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Sample records for human tumor-infiltrating lymphocytes

  1. Expansion of human tumor infiltrating lymphocytes for use in immunotherapy trials.

    PubMed

    Topalian, S L; Muul, L M; Solomon, D; Rosenberg, S A

    1987-08-24

    The potential utility of tumor-infiltrating lymphocytes (TIL) in the adoptive immunotherapy of human tumors has been suggested by murine experiments showing these cells to be 50-100 times more powerful than LAK cells in treating advanced metastatic disease. A method for the large-scale expansion of human TIL for the use of these cells in clinical trials is described in this report. TIL were successfully expanded on an experimental scale from 24 of 25 consecutive human tumors, including six melanomas, ten sarcomas, and eight adenocarcinomas. Tumors were digested enzymatically to yield single cell suspensions which were cultured in RPMI 1640 medium with 10% human serum and 1000 U/ml recombinant interleukin-2. Lymphocytes constituted from 3% to 74% of single cell tumor suspensions, and expanded from 2.9-fold to 9.1 X 10(8)-fold over a culture period ranging from 14 to 100 days. Nine of 24 TIL cultures lysed fresh autologous tumor targets in 4 h chromium release assays. Cell surface phenotyping identified cultured TIL as activated cytotoxic/suppressor T cells. Subsequently, large-scale expansion of TIL was successful in generating more than 10(10) lymphocytes in five of eight consecutive cases. Clinical trials employing the adoptive transfer of expanded TIL to patients with metastatic disease have begun. PMID:3305708

  2. Growth of tumor-infiltrating lymphocytes from human solid cancers: summary of a 5-year experience.

    PubMed

    Yannelli, J R; Hyatt, C; McConnell, S; Hines, K; Jacknin, L; Parker, L; Sanders, M; Rosenberg, S A

    1996-02-01

    Between 1989 and 1993, 255 tumor biopsies representing 4 tumor histologies (melanoma, breast cancer, colon cancer and renal cell cancer) were received by the Surgery Branch of the National Cancer Institute. Tumor-infiltrating lymphocytes (TIL) were grown from single-cell suspensions of tumor biopsies over the course of 30-45 days. The TIL were grown in medium containing IL-2. To obtain numbers suitable for therapy (>10(11)), TIL were expanded using a large-scale system of cell culture and harvesting. While the largest number of biopsies was obtained from melanoma patients, TIL were successfully grown from 160 of 255 tumor biopsies representing all 4 histologies. Under the culture conditions employed, several characteristics of TIL expansion were observed. The cell surface phenotype of TIL which grew out from the tumor biopsies was generally a mix of CD3+/CD4+ or CD3+/CD8+ lymphocytes. Only TIL from melanoma biopsies were found to be consistently cytolytic and, in many cases, lysed autologous tumor cells preferentially. Interestingly, TIL derived from extra-nodal sites of metastatic melanoma biopsies (subcutaneous, lung, bowel; 36 of 67, 54%) were more likely to have these cytolytic characteristics than TIL derived from tumor-involved lymph node biopsies (7 of 39, 18%). The present study summarizes 5 years of laboratory effort and validates the technologies developed for the large-scale growth and harvesting of TIL. In addition, it summarizes the laboratory effort supporting previously published clinical reports on TIL from our group. PMID:8621219

  3. Human gene transfer: Characterization of human tumor-infiltrating lymphocytes as vehicles for retroviral-mediated gene transfer in man

    SciTech Connect

    Kasid, A.; Morecki, S.; Aebersold, P.; Cornetta, K.; Culver, K.; Freeman, S.; Director, E.; Lotze, M.T.; Blaese, R.M.; Anderson, W.F.; Rosenberg, S.A. )

    1990-01-01

    Tumor-infiltrating lymphocytes (TILs) are cells generated from tumor suspensions cultured in interleukin 2 that can mediate cancer regression when adoptively transferred into mice or humans. Since TILs proliferate rapidly in vitro, recirculate, and preferentially localize at the tumor site in vivo, they provide an attractive model for delivery of exogenous genetic material into man. To determine whether efficient gene transfer into TILs is feasible. The authors transduced human TILs with the bacterial gene for neomycin-resistance (Neo{sup R}) using the retroviral vector N2. The transduced TIL populations were stable and polyclonal with respect to the intact Neo{sup R} gene integration and expressed high levels of neomycin phosphotransferase activity. The Neo{sup R} gene insertion did not alter the in vitro growth pattern and interleukin 2 dependence of the transduced TILs. Analyses of T-cell receptor gene rearrangement for {beta}- and {gamma}-chain genes revealed the oligoclonal nature of the TIL populations with no major change in the DNA rearrangement patterns or the levels of mRNA expression of the {beta} and {gamma} chains following transduction and selection of TILs in the neomycin analog G418. Human TILs expressed mRNA for tumor necrosis factors ({alpha} and {beta}) and interleukin 2 receptor P55. This pattern of cytokine-mRNA expression was not significantly altered following the transduction of TILs. The studies demonstrate the feasibility of TILs as suitable cellular vehicles for the introduction of therapeutic genes into patients receiving autologous TILs.

  4. Tumor infiltrating lymphocytes in ovarian cancer

    PubMed Central

    Santoiemma, Phillip P; Powell, Daniel J

    2015-01-01

    The accumulation of tumor infiltrating lymphocytes (TILs) in ovarian cancer is prognostic for increased survival while increases in immunosuppressive regulatory T-cells (Tregs) are associated with poor outcomes. Approaches that bolster tumor-reactive TILs may limit tumor progression. However, identifying tumor-reactive TILs in ovarian cancer has been challenging, though adoptive TIL therapy in patients has been encouraging. Other forms of TIL immunomodulation remain under investigation including Treg depletion, antibody-based checkpoint modification, activation and amplification using dendritic cells, antigen presenting cells or IL-2 cytokine culture, adjuvant cytokine injections, and gene-engineered T-cells. Many approaches to TIL manipulation inhibit ovarian cancer progression in preclinical or clinical studies as monotherapy. Here, we review the impact of TILs in ovarian cancer and attempts to mobilize TILs to halt tumor progression. We conclude that effective TIL therapy for ovarian cancer is at the brink of translation and optimal TIL activity may require combined methodologies to deliver clinically-relevant treatment. PMID:25894333

  5. Relevance of tumor-infiltrating lymphocytes in breast cancer.

    PubMed

    Dushyanthen, Sathana; Beavis, Paul A; Savas, Peter; Teo, Zhi Ling; Zhou, Chenhao; Mansour, Mariam; Darcy, Phillip K; Loi, Sherene

    2015-01-01

    While breast cancer has not been considered a cancer amenable to immunotherapeutic approaches, recent studies have demonstrated evidence of significant immune cell infiltration via tumor-infiltrating lymphocytes in a subset of patient tumors. In this review we present the current evidence highlighting the clinical relevance and utility of tumor-infiltrating lymphocytes in breast cancer. Retrospective and prospective studies have shown that the presence of tumor-infiltrating lymphocytes is a prognostic marker for higher responses to neoadjuvant chemotherapy and better survival, particularly in triple negative and HER2-positive early breast cancer. Further work is required to determine the immune subsets important in this response and to discover ways of encouraging immune infiltrate in tumor-infiltrating lymphocytes-negative patients. PMID:26300242

  6. [Research progress of tumor infiltrating lymphocytes in breast cancer].

    PubMed

    Huang, Jiahui; Chen, Xiaosong; Shen, Kunwei

    2015-09-01

    Breast cancer is a heterogeneous disease. The formation and progression of tumor and the sensitivity to treatment differs from patient to patient. In addition to the widely used molecular subtype, novel markers are needed to better personalize the treatment of breast cancer. Tumor infiltrating lymphocyte (TIL) have been consistently documented in breast cancer lesions especially in triple negative and human epidermal growth factor receptor-2 positive breast cancer. Several clinical trials have revealed that TIL are associated with prognosis and can predict therapeutic efficacy of special therapy. TIL could be divided to different subtypes including CD8 + TIL, CD4 + TIL, cytotoxic T lymphocyte-associated antigen-4 + TIL, programmed death-1 + TIL. They play different roles in the process of anti-tumor immunity and can predict different prognosis. Screening out special TIL subtype which is well associated with prognosis and therapeutic efficacy and developing targeting immunotherapy can help to improve outcomes of breast cancer patients. PMID:26654152

  7. Identification of a human melanoma antigen recognized by tumor-infiltrating lymphocytes associated with in vivo tumor rejection.

    PubMed Central

    Kawakami, Y; Eliyahu, S; Delgado, C H; Robbins, P F; Sakaguchi, K; Appella, E; Yannelli, J R; Adema, G J; Miki, T; Rosenberg, S A

    1994-01-01

    The cultured T-cell line TIL1200, established from the tumor-infiltrating lymphocytes (TILs) of a patient with advanced metastatic melanoma, recognized an antigen on most HLA-A2+ melanomas and on all HLA-A2+ cultured neonatal melanocytes in an HLA-A2 restricted manner but not on other types of tissues or cell lines tested. A cDNA encoding an antigen recognized by TIL1200 was isolated by screening an HLA-A2+ breast cancer cell line transfected with an expression cDNA library prepared from an HLA-A2+ melanoma cell line. The nucleotide and amino acid sequences of this cDNA were almost identical to the genes encoding glycoprotein gp100 or Pmel17 previously registered in the GenBank. Expression of this gene was restricted to melanoma and melanocyte cell lines and retina but was not expressed on other fresh or cultured normal tissues or other types of tumor tested. The cell line transfected with this cDNA also expressed antigen recognized by the melanoma-specific antibody HMB45 that bound to gp100. A synthetic 10-amino acid peptide derived from gp100 was recognized by TIL1200 in the context of HLA-A2.1. Since the administration of TIL1200 plus interleukin 2 resulted in regression of metastatic cancer in the autologous patient, gp100 is a possible tumor rejection antigen and may be useful for the development of immunotherapies for patients with melanoma. Images PMID:8022805

  8. [Principles of adoptive cell therapy based on "Tumor Infiltrating Lymphocytes"].

    PubMed

    Martins, Filipe; Orcurto, Angela; Michielin, Olivier; Coukos, George

    2016-05-18

    Adoptive cell therapy consists in the use of T lymphocytes for therapeutic purposes. Up to now, of limited use in clinical practice for logistical reasons, technical progress and substantial level of evidence obtained in the last decade allow its arrival in universitary hospitals. We will principally discuss the administration of expanded tumor infiltrating T cells in the treatment of metastatic melanoma. This treatment modality exploits the natural specificity of these cells and aims to potentiate their effectiveness. This personalized immunotherapy detains a potential for expansion to many other advanced tumor types. PMID:27424426

  9. Distribution and prognostic relevance of tumor-infiltrating lymphocytes (TILs) and PD-1/PD-L1 immune checkpoints in human brain metastases

    PubMed Central

    Harter, Patrick N.; Bernatz, Simon; Scholz, Alexander; Zeiner, Pia S.; Zinke, Jenny; Kiyose, Makoto; Blasel, Stella; Beschorner, Rudi; Senft, Christian; Bender, Benjamin; Ronellenfitsch, Michael W.; Wikman, Harriet; Glatzel, Markus; Meinhardt, Matthias; Juratli, Tareq A.; Steinbach, Joachim P.; Plate, Karl H.; Wischhusen, Jörg; Weide, Benjamin; Mittelbronn, Michel

    2015-01-01

    The activation of immune cells by targeting checkpoint inhibitors showed promising results with increased patient survival in distinct primary cancers. Since only limited data exist for human brain metastases, we aimed at characterizing tumor infiltrating lymphocytes (TILs) and expression of immune checkpoints in the respective tumors. Two brain metastases cohorts, a mixed entity cohort (n = 252) and a breast carcinoma validation cohort (n = 96) were analyzed for CD3+, CD8+, FOXP3+, PD-1+ lymphocytes and PD-L1+ tumor cells by immunohistochemistry. Analyses for association with clinico-epidemiological and neuroradiological parameters such as patient survival or tumor size were performed. TILs infiltrated brain metastases in three different patterns (stromal, peritumoral, diffuse). While carcinomas often show a strong stromal infiltration, TILs in melanomas often diffusely infiltrate the tumors. Highest levels of CD3+ and CD8+ lymphocytes were seen in renal cell carcinomas (RCC) and strongest PD-1 levels on RCCs and melanomas. High amounts of TILs, high ratios of PD-1+/CD8+ cells and high levels of PD-L1 were negatively correlated with brain metastases size, indicating that in smaller brain metastases CD8+ immune response might get blocked. PD-L1 expression strongly correlated with TILs and FOXP3 expression. No significant association of patient survival with TILs was observed, while high levels of PD-L1 showed a strong trend towards better survival in melanoma brain metastases (Log-Rank p = 0.0537). In summary, melanomas and RCCs seem to be the most immunogenic entities. Differences in immunotherapeutic response between tumor entities regarding brain metastases might be attributable to this finding and need further investigation in larger patient cohorts. PMID:26517811

  10. Tumor-infiltrating lymphocytes predict cutaneous melanoma survival.

    PubMed

    Fortes, Cristina; Mastroeni, Simona; Mannooranparampil, Thomas J; Passarelli, Francesca; Zappalà, Alba; Annessi, Giorgio; Marino, Claudia; Caggiati, Alessio; Russo, Nicoletta; Michelozzi, Paola

    2015-08-01

    Understanding differences in survival across distinct subgroups of melanoma patients may help with the choice of types of therapy. Tumor-infiltrating lymphocytes (TILs) are considered a manifestation of the host immune response to tumor, but the role of TILs in melanoma mortality is controversial. The aim of this study was to investigate independent prognostic factors for melanoma mortality. We carried out a 10-year cohort study on 4133 melanoma patients from the same geographic area (Lazio) with primary cutaneous melanoma diagnosed between January 1998 and December 2008. The probability of survival was estimated using Kaplan-Meier methods and prognostic factors were evaluated by multivariate analysis (Cox proportional hazards model). The 10-year survival rate for melanoma decreased with increasing Breslow thickness (Pfor trend<0.0001) and with age (Pfor trend<0.0001) whereas survival increased with increasing levels of TILs (Pfor trend=0.0001). The 10-year survival rate for melanoma divided into TILs intensity as scanty, moderate, and marked was 88.0, 92.2, and 97.0%, respectively. In the multivariate Cox model, the presence of high levels of TILs in primary invasive melanomas was associated with a lower risk of melanoma death (hazard ratio 0.32; 95% confidence interval 0.13-0.82) after controlling for sex, age, Breslow thickness, histological type, mitotic rate, and ulceration. After including lymph node status in the multivariate analysis, the protective effect of marked TILs on melanoma mortality remained (hazard ratio 0.37; 95% confidence interval 0.15-0.94). The results of this study suggest that the immune microenvironment affects melanoma survival. PMID:25933208

  11. Sunitinib pretreatment improves tumor-infiltrating lymphocyte expansion by reduction in intratumoral content of myeloid-derived suppressor cells in human renal cell carcinoma.

    PubMed

    Guislain, Aurelie; Gadiot, Jules; Kaiser, Andrew; Jordanova, Ekaterina S; Broeks, Annegien; Sanders, Joyce; van Boven, Hester; de Gruijl, Tanja D; Haanen, John B A G; Bex, Axel; Blank, Christian U

    2015-10-01

    Targeted therapy with sunitinib, pazopanib or everolimus has improved treatment outcome for patients with metastatic renal cell carcinoma patients (RCC). However, despite considerable efforts in sequential or combined modalities, durable remissions are rare. Immunotherapy like cytokine therapy with interleukin-2, T cell checkpoint blockade or adoptive T cell therapies can achieve long-term benefit and even cure. This raises the question of whether combining targeted therapy with immunotherapy could also be an effective treatment option for RCC patients. Sunitinib, one of the most frequently administered therapeutics in RCC patients has been implicated in impairing T cell activation and proliferation in vitro. In this work, we addressed whether this notion holds true for expansion of tumor-infiltrating lymphocytes (TILs) in sunitinib-treated patients. We compared resected primary RCC tumor material of patients pretreated with sunitinib with resection specimen from sunitinib-naïve patients. We found improved TIL expansion from sunitinib-pretreated tumor digests. These TIL products contained more PD-1 expressing TIL, while the regulatory T cell infiltration was not altered. The improved TIL expansion was associated with reduced intratumoral myeloid-derived suppressor cell (MDSC) content. Depletion of MDSCs from sunitinib-naïve RCC tissue-digest improved TIL expansion, proving the functional relevance of the MDSC alteration by sunitinib. Our in vivo results do not support previous in vitro observations of sunitinib inhibiting T cell function, but do provide a possible rationale for the combination of sunitinib with immunotherapy. PMID:26105626

  12. Simplified Method of the Growth of Human Tumor Infiltrating Lymphocytes (TIL) in Gas-Permeable Flasks to Numbers Needed for Patient Treatment

    PubMed Central

    Jin, Jianjian; Sabatino, Marianna; Somerville, Robert; Wilson, John R.; Dudley, Mark E.; Stroncek, David F.; Rosenberg, Steven A.

    2012-01-01

    Adoptive cell therapy (ACT) of metastatic melanoma with autologous tumor infiltrating lymphocytes (TIL) is clinically effective, but TIL production can be challenging. Here we describe a simplified method for initial TIL culture and rapid expansion in gas-permeable flasks. TIL were initially cultured from tumor digests and fragments in 40 mL capacity flasks with a 10 cm2 gas-permeable silicone bottom, G-Rex10. A TIL rapid expansion protocol (REP) was developed using 500 mL capacity flasks with a 100 cm2 gas-permeable silicone bottom, G-Rex100. TIL growth was successfully initiated in G-Rex10 flasks from tumor digests from 13 of 14 patients and from tumor fragments in all 11 tumor samples tested. TIL could then be expanded to 8–10×109 cells in a two-step REP which began by seeding 5 × 106 TIL into a G-Rex100 flask, followed by expansion at day 7 into 3 G-Rex100 flasks. To obtain the 30 to 60 × 109 cells used for patient treatment we seeded 6 G-Rex100 flasks with 5×106 cells and expanded into 18 G-Rex100 flasks. Large scale TIL REP in gas-permeable flasks requires approximately 9 to 10 liters of media, about 3 to 4 times less than other methods. In conclusion, TIL initiation and REP in gas-permeable G-Rex flasks require fewer total vessels, less media, less incubator space and less labor than initiation and REP in 24-well plates, tissue culture flasks and bags. TIL culture in G-Rex flasks will facilitate the production of TIL at the numbers required for patient treatment at most cell processing laboratories. PMID:22421946

  13. Current Issues and Clinical Evidence in Tumor-Infiltrating Lymphocytes in Breast Cancer

    PubMed Central

    Ahn, Sung Gwe; Jeong, Joon; Hong, SoonWon; Jung, Woo Hee

    2015-01-01

    With the advance in personalized therapeutic strategies in patients with breast cancer, there is an increasing need for biomarker-guided therapy. Although the immunogenicity of breast cancer has not been strongly considered in research or practice, tumor-infiltrating lymphocytes (TILs) are emerging as biomarkers mediating tumor response to treatments. Earlier studies have provided evidence that the level of TILs has prognostic value and the potential for predictive value, particularly in triple-negative and human epidermal growth factor receptor 2–positive breast cancer. Moreover, the level of TILs has been associated with treatment outcome in patients undergoing neoadjuvant chemotherapy. To date, no standardized methodology for measuring TILs has been established. In this article, we review current issues and clinical evidence for the use of TILs in breast cancer. PMID:26278518

  14. Reprogramming of Melanoma Tumor-Infiltrating Lymphocytes to Induced Pluripotent Stem Cells.

    PubMed

    Saito, Hidehito; Okita, Keisuke; Fusaki, Noemi; Sabel, Michael S; Chang, Alfred E; Ito, Fumito

    2016-01-01

    Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients hold great promise for autologous cell therapies. One of the possible applications of iPSCs is to use them as a cell source for producing autologous lymphocytes for cell-based therapy against cancer. Tumor-infiltrating lymphocytes (TILs) that express programmed cell death protein-1 (PD-1) are tumor-reactive T cells, and adoptive cell therapy with autologous TILs has been found to achieve durable complete response in selected patients with metastatic melanoma. Here, we describe the derivation of human iPSCs from melanoma TILs expressing high level of PD-1 by Sendai virus-mediated transduction of the four transcription factors, OCT3/4, SOX2, KLF4, and c-MYC. TIL-derived iPSCs display embryonic stem cell-like morphology, have normal karyotype, express stem cell-specific surface antigens and pluripotency-associated transcription factors, and have the capacity to differentiate in vitro and in vivo. A wide variety of T cell receptor gene rearrangement patterns in TIL-derived iPSCs confirmed the heterogeneity of T cells infiltrating melanomas. The ability to reprogram TILs containing patient-specific tumor-reactive repertoire might allow the generation of patient- and tumor-specific polyclonal T cells for cancer immunotherapy. PMID:27057178

  15. Reprogramming of Melanoma Tumor-Infiltrating Lymphocytes to Induced Pluripotent Stem Cells

    PubMed Central

    Saito, Hidehito; Okita, Keisuke; Fusaki, Noemi; Sabel, Michael S.; Chang, Alfred E.; Ito, Fumito

    2016-01-01

    Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients hold great promise for autologous cell therapies. One of the possible applications of iPSCs is to use them as a cell source for producing autologous lymphocytes for cell-based therapy against cancer. Tumor-infiltrating lymphocytes (TILs) that express programmed cell death protein-1 (PD-1) are tumor-reactive T cells, and adoptive cell therapy with autologous TILs has been found to achieve durable complete response in selected patients with metastatic melanoma. Here, we describe the derivation of human iPSCs from melanoma TILs expressing high level of PD-1 by Sendai virus-mediated transduction of the four transcription factors, OCT3/4, SOX2, KLF4, and c-MYC. TIL-derived iPSCs display embryonic stem cell-like morphology, have normal karyotype, express stem cell-specific surface antigens and pluripotency-associated transcription factors, and have the capacity to differentiate in vitro and in vivo. A wide variety of T cell receptor gene rearrangement patterns in TIL-derived iPSCs confirmed the heterogeneity of T cells infiltrating melanomas. The ability to reprogram TILs containing patient-specific tumor-reactive repertoire might allow the generation of patient- and tumor-specific polyclonal T cells for cancer immunotherapy. PMID:27057178

  16. A New Approach to the Adoptive Immunotherapy of Cancer with Tumor-Infiltrating Lymphocytes

    NASA Astrophysics Data System (ADS)

    Rosenberg, Steven A.; Spiess, Paul; Lafreniere, Rene

    1986-09-01

    The adoptive transfer of tumor-infiltrating lymphocytes (TIL) expanded in interleukin-2 (IL-2) to mice bearing micrometastases from various types of tumors showed that TIL are 50 to 100 times more effective in their therapeutic potency than are lymphokine-activated killer (LAK) cells. Therefore the use of TIL was explored for the treatment of mice with large pulmonary and hepatic metastatic tumors that do not respond to LAK cell therapy. Although treatment of animals with TIL alone or cyclophosphamide alone had little impact, these two modalities together mediated the elimination of large metastatic cancer deposits in the liver and lung. The combination of TIL and cyclophosphamide was further potentiated by the simultaneous administration of IL-2. With the combination of cyclophosphamide, TIL, and IL-2, 100% of mice (n = 12) bearing the MC-38 colon adenocarcinoma were cured of advanced hepatic metastases, and up to 50% of mice were cured of advanced pulmonary metastases. Techniques have been developed to isolate TIL from human tumors. These experiments provide a rationale for the use of TIL in the treatment of humans with advanced cancer.

  17. Tumor infiltration by chemokine receptor 7 (CCR7)+ T-lymphocytes is a favorable prognostic factor in metastatic colorectal cancer

    PubMed Central

    Correale, Pierpaolo; Rotundo, Maria Saveria; Botta, Cirino; Del Vecchio, Maria Teresa; Tassone, Pierfrancesco; Tagliaferri, Pierosandro

    2012-01-01

    The immune interactions occurring within the tumor microenvironment have a critical role in determining the outcome of colorectal cancer patients. We carried-out an immunohistochemical analysis of tumor infiltrating T-lymphocytes expressing chemokine receptor 7 (CCR7) in a series of colorectal cancer patients enrolled in a prospective clinical trial. We demonstrated that a high tumor infiltration score of this lymphocyte subset is predictive of longer progression free survival and overall survival. PMID:22754775

  18. Does famotidine enhance tumor infiltrating lymphocytes in breast cancer? Results of a randomized prospective pilot study.

    PubMed

    Parshad, Rajinder; Kapoor, Sorabh; Gupta, Siddhartha Datta; Kumar, Arvind; Chattopadhyaya, Tushar K

    2002-01-01

    Thirty patients with breast cancer were prospectively randomized into case and control groups receiving 40 mg famotidine preoperatively for 10-14 days and routine premedication, respectively. Surgical specimens were evaluated objectively for tumor infiltrating lymphocytes in the center and in the periphery of the tumor along with evaluation of metastatic lymph nodes for reactive changes. Ten famotidine-treated cases (67%) showed significant lymphocytic infiltration in the center compared to 4 controls (27%) (p = 0.03). Eleven cases (77%) had significant lymphocytic infiltration in the periphery (p = 0.03) compared to 5 controls (33%). Considering both sites, lymphocytic response was significant in 9 (60%) cases as opposed to only 3 (20%) controls (p = 0.03). This response did not correlate with the stage, grade of tumor or menopausal status of patients in either group. Seventy-eight percent (78%) of the cases showed significant reactive changes in the metastatic lymph nodes as compared to 22% in controls (p < 0.01). This study suggests that famotidine enhances tumor infiltrating lymphocytes in breast cancer and might have potential as an immunomodulator. A larger confirmatory study is suggested. PMID:12234028

  19. Characterization of Ex Vivo Expanded Tumor Infiltrating Lymphocytes from Patients with Malignant Melanoma for Clinical Application

    PubMed Central

    Junker, Niels; thor Straten, Per; Andersen, Mads Hald; Svane, Inge Marie

    2011-01-01

    Clinical trials of adoptive transfer of autologous tumor infiltrating lymphocytes (TILs) to patients with advanced malignant melanoma have shown remarkable results with objective clinical responses in 50% of the treated patients. In order to initiate a clinical trial in melanoma, we have established a method for expanding TILs to clinical relevant quantities in two steps with in 8 weeks. Further characterization of expanded TILs revealed an oligoclonal composition of T-cells with an effector memory like phenotype. When autologous tumor was available, TILs showed specific activity in all patients tested. TIL cultures contained specificity towards tumor cells as well as peptides derived from tumor-associated antigens (TAAs) during expansion procedures. PMID:21773037

  20. Tumor Infiltrating Lymphocytes – The Next Step in Assessing Outcome and Response to Treatment in Patients with Breast Cancer

    PubMed Central

    Wesolowski, Robert; Carson, William E

    2015-01-01

    Tumor infiltrating lymphocytes are studied for their potential as new clinically useful prognostic and predictive biomarkers in patients with triple negative and HER-2/neu amplified breast cancer. This area of research could also help guide the development of novel therapeutic approaches for these diseases. PMID:25750763

  1. Tumor-Infiltrating Lymphocytes in Triple Negative Breast Cancer: The Future of Immune Targeting

    PubMed Central

    García-Teijido, Paula; Cabal, María Luque; Fernández, Ignacio Peláez; Pérez, Yolanda Fernández

    2016-01-01

    Triple negative breast cancer (TNBC) is a highly heterogeneous tumor. There is increasing evidence of the role of tumor lymphocytic immune infiltrates in this subtype of breast cancer. Robust levels of tumor infiltrating lymphocytes (TILs) have been associated with improved disease-free and overall survival rates in TNBC patients with and without any treatment. Recent efforts have been made to develop a standardized methodology for evaluating TILs. The presence of TILs in the breast tumor microenvironment can also predict responses not only to neoadjuvant but also to adjuvant chemotherapy treatments. High numbers of TILs correlate with increased pathological complete responses (pCR) in TNBC. TILs are prognostic and predictive of response to standard therapies; thus, the immune system appears to play an active role in a subgroup of breast cancer. There is an increasing interest in directly targeting the immune system as part of breast cancer therapy, mainly in patients with TNBC. New immune modulatory agents, including immune checkpoints inhibitors, have shown promising activity in a subgroup of metastatic TNBC. Increased programmed cell death protein 1 ligand (PD-L1) expression on the surface of TNBC provides the rationale for implementing therapeutic strategies targeting the PD-1/PD-L1 axis in TNBC. The programmed cell death protein 1 (PD-1) inhibitor pembrolizumab, and the PD-L1 inhibitor atezolizumab have shown promising results in clinical trials. PMID:27081325

  2. Large Intrahepatic Cholangiocarcinoma with Tumor Infiltrative Lymphocytes and Autoimmune Hepatitis-Like Features

    PubMed Central

    Izumi, Sadanobu; Nakamura, Satoko; Mano, Shohei

    2010-01-01

    The development of a primary hepatic tumor associated with autoimmune hepatitis (AIH) has been rarely reported. This report describes a rare case of intrahepatic cholangiocarcinoma (ICC) that accompanied tumor infiltrative lymphocytes (TIL) and AIH-like features. Moreover, multiple early gastric cancers were recognized in synchrony. An 81-year-old male was admitted due to liver dysfunction. His laboratory data on admission showed an elevation of immunoglobulin G and a positive titer of antinuclear antibody. Biological tests for HBV and HCV were negative. Computed tomography showed a well-enhanced hepatic tumor and gastrointestinal fiberscopy revealed two early gastric cancers with mucosal invasion. Biopsies were obtained from the background liver and the hepatic tumor. Histologically, the tumor revealed adenocarcinoma and the liver showed piecemeal necrosis and interface hepatitis with lymphoplasmacytic infiltration. The patient underwent hepatectomy and distal gastrectomy. Finally, he was diagnosed to have a mass forming type ICC and early gastric cancers. Moreover, prominent TIL in the ICC was revealed. An analysis of the infiltrating lymphocytes by immunohistochemical staining suggested that there was a difference in the local immune response between the tumor and the background liver. Review of the literature showed that there are only three reports of ICC associated with AIH, if including the current case. PMID:21103227

  3. The Relationship between Obesity, Prostate Tumor Infiltrating Lymphocytes and Macrophages, and Biochemical Failure

    PubMed Central

    Zeigler-Johnson, Charnita; Morales, Knashawn H.; Lal, Priti; Feldman, Michael

    2016-01-01

    Background Obesity reflects a chronic inflammatory environment that may contribute to prostate cancer progression and poor treatment outcomes. However, it is not clear which mechanisms drive this association within the tumor microenvironment. The aim of this pilot study was to examine prostatic inflammation via tumor infiltrating lymphocytes and macrophages characterized by obesity and cancer severity. Methods We studied paraffin-embedded prostatectomy tissue from 99 participants (63 non-obese and 36 obese) from the Study of Clinical Outcomes, Risk and Ethnicity (University of Pennsylvania). Pathologists analyzed the tissue for type and count of lymphocytes and macrophages, including CD3, CD8, FOXP3, and CD68. Pathology data were linked to clinical and demographic variables. Statistical analyses included frequency tables, Kruskal-Wallis tests, Spearman correlations, and multivariable models. Results We observed positive univariate associations between the number of CD68 cells and tumor grade (p = 0.019). In multivariable analysis, CD8 counts were associated with time to biochemical failure (HR = 1.09, 95% CI = 1.004–1.192, p-value = 0.041.) There were no differences in lymphocytes or macrophages by obesity status or BMI. Conclusions The number of lymphocytes and macrophages in the tumor microenvironment did not differ by obesity status. However, these inflammation markers were associated with poor prostate cancer outcomes. Further examination of underlying mechanisms that influence obesity-related effects on prostate cancer outcomes is warranted. Such research will guide immunotherapy protocols and weight management as they apply to diverse patient populations and phenotypes. PMID:27487262

  4. Immune Checkpoint Blockade to Improve Tumor Infiltrating Lymphocytes for Adoptive Cell Therapy

    PubMed Central

    Kodumudi, Krithika N.; Siegel, Jessica; Weber, Amy M.; Scott, Ellen; Sarnaik, Amod A.; Pilon-Thomas, Shari

    2016-01-01

    Tumor-infiltrating lymphocytes (TIL) has been associated with improved survival in cancer patients. Within the tumor microenvironment, regulatory cells and expression of co-inhibitory immune checkpoint molecules can lead to the inactivation of TIL. Hence, there is a need to develop strategies that disrupt these negative regulators to achieve robust anti-tumor immune responses. We evaluated the blockade of immune checkpoints and their effect on T cell infiltration and function. We examined the ability of TIL to induce tumor-specific immune responses in vitro and in vivo. TIL isolated from tumor bearing mice were tumor-specific and expressed co-inhibitory immune checkpoint molecules. Administration of monoclonal antibodies against immune checkpoints led to a significant delay in tumor growth. However, anti-PD-L1 antibody treated mice had a significant increase in T cell infiltration and IFN-γ production compared to other groups. Adoptive transfer of in vitro expanded TIL from tumors of anti-PD-L1 antibody treated mice led to a significant delay in tumor growth. Blockade of co-inhibitory immune checkpoints could be an effective strategy to improve TIL infiltration and function. PMID:27050669

  5. Tumor-Infiltrating γδ T Lymphocytes: Pathogenic Role, Clinical Significance, and Differential Programing in the Tumor Microenvironment

    PubMed Central

    Lo Presti, Elena; Dieli, Franceso; Meraviglia, Serena

    2014-01-01

    There is increasing clinical evidence indicating that the immune system may either promote or inhibit tumor progression. Several studies have demonstrated that tumors undergoing remission are largely infiltrated by T lymphocytes [tumor-infiltrating lymphocytes (TILs)], but on the other hand, several studies have shown that tumors may be infiltrated by TILs endowed with suppressive features, suggesting that TILs are rather associated with tumor progression and unfavorable prognosis. γδ T lymphocytes are an important component of TILs that may contribute to tumor immunosurveillance, as also suggested by promising reports from several small phase-I clinical trials. Typically, γδ T lymphocytes perform effector functions involved in anti-tumor immune responses (cytotoxicity, production of IFN-γ and TNF-α, and dendritic cell maturation), but under appropriate conditions they may divert from the typical Th1-like phenotype and polarize to Th2, Th17, and Treg cells thus acquiring the capability to inhibit anti-tumor immune responses and promote tumor growth. Recent studies have shown a high frequency of γδ T lymphocytes infiltrating different types of cancer, but the nature of this association and the exact mechanisms underlying it remain uncertain and whether or not the presence of tumor-infiltrating γδ T lymphocytes is a definite prognostic factor remains controversial. In this paper, we will review studies of tumor-infiltrating γδ T lymphocytes from patients with different types of cancer, and we will discuss their clinical relevance. Moreover, we will also discuss on the complex interplay between cancer, tumor stroma, and γδ T lymphocytes as a major determinant of the final outcome of the γδ T lymphocyte response. Finally, we propose that targeting γδ T lymphocyte polarization and skewing their phenotype to adapt to the microenvironment might hold great promise for the treatment of cancer. PMID:25505472

  6. Detection of human brain tumor infiltration with quantitative stimulated Raman scattering microscopy

    PubMed Central

    Ji, Minbiao; Lewis, Spencer; Camelo-Piragua, Sandra; Ramkissoon, Shakti H.; Snuderl, Matija; Venneti, Sriram; Fisher-Hubbard, Amanda; Garrard, Mia; Fu, Dan; Wang, Anthony C.; Heth, Jason A.; Maher, Cormac O.; Sanai, Nader; Johnson, Timothy D.; Freudiger, Christian W.; Sagher, Oren; Xie, Xiaoliang Sunney; Orringer, Daniel A.

    2016-01-01

    Differentiating tumor from normal brain is a major barrier to achieving optimal outcome in brain tumor surgery. New imaging techniques for visualizing tumor margins during surgery are needed to improve surgical results. We recently demonstrated the ability of stimulated Raman scattering (SRS) microscopy, a non-destructive, label-free optical method, to reveal glioma infiltration in animal models. Here we show that SRS reveals human brain tumor infiltration in fresh, unprocessed surgical specimens from 22 neurosurgical patients. SRS detects tumor infiltration in near-perfect agreement with standard hematoxylin and eosin light microscopy (κ=0.86). The unique chemical contrast specific to SRS microscopy enables tumor detection by revealing quantifiable alterations in tissue cellularity, axonal density and protein:lipid ratio in tumor-infiltrated tissues. To ensure that SRS microscopic data can be easily used in brain tumor surgery, without the need for expert interpretation, we created a classifier based on cellularity, axonal density and protein:lipid ratio in SRS images capable of detecting tumor infiltration with 97.5% sensitivity and 98.5% specificity. Importantly, quantitative SRS microscopy detects the spread of tumor cells, even in brain tissue surrounding a tumor that appears grossly normal. By accurately revealing tumor infiltration, quantitative SRS microscopy holds potential for improving the accuracy of brain tumor surgery. PMID:26468325

  7. Detection of human brain tumor infiltration with quantitative stimulated Raman scattering microscopy.

    PubMed

    Ji, Minbiao; Lewis, Spencer; Camelo-Piragua, Sandra; Ramkissoon, Shakti H; Snuderl, Matija; Venneti, Sriram; Fisher-Hubbard, Amanda; Garrard, Mia; Fu, Dan; Wang, Anthony C; Heth, Jason A; Maher, Cormac O; Sanai, Nader; Johnson, Timothy D; Freudiger, Christian W; Sagher, Oren; Xie, Xiaoliang Sunney; Orringer, Daniel A

    2015-10-14

    Differentiating tumor from normal brain is a major barrier to achieving optimal outcome in brain tumor surgery. New imaging techniques for visualizing tumor margins during surgery are needed to improve surgical results. We recently demonstrated the ability of stimulated Raman scattering (SRS) microscopy, a nondestructive, label-free optical method, to reveal glioma infiltration in animal models. We show that SRS reveals human brain tumor infiltration in fresh, unprocessed surgical specimens from 22 neurosurgical patients. SRS detects tumor infiltration in near-perfect agreement with standard hematoxylin and eosin light microscopy (κ = 0.86). The unique chemical contrast specific to SRS microscopy enables tumor detection by revealing quantifiable alterations in tissue cellularity, axonal density, and protein/lipid ratio in tumor-infiltrated tissues. To ensure that SRS microscopic data can be easily used in brain tumor surgery, without the need for expert interpretation, we created a classifier based on cellularity, axonal density, and protein/lipid ratio in SRS images capable of detecting tumor infiltration with 97.5% sensitivity and 98.5% specificity. Quantitative SRS microscopy detects the spread of tumor cells, even in brain tissue surrounding a tumor that appears grossly normal. By accurately revealing tumor infiltration, quantitative SRS microscopy holds potential for improving the accuracy of brain tumor surgery. PMID:26468325

  8. Heterogeneous expansion of CD4+ tumor-infiltrating T-lymphocytes in clear cell renal cell carcinomas.

    PubMed

    Zhang, Qian; Jia, Qingzhu; Deng, Tianxing; Song, Bo; Li, Longkun

    2015-02-27

    Aberrant expression of tumor-associated antigens (TAAs) mediates the effective mounting of adaptive immunity in human solid tumors. The foundations of this tumor-host interaction strongly depend on specific recognition via TAA-cognate-receptors in T-cell repertoires. Previous studies focused on the phenotypic and functional properties of CD4+/CD8+ tumor-infiltrating T-lymphocytes (TILs), but the detailed composition of T-cell repertoires of these fundamental subsets remains largely unknown. This study recruited 10 clear cell renal cell carcinoma (ccRCC) patients and obtained samples from various tissues, including tumors, adjacent healthy renal tissue and peripheral blood. We utilized deep sequencing of T-cell receptor beta chains (TCRB), which serve as a unique identifier for each T clonotype, to characterize the CD4+/CD8+ TIL repertoire in ccRCC patients, assess the diversity and clonality of infiltrated T-cells in distinct tissues from patients and depict the clonal expansion events that occur in anti-tumor immune responses. We found that the CD4+ TIL repertoire exhibited signatures of heterogeneous T-cell expansion, which were characterized by divergent TRBV/J usage and an enrichment of expanded dominant clones. Taken together, our findings provide additional evidence of CD4+ T-cell-mediated anti-tumor immunity. The identification of the underlying molecular mechanisms of this process may provide novel avenues for targeted immunotherapeutic interventions. PMID:25637538

  9. Local morphologic scale: application to segmenting tumor infiltrating lymphocytes in ovarian cancer TMAs

    NASA Astrophysics Data System (ADS)

    Janowczyk, Andrew; Chandran, Sharat; Feldman, Michael; Madabhushi, Anant

    2011-03-01

    classes based on local structural properties. In this paper, we apply LMS to the specific problem of classifying regions of interest in Ovarian Cancer (OCa) histology images as either tumor or stroma. This approach is used to classify lymphocytes as either tumor infiltrating lymphocytes (TILs) or non-TILs; the presence of TILs having been identified as an important prognostic indicator for disease outcome in patients with OCa. We present preliminary results on the tumor/stroma classification of 11,000 randomly selected locations of interest, across 11 images obtained from 6 patient studies. Using a Probabilistic Boosting Tree (PBT), our supervised classifier yielded an area under the receiver operation characteristic curve (AUC) of 0.8341 +/-0.0059 over 5 runs of randomized cross validation. The average LMS computation time at every spatial location for an image patch comprising 2000 pixels with 24 particles at every location was only 18s.

  10. Tumor Infiltrating Lymphocytes Genetically Engineered with an Inducible Gene Encoding Interleukin-12 for the Immunotherapy of Metastatic Melanoma

    PubMed Central

    Zhang, Ling; Morgan, Richard A.; D.Beane, Joal; Zheng, Zhili; Dudley, Mark E.; Kassim, Sadik H.; Nahvi, Azam V.; Ngo, Lien T.; Sherry, Richard M.; Phan, Giao Q.; Hughes, Marybeth S.; Kammula, Udai S.; Feldman, Steven A.; Toomey, Mary Ann; Kerkar, Sid. P.; Restifo, Nicholas P.; Yang, James C.; Rosenberg, Steven A.

    2015-01-01

    Purpose Infusion of interleukin-12 (IL-12) can mediate anti-tumor immunity in animal models, yet its systemic administration to patients with cancer results in minimal efficacy and severe toxicity. Here, we evaluated the anti-tumor activity of adoptively transferred human tumor infiltrating lymphocytes (TIL) genetically engineered to secrete single-chain IL-12 selectively at the tumor site. Experimental design Thirty-three patients with metastatic melanoma were treated in a cell-dose escalation trial of autologous TIL transduced with a gene encoding a single chain IL-12 driven by a nuclear factor of activated T cells promoter (NFAT.IL12). No IL-2 was administered. Results The administration of 0.001-0.1 X 109 NFAT.IL12 transduced TIL to 17 patients resulted in a single objective response (5.9%). However, at doses between 0.3-3 X 109 cells, 10 of 16 patients (63%) exhibited objective clinical responses. The responses tended to be short and the administered IL-12 producing cells rarely persisted at one month. Increasing cell doses were associated with high serum levels of IL-12 and gamma-interferon as well as clinical toxicities including liver dysfunction, high fevers and sporadic life threatening hemodynamic instability. Conclusions In this first-in-man trial, administration of TIL transduced with an inducible IL-12 gene mediated tumor responses in the absence of IL-2 administration using cell doses 10-100 fold lower than conventional TIL. However, due to toxicities, likely attributable to the secreted IL-12, further refinement will be necessary before this approach can be safely utilized in the treatment of cancer patients. PMID:25695689

  11. Heparanase promotes tumor infiltration and antitumor activity of CAR-redirected T-lymphocytes

    PubMed Central

    Caruana, Ignazio; Savoldo, Barbara; Hoyos, Valentina; Weber, Gerrit; Liu, Hao; Kim, Eugene S.; Ittmann, Michael M.; Marchetti, Dario; Dotti, Gianpietro

    2015-01-01

    Adoptive transfer of chimeric antigen receptor (CAR)-redirected T lymphocytes (CAR-T cells) has had less striking effects in solid tumors1–3 than in lymphoid malignancies4, 5. Although active tumor-mediated immunosuppression may play a role in limiting efficacy6, functional changes in T lymphocytes following their ex vivo manipulation may also account for cultured CAR-T cells’ reduced ability to penetrate stroma-rich solid tumors. We therefore studied the capacity of human in vitro-cultured CAR-T cells to degrade components of the extracellular matrix (ECM). In contrast to freshly isolated T lymphocytes, we found that in vitro-cultured T lymphocytes lack expression of the enzyme heparanase (HPSE) that degrades heparan sulphate proteoglycans, which are main components of ECM. We found that HPSE mRNA is down regulated in in vitro-expanded T cells, which may be a consequence of p53 binding to the HPSE gene promoter. We therefore engineered CAR-T cells to express HPSE and showed improved capacity to degrade ECM, which promoted tumor T-cell infiltration and antitumor activity. Employing this strategy may enhance the activity of CAR-T cells in individuals with stroma-rich solid tumors. PMID:25849134

  12. Heparanase promotes tumor infiltration and antitumor activity of CAR-redirected T lymphocytes.

    PubMed

    Caruana, Ignazio; Savoldo, Barbara; Hoyos, Valentina; Weber, Gerrit; Liu, Hao; Kim, Eugene S; Ittmann, Michael M; Marchetti, Dario; Dotti, Gianpietro

    2015-05-01

    Adoptive transfer of chimeric antigen receptor (CAR)-redirected T lymphocytes (CAR-T cells) has had less striking therapeutic effects in solid tumors than in lymphoid malignancies. Although active tumor-mediated immunosuppression may have a role in limiting the efficacy of CAR-T cells, functional changes in T lymphocytes after their ex vivo manipulation may also account for the reduced ability of cultured CAR-T cells to penetrate stroma-rich solid tumors compared with lymphoid tissues. We therefore studied the capacity of human in vitro-cultured CAR-T cells to degrade components of the extracellular matrix (ECM). In contrast to freshly isolated T lymphocytes, we found that in vitro-cultured T lymphocytes lack expression of the enzyme heparanase (HPSE), which degrades heparan sulfate proteoglycans, the main components of ECM. We found that HPSE mRNA is downregulated in in vitro-expanded T cells, which may be a consequence of p53 (officially known as TP53, encoding tumor protein 53) binding to the HPSE gene promoter. We therefore engineered CAR-T cells to express HPSE and showed their improved capacity to degrade the ECM, which promoted tumor T cell infiltration and antitumor activity. The use of this strategy may enhance the activity of CAR-T cells in individuals with stroma-rich solid tumors. PMID:25849134

  13. Immunophenotypic features of tumor infiltrating lymphocytes from mammary carcinomas in female dogs associated with prognostic factors and survival rates

    PubMed Central

    2010-01-01

    Background The immune system plays an important role in the multifactorial biologic system during the development of neoplasias. However, the involvement of the inflammatory response in the promotion/control of malignant cells is still controversial, and the cell subsets and the mechanisms involved are poorly investigated. The goal of this study was to characterize the clinical-pathological status and the immunophenotyping profile of tumor infiltrating lymphocytes and their association with the animal survival rates in canine mammary carcinomas. Methods Fifty-one animals with mammary carcinomas, classified as carcinomas in mixed tumors-MC-BMT = 31 and carcinomas-MC = 20 were submitted to systematic clinical-pathological analysis (tumor size; presence of lymph node and pulmonary metastasis; clinical stage; histological grade; inflammatory distribution and intensity as well as the lymphocytic infiltrate intensity) and survival rates. Twenty-four animals (MC-BMT = 16 and MC = 8) were elected to the immunophenotypic study performed by flow cytometry. Results Data analysis demonstrated that clinical stage II-IV and histological grade was I more frequent in MC-BMT as compared to MC. Univariate analysis demonstrated that the intensity of inflammation (moderate/intense) and the proportion of CD4+ (≥ 66.7%) or CD8+ T-cells (<33.3%) were not associated with worse survival rate. Multivariate analysis demonstrated that only lymphocytic infiltrate intensity ≥ 600 (P = 0.02) remained as independent prognostic factor. Despite the clinical manifestation, the lymphocytes represented the predominant cell type in the tumor infiltrate. The percentage of T-cells was higher in animals with MC-BMT without metastasis, while the percentage of B-lymphocytes was greater in animals with metastasized MC-BMT (P < 0.05). The relative percentage of CD4+ T-cells was significantly greater in metastasized tumors (both MC-BMT and MC), (P < 0.05) while the proportion of CD8+ T-cells was higher in

  14. Tumor-infiltrating T lymphocytes improve clinical outcome of therapy-resistant neuroblastoma

    PubMed Central

    Mina, Marco; Boldrini, Renata; Citti, Arianna; Romania, Paolo; D'Alicandro, Valerio; De Ioris, Maretta; Castellano, Aurora; Furlanello, Cesare; Locatelli, Franco; Fruci, Doriana

    2015-01-01

    Neuroblastoma grows within an intricate network of different cell types including epithelial, stromal and immune cells. The presence of tumor-infiltrating T cells is considered an important prognostic indicator in many cancers, but the role of these cells in neuroblastoma remains to be elucidated. Herein, we examined the relationship between the type, density and organization of infiltrating T cells and clinical outcome within a large collection of neuroblastoma samples by quantitative analysis of immunohistochemical staining. We found that infiltrating T cells have a prognostic value greater than, and independent of, the criteria currently used to stage neuroblastoma. A variable in situ structural organization and different concurrent infiltration of T-cell subsets were detected in tumors with various outcomes. Low-risk neuroblastomas were characterized by a higher number of proliferating T cells and a more structured T-cell organization, which was gradually lost in tumors with poor prognosis. We defined an immunoscore based on the presence of CD3+, CD4+ and CD8+ infiltrating T cells that associates with favorable clinical outcome in MYCN-amplified tumors, improving patient survival when combined with the v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN) status. These findings support the hypothesis that infiltrating T cells influence the behavior of neuroblastoma and might be of clinical importance for the treatment of patients. PMID:26405592

  15. FOXP3 expression in tumor cells and tumor-infiltrating lymphocytes is associated with breast cancer prognosis

    PubMed Central

    TAKENAKA, MIKI; SEKI, NAOKO; TOH, UHI; HATTORI, SATOSHI; KAWAHARA, AKIHIKO; YAMAGUCHI, TOMOHIKO; KOURA, KEIKO; TAKAHASHI, RYUJI; OTSUKA, HIROKO; TAKAHASHI, HIROKI; IWAKUMA, NOBUTAKA; NAKAGAWA, SHINO; FUJII, TERUHIKO; SASADA, TETSURO; YAMAGUCHI, RIN; YANO, HIROHISA; SHIROUZU, KAZUO; KAGE, MASAYOSHI

    2013-01-01

    The forkhead box protein 3 (FOXP3) transcription factor is highly expressed in tumor cells as well as in regulatory T cells (Tregs). It plays a tumor-enhancing role in Tregs and suppresses carcinogenesis as a potent repressor of several oncogenes. The clinical prognostic value of FOXP3 expression has not yet been elucidated. In this study, immunohistochemistry was used to investigate the prognostic significance of FOXP3 expression in tumor cells and tumor-infiltrating lymphocytes (TILs) in breast cancer patients. Of the 100 tumor specimens obtained from primary invasive breast carcinoma, 63 and 57% were evaluated as FOXP3+ tumor cells and as being highly infiltrated by FOXP3+ lymphocytes, respectively. Although FOXP3 expression in tumor cells was of no prognostic significance, FOXP3+ lymphocytes were significantly associated with poor overall survival (OS) (n=98, log-rank test P=0.008). FOXP3 exhibited a heterogeneous subcellular localization in tumor cells (cytoplasm, 31%; nucleus, 26%; both, 6%) and, although cytoplasmic FOXP3 was associated with poor OS (P= 0.058), nuclear FOXP3 demonstrated a significant association with improved OS (P=0.016). Furthermore, when patients were grouped according to their expression of tumor cytoplasmic FOXP3 and lymphocyte FOXP3, there were notable differences in the Kaplan-Meier curves for OS (P<0.001), with a high infiltration of FOXP3+ lymphocytes accompanied by a cytoplasmic FOXP3+ tumor being the most detrimental phenotype. These findings indicated that FOXP3 expression in lymphocytes as well as in tumor cells may be a prognostic marker for breast cancer. FOXP3 in tumor cells may have distinct biological activities and prognostic values according to its localization, which may help establish appropriate cancer treatments. PMID:24649219

  16. The Prognostic Value of Tumor-Infiltrating Lymphocytes in Breast Cancer: A Systematic Review and Meta-Analysis

    PubMed Central

    Mao, Yan; Qu, Qing; Chen, Xiaosong; Huang, Ou; Wu, Jiayi; Shen, Kunwei

    2016-01-01

    Background The prognostic values of tumor-infiltrating lymphocytes (TILs) and TILs subsets in breast cancer (BC) are uncertain. Methods A systematic literature search (MEDLINE, Web of Science, EMBASE, and the Cochrane Library to August 2014) was conducted for studies which met the eligibility criteria. The primary clinical outcome was defined as disease-free survival (DFS), overall survival (OS), and BC-specific survival (BCSS). Random or fixed-effects model was adopted to estimate the summary hazard ratio (HR). Results Twenty-five published studies comprising 22,964 patients were reviewed. Pooled analysis indicated that TILs were not prognostic markers for DFS and OS in overall population, but related to improved DFS (HR, 0.82; 95% CI, 0.76–0.88) and OS (HR, 0.79; 95% CI, 0.71–0.87) in triple negative breast cancer (TNBC) patients. For TILs subsets, CD8+ lymphocytes were associated with improved DFS (HR, 0.69; 95% CI, 0.56–0.84) and BCSS (HR, 0.78; 95% CI, 0.71–0.86) in overall population, while FOXP3+ lymphocytes were associated with reduced DFS (HR, 1.47; 95% CI, 1.01–2.05) and OS (HR, 1.50; 95% CI, 1.15–1.97). In estrogen receptor (ER) negative patients, CD8+ lymphocytes was also related to better BCSS. In addition, the high density of CD20+, CD3+ or low level of PD-1+ or γδ T lymphocytes indicated increased OS in limited studies. Conclusion TILs and TILs subsets are promising prognostic biomarkers in breast cancer, especially in TNBC. PMID:27073890

  17. Tumor-infiltrating lymphocytes in glioblastoma are associated with specific genomic alterations and related to transcriptional class

    PubMed Central

    Rutledge, W. Caleb; Kong, Jun; Gao, Jingjing; Gutman, David A.; Cooper, Lee A.D.; Appin, Christina; Park, Yuna; Scarpace, Lisa; Mikkelsen, Tom; Cohen, Mark L.; Aldape, Kenneth D.; McLendon, Roger E.; Lehman, Norman L.; Miller, C. Ryan; Schniederjan, Matthew J.; Brennan, Cameron W.; Saltz, Joel H.; Moreno, Carlos S.; Brat, Daniel J.

    2013-01-01

    Purpose Tumor-infiltrating lymphocytes (TILs) have prognostic significance in many cancers, yet their roles in glioblastoma (GBM) have not been fully defined. We hypothesized TILs in GBM are associated with molecular alterations, histologies and survival. Experimental Design We used data from The Cancer Genome Atlas (TCGA) to investigate molecular, histologic and clinical correlates of TILs in GBMs. Lymphocytes were categorized as absent, present or abundant in histopathologic images from 171 TCGA GBMs. Associations were examined between lymphocytes and histologic features, mutations, copy number alterations, CpG island methylator phenotype, transcriptional class and survival. We validated histologic findings using CD3G gene expression. Results We found a positive correlation between TILs and GBMs with gemistocytes, sarcomatous cells, epithelioid cells and giant cells. Lymphocytes were enriched in the mesenchymal transcriptional class and strongly associated with mutations in NF1 and RB1. These mutations are frequent in the mesenchymal class and characteristic of gemistocytic, sarcomatous, epithelioid and giant cell histologies. Conversely, TILs were rare in GBMs with small cells and oligodendroglioma components. Lymphocytes were depleted in the classical transcriptional class and in EGFR-amplified and homozygous PTEN-deleted GBMs. These alterations are characteristic of GBMs with small cells and GBMs of the classical transcriptional class. No association with survival was demonstrated. Conclusions TILs were enriched in GBMs of the mesenchymal class, strongly associated with mutations in NF1 and RB1 and typical of histologies characterized by these mutations. Conversely, TILs were depleted in the classical class, EGFR-amplified and homozygous PTEN-deleted tumors and rare in histologies characterized by these alterations. PMID:23864165

  18. Inhibition of IL-17A in tumor microenvironment augments cytotoxicity of tumor-infiltrating lymphocytes in tumor-bearing mice.

    PubMed

    Hayata, Keiji; Iwahashi, Makoto; Ojima, Toshiyasu; Katsuda, Masahiro; Iida, Takeshi; Nakamori, Mikihito; Ueda, Kentaro; Nakamura, Masaki; Miyazawa, Motoki; Tsuji, Toshiaki; Yamaue, Hiroki

    2013-01-01

    It remains controversial whether IL-17A promotes or inhibits cancer progression. We hypothesized that IL-17A that is locally produced in the tumor microenvironment has an important role in angiogenesis and tumor immunity. We investigated the effect of inhibiting IL-17A at tumor sites on tumor growth and on local and systemic anti-tumor immunity. MC38 or B16 cells were inoculated subcutaneously into mice, and intratumoral injection of an adenovirus vector expressing siRNA against the mouse IL-17A gene (Ad-si-IL-17) significantly inhibited tumor growth in both tumor models compared with control mice. Inhibition of IL-17A at tumor sites significantly suppressed CD31, MMP9, and VEGF expression in tumor tissue. The cytotoxic activity of CD8(+) T cells from tumor-infiltrating lymphocytes in mice treated with Ad-si-IL-17 was significantly higher than in control mice; however, CD8(+) T cells from splenocytes had similar activity levels. Suppression of IL-17A at tumor sites led to a Th1-dominant environment, and moreover, eliminated myeloid-derived suppressor cells and regulatory T cells at tumor sites but not in splenocytes. In conclusion, blockade of IL-17A at tumor sites helped suppress tumor growth by inhibiting angiogenesis as well as cytotoxic T lymphocytes activation at tumor sites. PMID:23372655

  19. A phase I clinical trial utilizing autologous tumor-infiltrating lymphocytes in patients with primary hepatocellular carcinoma

    PubMed Central

    Weng, De-Sheng; Zhou, Zhong-Guo; Pan, Ke; Pan, Qiu-Zhong; Wang, Qi-Jing; Liu, Qing; He, Jia; Zhao, Jing-Jing; Li, Jiang; Chen, Min-Shan; Chang, Alfred E.; Li, Qiao; Xia, Jian-Chuan

    2015-01-01

    This report describes an ongoing Phase I clinical trial testing the safety of adoptive cell therapy (ACT) using autologous tumor-infiltrating lymphocytes (TIL) in patients with primary hepatocellular carcinoma (HCC). Fifteen HCC patients were treated with their activated and expanded TILs following tumor resection. From a total of 17 patients with HCC, TIL were successfully expanded from 15 patients (88%), whereas two patients showed minimal or no expansion of TIL. Transient increase in the frequency of T cells was observed after adoptive transfer who was found only associated with grade I flu-like symptoms and malaise. After a median follow-up of 14 months, 15 patients (100%) were alive; and 12 patients (80%) showed no evidence of disease, 3 patients (patient 1,11,12) had tumor recurrence. The time to the diagnosis of tumor recurrence following therapy ranged from 105 to 261 days. These results indicate that immunotherapy with activated and expanded autologous TIL could be successfully performed with low toxicity, thus would serve as a novel treatment modality for patients with HCC. PMID:26515587

  20. Tumor-infiltrating lymphocyte activity is enhanced in tumors with low IL-10 production in HBV-induced hepatocellular carcinoma

    SciTech Connect

    Shi, Yang Song, Qingwei; Hu, Dianhe; Zhuang, Xiaohu; Yu, Shengcai

    2015-05-22

    Hepatocellular carcinoma (HCC) is one of the most common cancers and can be induced by chronic HBV infection. The role of HBV-specific immune responses in mediating tumorigenesis and HCC prognosis is debated. The effect of intratumoral microenvironment on tumor-infiltrating lymphocytes (TILs) is also unclear. Here, we examined resected tumor tissue from 36 patients with HBV-induced HCC. We categorized study cohort based on ex vivo IL-10 secretion by tumor cells into high IL-10-secreting (Hi10) and low IL-10-secreting (Lo10) groups, and found that the Lo10 group was less sensitive to TLR ligand stimulation. TILs from the Lo10 group contained higher frequencies of HBV-specific IFN-g-producing cells and total IFN-g-producing cells, and possessed higher proliferative capacity. Moreover, the proliferative capacity of TILs from the Hi10 group was negatively correlated with IL-10 secretion from tumor cells. Together, our data demonstrated that low IL-10-producing capacity in HBV-induced HCC tumors is associated with enhanced TIL activity. - Highlights: • We examined intratumoral IL-10 production in HBV-induced HCC. • We grouped HCC tumors into Hi10 and Lo10 groups based on their IL-10 production. • Lo10 groups had better IFN-g response by TILs. • Lo10 groups had better TIL proliferative capacity. • Lo10 group tumor cells were refractory to TLR ligand stimulation.

  1. High-throughput sequencing of T cell receptors reveals a homogeneous repertoire of tumor-infiltrating lymphocytes in ovarian cancer

    PubMed Central

    Rieder, Mark J.; Guenthoer, Jamie; Williamson, David W.; Carlson, Christopher S.; Drescher, Charles W.; Tewari, Muneesh; Bielas, Jason H.; Robins, Harlan S.

    2014-01-01

    The cellular adaptive immune system mounts a response to many solid tumors mediated by tumor infiltrating T lymphocytes (TILs). Basic measurements of these TILs, including total count, show promise as prognostic markers for a variety of cancers, including ovarian and colorectal. In addition, recent therapeutic advances are thought to exploit this immune response to effectively fight melanoma with promising studies showing efficacy in additional cancers. However, many of the basic properties of TILs are poorly understood including specificity, clonality, and spatial heterogeneity of the T cell response. We utilize deep sequencing of rearranged T-cell receptor beta (TCRB) genes to characterize the basic properties of TILs in ovarian carcinoma. Due to somatic rearrangement during T cell development, the TCR beta chain sequence serves as a molecular tag for each T cell clone. Using these sequence tags, we assess similarities and differences between infiltrating T cells in discretely sampled sections of large tumors and compare to T cells from peripheral blood. Within the limits of sensitivity of our assay, the TIL repertoires show strong similarity throughout each tumor and are distinct from the circulating T cell repertoire. We conclude that the cellular adaptive immune response within ovarian carcinomas is spatially homogeneous and distinct from the T cell compartment of peripheral blood. PMID:24027095

  2. The Role of Tumor-Infiltrating Lymphocytes in Development, Progression, and Prognosis of Non-Small Cell Lung Cancer.

    PubMed

    Bremnes, Roy M; Busund, Lill-Tove; Kilvær, Thomas L; Andersen, Sigve; Richardsen, Elin; Paulsen, Erna Elise; Hald, Sigurd; Khanehkenari, Mehrdad Rakaee; Cooper, Wendy A; Kao, Steven C; Dønnem, Tom

    2016-06-01

    A malignant tumor is not merely an accumulation of neoplastic cells, but constitutes a microenvironment containing endothelial cells, fibroblasts, structural components, and infiltrating immune cells that impact tumor development, invasion, metastasis, and outcome. Hence, the evolution of cancers reflects intricate cellular and molecular interactions between tumor cells and constituents of the tumor microenvironment. Recent studies have shed new light on this complex interaction between tumor and host immune cells and the resulting immune response. The composition of the immune microenvironment differs across patients as well as in cancers of the same type, including various populations of T cells, B cells, dendritic cells, natural killer cells, myeloid-derived suppressor cells, neutrophils, and macrophages. The type, density, location, and organization of immune cells within solid tumors define the immune contexture, which has proved to be a major determinant of tumor characteristics and patient outcome. Lung cancer consists mostly of non-small cell lung cancer (85%); it is our most deadly malignant disease, with the 5-year survival rate being merely 15%. This review focuses on the immune contexture; the tumor-suppressing roles of tumor-infiltrating lymphocytes; and the relevance of this immune contexture for cancer diagnostics, prognostication, and treatment allocation, with an emphasis on non-small cell lung cancer. PMID:26845192

  3. Adoptive cell therapy with autologous tumor infiltrating lymphocytes and low-dose Interleukin-2 in metastatic melanoma patients

    PubMed Central

    2012-01-01

    Background Adoptive cell therapy may be based on isolation of tumor-specific T cells, e.g. autologous tumor infiltrating lymphocytes (TIL), in vitro activation and expansion and the reinfusion of these cells into patients upon chemotherapy induced lymphodepletion. Together with high-dose interleukin (IL)-2 this treatment has been given to patients with advanced malignant melanoma and impressive response rates but also significant IL-2 associated toxicity have been observed. Here we present data from a feasibility study at a Danish Translational Research Center using TIL adoptive transfer in combination with low-dose subcutaneous IL-2 injections. Methods This is a pilot trial (ClinicalTrials.gov identifier: NCT00937625) including patients with metastatic melanoma, PS ≤1, age <70, measurable and progressive disease and no involvement of the central nervous system. Six patients were treated with lymphodepleting chemotherapy, TIL infusion, and 14 days of subcutaneous low-dose IL-2 injections, 2 MIU/day. Results Low-dose IL-2 considerably decreased the treatment related toxicity with no grade 3–4 IL-2 related adverse events. Objective clinical responses were seen in 2 of 6 treated patients with ongoing complete responses (30+ and 10+ months), 2 patients had stable disease (4 and 5 months) and 2 patients progressed shortly after treatment. Tumor-reactivity of the infused cells and peripheral lymphocytes before and after therapy were analyzed. Absolute number of tumor specific T cells in the infusion product tended to correlate with clinical response and also, an induction of peripheral tumor reactive T cells was observed for 1 patient in complete remission. Conclusion Complete and durable responses were induced after treatment with adoptive cell therapy in combination with low-dose IL-2 which significantly decreased toxicity of this therapy. PMID:22909342

  4. HLA-E expression by gynecological cancers restrains tumor-infiltrating CD8⁺ T lymphocytes.

    PubMed

    Gooden, Marloes; Lampen, Margit; Jordanova, Ekaterina S; Leffers, Ninke; Trimbos, J Baptist; van der Burg, Sjoerd H; Nijman, Hans; van Hall, Thorbald

    2011-06-28

    HLA-E is a nonclassical HLA class I molecule, which differs from classical HLA molecules by its nonpolymorphic, conserved nature. Expression and function of HLA-E in normal tissues and solid tumors is not fully understood. We investigated HLA-E protein expression on tissue sections of 420 ovarian and cervical cancers and found equal or higher levels than normal counterpart epithelia in 80% of the tumors. Expression was strongly associated with components of the antigen presentation pathway, e.g., transporter associated with antigen processing (TAP), endoplasmic reticulum aminopeptide (ERAP), β2 microglobulin (β2m), HLA classes I and II, and for ovarian cancer with tumor infiltrating CD8(+) T lymphocytes (CTLs). This association argues against the idea that HLA-E would compensate for the loss of classical HLA in tumors. In situ detection of HLA-E interacting receptors revealed a very low infiltrate of natural killer (NK) cells, but up to 50% of intraepithelial CTLs expressed the inhibiting CD94/NKG2A receptor. In cervical cancer, HLA-E expression did not alter the prognostic effect of CTLs, most likely due to very high infiltrating CTL numbers in this virus-induced tumor. Overall survival of ovarian cancer patients, however, was strongly influenced by HLA-E, because the beneficial effect of high CTL infiltration was completely neutralized in the subpopulation with strong HLA-E expression. Interestingly, these results indicate that CTL infiltration in ovarian cancer is associated with better survival only when HLA-E expression is low and that intratumoral CTLs are inhibited by CD94/NKG2A receptors on CTLs in the tumor microenvironment. PMID:21670276

  5. Clinical Scale Zinc Finger Nuclease-mediated Gene Editing of PD-1 in Tumor Infiltrating Lymphocytes for the Treatment of Metastatic Melanoma

    PubMed Central

    Beane, Joal D; Lee, Gary; Zheng, Zhili; Mendel, Matthew; Abate-Daga, Daniel; Bharathan, Mini; Black, Mary; Gandhi, Nimisha; Yu, Zhiya; Chandran, Smita; Giedlin, Martin; Ando, Dale; Miller, Jeff; Paschon, David; Guschin, Dmitry; Rebar, Edward J; Reik, Andreas; Holmes, Michael C; Gregory, Philip D; Restifo, Nicholas P; Rosenberg, Steven A; Morgan, Richard A; Feldman, Steven A

    2015-01-01

    Programmed cell death-1 (PD-1) is expressed on activated T cells and represents an attractive target for gene-editing of tumor targeted T cells prior to adoptive cell transfer (ACT). We used zinc finger nucleases (ZFNs) directed against the gene encoding human PD-1 (PDCD-1) to gene-edit melanoma tumor infiltrating lymphocytes (TIL). We show that our clinical scale TIL production process yielded efficient modification of the PD-1 gene locus, with an average modification frequency of 74.8% (n = 3, range 69.9–84.1%) of the alleles in a bulk TIL population, which resulted in a 76% reduction in PD-1 surface-expression. Forty to 48% of PD-1 gene-edited cells had biallelic PD-1 modification. Importantly, the PD-1 gene-edited TIL product showed improved in vitro effector function and a significantly increased polyfunctional cytokine profile (TNFα, GM-CSF, and IFNγ) compared to unmodified TIL in two of the three donors tested. In addition, all donor cells displayed an effector memory phenotype and expanded approximately 500–2,000-fold in vitro. Thus, further study to determine the efficiency and safety of adoptive cell transfer using PD-1 gene-edited TIL for the treatment of metastatic melanoma is warranted. PMID:25939491

  6. Clinical Scale Zinc Finger Nuclease-mediated Gene Editing of PD-1 in Tumor Infiltrating Lymphocytes for the Treatment of Metastatic Melanoma.

    PubMed

    Beane, Joal D; Lee, Gary; Zheng, Zhili; Mendel, Matthew; Abate-Daga, Daniel; Bharathan, Mini; Black, Mary; Gandhi, Nimisha; Yu, Zhiya; Chandran, Smita; Giedlin, Martin; Ando, Dale; Miller, Jeff; Paschon, David; Guschin, Dmitry; Rebar, Edward J; Reik, Andreas; Holmes, Michael C; Gregory, Philip D; Restifo, Nicholas P; Rosenberg, Steven A; Morgan, Richard A; Feldman, Steven A

    2015-08-01

    Programmed cell death-1 (PD-1) is expressed on activated T cells and represents an attractive target for gene-editing of tumor targeted T cells prior to adoptive cell transfer (ACT). We used zinc finger nucleases (ZFNs) directed against the gene encoding human PD-1 (PDCD-1) to gene-edit melanoma tumor infiltrating lymphocytes (TIL). We show that our clinical scale TIL production process yielded efficient modification of the PD-1 gene locus, with an average modification frequency of 74.8% (n = 3, range 69.9-84.1%) of the alleles in a bulk TIL population, which resulted in a 76% reduction in PD-1 surface-expression. Forty to 48% of PD-1 gene-edited cells had biallelic PD-1 modification. Importantly, the PD-1 gene-edited TIL product showed improved in vitro effector function and a significantly increased polyfunctional cytokine profile (TNFα, GM-CSF, and IFNγ) compared to unmodified TIL in two of the three donors tested. In addition, all donor cells displayed an effector memory phenotype and expanded approximately 500-2,000-fold in vitro. Thus, further study to determine the efficiency and safety of adoptive cell transfer using PD-1 gene-edited TIL for the treatment of metastatic melanoma is warranted. PMID:25939491

  7. Complete Regression of Metastatic Cervical Cancer After Treatment With Human Papillomavirus–Targeted Tumor-Infiltrating T Cells

    PubMed Central

    Stevanović, Sanja; Draper, Lindsey M.; Langhan, Michelle M.; Campbell, Tracy E.; Kwong, Mei Li; Wunderlich, John R.; Dudley, Mark E.; Yang, James C.; Sherry, Richard M.; Kammula, Udai S.; Restifo, Nicholas P.; Rosenberg, Steven A.; Hinrichs, Christian S.

    2015-01-01

    Purpose Metastatic cervical cancer is a prototypical chemotherapy-refractory epithelial malignancy for which better treatments are needed. Adoptive T-cell therapy (ACT) is emerging as a promising cancer treatment, but its study in epithelial malignancies has been limited. This study was conducted to determine if ACT could mediate regression of metastatic cervical cancer. Patients and Methods Patients enrolled onto this protocol were diagnosed with metastatic cervical cancer and had previously received platinum-based chemotherapy or chemoradiotherapy. Patients were treated with a single infusion of tumor-infiltrating T cells selected when possible for human papillomavirus (HPV) E6 and E7 reactivity (HPV-TILs). Cell infusion was preceded by lymphocyte-depleting chemotherapy and was followed by administration of aldesleukin. Results Three of nine patients experienced objective tumor responses (two complete responses and one partial response). The two complete responses were ongoing 22 and 15 months after treatment, respectively. One partial response was 3 months in duration. The HPV reactivity of T cells in the infusion product (as measured by interferon gamma production, enzyme-linked immunospot, and CD137 upregulation assays) correlated positively with clinical response (P = .0238 for all three assays). In addition, the frequency of HPV-reactive T cells in peripheral blood 1 month after treatment was positively associated with clinical response (P = .0238). Conclusion Durable, complete regression of metastatic cervical cancer can occur after a single infusion of HPV-TILs. Exploratory studies suggest a correlation between HPV reactivity of the infusion product and clinical response. Continued investigation of this therapy is warranted. PMID:25823737

  8. Syngeneic syrian hamster tumors feature tumor-infiltrating lymphocytes allowing adoptive cell therapy enhanced by oncolytic adenovirus in a replication permissive setting.

    PubMed

    Siurala, Mikko; Vähä-Koskela, Markus; Havunen, Riikka; Tähtinen, Siri; Bramante, Simona; Parviainen, Suvi; Mathis, J Michael; Kanerva, Anna; Hemminki, Akseli

    2016-05-01

    Adoptive transfer of tumor-infiltrating lymphocytes (TIL) has shown promising yet sometimes suboptimal results in clinical trials for advanced cancer, underscoring the need for approaches improving efficacy and safety. Six implantable syngeneic tumor cell lines of the Syrian hamster were used to initiate TIL cultures. TIL generated from tumor fragments cultured in human interleukin-2 (IL-2) for 10 d were adoptively transferred into tumor-bearing hamsters with concomitant intratumoral injections of oncolytic adenovirus (Ad5-D24) for the assessment of antitumor efficacy. Pancreatic cancer (HapT1) and melanoma (RPMI 1846) TIL exhibited potent and tumor-specific cytotoxicity in effector-to-target (E/T) assays. MHC Class I blocking abrogated the cell killing of RPMI 1846 TIL, indicating cytotoxic CD8(+) T-cell activity. When TIL were combined with Ad5-D24 in vitro, HapT1 tumor cell killing was significantly enhanced over single agents. In vivo, the intratumoral administration of HapT1 TIL and Ad5-D24 resulted in improved tumor growth control compared with either treatment alone. Additionally, splenocytes derived from animals treated with the combination of Ad5-D24 and TIL killed autologous tumor cells more efficiently than monotherapy-derived splenocytes, suggesting that systemic antitumor immunity was induced. For the first time, TIL of the Syrian hamster have been cultured, characterized and used therapeutically together with oncolytic adenovirus for enhancing the efficacy of TIL therapy. Our results support human translation of oncolytic adenovirus as an enabling technology for adoptive T-cell therapy of solid tumors. PMID:27467954

  9. The clinical significance of tumor-infiltrating neutrophils and neutrophil-to-CD8+ lymphocyte ratio in patients with resectable esophageal squamous cell carcinoma

    PubMed Central

    2014-01-01

    Background The interaction between tumor cells and inflammatory cells has not been systematically investigated in esophageal squamous cell carcinoma (ESCC). The main aims of the study were to investigate the clinical significance of tumor-infiltrating neutrophils and neturophil-to-CD8+ lymphocyte ratio (NLR), and to analyze the distribution of tumor-infiltrating neutrophils and CD8+ lymphocytes in ESCC treated by curative resection. Methods The expressions of CD66b and CD8 were assessed with double staining immunohistochemistry in the surgical specimens from 90 patients with ESCC treated by curative surgery. Results We showed that increased intratumoral neutrophils were significantly associated with lymph node metastasis (P = 0.016), and advanced pathological stages (P = 0.013). Decreased peritumoral CD8+ lymphocyte density was more frequently observed in patients with single positive lymph node (p = 0.045). Peritumoral NLR was significantly associated with advanced T stages (p < 0.001), lymph node metastasis (p = 0.041) and a trend towards advanced pathological stages (p = 0.053). Increased intratumoral neutrophils were significantly associated with decreased disease-free survival (p < 0.001) and overall survival (p < 0.001) in univariate analysis and were identified as an independent prognostic factor for disease-free survival (p = 0.006) and overall survival (p = 0.037) in multivariate analysis. Neither the density nor the distribution of tumor-infiltrating neutrophils was significantly correlated with that of CD8+ lymphocytes. The density of intratumoral CD8+ lymphocytes was significantly lower than (P < 0.001) and moderately correlated with (r = 0.434, p < 0.001) that in peritumoral area. Conclusions Increased intratumoral neutrophils were an independent poor prognostic factor and peritumoral NLR was significantly associated with disease progression in ESCC treated by curative surgery, suggesting the possible

  10. Expression of programmed death-1 ligand (PD-L1) in tumor-infiltrating lymphocytes is associated with favorable spinal chordoma prognosis

    PubMed Central

    Zou, Ming-Xiang; Peng, An-Bo; Lv, Guo-Hua; Wang, Xiao-Bin; Li, Jing; She, Xiao-Ling; Jiang, Yi

    2016-01-01

    Aberrant expression of programmed death-1 (PD-1) receptor/PD-1 ligand (PD-L1) proteins alters human immunoresponse and promotes tumor development and progression. We assessed the expression status of PD-1 and PD-L1 in spinal chordoma tissue specimens and their association with clinicopathological characteristics of patients. Formalin-fixed paraffin-embedded tumor samples from 54 patients with spinal chordoma were collected for immunohistochemical analysis of PD-1 and PD-L1 expression. The association of the expression levels of PD-1 and PD-L1 with clinicopathological variables and survival data were statistically analyzed. Lymphocyte infiltrates were present in all 54 patient samples. Of 54 samples, 37 (68.5%) had both positive PD-1 and PD-L1 expression in tumor cell membrane. Moreover, 38 (70.4%) and 12 (22.2%) had positive PD-1 and PD-L1 expression in tumor-infiltrating lymphocytes (TILs), respectively. Tumors with positive PD-L1 expression were significantly associated with advanced stages of chordoma (p = 0.041) and TIL infiltration (p = 0.005), and had a borderline association with tumor grade (p = 0.051). However, positive tumor PD-L1 expression was not significantly associated with local recurrence-free survival (LRFS) or overall survival (OS). PD-1 expression in TILs was associated with poor LRFS (χ2 = 10.051, p = 0.002, log-rank test). Multivariate analysis showed that PD-L1 expression only in TILs was an independent predictor for LRFS (HR = 0.298, 95% CI: 0.098-0.907, p = 0.033), and OS (HR = 0.188, 95% CI: 0.051-0.687, p = 0.011) in spinal chordoma patients. In conclusion, PD-L1 expression in TILs was an independent predictor for both LRFS and OS in spinal chordoma patients. Our findings suggest that the PD-1/PD-L1 pathway may be a novel therapeutic target for the immunotherapy of chordoma. PMID:27508049

  11. PD-L1 expression in colorectal cancer is associated with microsatellite instability, BRAF mutation, medullary morphology and cytotoxic tumor-infiltrating lymphocytes.

    PubMed

    Rosenbaum, Matthew W; Bledsoe, Jacob R; Morales-Oyarvide, Vicente; Huynh, Tiffany G; Mino-Kenudson, Mari

    2016-09-01

    Programmed cell death 1 (PD-1) and its ligand (PD-L1) are key suppressors of the cytotoxic immune response. PD-L1 expression on tumor cells may be induced by the immune microenvironment, resulting in immune escape (adaptive immune resistance), and an adverse prognosis in many malignancies. In colorectal carcinoma the response to PD-1/PD-L1 inhibition is correlated with microsatellite instability. However, little is known about the clinicopathologic, molecular, and prognostic characteristics of colorectal carcinoma with PD-L1 expression. We performed immunohistochemistry for PD-L1 on 181 cases of colorectal carcinoma with known microsatellite instability and mutational status, and correlated PD-L1 expression with clinicopathologic features including tumor-infiltrating lymphocyte burden/immunophenotype, tumor mutational profile, and disease-specific survival. PD-L1 was expressed in tumors from 16 patients (9%) who were more often older (P=0.006) and female (P=0.035), with tumors exhibiting a larger size (P=0.013), but lower stage (P<0.001). PD-L1 expression was associated with increased CD8 and TBET-positive tumor-infiltrating lymphocytes, medullary phenotype, poor differentiation, microsatellite instability, BRAF mutation (P<0.001 for each), and a lower frequency of KRAS mutation (P=0.012). On multivariate analysis, PD-L1 expression was associated with medullary morphology and frequent CD8-positive tumor-infiltrating lymphocytes, suggesting adaptive immune resistance. PD-L1 positivity was not predictive of survival in the entire cohort, but it was associated with a lower disease-specific survival within the microsatellite-instability high cohort. PD-L1 expression in colorectal carcinoma is associated with clinicopathologic and molecular features of the serrated pathway of colorectal carcinogenesis, and is associated with a worse outcome within microsatellite-unstable tumors. These findings support the role of PD-L1 expression in providing normally immunogenic

  12. In vitro augmentation of the cytotoxic activity of peripheral blood mononuclear cells and tumor-infiltrating lymphocytes by famotidine in cancer patients.

    PubMed

    Tsunoda, T; Tanimura, H; Yamaue, H; Iwahashi, M; Tani, M; Tamai, M; Arii, K; Noguchi, K

    1992-01-01

    We investigated the in vitro effects of famotidine on the cytotoxic activity of peripheral blood mononuclear cells (PBMC) and tumor-infiltrating lymphocytes (TILs). The cytotoxic activity of PBMC was augmented by famotidine at a concentration of 10 ng/ml, which is equivalent to the serum level achieved by the intravenous administration of a dose of 20 mg. This response to famotidine was seen only in cancer patients. Both the cytotoxic activity and DNA synthesis of activated TILs were increased by the combination of interleukin-2 and 1 microgram/ml of famotidine. Augmentation of cytotoxic activity by famotidine occurred independently of any decrease in the population of suppressor T-cells. Thus, famotidine may have the potential to be used in adoptive immunotherapy with TILs for cancer patients. PMID:1582736

  13. Costimulated tumor-infiltrating lymphocytes are a feasible and safe alternative donor cell therapy for relapse after allogeneic stem cell transplantation

    PubMed Central

    Fellowes, Vicki; Rose, Jeremy J.; Odom, Jeanne; Pittaluga, Stefania; Steinberg, Seth M.; Blacklock-Schuver, Bazetta; Avila, Daniele N.; Memon, Sarfraz; Kurlander, Roger J.; Khuu, Hahn M.; Stetler-Stevenson, Maryalice; Mena, Esther; Dwyer, Andrew J.; Levine, Bruce L.; June, Carl H.; Reshef, Ran; Vonderheide, Robert H.; Gress, Ronald E.; Fowler, Daniel H.; Hakim, Frances T.; Bishop, Michael R.

