Sample records for lymphocyte cell line

  1. Differential expression of alkaline phosphatase gene in proliferating primary lymphocytes and malignant lymphoid cell lines. (United States)

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


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

  2. Clinical features of familial adenomas polyps in Chinese and establishment of its immortal lymphocyte cell lines

    Institute of Scientific and Technical Information of China (English)

    Shan-Rong Cai; Su-Zhang Zhang; Shu Zheng


    AIM:To reserve the rare Chinese familial adenomas polyp (FAP) family resource and to investigate the clinical features of FAP in Chinese for its diagnosis.METHODS: Clinical features of patients with FAP were investigated. If there is any question, their medical records were verified. Blood sample was taken and lymphocyte immortal cell lines were established with modified EB-transformation methods. Congenital hypertrophy of retinal pigment epithelium (CHRPE) was checked by an experienced ophthalmologist.RESULTS: Twenty seven families including 21 classical FAP (CFAP) families, 3 attenuated FAP (AFAP) families,and 3 suspected AFAP families were investigated. A total of 116 lymphocyte immortal cell lines were established from 26 families. In all the FAP families, colorectal cancer occurred at the mean age of 42.84 years. Of the 16 families checked, 15 (93.75%) had CHRPE. The mean number of patients suffering from colorectal neoplasm was 3.14 in CFAP families and 2.0 in AFAP families (P < 0.01). The mean oldest age at diagnosis of FAP was 41.75 years in CFAP families, and 58.67 years in AFAP families, respectively (P < 0.01). Mean age of development of colorectal cancer was 42.23 in CFAP and 57.33 years old in AFAP (P < 0.01). Mean of the earliest age at diagnosis of FAP was 29.95 years in the FAP families with a positive family history and 46.80 years in the FAP families with a negative family history (P <0.01). The ratio of extra-intestinal tumors to colorectal neoplasms was different in the two kinds of families with positive and negative family history (P < 0.01).CONCLUSION: Additional use of ciclosporin will effectively improve to establish lymphocyte immortal cell lines with modified EB- transformation methods. In Chinese FAP, there was a high frequency of CHRPE, and a later age at diagnosis and a later age of development of colorectal cancer in AFAP. And earlier age at diagnosis in FAP with positive family history was also found that will help to

  3. DNAM-1 mediates epithelial cell-specific cytotoxicity of aberrant intraepithelial lymphocyte lines from refractory celiac disease type II patients. (United States)

    Tjon, Jennifer M-L; Kooy-Winkelaar, Yvonne M C; Tack, Greetje J; Mommaas, A Mieke; Schreurs, Marco W J; Schilham, Marco W; Mulder, Chris J; van Bergen, Jeroen; Koning, Frits


    In refractory celiac disease (RCD), intestinal epithelial damage persists despite a gluten-free diet. Characteristic for RCD type II (RCD II) is the presence of aberrant surface TCR-CD3(-) intraepithelial lymphocytes (IELs) that can progressively replace normal IELs and eventually give rise to overt lymphoma. Therefore, RCD II is considered a malignant condition that forms an intermediate stage between celiac disease (CD) and overt lymphoma. We demonstrate in this study that surface TCR-CD3(-) IEL lines isolated from three RCD II patients preferentially lyse epithelial cell lines. FACS analysis revealed that DNAM-1 was strongly expressed on the three RCD cell lines, whereas other activating NK cell receptors were not expressed on all three RCD cell lines. Consistent with this finding, cytotoxicity of the RCD cell lines was mediated mainly by DNAM-1 with only a minor role for other activating NK cell receptors. Furthermore, enterocytes isolated from duodenal biopsies expressed DNAM-1 ligands and were lysed by the RCD cell lines ex vivo. Although DNAM-1 on CD8(+) T cells and NK cells is known to mediate lysis of tumor cells, this study provides, to our knowledge, the first evidence that (pre)malignant cells themselves can acquire the ability to lyse epithelial cells via DNAM-1. This study confirms previous work on epithelial lysis by RCD cell lines and identifies a novel mechanism that potentially contributes to the gluten-independent tissue damage in RCD II and RCD-associated lymphoma.

  4. Characterization of a new chronic lymphocytic leukemia cell line for mechanistic in vitro and in vivo studies relevant to disease.

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    Erin Hertlein

    Full Text Available Studies of chronic lymphocytic leukemia (CLL have yielded substantial progress, however a lack of immortalized cell lines representative of the primary disease has hampered a full understanding of disease pathogenesis and development of new treatments. Here we describe a novel CLL cell line (OSU-CLL generated by EBV transformation, which displays a similar cytogenetic and immunophenotype observed in the patient's CLL (CD5 positive with trisomy 12 and 19. A companion cell line was also generated from the same patient (OSU-NB. This cell line lacked typical CLL characteristics, and is likely derived from the patient's normal B cells. In vitro migration assays demonstrated that OSU-CLL exhibits migratory properties similar to primary CLL cells whereas OSU-NB has significantly reduced ability to migrate spontaneously or towards chemokine. Microarray analysis demonstrated distinct gene expression patterns in the two cell lines, including genes on chromosomes 12 and 19, which is consistent with the cytogenetic profile in this cell line. Finally, OSU-CLL was readily transplantable into NOG mice, producing uniform engraftment by three weeks with leukemic cells detectable in the peripheral blood spleen and bone marrow. These studies describe a new CLL cell line that extends currently available models to study gene function in this disease.

  5. Molecular characterization of neoplastic and normal "sister" lymphoblastoid B-cell lines from chronic lymphocytic leukemia

    DEFF Research Database (Denmark)

    Lanemo Myhrinder, Anna; Hellqvist, Eva; Bergh, Ann-Charlotte;


    /short tandem repeat (STR) fingerprinting. Innate B-cell features, i.e. natural Ab production and CD5 receptors, were present in most CLL cell lines, but in none of the normal LCLs. This panel of immortalized CLL-derived cell lines is a valuable reference representing a renewable source of authentic Abs and DNA....

  6. Combined Treatment with Low Concentrations of Decitabine and SAHA Causes Cell Death in Leukemic Cell Lines but Not in Normal Peripheral Blood Lymphocytes

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    Barbora Brodská


    Full Text Available Epigenetic therapy reverting aberrant acetylation or methylation offers the possibility to target preferentially tumor cells and to preserve normal cells. Combination epigenetic therapy may further improve the effect of individual drugs. We investigated combined action of demethylating agent decitabine and histone deacetylase inhibitor SAHA (Vorinostat on different leukemic cell lines in comparison with peripheral blood lymphocytes. Large decrease of viability, as well as huge p21WAF1 induction, reactive oxygen species formation, and apoptotic features due to combined decitabine and SAHA action were detected in leukemic cell lines irrespective of their p53 status, while essentially no effect was observed in response to the combined drug action in normal peripheral blood lymphocytes of healthy donors. p53-dependent apoptotic pathway was demonstrated to participate in the wtp53 CML-T1 leukemic cell line response, while significant influence of reactive oxygen species on viability decrease has been detected in p53-null HL-60 cell line.

  7. Zoledronic acid enhances Vδ2 T-lymphocyte antitumor response to human glioma cell lines. (United States)

    Cimini, E; Piacentini, P; Sacchi, A; Gioia, C; Leone, S; Lauro, G M; Martini, F; Agrati, C


    Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain tumor in humans, responds modestly to treatment: most patients survive less than one year after diagnosis, despite both classical and innovative treatment approaches. A recent paper focused on γδ T-cell response in GBM patients, suggesting the application of an immunomodulating strategy based on γδ T-cells which is already in clinical trials for other tumors. Human Vγ2 T-cells recognize changes in the mevalonate metabolic pathway of transformed cells by activating cytotoxic response, and by cytokine and chemokine release. Interestingly, this activation may also be induced in vivo by drugs, such as zoledronic acid, that induce the accumulation of Vγ2 T-cell ligand Isopentenyl-pyrophosphate by blocking the farnesyl pyrophosphate synthase enzyme. The aim of our work is to confirm whether bisphosphonate treatment would make glioma cell lines more susceptible to lysis by in vitro expanded γδ T-cells, improving their antitumor activity. We expanded in vitro human Vγ2 T-cells by phosphoantigen stimulation and tested their activity against glioma cell lines. Co-culture with glioma cells induced Vγ2 T-cell differentiation in effector/memory cells, killing glioma cells by the release of perforin. Interestingly, glioma cells were directly affected by zoledronic acid; moreover, treatment increased their activating ability on Vγ2 T-cells, inducing an effective antitumor cytotoxic response. Taken together, our results show that aminobisphosphonate drugs may play a dual role against GBM, by directly affecting tumor cells, and by enhancing the antitumor response of Vγ2 T-cells. Our results confirm the practicability of this approach as a new immunotherapeutic strategy for GBM treatment.

  8. Pulse and Trapezoidal Voltage Clamp Applied To Jurkat Cells: A T- Lymphocyte Cell Line (United States)


    clamp. (Upward deflection represents depolarization). Low frequency oscillations are clearly visible. Such oscillations have been described by Maltsev ...1952. (8) Maltsev , V. A. Oscillating and triggering properties of T cell membrane potential. Imun. Lett. 26:277-282, 1990. 13 (9) Gallin, E. K

  9. Individual radiosensitivity does not correlate with radiation-induced apoptosis in lymphoblastoid cell lines or CD{sup 3+} lymphocytes

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    Wistop, A.; Keller, U.; Grabenbauer, G.G.; Sauer, R.; Distel, L.V.R. [Dept. of Radiation Oncology, Friedrich Alexander Univ. Erlangen-Nuremberg, Erlangen (Germany); Sprung, C.N. [Div. of Research, Peter MacCallum Cancer Centre, East Melbourne, VIC (Australia)


    Background and purpose: spontaneous and radiation-induced apoptosis of lymphoblastoid cell lines (LCLs) derived from healthy donors, cancer patients and donors with radiosensitivity syndromes as well as CD{sup 3+} lymphocytes from patients with {>=} grade 3 late toxicity were investigated as a possible marker for the detection of individual radiosensitivity. These investigations are based on the hypothesis that hypersensitive patients have reduced levels of apoptosis after in vitro irradiation as a result of a defect in the signaling pathway. Material and methods: Epstein-Barr virus-(EBV-)transformed LCLs derived from five healthy donors, seven patients with heterozygous or homozygous genotype for ataxia-telangiectasia or Nijmegen breakage syndrome and five patients with {>=} grade 3 late toxicity (RTOG) were investigated. In addition, CD{sup 3+} lymphocytes from 21 healthy individuals and 18 cancer patients including five patients with a proven cellular hypersensitivity to radiation were analyzed. Cells were irradiated in vitro with a dose of 2 and 5 Gy and were incubated for 48 h. Apoptotic rates were measured by the TUNEL assay followed by customized image analysis. Results: four out of seven radiosensitivity syndrome patients were identified to have an increased cellular radiosensitivity as determined by reduced apoptotic rates after irradiation of their respective LCLs. Comparatively, only two of the five hypersensitive cancer patients were clearly identified by reduced apoptotic rates. Spontaneous apoptotic rates were very homogeneous among all 39 samples from controls and patients, while lymphocytes of all cancer patients showed significantly lower radiation-induced rates. Conclusion: only a subgroup of hypersensitive patients may be identified by reduction of radiation-induced apoptotic rate. It is concluded that the hypothesis according to which hypersensitive cells have reduced levels of apoptosis is only conditionally true. The authors suggest that this

  10. Malt1-dependent RelB cleavage promotes canonical NF-kappaB activation in lymphocytes and lymphoma cell lines. (United States)

    Hailfinger, Stephan; Nogai, Hendrik; Pelzer, Christiane; Jaworski, Maike; Cabalzar, Katrin; Charton, Jean-Enno; Guzzardi, Montserrat; Décaillet, Chantal; Grau, Michael; Dörken, Bernd; Lenz, Peter; Lenz, Georg; Thome, Margot


    The protease activity of the paracaspase Malt1 contributes to antigen receptor-mediated lymphocyte activation and lymphomagenesis. Malt1 activity is required for optimal NF-κB activation, but little is known about the responsible substrate(s). Here we report that Malt1 cleaved the NF-κB family member RelB after Arg-85. RelB cleavage induced its proteasomal degradation and specifically controlled DNA binding of RelA- or c-Rel-containing NF-κB complexes. Overexpression of RelB inhibited expression of canonical NF-κB target genes and led to impaired survival of diffuse large B-cell lymphoma cell lines characterized by constitutive Malt1 activity. These findings identify a central role for Malt1-dependent RelB cleavage in canonical NF-κB activation and thereby provide a rationale for the targeting of Malt1 in immunomodulation and cancer treatment.


    Institute of Scientific and Technical Information of China (English)

    仇一华; 陈诗书


    Using Southern blot, Northern blot and Quick blot methods, we examined the rearrangement and expression of TCR βgene in four early differentiation stage cell lines from human hemopoietic system, namely HL-60, Jurkat, Daudi and Raji cells as well as lymphocytes from 17 acute lymphocytic leukemia (ALL) patients. The results showed. Ⅰ) Rearrangement of TCR βgene was seen in Jurkat cells. A germline pattern was observed in HL-60, Daudi and Raji cells. 2) Eight of 9 patients with T-ALL had cells with rearranged TCR βgene. But two of 3 patients with B-ALL and three of 5 patients with nonT, nonB-ALL also had cells with rearranged TCR βgene. 3) A 1.3 kb full-length transcript and a 1.0 kb truncated transcript were detected in Jurkat cells by probing with 32P-TCR βcDNA. But some leukemic B cells also expressed an incompleted transcript. 4) TCR βmRNA was detected in six of 8 patients with T-ALL, four of 5 patients with nonT, nonB-ALL and one of 3 patients with B-ALL. But the level of expression was quite differ ent. The dual-rearrangement and the abnormal expression may give us a new clue for researching leukemogenesis.

  12. Absolute Quantification of the Host-To-Parasite DNA Ratio in Theileria parva-Infected Lymphocyte Cell Lines.

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    Hanzel T Gotia

    Full Text Available Theileria parva is a tick-transmitted intracellular apicomplexan pathogen of cattle in sub-Saharan Africa that causes East Coast fever (ECF. ECF is an acute fatal disease that kills over one million cattle annually, imposing a tremendous burden on African small-holder cattle farmers. The pathology and level of T. parva infections in its wildlife host, African buffalo (Syncerus caffer, and in cattle are distinct. We have developed an absolute quantification method based on quantitative PCR (qPCR in which recombinant plasmids containing single copy genes specific to the parasite (apical membrane antigen 1 gene, ama1 or the host (hypoxanthine phosphoribosyltransferase 1, hprt1 are used as the quantification reference standards. Our study shows that T. parva and bovine cells are present in similar numbers in T. parva-infected lymphocyte cell lines and that consequently, due to its much smaller genome size, T. parva DNA comprises between 0.9% and 3% of the total DNA samples extracted from these lines. This absolute quantification assay of parasite and host genome copy number in a sample provides a simple and reliable method of assessing T. parva load in infected bovine lymphocytes, and is accurate over a wide range of host-to-parasite DNA ratios. Knowledge of the proportion of target DNA in a sample, as enabled by this method, is essential for efficient high-throughput genome sequencing applications for a variety of intracellular pathogens. This assay will also be very useful in future studies of interactions of distinct host-T. parva stocks and to fully characterize the dynamics of ECF infection in the field.

  13. Alpharetroviral self-inactivating vectors produced by a superinfection-resistant stable packaging cell line allow genetic modification of primary human T lymphocytes. (United States)

    Labenski, Verena; Suerth, Julia D; Barczak, Elke; Heckl, Dirk; Levy, Camille; Bernadin, Ornellie; Charpentier, Emmanuelle; Williams, David A; Fehse, Boris; Verhoeyen, Els; Schambach, Axel


    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.

  14. Cytokines and Epstein Barr virus (EBV) genes expression in blood chronic lymphocytic leukaemia (CLL) cells and their immortalised CLL cell lines. (United States)

    Laytragoon-Lewin, Nongnit; Chen, Fu; Castro, Juan; Avila-Carino, Javier; Lewin, Freddi


    We have encountered two unique chronic lymphocytic leukaemia (CLL) patients, PG and NN. Some blood CLL cells of these patients have been infected and carry Epstein Barr virus (EBV) in vivo. In spite of their early-activated G0/G1 stage of post germinal center (GC) memory cells, ex vivo EBV-carrying blood CLL cells of PG clone expressed LMPs and used specific QUK splice for their EBNA1 expression, similar to the EBV-carrying cells of non-B origin. Interestingly, EBV-carrying CLL cells of NN clone expressed LMP2a and used UK-splice for their EBNA1 expression, similar to the in vivo EBV-carrying high density normal B cells in the blood of healthy individuals. The CLL-derived lines but not normal lymphoblastoid cell line (LCL) used QUK- and YUK-splice for their EBNA1 expression. As expected, LCL and their permanent CLL-derived lines used Cp promoter and up-regulated their EBNA2 expression. Blood CLL cells and the CLL-derived cell lines of these patients spontaneously produced cytokines as shown by microarray assay. The types and quantities of cytokines might relate to their CLL origin and viral strain in the given CLL cells. Neither blood CLL nor their CLL-derived cell lines express any detectable apoptosis-inducer ligands, CD95L or Apo 3L. As a consequence of cell cycle progression, CLL-derived cell lines up-regulated their co-stimulator molecules CD80 and apoptosis-related receptor CD95. Since only the rare EBV-carrying CLL cells grew in vitro, the combination of viral genome and cytokines seems to be critical for the outgrowth of EBV-carrying CLL cells over their EBV-negative counterpart in vitro but not in vivo.

  15. Effect of cisplatin alone or combined with monoclonal anti-programmed death ligand-1 anti-body on lung adenocarcinoma cell line SPCA-1 and T lymphocytes

    Institute of Scientific and Technical Information of China (English)



    Objective To observe the effect of cisplatin alone or combined with anti-programmed death ligand 1 monoclonal antibody(anti-PD-L1 mA b)on the co-culture system of lung adenocarcinoma SPCA-1 cells and T lymphocytes,and therefore to study the immunotherapeutic effect of anti-PD-L1 mA b on lung cancer.Methods Human adenocarcinoma SPCA-1 cell line was selected by

  16. miRNA-mediated relationships between Cis-SNP genotypes and transcript intensities in lymphocyte cell lines.

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    Wensheng Zhang

    Full Text Available In metazoans, miRNAs regulate gene expression primarily through binding to target sites in the 3' UTRs (untranslated regions of messenger RNAs (mRNAs. Cis-acting variants within, or close to, a gene are crucial in explaining the variability of gene expression measures. Single nucleotide polymorphisms (SNPs in the 3' UTRs of genes can affect the base-pairing between miRNAs and mRNAs, and hence disrupt existing target sites (in the reference sequence or create novel target sites, suggesting a possible mechanism for cis regulation of gene expression. Moreover, because the alleles of different SNPs within a DNA sequence of limited length tend to be in strong linkage disequilibrium (LD, we hypothesize the variants of miRNA target sites caused by SNPs potentially function as bridges linking the documented cis-SNP markers to the expression of the associated genes. A large-scale analysis was herein performed to test this hypothesis. By systematically integrating multiple latest information sources, we found 21 significant gene-level SNP-involved miRNA-mediated post-transcriptional regulation modules (SNP-MPRMs in the form of SNP-miRNA-mRNA triplets in lymphocyte cell lines for the CEU and YRI populations. Among the cognate genes, six including ALG8, DGKE, GNA12, KLF11, LRPAP1, and MMAB are related to multiple genetic diseases such as depressive disorder and Type-II diabetes. Furthermore, we found that ~35% of the documented transcript intensity-related cis-SNPs (~950 in a recent publication are identical to, or in significant linkage disequilibrium (LD (p<0.01 with, one or multiple SNPs located in miRNA target sites. Based on these associations (or identities, 69 significant exon-level SNP-MPRMs and 12 disease genes were further determined for two populations. These results provide concrete in silico evidence for the proposed hypothesis. The discovered modules warrant additional follow-up in independent laboratory studies.

  17. Alvocidib in Treating Patients With B-Cell Chronic Lymphocytic Leukemia or Small Lymphocytic Lymphoma (United States)


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

  18. Transcriptome analysis of the human T lymphocyte cell line Jurkat and human peripheral blood mononuclear cells exposed to deoxynivalenol (DON): New mechanistic insights

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    Katika, Madhumohan R. [RIKILT-Institute of Food Safety, Wageningen University and Research Centre, Wageningen (Netherlands); Department of Health Risk Analysis and Toxicology, Maastricht University (Netherlands); Netherlands Toxicogenomics Centre (Netherlands); Hendriksen, Peter J.M. [RIKILT-Institute of Food Safety, Wageningen University and Research Centre, Wageningen (Netherlands); Netherlands Toxicogenomics Centre (Netherlands); Shao, Jia [RIKILT-Institute of Food Safety, Wageningen University and Research Centre, Wageningen (Netherlands); Department of Health Risk Analysis and Toxicology, Maastricht University (Netherlands); Netherlands Toxicogenomics Centre (Netherlands); Loveren, Henk van [Department of Health Risk Analysis and Toxicology, Maastricht University (Netherlands); National Institute for Public Health and the Environment (RIVM), Bilthoven (Netherlands); Netherlands Toxicogenomics Centre (Netherlands); Peijnenburg, Ad, E-mail: [RIKILT-Institute of Food Safety, Wageningen University and Research Centre, Wageningen (Netherlands); Netherlands Toxicogenomics Centre (Netherlands)


    Deoxynivalenol (DON) or vomitoxin is a commonly encountered type-B trichothecene mycotoxin, produced by Fusarium species predominantly found in cereals and grains. DON is known to exert toxic effects on the gastrointestinal, reproductive and neuroendocrine systems, and particularly on the immune system. Depending on dose and exposure time, it can either stimulate or suppress immune function. The main objective of this study was to obtain a deeper insight into DON-induced effects on lymphoid cells. For this, we exposed the human T-lymphocyte cell line Jurkat and human peripheral blood mononuclear cells (PBMCs) to various concentrations of DON for various times and examined gene expression changes by DNA microarray analysis. Jurkat cells were exposed to 0.25 and 0.5 μM DON for 3, 6 and 24 h. Biological interpretation of the microarray data indicated that DON affects various processes in these cells: It upregulates genes involved in ribosome structure and function, RNA/protein synthesis and processing, endoplasmic reticulum (ER) stress, calcium-mediated signaling, mitochondrial function, oxidative stress, the NFAT and NF-κB/TNF-α pathways, T cell activation and apoptosis. The effects of DON on the expression of genes involved in ER stress, NFAT activation and apoptosis were confirmed by qRT-PCR. Other biochemical experiments confirmed that DON activates calcium-dependent proteins such as calcineurin and M-calpain that are known to be involved in T cell activation and apoptosis. Induction of T cell activation was also confirmed by demonstrating that DON activates NFATC1 and induces its translocation from the cytoplasm to the nucleus. For the gene expression profiling of PBMCs, cells were exposed to 2 and 4 μM DON for 6 and 24 h. Comparison of the Jurkat microarray data with those obtained with PBMCs showed that most of the processes affected by DON in the Jurkat cell line were also affected in the PBMCs. -- Highlights: ► The human T cell line Jurkat and human

  19. In vitro priming of tumor-specific cytotoxic T lymphocytes using allogeneic dendritic cells derived from the human MUTZ-3 cell line. (United States)

    Santegoets, Saskia J A M; Schreurs, Marco W J; Masterson, Allan J; Liu, Ying Poi; Goletz, Steffen; Baumeister, Hans; Kueter, Esther W M; Lougheed, Sinéad M; van den Eertwegh, Alfons J M; Scheper, Rik J; Hooijberg, Erik; de Gruijl, Tanja D


    The adoptive transfer of in vitro-induced and expanded tumor-specific cytotoxic T lymphocytes (CTL) presents a promising immunotherapeutic approach for the treatment of cancer. The in vitro induction of tumor-reactive CTL requires repeated stimulation of CTL precursors with dendritic cells (DC). To circumvent problems like scarcity of blood DC precursors and donor variability, it would be attractive to use DC from a non-autologous, unlimited source. DCs derived from the human acute myeloid leukemia (AML) cell line MUTZ-3 are attractive candidates since these DCs closely resemble monocyte-derived DC (MoDC) in terms of phenotype and T cell stimulatory capacity. Here we demonstrate that functional CTL clones could be generated against multiple tumor-associated antigens, i.e., human telomerase reverse transcriptase (hTERT), ErbB3-binding protein-1 (Ebp1), carcinoembryonic antigen (CEA) and Her-2/neu, by stimulating CD8beta(+) CTL precursors with peptide-loaded allogeneic, HLA-A2-matched MUTZ-3-derived DC. A consistent induction capacity, as determined by MHC tetramer-binding, was found in multiple donors and comparable to autologous peptide-loaded MoDC. Functional characterization at the clonal level revealed the priming of CTL that recognized endogenously processed epitopes on tumor cell lines in an HLA-A2-restricted fashion. Our data indicate that MUTZ-3-derived DC can be used as stimulator cells for in vitro priming and expansion of functional TAA-specific effector CTL. MUTZ-3-derived DCs thus represent a ready and standardized source of allogeneic DC to generate CTL for therapeutic adoptive transfer strategies.

  20. Up-regulation of lymphocyte antigen 6 complex expression in side-population cells derived from a human trophoblast cell line HTR-8/SVneo. (United States)

    Inagaki, Tetsunori; Kusunoki, Soshi; Tabu, Kouichi; Okabe, Hitomi; Yamada, Izumi; Taga, Tetsuya; Matsumoto, Akemi; Makino, Shintaro; Takeda, Satoru; Kato, Kiyoko


    The continual proliferation and differentiation of trophoblasts are critical for the maintenance of pregnancy. It is well known that the tissue stem cells are associated with the development of tissues and pathologies. It has been demonstrated that side-population (SP) cells identified by fluorescence-activated cell sorting (FACS) are enriched with stem cells. The SP cells in HTR-8/SVneo cells derived from human primary trophoblast cells were isolated by FACS. HTR-8/SVneo-SP cell cultures generated both SP and non-SP (NSP) subpopulations. In contrast, NSP cell cultures produced NSP cells and failed to produce SP cells. These SP cells showed self-renewal capability by serial colony-forming assay. Microarray expression analysis using a set of HTR-8/SVneo-SP and -NSP cells revealed that SP cells overexpressed several stemness genes including caudal type homeobox2 (CDX2) and bone morphogenic proteins (BMPs), and lymphocyte antigen 6 complex locus D (LY6D) gene was the most highly up-regulated in HTR-8/SVneo-SP cells. LY6D gene reduced its expression in the course of a 7-day cultivation in differentiation medium. SP cells tended to reduce its fraction by treatment of LY6D siRNA indicating that LY6D had potential to maintain cell proliferation of HTR-8/SVneo-SP cells. On ontology analysis, epithelial-mesenchymal transition (EMT) pathway was involved in the up-regulated genes on microarray analysis. HTR-SVneo-SP cells showed enhanced migration. This is the first report that LY6D was important for the maintenance of HTR-8/SVneo-SP cells. EMT was associated with the phenotype of these SP cells.

  1. Cytotoxicity of lymphocytes from melanoma patients against autologous tumor cells and its potentiation in vitro

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    Bykovskaya, S.N.; Iobadze, M.S.; Kupriyanova, T.A.; Demidov, L.V.


    The specific and natural cytotoxicity of peripheral blood lymphocytes from patients with melanomas was compared and stimulation with autologous tumor cells or a pool of allogeneic lymphocytes from five healthy blood donors also was used to potentiate the specific antitumor activity of the patients' lymphocytes. To assess cytolytic ability, cells of an autologous tumor, cells of the K-562 line, autologous peripheral blood lymphocytes, and blast cells obtained from these lymphocytes after stimulation by phytohemagglutinin were used as the target cells. The target cells were incubated in a medium containing sodium chromate and were labelled with the chromium 51 isotope.

  2. Inferring polymorphism-induced regulatory gene networks active in human lymphocyte cell lines by weighted linear mixed model analysis of multiple RNA-Seq datasets.

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    Wensheng Zhang

    Full Text Available Single-nucleotide polymorphisms (SNPs contribute to the between-individual expression variation of many genes. A regulatory (trait-associated SNP is usually located near or within a (host gene, possibly influencing the gene's transcription or/and post-transcriptional modification. But its targets may also include genes that are physically farther away from it. A heuristic explanation of such multiple-target interferences is that the host gene transfers the SNP genotypic effects to the distant gene(s by a transcriptional or signaling cascade. These connections between the host genes (regulators and the distant genes (targets make the genetic analysis of gene expression traits a promising approach for identifying unknown regulatory relationships. In this study, through a mixed model analysis of multi-source digital expression profiling for 140 human lymphocyte cell lines (LCLs and the genotypes distributed by the international HapMap project, we identified 45 thousands of potential SNP-induced regulatory relationships among genes (the significance level for the underlying associations between expression traits and SNP genotypes was set at FDR < 0.01. We grouped the identified relationships into four classes (paradigms according to the two different mechanisms by which the regulatory SNPs affect their cis- and trans- regulated genes, modifying mRNA level or altering transcript splicing patterns. We further organized the relationships in each class into a set of network modules with the cis- regulated genes as hubs. We found that the target genes in a network module were often characterized by significant functional similarity, and the distributions of the target genes in three out of the four networks roughly resemble a power-law, a typical pattern of gene networks obtained from mutation experiments. By two case studies, we also demonstrated that significant biological insights can be inferred from the identified network modules.

  3. T cell immunity using transgenic B lymphocytes (United States)

    Gerloni, Mara; Rizzi, Marta; Castiglioni, Paola; Zanetti, Maurizio


    Adaptive immunity exists in all vertebrates and plays a defense role against microbial pathogens and tumors. T cell responses begin when precursor T cells recognize antigen on specialized antigen-presenting cells and differentiate into effector cells. Currently, dendritic cells are considered the only cells capable of stimulating T lymphocytes. Here, we show that mature naïve B lymphocytes can be genetically programmed by using nonviral DNA and turned into powerful antigen-presenting cells with a dual capacity of synthesis and presentation of antigen to T cells in vivo. A single i.v. injection of transgenic lymphocytes activates T cell responses reproducibly and specifically even at very low cell doses (102). We also demonstrate that T cell priming can occur in the absence of dendritic cells and results in immunological memory with protective effector functions. These findings disclose aspects in the regulation of adaptive immunity and indicate possibilities for vaccination against viruses and cancer in humans.

  4. Establishment of Immortalized Human Lymphocytes Cell Lines with Multiple SNP Genotyping in Chinese Han Population%中国汉族人群代谢酶基因分型永生化淋巴细胞库的建立

    Institute of Scientific and Technical Information of China (English)

    张娟; 熊梦祯; 尹立红; 浦跃朴


    目的 建立汉族人群代谢酶基因分型的永生化淋巴细胞库,为单核苷酸多态(SNP)结构对毒物诱导的基因功能表达的影响建立研究平台.方法 用EB病毒(Epstein-Barr Virus,EBV)转化中国汉族健康成人外周血B淋巴细胞,通过对中国汉族人群CYP2E1和NQ01的单核苷酸多态(SNP)位点信息分析,选择中国人群特异性CYP2EI、NQ01基因SNP位点CYP2E1rs2070673,CYP2E1rs2031921,CYP2E13813866,NQO1rs 1800566,NQO1rs10517,应用适配器连接介导的等位基因特异性扩增法(ALM-SAS)对上述位点进行基因分型.将供体信息和细胞株基因分型的信息录入,构建中国汉族基因分型的永生化淋巴细胞库.结果 通过对细胞株进行上述5个SNP位点的基因分型,获得每株细胞的基因分型信息,获得CYP2E1rs2070673,CYP2E1rs2031921,CYP2E13813866,NQO1rs 1800566,NQO1rs10517不同基因分型的永生化淋巴细胞株.结论 通过永生化细胞库的建立、基因分型,初步建立中国的汉族人群代谢酶基因分型永生化淋巴细胞库,该细胞库的进一步建设,可以为研究中国人群环境毒物和药物代谢酶基因结构和功能的关系提供技术平台.%Objective To establish immortalized human lymphocytes cell lines with multiple SNP genotyping of Chinese Han population which could afford a platform to study gene function expression influenced by SNPs structure induced by chemical Methods EBV (Epstein-Bart Virus) were used to immortalize peripheral B lymphocytes derived from Chinese Hah population. After analyzing CYP2EI、NQOI SNP database of Chinese population, the five Chinese specific SNP sites: CYP2EIrs2070673,CYP2Elrs2031921, CYP2E13813866,NQOlrs 1800566, NQOl rs10517 were genotyped by an adapter-ligation mediated allele specific amplification (ALM-ASA) method. The information of providers and genotypes of cell lines were input to establish the database of immortalized human lymphocytes cell lines with multiple SNP genotyping

  5. ABT-737 resistance in B-cells isolated from chronic lymphocytic leukemia patients and leukemia cell lines is overcome by the pleiotropic kinase inhibitor quercetin through Mcl-1 down-regulation. (United States)

    Russo, Maria; Spagnuolo, Carmela; Volpe, Silvestro; Tedesco, Idolo; Bilotto, Stefania; Russo, Gian Luigi


    Chronic lymphocytic leukemia (CLL) is the most frequent form of leukemia in adult population and despite numerous studies, it is considered an incurable disease. Since CLL is characterized by overexpression of pro-survival Bcl-2 family members, treatments with their antagonists, such as ABT-737, represent a promising new therapeutic strategy. ABT-737 is a BH3 mimetic agent which binds Bcl-2, Bcl-XL and Bcl-w with high affinity, while weakly interacts with Mcl-1 and Bfl-1. Previous studies demonstrated that quercetin, a flavonoid naturally present in food and beverages, was able to sensitize B-cells isolated from CLL patients to apoptosis when associated with death ligands or fludarabine, through a mechanism involving Mcl-1 down-regulation. Here, we report that the association between ABT-737 and quercetin synergistically induces apoptosis in B-cells and in five leukemic cell lines (Combination Index quercetin treatment. The molecular pathways triggered by quercetin have been investigated in HPB-ALL cells, characterized by the highest resistance to both ABT-737 and quercetin when applied as single molecules, but highly sensitivity to the co-treatment. In this cell line, quercetin down-regulated Mcl-1 through the inhibition of PI3K/Akt signaling pathway, leading to Mcl-1 instability. The same mechanism was confirmed in B-cells. These results may open new clinical perspectives based on a translational approach in CLL therapy. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Fludarabine Phosphate and Total-Body Irradiation Before Donor Peripheral Blood Stem Cell Transplant in Treating Patients With Chronic Lymphocytic Leukemia or Small Lymphocytic Leukemia (United States)


    B-Cell Prolymphocytic Leukemia; Chronic Lymphocytic Leukemia; Prolymphocytic Leukemia; Recurrent Chronic Lymphocytic Leukemia; Recurrent Small Lymphocytic Lymphoma; Refractory Chronic Lymphocytic Leukemia; T-Cell Prolymphocytic Leukemia

  7. Accumulation of Cholesterol Esters in ex vivo Lymphocytes from Scrapie-susceptible Sheep and in Scrapie-infected Mouse Neuroblastoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Alessandra Pani


    Full Text Available Our studies on the role of cholesterol homeostasis in the pathogenesis of scrapie in sheep, revealed abnormal accumulation of cholesterol esters in brains and in ex vivo skin fibroblasts from genetically scrapie-susceptible, as compared to sheep with resistant genotype. We now report that PBMCs isolated from scrapie-susceptible sheep, as well as mouse neuroblastoma cell lines persistently infected with two different mouse-adapted strains of scrapie, showed similar alterations with up to 3-fold higher cholesterol ester levels than their resistant or uninfected counterparts. Treatments with drugs that interfere with intracellular cholesterol metabolism strongly reduced accumulation of cholesterol esters in scrapie-infected cell lines, whereas had significantly lower, or no effect, in uninfected cell line. These data add support to our hypothesis that accumulation of cholesterol esters may represent a biological marker of susceptibility to prion infection and a potential molecular target for prion inhibitors.

  8. Morphometric Characterization of Small Cell Lymphocytic Lymphoma

    Directory of Open Access Journals (Sweden)

    Chisoi Anca


    Full Text Available The morphometry in histopathology is used to characterize cell populations belonging to different tissues and to identify differences in their parameters with prognostic implications. To achieve morphometric examination were selected 6 of 24 cases identified as small cell lymphocytic lymphoma. For each case analysis was done on five fields, for each field measuring the parameters of 20 cells. The studied parameters were for cytoplasm: cytoplasmic area, maximum and minimum cytoplasmic diameter, cytoplasmic perimeter; for nucleus were measured: nuclear area, minimum and maximum nuclear diameter, nuclear perimeter, nuclear contour index, nuclear ellipticity index, nuclear irregularity index. Also the nucleocytoplasmic ratio was calculated in all studied cases. Small cell lymphocytic lymphoma is characterized in morphometric terms having a small cytoplasmic area (average 29.206 and also a small nuclear area (mean 28.939 having a nucleo-cytoplasmic ratio appearance suggestive for adult lymphocyte. A nuclear contour index small value (3.946, ellipticity index value also small (3.521 and small nuclear irregularity index (3.965. Standard deviations, in any of the studied morphometric categories, is around or below 1 suggesting monomorphic cell appearance. These morphometric and microscopic features characterized mainly by a small population of adult lymphocytes, monomorphic, with rounded hipercromic nuclei, dense chromatin, support the framing into indolent lymphoma group in terms of clinical outcome.

  9. Cell Death Mechanisms Induced by Cytotoxic Lymphocytes

    Institute of Scientific and Technical Information of China (English)

    Ch(a)vez-Gal(a)n L; Arenas-Del Angel MC; Zenteno E; Ch(a)vez R; Lascurain R


    One of the functions of the immune system is to recognize and destroy abnormal or infected cells to maintain homeostasis. This is accomplished by cytotoxic lymphocytes. Cytotoxicity is a highly organized multifactor process. Here, we reviewed the apoptosis pathways induced by the two main cytotoxic lymphocyte subsets, natural killer (NK) cells and CD8+T cells. In base to recent experimental evidence, we reviewed NK receptors involved in recognition of target-cell, as well as lytic molecules such as perforin, granzymes-A and -B, and granulysin. In addition, we reviewed the Fas-FasL intercellular linkage mediated pathway, and briefly the cross-linking of tumor necrosis factor (TNF) and TNF receptor pathway. We discussed three models of possible molecular interaction between lyric molecules from effector cytotoxic cells and target-cell membrane to induction of apoptosis.

  10. The clinical implications of mixed lymphocyte reaction with leukemic cells. (United States)

    Kim, Hee-Je; Kim, Tai-Gyu; Cho, Hyun Il; Han, Hoon; Min, Woo-Sung; Kim, Chun-Choo


    To evaluate the clinical implications of a mixed lymphocyte reaction between leukemic cells and lymphocytes from HLA-matched sibling donors, we attempted to generate donor-derived, graft-versus-leukemia-effective cells and to define their characteristics. We studied 8 patients with chronic myelogenous leukemia (CML), including 5 patients in the chronic phase (CP), 3 patients in the accelerated phase (AP), and 2 patients with acute myelogenous leukemia (AML) in their first complete remission. Cells from these patients were used as stimulators in a mixed lymphocyte reaction.The effects of natural killer (NK) cells and cytotoxic T-lymphocytes (CTLs) were separated by observing tests for cytotoxicity to target cells, including K562 cells, the patient's leukemic cells, and phytohemagglutinin (PHA) blasts. Donor-derived antileukemic CTLs againstthe patient's own leukemic cells are productive in vitro. The efficacy of generating CTLs against leukemic target cells was (in decreasing order) AML, CML-CP, and CML-AP. Cytotoxic activity against leukemic targets was prominent in 4 cases--2 CML-CP and the 2 AML cases. On the contrary, the 3 cases of CML-AP showed low CTL activity. In cases showing 1 positive result among 3 targets (K562 cells, the patient's leukemic cells, and PHA blasts), the relapse rate was significantly lower (P = .022) on follow-up (median, 33 months; 7-40 months) after hematopoietic stem cell transplantation. By a combined analysis of the cytotoxicity effects for all 3 target cells, we were able to demonstrate a correlation between leukemic relapse and the variable degree of the cytotoxicity test results. Although the total sample numbers for this study were low, we speculate that these results may come from differences in the individual characteristics of the leukemic cells that are in line with their clinical disease status.

  11. Human lymphocyte markers defined by antibodies derived from somatic cell hybrids. II. A hybridoma secreting antibody against an antigen expressed by human B and null lymphocytes. (United States)

    Beckman, I G; Bradley, J; Brooks, D A; Kupa, A; McNamara, P J; Thomas, M E; Zola, H


    A hybridoma (FMC4) has been derived which secretes antibody showing selective reaction with human B lymphocytes, monocytes and some null lymphocytes. Few, if any, T lymphocytes in normal blood are stained, although stimulation of lymphocytes with PHA leads to an increase in the proportion of cells reacting with the hybridoma antibody. The antibody reacts with B and null lymphoblastoid cell lines but not with T cell lines. B chronic lymphocytic leukaemia (CLL) cells but not T-CLLs are stained and null-type acute lymphoblastic leukaemia (ALL) cells but not T-type ALL also react. Normal blood myeloid cells do not react with FMC4 supernatant whilst some myeloid leukaemias do. The expression of the antigen reacting with FMC4 supernatant suggests that FMC4 may secrete an antibody against the human equivalent of the Ia antigen.

  12. Antiprion activity of cholesterol esterification modulators: a comparative study using ex vivo sheep fibroblasts and lymphocytes and mouse neuroblastoma cell lines. (United States)

    Pani, Alessandra; Norfo, Claudia; Abete, Claudia; Mulas, Claudia; Putzolu, Marirosa; Laconi, Sergio; Orrù, Christina Doriana; Cannas, M Dolores; Vascellari, Sarah; La Colla, Paolo; Dessì, Sandra


    Our studies on the role of cholesterol homeostasis in the pathogenesis of scrapie revealed abnormal accumulation of cholesterol esters in ex vivo peripheral blood mononuclear cells (PBMCs) and skin fibroblasts from healthy and scrapie-affected sheep carrying a scrapie-susceptible genotype compared to sheep with a resistant genotype. Similar alterations were observed in mouse neuroblastoma N2a cell lines persistently infected with mouse-adapted 22L and RML strains of scrapie that showed up to threefold-higher cholesterol ester levels than parental N2a cells. We now report that proteinase K-resistant prion protein (PrPres)-producing cell populations of subclones from scrapie-infected cell lines were characterized by higher cholesterol ester levels than clone populations not producing PrPres. Treatments with a number of drugs known to interfere with different steps of cholesterol metabolism strongly reduced the accumulation of cholesterol esters in ex vivo PBMCs and skin fibroblasts from scrapie-affected sheep but had significantly less or no effect in their respective scrapie-resistant or uninfected counterparts. In scrapie-infected N2a cells, inhibition of cholesterol esters was associated with selective antiprion activity. Effective antiprion concentrations of cholesterol modulators (50% effective concentration [EC(50)] range, 1.4 to 40 microM) were comparable to those of antiprion reference compounds (EC(50) range, 0.6 to 10 microM). These data confirm our hypothesis that abnormal accumulation of cholesterol esters may represent a biological marker of susceptibility to prion infection/replication and a novel molecular target of potential clinical importance.

  13. Fludarabine Phosphate, Radiation Therapy, and Rituximab in Treating Patients Who Are Undergoing Donor Stem Cell Transplant Followed by Rituximab for High-Risk Chronic Lymphocytic Leukemia or Small Lymphocytic Lymphoma (United States)


    Chronic Lymphocytic Leukemia; Prolymphocytic Leukemia; Recurrent Small Lymphocytic Lymphoma; Refractory Chronic Lymphocytic Leukemia; Stage III Chronic Lymphocytic Leukemia; Stage III Small Lymphocytic Lymphoma; Stage IV Chronic Lymphocytic Leukemia; Stage IV Small Lymphocytic Lymphoma; T-Cell Large Granular Lymphocyte Leukemia

  14. Dual character of interaction between lymphocytes and allogeneic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Petrov, R.V.; Dozmorov, I.M.; Kochetkova, M.O.; Nikolaeva, I.S.


    The mechanisms of stimulation of colony formation by small doses of allogeneic lymphocytes were studied in mice. When interaction of lymphocytes with allogeneic stem cells was studied, bone marrow cells of mice were injected into lethally irradiated recipients in the control, and mixtures of bone marrow cells with varied numbers of lymphocytes were injected in the experiment. Dependence of the inactivation indices on the number of lymphocytes injected, based on the results of counting macro- and microcolonies in the spleen, is shown.

  15. Influence of docosahexaenoic acid in vitro on intracellular adriamycin concentration in lymphocytes and human adriamycin-sensitive and -resistant small-cell lung cancer cell lines, and on cytotoxicity in the tumor cell lines

    NARCIS (Netherlands)

    Zijlstra, J G; de Vries, E G; Muskiet, F A; Martini, I A; Timmer-Bosscha, H; Mulder, N H


    An increase in the therapeutic effects of cancer chemotherapeutic agents and circumvention of drug resistance in cancer cells might result from an increase in the intracellular drug level. Alteration of the lipid domain of the cell membrane can result in a higher intracellular drug level. This alter

  16. Large-cell lymphocytic lymphoma

    African Journals Online (AJOL)


    Apr 9, 1983 ... marrow transplantation and immunological manipulation of the ... The clinical course in the patient with a biopsy-proven diagnosis of lymphoma ... in years, and in the majority of cases the tumour cell will be a small round or.

  17. Unsuitability of lymphoblastoid cell lines as surrogate of cryopreserved isolated lymphocytes for the analysis of DNA double-strand break repair activity

    Energy Technology Data Exchange (ETDEWEB)

    Zijno, Andrea [Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Rome (Italy); Porcedda, Paola [Department of Clinical and Biological Sciences, University of Turin (Italy); Saini, Francesca [Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Rome (Italy); Allione, Alessandra [Institute for Scientific Interchange (ISI) Foundation, Villa Gualino, Turin (Italy); Garofalo, Bruno; Marcon, Francesca [Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Rome (Italy); Guarrera, Simonetta [Institute for Scientific Interchange (ISI) Foundation, Villa Gualino, Turin (Italy); Turinetto, Valentina; Minieri, Valentina [Department of Clinical and Biological Sciences, University of Turin (Italy); Funaro, Ada [Department of Genetics, Biology and Biochemistry, University of Turin (Italy); Crebelli, Riccardo [Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Rome (Italy); Giachino, Claudia [Department of Clinical and Biological Sciences, University of Turin (Italy); Matullo, Giuseppe, E-mail: [Institute for Scientific Interchange (ISI) Foundation, Villa Gualino, Turin (Italy); Department of Genetics, Biology and Biochemistry, University of Turin (Italy)


    As first task of a comprehensive investigation on DNA repair genotype-phenotype correlations, the suitability of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) as surrogate of cryopreserved peripheral blood mononuclear cells (PBMCs) in DNA repair phenotypic assays was evaluated. To this aim the amount of DNA damage induced by {gamma}-rays and DNA repair capacity were evaluated in unstimulated (G{sub 0}) and mitogen-simulated (G{sub 2}) PBMC from 20 healthy subjects and in EBV-transformed LCL obtained from the same individuals. Phosphorylation of histone H2AX, micronuclei and chromosomal aberrations were the end-points investigated. The results obtained show higher basal frequencies of binucleated cells bearing micronuclei and nucleoplasmic bridge (NPB) in LCL with respect to PBMC, suggesting that EBV transformation may be associated with chromosomal instability. After irradiation, higher levels of micronuclei were induced in G{sub 0}-treated PBMC compared to cycling LCL; conversely, NPB were more frequent in LCL than in PBMC. Moreover, higher levels of chromosomal aberrations were observed in G{sub 2}-treated PBMC compared to LCL. Concerning {gamma}-H2AX measurements, phosphorylation levels 1 h after treatment and dephosphorylation kinetics were basically similar in LCL and in PBMC. However, while Spearman's test showed a strong correlation between the results obtained in replicated experiments with PBMC, high inter-experimental variability and poor reproducibility was observed in the experiments performed with LCL, possibly due to the intrinsic instability of LCL. In summary, both the analysis of {gamma}-H2AX and the evaluation of chromosome damage highlighted a larger inter-experimental variability in the results obtained with LCL compared to PBMC. Noteworthy, the two set of results proved to lack any significant correlation at the individual level. These results indicate that LCL may be unsuitable for investigating genotype

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

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


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

  19. Immune Escape for Renal Cell Carcinoma: CD70 Mediates Apoptosis in Lymphocytes

    Directory of Open Access Journals (Sweden)

    Julia Diegmann


    Full Text Available Tumors can escape immune recognition and destruction through the induction of apoptosis in lymphocytes. Although renal cell carcinoma (RCC is able to prevent immune recognition, only a few genes (such as FasL that are relevant for RCC immune escape have been identified so far. We have previously shown that some apoptosis-inducing genes are overexpressed in RCC. We hypothesized that these genes could be part of the immune-escape strategy of these tumors. Here we report that CD70, a cytokine overexpressed in RCC, promotes lymphocyte apoptosis through interaction with its receptor CD27 and with the intracellular receptor-binding protein SIVA. Apoptosis increased after cocultivating lymphocytes with the RCC cell lines A498 and CAKI2. The addition of recombinant soluble CD70 to both native lymphocytes and a T-cell cell line resulted in increased lymphocyte apoptosis as well. Furthermore, induced apoptosis could be partially blocked with anti-CD27 and anti-CD70 antibodies. Our results strongly indicate a role for CD70 and CD27 receptor in lymphocyte apoptosis within the tumor environment. Apoptosis mediated by exposure to the CD70 secreted by tumor cells may contribute to the failure of RCC patients to develop an effective lymphocyte-mediated antitumor response.

  20. In vitro radiosensitivity of human leukemia cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Weichselbaum, R.R.; Greenberger, J.S.; Schmidt, A.; Karpas, A.; Moloney, W.C.; Little, J.B.


    The in vitro radiobiologic survival values (anti n, D/sub 0/) of four tumor lines derived from human hematopoietic tumors were studied. These cell lines were HL60 promyelocytic leukemia; K562 erythroleukemia; 45 acute lymphocytic leukemia; and 176 acute monomyelogenous leukemia. More cell lines must be examined before the exact relationship between in vitro radiosensitivity and clinical radiocurability is firmly established.

  1. Safety and Tolerability Study of PCI-32765 in B Cell Lymphoma and Chronic Lymphocytic Leukemia (United States)


    B-cell Chronic Lymphocytic Leukemia; Small Lymphocytic Lymphoma; Diffuse Well-differentiated Lymphocytic Lymphoma; B Cell Lymphoma; Follicular Lymphoma; Mantle Cell Lymphoma; Non-Hodgkin's Lymphoma; Waldenstrom Macroglobulinemia; Burkitt Lymphoma; B-Cell Diffuse Lymphoma

  2. The effect of radiation on the apoptotic inducing ability of human breast milk (a-Lactalbumin) on a oesophageal and lung carcinoma cell line and lymphocytes



    M.Tech. Natural occurring components in human breast milk, cow milk and soy milk have shown anticarcinogenic abilities. The human breast milk protein, -lactalbumin, was found to induce apoptosis in cancer cells, embryonic cells and rapidly growing cells, when converted from its native form to a partial denatured apoptotic-inducing form. Moreover, radiation may cause irreversible changes of protein conformation at the molecular level. Native -lactalbumin is one protein that has shown a de...

  3. Storage of cell lines. (United States)

    Parker, Katharine A


    The successful storage of cell lines depends upon many factors, including the condition of the cells to be frozen and the experience of the operator. Attempting to freeze down unhealthy, contaminated or poorly labelled cells can have huge implications for a research laboratory. This chapter outlines the importance of good record keeping, vigilant monitoring, aseptic technique, and high-quality reagents in the successful storage and downstream propagation of cell lines.

  4. Cloning of aminopeptidase Npromoter and its activity in hematopoietic cell and different tumor cell lines

    Institute of Scientific and Technical Information of China (English)


    Aminopeptidase N (APN) promoter region was cloned and sequenced from peripheral blood mononuclear cells. The recombinant reporter construct containing the promoter and luciferase gene, designated pXP1-APNLuc, was introduced into myeloblastic cell line, T lymphocyte cell line and various tumor cell lines. Luciferase assay showed that APN upstream promoter is myeloid-specific for high expression in myeloblastic cell line and much lower expres sion in T lymphocyte cell line. The promoter activity was relatively high in lung adenoma cell line compared with other tumor cell lines including hepatoma cell line, tong cancer cell line and esophageal cancer cell line in which the promoter activity significantly diminished or was almost undetectable. The characteristics of APN promoter may pro vide a new strategy for specific myeloprotection while tumor patients are being treated with chemotherapy and/or radio therapy.


    Directory of Open Access Journals (Sweden)

    Luca Laurenti


    Full Text Available Currently standard first line therapy for fit patients with B-CLL/SLL are fludarabine-based regimens. Elderly patients or patients with comorbidities poorly tolerate purine analogue-based chemotherapy and they are often treated with Chlorambucil (Chl. However, complete response (CR and overall response (OR rates with Chl are relatively low. We now investigated whether the addition of Rituximab to Chl will improve the efficacy without impairing the tolerability in elderly and unfit patients. We included in our study 27 elderly or unfit patients that had not received prior therapy. All patients were treated with Chl (1mg/Kg per 28-day cycle for 8 cycles plus Rituximab (375 mg/m2 for the first course and 500 mg/m2 for subsequent cycles until the 6th cycle. We obtained an OR rate of 74%. The most frequent adverse effect was grade 3-4 neutropenia, which occurred in 18.5% of the patients. Infections or grade 3-4 extra-hematological side effects were not recorded. None of the patients required reduction of dose, delay of therapy or hospitalization. Overall, these data suggest that Chl-R is an effective and well tolerated regimen in elderly/unfit patients with CLL.

  6. Reprogramming T cell Lymphocytes to Induced Pluripotent Stem Cells (United States)

    Bared, Kalia

    The discovery of induced pluripotent stem cells (iPSC) provided a novel technology for the study of development and pharmacology and complement embryonic stem cells (ES) for cell therapy applications. Though iPSC are derived from adult tissue they are comparable to ES cells in their behavior; multi-lineage differentiation and self-renewal. This makes iPSC research appealing because they can be studied in great detail and expanded in culture broadly. Fibroblasts were the first cell type reprogrammed to an iPSC using a retrovirus vector, since then alternative cell types including lymphocytes have been used to generate iPSC. Different types of vectors have also been developed to enhance iPSC formation and quality. However, specific T lymphocyte subsets have not been shown to reprogram to a pluripotent state to date. Here, we proposed to derive iPSC from peripheral blood effector and central memory T cells, reasoning that the resultant iPSC will maintain the epigenetic memory of a T lymphocyte, including the T cell receptor (TCR) gene rearrangement. This epigenetic memory will enable the differentiation and expansion of T cell iPSC into professional T cells containing a specific TCR. These could then be used for cell therapy to target specific antigens, as well as to improve culture techniques to expand T cells in vitro. We studied different gene delivery methods to derive iPSC from different types of T lymphocytes. We assessed the viability of viral transduction using flow cytometry to detect green fluorescent marker contained in the viral construct and quantitative real time polymerase chain reaction (qRT-PCR) to detect Oct4, Klf4, Sox2, and c-Myc gene expression. Our results demonstrate that the Sendai virus construct is the most feasible platform to reprogram T lymphocytes. We anticipate that this platform will provide an efficient and safe approach to derive iPSC from different T cell subsets, including memory T cells.

  7. Elutriated lymphocytes for manufacturing chimeric antigen receptor T cells. (United States)

    Stroncek, David F; Lee, Daniel W; Ren, Jiaqiang; Sabatino, Marianna; Highfill, Steven; Khuu, Hanh; Shah, Nirali N; Kaplan, Rosandra N; Fry, Terry J; Mackall, Crystal L


    Clinical trials of Chimeric Antigen Receptor (CAR) T cells manufactured from autologous peripheral blood mononuclear cell (PBMC) concentrates for the treatment of hematologic malignancies have been promising, but CAR T cell yields have been variable. This variability is due in part to the contamination of the PBMC concentrates with monocytes and granulocytes. Counter-flow elutriation allows for the closed system separation of lymphocytes from monocytes and granulocytes. We investigated the use of PBMC concentrates enriched for lymphocytes using elutriation for manufacturing 8 CD19- and 5 GD2-CAR T cell products. When compared to PBMC concentrates, lymphocyte-enriched elutriation fractions contained greater proportions of CD3+ and CD56+ cells and reduced proportions of CD14+ and CD15+ cells. All 13 CAR T cell products manufactured using the elutriated lymphocytes yielded sufficient quantities of transduced CAR T cells to meet clinical dose criteria. The GD2-CAR T cell products contained significantly more T cells and transduced T cells than the CD19-CAR T cell products. A comparison of the yields of CAR T cells produced from elutriated lymphocytes with the yields of CAR T cells previous produced from cells isolated from PBMC concentrates by anti-CD3/CD28 bead selection or by anti-CD3/CD28 bead selection plus plastic adherence found that greater quantities of GD2-CAR T cells were produced from elutriated lymphocytes, but not CD19-CAR T cells. Enrichment of PBMC concentrates for lymphocytes using elutriation increased the quantity of GD2-CAR T cells produced. These results provide further evidence that CAR T cell expansion is inhibited by monocytes and granulocytes.

  8. Human liver sinusoidal endothelial cells promote intracellular crawling of lymphocytes during recruitment: A new step in migration. (United States)

    Patten, Daniel A; Wilson, Garrick K; Bailey, Dalan; Shaw, Robert K; Jalkanen, Sirpa; Salmi, Marko; Rot, Antal; Weston, Chris J; Adams, David H; Shetty, Shishir


    The recruitment of lymphocytes via the hepatic sinusoidal channels and positioning within liver tissue is a critical event in the development and persistence of chronic inflammatory liver diseases. The hepatic sinusoid is a unique vascular bed lined by hepatic sinusoidal endothelial cells (HSECs), a functionally and phenotypically distinct subpopulation of endothelial cells. Using flow-based adhesion assays to study the migration of lymphocytes across primary human HSECs, we found that lymphocytes enter into HSECs, confirmed by electron microscopy demonstrating clear intracellular localization of lymphocytes in vitro and by studies in human liver tissues. Stimulation by interferon-γ increased intracellular localization of lymphocytes within HSECs. Furthermore, using confocal imaging and time-lapse recordings, we demonstrated "intracellular crawling" of lymphocytes entering into one endothelial cell from another. This required the expression of intracellular adhesion molecule-1 and stabilin-1 and was facilitated by the junctional complexes between HSECs.

  9. Clonal expansion of renal cell carcinoma-infiltrating T lymphocytes

    DEFF Research Database (Denmark)

    Sittig, Simone; Køllgaard, Tania; Grønbæk, Kirsten


    T lymphocytes can mediate the destruction of cancer cells by virtue of their ability to recognize tumor-derived antigenic peptides that are presented on the cell surface in complex with HLA molecules and expand. Thus, the presence of clonally expanded T cells within neoplastic lesions...... is an indication of ongoing HLA-restricted T cell-mediated immune responses. Multiple tumors, including renal cell carcinomas (RCCs), are often infiltrated by significant amounts of T cells, the so-called tumor-infiltrating lymphocytes (TILs). In the present study, we analyzed RCC lesions (n = 13) for the presence...... of expanded T-cell clonotypes using T-cell receptor clonotype mapping. Surprisingly, we found that RCCs comprise relatively low numbers of distinct expanded T-cell clonotypes as compared with melanoma lesions. The numbers of different T-cell clonotypes detected among RCC-infiltrating lymphocytes were...

  10. Lymphocyte activation by purified HLA-DR molecules fused into autochthonous "stimulating cells". (United States)

    Diu, A; Abikar, K; Rode, H N; Gordon, J


    Affinity-purified Ia molecules derived from the Daudi cell line were reconstituted into vesicles with Sendai virus envelope glycoproteins. These vesicles inserted into human peripheral leukocytes could induce stimulation of autologous lymphocytes, as measured by thymidine uptake, 6 days later. It is suggested that this method could provide a means to study allostimulation at the molecular level.

  11. First-line treatment of chronic lymphocytic leukemia: role of alemtuzumab

    Directory of Open Access Journals (Sweden)

    Carmen Diana Schweighofer


    Full Text Available Carmen Diana Schweighofer1, Clemens-Martin Wendtner21Department of Hematopathology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA; 2Department of Internal Medicine I, University of Cologne, Cologne, GermanyAbstract: The CD52-targeting antibody alemtuzumab is established in clinical practice with convincing activity in relapsed and refractory chronic lymphocytic leukemia (CLL, particularly in patients with high-risk features and adverse prognosis. In the CAM307 study alemtuzumab was tested and finally approved as a first-line single agent, even though the hurdle with chlorambucil as the contender was not set very high. Within clinical trials, the drug demonstrated an excellent ability to eliminate minimal residual disease in blood and bone marrow, which has been correlated with a corresponding survival advantage in patients. However, in the maintenance setting, infectious complications due to severe T cell suppression have been highlighted and do not allow clinicans to use alemtuzumab outside of clinical trials. This review discusses potential therapeutic niches and future applications of alemtuzumab with a focus on CLL front-line treatment.Keywords: CLL, alemtuzumab, Campath, front-line, first-line treatment


    Directory of Open Access Journals (Sweden)

    Giovanni D'arena


    Full Text Available

    line-height: 200%;" class="MsoNormal">line-height: 200%; font-family: "Times New Roman","serif"; font-size: 12pt; mso-ansi-language: EN-US; mso-bidi-font-size: 11.0pt;">Regulatory T-cells (Tregs constitute a small subset of cells that are actively involved in maintaining self-tolerance, in immune homeostasis and in antitumor immunity. They are thought to play a significant role in the progression of cancer and are generally increased in patient with chronic lymphocytic leukemia (CLL. Their number correlates with more aggressive disease status and is predictive of the time to treatment, as well. Moreover, it is now clear that dysregulation in Tregs cell frequency and/or function may result in a plethora of autoimmune diseases, including multiple sclerosis, type 1 diabetes mellitus, myasthenia gravis, systemic lupus erythematosis, autoimmune lymphoproliferative disorders, rheumatoid arthritis, and psoriasis. Efforts are made aiming to develop approaches to deplete Tregs or inhibit their function in either cancer and autoimmune disorders.

  13. Outcomes of first-line treatment for chronic lymphocytic leukemia with 17p deletion. (United States)

    Strati, Paolo; Keating, Michael J; O'Brien, Susan M; Ferrajoli, Alessandra; Burger, Jan; Faderl, Stefan; Tambaro, Francesco Paolo; Jain, Nitin; Wierda, William G


    Although uncommon in treatment-naive patients with chronic lymphocytic leukemia, deletion 17p is a high-risk disease characteristic. We analyzed and reported outcomes for 63 patients with deletion 17p chronic lymphocytic leukemia who received first-line therapy at our institution; at time of first treatment, 81% had unmutated immunoglobulin heavy chain variable gene and 58% had complex karyotype. Forty-nine patients (76%) received first-line fludarabine, cyclophosphamide, rituximab-based therapy, 6 (11%) received rituximab-based and 8 (13%) received lenalidomide-based treatment. Overall, the complete plus nodular partial remission rate was 33%; on multivariable model, higher complete plus nodular partial remission rate was observed in patients with less than 50% cells positive for deletion 17p, and a higher probability of achieving at least a partial remission was observed with fludarabine, cyclophosphamide, rituximab-based treatment. After a median follow up of 33 months (range 1-89 months), the estimated median progression-free survival was 14 months (95% confidence interval 10-18) and estimated median overall survival was 63 months (95% confidence interval 43-83). In multivariable analysis, factors independently associated with longer progression-free survival were response to treatment and absence of complex karyotype. Achievement of complete plus nodular partial remission rate and mutated immunoglobulin heavy chain variable gene were independently associated with longer overall survival in multivariable model. Complex karyotype was associated with increased risk for Richter's transformation. New first-line strategies and agents must aim at both improving response and maintaining remission in patients with deletion 17p, particularly in the presence of complex karyotype.

  14. Activated allogeneic NK cells preferentially kill poor prognosis B-cell chronic lymphocytic leukemia cells

    Directory of Open Access Journals (Sweden)

    Diego Sanchez-Martinez


    Full Text Available Mutational status of TP53 together with expression of wild type (wt IGHV represents the most widely accepted biomarkers, establishing a very poor prognosis in B-cell chronic lymphocytic leukemia (B-CLL patients. Adoptive cell therapy using allogeneic HLA mismatched Natural Killer (NK cells has emerged as an effective and safe alternative in the treatment of acute myeloid and lymphoid leukemias that do not respond to traditional therapies. We have described that allogeneic activated NK cells eliminate hematological cancer cell lines with multidrug resistance acquired by mutations in the apoptotic machinery. This effect depends on the activation protocol, being B-lymphoblastoid cell lines (LCLs the most effective stimulus to activate NK cells. Here we have further analyzed the molecular determinants involved in allogeneic NK cell recognition and elimination of B-CLL cells, including the expression of ligands of the main NK cell activating receptors (NKG2D and NCRs and HLA mismatch. We present preliminary data suggesting that B-CLL susceptibility significantly correlates with HLA mismatch between NK cell donor and B-CLL patient. Moreover, we show that the sensitivity of B-CLL cells to NK cells depends on the prognosis based on TP53 and IGHV mutational status. Cells from patients with worse prognosis (mutated TP53 and wt IGHV are the most susceptible to activated NK cells. Hence, B-CLL prognosis may predict the efficacy of allogenic activated NK cells and, thus, NK cell transfer represents a good alternative to treat poor prognosis B-CLL patients who present a very short life expectancy due to lack of effective treatments.□

  15. Natural History Study of Monoclonal B Cell Lymphocytosis (MBL), Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (CLL/SLL), Lymphoplasmacytic Lymphoma (LPL)/Waldenstrom Macroglobulinemia (WM), and Splenic Marginal Zone Lymphoma (SMZL) (United States)


    B-Cell Chronic Lymphocytic Leukemia; Monoclonal B-Cell Lymphocytosis; Lymhoma, Small Lymphocytic; Chronic Lymphocytic Leukemia; Lymphoplasmacytic Lymphoma; Waldenstrom Macroglobulinemia; Splenic Marginal Zone Lymphoma

  16. Concanavalin A-induced activation of lymphocytic choriomeningitis virus memory lymphocytes into specifically cytotoxic T cells

    DEFF Research Database (Denmark)

    Marker, O; Thomsen, Allan Randrup; Andersen, G T


    When spleen cells, which have been primed to Lymphocytic Choriomeningitis (LCM) virus during a primary infection several months previously, are stimulated in vitro with Con A. highly specific secondary cytotoxic effector cells are generated. The degree of cytotoxicity revealed by such Con A......-stimulated cells is higher than that of non-incubated spleen cells harvested nine days following the primary infection, and the effect is totally inhibited by anti-theta serum plus complement treatment of the effector cells immediately before the cytotoxic test....

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

    Directory of Open Access Journals (Sweden)

    Halickman Isaac


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

  18. B cell acute lymphocytic leukemia in pregnancy. (United States)

    Bottsford-Miller, Justin; Haeri, Sina; Baker, Arthur M; Boles, Jeremiah; Brown, Mark


    Acute lymphocytic leukemia (ALL) is a rare occurrence in pregnancy and can be rapidly fatal if left untreated. The need for immediate treatment of ALL, coupled with the maternal-fetal risks from the chemotherapy regimen render a therapeutic dilemma in pregnant women with ALL. We report a case of ALL diagnosed in the 24th week of pregnancy to outline our management strategy, to demonstrate the feasibility of treatment with multi-agent chemotherapy, and to provide a review of the literature.

