretion in human peripheral T lymphocytes stimulated in response to CD3/CD28 co-ligation or the combination of PMA and ionomycin, as well as the proliferation of primary murine T cells stimulated with PMA and ionomycin. A BC12 analog that lacks PDE7 inhibitory activity (BC12-4) displays similar biological activity, suggesting that BC12 does not act via PDE7 inhibition. To investigate the mechanism of inhibition of IL-2 production by BC12, we performed microarray analyses using unstimulated and stimulated Jurkat T cells in the presence or absence of BC12 or BC12-4. Our studies show these compounds affect the transcriptional response to stimulation and act via one or more shared targets to produce both anti-inflammatory and pro-stress effects. These results demonstrate potent immunomodulatory activity for BC12 and BC12-4 in T lymphocytes and suggest a potential clinical use as an immunotherapeutic to treat T lymphocyte-mediated diseases.

  4. The Related Glycoside Monomer of Liuwei Dihuang Decoction and Proliferation of Splenic Lymphocytes in Mice%六味地黄汤相关苷类单体对小鼠脾淋巴细胞增殖作用的研究

    Institute of Scientific and Technical Information of China (English)

    刘继平; 兰洲; 傅强; 罗建光; 马世平; 孔令义

    2011-01-01

    . Results: Proliferative activities of cells that were treated with paeoniflorin (10-5M), loganin (10-7 ~10-5M) and morroniside (10-7 ~10-5 M) were promoted without ConA or LPS; LPS - induced proliferation of B lymphocytes was significantly promoted by paeoniflorin (10-5M), loganin (10-7M), morroniside (10-7 ~10-5M) and aucubin (10-5M); ConA - induced proliferation of T lymphocytes was markedly enhanced by paeoniflorin (10 -7 ~ 10-6 M) , morroniside (10~7 ~10-5M) and aucubin (10-7 ~10-3M), diosgenin played a certain role in promoting lymphocyte proliferation, but there was no significant difference. Conclusion; Paeoniflorin, diosgenin, loganin, morroniside and aucubin in Liuwei Dihuang decoction can enhance the proliferative activities of mice splenic lymphocytes to different degrees. The effect of morroniside on proliferative activity of lymphocytes is stronger than the effects of other compounds.

  5. Peripheral blood lymphocytes from low-grade squamous intraepithelial lesions patients recognize vaccine antigens in the presence of activated dendritic cells, and produced high levels of CD8 + IFNγ + T cells and low levels of IL-2 when induced to proliferate

    Directory of Open Access Journals (Sweden)

    Hernández-Montes Jorge

    2012-05-01

    Full Text Available Abstract Background Most infections with human papillomavirus (HPV are resolved without clinical intervention, but a minority evolves into chronic lesions of distinct grades, including cervical-uterine cancer. It is known that in most cases the immune system mediates elimination of HPV infection. However, the mechanism of immune evasion leading to HPV persistence and development of early cervical lesions is not fully understood. The aim of the present work was to evaluate the potential of peripheral blood leukocytes (PBL from low-grade squamous intraepithelial lesions (LSIL patients to be activated ex-vivo by vaccine antigens, the participation of cytotoxic lymphocytes and regulatory T cells, and to determine the secretion of Th1 and Th2 cytokines mediated by stimulation of T cell receptors. Results We found that PBL from LSIL patients showed a significantly lower proliferation rate to vaccine antigens as compared to that of healthy donors, even though there was not a difference in the presence of antibodies to those antigens in sera from both groups. We did not find differences in either the frequency of CD4 + CD25 + FoxP3+ in PBL, or the levels of IL-4, IL-5 and IL-10 in plasma or conditioned media from PBL incubated with TcR agonists in vitro, between the two groups. However, we detected a lower production of IL-2 and a higher proportion of CD8 + IFNγ + cells in PBL from LSIL patients as compared with PBL from normal donors. We also observed that PBL from patients infected by HPV-16 and −18 were not able to proliferate in the presence of soluble HPV antigens added to the culture; however, a high level of proliferation was attained when these antigens were presented by activated dendritic cells. Conclusions Our results suggest that the immunodeficiency reported in LSIL patients could be due to the inability of specific cytotoxic T lymphocytes that for some unknown reason are present but unable to mount a response when

  6. Activation of Signal Transducer and Activator of Transcription 5 (STAT5) in Splenocyte Proliferation of Asthma Mice Induced by Ovalbumin

    Institute of Scientific and Technical Information of China (English)

    GuopingLi; ZhigangLiu; PeixingRan; JingQiu; NanshanZhong

    2004-01-01

    To investigate the role of signal transducer and transcriptional activator 5 (STAT5) activated in ovalbumin (OVA)-induced splenocyte proliferation of asthma mice, an asthma mouse model was set up by intraperitoneal injection and aspiration of OVA with nebulizer. The proliferation of splenocytes isolated from the asthma mice was detected by [3H] thymidine incorporation. The phosphorytation of STAT5 was examined by Western blotting and STAT5-DNA binding was measured by electrophoretic mobility shift assay (EMSA). OVA could pronouncedly induce the splenocyte proliferation of asthma mice in a dose-dependent manner compared with control groups. Phosphorylation of STAT5 and STAT5-DNA binding were observed in splenocytes from asthma mice induced by OVA at 1 h and 3 h. These results indicated that STAT5 signal pathway played an important role in lymphocyte proliferation of asthma mice induced by OVA. Cellular & Molecular Immunology.2004;1(6):471-474.

  7. The Inhibitory Role of Lactacystin and β-Lactacystin on T-cell Activation and Proliferation

    Institute of Scientific and Technical Information of China (English)

    Peng-Hong SONG; Hai-Yang XIE; Shu-Sen ZHENG; Jian WU

    2004-01-01

    To evaluate the effects of proteasome inhibitors lactacystin (LAC) and β-lactacystin (β-LAC)on the proliferation and activation of T lymphocytes, flow cytometry was used to analyze the proliferationand the expression of CD69, CD25 and CD3 of T lymphocytes activated by PHA. Furthermore, theexpressions of PA28 and IL-2 mRNA were assayed by competitive RT-PCR. The results indicated that:(1) LAC and β-LAC significantly decreased the incorporation of BrdU and inhibited T lymphocytesproliferation in T lymphocytes activated by PHA; (2) although LAC and β-LAC did not affect the expressionof CD69 at any time, they significantly inhibited the expression of CD25 (48h, 72h, P<0.05);(3) in comparison with control, LAC and β-LAC significantly down-regulated the expression of PA28and IL-2 mRNA (48h, 72h, P<0.05). LAC and β-LAC significantly inhibited the proliferation and activationof T cells. Mechanisms involved are inhibition of CD25 and down-regulation of PA28 and IL-2 mRNAexpressions.

  8. The Inhibitory Role of Lactacystin and β-Lactacystinon T-cell Activation and Proliferation

    Institute of Scientific and Technical Information of China (English)

    Peng-HongSONG; Hai-YangXIE; Shu-SenZHENG; JianWU

    2004-01-01

    To evaluate the effects of proteasome inhibitors lactacystin (LAC) and β-1actacystin (β-LAC)on the proliferation and activation of T lymphocytes, flow cytometry was used to analyze the proliferationand the expression of CD69, CD25 and CD3 of T lymphocytes activated by PHA. Furthermore, theexpressions of PA28 and IL-2 mRNA were assayed by competitive RT-PCR. The results indicated that:(1) LAC and 13-LAC significantly decreased the incomoration of BrdU and inhibited T lymohocytesoroliferation in T lymphocytes activated by PHA; (2) although LAC and β-LAC did not affect the expressionof CD69 at any time, they significantly inhibited the expression of CD25 (48 h, 72 h, P<0.05);(3) in comoarison with control, LAC and β-LAC significantly down-regulated the expression of PA28and 1L-2 mRNA (48 h, 72 h, P<0.05). LAC and β-LAC significantly inhibited the proliferation and activationof T cells. Mechanisms involved are inhibition of CD25 and down-regulation of PA28 and IL-2 mRNAexpressions.

  9. Ofatumumab, Pentostatin, and Cyclophosphamide in Treating Patients With Untreated Chronic Lymphocytic Leukemia or Small Lymphocytic Lymphoma

    Science.gov (United States)

    2014-10-30

    Hematopoietic/Lymphoid Cancer; B-cell Chronic Lymphocytic Leukemia; Contiguous Stage II Small Lymphocytic Lymphoma; Noncontiguous Stage II Small Lymphocytic Lymphoma; Stage 0 Chronic Lymphocytic Leukemia; Stage I Chronic Lymphocytic Leukemia; Stage I Small Lymphocytic Lymphoma; Stage II Chronic Lymphocytic Leukemia; Stage III Chronic Lymphocytic Leukemia; Stage III Small Lymphocytic Lymphoma; Stage IV Chronic Lymphocytic Leukemia; Stage IV Small Lymphocytic Lymphoma

  10. Expression of B7-H4 gene from mouse in eukaryofic system and its suppressive effect on proliferation of lymphocytes cell%小鼠B7-H4基因的真核表达及其对淋巴细胞增殖的影响

    Institute of Scientific and Technical Information of China (English)

    李丽; 胡为民; 王朝莉; 杨致邦

    2012-01-01

    Objective To provide subjects for lucubrating the effect of B7-H4 on T cell activation and graft rejective reaction through cloning and constructing the eukaryotic expression vector encoding the gene of extracellular region of B7-H4 from mouse, and investigate the effect of B7-H4 on the proliferation of lymphocytes in vitro. Method The total RNAs of mouse lung and spleen were extracted and cDNA was transcribed from RNA using RT-PCR technique. The gene of extracellular region nf B7-H4 was amplified according to the template of cDNA by PCR. The amplified cDNA was imported into pGEM-T Easy vector to construct TA-mB7-H4 plasmid. The plasmid was cut by restriction enzyme of XbaⅠ and HindⅢ and was identified by the agarose gel electrophoresis and sequence scanning. Then the mB7-H4 corroborated by sequencing was inserted into the fluorescence expression vector MYC-HIS-EGFP-N after cut by the restriction enzymes to construct B7-H4-EGFP and control-EGFP eukaryotic expression vectors. The recombinant plasmids were transfected into JM109 competence bacteria, and were extracted and identified by the agarose gel electrophoresis and the sequencing after cut with the restriction enzymes. They were transfect-ed into CHO cell through lipofectamine? 2000, and the CHO cell lines expressing stably the fusion protein were obtained through G418 selection. MTT colorimetry was used to assess the effect of B7-H4 on the proliferation of lymphocyte in the culture of lymphocyte from BALB/c or C57 mouse respectively and in co-culture of lymphocyte from both BALB/c and C57 mouse. Result The gene sequences of B7-H4 cDNA cloned from mouse and TA-mB7-H4 constructed were correct by sequencing. The transfective CHO cells slahly expressed the recombinant transmembrane B7-H4 protein. The B7-H4 protein suppressed the lymphocyte proliferation either in the culture of lymphocyte respectively and in co-culture of lymphocyte from BALB/c and C57 mouse. Conclusion The B7-H4 eukaryotic expression vector

  11. Phytochemical screening of the dichloromethane–ethanolic extract of Eriosema campestre var. macrophylum roots and its antiproliferative effect on human peripheral blood lymphocytes

    Directory of Open Access Journals (Sweden)

    Michaelle G. Santos

    Full Text Available ABSTRACT Eriosema campestre var. macrophylum (Grear Fortunato, Fabaceae, is a native plant of the Brazilian Cerrado and the decoction of its roots has been used by folk medicine for the therapy of inflammatory diseases. In this study we aimed to investigate the effect of the dichloromethane–ethanolic extract of E. campestre roots on the proliferative response of lymphocytes and to examine the profile of IL-2 production. The effect of dichloromethane–ethanolic extract of E. campestre on the proliferation of phytohemagglutinin-stimulated lymphocytes was evaluated by using flow cytometry and the cell supernatants were assayed for IL-2 concentrations by using an enzyme-linked immunosorbent assay. The phytochemical screening of E. campestre roots was performed to determine the main secondary metabolites through chromogenic and precipitation reactions and by using HPLC-PAD. In addition to the presence of subclasses of flavonoids (flavones and flavonols in dichloromethane–ethanolic extract of E. campestre, we observed that the extract induced a concentration-dependent decrease in IL-2 levels on the supernatant of the cell cultures as well as an antiproliferative effect on T lymphocytes, including CD4+ and CD8+ cells. The anti-inflammatory effects attributed to E. campestre by folk medicine may partly be explained by its antiproliferative action on T lymphocytes.

  12. Determination of cell proliferation of peripheral blood lymphocytes from sheep by different assays%两种方法测定羊外周血淋巴细胞增殖的研究

    Institute of Scientific and Technical Information of China (English)

    张为宇; 黄维义; Chauvin Alain

    2003-01-01

    本试验比较3H胸腺嘧啶核苷(3H-TdR)渗入法和BrdU-ELISA方法测定羊外周血淋巴细胞增殖.分离健康羊外周血淋巴细胞,与不同浓度的非特异刺激因子刀豆素A(concanavalinA,1.0,0.5,0.25,0.125,0.0625,0.0313,0.0156,0.0078,0.0039 μg/孔)培养,分别用3H胸腺嘧啶核苷(3H-TdR)和5-溴-2′-脱氧尿苷(BrdU)标记细胞,测定刺激细胞和对照细胞的每分钟脉冲数cpm或光吸收值OD,计算刺激指数SI和光吸收值差ΔOD,统计学方法分析SI和ΔOD的相关程度.同样的方法测定羊试验感染肝片吸虫(Fasciola hepatica)后,在感染早期阶段(PIW0-PIW4)其外周血淋巴细胞在特异性抗原-片形吸虫分泌排泄产物(ESP,5 μg/孔)刺激下的增殖.结果表明,刺激指数SI和光吸收值差ΔOD在两个试验中的相关系数分别为0.946和0.924,SI和*OD均呈强正线性相关.BrdU-ELISA方法无同位素放射物,且敏感、快速、方便,和3H-TdR渗入法一样可以用于测定淋巴细胞增殖.

  13. Detection of TP53 dysfunction in chronic lymphocytic leukemia by an in vitro functional assay based on TP53 activation by the non-genotoxic drug Nutlin-3: a proposal for clinical application

    OpenAIRE

    Pozzo, Federico; Dal Bo, Michele; Peragine, Nadia; Bomben, Riccardo; Zucchetto, Antonella; Rossi, Francesca Maria; Degan, Massimo; Rossi, Davide; Chiarenza, Annalisa; Grossi, Alberto; Di Raimondo, Francesco; Zaja, Francesco; Pozzato, Gabriele; Secchiero, Paola; Gaidano, Gianluca

    2013-01-01

    Background TP53 defects, i.e. 17p13 deletion and/or nucleotide mutations, associate with short survival and chemorefractoriness in chronic lymphocytic leukemia (CLL). In this context, since direct sequencing of the TP53 gene does not evaluate TP53 functionality, a functional assessment of TP53 pathway may be of interest to identify high risk CLL. By taking advantage of a training cohort of 100 CLL and a validation cohort of 40 CLL with different patterns of TP53 mutation/deletion by FISH and ...

  14. T lymphocytes and NK cells in X-linked agammaglobulinemia.

    Science.gov (United States)

    Pituch-Noworolska, Anna; Zwonarz, Katarzyna; Błaut-Szlósarczyk, Anita; Szaflarska, Anna; Kowalczyk, Danuta; Siedlar, Maciej

    2013-01-01

    Seven boys with diagnosis of X-linked agammaglobulinemia on regular substitution of immunoglobulins were included into study. The patients showed episodes of infections but the clinical course was mild with good response to antibiotics. All patients developed, with time, the chronic sinusitis with proliferation of mucous membrane, two patients showed bronchiectases. The number of T lymphocytes, ratio of CD4:CD8 subpopulations, response to stimulation and NK number were assayed with flow cytometry and cell culture. Results showed CD4:CD8 ratio within normal value in majority of patients, reverse ratio in 2 boys, increased number of activated T cells (CD3/HLA-DR) in one of them. The number of NK cells was different from lack of these cells to high number. Response of T cells to stimulation (mitogens and CD3) were normal in majority of assays. There were no associations between clinical course and observed changes in T or NK cell populations. Further studies on number and function of NK cells are needed.

  15. Aryl hydrocarbon mono-oxygenase activity in human lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Griffin, G.D.; Schuresko, D.D.

    1981-06-01

    Aryl hydrocarbon mono-oxygenase (AHM), an enzyme of key importance in metabolism of xenobiotic chemicals such as polynuclear aromatic hydrocarbons (PNA), is present in human lymphocytes. Studies investing the relation of activity of AHM in human lymphocytes to parameters such as disease state, PNA exposure, in vitro mitogen stimulation, etc. have been summarized in this report. Some studies have demonstrated increased AHM activity in lymphocytes from cigarette smokers (compared to nonsmokers), and in lung cancer patients when compared to appropriate control groups. These observations are confused by extreme variability in human lymphocyte AHM activities, such variability arising from factors such as genetic variation in AHM activity, variation in in vitro culture conditions which affect AHM activity, and the problematical relationship of common AHM assays to actual PNA metabolism taking place in lymphocytes. If some of the foregoing problems can be adequately addressed, lymphocyte AHM activity could hold the promise of being a useful biomarker system for human PNA exposure.

  16. GABA, a natural immunomodulator of T lymphocytes

    DEFF Research Database (Denmark)

    Bjurstöm, Helen; Wang, Junyang; Ericsson, Ida

    2008-01-01

    gamma-aminobutyric acid (GABA) is the main neuroinhibitory transmitter in the brain. Here we show that GABA in the extracellular space may affect the fate of pathogenic T lymphocytes entering the brain. We examined in encephalitogenic T cells if they expressed functional GABA channels that could......M and higher GABA concentrations decreased T cell proliferation. The results are consistent with GABA being immunomodulatory....

  17. A new flow cytometric method for quantitative assessment of lymphocyte mitogenic potentials.

    Science.gov (United States)

    Yamamura, Y; Rodriguez, N; Schwartz, A; Eylar, E; Bagwell, B; Yano, N

    1995-01-01

    A new flow cytometric method was developed to quantitatively assess lymphocyte proliferation simultaneously for different subsets. The cells were stained with a fluorescent dye, PKH-26 and were stimulated with mitogens. The fluorescence intensities (FL2) of proliferating cells were measured by flow cytometry; and each subset was identified by the use of a monoclonal antibody (Mab)-fluorescein-isothiocyanate (FITC) (FL1). FL2 histograms were then analyzed by the cell proliferation model based on the ModFit software (Verity). This new method revealed information which could not be obtained by conventional mitogen assays. For example, the CD4+ and the CD4- T-subsets responded to phytohemagglutinin (PHA) quite differently from each other and it was indicated that activation of one population could significantly alter the response of the other. In addition, even within a subset, all activated cells did not proliferate uniformly. Some cells divided only once while others underwent further cellular division during the same time period. The method is, therefore, invaluable for studying the nature and the extent of interactions between different cellular subsets within a culture.

  18. Transfer of cholesterol from macrophages to lymphocytes in culture.

    Science.gov (United States)

    de Bittencourt Júnior, P I; Curi, R

    1998-02-01

    A major feature of macrophage metabolism is its capacity to produce and export cholesterol. Several reports have shown that the manipulation of lymphocyte cholesterol content elicits important changes in lymphocyte proliferation. These findings lead to an inquiry as to whether macrophage-derived cholesterol released into the lymphocyte surroundings may be transferred to the latter thus affecting lymphocyte function. In this study, cholesterol transfer from macrophages to lymphocytes was examined in vitro using rat cells in culture. The findings indicate that there may be a significant transfer of cholesterol from [4-14C]cholesterol labeled resident peritoneal macrophages to mesenteric lymph node resting lymphocytes (up to 173.9 +/- 2.7 pmol/10(7) lymphocytes/10(7) macrophages when co-cultivated for 48 h), in a lipoprotein-dependent manner. This represents the mass transfer of ca. 17 nmoles of cholesterol molecules per 10(7) lymphocytes from 10(7) macrophages (calculated on the basis of specific radioactivity incorporated into macrophages after the pre-labelling period), which suggests that macrophages are capable of replacing the whole lymphocyte cholesterol pool every 21 h. Moreover, an 111%-increase in the total cholesterol content of lymphocytes was found after co-cultivation with macrophages for 48 h. When compared to peritoneal cells, monocytes/macrophages obtained from circulating blood leukocytes presented a much higher cholesterol transfer capacity to lymphocytes (3.06 +/- 0.10 nmol/10(7) lymphocytes/10(7) macrophages co-cultivated for 24 h). Interestingly, inflammatory macrophages dramatically reduced their cholesterol transfer ability (by up to 91%, as compared to resident macrophages). Cholesterol transfer may involve a humoral influence, since it is not only observed when cells are co-cultivated in a single-well chamber system (cells in direct contact), but also in a two-compartment system (where cells can communicate but not by direct contact). Co

  19. Use of a soluble tetrazolium/formazan assay for chicken cells.

    Science.gov (United States)

    Tajima, T; Hironao, T; Kajikawa, T; Kawamura, H

    1992-12-01

    We evaluated a soluble tetrazolium/formazan assay using 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl ]-2H- tetrazolium hydroxide (XTT) for chicken cell growth. Fifty microliter of solution containing 1 mg/ml of XTT and 0.025 mM phenazine methosulfate was added to the cells in a well of 96-well microplate. After 4 hr incubation at 37 degrees C, the absorbance was measured at 490 nm. Under this condition, absorbances were well correlated with cell number of Marek's disease tumor cells and chicken embryo fibroblasts. Proliferation of chicken lymphocytes stimulated with mitogens was also effectively measured. The formazan of XTT is water-soluble and can be quantitated in culture medium without the necessity for extraction with organic solvents. Thus XTT assay is simple and useful for the quantity assay with chicken cells.

  20. BSAP/Pax-5在B淋巴细胞发育、增殖、分化中的作用%Effect of BSAP/Pax-5 on the Development, Proliferation and Differentiation of B Lymphocyte Cell

    Institute of Scientific and Technical Information of China (English)

    谭余庆; 张永祥

    2001-01-01

    BSAP, a B cell lineage-specific activator protein, is a nucleus transcription factor and is en coded by the Pax-5 gene. It is a critical modulator of B cell development, proliferation and differentia tion. BSAP also influences B cell immunoglobulin secretion at later stages of B cell differentiation.%BSAp,一个B细胞特异性激活蛋白,由Pax-5转录的核蛋白。作为核转录因子,其在B细胞的发育、增殖和分化中起重要作用。同时也影响B细胞分化晚期的Ig的分泌。

  1. Fluoride Stimulates the Proliferation of Osteoclasts in vitro by Upregulating MCM3

    Directory of Open Access Journals (Sweden)

    Shengbin Bai

    2016-06-01

    Full Text Available We have previously shown that the expression of the minichromosome maintenance protein 3 (MCM3 gene was upregulated in lymphocytes of patients with skeletal fluorosis. We speculated that increased MCM3 expression may be contribute to osteopathy in patients with skeletal fluorosis. Here, we investigated the effect of fluoride on the proliferation of osteoclasts derived from RAW264.7 cells and the involvement of MCM3. Our MTT assays showed that 0.25 mM NaF markedly stimulated the proliferation of RAW264.7 cells. The RT-PCR and immunoblotting assays revealed that 0.25 mM NaF upregulated MCM3 expression in RAW264.7 cells. The MTT assays additionally demonstrated that stimulation with MCM3 potentiated the effect of fluorine on the proliferation of RAW264.7 cells. These results demonstrated that fluoride at clinical relevant concentration upregulates MCM3 expression in osteoclasts in vitro. We are currently conducting a series of experiments to examine whether increased MCM3 in osteoclasts indeed contributes to osteopathy in skeletal fluorosis.

  2. The changes of T lymphocytes and cytokines in ICR mice fed with Fe3O4 magnetic nanoparticles

    Directory of Open Access Journals (Sweden)

    Wang J

    2011-04-01

    Full Text Available Jun Wang1, Baoan Chen1, Nan Jin1, Guohua Xia2, Yue Chen1, Ying Zhou1, Xiaohui Cai1,2, Jiahua Ding1, Xiaomao Li3, Xuemei Wang41Department of Hematology, Zhongda Hospital, 2Department of Medical Laboratory, Medical School, Southeast University, Nanjing, People's Republic of China; 3Department of Physics, University of Saarland, Saarbruecken, Germany; 4National Key Laboratory of Bioelectronics (Chien-Shiung Wu Laboratory, Southeast University, Nanjing, People’s Republic of ChinaAbstract: The aim of this article is to study the changes inhibited T lymphocytes and cytokines related to the cellular immunity in ICR (imprinting control region mice fed with Fe3O4 magnetic nanoparticles (Fe3O4-MNPs. The Fe3O4-MNPs were synthesized, and their characteristics such as particle size, zeta potential, and X-ray diffraction patterns were measured and determined. All ICR mice were sacrificed after being exposed to 0, 300, 600, and 1200 mg/kg of Fe3O4-MNPs by single gastric administration for 14 days. Splenocytes proliferation was indicated with stimulate index by MTT assay; release of cytokines in the serum of ICR mice was detected by enzyme-linked immunosorbent assay, and the phenotypic analyses of T-lymphocyte subsets were performed using flow cytometry. Our results indicated that there were no significant differences in splenocyte proliferation and release of cytokines between exposed and control groups. Furthermore, there was no significant difference in the proportions of T-lymphocyte subsets in the low-dose Fe3O4-MNPs group when compared to the control group, but the proportions of CD3+CD4+ and CD3+CD8+ T-lymphocyte subsets both in the medium- and high-dose Fe3O4-MNPs groups were higher than those in the control group. It is concluded that a high dose of Fe3O4-MNPs, to some extent, could influence in vivo immune function of normal ICR mice.Keywords: Fe3O4, magnetic nanoparticles, splenocyte proliferation, release of cytokines, T-lymphocyte subsets, ICR

  3. Bendamustine Plus Alemtuzumab for Refractory Chronic Lymphocytic Leukemia (CLL)

    Science.gov (United States)

    2013-08-20

    Recurrent Small Lymphocytic Lymphoma; Refractory Chronic Lymphocytic Leukemia; Stage III Chronic Lymphocytic Leukemia; Stage III Small Lymphocytic Lymphoma; Stage IV Chronic Lymphocytic Leukemia; Stage IV Small Lymphocytic Lymphoma

  4. What Is Chronic Lymphocytic Leukemia?

    Science.gov (United States)

    ... Chronic Lymphocytic Leukemia (CLL) About Chronic Lymphocytic Leukemia What Is Chronic Lymphocytic Leukemia? Cancer starts when cells ... body, including the lymph nodes, liver, and spleen. What is leukemia? Leukemia is a cancer that starts ...

  5. Alvocidib in Treating Patients With B-Cell Chronic Lymphocytic Leukemia or Small Lymphocytic Lymphoma

    Science.gov (United States)

    2013-07-01

    B-cell Chronic Lymphocytic Leukemia; Contiguous Stage II Small Lymphocytic Lymphoma; Noncontiguous Stage II Small Lymphocytic Lymphoma; Stage I Chronic Lymphocytic Leukemia; Stage I Small Lymphocytic Lymphoma; Stage II Chronic Lymphocytic Leukemia; Stage III Chronic Lymphocytic Leukemia; Stage III Small Lymphocytic Lymphoma; Stage IV Chronic Lymphocytic Leukemia; Stage IV Small Lymphocytic Lymphoma

  6. Does programmed CTL proliferation optimize virus control?

    DEFF Research Database (Denmark)

    Wodarz, Dominik; Thomsen, Allan Randrup

    2005-01-01

    CD8 T-cell or cytotoxic T-lymphocyte responses develop through an antigen-independent proliferation and differentiation program. This is in contrast to the previous thinking, which was that continuous antigenic stimulation was required. This Opinion discusses why nature has chosen the proliferati...