    2012-01-01

    Donor lymphocyte infusion (DLI), a standard relapse treatment after allogeneic stem cell transplantation (AlloSCT), has limited efficacy and often triggers GVHD. We hypothesized that after AlloSCT tumor-infiltrating donor lymphocytes could be costimulated ex vivo to preferentially activate/expand antitumor effectors. We tested the feasibility and safety of costimulated, tumor-derived donor lymphocyte (TDL) infusion in a phase 1 trial. Tumor was resected from 8 patients with B-cell malignancy progression post-AlloSCT; tumor cell suspensions were costimulated with anti-CD3/anti-CD28 Ab-coated magnetic beads and cultured to generate TDL products for each patient. Costimulation yielded increased proportions of T-bet+FoxP3− type 1 effector donor T cells. A median of 2.04 × 107 TDL/kg was infused; TDLs were well tolerated, notably without GVHD. Two transient positron emission tomography (PET) responses and 2 mixed responses were observed in these refractory tumors. TDL are a feasible, tolerable, and novel donor cell therapy alternative for relapse after AlloSCT. This trial is registered at clinicaltrials.gov as no. NCT00445666. PMID:22289893

  14. Landscape of tumor-infiltrating T cell repertoire of human cancers.

    PubMed

    Li, Bo; Li, Taiwen; Pignon, Jean-Christophe; Wang, Binbin; Wang, Jinzeng; Shukla, Sachet A; Dou, Ruoxu; Chen, Qianming; Hodi, F Stephen; Choueiri, Toni K; Wu, Catherine; Hacohen, Nir; Signoretti, Sabina; Liu, Jun S; Liu, X Shirley

    2016-07-01

    We developed a computational method to infer the complementarity-determining region 3 (CDR3) sequences of tumor-infiltrating T cells in 9,142 RNA-seq samples across 29 cancer types. We identified over 600,000 CDR3 sequences, including 15% that were full length. CDR3 sequence length distribution and amino acid conservation, as well as variable gene usage, for infiltrating T cells in many tumors, except in brain and kidney cancers, resembled those for peripheral blood cells from healthy donors. We observed a strong association between T cell diversity and tumor mutation load, and we predicted SPAG5 and TSSK6 as putative immunogenic cancer/testis antigens in multiple cancers. Finally, we identified three potential immunogenic somatic mutations on the basis of their co-occurrence with CDR3 sequences. One of them, a PRAMEF4 mutation encoding p.Phe300Val, was predicted to result in peptide binding strongly to both MHC class I and class II molecules, with matched HLA types in its carriers. Our analyses have the potential to simultaneously identify immunogenic neoantigens and tumor-reactive T cell clonotypes. PMID:27240091

  15. Tumors with high-density tumor infiltrating lymphocytes constitute a favorable entity in breast cancer: a pooled analysis of four prospective adjuvant trials

    PubMed Central

    Kotoula, Vassiliki; Chatzopoulos, Kyriakos; Lakis, Sotiris; Alexopoulou, Zoi; Timotheadou, Eleni; Zagouri, Flora; Pentheroudakis, George; Gogas, Helen; Galani, Eleni; Efstratiou, Ioannis; Zaramboukas, Thomas; Koutras, Angelos; Aravantinos, Gerasimos; Samantas, Epaminontas; Psyrri, Amanda; Kourea, Helen; Bobos, Mattheos; Papakostas, Pavlos; Kosmidis, Paris; Pectasides, Dimitrios; Fountzilas, George

    2016-01-01

    Background Tumor infiltrating lymphocytes (TILs) are considered in the prognosis of breast cancer (BC) patients. Here, we investigated the prognostic/predictive effect of TILs in patients treated in the frame of four prospective trials with adjuvant anthracycline-based chemotherapy in the pre- and post-trastuzumab era. Methods TILs density was histologically assessed as percentage of stromal area on whole routine sections of 2613 BC (1563 Luminal A/B; 477 Luminal HER2; 246 HER2-enriched; 327 triple negative [TNBC]) and were evaluated as high/low at three cut-offs (c/o; 50% [lymphocytic predominance, LP], 35% and 25%), in separate training and validation sets. Results High TILs were present in 3.5%, 6.5% and 11.5% of all tumors, using the 50%, 35% and 25% c/o, respectively. TILs status did not interact with BC subtypes or trastuzumab treatment. LPBC patient outcome was not affected by nodal status, while high TILs were favorable in TNBC with unfavorable nodal status. When adjusted for standard clinicopathological parameters and treatment, high TILs independently predicted for favorable outcome, e.g., disease-free survival with the 35% c/o in the entire cohort (HR = 0.44, 95% CI 0.28-0.69, p < 0.001) and in specific subtypes. Conclusions High TILs tumors, especially LPBC seem worthy validating as a separate entity of favorable prognosis in breast cancer. PMID:26506242

  16. Absence of Tumor-Infiltrating Lymphocyte Is a Reproducible Predictive Factor for Sentinel Lymph Node Metastasis: A Multicenter Database Study by the Brazilian Melanoma Group

    PubMed Central

    Duprat, João Pedreira; Brechtbülh, Eduard René; Costa de Sá, Bianca; Enokihara, Mauro; Fregnani, Jose Humberto; Landman, Gilles; Maia, Marcus; Riccardi, Felice; Belfort, Francisco Alberto; Wainstein, Alberto; Moredo, Luciana F.; Steck, Higino; Brandão, Miguel; Moreno, Marcelo; Miranda, Eduardo; Santos, Ivan Dunshee de Oliveira

    2016-01-01

    Aims The aim of this study is to confirm the function of tumor-infiltrating lymphocytes (TILs) in sentinel lymph node (SLN) metastasis. Materials and Methods This retrospective study included 633 patients with invasive melanoma who underwent sentinel lymph node biopsy in 7 referral centers certified by the Brazilian Melanoma Group. Independent risk factors of sentinel node metastasis (SNL) were identified by multiple logistic regression. Results SLN metastasis was detected in 101 of 633 cases (16.1%) and in 93 of 428 patients (21.7%) when melanomas ≤ 1mm were excluded. By multiple logistic regression, the absence of TILs was as an independent risk factor of SLN metastasis (OR = 1.8; 95%CI: 1.1–3.0), in addition to Breslow index (greater than 2.00 mm), lymph vascular invasion, and presence of mitosis. Conclusion SLNB can identify patients who might benefit from immunotherapy, and the determination of predictors of SLNB positivity can help select the proper population for this type of therapy. The absence of TILs is a reproducible parameter that can predict SLNB positivity in melanoma patients, since this study was made with several centers with different dermatopathologists. PMID:26859408

  17. Prognostic Value of Tumor-Infiltrating Lymphocytes in Triple-Negative Breast Cancers From Two Phase III Randomized Adjuvant Breast Cancer Trials: ECOG 2197 and ECOG 1199

    PubMed Central

    Adams, Sylvia; Gray, Robert J.; Demaria, Sandra; Goldstein, Lori; Perez, Edith A.; Shulman, Lawrence N.; Martino, Silvana; Wang, Molin; Jones, Vicky E.; Saphner, Thomas J.; Wolff, Antonio C.; Wood, William C.; Davidson, Nancy E.; Sledge, George W.; Sparano, Joseph A.; Badve, Sunil S.

    2014-01-01

    Purpose Recent studies suggest that tumor-infiltrating lymphocytes (TILs) are associated with disease-free (DFS) and overall survival (OS) in operable triple-negative breast cancer (TNBC). We seek to validate the prognostic impact of TILs in primary TNBCs in two adjuvant phase III trials conducted by the Eastern Cooperative Oncology Group (ECOG). Patients and Methods Full-face hematoxylin and eosin–stained sections of 506 tumors from ECOG trials E2197 and E1199 were evaluated for density of TILs in intraepithelial (iTILs) and stromal compartments (sTILs). Patient cases of TNBC from E2197 and E1199 were randomly selected based on availability of sections. For the primary end point of DFS, association with TIL scores was determined by fitting proportional hazards models stratified on study. Secondary end points were OS and distant recurrence–free interval (DRFI). Reporting recommendations for tumor marker prognostic studies criteria were followed, and all analyses were prespecified. Results The majority of 481 evaluable cancers had TILs (sTILs, 80%; iTILs, 15%). With a median follow-up of 10.6 years, higher sTIL scores were associated with better prognosis; for every 10% increase in sTILs, a 14% reduction of risk of recurrence or death (P = .02), 18% reduction of risk of distant recurrence (P = .04), and 19% reduction of risk of death (P = .01) were observed. Multivariable analysis confirmed sTILs to be an independent prognostic marker of DFS, DRFI, and OS. Conclusion In two national randomized clinical trials using contemporary adjuvant chemotherapy, we confirm that stromal lymphocytic infiltration constitutes a robust prognostic factor in TNBCs. Studies assessing outcomes and therapeutic efficacies should consider stratification for this parameter. PMID:25071121

  18. The Effects of Isoproterenol and Propranolol on Cytokine Profile Secretion by Cultured Tumor-infiltrating Lymphocytes Derived from Colorectal Cancer Patients

    PubMed Central

    Seyedi, Shahram; Andalib, Alireza; Rezaei, Abbas; Hosseini, Seyed Mohsen; Mohebbi, Seyed Reza; Zali, Mohammad Reza; Vafai, Mohamad; Behboo, Roubik; Tabatabaei, Seyed Abbas; Shahabi, Shahram

    2012-01-01

    Objective: Anti-tumor immunity and cytokine profiles have important roles in the development of cancer. Norepinephrine (NE) release due to sympathetic activation leads to a Th2 deviation via the beta-2 adrenergic receptor Beta-2 adrenergic receptor (β-2AR) and could increase cancer progression. This study intends to determine the effects of isoproterenol (ISO; beta-agonist) and propranolol (PRO; beta-antagonist) on the production of IFN-γ, IL-4, and IL-17. Cytokine levels have been examined in tumor-infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells (PBMCs) of patients with colorectal cancer (CRC). The β-2AR expression on lymphocyte subsets was also assessed. Materials and Methods: In this experimental study, TILs were isolated from fresh CRC tissue and patient PBMCs were obtained just prior to surgery. The cells were cultured in medium for 72 hours. Concomitantly, cells were stimulated with 10 µg/ml phytohemagglutinin (PHA) alone or in the presence of either 1 µmol/L of PRO or 1 µmol/L ISO. The concentration of cytokines in the supernatants was measured by ELISA. Three-color flow cytometry was used to determine the expression of β-2AR on the lymphocyte subsets. Statistical analyses were performed via paired or independent t-test. Results: Levels of IFN-γ, IL-4 and IL-17 were elevated after PHA-stimulation of PBMCs and TILs. However, the elevation of IFN-γ and IL-17 production by TILs in response to PHA was significantly lower than PBMCs. In the presence of ISO, the IFN-γ/IL-4 ratio reduced in all groups, but this reduction was very low in TILs. Interestingly, the effects of PRO on cytokine production were, at least partially, comparable to those of ISO. Depressed levels of β-2AR expression were demonstrated on CD4+IFN-γ+ and CD4+IL-17+ lymphocytes in patients' PBMCs and TILs. Conclusion: This study has demonstrated the effects of ISO and PRO on cytokine production by TILs and determined β-2AR expression on these cells. ISO failed

  19. The Value of Tumor Infiltrating Lymphocytes (TILs) for Predicting Response to Neoadjuvant Chemotherapy in Breast Cancer: A Systematic Review and Meta-Analysis

    PubMed Central

    Mao, Yan; Qu, Qing; Zhang, Yuzi; Liu, Junjun; Chen, Xiaosong; Shen, Kunwei

    2014-01-01

    Background We carried out a systematic review and meta-analysis to evaluate the predictive roles of tumor infiltrating lymphocytes (TILs) in response to neoadjuvant chemotherapy (NAC) in breast cancer. Method A PubMed and Web of Science literature search was designed. Random or fixed effect models were adopted to estimate the summary odds ratio (OR). Heterogeneity and sensitivity analyses were performed to explore heterogeneity among studies and to assess the effects of study quality. Publication bias was evaluated using a funnel plot, Egger's test and Begg's test. We included studies where the predictive significance of TILs, and/or TILs subset on the pathologic complete response (pCR) were determined in NAC of breast cancer. Results A total of 13 published studies (including 3251 patients) were eligible. In pooled analysis, the detection of higher TILs numbers in pre-treatment biopsy was correlated with better pCR to NAC (OR = 3.93, 95% CI, 3.26–4.73). Moreover, TILs predicted higher pCR rates in triple negative (OR = 2.49, 95% CI: 1.61–3.83), HER2 positive (OR = 5.05, 95% CI: 2.86–8.92) breast cancer, but not in estrogen receptor (ER) positive (OR = 6.21, 95%CI: 0.86–45.15) patients. In multivariate analysis, TILs were still an independent marker for high pCR rate (OR = 1.41, 95% CI: 1.19–1.66). For TILs subset, higher levels of CD8+ and FOXP3+ T-lymphocytes in pre-treatment biopsy respectively predicted better pathological response to NAC (OR = 6.44, 95% CI: 2.52–16.46; OR = 2.94, 95% CI: 1.05–8.26). Only FOXP3+ lymphocytes in post-NAC breast tissue were a predictive marker for low pCR rate in univariate (OR = 0.41, 95% CI: 0.21–0.80) and multivariate (OR = 0.36, 95% CI: 0.13–0.95) analysis. Conclusion Higher TILs levels in pre-treatment biopsy indicated higher pCR rates for NAC. TILs subset played different roles in predicting response to NAC. PMID:25501357

  20. Cloning of a new gene encoding an antigen recognized by melanoma-specific HLA-A24-restricted tumor-infiltrating lymphocytes

    SciTech Connect

    Robbins, P.F.; El-Gamil, M.; Li, Y.F.

    1995-06-01

    The role of tumor-specific T cells in mediating the regression of metastatic melanoma has been suggested by the clinical response of patients to treatment with tumor-infiltrating lymphocytes (TIL). A number of Ags recognized by class I-restricted melanoma-specific T cells have recently been isolated, raising the hope that this will lead to the development of improved therapies. In this study, we report the cloning of a tumor Ag recognized by T cells from melanoma patient 888. Previously, we reported that TIL 888, grown from the tumor of this patient, recognized tyrosinase in an HLA-A24 -restricted fashion. This line, when infused into the autologous patient, resulted in complete regression of multiple metastases. Three years later, a second TIL line, TIL 1290, was isolated from a recurrent pelvic tumor. Infusion of a mixture of TIL 888 and TIL 1290 cell lines into the patient resulted in complete regression of a residual abdominal mass and the patient remains disease-free 2 yr later. The TIL 1290 cell line, which recognized melanoma in an HLAA-A24-restricted manner, failed to recognize tyrosinase. TIL 1290 was then used to screen an 888 melanoma cDNA library, and an Ag was isolated that did not correspond to any found in sequence databases. This gene, termed p15, was found to be expressed in a variety of normal tissues, and a peptide epitope recognized by TIL 1290 was found to represent the product of an nonmutated gene. Screening of additional cDNA pools resulted in the isolation of a second clone which stimulated TIL 1290. This clone also appeared to represent a transcript of the p15 gene, indicating that this gene may encode the predominant Ag recognized by TIL 1290. 27 refs., 4 figs., 5 tabs.

  1. Prognostic and predictive value of tumor-infiltrating lymphocytes for clinical therapeutic research in patients with non-small cell lung cancer

    PubMed Central

    Zeng, Dong-Qiang; Yu, Yun-Fang; Ou, Qi-Yun; Li, Xiao-Yin; Zhong, Ru-Zhi; Xie, Chuan-Miao; Hu, Qiu-Gen

    2016-01-01

    Background Previous preclinical and clinical studies have shown that levels of tumor-infiltrating lymphocytes (TILs) significantly correlated with prognosis in non-small cell lung cancer (NSCLC), and survival after therapy; however, this finding remains controversial. We performed a meta-analysis, to evaluate, systematically, the clinical utilization of TIL subtypes in patients with NSCLC. Methods The PubMed, ISI Web of Science, EMBASE, and Cochrane Library databases were searched to identify relevant studies. We pooled estimates of treatment effects, and hazards were summarized using random or fixed effects models to evaluate survival outcomes. Results A total of 24 relevant studies involving 7,006 patients were eligible. The median percentage of lymph node positivity was 45.7% (95% confidence interval [CI], 37.1–56.4%). Pooled analysis shows that high levels of CD8+ TILs had a good prognostic effect on survival with a hazard ratio (HR) of 0.91 (P = 0.013) for death and 0.74 (P = 0.001) for recurrence, as did high levels of CD3+ and CD4+ TILs, with HRs of 0.77 (P = 0.009) and 0.78 (P = 0.005) for death, respectively. By contrast, high levels of FoxP3+ regulatory TILs had a worse prognostic effect for overall and recurrence-free survival, with HRs of 1.69 (P = 0.042) and 1.79 (P = 0.001), respectively. No individual study affected the results, and no publication bias was found. Conclusions Our findings support the hypothesis that TILs could be a prognostic marker in NSCLC. High-quality randomized studies are needed to verify statistically the effect of TILs on prognosis in future research. PMID:26871598

  2. Effects of TP53 and PIK3CA mutations in early breast cancer: a matter of co-mutation and tumor-infiltrating lymphocytes.

    PubMed

    Kotoula, Vassiliki; Karavasilis, Vasilios; Zagouri, Flora; Kouvatseas, George; Giannoulatou, Eleni; Gogas, Helen; Lakis, Sotiris; Pentheroudakis, George; Bobos, Mattheos; Papadopoulou, Kyriaki; Tsolaki, Eleftheria; Pectasides, Dimitrios; Lazaridis, Georgios; Koutras, Angelos; Aravantinos, Gerasimos; Christodoulou, Christos; Papakostas, Pavlos; Markopoulos, Christos; Zografos, George; Papandreou, Christos; Fountzilas, George

    2016-07-01

    The purpose of this study is to investigate whether the outcome of breast cancer (BC) patients treated with adjuvant chemotherapy is affected by co-mutated TP53 and PIK3CA according to stromal tumor-infiltrating lymphocytes (TILs). Paraffin tumors of all clinical subtypes from 1661 patients with operable breast cancer who were treated within 4 adjuvant trials with anthracycline-taxanes chemotherapy were informative for TP53 and PIK3CA mutation status (semiconductor sequencing genotyping) and for stromal TILs density. Disease-free survival (DFS) was examined. TP53 mutations were associated with higher (p < 0.001) and PIK3CA with lower (p = 0.004) TILs in an ER /PgR-specific manner (p < 0.001). Mutations did not affect the favorable DFS of patients with lymphocyte-predominant (LP) BC. Within non-LPBC, PIK3CA-only mutations conferred best, while TP53-PIK3CA co-mutations (6 % of all tumors) conferred worst DFS (HR 0.59; 95 % CI 0.44-0.79; p = 0.001 for PIK3CA-only). TP53-only mutations were unfavorable in patients with lower TILs, while patients with lower TILs performed worse if their tumors carried TP53-only mutations (interaction p = 0.046). Multivariate analysis revealed favorable PIK3CA-only mutations in non-LPBC (HR 0.64; 95 % CI 0.47-0.88; p = 0.007), and unfavorable TP53 mutations in ER/PgRpos/HER2neg (HR 1.55; 95 % CI 1.07-2.24; p = 0.021). Mutations did not interact with TILs in non-LP triple-negative and HER2-positive patients. TP53 and PIK3CA mutations appear to have diverse effects on the outcome of early BC patients, according to whether these genes are co-mutated or not, and for TP53 according to TILs density and ER/PgR-status. These findings need to be considered when evaluating the effect of these two most frequently mutated genes in the context of large clinical trials. PMID:27369359

  3. Prognostic value of tumor-infiltrating lymphocytes for patients with completely resected stage IIIA(N2) non-small cell lung cancer

    PubMed Central

    Shen, Lei; Cai, Xu-Wei; Zhu, Zheng-Fei; Chang, Jian-Hua; Xiang, Jia-Qing; Zhang, Ya-Wei; Chen, Hai-Quan; Fu, Xiao-Long

    2016-01-01

    Background The patient prognosis after complete resection for pathologic stage IIIA(N2) non-small cell lung cancer (NSCLC) remains a significant concern. The clinical relevance of the host immune response to NSCLC has yet to be established. We aimed to investigate the prognostic value of tumor-infiltrating lymphocytes (TILs) in a uniform cohort of patients with completely resected stage IIIA(N2) NSCLC. Methods From 2005 to 2012, consecutive patients with pathologic stage IIIA(N2) NSCLC who underwent complete resection at our institution were reviewed. For each case, full-face hematoxylin and eosin-stained sections from surgical specimens were evaluated for the TIL density. A published, recommended TIL scoring scale was followed. The patients were stratified into the TIL− or TIL+ group based on pathologic evaluation. Results Data from 320 patients were included in the analysis. Based on a median follow-up duration of 30.8 months, a higher density of TILs was associated with an improved postoperative survival time (P = 0.06). Subgroup analyses indicated that this positive effect was the greatest for patients with squamous cell carcinoma (SCC; P = 0.03). Among those with SCC, the TIL+ patients experienced a significantly increased 3-year distant metastasis-free survival (DMFS) compared to the TIL− patients (60.6% versus 42.7%, P = 0.02). Multivariate analyses of the 93 patients with SCC tumors confirmed that TIL+ was an independent prognostic factor for an increased DMFS (HR = 0.39, 95%CI 0.17–0.87, P = 0.02) and a prolonged overall survival (OS; HR = 0.47, 95%CI 0.22–1.00, P = 0.05). Conclusions Our data suggest a potential role of TILs in predicting the survival of patients with completely resected stage IIIA(N2) NSCLC. The beneficial effects of TILs were more pronounced in the prediction of the DMFS and the OS in patients with SCC. This parameter should be considered for prospective inclusion in clinical trials. PMID:26811495

  4. Density of tumor-infiltrating lymphocytes correlates with extent of brain edema and overall survival time in patients with brain metastases

    PubMed Central

    Berghoff, Anna S; Fuchs, Elisabeth; Ricken, Gerda; Mlecnik, Bernhard; Bindea, Gabriela; Spanberger, Thomas; Hackl, Monika; Widhalm, Georg; Dieckmann, Karin; Prayer, Daniela; Bilocq, Amelie; Heinzl, Harald; Zielinski, Christoph; Bartsch, Rupert; Birner, Peter; Galon, Jerome; Preusser, Matthias

    2016-01-01

    The immune microenvironment of the brain differs from that of other organs and the role of tumor-infiltrating lymphocytes (TILs) in brain metastases (BM), one of the most common and devastating complication of cancer, is unclear. We investigated TIL subsets and their prognostic impact in 116 BM specimens using immunohistochemistry for CD3, CD8, CD45RO, FOXP3, PD1 and PD-L1. The Immunoscore was calculated as published previously. Overall, we found TIL infiltration in 115/116 (99.1%) BM specimens. PD-L1 expression was evident in 19/67 (28.4%) BM specimens and showed no correlation with TIL density (p > 0.05). TIL density was not associated with corticosteroid administration (p > 0.05). A significant difference in infiltration density according to TIL subtype was present (p < 0.001; Chi Square); high infiltration was most frequently observed for CD3+ TILs (95/116; 81.9%) and least frequently for PD1+ TILs (18/116; 15.5%; p < 0.001). Highest TIL density was observed in melanoma, followed by renal cell cancer and lung cancer BM (p < 0.001). The density of CD8+ TILs correlated positively with the extent of peritumoral edema seen on pre-operative magnetic resonance imaging (p = 0.031). The density of CD3+ (15 vs. 6 mo; p = 0.015), CD8+ (15 vs. 11 mo; p = 0.030) and CD45RO+ TILs (18 vs. 8 mo; p = 0.006) showed a positive correlation with favorable median OS times. Immunoscore showed significant correlation with survival prognosis (27 vs. 10 mo; p < 0.001). The prognostic impact of Immunoscore was independent from established prognostic parameters at multivariable analysis (HR 0.612, p < 0.001). In conclusion, our data indicate that dense TILs infiltrates are common in BM and correlate with the amount of peritumoral brain edema and survival prognosis, thus identifying the immune system as potential biomarker for cancer patients with CNS affection. Further studies are needed to substantiate our findings. PMID:26942067

  5. Prognostic and predictive value of NanoString-based immune-related gene signatures in a neoadjuvant setting of triple-negative breast cancer: relationship to tumor-infiltrating lymphocytes.

    PubMed

    Lee, Hee Jin; Lee, Jeong-Ju; Song, In Hye; Park, In Ah; Kang, Jun; Yu, Jong Han; Ahn, Jin-Hee; Gong, Gyungyub

    2015-06-01

    The prognostic significance of tumor-infiltrating lymphocytes and immune signals has been described previously in triple-negative breast cancer (TNBC). Furthermore, recent studies have shown that immunologic parameters are relevant for the response to neoadjuvant chemotherapy (NAC) in breast cancer as well as for outcomes after adjuvant chemotherapy. However, immune signals are variable, and which signals are important is largely unknown. We, therefore, evaluated the expression of immune-related genes in TNBC treated with NAC. We retrospectively evaluated biopsy tissue from 55 patients with primary TNBC treated with NAC (anthracycline, cyclophosphamide, and docetaxel) against the NanoString nCounter GX Human Immunology Panel (579 immune-related genes). Higher expression of cytotoxic molecules, T cell receptor signaling pathway components, cytokines related to T helper cell type 1 (Th1), and B cell markers was associated with a pathologic complete response (pCR). Higher expression of NFKB1, MAPK1, TRAF1, CXCL13, GZMK, and IL7R was significantly associated with pCR, higher Miller-Payne grade, and lower residual cancer burden class. Expression of NFKB1, TRAF1, and CXCL13genes, in particular, was significantly correlated with a longer disease-free survival rate. Conversely, patients those who failed to achieve a pCR showed increased expression of genes related to neutrophils. Higher expression of cytotoxic molecules, T cell receptor signaling pathway components, Th1-related cytokines, and B cell markers is correlated with pCR and survival in TNBC patients treated with NAC. Our results suggest that the activation status of neutrophils may provide additional predictive information for TNBC patients treated with NAC. PMID:26006068

  6. Correlation of tumor-infiltrating lymphocytes to histopathological features and molecular phenotypes in canine mammary carcinoma: A morphologic and immunohistochemical morphometric study.

    PubMed

    Kim, Jong-Hyuk; Chon, Seung-Ki; Im, Keum-Soon; Kim, Na-Hyun; Sur, Jung-Hyang

    2013-04-01

    Abundant lymphocyte infiltration is frequently found in canine malignant mammary tumors, but the pathological features and immunophenotypes associated with the infiltration remain to be elucidated. The aim of the present study was to evaluate the relationship between lymphocyte infiltration, histopathological features, and molecular phenotype in canine mammary carcinoma (MC). The study was done with archived formalin-fixed, paraffin-embedded samples (n = 47) by histologic and immunohistochemical methods. The degree of lymphocyte infiltration was evaluated by morphologic analysis, and the T- and B-cell populations as well as the T/B-cell ratio were evaluated by morphometric analysis; results were compared with the histologic features and molecular phenotypes. The degree of lymphocyte infiltration was significantly higher in MCs with lymphatic invasion than in those without lymphatic invasion (P < 0.0001) and in tumors of high histologic grade compared with those of lower histologic grade (P = 0.045). Morphometric analysis showed a larger amount of T-cells and B-cells in MCs with a higher histologic grade and lymphatic invasion, but the T/B ratio did not change. Lymphocyte infiltration was not associated with histologic type or molecular phenotype, as assessed from the immunohistochemical expression of epidermal growth factor receptor 2, estrogen receptor, cytokeratin 14, and p63. Since intense lymphocyte infiltration was associated with aggressive histologic features, lymphocytes may be important for tumor aggressiveness and greater malignant behavior in the tumor microenvironment. PMID:24082407

  7. Expression of Sirt1 and FoxP3 in classical Hodgkin lymphoma and tumor infiltrating lymphocytes: Implications for immune dysregulation, prognosis and potential therapeutic targeting

    PubMed Central

    Quesada, Andrés E; Assylbekova, Binara; Jabcuga, Christine E; Zhang, Rongzhen; Covinsky, Michael; Rios, Adan; Nguyen, Nghia D; Brown, Robert E

    2015-01-01

    Background: Hodgkin Reed-Sternberg (HRS) cells may promote differentiation of CD4+ naïve T cells toward both FoxP3+ T regulatory (Treg) cells and TIA-1+ cytotoxic T lymphocytes (CTL). Previous studies suggest that an overabundance of cytotoxic TIA-1+ cells in relation to FoxP3+ T reg cells portends unfavorable outcomes in classical Hodgkin lymphoma (cHL), raising the possibility that its pathogenesis may be related to immune dysregulation. Sirt1 deacetylates FoxP3 and leads to decreased Treg functionality. Our objective was to compare Sirt1 and FoxP3 expressions in Hodgkin lymphoma infiltrating lymphocytes (HLIL) and confirm Sirt1 expression in HRS cells. Design: Immunohistochemical staining of paraffin-embedded tissue with antibodies to Sirt1, FoxP3, TIA-1, and CD8 was performed. Expression of Sirt1was assessed in both the HRS cells and in the HLILs in twenty-four cases. Adequate tissue was available in 13 cHL cases to permit the enumeration of FoxP3, TIA-1 and CD8 by giving their percent staining of HLILs. Results: In HLILs, nuclear expression of Sirt1 was 32-88% (mean 67%); FoxP3 expression was 9-40% (mean 23.9%); TIA-1 expression was 15-87% (mean 32%); and CD8 expression was 10-45% (mean = 31%). Sirt1 to FoxP3 ratio was 0.96-5.5 (mean 3.2). TIA-1 to FoxP3 ratio was 0.6-5.1 (mean 1.6). CD8 to FoxP3 ratio was 0.43-3.7 (mean 1.5). There was a difference of Sirt1 to FoxP3 ratios between remission and recurrence groups, being significantly higher in the recurrence group (P = 0.005). Sirt1 demonstrated high nuclear expression in the HRS cells of 21 out of 24 (88%) cases analyzed. Conclusion: The relative overexpression of Sirt1 to FoxP3 in HLILs may be considered possible targets for immune modulation. Histone deacetylase inhibitors may increase the efficacy of existing treatment regimens by downregulating SIRT1 gene mRNA/Sirt1 protein function and together with rapamycin could expand the T regulatory/FoxP3 population and functionality and improve prognosis for

  8. The ratio of CD8 to Treg tumor-infiltrating lymphocytes is associated with response to cisplatin-based neoadjuvant chemotherapy in patients with muscle invasive urothelial carcinoma of the bladder

    PubMed Central

    Baras, Alexander S.; Drake, Charles; Liu, Jen-Jane; Gandhi, Nilay; Kates, Max; Hoque, Mohamed O.; Meeker, Alan; Hahn, Noah; Taube, Janis M.; Schoenberg, Mark P.; Netto, George; Bivalacqua, Trinity J.

    2016-01-01

    ABSTRACT Introduction: Randomized controlled trials of platinum-based neoadjuvant chemotherapy (NAC) for bladder cancer have shown that patients who achieve a pathologic response to NAC exhibit 5 y survival rates of approximately 80–90% while NAC resistant (NR) cases exhibit 5 y survival rates of approximately 30–40%. These findings highlight the need to predict who will benefit from conventional NAC and the need for plausible alternatives. Methods: The pre-treatment biopsy tissues from a cohort of 41 patients with muscle invasive bladder who were treated with NAC were incorporated in tissue microarray and immunohistochemistry for PD-L1, CD8, and FOXP3 was performed. Percentage of PD-L1 positive tumor cells was measured. Tumor-infiltrating lymphocytes (TIL) densities, along with CD8 and Treg-specific TILs, were measured. Results: TIL density was strongly correlated with tumor PD-L1 expression, consistent with the mechanism of adaptive immune resistance in bladder cancer. Tumor cell PD-L1 expression was not a significant predictor of response. Neither was the CD8 nor Treg TIL density associated with response. Intriguingly though, the ratio of CD8 to Treg TIL densities was strongly associated with response (p = 0.0003), supporting the hypothesis that the immune system plays a role in the response of bladder cancer to chemotherapy. Discussion: To our knowledge, this is the first report in bladder cancer showing that the CD8 to Treg TIL density in the pre-treatment tissues is predictive for conventional NAC response. These findings warrant further investigations to both better characterize this association in larger cohorts and begin to elucidate the underlying mechanism(s) of this phenomenon.

  9. Prognostic impact of programmed cell death-1 (PD-1) and PD-ligand 1 (PD-L1) expression in cancer cells and tumor-infiltrating lymphocytes in ovarian high grade serous carcinoma

    PubMed Central

    Kulbe, Hagen; Sehouli, Jalid; Wienert, Stephan; Lindner, Judith; Budczies, Jan; Bockmayr, Michael; Dietel, Manfred; Denkert, Carsten; Braicu, Ioana; Jöhrens, Korinna

    2016-01-01

    Aims Antibodies targeting the checkpoint molecules programmed cell death 1 (PD-1) and its ligand PD-L1 are emerging cancer therapeutics. We systematically investigated PD-1 and PD-L1 expression patterns in the poor-prognosis tumor entity high-grade serous ovarian carcinoma. Methods PD-1 and PD-L1 protein expression was determined by immunohistochemistry on tissue microarrays from 215 primary cancers both in cancer cells and in tumor-infiltrating lymphocytes (TILs). mRNA expression was measured by quantitative reverse transcription PCR. An in silico validation of mRNA data was performed in The Cancer Genome Atlas (TCGA) dataset. Results PD-1 and PD-L1 expression in cancer cells, CD3+, PD-1+, and PD-L1+ TILs densities as well as PD-1 and PD-L1 mRNA levels were positive prognostic factors for progression-free (PFS) and overall survival (OS), with all factors being significant for PFS (p < 0.035 each), and most being significant for OS. Most factors also had prognostic value that was independent from age, stage, and residual tumor. Moreover, high PD-1+ TILs as well as PD-L1+ TILs densities added prognostic value to CD3+TILs (PD-1+: p = 0.002,; PD-L1+: p = 0.002). The significant positive prognostic impact of PD-1 and PD-L1 mRNA expression could be reproduced in the TCGA gene expression datasets (p = 0.02 and p < 0.0001, respectively). Conclusions Despite their reported immune-modulatory function, high PD-1 and PD-L1 levels are indicators of a favorable prognosis in ovarian cancer. Our data indicate that PD-1 and PD-L1 molecules are biologically relevant regulators of the immune response in high-grade serous ovarian carcinoma, which is an argument for the evaluation of immune checkpoint inhibiting drugs in this tumor entity. PMID:26625204

  10. Tumor-infiltrating lymphocytes (TILs) are a powerful prognostic marker in patients with triple-negative breast cancer enrolled in the IBCSG phase III randomized clinical trial 22-00.

    PubMed

    Pruneri, Giancarlo; Gray, Kathryn P; Vingiani, Andrea; Viale, Giuseppe; Curigliano, Giuseppe; Criscitiello, Carmen; Láng, István; Ruhstaller, Thomas; Gianni, Lorenzo; Goldhirsch, Aron; Kammler, Roswitha; Price, Karen N; Cancello, Giuseppe; Munzone, Elisabetta; Gelber, Richard D; Regan, Meredith M; Colleoni, Marco

    2016-07-01

    The purpose of this study was to assess the prognostic and predictive value of tumor-infiltrating lymphocytes (TILs) in the triple-negative breast cancer (TNBC) cohort of the phase III IBCSG trial 22-00, comparing low-dose oral 'metronomic' cyclophosphamide-methotrexate maintenance chemotherapy (CM-maintenance) to no-CM-maintenance in early breast cancer. TILs were evaluated in full-face hematoxylin-and-eosin-stained sections of tumor samples confirmed centrally as TNBC (< 1 % of ER and PgR immunoreactivity and absence of HER2 overexpression or amplification). Mononuclear cells were evaluated in the stromal area within the borders of the invasive tumor. The primary endpoint was breast cancer-free interval (BCFI). Cox proportional hazards regression model assessed the association of BCFI and secondary endpoints with TILs score. In the 647 tumor samples, the median percentage of TILs was 18 % (IQR = 8-40 %), with 18 % having TILs ≥ 50 % (lymphocyte-predominant breast cancer, LPBC). At a median follow-up of 6.9 years, TILs were associated with better prognosis. For every 10 % increase of TILs, BCFI risk reduction was 13 % (HR 0.87, 95 % CI 0.79-0.95,P = 0.003). DFS, DRFI, and OS risk reductions were 11 % (P = 0.005), 16 % (P = 0.003), and 17 % (P < 0.001), respectively. Multivariable analysis confirmed the independent prognostic value of TILs. No significant TILs-by-treatment interaction was observed (P = 0.39) for associations of TILs with BCFI, although patients with LPBC receiving CM-maintenance had a greater breast cancer risk reduction (HR 0.64,95 % CI 0.23-1.78) than those with non-LPBC (TILs < 50 %) (HR 0.96, 95 % CI 0.67-1.40). TILs score is a potent prognostic factor in patients with TNBC. Low-dose chemotherapy confers a greater (not statistically significant) clinical benefit in patients with LPBC. PMID:27372069

  11. Association and prognostic significance of BRCA1/2-mutation status with neoantigen load, number of tumor-infiltrating lymphocytes and expression of PD-1/PD-L1 in high grade serous ovarian cancer

    PubMed Central

    Strickland, Kyle C.; Howitt, Brooke E.; Shukla, Sachet A.; Rodig, Scott; Ritterhouse, Lauren L.; Liu, Joyce F.; Garber, Judy E.; Chowdhury, Dipanjan; Wu, Catherine J.; D'Andrea, Alan D.; Matulonis, Ursula A.; Konstantinopoulos, Panagiotis A.

    2016-01-01

    Immune checkpoint inhibitors (e.g., anti-PD-1 and anti-PD-L1 antibodies) have demonstrated remarkable efficacy against hypermutated cancers such as melanomas and lung carcinomas. One explanation for this effect is that hypermutated lesions harbor more tumor-specific neoantigens that stimulate recruitment of an increased number of tumor-infiltrating lymphocytes (TILs), which is counterbalanced by overexpression of immune checkpoints such as PD-1 or PD-L1. Given that BRCA1/2-mutated high grade serous ovarian cancers (HGSOCs) exhibit a higher mutational load and a unique mutational signature with an elevated number of larger indels up to 50 bp, we hypothesized that they may also harbor more tumor-specific neoantigens, and, therefore, exhibit increased TILs and PD-1/PD-L1 expression. Here, we report significantly higher predicted neoantigens in BRCA1/2-mutated tumors compared to tumors without alterations in homologous recombination (HR) genes (HR-proficient tumors). Tumors with higher neoantigen load were associated with improved overall survival and higher expression of immune genes associated with tumor cytotoxicity such as genes of the TCR, the IFN-gamma and the TNFR pathways. Furthermore, immunohistochemistry studies demonstrated that BRCA1/2-mutated tumors exhibited significantly increased CD3+ and CD8+ TILs, as well as elevated expression of PD-1 and PD-L1 in tumor-associated immune cells compared to HR-proficient tumors. Survival analysis showed that both BRCA1/2-mutation status and number of TILs were independently associated with outcome. Of note, two distinct groups of HGSOCs, one with very poor prognosis (HR proficient with low number of TILs) and one with very good prognosis (BRCA1/2-mutated tumors with high number of TILs) were defined. These findings support a link between BRCA1/2-mutation status, immunogenicity and survival, and suggesting that BRCA1/2-mutated HGSOCs may be more sensitive to PD-1/PD-L1 inhibitors compared to HR-proficient HGSOCs. PMID

  12. Immune signature of tumor infiltrating immune cells in renal cancer

    PubMed Central

    Geissler, Katharina; Fornara, Paolo; Lautenschläger, Christine; Holzhausen, Hans-Jürgen; Seliger, Barbara; Riemann, Dagmar

    2015-01-01

    Tumor-associated immune cells have been discussed as an essential factor for the prediction of the outcome of tumor patients. Lymphocyte-specific genes are associated with a favorable prognosis in colorectal cancer but with poor survival in renal cell carcinoma (RCC). Flow cytometric analyses combined with immunohistochemistry were performed to study the phenotypic profiles of tumor infiltrating lymphocytes (TIL) and the frequency of T cells and macrophages in RCC lesions. Data were correlated with clinicopathological parameters and survival of patients. Comparing oncocytoma and clear cell (cc)RCC, T cell numbers as well as activation-associated T cell markers were higher in ccRCC, whereas the frequency of NK cells was higher in oncocytoma. An intratumoral increase of T cell numbers was found with higher tumor grades (G1:G2:G3/4 = 1:3:4). Tumor-associated macrophages slightly increased with dedifferentiation, although the macrophage-to-T cell ratio was highest in G1 tumor lesions. A high expression of CD57 was found in T cells of early tumor grades, whereas T cells in dedifferentiated RCC lesions expressed higher levels of CD69 and CTLA4. TIL composition did not differ between older (>70 y) and younger (<58 y) patients. Enhanced patients’ survival was associated with a higher percentage of tumor infiltrating NK cells and Th1 markers, e.g. HLA-DR+ and CXCR3+ T cells, whereas a high number of T cells, especially with high CD69 expression correlated with a worse prognosis of patients. Our results suggest that immunomonitoring of RCC patients might represent a useful tool for the prediction of the outcome of RCC patients. PMID:25949868

  13. Diffusion tensor-based tumor infiltration index cannot discriminate vasogenic edema from tumor-infiltrated edema.

    PubMed

    Kinoshita, Manabu; Goto, Tetsu; Okita, Yoshiko; Kagawa, Naoki; Kishima, Haruhiko; Hashimoto, Naoya; Yoshimine, Toshiki

    2010-02-01

    Diffusion tensor imaging (DTI) by magnetic resonance imaging (MRI) is now used not only for delineating white matter fiber tracts, but also for assessing the histological characteristics of pathological tissues. Among these uses, predicting the extent or existence of tumor cell invasion into white matter by DTI is under extensive investigation. The previously reported tumor infiltration index (TII) holds great potential for the discrimination of pure vasogenic edema from tumor-infiltrated edema. However, conflicting data are being reported questioning the clinical value of TII. The present investigation reevaluated the utility of TII in patients with meningioma or glioma. We found that TII was unable to discriminate vasogenic from tumor-infiltrated edema. Conversely, detailed voxel-by-voxel comparison of TII and (11)C-methionie PET in the T2-hyperintense area of gliomas showed that TII and (11)C-methionie PET has a positive correlation, suggesting that, although TII is unable to discriminate the cause of edema, the extent of tumor cell invasion into white matter is depicted in gliomas by TII. These data suggest that TII involves both vasogenic and tumor-infiltrated factors, rather than only a single factor. A more intensive investigation is required to reach a complete understanding of TII. PMID:19696968

  14. Tumor infiltrating immune cells in gliomas and meningiomas.

    PubMed

    Domingues, Patrícia; González-Tablas, María; Otero, Álvaro; Pascual, Daniel; Miranda, David; Ruiz, Laura; Sousa, Pablo; Ciudad, Juana; Gonçalves, Jesús María; Lopes, María Celeste; Orfao, Alberto; Tabernero, María Dolores

    2016-03-01

    Tumor-infiltrating immune cells are part of a complex microenvironment that promotes and/or regulates tumor development and growth. Depending on the type of cells and their functional interactions, immune cells may play a key role in suppressing the tumor or in providing support for tumor growth, with relevant effects on patient behavior. In recent years, important advances have been achieved in the characterization of immune cell infiltrates in central nervous system (CNS) tumors, but their role in tumorigenesis and patient behavior still remain poorly understood. Overall, these studies have shown significant but variable levels of infiltration of CNS tumors by macrophage/microglial cells (TAM) and to a less extent also lymphocytes (particularly T-cells and NK cells, and less frequently also B-cells). Of note, TAM infiltrate gliomas at moderate numbers where they frequently show an immune suppressive phenotype and functional behavior; in contrast, infiltration by TAM may be very pronounced in meningiomas, particularly in cases that carry isolated monosomy 22, where the immune infiltrates also contain greater numbers of cytotoxic T and NK-cells associated with an enhanced anti-tumoral immune response. In line with this, the presence of regulatory T cells, is usually limited to a small fraction of all meningiomas, while frequently found in gliomas. Despite these differences between gliomas and meningiomas, both tumors show heterogeneous levels of infiltration by immune cells with variable functionality. In this review we summarize current knowledge about tumor-infiltrating immune cells in the two most common types of CNS tumors-gliomas and meningiomas-, as well as the role that such immune cells may play in the tumor microenvironment in controlling and/or promoting tumor development, growth and control. PMID:26216710

  15. Anti-CCR4 monoclonal antibody enhances antitumor immunity by modulating tumor-infiltrating Tregs in an ovarian cancer xenograft humanized mouse model

    PubMed Central

    Chang, De-Kuan; Peterson, Eric; Sun, Jiusong; Goudie, Calum; Drapkin, Ronny I.; Liu, Joyce F.; Matulonis, Ursula; Zhu, Quan; Marasco, Wayne A.

    2016-01-01

    ABSTRACT Recent studies have demonstrated that regulatory T cells (Tregs) are recruited to tumor sites where they can suppress antitumor immunity. The chemokine receptor CCR4 is expressed at high levels on functional CD4+CD25+FoxP3+ Tregs and production of the CCR4 ligand CCL22 by tumor cells and tumor-associated macrophages is associated with Treg recruitment to the tumor site. Here, we tested IgG1 and IgG4 isotypes of human anti-CCR4 mAb2-3 for their in vitro activity and in vivo capacity in a NSG mouse model bearing CCL22-secreting ovarian cancer (OvCA) xenograft to modulate Tregs and restore antitumor activity. Both mAb2-3 isotypes blocked in vitro chemoattraction of Tregs to CCL22-secreting OvCA cells. However, they differed in their in vivo mode of action with IgG1 causing Treg depletion and IgG4 blocking migration to the tumors. Primary T cells that were primed with OvCA-pulsed dendritic cells (DCs) demonstrated INFγ secretion that could be enhanced through Treg depletion by mAb2-3. Humanized mice reconstructed with allogeneic tumor-primed T cells (TP-T) were used to evaluate the restoration of OvCA immunity by depletion or blockade of Tregs with mAb2-3. We observed that IgG1 was more potent than IgG4 in inhibiting tumor growth. Mechanism studies demonstrated that mAb2-3 treatment lead to inhibition of IL-2 binding to its receptor. Further studies showed that mAb2-3 induced CD25 shedding (sCD25) from Tregs which lead to a decrease in IL-2-dependent survival. Together, the results demonstrate that mAb2-3 is an agonist antibody that can restore anti-OvCA immunity through modulation of Treg activity. PMID:27141347

  16. CD8+ lymphocyte infiltration is an independent favorable prognostic indicator in basal-like breast cancer

    PubMed Central

    2012-01-01

    Introduction Tumor infiltrating lymphocytes may indicate an immune response to cancer development, but their significance remains controversial in breast cancer. We conducted this study to assess CD8+ (cytotoxic T) lymphocyte infiltration in a large cohort of invasive early stage breast cancers, and to evaluate its prognostic effect in different breast cancer intrinsic subtypes. Methods Immunohistochemistry for CD8 staining was performed on tissue microarrays from 3992 breast cancer patients. CD8+ tumor infiltrating lymphocytes were counted as intratumoral when in direct contact with tumor cells, and as stromal in adjacent locations. Kaplan-Meier functions and Cox proportional hazards regression models were applied to examine the associations between tumor infiltrating lymphocytes and breast cancer specific survival. Results Among 3403 cases for which immunohistochemical results were obtained, CD8+ tumor infiltrating lymphocytes were identified in an intratumoral pattern in 32% and stromal pattern in 61% of the cases. In the whole cohort, the presence of intratumoral tumor-infiltrating lymphocytes was significantly correlated with young age, high grade, estrogen receptor negativity, human epidermal growth factor receptor-2 positivity and core basal intrinsic subtype, and was associated with superior breast cancer specific survival. Multivariate analysis indicated that the favorable prognostic effect of CD8+ tumor infiltrating lymphocytes was significant only in the core basal intrinsic subgroup (Hazard ratio, HR = 0.35, 95% CI = 0.23-0.54). No association with improved survival was present in those triple negative breast cancers that lack expression of basal markers (HR = 0.99, 95% CI = 0.48-2.04) nor in the other intrinsic subtypes. Conclusions CD8+ tumor infiltrating lymphocytes are an independent prognostic factor associated with better patient survival in basal-like breast cancer, but not in non-basal triple negative breast cancers nor in other intrinsic

  17. Human uterine lymphocytes.

    PubMed

    King, A; Burrows, T; Verma, S; Hiby, S; Loke, Y W

    1998-01-01

    During the luteal phase and the early months of pregnancy, there is a dense mucosal infiltration of CD56+ natural killer (NK) cells. These uterine NK cells have a phenotype (CD56bright, CD16-, mCD3-) which distinguishes them from peripheral blood NK cells (CD56dim, CD16bright, mCD3-). The uterine NK cells are in close association with extravillous trophoblast (EVT) cells which infiltrate into the decidua and maternal spiral arteries. This subpopulation of trophoblast expresses two human leukocyte antigen (HLA) class I molecules, HLA-G and HLA-C. Circulating NK cells express receptors for HLA class I molecules. We have recently found evidence that similar receptors are present on decidual NK cells belonging to both the Killer Inhibitory Receptor (KIR) and CD94 families. The repertoire of NK receptors expressed varies between different women. The findings indicate that decidual NK cells do have receptors for trophoblast HLA class I molecules. Experiments are underway to determine the effects of this interaction on NK cell function. PMID:10027599

  18. Differential regulation and function of tumor-infiltrating T cells in different stages of breast cancer patients.

    PubMed

    Zhu, Shiguang; Lin, Jun; Qiao, Guangdong; Xu, Yanping; Zou, Haidong

    2015-09-01

    Breast cancer survival was associated with higher frequencies of CD8(+) T cytotoxic T cells in infiltrating lymphocytes. On the other hand, the frequency of CD4(+)CD25(+)FoxP3(+) regulatory T cells was inversely correlated with clinical outcomes of breast cancer. The regulation and interaction of different types of tumor-infiltrating T cells in different stages of breast cancer patients are still unclear. In this study, we examined the functions and regulations of CD8(+) T cells and CD4(+)CD25(+)FoxP3(+) T cells from resected tumors from 12 stage I, 24 stage II, and 20 stage III untreated breast cancer patients. We found that tumor-infiltrating CD8(+) T cells from stage III patients were more refractory to T cell receptor (TCR) stimulation than those from stage I and stage II patients in terms of interferon gamma (IFN-γ) production and proliferation. On the other hand, tumor-infiltrating CD4(+)CD25(+)FoxP3(+) T cells had higher proliferation in stage III tumors than in stage I and stage II tumors. In addition, we found that tumor-infiltrating CD4(+)CD25(+) T cells can suppress CD8(+) T cell inflammation ex vivo. Altogether, our data demonstrated that stage III tumors in breast cancer patients had a more immunosuppressive microenvironment. PMID:25953262

  19. Transient exposure to proteins SOX2, Oct-4, and NANOG immortalizes exhausted tumor-infiltrating CTLs.

    PubMed

    Bhadurihauck, Anjuli; Li, Lei; Li, Qianqian; Wang, Jianjun; Xiao, Zhengguo

    2016-05-13

    Adoptive cell transfer therapy (ACT) is one of the most promising immunotherapies against cancer, using tumor-infiltrating lymphocytes (TILs) expanded in vitro. Tumor-infiltrating cytotoxic T lymphocytes (TICTLs) play a prominent role in cancer control. TILs terminally differentiate in response to immunosuppressive environments within tumors, and thus are slow to expand and challenging to maintain both in vitro and in patients. To reverse this exhaustion, we utilize a nuclear protein delivery system that exposes TICTLs to the SOX2, Oct-4, and NANOG (SON) proteins. Unlike activated naïve CTLs (effector CTLs), TICTLs respond favorably to SON treatment, exhibiting steady proliferation and extended survivability independent of cytokine and antigen stimulation. Though TICTLs treated with SON (STICTLs) still express T cell receptors as well as other critical downstream components, they are unresponsive to antigen challenge, suggesting that SON treatment regresses TICTLs into a state similar to that of an early double negative T cell. Our findings indicate the TICTL response to SON proteins is unique when compared to effector CTLs, suggesting TICTLs may be sensitive to regulation by other lineage-specific transcription factors and opening a promising new avenue into cancer immunotherapy. To our knowledge, this is the first report on lineage reprogramming of TILs using protein stem cell transcription factors delivered directly to the nucleus. PMID:27084449

  20. Different deoxyribonucleases in human lymphocytes

    PubMed Central

    Zöllner, E.Jürgen; Helm, Wolfgang; Zahn, Rudolf K.; Beck, Jörn; Reltz, Manfred

    1974-01-01

    The distribution pattern of deoxyribonuclease activities in human lymphocytes has been examined by micro-disc-electrophoresis. Four groups of deoxyribonuclease activities, differing in their electrophoretic mobility, in the nature of their optimal substrate and in their optimal incubation conditions, are characterized. There are two alkaline DNase-activities. One corresponds to DNase I (EC 3.1.4.5), the other having pH optimum of about pH 9.0, prefers denatured DNA as substrate and is not dependent on divalent cations. The fractions with an acid pH optimum can be subdivided into two groups, which differ in their activity towards native DNA, towards denatured DNA, in their activity when succinate is present and in their pH optimum. PMID:10793736

  1. Stimulation of human tonsillar lymphocytes in vitro

    PubMed Central

    Oettgen, H. F.; Silber, R.; Miescher, P. A.; Hirschhorn, K.

    1966-01-01

    We have studied the in vitro behaviour of cultured human tonsillar lymphocytes. In comparison with peripheral blood lymphocytes these cells show a higher degree of formation of large cells and mitoses in control cultures without any additive. They behave in a manner similar to peripheral blood lymphocytes when cultured with phytohaemagglutinin (PHA), streptolysin S (SLS) and specific antigens. The only exception is a lack of response to streptolysin O (SLO). PMID:5916348

  2. Biliary Ascariasis Mimicking Colonic Tumor Infiltration of the Biliary System.

    PubMed

    Sundriyal, Deepak; Mittal, Gyanendra; Kumar, Sushil; Manjunath, Suraj; Sharma, Navneet; Gupta, Mahesh

    2015-09-01

    Ascariasis is a common problem in developing countries with poor hygiene and sanitation. It is endemic in India and usually seen in the northern states. Biliary ascariasis is an uncommon cause of obstructive jaundice. We present a case of carcinoma of hepatic flexure of colon in which the patient developed biliary ascariasis and posed a diagnostic challenge as it mimicked tumor infiltration of the biliary system. PMID:27217679

  3. Transfection of Tumor-Infiltrating T Cells with mRNA Encoding CXCR2.

    PubMed

    Idorn, Manja; Thor Straten, Per; Svane, Inge Marie; Met, Özcan

    2016-01-01

    Adoptive T-cell therapy based on the infusion of patient's own immune cells after ex vivo culturing is among the most potent forms of personalized treatment among recent clinical developments for the treatment of cancer. However, despite high rates of successful initial clinical responses, only about 20 % of patients with metastatic melanoma treated with tumor-infiltrating lymphocytes (TILs) enter complete and long-term regression, with the majority either relapsing after initial partial regression or not benefiting at all. Previous studies have shown a positive correlation between the number infused T cells migrating to the tumor and the clinical response, but also that only a small fraction of adoptively transferred T cells reach the tumor site. In this chapter, we describe a protocol for transfection of TILs with mRNA encoding the chemokine receptor CXCR2 transiently redirecting and improving TILs migration toward tumor-secreted chemokines in vitro. PMID:27236805

  4. Tumor infiltration by Tbet+ effector T cells and CD20+ B cells is associated with survival in gastric cancer patients

    PubMed Central

    Hennequin, Audrey; Derangère, Valentin; Boidot, Romain; Apetoh, Lionel; Vincent, Julie; Orry, David; Fraisse, Jean; Causeret, Sylvain; Martin, François; Arnould, Laurent; Beltjens, Françoise; Ghiringhelli, François; Ladoire, Sylvain

    2016-01-01

    Tumor-infiltrating T and B lymphocytes could have the potential to affect cancer prognosis. The objective of this study was to investigate the prognostic significance of tumor infiltration by CD8 and CD4 T cells, and B lymphocytes in patients with localized gastric cancer. In a retrospective cohort of 82 patients with localized gastric cancer and treated by surgery we quantitatively assessed by immunohistochemistry on surgical specimen, immune infiltrates of IL-17+, CD8+, Foxp3+, Tbet+ T cells and CD20+ B cells both in the tumor core and at the invasive margin via immunohistochemical analyses of surgical specimens. We observed that CD8+ and IL17+ T-cell densities were not significantly associated with gastric cancer prognosis. In contrast, high infiltration of Tbet+ T cells, high numbers of CD20+ B-cell follicles, and low infiltration of Foxp3+ T cells, were associated with better relapse-free survival. Interestingly, treatment with neoadjuvant chemotherapy or histological tumor type (diffuse versus intestinal) did not influence type and density of immune infiltrates or their prognostic value. Immunohistochemical analysis of the gastric cancer stromal microenvironment revealed organized T and B cell aggregates, with strong structural analogies to normal secondary lymphoid organs and which could be considered as tertiary lymphoid structures. Using transcriptomic data from an independent cohort of 365 localized gastric cancer, we confirmed that a coordinated Th1, and B cell stromal gene signature is associated with better outcome. Altogether, these data suggest that tumor infiltration by B and Th1 T cells could affect gastric cancer prognosis and may be used to better define the outcome of patients with localized gastric cancer. PMID:27057426

  5. Early T cell signalling is reversibly altered in PD-1+ T lymphocytes infiltrating human tumors.

    PubMed

    Wang, Shu-Fang; Fouquet, Stéphane; Chapon, Maxime; Salmon, Hélène; Regnier, Fabienne; Labroquère, Karine; Badoual, Cécile; Damotte, Diane; Validire, Pierre; Maubec, Eve; Delongchamps, Nicolas B; Cazes, Aurélie; Gibault, Laure; Garcette, Marylène; Dieu-Nosjean, Marie-Caroline; Zerbib, Marc; Avril, Marie-Françoise; Prévost-Blondel, Armelle; Randriamampita, Clotilde; Trautmann, Alain; Bercovici, Nadège

    2011-01-01

    To improve cancer immunotherapy, a better understanding of the weak efficiency of tumor-infiltrating T lymphocytes (TIL) is necessary. We have analyzed the functional state of human TIL immediately after resection of three types of tumors (NSCLC, melanoma and RCC). Several signalling pathways (calcium, phosphorylation of ERK and Akt) and cytokine secretion are affected to different extents in TIL, and show a partial spontaneous recovery within a few hours in culture. The global result is an anergy that is quite distinct from clonal anergy induced in vitro, and closer to adaptive tolerance in mice. PD-1 (programmed death -1) is systematically expressed by TIL and may contribute to their anergy by its mere expression, and not only when it interacts with its ligands PD-L1 or PD-L2, which are not expressed by every tumor. Indeed, the TCR-induced calcium and ERK responses were reduced in peripheral blood T cells transfected with PD-1. Inhibition by sodium stibogluconate of the SHP-1 and SHP-2 phosphatases that associate with several inhibitory receptors including PD-1, relieves part of the anergy apparent in TIL or in PD-1-transfected T cells. This work highlights some of the molecular modifications contributing to functional defects of human TIL. PMID:21408177

  6. An infrared spectral signature of human lymphocyte subpopulations from peripheral blood.

    PubMed

    Wald, N; Legat, A; Meyer, C; Speiser, D E; Goormaghtigh, E

    2015-04-01

    Metastatic melanomas are frequently refractory to most adjuvant therapies such as chemotherapies and radiotherapies. Recently, immunotherapies have shown good results in the treatment of some metastatic melanomas. Immune cell infiltration in the tumor has been associated with successful immunotherapy. More generally, tumor infiltrating lymphocytes (TILs) in the primary tumor and in metastases of melanoma patients have been demonstrated to correlate positively with favorable clinical outcomes. Altogether, these findings suggest the importance of being able to identify, quantify and characterize immune infiltration at the tumor site for a better diagnostic and treatment choice. In this paper, we used Fourier Transform Infrared (FTIR) imaging to identify and quantify different subpopulations of T cells: the cytotoxic T cells (CD8+), the helper T cells (CD4+) and the regulatory T cells (T reg). As a proof of concept, we investigated pure populations isolated from human peripheral blood from 6 healthy donors. These subpopulations were isolated from blood samples by magnetic labeling and purities were assessed by Fluorescence Activated Cell Sorting (FACS). The results presented here show that Fourier Transform Infrared (FTIR) imaging followed by supervised Partial Least Square Discriminant Analysis (PLS-DA) allows an accurate identification of CD4+ T cells and CD8+ T cells (>86%). We then developed a PLS regression allowing the quantification of T reg in a different mix of immune cells (e.g. Peripheral Blood Mononuclear Cells (PBMCs)). Altogether, these results demonstrate the sensitivity of infrared imaging to detect the low biological variability observed in T cell subpopulations. PMID:25553786

  7. Methionine dependency of cultured human lymphocytes.

    PubMed

    Hall, C A; Begley, J A; Chu, R C

    1986-06-01

    Human peripheral blood lymphocytes stimulated with phytohemagglutinin and a lymphocyte model consisting of the RPMI 6410 cell, a human virus-transformed B cell, required added methionine (Met) for growth of the cultures. This failure to meet all needs for Met via endogenous synthesis, which is characteristic of oncogenic transformation, occurred even in the presence of adequate homocysteine, methylfolate (5-CH3-H4PteGlu) and cobalamin (Cbl)-dependent methionine synthetase activity. Folinic acid (5-CHO-H4PteGlu), which provides available folate independently of Cbl, improved growth only slightly in the absence of Met. Free Cbl at 222 nM, an amount great enough to alter other intracellular events, failed to increase growth in the absence of Met, but 0.22 nM Cbl bound to transcobalamin II did, however, enhance growth. PMID:3703873

  8. Reprogramming tumor-infiltrating dendritic cells for CD103+CD8+ mucosal T cell differentiation and breast cancer rejection

    PubMed Central

    Wu, Te-Chia; Xu, Kangling; Banchereau, Romain; Marches, Florentina; Yu, Chun I; Martinek, Jan; Anguiano, Esperanza; Pedroza-Gonzalez, Alexander; Snipes, G. Jackson; O’Shaughnessy, Joyce; Nishimura, Stephen; Liu, Yong-Jun; Pascual, Virginia; Banchereau, Jacques; Oh, Sangkon; Palucka, Karolina

    2014-01-01

    Our studies showed that tumor-infiltrating dendritic cells (DC) in breast cancer drive inflammatory T helper 2 (iTh2) cells and protumor inflammation. Here we show that intratumoral delivery of the β-glucan curdlan, a ligand of dectin-1, blocks the generation of iTh2 cells, and prevents breast cancer progression in vivo. Curdlan reprograms tumor-infiltrating DC via the ligation of dectin-1, enabling the DC to become resistant to cancer-derived thymic stromal lymphopoietin (TSLP), to produce IL12p70, and to favor the generation of T helper 1 (Th1) cells. DC activated via dectin-1, but not those activated with TLR-7/8 ligand or poly IC, induce CD8+ T cells to express CD103 (αE integrin), a ligand for cancer cells E-cadherin. Generation of these mucosal CD8+ T cells is regulated by DC-derived integrin αvβ8 and TGF-β activation in a dectin-1-dependent fashion. These CD103+CD8+ mucosal T cells accumulate in the tumors thereby increasing cancer necrosis and inhibiting cancer progression in vivo in a humanized mouse model of breast cancer. Importantly, CD103+CD8+ mucosal T cells elicited by reprogrammed DC can reject established cancer. Thus, reprogramming tumor-infiltrating DC represents a new strategy for cancer rejection. PMID:24795361

  9. Shaping the repertoire of tumor-infiltrating effector and regulatory T cells

    PubMed Central

    Savage, Peter A.; Leventhal, Daniel S.; Malchow, Sven

    2014-01-01

    Summary Many tumors express antigens that can be specifically or selectively recognized by T lymphocytes, suggesting that T cell-mediated immunity may be harnessed for the immunotherapy of cancer. However, since tumors originate from normal cells and evolve within the context of self tissues, the immune mechanisms that prevent the autoimmune attack of normal tissues function in parallel to restrict anti-tumor immunity. In particular, the purging of autoreactive T cells and the development of immune-suppressive regulatory T cells (Tregs) are thought to be major barriers impeding anti-tumor immune responses. Here, we discuss current understanding regarding the antigens recognized by tumor-infiltrating T cell populations, the mechanisms that shape the repertoire of these cells, and the role of the transcription factor autoimmune regulator (Aire) in these processes. Further elucidation of these principles is likely to be critical for optimizing emerging cancer immunotherapies, and for the rational design of novel therapies exhibiting robust anti-tumor activity with limited toxicity. PMID:24712470

  10. A Distinct Gene Module for Dysfunction Uncoupled from Activation in Tumor-Infiltrating T Cells.

    PubMed

    Singer, Meromit; Wang, Chao; Cong, Le; Marjanovic, Nemanja D; Kowalczyk, Monika S; Zhang, Huiyuan; Nyman, Jackson; Sakuishi, Kaori; Kurtulus, Sema; Gennert, David; Xia, Junrong; Kwon, John Y H; Nevin, James; Herbst, Rebecca H; Yanai, Itai; Rozenblatt-Rosen, Orit; Kuchroo, Vijay K; Regev, Aviv; Anderson, Ana C

    2016-09-01

    Reversing the dysfunctional T cell state that arises in cancer and chronic viral infections is the focus of therapeutic interventions; however, current therapies are effective in only some patients and some tumor types. To gain a deeper molecular understanding of the dysfunctional T cell state, we analyzed population and single-cell RNA profiles of CD8(+) tumor-infiltrating lymphocytes (TILs) and used genetic perturbations to identify a distinct gene module for T cell dysfunction that can be uncoupled from T cell activation. This distinct dysfunction module is downstream of intracellular metallothioneins that regulate zinc metabolism and can be identified at single-cell resolution. We further identify Gata-3, a zinc-finger transcription factor in the dysfunctional module, as a regulator of dysfunction, and we use CRISPR-Cas9 genome editing to show that it drives a dysfunctional phenotype in CD8(+) TILs. Our results open novel avenues for targeting dysfunctional T cell states while leaving activation programs intact. PMID:27610572

  11. Aryl hydrocarbon mono-oxygenase activity in human lymphocytes

    SciTech Connect

    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.

  12. OX40, PD-1 and CTLA-4 are selectively expressed on tumor-infiltrating T cells in head and neck cancer

    PubMed Central

    Montler, Ryan; Bell, R Bryan; Thalhofer, Colin; Leidner, Rom; Feng, Zipei; Fox, Bernard A; Cheng, Allen C; Bui, Tuan G; Tucker, Christopher; Hoen, Helena; Weinberg, Andrew

    2016-01-01

    The tumor microenvironment of squamous cell carcinoma of the head and neck (SCCHN) has been shown to be immune suppressive. Therefore, strategies aimed at overcoming this issue could have a positive therapeutic impact. Hence, we investigated the expression of the known immune-modulatory proteins OX40, programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) in SCCHN on different T-cell subsets of tumor-infiltrating lymphocytes (TIL) to ascertain whether these proteins could potentially be targeted alone or in combination for future clinical trials. T cells from peripheral blood (PBL) and tumor were analyzed for the expression of OX40, PD-1 and CTLA-4 in 29 patients undergoing surgery. These proteins were all expressed significantly higher in T-cell subsets isolated from tumors compared with PBL of the same patient. OX40 expression was significantly greater in the TIL regulatory T-cell (Treg) population relative to conventional CD4 and CD8 TIL or the Treg isolated from PBL. PD-1 expression was increased in all T-cell subsets relative to PBL. CTLA-4 was also increased in all TIL subsets relative to blood, and similar to OX40, its highest level of expression was observed in the Treg TIL. The highest frequency of PD-1, CTLA-4 and OX40 triple-positive cells were found in the Treg population isolated from the tumor. We analyzed both human papilloma virus-positive and -negative patients and found similar levels and expression patterns of these two patient populations for all three proteins. These data suggest that there may be therapeutic advantages of targeting these pathways independently or in combination for patients with this disease. PMID:27195113

  13. Analysis of tumor-infiltrating gamma delta T cells in rectal cancer

    PubMed Central

    Rong, Liang; Li, Ke; Li, Rui; Liu, Hui-Min; Sun, Rui; Liu, Xiao-Yan

    2016-01-01

    AIM: To investigate the regulatory effect of Vδ1 T cells and the antitumor activity of Vδ2 T cells in rectal cancer. METHODS: Peripheral blood, tumor tissues and para-carcinoma tissues from 20 rectal cancer patients were collected. Naïve CD4 T cells from the peripheral blood of rectal cancer patients were purified by negative selection using a Naive CD4+ T Cell Isolation Kit II (Miltenyi Biotec). Tumor tissues and para-carcinoma tissues were minced into small pieces and digested in a triple enzyme mixture containing collagenase type IV, hyaluronidase, and deoxyribonuclease for 2 h at room temperature. After digestion, the cells were washed twice in RPMI1640 and cultured in RPMI1640 containing 10% human serum supplemented with L-glutamine and 2-mercaptoethanol and 1000 U/mL of IL-2 for the generation of T cells. Vδ1 T cells and Vδ2 T cells from tumor tissues and para-carcinoma tissues were expanded by anti-TCR γδ antibodies. The inhibitory effects of Vδ1 T cells on naïve CD4 T cells were analyzed using the CFSE method. The cytotoxicity of Vδ2 T cells on rectal cancer lines was determined by the LDH method. RESULTS: The percentage of Vδ1 T cells in rectal tumor tissues from rectal cancer patients was significantly increased, and positively correlated with the T stage. The percentage of Vδ2 T cells in rectal tumor tissues from rectal cancer patients was significantly decreased, and negatively correlated with the T stage. After culture for 14 d with 1 μg/mL anti-TCR γδ antibodies, the percentage of Vδ1 T cells from para-carcinoma tissues was 21.45% ± 4.64%, and the percentage of Vδ2 T cells was 38.64% ± 8.05%. After culture for 14 d, the percentage of Vδ1 T cells from rectal cancer tissues was 67.45% ± 11.75% and the percentage of Vδ2 T cells was 8.94% ± 2.85%. Tumor-infiltrating Vδ1 T cells had strong inhibitory effects, and tumor-infiltrating Vδ2 T cells showed strong cytolytic activity. The inhibitory effects of Vδ1 T cells from para

  14. In vitro effects of flunarizine on human lymphocytes.

    PubMed

    Brohée, D; Piro, P; Kennes, B; Nève, P

    1986-01-01

    Flunarizine, a slow-channel calcium entry blocker used as a vasodilator, interferes in vitro with human lymphocyte functions. It prevents lymphocytes from capping sheep erythrocytes, an effect which is probably due to the disconnection of the membrane from its cytoskeletal control. Flunarizine antagonizes both colchicine and cytochalasin B effects upon capping. Although the mitogen-induced lymphocyte stimulation has been shown to be sensitive to calcium depletion or calcium entry blocking by Verapamil, an enhanced response to phytohaemagglutinin A was observed with flunarizine. This suggests a differential sensitivity of the lymphocytes to calcium-entry blockers. PMID:3731875

  15. Mechanisms of inhibition of Cryptococcus neoformans by human lymphocytes.

    PubMed Central

    Levitz, S M; North, E A; Dupont, M P; Harrison, T S

    1995-01-01

    Recently, our laboratory and others have demonstrated that human peripheral blood T and NK lymphocytes directly inhibit the growth of Cryptococcus neoformans. In this study, we further define the conditions under which lymphocyte-mediated fungistasis against C. neoformans occurs and examine whether mechanisms implicated in lymphocyte-mediated activities against other target cells are also involved in anticryptococcal activity. The addition of whole or broken heat-killed C. neoformans modestly inhibited lymphocyte-mediated fungistasis, whereas other particulates had no effect. The hydroxyl radical scavenger catechin, but not diethyl urea or propyl gallate, profoundly inhibited fungistasis. Salicylic acid inhibited fungistasis in a dose-dependent fashion. However, two other cyclooxygenase inhibitors, piroxicam and indomethacin, had no effect, suggesting that the mechanism of inhibition by salicylic acid was cyclooxygenase independent. Reagent prostaglandin E2, at concentrations shown by others to inhibit NK cell-mediated bactericidal and tumorlytic activities, had no effect on lymphocyte-mediated fungistasis. The addition of selected monoclonal antibodies or ligands reactive with receptors on human lymphocytes had no significant effect on lymphocyte-mediated fungistasis. Acapsular, small-capsuled, and large-capsuled C. neoformans organisms were inhibited by lymphocytes to an approximately equal extent. These data demonstrate that lymphocyte-mediated activity against C. neoformans proceeds regardless of the presence of capsule and by mechanisms at least in part dissimilar from those seen with other target cells. PMID:7642290

  16. Clinical significance of tumor-infiltrating immune cells focusing on BTLA and Cbl-b in patients with gallbladder cancer.

    PubMed

    Oguro, Seiji; Ino, Yoshinori; Shimada, Kazuaki; Hatanaka, Yutaka; Matsuno, Yoshihiro; Esaki, Minoru; Nara, Satoshi; Kishi, Yoji; Kosuge, Tomoo; Hiraoka, Nobuyoshi

    2015-12-01

    The host immune system plays a significant role in tumor control, although most cancers escape immune surveillance through a variety of mechanisms. The aim of the present study was to evaluate the clinicopathological significance of a novel co-inhibitory receptor, B and T lymphocyte attenuator (BTLA), the anergy cell marker Casitas-B-lineage lymphoma protein-b (Cbl-b), and clinical implications of tumor-infiltrating immune cells in gallbladder cancer (GBC) tissues. We investigated 211 cases of GBC, 21 cases of chronic cholecystitis (CC), and 11 cases of xanthogranulomatous cholecystitis (XGC) using immunohistochemistry to detect tissue-infiltrating immune cells and their expression of BTLA and Cbl-b, and carried out correlation and survival analyses. The density of infiltrating T cells was significantly higher in CC and XGC than in GBC. The density ratio of BTLA(+) cells to CD8(+) T cells (BTLA/CD8) and that of Cbl-b(+) cells to CD8(+) T cells (Cbl-b/CD8) were significantly higher in GBC than in CC and XGC. The FOXP3/CD4, BTLA/CD8, and Cbl-b/CD8 ratios were significantly correlated with each other, and also with malignant phenotypes. Survival analyses revealed that a lower density of tumor-infiltrating CD8(+) cells, and higher Foxp3/CD4, BTLA/CD8, and Cbl-b/CD8 ratios were significantly associated with shorter overall survival and disease-free survival in GBC patients. Multivariate analyses showed that M factor, perineural invasion, BTLA/CD8, and Cbl-b/CD8 were closely associated with shorter overall survival. These findings suggest that higher ratios of BTLA/CD8 and Cbl-b/CD8 are independent indicators of unfavorable outcome in GBC patients, and that upregulation of BTLA in cancer tissues is involved in inhibition of antitumor immunity. PMID:26395180

  17. A simple and rapid protocol to non-enzymatically dissociate fresh human tissues for the analysis of infiltrating lymphocytes.

    PubMed

    Garaud, Soizic; Gu-Trantien, Chunyan; Lodewyckx, Jean-Nicolas; Boisson, Anaïs; De Silva, Pushpamali; Buisseret, Laurence; Migliori, Edoardo; Libin, Myriam; Naveaux, Céline; Duvillier, Hugues; Willard-Gallo, Karen

    2014-01-01

    The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45(+) cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45(+) cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues. PMID:25548995

  18. Tempol protects human lymphocytes from genotoxicity induced by cisplatin.

    PubMed

    Khabour, Omar F; Alzoubi, Karem H; Mfady, Doa'a S; Alasseiri, Mohammed; Hasheesh, Taghrid F

    2014-01-01

    The use of cisplatin in treatments of human malignancies is limited by its side effects that include DNA damage and the subsequent risk of developing secondary cancer. In this study, we examined the possible protective effect of Tempol against DNA damage induced by cisplatin in human lymphocytes using chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs) assays. Cisplatin induced significant elevation in the frequencies of CAs and SCEs in cultured human lymphocytes (P < 0.01). Treatment of lymphocytes with Tempol significantly lowered CAs and SCEs induced by cisplatin. Tempol alone did not affect spontaneous levels of SCEs and CAs observed in the control group (P > 0.05). In conclusion, Tempol protects human lymphocytes against genotoxicity induced by the anticancer drug cisplatin. PMID:24955171

  19. Myeloperoxidase in human peripheral blood lymphocytes: Production and subcellular localization.

    PubMed

    Okada, Sabrina Sayori; de Oliveira, Edson Mendes; de Araújo, Tomaz Henrique; Rodrigues, Maria Rita; Albuquerque, Renata Chaves; Mortara, Renato Arruda; Taniwaki, Noemi Nosomi; Nakaya, Helder Imoto; Campa, Ana; Moreno, Ana Carolina Ramos

    2016-02-01

    Myeloperoxidase (MPO) is an important enzyme in the front-line protection against microorganisms. In peripheral blood, it is accepted that MPO is only produced by myeloid-lineage cells. Thus, MPO presence is unexpected in lymphocytes. We showed recently that B1-lymphocytes from mice have MPO. Here, we showed that subsets of human peripheral B, CD4(+) and CD8(+) T lymphocytes express MPO. The content of MPO in lymphocytes was very low compared to neutrophils/monocytes with a preferential distribution in the nucleus and perinuclear region. Also, we performed a MPO mRNA expression analysis from human blood cells derived from microarray raw data publicly available, showing that MPO is modulated in infectious disease. MPO was increased in CD4(+) T lymphocytes from HIV chronic infection and in CD8(+) T lymphocytes from HCV-positive patients. Our study points out MPO as a multifunctional protein due to its subcellular localization and expression modulation in lymphocytes indicating alternative unknown functions for MPO in lymphocytes. PMID:26632272

  20. Mitogenic effect of Parkia speciosa seed lectin on human lymphocytes.

    PubMed

    Suvachittanont, W; Jaranchavanapet, P

    2000-12-01

    Mitogenic activity of a lectin, purified from Parkia speciosa seeds, on the isolated peripheral blood lymphocytes taken from normal blood donors and patients with esophageal carcinoma was examined using [3H]thymidine incorporation. The lectin increases the incorporation of [3H]thymidine into DNA of human lymphocytes. The activity of the lectin increased as its concentration was increased and then declined once the concentration passed an optimum point. The stimulant effect was also expressed using a proliferation index (PI): the ratio of [3H]thymidine incorporated into lymphocytes in the presence and absence of the lectin. The mitogenic activity of the lectin is comparable to those of the known T-cell mitogens, such as concanavalin A, phytohaemagglutinin, and pokeweed mitogen. Only slightly less responsiveness was observed in the case of lymphocytes from esophageal cancer compared to lymphocytes from normal donors. PMID:11199124

  1. Adenosine metabolism in phytohemagglutinin-stimulated human lymphocytes.

    PubMed Central

    Snyder, F F; Mendelsohn, J; Seegmiller, J E

    1976-01-01

    The association of a human genetic deficiency of adenosine deaminase activity with combined immunodeficiency prompted a study of the effects of adenosine and of inhibition of adenosine deaminase activity on human lymphocyte transformation and a detailed study of adenosine metabolism throughout phytohemagglutinin-induced blastogenesis. The adenosine deaminase inhibitor, coformycin, at a concentration that inhibited adenosine deaminase activity more than 95%, or 50 muM adenosine, did not prevent blastogenesis by criteria of morphology or thymidine incorporation into acid-precipitable material. The combination of coformycin and adenosine, however, substantially reduced both the viable cell count and the incorporation of thymidine into DNA in phytohemagglutinin-stimulated lymphocytes. Incubation of lymphocytes with phytohemagglutinin for 72 h produced a 12-fold increase in the rate of deamination and a 6-fold increase in phosphorylation of adenosine by intact lymphocytes. There was no change in the apparent affinity for adenosine with either deamination or phosphorylation. The increased rates of metabolism, apparent as early as 3 h after addition of mitogen, may be due to increased entry of the nucleoside into stimulated lymphocytes. Increased adenosine metabolism was not due to changes in total enzyme activity; after 72 h in culture, the ratios of specific activities in extracts of stimulated to unstimulated lymphocytes were essentially unchanged for adenosine kinase, 0.92, and decreased for adenosine deaminase, 0.44. As much as 38% of the initial lymphocyte adenosine deaminase activity accumulated extracellularly after a 72-h culture with phytohemagglutinin. In phytohemagglutinin-stimulated lymphocytes, the principal route of adenosine metabolism was phosphorylation at less than 5 muM adenosine, and deamination at concentrations greater than 5 muM. In unstimulated lymphocytes, deamination was the principal route of adenosine metabolism over the range of adenosine

  2. Dopamine D2 receptor agonists inhibit lung cancer progression by reducing angiogenesis and tumor infiltrating myeloid derived suppressor cells.