  19. Human lymphocyte sub-populations and K cells. (United States)

    Sandilands, G; Gray, K; Cooney, A; Froebel, K; Anderson, J R


    Peripheral blood lymphocytes from 19 normal subjects were examined for surface Ig (SIg) and capacity to form rosettes with normal and neuraminidase-treated sheep erythrocytes and with chicken erythrocytes sensitised with IgG antibody. Information on the relationship between the presence of SIg and capacity to form rosettes was obtained by combined tests and depletion experiments. By these means, a population of lymphocytes with Fc receptors, but lacking SIg (mean 14.6%) was defined and shown to correlate closely with cytotoxic activity for antibody-sensitised target cells. Indirect evidence was also obtained that these lymphocytes, which are regarded as the major population of antibody-dependent cytotoxic cells, are capable of forming rosettes with normal and neuraminidase-treated sheep erythrocytes. The nature of these cells is briefly discussed.

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

    Energy Technology Data Exchange (ETDEWEB)

    Aro, H.; Eerola, E.; Aho, A.J.; Penttinen, R.


    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.

  1. Transformation of human fetal thymus and spleen lymphocytes by human t-cell leukemia virus type Ι

    Directory of Open Access Journals (Sweden)



    Full Text Available Co-cultivation of human thymus and spleen lymphocytes, which were obtained from 26-week and 27-week fetuses, with a lethally-irradiated human cord T-cell line harboring human T-cell leukemia virus type Ι(HTLV-Ι resultes in the establishment of T-cell lines positive for adult T-cell leukemia-associated antigens and producing HTLV-Ι. These cell lines had the phenotype of a helper/inducer subset of peripheral T-cells as evidenced by the reactivity with monoclonal antibodies to human T-cells.

  2. CLO : The cell line ontology

    NARCIS (Netherlands)

    Sarntivijai, Sirarat; Lin, Yu; Xiang, Zuoshuang; Meehan, Terrence F.; Diehl, Alexander D.; Vempati, Uma D.; Schuerer, Stephan C.; Pang, Chao; Malone, James; Parkinson, Helen; Liu, Yue; Takatsuki, Terue; Saijo, Kaoru; Masuya, Hiroshi; Nakamura, Yukio; Brush, Matthew H.; Haendel, Melissa A.; Zheng, Jie; Stoeckert, Christian J.; Peters, Bjoern; Mungall, Christopher J.; Carey, Thomas E.; States, David J.; Athey, Brian D.; He, Yongqun


    Background: Cell lines have been widely used in biomedical research. The community-based Cell Line Ontology (CLO) is a member of the OBO Foundry library that covers the domain of cell lines. Since its publication two years ago, significant updates have been made, including new groups joining the CLO

  3. CLO : The cell line ontology

    NARCIS (Netherlands)

    Sarntivijai, Sirarat; Lin, Yu; Xiang, Zuoshuang; Meehan, Terrence F.; Diehl, Alexander D.; Vempati, Uma D.; Schuerer, Stephan C.; Pang, Chao; Malone, James; Parkinson, Helen; Liu, Yue; Takatsuki, Terue; Saijo, Kaoru; Masuya, Hiroshi; Nakamura, Yukio; Brush, Matthew H.; Haendel, Melissa A.; Zheng, Jie; Stoeckert, Christian J.; Peters, Bjoern; Mungall, Christopher J.; Carey, Thomas E.; States, David J.; Athey, Brian D.; He, Yongqun


    Background: Cell lines have been widely used in biomedical research. The community-based Cell Line Ontology (CLO) is a member of the OBO Foundry library that covers the domain of cell lines. Since its publication two years ago, significant updates have been made, including new groups joining the CLO

  4. Expression of an accessory cell phenotype by hairy cells during lymphocyte colony formation in agar culture. (United States)

    Farcet, J P; Gourdin, M F; Testa, U; Andre, C; Jouault, H; Reyes, F


    Human T lymphocytes require the cooperation of accessory cells to generate lymphocyte colonies in agar culture under PHA stimulation. Various hairy cell enriched fractions, as well as normal monocytes, have been found to be able to initiate colony formation by normal lymphocytes. Leukemic monocytes from CMML patients were also effective, but not the leukemic lymphocytes from CLL patients. The phenotype expressed by HC in agar colonies was further studied using cell surface and enzymatic markers. We have concluded that HC in agar culture in the presence of both normal T lymphocytes and PHA lose the B phenotype that they express in vivo and function like an accessory cell in contrast to normal or leukemic B lymphocytes.

  5. Langerhans cells and subsets of lymphocytes in the nasal mucosa

    DEFF Research Database (Denmark)

    Hellquist-Dahl, B; Olsen, K E; Irander, K;


    Langerhans cells and different lymphocytes were studied in the nasal mucosa of 39 woodwork teachers and a control group of 14 healthy subjects. Ten of the woodwork teachers were sensitized as determined by skin prick test. A panel of different monoclonal antibodies was applied on the frozen nasal...

  6. Cyclophosphamide, Alvocidib, and Rituximab in Treating Patients With High Risk B-Cell Chronic Lymphocytic Leukemia or Small Lymphocytic Lymphoma (United States)


    Chronic Lymphocytic Leukemia; Prolymphocytic Leukemia; Recurrent Small Lymphocytic Lymphoma; Refractory Chronic Lymphocytic Leukemia; Stage I Chronic Lymphocytic Leukemia; Stage I Small Lymphocytic Lymphoma; Stage II Chronic Lymphocytic Leukemia; Stage II Small Lymphocytic Lymphoma; Stage III Chronic Lymphocytic Leukemia; Stage III Small Lymphocytic Lymphoma; Stage IV Chronic Lymphocytic Leukemia; Stage IV Small Lymphocytic Lymphoma

  7. Apoptosis of matured T lymphocytes induced by mouse sertoli cells in cocultures in vitro

    Institute of Scientific and Technical Information of China (English)

    LIU Yang; LIN Zi-hao; ZHU Xiao-hai; LIU Shan-rong


    Objective: To study whether mouse sertoli cells can induce the apoptosis of matured T lymphocytes in cocultures in vitro. Methods: With TUNEL, DNA electrophoresis, eleetro-mierography and flow cytometry, we examined the apoptosis and its rates of mouse matured T lymphocytes in control group (T lymphocytes only), group A (T lymphocytes + culture medium of sertoli cells), group B (T lymphocytes + sertoli cells). Results: Under electro-micrography, chromatin condensation, karyopyknosis, karyorhexis and apoptotic body were observed in some T lymphocytes in 3 groups; some nucleuses were stained dark blue with TUNEL; a typical DNA ladder was found with DNA electrophoresis. The apoptotic rates of T lymphocytes in group A and B were significantly higher than that in control group (P<0.01). The apoptotic rate of T lymphocytes in group B was significantly higher than that in group A (P<0.01). Conclusion: In coculture condition in vitro,mouse sertoli cells can induce the apoptosis of matured T lymphocytes.

  8. Apicobasal Polarity Controls Lymphocyte Adhesion to Hepatic Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Natalia Reglero-Real


    Full Text Available Loss of apicobasal polarity is a hallmark of epithelial pathologies. Leukocyte infiltration and crosstalk with dysfunctional epithelial barriers are crucial for the inflammatory response. Here, we show that apicobasal architecture regulates the adhesion between hepatic epithelial cells and lymphocytes. Polarized hepatocytes and epithelium from bile ducts segregate the intercellular adhesion molecule 1 (ICAM-1 adhesion receptor onto their apical, microvilli-rich membranes, which are less accessible by circulating immune cells. Upon cell depolarization, hepatic ICAM-1 becomes exposed and increases lymphocyte binding. Polarized hepatic cells prevent ICAM-1 exposure to lymphocytes by redirecting basolateral ICAM-1 to apical domains. Loss of ICAM-1 polarity occurs in human inflammatory liver diseases and can be induced by the inflammatory cytokine tumor necrosis factor alpha (TNF-α. We propose that adhesion receptor polarization is a parenchymal immune checkpoint that allows functional epithelium to hamper leukocyte binding. This contributes to the haptotactic guidance of leukocytes toward neighboring damaged or chronically inflamed epithelial cells that expose their adhesion machinery.

  9. Suiciding of lymphocytic precursor cells by tritiated nucleosides, in vitro. (United States)

    Uyeki, E M; Nishimura, T; Bisel, T U


    Differences in suiciding by various tritiated nucleosides were observed between two functional assays for in vitro lymphocytic precursor cell development, the hemolysin plaque-forming cell (PFC) assay and the B lymphocytic colony-forming cell (CFC-L) assay, using BDF1 mouse spleen cells. PFC growth was markedly reduced by an early (days 0-1) pulse of tritiated deoxyadenosine ([3H]dAdo), but relatively unaffected by a pulse of tritiated thymidine ([3H]dThd) during the same interval. In contrast, CFC-L formation significantly dropped after an early (day 0) [3H]dThd pulse, as well as after pulses of [3H]dAdo and the corresponding tritiated ribosides, uridine and adenosine. This implied a cycling state in an early lymphocytic precursor cell, as opposed to the PFC insensitivity to an early [3H]dThd pulse. The response pattern of colonies and clusters to [3H]dThd supported our notion of a delayed suiciding of CFC contributing to the increase in cluster numbers.

  10. Chronic lymphocytic leukemia/small lymphocytic lymphoma: another neoplasm related to the B-cell follicle? (United States)

    Tandon, Bevan; Swerdlow, Steven H; Hasserjian, Robert P; Surti, Urvashi; Gibson, Sarah E


    Although there has been increased attention paid to the critical nature of nodal involvement in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), the B-cell compartment it is most closely related to and its relationship to the follicle remain uncertain. A clinicopathologic investigation of 60 extramedullary biopsies of LEF1+ CLL/SLL, including 29 cases with perifollicular/follicular (PF/F) growth, was therefore performed. A subset of PF/F cases demonstrated inner mantle zone preservation or intra-mantle zone growth. All PF/F and 16/31 other cases contained CD21+ follicular dendritic cells. No cytogenetic, IGHV mutational or gene usage differences were seen between PF/F and diffuse cases. PF/F cases were more often kappa positive (p<0.03) and had fewer involved nodal sites (p=0.0004). These findings suggest that at least a subset of bona fide CLL/SLL is related to the follicle, most likely the outer mantle zone, and that at least a subset of the diffuse cases may represent "later" disease.

  11. The OP9-DL1 System: Generation of T-Lymphocytes from Embryonic or Hematopoietic Stem Cells In Vitro




    Authors: Roxanne Holmes and Juan Carlos Zúñiga-Pflücker Corresponding author: []() ### INTRODUCTION Differentiation of mouse embryonic stem cells (ESCs) or hematopoietic stem cells (HSCs) from fetal liver or bone marrow into T-lymphocytes can be achieved in vitro with the support of OP9-DL1 cells, a bone-marrow-derived stromal cell line that ectopically expresses the Notch ligand, Delta-like 1 (Dll1). This approach provides a simple, versat...

  12. HTLV-1-infected thymic epithelial cells convey the virus to CD4(+) T lymphocytes. (United States)

    Carvalho Barros, Luciana Rodrigues; Linhares-Lacerda, Leandra; Moreira-Ramos, Klaysa; Ribeiro-Alves, Marcelo; Machado Motta, Maria Cristina; Bou-Habib, Dumith Chequer; Savino, Wilson


    The human T-lymphotropic virus type-1 (HTLV-1) is the causative agent of adult T cell leukemia/lymphoma (ATL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). CD4(+)T cells are the main target of HTLV-1, but other cell types are known to be infected, including immature lymphocytes. Developing T cells undergo differentiation in the thymus, through migration and interaction with the thymic microenvironment, in particular with thymic epithelial cells (TEC) the major component of this three dimensional meshwork of non-lymphoid cells. Herein, we show that TEC express the receptors for HTLV-1 and can be infected by this virus through cell-cell contact and by cell-free virus suspensions. The expression of anti-apoptosis, chemokine and adhesion molecules genes are altered in HTLV-1-infected TEC, although gene expression of antigen presentation molecules remained unchanged. Furthermore, HTLV-1-infected TEC transmitted the virus to a CD4(+) T cell line and to CD4(+) T cells from healthy donors, during in vitro cellular co-cultures. Altogether, our data point to the possibility that the human thymic epithelial cells play a role in the establishment and progression of HTLV-1 infection, functioning as a reservoir and transmitting the virus to maturing CD4(+) T lymphocytes, which in turn will cause disease in the periphery. Copyright © 2017. Published by Elsevier GmbH.

  13. Cell-extrinsic defective lymphocyte development in Lmna(-/- mice.

    Directory of Open Access Journals (Sweden)

    J Scott Hale

    Full Text Available BACKGROUND: Mutations in the LMNA gene, which encodes all A-type lamins, result in a variety of human diseases termed laminopathies. Lmna(-/- mice appear normal at birth but become runted as early as 2 weeks of age and develop multiple tissue defects that mimic some aspects of human laminopathies. Lmna(-/- mice also display smaller spleens and thymuses. In this study, we investigated whether altered lymphoid organ sizes are correlated with specific defects in lymphocyte development. PRINCIPAL FINDINGS: Lmna(-/- mice displayed severe age-dependent defects in T and B cell development which coincided with runting. Lmna(-/- bone marrow reconstituted normal T and B cell development in irradiated wild-type recipients, driving generation of functional and self-MHC restricted CD4(+ and CD8(+ T cells. Transplantation of Lmna(-/- neonatal thymus lobes into syngeneic wild-type recipients resulted in good engraftment of thymic tissue and normal thymocyte development. CONCLUSIONS: Collectively, these data demonstrate that the severe defects in lymphocyte development that characterize Lmna(-/- mice do not result directly from the loss of A-type lamin function in lymphocytes or thymic stroma. Instead, the immune defects in Lmna(-/- mice likely reflect indirect damage, perhaps resulting from prolonged stress due to the striated muscle dystrophies that occur in these mice.

  14. Human lymphocyte markers defined by antibodies derived from somatic cell hybrids. III. A marker defining a subpopulation of lymphocytes which cuts across the normal T-B-null classification. (United States)

    Zola, H; Beckman, I G; Bradley, J; Brooks, D A; Kupa, A; McNamara, P J; Smart, I J; Thomas, M E


    A somatic cell hybrid line which secreted antibody reacting selectively with a proportion of the white cells in human blood was prepared. The hybridoma appeared to be monoclonal, and the antibody secreted stained 67% of the lymphocyte population in blood. It reacted less well with granulocytes and monocytes. The lymphocytes stained comprised 80% of the T cells and 50% of the B cells. The antibody showed no recognizable pattern in its reactivity with cell lines and leukaemic cells, although B cells tended to react less well than T cells, null cells, or myeloid leukaemic cells. The expression of the antigenic determinant is discussed in relation to the classification of leucocytes. This determinant and certain other markers exhibited differential expression on closely related cells, and yet were shared by more distantly related cells.

  15. Immune Killing Activity of Lymphocytes on Hela Cells Expressing Interleukin-12 In Vitro

    Institute of Scientific and Technical Information of China (English)

    Huiyan WANG; Suhua CHEN


    The killing effects of lymphocytes on Hela cells expressing intedeukin-12 (IL-12) in vitro were explored. By using gene transfection technique, full length IL-12 gene was transfected into Hela cells. The expression of IL-12 in Hela cells was detected quantitatively by ELISA; Changes in killing effects of lymphocytes on Hela cells expressing IL-12 were observed by MTT. It was found that Hela cells could express IL-12 between 24h and 72h after transfection. Killing activity of lymphocytes on Hela cells expressing IL-12 was significantly enhanced. It was concluded by cell transfection technique, Hela cells could express IL-12 and were more easily killed by lymphocytes.

  16. Isopentenyl pyrophosphate-activated CD56+ {gamma}{delta} T lymphocytes display potent antitumor activity toward human squamous cell carcinoma. (United States)

    Alexander, Alan A Z; Maniar, Amudhan; Cummings, Jean-Saville; Hebbeler, Andrew M; Schulze, Dan H; Gastman, Brian R; Pauza, C David; Strome, Scott E; Chapoval, Andrei I


    The expression of CD56, a natural killer cell-associated molecule, on alphabeta T lymphocytes correlates with their increased antitumor effector function. CD56 is also expressed on a subset of gammadelta T cells. However, antitumor effector functions of CD56(+) gammadelta T cells are poorly characterized. To investigate the potential effector role of CD56(+) gammadelta T cells in tumor killing, we used isopentenyl pyrophosphate and interleukin-2-expanded gammadelta T cells from peripheral blood mononuclear cells of healthy donors. Thirty to 70% of expanded gammadelta T cells express CD56 on their surface. Interestingly, although both CD56(+) and CD56(-) gammadelta T cells express comparable levels of receptors involved in the regulation of gammadelta T-cell cytotoxicity (e.g., NKG2D and CD94), only CD56(+) gammadelta T lymphocytes are capable of killing squamous cell carcinoma and other solid tumor cell lines. This effect is likely mediated by the enhanced release of cytolytic granules because CD56(+) gammadelta T lymphocytes expressed higher levels of CD107a compared with CD56(-) controls following exposure to tumor cell lines. Lysis of tumor cell lines is blocked by concanamycin A and a combination of anti-gammadelta T-cell receptor + anti-NKG2D monoclonal antibody, suggesting that the lytic activity of CD56(+) gammadelta T cells involves the perforin-granzyme pathway and is mainly gammadelta T-cell receptor/NKG2D dependent. Importantly, CD56-expressing gammadelta T lymphocytes are resistant to Fas ligand and chemically induced apoptosis. Our data indicate that CD56(+) gammadelta T cells are potent antitumor effectors capable of killing squamous cell carcinoma and may play an important therapeutic role in patients with head and neck cancer and other malignancies.

  17. Morphologic studies of lymphocyte nuclei in follicular and diffuse mixed small- and large-cell (lymphocytic-histiocytic) lymphoma. (United States)

    Dardick, I; Caldwell, D R; Moher, D; Jabi, M


    Twelve examples of mixed small- and large-cell lymphoma (eight follicular, one follicular and diffuse, and three diffuse) were investigated morphometrically using plastic-embedded tissue in order to study nuclear characteristics of lymphocyte populations in this form of non-Hodgkin's lymphoma (NHL) and to test morphologic bases for current NHL classification systems. This study illustrates that there are many inaccuracies, illusions, and misconceptions in the morphologic criteria currently used to classify mixed small- and large-cell lymphoma. A principal finding was that lymphocyte nuclear profiles in mixed-cell lymphomas tend to be smaller in size (P less than .005) and more irregular in shape (P = .0001) than the morphologically similar counterparts in germinal centers of lymph nodes with reactive hyperplasia. Intercase comparison of mixed small- and large-cell lymphomas revealed a considerable range of mean nuclear area values, some of which were within the size range of normal, small lymphocytes. At the magnifications used for morphometric assessment, a high proportion of lymphocyte nuclear profiles had shallow invaginations, but only a limited number of profiles (4% to 14%) had deep (cleaved) indentations. Contrary to current definitions for this subtype of NHL, lymphocytes with "small" nuclei had the same proportion of the nuclear diameter occupied by nuclear invaginations as lymphocytes with "large" nuclei and, in fact, mean nuclear invagination depth was shallower in "small" nuclei than in "large" nuclei. Furthermore, regardless of whether it is nuclear area or shape that is evaluated, lymphocytes in mixed-cell lymphoma do not separate into two populations of small-cleaved and large noncleaved cells. Morphometry reveals that only four of the 12 examples of mixed small- and large-cell lymphoma had a proportion of the lymphocytes in the size range of fully transformed germinal center lymphocytes that exceeded 25%, and none of the cases approached 50% even


    NARCIS (Netherlands)



    A population of B cells with characteristics of chronic lymphocytic leukaemia was found in the peripheral blood of four patients who presented with cutaneous infiltration of atypical CD4+ T cells with cerebriform nuclei. The B cells had a low density of immunoglobulin on their surface membrane, expr

  19. Radiation sensitivity of Merkel cell carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Leonard, J.H.; Ramsay, J.R.; Birrell, G.W. [Queensland Institute of Medical Research (Australia)] [and others


    Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT) assay was used for these lines, to estimate cell growth after {gamma} irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to {gamma} irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution. 25 refs., 3 figs., 1 tab.

  20. Activated allogeneic NK cells preferentially kill poor prognosis B-cell chronic lymphocytic leukemia cells



    Mutational status of TP53 together with expression of wild type (wt) IGHV represents the most widely accepted biomarkers, establishing a very poor prognosis in B-cell chronic lymphocytic leukemia (B-CLL) patients. Adoptive cell therapy using allogeneic HLA mismatched Natural Killer (NK) cells has emerged as an effective and safe alternative in the treatment of acute myeloid and lymphoid leukemias that do not respond to traditional therapies. We have described that allogeneic activated NK cell...

  1. In vitro radiosensitivity of human leukemia cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Weichselbaum, R.R.; Greenberger, J.S.; Schmidt, A.; Karpas, A.; Moloney, W.C.; Little, J.B.


    The in vitro radiobiologic survival values (n, D0) of four tumor lines derived from human hematopoietic tumors were studied. These cell lines were HL50 (n . 1.3, D0 . 117 rad(1.17 Gy)), promyelocytic leukemia; K562 (n . 1.4, D0 . 165 rad(1.65 Gy)), erythroleukemia; 45 (n . 1.1, D0 . 147 rad(1.47 Gy)), acute lymphocyte leukemia; and 176 (n . 4.0, D0 . 76 rad(0.76 Gy)), acute monomyelogenous leukemia. More cell lines must be examined before the exact relationship between in vitro radiosensitivity and clinical radiocurability is firmly established.

  2. The Importance of the Nurse Cells and Regulatory Cells in the Control of T Lymphocyte Responses

    Directory of Open Access Journals (Sweden)

    María Guadalupe Reyes García


    Full Text Available T lymphocytes from the immune system are bone marrow-derived cells whose development and activities are carefully supervised by two sets of accessory cells. In the thymus, the immature young T lymphocytes are engulfed by epithelial “nurse cells” and retained in vacuoles, where most of them (95% are negatively selected and removed when they have an incomplete development or express high affinity autoreactive receptors. The mature T lymphocytes that survive to this selection process leave the thymus and are controlled in the periphery by another subpopulation of accessory cells called “regulatory cells,” which reduce any excessive immune response and the risk of collateral injuries to healthy tissues. By different times and procedures, nurse cells and regulatory cells control both the development and the functions of T lymphocyte subpopulations. Disorders in the T lymphocytes development and migration have been observed in some parasitic diseases, which disrupt the thymic microenvironment of nurse cells. In other cases, parasites stimulate rather than depress the functions of regulatory T cells decreasing T-mediated host damages. This paper is a short review regarding some features of these accessory cells and their main interactions with T immature and mature lymphocytes. The modulatory role that neurotransmitters and hormones play in these interactions is also revised.

  3. Suppressor cell activity in a proliferative disorder of T lymphocytes. (United States)

    Kupa, A; Thomas, M E; Moore, H; Bradley, J; Zola, H; Hooper, M; Harding, P


    We report details of the immunological profile of a patient with the candidiasis endocrinopathy syndrome who has developed T-type chronic lymphocytic leukaemia. The patient is anergic to a panel of delayed hypersensitivity skin tests, and has poor in vitro mitogenic responses, but B cell function in vivo is not impaired. Subsequent functional studies have revealed that cells from the patient have a significant suppressive effect in coculture (P less than 0.05) on the responses of healthy donor lymphocytes (NR) to the mitogen phytohaemagglutinin (PHA). A degree of selectivity for the suppressive effect is suggested by the lack of similar effects on coculture responses to the mitogens concanavalin A (Con A) and pokeweed mitogen (PWM). Mitomycin C treatment of the patient's cells reduced their suppressive activity but significant suppression was still observed in the majority of PHA cocultures. The suppressor activity required the presence of the patient's cells in cocultures, as no suppression was observed when the patient's serum or cell culture supernatant were included instead of the patient's cells in NR cultures.

  4. Defining Molecular Phenotypes of Mesenchymal and hematopoietic Stem Cells derived from Peripheral blood of Acute Lymphocytic Leukemia patients for regenerative stem cell therapy. (United States)

    Potdar, Pd; Subedi, Rp


    Acute Lymphocytic Leukemia (ALL) is a clonal myeloid disorder affecting all age groups, characterized by accumulation of immature blast cells in bone marrow and in peripheral blood. Autologous Bone Marrow Transplantation is a present treatment for cure of ALL patients, which is very expensive, invasive process and may have possibility of transplantation of malignant stem cells to patients. In the present study, we hypothesized to isolate large number of normal Mesenchymal & Hematopoietic stem cells from peripheral blood of ALL patients, which will be further characterized for their normal phenotypes by using specific molecular stem cell markers. This is the first study, which defines the existing phenotypes of isolated MSCs and HSCs from peripheral blood of ALL patients. We have established three cell lines in which two were Mesenchymal stem cells designated as MSCALL and MSCnsALL and one was suspension cell line designated as HSCALL. The HSCALL cell line was developed from the lymphocyte like cells secreted by MSCALL cells. Our study also showed that MSCALL from peripheral blood of ALL patient secreted hematopoietic stem cells in vitro culture. We have characterized all three-cell lines by 14 specific stem cell molecular markers. It was found that both MSC cell lines expressed CD105, CD13, and CD73 with mixed expression of CD34 and CD45 at early passage whereas, HSCALL cell line expressed prominent feature of hematopoietic stem cells such as CD34 and CD45 with mild expression of CD105 and CD13. All three-cell lines expressed LIF, OCT4, NANOG, SOX2, IL6, and DAPK. These cells mildly expressed COX2 and did not express BCR-ABL. Overall it was shown that isolated MSCs and HSCs can be use as a model system to study the mechanism of leukemia at stem cell level and their use in stem cell regeneration therapy for Acute Lymphocytic Leukemia.

  5. MicroRNA-26a-mediated regulation of interleukin-2 expression in transformed avian lymphocyte lines

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    Smith Lorraine P


    Full Text Available Abstract Background Micro(miRNAs are a class of small non-coding RNAs that play critical roles in the induction of various cancers, including lymphomas induced by oncogenic viruses. While some of the miRNAs are oncogenic, miRNAs such as miR-26a are consistently downregulated in a number of cancers, demonstrating their potential tumor suppressor functions. Global miRNA expression profiles of a number of virus-transformed avian lymphoma cell lines have shown downregulation of gga-miR-26a expression, irrespective of molecular mechanisms of transformation or the viral aetiology. The neoplastic transformation of lymphocytes by many viruses accompanies high levels of proliferative responses, mostly mediated through cytokines such as IL-2. Chicken IL-2 can modulate T-cell proliferation and cytotoxicity in vitro and in vivo and dysregulation of IL-2 expression is observed in diseases such as leukaemia. Results The expression levels of gga-miR-26a in chicken lymphoma cells transformed by 3 distinct avian oncogenic viruses, viz Marek's disease virus (MDV, avian leukosis virus (ALV and Reticuloendotheliosis virus (REV were consistently downregulated compared to the levels in the normal lymphocytes. This downregulation of miR-26a regardless of the viral etiology and molecular mechanisms of transformation was consistent with the tumor suppressor role of this miRNA. Notwithstanding this well-established role in cancer, we demonstrate the additional role of this miRNA in directly targeting chicken IL-2 through reporter and biochemical assays. The downregulation of miR-26a can relieve the suppressive effect of this miRNA on IL-2 expression. Conclusions We show that miR-26a is globally downregulated in a number of avian lymphoma cells irrespective of the mechanisms of transformation, reiterating the highly conserved tumor suppressor function of this miRNA. However, with the potential for directly targeting chicken IL-2, the downregulation of miR-26a in these

  6. Regulation of DNA synthesis and the cell cycle in human prostate cancer cells and lymphocytes by ovine uterine serpin

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    Hansen Peter J


    Full Text Available Abstract Background Uterine serpins are members of the serine proteinase inhibitor superfamily. Like some other serpins, these proteins do not appear to be functional proteinase inhibitors. The most studied member of the group, ovine uterine serpin (OvUS, inhibits proliferation of several cell types including activated lymphocytes, bovine preimplantation embryos, and cell lines for lymphoma, canine primary osteosarcoma and human prostate cancer (PC-3 cells. The goal for the present study was to evaluate the mechanism by which OvUS inhibits cell proliferation. In particular, it was tested whether inhibition of DNA synthesis in PC-3 cells involves cytotoxic actions of OvUS or the induction of apoptosis. The effect of OvUS in the production of the autocrine and angiogenic cytokine interleukin (IL-8 by PC-3 cells was also determined. Finally, it was tested whether OvUS blocks specific steps in the cell cycle using both PC-3 cells and lymphocytes. Results Recombinant OvUS blocked proliferation of PC-3 cells at concentrations as low as 8 μg/ml as determined by measurements of [3H]thymidine incorporation or ATP content per well. Treatment of PC-3 cells with OvUS did not cause cytotoxicity or apoptosis or alter interleukin-8 secretion into medium. Results from flow cytometry experiments showed that OvUS blocked the entry of PC-3 cells into S phase and the exit from G2/M phase. In addition, OvUS blocked entry of lymphocytes into S phase following activation of proliferation with phytohemagglutinin. Conclusion Results indicate that OvUS acts to block cell proliferation through disruption of the cell cycle dynamics rather than induction of cytotoxicity or apoptosis. The finding that OvUS can regulate cell proliferation makes this one of only a few serpins that function to inhibit cell growth.

  7. Activation-induced cell death in B lymphocytes

    Institute of Scientific and Technical Information of China (English)


    Upon encountering the antigen (Ag), the immune system can either develop a specific immune response or enter a specific state of unresponsiveness, tolerance. The response of B cells to their specific Ag can be activation and proliferation, leading to the immune response, or anergy and activation-induced cell death (AICD), leading to tolerance. AICD in B lymphocytes is a highly regulated event initiated by crosslinking of the B cell receptor (BCR). BCR engagement initiates several signaling events such as activation of PLCγ, Ras, and PI3K, which generally speaking, lead to survival However, in the absence of survival signals (CD40 or IL-4R engagement), BCR crosslinking can also promote apoptotic signal transduction pathways such as activation of effector caspases, expression of pro-apoptotic genes, and inhibition of pro-survival genes. The complex interplay between survival and death signals determines the B cell fate and, consequently, the immune response.

  8. Donor lymphocyte infusion after allogeneic stem cell transplantation. (United States)

    Castagna, Luca; Sarina, Barbara; Bramanti, Stefania; Perseghin, Paolo; Mariotti, Jacopo; Morabito, Lucio


    Allogeneic stem cell transplantation (allo-SCT) is considered the cornerstone in the treatment of several malignant and not malignant hematological diseases. However, relapse of hematological disease after allo-SCT is considered the most challenging point in the field. The risk can be reduced through optimal patients, donor and disease selection before allo-SCT, but harnessing donor immune system is an appealing way to treat or avoid disease relapse. Donor lymphocyte infusion (DLI) is a simple and effective therapy after allo-SCT. In this paper, the efficacy of DLI will be analyzed in different hematological diseases, focusing also on their therapeutic or pre-emptive use.

  9. Activation of secretion and surface alteration of cytolytic T-lymphocytes interacting with target cells. (United States)

    Bykovskaya, S N; Shevelev, A A; Kupriyanova, T A


    Cells obtained in mixed lymphocyte culture (MLC) and memory cells adsorbed on the surface of target cells (TC) were examined using scanning and transmission electron microscopy depending on the time of interaction with TC. Three types of lymphocytes were revealed: type I - cells of spherical shape with a smooth surface or an insignificant amount of microvilli; predominantly small and medium-sized lymphocytes contacting TC with non significant involvement of their surface or by several microvilli; type II - oval or round-shaped lymphocytes evenly covered with microvilli with considerably enlarged region of contact; type III cells - predominantly large lymphocytes and lymphoblasts flattened (spread) on TC, with multiple microvilli, ridge-like projections, and ruffles on their surface. TEM revealed activation of the secretory apparatus in the cytoplasm of such lymphocytes. With increased time of interaction, type III cells increase in number (from 8.6% after 10 min to 90.2% after 60 min of incubation). Memory cells show no morphologic signs of secretion in correlation with the absence of lysis of TC on which they are adsorbed. The surface of the lymphocytes adsorbed on the substrate with poly-L-lysin is not noticeably altered. It is suggested that 3 morphological types of lymphocytes correspond to 3 stages of secretion activation. Lymphocyte contact with TC surface is evidently a specific stimulus for activating secretory apparatus of CTL. SEM can be used for quantitation of activated lymphocytes.

  10. Small B cell lymphocytic lymphoma presenting as obstructive sleep apnea

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    Chang Weng-Cheng


    Full Text Available Abstract Background Most lymphomas that involve the tonsil are large B cell lymphomas. Large B-cell lymphoma is a high grade malignancy which progresses rapidly. Tonsillar lymphoma usually presents as either a unilaterally enlarged palatine tonsil or as an ulcerative and fungating lesion over the tonsillar area. Small lymphocytic lymphomas (SLL of the Waldeyer's ring are uncommon. Case presentation We report a 41-year-old male who presented with a ten-year history of snoring. Physical examination revealed smooth bilateral symmetrically enlarged tonsils without abnormal surface change or cervical lymphadenopathy. Palatal redundancy and a narrowed oropharyngeal airway were also noted. The respiratory disturbance index (RDI was 66 per hour, and severe obstruction sleep apnea (OSA was suspected. No B symptoms, sore throat, odynophagia or dysphagia was found. We performed uvulopalatopharyngoplasty (UPPP and pathological examination revealed incidental small B-cell lymphocytic lymphoma (SLL. Conclusion It is uncommon for lymphoma to initially present as OSA. SLL is an indolent malignancy and is not easy to detect in the early stage. We conclude that SLL may be a contributing factor of OSA in the present case.


    Directory of Open Access Journals (Sweden)

    Giovanni D'arena


    Full Text Available Regulatory T-cells (Tregs constitute a small subset of cells that are actively involved in maintaining self-tolerance, in immune homeostasis and in antitumor immunity. They are thought to play a significant role in the progression of cancer and are generally increased in patient with chronic lymphocytic leukemia (CLL. Their number correlates with more aggressive disease status and is predictive of the time to treatment, as well. Moreover, it is now clear that dysregulation in Tregs cell frequency and/or function may result in a plethora of autoimmune diseases, including multiple sclerosis, type 1 diabetes mellitus, myasthenia gravis, systemic lupus erythematosis, autoimmune lymphoproliferative disorders, rheumatoid arthritis, and psoriasis. Efforts are made aiming to develop approaches to deplete Tregs or inhibit their function in either cancer and autoimmune disorders.

  12. Small lymphocytic lymphoma with Reed Sternberg cells: a diagnostic dilemma. (United States)

    Pervez, S; Abro, B; Shahbaz, H


    Reed Sternberg (RS) cells in the setting of small lymphocytic lymphoma (SLL) can complicate the histopathological diagnosis. We report a case of a man aged 54 years who presented with cervical lymphadenopathy. Resection of the lymph node was performed and sent for histopathological evaluation to a local laboratory. A diagnosis of SLL with Classic Hodgkin's lymphoma (CHL) was made. The medical oncologist who encountered this diagnosis for the first time sent the biopsy blocks to our laboratory for a second opinion. On review of the biopsy and immunohistochemical stains, it showed typical SLL morphology and immunophenotype. Focally, it showed large mononuclear RS type cells; however, no typical background of CHL was seen. The diagnosis was revised to 'SLL with RS like cells with no convincing evidence of CHL'. The patient was subsequently treated as a case of SLL and no progression was observed on a follow-up of 5 years. 2017 BMJ Publishing Group Ltd.

  13. Docosahexaenoic acid induces apoptosis in primary chronic lymphocytic leukemia cells

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    Romain Guièze


    Full Text Available Chronic lymphocytic leukemia is an indolent disorder with an increased infectious risk remaining one of the main causes of death. Development of therapies with higher safety profile is thus a challenging issue. Docosahexaenoic acid (DHA, 22:6 is an omega-3 fatty acid, a natural compound of normal cells, and has been shown to display antitumor potency in cancer. We evaluated the potential in vitro effect of DHA in primary CLL cells. DHA induces high level of in vitro apoptosis compared to oleic acid in a dose-dependent and time-dependent manner. Estimation of IC50 was only of 4.813 μM, which appears lower than those reported in solid cancers. DHA is highly active on CLL cells in vitro. This observation provides a rationale for further studies aiming to understand its mechanisms of action and its potent in vivo activity.


    Institute of Scientific and Technical Information of China (English)

    刘长安; 管增伟; 孙武; 邵玉霞; 李卓; 贾廷珍


    Objective To study on the expression and clinical significance of T lymphocyte subset and NK cell activity (NKA) in patients with colorectal cancer. Methods Fifty-seven cancer patients and 33 healthy controls were enrolled in this study. T lymphocyte subset was measured by SAP technique and NKA by LDH release assay based on K562 cells, which served as target cells.

  15. CD57+ T-cells are a subpopulation of T-follicular helper cells in nodular lymphocyte predominant Hodgkin lymphoma

    NARCIS (Netherlands)

    Sattarzadeh, Ahmad; Diepstra, Arjan; Rutgers, Bea; van den Berg, Anke; Visser, Lydia


    BACKGROUND: Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) is characterized by lymphocyte-predominant (LP) cells in a background of CD4+ CD57+ T-cells. These cells are normally present in the germinal center of lymphoid tissues. The cells rosetting LP cells are described to be PD-1 and

  16. CD57+ T-cells are a subpopulation of T-follicular helper cells in nodular lymphocyte predominant Hodgkin lymphoma

    NARCIS (Netherlands)

    Sattarzadeh, Ahmad; Diepstra, Arjan; Rutgers, Bea; van den Berg, Anke; Visser, Lydia


    BACKGROUND: Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) is characterized by lymphocyte-predominant (LP) cells in a background of CD4+ CD57+ T-cells. These cells are normally present in the germinal center of lymphoid tissues. The cells rosetting LP cells are described to be PD-1 and BCL-

  17. Gamma c-signaling cytokines induce a regulatory T cell phenotype in malignant CD4+ T lymphocytes

    DEFF Research Database (Denmark)

    Kasprzycka, Monika; Zhang, Qian; Witkiewicz, Agnieszka


    In this study, we demonstrate that malignant mature CD4(+) T lymphocytes derived from cutaneous T cell lymphomas (CTCL) variably display some aspects of the T regulatory phenotype. Whereas seven cell lines representing a spectrum of primary cutaneous T cell lymphoproliferative disorders expressed...... that the T regulatory cell features are induced in CTCL T cells by common gamma chain signaling cytokines such as IL-2 and do not represent a fully predetermined, constitutive phenotype independent of the local environmental stimuli to which these malignant mature CD4(+) T cells become exposed....

  18. The kinematics of cytotoxic lymphocytes influence their ability to kill target cells. (United States)

    Bhat, Purnima; Leggatt, Graham; Matthaei, Klaus I; Frazer, Ian H


    Cytotoxic lymphocytes (CTL) have been reported to show a range of motility patterns from rapid long-range tracking to complete arrest, but how and whether these kinematics affect their ability to kill target cells is not known. Many in vitro killing assays utilize cell lines and tumour-derived cells as targets, which may be of limited relevance to the kinetics of CTL-mediated killing of somatic cells. Here, live-cell microscopy is used to examine the interactions of CTL and primary murine skin cells presenting antigens. We developed a qualitative and quantitative killing assay using extended-duration fluorescence time-lapse microscopy coupled with large-volume objective software-based data analysis to obtain population data of cell-to-cell interactions, motility and apoptosis. In vivo and ex vivo activated antigen-specific cytotoxic lymphocytes were added to primary keratinocyte targets in culture with fluorometric detection of caspase-3 activation in targets as an objective determinant of apoptosis. We found that activated CTL achieved contact-dependent apoptosis of non-tumour targets after a period of prolonged attachment - on average 21 hours - which was determined by target cell type, amount of antigen, and activation status of CTL. Activation of CTL even without engagement of the T cell receptor was sufficient to mobilise cells significantly above baseline, while the addition of cognate antigen further enhanced their motility. Highly activated CTL showed markedly increased vector displacement, and velocity, and lead to increased antigen-specific target cell death. These data show that the inherent kinematics of CTL correlate directly with their ability to kill non-tumour cells presenting cognate antigen.

  19. The kinematics of cytotoxic lymphocytes influence their ability to kill target cells.

    Directory of Open Access Journals (Sweden)

    Purnima Bhat

    Full Text Available Cytotoxic lymphocytes (CTL have been reported to show a range of motility patterns from rapid long-range tracking to complete arrest, but how and whether these kinematics affect their ability to kill target cells is not known. Many in vitro killing assays utilize cell lines and tumour-derived cells as targets, which may be of limited relevance to the kinetics of CTL-mediated killing of somatic cells. Here, live-cell microscopy is used to examine the interactions of CTL and primary murine skin cells presenting antigens. We developed a qualitative and quantitative killing assay using extended-duration fluorescence time-lapse microscopy coupled with large-volume objective software-based data analysis to obtain population data of cell-to-cell interactions, motility and apoptosis. In vivo and ex vivo activated antigen-specific cytotoxic lymphocytes were added to primary keratinocyte targets in culture with fluorometric detection of caspase-3 activation in targets as an objective determinant of apoptosis. We found that activated CTL achieved contact-dependent apoptosis of non-tumour targets after a period of prolonged attachment - on average 21 hours - which was determined by target cell type, amount of antigen, and activation status of CTL. Activation of CTL even without engagement of the T cell receptor was sufficient to mobilise cells significantly above baseline, while the addition of cognate antigen further enhanced their motility. Highly activated CTL showed markedly increased vector displacement, and velocity, and lead to increased antigen-specific target cell death. These data show that the inherent kinematics of CTL correlate directly with their ability to kill non-tumour cells presenting cognate antigen.

  20. Thyroid cell lines in research on goitrogenesis. (United States)

    Gerber, H; Peter, H J; Asmis, L; Studer, H


    Thyroid cell lines have contributed a lot to the understanding of goitrogenesis. The cell lines mostly used in thyroid research are briefly discussed, namely the rat thyroid cell lines FRTL and FRTL-5, the porcine thyroid cell lines PORTHOS and ARTHOS, The sheep thyroid cell lines OVNIS 5H and 6H, the cat thyroid cell lines PETCAT 1 to 4 and ROMCAT, and the human thyroid cell lines FTC-133 and HTh 74. Chinese hamster ovary (CHO) cells and COS-7 cells, stably transfected with TSH receptor cDNA and expressing a functional TSH receptor, are discussed as examples for non-thyroidal cells, transfected with thyroid genes.

  1. Cell entry of lymphocytic choriomeningitis virus is restricted in myotubes. (United States)

    Iwasaki, Masaharu; Urata, Shuzo; Cho, Yoshitake; Ngo, Nhi; de la Torre, Juan C


    In mice persistently infected since birth with the prototypic arenavirus lymphocytic choriomeningitis viurs, viral antigen and RNA are readily detected in most organs and cell types but remarkably absent in skeletal muscle. Here we report that mouse C2C12 myoblasts that are readily infected by LCMV, become highly refractory to LCMV infection upon their differentiation into myotubes. Myotube's resistance to LCMV was not due to an intracellular restriction of virus replication but rather an impaired cell entry mediated by the LCMV surface glycoprotein. Our findings provide an explanation for the observation that in LCMV carrier mice myotubes, which are constantly exposed to blood-containing virus, remain free of viral antigen and RNA despite myotubes express high levels of the LCMV receptor alpha dystroglycan and do not pose an intracellular blockade to LCMV multiplication.

  2. T lymphocytes and NK cells in X-linked agammaglobulinemia. (United States)

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


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

  3. The Stromal Microenvironment Modulates Mitochondrial Oxidative Phosphorylation in Chronic Lymphocytic Leukemia Cells

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    Hima V. Vangapandu


    Full Text Available Peripheral blood chronic lymphocytic leukemia (CLL cells are replicationally quiescent mature B-cells. In short-term cultures, supporting stromal cells provide a survival advantage to CLL cells by inducing transcription and translation without promoting proliferation. We hypothesized that the stromal microenvironment augments malignant B cells' metabolism to enable the cells to cope with their energy demands for transcription and translation. We used extracellular flux analysis to assess the two major energy-generating pathways, mitochondrial oxidative phosphorylation (OxPhos and glycolysis, in primary CLL cells in the presence of three different stromal cell lines. OxPhos, measured as the basal oxygen consumption rate (OCR and maximum respiration capacity, was significantly higher in 28 patients' CLL cells cocultured with bone marrow–derived NK.Tert stromal cells than in CLL cells cultured alone (P = .004 and <.0001, respectively. Similar OCR induction was observed in CLL cells cocultured with M2-10B4 and HS-5 stromal lines. In contrast, heterogeneous changes in the extracellular acidification rate (a measure of glycolysis were observed in CLL cells cocultured with stromal cells. Ingenuity Pathway Analysis of CLL cells' metabolomics profile indicated stroma-mediated stimulation of nucleotide synthesis. Quantitation of ribonucleotide pools showed a significant two-fold increase in CLL cells cocultured with stromal cells, indicating that the stroma may induce CLL cellular bioenergy and the RNA building blocks necessary for the transcriptional requirement of a prosurvival phenotype. The stroma did not impact the proliferation index (Ki-67 staining of CLL cells. Collectively, these data suggest that short-term interaction (≤24 hours with stroma increases OxPhos and bioenergy in replicationally quiescent CLL cells.

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

    Energy Technology Data Exchange (ETDEWEB)

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


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

  5. PHA-induced cytotoxicity of human lymphocytes against adherent hela-cells

    NARCIS (Netherlands)

    Huges-Law, G.; de Gast, G. C.; The, T. Hauw


    The conditions for a phytohaemagglutinin(PHA)-induced cytotoxicity test of human peripheral blood lymphocytes were investigated. [3H]thymidine prelabelled HeLa cells were used as target cells. Stimulation with 10 μl PHA/ml during 24 h gave the best measure of lymphocyte cytotoxic capacity. Supernata

  6. Transcriptional Landscape of Human Tissue Lymphocytes Unveils Uniqueness of Tumor-Infiltrating T Regulatory Cells

    NARCIS (Netherlands)

    De Simone, M.; Arrigoni, A.; Rossetti, G.; Gruarin, P.; Ranzani, V.; Politano, C.; Bonnal, R.J.; Provasi, E.; Sarnicola, M.L.; Panzeri, I.; Moro, M.; Crosti, M.; Mazzara, S.; Vaira, V.; Bosari, S.; Palleschi, A.; Santambrogio, L.; Bovo, G.; Zucchini, N.; Totis, M.; Gianotti, L.; Cesana, G.; Perego, R.A.; Maroni, N.; Pisani Ceretti, A.; Opocher, E.; De Francesco, R.; Geginat, J.; Stunnenberg, H.; Abrignani, S.; Pagani, M.


    Tumor-infiltrating regulatory T lymphocytes (Treg) can suppress effector T cells specific for tumor antigens. Deeper molecular definitions of tumor-infiltrating-lymphocytes could thus offer therapeutic opportunities. Transcriptomes of T helper 1 (Th1), Th17, and Treg cells infiltrating colorectal or

  7. Transcriptional Landscape of Human Tissue Lymphocytes Unveils Uniqueness of Tumor-Infiltrating T Regulatory Cells

    NARCIS (Netherlands)

    De Simone, M.; Arrigoni, A.; Rossetti, G.; Gruarin, P.; Ranzani, V.; Politano, C.; Bonnal, R.J.; Provasi, E.; Sarnicola, M.L.; Panzeri, I.; Moro, M.; Crosti, M.; Mazzara, S.; Vaira, V.; Bosari, S.; Palleschi, A.; Santambrogio, L.; Bovo, G.; Zucchini, N.; Totis, M.; Gianotti, L.; Cesana, G.; Perego, R.A.; Maroni, N.; Pisani Ceretti, A.; Opocher, E.; De Francesco, R.; Geginat, J.; Stunnenberg, H.; Abrignani, S.; Pagani, M.


    Tumor-infiltrating regulatory T lymphocytes (Treg) can suppress effector T cells specific for tumor antigens. Deeper molecular definitions of tumor-infiltrating-lymphocytes could thus offer therapeutic opportunities. Transcriptomes of T helper 1 (Th1), Th17, and Treg cells infiltrating colorectal or

  8. Carotenoids located in human lymphocyte subpopulations and Natural Killer cells by Raman microspectroscopy

    NARCIS (Netherlands)

    Puppels, G.J.; Puppels, G.J.; Garritsen, H.S.P.; Garritsen, H.S.P.; Kummer, J.A.; Greve, Jan


    The presence and subcellular location of carotenoids in human lymphocyte sub-populations (CD4+, CD8+, T-cell receptor-γδ+, and CD19+ ) and natural killer cells (CD16+ ) were studied by means of Raman microspectroscopy. In CD4+ lymphocytes a high concentration (10-3M) of carotenoids was found in the

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


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

  10. Modeling cancer immunotherapy: Assessing the effects of lymphocytes on cancer cell growth and motility (United States)

    Ramos, R. A.; Zapata, Jair; Condat, C. A.; Deisboeck, Thomas S.


    A mesoscopic model is used to describe the effects of lymphocyte activity on a growing tumor. The model yields novel insights into the tumor-immune system interaction. In particular, we found that the presence of a putative chemotactic messenger that helps guide the lymphocytes towards the tumor is not critical to elicit the anti-tumor effects of the immune system, while lymphocytes that block tumor cell migration contribute to limit cancer expansion and thus have a more significant therapeutic impact.

  11. Activation of lymphocytes induced by bronchial epithelial cells with prolonged RSV infection.

    Directory of Open Access Journals (Sweden)

    Ling Qin

    Full Text Available Respiratory syncytial virus (RSV preferentially infects airway epithelial cells,which might be responsible for susceptibility to asthma; however, the underlying mechanism is not clear. This study determined the activation of lymphocytes and drift of helper T (Th subsets induced by RSV-infected human bronchial epithelial cells (HBECs in vitro. HBECs had prolonged infection with RSV, and lymphocytes isolated from human peripheral blood were co-cultured with RSV-infected HBECs. Four groups were established, as follows: lymphocytes (group L; lymphocytes infected with RSV (group RL; co-culture of lymphocytes with non-infected HBECs (group HL; and co-culture of lymphocytes with infected HBECs (group HRL. After co-culture with HBECs for 24 hours, lymphocytes were collected and the following were determined in the 4 groups: cell cycle status; apoptosis rate; and concentrations of IL-4, IFN-γ, and IL-17 in the supernatants. Cell cycle analysis for lymphocytes showed a significant increase in S phase cells, a decrease in G1 phase cells, and a higher apoptosis rate in group HRL compared with the other three groups. In group HRL, the levels of IL-4, IFN-γ, and IL-17 in supernatants were also higher than the other three groups. For further study, lymphocytes were individually treated with supernatants from non-infected and RSV-infected HBECs for 24 h. We showed that supernatants from RSV-infected HBECs induced the differentiation of Th2 and Th17 subsets, and suppressed the differentiation of Treg subsets. Our results showed that HBECs with prolonged RSV infection can induce lymphocyte proliferation and apoptosis, and enhance the release of cytokines by lymphocytes. Moreover, subset drift might be caused by RSV-infected HBECs.

  12. Idelalisib therapy of indolent B-cell malignancies: chronic lymphocytic leukemia and small lymphocytic or follicular lymphomas

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    Madanat YF


    Full Text Available Yazan F Madanat,1 Mitchell R Smith,2 Alexandru Almasan,3 Brian T Hill2 1Department of Internal Medicine, 2Department of Hematology and Medical Oncology, Taussig Cancer Institute, 3Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Abstract: Chronic lymphocytic leukemia, small lymphocytic lymphoma, and follicular lymphoma are indolent B-cell lymphoproliferative disorders that mainly affect an older population. Although the majority of patients in need of treatment derive significant benefit from conventional chemotherapeutic agents as well as monoclonal antibodies, less toxic and more effective treatments are needed. Novel agents that inhibit the B-cell receptor signaling pathway have shown promising outcomes in these disorders. Idelalisib is a potent selective oral inhibitor of phosphatidylinositol 3-kinase delta and has shown significant clinical activity in B-cell malignancies. In this review, we summarize the clinical trial data using idelalisib as monotherapy or in combination with rituximab for the treatment of relapsed/refractory disease. The adverse effect profile includes autoimmune disorders such as transaminitis, colitis, and pneumonitis. Given the efficacy and manageable toxicity profile of idelalisib, it is being increasingly incorporated into the management of indolent B-cell malignancies. Keywords: idelalisib, PI3Kδ inhibitors, chronic lymphocytic leukemia, follicular lymphoma

  13. Kefir induces cell-cycle arrest and apoptosis in HTLV-1-negative malignant T-lymphocytes

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    Katia Maalouf


    Full Text Available Katia Maalouf1, Elias Baydoun2, Sandra Rizk11Department of Natural Sciences, Lebanese American University, Beirut, Lebanon; 2Department of Biology, American University of Beirut, Beirut, LebanonBackground: Adult lymphoblastic leukemia (ALL is a malignancy that occurs in white blood cells. The overall cure rate in children is 85%, whereas it is only 40% in adults. Kefir is an important probiotic that contains many bioactive ingredients, which give it unique health benefits. It has been shown to control several cellular types of cancer.Purpose: The present study investigates the effect of a cell-free fraction of kefir on CEM and Jurkat cells, which are human T-lymphotropic virus type I (HTLV-1-negative malignant T-lymphocytes.Methods: Cells were incubated with different kefir concentrations. The cytotoxicity of the compound was evaluated by determining the percentage viability of cells. The effect of all the noncytotoxic concentrations of kefir on the proliferation of CEM and Jurkat cells was then assessed. The levels of transforming growth factor-alpha (TGF-α, transforming growth factor- beta1 (TGF-β1, matrix metalloproteinase-2 (MMP-2, and MMP-9 mRNA upon kefir treatment were then analyzed using reverse transcriptase polymerase chain reaction (RT-PCR. Finally, the growth inhibitory effects of kefir on cell-cycle progression/apoptosis were assessed by Cell Death Detection (ELISA and flow cytometry.Results: The maximum cytotoxicity recorded after 48-hours treatment with 80 µg/µL kefir was only 42% and 39% in CEM and Jurkat cells, respectively. The percent reduction in proliferation was very significant, and was dose-, and time-dependent. In both cell lines, kefir exhibited its antiproliferative effect by downregulating TGF-α and upregulating TGF- β1 mRNA expression. Upon kefir treatment, a marked increase in cell-cycle distribution was noted in the preG1 phase of CEM and Jurkat cells, indicating the proapoptotic effect of kefir, which was

  14. Kefir induces cell-cycle arrest and apoptosis in HTLV-1-negative malignant T-lymphocytes. (United States)

    Maalouf, Katia; Baydoun, Elias; Rizk, Sandra


    Adult lymphoblastic leukemia (ALL) is a malignancy that occurs in white blood cells. The overall cure rate in children is 85%, whereas it is only 40% in adults. Kefir is an important probiotic that contains many bioactive ingredients, which give it unique health benefits. It has been shown to control several cellular types of cancer. The present study investigates the effect of a cell-free fraction of kefir on CEM and Jurkat cells, which are human T-lymphotropic virus type I (HTLV-1)-negative malignant T-lymphocytes. Cells were incubated with different kefir concentrations. The cytotoxicity of the compound was evaluated by determining the percentage viability of cells. The effect of all the noncytotoxic concentrations of kefir on the proliferation of CEM and Jurkat cells was then assessed. The levels of transforming growth factor-alpha (TGF-α), transforming growth factor- beta1 (TGF-β1), matrix metalloproteinase-2 (MMP-2), and MMP-9 mRNA upon kefir treatment were then analyzed using reverse transcriptase polymerase chain reaction (RT-PCR). Finally, the growth inhibitory effects of kefir on cell-cycle progression/apoptosis were assessed by Cell Death Detection (ELISA) and flow cytometry. The maximum cytotoxicity recorded after 48-hours treatment with 80 μg/μL kefir was only 42% and 39% in CEM and Jurkat cells, respectively. The percent reduction in proliferation was very significant, and was dose-, and time-dependent. In both cell lines, kefir exhibited its antiproliferative effect by downregulating TGF-α and upregulating TGF-β1 mRNA expression. Upon kefir treatment, a marked increase in cell-cycle distribution was noted in the preG(1) phase of CEM and Jurkat cells, indicating the proapoptotic effect of kefir, which was further confirmed by Cell Death Detection ELISA. However, kefir did not affect the mRNA expression of metalloproteinases needed for the invasion of leukemic cell lines. In conclusion, kefir is effective in inhibiting proliferation and inducing

  15. HIV-1 infection and first line ART induced differential responses in mitochondria from blood lymphocytes and monocytes: the ANRS EP45 "Aging" study.

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    Sophie Perrin

    Full Text Available BACKGROUND: The ANRS EP45 "Aging" study investigates the cellular mechanisms involved in the accelerated aging of HIV-1 infected and treated patients. The data reported focus on mitochondria, organelles known to be involved in cell senescence. METHODS: 49 HIV-1 infected patients untreated with antiretroviral therapy, together with 49 seronegative age- and sex-matched control subjects and 81 HIV-1 infected and treated patients, were recruited by 3 AIDS centres (Marseille, Montpellier, Nice; France;, NCT01038999. In more than 88% of treated patients, the viral load was 500/mm(3. ROS (reactive oxygen species production and ΔΨm (inner membrane potential were measured by flow cytometry in blood lymphocytes and monocytes (functional parameters. Three mitochondrial network quantitative morphological parameters were computed using confocal microscopy and image analysis. Three PBMC mitochondrial proteins (porin and subunits 2 and 4 of cytochrome C oxidase encoded by mtDNA or nuclear DNA, respectively were analysed by western blotting. RESULTS: Quantitative changes in PBMC mitochondrial proteins were not induced by either HIV-1 infection or ART. Discriminant analysis integrating functional (ROS production and ΔΨm or morphological (network volume density, fragmentation and branching parameters revealed HIV-1 infection and ART differential effects according to cell type. First line ART tended to rescue lymphocyte mitochondrial parameters altered by viral infection, but induced slight changes in monocytes. No statistical difference was found between the effects of three ART regimens on mitochondrial parameters. Correlations between functional parameters and viral load confirmed the damaging effects of HIV-1 in lymphocyte mitochondria. CONCLUSIONS: In patients considered to be clinically stable, mitochondria exhibited functional and morphological modifications in PBMCs resulting from either direct or indirect effects of HIV-1

  16. Reduced lymphocyte activation in space - Role of cell-substratum interactions (United States)

    Gmuender, F. K.; Kiess, M.; Lee, J.; Cogoli, A.; Sonnenfeld, G.


    The effect of substratum adhesiveness on lymphocyte responsiveness was investigated by reducing and blocking cell adhesion with poly-HEMA in a simple on ground system. Cells grown on medium thick and thick poly-HEMA films were rounded in shape and displayed no signs of spreading. By contrast, on tissue culture plastic and very thin poly-HEMA films, they showed clear signs of spreading. The mitogenic response of lymphocytes grown on thick poly-HEMA films was reduced by up to 68 percent of the control (tissue culture plastic). Interferon gamma production was virtually nil when the cells were grown on the least adhesive substratum. These results show that activated lymphocytes need to anchor and spread prior to achieving an optimal proliferation response. It is concluded that decreased lymphocyte adhesion could contribute to the depressed in vitro lymphocyte responsiveness found in the microgravity conditions of space flight.

  17. Comparative In Vitro Immune Stimulation Analysis of Primary Human B Cells and B Cell Lines (United States)

    Van Belle, Kristien; Herman, Jean; Boon, Louis; Waer, Mark


    B cell specific immunomodulatory drugs still remain an unmet medical need. Utilisation of validated simplified in vitro models would allow readily obtaining new insights in the complexity of B cell regulation. For this purpose we investigated which human B lymphocyte stimulation assays may be ideally suited to investigate new B lymphocyte immunosuppressants. Primary polyclonal human B cells underwent in vitro stimulation and their proliferation, production of immunoglobulins (Igs) and of cytokines, and expression of cell surface molecules were analysed using various stimuli. ODN2006, a toll-like receptor 9 (TLR9) agonist, was the most potent general B cell stimulus. Subsequently, we investigated on which human B cell lines ODN2006 evoked the broadest immunostimulatory effects. The Namalwa cell line proved to be the most responsive upon TLR9 stimulation and hence may serve as a relevant, homogeneous, and stable B cell model in an in vitro phenotypic assay for the discovery of new targets and inhibitors of the B cell activation processes. As for the read-out for such screening assay, it is proposed that the expression of activation and costimulatory surface markers reliably reflects B lymphocyte activation. PMID:28116319

  18. Effects of dendritic cells from cord blood CD34+ cells on human hepatocarcinoma cell line BEL-7402 in vitro and in SCID mice

    Institute of Scientific and Technical Information of China (English)

    Zhong-Jing Su; Hai-Bin Chen; Jin-Kun Zhang; Lan Xu


    AIM: To develop a cancer vaccine of dendritic cells derived from human cord blood CD34+ cells and to investigate its cytotoxicity on human hepatocarcinoma cells in vitro and in sever combined immunodeficiency (SCTD) mice.METHODS: Lymphocytes from cord blood or peripheral blood were primed by DCs, which were derived from cord blood and pulsed with whole tumor cell lysates. Nonradiative neutral red uptake assay was adopted to detect the cytotoxicity of primed lymphocytes on human hepatocarcinoma cell line BEL-7402 in vitro. The anti-tumor effect of primed lymphocytes in vivo was detected in SCID mice, including therapeutic effect and vaccination effect.RESULTS: The cytotoxicity of DC vaccine primed lymphocytes from cord blood or peripheral blood on human hepatocarcinoma cell line BEL-7402 was significantly higher than that of unprimed lymphocytes in vitro (44.09% vs 14.69%,47.92% vs 19.44%, P<0.01). There was no significant difference between the cytotoxicity of primed lymphocytes from cord blood and peripheral blood (P>0.05). The tumor growth rate and tumor size were smaller in SCID mice treated or vaccinated with primed lymphocytes than those with unprimed lymphocytes. SCID mice vaccinated with primed lymphocytes had a lower tumor incidence (80%vs 100%, P<0.05) and delayed tumor latent period compared with mice vaccinated with unprimed lymphocytes (11 d vs 7 d, P<0.01).CONCLUSION: Vaccine of cord blood derived-DCs has an inhibitory activity on growth of human hepatocarcinoma cells in vitro and in SCID mice. The results also implicate the potential role of cord blood derived-DC vaccine in clinical tumor immunotherapy.

  19. Growth dynamics and cyclin expression in cutaneous T-cell lymphoma cell lines

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    Edyta Biskup


    Full Text Available We have investigated cell growth dynamics and cyclins B1 and E expression in cell lines derived from mycosis fungoides (MyLa, Sézary syndrome (SeAx, and CD30+ lympho-proliferative diseases (Mac1, Mac2a, JK. Mac1 and Mac2a had the highest growth rate (doubling time 18-28 h, >90% cycling cells whereas SeAx was proliferating slowly (doub-ling time 55 h, approximately 35% cycling cells. Expression of cyclin B1 correlated positively with doubling time whereas expression of cyclin E was unscheduled and constant across the investigated cell lines. All cell lines exhibited high expression of PCNA. Thus, we concluded that cyclin B1 could be used for rapid screening of cell proliferation in malignant lymphocytes derived from cutaneous T-cell lymphoma.

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

    NARCIS (Netherlands)

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


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

  1. Primary B Lymphocytes Infected with Kaposi's Sarcoma-Associated Herpesvirus Can Be Expanded In Vitro and Are Recognized by LANA-Specific CD4+ T Cells (United States)

    Nicol, Samantha M.; Sabbah, Shereen; Brulois, Kevin F.; Jung, Jae U.; Bell, Andrew I.


    ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) has tropism for B lymphocytes, in which it establishes latency, and can also cause lymphoproliferative disorders of these cells manifesting as primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). T cell immunity is vital for the control of KSHV infection and disease; however, few models of B lymphocyte infection exist to study immune recognition of such cells. Here, we developed a model of B lymphocyte infection with KSHV in which infected tonsillar B lymphocytes were expanded by providing mitogenic stimuli and then challenged with KSHV-specific CD4+ T cells. The infected cells expressed viral proteins found in PELs, namely, LANA and viral IRF3 (vIRF3), albeit at lower levels, with similar patterns of gene expression for the major latency, viral interleukin 6 (vIL-6), and vIRF3 transcripts. Despite low-level expression of open reading frame 50 (ORF50), transcripts for the immune evasion genes K3 and K5 were detected, with some downregulation of cell surface-expressed CD86 and ICAM. The vast majority of infected lymphocytes expressed IgM heavy chains with Igλ light chains, recapitulating the features seen in infected cells in MCD. We assessed the ability of the infected lymphocytes to be targeted by a panel of major histocompatibility complex (MHC) class II-matched CD4+ T cells and found that LANA-specific T cells restricted to different epitopes recognized these infected cells. Given that at least some KSHV latent antigens are thought to be poor targets for CD8+ T cells, we suggest that CD4+ T cells are potentially important effectors for the in vivo control of KSHV-infected B lymphocytes. IMPORTANCE KSHV establishes a latent reservoir within B lymphocytes, but few models exist to study KSHV-infected B cells other than the transformed PEL cell lines, which have likely accrued mutations during the transformation process. We developed a model of KSHV-infected primary B lymphocytes that

  2. mM-CSF 及其剪切体对淋巴细胞白血病Ramos 细胞增殖的抑制作用%Inhibitory effect of mM-CSF and its spliceosome on proliferation of lymphocytic leukemia cell line Ramos

    Institute of Scientific and Technical Information of China (English)

    马翠花; 种靖慧; 廖金凤; 林永敏; 卫佳; 郑国光


    目的:探讨膜结合型巨噬细胞集落刺激因子( mM-CSF)及其剪切体( mM-CSF-Δ)对淋巴细胞白血病Ramos细胞增殖的抑制作用。方法采用Overlap PCR法构建带有mM-CSF的真核表达质粒pTARGET-mM-CSF,再进一步构建胞内区截短30个氨基酸的表达质粒pTARGET-mM-CSF-Δ,并进行PCR及DNA双向测序鉴定。将空载体pTARGET、pTARGET-mM-CSF、pTARGET-mM-CSF-Δ质粒分别转染Ramos细胞,经G418筛选稳定表达细胞株,并用RT-PCR、Western blotting进行鉴定;MTT法检测细胞增殖,流式细胞仪检测细胞周期。结果成功构建了mM-CSF和 mM-CSF-Δ的真核表达载体,获得了稳定转染细胞株 Ramos-V、Ramos-M 和 Ramos-Δ。 Ramos-M、Ramos-Δ、Ramos-V细胞增殖能力的OD值分别为0.413±0.014、0.384±0.019、0.463±0.037,Ramos-M细胞与Ramos-Δ细胞比较,Ramos-M、Ramos-Δ细胞分别与Ramos-V细胞比较,P<0.05或<0.01。 Ramos-M、Ramos-Δ、Ramos-V细胞处于G0/G1期的比例分别为41.54%±1.22%、45.60%±1.09%、39.20%±1.53%,Ramos-M细胞与Ramos-Δ细胞比较, Ramos-M、Ramos-Δ细胞分别与 Ramos-V 细胞比较, P <0.05或<0.01。结论 mM-CSF、mM-CSF-Δ均能抑制淋巴细胞白血病Ramos细胞增殖,且后者抑制作用更强。%Objective To investigate the inhibitory effect of membrane-bound macrophage colony-stimulating factor ( mM-CSF) and its spliceosome ( mM-CSF-Δ) on proliferation of lymphocytic leukemia cell line Ramos.Methods The eukaryotic expression plasmid of pTARGET-mM-CSF with mM-CSF was constructed with Overlap PCR, and then pTAR-GET-mM-CSF-Δof 30 amino acide located in the intracellular region of brachytmema mutation was obtained; and mean-while, they were identified by PCR and DNA sequencing.The empty vector pTARGET, pTARGET-mM-CSF and pTAR-GET-mM-CSF-Δplasmid were transfected into Ramos cells, the cell line with stable expression was screened by G418 and


    Institute of Scientific and Technical Information of China (English)

    Zhu Zhenping; Yang Chunzheng; Tarunendu Ghose; Jaroslav Kralovec


    Objective: To analysis the uptake of free MTX and MTX conjugated to tumor specific monoclonal antibody by target and non-target cells. Methods: The folate antagonist methotrexate (MTX) was conjugated to two monoclonal antibodies (Mab) directed against human chronic lymphocytic leukemia (CLL), Dal B01 and Dal B02, by an active ester method. Both conjugates were more cytotoxic toward the target tumor cell line D10-1than to the non-target cell line MOLT-3, and Dal B02-MTX conjugate was more inhibitory to D10-1 cells than free MTX in a 6 h pulse exposure assay. Results: Drug uptake studies revealed that D10-1 cells took up much more Dal B01 and Dal B02-conjugated MTX than free MTX. The amounts of drug taken up by D10-1 cells incubated with Dal B01 and Dal B02-conjugated MTX were always 3 to 5-fold higher than that taken up by MOLT-3 cells, although the latter took up more drug when incubated with free MTX. Furthermore, tumor cells incubated with Dal B01 or Dal B02-conjugated MTX retained much larger amounts of drug for a prolonged period of time than those incubated with free MTX.Conclusion: The enhanced specific cytotoxicity of Dal B01 and Dal B02-MTX conjugates toward target tumor cells is therefore likely due to (Ⅰ) delivery of larger amounts of MTX to target cells when the drug is conjugated to Mab;(ii) longer retention of Mab-conjugated MTX by target cells; and (iii) slow, prolonged release of MTX from the surface-bound or endocytosed conjugates, rendering them into a sustained release dosage form.