  7. Effect of Oxymatrine on CD4+CD25+ regulatory T cells and lymphocyte proliferation%氧化苦参碱对CD4+CD25+调节性T细胞(Tr)和淋巴细胞增殖的影响

    Institute of Scientific and Technical Information of China (English)

    李建国; 伍斌; 谢红付; 张江林; 易梅; 李吉

    2008-01-01

    目的 通过分析氧化苦参碱(oxymatrine,OMT)各剂量组对小鼠外周血调节性T细胞(regulatory Tcells,Tr细胞)数量的影响,同时检测OMT对刀豆蛋白A(ConA)刺激的小鼠淋巴结T细胞增殖的影响.探讨OMT治疗ACD的免疫学作用机制.以期为临床用OMT治疗变态反应性疾病提供更深入的理论和实验依据.方法 建立DNFB诱发的小鼠ACD模型.以不同荆量的OMT、PBS、氢化可的松(HCT)进行腹腔注射(ip),在实验进程的第1、7、14、21和28天小鼠尾静脉采血,抗-CD3、抗-CD4、抗-CD25单抗进行三色免疫荧光标记,流式细胞术检测各组CD4+CD25+T细胞数量.利用羧基荧光素乙酰乙酸(CFDA-SE)染色,流式细胞术检测OMT对小鼠淋巴结T细胞增殖的影响.实验数据以SPSS 10.0以及CELLQuest软件进行处理.结果 体外实验证明,在500、125和31μg/mL组的OMT对小鼠淋巴结T细胞增殖呈剂量依赖性抑制,而在16、8、4和2μg/mL组的OMT对小鼠淋巴结T细胞增殖起促进作用,但其剂量依赖关系不明显.腹腔注射OMT能明显提高小鼠外周血中CD4+CD25+T细胞的数量,与HCT组比较(P<0.01),与PBS组比较(P<0.01).结论 OMT对小鼠淋巴结T细胞的增殖呈双向作用;腹腔注射OMT能明显提高小鼠外周血中CD4+CD25+T细胞的数量;即OMT对免疫系统的影响呈双向作用,OMT是一种双向免疫调节剂.%[Objective]To analyse Oxymatrine's(OMT)effects on the quantity of mouse's regulatory T cells(Tr cells)in the peripheral blood and mouse lymphocyte proliferation stimulated by con A,probe into the immunological mechanism that OMT treats allergic contact dermatitis(ACD).[Methods]Building up an ACD mouse medel stimulated by Dinitrofluorobenzene(DNFB),taking intraperitoneal injection (IP)by different dosage8 of OMT,PBS and Hydrocortisone(HCT),collecting blood In 1 d,7 d,14d,21 d and 28 d via mouse tail vein,then marking the T cells with anti-CD3,anti-CD4,anti-CD25 three-colored immune fluorescence

  8. Effects of Sargassum polysaccharide on Proliferation and Oxidative Stress of Splenic Lymphocytes in Chicken%马尾藻多糖对鸡脾脏淋巴细胞增殖及氧化应激影响的实验观察

    Institute of Scientific and Technical Information of China (English)

    张玲; 胡庭俊; 陈炳全

    2011-01-01

    The isolation and purification of Sargassum polysaccharides was carried out and the physicochemical property of Sargassum poly-saccharide was analyzed. The effects of S. Polysaccharide on glutathione concentration, nitric oxide production and cell proliferation were investigated in vitro. The results showed that S. Polysaccharide was obtained with a yield of 152.8 g/L (w/w). S. Polysaccharide at concentration of 100, 200 or 400 mg/L could significantly enhance the lymphocyte proliferation (P <0. 05). Hydrogen peroxide at 50 junol/L could induce oxidative stress of splenic lymphocytes in chicken. S. Polysaccharides at 400 mg/L could increase glutathione level significantly when the incubation time were 4 hours or 12 hours (P<0.05) and could decrease the level of nitric oxide in splenic lymphocytes significantly in chicken (P <0.05) at 8 hour. From the findings, it could be concluded that S. Polysaccharides markedly enhance antioxidant activity and may have potential immunomodulatory effect in chicken.%马尾藻具有很高的食用和药用价值,分离提取马尾藻多糖(Sargassum polysaccharides,SP),对其理化性质进行分析;研究马尾藻多糖对氧化应激态鸡脾脏淋巴细胞内谷胱甘肽(GSH)和一氧化氮(NO)分泌水平及淋巴细胞体外增殖的影响.结果表明:马尾藻多糖提取率为152.8 g/L;马尾藻多糖浓度为100、200、400 mg/L时,能显著促进体外培养的鸡脾脏淋巴细胞增殖;H2O2浓度为50 μmol/L时,能诱导体外培养的鸡脾脏淋巴细胞处于氧化应激状态.马尾藻多糖浓度为400 mg/L,与细胞共孵育4h或12h,能显著增加氧化应激态细胞内GSH含量;共孵育培养8h,能显著降低氧化应激态细胞的NO水平.马尾藻多糖能促进鸡脾脏淋巴细胞增殖,并通过调节细胞内GSH和NO水平,提高免疫细胞的抗氧化能力,发挥免疫调节作用.

  9. Potential Anti-proliferative and Immunomodulatory Effects of Marine Microalgal Exopolysaccharide on Various Human Cancer Cells and Lymphocytes In Vitro.

    Science.gov (United States)

    Park, Geon-Tae; Go, Ryeo-Eun; Lee, Hae-Miru; Lee, Geum-A; Kim, Cho-Won; Seo, Jeong-Woo; Hong, Won-Kyung; Choi, Kyung-Chul; Hwang, Kyung-A

    2017-02-04

    Marine microalgal exopolysaccharides (EPSs) have drawn great attention due to their biotechnological potentials such as anti-viral, anti-oxidant, anti-lipidemic, anti-proliferative, and immunomodulatory activities, etc. In the present study, the EPS derived from microalgae Thraustochytriidae sp.-derived mutant GA was investigated for its anti-proliferation and immunomodulation. Anti-cancer efficacy of the microalgal EPS was examined for the alterations in cell proliferation and cell cycle-related gene expression that occur in three types of human cancer cell lines, BG-1 ovarian, MCF-7 breast, and SW-620 colon cancer cell lines, by its treatment. Alterations in immunoreactivity by the microalgal EPS were examined by measuring its influence on the growth of T and B lymphocytes and cytokine production of T cells. In cell viability assay, the microalgal EPS inhibited cancer cell growth at the lowest concentration of 10(-11) dilution and in a dose-responsive manner within the range of dilution of 10(-11)~10(-3). In addition, the protein expression of cell cycle progression genes such as cyclin D1 and E in these cancer cell lines was significantly reduced by the microalgal EPS in a dose- and a time-dependant manner. In cell proliferation assay using T and B cells, the microalgal EPS induced B cell proliferation even at the lowest dilution of 10(-11), but not T cells. In cytokine assay, the microalgal EPS decreased the formation of IL-6 and INF-γ at 10(-3) dilution compared to the control and had no significant effects on TNF-α. Collectively, these findings suggest that the EPS derived from microalgae Thraustochytriidae sp. GA has an anti-proliferative activity against cancer cells and an immunomodulatory effect by having an influence on B cell proliferation and cytokine secretion of T cells.

  10. Effect of antimalarial drugs on stimulation and interleukin 2 production of human lymphocytes

    DEFF Research Database (Denmark)

    Bygbjerg, I C; Svenson, M; Theander, T G

    1987-01-01

    Effect of pyrimethamine, an antimalarial antifolate, and of mefloquine, chloroquine, and quinine, which belong to the quinoline group of antimalarials, on proliferation and interleukin 2 (IL-2) production of human lymphocytes was studied in vitro. Pyrimethamine at concentrations above therapeutic...

  11. Sister chromatid exchange in human lymphocytes induced by propoxur following plant activation by Vicia faba.

    Science.gov (United States)

    Gómez-Arroyo, S; Calderón-Segura, M E; Villalobos-Pietrini, R

    1995-01-01

    Because the carbamate insecticide propoxur induced sister chromatid exchanges (SCE) in Vicia faba but was ineffective in producing SCE in lymphocytes in culture, it was hardly suspected that plant metabolism was involved. Experiments were conducted in which metabolic activation was afforded by Vicia faba roots, and SCE in human lymphocytes in vitro was used to assess cytogenetic damage. Several concentrations of propoxur (250, 500, 1,000, 1,500, and 2,000 ppm) were applied for 4 hr to the roots of Vicia faba. Extracts prepared from these treatments were added to the lymphocyte cultures and a significant increase of SCE frequencies with a concentration-response relationship could be detected. The lymphocyte proliferation kinetics and the proliferation rate index (PRI) were not affected (except in the highest concentration, of 2,000 ppm). This general behavior was in agreement with the presence of an enzymatic system (S10 fraction) in Vicia roots capable of metabolizing or activating the propoxur. With 2,000 ppm, cell necrosis was produced in Vicia; therefore, this extract did not induce SCE in lymphocytes. However, lymphocyte proliferation kinetics were delayed and PRI was significantly decreased. Ethanol, a promutagen activated by this plant, was applied directly to the lymphocyte cultures as a positive control, and the response was negative. On the other hand, the extracts of roots treated with ethanol increased the SCE to more than twice that of the negative control, but the lymphocyte proliferation kinetics and PRI were not affected.

  12. A novel assay for high-throughput screening of anti-Alzheimer's disease drugs to determine their efficacy by real-time monitoring of changes in PC12 cell proliferation.

    Science.gov (United States)

    Hou, Xue-Qin; Yan, Rong; Yang, Cong; Zhang, Lei; Su, Ru-Yu; Liu, Si-Jun; Zhang, Shi-Jie; He, Wen-Qing; Fang, Shu-Huan; Cheng, Shu-Yi; Su, Zi-Ren; Chen, Yun-Bo; Wang, Qi

    2014-03-01

    Alzheimer's disease (AD) is a neurodegenerative disease that is characterized by the accumulation of senile plaque and neurofibrilary tangle formation in the brain, including the cerebral cortex and hippocampus. Nowadays, the first-line treatment for AD is the application of acetylcholinesterase inhibitors. However, acetylcholinesterase inhibitors are basically anti-symptomatic for a limited aspect of AD pathology and are associated with serious side-effects. With the advantage of multiple targets, pathways and systems, Chinese herbal compounds hold promising potential for the development of drugs for the treatment of AD. Over the past few years, with the development of Chinese herbal compounds and in vitro pharmacological studies, cell-based disease models are one of the main methods used to screen Chinese herbal compounds for potential efficacy. Testing the efficacy of possible anti-Alzheimer's disease drugs and the development of new drugs are hindered by the lack of objective high-throughput screening methods. Currently, the assessment of the effects of drugs is usually made by MTT assays, involving laborious, subjective, low-throughput methods. Herein, we suggest a novel application for a real-time cell monitoring device (xCELLigence) that can simply and objectively assess the effective composition of Chinese herbal compounds by assessing amyloid-β peptide Aβ1-42-induced apoptosis in PC12 cells. We detected the proliferation and motility of the cells using a fully automated high-throughput and real-time system. We quantitatively assessed cell motility and determined the real-time IC50 values of various anti-AD drugs that intervene in several developmental stages of Aβ1-42-induced apoptosis in PC12 cells, Then, we identified the optimal time phase by curative efficacy. Our data indicate that this technique may aid in the discovery and development of novel anti-Alzheimer's disease drugs. It is possible to utilize a similar technique to measure changes in

  13. Toll-Like Receptor 7 Agonist Therapy with Imidazoquinoline Enhances Cancer Cell Death and Increases Lymphocytic Infiltration and Proinflammatory Cytokine Production in Established Tumors of a Renal Cell Carcinoma Mouse Model

    Directory of Open Access Journals (Sweden)

    Eric C. Kauffman

    2012-01-01

    Full Text Available Imidazoquinolines are synthetic toll-like receptor 7 and 8 agonists and potent dendritic cell activators with established anticancer activity. Here we test the hypothesis that imidazoquinoline has in vivo efficacy within established renal cell carcinoma (RCC tumors. Immunocompetent mice bearing syngeneic RCC xenografts were treated with imidazoquinoline or placebo at two separate time points. Harvested tumors were assayed by TUNEL/caspase-3/Ki67 immunostains to evaluate cell death/apoptosis/proliferation, and CD3/B220/CD45 immunostains to evaluate T-cell lymphocyte/B-cell lymphocyte/pan-leukocyte tumor infiltration. ELISA measurement of tumor and serum levels of proinflammatory cytokines, IL-6 and MCP-1, was performed. A single imidazoquinoline dose significantly decreased RCC tumor growth by 50% and repeat dosing compounded the effect, without observed weight loss or other toxicity. Tumor immunostaining revealed significant increases in cell death and apoptosis without changes in cell proliferation, supporting induction of apoptosis as the primary mechanism of tumor growth suppression. Imidazoquinoline treatment also significantly enhanced peritumoral aggregation and intratumoral infiltration by T-cell lymphocytes, while increasing intratumoral (but not serum levels of proinflammatory cytokines. In conclusion, imidazoquinoline treatment enhances T-cell lymphocyte infiltration and proinflammatory cytokine production within established mouse RCC tumors, while suppressing tumor growth via induction of cancer cell apoptosis. These findings support a therapeutic role for imidazoquinoline in RCC.

  14. Acute Lymphocytic Leukemia

    Science.gov (United States)

    ... for information in your local library and on the Internet. Good sources include the National Cancer Institute, the ... mayoclinic.org/diseases-conditions/acute-lymphocytic-leukemia/basics/definition/CON-20042915 . Mayo Clinic Footer Legal Conditions and ...

  15. The lymphocyte transformation test in allergic contact dermatitis: New opportunities.

    Science.gov (United States)

    Popple, Amy; Williams, Jason; Maxwell, Gavin; Gellatly, Nichola; Dearman, Rebecca J; Kimber, Ian

    2016-01-01

    Allergic contact dermatitis (ACD) is driven by the activation and proliferation of allergen-specific memory T-lymphocytes and is currently diagnosed by patch testing with a selected panel of chemical allergens. The lymphocyte transformation test (LTT) can be used to monitor ex vivo T-lymphocyte responses to antigens, including contact allergens. The LTT is not viewed as being an alternative to patch testing, but it does seek to reflect experimentally skin sensitization to specific chemicals. The LTT is based on stimulation in vitro of antigen-driven T-lymphocyte proliferation. That is, exposure in culture of primed memory T-lymphocytes to the relevant antigen delivered in an appropriate configuration will provoke a secondary response that reflects the acquisition of skin sensitization. The technical aspects of this test and the utility of the approach for investigation of immune responses to contact allergens in humans are reviewed here, with particular emphasis on further development and refinement of the protocol. An important potential application is that it may provide a basis for characterizing those aspects of T-lymphocyte responses to contact allergens that have the greatest influence on skin sensitizing potency and this will be considered in some detail.

  16. Genotoxic effects of the carbamate insecticide Pirimor-50® in Vicia faba root tip meristems and human lymphocyte culture after direct application and treatment with its metabolic extracts.

    Science.gov (United States)

    Valencia-Quintana, Rafael; Gómez-Arroyo, Sandra; Sánchez-Alarcón, Juana; Milić, Mirta; Olivares, José Luis Gómez; Waliszewski, Stefan M; Cortés-Eslava, Josefina; Villalobos-Pietrini, Rafael; Calderón-Segura, María Elena

    2016-12-01

    The aim of the study was to evaluate genotoxic effects of Pirimor-50®, a pirimicarb-based formulation (50 % active ingredient), in human lymphocyte cultures and Vicia faba root meristems. Furthermore, the objective was to examine a combined influence of insecticide treatment with mammalian microsomal S9 and vegetal S10 metabolic fractions or S10 mix metabolic transformation extracts (after Vicia faba primary roots treatment with Pirimor-50®). We used sister chromatid exchange assay-SCE and measured cell cycle progression and proliferation (proportion of M1-M3 metaphases and replication index ratio-RI). Two processes were used for plant promutagen activation: in vivo activation-Pirimor-50® was applied for 4 h to the plant and then S10 mix was added to lymphocytes; and, in vitro activation-lymphocytes were treated with Pirimor-50® and S10 or S9 for 2 h. Direct treatment induced significantly higher SCE frequencies in meristems at 0.01 mg mL-1. In lymphocytes, significantly higher SCE was at 1 mg mL-1 with decrease in RI and M1-M3 metaphase proportions at 0.5 mg mL-1 and cell division stop at 2.5 mg mL1. S10 mix lymphocyte treatment showed significantly elevated SCE values at 2-2.5 mg mL-1, with cell death at 3 mg mL-1. Lymphocyte treatment with Pirimor-50® together with S9 or S10 showed slightly elevated SCE frequency but had a significant influence on RI decrease, with lowest values in S9 treatment. Since no data are available on the genotoxicity of Pirimor-50®, this study is one of the first to evaluate and compare its direct effect in two bioassays, animal and vegetal, and also the effect of plant and animal metabolism on its genotoxic potential.

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

    Science.gov (United States)

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

    2010-01-01

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

  18. CTL ELISPOT assay.

    Science.gov (United States)

    Ranieri, Elena; Popescu, Iulia; Gigante, Margherita

    2014-01-01

    Enzyme-linked immune absorbent spot (Elispot) is a quantitative method for measuring relevant parameters of T cell activation. The sensitivity of Elispot allows the detection of low-frequency antigen-specific T cells that secrete cytokines and effector molecules, such as granzyme B and perforin. Cytotoxic T cell (CTL) studies have taken advantage with this high-throughput technology by providing insights into quantity and immune kinetics. Accuracy, sensitivity, reproducibility, and robustness of Elispot resulted in a wide range of applications in research as well as in diagnostic field. Actually, CTL monitoring by Elispot is a gold standard for the evaluation of antigen-specific T cell immunity in clinical trials and vaccine candidates where the ability to detect rare antigen-specific T cells is of relevance for immune diagnostic. The most utilized Elispot assay is the interferon-gamma (IFN-γ) test, a marker for CD8(+) CTL activation, but Elispot can also be used to distinguish different subsets of activated T cells by using other cytokines such as T-helper (Th) 1-type cells (characterized by the production of IFN-γ, IL-2, IL-6, IL-12, IL-21, and TNF-α), Th2 (producing cytokines like IL-4, IL-5, IL-10, and IL-13), and Th17 (IL-17) cells. The reliability of Elispot-generated data, by the evaluation of T cell frequency recognizing individual antigen/peptide, is the core of this method currently applied widely to investigate specific immune responses in cancer, infections, allergies, and autoimmune diseases. The Elispot assay is competing with other methods measuring single-cell cytokine production, e.g., intracellular cytokine by FACS or Miltenyi cytokine secretion assay. Other types of lymphocyte frequency and function assays include limiting dilution assay (LDA), cytotoxic T cell assay (CTL), and tetramer staining. Compared with respect to sensitivity the Elispot assay is outranking other methods to define frequency of antigen-specific lymphocytes. The method

  19. In-vitro activation of cytotoxic T lymphocytes by fusion of mouse hepatocellular carcinoma cells and lymphotactin gene-modified dendritic cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: To investigate the in-vitro activation of cytotoxic T lymphocytes (CTLs) by fusion of mouse hepatocellular carcinoma (HCC) cells and lymphotactin gene-modified dendritic cells (DCs).METHODS: Lymphotactin gene modified DCs (DCLptn) were prepared by lymphotactin recombinant adenovirus transduction of mature DCs which differentiated from mouse bone marrow cells by stimulation with granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4) and tumor necrosis factor alpha (TNF-α). DCLptn and H22 fusion was prepared using 50% PEG. Lymphotactin gene and protein expression levels were measured by RT-PCR and ELISA, respectively. Lymphotactin chemotactic responses were examined by in-vitro chemotaxis assay. In-vitro activation of CTLs by DCLptn/H22 fusion was measured by detecting CD25 expression and cytokine production after autologous T cell stimulation. Cytotoxic function of activated T lymphocytes stimulated with DCLptn/H22 cells was determined by LDH cytotoxicity assay.RESULTS: Lymphotactin gene could be efficiently transduced to DCs by adenovirus vector and showed an effective biological activity. After fusion, the hybrid DCLptn/H22 cells acquired the phenotypes of both DCLptn and H22 cells. In T cell proliferation assay, flow cytometry showed a very high CD25 expression, and cytokine release assay showed a significantly higher concentration of IFN-γ and IL-2 in DCLptn/H22 group than in DCLptn, DCLptn+H22, DC/H22 or H22 groups. Cytotoxicity assay revealed that T cells derived from DCLptn/H22 group had much higher anti-tumor activity than those derived from DCLptn, H22, DCLptn + H22, DC/H22 groups.CONCLUSION: Lymphotactin gene-modified dendritoma induces T-cell proliferation and strong CTL reaction against allogenic HCC cells. Immunization-engineered fusion hybrid vaccine is an attractive strategy in prevention and treatment of HCC metastases.

  20. Assessment of cytotoxic and apoptotic effects of benzaldehyde using different assays.

    Science.gov (United States)

    Ulker, Z; Alpsoy, L; Mihmanli, A

    2013-08-01

    Benzaldehyde (BA) occurs naturally in a number of plants, including cherry, fig and peach fruit and carnation flowers at therapeutic doses. In addition, it is used in cosmetics, personal care products and food as a preservative. In this study, we aimed to determine the cytotoxic and apoptotic effects of different concentrations of BA on cultured human lymphocytes using lactate dehydrogenase assay, cell proliferation (water-soluble tetrazolium salts-1) assay and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) test (apoptotic test) as a group of cytotoxicity tests at 6th and 24th h on human lymphocyte cell culture. The cytotoxicity increased when cells were treated with 10, 25 and 50 μg/mL concentrations of BA (p < 0.05). Moreover, treatment of the cells with the same concentrations significantly decreased the cell number at the 6th and 24th hours (p < 0.05). TUNEL assay results also show that the concentration of BA at 10, 25 and 50 μg/mL caused DNA damage significantly (p < 0.05). According to our results, the toxic and genotoxic effects of BA have to be further evaluated before using in cosmetic and food products.

  1. Recent advances in chronic lymphocytic leukemia

    Directory of Open Access Journals (Sweden)

    N Vyas

    2012-01-01

    Full Text Available Chronic lymphocytic leukemia (CLL was largely considered to be a disease of slow progression, standard treatment with Chlorambucil and having almost similar prognosis. With the introduction of molecular methods for understanding the disease pathophysiology in CLL there has been a remarkable change in the approach towards the disease. The variation in B-cell receptor response and immunoglobulin heavy chain variable region (IGHV mutation, genetic aberration and defect in apoptosis and proliferation has had an impact on therapy initiation and prognosis. Early diagnosis of molecular variant is therefore necessary in CLL.

  2. Chemokines, lymphocytes, and HIV

    Directory of Open Access Journals (Sweden)

    Farber J.M.

    1998-01-01

    Full Text Available Chemokines are members of a family of more than 30 human cytokines whose best-described activities are as chemotactic factors for leukocytes and that are presumed to be important in leukocyte recruitment and trafficking. While many chemokines can act on lymphocytes, the roles of chemokines and their receptors in lymphocyte biology are poorly understood. The recent discoveries that chemokines can suppress infection by HIV-1 and that chemokine receptors serve, along with CD4, as obligate co-receptors for HIV-1 entry have lent urgency to studies on the relationships between chemokines and lymphocytes. My laboratory has characterized Mig and Crg-2/IP-10, chemokines that are induced by IFN-g and that specifically target lymphocytes, particularly activated T cells. We have demonstrated that the genes for these chemokines are widely expressed during experimental infections in mice with protozoan and viral pathogens, but that the patterns of mig and crg-2 expression differed, suggesting non-redundant roles in vivo. Our related studies to identify new chemokine receptors from activated lymphocytes resulted in the cloning of STRL22 and STRL33. We and others have shown that STRL22 is a receptor for the CC chemokine MIP-3a, and STRL22 has been re-named CCR6. Although STRL33 remains an orphan receptor, we have shown that it can function as a co-receptor for HIV-1 envelope glycoproteins, and that it is active with a broader range of HIV-1 envelope glycoproteins than the major co-receptors described to date. The ability of STRL33 to function with a wide variety of envelope glycoproteins may become particularly important if therapies are instituted to block other specific co-receptors. We presume that investigations into the roles of chemokines and their receptors in lymphocyte biology will provide information important for understanding the pathogenesis of AIDS and for manipulating immune and inflammatory responses for clinical benefit

  3. Lymphocytic proliferative response to outer-membrane proteins isolated from Salmonella.

    Science.gov (United States)

    González, C R; Isibasi, A; Ortiz-Navarrete, V; Paniagua, J; García, J A; Blanco, F; Kumate, J

    1993-01-01

    Porins isolated from Salmonella typhi have been demonstrated to protect against the challenge with this bacteria in mice. The mechanism has not been clarified, but could be associated with activation of both humoral and cellular immunity. In order to evaluate the induction of specific T cell responses, the lymphocytic proliferation to porins isolated from Salmonella typhimurium, Salmonella typhi and Escherichia coli was examined by 3H-thymidine incorporation assay in mice immunized with three different antigens: acetone-killed S. typhimurium, its porins, or outer-membrane proteins (OMPs) isolated from S. typhi. Higher proliferative responses were observed in mice immunized with porins and OMPs compared with those which received the acetone-killed bacteria. Although cross-reactivity was observed between porins, they were not mitogenic. Moreover, porins were able to activate T lymphocytes isolated from mice immunized with S. typhi OMPs. These results suggest that T cell activation, through the release of lymphokines, may play a role in the induction of protective immunity with porins.