    PubMed

    Hoeppner, Luke H; Wang, Ying; Sharma, Anil; Javeed, Naureen; Van Keulen, Virginia P; Wang, Enfeng; Yang, Ping; Roden, Anja C; Peikert, Tobias; Molina, Julian R; Mukhopadhyay, Debabrata

    2015-01-01

    We sought to determine whether Dopamine D2 Receptor (D2R) agonists inhibit lung tumor progression and identify subpopulations of lung cancer patients that benefit most from D2R agonist therapy. We demonstrate D2R agonists abrogate lung tumor progression in syngeneic (LLC1) and human xenograft (A549) orthotopic murine models through inhibition of tumor angiogenesis and reduction of tumor infiltrating myeloid derived suppressor cells. Pathological examination of human lung cancer tissue revealed a positive correlation between endothelial D2R expression and tumor stage. Lung cancer patients with a smoking history exhibited greater levels of D2R in lung endothelium. Our results suggest D2R agonists may represent a promising individualized therapy for lung cancer patients with high levels of endothelial D2R expression and a smoking history. PMID:25226814

  3. Dopamine D2 Receptor Agonists Inhibit Lung Cancer Progression by Reducing Angiogenesis and Tumor Infiltrating Myeloid Derived Suppressor Cells

    PubMed Central

    Hoeppner, Luke H.; Wang, Ying; Sharma, Anil; Javeed, Naureen; Van Keulen, Virginia P.; Wang, Enfeng; Yang, Ping; Roden, Anja C.; Peikert, Tobias; Molina, Julian R.; Mukhopadhyay, Debabrata

    2014-01-01

    We sought to determine whether Dopamine D2 Receptor (D2R) agonists inhibit lung tumor progression and identify subpopulations of lung cancer patients that benefit most from D2R agonist therapy. We demonstrate D2R agonists abrogate lung tumor progression in syngeneic (LLC1) and human xenograft (A549) orthotopic murine models through inhibition of tumor angiogenesis and reduction of tumor infiltrating myeloid derived suppressor cells. Pathological examination of human lung cancer tissue revealed a positive correlation between endothelial D2R expression and tumor stage. Lung cancer patients with a smoking history exhibited greater levels of D2R in lung endothelium. Our results suggest D2R agonists may represent a promising individualized therapy for lung cancer patients with high levels of endothelial D2R expression and a smoking history. PMID:25226814

  4. Activation of human lymphocytes by supernatants from human thymic epithelium.

    PubMed Central

    Goust, J M; Vesole, D H; Fudenberg, H H

    1979-01-01

    Supernatants from human thymic epithelial cells (TS) were found to have a mitogenic effect on cultured human peripheral blood mononuclear cells and to potentiate their responses to lectins. This was not observed with culture supernatants from the human cell lines AV-3 and HeLa or from the murine cell line L-929. The maximum potentiating effects were observed with pokeweed mitogen (PWM) and phytohaemagglutinin (PHA), whereas the response to concanavalin A (Con A) was only slightly enhanced. TS also potentiated the mixed lymphocyte culture (MLC) response of normal T cells and thymocytes cultured with mitomycin C-treated B lymphoid cell lines. The mitogenic effect of TS was time-dependent and paralleled the appearance of lymphoid colonies in semi-solid agar. Chromatographical separation of concentrated serum-free TS on Sephadex G-100 yielded an active fraction of molecular weight 15,000--25,000 which had all the activities of unseparated TS. PMID:160851

  5. Activation of human lymphocytes by supernatants from human thymic epithelium.

    PubMed

    Goust, J M; Vesole, D H; Fudenberg, H H

    1979-11-01

    Supernatants from human thymic epithelial cells (TS) were found to have a mitogenic effect on cultured human peripheral blood mononuclear cells and to potentiate their responses to lectins. This was not observed with culture supernatants from the human cell lines AV-3 and HeLa or from the murine cell line L-929. The maximum potentiating effects were observed with pokeweed mitogen (PWM) and phytohaemagglutinin (PHA), whereas the response to concanavalin A (Con A) was only slightly enhanced. TS also potentiated the mixed lymphocyte culture (MLC) response of normal T cells and thymocytes cultured with mitomycin C-treated B lymphoid cell lines. The mitogenic effect of TS was time-dependent and paralleled the appearance of lymphoid colonies in semi-solid agar. Chromatographical separation of concentrated serum-free TS on Sephadex G-100 yielded an active fraction of molecular weight 15,000--25,000 which had all the activities of unseparated TS. PMID:160851

  6. PD-L1 and Tumor Infiltrating Lymphocytes as Prognostic Markers in Resected NSCLC

    PubMed Central

    Ameratunga, Malaka; Asadi, Khashayar; Lin, Xihui; Walkiewicz, Marzena; Murone, Carmel; Knight, Simon; Mitchell, Paul; Boutros, Paul; John, Thomas

    2016-01-01

    Introduction Immune checkpoint inhibition has shifted treatment paradigms in non-small cell lung cancer (NSCLC). Conflicting results have been reported regarding the immune infiltrate and programmed death-ligand 1 (PD-L1) as a prognostic marker. We correlated the immune infiltrate and PD-L1 expression with clinicopathologic characteristics in a cohort of resected NSCLC. Methods A tissue microarray was constructed using triplicate cores from consecutive resected NSCLC. Immunohistochemistry was performed for CD8, FOXP3 and PD-L1. Strong PD-L1 expression was predefined as greater than 50% tumor cell positivity. Matched nodal samples were assessed for concordance of PD-L1 expression. Results Of 522 patients, 346 were node-negative (N0), 72 N1 and 109 N2; 265 were adenocarcinomas (AC), 182 squamous cell cancers (SCC) and 75 other. Strong PD-L1 expression was found in 24% cases. In the overall cohort, PD-L1 expression was not associated with survival. In patients with N2 disease, strong PD-L1 expression was associated with significantly improved disease-free (DFS) and overall survival (OS) in multivariate analysis (HR 0.49, 95%CI 0.36–0.94, p = 0.031; HR 0.46, 95%CI 0.26–0.80, p = 0.006). In this resected cohort only 5% harboured EGFR mutations, whereas 19% harboured KRAS and 23% other. KRAS mutated tumors were more likely to highly express PD-L1 compared to EGFR (22% vs 3%). A stromal CD8 infiltrate was associated with significantly improved DFS in SCC (HR 0.70, 95%CI 0.50–0.97, p = 0.034), but not AC, whereas FOXP3 was not prognostic. Matched nodal specimens (N = 53) were highly concordant for PD-L1 expression (89%). Conclusion PD-L1 expression was not prognostic in the overall cohort. PD-L1 expression in primary tumor and matched nodal specimens were highly concordant. The observed survival benefit in N2 disease requires confirmation. PMID:27104612

  7. Interaction of nanosilver particles with human lymphocyte cells

    NASA Astrophysics Data System (ADS)

    Zhornik, Alena; Baranova, Ludmila; Volotovski, Igor; Chizhik, Sergey; Drozd, Elizaveta; Sudas, Margarita; Buu Ngo, Quoc; Chau Nguyen, Hoai; Huynh, Thi Ha; Hien Dao, Trong

    2015-01-01

    The damaging effects of nanoparticles were hypothesized to be the oxidative stress caused by the formation of reactive oxygen species and initiation of inflammatory reactions. In this context a study on the effects of nanosilver particles on the formation of reactive oxygen species in human lymphocyte culture was carried out. The obtained results showed that fluorescence intensity considerably increased after cells had interacted with nanosilver particles of varying concentrations, indicating the formation of reactive oxygen species and their accumulation in lymphocyte cells. Morphological study of the lymphocyte cells under the effects of nanosilver particles showed that the change in morphology depends on the concentration and size of nanosilver particles: for a size ≤20 nm the lymphocyte cell significantly shrank with pronounced differences in the morphological structure of the cell membrane, but for a size ≥200 nm no change was observed.

  8. Tumor-Infiltrating Immune Cells Promoting Tumor Invasion and Metastasis: Existing Theories

    PubMed Central

    Man, Yan-gao; Stojadinovic, Alexander; Mason, Jeffrey; Avital, Itzhak; Bilchik, Anton; Bruecher, Bjoern; Protic, Mladjan; Nissan, Aviram; Izadjoo, Mina; Zhang, Xichen; Jewett, Anahid

    2013-01-01

    It is a commonly held belief that infiltration of immune cells into tumor tissues and direct physical contact between tumor cells and infiltrated immune cells is associated with physical destructions of the tumor cells, reduction of the tumor burden, and improved clinical prognosis. An increasing number of studies, however, have suggested that aberrant infiltration of immune cells into tumor or normal tissues may promote tumor progression, invasion, and metastasis. Neither the primary reason for these contradictory observations, nor the mechanism for the reported diverse impact of tumor-infiltrating immune cells has been elucidated, making it difficult to judge the clinical implications of infiltration of immune cells within tumor tissues. This mini-review presents several existing hypotheses and models that favor the promoting impact of tumor-infiltrating immune cells on tumor invasion and metastasis, and also analyzes their strength and weakness. PMID:23386907

  9. Effect of controlled ozone exposure on human lymphocyte function

    SciTech Connect

    Peterson, M.L.; Smialowicz, R.; Harder, S.; Ketcham, B.; House, D.

    1981-04-01

    The effects of ozone (O/sub 3/) on cell-mediated immunity were studied in 16 human subjects exposed to 1176 ..mu..g/m/sup 3/ O/sub 3/ (0.6 ppM) for 2 h in an environmentally controlled exposure chamber. Venous blood samples were taken before and immediately after controlled air and O/sub 3/ exposures, as well as at 72 h, 2 and 4 weeks, and at one random time at least 1 month after treatment. The relative frequency of T lymphocytes in blood and the in vitro blastogenic response of lymphocytes to phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM), and Candida albicans were determined. During the course of the experiment, no statistically significant changes were observed in the number of T lymphocytes that form spontaneous rosettes with sheep erythrocytes. The response of T lymphocytes to PHA was significantly reduced (P < 0.05) in samples taken at 2 and 4 weeks, following O/sub 3/ exposure. Normal response to PHA was observed at 2 months post-O/sub 3/ exposure. No statistically significant changes in lymphocyte responses to Con A, PWM, or Candida were seen. These results show that one 2 h exposure of humans to 0.6 ppM O/sub 3/ may lead to a transient suppression of the PHA-stimulated blastogenic transformation of peripheral blood lymphocytes. The data indicate that the blastogenic response to PHA of human lymphocytes is exquisitely sensitive to O/sub 3/ exposure and could serve as a bioassay for evaluating subtle changes in cellular immunity induced by O/sub 3/ and possibly other pollutants.

  10. Effects of Beryllium on Human Serum Immunoglobulin and Lymphocyte Subpopulation

    PubMed Central

    Kim, DaeSeong; Won, Yong Lim; Kang, Seong-Kyu

    2013-01-01

    To investigate the effects of short-term exposure of beryllium on the human immune system, the proportion of T-lymphocytes such as CD3+, CD4+, CD8+, CD95, and NK cells, andthe proportion of B cells and TNFα level in peripheral blood and immunoglobulins in the serum of 43 exposed workers and 34 healthy control subjects were studied. External exposure to beryllium was measured by atomic absorption spectrometer as recommended by the NIOSH analytical method 7300. T lymphocyte subpopulation analysis was carried out with flow cytometer. The working duration of exposed workers was less than 3 months and the mean ambient beryllium level was 3.4 μg/m3, 112.3 μg/m3, and 2.3 μg/m3 in molding (furnace), deforming (grinding), and sorting processes, respectively (cited from Kim et al., 2008). However, ambient beryllium level after process change was non-detectable (< 0.1 μg/m3). The number of T lymphocytes and the amount of immunoglobulins in the beryllium-exposed workers and control subjects were not significantly different, except for the total number of lymphocytes and CD95 (APO1/FAS). The total number of lymphocytes was higher in the beryllium-exposed individuals than in the healthy control subjects. Multiple logistic regression analysis showed lymphocytes to be affected by beryllium exposure (odd ratio = 7.293; p < 0.001). These results show that short-term exposure to beryllium does not induce immune dysfunction but is probably associated with lymphocytes proliferation. PMID:24278637

  11. Rabies virus infects mouse and human lymphocytes and induces apoptosis.

    PubMed Central

    Thoulouze, M I; Lafage, M; Montano-Hirose, J A; Lafon, M

    1997-01-01

    Attenuated and highly neurovirulent rabies virus strains have distinct cellular tropisms. Highly neurovirulent strains such as the challenge virus standard (CVS) are highly neurotropic, whereas the attenuated strain ERA also infects nonneuronal cells. We report that both rabies virus strains infect activated murine lymphocytes and the human lymphoblastoid Jurkat T-cell line in vitro. The lymphocytes are more permissive to the attenuated ERA rabies virus strain than to the CVS strain in both cases. We also report that in contrast to that of the CVS strain, ERA viral replication induces apoptosis of infected Jurkat T cells, and cell death is concomitant with viral glycoprotein expression, suggesting that this protein has a role in the induction of apoptosis. Our data indicate that (i) rabies virus infects lymphocytes, (ii) lymphocyte infection with the attenuated rabies virus strain causes apoptosis, and (iii) apoptosis does not hinder rabies virus production. In contrast to CVS, ERA rabies virus and other attenuated rabies virus vaccines stimulate a strong immune response and are efficient live vaccines. The paradoxical finding that a rabies virus triggers a strong immune response despite the fact that it infects lymphocytes and induces apoptosis is discussed in terms of the function of apoptosis in the immune response. PMID:9311815

  12. Influence of ethanol on human T-lymphocyte migration

    SciTech Connect

    Kaelin, R.M.; Semerjian, A.; Center, D.M.; Bernardo, J.

    1984-11-01

    Because ethanol consumption is associated with increased susceptibility to infection, an examination was made of the effects of ethanol and its metabolite acetaldehyde on human T-lymphocyte migration, an important functional component of cellular inflammatory responses. With a modified Boyden chamber system, ethanol at 0.25% and 0.50% (vol/vol) inhibited spontaneous motility of human T-lymphocytes, in a noncytotoxic manner, to 65% +/- 7% (mean +/- SEM) and 62% +/- 7% of control values of migration, respectively. When T-lymphocyte migration was stimulated by colchicine (10/sup -5/ mol/L), incubation with ethanol (0.25% and 0.50%, vol/vol) decreased migration to 80% +/- 4% and 66% +/- 8% of control values, respectively. Similar degrees of inhibition of migration were obtained with acetaldehyde at concentrations five to 10 times less than ethanol. Ethanol was similarly capable of inhibiting T cell migration induced by dibutyryl cyclic guanosine monophosphate, but it had no effect on stimulated migration induced by a human chemokinetic lymphokine. The study demonstrates that ethanol, at concentrations achievable in vivo, is capable of depressing T-lymphocyte migration. This effect might contribute to the immunosuppression associated with ethanol consumption. 36 references, 4 figures.

  13. Immunomodulating activity of seaweed extract on human lymphocytes in vitro.

    PubMed

    Shan, B E; Yoshida, Y; Kuroda, E; Yamashita, U

    1999-01-01

    Effect of eight kinds of seaweed extract (SWE) on human lymphocytes was studied in vitro. The extracts of Hizikiafusiformis and Meristotheca papulosa (green) markedly stimulated human lymphocytes to proliferate, whereas Eucheuma muricatum and Meristotheca papulosa (red) weakly stimulated proliferation. The responder cells are T cells, because T cells purified by sheep red blood cell (SRBC) rosette-formation were significantly stimulated with SWE, but B cells were not. These extracts enhanced the induction of cytotoxic T lymphocyte (CTL) activity, but failed to enhance natural killer (NK) cell activity. These extracts had a stimulatory effect on immunoglobulin (Ig) production by B cells and tumor necrosis factor (TNF) production by monocytes. The activity of Hizikia fusiformis associated with polysaccharides which were extracted with ethanol and purified by ion-exchange and gel filtration chromatography, whose molecular weight was about 100 kDa. These results suggest that SWE has an immunomodulating activity on human lymphocytes and this ability might be useful for clinical application to treat several diseases such as tumors. PMID:10411282

  14. Purine nucleoside modulation of functions of human lymphocytes.

    PubMed

    Priebe, T; Platsoucas, C D; Seki, H; Fox, F E; Nelson, J A

    1990-09-01

    The accumulation of endogenous substrates in patients with adenosine deaminase deficiency or purine nucleoside phosphorylase deficiency is believed to be responsible for the immunodeficiency observed in these patients. To identify the lymphocyte populations that are most susceptible to these substrates, we investigated the effect of their nucleoside analogs on a number of T and B cell functions of human lymphocytes. We found that tubercidin (Tub), 2-chloro 2'deoxyadenosine (2CldA), 2-fluoro adenine arabinoside-5'phosphate (FaraAMP), and 9-beta-D-arabinosyl guanine (AraGua) inhibited the proliferative responses of human peripheral blood mononuclear cells (PBMC) to polyclonal activators (PHA, OKT3 mab) or to allogeneic PBMC in mixed lymphocyte cultures (MLC). Addition of recombinant IL-2 from the beginning of the culture did not alter the inhibition by Tub of the proliferative responses of PBMC. These purine nucleoside analogs also inhibited the proliferative responses of purified human peripheral blood CD4+ and CD8+ T cells to PHA and of purified B cells to SAC. The concentrations of these nucleosides required to achieve a given degree of inhibition of proliferative responses of T lymphocyte subpopulations or B cells was similar, suggesting that these analogs do not exhibit any selectivity for these purified lymphocyte populations. Tub and FaraAMP, respectively, inhibited and enhanced, at the effector phase, both NK cytotoxicity and specific T cell-mediated cytotoxicity. In contrast to these findings, LAK cytotoxicity at the effector phase was not significantly inhibited by Tub, and was not enhanced by FaraAMP. Both analogs inhibited rIL-2-induced proliferative responses of PBMC, but did not affect the generation of LAK cytotoxicity (induction phase) against the K562 targets when added at the beginning of the culture. This suggests that DNA synthesis is not required for LAK cell induction. Both Tub and FaraAMP inhibited immunoglobulin production (IgG and IgM) by

  15. Stable growth transformation of human T lymphocytes by herpesvirus saimiri.

    PubMed Central

    Biesinger, B; Müller-Fleckenstein, I; Simmer, B; Lang, G; Wittmann, S; Platzer, E; Desrosiers, R C; Fleckenstein, B

    1992-01-01

    Herpesvirus saimiri induces T-cell lymphomas in various species of New World monkeys and in rabbits, and it is able to immortalize monkey T lymphocytes in vitro. Sequences responsible for these effects have been localized to a region of the genome that varies significantly among the virus subgroups A, B, and C. We now report that infection of human blood lymphocytes and thymocytes with strains of subgroup C, in contrast to viruses of the other subgroups, yields continuously proliferating T-cell lines with the phenotype of mature CD4- or CD8-positive cells. Infection with strains of Herpes-virus saimiri subgroup C can thus be used to generate human T-cell lines for a variety of immunological and developmental studies. Images PMID:1313581

  16. Telomerase Contributes to Fludarabine Resistance in Primary Human Leukemic Lymphocytes

    PubMed Central

    Shawi, May; Chu, Tsz Wai; Martinez-Marignac, Veronica; Yu, Y.; Gryaznov, Sergei M.; Johnston, James B.; Lees-Miller, Susan P.; Assouline, Sarit E.; Autexier, Chantal; Aloyz, Raquel

    2013-01-01

    We report that Imetelstat, a telomerase inhibitor that binds to the RNA component of telomerase (hTR), can sensitize primary CLL lymphocytes to fludarabine in vitro. This effect was observed in lymphocytes from clinically resistant cases and with cytogenetic abnormalities associated with bad prognosis. Imetelstat mediated-sensitization to fludarabine was not associated with telomerase activity, but with the basal expression of Ku80. Since both Imetelstat and Ku80 bind hTR, we assessed 1) if Ku80 and Imetelstat alter each other's binding to hTR in vitro and 2) the effect of an oligonucleotide complementary to the Ku binding site in hTR (Ku oligo) on the survival of primary CLL lymphocytes exposed to fludarabine. We show that Imetelstat interferes with the binding of Ku70/80 (Ku) to hTR and that the Ku oligo can sensitize CLL lymphocytes to FLU. Our results suggest that Ku binding to hTR may contribute to fludarabine resistance in CLL lmphocytes. This is the first report highlighting the potentially broad effectiveness of Imetelstat in CLL, and the potential biological and clinical implications of a functional interaction between Ku and hTR in primary human cancer cells. PMID:23922990

  17. Telomerase contributes to fludarabine resistance in primary human leukemic lymphocytes.

    PubMed

    Shawi, May; Chu, Tsz Wai; Martinez-Marignac, Veronica; Yu, Y; Gryaznov, Sergei M; Johnston, James B; Lees-Miller, Susan P; Assouline, Sarit E; Autexier, Chantal; Aloyz, Raquel

    2013-01-01

    We report that Imetelstat, a telomerase inhibitor that binds to the RNA component of telomerase (hTR), can sensitize primary CLL lymphocytes to fludarabine in vitro. This effect was observed in lymphocytes from clinically resistant cases and with cytogenetic abnormalities associated with bad prognosis. Imetelstat mediated-sensitization to fludarabine was not associated with telomerase activity, but with the basal expression of Ku80. Since both Imetelstat and Ku80 bind hTR, we assessed 1) if Ku80 and Imetelstat alter each other's binding to hTR in vitro and 2) the effect of an oligonucleotide complementary to the Ku binding site in hTR (Ku oligo) on the survival of primary CLL lymphocytes exposed to fludarabine. We show that Imetelstat interferes with the binding of Ku70/80 (Ku) to hTR and that the Ku oligo can sensitize CLL lymphocytes to FLU. Our results suggest that Ku binding to hTR may contribute to fludarabine resistance in CLL lmphocytes. This is the first report highlighting the potentially broad effectiveness of Imetelstat in CLL, and the potential biological and clinical implications of a functional interaction between Ku and hTR in primary human cancer cells. PMID:23922990

  18. Chrysin induces apoptosis in peripheral blood lymphocytes isolated from human chronic lymphocytic leukemia.

    PubMed

    Zaric, Milan; Mitrovic, Marina; Nikolic, Ivana; Baskic, Dejan; Popovic, Suzana; Djurdjevic, Predrag; Milosavljevic, Zoran; Zelen, Ivanka

    2015-01-01

    Chronic lymphocytic leukemia (CLL) develops due to an imbalance between apoptosis and proliferation of B lymphocytes. Chrysin induced apoptosis in leukemia cell lines such as U937, MO7e, THP-1 and HL-60, but there has not yet been data demonstrating the apoptotic effect of chrysin on CLL cells. Therefore, in our investigation we examined the cytotoxicity of chrysin against two leukemia cell lines, MOLT-4 and JVM-13, peripheral blood lymphocytes isolated from B-CLL patients and peripheral blood mononuclear cells (PBMC) from healthy individuals in vitro. The effect of chrysin on viability of MOLT-4 and JVM-13 cell lines, B-CLL cells derived from 28 patients and PBMC from 16 healthy subjects was determined by MTT assay. The type of cell death induced by chrysin was verified by Annexin V/7AAD assay and acridine orange and ethidium bromide (AO/EB) staining assay. Intracellular localisation and endogenic expression of apoptotic proteins including Bax, Bcl-2, cytochrome c and caspase-3 were determined by flow cytometry and fluorescent microscopy. Our results demonstrated that exposure of MOLT-4, JVM-13 cell lines and B-CLL cells to the concentration of chrysin of 10μM and higher selectively decreased viability of cells in this cell population, but not in the PBMC derived from healthy subjects; LC50 values of chrysin for B-CLL cells were 51μM for 24 hours and 32μM for 48 hours of incubation, respectively. Our findings demonstrated that chrysin induces the activation of proapoptotic Bax and decreases the expression of antiapoptotic Bcl-2 protein, releases cytochrome c from mitochondria into cytosol and cleavages/activates caspase-3, subsequently leading to the activation of apoptosis of B-CLL cells. Together, these findings suggest that chrysin selectively induces apoptosis of peripheral blood lymphocytes isolated from human chronic lymphocytic leukemia patients via mitochondrial pathway in vitro and that it might have a promising role as a potential future antileukemic

  19. Circulating and tumor-infiltrating mucosal associated invariant T (MAIT) cells in colorectal cancer patients

    PubMed Central

    Ling, Limian; Lin, Yuyang; Zheng, Wenwen; Hong, Sen; Tang, Xiuqi; Zhao, Pingwei; Li, Ming; Ni, Jingsong; Li, Chenguang; Wang, Lei; Jiang, Yanfang

    2016-01-01

    Mucosal associated invariant T (MAIT) cells are important for immune defense against infectious pathogens and regulate the pathogenesis of various inflammatory diseases. However, their roles in the development of colorectal cancer (CRC) are still unclear. This study examined the phenotype, distribution, clinical relevance and potential function of MAIT cells in CRC patients. We found that the percentages of circulating memory CD8+ MAIT cells were significantly reduced while tumor infiltrating MAIT cells were increased, especially in patients with advanced CRC. The serum CEA levels were positively correlated with the percentages of tumor infiltrating MAIT cells in CRC patients, but negatively correlated with the percentages of circulating MAIT in advanced CRC patients. Activated circulating MAIT cells from CRC patients produced lower IFN-γ, but higher IL-17. Furthermore, higher levels of Vα7.2-Jα33, IFN-γ and IL-17A were expressed in the CRC tissues. Co-culture of activated MAIT cells with HCT116 cells enhanced IL-17 expression and induced HCT116 cell cycle arrest at G2/M phase in a contact- and dose-dependent manner, which was abrogated by treatment with anti-MR1. Therefore, MAIT cells preferably infiltrate into the solid tumor in CRC patients and may participate in the immune surveillance of CRC. PMID:26837580

  20. Functional Alteration of Tumor-infiltrating Myeloid Cells in RNA Adjuvant Therapy.

    PubMed

    Seya, Tsukasa; Shime, Hiroaki; Matsumoto, Misako

    2015-08-01

    Macrophages, as well as dendritic cells (DCs), are derived from myeloid progenitor cells. Recent evidence suggests that tumor-infiltrating macrophages differ in many aspects from conventional tissue macrophages, including nature, function and markers. Tumors usually contain various myeloid lineage cells in their non-parenchymal environment. In immunotherapy for cancer, tumor cells and non-parenchymal cells are exposed to tumor-associated antigens (TAA) and tumor-cell-derived nucleic acids. In addition, a dsRNA mimic, polyinosinic:polycytidylic acid (polyI:C), exhibits strong adjuvant activity, which acts both on the immune system and tumor constituents. Herein we discuss the RNA recognition system and unique cellular output in tumor-associated myeloid cells in response to immunotherapy. We especially focus on the mechanism by which RNA adjuvant alters the tumor-supportive nature of tumor-infiltrated myeloid cells to those with tumoricidal activity. We discuss how RNA administration makes tumor cells collapse and its significance of evoking cell death signals in tumor cells and macrophages. This knowledge will be applicable to the development of an alternative immunotherapy for cancer. PMID:26168476

  1. Tracking of intertissue migration reveals the origins of tumor-infiltrating monocytes

    PubMed Central

    Shand, Francis H. W.; Ueha, Satoshi; Otsuji, Mikiya; Koid, Suang Suang; Shichino, Shigeyuki; Tsukui, Tatsuya; Kosugi-Kanaya, Mizuha; Abe, Jun; Tomura, Michio; Ziogas, James; Matsushima, Kouji

    2014-01-01

    Myeloid cells such as monocytes and monocyte-derived macrophages promote tumor progression. Recent reports suggest that extramedullary hematopoiesis sustains a sizable reservoir of tumor-infiltrating monocytes in the spleen. However, the influence of the spleen on tumor development and the extent to which spleen monocytes populate the tumor relative to bone marrow (BM) monocytes remain controversial. Here, we used mice expressing the photoconvertible protein Kikume Green-Red to track the redistribution of monocytes from the BM and spleen, and mice expressing fluorescent ubiquitination-based cell-cycle indicator proteins to monitor active hematopoiesis in these tissues. In mice bearing late-stage tumors, the BM, besides being the major site of monocyte production, supplied the expansion of the spleen reservoir, replacing 9% of spleen monocytes every hour. Deployment of monocytes was equally rapid from the BM and the spleen. However, BM monocytes were younger than those in the spleen and were 2.7 times more likely to migrate into the tumor from the circulation. Partly as a result of this intrinsic difference in migration potential, spleen monocytes made only a minor contribution to the tumor-infiltrating monocyte population. At least 27% of tumor monocytes had traveled from the BM in the last 24 h, compared with only 2% from the spleen. These observations highlight the importance of the BM as the primary hematopoietic tissue and monocyte reservoir in tumor-bearing mice, despite the changes that occur in the spleen monocyte reservoir during tumor development. PMID:24825888

  2. In vitro effect of fenthion on human lymphocytes

    SciTech Connect

    Rani, M.V.U. ); Rao, M.S. )

    1991-08-01

    Fenthion is an organophosphorus insecticide which is extensively used in control of leaf hoppers, cutworms, mites on vegetable crops. It has been reported that organophosphorus pesticides cause a significant increase in sister chromatid exchanges in mammalian cell lines. A significant increase of chromosomal aberrations has been reported in rural population exposed to pesticides. Organosphosphorus pesticides malathion, diazinon, dimethoate, phosdrin and dursban induced sister chromatid exchanges in human lymphoid cells. Exchange type of aberration has been reported in fluoriculturist who were exposed to organophosphorus, organochlorine pesticides. In the present investigation an attempt has been made to evaluate the cytogenetic effect of fenthion in human lymphocyte cultures in vitro.

  3. Electrostimulation of rat callus cells and human lymphocytes in vitro

    SciTech Connect

    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.

  4. Immunohistochemical identification of cytotoxic lymphocytes using human perforin monoclonal antibody.

    PubMed Central

    Hameed, A.; Olsen, K. J.; Cheng, L.; Fox, W. M.; Hruban, R. H.; Podack, E. R.

    1992-01-01

    Perforin is a potent cytolytic pore-forming protein expressed in cytoplasmic granules of cytotoxic T lymphocytes and natural killer cells. A new monoclonal antibody raised against human perforin was used to detect both in vitro and in vivo perforin expression in cytotoxic cells. Immunohistochemical analysis of human peripheral blood mononuclear cells cultured in recombinant interleukin-2 (rIL-2) showed strong granular cytoplasmic staining of the IL-2 activated cytotoxic cells. Fresh-frozen tissue sections from patients with heart allograft rejection were also stained. Strong granular cytoplasmic staining of the mononuclear inflammatory infiltrate characteristic for perforin in cardiac allograft rejection was observed. The detection and quantitative analysis of perforin-associated cytotoxic cells by the human anti-perforin monoclonal antibody will help to evaluate the significance of these functionally distinct cytotoxic cells in human tissue. Images Figure 1 PMID:1374586

  5. Human lymphocyte polymorphisms detected by quantitative two-dimensional electrophoresis

    SciTech Connect

    Goldman, D.; Merril, C.R.

    1983-09-01

    A survey of 186 soluble lymphocyte proteins for genetic polymorphism was carried out utilizing two-dimensional electrophoresis of /sup 14/C-labeled phytohemagglutinin (PHA)-stimulated human lymphocyte proteins. Nineteen of these proteins exhibited positional variation consistent with independent genetic polymorphism in a primary sample of 28 individuals. Each of these polymorphisms was characterized by quantitative gene-dosage dependence insofar as the heterozygous phenotype expressed approximately 50% of each allelic gene product as was seen in homozygotes. Patterns observed were also identical in monozygotic twins, replicate samples, and replicate gels. The three expected phenotypes (two homozygotes and a heterozygote) were observed in each of 10 of these polymorphisms while the remaining nine had one of the homozygous classes absent. The presence of the three phenotypes, the demonstration of gene-dosage dependence, and our own and previous pedigree analysis of certain of these polymorphisms supports the genetic basis of these variants. Based on this data, the frequency of polymorphic loci for man is: P . 19/186 . .102, and the average heterozygosity is .024. This estimate is approximately 1/3 to 1/2 the rate of polymorphism previously estimated for man in other studies using one-dimensional electrophoresis of isozyme loci. The newly described polymorphisms and others which should be detectable in larger protein surveys with two-dimensional electrophoresis hold promise as genetic markers of the human genome for use in gene mapping and pedigree analyses.

  6. The relationships between serum cytokine levels and tumor infiltrating immune cells and their clinical significance in colorectal cancer.

    PubMed

    Väyrynen, Juha P; Kantola, Tiina; Väyrynen, Sara A; Klintrup, Kai; Bloigu, Risto; Karhu, Toni; Mäkelä, Jyrki; Herzig, Karl-Heinz; Karttunen, Tuomo J; Tuomisto, Anne; Mäkinen, Markus J

    2016-07-01

    Increased inflammatory cell infiltration correlates to improved survival in colorectal cancer (CRC). Development and progression of CRC is associated with alterations in serum cytokine levels but their significance is not well defined. In this study, we investigated the relationships between the serum levels of 13 cytokines and the densities of eight types of tumor infiltrating inflammatory cells and their impact on disease-free survival (DFS), cancer-specific survival (CSS) and overall survival (OS) in a prospectively recruited group of 147 CRC patients. There were strong positive correlations between the serum concentrations of different cytokines, as well as between the different types of tumor infiltrating immune cells, whereas the associations between serum cytokines and tumor infiltrating immune cells were generally weak. High serum IL-12 levels associated with increased densities of peritumoral CD8(+) T cells, intraepithelial CD3(+) T cells and intratumoral neutrophils, while high serum CCL4 levels associated with increased densities of peritumoral CD68(+) cells. In multivariate survival models, increased infiltration of intraepithelial CD3(+) T cells and increased serum CCL4 associated with improved DFS, whereas higher intratumoral CD83(+) dendritic cell density and increased serum interferon gamma levels associated with improved CSS and OS. Also high density of peritumoral CD3(+) T cells associated with improved CSS. In conclusion, serum cytokines and tumor infiltrating immune cells in CRC represent entities with high intragroup correlations but relatively weak intergroup correlations. The results suggest that tumor infiltrating CD3(+) T cells, CD83(+) dendritic cells, serum CCL4 and serum interferon gamma represent relevant markers of disease outcome. PMID:26874795

  7. Monoclonal antibodies to antigens on human neutrophils, activated T lymphocytes, and acute leukemia blast cells

    SciTech Connect

    Miterev, G.Yu.; Burova, G.F.; Puzhitskaya, M.S.; Danilevich, S.V.; Bulycheva, T.I.

    1987-11-01

    The authors describe the production of two mouse hybridomas secreting monoclonal antibodies to antigenic determinants of the surface membranes of human neutrophils, activated T lymphocytes, and acute leukemic blast cells. The degree of lymphocyte stimulation was estimated from incorporation of /sup 3/H-thymidine with parallel microculture. Monoclonal antibodies of supernatants of hybridoma cultures shown here reacted in both immunofluorescence test and cytotoxicity test with surface membrane antigens on the majority of neutrophils and PHA-activated peripheral blood lymphocytes from healthy subjects, but did not give positive reactions with unactivated lymphocytes, adherent monocytes, erythrocytes, and alloantigen-stimulated lymphocytes.

  8. Evaluation of gamma-Induced Apoptosis in Human Peripheral Blood Lymphocytes

    SciTech Connect

    Baranova, Elena; Boreyko, Alla; Ravnachka, Ivanka; Saveleva, Maria

    2010-01-05

    Several experiments have been performed to study regularities in the induction of apoptotic cells in human lymphocytes by {sup 60}Cogamma-rays at different times after irradiation. Apoptosis induction by {sup 60}Cogamma-rays in human lymphocytes in different cell cycle phases (G{sub 0}, S, G{sub 1}, and G{sub 2}) has been studied. The maximal apoptosis output in lymphocyte cells was observed in the S phase. Modifying effect of replicative and reparative DNA synthesis inhibitors - 1-beta-D-arabinofuranosylcytosine (Ara-C) and hydroxyurea (Hu) - on the kinetics of {sup 60}Cogamma-rays induced apoptosis in human lymphocytes has been studied.

  9. Effects of budlein A on human neutrophils and lymphocytes

    PubMed Central

    KNOB, Carollinie Dias; SILVA, Milena; GASPAROTO, Thaís Helena; OLIVEIRA, Carine Ervolino; AMÔR, Nádia Ghinelli; ARAKAWA, Nilton Syogo; COSTA, Fernando Batista; CAMPANELLI, Ana Paula

    2016-01-01

    ABSTRACT Sesquiterpene lactones (SLs) are the active constituents of a variety of medicinal plants used in traditional medicine for the treatment of inflammatory diseases and other ailments. Objective In this study, we evaluated whether budlein A modulates the activation of innate and adaptive immune cells such as neutrophils and lymphocytes. Material and Methods Our research group has investigated several plant species and several compounds have been isolated, identified, and their medical potential evaluated. Budlein A is a SL isolated from the species Aldama buddlejiformis and A. robusta (Asteraceae) and shows anti-inflammatory and anti-nociceptive activities. Advances in understanding how plant-derived substances modulate the activation of innate and adaptive immune cells have led to the development of new therapies for human diseases. Results Budlein A inhibited MPO activity, IL-6, CXCL8, IL-10, and IL-12 production and induces neutrophil apoptosis. In contrast, budlein A inhibited lymphocyte proliferation and IL-2, IL-10, TGF-β, and IFN-γ production, but it did not lead to cell death. Conclusions Collectively, our results indicate that budlein A shows distinct immunomodulatory effects on immune cells. PMID:27383709

  10. Unstable high molecular weight inverted repetitive DNA in human lymphocytes.

    PubMed Central

    Rogers, J C; Rucinsky, T E

    1982-01-01

    About 1% of newly synthesized DNA from PHA-stimulated human lymphocytes can be isolated as large (up to 90 kilobase pairs) double stranded fragments that resist sequential alkali and heat denaturation steps but are not closed circular. By electron microscopy about 1% have single-strand hairpin loops at one end and therefore present inverted repetitive sequences (IR-DNA). Most of the remainder have a blunt-appearing double-strand terminus at both ends (78%) or one end (18%). Indirect evidence indicates that these also are inverted complementary structures with terminal hairpin loops too small to be visualized: (1) Treatment with either a 5' or 3' single-strand exonuclease generates essentially only fragments with a single strand at one end; (2) with partial denaturation, the number of fragments with identifiable single-strand hairpin loops increases (to about 20%); (3) after S1 nuclease digestion, greater than 95% can be fully heat denatured. Cot analysis indicates that these fragments are derived from dispersed sites throughout the genome. Up to 25% of DNA released from lymphocytes during growth similarly resists denaturation, and released DNA and IR-DNA are both enriched in the same set of repetitive sequences. Thus at least a portion of IR-DNA appears to be unstable. Images PMID:7145706

  11. Spaceflight alters microtubules and increases apoptosis in human lymphocytes (Jurkat)

    NASA Technical Reports Server (NTRS)

    Lewis, M. L.; Reynolds, J. L.; Cubano, L. A.; Hatton, J. P.; Lawless, B. D.; Piepmeier, E. H.

    1998-01-01

    Alteration in cytoskeletal organization appears to underlie mechanisms of gravity sensitivity in space-flown cells. Human T lymphoblastoid cells (Jurkat) were flown on the Space Shuttle to test the hypothesis that growth responsiveness is associated with microtubule anomalies and mediated by apoptosis. Cell growth was stimulated in microgravity by increasing serum concentration. After 4 and 48 h, cells filtered from medium were fixed with formalin. Post-flight, confocal microscopy revealed diffuse, shortened microtubules extending from poorly defined microtubule organizing centers (MTOCs). In comparable ground controls, discrete microtubule filaments radiated from organized MTOCs and branched toward the cell membrane. At 4 h, 30% of flown, compared to 17% of ground, cells showed DNA condensation characteristic of apoptosis. Time-dependent increase of the apoptosis-associated Fas/ APO-1 protein in static flown, but not the in-flight 1 g centrifuged or ground controls, confirmed microgravity-associated apoptosis. By 48 h, ground cultures had increased by 40%. Flown populations did not increase, though some cells were cycling and actively metabolizing glucose. We conclude that cytoskeletal alteration, growth retardation, and metabolic changes in space-flown lymphocytes are concomitant with increased apoptosis and time-dependent elevation of Fas/APO-1 protein. We suggest that reduced growth response in lymphocytes during spaceflight is linked to apoptosis.

  12. In vitro sensitization of human lymphocytes to a myeloma cell-related antigen

    SciTech Connect

    Whitson, M.E.; Griffin, G.D.; Novelli, G.D.; Solomon, A.

    1981-01-01

    Peripheral blood lymphocytes from normal human donors were cocultivated with cells from two established human multiple myeloma cell lines, RPMI 8226 and K-737, and with lymphoblastoid cells from a third B cell line, RAMM. After a comparison of three methods of lymphocyte sensitization, a 6-day incubation protocol with equal numbers of normal lymphocytes and mitomycin C-treated tumor cells was selected. Cells fom the RPMI 8226 myeloma line stimulated the differentiation of lymphocytes into cytotoxic effector cells as measured by /sup 51/Cr release from labeled target cells. The RPMI 8226-sensitized lymphocytes were cytotoxic for myeloma cells (RPMI 8226 and K-737) and for lymphoblastoid cells (RAMM) but not for cells from human lung tumor lines (A549, A427, MB9812), a breast carcinoma line (ALAB), a normal diploid fibroblast line (HSBP), or normal lymphocytes.

  13. Stimulation of human lymphocytes by Herpes simplex virus antigens.

    PubMed Central

    Starr, S E; Karatela, S A; Shore, S L; Duffey, A; Nahmias, A J

    1975-01-01

    Lymphocytes from individuals with laboratory evidence of prior infection with herpes simplex virus (HSV) type 1 or type 2 demonstrated transformation (av antigens. Higher stimulation indexes were obtained when lymphocytes were incubated with the homologous as compared with the heterologous antigen. Higher mean lymphocyte stimulation indexes were also demonstrated in seropositive as compared with seronegative individuals. Lymphocytes from children with HSV-1 stomatitis usually became responsive to HSV-1 antigen within 2 to 6 weeks after the onset of illness. Lymphocytes from infants with neonatal HSV-2 infection were stimulated by HSV-2 antigen. PMID:163788

  14. The effect of adherent and phagocytic cells on human lymphocyte PHA responsiveness.

    PubMed

    Potter, M R; Moore, M

    1977-01-01

    The effect of small numbers of adherent and phagocytic cells on the human peripheral blood lymphocyte response to PHA was examined by depleting these cells from lymphocyte preparations. Lymphocyte preparations obtained by centrifugation on Ficoll--Triosil, which contained on average 85% lymphocytes, responded well to PHA. Depletion of cells adhering to nylon fibre, giving a population containing on average 95% lymphocytes, resulted in a considerably reduced response. Depletion of cells that adhered to plastic or ingested iron powder to give populations containing on average 90% lymphocytes, also reduced the PHA response, but to a lesser extent. Reduction in PHA responsiveness correlated with increasing lymphocyte purity. The responsiveness of nylon-column-filtered cells could be restored by adding a small number of cells from a monocyte-rich population. PMID:300303

  15. Effect of steady magnetic field on human lymphocytes

    SciTech Connect

    Mileva, M.; Ivanov, B.; Bulanova, M.; Pantev, T.

    1983-01-01

    Exposure to steady magnetic field (SMF) for different periods of time did not elicit statistically reliable increase in chromosome aberrations in human peripheral blood lymphocytes. Metaphase analysis of Crepis capilaris cells revealed that SMF (9 k0e, 200 0e/cm) for 2 days did not induce chromosome aberrations. Nor were any changes demonstrated in roots of beans, onions and L-fibroblasts of subcutaneous tissue of mice and Chinese hamsters. The obtained data are indicative of absence of cytogenetic effect of SMF. The level and spectrum of chromosome aberrations did not exceed the values for spontaneous chromatic fragments in cultures. Cytogenetic analysis of DEDE cells of the Chinese hamster revealed a mild mutagenic effect of SMF. Chromosomal aberrations were also demonstrated after exposure (5 min) of garlic roots.

  16. Human cytotoxic T lymphocytes against the Plasmodium falciparum circumsporozoite protein.

    PubMed Central

    Malik, A; Egan, J E; Houghten, R A; Sadoff, J C; Hoffman, S L

    1991-01-01

    Cytotoxic T lymphocytes (CTL) against the circumsporozoite (CS) protein of malaria sporozoites protect against malaria in rodents. Although there is interest in developing human vaccines that induce CTL against the Plasmodium falciparum CS protein, humans have never been shown to produce CTL against any Plasmodium species protein or other parasite protein. We report that when peripheral blood mononuclear cells (PBMC) from three of four volunteers immunized with irradiated P. falciparum sporozoites were stimulated in vitro with a recombinant vaccinia virus expressing the P. falciparum CS protein or a peptide including only amino acids 368-390 of the P. falciparum CS protein [CS-(368-390)], the PBMC lysed autologous Epstein-Barr virus-transformed B cells transfected with the P. falciparum CS protein gene or incubated with CS-(368-390) tricosapeptide. Activity was antigen specific, genetically restricted, and dependent on CD8+ T cells. In one volunteer, seven peptides reflecting amino acids 311-400 were tested, and, as in B10.BR mice, CTL activity was only associated with the CS-(368-390) peptide. Development of an assay for studying human CTL against the CS and other malaria proteins and a method for constructing target cells by direct gene transfection provide a foundation for studying the role of CTL in protection against malaria. PMID:1707538

  17. Chromosome Aberration in Human Blood Lymphocytes Exposed to Energetic Protons

    NASA Technical Reports Server (NTRS)

    Hada, M.; George, Kerry A.; Cucinotta, F. A.

    2008-01-01

    During space flight, astronauts are exposed to a space radiation consisting of high-energy protons, high charge and energy (HZE) nuclei, as well as secondary particles that are generated when the primary particles penetrate the spacecraft shielding. Secondary particles have a higher LET value than primary protons and therefore expected to have a higher relative biological effectiveness (RBE). To investigate this theory, we exposed human peripheral blood lymphocytes to protons with energies of 250 MeV, 800MeV, 2 GeV, or 2.5 GeV. LET values for these protons ranged from 0.4 to 0.2 keV/micrometer. and doses ranged from 0.2 to 3 Gy. Over this energy the probability of nuclear reaction leading to secondary radiation, and the multiplicity of reaction produces such as neutrons and mesons increases substantially. The effect of aluminum and polyethylene shielding was also assessed using the 2 GeV and 2.5GeV proton beams. After exposure lymphocytes were stimulated to divide and chromosomes were collected from cells in the first G2 and metaphase cell cycle after exposure using a chemical induced premature chromosome condensation (PCC) technique. Dose response data for chromosome damage was analyzed using the fluorescence in situ hybridization (FISH) chromosome painting technique. Selected samples were also analyzed with multicolor FISH (mFISH) and multicolor banding FISH (mBAND) techniques. Data indicates that the dose response for simple-type exchanges is similar for proton and gamma exposure, whereas protons induce higher yields of complex exchanges that are LET dependent. RBE values will be presented for each proton energy, and the effects of shielding and possible cytogenetic signatures of proton exposure will be discussed.

  18. Modeling adenovirus latency in human lymphocyte cell lines.

    PubMed

    Zhang, Yange; Huang, Wen; Ornelles, David A; Gooding, Linda R

    2010-09-01

    Species C adenovirus establishes a latent infection in lymphocytes of the tonsils and adenoids. To understand how this lytic virus is maintained in these cells, four human lymphocytic cell lines that support the entire virus life cycle were examined. The T-cell line Jurkat ceased proliferation and died shortly after virus infection. BJAB, Ramos (B cells), and KE37 (T cells) continued to divide at nearly normal rates while replicating the virus genome. Viral genome numbers peaked and then declined in BJAB cells below one genome per cell at 130 to 150 days postinfection. Ramos and KE37 cells maintained the virus genome at over 100 copies per cell over a comparable period of time. BJAB cells maintained the viral DNA as a monomeric episome. All three persistently infected cells lost expression of the cell surface coxsackie and adenovirus receptor (CAR) within 24 h postinfection, and CAR expression remained low for at least 340 days postinfection. CAR loss proceeded via a two-stage process. First, an initial loss of cell surface staining for CAR required virus late gene expression and a CAR-binding fiber protein even while CAR protein and mRNA levels remained high. Second, CAR mRNA disappeared at around 30 days postinfection and remained low even after virus DNA was lost from the cells. At late times postinfection (day 180), BJAB cells could not be reinfected with adenovirus, even when CAR was reintroduced to the cells via retroviral transduction, suggesting that the expression of multiple genes had been stably altered in these cells following infection. PMID:20573817

  19. Modulation of human lymphocyte mitogen responsiveness and interleukin-2 production by polymorphonuclear leukocytes.

    PubMed

    Lyte, M

    1990-06-01

    The response of human peripheral blood lymphocytes to the mitogenic lectins phytohemagglutinin (PHA) and pokeweed mitogen (PWM) was examined in the presence of autologous polymorphonuclear leukocytes (PMN). Experiments were performed at sub-optimal and optimal mitogen concentrations employing lymphocyte: PMN ratios over a three log cell concentration range. Increases of up to 25,000-fold in mitogen stimulated lymphocyte proliferation as determined by 3H-thymidine incorporation were observed in PMN supplemented lymphocyte cultures as compared to lymphocytes cultured in the absence of PMN or with irradiated lymphocytes serving as filler cells. Similar results were obtained for PHA stimulated IL-2 production. The degree of enhancement of lymphocyte reactivity by PMN was also shown to be dependent on the source of serum supplementation (autologous versus xenogeneic). These results indicate that cell ratio is a critical factor in examining lymphocyte-PMN interactions as well as serum supplementation used. Early reports which have indicated a suppressive or no effect of PMN on lymphocyte reactivity based on a single lymphocyte: PMN cell ratio may need to be re-evaluated. PMID:1967045

  20. Effects of flavonoids on human lymphocyte proliferative responses

    SciTech Connect

    Mookerjee, B.K.; Lee, T.P.; Logue, G.P.; Lippes, H.A.; Middleton, E.

    1986-01-01

    Flavonoids reversibly inhibit lymphocyte proliferative responses to phytomitogens, soluble antigens and phorbol esters by blocking an early event or events that follow stimulation. Quercetin and tangeretin inhibit thymidine transport in stimulated lymphocytes. These flavonoids reversibly inhibit antigen processing by monocytes and inhibit the expression of class II histocompatibility (DR) antigens in PBM cells.

  1. Immune Profiling of Adenoid Cystic Carcinoma: PD-L2 Expression and Associations with Tumor-Infiltrating Lymphocytes.

    PubMed

    Sridharan, Vishwajith; Gjini, Evisa; Liao, Xiaoyun; Chau, Nicole G; Haddad, Robert I; Severgnini, Mariano; Hammerman, Peter; El-Naggar, Adel; Freeman, Gordon J; Hodi, F Stephen; Rodig, Scott J; Dranoff, Glenn; Schoenfeld, Jonathan D

    2016-08-01

    Adenoid cystic carcinoma (ACC) is among the most lethal salivary gland tumors, with no treatments for metastatic disease that prolong survival. We examined tissue from 28 primary and metastatic ACC deposits obtained from 21 patients for infiltrating immune cells and PD-L1/PD-L2 expression and determined mRNA profiles of over 1,400 oncogenic and immune-related genes. We also assessed the effect of chemoradiation on immune mediators in a patient who had serial biopsies available. Most tumors expressed PD-L2 but had few infiltrating immune cells. Lack of immune-cell infiltrate was associated with expression of genes in the β-catenin/Wnt and PI3K pathways. Additionally, certain transcripts linked to growth and invasion were differentially expressed among primary and metastatic deposits. Chemoradiation appeared to increase CD8(+) effector T cells, decrease regulatory T cells, and promote a systemic humoral response. These data suggest a potential role for PD-L2 inhibition and immune modulation as treatment for patients with ACC. Cancer Immunol Res; 4(8); 679-87. ©2016 AACR. PMID:27312343

  2. THE EFFECT OF OZONE ON HUMAN IMMUNITY: IN VITRO RESPONSIVENESS OF LYMPHOCYTES TO PHYTOHEMMAGGLUTININ

    EPA Science Inventory

    Peripheral blood lymphocytes from 20 human subjects exposed to 784 micrograms cu m ozone for 4 hours, and from 11 subjects exposed to clean air for the same length of time were studied for in vitro responsiveness to phytohemagglutinin (PHA). Thymus-derived (T) lymphocyte response...

  3. Viral Engineering of Chimeric Antigen Receptor Expression on Murine and Human T Lymphocytes.

    PubMed

    Hammill, Joanne A; Afsahi, Arya; Bramson, Jonathan L; Helsen, Christopher W

    2016-01-01

    The adoptive transfer of a bolus of tumor-specific T lymphocytes into cancer patients is a promising therapeutic strategy. In one approach, tumor specificity is conferred upon T cells via engineering expression of exogenous receptors, such as chimeric antigen receptors (CARs). Here, we describe the generation and production of both murine and human CAR-engineered T lymphocytes using retroviruses. PMID:27581020

  4. Vincristine-induced bystander effect in human lymphocytes.

    PubMed

    Testi, Serena; Azzarà, Alessia; Giovannini, Caterina; Lombardi, Sara; Piaggi, Simona; Facioni, Maria Sole; Scarpato, Roberto

    2016-07-01

    Bystander effect is a known radiobiological effect, widely described using ionizing radiations and which, more recently, has also been related to chemical mutagens. In this study, we aimed to assess whether or not a bystander response can be induced in cultured human peripheral lymphocytes by vincristine, a chemotherapeutic mutagen acting as spindle poison, and by mitomycin-C, an alkylating agent already known to induce this response in human lymphoblastoid cells. Designing a modified ad hoc protocol for the cytokinesis blocked micronucleus (MN) assay, we detected the presence of a dose-dependent bystander response in untreated cultures receiving the conditioned medium (CM) from mitomycin-C (MMC) or vincristine (VCR) treated cultures. In the case of MMC, MN frequencies, expressed as micronucleated binucleates, were: 13.5±1.41 at 6μM, 22±2.12 at 12μM or 28.25±5.13 at 15μM vs. a control value of 4.75±1.59. MN levels for VCR, expressed as micronucleated mononucleates were: 2.75±0.88 at 0.0μM, 27.25±2.30 at 0.4μM, 46.25±1.94 at 0.8μM, 98.25±7.25 at 1.6μM. To verify that no mutagen residual was transferred to recipient cultures together with the CM, we evaluated MN levels in cultures receiving the medium immediately after three washings following the chemical treatment (unconditioned medium). We further confirmed these results using a cell-mixing approach where untreated lymphocytes were co-cultured with donor cells treated with an effect-inducing dose of MMC or VCR. A distinct production pattern of both reactive oxygen species and soluble mediator proteins by treated cells may account for the differences observed in the manifestation of the bystander effect induced by VCR. In fact, we observed an increased level of ROS, IL-32 and TGF-β in the CM from VCR treated cultures, not present in MMC treated cultures. PMID:27050754

  5. T-lymphocyte induction of human monocyte angiotensin converting enzyme (ACE) is not dependent upon T-lymphocyte proliferation

    SciTech Connect

    Vuk-Pavlovic, Z.; Rohrbach, M.S.

    1986-03-05

    Human peripheral blood monocytes cultured in serum free media for seven days show a basal activity of the ectoenzyme ACE which is augmented 2-3 times by the presence of autologous peripheral blood T-lymphocytes. Since these two cell types are also involved in autologous mixed lymphocyte reaction if serum is present, the authors compared the ability of T-cells to stimulate ACE activity in the presence or absence of proliferation (measured by /sup 3/H-thymidine incorporation). By the seventh day, cultures with 5% AB/sup +/ serum showed significant increase in proliferation but no increase in ACE activity compared to the serum free cultures. Even higher proliferation rate achieved by co-culturing T-lymphocytes with allogeneic monocytes did not increase ACE production; on the contrary, ACE activity remained at the basal level. Monocyte-T-cell co-cultures stimulated with increasing concentrations of ConA or PHA showed dose dependent increases in proliferation but parallel decreases in ACE activity. Addition of soluble antigen (Candida albicans) also enhanced proliferation but not ACE synthesis. They conclude that T-lymphocyte induction of monocyte ACE is a result of cooperation between autologous cells which is not dependent upon T-cell proliferation.

  6. Specific binding of antigen onto human T lymphocytes

    SciTech Connect

    Durandy, A.; Fischer, A.; Charron, D.; Griscelli, C.

    1986-05-01

    Human T lymphocytes sensitized to Candida albicans (CA) were shown to proliferate in cultures induced with mannan, a ramified polysaccharide extracted from the cell well of CA. We presently describe that, when we used strongly labeled (/sup 3/H)mannan, antigen-specific T blast cells were able to bind the labeled mannan on their membrane. The observations that irrelevant blast cells did not bind (/sup 3/H)mannan, and that mannan-specific blast cells did not bind tritiated pneumococcal polysaccharide SIII, indicate the specificity of mannan binding. Mannan binding was reversible and saturable. Mannan binding on T blast cells was inhibited by preincubation with monoclonal antibodies to T3 but not to other T cell-related molecules. The characteristics of this receptor suggest its identity with the T cell receptor for antigen. The direct binding of mannan could be either due to a cross-linking of the receptor by multivalent mannan or to a recognition of mannan in association with HLA-DQ molecules, as suggested by partial blocking of mannan binding using anti-HLA-DQ monoclonal antibodies.

  7. The Human Leukocyte Antigen–presented Ligandome of B Lymphocytes*

    PubMed Central

    Hassan, Chopie; Kester, Michel G. D.; de Ru, Arnoud H.; Hombrink, Pleun; Drijfhout, Jan Wouter; Nijveen, Harm; Leunissen, Jack A. M.; Heemskerk, Mirjam H. M.; Falkenburg, J. H. Frederik; van Veelen, Peter A.

    2013-01-01

    Peptides presented by human leukocyte antigen (HLA) molecules on the cell surface play a crucial role in adaptive immunology, mediating the communication between T cells and antigen presenting cells. Knowledge of these peptides is of pivotal importance in fundamental studies of T cell action and in cellular immunotherapy and transplantation. In this paper we present the in-depth identification and relative quantification of 14,500 peptide ligands constituting the HLA ligandome of B cells. This large number of identified ligands provides general insight into the presented peptide repertoire and antigen presentation. Our uniquely large set of HLA ligands allowed us to characterize in detail the peptides constituting the ligandome in terms of relative abundance, peptide length distribution, physicochemical properties, binding affinity to the HLA molecule, and presence of post-translational modifications. The presented B-lymphocyte ligandome is shown to be a rich source of information by the presence of minor histocompatibility antigens, virus-derived epitopes, and post-translationally modified HLA ligands, and it can be a good starting point for solving a wealth of specific immunological questions. These HLA ligands can form the basis for reversed immunology approaches to identify T cell epitopes based not on in silico predictions but on the bona fide eluted HLA ligandome. PMID:23481700

  8. Blastogenic response of human lymphocytes to antigens of Rothia dentocariosa.

    PubMed

    Fotos, P G; Gerencser, V F; Gerencser, M A

    1982-05-01

    Peripheral blood lymphocytes were isolated from 20 individuals with varying degrees of periodontal health and classified as either normal, having acute gingivitis (GV), or chronic periodontitis (PD). Crude cell wall and cytoplasmic antigens were derived from Rothia dentocariosa (RD), were applied to lymphocyte microcultures, and subjected to radioactive thymidine; the resulting lymphocyte blastogenesis (LB) was surveyed with a scintillation counter. All three groups displayed statistically similar levels of stimulation (F = 0.71), demonstrating that crude antigens of RD are not appreciably active in vitro studies of cell-mediated immunity (CMI), as measured by LB. PMID:6953091

  9. IL-36 receptor is expressed by human blood and intestinal T lymphocytes and is dose-dependently activated via IL-36β and induces CD4+ lymphocyte proliferation.

    PubMed

    Penha, Rafael; Higgins, John; Mutamba, Shilla; Barrow, Paul; Mahida, Yashwant; Foster, Neil

    2016-09-01

    We show that IL-36R is expressed by T (CD4+ and CD8+) and B (CD19+) lymphocytes in human blood and also by CD4+ T lymphocytes in the intestinal lamina propria. IL-36R protein was mostly stored in the cytoplasm of CD4 lymphocytes and B cells, during steady state conditions and the greatest expression of IL-36R mRNA was measured in CD4+ (T helper) lymphocytes. IL-36 β, which functions via IL-36R induced rapid and significant (P<0.05) proliferation of CD4+ lymphocytes, within 48h. IL-36R expression was also maintained on the surface of circulating CD4+ lymphocytes which enter the intestinal lamina propria. In conclusion our study is the first to show that (1) all human blood lymphocytes express IL-36R; (2) IL-36R expression is maintained by circulating CD4+ lymphocytes which enter the intestinal lamina propria and (3) IL-36R/IL-36 β induces rapid CD4 lymphocyte proliferation. The possible significance of these results in the context of human disease is discussed. PMID:27269181

  10. Skewed Differentiation of Circulating Vγ9Vδ2 T Lymphocytes in Melanoma and Impact on Clinical Outcome

    PubMed Central

    Toia, Francesca; Buccheri, Simona; Anfosso, Ampelio; Moschella, Francesco; Dieli, Francesco; Meraviglia, Serena; Cordova, Adriana

    2016-01-01

    Objective The aim of this study was to evaluate over time circulating γδ T lymphocytes in melanoma patients in terms of frequency, effector functions, and relationship with clinical stage and evolution, by comparing preoperative values to those obtained at a mean follow-up of 36 months or in the event of recurrence or disease progression, and to those of healthy controls. Also, we correlated the presence of tumor-infiltrating γδ T lymphocytes with clinical evolution of melanoma. Results Mean frequencies of circulating γδ T cells before and after melanoma removal were very similar and comparable to healthy subjects, but patients who progressed to stage III or IV showed a significantly decreased frequency of circulating Vγ9Vδ2 T cells. The distribution of Vγ9Vδ2 memory and effector subsets was similar in healthy subjects and melanoma patients at diagnosis, but circulating γδ T cells of patients after melanoma removal had a skewed terminally-differentiated effector memory phenotype. Highly suggestive of progressive differentiation toward a cytotoxic phenotype, Vγ9Vδ2T cells from patients at follow up had increased cytotoxic potential and limited cytokine production capability, while the opposite pattern was detected in Vγ9Vδ2T cells from patients before melanoma removal. Conclusions Follow-up data also showed that tumor infiltrating γδ T cells were significantly associated with lower mortality and relapse rates, suggesting that they may serve as a prognostic biomarker, for human melanoma. PMID:26915072

  11. Human Lymphocytes Response to Low Gamma-ray Doses

    NASA Astrophysics Data System (ADS)

    Vega-Carrillo, Héctor René; Manzanares-Acuña, Eduardo; Bañuelos-Valenzuela, Rómulo

    2002-08-01

    Radiation and non-radiation workers lymphocytes were exposed to a low strength gamma-ray field to determine heat shock protein expression in function of radiation dose. Protein identification was carried out using mAb raised against Hsp25, Hsp60, Hsp70 and Hsp90; from these, only Hsp70 protein was detected before and after lymphocyte irradiation. In all cases, an increasing trend of relative amounts of Hsp70 in function to irradiation time was observed. After 70.5 uGy gamma-ray dose, radiation worker's lymphocytes expressed more Hsp70 protein, than non radiation workers' lymphocytes, indicating a larger tolerance to gamma rays (gammatolerance), due to an adaptation process developed by his labor condition.

  12. Experimental Study on Effect of Simulated Microgravity on Structural Chromosome Instability of Human Peripheral Blood Lymphocytes

    PubMed Central

    Wei, Lijun; Liu, Chuanpeng; Kang, Li; Liu, Yufeng; Shi, Shuliang; Wu, Qiong; Li, Yu

    2014-01-01

    Experimental study was made by keeping human peripheral blood lymphocytes under simulated microgravity in a Rotary Cell Culture System bioreactor to investigate the changes that occur in the number of chromosomes, the expression rate of chromosome fragile site, and the expressions of DNA replication- and repair-related genes. Experimental results indicate simulated microgravity has no effect on the numerical chromosome instability of human peripheral blood lymphocytes, but it enhances the structural chromosome instability of human peripheral blood lymphocytes through the inhibition of DNA replication and the reduction of DNA repair. So, the mechanism of chromosome fragile site induced by simulated microgravity can be explained using the changes that occur in the chromosome structure of human peripheral blood lymphocytes, the DNA replication and repair under the effect of simulated microgravity. PMID:24963972

  13. Experimental study on effect of simulated microgravity on structural chromosome instability of human peripheral blood lymphocytes.

    PubMed

    Wei, Lijun; Liu, Chuanpeng; Kang, Li; Liu, Yufeng; Shi, Shuliang; Wu, Qiong; Li, Yu

    2014-01-01

    Experimental study was made by keeping human peripheral blood lymphocytes under simulated microgravity in a Rotary Cell Culture System bioreactor to investigate the changes that occur in the number of chromosomes, the expression rate of chromosome fragile site, and the expressions of DNA replication- and repair-related genes. Experimental results indicate simulated microgravity has no effect on the numerical chromosome instability of human peripheral blood lymphocytes, but it enhances the structural chromosome instability of human peripheral blood lymphocytes through the inhibition of DNA replication and the reduction of DNA repair. So, the mechanism of chromosome fragile site induced by simulated microgravity can be explained using the changes that occur in the chromosome structure of human peripheral blood lymphocytes, the DNA replication and repair under the effect of simulated microgravity. PMID:24963972

  14. Phenotypic and functional consequences of herpesvirus saimiri infection of human CD8+ cytotoxic T lymphocytes.

    PubMed Central

    Berend, K R; Jung, J U; Boyle, T J; DiMaio, J M; Mungal, S A; Desrosiers, R C; Lyerly, H K

    1993-01-01

    Herpesvirus saimiri (HVS) was used to infect and transform human CD8+ cytotoxic T lymphocytes (CTL), and the phenotypic and functional consequences of HVS infection of CD8+ T lymphocytes were investigated. HVS-transformed CTL no longer require antigen restimulation yet maintain their phenotype and HLA-restricted cytolytic function and specificity. The ability of HVS to transform CTL may have an important role in the functional analysis of human antigen-specific CTL. Images PMID:8396687

  15. Signaling in Human and Murine Lymphocytes in Microgravity: Parallels and Contrasts

    NASA Technical Reports Server (NTRS)

    Neal, Pellis; Alamelu, Sundaresan; Kulkarni, A. D.; Yamauchi, K.

    2006-01-01

    Immune function in space undergoes dramatic changes, some of which are detrimental to lymphocyte function. These changes may lead to significant immune suppression. Studies with human lymphocytes both in space flight and with ground-based models (NASA in vitro ground-based microgravity analog) indicate that T cell activation is inhibited in microgravity. Other lymphocyte functions, such as locomotion, are also inhibited. There is about an 80 percent homology in the immune response of mice to that of humans. A murine model was investigated because of its ability to parallel some microgravity using hind limb suspension. In in vivo antiorthostatically (AOS)-suspended mice, T cell activation is greatly suppressed, with the majority of activation related cytokines being inhibited. PHA activation in lymphocytes derived from AOS mice (in vivo ground-based microgravity analog) is also suppressed. Calcium ionophore studies in human lymphocytes exposed to modeled microgravity indicate that the calcium pathways are probably unaffected in microgravity. IP3 (inositol triphosphate) receptor expression in both human and mouse lymphocytes cultured in modeled microgravity indicate no suppression of calcium signaling. In the human system, microgravity seems to inhibit signaling cascades either at the level of, or up-stream of, Protein Kinase C (PKC). In particular, a membrane event, such as phospholipase C gamma 1 activity in human lymphocytes is affected, with its direct upstream effector, LAT, being deficiently expressed. In the mouse pathway, LAT is undiminished while another critical intermediate, SLP-76, is diminished significantly. This study identifies critical stages in the human and mouse immune systems and in lymphocytes as a function of microgravity.

  16. Cryopreservation of human lymphocytes: a brief review and evaluation of an automated liquid nitrogen freezer.

    PubMed

    Glassman, A B; Bennett, C E

    1979-01-01

    Successful cryopreservation of human lymphocytes has been previously described. Cryopreserved lymphocytes are useful for a variety of in vitro immunologic studies. This study was performed to determine the applicability and/or advantages of using a programmable freezing system, and compares glycerol versus dimethyl sulfoxide (DMSO) at varying concentrations on post-thaw viability, E-rosetting and immunoglobulin fluorescence. Prefreeze T and B lymphocyte percentages were determined. Cells were then frozen in varying concentrations of glycerol and DMSO. Optimum cryoprotectant type and concentration was determined. Lymphocytes from seven individuals were frozen by the batch method in a mechanical freezer and with the automated liquid nitrogen injection system. Data on post-thaw T and B percentages and viability revealed 10% DMSO and liquid nitrogen control freezing method at 1 C/minute as the best conditions for lymphocyte preservation as reflected by post-thaw in vitro testing. PMID:373178

  17. Analysis of the in vitro effect of exogenous nitric oxide on human lymphocytes.

    PubMed

    Shoker, A S; Yang, H; Murabit, M A; Jamil, H; al-Ghoul, A; Okasha, K

    1997-06-01

    We investigated the role of endogenous or exogenous nitric oxide (NO) on human lymphocyte function. We used sodium nitroprusside, nitroglycerine, S-nitroso-N-acetylpenicillamine, sodium nitrite and S-nitroso-L-glutathione as NO-generating compounds. All agents were used at doses that do not produce direct cytotoxicity as measured by trypan blue exclusion as well as chromium-51 release assay. The immune responses examined were peripheral blood lymphocytes (PBL) proliferation and IL-2 production after activation with OKT3 and PHA; allogeneic mediated proliferation and cell mediated cytotoxicity (CML) in MLR; IgG and IgM production after PBL activation with Con-A; proliferation and expression of IFN-gamma and IL-4 mRNA after activation of allogeneic CD4+T cell clones. Cytokine mRNA expression was measured by reverse transcriptase PCR. Our results show that proliferating lymphocytes do not produce a detectable amount of NO as measured by the Griess reaction. In separate experiments, the addition of NG-monomethyl-L-arginine (L-NMMA) did not affect lymphocyte proliferation. Sodium nitroprusside and nitroglycerine exerted a dose dependent antimitogenic effect, inhibited cytokine production and expression, CML generation and antibody production. DNA gel electrophoresis showed no evidence for enhanced programmed cell death. The antimitogenic effect could not be blocked by the NO scavengers, hemoglobin or methylene blue. In contrast, the other nitric oxide generating compounds did not inhibit lymphocyte mitogenesis. The results suggest that human lymphocytes do not produce appreciable amounts of NO to affect lymphocyte mitogenesis. Sodium nitroprusside and nitroglycerine have a potent but nonspecific immunoinhibitory effect on human lymphocyte function by a mechanism other than NO production. In addition, pharmacological levels of NO do not inhibit human lymphocyte mitogenesis. PMID:9201699

  18. Clinically used selective oestrogen receptor modulators increase LDL receptor activity in primary human lymphocytes

    PubMed Central

    Cerrato, F; Fernández-Suárez, M E; Alonso, R; Alonso, M; Vázquez, C; Pastor, O; Mata, P; Lasunción, M A; Gómez-Coronado, D

    2015-01-01

    Background and Purpose Treatment with selective oestrogen receptor modulators (SERMs) reduces low-density lipoprotein (LDL) cholesterol levels. We assessed the effect of tamoxifen, raloxifene and toremifene and their combinations with lovastatin on LDL receptor activity in lymphocytes from normolipidaemic and familial hypercholesterolaemic (FH) subjects, and human HepG2 hepatocytes and MOLT-4 lymphoblasts. Experimental Approach Lymphocytes were isolated from peripheral blood, treated with different compounds, and 1,1′-dioctadecyl-3,3,3,3′-tetramethylindocarbocyanine perchlorate (DiI)-labelled LDL uptake was analysed by flow cytometry. Key Results Tamoxifen, toremifene and raloxifene, in this order, stimulated DiI-LDL uptake by lymphocytes by inhibiting LDL-derived cholesterol trafficking and subsequent down-regulation of LDL receptor expression. Differently to what occurred in HepG2 and MOLT-4 cells, only tamoxifen consistently displayed a potentiating effect with lovastatin in primary lymphocytes. The SERM-mediated increase in LDL receptor activity was not altered by the anti-oestrogen ICI 182 780 nor was it reproduced by 17β-oestradiol. However, the tamoxifen-active metabolite endoxifen was equally effective as tamoxifen. The SERMs produced similar effects on LDL receptor activity in heterozygous FH lymphocytes as in normal lymphocytes, although none of them had a potentiating effect with lovastatin in heterozygous FH lymphocytes. The SERMs had no effect in homozygous FH lymphocytes. Conclusions and Implications Clinically used SERMs up-regulate LDL receptors in primary human lymphocytes. There is a mild enhancement between SERMs and lovastatin of lymphocyte LDLR activity, the potentiation being greater in HepG2 and MOLT-4 cells. The effect of SERMs is independent of oestrogen receptors but is preserved in the tamoxifen-active metabolite endoxifen. This mechanism may contribute to the cholesterol-lowering action of SERMs. PMID:25395200

  19. The human peripheral lymph node vascular addressin. An inducible endothelial antigen involved in lymphocyte homing.

    PubMed Central

    Michie, S. A.; Streeter, P. R.; Bolt, P. A.; Butcher, E. C.; Picker, L. J.

    1993-01-01

    The extravasation of blood-borne lymphocytes into organized lymphoid tissues and sites of chronic inflammation is directed in part by interactions of lymphocyte surface adhesion molecules, known as homing receptors, with tissue-selective endothelial ligands called vascular addressins. In mice and humans, lymphocyte L-selectin and the peripheral lymph node addressin (PNAd) form a homing receptor-endothelial ligand pair involved in lymphocyte traffic to peripheral lymph node (PLN). We have examined the tissue distribution and function of human PNAd, using monoclonal antibody MECA-79 and in vitro assays of L-selectin-dependent lymphocyte binding. We demonstrate that PNAd is expressed by human high endothelial venules (HEV) in lymphoid tissues which support lymphocyte adhesion via a PLN-associated recognition system. MECA-79 inhibits adhesion to these HEV of a cell line that binds predominantly via the PLN-homing receptor, L-selectin, but has no effect on adhesion by a mucosal HEV-binding cell line. Furthermore, MECA-79 blocks binding of human peripheral blood mononuclear cells to both PLN and tonsil HEV, but not significantly to HEV in the appendix. In addition, we demonstrate PNAd induction on venules at chronic inflammatory sites in humans, particularly sites with severe or long-standing chronic inflammatory involvement. These results confirm that PNAd functions as a PLN vascular addressin in humans, and that in addition to directing normal lymphocyte recirculation to lymph nodes and tonsils, this addressin likely participates in lymphocyte recruitment to sites of chronic inflammation. Images Figure 1 Figure 4 PMID:8256856

  20. Divergence of human and nonhuman primate lymphocyte responses to bacterial superantigens.

    PubMed

    Bavari, S; Hunt, R E; Ulrich, R G

    1995-09-01

    We compared T cell responses of human, rhesus monkey (Macaca mulatta), and chimpanzee (Pan troglodytes) to four bacterial superantigens. When lymphocytes were cultured in media supplemented with species-specific sera, chimpanzee T cells were stimulated by lower doses of staphylococcal enterotoxin (SE) A and toxic shock syndrome toxin 1 (TSST1) than were human T cells, while chimpanzee responses to SEB and SEC1 were nearly equivalent to the human response. Interestingly, rhesus lymphocytes responded to 10,000 times lower amounts of SEA, SEB, and SEC1 and to 100 times lower concentrations of TSST1 than human cells. The greater sensitivity of rhesus T cells to these toxins was not a result of differences in class II binding affinities and was only partly attributable to the presence of anti-SE and TSST1 antibodies in human serum. These results suggest that rhesus T lymphocytes are more sensitive toward these bacterial superantigens than human T cells. PMID:7554446

  1. Metastatic spread in patients with non-small cell lung cancer is associated with a reduced density of tumor-infiltrating T cells.

    PubMed

    Müller, Philipp; Rothschild, Sacha I; Arnold, Walter; Hirschmann, Petra; Horvath, Lukas; Bubendorf, Lukas; Savic, Spasenija; Zippelius, Alfred

    2016-01-01

    Tumor-infiltrating lymphocytes play an important role in cell-mediated immune destruction of cancer cells and tumor growth control. We investigated the heterogeneity of immune cell infiltrates between primary non-small cell lung carcinomas (NSCLC) and corresponding metastases. Formalin-fixed, paraffin-embedded primary tumors and corresponding metastases from 34 NSCLC patients were analyzed by immunohistochemistry for CD4, CD8, CD11c, CD68, CD163 and PD-L1. The percentage of positively stained cells within the stroma and tumor cell clusters was recorded and compared between primary tumors and metastases. We found significantly fewer CD4(+) and CD8(+) T cells within tumor cell clusters as compared with the stromal compartment, both in primary tumors and corresponding metastases. CD8(+) T cell counts were significantly lower in metastatic lesions than in the corresponding primary tumors, both in the stroma and the tumor cell islets. Of note, the CD8/CD4 ratio was significantly reduced in metastatic lesions compared with the corresponding primary tumors in tumor cell islets, but not in the stroma. We noted significantly fewer CD11c(+) cells and CD68(+) as well as CD163(+) macrophages in tumor cell islets compared with the tumor stroma, but no difference between primary and metastatic lesions. Furthermore, the CD8/CD68 ratio was higher in primary tumors than in the corresponding metastases. We demonstrate a differential pattern of immune cell infiltration in matched primary and metastatic NSCLC lesions, with a significantly lower density of CD8(+) T cells in metastatic lesions compared with the primary tumors. The lower CD8/CD4 and CD8/CD68 ratios observed in metastases indicate a rather tolerogenic and tumor-promoting microenvironment at the metastatic site. PMID:26541588

  2. Identification and characterization of a tumor infiltrating CD56(+)/CD16 (-) NK cell subset with specificity for pancreatic and prostate cancer cell lines.

    PubMed

    Frankel, Timothy L; Burns, William; Riley, John; Morgan, Richard A; Davis, Jeremy L; Hanada, Kenichi; Quezado, Martha; Rosenberg, Steven A; Royal, Richard E

    2010-12-01

    In a recent clinical trial, a patient exhibited regression of several pancreatic cancer metastases following the administration of the immune modulator Ipilimumab (anti-CTLA-4 antibody). We sought to characterize the immune cells responsible for this regression. Tumor infiltrating lymphocytes (TIL-2742) and an autologous tumor line (TC-2742) were expanded from a regressing metastatic lesion excised from this patient. Natural killer (NK) cells predominated in the TIL (92% CD56(+)) with few T cells (12% CD3(+)). A majority (88%) of the NK cells were CD56(bright)CD16(-). TIL-2742 secreted IFN-γ and GM-CSF following co-culture with TC-2742 and major histocompatibility complex mismatched pancreatic tumor lines. After sorting TIL-2742, the purified CD56(+)CD16(-)CD3(-) subset showed reactivity similar to TIL-2742 while the CD56(-)CD16(-)CD3(+) cells exhibited no tumor recognition. In co-culture assays, TIL-2742 and the NK subset expressed high reactivity to several pancreatic and prostate cancer cell lines and could lyse the autologous tumor as well as pancreas and prostate cancer lines. Reactivity was partially abrogated by blockade of TRAIL. We thus identified a unique subset of NK cells (CD56(bright)CD16(dim)) isolated from a regressing metastatic pancreatic cancer in a patient responding to Ipilimumab. This represents the first report of CD56(+)CD16(-) NK cells with apparent specificity for pancreatic and prostate cancer cell lines and associated with tumor regression following the treatment with an immune modulating agent. PMID:20734041

  3. High-Efficiency Transfection of Primary Human and Mouse T Lymphocytes Using RNA Electroporation

    PubMed Central

    Zhao, Yangbing; Zheng, Zhili; Cohen, Cyrille J.; Gattinoni, Luca; Palmer, Douglas C.; Restifo, Nicholas P.; Rosenberg, Steven A.; Morgan, Richard A.

    2006-01-01

    The use of nonviral gene transfer methods in primary lymphocytes has been hampered by low gene transfer efficiency and high transfection-related toxicity. In this report, high gene transfection efficiency with low transfection-related toxicity was achieved by electroporation using in vitro-transcribed mRNA. Using these methods, >90% transgene expression with >80% viable cells was observed in stimulated primary human and murine T lymphocytes transfected with GFP or mCD62L. Electroporation of unstimulated human PBMCs or murine splenocytes with GFP RNA yielded 95 and 56% GFP+ cells, respectively. Electroporation of mRNA for NY-ESO-1, MART-1, and p53 antigen-specific TCRs into human T lymphocytes redirected these lymphocytes to recognize melanoma cell lines in an MHC-restricted manner. The onset of gene expression was rapid (within 30 min) and durable (up to 7 days postelectroporation) using both GFP and TCR-mediated recognition of target cells. There was no adverse effect observed on the T lymphocytes subjected to RNA electroporation evaluated by cell growth rate, annexin-V staining of apoptotic cells, BrdU incorporation, tumor antigen-specific recognition or antigen-specific TCR affinity. The results of this study indicate that mRNA electroporation provides a powerful tool to introduce genes into both human and murine primary T lymphocytes. PMID:16140584

  4. Migration of human lymphocytes. I. A model using the mouse as host.

    PubMed Central

    Morgan, K; Holt, P J

    1978-01-01

    The distribution of radioactivity after the intravenous injection of 51Cr-labelled human lymphocytes has been examined in normal mice, irradiated mice, mice treated with anti-platelet antiserum and in mice treated with colloidal carbon. Pre-treatment with carbon and anti-platelet antiserum appears to protect the human lymphocytes from uptake by the host's reticuloendothelial system (RES). Comparison of tissue radioactivity in carbon-treated mice after the injection of viable human lymphocytes with that found after the injection of dead cells and soluble or insoluble cell debris showed that radioactivity recovered in the spleen and lymph nodes is primarily due to the migration of viable lymphocytes into these tissues. Thus the measurement of radioactivity in lymph nodes of carbon-treated mice after the injection of 51Cr-labelled human lymphocytes can be used as a model of these lymphocytes' ability to migrate into the lymph nodes during recirculation and to study factors influencing this migration. PMID:721139

  5. Radioprotective effect of mefenamic acid against radiation-induced genotoxicity in human lymphocytes

    PubMed Central

    Nobakht, Reyhaneh; Ghasemi, Arash; Pourfallah, Tayyeb Allahverdi

    2015-01-01

    Purpose Mefenamic acid (MEF) as a non-steroidal anti-inflammatory drug is used as a medication for relieving of pain and inflammation. Radiation-induced inflammation process is involved in DNA damage and cell death. In this study, the radioprotective effect of MEF was investigated against genotoxicity induced by ionizing radiation in human blood lymphocytes. Materials and Methods Peripheral blood samples were collected from human volunteers and incubated with MEF at different concentrations (5, 10, 50, or 100 µM) for two hours. The whole blood was exposed to ionizing radiation at a dose 1.5 Gy. Lymphocytes were cultured with mitogenic stimulation to determine the micronuclei in cytokinesis blocked binucleated lymphocyte. Results A significant decreasing in the frequency of micronuclei was observed in human lymphocytes irradiated with MEF as compared to irradiated lymphocytes without MEF. The maximum decreasing in frequency of micronuclei was observed at 100 µM of MEF (38% decrease), providing maximal protection against ionizing radiation. Conclusion The radioprotective effect of MEF is probably related to anti-inflammatory property of MEF on human lymphocytes. PMID:26484310

  6. In vitro characteristics on human lymphocyte functions of a new immunomodulatory agent, a cyclic peptide, cyclomunine.

    PubMed Central

    Niaudet, P; Beaurain, G; Leibowitch, J; Bach, J F

    1980-01-01

    Cyclomunine, a cyclic peptide extracted from Fusarium equisiti, inhibits responses of human lymphocytes to mitogens, soluble antigens and allogeneic cells and the proliferation of lymphoblastoid cell lines. Cyclomunine has little effect on small lymphocytes but acts rather on lymphoblasts. It has no effect on fibroblasts and myeloid cells. Cyclomunine partially inhibits the generation of suppressor cells induced by Con A and the generation of cytotoxic T cells in a mixed lymphocyte culture and totally inhibits the in vitro synthesis of Ig by PBL. Cyclomunine merits consideration as a new in vitro anti-lymphoblastic agent. PMID:6451339

  7. Recognition of Major Histocompatibility Complex Antigens on Cultured Human Biliary Epithelial Cells by Alloreactive Lymphocytes

    PubMed Central

    Saidman, Susan L.; Duquesnoy, Rene J.; Zeevi, Adriana; Fung, John J.; Starzl, Thomas E.; Demetris, A. Jake

    2010-01-01

    We have developed an in vitro system to study the interactions between biliary epithelium and lymphocytes using cultured human biliary epithelial cells. No class II antigens were detected by immunoperoxidase staining of the normal biliary epithelial cells, but alloactivated lymphocyte culture supernatants were able to induce class II expression. The activity of the supernatants was blocked with an anti-γ-interferon monoclonal antibody. In addition, recombinant human γ-interferon alone induced the expression of class II antigens and increased the intensity of class I staining of cultured biliary epithelial cells. Biliary epithelial cell–induced proliferation of alloreactive T lymphocytes demonstrated that the major histocompatibility complex molecules carry functional lymphocyte-activating determinants. The recognition of major histocompatibility complex determinants was confirmed by monoclonal antibody–blocking studies and by stimulation of an alloreactive T-cell clone. However, the biliary epithelial cells were much less potent stimulators than arterial endothelial cells tested in the same assay system. PMID:1704868

  8. Cytotoxic human lymphocytes: from in vitro testing (1970s) to immunotherapy (1990s).

    PubMed

    Sinkovics, J; Horvath, J

    1993-01-01

    The senior author was the recipient of a contract (1-CP3-3292) from the National Cancer Institute, USA (NCI) in the early 1970s. The aim of NCI's targeted research program was the establishment of a tumour-specific human lymphocyte-mediated cytotoxicity assay. Neither lymphocyte growth factors nor monoclonal antibodies for lymphocyte typing were available. Tumour-specific populations of lymphocytes could not be maintained but their presence in ficoll-hypaque preparations of blood buffy coats or in primary cultures of tumours was clearly recognized. Another indiscriminately cytotoxic population of lymphocytes had usually overridden the tumour-specific population. In contradistinction to the ruling doctrine of the era, indiscriminately cytotoxic lymphocytes were readily found in the blood of tumour-bearing patients and healthy individuals (the senior author's lymphocytes were shown to practice indiscriminate cytotoxicity in 1971, an observation first interpreted as "immune surveillance at work" in an individual daily exposed to patients with metastatic cancers). Instead of converting the subject matter of the contract from a tumour-specific to a non-specific cytotoxicity assay, the NCI prematurely "phased it out" (but continued the project as intramural research). Nevertheless, many functions of cytotoxic lymphocytes that had become by now well established were foreshadowed during the early 1970s with the limited support of that NCI contract and funds from other sources. Here we recount those early observations; present the outlines of adoptive immunotherapy with various autologous lymphocyte populations and in a separate report in this volume give a technical description how these lymphocyte populations are prepared in the laboratory for therapeutic reinfusions into the patient. PMID:8191863

  9. Restricted expression of LW antigen on subsets of human B and T lymphocytes.

    PubMed

    Oliveira, O L; Thomas, D B; Lomas, C G; Tippett, P

    1984-01-01

    NIM-M8 is a monoclonal IgM antibody, specific for the LWab antigen as shown by its reaction with red cells of all donors except those lacking LWa, LWb and LWab. Indirect immunofluorescent staining and cell sorter analyses have shown that LWab is present on a subpopulation of human lymphocytes. Cell fractionation studies indicate that subsets of both B and T cells express LWab and it may, therefore, provide a further marker for heterogeneity in these lymphocyte populations. PMID:6443217

  10. A think tank of TINK/TANKs: tumor-infiltrating/tumor-associated natural killer cells in tumor progression and angiogenesis.