  4. Neurohypophysial Receptor Gene Expression by Thymic T Cell Subsets and Thymic T Cell Lymphoma Cell Lines

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    I. Hansenne


    transcribed in thymic epithelium, while immature T lymphocytes express functional neurohypophysial receptors. Neurohypophysial receptors belong to the G protein-linked seven-transmembrane receptor superfamily and are encoded by four distinct genes, OTR, V1R, V2R and V3R. The objective of this study was to identify the nature of neurohypophysial receptor in thymic T cell subsets purified by immunomagnetic selection, as well as in murine thymic lymphoma cell lines RL12-NP and BW5147. OTR is transcribed in all thymic T cell subsets and T cell lines, while V3R transcription is restricted to CD4+ CD8+ and CD8+ thymic cells. Neither V1R nor V2R transcripts are detected in any kind of T cells. The OTR protein was identified by immunocytochemistry on thymocytes freshly isolated from C57BL/6 mice. In murine fetal thymic organ cultures, a specific OTR antagonist does not modify the percentage of T cell subsets, but increases late T cell apoptosis further evidencing the involvement of OT/OTR signaling in the control of T cell proliferation and survival. According to these data, OTR and V3R are differentially expressed during T cell ontogeny. Moreover, the restriction of OTR transcription to T cell lines derived from thymic lymphomas may be important in the context of T cell leukemia pathogenesis and treatment.

  5. The correlation of lymphocyte subsets, natural killer cell, and Parkinson's disease: a meta-analysis. (United States)

    Jiang, Sen; Gao, Hua; Luo, Qin; Wang, Pengfei; Yang, Xinling


    The correlation between immunity and Parkinson's disease was presented in many papers, which also discussed lymphocyte and natural killer cell. But these studies have yielded inconsistent results. To systematically review the relationship between the lymphocyte subsets/natural killer cell and the risk of Parkinson's disease, we electronically searched the SpringerLink, Web of Science, Ebsco-medline with full text, Pubmed, Elsevier-ScienceDirect, Ovid-lww-oup, Wanfang Data for case-control trials on comparing the number of peripheral blood lymphocyte subsets and natural killer cell in Parkinson's patients and healthy controls. According to the Cochrane methods, the reviewers selected literature, extracted data, and assessed the quality. Then, a meta-analysis was performed using RevMan 5.2. Finally, 21 case-control trials including 943 cases of Parkinson's disease were fit into our data analysis. Meta-analysis showed that the decreased numbers of CD3+, CD4+ lymphocyte subsets and the increased number of natural killer cell were found in Parkinson's disease patients. In the intermediate and late stage of PD, CD8+ lymphocyte subsets had a significant decrement. However, the number of B lymphocyte subsets had no significant association with Parkinson's disease. The lymphocyte subsets and NK cell may be associated with the risk of Parkinson's disease.

  6. Early lymphocyte recovery as a predictor of outcome, including relapse, after hematopoieticstem cell transplantation

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    Juliane Morando


    Full Text Available BACKGROUND: Despite advances in the treatment of acute leukemia, many patients need to undergo hematopoietic stem cell transplantation. Recent studies show that early lymphocyte recovery may be a predictor of relapse and survival in these patients. OBJECTIVE: To analyze the influence of lymphocyte recovery on Days +30 and +100 post-transplant on the occurrence of relapse and survival. METHODS: A descriptive, retrospective study was performed of 137 under 21-year-old patients who were submitted to hematopoietic stem cell transplantation for acute leukemia between 1995 and 2008. A lymphocyte count 0.3 x 10(9/L were considered adequate. Lymphocyte recovery was also analyzed on Day +100 with < 0.75 x 10(9/Land < 0.75 x 10(9/L being considered inadequate and adequate lymphocyte recovery, respectively. RESULTS: There was no significant difference in the occurrence of relapse between patients with inadequate and adequate lymphocyte recovery on Day +30 post-transplant. However, the transplant-related mortality was significantly higher in patients with inadequate recovery on Day +30. Patients with inadequate lymphocyte recovery on Day +30 had worse overall survival and relapse-free survival than patients with adequate recovery. There was no significant difference in the occurrence of infections and acute or chronic graft-versus-host disease. Patients with inadequate lymphocyte recovery on Day +100 had worse overall survival and relapse-free survival and a higher cumulative incidence of relapse. CONCLUSION: The evaluation of lymphocyte recovery on Day +30 is not a good predictor of relapse after transplant however patients with inadequate lymphocyte recovery had worse overall survival and relapse-free survival. Inadequate lymphocyte recovery on Day +100 is correlated with higher cumulative relapse as well as lower overall survival and relapse-free survival.

  7. Laboratory Treated T Cells in Treating Patients With Relapsed or Refractory Chronic Lymphocytic Leukemia, Non-Hodgkin Lymphoma, or Acute Lymphoblastic Leukemia (United States)


    CD19-Positive Neoplastic Cells Present; Recurrent Adult Acute Lymphoblastic Leukemia; Recurrent Chronic Lymphocytic Leukemia; Recurrent Diffuse Large B-Cell Lymphoma; Recurrent Mantle Cell Lymphoma; Recurrent Non-Hodgkin Lymphoma; Recurrent Small Lymphocytic Lymphoma; Refractory Chronic Lymphocytic Leukemia; Refractory Diffuse Large B-Cell Lymphoma; Refractory Mantle Cell Lymphoma; Refractory Non-Hodgkin Lymphoma; Refractory Small Lymphocytic Lymphoma

  8. A critical appraisal of ibrutinib in the treatment of mantle cell lymphoma and chronic lymphocytic leukemia

    Directory of Open Access Journals (Sweden)

    Tucker DL


    Full Text Available David L Tucker, Simon A Rule Department of Haematology, Plymouth Hospitals NHS Trust, Plymouth, UK Abstract: Although chemo-immunotherapy remains at the forefront of first-line treatment for mantle cell lymphoma (MCL and chronic lymphocytic leukemia (CLL, small molecules, such as ibrutinib, are beginning to play a significant role, particularly in patients with multiply relapsed or chemotherapy-refractory disease and where toxicity is an overriding concern. Ibrutinib is a first-in-class, oral inhibitor of Bruton’s tyrosine kinase, which functions by irreversible inhibition of the downstream signaling pathway of the B-cell receptor, which normally promotes cell survival and proliferation. Early clinical trials have demonstrated excellent tolerability and a modest side-effect profile even in elderly and multiply pretreated patient cohorts. Although the majority of disease responses tend to be partial, efficacy data have also been encouraging with more than two-thirds of patients with CLL and MCL demonstrating a durable response, even in the high-risk disease setting. Resistance mechanisms are only partially understood and appear to be multifactorial, including the binding site mutation C481S, and escape through other common cell-signaling pathways. This article appraises the currently available data on safety and efficacy from clinical trials of ibrutinib in the management of MCL and CLL, both as a single agent and in combination with other therapies, and considers how this drug is likely to be used in future clinical practice. Keywords: ibrutinib, mantle cell lymphoma, chronic lymphocytic leukemia, Bruton’s tyrosine kinase, lymphoproliferative disorders

  9. 2-Methoxyestradiol induce the conversion of human peripheral blood memory B lymphocytes into plasma cells. (United States)

    Cayer, Marie-Pierre; Drouin, Mathieu; Proulx, Maryse; Jung, Daniel


    2-Methoxyestradiol (2ME), an end-metabolite of 17beta-estradiol, is an antiproliferative agent that is currently being tested in clinical trials for cancer treatment. We hereby report that sub-cytotoxic concentrations of 2ME influence the in vitro proliferation of human peripheral blood B lymphocytes. More surprisingly, we have observed that 2ME induces the conversion of CD138(-) B lymphocytes into CD138(+) cells of phenotype similar to immunoglobulin (Ig)-secreting plasma cells. Normal human B lymphocytes expressing CD138 increased in response to 2ME in a dose-dependent fashion, from 2% at baseline up to 31% in cells cultured in the presence of 0.75 microM 2ME. Moreover, most of the converted cells were also CD27(+) and secreted high levels of IgG (151 microg/10(6)cells/24h). IEF studies revealed that conversion occurred in a polyclonal manner. We then exploited this effect of 2ME to gain further insights into the molecular mechanisms that govern changes in transcription factors involved in plasma cells differentiation. Plasma cells generated by 2ME treatment of normal human B lymphocytes expressed elevated levels of IRF4 and reduced levels of Pax5 and Bcl-6. Similarly, levels of XBP-1 and Blimp-1 transcripts were increased. Our results suggest that the differentiation of peripheral blood B lymphocytes into plasma cells requires a similar modulation of transcription factors expression that for tonsil and bone marrow B lymphocytes.

  10. Composite mantle cell lymphoma and chronic lymphocytic leukemia/small lymphocytic lymphoma: a clinicopathologic and molecular study. (United States)

    Hoeller, Sylvia; Zhou, Yi; Kanagal-Shamanna, Rashmi; Xu-Monette, Zijun Y; Hoehn, Daniela; Bihl, Michel; Swerdlow, Steven H; Rosenwald, Andreas; Ott, German; Said, Jonathan; Dunphy, Cherie H; Bueso-Ramos, Carlos E; Lin, Pei; Wang, Michael; Miranda, Roberto N; Tzankov, Alexander; Medeiros, L Jeffrey; Young, Ken H


    Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) share many features and both arise from CD5+ B-cells; their distinction is critical as MCL is a more aggressive neoplasm. Rarely, cases of composite MCL and CLL/SLL have been reported. Little is known about the nature of these cases and, in particular, the clonal relationship of the 2 lymphomas. Eleven composite MCL and CLL/SLL cases were identified. The clinical, morphologic and immunophenotypic features of the MCL and CLL/SLL were characterized. IGH (immunoglobulin heavy chain) gene analysis was performed on microdissected MCL and CLL/SLL components to assess their clonal relationship. Ten patients had lymphadenopathy, and 7 patients had bone marrow involvement. The MCL component had the following growth patterns: in situ (n = 1), mantle zone (n = 3), nodular and diffuse (n = 3), diffuse (n = 3), and interstitial in the bone marrow (the only patient without lymphadenopathy) (n = 1); 6 MCLs had blastoid or pleomorphic and 5 small lymphocytic features. The CLL/SLL component was nodular (n = 9) or diffuse (n = 2). All MCL were CD5(+) and cyclin D1(+) with t(11;14) translocation. All CLL/SLL were CD5(+), CD23(+) and negative for cyclin D1 or t(11;14). IGH gene analysis showed that the MCL and CLL/SLL components displayed different sized fragments, indicating that the MCL and CLL/SLL are likely derived from different neoplastic B-cell clones. The lack of a clonal relationship between the MCL and CLL/SLL components suggests that MCL and CLL/SLL components represent distinct disease processes and do not share a common progenitor B-cell.

  11. Genetically Engineered Lymphocytes, Cyclophosphamide, and Aldesleukin in Treating Patients With Relapsed or Refractory Mantle Cell Lymphoma or Indolent B-Cell Non-Hodgkin Lymphoma (United States)


    B-cell Chronic Lymphocytic Leukemia; Extranodal Marginal Zone B-cell Lymphoma of Mucosa-associated Lymphoid Tissue; Nodal Marginal Zone B-cell Lymphoma; Recurrent Grade 1 Follicular Lymphoma; Recurrent Grade 2 Follicular Lymphoma; Recurrent Mantle Cell Lymphoma; Recurrent Marginal Zone Lymphoma; Recurrent Small Lymphocytic Lymphoma; Refractory Chronic Lymphocytic Leukemia; Splenic Marginal Zone Lymphoma; Waldenström Macroglobulinemia

  12. Spontaneous human squamous cell carcinomas are killed by a human cytotoxic T lymphocyte clone recognizing a wild-type p53-derived peptide

    DEFF Research Database (Denmark)

    Röpke, M; Hald, J; Guldberg, Per


    A cytotoxic T lymphocyte (CTL) clone generated in vitro from the peripheral blood of a healthy HLA-A2-positive individual against a synthetic p53 protein-derived wild-type peptide (L9V) was shown to kill squamous carcinoma cell lines derived from two head and neck carcinomas, which expressed mutant...

  13. 表面增强激光解吸/电离-飞行时间质谱技术检测不同来源淋巴瘤细胞株及健康人外周淋巴细胞的差异蛋白质%Detection of protein expression in lymphoma cell lines and peripheral blood lymphocyte of health persons by SELDI-TOF-MS

    Institute of Scientific and Technical Information of China (English)

    张健; 陶朝晖; 景莉; 韩丽英


    Objective To look for different expression of proteins between T and B lymphoma cell lines and normal peripheral blood lymphocytes by surface enhanced laser desorption/ionization-time of flightmass spectrometry (SELDI-TOF-MS) technology. Methods T and B lymphoma cells were conventionally cultivated, and logarithmic phase cells were collected for experiment. The normal lymphocytes were separated from peripheral blood of healthy persons. Proteins were extracted from the total protein of various cells, and were detected by SELDI-TOF-MS technology. SPSS 13.0 statistical software was used to statistical analysis and P <0.05 was considered as statistically difference. Results There are six differentially expressed proteins between T/B lymphoma cell lines and health human peripheral lymphocyte. There are seven differentially expressed proteins between B and T lymphoma cell lines. Conclusion The levels of proteomics were significantly different among T, B lymphoma cell line and normal lymphocyte. Application of SELDI-TOF-MS technology probably is some benefit on screening molecular markers of lymphoma. Moreover it is possible to lay the preliminary foundation for pathogenesis and targeted treatment of lymphoma.%目的 应用表面增强激光解吸/电离-飞行时间质谱(SELDI-TOF-MS)技术对体外培养的人类T、B淋巴瘤细胞株和健康人外周血淋巴细胞的蛋白质进行检测,寻找差异蛋白.方法 常规培养人类T、B淋巴瘤细胞株,并从健康人外周血中分离出淋巴细胞培养,取对数生长期的细胞进行试验,使用蛋白提取液提取蛋白质,应用SELDI-TOF-MS技术进行检测,采用SPSS 13.0统计软件包进行统计学分析.结果 T、B淋巴瘤细胞株与健康人外周淋巴细胞比较,均有6个差异蛋白质.T淋巴瘤细胞株与B淋巴瘤细胞株比较有7个差异蛋白质.结论 T、B淋巴瘤细胞间及二者与健康人外周淋巴细胞比较,蛋白质组学水平均有差异.应用SELDl-TOF-MS技

  14. Cost Effectiveness of Ofatumumab Plus Chlorambucil in First-Line Chronic Lymphocytic Leukaemia in Canada. (United States)

    Herring, William; Pearson, Isobel; Purser, Molly; Nakhaipour, Hamid Reza; Haiderali, Amin; Wolowacz, Sorrel; Jayasundara, Kavisha


    Our objective was to estimate the cost effectiveness of ofatumumab plus chlorambucil (OChl) versus chlorambucil in patients with chronic lymphocytic leukaemia for whom fludarabine-based therapies are considered inappropriate from the perspective of the publicly funded healthcare system in Canada. A semi-Markov model (3-month cycle length) used survival curves to govern progression-free survival (PFS) and overall survival (OS). Efficacy and safety data and health-state utility values were estimated from the COMPLEMENT-1 trial. Post-progression treatment patterns were based on clinical guidelines, Canadian treatment practices and published literature. Total and incremental expected lifetime costs (in Canadian dollars [$Can], year 2013 values), life-years and quality-adjusted life-years (QALYs) were computed. Uncertainty was assessed via deterministic and probabilistic sensitivity analyses. The discounted lifetime health and economic outcomes estimated by the model showed that, compared with chlorambucil, first-line treatment with OChl led to an increase in QALYs (0.41) and total costs ($Can27,866) and to an incremental cost-effectiveness ratio (ICER) of $Can68,647 per QALY gained. In deterministic sensitivity analyses, the ICER was most sensitive to the modelling time horizon and to the extrapolation of OS treatment effects beyond the trial duration. In probabilistic sensitivity analysis, the probability of cost effectiveness at a willingness-to-pay threshold of $Can100,000 per QALY gained was 59 %. Base-case results indicated that improved overall response and PFS for OChl compared with chlorambucil translated to improved quality-adjusted life expectancy. Sensitivity analysis suggested that OChl is likely to be cost effective subject to uncertainty associated with the presence of any long-term OS benefit and the model time horizon.

  15. Causes of upregulation of glycolysis in lymphocytes upon stimulation. A comparison with other cell types. (United States)

    Stark, Heiko; Fichtner, Maximilian; König, Rainer; Lorkowski, Stefan; Schuster, Stefan


    In this review, we revisit the metabolic shift from respiration to glycolysis in lymphocytes upon activation, which is known as the Warburg effect in tumour cells. We compare the situation in lymphocytes with those in several other cell types, such as muscle cells, Kupffer cells, microglia cells, astrocytes, stem cells, tumour cells and various unicellular organisms (e.g. yeasts). We critically discuss and compare several explanations put forward in the literature for the observation that proliferating cells adopt this apparently less efficient pathway: hypoxia, poisoning of competitors by end products, higher ATP production rate, higher precursor supply, regulatory effects, and avoiding harmful effects (e.g. by reactive oxygen species). We conclude that in the case of lymphocytes, increased ATP production rate and precursor supply are the main advantages of upregulating glycolysis.

  16. Expansion of activated lymphocytes obtained from renal cell carcinoma in an automated hollow fiber bioreactor. (United States)

    Hillman, G G; Wolf, M L; Montecillo, E; Younes, E; Ali, E; Pontes, J E; Haas, G P


    Immunotherapy using IL-2 alone or combined with activated lymphocytes has been promising for metastatic renal cell carcinoma. Cytotoxic lymphocytes can be isolated from tumors, expanded in vitro with IL-2, and adoptively transferred back into the tumor-bearing host. These cells can also be transduced with the genes coding for cytokines for local delivery to tumor sites. A major drawback in adoptive immunotherapy is the cumbersome and expensive culture technology associated with the growth of large numbers of cells required for their therapeutic effect. To reduce the cost, resources, and manpower, we have developed the methodology for lymphocyte activation and expansion in the automated hollow fiber bioreactor IMMUNO*STAR Cell Expander (ACT BIOMEDICAL, INC). Tumor Infiltrating Lymphocytes (TIL) isolated from human renal cell carcinoma tumor specimens were inoculated at a number of 10(8) cells in a small bioreactor of 30 ml extracapillary space volume. We have determined the medium flow rates and culture conditions to obtain a significant and repeated expansion of TIL at weekly intervals. The lymphocytes cultured in the bioreactor demonstrated the same phenotype and cytotoxic activity as those expanded in parallel in tissue culture plates. Lymphocyte expansion in the hollow fiber bioreactor required lower volumes of medium, human serum, IL-2 and minimal labor. This technology may facilitate the use of adoptive immunotherapy for the treatment of refractory malignancies.

  17. Molluscan cells in culture: primary cell cultures and cell lines. (United States)

    Yoshino, T P; Bickham, U; Bayne, C J


    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome.

  18. Initiation of lymphocyte DNA synthesis. (United States)

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


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

  19. MicroRNA expression profiling identifies activated B cell status in chronic lymphocytic leukemia cells.

    Directory of Open Access Journals (Sweden)

    Shuqiang Li

    Full Text Available Chronic lymphocytic leukemia (CLL is thought to be a disease of resting lymphocytes. However, recent data suggest that CLL cells may more closely resemble activated B cells. Using microRNA (miRNA expression profiling of highly-enriched CLL cells from 38 patients and 9 untransformed B cells from normal donors before acute CpG activation and 5 matched B cells after acute CpG activation, we demonstrate an activated B cell status for CLL. Gene set enrichment analysis (GSEA identified statistically-significant similarities in miRNA expression between activated B cells and CLL cells including upregulation of miR-34a, miR-155, and miR-342-3p and downregulation of miR-103, miR-181a and miR-181b. Additionally, decreased levels of two CLL signature miRNAs miR-29c and miR-223 are associated with ZAP70(+ and IgV(H unmutated status and with shorter time to first therapy. These data indicate an activated B cell status for CLL cells and suggest that the direction of change of individual miRNAs may predict clinical course in CLL.

  20. Inhibitory Effects of Berberine on the Activation and Cell Cycle Progression of Human Peripheral Lymphocytes

    Institute of Scientific and Technical Information of China (English)

    Lihui Xu; Yi Liu; Xianhui He


    The immunosuppressive property of berberine, an isoquinoline alkaloid, has been well documented, but the mechanism of its action on lymphocytes has not been completely elucidated. The present study is to investigate the effect of berberine on the activation and proliferation of lymphocytes, in particular T lymphocytes. Whole peripheral blood from healthy donors was stimulated with phytohemagglutinin (PHA) alone or phorbol dibutyrate (PDB) plus ionomycin, and the expression of CD69 and CD25 on T lymphocytes was evaluated with flow cytometry.The distribution of cell cycles and cell viability were analyzed by staining with propidium iodide (PI) and 7-aminoactinomycin D (7-AAD), respectively. The results showed that 100 μmol/L and 50 μmol/L of berberine significantly inhibited CD69 expression on T cells stimulated with PDB plus ionomycin or PHA, whereas the effect of 25 μmol/L berberine was not significant. As the incubation time increased, the extent of inhibition decreased.Similarly, the expression of CD25 was also reduced by berberine in a dose-dependent manner over the concentration range of 25-100 μmol/L. Besides, this alkaloid could block lymphocyte cell cycle progression from G0/G1 phase to S and G2/M phase without phase specificity. Moreover, analysis following 7-AAD staining revealed that berberine had no significant cytotoxicity on lymphocytes. Taken together, berberine significantly inhibits the expression of activation antigens on T lymphocytes and also blocks the progression of cell cycles of lymphocytes,suggesting that berberine may exert immunosuppressive effect through inhibiting the activation and proliferation of T cells.

  1. Pretreatment T lymphocyte numbers are contributing to the prognostic significance of absolute lymphocyte numbers in B-cell non-Hodgkins lymphomas. (United States)

    Gergely, Lajos; Váncsa, Andrea; Miltényi, Zsófia; Simon, Zsófia; Baráth, Sándor; Illés, Árpád


    Targeted immuno-chemotherapy resulted in greatly improved survival of B cell lymphoma patients. Several prognostic markers are investigated, amongst them the pretreatment absolute lymphocyte numbers. We investigated lymphocyte subpopulations and correlated this data with prognosis of patients. 88 patients (mean age: 56 years, 18-88, median follow up 32 months) with B cell lymphomas were investigated. There were 51 DLBCL, 16 Follicular NHL, 4 MALT, 7 Marginal Zone NHL, 10 Small lymphocytic cases were investigated. Our data showed that overall survival was statistically significant up to the 0.9 G/l absolute lymphocyte numbers as dividers between the subgroups. The CD19+ B cell numbers, or the CD56+/CD3- NK cell numbers did not represent any significant differences between subgroups. However CD3+, CD4+ and CD8+ T cells were differentiating statistically significant subgroups. Pretreatment CD3+ cell number less than 700/ul and CD8+ cell number less than 200/ul were corresponding with significantly inferior overall survival. These could be verified in the bad prognostic IPI group as well. Our data further support the importance of pretreatment lymphocyte numbers and highlight CD3+ and CD8+ lymphocytes to be the key factors in predicting outcome.

  2. Cytostatic and genotoxic effect of temephos in human lymphocytes and HepG2 cells. (United States)

    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


    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.

  3. Isopentenyl pyrophosphate activated CD56+ γδ T lymphocytes display potent anti-tumor activity towards human squamous cell carcinoma (United States)

    Alexander, Alan A.Z.; Maniar, Amudhan; Cummings, Jean-Saville; Hebbeler, Andrew M.; Schulze, Dan H.; Gastman, Brian R.; Pauza, C. David; Strome, Scott E.; Chapoval, Andrei I.


    Purpose The expression of CD56, a natural killer (NK) cell-associated molecule, on αβ T lymphocytes correlates with their increased anti-tumor effector function. CD56 is also expressed on a subset of γδ T cells. However, anti-tumor effector functions of CD56+ γδ T cells are poorly characterized. Experimental design To investigate the potential effector role of CD56+ γδ T cells in tumor killing, we employed isopentenyl pyrophosphate (IPP) and IL-2 expanded γδ T cells from PBMC of healthy donors. Results Thirty to 70% of IPP+IL-2 expanded γδ T cells express CD56 on their surface. Interestingly, while both CD56+ and CD56− γδ T cells express comparable levels of receptors involved in the regulation of γδ T cell cytotoxicity (e.g. NKG2D and CD94) only CD56+ γδ T lymphocytes are capable of killing squamous cell carcinoma (SCC) and other solid tumor cell lines. This effect is likely mediated by the enhanced release of cytolytic granules, since CD56+ γδ T lymphocytes expressed higher levels of CD107a compared to CD56− controls, following exposure to tumor cell lines. Lysis of tumor cell lines is blocked by concanomycin A and a combination of anti-γδTCR + anti-NKG2D mAb, suggesting that the lytic activity of CD56+ γδ T cells involves the perforin-granzyme pathway and is mainly γδTCR/NKGD2 dependent. Importantly, CD56 expressing γδ T lymphocytes are resistant to Fas ligand and chemically induced apoptosis. Conclusions Our data indicate that CD56+ γδ T cells are potent anti-tumor effectors capable of killing SCC and may play an important therapeutic role in patients with head and neck cancer and other malignancies. PMID:18594005

  4. Nuclear overexpression of lymphoid-enhancer-binding factor 1 identifies chronic lymphocytic leukemia/small lymphocytic lymphoma in small B-cell lymphomas. (United States)

    Tandon, Bevan; Peterson, Loann; Gao, Juehua; Nelson, Beverly; Ma, Shuo; Rosen, Steven; Chen, Yi-Hua


    Lymphoid-enhancer-binding factor 1 (LEF1), coupling with β-catenin, functions as a key nuclear mediator of WNT/β-catenin signaling, which regulates cell proliferation and survival. LEF1 has an important role in lymphopoiesis, and is normally expressed in T and pro-B cells but not mature B cells. However, gene expression profiling demonstrates overexpression of LEF1 in chronic lymphocytic leukemia, and knockdown of LEF1 decreases the survival of the leukemic cells. So far, the data on LEF1 expression in B-cell lymphomas are limited. This study represents the first attempt to assess LEF1 by immunohistochemistry in a large series (290 cases) of B-cell lymphomas. Strong nuclear staining of LEF1 was observed in virtually all neoplastic cells in 92 of 92 (100%) chronic lymphocytic leukemia/small lymphocytic lymphomas including two CD5- cases, with strongest staining in cells with Richter's transformation. LEF1 also highlighted the morphologically inconspicuous small lymphocytic lymphoma component in three composite lymphomas. All 53 mantle cell lymphomas, 31 low-grade follicular lymphomas and 31 marginal zone lymphomas, including 3 CD5+ cases, were negative. In 12 grade 3 follicular lymphomas, LEF1 was positive in a small subset (5-15%) of cells. Diffuse large B-cell lymphoma, however, demonstrated significant variability in LEF1 expression with overall positivity in 27 of 71 (38%) cases. Our results demonstrate that nuclear overexpression of LEF1 is highly associated with chronic lymphocytic leukemia/small lymphocytic lymphoma, and may serve as a convenient marker for differential diagnosis of small B-cell lymphomas. The expression of β-catenin, the coactivator of LEF1 in WNT signaling, was examined in 50 chronic lymphocytic leukemia/small lymphocytic lymphomas, of which 44 (88%) showed negative nuclear staining. The findings of universal nuclear overexpression of LEF1 but lack of nuclear β-catenin in the majority of chronic lymphocytic leukemia/small lymphocytic

  5. Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy. (United States)

    Themeli, Maria; Kloss, Christopher C; Ciriello, Giovanni; Fedorov, Victor D; Perna, Fabiana; Gonen, Mithat; Sadelain, Michel


    Progress in adoptive T-cell therapy for cancer and infectious diseases is hampered by the lack of readily available, antigen-specific, human T lymphocytes. Pluripotent stem cells could provide an unlimited source of T lymphocytes, but the therapeutic potential of human pluripotent stem cell-derived lymphoid cells generated to date remains uncertain. Here we combine induced pluripotent stem cell (iPSC) and chimeric antigen receptor (CAR) technologies to generate human T cells targeted to CD19, an antigen expressed by malignant B cells, in tissue culture. These iPSC-derived, CAR-expressing T cells display a phenotype resembling that of innate γδ T cells. Similar to CAR-transduced, peripheral blood γδ T cells, the iPSC-derived T cells potently inhibit tumor growth in a xenograft model. This approach of generating therapeutic human T cells 'in the dish' may be useful for cancer immunotherapy and other medical applications.

  6. Cell size variations of large granular lymphocyte leukemia: Implication of a small cell subtype of granular lymphocyte leukemia with STAT3 mutations. (United States)

    Tanahashi, Takahiro; Sekiguchi, Nodoka; Matsuda, Kazuyuki; Takezawa, Yuka; Ito, Toshiro; Kobayashi, Hikaru; Ichikawa, Naoaki; Nishina, Sayaka; Senoo, Noriko; Sakai, Hitoshi; Nakazawa, Hideyuki; Ishida, Fumihiro


    Large granular lymphocyte leukemia (LGL-L) has been morphologically defined as a group of lymphoproliferative disorders, including T-cell large granular lymphocytic leukemia (T-LGL-L), chronic lymphoproliferative disorders of NK cells (CLPD-NK) and aggressive NK cell leukemia. We investigated the morphological features of LGL leukemic cells in 26 LGL-L patients in order to elucidate relationships with current classifications and molecular backgrounds. LGL-L cells were mostly indistinguishable from normal LGL. Patients with STAT3 SH2 domain mutations showed significantly smaller cells compared with patients without STAT3 mutations. Four patients with T-LGL-L showed smaller granular lymphocytes with a median diameter of less than 13μm, which were rarely seen in normal subjects. This small subtype of T-LGL-L was recognized among rather young patients and was associated with D661Y mutations in the STAT3 gene SH2 domain. In addition, all of them showed anemia including two cases with pure red cell aplasia. These results suggest the heterogeneity of T-LGL-L and a specific subtype with small variants of T-LGL-L. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    Institute of Scientific and Technical Information of China (English)

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


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

  8. Transplantability of human lymphoid cell line, lymphoma, and leukemia in splenectomized and/or irradiated nude mice

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, S.; Shimosato, Y.; Kuroki, M.; Sato, Y.; Nakajima, T.


    The effects of splenectomy and/or whole-body irradiation of nude mice before xenotransplantation of lymphoid cell lines, lymphoma, and leukemia were studied. Transplantation after whole-body irradiation resulted in the increased ''take'' rate of three cultured cell lines (two of T-cell-derived acute lymphocytic leukemia and one of B-cell derived acute lymphocytic leukemia) and in the tumorous growth of Burkitt-derived Raji and spontaneously transformed lymphoblastoid cell lines. With splenectomy plus irradiation as a pretreatment, tumorous growth occurred in four other cell lines which were not transplantable after irradiation only (two cell lines of Epstein-Barr virus-transformed cord blood cells and one each of null acute lymphocytic leukemia and nodular lymphoma-derived cell lines). Direct transplantation of leukemia and lymphoma cells into the pretreated mice was successful in 7 of 24 cases (29%). B-cell-derived diffuse large lymphoid lymphoma was transplantable in three of seven cases (43%). However, lymphoma and leukemia of peripheral T-cell origin was difficult to transplant even with pretreatment, and only one pleomorphic T-cell lymphoma grew to a significant size (2 cm). One tumor each of B-cell-derived diffuse large lymphoid and T-cell diffuse lymphoblastic lymphoma became transplantable.

  9. A specific immune tolerance toward offspring cells is to exist after the mother lymphocyte infusion. (United States)

    Xing, Haizhou; Liu, Shiqin; Chen, Xue; Fang, Fang; Wu, Xueqiang; Zhu, Ping


    To examine immune tolerance between maternal lymphocytes and offspring tissue after a donor lymphocyte infusion. Mouse models were established by mating female BALB/c mice with male C57BL mice. Splenic lymphocytes from donors of different genetic backgrounds were labeled with carboxyfluorescein succinimidyl ester (CFSE), and 1×10(7) of the labeled cells were intravenously injected into a recipient. At 6h, 24h, 72h and 120h after the infusion, mononuclear cells in recipient spleen, liver, thymus, lymph nodes, and peripheral blood were collected. CFSE+, CFSE-, CD3+, CD8+, CD4+, CD19+, NK1.1+, CD25+, and CD127+ lymphocytes in those samples were analyzed by flow cytometry. The distribution of donor T cells, B cells, NK cells, helper T cells, cytotoxic T cells, and recipient regulatory T cells in the tissues were then analyzed. Maternal lymphocytes were more likely to survive in offspring. At 120h after infusion, the percentages of maternal cells in the offspring were 0.52±0.11% in lymph nodes, 0.97±0.04% in peripheral blood, and 0.97±0.11% in the spleen. Few donor cells, if any, were detected in these tissues at 120h after aunt to child, father to child, and unrelated allogeneic infusions were performed. The subtype proportion of donor lymphocytes changed significantly in the recipient tissues. Recipient Treg cells increased in the mother to child group, but not in the aunt to child, father to child, and unrelated allogeneic groups, suggesting a decreased cellular immune response to allogeneic cells in the mother to child group. At 120h after the infusion, no donor cells were detected in the recipient livers and thymuses of all groups, implying that donor cells were barely able to colonize in the liver and thymus. Specific immune tolerance to maternal lymphocytes exists in offspring. An infusion of maternal donor lymphocytes may produce a relatively persistent effect of adoptive immunotherapy with reduced side-effects. Copyright © 2017 Elsevier GmbH. All rights

  10. Chronic lymphocytic lymphoma and concomitant renal cell carcinoma (Clear Cell Type: Review of the literature

    Directory of Open Access Journals (Sweden)

    Burak Uz


    Full Text Available In the present report, a 73 years-old male patient who developed clear cell type renal cell carcinoma (RCC 5 years after the diagnosis of chronic lymphocytic lymphoma (CLL and plausible explanations for this association were discussed by the authors. The incidence of CLL and RCC occurring in the same patient is higher than that expected in the general population. Various explicative hypotheses of this concurrence include treatment-related development of a second malignancy, immunomodulatory mechanisms, viral aetiology, cytokine (interleukin 6 release from a tumor, and common genetic mutations. Further investigations are warranted.

  11. The Spectrum of Kidney Pathology in B-Cell Chronic Lymphocytic Leukemia / Small Lymphocytic Lymphoma: A 25-Year Multicenter Experience (United States)

    Poitou-Verkinder, Anne-Laure; Francois, Arnaud; Drieux, Fanny; Lepretre, Stéphane; Legallicier, Bruno; Moulin, Bruno; Godin, Michel; Guerrot, Dominique


    Background Chronic lymphocytic leukemia and small lymphocytic lymphoma are 2 different presentations of the most common B-cell neoplasm in western countries (CLL/SLL). In this disease, kidney involvement is usually silent, and is rarely reported in the literature. This study provides a clinicopathological analysis of all-cause kidney disease in CLL/SLL patients. Methods Fifteen CLL/SLL patients with kidney biopsy were identified retrospectively. Demographic, clinical, pathological and laboratory data were assessed at biopsy, and during follow-up. Results At biopsy 11 patients presented impaired renal function, 7 patients nephrotic syndrome, 6 patients dysproteinemia, and 3 patients cryoglobulinemia. Kidney pathology revealed CLL/SLL-specific monoclonal infiltrate in 10 biopsies, glomerulopathy in 9 biopsies (5 membranoproliferative glomerulonephritis, 2 minimal change disease, 1 glomerulonephritis with organized microtubular monoclonal immunoglobulin deposits, 1 AHL amyloidosis). Five patients presented interstitial granulomas attributed to CLL/SLL. After treatment of the hematological disease, improvement of renal function was observed in 7/11 patients, and remission of nephrotic syndrome in 5/7 patients. During follow-up, aggravation of the kidney disease systematically occurred in the absence of favorable response to hematological treatment. Conclusions A broad spectrum of kidney diseases is associated with CLL/SLL. In this setting, kidney biopsy can provide important information for diagnosis and therapeutic guidance. PMID:25811382

  12. Cytogenetic analyses of Azadirachtin reveal absence of genotoxicity but marked antiproliferative effects in human lymphocytes and CHO cells in vitro. (United States)

    Mosesso, Pasquale; Bohm, Lothar; Pepe, Gaetano; Fiore, Mario; Carpinelli, Alice; Gäde, Gerd; Nagini, Siddavaram; Ottavianelli, Alessandro; Degrassi, Francesca


    In this work we have examined the genotoxic potential of the bioinsecticide Azadirachtin A (AZA) and its influence on cell proliferation on human lymphocytes and Chinese Hamster ovary (CHO) cells. AZA genotoxicity was assessed by the analysis of chromosomal aberrations and sister chromatid exchanges (SCEs) in the absence and presence of rat liver S9 metabolism. Primary DNA damage was also investigated by means of the comet assay. The results obtained clearly indicate that AZA is not genotoxic in mammalian cells. On the other hand, AZA proved to interfere with cell cycle progression as shown by modulation of frequencies of first (M1) and second division (M2) metaphases detected by 5-Bromo-2'-deoxyuridine labeling. Accumulation of M1 metaphases were more pronounced in human lymphocytes. In the transformed CHO cell line, however, significant increases of multinucleated interphases and polyploid cells were observed at long treatment time. At higher dose-levels, the incidence of polyploidy was close to 100%. Identification of spindle structure and number of centrosomes by fluorescent immunostaining with α- and γ-tubulin antibodies revealed aberrant mitoses exhibiting multipolar spindles with several centrosomal signals. These findings suggest that AZA can act either through a stabilizing activity of microtubules or by inhibition of Aurora A, since both mechanisms are able to generate genetically unstable polyploid cells with multipolar spindles and multinucleated interphases. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  13. Caspase-3-dependent Cell Death in B lymphocyte Caused by Pseudomonas aeruginosa Pyocyanin

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    Heni Susilowati


    Full Text Available Objective: The aim of this study was to investigate cellular responses of B lymphocyte to the exposure of pyocyanin and the role of caspase-3 in its molecular mechanism. Methods: B lymphocytes (Raji cells were cultured overnight prior to the experiments. Cell culture in five replications were then exposed to various concentrations of pyocyanin and incubated for 24 h in antibiotics-free medium. MTT assay was performed to analyze the cytotoxicity effect of pyocyanin. In separated experiments, the cells were cultured with pyocyanin and addressed for cell morphological analysis using phase contrast microscope. To study the mechanism involved in pyocyanin-induced cellular damage, immunocytochemical analysis was run for the identification of active caspase-3 protein expression. Results: The results of this study showed that cell viability was decreased in pcyocyanin-treated groups. Statistical analysis using ANOVA (p < 0.05 demonstrated significant different between groups with significant value of 0.000. Pyocyanin induced cell death on B lymphocyte in dose-dependent manner. Nuclear fragmentation was observed in pyocyanin-induced cell death; furthermore, caspase-3 was expressed clearly in cell cytoplasm after 24 h incubation. Conclusion: It is concluded that pyocyanin is capable of inducing cell death on B lymphocyte. Caspase-3 may play important role in the molecular mechanism of pyocyanin-induced cell death.DOI: 10.14693/jdi.v22i2.403

  14. Active phagocytosis of Mycobacterium tuberculosis (H37Ra) by T lymphocytes (Jurkat cells). (United States)

    Zhang, Min; Zhu, Qi; Shi, Ming; Liu, Yang; Ma, Lei; Yang, Yining; Feng, Dongyun; Dai, Wen; Zhang, Lin; Kang, Tao; Chen, Ping; He, Ying; Liu, Tingting; Zhao, Qing; Wang, Wenjing; Zhi, Jin; Feng, Guodong; Zhao, Gang


    This study aimed to co-culture Jurkat T lymphocytes with inactivated Mycobacterium tuberculosis (Mtb H37Ra), explore whether T lymphocytes could phagocytose H37Ra cells, and determine the underlying mechanism. Jurkat T lymphocytes were co-cultured with H37Ra cells, and confocal laser scanning microscopy, electron microscopy, and flow cytometry techniques were used to identify phagocytosis and elucidate its mechanism. After Jurkat T lymphocytes phagocytosed H37Ra cells, the cell body became larger, with abundant cytoplasm, the portion of the nucleus closest to the bacterium deformed, long and short pseudopodia were extended, and the folds of the cell membrane formed depressions that created phagocytic vesicles surrounding the bacterium. The macropinocytosis inhibitor amiloride and the cytoskeletal inhibitor cytochalasin D were found to inhibit phagocytic efficacy; serum complements might enhance phagocytosis through opsonization. Jurkat T lymphocytes could actively phagocytose inactivated Mtb via the macropinocytotic mechanism. Actin remodeling played an important role in the macropinocytotic process. Serum complements may regulate phagocytosis.

  15. An experimental system for determining the influence of microgravity on B lymphocyte activation and cell fusion (United States)

    Sammons, D. W.; Zimmermann, U.; Klinman, N. R.; Gessner, P.; Humphreys, R. C.; Emmons, S. P.; Neil, G. A.

    The influence of microgravity on lymphocyte activation is central to the understanding of immunological function in space. Moreover, the adaptation of groundbased technologies to microgravity conditions presents opportunities for biotechnological applications including high efficiency production of antibody forming hybridomas. Because the emerging technology of microgravity hybridoma generation is dependent upon activation and cultivation of B lymphocytes during flight, we have adapted mitogen-driven B lymphocyte stimulation and culture that allows for the in vitro generation of large numbers of antibody forming cells suitable for cell fusion over a period of 1-2 weeks. We believe that this activation and cultivation system can be flown on near-term space flights to test fundamental hypotheses about mammalian cell activation, cell fusion, metabolism, secretion, growth, and bio-separation.

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

    Energy Technology Data Exchange (ETDEWEB)

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


    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.

  17. A novel method for producing target cells and assessing cytotoxic T lymphocyte activity in outbred hosts

    Directory of Open Access Journals (Sweden)

    Bendinelli Mauro


    Full Text Available Abstract Background Cytotoxic T lymphocytes play a crucial role in the immunological control of microbial infections and in the design of vaccines and immunotherapies. Measurement of cytotoxic T lymphocyte activity requires that the test antigen is presented by target cells having the same or compatible class I major hystocompatibility complex antigens as the effector cells. Conventional assays use target cells labeled with 51chromium and infer cytotoxic T lymphocyte activity by measuring the isotope released by the target cells lysed following incubation with antigen-specific cytotoxic T lymphocytes. This assay is sensitive but needs manipulation and disposal of hazardous radioactive reagents and provides a bulk estimate of the reporter released, which may be influenced by spontaneous release of the label and other poorly controllable variables. Here we describe a novel method for producing target in outbred hosts and assessing cytotoxic T lymphocyte activity by flow cytometry. Results The method consists of culturing skin fibroblasts, immortalizing them with a replication defective clone of simian virus 40, and finally transducing them with a bicistronic vector encoding the target antigen and the reporter green fluorescent protein. When used in a flow cytometry-based assay, the target cells obtained with this method proved valuable for assessing the viral envelope protein specific cytotoxic T lymphocyte activity in domestic cats acutely or chronically infected with feline immunodeficiency virus, a lentivirus similar to human immunodeficiency virus and used as animal model for AIDS studies. Conclusion Given the versatility of the bicistronic vector used, its ability to deliver multiple and large transgenes in target cells, and its extremely wide cell specificity when pseudotyped with the vesicular stomatitis virus envelope protein, the method is potentially exploitable in many animal species.

  18. Automated flagging influences the inconsistency and bias of band cell and atypical lymphocyte morphological differentials.

    NARCIS (Netherlands)

    Meer, W. van der; Scott, C.S.; Keijzer, M.H. de


    This study evaluated inter- and intra-observer variabilities of band cell and atypical lymphocyte differentials and the influence of instrument flagging information on resulting microscopic differentials. Five stained slides with a range of band cell counts and five with variable numbers of atypical

  19. Stepwise isolation of human peripheral erythrocytes, T lymphocytes, and monocytes for blood cell proteomics. (United States)

    Brosseron, Frederic; May, Caroline; Schoenebeck, Bodo; Tippler, Bettina; Woitalla, Dirk; Kauth, Marion; Brockmann, Kathrin; Meyer, Helmut E; Berg, Daniela; Bufe, Albrecht; Marcus, Katrin


    Density gradient centrifugation and magnetic- or fluorescence-activated cell sorting are common and robust techniques for the isolation of different types of blood cells. In this article, we give detailed description of a stepwise application of these methods as one isolation strategy for enrichment of different cell types from one blood sample. The workflow targeted erythrocytes, monocytes, and T lymphocytes. Pancoll® density gradient centrifugation was used together with subsequent MACS™ isolation. Purity of monocytes and T lymphocytes was controlled by fluorescence-activated cell sorting analysis, and cells were used for carrier-ampholine-based 2D-PAGE to confirm compatibility of the procedure to standard proteomic applications. Gradient centrifugation resulted in an average of 125 μL of packed erythrocytes per milliliter blood. MACS™ sorting reached purities of 90 ± 2% (monocytes) and 93 ± 2% (T lymphocytes), with an average yield of 12 × 10(4) monocytes or T lymphocytes. 2D-PAGE of isolated cells showed well-separated spot patterns. A combined isolation holds substantial advantages especially in clinical studies, as it allows for the comparison of findings not only between individuals, but also between different cell types derived from one donor. Our approach ensured high reproducibility, yields, and purities of cells as required for reliable proteome analysis. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Surface Ig on rabbit lymphocytes. Rabbit B and T cells are distinct populations

    NARCIS (Netherlands)

    Bast, B J; Catty, D; Manten-Slingerland, R; Jansen, J T; Veldhuis, Dick H.; Roholl, P; Ballieux, R E


    Rabbit peripheral blood lymphocytes (PBL) were analyzed by immunofluorescence using anti-T cell conjugates and anti-Fab, anti-a1 allotype, anti-IgM and anti-IgA conjugates. In addition, T cells were demonstrated by rosetting with papain-treated homologous erythrocytes. Control experiments, using aci

  1. Association of inclusion body myositis with T cell large granular lymphocytic leukaemia

    DEFF Research Database (Denmark)

    Greenberg, Steven A; Pinkus, Jack L; Amato, Anthony A


    SEE HOHLFELD AND SCHULZE-KOOPS DOI101093/BRAIN/AWW053 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: Inclusion body myositis and T cell large granular lymphocytic leukaemia are rare diseases involving pathogenic cytotoxic CD8+ T cells. After encountering four patients with both disorders, we prospe...

  2. Role of signaling lymphocytic activation molecule in T helper cell responses

    Directory of Open Access Journals (Sweden)

    Jan E. de Vries


    Full Text Available Signaling lymphocytic activation molecule (SLAM; CDw150 is a 70 kDa glycoprotein. Signaling lymphocytic activation molecule is constitutively expressed on memory T cells, CD56+ T cells, a subset of T cell receptor γδ+ cells, immature thymocytes and, at low levels, on a proportion of peripheral blood B cells. Signaling lymphocytic activation molecule is rapidly upregulated on all T and B cells after activation. Engagement of SLAM by F(ab’2 fragments of an anti-SLAM monoclonal antibody (mAb A12 enhances antigen-specific T cell proliferation. In addition, mAb A12 was directly mitogenic for T cell clones and activated T cells. T cell proliferation induced by mAb A12 is independent of interleukin (IL-2, IL-4, IL-12 and IL-15, but is cyclosporin A sensitive. Ligation of SLAM during antigen-specific T cell proliferation resulted in upregulation of interferon (IFN-γ production, even by allergen-specific T helper cell (Th 2 clones, whereas the levels of IL-4 and IL-5 production were only marginally affected. The mAb A12 was unable to induce IL-4 and IL-5 production by Th1 clones. Co-stimulation of skin-derived Der P1-specific Th2 cells from patients with atopic dermatitis via SLAM resulted in the generation of a population of IFN-γ-producing cells, thereby reverting their phenotype to a Th0 pattern. Signaling lymphocytic activation molecule is a high-affinity self ligand mediating homophilic cell interaction. In addition, soluble SLAM enhances both T and B cell proliferation. Collectively, these data indicate that SLAM molecules act both as receptors and ligands that are not only involved in T cell expansion but also drive the expanding T cells during immune responses into the Th0/Th1 pathway. This suggests that signaling through SLAM plays a role in directing Th0/Th1 development.

  3. Pre-malignant lymphoid cells arise from hematopoietic stem/progenitor cells in chronic lymphocytic leukemia. (United States)

    Kikushige, Yoshikane; Miyamoto, Toshihiro


    Human malignancies progress through a multistep process that includes the development of critical somatic mutations over the clinical course. Recent novel findings have indicated that hematopoietic stem cells (HSCs), which have the potential to self-renew and differentiate into multilineage hematopoietic cells, are an important cellular target for the accumulation of critical somatic mutations in hematological malignancies and play a central role in myeloid malignancy development. In contrast to myeloid malignancies, mature lymphoid malignancies, such as chronic lymphocytic leukemia (CLL), are thought to originate directly from differentiated mature lymphocytes; however, recent compelling data have shown that primitive HSCs and hematopoietic progenitor cells contribute to the pathogenesis of mature lymphoid malignancies. Several representative mutations of hematological malignancies have been identified within the HSCs of CLL and lymphoma patients, indicating that the self-renewing long-lived fraction of HSCs can serve as a reservoir for the development of oncogenic events. Novel mice models have been established as human mature lymphoma models, in which specific oncogenic events target the HSCs and immature progenitor cells. These data collectively suggest that HSCs can be the cellular target involved in the accumulation of oncogenic events in the pathogenesis of mature lymphoid and myeloid malignancies.

  4. Potential Anti-proliferative and Immunomodulatory Effects of Marine Microalgal Exopolysaccharide on Various Human Cancer Cells and Lymphocytes In Vitro. (United States)

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


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

  5. Antiproliferative, Cytotoxic, and Apoptotic Activity of Steroidal Oximes in Cervicouterine Cell Lines

    Directory of Open Access Journals (Sweden)

    Luis Sánchez-Sánchez


    Full Text Available Steroidal sapogenins have shown antiproliferative effects against several tumor cell lines; and their effects on human cancer cells are currently under study. Changes in the functionality on the steroidal structure make it possible to modify the biological activity of compounds. Herein, we report the synthesis and in vitro antitumor activity of two steroidal oxime compounds on cervical cancer cells. These derivatives were synthesized from the steroidal sapogenin diosgenin in good yields. The in vitro assays show that the steroidal oximes show significant antiproliferative activity compared to the one observed for diosgenin. Cell proliferation, cell death, and the cytotoxic effects were determined in both cervical cancer cells and human lymphocytes. The cancer cells showed apoptotic morphology and an increased presence of active caspase-3, providing the notion of a death pathway in the cell. Significantly, the steroidal oximes did not exert a cytotoxic effect on lymphocytes.

  6. Microfluidics for T- lymphocyte cell separation and inflammation monitoring in burn patients. (United States)

    Rosenbach, Alan E; Koria, Piyush; Goverman, Jeremy; Kotz, Kenneth T; Gupta, Amit; Yu, Ming; Fagan, Shawn P; Irimia, Daniel; Tompkins, Ronald G


    Severe burns result in T lymphocyte specific immunologic changes. In addition to decreased levels of circulating lymphocytes, changes in cytokine secretion and receptor expression also take place. Our finer understanding of the inflammatory response has led to the development of immune-targeted therapeutics, requiring specialized gene-expression monitoring. The emerging field of bio-micro-electromechanical systems can be used to isolate highly pure T lymphocytes in a clinically relevant and timely manner for downstream genomic analysis. Blood samples from healthy volunteers and burn-injured patients were introduced into microfluidic devices developed in our laboratory. Utilizing cell-affinity chromatography for positive selection of T lymphocytes, the devices served as a platform for RNA extraction and downstream cytokine analysis via quantitative real-time polymerase chain reaction (PCR). From a 0.5-mL whole blood sample, the microfluidic devices captured highly pure T lymphocytes from healthy volunteers and burn-injured patients. Cell capture was of sufficient quantity, and extracted RNA was of sufficient quality, for evaluating the gene expression of cytokines: interferon-gamma, interleukin-2, interleukin-4, and interleukin-10. Microfluidics is a useful tool in processing blood from burn-injured patients. Though in its very early stages of development, cell-specific information obtained by this platform/technology will likely be an important component of near-patient molecular diagnostics and personalized medicine.


    Institute of Scientific and Technical Information of China (English)

    He Xi; Qin Huilian; Xiang Rong; Zhang Yuejian; Ye Wenfei; He Qiuzao


    Although cervical carcinoma cells may express the human papillomavirus protein E6 and E7, they fail to induce an effective specific cytotoxic T lymphocyte response. Recent studies suggest that expression of CD-80(B7-1) on tumor cells is effective to induce anfitumor immune responses.1,2 In our study, CD-80 gene was transfected into human Hela cell line with a CD-80expression plasmid (B7-1+pcDNA3) by electroporation,then the immunogenecity of the modified Hela cell was tested in TLMC (tumor lymphocyte mixed culture)system. [3H]thymidine lymphocyte proliferation assays showed that the response of human peripheral blood lymphocytes (PBLS) to CD-80 positive Hela cells demonstrated a substantial increase in cell proliferation compared to the response to control cells. Cocultivation of allogeneic PBLs with CD-80 positive tumor cells for three days can induce an increased secretion of IL-2. Our results demonstrate an immunostimulatory effect of CD-80 expression on cervical cancer cells, which provides a basis for the development of a therapeutic tumor vaccine.

  8. Flow microfluorometric DNA content measurements of tissue culture cells and peripheral lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Cram, L.S. (Los Alamos Scientific Lab., NM); Lehman, J.M.


    The difference in DNA content of peripheral lymphocytes from normal males, normal females, and an individual with a 48 (xxxy) chromosome constitution was determined by rapid flow microfluorometric techniques. A similar comparison was performed using tissue culture fibroblasts derived from an individual with a 49 (xxxxy) chromosome constitution and WI-38 cells as a normal control. Less than 60 min were required to isolate the lymphocytes, to stain the cells fluorescently, and to measure the increased DNA content. The measured increase in DNA content is consistent with chromosome DNA analyses and chromosome length measurements.

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

    Directory of Open Access Journals (Sweden)

    Severin Zinöcker

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

  10. Engineered human embryonic stem cell-derived lymphocytes to study in vivo trafficking and immunotherapy. (United States)

    Knorr, David A; Bock, Allison; Brentjens, Renier J; Kaufman, Dan S


    Human embryonic stem cell (hESC)-derived natural killer (NK) cells are a promising source of antitumor lymphocytes for immunotherapeutics. They also provide a genetically tractable platform well suited for the study of antitumor immunotherapies in preclinical models. We have previously demonstrated the potency of hESC-derived NK cells in vivo. Here we use both bioluminescent and fluorescent imaging to demonstrate trafficking of hESC-derived NK cells to tumors in vivo. Our dual-imaging approach allowed us to more specifically define the kinetics of NK cell trafficking to tumor sites. NK cell persistence and trafficking were further evaluated by flow cytometry and immunohistochemistry. This integrated approach provides a unique system to apply the use of human pluripotent stem cells to study the kinetics and biodistribution of adoptively transferred lymphocytes, advances broadly applicable to the field of immunotherapy.

  11. CD4+ T Lymphocytes count in sickle cell anaemia patients attending a tertiary hospital

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    Omotola Toyin Ojo


    Full Text Available Background: Sickle cell haemoglobin (HbS is the commonest abnormal haemoglobin and it has a worldwide distribution. Reports have shown that patients with sickle cell anaemia (HbSS have an increased susceptibility to infection leading to increased morbidity and mortality. Impaired leucocyte function and loss of both humoral and cell-mediated immunity are some of the mechanisms that have been reported to account for the immunocompromised state in patients with sickle cell disease. This study was carried out to determine the CD4+ T lymphocytes count in patients with sickle cell anaemia. Materials and Methods: A comparative cross-sectional study of 40 sickle cell anaemia patients in steady state (asymptomatic for at least 4 weeks attending haematology clinic and 40 age and sex-matched healthy HbA control were recruited into the study. Both HbS patients and the controls were HIV negative. The blood samples obtained were analyzed for CD4+ T cell by Flow cytometry. Results: The study found that there was no significant difference in the number of CD4+ T lymphocyte count between individuals with sickle cell anaemia and HbA (1016 ± 513 cells/μL vs 920 ± 364cells/μL. Conclusion: It is recommended that the functionality of CD4+ T lymphocyte should be considered rather than the number in further attempt to elucidate the cellular immune dysfunction in patients with sickle cell anaemia.

  12. Posttranslational formation of hypusine in a single major protein occurs generally in growing cells and is associated with activation of lymphocyte growth. (United States)

    Cooper, H L; Park, M H; Folk, J E


    Growing lymphocytes perform a novel chemical modification of a single protein (Hy+: approximately 18 kd, pI approximately 5.1), resulting in the formation of the unusual amino acid, hypusine (N epsilon-[4-amino-2-hydroxybutyl]lysine). This posttranslational event occurs only following activation of lymphocyte growth. Hypusine formation increases at a rate parallel to protein synthesis during the first 24 hr of growth stimulation, beginning before 6 hr of growth. At all times, hypusine is restricted primarily to the single protein, Hy+. In resting cells, the unmodified substrate protein, Hy0, is continuously synthesized and maintained in a steady-state pool of significant size. In several other cell lines, hypusine formation was also observed in a single protein of approximately 18 kd, pI approximately 5.1, indistinguishable electrophoretically from the lymphocyte protein. Thus Hy+ is a ubiquitous protein showing significant conservation among divergent species. Maintenance by resting lymphocytes of a pool of unmodified protein and early activation during growth of the hypusine-forming enzyme system suggest that this posttranslational modification may be of importance to lymphocyte activation.

  13. Myxoma virus suppresses proliferation of activated T lymphocytes yet permits oncolytic virus transfer to cancer cells. (United States)

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


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

  14. Difference in membrane repair capacity between cancer cell lines and a normal cell line

    DEFF Research Database (Denmark)

    Frandsen, Stine Krog; McNeil, Anna K.; Novak, Ivana


    Electroporation-based treatments and other therapies that permeabilize the plasma membrane have been shown to be more devastating to malignant cells than to normal cells. In this study, we asked if a difference in repair capacity could explain this observed difference in sensitivity. Membrane...... repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique...... cancer cell lines (p cell line (p membrane permeabilization by electroporation. Viability in the primary normal cell line (98 % viable cells) was higher...

  15. Effect of insulin on functional status of cord blood-derived dendritic cells and on dendritic cell-induced CTL cytotoxicity against pancreatic cancer cell lines

    Institute of Scientific and Technical Information of China (English)

    Qiu-Liang Liu; Yi-Sheng Wang; Jia-Xiang Wang


    BACKGROUND: Dendritic cells (DCs) are the most important antigen-presenting cells in the human body, and DCs with different mature status possess different or even opposite functions. This study was designed to explore the influence of insulin on the functional status of cord blood-derived DCs and on DC-induced cytotoxic T lymphocyte (CTL) activity against pancreatic cancer cell lines. METHODS: Mononuclear cells were isolated from fresh cord blood. Interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were used to induce or stimulate the mononuclear cells. Insulin at different concentrations served to modify DCs, and then DC morphology, number, and growth status were assessed. The DC immunophenotype was detected with a flow cytometer. The IL-12 in DC supernatant was determined by ELISA. DC functional status was evaluated by the autologous mixed lymphocyte reaction. T lymphocytes were induced by insulin-modified DCs to become CTLs. The CTL cytotoxicity against pancreatic cancer cell lines was determined. RESULTS:  Mononuclear cells from cord blood can be differentiated into DCs by cytokine induction and insulin modification. With the increase in insulin concentration (2.5-25 mg/L), the expression of DC HLA-DR, CD1α, CD80, and CD83 was significantly increased, the DC ability to secrete IL-12 was significantly improved, DC function to activate autologous lymphocytes was significantly enhanced, and the cytotoxicity of CTLs induced by insulin-modified DCs against pancreatic cancer cell lines was significantly strengthened. CONCLUSIONS: Insulin may facilitate DC induction and maturation, and improve the reproductive activity of autologous lymphocytes. The cytotoxicity of CTLs induced by insulin-modified DCs against pancreatic cancer cell lines was significantly enhanced. Insulin may serve as a factor modifying DCs and inducing CTLs in vitro in insulin biotherapy.

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

    Directory of Open Access Journals (Sweden)

    Ochoa-Hernández Alejandra B


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

  17. Echinococcus granulosus-specific T-cell lines derived from patients at various clinical stages of cystic echinococcosis. (United States)

    Riganò, R; Buttari, B; De Falco, E; Profumo, E; Ortona, E; Margutti, P; Scottà, C; Teggi, A; Siracusano, A


    To investigate the role of T lymphocytes in the immune response to Echinococcus granulosus, using sheep hydatid fluid (SHF) and antigen B (AgB), we generated T-cell lines from patients with active, transitional and inactive hydatid cysts. We established 16 T-cell lines, eight specific to SHF and eight specific to AgB. At surface phenotyping 88-98% of cells displayed the helper/inducer CD4 antigen. In all patients, at all clinical stages of hydatid cyst disease, T-cell stimulation with SHF and AgB invariably amplified a large number of almost identical Vbeta subfamily fragments. Irrespective of antigen-specificity, the two cell lines from the patient with an inactive cyst had a Th1 profile, because they exclusively expressed and produced IFN-gamma. Conversely, the T-cell lines derived from the seven patients with active and transitional hydatid cysts had mixed Th1/Th2 and Th0 clones. The functional characteristics of the 16 T-cell lines differed markedly in the various clinical stages of cystic echinococcosis, thus providing new in vitro evidence that Th1 lymphocytes contribute decisively to the inactive stage of hydatid disease, Th2 lymphocytes in the active and transitional stages. The parasite-specific T-cell lines, especially the two Th1 lines from the patient with an inactive cyst, may help identify Th1 protective epitopes on SHF and AgB.

  18. Clinical scale rapid expansion of lymphocytes for adoptive cell transfer therapy in the WAVE® bioreactor (United States)


    Background To simplify clinical scale lymphocyte expansions, we investigated the use of the WAVE®, a closed system bioreactor that utilizes active perfusion to generate high cell numbers in minimal volumes. Methods We have developed an optimized rapid expansion protocol for the WAVE bioreactor that produces clinically relevant numbers of cells for our adoptive cell transfer clinical protocols. Results TIL and genetically modified PBL were rapidly expanded to clinically relevant scales in both static bags and the WAVE bioreactor. Both bioreactors produced comparable numbers of cells; however the cultures generated in the WAVE bioreactor had a higher percentage of CD4+ cells and had a less activated phenotype. Conclusions The WAVE bioreactor simplifies the process of rapidly expanding tumor reactive lymphocytes under GMP conditions, and provides an alternate approach to cell generation for ACT protocols. PMID:22475724

  19. Clinical scale rapid expansion of lymphocytes for adoptive cell transfer therapy in the WAVE® bioreactor

    Directory of Open Access Journals (Sweden)

    Somerville Robert PT


    Full Text Available Abstract Background To simplify clinical scale lymphocyte expansions, we investigated the use of the WAVE®, a closed system bioreactor that utilizes active perfusion to generate high cell numbers in minimal volumes. Methods We have developed an optimized rapid expansion protocol for the WAVE bioreactor that produces clinically relevant numbers of cells for our adoptive cell transfer clinical protocols. Results TIL and genetically modified PBL were rapidly expanded to clinically relevant scales in both static bags and the WAVE bioreactor. Both bioreactors produced comparable numbers of cells; however the cultures generated in the WAVE bioreactor had a higher percentage of CD4+ cells and had a less activated phenotype. Conclusions The WAVE bioreactor simplifies the process of rapidly expanding tumor reactive lymphocytes under GMP conditions, and provides an alternate approach to cell generation for ACT protocols.

  20. NFAT1 transcription factor regulates cell cycle progression and cyclin E expression in B lymphocytes. (United States)

    Teixeira, Leonardo K; Carrossini, Nina; Sécca, Cristiane; Kroll, José E; DaCunha, Déborah C; Faget, Douglas V; Carvalho, Lilian D S; de Souza, Sandro J; Viola, João P B


    The NFAT family of transcription factors has been primarily related to T cell development, activation, and differentiation. Further studies have shown that these ubiquitous proteins are observed in many cell types inside and outside the immune system, and are involved in several biological processes, including tumor growth, angiogenesis, and invasiveness. However, the specific role of the NFAT1 family member in naive B cell proliferation remains elusive. Here, we demonstrate that NFAT1 transcription factor controls Cyclin E expression, cell proliferation, and tumor growth in vivo. Specifically, we show that inducible expression of NFAT1 inhibits cell cycle progression, reduces colony formation, and controls tumor growth in nude mice. We also demonstrate that NFAT1-deficient naive B lymphocytes show a hyperproliferative phenotype and high levels of Cyclin E1 and E2 upon BCR stimulation when compared to wild-type B lymphocytes. NFAT1 transcription factor directly regulates Cyclin E expression in B cells, inhibiting the G1/S cell cycle phase transition. Bioinformatics analysis indicates that low levels of NFAT1 correlate with high expression of Cyclin E1 in different human cancers, including Diffuse Large B-cell Lymphomas (DLBCL). Together, our results demonstrate a repressor role for NFAT1 in cell cycle progression and Cyclin E expression in B lymphocytes, and suggest a potential function for NFAT1 protein in B cell malignancies.

  1. Emperipolesis-like invasion of neoplastic lymphocytes into hepatocytes in feline T-cell lymphoma. (United States)

    Suzuki, M; Kanae, Y; Kagawa, Y; Ano, N; Nomura, K; Ozaki, K; Narama, I


    Twelve cases of feline malignant lymphoma with emperipolesis-like invasion of neoplastic lymphocytes were examined microscopically, immunohistochemically and ultrastructurally. Intracytoplasmic invasion of neoplastic cells varied in severity between the cases, between hepatic lobules and between areas within the lobules. The number of infiltrating neoplastic cells ranged from one to several per hepatocyte. Neoplastic cells exhibited widely varying morphology from case-to-case and cell-to-cell within each case, and contained eosinophilic cytoplasmic granules in four cases. Immunohistochemical examination revealed that neoplastic cells in 11 of the 12 cases expressed one or both T-cell markers (CD3 and TIA-1). Diagnosis of T-cell lymphoma was also confirmed by assessment of clonality by polymerase chain reaction. Ultrastructural analysis revealed that the neoplastic lymphocytes were contained within an invagination of the cell membrane of the hepatocyte, rather than directly infiltrating into the cytoplasm of the cell. There was no evidence that the invasive neoplastic lymphocytes had a cytotoxic effect.

  2. Measuring melanoma-specific cytotoxic T lymphocytes elicited by dendritic cell vaccines with a tumor inhibition assay in vitro. (United States)

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


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

  3. Crosstalk between medulloblastoma cells and endothelium triggers a strong chemotactic signal recruiting T lymphocytes to the tumor microenvironment.