  4. Proliferation of latently infected CD4+ T cells carrying replication-competent HIV-1: Potential role in latent reservoir dynamics

    Science.gov (United States)

    Hosmane, Nina N.; Kwon, Kyungyoon J.; Bruner, Katherine M.; Capoferri, Adam A.; Rosenbloom, Daniel I.S.; Keele, Brandon F.; Ho, Ya-Chi

    2017-01-01

    A latent reservoir for HIV-1 in resting CD4+ T lymphocytes precludes cure. Mechanisms underlying reservoir stability are unclear. Recent studies suggest an unexpected degree of infected cell proliferation in vivo. T cell activation drives proliferation but also reverses latency, resulting in productive infection that generally leads to cell death. In this study, we show that latently infected cells can proliferate in response to mitogens without producing virus, generating progeny cells that can release infectious virus. Thus, assays relying on one round of activation underestimate reservoir size. Sequencing of independent clonal isolates of replication-competent virus revealed that 57% had env sequences identical to other isolates from the same patient. Identity was confirmed by full-genome sequencing and was not attributable to limited viral diversity. Phylogenetic and statistical analysis suggested that identical sequences arose from in vivo proliferation of infected cells, rather than infection of multiple cells by a dominant viral species. The possibility that much of the reservoir arises by cell proliferation presents challenges to cure. PMID:28341641

  5. Cyclophosphamide, Alvocidib, and Rituximab in Treating Patients With High Risk B-Cell Chronic Lymphocytic Leukemia or Small Lymphocytic Lymphoma

    Science.gov (United States)

    2015-11-10

    Chronic Lymphocytic Leukemia; Prolymphocytic Leukemia; Recurrent Small Lymphocytic Lymphoma; Refractory Chronic Lymphocytic Leukemia; Stage I Chronic Lymphocytic Leukemia; Stage I Small Lymphocytic Lymphoma; Stage II Chronic Lymphocytic Leukemia; Stage II Small Lymphocytic Lymphoma; Stage III Chronic Lymphocytic Leukemia; Stage III Small Lymphocytic Lymphoma; Stage IV Chronic Lymphocytic Leukemia; Stage IV Small Lymphocytic Lymphoma

  6. mM-CSF 及其剪切体对淋巴细胞白血病Ramos 细胞增殖的抑制作用%Inhibitory effect of mM-CSF and its spliceosome on proliferation of lymphocytic leukemia cell line Ramos

    Institute of Scientific and Technical Information of China (English)

    马翠花; 种靖慧; 廖金凤; 林永敏; 卫佳; 郑国光

    2015-01-01

    目的:探讨膜结合型巨噬细胞集落刺激因子( mM-CSF)及其剪切体( mM-CSF-Δ)对淋巴细胞白血病Ramos细胞增殖的抑制作用。方法采用Overlap PCR法构建带有mM-CSF的真核表达质粒pTARGET-mM-CSF,再进一步构建胞内区截短30个氨基酸的表达质粒pTARGET-mM-CSF-Δ,并进行PCR及DNA双向测序鉴定。将空载体pTARGET、pTARGET-mM-CSF、pTARGET-mM-CSF-Δ质粒分别转染Ramos细胞,经G418筛选稳定表达细胞株,并用RT-PCR、Western blotting进行鉴定;MTT法检测细胞增殖,流式细胞仪检测细胞周期。结果成功构建了mM-CSF和 mM-CSF-Δ的真核表达载体,获得了稳定转染细胞株 Ramos-V、Ramos-M 和 Ramos-Δ。 Ramos-M、Ramos-Δ、Ramos-V细胞增殖能力的OD值分别为0.413±0.014、0.384±0.019、0.463±0.037,Ramos-M细胞与Ramos-Δ细胞比较,Ramos-M、Ramos-Δ细胞分别与Ramos-V细胞比较,P<0.05或<0.01。 Ramos-M、Ramos-Δ、Ramos-V细胞处于G0/G1期的比例分别为41.54%±1.22%、45.60%±1.09%、39.20%±1.53%,Ramos-M细胞与Ramos-Δ细胞比较, Ramos-M、Ramos-Δ细胞分别与 Ramos-V 细胞比较, P <0.05或<0.01。结论 mM-CSF、mM-CSF-Δ均能抑制淋巴细胞白血病Ramos细胞增殖,且后者抑制作用更强。%Objective To investigate the inhibitory effect of membrane-bound macrophage colony-stimulating factor ( mM-CSF) and its spliceosome ( mM-CSF-Δ) on proliferation of lymphocytic leukemia cell line Ramos.Methods The eukaryotic expression plasmid of pTARGET-mM-CSF with mM-CSF was constructed with Overlap PCR, and then pTAR-GET-mM-CSF-Δof 30 amino acide located in the intracellular region of brachytmema mutation was obtained; and mean-while, they were identified by PCR and DNA sequencing.The empty vector pTARGET, pTARGET-mM-CSF and pTAR-GET-mM-CSF-Δplasmid were transfected into Ramos cells, the cell line with stable expression was screened by G418 and

  7. Inhibition of lipopolysaccharide-induced myeloid-derived suppressor cells in the proliferation of spleen T lymphocytes%脂多糖诱导的CD11b+Gr-1+髓源抑制性细胞对小鼠脾脏T细胞增殖的抑制作用

    Institute of Scientific and Technical Information of China (English)

    郑燕妮; 于化鹏; 陈新; 邓火金; 樊慧珍; 龚雨新; 刘俊芳

    2012-01-01

    Objective To explore the effects of lipopolysaccharide (LPS)-induced myeloid-derived suppressor cells (MDSCs) on the proliferation of spleen T lymphocytes.Methods BALB/c mice were randomly divided into two groups:LPS group and normal control group.They were injected intraperitoneally with LPS and normal saline solution respectively.MDSCs were separated with CD11b immunomagnetic beads from the spleen extract of mice. The morphological characteristics of MDCSs were observed by Wright-Giemsa staining and the characteristic molecules on cell surface identified by flow cytometry.And the effects of MDSCs on the in vitro proliferation of T cells were determined by methylthiazolyldiphenyl-tetrazolium bromide (MTT).Results The proportion of MDSCs in the spleen of the LPS group was much more than that of the normal control group ( 27.4% ± 6.6% vs 5.1% ± 3.8% ; t =5.06,P =0.007 ).CD11b + Gr-1 +MDSCs could be separated by CD11b immunomagnetic beads from the spleen of mice injected with LPS at a high purity of 84.0% +4.2%.MTT method showed that the proliferation of T cells decreased significantly after a co-cultivation with CD11 b+ M DSCs versus the control group.And it was positively correlated with the number of M DSCs ( F =46.26,P =0.000 ).Conclusions A high purity of LPS-induced myeloid-derived suppressor cells may be separated with CD11 h immunomagnetic beads.And it has dose-dependent inhibitory effects on the proliferation of the spleen T lymphocytes.%目的 观察脂多糖诱导的CD11b+Gr-1+髓源抑制性细胞(MDSCs)对小鼠脾脏T细胞增殖的影响,探讨其在免疫调控可能发挥的作用.方法 10只6~8周龄BALB/c小鼠随机数字表法随机分为脂多糖组和对照组各5只,分别予脂多糖或生理盐水腹腔注射;采用CD11b磁珠从脾脏组织中分选MDSCs,通过瑞氏-姬姆萨染色观察细胞形态,并用流式细胞术检测细胞表面特征分子表达情况;四唑盐(MTT)比色法测定与MDSCs在体外共培养

  8. Beryllium Lymphocyte Proliferation Test Surveillance Identifies Clinically Significant Beryllium Disease

    Science.gov (United States)

    Mroz, Margaret M.; Maier, Lisa A.; Strand, Matthew; Silviera, Lori; Newman, Lee S.

    2011-01-01

    Background Workplace surveillance identifies chronic beryllium disease (CBD) but it remains unknown over what time frame mild CBD will progress to a more severe form. Methods We examined physiology and treatment in 229 beryllium sensitization (BeS) and 171 CBD surveillance-identified cases diagnosed from 1982 to 2002. Never smoking CBD cases (81) were compared to never smoking BeS patients (83) to assess disease progression. We compared CBD machinists to non-machinists to examine effects of exposure. Results At baseline, CBD and BeS cases did not differ significantly in exposure time or physiology. CBD patients were more likely to have machined beryllium. Of CBD cases, 19.3% went on to require oral immunosuppressive therapy. At 30 years from first exposure, measures of gas exchange were significantly worse and total lung capacity was lower for CBD subjects. Machinists had faster disease progression as measured by pulmonary function testing and gas exchange. Conclusions Medical surveillance for CBD identifies individuals at significant risk of disease progression and impairment with sufficient time since first exposure. PMID:19681064

  9. Effects of low-dose carbon ion irradiation on the proliferation of splenocytes and the concentration of interferon in mice

    Science.gov (United States)

    Li, Ning

    AIM: To investigate the changes in the proliferation response of splenic lymphocytes and the concentration of serum interferon (IFN-γ) in mice induced by low doses carbon ion irradiation. METHODS: The experiment was carried out in the laboratory of physical medicine, Institute of Modern Physics, Chinese Academy of Sciences in November 2006. 1. Thirty Kunming mice were randomly divided into five groups with six animals in each group and irradiated with 0, 0.01, 0.03, 0.05 and 0.10 Gy carbon ion at Heavy Ion Research Facility Laboratory of Lanzhou. Twenty-four hours after irradiation, the eyeballs of mice were taken out under anesthesia and blood was harvested. 2. The concentration of IFN-γ in serum was detected by ELISA kit. After the mice were executed, the spleen was harvested under sterile condition to prepare spleen mononuclear cell suspension. The effects of concanavalin A(ConA) and lipopolysaccharide(LPS) on the proliferations of mononuclear cells was tested by MTT assay. RESULTS: All thirty mice were involved in the result analysis. 1. The concentration of IFN-γ in serum remarkably increased after irradiation with 0.01 Gy and 0.03 Gy compared with that in controls (p<0.05). However, the concentration of IFN-γ decreased after irradiation with 0.05 Gy and 0.1 Gy. 2. Compared with control group, the proliferation of T lymphocytes induced by ConA and B lymphocytes induced by LPS remarkably increased after irradiation with 0.01 Gy (p<0.001) and the effect was of significant difference compared with that of 0.03 Gy (p<0.01). The irradiation with 0.05 Gy presented an inhibition to the proliferation of splenic lymphocytes. This inhibition was also obvious when irradiated with 0.10 Gy. CONCLUSION: 0.01 Gy and 0.03 Gy carbon ion irradiation can stimulate the proliferation of splenocytes, induce the secretion of IFN-γ and, in consequence, enhance the immune function.

  10. LYMPHOCYTE APOPTOSIS IN PSORIASIS

    Directory of Open Access Journals (Sweden)

    О. M. Kapuler

    2006-01-01

    Full Text Available Abstract. Forty-two patients with progressive vulgar psoriasis (PASI = 19.7 ± 1.5 and 40 healthy volunteers were under investigation. Psoriatic patients were characterized by increased number of CD4+ CD95+ peripheral blood T lymphocytes, which correlates with clinical psoriatic score, and by increased levels of soluble Fas (sFas in serum, as compared to controls (resp., 1868.1 ± 186.8 pg/ml vs. 1281.4 ± 142.5 pg/ml, PLSD = 0.019. The levels of spontaneous lymphocyte apoptosis and anti-Fas (Mab-induced apoptosis in psoriatic patients did not differ from the controls. However, apoptosis induced by “oxidative stress” (50 M Н202, 4 hrs was depressed in the patients. Moreover, a simultaneous assessment of cell cycle structure (metachromatic staining with Acridine Orange, apoptosis and Fas receptor expression (AnnV-FITC/antiFas mAbs-PE staining following a short-term mitogenic stimulation (PHA-P, 5 µg/ml, 24 hrs were performed. We found no marked differences in mitogenic reactivity, activation-induced apoptosis, and activation-induced Fas receptor expression when studying lymphocytes from healthy donors and psoriatic patients. However, PHA-activated lymphocytes from psoriatic patients displayed a significantly decreased ratio of AnnV+CD95+ to the total AnnV+ subpopulation, thus suggesting a decreased role of Fas-dependent mechanisms of apoptosis during the cell activation. The data obtained confirm a view, that an abnormal lymphocyte “apoptotic reactivity”, which plays a crucial role in the mechanisms of autoimmunity, may also of importance in the pathogenesis of psoriasis.

  11. Preconditioning with basiliximab protects renal ischemia/reperfusion injury by inhibiting T lymphocytes proliferation%巴利昔单抗预处理抑制T淋巴细胞增殖对小鼠肾脏缺血再灌注损伤的保护作用

    Institute of Scientific and Technical Information of China (English)

    吕中桥; 范钰; 张艳涛; 邱镇; 胡建鹏; 崔飞伦; 王全兴

    2014-01-01

    vein 30 min before the operation].Then we occluded the renal pedicles on both sides for 45 min.Serum creatinine (Scr),blood urea nitrogen (BUN),fractional excretion of sodium (FENa),renal histology,CD4 + T lymphocytes,CD8 +T lymphocytes,interleukin (IL)-2,tumor necrosis factor (TNF)-α,and IL-6 in blood or the kidney were examined after reperfusion for 1,4,24 h.Results Compared with the Sham group,the levels of Scr [(174.33 ± 7.69),and (51.67 ± 6.56) μmol/L],BUN [(69.90 ± 1.68),and (23.97 ± 2.12) μ mol/L],FENa,the pathological injury scores,IL-2,TNF-α,and IL-6 were increased significantly in IR group and Basiliximab group after reperfusion for 24 h.CD4 + T lymphocytes were also increased in Basiliximab and IR groups after reperfusion for 4 h.All these variables were significantly decreased in Basiliximab group as compared with IR group.Conclusion Preconditioning with basiliximab alleviated renal ischemia/reperfusion injury probably by inhibiting CD4 +T lymphocytes proliferation and decreasing the expression of pro-inflammatory cytokines.

  12. Increased production of interleukin-6 by T lymphocytes from patients with multiple myeloma.

    Science.gov (United States)

    Lapeña, P; Prieto, A; Garcia-Suarez, J; Reyes, E; San Miguel, J; Jorda, J; Alvarez-Mon, M

    1996-01-01

    Alterations in T lymphocyte functions may affect other cellular components of the immune system. Several lymphokines produced by T cells are involved in the proliferation and differentiation of human B lymphocytes. Alterations in the secretion of these molecules may be implicated in the development of B cell lymphoproliferative diseases. We have investigated the production of interleukin-2 (IL-2) and interleukin-6 (IL-6) by T lymphocytes from 14 patients with multiple myeloma (MM) and 16 healthy controls. The phenotypical and functional characteristics of these T lymphocytes were also studied. The proliferative response to vegetal lectin phytohemagglutinin (PHA) stimulation was decreased in T lymphocytes from MM patients (p 0.05) but not by exogenous IL-6 (p lectin stimulation, the production of IL-2 by T lymphocytes from those patients was normal, while IL-6 secretion was increased.

  13. CLINICAL VALUE OF DETECTING T LYMPHOCYTE SUBSET AND NK CELL ACTIVITY IN PATIENTS WITH COLORECTAL CANCER

    Institute of Scientific and Technical Information of China (English)

    刘长安; 管增伟; 孙武; 邵玉霞; 李卓; 贾廷珍

    2001-01-01

    Objective To study on the expression and clinical significance of T lymphocyte subset and NK cell activity (NKA) in patients with colorectal cancer. Methods Fifty-seven cancer patients and 33 healthy controls were enrolled in this study. T lymphocyte subset was measured by SAP technique and NKA by LDH release assay based on K562 cells, which served as target cells.

  14. Protective effect of apigenin on radiation-induced chromosomal damage in human lymphocytes

    Science.gov (United States)

    Rithidech, Kanokporn Noy; Tungjai, Montree; Whorton, Elbert B.

    2005-01-01

    The potential use of flavonoids as a radioprotector is of increasing interest because of their high antioxidant activity and abundance in the diet. The aim of this study is to examine genotoxic and radioprotective effects of one of the most common flavonoids, apigenin, on radiation-induced chromosome aberrations in human lymphocytes. The cytokinesis-block micronucleus (CBMN) assay was used to evaluate such effects of apigenin. Blood samples were collected from two non-smoking healthy male volunteers who had no history of previous exposure to other clastogenic agents. Isolated lymphocytes were cultured. There were two tubes per concentration for all treatments. To evaluate the genotoxicity of apigenin, cells were first treated with different concentrations of apigenin (0, 2.5, 5, 10 and 25 microg/mL) at 24 h after culture initiation, followed by cytochalasin-B (Cyt-B) treatment (3 microg/mL) and cell harvest at 44 and 72 h, respectively. Secondly, to investigate the radioprotective effect, cell cultures were exposed to different concentrations of apigenin as described above for 30 min before being irradiated to 2 Gy of 137Cs gamma rays (at a dose rate of 0.75 Gy/min). In all instances, the frequency of MN was scored in binucleated (BN) cells. The nuclear proliferation index also was calculated. We did not detect an increase in the frequency of MN in non-irradiated human lymphocyte cultures treated with 2.5, 5.0 or 10 microg/mL apigenin; although, we did observe an increase in cultures treated with 25 microg/mL apigenin (the highest concentration of apigenin used in our study). We also observed a significant increase in the frequency of MN in irradiated cells overall; however, the frequency was decreased as the concentration of apigenin increased, suggesting a radioprotective effect. These findings provide a basis for additional studies to help clarify the potential use and benefit of apigenin as a radioprotector.

  15. Statins induce immunosuppressive effect on heterotopic limb allografts in rat through inhibiting T cell activation and proliferation.

    Science.gov (United States)

    Nie, Chunlei; Yang, Daping; Liu, Guofeng; Dong, Deli; Ma, Zhiqiang; Fu, Hailiang; Zhao, Zhengyu; Sun, Zhiyong

    2009-01-05

    Long-term use of immunosuppressive agents could bring many side effects. Recently, 3-Hydroxy-3-methyl-gutaryl coenzyme A reductase inhibitors (statins) have been reported to be immunomodulatory besides lowering serum cholesterol level. The aim of this study was to investigate the effects of statins on composite tissue allografts and T lymphocyte in vivo and in vitro. Rats were divided into 5 groups: syngeneic transplantation group (Lewis-Lewis); allogeneic control group (Brown Norway-Lewis, no treatment); low-dose statins group (15 mg /kg); high-dose statins group (30 mg /kg) and cyclosporin A group. In vivo, treatment of statins significantly prolonged allografts survival as compared to control group. Histological findings further supported these clinical results and demonstrated less extent of rejection. Immunohistochemical analysis showed that there was a remarkably reduced T cells infiltration in statins groups. Moreover, the serum levels of IL-2 and IFN-gamma were decreased after statins therapy, while these in control group increased significantly. Meanwhile, transcriptional activities of IL-2 and IFN-gamma were also dramatically down-regulated after statins treatment. In vitro, mixed lymphocyte reaction assay was performed and the results revealed lymphocyte proliferation was inhibited by statins in a dose-dependent manner. Furthermore, administration of statins exhibited inhibitory effects on CD3/CD28 mediated T cell activation and proliferation. Besides, the results demonstrated that statins significantly down-regulated mRNA expression and suppress cytokine production of IL-2 and IFN-gamma in vitro. In conclusion, our data demonstrated that application of statins could induce immunosuppressive effect and prolong allografts survival through inhibiting activation and proliferation of T cell and reducing production of IL-2 and IFN-gamma.

  16. Autologous hematopoietic stem cell transplantation in lymphoma patients is associated with a decrease in the double strand break repair capacity of peripheral blood lymphocytes.

    Science.gov (United States)

    Lacoste, Sandrine; Bhatia, Smita; Chen, Yanjun; Bhatia, Ravi; O'Connor, Timothy R

    2017-01-01

    Patients who undergo autologous hematopoietic stem cell transplantation (aHCT) for treatment of a relapsed or refractory lymphoma are at risk of developing therapy related- myelodysplasia/acute myeloid leukemia (t-MDS/AML). Part of the risk likely resides in inherent interindividual differences in their DNA repair capacity (DRC), which is thought to influence the effect chemotherapeutic treatments have on the patient's stem cells prior to aHCT. Measuring DRC involves identifying small differences in repair proficiency among individuals. Initially, we investigated the cell model in healthy individuals (primary lymphocytes and/or lymphoblastoid cell lines) that would be appropriate to measure genetically determined DRC using host-cell reactivation assays. We present evidence that interindividual differences in DRC double-strand break repair (by non-homologous end-joining [NHEJ] or single-strand annealing [SSA]) are better preserved in non-induced primary lymphocytes. In contrast, lymphocytes induced to proliferate are required to assay base excision (BER) or nucleotide excision repair (NER). We established that both NHEJ and SSA DRCs in lymphocytes of healthy individuals were inversely correlated with the age of the donor, indicating that DSB repair in lymphocytes is likely not a constant feature but rather something that decreases with age (~0.37% NHEJ DRC/year). To investigate the predictive value of pre-aHCT DRC on outcome in patients, we then applied the optimized assays to the analysis of primary lymphocytes from lymphoma patients and found that individuals who later developed t-MDS/AML (cases) were indistinguishable in their DRC from controls who never developed t-MDS/AML. However, when DRC was investigated shortly after aHCT in the same individuals (21.6 months later on average), aHCT patients (both cases and controls) showed a significant decrease in DSB repair measurements. The average decrease of 6.9% in NHEJ DRC observed among aHCT patients was much higher

  17. Autologous hematopoietic stem cell transplantation in lymphoma patients is associated with a decrease in the double strand break repair capacity of peripheral blood lymphocytes

    Science.gov (United States)

    Lacoste, Sandrine; Bhatia, Smita; Chen, Yanjun; Bhatia, Ravi; O’Connor, Timothy R.

    2017-01-01

    Patients who undergo autologous hematopoietic stem cell transplantation (aHCT) for treatment of a relapsed or refractory lymphoma are at risk of developing therapy related- myelodysplasia/acute myeloid leukemia (t-MDS/AML). Part of the risk likely resides in inherent interindividual differences in their DNA repair capacity (DRC), which is thought to influence the effect chemotherapeutic treatments have on the patient’s stem cells prior to aHCT. Measuring DRC involves identifying small differences in repair proficiency among individuals. Initially, we investigated the cell model in healthy individuals (primary lymphocytes and/or lymphoblastoid cell lines) that would be appropriate to measure genetically determined DRC using host-cell reactivation assays. We present evidence that interindividual differences in DRC double-strand break repair (by non-homologous end-joining [NHEJ] or single-strand annealing [SSA]) are better preserved in non-induced primary lymphocytes. In contrast, lymphocytes induced to proliferate are required to assay base excision (BER) or nucleotide excision repair (NER). We established that both NHEJ and SSA DRCs in lymphocytes of healthy individuals were inversely correlated with the age of the donor, indicating that DSB repair in lymphocytes is likely not a constant feature but rather something that decreases with age (~0.37% NHEJ DRC/year). To investigate the predictive value of pre-aHCT DRC on outcome in patients, we then applied the optimized assays to the analysis of primary lymphocytes from lymphoma patients and found that individuals who later developed t-MDS/AML (cases) were indistinguishable in their DRC from controls who never developed t-MDS/AML. However, when DRC was investigated shortly after aHCT in the same individuals (21.6 months later on average), aHCT patients (both cases and controls) showed a significant decrease in DSB repair measurements. The average decrease of 6.9% in NHEJ DRC observed among aHCT patients was much

  18. Selective loss of T cell functions in different stages of HIV infection. Early loss of anti-CD3-induced T cell proliferation followed by decreased anti-CD3-induced cytotoxic T lymphocyte generation in AIDS-related complex and AIDS.

    Science.gov (United States)

    Gruters, R A; Terpstra, F G; De Jong, R; Van Noesel, C J; Van Lier, R A; Miedema, F

    1990-05-01

    To investigate the effects of persistant human immunodeficiency virus (HIV) infection on T cell reactivity, functional properties of peripheral blood T cells from HIV-seropositive homosexual men in various stages of infection were studied. T cell activation via CD3 resulting in proliferation and differentiation was measured in a model system independent of accessory cells, using immobilized anti-CD3 monoclonal antibodies (mAb). T cells from HIV-infected asymptomatic men had a decreased proliferative response compared to HIV-negative controls. T cells from AIDS-related complex (ARC) and AIDS patients, compared to T cells from asymptomatic HIV-infected men, had a significantly lower proliferative response to anti-CD3 mAb. This diminished response to anti-CD3 mAb was shown to be due to decreased interleukin (IL) 2 production and could be enhanced by co-stimulation with anti-CD28 mAb or by adding IL 2. Anti-CD3-induced generation of cytotoxic T lymphocytes was fully intact in early infection but was severely decreased in T cells from ARC and AIDS patients. Cytotoxic activity could be restored to near normal levels after co-stimulation with either anti-CD28 mAb or IL 2. Our data demonstrate a differential loss of T cell functions in the course of HIV infection which is predominantly caused by a lack of IL 2 production after stimulation via the CD3/T cell receptor complex. In early HIV infection this seems to be predominantly caused by a specific loss of memory T cells. However, in later stages of infection when both naive and memory T cell subsets are depleted, resulting in a normal naive/memory T cell ratio, T cell functions further deteriorate probably due to intrinsic activation defects. These findings may be of pathogenic relevance since diminished T cell reactivity may facilitate spreading and replication of virulent HIV variants heralding development of ARC and AIDS.

  19. 2,4-二硝基氯苯诱导超敏反应性皮炎小鼠淋巴细胞增殖与活化研究%Research on the proliferation and activation of lymphocyte of 2,4-dinitrochlorobenzene-induced hypersensitivity dermatitis mice

    Institute of Scientific and Technical Information of China (English)

    纪玉青; 贾强; 赵雪铮; 孟涛; 李园园; 李红丽; 戴宇飞

    2014-01-01

    % vs(25.70±1.47)%,(13.38±0.98)% vs(18.53±0.93)%,(7.23±0.74)% vs (10.20±0.30)%,P <0.05],耳后淋巴结 T 淋巴细胞比例、CD4+细胞比例和 CD4+/CD8+比值均低于对照组[(25.20±1.22)% vs(39.40±2.53)%,(8.20±0.66)% vs(31.30±2.84)%,(0.76±0.35)vs(5.29±1.17), P<0.05],CD8+细胞比例高于对照组[(10.90±1.31)% vs(6.20±1.99)%,P<0.05]。高剂量组耳后淋巴结CD4+细胞比例和 CD4+/CD8+比值均低于低剂量组[(8.20±0.66)% vs(20.30±2.60)%,(0.76±0.35) vs (2.64±0.03),P<0.05]。结论 T淋巴细胞介导的细胞免疫在 DNCB诱导的超敏反应性皮炎中起重要作用, CD8+细胞是主要的效应性T淋巴细胞,CD44和CD62L表达可作为T淋巴细胞活化检测指标。%Objective To investigate the lymphocyte proliferation and activation in the 2,4-dinitrochlorobenzene( DNCB)-induced hypersensitivity dermatitis mice model. Methods Specific pathogens free 8-12 week-old female C57BL/6 mice were randomly divided into control group,low- and high-dose groups,6 mice per group. Mice in 3 groups were induced daily with 100 μl mass-to-volume ratio of 0. 00%,0. 50% and 1. 00% DNCB respectively on back skin on day 1-3,no special treatment on day 4,and then stimulated once on the dorsum of left ears on day 5 with 25μl mass-to-volume ratio of 0. 25%,0. 25% and 0. 50% DNCB respectively,24 hours later the auricle allergy symptoms were observed. Flow cytometry was adopted to detect the lymphocyte proliferation,activation and subpopulations of spleens( SPs)and auricular lymph nodes( ALNs). Results The left ears of mice were swelling and angiectatic in high dose group,while symptoms were not observed in the control and low dose groups. The grade of ear swelling in high dose group was higher than that in the control[(5. 30 ± 2. 04)vs(0. 50 ± 0. 49)mg,P<0. 05]. Both the organ coefficients of SPs and ALNs in low- and high

  20. At High Levels, Constitutively Activated STAT3 Induces Apoptosis of Chronic Lymphocytic Leukemia Cells.

    Science.gov (United States)

    Rozovski, Uri; Harris, David M; Li, Ping; Liu, Zhiming; Wu, Ji Yuan; Grgurevic, Srdana; Faderl, Stefan; Ferrajoli, Alessandra; Wierda, William G; Martinez, Matthew; Verstovsek, Srdan; Keating, Michael J; Estrov, Zeev

    2016-05-15

    In chronic lymphocytic leukemia (CLL), the increment in PBLs is slower than the expected increment calculated from the cells' proliferation rate, suggesting that cellular proliferation and apoptosis are concurrent. Exploring this phenomenon, we found overexpression of caspase-3, higher cleaved poly (ADP-ribose) polymerase levels (p < 0.007), and a higher apoptosis rate in cells from patients with high counts compared with cells from patients with low counts. Although we previously found that STAT3 protects CLL cells from apoptosis, STAT3 levels were significantly higher in cells from patients with high counts than in cells from patients with low counts. Furthermore, overexpression of STAT3 did not protect the cells. Rather, it upregulated caspase-3 and induced apoptosis. Remarkably, putative STAT3 binding sites were identified in the caspase-3 promoter, and a luciferase assay, chromatin immunoprecipitation, and an EMSA revealed that STAT3 activated caspase-3 However, caspase-3 levels increased only when STAT3 levels were sufficiently high. Using chromatin immunoprecipitation and EMSA, we found that STAT3 binds with low affinity to the caspase-3 promoter, suggesting that at high levels, STAT3 activates proapoptotic mechanisms and induces apoptosis in CLL cells.