    PubMed

    Bruno, Antonino; Ferlazzo, Guido; Albini, Adriana; Noonan, Douglas M

    2014-08-01

    Tumor-infiltrating leukocytes are often induced by the cancer microenvironment to display a protumor, proangiogenic phenotype. This "polarization" has been described for several myeloid cells, in particular macrophages. Natural killer (NK) cells represent another population of innate immune cells able to infiltrate tumors. The role of NK in tumor progression and angiogenesis has not yet been fully investigated. Several studies have shown that tumor-infiltrating NK (here referred to as "TINKs") and tumor-associated NK (altered peripheral NK cells, which here we call "TANKs") are compromised in their ability to lysew tumor cells. Recent data have suggested that they are potentially protumorigenic and can also acquire a proangiogenic phenotype. Here we review the properties of TINKs and TANKs and compare their activities to that of NK cells endowed with a physiological proangiogenic phenotype, in particular decidual NK cells. We speculate on the potential origins of TINKs and TANKs and on the immune signals involved in their differentiation and polarization. The TINK and TANK phenotype has broad implications in the immune response to tumors, ranging from a deficient control of cancer and cancer stem cells to an altered crosstalk with other relevant players of the immune response, such as dendritic cells, to induction of cancer angiogenesis. With this recently acquired knowledge that has not yet been put into perspective, we point out new potential avenues for therapeutic intervention involving NK cells as a target or an ally in oncology. PMID:25178695

  11. A Think Tank of TINK/TANKs: Tumor-Infiltrating/Tumor-Associated Natural Killer Cells in Tumor Progression and Angiogenesis

    PubMed Central

    Bruno, Antonino; Ferlazzo, Guido; Albini, Adriana; Noonan, Douglas M.

    2014-01-01

    Tumor-infiltrating leukocytes are often induced by the cancer microenvironment to display a protumor, proangiogenic phenotype. This “polarization” has been described for several myeloid cells, in particular macrophages. Natural killer (NK) cells represent another population of innate immune cells able to infiltrate tumors. The role of NK in tumor progression and angiogenesis has not yet been fully investigated. Several studies have shown that tumor-infiltrating NK (here referred to as “TINKs”) and tumor-associated NK (altered peripheral NK cells, which here we call “TANKs”) are compromised in their ability to lysew tumor cells. Recent data have suggested that they are potentially protumorigenic and can also acquire a proangiogenic phenotype. Here we review the properties of TINKs and TANKs and compare their activities to that of NK cells endowed with a physiological proangiogenic phenotype, in particular decidual NK cells. We speculate on the potential origins of TINKs and TANKs and on the immune signals involved in their differentiation and polarization. The TINK and TANK phenotype has broad implications in the immune response to tumors, ranging from a deficient control of cancer and cancer stem cells to an altered crosstalk with other relevant players of the immune response, such as dendritic cells, to induction of cancer angiogenesis. With this recently acquired knowledge that has not yet been put into perspective, we point out new potential avenues for therapeutic intervention involving NK cells as a target or an ally in oncology. PMID:25178695

  12. [Method for determining dopamine and morphine binding sites in lymphocytes from human peripheral blood].

    PubMed

    Gamaleia, N B; Kuz'mina, T I; Shostak, O A; Gamaleia, A A; Dmitrieva, I G

    2003-12-01

    A histochemical method was designed to detect the regions of binding the dopamine and morphine in human peripheral blood lymphocytes. It is based on incubating the suspension of lymphocytes and conjugated dopamine or morphine with bull serum albumin (BSA) marked by horse-radish peroxidase. After incubation, smears are prepared from the lymphocyte suspension, which are stained by diaminobenzidine in the presence of hydrogen peroxide for peroxidase. The light microscope with oil immersion is used to count the number of lymphocytes (from among 100 hundred of them), which contain the peroxidase granules. Smears from the lymphocyte suspension, which were incubated with the BSA-peroxidase conjugate, were controls. The binding of peroxidase-marked ligands of dopamine and mu-opioid receptors with lymphocytes was oppressed by the dose-dependant preliminary incubation with antagonists (haloperidol, naloxone), on the basis of which the presence of the ligand-receptor interaction can be suggested. The number of bindings of dopamine and morphine in lymphocytes was shown to be reliably higher in the alcoholic-intoxication state versus the healthy subjects without any signs of alcohol consumption. The designed method is simple enough in use and does not require any special equipment for the receptor detection in a moderate blood quantity. PMID:14971325

  13. Toxicity of methyl tertiary-butyl ether on human blood lymphocytes.

    PubMed

    Salimi, Ahmad; Vaghar-Moussavi, Mehrdad; Seydi, Enayatollah; Pourahmad, Jalal

    2016-05-01

    Methyl tertiary-butyl ether (MTBE) is a synthetic solvent widely used as oxygenate in unleaded gasoline. Few studies have addressed the cellular toxicity of MTBE on some cell lines, and so far, no comprehensive study has been conducted to investigate the probable immunotoxicity of this compound. In this study, the toxicity of MTBE on human blood lymphocytes was evaluated. Blood lymphocytes were isolated from healthy male volunteers' blood, using Ficoll polysaccharide followed by gradient centrifugation. Cell viability, reactive oxygen species (ROS) formation, lipid peroxidation, glutathione levels, and damage to mitochondria and lysosome were determined in blood lymphocytes after 6-h incubation with different concentrations of MTBE (0.1, 0.5, 1, and 2 mM). Our results showed that MTBE, in particular, decreased cell viability, which was associated with significant increase at intracellular ROS level and toxic alterations in mitochondria and lysosomes in human blood lymphocytes. Moreover, it was shown that MTBE strongly provoked lipid peroxidation and also depleted glutathione level at higher concentrations. Interestingly, MTBE exhibited its cytotoxic effects at low concentrations that may resemble to its concentrations in human blood following occupational and environmental exposure. It is therefore concluded that MTBE was capable of inducing oxidative stress and damage to mitochondria and lysosomes in human lymphocytes at concentrations ranging from 5 to 40 μg/L, which may be present in human blood as a result of environmental exposure. PMID:26797945

  14. Immunomodulation by neutrophil myeloperoxidase and hydrogen peroxide: differential susceptibility of human lymphocyte functions.

    PubMed

    el-Hag, A; Lipsky, P E; Bennett, M; Clark, R A

    1986-05-01

    The coexistence of activated polymorphonuclear leukocytes and lymphocytes in tumor masses and inflammatory tissues suggests the possibility of interaction between secreted neutrophil products and nearby lymphocytes. To test this hypothesis, we examined the effects of neutrophil myeloperoxidase and H2O2 on lymphocytes. Human peripheral blood mononuclear leukocytes were exposed to myeloperoxidase, an H2O2-generating system (glucose + glucose oxidase), and a halide, and were then tested for functional activities. Natural killer activity against K562 cells, lymphocyte proliferation in response to mitogens, and generation of immunoglobulin-secreting cells were all susceptible to oxidative injury by myeloperoxidase and H2O2. The degree as well as the mechanism of suppression was dependent on the glucose oxidase concentration (i.e., the rate of H2O2 delivery). At low H2O2 flux, myeloperoxidase was essential for induction of lymphocyte suppression; as the rate of H2O2 generation increased, suppression became myeloperoxidase-independent and was mediated by H2O2 alone. Various lymphocyte functions were differentially susceptible to oxidative injury by myeloperoxidase and H2O2. The proliferative response to poke-weed mitogen was the least sensitive, whereas antibody formation was the most sensitive. Proliferative responses to concanavalin A and phytohemagglutinin as well as natural killer activity displayed intermediate degrees of susceptibility. In all assays, lymphocyte viability was greater than 90%. Removal of monocytes from mononuclear leukocytes by adherence to glass increased susceptibility of lymphocytes to oxidative injury. Monocytes in proportions within the range present in peripheral blood mononuclear leukocytes protected lymphocyte functions against oxidative injury by myeloperoxidase and H2O2. This study demonstrates a differential susceptibility of various immune functions to oxidative injury by the neutrophil products myeloperoxidase and H2O2, and shows, in

  15. Specific high-affinity binding sites for a synthetic gliadin heptapeptide of human peripheral blood lymphocytes

    SciTech Connect

    Payan, D.G.; Horvath, K.; Graf, L.

    1987-03-23

    The synthetic peptide containing residues 43-49 of ..cap alpha..-gliadin, the major protein component of gluten, has previously been shown to inhibit the production of lymphokine activities by mononuclear leukocytes. The authors demonstrate using radiolabeled ..cap alpha..-gliadin(43-49) that human peripheral blood lymphocytes express approximately 20,000-25,000 surface receptors for this peptide, with a dissociation constant (K/sub D/) of 20 nM. In addition, binding is inhibited by naloxone and an enkephalin analog, thus confirming the functional correlate which demonstrates inhibition by these agents of ..cap alpha..-gliadin(43-49) functional effects. Furthermore, B-lymphocytes bind specifically a greater amount of (/sup 125/I)..cap alpha..-gliadin(43-49) than T-lymphocytes. The lymphocyte ..cap alpha..-gliadin(43-49) receptor may play an important role in mediating the immunological response to ..cap alpha..-gliadin. 16 references, 4 figures.

  16. COMPARATIVE GENOTOXIC RESPONSES TO ARSENITE IN GUINEA PIG, MOUSE, RAT AND HUMAN LYMPHOCYTES

    EPA Science Inventory

    Comparative genotoxic responses to arsenite in guinea pig, mouse, rat and human
    lymphocytes.

    Inorganic arsenic is a known human carcinogen causing skin, lung, and bladder cancer following chronic exposures. Yet, long-term laboratory animal carcinogenicity studies have ...

  17. Cytostatic and genotoxic effect of temephos in human lymphocytes and HepG2 cells.

    PubMed

    Benitez-Trinidad, A B; Herrera-Moreno, J F; Vázquez-Estrada, G; Verdín-Betancourt, F A; Sordo, M; Ostrosky-Wegman, P; Bernal-Hernández, Y Y; Medina-Díaz, I M; Barrón-Vivanco, B S; Robledo-Marenco, M L; Salazar, A M; Rojas-García, A E

    2015-06-01

    Temephos is an organophosphorus pesticide that is used in control campaigns against Aedes aegypti mosquitoes, which transmit dengue. In spite of the widespread use of temephos, few studies have examined its genotoxic potential. The aim of this study was to evaluate the cytotoxic, cytostatic and genotoxic effects of temephos in human lymphocytes and hepatoma cells (HepG2). The cytotoxicity was evaluated with simultaneous staining (FDA/EtBr). The cytostatic and genotoxic effects were evaluated using comet assays and the micronucleus technique. We found that temephos was not cytotoxic in either lymphocytes or HepG2 cells. Regarding the cytostatic effect in human lymphocytes, temephos (10 μM) caused a significant decrease in the percentage of binucleated cells and in the nuclear division index as well as an increase in the apoptotic cell frequency, which was not the case for HepG2 cells. The comet assay showed that temephos increased the DNA damage levels in human lymphocytes, but it did not increase the MN frequency. In contrast, in HepG2 cells, temephos increased the tail length, tail moment and MN frequency in HepG2 cells compared to control cells. In conclusion, temephos causes stable DNA damage in HepG2 cells but not in human lymphocytes. These findings suggest the importance of temephos biotransformation in its genotoxic effect. PMID:25746384

  18. Arctigenin from Arctium lappa inhibits interleukin-2 and interferon gene expression in primary human T lymphocytes

    PubMed Central

    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

  19. Assessment of in vitro genotoxic and cytotoxic effects of flurbiprofen on human cultured lymphocytes.

    PubMed

    Timocin, Taygun; Ila, Hasan Basri; Dordu, Tuba; Husunet, Mehmet Tahir; Tazehkand, Mostafa Norizadeh; Valipour, Ebrahim; Topaktas, Mehmet

    2016-07-01

    Flurbiprofen is non-steroidal anti-inflammatory drug which is commonly used for its analgesic, antipyretic, and anti-inflammatory effects. The purpose of the study was to explore the genotoxic and cytotoxic effects of flurbiprofen in human cultured lymphocytes by sister chromatid exchange, chromosome aberration, and cytokinesis-blocked micronucleus tests. 10, 20, 30, and 40 μg/mL concentrations of flurbiprofen (solvent is DMSO) were used to treatment of human cultured lymphocytes at two different treatment periods (24 and 48 h). Flurbiprofen had no significant genotoxic effect in any of these tests. But exposing to flurbiprofen for 24 and 48 h led to significant decrease on proliferation index, mitotic index, and nuclear division index (NDI). Also, all decreases were concentration-dependent (except NDI at 24 h treatment period). Consequently, the findings of this research showed that flurbiprofen had cytotoxic effects in human blood lymphocytes. PMID:26738809

  20. S15176 and S16950 interaction with Cyclosporin A antiproliferative effect on cultured human lymphocytes.

    PubMed

    Albengres, E; Le Louët, H; d'Athis, P; Tillement, J P

    2001-02-01

    S15176 and S16950 are trimetazidine derivatives that antagonize more strongly than the parent drug mitochondrial toxicity, which leads to cellular hypoxia and nephrotoxicity in kidneys experimentally exposed to cyclosporin A. We have investigated whether every derivative might interact or not with the inhibitory effect of Cyclosporin A on the proliferation of cultured human lymphocytes. S15176 significantly increased the antilymphoproliferative effect of Cyclosporin A, whereas S15176 by itself neither displayed any antilymphoproliferative effect, nor did it induce any apoptotic process in cultured human lymphocytes. The effect of S16950 was not significant. PMID:11468012

  1. Radioprotective effect of chicory seeds against genotoxicity induced by ionizing radiation in human normal lymphocytes.

    PubMed

    Hosseinimehr, S J; Ghaffari-Rad, V; Rostamnezhad, M; Ghasemi, A; Allahverdi Pourfallah, T; Shahani, S

    2015-01-01

    The search for less-toxic radioprotective agents has led to a growing trend towards natural products. Protective effect of the methanolic extract of chicory seeds (MCS) was investigated against genotoxicity induced by ionizing radiation in human lymphocytes. Human peripheral blood samples were collected and incubated with MCS at different concentrations (10, 50, 100, and 200 μg/mL) for two hours. The whole blood samples were exposed in vitro to X-ray at dose 2.5 Gy. Then, the lymphocytes were cultured with mitogenic stimulation to determine the micronucleus in cytokinesis blocked binucleated cell. The methanolic extract at all doses significantly reduced the frequency of micronuclei in binucleated lymphocytes, as compared with similarly irradiated lymphocytes without any extract treatment. The maximum protection was observed at 200 μg/mL of MCS, it completely protected genotoxicity induced by ionizing radiation in human lymphocytes. The extract exhibited a concentration-dependent radical scavenging activity on 1,1-diphenyl-2-picryl hydrazyl free radicals. HPLC analysis of MCS showed this extract is containing chlorogenic acid as a phenolic compound. These data suggest that the radioprotective effect of methanolic extract of chicory seeds can be attributed to the presence of phenolic compounds such as chlorogenic acid which act as antioxidant agents. PMID:26278267

  2. Radioprotective Effect of Achillea millefolium L Against Genotoxicity Induced by Ionizing Radiation in Human Normal Lymphocytes

    PubMed Central

    Shahani, Somayeh; Rostamnezhad, Mostafa; Ghaffari-rad, Vahid; Ghasemi, Arash; Allahverdi Pourfallah, Tayyeb

    2015-01-01

    The radioprotective effect of Achillea millefolium L (ACM) extract was investigated against genotoxicity induced by ionizing radiation (IR) in human lymphocytes. Peripheral blood samples were collected from human volunteers and incubated with the methanolic extract of ACM at different concentrations (10, 50, 100, and 200 μg/mL) for 2 hours. At each dose point, the whole blood was exposed in vitro to 2.5 Gy of X-ray and then the lymphocytes were cultured with mitogenic stimulation to determine the micronuclei in cytokinesis-blocked binucleated cell. Antioxidant capacity of the extract was determined using free radical-scavenging method. The treatment of lymphocytes with the extract showed a significant decrease in the incidence of micronuclei binucleated cells, as compared with similarly irradiated lymphocytes without any extract treatment. The maximum protection and decrease in frequency of micronuclei were observed at 200 μg/mL of ACM extract which completely protected genotoxicity induced by IR in human lymphocytes. Achillea millefolium extract exhibited concentration-dependent radical-scavenging activity on 1,1-diphenyl-2-picryl hydrazyl free radicals. These data suggest that the methanolic extract of ACM may play an important role in the protection of normal tissues against genetic damage induced by IR. PMID:26675116

  3. Genetic modification of human T lymphocytes for the treatment of hematologic malignancies

    PubMed Central

    Hoyos, Valentina; Savoldo, Barbara; Dotti, Gianpietro

    2012-01-01

    Modern chemotherapy regimens and supportive care have produced remarkable improvements in the overall survival of patients with hematologic malignancies. However, the development of targeted small molecules, monoclonal antibodies, and biological therapies that demonstrate greater efficacy and lower toxicity remains highly desirable in hematology, and oncology in general. In the context of biological therapies, T-lymphocyte based treatments have enormous potential. Donor lymphocyte infusion in patients relapsed after allogeneic hematopoietic stem cell transplant pioneered the concept that T lymphocytes can effectively control tumor growth, and this was then followed by the development of cell culture strategies to generate T lymphocytes with selective activity against tumor cells. Over the past decade, it has become clear that the adoptive transfer of ex vivo expanded antigen-specific cytotoxic T lymphocytes promotes sustained antitumor effects in patients with virus-associated lymphomas, such as Epstein-Barr virus related post-transplant lymphomas and Hodgkin's lymphomas. Because of this compelling clinical evidence and the concomitant development of methodologies for robust gene transfer to human T lymphocytes, the field has rapidly evolved, offering new opportunities to extend T-cell based therapies. This review summarizes the most recent biological and clinical developments using genetically manipulated T cells for the treatment of hematologic malignancies. PMID:22929977

  4. Lymphocyte-conditioned medium protects human monocyte-macrophages from cholesteryl ester accumulation.

    PubMed Central

    Fogelman, A M; Seager, J; Haberland, M E; Hokom, M; Tanaka, R; Edwards, P A

    1982-01-01

    Exposure of human monocyte-macrophages to as little as 50 microliters of cultured medium from lymphocytes stimulated by concanavalin A (Con A) resulted in a dramatic decrease in the activities of the low density lipoprotein (LDL) receptor pathway, the LDL-dextran sulfate pathway, and the scavenger receptor pathway. This effect was not seen when the monocyte-macrophages were exposed to culture medium from lymphocytes cultured without Con A or with Con A together with alpha-methyl mannoside or control medium without lymphocytes. The activity of 3-hydroxy-3-methyglutaryl-coenzyme A reductase also decreased in monocyte-macrophages exposed to culture medium from stimulated lymphocytes. Acyl-CoA:cholesterol O-acyltransferase activity, protein synthesis, protein content, phagocytosis of heat-killed yeast, and non-receptor-mediated endocytosis were not inhibited. Monocyte-macrophages exposed to malondialdehyde altered-LDL in the presence of stimulated lymphocyte culture medium accumulated substantially less cholesteryl esters than did cells in control medium. We propose that substances produced by stimulated lymphocytes may be useful in protecting macrophages from cholesteryl ester accumulation. Images PMID:6278500

  5. Choline deficiency increases lymphocyte apoptosis and DNA damage in humans2,3

    PubMed Central

    da Costa, Kerry-Ann; Niculescu, Mihai D; Craciunescu, Corneliu N; Fischer, Leslie M; Zeisel, Steven H

    2008-01-01

    Background: Whereas deficiency of the essential nutrient choline is associated with DNA damage and apoptosis in cell and rodent models, it has not been shown in humans. Objective: The objective was to ascertain whether lymphocytes from choline-deficient humans had greater DNA damage and apoptosis than did those from choline-sufficient humans. Design: Fifty-one men and women aged 18–70 y were fed a diet containing the recommended adequate intake of choline (control) for 10 d. They then were fed a choline-deficient diet for up to 42 d before repletion with 138–550 mg choline/d. Blood was collected at the end of each phase, and peripheral lymphocytes were isolated. DNA damage and apoptosis were then assessed by activation of caspase-3, terminal deoxynucleotide transferase–mediated dUTP nick end-labeling, and single-cell gel electrophoresis (COMET) assays. Results: All subjects fed the choline-deficient diet had lymphocyte DNA damage, as assessed by COMET assay, twice that found when they were fed the control diet. The subjects who developed organ dysfunction (liver or muscle) when fed the choline-deficient diet had significantly more apoptotic lymphocytes, as assessed by the activated caspase-3 assay, than when fed the control diet. Conclusions: A choline-deficient diet increased DNA damage in humans. Subjects in whom these diets induced liver or muscle dys-function also had higher rates of apoptosis in their peripheral lymphocytes than did subjects who did not develop organ dysfunction. Assessment of DNA damage and apoptosis in lymphocytes appears to be a clinically useful measure in humans (such as those receiving parenteral nutrition) in whom choline deficiency is suspected. PMID:16825685

  6. Single strand dna breaks in human lymphocytes exposed to para-phenylenediamine and its derivatives.

    PubMed

    Chye, Soi Mei; Hseu, You Cheng; Liang, Shih-Hsiung; Chen, Chin-Hui; Chen, Ssu Ching

    2008-01-01

    Para-Phenylenediamine (PPD), the main aromatic amines used in the hair dye formation, and its four derivatives (2-chloro-p-phenylenediamine, 4-chloro-o-phenylenediamine, 2-nitro-p-phenylenediamine, and 4-nitro-o-phenylenediamine) were examined for their potential to produce single strand DNA breaks in human lymphocytes using the alkaline comet assay. Results revealed that all the tested chemicals within the range of doses from 100 microM to 500 microM showed the genotoxicity in a dose-dependent manner after the incubation of lymphocytes with these chemicals for 2 h. In this study, we first reported that PPD and its four derivatives can elicit the type of single strand breaks in human lymphocytes. PMID:18058049

  7. Methionine synthetase activity of human lymphocytes both replete in and depleted of vitamin B12.

    PubMed

    Hall, C A; Begley, J A; Chu, R C

    1986-10-01

    The activity of the enzyme methionine synthetase (MS) (methyltetrahydrofolate:homocysteine methyltransferase) (EC 2.1.1.13) was measured in human lymphocytes of various types and cobalamin (vitamin B12) status. Total and holo MS activity was low in unstimulated peripheral blood lymphocytes from persons with tissue deficiency of cobalamin, but not in cells from those with low serum cobalamin levels for other reasons. The MS activity of the lymphocyte was increased by treatment of the patients with vitamin B12. The number of lymphocytes was often low or low normal in the circulation of those deficient in cobalamin. Holo MS activity was low in an established line of human B cells, RPMI 6410 cells, depleted of cobalamin. The total and holo MS activity of both RPMI 6410 cells, replete or depleted, and lymphocytes stimulated in culture was increased by cobalamin in vitro; 222 nmol/L free cobalamin was roughly the equivalent of 0.22 nmol/L cobalamin bound to transcobalamin II. Both lymphocytes and RPMI 6410 cells required folate for growth and could meet these needs via methylfolate, homocysteine, and the cobalamin-dependent MS reaction. Depleted RPMI 6410 cells, however, used cobalamin in some way in addition to the provision of available folate from methylfolate. The consequences of the reduced MS activity in deficient cells could include a reduction in available folate with diminished capacity for clonal expansion of lymphocytes in reaction to infection and impairment of essential methylations including those of protein synthesis. The prompt induction of MS activity by cobalamin, especially in the in vitro model, suggests an effect of therapeutic vitamin B12 well in advance of the numerical increase in cells of the blood. PMID:3760673

  8. Expression and regulation of normal and polymorphic epithelial sodium channel by human lymphocytes.

    PubMed

    Bubien, J K; Watson, B; Khan, M A; Langloh, A L; Fuller, C M; Berdiev, B; Tousson, A; Benos, D J

    2001-03-16

    Gene expression, protein expression, and function of amiloride-sensitive sodium channels were examined in human lymphocytes from normal individuals and individuals with Liddle's disease. Using reverse transcriptase polymerase chain reactions, expression of all three cloned epithelial sodium channel (ENaC) subunits was detected in lymphocytes. Polyclonal antibodies to bovine alpha-ENaC bound to the plasma membrane of normal and Liddle's lymphocytes. A quantitative analysis of fluorescence-tagged ENaC antibodies indicated a 2.5-fold greater surface binding of the antibodies to Liddle's lymphocytes compared with normal lymphocytes. The relative binding intensity increased significantly (25%; p < 0.001) for both normal and Liddle's cells after treatment with 40 microM 8-CPT-cAMP. Amiloride-sensitive whole cell currents were recorded under basal and cAMP-treated conditions for both cell types. Liddle's cells had a 4.5-fold larger inward sodium conductance compared with normal cells. A specific 25% increase in the inward sodium current was observed in normal cells in response to cAMP treatment. Outside-out patches from both cell types under both treatment conditions revealed no obvious differences in the single channel conductance. The P(open) was 4.2 +/- 3.9% for patches from non-Liddle's cells, and 27.7 +/- 5.4% in patches from Liddle's lymphocytes. Biochemical purification of a protein complex, using the same antibodies used for the immunohistochemistry, yielded a functional sodium channel complex that was inhibited by amiloride when reconstituted into lipid vesicles and incorporated into planar lipid bilayers. These four independent methodologies yielded findings consistent with the hypotheses that human lymphocytes express functional, regulatable ENaC and that the mutation responsible for Liddle's disease induces excessive channel expression. PMID:11113130

  9. Antibacterial activity of neem nanoemulsion and its toxicity assessment on human lymphocytes in vitro.

    PubMed

    Jerobin, Jayakumar; Makwana, Pooja; Suresh Kumar, R S; Sundaramoorthy, Rajiv; Mukherjee, Amitava; Chandrasekaran, Natarajan

    2015-01-01

    Neem (Azadirachta indica) is recognized as a medicinal plant well known for its antibacterial, antimalarial, antiviral, and antifungal properties. Neem nanoemulsion (NE) (O/W) is formulated using neem oil, Tween 20, and water by high-energy ultrasonication. The formulated neem NE showed antibacterial activity against the bacterial pathogen Vibrio vulnificus by disrupting the integrity of the bacterial cell membrane. Despite the use of neem NE in various biomedical applications, the toxicity studies on human cells are still lacking. The neem NE showed a decrease in cellular viability in human lymphocytes after 24 hours of exposure. The neem NE at lower concentration (0.7-1 mg/mL) is found to be nontoxic while it is toxic at higher concentrations (1.2-2 mg/mL). The oxidative stress induced by the neem NE is evidenced by the depletion of catalase, SOD, and GSH levels in human lymphocytes. Neem NE showed a significant increase in DNA damage when compared to control in human lymphocytes (P<0.05). The NE is an effective antibacterial agent against the bacterial pathogen V. vulnificus, and it was found to be nontoxic at lower concentrations to human lymphocytes. PMID:26491309

  10. Antibacterial activity of neem nanoemulsion and its toxicity assessment on human lymphocytes in vitro

    PubMed Central

    Jerobin, Jayakumar; Makwana, Pooja; Suresh Kumar, RS; Sundaramoorthy, Rajiv; Mukherjee, Amitava; Chandrasekaran, Natarajan

    2015-01-01

    Neem (Azadirachta indica) is recognized as a medicinal plant well known for its antibacterial, antimalarial, antiviral, and antifungal properties. Neem nanoemulsion (NE) (O/W) is formulated using neem oil, Tween 20, and water by high-energy ultrasonication. The formulated neem NE showed antibacterial activity against the bacterial pathogen Vibrio vulnificus by disrupting the integrity of the bacterial cell membrane. Despite the use of neem NE in various biomedical applications, the toxicity studies on human cells are still lacking. The neem NE showed a decrease in cellular viability in human lymphocytes after 24 hours of exposure. The neem NE at lower concentration (0.7–1 mg/mL) is found to be nontoxic while it is toxic at higher concentrations (1.2–2 mg/mL). The oxidative stress induced by the neem NE is evidenced by the depletion of catalase, SOD, and GSH levels in human lymphocytes. Neem NE showed a significant increase in DNA damage when compared to control in human lymphocytes (P<0.05). The NE is an effective antibacterial agent against the bacterial pathogen V. vulnificus, and it was found to be nontoxic at lower concentrations to human lymphocytes. PMID:26491309

  11. Diphtheria toxin resistance in human lymphocytes and lymphoblasts in the in vivo somatic cell mutation test

    SciTech Connect

    Tomkins, D.J.; Wei, L.; Laurie, K.E.

    1985-01-01

    It has been shown that circulating peripheral blood lymphocytes can be used for the enumeration of 6-thioguanine-resistant cells that presumably arise by mutation in vivo. This somatic cell mutation test has been studied in lymphocytes from human populations exposed to known mutagens and/or carcinogens. The sensitivity of the test could be further enhanced by including other gene markers, since there is evidence for locus-specific differences in response to mutagens. Resistance to diphtheria toxin (Dip/sup r/) seemed like a potential marker to incorporate into the test because the mutation acts codominantly, can readily be selected in human diploid fibroblasts and Chinese hamster cells with no evidence for cell density or cross-feeding effects, and can be assayed for in nondividing cells by measuring protein synthesis inhibition. Blood samples were collected from seven individuals, and fresh, cryopreserved, or Epstein-Barr virus (EBV)-transformed lymphocytes were tested for continued DNA synthesis (TH-thymidine, autoradiography) or protein synthesis (TVS-methionine, scintillation counting). Both fresh and cryopreserved lymphocytes, stimulated to divide with phytohemagglutinin (PHA), continued to synthesize DNA in the presence of high doses of diphtheria toxin (DT). Similarly, both dividing (PHA-stimulated) and nondividing fresh lymphocytes carried on significant levels of protein synthesis even 68 hr after exposure to 100 flocculating units (LF)/ml DT. The results suggest that human T and B lymphocytes may not be as sensitive to DT protein synthesis inhibition as human fibroblast and Chinese hamster cells. For this reason, Dip/sup r/ may not be a suitable marker for the somatic cell mutation test.

  12. Influence of macrophages on HSV-1 induced IL 2 production by human lymphocytes

    SciTech Connect

    Clouse, K.A.; Orosz, C.G.; Sheridan, J.F.

    1986-03-05

    Previous work has demonstrated that human peripheral blood mononuclear cells (PBMC) from HSV-1 seropositive individuals produce interleukin 2 (IL 2) following stimulation in vitro with uv-inactivated herpes simplex virus type 1 (HSV antigen). This study investigated the accessory macrophage (MO) and monokine requirements for IL 2 production by enriched T lymphocytes from HSV-1 seropositive individuals. Following removal of accessory MO populations, enriched T lymphocytes did not secrete IL 2 in response to HSV antigen. However, IL 2 production was restored by the addition of autologous, ..gamma..-irradiated (5000R) MO. HSV antigen-pulsed MO also induced IL 2 production by enriched T lymphocytes. Furthermore, when HSV-pulsed macrophages were treated with paraformaldehyde they no longer caused T lymphocytes to produce IL 2 unless exogenous monokines were provided. Neither exogenous monokines nor purified human IL 1 could support HSV antigen induced IL 2 production in the absence of MO. These studies demonstrated that MO are required for HSV-induced IL 2 production by T lymphocytes from HSV-1 seropositive individuals. Furthermore, these MO appear to provide two functions required for IL 2 production: viral antigen display and monokine production.

  13. Chemopreventive effect and lack of genotoxicity and mutagenicity of the exopolysaccharide botryosphaeran on human lymphocytes.

    PubMed

    Malini, M; Camargo, M S; Hernandes, L C; Vargas-Rechia, C G; Varanda, E A; Barbosa, A M; Dekker, R F H; Matsumoto, S T; Antunes, L M G; Cólus, I M S

    2016-10-01

    Carbohydrate biopolymers of fungal-origin are an important natural resource in the search for new bioagents with therapeutic and nutraceutical potential. In this study the mutagenic, genotoxic, antigenotoxic and antioxidant properties of the fungal exopolysaccharide botryosphaeran, a (1→3)(1→6)-β-D-glucan, from Botryosphaeria rhodina MAMB-05, was evaluated. The mutagenicity was assessed at five concentrations in Salmonella typhimurium by the Ames test. Normal and tumor (Jurkat cells) human T lymphocyte cultures were used to evaluate the genotoxicity and antigenotoxicity (Comet assay) of botryosphaeran alone and in combination with the mutagen methyl methanesulfonate (MMS). The ability of botryosphaeran to reduce the production of reactive oxygen and nitrogen species (RONS) generated by hydrogen peroxide was assessed using the CM-H2DCFDA probe in lymphocyte cultures under different treatment times. None of the evaluated botryosphaeran concentrations were mutagenic in bacteria, nor induced genotoxicity in normal and tumor lymphocytes. Botryosphaeran protected lymphocyte DNA against damage caused by MMS under simultaneous treatment and post-treatment conditions. However, botryosphaeran was not able to reduce the RONS generated by H2O2. Besides the absence of genotoxicity, botryosphaeran exerted a protective effect on human lymphocytes against genotoxic damage caused by MMS. These results are important in the validation of botryosphaeran as a therapeutic agent targeting health promotion. PMID:27387458

  14. Inorganic arsenic represses interleukin-17A expression in human activated Th17 lymphocytes

    SciTech Connect

    Morzadec, Claudie; Macoch, Mélinda; Robineau, Marc; Sparfel, Lydie; Fardel, Olivier; Vernhet, Laurent

    2012-08-01

    Trivalent inorganic arsenic [As(III)] is an efficient anticancer agent used to treat patients suffering from acute promyelocytic leukemia. Recently, experimental studies have clearly demonstrated that this metalloid can also cure lymphoproliferative and/or pro-inflammatory syndromes in different murine models of chronic immune-mediated diseases. T helper (Th) 1 and Th17 lymphocytes play a central role in development of these diseases, in mice and humans, especially by secreting the potent pro-inflammatory cytokine interferon-γ and IL-17A, respectively. As(III) impairs basic functions of human T cells but its ability to modulate secretion of pro-inflammatory cytokines by differentiated Th lymphocytes is unknown. In the present study, we demonstrate that As(III), used at concentrations clinically achievable in plasma of patients, has no effect on the secretion of interferon-γ from Th1 cells but almost totally blocks the expression and the release of IL-17A from human Th17 lymphocytes co-stimulated for five days with anti-CD3 and anti-CD28 antibodies, in the presence of differentiating cytokines. In addition, As(III) specifically reduces mRNA levels of the retinoic-related orphan receptor (ROR)C gene which encodes RORγt, a key transcription factor controlling optimal IL-17 expression in fully differentiated Th17 cells. The metalloid also blocks initial expression of IL-17 gene induced by the co-stimulation, probably in part by impairing activation of the JNK/c-Jun pathway. In conclusion, our results demonstrate that As(III) represses expression of the major pro-inflammatory cytokine IL-17A produced by human Th17 lymphocytes, thus strengthening the idea that As(III) may be useful to treat inflammatory immune-mediated diseases in humans. -- Highlights: ► Arsenic inhibits secretion of IL-17A from human naïve and memory Th17 lymphocytes. ► Arsenic represses early expression of IL-17A gene in human activated T lymphocytes. ► Arsenic interferes with activation of

  15. Lymphocytes as cellular vehicles for gene therapy in mouse and man

    SciTech Connect

    Culver, K.; Cornetta, K.; Morgan, R.; Morecki, S.; Aebersold, P.; Kasid, A.; Lotze, M.; Rosenberg, S.A.; Anderson, W.F.; Blaese, R.M. )

    1991-04-15

    The application of bone marrow gene therapy has been stalled by the inability to achieve stable high-level gene transfer and expression in the totipotent stem cells. The authors that retroviral vectors can stably introduce genes into antigen-specific murine and human T lymphocytes in culture. Murine helper T cells were transduced with the retroviral vector SAX to express both neomycin-resistance and human adenosine deaminase genes. To determine if cultured T cells might be used for gene therapy, their persistence and continued expression of the introduced genes was evaluated in nude mice transplanted with the SAX-transduced T cells. They studied cultured human tumor-infiltrating lymphocytes as a candidate cell for a trial of gene transfer in man. Gene insertion and subsequent G418 selection did not substantially alter the growth characteristics, interleukin 2 dependence, membrane phenotype, or cytotoxicity profile of the transduced T cells. These studies provided a portion of the experimental evidence supporting the feasibility of the presently ongoing clinical trials of lymphocyte gene therapy in cancer as well as in patients with adenosine deaminase deficiency.

  16. INDUCTION OF MICRONUCLEI BY X-RADIATION IN HUMAN, MOUSE, AND RAT PERIPHERAL BLOOD LYMPHOCYTES

    EPA Science Inventory

    We compared the radiosensitivity of human, rat, and mouse peripheral blood lymphocytes (PBLs) by analyzing micronuclei (MN) in cytochalasin B-induced binucleated (BN) cells. or each species and dose, 4 ml aliquots of whole blood were X-irradiated to obtain doses of 38, 75, 150, o...

  17. Transfection of an immunoglobulin kappa gene into mature human B lymphocytes

    SciTech Connect

    Bich-Thuy, L.T.; Queen, C.

    1988-01-01

    The authors show in this report that the transcription induced by interleukin-2 or pokeweed mitogens of the kappa MOPC 41 immunoglobulin light-chain gene transfected into primary human or murine B lymphocytes initiates from a previously unobserved start site about 26 base pairs upstream of the start site used in myeloma cell lines.

  18. The Effect of a Grape Seed Extract on Radiation-Induced DNA Damage in Human Lymphocytes

    NASA Astrophysics Data System (ADS)

    Dicu, Tiberius; Postescu, Ion D.; Foriş, Vasile; Brie, Ioana; Fischer-Fodor, Eva; Cernea, Valentin; Moldovan, Mircea; Cosma, Constantin

    2009-05-01

    Plant-derived antioxidants due to their phenolic compounds content are reported as potential candidates for reducing the levels of oxidative stress in living organisms. Grape seed extracts are very potent antioxidants and exhibit numerous interesting pharmacologic activities. Hydroethanolic (50/50, v/v) standardized extract was obtained from red grape seed (Vitis vinifera, variety Burgund Mare—BM). The total polyphenols content was evaluated by Folin-Ciocalteu procedure and expressed as μEq Gallic Acid/ml. The aim of this study was to evaluate the potential antioxidant effects of different concentrations of BM extract against 60Co γ-rays induced DNA damage in human lymphocytes. Samples of human lymphocytes were incubated with BM extract (12.5, 25.0 and 37.5 μEq GA/ml, respectively) administered at 30 minutes before in vitro irradiation with γ-rays (2 Gy). The DNA damage and repair in lymphocytes were evaluated using alkaline comet assay. Using the lesion score, the radiation-induced DNA damage was found to be significantly different (p<0.05) from control, both in the absence and presence of BM extract (except the lymphocytes treated with 37.5 μEq GA/ml BM extract). DNA repair analyzed by incubating the irradiated cells at 37° C and 5% CO2 atmosphere for 2 h, indicated a significant difference (p<0.05) in the lymphocytes group treated with 25.0 μEq GA/ml BM extract, immediately and two hours after irradiation. These results suggest radioprotective effects after treatment with BM extract in human lymphocytes.

  19. Inhibition of the lymphocyte metabolic switch by the oxidative burst of human neutrophils.

    PubMed

    Kramer, Philip A; Prichard, Lynn; Chacko, Balu; Ravi, Saranya; Overton, E Turner; Heath, Sonya L; Darley-Usmar, Victor

    2015-09-01

    Activation of the phagocytic NADPH oxidase-2 (NOX-2) in neutrophils is a critical process in the innate immune system and is associated with elevated local concentrations of superoxide, hydrogen peroxide (H2O2) and hypochlorous acid. Under pathological conditions, NOX-2 activity has been implicated in the development of autoimmunity, indicating a role in modulating lymphocyte effector function. Notably, T-cell clonal expansion and subsequent cytokine production requires a metabolic switch from mitochondrial respiration to aerobic glycolysis. Previous studies demonstrate that H2O2 generated from activated neutrophils suppresses lymphocyte activation but the mechanism is unknown. We hypothesized that activated neutrophils would prevent the metabolic switch and suppress the effector functions of T-cells through a H2O2-dependent mechanism. To test this, we developed a model co-culture system using freshly isolated neutrophils and lymphocytes from healthy human donors. Extracellular flux analysis was used to assess mitochondrial and glycolytic activity and FACS analysis to assess immune function. The neutrophil oxidative burst significantly inhibited the induction of lymphocyte aerobic glycolysis, caused inhibition of oxidative phosphorylation and suppressed lymphocyte activation through a H2O2-dependent mechanism. Hydrogen peroxide and a redox cycling agent, DMNQ, were used to confirm the impact of H2O2 on lymphocyte bioenergetics. In summary, we have shown that the lymphocyte metabolic switch from mitochondrial respiration to glycolysis is prevented by the oxidative burst of neutrophils. This direct inhibition of the metabolic switch is then a likely mechanism underlying the neutrophil-dependent suppression of T-cell effector function. PMID:25951298

  20. Loss of telomeric DNA during aging of normal and trisomy 21 human lymphocytes

    SciTech Connect

    Vaziri, H.; Uchida, I.; Lan Wei; Harley, C.B. ); Schaechter, F.; Cohen, D. ); Xiaoming Zhu; Effros, R. )

    1993-04-01

    The telomere hypothesis of cellular aging proposes that loss of telomeric DNA (TTAGGG) from human chromosomes may ultimately cause cell-cycle exit during replicative senescence. Since lymphocytes have a limited replicative capacity and since blood cells were previously shown to lose telomeric DNA during aging in vivo, the authors wished to determine (a) whether accelerated telomere loss is associated with the premature immunosenescence of lymphocytes in individuals with Down syndrome (DS) and (b) whether telomeric DNA is also lost during aging of lymphocytes in vitro. To investigate the effects of aging and trisomy 21 on telomere loss in vivo, genomic DNA was isolated from peripheral blood lymphocytes of 140 individuals (age 0--107 years), including 21 DS patients (age 0--45 years). Digestion with restriction enzymes HinfI and RsaI generated terminal restriction fragments (TRFs), which were detected by Southern analysis using a telomere-specific probe ([sup 32]P-(C[sub 3]TA[sub 2])[sub 3]). The rate of telomere loss was calculated from the decrease in mean TRF length, as a function of donor age. DS patients showed a significantly higher rate of telomere loss with donor age (133 [+-] 15 bp/year) compared with age-matched controls (41 [+-] 7.7 bp/year) (P < .0005), suggesting that accelerated telomere loss is a biomarker of premature immunosenescence of DS patients and that it may play a role in this process. Telomere loss during aging in vitro was calculated for lymphocytes from four normal individuals, grown in culture for 10--30 population doublings. The rate of telomere loss was [approximately]120 bp/cell doubling, comparable to that seen in other somatic cells. Moreover, telomere lengths of lymphocytes from centenarians and from older DS patients were similar to those of senescent lymphocytes in culture, which suggests that replicative senescence could partially account for aging of the immune system in DS patients and in elderly individuals. 31 refs., 3 figs.

  1. In vitro modeling of the interaction between human epithelial cells and lymphocytes upon influenza infection.

    PubMed

    Ilyushina, Natalia A; Wright, Peter F

    2016-09-01

    Influenza viruses are a continuous threat to humans because of their ability to cross species barriers and adapt to new hosts. Data from murine studies, along with limited human data, suggest that CD8(+) cytotoxic T lymphocytes (CTL) that recognize conserved epitopes of structural influenza proteins are the main mediators of influenza virus clearance. Additionally, the fact that many CTLs recognize epitopes shared between different influenza strains offers the potential for broad cross-strain immunity. However, the mechanisms of cellular immunity against influenza viruses are poorly defined in humans, where the CTL response has been hard to measure and interpret. We developed a novel CTL assay that utilizes fully differentiated nasal human epithelial cells taken from volunteers as permissive targets for autologous peripheral blood-derived influenza virus-specific cytotoxic T lymphocytes. This in vitro system of human lymphocyte-epithelial cell co-cultures can be considered as the closest approximation to events in vivo and can be employed for studying the interactions between the pathogen and human host. Modeling of the natural interaction process between the primary cell type that supports the productive replication of influenza and immune cells may allow us to put in perspective CTLs as a correlate of immunity to influenza in humans. PMID:27102577

  2. On-line studies of activation events in primary human T lymphocytes.

    PubMed

    Bental, M; Deutsch, C

    1994-04-01

    In this paper, we review our NMR studies of human peripheral blood T lymphocytes. These studies focus on the physiological and biochemical alterations accompanying cell cycle progression. In particular, we have characterized phosphorus metabolism, glucose utilization and lactate production, and pH regulation using 31P, 13C, and 19F NMR, respectively. These studies required developing new methods for monitoring on-line stimulation of quiescent T cells under sterile, physiological conditions (i.e., CO2/HCO3- buffer, 37 degrees C) for prolonged periods of time. A perfusion system optimized for T lymphocytes inside agarose beads is described. In addition, custom-designed 19F NMR pH indicators were synthesized, characterized, and used to determine intracellular pH in quiescent lymphocytes, stimulated lymphocytes, and lymphocytes undergoing the G0-G1 transition. These unique molecular probes are described in detail. Finally, the physiological relevance of our findings regarding carbon metabolism and pH regulation is considered in the context of mitogenesis. PMID:8069534

  3. Mutant Huntingtin Does Not Affect the Intrinsic Phenotype of Human Huntington’s Disease T Lymphocytes

    PubMed Central

    Miller, James R. C.; Träger, Ulrike; Andre, Ralph; Tabrizi, Sarah J.

    2015-01-01

    Huntington’s disease is a fatal neurodegenerative condition caused by a CAG repeat expansion in the huntingtin gene. The peripheral innate immune system is dysregulated in Huntington’s disease and may contribute to its pathogenesis. However, it is not clear whether or to what extent the adaptive immune system is also involved. Here, we carry out the first comprehensive investigation of human ex vivo T lymphocytes in Huntington’s disease, focusing on the frequency of a range of T lymphocyte subsets, as well as analysis of proliferation, cytokine production and gene transcription. In contrast to the innate immune system, the intrinsic phenotype of T lymphocytes does not appear to be affected by the presence of mutant huntingtin, with Huntington’s disease T lymphocytes exhibiting no significant functional differences compared to control cells. The transcriptional profile of T lymphocytes also does not appear to be significantly affected, suggesting that peripheral immune dysfunction in Huntington’s disease is likely to be mediated primarily by the innate rather than the adaptive immune system. This study increases our understanding of the effects of Huntington’s disease on peripheral tissues, while further demonstrating the differential effects of the mutant protein on different but related cell types. Finally, this study suggests that the potential use of novel therapeutics aimed at modulating the Huntington’s disease innate immune system should not be extended to include the adaptive immune system. PMID:26529236

  4. Toxicological Implications and Inflammatory Response in Human Lymphocytes Challenged with Oxytetracycline.

    PubMed

    Di Cerbo, A; Palatucci, A T; Rubino, V; Centenaro, S; Giovazzino, A; Fraccaroli, E; Cortese, L; Ruggiero, G; Guidetti, G; Canello, S; Terrazzano, G

    2016-04-01

    Antibiotics are widely used in zoo technical and veterinary practices as feed supplementation to ensure wellness of farmed animals and livestock. Several evidences have been suggesting both the toxic role for tetracyclines, particularly for oxytetracycline (OTC). This potential toxicity appears of great relevance for human nutrition and for domestic animals. This study aimed to extend the evaluation of such toxicity. The biologic impact of the drug was assessed by evaluating the proinflammatory effect of OTC and their bone residues on cytokine secretion by in vitro human peripheral blood lymphocytes. Our results showed that both OTC and OTC-bone residues significantly induced the T lymphocyte and non-T cell secretion of interferon (IFN)-γ, as cytokine involved in inflammatory responses in humans as well as in animals. These results may suggest a possible implication for new potential human and animal health risks depending on the entry of tetracyclines in the food-processing chain. PMID:26537863

  5. Potassium currents inhibition by gambierol analogs prevents human T lymphocyte activation.

    PubMed

    Rubiolo, J A; Vale, C; Martín, V; Fuwa, H; Sasaki, M; Botana, L M

    2015-07-01

    Gambierol is a marine polycyclic ether toxin, produced along with ciguatoxin congeners by the dinoflagellate Gambierdiscus toxicus. We have recently reported that two truncated skeletal analogs of gambierol comprising the EFGH- and BCDEFGH-rings of the parent compound showed similar potency to gambierol on voltage-gated potassium channels (Kv) inhibition in neurons. Gambierol and its truncated analogs share the main crucial elements for biological activity, which are the C28=C29 double bond within the H-ring and the unsaturated side chain. Since Kv channels are critical for the regulation of calcium signaling, proliferation, secretion and migration in human T lymphocytes, we evaluated the activity of both the tetracyclic and heptacyclic analogs of gambierol on potassium currents in resting T lymphocyte and their effects on interleukin-2 (IL-2) release and gene expression in activated T lymphocytes. The results presented in this work clearly demonstrate that both truncated analogs of gambierol inhibit Kv channels present in resting T lymphocytes (Kv1.3) and prevented lymphocyte activation by concanavalin A. The main effects of the heptacyclic and tetracyclic analogs of gambierol in human T cells are: (1) inhibition of potassium channels in resting and concanavalin-activated T cells in the nanomolar range, (2) inhibition of IL-2 release from concanavalin-activated T cells and (3) negatively affect the expression of genes involved in cell proliferation and immune response observed in concanavalin-activated lymphocytes. These results together with the lack of toxicity in this cellular model, indicates that both analogs of gambierol have additional potential for the development of therapeutic tools in autoimmune diseases. PMID:25155189

  6. Patient-Derived Tumor Xenografts Are Susceptible to Formation of Human Lymphocytic Tumors1

    PubMed Central

    Bondarenko, Gennadiy; Ugolkov, Andrey; Rohan, Stephen; Kulesza, Piotr; Dubrovskyi, Oleksii; Gursel, Demirkan; Mathews, Jeremy; O’Halloran, Thomas V.; Wei, Jian J.; Mazar, Andrew P.

    2015-01-01

    Patient-derived xenograft (PDX) tumor models have emerged as a new approach to evaluate the effects of cancer drugs on patients’ personalized tumor grafts enabling to select the best treatment for the cancer patient and providing a new tool for oncology drug developers. Here, we report that human tumors engrafted in immunodeficient mice are susceptible to formation of B-and T-cell PDX tumors. We xenografted human primary and metastatic tumor samples into immunodeficient mice and found that a fraction of PDX tumors generated from patients’ samples of breast, colon, pancreatic, bladder and renal cancer were histologically similar to lymphocytic neoplasms. Moreover, we found that the first passage of breast and pancreatic cancer PDX tumors after initial transplantation of the tumor pieces from the same human tumor graft could grow as a lymphocytic tumor in one mouse and as an adenocarcinoma in another mouse. Whereas subcutaneous PDX tumors resembling human adenocarcinoma histology were slow growing and non-metastatic, we found that subcutaneous PDX lymphocytic tumors were fast growing and formed large metastatic lesions in mouse lymph nodes, liver, lungs, and spleen. PDX lymphocytic tumors were comprised of B-cells which were Epstein-Barr virus positive and expressed CD45 and CD20. Because B-cells are typically present in malignant solid tumors, formation of B-cell tumor may evolve in a wide range of PDX tumor models. Although PDX tumor models show great promise in the development of personalized therapy for cancer patients, our results suggest that confidence in any given PDX tumor model requires careful screening of lymphocytic markers. PMID:26476081

  7. Epstein-Barr virus transformation induces B lymphocytes to produce human interleukin 10.

    PubMed

    Burdin, N; Péronne, C; Banchereau, J; Rousset, F

    1993-02-01

    Interleukin 10 (IL-10) is a pleiotropic factor that enhances proliferation of activated human B lymphocytes and induces them to secrete high amounts of immunoglobulins. Here we show that several human B cell lines were able to constitutively secrete human (h)IL-10. Whereas none of the pre-B nor the plasmocytic cell lines tested produced hIL-10, 25 of the 36 tested mature B cell lines (lymphoblastoid and Burkitt lymphoma cell lines) secreted hIL-10. Moreover, 24 of these 25 hIL-10-producing B cell lines contained the Epstein-Barr virus (EBV) genome, suggesting a relationship between hIL-10 production by human B cell lines and EBV expression. Accordingly, whereas polyclonal activation via triggering of surface immunoglobulins or CD40 antigen induced highly purified normal human B lymphocytes to produce only low (0.3-0.4 ng/ml) but significant amounts of hIL-10, EBV infection induced them to secrete high amounts of hIL-10 (4-9 ng/ml). Furthermore, addition of exogenous hIL-10, simultaneously to EBV infection, potentiated cell proliferation, whereas a blocking anti-IL-10 antiserum inhibited it. Thus, hIL-10 produced by infected human B lymphocytes appears to be involved in the mechanisms of EBV-induced B cell proliferation. PMID:8381152

  8. Inhibitory effects of extracellular products from oral bacteria on human fibroblasts and stimulated lymphocytes.

    PubMed Central

    Higerd, T B; Vesole, D H; Goust, J M

    1978-01-01

    Extracellular products of 12 strains of Streptococcus mutans and 5 additional species of oral bacteria were analyzed for their ability to inhibit proliferation of fibroblastoid cells (HeLa and AV3) and blast transformation of human peripheral blood lymphocytes obtained from normal individuals. Products from S. mutans strains AHT and BHT, Streptococcus intermedius, and Actinomyces viscosus inhibited [3H]thymidine uptake by fibroblastoid cells and phytohemagglutinin-stimulated lymphocytes. Products from S. mutans E49, Streptococcus salivarius, and Actinomyces naeslundii inhibited blast transformation of human lymphocytes but did not significantly inhibit the growth of fibroblastoid cells. Preparations from S. intermedius gave the greatest inhibitory activity against both target cell types; initial characterization of this preparation suggested a single factor active in both assays, in that the heat lability and Sephadex G-200 elution profile were similar for the inhibitory activity seen with the two cell types. The molecular weight of the inhibitor, estimated by gel filtration on Sephadex G-200 and Ultragel AcA34, was approximately 160,000. The results strongly suggest that oral bacteria produce heat-labile substances that interfere with fibroblast proliferation and alter the lymphocytic immunological response. Images PMID:689736

  9. Inhibitory effects of extracellular products from oral bacteria on human fibroblasts and stimulated lymphocytes.

    PubMed

    Higerd, T B; Vesole, D H; Goust, J M

    1978-08-01

    Extracellular products of 12 strains of Streptococcus mutans and 5 additional species of oral bacteria were analyzed for their ability to inhibit proliferation of fibroblastoid cells (HeLa and AV3) and blast transformation of human peripheral blood lymphocytes obtained from normal individuals. Products from S. mutans strains AHT and BHT, Streptococcus intermedius, and Actinomyces viscosus inhibited [3H]thymidine uptake by fibroblastoid cells and phytohemagglutinin-stimulated lymphocytes. Products from S. mutans E49, Streptococcus salivarius, and Actinomyces naeslundii inhibited blast transformation of human lymphocytes but did not significantly inhibit the growth of fibroblastoid cells. Preparations from S. intermedius gave the greatest inhibitory activity against both target cell types; initial characterization of this preparation suggested a single factor active in both assays, in that the heat lability and Sephadex G-200 elution profile were similar for the inhibitory activity seen with the two cell types. The molecular weight of the inhibitor, estimated by gel filtration on Sephadex G-200 and Ultragel AcA34, was approximately 160,000. The results strongly suggest that oral bacteria produce heat-labile substances that interfere with fibroblast proliferation and alter the lymphocytic immunological response. PMID:689736

  10. Protective Effect of Onion Extract on Bleomycin-Induced Cytotoxicity and Genotoxicity in Human Lymphocytes

    PubMed Central

    Cho, Yoon Hee; Lee, Joong Won; Woo, Hae Dong; Lee, Sunyeong; Kim, Yang Jee; Lee, Younghyun; Shin, Sangah; Joung, Hyojee; Chung, Hai Won

    2016-01-01

    Following one of the world’s largest nuclear accidents, occured at Fukushima, Japan in 2011, a significant scientific effort has focused on minimizing the potential adverse health effects due to radiation exposure. The use of natural dietary antioxidants to reduce the risk of radiation-induced oxidative DNA damage is a simple strategy for minimizing radiation-related cancer rates and improving overall health. The onion is among the richest sources of dietary flavonoids and is an important food for increasing their overall intake. Therefore, we examined the effect of an onion extract on cyto- and geno-toxicity in human lymphocytes treated with bleomycin (BLM), a radiomimetic agent. In addition, we measured the frequency of micronuclei (MN) and DNA damage following treatment with BLM using a cytokinesis-blocked micronucleus assay and a single cell gel electrophoresis assay. We observed a significant increase in cell viability in lymphocytes treated with onion extract then exposed to BLM compared to cells treated with BLM alone. The frequency of BLM induced MN and DNA damage increased in a dose-dependent manner; however, when lymphocytes were pretreated with onion extract (10 and 20 μL/mL), the frequency of BLM-induced MN was decreased at all doses of BLM and DNA damage was decreased at 3 μg/mL of BLM. These results suggest that onion extract may have protective effects against BLM-induced cyto- and genotoxicity in human lymphocytes. PMID:26907305

  11. Human malignant melanoma-derived progestagen-associated endometrial protein immunosuppresses T lymphocytes in vitro.

    PubMed

    Ren, Suping; Chai, Lina; Wang, Chunyan; Li, Changlan; Ren, Qiquan; Yang, Lihua; Wang, Fumei; Qiao, Zhixin; Li, Weijing; He, Min; Riker, Adam I; Han, Ying; Yu, Qun

    2015-01-01

    Progestagen-associated endometrial protein (PAEP) is a glycoprotein of the lipocalin family that acts as a negative regulator of T cell receptor-mediated activation. However, the function of tumor-derived PAEP on the human immune system in the tumor microenvironment is unknown. PAEP is highly expressed in intermediate and thick primary melanomas (Breslow's 2.5mm or greater) and metastatic melanomas, correlating with its expression in daughter cell lines established in vitro. The current study investigates the role of melanoma cell-secreted PAEP protein in regulating T cell function. Upon the enrichment of CD3+, CD4+ and CD8+ T cells from human peripheral blood mononuclear cells, each subset was then mixed with either melanoma-derived PAEP protein or PAEP-poor supernatant of gene-silenced tumor cells. IL-2 and IFN-γ secretion of CD4+ T cells significantly decreased with the addition of PAEP-rich supernatant. And the addition of PAEP-positive cell supernatant to activated lymphocytes significantly inhibited lymphocyte proliferation and cytotoxic T cell activity, while increasing lymphocyte apoptosis. Our result suggests that melanoma cell-secreted PAEP protein immunosuppresses the activation, proliferation and cytotoxicity of T lymphocytes, which might partially explain the mechanism of immune tolerance induced by melanoma cells within the tumor microenvironment. PMID:25785839

  12. Protective Effect of Onion Extract on Bleomycin-Induced Cytotoxicity and Genotoxicity in Human Lymphocytes.

    PubMed

    Cho, Yoon Hee; Lee, Joong Won; Woo, Hae Dong; Lee, Sunyeong; Kim, Yang Jee; Lee, Younghyun; Shin, Sangah; Joung, Hyojee; Chung, Hai Won

    2016-02-01

    Following one of the world's largest nuclear accidents, occured at Fukushima, Japan in 2011, a significant scientific effort has focused on minimizing the potential adverse health effects due to radiation exposure. The use of natural dietary antioxidants to reduce the risk of radiation-induced oxidative DNA damage is a simple strategy for minimizing radiation-related cancer rates and improving overall health. The onion is among the richest sources of dietary flavonoids and is an important food for increasing their overall intake. Therefore, we examined the effect of an onion extract on cyto- and geno-toxicity in human lymphocytes treated with bleomycin (BLM), a radiomimetic agent. In addition, we measured the frequency of micronuclei (MN) and DNA damage following treatment with BLM using a cytokinesis-blocked micronucleus assay and a single cell gel electrophoresis assay. We observed a significant increase in cell viability in lymphocytes treated with onion extract then exposed to BLM compared to cells treated with BLM alone. The frequency of BLM induced MN and DNA damage increased in a dose-dependent manner; however, when lymphocytes were pretreated with onion extract (10 and 20 μL/mL), the frequency of BLM-induced MN was decreased at all doses of BLM and DNA damage was decreased at 3 μg/mL of BLM. These results suggest that onion extract may have protective effects against BLM-induced cyto- and genotoxicity in human lymphocytes. PMID:26907305

  13. The tax gene of human T-cell leukemia virus type 2 is essential for transformation of human T lymphocytes.

    PubMed Central

    Ross, T M; Pettiford, S M; Green, P L

    1996-01-01

    The mechanism of human T-cell leukemia virus (HTLV)-mediated transformation and induction of malignancy is unknown; however, several studies have implicated the viral gene product, Tax. Conclusive evidence for the role of Tax in the HTLV malignant process has been impeded by the inability to mutate tax in the context of an infectious virus and dissociate viral replication from cellular transformation. To circumvent this problem we constructed a mutant of HTLV type 2 (HTLV-2) that replicates by a Tax-independent mechanism. For these studies, the Tax response element in the viral long terminal repeat was replaced with the cytomegalovirus immediate-early promoter enhancer (C-enh). Transcription of the chimeric HTLV-2 (HTLVC-enh) was efficiently directed by this heterologous promoter. Also, the chimeric virus transformed primary human T lymphocytes with an efficiency similar to that of wild-type HTLV-2. A tax-knockout virus, termed HTLVC-enhDeltaTax, was constructed to directly assess the importance of Tax in cellular transformation. Transfection and infection studies indicated that HTLVC-enhDeltaTax was replication competent; however, HTLVC-enhDeltaTax failed to transform primary human T lymphocytes. We conclude that Tax is essential for HTLV-mediated transformation of human T lymphocytes. Furthermore, this chimeric HTLV, that replicates in the absence of Tax, should facilitate studies to determine the precise mechanism of T-lymphocyte transformation by HTLV. PMID:8764028

  14. T Lymphocyte Density and Distribution in Human Colorectal Mucosa, and Inefficiency of Current Cell Isolation Protocols

    PubMed Central

    Preza, Gloria Cuevas; Yang, Otto O.; Elliott, Julie; Anton, Peter A.; Ochoa, Maria T.

    2015-01-01

    Mucosal tissues are critical immune effector sites containing complex populations of leukocytes in a tissue microenvironment that remains incompletely understood. We identify and quantify in human distal colorectal tissue absolute mucosal CD3+ lymphocytes, including CD4+ and CD8+ subsets, by direct visualization using immunohistochemistry (IHC), immunofluorescence (IF), and an automated counting protocol (r2=0.90). Sigmoid and rectal mucosal tissues are both densely packed with T lymphocytes in the mucosal compartment. Both compartments had similar densities of CD3+ T lymphocytes with 37,400 ± 2,801 cells/mm3 and 33,700 ± 4,324 cell/mm3, respectively. Sigmoid mucosa contained 57% CD3+CD4+ and 40% CD3+CD8+ T lymphocytes which calculates to 21,300 ± 1,476/mm3 and 15,000 ± 275/mm3 T lymphocytes, respectively. Rectal mucosa had 57% CD3+CD4+ and 42% CD3+CD8+ or 21,577 ± 332, and 17,090 ± 1,206 cells/mm3, respectively. By comparison, sigmoid mucosal biopsies subjected to conventional collagenase digestion, mononuclear cell (MMC) isolation and staining for flow cytometry yielded 4,549 ± 381/mm3 and 2,708 ± 245/mm3 CD4+ and CD8+ T lymphocytes. These data suggest only ~20.7% recovery compared to IHC results for these markers. Further studies will determine if this reflects a selective bias in only CD3+, CD4+ and CD8+ T cells or can be generalized to all flow-analyzed cells from mucosal tissues for phenotyping and functional testing. PMID:25856343

  15. B lymphocyte reconstitution after human bone marrow transplantation. Leu-1 antigen defines a distinct population of B lymphocytes.

    PubMed Central

    Antin, J H; Ault, K A; Rappeport, J M; Smith, B R

    1987-01-01

    Differences in the expression of Leu-1 (CD5) define two populations of recovering B cells after human marrow transplantation, Leu-1+ and Leu-1- B cells. The Leu-1+ B cells were polyclonal, of donor origin, and did not express detectable interleukin 2 receptor. Leu-1+ B cells generally appeared 2-4 wk after marrow grafting and often preceded the recovery of Leu-1- B cells. Acute and chronic graft vs. host disease (GvHD) resulted in the recovery of significantly fewer Leu-1+ B cells, whereas Leu-1- B cells were only decreased in acute GvHD. Multivariate analysis showed no significant effect of age, disease, prednisone or azathioprine, or ex vivo treatment of the marrow with anti-Leu-1 and complement on recovery of Leu-1+ and Leu-1- B cells, independent of the effects of GvHD. Leu-1+ B cells are a major lymphocyte population posttransplant. They may reflect a stage of differentiation of normal B cells or a separate B cell lineage. PMID:3112184

  16. Targeted delivery of let-7b to reprogramme tumor-associated macrophages and tumor infiltrating dendritic cells for tumor rejection.

    PubMed

    Huang, Zhen; Gan, Jingjing; Long, Ziyan; Guo, Guangxing; Shi, Xiafei; Wang, Chunming; Zang, Yuhui; Ding, Zhi; Chen, Jiangning; Zhang, Junfeng; Dong, Lei

    2016-06-01

    Both tumor associated macrophages (TAMs) and tumor infiltrating dendritic cells (TIDCs) are important components in the tumor microenvironment that mediate tumor immunosuppression and promote cancer progression. Targeting these cells and altering their phenotypes may become a new strategy to recover their anti-tumor activities and thereby restore the local immune surveillance against tumor. In this study, we constructed a nucleic acid delivery system for the delivery of let-7b, a synthetic microRNA mimic. Our carrier has an affinity for the mannose receptors on TAMs/TIDCs and is responsive to the low-pH tumor microenvironment. The delivery of let-7b could reactivate TAMs/TIDCs by acting as a TLR-7 agonist and suppressing IL-10 production in vitro. In a breast cancer mouse model, let-7b delivered by this system efficiently reprogrammed the functions of TAMs/TIDCs, reversed the suppressive tumor microenvironment, and inhibited tumor growth. Taken together, this strategy, designed based upon TAMs/TIDCs-targeting delivery and the dual biological functions of let-7b (TLR-7 ligand and IL-10 inhibitor), may provide a new approach for cancer immunotherapy. PMID:26994345

  17. Locomotor responses of human CD45 lymphocyte subsets: preferential locomotion of CD45RO+ lymphocytes in response to attractants and mitogens.

    PubMed Central

    Newman, I; Wilkinson, P C

    1993-01-01

    The CD45RO+ population of lymphocytes from human blood contains a higher proportion of locomotor cells than the CD45RA+ population. Direct from blood there were few locomotor lymphocytes (< 15%), but, among these, a higher proportion of CD45RO+ than of CD45RA+ cells responded to the chemotactic stimuli, foetal calf serum (FCS) and interleukin-2 (IL-2) in polarization assays. Likewise, after overnight culture, a higher proportion of CD45RO+ cells responded to IL-8. Culture for 24-72 hr in activators such as anti-CD3, purified protein derivative (PPD), phytohaemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM) or in an allogeneic mixed leucocyte reaction (AMLR) increased the proportion of locomotor lymphocytes to 20-60%, and the CD45RO+ subset showed proportionately more polarized cells than the CD45RA+ subset after culture with all the above activators. Preferential migration of CD45RO+ cells into collagen gels was also seen after culture in antigenic stimuli (PPD or AMLR) but not with polyclonal activators (alpha CD3 or Con A). Double labelling showed that, within the CD4+ and CD8+ subsets, antigen-stimulated CD45RO+ T cells invaded collagen gels in higher proportions than CD45RA+ T cells. Clustering of lymphocytes with accessory cells is an essential prerequisite for locomotion and, after culture in alpha CD3, CD45RO+ lymphocytes were found preferentially in clusters with monocytes. In all of the above populations, CD45RO+ lymphocytes were larger in size. These findings suggest that, not only selective adhesion to vascular endothelium as reported earlier, but also selective locomotion recruits CD45RO+ lymphocytes into sites of inflammation. PMID:8436407

  18. Studies of Two Subpopulations of Human Lymphocytes Differing in Responsiveness to Concanavalin A

    PubMed Central

    Boldt, David; Skinner, Sister Ann Marie; Kornfeld, Stuart

    1972-01-01

    We have identified two populations of human lymphocytes differing in responsiveness to the plant mitogen concanavalin A (Con-A). When peripheral blood lymphocytes are passed through a nylon column a population of lymphocytes highly responsive to Con-A adheres to the fibers while a second population of cells relatively unresponsive to Con-A emerges from the column. The untreated peripheral blood lymphocytes are termed “unfiltered” cells while the lymphocytes which pass through the column are termed “filtered” cells. Under standard assay conditions the Con-A-stimulated DNA synthesis is 6.5-fold greater, and the percentage blast formation is four-to fivefold greater in the unfiltered than in the filtered population. Mixing unfiltered with filtered cells fails to induce responsiveness in the latter indicating that a “helper” cell is not involved. The failure of filtered cells to respond to Con-A is specific for that mitogen since both populations respond nearly equally to erythroagglutinating phytohemagglutinin (E-PHA) and the poke weed mitogen (PWM). Binding studies with Con-A-131I demonstrate that the unfiltered population possesses approximately three times as many Con-A receptor sites per cell as the filtered cells, although both cell populations bind the mitogen with the same affinity (apparent association constant [K] of 1.67 × 106m−1). The relationship between Con-A binding and lymphocyte activation was determined by measuring the effect on DNA synthesis of incubating the two lymphocyte populations with increasing amounts of Con-A. The concentration of Con-A required for half-maximal stimulation of DNA synthesis was 5-14 times greater for the filtered cells. However in the presence of very high Con-A concentrations the filtered cells achieved a maximal rate of DNA synthesis approaching that of the unfiltered population. These data implicate the decreased number of Con-A receptor sites on the filtered cells in their failure to respond to low

  19. The Identification of Mitogen Responding Subpopulations of Human Lymphocytes by Flow Polarimeter Fluorescence Measurements.

    NASA Astrophysics Data System (ADS)

    Chan, Sandra Lynn

    I have developed a method to identify the mitogen responding subpopulation of human peripheral blood lymphocytes. This method employs a flow polarimeter to measure the distribution of the intensity and the polarization of intracellular fluorescein fluorescence in suspensions of mononuclear cells isolated on density gradients from the peripheral blood of donors. I have used the change in the fluorescence of cells exposed to the mitogens PHA and Con A to identify the responding cells and to quantitate this number. I have found that for most donors, the responding cells constitute about 20-40% of the lymphocyte population. The percent of responding cells decreases to zero in patients with acute lymphocytic leukemia (2 patients) and chronic lymphocyte leukemia (10 patients). For a variety of patients with other types of cancer, the responding fraction was not significantly different from healthy controls. Moreover, the number of responding cells does not appear to be age dependent in the age range of 20-80 years. I also found that the change in fluorescence polarization correlated strongly with changes in fluorescence intensity induced by mitogens--the number of responding cells, therefore can be estimated either from the intensity or polarization distributions. The shapes of fluorescence distributions depend strongly on a number of variables including the composition and density of the lymphocyte isolating medium, the mitogen and dye concentrations, the length of incubation with mitogen or dye, and the potassium, calcium, and magnesium concentrations in the medium. In the case of fluorescein, I have worked out a methodology that allows a consistent estimate of the responding lymphocyte number. I have also investigated the use of the dye carbocyanine for the same purpose. This dye presumably identifies the mitogen responding lymphocytes on the basis of changes in membrane potential. The results with carbocyanine were found to depend on a number of variables and I could

  20. Human gamma interferon production by cytotoxic T lymphocytes sensitized during hepatitis A virus infection

    SciTech Connect

    Maier, K.; Gabriel, P.; Koscielniak, E.; Stierhof, Y.D.; Wiedmann, K.H.; Flehmig, B.; Vallbracht, A.

    1988-10-01

    The production of interferon (IFN) during a chromium-51 release assay with hepatitis A virus (HAV)-infected fibroblasts and autologous peripheral blood lymphocytes from patients with acute HAV infection was studied to determine whether IFN plays a role in immunopathogenesis of hepatitis A infection in humans. Skin fibroblasts of eight patients after acute HAV infection and from two control persons without history of current of past HAV infection were infected with HAV. Peripheral blood lymphocytes were collected at different times after the onset of icterus and tested in a chromium-51 release assay against autologous HAV-infected skin fibroblasts for their cytolytic and IFN-producing activity. The IFN produced during the assay was characterized and found to have the properties of human gamma IFN. Cytotoxicity and gamma IFN release were virus specific. The cell types responsible for both functions were characterized and found to be in the HLA-dependent T8/sup +/ lymphocyte subset. Considering that gamma IFN has an antiviral effect on persistent HAV infection in vitro and that it probably accounts for stimulation of HLA class I antigen expression on hepatocytes, these experimental results presented here demonstrate that human gamma IFN produced by HAV-specific T cells may participate in pathogenesis of hepatitis A infection in humans.

  1. Activation of Ca2+-dependent K+ channels in human B lymphocytes by anti-immunoglobulin.

    PubMed Central

    MacDougall, S L; Grinstein, S; Gelfand, E W

    1988-01-01

    Many mammalian cell types exhibit Ca2+-dependent K+ channels, and activation of these channels by increasing intracellular calcium generally leads to a hyperpolarization of the plasma membrane. Their presence in B lymphocytes is as yet uncertain. Crosslinking Ig on the surface of B lymphocytes is known to increase the level of free cytoplasmic calcium ([Ca2+]i). However, rather than hyperpolarization, a depolarization has been reported to occur after treatment of B lymphocytes with anti-Ig. To determine if Ca2+-dependent K+ channels are present in B lymphocytes, and to examine the relationship between intracellular free calcium and membrane potential, we monitored [Ca2+]i by means of indo-1 and transmembrane potential using bis(1,3-diethylthiobarbituric)trimethine oxonol in human tonsillar B cells activated by anti-IgM. Treatment with anti-IgM induced a biphasic increase in [Ca2+]i and a simultaneous hyperpolarization. A similar hyperpolarization was induced by ionomycin, a Ca2+ ionophore. Delaying the development of the [Ca2+]i response by increasing the cytoplasmic Ca2+-buffering power delayed the hyperpolarization. Conversely, eliminating the sustained phase of the [Ca2+]i response by omission of external Ca2+ abolished the prolonged hyperpolarization. In fact, a sizable Na+-dependent depolarization was unmasked. This study demonstrates that in human B lymphocytes, Ca2+-dependent K+ channels can be activated by crosslinking of surface IgM. Moreover, it is likely that, by analogy with voltage-sensitive Ca2+ channels, Na+ can permeate through these ligand-gated Ca2+ "channels" in the absence of extracellular Ca2+. PMID:2448342

  2. Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

    PubMed Central

    Pachynski, Russell K.; Scholz, Alexander; Monnier, Justin; Butcher, Eugene C.; Zabel, Brian A.

    2015-01-01

    Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.  Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune “escape,” including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed. PMID:25938949

  3. Study of apoptosis in human lymphocytes by toxic substances implicated in toxic oil syndrome.

    PubMed

    Gallardo, S; Cárdaba, B; del Pozo, V; de Andrés, B; Cortegano, I; Jurado, A; Tramón, P; Palomino, P; Lahoz, C

    1997-03-14

    Toxic Oil Syndrome is a multisystemic disease that occurred in epidemic proportions in Spain in 1981 caused by the ingestion of rapeseed oil denatured with aniline. Several data implicate T cells in the pathogenesis of the disease. We evaluated the mechanisms of cytotoxicity in human lymphocytes of TOS-related products: aniline, 3-(N-phenylamino)-1,2-propanediol and its mono- and di-oleyl esters and eosinophilia myalgia-related product such as 3-(phenylamino)-L-alanine, which is chemically similar to 3-(N-phenylamino)-1,2-propanediol, and has been found in manufactured L-tryptophan. Our results show that only di-oleyl ester of 3-(N-phenylamino)-1,2-propanediol induces apoptosis in human lymphocytes, in a concentration and time-dependent way, confirmed by morphology, expression of phosphatidylserine in membrane and analysis of DNA degradation. PMID:9074655

  4. Protective effects of black tea extract against oxidative DNA damage in human lymphocytes.

    PubMed

    Ježovičová, Miriam; Koňariková, Katarína; Ďuračková, Zdeňka; Keresteš, Ján; Králik, Gabriel; Žitňanová, Ingrid

    2016-02-01

    The aim of the present study was to examine the genoprotective and radioprotective effects of black tea extract (BTE) against the induction of single strand DNA breaks in human lymphocytes subjected to hydrogen peroxide (H2O2) or gamma-rays (2 Gy dose). Lymphocytes were incubated with or without different concentrations of BTE (0.005-500 µg/ml) for 30 min, followed by treatment with or without H2O2 (0.088 µmol/l) for 5 min. To examine the radioprotective effect of BTE, the lymphocytes were incubated with or without BTE for 30 and 60 min prior to and following in vitro irradiation. Oxidative damage to DNA was monitored using a comet assay. BTE at lower concentrations prevented H2O2-induced DNA damage. An increase in BTE concentrations resulted in increased formation of single strand DNA breaks. BTE also exerted significant protective effects against gamma radiation-induced total DNA damage in healthy lymphocytes during their 30 or 60 min incubation with BTE prior to or following irradiation. Therefore, the protective effect of BTE against irradiation was time-dependent. The results contribute to the research on potential beneficial effects of natural compounds, such as BTE, in cancer and its protective effects of normal tissue during radiation therapy. PMID:26718244

  5. In vitro protection of human lymphocytes from toxic effects of chlorpyrifos by selenium-enriched medicines

    PubMed Central

    Navaei-Nigjeh, Mona; Asadi, Hamidreza; Baeeri, Maryam; Pedram, Sahar; Rezvanfar, Mohammad Amin; Mohammadirad, Azadeh; Abdollahi, Mohammad

    2015-01-01

    Objective(s): Chlorpyrifos (CP) is a broad-spectrum organophosphorus pesticide used extensively in agricultural and domestic pest control, accounting for 50% of the global insecticidal use. In the present study, protective effects of two selenium-enriched strong antioxidative medicines IMOD and Angipars were examined in human lymphocytes treated with CP in vitro. Materials and Methods: Isolated lymphocytes were exposed to 12 µg/ml CP either alone or in combination with effective doses (ED50) of IMOD (0.2 µg/ml) and Angipars (1 µg/ml). After 3 days incubation, the viability and oxidative stress markers including cellular lipid peroxidation (LPO), myeloperoxidase (MPO), total thiol molecules (TTM), and total antioxidant power (TAP) were evaluated. Also, the levels of tumor necrosis factor-α (TNF-α), as inflammatory index along with acetylcholinesterase (AChE) activity and cell apoptosis were assessed by flow cytometry. Results: Results indicated that effective doses of IMOD and Angipars reduced CP-exposed lymphocyte mortality rate along with oxidative stress. Both agents restored CP-induced elevation of TNF-α and protected the lymphocytes from CP-induced apoptosis and necrosis. Conclusion: Overall, results confirm that IMOD and Angipars reduce the toxic effects associated with CP through free radical scavenging and protection from apoptosis and necrosis. PMID:25945242

  6. Microgravity simulations with human lymphocytes in the free fall machine and in the random positioning machine

    NASA Technical Reports Server (NTRS)

    Schwarzenberg, M.; Pippia, P.; Meloni, M. A.; Cossu, G.; Cogoli-Greuter, M.; Cogoli, A.

    1998-01-01

    The purpose of this paper is to present the results obtained in our laboratory with both instruments, the FFM [free fall machine] and the RPM [random positioning machine], to compare them with the data from earlier experiments with human lymphocytes conducted in the FRC [fast rotating clinostat] and in space. Furthermore, the suitability of the FFM and RPM for research in gravitational cell biology is discussed.

  7. Ectopic lymphokine gene expression in human peripheral blood lymphocytes in vivo

    SciTech Connect

    Chambers, C.A.; Kang, Joonsoo; Hozumi, Nobumichi Mount Sinai Hospital, Toronto, Ontario )

    1992-02-01

    An animal model to study the effects of ectopic expression of cytokines involved in cell growth and differentiation has been established. Retrovirus vectors containing the human interleukin 6 cDNA were used to produce high titer virus-producing lines. Human peripheral blood lymphocytes (hPBLs) were successfully infected with the retrovirus and engrafted into severe combined immunodeficient mice. The majority of the animals were engrafted with hPBLs, as determined by the presence of human glucose phosphate isomerase. Furthermore, six of seven mice engrafted with hPBLs infected with high titer virus and detectable hPBLs present in the spleen expressed the retroviral human interleukin 6 gene. Importantly, human interleukin 6 protein was expressed at physiologically significant levels in these mice. These results demonstrate that models for human disease and immunotherapy involving retrovirus-mediated gene transfer into human cells can be developed in mice.