    Directory of Open Access Journals (Sweden)

    Vita S Salsman

    Full Text Available Cancer cells can live and grow if they succeed in creating a favorable niche that often includes elements from the immune system. While T lymphocytes play an important role in the host response to tumor growth, the mechanism of their trafficking to the tumor remains poorly understood. We show here that T lymphocytes consistently infiltrate the primary brain cancer, medulloblastoma. We demonstrate, both in vitro and in vivo, that these T lymphocytes are attracted to tumor deposits only after the tumor cells have interacted with tumor vascular endothelium. Macrophage Migration Inhibitory Factor (MIF" is the key chemokine molecule secreted by tumor cells which induces the tumor vascular endothelial cells to secrete the potent T lymphocyte attractant "Regulated upon Activation, Normal T-cell Expressed, and Secreted (RANTES." This in turn creates a chemotactic gradient for RANTES-receptor bearing T lymphocytes. Manipulation of this pathway could have important therapeutic implications.

  4. Characterizing T Cells in SCID Patients Presenting with Reactive or Residual T Lymphocytes

    Directory of Open Access Journals (Sweden)

    Atar Lev


    Full Text Available Introduction. Patients with severe combined immunodeficiency (SCID may present with residual circulating T cells. While all cells are functionally deficient, resulting in high susceptibility to infections, only some of these cells are causing autoimmune symptoms. Methods. Here we compared T-cell functions including the number of circulating CD3+ T cells, in vitro responses to mitogens, T-cell receptor (TCR repertoire, TCR excision circles (TREC levels, and regulatory T cells (Tregs enumeration in several immunodeficinecy subtypes, clinically presenting with nonreactive residual cells (MHC-II deficiency or reactive cells. The latter includes patients with autoreactive clonal expanded T cell and patients with alloreactive transplacentally maternal T cells. Results. MHC-II deficient patients had slightly reduced T-cell function, normal TRECs, TCR repertoires, and normal Tregs enumeration. In contrast, patients with reactive T cells exhibited poor T-cell differentiation and activity. While the autoreactive cells displayed significantly reduced Tregs numbers, the alloreactive transplacentally acquired maternal lymphocytes had high functional Tregs. Conclusion. SCID patients presenting with circulating T cells show different patterns of T-cell activity and regulatory T cells enumeration that dictates the immunodeficient and autoimmune manifestations. We suggest that a high-tolerance capacity of the alloreactive transplacentally acquired maternal lymphocytes represents a toleration advantage, yet still associated with severe immunodeficiency.

  5. Characterizing T cells in SCID patients presenting with reactive or residual T lymphocytes. (United States)

    Lev, Atar; Simon, Amos J; Trakhtenbrot, Luba; Goldstein, Itamar; Nagar, Meital; Stepensky, Polina; Rechavi, Gideon; Amariglio, Ninette; Somech, Raz


    Patients with severe combined immunodeficiency (SCID) may present with residual circulating T cells. While all cells are functionally deficient, resulting in high susceptibility to infections, only some of these cells are causing autoimmune symptoms. Here we compared T-cell functions including the number of circulating CD3(+) T cells, in vitro responses to mitogens, T-cell receptor (TCR) repertoire, TCR excision circles (TREC) levels, and regulatory T cells (Tregs) enumeration in several immunodeficinecy subtypes, clinically presenting with nonreactive residual cells (MHC-II deficiency) or reactive cells. The latter includes patients with autoreactive clonal expanded T cell and patients with alloreactive transplacentally maternal T cells. MHC-II deficient patients had slightly reduced T-cell function, normal TRECs, TCR repertoires, and normal Tregs enumeration. In contrast, patients with reactive T cells exhibited poor T-cell differentiation and activity. While the autoreactive cells displayed significantly reduced Tregs numbers, the alloreactive transplacentally acquired maternal lymphocytes had high functional Tregs. SCID patients presenting with circulating T cells show different patterns of T-cell activity and regulatory T cells enumeration that dictates the immunodeficient and autoimmune manifestations. We suggest that a high-tolerance capacity of the alloreactive transplacentally acquired maternal lymphocytes represents a toleration advantage, yet still associated with severe immunodeficiency.

  6. B-Cell Receptor Epitope Recognition Correlates With the Clinical Course of Chronic Lymphocytic Leukemia

    NARCIS (Netherlands)

    Binder, Mascha; Mueller, Fabian; Jackst, Antje; Lechenne, Barbara; Pantic, Milena; Bacher, Ulrike; Eulenburg, Christine Zu; Veelken, Hendrik; Mertelsmann, Roland; Pasqualini, Renata; Arap, Wadih; Trepel, Martin


    BACKGROUND: B-cell receptors (BCRs) and their recognition of specific epitopes may play a pivotal role in the development and progression of chronic lymphocytic leukemia (CLL). In this study, the authors set up a model system to explore epitope reactivity and its clinical relevance in CLL. METHODS:

  7. Peptide microarrays for the profiling of cytotoxic T-lymphocyte activity using minimum numbers of cells.

    NARCIS (Netherlands)

    Hoff, A.; Bagu, A.C.; Andre, T.; Roth, G.; Wiesmuller, K.H.; Guckel, B.; Brock, R.E.


    The identification of epitopes that elicit cytotoxic T-lymphocyte activity is a prerequisite for the development of cancer-specific immunotherapies. However, especially the parallel characterization of several epitopes is limited by the availability of T cells. Microarrays have enabled an unpreceden

  8. T-Cell Large Granular Lymphocyte Leukemia in the Lower Eyelid. (United States)

    Sia, Paul Ikgan; Figueira, Edwin; Kuss, Bryone; Craig, James; Selva, Dinesh

    The authors describe a case of T-cell large granular lymphocytic leukemia nodular lesion of the eyelid. To their knowledge, this has not been reported previously to occur in the eyelids. They have also reviewed previous literature reports on similar skin lesions in areas elsewhere.

  9. CD3 directed bispecific antibodies induce increased lymphocyte-endothelial cell interactions in vitro

    NARCIS (Netherlands)

    Molema, G; Tervaert, JWC; Kroesen, BJ; Helfrich, W; Meijer, DKF; de Leij, LFMH


    Bispecific antibody (BsMAb) BIS-1 has been developed to redirect the cytolytic activity of cytotoxic T lymphocytes (CTL) to epithelial glycoprotein-2 (EGP-2) expressing tumour cells; intravenous administration of BIS-1 F(ab')(2) to carcinoma patients in a phase I/II clinical trial, caused immunomodu

  10. Activity of vinorelbine on B-chronic lymphocytic leukemia cells in vitro. (United States)

    Bernabei, P A; Landini, I; Bartolozzi, B; Banchelli, I; Degli Innocenti o Nocentini, A; Santini, V; Ematologia, U O


    Vinorelbine (VNR) is a new semi-synthetic Vinca rosea alkaloid that has been employed both in combination and as a single agent, showing a significant antitumour activity. Since little is known about VNR in human leukemia, we studied the in vitro cytotoxic effect of VNR on peripheral blood lymphocytes from 18 patients affected by B-chronic lymphocytic leukemia (CLL), employing the INT assay. VNR inhibited fresh B-CLL cells from 15/18 patients in primary cultures, the ID50 doses ranging from 4 ng/ml to 83 micrograms/ml. These data strongly suggest that VNR could be effective in the treatment of B-CLL.

  11. Tumor infiltrating lymphocyte therapy for ovarian cancer and renal cell carcinoma

    DEFF Research Database (Denmark)

    Andersen, Rikke; Donia, Marco; Westergaard, Marie Christine Wulff


    Personalized cancer immunotherapy based on infusion of T cells holds the promise to specifically target a patient's individual tumor. Accumulating evidence indicates that the T cells mediating these tumor regressions after cancer immunotherapies may primarily target patient-specific mutations...... therapy in solid tumors other than melanoma have shown limited success, however none of these early trials used current preparative chemotherapy regimens, and the methods for in vitro lymphocyte expansion have changed considerably. New advances and understandings in T cell based immunotherapies have...... stimulated the interest in developing this approach for other indications. Here, we summarize the early clinical data in the field of adoptive cell transfer therapy (ACT) using tumor-infiltrating lymphocytes for patients with renal cell carcinoma (RCC) and ovarian cancer (OC). In addition we describe...

  12. Composite ALK-negative anaplastic large cell lymphoma and small lymphocytic lymphoma involving the right inguinal lymph node. (United States)

    Persad, Paul; Pang, Changlee S


    Anaplastic large cell lymphoma and small lymphocytic lymphoma are two lymphoid malignancies with completely distinct morphologies and natural histories. We present a rare case of composite anaplastic large cell lymphoma and small lymphocytic lymphoma in an inguinal lymph node of an otherwise healthy 47-year-old male patient. Immunohistochemical and molecular studies identified the two populations clearly. Their separation is imperative as anaplastic large cell lymphoma can be an aggressive neoplasm and easily overlooked in cases of small lymphocytic lymphoma with a small population of anaplastic large cell lymphoma cells.

  13. Autologous and allogeneic typing of human leukemia cells: definition of surface antigens restricted to lymphocytic leukemia cells.


    Naito, K.; Yamaguchi, H; Horibe, K; Shiku, H.; Takahashi, T.; Suzuki, S; Yamada, K.


    Serum from a patient (CO) with acute lymphoblastic leukemia was reactive in immunoadherence assays with autologous leukemia cells but not with autologous blood lymphocytes or bone marrow cells during complete remission. Extensive absorption tests with an array of leukemia cells and normal cells were performed in order to define the specificity of the reaction. The autologous leukemia reactivity was either completely or partially absorbed with acute lymphoblastic leukemia cells obtained from 1...

  14. Difference in Membrane Repair Capacity Between Cancer Cell Lines and a Normal Cell Line. (United States)

    Frandsen, Stine Krog; McNeil, Anna K; Novak, Ivana; McNeil, Paul L; Gehl, Julie


    Electroporation-based treatments and other therapies that permeabilize the plasma membrane have been shown to be more devastating to malignant cells than to normal cells. In this study, we asked if a difference in repair capacity could explain this observed difference in sensitivity. Membrane repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique, providing a sensitive index of repair capacity. The normal primary cell line of all tested cell lines exhibited the slowest rate of dye entry after laser disruption and lowest level of dye uptake. Significantly, more rapid dye uptake and a higher total level of dye uptake occurred in six of the seven tested cancer cell lines (p normal cell line (98 % viable cells) was higher than in the three tested cancer cell lines (81-88 % viable cells). These data suggest more effective membrane repair in normal, primary cells and supplement previous explanations why electroporation-based therapies and other therapies permeabilizing the plasma membrane are more effective on malignant cells compared to normal cells in cancer treatment.

  15. Apoptotic cell death, detected ex vivo in peripheral blood lymphocytes of HIV-1 infected persons

    Directory of Open Access Journals (Sweden)

    L. F. te Velde


    Full Text Available In HIV-1 infection the ongoing depletion of CD4+ T-lymphocytes is believed, to a large extent, to be due to apoptosis. Until now quantitative information about in vivo apoptosis of lymphocytes in HIV-patients is scarce because of the very nature of the apoptotic process. Successful detection of apoptosis ex vivo requires the recognition of the initial phase of this process, because at a later stage the cells may not remain any longer in the circulation. We measured quantitatively the amount of early apoptotic peripheral blood lymphocytes directly ex vivo in HIV-1 infected patients using a recently described flow cytometric assay. With this method we observed in an unselected heterogenous group of twelve HIV-infected individuals a median percentage of apoptotic lymphocytes to be significantly higher than in ten healthy controls. To the best of our knowledge this is the first report of ex vivo observed increased apoptosis of peripheral blood lymphocytes in HIV-infected persons.

  16. Reduced cell turnover in lymphocytic monkeys infected by human T-lymphotropic virus type 1. (United States)

    Debacq, Christophe; Héraud, Jean-Michel; Asquith, Becca; Bangham, Charles; Merien, Fabrice; Moules, Vincent; Mortreux, Franck; Wattel, Eric; Burny, Arsène; Kettmann, Richard; Kazanji, Mirdad; Willems, Luc


    Understanding cell dynamics in animal models have implications for therapeutic strategies elaborated against leukemia in human. Quantification of the cell turnover in closely related primate systems is particularly important for rare and aggressive forms of human cancers, such as adult T-cell leukemia. For this purpose, we have measured the death and proliferation rates of the CD4+ T lymphocyte population in squirrel monkeys (Saimiri sciureus) infected by human T-lymphotropic virus type 1 (HTLV-1). The kinetics of in vivo bromodeoxyuridine labeling revealed no modulation of the cell turnover in HTLV-1-infected monkeys with normal CD4 cell counts. In contrast, a substantial decrease in the proliferation rate of the CD4+ T population was observed in lymphocytic monkeys (e.g. characterized by excessive proportions of CD4+ T lymphocytes and by the presence of abnormal flower-like cells). Unexpectedly, onset of HTLV-associated leukemia thus occurs in the absence of increased CD4+ T-cell proliferation. This dynamics significantly differs from the generalized activation of the T-cell turnover induced by other primate lymphotropic viruses like HIV and SIV.

  17. Good syndrome presenting with CD8+ T-Cell large granular lymphocyte leukemia


    Caperton, Caroline; Agrawal, Sudhanshu; Gupta, Sudhir


    Good Syndrome is an adult-onset combined immunodeficiency defined by hypogammaglobulinemia, low or absent number of B cells, T cell deficiency and thymic tumor. We have characterized CD8+ T cells from a patient with Good syndrome that presented with CD8+T-cell large granular lymphocytic leukemia (LGL). Characterization of peripheral blood CD8+ T cells revealed that majority of CD8+ T cells were terminally differentiated effector memory phenotype (TEMRA; CD8+CCR7-CD45RA+), and were PD-1high (C...

  18. Tumor-infiltrating B lymphocytes as an efficient source of highly specific immunoglobulins recognizing tumor cells

    Directory of Open Access Journals (Sweden)

    Pelliccia Angela


    Full Text Available Abstract Background There is much evidence that tumor cells elicit a humoral immune response in patients. In most cases, the presence of antibodies in peripheral blood is detected only in small proportion of patients with tumors overexpressing the corresponding antigen. In the present study, we analyzed the significance of local humoral response provided by tumor-infiltrating lymphocytes in breast cancer patients. Methods The ability of a patient's immune system to produce specific antibodies inside tumor tissue, capable of recognizing tumor cells, was explored through analysis of the oligoclonality of antibodies derived from tumor-infiltrating lymphocytes and construction of a series of recombinant antibody libraries in scFv format, derived from breast tumor-infiltrating B lymphocytes. These libraries and one from peripheral blood lymphocytes of a single breast cancer patient were panned against three purified surface tumor antigens, such as CEA, MUC1 and ED-B domain, and against intact MCF7 breast carcinoma cells. Results Application of novel display vector, pKM19, allowed isolation of a large panel of breast cancer-specific antibodies against known tumor antigens, as well as against breast carcinoma cells. Reactivity of novel scFvs was confirmed by ELISA, immunohistochemistry, fluorescence staining and flow cytometry. We demonstrated that seven of ten primary breast tumor specimens, obtained using discarded surgical material, could be exploited as an appropriate source for generation of phage display libraries, giving highly specific antitumor antibodies which recognize heterologous tumor cells. Conclusion Local humoral immune response within tumor tissue in breast cancer patients frequently has an oligoclonal character. Efficient selection of specific antitumor antibodies from recombinant antibody libraries, derived from such oligoclonal tumor-infiltrated B lymphocytes, indicates the presence of natural immune response against tumor antigens

  19. Isoform of Vascular Endothelial Cell Growth Inhibitor (VEGI72-251) Increases Interleukin-2 Production by Activation of T Lymphocytes

    Institute of Scientific and Technical Information of China (English)

    Jing-Juan YAO; Min ZHANG; Xiao-Hui MIAO; Ping ZHAO; Shi-Ying ZHU; Hui DING; Zhong-Tian QI


    The objective of this study is to investigate the characteristics of the recombinant variant of human vascular endothelial cell growth inhibitor, VEGI72-251, and compare its biological activities with that of its prototype VEGI24-174. The recombinant plasmid containing the variant VEGI72-251 gene was constructed and expressed in Escherichia coli. The effects of the expressed VEGI72-251 on cell proliferations were checked in the human umbilical vein endothelial cell line and certain tumor cell lines (ECV304 and B 16). The inhibition of VEGI72-251 on angiogenesis was detected in the chorioallantoic membrane of chick embryos. In comparison with VEGI24-174, the recombinant human VEGI72-251 seems to have no effect on the proliferation of endothelial cells and the angiogenesis of the chorioallantoic membrane in vitro. An enzyme-linked immunosorbent assaybased method was used for the measurement of interleukin-2 (IL-2) production by peripheral blood monocytes (PBMCs) treated with VEGI72-251. PBMCs were pretreated with VEGI72-251 (1.25-12.50 μg/ml) for 24 h in vitro, and the IL-2 concentration in PBMC medium was increased from 354 pg/ml to 1256 pg/ml. It can be concluded that VEGI72-251 is able to increase the level of human IL-2 production by the activation of T lymphocytes. Differing from VEGI24-174 on anti-angiogenesis, VEGI72-251 may serve as an anti-cancer factor through its activation of T lymphocytes.

  20. In vitro cytotoxicity against Marek's disease lymphoblastoid cell lines after enzymatic removal of Marek's disease tumor-associated surface antigen.


    Schat, K A; Murthy, K K


    Marek's disease tumor-associated surface antigen (MATSA) has been claimed to be the target of cytotoxic lymphocytes in in vitro tests for Marek's disease immunity. Treatment with papain, but not with trypsin or mixed glycosidases, removed MATSA from certain Marek's disease lymphoblastoid cell lines. Tumor cells with and without MATSA were used as target cells for in vitro studies on cell-mediated immune responses with sensitized spleen cells in a chromium release assay. The removal of MATSA d...

  1. Stereotypical chronic lymphocytic leukemia B-cell receptors recognize survival promoting antigens on stromal cells.

    Directory of Open Access Journals (Sweden)

    Mascha Binder

    Full Text Available Chronic lymphocytic leukemia (CLL is the most common leukemia in the Western world. Survival of CLL cells depends on their close contact with stromal cells in lymphatic tissues, bone marrow and blood. This microenvironmental regulation of CLL cell survival involves the stromal secretion of chemo- and cytokines as well as the expression of adhesion molecules. Since CLL survival may also be driven by antigenic stimulation through the B-cell antigen receptor (BCR, we explored the hypothesis that these processes may be linked to each other. We tested if stromal cells could serve as an antigen reservoir for CLL cells, thus promoting CLL cell survival by stimulation through the BCR. As a proof of principle, we found that two CLL BCRs with a common stereotyped heavy chain complementarity-determining region 3 (previously characterized as "subset 1" recognize antigens highly expressed in stromal cells--vimentin and calreticulin. Both antigens are well-documented targets of autoantibodies in autoimmune disorders. We demonstrated that vimentin is displayed on the surface of viable stromal cells and that it is present and bound by the stereotyped CLL BCR in CLL-stroma co-culture supernatant. Blocking the vimentin antigen by recombinant soluble CLL BCR under CLL-stromal cell co-culture conditions reduces stroma-mediated anti-apoptotic effects by 20-45%. We therefore conclude that CLL BCR stimulation by stroma-derived antigens can contribute to the protective effect that the stroma exerts on CLL cells. This finding sheds a new light on the understanding of the pathobiology of this so far mostly incurable disease.

  2. Expression of Fas receptor on peripheral blood lymphocytes from patients with non-small cell lung cancer.

    Directory of Open Access Journals (Sweden)

    Joanna Domagała-Kulawik


    Full Text Available In recent years many data indicate that lymphocytes from cancer patients undergo increased apoptosis. The objective of this study was to evaluate the expression of Fas receptor on lymphocytes obtained from patients with lung cancer. Eighteen patients with non-small cell lung cancer and 18 healthy volunteers were investigated. Expression of Fas (CD95 on CD4+ and CD8+ blood lymphocytes was evaluated by flow cytometry. The proportion of blood Fas+ lymphocytes was significantly higher in lung cancer patients when compared with healthy individuals and in smokers when compared with nonsmokers.

  3. Role of rituximab in first-line treatment of chronic lymphocytic leukemia

    Directory of Open Access Journals (Sweden)

    Jeffrey Bryan


    Full Text Available Jeffrey Bryan, Gautam BorthakurDepartment of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, USAAbstract: Chronic lymphocytic leukemia (CLL is a biologically heterogeneous illness that primarily afflicts the elderly. For many decades, the initial therapy for most patients requiring treatment was limited to single-agent alkylator therapy. Within the last two decades, we have seen remarkable progress in understanding the biology of CLL and the development of more effective treatment strategies that have employed monoclonal antibodies, such as rituximab (anti-CD20. Furthermore, recognition of the synergy between fludarabine, cyclophosphamide, and rituximab (FCR prompted investigators to explore the clinical activity of FCR in Phase II and III trials in patients with relapsed/refractory or previously untreated CLL. On the basis of these findings, the US Food and Drug Administration (FDA recently approved rituximab in combination with fludarabine and cyclophosphamide for the treatment of patients with relapsed/refractory or previously untreated CD20-postive CLL. Recent data from a randomized Phase III trial has confirmed improved overall survival with FCR in patients with previously untreated CLL. However, FCR is not for everyone. More tolerable regimens using rituximab for the elderly and less fit patients are being pursued in clinical trials. Recent Phase II trials have explored potentially less myelosuppressive approaches by using lower doses of fludarabine and cyclophosphamide, replacing fludarabine with pentostatin, and combining rituximab with chlorambucil. Furthermore new biomarkers predictive of early disease progression have prompted investigators to explore the benefits of early treatment with rituximab combined with other agents. In addition to the proven utility of rituximab as a frontline agent for CLL, rituximab has a favorable toxicity profile both as a single agent and in combination with chemotherapy. The

  4. B Cell Acute Lymphocytic Leukemia Presenting as a Bile Duct Stricture Diagnosed With Cholangioscopy (United States)

    Bartel, Michael J.; Jiang, Liuyan; Lukens, Frank


    Indeterminate biliary strictures represent a diagnostic challenge requiring further work-up, which encompasses a variety of diagnostic modalities. We report a very rare case of B-cell acute lymphocytic leukemia presenting as a biliary stricture following remission of acute myeloid leukemia, which was initially treated with allogenic stem cell transplant. After multiple diagnostic modalities were implemented with no success, the use of cholangioscopy-guided biopsies was the key for the final diagnosis.

  5. Chaetoglobosin A preferentially induces apoptosis in chronic lymphocytic leukemia cells by targeting the cytoskeleton

    DEFF Research Database (Denmark)

    Knudsen, Peter Boldsen; Hanna, B.; Ohl, S.


    Chronic lymphocytic leukemia (CLL) is an incurable malignancy of mature B cells. One of the major challenges in treatment of CLL is the achievement of a complete remission to prevent relapse of disease originating from cells within lymphoid tissues and subsequent chemoresistance. In search for no...... with PI3K and BTK inhibitors, suggesting this compound as a novel potential drug for CLL.Leukemia accepted article preview online, 27 November 2013. doi:10.1038/leu.2013.360....


    Institute of Scientific and Technical Information of China (English)

    张鸿源; 王兰金; 等


    The chromosomoe and chromatid type aberration can be induced by benzene and the dicentric and ring ones were not observed in vitro experiment but observed in vivo one.In vitro experiment a good linear reression can be given between benzene concentrations and total aberration cells while power regression for radiation dose.The chromosome aberrations induced by benzene combined with radiation in rabbit blood lymphocytes are higher than in bone marryow cells.

  7. Bone marrow aplasia in B cell chronic lymphocytic leukaemia: successful treatment with antithymocyte globulin.


    Singal, R; Winfield, D A; Greaves, M.


    Pure red cell aplasia is a rare but well known association of chronic lymphocytic leukaemia (CLL). Pancytopenia due to bone marrow aplasia has not been previously described in CLL. A 42 year old man with B cell CLL became severely pancytopenic with bone marrow aplasia. Bone marrow culture resulted in a greatly reduced colony formation. High dose corticosteroids and intravenous immunoglobulin treatment were unsuccessful. Prompt and complete marrow recovery ensued after administration of antith...

  8. Metastatic Renal Cell Carcinoma to the Parotid Gland in the Setting of Chronic Lymphocytic Leukemia

    Directory of Open Access Journals (Sweden)

    Robert Deeb


    Full Text Available Renal cell carcinoma (RCC is infamous for its unpredictable behavior and metastatic potential. We report a case of a patient with a complex history of multifocal renal cell carcinoma and chronic lymphocytic leukemia (CLL, who subsequently developed a parotid mass. Total parotidectomy revealed this mass to be an additional site of metastasis which had developed 19 years after his initial diagnosis of RCC.

  9. Stochastic model for computer simulation of the number of cancer cells and lymphocytes in homogeneous sections of cancer tumors

    CERN Document Server

    Castellanos-Moreno, Arnulfo; Corella-Madueño, Adalberto; Gutiérrez-López, Sergio; Rosas-Burgos, Rodrigo


    We deal with a small enough tumor section to consider it homogeneous, such that populations of lymphocytes and cancer cells are independent of spatial coordinates. A stochastic model based in one step processes is developed to take into account natural birth and death rates. Other rates are also introduced to consider medical treatment: natural birth rate of lymphocytes and cancer cells; induced death rate of cancer cells due to self-competition, and other ones caused by the activated lymphocytes acting on cancer cells. Additionally, a death rate of cancer cells due to induced apoptosis is considered. Weakness due to the advance of sickness is considered by introducing a lymphocytes death rate proportional to proliferation of cancer cells. Simulation is developed considering different combinations of the parameters and its values, so that several strategies are taken into account to study the effect of anti-angiogenic drugs as well the self-competition between cancer cells. Immune response, with the presence ...

  10. Cell-mediated immune response of synovial fluid lymphocytes to ureaplasma antigen in Reiter's syndrome

    Directory of Open Access Journals (Sweden)

    Pavlica Ljiljana


    Full Text Available INTRODUCTION Reiter's syndrome (RS is an seronegative arthritis that occurs after urogenital or enteric infection which in addition with occular and/or mucocutaneous manifestations presents complete form of disease. According to previous understanding arthritis in the RS is the reactive one, which means that it is impossible to isolate its causative agent. However, there are the more and more authors suggesting that arthritis in the urogenital form of disease is caused by the infective agent in the affected joint. This suggestion is based on numerous studies on the presence of Chlmaydia trachomatis and Ureaplasma urealyticum in the inflamed joint by using new diagnostic methods in molecular biology published in the recent literature [1-3]. Besides, numerous studies of the humoral and cell-mediated immune response to "triggering" bacteria in the affected joint have supported previous suggestions [4-7]. Aim of the study was to determine whether synovial fluid T-cells specifically recognize the "triggering" bacteria presumably responsible for the Reiter's syndrome. METHOD The 3H-thymidine uptake procedure for measuring lymphocyte responses was applied to lymphocytes derived concurrently from synovial fluid (SF and from peripheral blood (PB [8]. Ureaplasma antigen and mitogen PHA stimulated lymphocytes in 24 RS patients (24 PB samples, 9 SF samples and the results were compared with those found in 10 patients with rheumatoid arthritis (RA (10 PB samples, 5 SF samples. Preparation of ureaplasma antigen. Ureaplasma was cultured on cell-free liquid medium [9]. Sample of 8 ml was heat-inactivated for 15 minutes at 601C and permanently stirred with magnetic mixer. The sample was centrifuged at 2000 x g for 40 minutes and than deposits carefully carried to other sterile glass tubes (Corex and recentrifuged at 9000 x g for 30 minutes. The deposit was washed 3 times in sterile 0.9% NaCl, and final sediment was resuspended in 1.2 ml sterile 0.9% Na


    Directory of Open Access Journals (Sweden)

    Abreu Velez Ana Maria


    Full Text Available Background: The clinical and histological presentation of spongiotic dermatitis and its inflammatory infiltrates warrant further investigation. In this case documentation of a patient with cutaneous spongiotic reactivity, we aim to characterize antigen presenting cells, as well as the skin-specific cutaneous lymphocyte antigen population by multiple techniques. Case report: A 30 year old Caucasian female presented with a two week history of blistering and erosions around the vaginal, rectal and axillary areas. Material and Methods: We utilized hematoxylin and eosin histology, direct immunofluorescence, immunohistochemistry and confocal microscopy methods to evaluate the immune reaction patterns of the cutaneous inflammatory cells. Results: In the primary histologic areas of spongiotic dermatitis, a mixed population of B and T lymphocytes was seen. Ki-67 antigen proliferative index staining was accentuated in these areas, correlating with the presence of large numbers of epidermal and dermal antigen presenting cells. Among the antigen presenting cell population, we detected strong positivities with CD1a, Factor XIIIa, myeloid/hystoid antigen, S100, HAM-56, and CD68. Interestingly, immunoglobulins G, D and M and Complement factors C1q and C3 were also strongly expressed in antigen presenting cell areas, including positivity within the spongiotic epidermis and around dermal vessels. Conclusions: We document a heterogeneous population of B and T lymphocytes and the presence of multiple classes of antigen presenting cells, immunoglobulins and complement in and surrounding histologically spongiotic areas; these findings further correlated with increased levels of expression of Ki-67.

  12. Lymphocyte, monocyte, and natural killer cell reference ranges in postpartal women. (United States)

    Gennaro, S; Fehder, W; Gallagher, P; Miller, S; Douglas, S D; Campbell, D E


    Normative values for immune-cell subsets in postpartal women, who are recovering from the relative immunosuppression of pregnancy, have not been established. Considerable differences in normative values for subsets of immune cells have been demonstrated based on sociodemographic factors, such as age and race. In order to make accurate clinical decisions about postpartal women, comparisons with normal reference ranges are necessary. Therefore, flow cytometric data for 51 healthy women at 4 months postpartum are presented and changes over the first 4 postpartal months are documented. The levels of some lymphocyte cell subsets, such as CD4+/CD45RA+ and Ia on lymphocytes, remained stable over time. The levels of other lymphocyte cell subsets, such as CD4+/CD29+, increased over the first 4 postpartal months, while those of other cell subsets, such as CD8 and CD11b, increased between delivery and 2 months postpartum and then dropped again by the fourth postpartal month. The levels of two natural killer cell subsets (CD3-/CD16+ and CD3-/CD57+) rose from delivery until 1 month postpartum and then plateaued. Comparisons were made with reference ranges of nonpostpartal groups provided in the literature and in a study of healthy women being conducted in the same laboratory, and postpartal women were found to have lower values of CD8, CD3-/CD16+, CD4+/CD45RA+, CD20, and CD11b than those reported in the literature.

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

    Directory of Open Access Journals (Sweden)

    Hidehito Saito


    Full Text Available 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.

  14. In vitro effects of mesenchymal stem cells on secreting function of T lymphocytes and CD4~+CD25~+T cells from patients with immune thrombo-cytopenia

    Institute of Scientific and Technical Information of China (English)



    Objective To analyze in vitro the effect of mesenchymal stem cells(MSCs)on secreting cytokines by T lymphocytes and ratio of CD4+CD25+T cells from patients with immune thrombocytopenia(ITP).Methods Human bone marrow-derived MSCs were isolated by Ficoll Hypaque and cultured for proliferating to passage cells.Allogeneic T lymphocytes

  15. P-glycoprotein-170 inhibition significantly reduces cortisol and ciclosporin efflux from human intestinal epithelial cells and T lymphocytes. (United States)

    Farrell, R J; Menconi, M J; Keates, A C; Kelly, C P


    To assess the role of P-glycoprotein-170 (P-gp) in transporting cortisol and ciclosporin from human intestinal epithelium and T lymphocytes. The effect of P-gp inhibitors (verapamil, 0-100 microM; PSC 833, 0-20 microM) on the intracellular accumulation of 3H-cortisol and 3H-ciclosporin was studied in confluent layers of human Caco-2 cells (n=6), a P-gp-dependent absorptive intestinal epithelial cell phenotype, and moderately resistant MDRhigh CEM/VBL 100 T cells (n=6). The transport of 3H-vinblastine, a strong multidrug resistance (MDR) substrate, and 3H-progesterone, a poor MDR substrate, was also studied. Caco-2 cells had a 2.4-, 6.6-, 6.7- and 1.03-fold higher net basal to apical transport (efflux) of 3H-cortisol, 3H-ciclosporin, 3H-vinblastine and 3H-progesterone, respectively. PSC 833 (20 microM) reduced cortisol efflux by 69% (0.23 +/- 0.04 to 0.07 +/- 0.01 pmol/cm2/h, P 0.1 microM) and verapamil (> 1 microM) restored the intracellular level of 3H-cortisol and 3H-ciclosporin in MDRhigh CEM/VBL 100 T cells to that of MDRlow CEM cells with little change in accumulation in the MDRlow parental cell line. P-gp inhibitors significantly increase intracellular cortisol and ciclosporin levels in human intestinal epithelium and T lymphocytes in a dose-dependent manner, demonstrating a potential mechanism for overcoming poor response to immunosuppressant therapy in refractory inflammatory bowel disease.

  16. Role of NOXA and its ubiquitination in proteasome inhibitor-induced apoptosis in chronic lymphocytic leukemia cells

    NARCIS (Netherlands)

    Baou, M.; Kohlhaas, S.L.; Butterworth, M.; Vogler, M.; Dinsdale, D.; Walewska, R.; Majid, A.; Eldering, E.; Dyer, M.J.S.; Cohen, G.M.


    Background Bortezomib has been successfully used in the treatment of multiple myeloma and has been proposed as a potential treatment for chronic lymphocytic leukemia. In this study we investigated the mechanism by which bortezomib induces apoptosis in chronic lymphocytic leukemia cells. Design and M

  17. Impaired removal of Vβ8(+) lymphocytes aggravates colitis in mice deficient for B cell lymphoma-2-interacting mediator of cell death (Bim). (United States)

    Leucht, K; Caj, M; Fried, M; Rogler, G; Hausmann, M


    We investigated the role of B cell lymphoma (BCL)-2-interacting mediator of cell death (Bim) for lymphocyte homeostasis in intestinal mucosa. Lymphocytes lacking Bim are refractory to apoptosis. Chronic colitis was induced in Bim-deficient mice (Bim(-/-) ) with dextran sulphate sodium (DSS). Weight loss and colonoscopic score were increased significantly in Bim(-/-) mice compared to wild-type mice. As Bim is induced for the killing of autoreactive cells we determined the role of Bim in the regulation of lymphocyte survival at mucosal sites. Upon chronic dextran sulphate sodium (DSS)-induced colitis, Bim(-/-) animals exhibited an increased infiltrate of lymphocytes into the mucosa compared to wild-type mice. The number of autoreactive T cell receptor (TCR) Vβ8(+) lymphocytes was significantly higher in Bim(-/-) mice compared to wild-type controls. Impaired removal of autoreactive lymphocytes in Bim(-/-) mice upon chronic DSS-induced colitis may therefore contribute to aggravated mucosal inflammation.

  18. Allorestricted T lymphocytes with a high avidity T-cell receptor towards NY-ESO-1 have potent anti-tumor activity. (United States)

    Krönig, Holger; Hofer, Kathrin; Conrad, Heinke; Guilaume, Philippe; Müller, Julia; Schiemann, Matthias; Lennerz, Volker; Cosma, Antonio; Peschel, Christian; Busch, Dirk H; Romero, Pedro; Bernhard, Helga


    The cancer-testis antigen NY-ESO-1 has been targeted as a tumor-associated antigen by immunotherapeutical strategies, such as cancer vaccines. The prerequisite for a T-cell-based therapy is the induction of T cells capable of recognizing the NY-ESO-1-expressing tumor cells. In this study, we generated human T lymphocytes directed against the immunodominant NY-ESO-1(157-165) epitope known to be naturally presented with HLA-A*0201. We succeeded to isolate autorestricted and allorestricted T lymphocytes with low, intermediate or high avidity TCRs against the NY-ESO-1 peptide. The avidity of the established CTL populations correlated with their capacity of lysing HLA-A2-positive, NY-ESO-1-expressing tumor cell lines derived from different origins, e.g. melanoma and myeloma. The allorestricted NY-ESO-1-specific T lymphocytes displayed TCRs with the highest avidity and best anti-tumor recognition activity. TCRs derived from allorestricted, NY-ESO-1-specific T cells may be useful reagents for redirecting primary T cells by TCR gene transfer and, therefore, may facilitate the development of adoptive transfer regimens based on TCR-transduced T cells for the treatment of NY-ESO-1-expressing hematological malignancies and solid tumors.

  19. Regulation of cell-fate decisions in T lymphocyte differenttiation

    NARCIS (Netherlands)

    M.C. Nawijn (Martijn)


    textabstractThe acquisition of protective immunity is essential for survival. Protective itmllunity can be divided in innate immunity and acquired immunity. These two parts of the immune system have evolved to closely interact. Cells of the innate immune system, such as dendritic cells and macrophag

  20. False leukemia-lymphoma cell lines: an update on over 500 cell lines. (United States)

    Drexler, H G; Dirks, W G; Matsuo, Y; MacLeod, R A F


    Human leukemia-lymphoma (LL) cell lines represent an extremely important resource for research in a variety of fields and disciplines. As the cell lines are used as in vitro model systems in lieu of primary cell material, it is crucial that the cells in the culture flasks faithfully correspond to the purported objects of study. Obviously, proper authentication of cell line derivation and precise characterization are indispensable requirements to use as model systems. A number of studies has shown an unacceptable level of LL cell lines to be false. We present here the results of authenticating a comprehensively large sample (n = 550) of LL cell lines mainly by DNA fingerprinting and cytogenetic evaluation. Surprisingly, near-identical incidences (ca 15%) of false cell lines were observed among cell lines obtained directly from original investigators (59/395: 14.9%) and from secondary sources (23/155: 14.8%) implying that most cross-contamination is perpetrated by originators, presumably during establishment. By comparing our data with those published, we were further able to subclassify the false cell lines as (1) virtual: cross-contaminated with and unretrievably overgrown by other cell lines during initiation, never enjoying independent existence; (2) misidentified: cross-contaminated subsequent to establishment so that an original prototype may still exist; or (3) misclassified: unwittingly established from an unintended (often normal) cell type. Prolific classic leukemia cell lines were found to account for the majority of cross-contaminations, eg CCRF-CEM, HL-60, JURKAT, K-562 and U-937. We discuss the impact of cross-contaminations on scientific research, the reluctance of scientists to address the problem, and consider possible solutions. These findings provide a rationale for mandating the procurement of reputably sourced LL cell lines and their regular authentication thereafter.

  1. What Is Chronic Lymphocytic Leukemia? (United States)

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

  2. Mercuric dichloride induces DNA damage in human salivary gland tissue cells and lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Schmid, Katharina; Kroemer, Susanne [University of Regensburg, Regensburg (Germany); Sassen, Andrea [University of Regensburg, Department of Pathology, Regensburg (Germany); Staudenmaier, Rainer [Technical University of Munich, Department of Otorhinolaryngology, Head and Neck Surgery, Munich (Germany); Reichl, Franz-Xaver [University of Munich, Institute of Pharmacology and Toxicology, Munich (Germany); Harreus, Ulrich [University of Munich, Department of Otorhinolaryngology, Head and Neck Surgery, Munich (Germany); Hagen, Rudolf; Kleinsasser, Norbert [University of Wuerzburg, Department of Otorhinolaryngology, Head and Neck Surgery, Wuerzburg (Germany)


    Amalgam is still one of the most frequently used dental filling materials. However, the possible adverse effects especially that of the mercuric component have led to continued controversy. Considering that mercury may be released from amalgam fillings into the oral cavity and also reach the circulating blood after absorption and resorption, it eventually may contribute to tumorigenesis in a variety of target cells. The present investigation focuses on genotoxic effects below a cytotoxic dose level of mercuric dichloride (HgCl{sub 2}) in human samples of salivary glands and lymphocytes to elucidate a possible role in tumor initiation. DNA migration due to single strand breaks, alkali labile sites and incomplete excision repair was quantified with the aid of the single cell microgel electrophoresis (Comet) assay. The concepts of Olive Tail Moment, percentage of DNA in the Tail and Tail Length were used as measures of DNA damage. To control for cytotoxic effects, the trypan blue exclusion test was applied. Human samples of the parotid salivary gland and lymphocytes of ten donors were exposed to HgCl{sub 2} concentrations from 1 to 50 {mu}M. N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and dimethyl sulfoxide (DMSO) served as controls. Increasing dose-dependent DNA migration could be demonstrated after exposure to HgCl{sub 2} in cells of the salivary glands and lymphocytes. In both cell types a significant increase in DNA migration could be shown starting from HgCl{sub 2} concentrations of 5 {mu}M in comparison to the negative control. The viability of the cell systems was not affected except at the highest concentration (50 {mu}M) tested. These data indicate genotoxic effects of mercuric dichloride in human salivary glands and lymphocytes at concentrations not leading to cytotoxic effects or cell death. Consequently, a contributory role in oral salivary gland tumor initiation warrants further investigation. (orig.)

  3. First-line treatment of chronic lymphocytic leukemia with three combined chemotherapy regimens

    Directory of Open Access Journals (Sweden)

    Matevž Škerget


    Conclusions: RFC is the first-line treatment of choice for younger fit patients without deletion 17. For older and frail patients, alternative newer chemotherapy regimens should be sought. Patients with deletion 17 should receive alemtuzumab treatment, and allogeneic bone marrow transplantation should be considered.

  4. Recirculation of lymphocyte subsets (CD5+, CD4+, CD8+, T19+ and B cells) through fetal lymph nodes. (United States)

    Kimpton, W G; Washington, E A; Cahill, R N


    The experiments reported in this paper examine the cell-surface phenotype (CD5, CD4, CD8, T19, MHC class II and sIg) and cell output of lymphocyte subsets circulating through a subcutaneous lymph node in the sheep fetus, in an environment unaffected by foreign antigen and circulating immunoglobulins. CD4+ lymphocytes were the major T-cell subset in fetal lymph and were clearly enriched in lymph compared with blood, whereas T19+, CD8+ and B lymphocytes were not. It seems likely that in the fetus CD4+ lymphocytes are extracted from the blood at a faster rate than are other T-cell subsets and B cells. There was a much higher percentage of CD8+ and T null cells and a lower percentage of MHC class II+ and B cells circulating in the fetal lymph than in adult lymph, while the percentage of T19+ lymphocytes in fetal blood was twice that in the adult. Although the hourly cell output from an adult prescapular lymph node was far higher than that from a fetal lymph node, the circulation of lymphocytes through fetal lymph nodes was much greater per gram lymph node weight than that through adult lymph nodes. The wholesale recirculation in the fetus of all the major T-cell subsets found in the adult is paradoxical because it is not known what function they serve in the fetus in the absence of antigen and ongoing immune responses, although clearly they are not memory cells. PMID:2481644

  5. The effects of phototherapy on the numbers of circulating natural killer cells and T lymphocytes in psoriasis.

    LENUS (Irish Health Repository)

    Tobin, A M


    The innate immune system is believed to be important in the pathogenesis of psoriasis and natural killer (NK) have been found in increased numbers in psoriatic plaques. Alterations in the numbers of NK cells in peripheral blood have been reported. We investigated the effect of phototherapy on levels of peripheral NK cells and lymphocytes in patients with psoriasis. In nine patients whom we followed before, during and after narrowband ultraviolet B (UVB) treatment there were no differences in the numbers of circulating lymphocytes, lymphocyte subsets or cells expressing NK markers and controls. Treatment with narrowband UVB did, however, significantly lower circulating CD4 counts which gradually recovered posttreatment.

  6. Regulatory T-cells in chronic lymphocytic leukemia: actor or innocent bystander? (United States)

    D’Arena, Giovanni; Simeon, Vittorio; D’Auria, Fiorella; Statuto, Teodora; Sanzo, Paola Di; Martino, Laura De; Marandino, Aurelio; Sangiorgio, Michele; Musto, Pellegrino; Feo, Vincenzo De


    Regulatory T (Treg) cells are now under extensive investigation in chronic lymphocytic leukemia (CLL). This small subset of T-cells has been, in fact, considered to be involved in the pathogenesis and progression of CLL. However, whether Treg dysregulation in CLL plays a key role or it rather represents a simple epiphenomenon is still matter of debate. In the former case, Treg cells could be appealing for targeting therapies. Finally, Treg cells have also been proposed as a prognostic indicator of the disease clinical course. PMID:23358515

  7. Virus Discovery Using Tick Cell Lines (United States)

    Bell-Sakyi, Lesley; Attoui, Houssam


    While ticks have been known to harbor and transmit pathogenic arboviruses for over 80 years, the application of high-throughput sequencing technologies has revealed that ticks also appear to harbor a diverse range of endogenous tick-only viruses belonging to many different families. Almost nothing is known about these viruses; indeed, it is unclear in most cases whether the identified viral sequences are derived from actual replication-competent viruses or from endogenous virus elements incorporated into the ticks’ genomes. Tick cell lines play an important role in virus discovery and isolation through the identification of novel viruses chronically infecting such cell lines and by acting as host cells to aid in determining whether or not an entire replication-competent, infective virus is present in a sample. Here, we review recent progress in tick-borne virus discovery and comment on the actual and potential applications for tick cell lines in this emerging research area. PMID:27679414

  8. Expression of the glucocorticoid receptor from the 1A promoter correlates with T lymphocyte sensitivity to glucocorticoid-induced cell death. (United States)

    Purton, Jared F; Monk, Julie A; Liddicoat, Douglas R; Kyparissoudis, Konstantinos; Sakkal, Samy; Richardson, Samantha J; Godfrey, Dale I; Cole, Timothy J


    Glucocorticoid (GC) hormones cause pronounced T cell apoptosis, particularly in immature thymic T cells. This is possibly due to tissue-specific regulation of the glucocorticoid receptor (GR) gene. In mice the GR gene is transcribed from five separate promoters designated: 1A, 1B, 1C, 1D, and 1E. Nearly all cells express GR from promoters 1B-1E, but the activity of the 1A promoter has only been reported in the whole thymus or lymphocyte cell lines. To directly assess the role of GR promoter use in sensitivity to glucocorticoid-induced cell death, we have compared the activity of the GR 1A promoter with GC sensitivity in different mouse lymphocyte populations. We report that GR 1A promoter activity is restricted to thymocyte and peripheral lymphocyte populations and the cortex of the brain. The relative level of expression of the 1A promoter to the 1B-1E promoters within a lymphocyte population was found to directly correlate with susceptibility to GC-induced cell death, with the extremely GC-sensitive CD4+CD8+ thymocytes having the highest levels of GR 1A promoter activity, and the relatively GC-resistant alphabetaTCR+CD24(int/low) thymocytes and peripheral T cells having the lowest levels. DNA sequencing of the mouse GR 1A promoter revealed a putative glucocorticoid-response element. Furthermore, GR 1A promoter use and GR protein levels were increased by GC treatment in thymocytes, but not in splenocytes. These data suggest that tissue-specific differences in GR promoter use determine T cell sensitivity to glucocorticoid-induced cell death. Copyright 2004 The American Association of Immunologists, Inc.

  9. The prognostic impact of minimal residual disease in patients with chronic lymphocytic leukemia requiring first-line therapy. (United States)

    Santacruz, Rodrigo; Villamor, Neus; Aymerich, Marta; Martínez-Trillos, Alejandra; López, Cristina; Navarro, Alba; Rozman, María; Beà, Sílvia; Royo, Cristina; Cazorla, Maite; Colomer, Dolors; Giné, Eva; Pinyol, Magda; Puente, Xose S; López-Otín, Carlos; Campo, Elías; López-Guillermo, Armando; Delgado, Julio


    A proportion of patients with chronic lymphocytic leukemia achieve a minimal residual disease negative status after therapy. We retrospectively evaluated the impact of minimal residual disease on the outcome of 255 consecutive patients receiving any front-line therapy in the context of a detailed prognostic evaluation, including assessment of IGHV, TP53, NOTCH1 and SF3B1 mutations. The median follow-up was 73 months (range, 2-202) from disease evaluation. The median treatment-free survival durations for patients achieving a complete response without or with minimal residual disease, a partial response and no response were 76, 40, 11 and 11 months, respectively (P<0.001). Multivariate analysis revealed that three variables had a significant impact on treatment-free survival: minimal residual disease (P<0.001), IGHV status (P<0.001) and β2-microglobulin levels (P=0.012). With regards to overall survival, factors predictive of an unfavorable outcome were minimal residual disease positivity (P=0.014), together with advanced age (P<0.001), unmutated IGHV status (P=0.001), TP53 mutations (P<0.001) and elevated levels of β2-microglobulin (P=0.003). In conclusion, for patients requiring front-line therapy, achievement of minimal residual disease negativity is associated with significantly prolonged treatment-free and overall survival irrespective of other prognostic markers or treatment administered.


    Directory of Open Access Journals (Sweden)

    Ashraf Mohabbati Mobarez


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

  11. The oncogene DEK promotes leukemic cell survival and is downregulated by both Nutlin-3 and chlorambucil in B-chronic lymphocytic leukemic cells. (United States)

    Secchiero, Paola; Voltan, Rebecca; di Iasio, Maria Grazia; Melloni, Elisabetta; Tiribelli, Mario; Zauli, Giorgio


    To characterize the role of the oncogene DEK in modulating the response to either Nutlin-3, a small-molecule inhibitor of the MDM2/p53 interaction, or chlorambucil in primary B-chronic lymphocytic leukemia (B-CLL) cells. DEK mRNA and protein levels were evaluated in primary B-CLL samples (n = 21), p53(wild-type) SKW6.4, p53(mutated) BJAB lymphoblastoid cell lines, and normal CD19(+) B lymphocytes-treated Nutlin-3 or chlorambucil (10 micromol/L, each). Knocking down experiments with either p53 or DEK small interfering RNA (siRNA) were done to investigate the potential role of p53 in controlling the expression of DEK and the role of DEK in leukemic cell survival/apoptosis. Both Nutlin-3 and chlorambucil downregulated DEK in primary B-CLL samples (n = 21) and SKW6.4 but not in BJAB cells. Knocking down p53 attenuated the effect of Nutlin-3 on DEK expression, whereas knocking down DEK significantly increased both spontaneous and Nutlin-3-induced apoptosis. Conversely, counteracting DEK downmodulation by using p53 small interfering RNA reduced Nutlin-3-mediated apoptosis. On the other hand, Nutlin-3 potently induced p53 accumulation, but it did not affect DEK levels in normal CD19(+) B lymphocytes. These data show that the downregulation of DEK in response to either Nutlin-3 or chlorambucil represents an important molecular determinant in the cytotoxic response of leukemic cells, and suggest that strategies aimed to downregulate DEK might improve the therapeutic potential of these drugs.

  12. Cell interactions in concanavalin A activated cation flux and DNA synthesis of mouse lymphocytes

    DEFF Research Database (Denmark)

    Owens, T; Kaplan, J G


    Co-culture at constant cell density of nude mouse spleen cells (by themselves unresponsive to the T-cell mitogen concanavalin A (Con A)), with congenic T-enriched lymphocyte suspensions and Con A caused anomalously high activation of K+ transport (measured by 86Rb uptake) and of incorporation...... of thymidine into DNA; the expected dilution of these two responses by nude spleen cells did not occur. However, if the nude splenocytes were added immediately prior to assay to the enriched T cells that had been precultured in presence of Con A, the expected dilution of the activated T-cell responses occurred......; both 86Rb uptake and thymidine incorporation were reduced proportionally to the degree of dilution of the T cells by the nonresponding cells. These data indicate that during co-culture in presence of Con A there is interaction between the T cells, capable of responding to mitogens, and the nude spleen...

  13. Paraflagellar rod protein-specific CD8+ cytotoxic T lymphocytes target Trypanosoma cruzi-infected host cells. (United States)

    Wrightsman, Ruth A; Luhrs, Keith A; Fouts, David; Manning, Jerry E


    Our previous studies show that in mice immunized with the paraflagellar rod (PFR) proteins of Trypanosoma cruzi protective immunity against this protozoan parasite requires MHC class I-restricted T cell function. To determine whether PFR-specific CD8+ T cell subsets are generated during T. cruzi infection, potential CTL targets in the PFR proteins were identified by scanning the amino acid sequences of the four PFR proteins for regions of 8-10 amino acids that conform to predicted MHC class I H-2b binding motifs. A subset of the peptide sequences identified were synthesized and tested as target antigen in 51Cr-release assays with effector cells from chronically infected T. cruzi mice. Short-term cytotoxic T lymphocyte (CTL) lines specific for two of the peptides, PFR-1(164-171) and PFR-3(123-130), showed high levels of lytic activity against peptide-pulsed target cells, secreted interferon (IFN)-gamma in response to parasite-infected target cells, and were found to be CD8+, CD4-, CD3+, TCRalphabeta+ cells of the Tc1 subset. Challenge of PFR immunized CD8-/- and perforin-deficient (PKO) mice confirmed that while CD8+ cells are required for survival of T. cruzi challenge infection, perforin activity is not required. Furthermore, while lytic activity of PFR-specific CD8+ T cell lines derived from PKO mice was severely impaired, the IFN-gamma levels secreted by CTLs from PKO mice were equivalent to that of normal mice, suggesting that the critical role played by CD8+ T cells in immunity to the parasite may be secretion of type 1 cytokines rather than lysis of parasite infected host cells.

  14. Human CD56+ cytotoxic lung lymphocytes kill autologous lung cells in chronic obstructive pulmonary disease.

    Directory of Open Access Journals (Sweden)

    Christine M Freeman

    Full Text Available CD56+ natural killer (NK and CD56+ T cells, from sputum or bronchoalveolar lavage of subjects with chronic obstructive pulmonary disease (COPD are more cytotoxic to highly susceptible NK targets than those from control subjects. Whether the same is true in lung parenchyma, and if NK activity actually contributes to emphysema progression are unknown. To address these questions, we performed two types of experiments on lung tissue from clinically-indicated resections (n = 60. First, we used flow cytometry on fresh single-cell suspension to measure expression of cell-surface molecules (CD56, CD16, CD8, NKG2D and NKp44 on lung lymphocytes and of the 6D4 epitope common to MICA and MICB on lung epithelial (CD326+ cells. Second, we sequentially isolated CD56+, CD8+ and CD4+ lung lymphocytes, co-cultured each with autologous lung target cells, then determined apoptosis of individual target cells using Annexin-V and 7-AAD staining. Lung NK cells (CD56+ CD3- and CD56+ T cells (CD56+ CD3+ were present in a range of frequencies that did not differ significantly between smokers without COPD and subjects with COPD. Lung NK cells had a predominantly "cytotoxic" CD56+ CD16+ phenotype; their co-expression of CD8 was common, but the percentage expressing CD8 fell as FEV1 % predicted decreased. Greater expression by autologous lung epithelial cells of the NKG2D ligands, MICA/MICB, but not expression by lung CD56+ cells of the activating receptor NKG2D, correlated inversely with FEV1 % predicted. Lung CD56+ lymphocytes, but not CD4+ or CD8+ conventional lung T cells, rapidly killed autologous lung cells without additional stimulation. Such natural cytotoxicity was increased in subjects with severe COPD and was unexplained in multiple regression analysis by age or cancer as indication for surgery. These data show that as spirometry worsens in COPD, CD56+ lung lymphocytes exhibit spontaneous cytotoxicity of autologous structural lung cells, supporting their

  15. Anti-HIV-1 activity of propolis in CD4(+) lymphocyte and microglial cell cultures. (United States)

    Gekker, Genya; Hu, Shuxian; Spivak, Marla; Lokensgard, James R; Peterson, Phillip K


    An urgent need for additional agents to treat human immunodeficiency virus type 1 (HIV-1) infection led us to assess the anti-HIV-1 activity of the natural product propolis in CD4(+) lymphocytes and microglial cell cultures. Propolis inhibited viral expression in a concentration-dependent manner (maximal suppression of 85 and 98% was observed at 66.6 microg/ml propolis in CD4(+) and microglial cell cultures, respectively). Similar anti-HIV-1 activity was observed with propolis samples from several geographic regions. The mechanism of propolis antiviral property in CD4(+) lymphocytes appeared to involve, in part, inhibition of viral entry. While propolis had an additive antiviral effect on the reverse transcriptase inhibitor zidovudine, it had no noticeable effect on the protease inhibitor indinavir. The results of this in vitro study support the need for clinical trials of propolis or one or more of its components in the treatment of HIV-1 infection.

  16. The Impact of ATRA on Shaping Human Myeloid Cell Responses to Epithelial Cell-Derived Stimuli and on T-Lymphocyte Polarization

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    Arunima Chatterjee


    Full Text Available Vitamin A plays an essential role in the maintenance of gut homeostasis but its interplay with chemokines has not been explored so far. Using an in vitro model system we studied the effects of human colonic epithelial cells (Caco2, HT-29, and HCT116 derived inflammatory stimuli on monocyte-derived dendritic cells and macrophages. Unstimulated Caco2 and HT-29 cells secreted CCL19, CCL21, and CCL22 chemokines, which could attract dendritic cells and macrophages and induced CCR7 receptor up-regulation by retinoic-acid resulting in dendritic cell migration. The chemokines Mk, CXCL16, and CXCL7 were secreted by all the 3 cell lines tested, and upon stimulation by IL-1β or TNF-α this effect was inhibited by ATRA but had no impact on CXCL1, CXCL8, and CCL20 secretion in response to IL-1β. In the presence of ATRA the supernatants of these cells induced CD103 expression on monocyte-derived dendritic cells and when conditioned by ATRA and cocultured with CD4+ T-lymphocytes they reduced the proportion of Th17 T-cells. However, in the macrophage-T-cell cocultures the number of these effector T-cells was increased. Thus cytokine-activated colonic epithelial cells trigger the secretion of distinct combinations of chemokines depending on the proinflammatory stimulus and are controlled by retinoic acid, which also governs dendritic cell and macrophage responses.

  17. Proteomic and protein interaction network analysis of human T lymphocytes during cell-cycle entry (United States)

    Orr, Stephen J; Boutz, Daniel R; Wang, Rong; Chronis, Constantinos; Lea, Nicholas C; Thayaparan, Thivyan; Hamilton, Emma; Milewicz, Hanna; Blanc, Eric; Mufti, Ghulam J; Marcotte, Edward M; Thomas, N Shaun B


    Regulating the transition of cells such as T lymphocytes from quiescence (G0) into an activated, proliferating state involves initiation of cellular programs resulting in entry into the cell cycle (proliferation), the growth cycle (blastogenesis, cell size) and effector (functional) activation. We show the first proteomic analysis of protein interaction networks activated during entry into the first cell cycle from G0. We also provide proof of principle that blastogenesis and proliferation programs are separable in primary human T cells. We employed a proteomic profiling method to identify large-scale changes in chromatin/nuclear matrix-bound and unbound proteins in human T lymphocytes during the transition from G0 into the first cell cycle and mapped them to form functionally annotated, dynamic protein interaction networks. Inhibiting the induction of two proteins involved in two of the most significantly upregulated cellular processes, ribosome biogenesis (eIF6) and hnRNA splicing (SF3B2/SF3B4), showed, respectively, that human T cells can enter the cell cycle without growing in size, or increase in size without entering the cell cycle. PMID:22415777

  18. Three-dimensional reconstruction of fluorescent lymphocyte cells (United States)

    Gaillard, Jean; Maire, Eric; Jacquey, Serge; Schultz, Guy; Jung, Georges


    The complete process, image acquisition, deblurring, 3D reconstruction and analysis has been tested on 160 normal T lymphoid cells marked with monoclonal antibodies CD4-CD8. The images are acquired from a conventional optical microscope illuminated by a mercury vapor lamp. A set of 16 serial image cuts is first grabbed by a high sensitivity Silicon Intensified Target (SIT) camera fitted on the microscope. The 2D image cuts are then deblurred using an original deconvolution method developed in the laboratory. The 3D reconstruction is processed slice by slice using a variant of the 2D Delaunay triangulation. To get a correct triangulation in the case of our specific application the following stringent requirements must be met. First, we exclude the possibility of joining two contours if we meet an empty cut between them, and secondly we must avoid branching points and finally, we state that reconstructed shapes may not have holes. The 3D reconstruction highlights some qualitative characteristics of the cells. So we can show that the fluorescent spots are either scattered on the membrane surface and present a regular radial arrangement or on the contrary are confined in a restricted area. Accordingly we propose to introduce a spatial coefficient in order to characterize such a volume distribution. In a further development we plan to obtain discriminant descriptors of the immunological status of hematopoietic cells.

  19. Postirradiation Changes of White Blood Cells and Lymphocyte Subpopulations in Cancer Patients

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    Ahn, Sung Ja; Chung, Woong Ki; Nam, Taek Keun; Nah, Byung Sik; Noh, Young Hee [Chonnam National University College of Medicine, Kwangju (Korea, Republic of)


    Purpose : Radiation-induced alteration in the immune function is well known phenomenon in cancer patients. Our purpose is to evaluate the extent of immune suppression immediately after mediastinal or pelvic irradiation, which include significant volume of active bone marrow in adults. Methods and Materials : 48 cancer patients with mediastinal(N=29) and pelvic irradiation(N=19) were the basis of this analysis. Age ranged from 36 to 76 and mean and median value was 57 years, respectively. Sex ratio was 1.3(M:F 27/21). The immunological parameters were the complete blood cell(CBC) with differential cell(D/C) count, T cell subset(CD3, CD4, CD8, CD19), NK cell test(CD16,CD56), and serum immunoglobulin (lgG, lgA, lgM) level. Results : The mean value of white blood cell(WBC) was reduced from 7017 to 4470 after irradiation (p=0.0000). In the differential count, the number of lymphocyte, neutrophil, and basophil was markedly reduced with statistical significance(p<0.01) and the number of monocyte was not changed and, on the contrary, that of eosinophil was increased by irradiation. In the lymphocyte subpopulation analysis, the number of all subpopulations, CD3(T cell), CD4(helper T cell), CD8(suppressor T cell), CD16(NK cell), CD19(B cell) was reduced with statistical significance. The mean ratio of CD4 to CD8 in all patients was 1.09 initially and reduced to 0.99 after radiotherapy(p = 0.34), but the proportional percentage of all subpopulations was not changed except CD19(B cell) after irradiation.In the immunoglobulin study, initial values of lg G, lg A, and lg M were relatively above the normal range and the only lg M was statistically significantly reduced after radiotherapy(p=0.02). Conclusion : Mediastinal and pelvic irradiation resulted in remarkable suppression of lymphocyte count in contrast to the relatively good preservation of other components of white blood cells. But the further study on the functional changes of lymphocyte after radiotherapy may be

  20. Cell Line Data Base: structure and recent improvements towards molecular authentication of human cell lines. (United States)

    Romano, Paolo; Manniello, Assunta; Aresu, Ottavia; Armento, Massimiliano; Cesaro, Michela; Parodi, Barbara


    The Cell Line Data Base (CLDB) is a well-known reference information source on human and animal cell lines including information on more than 6000 cell lines. Main biological features are coded according to controlled vocabularies derived from international lists and taxonomies. HyperCLDB ( is a hypertext version of CLDB that improves data accessibility by also allowing information retrieval through web spiders. Access to HyperCLDB is provided through indexes of biological characteristics and navigation in the hypertext is granted by many internal links. HyperCLDB also includes links to external resources. Recently, an interest was raised for a reference nomenclature for cell lines and CLDB was seen as an authoritative system. Furthermore, to overcome the cell line misidentification problem, molecular authentication methods, such as fingerprinting, single-locus short tandem repeat (STR) profile and single nucleotide polymorphisms validation, were proposed. Since this data is distributed, a reference portal on authentication of human cell lines is needed. We present here the architecture and contents of CLDB, its recent enhancements and perspectives. We also present a new related database, the Cell Line Integrated Molecular Authentication (CLIMA) database (, that allows to link authentication data to actual cell lines.

  1. Broad T-cell receptor repertoire in T-lymphocytes derived from human induced pluripotent stem cells.

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    Chia-Wei Chang

    Full Text Available 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.

  2. Differentially expressed cytosolic proteins in human leukemia and lymphoma cell lines correlate with lineages and functions. (United States)

    Gez, Swetlana; Crossett, Ben; Christopherson, Richard I


    Identification of cytosolic proteins differentially expressed between types of leukemia and lymphoma may provide a molecular basis for classification and understanding their cellular properties. Two-dimensional fluorescence difference gel electrophoresis (DIGE) and mass spectrometry have been used to identify proteins that are differentially expressed in cytosolic extracts from four human leukemia and lymphoma cell lines: HL-60 (acute promyelocytic leukemia), MEC1 (B-cell chronic lymphocytic leukemia), CCRF-CEM (T-cell acute lymphoblastic leukemia) and Raji (B-cell Burkitt's lymphoma). A total of 247 differentially expressed proteins were identified between the four cell lines. Analysis of the data by principal component analysis identified 22 protein spots (17 different protein species) differentially expressed at more than a 95% variance level between these cell lines. Several of these proteins were differentially expressed in only one cell line: HL-60 (myeloperoxidase, phosphoprotein 32 family member A, ras related protein Rab-11B, protein disulfide-isomerase, ran-specific GTPase-activating protein, nucleophosmin and S-100 calcium binding protein A4), and Raji (ezrin). Several of these proteins were differentially expressed in two cell lines: Raji and MEC1 (C-1-tetrahydrofolate synthase, elongation factor 2, alpha- and beta-tubulin, transgelin-2 and stathmin). MEC1 and CCRF-CEM (gamma-enolase), HL-60 and CCRF-CEM (ubiquitin-conjugating enzyme E2 N). The differentially expressed proteins identified in these four cell lines correlate with cellular properties and provide insights into the molecular basis of these malignancies.

  3. Influence of in vivo hypobaric hypoxia on function of lymphocytes, neutrocytes, natural killer cells, and cytokines

    DEFF Research Database (Denmark)

    Klokker, M; Kharazmi, A; Galbo, H


    of an increased concentration of lymphocytes. The absolute and relative concentration of CD16+ natural killer (NK) cells increased markedly during hypoxia and returned to pretest values after 2 h of recovery. The NK cell activity of blood mononuclear cells (BMNC, %lysis/fixed no. of BMNC) boosted with interferon......-alpha, interleukin-2 (IL-2), and indomethacin rose in parallel with unboosted NK cell activity during hypoxia. The percentage of CD4+ and CD8+ cells declined during hypoxia, whereas the absolute concentration of both CD8+ cells and CD14+ monocytes increased. Although the BMNC composition varied, the proliferative....... The chemiluminescence response of neutrocytes increased 2 h after hypoxia. It was concluded that acute hypoxia induced marked alterations in the immune system and that the NK cells are especially sensitive to the hypoxic stimulus....

  4. Curcumin inhibits prosurvival pathways in chronic lymphocytic leukemia B cells and may overcome their stromal protection in combination with EGCG. (United States)

    Ghosh, Asish K; Kay, Neil E; Secreto, Charla R; Shanafelt, Tait D


    Chronic lymphocytic leukemia (CLL) is incurable with current chemotherapy treatments. Curcumin (diferuloylmethane), an active ingredient in the spice turmeric, inhibits tumor metastasis, invasion, and angiogenesis in tumor cell lines. We evaluated the effects of curcumin on the viability of primary CLL B cells and its ability to overcome stromal mediated protection. The in vitro effect of curcumin on primary CLL B cells was evaluated using fluorescence activated cell sorter analysis and Western blotting. For some experiments, CLL B cells were cocultured with human stromal cells to evaluate the effects of curcumin on leukemia cells cultured in their microenvironment. Finally, the effect of curcumin in combination with the green tea extract epigallocatechin-3 gallate (EGCG) was evaluated. Curcumin induced apoptosis in CLL B cells in a dose-dependent (5-20 micromol/L) manner and inhibited constitutively active prosurvival pathways, including signal transducers and activators of transcription 3 (STAT3), AKT, and nuclear factor kappaB. Moreover, curcumin suppressed expression of the anti-apoptotic proteins Mcl-1 and X-linked inhibitor of apoptosis protein (XIAP), and up-regulated the pro-apoptotic protein BIM. Coculture of CLL B cells with stromal cells resulted in elevated levels of STAT3, increased expression of Mcl-1 and XIAP, and decreased sensitivity to curcumin. When curcumin was administered simultaneously with EGCG, antagonism was observed for most patient samples. In contrast, sequential administration of these agents led to substantial increases in CLL B-cell death and could overcome stromal protection. Curcumin treatment was able to overcome stromal protection of CLL B cells on in vitro testing and to synergize with EGCG when administered in a sequential fashion. Additional evaluation of curcumin as a potential therapeutic agent for treatment of CLL seems warranted.