  1. Flow cytometric analysis of lymphocyte proliferative responses to food allergens in dogs with food allergy.

    Science.gov (United States)

    Fujimura, Masato; Masuda, Kenichi; Hayashiya, Makio; Okayama, Taro

    2011-10-01

    Two different allergy tests, antigen-specific immunoglobulin E quantification (IgE test) and flow cytometric analysis of antigen-specific proliferation of peripheral lymphocytes (lymphocyte proliferation test), were performed to examine differences in allergic reactions to food allergens in dogs with food allergy (FA). Thirteen dogs were diagnosed as FA based on clinical findings and elimination diet trials. Seven dogs clinically diagnosed with canine atopic dermatitis (CAD) were used as a disease control group, and 5 healthy dogs were used as a negative control group. In the FA group, 19 and 33 allergen reactions were identified using the serum IgE test and the lymphocyte proliferation test, respectively. Likewise, in the CAD group, 12 and 6 allergen reactions and in the healthy dogs 3 and 0 allergen reactions were identified by each test, respectively. A significant difference was found between FA and healthy dogs in terms of positive allergen detection by the lymphocyte proliferation test, suggesting that the test can be useful to differentiate FA from healthy dogs but not from CAD. Both tests were repeated in 6 of the dogs with FA after a 1.5- to 5-month elimination diet trial. The IgE concentrations in 9 of 11 of the positive reactions decreased by 20-80%, whereas all the positive reactions in the lymphocyte proliferation test decreased to nearly zero (Pfood allergens may be involved in the pathogenesis of canine FA.

  2. Arctigenin from Arctium lappa inhibits interleukin-2 and interferon gene expression in primary human T lymphocytes

    Science.gov (United States)

    2011-01-01

    Background Arctium lappa (Niubang), a Chinese herbal medicine, is used to treat tissue inflammation. This study investigates the effects of arctigenin (AC), isolated from A. lappa, on anti-CD3/CD28 Ab-stimulated cell proliferation and cytokine gene expression in primary human T lymphocytes. Methods Cell proliferation was determined with enzyme immunoassays and the tritiated thymidine uptake method. Cytokine production and gene expression were analyzed with reverse transcription-polymerase chain reaction. Results AC inhibited primary human T lymphocytes proliferation activated by anti-CD3/CD28 Ab. Cell viability test indicated that the inhibitory effects of AC on primary human T lymphocyte proliferation were not due to direct cytotoxicity. AC suppressed interleukin-2 (IL-2) and interferon-γ (IFN-γ) production in a concentration-dependent manner. Furthermore, AC decreased the IL-2 and IFN-γ gene expression in primary human T lymphocytes induced by anti-CD3/CD28 Ab. Reporter gene analyses revealed that AC decreased NF-AT-mediated reporter gene expression. Conclusion AC inhibited T lymphocyte proliferation and decreased the gene expression of IL-2, IFN-γ and NF-AT. PMID:21435270

  3. Arctigenin from Arctium lappa inhibits interleukin-2 and interferon gene expression in primary human T lymphocytes.

    Science.gov (United States)

    Tsai, Wei-Jern; Chang, Chu-Ting; Wang, Guei-Jane; Lee, Tzong-Huei; Chang, Shwu-Fen; Lu, Shao-Chun; Kuo, Yuh-Chi

    2011-03-25

    Arctium lappa (Niubang), a Chinese herbal medicine, is used to treat tissue inflammation. This study investigates the effects of arctigenin (AC), isolated from A. lappa, on anti-CD3/CD28 Ab-stimulated cell proliferation and cytokine gene expression in primary human T lymphocytes. Cell proliferation was determined with enzyme immunoassays and the tritiated thymidine uptake method. Cytokine production and gene expression were analyzed with reverse transcription-polymerase chain reaction. AC inhibited primary human T lymphocytes proliferation activated by anti-CD3/CD28 Ab. Cell viability test indicated that the inhibitory effects of AC on primary human T lymphocyte proliferation were not due to direct cytotoxicity. AC suppressed interleukin-2 (IL-2) and interferon-γ (IFN-γ) production in a concentration-dependent manner. Furthermore, AC decreased the IL-2 and IFN-γ gene expression in primary human T lymphocytes induced by anti-CD3/CD28 Ab. Reporter gene analyses revealed that AC decreased NF-AT-mediated reporter gene expression. AC inhibited T lymphocyte proliferation and decreased the gene expression of IL-2, IFN-γ and NF-AT.

  4. Arctigenin from Arctium lappa inhibits interleukin-2 and interferon gene expression in primary human T lymphocytes

    Directory of Open Access Journals (Sweden)

    Chang Shwu-Fen

    2011-03-01

    Full Text Available Abstract Background Arctium lappa (Niubang, a Chinese herbal medicine, is used to treat tissue inflammation. This study investigates the effects of arctigenin (AC, isolated from A. lappa, on anti-CD3/CD28 Ab-stimulated cell proliferation and cytokine gene expression in primary human T lymphocytes. Methods Cell proliferation was determined with enzyme immunoassays and the tritiated thymidine uptake method. Cytokine production and gene expression were analyzed with reverse transcription-polymerase chain reaction. Results AC inhibited primary human T lymphocytes proliferation activated by anti-CD3/CD28 Ab. Cell viability test indicated that the inhibitory effects of AC on primary human T lymphocyte proliferation were not due to direct cytotoxicity. AC suppressed interleukin-2 (IL-2 and interferon-γ (IFN-γ production in a concentration-dependent manner. Furthermore, AC decreased the IL-2 and IFN-γ gene expression in primary human T lymphocytes induced by anti-CD3/CD28 Ab. Reporter gene analyses revealed that AC decreased NF-AT-mediated reporter gene expression. Conclusion AC inhibited T lymphocyte proliferation and decreased the gene expression of IL-2, IFN-γ and NF-AT.

  5. B-Lymphocytes from a Population of Children with Autism Spectrum Disorder and Their Unaffected Siblings Exhibit Hypersensitivity to Thimerosal

    Directory of Open Access Journals (Sweden)

    Martyn A. Sharpe

    2013-01-01

    Full Text Available The role of thimerosal containing vaccines in the development of autism spectrum disorder (ASD has been an area of intense debate, as has the presence of mercury dental amalgams and fish ingestion by pregnant mothers. We studied the effects of thimerosal on cell proliferation and mitochondrial function from B-lymphocytes taken from individuals with autism, their nonautistic twins, and their nontwin siblings. Eleven families were examined and compared to matched controls. B-cells were grown with increasing levels of thimerosal, and various assays (LDH, XTT, DCFH, etc. were performed to examine the effects on cellular proliferation and mitochondrial function. A subpopulation of eight individuals (4 ASD, 2 twins, and 2 siblings from four of the families showed thimerosal hypersensitivity, whereas none of the control individuals displayed this response. The thimerosal concentration required to inhibit cell proliferation in these individuals was only 40% of controls. Cells hypersensitive to thimerosal also had higher levels of oxidative stress markers, protein carbonyls, and oxidant generation. This suggests certain individuals with a mild mitochondrial defect may be highly susceptible to mitochondrial specific toxins like the vaccine preservative thimerosal.

  6. Effect of Spatholobus suberectus Dunn on proliferation of progenitor cells in mice with bone marrow depression

    Institute of Scientific and Technical Information of China (English)

    Chen Donghui; Luo Xia; Yu Mengyao; Zhao Yiqing; Yang Zhirong

    2005-01-01

    AIM: To study the effect of Spatholobus suberectus Dunn on the proliferation and hematonic mechanism of Spatholobus suberectus Dunn. Methods: The techniques of culture of hematopoietic cell and hematopoietic growth factor (HGF) assay were used. The method of semi-solid culture with methylcellulose of CFU-GM, CFU-E, BFU-E,CFU-Meg was adopted in bone marrow depressed mice which treated with Spatholobus suberectus Dunn for a long time. Results: Spatholobus suberectus Dunn could obviously promote the proliferation of bone marrow cells and spleen lymphocytes in healthy and anaemic mice. The culture medium of spleen cell, macrophage, lung and skeletal muscle treated with Spatholobus suberectus Dunn had much stronger stimulating effects on hematopoietic cells. The numbers of CFU-GM, CFU-E,BFU-E,CFU-Meg in bone marrow depressed mice were raised distinctly under the control of Spatholobus suberectus Dunn as compared with those of contrast group. Conclusions: Spatholobus suberectus Dunn may enhance hematopoiesis by stimulating directly and/or indirectly stroma cell in hematopoietic inductive microenvironment and muscle tissue to secrete some HGF (Epo, GM-CSF, IL, and MK-CSF). It can promote the proliferation and differentiation of hematopoietic cells in anaemic mice. This is one of the biological mechanisms for hematonic effect of Spatholobus suberectus Dunn.

  7. D-ribose inhibits DNA repair synthesis in human lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Zunica, G.; Marini, M.; Brunelli, M.A.; Chiricolo, M.; Franceschi, C.

    1986-07-31

    D-ribose is cytotoxic for quiescent human lymphocytes and severely inhibits their PHA-induced proliferation at concentrations (25-50 mM) at which other simple sugars are ineffective. In order to explain these effects, DNA repair synthesis was evaluated in PHA-stimulated human lymphocytes treated with hydroxyurea and irradiated. D-ribose, in contrast to other reducing sugars, did not induce repair synthesis and therefore did not apparently damage DNA in a direct way, although it markedly inhibited gamma ray-induced repair. Taking into account that lymphocytes must rejoin physiologically-formed DNA strand breaks in order to enter the cell cycle, we suggest that D-ribose exerts its cytotoxic activity by interfering with metabolic pathways critical for the repair of DNA breaks.

  8. Herpesvirus sylvilagus infects both B and T lymphocytes in vivo.

    Science.gov (United States)

    Kramp, W J; Medveczky, P; Mulder, C; Hinze, H C; Sullivan, J L

    1985-01-01

    Herpesvirus sylvilagus infection of cottontail rabbits (Sylvilagus floridanus) was studied as a model of herpesvirus-induced lymphoproliferative disorders. Leukocytosis, splenomegaly, proliferation of T cells and virus production by lymphocytes characterized this infectious mononucleosis-like disease. Approximately two copies of circular herpesvirus sylvilagus genomes per cell were detected in spleen cells at 2 weeks postinfection, and circular genomes could still be observed after 4 months. Circular viral genomes were found in both B and T lymphocytes. Small amounts of linear viral DNA (0.1 to 0.3 copies per cell) were also detected in both B and T cells. These results indicated that the virus did not replicate in the majority of lymphocytes in vivo. Herpesvirus sylvilagus infection in cottontail rabbits could be useful as a model for studying the complex virus-host relationships of lymphotropic herpesviruses and perhaps as an animal model for Epstein-Barr virus infection in humans. Images PMID:2993667

  9. miRNA expression profiles in chronic lymphocytic and acute lymphocytic leukemia

    Directory of Open Access Journals (Sweden)

    D.L. Zanette

    2007-11-01

    Full Text Available MicroRNAs (miRNAs are a class of small endogenous RNAs that play important regulatory roles by targeting mRNAs for cleavage or translational repression. miRNAs act in diverse biological processes including development, cell growth, apoptosis, and hematopoiesis, suggesting their association with cancer. We determined the miRNA expression profile of chronic and acute lymphocytic leukemias (CLL and ALL using the TaqMan® MicroRNA Assays Human Panel (Applied Biosystems. Pooled leukemia samples were compared to pooled CD19+ samples from healthy individuals (calibrator by the 2-DDCt method. Total RNA input was normalized based on the Ct values obtained for hsa-miR-30b. The five most highly expressed miRNAs were miR-128b, miR-204, miR-218, miR-331, and miR-181b-1 in ALL, and miR-331, miR-29a, miR-195, miR-34a, and miR-29c in CLL. To our knowledge, this is the first report associating miR-128b, miR-204 and miR-331 to hematological malignancies. The miR-17-92 cluster was also found to be up-regulated in ALL, as previously reported for some types of lymphomas. The differences observed in gene expression levels were validated for miR-331 and miR-128b in ALL and CD19+ samples. These miRNAs were up-regulated in ALL, in agreement with our initial results. A brief target analysis was performed for miR-331. One of its putative targets, SOCS1, promotes STAT activation, which is a known mediator of cell proliferation and survival, suggesting the possibility of an association between miR-331 and these processes. This initial screening provided information on miRNA differentially expressed in normal and malignant B-cells that could suggest the potential roles of these miRNAs in hematopoiesis and leukemogenesis.

  10. T-lymphocyte and B-lymphocyte dichotomy in anuran amphibians: III. Assessment and identification of inducible killer T-lymphocytes (IKTL) and spontaneous killer T-lymphocytes (SKTL).

    Science.gov (United States)

    Klempau, A E; Cooper, E L

    1984-01-01

    We have established the existence of alloreactive inducible killer (IK) T-lymphocytes in Rana pipiens by injecting immunogenic concentrations of allogeneic frog erythrocytes (RBC). Assessment of specific IK activity was determined microscopically, observing effector-target conjugate formation, and spectrophotometrically as released hemoglobin (Hb) from lysed targets (RBC). The presence of spontaneous killer (SK) T-lymphocyte activity was also determined using unimmunized frogs and similar assay conditions. Assays using rabbit anti-frog Thy-1.1 antiserum inhibition, but not E-rosetted T-lymphocyte depletion, confirmed the T-lymphocyte category of both effector cell populations in Rana pipiens. For IK activity, we determined the 1) best priming doses, 2) best effector cell source (peripheral blood), 3) best priming route (intraperitoneal), 4) kinetics of immunity development, and 5) kinetics of lysis. Kinetics of lysis and organ distribution for spontaneous killer cells were also determined. Our results may assist 1) in establishing the evolutionary origin of cytotoxic T-lymphocytes (CTL) and natural killer (NK) cells, and 2) in predicting where the capacity of immuno-surveillance against modified-self appeared in phylogeny. The implications are important for understanding origins of mechanisms of resistance against neoplastic conditions.

  11. Curcumin and Cholecalciferol in Treating Patients With Previously Untreated Stage 0-II Chronic Lymphocytic Leukemia or Small Lymphocytic Lymphoma

    Science.gov (United States)

    2016-10-04

    Contiguous Stage II Small Lymphocytic Lymphoma; Noncontiguous Stage II Small Lymphocytic Lymphoma; Stage 0 Chronic Lymphocytic Leukemia; Stage I Chronic Lymphocytic Leukemia; Stage I Small Lymphocytic Lymphoma; Stage II Chronic Lymphocytic Leukemia

  12. Radiosensitivity of peripheral blood lymphocytes in autoimmune disease

    Energy Technology Data Exchange (ETDEWEB)

    Harris, G. (Kennedy Inst. of Rheumatology, London (UK). Div. of Experimental Pathology); Cramp, W.A.; Edwards, J.C.; George, A.M.; Sabovljev, S.A.; Hart, L.; Hughes, G.R.V. (Hammersmith Hospital, London (UK)); Denman, A.M. (Northwich Park Hospital, Harrow (UK)); Yatvin, M.B. (Wisconsin Clinical Cancer Center, Madison (USA))

    1985-06-01

    The proliferation of peripheral blood lymphocytes, cultured with Con A, can be inhibited by ionizing radiation. Lymphocytes from patients with conditions associated with autoimmunity, such as rheumatoid arthritis, systemic lupus erythematosus and polymyositis, are more radiosensitive than those from healthy volunteers or patients with conditions not associated with autoimmunity. Nuclear material isolated from the lymphocytes of patients with autoimmune diseases is, on average, lighter in density than the nuclear material from most healthy controls. This difference in density is not related to increased sensitivity to ionizing radiation but the degree of post-irradiation change in density (lightening) is proportional to the initial density, i.e. more dense nuclear material always shows a greater upward shift after radiation. The recovery of pre-irradiation density of nuclear material, 1 h after radiation exposure, taken as an indication of DNA repair, correlates with the radiosensitivity of lymphocyte proliferation (Con A response); failure to return to pre-irradiation density being associated with increased sensitivity of proliferative response. These results require extension but, taken with previously reported studied of the effects of DNA methylating agents, support the idea that DNA damage and its defective repair could be important in the aetio-pathogenesis of autoimmune disease.

  13. Lactobacillus casei胞外多糖对BALB/c小鼠肠相关淋巴细胞调控的初步研究%Effect of exopolysaccharide from Lactobacillus casei on murine gut-associated lymphocytes

    Institute of Scientific and Technical Information of China (English)

    包鹏; 唐彦君; 刘宁

    2013-01-01

    Objective: To study the effect of exopolysaocharide (EPS) from Lactobacillus casei on murine gut-associated lymphocytes proliferation and production of TNF-α, IL-17 and IFN-γ Methods: intraepithelial lymphocytes (IEL) , lamina propria lymphocytes (LPL) , Peyer's Patches lymphocytes (PPL) and mesenteric lymph nodes lymphocytes (MLNL) were isolated from small intestine of mice by using Percoll discontinuous density gradient centrifugation. WST-1 assay was used to determine the proliferation of lymphocyte. The secretion of TNF-α, IL-17, IFN-γ in cell culture supernatant was assayed by ELISA. Results; EPS could induce IEL, LPL, and PPL proliferation, and increased the proliferation obviously at 100 μg/ml, EPS could promote the proliferation MNLN at 50-200 μg/ml, which was dose-dependent. EPS demonstrated inhibitory activity to the secretion of TNF-α in IEL, LPL and PPL and there were significant differences in the control group at 50-200 μg/ml. EPS could not depress the secretion of TNF-α in MLNL, and increased the secretion of TNF-α obviously at 200 μg/ml EPS demonstrated facilitative activity to the secretion of IL-17 in IEL, LPL, PPL and MLNL EPS could not influence the secretion of IFN-γ. Conclusion; EPS can induce the proliferation of gut-associated lymph-oid tissues' lymphocytes, and regulate TNF-α, IL-17 secretion.%目的:探讨干酪乳杆菌胞外多糖(EPS)对BALB/c小鼠小肠相关淋巴细胞增殖及细胞因子TNF-α、IL-17及IFN-γ分泌的影响.方法:Percoll不连续密度梯度分离法从小鼠小肠中分离上皮内淋巴细胞(IEL)、固有层淋巴细胞(LPL)、派氏集合淋巴结淋巴细胞(PPL)和肠系膜淋巴结淋巴细胞(MLNL).实验组分别加入10、25、50、100和200 μg/ml浓度的干酪乳杆菌胞外多糖.水溶性四氮唑(WST-1)法检测淋巴细胞增殖;ELISA法测定细胞上清液中细胞因子TNF-α、IL-17及IFN-γ的含量.结果:EPS能够一定程度地促进IEL、LPL、PPL的增殖,且均在100

  14. Lymphocyte 'homing' and chronic inflammation.

    Science.gov (United States)

    Sakai, Yasuhiro; Kobayashi, Motohiro

    2015-07-01

    Chronic inflammation is a response to prolonged exposure to injurious stimuli that harm and destroy tissues and promote lymphocyte infiltration into inflamed sites. Following progressive accumulation of lymphocytes, the histology of inflamed tissue begins to resemble that of peripheral lymphoid organs, which can be referred to as lymphoid neogenesis or formation of tertiary lymphoid tissues. Lymphocyte recruitment to inflamed tissues is also reminiscent of lymphocyte homing to peripheral lymphoid organs. In the latter, under physiological conditions, homing receptors expressed on lymphocytes adhere to vascular addressin expressed on high endothelial venules (HEVs), initiating a lymphocyte migration process composed of sequential adhesive interactions. Intriguingly, in chronic inflammation, HEV-like vessels are induced de novo, despite the fact that the inflamed site is not originally lymphoid tissue, and these vessels contribute to lymphocyte recruitment in a manner similar to physiological lymphocyte homing. In this review, we first describe physiological lymphocyte homing mechanisms focusing on vascular addressins. We then describe HEV-like vessel-mediated pathogenesis seen in various chronic inflammatory disorders such as Helicobacter pylori gastritis, inflammatory bowel disease (IBD), autoimmune pancreatitis and sclerosing sialadenitis, as well as chronic inflammatory cell neoplasm MALT lymphoma, with reference to our work and that of others.

  15. Lymphocyte Trafficking to Mucosal Tissues

    DEFF Research Database (Denmark)

    Mikhak, Zamaneh; Agace, William Winston; Luster, Andrew D.

    2015-01-01

    Lymphocytes are the key cells of the adaptive immune system that provide antigen-specific responses tailored to the context of antigen exposure. Through cytokine release and antibody production, lymphocytes orchestrate and amplify the recruitment and function of other immune cells and contribute...... to host defense against invading pathogens and the pathogenesis of many inflammatory diseases. Lymphocyte function is critically dependent on their ability to traffic into the correct anatomic locations at the appropriate times. This process is highly regulated and requires that lymphocytes interact...

  16. Enzyme assays.

    Science.gov (United States)

    Reymond, Jean-Louis; Fluxà, Viviana S; Maillard, Noélie

    2009-01-07

    Enzyme assays are analytical tools to visualize enzyme activities. In recent years a large variety of enzyme assays have been developed to assist the discovery and optimization of industrial enzymes, in particular for "white biotechnology" where selective enzymes are used with great success for economically viable, mild and environmentally benign production processes. The present article highlights the aspects of fluorogenic and chromogenic substrates, sensors, and enzyme fingerprinting, which are our particular areas of interest.

  17. Lymphocytic Interstitial Pneumonia.

    Science.gov (United States)

    Panchabhai, Tanmay S; Farver, Carol; Highland, Kristin B

    2016-09-01

    Lymphocytic interstitial pneumonia (LIP) is a rare lung disease on the spectrum of benign pulmonary lymphoproliferative disorders. LIP is frequently associated with connective tissue diseases or infections. Idiopathic LIP is rare; every attempt must be made to diagnose underlying conditions when LIP is diagnosed. Computed tomography of the chest in patients with LIP may reveal ground-glass opacities, centrilobular and subpleural nodules, and randomly distributed thin-walled cysts. Demonstrating polyclonality with immunohistochemistry is the key to differentiating LIP from lymphoma. The 5-year mortality remains between 33% and 50% and is likely to vary based on the underlying disease process.

  18. Stem cell survival is severely compromised by the thymidineanalog EdU (5-ethynyl-2'-deoxyuridine), an alternative to BrdU for proliferation assays and stem cell tracing

    DEFF Research Database (Denmark)

    Andersen, Ditte C; Skovrind, Ida; Christensen, Marlene Louise

    2013-01-01

    Stem cell therapy has opened up the possibility of treating numerous degenerating diseases. However, we are still merely at the stage of identifying appropriate sources of stem cells and exploring their full differentiation potential. Thus, tracking the stem cells upon in vivo engraftment...... and during in vitro co-culture is very important and is an area of research embracing many pitfalls. 5-Ethynyl-2'-deoxyuridine (EdU), a rather new thymidine analog incorporated into DNA, has recently been suggested to be a novel highly valid alternative to other dyes for labeling of stem cells and subsequent...... tracing of their proliferation and differentiation ability. However, our results herein do not at any stage support this recommendation, since EdU severely reduces the viability of stem cells. Accordingly, we found that transplanted EdU-labeled stem cells hardly survive upon in vivo transplantation...

  19. The development of in vitro mutagenicity testing systems using T-lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Albertini, R.J.

    1992-05-01

    This work has focused on the development of in vitro T-cell mutation assays. Conditions have been defined to measure the in vitro induction of mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in human T-lymphocytes. This assay is a parallel to our in vivo hprt assay, in that the same cells are utilized. However, the in vitro assay allows for carefully controlled dose response studies. 21 refs., 16 figs., 13 tabs.

  20. Rituximab for the treatment of patients with chronic lymphocytic leukemia

    Directory of Open Access Journals (Sweden)

    M Gentile

    2010-03-01

    Full Text Available M Gentile, E Vigna, C Mazzone, E Lucia, AG Recchia, L Morabito2, MG Bisconte, C Gentile, F Morabito1UOC di Ematologia, Azienda Ospedaliera di Cosenza, Italy; 2Servicio de Hematología y Hemoterapia, Hospital Universitario de Canarias, La Laguna, Tenerife, SpainAbstract: Chronic lymphocytic leukemia (CLL is a lymphoproliferative disorder that originates from antigen-experienced B lymphocytes that do not die and hence accumulate due to external survival signals or undergo apoptosis and are replenished by proliferating precursors. These neoplastic lymphocytes exhibit a characteristic immunophenotype of CD5+/CD19+/CD20+/HLA-DR+/CD23+/sIgdim. Thus, the CD20 antigen has been an appealing target for therapy. The introduction of the monoclonal antibody rituximab (anti-CD20 enabled an outstanding advance in CLL treatment. The introduction of this monoclonal antibody into chemotherapy regimens has dramatically improved complete response rates and progression-free survival in patients with both untreated and relapsed CLL. Although only preliminary data from phase III confirmatory trials have been reported, the FCR regimen, which combines fludarabine and cyclophosphamide with rituximab, is currently the most effective treatment regimen for CLL patients, and has also been demonstrated to significantly improve overall survival . The success of rituximab and the identification of other CLL lymphocyte surface antigens have spurred the development of a multitude of monoclonal antibodies targeting distinct proteins and epitopes in an attempt to target CLL cells more effectively.Keywords: rituximab, chronic lymphocytic leukemia, chemotherapy

  1. Chronic lymphocytic leukaemia

    Science.gov (United States)

    Kipps, Thomas J.; Stevenson, Freda K.; Wu, Catherine J.; Croce, Carlo M.; Packham, Graham; Wierda, William G.; O’Brien, Susan; Gribben, John; Rai, Kanti

    2017-01-01

    Chronic lymphocytic leukaemia (CLL) is a malignancy of CD5+ B cells that is characterized by the accumulation of small, mature-appearing lymphocytes in the blood, marrow and lymphoid tissues. Signalling via surface immunoglobulin, which constitutes the major part of the B cell receptor, and several genetic alterations play a part in CLL pathogenesis, in addition to interactions between CLL cells and other cell types, such as stromal cells, T cells and nurse-like cells in the lymph nodes. The clinical progression of CLL is heterogeneous and ranges from patients who require treatment soon after diagnosis to others who do not require therapy for many years, if at all. Several factors, including the immunoglobulin heavy-chain variable region gene (IGHV) mutational status, genomic changes, patient age and the presence of comorbidities, should be considered when defining the optimal management strategies, which include chemotherapy, chemoimmunotherapy and/or drugs targeting B cell receptor signalling or inhibitors of apoptosis, such as BCL-2. Research on the biology of CLL has profoundly enhanced our ability to identify patients who are at higher risk for disease progression and our capacity to treat patients with drugs that selectively target distinctive phenotypic or physiological features of CLL. How these and other advances have shaped our current understanding and treatment of patients with CLL is the subject of this Primer. PMID:28102226

  2. Genotoxic effects of textile printing dye exposed workers in India detected by micronucleus assay.

    Science.gov (United States)

    Sellappa, Sudha; Prathyumnan, Shibily; Joseph, Shyn; Keyan, Kripa S; Balachandar, Vellingiri

    2010-01-01

    The textile printing industry in South India employs a great number of workers that may possibly be exposed to toxic compounds. In the present study, subjects from textile printing units were investigated for the presence of genetic damage in their peripheral blood lymphocytes using micronucleus assay. Proliferation was also investigated using a nuclear division index. It was found that the micronucleus frequency was considerably greater in exposed subjects than in non exposed control subjects, but division was not increased in a statistically significant way. For the time being, this investigation should be considered as a preliminary study in which the influence of potential confounders could be adequately assessed. However, our results are non-ambiguous, indicating a potential health risk in these workers.

  3. Decreased deformability of lymphocytes in chronic lymphocytic leukemia

    Science.gov (United States)

    Zheng, Yi; Wen, Jun; Nguyen, John; Cachia, Mark A.; Wang, Chen; Sun, Yu

    2015-01-01

    This paper reports the first study of stiffness/deformability changes of lymphocytes in chronic lymphocytic leukemia (CLL) patients, demonstrating that at the single cell level, leukemic metastasis progresses are accompanied by biophysical property alterations. A microfluidic device was utilized to electrically measure cell volume and transit time of single lymphocytes from healthy and CLL patients. The results from testing thousands of cells reveal that lymphocytes from CLL patients have higher stiffness (i.e., lower deformability), as compared to lymphocytes in healthy samples, which was also confirmed by AFM indentation tests. This observation is in sharp contrast to the known knowledge on other types of metastatic cells (e.g., breast and lung cancer cells) whose stiffness becomes lower as metastasis progresses.