  8. The role of the enzymatic antioxidant system in cylindrospermopsin-induced toxicity in human lymphocytes.

    PubMed

    Poniedziałek, Barbara; Rzymski, Piotr; Karczewski, Jacek

    2015-08-01

    Cylindrospermopsin (CYN) is known to induce cytotoxic effects in eukaryotic cells although the exact mechanism underlying these alterations is not fully explained. Given that CYN was previously found to decrease the proliferation of human lymphocytes through DNA damage and cell cycle arrest followed by an increase in the apoptotic rate, the present study evaluated the possible involvement of reactive oxygen species (ROS) and oxidative stress in these cytopathic responses. The status of enzymatic antioxidants: superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) as well as level of lipid peroxidation (LO) under CYN influence in human lymphocytes were also studied. It was found that CYN exposure (0.01-1.0 μg/ml) induces a concentration-dependent increase in H2O2 content within a time as short as 0.5h, reaching its maximum level after 3 and 6h. The highest H2O2 content was accompanied by a significant decrease of SOD and CAT activity and an elevated level of GPx. Moreover, CYN treatment resulted in a detectable increase in LO. We conclude that ROS and the products of LO play an essential role in CYN-induced toxicity in human lymphocytes. Our study helps to elucidate the sequence of events caused by CYN in eukaryotic cells and explain the background for previously observed cytopathic responses. PMID:25863213

  9. Deoxynivalenol induced oxidative stress and genotoxicity in human peripheral blood lymphocytes.

    PubMed

    Yang, Wei; Yu, Miao; Fu, Juan; Bao, Wei; Wang, Di; Hao, Liping; Yao, Ping; Nüssler, Andreas K; Yan, Hong; Liu, Liegang

    2014-02-01

    Deoxynivalenol (DON) is one of the most common mycotoxins. The aim of this study consists in using diverse cellular and molecular assays to evaluate cytotoxicity, genotoxicity as well as oxidative damage and to investigate their mechanisms in human peripheral blood lymphocytes. The human lymphocytes were cultured in eight different doses of DON (0, 6.25, 12.5, 25, 50, 100, 250 and 500 ng/mL) during 6, 12 and 24 h. DON was able to decrease cell viability and cause damage to the membrane, the chromosomes or the DNA at all times of culture. It was also able to induce lipid peroxidation and raise the levels of 8-OHdG and ROS in 6, 12 and 24 h. The results of the RT-PCR and the Western Blot indicated that DON is able to enhance mRNA or protein expressions of DNA repair genes and HO-1 in 6 h and to inhibit these expressions in 24 h. DON potentially triggers genotoxicity in human lymphocytes. This mechanism is probably related to depletion of antioxidase and oxidative damage to the DNA that reduced expression of HO-1, thereby inhibiting the ability of DNA repair. PMID:24355168

  10. Physical association and functional interaction between beta1 integrin and CD98 on human T lymphocytes

    NASA Technical Reports Server (NTRS)

    Miyamoto, Yuko J.; Mitchell, Jason S.; McIntyre, Bradley W.

    2003-01-01

    CD98 is a cell surface protein previously characterized as a cell activation marker, an amino acid transporter, and has recently been implicated in integrin-related functions. Integrins are cell surface proteins, important for homotypic cell aggregation, cell adhesion, and coactivation of T lymphocytes. We have previously shown that the anti-CD98 mAb 80A10, when coimmobilized with anti-CD3 mAb OKT3, is able to mediate human T cell coactivation that is inhibited by anti-beta1 integrin specific mAb 18D3. These results indicated a functional association of CD98 and beta1 integrin signaling but left open the question of a physical association. We now show the induction of homotypic aggregation through CD98 among human T cells and this aggregation was inhibited by anti-beta1 integrin mAb. Therefore, CD98-dependent lymphocyte proliferation and adhesion may involve integrins. Competitive binding assays and fluorescence colocalization analysis suggested that CD98 and beta1 integrin were physically associated. Differential extraction techniques and immunoprecipitations provided the first evidence that the alpha4beta1 integrin and CD98 are specifically associated on human T lymphocytes.

  11. Enhanced lymphocyte longevity and absence of proliferation and lymphocyte apoptosis in Quilty effects of human heart allografts.

    PubMed Central

    Dong, C.; Winters, G. L.; Wilson, J. E.; McManus, B. M.

    1997-01-01

    "Quilty effect" (QE) is a common and problematic observation in endomyocardial biopsy specimens from patients after cardiac transplantation. The origin, fate, and significance of QE cellular elements are unknown. Twenty-six paraffin-embedded endomyocardial biopsy specimens with QE (five QE As and twenty-one QE Bs) from twenty-two cardiac allografts were studied by immunohistochemistry for expression of Bcl-2, Fas antigen, proliferating cell nuclear antigen (PCNA), perforin, T cells (UCHL-1), macrophages (CD68), and apoptosis by in situ terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL). Approximately 50% of the lymphocytes present, mainly in the deeper region of 20 of 21 QE Bs and all 5 QE As, expressed Bcl-2 in a pseudo-nodular pattern surrounding high endothelial venules. Fas expression was detected in lymphocytes in 20 of 21 QE Bs and 5 QE As in a similar pattern to Bcl-2. However, endothelial cells and macrophages were Bcl-2 negative, whereas both cell types were Fas positive. Perforin was negative in nearly all lymphocytes. TUNEL staining revealed that lymphocytes in QEs did not undergo apoptosis; however, TUNEL positivity was observed in approximately 70% of endothelial cells and macrophages and certain adjacent cardiac myocytes in 20 of 21 QE Bs and 5 QE As. One large QE B with a germinal center was noted. Germinal center cells expressed PCNA intensely but were negative for Bcl-2, Fas, and TUNEL. Cells surrounding the germinal center expressed abundant Bcl-2. The following conclusions were drawn. 1) Apoptosis does not occur in lymphocytes in QE where enhanced Bcl-2 (apoptosis inhibitor) and Fas antigen (apoptosis inducer) are expressed. 2) PCNA negativity indicates that QE lymphocytes may not proliferate, and perforin negativity indicates that they may not exhibit perforin-based cytotoxicity. We propose that there may be a relationship between the longevity of lymphocytes in QE and the absence of apoptosis. Images Figure 1

  12. Modeled microgravity-induced protein kinase C isoform expression in human lymphocytes

    NASA Technical Reports Server (NTRS)

    Sundaresan, A.; Risin, D.; Pellis, N. R.

    2004-01-01

    In long-term space travel, the crew is exposed to microgravity and radiation that invoke potential hazards to the immune system. T cell activation is a critical step in the immune response. Receptor-mediated signaling is inhibited in both microgravity and modeled microgravity (MMG) as reflected by diminished DNA synthesis in peripheral blood lymphocytes and their locomotion through gelled type I collagen. Direct activation of protein kinase C (PKC) bypassing cell surface events using the phorbol ester PMA rescues MMG-inhibited lymphocyte activation and locomotion, whereas the calcium ionophore ionomycin had no rescue effect. Thus calcium-independent PKC isoforms may be affected in MMG-induced locomotion inhibition and rescue. Both calcium-dependent isoforms and calcium-independent PKC isoforms were investigated to assess their expression in lymphocytes in 1 g and MMG culture. Human lymphocytes were cultured and harvested at 24, 48, 72, and 96 h, and serial samples were assessed for locomotion by using type I collagen and expression of PKC isoforms. Expression of PKC-alpha, -delta, and -epsilon was assessed by RT-PCR, flow cytometry, and immunoblotting. Results indicated that PKC isoforms delta and epsilon were downregulated by >50% at the transcriptional and translational levels in MMG-cultured lymphocytes compared with 1-g controls. Events upstream of PKC, such as phosphorylation of phospholipase Cgamma in MMG, revealed accumulation of inactive enzyme. Depressed calcium-independent PKC isoforms may be a consequence of an upstream lesion in the signal transduction pathway. The differential response among calcium-dependent and calcium-independent isoforms may actually result from MMG intrusion events earlier than PKC, but after ligand-receptor interaction.

  13. Modeled Microgravity-Induced Protein Kinase C Isoform Expression in Human Lymphocytes

    NASA Technical Reports Server (NTRS)

    Sundaresan, A.; Risin, D.; Pellis, N. R.

    2003-01-01

    In long-term space travel, the crew is exposed to microgravity and radiation that invoke potential hazards to the immune system. T cell activation is a critical step in the immune response. Receptor-mediated signaling is inhibited both in microgravity and modeled microgravity (MMG) as reflected in diminished DNA synthess in peripheral blood lymphocytes and their locomotion through gelled type 1 collagen. Direct activation of Protein Kinase C (PKC) bypassing cell surface events using the phorbol ester PMA rescues MMG-inhibited lymphocyte activation and locomotion, whereas calcium ionophore ionomycin had no rescue effect. Thus calcium-independent PKC isoforms may be affected in MMG-induced locomotion inhibition and rescue. Both calcium-dependent isoforms and calcium-independent PKC isoforms were investigated to assess their expression in lymphocytes in 19 and MMG-culture. Human lymphocytes were cultured and harvested at 24, 48, 72 and 96 hours and serial samples assessed for locomotion using type I collagen and expression of PKC isoforms. Expression of PKC-alpha, -delta and -epsilon was assessed by RT-PCR, flow cytometry and immunoblotting. Results indicated that PKC isoforms delta and epsilon were down-regulated by more than 50% at the transcriptional and translational levels in MMG-cultured lymphocytes compared with 19 controls. Events upstream of PKC such as phosphorylation of Phospholipase C(gamma) (PLC-gamma) in MMG, revealed accumulation of inactive enzyme. Depressed Ca++ -independent PKC isoforms may be a consequence of an upstream lesion in the signal transduction pathway. The differential response among calcium-dependent and calcium-independent isoforms may actually result from MMG intrusion events earlier than, but after ligand-receptor interaction. Keywords: Signal transduction, locomotion, immunity

  14. Long Intergenic Non-Coding RNAs: Novel Drivers of Human Lymphocyte Differentiation

    PubMed Central

    Panzeri, Ilaria; Rossetti, Grazisa; Abrignani, Sergio; Pagani, Massimiliano

    2015-01-01

    Upon recognition of a foreign antigen, CD4+ naïve T lymphocytes proliferate and differentiate into subsets with distinct functions. This process is fundamental for the effective 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 re-direct 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 non-coding 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 non-coding RNAs as key players in these differentiation events. We will also present here new data from our laboratory highlighting the contribution of long non-coding RNAs in driving human CD4+ T lymphocytes differentiation. PMID:25926836

  15. Fas-mediated apoptosis of melanoma cells and infiltrating lymphocytes in human malignant melanomas.

    PubMed

    Shukuwa, Tetsuo; Katayama, Ichiro; Koji, Takehiko

    2002-04-01

    In a rodent system, melanoma cells expressing Fas ligand (FasL) could kill Fas-positive lymphocytes, suggesting that FasL expression was an essential factor for melanoma cell survival in vivo. These findings led us to investigate apoptosis, and to histochemically analyze involvement of Fas and FasL in the induction of apoptosis, in human malignant melanoma tissues. The percentages of terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick end-labeling (TUNEL)-positive melanoma cells and of proliferating cell nuclear antigen (PCNA)-positive melanoma cells in melanoma tissues (n = 22) were greater than those in melanocytes in uninvolved skin (n = 6) and nevus cells in nevi tissues (n = 9). The infiltrating lymphocytes around melanomas were also TUNEL positive. Immunohistochemistry revealed expression of Fas and FasL in melanoma cells and lymphocytes, whereas no Fas or FasL expression was detected in normal skin melanocytes and nevus cells. There was significant correlation between Fas-positive indices and TUNEL indices in melanoma tissues. Moreover, TUNEL-, Fas-, and FasL-positive indices of melanoma cells from patients with Stage 3 melanomas were significantly lower than those with Stage 2 melanomas. The PCNA index of Stage 1 melanoma was significantly lower than that of the other stages, although the difference of PCNA index was insignificant among Stages 2 to 4. Among Stages 1 to 4, there was no difference in the PCNA, TUNEL-, and Fas-positive indices of lymphocytes, although the FasL-positive index of lymphocytes from Stage 3 melanomas was significantly lower than in that from Stage 2. These data reveal that melanoma cells and infiltrating lymphocytes have the potential to induce their own apoptosis regulated by Fas and FasL in an autocrine and/or paracrine fashion and that the decline of Fas-mediated apoptosis of melanoma cells, rather than the apoptosis of infiltrating lymphocytes, may affect the prognosis of melanoma patients, possibly through the

  16. Cardiomyocyte marker expression in a human lymphocyte cell line using mouse cardiomyocyte extract.

    PubMed

    Vojdani, Zahra; Tavakolinejad, Sima; Talaei-Khozani, Tahereh; Esmaeilpour, Tahereh; Rasooli, Manuchehr

    2011-03-01

    Cell transplantation shows potential for the treatment of cardiac diseases. Embryonic stem cells, cord blood and mesenchymal stem cells have been suggested as sources for transplantation therapy. Because of some technical limitations with the use of stem cells, transdifferentiation of fully differentiated cells is a potentially useful alternative. We investigated whether human peripheral blood cells could transdifferentiate into cardiomyocyte. Transdifferentiation was induced in a human B lymphocyte cell line (Raji). Cardiomyocyte extract was prepared from adult mouse cardiomyocytes. The cells were treated with 5-aza-2-deoxycytidine and trichostatin A, permeabilized with streptolysin O, and exposed to the mouse cardiomyocyte extract. They were cultured for 10 days, 3 weeks and 4 weeks. Cardiomyocyte markers were detected with immunohistochemistry and flow cytometry. Immunocytochemistry revealed that some cells expressed myosin heavy chain, α-actinin and cardiac troponin T after 3 and 4 weeks. Flow cytometry confirmed these data. In cells exposed to trichostatin A and 5-aza-2-deoxycytidine and permeabilized in the presence of the cardiomyocyte extract, troponin T expression was seen in 3.53% of the cells and 3.11% of them expressed α-actinin. After exposure to the cardiomyocyte extract, some permeabilized cells adhered to the plate loosely; however, the morphology did not change significantly, and they continued to show a rounded shape after 4 weeks. Our treated lymphocytes expressed cardiomyocyte markers. Our results suggest that lymphocytes may be useful in future research as a source of cells for reprogramming procedures. PMID:21547694

  17. A secreted form of the human lymphocyte cell surface molecule CD8 arises from alternative splicing

    SciTech Connect

    Giblin, P.; Kavathas, P. ); Ledbetter, J.A. )

    1989-02-01

    The human lymphocyte differentiation antigen CD8 is encoded by a single gene that gives rise to a 33- to 34-kDa glycoprotein expressed on the cell surface as a dimer and in higher molecular mass forms. The authors demonstrate that the mRNA is alternatively spliced so that an exon encoding a transmembrane domain is deleted. This gives rise to a 30-kDa molecule that is secreted and exists primarily as a monomer. mRNA corresponding to both forms is present in peripheral blood lymphocytes, Con A-activated peripheral blood lymphocytes, and three CD8{sup +} T-cell lines, with the membrane form being the major species. However, differences in the ratio of mRNA for membrane CD8 and secreted CD8 exist. In addition, the splicing pattern observed differs from the pattern found for the mouse CD8 gene. This mRNA is also alternatively spliced, but an exon encoding a cytoplasmic region is deleted, giving rise to a cell surface molecule that differs in its cytoplasmic tail from the protein encoded by the longer mRNA. Neither protein is secreted. This is one of the first examples of a different splicing pattern between two homologous mouse and human genes giving rise to very different proteins. This represents one mechanism of generating diversity during speciation.

  18. Cell surface Glut1 levels distinguish human CD4 and CD8 T lymphocyte subsets with distinct effector functions

    PubMed Central

    Cretenet, Gaspard; Clerc, Isabelle; Matias, Maria; Loisel, Severine; Craveiro, Marco; Oburoglu, Leal; Kinet, Sandrina; Mongellaz, Cédric; Dardalhon, Valérie; Taylor, Naomi

    2016-01-01

    CD4 and CD8 T lymphocyte activation requires the generation of sufficient energy to support new biosynthetic demands. Following T cell receptor (TCR) engagement, these requirements are met by an increased glycolysis, due, at least in part, to induction of the Glut1 glucose transporter. As Glut1 is upregulated on tumor cells in response to hypoxia, we assessed whether surface Glut1 levels regulate the antigen responsiveness of human T lymphocytes in both hypoxic and atmospheric oxygen conditions. Notably, Glut1 upregulation in response to TCR stimulation was significantly higher in T lymphocytes activated under hypoxic as compared to atmospheric oxygen conditions. Furthermore, TCR-stimulated human T lymphocytes sorted on the basis of Glut1-Lo and Glut1-Hi profiles maintained distinct characteristics, irrespective of the oxygen tension. While T cells activated in hypoxia divided less than those activated in atmospheric oxygen, Glut1-Hi lymphocytes exhibited increased effector phenotype acquisition, augmented proliferation, and an inverted CD4/CD8 ratio in both oxygen conditions. Moreover, Glut1-Hi T lymphocytes exhibited a significantly enhanced ability to produce IFN-γ and this secretion potential was completely dependent on continued glycolysis. Thus, Glut1 surface levels identify human T lymphocytes with distinct effector functions in both hypoxic and atmospheric oxygen tensions. PMID:27067254

  19. Metabolic and cytoskeletal modulation of transferrin receptor mobility in mitogen-activated human lymphocytes.

    PubMed Central

    Galbraith, G M; Galbraith, R M

    1980-01-01

    The transferrin receptors which appear on mitogen-activated human peripheral blood lymphocytes were found by the use of immunofluorescence techniques to display temperature-dependent patching and capping reactions upon binding of transferrin. Lateral mobility of ligand-occupied membrane sites was accompanied by both shedding and endocytosis of receptor-transferrin complexes. In the presence of sodium azide or the microfilament inhibitor cytochalasin B, cap formation and shedding were markedly inhibited. In contrast, endocytosis of patched receptor-ligand complexes was inhibited by azide and microtubule inhibitors, including colchicine, vinblastine and vincristine. Co-capping experiments performed to elucidate further the alterations in membrane configuration involved in these reactions failed to reveal any topographical relationship between transferrin receptors and lectin-binding sites in these cells. These studied indicate that temperature-dependent mobility of transferrin receptors upon mitogen-activated peripheral blood lymphocytes is dependent upon the integrity of the cytoskeletal system and metabolic function of the cell. PMID:6258830

  20. Seasonal variations of DNA damage in human lymphocytes: correlation with different environmental variables.

    PubMed

    Giovannelli, Lisa; Pitozzi, Vanessa; Moretti, Silvia; Boddi, Vieri; Dolara, Piero

    2006-01-29

    Several types of DNA damage, including DNA breaks and DNA base oxidation, display a seasonal trend. In the present work, a sample of 79 healthy subjects living in the city of Florence, Italy, was used to analyse this effect. Three possible causative agents were taken into consideration: solar radiation, air temperature and air ozone level. DNA damage was measured in isolated human lymphocytes at different times during the year and the observed damage was correlated with the levels of these three agents in the days preceding blood sampling. Three time windows were chosen: 3, 7 and 30 days before blood sampling. DNA strand breaks and the oxidized purinic bases cleaved by the formamidopyrimidine glycosylase (FPG sites) were measured by means of the comet assay. The results of multivariate regression analysis showed a positive correlation between lymphocyte DNA damage and air temperature, and a less strong correlation with global solar radiation and air ozone levels. PMID:16095632

  1. Human EBV-transformed lymphocytes of patients with Schistosoma japonicum infection secrete idiotypically related immunoregulatory antibodies.

    PubMed

    Kresina, T F; Cheever, L W; Chireau, M; Johnson, J; Ramirez, B; Peters, P; Olds, G R

    1992-12-01

    Lymphocytes derived from the peripheral blood of individuals infected with Schistosoma japonica were transformed in vitro with Ebstein-Barr virus (EBV). Serological characterization of antibody molecules revealed both antigen reactive (idiotypic) and anti-idiotypic transformants. One idiotypic EBV transformant, LO2C2, describes a major cross-reactive idiotype associated with anti-antigen binding molecules. Other antibody populations expressing idiotypic cross-reactivity were derived from separate individuals showing shared idiotypy in S. japonicum field study populations in the Republic of Philippines. Both idiotypic and anti-idiotypic molecules suppressed parasite antigen-driven blastogenesis of heterologous human peripheral blood lymphocytes. The data show a serologically related immunoregulatory immune network in patients in the Republic of the Philippines which is serologically distinct from idiotypy expressed in other selected S. japonicum endemic areas in the Far East. PMID:1333380

  2. Assessment of genomic damage and repair on human lymphocytes by paint thinner in vitro.

    PubMed

    Londoño-Velasco, Elizabeth; Hidalgo-Cerón, Victor; Escobar-Hoyos, Luisa F; Hoyos-Giraldo, Luz Stella

    2014-05-01

    Paint thinners are organic-solvent complex mixtures frequently used by car painters around the world in industries and shops. Some studies have revealed the oxidative effect induced by thinner inhalation; however, its genotoxic effect is poorly studied. The aim of this study was to assess the cytotoxicity, genomic damage and DNA repair in vitro induced by commercial paint thinner 0.14 in human lymphocytes. Cytotoxicity was determined by cell-viability analysis with trypan blue after 4 h treatment with different thinner concentrations (0.025 to 1.2 µL/mL). Genomic damage was evaluated by means of the alkaline single-cell gel electrophoresis (SCGE; pH > 13) in treated cultures after 1 h with three low-cytotoxic thinner concentrations (0.05, 0.1 and 0.2 µL/mL). In order to evaluate the genomic DNA repair, one set of SCGE slides was prepared immediately after treatment, and another one was prepared after 4 h of liquid-holding recovery. A significant level of cytotoxicity was observed over the entire concentration range of paint thinner in lymphocytes (F = 175.98; p ≤ 0.001). In the SCGE % tail DNA assessment, a significant increase of lymphocyte genomic damage was evidenced (F = 72.32; p < 0.001). In addition, we found a significant decrease in the % tail DNA in thinner-treated cells after liquid-holding recovery period (all p < 0.05), demonstrating that DNA primary lesions induced by low-cytotoxic thinner concentrations are efficiently repaired. In conclusion, thinner components induce cytotoxicity and genomic damage in human lymphocytes under the study conditions, possibly by oxidative and alkylative DNA damage. PMID:24236478

  3. An ATP-activated channel is involved in mitogenic stimulation of human T lymphocytes.

    PubMed

    Baricordi, O R; Ferrari, D; Melchiorri, L; Chiozzi, P; Hanau, S; Chiari, E; Rubini, M; Di Virgilio, F

    1996-01-15

    We investigated the effect of pharmacologic modulation of the ATP receptor on intracellular ion changes and proliferative response of human peripheral blood lymphocytes (PBLs) and purified T lymphocytes. Extracellular ATP (ATPe) triggered in these cells an increase in the cytoplasmic Ca2+ concentration ([Ca2+]i) and plasma membrane depolarization. Whereas both Ca2+ release from intracellular stores and influx across the plasma membrane were detected in the whole PBL population, only Ca2+ influx was observed in T cells. In the presence of near physiologic extracellular Na+ concentrations (125 mmol/L), Ca2+ permeability through the ATPe-gated channel was very low, suggesting a higher selectivity for monovalent over divalent cations. The selective P2Z agonist benzoylbenzoic ATP (BzATP) increased [Ca2+]i in the presence but not the absence of extracellular Ca2+ and also caused plasma membrane depolarization. The covalent blocker oxidized ATP (oATP), an inhibitor of P2X and P2Z receptors, prevented Ca2+ influx and plasma membrane depolarization, but had no effect on Ca2+ release from stores. Stimulation with ATPe alone had no significant effects on PBL 3H-thymidine incorporation. On the contrary, ATPe or BzATP had a synergistic effect on DNA synthesis stimulated by selective T-cell mitogens such as phytohemagglutinin, anti-CD3 monoclonal antibody, or allogenic PBLs (mixed lymphocyte cultures). Treatment with oATP inhibited mitogenic stimulation by these receptor-directed agents but not by the combined application of the Ca2+ ionophore ionomycin and phorbol myristate acetate. Interleukin-2 partially relieved inhibition by oATP. These results suggest that human T lymphocytes express a plasma membrane channel gated by ATPe that is involved in mitogenic stimulation. PMID:8555491

  4. Lymphocyte trafficking and HIV infection of human lymphoid tissue in a rotating wall vessel bioreactor

    NASA Technical Reports Server (NTRS)

    Margolis, L. B.; Fitzgerald, W.; Glushakova, S.; Hatfill, S.; Amichay, N.; Baibakov, B.; Zimmerberg, J.

    1997-01-01

    The pathogenesis of HIV infection involves a complex interplay between both the infected and noninfected cells of human lymphoid tissue, the release of free viral particles, the de novo infection of cells, and the recirculatory trafficking of peripheral blood lymphocytes. To develop an in vitro model for studying these various aspects of HIV pathogenesis we have utilized blocks of surgically excised human tonsils and a rotating wall vessel (RWV) cell culture system. Here we show that (1) fragments of the surgically excised human lymphoid tissue remain viable and retain their gross cytoarchitecture for at least 3 weeks when cultured in the RWV system; (2) such lymphoid tissue gradually shows a loss of both T and B cells to the surrounding growth medium; however, this cellular migration is reversible as demonstrated by repopulation of the tissue by labeled cells from the growth medium; (3) this cellular migration may be partially or completely inhibited by embedding the blocks of lymphoid tissue in either a collagen or agarose gel matrix; these embedded tissue blocks retain most of the basic elements of a normal lymphoid cytoarchitecture; and (4) both embedded and nonembedded RWV-cultured blocks of human lymphoid tissue are capable of productive infection by HIV-1 of at least three various strains of different tropism and phenotype, as shown by an increase in both p24 antigen levels and free virus in the culture medium, and by the demonstration of HIV-1 RNA-positive cells inside the tissue identified by in situ hybridization. It is therefore reasonable to suggest that gel-embedded and nonembedded blocks of human lymphoid tissue, cocultured with a suspension of tonsillar lymphocytes in an RWV culture system, constitute a useful model for simulating normal lymphocyte recirculatory traffic and provide a new tool for testing the various aspects of HIV pathogenesis.

  5. Human Lymphocytes

    NASA Technical Reports Server (NTRS)

    2004-01-01

    Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. The objective of the research was to define a way to differentiate between effects due to microgravity and those due to possible stress from non-optimal spaceflight conditions.

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

    PubMed

    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. PMID:27331299

  7. Differential expression of three T lymphocyte-activating CXC chemokines by human atheroma-associated cells

    PubMed Central

    Mach, François; Sauty, Alain; Iarossi, Albert S.; Sukhova, Galina K.; Neote, Kuldeep; Libby, Peter; Luster, Andrew D.

    1999-01-01

    Activated T lymphocytes accumulate early in atheroma formation and persist at sites of lesion growth and rupture, suggesting that they may play an important role in the pathogenesis of atherosclerosis. Moreover, atherosclerotic lesions contain the Th1-type cytokine IFN-γ, a potentiator of atherosclerosis. The present study demonstrates the differential expression of the 3 IFN-γ–inducible CXC chemokines — IFN-inducible protein 10 (IP-10), monokine induced by IFN-γ (Mig), and IFN-inducible T-cell α chemoattractant (I-TAC) — by atheroma-associated cells, as well as the expression of their receptor, CXCR3, by all T lymphocytes within human atherosclerotic lesions in situ. Atheroma-associated endothelial cells (ECs), smooth muscle cells (SMCs), and macrophages (MØ) all expressed IP-10, whereas Mig and I-TAC were mainly expressed in ECs and MØ, as detected by double immunofluorescence staining. ECs of microvessels within lesions also expressed abundant I-TAC. In vitro experiments supported these results and showed that IL-1β, TNF-α, and CD40 ligand potentiated IP-10 expression from IFN-γ–stimulated ECs. In addition, nitric oxide (NO) treatment decreased IFN-γ induction of IP-10. Our findings suggest that the differential expression of IP-10, Mig, and I-TAC by atheroma-associated cells plays a role in the recruitment and retention of activated T lymphocytes observed within vascular wall lesions during atherogenesis. PMID:10525042

  8. Visualization of antigen-specific human cytotoxic T lymphocytes labeled with superparamagnetic iron-oxide particles.

    PubMed

    Beer, Ambros J; Holzapfel, Konstantin; Neudorfer, Juliana; Piontek, Guido; Settles, Marcus; Krönig, Holger; Peschel, Christian; Schlegel, Jürgen; Rummeny, Ernst J; Bernhard, Helga

    2008-06-01

    New technologies are needed to characterize the migration and survival of antigen-specific T cells in vivo. In this study, we developed a novel technique for the labeling of human cytotoxic T lymphocytes with superparamagnetic iron-oxide particles and the subsequent depiction with a conventional 1.5-T magnetic resonance scanner. Antigen-specific CD8(+) T lymphocytes were labeled with ferucarbotran by lipofection. The uptake of ferucarbotran was confirmed by immunofluorescence microscopy using a dextran-specific antibody, and the intracellular enrichment of iron was measured by atomic absorption spectrometry. The imaging of T cells was performed by magnetic resonance on day 0, 2, 7 and 14 after the labeling procedure. On day 0 and 2 post labeling, a pronounced shortening of T2*-relaxation times was observed, which diminished after 7 days and was not detectable anymore after 14 days, probably due to the retained mitotic activity of the labeled T cells. Of importance, the antigen-specific cytolytic activity of the T cells was preserved following ferucarbotran labeling. Efficient ferucarbotran labeling of functionally active T lymphocytes and their detection by magnetic resonance imaging allows the in vivo monitoring of T cells and, subsequently, will impact the further development of T cell-based therapies. PMID:18286290

  9. Early and Late Damages in Chromosome 3 of Human Lymphocytes After Radiation Exposure

    NASA Technical Reports Server (NTRS)

    Sunagawa, Mayumi; Mangala, Lingegowda; Zhang, Ye; Kahdim, Munira; Wilson, Bobby; Cucinotta, Francis A.; Wu, Honglu

    2011-01-01

    Tumor formation in humans or animals is a multi-step process. An early stage of cancer development is believed to be genomic instability (GI) which accelerates the mutation rate in the descendants of the cells surviving radiation exposure. GI is defined as elevated or persistent genetic damages occurring many generations after the cells are exposed. While early studies have demonstrated radiation-induced GI in several cell types as detected in endpoints such as mutation, apoptosis and damages in chromosomes, the dependence of GI on the quality of radiation remains uncertain. To investigate GI in human lymphocytes induced by both low- and high-LET radiation, we initially exposed white blood cells collected from healthy subjects to gamma rays in vitro, and cultured the cells for multiple generations. Chromosome aberrations were analyzed in cells collected at first mitosis post irradiation and at several intervals during the culture period. Among a number of biological endpoints planned for the project, the multi-color banding fluorescent in situ hybridization (mBAND) allows identification of inversions that were expected to be stable. We present here early and late chromosome aberrations detected with mBAND in chromosome 3 after gamma exposure. Comparison of chromosome damages in between human lymphocytes and human epithelial cells is also discussed

  10. Monocyte chemotactic protein-1 (MCP-1), -2, and -3 are chemotactic for human T lymphocytes.

    PubMed Central

    Taub, D D; Proost, P; Murphy, W J; Anver, M; Longo, D L; van Damme, J; Oppenheim, J J

    1995-01-01

    Monocyte chemotactic protein (MCP)-1, -2, and -3 all have been shown to induce monocyte/macrophage migration in vitro and MCP-1, also known as MCAF, chemoattracts basophils and mast cells. We report here that natural MCP-1 as well as synthetic preparations of MCP-2 and MCP-3 stimulate significant in vitro chemotaxis of human peripheral blood T lymphocytes. This MCP-induced migration was dose-dependent and directional, but not chemokinetic. Phenotypic analysis of the T cell population responsive to MCP-1, MCP-2, and MCP-3 demonstrates that both CD4+ and CD8+ T cells migrated in response to these chemokines. Similar results were observed using human CD4+ and CD8+ T cell clones. Neutralizing antisera to MCAF or MCP-2 abrogated T cell migration in response to MCP-1 and MCP-2, respectively, but not to RANTES. Subcutaneous administration of purified MCP-1 into the hind flanks of SCID mice engrafted with human peripheral blood lymphocytes (PBL) induced significant human CD3+ T cell infiltration into the site of injection at 4 h. These results demonstrate that MCP-1, MCP-2, and MCP-3 are inflammatory mediators that specifically stimulate the directional migration of T cells as well as monocytes and may play an important role in immune cell recruitment into sites of antigenic challenge. Images PMID:7883984

  11. The pyrimidin analogue cyclopentenyl cytosine induces alloantigen-specific non-responsiveness of human T lymphocytes

    PubMed Central

    Nikolaeva, N; Bemelman, F J; Yong, S-L; Verschuur, A; van Lier, R A W; ten Berge, I J M

    2008-01-01

    Cyclopentenyl cytosine (CPEC) has been shown to induce apoptosis in human T lymphoblastic cell lines and T cells from leukaemia patients. In this study we have addressed the question of whether CPEC is able to decrease proliferation and effector functions of human alloresponsive T lymphocytes and induce T cell anergy. The proliferative capacity of human peripheral blood mononuclear cells in response to allogeneic stimulation was measured by 5,6-carboxy-succinimidyl-diacetate-fluorescein-ester staining. Flow cytometric analysis was performed using surface CD4, CD8, CD25, CD103 and intracellular perforin, granzyme A, granzyme B, caspase-3 and forkhead box P3 (FoxP3) markers. The in vivo immunosuppressive capacity was tested in a murine skin graft model. Addition of CPEC at a concentration of 20 nM strongly decreased the expansion and cytotoxicity of alloreactive T cells. Specific restimulation in the absence of CPEC showed that the cells became anergic. The drug induced caspase-dependent apoptosis of alloreactive T lymphocytes. Finally, CPEC increased the percentage of CD25high FoxP3+ CD4+ and CD103+ CD8+ T cells, and potentiated the effect of rapamycin in increasing the numbers of alloreactive regulatory T cells. Treatment with CPEC of CBA/CA mice transplanted with B10/Br skin grafts significantly prolonged graft survival. We conclude that CPEC inhibits proliferation and cytotoxicity of human alloreactive T cells and induces alloantigen non-responsiveness in vitro. PMID:18062797

  12. Serine leucocyte proteinase inhibitor-treated monocyte inhibits human CD4(+) lymphocyte proliferation.

    PubMed

    Guerrieri, Diego; Tateosian, Nancy L; Maffía, Paulo C; Reiteri, Romina M; Amiano, Nicolás O; Costa, María J; Villalonga, Ximena; Sanchez, Mercedes L; Estein, Silvia M; Garcia, Verónica E; Sallenave, Jean-Michel; Chuluyan, Héctor E

    2011-08-01

    Serine leucocyte proteinase inhibitor (SLPI) is the main serine proteinase inhibitor produced by epithelial cells and has been shown to be a pleiotropic molecule with anti-inflammatory and microbicidal activities. However, the role of SLPI on the adaptive immune response is not well established. Therefore, we evaluated the effect of SLPI on lymphocyte proliferation and cytokine production. Human peripheral blood mononuclear cells (PBMC) were treated with mitogens plus SLPI and proliferation was assessed by [(3) H]thymidine uptake. The SLPI decreased the lymphocyte proliferation induced by interleukin-2 (IL-2) or OKT3 monoclonal antibodies in a dose-dependent manner. Inhibition was not observed when depleting monocytes from the PBMC and it was restored by adding monocytes and SLPI. SLPI-treated monocyte slightly decreased MHC II and increased CD18 expression, and secreted greater amounts of IL-4, IL-6 and IL-10 in the cell culture supernatants. SLPI-treated monocyte culture supernatant inhibited the CD4(+) lymphocyte proliferation but did not affect the proliferation of CD8(+) cells. Moreover, IL-2 increased T-bet expression and the presence of SLPI significantly decreased it. Finally, SLPI-treated monocyte culture supernatant dramatically decreased interferon-γ but increased IL-4, IL-6 and IL-10 in the presence of IL-2-treated T cells. Our results demonstrate that SLPI target monocytes, which in turn inhibit CD4 lymphocyte proliferation and T helper type 1 cytokine secretion. Overall, these results suggest that SLPI is an alarm protein that modulates not only the innate immune response but also the adaptive immune response. PMID:21574992

  13. Serine leucocyte proteinase inhibitor-treated monocyte inhibits human CD4+ lymphocyte proliferation

    PubMed Central

    Guerrieri, Diego; Tateosian, Nancy L; Maffía, Paulo C; Reiteri, Romina M; Amiano, Nicolás O; Costa, María J; Villalonga, Ximena; Sanchez, Mercedes L; Estein, Silvia M; Garcia, Verónica E; Sallenave, Jean-Michel; Chuluyan, Héctor E

    2011-01-01

    Serine leucocyte proteinase inhibitor (SLPI) is the main serine proteinase inhibitor produced by epithelial cells and has been shown to be a pleiotropic molecule with anti-inflammatory and microbicidal activities. However, the role of SLPI on the adaptive immune response is not well established. Therefore, we evaluated the effect of SLPI on lymphocyte proliferation and cytokine production. Human peripheral blood mononuclear cells (PBMC) were treated with mitogens plus SLPI and proliferation was assessed by [3H]thymidine uptake. The SLPI decreased the lymphocyte proliferation induced by interleukin-2 (IL-2) or OKT3 monoclonal antibodies in a dose-dependent manner. Inhibition was not observed when depleting monocytes from the PBMC and it was restored by adding monocytes and SLPI. SLPI-treated monocyte slightly decreased MHC II and increased CD18 expression, and secreted greater amounts of IL-4, IL-6 and IL-10 in the cell culture supernatants. SLPI-treated monocyte culture supernatant inhibited the CD4+ lymphocyte proliferation but did not affect the proliferation of CD8+ cells. Moreover, IL-2 increased T-bet expression and the presence of SLPI significantly decreased it. Finally, SLPI-treated monocyte culture supernatant dramatically decreased interferon-γ but increased IL-4, IL-6 and IL-10 in the presence of IL-2-treated T cells. Our results demonstrate that SLPI target monocytes, which in turn inhibit CD4 lymphocyte proliferation and T helper type 1 cytokine secretion. Overall, these results suggest that SLPI is an alarm protein that modulates not only the innate immune response but also the adaptive immune response. PMID:21574992

  14. Impact of different concentrations of human recombinant growth hormone on T lymphocytes.

    PubMed

    Borrione, P; Grasso, L; Pautasso, M; Parisi, A; Quaranta, F; Ciminelli, E; Di Gianfrancesco, A; Di Luigi, L; Pigozzi, F

    2012-01-01

    The aim of the present study is to evaluate the effects induced by increasing concentrations of human recombinant growth hormone on T lymphocytes. Ten healthy volunteers and twelve subjects with symptomatic allergies were enrolled in the study. Peripheral blood mononuclear cells and purified T lymphocytes were cultured in the presence of graded concentrations of growth hormone. Following appropriate in vitro stimulations, the proportion of apoptotic T cells, the percentage of activated T lymphocyte subpopulations, the phytohemagglutinin responsiveness and the Th2 response were assessed by flow cytometry analysis. Moreover, in order to evaluate the phosphoinositol-3-kinase signaling pathway involvement, cells were also analyzed after treatment with LY294002. The treatment with different concentrations of growth hormone did not influence the activation pattern of un-stimulated T lymphocytes. On the contrary, growth hormone was able to modify the CD38/HLA-DR co-expression of T cells activated with phytohemoagglutinin. A different response was observed when samples obtained from healthy donors and from subjects with symptomatic allergies were analysed. Moreover, growth hormone treatment was able to increase the Th2 response in the samples obtained from healthy donors only. The results of the present study strongly support the hypothesis that growth hormone administration may play an important role in conditions of impaired/activated immune systems. The observation that growth hormone administration at high doses may reverse its effects and that it may promote a Th2-oriented response have significant clinical implications when considering the use of this hormone for artificially enhancing the physical performances of healthy athletes. PMID:22507321

  15. Impact of the New Generation Reconstituted Surfactant CHF5633 on Human CD4+ Lymphocytes

    PubMed Central

    Fehrholz, Markus; Glaser, Kirsten; Seidenspinner, Silvia; Ottensmeier, Barbara; Curstedt, Tore; Speer, Christian P.; Kunzmann, Steffen

    2016-01-01

    Background Natural surfactant preparations, commonly isolated from porcine or bovine lungs, are used to treat respiratory distress syndrome in preterm infants. Besides biophysical effectiveness, several studies have documented additional immunomodulatory properties. Within the near future, synthetic surfactant preparations may be a promising alternative. CHF5633 is a new generation reconstituted synthetic surfactant preparation with defined composition, containing dipalmitoyl-phosphatidylcholine, palmitoyl-oleoyl-phosphatidylglycerol and synthetic analogs of surfactant protein (SP-) B and SP-C. While its biophysical effectiveness has been demonstrated in vitro and in vivo, possible immunomodulatory abilities are currently unknown. Aim The aim of the current study was to define a potential impact of CHF5633 and its single components on pro- and anti-inflammatory cytokine responses in human CD4+ lymphocytes. Methods Purified human CD4+ T cells were activated using anti CD3/CD28 antibodies and exposed to CHF5633, its components, or to the well-known animal-derived surfactant Poractant alfa (Curosurf®). Proliferative response and cell viability were assessed using flow cytometry and a methylthiazolyldiphenyltetrazolium bromide colorimetric assay. The mRNA expression of IFNγ, IL-2, IL-17A, IL-22, IL-4, and IL-10 was measured by quantitative PCR, while intracellular protein expression was assessed by means of flow cytometry. Results Neither CHF5633 nor any of its phospholipid components with or without SP-B or SP-C analogs had any influence on proliferative ability and viability of CD4+ lymphocytes under the given conditions. IFNγ, IL-2, IL-17A, IL-22, IL-4, and IL-10 mRNA as well as IFNγ, IL-2, IL-4 and IL-10 protein levels were unaffected in both non-activated and activated CD4+ lymphocytes after exposure to CHF5633 or its constituents compared to non-exposed controls. However, in comparison to Curosurf®, expression levels of anti-inflammatory IL-4 and IL-10 m

  16. Dopamine Receptors in Human Lymphocytes: Radioligand Binding and Quantitative RT-PCR Assays

    PubMed Central

    Kirillova, Galina P.; Hrutkay, Rebecca J.; Shurin, Michael R.; Shurin, Galina V.; Tourkova, Irina L.; Vanyukov, Michael M.

    2008-01-01

    Analysis of dopamine receptors (DR) in lymphocytes of the human peripheral blood mononuclear cell (PBMC) fraction is an attractive tool for evaluation of functional properties of dopaminergic function underlying variation in complex psychological/psychopathological traits. Receptor binding assays (RBA) with selective radioligands, which are widely used in CNS studies, have not produced consistent results when applied to isolated PBMC. We tested the assay conditions that could be essential for detection of DR in human PBMC and their membrane preparations. Using [3H]SCH23390, a dopamine D1-like receptor antagonist, we demonstrated the presence of two binding sites in PBMC-derived membrane fraction. One of them is characterized by the Kd value consistent with that reported for D5 dopamine receptors in human lymphocytes, whereas the other Kd value possibly corresponds to serotonin receptor(s). Although D5 receptor binding sites in PBMC membranes could be characterized by binding assays, the low protein expression and the large volume of blood needed for membrane preparation render the binding method impracticable for individual phenotyping. In contrast, real-time RT-PCR may be used for this purpose, contingent on the relationship between DR expression in the brain and in lymphocytes. The expression of the DRD2-DRD5 genes, as detected by this method, varied widely among samples, whereas the DRD1 expression was not detected. The expression levels were comparable with those in the brain for DRD3 and DRD4, and were significantly lower for DRD2 and DRD5. PMID:18721826

  17. Effects of increasing docosahexaenoic acid intake in human healthy volunteers on lymphocyte activation and monocyte apoptosis

    PubMed Central

    Mebarek, Saïda; Ermak, Natalia; Benzaria, Amal; Vicca, Stéphanie; Dubois, Madeleine; Némoz, Georges; Laville, Martine; Lacour, Bernard; Véricel, Evelyne; Lagarde, Michel; Prigent, Annie-France

    2009-01-01

    Dietary intake of long-chain n-3 polyunsaturated fatty acids (n-3 PUFA) has been reported to decrease several markers of lymphocyte activation and modulate monocyte susceptibility to apoptosis. However most human studies examined the combined effect of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) using relatively high daily amounts of n-3 PUFA. The present study investigated the effects of increasing doses of DHA added to the regular diet of human healthy volunteers on lymphocyte response to tetradecanoylphorbol acetate (TPA) plus ionomycin activation, and on monocyte apoptosis induced by oxidized LDL (oxLDL). Eight subjects were supplemented with increasing daily doses of DHA (200, 400, 800 and 1600mg) in a triacylglycerol form containing DHA as the only PUFA, for two weeks each dose. DHA intake dose-dependently increased the proportion of DHA in mononuclear cell phospholipids, the augmentation being significant after 400mg DHA/day. The TPA plus ionomycin-stimulated IL-2 mRNA level started to increase after ingestion of 400mg DHA/day, with a maximum after 800mg intake, and was positively correlated (P<0.003) with DHA enrichment in cell phospholipids. The treatment of monocytes by oxLDL before DHA supplementation drastically reduced mitochondrial membrane potential as compared with native LDL treatment. OxLDL apoptotic effect was significantly attenuated after 400mg DHA/day and the protective effect was maintained throughout the experiment, although to a lesser extent at higher doses. The present results show that supplementation of the human diet with low DHA dosages improves lymphocyte activability. It also increases monocyte resistance to oxLDL-induced apoptosis, which may be beneficial in the prevention of atherosclerosis. PMID:18710607

  18. Brucella fractions behave as nonspecific mitogens and polyclonal B-cell activators for human lymphocytes.

    PubMed Central

    Vendrell, J P; Rabesandratana, H; Huguet, M F; Cannat, A; Serre, A

    1985-01-01

    Two lipid-A-free fractions which were extracted from Brucella melitensis and were designated PI and SF stimulated human unsensitized mononuclear cells to proliferate and to secrete immunoglobulins. Both of these effects were observed in cultures of peripheral blood, tonsils, and cord blood lymphocytes. Neither B cells nor T cells alone proliferated in the presence of these fractions, whereas the proliferative response of T cells plus B cells was largely independent of accessory cells. Polyclonal activation was estimated by counting the cells which secreted immunoglobulins of different isotypes into culture supernatants. This phenomenon was strongly T dependent. PMID:3876286

  19. The effects of lipid A on gamma-irradiated human peripheral blood lymphocytes in vitro

    NASA Astrophysics Data System (ADS)

    Dubničková, M.; Kuzmina, E. A.; Chausov, V. N.; Ravnachka, I.; Boreyko, A. V.; Krasavin, E. A.

    2016-03-01

    The modulatory effects of lipid A (diphosphoryl lipid A (DLA) and monophosphoryl lipid A (MLA)) on apoptosis induction and DNA structure damage (single and double-strand breaks (SSBs and DSBs, respectively)) in peripheral human blood lymphocytes are studied for 60Co gamma-irradiation. It is shown that in the presence of these agents the amount of apoptotic cells increases compared with the irradiated control samples. The effect is most strongly pronounced for DLA. In its presence, a significant increase is observed in the number of radiation-induced DNA SSBs and DSBs. Possible mechanisms are discussed of the modifying influence of the used agents on radiation-induced cell reactions

  20. Suppression of human lymphocyte responses to specific and non-specific stimuli in human onchocerciasis.

    PubMed Central

    Elkhalifa, M Y; Ghalib, H W; Dafa'Alla, T; Williams, J F

    1991-01-01

    Characterization of in vitro lymphocyte responsiveness was performed on selected groups of onchocerciasis patients from Sudan and Sierra Leone. These patients manifested a very broad range of clinical signs and showed widely divergent parasite infection intensities. Lymphocyte proliferative responses to soluble Onchocerca volvulus antigen (sAg) were poor in infected persons; mitogen and PPD responses were maintained in the normal range in one group of patients from southwestern Sudan, but were profoundly depressed in a group from N.E. Sudan. Proliferative responses and interferon-gamma (INF-gamma) secretion were very significantly depressed in the presence of live microfilariae of O. volvulus or secretions/excretions (S/E) from microfilariae (mf) or from female, but not male, adult parasites. Lymphocyte responses were maintained near normal when exogenous IL-2 was added to these cultures. The results indicate that O. volvulus infection and its clinical consequences are not consistently associated with systemic deficits in immune responsiveness. However, suppression of lymphocyte reactivity by mf and S/E in vitro suggests that direct parasite intervention in host cell responses could be taking place in vivo, perhaps at the local microenvironment level; mediated by effects on cytokine production. PMID:1747951

  1. Erythropoietin exerts direct immunomodulatory effects on the cytokine production by activated human T-lymphocytes.

    PubMed

    Todosenko, N M; Shmarov, V A; Malashchenko, V V; Meniailo, M E; Melashchenko, O B; Gazatova, N D; Goncharov, A G; Seledtsov, V I

    2016-07-01

    The effect of erythropoietin-β (Epo-β) on the functional profile of activated human T-lymphocytes remains largely unknown, which hinders clinical application of Epo as an immunomodulatory agent. We studied the direct impact of Epo on the activation status of human T lymphocytes following activation by particles loaded with antibodies (Abs) against human CD2, CD3, and CD28. T cell activation was assessed by the surface expression of CD38 activation marker. Epo did not significantly affect activation status of both CD4(+) and CD4(-) T cells, as well as of naive (CD45RA(+)CD197(+)), central memory (CD45RA(-)CD197(+)), effector memory (CD45RA(-)CD197(-)), and terminally-differentiated (CD45RA(+)CD197(-)) T cells. However, Epo markedly augmented production of IL-2, IL-4 and IL10 by activated T cells with concomitant reduction in IFN-γ secretion. Taken together, our data showed that Epo could directly down-regulate pro-inflammatory T cell responses without affecting T cell activation status. PMID:27208431

  2. Interaction of Epstein-Barr virus (EBV) with human B-lymphocytes

    SciTech Connect

    Klein, George; Klein, Eva; Kashuba, Elena

    2010-05-21

    Epstein-Barr virus, EBV, and humans have a common history that reaches back to our primate ancestors. The virus co-evolved with man and has established a largely harmless and highly complex co-existence. It is carried as silent infection by almost all human adults. A serendipitous discovery established that it is the causative agent of infectious mononucleosis. Still, EBV became known first in 1964, in a rare, geographically prevalent malignant lymphoma of B-cell origin, Burkitt lymphoma BL. Its association with a malignancy prompted intensive studies and its capacity to immortalize B-lymphocytes in vitro was soon demonstrated. Consequently EBV was classified therefore as a potentially tumorigenic virus. Despite of this property however, the virus carrier state itself does not lead to malignancies because the transformed cells are recognized by the immune response. Consequently the EBV induced proliferation of EBV carrying B-lymphocytes is manifested only under immunosuppressive conditions. The expression of EBV encoded genes is regulated by the cell phenotype. The virus genome can be found in malignancies originating from cell types other than the B-lymphocyte. Even in the EBV infected B-cell, the direct transforming capacity is restricted to a defined window of differentiation. A complex interaction between virally encoded proteins and B-cell specific cellular proteins constitute the proliferation inducing program. In this short review we touch upon aspects which are the subject of our present work. We describe the mechanisms of some of the functional interactions between EBV encoded and cellular proteins that determine the phenotype of latently infected B-cells. The growth promoting EBV encoded genes are not expressed in the virus carrying BL cells. Still, EBV seems to contribute to the etiology of this tumor by modifying events that influence cell survival and proliferation. We describe a possible growth promoting mechanism in the genesis of Burkitt lymphoma

  3. Protective effect of apigenin on radiation-induced chromosomal damage in human lymphocytes

    NASA Technical Reports Server (NTRS)

    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.

  4. Activated human B lymphocytes express cyclooxygenase-2 and cyclooxygenase inhibitors attenuate antibody production.

    PubMed

    Ryan, Elizabeth P; Pollock, Stephen J; Pollack, Stephen J; Murant, Thomas I; Bernstein, Steven H; Felgar, Raymond E; Phipps, Richard P

    2005-03-01

    Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of inflammatory diseases and target cyclooxygenases 1 and 2 (Cox-1, Cox-2) that are responsible for PG production. Newer Cox-2-selective drugs have been heavily prescribed to quench inflammation. Little is known about whether or not these drugs influence human B lymphocytes and their ability to produce Ab. We report herein that activated human B cells not only highly express Cox-2 and produce PGs, but that the NSAID indomethacin and Cox-2-selective drugs profoundly inhibit the ability of human B cells to produce IgG and IgM in vitro. Human blood B cells highly express Cox-2 mRNA and protein and produce PGs after activation with CD40L, pansorbin, or CD40L plus BCR engagement. Cox-2 is also highly expressed by human tonsil B cells, as shown by immunohistochemistry. Cox-inhibiting drugs modestly affect purified B cell proliferation but profoundly reduce Ab production. The ability of whole blood to produce IgM and IgG following stimulation is also strongly inhibited. In support that Cox-2 plays a seminal role in B lymphocyte Ab production, Cox-2 knockout mice have 64% less IgM and 35% less IgG than normal littermate controls. These findings support that NSAIDs and the new Cox-2-selective drugs have an unsuspected target, the B cell, and attenuate Ab production in humans. Use of NSAIDs may therefore influence autoantibody production in autoimmune diseases and may dampen humoral immunity in response to antigenic challenge/vaccination. PMID:15728468

  5. Development of a chemically defined serum- and protein-free medium for growth of human peripheral lymphocytes.

    PubMed

    Shive, W; Pinkerton, F; Humphreys, J; Johnson, M M; Hamilton, W G; Matthews, K S

    1986-01-01

    A chemically defined, protein-free medium (designated CFBI 1000, where CFBI = Clayton Foundation Biochemical Institute) that supports human peripheral lymphocyte proliferation has been developed. This medium allows exploration of individual metabolic differences by varying the medium composition as well as providing a base to explore further the mechanisms of lymphocyte activation in a system initially free of added macromolecular species other than mitogen. The peripheral blood lymphocyte is an ideal system for metabolic studies because it is easily obtained, is a primary resting cell that can be activated to proliferate, and presumably reflects both the genetic makeup and biochemical environmental history of the individual at the time the cells were formed. Examination of the role of various factors in lymphocyte activation and subsequent events may be simplified by the utilization of a medium that is protein-free and chemically defined. The CFBI 1000 medium supports the growth response of human peripheral lymphocytes to mitogen as measured by [3H]thymidine incorporation to an extent comparable to other media used widely in assessment of lymphocyte proliferation. PMID:3079905

  6. Blackberry seed extracts and isolated polyphenolic compounds showing protective effect on human lymphocytes DNA.

    PubMed

    Gođevac, Dejan; Tešević, Vele; Vajs, Vlatka; Milosavljević, Slobodan; Stanković, Miroslava

    2011-09-01

    The tentative identification of seed extracts from 3 cultivars of blackberry (blackberry seed extracts [BSEs]) constituents was performed by LC/UV/MS technique. The identified compounds belonged to ellagitannins, galic acid derivatives, and ellagic acid derivatives. Two ellagitannins, Lambertianin C and Sanguiniin H-6, and an ellagic acid derivative, 4-α-L-arabinofuranosylellagic acid, were isolated using semipreparative High-performance liquid chromatography. The structure elucidations were based on high resolution-mass spectrometry and nuclear magnetic resonance studies. The BSEs and 3 isolated pure compounds were tested for in vitro protective effect on chromosome aberrations in peripheral human lymphocytes using cytochalasin-B blocked micronucleus (MN) assay. The frequency of MN was scored in binucleated cells, and nuclear proliferation index was calculated. Among the tested extracts, the seeds of cv. Thornfree at concentration of 1 μg/mL exhibit the most prominent effect decreasing the frequency of MN by 62.4%, when compared with the controls cell cultures. Antioxidant potential of pure ellagitannins cannot explain the strong effect of BSEs. The assumption was that better antioxidant effect of BSEs result from synergistic effects of individual compounds contained in the extracts and/or some minor components possessed strong activity. PraCTICAL APPLICATION: Our results provide evidence of protective effects of BSEs and isolated pure compounds on cytogenetic damages in human lymphocytes. Thus, BSEs could exert beneficial effects in quite a few diseases, because many of the biological actions have been attributed to their antioxidant properties. PMID:21824137

  7. Effects of copaene, a tricyclic sesquiterpene, on human lymphocytes cells in vitro.

    PubMed

    Türkez, Hasan; Celik, Kübra; Toğar, Başak

    2014-08-01

    In this study, the cytotoxic, genotoxic/antigenotoxic and antioxidant/oxidant activity of copaene (COP), a plant-derived tricyclic sesquiterpene, on human lymphocyte cultures (n = 5) was investigated. COP was added into culture tubes at various concentrations (0, 10, 25, 50, 100, 200 and 400 mg/L). While the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays were used for viability and cytotoxic evaluations, the micronucleus (MN) and sister chromatid exchange (SCE) assays were used for genetic evaluations. Moreover, total antioxidant capacity (TAC) and total oxidative status analysis were used for biochemical evaluations. According to LDH and MTT assays COP significantly reduced cell proliferation at high concentrations (200 and 400 mg/L). In addition, there was no significant increase (P < 0.05) in both SCE and MN frequencies of cultures treated with COP as compared to controls. We have also concluded that concentrations of COP of 50 and 100 mg/L increased TAC level when compared to the controls. In conclusion, in this study it has been reported for the first time that copaene is not genotoxic and it increases the antioxidant capacity in human lymphocyte cultures. PMID:24287609

  8. 50 Hz sinusoidal magnetic fields do not affect human lymphocyte activation and proliferation in vitro

    NASA Astrophysics Data System (ADS)

    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.

  9. Evaluation of Possible Genotoxic Activity of Dirithromycin in Cultured Human Lymphocytes

    PubMed Central

    Kayraldız, Ahmet; Dönbak, Lale; Yavuz Kocaman, Ayşe; Köker, Esra; Gökçe, Şule

    2015-01-01

    Dirithromycin antibiotic is a 14-membered lactone ring macrolide and is widely used in medicine to treat many different types of bacterial infections. In the present study, the possible genotoxicity of dirithromycin was evaluated in cultured human lymphocytes by using sister chromatid exchanges (SCEs), chromosome aberration (CA), and micronucleus (MN) tests and also cell proliferation kinetics such as mitotic index (MI), replication index (RI), and nuclear division index (NDI) were analyzed for cytotoxicity. Cell cultures were treated with four different concentrations of dirithromycin (37.75, 67.50, 125, and 250 µg/mL) for 24 and 48 h periods. Dirithromycin significantly induced SCE and MN frequency at all concentrations in both 24 and 48 h treated cells. In addition, CA level has been markedly increased in the cells treated with almost all concentrations of dirithromycin for 24 (except 37.75 µg/mL) and 48 h treatment periods as compared to control. However, MI, RI, and NDI values were not affected by the dirithromycin treatment (p > 0.05). The results of this study indicated that dirithromycin treatment caused genetic damage by increasing the level of cytogenetic endpoints, suggesting its genotoxic and mutagenic action on human lymphocytes in vitro. PMID:26576152

  10. Effects of sotrastaurin, mycophenolic acid and everolimus on human B-lymphocyte function and activation.

    PubMed

    Matz, Mareen; Lehnert, Martin; Lorkowski, Christine; Fabritius, Katharina; Unterwalder, Nadine; Doueiri, Salim; Weber, Ulrike A; Mashreghi, Mir-Farzin; Neumayer, Hans-H; Budde, Klemens

    2012-10-01

    Humoral rejection processes may lead to allograft injury and subsequent dysfunction. Today, only one B-cell-specific agent is in clinical use and the effects of standard and new immunosuppressant substances on B-cell activation and function are not fully clarified. The impact of sotrastaurin, mycophenolic acid and everolimus on human B-lymphocyte function was assessed by analysing proliferation, apoptosis, CD80/CD86 expression and immunoglobulin and IL-10 production in primary stimulated B cells. In addition, B-cell co-cultures with pre-activated T cells were performed to evaluate the effect of the different immunosuppressive agents on T-cell-dependent immunoglobulin production. Sotrastaurin did not inhibit B-cell proliferation, CD80/CD86 expression, and IgG production and had only minor effects on IgM levels at the highest concentration administered. In contrast, mycophenolic acid and everolimus had strong effects on all B-cell functions in a dose-dependent manner. All immunosuppressive agents caused decreased immunoglobulin levels in T-cell-dependent B-cell cultures. The data provided here suggest that mycophenolic acid and everolimus, but not sotrastaurin, are potent inhibitors of human B-lymphocyte function and activation. PMID:22816666

  11. The effect of stem cell from human exfoliated deciduous teeth on T lymphocyte proliferation

    PubMed Central

    Alipour, Razieh; Adib, Minoo; Hashemi-Beni, Batool; Sadeghi, Farzaneh

    2014-01-01

    Background: Mesenchymal stem cells (MSC), a specific type of adult tissue stem cell; have the immunosuppressive effects that make them valuable targets for regenerative medicine and treatment of many human illnesses. Hence, MSC have been the subject of numerous studies. The classical source of MSC is adult bone marrow (BM). Due to many shortcomings of harvesting MSC from BM, finding the alternative sources for MSC is an urgent. Stem cells from human exfoliated deciduous teeth (SHED) are relative new MSC populations that fulfill these criteria but their potential immunosuppressive effect has not been studied enough yet. Thus, in this work the effect of SHED on the proliferation of in vitro activated T lymphocytes were explored. Materials and Methods: In this study, both mitogen and alloantigen activated T cells were cultured in the presence of different numbers of SHED. In some co-cultures, activated T cells were in direct contact to MSCs and in other co-cultures; they were separated from SHED by a permeable membrane. In all co-cultures, the proliferation of T cells was measured by ELISA Bromodeoxyuridine proliferation assay. Results: In general, our results showed that SHED significantly suppress the proliferation of activated T cells in a dose-dependent manner. Moreover, the suppression was slightly stronger when MSCs were in physical contact to activated T cells. Conclusion: This study showed that SHED likewise other MSC populations can suppress the activation of T lymphocytes, which can be used instead of BM derived MSCs in many investigational and clinical applications. PMID:25337532

  12. Lead and cadmium at very low doses affect in vitro immune response of human lymphocytes

    SciTech Connect

    Borella, P.; Giardino, A. )

    1991-08-01

    The effect of lead chloride and cadmium chloride on in vitro immunoglobulin (Ig) production by human lymphocytes was investigated. After 7 days in culture, lead added in the range of human exposure (207-1035 {mu}g/liter) significantly enhanced Ig production either when cells were activated by pokeweed mitogen (PWM) or not. The effect was dose-dependent and was related to the Pb were measured in the extracellular medium and in the cells. Independently of the mitogen addition, about 2% of the Pb added was accumulated in the cells, most being associated with the nuclear fraction. Those findings suggest that the Pb effects could depend on its uptake and distribution in the cells. Cadmium added in the 50-500 nM range exhibited a dose-independent mitogenic activity in unstimulated cells, whereas the Ig secretion was not significantly affected by Cd when cells were PWM-activated. A considerable intraindividual variability, however, was observed when blood donors were separately examined, with both an increase, a decrease, or no variation on Ig production. Furthermore, higher percentages of Cd were accumulated in the nuclear fraction, and lower in the cytosol and precipitate, in PWM-activated compared to resting lymphocytes. Genetic factors could be of importance for the observed variability of the immune response to cadmium, and the authors support the hypothesis that differences in the metallothionein (MT) inducibility could play a role.

  13. Synergistic effect of DHT and IGF-1 hyperstimulation in human peripheral blood lymphocytes.

    PubMed

    Imperlini, Esther; Spaziani, Sara; Mancini, Annamaria; Caterino, Marianna; Buono, Pasqualina; Orrù, Stefania

    2015-06-01

    The abuse of mixed or combined performance-enhancing drugs is widespread among athletes and amateurs, adults and adolescents. Clinical studies demonstrated that misuse of these doping agents is associated with serious adverse effects to many organs in human. Previously, we demonstrated in human peripheral blood lymphocytes that high doses of anabolic androgenic steroids, such as dihydrotestosterone (DHT) and growth factors, such as insulin-like growth factor-1 (IGF-1), have effects at gene and protein levels. Supraphysiological treatments of DHT and IGF-1 affected the expression of genes involved in skeletal muscle disorders as well as in cell-mediated immunological response. At protein level, DHT hyperdosage affects cell motility and apoptosis; IGF-1 hyperstimulation triggers an active cytoskeletal reorganization and an overproduction of immune response- and inflammation-related cytokines. In this study, we investigate the combined effects of DHT and IGF-1 hyperdosage in peripheral blood lymphocytes using a differential proteomic approach. DHT and IGF-1 combined treatment affects cell adhesion, migration, and survival through modulation of expression levels of cytokines and paxillin-signaling-related proteins, and activation of several pathways downstream focal adhesion kinase. Our results indicate a synergistic effect of DHT and IGF-1 which has potential implications for health risk factors. PMID:25669835

  14. Cytogenetic response to coffee in Chinese hamster ovary AUXB1 cells and human peripheral lymphocytes.

    PubMed

    Tucker, J D; Taylor, R T; Christensen, M L; Strout, C L; Hanna, M L

    1989-09-01

    We have investigated the genotoxic effects of three different brands and three types of coffee (freshly brewed regular, instant regular and freshly brewed decaffeinated) in two mammalian systems: the Chinese hamster ovary (CHO) AUXB1 cell line and human peripheral lymphocytes. Sister-chromatid exchanges (SCEs) and endoreduplicated cells (ERCs) were used as the endpoints. Coffee was prepared according to the manufacturer's suggestions, and after cooling, administered to cultured cells at dilutions ranging up to 11% that of full-strength coffee. Each brand and type of coffee induced significant levels of SCEs and ERCs in AUXB1 cells. SCEs, but not ERCs, were induced in human peripheral lymphocytes. Bisulfite, which complexes with carbonyls and reduces their genotoxicity, significantly diminished the number of SCEs and ERCs found after administration of coffee. Catalase and peroxidase, enzymes that destroy hydrogen peroxide activity, had no significant effect upon the SCE and ERC frequencies in AUXB1 cultures treated with freshly brewed regular coffee. These experiments indicate that different brands and types of coffee have sufficient genotoxic activity to increase SCEs and ERCs at levels only a fraction of those normally consumed. 1,2-Dicarbonyls alone and peroxides alone do not appear to be responsible for the majority of SCEs and ERCs that were observed to be induced by dilute coffee. PMID:2687627

  15. Micronucleus assay in human lymphocytes after exposure to alloxydim sodium herbicide in vitro.

    PubMed

    Akyıl, Dilek; Özkara, Arzu; Erdoğmuş, S Feyza; Eren, Yasin; Konuk, Muhsin; Sağlam, Esra

    2015-12-01

    This study evaluates the cytotoxic and genotoxic potential of alloxydim sodium using micronucleus (MN) assay, in human peripheral lymphocytes. MN assay was used to investigate the genotoxic effects of alloxydim sodium in human peripheral lymphocytes treated with 250, 500, 750, 1,000 µg/ml concentrations of alloxydim sodium for 24 and 48 h. Solvent, negative and positive controls were also used in the experiments in parallel. The obtained results were evaluated in statistical analyses by using Dunnett-t test (two sided) and p < 0.05 was accepted as significant. Alloxydim sodium significantly increased the MN formation compared with the negative control, at both 750 and 1,000 µg/ml concentrations and treatment periods. We also evaluated the nuclear division index (NDI) for cytotoxicity of this pesticide in the experiment, and finally observed a significant decrease of the NDI values at all concentrations of alloxydim sodium and at both treatment periods. PMID:25017922

  16. Grapevine fruit extract protects against radiation-induced oxidative stress and apoptosis in human lymphocyte.

    PubMed

    Singha, Indrani; Das, Subir Kumar

    2015-11-01

    Ionizing radiation (IR) causes oxidative stress through overwhelming generation of reactive oxygen species (ROS) in the living cells leading the oxidative damage further to biomolecules. Grapevine (Vitis vinifera L.) posses several bioactive phytochemicals and is the richest source of antioxidants. In this study, we investigated V. vinifera for its phytochemical content, enzymes profile and, ROS- and oxidant-scavenging activities. We have also studied the fruit extract of four different grapevine viz., Thompson seedless, Flame seedless, Kishmish chorni and Red globe for their radioprotective actions in human lymphocytes. The activities of ascorbic acid oxidase and catalase significantly (P < 0.01) differed among extracts within the same cultivar, while that of peroxidase and polyphenol oxidase did not differ significantly. The superoxide radical-scavenging activity was higher in the seed as compared to the skin or pulp of the same cultivar. Pretreatment with grape extracts attenuated the oxidative stress induced by 4 Gy γ-radiation in human lymphocytes in vitro. Further, γ-radiation-induced increase in caspase 3/7 activity was significantly attenuated by grape extracts. These results suggest that grape extract serve as a potential source of natural antioxidants against the IR-induced oxidative stress and also inhibit apoptosis. Furthermore, the protective action of grape depends on the source of extract (seed, skin or pulp) and type of the cultivars. PMID:26669019

  17. The effects of boric acid on sister chromatid exchanges and chromosome aberrations in cultured human lymphocytes

    PubMed Central

    Arslan, Mehmet; Topaktas, Mehmet

    2007-01-01

    The aim of this study was to determine the possible genotoxic effects of boric acid (BA) (E284), which is used as an antimicrobial agent in food, by using sister chromatid exchange (SCEs) and chromosome aberration (CAs) tests in human peripheral lymphocytes. The human lymphocytes were treated with 400, 600, 800, and 1000 μg/mL concentrations of BA dissolved in dimethyl sulfoxide (DMSO), for 24 h and 48 h treatment periods. BA did not increase the SCEs for all the concentrations and treatment periods when compared to control and solvent control (DMSO). BA induced structural and total CAs at all the tested concentrations for 24 and 48 h treatment periods. The induction of the total CAs was dose dependent for the 24 h treatment period. However, BA did not cause numerical CAs. BA showed a cytotoxic effect by decreasing the replication index (RI) and mitotic index (MI). BA decreased the MI in a dose-dependent manner for the 24 h treatment period. PMID:19002846

  18. Evaluation of DNA damage induced by norcantharidin in human cultured lymphocytes.

    PubMed

    Khabour, Omar F; Enaya, Fatima M; Alzoubi, Karem; Al-Azzam, Sayer I

    2016-07-01

    Norcantharidin (NCTD) is currently used in the treatment of several cancers such as leukemia, melanoma and hepatoma. The mechanism of action of NCTD is suggested to involve induction of apoptosis of cancer cells via production of reactive oxygen species. In this study, the genotoxic effect of different concentrations of NCTD (1, 10 and 20 μm) in human lymphocytes was investigated using sister chromatid exchanges (SCEs) and chromosomal aberrations (CAs) assays. The results revealed that NCTD significantly increased the rate of SCEs (p < 0.05) in a dose-dependent manner. In addition, NCTD significantly increased the number of high-frequency cells (SCEs ≥ 8, p < 0.05). However, NCTD did not have any significant effect on the rate of CAs (p > 0.05). In addition, no significant differences were detected in the mitotic index or proliferative index at examined doses (up to 20 μm). In conclusion, NCTD is genotoxic to human cultured lymphocytes as measured by SCE assay. PMID:26599593

  19. T Cells and Stromal Fibroblasts in Human Tumor Microenvironments Represent Potential Therapeutic Targets

    PubMed Central

    Barnas, Jennifer L.; Simpson-Abelson, Michelle R.; Yokota, Sandra J.; Kelleher, Raymond J.

    2010-01-01

    The immune system of cancer patients recognizes tumor-associated antigens expressed on solid tumors and these antigens are able to induce tumor-specific humoral and cellular immune responses. Diverse immunotherapeutic strategies have been used in an attempt to enhance both antibody and T cell responses to tumors. While several tumor vaccination strategies significantly increase the number of tumor-specific lymphocytes in the blood of cancer patients, most vaccinated patients ultimately experience tumor progression. CD4+ and CD8+ T cells with an effector memory phenotype infiltrate human tumor microenvironments, but most are hyporesponsive to stimulation via the T cell receptor (TCR) and CD28 under conditions that activate memory T cells derived from the peripheral blood of the cancer patients or normal donors. Attempts to identify cells and molecules responsible for the TCR signaling arrest of tumor-infiltrating T cells have focused largely upon the immunosuppressive effects of tumor cells, tolerogenic dendritic cells and regulatory T cells. Here we review potential mechanisms by which human T cell function is arrested in the tumor microenvironment with a focus on the immunomodulatory effects of stromal fibroblasts. Determining in vivo which cells and molecules are responsible for the TCR arrest in human tumor-infiltrating T cells will be necessary to formulate and test strategies to prevent or reverse the signaling arrest of the human T cells in situ for a more effective design of tumor vaccines. These questions are now addressable using novel human xenograft models of tumor microenvironments. PMID:21209773

  20. MAP3K3 expression in tumor cells and tumor-infiltrating lymphocytes is correlated with favorable patient survival in lung cancer

    PubMed Central

    He, Yanli; Wang, Lihui; Liu, Weijun; Zhong, Jinjie; Bai, Shengbin; Wang, Zhuwen; Thomas, Dafydd G.; Lin, Jules; Reddy, Rishindra M.; Ramnath, Nithya; Carrott, Philip W.; Lynch, William R.; Orringer, Mark B.; Chang, Andrew C.; Beer, David G.; Chen, Guoan

    2015-01-01

    MAP3K3 is involved in both the immune response and in tumor progression. Its potential biological role in vitro in lung cancer cell lines and the association of mRNA/protein expression patterns with clinical outcome of primary lung tumors were investigated in this study. Silencing MAP3K3 using siRNA in lung cancer cell lines resulted in decreased cell proliferation, migration and invasion. These effects were associated with down-regulation of the JNK, p38, AKT, and GSK3β pathways as determined using phospho-protein and gene expression array analyses. However, MAP3K3 mRNA and protein overexpression in primary lung tumors correlated significantly with favorable patient survival. Gene cluster and pathway analyses of primary tumor datasets indicated that genes positively-correlated with MAP3K3 are significantly involved in immune response rather than the cell cycle regulators observed using in vitro analyses. These results indicate that although MAP3K3 overexpression has an oncogenic role in vitro, in primary lung adenocarcinomas it correlates with an active immune response in the tumor environment that correlates with improved patient survival. MAP3K3 may potentially not only serve as diagnostic/prognostic markers for patients with lung cancer but also provide an indicator for future investigations into immunomodulatory therapies for lung cancer. PMID:26088427

  1. Evaluation of toxicity of essential oils palmarosa, citronella, lemongrass and vetiver in human lymphocytes.

    PubMed

    Sinha, Sonali; Jothiramajayam, Manivannan; Ghosh, Manosij; Mukherjee, Anita

    2014-06-01

    The present investigation was undertaken to study the cytotoxic and genotoxic potential of the essential oils (palmarosa, citronella, lemongrass and vetiver) and monoterpenoids (citral and geraniol) in human lymphocytes. Trypan blue dye exclusion and MTT test was used to evaluate cytotoxicity. The genotoxicity studies were carried out by comet and DNA diffusion assays. Apoptosis was confirmed by Annexin/PI double staining. In addition, generation of reactive oxygen species was evaluated by DCFH-DA staining using flow cytometry. The results demonstrated that the four essential oils and citral induced cytotoxicity and genotoxicity at higher concentrations. The essential oils were found to induce oxidative stress evidenced by the generation of reactive oxygen species. With the exception of geraniol, induction of apoptosis was confirmed at higher concentrations of the test substances. Based on the results, the four essential oils are considered safe for human consumption at low concentrations. PMID:24650756

  2. Loss of Telomeres in the Progeny of Human Lymphocytes Exposed to Energetic Heavy Ions

    NASA Technical Reports Server (NTRS)

    Cucinotta, F.A.; George, K.; Durante, M.

    2006-01-01

    We have used cross-species multi-color banding (RxFISH) combined with telomere FISH probes, to measure chromosomal aberrations in the progeny of human peripheral blood lymphocytes exposed to ionizing radiation. Accelerated iron particles (energy 1 GeV/nucleon) induced many more terminal deletions than the same dose of gamma-rays. We found that truncated chromosomes without telomeres could be transmitted for at least three cell cycles following exposure, and represented about 10% of all aberrations observed in the progeny of cells exposed to iron ions. High energy heavy ions generate the most significant health risk for human space exploration and the results suggest that telomere loss may be the leading mechanism for their high efficiency in the induction of late effects.

  3. Beta-casomorphin (BCM) and human colonic lamina propria lymphocyte proliferation.

    PubMed Central

    Elitsur, Y; Luk, G D

    1991-01-01

    BCM is a milk-derived peptide with opiate-like properties which is absorbed through the gastrointestinal mucosa. It has been shown to affect gastrointestinal motility, absorption and secretion. Recently, modulation of the immune system by BCM was also reported. In this study we investigated the in vitro effect of BCM on the human mucosal immune response as represented by lamina propria lymphocyte (LPL) proliferation. Results show that BCM significantly inhibited concanavalin A (ConA) stimulated LPL DNA synthesis. BCM also inhibited ornithine decarboxylase activity (ODC) in ConA-stimulated LPL. Although BCM also inhibited 12-O-tetradecanoyl phorbol-13-acetate (TPA) stimulated LPL DNA synthesis, the degree of inhibition was much lower than in ConA-stimulated LPL. The anti-proliferative effect of BCM was reversed by the opiate receptor antagonist, neloxone. Our results suggest that BCM may affect the human mucosal immune system, possibly via the opiate receptor. PMID:1893631

  4. p53 mutations in human lymphoid malignancies: Association with Burkitt lymphoma and chronic lymphocytic leukemia

    SciTech Connect

    Gaidano, G.; Ballerini, P.; Gong, J.Z.; Inghirami, G.; Knowles, D.M.; Dalla-Favera, R. ); Neri, A, Centro Malattie del Sangue G. Marcora, Milan ); Newcomb, E.W. ); Magrath, I.T. )

    1991-06-15

    The authors have investigated the frequency of p53 mutations in B- and T-cell human lymphoid malignancies, including acute lymphoblastic leukemia, the major subtypes of non-Hodgkin lymphoma, and chronic lymphocytic leukemia. p53 exons 5-9 were studied by using genomic DNA from 197 primary tumors and 27 cell lines by single-strand conformation polymorphism analysis and by direst sequencing of PCR-amplified fragments. Mutations were found associated with (i) Burkitt lymphoma (9/27 biopsoes; 17/27 cell lines) and its leukemic counterpart L{sub 3}-type B-cell acute lymphoblastic leukemia (5/9), both of which also carry activated c-myc oncogenes, and (ii) B-cell chronic lymphocytic leukemia (6/40) and, in particular, its stage of progression known as Richter's transformation (3/7). Mutations were not found at any significant frequency in other types of non-Hodgkin lymphoma or acute lymphoblastic leukemia. In many cases, only the mutated allele was detectable, implying loss of the normal allele. These results suggest that (1) significant differences in the frequency of p53 mutations are present among subtypes of neoplasms derived from the same tissue; (2) p53 may play a role in tumor progression in B-cell chronic lymphocytic leukemia; (3) the presence of both p53 loss/inactivation and c-myc oncogene activation may be important in the pathogenesis of Burkitt lymphoma and its leukemia form L{sub 3}-type B-cell acute lymphoblastic leukemia.

  5. Induction of adaptive response in human blood lymphocytes exposed to radiofrequency radiation.

    PubMed

    Sannino, Anna; Sarti, Maurizio; Reddy, Siddharth B; Prihoda, Thomas J; Vijayalaxmi; Scarfì, Maria Rosaria

    2009-06-01

    The incidence of micronuclei was evaluated to assess the induction of an adaptive response to non-ionizing radiofrequency (RF) radiation in peripheral blood lymphocytes collected from five different human volunteers. After stimulation with phytohemagglutinin for 24 h, the cells were exposed to an adaptive dose of 900 MHz RF radiation used for mobile communications (at a peak specific absorption rate of 10 W/kg) for 20 h and then challenged with a single genotoxic dose of mitomycin C (100 ng/ml) at 48 h. Lymphocytes were collected at 72 h to examine the frequency of micronuclei in cytokinesis-blocked binucleated cells. Cells collected from four donors exhibited the induction of adaptive response (i.e., responders). Lymphocytes that were pre-exposed to 900 MHz RF radiation had a significantly decreased incidence of micronuclei induced by the challenge dose of mitomycin C compared to those that were not pre-exposed to 900 MHz RF radiation. These preliminary results suggested that the adaptive response can be induced in cells exposed to non-ionizing radiation. A similar phenomenon has been reported in cells as well as in animals exposed to ionizing radiation in several earlier studies. However, induction of adaptive response was not observed in the remaining donor (i.e., non-responder). The incidence of micronuclei induced by the challenge dose of mitomycin C was not significantly different between the cells that were pre-exposed and unexposed to 900 MHz RF radiation. Thus the overall data indicated the existence of heterogeneity in the induction of an adaptive response between individuals exposed to RF radiation and showed that the less time-consuming micronucleus assay can be used to determine whether an individual is a responder or non-responder. PMID:19580480

  6. Stability of Radiation Induced Chromosome Damage in Human Peripheral Blood Lymphocytes

    NASA Technical Reports Server (NTRS)

    Cucinotta, F. A.; George, K.; Willingham, V.

    2006-01-01

    Chromosome damage in an individual's peripheral blood lymphocytes can be an indicator of radiation exposure and this data can be used to evaluate dose after accidental or occupational exposure. Evidence suggests that the yield of chromosome damage in lymphocytes is also a relevant biomarker of cancer risk in humans that reflects individual cancer susceptibility. It follows that biomonitoring studies can be used to uncover subjects who are particularly susceptible to radiation damage and therefore at higher risk of cancer. Translocations and other stable aberrations are commonly believed to persist in peripheral blood cells for many years after exposure, and it has been suggested that translocations can be used for assessing retrospective radiation doses or chronic exposures. However, recent investigations suggest that translocations might not always persist indefinitely. We measured chromosome aberrations in the blood lymphocytes of six astronauts before their respective missions of approximately 3 to 6 months onboard the international space station, and again at various intervals up to 5 years after flight. In samples collected a few days after return to earth, the yield of chromosome translocations had significantly increased compared with preflight values, and results indicate that biodosimetry estimates lie within the range expected from physical dosimetry. However, for five of the astronauts, follow up analysis revealed a temporal decline in translocations with half-lives ranging from 10 to 58 months. The yield of exchanges remained unchanged for the sixth astronaut during an observation period of 5 months post-flight. These results may indicate complications with the use of stable aberrations for retrospective dose reconstruction and could affect cancer risk predictions that are estimated from yields of chromosome damage obtained shortly after exposure.