  5. Salinomycin inhibits Wnt signaling and selectively induces apoptosis in chronic lymphocytic leukemia cells. (United States)

    Lu, Desheng; Choi, Michael Y; Yu, Jian; Castro, Januario E; Kipps, Thomas J; Carson, Dennis A


    Salinomycin, an antibiotic potassium ionophore, has been reported recently to act as a selective breast cancer stem cell inhibitor, but the biochemical basis for its anticancer effects is not clear. The Wnt/β-catenin signal transduction pathway plays a central role in stem cell development, and its aberrant activation can cause cancer. In this study, we identified salinomycin as a potent inhibitor of the Wnt signaling cascade. In Wnt-transfected HEK293 cells, salinomycin blocked the phosphorylation of the Wnt coreceptor lipoprotein receptor related protein 6 (LRP6) and induced its degradation. Nigericin, another potassium ionophore with activity against cancer stem cells, exerted similar effects. In otherwise unmanipulated chronic lymphocytic leukemia cells with constitutive Wnt activation nanomolar concentrations of salinomycin down-regulated the expression of Wnt target genes such as LEF1, cyclin D1, and fibronectin, depressed LRP6 levels, and limited cell survival. Normal human peripheral blood lymphocytes resisted salinomycin toxicity. These results indicate that ionic changes induced by salinomycin and related drugs inhibit proximal Wnt signaling by interfering with LPR6 phosphorylation, and thus impair the survival of cells that depend on Wnt signaling at the plasma membrane.

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

    Institute of Scientific and Technical Information of China (English)

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


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

  7. Human T-lymphotropic virus type-1 p30 alters cell cycle G2 regulation of T lymphocytes to enhance cell survival

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    Silverman Lee


    Full Text Available Abstract Background Human T-lymphotropic virus type-1 (HTLV-1 causes adult T-cell leukemia/lymphoma and is linked to a number of lymphocyte-mediated disorders. HTLV-1 contains both regulatory and accessory genes in four pX open reading frames. pX ORF-II encodes two proteins, p13 and p30, whose roles are still being defined in the virus life cycle and in HTLV-1 virus-host cell interactions. Proviral clones of HTLV-1 with pX ORF-II mutations diminish the ability of the virus to maintain viral loads in vivo. p30 expressed exogenously differentially modulates CREB and Tax-responsive element-mediated transcription through its interaction with CREB-binding protein/p300 and while acting as a repressor of many genes including Tax, in part by blocking tax/rex RNA nuclear export, selectively enhances key gene pathways involved in T-cell signaling/activation. Results Herein, we analyzed the role of p30 in cell cycle regulation. Jurkat T-cells transduced with a p30 expressing lentivirus vector accumulated in the G2-M phase of cell cycle. We then analyzed key proteins involved in G2-M checkpoint activation. p30 expression in Jurkat T-cells resulted in an increase in phosphorylation at serine 216 of nuclear cell division cycle 25C (Cdc25C, had enhanced checkpoint kinase 1 (Chk1 serine 345 phosphorylation, reduced expression of polo-like kinase 1 (PLK1, diminished phosphorylation of PLK1 at tyrosine 210 and reduced phosphorylation of Cdc25C at serine 198. Finally, primary human lymphocyte derived cell lines immortalized by a HTLV-1 proviral clone defective in p30 expression were more susceptible to camptothecin induced apoptosis. Collectively these data are consistent with a cell survival role of p30 against genotoxic insults to HTLV-1 infected lymphocytes. Conclusion Collectively, our data are the first to indicate that HTLV-1 p30 expression results in activation of the G2-M cell cycle checkpoint, events that would promote early viral spread and T-cell

  8. Idelalisib and bendamustine combination is synergistic and increases DNA damage response in chronic lymphocytic leukemia cells. (United States)

    Modi, Prexy; Balakrishnan, Kumudha; Yang, Qingshan; Wierda, William G; Keating, Michael J; Gandhi, Varsha


    Idelalisib is a targeted agent that potently inhibits PI3Kδ which is exclusively expressed in hematological cells. Bendamustine is a well-tolerated cytotoxic alkylating agent which has been extensively used for treatment of chronic lymphocytic leukemia (CLL). Both these agents are FDA-approved for CLL. To increase the potency of idelalisib and bendamustine, we tested their combination in primary CLL lymphocytes. While each compound alone produced a moderate response, combination at several concentrations resulted in synergistic cytotoxicity. Idelalisib enhanced the bendamustine-mediated DNA damage/repair response, indicated by the phosphorylation of ATM, Chk2, and p53. Each drug alone activated γH2AX but combination treatment further increased the expression of this DNA damage marker. Compared with the control, idelalisib treatment decreased global RNA synthesis, resulting in a decline of early-response and short-lived MCL1 transcripts. In concert, there was a decline in total Mcl-1 protein in CLL lymphocytes. Isogenic mouse embryonic fibroblasts lacking MCL1 had higher sensitivity to bendamustine alone or in combination compared to MCL1 proficient cells. Collectively, these data indicate that bendamustine and idelalisib combination therapy should be investigated for treating patients with CLL.

  9. Effect of dendritic cell vaccine therapy on lymphocyte subpopulation in refractory primary brain tumor

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    J Niu


    Full Text Available BACKGROUND: Dendritic cell (DC-based immunotherapy has the potential to induce an antitumor response within the immunologically privileged brain. AIMS: The aim of this study was to evaluate the short-term effect of DC vaccine therapy on lymphocyte subsets in patients with refractory primary brain tumor. MATERIALS AND METHODS: Eighteen cases with refractory primary brain tumor who refused any treatment against tumor within 6 months of the therapy, were referred to one medicine center, from January 2011 to October 2012. All patients received 1 × 107 tumor lysate–pulsed DC vaccinations both intradermal injection and intravenous infusion 3 times/week. RESULTS: There were increases of lymphocytes CD8+ (P = 0.002 and CD56+ (P = 4.207E-10, but no change of lymphocytes CD3+ (P = 0.651. Six patients were positive response of delayed-type hypersensitivity. There were improving of appetite in 14 cases and increasing of physical strength 17 cases. CONCLUSIONS: DC vaccine has the potential for inducing an immune cytotoxic effect directed toward tumor cells.

  10. Effect of anti-carbohydrate antibodies on HIV infection in a monocytic cell line (U937)

    DEFF Research Database (Denmark)

    Hansen, J E; Nielsen, C; Clausen, H;


    . This study therefore investigated the neutralization of HIV in a monocytic cell line (U937) using mAbs against these carbohydrate gp120-epitopes. While antibodies against one of the epitopes (AI) neutralized infection of U937 cells despite binding to the Fc-receptor, one mAb against the sialosyl-Tn epitope...... enhanced infection. This enhancement was independent of complement and could be blocked by mAb Leu3a against the CD4-receptor. The study indicated that enhancement of infection in monocytic cells can occur by the same anti-carbohydrate antibodies that neutralize infection in lymphocytes, and that antibody...

  11. B-cell differentiation in the chicken: expression of immunoglobulin genes in the bursal and peripheral lymphocytes. (United States)

    Mansikka, A; Veromaa, T; Vainio, O; Toivanen, P


    We have studied the expression of immunoglobulin genes in the chicken B-cell precursors, and of a B-cell surface marker (Bu-1) on the bursal and peripheral B cells during normal ontogeny. Since there is no way of distinguishing the precursor cells from the more mature bursal lymphocytes on the basis of surface markers, we chose to study the total bursal lymphocyte population at ages when the numbers of the various precursor cells (bursal, early post-bursal, and post-bursal stem cells) in the bursa are estimated to be at their highest. Thereafter, comparisons with the more mature lymphocytes in the peripheral organs were made. As a result, levels of the lambda and mu transcripts and expression of Bu-1 antigen in the chicken B-cell precursors were found to be unchanged during the post-hatching period. In the light of these experiments, the later events of B-cell differentiation, i.e. the development from the bursal to post-bursal B lymphocytes, occurs without the lambda, mu, and Bu-1 gene loci involved. On the other hand, the higher level of lambda and mu expression in the splenic B lymphocytes indicates that the post-bursal stem cells mature into highly active plasma cells after seeding to the peripheral organs.


    Directory of Open Access Journals (Sweden)

    Soeyoko Soeyoko


    Full Text Available Wuchereria bancrofti, Brugia malayi, and Brugia timori are the causative agents of lymphatic hiariasis in Indonesia, but in some endemic areas, Brugia malayi is the most commonly found,Diagnosis of hiariasis is normally based on clinical and parasitological examinations, but both have limitations. Therefore now the immunological examination plays an important role in the diagnosis of filariasis. The discovery of monoclonal antibodies recently may probably give a firm scientific basis in immunology and add a new dimension to the efforts of developing a specific and sensitive immunological test for various stages of filarial infection. In this study, the production of hybridoma cells to develop monoclonal antibodies against B. malayi integrated a number of techniques: preparations of B. malayi surface antigen, immune lymphocyte cells, NSI myeloma cells and macrophage feeder layers, and a fusion of immune lymphocytes with myeloma cells. The results of this study can be concluded in three points: Protein analysis of the surface antigen was examined by Biureet and SDS-PAGE. A total of fourteen examinations were conducted by using 4000 L3 for each experiment. Three were not detected by Biureet method, but showed five protein fractions by SDS-PAGE. The protein concentration of the surface L3 was varied from 85.0/µg/ml to 769.23/µg/ml, with an average of 297.04/µg/ml.The immunoreactivifies of Balb/c mice antibodies to B. malayi L3 surface antigen were tested by ELISA and showed a gradual increase after four times immunizations at two weeks interval. The optical density (OD after four times immunizations was varied from 0.363 to 0.878 each mouse, where as the positive control sera OD was 0.570.Hybridization using immune lymphocytes against B. malayi L3 surface antigen with myeloma cells yielded 60.41% hybrid cells and none of them produced monoclonal antibodies tested of ELISA.

  13. Hyperforin induces apoptosis of chronic lymphocytic leukemia cells through upregulation of the BH3-only protein Noxa. (United States)

    Zaher, Murhaf; Tang, Ruoping; Bombarda, Isabelle; Merhi, Faten; Bauvois, Brigitte; Billard, Christian


    We previously reported that hyperforin, a phloroglucinol purified from Hypericum perforatum, induces the mitochondrial pathway of caspase-dependent apoptosis in chronic lymphocytic leukemia (CLL) cells ex vivo, and that this effect is associated with upregulation of Noxa, a BH3-only protein of the Bcl-2 family. Here, we investigated the role of this upregulation in the pro-apoptotic activity of hyperforin in the cells of CLL patients and MEC-1 cell line. We found that the increase in Noxa expression is a time- and concentration-dependent effect of hyperforin occurring without change in Noxa mRNA levels. A post-translational regulation is suggested by the capacity of hyperforin to inhibit proteasome activity in CLL cells. Noxa silencing by siRNA reduces partially hyperforin-elicited apoptosis. Furthermore, treatment with hyperforin, which has no effect on the expression of the prosurvival protein Mcl-1, induces the interaction of Noxa with Mcl-1 and the dissociation of Mcl-1/Bak complex, revealing that upregulated Noxa displaces the proapoptotic protein Bak from Mcl-1. This effect is accompanied with Bak activation, known to allow the release of apoptogenic factors from mitochondria. Our data indicate that Noxa upregulation is one of the mechanisms by which hyperforin triggers CLL cell apoptosis. They also favor that new agents capable of mimicking specifically the BH3-only protein Noxa should be developed for apoptosis-based therapeutic strategy in CLL.

  14. Investigation of Cytocidal Activity of Bacillus Thuringiensis Parasporal Toxin on CCRF-CEM Cell Line

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    Elham Moazamian


    Full Text Available Background & Objective: Parasporin is a parasporal protein of Bacillus thuringiensis and exhibits special cytocidal activity against human cancer cells. Similar to other insecticidal Bacillus thuringiensis crystal toxins, parasporin shows target specificity and damages the cellular membrane. In this study, different strains of Bacillus thuringiensis isolated from various regions of Iran and their cytocidal activity against CCRF-CEM cell line and human erythrocyte were investigated.   Materials & Methods: Fifty soil samples were collected from different Iranian provinces, and characterization was performed based on protein crystal morphology by phase-contrast microscope and variations of Cry protein toxin using SDS-PAGE. After parasporin was processed with proteinase K, the active form was produced and protein activity on the cell line was evaluated. Results: Parasporal inclusion proteins showed different cytotoxicity against acute lymphoblastic leukemia cells (ALL, but not against normal lymphocyte. Isolated parasporin demonstrated no hemolytic activity against human erythrocyte. It appears that these proteins have the ability to differentiate between normal lymphocytes and leukemia cells and have specific receptors on specific cancer cell lines. Conclusion: Our results provide evidence that the parasporin-producing organism is a common member in Bacillus thuringiensis populations occurring in the natural environments of Iran.

  15. Macropinocytosis is responsible for the uptake of pathogenic and non-pathogenic mycobacteria by B lymphocytes (Raji cells

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    García-Pérez Blanca Estela


    Full Text Available Abstract Background The classical roles of B cells include the production of antibodies and cytokines and the generation of immunological memory, these being key factors in the adaptive immune response. However, their role in innate immunity is currently being recognised. Traditionally, B cells have been considered non-phagocytic cells; therefore, the uptake of bacteria by B cells is not extensively documented. In this study, we analysed some of the features of non-specific bacterial uptake by B lymphocytes from the Raji cell line. In our model, B cells were infected with Mycobacterium tuberculosis (MTB, Mycobacterium smegmatis (MSM, and Salmonella typhimurium (ST. Results Our observations revealed that the Raji B cells were readily infected by the three bacteria that were studied. All of the infections induced changes in the cellular membrane during bacterial internalisation. M. smegmatis and S. typhimurium were able to induce important membrane changes that were characterised by abundant filopodia and lamellipodia formation. These membrane changes were driven by actin cytoskeletal rearrangements. The intracellular growth of these bacteria was also controlled by B cells. M. tuberculosis infection also induced actin rearrangement-driven membrane changes; however, the B cells were not able to control this infection. The phorbol 12-myristate 13-acetate (PMA treatment of B cells induced filopodia and lamellipodia formation, the production of spacious vacuoles (macropinosomes, and the fluid-phase uptake that is characteristic of macropinocytosis. S. typhimurium infection induced the highest fluid-phase uptake, although both mycobacteria also induced fluid uptake. A macropinocytosis inhibitor such as amiloride was used and abolished the bacterial uptake and the fluid-phase uptake that is triggered during the bacterial infection. Conclusions Raji B cells can internalise S. typhimurium and mycobacteria through an active process, such as

  16. Asymptomatic T-cell large granular lymphocyte leukemia with an unusual immunophenotype

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    Panagiota K. Petsa


    Full Text Available T-cell large granular lymphocyte (T-LGL leukemia represents a clonal proliferation of cytotoxic T-cells which etiology has not been entirely elucidated. However, CD4+, CD4–,CD8–, CD4+, CD8+ cases have been described. The disease is usually characterized by cytopenias and a modest lymphocytosis. The majority of patients with T-LGL leukemia remains asymptomatic for a long period and will require treatment later during the course of their disease. Hereby we describe a case of T-LGL leukemia diagnosed by flow cytometry, which presented indolent course and required no treatment so far.

  17. NK cell function is markedly impaired in patients with chronic lymphocytic leukaemia but is preserved in patients with small lymphocytic lymphoma. (United States)

    Parry, Helen M; Stevens, Tom; Oldreive, Ceri; Zadran, Bassier; McSkeane, Tina; Rudzki, Zbigniew; Paneesha, Shankara; Chadwick, Caroline; Stankovic, Tatjana; Pratt, Guy; Zuo, Jianmin; Moss, Paul


    Chronic lymphocytic leukemia (B-CLL) and small lymphocytic lymphoma (SLL) are part of the same disease classification but are defined by differential distribution of tumor cells. B-CLL is characterized by significant immune suppression and dysregulation but this is not typical of patients with SLL. Natural killer cells (NK) are important mediators of immune function but have been poorly studied in patients with B-CLL/SLL. Here we report for the first time the NK cell phenotype and function in patients with B-CLL and SLL alongside their transcriptional profile. We show for the first time impaired B-CLL NK cell function in a xenograft model with reduced activating receptor expression including NKG2D, DNAM-1 and NCRs in-vitro. Importantly, we show these functional differences are associated with transcriptional downregulation of cytotoxic pathway genes, including activating receptors, adhesion molecules, cytotoxic molecules and intracellular signalling molecules, which remain intact in patients with SLL. In conclusion, NK cell function is markedly influenced by the anatomical site of the tumor in patients with B-CLL/SLL and lymphocytosis leads to marked impairment of NK cell activity. These observations have implications for treatment protocols which seek to preserve immune function by limiting the exposure of NK cells to tumor cells within the peripheral circulation.

  18. Stem cell characteristics in prostate cancer cell lines.

    NARCIS (Netherlands)

    Pfeiffer, M.J.; Schalken, J.A.


    BACKGROUND: Recent studies indicate the presence of a small, stem-like cell population in several human cancers that is crucial for the tumour (re)population. OBJECTIVE: Six established prostate cancer (PCa) cell lines-DU145, DuCaP, LAPC-4, 22Rv1, LNCaP, and PC-3-were examined for their stem cell pr

  19. The antihistamines clemastine and desloratadine inhibit STAT3 and c-Myc activities and induce apoptosis in cutaneous T-cell lymphoma cell lines. (United States)

    Döbbeling, Udo; Waeckerle-Men, Ying; Zabel, Franziska; Graf, Nicole; Kündig, Thomas M; Johansen, Pål


    Mycosis fungoides and its leukaemic counterpart Sézary syndrome are the most frequent cutaneous T-cell lymphomas (CTCL), and there is no cure for these diseases. We evaluated the effect of clinically approved antihistamines on the growth of CTCL cell lines. CTCL cell lines as well as blood lymphocytes from patients with Sézary syndrome were cultured with antihistamines, and the cell were analysed for proliferation, apoptosis and expression of programmed death molecules and transcription factors. The two antihistamines clemastine and desloratadine, currently used for symptom alleviation in allergy, induced potent reduction of the activities of the constitutively active transcription factors c-Myc, STAT3, STAT5a and STAT5b in mycosis fungoides and Sézary syndrome cell lines. This inhibition was followed by apoptosis and cell death, especially in the Sézary syndrome-derived cell line Hut78 that also showed increased expression of the programmed death-1 (PD-1) after clemastine treatment. In lymphocytes isolated from Sézary syndrome patients, the CD4-positive fraction underwent apoptosis after clemastine treatment, while CD4-negative lymphocytes were little affected. Because both c-Myc and STAT transcription factors are highly expressed in proliferating tumours, their inhibition by clemastine, desloratadine and other inhibitors could complement established chemotherapies not only for cutaneous T-cell lymphomas but perhaps also other cancers.

  20. Exposure of human nasal epithelial cells to formaldehyde does not lead to DNA damage in lymphocytes after co-cultivation. (United States)

    Neuss, Simone; Moepps, Barbara; Speit, Günter


    We performed in vitro co-cultivation experiments with primary human nasal epithelial cells (HNEC) and isolated lymphocytes to investigate whether reactive formaldehyde (FA) can be passed on from nasal epithelial cells (site of first contact) to lymphocytes located in close proximity and induce DNA damage in these cells. A modified comet assay was used as a sensitive method for the detection of FA-induced DNA-protein cross links (DPX) because DPX are the most relevant type of FA-induced DNA damage. Our results clearly indicate that co-cultivation of lymphocytes with HNEC exposed to FA for 1 h causes a concentration-related induction of DPX in lymphocytes when co-cultivation takes place in the exposure medium. However, when the exposure medium is changed after FA treatment of HNEC and before lymphocytes are added, no induction of DPX is measured in lymphocytes even after exposure of HNEC to high FA concentrations (300 microM) and extended co-cultivation (4 h). Direct measurement of FA in the cell culture medium by a sensitive fluorescent detection kit indicated that FA is actually not released even from highly exposed cells into the cell culture medium. These results suggest that FA that has entered nasal epithelial cells is not released and does not damage other cells in close proximity to the epithelial cells. If these results also apply to the in vivo situation, FA would only be genotoxic towards directly exposed cells (site of first contact) and there should be no significant delivery of inhaled FA to other cells and distant sites. Our results do not support a recently proposed hypothetic mechanism for FA-induced leukaemia by damaging circulating haematopoietic stem cells or haematopoietic progenitor cells in nasal passages, which then travel to the bone marrow and become initiated leukaemic stem cells.

  1. Scanning electron microscopy of interaction of peripheral blood lymphocytes from colonic cancer patients with human colonic cancer-derived cells; P-4788.

    Directory of Open Access Journals (Sweden)



    Full Text Available Peripheral blood lymphocytes and the various lymphocyte fractions from patients with cancer of the colon were cultivated with target cells (P-4788 derived from the colon cancer. Changes in the surface ultrastructure during tumor cell destruction were studied by scanning electron microscopy (SEM. P-4788 cells adhering to the coverslip showed various surface activity. The surfaces of some cells were relatively flat; others were smooth or had fine granules. Still other cells were villous, round or had marked blebs. When host lymphocytes were added to the target cells, adhesion of the two cell groups began by many fine projections. After incubation for 6 h, some lymphocytes had adhered to the target cells. Many lymphocytes had adhered to the target tumor cells by 24--48 h incubation. Ultimately the tumor cells became swollen and disrupted. Most lymphocytes adherent to the target cells had few microvilli. Lymphocytes after elimination of phagocytes by carbonyl iron treatment also adhered readily. Some target cells showed adhesion with lymphocytes passed through nylon-wool columns, although the number of lymphocytes adhering was fewer than in the case of lymphocytes not passed through nylon-wool columns. T cells were collected from lymphocytes that form rosettes with SRBC by isolation with NH4Cl. They had markedly elongated microvilli which in places were sparsely scattered and tended to be localized on the side, a finding which suggests loss of cell activity by the time of SEM. Only a few T cells adhered to target cells and they seemed to be T cells without activity. It was thought that there are cytotoxic cells among T cells and that the co-existence of T cells, non-T cells and monocytes caused target cell destruction.

  2. Genetic difference in the proliferative response to T mitogens between Hi/PHA and Lo/PHA lymphocytes is independent of accessory cell function. (United States)

    Stiffel, C; Liacopoulos-Briot, M; Decreusefond, C; Parlebas, J


    The role of the macrophage as accessory cell in the proliferative response of lymphocytes to phytohemagglutinin (PHA) was studied in two lines of mice genetically selected for high and low responsiveness to T mitogens. Adherent cell depletion of lymph node cells abrogated the low (Lo)/PHA response, but only partially inhibited the high (Hi)/PHA response. Addition of peritoneal cells provided either by Hi/PHA or by Lo/PHA mice equally restored Hi/PHA responsiveness but had only a slight reconstituting effect on the inhibited Lo/PHA response. Equivalent enhancement or suppression of proliferation of untreated lymph node cells was obtained by the addition of increasing percentages of each of the two peritoneal cell populations. However, the maximum level of the Lo/PHA response never reached that of Hi/PHA cells. These data indicate that the bidirectional selective breeding has not modified the potentialities of the macrophages as accessory cells but has resulted in an impaired response of Lo/PHA lymphocytes to the signals delivered either by accessory cells or by T mitogens.

  3. Serglycin determines secretory granule repertoire and regulates natural killer cell and cytotoxic T lymphocyte cytotoxicity. (United States)

    Sutton, Vivien R; Brennan, Amelia J; Ellis, Sarah; Danne, Jill; Thia, Kevin; Jenkins, Misty R; Voskoboinik, Ilia; Pejler, Gunnar; Johnstone, Ricky W; Andrews, Daniel M; Trapani, Joseph A


    The anionic proteoglycan serglycin is a major constituent of secretory granules in cytotoxic T lymphocyte (CTL)/natural killer (NK) cells, and is proposed to promote the safe storage of the mostly cationic granule toxins, granzymes and perforin. Despite the extensive defects of mast cell function reported in serglycin gene-disrupted mice, no comprehensive study of physiologically relevant CTL/NK cell populations has been reported. We show that the cytotoxicity of serglycin-deficient CTL and NK cells is severely compromised but can be partly compensated in both cell types when they become activated. Reduced intracellular granzyme B levels were noted, particularly in CD27(+) CD11b(+) mature NK cells, whereas serglycin(-/-) TCR-transgenic (OTI) CD8 T cells also had reduced perforin stores. Culture supernatants from serglycin(-/-) OTI T cells and interleukin-2-activated NK contained increased granzyme B, linking reduced storage with heightened export. By contrast, granzyme A was not significantly reduced in cells lacking serglycin, indicating differentially regulated trafficking and/or storage for the two granzymes. A quantitative analysis of different granule classes by transmission electronmicroscopy showed a selective loss of dense-core granules in serglycin(-/-) CD8(+) CTLs, although other granule types were maintained quantitatively. The findings of the present study show that serglycin plays a critical role in the maturation of dense-core cytotoxic granules in cytotoxic lymphocytes and the trafficking and storage of perforin and granzyme B, whereas granzyme A is unaffected. The skewed retention of cytotoxic effector molecules markedly reduces CTL/NK cell cytotoxicity, although this is partly compensated for as a result of activating the cells by physiological means.


    Directory of Open Access Journals (Sweden)

    Fabienne McClanahan


    Full Text Available Chemoimmunotherapy with fludarabine, cyclophosphamide, and rituximab (FCR has been established as the current standard of care for young and fit patients with chronic lymphocytic leukemia (CLL. In the early nineties of the last century, long before the advent of fludarabine or antibody-based strategies, there was realistic hope that myeloablative therapy followed by autologous stem cell transplantation (autoSCT might be an effective and potentially curative front-line treatment option for suitable patients with CLL. Since then, several prospective trials have disenthralled this hope: although autoSCT can prolong event and progression-free survival if used as part of early front-line treatment, it does not improve overall survival, while it is associated with an increased risk of late adverse events such as secondary malignancies. In addition, autoSCT lacks the potential to overcome the negative impact of biomarkers that confer resistance to chemotherapy or early relapse. The role of autoSCT has also been explored in the context of FCR, and it was demonstrated that its effect is inferior to the currently established optimal treatment regimen. In view of ongoing attempts to improve on FCR, promising clinical activity of new substances even in relapsed/ refractory CLL patients, exciting novel cell therapy approaches and advantages in the understanding of the disease and detection of Minimal Residual Disease (MRD, autoSCT has lost its place as a standard treatment option for CLL.

  5. Network signatures of cellular immortalization in human lymphoblastoid cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Shim, Sung-Mi; Jung, So-Young; Nam, Hye-Young; Kim, Hye-Ryun; Lee, Mee-Hee; Kim, Jun-Woo; Han, Bok-Ghee [National Biobank of Korea, Center for Genome Science, Korea National Institute of Health, Osong 363-951 (Korea, Republic of); Jeon, Jae-Pil, E-mail: [Division of Brain Diseases, Center for Biomedical Science, Korea National Institute of Health, Osong 363-951 (Korea, Republic of)


    Highlights: •We identified network signatures of LCL immortalization from transcriptomic profiles. •More than 41% of DEGs are possibly regulated by miRNAs in LCLs. •MicroRNA target genes in LCLs are involved in apoptosis and immune-related functions. •This approach is useful to find functional miRNA targets in specific cell conditions. -- Abstract: Human lymphoblastoid cell line (LCL) has been used as an in vitro cell model in genetic and pharmacogenomic studies, as well as a good model for studying gene expression regulatory machinery using integrated genomic analyses. In this study, we aimed to identify biological networks of LCL immortalization from transcriptomic profiles of microRNAs and their target genes in LCLs. We first selected differentially expressed genes (DEGs) and microRNAs (DEmiRs) between early passage LCLs (eLCLs) and terminally differentiated late passage LCLs (tLCLs). The in silico and correlation analysis of these DEGs and DEmiRs revealed that 1098 DEG–DEmiR pairs were found to be positively (n = 591 pairs) or negatively (n = 507 pairs) correlated with each other. More than 41% of DEGs are possibly regulated by miRNAs in LCL immortalizations. The target DEGs of DEmiRs were enriched for cellular functions associated with apoptosis, immune response, cell death, JAK–STAT cascade and lymphocyte activation while non-miRNA target DEGs were over-represented for basic cell metabolisms. The target DEGs correlated negatively with miR-548a-3p and miR-219-5p were significantly associated with protein kinase cascade, and the lymphocyte proliferation and apoptosis, respectively. In addition, the miR-106a and miR-424 clusters located in the X chromosome were enriched in DEmiR–mRNA pairs for LCL immortalization. In this study, the integrated transcriptomic analysis of LCLs could identify functional networks of biologically active microRNAs and their target genes involved in LCL immortalization.

  6. The identification of lymphocyte-like cells and lymphoid-related genes in amphioxus indicates the twilight for the emergence of adaptive immune system.

    Directory of Open Access Journals (Sweden)

    Gonghua Huang

    Full Text Available To seek evidence of a primitive adaptive immune system (AIS before vertebrate, we examined whether lymphocytes or lymphocyte-like cells and the related molecules participating in the lymphocyte function existed in amphioxus. Anatomical analysis by electron microscopy revealed the presence of lymphocyte-like cells in gills, and these cells underwent morphological changes in response to microbial pathogens that are reminiscent of those of mammalian lymphocytes executing immune response to microbial challenge. In addition, a systematic comparative analysis of our cDNA database of amphioxus identified a large number of genes whose vertebrate counterparts are involved in lymphocyte function. Among these genes, several genes were found to be expressed in the vicinity of the lymphocyte-like cells by in situ hybridization and up-regulated after exposure to microbial pathogens. Our findings in the amphioxus indicate the twilight for the emergence of AIS before the invertebrate-vertebrate transition during evolution.

  7. Relation between clinical mature and immature lymphocyte cells in human peripheral blood and their spatial label free scattering patterns (United States)

    Zhang, Lu; Zhao, Xin; Zhang, Zhenxi; Zhao, Hong; Chen, Wei; Yuan, Li


    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.

  8. Rho and Rap guanosine triphosphatase signaling in B cells and chronic lymphocytic leukemia. (United States)

    Mele, Silvia; Devereux, Stephen; Ridley, Anne J


    Chronic lymphocytic leukemia (CLL) cells proliferate predominantly in niches in the lymph nodes, where signaling from the B cell receptor (BCR) and the surrounding microenvironment are critical for disease progression. In addition, leukemic cells traffic constantly from the bloodstream into the lymph nodes, migrate within lymphatic tissues and egress back to the bloodstream. These processes are driven by chemokines and their receptors, and depend on changes in cell migration and integrin-mediated adhesion. Here we describe how Rho and Rap guanosine triphosphatases (GTPases) contribute to both BCR signaling and chemokine receptor signaling, particularly by regulating cytoskeletal dynamics and integrin activity. We propose that new inhibitors of BCR-activated kinases are likely to affect CLL cell trafficking via Rho and Rap GTPases, and that upstream regulators or downstream effectors could be good targets for therapeutic intervention in CLL.

  9. Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Felthaus, O. [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany); Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Ettl, T.; Gosau, M.; Driemel, O. [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Brockhoff, G. [Department of Gynecology and Obstetrics, University of Regensburg (Germany); Reck, A. [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Zeitler, K. [Institute of Pathology, University of Regensburg (Germany); Hautmann, M. [Department of Radiotherapy, University of Regensburg (Germany); Reichert, T.E. [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Schmalz, G. [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany); Morsczeck, C., E-mail: [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany)


    Research highlights: {yields} Four oral squamous cancer cell lines (OSCCL) were analyzed for cancer stem cells (CSCs). {yields} Single cell derived colonies of OSCCL express CSC-marker CD133 differentially. {yields} Monoclonal cell lines showed reduced sensitivity for Paclitaxel. {yields} In situ CD133{sup +} cells are slow cycling (Ki67-) indicating a reduced drug sensitivity. {yields} CD133{sup +} and CSC-like cells can be obtained from single colony forming cells of OSCCL. -- Abstract: Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simple method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133{sup +} cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.

  10. Increased IMP dehydrogenase gene expression in solid tumor tissues and tumor cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Collart, F.R.; Chubb, C.B.; Mirkin, B.L.; Huberman, E.


    IMP dehydrogenase, a regulatory enzyme of guanine nucleotide biosynthesis, may play a role in cell proliferation and malignancy. To assess this possibility, we examined IMP dehydrogenase expression in a series of human solid tumor tissues and tumor cell lines in comparison with their normal counterparts. Increased IMP dehydrogenase gene expression was observed in brain tumors relative to normal brain tissue and in sarcoma cells relative to normal fibroblasts. Similarly, in several B- and T-lymphoid leukemia cell lines, elevated levels of IMP dehydrogenase mRNA and cellular enzyme were observed in comparison with the levels in peripheral blood lymphocytes. These results are consistent with an association between increased IMP dehydrogenase expression and either enhanced cell proliferation or malignant transformation.

  11. TCR gamma delta cytotoxic T lymphocytes expressing the killer cell-inhibitory receptor p58.2 (CD158b) selectively lyse acute myeloid leukemia cells. (United States)

    Dolstra, H; Fredrix, H; van der Meer, A; de Witte, T; Figdor, C; van de Wiel-van Kemenade, E


    Cytotoxic T lymphocytes (CTL) are thought to play an important role in the graft-versus-leukemia (GVL) response. Unfortunately, GVL reactivity is often associated with life-threatening graft-versus-host disease (GVHD). Characterization of CTL that selectively attack leukemic cells but not normal cells may lead to the development of adjuvant immunotherapy that separates GVL from GVHD. Here, we describe TCR gamma delta (V gamma 9/V delta 1) CTL, isolated from the peripheral blood of an AML patient after stem cell transplantation (SCT), that very efficiently lysed freshly isolated acute myeloid leukemia (AML) cells and AML cell lines. Interestingly, HLA-matched non-malignant hematopoietic cells were not killed. We revealed that the killer cell-inhibitory receptor (KIR) p58.2 (CD158b) specific for group 2 HLA-C molecules negatively regulates the cytotoxic effector function displayed by these TCR gamma delta CTL. First, an antibody against HLA-C enhances lysis of non-malignant cells. Secondly, stable transfection of HLA-Cw*0304 into the class I-negative cell line 721.221 inhibited lysis. Finally, engagement of p58.2 by antibodies immobilized on Fc gamma R-expressing murine P815 cells inhibits CD3- and TCR gamma delta-directed lysis. Compared to non-malignant hematopoietic cells, AML cells express much lower levels of MHC class I molecules making them susceptible to lysis by p58.2(+) TCR gamma delta CTL. Such KIR-regulated CTL reactivity may have a role in the GVL response without affecting normal tissues of the host and leading to GVHD.

  12. Analysis of Vδ1 T cells in clinical grade melanoma-infiltrating lymphocytes

    DEFF Research Database (Denmark)

    Donia, Marco; Ellebaek, Eva; Andersen, Mads Hald


    γδ T cells, including Vδ1 and Vδ2 T cells, can recognize tumor-associated ligands neglected by conventional αβ T cells in a MHC-independent manner. Little is known regarding the anticancer potential and the possibility to isolate and expand Vδ1 T cells to therapeutically relevant numbers....... In this study, we have detected low frequencies of Vδ1 T cells among tumor-infiltrating lymphocyte (TIL) products for adoptive cell transfer generated from melanoma metastases. An increased frequency of Vδ1 T cells was found among the cell products from patients with an advanced disease stage. Vδ1 T cells...... displayed in vitro antitumor activities and sufficient proliferative potential to generate over 1 × 10(9) cells using current protocols for T cell transfer. Infusion of Vδ1 T cells together with high numbers of αβ TILs in a clinical trial was safe and well tolerated. These data suggest that Vδ1 T cells...

  13. ATR inhibition induces synthetic lethality and overcomes chemoresistance in TP53- or ATM-defective chronic lymphocytic leukemia cells. (United States)

    Kwok, Marwan; Davies, Nicholas; Agathanggelou, Angelo; Smith, Edward; Oldreive, Ceri; Petermann, Eva; Stewart, Grant; Brown, Jeff; Lau, Alan; Pratt, Guy; Parry, Helen; Taylor, Malcolm; Moss, Paul; Hillmen, Peter; Stankovic, Tatjana


    TP53 and ataxia telangiectasia mutated (ATM) defects are associated with genomic instability, clonal evolution, and chemoresistance in chronic lymphocytic leukemia (CLL). Currently, therapies capable of providing durable remissions in relapsed/refractory TP53- or ATM-defective CLL are lacking. Ataxia telangiectasia and Rad3-related (ATR) mediates response to replication stress, the absence of which leads to collapse of stalled replication forks into chromatid fragments that require resolution through the ATM/p53 pathway. Here, using AZD6738, a novel ATR kinase inhibitor, we investigated ATR inhibition as a synthetically lethal strategy to target CLL cells with TP53 or ATM defects. Irrespective of TP53 or ATM status, induction of CLL cell proliferation upregulated ATR protein, which then became activated in response to replication stress. In TP53- or ATM-defective CLL cells, inhibition of ATR signaling by AZD6738 led to an accumulation of unrepaired DNA damage, which was carried through into mitosis because of defective cell cycle checkpoints, resulting in cell death by mitotic catastrophe. Consequently, AZD6738 was selectively cytotoxic to both TP53- and ATM-defective CLL cell lines and primary cells. This was confirmed in vivo using primary xenograft models of TP53- or ATM-defective CLL, where treatment with AZD6738 resulted in decreased tumor load and reduction in the proportion of CLL cells with such defects. Moreover, AZD6738 sensitized TP53- or ATM-defective primary CLL cells to chemotherapy and ibrutinib. Our findings suggest that ATR is a promising therapeutic target for TP53- or ATM-defective CLL that warrants clinical investigation.

  14. Autologous hematopoietic stem cell transplantation in lymphoma patients is associated with a decrease in the double strand break repair capacity of peripheral blood lymphocytes. (United States)

    Lacoste, Sandrine; Bhatia, Smita; Chen, Yanjun; Bhatia, Ravi; O'Connor, Timothy R


    Patients who undergo autologous hematopoietic stem cell transplantation (aHCT) for treatment of a relapsed or refractory lymphoma are at risk of developing therapy related- myelodysplasia/acute myeloid leukemia (t-MDS/AML). Part of the risk likely resides in inherent interindividual differences in their DNA repair capacity (DRC), which is thought to influence the effect chemotherapeutic treatments have on the patient's stem cells prior to aHCT. Measuring DRC involves identifying small differences in repair proficiency among individuals. Initially, we investigated the cell model in healthy individuals (primary lymphocytes and/or lymphoblastoid cell lines) that would be appropriate to measure genetically determined DRC using host-cell reactivation assays. We present evidence that interindividual differences in DRC double-strand break repair (by non-homologous end-joining [NHEJ] or single-strand annealing [SSA]) are better preserved in non-induced primary lymphocytes. In contrast, lymphocytes induced to proliferate are required to assay base excision (BER) or nucleotide excision repair (NER). We established that both NHEJ and SSA DRCs in lymphocytes of healthy individuals were inversely correlated with the age of the donor, indicating that DSB repair in lymphocytes is likely not a constant feature but rather something that decreases with age (~0.37% NHEJ DRC/year). To investigate the predictive value of pre-aHCT DRC on outcome in patients, we then applied the optimized assays to the analysis of primary lymphocytes from lymphoma patients and found that individuals who later developed t-MDS/AML (cases) were indistinguishable in their DRC from controls who never developed t-MDS/AML. However, when DRC was investigated shortly after aHCT in the same individuals (21.6 months later on average), aHCT patients (both cases and controls) showed a significant decrease in DSB repair measurements. The average decrease of 6.9% in NHEJ DRC observed among aHCT patients was much higher

  15. Autologous hematopoietic stem cell transplantation in lymphoma patients is associated with a decrease in the double strand break repair capacity of peripheral blood lymphocytes (United States)

    Lacoste, Sandrine; Bhatia, Smita; Chen, Yanjun; Bhatia, Ravi; O’Connor, Timothy R.


    Patients who undergo autologous hematopoietic stem cell transplantation (aHCT) for treatment of a relapsed or refractory lymphoma are at risk of developing therapy related- myelodysplasia/acute myeloid leukemia (t-MDS/AML). Part of the risk likely resides in inherent interindividual differences in their DNA repair capacity (DRC), which is thought to influence the effect chemotherapeutic treatments have on the patient’s stem cells prior to aHCT. Measuring DRC involves identifying small differences in repair proficiency among individuals. Initially, we investigated the cell model in healthy individuals (primary lymphocytes and/or lymphoblastoid cell lines) that would be appropriate to measure genetically determined DRC using host-cell reactivation assays. We present evidence that interindividual differences in DRC double-strand break repair (by non-homologous end-joining [NHEJ] or single-strand annealing [SSA]) are better preserved in non-induced primary lymphocytes. In contrast, lymphocytes induced to proliferate are required to assay base excision (BER) or nucleotide excision repair (NER). We established that both NHEJ and SSA DRCs in lymphocytes of healthy individuals were inversely correlated with the age of the donor, indicating that DSB repair in lymphocytes is likely not a constant feature but rather something that decreases with age (~0.37% NHEJ DRC/year). To investigate the predictive value of pre-aHCT DRC on outcome in patients, we then applied the optimized assays to the analysis of primary lymphocytes from lymphoma patients and found that individuals who later developed t-MDS/AML (cases) were indistinguishable in their DRC from controls who never developed t-MDS/AML. However, when DRC was investigated shortly after aHCT in the same individuals (21.6 months later on average), aHCT patients (both cases and controls) showed a significant decrease in DSB repair measurements. The average decrease of 6.9% in NHEJ DRC observed among aHCT patients was much

  16. The white blood cell line: changes induced in mice by hypergravity (United States)

    Goldstein, Orna; Ishay, Jacob S.

    The effect of hypergravity on the white blood cell (WBC) line of mice was investigated by use of horizontal centrifuge. Several sets of experiments were performed, in which the parameters measured were the WBC and differential cell count in the peripheral blood. In another experiment, lymphocyte counts from the spleen, lymph nodes, and the thymus were measured. The needed samples were taken from the mice during a stay of 7-40 days under a hypergravity of 1.6G. The test groups that were placed on the arms of the centrifuge (1.6G) were compared with stationary control groups (1G) and a rotating control group located at the center of the centrifuge (1G). Such a comparison revealed the test animals to be deficient on all counts, to wit, showing a decrease in total number of WBC's, a decrease in lymphocyte number in the peripheral blood and a decrease in the number of lymphocyte in the spleen and thymus. The decrease of lymphocytes in peripheral blood was characterized by two different slopes - an early and temporary decrease at the first days of the experiment evident in both test and rotating control groups followed by a temporary increase, and a later persistent decrease, evident only in the test group, while in the rotating control lymphocyte counts reverted to normal. There were no significant differences in monocyte or neutrophil counts, except for a temporary increase in the number of neutrophils which peaked on the seventh day. In order to evaluate the effect of hypergravity on restoration of hematopoiesis following hematopoietic suppression, 5-fluoro-uracil (5-FU) was administered i.v. to both the experimental and control mice. Suppression of bone marrow was observed in all groups injected with 5-FU, but while there was later an increase in cell counts in the control groups, there was no such increase in the test group subjected to hypergravity.

  17. Specific induction of anti-leukemia effects by umbilical cord cell-derived CD8~+ T cytotoxic lymphocytes

    Institute of Scientific and Technical Information of China (English)



    Objective To explore the specific anti-leukemia immune response of CD8+ cytotoxic T lymphocyte (CTL) derived from cord blood (CB) ex vivo and evaluate the feasibilities and values of the CTL for specific immunotherapy. Methods Dendritic cells (DC) were induced from mononuclear cells (MNC) by combination cytokines in 10 CB samples. Loading U937 cell lysate antigen on

  18. Achievements and challenges of adoptive T cell therapy with tumor-infiltrating or blood-derived lymphocytes for metastatic melanoma

    DEFF Research Database (Denmark)

    Svane, Inge Marie; Verdegaal, Els M


    Adoptive cell therapy (ACT) based on autologous T cell derived either from tumor as tumor-infiltrating lymphocytes (TILs) or from peripheral blood is developing as a key area of future personalized cancer therapy. TIL-based ACT is defined as the infusion of T cells harvested from autologous fresh...... review....

  19. The association between DRESS and the diminished numbers of peripheral B lymphocytes and natural killer cells. (United States)

    Yazicioglu, Mehtap; Elmas, Reyhan; Turgut, Burhan; Genchallac, Tugba


    Drug reaction with eosinophilia and systemic symptoms (DRESS) is a drug-induced, severe multiorgan system reaction whose exact pathogenesis remains unknown. This study aimed at evaluating specific changes in peripheral blood lymphocyte subtypes associated with DRESS during antibiotic treatment. We analyzed six patients with DRESS. A complete blood count and peripheral blood lymphocytes immunophenotyping were carried out at symptom onset and at follow-up visits. Acute-phase reactants and liver enzymes were measured in all patients. Other tests - viral serology, serum immunoglobulin levels, and skin tests were performed when possible. B-cell counts were low in all patients at the onset of DRESS, and natural killer (NK) cells were low in all cases except one. During recovery, B-cell numbers were within a normal range in five patients. In one, there was even a 10-fold increase in B-cell counts, although the level was mildly low after 3 months. NK-cell numbers were within a normal range in three patients. The mean numbers of B cells and NK cells were significantly higher in the second samples compared to the values on admission. Serum IgA and IgM levels were low in one patient. The drug provocation test was positive with cefotaxime in one patient. Viral serology, performed on five patients, was negative. A decrease in B-cell and NK-cell counts was the most consistent finding associated with the onset of antibiotic-induced DRESS in our patients. This immunologic alteration might be a useful predictor of DRESS development.

  20. Blister fluid T lymphocytes during toxic epidermal necrolysis are functional cytotoxic cells which express human natural killer (NK) inhibitory receptors (United States)

    Le Cleach, L; Delaire, S; Boumsell, L; Bagot, M; Bourgault-Villada, I; Bensussan, A; Roujeau, J C


    Toxic epidermal necrolysis (TEN) is a rare life-threatening adverse drug reaction characterized by a massive destruction of the epidermis. Immunohistological studies of skin biopsies of TEN showed infiltrates of predominantly CD8+ T lymphocytes even though other authors reported a prominent involvement of cells of the monocyte-macrophage lineage. The aim of this study was to characterize phenotypically and functionally the cells present in the cutaneous blister fluid of four patients with TEN. We first determined that lymphocytes were predominant in blister fluid obtained early, while monocytes/macrophages later became the most important population. We then showed that this lymphocyte population, mainly CD3+CD8+, corresponded to a peculiar cell subset as they expressed cutaneous leucocyte antigen, killer inhibitory receptors KIR/KAR and failed to express CD28 molecule. Functionally, we determined that blister T lymphocytes had a cytotoxic T lymphocyte (CTL)- and NK-like cytotoxicity. The role of this cytotoxic lymphocyte population present at the site of lesions during TEN remains to be understood. PMID:10606987

  1. Selection of reference genes for quantitative PCR studies in purified B cells from B cell chronic lymphocytic leukaemia patients


    Valceckiene, Vilma; Kontenyte, Rima; Jakubauskas, Arturas; Griskevicius, Laimonas


    Abstract Clinical heterogeneity of B-cell chronic lymphocytic leukaemia (B-CLL) makes it necessary to identify potent prognostic indicators to predict individual clinical course and select risk-adapted therapy. During the last years numerous gene expression models have been suggested as prognostic factors of B-CLL. Today quantitative polymerase chain reaction (qPCR) is a preferred method for rapid quantification of gene expression and validation of microarray data. Reliability of q...

  2. Umbilical Cord Tissue-Derived Mesenchymal Stem Cells Induce T Lymphocyte Apoptosis and Cell Cycle Arrest by Expression of Indoleamine 2, 3-Dioxygenase

    Directory of Open Access Journals (Sweden)

    Xiuying Li


    Full Text Available It has been reported that human mesenchymal stem cells are able to inhibit T lymphocyte activation; however, the discrepancy among different sources of MSCs is not well documented. In this study, we have compared the MSCs from bone marrow (BM, adipose tissue (AT, placenta (PL, and umbilical cord (UC to determine which one displayed the most efficient immunosuppressive effects on phytohemagglutinin-induced T cell proliferation. Among them we found that hUC-MSC has the strongest effects on inhibiting T cell proliferation and is chosen to do the further study. We observed that T lymphocyte spontaneously released abundant IFN-γ. And IFN-γ secreted by T lymphocyte could induce the expression of indoleamine 2, 3-dioxygenase (IDO in hUC-MSCs. IDO was previously reported to induce T lymphocyte apoptosis and cell cycle arrest in S phase. When cocultured with hUC-MSCs, T lymphocyte expression of caspase 3 was significantly increased, while Bcl2 and CDK4 mRNA expression decreased dramatically. Addition of 1-methyl tryptophan (1-MT, an IDO inhibitor, restored T lymphocyte proliferation, reduced apoptosis, and induced resumption of the cell cycle. In addition, the changes in caspase 3, CDK4, and Bcl2 expression were reversed by 1-MT. These findings demonstrate that hUC-MSCs induce T lymphocyte apoptosis and cell cycle arrest by expressing abundant IDO and provide an explanation for some of the immunomodulatory effects of MSCs.

  3. Chlorambucil plus rituximab with or without maintenance rituximab as first-line treatment for elderly chronic lymphocytic leukemia patients. (United States)

    Foà, Robin; Del Giudice, Ilaria; Cuneo, Antonio; Del Poeta, Giovanni; Ciolli, Stefania; Di Raimondo, Francesco; Lauria, Francesco; Cencini, Emanuele; Rigolin, Gian Matteo; Cortelezzi, Agostino; Nobile, Francesco; Callea, Vincenzo; Brugiatelli, Maura; Massaia, Massimo; Molica, Stefano; Trentin, Livio; Rizzi, Rita; Specchia, Giorgina; Di Serio, Francesca; Orsucci, Lorella; Ambrosetti, Achille; Montillo, Marco; Zinzani, Pier Luigi; Ferrara, Felicetto; Morabito, Fortunato; Mura, Maria Angela; Soriani, Silvia; Peragine, Nadia; Tavolaro, Simona; Bonina, Silvia; Marinelli, Marilisa; De Propris, Maria Stefania; Starza, Irene Della; Piciocchi, Alfonso; Alietti, Alessandra; Runggaldier, Eva Josephine; Gamba, Enrica; Mauro, Francesca Romana; Chiaretti, Sabina; Guarini, Anna


    In a phase II trial, we evaluated chlorambucil and rituximab (CLB-R) as first-line induction treatment with or without R as maintenance for elderly chronic lymphocytic leukemia (CLL) patients. Treatment consisted of eight 28-day cycles of CLB (8 mg/m(2) /day, days 1-7) and R (day 1 of cycle 3, 375 mg/m(2) ; cycles 4-8, 500 mg/m(2) ). Responders were randomized to 12 8-week doses of R (375 mg/m(2) ) or observation. As per intention-to-treat analysis, 82.4% (95% CI, 74.25-90.46%) of 85 patients achieved an overall response (OR), 16.5% a complete response (CR), 2.4% a CR with incomplete bone marrow recovery. The OR was similar across Binet stages (A 86.4%, B 81.6%, and C 78.6%) and age categories (60-64 years, 92.3%; 65-69, 85.2%; 70-74, 75.0%; ≥75, 81.0%). CLB-R was well tolerated. After a median follow-up of 34.2 months, the median progression-free survival (PFS) was 34.7 months (95% CI, 33.1-39.5). TP53 abnormalities, complex karyotype, and low CD20 gene expression predicted lack of response; SF3B1 mutation and BIRC3 disruption low CR rates. IGHV mutations significantly predicted PFS. R maintenance tended towards a better PFS than observation and was safe and most beneficial for patients in partial response and for unmutated IGHV cases. CLB-R represents a promising option for elderly CLL patients. Copyright © 2014 Wiley Periodicals, Inc.

  4. Fludarabine nucleoside modulates nuclear "survival and death" proteins in resistant chronic lymphocytic leukemia cells. (United States)

    Henrich, Silke; Mactier, Swetlana; Best, Giles; Mulligan, Stephen P; Crossett, Ben; Christopherson, Richard Ian


    The nuclear mechanisms by which fludarabine nucleoside (F-ara-A) induces apoptosis have been investigated in human MEC1 cells derived from B-cell chronic lymphocytic leukemia. Upon treatment of cells with F-ara-A (100 μM, 72 hours), 15 nuclear proteins changed in abundance by more than 2-fold. Nuclear proteins up-regulated included calmodulin (4.3-fold), prohibitin (3.9-fold), β-actin variant (3.7-fold), and structure-specific recognition protein 1 (3.7-fold); those down-regulated included 60S ribosomal protein P2B (0.12-fold), fumarate hydratase (0.19-fold), splicing factor arginine/serine-rich 3 (0.35-fold), and replication protein A2 (0.42-fold). These changes in the levels of specific proteins promote survival or apoptosis; because the end result is apoptosis of MEC1 cells, apoptotic effects predominate.

  5. Perforin-deficient CD8+ T cells mediate fatal lymphocytic choriomeningitis despite impaired cytokine production

    DEFF Research Database (Denmark)

    Storm, Pernille; Bartholdy, Christina; Sørensen, Maria Rathmann


    invariably succumb to i.c. infection with LCMV strain Armstrong, although a few days later than matched wild-type mice. Upon further investigation, we found that this delay correlates with the delayed recruitment of inflammatory cells to the central nervous system (CNS). However, CD8(+) effector T cells were......Intracerebral (i.c.) infection with lymphocytic choriomeningitis virus (LCMV) is one of the most studied models for virus-induced immunopathology, and based on results from perforin-deficient mice, it is currently assumed that fatal disease directly reflects perforin-mediated cell lysis. However......, recent studies have revealed additional functional defects within the effector T cells of LCMV-infected perforin-deficient mice, raising the possibility that perforin may not be directly involved in mediating lethal disease. For this reason, we decided to reevaluate the role of perforin in determining...

  6. Effect of anti-carbohydrate antibodies on HIV infection in a monocytic cell line (U937)

    DEFF Research Database (Denmark)

    Hansen, J E; Nielsen, C; Clausen, H


    Monoclonal antibodies (mAbs) against carbohydrate epitopes of gp120 have recently been found to inhibit HIV infection of lymphocytes in vitro thereby opening new possibilities for vaccine considerations. Antibody-dependent enhancement of infection has however come increasingly into focus....... This study therefore investigated the neutralization of HIV in a monocytic cell line (U937) using mAbs against these carbohydrate gp120-epitopes. While antibodies against one of the epitopes (AI) neutralized infection of U937 cells despite binding to the Fc-receptor, one mAb against the sialosyl-Tn epitope...

  7. Early interferon-γ production in human lymphocyte subsets in response to nontyphoidal Salmonella demonstrates inherent capacity in innate cells. (United States)

    Nyirenda, Tonney S; Seeley, Anna E; Mandala, Wilson L; Drayson, Mark T; MacLennan, Calman A


    Nontyphoidal Salmonellae frequently cause life-threatening bacteremia in sub-Saharan Africa. Young children and HIV-infected adults are particularly susceptible. High case-fatality rates and increasing antibiotic resistance require new approaches to the management of this disease. Impaired cellular immunity caused by defects in the T helper 1 pathway lead to intracellular disease with Salmonella that can be countered by IFNγ administration. This report identifies the lymphocyte subsets that produce IFNγ early in Salmonella infection. Intracellular cytokine staining was used to identify IFNγ production in blood lymphocyte subsets of ten healthy adults with antibodies to Salmonella (as evidence of immunity to Salmonella), in response to stimulation with live and heat-killed preparations of the D23580 invasive African isolate of Salmonella Typhimurium. The absolute number of IFNγ-producing cells in innate, innate-like and adaptive lymphocyte subpopulations was determined. Early IFNγ production was found in the innate/innate-like lymphocyte subsets: γδ-T cells, NK cells and NK-like T cells. Significantly higher percentages of such cells produced IFNγ compared to adaptive αβ-T cells (Student's t test, Peffect on IFNγ production of depletion of Salmonella-specific CD4(+)-T lymphocytes in HIV infection.

  8. Leishmania infantum antigens modulate memory cell subsets of liver resident T lymphocyte. (United States)

    Rodrigues, A; Claro, M; Alexandre-Pires, G; Santos-Mateus, D; Martins, C; Valério-Bolas, A; Rafael-Fernandes, M; Pereira, M A; Pereira da Fonseca, I; Tomás, A M; Santos-Gomes, G


    In the recent years, the liver has been recognized as an important immune organ with major regulatory functions and immune memory, adding to the well-described vital metabolic functions. There are evidences from experimental infections performed with visceral Leishmania species that immune responses to parasite infection can be organ-specific. The liver is the compartment of acute resolving infection, with minimal tissue damage and resistance to reinfection, whereas the spleen is the compartment of parasite persistence. Control of hepatic infection in mice requires a coordinated immune response that involves the development of inflammatory granulomas. It is also described that the liver harbors populations of resident lymphocytes, which may exhibit memory characteristics. Therefore, the present study aims to address the role of the liver as an immune memory organ in the context of Leishmania infantum infection, by characterizing phenotypically resident liver T lymphocytes. The dynamics of memory T cells in L. infantum infected BALB/c mice and the effect of anti-leishmanial treatment in the differentiation of memory cell subsets were analyzed. The potential of recognition, differentiation and selection of memory lymphocytes by three L. infantum recombinant proteins were also explored. L. infantum infection generates effector and central memory T cells, but the cells did not expand when recalled, demonstrating a possible parasite silencing effect. The treatment with a leishmanicidal drug (antimoniate meglumine) increases the levels of memory and effector T cells, eliciting a more robust hepatic immune response. L. infantum parasites with a decreased sensitivity to the leishmanicidal drug favor the expansion of memory CD8(+) T cell subset, but inhibit the proliferation of CD8(+) T effector cells, possibly assuring their own survival. The recombinant proteins LirCyp1 and LirSOD are strongly recognized by memory cells of treated mice, indicating that these proteins

  9. Practical Aspects of Allogeneic Hematopoietic Cell Transplantation for Patients with Poor-Risk Chronic Lymphocytic Leukemia

    Directory of Open Access Journals (Sweden)

    Julio Delgado


    Full Text Available Allogeneic hematopoietic cell transplantation has become a viable option for younger patients with poor-risk chronic lymphocytic leukemia. The results obtained with either conventional or reduced-intensity conditioning regimens have been recently evaluated and compared with alternative nontransplant strategies. This manuscript deals with practical aspects of the procedure, including patient and donor selection, conditioning regimen, GVHD prophylaxis, disease monitoring, infectious and noninfectious complications, and timing of the procedure. Finally, we speculate on how we could improve the results obtained with the procedure and new advances currently in clinical trials.

  10. Cutaneous B-cell chronic lymphocytic leukaemia resembling a granulomatous rosacea. (United States)

    di Meo, Nicola; Stinco, Giuseppe; Trevisan, Giusto


    B-cell chronic lymphocytic leukemia (B-CLL) is a low-grade lymphoproliferative disease. Cutaneous involvement of B-CLL is limited and, in most cases, it represents non-specific manifestations related to an impaired immune system. Leukemic skin infiltrates (leukemia cutis) occur in 4-20% of patients. Herein we report the case of a 65-year-old woman with B-CLL presenting with papular, nodular, and plaque skin infiltrates affecting the nose, mimicking granulomatous rosacea. We discuss several aspects of rare cutaneous manifestations of B-CLL involving the face.

  11. Lymphocyte subsets, dendritic cells and cytokine profiles in mice with melanoma treated with Uncaria tomentosa


    Lozada-Requena, Iván; Laboratorio de Inmunología. Departamento de Ciencias Celulares y Moleculares. Facultad de Ciencias y Filosofía. Universidad Peruana Cayetano Heredia. Lima, Perú. EMINDES SAC (empresa de investigación y desarrollo en cáncer). Lima, Perú.; Núñez, César; Laboratorio de Inmunología. Departamento de Ciencias Celulares y Moleculares. Facultad de Ciencias y Filosofía. Universidad Peruana Cayetano Heredia. Lima, Perú. EMINDES SAC (empresa de investigación y desarrollo en cáncer). Lima, Perú.; Alvárez, Yubell; Laboratorio de Inmunología. Departamento de Ciencias Celulares y Moleculares. Facultad de Ciencias y Filosofía. Universidad Peruana Cayetano Heredia. Lima, Perú.; Kahn, Laura; Laboratorio de Inmunología. Departamento de Ciencias Celulares y Moleculares. Facultad de Ciencias y Filosofía. Universidad Peruana Cayetano Heredia. Lima, Perú.; Aguilar, José; Laboratorio de Inmunología. Departamento de Ciencias Celulares y Moleculares. Facultad de Ciencias y Filosofía. Universidad Peruana Cayetano Heredia. Lima, Perú.


    Objectives. To evaluate the immunomodulatory effect on lymphocyte subsets, dendritic cells (DC), Th1 / Th2 / Th17 and inflammatory cytokines on systemic level and/or in the tumor microenvironment of mice with or without melanoma. Materials and methods: Peripheral blood and/or primary tumors samples were obtained of mice with B16 melanoma treated or not with a hydroalcoholic extract of Uncaria tomentosa (UT) with 5.03% of pentacyclic oxindole alkaloids (UT-POA) obtained from the bark of the pl...

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

    Directory of Open Access Journals (Sweden)

    Thiel Cora S


    Full Text Available Abstract 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.

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

    Institute of Scientific and Technical Information of China (English)

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


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

  14. The immunodeficiency of bone marrow-transplanted patients. II. CD8-related suppression by patient lymphocytes of the response of donor lymphocytes to mitogens, antigens, and allogeneic cells

    DEFF Research Database (Denmark)

    Ødum, Niels; Hofmann, B; Jacobsen, N


    Lymphocytes from 21 patients sampled 1-6 months after bone marrow transplantation (BMT) were tested for functional suppressor activity against marrow-donor lymphocytes in the lymphocyte transformation test. Suppression of donor responses to allogeneic (i.e. mixed lymphocyte reaction, MLR...

  15. [Lymphocyte subsets, dendritic cells and cytokine profiles in mice with melanoma treated with Uncaria tomentosa]. (United States)

    Lozada-Requena, Iván; Núñez, César; Alvárez, Yubell; Kahn, Laura; Aguilar, José


    To evaluate the immunomodulatory effect on lymphocyte subsets, dendritic cells (DC), Th1 / Th2 / Th17 and inflammatory cytokines on systemic level and/or in the tumor microenvironment of mice with or without melanoma. Peripheral blood and/or primary tumors samples were obtained of mice with B16 melanoma treated or not with a hydroalcoholic extract of Uncaria tomentosa (UT) with 5.03% of pentacyclic oxindole alkaloids (UT-POA) obtained from the bark of the plant. All cell assays and cytokine measurements were performed by flow cytometry. UT-POA systemically increased CD4/CD8a relation while cell activation was inversely proportional; increased the proportion of DCm; induced a pro-inflammatory Th1 profile and reduced Th17 response. TNF-α and IL-17A positively and negatively correlated with CD4/CD8a relation. The increase of Th1 (TNF-α) may result in the increase of CD4 or M1 macrophage activation. Although UT-POA shows increased DCm, is not dose-dependent. Th17(IL-17A) decreased can support the function of CD8a lymphocytes. UT-POA shows better systemic immunomodulatory effects than intratumoral.

  16. Monitoring of WT1-specific cytotoxic T lymphocytes after allogeneic hematopoietic stem cell transplantation. (United States)

    Morita, Yuriko; Heike, Yuji; Kawakami, Mami; Miura, Osamu; Nakatsuka, Shin-Ichi; Ebisawa, Michiko; Mori, Shin-Ichiro; Tanosaki, Ryuji; Fukuda, Takahiro; Kim, Sung-Won; Tobinai, Kensei; Takaue, Yoichi


    Donor-derived cytotoxic T lymphocytes (CTL) that respond to tumor antigens emerge after hematopoietic stem cell transplantation (HSCT), particularly in association with the status of immune recovery. To analyze the frequency of CTL against PR1, PRAME and WT1 after HSCT, a tetramer-based analysis was performed in 97 samples taken from 35 patients (9 AML, 11 MDS, 2 CML, 4 ALL, 7 lymphoma and 2 renal cell carcinoma [RCC]) with the HLA-A02 phenotype. Regarding PR1, only 1 sample showed the presence of tetramer-positive cells (0.04%/lymphocyte). Similarly, in PRAME, only 10 of 97 samples were sporadically positive with low titers. For WT1, positive results were detected in 39 of 97 samples and 7 (2 CML, 1 ALL, 2 lymphoma and 2 RCC) patients clearly showed positive results more than once. On the basis of these results, we performed serial analyses of WT1-specific CTL during the clinical course in 2 patients with RCC, who underwent HSCT with a reduced-intensity regimen, to examine the precise correlation between the kinetics of CTL, the occurrence of GVHD and the observed clinical response. A higher positive rate for WT1-specific CTL and a correlation with the clinical response suggest that WT1 may be a useful antigen for a wider monitoring application.

  17. Constitutive activation of extracellular signal-regulated kinase predisposes diffuse large B-cell lymphoma cell lines to CD40-mediated cell death

    DEFF Research Database (Denmark)

    Hollmann, C Annette; Owens, Trevor; Nalbantoglu, Josephine;


    CD40 promotes survival, proliferation, and differentiation of normal B cells but can cause activation-induced cell death in malignant B lymphocytes. CD40 ligand and anti-CD40 antibodies have been used successfully to induce apoptosis in lymphoma lines both in vitro and in xenograft tumor models...... a specific cell line or tumor will undergo apoptosis when stimulated with CD40 and to identify targets downstream of CD40 that affect only the apoptotic arm of CD40 signaling. We have analyzed gene expression patterns in CD40-sensitive and CD40-resistant diffuse large B-cell lymphoma (DLBCL) cell lines...... and no increase in ERK activity in response to CD40 stimulation. Our results suggest that constitutive activation of ERK may be required for death signaling by CD40....

  18. Anti-T-lymphocyte globulin-based non-myeloablative stem cell transplantation followed by HLA-identical donor lymphocyte infusion for hematologic malignancies

    Institute of Scientific and Technical Information of China (English)

    毛平; 王顺清; 王汉平; 李庆山; 许艳丽; 莫文健; 应逸; 朱志刚


    Objective To evaluate the application of anti-T-lymphocyte globulin (ATG) based nonmyeloablative but profoundly immunosuppressive regimens followed by donor lymphocyte infusion (DLI) for the treatment of hematologic malignancies.Methods The protocol was designed to minimize the intensity of the conditioning regimen to the range of nonmyeloablative therapies based on ATG with low-dose busulfan (Bu) and Cytoxan (CTX) (15-19.5 mg/kg, 8 mg/kg and 80 mg/kg, respectively). The patients received the first lymphocytic infusion from HLA-identical sibling donors on days 28-30 after transplant, and the first T cell dosage of 106/kg followed by the escalated dosage in the range of (0.5-1.5)×108/kg. The total number of procedures were performed at a median of 4.2 procedures (range of 2-8 procedures).Results Engraftment was documented in all six patients in the form of donor-recipient hematopoietic cells mixed chimera at early-stage posttransplant, which was converted gradually into complet chimera by DLI in four patients. Graft-versus-host disease (GVHD) developed in three of six cases, only one of which was severe. To date,four patients are disease free and alive.Conclusions Allogeneic donor stem cell engraftment into host can be achieved by nonmyeloablative conditioning regimen based on ATG. Transient mixed donor-recipient hematopoietic cell mixed with chimeras may be successfully converted into complete chimerism by DLI posttransplant. GVHD remains major clinical concern in our study.