  4. Reduced lymphocyte activation in space - Role of cell-substratum interactions

    Science.gov (United States)

    Gmuender, F. K.; Kiess, M.; Lee, J.; Cogoli, A.; Sonnenfeld, G.

    1992-01-01

    The effect of substratum adhesiveness on lymphocyte responsiveness was investigated by reducing and blocking cell adhesion with poly-HEMA in a simple on ground system. Cells grown on medium thick and thick poly-HEMA films were rounded in shape and displayed no signs of spreading. By contrast, on tissue culture plastic and very thin poly-HEMA films, they showed clear signs of spreading. The mitogenic response of lymphocytes grown on thick poly-HEMA films was reduced by up to 68 percent of the control (tissue culture plastic). Interferon gamma production was virtually nil when the cells were grown on the least adhesive substratum. These results show that activated lymphocytes need to anchor and spread prior to achieving an optimal proliferation response. It is concluded that decreased lymphocyte adhesion could contribute to the depressed in vitro lymphocyte responsiveness found in the microgravity conditions of space flight.

  5. Effect of Excessive Iodine on Immune Function of Lymphocytes and Intervention with Selenium

    Institute of Scientific and Technical Information of China (English)

    CHEN Xiaoyi; LIU Liegang; YAO Ping; YU Dong; HAO Liping; SUN Xiufa

    2007-01-01

    In order to study the effect of excessive iodine on immune function of lymphocytes and the role of selenium supplementation with excessive iodine intake, the changes of T lymphocyte number, ratio of subsets, activity of natural killer (NK) cells and lymphocytes proliferation response were investigated. 150 female BALB/C mice were randomly divided into 5 groups in terms of their body weight (n=30 in each group), and 10 of each group were taken as one batch for test. Mice in the 5 groups were orally administrated with iodine 0 (group Ⅰ ), 1500 (group Ⅱ), 3000 (group Ⅲ),6000 μg/L (group Ⅳ), iodine 6000 μg/L plus selenium 0.3 mg/L (group Ⅴ) respectively for 30 days.Lymphocyte proliferation response, CD4+/CD8+, Th1/Th2 and the activity of NK cells were measured. CD4+/CD8+ was significantly lower, while lymphocyte proliferation response stronger, and Th1/Th2 and the activity of NK cells significantly higher in group Ⅳ than in group Ⅰ (P<0.01).There was no significant difference in all indexes between group Ⅴ and group Ⅰ (P>0.05). It was suggested that excessive iodine as exogenous chemical materials can induce disorders of T lymphocyte immune function in mice. 0.3 mg/L selenium supplementation can protect mice against toxicity induced by 6000 μg/L iodine.

  6. Assessment of cytotoxic and genotoxic potential of pyracarbolid by Allium test and micronucleus assay.

    Science.gov (United States)

    Özkara, Arzu; Akyıl, Dilek; Eren, Yasin; Erdoğmuş, S Feyza; Konuk, Muhsin; Sağlam, Esra

    2015-01-01

    The present study evaluates the cytotoxic and genotoxic potential of pyracarbolid using both micronuleus (MN) assay, in human lymphocytes, and Allium cepa assay, in the root meristem cells. In Allium test, EC50 value was determined in order to selecting the test concentrations for the assay and the root tips were treated with 25 ppm (EC50/2), 50 ppm (EC50) and 100 ppm (EC50 × 2) concentrations of pyracarbolid. One percent of dimethyl sulphoxide (DMSO) and methyl methane sulfonate (MMS) were used as negative and positive controls, respectively. In the micronucleus assay, the cultures were treated with four concentrations (250, 500, 750 and 1000 µg/ml) of pyracarbolid for 24 and 48 h, negative and positive controls were also used in the experiment parallely. The results showed that mitotic index (MI) significantly reduced with increasing the pyracarbolid concentration at each exposure time. It was also obtained that prophase and metaphase index decreased significantly in all concentration at each exposure time. Anaphase index decreased as well and results were found to be statistically significant, except 24 h. A significant increase was observed in MN frequency in all concentrations and both treatment periods when compared with the controls. Pyracarbolid also caused a significant reduction in the cytokinesis block proliferation index (CBPI) in all concentration and both exposure time.

  7. Dendritic cells transduced with Rsf-1/HBXAP gene generate specific cytotoxic T lymphocytes against ovarian cancer in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Li [Department of Gynecology Oncology, Shan Dong Tumor Hospital, Jinan, Shandong (China); Department of Obstetrics and Gynecology, Qilu Hospital, Shandong University, Jinan, Shandong (China); Kong, Beihua, E-mail: kongbeihua@sdu.edu.cn [Department of Obstetrics and Gynecology, Qilu Hospital, Shandong University, Jinan, Shandong (China); Sheng, Xiugui [Department of Gynecology Oncology, Shan Dong Tumor Hospital, Jinan, Shandong (China); Sheu, Jim Jinn-Chyuan [Human Genetic Center, China Medical University Hospital and Graduate Institute of Chinese Medical Science, China Medical University, Taichung City, Taiwan (China); Shih, Ie-Ming [Departments of Pathology, Oncology, and Gynecology and Obstetrics, Johns Hopkins Medical Institutions, Baltimore, MD 21231 (United States)

    2010-04-09

    Recently, some studies have indicated that Rsf-1/HBXAP plays a role in chromatin remodeling and transcriptional regulation that may contribute to tumorigenesis in ovarian cancer. The present study demonstrates that using dendritic cells (DCs) from human cord blood CD34{sup +} cells transduced with Rsf-1/HBXAP DNA plasmids by nucleofection generate specific cytotoxic T lymphocytes (CTL) against ovarian cancer in vitro. After transfection, DCs were analyzed for Rsf-1/HBXAP mRNA expression by RT-PCR and protein expression by Western blot. Then the DC phenotypes, T-cell stimulatory capacity, endocytic activity and migration capacity were explored by flow cytometry analysis, allogeneic mixed lymphocyte reaction, endocytosis and transwell chemotaxis assay, respectively. After transfection, Rsf-1/HBXAP expression was detected at mRNA and protein levels. Allogeneic T-cell proliferation induced by transfected DCs was obviously higher than non-transfected DCs, but the endocytosis capacity and migratory ability were not different. Rsf-1/HBXAP gene-transduced DCs could induce antigen-specific CTL and generate a very potent cytotoxicity to OVCAR3 cells. These data suggest that Rsf-1/HBXAP gene-transduced DCs may be a potential adjuvant immunotherapy for ovarian cancer in clinical applications.

  8. Effect of North American ginseng on 137Cs-induced micronuclei in human lymphocytes: a comparison with WR-1065.

    Science.gov (United States)

    Lee, Tung-Kwang; Wang, Weidong; O'Brien, Kevin F; Johnke, Roberta M; Wang, Tao; Allison, Ron R; Diaz, Angelica L

    2008-12-01

    To explore the radioprotective effect of a standardized North American ginseng extract (NAGE) on human peripheral blood lymphocytes (PBL), a micronuclei (MN) assay was conducted in PBL obtained from 12 volunteers. NAGE (50-1000 microg/mL) and WR-1065 (1 mM and 3 mM) were applied to PBL cultures at 0 h and 90 min post-irradiation. It was found that (1) the baseline MN yield of PBL ranged from 14.4 +/- 1.5 to 15.9 +/- 1.5 per 1000 binucleated cells (p > 0.05); after irradiation (1 Gy and 2 Gy), the MN yield increased sharply; (2) MN yields declined with increasing concentrations of NAGE and WR-1065. Even at 90 min post-irradiation of 1 Gy, the maximum level of MN reduction rate caused by NAGE and WR-1065 was 53.8% and 59.2%, respectively; after 2 Gy irradiation, it was 37.3% and 42%, respectively; (3) the MN distribution in PBL followed a non-Poisson distribution in all cases; and (4) both NAGE and WR-1065 showed no significant effect on the proliferation index of lymphocytes. The results indicate that NAGE is a relatively non-toxic natural product, which can be administered as a dietary supplement and has the potential to be a radiation countermeasure. (c) 2008 John Wiley & Sons, Ltd.

  9. Cytogenetic analyses of Azadirachtin reveal absence of genotoxicity but marked antiproliferative effects in human lymphocytes and CHO cells in vitro.

    Science.gov (United States)

    Mosesso, Pasquale; Bohm, Lothar; Pepe, Gaetano; Fiore, Mario; Carpinelli, Alice; Gäde, Gerd; Nagini, Siddavaram; Ottavianelli, Alessandro; Degrassi, Francesca

    2012-09-18

    In this work we have examined the genotoxic potential of the bioinsecticide Azadirachtin A (AZA) and its influence on cell proliferation on human lymphocytes and Chinese Hamster ovary (CHO) cells. AZA genotoxicity was assessed by the analysis of chromosomal aberrations and sister chromatid exchanges (SCEs) in the absence and presence of rat liver S9 metabolism. Primary DNA damage was also investigated by means of the comet assay. The results obtained clearly indicate that AZA is not genotoxic in mammalian cells. On the other hand, AZA proved to interfere with cell cycle progression as shown by modulation of frequencies of first (M1) and second division (M2) metaphases detected by 5-Bromo-2'-deoxyuridine labeling. Accumulation of M1 metaphases were more pronounced in human lymphocytes. In the transformed CHO cell line, however, significant increases of multinucleated interphases and polyploid cells were observed at long treatment time. At higher dose-levels, the incidence of polyploidy was close to 100%. Identification of spindle structure and number of centrosomes by fluorescent immunostaining with α- and γ-tubulin antibodies revealed aberrant mitoses exhibiting multipolar spindles with several centrosomal signals. These findings suggest that AZA can act either through a stabilizing activity of microtubules or by inhibition of Aurora A, since both mechanisms are able to generate genetically unstable polyploid cells with multipolar spindles and multinucleated interphases. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  10. Characterization of CRTAM gene promoter: AP-1 transcription factor control its expression in human T CD8 lymphocytes.

    Science.gov (United States)

    Valle-Rios, Ricardo; Patiño-Lopez, Genaro; Medina-Contreras, Oscar; Canche-Pool, Elsy; Recillas-Targa, Felix; Lopez-Bayghen, Esther; Zlotnik, Albert; Ortiz-Navarrete, Vianney

    2009-10-01

    Class-I MHC-restricted T-cell associated molecule (CRTAM) is a member of the Nectin-like adhesion molecule family. It is rapidly induced in NK, NKT and CD8(+) T cells. Interaction with its ligand Nectin-like 2 results in increased secretion of IFN-gamma by activated CD8(+) T lymphocytes. Through sequential bioinformatic analyses of the upstream region of the human CRTAM gene, we detected cis-elements potentially important for CRTAM gene transcription. Analyzing 2kb upstream from the ATG translation codon by mutation analysis in conjunction with luciferase reporter assays, electrophoretic mobility shify assay (EMSA) and supershift assays, we identified an AP-1 binding site, located at 1.4kb from the ATG translation codon of CRTAM gene as an essential element for CRTAM expression in activated but not resting human CD8(+) T cells. CRTAM expression was reduced in activated CD8(+) T cells treated with the JNK inhibitor SP600125, indicating that CRTAM expression is driven by the JNK-AP-1 signaling pathway. This study represents the first CRTAM gene promoter analysis in human T cells and indicates that AP-1 is a positive transcriptional regulator of this gene, a likely important finding because CRTAM has recently been shown to play a role in IFN-gamma and IL-17 production and T cell proliferation.

  11. Obinutuzumab in chronic lymphocytic leukemia.

    Science.gov (United States)

    Dupuis, Jehan

    2015-09-01

    Obinutuzumab is the second next-generation monoclonal anti-CD20 antibody (after ofatumumab) to enter clinical practice in chronic lymphocytic leukemia. Its superiority in association with chlorambucil as compared with chlorambucil alone has led to its approval as a first-line treatment for chronic lymphocytic leukemia, for patients who are not candidates for a more intensive treatment.

  12. SUBTYPES OF B LYMPHOCYTES IN PATIENTS WITH AUTOIMMUNE HEMOCYTOPENIA

    Institute of Scientific and Technical Information of China (English)

    Li-min Xing; Hai-rong Jia; Juan Sun; Chong-li Yang; Zong-hong Shao; Rong Fu; Hong Liu; Jun Shi; Lie Bai; Mei-feng Tu; Hua-quan Wang; Zhen-zhu Cui

    2007-01-01

    Objective To investigate the quantities of bone marrow CD5+ B lymphocytes in the patients with autoimmune hemocytopenia and the relationship between quantities of CD5+ B lymphocytes and clinical or laboratorial parameters.Methods Quantities of CD5+ B lymphocytes in the bone marrow of 14 patients with autoimmune hemolytic anemia (AIHA) or Evans syndrome, 22 immunorelated pancytopenia (IRP) patients, and 10 normal controls were assayed by flow cytometry. The correlation between their clinical or laboratorial parameters and CD5+ B lymphocytes was analyzed.Results The quantity of CD5+B lymphocytes of AIHA/Evans syndrome (34. 64% ± 19. 81% ) or IRP patients (35.81% ±16.83% ) was significantly higher than that of normal controls (12.00% ±1.97% , P<0. 05). However, there was no significant difference between AIHA/Evans syndrome and IRP patients (P > 0. 05). In all hemocytopenic patients, the quantity of bone marrow CD5+ B lymphocytes showed significantly negative correlation with serum complement C3 level (r = - 0. 416, P< 0. 05). In the patients with AIHA/Evans syndrome, the quantity of bone marrow CD5+ B lymphocytes showed significantly positive correlation with serum indirect bilirubin level (r = 1. 00, P<0. 05). In Evans syndrome patients, the quantity of CD5+ B lymphocytes in bone marrow showed significantly positive correlation with platelet-associated immunoglobulin G (r = 0. 761, P< 0. 05) and platelet-associated immunoglobulin M (r = 0. 925, P< 0. 05). The quantity of CD5+ B lymphocytes in bone marrow of all hemocytopenic patients showed significantly negative correlation with treatment response (tau-b = - 0. 289, P< 0. 05), but had no correlation with colony forming unit-erythroid ( r = - 0. 205, P > 0. 05 ) or colony forming unit-granulocyte-macrophage colonies ( r = -0.214, P>0.05).Conclusions The quantity of bone marrow CD5+ B lymphocytes in the patients with autoimmune hemocytopenia significantly increases and is correlated with disease

  13. The azo dyes Disperse Red 1 and Disperse Orange 1 increase the micronuclei frequencies in human lymphocytes and in HepG2 cells.

    Science.gov (United States)

    Chequer, Farah Maria Drumond; Angeli, José Pedro Friedmann; Ferraz, Elisa Raquel Anastácio; Tsuboy, Marcela Stefanini; Marcarini, Juliana Cristina; Mantovani, Mário Sérgio; de Oliveira, Danielle Palma

    2009-05-31

    The use of azo dyes by different industries can cause direct and/or indirect effects on human and environmental health due to the discharge of industrial effluents that contain these toxic compounds. Several studies have demonstrated the genotoxic effects of various azo dyes, but information on the DNA damage caused by Disperse Red 1 and Disperse Orange 1 is unavailable, although these dyes are used in dyeing processes in many countries. The aim of the present study was to evaluate the mutagenic activity of Disperse Red 1 and Disperse Orange 1 using the micronucleus (MN) assay in human lymphocytes and in HepG2 cells. In the lymphocyte assay, it was found that the number of MN induced by the lowest concentration of each dye (0.2 microg/mL) was similar to that of the negative control. At the other concentrations, a dose response MN formation was observed up to 1.0 microg/mL. At higher dose levels, the number of MN decreased. For the HepG2 cells the results were similar. With both dyes a dose dependent increase in the frequency of MN was detected. However for the HepG2, the threshold for this increase was 2.0 microg/mL, while at higher doses a reduction in the MN number was observed. The proliferation index was also calculated in order to evaluate acute toxicity during the test. No differences were detected between the different concentrations tested and the negative control.

  14. Stem cell survival is severely compromised by the thymidineanalog EdU (5-ethynyl-2'-deoxyuridine), an alternative to BrdU for proliferation assays and stem cell tracing.

    Science.gov (United States)

    Andersen, Ditte C; Skovrind, Ida; Christensen, Marlene Louise; Jensen, Charlotte H; Sheikh, Søren P

    2013-11-01

    Stem cell therapy has opened up the possibility of treating numerous degenerating diseases. However, we are still merely at the stage of identifying appropriate sources of stem cells and exploring their full differentiation potential. Thus, tracking the stem cells upon in vivo engraftment and during in vitro co-culture is very important and is an area of research embracing many pitfalls. 5-Ethynyl-2'-deoxyuridine (EdU), a rather new thymidine analog incorporated into DNA, has recently been suggested to be a novel highly valid alternative to other dyes for labeling of stem cells and subsequent tracing of their proliferation and differentiation ability. However, our results herein do not at any stage support this recommendation, since EdU severely reduces the viability of stem cells. Accordingly, we found that transplanted EdU-labeled stem cells hardly survive upon in vivo transplantation into regenerating muscle, whereas stem cells labeled in parallel with another dye survived very well and also participated in myofiber formation. Similar data were obtained upon in vitro myogenic culture, and further analysis showed that EdU reduced cell numbers by up to 88 % and increased the cell volume of remaining cells by as much as 91 %. Even at low EdU concentrations, cell survival and phenotype were substantially compromised, and the myogenic differentiation potential was inhibited. Since we examined both primary derived cells and cell lines from several species with the same result, this appears to be a common trait of EdU. We therefore suggest that EdU labeling should be avoided (or used with precaution) for stem cell tracing purposes.

  15. Use of cytogenetic endpoints in human lymphocytes as indicators of exposure to genotoxicants

    Energy Technology Data Exchange (ETDEWEB)

    Preston, R.J.

    1983-01-01

    The utility of the lymphocyte assay for chemical exposures is discussed, with reference to the underlying mechanisms of induction of chromosome alterations. Possible ways of increasing the sensitivity of the assay in order to make it more useful for population monitoring are also discussed. 28 references. (ACR)

  16. In Vitro Influence of Mycophenolic Acid on Selected Parameters of Stimulated Peripheral Canine Lymphocytes.

    Directory of Open Access Journals (Sweden)

    Maciej Guzera

    Full Text Available Mycophenolic acid (MPA is an active metabolite of mycophenolate mofetil, a new immunosuppressive drug effective in the treatment of canine autoimmune diseases. The impact of MPA on immunity is ambiguous and its influence on the canine immune system is unknown. The aim of the study was to determine markers of changes in stimulated peripheral canine lymphocytes after treatment with MPA in vitro. Twenty nine healthy dogs were studied. Phenotypic and functional analysis of lymphocytes was performed on peripheral blood mononuclear cells cultured with mitogens and different MPA concentrations- 1 μM (10-3 mol/m3, 10 μM or 100 μM. Apoptotic cells were detected by Annexin V and 7-aminoactinomycin D (7-AAD. The expression of antigens (CD3, CD4, CD8, CD21, CD25, forkhead box P3 [FoxP3] and proliferating cell nuclear antigen [PCNA] was assessed with monoclonal antibodies. The proliferation indices were analyzed in carboxyfluorescein diacetate succinimidyl ester (CFSE-labeled cells. All analyses were performed using flow cytometry. The influence of MPA on apoptosis was dependent on the mechanism of cell activation and MPA concentration. MPA caused a decrease in the expression of lymphocyte surface antigens, CD3, CD8 and CD25. Its impact on the expression of CD4 and CD21 was negligible. Its negative influence on the expression of FoxP3 was dependent on cell stimulation. MPA inhibited lymphocyte proliferation. In conclusion, MPA inhibited the activity of stimulated canine lymphocytes by blocking lymphocyte activation and proliferation. The influence of MPA on the development of immune tolerance-expansion of Treg cells and lymphocyte apoptosis-was ambiguous and was dependent on the mechanism of cellular activation. The concentration that MPA reaches in the blood may lead to inhibition of the functions of the canine immune system. The applied panel of markers can be used for evaluation of the effects of immunosuppressive compounds in the dog.

  17. The state of T cells before cryopreservation: Effects on post-thaw proliferation and function.

    Science.gov (United States)

    Luo, Ying; Wang, Peng; Liu, Hui; Zhu, Zhengyan; Li, Chenglong; Gao, Yingtang

    2017-08-31

    We aim to assess the effect of the state of T cells before cryopreservation on the post-thaw proliferative capacity, phenotype and functional response. Peripheral blood mononuclear cells (PBMCs) were isolated from a hepatocellular carcinoma (HCC) patient, and the T cells were frozen during cell culture according to our experimental design. After a period of re-culture, the proliferative capacity of the cryopreserved cells, the expression of T cell surface markers and the secretion of IFN-γ and IL-10 were assayed. There was >90% cell viability after thaw in every group. Lymphocytes cryopreserved at day 4, 8 or 12 during the cell culture were allowed to recover for 24 h, whereas lymphocytes cryopreserved while freshly isolated were allowed to recover for 72 h. After the period of re-culture, cryopreservation at day 4, 8 or 12 during T cell culture was not found to alter the T cell subpopulation. The proportions of NKT and Treg cells were unchanged when cells were cryopreserved at day 12 during T cell culture. IFN-γ secretion was not impacted by cryopreservation, and IL-10 secretion was significantly decreased when cells were cryopreserved at day 8 or 12 during T cell culture. The state of T cells before cryopreservation has effects on the post-thaw proliferation capacity, the phenotype and the secretion of IFN-γ and IL-10. Cryopreservation of lymphocytes at day 8 or 12 during the cell culture may be the best choice for T cell immunotherapy. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Lack of direct immunosuppressive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on human peripheral blood lymphocyte subsets in vitro

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    Lang, D.S. (Center for Environmental Medicine and Lung Biology, Univ. of North Carolina, Chapel Hill, NC (United States)); Becker, S. (TCR-Environmental Corp., Inc., Chapel Hill, NC (United States)); Clark, G.C. (National Inst of Environmental Health Science, Lab. of Biochemical Risk Analysis, RTP, NC (United States)); Devlin, R.B. (US Environmental Protection Agency, Health Effects Research Lab., RTP, NC (United States)); Koren, H.S. (US Environmental Protection Agency, Health Effects Research Lab., RTP, NC (United States))

    1994-05-01

    The direct effects of dioxin on human PBL subpopulations have been studied, in order to determine their usefulness as sensitive biomarkers for human dioxin exposure. Lymphocyte cultures from healthy individuals were treated with 10[sup -7] M-10[sup -14] M TCDD in the absence and presence of stimulation with pokeweed mitogen (PWM) or anti-CD3 monoclonal antibody (moAb; OKT3) for 3 days. Cytochrome P450 (CYP1A1) enzyme induction, one of the best studied direct biological effects of TCDD on numerous cell types, was assayed in parallel by ethoxyresorufin-O-deethylase (EROD) activity. Percentages of the different lymphocytes subsets, including CD2 (T cells); CD4; CD45 RA (subpressor-inducer/virgin T cells); CD4 CD29; CD8; CD19 (B cells) as well as interleukin 2 (IL-2) receptor (CD25) and class II antigen (HLA-DR) expression, were analyzed by flow cytometry. DNA synthesis was determined by [sup 3]H-thymidine uptake after 3 days of culture. In the present study, all stimulated lymphocyte cultures showed a dose-dependent significant increase of CYP1A1 activity at dioxin concentrations of 10[sup -7] and 10[sup -9] M. No enzyme activity could be detected at lower concentrations of TCDD. On the other hand, neither alteration in surface marker distribution nor suppression of lymphocyte proliferation could be demonstrated in mitogen-activated cells following any concentration of TCDD treatment. These data suggest that the inducibility of CYP1A1 enzyme activity is not correlated with direct immunotoxic effects in vitro in human PBL. (orig./MG)

  19. Determination of cell survival after irradiation via clonogenic assay versus multiple MTT Assay - A comparative study

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    Buch Karl

    2012-01-01

    Full Text Available Abstract For studying proliferation and determination of survival of cancer cells after irradiation, the multiple MTT assay, based on the reduction of a yellow water soluble tetrazolium salt to a purple water insoluble formazan dye by living cells was modified from a single-point towards a proliferation assay. This assay can be performed with a large number of samples in short time using multi-well-plates, assays can be performed semi-automatically with a microplate reader. Survival, the calculated parameter in this assay, is determined mathematically. Exponential growth in both control and irradiated groups was proven as the underlying basis of the applicability of the multiple MTT assay. The equivalence to a clonogenic survival assay with its disadvantages such as time consumption was proven in two setups including plating of cells before and after irradiation. Three cell lines (A 549, LN 229 and F 98 were included in the experiment to study its principal and general applicability.

  20. T lymphocyte insensitivity to corticosteroids in chronic obstructive pulmonary disease

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    Kaur Manminder

    2012-03-01

    Full Text Available Abstract Background There are increased numbers of activated lymphocytes in the lungs of chronic obstructive pulmonary disease (COPD patients. The clinical benefits of corticosteroids in COPD patients are limited. Our hypothesis is that lymphocytes play a role in this corticosteroid insensitivity. Objectives To investigate the effects of the corticosteroid dexamethasone on lung lymphocyte cytokine production from patients with COPD compared to controls. Methods Cultured airway lymphocytes obtained by bronchoscopy from healthy non-smokers (HNS, smokers (S and COPD patients were stimulated with phytohaemagglutinin (PHA & phorbol myristate acetate (PMA, +/- dexamethasone. Supernatants were assayed for interleukin (IL-2 and interferon (IFNγ. Immunofluoresence was used to analyse changes in CD8 glucocorticoid receptor (GRα and GRβ expression. Results The inhibition of PHA/PMA stimulated IFNγ production by dexamethasone was reduced in COPD patients compared to HNS (p p Conclusions IFNγ production from COPD airway lymphocytes is corticosteroid insensitive. This phenomenon may be important in the poor clinical response often observed with corticosteroids.

  1. Ivabradine Reduces Chemokine-Induced CD4-Positive Lymphocyte Migration

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    Thomas Walcher

    2010-01-01

    Full Text Available Aims. Migration of CD4-positive lymphocytes into the vessel wall is a critical step in atherogenesis. Recent data suggest that ivabradine, a selective I(f-channel blocker, reduces atherosclerotic plaque formation in apolipoprotein E-deficient mice, hitherto nothing is known about the mechanism by which ivabradine modulates plaque formation. Therefore, the present study investigated whether ivabradine regulates chemokine-induced migration of lymphocytes. Methods and results. Stimulation of CD4-positive lymphocytes with SDF-1 leads to a 2.0±0.1 fold increase in cell migration (P<.01; n=7. Pretreatment of cells with ivabradine reduces this effect to a maximal 1.2±0.1 fold induction at 0.1 µmol/L ivabradine (P<.01 compared to SDF-1-treated cells, n=7. The effect of ivabradine on CD4-positive lymphocyte migration was mediated through an early inhibition of chemokine-induced PI-3 kinase activity as determined by PI-3 kinase activity assays. Downstream, ivabradine inhibits activation of the small GTPase Rac and phosphorylation of the Myosin Light Chain (MLC. Moreover, ivabradine treatment reduces f-actin formation as well as ICAM3 translocation to the uropod of the cell, thus interfering with two important steps in T cell migration. Conclusion. Ivabradine inhibits chemokine-induced migration of CD4-positive lymphocytes. Given the crucial importance of chemokine-induced T-cell migration in early atherogenesis, ivabradine may be a promising tool to modulate this effect.

  2. Laboratorial diagnosis of lymphocytic meningitis

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    Sérgio Monteiro de Almeida

    Full Text Available Meningitis is the main infectious central nervous system (CNS syndrome. Viruses or bacteria can cause acute meningitis of infectious etiology. The term "Aseptic Meningitis" denotes a clinical syndrome with a predominance of lymphocytes in the cerebrospinal fluid (CSF, with no common bacterial agents identified in the CSF. Viral meningitis is considered the main cause of lymphocyte meningitis. There are other etiologies of an infectious nature. CSF examination is essential to establish the diagnosis and to identify the etiological agent of lymphocytic meningitis. We examined CSF characteristics and the differential diagnosis of the main types of meningitis.