  7. Rapid alterations of cell cycle control proteins in human T lymphocytes in microgravity

    PubMed Central

    2012-01-01

    In our study we aimed to identify rapidly reacting gravity-responsive mechanisms in mammalian cells in order to understand if and how altered gravity is translated into a cellular response. In a combination of experiments using "functional weightlessness" provided by 2D-clinostats and real microgravity provided by several parabolic flight campaigns and compared to in-flight-1g-controls, we identified rapid gravity-responsive reactions inside the cell cycle regulatory machinery of human T lymphocytes. In response to 2D clinorotation, we detected an enhanced expression of p21 Waf1/Cip1 protein within minutes, less cdc25C protein expression and enhanced Ser147-phosphorylation of cyclinB1 after CD3/CD28 stimulation. Additionally, during 2D clinorotation, Tyr-15-phosphorylation occurred later and was shorter than in the 1 g controls. In CD3/CD28-stimulated primary human T cells, mRNA expression of the cell cycle arrest protein p21 increased 4.1-fold after 20s real microgravity in primary CD4+ T cells and 2.9-fold in Jurkat T cells, compared to 1 g in-flight controls after CD3/CD28 stimulation. The histone acetyltransferase (HAT) inhibitor curcumin was able to abrogate microgravity-induced p21 mRNA expression, whereas expression was enhanced by a histone deacetylase (HDAC) inhibitor. Therefore, we suppose that cell cycle progression in human T lymphocytes requires Earth gravity and that the disturbed expression of cell cycle regulatory proteins could contribute to the breakdown of the human immune system in space. PMID:22273506

  8. Immunogenicity of somatic mutations in human gastrointestinal cancers.

    PubMed

    Tran, Eric; Ahmadzadeh, Mojgan; Lu, Yong-Chen; Gros, Alena; Turcotte, Simon; Robbins, Paul F; Gartner, Jared J; Zheng, Zhili; Li, Yong F; Ray, Satyajit; Wunderlich, John R; Somerville, Robert P; Rosenberg, Steven A

    2015-12-11

    It is unknown whether the human immune system frequently mounts a T cell response against mutations expressed by common epithelial cancers. Using a next-generation sequencing approach combined with high-throughput immunologic screening, we demonstrated that tumor-infiltrating lymphocytes (TILs) from 9 out of 10 patients with metastatic gastrointestinal cancers contained CD4(+) and/or CD8(+) T cells that recognized one to three neo-epitopes derived from somatic mutations expressed by the patient's own tumor. There were no immunogenic epitopes shared between these patients. However, we identified in one patient a human leukocyte antigen-C*08:02-restricted T cell receptor from CD8(+) TILs that targeted the KRAS(G12D) hotspot driver mutation found in many human cancers. Thus, a high frequency of patients with common gastrointestinal cancers harbor immunogenic mutations that can potentially be exploited for the development of highly personalized immunotherapies. PMID:26516200

  9. DNA damage and repair in human peripheral blood lymphocytes following treatment with microcystin-LR.

    PubMed

    Lankoff, Anna; Krzowski, Łukasz; Głab, Joanna; Banasik, Anna; Lisowska, Halina; Kuszewski, Tomasz; Góźdź, Stanisław; Wójcik, Andrzej

    2004-04-11

    The purpose of this study was to find a possible explanation of the inconsistency of data regarding the genotoxicity of microcystin-LR (MC-LR). We compared the results of the comet assay with the results of the analysis of chromosome aberrations and apoptosis. In order to investigate the influence of MC-LR on DNA damage in human lymphocytes, cells were treated with MC-LR at different concentrations (1, 10 and 25 microg/ml) for 6, 12, 18 and 24 h. Analyses of Olive Tail Moment (OTM) as an indicator of DNA damage showed that MC-LR treatment induced DNA damage in a time-dependent manner, reaching its maximum after 18 h. The lowest values of OTM were observed after 24 h. MC-LR had no effect on the frequency of chromosome aberrations in lymphocytes. Since some data available in the literature indicate that apoptosis may lead to overestimated or false positive results regarding the genotoxicity of mutagens in the comet assay, we measured the frequency of late apoptotic cells by use of the comet assay and the frequency of early apoptotic cells with the TUNEL method. The comet assay results revealed that the highest level of apoptosis was observed after 24 h and the lowest after 18 h. The comparison of the frequency of apoptotic cells determined by the comet assay with DNA damage (OTM) examined by the comet assay revealed a statistically significant, negative correlation. The TUNEL results showed that the frequency of apoptotic cells progressively increased in a dose- and time-dependent manner. The comparison of the frequency of apoptotic cells determined by TUNEL method with DNA damage (OTM) examined by the comet assay showed a significant positive correlation for lymphocytes treated with MC-LR for 6, 12 and 18 h. Therefore, our findings indicate that microcystin-LR-induced DNA damage observed in the comet assay may be related to the early stages of apoptosis due to cytotoxicity but not genotoxicity. In addition, we examined the DNA repair kinetics in lymphocytes

  10. Genotoxicity of di-butyl-phthalate and di-iso-butyl-phthalate in human lymphocytes and mucosal cells.

    PubMed

    Kleinsasser, N H; Wallner, B C; Kastenbauer, E R; Weissacher, H; Harréus, U A

    2001-01-01

    The genotoxicity of phthalates, widely used plasticizers, has been shown previously for di-butyl-phthalate (DBP) and di-iso-butyl-phthalate (DBP) in human mucosal cells of the upper aerodigestive tract in a previous study using the Comet assay. Furthermore, higher genotoxic sensitivities of patients with squamous cell carcinomas of either the larynx or the oropharynx compared to non-tumor patients were described. Other authors have demonstrated DNA damage by a different phthalate in human lymphocytes. It was the aim of the present study to determine whether there is a correlation between the genotoxic sensitivities to DBP and its isomer DiBP in either mucosal cells or lymphocytes. The single-cell microgel electrophoresis assay (Comet assay) was applied to detect DNA strand breaks in human epithelial cells of the upper aerodigestive tract (n=132 specimens). Human mucosa was harvested from the oropharynx in non-tumor patients and patients with squamous cell carcinomas of the oropharynx. Laryngeal mucosa of patients with laryngeal squamous cell carcinomas was harvested as well. Peripheral lymphocytes (n=49 specimens) were separated from peripheral blood. Xenobiotics investigated were DBP, DiBP, and N'methyl-N'-nitro-N-nitrosoguanidine (MNNG) as positive control, respectively. For statistical analysis, the SPSS correlation analysis according to Pearson and the Wilcoxon test were performed. Genotoxicity was found for DBP and DiBP in epithelial cells and lymphocytes (P<0.001). MNNG caused severe DNA damage. In analyzing DBP and DiBP results, genotoxic impacts in mucosal cells showed an intermediate correlation (r=0.570). Correlation in lymphocytes was the same (r=0.570). Phthalates have been investigated as a potential health hazard for a variety of reasons, including possible xenoestrogenic impact, peroxisome proliferation, and membrane destabilization. The present investigation suggests a correlated DNA-damaging impact of DBP and DiBP in human mucosal cells and in

  11. Correlation between radiation dose and p53 protein expression levels in human lymphocytes.

    PubMed

    Cavalcanti, Mariana B; Fernandes, Thiago S; Silva, Edvane B; Amaral, Ademir

    2015-09-01

    The aim of this research was to evaluate the relationship between p53 protein levels and absorbed doses from in vitro irradiated human lymphocytes. For this, samples of blood from 23 donors were irradiated with 0.5; 1; 2; and 4 Gy from a Cobalt-60 source, and the percentages of lymphocytes expressing p53 were scored using Flow Cytometry. The subjects were divided into 3 groups, in accordance with the p53 levels expressed per radiation dose: low (Group I), high (Group II), and excessive levels (Group III). For all groups, the analyses showed that the p53 expression levels increase with the absorbed dose. Particularly for groups I and II, the correlation between this protein expression and the dose follows the linear-quadratic model, such as for radioinduced chromosomal aberrations. In conclusion, our findings indicate possible applications of this approach in evaluating individual radiosensitivity prior to radiotherapeutical procedures as well as in medical surveillance of occupationally exposed workers. Furthermore, due to the rapidity of flow-cytometric analyses, the methodology here employed would play an important role in emergency responses to a large-scale radiation incident where many people may have been exposed. PMID:26312422

  12. Micronuclei Assessment of The Radioprotective Effects of Melatonin and Vitamin C in Human Lymphocytes

    PubMed Central

    Rostami, Aram; Moosavi, Seyed Akbar; Dianat Moghadam, Hassan; Bolookat, Eftekhar Rajab

    2016-01-01

    Objective Critical macromolecules such as DNA maybe damaged by free radicals that are generated from the interaction of ionizing radiation with biological systems. Melatonin and vitamin C have been shown to be direct free radical scavengers. The aim of this study was to investigate the in vivo/in vitro radioprotective effects of melatonin and vitamin C separately and combined against genotoxicity induced by 6 MV x-ray irradiation in human cultured blood lymphocytes. Materials and Methods In this experimental study, fifteen volunteers were divided into three groups of melatonin, vitamin C and melatonin plus vitamin C treatment. Peripheral blood samples were collected from each group before, and 1, 2 and 3 hours after melatonin and vitamin C administration (separately and combined). The blood samples were then irradiated with 200 cGy of 6 MV x-ray. In order to characterize chromosomal aberrations, the lymphocyte samples were cultured with mitogenic stimulus on cytokinesisblocked binucleated cells. Results The samples collected 1hour after melatonin and vitamin C (separately and combined) ingestion exhibited a significant decrease in the incidence of micronuclei compared with their control group (P<0.05). The maximum synergic protection and reduction in frequency of micronuclei (57%) was observed 1 hour after vitamin C and melatonin administration combined. Conclusion We conclude that simultaneous administration of melatonin and vitamin C as radioprotector substances before irradiation may reduce genotoxicity caused by x-ray irradiation. PMID:27054118

  13. Proliferative kinetics of human lymphocytes in culture measured by autoradiography and sister chromatid differential staining

    SciTech Connect

    Morimoto, K.; Sato, M.; Koizumi, A.

    1983-01-01

    A simple combination of autoradiography, to determine when a cell synthesized DNA, and sister chromatid differential staining, to determine how many times a cell has divided, was used to follow up the proliferating fate of human lymphocytes in culture. Cells were incubated continuously with 5-bromodeoxyuridine (BrdU) and pulse-labelled with 0.1 ..mu..Ci/ml (/sup 3/H)thymidine at various times after stimulation with phytohemagglutinin (PHA). The cells were then harvested at 4 h intervals up to 72 h, and the percentage of labelled mitoses was determined separately in first, second, or third division cells. The data showed that the cycling cells, whether they began cycling at earlier or later times after stimulation, had about the same generation times of 12-14 h. This confirms that the heterogeneity of cell generations seen in short-term lymphocyte cultures is in large part due to the difference in the times when cells began cell cycling in response to PHA. 34 references, 2 figures, 1 table.

  14. Effects of ascorbic acid and sodium ascorbate on cyclic nucleotide metabolism in human lymphocytes.

    PubMed

    Atkinson, J P; Weiss, A; Ito, M; Kelly, J; Parker, C W

    1979-01-01

    L-ascorbic acid (LAA) augmented cGMP many-fold in highly purified human peripheral blood lymphocytes. The cGMP response occurred within 10 sec and persisted for at least 60 min. D-ascorbic acid (DAA) and dehydroascorbic acid (DHAA) were also equally active in enhancing cGMP concentrations but metabolic precursors of ascorbic acid and other inorganic acids did not increase cGMP levels. Determination of the amount of DHAA contaminating the LAA precluded the possibility that it was solely responsible for the enhanced cGMP levels. The sodium or calcium salts of ascorbic acid did not increase cGMP concentrations. If these neutralized preparations were acidified, increased cGMP concentrations were then noted. In broken cell preparations, LAA, DAA, and DHAA and to a lesser extent sodium ascorbate (NaA) enhanced guanylate cyclase activity while neither inhibited cAMP or cGMP phosphodiesterase (PDE) activity. The possible role of H2O2, fatty acid liberation, prostaglandin production, oxidizing-reducing agents, and free radical formation in mediating the effects of ascorbic acid on cGMP levels were evaluated, but none of these potential mechanisms were definitively proven to be a required intermediary for the cGMP enhancing activity of ascorbic acid. LAA, DHAA or NaA did not induce lymphocyte transformation or modulate lectin-induced mitogenesis. PMID:36416

  15. Frequency domain electrical conductivity measurements of the passive electrical properties of human lymphocytes.

    PubMed

    Bordi, F; Cametti, C; Rosi, A; Calcabrini, A

    1993-11-21

    An extensive set of electrical conductivity measurements of human lymphocyte suspensions has been carried out in the frequency range from 1 kHz to 100 MHz, where the surface polarization due to the Maxwell-Wagner effect occurs. The data have been analyzed according to well-established heterogeneous system theories and the passive electrical parameters of both the cytoplasmic and nuclear membranes have been obtained. Moreover, a further analysis to take into account the roughness of the membrane surface on the basis of a fractal model yields new estimates for the membrane conductivity and the membrane permittivity, independently of the surface architecture of the cell. These findings are confirmed by measurements carried out at higher frequencies, in the range from 1 MHz to 1 GHz, on lymphocytes dispersed in both hyperosmotic and hypoosmotic media, that influence the surface complexity of the membrane due to the microvillous protrusions. The surface roughness of the cell is described by a macroscopic parameter (the fractal dimension) whose variations are associated to the progressive swelling of the cell, as the osmolality of the solution is changed. PMID:8241253

  16. Effects of halothane on the human beta-adrenergic receptor of lymphocyte membranes

    SciTech Connect

    Marty, J.; Nivoche, Y.; Nimier, M.; Rocchiccioli, C.; Luscombe, F.; Henzel, D.; Loiseau, A.; Desmonts, J.M.

    1987-12-01

    The effects of halothane on beta-adrenergic receptor antagonist interaction were studied using the membranes of human lymphocytes as a model. Membrane preparations of lymphocytes were obtained from blood samples withdrawn from seven healthy young volunteers. Beta-receptor studies were performed using (-)/sup 125/I iodocyanopindolol (/sup 125/ICP) binding. Non-specific binding was determined in the presence of (-)isoproterenol. Beta-receptor density (Bmax) and the dissociation constant (KD) for /sup 125/ICP were determined from saturation curves. Beta-receptor affinity for agonists evaluated by the IC50 (the concentration of isoproterenol required to inhibit 50% of specific /sup 125/ICP binding) and the dissociation constant (KL) for isoproterenol was established from competition curves. The effect of halothane 1%, in an air oxygen mixture (oxygen fraction: 0.3) administered by tonometry during ligand membrane incubation, on beta-adrenergic receptor, was compared to that of control experiments not exposed to halothane. Halothane produced a moderate but significant decrease of Bmax (-10%) and a significant increase in non-specific binding (+30%), while KD, IC50, and KL were unchanged. The authors conclude that halothane, in vitro, decreases beta-adrenergic receptor density. This effect could be mediated by an alteration of the receptor in the membrane due to action of halothane on the lipid phase of the membrane.

  17. Induction of sister chromatid exchanges by coal dust and tobacco snuff extracts in human peripheral lymphocytes

    SciTech Connect

    Tucker, J.D.; Ong, T.

    1985-01-01

    The organic solvent extracts of sub-bituminous coal dust and tobacco snuff, both together and separately, were tested for the induction of sister chromatid exchanges (SCEs) in human peripheral lymphocytes. The results indicate that these extracts induced SCEs, and that when tested together synergistically induced SCEs in two of three donors. Studies with the organic solvent extracts of all five ranks of coal indicate that the extracts of bituminous, lignite, and peat, but not anthracite, induced SCEs. Similar experiments conducted with water extracts, induced SCEs, and that anthracite was equivocal. To determine whether individuals differed in their SCE responses to coal dust extracts, lymphocytes from five donors were tested with organic solvent extracts of bituminous and sub-bituminous coal. An analysis of variance indicates that the SCE response was significantly influenced by the donor and each of the two coal extracts. The findings presented here suggest that coal dust, with or without tobacco snuff, may play a role in the elevated incidence of gastric cancer in coal miners. Because water extracts of some ranks of coal induced SCEs, there exists the possibility of adverse environmental effects due to coal leachates.

  18. Growth hormone protects human lymphocytes from irradiation-induced cell death

    PubMed Central

    Lempereur, Laurence; Brambilla, Daria; Maria Scoto, Giovanna; D'Alcamo, Maria; Goffin, Vincent; Crosta, Lucia; Palmucci, Tullio; Rampello, Liborio; Bernardini, Renato; Cantarella, Giuseppina

    2003-01-01

    Undesired effects of cancer radiotherapy mainly affect the hematopoietic system. Growth hormone (GH) participates in both hematopoiesis and modulation of the immune response. We report both r-hGH cell death prevention and restoration of secretory capacities of irradiated human peripheral blood lymphocytes (PBL) in vitro. r-hGH induced cell survival and increased proliferation of irradiated cells. Western blot analysis indicated that these effects of GH were paralleled by increased expression of the antiapoptotic protein Bcl-2. r-hGH restored mitogen-stimulated release of IL-2 by PBL. Preincubation of irradiated lymphocytes with the growth hormone receptor (GHR) antagonists B2036 and G120 K abrogated r-hGH-dependent IL-2 release. These results demonstrate that r-hGH protects irradiated PBL from death in a specific, receptor-mediated manner. Such effect of r-hGH on PBL involves activation of the antiapoptotic gene bcl-2 and prevention of cell death, associated with preserved functional cell capacity. Finally, potential use of GH as an immunopotentiating agent could be envisioned during radiation therapy of cancer. PMID:12721095

  19. Biodosimetry of ionizing radiation by selective painting of prematurely condensed chromosomes in human lymphocytes

    NASA Technical Reports Server (NTRS)

    Durante, M.; George, K.; Yang, T. C.

    1997-01-01

    Painting of interphase chromosomes can be useful for biodosimetric purposes in particular cases such as radiation therapy, accidental exposure to very high radiation doses and exposure to densely ionizing radiation, for example during space missions. Biodosimetry of charged-particle radiation is analyzed in the present paper. Target cells were human peripheral blood lymphocytes irradiated in vitro with gamma rays, protons and iron ions. After exposure, lymphocytes were incubated for different times to allow repair of radiation-induced damage and then fused to mitotic hamster cells to promote premature condensation in the interphase chromosomes. Chromosome spreads were then hybridized with whole-chromosome DNA probes labeled with fluorescent stains. Dose-response curves for the induction of chromatin fragments shortly after exposure, as well as the kinetics of rejoining and misrejoining, were not markedly dependent on linear energy transfer. However, after exposure to heavy ions, more aberrations were scored in the interphase cells after incubation for repair than in metaphase samples harvested at the first postirradiation mitosis. On the other hand, no significant differences were observed in the two samples after exposure to sparsely ionizing radiation. These results suggest that interphase chromosome painting can be a useful tool for biodosimetry of particle radiation.

  20. Karyotypes of Human Lymphocytes Exposed to High-Energy Iron Ions

    NASA Technical Reports Server (NTRS)

    Durante, M.; George, K.; Wu, H.; Cucinotta, F. A.

    2002-01-01

    Chromosomal aberrations were analyzed using multicolor fluorescence in situ hybridization (mFISH) in human peripheral blood lymphocytes after in vitro exposure to gamma rays or accelerated (56)Fe ions (1 GeV/nucleon, 145 keV/microm) at Brookhaven National Laboratory (Upton, NY). Doses of 0.3 and 3 Gy were used for both radiation types. Chromosomes were prematurely condensed by a phosphatase inhibitor (calyculin A) to avoid the population selection bias observed at metaphase as a result of the severe cell cycle delays induced by heavy ions. A total of 1053 karyotypes (G(2) and M phases) were analyzed in irradiated lymphocytes. Results revealed different distribution patterns for chromosomal aberrations after low- and high-LET radiation exposures: Heavy ions induced a much higher fraction of cells with multiple aberrations, while the majority of the aberrant cells induced by low doses of gamma rays contained a single aberration. The high fraction of complex-type exchanges after heavy ions leads to an overestimation of simple-type asymmetrical interchanges (dicentrics) from analysis of Giemsa-stained samples. However, even after a dose of 3 Gy iron ions, about 30% of the cells presented no complex-type exchanges. The involvement of individual chromosomes in exchanges was similar for densely and sparsely ionizing radiation, and no statistically significant evidence of a nonrandom involvement of specific chromosomes was detected.

  1. Reduction of chrysotile asbestos-induced genotoxicity in human peripheral blood lymphocytes by garlic extract.

    PubMed

    Bhattacharya, Kunal; Yadava, Santosh; Papp, Thilo; Schiffmann, Dietmar; Rahman, Qamar

    2004-11-28

    Asbestos fibers are well known environmental carcinogen, however, the underlying mechanisms of their action have still not clearly been identified. Asbestos is capable of depleting glutathione and generating reactive oxygen species (ROS), which are important mediators of damage in biological system. Asbestos-induced mutagenecity, may be mediated by the generation. It is known that a number of scavengers and antioxidants attenuate asbestos-induced ROS release. Furthermore, it is known that garlic, contains numerous sulfur compounds and glutathione precursors which act as antioxidants and also demonstrate anticarcinogenic properties. The aim of this study was to investigate whether garlic extract has any influence on asbestos-mediated genotoxicity. As an assay system, we applied the micronucleus assay, sister chromatid exchanges, and chromosomal aberrations with human peripheral blood lymphocytes, which has already been used to analyze the genotoxicity of asbestos fibers. Our results indicate that garlic extract, when administered to the lymphocytes cell culture simultaneously with chrysotile reduced the rates of micronucleus formation, sister chromatid exchanges, and chromosomal aberrations significantly. We conclude that garlic extract may be an efficient, physiologically tolerable quencher of asbestos-mediated genotoxicity. PMID:15454308

  2. Visualization of microvascular proliferation as a tumor infiltration structure in rat glioma specimens using the diffraction-enhanced imaging in-plane CT technique

    NASA Astrophysics Data System (ADS)

    Seo, Seung-Jun; Sunaguchi, Naoki; Yuasa, Tetsuya; Huo, Qingkai; Ando, Masami; Choi, Gi-Hwan; Kim, Hong-Tae; Kim, Ki-Hong; Jeong, Eun-Ju; Chang, Won-Seok; Kim, Jong-Ki

    2012-03-01

    In order to study potent microenvironments of malignant gliomas with a high- resolution x-ray imaging technique, an injection orthotopic glioma model was made using the Sprague-Dawley rat. Total brain tissue, taken out as an ex vivo model, was examined with diffraction-enhanced imaging (DEI) computed tomography (CT) acquired with a 35 keV monochromatic x-ray. In the convolution-reconstructed 2D/3D images with a spatial resolution of 12.5 × 12.5 × 25 µm, distinction among necrosis, typical ring-shaped viable tumors, edemas and healthy tissues was clearly observed near the frontal lobe in front of the rat's caudate nucleus. Multiple microvascular proliferations (MVPs) were observed surrounding peritumoral edemas as a tumor infiltration structure. Typical dimensions of tubular MVPs were 130 (diameter) ×250 (length) µm with a partial sprout structure revealed in the 3D reconstructed image. Hyperplasia of cells around vessel walls was revealed with tumor cell infiltration along the perivascular space in microscopic observations of mild MVP during histological analysis. In conclusion, DEI-CT is capable of imaging potent tumor-infiltrating MVP structures surrounding high-grade gliomas.

  3. Smad7 induces plasticity in tumor-infiltrating Th17 cells and enables TNF-alpha-mediated killing of colorectal cancer cells.

    PubMed

    Rizzo, Angelamaria; De Mare, Vincenzo; Rocchi, Chiara; Stolfi, Carmine; Colantoni, Alfredo; Neurath, Markus F; Macdonald, Thomas T; Pallone, Francesco; Monteleone, Giovanni; Fantini, Massimo C

    2014-07-01

    Transforming growth factor-beta (TGF-β) is deeply involved in colorectal cancer development and the disruption of the TGF-β signaling in dysplastic cells is required for tumor to grow. Nevertheless, tumor cells express TGF-β to escape the immune surveillance mediated by T cells. T-cell expression of Smad7, an intracellular inhibitor of the TGF-β signaling, protects against colitis-associated colorectal cancer. However, whether Smad7 in T cells might influence colorectal cancer growth independently of chronic inflammation and which T-cell subset is involved in this process is unknown. To address this issue, T-cell-specific Smad7 transgenic mice and wild-type (WT) littermates were subcutaneously transplanted with syngenic MC38 colon carcinoma cells. Smad7Tg mice were resistant to tumor development compared with WT mice and protection was dependent on CD4(+) T cells. Smad7 expression in T cells increased the number of tumor-infiltrating Tbet/ROR-γ-t double-positive CD4 T cells characterized by the expression of tumor necrosis factor-alpha (TNF-α) and interferon-gamma but lower IL17A. The low expression of IL17A caused by the Smad7 expression in tumor-infiltrating CD4(+) T cells enabled the TNF-α-mediated killing of cancer cells both in vitro and in vivo, thus indicating that the Smad7-mediated plastic effect on T-cell phenotype induces protection against colorectal cancer. PMID:24480808

  4. Rejoining and misrejoining of radiation-induced chromatin breaks. I. experiments with human lymphocytes

    NASA Technical Reports Server (NTRS)

    Durante, M.; George, K.; Wu, H.; Yang, T. C.

    1996-01-01

    Fluorescence in situ hybridization with a composite probe for human chromosome 4 and a probe that stained all centromeres was used to study gamma-ray induced breakage, rejoining and misrejoining in prematurely condensed chromosomes in human lymphocytes. Dose-response curves for the induction of all types of aberrations in prematurely condensed human chromosomes 4 were determined immediately after irradiation and after 8 h postirradiation incubation. In addition, aberrations were measured after various incubation times from 0 to 18 h after a dose of 7 Gy. Unrejoined chromosome breaks were the most frequent type of aberration observed immediately after irradiation. Approximately 15% of total aberrations observed were chromosome exchanges. After 8 h postirradiation incubation, the frequency of breaks in prematurely condensed chromosomes declined to about 20% of the initial value, and chromosomal exchanges became the most frequent aberration. Results of metaphase analysis were similar to those for prematurely condensed chromosomes after 8 h incubation with the exception that a significantly lower frequency of fragments was observed. Symmetrical and asymmetrical interchanges were found at similar frequencies at all doses. No complex exchanges were observed in lymphocyte chromosomes immediately after exposure. They accounted for about 1% of total exchanges in metaphase chromosomes at doses <3 Gy and about 14% at 7 Gy. Incomplete exchanges amounted to approximately 15% of total exchanges at all doses. The kinetics of break rejoining was exponential, and the frequency of exchanges increased with kinetics similar to that observed for the rejoining of the breaks. This increase in the total exchanges as a function of the time between irradiation and fusion was due to a rapid increase in reciprocal interchanges, and a slower increase in complex exchanges; the frequency of incomplete exchanges increased initially, then decreased with prolonged incubation to the level observed

  5. Cytogenetic Low-Dose Hyperradiosensitivity Is Observed in Human Peripheral Blood Lymphocytes

    SciTech Connect

    Seth, Isheeta; Joiner, Michael C.; Tucker, James D.

    2015-01-01

    Purpose: The shape of the ionizing radiation response curve at very low doses has been the subject of considerable debate. Linear-no-threshold (LNT) models are widely used to estimate risks associated with low-dose exposures. However, the low-dose hyperradiosensitivity (HRS) phenomenon, in which cells are especially sensitive at low doses but then show increased radioresistance at higher doses, provides evidence of nonlinearity in the low-dose region. HRS is more prominent in the G2 phase of the cell cycle than in the G0/G1 or S phases. Here we provide the first cytogenetic mechanistic evidence of low-dose HRS in human peripheral blood lymphocytes using structural chromosomal aberrations. Methods and Materials: Human peripheral blood lymphocytes from 2 normal healthy female donors were acutely exposed to cobalt 60 γ rays in either G0 or G2 using closely spaced doses ranging from 0 to 1.5 Gy. Structural chromosomal aberrations were enumerated, and the slopes of the regression lines at low doses (0-0.4 Gy) were compared with doses of 0.5 Gy and above. Results: HRS was clearly evident in both donors for cells irradiated in G2. No HRS was observed in cells irradiated in G0. The radiation effect per unit dose was 2.5- to 3.5-fold higher for doses ≤0.4 Gy than for doses >0.5 Gy. Conclusions: These data provide the first cytogenetic evidence for the existence of HRS in human cells irradiated in G2 and suggest that LNT models may not always be optimal for making radiation risk assessments at low doses.

  6. Gene expression profile of human lymphocytes exposed to (211)At alpha particles.

    PubMed

    Turtoi, A; Brown, I; Schläger, M; Schneeweiss, F H A

    2010-08-01

    In this study, the Whole Human Genome 44K DNA microarray assay was used for the first time to obtain gene expression profiles in human peripheral blood lymphocytes 2 h after exposure (in suspension) to 6.78 MeV mean energy alpha particles from extracellular (211)At. Lymphocytes were exposed to fluences of 0.3-9.6 x 10(6) alpha particles/cm(2) [corresponding to mean absorbed alpha-particle doses (D(alpha)) of 0.05-1.60 Gy] over 30 min. Significantly modulated expression was identified in 338 early-response genes. Up-regulated expression was evident in 183 early-response genes, while the remaining 155 were down-regulated. Over half of the up-regulated genes and 40% of the down-regulated genes had a known biological process related primarily to cell growth and maintenance and cell communication. Genes associated with cell death were found only in the up-regulated genes and those with development only in the down-regulated genes. Eight selected early-response genes that displayed a sustained up- or down-regulation (CD36, HSPA2, MS4A6A, NFIL3, IL1F9, IRX5, RASL11B and SULT1B1) were further validated in alpha-particle-irradiated lymphocytes of two human individuals using the TaqMan(R) RT-qPCR technique. The results confirmed the observed microarray gene expression patterns. The expression modulation profiles of IL1F9, IRX5, RASL11B and SULT1B1 genes demonstrated similar trends in the two individuals studied. However, no significant linear correlation between increasing relative gene expression and the alpha-particle dose was evident. The results suggest the possibility that a panel of genes that react to alpha-particle radiation does exist and that they merit further study in a greater number of individuals to determine their possible value regarding alpha-particle biodosimetry. PMID:20681779

  7. The impact of lymphocyte isolation on induced DNA damage in human blood samples measured by the comet assay.

    PubMed

    Bausinger, Julia; Speit, Günter

    2016-09-01

    The comet assay is frequently used in human biomonitoring for the detection of exposure to genotoxic agents. Peripheral blood samples are most frequently used and tested either as whole blood or after isolation of lymphocytes (i.e. peripheral blood mononuclear cells, PBMC). To investigate a potential impact of lymphocyte isolation on induced DNA damage in human blood samples, we exposed blood ex vivo to mutagens with different modes of genotoxic action. The comet assay was performed either directly with whole blood at the end of the exposure period or with lymphocytes isolated directly after exposure. In addition to the recommended standard protocol for lymphocyte isolation, a shortened protocol was established to optimise the isolation procedure. The results indicate that the effects of induced DNA strand breaks and alkali-labile sites induced by ionising radiation and alkylants, respectively, are significantly reduced in isolated lymphocytes. In contrast, oxidative DNA base damage (induced by potassium bromate) and stable bulky adducts (induced by benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide; BPDE) seem to be less affected. Our findings suggest that in vivo-induced DNA damage might also be reduced in isolated lymphocytes in comparison with the whole blood depending of the types of DNA damage induced. Because only small genotoxic effects can generally be expected in human biomonitoring studies with the comet assay after occupational and environmental exposure to genotoxic agents, any loss might be relevant and should be avoided. The possibility of such effects and their potential impact on variability of comet assay results in human biomonitoring should be considered when performing or evaluating such kind of studies. PMID:27154923

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

    PubMed

    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. PMID:25531291

  9. Modulation of radiation-induced and mitomycin C-induced chromosome damage by apigenin in human lymphocytes in vitro

    PubMed Central

    Sharma, Narinder K.

    2013-01-01

    Apigenin (APG), a flavone, is known to exhibit antioxidant, antimutagenic and antitumorigenic activity, both in vivo and in vitro. The aim of this study is to investigate the modulatory effects of APG on human lymphocytes after irradiation with gamma rays (3 Gy) or treatment with the antineoplastic agent, mitomycin C (MMC), in vitro. Cytogenetic biomarkers such as chromosome aberrations (CAs), sister chromatid exchanges (SCEs) and cytochalasin-B blocked micronuclei (CBMN), were studied in blood lymphocytes treated with radiation, or antineoplastic agent (MMC), and APG. Whole blood lymphocytes were cultured in vitro using a standard protocol. No significant differences were found in the frequency of CAs or micronuclei (MN) in human peripheral blood lymphocytes irradiated with gamma rays (3 Gy) and then post-treated with APG. There was an increase in the frequency of SCEs per cell in APG-treated samples compared with the controls. Lymphocytes treated with MMC in the presence of APG exhibited a significant decrease (P < 0.01) in the frequency of SCEs compared with MMC treatment alone. The data for the MN test indicated that APG treatment significantly reduced (P < 0.01) the frequency of MMC-induced MN. PMID:23764456

  10. Setae from Larvae of the Northern Processionary Moth (Thaumetopoea pinivora, TP) Stimulate Proliferation of Human Blood Lymphocytes In Vitro

    PubMed Central

    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. PMID:25531291

  11. [Physicochemical properties of the human lymphocyte mitogenic factor induced by phytohemagglutinin].

    PubMed

    Voĭtenok, N N; Varivotskaia, N V; Murzenok, P P; Potemkina, N D

    1976-08-01

    Some physical and chemical properties of the mitogenic factor (MF) produced in vitro by PHA-stimulated human lymphocytes were investigated. Treatment of the culture medium with proteases proved to decrease the MF activity sharply. The MF was found to have an elution pattern in Sephadex and Bio-gel gels similar to proteins with the molecular weight of 20 000--30 000 daltons (peak--25 000). In experiments with the MF fractionation by disc-electrophoresis on 5% polyacylamide gel the MF formed a few discrete peaks in the fractions corresponding to the mobility of alpha1- and alpha2-globulins and transferrin. In isoelectric focusing the MF formed 3 fractions in the pH range of 4.5--8.3. Functional heterogeneity of the MF is suggested. PMID:1026298

  12. Telomerase-based pharmacologic enhancement of antiviral function of human CD8+ T lymphocytes.

    PubMed

    Fauce, Steven Russell; Jamieson, Beth D; Chin, Allison C; Mitsuyasu, Ronald T; Parish, Stan T; Ng, Hwee L; Kitchen, Christina M Ramirez; Yang, Otto O; Harley, Calvin B; Effros, Rita B

    2008-11-15

    Telomerase reverse transcribes telomere DNA onto the ends of linear chromosomes and retards cellular aging. In contrast to most normal somatic cells, which show little or no telomerase activity, immune cells up-regulate telomerase in concert with activation. Nevertheless, during aging and chronic HIV-1 infection, there are high proportions of dysfunctional CD8(+) CTL with short telomeres, suggesting that telomerase is limiting. The present study shows that exposure of CD8(+) T lymphocytes from HIV-infected human donors to a small molecule telomerase activator (TAT2) modestly retards telomere shortening, increases proliferative potential, and, importantly, enhances cytokine/chemokine production and antiviral activity. The enhanced antiviral effects were abrogated in the presence of a potent and specific telomerase inhibitor, suggesting that TAT2 acts primarily through telomerase activation. Our study is the first to use a pharmacological telomerase-based approach to enhance immune function, thus directly addressing the telomere loss immunopathologic facet of chronic viral infection. PMID:18981163

  13. Calcium transport by ionophorous peptides in dog and human lymphocytes detected by quin-2 fluorescence.

    PubMed

    Deber, C M; Hsu, L C

    1986-01-29

    Synthetic peptides of structure cyclo(Glu(OBz)-Sar-Gly-(N-R)Gly)2 (I), electrogenic Ca2+-selective carriers in phospholipid vesicle membranes, are shown to mediate the uptake of Ca2+ ions into the cytoplasm of dog and human lymphocytes. Ca2+ transport by DECYL-2E (I, R = n-decyl) - monitored by measurements of the fluorescence of an intracellular dye, quin-2 - occurred at a rate comparable to that produced by electroneutral Ca2+ ionophores ionomycin and Br-A23187. Fluorescence quenching experiments using Mn2+ suggested a greater selectivity by DECYL-2E for Ca2+/Mn2+ vs. the other two ionophores. The result that Ca2+ ions can traverse biological membranes bound in a neutral cavity consisting exclusively of peptide carbonyl ligands may imply the functional significance of binding sites of similar structures in membrane transport proteins. PMID:3947349

  14. Radiosensitivity of chromosomes in two successive mitotic cycles of human lymphocytes

    SciTech Connect

    Luchnik, N.V.; Poryadkova, N.A.

    1988-11-01

    A culture of human lymphocytes was irradiated with /gamma/-quanta in a dose of 0.5 Gy with different ratios of cells in first (M1) and second (M2) mitotic cycle and the frequency of aberrations induced at stage G2 was analyzed. With increase in interval of time between the start of culturing and irradiation, total yield of aberrations increased in a regular way. However, if the M1:M2 ratio is considered, then it turns out that in M2 chromosomes are /approximately/1.5 times more sensitive than in M1: within the limits of each cycle, radiosensitivity is constant and does not depend on its duration. It was established in accordance with data of other authors that 5-bromodeoxyuridine (5-BdU) increases radiosensitivity materially.

  15. Portable device for magnetic stimulation: Assessment survival and proliferation in human lymphocytes

    NASA Astrophysics Data System (ADS)

    Pérez, H.; Cordova-Fraga, T.; López-Briones, S.; Martínez-Espinosa, J. C.; Rosas, E. F.; Espinoza, A.; Villagómez-Castro, J. C.; Sosa, M.; Topsu, S.; Bernal-Alvarado, J. J.

    2013-09-01

    A device's instrumentation for magnetic stimulation on human lymphocytes is presented. This is a new procedure to stimulate growing cells with ferrofluid in vortices of magnetic field. The stimulation of magnetic vortices was provided at five different frequencies, from 100 to 2500 Hz and intensities from 1.13 to 4.13 mT. To improve the stimulation effects, a paramagnetic ferrofluid was added on the cell culture medium. The results suggest that the frequency changes and the magnetic field variation produce an important increase in the number of proliferating cells as well as in the cellular viability. This new magnetic stimulation modality could trigger an intracellular mechanism to induce cell proliferation and cellular survival only on mitogen stimulated cells.

  16. Chromosome aberrations induced in human lymphocytes by D-T neutrons

    SciTech Connect

    Lloyd, D.C.; Edwards, A.A.; Prosser, J.S.; Bolton, D.; Sherwin, A.G.

    1984-06-01

    Unstable chromosome aberrations induced by in vitro irradiation with D-T neutrons have been analyzed in human blood lymphocytes. With respect to 250 kVp X rays a maximum limiting RBE at low doses of 4.1 was obtained for dicentric aberrations. Using aberrations as markers in mixed cultures of irradiated and unirradiated cells permits an assessment of interphase death plus mitotic delay. The low-dose RBE for this effect is 2.5. Assuming all unstable aberrations observed at metaphase would lead to cell death by nondisjunction allows an assessment of mitotic death. The low-dose RBE for this effect is 4.5. The data are compared with similar work obtained earlier with /sup 242/Cm ..cap alpha.. particles. The application of the present work to cytogenetic assessment of dose after accidental exposure to D-T neutrons is discussed.

  17. Alteration of membrane transductive mechanisms induced by ethanol in human lymphocyte cultures.

    PubMed

    Fanò, G; Belia, S; Mariggiò, M A; Antonica, A; Agea, E; Spinozzi, F

    1993-03-01

    Ethanol, in millimolar concentrations, significantly modifies different transductive systems in human lymphocyte cultures. In particular, the presence of alcohol in the medium more than doubles the [Ca2+]i (from 70-90 to 200-250 nM), increasing Ca2+ fluxes from outside, and inhibits the active transport carried out by the calcium pump. The Ca2+ release from intracellular stores is not involved because 10 mM EGTA in the medium completely abolished the rise of [Ca2+]i. Since IP3 levels and cAMP concentrations are also involved in ethanol events (although with opposite effects), it seems that the alcohol may have a specific target on cell membranes (G-proteins) which influence many transductive pathways. PMID:8388700

  18. Clastogenic effects of food additive citric acid in human peripheral lymphocytes

    PubMed Central

    Ünal, Fatma; Yüzbaşıoğlu, Deniz; Aksoy, Hüseyin

    2008-01-01

    Clastogenic properties of the food additive citric acid, commonly used as an antioxidant, were analysed in human peripheral blood lymphocytes. Citric acid induced a significant increase of chromosomal aberrations (CAs) at all the concentrations and treatment periods tested. Citric acid significantly decreased mitotic index (MI) at 100 and 200 μg ml−1 concentrations at 24 h, and in all concentrations at 48 h. However, it did not decrease the replication index (RI) significantly. Citric acid also significantly increased sister chromatid exchanges (SCEs) at 100 and 200 μg ml−1 concentrations at 24 h, and in all concentrations at 48 h. This chemical significantly increased the micronuclei frequency (MN) compared to the negative control. It also decreased the cytokinesis-block proliferation index (CBPI), but this result was not statistically significant. PMID:19002851

  19. Comparison of histamine release from human blood monocytes, lymphocytes, adenoidal and skin mast cells.

    PubMed

    Schmutzler, W; Bolsmann, K; Zwadlo-Klarwasser, G

    1995-01-01

    Monocytes and lymphocytes from human blood contain 0.043 +/- 0.007 and 0.053 +/- 0.014 pg histamine/cell, respectively, which can be released by a number of stimulants (A 23187, C5a, substance P, specific allergen). The release process takes 60-120 min to reach its end point, in contrast to tissue mast cells which complete the release within 1-3 min. Both, ketotifen (10(-7) - 10(-5) M) and disodium cromoglycate (10(-5) - 10(-3) M) inhibited histamine release dose dependently up to 40-45%, which might be particularly relevant during the later stages of acute allergic or pseudoallergic reactions. PMID:7542070

  20. Monoclonal antibody (H107) inhibiting IgE binding to Fc epsilon R(+) human lymphocytes.

    PubMed

    Noro, N; Yoshioka, A; Adachi, M; Yasuda, K; Masuda, T; Yodoi, J

    1986-08-15

    A hybridoma-producing monoclonal antibody blocking the binding of human IgE to lymphocytes Fc receptor (Fc epsilon R) was established by the fusion of murine myeloma cells. P3X63.653.Ag8, with BALB/c spleen cells immunized with Fc epsilon R(+) human B lymphoblastoid cell line cells, RPMI1788. A clone of the hybridoma (H107) produced a monoclonal IgG2b antibody that inhibited the rosette formation of Fc epsilon R(+) human B lymphoblastoid cell line cells (RPMI1788, RPMI8866, CESS, Dakiki, and IM9) with fixed ox red blood cells (ORBC) conjugated with human IgE (IgE-ORBC). In contrast, the rosette formation with IgG-conjugated ORBC (IgG-ORBC) on Fc gamma R(+), Fc epsilon R(-) Daudi cells were not affected by the H107 antibodies. A close association of Fc epsilon R and the antigenic determinant recognized by H107 antibody was suggested by the following results. First, the bindings of 125I-labeled IgE (125I-IgE) or 125I-labeled H107 IgG2b antibody (125I-H107) to RPMI8866 cells were inhibited by cold human IgE and H107 IgG2b but not by other classes of human Ig (IgA and IgG), MPC11 IgG2b, or unrelated monoclonal antibodies. Second, H107 antibody reacted with Fc epsilon R(+) B cell lines but not with Fc epsilon R(-) B cell lines as determined by an indirect immunofluorescence. Third, Fc epsilon R(+) cells were depleted by the incubation in the dish coated with H107 antibody or IgE but not in the dish coated with unrelated antibodies. Finally, there was a correlation between the increase of Fc epsilon R(+) cells and that of H107(+) cells in the peripheral blood lymphocytes of the patients with atopic dermatitis. The surface antigens on Fc epsilon R(+) RPMI8866 cells recognized by H107 antibodies had the molecular size of 45,000 as determined by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. PMID:2942602

  1. Age-related increases in human lymphocyte DNA damage: is there a role of aerobic fitness?

    PubMed

    Soares, Jorge Pinto; Mota, Maria Paula; Duarte, José Alberto; Collins, Andrew; Gaivão, Isabel

    2013-12-01

    Oxidative stress has been advanced as one of the major causes of damage to DNA and other macromolecules. Although physical exercise may also increase oxidative stress, an important role has been recognized for regular exercise in improving the overall functionality of the body, as indicated by an increase in maximal aerobic uptake ((V)O2max), and in resistance to cell damage. The aims of this study were 1) to evaluate the association between DNA damage in human lymphocytes and age and 2) to evaluate the association between DNA damage in human lymphocytes and ((V)O2max. The sample was composed of 36 healthy and nonsmoking males, aged from 20 to 84 years. ((V)O2max was evaluated through the Bruce protocol with direct measurement of oxygen consumption. The comet assay was used to evaluate the DNA damage, strand breaks and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites. We found a positive correlation of age with DNA strand breaks but not with FPG-sensitive sites. ((V)O2max was significantly inversely related with DNA strand breaks, but this relation disappeared when adjusted for age. A significantly positive relation between ((V)O2max and FPG-sensitive sites was verified. In conclusion, our results showed that younger subjects have lower DNA strand breaks and higher (V)O2max compared with older subjects and FPG-sensitive sites are positively related with ((V)O2max, probably as transient damage due to the acute effects of daily physical activity. PMID:24446564

  2. CD57 in human natural killer cells and T-lymphocytes.

    PubMed

    Kared, Hassen; Martelli, Serena; Ng, Tze Pin; Pender, Sylvia L F; Larbi, Anis

    2016-04-01

    The CD57 antigen (alternatively HNK-1, LEU-7, or L2) is routinely used to identify terminally differentiated 'senescent' cells with reduced proliferative capacity and altered functional properties. In this article, we review current understanding of the attributes of CD57-expressing T-cells and NK cells in both health and disease and discuss how this marker can inform researchers about their likely functions in human blood and tissues in vivo. While CD57 expression on human lymphocytes indicates an inability to proliferate, these cells also display high cytotoxic potential, and CD57(pos) NK cells exhibit both memory-like features and potent effector functions. Accordingly, frequencies of CD57-expressing cells in blood and tissues have been correlated with clinical prognosis in chronic infections or various cancers and with human aging. Functional modulation of senescent CD57(pos) T-cells and mature CD57(pos) NK cells may therefore represent innovative strategies for protection against human immunological aging and/or various chronic diseases. PMID:26850637

  3. Expression profile of Eph receptors and ephrin ligands in healthy human B lymphocytes and chronic lymphocytic leukemia B-cells.

    PubMed

    Alonso-C, Luis M; Trinidad, Eva M A; de Garcillan, Beatriz; Ballesteros, Monica; Castellanos, Milagros; Cotillo, Ignacio; Muñoz, Juan J; Zapata, Agustin G

    2009-03-01

    Increasing information relates some Eph receptors and their ligands, ephrins (EFN), with the immune system. Herein, we found that normal B-cells from peripheral blood (PB) and lymph nodes (LN) showed a differential expression of certain Eph/EFN members, some of them being modulated upon in vitro stimulation including EFNA1, EFNA4, EphB6 and EphA10. In contrast, PB CLL B-cells showed a more heterogeneous Eph/EFN profile than their normal PB B-cell counterparts, expressing Eph/EFN members frequently found within the LN and activated B-cells, specially EFNA4, EphB6 and EphA10. Two of them, EphB6 and EFNA4 were further related with the clinical course of CLL patients. EphB6 expression correlated with a high content of ZAP-70 mRNA and a poor prognosis. High serum levels of a soluble EFNA4 isoform positively correlated with increasing peripheral blood lymphocyte counts and lymphadenopathy. These findings suggest that Eph/EFN might be relevant in normal B-cell biology and could represent new potential prognostic markers and therapeutic targets for CLL. PMID:18819711

  4. Human T lymphocyte migration towards the supernatants of human rhinovirus infected airway epithelial cells: influence of exercise and carbohydrate intake.

    PubMed

    Bishop, Nicolette C; Walker, Gary J; Gleeson, Michael; Wallace, Fiona A; Hewitt, Colin R A

    2009-01-01

    Physical stress induces a marked redistribution of T lymphocytes that may be influenced by carbohydrate (CHO) availability, yet the effect of these on T lymphocyte migration towards infected tissue is unknown. Therefore, the aim of this study was to determine the effect of strenuous exercise and CHO ingestion on subsequent ex vivo lymphocyte migration towards the supernatants of a Human Rhinovirus (HRV)-infected bronchial epithelial cell line. In a randomised, cross-over, double-blind design, 7 trained males ran for 2 h at 60% VO2peak on two occasions with regular ingestion of either a 6.4% w/v glucose and maltodextrin solution (CHO trial) or placebo solution (PLA trial). Plasma glucose concentration was higher on CHO than PLA after exercise (P<0.05). Migration of CD4+ and CD8+ cells and their CD45RA+ and CD45RO+ subpopulations towards supernatants from HRV-infected cells decreased following exercise (main effect for exercise, P<0.01 for CD4+, CD4+CD45RA+ and CD4+CD45RO+; P<0.05 for CD8+, CD8+CD45RA+ and CD8+CD45RO+). Migration of CD4+ cells and CD4+CD45RA+ cells was approximately 35% and approximately 30% higher, respectively, on CHO than PLA at 1 h post-exercise (interaction, P<0.05 for both) and was higher on CHO than PLA for all other subpopulations (P<0.05, main effect for trial). There was little effect of exercise or CHO on migration of these cells towards uninfected (control) cell supernatants or on the proportion of these cells within the peripheral blood mononuclear cell population. The findings of this study suggest that physical stress reduces T cell migration towards HRV-infected cell supernatants and that ingestion of CHO can lessen this effect. PMID:19957874

  5. LARGE DELETIONS ARE TOLERATED AT THE HPRT LOCUS OF IN-VIVO DERIVED HUMAN T-LYMPHOCYTES

    EPA Science Inventory

    A cloning assay was used to recover hprt T-lymphocytes from adult human males. nalysis of crude cellular extracts by polymerase chain reactions (PCRs) demonstrated that 8% (18/218) of the hprt mutations were due to total deletion of the hprt gene. ourteen of the 18 mutants were e...

  6. CYTOGENETIC COMPARISON OF THE RESPONSES OF MOUSE AND HUMAN PERIPHERAL BLOOD LYMPHOCYTES TO 60CO GAMMA RADIATION (JOURNAL VERSION)

    EPA Science Inventory

    Experiments were conducted to compare the chromosome damaging effects of (60)Co gamma radiation on mouse and human peripheral blood lymphocytes (PBLs). Either whole blood or isolated and pelleted mononuclear leucocytes (MNLs) were irradiated with a (60)Co unit to yield exposures ...

  7. MULTIPLEX PCR ANALYSIS OF IN VIVO-ARISING DELETION MUTATIONS IN THE HPRT GENE OF HUMAN T-LYMPHOCYTES

    EPA Science Inventory

    A multiplex polymerase chain reaction (PCR) procedure was adapted for the rapid and efficient evaluation of the hypoxanthine guanine phosphoribosyltransferase (hprt) gene in human T-lymphocytes for deletions. he hprt clonal assay was used to isolate in-vivo-arising hprt-deficient...

  8. DELETION MUTATIONS IN THE HPRT GENE OF T-LYMPHOCYTES AS A BIOMARKER FOR GENOMIC REARRANGEMENTS IMPORTANT IN HUMAN CANCERS

    EPA Science Inventory

    The DNA sequence of 11 in vivo-arising intragenic deletion breaksite junctions occurring in the hypoxanthine guanine phosphoribosyltransferase gene of human T-lymphocytes was determined and deletions ranged in size from 16 bp to 4057 bp. o extensive homology was found at the dele...

  9. Effect of environmental exposures to lead and cadmium on human lymphocytic detoxifying enzymes

    SciTech Connect

    D'Souza, S.J.; Narurkar, L.M.; Narurkar, M.V. )

    1994-09-01

    Lead (Pb) is among the most toxic heavy elements in the atmosphere. Aerosol lead enters the human blood stream by way of the respiratory tract and indirectly, by surface disposition in the alimentary tract followed by adsorption. Lead pollution is also known to occur through its presence in petrol, pain, glazed vessels and solder. Atmospheric lead pollution may be predominantly high around factories manufacturing Pb alloys. Lead toxicity is associated with inhibition of [alpha]-aminolevulinic acid dehydrase (ALAD) activity, rise in the blood porphyrin, inhibition of ATPase in erthrocytes, decreased blood haemoglobin and anemia. Elevated lead concentrations in pregnant women have been shown to cause hypertension and birth defects. Lead is also known to interact with other elements such as Fe, Zn, Ca and Cu in biological systems. Cadmium (Cd) is not essential for human body. It enters the human environment as a contaminant. Human intake of Cd is chiefly through the food chain (about 400-500 [mu]g/wk). Analysis of neuropsy material shows that smokers accumulate much more Cd than nonsmokers. Chronic Cd poisoning produces proteinuere and affects the proximal tubules of kidney, causing the formation of kidney stones. The reported hypertensive effect of Cd in man has been associated with high Cd/Zn ratio in kidney. Studies on air pollution have shown that Cd concentration in air could be positively correlated with heart disease, hypertension and arteriosclerosis. The present investigation was aimed at assessing the usefulness of human lymphocytic detoxicating enzyme activities and their ratios in an assessment of human health-risks during environmental exposures to Pb and Cd. The human subjects investigated comprised those exposed to highly contaminated lead and cadmium areas in the state of Maharashtra, India. 17 refs., 2 figs.

  10. Relation between clinical mature and immature lymphocyte cells in human peripheral blood and their spatial label free scattering patterns

    NASA Astrophysics Data System (ADS)

    Zhang, Lu; Zhao, Xin; Zhang, Zhenxi; Zhao, Hong; Chen, Wei; Yuan, Li

    2016-07-01

    A single living cell's light scattering pattern (LSP) in the horizontal plane, which has been denoted as the cell's "2D fingerprint," may provide a powerful label-free detection tool in clinical applications. We have recently studied the LSP in spatial scattering planes, denoted as the cell's "3D fingerprint," for mature and immature lymphocyte cells in human peripheral blood. The effects of membrane size, morphology, and the existence of the nucleus on the spatial LSP are discussed. In order to distinguish clinical label-free mature and immature lymphocytes, the special features of the spatial LSP are studied by statistical method in both the spatial and frequency domains. Spatial LSP provides rich information on the cell's morphology and contents, which can distinguish mature from immature lymphocyte cells and hence ultimately it may be a useful label-free technique for clinical leukemia diagnosis.

  11. [Structural and Functional Modifications of Human Lymphocytes in Dynamics of UV-Induced Development of Their Apoptosis].

    PubMed

    Nakvasina, M A; Lidokhova, O V; Domanskya, T L; Artyukhov, V G; Ryazantsev, S V

    2015-01-01

    The flow-cytometric analysis of condition of human peripheral blood lymphocytes in dynamics of development of the apoptosis induced by exposure to UV-light (240-390 nanometers) at a dose of 1510 J/m2 was carried out. Superficial architectonics and changes of the level of a membrane potential and functional activity of succinatedehydrogenase of mitochondrions are studied. The contribution of extracellular calcium to the processes of lymphocyte cellular death is revealed. The ideas about dynamics of the intracellular events leading to the death of photomodified lymphocytes 1-6 h after their UV-radiation exposure are specified and added on the basis of the analysis of our new and already published results. PMID:26964347

  12. Relation between clinical mature and immature lymphocyte cells in human peripheral blood and their spatial label free scattering patterns.

    PubMed

    Zhang, Lu; Zhao, Xin; Zhang, Zhenxi; Zhao, Hong; Chen, Wei; Yuan, Li

    2016-07-01

    A single living cell's light scattering pattern (LSP) in the horizontal plane, which has been denoted as the cell's "2D fingerprint," may provide a powerful label-free detection tool in clinical applications. We have recently studied the LSP in spatial scattering planes, denoted as the cell's "3D fingerprint," for mature and immature lymphocyte cells in human peripheral blood. The effects of membrane size, morphology, and the existence of the nucleus on the spatial LSP are discussed. In order to distinguish clinical label-free mature and immature lymphocytes, the special features of the spatial LSP are studied by statistical method in both the spatial and frequency domains. Spatial LSP provides rich information on the cell's morphology and contents, which can distinguish mature from immature lymphocyte cells and hence ultimately it may be a useful label-free technique for clinical leukemia diagnosis. PMID:27475572

  13. Expression of Thomsen-Friedenreich (TF) antigens on lymphocytes. II. Loss of cryptic TF antigens during mitogenic activation of human T and B lymphocytes.

    PubMed

    Wolf, M F; Schmitt, H R; Schumacher, K

    1989-07-01

    Human peripheral blood lymphocytes (HPBL) were examined for the presence of cryptic Thomsen-Friedenreich (TF) antigens as detected by PNA or an anti-TF mAb (49H8) after neuraminidase treatment of the cell surface. Neither PNA nor the mAb bound to the cells before treatment with neuraminidase. After removal of surface sialic acid, all lymphocytes were PNA-reactive, and 85% of HPBL reacted with the mAb 49H8. Seventy-seven percent of nylon wool (NW)-eluted T cells (96% Leu 1+), 80% of enriched helper T cells (83% Leu 3a+), and 78% of suppressor/cytotoxic T cells (63% Leu 2a+) carried the cryptic TF determinant recognized by the mAb 49H8. Ninety-one percent of NW-adherent cells (68% Leu 10+, 5% Leu 1+) were also TF positive. In contrast to NW-eluted T cells which showed low to moderate mAb 49H8 binding, 48% of NW-adherent cells revealed strong binding of anti-TF mAb. With progressive activation of T cells by PHA, binding of mAb to the cryptic TF antigen completely disappeared on blast cells. The presence of TF antigens on small cells in the culture was only poorly affected. The same was observed for activation of B cells with PWM. On the other hand, binding sites for PNA did not change during blastogenesis. The disappearance of the particular, mAb 49H8-reactive TF antigen on T blast cells is not due to the loss of antigen in a distinct T cell subset, but occurs to an equal extent in the helper and suppressor/cytotoxic T cell subpopulations. Thus, the majority of peripheral T and B lymphocytes carries cryptic TF antigens. Activated T or B cell blasts, on the other hand, are deficient for the particular TF antigen detected by the mAb 49H8. We interpret these data as a modulation of certain TF antigens on effector cells in the course of lymphocyte activation. PMID:2786763

  14. Inhibition of human peripheral blood lymphocyte function by protoporphyrin and longwave ultraviolet light

    SciTech Connect

    Barrett, K.E.; Yen, A.; Montisano, D.; Gigli, I.; Bigby, T.D.

    1994-10-01

    Modulation of immunologic effector cells by exogenous photoactive substances has been advanced as an underlying mechanism for the efficacy of various photochemotherapeutic regimens. It is also possible that endogenous photosensitizers, such as protoporphyrin, could similarly modify the function of immune cell types. The authors examined the effects of protoporphyrin plus longwave UV light on the ability of human PBL to proliferate in response to mitogens. Noncytotoxic dosages of protoporphyrin plus UV light suppressed PHA-stimulated proliferation of both PBMC and enriched T cells. CD8{sup +} cells were more sensitive to this inhibitory effect than CD4{sup +} cells. The inhibitory effect was also observed when proliferation was induced by the combination of a phorbol ester and ionomycin. Inhibition of PBMC proliferation was associated with inhibition of IL-2 secretion but proliferation was not restored with exogenous IL-2. Instead, the effect of protoporphyrin plus UV light may be on IL-2R. Cells treated with protoporphyrin and UV light did not display the increase in CD25 and {beta}-chain of the IL-2R induced by PHA in control cells. In contrast to the effects of protoporphyrin and UV light on IL-2 and IL-2R {alpha}-chain protein expression, the accumulation of mRNA for these proteins induced by PHA was unaffected. None of the effects of protoporphyrin plus UV light on lymphocytes were observed in control experiments where cells were treated with either protoporphyrin or UV light alone. They conclude that biologically relevant dosages of protoporphyrin and UV light modify the function of circulating lymphocytes. 26 refs., 8 figs., 1 tab.

  15. Glucocorticoids increase the synthesis of immunoglobulin E by interleukin 4-stimulated human lymphocytes.

    PubMed Central

    Wu, C Y; Sarfati, M; Heusser, C; Fournier, S; Rubio-Trujillo, M; Peleman, R; Delespesse, G

    1991-01-01

    This study indicates that hydrocortisone (HC) markedly increases the synthesis of immunoglobulin E (IgE) by interleukin 4 (IL-4)-stimulated human lymphocytes. The effect is glucocorticoid specific and is obtained with low concentrations of HC (0.1-10 microM). In both the early and the late phase of the IL-4-induced response HC exerts its effects which are respectively IL-4 dependent and IL-4 independent. The IgE potentiation cannot be explained by the inhibition of interferon-gamma (IFN-gamma) production since it is observed in the absence of endogenous secretion of IFN-gamma. HC inhibits the production of IgE-binding factors (soluble CD23) and the expression of the low-affinity receptor for IgE, also known as the (Fc epsilon RII) CD23 antigen; however, the residual expression of Fc epsilon RII by IL-4- and HC-treated peripheral blood mononuclear cells (PBMCs) is important since the IgE response of these cells is markedly inhibited by anti-CD23 monoclonal antibody. HC acts mainly by amplifying the cellular interactions between monocytes and lymphocytes; indeed, HC has no effect on monocyte-depleted PBMCs, and moreover, monocytes cannot be replaced by soluble factors. Most importantly, T cells are not required for the induction of IgE synthesis by costimulation with IL-4 and HC. However, the IgE response of rigorously T cell-depleted PBMCs may be further increased by the addition of T cells. Further analysis of the permissive effect of HC on the synthesis of IgE by T cell-depleted PBMCs suggests that HC acts in synergy with IL-4 to trigger the activation and the differentiation of B cells into IgE-producing cells. PMID:1825666

  16. Melittin induced cytogenetic damage, oxidative stress and changes in gene expression in human peripheral blood lymphocytes.

    PubMed

    Gajski, Goran; Domijan, Ana-Marija; Žegura, Bojana; Štern, Alja; Gerić, Marko; Novak Jovanović, Ivana; Vrhovac, Ivana; Madunić, Josip; Breljak, Davorka; Filipič, Metka; Garaj-Vrhovac, Vera

    2016-02-01

    Melittin (MEL) is the main constituent and principal toxin of bee venom. It is a small basic peptide, consisting of a known amino acid sequence, with powerful haemolytic activity. Since MEL is a nonspecific cytolytic peptide that attacks lipid membranes thus leading to toxicity, the presumption is that it could have significant therapeutic benefits. The aim was to evaluate the cyto/genotoxic effects of MEL in human peripheral blood lymphocytes (HPBLs) and the molecular mechanisms involved using a multi-biomarker approach. We found that MEL was cytotoxic for HPBLs in a dose- and time-dependent manner. It also induced morphological changes in the cell membrane, granulation and lysis of exposed cells. After treating HPBLs with non-cytotoxic concentrations of MEL, we observed increased DNA damage including oxidative DNA damage as well as increased formation of micronuclei and nuclear buds, and decreased lymphocyte proliferation determined by comet and micronucleus assays. The observed genotoxicity coincided with increased formation of reactive oxygen species, reduction of glutathione level, increased lipid peroxidation and phospholipase C activity, showing the induction of oxidative stress. MEL also modulated the expression of selected genes involved in DNA damage response (TP53, CDKN1A, GADD45α, MDM), oxidative stress (CAT, SOD1, GPX1, GSR and GCLC) and apoptosis (BAX, BCL-2, CAS-3 and CAS-7). Results indicate that MEL is genotoxic to HPBLs and provide evidence that oxidative stress is involved in its DNA damaging effects. MEL toxicity towards normal cells has to be considered if used for potential therapeutic purposes. PMID:26704293

  17. Chromosome aberration yields and apoptosis in human lymphocytes irradiated with Fe-ions of differing LET

    NASA Astrophysics Data System (ADS)

    Lee, R.; Nasonova, E.; Ritter, S.

    In the present paper the relationship between cell cycle delays induced by Fe-ions of differing LET and the aberration yield observable in human lymphocytes at mitosis was examined. Cells of the same donor were irradiated with 990 MeV/n Fe-ions (LET = 155 keV/μm), 200 MeV/n Fe-ions (LET = 440 keV/μm) and X-rays and aberrations were measured in first cycle mitoses harvested at different times after 48 84 h in culture and in prematurely condensed G2-cells (PCCs) collected at 48 h using calyculin A. Analysis of the time-course of chromosomal damage in first cycle metaphases revealed that the aberration frequency was similar after X-ray irradiation, but increased two and seven fold after exposure to 990 and 200 MeV/n Fe-ions, respectively. Consequently, RBEs derived from late sampling times were significantly higher than those obtained at early times. The PCC-data suggest that the delayed entry of heavily damaged cells into mitosis results especially from a prolonged arrest in G2. Preliminary data obtained for 4.1 MeV/n Cr-ions (LET = 3160 keV/μm) revealed, that these delays are even more pronounced for low energy Fe-like particles. Additionally, for the different radiation qualities, BrdU-labeling indices and apoptotic indices were determined at several time-points. Only the exposure to low energy Fe-like particles affected the entry of lymphocytes into S-phase and generated a significant apoptotic response indicating that under this particular exposure condition a large proportion of heavily damaged cells is rapidly eliminated from the cell population. The significance of this observation for the estimation of the health risk associated with space radiation remains to be elucidated.

  18. Human lymphocytes express the transcriptional regulator, Wilms tumor 1: The role of WT1 in mediating nitric oxide-dependent repression of lymphocyte proliferation

    SciTech Connect

    Marcet-Palacios, Marcelo; Davoine, Francis; Adamko, Darryl J.; Moqbel, Redwan; Befus, A. Dean

    2007-11-16

    The inhibitory roles of nitric oxide (NO) in T cell proliferation have been observed and studied extensively over the last two decades. Despite efforts, the fundamental pathway by which NO exerts its inhibitory actions remains to be elucidated although recent evidence suggests that the transcription factor Wilms tumor 1 (WT1) may be important. WT1 has been linked to numerous developmental pathways in particular nephrogenesis. Due to its roles in development and cell proliferation, polymorphisms within the WT1 gene can result in malignancies such as leukemia and Wilms tumor. WT1 functions as a transcriptional regulator and its activity is controlled through phosphorylation by protein kinase A (PKA). PKA-dependent WT1 phosphorylation results in translocation of WT1 from the nucleus to the cytosol, a process that interferes with WT1 transcriptional activities. In the current study we demonstrate that WT1 is expressed in human lymphocytes. Using the proliferative compound PHA we induced T cell proliferation and growth correlated with an increase in the expression of WT1 measured by RT-PCR, flow cytometry and immunoblot. Co-stimulation with the NO donor SNOG at concentrations of 0, 100, 300 and 600 {mu}M reduced in a concentration dependent way the PHA-induced upregulation of WT1 that correlated with a reduction in T cell proliferation. We conclude that WT1 might be an important component of the NO-dependent regulation of T lymphocyte proliferation and potential function.

  19. Results of space experiment program "Interferon". I. Production of interferon in vitro by human lymphocytes aboard space laboratory Solyut-6 ("Interferon I") and influence of space flight on lymphocyte functions of cosmonauts ("Interferon III").

    PubMed

    Tálas, M; Bátkai, L; Stöger, I; Nagy, L; Hiros, L; Konstantinova, I; Rykova, M; Mozgovaya, I; Guseva, O; Kozharinov, V

    1983-01-01

    The results of the biological space experiment "Interferon" performed by two international cosmonaut crews aboard the space laboratory Solyut-6 are reported. Human lymphocytes separated from the blood of healthy donors and placed into "Interferon I" equipment could be kept for 7 days in suspension culture under spaceflight conditions. Interferon production could be induced in human lymphocytes by preparations of different origin, such as virus, synthetic polyribonucleotides, bacterial protein and plant pigment. An increased lymphocyte interferon production was observed in the space laboratory as compared to the ground control. A decrease of induced interferon production and natural killer cell activity was observed in the cosmonauts' lymphocytes on the 1st day on Earth after 7 days spaceflight. PMID:6659855

  20. Effects of ISS equivalent ionizing radiation dose on Human T-lymphocytes

    NASA Astrophysics Data System (ADS)

    Meloni, Maria Antonia; Pani, Giuseppe; Benotmane, Rafi; Mastroleo, Felice; Aboul-El-Ardat, Khalil; Janssen, Ann; Leysen, Liselotte; Vanhavere, Filip; Leys, Natalie; Galleri, Grazia; Pippia, Proto; Baatout, Sarah

    One of the objectives of the current international space programs is to investigate the effects of cosmic environment on Humans. It is known that during a long exposure to the space conditions, including ionizing radiations and microgravity, the immune system of the astronauts is impaired. In past years several experiments were performed to identify responsible factors of in vitro mitogenic activation process in human T-lymphocytes under simulated microgravity effect and during dedicated space missions. It come out that the lack of immune response in microgravity occurs at the cellular and molecular level. In order to evaluate effects on pure primary T-lymphocytes from peripheral blood exposed to International Space Station (ISS)-like ionizing radiation, we applied a mixture of Cesium-137, as representative of low energy particles, and Californium-252, as representative of hight energy particles, with rate similar to those monitored inside the ISS during previous space mission (Goossens et all. 2006). This facility is available at SCK•CEN (Belgium) (Mastroleo et al., 2009). Although the dose received by the cells was relatively low, flow cytometry analysis 24 hours after irradiation showed a decrease in cell viability coupled with the increase of the caspase-3 activity. However, Bcl-2 activity did not seem to be affected by the radiation. Furthermore, activation of cells induced an increase of the cell size and alteration of cellular morphology. Cell cycle as well as 8-oxo-G were also modified upon radiation and activation. Gene expression analysis shows a modulation of genes rather as a consequence of exposure than with the activation status. 330 genes have been identified to be significantly modulated in function of the time and have been grouped in four different cluster representing significant expression profiles. Preliminary functional analysis shows mainly genes involved in the immune response and inflammatory diseases as well as oxidative stress and

  1. Cytogenetic effects of space-relevant hze-particles in human blood lymphocytes

    NASA Astrophysics Data System (ADS)

    Lee, R.; Nasonova, E.; Ritter, S.

    The analysis of aberrations in human lymphocytes collected 48 h after exposure is used since the 1960s to estimate the radiation risk. However, evidence is increasing that this protocol is not reliable in the case of high LET exposure, because particle induced cell cycle delays influence the aberration yield. To contribute to this issue lymphocytes obtained from a healthy donor were irradiated with Fe-ions (200 MeV/n, 440 keV/μ m), iron-like particles (˜ 4 MeV/n Ni- and Cr-ions, ˜ 4000 keV/μ m) and X-rays. Directly after irradiation PHA and BrdU was added to the cell culture medium. Aberrations were measured in first mitoses collected at 48, 60 and 72 h post-irradiation following colcemid treatment and in prematurely condensed G2-cells (PCCs) at 48 h using calyculin A. Samples were stained with the FPG-technique to allow cell cycle discrimination. Additionally, the mitotic index, the BrdU-labelling index and the number of apoptotic cells were determined at several time-points. Analysis of the BrdU-labelling indices and the mitotic indices revealed a dose- and LET-dependent delay in the cell cycle progression. Cells that reached the first mitosis 48 h after high LET exposure carried only a few aberrations. However, cells that entered the first mitosis 60 to 72 h after high LET exposure carried at least five times more aberrations than those collected at 48 h. The analysis of chromosomal damage in G2-PCCs showed that the delayed entry of severely damaged cells into mitosis results from a prolonged arrest in G2. Conversely, after X-ray exposure a stable aberration-yield was observed in lymphocytes collected at different time-points post-irradiation and the number of aberrations measured in G2-PCCs was only slightly higher than in metaphase cells. Furthermore, only in samples exposed to stopping heavy charged particles a high frequency of apoptotic cells was detected indicating that under this exposure conditions a large proportion of heavily damaged cells is

  2. Erythropoietin receptor is expressed on human peripheral blood T and B lymphocytes and monocytes and is modulated by recombinant human erythropoietin treatment.

    PubMed

    Lisowska, Katarzyna A; Debska-Slizień, Alicja; Bryl, Ewa; Rutkowski, Bolesław; Witkowski, Jacek M

    2010-08-01

    Erythropoietin receptor (EPO-R) appears on the cell surface in the early stages of erythropoiesis. It has also been found on endothelial cells and polymorphonuclear leukocytes, suggesting erythropoietin (EPO) role beyond erythropoiesis itself. Earlier reports have shown that treatment with recombinant human erythropoietin (rhEPO) in chronic renal failure (CRF) patients improves interleukin-2 production and restores the T lymphocyte function. We decided to investigate possible expression of EPO-R on circulating peripheral blood lymphocytes and monocytes of CRF patients in order to assess the possibility of rhEPO direct action on these cells. Flow cytometry was used for detection and quantification of EPO-R, and reverse transcription polymerase chain reaction for detection of the EPO receptor mRNA. Our results show for the first time the existence of EPO-R on cell surface of human T and B lymphocytes and monocytes as well as at the transcriptional activity of the EPO-R gene in these cells, both in healthy and CRF individuals. We have also found significant differences between the numbers of EPO-R molecules on T and B lymphocytes of CRF patients not treated and treated with rhEPO and healthy control. Discovery of EPO-R expression on human lymphocytes suggests that EPO is probably able to directly modulate some signaling pathways important for these cells. PMID:20528849

  3. Human autoimmunity after lymphocyte depletion is caused by homeostatic T-cell proliferation

    PubMed Central

    Jones, Joanne L.; Thompson, Sara A. J.; Loh, Priscilla; Davies, Jessica L.; Tuohy, Orla C.; Curry, Allison J.; Azzopardi, Laura; Hill-Cawthorne, Grant; Fahey, Michael T.; Compston, Alastair; Coles, Alasdair J.

    2013-01-01

    The association between lymphopenia and autoimmunity is recognized, but the underlying mechanisms are poorly understood and have not been studied systematically in humans. People with multiple sclerosis treated with the lymphocyte-depleting monoclonal antibody alemtuzumab offer a unique opportunity to study this phenomenon; one in three people develops clinical autoimmunity, and one in three people develops asymptomatic autoantibodies after treatment. Here, we show that T-cell recovery after alemtuzumab is driven by homeostatic proliferation, leading to the generation of chronically activated (CD28−CD57+), highly proliferative (Ki67+), oligoclonal, memory-like CD4 and CD8 T cells (CCR7−CD45RA− or CCR7−CD45RA+) capable of producing proinflammatory cytokines. Individuals who develop autoimmunity after treatment are no more lymphopenic than their nonautoimmune counterparts, but they show reduced thymopoiesis and generate a more restricted T-cell repertoire. Taken together, these findings demonstrate that homeostatic proliferation drives lymphopenia-associated autoimmunity in humans. PMID:24282306

  4. Studies on the cytotoxicity of diamond nanoparticles against human cancer cells and lymphocytes.

    PubMed

    Adach, Kinga; Fijalkowski, Mateusz; Gajek, Gabriela; Skolimowski, Janusz; Kontek, Renata; Blaszczyk, Alina

    2016-07-25

    Detonation nanodiamonds (DND) are a widely studied group of carbon nanomaterials. They have the ability to adsorb a variety of biomolecules and drugs onto their surfaces, and additionally their surfaces may be subjected to chemical functionalization by covalent bonds. We present a procedure for the purification and surface oxidation of diamond nanoparticles, which were then tested by spectroscopic analysis such as ATR-FTIR, Raman spectroscopy, and thermogravimetric analysis. We also examined the zeta potential of the tested material. Analysis of the cytotoxic effect of nanodiamonds against normal lymphocytes derived from human peripheral blood, the non-small cell lung cancer cell line (A549) and the human colorectal adenocarcinoma cell line (HT29) was performed using MTT colorimetric assay. Evaluation of cell viability was performed after 1-h and 24-h treatment with the tested nanoparticles applied at concentrations ranging from 1 μg/ml to 100 μg/ml. We found that the survival of the examined cells was strongly associated with the presence of serum proteins in the growth medium. The incubation of cells with nanodiamonds in the presence of serum did not exert a significant effect on cell survival, while the cell treatment in a serum-free medium resulted in a decrease in cell survival compared to the negative control. The role of purification and functionalization of nanodiamonds on their cytotoxicity was also demonstrated. PMID:27270448

  5. Chaga mushroom extract inhibits oxidative DNA damage in human lymphocytes as assessed by comet assay.

    PubMed

    Park, Yoo Kyoung; Lee, Hyang Burm; Jeon, Eun-Jae; Jung, Hack Sung; Kang, Myung-Hee

    2004-01-01

    The Chaga mushroom (Inonotus obliquus) is claimed to have beneficial properties for human health, such as anti-bacterial, anti-allergic, anti-inflammatory and antioxidant activities. The antioxidant effects of the mushroom may be partly explained by protection of cell components against free radicals. We evaluated the effect of aqueous Chaga mushroom extracts for their potential for protecting against oxidative damage to DNA in human lymphocytes. Cells were pretreated with various concentrations (10, 50, 100 and 500 microg/mL) of the extract for 1 h at 37 degrees C. Cells were then treated with 100 microM of H2O2 for 5 min as an oxidative stress. Evaluation of oxidative damage was performed using single-cell gel electrophoresis for DNA fragmentation (Comet assay). Using image analysis, the degree of DNA damage was evaluated as the DNA tail moment. Cells pretreated with Chaga extract showed over 40% reduction in DNA fragmentation compared with the positive control (100 micromol H2O2 treatment). Thus, Chaga mushroom treatment affords cellular protection against endogenous DNA damage produced by H2O2. PMID:15630179

  6. Protective Effect of Prolactin against Methylmercury-Induced Mutagenicity and Cytotoxicity on Human Lymphocytes

    PubMed Central

    Silva-Pereira, Liz Carmem; da Rocha, Carlos Alberto Machado; Cunha, Luiz Raimundo Campos da Silva e; da Costa, Edmar Tavares; Guimarães, Ana Paula Araújo; Pontes, Thais Brilhante; Diniz, Domingos Luiz Wanderley Picanço; Leal, Mariana Ferreira; Moreira-Nunes, Caroline Aquino; Burbano, Rommel Rodríguez

    2014-01-01

    Mercury exhibits cytotoxic and mutagenic properties as a result of its effect on tubulin. This toxicity mechanism is related to the production of free radicals that can cause DNA damage. Methylmercury (MeHg) is one of the most toxic of the mercury compounds. It accumulates in the aquatic food chain, eventually reaching the human diet. Several studies have demonstrated that prolactin (PRL) may be differently affected by inorganic and organic mercury based on interference with various neurotransmitters involved in the regulation of PRL secretion. This study evaluated the cytoprotective effect of PRL on human lymphocytes exposed to MeHg in vitro, including observation of the kinetics of HL-60 cells (an acute myeloid leukemia lineage) treated with MeHg and PRL at different concentrations, with both treatments with the individual compounds and combined treatments. All treatments with MeHg produced a significant increase in the frequency of chromatid gaps, however, no significant difference was observed in the chromosomal breaks with any treatment. A dose-dependent increase in the mitotic index was observed for treatments with PRL, which also acts as a co-mitogenic factor, regulating proliferation by modulating the expression of genes that are essential for cell cycle progression and cytoskeleton organization. These properties contribute to the protective action of PRL against the cytotoxic and mutagenic effects of MeHg. PMID:25247425

  7. Protective effect of prolactin against methylmercury-induced mutagenicity and cytotoxicity on human lymphocytes.

    PubMed

    Silva-Pereira, Liz Carmem; da Rocha, Carlos Alberto Machado; Cunha, Luiz Raimundo Campos da Silva E; da Costa, Edmar Tavares; Guimarães, Ana Paula Araújo; Pontes, Thais Brilhante; Diniz, Domingos Luiz Wanderley Picanço; Leal, Mariana Ferreira; Moreira-Nunes, Caroline Aquino; Burbano, Rommel Rodríguez

    2014-09-01

    Mercury exhibits cytotoxic and mutagenic properties as a result of its effect on tubulin. This toxicity mechanism is related to the production of free radicals that can cause DNA damage. Methylmercury (MeHg) is one of the most toxic of the mercury compounds. It accumulates in the aquatic food chain, eventually reaching the human diet. Several studies have demonstrated that prolactin (PRL) may be differently affected by inorganic and organic mercury based on interference with various neurotransmitters involved in the regulation of PRL secretion. This study evaluated the cytoprotective effect of PRL on human lymphocytes exposed to MeHg in vitro, including observation of the kinetics of HL-60 cells (an acute myeloid leukemia lineage) treated with MeHg and PRL at different concentrations, with both treatments with the individual compounds and combined treatments. All treatments with MeHg produced a significant increase in the frequency of chromatid gaps, however, no significant difference was observed in the chromosomal breaks with any treatment. A dose-dependent increase in the mitotic index was observed for treatments with PRL, which also acts as a co-mitogenic factor, regulating proliferation by modulating the expression of genes that are essential for cell cycle progression and cytoskeleton organization. These properties contribute to the protective action of PRL against the cytotoxic and mutagenic effects of MeHg. PMID:25247425

  8. De novo transcriptome profiling of highly purified human lymphocytes primary cells

    PubMed Central

    Bonnal, Raoul J.P.; Ranzani, Valeria; Arrigoni, Alberto; Curti, Serena; Panzeri, Ilaria; Gruarin, Paola; Abrignani, Sergio; Rossetti, Grazisa; Pagani, Massimiliano

    2015-01-01

    To help better understand the role of long noncoding RNAs in the human immune system, we recently generated a comprehensive RNA-seq data set using 63 RNA samples from 13 subsets of T (CD4+ naive, CD4+ TH1, CD4+ TH2, CD4+ TH17, CD4+ Treg, CD4+ TCM, CD4+ TEM, CD8+ TCM, CD8+ TEM, CD8+ naive) and B (B naive, B memory, B CD5+) lymphocytes. There were five biological replicates for each subset except for CD8+ TCM and B CD5+ populations that included 4 replicates. RNA-Seq data were generated by an Illumina HiScanSQ sequencer using the TruSeq v3 Cluster kit. 2.192 billion of paired-ends reads, 2×100 bp, were sequenced and after filtering a total of about 1.7 billion reads were mapped. Using different de novo transcriptome reconstruction techniques over 500 previously unknown lincRNAs were identified. The current data set could be exploited to drive the functional characterization of lincRNAs, identify novel genes and regulatory networks associated with specific cells subsets of the human immune system. PMID:26451251

  9. Mouse and human CD8(+) CD28(low) regulatory T lymphocytes differentiate in the thymus.

    PubMed

    Vuddamalay, Yirajen; Attia, Mehdi; Vicente, Rita; Pomié, Céline; Enault, Geneviève; Leobon, Bertrand; Joffre, Olivier; Romagnoli, Paola; van Meerwijk, Joost P M

    2016-06-01

    Regulatory T (Treg) lymphocytes play a central role in the control of immune responses and so maintain immune tolerance and homeostasis. In mice, expression of the CD8 co-receptor and low levels of the co-stimulatory molecule CD28 characterizes a Treg cell population that exerts potent suppressive function in vitro and efficiently controls experimental immunopathology in vivo. It has remained unclear if CD8(+) CD28(low) Treg cells develop in the thymus or represent a population of chronically activated conventional T cells differentiating into Treg cells in the periphery, as suggested by their CD28(low) phenotype. We demonstrate that functional CD8(+) CD28(low) Treg cells are present in the thymus and that these cells develop locally and are not recirculating from the periphery. Differentiation of CD8(+) CD28(low) Treg cells requires MHC class I expression on radioresistant but not on haematopoietic thymic stromal cells. In contrast to other Treg cells, CD8(+) CD28(low) Treg cells develop simultaneously with CD8(+) CD28(high) conventional T cells. We also identified a novel homologous naive CD8(+) CD28(low) T-cell population with immunosuppressive properties in human blood and thymus. Combined, our data demonstrate that CD8(+) CD28(low) cells can develop in the thymus of mice and suggest that the same is true in humans. PMID:26924728

  10. Identification of aneuploidy-inducing agents using cytokinesis-blocked human lymphocytes and an antikinetochore antibody

    SciTech Connect

    Eastmond, D.A.; Tucker, J.D.