  19. Estimating the number of hematopoietic or lymphoid stem cells giving rise to clonal chromosome aberrations in blood T lymphocytes. (United States)

    Nakano, M; Kodama, Y; Ohtaki, K; Itoh, M; Awa, A A; Cologne, J; Kusunoki, Y; Nakamura, N


    Quantifying the proliferative capacity of long-term hematopoietic stem cells in humans is important for bone marrow transplantation and gene therapy. Obtaining appropriate data is difficult, however, because the experimental tools are limited. We hypothesized that tracking clonal descendants originating from hematopoietic stem cells would be possible if we used clonal chromosome aberrations as unique tags of individual hematopoietic stem cells in vivo. Using FISH, we screened 500 blood T lymphocytes from each of 513 atomic bomb survivors and detected 96 clones composed of at least three cells with identical aberrations. The number of clones was inversely related to their population size, which we interpreted to mean that the progenitor cells were heterogeneous in the number of progeny that they could produce. The absolute number of progenitor cells contributing to the formation of the observed clones was estimated as about two in an unexposed individual. Further, scrutiny of ten clones revealed that lymphocyte clones could originate roughly equally from hematopoietic stem cells or from mature T lymphocytes, thereby suggesting that the estimated two progenitor cells are shared as one hematopoietic stem cell and one mature T cell. Our model predicts that one out of ten people bears a non- aberrant clone comprising >10% of the total lymphocytes, which indicates that clonal expansions are common and probably are not health-threatening.

  20. Leukoreduction system chambers are an efficient, valid, and economic source of functional monocyte-derived dendritic cells and lymphocytes. (United States)

    Pfeiffer, Isabell A; Zinser, Elisabeth; Strasser, Erwin; Stein, Marcello F; Dörrie, Jan; Schaft, Niels; Steinkasserer, Alexander; Knippertz, Ilka


    The demand for human monocyte-derived dendritic cells (moDCs), as well as for primary human B and T lymphocytes for immunological research purposes has been increased in recent years. Classically, these monocytes are isolated from blood, leukapheresis products or buffy coats of healthy donors by plastic adherence of peripheral blood mononuclear cells (PBMCs), followed by stimulation with granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4, while lymphocytes are usually isolated from the non-adherent fraction (NAF) by magnetic cell sorting. However, donor-blood is a limited resource and not every blood bank offers leukapheresis products or buffy coats for laboratory use. Additionally, a leukapheresis is very expensive and also the generation/isolation of cells is time- and cost-intensive. To overcome some of these obstacles, we evaluated if low-cost leukoreduction system chambers (LRSCs), which arise after routine donor plateletpheresis procedures, and are usually discarded, would be an alternative and appropriate source of PBMCs to generate moDCs and to isolate lymphocytes. By analyzing the number and phenotype of immature and mature dendritic cells (DCs), as well as of B and T lymphocytes derived from LRSCs, we found all cells to be of high quantity and quality. Further investigations on DCs comprising transwell migration assays, allogeneic mixed lymphocyte reactions (MLR), cytokine secretion assays, and cytotoxic T cell induction assays revealed high migratory, as well as stimulatory capacity of these cells. In addition, DCs and T cells were efficiently electroporated with mRNA and showed characteristic cytokine production after co-culture, demonstrating LRSCs as an efficient, valid, and economic source for generation of moDCs and lymphocytes for research purposes.

  1. Isolation of murine hepatic lymphocytes using mechanical dissection for phenotypic and functional analysis of NK1.1 + cells

    Institute of Scientific and Technical Information of China (English)

    Zhong-Jun Dong; Hai-Ming Wei; Rui Sun; Bin Gao; Zhi-Gang Tian


    AIM: To choose an appropriate methods for the isolation of hepatic lymphocytes between the mechanical dissection and the enzymatic digestion and investigate the effects of two methods on phenotype and function of hepatic lymphocytes.METHODS: Hepatic lymphocytes were isolated from untreated, poly (I:C)-stimulated or ConA-stimulated mice using the two methods, respectively. The cell yield per liver was evaluated by direct counting under microscope.Effects of digestive. enzymes on the surface markers involved in hepatic lymphocytes were represented by relative change rate [(percentage of post-digestion -percentage of pre-digestion)/percentage of pre-digestion].Phenotypic analyses of the subpopulations of hepatic lymphocytes and intracellular cytokines were detected by flow cytometry. The cytotoxicity of NK cells from wild C57BL/6 or poly (I:C)-stimulated C57BL/6 mice was analyzed with a 4-h 51Cr release assay.RESULTS: NK1.1+ cell markers, NK1.1 and DX5, were significantly down-expressed after enzymatic digestion and their relative change rates were about 28% and 32%,respectively. Compared with the enzymatic digestion, the cell yield isolated from unstimulated, poly (I:C)-treated or ConA-treated mice by mechanical dissection was not significantly decreased. Hepatic lymphocytes isolated by the mechanical dissection comprised more innate immune cells like NK, NKT and γδ cells in normal C57BL/6 mice.After poly (I:C) stimulation, hepatic NK cells rose to about 35%, while NKT cells simultaneously decreased. Following ConA injection, the number of hepatic NKT cells was remarkably reduced to 3.67%. Higher ratio of intracellular IFN-γ+(68%) or TNF-α+(15%) NK1.1+ cells from poly (I:C)-treated mice was obtained using mechanical dissection method than control mice. There was no difference in viability between the mechanical dissection and the enzymatic digestion, and hepatic lymphocytes obtained with the two methods had similar cytotoxicity against YAC-1cells

  2. STAT1 mediates differentiation of chronic lymphocytic leukemia cells in response to Bryostatin 1. (United States)

    Battle, Traci E; Frank, David A


    Bryostatin 1 is known to exhibit in vitro and in vivo activity against chronic lymphocytic leukemia (CLL) cells by inducing their further maturation into plasma-like cells. Signal transducer and activator of transcription (STAT) proteins play a central role in B-lymphocyte growth and function and are aberrantly phosphorylated on serine residues in CLL cells. To determine whether STAT transcription factors are important in Bryostatin 1-induced differentiation of CLL cells, primary CLL cells were examined for signaling events following exposure to Bryostatin 1 in vitro. Western analysis and electrophoretic mobility shift assays revealed that Bryostatin 1 induced tyrosine phosphorylation and DNA binding of STAT1, yet there was no effect on constitutive serine phosphorylation of STAT1. Bryostatin 1-induced STAT1 activation occurred in a manner that was dependent on protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and Janus tyrosine kinase (JAK) activation. Evidence indicates that Bryostatin 1 induces STAT1 activation through an interferon gamma (IFN gamma) autocrine loop. However, STAT1 activation by IFN gamma stimulation alone was not sufficient to induce differentiation. This insufficiency is due to the broader effect on gene expression caused by Bryostatin 1 compared with IFN gamma, as demonstrated by microarray analysis. Both up-regulation of CD22 expression and immunoglobulin M (IgM) production, markers of CLL differentiation, were inhibited by a decoy oligonucleotide for STAT1, indicating that STAT1 is necessary for Bryostatin 1-induced differentiation of CLL cells. This study implicates STAT transcription factors as important mediators of Bryostatin 1-induced differentiation of CLL cells and could possibly lead to improved therapeutic approaches for the treatment of CLL.

  3. Single-Pass, Closed-System Rapid Expansion of Lymphocyte Cultures for Adoptive Cell Therapy (United States)

    Klapper, Jacob A.; Thomasian, Armen A.; Smith, Douglas M.; Gorgas, Gayle C.; Wunderlich, John R.; Smith, Franz O.; Hampson, Brian S.; Rosenberg, Steven A.; Dudley, Mark E.


    Adoptive cell therapy (ACT) for metastatic melanoma involves the ex vivo expansion and re-infusion of tumor infiltrating lymphocytes (TIL) obtained from resected specimens. With an overall objective response rate of fifty-six percent, this T-cell immunotherapy provides an appealing alternative to other therapies, including conventional therapies with lower response rates. However, there are significant regulatory and logistical concerns associated with the ex vivo activation and large scale expansion of these cells. The best current practice uses a rapid expansion protocol (REP) consisting of an ex vivo process that occurs in tissue culture flasks (T-flasks) and gas-permeable bags, utilizes OKT3 (anti-CD3 monoclonal antibody), recombinant human interleukin-2, and irradiated peripheral blood mononuclear cells to initiate rapid lymphocyte growth. A major limitation to the widespread delivery of therapy to large numbers of melanoma patients is the open system in which a REP is initiated. To address this problem, we have investigated the initiation, expansion and harvest at clinical scale of TIL in a closed-system continuous perfusion bioreactor. Each cell product met all safety criteria for patient treatment and by head-to-head comparison had a similar potency and phenotype as cells grown in control T-flasks and gas-permeable bags. However, the currently available bioreactor cassettes were limited in the total cell numbers that could be generated. This bioreactor may simplify the process of the rapid expansion of TIL under stringent regulatory conditions thereby enabling other institutions to pursue this form of ACT. PMID:19389403

  4. Piperine blocks interleukin-2-driven cell cycle progression in CTLL-2 T lymphocytes by inhibiting multiple signal transduction pathways. (United States)

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


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

  5. CD161 Expression Defines a Th1/Th17 Polyfunctional Subset of Resident Memory T Lymphocytes in Bronchoalveolar Cells.

    Directory of Open Access Journals (Sweden)

    Yolanda Gonzalez

    Full Text Available Alveolar resident memory T cells (T(RM comprise a currently uncharacterized mixture of cell subpopulations. The CD3(+CD161(+ T cell subpopulation resides in the liver, intestine and skin, but it has the capacity for tissue migration; however, the presence of resident CD3(+CD161(+ T cells in the bronchoalveolar space under normal conditions has not been reported. Bronchoalveolar cells (BACs from healthy volunteers were evaluated and found that 8.6% (range 2.5%-21% of these cells were CD3(+ T lymphocytes. Within the CD3(+ population, 4.6% of the cells (2.1-11.3 expressed CD161 on the cell surface, and 74.2% of the CD161(+CD3(+ T cells expressed CD45RO. The number of CD3(+CD161(+ T cells was significantly lower in the bronchoalveolar space than in the blood (4.6% of BACs vs 8.4% of peripheral blood mononuclear cells (PBMCs; P<0.05. We also found that 2.17% of CD4(+ T lymphocytes and 1.52% of CD8(+ T lymphocytes expressed CD161. Twenty-two percent of the alveolar CD3(+CD161(+ T lymphocytes produced cytokines upon stimulation by PMA plus ionomycin, and significantly more interferon gamma (IFN-γ was produced compared with other cytokines (P = 0.05. Most alveolar CD3(+CD161(+ T cells produced interleukin-17 (IL-17 and IFN-γ simultaneously, and the percentage of these cells was significantly higher than the percentage of CD3(+CD161- T cells. Moreover, the percentage of alveolar CD3(+CD161(+ T lymphocytes that produced IFN-γ/IL-17 was significantly higher than those in the peripheral blood (p<0.05. In conclusion, Th1/Th17-CD3(+CD161(+ TRM could contribute to compartment-specific immune responses in the lung.

  6. The sphingosine-1-phosphate transporter Spns2 expressed on endothelial cells regulates lymphocyte trafficking in mice. (United States)

    Fukuhara, Shigetomo; Simmons, Szandor; Kawamura, Shunsuke; Inoue, Asuka; Orba, Yasuko; Tokudome, Takeshi; Sunden, Yuji; Arai, Yuji; Moriwaki, Kazumasa; Ishida, Junji; Uemura, Akiyoshi; Kiyonari, Hiroshi; Abe, Takaya; Fukamizu, Akiyoshi; Hirashima, Masanori; Sawa, Hirofumi; Aoki, Junken; Ishii, Masaru; Mochizuki, Naoki


    The bioactive lysophospholipid mediator sphingosine-1-phosphate (S1P) promotes the egress of newly formed T cells from the thymus and the release of immature B cells from the bone marrow. It has remained unclear, however, where and how S1P is released. Here, we show that in mice, the S1P transporter spinster homolog 2 (Spns2) is responsible for the egress of mature T cells and immature B cells from the thymus and bone marrow, respectively. Global Spns2-KO mice exhibited marked accumulation of mature T cells in thymi and decreased numbers of peripheral T cells in blood and secondary lymphoid organs. Mature recirculating B cells were reduced in frequency in the bone marrow as well as in blood and secondary lymphoid organs. Bone marrow reconstitution studies revealed that Spns2 was not involved in S1P release from blood cells and suggested a role for Spns2 in other cells. Consistent with these data, endothelia-specific deletion of Spns2 resulted in defects of lymphocyte egress similar to those observed in the global Spns2-KO mice. These data suggest that Spns2 functions in ECs to establish the S1P gradient required for T and B cells to egress from their respective primary lymphoid organs. Furthermore, Spns2 could be a therapeutic target for a broad array of inflammatory and autoimmune diseases.

  7. MG132 Induced Apoptosis Pathway in HL-60 Cells and Impact of Allogeneic Mixed Lymphocyte Reaction

    Institute of Scientific and Technical Information of China (English)

    Yong-ming Zhou; Wei Guo; Hao Zhou; Jin-hua Zhang; Zhi-ping Liu; Mei-xia Yu


    Objective: To investigate the proteasome inhibitor MG132-induced apoptosis pathway in HL-60 cells and the role of allogeneic mixed lymphocyte reaction.Methods: Cell apoptosis was analyzed by flow cytometry. The expressions of p21 protein, p27 protein and p53 protein in HL-60 cells treated with MG132 were measured by Western blot. The proliferation of, peripheral blood mononuclear cells (PBMNCs) after treatment with 75 Gy irradiated HL-60 cells treated with MG132 was measured with CCK-8.Results: High-dose MG132 induced apoptosis in HL-60 cells. No significant change was observed in MG132-induced apoptosis after inhibiting caspase-8 and caspase-9 pathway. The expressions of p21 protein and p27 protein increased in MG132-induced apoptosis. HL-60 cells treated with low-dose MG132 improved the proliferation of PBMNCs from healthy volunteers.Conclusion: High-dose MG132 induced apoptosis and directly killed HL-60 cells. MG132 induced apoptosis in a caspase-8- and caspase-9-independent pathway. p21 protein and p27 protein were involved in MG132-induced apoptosis in HL-60 cells. HL-60 cells treated with Low-dose MG132 improved the effect of promoting the proliferation of PBMNCs from healthy volunteers.

  8. T lymphocytes and dendritic cells are activated by the deletion of peroxiredoxin II (Prx II) gene. (United States)

    Moon, Eun-Yi; Noh, Young-Wook; Han, Ying-Hao; Kim, Sun-Uk; Kim, Jin-Man; Yu, Dae-Yeul; Lim, Jong-Seok


    Peroxiredoxin II (Prx II) is a member of antioxidant enzyme family and it plays a protective role against oxidative damage. Constitutive production of endogenous reactive oxygen species was detected in spleen and bone marrow cells lacking Prx II. Here, we investigated the role of Prx II in immune responses. The total number of splenocytes (especially, the population of S-phase cells and CD3(+) T cells) was significantly higher in Prx II(-/-) mice than in wild type. Number of peripheral blood mononuclear cells (PBMCs) in Prx II(-/-) mice was also higher than wild type. Differentiation of Prx II(-/-) mouse bone marrow cells into CD11c-positive dendritic cells was greater than that of wild type. Transplantation of Prx II(-/-) bone marrow cells into wild type mice increased PBMCs in blood and bone marrow-derived dendritic cells. Prx II deletion enhances concanavalin A (ConA)-induced splenocyte proliferation and mixed lymphocyte reaction (MLR) activity of bone marrow-derived CD11c-positive dendritic cells to stimulate recipient splenocytes. Collectively, these data suggest that Prx II inhibits the immune cell responsiveness, which may be regulated by scavenging the low amount of reactive oxygen species (ROS).

  9. High mitochondrial DNA stability in B-cell chronic lymphocytic leukemia.

    Directory of Open Access Journals (Sweden)

    María Cerezo

    Full Text Available BACKGROUND: Chronic Lymphocytic Leukemia (CLL leads to progressive accumulation of lymphocytes in the blood, bone marrow, and lymphatic tissues. Previous findings have suggested that the mtDNA could play an important role in CLL. METHODOLOGY/PRINCIPAL FINDINGS: The mitochondrial DNA (mtDNA control-region was analyzed in lymphocyte cell DNA extracts and compared with their granulocyte counterpart extract of 146 patients suffering from B-Cell CLL; B-CLL (all recruited from the Basque country. Major efforts were undertaken to rule out methodological artefacts that would render a high false positive rate for mtDNA instabilities and thus lead to erroneous interpretation of sequence instabilities. Only twenty instabilities were finally confirmed, most of them affecting the homopolymeric stretch located in the second hypervariable segment (HVS-II around position 310, which is well known to constitute an extreme mutational hotspot of length polymorphism, as these mutations are frequently observed in the general human population. A critical revision of the findings in previous studies indicates a lack of proper methodological standards, which eventually led to an overinterpretation of the role of the mtDNA in CLL tumorigenesis. CONCLUSIONS/SIGNIFICANCE: Our results suggest that mtDNA instability is not the primary causal factor in B-CLL. A secondary role of mtDNA mutations cannot be fully ruled out under the hypothesis that the progressive accumulation of mtDNA instabilities could finally contribute to the tumoral process. Recommendations are given that would help to minimize erroneous interpretation of sequencing results in mtDNA studies in tumorigenesis.

  10. Feasibility of Telomerase-Specific Adoptive T-cell Therapy for B-cell Chronic Lymphocytic Leukemia and Solid Malignancies. (United States)

    Sandri, Sara; Bobisse, Sara; Moxley, Kelly; Lamolinara, Alessia; De Sanctis, Francesco; Boschi, Federico; Sbarbati, Andrea; Fracasso, Giulio; Ferrarini, Giovanna; Hendriks, Rudi W; Cavallini, Chiara; Scupoli, Maria Teresa; Sartoris, Silvia; Iezzi, Manuela; Nishimura, Michael I; Bronte, Vincenzo; Ugel, Stefano


    Telomerase (TERT) is overexpressed in 80% to 90% of primary tumors and contributes to sustaining the transformed phenotype. The identification of several TERT epitopes in tumor cells has elevated the status of TERT as a potential universal target for selective and broad adoptive immunotherapy. TERT-specific cytotoxic T lymphocytes (CTL) have been detected in the peripheral blood of B-cell chronic lymphocytic leukemia (B-CLL) patients, but display low functional avidity, which limits their clinical utility in adoptive cell transfer approaches. To overcome this key obstacle hindering effective immunotherapy, we isolated an HLA-A2-restricted T-cell receptor (TCR) with high avidity for human TERT from vaccinated HLA-A*0201 transgenic mice. Using several relevant humanized mouse models, we demonstrate that TCR-transduced T cells were able to control human B-CLL progression in vivo and limited tumor growth in several human, solid transplantable cancers. TERT-based adoptive immunotherapy selectively eliminated tumor cells, failed to trigger a self-MHC-restricted fratricide of T cells, and was associated with toxicity against mature granulocytes, but not toward human hematopoietic progenitors in humanized immune reconstituted mice. These data support the feasibility of TERT-based adoptive immunotherapy in clinical oncology, highlighting, for the first time, the possibility of utilizing a high-avidity TCR specific for human TERT. Cancer Res; 76(9); 2540-51. ©2016 AACR.

  11. Regulatory T-Cells in Chronic Lymphocytic Leukemia and Autoimmune Diseases (United States)

    D’Arena, Giovanni; Rossi, Giovanni; Vannata, Barbara; Deaglio, Silvia; Mansueto, Giovanna; D’Auria, Fiorella; Statuto, Teodora; Simeon, Vittorio; De Martino, Laura; Marandino, Aurelio; Del Poeta8, Giovanni; De Feo, Vincenzo; Musto, Pellegrino


    Regulatory T-cells (Tregs) constitute a small subset of cells that are actively involved in maintaining self-tolerance, in immune homeostasis and in antitumor immunity. They are thought to play a significant role in the progression of cancer and are generally increased in patient with chronic lymphocytic leukemia (CLL). Their number correlates with more aggressive disease status and is predictive of the time to treatment, as well. Moreover, it is now clear that dysregulation in Tregs cell frequency and/or function may result in a plethora of autoimmune diseases, including multiple sclerosis, type 1 diabetes mellitus, myasthenia gravis, systemic lupus erythematosus, autoimmune lymphoproliferative disorders, rheumatoid arthritis, and psoriasis. Efforts are made aiming to develop approaches to deplete Tregs or inhibit their function in cancer and autoimmune disorders, as well. PMID:22973497

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

    Directory of Open Access Journals (Sweden)

    Aysun Adan Gökbulut


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

  13. Susceptibility testing of fish cell lines for virus isolation

    DEFF Research Database (Denmark)

    Ariel, Ellen; Skall, Helle Frank; Olesen, Niels Jørgen


    compare susceptibility between cell lines and between lineages within a laboratory and between laboratories (Inter-laboratory Proficiency Test). The objective being that the most sensitive cell line and lineages are routinely selected for diagnostic purposes.In comparing cell lines, we simulated "non......-cell-culture-adapted" virus by propagating the virus in heterologous cell lines to the one tested. A stock of test virus was produced and stored at - 80 °C and tests were conducted biannually. This procedure becomes complicated when several cell lines are in use and does not account for variation among lineages. In comparing...... cell lineages, we increased the number of isolates of each virus, propagated stocks in a given cell line and tested all lineages of that line in use in the laboratory. Testing of relative cell line susceptibility between laboratories is carried out annually via the Inter-laboratory Proficiency Test...

  14. Susceptibility testing of fish cell lines for virus isolation

    DEFF Research Database (Denmark)

    Ariel, Ellen; Skall, Helle Frank; Olesen, Niels Jørgen


    compare susceptibility between cell lines and between lineages within a laboratory and between laboratories (Inter-laboratory Proficiency Test). The objective being that the most sensitive cell line and lineages are routinely selected for diagnostic purposes.In comparing cell lines, we simulated "non......-cell-culture-adapted" virus by propagating the virus in heterologous cell lines to the one tested. A stock of test virus was produced and stored at - 80 °C and tests were conducted biannually. This procedure becomes complicated when several cell lines are in use and does not account for variation among lineages. In comparing...... cell lineages, we increased the number of isolates of each virus, propagated stocks in a given cell line and tested all lineages of that line in use in the laboratory. Testing of relative cell line susceptibility between laboratories is carried out annually via the Inter-laboratory Proficiency Test...

  15. Non-small cell lung cancer-derived soluble mediators enhance apoptosis in activated T lymphocytes through an I kappa B kinase-dependent mechanism. (United States)

    Batra, Raj K; Lin, Ying; Sharma, Sherven; Dohadwala, Mariam; Luo, Jie; Pold, Mehis; Dubinett, Steven M


    T lymphocyte survival is critical for the development and maintenance of an effective host antitumor immune response; however, the tumor environment can negatively impact T-cell survival. Lymphocytes exposed to tumor supernatants (TSNs) were evaluated for apoptosis after mitogen stimulation. TSN was observed to significantly enhance phorbol 12-myristate 13-acetate/ionomycin- and anti-CD3-stimulated lymphocyte apoptosis. Enhanced lymphocyte apoptosis was associated with an impairment of nuclear factor kappa B nuclear translocation and diminished I kappa B alpha degradation. In lymphocytes stimulated after exposure to TSNs, cytoplasmic I kappa B alpha persisted as a result of alterations in I kappa B kinase (IKK) activity. Accordingly, although there were no apparent differences in IKK component concentrations, lymphocytes preexposed to TSNs exhibited markedly reduced IKK activity. We conclude that non-small cell lung cancer-derived soluble factors promote apoptosis in activated lymphocytes by an IKK-dependent pathway.

  16. A general functional response of cytotoxic T lymphocyte-mediated killing of target cells. (United States)

    Gadhamsetty, Saikrishna; Marée, Athanasius F M; Beltman, Joost B; de Boer, Rob J


    Cytotoxic T lymphocytes (CTLs) kill virus-infected cells and tumor cells, and play a critical role in immune protection. Our knowledge of how the CTL killing efficiency varies with CTL and target cell numbers is limited. Here, we simulate a region of lymphoid tissue using a cellular Potts model to characterize the functional response of CTL killing of target cells, and find that the total killing rate saturates both with the CTL and the target cell densities. The relative saturation in CTL and target cell densities is determined by whether a CTL can kill multiple target cells at the same time, and whether a target cell can be killed by many CTLs together. We find that all the studied regimes can be well described by a double-saturation (DS) function with two different saturation constants. We show that this DS model can be mechanistically derived for the cases where target cells are killed by a single CTL. For the other cases, a biological interpretation of the parameters is still possible. Our results imply that this DS function can be used as a tool to predict the cellular interactions in cytotoxicity data.

  17. Ibrutinib Therapy Increases T Cell Repertoire Diversity in Patients with Chronic Lymphocytic Leukemia. (United States)

    Yin, Qingsong; Sivina, Mariela; Robins, Harlan; Yusko, Erik; Vignali, Marissa; O'Brien, Susan; Keating, Michael J; Ferrajoli, Alessandra; Estrov, Zeev; Jain, Nitin; Wierda, William G; Burger, Jan A


    The Bruton's tyrosine kinase inhibitor ibrutinib is a highly effective, new targeted therapy for chronic lymphocytic leukemia (CLL) that thwarts leukemia cell survival, growth, and tissue homing. The effects of ibrutinib treatment on the T cell compartment, which is clonally expanded and thought to support the growth of malignant B cells in CLL, are not fully characterized. Using next-generation sequencing technology, we characterized the diversity of TCRβ-chains in peripheral blood T cells from 15 CLL patients before and after 1 y of ibrutinib therapy. We noted elevated CD4(+) and CD8(+) T cell numbers and a restricted TCRβ repertoire in all pretreatment samples. After 1 y of ibrutinib therapy, elevated peripheral blood T cell numbers and T cell-related cytokine levels had normalized, and T cell repertoire diversity increased significantly. Dominant TCRβ clones in pretreatment samples declined or became undetectable, and the number of productive unique clones increased significantly during ibrutinib therapy, with the emergence of large numbers of low-frequency TCRβ clones. Importantly, broader TCR repertoire diversity was associated with clinical efficacy and lower rates of infections during ibrutinib therapy. These data demonstrate that ibrutinib therapy increases diversification of the T cell compartment in CLL patients, which contributes to cellular immune reconstitution.

  18. Differential expression and function of CD27 in chronic lymphocytic leukemia cells expressing ZAP-70. (United States)

    Lafarge, Sandrine T; Hou, Sen; Pauls, Samantha D; Johnston, James B; Gibson, Spencer B; Marshall, Aaron J


    Chronic lymphocytic leukemia is a malignancy driven by abberant B cell signaling and survival. Leukemic B cells accumulate in the peripheral blood and the lymphoid organs where contact with stromal cells and T cells provide critical survival signals. Clinical severity of CLL is associated with several prognostic markers including expression of the kinase ZAP-70. ZAP-70 expression enhances signaling via the B cell antigen receptor and is associated with increased cell adhesion and migration capacity. Here we report that ZAP-70-positive CLL patients display significantly higher expression of the TNF superfamily receptor and memory marker CD27 than do ZAP-70 negative patients. CD27 expression by CLL was acutely elevated upon BCR cross-linking, or upon ectopic expression of ZAP-70. CD27 expression correlated with functional capacity to adhere to stromal cells and antibody blockade of CD27 impaired CLL binding to stroma. These results provide the first evidence for differential expression of CD27 among CLL prognostic groups, suggest a role for ZAP-70 dependent signaling in CD27 induction and implicate CD27 in cell-cell interactions with the lymphoid tissue microenvironment.

  19. Induced pluripotent stem cell lines derived from human somatic cells. (United States)

    Yu, Junying; Vodyanik, Maxim A; Smuga-Otto, Kim; Antosiewicz-Bourget, Jessica; Frane, Jennifer L; Tian, Shulan; Nie, Jeff; Jonsdottir, Gudrun A; Ruotti, Victor; Stewart, Ron; Slukvin, Igor I; Thomson, James A


    Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors (OCT4, SOX2, NANOG, and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells. These induced pluripotent human stem cells have normal karyotypes, express telomerase activity, express cell surface markers and genes that characterize human ES cells, and maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers. Such induced pluripotent human cell lines should be useful in the production of new disease models and in drug development, as well as for applications in transplantation medicine, once technical limitations (for example, mutation through viral integration) are eliminated.

  20. Mutagenicity of hydroxyurea in lymphocytes from patients with sickle cell disease

    Directory of Open Access Journals (Sweden)

    Khayat André Salim


    Full Text Available Hydroxyurea is commonly used in the treatment of myeloproliferative diseases and in patients with sickle cell disease (SCD. The use of this antineoplastic agent in patients with SCD is justified because of the drug's ability to increase fetal hemoglobin levels, thereby decreasing the severity of SCD. However, high doses or prolonged treatment with hydroxyurea can be cytotoxic or genotoxic for these patients, with an increased risk of developing acute leukemia. This danger can be avoided by monitoring the lymphocytes of patients treated with hydroxyurea. Cytogenetic tests are important endpoints for monitoring the physiological effects of physical and chemical agents, including drugs. In this work, we assessed the genotoxicity of hydroxyurea in short-term cultures of lymphocytes from SCD patients. Hydroxyurea was not cytotoxic or genotoxic at the concentrations tested in the G2 phase of the cell cycle. These results support the use of hydroxyurea in the treatment of SCD, although further work is necessary to understand the effects of this drug in vivo.

  1. Molecular cloning and characterization of lymphocyte cell kinase from humphead snapper (Lutjanus sanguineus). (United States)

    Huang, Y; Cai, J; Wang, B; Tang, J-F; Jian, J-C; Wu, Z-H; Gan, Z; Lu, Y-S


    Lymphocyte cell kinase (LCK) belongs to the Src family of tyrosine kinases, which involves in the proliferation control of lymphocytes. In this study, we cloned the LCK gene of humphead snapper (Lutjanus sanguineus) (designed as LsLCK). Sequence analysis showed that the full-length cDNA of LsLCK was 2279 bp, contained a 1506-bp open reading frame (ORF), encoding a polypeptide of 501 amino acids. The deduced amino acid possessed the typical structural features of known LCK proteins, including four Src homology (SH) domains arranged as the SH1 domain followed by a regulatory C-terminal tail (COOH-domain), SH2 and SH3 adapter domains and SH4 domain which required for membrane attachment and CD4/CD8 binding. Fluorescent quantitative real-time PCR analysis indicated that LsLCK transcripts were expressed mainly in thymus, spleen and head kidney in healthy fish. Moreover, the mRNA expressions in these tissues were significantly up-regulated after challenge with Vibrio harveyi. The results of immunohistochemistry showed that LsLCK protein localized distinctly in cytoplasm of cell in thymus, spleen and head kidney. Taken together, these findings indicated that LsLCK may play an important role in the immune response of humphead snapper against bacterial infection.

  2. High susceptibility of activated lymphocytes to oxidative stress-induced cell death

    Directory of Open Access Journals (Sweden)

    Giovanna R. Degasperi


    Full Text Available The present study provides evidence that activated spleen lymphocytes from Walker 256 tumor bearing rats are more susceptible than controls to tert-butyl hydroperoxide (t-BOOH-induced necrotic cell death in vitro. The iron chelator and antioxidant deferoxamine, the intracellular Ca2+ chelator BAPTA, the L-type Ca2+ channel antagonist nifedipine or the mitochondrial permeability transition inhibitor cyclosporin A, but not the calcineurin inhibitor FK-506, render control and activated lymphocytes equally resistant to the toxic effects of t-BOOH. Incubation of activated lymphocytes in the presence of t-BOOH resulted in a cyclosporin A-sensitive decrease in mitochondrial membrane potential. These results indicate that the higher cytosolic Ca2+ level in activated lymphocytes increases their susceptibility to oxidative stress-induced cell death in a mechanism involving the participation of mitochondrial permeability transition.O presente estudo demonstra que linfócitos ativados de baço de ratos portadores do tumor de Walker 256 são mais susceptíveis à morte celular necrótica induzida por tert-butil hidroperóxido (t-BOOH in vitro quando comparados aos controles. O quelante de ferro e antioxidante deferoxamina, o quelante intracelular de Ca2+ BAPTA, o antagonista de canal de Ca2+ nifedipina ou o inibidor da transição de permeabilidade mitocondrial ciclosporina-A, mas não o inibidor de calcineurina FK-506, inibiram de maneira similar a morte celular induzida por t-BOOH em linfócitos ativados e controles. Os linfócitos ativados apresentaram redução do potencial de membrana mitocondrial induzida por t-BOOH num mecanismo sensível a ciclosporina-A. Nossos resultados indicam que o aumento da concentração de Ca2+ citosólico em linfócitos ativados aumenta a susceptibilidade dos mesmos à morte celular induzida por estresse oxidativo, num mecanismo envolvendo a participação do poro de transição de permeabilidade mitocondrial.

  3. Autoantigenic targets of B-cell receptors derived from chronic lymphocytic leukemias bind to and induce proliferation of leukemic cells. (United States)

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


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

  4. Investigation of the selenium metabolism in cancer cell lines

    DEFF Research Database (Denmark)

    Lunøe, Kristoffer; Gabel-Jensen, Charlotte; Stürup, Stefan


    incubated with cells for 24 h and the induction of cell death was measured using flow cytometry. The amounts of total selenium in cell medium, cell lysate and the insoluble fractions was determined by ICP-MS. Speciation analysis of cellular fractions was performed by reversed phase, anion exchange and size......The aim of this work was to compare different selenium species for their ability to induce cell death in different cancer cell lines, while investigating the underlying chemistry by speciation analysis. A prostate cancer cell line (PC-3), a colon cancer cell line (HT-29) and a leukaemia cell line...... exclusion chromatography and ICP-MS detection. The selenium compounds exhibited large differences in their ability to induce cell death in the three cell lines and the susceptibilities of the cell lines were different. Full recovery of selenium in the cellular fractions was observed for all Se compounds...

  5. SOCS5 is expressed in primary B and T lymphoid cells but is dispensable for lymphocyte production and function

    DEFF Research Database (Denmark)

    Brender, Christine; Columbus, Ruth; Metcalf, Donald


    Suppressors of cytokine signaling (SOCSs) are key regulators of cytokine-induced responses in hematopoietic as well as nonhematopoietic cells. SOCS1 and SOCS3 have been shown to modulate T-cell responses, whereas the roles of other SOCS family members in the regulation of lymphocyte function...... are less clear. Here, we report the generation of mice with a targeted disruption of the Socs5 gene. Socs5(-/-) mice were born in a normal Mendelian ratio and were healthy and fertile. We found that SOCS5 is expressed in primary B and T cells in wild-type mice. However, no abnormalities in the lymphocyte...... to be dispensable for the regulation of lymphocyte function....

  6. At High Levels, Constitutively Activated STAT3 Induces Apoptosis of Chronic Lymphocytic Leukemia Cells. (United States)

    Rozovski, Uri; Harris, David M; Li, Ping; Liu, Zhiming; Wu, Ji Yuan; Grgurevic, Srdana; Faderl, Stefan; Ferrajoli, Alessandra; Wierda, William G; Martinez, Matthew; Verstovsek, Srdan; Keating, Michael J; Estrov, Zeev


    In chronic lymphocytic leukemia (CLL), the increment in PBLs is slower than the expected increment calculated from the cells' proliferation rate, suggesting that cellular proliferation and apoptosis are concurrent. Exploring this phenomenon, we found overexpression of caspase-3, higher cleaved poly (ADP-ribose) polymerase levels (p < 0.007), and a higher apoptosis rate in cells from patients with high counts compared with cells from patients with low counts. Although we previously found that STAT3 protects CLL cells from apoptosis, STAT3 levels were significantly higher in cells from patients with high counts than in cells from patients with low counts. Furthermore, overexpression of STAT3 did not protect the cells. Rather, it upregulated caspase-3 and induced apoptosis. Remarkably, putative STAT3 binding sites were identified in the caspase-3 promoter, and a luciferase assay, chromatin immunoprecipitation, and an EMSA revealed that STAT3 activated caspase-3 However, caspase-3 levels increased only when STAT3 levels were sufficiently high. Using chromatin immunoprecipitation and EMSA, we found that STAT3 binds with low affinity to the caspase-3 promoter, suggesting that at high levels, STAT3 activates proapoptotic mechanisms and induces apoptosis in CLL cells.

  7. Derivation of induced pluripotent stem cells from human peripheral blood T lymphocytes.

    Directory of Open Access Journals (Sweden)

    Matthew E Brown

    Full Text Available Induced pluripotent stem cells (iPSCs hold enormous potential for the development of personalized in vitro disease models, genomic health analyses, and autologous cell therapy. Here we describe the generation of T lymphocyte-derived iPSCs from small, clinically advantageous volumes of non-mobilized peripheral blood. These T-cell derived iPSCs ("TiPS" retain a normal karyotype and genetic identity to the donor. They share common characteristics with human embryonic stem cells (hESCs with respect to morphology, pluripotency-associated marker expression and capacity to generate neurons, cardiomyocytes, and hematopoietic progenitor cells. Additionally, they retain their characteristic T-cell receptor (TCR gene rearrangements, a property which could be exploited for iPSC clone tracking and T-cell development studies. Reprogramming T-cells procured in a minimally invasive manner can be used to characterize and expand donor specific iPSCs, and control their differentiation into specific lineages.

  8. Augmented lymphocyte expansion from solid tumors with engineered cells for costimulatory enhancement. (United States)

    Friedman, Kevin M; Devillier, Laura E; Feldman, Steven A; Rosenberg, Steven A; Dudley, Mark E


    Treatment of patients with adoptive T-cell therapy requires expansion of unique tumor-infiltrating lymphocyte (TIL) cultures from single-cell suspensions processed from melanoma biopsies. Strategies which increase the expansion and reliability of TIL generation from tumor digests are necessary to improve access to TIL therapy. Previous studies have evaluated artificial antigen presenting cells for their antigen-specific and costimulatory properties. We investigated engineered cells for costimulatory enhancement (ECCE) consisting of K562 cells that express 4-1BBL in the absence of artificial antigen stimulation. ECCE accelerated TIL expansion and significantly improved TIL numbers (P=0.001) from single-cell melanoma suspensions. TIL generated with ECCE contain significantly more CD8CD62L and CD8CD27 T cells then comparable interleukin-2-expanded TIL and maintained antitumor reactivity. Moreover, ECCE improved TIL expansion from nonmelanoma-cell suspensions similar to that seen with melanoma tumors. These data demonstrate that the addition of ECCE to TIL production will enable the treatment of patients that are ineligible using current methods.

  9. Adoptive transfer of MART-1 T cell receptor transgenic lymphocytes and dendritic cell vaccination in patients with metastatic melanoma (United States)

    Chodon, Thinle; Comin-Anduix, Begonya; Chmielowski, Bartosz; Koya, Richard C; Wu, Zhongqi; Auerbach, Martin; Ng, Charles; Avramis, Earl; Seja, Elizabeth; Villanueva, Arturo; McCannel, Tara A.; Ishiyama, Akira; Czernin, Johannes; Radu, Caius G.; Wang, Xiaoyan; Gjertson, David W.; Cochran, Alistair J.; Cornetta, Kenneth; Wong, Deborah J.L.; Kaplan-lefko, Paula; Hamid, Omid; Samlowski, Wolfram; Cohen, Peter A.; Daniels, Gregory A.; Mukherji, Bijay; Yang, Lili; Zack, Jerome A.; Kohn, Donald B.; Heath, James R.; Glaspy, John A.; Witte, Owen N.; Baltimore, David; Economou, James S.; Ribas, Antoni


    Purpose It has been demonstrated that large numbers of tumor-specific T cells for adoptive cell transfer (ACT) can be manufactured by retroviral genetic engineering of autologous peripheral blood lymphocytes and expanding them over several weeks. In mouse models, this therapy is optimized when administered with dendritic cell (DC) vaccination. We developed a short one-week manufacture protocol to determine the feasibility, safety and antitumor efficacy of this double cell therapy. Experimnetal Design A clinical trial (NCT00910650) adoptively transferring MART-1 T cell receptor (TCR) transgenic lymphocytes together with MART-1 peptide pulsed DC vaccination in HLA-A2.1 patients with metastatic melanoma. Autologous TCR transgenic cells were manufactured in 6 to 7 days using retroviral vector gene transfer, and re-infused with (n = 10) or without (n = 3) prior cryopreservation. Results 14 patients with metastatic melanoma were enrolled and nine out of 13 treated patients (69%) showed evidence of tumor regression. Peripheral blood reconstitution with MART-1-specific T cells peaked within two weeks of ACT indicating rapid in vivo expansion. Administration of freshly manufactured TCR transgenic T cells resulted in a higher persistence of MART-1-specific T cells in the blood as compared to cryopreserved. Evidence that DC vaccination could cause further in vivo expansion was only observed with ACT using non-cryopreserved T cells. Conclusion Double cell therapy with ACT of TCR engineered T cells with a very short ex vivo manipulation and DC vaccines is feasible and results in antitumor activity, but improvements are needed to maintain tumor responses. PMID:24634374

  10. A human follicular lymphoma B cell line hypermutates its functional immunoglobulin genes in vitro. (United States)

    Wu, H; Pelkonen, E; Knuutila, S; Kaartinen, M


    The functional immunoglobulin (Ig) genes of B lymphocytes undergo somatic mutations during immune responses. These mutations modify the antigen binding site of the immunoglobulins, thereby enhancing the average affinity of the antibodies produced. The molecular mechanism underlying these B cell hypermutations remains unresolved, partly because it is difficult to grow normal B cells in long-term cell cultures and because there is no suitable transformed or malignant B cell line which generates mutations in its immunoglobulin genes in vitro. Here, we show that the recently established follicular lymphoma line HF-1.3.4 generates somatic hypermutations in vitro at a high frequency of 0.7 x 10(-6) mutations per base pair per generation in standard cell cultures (RPMI 1640 + 5% fetal calf serum). This shows for the first time that B cell hypermutation can occur without T cells or T cell factors. The mutation frequency increased approximately tenfold to 1 x 10(-5) mutations/base pair/generation with B cell-specific growth factors (interleukins-2 and -4 and three antibodies stimulatory to HF-1.3.4 cells). This HF-1.3.4 lymphoma line may help to elucidate the molecular mechanism of Ig gene hypermutation.

  11. Protective effects of hesperidin against genotoxicity induced by {sup 99m}Tc-MIBI in human cultured lymphocyte cells

    Energy Technology Data Exchange (ETDEWEB)

    Hosseinimehr, Seyed Jalal [Department of Radiopharmacy, Faculty of Pharmacy and Traditional and Complementary Medicine Research Center, Mazandaran University of Medical Sciences, Sari (Iran, Islamic Republic of)], E-mail:; Ahmadi, Amirhossein [Department of Radiopharmacy, Faculty of Pharmacy and Traditional and Complementary Medicine Research Center, Mazandaran University of Medical Sciences, Sari (Iran, Islamic Republic of); Beiki, Davood [Research Institute for Nuclear Medicine, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Habibi, Emran [Department of Radiopharmacy, Faculty of Pharmacy and Traditional and Complementary Medicine Research Center, Mazandaran University of Medical Sciences, Sari (Iran, Islamic Republic of); Mahmoudzadeh, Aziz [Laboratory of Cytogenetics, Novin Radiation Institute, Tehran (Iran, Islamic Republic of)


    Introduction: Radiopharmaceuticals have been widely used as nuclear tracers for myocardial perfusion imaging. The purpose of this study was to investigate the radioprotective effects of hesperidin as a flavonoid which protects against the genotoxic effects of {sup 99m}Tc-MIBI in human cultured lymphocytes. Methods: Whole blood samples from human volunteers were incubated with hesperidin at doses of 10, 50 and 100 {mu}mol. After 1 h of incubation, the lymphocytes were incubated with {sup 99m}Tc-MIBI (200 {mu}Ci/2 ml) for 3 h. The lymphocyte cultures were then mitogenically stimulated to allow for evaluation of the number of micronuclei in cytokinesis-blocked binucleated cells. Results: Incubation of lymphocytes with {sup 99m}Tc-MIBI at this high dose induces additional genotoxicity and shown by increases in micronuclei frequency in human lymphocytes. Hesperidin at these doses significantly reduced the micronuclei frequency in cultured lymphocytes. The maximum protective effect and greatest decrease in micronuclei frequency occurred when cultures were incubated with a 100-{mu}mol dose of 65% hesperidin. Conclusion: This study has important implications for patients undergoing nuclear medicine procedures. The results indicate a protective role for hesperidin against the genetic damage and side effects induced by radiopharmaceutical administration.

  12. A bioinformatics analysis of the cell line nomenclature. (United States)

    Sarntivijai, Sirarat; Ade, Alexander S; Athey, Brian D; States, David J


    Cell lines are used extensively in biomedical research, but the nomenclature describing cell lines has not been standardized. The problems are both linguistic and experimental. Many ambiguous cell line names appear in the published literature. Users of the same cell line may refer to it in different ways, and cell lines may mutate or become contaminated without the knowledge of the user. As a first step towards rationalizing this nomenclature, we created a cell line knowledgebase (CLKB) with a well-structured collection of names and descriptive data for cell lines cultured in vitro. The objectives of this work are: (i) to assist users in extracting useful information from biomedical text and (ii) to highlight the importance of standardizing cell line names in biomedical research. This CLKB contains a broad collection of cell line names compiled from ATCC, Hyper CLDB and MeSH. In addition to names, the knowledgebase specifies relationships between cell lines. We analyze the use of cell line names in biomedical text. Issues include ambiguous names, polymorphisms in the use of names and the fact that some cell line names are also common English words. Linguistic patterns associated with the occurrence of cell line names are analyzed. Applying these patterns to find additional cell line names in the literature identifies only a small number of additional names. Annotation of microarray gene expression studies is used as a test case. The CLKB facilitates data exploration and comparison of different cell lines in support of clinical and experimental research. The web ontology file for this cell line collection can be downloaded at

  13. Expression of Cyclooxygenase-2 in Ovarian Cancer Cell Lines

    Institute of Scientific and Technical Information of China (English)


    To investigate the expression of cyclooxygenase-2 (COX-2) in ovarian cancer cell lines,RT-PCR and immunocytochemistry were used to detect the expression of COX-2 in 5 ovarian cancer cell lines. The expression of COX-2 mRNA and protein was detected in all 5 cell lines. It is suggested that COX-2 is expressed in ovarian cancer cell lines, which provides a basis for the chemoprevention of ovarian cancer.

  14. Effects of cis-malonato-diammino platinum (II) on P-388 lymphocytic leukemia cell metabolism. (United States)

    Hall, I H; Holshouser, M H; Loeffler, L J


    cis-Malonato-diammino platinum(II) significantly inhibited P-388 lymphocytic leukemia cell proliferation at 10 mg/kg/day. Incorporation studies showed that DNA synthesis was inhibited following in vivo drug therapy. The major inhibitory effects appeared to be on thymidine kinase and dihydrofolate reductase activities and on overall purine synthesis, with marginal effects on DNA polymerase and ribonucleotide reductase activities. In addition to the DNA inhibition, a marked increase in cyclic adenosine 3',5'-monophosphate levels was noted, which correlated with a rapid decrease in histone phosphorylation. Other minor effects of the drug included significant reduction of proteolytic activity, suppression of States 4 and 3 respiration, and an increase in adenosine triphosphatase and acid phosphatase activities of P-388 cells.

  15. In vitro generation of human cytotoxic lymphocytes by virus. Viral glycoproteins induce nonspecific cell-mediated cytotoxicity without release of interferon



    Purified hemagglutinin and fusion glycoproteins of measles virus either in soluble form or inserted in artifical membranes bind to human peripheral blood lymphocytes and induce cell-mediated cytotoxicity (CMC) in a dose-response fashion. Both autologous and heterologous noninfected target cells are lysed in vitro. The expression of CMC is not inhibited by anti-measles virus antibody added to lymphocytes previously exposed to viral glycoproteins. THe killer lymphocytes are Fc receptor positive...

  16. On the ontology based representation of cell lines.

    Directory of Open Access Journals (Sweden)

    Matthias Ganzinger

    Full Text Available Cell lines are frequently used as highly standardized and reproducible in vitro models for biomedical analyses and assays. Cell lines are distributed by cell banks that operate databases describing their products. However, the description of the cell lines' properties are not standardized across different cell banks. Existing cell line-related ontologies mostly focus on the description of the cell lines' names, but do not cover aspects like the origin or optimal growth conditions. The objective of this work is to develop an ontology that allows for a more comprehensive description of cell lines and their metadata, which should cover the data elements provided by cell banks. This will provide the basis for the standardized annotation of cell lines and corresponding assays in biomedical research. In addition, the ontology will be the foundation for automated evaluation of such assays and their respective protocols in the future. To accomplish this, a broad range of cell bank databases as well as existing ontologies were analyzed in a comprehensive manner. We identified existing ontologies capable of covering different aspects of the cell line domain. However, not all data fields derived from the cell banks' databases could be mapped to existing ontologies. As a result, we created a new ontology called cell culture ontology (CCONT integrating existing ontologies where possible. CCONT provides classes from the areas of cell line identification, origin, cell line properties, propagation and tests performed.

  17. Radiosensitivity of Human Melanoma Cell Lines

    Energy Technology Data Exchange (ETDEWEB)

    Bergoc, R. M.; Medina, V.; Cricco, G.; Mohamed, N.; Martin, G.; Nunez, M.; Croci, M.; Crescenti, E. J.; Rivera, E. S.


    Cutaneous melanoma is a skin cancer resulting from the malign transformation of skin-pigment cells, the melanocytes. The radiotherapy, alone or in combination with other treatment, is an important therapy for this disease. the objective of this paper was to determine in vitro the radiosensitivity of two human melanoma cell lines with different metastatic capability: WM35 and MI/15, and to study the effect of drugs on radiobiological parameters. The Survival Curves were adjusted to the mathematical Linear-quadratic model using GrapsPad Prism software. Cells were seeded in RPMI medium (3000-3500 cells/flask), in triplicate and irradiated 24 h later. The irradiation was performed using an IBL 437C H Type equipment (189 TBq, 7.7 Gy/min) calibrated with a TLD 700 dosimeter. The range of Doses covered from 0 to 10 Gy and the colonies formed were counted at day 7th post-irradiation. Results obtained were: for WM35, {alpha}=0.37{+-}0.07 Gy''-1 and {beta}=0.06{+-}0.02 Gy''-2, for M1/15m {alpha}=0.47{+-}0.03 Gy''-1 and {beta}=0.06{+-}0.01 Gy''-2. The {alpha}/{beta} values WM35: {alpha}/{beta} values WM35: {alpha}/{beta}=6.07 Gy and M1/15: {alpha}/{beta}{sub 7}.33 Gy were similar, independently of their metastatic capabillity and indicate that both lines exhibit high radioresistance. Microscopic observation of irradiated cells showed multinuclear cells with few morphologic changes non-compatible with apoptosis. By means of specific fluorescent dyes and flow cytometry analysis we determined the intracellular levels of the radicals superoxide and hydrogen peroxide and their modulation in response to ionizing radiation. The results showed a marked decreased in H{sub 2}O{sub 2} intracellular levels with a simultaneous increase in superoxide that will be part of a mechanism responsible for induction of cell radioresistance. This response triggered by irradiated cells could not be abrogated by different treatments like histamine or the

  18. The meaning and relevance of B-cell receptor structure and function in chronic lymphocytic leukemia. (United States)

    Stevenson, Freda K; Forconi, Francesco; Packham, Graham


    The B-cell receptor (BCR) is of critical importance for normal B cells and for the majority of B-cell malignancies, especially chronic lymphocytic leukemia (CLL). The two major subsets of CLL are biologically distinct, being derived from B cells at different stages of differentiation and carrying unmutated (U-CLL) or mutated (M-CLL) IGHV genes. U-CLL, which has a poorer prognosis, often has relatively conserved (stereotypic) IGHV-HD-HJ sequences, indicative of interaction with large (super)antigens and similar to those in normal naive innate B cells. Conserved sequences are less evident in M-CLL, in keeping with its postfollicular origin. However, both subsets exhibit features of chronic antigen exposure in tissue sites, with local proliferative events, but also downregulation of surface immunoglobulin M but not surface immunoglobulin D, a characteristic of normal anergic B cells. BCR-mediated anergy can spread to other receptors such as CXCR4. Circulating CLL cells retain a shadow of tissue-based events that can reverse over time, but the overall extent of anergy is greater in M-CLL. Despite this stereotypic variety and more genomic complexity, BCR-mediated responses in vitro appear relatively homogeneous in U-CLL, but M-CLL is more heterogeneous. The differential balance between antigen-induced proliferation or anergy is the likely determinant of clinical behavior and possibly of response to kinase inhibitors.

  19. High densities of serotonin and peptide YY cells in the colon of patients with lymphocytic colitis

    Institute of Scientific and Technical Information of China (English)

    Magdy El-Salhy; Doris Gundersen; Jan Gunnar Hatlebakk; Trygve Hausken


    AIM:To investigate colonic endocrine cells in lymphocytic colitis (LC) patients.METHODS:Fifty-seven patients with LC were included.These patients were 41 females and 16 males,with an average age of 49 years (range 19-84 years).Twenty-seven subjects that underwent colonoscopy with biopsies were used as controls.These subjects underwent colonoscopy because of gastrointestinal bleeding or health worries,where the source of bleeding was identified as haemorrhoids or angiodysplasia.They were 19 females and 8 males with an average age of 49 years (range 18-67 years).Biopsies from the right and left colon were obtained from both patients and controls during colonoscopy.Biopsies were fixed in 4% buffered paraformaldehyde,embedded in paraffin and cut into 5 μm-thick sections.The sections immunostained by the avidin-biotin-complex method for serotonin,peptide YY (PYY),pancreatic polypeptide (pP)enteroglucagon and somatostatin cells.The cell densities were quantified by computerised image analysis using Olympus software.RESULTS:The colon of both the patient and the control subjects were macroscopically normal.Histopathological examination of colon biopsies from controis revealed normal histology.All patients fulfilled the diagnosis criteria required for of LC:an increase in intraepithelial lymphocytes (> 20 lymphocytes/100 epithelial cells) and surface epithelial damage with increased lamina propria plasma cells and absent or minimal crypt architectural distribution.In the colon of both patients and control subjects,serotonin-,PYY-,PP-,enteroglucagon-and somatostatin-immunoreactive cells were primarily located in the upper part of the crypts of Lieberkühn.These cells were basket-or flask-shaped.There was no statistically significant difference between the right and left colon in controls with regards to the densities of serotonin-and PYY-immunoreactive cells (P =0.9 and 0.1,respectively).Serotonin cell density in the right colon in controls was 28.9 ± 1.8 and in LC

  20. Study on T lymphocyte subsets and NK cells in patients with Graves disease combined with type 2 diabetes

    Institute of Scientific and Technical Information of China (English)

    魏枫; 杜婧; 苏秀兰; 乌兰; 王津京; 霍晓静


    Objective To investigate changes in T lymphocyte subsets and NK cells in patients with simple Graves' disease(GD)and Graves' disease combined with type 2 diabetes mellitus(GD/T2DM).Methods Fifteen cases of GD/T2DM were selected from our hospital from November 2001 to November 2004.Before and after therapy thyroid function,thyroglobulin antibody(TGA),thyroid microsomal antibody(TMA)and blood glucose level were measured,and T lymphocyte subsets(CD3,CD4,CD8,CD4/CD8)and NK cells(CD56)were measured by immunofluo...

  1. Autologous Peripheral Blood Stem Cell Transplant Followed by Donor Bone Marrow Transplant in Treating Patients With High-Risk Hodgkin Lymphoma, Non-Hodgkin Lymphoma, Multiple Myeloma, or Chronic Lymphocytic Leukemia (United States)


    B-Cell Prolymphocytic Leukemia; Plasma Cell Leukemia; Progression of Multiple Myeloma or Plasma Cell Leukemia; Recurrent Adult Hodgkin Lymphoma; Recurrent Adult Non-Hodgkin Lymphoma; Recurrent Childhood Hodgkin Lymphoma; Recurrent Childhood Non-Hodgkin Lymphoma; Recurrent Chronic Lymphocytic Leukemia; Recurrent Plasma Cell Myeloma; Recurrent Small Lymphocytic Lymphoma; Refractory Childhood Hodgkin Lymphoma; Refractory Chronic Lymphocytic Leukemia; Refractory Non-Hodgkin Lymphoma; Refractory Plasma Cell Myeloma; Refractory Small Lymphocytic Lymphoma; T-Cell Prolymphocytic Leukemia; Waldenstrom Macroglobulinemia

  2. FMC46, a cell protrusion-associated leukocyte adhesion molecule-1 epitope on human lymphocytes and thymocytes. (United States)

    Pilarski, L M; Turley, E A; Shaw, A R; Gallatin, W M; Laderoute, M P; Gillitzer, R; Beckman, I G; Zola, H


    In this report, we describe a 76-kDa glycoprotein recognized by mAb FMC46 that, by virtue of its concentration on cell protrusions involved in motility, may be important in lymphoid cell locomotion. FMC46 detects an epitope of the leukocyte adhesion molecule-1 (LAM-1), a member of the selecting family (LAM-1, Endothelial Leukocyte Adhesion Molecular-1 (ELAM-1), and Granule Membrane Protein-140 (GMP-140), that is expressed on LAM-1-transfected cell lines, is a glycosylation epitope based on its loss after culture in tunicamycin, and is closely related to the LAM-1.2 epitope. FMC46 is expressed at high density on the majority of CD45RA+ and CD45RO+ peripheral blood T cells (60 to 70%) and on a subset of thymocytes that includes the multinegative CD3- CD4- CD8- progenitor cells (100% FMC46hi) and the CD45R0- presumptive thymic generative lineage (70% FMC46hi). It appears at reduced density and frequency on CD45RA- thymocytes (50% FMC46lo), comprised mainly of death-committed thymocytes. Among thymic subsets defined by expression of CD4 and/or CD8, FMC46 is expressed at high density predominantly on a subset of single-positive cells and not on double-positive cells. These results suggest a fundamental role for LAM-1 in thymic development, with a high density preferentially expressed on cells involved in thymic generative processes and a low density on cells progressing to intrathymic death. A major subset of peripheral blood B cells and thymic B cells also express FMC46. Immunohistochemistry on frozen sections indicated strong staining in splenic follicles and around blood vessels, staining of the thymic medulla and subcapsular areas, and staining of the mantle zone of germinal centers of the lymph node. FMC46+ lymphocytes accumulated along high endothelial venules in the lymph node. On locomoting multinegative thymocytes, FMC46 is concentrated on the leading tip of extended processes, on pseudopods, and on ruffles, unlike the distribution of either CD44 or TQ1 (LAM 1

  3. Toxicological effects of ultraviolet radiation on lymphocyte cells with different manganese superoxide dismutase Ala16Val polymorphism genotypes. (United States)

    dos Santos Montagner, Greice Franciele Feyh; Sagrillo, Michele; Machado, Michel Mansur; Almeida, Renata Chequeller; Mostardeiro, Clarice Pinheiro; Duarte, Marta Maria Medeiros Frescura; da Cruz, Ivana Beatrice Mânica


    The aim of this study was to investigate whether there is a differential response of lymphocytes from healthy MnSOD genotype subjects to oxidative stress. We used UV radiation as a toxic agent due to its genotoxic effects associated with chromosome aberrations caused by breaks in the DNA strands. Cellular growth rate, cell viability, mitotic index, chromosomal instability and biomarkers of oxidative metabolism were analysed in lymphocyte cells from healthy adults with different Ala16Val MnSOD polymorphisms that produce tree genotypes: AA, VV and AV. We found a differential response to UV exposure in cultures of lymphocyte cells from Ala16Val genotype donors. In general, AA cell cultures presented higher viability and mitotic index and lower TBARS levels than VV and AV cells for both the control and UV exposure groups. However, when we compared the DNA damage among the three genotypes, AA lymphocyte cells presented the highest damage from UV exposure. These data suggest that the Ala16Val polymorphism affects the response of cellular oxidative metabolism in different ways.

  4. An in vitro study of liposomal curcumin: stability, toxicity and biological activity in human lymphocytes and Epstein-Barr virus-transformed human B-cells. (United States)

    Chen, Changguo; Johnston, Thomas D; Jeon, Hoonbae; Gedaly, Roberto; McHugh, Patrick P; Burke, Thomas G; Ranjan, Dinesh


    Curcumin is a multi-functional and pharmacologically safe natural agent. Used as a food additive for centuries, it also has anti-inflammatory, anti-virus and anti-tumor properties. We previously found that it is a potent inhibitor of cyclosporin A (CsA)-resistant T-cell co-stimulation pathway. It inhibits mitogen-stimulated lymphocyte proliferation, NFkappaB activation and IL-2 signaling. In spite of its safety and efficacy, the in vivo bioavailability of curcumin is poor, and this may be a major obstacle to its utility as a therapeutic agent. Liposomes are known to be excellent carriers for drug delivery. In this in vitro study, we report the effects of different liposome formulations on curcumin stability in phosphate buffered saline (PBS), human blood, plasma and culture medium RPMI-1640+10% FBS (pH 7.4, 37 degrees C). Liposomal curcumin had higher stability than free curcumin in PBS. Liposomal and free curcumin had similar stability in human blood, plasma and RPMI-1640+10% FBS. We looked at the toxicity of non-drug-containing liposomes on (3)H-thymidine incorporation by concanavalin A (Con A)-stimulated human lymphocytes, splenocytes and Epstein-Barr virus (EBV)-transformed human B-cell lymphoblastoid cell line (LCL). We found that dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) were toxic to the tested cells. However, addition of cholesterol to the lipids at DMPC:DMPG:cholesterol=7:1:8 (molar ratio) almost completely eliminated the lipid toxicity to these cells. Liposomal curcumin had similar or even stronger inhibitory effects on Con A-stimulated human lymphocyte, splenocyte and LCL proliferation. We conclude that liposomal curcumin may be useful for intravenous administration to improve the bioavailability and efficacy, facilitating in vivo studies that could ultimately lead to clinical application of curcumin.

  5. Cell surface phenotype of cytolytic T lymphocyte precursors in aged nude mice. (United States)

    Maryanski, J L; MacDonald, H R; Sordat, B; Cerottini, J C


    The cell surface phenotype of cytolytic T lymphocyte precursors (CTL-P) in congenitally athymic C57BL/6 nu/nu mice has been investigated. CTL-P were detected and quantitated in a limited dilution mixed leukocyte microculture assay system supplemented with interleukin 2. Minimal estimates of the frequency of CTL-P among nylon wool passed (NWP) nude spleen cells were obtained following elimination of Thy-1-bearing or Lyt-2-bearing cells with monoclonal antibodies plus complement. Alternatively, NWP spleen cells bearing Thy-1 or Lyt-2 were positively selected on a cell sorter and assayed for CTL-P frequency. Both positive and negative selection techniques demonstrated that essentially all (greater than 98%) CTL-P in NWP nude spleen expressed Thy-1 and that the majority (80-90%) expressed Lyt-2. In control NWP spleen cells from normal C57BL/6 mice, greater than 98% of CTL-P were positive for both Thy-1 and Lyt-2. These data demonstrate that most functional alloreactive CTL-P developing in the apparent absence of thymic influence already express both Thy-1 and Lyt-2 prior to exposure to antigen.

  6. Cytotoxic T-lymphocyte-mediated killing of human pancreatic islet cells in vitro. (United States)

    Campbell, Peter D; Estella, Eugene; Dudek, Nadine L; Jhala, Gaurang; Thomas, Helen E; Kay, Thomas W H; Mannering, Stuart I


    Cytotoxic T lymphocytes (CTL) are believed to play an essential role in beta-cell destruction leading to development of type 1 diabetes and allogeneic islet graft failure. We aimed to identify the mechanisms used by CTL to kill human beta cells. CTL clones that recognize epitopes from influenza virus and Epstein-Barr virus restricted by human leukocyte antigen (HLA)-A0201 and -B0801, respectively, were used to investigate the susceptibility of human beta cells to CTL. In a short-term (5-hour) assay, CTL killed human islet cells of the appropriate major histocompatibility complex (MHC) class I type that had been pulsed with viral peptides. Killing was increased by pretreating islets with interferon gamma that increases MHC class I on target cells. Killing was abolished by incubation of CTL with the perforin inhibitor concanamycin A. The Fas pathway did not contribute to killing because blocking with neutralizing anti-Fas ligand antibody did not significantly reduce beta-cell killing. In conclusion, we report a novel way of investigating the interaction between CTL and human islets. Human islets were rapidly killed in vitro by MHC class I-restricted CTL predominantly by the granule exocytosis pathway.

  7. HLA-G inhibits xenogenetic cytotoxicity mediated by human NK cells and T lymphocytes against PECs

    Institute of Scientific and Technical Information of China (English)


    In order to investigate whether the non-classi-cal HLA-G classⅠmolecule protects the porcine endothelial cells (PECs) from the lysis mediated by human immune cells in pig to human discordant xenotransplantation, we have cloned HLA-G cDNA from a human placenta by RT-PCR. Mammalian expression vector, pEFG-neo, was constructed by insertion of HLA-G cDNA in pEF-neo. We obtained efficiently expressed PECs by stable transfection. Cytotoxicity assay showed that overexpression of HLA-G on PECs was sufficient to inhibit human NK-92 cell lysis. The level of lysis was equal to or less than that of the lysis of human umbilical vein endothelial cells mediated by human NK-92 cells. It also indicated that HLA-G inhibited the lysis of PECs mediated by xeno-antigen specific T lymphocytes. The reduction of lysis ranged between 59.1% and 88.9%. These findings suggest that the transgenic approach to overexpress HLA-G is believed to be a new immunotherapy in overcoming the immune rejections in xenotransplantion, including delayed xenograft rejection and cell-mediated rejection.

  8. Early interferon-γ production in human lymphocyte subsets in response to nontyphoidal Salmonella demonstrates inherent capacity in innate cells.

    Directory of Open Access Journals (Sweden)

    Tonney S Nyirenda

    Full Text Available BACKGROUND: Nontyphoidal Salmonellae frequently cause life-threatening bacteremia in sub-Saharan Africa. Young children and HIV-infected adults are particularly susceptible. High case-fatality rates and increasing antibiotic resistance require new approaches to the management of this disease. Impaired cellular immunity caused by defects in the T helper 1 pathway lead to intracellular disease with Salmonella that can be countered by IFNγ administration. This report identifies the lymphocyte subsets that produce IFNγ early in Salmonella infection. METHODOLOGY: Intracellular cytokine staining was used to identify IFNγ production in blood lymphocyte subsets of ten healthy adults with antibodies to Salmonella (as evidence of immunity to Salmonella, in response to stimulation with live and heat-killed preparations of the D23580 invasive African isolate of Salmonella Typhimurium. The absolute number of IFNγ-producing cells in innate, innate-like and adaptive lymphocyte subpopulations was determined. PRINCIPAL FINDINGS: Early IFNγ production was found in the innate/innate-like lymphocyte subsets: γδ-T cells, NK cells and NK-like T cells. Significantly higher percentages of such cells produced IFNγ compared to adaptive αβ-T cells (Student's t test, P<0.001 and ≤0.02 for each innate subset compared, respectively, with CD4(+- and CD8(+-T cells. The absolute numbers of IFNγ-producing cells showed similar differences. The proportion of IFNγ-producing γδ-T cells, but not other lymphocytes, was significantly higher when stimulated with live compared with heat-killed bacteria (P<0.0001. CONCLUSION/SIGNIFICANCE: Our findings indicate an inherent capacity of innate/innate-like lymphocyte subsets to produce IFNγ early in the response to Salmonella infection. This may serve to control intracellular infection and reduce the threat of extracellular spread of disease with bacteremia which becomes life-threatening in the absence of protective antibody

  9. Oropharyngeal malignant epithelial cell, lymphocyte and macrophage CD44 surface receptors for hyaluronate are expressed in sustained EBV infection: immunohistochemical data and EBV DNA tissue indices. (United States)

    Groma, Valerija; Kazanceva, Anna; Nora-Krukle, Zaiga; Murovska, Modra


    The role of CD44 in Epstein-Barr virus (EBV)-related epithelial tumors is poorly understood. We studied the expression of CD44 in EBV infection in patients with oral squamous cell carcinoma (SCC) and nasopharyngeal carcinoma (NPC) and measured the EBV DNA. Whole blood, plasma and tissue samples from 8 male and 2 female patients with oral SCC, NPC, salivary gland lymphoepithelioma, normal salivary gland and buccal mucosa were assayed for EBV DNA. Expression of CD44, latent membrane protein (LMP), and labeling of lymphocytes, macrophages and dendritic cells were estimated by immunohistochemistry. Tissue EBV DNA was detected in 7 of 8 cases (87.5%) of oral malignant, benign and border-line lesions. LMP expression levels in tumors varied from absence and minimal to moderate - 50.3, 43.6, 6.0% and 91.1, 6.7, 2.2% for SCC and NPC, respectively. Levels of CD44 positivity in neoplasms were minimal (15.5 and 16.7%), moderate (30.3 and 47.8%), and diffuse (54.2 and 35.5%) for SCC and NPC, respectively, thus deviating from normal oral mucosa revealing heavily stained (100.0%) epithelial contours. CD19-positive B lymphocytes and S100-positive dendritic cells were intermixed with neoplastic cells. Collectively, CD44 mediated signaling may be implicated in EBV infection associated with the pathogenesis of oral SCC and NPC.