  3. Laboratorial diagnosis of lymphocytic meningitis

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    Sérgio Monteiro de Almeida

    2007-10-01

    Full Text Available Meningitis is the main infectious central nervous system (CNS syndrome. Viruses or bacteria can cause acute meningitis of infectious etiology. The term "Aseptic Meningitis" denotes a clinical syndrome with a predominance of lymphocytes in the cerebrospinal fluid (CSF, with no common bacterial agents identified in the CSF. Viral meningitis is considered the main cause of lymphocyte meningitis. There are other etiologies of an infectious nature. CSF examination is essential to establish the diagnosis and to identify the etiological agent of lymphocytic meningitis. We examined CSF characteristics and the differential diagnosis of the main types of meningitis.

  4. Activation of lymphocytes induced by bronchial epithelial cells with prolonged RSV infection.

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    Ling Qin

    Full Text Available Respiratory syncytial virus (RSV preferentially infects airway epithelial cells,which might be responsible for susceptibility to asthma; however, the underlying mechanism is not clear. This study determined the activation of lymphocytes and drift of helper T (Th subsets induced by RSV-infected human bronchial epithelial cells (HBECs in vitro. HBECs had prolonged infection with RSV, and lymphocytes isolated from human peripheral blood were co-cultured with RSV-infected HBECs. Four groups were established, as follows: lymphocytes (group L; lymphocytes infected with RSV (group RL; co-culture of lymphocytes with non-infected HBECs (group HL; and co-culture of lymphocytes with infected HBECs (group HRL. After co-culture with HBECs for 24 hours, lymphocytes were collected and the following were determined in the 4 groups: cell cycle status; apoptosis rate; and concentrations of IL-4, IFN-γ, and IL-17 in the supernatants. Cell cycle analysis for lymphocytes showed a significant increase in S phase cells, a decrease in G1 phase cells, and a higher apoptosis rate in group HRL compared with the other three groups. In group HRL, the levels of IL-4, IFN-γ, and IL-17 in supernatants were also higher than the other three groups. For further study, lymphocytes were individually treated with supernatants from non-infected and RSV-infected HBECs for 24 h. We showed that supernatants from RSV-infected HBECs induced the differentiation of Th2 and Th17 subsets, and suppressed the differentiation of Treg subsets. Our results showed that HBECs with prolonged RSV infection can induce lymphocyte proliferation and apoptosis, and enhance the release of cytokines by lymphocytes. Moreover, subset drift might be caused by RSV-infected HBECs.

  5. ROLE OF B LYMPHOCYTE AND ITS SUBPOPULATIONS IN PATHOGENESIS OF IMMUNORELATED PANCYTOPENIA

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To measure the quantities and apoptosis-related protein levels of B lymphocyte in the patients with immunorelated pancytopenia (IRP) and explore the action of B lymphocyte in the pathogenic mechanism of IRP.Methods Quantities of whole B lymphocytes and CD5 + B lymphocytes as well as the expressions of Fas and Bcl-2in B lymphocytes in 35 patients with untreated IRP, 15 IRP patients in complete remission (CR), and 10 normal controls were assayed by flow cytometry.Results The percentages of B lymphocyte and CD5 + B lymphocyte were significantly higher in untreated IRP patients than in CR IRP patients and normal controls ( P < 0. 05 ), and there was no significant difference between the latter two groups (P > 0. 05). There was no significant difference of Fas expression in B lymphocyte among three groups (P >0. 05). The expression of Bcl-2 in B lymphocyte was significantly higher in untreated patients than in CR patients or normal controls (P <0. 01 ), and significantly higher in CR patients than in normal controls (P <0. 01 ). The apoptosisrelated index was significantly lower in untreated patients than in CR patients or normal controls ( P < 0. 05 ), and significantly lower in CR patients than in normal controls ( P < 0. 05 ). The percentage of B lymphocyte was positively correlated with post-treated response time ( r = 0. 53, P < 0. 01 ).Conclusion The production of auto-antibodies in IRP patients probably has some relationship with the abnormal quantities of B lymphocyte and its subpopulations as well as with the inhibition of B lymphocyte apoptosis.

  6. In vitro effects of mesenchymal stem cells on secreting function of T lymphocytes and CD4~+CD25~+T cells from patients with immune thrombo-cytopenia

    Institute of Scientific and Technical Information of China (English)

    赵霞

    2014-01-01

    Objective To analyze in vitro the effect of mesenchymal stem cells(MSCs)on secreting cytokines by T lymphocytes and ratio of CD4+CD25+T cells from patients with immune thrombocytopenia(ITP).Methods Human bone marrow-derived MSCs were isolated by Ficoll Hypaque and cultured for proliferating to passage cells.Allogeneic T lymphocytes

  7. Electrostimulation of rat callus cells and human lymphocytes in vitro

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    Aro, H.; Eerola, E.; Aho, A.J.; Penttinen, R.

    1984-01-01

    Asymmetrical pulsing low voltage current was supplied via electrodes to cultured rat fracture callus cells and human peripheral blood lymphocytes. The (/sup 3/H)thymidine incorporation of the callus cells and 5-(/sup 125/I)iodo-2'-deoxyuridine incorporation of the lymphocytes were determined. The growth pattern of callus cells (estimated by cellular density) did not respond to electrical stimulation. However, the uptake of (/sup 3/H)thymidine was increased at the early phase of cell proliferation and inhibited at later phases of proliferation. The (/sup 3/H)thymidine uptake of confluent callus cell cultures did not respond to electrical stimulation. Lymphocytes reacted in a similar way; stimulated cells took up more DNA precursor than control cells at the early phase of stimulation. During cell division, induced by the mitogens phytohemagglutinin and Concanavalin-A, the uptake of DNA precursor by stimulated cells was constantly inhibited. The results suggest that electrical stimuli affect the uptake mechanisms of cell membranes. The duality of the effect seems to be dependent on the cell cycle.

  8. C/EBPβ regulates transcription factors critical for proliferation and survival of multiple myeloma cells

    Science.gov (United States)

    Pal, Rekha; Janz, Martin; Galson, Deborah L.; Gries, Margarete; Li, Shirong; Jöhrens, Korinna; Anagnostopoulos, Ioannis; Dörken, Bernd; Mapara, Markus Y.; Borghesi, Lisa; Kardava, Lela; Roodman, G. David; Milcarek, Christine

    2009-01-01

    CCAAT/enhancer-binding protein β (C/EBPβ), also known as nuclear factor–interleukin-6 (NF-IL6), is a transcription factor that plays an important role in the regulation of growth and differentiation of myeloid and lymphoid cells. Mice deficient in C/EBPβ show impaired generation of B lymphocytes. We show that C/EBPβ regulates transcription factors critical for proliferation and survival in multiple myeloma. Multiple myeloma cell lines and primary multiple myeloma cells strongly expressed C/EBPβ, whereas normal B cells and plasma cells had little or no detectable levels of C/EBPβ. Silencing of C/EBPβ led to down-regulation of transcription factors such as IRF4, XBP1, and BLIMP1 accompanied by a strong inhibition of proliferation. Further, silencing of C/EBPβ led to a complete down-regulation of antiapoptotic B-cell lymphoma 2 (BCL2) expression. In chromatin immunoprecipitation assays, C/EBPβ directly bound to the promoter region of IRF4, BLIMP1, and BCL2. Our data indicate that C/EBPβ is involved in the regulatory network of transcription factors that are critical for plasma cell differentiation and survival. Targeting C/EBPβ may provide a novel therapeutic strategy in the treatment of multiple myeloma. PMID:19717648

  9. Benzene metabolites induce apoptosis in lymphocytes.

    Science.gov (United States)

    Martínez-Velázquez, M; Maldonado, V; Ortega, A; Meléndez-Zajgla, J; Albores, A

    2006-08-01

    Benzene is an important environmental pollutant with important health implications. Exposure to this aromatic hydrocarbon is associated with hematotoxicity, and bone marrow carcinogenic effects. It has been shown that benzene induces oxidative stress, cell cycle alterations, and programmed cell death in cultured cells. Hepatic metabolism of benzene is thought to be a prerequisite for its bone marrow toxicity. Nevertheless, there are no reports on the cellular effects of reactive intermediates derived from hepatic metabolism of benzene. Thus, the goal of this project was to determine the cellular alterations of benzene metabolites produced by the cultured hepatic cell line HepG2. Supernatants collected from these cells were applied to a culture of freshly isolated lymphocytes. A higher decrease in cell viability was found in cells exposed to these supernatants than to unmetabolized benzene. This viability decrease was due to apoptosis, as determined by Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) assay and internucleosomal fragmentation of DNA. When supernatants were analyzed by HPLC, we found that not all the hydrocarbon was biotransformed, since a 28 microM concentration (37%) remained. The only metabolite found in the culture medium was muconic acid. The present results show that muconic acid derived from benzene metabolism is able to cooperate with the pollutant for the induction of apoptosis in rat lymphocytes.

  10. Artificial antigen-presenting cells plus IL-15 and IL-21 efficiently induce melanoma-specific cytotoxic CD8+CD28+ T lymphocyte responses

    Institute of Scientific and Technical Information of China (English)

    Xia Yu; Yuan Fang; Xi Li; Nuo Zhou; Yong-Xiang Zhao; Xiao-Ling Lu; Jian He; Sodaly Mongkhoune; Yi Peng; Yuan Xie; Jing Su; Su-Fang Zhou; Xiao-Xun Xie; Guo-Rong Luo

    2013-01-01

    To develop a novel artificial antigen-presenting system for efficiently inducing melanoma-specific CD8+CD28+ cytotoxic T lymphocyte (CTL) responses. Methods: Cell-sized Dynabeads® M-450 Epoxy beads coated with H-2Kb:Ig-TRP2180-188 and anti-CD28 antibody were used as artificial antigen-presenting cells (aAPCs) to induce melanoma-specific CD8+CD28+CTL responses with the help of IL-21 and IL-15. Dimer staining, proliferation, ELISPOT, and cytotoxicity experiments were conducted to evaluate the frequency and activity of induced CTLs. Results: Dimer staining demonstrated that the new artificial antigen-presenting system efficiently induced melanoma TRP2-specific CD8+CD28+ CTLs. Proliferation and ELISPOT assays indicated that the induced CTLs rapidly proliferate and produce increased IFN-γ under the stimulation of H-2Kb:Ig-TRP2-aAPCs, IL-15, and IL-21. In addition, cytotoxicity experiments showed that induced CTLs have specific killing activity of target cells. Conclusions: The new artificial antigen-presenting system including aAPCs plus IL-21 and IL-15 can induce a large number of antigen-specific CD8+CD28+ CTLs against the melanoma. Our study provides evidence for a novel adoptive immunotherapy against tumors.

  11. Suiciding of lymphocytic precursor cells by tritiated nucleosides, in vitro.

    Science.gov (United States)

    Uyeki, E M; Nishimura, T; Bisel, T U

    1978-02-01

    Differences in suiciding by various tritiated nucleosides were observed between two functional assays for in vitro lymphocytic precursor cell development, the hemolysin plaque-forming cell (PFC) assay and the B lymphocytic colony-forming cell (CFC-L) assay, using BDF1 mouse spleen cells. PFC growth was markedly reduced by an early (days 0-1) pulse of tritiated deoxyadenosine ([3H]dAdo), but relatively unaffected by a pulse of tritiated thymidine ([3H]dThd) during the same interval. In contrast, CFC-L formation significantly dropped after an early (day 0) [3H]dThd pulse, as well as after pulses of [3H]dAdo and the corresponding tritiated ribosides, uridine and adenosine. This implied a cycling state in an early lymphocytic precursor cell, as opposed to the PFC insensitivity to an early [3H]dThd pulse. The response pattern of colonies and clusters to [3H]dThd supported our notion of a delayed suiciding of CFC contributing to the increase in cluster numbers.

  12. Angiogenesis Assays.

    Science.gov (United States)

    Nambiar, Dhanya K; Kujur, Praveen K; Singh, Rana P

    2016-01-01

    Neoangiogenesis constitutes one of the first steps of tumor progression beyond a critical size of tumor growth, which supplies a dormant mass of cancerous cells with the required nutrient supply and gaseous exchange through blood vessels essentially needed for their sustained and aggressive growth. In order to understand any biological process, it becomes imperative that we use models, which could mimic the actual biological system as closely as possible. Hence, finding the most appropriate model is always a vital part of any experimental design. Angiogenesis research has also been much affected due to lack of simple, reliable, and relevant models which could be easily quantitated. The angiogenesis models have been used extensively for studying the action of various molecules for agonist or antagonistic behaviour and associated mechanisms. Here, we have described two protocols or models which have been popularly utilized for studying angiogenic parameters. Rat aortic ring assay tends to bridge the gap between in vitro and in vivo models. The chorioallantoic membrane (CAM) assay is one of the most utilized in vivo model system for angiogenesis-related studies. The CAM is highly vascularized tissue of the avian embryo and serves as a good model to study the effects of various test compounds on neoangiogenesis.

  13. Expression of p13MTCP1 is restricted to mature T-cell proliferations with t(X;14) translocations.

    Science.gov (United States)

    Madani, A; Choukroun, V; Soulier, J; Cacheux, V; Claisse, J F; Valensi, F; Daliphard, S; Cazin, B; Levy, V; Leblond, V; Daniel, M T; Sigaux, F; Stern, M H

    1996-03-01

    T-cell prolymphocytic leukemia (T-PLL), a rare form of mature T-cell leukemias, and ataxia telangiectasia clonal proliferation, a related condition occurring in patients suffering from ataxia telangiectasia, have been associated to translocations involving the 14q32.1 or Xq28 regions, where are located the TCL1 and MTCP1 putative oncogenes, respectively. The MTCP1 gene is involved in the t(X;14)(q28;q11) translocation associated with these T-cell proliferations. Alternative splicing generates type A and B transcripts that potentially encode two entirely distinct proteins; type A transcripts code for a small mitochondrial protein, p8MTCP1, and type B transcripts, containing an additional open reading frame, may code for 107 amino-acid protein, p13MTCP1. The recently cloned TCL1 gene, also involved in translocations and inversions associated with T-cell proliferations, codes for a 14-kD protein that displays significant homology with p13MTCP1. We have generated rabbit antisera against this putative p13MTCP1 protein and screened for expression of p13MTCP1 normal lymphoid tissues and 33 cases of immature and mature lymphoid T-cell proliferations using a sensitive Western blot assay. We also investigated the MTCP1 locus configuration by Southern blot analysis. The p13MTCP1 protein was detected in the three T-cell proliferations with MTCP1 rearrangements because of t(X;14) translocations, but neither in normal resting and activated lymphocytes nor in the other T-cell leukemias. Our data support the hypothesis that p13MTCP1 and p14TCL1 form a new protein family that plays a key role in the pathogenesis of T-PLL and related conditions.

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

    Science.gov (United States)

    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 ...

  15. SERUM HAPTOGLOBIN SUPPRESSES T-LYMPHOCYTE FUNCTIONS FOLLOWING BURNS

    Institute of Scientific and Technical Information of China (English)

    王凤君; 黄文华; 黎鳌

    1996-01-01

    It is well known that serum immunosuppressive factors play an important role in the mechanismi of postburn immunosuppression.This study was intended to investigate the effect of haptoglobin,purified from the serum of burned patients by affinity chromatography,on the proliferation and interleukin-2(IL-2)secretion of normal nurine thymocytes induced by conA and the proliferation of IL-2 dependent cell line (CTLL-2) stimulated by recombinant human IL-2,so as to elucidate the role of serum haptoglobin in postburn T-lymphocyte dysfunction.The results showed that purified haptoglobin,at the level equivalent to the concentration found in serum of burned patients,significantly inhibited the prolifration and IL-2 secretion of normal murine thymocytes as well as CTLL-2 proliferation;wheres it exhibited no immunosuppressive effects at the level equivalent to the concentration found in serum of normal vohmteers.According to the results reported here,it is suggested that extraordinary increase in serum haptoglobin level may be an important factor of impaired T-lymphocyte responses following burns.

  16. Potentiation of lymphocyte proliferative responses by nickel sulfide

    Science.gov (United States)

    Jaramillo, A.; Sonnenfeld, G.

    1992-01-01

    Crystalline nickel sulfide (NiS) induced a spleen cell proliferation that resembles a mixed lymphocyte reaction (MLR). It depended on cell-cell interaction, induced high levels of interleukin-1 (IL-1) and interleukin-2 (IL-2) and the responding cell subpopulation was composed of CD4+ T lymphocytes. Furthermore, the proliferation was inhibited in a dose-dependent manner by magnesium. Crystalline NiS also increased significantly the spleen cell proliferative response to concanavalin A (Con A) and lipopolysaccharide (LPS) with magnesium potentiating the combined effects of crystalline NiS and mitogens. Interestingly, crystalline NiS did not show any effect on the induction of IL-2 by Con A. The results described herein suggest that crystalline NiS can potentiate both antigenic (MLR) and mitogenic (Con A and LPS) proliferative responses in vitro. Crystalline NiS appears to potentiate these responses by acting in the form of ionic nickel on several intracellular targets for which magnesium ions have different noncompetitive interactions. The effects of magnesium on the potentiating action of crystalline NiS are different depending upon the type of primary stimulatory signal for proliferation (mitogenic or antigenic).

  17. Human liver sinusoidal endothelial cells promote intracellular crawling of lymphocytes during recruitment: A new step in migration.

    Science.gov (United States)

    Patten, Daniel A; Wilson, Garrick K; Bailey, Dalan; Shaw, Robert K; Jalkanen, Sirpa; Salmi, Marko; Rot, Antal; Weston, Chris J; Adams, David H; Shetty, Shishir

    2017-01-01

    The recruitment of lymphocytes via the hepatic sinusoidal channels and positioning within liver tissue is a critical event in the development and persistence of chronic inflammatory liver diseases. The hepatic sinusoid is a unique vascular bed lined by hepatic sinusoidal endothelial cells (HSECs), a functionally and phenotypically distinct subpopulation of endothelial cells. Using flow-based adhesion assays to study the migration of lymphocytes across primary human HSECs, we found that lymphocytes enter into HSECs, confirmed by electron microscopy demonstrating clear intracellular localization of lymphocytes in vitro and by studies in human liver tissues. Stimulation by interferon-γ increased intracellular localization of lymphocytes within HSECs. Furthermore, using confocal imaging and time-lapse recordings, we demonstrated "intracellular crawling" of lymphocytes entering into one endothelial cell from another. This required the expression of intracellular adhesion molecule-1 and stabilin-1 and was facilitated by the junctional complexes between HSECs.

  18. Blood and alveolar lymphocyte subsets in pulmonary cytomegalovirus infection after lung transplantation

    Directory of Open Access Journals (Sweden)

    Grenet Dominique

    2001-09-01

    Full Text Available Abstract Background Cytomegalovirus (CMV pneumonitis has been shown to be associated with lymphocytic alveolitis after lung transplantation. In the present study, we investigated a series of bronchoalveolar (BAL and blood samples, collected in the absence of rejection or acute infectious episodes. in order -1: to evaluate intra-alveolar cell population changes concomitant with CMV replication and -2: to reappraise the value of cell population analysis in the management of patients after lung transplantation. Methods We used flow cytometry to investigate modifications of lymphocyte subpopulations related to pulmonary cytomegalovirus infections in blood and BAL samples from a series of 13 lung transplant recipients. After exclusion of samples obtained during pulmonary rejection, bronchiolitis obliterans or acute bacterial infection, 48 blood and BAL samples were retained for analysis: 17 were CMV positive by shell-vial assay and 31 were CMV negative in blood and BAL. Results Our results demonstrate that pulmonary CMV infection is associated with a significant increase in the total lymphocyte population in BAL samples, but with minor modifications of the various lymphocyte subpopulations and a significantly higher absolute number of B lymphocytes in blood samples. Conclusions Cytomegalovirus pulmonary infection is accompanied by only minor changes in BAL lymphocyte subpopulations. The study of BAL lymphocyte subpopulations therefore appears to be of limited clinical value in the diagnosis of pulmonary CMV infection. However, increased blood B-lymphocytes seems to be a clinical feature associated with CMV infection.

  19. Is the Oxidative DNA Damage Level of Human Lymphocyte Correlated with the Antioxidant Capacity of Serum or the Base Excision Repair Activity of Lymphocyte?

    Directory of Open Access Journals (Sweden)

    Yi-Chih Tsai

    2013-01-01

    Full Text Available A random screening of human blood samples from 24 individuals of nonsmoker was conducted to examine the correlation between the oxidative DNA damage level of lymphocytes and the antioxidant capacity of serum or the base excision repair (BER activity of lymphocytes. The oxidative DNA damage level was measured with comet assay containing Fpg/Endo III cleavage, and the BER activity was estimated with a modified comet assay including nuclear extract of lymphocytes for enzymatic cleavage. Antioxidant capacity was determined with trolox equivalent antioxidant capacity assay. We found that though the endogenous DNA oxidation levels varied among the individuals, each individual level appeared to be steady for at least 1 month. Our results indicate that the oxidative DNA damage level is insignificantly or weakly correlated with antioxidant capacity or BER activity, respectively. However, lymphocytes from carriers of Helicobacter pylori (HP or Hepatitis B virus (HBV tend to give higher levels of oxidative DNA damage (P<0.05. Though sera of this group of individuals show no particular tendency with reduced antioxidant capacity, the respective BER activities of lymphocytes are lower in average (P<0.05. Thus, reduction of repair activity may be associated with the genotoxic effect of HP or HBV infection.

  20. 破布木果提取物对小鼠脾淋巴细胞的增殖作用%Proliferative effects of extracts from Cordia dichotoma seeds on spleen lymphocytes in mice

    Institute of Scientific and Technical Information of China (English)

    朱明; 欧亮苗; 何巧丽; 哈木拉提·吾甫尔; 田树革

    2015-01-01

    Objective To investigate the proliferative effects of extracts from Cordia dichotoma seeds on spleen lymphocytes of mice.Methods Detect the effects of the extracts with different concentrations from Cordia dichotoma seeds on mouse spleen lymphocytes by concanavalin A (ConA)and lipopolysaccharide (LPS)in vitro treatment;Detect the proliferation ability of lymphocyte by MTT assay.Results The ex-tracts of Cordia dichotoma seeds in the range of 2.69-86 mg/mL had a synergistic effect in vitro on the mouse spleen lymphocytes proliferation induced by ConA and LPS;polysaccharides of Cordia dichotoma seeds in the range of 21.50-86 mg/mL had synergistic effects in vitro on the spleen lymphocyte prolifera-tion induced by ConA;polysaccharides in the range of 10.75-86 mg/mL had synergistic effects in vitro on the spleen lymphocyte proliferation induced by LPS;rosmarinic acid standard,used as the positive control drug,had a synergistic effect in vitro on the mouse spleen lymphocytes proliferation induced by ConA and LPS.Conclusion This primary study showed that the extracts from Cordia dichotoma seeds,polysaccha-rides of Cordia dichotoma seeds and rosmarinic acid standards had some immunomodulatory effects.%目的:研究破布木果提取物对小鼠脾淋巴细胞的增殖作用。方法不同浓度的破布木果提取物对刀豆蛋白 A(concanavalin A,ConA)、脂多糖试剂(LPS)体外处理的小鼠脾淋巴细胞的作用;采用 MTT法测定淋巴细胞的增殖能力。结果破布木果提取液在2.69~86 mg/mL范围内,均与 ConA、LPS诱导的小鼠脾淋巴细胞的增殖在体外具有协同作用;破布木果多糖在21.50~86 mg/mL范围内,与 ConA诱导的小鼠脾淋巴细胞的增殖在体外具有协同作用;在10.75~86 mg/mL范围内,与 LPS诱导的小鼠脾淋巴细胞的增殖在体外具有协同作用;迷迭香酸标准品作为药物的阳性对照,均与 ConA、LPS诱导的小鼠脾淋巴细胞的增殖

  1. Glucose-dependent de Novo Lipogenesis in B Lymphocytes

    Science.gov (United States)

    Dufort, Fay J.; Gumina, Maria R.; Ta, Nathan L.; Tao, Yongzhen; Heyse, Shannon A.; Scott, David A.; Richardson, Adam D.; Seyfried, Thomas N.; Chiles, Thomas C.

    2014-01-01

    Bacterially derived lipopolysaccharide (LPS) stimulates naive B lymphocytes to differentiate into immunoglobulin (Ig)-secreting plasma cells. Differentiation of B lymphocytes is characterized by a proliferative phase followed by expansion of the intracellular membrane secretory network to support Ig production. A key question in lymphocyte biology is how naive B cells reprogram metabolism to support de novo lipogenesis necessary for proliferation and expansion of the endomembrane network in response to LPS. We report that extracellularly acquired glucose is metabolized, in part, to support de novo lipogenesis in response to LPS stimulation of splenic B lymphocytes. LPS stimulation leads to increased levels of endogenous ATP-citrate lyase (ACLY), and this is accompanied by increased ACLY enzymatic activity. ACLY produces cytosolic acetyl-CoA from mitochondrially derived citrate. Inhibition of ACLY activity in LPS-stimulated B cells with the selective inhibitor 2-hydroxy-N-arylbenzenesulfonamide (compound-9; C-9) blocks glucose incorporation into de novo lipid biosynthesis, including cholesterol, free fatty acids, and neutral and acidic phospholipids. Moreover, inhibition of ACLY activity in splenic B cells results in inhibition of proliferation and defective endomembrane expansion and reduced expression of CD138 and Blimp-1, markers for plasma-like B cell differentiation. ACLY activity is also required for LPS-induced IgM production in CH12 B lymphoma cells. These data demonstrate that ACLY mediates glucose-dependent de novo lipogenesis in response to LPS signaling and identify a role for ACLY in several phenotypic changes that define plasma cell differentiation. PMID:24469453

  2. Investigating the in vitro effect of taurine on the infant lymphocytes by sister chromatid exchange.

    Science.gov (United States)

    Ergun, Mehmet Ali; Soysal, Yasemin; Kismet, Erol; Akay, Cemal; Dundaroz, Rusen; Ilhan, Mustafan; Imirzalioglu, Necat

    2006-06-01

    Taurine (2-aminoethane sulphonic acid) is normally present in most mammalian tissues and the most abundant free amino acid in lymphocytes. It participates in various important physiological activities including modulation of the functioning of the central nervous system, cell proliferation, viability and prevention of oxidant-induced injury in many tissues. Its levels in human milk are very high which may be the most important difference from cow's milk. In contrast, an inverse association between breast-feeding and carcinogenesis in childhood or later in life has been suggested by several studies. The study group consisted of eight healthy infants. Peripheral blood was collected and lymphocytes were cultured with either Taurine or Mitomycin C (MMC). Sister chromatid exchange in lymphocytes of the infants were calculated. Statistical differences were found between untreated and MMC-treated lymphocytes, untreated and MMC plus taurine-treated lymphocytes, and between MMC and MMC plus taurine-treated lymphocytes (P = 0.012). The results indicated that taurine plays a protective role in MMC-induced sister chromatid exchange in human lymphocytes. The authors suggest that the high levels of taurine found in human milk may induce protecting effects from breast-feeding against DNA damage and malignancy.

  3. Abnormal lipid rafts related ganglioside expression and signaling in T lymphocytes in immune thrombocytopenia patients.