    1989-01-01

    The identification of agents causing aneuploidy in humans, a condition associated with carcinogenesis and birth defects, is currently limited due to the highly skilled and time-consuming nature of cytogenetic analyses. We report the development of a new simple and rapid assay to identify aneuploidy-inducing agents (aneuploidogens). The assay involves the chemical- or radiation-induced formation of micronuclei in cytokinesis-blocked human lymphocytes and the use of an antikinetochore antibody to determine whether the micronuclei contain centromeres--a condition indicating a high potential for aneuploidy. All agents tested produced dose-related increases in the frequency of micronucleated cells. The micronucleated cells induced by the known aneuploidogens--colchicine, vincristine sulfate, and diethylstilbestrol--contained kinetochore-positive micronuclei 92, 87, and 76% of the time, respectively. In contrast, the micronucleated cells induced by the potent clastogens--ionizing radiation and sodium arsenite--contained kinetochore-positive micronuclei only 3 and 19% of the time, respectively. These results indicate that this relatively simple assay can discriminate between aneuploidogens and clastogens and may allow a more rapid identification of environmental and therapeutic agents with aneuploidy-inducing potential.

  11. The effect of brevenal on brevetoxin-induced DNA damage in human lymphocytes

    PubMed Central

    Sayer, Andrew; Hu, Qing; Bourdelais, Andrea J.; Baden, Daniel G.; Gibson, James E.

    2008-01-01

    Brevenal is a nontoxic short-chain trans-syn polyether that competes with brevetoxin (PbTx) for the active site on voltage-sensitive sodium channels. The PbTxs are highly potent polyether toxins produced during blooms of several species of marine dinoflagellates, most notably Karenia brevis. Blooms of K. brevis have been associated with massive fish kills, marine mammal poisoning, and are potentially responsible for adverse human health effects such as respiratory irritation and airway constriction in beach-goers. Additionally, the consumption of shellfish contaminated with PbTxs results in neurotoxic shellfish poisoning (NSP). The purpose of the present study was to determine whether PbTx could induce DNA damage in a human cell type, the lymphocyte, and if so, whether the damage could be antagonized or ameliorated by brevenal, a brevetoxin antagonist. The DNA damage may occur through both endogenous and exogenous physiological and pathophysiological processes. Unrepaired or erroneously repaired DNA damage may result in gene mutation, chromosome aberration, and modulation of gene regulation, which have been associated with immunotoxicity and carcinogenesis. A single-cell gel electrophoresis assay, or comet assay, was used to determine and compare DNA damage following various treatments. The data were expressed as tail moments, which is the percentage of DNA in the tail multiplied by the length between the center of the head and center of the tail (in arbitrary units). The negative control tail moment was 29.2 (SE=±0.9), whereas the positive control (hydrogen peroxide) was 72.1 (1.5) and solvent (ethanol) was 24.2 (2.1). The PbTx-2 (from Sigma, St. Louis, MO, USA), 10−8 M was 41.3 (3.6), PbTx-9 (Sigma), 10−8 M was 57.0 (5.3), PbTx-2 (from University of North Carolina at Wilmington, UNCW), 10−8 M was 49.4 (9.9), and PbTx-3 (UNCW), 10−8 M was 64.0 (6.4). 1.0 μg/ml brevenal applied 1 h before the PbTxs protected the lymphocytes from DNA damage; PbTx-2 (Sigma

  12. The effect of brevenal on brevetoxin-induced DNA damage in human lymphocytes.

    PubMed

    Sayer, Andrew; Hu, Qing; Bourdelais, Andrea J; Baden, Daniel G; Gibson, James E

    2005-11-01

    Brevenal is a nontoxic short-chain trans-syn polyether that competes with brevetoxin (PbTx) for the active site on voltage-sensitive sodium channels. The PbTxs are highly potent polyether toxins produced during blooms of several species of marine dinoflagellates, most notably Karenia brevis. Blooms of K. brevis have been associated with massive fish kills, marine mammal poisoning, and are potentially responsible for adverse human health effects such as respiratory irritation and airway constriction in beach-goers. Additionally, the consumption of shellfish contaminated with PbTxs results in neurotoxic shellfish poisoning (NSP). The purpose of the present study was to determine whether PbTx could induce DNA damage in a human cell type, the lymphocyte, and if so, whether the damage could be antagonized or ameliorated by brevenal, a brevetoxin antagonist. The DNA damage may occur through both endogenous and exogenous physiological and pathophysiological processes. Unrepaired or erroneously repaired DNA damage may result in gene mutation, chromosome aberration, and modulation of gene regulation, which have been associated with immunotoxicity and carcinogenesis. A single-cell gel electrophoresis assay, or comet assay, was used to determine and compare DNA damage following various treatments. The data were expressed as tail moments, which is the percentage of DNA in the tail multiplied by the length between the center of the head and center of the tail (in arbitrary units). The negative control tail moment was 29.2 (SE=+/-0.9), whereas the positive control (hydrogen peroxide) was 72.1 (1.5) and solvent (ethanol) was 24.2 (2.1). The PbTx-2 (from Sigma, St. Louis, MO, USA), 10(-8) M was 41.3 (3.6), PbTx-9 (Sigma), 10(-8) M was 57.0 (5.3), PbTx-2 (from University of North Carolina at Wilmington, UNCW), 10(-8) M was 49.4 (9.9), and PbTx-3 (UNCW), 10(-8) M was 64.0 (6.4). 1.0 microg/ml brevenal applied 1 h before the PbTxs protected the lymphocytes from DNA damage; PbTx-2

  13. Studies on human blood lymphocytes with iC3b (type 3) complement receptors. II. Characterization of subsets which regulate pokeweed mitogen-induced lymphocyte proliferation and immunoglobulin synthesis.

    PubMed Central

    Abo, W; Gray, J D; Bakke, A C; Horwitz, D A

    1987-01-01

    Human blood lymphocytes that express Type 3 complement receptors (CR3) can be divided into a major subset with high density Fc receptors for IgG (FcR) identified with the monoclonal antibody Leu 11 and two minor subsets which display either CD8 (Leu 2) or CD4 (Leu 3) markers. We isolated CR3+ lymphocyte subsets and examined them for regulatory effects on pokeweed mitogen (PWM) stimulated cells. The FCR CR3+ cell suppressed PWM-induced proliferation and Ig production. Pretreatment of these lymphocytes with immune complexes was required to suppress proliferation, but not IgG production. The CR3+ Leu 2+ FCR- subset also had suppressive activity, but this effect was not observed unless the CR3+ Leu 3+ enriched subset was removed. In fact, the CR3+ Leu 3+ enriched subset enhanced IgG synthesis. Brief exposure of CR3+ lymphocytes to recombinant interleukin 2, recombinant alpha-interferon, but not gamma-interferon, markedly enhanced the inhibitory effect. Time course studies and a comparison of inhibition of Ig synthesis with natural killer cell activity suggested that CR3+ lymphocytes act shortly after lymphocytes are exposed to PWM and that Ig production was regulated by suppression rather than cytotoxicity. These CR3+ lymphocyte subsets may have broad antigen non-specific effects on immunoglobulin synthesis. PMID:2955973

  14. Exogenous Interleukin-2 Administration Corrects the Cell Cycle Perturbation of Lymphocytes from Human Immunodeficiency Virus-Infected Individuals

    PubMed Central

    Paiardini, Mirko; Galati, Domenico; Cervasi, Barbara; Cannavo, Giuseppe; Galluzzi, Luca; Montroni, Maria; Guetard, Denise; Magnani, Mauro; Piedimonte, Giuseppe; Silvestri, Guido

    2001-01-01

    Human immunodeficiency virus (HIV)-induced immunodeficiency is characterized by progressive loss of CD4+ T cells associated with functional abnormalities of the surviving lymphocytes. Increased susceptibility to apoptosis and loss of proper cell cycle control can be observed in lymphocytes from HIV-infected individuals and may contribute to the lymphocyte dysfunction of AIDS patients. To better understand the relation between T-cell activation, apoptosis, and cell cycle perturbation, we studied the effect of exogenous interleukin-2 (IL-2) administration on the intracellular turnover of phase-dependent proteins. Circulating T cells from HIV-infected patients display a marked discrepancy between a metabolic profile typical of G0 and a pattern of expression of phase-dependent proteins that indicates a more-advanced position within the cell cycle. This discrepancy is enhanced by in vitro activation with ConA and ultimately results in a marked increase of apoptotic events. Conversely, treatment of lymphocytes with IL-2 alone restores the phase-specific pattern of expression of cell cycle-dependent proteins and is associated with low levels of apoptosis. Interestingly, exogenous IL-2 administration normalizes the overall intracellular protein turnover, as measured by protein synthesis, half-life of newly synthesised proteins, and total protein ubiquitination, thus providing a possible explanation for the effect of IL-2 on the intracellular kinetics of cell cycle-dependent proteins. The beneficial effect of IL-2 administration is consistent with the possibility of defective IL-2 function in vivo, which is confirmed by the observation that lymphocytes from HIV-infected patients show abnormal endogenous IL-2 paracrine/autocrine function upon in vitro mitogen stimulation. Overall these results confirm that perturbation of cell cycle control contributes to HIV-related lymphocyte dysfunction and, by showing that IL-2 administration can revert this perturbation, suggest a new

  15. Infection frequency of dendritic cells and CD4+ T lymphocytes in spleens of human immunodeficiency virus-positive patients.

    PubMed Central

    McIlroy, D; Autran, B; Cheynier, R; Wain-Hobson, S; Clauvel, J P; Oksenhendler, E; Debré, P; Hosmalin, A

    1995-01-01

    Dendritic cells (DC) are specialized antigen-presenting leukocytes that are responsible for the activation of naive as well as memory T lymphocytes. If infected by human immunodeficiency virus (HIV), DC may transfer virus to CD4+ lymphocytes. However, the question of whether DC are infected in vivo is controversial. As HIV infection is more active in secondary lymphoid organs than in blood, infection of splenic DC isolated from HIV-seropositive patients was investigated. Splenic DC were first enriched and characterized by flow cytometry from HIV- donors. After direct isolation, they were negative for monocyte and T- and B-lymphocyte markers, negative for CD1a, but positive for major histocompatibility complex class II and CD4. After in vitro maturation, major histocompatibility complex class II expression increased, while CD4 expression was lost. Extensive purification from the spleens of seven HIV+ patients was performed by fluorescence-activated cell sorting. The frequency of cells harboring HIV DNA in purified populations was quantified by limiting-dilution PCR. Directly isolated DC (average, 1/3,000; range, 1/720 to 1/18,000) were in each patient 10 to 100 times less infected than CD4+ T lymphocytes (average, 1/52; range, 1/17 to 1/190). On average, 1/1,450 (1/320 to 1/6,100) unseparated mononuclear splenocytes (containing 5% CD4+ lymphocytes) harbored HIV DNA. In conclusion, in these HIV+ patient spleens, DC seem to be infected, but HIV-DNA positive CD4+ T lymphocytes accounted for the vast majority of infected mononuclear splenocytes. PMID:7609039

  16. Differential Activation of Human Monocytes and Lymphocytes by Distinct Strains of Trypanosoma cruzi

    PubMed Central

    Magalhães, Luísa M. D.; Viana, Agostinho; Chiari, Egler; Galvão, Lúcia M. C.; Gollob, Kenneth J.; Dutra, Walderez O.

    2015-01-01

    Background Trypanosoma cruzi strains are currently classified into six discrete typing units (DTUs) named TcI to VI. It is known that these DTUs have different geographical distribution, as well as biological features. TcI and TcII are major DTUs found in patients from northern and southern Latin America, respectively. Our hypothesis is that upon infection of human peripheral blood cells, Y strain (Tc II) and Col cl1.7 (Tc I), cause distinct immunological changes, which might influence the clinical course of Chagas disease. Methodology/Principal Findings We evaluated the infectivity of CFSE-stained trypomastigotes of Col cl1.7 and Y strain in human monocytes for 15 and 72 hours, and determined the immunological profile of lymphocytes and monocytes exposed to the different isolates using multiparameter flow cytometry. Our results showed a similar percentage and intensity of monocyte infection by Y and Col cl1.7. We also observed an increased expression of CD80 and CD86 by monocytes infected with Col cl1.7, but not Y strain. IL-10 was significantly higher in monocytes infected with Col cl1.7, as compared to Y strain. Moreover, infection with Col cl1.7, but not Y strain, led to an increased expression of IL-17 by CD8+ T cells. On the other hand, we observed a positive correlation between the expression of TNF-alpha and granzyme A only after infection with Y strain. Conclusion/Significance Our study shows that while Col cl1.7 induces higher monocyte activation and, at the same time, production of IL-10, infection with Y strain leads to a lower monocyte activation but higher inflammatory profile. These results show that TcI and TcII have a distinct immunological impact on human cells during early infection, which might influence disease progression. PMID:26147698

  17. Characterization of a cellular inhibitory activity affecting the human autologous mixed lymphocyte reaction

    SciTech Connect

    Haynes, M.K.

    1987-01-01

    The human autologous mixed lymphocyte reaction, peripheral blood T cells are stimulated to proliferate when co-cultured with irradiated, autologous, non-T cells was studied. ({sup 3}H)-thymidine uptake was maximal on day 7 and observable on day 9. Non-adherent and adherent fractions of non-T population and lectin-activated T cell blasts contributed. Unseparated non-T stimulator cells were used. Autologous T cells, when added as irradiated third party cells, reduced AMLR proliferative responses. Depletion of Fc IgG receptor-bearing cells (T{gamma}) from T cell population by (a) IgG-sensitized ox erythrocyte rosetting (EA-rosettes) or (b) adsorption of the T{gamma} cells onto a Sepharose 6MB {approximately} BSA/anti-BSA immune complex column resulted in enhanced AMLR proliferation not attributed solely to an increase in CD8+ AMLR responder cell phenotype. Isolated T{gamma} populations exhibited a reversed helper/suppressor phenotype containing HLA DR+ and CD8+ cells. Dual labeling studies demonstrated 7-24% of CD8+ cells expressed DR antigens. After separation, AMLR proliferative capacity was localized in T-depleted population.

  18. Synthesis of novel tetrahydrobenzazepine derivatives and their cytoprotective effect on human lymphocytes.

    PubMed

    Simic, Milena R; Stankovic, Miroslava; Mandic, Boris M; Tesevic, Vele V; Savic, Vladimir M

    2015-02-01

    Cytoprotective compounds such as amifostine play an important role in chemo- and radiotherapy due to their ability to reduce the side effects of these treatments. Our work was initiated with the intention to design, synthesise and test a new class of heterocyclic compounds that would have an antioxidative profile with the potential to be further developed as cytoprotective agents. The design was based on the privileged tetrahydrobenzazepine scaffold found in many natural products with a wide range of biological properties. This structure was further functionalised with moieties known to possess antioxidative features such as tertiary amine and styrene double bond. A series of eight tetrahydrobenzazepine derivatives of isoquinoline, 3,4-dihydro-β-carboline and pyridine were synthesised employing the Heck reaction as a key transformation. Some of the prepared compounds were tested for their in vitro effects on chromosome aberrations in peripheral human lymphocytes using the cytochalasin-B blocked micronucleus (MN) assay. Three tetrahydrobenzoazepine derivatives showed significant cytoprotective properties, comparable or even better to those of the radioprotective agent amifostine. PMID:25664628

  19. Functional changes in astrocytes by human T-lymphotropic virus type-1 T-lymphocytes.

    PubMed

    Akaoka, H; Szymocha, R; Beurton-Marduel, P; Bernard, A; Belin, M F; Giraudon, P

    2001-10-30

    The human T-lymphotropic virus type-1 (HTLV-1) is the causative agent of a chronic progressive myelopathy (TSP/HAM) in which lesions of the central nervous system (CNS) are associated with infiltration of HTLV-1-infected T-cells. In a model that mimics the interaction between glial and T-cells, we show that transient contact with T-lymphocytes chronically infected with HTLV-1 induce profound metabolic alterations in astrocytes. Within the first week post-contact, an overall activation of astrocyte metabolism was observed as assessed by enhanced uptake of glutamate and glucose, and lactate release. In contrast, longer examination showed a reduced astrocytic accumulation of glutamate. The time course of the change in glutamate uptake was in fact biphasic. Previous observations indicated that HTLV-1 protein Tax-1 was involved in this delayed decrease, via the induction of TNF-alpha. The expression of the glial glutamate transporters, GLAST and GLT-1 decreased in parallel. These decreases in glutamate uptake and transporters' expression were associated with an imbalance in the expression of the catabolic enzymes of glutamate, GS and GDH, presumably due to Tax-1. Given the fact that impairment of glutamate management in astrocytes is able to compromise the functional integrity of neurons and oligodendrocytes, our results altogether give new insights into the physiopathology of TSP/HAM. PMID:11520580

  20. Dose rate effect of pulsed electron beam on micronucleus frequency in human peripheral blood lymphocytes.

    PubMed

    Acharya, Santhosh; Sanjeev, Ganesh; Bhat, Nagesh N; Narayana, Yerol

    2010-03-01

    The micronucleus assay in human peripheral blood lymphocytes is a sensitive indicator of radiation damage and could serve as a biological dosimeter in evaluating suspected overexposure to ionising radiation. Micronucleus (MN) frequency as a measure of chromosomal damage has also extensively been employed to quantify the effects of radiation dose rate on biological systems. Here we studied the effects of 8 MeV pulsed electron beam emitted by Microtron electron accelerator on MN induction at dose rates between 35 Gy min-1 and 352.5 Gy min-1. These dose rates were achieved by varying the pulse repetition rate (PRR). Fricke dosimeter was employed to measure the absorbed dose at different PRR and to ensure uniform dose distribution of the electron beam. To study the dose rate effect, blood samples were irradiated to an absorbed dose of (4.7+/-0.2) Gy at different rates and cytogenetic damage was quantified using the micronucleus assay. The obtained MN frequency showed no dose rate dependence within the studied dose rate range. Our earlier dose effect study using 8 MeV electrons revealed that the response of MN was linear-quadratic. Therefore, in the event of an accident, dose estimation can be made using linear-quadratic dose response parameters, without adding dose rate as a correction factor. PMID:20338871

  1. Characterization of protein kinase C and its isoforms in human T lymphocytes.

    PubMed

    Beyers, A D; Hanekom, C; Rheeder, A; Strachan, A F; Wooten, M W; Nel, A E

    1988-11-15

    Protein kinase C (PKC) regulates numerous T cell functions and is present in abundance in normal human T cells and certain T cell lines. Although crude Triton X-100 soluble material obtained from T cell pellets contains minimal PKC activity, DEAE chromatography revealed that 12 to 37% of cellular PKC was membrane associated, probably due to removal of an inhibitor through column chromatography. As in other tissues, PKC from lymphoid tissue was phospholipid and Ca2+ dependent and diolein reduced the Ca2+ requirements for enzyme activity. Hydroxylapatite chromatography revealed that T cells possess two major peaks of PKC activity. Although, the enzyme in these peaks had similar m.w. and identical iso-electric mobility, the proteins differed with respect to their autophosphorylation sites and immunoreactivity toward an isoform specific antibody. Furthermore, differences in their activities in the presence of phospholipid, diolein, and limiting amounts of Ca2+ imply that these isoforms may be differentially activated. We discuss optimal conditions for activation of PKC and its isoforms for study of T lymphocyte cellular function. PMID:3263426

  2. Apigenin ameliorates gamma radiation-induced cytogenetic alterations in cultured human blood lymphocytes.

    PubMed

    Begum, Naziya; Prasad, N Rajendra; Kanimozhi, G; Hasan, Annie Q

    2012-08-30

    The aim of the present study was to assess the protective effect of apigenin, a dietary flavone, against cytogenetic alterations in human peripheral blood lymphocytes (HPBL) induced by Cobalt-60 radiation (3Gy). Results of MTT [3-(4, 5-dimethyl-2-thiaozolyl)-2,5-diphenyl-2H tetrazolium bromide] assay revealed that 37.2μM of apigenin was found to be non-toxic in HPBL. At this dose (37.2μM) of apigenin, the LD(50) radiation dose of HPBL increased from 2.9Gy to 3.4Gy, which resulted in a DMF of 1.17. Apigenin (37.2μM) treatment 1h before irradiation significantly (p<0.05) reduced DNA damage in irradiated HPBL as measured by comet assay (% tail DNA, tail length, tail moment, and olive tail moment). Moreover, apigenin treatment significantly decreased the frequencies of dicentric (DC), acentric fragments (AF), and acentric rings (AR) in irradiated HPBL. Apigenin pretreatment also reduced the radiation-induced CBMN (cytokinesis blocked micronuclei) anomalies such as micronuclei (MNi), nucleoplasmic bridges (NPB) and nuclear buds (NBUD) in HPBL. These results also showed that there was a significant correlation between NPB and DC frequencies and MNi and AF+AR. Treatment with apigenin alone had no significant effect on DNA damage and chromosomal aberrations in HPBL. Thus, the current studies indicate that apigenin protects HPBL from radiation-induced cytogenetic alterations. PMID:22516036

  3. Immunoresistant human glioma cell clones selected with alloreactive cytotoxic T lymphocytes

    PubMed Central

    Gomez, German G.; Hickey, Michelle J.; Tritz, Richard; Kruse, Carol A.

    2008-01-01

    Summary We previously reported the cellular, functional and cytogenetic characterization of immunoresistant (IR) 13-06-IR29 and 13-06-IR30 human glioma cell clones isolated after immunoselection with alloreactive cytotoxic T lymphocytes (aCTL). Relative to the 13-06-MG parental cells, both clones resisted aCTL lysis at multiple effector to target ratios; the resistant phenotype was maintained for 13-41 cell doublings after cloning and when selective pressure was removed; cross-resistance to other inducers of apoptosis/cell death was also observed (Gomez et al, 2006; Gomez and Kruse, 2007). In this study we further characterize the IR clones for factors that may contribute to the resistance. Data obtained by in-vitro quantitative morphologic and 7-amino actinomycin D flow cytometric assays revealed reduced apoptotic cell death when IR clones were coincubated with aCTL, relative to the parental cells. Since changes in apoptosis were observed, we examined the expression patterns of apoptosis-related genes in several extracts of parental cells and IR clones using pathway-specific cDNA microarray analysis. In general, the apoptotic factors were downregulated in the IR clones. From three separate extracts analyzed separately on microarrays, three factors, ATM, caspases 3 and 8, were statistically downregulated in both IR clones. Immunoblotting of the proteins confirmed the findings. Therefore, a possible mechanism for immunoresistance in gliomas may be achieved by the downregulation of one or more genes in the apoptotic pathway. PMID:19066635

  4. Genomic Instability in Human Lymphocytes from Male Users of Crack Cocaine

    PubMed Central

    de Freitas, Thiago Aley Brites; Palazzo, Roberta Passos; de Andrade, Fabiana Michelsen; Reichert, César Luis; Pechansky, Flávio; Kessler, Félix; de Farias, Caroline Brunetto; de Andrade, Gisele Gomes; Leistner-Segal, Sandra; Maluf, Sharbel Weidner

    2014-01-01

    Recent research suggests that crack cocaine use alters systemic biochemical markers, like oxidative damage and inflammation markers, but very few studies have assessed the potential effects of crack cocaine at the cellular level. We assessed genome instability by means of the comet assay and the cytokinesis-block micronucleus technique in crack cocaine users at the time of admission to a rehabilitation clinic and at two times after the beginning of withdrawal. Thirty one active users of crack cocaine and forty control subjects were evaluated. Comparison between controls and crack cocaine users at the first analysis showed significant differences in the rates of DNA damage (p = 0.037). The frequency of micronuclei (MN) (p < 0.001) and nuclear buds (NBUDs) (p < 0.001) was increased, but not the frequency of nucleoplasmic bridges (NPBs) (p = 0.089). DNA damage decreased only after the end of treatment (p < 0.001). Micronuclei frequency did not decrease after treatment, and nuclear buds increased substantially. The results of this study reveal the genotoxic and mutagenic effects of crack cocaine use in human lymphocytes and pave the way for further research on cellular responses and the possible consequences of DNA damage, such as induction of irreversible neurological disease and cancer. PMID:25264678

  5. Very short-lived and stable mRNAs from resting human lymphocytes.

    PubMed Central

    Berger, S L; Cooper, H L

    1975-01-01

    The kinetics of degradation of newly synthesized cytoplasmic poly(A)-bearing RNA have been examined in resting human lymphocytes. Two classes were identified, a very labile component with a half-life of less than 17 min and a stable component which remains apparently undiminished during 24 hr of observation. Both classes have molecular weights between 2.5 and 3.5 x 10(5) but the stable material has a narrower size distribution and a slightly lower average molecular weight than the short-lived component. The fate of stable RNA synthesized in the resting cell was also examined after growth stimulation with phytohemagglutinin after 2 and 6 hr of treatment. No transfer of stable material into the labile pool could be discerned; the amount of stable material remained constant. The existence of two species of mRNAs with different lifetimes in animal cells provides a potential means for regulation of protein synthesis by controlling the supply of specific messages. Furthermore, such a short-lived mRNA species may explain the observed disparity between the amount of poly(A)-bearing heterogeneous RNA produced in the nucleus and the amount of mature message found in the cytoplasm. PMID:1060069

  6. Large granular lymphocytes are universally increased in human, macaque, and feline lentiviral infection.

    PubMed

    Sprague, Wendy S; Apetrei, Cristian; Avery, Anne C; Peskind, Robert L; Vandewoude, Sue

    2015-10-15

    Large granular lymphocytes (LGLs) have only been anecdotally reported in HIV infection. We previously reported an LGL lymphocytosis in FIV-infected cats associated with a rise in FIV proviral loads and a marked neutropenia that persisted during chronic infection. Extensive immunophenotyping of peripheral blood mononuclear cells in cats chronically infected with FIV were identified LGLs as CD8lo(+)FAS(+); this cell population expanded commensurate with viral load. CD8lo(+)FAS(+) cells expressed similar levels of interferon-γ compared to CD8lo(+)FAS(+) cells from FIV-naive control animals, yet CD3ɛ expression, which was increased on total CD8(+) T cells in FIV-infected cats, was decreased on CD8lo(+)FAS(+) cells. Down-modulation of CD3 expression was reversed after culturing PBMC for 3 days in culture with ConA/IL-2. We identified CD8lo(+)FAS(+) LGLs to be polyclonal T cells lacking CD56 expression. Blood smears from HIV-infected individuals and SIVmac239-infected rhesus macaques revealed increased LGLs compared to HIV/SIV negative counterparts. In humans, there was no correlation with viral load or treatment and in macaques the LGLs arose in acute SIV infection with increases in viremia. This is the first report describing and partially characterizing LGL lymphocytosis in association with lentiviral infections in three different species. PMID:26292765

  7. Effects of anti-human T lymphocyte immune globulins in patients: new or old.

    PubMed

    Wang, Diane C; Wang, Xiangdong; Chen, Chengshui

    2016-09-01

    Multiple studies demonstrated that anti-human T lymphocyte immune globulins (ATG) can decrease the incidence of acute and chronic graft rejection in cell or organ transplants. However, further in-depth study indicates that different subgroups may benefit from either different regimes or alteration of them. Studies among renal transplant patients indicate that low immunological risk patients may not gain the same amount of benefit and thus tilt the risk versus benefit consideration. This may hold true for low immunological risk patients receiving other organ transplants and would be worth further investigation. The recovery time of T cells and natural killer (NK) cells also bears consideration and the impact that it has on the severity and incidence of opportunistic infections closely correlated with the dosage of ATG. The use of lower doses of ATG in combination with other induction medications may offer a solution. The finding that ATG may lose efficacy in cases of multiple transplants or re-transplants in the case of heart transplants may hold true for other transplantations. This may lead to reconsideration of which induction therapies would be most beneficial in the clinical setting. These studies on ATG done on different patient groups will naturally not be applicable to all, but the evidence accrued from them as a whole may offer us new and different perspectives on how to approach and potentially solve the clinical question of how to best reduce the mortality associated with chronic host-versus-graft disease. PMID:27084794

  8. Effects on DNA repair in human lymphocytes exposed to the food dye tartrazine yellow.

    PubMed

    Soares, Bruno Moreira; Araújo, Taíssa Maíra Thomaz; Ramos, Jorge Amando Batista; Pinto, Laine Celestino; Khayat, Bruna Meireles; De Oliveira Bahia, Marcelo; Montenegro, Raquel Carvalho; Burbano, Rommel Mario Rodríguez; Khayat, André Salim

    2015-03-01

    Tartrazine is a food additive that belongs to a class of artificial dyes and contains an azo group. Studies about its genotoxic, cytotoxic and mutagenic effects are controversial and, in some cases, unsatisfactory. This work evaluated the potential in vitro cytotoxicity, genotoxicity and effects on DNA repair of human lymphocytes exposed to the dye. We assessed the cytotoxicity of tartrazine by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide test and the response of DNA repair through comet assay (alkaline version). We used different concentrations of the dye, ranging from 0.25-64.0 mM. The results demonstrated that tartrazine has no cytotoxic effects. However, this dye had a significant genotoxic effect at all concentrations tested. Although most of the damage was amenable to repair, some damage remained higher than positive control after 24 h of repair. These data demonstrate that tartrazine may be harmful to health and its prolonged use could trigger carcinogenesis. PMID:25750299

  9. Study on the cytogenetic changes induced by benzene and hydroquinone in human lymphocytes.

    PubMed

    Peng, D; Jiaxing, W; Chunhui, H; Weiyi, P; Xiaomin, W

    2012-04-01

    Benzene (BN) is a prototypical hematotoxicant, genotoxic carcinogen, and ubiquitous environmental pollutant. Although the molecular mechanisms of BN-induced cytotoxicity and genotoxic damage are poorly understood in humans, previous studies suggested that bioactivated BN metabolites are capable of oxidative stress, cell cycle arrest, apoptosis, and DNA damage. The objective of the current study was to investigate the BN-induced cytogenetic changes and underlying mechanisms based on these hypotheses. Peripheral blood lymphocytes (PBLs) might be the targets for BN-induced cytotoxicity and genotoxicity, and therefore DNA damage responses of PBLs after exposure to different concentrations of BN (0.25, 3.5, 50 μmol/L) or BN metabolite, hydroquinone (HQ; 50, 150, 450 μmol/L) were studied in vitro. Microculture tetrazolium assay, flow cytometry, 2',7'-dichlorodihydrofluorescein-diacetate assay, comet assay, micronuclei assay, and attenuated total reflectance microspectroscope were chosen for this study. Based on the results, we reached the conclusion that different concentrations of BN or HQ significantly inhibited cell growth, induced the arrest of S phase and G2/M phase, and increased late apoptosis in a concentration-dependent manner. Furthermore, evidence was also provided to support the conclusion that BN and HQ induced DNA strand breaks and chromosomal mutations in PBL, which indicated the genotoxicity of BN and HQ. Current evidence has indicated that multiple mechanisms including dysfunction of cell cycle, programmed cell death, oxidative stress, and DNA lesions are likely to contribute to BN-induced cytogenetic changes. PMID:22297702

  10. Human Herpesvirus 8 Induces Polyfunctional B Lymphocytes That Drive Kaposi’s Sarcoma

    PubMed Central

    Knowlton, Emilee R.; Rappocciolo, Giovanna; Piazza, Paolo; Lepone, Lauren M.; Nadgir, Sagar V.; Bullotta, Arlene; Berendam, Stella J.; Li, Jun; Reinhart, Todd A.; Jenkins, Frank J.

    2014-01-01

    ABSTRACT Kaposi’s sarcoma (KS) is an unusual neoplasia wherein the tumor consists primarily of endothelial cells infected with human herpesvirus 8 (HHV-8; Kaposi’s sarcoma-associated herpesvirus) that are not fully transformed but are instead driven to excess proliferation by inflammatory and angiogenic factors. This oncogenic process has been postulated but unproven to depend on a paracrine effect of an abnormal excess of host cytokines and chemokines produced by HHV-8-infected B lymphocytes. Using newly developed measures for intracellular detection of lytic cycle proteins and expression of cytokines and chemokines, we show that HHV-8 targets a range of naive B cell, IgM memory B cell, and plasma cell-like populations for infection and induction of interleukin-6, tumor necrosis factor alpha, macrophage inhibitory protein 1α, macrophage inhibitory protein 1β, and interleukin-8 in vitro and in the blood of HHV-8/HIV-1-coinfected subjects with KS. These B cell lineage subsets that support HHV-8 infection are highly polyfunctional, producing combinations of 2 to 5 of these cytokines and chemokines, with greater numbers in the blood of subjects with KS than in those without KS. Our study provides a new paradigm of B cell polyfunctionality and supports a key role for B cell-derived cytokines and chemokines produced during HHV-8 infection in the development of KS. PMID:25182322

  11. Effect of insulin on low-density-lipoprotein metabolism in human lymphocytes in vitro.

    PubMed Central

    Suresh, S; Warty, V; Virji, M; Sanghvi, A

    1986-01-01

    The metabolism of low-density lipoproteins (LDL) in vitro in the presence of insulin was studied in freshly isolated human peripheral-blood lymphocytes. Insulin appeared to decrease the binding affinity of 125I-LDL to its cell-surface receptor, without any change in apparent Vmax or in the number of LDL receptors. As a consequence, the absolute amounts of 125I-LDL internalized and degraded were lower in the presence of insulin than in its abscence, although the fraction of internalized 125I-LDL degraded in either instance was quite similar. 3-Hydroxy-3-methylglutaryl-CoA reductase activity, and hence cholesterol synthesis, were stimulated by insulin. This effect of insulin was independent of the inhibitory effect of LDL on cholesterol synthesis. At the same time, acid cholesterol esterase and acyl-CoA: cholesterol O-acetyltransferase activities were lower in cells incubated with insulin than in controls. The net effect of these metabolic alterations seems to be that cells accumulate greater quantities of free and esterified cholesterol when treated with insulin. PMID:3513764

  12. Interaction between nitric oxide and subsets of human T lymphocytes with differences in glutathione metabolism

    PubMed Central

    Roozendaal, Ramon; Kauffman, Henk F; Dijkhuis, Anne-Jan; Ommen, Elisabeth T V; Postma, Dirkje S; De Monchy, Jan G R; Vellenga, Edo

    2002-01-01

    Nitric oxide (NO) modulates human T-lymphocyte responses through several mechanisms. In the current study we show that interactions between NO and glutathione (GSH) metabolism are related to the selective persistent inhibition of interferon-γ (IFN-γ) production by NO, which we previously identified. T cells were exposed to NO using the NO-donor compound Spermine-nonoate (Sper) and activated using anti-CD3 plus anti-CD28 monoclonal antibodies. Persistent inhibition of IFN-γ by Sper was prevented by addition of the GSH precursor l-cysteine, which inhibits Sper induced GSH depletion. Subsets of cells were either susceptible (GSHlow) or resistant (GSHhigh) to NO-induced GSH depletion. The GSHlow subset was characterized by enhanced numbers of CD4+ cells, reduced numbers of activated cells as characterized by CD25 and CD69, and reduced numbers of memory (CD45RO+) cells relative to the GSHhigh population. Rather than directly affecting susceptibility to NO, these surface markers reflected different expression patterns. Particularly, the GSHlow subset was further characterized by decreased activity of the GSH synthesis related enzymes multi-drug resistance related protein (MRP)-1 and γ-glutamyltranspeptidase (γ-GT). Blocking γ-GT, using acivicin was shown to exacerbate NO-induced GSH depletion and NO-induced apoptosis. Since NO induced apoptosis selectively affects IFN-γ production these findings implicate GSH metabolism in the modulation and maintenance of the T helper (Th)1/Th2 balance. PMID:12423309

  13. Biochemical manipulation of intracellular glutathione levels influences cytotoxicity to isolated human lymphocytes by sulfur mustard

    SciTech Connect

    Gross, C.L.; Innace, J.K.; Hovatter, R.C.; Meier, H.L.; Smith, W.J.

    1993-12-31

    Glutathione (GSH) is the major nonprotein thiol that can protect cells from damage due to electrophilic alkylating agents by forming conjugates with the agent. Sulfur mustard (HD) is an electrophilic alkylating agent that has potent mutagenic, carcinogenic, cytotoxic, and vesicant properties. Compounds that elevate or reduce intracellular levels of GSH may produce changes in cytotoxicity induced by sulfur mustard. Pretreatment of human peripheral blood lymphocytes (PBL) for 72 hr with 1 mM buthionine sulfoximine (BSO), which reduces intracellular GSH content to approximately 26% of control, appears to sensitize these in vitro cells to the cytotoxic effects of 10 AM HD but not to higher HD concentrations. Pretreatment of PBL for 48 hr with 10 mM N-acetyl cysteine (NA C), which elevates intracellular glutathione levels to 122% of control, appears to partially protect these in vitro cells from the cytotoxic effects of 10 LAIHD but not to higher HD concentrations. Augmentation of intracellular levels of glutathione may provide partial protection against cytotoxicity of sulfur mustard.

  14. [Cytogenetic effect of thaliblastine in a culture of human peripheral blood lymphocytes].

    PubMed

    Khadzhidekova, V; Krusteva, E; Bulanova, M; Ivanov, V; Benova, D

    1988-12-01

    The mutagenicity of thaliblastine (Bulgarian potential antitumor drug) was investigated in vitro in lymphocytes from healthy donors, and in vivo in lymphocytes of oncological patients after thaliblastine administration. No increase in the rate of chromosome aberrations was noted with increasing thaliblastine concentrations in vitro and in the course of therapy in vivo. Some polyploid metaphases were found in the lymphocytes of the patients treated with thaliblastine, as a result of the statmokinetic effect of the drug. Thaliblastine exerts extraordinarily slight mutagenic effect, as compared with other cytostatics. PMID:2977980

  15. The assessment of genotoxicity of carbamazepine using cytokinesis-block (CB) micronucleus assay in cultured human blood lymphocytes.

    PubMed

    Celik, Ayla

    2006-01-01

    The genotoxic effect of CBZ has been investigated in few studies. There is little evidence linking carbamazepine (CBZ) with any genotoxic effects, particularly in vitro micronucleus test using cytogenesis-block technique. In this study, the genotoxicity of the antiepileptic drug, carbamazepine, was tested using cytokinesis-block (CB) micronucleus assay. In vitro analysis was performed in human blood lymphocytes from four healthy persons at five different concentrations of carbamazepine (6, 8, 10, 12, 14 microg/mL). Genotoxic potential and cytotoxic effects of carbamazepine were evaluated by using micronucleus assay and cytokinesis-block proliferation index (CBPI), called the parameter of cytotoxicity in human peripheral blood lymphocyte cultures, respectively. The results of this study indicate that CBZ caused the genotoxic effect under in vitro conditions, except at the dose of 6 microg/mL, and cytotoxic effects of carbamazepine were revealed by a decrease in the cytokinesis-block proliferation index at all the concentrations. PMID:16707330

  16. Radiosensitivity of CD4 or CD8 positive human T-lymphocytes by an in vitro colony formation assay

    SciTech Connect

    Nakamura, N.; Kusunoki, Y.; Akiyama, M. )

    1990-08-01

    The recent development of an in vitro lymphocyte colony assay makes it possible to examine variations in the radiosensitivity of humans using peripheral blood lymphocytes (PBL) instead of the skin fibroblast assay. Our recent study showed that most of the colonies consisted of lymphocytes bearing CD4 or CD8 antigens. Since the fraction of CD4+ and CD8+ cells in PBL differs among individuals, we suspected that individual radiosensitivity might be biased by the different subset frequencies if the dose-survival curves of the CD4+ and CD8+ cells were different from each other. In the present study, CD4+ (helper/inducer T) and CD8+ (suppressor/cytotoxic T) lymphocytes were isolated from PBL and their dose-survival curves were determined. The results showed that the D10 (dose required to reduce the surviving fraction to 10%) was similar for these two types of cells (3.13 +/- 0.10 Gy (mean +/- SD) for CD4+, 3.34 +/- 0.50 Gy for CD8+ and 3.14 +/- 0.17 Gy for the unsorted cells), supporting the use of the whole PBL population for the screening of individuals with altered radiosensitivity.

  17. Human T-lymphotropic virus type I-associated myelopathy and tax gene expression in CD4+ T lymphocytes.

    PubMed

    Moritoyo, T; Reinhart, T A; Moritoyo, H; Sato, E; Izumo, S; Osame, M; Haase, A T

    1996-07-01

    Infection by human T-lymphotropic virus type I (HTLV-I) is associated with adult T-cell leukemia and a slowly progressive disease of the central nervous system (CNS), HTLV-I-associated myelopathy/tropical spastic paraparesis, characterized pathologically by inflammation and white matter degeneration in the spinal cord. One of the explanations for the tissue destruction is that HTLV-I infects cells in the CNS, or HTLV-I-infected CD4+ T lymphocytes enter the CNS, and this drives local expansion of virus-specific CD8+ cytotoxic T lymphocytes, which along with cytokines cause the pathological changes. Because both in the circulation and in the cerebrospinal fluid, CD8+ cytotoxic T lymphocytes are primarily reactive to the product of the HTLV-I tax gene, we sought evidence of expression of this gene within cells in the inflammatory lesions. After using double-label in situ hybridization techniques, we now report definitive localization of HTLV-I tax gene expression in CD4+ T lymphocytes in areas of inflammation and white matter destruction. These findings lend support to a hypothetical scheme of neuropathogenesis in which HTLV-I tax gene expression provokes and sustains an immunopathological process that progressively destroys myelin and axons in the spinal cord. PMID:8687197

  18. Protective Effect of Carvacrol on Oxidative Stress and Cellular DNA Damage Induced by UVB Irradiation in Human Peripheral Lymphocytes.

    PubMed

    Aristatile, Balakrishnan; Al-Numair, Khalid S; Al-Assaf, Abdullah H; Veeramani, Chinnadurai; Pugalendi, Kodukkur Viswanathan

    2015-11-01

    Exposure to ultraviolet B (UVB; 280-320 nm) radiation induces the formation of reactive oxygen species (ROS) in the biological system. In this study, we examined the protective effect of carvacrol on UVB-induced lipid peroxidation and oxidative DNA damage with reference to alterations in cellular an-tioxidant status in human lymphocytes. A series of in vitro assays (hydroxyl radical, superoxide, nitric oxide, DPPH (2,2-Diphenyl-1-picryl hydrazyl), and ABTS (2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging assays) demonstrate antioxidant property of carvacrol in our study. UVB exposure significantly increased thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides (LHPs), % tail DNA and tail moment; decreased % cell viability and antioxidant status in UVB-irradiated lymphocytes. Treatment with carvacrol 30 min prior to UVB-exposure resulted in a significant decline of TBARS, LHP, % tail DNA, and tail moment and increased % cell viability as carvacrol concentration increased. UVB irradiated lymphocytes with carvacrol alone (at 10 μg/mL) gave no significant change in cell viability, TBARS, LHP, % tail DNA, and tail moment when compared with normal lymphocytes. On the basis of our results, we conclude that carvacrol, a dietary antioxidant, mediates its protective effect through modulation of UVB-induced ROS. PMID:26768646

  19. Relation of impaired lymphocyte proliferative function to other major human immunodeficiency virus type 1-induced immunological changes.

    PubMed Central

    Bass, H Z; Fahey, J L; Nishanian, P; Detels, R; Cumberland, W; Kemeny, M; Plaeger, S

    1997-01-01

    Human immunodeficiency virus (HIV) type 1 (HIV-1) induces impairment of immune function reflected in reduced lymphocyte proliferative responses. Many other immune changes are induced by HIV-1, but their relationship to lymphocyte functional defects is not known. The present study was designed to correlate functional defects with other HIV disease parameters. Cryopreserved samples from 118 HIV-1-positive subjects and 40 seronegative individuals were examined. The main findings were that impaired proliferative responses to mitogens correlated with (i) decreased cell surface expression of the interleukin-2 receptor (CD25), (ii) increased expression of HLA-DR antigens on CD4 cells, (iii) reduced CD4 and increased CD8 cell numbers, and (iv) increased levels of serum immune complex dissociated p24 antigen. However, impaired function was not associated with increased serum neopterin, beta2-microglobulin, or soluble interleukin-2 receptor or with CD38 antigen expression on lymphocytes. In summary, proliferative functional impairment correlated with some, but not all, immunological changes associated with HIV-1 infection. Most of the phenotypic markers that correlated with altered function are cell surface molecules with significant roles in lymphocyte proliferation and were associated primarily with CD4 cells, compatible with the view that dysregulation of CD4 cells is responsible for impaired function. PMID:9008283

  20. Effects of synthetic and naturally occurring flavonoids on mitogen-induced proliferation of human peripheral-blood lymphocytes

    SciTech Connect

    Hirano, Toshihiko; Oka, Kitaro; Kawashima, Etsuko; Akiba, Mitsuo )

    1989-01-01

    Examination was made of the effects of 17 synthetic and naturally occurring flavonoids on human lymphocyte proliferation in the presence of concanavalin A as a mitogen. Twelve of the flavonoids examined were mono-hydroxy of methoxy derivatives. The mitogen-induced response of lymphocytes was evaluated from the extent of the incorporation of ({sup 3}H)thymidine into cells in vitro. All the compounds showed inhibitory effects; 4.5-77.7% of ({sup 3}H) thymidine incorporation was blocked by an 1.0 {mu}g/ml concentration. The viability of lymphocytes before and after treatment, as assessed by a dye exclusion test, indicated no change, and thus the flavonoids may inhibit DNA synthesis. The flavonoids possessing 5-hydroxyl, 5-methoxyl and 6-methoxyl groups, and those with cyclohexyl instead of phenyl substituent (i.e. 2-cyclohexyl-benzopyran-4-one), showed the greatest inhibition. The inhibitory effect of any one of them was less than one half that of prednisolone, but essentially the same or somewhat exceeding that of bredinine of azathioprine. It would thus appear that the well-known anti-inflammatory effects of flavonoids may possibly arise in part from the inhibition of the proliferative response of lymphocytes.

  1. In vitro genotoxicity and cytotoxicity of a particular combination of pemetrexed and cefixime in human peripheral blood lymphocytes.

    PubMed

    Istifli, Erman Salih; Topaktaş, Mehmet

    2015-01-01

    This study aims to find the genotoxic and cytotoxic effects of a particular combination of pemetrexed (PMX) and cefixime (CFX) in human peripheral blood lymphocytes. Chromosome aberration (CA), sister chromatid exchange (SCE), and micronucleus (MN) tests were used to assess genotoxicity. Whereas, the cytotoxicity was evaluated by using mitotic index (MI), proliferation index (PI), and nuclear division index (NDI). Our tests were proceeded with concentrations of 12.5 + 450, 25 + 800, 37.5 + 1150, and 50 + 1500 μg/mL of a mixture of PMX and CFX separately for 24 hr and 48 hr. The combination of PMX + CFX did not induce the CA or SCE in human peripheral blood lymphocytes when compared with both the control and the solvent control. MN in human peripheral blood lymphocytes was not significantly increased after treatment with a particular combination of PMX + CFX. However, PMX + CFX significantly decreased the MI, PI and NDI at all concentrations for 24- and 48-hr treatment periods when compared with both controls. Generally, PMX + CFX inhibited cell proliferation more than positive control (MMC) and showed a higher cytotoxic effect than MMC at both treatment periods. These results were compared with individual effects of PMX and CFX. As a result, it was observed that a particular combination of PMX + CFX was not genotoxic. However, the combination synergistically increase cytotoxicity in human peripheral blood lymphocytes. PMID:25653913

  2. Behavior and Properties of Mature Lytic Granules at the Immunological Synapse of Human Cytotoxic T Lymphocytes

    PubMed Central

    Ming, Min; Schirra, Claudia; Becherer, Ute; Stevens, David R.; Rettig, Jens

    2015-01-01

    Killing of virally infected cells or tumor cells by cytotoxic T lymphocytes requires targeting of lytic granules to the junction between the CTL and its target. We used whole-cell patch clamp to measure the cell capacitance at fixed intracellular [Ca2+] to study fusion of lytic granules in human CTLs. Expression of a fluorescently labeled human granzyme B construct allowed identification of lytic granule fusion using total internal reflection fluorescence microscopy. In this way capacitance steps due to lytic granule fusion were identified. Our goal was to determine the size of fusing lytic granules and to describe their behavior at the plasma membrane. On average, 5.02 ± 3.09 (mean ± s.d.) lytic granules were released per CTL. The amplitude of lytic granule fusion events was ~ 3.3 fF consistent with a diameter of about 325 nm. Fusion latency was biphasic with time constants of 15.9 and 106 seconds. The dwell time of fusing lytic granules was exponentially distributed with a mean dwell time of 28.5 seconds. Fusion ended in spite of the continued presence of granules at the immune synapse. The mobility of fusing granules at the membrane was indistinguishable from that of lytic granules which failed to fuse. While dwelling at the plasma membrane lytic granules exhibit mobility consistent with docking interspersed with short periods of greater mobility. The failure of lytic granules to fuse when visible in TIRF at the membrane may indicate that a membrane-confined reaction is rate limiting. PMID:26296096

  3. Cannibalism of live lymphocytes by human metastatic but not primary melanoma cells.

    PubMed

    Lugini, Luana; Matarrese, Paola; Tinari, Antonella; Lozupone, Francesco; Federici, Cristina; Iessi, Elisabetta; Gentile, Massimo; Luciani, Francesca; Parmiani, Giorgio; Rivoltini, Licia; Malorni, Walter; Fais, Stefano

    2006-04-01

    The phenomenon of cell cannibalism, which generally refers to the engulfment of cells within other cells, was described in malignant tumors, but its biological significance is still largely unknown. In the present study, we investigated the occurrence, the in vivo relevance, and the underlying mechanisms of cannibalism in human melanoma. As first evidence, we observed that tumor cannibalism was clearly detectable in vivo in metastatic lesions of melanoma and often involved T cells, which could be found in a degraded state within tumor cells. Then, in vitro experiments confirmed that cannibalism of T cells was a property of metastatic melanoma cells but not of primary melanoma cells. In particular, morphologic analyses, including time-lapse cinematography and electron microscopy, revealed a sequence of events, in which metastatic melanoma cells were able to engulf and digest live autologous melanoma-specific CD8(+) T cells. Importantly, this cannibalistic activity significantly increased metastatic melanoma cell survival, particularly under starvation condition, supporting the evidence that tumor cells may use the eating of live lymphocytes as a way to "feed" in condition of low nutrient supply. The mechanism underlying cannibalism involved a complex framework, including lysosomal protease cathepsin B activity, caveolae formation, and ezrin cytoskeleton integrity and function. In conclusion, our study shows that human metastatic melanoma cells may eat live T cells, which are instead programmed to kill them, suggesting a novel mechanism of tumor immune escape. Moreover, our data suggest that cannibalism may represent a sort of "feeding" activity aimed at sustaining survival and progression of malignant tumor cells in an unfavorable microenvironment. PMID:16585188

  4. Human ecalectin, a variant of human galectin-9, is a novel eosinophil chemoattractant produced by T lymphocytes.

    PubMed

    Matsumoto, R; Matsumoto, H; Seki, M; Hata, M; Asano, Y; Kanegasaki, S; Stevens, R L; Hirashima, M

    1998-07-01

    A 1.6-kilobase pair cDNA was isolated from a human T-cell-derived expression library that encodes a novel eosinophil chemoattractant (designated ecalectin) expressed during allergic and parasitic responses. Based on its deduced amino acid sequence, ecalectin is a 36-kDa protein consisting of 323 amino acids. Although ecalectin lacks a hydrophobic signal peptide, it is secreted from mammalian cells. Ecalectin is not related to any known cytokine or chemokine but rather is a variant of human galectin-9, a member of the large family of animal lectins that have affinity for beta-galactosides. Recombinant ecalectin, expressed in COS cells and insect cells, exhibited potent eosinophil chemoattractant activity and attracted eosinophils in vitro and in vivo in a dose-dependent manner but not neutrophils, lymphocytes, or monocytes. The finding that the ecalectin transcript is present in abundance in various lymphatic tissues and that its expression increases substantially in antigen-activated peripheral blood mononuclear cells suggests that ecalectin is an important T-cell-derived regulator of eosinophil recruitment in tissues during inflammatory reactions. We believe that this is the first report of the expression of an immunoregulatory galectin expressed by a T-cell line that is selective for eosinophils. PMID:9642261

  5. Evaluation of T and B lymphocyte membrane markers in human non-Hodgkin malignant lymphomata.

    PubMed Central

    Brouet, J. C.; Labaume, S.; Seligmann, M.

    1975-01-01

    Lymphoma cells from 25 patients were studied for the presence of B lymphocytes (membrane bound Ig and Fc receptor) and T lymphocytes (rosette formation with sheep erythrocytes) membrane markers. All cases of well differentiated lymphocytic lymphoma and of acute lymphosarcoma cell leukaemia and most cases of poorly differentiated lymphocytic lymphoma behaved as B cell monoclonal malignancies. However, the malignant cells of some patients were not definitely classified according to their B or T cell origin or lacked these membrane markers. The latter situation was encountered in 4 reticulum cell sarcomata. Polyclonal Ig were found on the surface of B cells in a case of hyperbasophilic undifferentiated lymphoma. The need for using several membrane markers to study the abnormal lymphoma cells is outlined. Such studies improve our understanding of these malignancies and may lead in the future to a satisfactory classification of non-Hodgkin lymphomata. PMID:1081001

  6. Molecular cloning and expression of Pgp-1. The mouse homolog of the human H-CAM (Hermes) lymphocyte homing receptor.

    PubMed

    Zhou, D F; Ding, J F; Picker, L J; Bargatze, R F; Butcher, E C; Goeddel, D V

    1989-11-15

    Mouse phagocytic glycoprotein-1 (Pgp-1; Ly-24) is a 95-kDa glycoprotein of unknown function that has served as an important T cell/leukocyte differentiation marker. Recent work has suggested that it may be related to a human 85- to 95-kDa glycoprotein (termed variously the Hermes Ag/lymphocyte homing receptor, ECMRIII, P80, and CD44) that is involved in lymphocyte binding to high endothelial venules in the process of lymphocyte homing, and has been implicated in other cell adhesion events. The widespread expression of this molecular class in diverse organ systems suggests a broad role in cellular adhesion, and has led to the unifying designation homing-cellular adhesion molecule (H-CAM). By using human H-CAM cDNA probes, we have isolated a full-length cDNA for the mouse homolog. Comparison of the human and mouse sequences reveals that an N-terminal domain homologous to cartilage proteoglycan core and link proteins, as well as the C-terminal transmembrane and cytoplasmic sequences, are highly conserved (89% and 86% identity, respectively). In contrast, a proximal extracellular domain thought to serve as a target for O-glycosylation and chondroitin sulfate attachment has undergone substantial divergence (only 42% identity). Transient expression of the cDNA in CHO cells followed by immunologic staining confirms that this mouse H-CAM cDNA encodes Pgp-1.1, one of two known Pgp-1 alloantigens. PMID:2681416

  7. Evaluating the effects of galbanic acid on hydrogen peroxide-induced oxidative DNA damage in human lymphocytes

    PubMed Central

    Shirani, Kobra; Behravan, Javad; Mosaffa, Fatemeh; Iranshahi, Mehrdad; Mehmankhah, Babak; Razavi-Azarkhiavi, Kamal; Karimi, Gholamreza

    2014-01-01

    Objective: Ferula szowitsiana has been widely used for medicinal purposes around the world. The anti-oxidant effect of F. szowitsiana had been proved. The current study aims to determine the protective effects of galbanic acid, a sesquiterpene coumarin from F. szowitsiana, against hydrogen peroxide (H2O2) - induced oxidative DNA damage in human lymphocytes. Materials and Methods: Human lymphocytes were incubated with H2O2 (0, 25, 50, 100, and 200 µM), galbanic acid (200 and 400 µM) and a combination of galbanic acid (200 and 400 µM) and H2O2 (25 µM) at 4 C for 30 minutes. Solvents of galbanic acid without H2O2 were used as negative controls. Results: The findings of this study demonstrated that H2O2 exposure leads to a significant concentration-dependent increase in DNA damage. Galbanic acid did not cause DNA damage compared with the control cells. Data showed that galbanic acid does not have a protective effect against H2O2-induced oxidative DNA damage in human lymphocytes. Conclusion: According to the results, it is concluded that the capability of F. szowitsiana in reducing reactive oxygen species and the anti-inflammatory property of its methanolic extract may be due to its other ingredients. PMID:25386396

  8. Phenytoin influence on human lymphocyte mitogen response: a prospective study of epileptic and nonepileptic patients.

    PubMed

    Gabourel, J D; Davies, G H; Bardana, E J; Ratzlaff, N A

    1982-08-01

    The results of this prospective study fail to confirm previously reported phenytoin suppression of lymphocyte responsiveness to mitogens. Our data show a significantly greater than expected percentage (p less than 0.0001) of patients requiring phenytoin treatment have low lymphocyte responsiveness to mitogens prior to phenytoin therapy. Analysis of changes in each individual's response during phenytoin treatment as compared with their pre-phenytoin responses shows a consistent trend to increased responsiveness to concanavalin A, pokeweed mitogen, and to a suboptimal concentration of phytohemagglutinin. This trend was most pronounced for patients whose serum IgA concentration was decreased while taking phenytoin, whereas there was no such trend for individuals whose serum IgA levels were not decreased. This phenomenon was not related to neurological disease classification. Phenytoin added directly to lymphocyte cultures depressed lymphocyte responses to all mitogens in a small (less than 20%) but significant degree, confirming similar in vitro studies by other investigators. Because of limited serum proteins for phenytoin binding in culture medium, these in vitro studies have little application to possible phenytoin effects on lymphocytes of patients taking it to prevent seizures. Thus, the suggestion that phenytoin causes depressed lymphocyte responses to mitogens in epileptic patients appears unwarranted. PMID:7094904

  9. Cytotoxic T lymphocyte antigen 4 expression in human breast cancer: implications for prognosis.

    PubMed

    Yu, Haiming; Yang, Junlan; Jiao, Shunchang; Li, Ying; Zhang, Wei; Wang, Jiandong

    2015-07-01

    To examine the relationship between cytotoxic T lymphocyte antigen 4 (CTLA-4) expression and breast cancer prognosis, CTLA-4 expression was immunohistochemically detected in paraffin-embedded specimens of primary tumors from 130 patients with breast cancer who had a mean follow-up period of 112 months. CTLA-4 expressed in cytoplasm of breast cancer cells and in cytoplasm and cell membranes of interstitial lymphocytes. Univariate analysis (log-rank) associated higher density of interstitial CTLA-4(+) lymphocytes with longer DFS and OS, but higher tumor CTLA-4 expression with shorter OS. After controlling for age, clinical stage, Scarff-Bloom-Richardson grade, tumor thrombus, ER, PR, HER2 and Ki-67, multivariate analysis (Cox) showed that density of interstitial CTLA-4(+) lymphocytes independently predicted longer DFS (HR 0.315, P = 0.002) and OS (HR 0.313, P = 0.005), whereas tumor CTLA-4 expression independently predicted shorter DFS (HR 2.176, P = 0.029) and OS (HR 2.820, P = 0.007), i.e., patients with high CTLA-4(+) lymphocyte density and CTLA-4(low) tumor cells had the best prognoses. These results indicated that CTLA-4 expression in lymphocytes was associated with better prognosis, but that in tumor cells was associated with worse prognosis. Patients' CTLA-4 profiles might thus be used to predict the benefits and toxicity of CTLA-4 blockade. PMID:25893809

  10. Fc block treatment, dead cells exclusion, and cell aggregates discrimination concur to prevent phenotypical artifacts in the analysis of subpopulations of tumor-infiltrating CD11b(+) myelomonocytic cells.

    PubMed

    Kuonen, Francois; Touvrey, Cedric; Laurent, Julien; Ruegg, Curzio

    2010-11-01

    It is well established that cancer cells can recruit CD11b(+) myeloid cells to promote tumor angiogenesis and tumor growth. Increasing interest has emerged on the identification of subpopulations of tumor-infiltrating CD11b(+) myeloid cells using flow cytometry techniques. In the literature, however, discrepancies exist on the phenotype of these cells (Coffelt et al., Am J Pathol 2010;176:1564-1576). Since flow cytometry analysis requires particular precautions for accurate sample preparation and trustable data acquisition, analysis, and interpretation, some discrepancies might be due to technical reasons rather than biological grounds. We used the syngenic orthotopic 4T1 mammary tumor model in immunocompetent BALB/c mice to analyze and compare the phenotype of CD11b(+) myeloid cells isolated from peripheral blood and from tumors, using six-color flow cytometry. We report here that the nonspecific antibody binding through Fc receptors, the presence of dead cells and cell doublets in tumor-derived samples concur to generate artifacts in the phenotype of tumor-infiltrating CD11b(+) subpopulations. We show that the heterogeneity of tumor-infiltrating CD11b(+) subpopulations analyzed without particular precautions was greatly reduced upon Fc block treatment, dead cells, and cell doublets exclusion. Phenotyping of tumor-infiltrating CD11b(+) cells was particularly sensitive to these parameters compared to circulating CD11b(+) cells. Taken together, our results identify Fc block treatment, dead cells, and cell doublets exclusion as simple but crucial steps for the proper analysis of tumor-infiltrating CD11b(+) cell populations. PMID:20824631

  11. Cetuximab Reconstitutes Pro-Inflammatory Cytokine Secretions and Tumor-Infiltrating Capabilities of sMICA-Inhibited NK Cells in HNSCC Tumor Spheroids.

    PubMed

    Klöss, Stephan; Chambron, Nicole; Gardlowski, Tanja; Weil, Sandra; Koch, Joachim; Esser, Ruth; Pogge von Strandmann, Elke; Morgan, Michael A; Arseniev, Lubomir; Seitz, Oliver; Köhl, Ulrike

    2015-01-01

    Immunosuppressive factors, such as soluble major histocompatibility complex class I chain-related peptide A (sMICA) and transforming growth factor beta 1 (TGF-β1), are involved in tumor immune escape mechanisms (TIEMs) exhibited by head and neck squamous cell carcinomas (HNSCCs) and may represent opportunities for therapeutic intervention. In order to overcome TIEMs, we investigated the antibody-dependent cellular cytotoxicity (ADCC), cytokine release and retargeted tumor infiltration of sMICA-inhibited patient NK cells expressing Fcγ receptor IIIa (FcγRIIIa, CD16a) in the presence of cetuximab, an anti-epidermal growth factor receptor (HER1) monoclonal antibody (mAb). Compared to healthy controls, relapsed HNSCC patients (n = 5), not currently in treatment revealed decreased levels of circulating regulatory NK cell subsets in relation to increased cytotoxic NK cell subpopulations. Elevated sMICA and TGF-β1 plasma levels correlated with diminished TNFα and IFN-γ release and decreased NKG2D (natural killer group 2 member D)-dependent killing of HNSCC cells by NK cells. Incubation of IL-2-activated patient NK cells with patient plasma containing elevated sMICA or sMICA analogs (shed MICA and recombinant MICA) significantly impaired NKG2D-mediated killing by down-regulation of NKG2D surface expression. Of note, CD16 surface expression levels, pro-apoptotic and activation markers, and viability of patient and healthy donor NK cell subpopulations were not affected by this treatment. Accordingly, cetuximab restored killing activity of sMICA-inhibited patient NK cells against cetuximab-coated primary HNSCC cells via ADCC in a dose-dependent manner. Rapid reconstitution of anti-tumor recognition and enhanced tumor infiltration of treated NK cells was monitored by 24 h co-incubation of HNSCC tumor spheroids with cetuximab (1 μg/ml) and was characterized by increased IFN-γ and TNFα secretion. This data show that the impaired NK cell-dependent tumor

  12. Cetuximab Reconstitutes Pro-Inflammatory Cytokine Secretions and Tumor-Infiltrating Capabilities of sMICA-Inhibited NK Cells in HNSCC Tumor Spheroids

    PubMed Central

    Klöss, Stephan; Chambron, Nicole; Gardlowski, Tanja; Weil, Sandra; Koch, Joachim; Esser, Ruth; Pogge von Strandmann, Elke; Morgan, Michael A.; Arseniev, Lubomir; Seitz, Oliver; Köhl, Ulrike

    2015-01-01

    Immunosuppressive factors, such as soluble major histocompatibility complex class I chain-related peptide A (sMICA) and transforming growth factor beta 1 (TGF-β1), are involved in tumor immune escape mechanisms (TIEMs) exhibited by head and neck squamous cell carcinomas (HNSCCs) and may represent opportunities for therapeutic intervention. In order to overcome TIEMs, we investigated the antibody-dependent cellular cytotoxicity (ADCC), cytokine release and retargeted tumor infiltration of sMICA-inhibited patient NK cells expressing Fcγ receptor IIIa (FcγRIIIa, CD16a) in the presence of cetuximab, an anti-epidermal growth factor receptor (HER1) monoclonal antibody (mAb). Compared to healthy controls, relapsed HNSCC patients (n = 5), not currently in treatment revealed decreased levels of circulating regulatory NK cell subsets in relation to increased cytotoxic NK cell subpopulations. Elevated sMICA and TGF-β1 plasma levels correlated with diminished TNFα and IFN-γ release and decreased NKG2D (natural killer group 2 member D)-dependent killing of HNSCC cells by NK cells. Incubation of IL-2-activated patient NK cells with patient plasma containing elevated sMICA or sMICA analogs (shed MICA and recombinant MICA) significantly impaired NKG2D-mediated killing by down-regulation of NKG2D surface expression. Of note, CD16 surface expression levels, pro-apoptotic and activation markers, and viability of patient and healthy donor NK cell subpopulations were not affected by this treatment. Accordingly, cetuximab restored killing activity of sMICA-inhibited patient NK cells against cetuximab-coated primary HNSCC cells via ADCC in a dose-dependent manner. Rapid reconstitution of anti-tumor recognition and enhanced tumor infiltration of treated NK cells was monitored by 24 h co-incubation of HNSCC tumor spheroids with cetuximab (1 μg/ml) and was characterized by increased IFN-γ and TNFα secretion. This data show that the impaired NK cell-dependent tumor

  13. IL-15 induces strong but short-lived tumor-infiltrating CD8 T cell responses through the regulation of Tim-3 in breast cancer

    SciTech Connect

    Heon, Elise K.; Wulan, Hasi; Macdonald, Loch P.; Malek, Adel O.; Braunstein, Glenn H.; Eaves, Connie G.; Schattner, Mark D.; Allen, Peter M.; Alexander, Michael O.; Hawkins, Cynthia A.; McGovern, Dermot W.; Freeman, Richard L.; Amir, Eitan P.; Huse, Jason D.; Zaltzman, Jeffrey S.; Kauff, Noah P.; Meyers, Paul G.; Gleason, Michelle H.; Overholtzer, Michael G.; Wiseman, Sam S.; and others

    2015-08-14

    IL-15 has pivotal roles in the control of CD8{sup +} memory T cells and has been investigated as a therapeutic option in cancer therapy. Although IL-15 and IL-2 share many functions together, including the stimulation of CD8 T cell proliferation and IFN-γ production, the different in vivo roles of IL-15 and IL-2 have been increasingly recognized. Here, we explored the different effects of IL-15 and IL-2 on tumor-infiltrating (TI) T cells from resected breast tumors. We found that neither IL-2 nor IL-15 induced intratumoral CD8 T cell proliferation by itself, but after CD3/CD28-stimulation, IL-15 induced significantly higher proliferation than IL-2 during early time points, at day 2, day 3 and day 6. However, the IL-15-induced proliferation leveled off at day 9 and day 12, whereas IL-2 induced lower but progressive proliferation at each time point. Furthermore, IL-15 caused an early and robust increase of IFN-γ in the supernatant of TI cell cultures, which diminished at later time points, while the IL-2-induced IFN-γ production remained constant over time. In addition, the IL-15-costimulated CD8 T cells presented higher frequencies of apoptotic cells. The diminishing IL-15-induced response was possibly due to regulatory and/or exhaustion mechanisms. We did not observe increased IL-10 or PD-1 upregulation, but we have found an increase of Tim-3 upregulation on IL-15-, but not IL-2-stimulated cells. Blocking Tim-3 function using anti-Tim-3 antibodies resulted in increased IL-15-induced proliferation and IFN-γ production for a prolonged period of time, whereas adding Tim-3 ligand galectin 9 led to reduced proliferation and IFN-γ production. Our results suggest that IL-15 in combination of Tim-3 blocking antibodies could potentially act as an IL-2 alternative in tumor CD8 T cell expansion in vitro, a crucial step in adoptive T cell therapy. - Highlights: • We explored the effects of IL-15 and IL-2 on tumor-infiltrating (TI) T cells of breast cancer. • IL-15

  14. Human spleen contains different subsets of dendritic cells and regulatory T lymphocytes

    PubMed Central

    Velásquez-Lopera, M M; Correa, L A; García, L F

    2008-01-01

    Most knowledge about dendritic cells (DCs) and regulatory T cells in humans has been gathered from circulating cells but little is known about their frequency and distribution in lymphoid organs. This report shows the frequency, phenotype and location of DCs and regulatory T cells in deceased organ donors' spleens. As determined by flow cytometry, conventional/myeloid DCs (cDCs) CD11chighHLA-DR+CD123−/low were 2·3 ± 0·9% and LIN- HLA-DR+CD11chigh 2·1 ± 0·3% of total spleen cells. Mature CD11chighHLA-DR+CD83+ were 1·5 ± 0·8% and 1·0 ± 1·6% immature CD11chighHLA-DR+CD83- cDC. There were 0·3 ± 0·3% plasmacytoid DCs (pDC) CD11c−/lowHLA-DR+CD123high and 0·3 ± 0·1% LIN-HLA-DR+CD123high. Cells expressing cDCs markers, BDCA-1 and BDCA-3, and pDCs markers BDCA-2 and BDCA-4 were observed in higher frequencies than DCs with other phenotypes evaluated. CD11c+, CD123+ and CD83+ cells were located in subcapsular zone, T cells areas and B-cell follicles. CD4+CD25high Tregs were 0·2 ± 0·2% and CD8+CD28- comprised 11·5 ± 8·1% of spleen lymphocytes. FOXP3+ cells were found in T- and B-cell areas. The improvement in cell separation, manipulation and expansion techniques, will facilitate the manipulation of donor spleen cells as a part of protocols for induction and maintenance of allograft tolerance or treatment of autoimmune diseases. PMID:18727627

  15. Early and Late Chromosome Damages in Human Lymphocytes Induced by Gamma Rays and Fe Ions

    NASA Technical Reports Server (NTRS)

    Sunagawa, Mayumi; Zhang, Ye; Yeshitla, Samrawit; Kadhim, Munira; Wilson, Bobby; Wu, Honglu

    2014-01-01

    Chromosomal translocations and inversions are considered stable, and cells containing these types of chromosome aberrations can survive multiple cell divisions. An efficient method to detect an inversion is multi-color banding fluorescent in situ hybridization (mBAND) which allows identification of both inter- and intrachromosome aberrations simultaneously. Post irradiation, chromosome aberrations may also arise after multiple cell divisions as a result of genomic instability. To investigate the stable or late-arising chromosome aberrations induced after radiation exposure, we exposed human lymphocytes to gamma rays and Fe ions ex vivo, and cultured the cells for multiple generations. Chromosome aberrations were analyzed in cells collected at first mitosis and at several time intervals during the culture period post irradiation. With gamma irradiation, about half of the damages observed at first mitosis remained after 7 day- and 14 day- culture, suggesting the transmissibility of damages to the surviving progeny. Detailed analysis of chromosome break ends participating in exchanges revealed a greater fraction of break ends involved in intrachromosome aberrations in the 7- and 14-day samples in comparison to the fraction at first mitosis. In particular, simple inversions were found at 7 and 14 days, but not at the first mitosis, suggesting that some of the aberrations might be formed days post irradiation. In contrast, at the doses that produced similar frequencies of gamma-induced chromosome aberrations as observed at first mitosis, a significantly lower yield of aberrations remained at the same population doublings after Fe ion exposure. At these equitoxic doses, more complex type aberrations were observed for Fe ions, indicating that Fe ion-induced initial chromosome damages are more severe and may lead to cell death. Comparison between low and high doses of Fe ion irradiation in the induction of late damages will also be discussed.

  16. Cyclotides Suppress Human T-Lymphocyte Proliferation by an Interleukin 2-Dependent Mechanism

    PubMed Central

    Gründemann, Carsten; Thell, Kathrin; Lengen, Karin; Garcia-Käufer, Manuel; Huang, Yen-Hua; Huber, Roman; Craik, David J.; Schabbauer, Gernot; Gruber, Christian W.

    2013-01-01

    Cyclotides are a diverse and abundant group of ribosomally synthesized plant peptides containing a unique cyclic cystine-knotted topology that confers them with remarkable stability. Kalata B1, a representative member of this family of mini-proteins, has been found to inhibit the proliferation of human peripheral blood mononuclear cells. Analysis of T-cell proliferation upon treatment with chemically synthesized kalata B1 mutants revealed a region comprising inter-cysteine loops 1 and 2 of the cyclotide framework to be important for biological activity. Cytokine signaling analysis using an ‘active’ kalata B1 mutant [T20K], and the reference drug cyclosporin A (CsA) demonstrated that treatment of activated T-lymphocytes with these compounds decreased the expression of the interleukin-2 (IL-2) surface receptor as well as IL-2 cytokine secretion and IL-2 gene expression, whereas the ‘inactive’ kalata B1 mutant [V10K] did not cause any effects. The anti-proliferative activity of [T20K] kalata B1 was antagonized by addition of exogenous IL-2. Furthermore, treatment with [T20K] kalata B1 led to an initial reduction of the effector function, as indicated by the reduced IFN-γ and TNF-α production, but the levels of both cytokines stabilized over time and returned to their normal levels. On the other hand, the degranulation activity remained reduced. This indicated that cyclotides interfere with T-cell polyfunctionality and arrest the proliferation of immune-competent cells through inhibiting IL-2 biology at more than one site. The results open new avenues to utilize native and synthetically-optimized cyclotides for applications in immune-related disorders and as immunosuppressant peptides. PMID:23840803

  17. The Biological Effectiveness of Different Radiation Qualities for the Induction of Chromosome Damage in Human Lymphocytes

    NASA Technical Reports Server (NTRS)

    Hada, M.; George, K.; Cucinotta, F. A.

    2010-01-01

    Chromosome aberrations were measured in human peripheral blood lymphocytes after in vitro exposure to 28Si- ions with energies ranging from 90 to 600 MeV/u, or to 56Fe-ions with energies ranging from 200 to 5,000 MeV/u. The LET of the various Fe beams in this study ranged from 145 to 440 keV/micron and the LET of the Si ions ranged from 48 to 158 keV/ m. Doses delivered were in the 10- to 200-cGy range. Dose-response curves for chromosome exchanges in cells at first division after exposure, measured using fluorescence in situ hybridization (FISH) with whole-chromosome probes, were fitted with linear or linear-quadratic functions. The relative biological effectiveness (RBE) was estimated from the initial slope of the dose-response curve for chromosome damage with respect to -rays. The estimates of RBE(sub max) values for total chromosome exchanges ranged from 4.4+/-0.4 to 31.5+/-2.6 for Fe ions, and 11.8+/-1.0 to 42.2+/-3.3 for Si ions. The highest RBE(sub max) value for Fe ions was obtained with the 600-Mev/u beam, and the highest RBE(sub max) value for Si ions was obtained with the 170 MeV/u beam. For both ions the RBEmax values increased with LET, reaching a maximum at about 180 keV/micron for Fe and about 100 keV/ m for Si, and decreasing with further increase in LET. Additional studies for low doses 28Si-ions down to 0.02 Gy will be discussed.

  18. Expression of transferrin receptors on mitogen-stimulated human peripheral blood lymphocytes: relation to cellular activation and related metabolic events.

    PubMed Central

    Galbraith, R M; Galbraith, G M

    1981-01-01

    Mitogen-activated normal human peripheral blood lymphocytes bind transferrin to specific membrane receptors. In this study, lymphocytes stimulated with phytohaemagglutinin for 0-66 hr were examined to determine the relation of this phenomenon to cellular activation and related metabolic events. Transferrin receptors were first detected at 20-24 hr. This event was consistently preceded by RNA and protein turnover which commenced during the first 6 hr of culture, whereas initiation of DNA synthesis was detected concurrently with the appearance of receptors or slightly later (24-30 hr). Exposure of cells to inhibitors of RNA and protein synthesis early during culture (at 0 or 24 hr) prevented the expression of transferrin receptors, but also caused generalized metabolic failure, and abrogated cellular activation. In contrast, later addition of these agents at 48 hr did not interfere significantly with the process of activation, but did suppress the terminal increase in receptor-bearing cells observed during the final 18 hr in control cultures lacking inhibitor. After deliberate thermal stripping of receptors from activated cells, the reappearance of membrance binding sites which normally occurred within 30 min, was also blocked by cycloheximide, puromycin and actinomycin D. However, similar inhibition of DNA which was induced by hydroxyurea had much less effect upon both the initial appearance of receptors and their reappearance after ligand-induced depletion. These results demonstrate that the appearance of transferrin receptors upon human lymphocytes is dependent upon cellular activation and requires synthesis of protein and RNA. PMID:6172372

  19. gp140, the C3d receptor of human B lymphocytes, is also the Epstein-Barr virus receptor.

    PubMed

    Frade, R; Barel, M; Ehlin-Henriksson, B; Klein, G

    1985-03-01

    The relationship between gp140, the membrane C3d receptor (CR2) of human B lymphocytes, and the Epstein-Barr virus receptor (EBVR) was analyzed by using the polyclonal anti-gp140, previously prepared by immunizing rabbits with highly purified gp140 (isolated by some of us) from CR2/EBVR-positive Raji cells. Polyclonal anti-gp72, a C3-binding membrane component, not related to the EBVR but also expressed on the Raji cell surface, was used as a control. Binding of rabbit IgG and EBV on cells was assessed by using immunofluorescence techniques with analysis by flow cytofluorometry. A semiquantitative bioassay was also used to measure the EBV binding. Polyclonal monospecific anti-gp140 IgG inhibits directly the binding of EBV to Raji cells at the same concentration that inhibits the binding of EC3d on cells, whereas a 35 times higher concentration of anti-gp72 IgG or preimmune serum IgG does not. Anti-gp140 IgG treatment also inhibits the induction of EBV-determined nuclear antigen in normal tonsil B lymphocytes or in EBV-negative Ramos cells, whereas high concentrations of anti-gp72 IgG or preimmune serum IgG have no effect. These data strongly suggest that gp140, the CR2 of human B lymphocytes, is also the EBVR. PMID:2983347

  20. Primary human alveolar epithelial cells can elicit the transendothelial migration of CD14+ monocytes and CD3+ lymphocytes

    PubMed Central

    Eghtesad, M; Jackson, H E; Cunningham, A C

    2001-01-01

    The ability of freshly isolated primary human alveolar epithelial cells (type II pneumocytes) to induce leucocyte migration across an endothelial monolayer was investigated. Three-way factorial analysis of variance (anova) demonstrated that resting alveolar endothelial cells (AEC) could produce detectable quantities of monocyte chemoattractant protein 1 (MCP-1), which was upregulated in response to tumour necrosis factor-α (TNF-α) in a dose- and time-dependent fashion. Interferon-γ (IFN-γ) had no significant effect on this process. TNF-α and IFN-γ both induced AEC to provoke migration of CD14+ monocytes and CD3+ lymphocytes across endothelium. IFN-γ and TNF-α synergized in their ability to induce production of T lymphocyte, but not monocyte, chemoattractants from AEC. Leucocyte transendothelial migration was inhibited by anti-MCP-1 neutralizing antibody and by heparin, a polyanionic glycosaminoglycan (GAG). These data suggest that human AEC play a role in the multiple mechanisms that facilitate monocyte and T lymphocyte migration into the alveolar compartment of the lung under homeostasis and inflammatory conditions. One of these mechanisms is mediated via constitutive MCP-1 production by alveolar epithelial cells, which is upregulated by TNF-α. PMID:11260320

  1. In vitro hemocompatibility and toxic mechanism of graphene oxide on human peripheral blood T lymphocytes and serum albumin.

    PubMed

    Ding, Zhijia; Zhang, Zhijun; Ma, Hongwei; Chen, Yanyan

    2014-11-26

    Graphene oxide (GO) has shown tremendous application potential as a biomedical material. However, its interactions with blood components are not yet well understood. In this work, we assess the toxicity of pristine GO (p-GO) and functionalized GO (GO-COOH and GO-PEI) to primary human peripheral blood T lymphocytes and human serum albumin (HSA), and also study the underlying toxic mechanism. Our results indicate that p-GO and GO-COOH have good biocompatibility to T lymphocytes at the concentration below 25 μg mL(-1), but notable cytotoxicity above 50 μg mL(-1). By contrast, GO-PEI exhibits significant toxicity even at 1.6 μg mL(-1). Further investigations show that although p-GO does not enter into the cell or damage the membrane, its presence leads to the increase in reactive oxygen species (ROS), moderate DNA damage, and T lymphocyte apoptosis. Interestingly, little effect on T lymphocyte immune response suppression is observed in this process despite p-GO inflicting cell apoptosis. The toxic mechanism is that p-GO interacts directly with the protein receptors to inhibit their ligand-binding ability, leading to ROS-dependent passive apoptosis through the B-cell lymphoma-2 (Bcl-2) pathway. Compared with p-GO, GO-COOH exhibits a similar toxic effect on T lymphocytes except keeping a normal ROS level. A proposed toxic mechanism is that GO-COOH inhibits protein receptor-ligand binding, and passes the passive apoptosis signal to nucleus DNA through a ROS-independent mechanism. On the other hand, GO-PEI shows severe hematotoxicity to T lymphocytes by inducing membrane damage. For plasma protein HSA, the binding of GO-COOH results in minimal conformational change and HSA's binding capacity to bilirubin remains unaffected, while the binding of p-GO and GO-PEI exhibits strong toxicity on HSA. These findings on the interactions of two-dimensional nanomaterials and biological systems, along with the enquiry of the mechanisms, would provide essential support for further

  2. Analysis of purine metabolic enzymes in human CD4 Leu 8- and CD4 Leu 8+ lymphocyte subpopulations.

    PubMed

    Fernandez-Mejia, C; Polmar, S H; Peralta-Zaragoza, O; Madrid-Marina, V

    1993-02-01

    1. Specific activities of adenosine deaminase, purine nucleoside phosphorylase, adenosine kinase, 5'-nucleotidase, S-adenosyl-L-homocysteine hydrolase, AMP deaminase, adenine phosphoribosyl transferase, and hypoxanthine phosphoribosyl transferase were analyzed in human CD4 T-lymphocyte subsets. 2. CD4 Leu 8- (helper/inducer) and CD4 Leu 8+ (suppressor/inducer) subpopulations were obtained by panning or fluorescence activated cell sorting techniques using specific monoclonal antibodies. 3. A 45% decrease of 5'-NT AMP activity in the CD4 Leu 8- cells (suppressor/inducer) compared with CD4 total cell population. 4. No statistical significant differences in enzyme activity were found between the subsets analyzed in other purine enzymes. 5. These results suggest that the distribution of purine metabolic enzymes is homogeneous in CD4 Leu 8- and CD4 Leu 8+ T-lymphocyte subpopulations. PMID:8444317

  3. Metabolic activation of herbicide products by Vicia faba detected in human peripheral lymphocytes using alkaline single cell gel electrophoresis.