  10. Radiation Sensitivity of Human CD34(+) Cells Versus Peripheral Blood T Lymphocytes of Newborns and Adults: DNA Repair and Mutagenic Effects. (United States)

    Vandevoorde, C; Vral, A; Vandekerckhove, B; Philippé, J; Thierens, H


    As hematopoietic stem and progenitor cells (HSPCs) self-renew throughout life, accumulation of genomic alterations can potentially give rise to radiation carcinogenesis. In this study we examined DNA double-strand break (DSB) induction and repair as well as mutagenic effects of ionizing radiation in CD34(+) cells and T lymphocytes from the umbilical cord of newborns. The age dependence of DNA damage repair end points was investigated by comparing newborn T lymphocytes with adult peripheral blood T lymphocytes. As umbilical cord blood (UCB) contains T lymphocytes that are practically all phenotypically immature, we examined the radiation response of separated naive (CD45RA(+)) and memory (CD45RO(+)) T lymphocytes. The number of DNA DSBs was assessed by microscopic scoring of γ-H2AX/53BP1 foci 0.5 h after low-dose radiation exposure, while DNA repair was studied by scoring the number of residual γ-H2AX/53BP1 foci 24 h after exposure. Mutagenic effects were studied by the cytokinesis block micronucleus (CBMN) assay. No significant differences in the number of DNA DSBs induced by low-dose (100-200 mGy) radiation were observed among the three different cell types. However, residual γ-H2AX/53BP1 foci levels 24 h postirradiation were significantly lower in CD34(+) cells compared to newborn T lymphocytes, while newborn T lymphocytes showed significantly higher foci yields than adult T lymphocytes. No significant differences in the level of radiation-induced micronuclei at 2 Gy were observed between CD34(+) cells and newborn T lymphocytes. However, newborn T lymphocytes showed a significantly higher number of micronuclei compared to adult T lymphocytes. These results confirm that CD34(+) cell quiescence promotes mutagenesis after exposure. Furthermore, we can conclude that newborn peripheral T lymphocytes are significantly more radiosensitive than adult peripheral T lymphocytes. Using the results from the comparative study of radiation-induced DNA damage repair end

  11. Quantitative profiling of housekeeping and Epstein-Barr virus gene transcription in Burkitt lymphoma cell lines using an oligonucleotide microarray

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    Niggli Felix K


    Full Text Available Abstract Background The Epstein-Barr virus (EBV is associated with lymphoid malignancies, including Burkitt's lymphoma (BL, and can transform human B cells in vitro. EBV-harboring cell lines are widely used to investigate lymphocyte transformation and oncogenesis. Qualitative EBV gene expression has been extensively described, but knowledge of quantitative transcription is lacking. We hypothesized that transcription levels of EBNA1, the gene essential for EBV persistence within an infected cell, are similar in BL cell lines. Results To compare quantitative gene transcription in the BL cell lines Namalwa, Raji, Akata, Jijoye, and P3HR1, we developed an oligonucleotide microarray chip, including 17 housekeeping genes, six latent EBV genes (EBNA1, EBNA2, EBNA3A, EBNA3C, LMP1, LMP2, and four lytic EBV genes (BZLF1, BXLF2, BKRF2, BZLF2, and used the cell line B95.8 as a reference for EBV gene transcription. Quantitative polymerase chain reaction assays were used to validate microarray results. We found that transcription levels of housekeeping genes differed considerably among BL cell lines. Using a selection of housekeeping genes with similar quantitative transcription in the tested cell lines to normalize EBV gene transcription data, we showed that transcription levels of EBNA1 were quite similar in very different BL cell lines, in contrast to transcription levels of other EBV genes. As demonstrated with Akata cells, the chip allowed us to accurately measure EBV gene transcription changes triggered by treatment interventions. Conclusion Our results suggest uniform EBNA1 transcription levels in BL and that microarray profiling can reveal novel insights on quantitative EBV gene transcription and its impact on lymphocyte biology.

  12. A high rate of telomeric sister chromatid exchange occurs in chronic lymphocytic leukaemia B-cells. (United States)

    Medves, Sandrine; Auchter, Morgan; Chambeau, Laetitia; Gazzo, Sophie; Poncet, Delphine; Grangier, Blandine; Verney, Aurélie; Moussay, Etienne; Ammerlaan, Wim; Brisou, Gabriel; Morjani, Hamid; Géli, Vincent; Palissot, Valérie; Berchem, Guy; Salles, Gilles; Wenner, Thomas


    Cancer cells protect their telomere ends from erosion through reactivation of telomerase or by using the Alternative Lengthening of Telomere (ALT) mechanism that depends on homologous recombination. Chronic lymphocytic leukaemia (CLL) B cells are characterized by almost no telomerase activity, shelterin deregulation and telomere fusions. To characterize telomeric maintenance mechanisms in B-CLL patients, we measured their telomere length, telomerase expression and the main hallmarks of the ALT activity i.e. C-circle concentration, an extra-chromosomal telomere repeat (ECTR), and the level of telomeric sister chromatid exchange (T-SCE) rate. Patients showed relative homogenous telomere length although almost no TERT transcript and nearly no C-circle were evidenced. Nevertheless, compared with normal B cells, B-CLL cells showed an increase in T-SCE rate that was correlated with a strong down-regulation of the topoisomerase III alpha (TOP3A) expression, involved in the dissolution of Holliday Junctions (HJ), together with an increased expression of SLX1A, SLX4, MUS81 and GEN1, involved in the resolution of HJ. Altogether, our results suggest that the telomere maintenance mechanism of B-CLL cells do not preferentially use telomerase or ALT. Rather, the rupture of the dissolvasome/resolvasome balance may increase telomere shuffling that could homogenize telomere length, slowing telomere erosion in this disease.

  13. Tolerogenic dendritic cells for reprogramming of lymphocyte responses in autoimmune diseases. (United States)

    García-González, Paulina; Ubilla-Olguín, Gabriela; Catalán, Diego; Schinnerling, Katina; Aguillón, Juan Carlos


    Dendritic cells (DCs) control immune responses by driving potent inflammatory actions against external and internal threats while generating tolerance to self and harmless components. This duality and their potential to reprogram immune responses in an antigen-specific fashion have made them an interesting target for immunotherapeutic strategies to control autoimmune diseases. Several protocols have been described for in vitro generation of tolerogenic DCs (tolDCs) capable of modulating adaptive immune responses and restoring tolerance through different mechanisms that involve anergy, generation of regulatory lymphocyte populations, or deletion of potentially harmful inflammatory T cell subsets. Recently, the capacity of tolDCs to induce interleukin (IL-10)-secreting regulatory B cells has been demonstrated. In vitro assays and rodent models of autoimmune diseases provide insights to the molecular regulators and pathways enabling tolDCs to control immune responses. Here we review mechanisms through which tolDCs modulate adaptive immune responses, particularly focusing on their suitability for reprogramming autoreactive CD4(+) effector T cells. Furthermore, we discuss recent findings establishing that tolDCs also modulate B cell populations and discuss clinical trials applying tolDCs to patients with autoimmune diseases.

  14. Impaired Lymphocytes Development and Xenotransplantation of Gastrointestinal Tumor Cells in Prkdc-Null SCID Zebrafish Model

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    In Hye Jung


    Full Text Available Severe combined immunodeficiency (SCID mice have widely been used as hosts for human tumor cell xenograft study. This animal model, however, is labor intensive. As zebrafish is largely emerging as a promising model system for studying human diseases including cancer, developing efficient immunocompromised strains for tumor xenograft study are also demanded in zebrafish. Here, we have created the Prkdc-null SCID zebrafish model which provides the stable immune-deficient background required for xenotransplantation of tumor cell. In this study, the two transcription activator-like effector nucleases that specifically target the exon3 of the zebrafish Prkdc gene were used to induce a frame shift mutation, causing a complete knockout of the gene function. The SCID zebrafish showed susceptibility to spontaneous infection, a well-known phenotype found in the SCID mutation. Further characterization revealed that the SCID zebrafish contained no functional T and B lymphocytes which reflected the phenotypes identified in the mice SCID model. Intraperitoneal injection of human cancer cells into the adult SCID zebrafish clearly showed tumor cell growth forming into a solid mass. Our present data show the suitability of using the SCID zebrafish strain for xenotransplantation experiments, and in vivo monitoring of the tumor cell growth in the zebrafish demonstrates use of the animal model as a new platform of tumor xenograft study.

  15. Spontaneous regression of Merkel cell carcinoma in a patient with chronic lymphocytic leukemia: a case report

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    Turk Tamara


    Full Text Available Abstract Introduction Merkel cell carcinoma is a rare and aggressive primary cutaneous neuroendocrine malignant tumor. The tumor has a high rate of local recurrence after surgical removal. Spontaneous regression appears to be relatively common in this rare type of tumor. Case presentation We describe the clinical course, cytological and histological findings of a Merkel cell carcinoma in a 70-year-old Caucasian woman, simultaneously diagnosed with chronic lymphatic leukemia. The tumor showed clinical regression after fine needle aspiration. At primary presentation, the tumor had no apparent leukocyte infiltration, but was completely cleared by T-cell mediated immunity within 3 weeks after fine needle aspiration. Conclusion Fine needle aspiration may have acted as a mechanical trigger involved in the activation of cell-mediated immunity, leading to the clinical and histological regression of the tumor. To the best of our knowledge, this is the first case report of spontaneous regression of Merkel cell carcinoma in a patient with a co-malignancy, that is to say, chronic lymphocytic leukemia.

  16. Inhibitors of XIAP sensitize CD40-activated chronic lymphocytic leukemia cells to CD95-mediated apoptosis (United States)

    Kater, Arnon P.; Dicker, Frank; Mangiola, Massimo; Welsh, Kate; Houghten, Richard; Ostresh, John; Nefzi, Adel; Reed, John C.; Pinilla, Clemencia; Kipps, Thomas J.


    Patients with chronic lymphocytic leukemia (CLL) treated with adenovirus CD154 (Ad-CD154, CD40 ligand [CD40L]) gene therapy experienced rapid reductions in leukemia cell counts and lymph node size associated with the induced expression of Fas (CD95). However, CLL cells initially resist CD95-mediated apoptosis within the first 3 days after CD40 ligation in vitro. Thereafter, they become sensitive, which is associated with the CD40-induced expression of the proapoptotic protein B-cell leukemia 2 homology 3 (BH3) interacting domain death agonist (Bid). We hypothesized that the initial resistance to CD95-mediated apoptosis may be due to the high-level expression of X-linked inhibitor of apoptosis protein (XIAP) by CLL cells. Consistent with this, CLL cells from patients 1 day after treatment with autologous Ad-CD154-transduced CLL cells became sensitive to CD95-mediated apoptosis following treatment with a novel XIAP inhibitor, 1540-14. Similarly, 1540-14 specifically enhanced CD95-mediated apoptosis of CLL cells following CD40 ligation in vitro. Immunoblot analyses demonstrated that treatment with 1540-14 allowed CD40-stimulated CLL cells to experience high-level activation of caspases-8 and -3 and cleavage of poly(adenosine diphosphate [ADP]-ribose) polymerase following CD95 ligation. This study demonstrates that distal apoptosis regulators contribute to the initial resistance of CD40-activated CLL cells to CD95-mediated apoptosis and suggests that XIAP inhibitors might enhance the effectiveness of immune-based treatment strategies that target CD40, such as CD154 gene therapy. (Blood. 2005;106:1742-1748) PMID:15914559

  17. Clinical-Grade Human Multipotent Adult Progenitor Cells Block CD8+ Cytotoxic T Lymphocytes. (United States)

    Plessers, Jeroen; Dekimpe, Emily; Van Woensel, Matthias; Roobrouck, Valerie D; Bullens, Dominique M; Pinxteren, Jef; Verfaillie, Catherine M; Van Gool, Stefaan W


    : MultiStem cells are clinical-grade multipotent adult bone marrow-derived progenitor cells (MAPCs), with extensive replication potential and broader differentiation capacity compared with mesenchymal stem cells. Human MAPCs suppress T-cell proliferation induced by alloantigens and mutually interact with allogeneic natural killer cells. In this study, the interaction between MultiStem and CD8(+) cytotoxic T lymphocytes (CTLs) was addressed for the first time. In an in vitro setting, the immunogenicity of MultiStem, the susceptibility of MultiStem toward CTL-mediated lysis, and its effects on CTL function were investigated. MultiStem was nonimmunogenic for alloreactive CTL induction and was-even after major histocompatibility complex class I upregulation-insensitive to alloantigen-specific CTL-mediated lysis. Furthermore, MultiStem reduced CTL proliferation and significantly decreased perforin expression during the T-cell activation phase. As a consequence, MultiStem dose-dependently impaired the induction of CTL function. These effects of MultiStem were mediated predominantly through contact-dependent mechanisms. Moreover, MultiStem cells considerably influenced the expression of T-cell activation markers CD25, CD69, and human leukocyte antigen-DR. The MultiStem-induced CD8(-)CD69(+) T-cell population displayed a suppressive effect on the induction of CTL function during a subsequent mixed-lymphocyte culture. Finally, the killer activity of activated antigen-specific CTLs during their cytolytic effector phase was also diminished in the presence of MultiStem. This study confirms that these clinical-grade MAPCs are an immune-modulating population that inhibits CTL activation and effector responses and are, consequently, a highly valuable cell population for adoptive immunosuppressive therapy in diseases where damage is induced by CTLs. Because multipotent adult progenitor cells (MAPCs) are among the noteworthy adult mesenchymal stem cell populations for immune

  18. Successful alemtuzumab retreatment in progressive B-cell chronic lymphocytic leukemia: a multicenter survey in 30 patients. (United States)

    Fiegl, Michael; Falkner, Florian; Steurer, Michael; Zojer, Niklas; Hopfinger, Georg; Haslbauer, Ferdinand; Winder, Guntram; Voskova, Daniela; Andel, Johannes; Lang, Alois; Brychtova, Yvona; Mayer, Jiri; Greil, Richard; Gastl, Günther


    Although retreatment with alemtuzumab in relapsing B-cell chronic lymphocytic leukemia (CLL) may be beneficial, there has thus far been no thorough analysis available on this topic. Data were collected from 30 chemotherapy-pretreated patients with progressive CLL who had received alemtuzumab twice in consecutive, distinct therapy lines. The median dose of alemtuzumab retreatment was 402 mg (range, 43-1,090 mg). Retreatment with alemtuzumab induced an overall response rate of 47%. From the start of alemtuzumab retreatment, median progression-free survival (PFS) and overall survival (OS) were 6.3 and 20.0 months, respectively. Response rates, PFS and OS upon alemtuzumab retreatment were correlated with response to initial alemtuzumab treatment, the time interval between the initial course of alemtuzumab and start of retreatment, and the hemoglobin concentration prior to retreatment. Reported toxicities from 24 cases included infections (50%), febrile reactions upon alemtuzumab administration (38%), exanthema (21%), and grade 4 neutropenia (13%) and thrombocytopenia (17%). We conclude that alemtuzumab retreatment represents an effective and tolerable therapeutic option for selected patients with CLL.

  19. A Neuroblastoma × Glioma Hybrid Cell Line with Morphine Receptors (United States)

    Klee, Werner A.; Nirenberg, Marshall


    A neuroblastoma × glioma hybrid cell line with well-developed neural properties was found that has high-affinity morphine receptors. The average cell contains approximately 3 × 106 receptors. In contrast, parent cells and other neuroblastoma or hybrid cell lines tested had few or no morphine receptors. PMID:4530316

  20. Allogeneic effector/memory Th-1 cells impair FoxP3+ regulatory T lymphocytes and synergize with chaperone-rich cell lysate vaccine to treat leukemia


    Janikashvili, Nona; LaCasse, Collin J.; Larmonier, Claire; Trad, Malika; Herrell, Amanda; Bustamante, Sara; Bonnotte, Bernard; Har-Noy, Michael; Larmonier, Nicolas; Katsanis, Emmanuel


    Therapeutic strategies combining the induction of effective antitumor immunity with the inhibition of the mechanisms of tumor-induced immunosuppression represent a key objective in cancer immunotherapy. Herein we demonstrate that effector/memory CD4+ T helper-1 (Th-1) lymphocytes, in addition to polarizing type-1 antitumor immune responses, impair tumor-induced CD4+CD25+FoxP3+ regulatory T lymphocyte (Treg) immunosuppressive function in vitro and in vivo. Th-1 cells also inhibit the generatio...

  1. Absolute lymphocyte count as a surrogate marker for CD4+ cell count in monitoring of antiretroviral therapy, Northwest Ethiopia: retrospective evaluation

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    Aschalew Gelaw


    Full Text Available Objective: To determine the use of total lymphocyte count as a surrogate marker for CD4+ cell count among HIV infected patients at the University of Gondar Hospital. Methods: A retrospective cross sectional study was conducted at the University of Gondar Hospital antiretroviral therapy laboratory from December 2011 to May 2012. Data on CD4+ cell count, total lymphocyte count, sex, and age were collected from 2964 HIV infected patients and analyzed using SPSS version 16 computer software. Results: Total lymphocyte count was significantly correlated with CD4+ cell count (P<0.001; r 2 =0.434. The sensitivity, specificity, positive predictive value, negative predictive value of total lymphocyte count<1 200 cells/mm3 to predict CD4+ cell count <200 cells/mm3 was 57.8%, 86.4%%, 34.1%, 86.39%, respectively. A total lymphocyte count<1 000cells/mm3 was found to have suboptimal sensitivity (69.0%, and specificity (85.0% for predicting a CD4+ cell count <200 cells/ mm 3 . Conclusions: Total lymphocyte count and CD4+ cell count was positively correlated. Hence, lymphocyte count less than or equal to 1 000/mm3 can be used as a cutoff value in place where there is no CD4+ cell counting machine.


    Institute of Scientific and Technical Information of China (English)

    张建军; 丁尔迅; 王强; 陈学云; 付志仁


    To investigate the expression of Fas ligand in human colon carcinoma cell lines. Methods: A total of six human colon cancer cell lines were examined for the expression of Fas ligand mRNA and cell surface protein by using RT-PCR and flow cytometry respectively. Results: The results showed that Fas ligand mRNA was expressed in all of the six cancer cell lines and Fas ligand cell surface protein was expressed in part of them. Conclusion: These data suggest that Fas ligand was expressed, at least in part, in human colon cancer cell lines and might facilitate to escape from immune surveillance of the host.

  3. Effect of Piper chaba Hunter, Piper sarmentosum Roxb. and Piper interruptum Opiz. on natural killer cell activity and lymphocyte proliferation. (United States)

    Panthong, Sumalee; Itharat, Arunporn


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

  4. Ongoing in vivo immunoglobulin class switch DNA recombination in chronic lymphocytic leukemia B cells. (United States)

    Cerutti, Andrea; Zan, Hong; Kim, Edmund C; Shah, Shefali; Schattner, Elaine J; Schaffer, András; Casali, Paolo


    Chronic lymphocytic leukemia (CLL) results from the expansion of malignant CD5(+) B cells that usually express IgD and IgM. These leukemic cells can give rise in vivo to clonally related IgG(+) or IgA(+) elements. The requirements and modalities of this process remain elusive. Here we show that leukemic B cells from 14 of 20 CLLs contain the hallmarks of ongoing Ig class switch DNA recombination (CSR), including extrachromosomal switch circular DNAs and circle transcripts generated by direct S micro -->Sgamma, S micro -->Salpha, and S micro -->Sepsilon as well as sequential Sgamma-->Salpha and Sgamma-->Sepsilon CSR. Similar CLL B cells express transcripts for activation-induced cytidine deaminase, a critical component of the CSR machinery, and contain germline I(H)-C(H) and mature V(H)DJ(H)-C(H) transcripts encoded by multiple Cgamma, Calpha, and Cepsilon genes. Ongoing CSR occurs in only a fraction of the CLL clone, as only small proportions of CD5(+)CD19(+) cells express surface IgG or IgA and lack IgM and IgD. In vivo class-switching CLL B cells down-regulate switch circles and circle transcripts in vitro unless exposed to exogenous CD40 ligand and IL-4. In addition, CLL B cells that do not class switch in vivo activate the CSR machinery and secrete IgG, IgA, or IgE upon in vitro exposure to CD40 ligand and IL-4. These findings indicate that in CLL at least some members of the malignant clone actively differentiate in vivo along a pathway that induces CSR. They also suggest that this process is elicited by external stimuli, including CD40 ligand and IL-4, provided by bystander immune cells.

  5. T cell receptor transgenic lymphocytes infiltrating murine tumors are not induced to express foxp3

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    Quatromoni Jon G


    Full Text Available Abstract Regulatory T cells (Treg that express the transcription factor Foxp3 are enriched within a broad range of murine and human solid tumors. The ontogeny of these Foxp3 Tregs - selective accumulation or proliferation of natural thymus-derived Treg (nTreg or induced Treg (iTreg converted in the periphery from naïve T cells - is not known. We used several strains of mice in which Foxp3 and EGFP are coordinately expressed to address this issue. We confirmed that Foxp3-positive CD4 T cells are enriched among tumor-infiltrating lymphocytes (TIL and splenocytes (SPL in B16 murine melanoma-bearing C57BL/6 Foxp3EGFP mice. OT-II Foxp3EGFP mice are essentially devoid of nTreg, having transgenic CD4 T cells that recognize a class II-restricted epitope derived from ovalbumin; Foxp3 expression could not be detected in TIL or SPL in these mice when implanted with ovalbumin-transfected B16 tumor (B16-OVA. Likewise, TIL isolated from B16 tumors implanted in Pmel-1 Foxp3EGFP mice, whose CD8 T cells recognize a class I-restricted gp100 epitope, were not induced to express Foxp3. All of these T cell populations - wild-type CD4, pmel CD8 and OTII CD4 - could be induced in vitro to express Foxp3 by engagement of their T cell receptor (TCR and exposure to transforming growth factor β (TGFβ. B16 melanoma produces TGFβ and both pmel CD8 and OTII CD4 express TCR that should be engaged within B16 and B16-OVA respectively. Thus, CD8 and CD4 transgenic T cells in these animal models failed to undergo peripheral induction of Foxp3 in a tumor microenvironment.

  6. Histone deacetylase inhibitors impair the elimination of HIV-infected cells by cytotoxic T-lymphocytes.

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    Richard Brad Jones


    Full Text Available Resting memory CD4+ T-cells harboring latent HIV proviruses represent a critical barrier to viral eradication. Histone deacetylase inhibitors (HDACis, such as suberanilohydroxamic acid (SAHA, romidepsin, and panobinostat have been shown to induce HIV expression in these resting cells. Recently, it has been demonstrated that the low levels of viral gene expression induced by a candidate HDACi may be insufficient to cause the death of infected cells by viral cytopathic effects, necessitating their elimination by immune effectors, such as cytotoxic T-lymphocytes (CTL. Here, we study the impact of three HDACis in clinical development on T-cell effector functions. We report two modes of HDACi-induced functional impairment: i the rapid suppression of cytokine production from viable T-cells induced by all three HDACis ii the selective death of activated T-cells occurring at later time-points following transient exposures to romidepsin or, to a lesser extent, panobinostat. As a net result of these factors, HDACis impaired CTL-mediated IFN-γ production, as well as the elimination of HIV-infected or peptide-pulsed target cells, both in liquid culture and in collagen matrices. Romidepsin exerted greater inhibition of antiviral function than SAHA or panobinostat over the dose ranges tested. These data suggest that treatment with HDACis to mobilize the latent reservoir could have unintended negative impacts on the effector functions of CTL. This could influence the effectiveness of HDACi-based eradication strategies, by impairing elimination of infected cells, and is a critical consideration for trials where therapeutic interruptions are being contemplated, given the importance of CTL in containing rebound viremia.

  7. Epstein-Barr virus-transformed B-cells as efficient antigen presenting cells to propagate Aspergillus-specific cytotoxic T-lymphocytes. (United States)

    Ramadan, Gamal


    To overcome the cytotoxic T-lymphocytes (CTL) expansion limitations imposed by the lack of sufficient dendritic cells (DC) alternative sources of autologous antigen presenting cells (APC) such as Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines (BLCL), which are easy to establish in vitro, have been considered and studied in the present work. Non-adherent peripheral blood mononuclear cells of three healthy donors were repeatedly primed with autologous Aspergillus fumigatus commercial culture-filtrate antigen-pulsed fast monocyte-derived DC (Aspf-CFA-DC) alone, Aspf-CFA-pulsed BLCL (Aspf-CFA-BLCL) alone or Aspf-CFA-BLCL after one, two, or three primings with Aspf-CFA-DC (1DC/BLCL, 2DC/BLCL or 3DCIBLCL; respectively). After 5th priming, lines generated by Aspf-CFA-BLCL only showed strong/weak lytic activity for EBV/Aspf; respectively. Aspf-specific lytic activity in all donors was increased by increasing the number of primings with Aspf-CFA-DC before switching to Aspf-CFA-BLCL (18.20 +/- 1.65% versus 35.67 +/- 1.02% and 40.03 +/- 1.41% in bulk cultures generated by 1DC/BLCL versus 2DC/BLCL and 3DC/BLCL, respectively). Bulk cultures generated by Aspf-CFA-BLCL after at least two primings with Aspf-CFA-DC showed approximately the same Aspf-specific lytic activity, effector cell phenotype, expansion level and percentage expression of IFN-gamma, CD69 and CD107a without any significant differences (p > 0.05) as standard bulk cultures generated by only Aspf-CFA-DC. Thus, this study explored the use of a combined DC/BLCL protocol to establish/propagate Aspf-specific CTL for adoptive immunotherapy to prevent or treat invasive pulmonary aspergillosis.

  8. Biological characteristics of cell lines of human dental alveolus

    Institute of Scientific and Technical Information of China (English)

    陈世璋; 黄靖香; 孙明学; 赵斌


    Objective To investigate the biological characteristics of cell lines of healthy and diseased human dental alveoli. Methods Primary cell lines from either healthy or diseased human dental alveoli were obtained. Two cell lines, H-258 and H-171 derived from healthy and diseased human tissues respectively, were selected for morphological study and research on their growth and aging, using cell counting, and histochemical and immunohistochemical staining. Results Primary cell lines were successfully established from innormal dental alveoli. After freezing and thawing for three times, cell growth was continued and no morphological alterations were observed. The doubling time was 53.4 hours and mean division index (MDI) was 4‰. Cells were kept normal after twenty generations with no obvious reduction of doubling time and MDI. Of twenty-six primary cell lines derived from healthy human dental alveoli, only three cell lines achieved generation. After freezing and thawing for twice, cultured cells were still alive at a decreased growth speed, with doubling time of 85.9 hours and MDI of 3‰. Both cell lines, H-171 and H-258, shared the characteristics of osteoblast. Conclusions Primary cell lines of diseased human dental alveoli show greater growth potential. All cell lines of dental alveoli share characteristics of osteoblast. The technique we developed may be put into practice for the treatment of abnormal dental alveoli.

  9. Fetal Hematopoietic Stem Cell Transplantation Fails to Fully Regenerate the B-Lymphocyte Compartment. (United States)

    Ghosn, Eliver Eid Bou; Waters, Jeffrey; Phillips, Megan; Yamamoto, Ryo; Long, Brian R; Yang, Yang; Gerstein, Rachel; Stoddart, Cheryl A; Nakauchi, Hiromitsu; Herzenberg, Leonore A


    B cells are key components of cellular and humoral immunity and, like all lymphocytes, are thought to originate and renew from hematopoietic stem cells (HSCs). However, our recent single-HSC transfer studies demonstrate that adult bone marrow HSCs do not regenerate B-1a, a subset of tissue B cells required for protection against pneumonia, influenza, and other infections. Since B-1a are regenerated by transfers of fetal liver, the question arises as to whether B-1a derive from fetal, but not adult, HSCs. Here we show that, similar to adult HSCs, fetal HSCs selectively fail to regenerate B-1a. We also show that, in humanized mice, human fetal liver regenerates tissue B cells that are phenotypically similar to murine B-1a, raising the question of whether human HSC transplantation, the mainstay of such models, is sufficient to regenerate human B-1a. Thus, our studies overtly challenge the current paradigm that HSCs give rise to all components of the immune system.

  10. Effects of Radiofrequency Ablation on Lymphocyte Subsets and Type 1/Type 2 T Cell Subpopulations in Patients with Hepatocellular Carcinoma

    Institute of Scientific and Technical Information of China (English)

    Yan-bin Wang; Wei-Guo Xu; He-liang Liu; Kun Yan; Lin Ma; Wan-hou Guo


    Objective: To evaluate whether radiofrequency ablation (RFA) might have an influence on immune status in hepatocellular carcinoma (HCC) patients. Methods: We measured the T lymphocytes, B lymphocyte and NK cells, and determined the population of Th1, Th2, Tc1 and Tc2 of peripheral blood samples taken from 26 HCC patients before and after RFA. Results: The proportion of Type1 cells (Th1 and Tc1) and NK cells were significantly increased after RFA, especially in patients of the following subgroups: male, age>55 years, pathological grade I-II tumor, clinical stage I-II or Child-Pugh A and B. Conclusion: Type1 cells and NK cells in HCC patients were increased in a short period after RFA.

  11. Targeting chronic lymphocytic leukemia cells in the tumor microenviroment: A review of the in vitro and clinical trials to date



    Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. Despite significant advances in therapy over the last decade CLL remains incurable. Current front-line therapy often consists of chemoimmunotherapy-based regimens, most commonly the fludarabine, cyclophosphamide plus rituximab combination, but rates of relapse and refractory disease are high among these patients. Several key signaling pathways are now known to mediate the survival and proliferation of CLL cel...

  12. Molluscan cells in culture: primary cell cultures and cell lines



    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as bi...

  13. Attachment of an anti-receptor antibody to non-target cells renders them susceptible to lysis by a clone of cytotoxic T lymphocytes. (United States)

    Kranz, D M; Tonegawa, S; Eisen, H N


    The molecular basis for the dependence of antigen recognition by T cells on products of the major histocompatibility complex (MHC) is unknown, and the antigenic structures that are actually bound by T-cell receptors are ill-defined. In this study, we asked whether a monoclonal antibody (mAb) that reacts with the T-cell receptor of a clone of murine cytotoxic T lymphocytes (CTL) and not with the receptors of other CTL clones can substitute for that clone's natural ligand in specific cytolytic reactions. To answer the question, a mAb (1B2) to the receptor of a CTL clone (2C) was attached covalently to 51Cr-labeled cells that were not otherwise susceptible to lysis by clone 2C, and the cells thus modified were then tested as targets for clone 2C and other CTL clones of similar specificity. All labeled cells modified in this way, including a murine cell line that expresses no cell-surface MHC class I molecules and a human cell line, were lysed by clone 2C but not by other CTL clones. If, however, instead of attaching the mAb to the receptor of clone 2C, the cells were modified by attaching to them mAbs to other surface antigens on CTL [lymphocyte function-associated antigen (LFA-1), Thy-1.2], they were not lysed. In cytolytic titrations, the cells that had been converted by attachment of mAb 1B2 into specific targets for clone 2C were just as susceptible to lysis by that clone as the clone's natural H-2d targets (e.g., P815 cells). However, some accessory surface molecules (LFA-1, Lyt-2) that are required for clone 2C to lyse its natural H-2d targets seemed not to be required for this clone to lyse the mAb-converted target cells. By demonstrating that a variety of different cell types can be thus converted into target cells for CTL, the approach described in this study may provide opportunities to analyze further the mechanisms by which CTL destroy target cells.

  14. Single-channel and whole-cell recordings of mitogen-regulated inward currents in human cloned helper T lymphocytes. (United States)

    Kuno, M; Goronzy, J; Weyand, C M; Gardner, P

    Cytoplasmic free Ca2+ [( Ca2+]i) appears to be an important signal for DNA synthesis in early stages of lymphocyte activation. In spite of many experimental studies which employ fluorescent Ca2+ indicator dye to demonstrate an early increase of [Ca2+]i in T-lymphocytes after stimulation with lectins, specific antigens, and monoclonal antibodies to T-lymphocyte receptors, the mechanism responsible for the rise of [Ca2+]i is unknown. We have used the extracellular patch clamp technique to investigate this mechanism. Unitary inward currents, mediated by Ca2+ or Ba2+, were recorded in the membrane of T-lymphocytes. The inward current channel was characterized by a conductance of 7 pS and extrapolated reversal potential (Erev) 110 mV positive to resting potential (Vr). While gating kinetic parameters were not affected by membrane potential changes, the probability of channel opening markedly increased upon activation of the T-lymphocyte by the mitogenic lectin, phytohaemagglutinin (PHA). PHA also evoked a cadmium-sensitive, inward Ba2+ current on whole-cell clamp. We suggest that this mitogen-regulated channel introduces Ca2+ into the cytoplasm upon activation and represents a new class of voltage-independent Ca2+ channels.

  15. Increase in DNA damage in lymphocytes and micronucleus frequency in buccal cells in silica-exposed workers

    Directory of Open Access Journals (Sweden)

    Ajanta Halder


    Full Text Available The alkaline single cell gel electrophoresis (comet assay was applied to study the genotoxic properties of silica in human peripheral blood lymphocytes (PBL. The study was designed to evaluate the DNA damage of lymphocytes and the end points like micronuclei from buccal smears in a group of 45 workers, occupationally exposed to silica, from small mines and stone quarries. The results were compared to 20 sex and age matched normal individuals. There was a statistically significant difference in the damage levels between the exposed group and the control groups. The types of damages (type I -type 1V were used to measure the DNA damage. The numbers of micronuclei were higher in the silica-exposed population. The present study suggests that the silica exposure can induce lymphocyte DNA damage and produces significant variation of micronuclei in buccal smear.

  16. Exosomes released by chronic lymphocytic leukemia cells induce the transition of stromal cells into cancer-associated fibroblasts. (United States)

    Paggetti, Jerome; Haderk, Franziska; Seiffert, Martina; Janji, Bassam; Distler, Ute; Ammerlaan, Wim; Kim, Yeoun Jin; Adam, Julien; Lichter, Peter; Solary, Eric; Berchem, Guy; Moussay, Etienne


    Exosomes derived from solid tumor cells are involved in immune suppression, angiogenesis, and metastasis, but the role of leukemia-derived exosomes has been less investigated. The pathogenesis of chronic lymphocytic leukemia (CLL) is stringently associated with a tumor-supportive microenvironment and a dysfunctional immune system. Here, we explore the role of CLL-derived exosomes in the cellular and molecular mechanisms by which malignant cells create this favorable surrounding. We show that CLL-derived exosomes are actively incorporated by endothelial and mesenchymal stem cells ex vivo and in vivo and that the transfer of exosomal protein and microRNA induces an inflammatory phenotype in the target cells, which resembles the phenotype of cancer-associated fibroblasts (CAFs). As a result, stromal cells show enhanced proliferation, migration, and secretion of inflammatory cytokines, contributing to a tumor-supportive microenvironment. Exosome uptake by endothelial cells increased angiogenesis ex vivo and in vivo, and coinjection of CLL-derived exosomes and CLL cells promoted tumor growth in immunodeficient mice. Finally, we detected α-smooth actin-positive stromal cells in lymph nodes of CLL patients. These findings demonstrate that CLL-derived exosomes actively promote disease progression by modulating several functions of surrounding stromal cells that acquire features of cancer-associated fibroblasts.

  17. Rearrangement and expression of beta-T-cell receptor and immunoglobulin genes in established Ph1 chronic myelogenous leukemia cell lines. (United States)

    Berenson, J; Koeffler, H P


    We have determined the arrangement and expression of immunoglobulin (Ig) and beta-T-cell receptor (TCR) genes in six established Philadelphia chromosome-positive (Ph1) chronic myelogenous leukemia (CML) cell lines, and correlated these results with their phenotypic characteristics. Three cell lines with nonlymphoid characteristics, EM2, EM3, and K562, did not demonstrate rearrangement or expression of Ig or beta-TCR genes. A new cell line, MB, with a mature B-cell phenotype recently established in our laboratory, contained light and heavy chain immunoglobulin gene rearrangements and expressed mature Ig RNA. In a cell line with an early lymphoid phenotype, BV173, this analysis showed rearrangement of Ig heavy chain and beta-TCR genes, unrearranged Ig light chain DNA, and expression of only an immature beta-TCR transcript. This line provides evidence for T-cell lineage involvement in Ph1 CML. One cell line without markers of any cell type, KCL-22, demonstrated rearranged, unexpressed Ig heavy chain genes, suggesting these cells are at the very earliest stages of lymphoid differentiation. These lines should provide valuable tools to dissect the molecular biology of differentiation in CML and in early lymphocytes.

  18. CCR5 susceptibility to ligand-mediated down-modulation differs between human T lymphocytes and myeloid cells. (United States)

    Fox, James M; Kasprowicz, Richard; Hartley, Oliver; Signoret, Nathalie


    CCR5 is a chemokine receptor expressed on leukocytes and a coreceptor used by HIV-1 to enter CD4(+) T lymphocytes and macrophages. Stimulation of CCR5 by chemokines triggers internalization of chemokine-bound CCR5 molecules in a process called down-modulation, which contributes to the anti-HIV activity of chemokines. Recent studies have shown that CCR5 conformational heterogeneity influences chemokine-CCR5 interactions and HIV-1 entry in transfected cells or activated CD4(+) T lymphocytes. However, the effect of CCR5 conformations on other cell types and on the process of down-modulation remains unclear. We used mAbs, some already shown to detect distinct CCR5 conformations, to compare the behavior of CCR5 on in vitro generated human T cell blasts, monocytes and MDMs and CHO-CCR5 transfectants. All human cells express distinct antigenic forms of CCR5 not detected on CHO-CCR5 cells. The recognizable populations of CCR5 receptors exhibit different patterns of down-modulation on T lymphocytes compared with myeloid cells. On T cell blasts, CCR5 is recognized by all antibodies and undergoes rapid chemokine-mediated internalization, whereas on monocytes and MDMs, a pool of CCR5 molecules is recognized by a subset of antibodies and is not removed from the cell surface. We demonstrate that this cell surface-retained form of CCR5 responds to prolonged treatment with more-potent chemokine analogs and acts as an HIV-1 coreceptor. Our findings indicate that the regulation of CCR5 is highly specific to cell type and provide a potential explanation for the observation that native chemokines are less-effective HIV-entry inhibitors on macrophages compared with T lymphocytes.

  19. Analysis of renal cancer cell lines from two major resources enables genomics-guided cell line selection (United States)

    Sinha, Rileen; Winer, Andrew G.; Chevinsky, Michael; Jakubowski, Christopher; Chen, Ying-Bei; Dong, Yiyu; Tickoo, Satish K.; Reuter, Victor E.; Russo, Paul; Coleman, Jonathan A.; Sander, Chris; Hsieh, James J.; Hakimi, A. Ari


    The utility of cancer cell lines is affected by the similarity to endogenous tumour cells. Here we compare genomic data from 65 kidney-derived cell lines from the Cancer Cell Line Encyclopedia and the COSMIC Cell Lines Project to three renal cancer subtypes from The Cancer Genome Atlas: clear cell renal cell carcinoma (ccRCC, also known as kidney renal clear cell carcinoma), papillary (pRCC, also known as kidney papillary) and chromophobe (chRCC, also known as kidney chromophobe) renal cell carcinoma. Clustering copy number alterations shows that most cell lines resemble ccRCC, a few (including some often used as models of ccRCC) resemble pRCC, and none resemble chRCC. Human ccRCC tumours clustering with cell lines display clinical and genomic features of more aggressive disease, suggesting that cell lines best represent aggressive tumours. We stratify mutations and copy number alterations for important kidney cancer genes by the consistency between databases, and classify cell lines into established gene expression-based indolent and aggressive subtypes. Our results could aid investigators in analysing appropriate renal cancer cell lines.

  20. A Sigmoid Functional Response Emerges When Cytotoxic T Lymphocytes Start Killing Fresh Target Cells. (United States)

    Gadhamsetty, Saikrishna; Marée, Athanasius F M; Beltman, Joost B; de Boer, Rob J


    Cytotoxic T lymphocyte (CTL)-mediated killing involves the formation of a synapse with a target cell, followed by delivery of perforin and granzymes. Previously, we derived a general functional response for CTL killing while considering that CTLs form stable synapses (i.e., single-stage) and that the number of conjugates remains at steady state. However, the killing of target cells sometimes requires multiple engagements (i.e., multistage). To study how multistage killing and a lack of steady state influence the functional response, we here analyze a set of differential equations as well as simulations employing the cellular Potts model, in both cases describing CTLs that kill target cells. We find that at steady state the total killing rate (i.e., the number of target cells killed by all CTLs) is well described by the previously derived double saturation function. Compared to single-stage killing, the total killing rate during multistage killing saturates at higher CTL and target cell densities. Importantly, when the killing is measured before the steady state is approached, a qualitatively different functional response emerges for two reasons: First, the killing signal of each CTL gets diluted over several targets and because this dilution effect is strongest at high target cell densities; this can result in a peak in the dependence of the total killing rate on the target cell density. Second, the total killing rate exhibits a sigmoid dependence on the CTL density when killing is a multistage process, because it takes typically more than one CTL to kill a target. In conclusion, a sigmoid dependence of the killing rate on the CTLs during initial phases of killing may be indicative of a multistage killing process. Observation of a sigmoid functional response may thus arise from a dilution effect and is not necessarily due to cooperative behavior of the CTLs. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  1. Alteration of CD44 expression in HIV type 1-infected T cell lines. (United States)

    Giordanengo, V; Limouse, M; Doglio, A; Lesimple, J; Lefebvre, J C


    CD44 is known to interfere in HIV replication and to participate in many physiological processes such as lymphocyte binding to high endothelial venules of lymphoid tissue, lymph nodes, and mucosal endothelium. The T cell lines MOLT-4 and CEM, and CEM subclones were infected with the HIV-1 LAI strain and monitored for the expression of CD44 during the course of chronic virus production until the infected cells were at the stage of latent infection. The levels of CD44 protein expression were quantified using cell surface immunostaining and biotinylation. The maturation of CD44 molecules was evaluated by metabolic sulforadiolabeling and CD44 mRNA was visualized by Northern blot analysis. We show a downmodulation of CD44 expression in infected T cell lines and subclones. This phenomenon was most evident at the stage of latent infection. Then, CD44 molecules were undetectable at both the protein and mRNA levels in latently infected CEM cells and CEM subclones. In addition, the 97-kDa standard CD44 isoform showed a shift upward, while detectable during the stage of chronic virus production. In latently infected MOLT-4 cells, the CD44 protein levels were dramatically decreased; CD44 mRNA was detected, but the sizes differed from the mRNA in uninfected cells. Since CD44 is known to regulate in part lymphocyte homing and HIV replication, the alterations that were observed in the expression of this molecule could interfere with the particular homing of HIV-infected cells and/or viral latency.

  2. Biological effects of extract from newborn porcine liver on hepatocytes, hepatic stellate cells, and hepatoma cell line

    Institute of Scientific and Technical Information of China (English)


    Objective: Porcine liver extract has been shown to be effective in the clinical treatment of severe hepatitis. The aim of the present study was to study its antifibrotic as well as immune regulatory effect in vitro. Methods: Hepatocytes, hepatic stellate cells (HSCs), hepatoma cell line (HepG2) and human peripheral blood mononuclear cells (PMNCs) were studied with respect to proliferation, extracellular matrix production and apoptotic activities by proliferation assay, radioimmunoassay, gene transfection, reporter gene analysis and flow cytometry, respectively. Results: A strong stimulatory proliferation effect was observed in hepatocytes, and an inhibitory effect was found in HSCs. Hyaluronic acid (HA) production and reporter gene activities driven by various α1(Ⅰ) procollagen gene promoters in HSC-T6 were significantly decreased after treatment with the extract. Fluo-Anexin V binding apoptotic HepG2 cells were more prominent in the presence of 60 μg/ml extract. More CD4+/CD69+ positive T lymphocytes existed in the presence of the extract. Conclusion: Porcine liver extract is effective for antifibrogenesis via hepatocyte regeneration, HSC and hepatoma cell inhibition in vitro. The elevation of active T lymphocytes is helpful for immune surveillance. Fine mapping of the extract is necessary in order to get definite molecules which are essential in all described functions.

  3. In vitro viability of lymphoid cells from lines of mice genetically selected for high or low responsiveness to phytohemagglutinin. (United States)

    Stiffel, C; Liacopoulos-Briot, M; Decreusefond, C; Lambert, F


    The kinetics of viability of lymph node and spleen cells of mice genetically selected for "high" or "low" in vitro lymphocyte responsiveness to PHA were studied in PHA or PPD-stimulated short-term cultures. Lo/PHA cells were found to be less viable than Hi/PHA cells in unstimulated control cultures. PHA improved the viability of Lo/PHA cells while inducing proliferation of Hi/PHA cells with the appearance of more and larger lymphoblasts in the latter. PPD only improved the viability of spleen cell cultures, more so for the Hi/PHA line. The interline difference in thymidine uptake was smaller after PPD than after PHA stimulation. Modifications of culture conditions designed to decrease the interline difference in cell viability lessened but did not abolish the separation between the two lines for the PHA response as measured by thymidine uptake.

  4. Unraveling the equine lymphocyte proteome: differential septin 7 expression associates with immune cells in equine recurrent uveitis. (United States)

    Degroote, Roxane L; Hauck, Stefanie M; Amann, Barbara; Hirmer, Sieglinde; Ueffing, Marius; Deeg, Cornelia A


    Equine recurrent uveitis is a spontaneous, lymphocyte-driven autoimmune disease. It affects horses worldwide and presents with painful remitting-relapsing inflammatory attacks of inner eye structures eventually leading to blindness. Since lymphocytes are the key players in equine recurrent uveitis, we were interested in potential changes of their protein repertoire which may be involved in disease pathogenesis. To create a reference for differential proteome analysis, we first unraveled the equine lymphocyte proteome by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently identified 352 protein spots. Next, we compared lymphocytes from ERU cases and healthy horses with a two-dimensional fluorescence difference in gel electrophoresis approach. With this technique, we identified seven differentially expressed proteins between conditions. One of the significantly lower expressed candidates, septin 7, plays a role in regulation of cell shape, motility and migration. Further analyses revealed T cells as the main cell type with decreased septin 7 abundance in equine recurrent uveitis. These findings point to a possible pathogenetic role of septin 7 in this sight-threatening disease.

  5. Unraveling the equine lymphocyte proteome: differential septin 7 expression associates with immune cells in equine recurrent uveitis.

    Directory of Open Access Journals (Sweden)

    Roxane L Degroote

    Full Text Available Equine recurrent uveitis is a spontaneous, lymphocyte-driven autoimmune disease. It affects horses worldwide and presents with painful remitting-relapsing inflammatory attacks of inner eye structures eventually leading to blindness. Since lymphocytes are the key players in equine recurrent uveitis, we were interested in potential changes of their protein repertoire which may be involved in disease pathogenesis. To create a reference for differential proteome analysis, we first unraveled the equine lymphocyte proteome by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently identified 352 protein spots. Next, we compared lymphocytes from ERU cases and healthy horses with a two-dimensional fluorescence difference in gel electrophoresis approach. With this technique, we identified seven differentially expressed proteins between conditions. One of the significantly lower expressed candidates, septin 7, plays a role in regulation of cell shape, motility and migration. Further analyses revealed T cells as the main cell type with decreased septin 7 abundance in equine recurrent uveitis. These findings point to a possible pathogenetic role of septin 7 in this sight-threatening disease.

  6. Study of Safety,Efficacy and Pharmacokinetics of CT-1530 in Patients With Relapsed or Refractory B Cell Non-Hodgkin Lymphoma, Chronic Lymphocytic Leukemia, and Waldenstrom's Macroglobulinemia (United States)


    Relapsed or Refractory B Cell Non-Hodgkin Lymphoma; Chronic Lymphocytic Leukemia; Waldenstrom's Macroglobulinemia; Mantle Zone Lymphoma Refractory/Recurrent; Follicle Centre Lymphoma Diffuse; Diffuse Large B Cell Lymphoma

  7. Matrix metalloproteinase-9 is involved in chronic lymphocytic leukemia cell response to fludarabine and arsenic trioxide.

    Directory of Open Access Journals (Sweden)

    Irene Amigo-Jiménez

    Full Text Available BACKGROUND: Matrix metalloproteinase-9 (MMP-9 contributes to chronic lymphocytic leukemia (CLL pathology by regulating cell migration and preventing spontaneous apoptosis. It is not known if MMP-9 is involved in CLL cell response to chemotherapy and we address this in the present study, using arsenic trioxide (ATO and fludarabine as examples of cytotoxic drugs. METHODS: We used primary cells from the peripheral blood of CLL patients and MEC-1 cells stably transfected with an empty vector or a vector containing MMP-9. The effect of ATO and fludarabine was determined by flow cytometry and by the MTT assay. Expression of mRNA was measured by RT-PCR and qPCR. Secreted and cell-bound MMP-9 was analyzed by gelatin zymography and flow cytometry, respectively. Protein expression was analyzed by Western blotting and immunoprecipitation. Statistical analyses were performed using the two-tailed Student's t-test. RESULTS: In response to ATO or fludarabine, CLL cells transcriptionally upregulated MMP-9, preceding the onset of apoptosis. Upregulated MMP-9 primarily localized to the membrane of early apoptotic cells and blocking apoptosis with Z-VAD prevented MMP-9 upregulation, thus linking MMP-9 to the apoptotic process. Culturing CLL cells on MMP-9 or stromal cells induced drug resistance, which was overcome by anti-MMP-9 antibodies. Accordingly, MMP-9-MEC-1 transfectants showed higher viability upon drug treatment than Mock-MEC-1 cells, and this effect was blocked by silencing MMP-9 with specific siRNAs. Following drug exposure, expression of anti-apoptotic proteins (Mcl-1, Bcl-xL, Bcl-2 and the Mcl-1/Bim, Mcl-1/Noxa, Bcl-2/Bax ratios were higher in MMP-9-cells than in Mock-cells. Similar results were obtained upon culturing primary CLL cells on MMP-9. CONCLUSIONS: Our study describes for the first time that MMP-9 induces drug resistance by modulating proteins of the Bcl-2 family and upregulating the corresponding anti-apoptotic/pro-apoptotic ratios. This

  8. Nonmyeloablative Allogeneic Stem Cell Transplantation in Relapsed/Refractory Chronic Lymphocytic Leukemia (United States)

    Khouri, Issa F.; Bassett, Roland; Poindexter, Nancy; O'Brien, Susan; Bueso-Ramos, Carlos E.; Hsu, Yvonne; Ferrajoli, Alessandra; Keating, Michael J.; Champlin, Richard; Fernandez-Vina, Marcelo


    BACKGROUND The role of nonmyeloablative allogeneic stem cell transplantation (NST) in the treatment of chronic lymphocytic leukemia (CLL) is not well established. The authors report on long-term experience with NST in relapsed/refractory CLL and define prognostic factors associated with outcome. METHODS The authors reviewed the outcome of 86 patients with relapsed/relapsed CLL enrolled in sequential NST protocols. RESULTS The median patient age was 58 years. Patients were heavily pretreated before transplantation, and 43 required immunomanipulation after NST for persistent or recurrent disease. Immunomanipulation included withdrawal of immunosuppression, rituximab, and step-wise donor lymphocyte infusions. Of 43 patients receiving immunomanipulation, 20 (47%) experienced a complete remission. Patients with human leukocyte antigen (HLA) genotype A1+/A2−/B44− were more likely to experience a complete remission (P ¼ .0009), with rates of 9%, 36%, 50%, and 91%, respectively, for 0, 1, 2, and 3 of these HLA factors. This resulted in significant improvement in progression-free-survival rates of 68.2% at 5 years for patients with all 3 HLA factors. Overall, the estimated 5-year survival rate was 51%. In a multivariate model, a CD4 count of <100/mm3 and a below normal serum immunoglobulin G level at study entry were associated with a short survival duration (P < .0001). CONCLUSIONS These results confirm the potential cure of relapsed/refractory CLL with NST and provide the first evidence that immunoglobulin G and CD4 levels are predictive of overall survival after NST in CLL and that human leukocyte antigen alleles predict response to immunomanipulation. PMID:21455998

  9. Stimulation of the B-cell receptor activates the JAK2/STAT3 signaling pathway in chronic lymphocytic leukemia cells. (United States)

    Rozovski, Uri; Wu, Ji Yuan; Harris, David M; Liu, Zhiming; Li, Ping; Hazan-Halevy, Inbal; Ferrajoli, Alessandra; Burger, Jan A; O'Brien, Susan; Jain, Nitin; Verstovsek, Srdan; Wierda, William G; Keating, Michael J; Estrov, Zeev


    In chronic lymphocytic leukemia (CLL), stimulation of the B-cell receptor (BCR) triggers survival signals. Because in various cells activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway provides cells with survival advantage, we wondered whether BCR stimulation activates the JAK/STAT pathway in CLL cells. To stimulate the BCR we incubated CLL cells with anti-IgM antibodies. Anti-IgM antibodies induced transient tyrosine phosphorylation and nuclear localization of phosphorylated (p) STAT3. Immunoprecipitation studies revealed that anti-JAK2 antibodies coimmunoprecipitated pSTAT3 and pJAK2 in IgM-stimulated but not unstimulated CLL cells, suggesting that activation of the BCR induces activation of JAK2, which phosphorylates STAT3. Incubation of CLL cells with the JAK1/2 inhibitor ruxolitinib inhibited IgM-induced STAT3 phosphorylation and induced apoptosis of IgM-stimulated but not unstimulated CLL cells in a dose- and time-dependent manner. Whether ruxolitinib treatment would benefit patients with CLL remains to be determined.

  10. The characterization of equine herpes virus-1-infected cell polypeptides recognized by equine lymphocytes. (United States)

    Bridges, C G; Ledger, N; Edington, N


    Ponies, without evidence of previous exposure to Equine herpes virus-1 (EHV-1), were experimentally infected with EHV-1 subtype 2 and investigated for lymphocyte transformation to virus-infected cell polypeptides, as shown by separation with gel electrophoresis. Animals made significant responses to Western blot fractions that corresponded to molecular weights of approximately 30,000, 40,000-45,000, 60,000-65,000, 80,000-95,000 and 100,000-140,000 MW. These molecular weight ranges correlated with the positions of major EHV-1 subtype 2 glycoproteins that were found at migration distances approximating to 137,000, 111,000, 90,000, 65,000 and 47,000 MW. Responses were also made to a subset of similar points on the subtype 1 profile. Hyperimmune equine serum precipitated numerous infected-cell proteins of both subtypes; in particular the recognition of polypeptides with MW of 142,000, 132,000, 114,000, and 46,000 was in agreement with the mitogenic responses. Labelling with 125I indicated that immunoprecipitated greater than 250,000, 182,000, 142,000, 132,000, 75,000, 46,000 and 32,000/34,000 MW products were exposed on the surface of infected cells.

  11. Minimal residual disease surveillance in chronic lymphocytic leukemia by fluorescence-activated cell sorting. (United States)

    Ringelstein-Harlev, Shimrit; Fineman, Riva


    Achievement of complete response (CR) to therapy in chronic lymphocytic leukemia (CLL) has become a feasible goal, directly correlating with prolonged survival. It has been established that the classic definition of CR actually encompasses a variety of disease loads, and more sensitive multiparameter flow cytometry and polymerase chain reaction methods can detect the disease burden with a much higher sensitivity. Detection of malignant cells with a sensitivity of 1 tumor cell in 10,000 cells (10(-4)), using the abovementioned sophisticated techniques, is the current cutoff for minimal residual disease (MRD). Tumor burdens lower than 10(-4) are defined as MRD-negative. Several studies in CLL have determined the achievement of MRD negativity as an independent favorable prognostic factor, leading to prolonged disease-free and overall survival, regardless of the treatment protocol or the presence of other pre-existing prognostic indicators. Minimal residual disease evaluation using flow cytometry is a sensitive and applicable approach which is expected to become an integral part of future prospective trials in CLL designed to assess the role of MRD surveillance in treatment tailoring.

  12. Minimal Residual Disease Surveillance in Chronic Lymphocytic Leukemia by Fluorescence-Activated Cell Sorting

    Directory of Open Access Journals (Sweden)

    Shimrit Ringelstein-Harlev


    Full Text Available Achievement of complete response (CR to therapy in chronic lymphocytic leukemia (CLL has become a feasible goal, directly correlating with prolonged survival. It has been established that the classic definition of CR actually encompasses a variety of disease loads, and more sensitive multiparameter flow cytometry and polymerase chain reaction methods can detect the disease burden with a much higher sensitivity. Detection of malignant cells with a sensitivity of 1 tumor cell in 10,000 cells (10–4, using the abovementioned sophisticated techniques, is the current cutoff for minimal residual disease (MRD. Tumor burdens lower than 10–4 are defined as MRD-negative. Several studies in CLL have determined the achievement of MRD negativity as an independent favorable prognostic factor, leading to prolonged disease-free and overall survival, regardless of the treatment protocol or the presence of other pre-existing prognostic indicators. Minimal residual disease evaluation using flow cytometry is a sensitive and applicable approach which is expected to become an integral part of future prospective trials in CLL designed to assess the role of MRD surveillance in treatment tailoring.

  13. Deregulated expression of circadian clock and clock-controlled cell cycle genes in chronic lymphocytic leukemia. (United States)

    Rana, Sobia; Munawar, Mustafa; Shahid, Adeela; Malik, Meera; Ullah, Hafeez; Fatima, Warda; Mohsin, Shahida; Mahmood, Saqib


    Circadian rhythms are endogenous and self-sustained oscillations of multiple biological processes with approximately 24-h rhythmicity. Circadian genes and their protein products constitute the molecular components of the circadian oscillator that form positive/negative feedback loops and generate circadian rhythms. The circadian regulation extends from core clock genes to various clock-controlled genes that include various cell cycle genes. Aberrant expression of circadian clock genes, therefore, may lead to genomic instability and accelerated cellular proliferation potentially promoting carcinogenesis. The current study encompasses the investigation of simultaneous expression of four circadian clock genes (Bmal1, Clock, Per1 and Per2) and three clock-controlled cell cycle genes (Myc, Cyclin D1 and Wee1) at mRNA level and determination of serum melatonin levels in peripheral blood samples of 37 CLL (chronic lymphocytic leukemia) patients and equal number of age- and sex-matched healthy controls in order to indicate association between deregulated circadian clock and manifestation of CLL. Results showed significantly down-regulated expression of Bmal1, Per1, Per2 and Wee1 and significantly up-regulated expression of Myc and Cyclin D1 (P circadian clock genes can lead to aberrant expression of their downstream targets that are involved in cell proliferation and apoptosis and hence may result in manifestation of CLL. Moreover, shift-work and low melatonin levels may also contribute in etiology of CLL by further perturbing of circadian clock.

  14. Artificial antigen-presenting cells expressing AFP(158-166) peptide and interleukin-15 activate AFP-specific cytotoxic T lymphocytes. (United States)

    Sun, Longhao; Guo, Hao; Jiang, Ruoyu; Lu, Li; Liu, Tong; Zhang, Zhixiang; He, Xianghui


    Professional antigen-presenting cells (APCs) are potent generators of tumor antigen-specific cytotoxic T lymphocytes (CTLs) for adoptive immunotherapy; however, generation of APCs is cumbersome, expensive, and subject to the tumor microenvironment. Artificial APCs (aAPCs) have been developed as a cost-effective alternative to APCs. We developed a cellular aAPC that efficiently generated alpha-fetoprotein (AFP)-specific CTLs. We genetically modified the human B cell lymphoma cell line BJAB with a lentiviral vector to establish an aAPC called BA15. The expression of AFP(158-166)-HLA-A*02:01 complex, CD80, CD86, and interleukin (IL)-15 in BA15 cells was assessed. The efficiency of BA15 at generating AFP-specific CTLs and the specific cytotoxicity of CTLs against AFP+ cells were also determined. BA15 cells expressed high levels of AFP(158-166) peptide, HLA-A2, CD80, CD86, and IL-15. BA15 cells also exhibited higher efficiency in generating AFP-specific CTLs than did dendritic cells. These CTLs had greater cytotoxicity against AFP+ hepatocellular carcinoma cells than did CTLs obtained from dendritic cells in vitro and in vivo. Our novel aAPC system could provide a robust platform for the generation of functional AFP-specific CTLs for adoptive immunotherapy of hepatocellular carcinoma.

  15. A Well-Controlled Experimental System To Study Interactions Of Cytotoxic T Lymphocytes With Tumor Cells

    Directory of Open Access Journals (Sweden)

    Natalie Jessica Neubert


    Full Text Available While T cell-based immunotherapies are steadily improving, there are still many patients who progress, despite T cell-infiltrated tumors. Emerging evidence suggests that T cells themselves may provoke immune escape of cancer cells. Here we describe a well-controlled co-culture system for studying the dynamic T cell - cancer cell interplay, using human melanoma as a model. We explain starting material, controls and culture parameters to establish reproducible and comparable cultures with highly heterogeneous tumor cells. Low passage melanoma cell lines and melanoma-specific CD8+ T cell clones generated from patient blood were cultured together for up to three days. Living melanoma cells were isolated from the co-culture system by fluorescence-activated cell sorting. We demonstrate that the characterization of isolated melanoma cells is feasible using flow cytometry for protein expression analysis as well as an Agilent whole human genome microarray and the NanoString Technology for differential gene expression analysis. In addition, we identify five genes (ALG12, GUSB, RPLP0, KRBA2 and ADAT2 that are stably expressed in melanoma cells independent of the presence of T cells or the T cell-derived cytokines IFNγ and TNFα. These genes are essential for correct normalization of gene expression data by NanoString. Further to the characterization of melanoma cells after exposure to CTLs, this experimental system might be suitable to answer a series of questions including how the affinity of CTLs for their target antigen influences the melanoma cell response and whether CTL-induced gene expression changes in melanoma cells are reversible. Taken together, our human T cell - melanoma cell culture system is well suited to characterize immune-related mechanisms in cancer cells.


    Directory of Open Access Journals (Sweden)

    Maria Raffaella eZocchi


    Full Text Available Among γδT cells, the Vδ1 subset, resident in epithelial tissues, is implied in the defense against viruses, fungi and certain hematological malignancies, while the circulating Vδ2 subpopulation mainly respond to mycobacteria and solid tumors. Both subsets can be activated by stress-induced molecules (MIC-A, MIC-B, ULBPs to produce pro-inflammatory cytokines and lytic enzymes and destroy bacteria or damaged cells. γδT lymphocytes can also recognize lipids, as those associated to M. tuberculosis, presented by the CD1 molecule, or phosphoantigens (P-Ag, either autologous, which accumulates in virus-infected cells, or microbial produced by prokaryotes and parasites. In cancer cells P-Ag accumulate due to alterations in the mevalonate pathway; recently, butyrophilin 3A1 has been shown to be the presenting molecule for P-Ag. Of interest, aminobisphosphonates indirectly activate Vδ2 T cells inducing the accumulation of P-Ag. Based on these data, γδT lymphocytes are attractive effectors for cancer immunotherapy. However, the results obtained in clinical trials so far have been disappointing: this review will focus on the possible reasons of this failure as well as on suggestions for implementation of the therapeutic strategies.

  17. Genotoxic Effect of Chronic Exposure to DDT on Lymphocytes, Oral Mucosa and Breast Cells of Female Rats

    Directory of Open Access Journals (Sweden)

    Ruth De Celis


    Full Text Available The genotoxicity of some environmental contaminants may affect human health directly by damaging genetic material and thus plays an important role in cancer development. Xenoestrogens are one kind of environmental pollutants that may alter hormonal routes or directly affect DNA. The number of available biomarkers used to assess genetic risk and cancer is very extensive. The present study evaluated genotoxicity produced by the pesticide DDT on systemic and mammary gland cells obtained from adult female Wistar rats. Oral mucosa cells micronuclei were assessed; the comet assay in peripheral blood-isolated lymphocytes and mammary epithelial cells was also carried out. Additionally, oxidative stress was studied in mammary tissue through a lipid peroxidation assay. Our data showed an increase in lipid peroxidation, product of an increase in free oxygen radical levels, which leads to an oxidative stress status. Our results suggest that DDT is genotoxic, not only for lymphocytes but also to mammary epithelial cells.

  18. Genotoxic effect of chronic exposure to DDT on lymphocytes, oral mucosa and breast cells of female rats. (United States)

    Canales-Aguirre, Alejandro; Padilla-Camberos, Eduardo; Gómez-Pinedo, Ulises; Salado-Ponce, Hugo; Feria-Velasco, Alfredo; De Celis, Ruth


    The genotoxicity of some environmental contaminants may affect human health directly by damaging genetic material and thus plays an important role in cancer development. Xenoestrogens are one kind of environmental pollutants that may alter hormonal routes or directly affect DNA. The number of available biomarkers used to assess genetic risk and cancer is very extensive. The present study evaluated genotoxicity produced by the pesticide DDT on systemic and mammary gland cells obtained from adult female Wistar rats. Oral mucosa cells micronuclei were assessed; the comet assay in peripheral blood-isolated lymphocytes and mammary epithelial cells was also carried out. Additionally, oxidative stress was studied in mammary tissue through a lipid peroxidation assay. Our data showed an increase in lipid peroxidation, product of an increase in free oxygen radical levels, which leads to an oxidative stress status. Our results suggest that DDT is genotoxic, not only for lymphocytes but also to mammary epithelial cells.

  19. Distinct regulation of cell cycle and survival in lymphocytes from patients with Alzheimer's disease and amyotrophic lateral sclerosis. (United States)

    Bartolomé, Fernando; Muñoz, Ursula; Esteras, Noemí; Esteban, Jesús; Bermejo-Pareja, Félix; Martín-Requero, Angeles


    Alterations in cell cycle progression seem to be associated with neuronal death in Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). We previously reported disturbances in the control of cell survival/death fate in immortalized lymphocytes from AD patients. These cell cycle dysfunction and impaired apoptosis were considered systemic manifestations of AD disease. The purpose of this study was to evaluate whether these abnormalities are characteristic of AD, or they may be seen in other neurodegenerative disorders such ALS. Our results indicate that alterations in signaling molecules, Akt and ERK1/2, and in the cyclin-dependent kinase complex inhibitors (CDKis) p21(Cip1) and p27(Kip1) are detectable in lymphoblasts from AD patients, but not in ALS patients, suggesting that these variables may be considered for the development of biomarkers of AD. However, lymphocytes from ALS patients do not represent a useful model to study cell cycle-related events associated with neurodegeneration of motoneurons.