    Science.gov (United States)

    Zhang, Xian; Zhang, Donglei; Liu, Wenjie; Li, Huiyuan; Fu, Rongfeng; Liu, Xiaofan; Xue, Feng; Yang, Renchi

    2016-01-01

    Aberrant T lymphocytes signaling is considered to play a crucial role in the abnormal immune state of primary immune thrombocytopenia (ITP). Lipid raft has been verified to engage in the T cell receptor (TCR)-mediated T lymphocytes signal transduction. Whether lipid raft-associated T cells signal transduction has impact on the pathogenesis of ITP is still unconfirmed. In this study, we aimed to reveal the abnormality in structure and function of lipid rafts (LRs) in CD4(+) and CD8(+) T lymphocytes of patients with ITP. Our results showed that there was an increased lipid raft aggregation in ITP patients, while this kind of increase would not be influenced by platelet counts or therapeutic regimes. Stimulation by anti-CD3/CD28 monoclonal antibodies promoted enhanced lipid raft clustering in T lymphocytes of ITP patients compared with negative controls. Methyl-β-cyclodextrin (MβCD) could block the abnormal lipid raft aggregation and disrupt the TCR-mediated T cells proliferation and cytokines secretion, including both proinflammatory cytokines and anti-inflammatory cytokines. The spontaneous activation of T lymphocytes from ITP patients might be due to the elevated co-localization of protein tyrosine phosphatase (PTP) CD45 and lipid rafts in patients' CD4(+) and CD8(+) T lymphocytes. These findings suggest that the autoactivation of T lymphocytes from ITP patients may lead to the abnormality in lipid raft structure and raft-anchored proteins, and the changes conversely promote the TCR-mediated T cells activation of ITP patients.

  4. Mechanisms of T-Lymphocyte Accumulation during Experimental Pleural Infection Induced by Mycobacterium bovis BCG▿

    Science.gov (United States)

    Souza, Mariana C.; Penido, Carmen; Costa, Maria F. S.; Henriques, Maria Graças

    2008-01-01

    Tuberculous pleurisy is a frequent extrapulmonary manifestation characterized by accumulation of fluid and inflammatory cells in the pleural space. Here, we investigated the mechanisms of T-lymphocyte accumulation in the pleural space by using a murine model of pleurisy induced by Mycobacterium bovis BCG. Intrathoracic (i.t.) injection of BCG (4.5 × 105 bacteria/cavity) induced accumulation of T lymphocytes in the pleural cavities of C57BL/6 mice. We observed the presence of CFU in pleural washes conducted 1, 2, 3, 7, and 15 days after pleurisy induction. Pretreatment with fucoidan inhibited T-lymphocyte accumulation at 1 day, but not at 15 days, after BCG-induced pleurisy. Accordingly, adoptive transfer of fluorescein isothiocyanate-labeled blood mononuclear cells to infected mice showed that T lymphocytes migrated into the pleural cavity 1 day (but not 15 days) after BCG injection. Cell-free pleural wash fluids recovered from mice 1 day after BCG i.t. stimulation (day 1 BCG-PW), but not day 7 or day 15 BCG-PW, induced in vitro T-cell transmigration, which was dependent on L-, P-, and E-selectins. In contrast, day 7 BCG-PW (but not day 1 BCG-PW) induced in vitro T-lymphocyte proliferation via interleukin-2 (IL-2) and gamma interferon (IFN-γ). Accordingly, in vivo IL-2 or IFN-γ neutralization abolished T-lymphocyte accumulation 7 days after pleurisy induction. Our results demonstrate that pleural infection induced by BCG leads to T-lymphocyte accumulation in two waves. The acute phase depends on selectin-mediated migration, while the second wave of T-lymphocyte accumulation seems to depend on a local proliferation induced by cytokines produced in situ. PMID:18809659

  5. Inhibitory Effects of Berberine on the Activation and Cell Cycle Progression of Human Peripheral Lymphocytes

    Institute of Scientific and Technical Information of China (English)

    Lihui Xu; Yi Liu; Xianhui He

    2005-01-01

    The immunosuppressive property of berberine, an isoquinoline alkaloid, has been well documented, but the mechanism of its action on lymphocytes has not been completely elucidated. The present study is to investigate the effect of berberine on the activation and proliferation of lymphocytes, in particular T lymphocytes. Whole peripheral blood from healthy donors was stimulated with phytohemagglutinin (PHA) alone or phorbol dibutyrate (PDB) plus ionomycin, and the expression of CD69 and CD25 on T lymphocytes was evaluated with flow cytometry.The distribution of cell cycles and cell viability were analyzed by staining with propidium iodide (PI) and 7-aminoactinomycin D (7-AAD), respectively. The results showed that 100 μmol/L and 50 μmol/L of berberine significantly inhibited CD69 expression on T cells stimulated with PDB plus ionomycin or PHA, whereas the effect of 25 μmol/L berberine was not significant. As the incubation time increased, the extent of inhibition decreased.Similarly, the expression of CD25 was also reduced by berberine in a dose-dependent manner over the concentration range of 25-100 μmol/L. Besides, this alkaloid could block lymphocyte cell cycle progression from G0/G1 phase to S and G2/M phase without phase specificity. Moreover, analysis following 7-AAD staining revealed that berberine had no significant cytotoxicity on lymphocytes. Taken together, berberine significantly inhibits the expression of activation antigens on T lymphocytes and also blocks the progression of cell cycles of lymphocytes,suggesting that berberine may exert immunosuppressive effect through inhibiting the activation and proliferation of T cells.

  6. Graft-versus-leukemia reactivity involves cluster formation between superantigen-reactive donor T lymphocytes and host macrophages.

    Science.gov (United States)

    Müerköster, S; Wachowski, O; Zerban, H; Schirrmacher, V; Umansky, V; Rocha, M

    1998-12-01

    T-cell-mediated antitumor effects play an important role clinically in allogeneic graft-versus-leukemia (GvL) reactivity, whereas T-cell-mediated antihost effects are associated with a risk of developing graft-versus-host (GvH) disease. GvL and GvH were compared in an animal tumor model system after the systemic transfer of allogeneic antitumor immune T lymphocytes from B10.D2 [H-2d; minor lymphocyte-stimulating antigen (Mls)b] mice into ESb-MP tumor-bearing or normal DBA/2 (H-2d; Mls(a)) mice. Here we demonstrate that this T-cell-mediated therapy involves the formation of clusters of donor CD4 and CD8 T cells with host macrophages, in particular, with a subpopulation expressing the lymphocyte adhesion molecule sialoadhesin. DBA/2 mice and the derived tumor ESb-MP express viral superantigen 7 (Mls(a)), an endogenous viral superantigen that is absent from B10.D2 mice. To test the contribution of viral superantigen 7-reactive Vbeta6 donor T cells in the GvL-mediated eradication of liver metastases, we performed immunohistological and transmission electron microscopy studies. Vbeta6+ CD4 and CD8 T cells from B10.D2 donors formed tight clusters with host sialoadhesin-positive macrophages, and transmission electron microscopy pictures revealed direct membrane-membrane interactions between T cells and macrophages. Clusters were more abundant and consisted of more cells in tumor-bearing hosts (GvL model) than in non-tumor-bearing hosts (GvH model). In addition, Vbeta6 T cells within the clusters showed a strong proliferation activity, indicating stimulation. Moreover, in an in vitro tumor cytostasis assay, primed as well as nonprimed purified Vbeta6 T cells from donor mice were able to inhibit the proliferation of superantigen-expressing ESb-MP lymphoma cells. This suggests that the transferred superantigen-reactive Vbeta6 T cells contribute to the eradication of metastases. The observed cell clusters might be sites for antigen presentation and the activation of tumor

  7. Changes in count and function of splenic lymphocytes from patients with portal hypertension

    Institute of Scientific and Technical Information of China (English)

    Zong-Fang Li; Shu Zhang; Gao-Bo Lv; Ying Huang; Wei Zhang; Song Ren; Jun Yang; Shuang-Suo Dang

    2008-01-01

    AIM: To investigate changes in numbers and proliferative function of splenic lymphocytes in patients with hypersplenism due to portal hypertension (PH), to provide evidence for further study of immune status of the spleen during PH.METHODS: Twelve spleens from patients with hypersplenism due to PH served as the PH group, and four spleens from cases of traumatic spleen rupture were regarded as the control group. After weighing the spleen, lymphocytes were separated and counted using a cell counting plate to calculate the lymphocyte count per gram of spleen tissue (relative quantity) and total lymphocyte count in whole spleen (absolute quantity). The immunohistochemical SP method was used to observe the density and distribution of lymphocytes in the spleen. The MTT method was used to observe changes in lymphocyte proliferative function.RESULTS: As compared to the control group, the splenic lymphocytes in the PH group showed that: (1) There was no difference in distribution but a significant decrease in density; (2) the number of lymphocytes per gram of spleen (relative quantity) decreased significantly [(0.822±0.157) × 108 vs (1.174±0.254) × 108, P < 0.01];(3) with the significant increase in the weight of the PH spleen (832.6±278.2 g vs 211.7±85.6 g, P < 0.01),the total quantity of lymphocytes (absolute quantity)increased significantly [(0.685±0.072) × 1011 vs (0.366±0.057) × 1011, P < 0.01]; and (4) the proliferative function of lymphocytes was enhanced: T lymphocytes,(0.022±0.005 vs 0.015±0.003, P < 0.05), and B lymphocytes (0.034±0.006 vs 0.023±0.001, P < 0.01).CONCLUSION: Although lymphocyte density in the spleen decreased in patients with PH, the total quantity of lymphoytes increased because spleen weight increased greatly, along with the proliferating function. With respect to changes in lymphocytes, PH spleens may still have immune function, although it may be disordered. However,complete evaluation of the immune function of the spleenin PH

  8. Long intergenic noncoding RNAs: novel drivers of human lymphocyte differentiation

    Directory of Open Access Journals (Sweden)

    Ilaria ePanzeri

    2015-04-01

    Full Text Available Upon recognition of a foreign antigen, CD4+ naïve T lymphocytes proliferate and differentiate into subsets with distinct functions. This process is fundamental for the proper immune system function, as CD4+ T cells orchestrate both the innate and adaptive immune response. Traditionally, this differentiation event has been regarded as the acquisition of an irreversible cell fate so that memory and effector CD4+ T subsets were considered terminally differentiated cells or lineages. Consequently, these lineages are conventionally defined thanks to their prototypical set of cytokines and transcription factors. However, recent findings suggest that CD4+ T lymphocytes possess a remarkable phenotypic plasticity, as they can often redirect their functional program depending on the milieu they encounter. Therefore new questions are now compelling such as which are the molecular determinants underlying plasticity and stability and how the balance between these two opposite forces drives the cell fate. As already mentioned, in some cases the mere expression of cytokines and master regulators could not fully explain lymphocytes plasticity. We should consider other layers of regulation, including epigenetic factors such as the modulation of chromatin state or the transcription of noncoding RNAs, whose high cell-specificity give a hint on their involvement in cell fate determination. In this review, we will focus on the recent advances in understanding CD4+ T lymphocytes subsets specification from an epigenetic point of view. In particular, we will emphasize the emerging importance of noncoding RNAs as key players in these differentiation events. We will also present here new data from our laboratory highlighting the contribution of long noncoding RNAs in driving human CD4+ T lymphocytes differentiation.

  9. The inhibition of DNA synthesis in chronic lymphocytic leukaemia cells by chlorambucil in vitro.

    Science.gov (United States)

    Bentley, D. P.; Blackmore, J. A.

    1992-01-01

    The inhibition of 3H-thymidine incorporation into the DNA of mitogen-stimulated lymphocytes from patients with chronic lymphocytic leukaemia by chlorambucil was measured in vitro and the results related to clinical drug resistance. The assay proved to be both sensitive and specific showing a clear separation of those patients with responsive disease from those with disease resistant to treatment. There was evidence of primary drug resistance in untreated patients. In almost all patients who received treatment this led to increasing resistance to chlorambucil in vitro. The assay is predictive of clinical responsiveness and provides a potential means whereby new therapeutic agents and treatment modifiers may be investigated. PMID:1739612

  10. Regulation of human natural killer cell migration and proliferation by the exodus subfamily of CC chemokines.

    Science.gov (United States)

    Robertson, M J; Williams, B T; Christopherson, K; Brahmi, Z; Hromas, R

    2000-01-10

    Natural killer (NK) cells play an important role in innate and adaptive immune responses to obligate intracellular pathogens. Nevertheless, the regulation of NK cell trafficking and migration to inflammatory sites is poorly understood. Exodus-1/MIP-3alpha/LARC, Exodus-2/6Ckine/SLC, and Exodus-3/MIP-3beta/ELC/CKbeta-11 are CC chemokines that share a unique aspartate-cysteine-cysteine-leucine motif near their amino terminus and preferentially stimulate the migration of T lymphocytes. The effects of Exodus chemokines on human NK cells were examined. Exodus-1, -2, and -3 did not induce detectable chemotaxis of resting peripheral blood NK cells. In contrast, Exodus-2 and -3 stimulated migration of polyclonal activated peripheral blood NK cells in a dose-dependent fashion. Exodus-2 and -3 also induced dose-dependent chemotaxis of NKL, an IL-2-dependent human NK cell line. Results of modified checkerboard assays indicate that migration of NKL cells in response to Exodus-2 and -3 represents true chemotaxis and not simply chemokinesis. Exodus-1, -2, and -3 did not induce NK cell proliferation in the absence of other stimuli. Nevertheless, Exodus-2 and -3 significantly augmented IL-2-induced proliferation of normal human CD56(dim) NK cells. In contrast, Exodus-1, -2, and -3 did not affect the cytolytic activity of resting or activated peripheral blood NK cells. Expression of message for CCR7, a shared receptor for Exodus-2 and -3, was detected in activated polyclonal NK cells and NKL cells but not resting NK cells. Taken together, these results indicate that Exodus-2 and -3 can participate in the recruitment and proliferation of activated NK cells. Exodus-2 and -3 may regulate interactions between T cells and NK cells that are crucial for the generation of optimal immune responses.

  11. Effects of cobalt and chromium ions on lymphocyte migration.

    Science.gov (United States)

    Baskey, Stephen J; Lehoux, Eric A; Catelas, Isabelle

    2017-04-01

    A T cell-mediated hypersensitivity reaction has been reported in some patients with CoCrMo-based implants. However, the role of cobalt and chromium ions in this reaction remains unclear. The objective of the present study was to analyze the effects of Co(2+) and Cr(3+) in culture medium, as well as the effects of culture supernatants of macrophages exposed to Co(2+) or Cr(3+) , on the migration of lymphocytes. The release of cytokines/chemokines by macrophages exposed to Co(2+) and Cr(3+) was also analyzed. The migration of murine lymphocytes was quantified using the Boyden chamber assay and flow cytometry, while cytokine/chemokine release by J774A.1 macrophages was measured by ELISA. Results showed an ion concentration-dependent increase in TNF-α and MIP-1α release and a decrease in MCP-1 and RANTES release. Migration analysis showed that the presence of Co(2+) (8 ppm) and Cr(3+) (100 ppm) in culture medium increased the migration of T lymphocytes, while it had little or no effect on the migration of B lymphocytes, suggesting that Co(2+) and Cr(3+) can stimulate the migration of T but not B lymphocytes. Levels of T lymphocyte migration in culture medium containing Co(2+) or Cr(3+) were not statistically different from those in culture supernatants of macrophages exposed to Co(2+) or Cr(3+) , suggesting that the effects of the ions and chemokines were not additive, possibly because of ion interference with the chemokines and/or their cognate receptors. Overall, results suggest that Co(2+) and Cr(3+) are capable of stimulating the migration of T (but not B) lymphocytes in the absence of cytokines/chemokines, and could thereby contribute to the accumulation of more T than B lymphocytes in periprosthetic tissues of some patients with CoCrMo-based implants. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:916-924, 2017. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  12. Inhibitory Effect of Cantharidin on Proliferation of A549 Cells

    Institute of Scientific and Technical Information of China (English)

    WANG Xiao-hua; YIN Yuan-qin; SUI Cheng-guang; MENG Fan-dong; MA Ping; JIANG You-hong

    2007-01-01

    Objective: To study the inhibition of Cantharidin against the proliferation of human lung cancer A549 cells and its mechanism. Methods: MTT assay was employed to determine the inhibition of Cantharidin against proliferation of A549 cells and flow Cytometry was applied to analyze A549 cell cycle and the effect of Cantharidin on cell cycle. Results: Cantharidin showed inhibition against the proliferation of A549 cells, and the inhibition was mediated by blocking A549 cell cycle at G2/M phase significantly. Conclusion: Cantharidin exhibits inhibition against the proliferation of human lung cancer A549 cells.

  13. Usefulness of targeting lymphocyte Kv1.3-channels in the treatment of respiratory diseases.

    Science.gov (United States)

    Kazama, Itsuro; Tamada, Tsutomu; Tachi, Masahiro

    2015-10-01

    T lymphocytes predominantly express delayed rectifier K(+)-channels (Kv1.3) in their plasma membranes. Patch-clamp studies revealed that the channels play crucial roles in facilitating the calcium influx necessary to trigger lymphocyte activation and proliferation. Using selective channel inhibitors in experimental animal models, in vivo studies further revealed the clinically relevant relationship between the channel expression and the development of chronic respiratory diseases, in which chronic inflammation or the overstimulation of cellular immunity in the airways is responsible for the pathogenesis. In chronic respiratory diseases, such as chronic obstructive pulmonary disease, asthma, diffuse panbronchiolitis and cystic fibrosis, in addition to the supportive management for the symptoms, the anti-inflammatory effects of macrolide antibiotics were shown to be effective against the over-activation or proliferation of T lymphocytes. Recently, we provided physiological and pharmacological evidence that macrolide antibiotics, together with calcium channel blockers, HMG-CoA reductase inhibitors, and nonsteroidal anti-inflammatory drugs, effectively suppress the Kv1.3-channel currents in lymphocytes, and thus exert anti-inflammatory or immunomodulatory effects. In this review article, based on the findings obtained from recent in vivo and in vitro studies, we address the novel therapeutic implications of targeting the lymphocyte Kv1.3-channels for the treatment of chronic or acute respiratory diseases.

  14. REPRESENTATION OF DIFFERENT LYMPHOCYTES' POPULATIONS IN PERIPHERAL BLOOD OF PATIENTS WITH UTERINE MYOMA

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    Ye. E. Zueva

    2014-07-01

    Full Text Available Abstract. Uterine myoma is one of the most widespread gynecological pathology among reproductive women older than 30 years. It is known, that often progress of this pathology is associated with genetic and endocrinologic factors. The immune system is not evident still. The aim of this study was to analyze the state of patient's immune system using flow cytometry assessment of different subpopulations of lymphocytes in peripheral blood. We have examined 46 patients with simple and proliferating forms of the myoma, with different variants of clinical symptoms. Absolute and relative content of different subpopulations of lymphocytes was not differed from normal population's standard. Significant differences of B-lymphocytes and natural killers content were observed between groups with simple and proliferating forms of disease. It was shown that metrorrhagia is associated with high level of T-lymphocytes and T-killers. It was noted that decreasing of B-lymphocytes content took place in cases with large number of uterine nodes. Obtained data are not sufficient for complete understanding of the role of immune system in pathogenesis of this disease, but they confirm that using of immunomodulating therapy is expedient for complex treatment of uterine myoma.

  15. Lymphocyte function following radium-223 therapy in patients with metastasized, castration-resistant prostate cancer

    Energy Technology Data Exchange (ETDEWEB)

    Barsegian, Vahe; Moeckel, Daniel [Helios Kliniken, Institute of Nuclear Medicine, Schwerin (Germany); Mueller, Stefan P.; Bockisch, Andreas [University Hospital Essen, Department of Nuclear Medicine, Essen (Germany); Horn, Peter A.; Lindemann, Monika [University Hospital Essen, Institute for Transfusion Medicine, Essen (Germany)

    2017-02-15

    Therapy with the alpha-emitter radium-223 chloride ({sup 223}Ra) is an innovative therapeutic option in patients with metastasized, castration-resistant prostate cancer. However, radiotherapy can lead to hematopoietic toxicity. The aim of this study was to determine if {sup 223}Ra therapy induces an impairment of cellular antimicrobial immune responses. In 11 patients receiving {sup 223}Ra treatment, lymphocyte proliferation and the production of pro- and anti-inflammatory cytokines (interferon-γ and interleukin-10) were determined, using lymphocyte transformation testing and ELISpot, respectively. Lymphocyte function after stimulation with mitogens and microbial antigens was assessed prior to therapy and at day 1, 7 and 28 after therapy. Lymphocyte proliferation and the production of interferon-γ and interleukin-10 towards mitogens and antigens remained unchanged after therapy. Consistent with these in vitro data, we did not observe infectious complications after treatment. The results argue against an impairment of lymphocyte function after {sup 223}Ra therapy. Thus, immune responses against pathogens should remain unaffected. (orig.)

  16. Emerging roles of L-type voltage gated and other calcium channels in T lymphocytes.

    Directory of Open Access Journals (Sweden)

    Abdallah eBadou

    2013-08-01

    Full Text Available In T lymphocytes, calcium ion controls a variety of biological processes including development, survival, proliferation, and effector functions. These distinct and specific roles are regulated by different calcium signals, which are generated by various plasma membrane calcium channels. The repertoire of calcium-conducting proteins in T lymphocytes includes store-operated CRAC channels, transient receptor potential (TRP channels, P2X channels, and L-type voltage-gated calcium (Cav1 channels. In this paper, we will focus mainly on the role of the Cav1 channels found expressed by T lymphocytes, where these channels appear to operate in a TCR stimulation-dependent and voltage-sensor independent manner. We will review their expression profile at various differentiation stages of CD4 and CD8 T lymphocytes. Then, we will present crucial genetic evidence in favor of a role of these Cav1 channels and related regulatory proteins in both CD4 and CD8 T cell functions such as proliferation, survival, cytokine production and cytolysis. Finally, we will provide evidence and speculate on how these voltage-gated channels might function in the T lymphocyte, a non-excitable cell.

  17. Studies on rabbit lymphocytes in vitro

    Science.gov (United States)

    Sell, S.; Gell, P. G. H.

    1969-01-01

    Anti-allotypic sera that have no known allotypic determinants other than those also present in the genotype of the lymphocyte donor are as able to induce lymphocyte `blast' transformation in vitro as are anti-allotypic sera that do have allotypic determinants that are not present in the lymphocyte donor. Therefore, anti-allotypic sera do not appear to function in the stimulation of blast transformation by providing access for any of the known allotypic determinants into lymphocytes. PMID:5769980

  18. Effects of benzene inhalation on lymphocyte subpopulations and immune response in mice.

    Science.gov (United States)

    Aoyama, K

    1986-08-01

    To clarify the immunotoxicity of benzene, the effects of benzene inhalation on T and B lymphocytes and immune responses in mice were examined. BALB/c male mice were exposed to 50 or 200 ppm benzene vapor, 6 hr/day for 7 or 14 consecutive days. T and B lymphocytes, in blood and spleen, were detected by the cytotoxicity assay with anti-Thy-1.2 monoclonal antibody and the membrane immunofluorescence test with anti-immunoglobulin antibody, respectively. Humoral immune response to sheep red blood cells was determined by the hemolytic plaque-forming cell assay. Cell-mediated immune response was measured by contact sensitivity (CS) to picryl chloride. The activity of suppressor cells was evaluated in spleen by the suppressive effect on passive transfer of CS. The ratio and absolute number of T and B lymphocytes in blood and spleen were depressed after a 7-day exposure at 50 ppm benzene. The depression of B lymphocytes was dose dependent and more intense than that of T lymphocytes. The ability to form antibodies was suppressed by benzene at all exposure levels, but the CS response was resistant to benzene inhalation and rather enhanced at 200 ppm exposure for 14 days. The activity of suppressor cells could not be detected at this dose level. These data show that benzene inhalation effects on humoral and cell-mediated immune responses are a result of the selective toxicity of benzene to B lymphocytes and suppressor T cells.

  19. Rapid exacerbation of lymphocytic infundibuloneurohypophysitis

    Science.gov (United States)

    Shibue, Kimitaka; Fujii, Toshihito; Goto, Hisanori; Yamashita, Yui; Sugimura, Yoshihisa; Tanji, Masahiro; Yasoda, Akihiro; Inagaki, Nobuya

    2017-01-01

    Abstract Rationale: Lymphocytic hypophysitis is a relatively rare autoimmune disease defined by lymphocytic infiltration to the pituitary. Its rarity and wide spectrum of clinical manifestations make clarification of the pathology difficult. Here, we describe a case we examined from the primary diagnosis to final discharge, showing the serial progression of lymphocytic infundibuloneurohypophysitis (LINH) to panhypopituitarism with extrapituitary inflammatory invasion in a short period, and responding favorably to high-dose glucocorticoid treatment. Patient concerns: Polyuria, General fatigue and Nausea/Vomiting. Diagnoses: Central diabetes insipidus (CDI), Lymphocytic infundibuloneurohypophysitis (LINH). Interventions: Desmopressin acetate, High-dose glucocorticoid (GC) treatment. Outcomes: He was prescribed desmopressin acetate and subsequently discharged. A month later, he revisited our hospital with general fatigue and nausea/vomiting. A screening test disclosed hypopituitarism with adrenal insufficiency. MRI revealed expanded contrast enhancement to the peripheral extrapituitary lesion. He received high-dose GC treatment and the affected lesion exhibited marked improvement on MRI, along with the recovery of the anterior pituitary function. Lessons: This case demonstrates the potential for classical LINH to develop into panhypopituitarsim. We consider this is the first documentation of approaching the cause of atypical LINH with progressive clinical course from the pathological viewpoint. PMID:28248860

  20. Treatment of chronic lymphocytic leukemia.

    Science.gov (United States)

    Ferrajoli, Alessandra; O'Brien, Susan M

    2004-04-01

    Treatment options for patients with chronic lymphocytic leukemia have changed over the past two decades. This article reviews the experience accumulated with the use of alkylating agents alone and in combination; purine analogues alone and in combination and monoclonal antibodies such as rituximab, and alemtuzumab alone and in combination. The results obtained with different treatment strategies are summarized, compared, and reviewed.

  1. Integration Analysis of MicroRNA and mRNA Expression Profiles in Human Peripheral Blood Lymphocytes Cultured in Modeled Microgravity

    Directory of Open Access Journals (Sweden)

    C. Girardi

    2014-01-01

    Full Text Available We analyzed miRNA and mRNA expression profiles in human peripheral blood lymphocytes (PBLs incubated in microgravity condition, simulated by a ground-based rotating wall vessel (RWV bioreactor. Our results show that 42 miRNAs were differentially expressed in MMG-incubated PBLs compared with 1 g incubated ones. Among these, miR-9-5p, miR-9-3p, miR-155-5p, miR-150-3p, and miR-378-3p were the most dysregulated. To improve the detection of functional miRNA-mRNA pairs, we performed gene expression profiles on the same samples assayed for miRNA profiling and we integrated miRNA and mRNA expression data. The functional classification of miRNA-correlated genes evidenced significant enrichment in the biological processes of immune/inflammatory response, signal transduction, regulation of response to stress, regulation of programmed cell death, and regulation of cell proliferation. We identified the correlation of miR-9-3p, miR-155-5p, miR-150-3p, and miR-378-3p expression with that of genes involved in immune/inflammatory response (e.g., IFNG and IL17F, apoptosis (e.g., PDCD4 and PTEN, and cell proliferation (e.g., NKX3-1 and GADD45A. Experimental assays of cell viability and apoptosis induction validated the results obtained by bioinformatics analyses demonstrating that in human PBLs the exposure to reduced gravitational force increases the frequency of apoptosis and decreases cell proliferation.

  2. Development of an Easy and High-Throughput Cell Assay System with a Culture Chip and an Assay Chip

    Science.gov (United States)

    Sugiura, Kanako; Kaji, Noritada; Okamoto, Yukihiro; Tokeshi, Manabu; Baba, Yoshinobu

    High throughput cell assay is significantly important in drug screening, assessment of toxicity etc. Cell assay with a microchip is one of the candidates for high throughput cell assay. However, reported cell assay system with the microchip requires expensive apparatus for refluxing medium and investigation of optimum experimental condition for steady data. For an inexpensive, easy and high throughput cell assay, we introduce a new cell assay system combined with a culture chip and an assay chip made of poly(dimethyl siloxane). Cell culture chips enabled cell to proliferate along the microchannel without refluxing medium and permitted to prepare cell patterning easily. Also, assay chips formed concentration gradient inside the chip and allowed the cell assay with different concentrations of drug at the same time. Thus, our developed cell assay system can overcome the problems of the present cell assay and would promote the drug discovery, assessment of toxicity etc.