    PubMed

    Calderón-Segura, María Elena; Gómez-Arroyo, Sandra; Molina-Alvarez, Bertha; Villalobos-Pietrini, Rafael; Calderón-Ezquerro, Carmen; Cortés-Eslava, Josefina; Valencia-Quintana, Pedro Rafael; López-González, Lucina; Zúñiga-Reyes, Rubén; Sánchez-Rincón, José

    2007-09-01

    Ametryn and metribuzin S-triazines derivatives and EPTC thiocarbamate are herbicides used extensively in Mexican agriculture, for example in crops such as corn, sugar cane, tomato, wheat, and beans. The present study evaluated the DNA damage and cytotoxic effects of three herbicides after metabolism by Vicia faba roots in human peripheral lymphocytes using akaline single cell gel electrophoresis. Three parameters were scored as indicators of DNA damage: tail length, percentage of cells with DNA damage (with comet), and level DNA damage. The lymphocytes were treated for 2 h with 0.5-5.0 microg/ml ametryn or metribuzin and 1.5-10 microg/ml EPTC. Lymphocytes also were coincubated for 2 h with 20 microl V. faba roots extracts that had been treated for 4 h with 50-500 mg/l of the two triazines or with the thiocarbamate herbicide or with ethanol (3600 mg/l), as positive control. The lymphocytes treated with three pesticides without in vivo metabolic activation by V. faba root did not show significant differences in the mean values between genotoxic parameters compared with negative control. But when human cells were exposed to three herbicides after they had been metabolized the frequency of cell comet, tail length and level DNA damage all increased. At highest concentrations of the three herbicides produced severe DNA damage compared with S10 fraction and negative control. The linear regression analysis of the tail length values of three herbicides indicated that there was genotoxic effect concentration-response relationship with ametryn and ametribuzin but no EPTC. The ethanol induced major increase DNA damage compared with S10 fraction and the three pesticides. There were not effects in cell viability with treatment EPTC and metribuzin whether or not it had been metabolized. High concentrations of ametryn alone and after it had been metabolized decreased cell viability compared with the negative control. The results demonstrated that the three herbicides needed to be

  4. Diminution of Oxidative Damage to Human Erythrocytes and Lymphocytes by Creatine: Possible Role of Creatine in Blood

    PubMed Central

    Qasim, Neha; Mahmood, Riaz

    2015-01-01

    Creatine (Cr) is naturally produced in the body and stored in muscles where it is involved in energy generation. It is widely used, especially by athletes, as a staple supplement for improving physical performance. Recent reports have shown that Cr displays antioxidant activity which could explain its beneficial cellular effects. We have evaluated the ability of Cr to protect human erythrocytes and lymphocytes against oxidative damage. Erythrocytes were challenged with model oxidants, 2, 2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and hydrogen peroxide (H2O2) in the presence and absence of Cr. Incubation of erythrocytes with oxidant alone increased hemolysis, methemoglobin levels, lipid peroxidation and protein carbonyl content. This was accompanied by decrease in glutathione levels. Antioxidant enzymes and antioxidant power of the cell were compromised while the activity of membrane bound enzyme was lowered. This suggests induction of oxidative stress in erythrocytes by AAPH and H2O2. However, Cr protected the erythrocytes by ameliorating the AAPH and H2O2 induced changes in these parameters. This protective effect was confirmed by electron microscopic analysis which showed that oxidant-induced cell damage was attenuated by Cr. No cellular alterations were induced by Cr alone even at 20 mM, the highest concentration used. Creatinine, a by-product of Cr metabolism, was also shown to exert protective effects, although it was slightly less effective than Cr. Human lymphocytes were similarly treated with H2O2 in absence and presence of different concentrations of Cr. Lymphocytes incubated with oxidant alone had alterations in various biochemical and antioxidant parameters including decrease in cell viability and induction of DNA damage. The presence of Cr attenuated all these H2O2-induced changes in lymphocytes. Thus, Cr can function as a blood antioxidant, protecting cells from oxidative damage, genotoxicity and can potentially increase their lifespan. PMID

  5. TGF-β modulates the functionality of tumor-infiltrating CD8+ T cells through effects on TCR signaling and Spred1 expression

    PubMed Central

    di Bari, Maria Giovanna; Lutsiak, M.E. Christine; Takai, Shinji; Mostböck, Sven; Farsaci, Benedetto; Semnani, Roshanak Tolouei; Wakefield, Lalage M.; Schlom, Jeffrey; Sabzevari, Helen

    2012-01-01

    This study demonstrates that CD8+ T cells in the tumor microenvironment display reduced functionality and hyporesponsiveness. TGF-β contributed markedly to the tumor-infiltrating CD8+ T cells’ (TILs) reduced functionality, which could be reversed using a small molecule TGF-β inhibitor. Upon T-cell receptor (TCR) activation, the activation of ITK and ERK kinases were reduced in CD8+ TILs, as compared to splenic CD8+ T cells: TGF-β inhibitor could reverse this phenomenon. This study demonstrates for the first time the association of the Spred-1 gene, an inhibitor of the Ras/MAPK pathway, with CD8+ TILs and TGF-β activity. Spred-1 was upregulated in CD8+ TILs and TGF-β enhanced the expression of Spred-1 in effector/memory CD8+ T cells and not in rested/memory CD8+ T cells. Based on these findings, this study supports the hypothesis that TGF-β mediates an inhibitory mechanism on CD8+ TILs involving TCR-signaling blockade and the upregulation of Spred-1, thus implicating Spred-1 as a potential new target for future anti-tumor immune studies. PMID:19319531

  6. TGF-beta modulates the functionality of tumor-infiltrating CD8+ T cells through effects on TCR signaling and Spred1 expression.

    PubMed

    di Bari, Maria Giovanna; Lutsiak, M E Christine; Takai, Shinji; Mostböck, Sven; Farsaci, Benedetto; Semnani, Roshanak Tolouei; Wakefield, Lalage M; Schlom, Jeffrey; Sabzevari, Helen

    2009-11-01

    This study demonstrates that CD8+ T cells in the tumor microenvironment display reduced functionality and hyporesponsiveness. TGF-beta contributed markedly to the tumor-infiltrating CD8+ T cells' (TILs) reduced functionality, which could be reversed using a small molecule TGF-beta inhibitor. Upon T-cell receptor (TCR) activation, the activation of ITK and ERK kinases were reduced in CD8+ TILs, as compared to splenic CD8+ T cells: TGF-beta inhibitor could reverse this phenomenon. This study demonstrates for the first time the association of the Spred-1 gene, an inhibitor of the Ras/MAPK pathway, with CD8+ TILs and TGF-beta activity. Spred-1 was upregulated in CD8+ TILs and TGF-beta enhanced the expression of Spred-1 in effector/memory CD8+ T cells and not in rested/memory CD8+ T cells. Based on these findings, this study supports the hypothesis that TGF-beta mediates an inhibitory mechanism on CD8+ TILs involving TCR-signaling blockade and the upregulation of Spred-1, thus implicating Spred-1 as a potential new target for future anti-tumor immune studies. PMID:19319531

  7. Enrichment of CD56dimKIR+CD57+ highly cytotoxic NK cells in tumor infiltrated lymph nodes of melanoma patients

    PubMed Central

    Mortarini, Roberta; Anichini, Andrea; Garofalo, Cinzia; Tallerico, Rossana; Santinami, Mario; Gulletta, Elio; Ietto, Caterina; Galgani, Mario; Matarese, Giuseppe; Bifulco, Maurizio; Ferrone, Soldano; Colucci, Francesco; Moretta, Alessandro; Kärre, Klas; Carbone, Ennio

    2014-01-01

    An important checkpoint in the progression of melanoma is the metastasis to lymph nodes. Here, to investigate the role of lymph node NK cells in disease progression, we analyze frequency, phenotype and functions of NK cells from tumor-infiltrated (TILN) and tumor-free ipsilateral lymph nodes (TFLN) of the same patients. We show an expansion of CD56dimCD57dimCD69+CCR7+KIR+ NK cells in TILN. TILN NK cells display robust cytotoxic activity against autologous melanoma cells. In the blood of metastatic melanoma patients the frequency of NK cells expressing the receptors for CXCL8 receptor is increased compared to healthy subjects, and blood NK cells also express the receptors for CCL2 and IL6. These factors are produced in high amount in TILN and in vitro switch the phenotype of blood NK cells from healthy donors to the phenotype associated with TILN. Our data suggest that the microenvironment of TILN generates and/or recruits a particularly effective NK cell subset. PMID:25472612

  8. Non-classical HLA-class I expression in serous ovarian carcinoma: Correlation with the HLA-genotype, tumor infiltrating immune cells and prognosis

    PubMed Central

    Andersson, Emilia; Poschke, Isabel; Villabona, Lisa; Carlson, Joseph W; Lundqvist, Andreas; Kiessling, Rolf; Seliger, Barbara; Masucci, Giuseppe V

    2016-01-01

    In our previous studies, we have shown that patients with serous ovarian carcinoma in advanced surgical stage disease have a particularly poor prognosis if they carry the HLA-A*02 genotype. This represent a stronger prognostic factor than loss or downregulation of the MHC class I heavy chain (HC) on tumor cells. In this study, we investigated the expression of the non-classical, immune tolerogenic HLA -G and -E on the tumor cells along with the infiltration of immune cells in the tumor microenvironment. FFPE primary tumors from 72 patients with advanced stages of serous adenocarcinoma and metastatic cells present in ascites fluid from 8 additional patients were included in this study. Both expression of HLA-G and aberrant expression of HLA-E were correlated to a significant worse prognosis in patients with HLA-A*02, but not with different HLA genotypes. Focal cell expression of HLA-G correlated to a site-specific downregulation of classical MHC class I HC products and aberrant HLA-E expression, showing a poor survival. HLA-G was more frequently expressed in metastatic cells than in primary tumor lesions and the expression of HLA-G inversely correlated with the frequency of tumor infiltrating immune cells. All these parameters can contribute together to identify and discriminate subpopulations of patients with extremely poor prognosis and can give them the opportunity to receive, and benefit of individually tailored treatments. PMID:26942060

  9. Tasquinimod modulates tumor-infiltrating myeloid cells and improves the antitumor immune response to PD-L1 blockade in bladder cancer

    PubMed Central

    Nakhlé, Jessica; Pierron, Valérie; Bauchet, Anne-Laure; Plas, Pascale; Thiongane, Amath; Meyer-Losic, Florence; Schmidlin, Fabien

    2016-01-01

    ABSTRACT The infiltration of myeloid cells helps tumors to overcome immune surveillance and imparts resistance to cancer immunotherapy. Thus, strategies to modulate the effects of these immune cells may offer a potential therapeutic benefit. We report here that tasquinimod, a novel immunotherapy which targets S100A9 signaling, reduces the immunosuppressive properties of myeloid cells in preclinical models of bladder cancer (BCa). As single anticancer agent, tasquinimod treatment was effective in preventing early stage tumor growth, but did not achieve a clear antitumor effect in advanced tumors. Investigations of this response revealed that tasquinimod induces an increase in the expression of a negative regulator of T cell activation, Programmed-death-ligand 1 (PD-L1). This markedly weakens its antitumor immunity, yet provokes an “inflamed” milieu rendering tumors more prone to T cell-mediated immune attack by PD-L1 blockade. Interestingly, the combination of tasquinimod with an Anti-PD-L1 antibody enhanced the antitumor immune response in bladder tumors. This combination synergistically modulated tumor-infiltrating myeloid cells, thereby strongly affecting proliferation and activation of effector T cells. Together, our data provide insight into the rational combination of therapies that activate both innate and adaptive immune system, such as the association of S100A9-targeting agents with immune checkpoints inhibitors, to improve the response to cancer immunotherapeutic agents in BCa. PMID:27471612

  10. Epstein-Barr virus-transformed human B lymphocytes produce natural antibodies to histones.

    PubMed

    Garzelli, C; Incaprera, M; Bazzichi, A; Manunta, M; Rognini, F; Falcone, G

    1994-03-01

    To study the mechanism(s) responsible for the appearance of Epstein-Barr virus (EBV)-induced anti-histone autoantibodies, peripheral blood B lymphocytes from healthy donors were infected with EBV and the resulting lymphoblastoid cell lines were tested for secretion of antibodies reacting with histones. It was found that EBV-transformed cells produce IgM antibody reactive with histones and that the frequency of EBV-inducible circulating B lymphocytes that produce antibodies to histones is at least 10(-5). Moreover, in cultures of tonsillar lymphoid cells, the enrichment in CD5+ B lymphocytes increases the percentage of EBV-transformed cultures making anti-histone IgM antibodies. EBV may therefore, also in vivo, induce natural anti-histone antibody by polyclonal B-cell activation without any requirement of antigen to trigger antibody response. PMID:7518420

  11. Gene expression of inflammatory molecules in circulating lymphocytes from arsenic-exposed human subjects.

    PubMed Central

    Wu, Meei-Maan; Chiou, Hung-Yi; Ho, I-Ching; Chen, Chien-Jen; Lee, Te-Chang

    2003-01-01

    Long-term arsenic exposure is associated with an increased risk of vascular diseases including ischemic heart disease, cerebrovascular disease, and carotid atherosclerosis. The pathogenic mechanisms of arsenic atherogenicity are not completely clear. A fundamental role for inflammation in atherosclerosis and its complications has become appreciated recently. To investigate molecular targets of inflammatory pathway possibly involved in arsenic-associated atherosclerosis, we conducted an exploratory study using cDNA microarray and enzyme-linked immunosorbent assay to identify genes with differential expression in arsenic-exposed yet apparently healthy individuals. As an initial experiment, array hybridization was performed with mRNA isolated from activated lymphocytes of 24 study subjects with low (0-4.32 microg/L), intermediate (4.64-9.00 microg/L), and high (9.60-46.5 microg/L) levels of blood arsenic, with each group comprising eight age-, sex-, and smoking frequency-matched individuals. A total of 708 transcripts of known human genes were analyzed, and 62 transcripts (8.8%) showed significant differences in the intermediate or high-arsenic groups compared with the low-level arsenic group. Among the significantly altered genes, several cytokines and growth factors involving inflammation, including interleukin-1 beta, interleukin-6, chemokine C-C motif ligand 2/monocyte chemotactic protein-1 (CCL2/MCP1), chemokine C-X-C motif ligand 1/growth-related oncogene alpha, chemokine C-X-C motif ligand 2/growth-related oncogene beta, CD14 antigen, and matrix metalloproteinase 1 (interstitial collagenase) were upregulated in persons with increased arsenic exposure. Multivariate analyses on 64 study subjects of varying arsenic exposure levels showed that the association of CCL2/MCP1 plasma protein level with blood arsenic remained significant after adjustment for other risk factors of cardiovascular diseases. The results of this gene expression study indicate that the

  12. The use of decompression to simulate the effect of extravehicular activity on human lymphocyte transformation

    NASA Technical Reports Server (NTRS)

    Meehan, R. T.; Duncan, U.; Neale, L.; Waligora, J.; Taylor, G. R.

    1986-01-01

    Lymphocytes from 35 subjects participating in a chamber study simulating extravehicular activity (EVA) conditions were studied. No significant differences in H3 thymidine uptake between pre chamber and post chamber response to any mitogens autologous plasma, or among circulating mononuclear cells by flow cytometry are observed. The studies could not identify the subjects who developed venous bubbles. Data from eight subjects suggests that acute stress associated with participating in the study augments in vitro lymphocyte proliferation. Results indicate EVA exposure does not greatly influence space-flight induced alterations in immune effector cell function.

  13. Differential expression of CC chemokines (CCLs) and receptors (CCRs) by human T lymphocytes in response to different Aggregatibacter actinomycetemcomitans serotypes

    PubMed Central

    ALVAREZ, Carla; BENÍTEZ, Alvaro; ROJAS, Leticia; PUJOL, Myriam; CARVAJAL, Paola; DÍAZ-ZÚÑIGA, Jaime; VERNAL, Rolando

    2015-01-01

    ABSTRACT In Aggregatibacter actinomycetemcomitans, different serotypes have been described based on LPS antigenicity. Recently, our research group has reported a differential immunogenicity when T lymphocytes were stimulated with these different serotypes. In particular, it was demonstrated that the serotype b of A. actinomycetemcomitans has a stronger capacity to trigger Th1- and Th17-type cytokine production. Objective This study aimed to quantify the expression of different CC chemokines (CCLs) and receptors (CCRs) in T lymphocytes stimulated with the different A. actinomycetemcomitans serotypes. In addition, the expression of the transcription factors T-bet, GATA-3, RORC2, and Foxp3, master-switch genes implied in the Th1, Th2, Th17, and T-regulatory differentiation, respectively, was analyzed in order to determine T-cell phenotype-specific patterns of CCL and CCR expression upon A. actinomycetemcomitans stimulation. Material and Methods Human naïve CD4+ T lymphocytes were obtained from healthy subjects and stimulated with autologous dendritic cells primed with the different A. actinomycetemcomitans serotypes. The expression levels for the chemokines CCL1, CCL2, CCL3, CCL5, CCL11, CCL17, CCL20, CCL21, CCL25, and CCL28, as well as the chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, and CCR10 were quantified by qPCR. Similarly, the expression levels for the transcription factors T-bet, GATA-3, RORC2, and Foxp3 were quantified and correlated with the CCL and CCR expression levels. Results Higher expression levels of CCL2, CCL3, CCL5, CCL20, CCL21, CCL28, CCR1, CCR2, CCR5, CCR6, CCR7, and CCR9 were detected in T lymphocytes stimulated with the serotype b of A. actinomycetemcomitans compared with the other serotypes. In addition, these higher expression levels of CCLs and CCRs positively correlated with the increased levels of T-bet and RORC2 when T lymphocytes were stimulated with the serotype b. Conclusion A T-lymphocyte response biased

  14. Differential expression of CC chemokines (CCLs) and receptors (CCRs) by human T lymphocytes in response to different Aggregatibacter actinomycetemcomitans serotypes.

    PubMed

    Alvarez, Carla; Benítez, Alvaro; Rojas, Leticia; Pujol, Myriam; Carvajal, Paola; Díaz-Zúñiga, Jaime; Vernal, Rolando

    2015-01-01

    In Aggregatibacter actinomycetemcomitans, different serotypes have been described based on LPS antigenicity. Recently, our research group has reported a differential immunogenicity when T lymphocytes were stimulated with these different serotypes. In particular, it was demonstrated that the serotype b of A. actinomycetemcomitans has a stronger capacity to trigger Th1- and Th17-type cytokine production. Objective This study aimed to quantify the expression of different CC chemokines (CCLs) and receptors (CCRs) in T lymphocytes stimulated with the different A. actinomycetemcomitans serotypes. In addition, the expression of the transcription factors T-bet, GATA-3, RORC2, and Foxp3, master-switch genes implied in the Th1, Th2, Th17, and T-regulatory differentiation, respectively, was analyzed in order to determine T-cell phenotype-specific patterns of CCL and CCR expression upon A. actinomycetemcomitans stimulation. Material and Methods Human naïve CD4+ T lymphocytes were obtained from healthy subjects and stimulated with autologous dendritic cells primed with the different A. actinomycetemcomitans serotypes. The expression levels for the chemokines CCL1, CCL2, CCL3, CCL5, CCL11, CCL17, CCL20, CCL21, CCL25, and CCL28, as well as the chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, and CCR10 were quantified by qPCR. Similarly, the expression levels for the transcription factors T-bet, GATA-3, RORC2, and Foxp3 were quantified and correlated with the CCL and CCR expression levels. Results Higher expression levels of CCL2, CCL3, CCL5, CCL20, CCL21, CCL28, CCR1, CCR2, CCR5, CCR6, CCR7, and CCR9 were detected in T lymphocytes stimulated with the serotype b of A. actinomycetemcomitans compared with the other serotypes. In addition, these higher expression levels of CCLs and CCRs positively correlated with the increased levels of T-bet and RORC2 when T lymphocytes were stimulated with the serotype b. Conclusion A T-lymphocyte response biased towards a

  15. Tumor-infiltrating macrophages express interleukin-25 and predict a favorable prognosis in patients with gastric cancer after radical resection

    PubMed Central

    Li, Jinqing; Liao, Yuan; Ding, Tong; Wang, Bo; Yu, Xingjuan; Chu, Yifan; Xu, Jing; Zheng, Limin

    2016-01-01

    Interleukin-25 (IL-25) is a recently identified member of the proinflammatory IL-17 cytokine family; however, its role in human tumors remains largely unknown. The aim of this study was to investigate the cellular source and clinical significance of IL-25 in gastric cancer (GC) in situ. The results demonstrated that macrophages (Mφs) were the primary IL-25-expressing cells (IL-25+) in GC in situ. Moreover, IL-25+ cells were highly enriched in the intra-tumoral (IT) region of GC tissues (p < 0.001). The production of IL-25 in Mφs exposed to culture supernatant from gastric cancer cell line SGC7901 in vitro was induced by transforming growth factor-β1, and their density in the IT region was positively associated with those of other effector immune cells, namely, CD4+ T cells, CD8+ T cells and CD103+T cells (p < 0.01). This suggested that macrophages might produce IL-25 to create an antitumor micromilieu in GC tissues. The level of IL-25+IT cells was positively associated with histological grade (p < 0.001) and found to be an independent predictor of favorable survival (p = 0.024) in patients with GC after radical resection. These findings suggest that IL-25+IT cells may be a novel therapeutic target in those patients. PMID:26840565

  16. [Neoadjuvant treatment in human epidermal growth factor receptor 2-positive breast cancer].

    PubMed

    Liu, Yinhua; Liu, Shiwei; Zhang, Hong; Xu, Ling; Li, Ting; Duan, Xuening

    2015-12-01

    Breast cancer is the most prevalent malignancy among females worldwide. Human epidermal growth factor receptor 2 (HER2)-positive breast cancer represents a subtype with aggressive behavior, poor response to treatment and unfavorable prognosis. Anti-HER2-based neoadjuvant treatment has improved clinical outcomes of patients with HER2-positive disease. Pathological complete response (pCR) after neoadjuvant treatment indicates a favorable prognosis. With the development of HER2-targeted therapy and neoadjuvant treatment, numerous studies focus on the predictive factors of pCR or therapeutic resistance of anti-HER2 therapy. Identification of novel predictive factors in HER2-positive breast cancer, such as tumor-infiltrating lymphocytes, will be helpful for clinical decision. PMID:26850663

  17. Induction of monocyte chemoattractant protein-1 and interleukin-10 by TGFbeta1 in melanoma enhances tumor infiltration and immunosuppression.

    PubMed

    Díaz-Valdés, Nancy; Basagoiti, María; Dotor, Javier; Aranda, Fernando; Monreal, Iñaki; Riezu-Boj, José Ignacio; Borrás-Cuesta, Francisco; Sarobe, Pablo; Feijoó, Esperanza

    2011-02-01

    Melanoma progression is associated with the expression of different growth factors, cytokines, and chemokines. Because TGFβ1 is a pleiotropic cytokine involved not only in physiologic processes but also in cancer development, we analyzed in A375 human melanoma cells, the effect of TGFβ1 on monocyte chemoattractant protein-1 (MCP-1) and interleukin-10 (IL-10) expression, two known factors responsible for melanoma progression. TGFβ1 increased the expression of MCP-1 and IL-10 in A375 cells, an effect mediated by the cross-talk between Smad, PI3K (phosphoinositide 3-kinase)/AKT, and BRAF-MAPK (mitogen activated protein kinase) signaling pathways. Supernatants from TGFβ1-treated A375 cells enhanced MCP-1-dependent migration of monocytes, which, in turn, expressed high levels of TGF,β1, bFGF, and VEGF mRNA. Moreover, these supernatants also inhibited functional properties of dendritic cells through IL-10-dependent mechanisms. When using in vitro, the TGFβ1-blocking peptide P144, TGFβ1-dependent Smad3 phosphorylation, and expression of MCP-1 and IL-10 were inhibited. In vivo, treatment of A375 tumor-bearing athymic mice with P144 significantly reduced tumor growth, associated with a lower macrophage infiltrate and decreased intratumor MCP-1 and VEGF levels, as well as angiogenesis. Finally, in C57BL/6 mice with B16-OVA melanoma tumors, when administered with immunotherapy, P144 decreased tumor growth and intratumor IL-10 levels, linked to enhanced activation of dendritic cells and natural killer cells, as well as anti-OVA T-cell responses. These results show new effects of TGFβ1 on melanoma cells, which promote tumor progression and immunosuppression, strongly reinforcing the relevance of this cytokine as a molecular target in melanoma. PMID:21159663

  18. Relationship of the CD5 B cell to human tonsillar lymphocytes that express autoantibody-associated cross-reactive idiotypes.

    PubMed Central

    Kipps, T J; Duffy, S F

    1991-01-01

    We examined human tonsillar B cells for expression of autoantibody heavy-chain or kappa light-chain cross-reactive idiotypes (CRIs), respectively defined by murine MAbs G6 or 17.109. We find 17.109 or G6 each specifically binds a subpopulation of B cells, respectively reacting with 3.8 +/- 3% (mean +/- SD) or 2.0 +/- 1.2% of all tonsillar lymphocytes. Cells reactive with both 17.109 and G6 comprise only 0.4 +/- 0.3% of tonsillar lymphocytes. Although each tested specimen had 17.109-positive cells, 2 of 19 tonsils (11%) did not have any G6-reactive cells. We find that CRI-positive cells and CD5 B cells both co-express slgD but fail to bind peanut agglutinin or MAbs specific for CD10, indicating that both cell types reside in the mantle zones of secondary B cell follicles. However, less than half of the B cells bearing one or both of these CRIs express detectable levels of CD5. Nevertheless, we find that G6-reactive lymphocytes constitute a multiclonal population of cells that express homologous heavy chain variable region genes, each rearranged to one of several distinct and apparently nonmutated D and JH gene segments. Collectively, these studies indicate that expression of nondiversified autoantibody-encoding variable region genes may not be an exclusive property of B cells that bear detectable levels of the CD5 surface antigen. PMID:1710233

  19. Effects of strong magnetic fields on cell growth and radiation response of human T-lymphocytes in culture.

    PubMed

    Norimura, T; Imada, H; Kunugita, N; Yoshida, N; Nikaido, M

    1993-06-01

    Experiments were undertaken in order to verify whether or not a strong magnetic field would have any biological effects on the cell growth, viability and radiation response of mammalian cells. Magnetic field exposures were conducted using a superconducting magnet with freshly-isolated human peripheral blood T-lymphocytes maintained at their normal growing temperature of 37 degrees C. The static magnetic fields with intensities up to 6.3-tesla (T) exerted little influence on the cell growth and viability of actively-growing T-lymphocytes under normal cell-culture conditions. On the other hand, the T cells exposed to the magnetic fields (4 T-6.3 T) during PHA stimulation were inhibited in their cell growth when compared to controls. The effects of the magnetic fields with intensities up to 2 T on cell growth properties, however, were minimal in this system. Also, the radiosensitivity of T-lymphocytes previously exposed to the strong magnetic fields was more sensitive than that of control cells. These results suggest that exposure to a static magnetic field of 4 T or stronger might lead to physiological and growth abnormalities at the cellular level. PMID:8316709

  20. Effects of folic acid deficiency and MTHFRC677T polymorphisms on cytotoxicity in human peripheral blood lymphocytes

    SciTech Connect

    Wu Xiayu; Liang Ziqing; Zou Tianning; Wang Xu

    2009-02-13

    Apoptosis (APO) and necrosis (NEC) are two different types of cell death occurring in response to cellular stress factors. Cells with DNA damage may undergo APO or NEC. Folate is an essential micronutrient associated with DNA synthesis, repair and methylation. Methylenetetrahydrofolate reductase (MTHFR) regulates intracellular folate metabolism. Folate deficiency and MTHFR C677T polymorphisms have been shown to be related to DNA damage. To verify the cytotoxic effects of folate deficiency on cells with different MTHFR C677T genotypes, 15 human peripheral lymphocyte cases with different MTHFR C677T genotypes were cultured in folic acid (FA)-deficient and -sufficient media for 9 days. Cytotoxicity was quantified using the frequencies of APO and NEC as endpoints, the nuclear division index (NDI), and the number of viable cells (NVC). These results showed that FA is an important factor in reducing cytotoxicity and increasing cell proliferation. Lymphocytes with the TT genotype proliferated easily under stress and exhibited different responses to FA deficiency than lymphocytes with the CC and CT genotypes. A TT individual may accumulate more cytotoxicity under cytotoxic stress, suggesting that the effects of FA deficiency on cytotoxicity are greater than the effects in individuals with the other MTHFR C677T variants.

  1. Broad T-Cell Receptor Repertoire in T-Lymphocytes Derived from Human Induced Pluripotent Stem Cells

    PubMed Central

    Chang, Chia-Wei; Lai, Yi-Shin; Lamb, Lawrence S.; Townes, Tim M.

    2014-01-01

    Human induced pluripotent stem cells (hiPSCs) have enormous potential for the treatment of inherited and acquired disorders. Recently, antigen-specific T lymphocytes derived from hiPSCs have been reported. However, T lymphocyte populations with broad T cell receptor (TCR) diversity have not been generated. We report that hiPSCs derived from skin biopsy are capable of producing T lymphocyte populations with a broad TCR repertoire. In vitro T cell differentiation follows a similar developmental program as observed in vivo, indicated by sequential expression of CD7, intracellular CD3 and surface CD3. The γδ TCR locus is rearranged first and is followed by rearrangement of the αβ locus. Both γδ and αβ T cells display a diverse TCR repertoire. Upon activation, the cells express CD25, CD69, cytokines (TNF-α, IFN-γ, IL-2) and cytolytic proteins (Perforin and Granzyme-B). These results suggest that most, if not all, mechanisms required to generate functional T cells with a broad TCR repertoire are intact in our in vitro differentiation protocol. These data provide a foundation for production of patient-specific T cells for the treatment of acquired or inherited immune disorders and for cancer immunotherapy. PMID:24828440

  2. Comparison of Changes in Immunological Parameters in Human Lymphocytes in 2D Versus 3D Clinostats-Goal Towards Microgravity Analog Calibration for Future Space Experiments

    NASA Astrophysics Data System (ADS)

    Sundaresan, Alamelu; Russomano, Thais; Pellis, Neal R.

    2008-06-01

    Exposure to microgravity may produce changes in the performance of the immunological system at the cellular level as well as in the major physiological systems of the body. Studies in true spaceflight and similar studies in 2D clinostats (Rotating wall vessels) related to decreased immune function in astronaut blood and normal human lymphocytes indicate a decrease in cell proliferation, T cell activation, locomotion and altered lymphocyte signal transduction (Sundaresan and Pellis, 2008, Sundaresan et al., 2004). The present study was designed to investigate whether the proliferation and viability of lymphocytes are reduced by exposure to rotation in a 3D-Clinostat, which is used to simulate microgravity for cells.

  3. Prospective study of cytotoxic T lymphocyte responses to influenza and antibodies to human T lymphotropic virus-III in homosexual men. Selective loss of an influenza-specific, human leukocyte antigen-restricted cytotoxic T lymphocyte response in human T lymphotropic virus-III positive individuals with symptoms of acquired immunodeficiency syndrome and in a patient with acquired immunodeficiency syndrome.

    PubMed Central

    Shearer, G M; Salahuddin, S Z; Markham, P D; Joseph, L J; Payne, S M; Kriebel, P; Bernstein, D C; Biddison, W E; Sarngadharan, M G; Gallo, R C

    1985-01-01

    Peripheral blood leukocytes (PBL) from 18 homosexual men who did not have acquired immunodeficiency syndrome (AIDS) and from 9 heterosexual men were repetitively tested for their ability to generate HLA self-restricted cytotoxic T lymphocyte responses to influenza virus (flu-self) over a 2-yr period. The sera of the same donors were tested for antibodies to human T lymphotropic virus-III (HTLV-III). Six of the homosexual and none of the heterosexual donors consistently generated weak cytotoxic T lymphocyte responses to flu-self. Seven of the homosexual and none of the heterosexual donors were seropositive for antibodies to HTLV-III. No obvious correlation was detected between weak flu-self cytotoxic T lymphocyte responses and antibodies to HTLV-III. However, one homosexual donor generated no detectable cytotoxic T lymphocyte activity to flu-self, although he was a strong responder to HLA-alloantigens. This donor had an OKT4:OKT8 ratio of 0.4 and was seropositive for HTLV-III antigens; HTLV-III virus was identified in his PBL; and he developed AIDS during the course of this study. A second donor with lymphadenopathy and who was seropositive for HTLV-III antigens exhibited marginal cytotoxic T lymphocyte activity to flu-self which he subsequently lost. PBL from two patients, one with Kaposi's sarcoma and one with generalized lymphadenopathy, were also tested for cytotoxic T lymphocyte responses to flu-self and to alloantigens. Both donors failed to generate cytotoxic T lymphocyte to flu-self, but generated strong cytotoxic T lymphocyte responses to alloantigens. The selective loss of an HLA-restricted cytotoxic T lymphocyte response without loss of HLA alloantigenic cytotoxic T lymphocyte activity may be an important functional immunologic characteristic in the development of AIDS. PMID:2997287

  4. Heritability of Susceptibility to Ionizing Radiation-Induced Apoptosis of Human Lymphocyte Subpopulations

    SciTech Connect

    Schmitz, Annette; Dechamps, Nathalie; Goldin, Lynn; Thomas, Gilles

    2007-07-15

    Purpose: To evaluate the heritability of intrinsic radiosensitivity, the induction of apoptosis in lymphocyte subpopulations was determined on samples from related individuals belonging to large kindred families. Methods and Materials: Quiescent lymphocytes from 334 healthy individuals were gamma-irradiated in vitro. Apoptosis was determined 18 h after irradiation by eight-color flow cytometry. Radiosensitivity was quantified from dose-effect curves. Intrafamilial correlations and heritability were computed for 199 father-mother-offspring trios using the programs SOLAR (Sequential Oligogenic Linkage Analysis Routines) and SAGE (Statistical Analysis for Genetic Epidemiology). Segregation analyses were conducted using SAGE. Results: Marked differential susceptibility of naive and memory T lymphocytes was demonstrated. Also, although age and gender were significant covariates, their effects only accounted for a minor part of the inter-individual variation. Parent-offspring and sib-sib correlations were significant for the radiosensitivity of B cells, T4, and T8 and of effector memory T4 and T8 subpopulations. In the T4-effector memory subpopulation, the phenotype showed correlations most consistent with dominant or additive genetic effects, and the results of the segregation analysis were consistent with the contribution of a bi-allelic dominant locus. Conclusions: Heritability was demonstrated for the susceptibility to ionizing radiation-induced apoptosis of lymphocyte populations, and the segregation of the T4-effector memory radiosensitivity phenotype was consistent with a simple mendelian transmission model involving one major gene.

  5. Taurine concentration in human blood peripheral lymphocytes: major depression and treatment with the antidepressant mirtazapine.

    PubMed

    Lima, Lucimey; Obregón, Francisco; Urbina, Mary; Carreira, Isabel; Baccichet, Edith; Peña, Solisbella

    2003-01-01

    Major depression is a serious disease with various systemic effects, including dysfunction of the immune response. Taurine has been known to be related to certain modifications of the immune system. The aim of this study was to determine the taurine concentration in lymphocytes of patients with major depression and to evaluate the influence of the antidepressant treatment with mirtazapine for six weeks on the levels of taurine. Gamma-aminobutyric acid, aspartate, glutamate and glutamine were also determined. Taurine, aspartate and glutamine levels were increased in the lymphocytes of depressed patients before mirtazapine treatment compared to the control group, and were normalized after treatment. Gamma-aminobutyric acid and glutamate did not differ between patients and controls. There was a significant and positive correlation between the severity of the disorder, measured by the Hamilton Rating Scale, and the concentration of taurine in the lymphocytes of depressed patients before treatment. This correlation was not observed after treatment and neither was there a correlation observed for the other amino acids. The present observations could be an indication of the relevance of taurine as a protective agent in the lymphocytes of patients with severe depression, and could be the result of modifications of taurine transport or efflux processes. PMID:12908614

  6. CHARACTERIZATION OF NORMAL HUMAN LUNG LYMPHOCYTES AND INTERLEUKIN-2-INDUCED LUNG T CELL LINES

    EPA Science Inventory

    Lymphocytes from the lower respiratory tract were obtained by bronchoalveolar lavage of healthy, non-smoking individuals. arious monoclonal antibodies characterizing activated T cells, helper-inducer and suppressor-inducer T cell subsets, and naive versus memory cells were used t...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  8. Use of cytogenetic endpoints in human lymphocytes as indicators of exposure to genotoxicants

    SciTech Connect

    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)

  9. Difference in constitutive heterochromatin behaviour between human amniocytes and lymphocytes detected by a sequential in situ exonuclease III digestion-random primer extension procedure.

    PubMed Central

    Fernández, J L; Campos, A; López-Fernández, C; Gosálvez, J; Goyanes, V

    1995-01-01

    Fixed chromosomes from human amniotic fluid cells and peripheral blood lymphocytes were digested in situ with exonuclease III and the single stranded DNA obtained was used as template for an in situ random primer extension. Under these conditions an R banding pattern, more evident in lymphocytes than in amniocytes, was obtained. Nevertheless, constitutive heterochromatin of chromosomes 1, 16, Yq, and mainly the pericentromeric region of chromosome 9 was far more intensely labelled in amniocytes than in lymphocytes. Fluorescence in situ hybridisation with a specific classical satellite DNA probe, showed that this differential labelling was dependent on a greater sensitivity of chromosome 9 constitutive heterochromatin to exonuclease III digestion in amniocytes than in lymphocytes, thus indicating qualitative differences in this region between both human cellular materials. Images PMID:7897623

  10. Induction of micronuclei and sister chromatid exchanges by polycyclic and N-heterocyclic aromatic hydrocarbons in cultured human lymphocytes

    SciTech Connect

    Warshawsky, D.; Livingston, G.K.; LaDow, K.

    1995-12-31

    Many natural environments are contaminated with carcinogenic polycyclic aromatic hydrocarbons (PAHs) and N-heterocyclic aromatic hydrocarbons (NHAs) as complex mixtures of coal tar, petroleum, and shale oil. These potentially hazardous substances are prevalent at many former tar production and coal gasification sites. Three polycyclic [benzo(a)pyrene (BaP), benz(a)anthracene (BAA), and 7, 12-dimethylbenz(a)anthracene (DMBA)] and two N-heterocyclic [7H-dibenzo(c,g)carbazole (DBC), and dibenz(a,j)acridine (DBA)] aromatic hydrocarbons were analyzed for cytotoxic and genotoxic effects on human lymphocytes. All of these polyaromatic compounds are normally present in the environment, except for DMBA. Lymphocytes from healthy donors were isolated from whole blood. The 5-ring polycyclic aromatic BaP consistently induced micronuclei in a linear dose-dependent manner with doses from 0.1-10.0 {mu}g/ml, whereas the 4-ring compounds (BAA and DMBA) had no effect on the induction of micronuclei above controls except at 5 and 10 {mu}g/ml. Of the two N-heterocyclic compounds DBC produced a significant increase in micronuclei in lymphocytes, but the dose response tended to plateau above 0.1 {mu}g/ml. DBA showed an effect on the frequency of micronuclei above controls only at high doses of 5 and 10 {mu}g/ml. The average background frequency of micronuclei for 7 lymphocyte donors averaged 3.1 per 1,000 stimulated cells, whereas the average frequency of micronuclei at 10 {mu}g/ml BaP was 36.8 per 1,000 stimulated cells. The lowest effective dose in 2 donors for BaP occurred at 0.1 {mu}g/ml. 61 refs., 2 figs., 6 tabs.

  11. The Biological Effectiveness of Different Radiation Qualities for the Induction of Chromosome Damage in Human Lymphocytes

    NASA Technical Reports Server (NTRS)

    Hada, M.; George, Kerry; Cucinotta, F. A.

    2011-01-01

    Chromosome aberrations were measured in human peripheral blood lymphocytes after in vitro exposure to Si-28-ions with energies ranging from 90 to 600 MeV/u, Ti-48-ions with energies ranging from 240 to 1000 MeV/u, or to Fe-56-ions with energies ranging from 200 to 5,000 MeV/u. The LET of the various Si beams in this study ranged from 48 to 158 keV/ m, the LET of the Ti ions ranged from 107 to 240 keV/micron, and the LET of the Fe-ions ranged from 145 to 440 keV/ m. Doses delivered were in the 10- to 200-cGy range. Dose-response curves for chromosome exchanges in cells at first division after exposure, measured using fluorescence in situ hybridization (FISH) with whole-chromosome probes, were fitted with linear or linear-quadratic functions. The relative biological effectiveness (RBE) was estimated from the initial slope of the dose-response curve for chromosome damage with respect to gamma-rays. The estimates of RBEmax values for total chromosome exchanges ranged from 4.4+/-0.4 to 31.5+/-2.6 for Fe ions, 21.4+/-1.7 to 28.3+/-2.4 for Ti ions, and 11.8+/-1.0 to 42.2+/-3.3 for Si ions. The highest RBEmax value for Fe ions was obtained with the 600 MeV/u beam, the highest RBEmax value for Ti ions was obtained 1000 MeV/u beam, and the highest RBEmax value for Si ions was obtained with the 170 MeV/u beam. For Si and Fe ions the RBEmax values increased with LET, reaching a maximum at about 180 keV/micron for Fe and about 100 keV/micron for Si, and decreasing with further increase in LET. Additional studies for low doses Si-28-ions down to 0.02 Gy will be discussed.

  12. p-Aramid RFP do not induce chromosomal aberrations in a standardized in vitro genotoxicity assay using human lymphocytes.

    PubMed

    Warheit, D B; Donner, M; Murli, H

    2001-12-01

    Genotoxicity evaluations have been proposed as regulatory requirements for establishing German MAK values for inhaled fibrous dusts. The objective of this in vitro assay was to assess the potential for para-aramid (p-aramid) respirable-sized, fiber-shaped particulates (RFP) to induce chromosomal aberrations in cultured human peripheral blood lymphocytes without metabolic activation. The highest concentration tested in this assay was limited by the physical characteristics of p-aramid RFP. The test substance was suspended in fully supplemented RPMI culture medium with 1% Pluronic F68. All dosing was achieved using a dosing volume of 90% (900 microl/ml), and the vehicle control cultures were treated with 900 microl/ml of fully supplemented RPMI culture medium with 1% Pluronic F68. In the chromosomal aberrations assay, the treatments were either 3 or 19 h without metabolic activation. Cultures were harvested 22 h from the initiation of treatment. Replicated cultures of human whole blood lymphocytes were incubated with p-aramid RFP concentrations of 6.30, 12.6, 25.2, 50.4, 101, 201, and 401 microg/ml. Cultures treated with concentrations to 50.4 microg/ml for 3 h and 6.30, 12.6, 25.2, and 201 microg/ml for 19 h were analyzed for structural and numerical chromosomal aberrations. No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed in the cultures analyzed. The results demonstrated that p-aramid RFP was negative for inducing chromosomal aberrations in cultured human peripheral blood lymphocytes without metabolic activation. In addition, we conclude that the utility of these tests for evaluating the genotoxicity of fibrous or particulate materials is questionable. PMID:11696875

  13. Altered lymphocyte trafficking and diminished airway reactivity in transgenic mice expressing human MMP-9 in a mouse model of asthma.

    PubMed

    Mehra, Divya; Sternberg, David I; Jia, Yuxia; Canfield, Stephen; Lemaitre, Vincent; Nkyimbeng, Takwi; Wilder, Julie; Sonett, Joshua; D'Armiento, Jeanine

    2010-02-01

    Matrix metalloproteinase-9 (MMP-9) is hypothesized to facilitate leukocyte extravasation and extracellular remodeling in asthmatic airways. Careful descriptive studies have shown that MMP-9 levels are higher in the sputum of asthmatics; however, the consequence of increased MMP-9 activity has not been determined in this disease. We induced asthma in transgenic mice that express human MMP-9 in the murine lung tissue macrophage to determine the direct effect of human MMP-9 expression on airway inflammation. Transgenic (TG) and wild-type (WT) mice were immunized and challenged with ovalbumin. Forty-eight hours after the ovalbumin challenge, airway hyperresponsiveness (AHR) was measured, and inflammatory cell infiltration was evaluated in bronchoalveolar lavage fluid (BALF) and lung tissue. Baseline levels of inflammation were similar in the TG and WT groups of mice, and pulmonary eosinophilia was established in both groups by sensitization and challenge with ovalbumin. There was a significant reduction in AHR in sensitized and challenged trangenics compared with WT controls. Although total BALF cell counts were similar in both groups, the lymphocyte number in the lavage of the TG group was significantly diminished compared with the WT group (0.25 +/- 0.08 vs. 0.89 +/- 0.53; P = 0.0032). In addition, the draining lymphocytes were found to be larger in the TG animals compared with the WT mice. Equal numbers of macrophages, eosinophils, and neutrophils were seen in both groups. IL-13 levels were found to be lower in the sensitized TG compared with the WT mice. These results demonstrate an inverse relationship between human MMP-9 and AHR and suggest that MMP-9 expression alters leukocyte extravasation by reducing lymphocyte accumulation in the walls of asthmatic airways. PMID:19940022

  14. Altered lymphocyte trafficking and diminished airway reactivity in transgenic mice expressing human MMP-9 in a mouse model of asthma

    PubMed Central

    Mehra, Divya; Sternberg, David I.; Jia, Yuxia; Canfield, Stephen; Lemaitre, Vincent; Nkyimbeng, Takwi; Wilder, Julie; Sonett, Joshua

    2010-01-01

    Matrix metalloproteinase-9 (MMP-9) is hypothesized to facilitate leukocyte extravasation and extracellular remodeling in asthmatic airways. Careful descriptive studies have shown that MMP-9 levels are higher in the sputum of asthmatics; however, the consequence of increased MMP-9 activity has not been determined in this disease. We induced asthma in transgenic mice that express human MMP-9 in the murine lung tissue macrophage to determine the direct effect of human MMP-9 expression on airway inflammation. Transgenic (TG) and wild-type (WT) mice were immunized and challenged with ovalbumin. Forty-eight hours after the ovalbumin challenge, airway hyperresponsiveness (AHR) was measured, and inflammatory cell infiltration was evaluated in bronchoalveolar lavage fluid (BALF) and lung tissue. Baseline levels of inflammation were similar in the TG and WT groups of mice, and pulmonary eosinophilia was established in both groups by sensitization and challenge with ovalbumin. There was a significant reduction in AHR in sensitized and challenged trangenics compared with WT controls. Although total BALF cell counts were similar in both groups, the lymphocyte number in the lavage of the TG group was significantly diminished compared with the WT group (0.25 ± 0.08 vs. 0.89 ± 0.53; P = 0.0032). In addition, the draining lymphocytes were found to be larger in the TG animals compared with the WT mice. Equal numbers of macrophages, eosinophils, and neutrophils were seen in both groups. IL-13 levels were found to be lower in the sensitized TG compared with the WT mice. These results demonstrate an inverse relationship between human MMP-9 and AHR and suggest that MMP-9 expression alters leukocyte extravasation by reducing lymphocyte accumulation in the walls of asthmatic airways. PMID:19940022

  15. Lysophosphatidylcholine hydrolases of human erythrocytes, lymphocytes, and brain: Sensitive targets of conserved specificity for organophosphorus delayed neurotoxicants

    SciTech Connect

    Vose, Sarah C.; Holland, Nina T.; Eskenazi, Brenda; Casida, John E.

    2007-10-01

    Brain neuropathy target esterase (NTE), associated with organophosphorus (OP)-induced delayed neuropathy, has the same OP inhibitor sensitivity and specificity profiles assayed in the classical way (paraoxon-resistant, mipafox-sensitive hydrolysis of phenyl valerate) or with lysophosphatidylcholine (LysoPC) as the substrate. Extending our earlier observation with mice, we now examine human erythrocyte, lymphocyte, and brain LysoPC hydrolases as possible sensitive targets for OP delayed neurotoxicants and insecticides. Inhibitor profiling of human erythrocytes and lymphocytes gave the surprising result of essentially the same pattern as with brain. Human erythrocyte LysoPC hydrolases are highly sensitive to OP delayed neurotoxicants, with in vitro IC{sub 50} values of 0.13-85 nM for longer alkyl analogs, and poorly sensitive to the current OP insecticides. In agricultural workers, erythrocyte LysoPC hydrolyzing activities are similar for newborn children and their mothers and do not vary with paraoxonase status but have high intersample variation that limits their use as a biomarker. Mouse erythrocyte LysoPC hydrolase activity is also of low sensitivity in vitro and in vivo to the OP insecticides whereas the delayed neurotoxicant ethyl n-octylphosphonyl fluoride inhibits activity in vivo at 1-3 mg/kg. Overall, inhibition of blood LysoPC hydrolases is as good as inhibition of brain NTE as a predictor of OP inducers of delayed neuropathy. NTE and lysophospholipases (LysoPLAs) both hydrolyze LysoPC, yet they are in distinct enzyme families with no sequence homology and very different catalytic sites. The relative contributions of NTE and LysoPLAs to LysoPC hydrolysis and clearance from erythrocytes, lymphocytes, and brain remain to be defined.

  16. [Current Cancer Immunotherapy Using Activated Lymphocytes - Do Lymphocytes Actually Recognize Cancer Cells ?].

    PubMed

    Yamaguchi, Yoshiyuki; Katata, Yousuke; Okawaki, Makoto; Yamamura, Masahiro; Sawaki, Akira

    2015-09-01

    Molecular cloning of interleukin-2(IL-2)has enabled adoptive cell therapy(ACT)to be established by using autologous activated lymphocytes. The low of regenerative medicine will promote the active development of ACT for public use, and ACTs that utilize tumor-infiltrating lymphocytes(TIL), in vitro tumor-sensitized lymphocytes, natural killer T cells, and gammadelta T cells are being evaluated as advanced medical treatments in Japan. In addition, chimeric antigen receptor genemodified T(CAR-T)cells and T cell receptor gene-modified T(TCR-T)cells are available for investigational clinical use. CART and TCR-T cells have been associated with serious adverse events as well as drastic clinical efficacies, indicating the importance of choosing the antigens to be targeted. Presently, it is accurate to state that lymphocytes do recognize cancer cells. Clinical ACT research focusing on TIL and mutated cancer antigens will be initiated for the development of personalized immunotherapy for cancer in the future. PMID:26469157

  17. Real-Time Imaging of Resident T Cells in Human Lung and Ovarian Carcinomas Reveals How Different Tumor Microenvironments Control T Lymphocyte Migration

    PubMed Central

    Bougherara, Houcine; Mansuet-Lupo, Audrey; Alifano, Marco; Ngô, Charlotte; Damotte, Diane; Le Frère-Belda, Marie-Aude; Donnadieu, Emmanuel; Peranzoni, Elisa

    2015-01-01

    T cells play a key role in the battle against cancer. To perform their antitumor activities, T cells need to adequately respond to tumor antigens by establishing contacts with either malignant cells or antigen-presenting cells. These latter functions rely on a series of migratory steps that go from entry of T cells into the tumor followed by their locomotion in the tumor stroma. Our knowledge of how T cells migrate within tumors mainly comes from experiments performed in mouse models. Whereas such systems have greatly advanced our understanding, they do not always faithfully recapitulate the disease observed in cancer patients. We previously described a technique based on tissue slices that enables to track with real-time imaging microscopy the motile behavior of fluorescent T cells plated onto fresh sections of human lung tumors. We have now refined this approach to monitor the locomotion of resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies. Using this approach, our findings reveal that CD8 T cells accumulate in the stroma of ovarian and lung carcinomas but move slowly in this compartment. Conversely, even though less populated, tumors islets were found to be zones of faster migration for resident CD8 T cells. We also confirm the key role played by collagen fibers, which, by their orientation, spacing and density, control the distribution and migration of resident CD8 T cells within the tumor stroma. We have subsequently demonstrated that, under some physical tissue constraints, CD8 T cells exhibited a mode of migration characterized by alternate forward and backward movements. In sum, using an ex vivo assay to track CD8 T cells in fresh human tumor tissues, we have identified the extracellular matrix as a major stromal component in influencing T cell migration, thereby impacting the control of tumor growth. This approach will aid in the development and testing of novel immunotherapy strategies to promote T cell migration in

  18. Comparative cytotoxicity and genotoxicity of cobalt (II, III) oxide, iron (III) oxide, silicon dioxide, and aluminum oxide nanoparticles on human lymphocytes in vitro.

    PubMed

    Rajiv, S; Jerobin, J; Saranya, V; Nainawat, M; Sharma, A; Makwana, P; Gayathri, C; Bharath, L; Singh, M; Kumar, M; Mukherjee, A; Chandrasekaran, N

    2016-02-01

    Despite the extensive use of nanoparticles (NPs) in various fields, adequate knowledge of human health risk and potential toxicity is still lacking. The human lymphocytes play a major role in the immune system, and it can alter the antioxidant level when exposed to NPs. Identification of the hazardous NPs was done using in vitro toxicity tests and this study mainly focuses on the comparative in vitro cytotoxicity and genotoxicity of four different NPs including cobalt (II, III) oxide (Co3O4), iron (III) oxide (Fe2O3), silicon dioxide (SiO2), and aluminum oxide (Al2O3) on human lymphocytes. The Co3O4 NPs showed decrease in cellular viability and increase in cell membrane damage followed by Fe2O3, SiO2, and Al2O3 NPs in a dose-dependent manner after 24 h of exposure to human lymphocytes. The oxidative stress was evidenced in human lymphocytes by the induction of reactive oxygen species, lipid peroxidation, and depletion of catalase, reduced glutathione, and superoxide dismutase. The Al2O3 NPs showed the least DNA damage when compared with all the other NPs. Chromosomal aberration was observed at 100 µg/ml when exposed to Co3O4 NPs and Fe2O3 NPs. The alteration in the level of antioxidant caused DNA damage and chromosomal aberration in human lymphocytes. PMID:25829403

  19. Fluorescent methods in the study of UV-induced changes in structural and functional state of human blood lymphocytes.

    PubMed

    Artyukhov, V G; Putintseva, O V; Vdovina, V A; Pashkov, M V; Vasilenko, D V

    2012-10-01

    Structural and functional state of human blood lymphocytes after exposure to UV light (240-390 nm) in doses of 151-1359 J/m(2) was studied by methods of laser flow cytofluorometry, indirect immunofluorescence, and fluorescent probes. Using a combination of these methods, we have showed that UV light in the specified doses induced changes in the surface phenotype of T cells: stimulation or suppression of the expression of antigen-recognizing receptor complex molecules (CD3, CD4, and CD8 markers) and their redistribution on the surface of immunocompetent cells (capping effect) with the formation of receptor clusters of various types. PMID:23113315

  20. Antibodies to Hepatitis B Surface Antigen Potentiate the Response of Human T Lymphocyte Clones to the Same Antigen

    NASA Astrophysics Data System (ADS)

    Celis, Esteban; Chang, Tse Wen

    1984-04-01

    Human T-helper lymphocyte clones specific for hepatitis B virus surface antigen (HBsAg) proliferate on stimulation with HBsAg in vitro. Antibodies specific for HBsAg, but no other antibodies, augment this proliferative response. In the presence of antibodies to HBsAg, the maximum response could be achieved at HBsAg concentrations that were 1 percent of those required in the absence of the antibodies. These findings suggest that antigen-specific antibodies exert regulatory controls on T cells that recognize the same antigens.

  1. Electron microscopic in situ hybridization and autoradiography: Localization and transcription of rDNA in human lymphocyte nucleoli

    SciTech Connect

    Wachtler, F.; Mosgoeller, W.S.; Schwarzacher, H.G. )

    1990-04-01

    The distribution of ribosomal DNA (rDNA) in the nucleoli of human lymphocytes was revealed by in situ hybridization with a nonautoradiographic procedure at the electron microscopic level. rDNA is located in the dense fibrillar component of the nucleolus but not in the fibrillar centers. In the same cells the incorporation of tritiated uridine takes place in the dense fibrillar component of the nucleolus as seen by autoradiography followed by gold latensification. From these findings it can be concluded that the transcription of ribosomal DNA takes place in the dense fibrillar component of the nucleolus.

  2. In vitro and occupational induction of sister-chromatid exchanges in human lymphocytes with furfuryl alcohol and furfural.

    PubMed

    Gomez-Arroyo, S; Souza, V

    1985-06-01

    Sister-chromatid exchanges (SCEs) in human lymphocytes were studied using the FPG technique in order to determine the cytogenetic effect of furfural and furfuryl alcohol. The induction of SCEs was also investigated in workers occupationally exposed to these solvents that are commonly used in the manufacture of furoic resins. The results obtained from the in vitro treatments show that furfural increased the number of SCEs, while furfuryl alcohol did not. In exposed workers, neither of these solvents increased the spontaneous frequency of SCEs per metaphase. PMID:4000179

  3. Effects of 8-aminoguanosine and 2'-deoxyguanosine on the human mixed lymphocyte reaction (MLR). I. Description of the inhibition

    SciTech Connect

    Scott, M.E.; Gilbertsen, R.B.

    1986-03-05

    Patients deficient in purine nucleoside phosphorylase (PNP) generally have a pronounced deficiency in T cell function with no deleterious effect on B cell function. It has been demonstrated that high concentrations of 8-amino-guanosine (8-AG), an inhibitor of PNP, in combination with the PNP substrate 2'-deoxyguanosine (dGuo) can inhibit mitogen and antigen responsiveness of human lymphocytes in culture. The studies described here evaluated the effects of 8-AG and dGuo on the human MLR. Normal human lymphocytes were isolated from venous blood using Ficoll-Hypaque gradients. Stimulator cells, pretreated with mitomycin C (50 ..mu..g/ml), were added with an equal number of responder cells (2 x 10/sup 5/) to the wells of flat-bottomed microtiter plates. Cell proliferation was quantitated by the uptake of /sup 3/H-thymidine (TdR) during the final 4 hrs of six-day cultures. dGuo at 10-50 ..mu..M had no effect or stimulated proliferation, while dGuo at > 75 ..mu..M was markedly inhibitory. 8-AG alone at 100 ..mu..M had essentially no effect on TdR uptake. 8-AG (10-100 ..mu..M) plus dGuo (50 ..mu..M) produced a concentration-dependent inhibition of the MLR resulting in 8-AG IC/sub 50/s generally < 60 ..mu..M. Coaddition of 50 ..mu..M 2'-deoxycytidine, hypoxanthine or adenine partially prevented inhibition of the MLR.

  4. Human T lymphocytes express N-methyl-D-aspartate receptors functionally active in controlling T cell activation

    SciTech Connect

    Miglio, Gianluca; Varsaldi, Federica; Lombardi, Grazia . E-mail: lombardi@pharm.unipmn.it

    2005-12-30

    The aim of this study was to investigate the expression and the functional role of N-methyl-D-aspartate (NMDA) receptors in human T cells. RT-PCR analysis showed that human resting peripheral blood lymphocytes (PBL) and Jurkat T cells express genes encoding for both NR1 and NR2B subunits: phytohemagglutinin (PHA)-activated PBL also expresses both these genes and the NR2A and NR2D genes. Cytofluorimetric analysis showed that NR1 expression increases as a consequence of PHA (10 {mu}g/ml) treatment. D-(-)-2-Amino-5-phosphonopentanoic acid (D-AP5), and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine [(+)-MK 801], competitive and non-competitive NMDA receptor antagonists, respectively, inhibited PHA-induced T cell proliferation, whereas they did not affect IL-2 (10 U/ml)-induced proliferation of PHA blasts. These effects were due to the prevention of T cell activation (inhibition of cell aggregate formation and CD25 expression), but not to cell cycle arrest or death. These results demonstrate that human T lymphocytes express NMDA receptors, which are functionally active in controlling cell activation.

  5. The influence of some vegetable extracts on the in vitro adherence of mouse and human lymphocytes to nylon fibers.

    PubMed

    Lenghel, V; Radu, D L; Chirilă, P; Olinescu, A

    1995-01-01

    We found that the total watery extracts obtained from roots of various plants such as Symphytum officinale, Phytolacca americana etc, precipitate human glycoproteins, agglutinate sheep red blood cells (SRBC) and stimulate lymphocyte adherence to nylon fibers. Five out of seven extracts precipitated human gammaglobulins and one of seven obviously agglutinate SRBC. If these cells were pretreated with rabbit antibodies against SRBC, all extracts agglutinated the cells at various degrees of intensity, the most active being Phytolacca americana. The adherence of mouse but not human lymphocytes to nylon fibers was stimulated by extracts of Symphytum officinale and Phytolacca americana. This process was neither stimulated nor inhibited by Mannose (Man), Galactose (Gal), Glucose (Glc), N-acethyl Galactose (GalNAc) and N-acethyl Glucose (Glc-NAc). These biological effects of the plant extracts could be the expression of a lectin-like ability to bind various sugars other than those mentioned. The results suggest the possibility of using different extracts as means to point out the presence in serum or at the cellular level of some carbohydrates influencing the cellular adhesion, phenomenon which plays an important role in the functions of hematopoietic cells. PMID:8993111

  6. Cell-to-cell distances between tumor-infiltrating inflammatory cells have the potential to distinguish functionally active from suppressed inflammatory cells.

    PubMed

    Nagl, S; Haas, M; Lahmer, G; Büttner-Herold, M; Grabenbauer, G G; Fietkau, R; Distel, L V

    2016-05-01

    Beyond their mere presence, the distribution pattern of inflammatory cells is of special interest. Our hypothesis was that random distribution may be a clear indicator of being non-functional as a consequence of lack of interaction. Here, we have assessed the implication of cell-to-cell distances among inflammatory cells in anal squamous cell carcinoma and a possible association with survival data. Thirty-eight patients suffering from anal carcinoma were studied using tissue microarrays, double staining immunohistochemistry, whole slide scanning and image analysis software. Therapy consisted of concurrent radiochemotherapy. Numbers of stromal and intraepithelial tumor-infiltrating inflammatory cells (TIC) and the distances between cells were quantified. Double-staining of FoxP3(+) cells with either CD8(+), CD1a(+) or CD20(+) cells was performed. Measured cell-to-cell distances were compared to computer simulated cell-to-cell distances leading to the assumption of non-randomly distributed and therefore functional immune cells. Intraepithelial CD1a(+) and CD20(+) cells were randomly distributed and therefore regarded as non-functional. In contrary, stromal CD20(+) cells had a non-random distribution pattern. A non-random distance between CD20(+) and FoxP3(+) cells was associated with a clearly unfavorable outcome. Measured distances between FoxP3(+) cells were distinctly shorter than expected and indicate a functional active state of the regulatory T cells (Treg). Analysis of cell-to-cell distances between TIC has the potential to distinguish between suppressed non-functional and functionally active inflammatory cells. We conclude that in this tumor model most of the CD1a(+) cells are non-functional as are the intraepithelial CD20(+) cells, while stromal CD20(+) cells and FoxP3(+) cells are functional cells. PMID:27467940

  7. Methamphetamine induces trace amine-associated receptor 1 (TAAR1) expression in human T lymphocytes: role in immunomodulation.

    PubMed

    Sriram, Uma; Cenna, Jonathan M; Haldar, Bijayesh; Fernandes, Nicole C; Razmpour, Roshanak; Fan, Shongshan; Ramirez, Servio H; Potula, Raghava

    2016-01-01

    The novel transmembrane G protein-coupled receptor, trace amine-associated receptor 1 (TAAR1), represents a potential, direct target for drugs of abuse and monoaminergic compounds, including amphetamines. For the first time, our studies have illustrated that there is an induction of TAAR1 mRNA expression in resting T lymphocytes in response to methamphetamine. Methamphetamine treatment for 6 h significantly increased TAAR1 mRNA expression (P < 0.001) and protein expression (P < 0.01) at 24 h. With the use of TAAR1 gene silencing, we demonstrate that methamphetamine-induced cAMP, a classic response to methamphetamine stimulation, is regulated via TAAR1. We also show by TAAR1 knockdown that the down-regulation of IL-2 in T cells by methamphetamine, which we reported earlier, is indeed regulated by TAAR1. Our results also show the presence of TAAR1 in human lymph nodes from HIV-1-infected patients, with or without a history of methamphetamine abuse. TAAR1 expression on lymphocytes was largely in the paracortical lymphoid area of the lymph nodes with enhanced expression in lymph nodes of HIV-1-infected methamphetamine abusers rather than infected-only subjects. In vitro analysis of HIV-1 infection of human PBMCs revealed increased TAAR1 expression in the presence of methamphetamine. In summary, the ability of methamphetamine to activate trace TAAR1 in vitro and to regulate important T cell functions, such as cAMP activation and IL-2 production; the expression of TAAR1 in T lymphocytes in peripheral lymphoid organs, such as lymph nodes; and our in vitro HIV-1 infection model in PBMCs suggests that TAAR1 may play an important role in methamphetamine -mediated immune-modulatory responses. PMID:26302754

  8. Acetaminophen induces a caspase-dependent and Bcl-XL sensitive apoptosis in human hepatoma cells and lymphocytes.

    PubMed

    Boulares, A Hamid; Zoltoski, Anna J; Stoica, Bogdan A; Cuvillier, Olivier; Smulson, Mark E

    2002-01-01

    Acetaminophen is a widely used analgesic and antipyretic drug that exhibits toxicity at high doses to the liver and kidneys. This toxicity has been attributed to cytochrome P-450-generated metabolites which covalently modify target proteins. Recently, acetaminophen, in its unmetabolized form, has been shown to affect a variety of cells and tissues, for instance, testicular and lymphoid tissues and lymphocyte cell lines. The effects on cell viability of acetaminophen at a concentration comparable to that achieved in plasma during acetaminophen toxicity have now been examined with a hepatoma cell line SK-Hep1, primary human peripheral blood lymphocytes and human Jurkat T cells. Acetaminophen reduced cell viability in a time-dependent manner. Staining of cells with annexin-V also revealed that acetaminophen induced, after 8 hr of treatment, a loss of the asymmetry of membrane phospholipids, which is an early event associated with apoptosis. Acetaminophen triggered the release of cytochrome c from mitochondria into the cytosol, activation of caspase-3, 8, and 9, cleavage of poly(ADP-ribose) polymerase, and degradation of lamin B1 and DNA. Whereas cleavage of DNA into internucleosomal fragments was apparent in acetaminophen treated SK-Hep1 and primary lymphocytes, DNA was only degraded to 50-kb fragments in treated Jurkat cells. Overexpression of the antiapoptotic protein Bcl-XL prevented these various apoptotic events induced by acetaminophen in Jurkat cells. Caspase-8 activation was a postmictochondrial event and occurred in a Fas-independent manner. These results demonstrate that acetaminophen induces caspases-dependent apoptosis with mitochondria as a primary target. These results also reiterate the potential role of apoptosis in acetaminophen hepatic and extrahepatic toxicity. PMID:12005112

  9. Effects of human immunodeficiency virus type 1 on CD4 lymphocyte subset activation.

    PubMed

    Masci, A M; Paz, F L; Borriello, A; Cassano, S; Della Pietra, V; Stoiber, H; Matarese, G; Della Ragione, F; Zappacosta, S; Racioppi, L

    1999-06-01

    The pathogenesis of the decline of CD4 lymphocyte counts accompanying the typical course of HIV-1 infection is not completely defined and might be related to a differential susceptibility of naive and memory cells to HIV-1 exposure. Here, we examined the effects induced by heat-inactivated HIV-1 virions on these lymphocyte populations. Exposure of CD45RA naive T cells to inactivated viral particles induced a marked decrease of both mitogenic responses and activation-induced apoptosis. Conversely, the growth of CD45RO cells was less severely restrained. Analysis of intracellular levels of cell cycle regulatory proteins revealed an arrest at the G1/S restriction point of the naive but not memory subset. This effect was associated with alterations in phosphotyrosine profile and with a marked decrease of ERK and NJK kinase activation. Finally, up-regulation of the cAMP-dependent protein kinase A (PKA) activity induced by mitogens was not affected by virus. Altogether, these findings show that interaction of HIV-1 with the T cell surface is sufficient to inhibit the proliferative response of the CD4CD45RA subset by disturbing proximal TCR signaling. This mechanism would affect renewal of naive lymphocytes, contributing in such a way to the impairment of T cell turnover during the course of HIV-1 infection. PMID:10382750

  10. Fluorometric determination of 2'-beta-fluoro-2',3'-dideoxyadenosine 5'-triphosphate, the active metabolite of a new anti-human immunodeficiency virus drug, in human lymphocytes.

    PubMed

    Dai, F; Kelley, J A; Zhang, H; Malinowski, N; Kavlick, M F; Lietzau, J; Welles, L; Yarchoan, R; Ford, H

    2001-01-01

    A sensitive precolumn derivatization method has been developed to measure the 5'-triphosphate of 2'-beta-fluoro-2',3'-dideoxyadenosine (F-ddA, lodenosine), a new anti-HIV drug, in human lymphocytes by HPLC using fluorescence detection. Reaction of chloroacetaldehyde with F-ddA triphosphate in extracts from human lymphocytes produces a highly fluorescent etheno adduct. This derivative is then separated and quantitated by reverse-phase paired-ion chromatography. Degradation of natural nucleic acid ribosides, such as ATP, using periodate oxidation simplifies the chromatogram and minimizes interference with detection of the target analyte. This method, modeled using cultured MOLT-4 T-lymphocytes, achieves a linear detector response for peak area measurements over the range 2.5 to 22.5 pmol (50-450 nM using 50 microl sample). Analyte recovery is greater than 90%, and the method achieves a limit of detection and limit of quantitation of 1.4 and 2.5 pmol per HPLC injection (50 microl sample containing cellular extract from 2.5 x 10(6) cells), respectively. Application of this method to measure F-ddATP in peripheral blood mononuclear cells from HIV-infected patients treated with F-ddA at 3.2 mg/kg twice daily for 22 days shows F-ddATP levels which range from 1.5 to 3.5 pmol/10(6) cells. PMID:11141306

  11. "Up-dating the monograph." [corrected] Cytolytic immune lymphocytes in the armamentarium of the human host.

    PubMed

    Sinkovics, J G

    2008-12-01

    The author of the monograph "Cytolytic Immune Lymphocytes..." (published in 2008 by Schenk Buchverlag Campus Dialog, Budapest, Passau, Pécs) proposed several research projects and described certain clinical events that require further elaboration and documentation. In this article the author provides what is required and has since become available. The first subject matter in question concerns the fusogenic viruses. The ancient fusogenic viruses might have created the first eukaryotic cell(s) by uniting archaeabacterial and prokaryotic/protobacterial protospheroplasts. Extant fusogenic viruses either produce tumor cell syncytia and lyse them, thus practicing viral oncolysis. Or, create chimaeric fusion products, the so-called "natural hybridomas", of lymphoma cells exhibiting transmembrane budding of retrovirus particles or envelope proteins, and anti-viral specific antibody-producing plasma cells. The second topic concerns the horizontal-lateral mode of acquisition of those genes, which were "present in the waiting" in the amphioxus, sea urchin, and the agnathans, and met in the primitive gnatostomata sharks to encode in unison the entire adaptive immune system. The consensus of opinion is such that these genes derived from newly acquired transposons/retrotransposons. The author points out that the extant Epstein-Barr virus harbors genes displaying sequence homology with those genes from the sharks up to mammals that regulate the somatic hypermutation of specific antibody production. The author proposes that an ancient herpesvirus might have propagated the V(D)J and RAG genes from sea urchins to sharks. The third area is that of lymphocytes cytotoxic/cytolytic to virally infected or malignantly transformed host cells. This discovery led to the adoptive immune lymphocyte therapy of tumors. Installed in the adaptive immune system are regulatory T cells and myeloid-derived suppressor cells for he protection of "self". Tumor cells masquerading as "self" are protected

  12. Targeting miR-23a in CD8+ cytotoxic T lymphocytes prevents tumor-dependent immunosuppression

    PubMed Central

    Lin, Regina; Chen, Ling; Chen, Gang; Hu, Chunyan; Jiang, Shan; Sevilla, Jose; Wan, Ying; Sampson, John H.; Zhu, Bo; Li, Qi-Jing

    2014-01-01

    CD8+ cytotoxic T lymphocytes (CTLs) have potent antitumor activity and therefore are leading candidates for use in tumor immunotherapy. The application of CTLs for clinical use has been limited by the susceptibility of ex vivo–expanded CTLs to become dysfunctional in response to immunosuppressive microenvironments. Here, we developed a microRNA-targeting (miRNA-targeting) approach that augments CTL cytotoxicity and preserves immunocompetence. Specifically, we screened for miRNAs that modulate cytotoxicity and identified miR-23a as a strong functional repressor of the transcription factor BLIMP-1, which promotes CTL cytotoxicity and effector cell differentiation. In a cohort of advanced lung cancer patients, miR-23a was upregulated in tumor-infiltrating CTLs, and expression correlated with impaired antitumor potential of patient CTLs. We determined that tumor-derived TGF-β directly suppresses CTL immune function by elevating miR-23a and downregulating BLIMP-1. Functional blocking of miR-23a in human CTLs enhanced granzyme B expression, and in mice with established tumors, immunotherapy with just a small number of tumor-specific CTLs in which miR-23a was inhibited robustly hindered tumor progression. Together, our findings provide a miRNA-based strategy that subverts the immunosuppression of CTLs that is often observed during adoptive cell transfer tumor immunotherapy and identify a TGF-β–mediated tumor immune-evasion pathway. PMID:25347474

  13. Synergy between anti-CD40 MAb and Epstein-Barr virus in activation and transformation of human B lymphocytes.

    PubMed

    Tsuchiyama, L; Kieran, J; Boyle, P; Wetzel, G D

    1997-01-01

    For human B lymphocytes, Epstein-Barr virus (EBV) is a polyclonal activator, inducing both proliferation and Ig secretion. It is also a transforming virus capable of generating immortalized B cell lines. These early and late functions of EBV are not apparently connected. The receptor for EBV, CD21, also serves as a receptor for some complement components and is called CR2. This molecule associates with CD19 and TAPA-1 on the surface of B cells. This complex is involved in signaling B cells and participates in many responses. We have observed that simultaneous ligation of CD40 and the CD21 complex, by exposure to anti-CD40 MAbs and EBV, enhances both the short-term proliferation as well as the long-term transformation rate of human B lymphocytes. B cell proliferation shows synergy between anti-CD40 MAb and EBV. CD19 also appears to be involved in the synergistic activation of B cells through CD40 and CD21, since ligation of CD19 with anti-CD19 MAbs, either prior to or concomitant with exposure to anti-CD40 and EBV, markedly inhibits both proliferation and subsequent B cell transformation. These observations do not elucidate the mechanisms of B cell transformation employed by EBV but the do suggest a relationship between early proliferation and later transformation induced by the virus. Anti-CD40 enhances both these effects and anti-CD19 is capable of inhibiting both. PMID:9265505

  14. Influence of dehydroepiandrosterone on G-6-PD activity and /sup 3/H-thymidine uptake of human lymphocytes in vitro

    SciTech Connect

    Ennas, M.G.; Laconi, S.; Dessi, S.; Milia, G.; Murru, M.R.; Manconi, P.E.

    1987-01-01

    Dehydroepiandrosterone (DHEA) was found to inhibit experimental cancer development in mouse and rat lung, colon and mammary gland. Since DHEA is a potent inhibitor of mammalian G-6-PD, the hypothesis that the compound could inhibit cell proliferation through an inhibition of the pentose phosphate pathway has been formulated. We studied the effects of DHEA on the proliferation in vitro of human lymphocytes induced by several mitogens (PHA, ConA and PWM), measuring /sup 3/H-thymidine uptake. DHEA inhibited /sup 3/H-thymidine uptake of mitogen-stimulated cells from both G-6-PD+ and G-6-PD- (mediterranean type deficiency) individuals in a dose-dependent and reversible fashion. The inhibitory effect was found even if DHEA was added to cells in the last hours of culture, simultaneously with the addition of /sup 3/H-thymidine. These data suggest that the inhibition of thymidine uptake induced by DHEA on human lymphocytes probably does not depend on the inhibition of G-6-PD.

  15. Jatropha curcas leaf and bark fractions protect against ultraviolet radiation-B induced DNA damage in human peripheral blood lymphocytes.

    PubMed

    Sundari, J; Selvaraj, R; Rajendra Prasad, N; Elumalai, R

    2013-11-01

    The present study is conducted to investigate the antioxidant potential of Jatropha curcas root bark extract (RB4 fraction) and leaf extract (L1 fraction), and to study their effects on UVB-radiation-induced DNA damage in cultured human blood lymphocytes. In this study, J. curcas showed strong antioxidant property in different free radical scavenging systems. Both the fractions effectively scavenged hydroxyl (OH), superoxide anion (O₂(·-)), 1,1-diphenyl-2-picrylhydrazyl (DPPH·) and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid radical cation (ABTS(·+)) in a concentration-dependent manner. The IC₅₀ (Inhibitory Concentration 50) values of J. curcas fractions were compared to standard ascorbic acid used in this study. The antioxidant potential of a compound was directly proportional to the photoprotective effect. In this study, human peripheral blood lymphocytes (HPBL) were exposed to UVB-radiation and there was an increase in comet attributes (% tail DNA, tail length, tail movement and Olive tail moment). Jatropha curcas RB4 fraction and L1 fraction treatment before UVB-irradiation significantly decreased the % tail DNA, tail length, tail moment and Olive tail moment in irradiated HPBL. These results suggested that J. curcas exhibited strong antioxidant property and RB4 and L1 fractions protected UVB-radiation-induced DNA damage in HPBL. PMID:23995553

  16. Cytogenetic analyses of Azadirachtin reveal absence of genotoxicity but marked antiproliferative effects in human lymphocytes and CHO cells in vitro.

    PubMed

    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. PMID:22885097

  17. Induction and prevention of micronuclei and chromosomal aberrations in cultured human lymphocytes exposed to the light of halogen tungsten lamps.

    PubMed

    D'Agostini, F; Caimo, A; De Filippi, S; De Flora, S

    1999-07-01

    Previous studies have shown that the light emitted by halogen tungsten lamps contains UV radiation in the UV-A, UV-B and UV-C regions, induces mutations and irreparable DNA damage in bacteria, enhances the frequency of micronuclei in cultured human lymphocytes and is potently carcinogenic to the skin of hairless mice. The present study showed that the light emitted by an uncovered, traditional halogen lamp induces a significant, dose-related and time-related increase not only in micronuclei but also in chromosome-type aberrations, such as breaks, and even more in chromatid-type aberrations, such as isochromatid breaks, exchanges and isochromatid/chromatid interchanges, all including gaps or not, in cultured human lymphocytes. All these genotoxic effects were completely prevented by shielding the same lamp with a silica glass cover, blocking UV radiation. A new model of halogen lamp, having the quartz bulb treated in order to reduce the output of UV radiation, was considerably less genotoxic than the uncovered halogen lamp, yet induction of chromosomal alterations was observed at high illuminance levels. PMID:10390512

  18. Activities of the four optical isomers of 2',3'-dideoxy-3'-thiacytidine (BCH-189) against human immunodeficiency virus type 1 in human lymphocytes.

    PubMed Central

    Schinazi, R F; Chu, C K; Peck, A; McMillan, A; Mathis, R; Cannon, D; Jeong, L S; Beach, J W; Choi, W B; Yeola, S

    1992-01-01

    Four different isomers of 2',3'-dideoxy-3'-thiacytidine [beta-DL-(+-)-BCH-189] were evaluated in primary human lymphocytes infected with human immunodeficiency virus type 1. The beta-L-(-) isomer was the most potent enantiomer, with a median effective concentration of 1.8 nM and no discernible cytotoxicity up to 100 microM. The relative order of potencies for the isomers was beta-L-(-) greater than beta-DL-(+-) racemic greater than beta-D-(+) greater than alpha-L-(+) greater than alpha-D-(-). The beta-L-(-) enantiomer was as potent as 3'-azido-3'-deoxythymidine. PMID:1320365

  19. Nivolumab and Urelumab Enhance Antitumor Activity of Human T Lymphocytes Engrafted in Rag2-/-IL2Rγnull Immunodeficient Mice.

    PubMed

    Sanmamed, Miguel F; Rodriguez, Inmaculada; Schalper, Kurt A; Oñate, Carmen; Azpilikueta, Arantza; Rodriguez-Ruiz, Maria E; Morales-Kastresana, Aizea; Labiano, Sara; Pérez-Gracia, Jose L; Martín-Algarra, Salvador; Alfaro, Carlos; Mazzolini, Guillermo; Sarno, Francesca; Hidalgo, Manuel; Korman, Alan J; Jure-Kunkel, Maria; Melero, Ignacio

    2015-09-01

    A current pressing need in cancer immunology is the development of preclinical model systems that are immunocompetent for the study of human tumors. Here, we report the development of a humanized murine model that can be used to analyze the pharmacodynamics and antitumor properties of immunostimulatory monoclonal antibodies (mAb) in settings where the receptors targeted by the mAbs are expressed. Human lymphocytes transferred into immunodeficient mice underwent activation and redistribution to murine organs, where they exhibited cell-surface expression of hCD137 and hPD-1. Systemic lymphocyte infiltrations resulted in a lethal CD4(+) T cell-mediated disease (xenograft-versus-host disease), which was aggravated when murine subjects were administered clinical-grade anti-hCD137 (urelumab) and anti-hPD-1 (nivolumab). In mice engrafted with human colorectal HT-29 carcinoma cells and allogeneic human peripheral blood mononuclear cells (PBMC), or with a patient-derived gastric carcinoma and PBMCs from the same patient, we found that coadministration of urelumab and nivolumab was sufficient to significantly slow tumor growth. Correlated with this result were increased numbers of activated human T lymphocytes producing IFNγ and decreased numbers of human regulatory T lymphocytes in the tumor xenografts, possibly explaining the efficacy of the therapeutic regimen. Our results offer a proof of concept for the use of humanized mouse models for surrogate efficacy and histology investigations of immune checkpoint drugs and their combinations. PMID:26113085

  20. Alpharetroviral self-inactivating vectors produced by a superinfection-resistant stable packaging cell line allow genetic modification of primary human T lymphocytes.

    PubMed

    Labenski, Verena; Suerth, Julia D; Barczak, Elke; Heckl, Dirk; Levy, Camille; Bernadin, Ornellie; Charpentier, Emmanuelle; Williams, David A; Fehse, Boris; Verhoeyen, Els; Schambach, Axel

    2016-08-01

    Primary human T lymphocytes represent an important cell population for adoptive immunotherapies, including chimeric-antigen and T-cell receptor applications, as they have the capability to eliminate non-self, virus-infected and tumor cells. Given the increasing numbers of clinical immunotherapy applications, the development of an optimal vector platform for genetic T lymphocyte engineering, which allows cost-effective high-quality vector productions, remains a critical goal. Alpharetroviral self-inactivating vectors (ARV) have several advantages compared to other vector platforms, including a more random genomic integration pattern and reduced likelihood for inducing aberrant splicing of integrated proviruses. We developed an ARV platform for the transduction of primary human T lymphocytes. We demonstrated functional transgene transfer using the clinically relevant herpes-simplex-virus thymidine kinase variant TK.007. Proof-of-concept of alpharetroviral-mediated T-lymphocyte engineering was shown in vitro and in a humanized transplantation model in vivo. Furthermore, we established a stable, human alpharetroviral packaging cell line in which we deleted the entry receptor (SLC1A5) for RD114/TR-pseudotyped ARVs to prevent superinfection and enhance genomic integrity of the packaging cell line and viral particles. We showed that superinfection can be entirely prevented, while maintaining high recombinant virus titers. Taken together, this resulted in an improved production platform representing an economic strategy for translating the promising features of ARVs for therapeutic T-lymphocyte engineering. PMID:27162078

  1. Biochemical and molecular evidences on the protection by magnesium oxide nanoparticles of chlorpyrifos-induced apoptosis in human lymphocytes

    PubMed Central

    Heydary, Vida; Navaei-Nigjeh, Mona; Rahimifard, Mahban; Mohammadirad, Azadeh; Baeeri, Maryam; Abdollahi, Mohammad

    2015-01-01

    Background: Chlorpyrifos (CP) is one of the most widely used organophosphate (OP) in