  20. The tyrosine kinase receptor ROR1 is constitutively phosphorylated in chronic lymphocytic leukemia (CLL cells.

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    Mohammad Hojjat-Farsangi

    Full Text Available Phosphorylation of receptor tyrosine kinases (RTKs has a key role in cellular functions contributing to the malignant phenotype of tumor cells. We and others have previously demonstrated that RTK ROR1 is overexpressed in chronic lymphocytic leukemia (CLL. Silencing siRNA downregulated ROR1 and induced apoptosis of CLL cells. In the present study we analysed ROR1 isoforms and the phosphorylation pattern in CLL cells (n=38 applying western blot and flow-cytometry using anti-ROR1 antibodies and an anti-phospho-ROR1 antibody against the TK domain. Two major ROR1 bands with the size of 105 and 130 kDa respectively were identified, presumably representing unglycosylated (immature and glycosylated (mature ROR1 respectively as well as a 260 kDa band which may represent dimerized ROR1. A ROR1 band of 64 kDa that may correspond to a C-terminal fragment was also noted, present only in the nucleus. The 105 kDa ROR1 isoform was more frequently expressed in non-progressive as compared to progressive CLL patients (p=0.03. The 64, 105, 130 and 260 kDa bands were constitutively phosphorylated both at tyrosine and serine residues. Phosphorylation intensity of the mature (130 kDa isoform was significantly higher in progressive than in non-progressive disease (p<0.001. Incubation of CLL cells with a mouse anti-ROR1 KNG or an anti-ROR1 CRD mAb respectively induced dephosphorylation of ROR1 before entering apoptosis. In conclusion CLL cells expressed different isoforms of ROR1 which were constitutively phosphorylated. The mature, phosphorylated ROR1 isoform was associated with a progressive disease stage. Targeting ROR1 by mAbs induced specific dephosphorylation and leukemic cell death. ROR1 might be an interesting therapeutic target.

  1. Major proteins in normal human lymphocyte subpopulations separated by fluorescence-activated cell sorting and analyzed by two-dimensional gel electrophoresis

    DEFF Research Database (Denmark)

    Madsen, P S; Hokland, M; Ellegaard, J


    We have compared the overall patterns of protein synthesis of normal human lymphocyte subpopulations taken from five volunteers using high resolution two-dimensional gel electrophoresis. The lymphocytes were isolated using density gradient centrifugation, labeled with subtype-specific Mo......Abs, and separated to a high degree of homogeneity by FACS into CD4+ helper T cells, CD8+ suppressor T cells, CD20+ B cells, and N901 (NHK-1)+ NK cells. The four lymphocyte subpopulations were labeled with [35S]methionine for 14 hr, solubilized in lysis buffer, and analyzed by two-dimensional gel electrophoresis...

  2. [Changes of T-cell clonality after induction-cultivation of peripheral T lymphocytes in adoptive immunotherapy for leukemias]. (United States)

    Liu, Yan; Gu, Jiang-Ying; Ou, Yuan; Li, Mian-Yang; Wang, He; Jin, Xian; Tao, Xiu-Yan; Liu, Zhao-Li; Ma, Xing-Fan; Wang, Xiu-Li; Ma, Si-Kun; Kang, Rui; Cai, Peng; Tong, Chun-Rong; Zhu, Ping


    This study was purposed to analyze the changes of T-cell clonality after induction of peripheral T lymphocytes by autogenous DC and cytokines in the preparation of adoptive immunotherapy for leukemias. The bone marrow and peripheral blood from 21 leukemia patients at remission stage after treatment and subjected to adoptive immunotherapy were collected. Their DCs and T-cells were stimulated with cytokines and then were mixed to activate T-cells. T-cell receptor beta variable region (TCRBV) families were amplified by RT-PCR, and genescan method and sequencing of the PCR products were used to observe the clonality changes of T-cells before and after the induction and cultivation of T-cells. The flow cytometry was used to identify CD3(+), CD4(+), CD8(+), CD3(+)CD56(+) and CD4(+)CD25str(+)FOXP3(+) cells to disclose the ratio change of cytotoxic T lymphocytes (CTL), helper T-cells, regulatory T-cells and NK T-cells before and after induction and cultivation of T-cells. The results showed that in the 21 patients, most of the 24 TCRBV families presented as oligoclonal distribution on genescan, several families were not expressed, and only a few families remained polyclonal. TCRBV24 was found to be oligoclonal in all of the 21 patients. DNA sequence analysis of TCRBV24 revealed a common motif of VAG in CDR3 in 3 cases and a common motif of GGG in CDR3 in 2 cases. In patient 5, both TCRBV 24 and TCRBV8 contained the same motif of GGG in CDR3. The identical motif in these patients may suggest that these T-cells recognize the same antigen. The peripheral lymphocytes demonstrated recovery of clonal profile on genescan from oligoclonal profile and absence of several families before the induction and cultivation to typical polyclonal profile in all TCRBV families after the induction by DC and cytokines for 13 days. After the induction and cultivation, the number of lymphocytes increased to 3.38 +/- 1.20 times. CD3(+), CD4(+), CD8(+), CD3(+)CD56(+) and CD4(+)CD25str(+)FOX P3

  3. Regulation of T lymphocyte apoptotic markers is associated to cell activation during the acute phase of dengue. (United States)

    Torrentes-Carvalho, Amanda; Marinho, Cintia Ferreira; de Oliveira-Pinto, Luzia Maria; de Oliveira, Débora Batista; Damasco, Paulo Vieira; Cunha, Rivaldo Venâncio; de Souza, Luiz José; de Azeredo, Elzinandes Leal; Kubelka, Claire Fernandes


    Dengue fever, a public health problem in Brazil, may present severe clinical manifestations as result of an increased vascular permeability and coagulation disorders. T cell activation is a critical event for an effective immune response against infection, including the production of cytokines. We aim to reveal mechanisms that modulate the virus-cell interaction, with an emphasis on cell death. Apoptosis is involved in lymphocyte homeostasis, contributes to the clearance of virus-infected cells but also may play a role in the pathogenesis. Phosphatidylserine exposure on CD8T lymphocytes from dengue patients support early apoptotic processes and loss of genomic integrity, observed by DNA fragmentation in T lymphocytes and indicating late apoptosis. These T cells express activation and cytotoxic phenotypes as revealed by CD29 and CD107a upregulation. Higher frequencies of CD95 were detected in T lymphocytes mainly in those with the cytotoxic profile (CD107a+) and lower levels of anti-apoptotic molecule Bcl-2, suggesting that both CD4+ and CD8+ T cell subsets are more susceptible to apoptosis during acute dengue. The analysis of apoptosis-related protein expression profile showed that not only molecules with pro- but also those with anti-apoptotic functions are overexpressed, indicating that survival mechanisms could be possibly protecting cells against apoptosis caused by viral, immune, oxidative and/or genotoxic stresses. These observations led us to propose that in dengue patients there is an association between T cell susceptibility to apoptosis and the activation state. The mechanisms for understanding the immunopathogenesis during dengue infection are discussed.

  4. Chronic Lymphocytic Leukemia With Hodgkin and Reed-Sternberg (HRS) Cells: A Potential Diagnostic Pitfall in Lymph Node Biopsies. (United States)

    Agrawal, Parimal; Bal, Amanjit; Das, Ashim; Sachdeva, ManUpdesh; Prakash, Gaurav


    Chronic lymphocytic leukemia (CLL) is known to undergo Richter transformation in a proportion of cases. Transformation into Hodgkin lymphoma has been described in a minority of the cases. However, CLL rarely also shows Hodgkin and Reed-Sternberg cells with a classic morphology and the immunophenotype of Hodgkin lymphoma, even when not in transformation. The presence of these Hodgkin and Reed-Sternberg cells in CLL can cause a diagnostic dilemma.

  5. Immunoregulatory effects on T lymphocytes by human mesenchymal stromal cells isolated from bone marrow, amniotic fluid, and placenta. (United States)

    Mareschi, Katia; Castiglia, Sara; Sanavio, Fiorella; Rustichelli, Deborah; Muraro, Michela; Defedele, Davide; Bergallo, Massimiliano; Fagioli, Franca


    Mesenchymal stromal cells (MSCs) are a promising tool in cell therapies because of their multipotent, bystander, and immunomodulatory properties. Although bone marrow represents the main source of MSCs, there remains a need to identify a stem cell source that is safe and easily accessible and yields large numbers of cells without provoking debates over ethics. In this study, MSCs isolated from amniotic fluid and placenta were compared with bone marrow MSCs. Their immunomodulatory properties were studied in total activated T cells (peripheral blood mononuclear cells) stimulated with phytohemagglutinin (PHA-PBMCs). In particular, an in vitro co-culture system was established to study: (i) the effect on T-lymphocyte proliferation; (ii) the presence of T regulatory lymphocytes (Treg); (iii) the immunophenotype of various T subsets (Th1 and Th2 naïve, memory, effector lymphocytes); (iv) cytokine release and master gene expression to verify Th1, Th2, and Th17 polarization; and (v) IDO production. Under all co-culture conditions with PHA-PBMCs and MSCs (independently of tissue origin), data revealed: (i) T proliferation inhibition; (ii) increase in naïve T and decrease in memory T cells; (iii) increase in T regulatory lymphocytes; (iv) strong Th2 polarization associated with increased interleukin-10 and interleukin-4 levels, Th1 inhibition (significant decreases in interleukin-2, tumor necrosis factor-α, interferon-γ, and interleukin-12) and Th17 induction (production of high concentrations of interleukins-6 and -17); (v) indoleamine-2,3-dioxygenase mRNA induction in MSCs co-cultured with PHA-PBMCs. AF-MSCs had a more potent immunomodulatory effect on T cells than BM-MSCs, only slightly higher than that of placenta MSCs. This study indicates that MSCs isolated from fetal tissues may be considered a good alternative to BM-MSCs for clinical applications.

  6. Lenalidomide With or Without Rituximab in Treating Patients With Progressive or Relapsed Chronic Lymphocytic Leukemia, Small Lymphocytic Lymphoma, Prolymphocytic Leukemia, or Non-Hodgkin Lymphoma Previously Treated With Donor Stem Cell Transplant (United States)


    Adult Nasal Type Extranodal NK/T-cell Lymphoma; Anaplastic Large Cell Lymphoma; Angioimmunoblastic T-cell Lymphoma; Cutaneous B-cell Non-Hodgkin Lymphoma; Extranodal Marginal Zone B-cell Lymphoma of Mucosa-associated Lymphoid Tissue; Hepatosplenic T-cell Lymphoma; Intraocular Lymphoma; Nodal Marginal Zone B-cell Lymphoma; Noncutaneous Extranodal Lymphoma; Peripheral T-cell Lymphoma; Prolymphocytic Leukemia; Recurrent Adult Burkitt Lymphoma; Recurrent Adult Diffuse Large Cell Lymphoma; Recurrent Adult Diffuse Mixed Cell Lymphoma; Recurrent Adult Diffuse Small Cleaved Cell Lymphoma; Recurrent Adult Grade III Lymphomatoid Granulomatosis; Recurrent Adult Immunoblastic Large Cell Lymphoma; Recurrent Adult Lymphoblastic Lymphoma; Recurrent Adult T-cell Leukemia/Lymphoma; Recurrent Cutaneous T-cell Non-Hodgkin Lymphoma; Recurrent Grade 1 Follicular Lymphoma; Recurrent Grade 2 Follicular Lymphoma; Recurrent Grade 3 Follicular Lymphoma; Recurrent Mantle Cell Lymphoma; Recurrent Marginal Zone Lymphoma; Recurrent Mycosis Fungoides/Sezary Syndrome; Recurrent Small Lymphocytic Lymphoma; Refractory Chronic Lymphocytic Leukemia; Refractory Hairy Cell Leukemia; Small Intestine Lymphoma; Splenic Marginal Zone Lymphoma; T-cell Large Granular Lymphocyte Leukemia; Testicular Lymphoma; Waldenström Macroglobulinemia


    NARCIS (Netherlands)



    In the present study the cytotoxicity of 16 proanthocyanidins was evaluated in GLC(4), a human small cell lung carcinoma cell line, and in COLO 320, a human colorectal cancer cell line, using the microculture tetrazolium (MTT) assay. With IC50 values ranging from 18 to >200 mu m following continuous

  8. Alloantigens of human lymphoid cell lines; `human Ia-like antigens' (United States)

    Koyama, K.; Nakamuro, K.; Tanigaki, N.; Pressman, D.


    Membrane glycoproteins that appear to belong to a new alloantigen system were partially purified by gel filtration and lentil-lectin affinity chromatography from a non-ionic detergent (Renex 30) solubilized membrane fraction of each of two Burkitt lymphoma cell lines, B46M and Daudi. The preparations were radioiodinated and further purified by gel filtration, lentil—lectin affinity chromatography and anti-HLA antibody affinity chromatography. The labelled preparations thus obtained did not have binding activity with any of rabbit anti-HLA, rabbit anti-human β2-microglobulin and rabbit anti-human IgG-Fab antisera, but did have a high level of binding activity with rabbit anti-B-cell membrane antiserum. Moreover, the labelled preparations showed relatively high binding activity with some conventional HLA typing sera. Out of sixty-eight human tissue typing alloantisera tested, three (Berlin 373, Betz and TO-29-01) gave especially high binding with both of the labeled preparations. The antigens involved in reaction with these alloantisera and also with the rabbit anti-B-cell membrane antiserum contained two components, one 31,000 ∼ 32,000 daltons and another 24,000 ∼ 25,000 daltons, bound non-covalently. The alloantigens were specific to B cell type cell lines (B-lymphoid cell lines and Burkitt-lymphoma cell lines) in cultured cell lines and also to B lymphocytes in peripheral blood. In organs and tissues, however, they were found to be present widely distributed in lymphoid organs (thymus as well as spleen and lymph node) and in non-lymphoid organs (including liver, kidney, testis and heart). PMID:24587

  9. Evidence for progenitors of chronic lymphocytic leukemia B cells that undergo intraclonal differentiation and diversification. (United States)

    Dono, M; Hashimoto, S; Fais, F; Trejo, V; Allen, S L; Lichtman, S M; Schulman, P; Vinciguerra, V P; Sellars, B; Gregersen, P K; Ferrarini, M; Chiorazzi, N


    Peripheral blood mononuclear cells from five patients with IgG+ B-type chronic lymphocytic leukemia (B-CLL) were analyzed for the presence of clone-specific Ig H chain variable region gene mRNA transcripts linked to C mu and/or C alpha. This was assessed by (1) comparing the lengths of portions of the VHDJH of the IgG+ CLL clones with those of the mu and alpha isotype-expressing B cells, (2) performing clone-specific endonuclease digestion studies, and (3) determining the DNA sequences of the mu and alpha isotype-expressing cDNA. Thus, when B-cell mRNA from these five patients were reverse transcribed with C gamma-specific primers and then amplified by polymerase chain reaction, dominant cDNA were found with lengths corresponding to those of the IgG+ CLL B cell. In addition, in four cases, cDNA of lengths identical to those of the CLL B cell were detected when mRNA was reverse transcribed and amplified using c mu- and/or C alpha-specific primers, strongly suggesting clonal relatedness. These CLL-related mu- and alpha-expressing cDNA were present in greater amounts that unrelated (non-CLL) mu- and alpha-expressing cDNA from normal B cells that used genes of the same VH family. When the sequences of these CLL-related C mu- and C alpha-expressing cDNA were compared with those of the IgG+ CLL clones, it was clear that they were derived from the same ancestral gene as the IgG-expressing CLL B cell, thus documenting their common origin. Finally, nucleotide point mutations were observed in the mu- and alpha-expressing cDNA of certain patients, indicating divergence with the CLL. These data suggest that IgM+ B cells, which are precursors of the leukemic B cells, exist in increased numbers in the blood of most patients with IgG+ B-CELL and that these cells may differentiate, accumulate V genes mutations, and undergo isotype switching in vivo. In addition, the data are consistent with a sequential-hit model for the evolution of CLL.

  10. Ouabain exacerbates activation-induced cell death in human peripheral blood lymphocytes

    Directory of Open Access Journals (Sweden)

    Mabel B. Esteves


    Full Text Available Lymphocytes activated by mitogenic lectins display changes in transmembrane potential, an elevation in the cytoplasmic Ca2+ concentrations, proliferation and/or activation induced cell death. Low concentrations of ouabain (an inhibitor of Na+,K+-ATPase suppress mitogen-induced proliferation and increases cell death. To understand the mechanisms involved, a number of parameters were analyzed using fluorescent probes and flow cytometry. The addition of 100nM ouabain to cultures of peripheral blood lymphocytes activated with 5µg/ml phytohemagglutinin (PHA did not modify the increased expression of the Fas receptor or its ligand FasL induced by the mitogen. However, treatment with ouabain potentiated apoptosis induced by an anti-Fas agonist antibody. A synergy between ouabain and PHA was also observed with regard to plasma membrane depolarization. PHA per se did not induce dissipation of mitochondrial membrane potential but when cells were also exposed to ouabain a marked depolarization could be observed, and this was a late event. It is possible that the inhibitory effect of ouabain on activated peripheral blood lymphocytes involves the potentiation of some of the steps of the apoptotic process and reflects an exacerbation of the mechanism of activation-induced cell death.Quando linfócitos são ativados por lectinas mitogênicas apresentam mudanças do potencial de membrana, elevação das concentrações citoplasmáticas de cálcio, proliferação e/ou morte celular induzida por ativação (AICD. Concentrações baixas de ouabaína (um inibidor da Na,K-ATPase suprimem a proliferação induzida por mitógenos e aumentam a morte celular. Para entender os mecanismos envolvidos, uma série de parâmetros foram avaliados usando sondas fluorescentes e citometria de fluxo. A adição de 100nM de ouabaína para culturas de linfócitos de sangue periférico ativadas por fitohemaglutinina (PHA não modificou o aumento de expressão do receptor Fas ou de

  11. ER-mitochondria contacts control surface glycan expression and sensitivity to killer lymphocytes in glioma stem-like cells. (United States)

    Bassoy, Esen Yonca; Kasahara, Atsuko; Chiusolo, Valentina; Jacquemin, Guillaume; Boydell, Emma; Zamorano, Sebastian; Riccadonna, Cristina; Pellegatta, Serena; Hulo, Nicolas; Dutoit, Valérie; Derouazi, Madiha; Dietrich, Pierre Yves; Walker, Paul R; Martinvalet, Denis


    Glioblastoma is a highly heterogeneous aggressive primary brain tumor, with the glioma stem-like cells (GSC) being more sensitive to cytotoxic lymphocyte-mediated killing than glioma differentiated cells (GDC). However, the mechanism behind this higher sensitivity is unclear. Here, we found that the mitochondrial morphology of GSCs modulates the ER-mitochondria contacts that regulate the surface expression of sialylated glycans and their recognition by cytotoxic T lymphocytes and natural killer cells. GSCs displayed diminished ER-mitochondria contacts compared to GDCs. Forced ER-mitochondria contacts in GSCs increased their cell surface expression of sialylated glycans and reduced their susceptibility to cytotoxic lymphocytes. Therefore, mitochondrial morphology and dynamism dictate the ER-mitochondria contacts in order to regulate the surface expression of certain glycans and thus play a role in GSC recognition and elimination by immune effector cells. Targeting the mitochondrial morphology, dynamism, and contacts with the ER could be an innovative strategy to deplete the cancer stem cell compartment to successfully treat glioblastoma. © 2017 The Authors.

  12. EBI2 overexpression in mice leads to B1 B cell expansion and chronic lymphocytic leukemia-(CLL)-like B cell malignancies

    DEFF Research Database (Denmark)

    Niss Arfelt, Kristine; Barington, Line; Benned-Jensen, Tau


    Human and mouse chronic lymphocytic leukemia (CLL) develop from CD5+ B cells that in mice and macaques are known to define the distinct B1a B cell lineage. B1a cells are characterized by lack of germinal center development and the B1a cell population is increased in mice with reduced germinal...... center formation. As a major mediator of follicular B cell migration, the G protein-coupled receptor (GPCR) Epstein Barr virus-induced gene 2 (EBI2 or GPR183) directs B cell migration in the lymphoid follicles in response to its endogenous ligands, oxysterols. Thus, upregulation of EBI2 drives the B...... cells towards the extrafollicular area, whereas downregulation is essential for germinal center formation. We therefore speculated whether increased expression of EBI2 would lead to an expanded B1 cell subset and, ultimately, progression to chronic lymphocytic leukemia. Here we demonstrate that B cell...

  13. Synergistic Activity of Deguelin and Fludarabine in Cells from Chronic Lymphocytic Leukemia Patients and in the New Zealand Black Murine Model (United States)

    Rebolleda, Nerea; Losada-Fernandez, Ignacio; Perez-Chacon, Gema; Castejon, Raquel; Rosado, Silvia; Morado, Marta; Vallejo-Cremades, Maria Teresa; Martinez, Andrea; Vargas-Nuñez, Juan A.


    B-cell chronic lymphocytic leukemia (CLL) remains an incurable disease, and despite the improvement achieved by therapeutic regimes developed over the last years still a subset of patients face a rather poor prognosis and will eventually relapse and become refractory to therapy. The natural rotenoid deguelin has been shown to induce apoptosis in several cancer cells and cell lines, including primary human CLL cells, and to act as a chemopreventive agent in animal models of induced carcinogenesis. In this work, we show that deguelin induces apoptosis in vitro in primary human CLL cells and in CLL-like cells from the New Zealand Black (NZB) mouse strain. In both of them, deguelin dowregulates AKT, NFκB and several downstream antiapoptotic proteins (XIAP, cIAP, BCL2, BCL-XL and survivin), activating the mitochondrial pathway of apoptosis. Moreover, deguelin inhibits stromal cell-mediated c-Myc upregulation and resistance to fludarabine, increasing fludarabine induced DNA damage. We further show that deguelin has activity in vivo against NZB CLL-like cells in an experimental model of CLL in young NZB mice transplanted with spleen cells from aged NZB mice with lymphoproliferation. Moreover, the combination of deguelin and fludarabine in this model prolonged the survival of transplanted mice at doses of both compounds that were ineffective when administered individually. These results suggest deguelin could have potential for the treatment of human CLL. PMID:27101369

  14. In vitro and in vivo evaluation of direct rhenium-188-labeled anti-CD52 monoclonal antibody alemtuzumab for radio immunotherapy of B-cell chronic lymphocytic leukemia

    NARCIS (Netherlands)

    De Decker, Mario; Bacher, Klaus; Thierens, Hubert; Slegers, Guido; Dierckx, Rudi A.; De Vos, Filip


    Alemtuzumab (Campath, Berlex) is a humanized IgG1 rat monoclonal antibody directed against the cell surface CD52 antigen, found on lymphocytes and monocytes. It is being developed for the treatment of chronic lymphocytic leukemia (CLL), autoinumme disease and for the prevention of transplant rejecti

  15. T-cell-mediated immunity to lymphocytic choriomeningitis virus in beta2-integrin (CD18)- and ICAM-1 (CD54)-deficient mice

    DEFF Research Database (Denmark)

    Christensen, Jan Pravsgaard; Marker, O; Thomsen, Allan Randrup


    The T-cell response to lymphocytic choriomeningitis virus was studied in mice with deficient expression of beta2-integrins or ICAM-1. In such mice, the generation of virus-specific cytotoxic T lymphocytes was only slightly impaired and bystander activation was as extensive as that observed in wild...

  16. An association, in adult Japanese, between the occurrence of rogue cells among cultured lymphocytes (JC virus activity) and the frequency of simple chromosomal damage among the lymphocytes of persons exhibiting these rogue cells

    Energy Technology Data Exchange (ETDEWEB)

    Neel, J.V. [Univ. of Michigan, Ann Arbor, MI (United States). Dept. of Human Genetics


    Data from a previous study of the cytogenetic effects, in cultured lymphocytes, of exposure to the atomic bomb in Hiroshima have been reanalyzed to determine the relationship between the occurrence of rogue cells in an individual and the frequency of simple chromosomal damage in the nonrogue cells of the same individual. Rogue cells are cells with complex chromosomal damage, currently believed to be a manifestation of the activity of a human polyoma virus termed JC. Among a total of 1,835 persons examined, there were 45 exhibiting rogue cells. A total of 179,599 cells were scored for simple chromosomal damage. In both the exposed and the control populations, there was an absolute increase of {approximately}1.5% in the frequency of simple chromosomal damage in the nonrogue cells of those exhibiting rogue cells, when compared with the frequencies observed in those not exhibiting rogue cells, which is a statistically significant difference. It is argued that this phenomenon, occurring not only in lymphocytes but possibly also in other cells/tissues, may play a contributory role in the origin of malignancies characterized by clonal chromosome abnormalities. Unexpectedly, among those exhibiting rogue cells, there was a disproportionately greater representation of persons who had received relatively high radiation exposures from the bomb. The reason for this is unclear, but it is tempting to relate the finding to some lingering effect of the exposure (or the circumstances surrounding the exposure) on immunocompetence.

  17. Protection of stem cell-derived lymphocytes in a primate AIDS gene therapy model after in vivo selection.

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    Grant D Trobridge

    Full Text Available BACKGROUND: There is currently no effective AIDS vaccine, emphasizing the importance of developing alternative therapies. Recently, a patient was successfully transplanted with allogeneic, naturally resistant CCR5-negative (CCR5Delta32 cells, setting the stage for transplantation of naturally resistant, or genetically modified stem cells as a viable therapy for AIDS. Hematopoietic stem cell (HSC gene therapy using vectors that express various anti-HIV transgenes has also been attempted in clinical trials, but inefficient gene transfer in these studies has severely limited the potential of this approach. Here we evaluated HSC gene transfer of an anti-HIV vector in the pigtailed macaque (Macaca nemestrina model, which closely models human transplantation. METHODS AND FINDINGS: We used lentiviral vectors that inhibited both HIV-1 and simian immunodeficiency virus (SIV/HIV-1 (SHIV chimera virus infection, and also expressed a P140K mutant methylguanine methyltransferase (MGMT transgene to select gene-modified cells by adding chemotherapy drugs. Following transplantation and MGMT-mediated selection we demonstrated transgene expression in over 7% of stem-cell derived lymphocytes. The high marking levels allowed us to demonstrate protection from SHIV in lymphocytes derived from gene-modified macaque long-term repopulating cells that expressed an HIV-1 fusion inhibitor. We observed a statistically significant 4-fold increase of gene-modified cells after challenge of lymphocytes from one macaque that received stem cells transduced with an anti-HIV vector (p<0.02, Student's t-test, but not in lymphocytes from a macaque that received a control vector. We also established a competitive repopulation assay in a second macaque for preclinical testing of promising anti-HIV vectors. The vectors we used were HIV-based and thus efficiently transduce human cells, and the transgenes we used target HIV-1 genes that are also in SHIV, so our findings can be rapidly


    Directory of Open Access Journals (Sweden)

    Mohan Durga


    Full Text Available In India, the unprecedented growth rate and urbanization along with the rapid increase in motor vehicle activity and industrialization are contributing to high levels of urban air pollution. The population is mainly exposed to high air pollution concentrations, where motor vehicle emissions constitute the main source of fine and ultrafine particles. Motor exhaust emissions is a mixture of gases and Particulate Matter (PM. Diesel and petrol fuels in vehicles produce combustion-derived particles as a result of combustion. Vehicle exhaust particles are the main constituents of environmental nanoparticles. In the present investigation, environmental nanoparticles such as Diesel Exhaust Particles (DEP and Petrol Exhaust Particles (PEP were collected from on-road vehicles using a specially designed collection chamber. The surface morphology of the collected particles was analyzed through Transmission Electron Microscope (TEM, and the elemental mapping was performed through EDAX analysis. Results indicated the presence of nanometer-size particles in both the categories of vehicle exhaust. These small-size particles of respirable range can enter the respiratory tract of humans and get deposited in the lungs and cause various effects inside the human body. The aim of this study is to assess the cytotoxicity of the collected Diesel Exhaust Nanoparticles (DENPs and Petrol Exhaust Nanoparticles (PENPs. Cytotoxicity endpoint, such as IC50 (50% Inhibitory Concentration, was determined after a 24-h exposure. Results of this study indicated that all five cell lines were sensitive to these vehicle exhaust nanoparticles at varying levels.

  19. Separate Developmental Programs for HLA-A and -B Cell Surface Expression during Differentiation from Embryonic Stem Cells to Lymphocytes, Adipocytes and Osteoblasts

    DEFF Research Database (Denmark)

    Sabir, Hardee J; Nehlin, Jan O; Qanie, Diyako


    hematopoietic stem cells (hHSC), human mesenchymal stem cells (hMSC) and their fully-differentiated progenies such as lymphocytes, adipocytes and osteoblasts. hESC showed extremely low levels of HLA-A and no -B. In contrast, multipotent hMSC and hHSC generally expressed higher levels of HLA-A and clearly HLA...

  20. Cell-surface Associated p43/Endothelial-monocyte-activating-polypeptide-II in Hepatocellular Carcinoma Cells Induces Apoptosis in T-lymphocytes

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    Wasek Faisal


    Conclusion: Our data suggest that membrane-bound EMAP-II is cytotoxic to lymphocytes and, therefore, might constitute a component of a novel, immunosuppressive pathway by which HCC cells may eliminate attacking T-cells and evade the immune system. The mechanism by which it does so is currently under investigation.

  1. Bone Marrow Mesenchymal Stem Cells Enhance the Differentiation of Human Switched Memory B Lymphocytes into Plasma Cells in Serum-Free Medium (United States)

    Gervais-St-Amour, Catherine


    The differentiation of human B lymphocytes into plasma cells is one of the most stirring questions with regard to adaptive immunity. However, the terminal differentiation and survival of plasma cells are still topics with much to be discovered, especially when targeting switched memory B lymphocytes. Plasma cells can migrate to the bone marrow in response to a CXCL12 gradient and survive for several years while secreting antibodies. In this study, we aimed to get closer to niches favoring plasma cell survival. We tested low oxygen concentrations and coculture with mesenchymal stem cells (MSC) from human bone marrow. Besides, all cultures were performed using an animal protein-free medium. Overall, our model enables the generation of high proportions of CD38+CD138+CD31+ plasma cells (≥50%) when CD40-activated switched memory B lymphocytes were cultured in direct contact with mesenchymal stem cells. In these cultures, the secretion of CXCL12 and TGF-β, usually found in the bone marrow, was linked to the presence of MSC. The level of oxygen appeared less impactful than the contact with MSC. This study shows for the first time that expanded switched memory B lymphocytes can be differentiated into plasma cells using exclusively a serum-free medium. PMID:27872867

  2. Tumour infiltrating lymphocytes correlate with improved survival in patients with esophageal squamous cell carcinoma. (United States)

    Jiang, Dongxian; Liu, Yalan; Wang, Hao; Wang, Haixing; Song, Qi; Sujie, Akesu; Huang, Jie; Xu, Yifan; Zeng, Haiying; Tan, Lijie; Hou, Yingyong; Xu, Chen


    We undertook a study of tumour infiltrating lymphocytes (TILs) in a large and relatively homogeneous group of patients with completely resected esophageal squamous cell carcinoma (ESCC). Hematoxylin and eosin-stained sections of 235 ESCC tumours were evaluated for density of TILs in intratumoural (iTIL) and stromal compartments (sTIL). Foxp3+, CD4+, and CD8+ T cells in tumoural and stromal areas were evaluated by immunohistochemistry. Of the 235 tumours, high sTIL (>10%), and iTIL (>10%) were observed in 101 (43.0%) and 98 (41.7%), respectively. The median follow-up period was 36.0 months (95% CI 29.929-42.071). Univariate analysis revealed that sTIL (>10%), iTIL (>20%), vessels involvement, lymph node metastasis, and clinical stage were significantly associated with postoperative outcome. In multivariate analysis, high sTIL (HR: 0.664, P = 0.019 for Disease free survival; HR: 0.608, P = 0.005 for Overall survival) was identified as independent better prognostic factor. Further analysis, sTIL was identified as independently prognostic factor in Stage III-IVa disease, which was not found in Stage I-II disease. Our study demonstrated that sTIL was associated with better ESCC patients' survival, especially in Stage III-IVa disease. Assessment of sTIL could be useful to discriminate biological behavior for ESCC patients.

  3. Treatment With Lenalidomide Modulates T-Cell Immunophenotype and Cytokine Production in Patients With Chronic Lymphocytic Leukemia (United States)

    Lee, Bang-Ning; Gao, Hui; Cohen, Evan N.; Badoux, Xavier; Wierda, William G.; Estrov, Zeev; Faderl, Stefan H.; Keating, Michael J.; Ferrajoli, Alessandra; Reuben, James M.


    BACKGROUND Lenalidomide, an immunomodulatory agent, has activity in lymphoproliferative disorders. The authors, therefore, evaluated its effects on T-cell immunophenotype and cytokine production in patients with chronic lymphocytic leukemia (CLL). METHODS To study the immunomodulatory effects of lenalidomide in CLL, the authors recruited 24 patients with untreated CLL enrolled in a phase 2 clinical trial of lenalidomide and obtained peripheral blood specimens for immunologic studies consisting of enumeration of T cells and assessing their ability to synthesize cytokines after activation through T-cell receptor (TCR). RESULTS After 3 cycles of therapy, patients had a significant reduction in percentage (%) and absolute lymphocyte count (ALC) and an increase in percentage of T cells, percentage of activated CD8+ T cells producing IFN-γ, and percentage of regulatory T (TR) cells when compared with their respective levels before treatment. After 15 cycles of treatment, responder patients had significant reduction in percentage of lymphocytes and ALC, percentage of activated CD4+ T cells producing IL-2, IFN-γ, or TNF-α, and percentage of TR cells when compared with their perspective levels after 3 cycles of treatment. Furthermore, the numbers of activated CD4+ T cells producing IL-2, IFN-γ, or TNF-α, activated CD8+ T cells producing IFN-γ, and TR cells normalized to the range of healthy subjects. CONCLUSIONS Treatment with lenalidomide resulted in the normalization of functional T-cell subsets in responders, suggesting that lenalidomide may modulate cell-mediated immunity in patients with CLL. PMID:21858802

  4. Derivation and Utilization of Functional CD8(+) Dendritic Cell Lines. (United States)

    Pigni, Matteo; Ashok, Devika; Acha-Orbea, Hans


    It is notoriously difficult to obtain large quantities of non-activated dendritic cells ex vivo. For this reason, we produced and characterized a mouse model expressing the large T oncogene under the CD11c promoter (Mushi mice), in which CD8α(+) dendritic cells transform after 4 months. We derived a variety of stable cell lines from these primary lines. These cell lines reproducibly share with freshly isolated dendritic cells most surface markers, mRNA and protein expression, and all tested biological functions. Cell lines can be derived from various strains and knockout mice and can be easily transduced with lentiviruses. In this article, we describe the derivation, culture, and lentiviral transduction of these dendritic cell lines.

  5. Cut-off value for absolute lymphocytes as an alternative for the immunophenotypic analysis of CD3+ T cells in the monitoring of immunosuppressive therapy with thymoglobulin

    Directory of Open Access Journals (Sweden)

    Gislaine Furlanetto

    Full Text Available Abstract Introduction: Immunosuppression of T lymphocytes is required for preventing acute rejection after transplantation and for the treatment of chronic autoimmune and inflammatory diseases. The laboratory monitoring for this therapy is the measurement of T cells by immunophenotyping, aiming the target value of less than 20 cells per µL. Objective: To establish a cut-off point for the total number of lymphocytes in the automated blood cell count that reflects less than twenty T cells µL by immunophenotyping. Methods: We studied and evaluated 242 kidney transplant patients that had results of automated blood cell count and quantification of T cells by immunophenotyping technique. The patients were divided into two groups, depending on the T lymphocyte immunophenotyping rates established by lower and higher than 20 cells per µL. After, we evaluated the cut-off point for lymphocytes in the blood cell count with a specificity of 100% to exclude patients with high levels of T lymphocytes. Results: We found that the cut-off point of 70 lymphocytes per µL obtained by automated blood cell count showed 100% of specificity to exclude patients with T-cell counts higher than 20 cells per µL by immunophenotyping. Conclusion: The results found in this study may be helpful to monitor the immunosuppressive therapy in kidney transplant patients in places where a flow cytometer is not available, or when this equipment is not present in the full routine.

  6. Transcriptional Reprogramming of Mature CD4+ T helper Cells generates distinct MHC class II-restricted Cytotoxic T Lymphocytes (United States)

    Mucida, Daniel; Husain, Mohammad Mushtaq; Muroi, Sawako; van Wijk, Femke; Shinnakasu, Ryo; Naoe, Yoshinori; Reis, Bernardo Sgarbi; Huang, Yujun; Lambolez, Florence; Docherty, Michael; Attinger, Antoine; Shui, Jr-Wen; Kim, Gisen; Lena, Christopher J.; Sakaguchi, Shinya; Miyamoto, Chizuko; Wang, Peng; Atarashi, Koji; Park, Yunji; Nakayama, Toshinori; Honda, Kenya; Ellmeier, Wilfried; Kronenberg, Mitchell; Taniuchi, Ichiro; Cheroutre, Hilde


    TCRαβ thymocytes differentiate to either CD8αβ cytotoxic T lymphocytes or CD4+ T helper cells. This functional dichotomy is controlled by key transcription factors, including the T helper master regulator, ThPOK, which suppresses the cytolytic program in MHC class II-restricted CD4+ thymocytes. ThPOK continues to repress CD8-lineage genes in mature CD4+ T cells, even as they differentiate to T helper effector subsets. Here we show that the T helper-fate was not fixed and that mature antigen-stimulated CD4+ T cells could terminate Thpok expression and reactivate CD8-lineage genes. This unexpected plasticity resulted in the post-thymic termination of the T helper-program and the functional differentiation of distinct MHC class II-restricted CD4+ cytotoxic T lymphocytes. PMID:23334788

  7. Colchicum autumnale agglutinin activates all murine T-lymphocytes but does not induce the proliferation of all activated cells. (United States)

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


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

  8. Mesenchymal stromal cells improve salivary function and reduce lymphocytic infiltrates in mice with Sjogren's-like disease.

    Directory of Open Access Journals (Sweden)

    Saeed Khalili

    Full Text Available BACKGROUND: Non-obese diabetic (NOD mice develop Sjögren's-like disease (SS-like with loss of saliva flow and increased lymphocytic infiltrates in salivary glands (SGs. There are recent reports using multipotent mesenchymal stromal cells (MSCs as a therapeutic strategy for autoimmune diseases due to their anti-inflammatory and immunomodulatory capabilities. This paper proposed a combined immuno- and cell-based therapy consisting of: A an injection of complete Freund's adjuvant (CFA to eradicate autoreactive T lymphocytes, and B transplantations of MSCs to reselect lymphocytes. The objective of this was to test the effectiveness of CD45(-/TER119(- cells (MSCs in re-establishing salivary function and in reducing the number of lymphocytic infiltrates (foci in SGs. The second objective was to study if the mechanisms underlying a decrease in inflammation (focus score was due to CFA, MSCs, or CFA+MSCs combined. METHODOLOGY/PRINCIPAL FINDINGS: Donor MSCs were isolated from bones of male transgenic eGFP mice. Eight week-old female NOD mice received one of the following treatments: insulin, CFA, MSC, or CFA+MSC (combined therapy. Mice were followed for 14 weeks post-therapy. CD45(-/TER119(- cells demonstrated characteristics of MSCs as they were positive for Sca-1, CD106, CD105, CD73, CD29, CD44, negative for CD45, TER119, CD11b, had high number of CFU-F, and differentiated into osteocytes, chondrocytes and adipocytes. Both MSC and MSC+CFA groups prevented loss of saliva flow and reduced lymphocytic infiltrations in SGs. Moreover, the influx of T and B cells decreased in all foci in MSC and MSC+CFA groups, while the frequency of Foxp3(+ (T(reg cell was increased. MSC-therapy alone reduced inflammation (TNF-α, TGF-β, but the combination of MSC+CFA reduced inflammation and increased the regenerative potential of SGs (FGF-2, EGF. CONCLUSIONS/SIGNIFICANCE: The combined use of MSC+CFA was effective in both preventing saliva secretion loss and reducing

  9. Expansion of NK cells and reduction of NKG2D expression in chronic lymphocytic leukemia. Correlation with progressive disease.

    Directory of Open Access Journals (Sweden)

    Leticia Huergo-Zapico

    Full Text Available The immune system may mediate anti-tumor responses in chronic lymphocytic leukemia (CLL which may affect disease progression and survival. In this study, we analyzed the immune characteristics of 99 consecutive previously diagnosed CLL patients and 50 healthy controls. The distribution of lymphocyte subsets at diagnosis was retrospectively analyzed. Compared with controls, leukemia patients showed an expansion of NK and CD8 T cells at diagnosis. The relative number of CD8 T cells at diagnosis was associated with time to treatment, suggesting that CD8 T cells may modify disease progression. The distribution of lymphocyte subsets was analyzed again when patients were enrolled in this study. The median time since these patients were diagnosed was 277 weeks. Compared with diagnosis, the absolute number of CD8 T cells significantly decreased in these patients, reaching similar values to healthy controls; however NK cells kept significantly elevated overtime. Nevertheless, NK cells showed an impaired expression of NKG2D receptor and a defective cytotoxic activity. This down-regulation of NKG2D expression was further enhanced in patients with advanced and progressive disease. Additionally, membrane NKG2D levels significantly decreased on CD8 T cells, but a significant increase of NKG2D+CD4+ T cells was observed in CLL patients. The cytotoxic activity of NK cells was diminished in CLL patients; however the treatments with IL-2, IL-15, IL-21 and lenalidomide were able to restore their activity. The effect of IL-2 and IL-15 was associated with the increase of NKG2D expression on immune cells, but the effect of IL-21 and lenalidomide was not due to NKG2D up-regulation. The expansion of NK cells and the reversibility of NK cell defects provide new opportunities for the immunotherapeutic intervention in CLL.

  10. Lymphocytes and macrophages are infected by Theileria equi, but T cells and B cells are not required to establish infection in vivo. (United States)

    Ramsay, Joshua D; Ueti, Massaro W; Johnson, Wendell C; Scoles, Glen A; Knowles, Donald P; Mealey, Robert H


    Theileria equi has a biphasic life cycle in horses, with a period of intraleukocyte development followed by patent erythrocytic parasitemia that causes acute and sometimes fatal hemolytic disease. Unlike Theileria spp. that infect cattle (Theileria parva and Theileria annulata), the intraleukocyte stage (schizont) of Theileria equi does not cause uncontrolled host cell proliferation or other significant pathology. Nevertheless, schizont-infected leukocytes are of interest because of their potential to alter host cell function and because immune responses directed against this stage could halt infection and prevent disease. Based on cellular morphology, Theileria equi has been reported to infect lymphocytes in vivo and in vitro, but the specific phenotype of schizont-infected cells has yet to be defined. To resolve this knowledge gap in Theileria equi pathogenesis, peripheral blood mononuclear cells were infected in vitro and the phenotype of infected cells determined using flow cytometry and immunofluorescence microscopy. These experiments demonstrated that the host cell range of Theileria equi was broader than initially reported and included B lymphocytes, T lymphocytes and monocyte/macrophages. To determine if B and T lymphocytes were required to establish infection in vivo, horses affected with severe combined immunodeficiency (SCID), which lack functional B and T lymphocytes, were inoculated with Theileria equi sporozoites. SCID horses developed patent erythrocytic parasitemia, indicating that B and T lymphocytes are not necessary to complete the Theileria equi life cycle in vivo. These findings suggest that the factors mediating Theileria equi leukocyte invasion and intracytoplasmic differentiation are common to several leukocyte subsets and are less restricted than for Theileria annulata and Theileria parva. These data will greatly facilitate future investigation into the relationships between Theileria equi leukocyte tropism and pathogenesis, breed

  11. Lymphocytes and macrophages are infected by Theileria equi, but T cells and B cells are not required to establish infection in vivo.

    Directory of Open Access Journals (Sweden)

    Joshua D Ramsay

    Full Text Available Theileria equi has a biphasic life cycle in horses, with a period of intraleukocyte development followed by patent erythrocytic parasitemia that causes acute and sometimes fatal hemolytic disease. Unlike Theileria spp. that infect cattle (Theileria parva and Theileria annulata, the intraleukocyte stage (schizont of Theileria equi does not cause uncontrolled host cell proliferation or other significant pathology. Nevertheless, schizont-infected leukocytes are of interest because of their potential to alter host cell function and because immune responses directed against this stage could halt infection and prevent disease. Based on cellular morphology, Theileria equi has been reported to infect lymphocytes in vivo and in vitro, but the specific phenotype of schizont-infected cells has yet to be defined. To resolve this knowledge gap in Theileria equi pathogenesis, peripheral blood mononuclear cells were infected in vitro and the phenotype of infected cells determined using flow cytometry and immunofluorescence microscopy. These experiments demonstrated that the host cell range of Theileria equi was broader than initially reported and included B lymphocytes, T lymphocytes and monocyte/macrophages. To determine if B and T lymphocytes were required to establish infection in vivo, horses affected with severe combined immunodeficiency (SCID, which lack functional B and T lymphocytes, were inoculated with Theileria equi sporozoites. SCID horses developed patent erythrocytic parasitemia, indicating that B and T lymphocytes are not necessary to complete the Theileria equi life cycle in vivo. These findings suggest that the factors mediating Theileria equi leukocyte invasion and intracytoplasmic differentiation are common to several leukocyte subsets and are less restricted than for Theileria annulata and Theileria parva. These data will greatly facilitate future investigation into the relationships between Theileria equi leukocyte tropism and pathogenesis

  12. Derivation and characterization of a pig embryonic stem cell-derived exocrine pancreatic cell line (United States)

    The establishment and initial characterization of a pig embryonic stem cell-derived pancreatic cell line, PICM-31, and a colony-cloned derivative cell line, PICM-31A, is described. The cell lines were propagated for several months at split ratios of 1:3 or 1:5 at each passage on STO feeder cells af...

  13. Occurrence of nodular lymphocyte-predominant hodgkin lymphoma in hermansky-pudlak type 2 syndrome is associated to natural killer and natural killer T cell defects.

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    Luisa Lorenzi

    Full Text Available Hermansky Pudlak type 2 syndrome (HPS2 is a rare autosomal recessive primary immune deficiency caused by mutations on β3A gene (AP3B1 gene. The defect results in the impairment of the adaptor protein 3 (AP-3 complex, responsible for protein sorting to secretory lysosomes leading to oculo-cutaneous albinism, bleeding disorders and immunodeficiency. We have studied peripheral blood and lymph node biopsies from two siblings affected by HPS2. Lymph node histology showed a nodular lymphocyte predominance type Hodgkin lymphoma (NLPHL in both HPS2 siblings. By immunohistochemistry, CD8 T-cells from HPS2 NLPHL contained an increased amount of perforin (Prf + suggesting a defect in the release of this granules-associated protein. By analyzing peripheral blood immune cells we found a significant reduction of circulating NKT cells and of CD56(brightCD16(- Natural Killer (NK cells subset. Functionally, NK cells were defective in their cytotoxic activity against tumor cell lines including Hodgkin Lymphoma as well as in IFN-γ production. This defect was associated with increased baseline level of CD107a and CD63 at the surface level of unstimulated and IL-2-activated NK cells. In summary, these results suggest that a combined and profound defect of innate and adaptive effector cells might explain the susceptibility to infections and lymphoma in these HPS2 patients.

  14. Dynamic visualization the whole process of cytotoxic T lymphocytes killing the B16 tumor cells in vitro (United States)

    Qi, Shuhong; Zhang, Zhihong


    Cytotoxic T lymphocytes (CTLs) played a key role in the immune system to destroy the tumor cells. Although some mechanisms of CTLs killing the tumor cells are revealed already, the dynamic information of CTLs interaction with tumor cells are still not known very clearly. Here we used confocal microscopy to visualize the whole process of CTLs killing the tumor cells in vitro. The imaging data showed that CTLs destroyed the target tumor cells rapidly and efficiently. Several CTLs surrounded one or some tumor cells and the average time for CTLs destroying one tumor cell is just a few minutes in vitro. The study displayed the temporal events of CTLs interacting with tumor cells at the beginning and finally killing them and directly presented the efficient tumor cell cytotoxicity of the CTLs. The results helped us to deeply understand the mechanism of the CTLs destroying the tumor cells and to develop the cancer immunotherapy.

  15. In vitro and in vivo analyses of a genetically—restricted antigen specific factor from mixed cell cultures of macrophage,T and B lymphocytes

    Institute of Scientific and Technical Information of China (English)



    An immunostimulatory factor was identified to be secreted by antigen-pulsed macrophages.This factor was able to induce the generation of antigen specific T helper lymphocytes in vitro as well as in vivo.Further in vitro experiments testing for the genetic restriction of this factor indicated that it is a geneticallyrestricted antigen specific factor (ASF).The Cunningham plaque assay was used to quantify the generation of T helper lymphocytes by measuring the number of plaque forming cells after sequential incubations of antigen-qulsed macrophages with T lymphocytes,and then spleen cells,and finally the TNP-coated sheep red blood cells.

  16. Tumor infiltrating lymphocytes in melanoma comprise high numbers of T-cell clonotypes that are lost during in vitro culture

    DEFF Research Database (Denmark)

    thor Straten, P; Kirkin, A F; Siim, E


    Melanoma is generally accepted as being an antigenic tumor capable of eliciting T-cell responses that, however, in most cases are inadequate to control tumor growth. Tumor-infiltrating lymphocytes (TIL) in melanoma lesions comprise clonotypic T cells, indicating the in situ recognition of melanoma......-associated peptide epitopes. Cultured TIL have been studied in order to unveil characteristics of TIL and the interactions of TIL and melanoma cells. Whether in vitro cultured TIL mirrors the in situ situation has, however, been questioned. In the present study we have taken advantage of T-cell receptor clonotype...

  17. UV-induced chromosome aberrations, sister-chromatid exchanges and cell survival in cultured lymphocytes from malnourished children

    Energy Technology Data Exchange (ETDEWEB)

    Murthy, P.B.; Rahiman, M.A.; Tulpule, P.G.


    Cultured lymphocytes from children with kwashiorkor and from normal children were examined for their susceptibility to ultraviolet (UV)-induced chromosome aberrations, sister-chromatid exchanges and cell survival. Cells from kwashiorkor exhibited increased chromosome aberrations, but not sister-chromatid exchanges, when exposed to higher doses of UV. Furthermore, when cells from these patients were exposed to higher doses of UV, there was a significant reduction in viability. These results indicate that, as compared to normals, cells from kwashiorkor were more sensitive to the lethal effects of UV.

  18. Two structurally identical mannose-specific jacalin-related lectins display different effects on human T lymphocyte activation and cell death. (United States)

    Benoist, Hervé; Culerrier, Raphaël; Poiroux, Guillaume; Ségui, Bruno; Jauneau, Alain; Van Damme, Els J M; Peumans, Willy J; Barre, Annick; Rougé, Pierre


    Plant lectins displaying similar single sugar-binding specificity and identical molecular structure might present various biological effects. To explore this possibility, the effects on human lymphocytes of two mannose-specific and structurally closely related lectins, Morniga M from Morus nigra and artocarpin from Artocarpus integrifolia were investigated. In silico analysis revealed that Morniga M presents a more largely open carbohydrate-binding cavity than artocarpin, probably allowing interactions with a broader spectrum of carbohydrate moieties. In vitro, Morniga M interacted strongly with the lymphocyte surface and was uptaken quickly by cells. Morniga M and artocarpin triggered the proliferation and activation of human T and NK lymphocytes. A minority of B lymphocytes was activated in artocarpin-treated culture, whereas Morniga M favored the emergence of CD4+ CD8+ T lymphocytes. Moreover, cell death occurred in activated PBMC, activated T lymphocytes, and Jurkat T leukemia cells incubated with Morniga M only. The biological effects of both lectins were dependent on carbohydrate recognition. The Morniga M-induced cell death resulted, at least in part, from caspase-dependent apoptosis and FADD-dependent receptor-mediated cell death. Finally, Morniga M, but not artocarpin, triggered AICD of T lymphocytes. In conclusion, both lectins trigger lymphocyte activation, but only Morniga M induces cell death. In spite of similar in vitro mannose-binding specificities and virtually identical structure, only Morniga M probably interacts with carbohydrate moieties bound to molecules able to induce cell death. The present data suggest that subtle alterations in N-glycans can distinguish activation and cell death molecules at the lymphocyte surface.

  19. Heterogeneous X-ray survival characteristics of lymphocytes in prolymphocytic leukaemia. Mathematical analysis distinguishing delayed cell death and true radioresistance

    Energy Technology Data Exchange (ETDEWEB)

    Thomson, A.E.R.; O' Connor, T.W.E.; Slater, N.G.P. (Division of Haematology, United Medical and Dental Schools of Guy' s and St. Thomas' Hospitals, London (United Kingdom)); Peel, W.E. (Computing Services, Manchester Metropolitan University, Manchester (United Kingdom))


    The survival of non-dividing (G[sub 0]) leukaemic lymphocytes in culture is generally too short for their radiosensitivity to be quantitatively assessed, since lethally X-irradiated cells may show a long delay before manifestations of cell death ('interphase death') are seen. Counts of surviving cells well therefore include both lethally-hit cells (apparent survivors), and real survivors which have not been lethally hit. Death rates of irradiated leukaemic and normal cells show great variation between individuals, so that comparisons of radiosensitivity between different cell populations based on surviving cell counts at a single time-point are invalid. In this study the supposed radioresistance of prolymphocytic leukaemia lymphocytes was examined in 6 patients with B-cell disease. Survival curves were plotted from serial observations made over several days after graded X-irradiation (0-1000 cGy). We attempted to interpret these radiation responses in terms of their dose dependence (intrinsic radiosensitivity) and time dependence (cell death rate) characteristics using the best-fitting of four mathematical models, all based on classical 'single-hit' target theory. The apparent radioresistance shown in 4 cases could be explained by very slow death rates (T[sub 172] values 55-205 h) of cells proving otherwise radiosensitive (D37 values 38-123 cGy). Genuine radioresistance was found in only 1 case (actual D37 value above 2000 cGy). B ignoring delayed cell death in clinical assessments, pathological lymphocytes could be mistakenly categorized as resistant to elimination by radiotherapy. (au) (30 refs.).

  20. Cellular radiosensitivity of small-cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Krarup, M; Poulsen, H S; Spang-Thomsen, M


    . The multitarget single hit model was applied to calculate the cellular radiosensitivity (D0), the capacity for sublethal damage repair (Dq), and the extrapolation number (n). Values for alpha and beta were determined from best-fit curves according to the linear-quadratic model and these values were applied...... to calculate the surviving fraction after 2-Gy irradiation (SF2). RESULTS: In our investigation, the extrapolation method proved to be inappropriate for the study of in vitro cellular radiosensitivity due to lack of reproducibility. The results obtained by the clonogenic assay showed that the cell lines...

  1. Authentication of the R06E Fruit Bat Cell Line

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    Ingo Jordan


    Full Text Available Fruit bats and insectivorous bats are believed to provide a natural reservoir for a wide variety of infectious diseases. Several lines of evidence, including the successful isolation of infectious viruses, indicate that Marburg virus and Ravn virus have found a major reservoir in colonies of the Egyptian rousette (Rousettus aegyptiacus. To facilitate molecular studies on virus-reservoir host interactions and isolation of viruses from environmental samples, we established cell lines from primary cells of this animal. The cell lines were given to several laboratories until we realized that a contamination with Vero cells in one of the cultures had occurred. Here we describe a general diagnostic procedure for identification of cross-species contamination with the focus on Vero and Rousettus cell lines, and summarize newly discovered properties of the cell lines that may pertain to pathogen discovery.

  2. The immunodeficiency of bone marrow-transplanted patients. The effect of patient lymphocytes on the response of donor lymphocytes to mitogens and allogeneic cells

    DEFF Research Database (Denmark)

    Ødum, Niels; Hofmann, B; Platz, P


    Lymphocytes from patients after bone marrow transplantation (BMT) are in most cases predominantly of the Leu-2+ (cytotoxic/suppressor) phenotypes and are almost unresponsive to mitogens. In contrast, normal Leu-3+-depleted, Leu-2+-enriched lymphocyte suspensions retain approximately 50...

  3. Iso-suillin from Suillus flavus Induces Apoptosis in Human Small Cell Lung Cancer H446 Cell Line. (United States)

    Zhao, Jun-Xia; Zhang, Qing-Shuang; Chen, Ying; Yao, Sheng-Jie; Yan, Yong-Xin; Wang, Ying; Zhang, Jin-Xiu; Wang, Li-An


    The suillin isoform iso-suillin is a natural substance isolated from a petroleum ether extract of the fruiting bodies of the mushroom Suillus flavus. Previous studies have found its inhibition effect on some cancer cells, and we aimed to study its effects on human small cell lung cancer H446 cell line. Cell viability was measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. Cellular morphological changes (apoptosis and necrosis) were evaluated using an electron microscope and Hoechst 33258 staining detected by the inverted microscope. Flow cytometry was used to detect cell apoptosis, cell cycle distribution, and mitochondrial membrane potential. Protein expression was determined by Western blotting analysis. Here, we describe the ability of iso-suillin to inhibit the growth of H446 cells in time- and dose-dependent way. Iso-suillin had no obvious impact on normal human lymphocyte proliferation at low concentrations (9.09, 18.17, or 36.35 μmol/L) but promoted lymphocyte proliferation at a high concentration (72.70 μmol/L). After treatment of different concentrations of iso-suillin (6.82, 13.63, or 20.45 μmol/L), the apoptosis rate of H446 cells increased with increasing concentrations of iso-suillin (16.70%, 35.54%, and 49.20%, respectively, all P iso-suillin could induce H446 cell apoptosis through the mitochondrial pathway and the death-receptor pathway. Therefore, iso-suillin might have a potential application as a novel drug for lung cancer treatment.

  4. [Epstein-Barr Virus LMP1 oncogene variants in cell lines of different origin]. (United States)

    Yakovleva, L S; Senyuta, N B; Goncharova, E V; Scherback, L N; Smirnova, R V; Pavlish, O A; Gurtsevitch, V E


    It is well known that the Epstein-Barr virus (EBV) is a widespread infection in the human population. Typically, infection occurs in early childhood without serious consequences for infected people. At the same time, a secondary infection with an additional EBV strain occurs quite often. During the in vitro cultivation of peripheral blood lymphocytes from persons infected with multiple strains of the virus, only one of these strains with higher transforming potential becomes dominant, while the others are eliminated. Under certain conditions, such a highly transforming EBV strain apparently is able to be the etiologic agent of EBVassociated diseases. To find out the range of highly transforming EBV strains prevalent among Russians, cell lines from patients with EBV-associated and non-associated tumors, as well as healthy individuals, were established. The structural analysis of the latent membrane protein 1 gene (LMP1), a key oncogene of the virus, isolated from established cell lines and peripheral blood lymphocytes of blood donors was carried out, and data obtained were compared with the respective data for LMP1 isolates, amplified from cell lines established from African and Japanese patients with Burkitt's lymphoma. The data obtained show a genetic relationship between Russian LMP1 isolates regardless the fact whether they come from patients with tumors or healthy individuals and differ significantly from LMP1 isolates from Burkitt's lymphoma patients. Thus, the results of the study suggest that in nonendemic region for EBV-associated pathology, Russia, any strain of EBV with any structure of LMP1 with concomitant effect of additional factors may become an etiologic agent for EBV-associated neoplasia.

  5. Cellular Immunotherapy Following Chemotherapy in Treating Patients With Recurrent Non-Hodgkin Lymphomas, Chronic Lymphocytic Leukemia or B-Cell Prolymphocytic Leukemia (United States)


    Post-transplant Lymphoproliferative Disorder; B-Cell Prolymphocytic Leukemia; Recurrent Adult Burkitt Lymphoma; Recurrent Adult Diffuse Large Cell Lymphoma; Recurrent Grade 1 Follicular Lymphoma; Recurrent Grade 2 Follicular Lymphoma; Recurrent Grade 3 Follicular Lymphoma; Recurrent Mantle Cell Lymphoma; Recurrent Marginal Zone Lymphoma; Recurrent Small Lymphocytic Lymphoma; Refractory Chronic Lymphocytic Leukemia; Refractory Hairy Cell Leukemia; B-Cell Lymphoma, Unclassifiable, With Features Intermediate Between Diffuse Large B-Cell Lymphoma and Burkitt Lymphoma; B-Cell Lymphoma, Unclassifiable, With Features Intermediate Between Diffuse Large B-Cell Lymphoma and Classical Hodgkin Lymphoma; Recurrent Lymphoplasmacytic Lymphoma

  6. Distribution of T-cell receptor-bearing lymphocytes in the synovial membrane from patients with rheumatoid arthritis. (United States)

    Chaouni, I; Radal, M; Simony-Lafontaine, J; Combe, B; Sany, J; Rème, T


    Using immunohistology and monoclonal antibodies directed to the T-cell receptor (TCR) chains, we have analysed the distribution of TCR-bearing lymphocytes within the membrane of rheumatoid arthritis (RA) patients. Alkaline phosphatase staining for TCR alpha beta-bearing lymphocytes showed a distribution paralleling that of the total T cells. Staining for the TCR gamma delta chains revealed a moderate and rather homogeneous distribution of T gamma delta lymphocytes within the RA synovium. As evidenced by simultaneous staining for alpha beta and gamma delta receptors, the relative count of T gamma delta to alpha beta-expressing cells is close to the peripheral count (e.g.5%), and lower than that previously observed in the synovial fluid. Interestingly, the peripheral type V gamma 9-J gamma P rearrangement using the T gamma delta cell subset was relatively decreased in the synovial membrane, as compared to synovial fluid and peripheral blood, suggesting that the T gamma delta distribution in the rheumatoid synovium resembles a thymic-like situation.

  7. Mechanisms of action of the anti-VEGF monoclonal antibody bevacizumab on chronic lymphocytic leukemia cells

    Directory of Open Access Journals (Sweden)

    Jakub Bogusz


    Full Text Available Introduction: Chronic lymphocytic leukemia (CLL remains incurable; therefore searching for new therapeutic strategies in this disease is necessary. An important mechanism of tumor development is neoangiogenesis. A potent antiangiogenic factor, bevacizumab (Avastin, AVA, has been poorly explored in CLL so far. In the current study we assessed cytotoxic activity of AVA alone or in combinations with drugs routinely used in this disease.Matherials and Methods: Cells isolated from 60 CLL patients were treated with AVA alone or in combination with anti-CD20 monoclonal antibody (MoAb, rituximab (RIT, anti-CD52 MoAb, alemtuzumab (ALT, 2-CdA (2-chlorodeoxyadenosine, FA (fludarabine, MAF (mafosfamide or RAPA (rapamycin. Cytotoxicity was assessed by propidium iodide staining. Apoptosis was evaluated using annexin-V and TUNEL assays. Additionally, a drop of mitochondrial potential (DYm as well as expression of apoptosis-regulating proteins Bax, Bak, Bid, Bad, Bcl-2, Mcl-2, XIAP, FLIP, Akt and Bcl-2-A1 were determined by flow cytometry.Results: At the dose of 40 μg/ml, after 48 hours of incubation, AVA induced significant cytotoxicity against CLL cells. The drug triggered apoptosis, with activation of caspase-3 and -9, but not caspase-8, along with a drop of DYm. Incubation with AVA induced significant overexpression of proapoptotic Bak and Bad as well as downregulation of antiapoptotic Mcl-2 and Akt proteins. Combination of AVA with RIT, ALT or RAPA significantly increased cytotoxicity when compared with the effects of single drugs.Discussion: In conclusion, this is the first report showing proapoptotic activity of AVA against CLL cells. Combination of AVA with RIT, ALT or RAPA may be a promising therapeutic strategy, which requires confirmation in further studies.

  8. Mutation Pattern of Paired Immunoglobulin Heavy and Light Variable Domains in Chronic Lymphocytic Leukemia B Cells

    KAUST Repository

    Ghiotto, Fabio


    B-cell chronic lymphocytic leukemia (CLL) patients display leukemic clones bearing either germline or somatically mutated immunoglobulin heavy variable (IGHV ) genes. Most information on CLL immunoglobulins (Igs), such as the definition of stereotyped B-cell receptors (BCRs), was derived from germline unmutated Igs. In particular, detailed studies on the distribution and nature of mutations in paired heavy- and light-chain domains of CLL clones bearing mutated Igs are lacking. To address the somatic hyper-mutation dynamics of CLL Igs, we analyzed the mutation pattern of paired IGHV-diversity-joining (IGHV-D-J ) and immunoglobulin kappa/lambda variable-joining (IGK/LV-J ) rearrangements of 193 leukemic clones that displayed ≥ 2% mutations in at least one of the two immunoglobulin variable (IGV ) genes (IGHV and/or IGK/LV ). The relationship between the mutation frequency in IGHV and IGK/LV complementarity determining regions (CDRs) and framework regions (FRs) was evaluated by correlation analysis. Replacement (R) mutation frequency within IGK/LV chain CDRs correlated significantly with mutation frequency of paired IGHV CDRs in λ but not κ isotype CLL clones. CDRs of IGKV-J rearrangements displayed a lower percentage of R mutations than IGHVs. The frequency/pattern of mutations in kappa CLL Igs differed also from that in κ-expressing normal B cells described in the literature. Instead, the mutation frequency within the FRs of IGHV and either IGKV or IGLV was correlated. Notably, the amount of diversity introduced by replaced amino acids was comparable between IGHVs and IGKVs. The data indicate a different mutation pattern between κ and λ isotype CLL clones and suggest an antigenic selection that, in κ samples, operates against CDR variation.

  9. TAP-deficient human iPS cell-derived myeloid cell lines as unlimited cell source for dendritic cell-like antigen-presenting cells. (United States)

    Haruta, M; Tomita, Y; Yuno, A; Matsumura, K; Ikeda, T; Takamatsu, K; Haga, E; Koba, C; Nishimura, Y; Senju, S


    We previously reported a method to generate dendritic cell (DC)-like antigen-presenting cells (APC) from human induced pluripotent stem (iPS) cells. However, the method is relatively complicated and laborious. In the current study, we attempted to establish a method through which we could obtain a large number of functional APC with a simple procedure. We transduced iPS cell-derived CD11b(+) myeloid cells with genes associated with proliferative or anti-senescence effects, enabling the cells to propagate for more than 4 months in a macrophage colony-stimulating factor (M-CSF)-dependent manner while retaining their capacity to differentiate into functional APC. We named these iPS cell-derived proliferating myeloid cells 'iPS-ML', and the iPS-ML-derived APC 'ML-DC'. In addition, we generated TAP2-deficient iPS cell clones by zinc finger nuclease-aided targeted gene disruption. TAP2-deficient iPS cells and iPS-ML avoided recognition by pre-activated allo-reactive CD8(+) T cells. TAP2-deficient ML-DC expressing exogenously introduced HLA-A2 genes stimulated HLA-A2-restricted MART-1-specific CD8(+) T cells obtained from HLA-A2-positive allogeneic donors, resulting in generation of MART-1-specific cytotoxic T lymphocyte (CTL) lines. TAP-deficient iPS-ML introduced with various HLA class I genes may serve as an unlimited source of APC for vaccination therapy. If administered into allogeneic patients, ML-DC with appropriate genetic modifications may survive long enough to stimulate antigen-specific CTL and, after that, be completely eliminated. Based on the present study, we propose an APC-producing system that is simple, safe and applicable to all patients irrespective of their HLA types.

  10. Kisspeptin Effect on Endothelial Monocyte Activating Polypeptide II (EMAP-II)-Associated Lymphocyte Cell Death and Metastases in Colorectal Cancer Patients (United States)

    Stathaki, Martha; Armakolas, Athanasios; Dimakakos, Andreas; Kaklamanis, Loukas; Vlachos, Ioannis; Konstantoulakis, Manoussos M; Zografos, George; Koutsilieris, Michael


    Kisspeptin is an antimetastatic agent in some cancers that has also been associated with lymphoid cell apoptosis, a phenomenon favoring metastases. Our aim was to determine the association of kisspeptin with lymphocyte apoptosis and the presence of metastases in colorectal cancer patients. Blood was drawn from 69 colon cancer patients and 20 healthy volunteers. Tissue specimens from healthy and pathological tissue were immunohistochemically analyzed for kisspeptin and endothelial monocyte activating polypeptide II (EMAP-II) expression. Blood EMAP-II and soluble Fas ligand (sFasL) le