  3. A high-content analysis toolbox permits dissection of diverse signaling pathways for T lymphocyte polarization.

    Science.gov (United States)

    Freeley, Michael; Bakos, Gabor; Davies, Anthony; Kelleher, Dermot; Long, Aideen; Dunican, Dara J

    2010-06-01

    RNA interfering (RNAi) screening strategies offer the potential to elucidate the signaling pathways that regulate integrin and adhesion receptor-mediated changes in T lymphocyte morphology. Of crucial importance, however, is the definition of key sets of parameters that will provide accurate, quantitative, and nonredundant information to flag relevant hits in such assays. In this study, the authors have used an image-based high-content analysis (HCA) technology platform and a panel of 24 pharmacological inhibitors, at a range of concentrations, to define key sets of parameters that enables sensitive and quantitative effects on integrin (LFA-1)-mediated lymphocyte morphology to be evaluated. In particular, multiparametric analysis of lymphocyte morphology that was based on intracellular staining of both the F-actin and alpha-tubulin cytoskeleton resulted in improved ability to discriminate morphological behavior compared to F-actin staining alone. Morphological and fluorescence intensity/distribution profiling of pharmacologically treated lymphocytes stimulated with integrin (LFA-1) and adhesion receptors (CD44) also revealed notable differences in their sensitivity to inhibitors. The assay described here may be used in HCA strategies such as RNAi screening assays to elucidate the signaling pathways and molecules that regulate integrin/adhesion receptor-mediated T lymphocyte polarization.

  4. Genotoxicity of food preservative sodium sorbate in human lymphocytes in vitro.

    Science.gov (United States)

    Mamur, Sevcan; Yüzbaşıoğlu, Deniz; Unal, Fatma; Aksoy, Hüseyin

    2012-10-01

    The genotoxic effects of antimicrobial food additive sodium sorbate (SS) was assessed by using chromosome aberrations (CAs), sister-chromatid exchanges (SCEs), and micronucleus (MN) in cultured human lymphocytes and comet assay in isolated human lymphocytes. Lymphocytes were treated with four concentrations (100, 200, 400 and 800 μg/ml) of SS as well as a negative (sterile distilled water) and a positive control (Mitomycin-C: MMC for cultured lymphocytes and H(2)O(2) for isolated lymphocytes). The result of this study indicated that SS increased the frequency of CAs at both 24 and 48 h period compared to control. When gaps were included, this increase was significant at 200, 400 and 800 μg/ml concentrations at 24 h and, at all concentrations at 48 h treatment time. When gaps were excluded, this increase was significant at only 800 μg/ml concentration at both 24 and 48 h treatments. In addition, SS increased SCEs/cell and MN frequency at 400 and 800 μg/ml concentrations at both 24 and 48 h compared to negative control. Furthermore, this additive caused DNA damage at all concentrations in isolated human lymphocytes after 1 h in vitro exposure. The present results show that SS is genotoxic to the human peripheral blood lymphocytes in vitro at the highest concentrations.

  5. Hepatocyte damage induced by lymphocytes from patients with chronic liver diseases, as detected by LDH release.

    Science.gov (United States)

    Fernandez-Cruz, E; Escartin, P; Bootello, A; Kreisler, M; Segovia de Arana, J M

    1978-01-01

    We have used a cytoplasmic enzyme system in the study of the in vitro cytotoxic activity of human peripheral blood leucocytes against isolated liver cells in patients with chronic liver diseases. Lymphocytes from primary biliary cirrhosis and chronic active liver disease patients were shown to have an in vitro capacity to induce a cytolitic effect on isolated hepatocytes, as demonstrated by the enhanced release of lactate dehydrogenase (LDH), a cytoplasmic marker enzyme. No significant LDH release was seen with control lymphocytes of normal persons or with lymphocytes from patients with alcoholic cirrhosis. Our results corroborate, in a different assay system, by a simple, reproducible and different method, that lymphocyte-mediated liver cell damage "in vitro" occurs in both primary biliary cirrhosis and chronic active liver disease. PMID:657588

  6. Genotoxic Effect in Autoimmune Diseases Evaluated by the Micronucleus Test Assay: Our Experience and Literature Review

    Science.gov (United States)

    Torres-Bugarín, Olivia; Macriz Romero, Nicole; Ramos Ibarra, María Luisa; Flores-García, Aurelio; Valdez Aburto, Penélope; Zavala-Cerna, María Guadalupe

    2015-01-01

    Autoimmune diseases (AD) are classified into organ-specific, systemic, and mixed; all forms of AD share a high risk for cancer development. In AD a destructive immune response induced by autoreactive lymphocytes is started and continues with the production of autoantibodies against different targets; furthermore apoptosis failure and loss of balance in oxidative stress as a consequence of local or systemic inflammation are common features seen in AD as well. Micronucleus (MN) assay can be performed in order to evaluate loss of genetic material in a clear, accurate, fast, simple, and minimally invasive test. The MN formation in the cytoplasm of cells that have undergone proliferation is a consequence of DNA fragmentation during mitosis and the appearance of small additional nuclei during interphase. The MN test, widely accepted for in vitro and in vivo genotoxicity research, provides a sensitive marker of genomic damage associated to diverse conditions. In here, we present a review of our work and other published papers concerning genotoxic effect in AD, identified by means of the MN assay, with the aim of proposing this tool as a possible early biomarker for genotoxic damage, which is a consequence of disease progression. Additionally this biomarker could be used for follow-up, to asses genome damage associated to therapies. PMID:26339592

  7. Genotoxic Effect in Autoimmune Diseases Evaluated by the Micronucleus Test Assay: Our Experience and Literature Review

    Directory of Open Access Journals (Sweden)

    Olivia Torres-Bugarín

    2015-01-01

    Full Text Available Autoimmune diseases (AD are classified into organ-specific, systemic, and mixed; all forms of AD share a high risk for cancer development. In AD a destructive immune response induced by autoreactive lymphocytes is started and continues with the production of autoantibodies against different targets; furthermore apoptosis failure and loss of balance in oxidative stress as a consequence of local or systemic inflammation are common features seen in AD as well. Micronucleus (MN assay can be performed in order to evaluate loss of genetic material in a clear, accurate, fast, simple, and minimally invasive test. The MN formation in the cytoplasm of cells that have undergone proliferation is a consequence of DNA fragmentation during mitosis and the appearance of small additional nuclei during interphase. The MN test, widely accepted for in vitro and in vivo genotoxicity research, provides a sensitive marker of genomic damage associated to diverse conditions. In here, we present a review of our work and other published papers concerning genotoxic effect in AD, identified by means of the MN assay, with the aim of proposing this tool as a possible early biomarker for genotoxic damage, which is a consequence of disease progression. Additionally this biomarker could be used for follow-up, to asses genome damage associated to therapies.

  8. Genotoxic Effect in Autoimmune Diseases Evaluated by the Micronucleus Test Assay: Our Experience and Literature Review.

    Science.gov (United States)

    Torres-Bugarín, Olivia; Macriz Romero, Nicole; Ramos Ibarra, María Luisa; Flores-García, Aurelio; Valdez Aburto, Penélope; Zavala-Cerna, María Guadalupe

    2015-01-01

    Autoimmune diseases (AD) are classified into organ-specific, systemic, and mixed; all forms of AD share a high risk for cancer development. In AD a destructive immune response induced by autoreactive lymphocytes is started and continues with the production of autoantibodies against different targets; furthermore apoptosis failure and loss of balance in oxidative stress as a consequence of local or systemic inflammation are common features seen in AD as well. Micronucleus (MN) assay can be performed in order to evaluate loss of genetic material in a clear, accurate, fast, simple, and minimally invasive test. The MN formation in the cytoplasm of cells that have undergone proliferation is a consequence of DNA fragmentation during mitosis and the appearance of small additional nuclei during interphase. The MN test, widely accepted for in vitro and in vivo genotoxicity research, provides a sensitive marker of genomic damage associated to diverse conditions. In here, we present a review of our work and other published papers concerning genotoxic effect in AD, identified by means of the MN assay, with the aim of proposing this tool as a possible early biomarker for genotoxic damage, which is a consequence of disease progression. Additionally this biomarker could be used for follow-up, to asses genome damage associated to therapies.

  9. Fludarabine Phosphate, Radiation Therapy, and Rituximab in Treating Patients Who Are Undergoing Donor Stem Cell Transplant Followed by Rituximab for High-Risk Chronic Lymphocytic Leukemia or Small Lymphocytic Lymphoma

    Science.gov (United States)

    2017-03-27

    Chronic Lymphocytic Leukemia; Prolymphocytic Leukemia; Recurrent Small Lymphocytic Lymphoma; Refractory Chronic Lymphocytic Leukemia; Stage III Chronic Lymphocytic Leukemia; Stage III Small Lymphocytic Lymphoma; Stage IV Chronic Lymphocytic Leukemia; Stage IV Small Lymphocytic Lymphoma; T-Cell Large Granular Lymphocyte Leukemia

  10. Fludarabine Phosphate and Total-Body Irradiation Before Donor Peripheral Blood Stem Cell Transplant in Treating Patients With Chronic Lymphocytic Leukemia or Small Lymphocytic Leukemia

    Science.gov (United States)

    2016-07-18

    B-Cell Prolymphocytic Leukemia; Chronic Lymphocytic Leukemia; Prolymphocytic Leukemia; Recurrent Chronic Lymphocytic Leukemia; Recurrent Small Lymphocytic Lymphoma; Refractory Chronic Lymphocytic Leukemia; T-Cell Prolymphocytic Leukemia

  11. Eosinophils induce airway smooth muscle cell proliferation.

    Science.gov (United States)

    Halwani, Rabih; Vazquez-Tello, Alejandro; Sumi, Yuki; Pureza, Mary Angeline; Bahammam, Ahmed; Al-Jahdali, Hamdan; Soussi-Gounni, Abdelillah; Mahboub, Bassam; Al-Muhsen, Saleh; Hamid, Qutayba

    2013-04-01

    Asthma is characterized by eosinophilic airway inflammation and remodeling of the airway wall. Features of airway remodeling include increased airway smooth muscle (ASM) mass. However, little is known about the interaction between inflammatory eosinophils and ASM cells. In this study, we investigated the effect of eosinophils on ASM cell proliferation. Eosinophils were isolated from peripheral blood of mild asthmatics and non-asthmatic subjects and co-cultured with human primary ASM cells. ASM proliferation was estimated using Ki-67 expression assay. The expression of extracellular matrix (ECM) mRNA in ASM cells was measured using quantitative real-time PCR. The role of eosinophil derived Cysteinyl Leukotrienes (CysLTs) in enhancing ASM proliferation was estimated by measuring the release of leukotrienes from eosinophils upon their direct contact with ASM cells using ELISA. This role was confirmed either by blocking eosinophil-ASM contact or co-culturing them in the presence of leukotrienes antagonist. ASM cells co-cultured with eosinophils, isolated from asthmatics, but not non-asthmatics, had a significantly higher rate of proliferation compared to controls. This increase in ASM proliferation was independent of their release of ECM proteins but dependent upon eosinophils release of CysLTs. Eosinophil-ASM cell to cell contact was required for CysLTs release. Preventing eosinophil contact with ASM cells using anti-adhesion molecules antibodies, or blocking the activity of eosinophil derived CysLTs using montelukast inhibited ASM proliferation. Our results indicated that eosinophils contribute to airway remodeling during asthma by enhancing ASM cell proliferation and hence increasing ASM mass. Direct contact of eosinophils with ASM cells triggers their release of CysLTs which enhance ASM proliferation. Eosinophils, and their binding to ASM cells, constitute a potential therapeutic target to interfere with the series of biological events leading to airway remodeling

  12. IFN-γ upregulates survivin and Ifi202 expression to induce survival and proliferation of tumor-specific T cells.

    Directory of Open Access Journals (Sweden)

    Mary Zimmerman

    Full Text Available BACKGROUND: A common procedure in human cytotoxic T lymphocyte (CTL adoptive transfer immunotherapy is to expand tumor-specific CTLs ex vivo using CD3 mAb prior to transfer. One of the major obstacles of CTL adoptive immunotherapy is a lack of CTL persistence in the tumor-bearing host after transfer. The aim of this study is to elucidate the molecular mechanisms underlying the effects of stimulation conditions on proliferation and survival of tumor-specific CTLs. METHODOLOGY/PRINCIPAL FINDINGS: Tumor-specific CTLs were stimulated with either CD3 mAb or cognate Ag and analyzed for their proliferation and survival ex vivo and persistence in tumor-bearing mice. Although both Ag and CD3 mAb effectively induced the cytotoxic effecter molecules of the CTLs, we observed that Ag stimulation is essential for sustained CTL proliferation and survival. Further analysis revealed that Ag stimulation leads to greater proliferation rates and less apoptosis than CD3 mAb stimulation. Re-stimulation of the CD3 mAb-stimulated CTLs with Ag resulted in restored CTL proliferative potential, suggesting that CD3 mAb-induced loss of proliferative potential is reversible. Using DNA microarray technology, we identified that survivin and ifi202, two genes with known functions in T cell apoptosis and proliferation, are differentially induced between Ag- and CD3 mAb-stimulated CTLs. Analysis of the IFN-γ signaling pathway activation revealed that Ag stimulation resulted in rapid phosphorylation of STAT1 (pSTAT1, whereas CD3 mAb stimulation failed to activate STAT1. Chromatin immunoprecipitation revealed that pSTAT1 is associated with the promoters of both survivin and ifi202 in T cells and electrophoresis mobility shift assay indicated that pSTAT1 directly binds to the gamma activation sequence element in the survivin and ifi202 promoters. Finally, silencing ifi202 expression significantly decreased T cell proliferation. CONCLUSIONS/SIGNIFICANCE: Our findings delineate a new

  13. 2-Methoxyestradiol induce the conversion of human peripheral blood memory B lymphocytes into plasma cells.

    Science.gov (United States)

    Cayer, Marie-Pierre; Drouin, Mathieu; Proulx, Maryse; Jung, Daniel

    2010-04-15

    2-Methoxyestradiol (2ME), an end-metabolite of 17beta-estradiol, is an antiproliferative agent that is currently being tested in clinical trials for cancer treatment. We hereby report that sub-cytotoxic concentrations of 2ME influence the in vitro proliferation of human peripheral blood B lymphocytes. More surprisingly, we have observed that 2ME induces the conversion of CD138(-) B lymphocytes into CD138(+) cells of phenotype similar to immunoglobulin (Ig)-secreting plasma cells. Normal human B lymphocytes expressing CD138 increased in response to 2ME in a dose-dependent fashion, from 2% at baseline up to 31% in cells cultured in the presence of 0.75 microM 2ME. Moreover, most of the converted cells were also CD27(+) and secreted high levels of IgG (151 microg/10(6)cells/24h). IEF studies revealed that conversion occurred in a polyclonal manner. We then exploited this effect of 2ME to gain further insights into the molecular mechanisms that govern changes in transcription factors involved in plasma cells differentiation. Plasma cells generated by 2ME treatment of normal human B lymphocytes expressed elevated levels of IRF4 and reduced levels of Pax5 and Bcl-6. Similarly, levels of XBP-1 and Blimp-1 transcripts were increased. Our results suggest that the differentiation of peripheral blood B lymphocytes into plasma cells requires a similar modulation of transcription factors expression that for tonsil and bone marrow B lymphocytes.

  14. Oxidative Damage in Lymphocytes of Copper Smelter Workers Correlated to Higher Levels of Excreted Arsenic

    Directory of Open Access Journals (Sweden)

    Jorge Escobar

    2010-01-01

    Full Text Available Arsenic has been associated with multiple harmful effects at the cellular level. Indirectly these defects could be related to impairment of the integrity of the immune system, in particular in lymphoid population. To characterize the effect of Arsenic on redox status on this population, copper smelter workers and arsenic unexposed donors were recruited for this study. We analyzed urine samples and lymphocyte enriched fractions from donors to determinate arsenic levels and lymphocyte proliferation. Moreover, we studied the presence of oxidative markers MDA, vitamin E and SOD activity in donor plasma. Here we demonstrated that in human beings exposed to high arsenic concentrations, lymphocyte MDA and arsenic urinary levels showed a positive correlation with SOD activity, and a negative correlation with vitamin E serum levels. Strikingly, lymphocytes from the arsenic exposed population respond to a polyclonal stimulator, phytohemaglutinin, with higher rates of thymidine incorporation than lymphocytes of a control population. As well, similar in vitro responses to arsenic were observed using a T cell line. Our results suggest that chronic human exposure to arsenic induces oxidative damage in lymphocytes and could be considered more relevant than evaluation of T cell surveillance.

  15. Oxidative Damage in Lymphocytes of Copper Smelter Workers Correlated to Higher Levels of Excreted Arsenic

    Science.gov (United States)

    Escobar, Jorge; Varela-Nallar, Lorena; Coddou, Claudio; Nelson, Pablo; Maisey, Kevin; Valdés, Daniel; Aspee, Alexis; Espinosa, Victoria; Rozas, Carlos; Montoya, Margarita; Mandiola, Cristian; Rodríguez, Felipe E.; Acuña-Castillo, Claudio; Escobar, Alejandro; Fernández, Ricardo; Diaz, Hernán; Sandoval, Mario; Imarai, Mónica; Rios, Miguel

    2010-01-01

    Arsenic has been associated with multiple harmful effects at the cellular level. Indirectly these defects could be related to impairment of the integrity of the immune system, in particular in lymphoid population. To characterize the effect of Arsenic on redox status on this population, copper smelter workers and arsenic unexposed donors were recruited for this study. We analyzed urine samples and lymphocyte enriched fractions from donors to determinate arsenic levels and lymphocyte proliferation. Moreover, we studied the presence of oxidative markers MDA, vitamin E and SOD activity in donor plasma. Here we demonstrated that in human beings exposed to high arsenic concentrations, lymphocyte MDA and arsenic urinary levels showed a positive correlation with SOD activity, and a negative correlation with vitamin E serum levels. Strikingly, lymphocytes from the arsenic exposed population respond to a polyclonal stimulator, phytohemaglutinin, with higher rates of thymidine incorporation than lymphocytes of a control population. As well, similar in vitro responses to arsenic were observed using a T cell line. Our results suggest that chronic human exposure to arsenic induces oxidative damage in lymphocytes and could be considered more relevant than evaluation of T cell surveillance. PMID:21253489

  16. An in vitro immune response model to determine tetanus toxoid antigen (vaccine) specific immunogenicity: Selection of sensitive assay criteria.

    Science.gov (United States)

    Piersma, Sytse J; Leenaars, Marlies P P A M; Guzylack-Piriou, Laurence; Summerfield, Artur; Hendriksen, Coenraad F M; McCullough, Ken C

    2006-04-12

    Many vaccines employed in childhood vaccination programmes are produced by conventional techniques, resulting in complex biological mixtures for which batch-related quality control requires in vivo potency testing. Monitoring consistency via in vitro tests during the vaccine production has the capacity to replace certain of the in vivo methods. In this respect, determining vaccine antigen immunogenicity through functional immunological tests has high potential. Advances in immunology have made it possible to analyse this biological activity by in vitro means. The present study established such an in vitro test system for tetanus toxoid (TT). This measured vaccine immunogenicity through an antigen-specific secondary (recall) response in vitro, using a porcine model growing in value for its closeness to human immune response characteristics. Discrimination between the specific recall TT antigen and diphtheria toxoid (DT) was possible using both peripheral blood mononuclear cell cultures and monocyte-derived dendritic cells in co-culture with autologous specific lymphocytes. TT-specific activation was detected with highest discrimination capacity using proliferation assays, as well as IFN-gamma and TT-specific antibody ELISPOTS (measuring secreting T and B lymphocytes, respectively). These in vitro systems show a high potential for replacing animal experimentation to evaluate the immunogenicity of complex vaccines.

  17. Umbilical Cord Tissue-Derived Mesenchymal Stem Cells Induce T Lymphocyte Apoptosis and Cell Cycle Arrest by Expression of Indoleamine 2, 3-Dioxygenase

    Directory of Open Access Journals (Sweden)

    Xiuying Li

    2016-01-01

    Full Text Available It has been reported that human mesenchymal stem cells are able to inhibit T lymphocyte activation; however, the discrepancy among different sources of MSCs is not well documented. In this study, we have compared the MSCs from bone marrow (BM, adipose tissue (AT, placenta (PL, and umbilical cord (UC to determine which one displayed the most efficient immunosuppressive effects on phytohemagglutinin-induced T cell proliferation. Among them we found that hUC-MSC has the strongest effects on inhibiting T cell proliferation and is chosen to do the further study. We observed that T lymphocyte spontaneously released abundant IFN-γ. And IFN-γ secreted by T lymphocyte could induce the expression of indoleamine 2, 3-dioxygenase (IDO in hUC-MSCs. IDO was previously reported to induce T lymphocyte apoptosis and cell cycle arrest in S phase. When cocultured with hUC-MSCs, T lymphocyte expression of caspase 3 was significantly increased, while Bcl2 and CDK4 mRNA expression decreased dramatically. Addition of 1-methyl tryptophan (1-MT, an IDO inhibitor, restored T lymphocyte proliferation, reduced apoptosis, and induced resumption of the cell cycle. In addition, the changes in caspase 3, CDK4, and Bcl2 expression were reversed by 1-MT. These findings demonstrate that hUC-MSCs induce T lymphocyte apoptosis and cell cycle arrest by expressing abundant IDO and provide an explanation for some of the immunomodulatory effects of MSCs.

  18. Desensitization oft lymphocyte function by CXCR3 ligands in human hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Yu-Qing Liu; Ronnie T. Poon; Jeremy Hughes; Qin-Yu Li; Wan-Ching Yu; Sheung-Tat Fan

    2005-01-01

    AIM: Despite the presence of lymphocyte infiltration, human hepatocellular carcinoma (HCC) is typically a rapidly progressive disease. The mechanism of regulation of lymphocyte migration is poorly understood. In this study,we investigated various factors regulating T cell migration in HCC patients. We examined serum CXC chemokine levels in HCC patients and demonstrated the production of CXC chemokines by HCC cell lines. We determined the effect of both HCC patient serum and tumor cell conditioned supernatant upon lymphocyte expression of chemokine receptor CXCR3 as well as lymphocyte migration. Lastly,we examined the chemotactic responses of lymphocytes derived from HCC patients.METHODS: The serum chemokines IP-10 (CXCL10) and Mig (CXCL9) levels were measured by cytometric bead array (CBA) and the tumor tissue IP-10 concentration was measured by ELISA. The surface expression of CXCR3 on lymphocytes was determined by flow cytometry. The migratory function of lymphocytes to the corresponding chemokines was assessed using an in vitro chemotactic assay. Phosphorylation of extracellular signal-regulated kinase (ERK) was determined by Western blot analysis.RESULTS: Increased levels of IP-10 and Mig were detected in HCC patient serum and culture supernatants of HCC cell lines. The IP-10 concentration in the tumor was significantly higher than that in the non-involved adjacent liver tissues.HCC cell lines secreted functional chemokines that induced a CXCR3-specific chemotactic response of lymphocytes.Furthermore, tumor-cell-derived chemokines induced initial rapid phosphorylation of lymphocyte ERK followed by later inhibition of ERK phosphorylation. The culture of normal lymphocytes with HCC cell line supernatants or medium containing serum from HCC patients resulted in a significant reduction in the proportion of lymphocytes exhibiting surface expression of CXCR3. The reduction in T cell expression of CXCR3 resulted in reduced migration toward the ligand IP-10, and both

  19. In vitro susceptibilities in lymphocytes from mothers and cord blood to the monofunctional alkylating agent EMS

    DEFF Research Database (Denmark)

    Wyatt, N P; Falque-Gonzalez, C; Farrar, D;

    2007-01-01

    at the Bradford Royal Infirmary collected venous blood samples from mothers at the time of birth and venous cord blood post-delivery. Lymphocytes were isolated from both blood types and examined in the alkaline comet assay using the monofunctional alkylating agent ethyl methanesulphonate (EMS). There were...

  20. SFTG international collaborative study on in vitro micronucleus test. II. Using human lymphocytes

    NARCIS (Netherlands)

    Clare, M.G.; Lorenzon, G.; Akhurst, L.C.; Marzin, D.; Delft, J. van; Montero, R.; Botta, A.; Bertens, A.; Cinelli, S.; Thybaud, V.; Lorge, E.

    2006-01-01

    This study on the in vitro micronucleus assay, comprising 11 laboratories using human lymphocytes, was coordinated by an organizing committee supported by the SFTG (the French branch of the European Environmental Mutagen Society). Nine coded substances were assessed for their ability to induce micro

  1. JPRS Report, Proliferation Issues

    Science.gov (United States)

    2016-03-24

    sinister image and is today a busi- ness-oriented body whose function is to produce quality industrial solutions and adapt technology, products and...people’s image of the reactor by regular monthly visits." We will soon find out whether they will succeed in this. The reactor is ready for start. JPRS...lymphocytic choriomeningitis 300290500 1.13 Monkey Pox Pathogen 300290500 1.14 White Pox Pathogen 300290500 1.15 Pathogen of Kyasanurskaya [as

  2. Nuclear overexpression of lymphoid-enhancer-binding factor 1 identifies chronic lymphocytic leukemia/small lymphocytic lymphoma in small B-cell lymphomas.

    Science.gov (United States)

    Tandon, Bevan; Peterson, Loann; Gao, Juehua; Nelson, Beverly; Ma, Shuo; Rosen, Steven; Chen, Yi-Hua

    2011-11-01

    Lymphoid-enhancer-binding factor 1 (LEF1), coupling with β-catenin, functions as a key nuclear mediator of WNT/β-catenin signaling, which regulates cell proliferation and survival. LEF1 has an important role in lymphopoiesis, and is normally expressed in T and pro-B cells but not mature B cells. However, gene expression profiling demonstrates overexpression of LEF1 in chronic lymphocytic leukemia, and knockdown of LEF1 decreases the survival of the leukemic cells. So far, the data on LEF1 expression in B-cell lymphomas are limited. This study represents the first attempt to assess LEF1 by immunohistochemistry in a large series (290 cases) of B-cell lymphomas. Strong nuclear staining of LEF1 was observed in virtually all neoplastic cells in 92 of 92 (100%) chronic lymphocytic leukemia/small lymphocytic lymphomas including two CD5- cases, with strongest staining in cells with Richter's transformation. LEF1 also highlighted the morphologically inconspicuous small lymphocytic lymphoma component in three composite lymphomas. All 53 mantle cell lymphomas, 31 low-grade follicular lymphomas and 31 marginal zone lymphomas, including 3 CD5+ cases, were negative. In 12 grade 3 follicular lymphomas, LEF1 was positive in a small subset (5-15%) of cells. Diffuse large B-cell lymphoma, however, demonstrated significant variability in LEF1 expression with overall positivity in 27 of 71 (38%) cases. Our results demonstrate that nuclear overexpression of LEF1 is highly associated with chronic lymphocytic leukemia/small lymphocytic lymphoma, and may serve as a convenient marker for differential diagnosis of small B-cell lymphomas. The expression of β-catenin, the coactivator of LEF1 in WNT signaling, was examined in 50 chronic lymphocytic leukemia/small lymphocytic lymphomas, of which 44 (88%) showed negative nuclear staining. The findings of universal nuclear overexpression of LEF1 but lack of nuclear β-catenin in the majority of chronic lymphocytic leukemia/small lymphocytic

  3. Comparative study of lymphocytes from individuals that were vaccinated and unvaccinated against the pandemic 2009-2011 H1N1 influenza virus in Southern Brazil